The use of Diathermic Syncope® for stunning cattle

TL;DR

EFSA evaluated Diathermic Syncope® for stunning cattle and found it does not meet current animal welfare standards.

Contribution

This paper provides a scientific assessment of a novel cattle stunning method against established welfare criteria.

Findings

The data provided only partially fulfilled the necessary criteria for approval.

DTS does not ensure a level of welfare equivalent to currently allowed methods.

The assessment followed EFSA's guidance and included expert elicitation and literature review.

Abstract

The EFSA Panel on Animal Health and Welfare (AHAW) was asked to deliver a scientific opinion on the use of Diathermic Syncope® (DTS) for stunning cattle. A dossier was provided by the applicant as the basis for an assessment of the extent to which the method is able to provide a level of animal welfare at least equivalent to that ensured by the currently allowed methods for stunning cattle. This scientific opinion followed the EFSA Guidance (2018) on the assessment criteria for applications for new or modified stunning methods regarding animal protection at time of killing. Under Council Regulation (EC) No 1099/2009, approval of novel stunning methods requires demonstration of (1) the absence of pain, distress or suffering until the onset of unconsciousness and (2) that the animal remains unconscious until death. An ad hoc Working Group (WG) by EFSA performed the assessment as follows: (1) check of provided data against the criteria laid down in the EFSA Guidance; (2) extensive literature search; (3) data extraction and quantitative assessment; (4) exercise based on non‐formal expert elicitation and qualitative assessment. Although the data and studies provided in the dossier only partially fulfilled the necessary criteria, they were sufficient to proceed with the animal welfare risk assessment. According to the data and the use of DTS parameterised by the applicant (delivering 160–200 kJ of energy and an incident power of 16–20 kW for 10 s), DTS does not ensure a level of welfare at least equivalent to one or more of the currently allowed methods listed in Annex I of Council Regulation (EC) No 1099/2009.

Figures

DTS: Diathermic Syncope®: Flowchart representation of development timeline, identifying the nature of each STEP. (Source: Figure 21 of the dossier).

Apparatus Overview (animal restraint not shown). (Source: Figure 1 of the dossier).

Interface panel and close‐up of result screen following a DTS stun. (Source: Figure 2 of the dossier).

Schematic diagram of rotating box and ejector system. This front view shows the restraint and transfer from ejection system onto bleed conveyor. The position of the animal depicted while being upright prior to DTS application (right) and neck cutting applied in inverted position, and while being ejected onto the bleed conveyor (left) in the tipped catching cradle post exsanguination (middle). (Source: Figure 3 of the dossier: ‘Schematic diagram of restraint and ejection system onto bleed conveyor, front elevation. One animal is upright prior to DTS application (right), one is in the catching cradle post exsanguination (middle) and a third is on the bleed conveyor (left)’).

Rear view of the animal entrance to the rotating restraint box. The upright side is shown on the left and the inverted side on the right. Image taken during workshop construction by the applicant.

View of the neck capture area with the side and the ‘top’ doors opened to allow animal ejection when inverted. In this image, the upright side is on the right, and the inverted side is on the left. Image taken during workshop construction by the applicant.

View of the inverted part of the rotating restraint box, showing the side and ‘top’ doors opened to allow animal ejection when inverted. Image taken during workshop construction by the applicant. (Source: Figure 8 of the dossier).

3D representation of the rotating restraint box within the Faraday cage. Personnel access steps are shown on the right, and the bleed conveyor is positioned on the left. (Source: Figure 9 of the dossier).

3D representation of the rotating restraint box within the Faraday cage shown from the rear to illustrate personnel access. (Source: Figure 10 of the dossier).

Diagram showing the placement of the applicator on the bovine head. Left: Frontal view, hatched area shows applicator position; Right: sagittal view, with the hatched area representing the applicator and its position relative to the brain and frontal sinuses (black). (Source: Figure 11 of the dossier).

Applicator positioning on the forehead of the animal. (Source: Figure 12 of the dossier). (A) Upper left: Positioning of the head in relation to reference points. (B) Upper right: Head positioned and ready for applicator placement. (C) Lower left: Placing the applicator through the hatch in the Faraday cage. (D) Lower right: Final applicator position.

Animal secured in the restraint with the chin lift activated and the applicator in position, immediately prior to DTS energy application. (Source: Figure 13 of the dossier).

Cumulative energy delivered during 12 s of DTS application (Source: Figure 14 of the dossier: ‘Application of microwave during DTS stunning – power (kJ/sec), and total energy delivery over time’).

Animal being exsanguinated through a hatch in the Faraday cage. (Source: Figure 16 of the dossier).

Diagram representing a sinusoidal wave in the Time IQ plane. The I component is called inphase because it is in phase with cos(⋅), while Q component is called quadrature because sin(⋅) is in quadrature, i.e. 90° apart, with cos(⋅). Source: © Wireless Pi 2026 https://wirelesspi.com(https://wirelesspi.com/the‐concept‐of‐frequency/ Figure 18 of the dossier).

Schematic diagram of energy application (orange) and expanding neuro‐electrical field (purple) (Source: Figure 10 of the dossier).

Schematic diagrams of heat transfer within the brain (A) via conduction and (B) via circulation (Source: Figure 20 of the dossier).

Fixed eyes or display of eye white occurs in conscious cattle during heat stroke. Source: Courtesy of Dr. Shanker K. Singh. Heat Stroke in Cow? Hyperthermia! Cause and Treatment? Dr. Shanker – VetVirtual Classes. https://www.youtube.com/watch?v=G38Izvaot6Q, accessed on 31 October 2025.

Tables

TABLE 1. Overview of translation of the mandate assessment questions into sub‐questions.

Assessment question	Sub‐questions
1. Describe the new method	1.1 What is the primary objective/claim of the method?
	1.2 How does the new method work and what are key steps to achieve its primary objective?
	1.3 What materials and tools are required for the new method?
2. Does the proposed method and information provided with the application comply with the eligibility criteria of EFSA's guidelines?
3. Can the proposed method provide a level of animal welfare at least equivalent to that ensured by the existing methods in the legislation?	3.1 Can the proposed method provide a level of AW at least equivalent with the methods that exist in Annex I of Council Regulation (EC) 1099/2009 for simple stunning of cattle?
	3.2 Does the proposed method ensure that cattle are spared of avoidable pain, distress or suffering during the killing as referred to in paragraph 1 of Article 3 of the killing Regulation?
	3.3 Does the proposed method maintain the loss of consciousness and sensibility until death of cattle, as referred to in paragraph 1 of Article 4 of Council Regulation (EC) 1099/2009?
4. To what extent the information provided by the applicant is taken into account for the final assessment and if the method is evaluated by other sources of information.
5. To what extent the method proposed for simple stunning of cattle is able to provide a level of AW in compliance with the above‐mentioned legislation and, if yes, under which conditions.

TABLE 2. List of publications retrieved after literature search.

	Authors	Title	Journal/Thesis	Year	Doi
1	Gibson, TJ; Oliveira, SEO; Dalla Costa, FA; Gregory, NG	Electroencephalographic assessment of pneumatically powered penetrating and non‐penetrating captive‐bolt stunning of bulls	Meat Science	2019	https://doi.org/10.1016/j.meatsci.2019.01.006
2	Lücking, A; Louton, H; von Wenzlawowicz, M; Erhard, M; von Holleben, K	Movements after Captive Bolt Stunning in Cattle and Possible Animal‐ and Process‐Related Impact Factors‐A Field Study	Animals	2024	https://doi.org/10.3390/ani14071112
3	Neves, JEG; da Costa, MJRP; Roça, RO; Faucitano, L; Gregory, NG	A note comparing the welfare of Zebu cattle following three stunning‐slaughter methods	Meat Science	2016	https://doi.org/10.1016/j.meatsci.2016.02.033
4	Nielsen, SS; Alvarez, J; Bicout, DJ; Calistri, P; Depner, K; Drewe, JA; Garin‐Bastuji, B; Rojas, JLG; Schmidt, CG; Michel, V; Chueca, MAM; Roberts, HC; Sihvonen, LH; Spoolder, H; Stahl, K; Velarde, A; Viltrop, A; Candiani, D; Van der Stede, Y; Winckler, C	Welfare of cattle at slaughter	EFSA Journal	2020	https://doi.org/10.2903/j.efsa.2020.6275
5	Oliveira, SEO; Dalla Costa, FA; Gibson, TJ; Dalla Costa, OA; Coldebella, A; Gregory, NG	Evaluation of brain damage resulting from penetrating and non‐penetrating stunning in Nelore Cattle using pneumatically powered captive bolt guns	Meat Science	2022	https://doi.org/10.1111/asj.13728
6	Vecerek, V; Kamenik, J; Voslarova, E; Vecerkova, L; Machovcova, Z; Volfova, M; Konvalinova, J	The occurrence of reflexes and reactions in cattle following stunning with a captive bolt at the slaughterhouse	Animal Science Journal	2020	https://doi.org/10.1111/asj.13373
7	Vecerek, V; Kamenik, J; Voslarova, E; Volfova, M; Machovcova, Z; Konvalinova, J; Vecerkova, L	The Impact of Deviation of the Stun Shot from the Ideal Point on Motor Paralysis in Cattle	Animals	2020	https://doi.org/10.3390/ani10020280
8	Verhoeven, MTW; Gerritzen, MA; Hellebrekers, LJ; Kemp, B	Validation of indicators used to assess unconsciousness in veal calves at slaughter	Animal	2021	https://www.sciencedirect.com/science/article/pii/S1751731116000422?via%3Dihub
9	von Wenzlawowicz, M; von Holleben, K; Eser, E	Identifying reasons for stun failures in slaughterhouses for cattle and pigs: a field study	Animal welfare	2017	https://doi.org/10.7120/096272812X13353700593527
10	Blackmore, DK; Newhook, JC	Electroencephalographic studies of stunning and slaughter of sheep and calves. 3. The duration of insensibility induced by electrical stunning in sheep and calves	Meat Science	2013	https://doi.org/10.7120/09627286.22.2.287
11	Endres, JM	Effectiveness of Concussion Stunning in Comparison to Captive Bolt Stunning in Routine Slaughtering of Cattle.	PhD Thesis, Ludwig‐Maximilians‐Universität München, Munich, Germany	2005	https://edoc.ub.uni‐muenchen.de/4344/1/Endres_Josef_Markus.pdf
12	Gregory, NG; Lee, CJ; Widdicombe, JP	Depth of concussion in cattle shot by penetrating captive bolt.	Meat Science	2007	https://doi.org/10.1016/j.meatsci.2007.04.026
13	Oliveira, SEO; Gregory, NG, Dalla Costa, FA; Gibson, TJ; Dalla Costa, OA; Paranhos da Costa, MJR	Effectiveness of pneumatically powered penetrating and non‐penetrating captive bolts in stunning cattle.	Meat Science	2018	https://doi.org/10.1016/j.meatsci.2018.02.010
14	Dörfler, K	Assessment of the Efficiency of Different Captive Bolt Stunning Devices in Cattle and Their Influence on the Degree of Bleeding.	Ph.D. Thesis, Universität Leipzig, Leipzig, Germany	2015

TABLE 3. Scoring method used to elicit the impact to cattle caused by each hazard in each of the identified stunning methods.

Score	Impact on animal welfare	Interpretation from the experts
1	No negative impact	No stress
2		Mild stress and no pain regardless of the duration and prevalence
3		Moderate stress and no pain regardless of the duration and prevalence
4		Mild pain with short duration (< 2 s) and with a low prevalence (< 10%).
5		Moderate pain with a short duration (< 2 s) and low prevalence (< 10%) or mild pain for a long duration (> 2 s) or high prevalence (> 10%).
6		Severe pain for short duration (< 2 s) and low prevalence (< 10%) or moderate pain with long duration (> 2 s) or high prevalence (> 10%).
7	Very negative impact	Severe pain with long duration (> 2 s) or/and high prevalence (> 10%).

TABLE 4. Results of the data check of the provided studies (Section 3.2) for risk assessment of the use of Diathermic Syncope® for stunning cattle according to EFSA Guidelines (2018).

	STEP 3	STEP 5	STEP 7	STEP 9	STEP 11	SUB‐STEP 13.2	SUB‐STEP 13.3	SUB‐STEP 13.4
Method
Study population
Sampling strategy
Experimental design
Ethical considerations
Randomisation and blinding
Reporting the methods of analysis
Measurement of the outcomes
Onset and duration of unconsciousness
Time to death	NA	NA	NA	NA	NA		NA	NA
EEG parameters							NA	NA
ABMs Vocalisation	NA	NA	NA			NA	NA	NA
ABMs Postures and movements	NA	NA	NA
ABMs General behaviour	NA	NA	NA
ABMs Hormone concentrations	NA	NA	NA			NA	NA	NA
ABMs Blood metabolites	NA	NA	NA			NA	NA	NA
ABMs Autonomic responses	NA		NA			NA	NA	NA
Pain magnitude (duration and severity)	NA	NA	NA			NA		NA
Correlation neurological and other ABMs	NA	NA	NA					NA
Reporting outcomes and estimations
Proportion of animals mis‐stunned	NA	NA		NA
Time to onset of unconsciousness
Duration of pain, distress and suffering	NA	NA	NA					NA
Magnitude of pain, distress and suffering	NA	NA	NA					NA
Duration unconsciousness
Frequency of animals recovering consciousness before death	NA	NA	NA		NA	NA	NA	NA
Time to onset of death due to bleeding	NA	NA	NA	NA	NA		NA	NA
Proportion of death animals due to DTS	NA	NA	NA	NA	NA	NA	NA	NA
Stun‐to‐stick interval	NA	NA	NA	NA	NA		NA	NA
Adverse events

TABLE 5. Hazard outcome table on application of energy, according to the dossier.

Hazard	WC(s) occurring to the cattle due to the hazard	Hazard origin(s)	Hazard origin specification	Preventive measures	Corrective measures
Restraint and/or inappropriate restraint	Pain, fear	Staff, equipment	– Immobilisation of the animal and presentation of the head of the animal to the operator are required – Lack of skilled operators	Use optimum pressure on the head and the body according to the size of the animal	– Keep the duration of restraint to minimum – Adjust the chin lift
Incorrect placement of applicator	Pain, fear	Staff	– Lack of skilled operators – Operator fatigue – Poor restraint – Inappropriate placement of the applicator due to the shape of the head	– Staff training and rotation – Appropriate restraint of the animal – Applicator size suited to the animal – The equipment is fitted with automatic shut‐off of energy flow if there is energy leakage	– Correct placement of applicator – Change applicator size if needed
Incorrect electromagnetic stunning parameters	Pain, fear	Staff, equipment	– Wrong choice of settings – Lack of skilled operators	– Staff training – Ensure settings are suited to the class of animal processed – Monitor stun quality routinely and adjust the settings accordingly – Equipment maintenance	Select the correct parameters or apply back‐up stunning method

TABLE 6. Incidence of ABMs indicative of pain, distress or suffering in STEP 11 studies.

Phase	ABM	Observed in development STEP 11.1 n = 215	Observed in development STEP 11.2 n = 35
Restraint	Vocalise	NR	0
	Voluntary movement	10	2
	Escape attempt	0	0
	Injury	0	NR
DTS energy application	Vocalise	NR	0
	Voluntary movement	2	2
	Escape attempt	5	0
	Injury	0	NR

TABLE 7. ABMs indicative of pain, fear and distress in STEP 13.2 study.

Phase	ABM	Observed in STEP 13.2 n = 12
Restraint	Back up	2
	Pull back	1 (2)*
	Jump forward	0
	Toss head	0
	Struggle	0
	Bellow	0
	Other vocal	1
	Pain face	0
DTS energy application	Pull back	0
	Jump forward	0
	Toss head	0
	Struggle	0
	Bellow	0
	Pain face	0

TABLE 8. Summary of the assessments of feasibility, sensitivity and specificity of each of the identified ABMs for assessment of electromagnetic stunning, at each of the three Key Stages of the process, according to the dossier.

ABMs	Outcomes of consciousness	After stunning			At neck cutting			During bleeding
		Sensitivity	Specificity	Feasibility	Sensitivity	Specificity	Feasibility	Sensitivity	Specificity	Feasibility
Posture	Failure to collapse or attempts to regain posture	Very Likely 90% or more	Almost Certain 99%–100%	Difficult	Very Likely 90% or more	Almost Certain 99%–100%	Almost impossible	Very Likely 90% or more	Almost Certain 99%–100%	Easy or moderately easy
Breathing	Presence	Almost Certain 99%–100%	Almost Certain 99%–100%	Very easy	Almost Certain 99%–100%	Unlikely 33% or less	Very easy	Almost Certain 99%–100%	Unlikely 33% or less	Very easy
Tonic–clonic seizures	Absence of tonic–clonic seizures	Almost Certain 99%–100%	Extremely Likely 95%	Very easy	Almost Certain 99%–100%	Extremely Likely 95%	Easy	Not applicable	Not applicable	Not applicable
Palpebral and/or corneal reflex	Presence	Almost Certain 99%–100%	Likely 66% or more	Moderately difficult	Almost Certain 99%–100%	Likely 66% or more	Difficult	Almost Certain 99%–100%	Almost Certain 99%–100%	Moderately difficult to easy
Spontaneous blinking	Presence	Extremely Likely 95%	Likely 66% or more	Moderately difficult	Extremely Likely 95%	Likely 66% or more	Difficult	Extremely Likely 95%	Almost Certain 99%–100%	Moderately difficult to easy
Eye movements	Presence	Very Likely 90% or more	Very Likely 90% or more	Moderately difficult	Very Likely 90% or more	Very Likely 90% or more	Difficult	Very Likely 90% or more	Very Likely 90% or more	Moderately difficult to easy
Vocalisations	Presence	10%–33%	Very Likely 90% or more	Moderately easy	10%–33%	Very Likely 90% or more	Moderately easy	Not applicable	Very Likely 90% or more	Moderately easy
Muscle Tone	Presence of ‘normal’ tone	Likely 66% or more	Likely 66% or more	Easy	Likely 66% or more	Likely 66% or more	Easy	Likely 66% or more	Likely 66% or more	Easy
Response to nose pinch	Presence	Likely 66% or more	Likely 66% or more	Easy	Likely 66% or more	Likely 66% or more	Easy	Almost Certain 99%–100%	Likely 66% or more	Easy

TABLE 9. Parameters considered critical for DTS stunning method provided in the dossier.

Parameter	Component	Description	Reference in the dossier
Position and application of the Applicator	Restraining system	Commercial rotating or tipping knocking box	Section 3.1.2.4
		Head restraint with chin lift is used, which fixes the head and holds the neck in extension
		Rear pusher may be used to help push smaller animals into the neck yoke
	Applicator	Purpose‐built device that is pressed firmly onto the animal's head and directs DTS energy to the correct region	Section 3.1.2.5
			SS04
	Applicator positioning	Applicator positioned directly over the frontal bones on the skull. Operation aborted if the animal's head is not pressed firmly against the applicator.	Section 3.1.2.5
	Energy penetration	Energy penetration through the skin and bone into frontal lobes	Sections 3.1.2.5, 3.1.4, SS04
Appropriate energy according to animal size and species	Output frequency of the microwave generator	922 MHz magnetron has been used, but magnetrons within the region of 890–925 MHz would be suitable	STEP 2 SS04
	Microwave generator minimum energy output	16–20 kW	Section 3.1.2.5 SS05, SS07, PR08, PR12, PR15, PR22, PR24
	Safety mechanism	Safety interlocks throughout includes: microwave leakage monitors in appropriate areas, screened animal stunning space (Faraday cage), interlocks on all doors and openings. Warning lights fitted on the cage and generator. Auto cut‐off mechanism which operates if the applicator is not in contact with the animal's head	Section 3.1.2, Section 3.1.2.4
	Animal Type (e.g. beef or dairy cattle), size of animal, horned or polled	Validated on cattle ranging from 267 to 800 kg liveweight. Bos taurus, beef, dairy and cross‐bred types. Heifers, steers, cows and bulls were represented; some were horned, some naturally polled and some dehorned. Five Brahman (Bos indicus) animals	STEP 11, Section 3.2.3.1; STEP 13.1, 13.5, PR12, PR24
	Microwave energy delivered (KJ)	Minimum 160 kJ for simple stun Recommended maximum 200 kJ for a simple stun where recovery is desirable	SS05; PR08, PR12 Section 3.1.2.5, 3.1.2.8 STEP11, Section 3.2.3.1 Part 2 STEP 13, Section 3.2.3.1 SS05, SS07; PR08, PR12, PR22, PR24
	Animal skin condition	Wet, dry or damp	Section 3.1.2.5 STEP 14, SS08
Appropriate energy according to animal size and species	Depth of penetration	A function of the distance that microwaves will travel through the animal's skin, bone and brain and their ability to absorb the applied energy. Energy frequency will impact the penetration depth. Energy frequency and penetration depth, are determined by the magnetron used.	Section 3.1.4 STEP 2 SS04
Duration of intervention	Minimum time of exposure	The duration is a function of incident power, desired energy delivery and operation of the system. For example, 180 kJ energy delivered with power 18 kW, will take approximately 10 s to apply.	Section 3.1.2.5 STEP 7, 3.2.3.1 STEP 9, 3.2.3.1 STEP 11, 3.2.3.1 Part 2 STEP 13, 3.2.3.1 SS05, SS07 PR12, PR22, PR24
Equipment maintenance, cleaning and storage conditions		Generator Care	Section 3.1.2.3
		Daily:
		Remove any build‐up of rubbish from within and around the generator
		Ensure that all air intakes and outlets are clear
		Weekly:
		Check water flow and arc detection devices are functional
		Check for water leaks inside the generator
		Check and replace (if required) air filters on generator side walls
		Monthly:
		Check generator doors, limit switches and adjust to suit door stroke
		Replace/wash air filters on generator side walls
		Applicator Care
		Daily:
		Wipe out any moisture, dust or hair
		Visually inspect for cracks or other damage
		Weekly:
		Check waveguide and applicator for loose bolts and connections
Equipment maintenance, cleaning and storage conditions		Restraint Unit Care	Section 3.1.2.3
		Daily:
		Wash out any faecal matter, blood, hair or excreta
		Visually inspect for cracks or other damage
		Grease moving parts
		Visually inspect for damage
		Visually inspect Faraday Cage for damage
Frequency of calibration of the equipment		Calibration of the auto‐tuner is required every 2 years (manufacturer's recommendation). Assessment of tuning should be carried out by a specialist technician annually or in the event of a change in the size of the applicator (e.g. for use on another species)	Section 3.1.2.3
Maximum stun‐to‐stick interval(s)		Maximum stun‐to‐stick interval of 45 s.	Section 3.1.2.5, 3.1.2.8 Section 3.1.4 STEP 7, Section 3.2.3.1 STEP 9, Section 3.2.3.1 STEP 11, Section 3.2.3.1 Parts 1 and 2 STEP 13, Section 3.2.3.1 SS05; SS07; PR08, PR12, PR15, PR22, PR24

TABLE 10. Determination and specification of intervention parameters for DTS, according to the dossier. Establishing equivalency with the parameters to be provided when applying a mechanical or electrical stunning intervention, based on Annex I of Council Regulation (EC) No 1099/2009

Intervention	Parameters	Component	Equivalent DTS parameters	Equivalent DTS component	Exact value	Explanation according to the dossier
Mechanical stunning – Penetrative captive bolt	Position and direction of the shot	Restraining system	Position and application of the applicator	Restraining system	Neck capture with chin lift based on the ASPCA pen design	3.1.2.1
		Position of the captive bolt gun		Position and contact of the applicator	The applicator is positioned directly over the frontal bones. The applicator is pressed firmly against the animal's head.	3.1.2.5
		Bolt penetration site		Wave penetration site	Frontal bones	3.1.2.5
	Appropriate velocity, bolt length and diameter of the bolt according to animal size and species	Captive bolt gun characteristics	Appropriate power (kW) and energy (kJ) according to animal size and species	Design of applicator	The applicator is approximately square in cross section, tapering at the nose end, outside dimension of 16.5 × 16.5 cm. Contouring within the Applicator head interface end enables uniform and continuous contact with the skin covering the frontal bones.	3.1.2.5
		Cartridge or compressed air specifications		Microwave generator output	Frequency: 922 MHz (890–925 MHz) Power: minimum 16–20 kW, Energy: 160–200 kJ	3.1.2.1, 3.1.2.5
		Type (e.g. beef or dairy cattle), size of animal		Type (e.g. beef or dairy cattle), size of animal	Any animal large enough to accommodate the footprint of the applicator (square cross section, tapered slightly at the nose end, approximately 16.5 × 16.5 cm, above its frontal bone can be suitably stunned using DTS. Animal type does not influence efficacy of induction of unconsciousness, providing that the applicator could be placed in contact with the forehead with no air gaps	3.1.2.6
		Equipment maintenance, cleaning and storage conditions		Equipment maintenance, cleaning and storage conditions	No specific maintenance is required to ensure that energy is delivered correctly. General cleaning, maintenance only is required and infrequent calibration of the auto‐tuner	3.1.2.3
	Maximum stun‐to‐stick/kill interval(s)1		Maximum stun‐to‐stick/kill interval(s)		45 s	3.1.2.5
Mechanical stunning – Non‐penetrative captive bolt	Position and direction of the shot	Restraining system	Position and application of the waveguide	Restraining system	As above	As above
		Position of the captive bolt gun		Position and contact of the applicator	As above	As above
		Bolt impact site		Wave penetration site	As above	As above
	Appropriate velocity and shape of the bolt according to animal size and species	Captive bolt gun characteristics	Appropriate energy according to animal size and species	Design of applicator	As above	As above
		Cartridge or compressed air specifications		Microwave generator output	As above	As above
		Bolt dimensions, mass and velocity		Design of applicator	As above	As above
		Type (e.g. beef or dairy cattle), size of animal		Type (e.g. beef or dairy cattle), size of animal	As above	As above
		Equipment maintenance, cleaning and storage conditions		Equipment maintenance, cleaning and storage conditions	As above	As above
Head‐only and head‐to‐body electrical stunning	Maximum stun‐to‐stick/kill interval(s)		Maximum stun‐to‐stick/kill interval(s)		As above	As above
	Minimum current (A or mA)	Current type	Minimum energy (kJ)	Microwave energy	160–200 kJ	3.1.2.5
		Waveform			Sine, 922 MHz (890–925 MHz)	3.1.4.1, 3.1.2.1
		Minimum current
		Latency
	Minimum voltage (V)	Exposed minimum voltage (V)	Minimum power (kW)		16 kW	3.1.2.5
		Delivered minimum voltage (V)
	Maximum frequency (Hz)	Maximum frequency (Hz)	Maximum frequency (Hz)		925 MHz	3.1.2.1
		Minimum frequency (Hz)	Minimum frequency (Hz)		890 MHz	3.1.2.1
	Minimum time exposure		Duration of intervention
	Minimum stun‐to‐stick interval		Minimum stun‐to‐stick interval		As above	As above
	Frequency of calibration		Frequency of calibration		Auto‐tuner: 2 years	3.1.2.3
	Optimisation of the current flow	Electrode characteristics	Optimisation of the energy applied	Applicator characteristics	As above	As above
		Electrode appearance
		Animal restraining		Restraining system	As above	As above
	Prevention of electrical shocks before stunning
		Position of the electrodes	Position and application of the waveguide	Position and application of the waveguide	As above	As above
		Type of electrode		Design of applicator	As above	As above
	Position and contact surface area of electrodes	Animal skin condition		Animal skin condition	Both wet and dry animals should be processed with equivalent efficiency	3.1.2.6

TABLE 11. List of related specifications as per Annex I of EC 1099/2009, according to the dossier.

Name	Description	Conditions of use	Key parameters
DTS: Diathermic Syncope ®	Exposure of the brain to electromagnetic energy generating an increase in brain temperature and a generalised epileptic form on the electro‐encephalogram (EEG). Simple stunning.	Cattle, sheep Slaughter	Frequency Energy Power Applicator size and position Maximum stun‐to‐stick interval for simple stunning

TABLE 12. Determination and specification of intervention parameters for DTS (Boon & Kok, 1988).

Material	Frequency	ε′	ε″	Dp (mm)
Skin	2.45 GHz	49.5	10.1	13.6
	922 MHz	53.5	13.5	28
Bone	2.45 GHz	7.5	0.65	85
	922 MHz	8	0.49	300
Bone marrow	2.45 GHz	4.8	0.61	70
	922 MHz	5.8	0.69	180
Brain	2.45 GHz	37	6.6	17.9
	922 MHz	41	8.5	39

TABLE 13. Estimated dielectric properties* (humans) (Gandhi et al., 1996).

Material	Frequency	ε′	Σ	Dp (mm)
Skin	2.45 GHz	32	0.57	53
	922 MHz	35	0.6	52
Bone	2.45 GHz	8	0.15	100
	922 MHz	8	0.11	136
Brain	2.45 GHz	47	1.42	26
	922 MHz	49	1.1	34

TABLE 14. Approximate material thicknesses.

Animal	Skin thickness (mm)	Bone thickness (mm)	Brain thickness (mm)
Cattle	5	12	60

TABLE 15. Effective brain penetration depth – estimate for Cattle at 2450 MHz computed using dielectric properties of human tissue provided in referenced publications.

Material	Reference	% incident energy used	Dp (mm) into brain
Skin	Boon and Kok (1988)	23.2
	Gandhi et al. (1996)	6.0
Bone	Boon and Kok (1988)	7.4
	Gandhi et al. (1996)	7.1
Brain	Boon and Kok (1988)		9.2
	Gandhi et al. (1996)		20.3

TABLE 16. Effective brain penetration depth – estimate for Cattle at 922 MHz computed using dielectric properties of human tissue provided in referenced publications.

Material	Reference	% incident energy used	Dp (mm) into brain
Skin	Boon and Kok (1988)	11.3
	Gandhi et al. (1996)	6.0
Bone	Boon and Kok (1988)	2.9
	Gandhi et al. (1996)	11.3
Brain	Boon and Kok (1988)		30.2
	Gandhi et al. (1996)		27.7

TABLE 17. Potential impact of process failures on AW hazards, including their causes, likelihood of occurrence and corrective measures, as described in the dossier. The WG complemented this table with four additional potential problems (nrs 19, 20, 21 and 22).

	Category	Potential problem	Cause	Current controls	Likelihood of occurrence	Animal WCs	Correction	Action to prevent recurrence	Residual likelihood of adverse animal WCs
1	Animal	Animal too small to be positioned correctly for an effective stun to be produced.	No testing has been completed on animals < 270 kg (3.1.2.6), therefore effectiveness on small animals has not been demonstrated Operator error	Process procedure requires that only animals > 270 kg are stunned using the system, therefore these animals would be identified prior to processing and stunned using an alternative method.	Medium High	No adverse effects on animal welfare as animals < 270 kg would be identified prior to processing and DTS would not be applied. An alternative stunning method used. In the event of an animal being too small to accommodate the applicator (and not being identified by the operator), the equipment would not function and energy would not be applied. Misadventure will cause fear.	Small animals that have been moved into the system identified by the stunning operator and stunned using the back‐up device.	Documented procedure to state that small animals < 270 kg are not to be processed using the system. Retraining of operatives to recognise suitable and unsuitable animals prior to the animals entering the restraint unit.	Low
			The head of the animal is too small to accommodate the applicator (about 16.5 × 16.5 cm) (3.1.2.6) Operator error	System would not operate or would shut down due to inadequate contact with the head.	Medium	No adverse effects on animal welfare ‐ In the event of an animal being too small to accommodate the applicator, the equipment would not function and energy would not be applied.	Small animals that have been moved into the system identified by the stunning operator and stunned using the back‐up device.	Documented procedure to state that small animals (when the head is too small to accommodate the applicator) are not to be processed using the system. Retraining of operators to recognise suitable and unsuitable animals prior to the animals entering the restraint unit.	Rare
2		Animal too large to fit into the restraint unit, head restraint and chin lift such that it can be stunned effectively.	No testing has been completed on animals > 800 kg (3.1.2.6) Operator error	Process procedure requires that only animals < 800 kg are stunned using the system, therefore these animals would be identified prior to processing and stunned using an alternative method. System would not operate if the animal could not be positioned to allow correct contact between the head and the applicator.	Medium	No adverse effects on animal welfare ‐ In the event of an animal being too big to allow for correct positioning, the equipment would not function and energy would not be applied. Misadventure will cause fear and distress.	Large animals that have been moved into the system identified by the stunning operator and stunned using the back‐up device.	Documented procedure to state that large animals (when they are too big to fit into the restraint unit, head restraint and chin lift) are not to be processed using the system. Retraining of operators to recognise suitable and unsuitable animals prior to the animals entering the restraint unit.	Rare
3		Animal too heavy (> 690 kg) for applied energy to produce an effective stun due to increased brain size.	No testing has been completed on animals > 800 kg; however, brain size will only vary about 5% from the mean (3.1.2.5) but it is possible that the animal may not be stunned for a sufficient duration. Operator error	Process procedure requires that only animals < 800 kg are stunned using the system, therefore these animals would be identified prior to processing and stunned using an alternative method.	Rare	If the energy applied is too low, then the animal is ineffectively stunned and therefore exposed to potential pain, fear and distress.	Large animals that have been moved into the system identified by the stunning operative and stunned using the back‐up device.	Documented procedure to state that large animals (when they are too big to fit into the restraint unit, head restraint and chin lift) are not to be processed using the system. Retraining of operatives to recognise suitable and unsuitable animals prior to the animals entering the restraint unit. Increase energy application for very heavy animals (validation required).	Negligible
4		Presence of horns prevent the animal from being positioned correctly for an effective stun to be produced.	Animals with very wide horns may not fit onto the restraint unit and head restraint (3.1.2.6). Operator error	Process procedure requires that animals with large horns are not stunned using the system, therefore these animals would be identified prior to processing and stunned using an alternative method.	Rare	No adverse effects on animal welfare ‐ In the event of an animal being presented where the horns do not allow for correct positioning, the equipment would not function and energy would not be applied. Pain, fear and distress.	Animals with large horns that have been moved into the system identified by the stunning operator and stunned using the back‐up device.	Documented procedure to state that animals with wide horns are not to be processed using the system. Retraining of operators to recognise suitable and unsuitable animals prior to the animals entering the restraint unit.	Negligible
5		Presence of large horns may cause an increase in the stun‐to‐stick time.	The presence of large horns may prevent the box from rotating properly and consequently increase the time taken to present the animal for sticking. Operator error	Process procedure requires that animals with large horns are not stunned using the system, therefore these animals would be identified prior to processing and stunned using an alternative method.	Rare	Animal could get stuck within the equipment prior or post‐stunning. Pain, fear and distress.	Animals with large horns that are stuck in the equipment pre‐stun to be identified by the stunning operator and stunned using the back‐up device. Animals stuck in the equipment post‐stun to be accessed by the stunning or sticking operative and stunned with a penetrative captive bolt prior to sticking.	Documented procedure to state that animals with wide horns are not to be processed using the system. Retraining of operators to recognise suitable and unsuitable animals prior to the animals entering the restraint unit.	Negligible
6		The age of the animal prevents the production of an effective stun.	No testing has been completed on calves, i.e. animals weighing less than 270 kg. Operator error	Process procedure requires that calves are not stunned using the system, therefore these animals would be identified prior to processing and stunned using an alternative method. System would not operate if the animal could not be positioned to allow the applicator to be positioned firmly on the animal's head (3.1.2.5).	Rare	No adverse effects on animal welfare ‐ In the event of an animal being too small to accommodate the applicator, the equipment would not function and energy would not be applied. Fear and distress	Calves that have been moved into the system identified by the stunning operator and stunned using the back‐up device.	Documented procedure to state that calves are not to be processed using the system. Retraining of operators to recognise suitable and unsuitable animals prior to the animals entering the restraint unit.	Negligible
7		The breed of animal prevents sufficient energy from being applied to produce an effective stun.	Animal type does not influence efficacy of induction of unconsciousness; however the breed of the animal may prevent the applicator from being positioned firmly on the animal's head (3.1.2.5, 3.1.2.6). Operator error	System would not operate if the applicator could not be positioned correctly on the head.	Rare	No adverse effects on animal welfare ‐ In the event breed preventing the correct positioning of the applicator, the equipment would not function and energy would not be applied. Fear and distress	When the applicator cannot be positioned correctly, the operator uses the back‐up device to stun the animal.	Documented procedure to state that in the event of insufficient contact between the head and applicator, an alternative stunning method is used.	Negligible
8	DTS Equipment	Equipment fails to operate correctly and the animal is not stunned effectively.	Power supply to plant fails. Facility	Alarmed failure. Back‐up generator installed which enables processing to continue but may take a short period of time to activate.	Low	(1) Animal held in the restraint box without energy application – No adverse effects on welfare (2) Any animal which has had partial energy applied ‐ Ineffectively stunned therefore potential pain (3) Any animal that has had full energy applied – No adverse effects on welfare.	Alarm of equipment failure prompts the stunning operator to stun the animal using the back‐up device.	Documented procedure to outlining the action to be taken in the event of equipment failure. Training of operatives in emergency procedures.	Rare
9		Equipment fails to operate correctly and the animal is not stunned effectively	Electrical fault in generator cabinet Equipment	Pre‐operational checks. Routine maintenance of equipment in accordance with manufacturer's instructions.	Low	(1) Animal held in the restraint box without energy application – No adverse effects on welfare. However, restraint will cause fear and distress. (2) Any animal which has had partial energy applied ‐ Ineffectively stunned therefore potential pain, fear and distress (3) Any animal that has had full energy applied – No adverse effects on welfare.	Alarm of equipment failure prompts the stunning operative to stun the animal using the back‐up device.	Documented procedure to outlining the action to be taken in the event of equipment failure. Training of operatives to in emergency procedures. Routine maintenance of equipment.	Rare
10		Equipment fails to operate correctly and the animal is not stunned effectively.	Energy not delivered to applicator – Failure of coaxial cable. Equipment	Pre‐operational checks. Routine maintenance of equipment in accordance with manufacturer's instructions.	Low	(1) Animal held in the restraint box without energy application No adverse effects on welfare. However, restraint will cause fear and distress. (2) Any animal which has had partial energy applied ‐ Ineffectively stunned therefore, potential pain, fear and distress. (3) Any animal that has had full energy applied – No adverse effects on welfare.	Alarm of equipment failure prompts the stunning operative to stun the animal using the back‐up device.	Documented procedure to outlining the action to be taken in the event of equipment failure. Training of operatives to in emergency procedures. Routine maintenance of equipment.	Rare
11		Equipment fails to operate correctly and the animal is not stunned effectively	Software algorithm terminates stun prematurely Equipment	Pre‐op checks. Routine maintenance of equipment in accordance with manufacturer's instructions.	Rare	Any animal which has had partial energy applied – Ineffectively stunned therefore exposed to potential pain, fear and distress.	Alarm indicating failed stun prompts the stunning operator to stun the animal using the back‐up device.	Documented procedure to outlining the action to be taken in the event of equipment failure. Training of operators in emergency procedures. Routine checking maintenance of equipment.	Negligible
12	Process	Incorrect neck capture and subsequent head restraint and system operation cannot proceed.	(1) Operator error – not competent or inexperienced (2) Flighty animal (3) Distractions resulting in animal movement. Operator error, Equipment, Facility	Equipment allows neck capture to be attempted again in the event of a failure on first attempt.	Medium High	Increased duration in the box possibly leading to increased fear, potential pain and distress.	Operator should attempt to reapply neck capture/head restraint. If animal becomes agitated and distressed and equipment cannot be applied appropriately for system to operate, back‐up stunning should be applied.	Clear work instruction detailing the process to be followed and action to be taken in the event of incorrect restraint of the animal. Training of operators (same skills required as for other restraint systems).	Low
13		Incorrect applicator positioning and the animal is not stunned.	Operator error – not competent or inexperienced.	Equipment is designed and manufactured so that positioning of the applicator is a straightforward process.	Medium	No adverse effects on animal welfare – In the event of the applicator being in the incorrect position, the equipment would not function and energy would not be applied. Fear and distress	Operator to reposition waveguide, then re‐start application. If animal becomes agitated and distressed and equipment cannot be applied appropriately for system to operate, back‐up stunning should be applied.	Clear work instruction detailing the process to be followed. Training of operators (skills required are considered to be less demanding than positioning for mechanical stunning).	Rare
14		Door interlocks not activated and system operation cannot proceed.	Operator error – not competent or inexperienced.	Equipment is designed and manufactured so door activation is a straightforward process.	Rare	Animal may be held for slightly longer in restraint, possible increased fear and distress.	Operator to check that doors are fully closed, then re‐start application If animal becomes agitated and distressed and equipment cannot be applied appropriately for system to operate, back‐up stunning should be applied.	Clear work instruction detailing the process to be followed. Training of operators.	Negligible
15		Selection of incorrect energy parameters resulting in an ineffective stun.	Operator error – not competent or inexperienced.	In the experimental units, manual selection is possible – This will not be available in the commercial units.	Negligible	Ineffectively stunned therefore potential pain or short period of unconsciousness, therefore fear and distress.	Not relevant to commercial units.	Not relevant to commercial units	Negligible
16		Overheating and blistering of the skin before unconsciousness is induced.	Animal and environmental factors. Equipment operating correctly. Equipment	Equipment designed and manufactured to minimise risk of overheating prior to the induction of an effective stun.	Medium	Short period of pain immediately prior to induction of unconsciousness, fear and distress.	Note the event and proceed with confirmation of effective stun.	Clear work instruction detailing the process to be followed. Training of operators. Check equipment of possible faults before application to next animal.	Low
17		Delay between end of stun and sticking.	(1) Equipment failure leading to difficulties in accessing animal to bleed it Equipment (2) Slaughterman not ready to bleed the animal Operator error	Pre‐operational checks. Routine maintenance of equipment in accordance with manufacturer's instructions. Process procedure requires that animals are bled without delay.	Medium	Animal may regain consciousness before death through loss of blood. Pain, fear and distress.	If signs of returning consciousness are present (end of tonic –clonic and animal moving into recovery phase), the operator should re‐stun the animal using the back‐up device prior to bleeding.	Clear work instruction detailing the process to be followed. Retraining of operators.	Low
18		Poor bleedout	(1) The position of the animal impedes bleedout Equipment (2) Incorrect cutting of blood vessels Operator error	Process procedure details the correct blood vessels to cut and the required methodology. Head restraint equipment exposes the neck for cutting. Pre‐operational checks. Routine maintenance of equipment in accordance with manufacturer's instructions to ensure that the equipment operating correctly allowing access to the animal post‐stun.	Medium	Animal may regain consciousness before death through loss of blood. Pain, fear and distress	(1) Re‐cut if animal is unconscious. (2) If signs of returning consciousness are present (end of tonic–clonic and animal moving into recovery phase), the operator should re‐stun the animal using the back‐up device prior to bleeding.	Clear work instruction detailing the process to be followed. Retraining of operators.	Low
19		Energy leakage	Problems with maintaining contact between the waveguide and the forehead, resulting in leakage of energy into the environment, rather than penetration into the brain. Operator error Equipment		Medium	If the energy applied to the brain is too low, then the animal is ineffectively stunned and therefore exposed to potential pain, distress and suffering.	The equipment is fitted with automatic shut‐off of energy flow if there is energy leakage. However, the duration of exposure prior to detection of leakage and system shut‐off is not known.	Ensure the applicator is secured correctly on the head	Unkown
20		Wetness of the skin	As water is heated rapidly by microwaves, it is very likely that wetness of the fleece/skin due to sweat or water will lead to localised overheating and cause skin burns (Waldman et al., 2009 ). Equipment		Medium	Pain, distress and suffering prior to loss of consciousness		Ensure the head of the animal is clean and dry	Unknown
21		Lack of cooling system	Overheating of the applicator leading to skin burn Equipment	Modern Diathermy equipment used in clinical practice is fitted with surface cooling system to prevent overheating of the applicator, The ‘water cooling system’ incorporated in this device may be inadequate to prevent this from happening.	Medium	Pain, distress and suffering		Develop more efficient surface cooling system to prevent overheating	Unknown
22		Carotid occlusion or false aneurism	Carotid occlusion due to the development of false aneurisms leading to recovery of consciousness and prolonged time to onset of death Operator error	None	Medium	Development of false aneurism leading to sustained supply of oxygenated blood to the brain via vertebral arteries, which would also lead to rapid recovery of consciousness by overcoming the heating effect of DTS by establishing homeothermy. Pain, distress and suffering.		Since the animal is rotated 180° and the frontal bone is facing downwards, application of a back‐up stunning method, i.e. penetrating captive bolt, will require rotating the animal back to upright position. Perform chest sticking/cut brachiocephalic trunk swiftly.	Unknown

TABLE 18. Peer‐reviewed publications.

ID	Author	Title	Journal	Year published
SS01	Small, A., McLean, D., Owen., J. S., Ralph, J.	Electromagnetic induction of insensibility in animals: a review	Animal Welfare 22: 287–290	2013
SS02	Small, A., McLean, D., Keates, H., Owen., J. S., Ralph, J.	Preliminary investigations into the use of microwave energy for reversible stunning of sheep	Animal Welfare 22: 291–296	2013
SS03	Rault, J.‐L., Hemsworth, P., Cakebread, P., Mellor, D., Johnson, C.	Evaluation of microwave application as a humane stunning technique based on electroencephalography (EEG) of anaesthetised cattle	Animal Welfare 23: 391–400	2014
SS04	McLean, D., Meers, L., Ralph, J., Owen, J. S., Small, A.	Development of a microwave energy delivery system for reversible stunning of cattle	Research in Veterinary Science 112: 13–17	2017
SS05	Small, A., Lea, J., Niemeyer, D., Hughes, J., McLean, D., Ralph, J.	Development of a microwave stunning system for cattle 2: Preliminary observations on behavioural responses and EEG	Research in Veterinary Science 122: 72–80	2019
SS06	Hughes, J., Small, A.	A preliminary comparison of beef carcases stunned using DTS: Diathermic Syncope® or captive bolt in terms of selected meat quality attributes and plasma biomarker concentrations	Animal Review 9(1): 13–23	2022
SS07	Small A, Jenson I, Phillips A, McLean D, Kalinowski T, Ralph J.	Cattle recover completely from unconsciousness induced by controlled application of 150–180 kJ of 915 MHz microwave energy to the forehead.	Veterinary and Animal Science Vol. 29 Pages 100466	2025a
SS08	Small A, Jenson I, Fiszon B, Neindre PL, Phillips A, McLean D, McLean J, Kalinowski T, Ralph J.	Tissue integrity impacts of application of 160–200 kJ of 915 MHz microwave energy, using the DTS: Diathermic Syncope® system, to the forehead of cattle and alignment with the requirements of religious slaughter markets	Veterinary and Animal Science Vol. 29 Pages 100464	2025b

TABLE 19. Project reports – not peer reviewed – CONFIDENTIAL.

ID	Title	Date of production of report	Status
PR01	SRP.003 Investigate the Use of Microwave Technology for Stunning Beef.	2005	CONFIDENTIAL
PR02	Report on Microwave Technology Development Stage 1.	2009	CONFIDENTIAL
PR03	Microwave induced insensibility for animals Stage 2.	2010	CONFIDENTIAL
PR04	Development of applicator and auto‐tuner	2011–2012	CONFIDENTIAL
PR05	Evaluation of microwave application as a humane stunning technique based on electroencephalography (EEG) of anaesthetised cattle.	2013	CONFIDENTIAL
PR06	Microwave results from the live testing conducted at ■■■■■ in April 2012. Interim project report	2012	CONFIDENTIAL
PR07	Refinement of delivery apparatus and development of user interface software.	2014	CONFIDENTIAL
PR08	Dielectric induction of temporary insensibility in cattle – animal trials.	2015	CONFIDENTIAL
PR09	Meat quality from microwave stunned cattle.	2015	CONFIDENTIAL
PR10	Refinements to delivery system: applicators and coaxial cabling. Interim project report.	2016–2019	CONFIDENTIAL
PR11	DTS: Diathermic Syncope® – commercial validation trials.	2018	CONFIDENTIAL
PR12	DTS: Diathermic Syncope® controlled trials.	2021	CONFIDENTIAL
PR13	Refinements to delivery system: applicators, coaxial cabling and tuning.	2018–2021	CONFIDENTIAL
PR15	Repeatability of induction of insensibility in cattle using DTS: Diathermic Syncope®; and recovery of consciousness from DTS‐induced insensibility	2024	CONFIDENTIAL
PR16	Wet scalp impact on energy transfer to the head	2023	CONFIDENTIAL
PR17	DTS Process (video)	2024	CONFIDENTIAL
PR18	Video providing supplementary data to SS07	2024	CONFIDENTIAL
PR19	Compilation of EEG data for DTS: Diathermic Syncope® treated cattle	2024	CONFIDENTIAL
PR20	Raw EEG data files for PR19	2024	CONFIDENTIAL
PR21	DTS: Diathermic Syncope® – process times and practical animal welfare	2024	CONFIDENTIAL
PR22	Manuscript: EEG, behavioural observations, bleedout and temperature at the point of application in cattle receiving DTS: Diathermic Syncope® at 160–200 kJ	2025	CONFIDENTIAL
PR23	EEG data files for STEP 13 and 14	2025	CONFIDENTIAL
PR24	Line list of animals processed at 210 kJ or less	2025	CONFIDENTIAL

TABLE 20. Published data on endocrine parameters measured in the DTS pilot study (STEP 7). The first value refers to the basal level and the second to the level after the treatment.

	Rulofson et al. (1988)	Mitchell et al. (1988)	Dunn (1990)	Shaw and Tume (1990)	Tume and Shaw (1992)	Zulkifli et al. (2014)	DTS pilot study
Cortisol (nmol/L)		25.0 ± 13.7 to 176.7 ± 65.5	124.8 ± 10.7 to 259.6 ± 104.0	44.9 ± 6.9 to 88.3 ± 10.8	41.0 ± 3.7 to 123.2 ± 5.3	175.11 to 274.08	14.36 to 212.46
ACTH (pmol/L)		20.6 ± 7.9 to 301.8 ± 142.9				922.1 to 4092.9	3.7 to 125.4
Adrenaline (nmol/L)	1.2 ± 0.1 to 81.3 ± 20.7	9.28 ± 0.44 to 49.12 ± 89.51				102.28 to 519.6	22.33 to 420.12
Noradrenaline (nmol/L)	1.9 ± 0.3 to 135.8 ± 31.6	0 to 37.82 ± 54.37				271.21 to 754.3	< 0.12 to 503.64
b‐Endorphin (pmol/L)				14.6 ± 1.8 to 30.7 ± 4.3	19.2 ± 1.5 to 20.9 ± 1.2	259.9 to 441.8	4.5 to 1559.8

TABLE 21. Summary of change in plasma biomarker concentrations between pre‐ and post‐treatment samples (Mean ± standard deviation). Data from SS06.

		DTS		Captive bolt
		Baseline	Post‐treatment	Baseline	Post‐treatment
Cortisol (nmol/L)	Mean	33.20	150.38	61.43	160.64
	Range	14.36–65.20	94.09–198.42	21.45–169.01	85.82–212.46
ACTH (pmol/L)	Mean	25.69	11.25	42.21	26.6
	Range	14.4–47.2	3.7–33.1	11.9–125.4	6.6–55.7
Adrenaline (nmol/L)	Mean	67.76	78.23	50.37	151.35
	Range	22.33–70.40	45.67–420.12	25.48–161.33	25.08–248.16
Noradrenaline (nmol/L)	Mean	17.84*	138.10	24.14*	80.32**
	Range	< 0.12–56.39	19.18–503.64	< 0.12–110.79	< 0.12–262.88
b‐endorphin (pmol/L)	Mean	794.8	646.1	686.8	614.1
	Range	11.8–1334.0	313.5–1059.9	177.8–1100.2	4.5–1559.8

TABLE 22. Indicators of an effective mechanical stun.

Behavioural sign	DTS	OIE terrestrial animal health code	EFSA scientific opinion 2004	AMIC animal welfare standard	USDA humane handling guidebook**
Immediate collapse	x	x	x	x
No attempt to stand up *	x	x			x
Body and muscles immediately rigid (tonic)	x	x	x	x
Lack of normal rhythmic breathing	x	x	x	x
Eye open and staring straight ahead/glazed expression	x	x		x
No attempt to rise head	x			x
Ears relaxed and drooping				x
Tongue loose and flapping/hanging out				x
Corneal reflex absent	x		x	x
No spontaneous eye blinking	x			x
Straight back and floppy head					x
No vocalisation of any kind					x
No vocalisation in response to stimulus	x
No response to painful stimulus	x		x
Gradual pupillary dilation			x

TABLE 23. Indicators of an effective electrical stun.

Behavioural sign	DTS	EFSA 2004 1	AMIC AW standard
Immediate collapse	x*	X	x
Epileptiform seizure (described in detail)	x	X †	x
Lack of normal rhythmic breathing	x #	X	x
No spontaneous eye blinking	x		x
Gasping (breathing in without breathing out) sometimes occurs	x		x
Upward rotation of eyes		X
Dilated pupils		X
No response to nose prick (painful stimulus)	x	X

TABLE 24. Hazards associated with DTS for stunning cattle.

Hazard	Description
Inappropriate handling	Handling animals for the purpose of restraining them into the box of the DTS system in an inappropriate way causing pain and fear.
Inappropriate restraint	Excessive pressure applied during restraint to the animal's body and/or head could lead to pain and fear. Lack of adequate pressure leading to poor contact can cause ineffective stunning.
Release from the restraint while conscious	Cattle released from the restraining device conscious or recovering consciousness.
Incorrect stunning positioning	Improper placement of the stunning device, being either too far from the intended area or at an incorrect angle, leading to ineffective stunning.
Wetness of the skin	Sweat or water that leads to localised overheating and causes skin burns.
Presence of horns	Bony extensions from the heads of specific cattle breeds.
Unexpected loud noise	Sudden and unanticipated sound that is significantly louder than the surrounding environment, potentially leading to fear.
Inappropriate stunning parameters	Selection of too low energy parameters resulting in an ineffective induction to unconsciousness, or two high energy parameters resulting in overheating and blistering of the skin before unconsciousness is induced.
Too short exposure time	The duration of exposure to DTS applicator is too short to result in epileptiform activity in the brain indicative of unconsciousness.
Energy leakage	Uncontrolled loss of energy during exposure to DTS causing overheating of the skin.
Overheating of applicator/tong	Excessive heating of the equipment used may cause burns or other injuries, ineffective stunning and potential damage to the equipment.
Prolonged stun‐to‐stick interval	The interval between the end of stunning and sticking is too long to sustain unconsciousness until death occurs due to bleeding.
Ineffective sticking	Inadequate or incomplete severing of the major blood vessels, leading to insufficient bleeding.
Sticking of conscious animals	Sticking comprises the incision of the skin, soft tissues, nerves and of the brachiocephalic trunk. This hazard applies only to ineffectively stunned cattle or those recovering consciousness during sticking. Lack of skilled operators and lack of monitoring consciousness at the time of sticking are hazard origins.

TABLE 25. ABMs for the assessment of WCs pain, and fear related to handling, restraint and induction to unconsciousness during DTS stunning process in cattle.

ABM	Description	WCs	Hazards associated with WCs
Vocalisations	An animal's vocalising response in terms of mooing, bellowing or roaring (modified by EFSA AHAW Panel, 2020 after Grandin, 2012)	Pain, fear	Handling, restraint and/or inappropriate restraint, loud noise
Escape attempts	Attempts to go through, under or over gates and other barriers. Head and neck stretched forward and either slightly raised above back, slightly lowered or level with back (modified by EFSA AHAW Panel, 2020 after Lanier et al., 2001), move any part of the body as far as restraint allows for it	Pain, fear	Handling, restraint and/or inappropriate restraint, loud noise
Injuries	Tissue damage (bruises, scratches, broken bones, dislocations) (EFSA AHAW Panel, 2012, 2020)	Pain	Restraint and/or inappropriate restraint, loud noise
Facial expression	‘Pain face’: (1) Ears are tense and backwards or low/lamb's‐ears. (2) Eyes have a tense stare or a withdrawn appearance. Tension of the muscles above the eyes may be seen as ‘furrow lines’. (3) Tension of the facial muscles on the side of the head. (4) Strained nostrils, the nostrils may be dilated and there may be ‘lines’ above the nostrils. There is increased tonus of the lips (Gleerup et al., 2015)	Pain	Handling, restraint and/or inappropriate restraint
Skin burns	Injury to the skin or other tissues caused by exposure to DTS	Pain	Incorrect applicator positioning, too high energy parameters, energy leakage, wetness of the skin

TABLE 26. Animal‐based measures (ABMs) for the assessment of the state of consciousness, description of the outcomes of consciousness and when to assess them (key stage) after DTS stunning.

ABMs	Description of the outcome of consciousness	Key stage
Posture	Conscious animals will maintain posture. However, during the application of DTS, when animals are restrained in neck bails with chin capture, the forequarter is held in place and loss of posture could only be evidenced as a dropping of the hindquarters towards the floor of the box (hindquarter collapse), rather than complete and permanent loss of posture on all four limbs	–
Breathing	Rapid breathing, flared nostrils. Breathing alters in response to stimulation, e.g. through incision of the skin.	Between stun and shackle, during neck cutting/sticking, during bleeding
Corneal reflex	Presence of corneal reflex response elicited by touching or tapping the cornea with a finger or paint brush.	Between stun and shackle, during neck cutting/sticking, during bleeding
Palpebral reflex	Presence of palpebral response elicited by touching or tapping the inner/outer eye canthus or eyelashes with a finger.	Between stun and shackle, during neck cutting/sticking, during bleeding
Body movement	Intentional or purposeful kicking or body or head movements as a response to incision of the skin and/or insertion of the knife.	During neck cutting/sticking
Spontaneous blinking	Animal opens/closes eyelid on its own without stimulation. Involuntary flicking of the eyelids and third eyelid may be seen in the unconscious animal.	Between stun and shackle, during neck cutting/sticking, during bleeding
Vocalisations	Grunting, bellowing or mooing. Passive vocalisation may occur when the unconscious animal is inverted, due to the tension in the vocal cords. Noise associated with deep, slow breathing (snore) is not considered to be vocalisation	Between stun and shackle, during neck cutting/sticking, during bleeding
Muscle tone	Righting reflex (when shackled and hoisted) and attempts to raise the head.	During neck cutting/sticking

TABLE 27. Hazards selected as highly relevant related to DTS and other stunning methods in cattle. X = the hazard is present for the respective stunning method.

Hazards	DTS	Penetrative captive bolt	Head‐only electrical	Head‐to‐body electrical
Pre‐stunning shocks			X	X
Incorrect stun position		X
Wet skin	X
Inappropriate stunning parameters		X
Too short exposure time	X		X
Energy leakage	X
Prolonged stun‐to‐stick interval	X	X	X	X
Ineffective sticking	X	X	X	X
Sticking of conscious animals	X	X	X	X

Full text

SUMMARY

Council Regulation (EC) No 1099/2009 on the protection of animals at the time of killing lists in Annex I the stunning applications currently allowed in the European Union (EU), together with the conditions under which those applications can be implemented. With the aim of improving animal welfare, the European Commission (EC) can amend the list of approved methods in Annex I, considering scientific and technical progress. For a new or modified stunning application to be listed as an approved method in Annex 1, evidence that it ensures a level of animal welfare at least equivalent to that ensured by the currently approved methods must be provided.

EFSA was requested to assess the use of a new method described as Diathermic Syncope® (DTS) for simple stunning of cattle followed by neck cutting. For this assessment, the EFSA Guidance (EFSA AHAW Panel, 2018) on the assessment criteria for applications for new or modified stunning methods regarding animal protection at time of killing was followed. The stunning method involves microwave irradiation of the brain of cattle weighing 270–690 kg (live weight) with a microwave frequency range of 890–925 MHz delivering 160–200 kJ of energy and an incident power of 16–20 kW for 10 s. DTS applies electromagnetic energy consisting of both a magnetic and an electrical field in sine waves perpendicular to one another. In the DTS stunning procedure, a bovine is restrained in an upright position, the head is restrained to prevent lateral and vertical movements, and the applicator is positioned on the frontal bone before switching on the completely automated system. After the application, the animal is rotated within 10 s onto the spine and presented for neck cutting (i.e. severing carotid arteries and jugular veins in the neck). It is claimed that the electrical field induces an epileptiform seizure in the brain, while the changing electromagnetic field induces heat in the brain tissue through a process called diathermy.

An ad hoc Working Group (WG) was set up by EFSA to address the terms of reference of the mandate received from the EC. The welfare risk assessment focused on the hazards associated with the restraint, and on neurological and behavioural animal‐based measures (ABMs) for assessing the state of unconsciousness during and after the application of DTS, and until death occurs. For assessment Phase 1, which is a check of data for the risk assessment, the WG assessed the scientific papers and the related annexes provided by the applicant in a dossier following the procedure of EFSA Guidance on the assessment criteria for applications for new or modified stunning methods regarding animal protection at the time of killing (EFSA AHAW Panel, 2018). According to the provided scientific papers, DTS partially fulfils the requirements for assessment Phase 1. The term ‘partially’ is used because the dossier, for example, failed to adequately correlate neurological with behavioural measures. This lack of specific scientific data prevented the WG from concluding on the occurrence and prevalence of the hazards associated with the stunning method, as well as on the time during which the animals could be subjected to these hazards. The WG sought further clarifications from the applicant in order to facilitate an animal welfare risk assessment. The replies and revised dossier also contained information from field data, i.e. animals stunned using DTS in abattoir conditions. The method was assessed only for animal categories for which data were provided.

The mechanism associated with induction of unconsciousness was claimed to be hyperthermia and epileptiform seizure induced in the brain. However, the brain temperature was not measured in live cattle and the critical temperature, at which syncope is expected to occur, is unknown. In rats, it is reported that the heat produced leads to cessation of neurotransmitter function, maintaining unconsciousness until the brain temperature returns to within 1°C of its normal temperature. However, this has not been confirmed in cattle.

The WG concluded that the dossier and answers provided by the applicant failed to inform about important animal welfare concerns, in particular induction and maintenance of unconsciousness without causing avoidable pain, distress and suffering.

Based on the information provided in the dossier, the EFSA experts concluded that it is very unlikely that DTS applied to cattle at 160–200 kJ energy fulfils the requirements for the maintenance of unconsciousness until death. Therefore, this stunning method with the specific proposed energy values does not provide a level of welfare at least equivalent to one or more of the currently allowed methods listed in Annex I of Council Regulation (EC) No 1099/2009.

INTRODUCTION

1

The European Commission (EC) received a request from a business operator to allow the use of Diathermic Syncope® for stunning cattle. With this application, the EC has received a series of publications and technical information in order to perform a full assessment of the method. This information is attached to the aforementioned request.

Background and Terms of Reference as provided by the requestor

1.1

Background

1.1.1

Council Regulation (EC) No 1099/2009 on the protection of animals at the time of killing (later mentioned as “the killing Regulation”) defines stunning in Article 2(f) as any intentionally induced process, which causes loss of consciousness and sensibility without pain, including any process resulting in instantaneous death.

Article 3(1) requires that animals shall be spared any avoidable pain, distress or suffering during their killing and related operations.

Article 4(1) requires that animals shall only be killed after stunning in accordance with the methods and specific requirements related to the application of those methods set out in Annex I of the Regulation and that the loss of consciousness and sensibility shall be maintained until the death of the animal.

Annex I to the killing Regulation lists the stunning methods and related specifications.

Article 4(2) allows the Commission to amend Annex I as to take into account scientific and technical progress on the basis of an opinion of the EFSA. Any such amendments shall ensure a level of animal welfare at least equivalent to that ensured by the existing methods.

At present, the use of Diathermic Syncope® for stunning cattle is not listed in Annex I and therefore not allowed for stunning animals.

Terms of Reference

1.1.2

In accordance with Article 29 (1) (a) of Regulation (EC) No 178/2004,1 EFSA is requested to evaluate the use of Diathermic Syncope® for stunning cattle as proposed in the application (later called “the proposed method”).

In particular, EFSA shall assess:

1. Whether the proposed method and information provided with the application meet the eligibility criteria of EFSA's guidelines.2
2. Whether the proposed method can provide a level of animal welfare at least equivalent to that ensured by the existing methods in the legislation and especially:

• whether the proposed method ensures that cattle are spared of avoidable pain, distress or suffering during the killing as referred to in paragraph 1 of Article 3 of the killing Regulation and,
• whether the proposed method maintains the loss of consciousness and sensibility until death of cattle as referred to in paragraph 1 of Article 4 of the killing Regulation.

Interpretation of the Terms of Reference

1.2

Considering the background and the Terms of Reference (ToR) as provided by the EC, this Scientific Opinion (SO):

1. Is based on the most recent updated (3rd update) dossier submitted to EFSA, which included all the files and documents provided by the applicant. The applicant specifically requested an evaluation of the DTS method: Diathermic Syncope® for stunning cattle, for simple stunning of cattle according to the following parameters: delivering 160–200 kJ of energy and an incident power of 16–20 kW for 10 s. According to the dossier (page 38), the animal types for the application of DTS include cattle within a liveweight range of approximately 270–690 kg.
2. Assesses whether the proposed method and information provided with the application meet the eligibility criteria of EFSA's guidelines (ToR1), which is the ‘Guidance on the assessment criteria for applications for new or modified stunning methods regarding animal protection at the time of killing’ (EFSA, 2018). This guidance was also the protocol that was followed for the SO.
3. Assesses whether the proposed method can provide a level of animal welfare (AW) at least equivalent to that ensured by the methods listed in Annex I of Council Regulation (EC) 1099/2009 for simple stunning of cattle (ToR2), i.e.:

• whether the proposed method ensures that cattle are spared of avoidable pain, distress or suffering during the killing as referred to in paragraph 1 of Article 3 of the killing Regulation, (ToR2i).
• whether the proposed method maintains the loss of consciousness and sensibility until death of cattle as referred to in paragraph 1 of Article 4 of the killing Regulation, that is from the onset of unconsciousness until death (ToR2ii)

4. Uses ‘pain, distress and/or suffering’ together, because this is how it is used in EFSA Guidance 2018 and also in the killing Regulation.
5. Uses the terminology on pain, fear, distress and/or suffering following the approach used in previous EFSA assessments on cattle slaughter (EFSA AHAW Panel, 2020) and on high expansion foam (EFSA AHAW Panel, 2024).
6. According to EUPAHW (European Partnership for Animal Health and Welfare, a collaborative research effort of over 90 entities across Europe), ‘distress is a negative affective state of an animal when it has been unable to adapt to stressors. It may manifest as physiological and/or behavioural responses. Distress results from severe, prolonged or cumulative stress and has effects on biological function, welfare and health. Distress can be acute (short‐term) or chronic (long‐term) with consequences such as impaired immune function potentially leading to increased vulnerability to diseases’.
7. In this scientific opinion, pain, fear and distress are considered the main welfare consequences to be assessed as they represent the most immediate and clearly defined welfare responses to the hazards listed for DTS. Additionally, pain, fear and distress are the three welfare consequences also defined in the SO Welfare of cattle at slaughter (EFSA AHAW Panel, 2020) and by the applicant as well.
8. The term suffering is not listed separately as a welfare consequence, as suffering generally results from prolonged or intense negative affective states such as experiences of pain, fear and distress. Therefore, consistent with EFSA practice and with EU legislation (Regulation No (EC) 1099/2009) requiring the avoidance of unnecessary pain, distress and suffering, this opinion focuses primarily on the assessment of pain, fear and distress as welfare consequences and uses pain, distress and/or suffering for phraseology reasons mentioned in Point 4.
9. Bases its evaluation on 8 published scientific papers, 24 confidential non‐peer reviewed project reports and 10 other supplementary materials provided by the applicant as well as other published scientific papers after extensive literature search on Diathermic Syncope® stunning method. For more information see Sections 2 and 3.2.
10. Concludes if the use of the new proposed method is valid under commercial conditions, stating to what extent the method proposed for stunning and killing cattle is able to provide a level of AW in compliance with the above‐mentioned legislation and, if yes, under which conditions.

Additional information

1.3

During the assessment process as defined in the EFSA guidance, it became apparent that some of the information submitted by the applicant lacked sufficient details to clearly outline the potential welfare concerns.

For these reasons, and with the specific aim of gathering all necessary information and data to assess the exact sequence of the events during the Diathermic Syncope® process, EFSA asked the applicant for additional data and information on three occasions (see Section 2.1 for more information).

DATA AND METHODOLOGY

2

Data

2.1

The applicant, after receiving three requests for additional information to the initial submitted dossier, provided EFSA with the following material considered in this assessment: 8 scientific publications, 24 confidential not peer‐reviewed project reports (PR), unpublished field trial data and 10 other supplementary materials (9 conference contributions, thereof 8 were peer reviewed and 1 not peer reviewed, i.e. a factsheet).

Specifically, EFSA requested additional information and/or clarifications from the applicant:

• On 20.06.2023 regarding (1) Description of the stunning method, (2) Description of the individual studies submitted and (3) Overall integration of findings from all studies. The reply, along with the updated dossier of the applicant, was received on 20.11.2023.
• On 06.03.2024 concerning the Description of the stunning method: Description of the method including potential sources of pain, distress and suffering, Key parameters of the effective use of the method, Scientific basis of induction and maintenance of unconsciousness for this method, Potential causes of system failure and chances of occurrence. The reply, along with the second updated dossier of the applicant, was received on 22.05.2024.
• On 06.11.2024 concerning the Description of the stunning method: Description of the method, including potential sources of pain, distress and suffering, Potential causes of system failure and chances of occurrence. The reply, along with the latest updated dossier of the applicant, was received on 2.9.2025.

Methodology

2.2

This SO follows the protocol detailed in the methodological guidance developed by the EFSA AHAW Panel for mandates to assess new or modified stunning method applications regarding animal protection at the time of killing (EFSA AHAW Panel, 2018). Sections 3.1 and 3.3 contain data and information from the dossier, and when there is a comment or assessment in these Sections from EFSA, it is clearly indicated. Section 4 describes the independent assessment of EFSA for the stunning method.

Literature search

2.2.1

An extensive literature search was conducted by EFSA to identify studies describing the stunning systems used for cattle at slaughterhouses, with a special focus on their effects on AW. The search was performed using the Web of Science (WoS). The search included the period from 1975 until September 2024, and scientific publications written in English.

The query to the WoS database used the following search terms:

“(((TS=(cow* OR cattle OR calf OR calv* OR bull OR “bos taurus” OR bovine*) AND TS = (stun* OR unconscious*OR insensibility)”.

From a total of 380 retrieved scientific publications, 371 were excluded for several reasons: species other than cattle (n = 71), not related to stunning effect on welfare (n = 178), full text not available (n = 14), stunning methods not allowed for cattle in general or not allowed for cattle of more than 10 kg in Council Regulation (EC) No 1099/2009 (e.g. controlled atmosphere stunning (CAS), puntilla, non‐penetrating captive bolt; n = 15), papers not related to the effectiveness of stunning and indicators of unconsciousness (e.g. study on eligibility of devices; n = 15), related to other challenges to AW than stunning (e.g. blood in trachea, false aneurysm, stress during transport; n = 8), other reasons (e.g. ritual slaughter, reviews; n = 70). Therefore, only nine scientific publications were retrieved.

In addition, the references of these nine scientific publications led to five additional relevant sources of information (three scientific publications and two PhD theses). The final 14 publications used for this assessment are listed in Table 2.

EFSA guidance on the assessment criteria for studies evaluating the effectiveness of stunning interventions regarding animal protection at the time of killing (EFSA AHAW Panel, 2018)

2.2.2

The first part of the assessment process involved the assessment of the submitted documentation against the criteria laid down in EFSA Guidance (2018). In case the criteria are not fulfilled, the assessment report must highlight the shortcomings and indicate where improvements are required before the study/method can be further assessed. It should be noted that the EFSA Guidance is applicable to the individual studies. As mentioned in Section 2.1, the WG experts identified a lack of some specific information in the submitted papers. Consequently, considering these limitations with regard to at least one of the criteria laid down in the guidance (see following sections), none of the individual studies would have passed the first assessment phase (ToR1).

However, the WG experts, the AHAW Panel and EC representatives agreed that, in order to address ToR2 and to support the development or refinement of stunning methods, it was necessary to evaluate the entire combined information and body of evidence provided by the applicant (including the submitted papers), other existing literature and expert knowledge elicitation/evaluation rather than applying the criteria to the individual and/or unpublished studies. This approach remains consistent with EFSA Guidance 2018, which allows an integrated assessment of all information available when appropriate. Additionally, the AHAW Panel and the WG experts decided to ask the applicant for additional data and information in order to perform ad hoc analyses with the aim of improving clarity of the provided information and studies and to facilitate the assessment (see Section 2.1).

Assessment of the level of animal welfare provided by the Diathermic Syncope®

2.2.3

Quantitative assessment

2.2.3.1

To compare DTS with other killing methods, a scientific literature review (see Section 2.2.1) was conducted to gather data on neurological and behavioural indicators of the onset of unconsciousness and death (see Section 3.3.1.1). For a full explanation regarding the use of indicators and comparisons among the methods, see Section 4.2.1.

Qualitative assessment and hazard ranking

2.2.3.2

ToR2 involved assessing the equivalence of DTS with currently legally allowed stunning methods. To address this, a comparison between the proposed method and existing ones in terms of AW was necessary. Various methodologies could have been employed, including a comparative analysis of welfare outcome measures (ABMs) reflecting animals' responses to the method, or an assessment of welfare hazards associated with each method. However, conducting a full quantitative comparison based on ABMs was deemed unfeasible due to the following reasons: the welfare outcomes for the quantitative assessment of equivalence with existing methods (penetrative captive bolt, electrical head‐only and electrical head‐to‐body) are biomarkers of stress, ABMs of the state of unconsciousness, latency to insensibility and aversiveness of the system. The studies on biomarkers of stress associated with restraint, positioning of the head into the head capture and stunning with DTS and penetrating captive bolt are not comparable. For instance, sources of variation in endocrine markers include not only stress levels, but also the time of day, age and sex, health status, diet, genetic factors, the method of sampling and handling, as well as the procedures for the laboratory analysis. None of the ABMs proposed (see Section 4.2.1) to assess the equivalence could be reliably used for monitoring the state of consciousness during or following the application of DTS because of the uncertainty and lack of direct correlation with the EEG evidence of unconsciousness. In addition, these ABMs have low sensitivity, specificity or feasibility on their own for DTS (see Table 8). Furthermore, there is no EEG evidence of occurrence of generalised epileptiform activity in the brain following the application of DTS (16 to 20 kW for 10 s) and, owing to this, correlation between EEG and the ABMs is lacking. Hence, the equivalence assessment was based on a non‐formal expert elicitation utilising a semi‐qualitative approach and expert opinions. For more information see Sections 2.2.2 and 4.2.1.

This elicitation exercise was structured in five steps:

1. Identification of comparable stunning methods;
2. Identification of a list of hazards for each stunning method, along with their associated WCs;
3. Evaluation of the welfare impact caused by these hazards via expert elicitation tailored for each identified method;
4. Group discussion on the individual evaluations to reach a consensus judgement;
5. Identification & selection of highly relevant hazards for each method and presentation of the results.

1. Identification of comparable stunning methods

For the identification of comparable stunning methods, descriptions provided in the more recent EFSA scientific opinion on the welfare aspects of the main systems for stunning and killing of cattle (EFSA AHAW Panel, 2020) were taken into consideration.

Non‐penetrative captive bolt was excluded since this method is only used for ruminants of less than 10 kg of live weight. Firearm with free projectile was also excluded, as it causes irreversible and instantaneous damage to the brain and was not considered suitable for comparison with DTS, since DTS is a simple stunning method and does not cause irreversible death.

The remaining methods included in comparison to DTS were penetrative captive bolt device, head‐only electrical stunning and head‐to‐body electrical stunning. The list of comparable stunning methods in cattle is reported in Annex A Table A.1.

2. Identification of hazards and associated WCs

EFSA experts identified, for each of the comparable methods, a list of hazards associated with the WCs for the animal. Hazards were included considering all phases: pre‐stunning, stunning and the actual process of killing, which encompasses the induction of unconsciousness and the onset of death.

Hazards with their corresponding WCs related to DTS and ABMs for the assessment of the associated WCs were identified in this opinion as described in Section 4.1.2 (Tables 24, 25 and 26) and summarised in Annex A (Table A.3).

The hazards and associated WCs related to comparable stunning methods have been identified and modified in previous EFSA opinion (EFSA AHAW Panel, 2020) and are summarised in Annex A (Table A.3).

3. Evaluation by experts of the welfare impact caused by the hazards

To assess the magnitude of the WCs (pain, fear and distress) caused by the hazards in relation to each stunning method, five EFSA experts were asked to estimate the following:

• Prevalence (%) of cattle exposed to the hazard in each of the methods during routine operation. The experts were requested to provide an estimate of the prevalence of animals exposed to each hazard within each method by providing the minimum, the maximum and the mean estimated value. The minimal percentage of animals is the mean value in the slaughterhouses with the best expected practices, and the maximal value corresponds to the mean value of the slaughterhouses with the worst conditions from the AW point of view. The experts discussed and agreed on the prevalence ranges for each hazard and for each stunning method.
• Duration (s) of the exposure to pain, fear and distress due to each hazard for each stunning method. The experts discussed and agreed on the values of duration for each hazard and for each stunning method. Where needed and due to variation, a range of possible durations is indicated.
• Impact (WCs) caused by each hazard, using the scoring system reported in Table 1, 3. In short, the scale describes a stepwise increase in the impact on AW, from a situation characterised by no pain or stress to a situation characterised by severe pain lasting more than 2 s and with a high prevalence.

Each expert provided individual judgements using the Likert scale for each hazard and each stunning method.

4. Expert discussion on the individual evaluations to reach a consensus judgement

The individual judgements of hazards associated with each stunning method were discussed in the expert group. Each expert was asked to provide supporting arguments for their evaluation. Subsequently, all experts were asked whether, in light of the reasoning that was brought up during the group discussion, they were inclined to reconsider their evaluation. These discussions allowed refinement of the individual assessments and led to a consensus score for each hazard.

5. Identification of highly relevant hazards for each method and presentation of the results

A mean score was calculated based on the consensus score for each hazard. The mean scores for each hazard ranged between 2.8 and 6.6. Hazards with final scores of 6 or higher were classified as highly relevant to facilitate comparison between methods (see Table 27). The final list of highly relevant hazards and the results of the analysis are detailed in Section 4.2.2. These results were then discussed and used to compare four main stunning methods for cattle: DTS, penetrative captive bolt, electrical head‐only and electrical head‐to‐body (see Section 4.2.2).

Limitations of the exercise and uncertainty analysis

2.2.3.3

Some limitations in the exercise were identified. Because the number of hazards to be scored for each method was not the same, it could inadvertently lead to a potential bias. However, the number of hazards was not considered as a factor for the comparison. In addition, more uncertainty could be expected around methods for which limited information exists versus others with a lot of scientific evidence available. These limitations were discussed and accounted for as much as possible in the third phase of the exercise (group discussion).

For conclusions reached through non‐formal expert elicitation, EFSA experts provided their individual judgement on the certainty for a rephrased question referring to a well‐defined quantity of interest (see STEPS 3 and 4 of Section 2.2.3.2). For assessing the equivalence with an already approved method, the experts were presented with the hazards and their associated WCs identified for DTS and each alternative method. Since the objective was to assess if DTS was equivalent to or better than each method in terms of the overall welfare of animals subjected to it, experts were asked how certain they were that the prevalence of animals experiencing the identified WCs associated with the hazards for DTS was lower, equal or higher than the respective prevalence for each alternative method. The experts were also asked to take into consideration the magnitude of the hazard's impact and its associated WCs. For instance, a low proportion of animals experiencing a highly severe WC could indicate poorer welfare than a higher proportion of animals experiencing a milder WC. The group discussed and agreed on a certainty range, which is reported in the assessment section and in the key conclusions.

The main question posed to the experts after reading the dossier was ‘How likely is it that cattle subjected to DTS will be induced to unconsciousness after its application and they will maintain in this state until death?’

ASSESSMENT PHASE 1: CHECK OF DATA FOR RISK ASSESSMENT

3

Figure 1 shows the development timeline of DTS, identifying the nature of each STEP and Table 4 shows how the different studies in the dossier, addressing the Diathermic Syncope® for stunning cattle, meet the eligibility risk assessment criteria according to the EFSA Guidelines (2018). Only peer‐reviewed scientific studies (SS) (i.e. SS02, SS03, SS05, SS06, SS07 and SS08) were considered. The descriptions of the individual studies submitted can be found in Section 3.2. In the same section, Table 18 shows the titles of the peer‐reviewed publications and Figure 22 of the dossier illustrates which STEPS contributed to each of the submitted peer‐reviewed publications. The full dossier of the applicant can be seen at this link https://open.efsa.europa.eu/questions/EFSA‐Q‐2023‐00085?search=Diathermic+stunning.

DTS: Diathermic Syncope®: Flowchart representation of development timeline, identifying the nature of each STEP. (Source: Figure 21 of the dossier).

Description of the stunning method according to the dossier

3.1

In line with EFSA (2018) guidance, Section 3.1 presents information and data from the submitted dossier. This section includes all relevant details provided by the applicant, supplemented by comments, analyses and assessments from the ad hoc EFSA WG. WG‐contributions are explicitly identified and clearly labelled as ‘Assessment by the WG’ to distinguish them from the applicant's material. A comprehensive and consolidated assessment prepared by the WG is provided separately in Section 4, offering an integrated evaluation of the evidence and conclusions.

The applicant has developed the key parameters, such as the energy level and duration of exposure to come up with a simple stunning method that may be acceptable to some religious groups. However, the assessment of religious requirements is beyond the scope of the EFSA remit.

Name of the method

3.1.1

DTS: Diathermic Syncope® hereafter referred to as ‘DTS’.

Description of the method including potential sources of pain, distress and suffering

3.1.2

The potential sources described here are sources of the negative affective states pain, fear and distress, which are considered the main WCs in this scenario. DTS applies electromagnetic energy consisting of both a magnetic and an electrical field, moving as sine waves, perpendicular to one another. The electrical field induces an epileptiform seizure in the brain, while the changing electromagnetic field induces heat in the brain tissue through a process called diathermy. This heat leads to cessation of neurotransmitter function (Ikarashi et al., 1984), maintaining unconsciousness until the brain temperature returns to within 1°C of normal core temperature of the animal (Guy & Chou, 1982). The onset of the epileptiform seizure is calculated to occur prior to the onset of unconsciousness as a result of hyperthermia in the zone of application, and before the onset of any physical sensation of overheating of the skin (see Section 3.1.2.7). The EEG data collected throughout the development process for STEPS 5, 7, 9, 11 and 13.1 have been compiled (PR19) and raw data files provided where available (PR20 STEPS 5, 7, 9, 11 and 13, PR23 STEP 13.2).

Technical description of the apparatus

3.1.2.1

The stunning system apparatus includes six critical components, which are shown in Figure 2 and are listed below:

1. DTS Generator
2. User interface panel
3. Waveguide system
4. Applicator
5. Animal Restraint (not shown in Figure 2)
6. Faraday cage

1. DTS generator. This water‐cooled generator contains a magnetron (an evacuated tube for generating microwaves, with the flow of electrons controlled by an external magnetic field), high power components, automation hardware and the electronics required for generating electromagnetic energy at a frequency of 922 MHz (or within the region of 890–925 MHz). Generator output is a minimum of 10 kW and a maximum of 40 kW.

2. User interface panel. A software‐based user control panel which allows pre‐selection of the total amount of energy to be delivered to the animal. Energy calculations are based on variations of power (kW) and duration of applied energy (seconds). Each dose of energy delivered to the animal is monitored and recorded. The interface unit includes emergency stop capability; indicators of magnetron status (standby, warming, on); fault alerts and reset. Fault diagnostics and permanent storage of energy transmission data are included in the software.

3. Waveguide system. This transports the energy from the DTS generator to the Applicator. Electromagnetic energy leaves the DTS generator via rectangular aluminium tubing (waveguide). The cross‐sectional dimensions of the waveguide are sized according to the frequency of the electromagnetic energy being produced. The rectangular waveguide is rigid and thus requires the energy to be transitioned to a more flexible apparatus (waveguide rotary and telescopic joints and/or coaxial cable) to allow for accurate positioning of the Applicator on the animal's head (Section 3.1.2.5). There is an auto‐tuner located on the generator side of the coaxial cable in the waveguide section to optimise the energy transfer to the animal's head for different head sizes and shapes.

4. Applicator. This is the direct interface between the DTS system and the animal's head. The design is specific for the electromagnetic energy frequency and the type of animal being processed (e.g. cattle, calves, sheep). The penetrative energy from the waveguide applicator is focused on a target region. Fixed tuning stubs are located just prior to the applicator to improve the focus of the energy within the head for a range of animals. The auto‐tuner corrects small mismatches in tuning that may arise due to animal head variations. A quartz window protects the coaxial to waveguide applicator transition piece from water, hair and foreign material.

NB: According to the dossier, poor tuning results in inefficient energy transfer, which could lead to failure to induce unconsciousness, or inappropriate surface heating. However, the prevalence is not known, i.e. not reported in the dossier.

5. Animal restraint. (Section 3.1.2.6) The animal is restrained in an upright position in a mechanism that will allow the animal to be rotated, e.g. in a tipping or rotating box (to allow rotation and ejection after unconsciousness has been confirmed). A rear pusher may be used to ensure that the animal is positioned with the head protruding through the neck yoke aperture. A neck capture with chin lift (based on the American Society for the Prevention of Cruelty to Animals (ASPCA) Pen design) is applied. The chin lift is used to position the head such that the frontal bones are between 0 and 30 degrees from horizontal. The head capture unit has hydraulic rams, which are more rigid than the pneumatic rams, thus reducing the risk of compression during application.

6. Faraday cage. The applicator and the entire rotary box are enclosed in a Faraday cage (an enclosure that blocks static and non‐static electric fields) to protect operators in the case of energy leakage. The Faraday cage consists of a steel mesh with holes less than 12 mm in diameter. Personnel doors for access to the restraint unit are fitted at the level of the animal's head: from one side when in the upright position, and from the front when in the rotated position. These doors seal using electromagnets when the energy application is underway. The rear door, through which the animal enters, is also sealed using electromagnets. Safety switches are installed at the doors such that energy application cannot proceed if the doors are not properly sealed (these safety interlocks cannot be manually overridden). An emergency stop button is fitted inside the Faraday cage in case of the unlikely event of personnel being trapped within to prevent the risk of injury by the rotating mechanical restraint unit. DTS Active warning lights are fitted in prominent positions on the DTS generator and the Faraday Cage.

Apparatus Overview (animal restraint not shown). (Source: Figure 1 of the dossier).

Stunning system operation sequence

3.1.2.2

The application of energy is controlled from the interface panel in the vicinity of the animal and outside the Faraday cage (Figure 3).

Interface panel and close‐up of result screen following a DTS stun. (Source: Figure 2 of the dossier).

The start‐up sequence is the following:

1. Turn on Cooling Water.
2. Switch on Mains Power.
3. Use the key to turn Key‐Switch to 0
4. Wait for the computer to start up and the DTS control program to initialise
5. Press ‘Continue’ on the ‘Open Com Port’ box
6. Press ‘STANDBY’ to begin the 3 min Warm Up Sequence

The warmup is complete when STANDBY LED stays illuminated and Generator Status Reads ‘Generator Ready (Standby)’.

Once the Start‐Up Sequence is completed, DTS energy can be applied and reapplied continuously without the need to go through the Start‐Up Sequence again, until the system is shut down, either manually, as a result of an electrical fault or in the event of a power outage.

To apply energy, the following steps need to be followed:

1. Select the correct DTS Profile (Power & Time Combination) on the Main Operations Page. For a commercial unit, the range of options will be limited to ensure that the risk of operator error is minimised.
2. Put the Applicator in place manually (fitted against the animal's head).
3. When the animal is ready, push one of the ‘APPLY ENERGY’ buttons to initiate energy application. To cancel the energy application, push the ‘CANCEL’ button.

Note: Energy application is only possible when there are no generator faults, the animal is in place and all safety interlocks (E‐Stop, gates and gen doors) read OK. Some faults may result in the need to restart the warm‐up sequence. If the applicator is not positioned on the forehead of an animal, energy escaping from the applicator will trigger the automatic safety cut‐out and energy delivery will immediately cease.

The shut‐down sequence is the following:

1. Exit Standby mode by pressing the STANDBY push button
2. Press the touch screen Shutdown button to close the program
3. Remove the key from the key switch
4. Shut down the computer via the Windows start menu
5. Allow 10 min to elapse for cooling
6. Turn off mains power
7. Turn off cooling water.

Stunning apparatus cleaning and maintenance

3.1.2.3

This section provides, according to the applicant, practical guidance on the routine care, maintenance and safety checks required for the generator, applicator and restraint unit of the system, as well as essential information on microwave safety and calibration.

Generator care

Daily:

• Remove any build‐up of rubbish from within and around the generator.s
• Ensure that all air intakes and outlets are clear.

Weekly:

• Check water flow and arc detection devices are functional.
• Check for water leaks inside the generator.
• Check and replace (if required) air filters on generator side walls.
• Check waveguide for loose fittings and bolts.

Monthly:

• Check generator doors, limit switches and adjust to suit door stroke.
• Replace/wash air filters on generator side walls.

Applicator care

Daily:

• Wipe out any moisture, dust or hair.
• Visually inspect for cracks or other damage.

Weekly:

• Check waveguide and applicator for loose fittings and bolts.

Restraint unit care

Daily:

• Wash out any faecal matter, blood, hair or excreta.
• Visually inspect for cracks or other damage.
• Grease moving parts.
• Visually inspect unit and Faraday cage for damage.

Microwave safety

Primary microwave safety lies in the design of the Faraday cage, which prevents any microwaves from escaping into the surrounding environment. All hatches on the Faraday cage, which allow access for placement of the applicator, and for exsanguination, are fitted with specially designed microwave chokes which reflect energy back into the cage. The legal requirements pertaining to this screening differ in different countries, as do the requirements around the specific frequencies generated by the magnetron. Alternative frequencies should be validated prior to commercial use. In the case of a cage or choke failure, a radiation interlock system is placed on the outside of the cage and will instantly shut the DTS system down and prevent further operation, if an unsafe level of radiation is detected. All Faraday cage hatches are to be fitted with double interlocks to prevent operation if a hatch is not correctly closed/sealed. An optional interlock may be fitted on the applicator to check that head placement is correct before energy can be applied.

Calibration of the apparatus

Except for the auto‐tuner for which calibration is required every 2 years (manufacturer's recommendation), the stunning apparatus does not need regular calibration. However, care should be taken to inspect the waveguide, applicator and Faraday cage each day for damage and ensure everything is in good condition. Assessment of tuning should be carried out by a specialist technician annually, or in the event of a change in size of the applicator (e.g. for use on another species).

Technical description of restraining system construction and operation

3.1.2.4

The restraining system does not deviate substantially from that associated with conventional stunning and slaughter processes for cattle, e.g. using captive bolt or electric stun. The restraint unit is supplied via a crowd pen and race like for existing commercial restraint boxes used for mechanical or electrical stunning. The design of the restraining system allows for prompt ejection and immediate slaughter. Schematic diagrams of the rotating box restraining system are shown in Figure 4. Additional pictures of a rotating box restraining system under development are presented in Figures 5, 6, 7 and 3D representations of the rotating restraint box within the Faraday cage are shown in Figures 8 and 9.

Schematic diagram of rotating box and ejector system. This front view shows the restraint and transfer from ejection system onto bleed conveyor. The position of the animal depicted while being upright prior to DTS application (right) and neck cutting applied in inverted position, and while being ejected onto the bleed conveyor (left) in the tipped catching cradle post exsanguination (middle). (Source: Figure 3 of the dossier: ‘Schematic diagram of restraint and ejection system onto bleed conveyor, front elevation. One animal is upright prior to DTS application (right), one is in the catching cradle post exsanguination (middle) and a third is on the bleed conveyor (left)’).

Rear view of the animal entrance to the rotating restraint box. The upright side is shown on the left and the inverted side on the right. Image taken during workshop construction by the applicant.

View of the neck capture area with the side and the ‘top’ doors opened to allow animal ejection when inverted. In this image, the upright side is on the right, and the inverted side is on the left. Image taken during workshop construction by the applicant.

View of the inverted part of the rotating restraint box, showing the side and ‘top’ doors opened to allow animal ejection when inverted. Image taken during workshop construction by the applicant. (Source: Figure 8 of the dossier).

3D representation of the rotating restraint box within the Faraday cage. Personnel access steps are shown on the right, and the bleed conveyor is positioned on the left. (Source: Figure 9 of the dossier).

3D representation of the rotating restraint box within the Faraday cage shown from the rear to illustrate personnel access. (Source: Figure 10 of the dossier).

Prolonged restraint in any system is stressful to animals (Ewbank, 1992). According to the applicant, quiet handling and smooth application of the neck capture and chin lift apparatus is well tolerated by cattle (Dunn, 1990; Grandin, 1992) and the head position achieved allows accurate placement of the applicator onto the forehead of the animal. The animal is held in the chin lift for a maximum of 30 s while the applicator is positioned and DTS energy delivery is initiated.

Technical description of the DTS applicator's position and application of energy

3.1.2.5

Contact between the applicator and the animal's head

The applicator is attached to a flexible waveguide system, allowing it to be applied at different heights and angles to match various head shapes. The applicator is approximately square in cross section, tapering towards the interface end, with outside dimensions of 16.5 × 16.5 cm. Contouring within the Applicator head interface end, that is an interface at the tip of the applicator, also enables a uniform and continuous contact between the applicator and the head.

Position of the Applicator on the animal's head

The animal is held in the neck restraint and chin lift, ensuring that the poll is just in front of, and against, the fixed bar at the back of the head and the forehead is nearly horizontal (Figure 10, 11). The applicator is lowered onto the head ensuring that it is placed against the rubber cushion and is placed high on the forehead of the animal, on the frontal bones in front of the poll, held firmly using a rubber strap (Figure 11C,D) such that the opportunity for movement is minimised and energy is directed into the brain mass (Figure 12).

Diagram showing the placement of the applicator on the bovine head. Left: Frontal view, hatched area shows applicator position; Right: sagittal view, with the hatched area representing the applicator and its position relative to the brain and frontal sinuses (black). (Source: Figure 11 of the dossier).

Applicator positioning on the forehead of the animal. (Source: Figure 12 of the dossier). (A) Upper left: Positioning of the head in relation to reference points. (B) Upper right: Head positioned and ready for applicator placement. (C) Lower left: Placing the applicator through the hatch in the Faraday cage. (D) Lower right: Final applicator position.

Animal secured in the restraint with the chin lift activated and the applicator in position, immediately prior to DTS energy application. (Source: Figure 13 of the dossier).

Recommended energy (kJ) application according to the dossier

Based on the dossier and different stages of development of the method, DTS was applied to animals using energy doses approximating the recommended levels to animals within a liveweight range of approximately 270–690 kg (STEP 3.2 STEP 11.1, PR12, PR13, STEP 13, SS07, SS08 PR22). Linear regression of body weight to brain weight, following Ballarin et al. (2016), was used to estimate brain weights, which were 443–490 g (around 5% variation from the mean). According to the dossier, no variation was found in the animals' responses applying DTS according to the type (size, age, breed) of animals (see Section 3.1.2.6).

For commercial processing, energy levels of 160–200 kJ (180 ± 20 kJ) were recommended for a simple stun (Table 9, STEP 11 (PR12), STEP 12 (PR13), STEP 13 (PR15 SS07, SS08)). The dossier expressed a range of energy values and stated having evidence that a 16 kW incident power is sufficient to induce unconsciousness within 5 s of the onset of energy application, and that 160 kJ is adequate to maintain AW requirements throughout the process (duration of unconsciousness being sufficient to achieve death through exsanguination without return to consciousness and without causing physical damage to the brain), and that no signs of compromised welfare were observed within this range of energy application. Further investigations and experiences determined that one specification for energy application is preferable. Therefore, for routine commercial processing, it was recommended in the dossier to use 160–200 kJ, delivered with 18–20 kW incident power.

Although energy levels of less than 100 kJ have been shown to result in unconsciousness in some animals, the achievement and duration of unconsciousness were unreliable. Occasionally, animals were subjected to low energy application due to equipment failure as described at different stages of the development of the method (STEP 7‐SS05, Animals 1.10, 1.14, 1.17; STEP 11 Parts 1 and 2). Responses were variable, with animals ranging from seemingly unaffected, through being sedated, to unconscious for variable times. Therefore, energy application at levels below 100 kJ was considered to be a risk for AW.

In addition, the dossier described that energy levels above 220 kJ can lead to damage to brain tissue (PR12, SS08, STEP 14.), while energy levels of 300 kJ and above will result in death (STEP 9).

Duration of energy application

The emitted energy is equal to incident power multiplied by the duration of energy application. According to the dossier, a higher incident power, will reduce the time required to reach the desired energy delivery. However, the higher power setting may predispose to focal overheating, while a very low incident power may be insufficient to raise the temperature of the brain sufficiently rapidly to overcome the natural cooling provided by the blood circulation, and therefore fail to induce unconsciousness.

Based on the dossier, the data set indicated that a power setting of 16–20 kW is suitable. 18 kW delivered 180 kJ in a period of 10 s, while 20 kW delivered 180 kJ in a period of 9 s. In practice, delivery of energy takes slightly longer, because it takes time for the power to reach the desired level and some energy is reflected rather than being directed to the target surface (Figure 13). In addition, the machine runs through a start‐up sequence prior to energy being directed to the animal's forehead.

Cumulative energy delivered during 12 s of DTS application (Source: Figure 14 of the dossier: ‘Application of microwave during DTS stunning – power (kJ/sec), and total energy delivery over time’).

Effect on animal and process time

According to the dossier, DTS that delivers 160–220 kJ at an incident power (total forward power) of 18–20 kW induces loss of consciousness, as evidenced by the tonic–clonic behavioural response within 3 (tonic) to 11 (tonic–clonic) s of onset of energy application (PR 15, SS07, SS08 and PR21). The duration of unconsciousness recorded during a study on full recovery (STEP 13) was between 90 and 180 s, based on absence of corneal reflex and duration of EEG changes, whereas full consciousness was regained between 152 and 369 s after the onset of the tonic response.

The dossier (PR 24) generated a list for all animals (n = 195) processed using between 50 and 210 kJ since October 2019 (STEP 11 and STEP13), such that an overall analysis of first stun effectiveness, and average + SD time to onset of unconsciousness and duration of unconsciousness could be calculated. October 2019 was chosen as the starting point for these data, because from this point onwards a number of refinements to the delivery system had been conducted, including the incorporation of an autotuner and coaxial cabling to improve applicator manoeuvrability (STEPs 6, 8 and 10). The list contains 126 animals that were processed commercially, for which an assessment of first stun effectiveness was conducted by trained abattoir personnel, plus 69 animals on which detailed annotation of video footage was conducted to measure time to onset of unconsciousness. Overall, 192/195 (98.5%) of animals were effectively stunned on the first attempt.

In October 2019, in a sample of 9 animals, onset of the tonic seizure occurred between 1 and 6 s after the commencement of energy application (1.89 + 1.62 s), and the tonic seizure lasted for 0–32 s (14.89 + 14.11 s) before the clonic seizures began. In 2019, the minimum duration of unconsciousness was 62 s, based on a return of corneal reflex. The animals that were observed for signs of recovery were processed at 20 kW, 200 kJ.

Refinements to the applicator and coupling to the forehead continued, which has improved consistency of induction of unconsciousness. In December 2020, onset of the tonic seizure occurred within 1 (+ 0) s after the commencement of energy application (n = 30), and the tonic seizure lasted for 0–7 s (1.76 + 1.74 s) before the clonic seizures began. In 2020, no animal was allowed to recover, and no evidence of returning reflexes were recorded during bleedout.

In 2022, the formal assessment of recovery was carried out (SS07), onset of the tonic phase occurred within 6 s (3.17 + 2.56, n = 6) with the exception of one animal which had an interrupted, and therefore prolonged, energy delivery. Duration of the tonic phase was not recorded, and the earliest sign of recovery was shown at 63 s based on return of spontaneous blinking of the eyes. One animal which received 150–160 kJ recovered sooner than animals that received 180 kJ in that study. No animals have been assessed for time to recovery since 2022.

Refinements to the applicator have continued, and the most recent data (2024 and 2025) indicate that onset of the tonic seizure occurred within 4 s (1.76 + 1.00) after the commencement of energy application (n = 21), and the tonic seizure lasted for 0–5 s (2.42 + 1.38 s) before the clonic seizures began (n = 12). It is important to note that, in 2024 and 2025, application power was 16–18 kW, rather than 18–20 kW, which was used in 2019 and 2020. The reduced power could account for a slightly slower induction of the tonic phase.

The summary tables of data pertaining to the average (and STDEV) time to onset of unconsciousness and duration of unconsciousness for each study (STEPS 11, 13) (Table 1 of the dossier), each energy setting (Tables 2 and 3 of the dossier) and each power setting (Tables 4 and 5 of the dossier) have been presented in the dossier as confidential information.

At the current recommended parameters (16–20 kW, 160–200 kJ), the animals are described to regain consciousness at 180 s after the start of energy application, although early signs of returning consciousness (return of corneal reflex and spontaneous blinking) have been observed after 63 s from the start of energy application (STEP 13, SS07). In studies with unstunned animals during slaughter, the loss of eye responsiveness (palpebral/corneal reflex) as a result of exsanguination occurred between 95 and 103 s after the ritual cut (Gibson et al., 2015); while an isoelectric state in EEG has been demonstrated to occur 75 s (maximum 126 s in the absence of false aneurysm) after the ritual cut (Daly et al., 1988). To allow a margin of safety, it is proposed in the dossier to use the maximum time to isoelectric EEG as defined by Daly et al. (1988) to estimate the maximum stun‐to‐stick interval. Thus, according to the dossier, 180 minus 126 s indicates that a stun‐to‐stick interval of 54 s will prevent recovery of consciousness during bleeding. Figure 15 of the dossier timeline showed that the duration of bleed is shorter after applying DTS (55 + 8.7 s) compared to the duration of bleed after ritual cut of unstunned animals (76 + 7.9 s, Njisane & Muchenje, 2017). This may be a result of increased blood pressure and the inverted position of the unconscious animal. Furthermore, the dossier mentioned that the incidence of false aneurysm with DTS (one incident associated with operator error, across 350 cattle), is substantially lower than the incidence observed during ritual slaughter, reported in different papers, such as 7%, Gregory et al., 2010; 7.25%–10.25%, Bozzo et al., 2020; 38%–40%, Gibson et al., 2015; 60% Supratikno et al., 2022. Because DTS bleedout times are around 20 s shorter than those observed during ritual slaughter, and because false aneurysms occur less frequently with DTS, it was inferred in the dossier that an additional 20 s safety margin could be added when defining the maximum stun‐to‐stick interval. Therefore, the dossier recommends a maximum stun‐to‐stick interval for commercial processing of 45 s from the onset of energy application (Figure 15 of the dossier, indicated as confidential).

Figure 15 of the dossier shows the timing (range, in s) of occurrence of the major process steps, relative to commencement of energy application (time 0 s), recorded during STEPs 13.2 and 13.4. A number of these steps and transitions between steps were slower than would be expected under commercial processing conditions because of experimental procedures. For example: between positioning of the applicator and commencement of energy application, the experimental procedures included double‐checking and recording of applicator positioning; between end of energy application and the start of rotation of the box, the experimental procedures included detailed assessment and recording of ABMs (corneal reflex, palpebral reflex, eye position and response to nose pinch) by the researcher as well as the operator; rotation of the box itself was slow (22 s) so that EEG leads were not dislodged; and there was a delay between rotation end and application of cutting both carotid arteries and jugular veins to allow for detailed assessment and recording of ABMs (corneal reflex, palpebral reflex, eye position and response to nose pinch) by the researcher as well as the operator.

Post‐energy application observations were conducted by different operators at each of STEP 13.2 and STEP 13.4; thus, the estimation of ‘end of tonic–clonic’ and ‘blood flow slowed’ is not consistent between studies as both are subjective descriptors, according to the dossier.

In Figure 15 of the dossier, below the observations, the inferred state of consciousness, based on the data collected during STEP 13.3 (SS07) is shown (superscript label 1), followed by data extracted from published literature on state of unconsciousness after electrical stunning of cattle (von Holleben et al., 2010, Warrington, 1974, Weaver & Wotton, 2009, Wotton 2000; superscript label *), time to collapse after ritual slaughter (Gibson et al., 2015, Gregory et al., 2010; superscript labels 2 and 3), EEG changes after ritual slaughter (Daly et al., 1988; superscript label 4) and end of bleedout (Njisane & Muchenje, 2017; superscript label 5).

Under the experimental conditions of STEPS 13.2 and 13.4, the Halal cut was applied between 43 and 67 s after the commencement of energy application, and no evidence of returning consciousness during bleeding was observed, but this was for a relatively small number of animals. So, to ensure that animal welfare is protected during bleedout, it is recommended in the dossier that under commercial conditions, the target maximum stun‐to‐stick interval should be 45 s (blue dashed vertical line in Figure 15 of the dossier). This is achievable because the rotation speed of the box can be adjusted (to an estimated 10 s inversion time) to accommodate the recommendation for a shorter stun‐to‐stick interval. Furthermore, under commercial operating conditions, the delays incurred between steps as a result of detailed experimental observations and recording would not occur.

Assessment by the WG

The recommended energy (kJ) application, as described above, lacks a scientific rationale and the duration of energy application is not based on a sound scientific basis.

It is reported in the dossier that cattle continue to breathe during and following the application of DTS (Small et al., 2019). This finding suggests that DTS does not induce tonic–clonic seizures, and therefore, the occurrence of a grand mal epilepsy in the brain is very unlikely and is based on behavioural manifestations that are not necessarily indicative of unconsciousness.

It is also stated that ‘most recent data (2024 and 2025) indicate that onset of the tonic seizure occurs within a maximum of 4 (1.76 + 1.00) s after the commencement of energy application (n = 21), and the tonic seizure lasted for 0‐5 s (2.42 + 1.38 s) before the clonic seizures began (n = 12)’. The disclosure that tonic seizure lasted for ‘0 s’ implies that tonic seizure did not occur in some cattle exposed to DTS. In addition, the behavioural events used to ascertain tonic–clonic seizures have not been correlated with the EEG. Owing to these reasons, it is inferred that the stepping behaviour (kicking of limbs, Idris et al., 2024) occurring during heat stress, which is an indicator of attempt to escape a stressful situation according to the literature submitted (see studies SS07, SS08 in Section 3.2 and dossier reference PR21), begins within 3–11 s after the application of DTS when the animal is still conscious.

For more details on assessment regarding the applied energy levels, the duration of the application and the effect on the animals, see Section 4.

Additional commercial considerations

3.1.2.6

Animal characteristics

The DTS system has been validated on over 420 cattle ranging from 270 to 690 kg (estimated based on carcase weight); 262 of these received energy levels of 200 kJ or less (3.2 STEP 11, 3.2 STEP 13, PR24). Any animal large enough to accommodate the footprint of the applicator above its frontal bone (square cross section, tapered slightly at the nose end, approximately 16.5 × 16.5 cm) could be suitably stunned using DTS. The cattle were predominantly Bos taurus: beef, dairy and cross‐bred types. Heifers, steers, cows and bulls were represented; some were horned, some naturally polled and some dehorned. Five Brahman (Bos indicus) animals were included in the validation. Three mature bulls were successfully stunned using 200–250 kJ (lower power application was not tested). According to the dossier, animal type did not influence efficacy of induction of unconsciousness, providing that the applicator could be placed in contact with the forehead with no air gaps. Animals with very wide horns may not fit onto the restraint unit, and according to the applicant this is also the case with existing restraint units for captive bolt or electrical stun application.

The design of the applicator has been refined during the course of development to maximise the range of forehead shapes that can be presented to the applicator. According to the dossier, the DTS system is designed to be used for any medium to large animal (270–800 kg liveweight) with a forehead able to accommodate the applicator footprint approximately 16.5 × 16.5 cm. It is stated that applicators may be designed to suit specific bovine head types. Custom applicators will need to be designed and validated for use with other animals, such as sheep or calves.

In document SSO4 of the dossier, the penetration depth can be predicted based on the dielectric properties of the tissues through which the microwaves pass (SS04 McLean et al., 2017). It describes a thickness of 5 mm of skin and 12 mm of bone, and therefore the brain penetration depth has been predicted to be approximately 38 mm. Penetration depth may vary depending on factors such as the size and shape of the bovine head. Penetration depth is also affected by the frequency of the microwaves, which is a function of the magnetron (microwave generator) used. Heat is distributed through the brain mass via direct electromagnetic heating, conduction and circulating cerebrospinal fluid and blood.

All animals processed via DTS to date had been lairaged, so the skin of the forehead was dry. Some animals had a layer of dew in the hair coat. This moisture rapidly evaporated when energy application began. Both practical observations and theoretical calculations indicate that this moisture does not affect the efficacy of induction of unconsciousness (3.2 STEP 14, PR16, SS08). There has been no evidence of differences in efficiency related to thickness of the hair coat.

Cycle time

The cycle time for processing an animal was measured during trial operations, which were modified so that only a single animal was processed at any point in time (Figure 15 of the dossier). The times were measured while investigational work was also being conducted, and the ‘investigational time’ was subtracted from the process times where possible. The process was not operating with the intent of being as rapid as possible. According to the dossier, some fixed aspects of the process (such as the time taken for the box to rotate from the stunning to the sticking position) could also have operated at a faster rate.

Commercial throughput

Using a rotating box for animal restraint allowed for a second animal to be loaded while the first animal was being exsanguinated (Figure 14). Under these conditions, the dossier stated that animals have been processed at a rate of one per minute (60 per h). The engineering team have been working on an alternative box design to increase the speed of ejection of the exsanguinated animal from the restraint, with a view to achieving a processing rate of 2 per minute (120 per h). This is in line with current processing rates in commercial beef abattoirs in Australia (60–120 per h per restraint box).

Animal being exsanguinated through a hatch in the Faraday cage. (Source: Figure 16 of the dossier).

Handling of exsanguinated blood

According to the dossier, exsanguinated blood flows into the abattoir blood drain and is disposed of according to the local procedures (e.g. DAFS processing, settlement ponds, rendering).

Potential sources of ‘pain, distress and suffering’

3.1.2.7

According to the dossier, the hazards identified during this process are:

1. Restraint and/or inappropriate restraint
2. Incorrect position of applicator
3. Incorrect power and energy parameters
4. Undue delay between stunning and bleeding
5. Overheating of the skin prior to onset of unconsciousness, or from an overheated applicator rim.

These hazards can lead to the WCs of pain and fear and may also result in failure in the onset of unconsciousness or in too early recovery before or during bleeding.

Welfare hazards and consequences: Prevention and correction of WCs and their related hazards

A hazard outcome table is presented below (Table 5), with detailed descriptions of each hazard presented in the subsequent sections.

Restraint and/or inappropriate restraint

According to the dossier, if the animal's head is not secured in an appropriate position that permits the applicator to be in contact with the forehead, energy leakage at the applicator interface can occur. The DTS system is fitted with sensors to detect energy leakage and will shut down the energy application before leaked energy reaches a level that could cause overheating of skin without induction of unconsciousness. However, there is a risk that excessive pressure may be applied during restraint. The chin lift should not be applied until the operator is ready to position the applicator for energy delivery. Animals should not be left in restraint during breaks or stoppages. Procedures must be in place to enable the evacuation of animals from the restraint in case of an emergency (HSA (Humane Slaughter Association) 2016).

Based on the dossier, some animals may be too small to be positioned correctly for an effective stun to be produced: No testing has been completed on animals < 250 kg (Section 3.1.2.6); therefore, effectiveness on small animals has not been demonstrated. If the animal's head is too small to allow adequate contact with the applicator, the system would not operate (interlock sensor devices).

Also, some animals may be too large to fit into the restraint unit, which includes head restraint and chin lift, such that they can't be stunned effectively. No testing has been completed on animals > 800 kg (Section 3.1.2.6). The current restraint box has been designed to accommodate animals in the 250–800 kg range. The system would not operate if the animal could not be positioned to allow correct contact between the head and the applicator.

Incorrect neck capture and head restraint, difficulties in positioning the head and/or applicator can lead to increased time in the restraint box, possibly leading to increased fear. The head restraining equipment is designed to assist the operators; however, the issues relating to livestock restraint and head capture that are present in other cattle handling/stunning systems do remain. For example, ‘stun boxes’ are used in cattle slaughterhouses to restrict the forward, backward and lateral movements of the animal and head restraining devices are used to restrict vertical and lateral movements of the head of the animal to facilitate effective penetrating captive bolt or electrical stunning. Positioning of the DTS applicator on the head of the animal is carried out manually, and this requires a competent operator. Incorrect positioning of the applicator can lead to energy leaks and further delay in the application of DTS.

Incorrect position of the applicator

According to the dossier, the applicator must be positioned on the flat frontal bones of the forehead, above a line drawn between the lateral canthi of the eyes. If the applicator is placed too far forward, between the eyes, there is a risk that the sensitive ethmoid turbinates of the nasal cavity are heated, which would cause pain. If the applicator is placed at an angle such that there is a gap between the applicator rim and the forehead, energy leakage can occur. The DTS system is fitted with sensors to detect energy leakage and will shut down the energy application before leaked energy reaches a level that could cause overheating of skin without induction of unconsciousness. The applicator footprint should fit the forehead of the size of animal processed – i.e. if small cattle or calves were to be processed, a smaller applicator would be required than when processing full‐grown cattle.

Incorrect power and energy parameters

Power (kW) and energy (kJ) determine the effectiveness of induction of unconsciousness using the DTS system. Total energy determines the duration of unconsciousness. In adult cattle, power settings of 18 kW and above lead to unconsciousness within 3 s (onset of tonic seizure) to 8 s (onset of clonic seizure) from the commencement of energy application (Small et al., 2019;Small, Jenson, Fiszon, et al., 2025 ; Small, Jenson, Phillips, et al., 2025) (SS07, SS05, PR22 (DTS‐ABMs‐manuscript 2025), while power settings below 16 kW may not be sufficient to overcome the animal's cerebral thermoregulatory response leading to slow or no induction of unconsciousness and a risk of pain resulting from overheating of the skin.

Small et al. (2019) (SS05) demonstrated that an energy delivery of 45.87 kJ could result in unconsciousness, but the animal showed signs of recovering consciousness approximately 1.5 s after the end of energy application, and similarly an animal that received 85 kJ showed signs of recovering consciousness approximately 3 s after the end of energy application (Small et al., 2019; Small, Jenson, Fiszon, et al., 2025 ; Small, Jenson, Phillips, et al., 2025) (SS07, SS05). Although the sample sizes were limited, recent studies indicate that total energy deliveries of 160–180 kJ can provide a duration of unconsciousness that may be commercially acceptable, while still preserving the integrity of brain tissue (Small, Jenson, Fiszon, et al., 2025; Small, Jenson, Phillips, et al., 2025) (SS07, SS08, PR22). If the criterion of ‘no damage to brain tissues’ was not required for acceptability of the method to the proposed market, total energy packages of 200–220 kJ could be used, providing a longer duration of unconsciousness.

Undue delay between stunning and bleeding

According to the dossier, a delay between the end of stun and sticking leads to a potential for return of consciousness during bleedout, causing pain and distress. A maximum stun‐to‐stick interval of 45 s from the start of energy delivery is recommended, which provides a substantial safety interval before full recovery could be expected (Section 3.1.2.5). Monitoring of ABMs prior to sticking and during bleedout is essential.

Poor bleedout can occur when both carotid arteries and both jugular veins are not fully severed. The restraint unit presents the neck extended to optimise the operator's access to the appropriate cut region, high on the neck behind the jaw. After completing the cut, the operator should check that all four vessels have been cleanly cut and check for the presence of false aneurysm; if deficiencies are detected, a re‐cut must be performed.

Three incidents of a unilateral clot occluding one carotid artery were recorded among 424 animals receiving DTS application at any energy level up to 360 kJ (0.6%). All three were attributable to operator error, specifically incomplete neck cutting. These cases all occurred in the subset of animals receiving energy applications of 200 kJ or less, when observers from religious certification authorities were crowding the sticking area. The operator reported being concerned about the risk of injuring a visitor.

Preventive measures listed in the dossier are:

• Ensure that the operator has adequate space in which to perform a full cut of the neck;
• Ensure that the blade is sufficiently long for the task;
• Ensure that the blade is sharp and free of nicks.

Corrective measures listed in the dossier are:

• As soon as the cut is complete, inspect the gash and if the neck is not fully opened, re‐cut the vessels;
• Apply a brachiocephalic/thoracic sticking procedure if blood is not flowing strongly;
• Apply a back‐up captive bolt stun if breathing movements do not cease within 90 s of the halal cut;
• Apply a back‐up captive bolt stun if righting reflex is noted during bleedout.

Overheating of the skin prior to onset of unconsciousness, or from an overheated applicator rim

Overheating of skin was a concern early in the development of the system, and refinements to the application of DTS continued.

Under the current recommended operating parameters and with the current applicator, waveguide, and tuning device design, the onset of unconsciousness, indicated by the onset of the tonic behavioural response, occurs 0–3 s after the onset of energy delivery (onset of tonic–clonic response: 3–8 s after the onset of energy delivery, Figure 15 of the dossier). Accordingly, the mean brain temperature at the point of loss of consciousness is predicted to be no more than 40°C (corresponding to the latest onset of the tonic response at 4 s), with local hotspots not exceeding 53°C (PR22).

In all animals, forehead heating occurs during energy application, and warmth can be felt on the forehead when the waveguide is removed. At this stage, the hair remains attached to the skin. At the end of bleeding, depilation of the hair is evident and visible skin changes are present. Histologically, this skin change resembles a second‐degree burn, extending partially into the dermis (Small, Jenson, Fiszon, et al., 2025; Small, Jenson, Phillips, et al., 2025) (SS08).

Measurement of the skin temperature changes at the point of contact of the applicator with the forehead, done by fibre optic temperature sensors, indicates that temperature begins to rise some 8 s after the start of energy application and continues to increase thereafter. In all animals measured, the tonic–clonic response began within 3 s of energy application, so cattle were unconscious before temperature increase of the forehead (PR22).

The applicator currently used is fabricated from mild steel, which is susceptible to warming. According to the dossier, for a commercial system, it would be preferable to construct the applicator from aluminium, which will not retain heat.

Assessment by the WG:

Overheating of the skin occurs in all the animals exposed to DTS applications. In addition, the time to onset of skin burn was not reported. The assessment concluded that it is difficult to assume that unconsciousness occurs prior to burning of the skin. More details can be retrieved in Section 4.1.2.

Evidence of pain, distress and suffering in the operating DTS system

According to the dossier, studies of near‐to‐commercial operating system (Section 3.2 of the dossier, STEPS 11, 13, 14) have reported animal WCs when observed, with the nature of these observations becoming more detailed as work has progressed.

First stun effectiveness is significant to AW because it reduces the duration of restraint and activity around the animal. Across STEP 11 and STEP 13 studies, the first stun effectiveness was calculated at 98.5% (192/195) (Section 3.2 of the dossier, STEP 13.1). The three failures were due to an incorrectly positioned applicator, causing the system to shut down. In these cases, the only negative welfare impact was the longer time in restraint (~10–20 s) due to the need to reposition the applicator.

Since the reporting of STEP 11, 7 incidents of energy leakage and automatic shut‐down have been recorded among 262 animals exposed to 200 kJ energy application or less (PR24), representing 2.7%. Three of these incidents (1.1%) resulted in energy applications of less than 95 kJ and did not induce the tonic–clonic response. Four animals receiving between 95 and 128 kJ did enter the tonic–clonic phase, but the duration of unconsciousness was shorter than desired. In these cases, back‐up stunning was performed using a captive bolt.

In STEP 11, ABMs indicative of serious pain, distress or suffering were observed only infrequently during restraint or application of DTS (Table 6).

In development STEP 13.2 (PR22), responses potentially indicative of pain, fear and distress of 12 animals to restraint and induction of unconsciousness were studied. Several ABMs were included. Due to the experimental nature of this work, some ABMs were observed at time points that would not occur under commercial processing. Only very minor discomfort was noted during restraint. None of the animals showed pull back, jump forward, toss head, struggle, bellow or pain face. During energy application the maximum time to unconsciousness, based on onset of the tonic phase, is 3 s and in this phase no negative ABMs were observed (Table 7). It is concluded in the dossier that the animals do not experience pain, fear or distress during restraint and induction of unconsciousness.

Assessment by the WG:

The WG noted that because animals are physically restrained during DTS energy application, behaviours such as pull back, jump forward, toss head and struggle may be prevented. Therefore, the absence of these ABMs cannot be interpreted as evidence of absence of pain, fear and distress.

Assessment of animal welfare when DTS is used

3.1.2.8

For the assessment of AW when applying DTS, the applicant provided the following definitions for pain, fear and consciousness:

Pain is defined by the International Association for the Study of Pain (ISAP) as ‘an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage’ (Raja et al., 2020).

Fear is defined as an emotional state induced by the perception of a danger or a potential danger that threatens the integrity of the animal (Boissy, 1995).

Consciousness is defined as the capacity to receive, process and respond to information from internal and external environments and therefore the ability to experience emotions, leading to pain and fear (Andrews et al., 2025; Boly et al., 2013; Le Neindre et al., 2017; Mashour & Alkire, 2013; Terlouw et al., 2016; Terlouw & Le Neindre, 2024; Zeman, 2001).

In addition, and as part of the request for additional information, the applicant was asked to provide the ad hoc WG of EFSA with the percentage of certainty that individual ABMs or a combination thereof indicate unconsciousness. The applicant's reply consisted of the table below (Table 8), which reports ABMs associated with consciousness rather than unconsciousness for the 3 key stages: after stunning, at neck cutting and during bleeding. The WG's assessment of these ABMs – used to assess the AW after applying DTS – is presented in Section 4.

For more analytical information please see Section 3.1.2.8 of the dossier.

Assessment by the WG

According to the WG, the potential WCs during restraint are pain and fear. If the stunning is ineffective, the potential WCs are pain and fear due to the persistence of consciousness during possible skin burn from the application of DTS and following sticking and bleeding. Since DTS application is proposed as a method to induce heatstroke and epileptiform seizure in the brain of cattle, and since cattle exhibit several behavioural changes (depending upon the stages of heatstroke) indicative of pain, distress and suffering, assessment of AW should be based on the behavioural signs of escape attempts or struggle. In this regard, the WG believes that previously published literature concerning heatstroke in cattle is required to fully assess the WCs.

In addition, in Appendix 2 of the dossier, a table lists hazards identified for each phase of different stunning methods. For the unconscious phase of DTS, it is written that ‘Unconscious animal is rolled out of the restraint unit and exsanguinated’. According to the WG, this sentence is inaccurate for two reasons. First, unconsciousness induced by DTS has not been unequivocally demonstrated. Second, the dossier states that animals are rotated 180 degrees within 10 s after the application of DTS whilst remaining restrained inside the faraday cage and that neck cutting is performed through a hatch (see Figure 14). Therefore, animals are exsanguinated within the restraint and held until the bleeding is complete.

For EFSA's WG detailed assessment of AW when DTS is used, see Section 4.

Key parameters of the effective use of the proposed method

3.1.3

According to the dossier, DTS uses electromagnetic energy that results in volumetric heating of the brain to a level that sustains unconsciousness (hyperthermic syncope). The temperature of cattle (SS04 – McLean et al., 2017) and sheep (SS02 – Small, Mclean, Owen, & Ralph, 2013; Small, Mclean, Keates, et al., 2013) brains could be raised by the required amount (5°C–12°C) to achieve induction of unconsciousness.

The applicant states that at the initial stages of development (3.2 STEPS 1 and 2), the working hypothesis posited that unconsciousness was induced and maintained through the volumetric heating effect alone. As development proceeded, it was recognised that an electrical component is also present. This was evidenced by the tonic–clonic epileptic behavioural responses observed in conscious animals exposed to energy packages up to 250 kJ (3.2 STEPS 9 and 11).

In humans, hyperthermic loss of consciousness or fainting, typically occurs when core body temperatures reach 40°C–45°C (Ohshima et al., 1992), while rats have been observed to lose consciousness based on behavioural indicators, following an increase in brain temperature of 8°C compared to normal (Guy & Chou, 1982). Ikarashi et al. (1984) demonstrated that neurotransmission, as measured by acetylcholinesterase activity, is abruptly diminished in rodent brain tissue when brain temperatures exceed 45°C. Bench‐top cadaver work conducted as part of the development of the DTS system indicated that it is possible to raise brain temperatures in cattle (SS04 – McLean et al., 2017) and in sheep (SS02 – Small, Mclean, Owen, & Ralph, 2013; Small, Mclean, Keates, et al., 2013) by 5–12°C to induce unconsciousness. The microwaves are delivered at a frequency selected to maximise penetration into the brain and can be visualised as a column of energy entering through the applicator. Heat generated within that column is transferred throughout the entire brain via direct conduction, as well as through the circulation of warmed blood and cerebrospinal fluid. It is expected that the skull acts as a heat insulator, reducing radiant heat loss and maintaining the increased brain temperature (PR02: Report on Microwave Technology Development Stage 1). The effect of brain heating was then confirmed using electroencephalography (EEG) in anaesthetised sheep (SS02 – Small, Mclean, Owen, & Ralph, 2013; Small, Mclean, Keates, et al., 2013; n = 3) and anaesthetised cattle (SS03 – Rault et al., 2014; n = 2); and subsequently in conscious cattle (SS05 – Small et al., 2019; n = 3 or 4 per treatment).

Assessment by the WG

The dossier states that a maximum stun‐to‐stick interval of 45 s is recommended in all cases. This recommendation is based on EEG findings and ABMs indicative for unconsciousness, the practical operating speed of rotary boxes and the absence of ABMs post sticking. It is also assumed that death through exsanguination occurs within 4 min or less in cattle (Gregory et al., 2010; Newhook & Blackmore, 1982).

Table 9 provides a summary of the key parameters associated with the use of DTS and, where applicable, provides values for these key parameters. References were made to the relevant sections of the dossier, peer‐reviewed publications and project reports that contain further information and justification for the parameters. Nevertheless, according to the WG, it would be helpful to define the temperature in critical brain regions necessary for the induction of unconsciousness, but according to the submission of additional information, it is argued that temperature measurements of the brain in live animals have not been possible because it would require implantation of fibreoptic thermoprobes under general anaesthesia before applying the process.

Key parameters for existing methods are provided in Annex A of the EFSA guidance (EFSA, 2018). As DTS represents a completely novel stunning method, key parameters have been established using a similar approach and based on the research available to date. Table 10 provides, according to the applicant, the values for the key parameters identified as being analogous to those used for other cattle stunning methods. The other cattle stunning methods listed in Annex I of Council Regulation (EC) No 1099/2009 were mechanical or electrical stunning. References are provided to sections of the applicant's dossier, peer‐reviewed publications and project reports that contain further information and justification for the parameters. Further to the references to scientific studies (STEPS), Figure 22 of the dossier (Section 3.2) provides a summary of the steps involving conscious animals stunned with DTS, some of the key parameters investigated and outcomes obtained.

List of related specifications (as per Annex I of EC 1099/2009)

3.1.3.1

Table 11 lists the specifications for DTS applications according to the requirements of Annex I of Regulation (EC) No 1099/2009, as reported by the applicant. These specifications will be assessed by EFSA's ad hoc WG.

Scientific basis of induction and maintenance of unconsciousness for this method

3.1.4

According to the dossier, the initial working hypothesis (Section 3.2 of the dossier, STEPS 1 and 2) was that unconsciousness could be induced and sustained solely through the diathermic heating effect. As development proceeded, evidence emerged indicating that there is also an electrical effect, evidenced by the tonic–clonic epileptic behavioural responses observed in conscious animals exposed to energy packages of up to 250 kJ (3.2, STEPS 9 and 11). Therefore, the dossier concluded that the induction and the maintenance of unconsciousness result from the combined action of electrical effects and volumetric (whole tissue mass) heating of the brain tissue via diathermy, conduction and circulation, which occur simultaneously.

Electromagnetic effects, according to the dossier

3.1.4.1

The mechanisms described here are based on theoretical considerations, with inferences from research performed in other stunning methods (e.g. electrical stunning), diathermic therapies (e.g. brain cancer ablation) and electrophysics (e.g. Gauss' theorem, Stokes' theorem and Faraday's law of Electromagnetic induction). Measurement of electrical activity within the brain during energy application is not currently possible because the applied microwave energy saturates detection systems, while measurement of internal brain temperature is complicated in conscious cattle in an abattoir situation, because implantation of fibreoptic thermoprobes into brain tissue is required.

Electromagnetic energy is applied to the brain as a sine wave, essentially a spiral of moving energy (Figure 15). At each point of the spiral, there are perpendicular electric and magnetic fields, meaning that the electrical field and the magnetic field are constantly changing. These constantly fluctuating electrical fields generate current within the tissue, leading to the diathermic heating effect.

Diagram representing a sinusoidal wave in the Time IQ plane. The I component is called inphase because it is in phase with cos(⋅), while Q component is called quadrature because sin(⋅) is in quadrature, i.e. 90° apart, with cos(⋅). Source: © Wireless Pi 2026 https://wirelesspi.com(https://wirelesspi.com/the‐concept‐of‐frequency/ Figure 18 of the dossier).

Electrical effect

Schematic diagram of energy application (orange) and expanding neuro‐electrical field (purple) (Source: Figure 10 of the dossier).

The high frequency electromagnetic energy directly affects neurones in the path of application, as occurs in electrical stunning (Figure 16). This disrupts neuronal function by passing sufficient current flux across neurones to disrupt their membrane potential and make normal activity impossible. Once the current flux stops, the neurones remain uncoordinated for a period (the epileptiform activity) and then either resume coordinated function or move to isoelectric (non)activity. Although the electromagnetic energy itself is attenuated as it penetrates into the brain mass, according to the dossier it is hypothesised that, similar to the situation in electrical stunning, release of neurotransmitters results in sequential recruitment of other neurones, resulting in rapid expansion of the neuro‐electrical response to encompass the entire brain and upper spinal cord. This leads to the behavioural responses observed, particularly the tonic–clonic epileptiform behaviours indicative of a generalised epilepsy, which are observed within 3–11 s of energy application at 18 kW. Hindlimb extension, which indicates expansion of the neuro‐electrical response into the upper thoracic spinal cord, is observed and occurs during energy application making loss of posture an unreliable ABM for DTS. Hindlimb extension is also observed in electrical stunning when the current is applied for longer periods, leading to expansion of the neuro‐electrical response into the thoracic spinal cord. In electrical stunning, very short applications lead to partial epilepsy and a flaccid state which is not observed with DTS at the recommended power and energy delivery.

Heating effect

When electromagnetic energy is applied to tissue, diathermic heating occurs due to mechanisms like ionic conduction and dipole rotation, which dominate at microwave frequencies for most biological tissues, providing energy storage and conversion to heat (Boon & Kok, 1988).

The dielectric properties of any material are the main parameters that influence how microwave energy is coupled into the material and how efficiently this energy is then converted into thermal energy. These properties are described by the following equation:

$$[eqn]$$

The real part of the equation (ε′), the dielectric constant, represents the capacity of the material to store energy under a microwave field while the imaginary component (ε″), or the loss factor, reflects how efficiently the stored energy is converted into heat within the material.

The dielectric properties of bone, skin, fat and brain tissue are influenced by many factors including electric field frequency, temperature, density and moisture content. As a result, during any microwave heating process, the dielectric properties will inevitably change. The nature of this change depends on the influences mentioned above. Consequently, as microwave heating progresses, the tissue temperature increases and dielectric loss factor increases as well. This can lead to an effect known as thermal runaway, where hotter parts of the workload are heated preferentially, which in turn increases the loss factor, thus promoting non‐uniform heating (Metaxas & Meredith, 1983).

Knowledge of dielectric data is essential in the design of any microwave heating system because it enables accurate estimates of power density, the associated electric field stress and the penetration depth within the material being processed.

The starting point for all mathematical analysis of bioheat transfer in the living tissues is the Pennes' equation:

$$[eqn]$$

The Pennes' equation represents the energy balance between the conductive heat transfer, the endogenous heat generated by the metabolic processes and the effects due to the blood vessels acting as a thermal sinks or sources. The left‐hand side of the equation features ρ and C, denoting the density and the specific heat capacity of the tissue, respectively, Wb represents the volumetric perfusion rate, Cpb is the specific heat capacity of blood and Qm is the volumetric heat generated by the specific tissue. The bioheat equation features an additional term, H, representing the heat deposited in the tissue from external sources, e.g. electromagnetic radiation.

The penetration depth (D_p_) refers to a wave passing into a dielectric material, whose amplitude is diminishing owing to power absorption as heat into the material. The wave's field intensity and power flux density fall exponentially with distance from the surface. The rate of decay is a function of the complex dielectric constant ε, which comprises the real component ε′ and the loss factor ε″ as shown in Equation 1.

The penetration depth is defined as the depth into the material at which the power flux has fallen to 37% (1/e) of its value on the surface. The dielectric constants of the material being processed will provide insight into the electromagnetic behaviour of the wave inside the material and allow design criteria to be defined.

The relationship between D_p_ and the dielectric properties of a material can be described using the following formula for low loss dielectrics.

$$[eqn]$$

where: λ = wavelength.

Equation (3) reveals that penetration depth is proportional to the wavelength of the incident microwave energy, thus higher frequencies will achieve lower penetration into the workload given the dielectric properties are similar at both frequencies. In commercial applications in Australia the two frequencies available are 2.45 GHz and 922 MHz (SS04; Mclean et al., 2017), and these were the frequencies first investigated during development of the DTS: Diathermic Syncope® technology. Dielectric properties for a variety of tissues (human) and penetration depths are presented for both frequencies in Table 12 below and calculations for cattle assume similar dielectric properties. This assumption is based on similarity of dielectric properties of tissues in different animals including humans reviewed in (Gabriel, Gabriel, & Corthout, 1996; Gabriel, Lau, & Gabriel, 1996), although some inconsistences can be found in the literature. Human dielectric data are taken because they are completer and more consistent than animal data.

The vast majority of the literature on the dielectric properties of biological tissues described them in terms of dielectric constant and conductivity (Axer et al., 2002; Bao & Hurt, 1997; Cook, 1951; Foster et al., 1979; Gabriel, Gabriel, & Corthout, 1996; Gabriel, Lau, & Gabriel, 1996). These data can be used to determine penetration depth using Equation 4 below.

$$[eqn]$$

where:

c = 299,792,458 (m/s),

ε _ 0 _ = 10^9^/36π (Farad/m),

σ = Conductivity (Siemens/m).

From Equation 4, another set of penetration depth data was calculated as shown in Table 13 below.

The main area of concern is the penetration depth into the brain. To estimate this, the approximate percentage of incident power absorbed into the skin and bone was calculated based on approximate thicknesses (Table 14) and the assumption that power travels perpendicular to the plane of each material. This is shown in Tables 15 and 16 below for cattle heads at 2.45 GHz and 922 MHz, respectively.

For more information see also Section 3.1.4.1 of the dossier.

Summary according to the dossier

One of the primary concerns in any microwave heating system is the penetration depth of the power flux into the material, which directly influences the heat distribution and the overall uniformity of heating. Penetration depth refers specifically to the distance from the surface at which the power flux density has decreased to 37% of its initial value. As power decays exponentially with depth, the corresponding temperature also declines with increasing distance from the surface of the animal's head.

The following are some general conclusions that may be derived from the dielectric values of brain, bone and skin:

• These sets of results represent a generalised set of loss factors ε′ that may be encountered. For a given electric field E the absorption of energy depends on ε′. The effectiveness of heating will depend as much on the design of the applicator as on the loss factor of the material.
• Tables 12, 13, 14, 15, 16 show that penetration depths calculated from both dielectric data sources match quite closely at 922 MHz but differ considerably at 2.45 GHz. Since DTS application deals with frequency of 922 MHz, non‐matching data at higher frequency 2.45 GHz is not important for consideration. Penetration depths are significantly greater at 922 MHz than at 2.45 GHz. Penetration depth at 2.45 GHz is approximately 30% of that at 922 MHz mainly due to frequency effects.

The above calculations formed the theoretical basis on which development of the DTS technology ensued. Animal research began using high power/energy parameters (12–30 kW, 150–300 kJ) in anaesthetised animals (STEP 5; n = 2 per treatment) to confirm that electroencephalographic changes that were not compatible with consciousness could be achieved. Subsequently, similar parameters were tested in conscious cattle (20–30 kW, 100–360 kJ) followed by a stepwise reduction in power/energy parameters over a prolonged period to reach the current recommendations of 18–20 kW, 160–200 kJ. The stepwise reduction was performed to identify the parameters that would provide opportunities for commercial processing, without undue compromise of AW.

When the electromagnetic energy enters the brain, the tissue is heated along the path of energy penetration. Heat is then distributed through the brain mass both by direct conduction and, in the live animal, by circulation via the active vascular system and circulating cerebrospinal fluid (Figure 17).

Schematic diagrams of heat transfer within the brain (A) via conduction and (B) via circulation (Source: Figure 20 of the dossier).

Although brain temperatures can be measured in cadaver heads and were measured as part of the early development process (SS04, McLean et al., 2017), temperature measurements of the brain in the live animal have not been possible. This would require implantation of fibreoptic thermoprobes under general anaesthesia, which would result in anaesthetic residues in the tissues of the carcass processed. Infrared thermal imaging also cannot show temperatures within the brain, only surface temperatures after application. According to the applicant, subsequent to the early development work described in McLean et al. (2017), all assessments of efficacy have been based on behavioural responses and EEG.

Theory of thermal unconsciousness

3.1.4.2

According to the dossier, for DTS, insensibility is maintained by heating of the brain tissue to a temperature above which neurotransmission cannot occur. Brain metabolism and neurotransmitter activity is affected by temperature. This implies that volumetric heating of the brain, resulting in inhibition of neurotransmitter activity (including excitatory neurotransmitters such as glutamate), is incompatible with the induction of epileptiform activity in the brain.

Assessment by the WG

The WG does not support the hypothesis that tonic–clonic seizures are indicative of unconsciousness. Instead, it is more likely that the inhibition of brain metabolism and neurotransmitter function results in suppressed EEG patterns rather than seizure‐like (epileptiform) activity. In rats, at a brain temperature of 41°C (5.4°C above the control brain temperature of 35.6°C), adenosine triphosphate (ATP) concentration was reduced by 29.2% and creatine phosphate (CP) concentration was reduced by 44% (Sanders & Joines, 1984). In the same study, nicotinamide adenine dinucleotide (NADH) fluorescence increased during microwave application and returned to baseline levels within 1 min after cessation of energy application. Another study (Ikarashi et al., 1984) demonstrated a rapid reduction in acetylcholinesterase activity in rat brains once a temperature of 50°C was reached.

Thermal unconsciousness, such as that induced by exercise heat stress or fever, is reported to occur when core body temperatures of mammalian species, including rats, dogs, sheep and humans reach between 40°C and 45°C (McDaniel et al., 1991 rats; Ohshima et al., 1992 human; Mohanty et al., 1997 sheep; Roccatto et al., 2010 human; Lerman et al., 2014 dog; Yoshizawa et al., 2016 human; Hjeresen et al., 1983 rats). Similar data are not available for cattle. In a study on rats, Guy and Chou (1982) demonstrated that after rendering the animals unconscious using diathermy, they recovered consciousness when the brain temperature returned to within 1°C of normal.

In cattle, in which core body temperature (38.6°C–39.1°C) is comparable to that of sheep (38.3°C–40.5°C) and dogs (37.5°C–39.2°C), but 1–2 degrees greater than rats (37.5°C–37.8°C) or humans (36.5°C–37.5°C), normal brain temperature would be in the range of 36.6°C–38.1°C and therefore this suggests that thermal unconsciousness in cattle should be achieved when the brain temperature reaches between 39°C and 44°C.

Experimental work has shown that, using 2–10 kW of 915 MHz microwave radiation, rats could be stunned (Guy & Chou, 1982). The rats would be unconscious after a brain temperature rise of 8°C and would remain unconscious for 4–5 min post‐exposure. Consciousness was regained when brain temperature returned to within 1°C of normal values. Also, Lambooy et al. (1989) successfully induced unconsciousness in rats using a low‐power microwave (2 kW): application for 1.5–2.0 s resulted in unconsciousness in 100% of the rats tested. In the absence of cattle‐specific data, based on an extrapolation from the data for other mammalian species, that in cattle thermal unconsciousness would occur when core body temperature was in the range 42°C–47°C. Thus, using a heat transfer model for calculation, the required rise in temperature to achieve insensibility is in the range of 3.4–8.4 degrees.

However, according to the WG, temperature changes in different parts of the brain of rodents have been measured after microwave irradiation using previously implanted cannulas (e.g. Butcher & Butcher, 1976). Scientifically sound and robust data are needed for cattle brain temperature, specifically regarding the time needed to reach critical brain temperature following the application of DTS at 160–180 kJ for 10 s. It should also be noted that, in cattle, the time to reach the critical brain temperature will be prolonged due to the additional blood supply to the brain via the vertebral artery, and therefore direct extrapolation of data from humans and rodents may not be reliable. Furthermore, another disconcerting fact is that Ikarashi et al. (1984) reported that two out of six rats remained conscious after microwave irradiation of their brain and attaining a brain temperature of 45°C. Based on rodent studies (Guy & Chou, 1982), unconsciousness would be expected only when brain temperature increases by more than 8°C above normal. Overall, sound scientific evidence is lacking to demonstrate that thermal unconsciousness can be induced or achieved in cattle without causing pain, fear and distress.

In live animals, brain circulation will increase to maintain homeothermy in response to the heating effect of the application of microwave energy. Therefore, brain heating rates by microwave will have to be greater than the cooling effect of the increased cerebral blood circulation, which would be determined by both the energy level (dose) and the duration of exposure. Rault et al. (2014) investigated this in anaesthetized cattle (n = 10) and reported that a minimum of 20 kW delivered for 10s resulted in suppressed EEG (as an indicator of unconsciousness) at 13 s, maximum EEG suppression at 25s and lasted for a minimum of 78 s (n = 2) after the application. Although the magnitude of EEG suppression (% of pre‐stun EEG power) considered to be indicative of unconsciousness following microwave irradiation is not reported anywhere in the submitted dossier, the time to onset of maximum EEG suppression could be considered as the time taken to reach the critical brain temperature and hence the time to onset of thermal unconsciousness. The time to onset of EEG suppression in conscious cattle after application of DTS is unknown.

Additionally, it is important to reiterate that cattle, unlike the other animal species used in different studies cited in the dossier, have a unique feature as their vertebral arteries provide additional blood supply to the brain. This enhanced circulation increases cooling of the brain during diathermy thereby prolonging the time needed to reach the critical brain temperature and also explains the faster rate of recovery from the effects of diathermy.

Furthermore, the WG took into account experimental work conducted during DTS development. During early development and testing of the DTS equipment utilised in STEP 7, the temperature profiles of cadaver heads were tested, and a predictive equation describing the heating rate within the brain was generated (0.124°C/kW^.^s). From this equation, according to the dossier, it is inferred that, for a 30‐kW application, the time required to achieve an 8‐degree rise in temperature would be 2.15 s (8/30/0.124); and for a 20‐kW application, the time required would be 3.23 s (8/20/0.124) (SS04, McLean et al., 2017). Based on the data provided, it is possible to infer that unconsciousness can be achieved in cattle within 1 s by using a power output of more than 30 kW. In STEP 7 of the development of DTS application, tonic–clonic responses were first observed at 4–8 s after the onset of energy application – a longer time than predicted by the data collected on cadaver heads. However, the occurrence of tonic–clonic seizure was not supported by EEG evidence of generalised epilepsy in the brain. This may be a result of the inherent cooling effect of circulating cerebral blood flow. However, the dossier does not appear to account for the additional energy that would be required to overcome this cooling effect.

Subsequently, refinements to the applicator system and addition of the auto‐tuner have resulted in improved electromagnetic coupling, reduced reflectance and far more efficient energy transfer into the tissues, such that the tonic–clonic response is then observed within 2 s after the onset of energy delivery (STEP 13).

Potential causes of system failure and chances of occurrence

3.1.5

Potential causes of system failure were discovered during the simultaneous development of the system with animal applications (Section 3.2 of the dossier introduction to Description of the individual scientific studies). In general terms, potential causes of system failure could only be solved sequentially, starting with the generation of microwaves and the transfer of these to the required energy to sufficiently heat the brain. Many of the issues encountered in the early stages of development (STEPs 1–11) have been corrected through refinements to the delivery system.

An analysis of system failure, the chance of occurrence, animal welfare implications and responses are summarised in Table 17. The potential problems are based on experience with the system, and anticipated problems that may not have occurred. The likelihood of occurrence is based on a consensus of project team members based on the operation of the system at the time of submission. Each row of the table deals only with issues arising directly from the identified potential problem (resulting problems are dealt with in their own row of the table).

For more related information according to the applicant, see Section 3.1.5 of the dossier.

For the first 18 potential problems, the WG supplemented Table 17 with additional causes and WCs beyond those reported in the dossier. The last 4 potential problems – including their associated analyses in all relevant columns – were identified and added entirely by the WG. All additions by the WG are indicated in italics. Based on the information in Table 17, the WG identified a total of 22 causes of potential problems. Of these, 16 were attributed to operator error, 10 to equipment failure and 2 to facility‐related issues (i.e. lack of appropriate infrastructure at the slaughterhouse).

Assessment of Table 17 by the WG:

As in the EFSA AHAW Panel (2020) report, many identified problems involved decisions and actions taken by the operator involved in the application of DTS. The applicant claims that DTS requires fewer operator decisions and a lower level of skills than for mechanical stunning methods. However, in the view of the WG, this statement is not supported. The dossier does not adequately consider the level of staff skills and competence required for the application of the DTS. The lack of standard operating procedures (SOPs) or operating instructions leads to doubts, for example regarding recognition of energy leaks due to inappropriate positioning of the applicator, methods for the prevention of overheating of the skin due to wetness or lack of mitigation to overcome second‐degree burning of the skin. It is also worth noting that 16 out of 22 causes of potential problems leading to system failure (see Table 17) have also been attributed to operator error.

Description of the individual studies submitted

3.2

DTS was developed over several years, through multiple steps (Figure 22 of the dossier) as the technology was explored, refined and data were collected to validate it as a method for simple stunning at the time of slaughter. Giving consideration to the ethical treatment of animals in research, development of the system of intervention and measurement of outcomes occurred simultaneously. The outcomes determined included:

• onset of unconsciousness and insensibility;
• absence of pain, distress and suffering;
• duration of unconsciousness and insensibility.

According to the dossier, the stepwise description of the development process provides transparency; however, not all DTS trials on cattle yielded data suitable for establishing reliable operating parameters because the system was not yet functioning under its final settings. In early trials on conscious animals, key parameters were still being defined and animal welfare outcomes and causes of equipment failure were assessed. Only in later studies were the intervention parameters applied with sufficient consistency to contribute meaningfully to determining operational settings. Throughout the development process, the effectiveness of the first stun and the causes of ineffective or compromised stunning were evaluated.

The scientific studies (SS) submitted are outlined in Table 18, and project reports (PR) in Table 19. A list of supplementary information (SUP1–SUP10) is also provided. Figure 22 of the dossier indicates which steps contributed to each of the submitted peer‐reviewed publications.

Supplementary material (SUP)

SUP1: Conference material. Evaluation of microwave application as a humane stunning technique based on electroencephalography. International Society for Applied Ethology 2013. Peer‐reviewed. PUBLIC.

SUP2: Conference material. DTS®: a novel method for stunning cattle. Impacts on cortisol response and post slaughter meat quality attributes. Pan‐Commonwealth Veterinary Conference 2015. Peer‐reviewed. PUBLIC.

SUP3: Conference material. Meat quality attributes of beef carcases slaughtered using DTS: Diathermic Syncope®. International Congress of Meat Science and Technology 2015. Peer‐reviewed. PUBLIC.

SUP4: Conference material. DTS®: Diathermic Syncope for cattle stunning. Humane Slaughter Association 2015. Peer‐reviewed. PUBLIC.

SUP5: Conference material. DTS: Diathermic Syncope® – induction of insensibility in cattle. Presentation Slides used for a number of stakeholder meetings. Not Peer‐reviewed. PUBLIC.

SUP6: Conference material. DTS: Diathermic Syncope® – a new technology for pre‐slaughter induction of insensibility. Royal Society for the Prevention of Cruelty to Animals Australia Conference 2018. Peer‐reviewed. PUBLIC.

SUP7: Conference material. Applicator development for animal stunning. AMPERE Conference 2017. Peer‐reviewed. PUBLIC.

SUP8: Factsheet. DTS: Diathermic Syncope®. Not peer‐reviewed. PUBLIC.

SUP9: Conference material. DTS: Diathermic Syncope® – A new technology for pre‐slaughter induction of reversible unconsciousness. North American Meat Industry Animal Care Conference 2021. Peer‐reviewed. PUBLIC.

SUP10: Conference presentation recording. DTS: Diathermic Syncope® – A new technology for pre‐slaughter induction of reversible unconsciousness. North American Meat Industry Animal Care Conference 2021. Peer‐reviewed. PUBLIC.

In this section the peer‐reviewed publications SS03, SS05, SS06, SS07, SS08 are summarised and assessed by the WG. SS01 and SS02 contributed only to literature review and to laboratory scale studies in anaesthetised sheep while SS04 was limited to laboratory bench‐top evaluations using cadaver material. Therefore, they will not be described (see also Figure 1 of this SO and Figure 22 of the dossier). For more information regarding all the studies and documents submitted, see Section 3.2.1 of the dossier.

SS03 (Rault et al., 2014)

3.2.1

Introduction (SS03)

3.2.1.1

• Background and rationale

Microwave energy (also known under the terms ‘high energy microwave’ or ‘focused beam microwave irradiation’) has been reported to induce loss of consciousness when applied to conscious rats (Rattus norvegicus), causing petit or grand mal seizures for 1 min after exposure and an unconscious state for the following 4–5 min with the animal ultimately recovering (Guy & Chou, 1982; Lambooy et al., 1989). However, Lambooy et al. (1989) deemed this technique unsuitable for pigs (Sus scrofa) at that time, partly because of the capacity of the microwave generator being too low to deliver sufficient power. In recent years, microwave technology has developed to the point that high power equipment is available that can focus the energy to produce a rapid rise in temperature in cattle brains (Ralph et al., 2011). It is expected that raising the brain temperature will stop brain function and result in insensibility, whilst still allowing the animal to regain consciousness after a period of time (for a review, see Small, Mclean, Owen, & Ralph, 2013). This is supported by preliminary evidence in sheep (Small, Mclean, Keates, et al., 2013).

• Objective

The aim of this project was to evaluate the effectiveness of varying microwave energy delivery settings, such as power levels and application durations, in inducing insensibility in minimally anesthetised cows.

Materials and methods (SS03)

3.2.1.2

• Method

Study population: Ten Bos taurus Hereford × Angus crossbred female cows with estimated live weight 180 kg each.

Sampling strategy: Not reported.

Experimental design: The experimental unit was the cow with a single treatment of DTS application. While theoretical determination of microwave power necessary for stunning is difficult, efficiency of three power levels 30, 20 and 12 kW was tested experimentally. Microwave power 30 kW was applied for 10 s on three animals and for 5 s on one animal. Microwave power 20 kW was applied for 15 s on two animals and for 10 s on other two. Microwave power 12 kW was applied for 25 s on one animal.

Ethical considerations: The project was approved by the University of Melbourne Animal Ethics Committee (approval number 1212620.1) in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NHMRC, 2013). The date for animal ethics approval was not reported.

Randomisation and blinding: Treatments were pre‐determined, so randomisation was related to the order in which animals presented themselves to the restraint unit. Observers were not blinded to treatment.

Reporting the methods of analysis: This was an observational study, with no statistical analysis performed.

• Measurement of the outcomes

All animals used were anaesthetized prior to being subjected to the experimental treatment and kept under anaesthesia for the whole procedure. Therefore, onset and duration of unconsciousness, neurological, behavioural and physiological measures, as well as physical reflexes and correlation of neurological with other ABMs for determining onset of unconsciousness and the recovery of consciousness or time to death, and magnitude of pain, distress and suffering were not assessed.

Use of neurological measures: The EEG signals were amplified using isolated differential signal amplifiers (Iso‐Dam isolated physiological signal amplifiers; World Precision Instruments, Sarasota FL, USA), with a gain of 1000, a passband of 0.1 to 500 Hz and digitised at a rate of 1 kHz (Powerlab/4sp; ADInstruments Ltd., Sydney, Australia).

Data were analysed offline after completion of the experiment. Insensibility was assessed by the appearance of seizure‐like complexes in the EEG. Four variables were derived from combinations of the raw data and/or the variables derived from the frequency spectra:

• time to onset of EEG suppression (raw data), measured from the start of the DTS application until the first appearance of seizure‐like complexes in the EEG, hence including the duration of DTS application;
• time to lowest EEG high frequencies intensity estimated as frequency (nHz), below which 95% of total EEG power lies (95% spectral edge), the time is calculated from the start of the DTS application to when the EEG high frequency activity is the lowest (most suppressed, nadir of 95% spectral edge);
• duration of EEG suppression (combination of raw data, 95% spectral edge and total EEG power), measured from the time of onset until the re‐emergence of a normal EEG pattern similar to that seen prior to the application of DTS; and
• maximum effect (95% spectral edge), which measures the extent of EEG suppression and often quantified as a percentage reduction in spectral edge frequency (SEF) or total EEG power.

Use of physiological measures: Basal heart rate and changes in heart rate after DTS.

Reporting the results (SS03)

3.2.1.3

• Reporting outcomes and estimations

Since DTS was applied in anaesthetized cows, outcomes and estimations on the proportion of mis‐stunned animals, the time to onset of unconsciousness, duration and magnitude of pain, distress and suffering, duration of unconsciousness and frequency of animals recovering consciousness before death, time to death, proportion of dead animals and stun‐to stick interval could not be described.

Adverse events: Post‐mortem examinations revealed that at the point of application there was an area of complete skin loss, surrounded by a larger region displaying grey‐tan discolouration of the subcutaneous tissue. The skin within this area displayed full‐thickness coagulative necrosis extending down to the skull. Beyond this area, there was a rapid progressive decrease in the severity of necrosis, with normal, unaffected skin present within 0.5 cm of the margin. In the brain, Animal 8 (single application, 30 kW, 10 s) had medium to severe lesions in the frontal and parietal lobes, and Animal 9 (single application, 30 kW, 5 s) had relatively minor to no lesions in the same regions. For both animals, the basal nuclei, thalamus, hypothalamus and the caudal colliculus were relatively unaltered except for mild to moderate vascular congestion. One animal that had two microwave applications showed severe lesions at various levels (meninges, cortex and white matter) in the frontal and parietal lobes, with severe tissue and vascular necrosis, cavitation, haemorrhage and vascular congestion. Minor or no changes were apparent in the basal nuclei, thalamus, hypothalamus and the caudal colliculus. However, these changes are described in the dossier (SS003), but no pictorial representations of macroscopic or microscopic findings are presented for scientific scrutiny.

Discussion and conclusions (SS03)

3.2.1.4

Assessment by the WG

The DTS method requested for approval consists of an application of 16–20 kW energy for 10 s to the head, followed by neck cutting (severing both carotid arteries and jugular veins). Only two animals (animal ID 3 and 6 – Tables 1 and 2 of SS03) subjected to DTS in this study received 20 kW for 10 s, i.e. the highest energy level and duration of exposure requested by the applicant. Therefore, the discussion in the dossier should have focused on findings (i.e. EEG, ECG, skin temperature and post‐mortem lesions) from these two animals which received the suggested range of energy and not on the entire study population. Thus, the discussion was too general and disconnected from the key parameters.

Nevertheless, the results of animals ID 3 and 6 (Treatment 2 in Table 2 of SS03) indicated that the average times (s) to onset of EEG changes (appearance of seizure complex) and nadir is 13 and 25 s, respectively. These parameters are considered to be indicators of unconsciousness and should have been discussed in comparison with the other EU authorised stunning methods approved for cattle. In addition, the duration of nadir effect, considered to be the duration of unconsciousness, in the two animals, was also highly variable (78 and 140 s).

In contrast to the claim made in the discussion, based on the time to onset of EEG changes it can be inferred that application of DTS at 20 kW for 10 s is not quicker than non‐penetrative captive bolt stunning (8 s). It is well known that effective head‐only electrical stunning induces immediate loss of consciousness (EFSA, 2004). Comparison with carbon dioxide stunning is not relevant, because it is not an approved method in the EU for stunning cattle.

It is stated that the duration of EEG suppression lasted for at least 37 s, which is shorter than the stun‐to‐neck cutting interval of 45 s requested in the dossier. This is a serious AW concern, because the presumed duration of unconsciousness is shorter than the stun‐to‐neck cutting interval. However, there is a high uncertainty in neurological knowledge with regard to the magnitude of EEG suppression required to assume unconsciousness.

Furthermore, key ECG parameter data were only reported for one animal (Animal 6, Table 3 of SS03), which is too few observations to make any statement or conclusion. In addition, exposure to DTS with 20–30 kW for 5–10 s (n = 4 animals) resulted in bradycardia in all the animals. According to the discussion, the welfare implications of the onset of bradycardia during the application of DTS are similar to those associated with noxious stimuli (e.g. scoop dehorning in calves or velvet removal in red deer), but the welfare implications of this important finding were not discussed.

It is stated that the results of histological examination of brain sections from animals exposed to DTS indicated non‐uniform dissipation of heat throughout the brain, which contradicts the claim that application of a combination of electromagnetic field and microwaves facilitated uniform dissipation of heat in the brain of rodents (Ikarashi et al., 1984). This is possibly because the key parameters (e.g. energy levels) used in these studies are different and because the brain volumes are different. These elements were not discussed in the scientific publication/dossier.

The changes in the EEG indicated that application of DTS at 20 kW for 10 s did not induce immediate loss of consciousness and that the onset of bradycardia before the onset of EEG changes was equivalent to that reported during the application of noxious stimuli. In contrast to this important finding, it was stated that there is a window of uncertainty regarding the aversiveness of the microwave application and the experience of the animal during that time (10 s). Based on these results it is very likely that cattle will experience pain during exposure to DTS applied with these parameters.

Conflicts of interest (SS03)

3.2.1.5

No conflict of interest reported in SS03.

SS05 (Small et al., 2019)

3.2.2

Introduction (SS05)

3.2.2.1

• Background and rationale

According to the introduction of study SS05, preliminary research has shown that electromagnetic energy technology is likely to induce recoverable insensibility in animals and could result in an effective reversible stunning method that may be suitable for religious slaughter (McLean et al., 2017), and a prototype delivery system, trademarked DTS: Diathermic Syncope® has been developed. The aim of the system is to selectively increase the temperature of the brain to the point that hyperthermic syncope (fainting) occurs, but below the point at which irreversible brain damage and death occur. Research using anaesthetised sheep (Small, Mclean, Keates, et al., 2013; Small, Mclean, Owen, & Ralph, 2013) and lightly anaesthetised cattle (Rault et al., 2014) showed that DTS application induced EEG changes incompatible with consciousness. Further development of the system, reported here, involved a proof‐of‐concept demonstration of the humane induction of unconsciousness in awake cattle, using fixed, staring eye and loss of corneal and pain reflexes as real‐time behavioural indicators and EEG as the reference objective measure. The behavioural indicators of insensibility used are described for other methods of assessing pre‐slaughter stunning (AMIC, 2009; EFSA, 2004; OIE, 2015; USDA‐FSIS, 2009) and may eventually be utilised to monitor the outcomes of energy application in a commercial slaughterhouse situation, bearing in mind that the mechanism of action of DTS is different from the existing methods of stunning.

• Objective

Information on the objective was not reported in SS05. However, according to the submitted dossier, the main objectives were as follows:

• To provide a proof‐of‐concept demonstration of induction of unconsciousness in cattle;
• To identify which signs, comparable to other validated stunning processes, could be experimentally examined in future assessments, in order to evaluate the effectiveness of the device in rendering the animal unconscious.

Materials and methods (SS05)

3.2.2.2

• Method

NOTE: SS05 includes two trials. Trial 1 was performed in September 2014 and Trial 2 in October 2017. Trial 1 was the initial proof‐of‐concept, and Trial 2, with modifications to the energy delivery system and restraint. Trial 1 and Trial 2 were included separately in the dossier (STEP 7 and STEP 9, respectively).

Study population:

Trial 1: Eighteen Bos taurus Aberdeen Angus crossbred heifers (weight range 350–400 kg) with a quiet temperament.

Trial 2: A total of 20 cattle were processed. The animals were Bos taurus mixed breeds, predominantly dairy crosses; some were aged cull cows, and others were poor‐quality dairy cross steers, randomly drafted from the normal intake at the abattoir. Age and sex of individuals were not recorded.

Sampling strategy: Sample size determination was justified through the three Rs in order to reduce animal numbers, since according to Article 4 of Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes, the principle of replacement, reduction and refinement (3Rs) should be used. Specifically, for ‘reduction’, it is stated that the number of animals used in projects should be reduced to a minimum without compromising the objectives of the project. In both Trial 1 and Trial 2 the experimental unit was the individual animal, and 7 animals were used in control group (captive bolt) and 10 animals in DTS group.

Experimental design

Trial 1: There were four experimental treatments: captive bolt stunning serving as control and three DTS based on three energy delivery classes:

• Control (captive bolt) (n = 7)
• DTS:
• High energy DTS, receiving greater than 290 kJ (n = 3)
• Intermediate, receiving 200–290 kJ (n = 3)
• Low energy DTS, receiving 43 to 191 kJ (n = 4)

One animal without treatment assigned in material and methods, received insufficient energy to induce unconsciousness (38.55 kJ), due to poor waveguide contact and short duration of application (see Table 2 of SS05 in the dossier).

In six of the 11 heifers, a power setting of 20 kW was applied, while a power setting of 30 kW was applied to the remaining five heifers.

Trial 2: The experimental unit was the individual, and there was only one treatment (DTS application in the range 200–360 kJ). Three different power settings (kW) were applied: 15 kW in one animal, 20 kW in five animals and 30 kW in 14 animals. No rationale was provided for this design.

Ethical considerations: According to SS05, both Trial 1 and Trial 2 were carried out under the authority of the Victorian State Government Wildlife and Small Institutions Animal Ethics Committee, Authority 29.13 (Trial 1) and Authority 13.16 (Trial 2). Nonetheless, according to the dossier, the protocol and conduct of Trial 2 was approved by the DEDJTR Wildlife and Small Institutions Animal Ethics Committee under the VIC Prevention of Cruelty to Animals Act, 1986 (Animal Research Authority 30.16). Dates for animal ethics approval were not reported.

Randomisation and blinding:

Trial 1: Animals were randomly assigned to the treatments based on the order in which they entered the restraint box, the treatments being conducted as 2x captive bolt to start the trial and ensure all team members were correctly positioned for data collection; followed by two DTS; and then randomisation of treatments by selection of a card from a pre‐prepared stack.

Trial 2: The order of treatment application was pre‐determined; randomisation was based on the order in which the animals presented themselves to the lead‐in race to the restraint unit. Personnel carrying out offline video behaviour recording, EEG and thermal imaging analysis were blinded to the treatment applied.

Reporting the methods of analysis: The EEG data in both Trials 1 and 2 were analysed offline using LabChart 8 (ADInstruments, Sydney, Australia). The Spectral Analysis Package within LabChart 8 was used to apply fast Fourier transformation (FFT), with multiplication using a Hann window in 1‐s epochs with a 25% overlap. Total power (Ptot), median frequency (F50) and 95% spectral edge frequency (F95) were extracted. Epochs (short, predefined segments of continuous data to be analysed separately) containing artefacts were manually identified and rejected, using video footage to identify event‐related artefacts such as animal movements, eye/ear movements, personnel movement or movement of leads. The first and last 2 s of each recording were removed to eliminate edge artefacts. Heavily contaminated recordings and recordings in which poor electrode contact was present were discarded entirely. For each animal, the median value of the total power (Ptot) during the initial period (T1) was calculated and used as the baseline value. Baseline normalisation was performed by transforming data for each 1‐s epoch into a decibel change from baseline using the formula: dB = 10 × log10(value/baseline), allowing all data sets to be compared uniformly. These data, and data for edge frequency F95 and F50, were charted and inspected for evidence of EEG suppression and epileptiform activity. When possible, the time to resolution of EEG suppression was also recorded.

• Measurement of the outcomes

Onset and duration of unconsciousness and death: The onset of unconsciousness could not be determined by EEG as there is a time lag between the animal being stunned by DTS, and the electrodes pads being placed for EEG recording. Duration of unconsciousness is estimated through the elapsed time between loss of posture and time to return corneal reflex. In some animals, the duration of unconsciousness was greater than 7 min. In such cases, animals were exsanguinated to allow them to bleed to death. Onset of death was not considered since DTS is intended to cause unconsciousness but not death.

Use of neurological measures: Once the animal's head was captured, but prior to the application of the waveguide, a baseline (T1) EEG was recorded using PowerLab and LabChart (ADInstruments, Sydney, Australia), applying a low‐pass filter of 50 Hz, for a period of up to 2 min. The electrode montage was prepared using low impedance (< 5 kΩ) electrode pads (RedDotMini, 3M Australia, North Ryde, NSW). Good contact with skin was achieved by fixing the pad to the skin using cyanoacrylate (Loctite 454, Loctite Australia, Caringbah, NSW) without shaving the hair to minimise stress to the animal. Electrode gel was not applied, as prior findings indicated that strong adhesion via cyanoacrylate was sufficient for reliable EEG data collection. In Trial 1, a single channel, semi‐hemispheric, three‐electrode EEG montage (ground electrode on the bony protuberance of the atlas behind the ear; reference electrode midline on the nose, midway between the nostrils and the eyes; and the inverting electrode on the left frontal bone, between the eye and the poll) was used. A second channel was not used, as access to the right frontal bone was restricted due to the head capture and waveguide apparatus and it was felt that attempting to apply an electrode here would add to the stress for the captured animal. In Trial 2, a four‐electrode montage was prepared, as access to the right frontal bone was facilitated with the redesign of the restraint. Once the baseline T1 EEG recording was complete, the EEG leads were then removed, leaving the electrodes in situ and the waveguide applied to the forehead. EEG cannot be recorded during, and for at least 3 s after the end of, DTS application, as the high‐power microwave energy interferes with the recording (Rault et al., 2014) and the leads were removed to protect the equipment from damage either physically from the faraday cage apparatus or electronically from a high energy microwave burst.

Post‐treatment (T2) EEG was recorded while the animal remained in the head restraint, for at least 2 min. The time from DTS application to EEG recording varied due to time taken to open the faraday cage, remove the waveguide from the head and reattach the electrode leads. In Trial 1, the mean time to start of T2 EEG recording was 72.82 s (range 38–124 s) and in Trial 2 51.82 s (range 12–77 s) from the start of DTS application. In Trial 2, T1 and T2 EEG recordings were made only from the first 13 animals. During processing of the remaining seven animals, an assessment of the duration of time required to roll out and exsanguinate the animal (the stun‐to‐stick interval) was made, which would have further delayed EEG recording. The EEG data were analysed offline using LabChart 8 (ADInstruments, Sydney, Australia). The Spectral Analysis Package within LabChart 8 was used to apply FFT, with multiplication using a Hann window in 1‐s epochs with a 25% overlap. Total power (Ptot), median frequency (F50) and 95% spectral edge frequency (F95) were extracted. Epochs containing artefacts were identified and rejected manually, with reference to video footage to identify event‐related artefact (e.g. animal movements, eye/ear movements, personnel movement or movement of leads), and the first and last 2 s of each recording were removed to eliminate edge artefacts. Heavily contaminated recordings and recordings in which poor electrode contact was present were discarded in entirety. Post‐treatment (T2) EEG recordings in Trial 1 were heavily contaminated with movement artefact, a problem which was less notable in Trial 2, and may be associated with better energy transfer and better‐quality stunning. Twelve usable recordings were obtained in Trial 1, with poor electrode contact noted in Animal 1.9 during T2, and recordings from Animals 1.12, 1.13, 1.14, 1.15 and 1.18 being discarded due to contamination by movement artefacts. In Trial 2, 11 usable recordings were obtained, with no T2 recording from Animal 2.2 due to a laptop malfunction, and Animal 2.13 was stunned with a backup captive bolt shortly after opening the faraday cage due to the presence of corneal reflexes. For each animal the median value of Ptot during T1 was calculated and this was used as the baseline value. Baseline normalisation was then carried out by transforming data for each 1‐s epoch into decibel change from baseline according to the formula: dB = 10 × log10 (value/baseline), to bring all data sets into a comparable format. These data, and data for F95 and F50 were charted and inspected for EEG suppression and epileptiform activity, and where possible time to resolution of EEG suppression was recorded.

Use of behavioural measures, physiological measures and physical reflexes

In SS05, the ABMs initially used immediately after DTS to assess insensibility were corneal reflex (fingertip touched to the medial canthus of the eye), assessment of visual function (a hand passed across the field of vision, observing for focussing or following movements) and flinch response to a painful stimulus (16G hypodermic needle) to the nose, and subsequently observations were made against the list of possible indicators of insensibility described for other methods of pre‐slaughter stunning (AMIC, 2009; EFSA, 2004; Manteca et al., 2013; OIE, 2015; USDA‐FSIS, 2009), namely: Immediate collapse; No attempt to stand up; Body and muscles immediately rigid (tonic); Epileptiform seizure; Loss of normal rhythmic breathing; Gasping (breathing in without breathing out); No attempt to raise head; Straight back and floppy head; Ears relaxed and drooping; Tongue loose and flapping/hanging out; Eye open and staring straight ahead/glazed expression; Upward rotation of eyes; Gradual pupillary dilation; Corneal reflex absent; No spontaneous eye blinking; No vocalisation; No vocalisation in response to stimulus; No response to painful stimulus (pin prick). Assessments were carried out by a single, trained operator, with over 20 years' experience in slaughter using mechanical and electrical stunning methods. While the mechanism of action of DTS is different from the existing methods of stunning, i.e. the elevation of brain temperature and deactivation of enzymatic function by increasing the temperature (Ikarashi et al., 1984), the behavioural outcomes are likely to differ from the existing methods of stunning. No clear descriptions of the behavioural responses of cattle to rapid hyperthermic change were found, other than panting, salivation and coma being associated with heat stress. Therefore, known indicators of unconsciousness from abattoir settings were used. In the end, the behavioural and physical ABMs used to assess unconsciousness following DTS application were primarily loss of corneal response, loss of withdrawal response, eye staring without following movement and lack of response to a painful stimulus (e.g. nose prick).

Correlation of neurological and other ABMs: no correlation of neurological and other ABMs was provided.

Magnitude of pain, distress and suffering: No potential sources of pain, distress and/or suffering, including those related to restraint, were described. The measurements aimed at assessing the magnitude of pain, distress and suffering were based on an ethogram of behaviours and postures (see Table 1 in SS05 of the dossier). However, the validity of these behaviours and postures as ABMs to measure pain, distress or suffering was not provided. Moreover, the ABMs were not described in full detail.

Reporting the results (SS05)

3.2.2.3

• Reporting outcomes and estimations

Proportion of mis‐stunned animals: DTS failed to induce unconsciousness in one animal in Trial 1 and three animals in Trial 2. According to the dossier, this failure was due to energy leakage occurring as a result of animal factors such as cattle with dished forehead and onset of strong convulsions.

Time to onset of unconsciousness: In both Trial 1 and Trial 2, onset of unconsciousness is shown as time to loss of posture, loss of corneal reflex, loss of withdrawal reflex to nose prick and occurrence of fixed, staring eyes.

Duration of ‘pain, distress and suffering’: In both Trial 1 and Trial 2, duration of pain, distress and suffering might be reported in Table 2 of SS05 as ‘time to physical response’ but the timing of the appearance of the different behavioural events remains unclear, not enabling determination of the sequence per animal.

Magnitude of ‘pain, distress and suffering’: according to SS05: ‘From video footage, there was no evidence of an aversive response (e.g. escape attempts, salivation, grimace or startle expression) in any animal’. However, a general description of what behaviours were observed from DTS application to loss of posture was provided, but more quantitative and qualitative information per animal would be needed for a proper assessment. Such data should include measures like behaviour intensity and frequency, as well as post‐mortem findings (e.g. necropsy lesions). This is because the authors pointed out that ‘loss of posture occurred between 1 and 19 s after onset of energy delivery, although 18 of 20 animals lost posture within 10 s of DTS application. It is unclear as to why such a large range in latency to loss of posture was observed, as it did not appear to be related to either power of application or total energy delivered. It may reflect individual variation in the ability of the circulatory system to cool the brain and counteract the heating effect of DTS, or it may be related to inefficient transfer of energy from the waveguide to the brain, longer latencies being associated with energy leakage and this would have reduced the heating rate within the brain’.

‘In some animals (particularly Animals 1.10, 1.14 and 1.16) the back arched, and the muscles of the neck contracted, pulling the chin down into the chin lift. This occurred at about 2–3 s following the start of treatment and was followed by a period of convulsive movements, lasting 10–20 s, then ataxia, noted as the hind limbs slipping out from behind the animal, with attempts to replace the foot showing incomplete stride length, such that the foot remained behind the centre of balance. Following this ataxic phase, the animal lost posture, then progressed to tetany and convulsions as described above’.

Duration of unconsciousness: In the DTS animals in Trial 1, the period of insensibility as indicated by lack of responsiveness to stimuli lasted for at least 3 to 4 min post energy application (Table 2 in SS05 of the dossier), and the animal was either pronounced dead or exsanguinated prior to return of reflexes in two animals (animal 1.4, receiving 295.36 kJ and Animal 1.11, receiving 295.37 kJ). In animal 1.5 a wide range of energy levels was applied (47.55 + 192.31 kJ) due to poor waveguide contact and animal 1.18 received 217.62 kJ. For both these animals the corneal reflexes reappeared, with video footage showing their return at 242 and 238 s after the loss of posture, respectively. Duration of unconsciousness was provided although they consider loss of corneal response, loss of withdrawal response and eye staring, not following movement and response to a painful event (e.g. nose prick) as indicators of unconsciousness. Nevertheless, after DTS application, the animals kept breathing and ‘made sounds’. According to the dossier, cattle continued breathing during and following the application of DTS and therefore breathing is not associated with ineffective DTS stunning.

According to the WG, it is confusing, at least, to read that 17 out of 20 cattle in Trial 2 exposed to DTS showed loss of rhythmic breathing. It is worth reiterating the fact that if effective application of DTS results in tonic–clonic seizures, then breathing should cease (result in apnoea), due to tetanus occurring during tonic phase involving all the muscles, including respiratory muscles. Evidently, contradictory interpretations are made on this ABM with high uncertainty.

Frequency of animals recovering consciousness before death varied between trials**:** In Trial 1, rhythmic breathing continued throughout DTS application, characterised by a slow and deep pattern, suggesting that although medullary function was not fully disrupted, there was some alteration in its breathing activity during the transition of animals from conscious to unconscious. As mentioned above, according to the WG, the observation and interpretation of breathing is contradictory and has high uncertainty.

In Trial 2, using the improved restraint and energy delivery apparatus, the 17 animals that were assessed as insensible following DTS application, demonstrated behavioural signs consistent and associated with an electrical stunning including rapid blinking or flicking of the eyelids including the nictitans membrane (third eyelid), abrupt loss of posture, tonic (stiff) and clonic (convulsive) phases. Loss of posture occurred between 1 and 19 s after start of energy application. In 4 animals, which received 300–360 kJ, this insensibility progressed to death. In the remaining 13 animals, absence of corneal reflex persisted for between 100 and 170 s, and captive bolt was administered in case of returning consciousness. Slow, deep breathing had returned prior to return of corneal reflex. According to the dossier, animals subjected to DTS continue to breathe, however the pattern of this breathing has not been described clearly to recognise this changing pattern of breathing is related to unconsciousness. In three animals (2.12, 2.13 and 2.20) that were not deemed fully insensible, there had been problems with maintaining contact between the waveguide and the forehead, resulting in leakage of energy into the environment, rather than penetration into the brain. All three had transiently lost posture, but at assessment were standing and appeared partially conscious, with loss of eye focus, no visual following of movement and a slow response to a touch on the cornea. The interpretation of the WG is that these animals had possibly corneal reflex indicative of remaining conscious after the treatment.

Time to death: DTS is intended to cause reversible stunning. Nonetheless, in Trial 2, four animals that received 300–360 kJ progressed to death, although the time to death was not provided.

Proportion of dead animals: DTS is not intended to cause death, yet in Trial 2, 20% (four out of 20 heifers) died after receiving a 300–360 kJ dose.

Stun‐to‐stick interval: the interval that will prevent recovery of consciousness prior to or during bleeding was not provided.

• Reporting uncertainty

Trial 1: According to the dossier, a potential source of bias is the small group size (n = 18), animals being sourced from a single mob presented at the abattoir. All animals were female Australian Angus cattle. Good contact between the microwave applicator and the forehead of the animal is required for transfer of the microwave energy into the brain without leakage. Some variation to expected treatments was observed. The microwave application caused artefacts that rendered the EEG unreadable, leaving a period in which discomfort or pain could only be measured by ABMs.

Overheating of the skin at the point of application was observed. At this stage in development, it remains unclear whether this caused discomfort or occurred after the loss of consciousness.

Trial 2: According to the dossier, a potential source of bias is the small group size (n = 20), animals being sourced from a single mob presented at the abattoir. A variety of breeds, sexes and ages were presented.

Discussion and conclusions (SS05)

3.2.2.4

• Reporting interpretation of results

Trial 1: EEG data indicated that DTS‐induced insensibility. Particularly, application of 30 kW power resulted either in immediate EEG suppression or in immediate seizures (n = 2). In both cases, normal electrical brain activity was essentially disrupted. The brain functions (sensation, etc.) were most probably disrupted as well, leading to unconsciousness. DTS animals in the current study remained unresponsive to stimuli and showed evidence of EEG suppression for 3–4 min post energy application. In a commercial situation, it would be expected that exsanguination would be carried out within 60 s from the start of the energy application.

Video footage demonstrated a convulsive phase during the application of low energy DTS (< 200 kJ according to SS05). Scientific literature concerning heat stroke in cattle reveals that kicking of limbs occurs as escape attempts.

Throughout the period of insensibility, which lasted for at least 3–4 min post application of energy, DTS‐treated animals maintained rhythmic breathing and strong cardiac activity. During this time, no corneal reflex, no response to a painful stimulus of the muzzle and no evidence of the eye beginning to focus and follow hand movement were observed. There was a temporary cessation of breathing during energy application, while the animal undergoes the epileptiform phase. This cessation quickly passes, and breathing recommences, but slow and deep. The respiratory and cardiac centres though impacted were not obliterated, similar to the effects seen in chemical general anaesthesia, where loss and subsequent return of reflexes occur in a defined sequence that correlates with the depth of anaesthesia and also to the order in which cranial nerves leave the brainstem. The cranial nerves, in descending order are: I Olfactory nerve; II Optic nerve; III Oculomotor nerve; IV Trochlear nerve; V Trigeminal nerve; VI Abducent nerve; VII Facial nerve; VIII Vestibulocochlear nerve; IX Glossopharyngeal nerve; X Vagus nerve; XI Spinal division of accessory nerve; XII Hypoglossal nerve.

Thus, under general anaesthesia, the earliest signs of returning to consciousness are voluntary tongue movements (Cranial nerve XII) and swallowing or gag reflex (Cranial nerves IX and X). Conversely, the cessation of spontaneous breathing is an early indicator that anaesthetic depth may be excessive. The vagus nerve (Cranial nerve X) also modulates cardiac and respiratory rate – lesions in cranial nerve X can result in tachycardia and hyperventilation, while stimulation of cranial nerve X can result in bradycardia and deeper/slower breathing.

Following the return of the gag reflex, the next sense to return is hearing (cranial nerve VIII), which is challenging to assess in an abattoir situation. Subsequently, eye‐related reflexes return. Cranial nerves III to VII coordinate muscular responses to stimuli detected by cranial nerves I and II. However, until Cranial nerves I and II regain function, evaluating the activity of Cranial nerves III to VII remains limited.

Cranial nerve II is the afferent, or sensory nerve associated with the eye responses: pupillary reflex, menace reflex, corneal reflex and fixating response (ability to focus and follow movement with the eyes). Therefore, the confirmation of a corneal reflex strongly suggests that full consciousness is near. On the other hand, corneal reflex depends on two different cranial nerves, both located in the pontomedullary region: on the one hand, the noxious stimulus is received by the trigeminal nerve, and more specifically by the ophthalmic division of this nerve, and on the other hand, the animal's reaction by blinking the eye is carried out by the facial nerve, and more specifically by the auricopalpebral division of this nerve. Both nerves, the ophthalmic and auricopalpebral, have their end close to the frontal bone, where the DTS is applied, so it cannot be discarded that, instead of an effective stunning by DTS application, the corneal reflex is affected because of a local effect on the nerves in this area due to the high temperatures. In fact, this is a typical place where local anaesthesia is applied to perform dehorning (ophthalmic division of the trigeminal nerve) or to prevent the eyelids closure during examination of eyeball (auricopalpebral division of facial nerve). Therefore, the corneal reflex may be absent when the afferent trigeminal nerve (Cranial Nerve V), the efferent facial nerve (Cranial nerve VII) or their reflex connections within the pons and medulla oblongata are damaged.

It is reported in the dossier that:

Animal 1.16 received 184.68 kJ and return of blink and corneal reflexes were noted prior to captive bolt application, 228 s after DTS application.

Animal 1.18, which received 217.62 kJ, was unresponsive for 90 s post‐treatment, after which it entered a clonic or convulsive phase. Following this, the animal lay quietly, unresponsive for stimuli, for a further 90 s. Towards the end of this period the animal's eye began to regain focus, followed closely by the return of the corneal reflex and within 15 s the return of the righting reflex.

Overheating of the skin at the point of application was observed. At this stage in development (Trial 1 of S005), it is unclear whether the animal experienced discomfort as a result of this overheating or if the overheating occurred after loss of consciousness.

The main conclusions of TRIAL 1 in study SS05 according to the dossier were:

• The application of DTS with a dose above 45 kJ resulted in signs of unconsciousness immediately following energy application.
• In the absence of EEG evidence of epileptiform activity in the brain, it is inferred that excitation of neurones required to inducing tonic–clonic seizures may not occur, as heating of the brain is expected to cause inhibition of neurotransmitter.
• The duration of unconsciousness was at least 3 min after energy application. No signs of pain, distress and suffering occur during the application of the intervention.
• Endocrine data indicated no differences between DTS and captive bolt stunning.

For WG's assessment and conclusions, see Section 4.

Trial 2: Seventeen out of 20 cattle were assessed as insensible following DTS application. It is reported that loss of posture occurred between 1 and 19 s after onset of energy delivery, with 18 of 20 animals losing posture within 10 s of DTS application. It is stated that it is unclear as to why such a large range in latency to loss of posture was observed, as it did not appear to be related to either power of application or total energy delivered. It may reflect individual variation in the ability of the circulatory system to cool the brain and counteract the heating effect of DTS, or it may be related to inefficient transfer of energy from the waveguide to the brain, longer latencies being associated with energy leakage. This would have reduced the heating rate within the brain. In addition, it may not be possible to accurately determine the onset of loss of posture in severely restrained animal, as the animal is obscured within the stun‐box. Onset of unconsciousness was assessed based on onset of the tonic, then tonic–clonic behavioural responses, which occurred within 3–8 s after the start of energy delivery. These animals demonstrated behavioural signs consistent with an electrical stun, e.g. loss of posture. In four animals, which received 300–360 kJ, this insensibility progressed to death. In the remaining 13 animals, absence of corneal reflex persisted for between 100 and 170 s, at which point captive bolt was administered in case of returning consciousness. When these timeframes are compared with those reported for head‐only electrical stunning in cattle (application of 4–> 20 s, duration 31–90 s), those of DTS application are shorter.

For each of the three animals that were not deemed insensible or unconscious, there had been problems with maintaining contact between the waveguide and the forehead, resulting in leakage of energy into the environment, rather than penetration into the brain. All three appeared partially conscious, with loss of eye focus and no visual following of movement and a slow response to a touch on the cornea. All three were stunned using captive bolt immediately following assessment of consciousness.

Thermal imaging revealed that the average forehead surface temperature at the point of loss of posture ranged between 26°C and 49°C. Notably, some animals (Animals 6 and 7), which exhibited a delayed loss of posture, displayed localised hotspots of up to 109°C. In context, Australian cattle may experience ambient air temperatures of 45°C in summer and could experience higher surface temperatures when standing in direct sunlight. While temperatures above 50°C may be uncomfortable for cattle, no specific literature on thermal contact sensitivity in cattle was available to confirm this. Temperatures exceeding 60°C are known to cause skin tissue damage, as observed with hot branding, a common practice, however painful, for cattle identification.

The results gathered were confounded by problems with waveguide to forehead contact, resulting in loss of energy to the environment instead of being transmitted into the brain, variability in surface temperatures and the presence of hot spots on the forehead. This variability in energy delivery may account for the variability in latency to loss of consciousness (using loss of posture as the proxy indicator).

The main conclusions of TRIAL 2 in study SS05 according to the dossier were:

• The application of DTS with a minimum dose of 200 kJ, delivered with 20 kW incident power within a minimum period of 10 s results in signs of unconsciousness immediately following energy application.
• The duration of unconsciousness is between 100 and 225 s based on duration from loss of posture to return of corneal reflex.
• No signs of pain, distress and suffering were observed during the application of the intervention.

However, for the reasons stated above, according to the WG any interpretation based on the loss of posture has high uncertainty.

Conflicts of interest (SS05)

3.2.2.5

Trial 1: not provided in SS05 but according to the dossier: ‘■■■■■

Trial 2: not provided in SS05 but according to the dossier: ‘■■■■■

SS06 (Hughes et al., 2022)

3.2.3

Introduction

3.2.3.1

• Background and rationale

The study investigates the impacts of using DTS in cattle processing on meat quality attributes of the carcases as well as the endocrine responses of the animals. Background and rationale in relation to endocrine response and its relationship with animal welfare were not provided.

• Objective

To conduct a preliminary evaluation of the meat quality and endocrine (cortisol, adrenocorticotropic hormone, β‐endorphin and catecholamines) attributes of cattle carcasses stunned prior to slaughter using the DTS, as compared with cattle stunned prior to slaughter using penetrative captive bolt (CB).

Materials and methods (SS06)

3.2.3.2

• Method

** NOTE:** blood samples were collected during Trial 1 of SS05. Therefore, the study population, the sampling strategy, the experimental design, ethical considerations and randomisation and blinding are the same as those reported in SS05 Methods in previous sections.

Reporting the methods of analysis: Statistical methods used to summarise the data and test the hypotheses were provided.

• Measurement of the outcomes

Measures relating to the onset and duration of unconsciousness, time to death, neurological assessments, behavioural or physiological indicators, and correlations between neurological and other ABMs were all considered not applicable according to the study.

Magnitude of pain, distress and suffering**:** Physiological response of cattle to DTS stunning (i.e. plasma concentrations of cortisol, adrenocorticotropic hormone (ACTH), β‐endorphin, epinephrine and norepinephrine) was measured and compared to that produced by captive bolt to assess the magnitude of pain, distress and suffering. The trial did not include a sham control group.

Reporting the results (SS06)

3.2.3.3

• Reporting outcomes and estimations

It is reported in this dossier that there were no significant differences between DTS (n = 6) and captive bolt (n = 7) animals in terms of cortisol, ACTH, β‐endorphin and catecholamine responses in the blood samples collected at exsanguination. The study was not designed as a non‐inferiority study. Therefore, the endocrine outcomes cannot be directly compared between this study and those reported in the literature. Both treatments resulted in an increase in cortisol from baseline (DTS 33.19 ± 16.89 nmol/L; Captive bolt 61.43 ± 12.59 nmol/L) to post‐stun levels (DTS 150.38 ± 17.79 nmol/L; Captive bolt 160.64 ± 13.26 nmol/L), indicating physiological stress. It remains unclear whether the observed stress is attributable to the stunning methods themselves, to the head capture and restraint, which was longer than in commercial settings due to the need to take pre‐stun EEG recordings or to a combination of both restraint and stunning. It is important to note that the technique and duration of the restraint differ between the captive bolt and DTS methods. If the restraint procedure already elicits maximum physiological stress responses, any additional increases following DTS application may not be detectable.

Data were analysed using a mixed model with the individual animals treated as a random effect. Nevertheless, results shown are not consistent with the statistical model used. Level of variation should be reported as standard error in all variables. Exact p‐values are missing and data at individual animal level of variation would be of interest. Since some parameters are measured twice (pre‐ and post‐stun) and there are three different treatments (penetrative captive bolt, DTS at 30 kW and DTS at 20 kW), the statistical model should be mixed with the individual as random effect as the authors reported they did in material and methods. However, the results are shown as Student's t test for comparison of two treatments, and not three (penetrative captive bolt vs. DTS) without considering the effect of time (pre and post) but the difference between pre and post. Another potential source of error is that technical faults during DTS application led to considerable variation in total energy (kJ) delivered to each animal, with values ranging from 3.55 to 297.97 kJ.

• Reporting uncertainty

According to the dossier, a potential source of bias is the small group size (n = 18), animals being sourced from a single mob presented at the abattoir. All animals were female Australian Angus cattle.

Discussion and conclusions (SS06)

3.2.3.4

• Reporting interpretation of results

According to the dossier: ‘Endocrine data indicated no differences between DTS and captive bolt’.

Conflicts of interest (SS06)

3.2.3.5

■■■■■

SS07 (Small, Jenson, Fiszon, et al., 2025)

3.2.4

Introduction

3.2.4.1

SS07 investigates the recovery of consciousness in cattle stunned by controlled application of 150–180 kJ of 915 MHz microwave energy to the forehead.

• Background and rationale

Some religious practices require the animal to be alive at the time of exsanguination, and capable of recovering if stunned before slaughter. Mechanical stunning causes physical damage in the brain and sometimes death, making them unsuitable for religious compliance.

• Objective

The aim of this study was to demonstrate to the religious authorities if the application 150–180 kJ of 915 MHz microwave energy to the forehead allows the recovery of cattle.

Materials and methods (SS07)

3.2.4.2

• Method

Study population:

Seven Bos taurus cattle (four heifers and three steers designated as R1–R7) were used for the different treatments but the number of heifers and steers per treatment is unspecified. Liveweight range was 470–560 kg and age 12–16 months. Breeds represented were Australian Angus, Murray Grey and Limousin. The animals were randomly selected at the abattoir. Application of DTS at a power level of 18 kW.

Three different energy application levels and durations:

• –R1, R2 and R6: 180 kJ during approximately 10 s
• –R3, R4 and R7: 160 kJ during approximately 9 s
• –R5: 150 kJ during approximately 8 s.

R1 received an interrupted stun due to an operator error (a waveguide restraining strap had not been secured), the waveguide slipped to one side and the automatic safety cut‐out switches were triggered after 20 kJ had been delivered. The waveguide was repositioned, the restraining strap secured and a further 160 kJ were delivered to meet the 180 kJ target. R1 and R2 refused to reverse from the restraint and therefore they were re‐stunned and slaughtered. Between 2 and 7 min after DTS application, R3 to R7 were released from the restraint and placed in a holding pen with a companion animal, where hay and water were available. Their behaviour was then recorded.

Observational study with no control group.

• Measurement of the outcomes

Observational study conducted over 2 days.

Electroencephalography:

Collected in six animals (no in R7) for 3–5 min beginning 12 s after the start of DTS, applying a low‐pass filter of 30 Hz.

• –Root mean Square (RMS) Power was calculated from the untransformed, filtered recording.
• –Fast Fourier Transformation (FFT)
• –Total power (Ptot) in the entire frequency range (0–30 Hz) also baseline value.
• –Median frequency (SEF50)
• –95% Spectral Edge frequency (SEF95)
• –Power in: Alpha (20.1–30 Hz); Beta (8.1–20 Hz); Theta (4.1–8 Hz) and Delta (0.1–4 Hz)

Assessment by the WG: The WG noted that the EEG data provided by the applicant (PR23) contained artefacts due to background noise and animal movements and therefore deemed them challenging and non‐interpretable.

Behaviour after DTS application:

Indicators of unconsciousness:

• –Tonic phase: Rigid posture of the body with flexion of front and hind legs and/or extension of front legs and neck and flexion of hind legs
• –Clonic phase: Uncontrolled jerking activity, involuntary running or kicking movements of hind legs and extension with some paddling of front legs. May occur in several phases following tonic convulsive phase
• –Tonic Facial muscles: Tensing of muscles around eyes, nose, upper jaw; eyelids held wide open or shut tight, cessation of rhythmic breathing movements at the corner of the nostril.
• –Eyelid flutter: Uncontrolled rapid movement of eyelids, rapid blinking.
• –Ear flicking: Uncontrolled rapid back and forth movements of the ears
• –Nose twitching: Uncontrolled rapid movement of the soft end part of the nose adjacent to the nostrils
• –Convulsive body/limb movement: Full body jerking motions and/or uncontrolled paddling of the limbs; kicking or walking action

Assessment by the WG: The scientific literature on heat stress and heatstroke in cattle identifies kicking of limbs as stepping behaviour in conscious cattle and an indicator of attempts to escape from the stressful situation (Idris et al., 2024). Moreover, none of the behaviours showed any correlation with the EEG changes induced by DTS application. Given these uncertainties, the applicant excluded several of these behaviours from the flowchart for monitoring the state of consciousness.

Indicators of return to consciousness:

Return of brainstem reflexes:

• –Rhythmic breathing: Regular movements of the corner of the nostril. Time of return of rhythmic breathing was noted when three evenly spaced inspirations in series were observed at the nostrils
• –Eyelid blink: Animal opens/closes eyelid on its own without stimulation
• –Corneal reflex: Presence of corneal reflex response: retraction of the eyeball, or closure of the eyelids elicited by touching or tapping the cornea with a finger
• –Awareness of surroundings: Eye tracks movement passed in front of the eyeball, ears swivel towards sound
• –Alertness: Animal lifts and moves head, focuses on personnel activity

Resumption of postural control:

• –Standing attempt: Movement of limbs after clonic convulsions have ceased.
• –Standing unsupported: Hind limbs in a position to support the weight of the hind quarter

Assessment by the WG: These behaviours and reflexes may suggest recovery from heatstroke.

Behaviour of the animal in the holding pen:

For a period of 20–30 min. No information about the ABMs.

The dossier states that the onset and duration of unconsciousness, and magnitude of pain, distress and suffering were assessed through live observations, testing of brain stem reflexes and detailed video annotation.

Assessment by the WG: The WG states that no information on the magnitude of pain is provided.

Reporting the results (SS07)

3.2.4.3

• Reporting outcomes and estimations

Number of mis‐stunned animals: 1 animal, R1 for which energy application was interrupted.

Time to onset of unconsciousness: Between 1 and 6 s after the start energy delivery began the tonic phase. R1 at 10s. The clonic convulsive phase began between 0 and 12 s after the start of energy delivery and lasted for 43–119 s. There is an overlap within tonic and clonic phases. It is not clear how this overlap can occur (e.g. R5). According to the dossier (page 40) the duration of the tonic phase was not recorded.

Convulsive movements were not continuous, rather occurring in bursts of activity, with the movement of facial muscles notably diminishing in intensity towards the end of the convulsive phase. Two animals (R1 and R4) opened their mouths wide at the end of the convulsive phase.

Duration and magnitude of pain, distress and suffering: The duration of pain, distress and suffering might be reported in Table 2 of SS07 as before the ‘time to tonic convulsive phase’. Behaviours during this period are not reported.

Duration of unconsciousness:

• –Return of rhythmic breathing occurred shortly before the end of the convulsive phase, between 45 and 85 s after the start of energy delivery (when three evenly spaced breaths were performed in series).
• –Corneal and palpebral reflexes returned 63 s after the start of energy application. According to the dossier (page 40) the return of spontaneous blink was considered the earliest sign of recovery. However, conscious animals might not blink.
• –Awareness: Some animals appeared to regain awareness as early as 83 s after the start of energy application, then lapsed once more into an unresponsive or drowsy state.
• –Full alertness was present between 119 and 273 s after the start of energy application.

According to the dossier (page 40), one animal which received 150–160 kJ recovered sooner than animals that received 180 kJ in that study.

Table 37 in the dossier indicated that the three animals with an average carcass weight of 306.3 ± 27.4 kg, and estimated liveweight of 508.5 ± 45.5 kg, were stunned with a power of 18 kw and energy of 180 kJ. The time from commencement of energy application to onset of tonic seizure was 6.00 ± 4.00 s, and the duration of unconsciousness (time from onset of tonic seizure to first return of an indicator of consciousness) was 108.00 ± 38.55 s. On the other hand, the 3 animals with an average carcass weight of 295.33 ± 14.6 kg, and estimated liveweight of 490.3 ± 24.2 kg, stunned with a power of 18 kw and energy of 160 kJ, while the time from commencement of energy application to onset of tonic seizure was 3.67 ± 2.52 s, and the duration of unconsciousness (time from onset of tonic seizure to first return of an indicator of consciousness) was 116.67 ± 86.12 s.

EEG

• –dB change in power in frequency bands (Alpha 20.1–30 Hz, Beta 8.1–20 Hz, Theta 4.1–8 Hz and Delta 0.1–4 Hz) over time.
• –Root mean square (RMS) power was initially increased relative to baseline in animals R2, R3, R5 and R6 (Table 4 of SS07). In animals R2 and R3, a, RMS dropped to below baseline after the 42–72 s and 72–102 s intervals, while in animal R5, RMS remained markedly elevated until the 162–192 s interval had elapsed, and less markedly elevated between 192 s and the end of recording at 282 s. RMS in animal R6 was variable throughout recording, with peaks in the 12–42 s, 72–102 s and 132–192 s intervals and RMS below baseline in the 102–132 s and 222–252 s intervals, recording terminated after 252 s. Decreased in animals R1 and R4 and remained suppressed in throughout post‐DTS recording (until 282 s post energy application for R1 and until 312 s post energy application for R4).
• –Shift in frequency: power, with reductions in the higher frequencies in the EEG relative to lower frequencies, particularly after the initial 42 s (Supplementary File 2).
• • Reporting uncertainty

According to the dossier, a potential source of bias is the small group size (n = 7). Animals were males and females but no information on the sex by treatment were provided. The environment (commercial slaughterhouse) and the microwave application caused artefacts that rendered the EEG unreadable. The exact time of return of the crisp response was difficult to quantify as it depended on the operator checking for a response.

Discussion and conclusions (SS07)

3.2.4.4

• Reporting interpretation of the results

Using energy applications of between 150 and 180 kJ at 18 kW ensures loss of consciousness within 10 s (the dossier states 3 s for tonic seizure) from the start of energy application, of a duration of 63 s or more (based on returning palpebral or corneal reflexes). The paper stated that the exact mechanism by which unconsciousness is induced and then maintained is not completely understood.

All the animals (n = 6) recovered consciousness and responded normally to environmental stimuli.

Assessment by the WG: It was noted that according to SS07, EEG recorded prior to and after 10 s application of DTS was used to demonstrate the ‘return of consciousness’ to religious authorities, which is not an animal welfare concern. Secondly, the EEG data provided by the applicant were deemed to be unreadable by the experts and therefore invalid. The authors of the study also stated that behaviour was used to assume unconsciousness and recovery of consciousness. The behavioural signs of unconsciousness used in this study (corneal reflex, eye position and eye tracking), are unreliable, i.e. have low sensitivity, specificity and feasibility according to the dossier. Owing to these reasons, SS007 is considered irrelevant for the ToRs provided in this SO.

Conflicts of interest (SS07)

3.2.4.5

■■■■■

SS08 (Small, Jenson, Phillips, et al., 2025)

3.2.5

Introduction

3.2.5.1

SS08 investigates tissue integrity impacts of application of 160–200 kJ of 915 MHz microwave energy, using the DTS, applied to the forehead of cattle.

• Background and rationale

The DTS system has been designed to render the animal unconscious within 0–5 s, by applying 160–200 kJ of 915 MHz electromagnetic energy to the forehead, while limiting damage or changes to the tissues. To investigate this latter aspect, surface temperature at the point of application was measured using fibre optic sensors. Skin under the application point and brains were examined histologically.

• Objective

The aim of this study was to determine whether the application 160–200 kJ of 915 MHz microwave energy to the forehead results in visible damage to brain tissue and potential for skin burn.

Materials and methods (SS08)

3.2.5.2

• Method

Study population:

Twelve Australian Angus steers, liveweight range 430–500 kg, were used for skin temperature profile measurement at the place of DTS application 1 day in February 2025. The first three received 200kJ energy at 18 kW but were excluded from the analysis because the hair was not clipped and the temperature sensors were too far from the skin surface. Three further animals received 200 kJ, three received 180 kJ and three received 160 kJ, all applications at 18 kW power. 160 kJ of energy was delivered over a 10 s period, 180 kJ over 11 s and 200 kJ over 12 s, including the generator‐start‐up sequence.

Four Murray Grey cattle, liveweight 350–500 kg, in a single batch processed in August 2022, with energy delivered at 180 kJ and 18 kW, were opened to allow inspection of the brain and histological assessment.

A further seven brains from a single batch of Australian Angus steers, estimated liveweight 400–530 kg, were visually inspected in February 2025. Four were from cattle that had received energy at 160 kJ, one at 180 kJ, one at 200 kJ and one at 220 kJ. All applications were at 18 kW power.

Three skin pieces from a single batch of Hereford cross animals (carcass weights not recorded) processed using 180 kJ energy application, at 18 kW power in September 2023 were evaluated.

• Measurement of the outcomes

To measure temperature changes at the point of application, the fibre optic temperature sensors (FISO Technologies, Quebec QC, Canada) were inserted into a Teflon plate at the tip of the applicator, where it contacts the forehead of the animals (n = 12). Three temperature sensors were placed under the wave guide where electromagnetic fields were applied. However, only one of them gave a reasonable readout that was used. In the other two, only small temperature changes were observed. The hair of the forehead was closely clipped to minimise any insulating effect of the hair coat, and the tips of the sensors were protruded 1–2 mm from the surface of the Teflon plate pushed towards animal head.

To assess effects on skin at the DTS application point three skin pieces from a single batch of Hereford cross animals were used. Post‐mortem skin samples were collected from the forehead at the site of DTS application from three animals and subjected to histological investigation. They had been excised from the cattle heads approximately 30 min after DTS energy application, during carcass dressing, refrigerated and shipped with ice packs, arriving at the laboratory 4 days after slaughter. Animal H1 had been processed with no prior preparation; Animal H2 had the hair clipped close (no cover comb attached to the clipper blades); Animal H3 had a water‐soaked hide piece laid over the top of the forehead, under the applicator aperture, such that the area remained thoroughly wetted and steam could be retained in the area during DTS application. Three punch biopsies were collected from each skin piece: one from the centre of the application area, one from the margin of the application area and one approximately 4 cm outside the application area. Samples collected were full‐thickness and ranged from 9 mm to 14 mm thick. Samples were immediately placed in histology cassettes (Techno‐Plas, St Marys SA, Australia) and immersed in 10% neutral buffered formalin solution (Hurst Scientific, Forrestdale WA, Australia). After 7 days they were rinsed twice in 80% ethanol solution (ChemSupply Australia, Port Adelaide SA, Australia), then stored in 80% ethanol solution until processed. Stored samples were embedded in wax, sliced and mounted, then stained with Haematoxylin and Eosin (H&E) for evaluation by light microscopy.

To assess the effects on the brain following DTS application, the skulls of four Murray Grey cattle were opened to allow inspection of the brain, approximately 45–60 min post energy application (after carcass processing) and three samples, each 5 mm × 5 mm × 5 mm were collected from each brain: one from the cerebral cortex close to the poll, approximately 2 cm from the cerebellum, one from the application point, approximately 3 cm from the proximal tip of the cerebral cortex and one from the underside of the brainstem, in the region of the medulla oblongata. All samples were no more than 5 mm from midline. Samples were immediately placed in histology cassettes and processed.

Reporting the results (SS08)

3.2.5.3

• Reporting outcomes and estimations

Average values of skin temperature over three animals in each of three group (160 kJ, 180 kJ and 200 kJ) were provided together with variances. Temperature was measured at a single point in the brain, but the choice of this location was not justified. Measurements taken at two additional sites did not show any meaningful increase in temperature. In these groups skin temperature started to rise at 8, 9 and 9 s after microwave application. Maximum observed temperatures were 48.43 (± 11.14)°C at 12 s, 54.27 (± 6.56)°C at 14 s for the first two energies 160 kJ and 180 kJ. Maximum temperature for the last 200 kJ group is not given in the table. The maximum recorded temperature was 69.1°C observed in animal 11 at 19 s, i.e. 4 s after termination of electric field.

On visual inspection, the applicator footprint was evident as an area of hair loss (depilation) on all three skin pieces. The depilated area was flexible and rubbery in texture. At the edge of the depilated area, the suint (grease and dandruff) could be seen lifting away from the skin surface. The underside of the pieces appeared normal, with visible connective tissue and intact blood vessels.

Histology slides were assessed with reference to standard histology textbooks. From each slide, three areas were examined and photographed: the epidermis (top surface); the papillary layer of the dermis (within 2 mm from the surface) and the reticular layer of the dermis (5–8 mm from the surface). Histological examples are provided without statistical evaluation. At the margins of the application point, there is patchy loss of the epidermis and the connective tissue supporting the cells in the dermis is less well‐defined. However, the reticular layer appeared unchanged, and intact blood vessels and red blood cells were evident. In another sample there was more marked loss of the epidermis, and further coagulation of the tissues of the papillary layer of the dermis, with loss of definition around the remaining hair follicles. However, the reticular layer appeared unchanged, and intact blood vessels and red blood cells are evident. Subjectively, skin sample 3 (in which moisture and steam were trapped between the applicator aperture and the forehead) appeared to have greater epidermal loss than Samples 1 and 2.

On gross inspection there was no physical damage to brains from 160 kJ, 180 kJ and 200 kJ applications. Brains that had been harvested with a delay of 30–45 min from energy application showed superficial hyperaemia and engorgement of blood vessels on the rostral parts of the cerebral hemispheres. This was not evident on brains that were harvested within 10 min of energy application. The brain from the 220‐kJ application given for comparison showed disruption of the meninges covering the brain and liquefaction of the superficial tissues. In essence, it is stated that there were no gross morphological changes to the brain, and limited evidence of changes on histological examination. Maximum recorded temperature was 54.27 (± 6.56)°C, which occurred after energy application had ceased.

Histological evaluation of brain samples from the four animals processed at 180 kJ showed minimal changes to tissue. Meninges (arachnoid mater and pia mater) were intact on the surface of each sample and within sulci, there was no evidence of malacia within the tissues or coagulation of brain tissue or blood within subarachnoid or penetrating vessels. Small areas of lucency were evident immediately below the pia mater in some fields of view of the cerebral surface. It is unclear whether these are a result of processing artefact, or a result of the energy application. Brainstem tissue appeared normal.

• Reporting uncertainty

A potential source of bias is the small group size (n = 3) per energy level in skin temperature measurement. However, the consistency of results between groups decreases uncertainty.

Assessment by the WG

Only selected examples of histological material are presented and discussed. The absence of essential structural changes in the brain in investigated samples is used as an argument in favour of the proposed method. In addition, in the dossier it is inferred that the animals are rendered unconscious in 0–5 s and a rise in skin temperature occurred between 8 and 12 s after the start of energy application, and hence, overheating of the skin is not an AW concern since it appears after the onset of unconsciousness. However, according to the WG, there is a high uncertainty associated with the assertion of unconsciousness based on the behavioural changes such as tightening of facial muscles and ocular reflexes as indicators of tonic–clonic seizures. Because these behaviours are also reported in the literature in heat‐stressed conscious cattle. In addition, there is a high uncertainty associated with the interpretation of kicking of the limbs as clonic seizure, because this behaviour is reported to be an attempt to escape from stressful situation in heat‐stressed cattle. Even if the clonic seizure is a manifestation of heat stroke in cattle, the animals will have to go through several stages of heat stroke that would lead to pain and fear, and there is no sound scientific data in the dossier to assume unconsciousness during the onset of these movements.

Further uncertainty emerges from the fact that gross and histological investigations of the brain samples were inconclusive, and no reference is made to pathognomonic lesions of heat stroke reported in the literature (e.g. Yoneda et al., 2024). Although some of the lesions may have been found in extreme cases of heat stroke, leading to chronic debilitation or death, according to these authors, some gross and histological changes may indicate early onset of heat stroke (e.g. cerebral oedema, haemorrhaging suggestive of syncope).

Discussions and conclusions (SS08)

3.2.5.4

• Reporting interpretation of the results

Based on a review of previously published literature on the development of the DTS system, concerns were expressed about lasting damage to brain tissue, due to the thermal tolerance of brain cells. The current study failed to demonstrate pathognomonic lesions in the brain tissue indicative of heat stroke (i.e. thermal unconsciousness) on both a gross and histological level, if energy applications were within the 160–200 kJ range. This is likely to be a result of the short duration of heating resulting from using the DTS system. In the skin pieces, the histological changes observed in the centre of the depilated area are consistent with a second‐degree burn as described by Garcia‐Espinoza et al. (2017), and it would be expected to heal within 1–3 weeks if the animal had been allowed to recover. According to the authors, the temperature profiles at the point of application, however, would suggest that this degree of tissue change would not be present in the conscious animal, as temperatures reached potentially damaging levels late in energy application after it had lapsed into unconsciousness (loss of consciousness occurred between 0 and 5 s after the onset of energy application, based on presence of tonic–clonic response and absence of ocular reflexes) and rapidly fell once energy application ceased.

Assessment by the WG

The WG noted that the results of SS08 indicated that the skin temperature remained close to ambient (i.e. no heating effect at the skin level) for up to 12 s. The study authors stated that the animals became unconscious within 3 s after the start of the energy application based on the occurrence of ‘tonic seizures’, manifested as rapid blinking and contraction of facial muscles, as well as the occurrence of clonic seizures manifested as kicking of legs after the initial 3 s exposure. Therefore, the interpretation of SS08 by the applicant is that the animals were rendered unconscious before skin temperature started to rise. However, the WG's opinion is, that the ‘kicking behaviour’ more likely can be seen as an attempt to escape from a stressful/painful situation as reported in the literature about heat stress in cattle (Idris et al., 2024). This interpretation is also supported by the fact that the cattle displayed whites in their eyes, which is also a sign of affective states such as pain, fear and distress (Gerritzen et al., 2014).

Lastly, this study clearly demonstrated that second‐degree skin burns occurred during the DTS application, causing pain, fear and distress, but the burn lesion becomes visible only during bleeding. It is worth noting that the sample size is too small (n = 3) and two out of three temperature probes failed to produce reliable data, thus producing uncertain results.

Conflicts of interest (SS08)

3.2.5.5

■■■■■

Overall integration of findings from all studies

3.3

In Section 3.3, the integration of findings from all studies is provided by the applicant, and when there is a comment or assessment by EFSA, it is clearly indicated. To see the whole related information from the applicant, see Section 3.3 of the dossier.

Demonstration of equivalence with existing methods

3.3.1

Article 4 (2) of Council Regulation 1099/2009 requires that the new or modified stunning method ensures a level of animal welfare which is at least equivalent to that ensured by the existing methods. The DTS stunning method was submitted to EFSA for evaluation for use in cattle weighing 270–690 kg. The dossier provided the applicant's interpretation of equivalence with existing methods. However, the assessment was incomplete and did not follow completely EFSA guidelines (2018). Therefore, the WG summarised the information provided and included additional considerations wherever judged as necessary. The independent assessment was performed by EFSA in Section 4.2. Hereto, several approaches can be employed to compare the stunning methods in terms of animal welfare. Ideally, these should rely on the comparison of ABMs that reflect the animals' WCs, or on ranking the associated welfare hazards (EFSA AHAW Panel, 2017).

The applicant claims and states: ‘Using the recommended parameters for DTS, animal welfare is maintained through restraint, induction of unconsciousness, through to death by exsanguination. It delivers an animal welfare outcome that is at least equivalent to that observed with other stunning methods approved for cattle (Table 51 of the dossier)’, i.e. to mechanical stunning (penetrative captive bolt), electrical head‐only and electrical head‐to‐body stunning.

Assessment by the WG: The WG does not agree with the claim above. See Section 4 and its sub‐sections for the full justification.

Quantitative approaches

3.3.1.1

Biomarkers of stress

According to the dossier, the endocrine stress responses measured indicate activation of two physiological response systems. One is the ‘fight – or – flight’ response initiated by all animals when a threatening or ‘emergency’ situation is perceived: activation of this response leads to increases in catecholamines (adrenaline and noradrenaline). The catecholamines speed blood circulation and divert blood, and therefore oxygen, from internal organs to the brain and muscle, readying the animal for action. The second response is the HPA axis (Hypothalamic–Pituitary–Adrenal axis): activation of this response leads to the release of ACTH which leads to increased cortisol levels. This in turn increases the rate of production of glucose from glycogen reserves, so that the energy required for action is available. This speeding up of glycogen breakdown, circulation and metabolism generally can have detrimental effects on meat quality: pre‐slaughter stress at the point of slaughter can decrease pH rapidly while muscle temperature is still high, reaching pH below 5.8–6.0 at 45 min post mortem and can lead to pale meat colour or heat toughening; prolonged raised metabolism leads to reductions in glycogen stores, which in turn lead to dark cutting.

In a commercial slaughterhouse situation, the stress associated with movement of the animal along the race into the restraint or stun box and positioning the head into the head capture unit are common to all cattle, regardless of whether they are subjected to DTS, electrical stunning or captive bolt stunning. Even differences between abattoirs can strongly influence the stress response (Hemsworth & Barnett, 2001). For example, Zulkifli et al. (2014), working in a large commercial slaughterhouse, report higher and a wider range in cortisol concentrations than did Tume and Shaw (1992), from a small experimental facility. This is why baseline samples are taken prior to treatment, against which the post‐treatment samples are compared. Examples of published data are presented in Table 20 alongside the ranges found in the detailed evaluation of 18 cattle subjected to either DTS or penetrative captive bolt (STEP 7), which align with the published values. The large range in values makes it difficult to draw firm statistical conclusions from small numbers of animals.

For the DTS pilot study (STEP 7), group mean and range (converted to SI (International System of Units)) for each sample point are summarised in Table 21. While baseline cortisol levels in the DTS group are significantly lower than post‐treatment cortisol levels within the same group, they are not significantly different from baseline cortisol levels in the captive bolt group. Furthermore, post‐treatment cortisol levels in both groups are not significantly different from the aggregate of all baseline cortisol results, suggesting that the level of stress in captive bolt and DTS groups did not differ.

Assessment by the WG: The significant increase of cortisol levels post‐treatment compared to baseline in the DTS group indicated that DTS procedure was stressful for the animals. However, direct comparison of level of stress with captive bolt stunning based on cortisol levels is difficult because the baseline cortisol levels were different.

According to the dossier, both stunning methods resulted in an increase in cortisol from baseline (DTS 33.19 ± 16.89 nmol/L; Captive bolt 61.43 ± 12.59 nmol/L) to post‐treatment levels (DTS 150.38 ± 17.79 nmol/L; Captive bolt 160.64 ± 13.26 nmol/L), indicating a physiological response. However, it is unclear if this response is due to the methods; or to the head capture and restraint, which was longer than in a commercial situation due to the need to take baseline EEG recordings; or to a combination of both restraint and treatment. Shaw and Tume (1990) and Zulkifli et al. (2014) both report that cortisol levels in cattle are not affected by stunning (captive bolt), however, the former carried out their study in a highly controlled research abattoir environment, in which external stimuli are likely to have been minimised, while the latter carried out their study in a commercial slaughterhouse, and baseline cortisol levels prior to slaughter were already high, as a result of the pre‐slaughter handling and environment. Both Dunn (1990) and Zulkifli et al. (2014) report post‐stun cortisol levels greater than those measured in the DTS pilot study, but it is important to note that these both relate to the case of slaughter without stunning. Indeed, the latter authors demonstrated an increase in cortisol levels associated with slaughter without stunning, but not in the case of captive bolt stunned slaughter. Mitchell et al. (1988) did find that slaughter (captive bolt) resulted in an increase in cortisol levels (+61.2 nmol/L), but this increase was less than in cattle that were handled through a race (+151.7 nmol/L). By inference, it is likely that the cortisol response seen in the DTS pilot study is predominantly due to the head capture and restraint. A tightly clamped head is required to ensure delivery of the DTS energy; however, prolonged tight head capture is likely to be very stressful to the animal and struggling while restrained is considered to be an indication of excessive pressure (Grandin & Regenstein, 1994). EFSA recommends that ‘all restraining devices should use the concept of optimal pressure’ (EFSA, 2004), however, the parameters that constitute ‘optimal pressure’ have not been determined. In this context, ‘optimal pressure’ refers to the level of restraint that is sufficient to allow effective stunning without being excessive. The evidence on physiological stress responses is inconclusive, as the slaughter conditions to which the animals were exposed were not comparable.

In the DTS pilot study, there was neither a difference in ACTH levels between baseline and post‐treatment samples, nor was there a treatment effect. The values generated in the DTS pilot study were lower than those reported by Zulkifli et al. (2014), but did not differ from those reported by Mitchell et al. (1988), who found that ACTH was, like cortisol, affected more by transportation and handling than by stunning itself.

Catecholamines, specifically adrenaline and noradrenaline have been reported to increase as a result of stunning (Mitchell et al., 1988; Rulofson et al., 1988; Zulkifli et al., 2014). Zulkifli et al. (2014) report no significant changes in adrenaline levels as a result of penetrative captive bolt stunning, or high‐powered percussive stunning, but an increase as a result of low powered percussive stunning and unstunned slaughter, while noradrenaline levels increased in all treatment groups; findings that concur with the DTS pilot study. However, Mitchell et al. (1988) and Rulofson et al. (1988) both report an increase in both adrenaline and noradrenaline associated with captive bolt slaughter. In the DTS pilot study, adrenaline levels tended to decrease between pre‐ and post‐slaughter samples, while noradrenaline levels tended to increase, with no significant differences observed between the treatments.

β‐endorphins have a role in modulating the physiological stress response, and increases are associated with painful stimuli, fear and excitement. In the DTS pilot study, there was no significant effect of treatment on β‐endorphin concentrations in either group, although the mean value appeared to decrease slightly. This finding concurs with that of Zulkifli et al. (2014), who demonstrated no significant change in concentrations as a result of penetrative captive bolt, percussive captive bolt and unstunned slaughter at a commercial abattoir. The slight decline in β‐endorphin concentrations also reflects the findings of Shaw and Tume (1990), who demonstrated a significantly lower concentration in captive bolt slaughtered cattle than in the live animals. Those authors, however, carried out their study in a small research abattoir, and the live animal blood samples were collected on a separate occasion within a month prior to slaughter. Therefore, their live animal data may not be directly comparable to the baseline samples collected immediately prior to stunning in the restraint box, as in the DTS pilot study and Zulkifli et al. (2014).

In summary, it was concluded in the dossier (SS06) that the differences in endocrine or physiological stress biomarkers in cattle subjected to captive bolt stunning (n = 7) or DTS (n = 11) were not statistically significant (p > 0.05).

Assessment by the WG

The WG noted that the results have not been presented clearly enough to allow deeper assessment. Firstly, the experimental design was intended to apply captive bolt stunning to 7 and DTS to 11 cattle. Within the DTS group, 5 and 6 cattle were intended to receive 30 kW and 20 kW, respectively. Although captive bolt stunning was performed using the same gun and cartridge power to all 7 cattle, the application of DTS power content varied between 3.55 kW to 297.97 kW due to technical fault, i.e. 10 times lower to 10 times higher than planned. Owing to the lack of control over the treatment parameters, interpretation of the results is invalid. Secondly, it is reported in the dossier that plasma norepinephrine levels were below the detection limits in 10 out of 14 samples, but the number of samples in each treatment has not been disclosed. Thirdly, the duration of DTS application was not disclosed. Fourthly, it is reported that animals were exsanguinated at 60 to 120 s after the treatments and blood samples were collected for post‐treatment determination of physiological stress indicators. It is known that some of these hormones have different release time profiles and therefore the time to reach peak responses are likely to differ and peak concentrations may not have been captured reliably using the 60–120 s interval after application of the DTS. It is also possible that, if the peak response was induced by the pre‐slaughter handling and restraining procedures, further treatment‐induced increases may not occur.

Behavioural indicators of insensibility, according to the dossier

In research, the gold standard of assessing consciousness is electroencephalography (EEG, in which electrodes are placed on or into the skin of the scalp), or electrocorticography (ECoG, in which the electrodes are implanted into the superficial parts of the brain). However, in a commercial situation, it is impossible to utilise techniques such as EEG or ECoG to monitor the effectiveness of stunning, and behavioural indicators and responses to certain stimuli are used as surrogates. For example, captive bolt stunning is considered effective if the observer notes:

• Immediate collapse, hind legs tucked in then slowly extend, forelegs rigidly extended,
• Immediate and sustained absence of rhythmic breathing
• Fixed, staring eye with no corneal of palpebral reflex
• No righting reflex, no response to ear or nose pinch, no vocalisation

An effective electrical stun results in:

• Immediate collapse, hind legs tucked in, forelimbs rigidly extended
• Immediate onset of tonic (stiff) seizure that lasts for several seconds, followed by clonic (convulsing) seizure
• No rhythmic breathing
• Eyes rotated upwards, dilated pupils
• No response to nose prick

Table 22 (mechanical stunning) and Table 23 (electrical stunning) were provided in the dossier in order to give a summary of the indicators recommended in various international standards and guidelines, along with an assessment of whether these indicators are present in DTS‐stunned animals.

Assessment by the WG: The WG considers the DTS indicators outlined in Tables 22 and 23 to be inconsistent with information presented in other sections of the submitted dossier. For instance, the absence of rhythmic breathing is reported in these tables as a sign of effective DTS application, while elsewhere in the dossier it is noted that animals continue to breathe during and after the application of DTS. Similarly, it is indicated in the tables that immediate collapse occurred during the application of DTS, however the time to loss of posture (LOP) reported elsewhere in the dossier ranges between 1 and 19 s after the start of DTS application, depending on the energy delivered to the head. It is claimed by the applicant that the animals exhibiting tonic seizures lasting 0 to 3 s, followed by clonic seizures, would not be conscious, also manifested as LOP. However, the WG doubts whether these seizures ascertained on the basis of occurrence of rapid blinking, tightening of the facial muscles and kicking of limbs could be interpreted as the signs of grand mal epilepsy in the brain and therefore tonic–clonic seizures. This interpretation is supported by the report involving rats that were effectively stunned using microwaves and exhibited tonic seizure manifested as rigidly extended body and limbs followed by clonic seizures manifested as jumping or twitching (Guy & Chou, 1982). The reason for this discrepancy, i.e. manifestation of tonic seizure as fully blown tetanus involving all the skeletal muscles, including respiratory muscles, in rats and lack of such manifestation in cattle, has not been addressed in the dossier. In addition, since cattle exposed to DTS continue breathing during and after exposure to 16–20 kW for 10 s, it is very likely that these parameters do not induce tonic seizure.

The applicant continues that for mechanical stun, Gracey and Collins (1992) cite ‘no rhythmic breathing’ as the cardinal sign that the stun has worked, while Grandin (2002) advises observers to focus on ‘limp head, tongue protruding from the mouth and blank stare’. Vocalisation during application of the stun is indicative of pain or distress (EFSA, 2004), but lack of vocalisation does not guarantee absence of pain or distress, some animals may be disinclined to vocalise. Indeed, in the DTS pilot study, two animals vocalised during application of the head restraint and chin lift, but once in position, and not being handled by people, the animals fell silent and remained so throughout the stun process. Similarly, the corneal reflex does not distinguish accurately between consciousness and unconsciousness, but if there is no corneal reflex, it is likely that the animal is unconscious (Anil & Mckinstry, 1991; Gregory and Grandin, 2007). In the DTS pilot study, corneal reflex was present in animals prior to treatment, and absent following treatment, in both captive bolt and DTS groups, suggesting effective stunning in both cases.

DTS, delivering 160–200 kJ at an incident power of 18–20 kW, induces loss of consciousness, as evidenced by the tonic–clonic behavioural response within 3 (tonic) to 11 (tonic–clonic) s of onset of energy application (STEP11, STEP 13, SS07, SS08, PR21, PR24), and the duration of unconsciousness recorded during a study on full recovery (STEP 13) was between 90 and 180 ss, based on absence of corneal reflex, and duration of EEG changes, whereas full consciousness was regained between 152 and 369 s after the onset of the tonic response.

For proper slaughter purposes, the insensibility should be of a duration that allows death through exsanguination without recovery of consciousness. At the current recommended parameters, the animal will regain consciousness at 180 s after the start of energy application. In studies of slaughter without stunning, the ABM of unconsciousness ‘loss of eye responsiveness’ as a result of exsanguination has been demonstrated to occur between 95 and 103 s after the ritual cut (Gibson et al., 2015), while an isoelectric state in EEG has been demonstrated to occur 75 s (maximum 126 s in the absence of false aneurysm) after the neck cut (Daly et al., 1988). To allow a margin of safety, according to the dossier, it is proposed to use the maximum time to isoelectric EEG as defined by Daly et al. (1988) to estimate the maximum stun‐stick interval. Thus, 180 minus 126 s indicates that a stun‐to‐stick interval of 54 s will maintain unconsciousness until death occurs. It is evident from Figure 15 of the dossier (timeline) that the duration of bleeding is shorter with DTS (55 + 8.7 s) than ritual cut (76 + 7.9 s, Njisane & Muchenje, 2017), perhaps as a result of increased blood pressure and the inverted position of the unconscious animal. Furthermore, the incidence of false aneurysm with DTS (one incident, associated with operator error, across 350 cattle) is substantially lower than that of ritual slaughter (7%, Gregory et al. (2010); 7.25%–10.25%, Bozzo et al. (2020); 38%–40%, Gibson et al. (2015); 60% Supratikno et al. (2022)). Because DTS bleeding times are around 20 s shorter than those observed in ritual slaughter, and the incidence of false aneurysm are much lower compared to ritual slaughter, it can be inferred that an additional 20 s margin of confidence (inadvertent delay) can be applied when determining the maximum stun‐stick interval, as this suggests a greater safety buffer in ensuring effective stunning. Therefore, it could be argued that the maximum stun‐stick interval for DTS could be greater than 54 s, perhaps up to 74 s.

Thus, it is recommended in the dossier that the maximum stun‐to‐stick interval for commercial processing is 45 s from onset of energy application, or 35 s after the energy application of 10 s, respectively. In comparison, electrical stunning has much shorter durations of unconsciousness (Figure 15 of the dossier – confidential) and therefore shorter maximum stun‐to‐stick interval.

The response to a painful event, such as a nose prick, is a good indicator of consciousness and sensibility to pain. Anil and McKinstry (1991) demonstrated that an animal that perceives pain will draw back from the nose prick, and during recovery from electrical head‐only stunning, sheep that demonstrated this withdrawal reflex soon regained the righting reflex. With DTS, prior to treatment, animals visibly flinched in response to the nose prick, but this response was absent post‐treatment in both captive bolt and DTS animals.

Other authors have taken a reverse approach by identifying indicators of ineffective stunning. For example, Gouveia et al. (2009), studying 2800 cattle stunned using captive bolt in a commercial Portuguese slaughterhouse, found that the most common signs of ineffective stunning were muscle tone of the ears (17.8%), absence of muscle spasms in the back and legs (11.5%), presence of rhythmic breathing (9.4%) and vocalisation (7.9%). They also observed animals that showed signs of recovery (e.g. corneal response and righting reflex returning) and suggested that this might be predicted by lack of immediate collapse (100%), eyes rotated rather than fixed (91.3%), rhythmic breathing (91%) and response to nose or ear pinch (84.6%).

For DTS animals, rhythmic breathing returns after the first clonic phase. The applicant suggests that this makes it an unsuitable indicator of effective induction of insensibility for this method. The eyes were fixed and staring, with no ocular following of movement, and there was no withdrawal response to nose prick. Initially, rapid twitching of ears and the third eyelid (nictitating membrane) was evident, with ataxia and loss of posture, particularly in the hindquarters. This was noted as the hind limbs slipping out from behind the animal and attempts to replace the foot showed incomplete stride length, such that the foot remained behind the centre of balance. The limbs did not suddenly tuck in tightly to the body as seen in mechanical and electrical stun (the main reason for ‘immediate collapse’, as there is no longer any support for the body): in high energy applications (STEP 7) the effect was more similar to a heavily sedated animal attempting to maintain posture, similar to that seen in pigs stunned using gas inhalation. It is probable that, had the animal not been supported by the head restraint unit and the close confines of the restraint box, the animals would indeed have collapsed.

According to the dossier, DTS‐stunned animals demonstrated behavioural ABMs consistent with induction of a grand mal epileptic seizure. These include – rapid blinking or flicking of the eyelids including the membrana nictitans (third eyelid) during application, loss of posture, tonic (stiff) and clonic (convulsive) phases (Table 50 of the dossier), so it is of value to compare the outcome of DTS against those reported for electrical stunning. In cattle, electrical stunning can be applied as head‐only, which affects the brain, resulting in an epileptiform seizure, from which the animal can regain consciousness; or as head‐to‐chest, which incorporates a current passed through the heart, resulting in cardiac arrest and the death of the animal. As DTS does not directly affect cardiac function, the comparison should be against head‐only electrical stunning.

Head‐only electrical stunning of cattle is characterised by a very short duration of epilepsy, followed by strong convulsions (EFSA, 2004). For effective stunning, the electrical current must be applied across the brain for a sufficient duration to overcome the inherent impedance (resistance) across the head, and application of electrical stun ranges from 4 to more than 20 s (Devine et al., 1986; Schatzmann & Jaggin‐Schmucker, 2000; von Mickwitz et al., 1989). The duration of unconsciousness in those studies lasts between 31 and 90 s. In contrast, DTS induces loss of posture within 1–19 s after the onset of energy application and the duration of unconsciousness during the DTS validation study was 100–240 s based on absence of corneal reflex and duration of EEG changes.

Return of reflexes is considered to be the earliest indication that consciousness is returning and was the point at which penetrative captive bolt was applied during the DTS validation study for AW reasons.

In the initial stages of the project, overheating of the skin surface was observed, particularly when incident power levels exceeded 25 kW. This was eliminated in the later stages of the development, through optimisation of the auto‐tuning device and applicator apparatus such that incident power levels ranged from 18 kW to 20 kW. Australian cattle may experience ambient air temperatures of 45°C in summer and could experience higher surface temperatures when standing in direct sunlight. Surface temperatures above 50°C may be uncomfortable to cattle, although no literature on thermal contact sensitivity in cattle could be located to confirm or refute this supposition. There are a few studies investigating the nociceptive response to heat in cattle. Machado Filho et al. (1998) identified a nociceptive threshold of 62°C–70°C in adult cattle, using a radiant heat source applied to the skin above the hoof, while Veissier et al. (2000) used a laser‐based heat source on the legs of calves, which have thinner skin that adult cattle, suggesting a nociceptive threshold of between 45°C and 55°C. Thus, for the purposes of the assessment, 60°C is defined. It is stated in the dossier that using the recommended parameters for DTS (18 kW, 160–200 kJ), the minimum time that has been recorded from the start of energy application to 60°C was 12 s (3.2 STEP 14, PR22). According to the dossier, the measurements indicated that temperature begins to rise approximately 8 s after energy application commences and reaches maximum temperature towards the end of energy application (PR22). The applicant concludes that because the tonic–clonic response began within 3 s of the start of energy application in all animals measured (PR22) the cattle were unconscious before the temperature increased on the forehead.

Latency to insensibility, according to the dossier

With regards to induction of insensibility prior to slaughter, there is discussion over the ‘immediacy’ of the lapse into unconsciousness. Indeed, for both mechanical and electrical stun, normal neurological function is disrupted almost instantaneously following application of the equipment (Daly et al., 1987; Daly & Whittington, 1989). Estimates of ‘immediacy’ in these situations suggest loss of sensibility within less than 1 second following application, although in electrical stunning, the current must flow for a period of at least 2 (sheep) or 5 (cattle) s in order to ensure a sustained loss of consciousness (Von Holleben et al., 2010; Warrington, 1974). Even using EEG assessment, in those situations in which EEG can be measured, according to the dossier, traces can be identified that are consistent with awareness and traces that are inconsistent with awareness, but these are separated by a transition trace (McKeegan et al., 2007), during which the ability of the animal to perceive external stimuli is unknown. The most reliable method currently available is to calculate or infer the time taken to achieve EEG changes indicative of insensibility. For example, in electrical stunning, Wotton et al. (2000) demonstrated that a minimum current of 1.15 A was required to induce EEG changes indicative of insensibility in cattle. Subsequently, Weaver and Wotton (2009) applied a current of 3.28 A or more to 287 cattle in a commercial situation, measuring the current flow between the electrodes, across the animal's brain. In all animals, a current flow of 1.15 A was achieved within 100 ms, and this figure has been accepted as the ‘time to unconsciousness’ for electrical stunning of cattle. In practice, the current flows for longer than 100 ms, to ensure that the insensibility is maintained until death through exsanguination.

In contrast, it is well known that gaseous inhalation (CO_2_ and CO_2_/N_2_ mixtures) is not as dramatically ‘immediate’: the induction of unconsciousness is a gradual process as the inhaled CO_2_ is absorbed into the circulation, progressively lowering the pH of the cerebrospinal fluid (CSF) to a threshold level at which neurotransmission fails (Woodbury & Karler, 1960) and the animal loses consciousness. At this threshold, the lapse from consciousness to unconsciousness is considered to be ‘immediate’. In comparison, DTS utilises electromagnetic energy to induce unconsciousness, with sustained unconsciousness as a result of an increase in brain temperature to achieve the point at which neurotransmission fails (Small, Mclean, Keates, et al., 2013: SS02). In cattle, the core body temperature tends to be 1–2 degrees higher than in humans (38.6°C in resting cattle, 37°C in humans), so it is expected that in cattle thermal unconsciousness would occur in the range 42°C–47°C. Thus, the required rise in temperature to achieve insensibility is in the range of 3.4–8.4 degrees. During development and testing of the DTS equipment utilised, the temperature profiles of cadaver heads was assessed and a predictive equation describing the heating rate within the brain was generated (0.124°C/kW^.^s). From this equation, according to the dossier, it can be inferred that for a 30‐kW application, the time taken to achieve an 8 degree rise in temperature would be 2.15 s (8/30/0.124), and for a 20‐kW application, the time taken would be 3.23 s (8/20/0.124). In a commercial situation, the real‐time monitoring system incorporated within the control panel will allow an objective record of energy delivered and rate of energy delivery to be maintained, and an alarm feature could be incorporated to use in conjunction with animal‐based indicators (e.g. response to reflexes) when confirming insensibility prior to exsanguination.

According to the dossier, it is useful to compare the application of DTS with the use of inhalational agents used for stunning, as both approaches do not produce unconsciousness ‘immediately’. The gradual induction of insensibility by gaseous inhalation has been demonstrated to expose animals to a period of discomfort of 20 s or more prior to loss of consciousness. Llonch et al. (2012) described retreat attempts by pigs 10 s after entry into a commercial Dip‐Lift gas chamber mixtures; followed by escape attempts and gasping at 25 s exposure, with loss of balance at 30–32 s, muscular excitation at 33 s and vocalisations at 35 s. Raj and Gregory (1996) described loss of posture occurring at 15–18 s of exposure to gas mixtures, vocalisations at 26–30 s and convulsions at 21–22 s (persisting for 17–33 s); while Raj et al. (1997) measured ECoG suppression beginning between 15 and 25 s of exposure, and an isoelectric trace (maximum effect) between 23 and 45 s. In comparison, for DTS energy exposures of 140–210 kJ (based on prototype 2 delivering only 70% energy as compared with prototype 3), Rault et al. (2014: SS03) estimated EEG changes to occur within 3 s of the end of each application and the nadir (maximum effects) occurring within 4–22 s post application. In the DTS pilot study (STEP 7), by the time the cattle were removed from the Faraday cage (approximately 20–30 s post energy application), DTS animals were unresponsive to stimuli and the EEG recorded was suppressed. With refinements to the system, the current apparatus and recommended parameters result in unconsciousness based on onset of the tonic phase within 1–3 s of energy application (STEP 13, SS07, PR22). Although DTS induces insensibility gradually, as the neuroelectric field expands and brain temperature rises to a threshold at which unconsciousness occurs, this process is significantly faster than that experienced by animals subjected to gas inhalation.

Aversiveness of the system, according to the dossier

The overall aversiveness of any stunning system can be considered to be made up of three main components:

• Pre‐restraint handling;
• Restraint;
• The stunning method itself

To minimise the adverse impacts of pre‐restraint handling, low‐stress stock handling by trained and competent personnel are recommended, according to normal commercial practice. Similarly, to minimise the adverse impacts of restraint, the units utilised of DTS are designed based on current best practice designs, specifically the ASPCA Pen design described by Grandin (1992) and Marshall et al. (1963). Quiet handling and smooth application of the neck capture and chin lift apparatus is well tolerated by cattle (Dunn, 1990; Grandin, 1992) and the head position achieved allows accurate placement of the applicator onto the forehead of the animal. For DTS application, the head is held in the chin lift for a maximum of 30 s while the applicator is positioned and DTS energy delivery initiated, as it is known that prolonged restraint in any system is stressful to animals (Ewbank et al., 1992).

Assessment of aversiveness of the final component, the stunning method itself, separate from the impacts of restraint and handling, is challenging. However, the fact that pain‐related complexes were not evident in the EEG traces of minimally anaesthetised animals (STEP 5; Rault et al., 2014); and that endocrine and meat quality markers from DTS cattle and carcases were not significantly different from captive bolt cattle and carcases when handled using the same pre‐restraint handling and restraint unit (STEP 7). These findings indicate that the DTS system does not differ from captive‐bolt stunning in terms of aversiveness. Indeed, in an assessment of full recovery from unconsciousness induced by DTS, all animals were easily returned to the restraint unit without baulking, backing up or vocalisation. The animals behaved in a similar manner to any cattle processed in an abattoir lairage, and there was no evidence of distress or aversion, supporting the conclusion that DTS does not impose additional welfare concerns relative to conventional stunning methods.

Commentary by the WG: For the assessment of EFSA regarding the above quantitative approaches provided in the dossier see Section 4.2.1.

Qualitative approaches

3.3.1.2

The dossier does not include any qualitative assessment. For example, it does not compare the methods by assigning scores to different measures and ranking them by severity, which would then allow a quantitative comparison.

Overall discussion and conclusions

3.3.2

Benefits for the industry, according to the dossier

The applicant claims that DTS is an effective means of inducing insensibility in cattle, with a duration of insensibility that is well‐suited to exsanguination via neck cut as required in religious slaughter. Key parameters expected to be favourable compared with the current stunning methods are:

• There is no need for a back‐up ventral cut (thoracic stick), as the blood flow was strong and exsanguination was rapid;
• Blood flow was visibly strong, and the pulsations associated with heart function were visible in the early stages of bleedout;
• Exsanguination could be performed safely, as the animal is immobilised;
• Visual inspection of the brains of carcases indicated no visible damage when energy deliveries of less than 220 kJ were applied, and the stun did not cause cracks in the skull, so eliminates a source of rejection against religious requirements, and eliminates a potential source of breaches noted on audit when the auditors' interpretation of ‘cracked’ differs from the regular inspector;
• The stun is effective in heavy animals and bulls, which currently and often present challenges for percussive stunning methods;
• The DTS method allows for the possibility of the animal returning to consciousness, making it more likely to be acceptable to the Halal and Kosher market.

The dossier concludes with the following:

1. Using the recommended parameters for DTS, AW is maintained through restraint, induction of unconsciousness, through to death by exsanguination. It delivers an AW outcome that is at least equivalent to that observed with other stunning methods approved for cattle (Table 51 of the dossier).
2. ABMs assessed during restraint and application of DTS indicated only minor discomfort or distress in a small number of animals, not unexpected in any form of restraint or stunning.
3. In the absence of technical problems leading to interrupted delivery of energy, all animals were rendered unconscious using DTS when the recommended parameters for administration of DTS are used.
4. Administration of between 160 and 200 kJ energy at 18 kW (following engineering modifications) ensures rapid loss of consciousness without overheating the skin of the forehead (prior to loss of consciousness) or inducing extreme convulsions.
5. Animals are unconscious for at least 60s following the induction of unconsciousness. EEG data indicate at least 60s unconsciousness with long transition to return of consciousness (as indicated by behavioural ABMs).
6. ABMs used to detect consciousness and unconsciousness are confirmed to correlate with EEG. ABMs to monitor the state of consciousness are suggested in Table 52 of the dossier, to be used at three key stages (after stunning, at neck cutting and during bleeding), as depicted in the flowchart that follows (Figure 28 of the dossier). For each key stage, three or four ABMs that are reliable in monitoring consciousness are suggested.
7. Use of the rotary box optimised handling of the stunned body, allowing a short stun‐to‐stick interval without the use of an immobiliser. The updated applicator configuration allowed consistent repeatable induction of insensibility, which was sustained during Halal bleeding, without the need to apply an additional ventral cut.
8. There was no requirement to use an immobiliser during application of the exsanguination cuts, and the animal bled rapidly to brain death using the Halal cut alone. The blood flow on exsanguination was strong and rapid.
9. Using the recommended parameters for DTS there are no significant physical effects on the brain and therefore normal recovery can occur in animals that are not exsanguinated.

Commentary from the WG: These conclusions are provided in the dossier and do not represent the views of the WG. The WG carried out an independent assessment in Section 4. This included an evaluation of Table 51 of the dossier ‘Equivalence of DTS: Diathermic Syncope® with existing stunning methods for cattle’, Table 52 of the dossier ‘ABMs for assessment of “outcome of consciousness” after DTS’ as well as the relevant Figure 28 of the dossier ‘Toolbox approach to assessing unconsciousness immediately after stunning through sticking and bleeding’.

ASSESSMENT PHASE 2: ANIMAL WELFARE RISK ASSESSMENT OF THE DIATHERMIC SYNCOPE® STUNNING METHOD

4

The EFSA experts assessed the DTS method, focusing on two main aspects as outlined in the EFSA guidance (EFSA AHAW Panel, 2018): (i) the AW risk based on the assessment of the outcomes resulting from the stunning method and (ii) the validation of the equivalence of the proposed stunning methods with existing approved methods. A similar evaluation process was employed for the assessment of the Low Atmospheric Pressure Stunning (LAPS; EFSA AHAW Panel, 2017) and Nitrogen Expansion Foam Stunning (NEFS; EFSA AHAW Panel, 2024).

Animal welfare risk assessment

4.1

The DTS method involves microwave irradiation of the brain of cattle weighing 270 to 690 kg (live weight – claimed to be validated for 270–690 kg and designed to be used for 270–800 kg) with a microwave frequency range of 890–925 MHz delivering 160–200 kJ energy level and an incident power of 18–20 kW for 10 s. According to the dossier, Diathermic Syncope® applies electromagnetic energy consisting of both a magnetic and electrical field, moving as sine waves, perpendicular to one another.

Based on the EFSA guidelines (EFSA AHAW Panel, 2018) for the assessment of pain, distress and/or suffering and the onset and duration of unconsciousness or death, the measures chosen in the submitted dossier were scrutinised in terms of validity and reliability. This evaluation was based on the applicant's justification for the selected measures and will be compared with the scientific state of the art for cattle.

Assessment of onset and duration of unconsciousness

4.1.1

The assessment of DTS involves evaluating the methodology and criteria used to determine unconsciousness and death as well as evaluating the associated AW outcomes.

Brain mechanism associated with the induction of unconsciousness

Guy and Chou (1982) investigated this principle using pulsed microwave (pulse width ranging from 1 to 360 ms) and focal application of microwave energy at 2–10 kW levels to Wistar rats' heads. The results indicated that a maximum temperature rise of 8°C or final maximum brain temperature of 46°C to 46.5°C resulted in petit or grand mal seizures. However, two out of six (33%) awake (unanaesthetised) rats exposed to microwave attained brain temperatures of 45.8°C and 46.2 C but they were not stunned by the treatment, which is disconcerting from the AW point of view as unconsciousness was not induced. Another concern is that, unlike rats, cattle have a unique brain circulation via carotid rete and vertebral artery that helps regulate brain temperature ‐ within the range of 38°C–40°C ‐ by enabling selective brain cooling to maintain a temperature below core body temperature. In addition, there is no scientifically sound data regarding brain temperature profile induced by the DTS method and thermal unconsciousness in cattle.

Assessments of increases in brain temperature (SS02 and SS04) were carried out in cadavers' head without brain blood circulation and its impact on maintaining brain temperature (cooling effect). These studies also involved application of energy levels lower than the range of energy levels requested.

Application of DTS is claimed in the dossier to result in inhibition of neurotransmitters leading to unconsciousness. This would mean that the induction of tonic–clonic seizures by DTS would not be expected to occur, because these generalised seizures could occur only when neuronal excitation, activated by the release of excitatory neurotransmitters such as glutamate and aspartate, occurs in the brain. This scenario is completely opposite to neuronal inhibition.

Nevertheless, if neurotransmitter inhibition is expected to occur due to the application of DTS, then loss of spontaneous firing of neurons would be expected and, consequently, occurrence of a profoundly suppressed EEG. In this regard, the time to onset of nadir of EEG suppression (i.e. lowest EEG activity estimated qualitatively) as an indicator of neuronal inhibition suggestive of unconsciousness provided by the applicant in PR22 Extra document, ranges between ■■■■■ The disconcerting fact from the AW point of view is that ■■■■■ after the recommended (in the dossier) stun‐to‐stick interval of 45 s from the start of energy application. This raises a serious AW concern, i.e. whether cattle are rendered unconscious by the proposed energy levels (160 to 200 kJ) and duration of exposure (10 s) and whether neck cutting is performed in conscious animals.

Methodological aspects

4.1.1.1

The methodologies used in the evaluation of DTS are assessed for validity and reliability, including the criteria and thresholds for determining unconsciousness. Specifically, the brain mechanisms involved in inducing unconsciousness and the scientific rationale for selecting neurological measures are examined. Additionally, the use of ABMs for assessing the state of consciousness through behavioural (e.g. loss of posture) and physical (e.g. corneal reflex) reflexes is reviewed. Lastly, the approach used to establish the association between neurological measures and ABMs is evaluated.

There are several aspects of DTS that have not been clearly explained in the dossier, and have raised several questions:

• What is the brain temperature at which syncope is expected to occur in cattle?
• How long does it take to achieve this threshold temperature during the application of DTS at energy levels 16–20 kW?
• What are the impacts/consequences of the presence of carotid rete and blood supply to the brain via the vertebral artery in cattle in terms of maintaining homeothermy in the brain and delaying the onset of syncope?
• What is the variability in a cattle population, due to age, sex and breed (tropical vs. temperate) of cattle in terms of maintaining homeothermy in the brain?
• How long does the syncope last?
• How soon after the termination of DTS will brain temperature return to normal due to continuous blood supply via the vertebral artery?
• How is the brain temperature maintained to sustain syncope until death occurs through exsanguination?
• What are the EEG parameters/criteria for determining syncope and how do they correlate with the clinical signs of syncope in cattle that could be used as ABMs for monitoring purposes?
• What are the clinical signs of recovery from syncope?
• It is known that carotid occlusion leads to increased blood flow to the brain. What are the associated animal WCs of this?

Neurological measures

According to EFSA guidelines (EFSA AHAW Panel, 2018), it is recommended to use EEG to determine the onset and duration of unconsciousness, as well as the time to death. EEG methods are endorsed because they are well‐established in internationally recognised and peer‐reviewed literature, providing a robust basis for evaluating the effectiveness of a stunning method.

The applicant claimed that direct measurement of electrical activity within the brain during energy application is currently not feasible due to the saturation of EEG recording systems by the applied microwave energy. Similarly, measurement of internal brain temperature in conscious cattle within an abattoir setting is impractical, as it would require the implantation of fibreoptic thermoprobes into brain tissue, which is not feasible in this context. Owing to this, the applicant stated in the first set of dossiers that EEG could not be recorded/evaluated to ascertain the impact of DTS on the brain activity in cattle. However, when DTS was applied in anaesthetized cattle in laboratory situation (see SS03), it is stated that EEG was recordable/readable 3 s after the application. The results of this experiment indicated that application of DTS resulted in seizure‐like activity in all the animals, leading to EEG suppression. The time to onset of EEG suppression depended on the amount of microwave energy delivered, with higher power resulting in longer time to EEG suppression (30 kW > 20 kW > 12 kW). The time to onset of maximum EEG suppression (time to nadir) varied between 4 and 22 s, and the duration of the maximum suppression lasted 37 to 162 s depending on the amount of energy delivered to the animal (12 kW – 37 s, n = 1; 20 kW – 107 s, n = 4; and 30 kW – 162s, n = 2).

The applicant provided EEG records from ■■■■■ (data from PR 22). However, ■■■■■ did not permit to elucidate the times of unconsciousness and death in any of the ■■■■■ records provided. The post‐exposure EEG patterns, as observed in electric stunning, were not visible in the records and no information about them was provided in spite of the fact that electrical head‐only stunning was used by the applicant for comparison. The most valid measures available (e.g. EEG) should be used and correlations between these measurements and non‐invasive ABMs that can be applied in a slaughterhouse context should be established.

No robust evidence on induction and maintenance of unconsciousness is provided by the EEG data as these were incomplete or inconsistent: EEG was recorded before and after stunning, and not during DTS application due to incompatibility between DTS and electrodes. Post‐exposure EEG is unreadable due to artefacts originating from background noise, movement of animals and blood flowing over the head following neck cutting (sticking). Thus, the onset of unconsciousness cannot be directly determined from the EEG. In addition, the correlation between EEG and ABMs has not been demonstrated for this new method. Therefore, due to the absence of EEG evidence of unconsciousness and lack of demonstration of correlation between EEG and behavioural and physical measures of unconsciousness (ABMs), induction and maintenance of unconsciousness until death remains to be demonstrated unequivocally.

• Behavioural and physical measures for determination of unconsciousness

Between the end of stunning and shackling, the recommended behavioural measures for assessing unconsciousness in cattle include immediate collapse, apnoea, presence of tonic–clonic seizure and absence of corneal reflex in either mechanically or electrically stunned cattle (EFSA, 2020). Other indicators, such as loss of muscle tone, fixed eyes and absence of vocalisations, are considered less reliable. During bleeding, loss of muscle tone and apnoea are indicators of unconsciousness. In DTS, according to the dossier, tonic–clonic seizure was used to determine the time to onset of unconsciousness in cattle, while the absence of the corneal reflex and lack of response to painful stimuli after tonic and clonic seizure were used to assess the duration of unconsciousness (SS05). In the absence of EEG evidence of generalised epileptic activity in the brain, the tonic–clonic seizure activity is unsubstantiated and is likely to be the stepping behaviour reported in heat‐stressed cattle (Idris et al., 2024; see also Section 4.1.2.2 for details).

Nonetheless, it should be highlighted that the animals stunned by DTS were placed and restrained in a box, making the assessment of certain behaviours, such as the indicators for tonic–clonic seizures, challenging. It is difficult to know whether the animal is kicking the box because of the clonic seizures or because of escape attempts caused by e.g. pain or distress during induction to unconsciousness. Therefore, the interpretation of the presence of tonic–clonic seizures as evidence of grand mal seizures during DTS application leads to high uncertainty.

• Association between neurological measures and other ABMs

It is not possible to correlate the onset of unconsciousness determined by EEG with other ABMs from the data provided (e.g. loss of posture, corneal reflex), as EEG recordings were made before and after stunning but not during the application, due to the incompatibility between electrode readings and the use of DTS. The post‐exposure EEG might indicate the magnitude of suppression in the brain activity, suggesting unconsciousness, but unfortunately, this post‐exposure EEG is not visible in the provided records because of noise. In addition, it is clearly stated that blood flowing over the head made EEG data collection challenging and generated artefacts leading to unreadable EEGs. In addition, the EEG record provided in the publication has wrong units: mV instead of μV. It is unclear whether this is just a misprint, or the signal indeed was 1000 times larger than expected. There are artefacts leading to unreadable EEGs.

Results regarding onset and duration of unconsciousness and death

4.1.1.2

The assessment of the effectiveness of DTS in relation to unconsciousness and death should take into account the following variables: frequency of correctly stunned animals, time to onset of unconsciousness during exposure to DTS, time to recovery of consciousness, duration of unconsciousness, maximum permissible time between the end of exposure and exsanguination and time to death due to exsanguination.

Owing to the absence of EEG evidence of unconsciousness and lack of correlation between the EEG and behavioural and physical measures of unconsciousness (ABMs), induction and maintenance of unconsciousness until death remains unclear. Under this circumstance, the possibility that kicking of limbs, which has been interpreted as continuation of grand mal seizure, could also be due to the stimulation of motor neurones in the conscious animals could not be ruled out. More importantly, animals will experience pain, fear and distress due to such convulsions occurring in conscious state induced by the stimulation of motor neurones in the brain.

Assessment of ‘pain, distress and suffering’ associated with the pre‐stunning process, during induction of unconsciousness and due to mis‐stunning

4.1.2

Methodological aspects

4.1.2.1

The identified hazards by EFSA's ad hoc WG associated with DTS applied to cattle is detailed in Table 24 below:

ABMs to assess the WCs of hazards related to handling, restraint and induction to unconsciousness during DTS stunning process are shown in Table 25.

A list and description of ABMs for monitoring the state of consciousness is provided. The compilation of the ABMs and the key stages that can be used for the assessment of the state of consciousness after DTS stunning process is shown in Table 26.

Evidence of ‘pain, distress and suffering’ from the Diathermic Syncope® method (ToR 2i)

4.1.2.2

The DTS system for delivering energy can be considered linear. Thus, the field intensity 𝐸 at 0.1% power can be measured and scaled to 100% by multiplying by square root of 1000, since power 𝑃 is proportional to 𝐸^2. However, the lack of field intensity measurements in the current system poses a challenge. It is not known what part of generated energy goes inside the body (the skull), and which is reflected and is wasted in the wave guide and the environment. The rate of brain heating measured in the experiment indicates that a significant part of energy goes into the animals' head, but this estimate remains imprecise. The various tissues in the pathway will absorb the microwave energy and therefore the strength of the microwave field will decrease, limiting the depth of the heating effect (see Section 3.1.2 for details).

The potential sources of pain, distress and suffering during exposure to DTS are described in detail under Section 3. Consequently, the focus in this section will be on the evidence submitted by the applicant to ascertain that these WCs are prevented or largely mitigated.

According to the EFSA guidelines (EFSA AHAW Panel, 2018), two criteria must be fulfilled before a stunning method is considered not to induce avoidable pain, distress or suffering prior to the onset of unconsciousness. First, the ABMs chosen by the applicant should not indicate a greater magnitude of pain, distress and suffering in the treatment group compared to the appropriate control group. Second, the responses of cattle undergoing the procedure without stunning (control or sham operation) should be similar to those exposed to DTS.

In general, the outcomes of the different ABMs in an individual animal should be consistent, enabling a clear interpretation of their impact on AW. If evidence indicates that a method causes signs of pain, distress or suffering, the evaluation will consider both the proportion of affected animals and, where feasible, the severity and duration of the negative affective state(s) involved. For this purpose, the existing literature and/or expert opinions will be used for data interpretation.

The risk of overheating of the skin of the forehead prior to induction of unconsciousness causing skin burns was identified in the early stages of DTS development. The updated Report PR15 declared that the ■■■■■ However, the WG believes that this conclusion based on skin samples collected from ■■■■■ involves high uncertainty.

Additionally, during DTS energy application, a temporary cessation of breathing occurs as the animal enters the epileptiform phase. This phase resolves quickly, and breathing recommences in a slow and deep pattern. However, the implications of this on the animal's anaesthetic plane (i.e. depth of unconsciousness) and its experience of pain sensation is unclear. In contrast with these interpretations, Small et al. (2019) reported that breathing continued during and after DTS application.

Sources of pain, distress and suffering

Rault et al. (2014) reported that all the animals subjected to microwave energy exposure (20 kW for 10 s; n = 2; or 15 s n = 2; or 30 kW for 10 s n = 2) had skin burns over the forehead. The authors described the lesions as ‘there was an area of complete skin loss, surrounded by a larger region displaying grey‐tan discolouration of the subcutaneous tissue. The skin within this area displayed full‐thickness, coagulative necrosis extending down to the skull’. Unfortunately, the time to onset of skin burn was not reported, and it is also difficult to assume whether unconsciousness occurs prior to burning of the skin. This is supported by the report by Small, Mclean, Owen, and Ralph (2013); Small, Mclean, Keates, et al. (2013) that ‘the skin experienced greater heating than the brain, and heating rate decreased with depth into the brain’. It is stated in the recently submitted additional dossier, PR22, that the ■■■■■ using ■■■■■ rather than epileptiform activity in the EEG.

In the dossier, it is stated under Section 3.1.2.1. Point 4. Applicator: ‘Poor tuning results in inefficient energy transfer, which could lead to failure to induce unconsciousness, or inappropriate surface heating’. This is a potential source of system failure/source of pain, distress and suffering, and the prevalence of this improper tuning (i.e. the process of adjusting the microwave device's physical or electrical properties to optimise performance) is not known. In addition, it is stated that ‘assessment of tuning should be carried out by a specialist technician annually or in the event of a change in size of the applicator (e.g. use on another species)’. From an AW point of view, every incidence of poor tuning should be investigated and corrected before using the applicator on another animal.

According to the original dossier (May 2023), the criteria initially used to determine unconsciousness were time to loss of posture (collapse in severely restrained animals) as time to onset of unconsciousness, absence of corneal reflex and absence of response to painful stimulus (nose prick with a hypodermic needle) as indicators of unconsciousness. AW concerns originated from the fact that it is difficult to see the animal restrained inside the metal box to ascertain loss of posture. The absence of the corneal reflex (false positive) could be misleading, as it may result from damage to the fifth or seventh cranial nerves, the cornea or the muscles controlling the eyelid (which can occur due to the proximity of the applicator), rather than due to loss of consciousness, which leads to high uncertainty. This interpretation is supported by the fact that Small et al. (2019) reported that delivery of 45.87 kJ applied for 1.5 s, which is substantially lower than the energy and duration requested in the recent dossier, to a bovine (animal 1.14, Table 2 of the study) resulted in abolition of corneal reflex and loss of response to painful stimulus (nose prick), with fixed and staring eyes, but the animal failed to collapse (i.e. remained standing). Clearly, this observation raises serious welfare concerns about the DTS method and the use of these ABMs to determine unconsciousness in the submitted dossier. Lastly, the applicant reported that the onset of unconsciousness is assessed based on the onset of the tonic, then tonic–clonic behavioural responses, which occur within 3–8 s after the onset of energy delivery. However, tonic seizures occurring during generalised epilepsy (grand mal) is expected to cause tetanus in all the muscles, including respiratory muscles, leading to apnoea (absence of breathing). But Small et al. (2019) reported that rhythmic breathing continued throughout DTS application. This suggests that the movement or behavioural changes in cattle is unlikely to be due to tonic ‐ clonic seizures, because tonic seizure would result in tetanus involving all the muscles, including respiratory muscles. In this context, it is important from AW point of view to note that Guy and Chou (1982) reported that rats exposed to microwave exhibited grand mal seizures in which tonic seizure was manifested as rigidly stretched out body for 1 min after exposure and clonic seizure manifested as jumping or twitching leading to a quiescent, unconscious period lasting for 4–5 min. This observed grand mal seizures in rats is in complete contradiction with the description of grand mal seizures in cattle reported in the dossier.

In the first set of dossiers submitted (May 2023 to May 2024) for assessment, the behavioural changes in cattle during the application of DTS were described differently from the descriptions in the more recent dossier (September 2025). For example, during the application of DTS, cattle held their ears stiff pointing backwards along the neck, blinked rapidly and displayed whites in the eyes. Background literature suggests that cattle subjected to heat stress (i.e. at different stages prior to the onset of heatstroke) and those subjected to highly stressful handling at slaughter held their ears in this position, and this has been interpreted as evidence of pain, fear and distress (Boissy et al., 2011; Hemsworth et al., 2011). It is known that cattle subjected to pain, distress or suffering also display eye whites (Gerritzen et al., 2014). Based on the available evidence, causing pain, distress and suffering to cattle during the induction of unconsciousness by DTS could not be ruled out. This interpretation is also supported by the fact that cattle exposed to heat stress in feedlot and experimental conditions also stamp their limbs (stepping behaviour), which has been described as an attempt to get away from the stressful situation (Idris et al., 2024). Indeed, it is clearly audible in the video clips of the dossier that cattle exposed to DTS were kicking their limbs against the metal floor or sides of the restraint. The WG assessed that, in the absence of EEG evidence of unconsciousness this stepping behaviour cannot be interpreted as clonic seizures indicative of unconsciousness. In addition, the rigidly extended body and limbs reported prior to occurrence of twitching or jumping and interpreted as clonic seizures in rats subjected to microwave stunning had not been observed in cattle following DTS application. It is also worth noting that there are several stages of heatstroke. The behavioural and clinical symptoms vary according to the stage (guidelines provided as factsheets by the Michigan State University https://www.canr.msu.edu/news/heat_stress_cattle_tips_to_keep_your_cattle_cool), finally resulting in ataxia and death (https://www.vff.org.au/stock‐sense/resources‐factsheets/heat‐stress/; How to Treat Heat Stroke & Hyperthermia FAST; https://www.youtube.com/watch?v=‐mMORd4qwpA). It is worth cautioning that ataxia is not an indicator of unconsciousness, as reported in the previous EFSA opinions.

The recently submitted report PR22 from 2025 reports a study carried out in ■■■■■ The occurrence of seizures is not based on EEG data but on arhythmic movements of the facial muscles and eyelids, eyes fixed and staring or rolling down and then returning to centre. A search on the internet (heat stroke in cattle) revealed that fixed eyes or display of eye white occurs in conscious cattle during heat stroke as well (Figure 18).

Fixed eyes or display of eye white occurs in conscious cattle during heat stroke. Source: Courtesy of Dr. Shanker K. Singh. Heat Stroke in Cow? Hyperthermia! Cause and Treatment? Dr. Shanker – VetVirtual Classes. https://www.youtube.com/watch?v=G38Izvaot6Q, accessed on 31 October 2025.

It is also reported in the dossier that the assessment of corneal and palpebral reflexes was not possible due to the uncontrolled blinking and twitching of the eyelids and facial muscles. Typically, grand mal epilepsy is manifested in the EEG by the occurrence of high amplitude (more than 100 μV) activity typically in the frequency range of 8–14 Hz (EFSA, 2004). But this is not evident in the EEG data presented in PR22. In addition, it is reported in the dossier that the latency to onset of EEG suppression, onset of nadir and duration of suppression were measured. However, the interpretation of data was challenged by the blood flowing over the EEG leads and the occurrence of agonal convulsions. The WG experts also deemed EEG data unreadable. It is very likely that blood flow over the eyes would also make the elicitation of corneal and palpebral reflexes difficult, if not impossible.

It is argued in the dossier that a transitional EEG from a conscious state to an unconscious state can occur. However, this is incompatible with the claim that the application of DTS induces grand mal epilepsy, which is an all‐or‐none neurological event that does not involve a transitional phase. In addition, some of the references cited as supporting this claim (e.g. McKeegan et al., 2007) concern poultry stunned using CAS, a method in which induction of grand mal epilepsy does not occur and which is therefore not comparable with DTS.

It is reported in the dossier that there was no rise in forehead temperature while the animals were conscious. In the absence of convincing EEG evidence of unconsciousness, it is not possible to substantiate this statement. Similarly, the lack of sound scientific evidence demonstrating the occurrence of grand mal epilepsy renders the argument presented in the dossier against the occurrence of absence seizures invalid.

Assessment of external validity

4.1.3

This section of the assessment evaluates the extent to which the findings from laboratory studies are consistent with those from studies in pilot‐plants, under commercial conditions or in different settings and can thus be extrapolated beyond the study population and experimental conditions. Appraisal of a study's external validity (i.e. its applicability beyond the study population) requires that its results are compared with those of comparable studies.

The technical document of the dossier does not provide an external validity assessment for the submitted studies. However, external validity can be considered from the pilot‐plant scale studies in SS05 (Section 3.2.2.1) and SS07 (Section 3.2.4.3). SS05 comprised two trials with 37 non‐anaesthetized adult cattle to gather data on behavioural outcomes and EEG changes following DTS energy application (n = 30) and captive bolt stunning (n = 7). In SS07, 7 cattle were stunned with DTS at 150–180 kJ of energy, and EEG was recorded prior to and after DTS energy application on six animals which were allowed to recover consciousness. The seventh animal was handled in accordance with commercial processing procedures without the delays caused by collecting EEG data.

In addition to the pilot studies, the updated PR15 describes two separate studies conducted in a slaughterhouse. Study 1 involved three separate investigations.

Investigation one has a serious weakness. ■■■■■ which have not been scientifically validated or correlated with the EEG manifestations. In fact, it is reported that all the EEGs collected in these animals were ■■■■■. It is well‐established that tonic seizure accompanying a generalised epileptiform activity in the brain will lead to tetanus involving all the muscles (see EFSA, 2004). Therefore, the ABMs used in this study are not scientifically sound.

The results of investigation two indicated ■■■■■, which is surprising because increase in brain temperature would be expected to cause oedema and hyperaemia as physiological response to hyperthermia.

The results of investigation three indicated that, ■■■■■ Clearly, this should be seen as evidence to overheating of the skin by the applicator, especially in the absence of an effective cooling system. In addition, the conclusion in the dossier is based on one skin sample in each treatment.

The ABMs used to ascertain onset of tonic seizure in Study 2 are the same as in investigation one of Study 1, which are not based on sound scientific principles.

Overall, the findings from the studies are consistent with those under laboratory conditions. However, as in the laboratory studies, the assessment of the state of consciousness has been carried out using poor‐quality EEG data and did not include ABMs of behaviour and physical reflexes validated under this specific method.

Assessment of equivalence of the method with existing stunning methods (TOR 2ii)

4.2

The comparison of DTS with other authorised stunning methods used for slaughter was based on a non‐formal expert elicitation utilising a semi‐qualitative approach and expert opinions.

A scientific literature review was conducted, to identify a list of hazards for each stunning method, along with their associated WCs. Hazards were included considering all phases: pre‐stunning, stunning and the actual process of killing, which encompasses the induction of unconsciousness and the onset of death.

It is stated in the dossier (see Section 3.1.2.4) that the restraint used for the application of DTS is equivalent to those used for captive bolt or electrical stunning of cattle. Therefore, AW risk assessment has not been carried out for the restraint.

Quantitative assessment

4.2.1

The applicant opted for a quantitative approach to demonstrate equivalence with existing methods (penetrative captive bolt, electrical head‐only, and electrical head‐to‐body) based on biomarkers of stress, ABMs of the state of unconsciousness, latency to insensibility, impact on meat quality attributes and aversiveness of the system. It is worth noting that unconsciousness itself was not included. Impact on meat quality will not be evaluated as it is not relevant for the assessment. EFSA assessed the relevance of the information provided in the dossier in the following sub‐sections:

• Biomarkers of stress

The dossier shows two tables comparing biomarkers of stress associated with restraint, positioning the head into the head capture and stunning with DTS and penetrating captive bolt: Tables 20 and 21.

Table 20 summarises the endocrine markers quantified in SS06 and the same markers found in six other scientific studies.

The results from the remaining studies considered in the dossier are not comparable. For instance, sources of variation in endocrine markers include not only stress levels, but also the time of day (since hormone secretion follows a diurnal rhythm, leading to variations depending on the time of sampling), age and sex, health status, diet, genetic factors (differences between species and breeds), the method of sampling and handling as well as the procedures for the laboratory analysis.

For adrenaline and noradrenaline, the results are not comparable because in Rulofson et al. (1988) blood samples were taken from five yearling Red Angus bulls using a catheter placed in the jugular vein. Sampling occurred 1 week before slaughter, with seven samples taken at 10‐min intervals. After 1 week of acclimatation in box stalls and following shipment of the bulls 320 km to the abattoir, additional samples were taken, including one during exsanguination the following day. Although all samples were taken from the same animals, the sample size was limited to only five animals. In addition, the study fails to provide crucial details regarding key parameters of the captive bolt, the effectiveness of stunning and whether blood samples were collected at consistent times. In Mitchell et al. (1988), 40 crossbred steers (castrated) and 40 crossbred heifers (Brahman × Hereford × Afrikander) of similar age and body weight (15‐ to 18‐month‐old and 500–600 kg) were divided into 4 balanced groups. Blood samples were taken after handling (being driven into a race), after transport and after slaughter in subsets of the total sample size. The adrenaline concentration reported in Table 20 for Mitchell et al. (1988) is incorrect. For instance, it was not measured in nmol/L but in ng/mL and the concentration of adrenaline after stunning did not differ from after transport (after transport: 9.6 ± 6.5 ng/mL; after stunning: 9.0 ± 16.4 ng/mL). Similarly, erroneous reporting occurred for noradrenaline (after transport: 1.1 ± 1.9 ng/mL; after stunning: 6.4 ± 9.2 ng/mL). Therefore, the conclusion found in the dossier that catecholamines have been reported to increase as a result of stunning is not correct in this specific study. It should also be noted that measurements occurred from 14:00 to 16:00 h after transport and from 07:00 to 9:00 h after slaughter, and thus results can be affected by the circadian rhythm.

For cortisol, Mitchell et al. (1988) reports an increase in cortisol of 61.2 nmol/L after stunning with captive bolt and +151.7 nmol/L when cattle were handled through a race prior to transport. Therefore, the lower cortisol concentration found after captive bolt stunning suggests that restraining and application of captive bolt stunning could be less stressful than handling a race.

Caution should be exercised when interpreting data from Zulkifli et al. (2014). The study measured cortisol, ACTH, adrenaline, noradrenaline and b‐endorphin from blood samples collected from 10 Bos taurus × Bos indicus heifers and steers three times per animal (pre‐stunning, post‐stunning and post‐slaughter) on three different days. Thus, the sources of variation are multiple, and the sample size is quite low for this purpose. In addition, the hormone concentrations (biomarkers of stress) are reported as percentages of pre‐stunning sample values, and therefore, the values shown in Table 20 are not only absent from the scientific paper but also cannot be deduced or calculated.

Table 21 reports changes in endocrine markers between pre‐treatment (at the knocking box before restraining) and post‐stunning in both captive bolt and DTS‐stunned Angus crossbred heifers. According to the dossier, no statistical difference was found between captive bolt and DTS for any of the biomarkers. However, it should be highlighted that:

1. The sample size was too low to reliably detect statistical differences, if any (captive bolt: n = 7; DTS: n = 11);
2. Variability in pre‐stunning sampling: checking the material and methods in both SS05 and SS06, the blood samples from the pre‐stunning phase did not include the effects of restraining, and duration of restraining varied from 2 to 5 min which would increase the variability of biomarker concentrations;
3. SS06 states that changes in biomarkers were analysed using a mixed model that included as fixed effects the experimental day (experimental study lasted 2 days so 7 animals were stunned on day 1 and 11 on day 2), the treatment (three levels: captive bolt (n = 7), DTS at 20 kW (n = 6) and DTS at 30 kW (n = 5)) and the animal as random effect. However, as the experimental day was not a factor of scientific interest, it is more appropriate to include it in the model as a random instead of a fixed factor;
4. For noradrenaline, some samples were below the detection level and therefore excluded from the model. Considering the extremely low sample size and the flaws in the model chosen (i.e. including the experimental day as fixed effect rather than a random effect), it is quite difficult to detect statistical differences in plasma biomarkers, if any. Therefore, Table 21 does not provide relevant information.

It is concluded that although biomarkers of stress could be useful for demonstrating equivalence with existing methods, they are not relevant based on the evidence provided.

• ABMs used to assess the state of consciousness

The analysis of the provided raw EEG data revealed that contamination by motion artefact does not allow determination of unconsciousness/insensibility by EEG in most cases. An exception is two animals in which 30 kW of applied energy led to immediate EEG suppression or immediate tonic/clonic seizures. Thus, these animals fell into an unconsciousness/insensibility state rapidly. However, the reason for the highly different EEG reactions in two animals to identical stimulation is unknown. In any case, even if these animals have lost consciousness rapidly, the extrapolation of this data to lower energy levels is difficult. Therefore, only ABMs related to reflexes are used to assess the state of consciousness. These ABMs are assessed through the three stages of monitoring during the slaughter process, between the end of energy application and neck cutting, during neck cutting and during bleeding.

Posture (collapse), breathing, tonic–clonic seizures, palpebral and/or corneal reflex, spontaneous blinking, eye movement, vocalisations, muscle tone and response to nose pinch are the nine ABMs used to ascertain unconsciousness.

Nonetheless, in studies where DTS is applied, animals show rhythmic breathing during and following the application of DTS. According to EFSA AHAW Panel (2020), this indicates that the brain stem is not affected when using the key parameters recommended by the applicant. Hence, the achieved level of unconsciousness is not deep, so animals are at high risk of remaining conscious at the time of slaughter or regaining consciousness during bleeding. In this sense, information about the exact brain parts affected by DTS is not available, and the relationship between changes in different parts of the brain and the anaesthetic plane achieved by means of DTS is not disclosed/demonstrated in the dossier. In addition, scientific literature on focused high energy microwave irradiation of rodent brains involved increases in brain temperatures between 8°C and 12°C above normal within ms of application. In this regard, it is worth noting that the American Veterinary Medical Association (AVMA) guidelines on euthanasia of animals (AVMA, 2020) states that a 10‐kW, 2450‐MHz instrument operated at a power of 9 kW will increase the brain temperature of 18‐ to 28‐g mice to 79°C in 330 ms, and the brain temperature of 250‐ to 420‐g rats to 94°C in 800 ms (Ikarashi et al., 1984).

Although brain temperature has not been measured or reported in cattle, it is implied in the dossier that a brain temperature of 40 C–45 C would be sufficient to induce unconsciousness by DTS, which is unlikely because 40 C is the upper limit of the normal range of core body temperature in cattle (Idris et al., 2024), which is similar to the temperature of the main organs such as the brain in cattle (Godyn et al., 2019).

The dossier states that response to nose prick is a good indicator of consciousness. However, it is stated in the dossier that the interpretation of response to nose pinch/prick is challenging during key stages 1 (after stunning) and 2 (during neck‐cutting) due to the presence of clonic seizure, and during key stage 3 (bleeding) due to agonal movements. Therefore, the assessment of the outcome of application of DTS is based on the occurrence of seizure, and the interpretation of seizure has high uncertainty (tonic/clonic in unconscious animals or stepping behaviour in conscious animals). This interpretation is supported by the fact that grand mal seizures occurring as a consequence of exposure of rats to microwave result in rigidly extended body (tetanic state) lasting for 1 min followed by clonic phase (twitching and jumping) leading to a quiescent period lasting for 4 to 5 min after exposure (Guy & Chou, 1982). From the AW monitoring point of view, it is logical to expect the tonic seizures to last for a period of time after exposure to DTS during which it should be possible to elicit response to nose prick or a painful stimulus.

A major concern is that the historic data presented in this section involved energy levels well above the requested level of 160–200 kJ, therefore most of the text/evidence is invalid.

Loss of posture (LOP): LOP could not be used because the restraint prevents collapse of the forequarter of the animal and hindquarter is obscured by the metal box. LOP cannot be used for monitoring the state of consciousness during or following the application of DTS in restrained animals.

Breathing: All the references cited in the dossier refer to electrical stunning, in which breathing is absent during the tonic phase and the first phase of clonic seizures. In contrast, Small, Mclean, Owen, and Ralph (2013); Small, Mclean, Keates, et al. (2013) reported that animals continued breathing throughout and following the application of DTS. Therefore, the presence of breathing during and following DTS indicates tonic–clonic seizures do not occur or have finished when breathing starts. The EFSA experts argue that breathing can be used for monitoring the state of consciousness, as it is present when the animal is conscious or in light anaesthesia. After the first clonic phase, animals start breathing as they might start to recover consciousness. Apnoea is listed in the flow chart (Figure 17 of the dossier) as an indicator of unconsciousness. Therefore, it is doubtful whether breathing can be used for monitoring the state of consciousness during or following the application of DTS.

Tonic–clonic seizures: In the absence of EEG evidence and due to the presence of continuous breathing, the occurrence of generalised seizures is not valid. Tonic–clonic seizures cannot be used for monitoring the state of consciousness during or following the application of DTS. Similarly, in sheep, physical activity after electrical stunning includes one tonic and two clonic phases (Velarde et al., 2002). In fact, scientific literature on head‐only electrical stunning of cattle, sheep and pigs suggests that delivery of a sufficient current to evoke a grand mal epilepsy in the brain induces tonic seizure (tetanus) lasting for about 10–15 s, followed by clonic seizures lasting for 10–15 s (see EFSA, 2004). In other words, in contrast with DTS, grand mal induced tonic–clonic seizures last for about 30 s following head‐only electrical stunning and this results in the immediate onset of unconsciousness, as proven by EEG. According to the dossier, EEG results suggested that during the tonic phase and the first clonic phase, the animal was unconscious, whereas during the second clonic phase it became conscious again. Occurrence of tonic–clonic seizures is considered to indicate a grand mal epilepsy in the brain. During the tonic phase all the muscles in the body, including those controlling respiration, are in a state of tetanus. It is worth noting that electrical stunning induces tonic seizure lasting for several seconds before it was followed by the clonic phase and animals resumed breathing rhythmically during the onset of the second episode of the clonic phase, and hence regained consciousness. In contrast with this, it is reported in the dossier that cattle continue breathing during and following the application of DTS (Small et al., 2019). This finding suggests that DTS does not induce tonic seizures, and therefore occurrence of a grand mal epilepsy in the brain is ambiguous and is based on behavioural manifestations that are not necessarily indicative of unconsciousness.

Palpebral and/or corneal reflex: The absence of, or ‘lazy’ positive responses is not valid due to the absence of EEG evidence of tonic–clonic seizures and the possibility that these reflexes could be abolished due to overheating of the superficial nerves controlling these reflexes and this aspect has not been ruled out in the dossier. Therefore, palpebral and/or corneal reflex cannot be used for monitoring the state of consciousness during or following the application of DTS.

Spontaneous blinking: Text presented in the dossier overrules the use of this response. It is also possible that rapid blinking occurs as a result of stimulation of the superficial branches of the facial nerve during the application of DTS, but this has not been ruled out in the dossier.

Eye movements: It is stated ‘With effective electromagnetic stunning, the eyeballs will be fixed and staring, or may roll up or down, showing the whites of the eyes. Side‐to‐side movement of the eyes (nystagmus) is associated with ineffective electromagnetic stunning’. Firstly, fixed eye could be an indication of absence seizures, and background literature suggests that pain sensitivity is increased during absence seizures in humans (Velioglu et al., 2018). Secondly, display of eye white is interpreted as an ABM for monitoring pain and fear in cattle rather than a sign of unconsciousness (EFSA AHAW Panel, 2020).

Vocalisation: The dossier reaffirms that vocalisation cannot be used for monitoring the state of consciousness. ‘Snorting’ noises observed during ‘clonic’ seizures are disconcerting from the AW point of view as this behaviour may suggest forced expiration due to pain or struggle.

Muscle tone: The presence of muscle tone during Key Stages 1 and 2, namely after stunning (between the end of stunning and rotation in the restraint) and during sticking (cutting of the brachiocephalic trunk) could be interpreted as a clear indication of failure to induce a generalised seizure leading to unconsciousness due to the application of DTS.

Response to nose pinch/prick: It is stated that this ABM is difficult to interpret due to the presence of ‘clonic’ seizures during Key Stages 1 (after stunning) and 2 (during sticking). Response to nose pinch cannot be used for monitoring the state of consciousness during or following the application of DTS. During the second clonic phase animals are already conscious but insensible to pain.

The interpretation of the EFSA experts is that none of the ABMs listed in the tables and flowchart of the dossier could be reliably used for monitoring the state of consciousness during or following the application of DTS.

• Combination of ABMs used to assess unconsciousness

As stated above, the ABMs could not be used individually to assess the state of unconsciousness because of the uncertainty and lack of direct correlation with the EEG evidence of unconsciousness. In addition, these ABMs have low sensitivity, specificity or feasibility on their own. Therefore, any attempt to use them in various combinations as stated in Table 12 of the dossier is considered futile.

• Assessment of the effectiveness of DTS in relation to unconsciousness and death

Assessment of the effectiveness of DTS overwhelmingly relies on ABMs, rather than EEG data, which limits the robustness of the assessment. As stated previously, these ABMs are unreliable and have low sensitivity, specificity or feasibility (see Table 8), and using them in any combination is unlikely to be fruitful.

If neurotransmitter inhibition is expected to occur due to the application of DTS, then loss of spontaneous firing of neurons and, consequently, occurrence of a profoundly suppressed EEG would be expected, which could be used to determine the time to onset of unconsciousness. In this regard, the time to onset of nadir of EEG suppression is provided by the applicant in PR22 Extra document, which ranges ■■■■■. The disconcerting fact from the AW point of view is that ■■■■■ after the stun‐to‐stick interval of 45 s from the start of energy application recommended (in the dossier). This raises a serious AW concern, i.e. whether cattle are rendered unconscious by the proposed energy levels (160–200 kJ) and duration of exposure (10 s) and whether neck cutting is performed in conscious animals.

The time to onset of death following neck cutting is also an important aspect of AW risk assessment.

• Latency to insensibility

A well‐documented and widely accepted scientific observation is that, during head‐only electrical stunning of cattle, electrical stimulation of the brain with a current of sufficient magnitude for less than 1 second results in generalised epileptiform activity in the brain (involving both cerebral hemispheres and proven by EEG) and this results in the immediate onset of unconsciousness. The onset of epileptiform activity in the brain is associated with certain ABMs: immediate collapse of the animal, tonic–clonic seizures, apnoea (absence of breathing due to a state of tetanus during the tonic seizure), fixed eyes, abolition of corneal reflex and lack of response to nose prick (see EFSA, 2004 for further details). In contrast, there is no EEG evidence of the occurrence of generalised epileptiform activity in the brain following the application of DTS (16–20 kW for 10s) and, owing to this, correlation between EEG and the ABMs is lacking.

In addition, microwave irradiation of rodent brains has been reported to cause inactivation of neurotransmitters, which would be incompatible with the activation of the excitatory neurotransmitter system required for epileptiform activity. Therefore, the claim that DTS leads to tonic–clonic seizure, as in electrical stunning, is physiologically impossible because neurotransmitters are inactivated. The claim made in the dossier that incorporation of electromagnetic field (with microwave stimulation) in DTS leads to tonic–clonic seizure is also unfounded. It is worth mentioning that the original claim was that this combination results in more uniform heating of the brain in rodents (see Ikarashi et al., 1984). The worrying fact from an AW point of view is that two out of six rodents exposed to irradiation in the same publication remained conscious but exhibited seizures.

It is also well‐documented in the scientific literature that captive bolt stunning of cattle leads to immediate collapse of the animal with the onset of tonic seizure, and all the ABMs applicable to electrical stunning are also applicable to effective captive bolt stunning (see EFSA, 2004 for details). However, it is argued in the dossier that captive bolt stunning results in an unaltered EEG for a few seconds before a profoundly suppressed EEG is manifested, and this scenario could be considered equivalent to DTS. In this regard, it is worth noting that loss of posture occurs in cattle about 8 s after the start of application of DTS at 16–20 kW, but this behavioural event is not correlated with the EEG evidence of unconsciousness. The inference is that captive bolt stunning leads to the immediate onset of unconsciousness, but the manifestation of suppressed EEG requires disturbances in the blood circulation to the brain, which could take time (see EFSA, 2004 for details).

It is also argued by the applicant that exposure of pigs and poultry to a gas mixture takes time to induce unconsciousness, and by implication, DTS could be considered equivalent to this method. Exposure to gas mixtures is not a killing method approved for cattle in the killing Regulation, but only for pigs and poultry and it is applied in unrestrained group stunning (see also, EFSA, 2004).

• Aversiveness of the system

The dossier provides a list and description of ABMs used to assess pain, fear and distress (i.e. vocalisation, voluntary movement, escape attempts and injuries). Since the stunning process involves the induction to unconsciousness through brain hyperthermia and the loss of consciousness is not instantaneous but gradual, facial expression should be considered as an ABM for these negative affective states in cattle, especially if there is risk of overheating of the skin of the forehead prior to the loss of consciousness. In the video provided by the applicant ‘PR17 DTS Process (video)’, just after energy application, it can be seen how the animal tightly closes the eyes so the orbital tightening is obviously present, the ears are held stiffly and turned backwards, straining masseter muscles are in clearly visible tension and the chin lift moves towards the ground because the animal is applying a lot of force downwards with the neck. According to the applicant, this is part of a tonic–clonic phase produced by the stunning system on the brain, but this suggestion is not supported by any of the data sent with the dossier.

Additionally, this ear position has been used as an indicator of stress in cattle and sheep (Boissy et al., 2011; Hemsworth et al., 2011) and also as an indicator of heat stress in conscious cattle (Idris et al., 2024). Furthermore, the animal in the video ‘PR18 Video providing supplementary data to SS07’ shows clearly visible eye whites at the application of the stunning system from 00:45 to 00:58, which is an indicator of negative affective states such as fear and pain in cattle (Proctor & Carder, 2015).

It should be noted that in video ‘PR18 Video providing supplementary data to SS07’, at 00:55 (or 0:00:08:11 since the energy application in progress sign appears), smoke or steam can be seen emanating from the head of the bovine. At 01:16 (or 0:00:29:12) spontaneous blinking can be observed, although the return of deliberate blink sign does not appear until 01:41 (or 0:00:55:03). At 01:57 (or 0:01:10:21) positive corneal reflex can be observed, but it is never tested prior to this moment. This spontaneous blinking appearing in one moment while the video annotation states that the animal is not conscious is unclear. In addition, the video does not allow to assess breathing rhythm, and corneal reflex is not assessed. Therefore, it is not possible to confirm, based on the images, that the animal was truly unconscious at that time.

Lastly, it has also not been discussed by the applicant whether the changes in temperature profiles can also be used to predict the time to regain consciousness. In the dossier, it is argued that temperature measurements of the brain in live animals have not been possible because it would require implantation of fibreoptic thermoprobes under general anaesthesia. But it is worth mentioning that brain temperature has been measured in rodents using previously implanted canulae (Butcher & Butcher, 1976). Furthermore, infrared or thermal imaging camera has been used to measure the temperature of the eye during the assessment of heat stress in cattle, and such a non‐invasive technique could have been employed (Idris et al., 2024).

To compare DTS with other authorised stunning methods in cattle, data provided by the applicant were combined with data from a scientific literature review to inform on restraint duration, duration of application and neurological and behavioural indicators of onset of unconsciousness and death, duration of unconsciousness and failure rate in cattle.

Equivalence of DTS with other stunning methods used in cattle is presented in Table 51 of the dossier based on the assumption that DTS induces unconsciousness without causing pain, fear and distress, but sound scientific evidence to support this assumption is lacking. The occurrence of tonic–clonic seizure based on facial expressions (behaviour) and kicking of limbs is presented as evidence to support DTS, but these are considered in the scientific literature to be indicators of pain and fear or attempts to escape from stressful situations in heat‐stressed cattle. Additionally, immediate collapse of the animal based on loss of posture has been well reported following the application of all the stunning methods in this table, except DTS (see EFSA, 2004 Report). Latency to EEG changes presented in this table refers to the time to onset of EEG suppression (12–50 s) following the application of DTS. But the magnitude of EEG suppression required to assume unconsciousness is not clearly defined. In addition, the time to nadir EEG is reported to be as long as 144 s in DTS. It is also worth noting that only insensibility is mentioned in this table and not unconsciousness.

According to the EEG data provided and the proposed energy values of the dossier, EFSA experts consider these EEG data not reliable and/or readable; combined with the information that the animals did not remain unconscious until death, the comparison of DTS with other stunning methods is already unavailing. Nevertheless, it is useful to state some findings and also highlight other challenges and limitations according to the literature search.

The characteristics of the captive bolt device and settings, and the restraining methods used in scientific studies show considerable variability, which should be emphasised. For example, Gibson et al. (2019) employed a pneumatically powered penetrative captive bolt (PCB) gun, while Lücking et al. (2024) utilised two methods: 2160 animals were stunned using a pneumatically powered PCB, and 731 with a cartridge‐powered device. Cartridge‐powered device was reported to be less effective than pneumatically powered (failure rate: 4% vs. 36%) in an audit of Swedish cattle slaughterhouses (Atkinson, 2016). In the study by Vecerek et al. (2020), animals were stunned in a stunning box without mechanical head or body fixation, which may explain the higher failure rate observed compared to Neves et al. (2016), where animals were stunned using both head and body restraint, since angular or positional deviation of more than 20° and/or above 2 cm from the optimal shot place can cause ineffective induction of unconsciousness (Lücking et al., 2024; Vecerek et al., 2020). The higher the deviation of the angle and/or the position in relation to the optimal place, the higher the odds of failure.

Despite the scarce literature on this topic, it seems that PCB causes immediate loss of consciousness (< 1 s) measured by either EEG (transitional pattern) or behavioural (collapse) indicators. According to Gibson et al. (2019), PCB stunned cattle die on average at 12 s post‐stunning with a maximum of 27 s. Failure rate of PCB is reported to be from 0.6% to 9.6% (Endres, 2005; Gregory et al., 2007; Marzin et al., 2008; Troeger et al., 2012; Von Wenzlawowicz et al., 2012; Lücking et al., 2024). Dörfler (2015) mentions 0.9%–1.9%, rising to 5.7% in exceptional cases.

Even if DTS with the proposed values (energy: 160–200 kJ; power: 16–20 kW) induced unconsciousness, it would not be immediate, as it takes several seconds to cause convulsions in bovines.

A comparison between DTS, even with higher energy values than proposed in the dossier, and electrical stunning (including both head‐only and head‐to‐body methods) has not been possible due to the limited literature on welfare assessment in cattle undergoing electrical stunning. Only two scientific studies have evaluated the elapsed time to onset of unconsciousness and death in electrical head‐to‐body stunned cattle. However, both studies focused on calves younger than 1 week of age, and the electrical currents applied were below the minimum required by Regulation 1099/2009 (0.9 A in Blackmore & Newhook, 1982; 1 A in Beausoleil et al., 2024). Consequently, these studies have not been included in the quantitative assessment.

Qualitative assessment

4.2.2

According to information presented in Section 4, both for AW risk assessment (Section 4.1 with its sub‐sections) and for assessment of equivalence of the method with existing stunning methods by quantitative assessment (Section 4.2.1), the available EEG data are not sufficiently reliable for thorough consideration, and the ABMs provided do not demonstrate that cattle remain unconscious until death with the specific proposed energy values. As a result, a robust quantitative comparison of DTS with existing stunning methods is not feasible. However, because quantitative comparisons are generally constrained by limited data and differences in measurement methods, a qualitative comparison of DTS with other stunning methods was undertaken.

Hereto, a non‐formal expert consultation (EFSA, 2014) was used to assess the equivalence of DTS with authorised stunning methods based on a list of hazards that detail the negative welfare impacts of cattle stunning. A full explanation of the methodology used for this qualitative assessment can be found in Section 2.2.3.2 of the methodology.

For the exercise, DTS was compared with three other stunning systems for cattle authorised under Council Regulation (EC) 1099/2009.

A total of 16 hazards for cattle were assessed by five experts from the EFSA ad hoc WG. From these, the hazards considered highly relevant were selected (see Section 2.2.3.2). The WCs of these highly relevant hazards were discussed in order to compare DTS with other authorised stunning methods.

The experts agreed on the final scores of most of the hazards, with only one expert differing in several cases due to a different interpretation of hazard prevalence. After discussion, the group reached consensus on the final scores for each method and on whether DTS could be considered equivalent to other stunning methods. The experts evaluated whether DTS ensures that cattle are spared of avoidable pain, distress or suffering during the killing and whether the method maintains the loss of consciousness and sensibility until death at least at the same level with at least one of the authorised methods and as described in the killing regulation.

All the experts agreed that comparing different stunning methods is challenging, especially when some methods have not been extensively studied. However, they also agreed that an evaluation starting from the welfare implications for the animals was still possible.

Comparison of DTS with other stunning methods

The selected highly relevant hazards and the results of the comparison of DTS with other stunning methods are shown in Table 27.

• Hazards specific for Penetrative Captive Bolt

Incorrect stun position and inappropriate stunning parameters are highly relevant hazards for Penetrative Captive Bolt.

The correct position of the Penetrative Captive Bolt should target the brain. Incorrect position will lead to persistence of consciousness and, as a consequence, to pain and fear. When inappropriate stunning parameters are used, there is a risk of producing painful injury to the head with the animal being conscious.

• Hazard specific for head‐only and head‐to‐body electrical stunning

Pre‐stunning shocks are a highly relevant hazard for both head‐only and head‐to‐body electrical stunning. They can produce a painful stimulus by triggering nociceptors without affecting the animal's conscious state.

• Hazards specific to DTS

Wet skin and energy leakage are two hazards identified only in DTS. Energy leakage can cause overheating of non‐target surface areas of the animal and ineffective stunning, resulting in negative affective states such as pain and fear. Wet skin increases the risk of burning the skin, leading to pain. Across STEP 11 (PR12) and STEP 13 studies (3.2, STEP 13.1), stun ineffectiveness was calculated as 1.5% (3/195). The three failures were due to an incorrectly positioned applicator, causing the system to shut down. Since reporting of STEP 11, out of 262 animals exposed to energy application of 200 kJ or less (PR24), 7 incidents of energy leakage and automatic shut‐down were recorded (2.7%). Three of these incidents (1.1%) resulted in energy applications of less than 95 kJ and did not induce the tonic–clonic response. Four animals receiving between 95 and 128 kJ did enter the tonic–clonic phase, but duration of unconsciousness was shorter than desired. In these cases, back‐up stunning was performed using captive bolt.

• Too short exposure time

Too short exposure time is a common hazard for DTS and electrical head‐only, which results in persistence of consciousness and thus WCs.

• Common hazards to all methods

The DTS stunning system and authorised stunning systems present at least three common hazards: prolonged stun‐to‐stick interval, ineffective sticking and sticking of conscious animals.

A delay between the end of stunning and sticking poses a risk of the animal regaining consciousness either prior to neck cutting or during exsanguination, which could lead to pain, fear and distress.

Unconscious cattle are bled out by using two methods: (i) cutting both the carotid arteries and jugular veins just behind the mandibles (known as neck cutting); or (ii) cutting the brachiocephalic artery at the thoracic inlet (known as chest sticking). The risk of an animal regaining consciousness during bleeding is well known (see EFSA, 2004) following neck cutting when bleed out is hindered due to the formation of false aneurism at the severed end of the carotid arteries (known as carotid occlusion). Under this situation, blood flow to the brain via the vertebral artery is increased, in worst case scenario, when compared with animals that do not have false aneurism or those animals subjected to cutting brachiocephalic trunk (chest sticking) (Anil, McKinstry, Gregory, et al., 1995; Anil, McKinstry, Wotton, & Gregory, 1995) and, consequently, there are increased risks of recovery of consciousness. The vertebral artery originates from the subclavian artery which in turn originates from the common carotid artery. This hazard is eliminated following chest sticking because severing brachiocephalic artery involves making the cut before the origin of vertebral artery (caudal to the origin of vertebral artery). Neck cutting is described as the norm following the application of DTS in cattle, which raises potential AW concern associated with this procedure (see EFSA, 2004 and Von Holleben et al., 2010 for further details).

When sticking of conscious animals occurs, they feel pain. The onset of unconsciousness is almost immediate in electrical stunning (Von Holleben et al., 2010; Weaver & Wotton, 2009) and mechanical stunning. According to the dossier, DTS induces a gradual consciousness as brain temperature increases due to a critical level. The welfare concern is that the critical brain temperature required to induce unconsciousness in cattle is not known or disclosed in the dossier. In addition, the time taken to achieve this critical brain temperature and whether it can be achieved without causing negative affective states such as pain, fear and distress remain to be demonstrated. It is also reported in the dossier that DTS, delivering 160–220 kJ at an incident power of 18–20 kW induces loss of consciousness, as evidenced by the tonic–clonic behavioural response within 3 (tonic) to 11 (tonic–clonic) s of onset of energy application. The problem is that the evidence submitted to date does not support this assumption, i.e. occurrence of tonic–clonic seizure. For example, changes in facial expression, such as tightening of muscles and rapid blinking of eyelids, are used as a sign of tonic seizure, but literature suggests that tonic seizure associated with unconsciousness is manifested as tetanus involving all the muscles, including respiratory muscles. Therefore, tonic seizure would be expected to cause cessation of breathing, i.e. induce apnoea, but the dossier reveals that cattle continue to breathe during and following DTS application. In the absence of reliable EEG evidence of unconsciousness, any interpretation of behavioural manifestation involves high uncertainty.

Furthermore, unlike the other stunning methods used in cattle, there is a high level of uncertainty regarding the effectiveness of DTS in producing unconsciousness in the animal.

The system itself is affecting the brain from the most superficial to the deepest area, so it is possible that the absence of the corneal reflex (false positive) may be misleading, as it may be the result of damage to the fifth or seventh cranial nerves, the cornea or the muscles controlling the eyelid, which may occur due to the proximity of the applicator, rather than due to loss of consciousness, leading to high uncertainty. This interpretation is supported by the fact that Small et al. (2019) reported that administration of 45.87 kJ for 1.5 s, which is substantially less than the energy and duration of exposure required to reach critical brain temperature, to a cattle (animal 1.14, Table 2 of this paper) resulted in abolition of the corneal reflex and loss of response to painful stimulus (nose prick), with eyes fixed and staring, but the animal did not collapse (i.e. remained standing).

More importantly, the claim that application of DTS induces tonic seizures within 3 s is based on changes in facial expressions such as tightening of muscles. By contrast, some of these signs, especially tightening of facial muscles, have been used as indicators of negative affective states such as pain and fear in other species of animals as stated previously. The claim that clonic seizures occur within 8 s of DTS application is also misleading because kicking has been reported to occur in conscious cattle exposed to heat stress and is a sign of animals attempting to escape from a stressful situation, as stated previously.

Additionally, although EEG suppression has been reported to occur in anaesthetised cattle subjected to DTS, the magnitude of suppression required to assume unconsciousness is missing in the dossier. If nadir (i.e. strongest suppression) in the EEG is considered an indicator of unconsciousness, application of DTS takes up to 144 s to induce this state. If the recommended stun‐to‐stick interval is 45 s, then the animals are likely to remain conscious during neck cutting.

Lastly, as mentioned earlier, it has been reported that two out of six rodents exposed to microwave irradiation of the brain remained conscious (unstunned) despite their brain temperature being 45°C. Therefore, the assumption that heating cattle brain via DTS to 40–45°C will be sufficient to induce thermal unconsciousness carries huge uncertainty because 40°C is the upper value of the range of normal brain temperature. In addition, the brain temperature, at which syncope is expected to occur, is not unequivocally demonstrated.

Main result of the comparison of DTS with other stunning methods

Based on the evidence available, the EFSA experts concluded that it is very unlikely that DTS applied to cattle at 160–200 kJ energy fulfils the requirements for the maintenance of unconsciousness until death. For all the above reasons, DTS has not been documented sufficiently to be at least equivalent with one of the authorised stunning methods.

CONCLUSIONS

5

General summary of the results of the assessment of the Diathermic Syncope® procedure

5.1

1. The assessment of the state of consciousness after the DTS application is not possible due to the poor quality of the EEG data provided and the lack of validity of the used ABMs of behaviour and physical reflexes (e.g. tonic–clonic seizures).
2. The quality of EEG data is poor due to the background noise and animal movement artefacts. Therefore, there is insufficient EEG evidence of epileptiform activity in the brain after application of 160–200 kJ of energy for 10 s to conduct scientific validation.
3. The ABMs of behaviour and physical reflexes used to assess unconsciousness following DTS application in key stage 1 (after stunning) and key stage 2 (during neck‐cutting) are invalid for ascertaining the state of consciousness as they have low sensitivity, specificity and feasibility. For example, according to current knowledge, tightening of the facial muscles and kicking can indicate pain, fear and escape attempts from a stressful situation and not necessarily tonic or clonic seizures.
4. Other ABMs, such as rapid blinking and arched back, are interpreted by the applicant as indicators of the onset of tonic–clonic seizures suggestive of unconsciousness, but these have neither been correlated with the EEGs, nor scientifically validated during DTS usage.
5. In the absence of scientifically sound data, induction of unconsciousness by the application of DTS at 160–200 kJ of energy for 10 s is speculative. The occurrence of bradycardia prior to the onset of EEG changes indicates that cattle exposed to these parameters may experience pain.
6. Skin burn occurrence in conscious animals and associated pain cannot be ruled out.
7. Exsanguination is the likely cause of unconsciousness and death in DTS‐stunned animals, but the times to onset of these are not known. Therefore, it is likely that neck cutting is performed in conscious, but severely restrained and inverted animals.
8. It is known that false aneurysms/carotid occlusion lead to increased blood flow to the brain of cattle, in worst case scenario, when compared with animals that do not have false aneurism or those animals subjected to cutting brachiocephalic trunk, but the WCs of this have not been clarified. Occurrence of false aneurysms/carotid occlusion following neck cutting will prolong the time to loss of consciousness and onset of death.
9. The applicant has not provided sound scientific evidence on the following:

• The brain temperature at which syncope is expected to occur in cattle;
• The duration to achieve the threshold temperature required to induce syncope in cattle during the application of DTS at energy levels of 16–20 kW;
• The impacts/consequences of the presence of carotid rete and blood supply to the brain via the vertebral artery in cattle in terms of maintaining homeothermy in the brain and delaying the onset of unconsciousness;
• The variability in a cattle population of maintaining homeothermy in the brain due to age, sex and breed;
• The duration of the syncope in cattle after delivering 16–20 kW energy levels;
• How quickly the brain temperature returns to normal either prior to neck cutting due to continuous blood supply after the termination of DTS with 16–20 kW or during exsanguination in case carotid occlusion occurs;
• How the increased brain temperature needed for syncope is maintained throughout exsanguination until death following the application of 16–20 kW energy;
• Significant WCs such as heatstroke‐type responses and skin burns and also mis‐stunning;
• EEG parameters/criteria for determining syncope and their correlation with the clinical signs of syncope that could be used as ABMs for monitoring purposes;
• The clinical signs of recovery from syncope in cattle.

Extent to which the proposed method and the information provided with the application meet the eligibility criteria of EFSA's guidelines (ToR 1)

5.2

• 1The name of the method was provided.
• 2Description of the method including potential sources of pain, distress and/or suffering was provided.
• 3Key parameters required for the use of the method were provided, but neither the criteria used to derive these parameters nor their effectiveness in terms of inducing unconsciousness were demonstrated unequivocally.
• 4The scientific basis of induction and maintenance of unconsciousness for the method is described using data from rodents, but sound scientific evidence is lacking for cattle.
• 5Potential causes of system failure and chances of occurrence have been identified.
• 6Individual studies submitted partially fulfil the eligibility criteria of EFSA's guidelines. Specifically, there were some deficiencies regarding the method of measurement of the outcomes and the reporting of outcomes and estimations (see also Table 4).
• 2Scientific publications provided by the applicant contained information to partially fulfil the requirements for assessment phase one (eligibility criteria of EFSA guidelines). Submitted papers had inadequacies, notably either failed to record EEGs sufficiently in order to be interpreted properly or failed to correlate neurological measures with the behavioural ABMs (see also Point 9 in Section 5.1 for issues where the applicant did not provide clarifications).
• 3The dossier lacked detailed data on the identified hazards and the duration of their occurrence.
• 4The submitted dossier lacked comprehensive welfare outcome data (neurological, physiological, behavioural and/or physical reflexes) for the DTS method across the proposed types of cattle. Although the pathophysiology of heatstroke is described in the literature, it has not been elucidated in this context.
• 5The dossier contained implementation of the method on a pilot scale, aiming at but not achieving external validity because the assessment of the state of consciousness was carried out using poor‐quality EEG data and ABMs of behaviour and physical reflexes not validated for DTS.
• 6The dossier provided sufficient information to perform assessment phase two (risk assessment of the proposed method), particularly about pain, distress or suffering caused to cattle exposed to DTS.

Extent to which Diathermic Syncope® can provide a level of animal welfare at least equivalent to that ensured by the existing methods in the legislation (ToR 2)

5.3

Diathermic Syncope® (DTS), as described and parameterised by the applicant (160–200 kJ, 16–20 kW, 10 s), does not provide a level of animal welfare at least equivalent to one or more methods currently listed for cattle in Annex I of Regulation (EC) 1099/2009. DTS very likely fails to induce unconsciousness and/or maintain unconsciousness until death.

A qualitative comparison was considered necessary because EEG data were unreliable, and ABMs did not show that cattle remain unconscious until death at the proposed DTS settings.

Head‐only electrical stunning reliably induces generalised epileptiform activity and immediate unconsciousness in cattle, as confirmed by EEG and associated ABMs. In contrast, DTS shows no EEG evidence of such activity, and the ABMs used cannot be equated with those of effective electrical stunning.

Does the Diathermic Syncope® method ensure that cattle are spared of avoidable pain, distress or suffering during the killing, to what extent and under which conditions? (ToR 2i)

5.3.1

According to the evidence provided, DTS applied at 160–200 kJ, 16–20 kW, for 10 s does not ensure that cattle are spared avoidable pain, distress or suffering throughout the procedure of killing, particularly during the induction phase.

Pain, distress or suffering cannot be excluded for the following reasons:

• Grand mal epilepsy cannot be confirmed from the EEG, as the characteristic high‐amplitude, low‐frequency activity is absent from the data provided, contradicting the behavioural interpretation of seizure‐like movements.
• Claims that forehead temperature does not rise while animals are conscious are unsupported, as unconsciousness cannot be verified without valid EEG data, making these statements scientifically unsubstantiated. Due to lacking EEG evidence, it cannot be determined whether skin burns occur after the loss of consciousness, which means painful heating may occur while animals remain conscious.
• Behavioural signs reported during DTS (ear position, eye whites, stepping) resemble heat stress and/or pain responses observed in conscious cattle, supporting the possibility that painful heating occurs in conscious animals.
• The risk of skin burns during DTS application cannot be excluded, as previous studies documented full‐thickness forehead burns and tissue necrosis under comparable microwave exposures, indicating that heating can exceed safe thresholds.
• Poor tuning of the DTS applicator is acknowledged to exacerbate inappropriate surface heating.
• DTS method uniquely presents risks of wet skin and energy leakage, which may cause skin burns, overheating and ineffective stunning, as demonstrated by reported leakage incidents and stun failures. In addition, too short exposure time is a hazard for DTS because insufficient duration prolongs the period during which animals can experience negative WCs.
• The DTS method is likely aversive because facial expressions, eye whites, ear position and other behaviours in the submitted videos resemble established ABMs of pain and fear, and the dossier provides no valid EEG evidence that these signs represent tonic–clonic seizures.
• The effectiveness of DTS cannot be established because EEG evidence and EEG & ABM correlations are absent, making it impossible to confirm unconsciousness or its duration, and movements interpreted as seizures may reflect conscious motor activity, indicating risk of pain, distress and suffering.

Does the Diathermic Syncope® method maintain the loss of consciousness and sensibility until the death of cattle, to what extent and under which conditions? (ToR 2ii)

5.3.2

According to the evidence and the specific application parameters provided in the dossier, there is a high uncertainty associated with the induction of unconsciousness by the method and, if it does, whether the method maintains loss of consciousness and sensibility until the death of cattle.

It is very likely that DTS does not induce unconsciousness or maintain the loss of consciousness and sensibility until the death of cattle, for the following reasons:

• The ABMs of corneal and palpebral reflexes cannot be reliably used to assess the state of consciousness because of uncontrolled eyelid twitching and likely blood contamination, which impair reliable elicitation. In addition, these ABMs were not correlated with EEG indications of unconsciousness.
• Occurrence, onset and duration of unconsciousness induced by DTS (applied at 160–200 kJ, 16–20 kW, for 10 s) cannot be reliably established because available evidence (brain temperature data, EEG suppression timing and species‐specific brain cooling mechanisms) shows inconsistent or delayed effects relative to the claimed stun‐to‐stick interval.
• DTS cannot be confirmed to induce unconsciousness via thermal or neurotransmitter‐inhibition/overstimulation mechanisms, because brain temperature thresholds that induce unconsciousness are unknown in cattle, species‐specific brain cooling likely limits heating, and EEG suppression as a sign of unconsciousness could not be detected after brain heating or occurred too late after brain heating in most animals.
• No association or correlation between EEG indicators and ABMs can be established in relation to the state of consciousness, because EEG during DTS application was unavailable and post‐exposure EEG is obscured by artefacts, preventing validation of behavioural or physical reflex measures as indicators of unconsciousness.
• None of the ABMs provided can reliably assess unconsciousness after DTS application, because breathing persists, EEG evidence is largely unreadable and behavioural indicators are ambiguous or confounded by seizures, heat effects or restraint. Combining ABMs does not improve assessment reliability, since each ABM lacks sensitivity, specificity or feasibility.
• The ABMs used (behavioural indicators and physical reflexes) cannot verify unconsciousness in DTS‐stunned cattle, as tonic–clonic seizures are unsubstantiated without EEG confirmation and observed movements may reflect pain and/or fear (e.g. attempted escape) rather than epileptic activity during DTS application.

DOCUMENTATION AS PROVIDED TO EFSA

6

Diathermic Syncope®. May 2023.

First additional data and updated dossier. November 2023.

Second additional data and updated dossier. May 2024.

Third additional data and updated dossier. September 2025. Submitted by ■■■■■

ABBREVIATIONSABManimal based measureACTHadrenocorticotropic hormoneAHAW PanelEFSA Panel on Animal Health and WelfareASPCAAmerican Society for the Prevention of Cruelty to AnimalsAWAnimal WelfareCAScontrolled atmosphere stunningCO_2_ carbon dioxideCPcreatine phosphateDAFSDirect Animal Food SafetyD_p_ penetration depthDTSDiathermic Syncope®ECoGelectrocorticographyEEGelectroencephalographyF50median frequencyF9595% spectral edge frequencyHPA axishypothalamic–pituitary–adrenal (hpa) axisIASPInternational Association for the Study of PainLAPSlow atmospheric pressure stunningLOPloss of postureNAnot availableN_2_ nitrogenNB Nota bene NADHnicotinamide adenine dinucleotideNEFSnitrogen expansion foam stunningNGOsNon‐Government OrganisationsNPCBnon‐penetrative captive boltPCBpenetrative captive boltPRproject reportPtottotal powerSIInternational System of UnitsSOScientific OpinionSOPStandard Operating ProcedureSUPsupplementary materialSSscientific studyToRTerm of ReferenceWCWelfare ConsequenceWGWorking GroupWoSWeb of Science

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00085

COPYRIGHT FOR NON‐EFSA CONTENT

EFSA may include images or other content for which it does not hold copyright. In such cases, EFSA indicates the copyright holder and users should seek permission to reproduce the content from the original source.

PANEL MEMBERS

Søren Saxmose Nielsen, Julio Alvarez, Anette Boklund, Sabine Dippel, Fernanda Dorea, Jordi Figuerola, Mette S. Herskin, Miguel Angel Miranda Chueca, Eleonora Nannoni, Romolo Nonno, Anja B. Riber, Karl Stahl, Jan Arend Stegeman, Hans‐Hermann Thulke, Frank Tuyttens, Virginie Michel, Christoph Winckler.

LEGAL NOTICE

Relevant information or parts of this scientific output have been blackened in accordance with the confidentiality requests formulated by the applicant pending a decision thereon by the European Food Safety Authority. The full output has been shared with the European Commission and the applicant. The blackening will be subject to review once the decision on the confidentiality requests is adopted by the European Food Safety Authority.