Safety and efficacy of a feed additive consisting of Macleaya cordata (Willd.) R. Br. extract and leaves (Sangrovit® Extra) for suckling and weaned piglets and other growing Suidae (Phytobiotics Futterzusatzstoffe GmbH)

TL;DR

This study evaluates the safety and effectiveness of a plant extract feed additive for piglets and other growing pigs.

Contribution

The paper provides a scientific assessment of Sangrovit® Extra's safety and efficacy as a zootechnical feed additive for pigs.

Findings

The additive is safe for target species at recommended levels but raises concerns for consumer safety due to DNA-reactive compounds.

The additive may irritate eyes and could potentially be a respiratory sensitiser.

The additive is considered safe for the environment and may improve piglet performance.

Abstract

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of Macleaya cordata (Willd.) R. Br. extract and leaves (Sangrovit® Extra) as a zootechnical feed additive for suckling and weaned piglets and other growing Suidae. The additive is standardised to contain a concentration of the sum of the four alkaloids sanguinarine, chelerythrine, protopine and allocryptopine of 1.25%, with 0.5% sanguinarine. Owing to the presence of the DNA intercalators sanguinarine and chelerythrine, a concern for genotoxicity was identified. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) had no safety concerns for the target species when the additive is used at the recommended level of 0.750 mg sanguinarine/kg complete feed for suckling and weaned piglets and other growing Suidae. Since in all consumer categories the exposure to sanguinarine and chelerythrine via the use of Sangrovit® Extra exceeds the threshold of toxicological concern of 0.0025 μg/kg bw per day for DNA reactive mutagens and/or carcinogens, the FEEDAP Panel could not conclude on the safety for the consumers. The additive was shown to be irritant to the eyes but not irritant to skin or a skin sensitiser. The FEEDAP Panel could not exclude the potential of the additive to be a respiratory sensitiser. When handling the additive, exposure of unprotected users to sanguinarine and chelerythrine may occur. Therefore, to reduce the risk, the exposure of users should be reduced. The use of Sangrovit® Extra as a feed additive under the proposed conditions of use was considered safe for the environment. The additive Sangrovit® Extra had the potential to be efficacious in improving performance of weaned piglets at 0.600 mg sanguinarine/kg complete feed. This conclusion was extended to suckling piglets and extrapolated to other growing Suidae.

Tables

TABLE 1. Residue data of sanguinarine, chelerythrine, protopine and allocryptopine in tissues (skin + fat, kidney, liver and muscle) and excreta samples collected at the end of the efficacy study (after 70 days) from the animals administered with the maximum recommended level (0.750 mg SG/kg feed). The results are presented as the mean (SD) for n ≥ 6 or mean (range) when n < 6.

Sample	Sanguinarine μg/kg	Chelerythrine μg/kg	Protopine μg/kg	Allocryptopine μg/kg
Skin+fat	1.38 (0.70) ■■■■■	■■■■■ ■■■■■	< 0.5 (n = 3)	n.d.
Kidney	0.63 ■■■■■ ■■■■■	■■■■■ ■■■■■	n.d.	n.d.
Liver	1.13 (0.62) ■■■■■	■■■■■ ■■■■■	0.59 (< 0.5–1.10) (n = 4)	< 0.5 (n = 1)
Muscle	0.7 ■■■■■	■■■■■ ■■■■■	< 0.5 (n = 6)	< 0.5 (n = 8)

TABLE 2. Input values used to calculate consumer exposure to sanguinarine and chelerythrine residues (mg/kg) in mammal tissues (pigs).

	Input values sanguinarine + chelerythrine mg/kg wet tissue
Mammal fat tissue	0.00368
Mammal liver	0.00261
Mammal meat a	0.00150
Mammal offal and slaughtering products (other than liver)	0.00165

TABLE 3. Chronic exposure of consumers to sanguinarine and chelerythrine residues based on residue data in pig tissues.

Population class	Highest exposure estimate a ng/kg bw/day	% TTC value b
Infants	10.53	421
Toddlers	13.18	527
Other children	14.64	586
Adolescents	11.95	478
Adults	9.18	367
Elderly	6.38	255
Very elderly	5.96	239

TABLE 4. Input values used to calculate consumer exposure to protopine and allocryptopine residues (mg/kg) in mammal tissues (pigs).

	Input values protopine + allocryptopine mg/kg wet tissue
Pigs
Mammal fat tissue	0.00025
Mammal liver	0.00135
Mammal meat a	0.00045
Mammal offal and slaughtering products (other than liver)	n.d.

TABLE 5. Chronic exposure of consumers to protopine and allocryptopine residues based on residue data in pig tissues.

Population class	Highest exposure estimate a ng/kg bw per day	% TTC value b
Infants	2.79	0.19
Toddlers	3.64	0.24
Other children	3.96	0.26
Adolescents	3.11	0.21
Adults	2.30	0.15
Elderly	1.63	0.11
Very elderly	1.59	0.11

TABLE 6. Trial design and use level of the efficacy trials performed in weaned piglets.

Trial	Total no animals (animals × replicate) replicates × treatment	Breed sex (duration, days)	Feed composition (form)	Groups
				mg Sangrovit® extra/kg complete feed	mg sanguinarine/kg complete feed		mg chelerythrine/kg complete feed
					Intended	Analysed*	Intended	Analysed*
1 29	42 (2) 7	DanBred × Piétrain 1:1 ♂/♀ (42)	Maize, wheat and soybean meal (mashed)	0 60 120	0 0.29 0.58	< 0.05 0.39 0.81	0 0.22 0.45	< 0.05 0.16 0.25
2 30	80 (5) 8	DanBred × Piétrain 1:1 ♂/♀ (42)	Maize, wheat and soybean meal (mashed)	0 120	0 0.67	< 0.05 0.95	0 0.53	< 0.05 0.29
3 31	120 (15) 2	DanBred × Piétrain 1:1 ♂/♀ (42)	Wheat, soybean meal and maize (mashed)	0 90 120 150	0 0.49 0.65 0.81	< 0.05 0.48 0.57 0.74	0 0.24 0.32 0.40	< 0.05 0.19 0.22 0.25
4 32	1440 (24) 12	DanBred 1:1 ♂/♀ (42)	Wheat, soya and barley (mashed and pelleted)	0 60 90 120 150	0 0.33 0.49 0.65 0.81	< 0.05 0.27 0.43 0.59 0.72	0 0.16 0.24 0.32 0.40	< 0.05 0.19 0.27 0.33 0.47

TABLE 7. Effects of Sangrovit® Extra on the zootechnical performance of weaned piglets.

Trial	Groups (mg Sangrovit® extra/kg complete feed)	Feed intake 1 (kg or kg/day)	Final body weight (kg)	Weight gain 2 (kg or g/day)	Feed‐to‐gain ratio	Mortality and culling (%)
1	0	25.9	24.1	17.7a	1.46a	0
	60	26.1	24.6	18.2b	1.43b	0
	120	26.1	25.1	18.7b	1.40c	7.1
2	0	0.69	26.6	480a	1.46a	0
	120	0.73	28.5	520b	1.40b	2.5
3	0	0.71	25.8a	442a	1.62a	0
	90	0.72	26.2ab	453ab	1.59ab	3.3
	120	0.74	26.9b	470b	1.56b	0
	150	0.75	27.2b	478b	1.57b	3.3
4	0	0.72	25.7	464.9a	1.53a	3.5
	75	0.73	26.2	477.9ab	1.52ab	2.1
	90	0.73	26.4	483.7ab	1.50ab	2.1
	120	0.72	26.7	488.5b	1.48b	2.4
	150	0.73	26.7	491.4b	1.49b	2.1

Full text

INTRODUCTION

1

Background and Terms of Reference

1.1

Regulation (EC) No 1831/20031 establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lays down that any person seeking authorisation for a feed additive or for a new use of feed additive shall submit an application in accordance with Article 7.

The European Commission received a request from Phytobiotics Futterzusatzstoffe GmbH2 for the authorisation of the additive consisting of Macleaya cordata extract (Sangrovit® Extra), when used as a feed additive for suckling and weaned piglets and other growing Suidae (category: zootechnical additive; functional group: other zootechnical additives).

According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the application to the European Food Safety Authority (EFSA) as an application under Article 4(1) (authorisation of a feed additive or new use of a feed additive). EFSA received directly from the applicant the technical dossier in support of this application. The particulars and documents in support of the application were considered valid by EFSA as of 20 March 2023.

According to Article 8 of Regulation (EC) No 1831/2003, EFSA, after verifying the particulars and documents submitted by the applicant, shall undertake an assessment in order to determine whether the feed additive complies with the conditions laid down in Article 5. EFSA shall deliver an opinion on the safety for the target animals, consumer, user and the environment and on the efficacy of the feed additive consisting of Macleaya cordata extract (Sangrovit® Extra), when used under the proposed conditions of use (see Section 3.1.1).

Additional information

1.2

The additive under assessment, Sangrovit® Extra, consists of Macleaya cordata extract and leaves. The FEEDAP Panel issued an opinion on the safety and efficacy of Sangrovit® Extra for all poultry species (excluding laying and breeding birds) (EFSA FEEDAP Panel, 2023a).

It has not been previously authorised as a feed additive in the European Union.

DATA AND METHODOLOGIES

2

Data

2.1

The present assessment is based on data submitted by the applicant in the form of a technical dossier3 in support of the authorisation request for the use of Macleaya cordata extract and leaves (Sangrovit® Extra) as a feed additive. The dossier was received on 08/06/2022 and the general information and supporting documentation is available at https://open.efsa.europa.eu/questions/EFSA‐Q‐2022‐00357.

The confidential version of the technical dossier was subject to a target consultation of the interested Member States from 20 March 2023 to 20 June 2023 for which the received comments were considered for the assessment.

In accordance with Article 38 of the Regulation (EC) No 178/20024 and taking into account the protection of confidential information and of personal data in accordance with Articles 39 to 39e of the same Regulation, and of the Decision of EFSA's Executive Director laying down practical arrangements concerning transparency and confidentiality,5 a non‐confidential version of the dossier has been published on Open.EFSA.

According to Article 32c(2) of Regulation (EC) No 178/2002 and to the Decision of EFSA's Executive Director laying down the practical arrangements on pre‐submission phase and public consultations, EFSA carried out a public consultation on the non‐confidential version of the technical dossier from 2 February to 23 February 2024 for which no comments were received.

The FEEDAP Panel used the data provided by the applicant together with data from other sources, such as previous risk assessments by EFSA or other expert bodies, peer‐reviewed scientific papers, other scientific reports and experts' knowledge, to deliver the present output.

EFSA has verified the European Union Reference Laboratory (EURL) report as it relates to the methods used for the control of the sanguinarine (SG) in Sangrovit® Extra and in premixtures and feedingstuffs.6

Methodologies

2.2

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of Macleaya cordata extract and leaves (Sangrovit® Extra) is in line with the principles laid down in Regulation (EC) No 429/20087 and the relevant guidance documents: Guidance on the assessment of the safety of feed additives for the consumer (EFSA FEEDAP Panel, 2017a), Guidance on the identity, characterisation and conditions of use of feed additives (EFSA FEEDAP Panel, 2017b), Guidance on the assessment of the safety of feed additives for the target species (EFSA FEEDAP Panel, 2017c), Guidance on the assessment of the efficacy of feed additives (EFSA FEEDAP Panel, 2018), Guidance on the assessment of the safety of feed additives for the environment (EFSA FEEDAP Panel, 2019), Guidance on the assessment of the safety of feed additives for the users (EFSA FEEDAP Panel, 2023b), Statement on the genotoxicity assessment of chemical mixtures (EFSA Scientific Committee, 2019a) Guidance on the use of the Threshold of Toxicological Concern approach in food safety assessment (EFSA Scientific Committee, 2019b) and General approach to assess the safety for the target species of botanical preparations which contain compounds that are genotoxic and/or carcinogenic (EFSA FEEDAP Panel, 2021).8

ASSESSMENT

3

The additive under assessment (Sangrovit® Extra) consists of a M. cordata (Willd.) R. Br. extract and M. cordata processed leaves. It is intended for use as a zootechnical additive (functional group: other zootechnical additives) in feed for suckling and weaned piglets and other growing Suidae.

Characterisation

3.1

The additive and the active substances were fully characterised in a previous opinion of the FEEDAP Panel (EFSA FEEDAP Panel, 2023a). The applicant stated that no changes in the manufacturing or composition of the additive have been introduced since the previous assessment.

The additive Sangrovit® Extra consists of M. cordata extract (■■■■■) and M. cordata processed leaves (■■■■■).

The additive is formulated with whole wheat flour (■■■■■), lignosulfonate (■■■■■) and sorbic acid (■■■■■) in order to achieve a target concentration of the sum of four M. cordata alkaloids of 1.25%, with 0.5% SG (selected as the marker substance).

The additive is specified to contain a minimum of 4000 mg SG/kg additive and a maximum 7000 mg SG/kg additive. Analysis of 10 batches of the additive (manufactured in 2019–2023) showed compliance with these specifications ■■■■■.9

All data pertaining to composition, purity, physicochemical properties and stability described thereof are considered valid also for this application (EFSA FEEDAP Panel, 2023a).

Conditions of use

3.1.1

The additive is intended for use in feed for suckling and weaned piglets and other growing Suidae up to the age of 120 days or a body weight of 35 kg to provide a minimum SG content in complete feed of 0.600 mg/kg feed and a maximum SG content of 0.750 mg/kg feed. These levels are achieved with varying amount of the additive ranging between 90 and 150 mg Sangrovit® Extra/kg complete feed, depending on the SG concentration in the additive between 4000 and 7000 mg SG/kg Sangrovit® Extra.

Safety

3.2

The applicant submitted the same data set that was already evaluated by the FEEDAP Panel in its previous assessment (EFSA FEEDAP Panel, 2023a). This included: (i) a structured literature search on the absorption, distribution, metabolism and excretion (ADME) of the alkaloids present in M. cordata, and the safety of the additive; (ii) toxicological studies, including genotoxicity, sub‐chronic toxicity studies and studies aimed at demonstrating the safety of the additive for the user (skin and eye irritancy and skin sensitisation).

The previous conclusions on the ADME and toxicology of the M. cordata alkaloids, SG, chelerythrine (CH), protopine (PRO) and allocryptopine (ALL), are briefly summarised below.

In rat and in food‐producing animals, SG and CH are poorly absorbed as such after oral administration. The two compounds are extensively metabolised in the gut mainly by reductive reactions. The resulting dihydrometabolites are absorbed, metabolised in the liver to conjugated derivatives and excreted via bile to the intestine and subsequently in faeces. Very low levels of the compounds or their metabolites were found in plasma, urine and in tissues. The biotransformation of SG and CH was similar in rat liver fractions and in human hepatocytes. PRO and ALL are absorbed in the rat to a greater extent than SG and CH, distributed in various tissues, extensively metabolised after absorption, and excreted in urine and faeces.

Although genotoxicity studies performed with M. cordata extract (in vitro) and with the formulated additive Sangrovit® Extra (in vitro and in vivo) gave negative results, based on the available information on individual components of the extract, the FEEDAP Panel concluded that SG ‘has the potential to (i) intercalate between DNA base pairs; (ii) induce DNA strand breaks; (iii) and also induce DNA strand breaks associated with oxidative damage and the formation of reactive oxygen species (ROS). Therefore, SG has the potential to produce ROS in proximity of the double helix of DNA and to induce oxidative DNA damage. Based on the structural similarity with SG, the same conclusions apply to CH’. For the other two alkaloids, the FEEDAP Panel concluded that ‘a direct interaction of PRO with DNA is not supported by the experimental data available. Based on the structural similarity between PRO and ALL, this conclusion can be extended to ALL’. Overall, a concern for genotoxicity was identified owing to the presence of the DNA intercalators SG and CH.

From a 90‐day study with Sangrovit® Extra in Wistar rats,10 the FEEDAP Panel identified a no observed adverse effect level (NOAEL) for SG of 95 mg/kg feed (corresponding to 7.7 mg/kg body weight (bw) per day) (EFSA FEEDAP Panel, 2023a).

In the next sections, only the data in pigs are described. These include ADME studies retrieved from the literature, a residue study and a tolerance study in weaned piglets conducted with the additive under assessment (Sangrovit® Extra).

Absorption, distribution, metabolism and excretion in pigs

3.2.1

In the publication from Wu et al. (2020), which describes an in vitro metabolic study and an in vivo pharmacokinetics study of SG in pigs, incubation of SG with microsomes prepared from intestinal mucosa (added with nicotinamide adenine dinucleotide phosphate, NADPH), cytosol (added with nicotinamide adenine dinucleotide, NADH) and gut flora of pigs showed its reductive biotransformation to dihydrosanguinarine (DHSG). When pigs were administered a single oral dose of SG at 0.1 mg/kg bw, plasma SG and DHSG reached C max (3.41 and 2.41 μg/L, respectively) at 2.75 h. For SG and DHSG, the area under the curve (AUC) was 15.6 and 9.1 μg h/L, and the half‐life was 2.33 h and 2.20 h, respectively. A repeated dose administration was also carried out by orally giving to six pigs SG at a dose of 0.1 mg/kg bw three times a day, each dose interval of 8 h, for three consecutive days. Blood samples were collected at several time points up to 24 h post the last dose. AUC for SG and DHSG were 31 and 13 μg h/L, respectively, higher than after a single dose. Also, plasma C max was higher after repeated doses, 5.9 and 2.9 μg/L for SG and DHSG, respectively, although attained at a similar T max as for single dose (2.6 h). T 1/2 of SG and DHSG was 3.2 h and 2.4 h, respectively.

In the study from Kosina et al. (2004), three groups of pigs (three males and three females per group) were fed a standard diet (control group) or a diet added with an extract of M. cordata at 2 or 100 mg extract/kg feed,11 (SG:CH of 3:1) for 90 days. The lower concentration tested contained 1.28 mg SG/kg feed and 0.44 mg CH/kg feed and the higher contained 64 mg SG/kg feed and 22 mg CH/kg feed. Blood was collected at day zero, and throughout the experiment at days 30, 60, and 90, and faeces at day 90. On day 91, the animals were killed and liver, muscle, gingiva, tongue, stomach and intestines collected for analysis of SG and CH by high‐performance liquid chromatography (HPLC) with diode array and/or fluorimetric detectors (LOD: 0.001 μg/mL). SG was present in plasma in a concentration‐dependent manner (4 and 108 μg/L for the low and high diet concentration); CH was not detected in plasma of the low concentration group, being present at 24 μg/L in the high concentration group. Plasma levels were constant throughout the experiment. The highest levels of SG in organs/tissues for both diet concentrations were reported in the gingiva (79 and 514 μg/kg), followed by the intestine (15 and 124 μg/kg), liver (13 and 113 μg/kg), tongue (10 and 32 μg/kg) and stomach (7 and 52 μg/kg). In the low concentration diet group, CH was only detected in the gingiva and liver (36 and 5 μg/kg, respectively) and in the gingiva, intestine and liver (50, 49 and 40 μg/kg, respectively) in the high concentration group. Both SG and CH were not detected in the muscle. The method of analysis did not include the search for metabolites. The very high contents of both compounds in faeces (1180 μg SG/kg and 842 μg CH/kg at the lowest diet concentration; 16,110 μg SG/kg and 8412 μg CH/kg at the highest diet concentration) indicate that this is the principal route of excretion.

The metabolism of SG was studied in microsomes and cytosol of pig liver in the presence of NADPH (Zhang et al., 2013). After incubation, the supernatant was analysed by liquid chromatography ion trap/time of flight mass spectrometry (LC‐IT/TOFMS). In microsomes, seven metabolites were identified, while DHSG was the only metabolite identified in cytosol. The reduction of the iminium bond and O‐demethylenation of SG were the main metabolic elucidated pathways. The amount of DHSG formed in liver cytosol was higher than that in liver microsomes in the presence of NADPH. This indicates that cytosolic enzymes are mainly responsible for the reduction of SA in the pig liver. The hydroxylated metabolites if formed in vivo are expected to be conjugated and excreted.

Conclusions

3.2.1.1

In pigs fed a diet with M. cordata extract for 90 days, the gingiva showed the highest contents of SG followed by the intestine, liver, tongue and stomach. CH was only detected in the gingiva, intestine and liver. Both SG and CH were not detected in the muscle. Based on the high contents of both compounds in faeces together with their absence in the muscle, the FEEDAP Panel concludes that faecal excretion is the principal route. The results from the studies in pigs are consistent with the results in laboratory animals described in a previous opinion (EFSA FEEDAP Panel, 2023a).

Residue study in pigs

3.2.2

The applicant submitted an efficacy study in weaned piglets, from which some information on residues of the four alkaloids SG, CH, PRO and ALL can be obtained (see Section 3.3.1).

Tissue samples were taken from eight weaned piglets per treatment (four males/four females).12 The tissues collected at the end of the study (after 70 days) were muscle and skin+fat in natural proportion and organs (liver and kidneys) from the not supplemented (control) group and from the group fed with the maximum recommended level (0.750 mg SG/kg feed). All alkaloids were quantified by ultraperformance liquid chromatography tandem mass spectrometry (UPLC–MS/MS).

In samples from the control group, ■■■■■

The results of the analyses of the samples of tissues and organs of the animals fed with the maximum recommended level are summarised in Table 1.13

TABLE 1: Residue data of sanguinarine, chelerythrine, protopine and allocryptopine in tissues (skin + fat, kidney, liver and muscle) and excreta samples collected at the end of the efficacy study (after 70 days) from the animals administered with the maximum recommended level (0.750 mg SG/kg feed). The results are presented as the mean (SD) for n ≥ 6 or mean (range) when n < 6.

■■■■■

■■■■■14

PRO was below the LOQ in all samples, except in four liver samples and it was detectable in few skin+fat (n = 3) ad muscle samples (n = 6). ALL was < LOQ in all samples and was detectable in one liver sample and in all muscle samples (n = 8).15

Safety for the target species

3.2.3

According to the General approach to assess the safety for the target species of botanical preparations which contain compounds that are genotoxic and/or carcinogenic (EFSA FEEDAP Panel, 2021), genotoxicity and carcinogenicity endpoints are not considered relevant for short‐living animals. Short‐living animals are defined as those animals raised for fattening whose lifespan under farming conditions makes it very unlikely to develop cancer as a result of the exposure to genotoxic and/or carcinogenic substances in the diet. Therefore, for these species, the safety evaluation of additives containing substances which are genotoxic and carcinogenic can be based on the outcome of the tolerance trials in the target species (EFSA FEEDAP Panel, 2021). In the context of this assessment, the definition of short‐living animals includes growing pigs and other growing Suidae but does not include pigs and Suidae reared for reproduction which are considered to be long‐living/reproductive animals and for which genotoxicity and carcinogenicity endpoints are considered relevant.

Tolerance trial in weaned piglets

3.2.3.1

To support the safety for the target species, the applicant submitted a tolerance‐efficacy study with the additive under assessment.16

A total of 800 hybrid17 weaned piglets (26 days old, initial body weight ≈ 6.1 kg; 50%♀: ♂) coming from four consecutive batches were categorised by body weight, distributed in 80 single‐sex pens of 10 animals each, and randomly allocated to five groups (16 replicates per group, eight of each sex).

Two basal diets (pre‐starter, from day 1 to 14; starter from day 15 to 42) based on maize, soybean meal and wheat were either not supplemented (control) or supplemented with the additive to provide 0.45 (0.6× maximum proposed use level), 0.60 (0.8×), 0.75 (1×) and 15 (20×) mg SG/kg complete feed (equivalent to 90, 120, 150 or 3000 mg Sangrovit® Extra/kg complete feed).18 The diets were offered ad libitum as pellets for 42 days.

Mortality and health status were checked every day and the most likely reason of death/culling provided. The animals were individually weighed at the start of the trial. Thereafter, the body weight and the feed intake were recorded on days 14 and 42. The average daily feed intake, average daily weight gain and feed‐to‐gain ratio were calculated and corrected for mortality for the overall period. Blood samples were obtained from two randomly selected piglets per pen on day 42 for haematology and biochemistry analysis.19

The zootechnical data were analysed with a general linear model with the weaning batch and the diet as fixed effects and the initial body weight as a covariate, including the pen as the experimental unit. For the blood haematology and biochemistry, a generalised linear model was used, with the diet as a fixed effect and the animal as the experimental unit. When differences were observed, group means were compared with Tukey test. The significance level was set at 0.05. The piglets' zootechnical data were subjected to a non‐inferiority test. For that purpose, the lower limits of the 95% confidence interval of the 1× and 20× diets mean minus the control were compared with the established margins for each parameter (body weight = −1.73 kg; average daily weight gain = −30.46 g; average daily feed intake = −40.83 g; feed‐to‐gain ratio = 0.055). The significance level was set at 0.05.

The overall mortality and culling rate were 5.4% on average (4.5%–6.3%) and no statistical difference was observed between groups. The inclusion of the additive up to 20× the maximum proposed use level showed no significant differences in any of the zootechnical performance and blood haematology parameters recorded in comparison with the control. Regarding the blood biochemistry data, the 0.6×, 0.8× and 1× groups showed lower serum cholesterol content compared to the control; the creatine kinase serum concentration was significantly decreased in the 1× group; and the serum C‐reactive protein and potassium concentration and the lactate dehydrogenase (LDH) activity were higher in the 0.8× group compared to the control. The differences observed in the biochemistry parameters were small and not dose related. Therefore, those were not considered relevant for the safety assessment.

Based on the confidence intervals obtained in the non‐inferiority test for body weight (−0.66 to 1.01), average daily weight gain (−18.84 to 24.52), average daily feed intake (−16.77 to 36.23) and feed‐to‐gain ratio (−0.03 to 0.06), the 20× group was not inferior to the control group.

To complement the safety for the target species, the applicant provided six published studies involving the dietary supplementation of weaned piglets either with M. cordata extracts or SG. All the studies showed limitations that prevent using them as evidence for the safety of growing pigs. Four of them20 included no overdose levels (maximum level applied of 0.75 mg SG/kg complete feed), recorded a limited number of endpoints relevant for the safety (no data on blood haematology/biochemistry or gross pathology) and two of them had a short duration (21 days). One of the studies21 was designed as a challenge trial in which all the animals from all groups were inoculated with Salmonella Typhimurium. The other study22 included one group supplemented with a high level of SG (up to 53 times the content supplied in the maximum proposed use level); however, the Panel notes that the test item used might not be representative of Sangrovit® Extra, the duration of the study was limited to 14 days, and no blood data or gross pathology evaluation were monitored. In any case, no adverse effect was observed in any of the parameters evaluated in growing pigs in any of the trials reported.

Conclusions on safety for the target species

3.2.3.2

Based on the results of the tolerance trial in weaned piglets, the Panel concludes that Sangrovit Extra® is safe for weaned piglets at 0.750 mg SG/kg complete feed (achieved with 150 mg Sangrovit® Extra/kg complete feed) with a wide margin of safety. This conclusion is extended to suckling piglets for the period in which solid feed is given and other growing Suidae up to an age of 120 days or a body weight of 35 kg.

The Panel notes that these conclusions do not cover pigs and minor porcine species reared for reproduction, which are considered long‐living/reproductive animals.

Safety for the consumer

3.2.4

Assessment of consumer exposure

The FEEDAP Panel performed an exposure assessment following the methodology described in the Guidance on consumer safety (EFSA FEEDAP Panel, 2017a) (Appendix A).

The residue values for SG and CH from the residue study in weaned piglets (see Section 3.2.2) were used as input data for the exposure calculation and are reported in Table 2. For SG, quantified in ■■■■■, the arithmetic mean plus two standard deviations was calculated. For SG in ■■■■■ the highest value was considered. CH was quantified ■■■■■ (the highest analysed value was used). ■■■■■ where CH was detected (> LOD) but not quantified (< LOQ), a value corresponding to half the LOQ was used.

The values of chronic exposure of consumers to SG + CH for the different population categories are reported in Table 3.

In the absence of a reference point for SG and CH, the estimated exposure can be related to the value that, according to threshold of toxicological concern (TTC) approach, is considered of low probability of adverse health effects (EFSA Scientific Committee, 2019b). For substances that have the potential to be DNA reactive mutagens and/or carcinogens, the TTC value corresponds to 0.0025 μg/kg bw per day (equal to 2.5 ng/kg bw per day). Table 3 reports the estimated exposure related to the TTC of 2.5 ng/kg bw per day.

When considering the residue data in pig tissues, the estimated 95th percentile ranged between 2.31 and 14.64 ng/kg bw per day. The highest exposure at the 95th percentile occurs in the category 'other children'.

For all consumer categories, exposure in the individual countries was constantly above the TTC value of 2.5 ng/kg bw per day. Above this threshold, a non‐TTC approach (e.g. substance‐specific risk assessment) is required in order to reach a conclusion on potential adverse health effects.

Based on the available data, the FEEDAP Panel cannot conclude on the safety of the additive for the consumers.

The Panel notes that there was an application for the authorisation of this additive in growing poultry. When considering the combined exposure of consumers to SG and CH residues from pig and poultry tissues,23 the estimated exposure24 was comparable to the exposure from pig tissues only. The FEEDAP Panel notes that consumer exposure to SG + CH is essentially due to the consumption of pig tissues, in particular to the consumption of fat.

The residue values for PRO and ALL from the residue study in weaned piglets (see Section 3.2.2)25 were used as input data for the exposure calculation and are reported in Table 4. PRO was quantified only in four liver samples (the highest value was considered). In the other samples, where PRO was detected (> LOD) but not quantified (< LOQ), a value corresponding to half the LOQ was used. PRO and ALL were not detected in the kidney; ALL was not detected in fat.

Chronic exposure of consumers to PRO and ALL for the different population categories are reported in Table 5.

In the absence of a reference point for PRO and ALL, the estimated exposure can be related to the value that, according to TTC approach, is considered of low probability of adverse health effects (EFSA Scientific Committee, 2019b). For substances belonging to Cramer class III, the TTC value corresponds to 1.5 μg/kg bw per day (equal to 1500 ng/kg bw per day). For all consumer categories, exposure in the individual countries was consistently below 4 ng/kg bw per day, corresponding to < 0.26% of the value of TTC. Below this threshold, the probability of adverse effects is low.

When considering the residue data in pig tissues, the 95th percentile ranged between 0.007 and 3.96 ng/kg bw per day. The highest exposure at the 95th percentile occurs in the category 'other children'.

When considering the combined exposure of consumers to PRO and ALL residues from pig and poultry tissues,26 the estimated exposure27 was more than doubled compared to the exposure from pig tissues only and was comparable to the exposure calculated for poultry tissues only (EFSA FEEDAP Panel, 2023a). The FEEDAP Panel notes that consumer exposure to PRO + ALL is essentially due to the consumption of poultry liver. However, since consumer exposure is < 1% of the TTC value for Cramer Class III compounds, the probability of adverse effects is low.

Conclusions on safety for the consumer

In all consumer categories, the exposure to SG and CH exceeds the threshold of toxicological concern of 0.0025 μg/kg bw per day (equal to 2.5 ng/kg bw per day) for DNA reactive mutagens and/or carcinogens. Therefore, the FEEDAP Panel cannot conclude on the safety of Sangrovit® Extra for the consumers.

Safety for the user

3.2.5

The safety for the user was previously evaluated (EFSA FEEDAP Panel, 2023a, 2023b). The FEEDAP Panel is not aware of any additional data or information that would lead to a change of the conclusions previously reached. The additive is considered irritant to the eyes but not to the skin. The additive is not a skin sensitiser; however, the FEEDAP Panel cannot exclude the potential of the additive to be a respiratory sensitiser. When handling the additive, exposure of unprotected users to SG and CH may occur. Therefore, to reduce the risk, the exposure of users should be minimised.

Safety for the environment

3.2.6

M. cordata is a natural occurring plant primarily distributed in temperate areas in North America and eastern Asia. It was introduced in Europe in the late part of the 18th century, where it is widely distributed as an ornamental garden plant.

SG, the main component of M. cordata extract, is widely distributed in nature and can be found in plants of the families Papaveraceae, Fumariaceae and Rutaceae.

The use of Sangrovit® Extra in animal nutrition at the proposed conditions of use is not expected to increase the concentration of SG and other alkaloids in the environment, and therefore is considered of no concern for the environment.

Efficacy

3.3

Efficacy in weaned piglets

3.3.1

The applicant submitted five trials aiming at supporting the efficacy of Sangrovit® Extra to improve the performance of weaned piglets.

One of the trials28 showed a non‐optimal animal health status (reflected by the outspread of colibacillosis in some pens of the control group and a mortality rate up to 9.5% in the group supplemented with 90 mg Sangrovit® Extra/kg complete feed). Owing to this limitation, the study was not further considered for the assessment of the efficacy.

The other four trials shared a similar design (see Table 6). In all trials, the animals were fed two basal diets (starter, days 1–14; grower days 15–42), either not supplemented (control) or supplemented with Sangrovit® Extra from 60 to 150 mg/kg complete feed. The level of Sangrovit® Extra in the experimental feeds was checked based on the analysis of SG and CH as primary and secondary markers, respectively. In all trials, the experimental feeds were offered ad libitum for 42 days. The first trial included an external marker in the grower diet to measure the apparent ileal digestibility of phosphorus and total amino acids.

In all trials, the mortality and health status were checked every day. The animals were individually weighed at the start of the trial. Thereafter, the body weight and feed intake were recorded on a pen basis once a week and the average feed intake, average weight gain and feed‐to‐gain ratio were calculated and corrected for mortality for the overall period.

The experimental data were analysed with a generalised linear model with the pen as the experimental unit and the diet as the fixed effect. When differences were observed, group means were compared with Tukey test. The significance level was set at 0.05.

The results of the four trials are presented in Table 7. Mortality values were within commercial standards in all trials, with no difference between groups. In all trials, the piglets that received Sangrovit® Extra at the minimum use level (120 mg Sangrovit® Extra/kg complete feed) showed improved performance (higher body weight gain and better feed‐to‐gain ratio) in comparison with the control. In trial 3, the piglets supplemented with the proposed use level also showed a higher final body weight. In trial 1, the results of the digestibility trial showed that the apparent ileal digestibility of phosphorus and total amino acids was significantly higher in the group supplemented with 120 mg Sangrovit® Extra/kg complete feed. These results support the findings observed in the zootechnical data.

Conclusions on efficacy

3.3.2

The additive has the potential to be efficacious for weaned piglets at the minimum use level of 0.600 mg SG/kg complete feed (achieved with 120 mg Sangrovit® Extra/kg complete feed). This conclusion is extended to sucking piglets for the period in which solid feed is given and extrapolated to other growing Suidae up to an age of 120 days or a body weight of 35 kg.

Post‐market monitoring

3.4

The FEEDAP Panel considers that there is no need for specific requirements for a post‐market monitoring plan other than those established in the Feed Hygiene Regulation33 and Good Manufacturing Practice.

CONCLUSIONS

4

The following conclusions apply to Sangrovit® Extra, which consists of a M. cordata extract (0.5%–1.2%) ■■■■■ and of M. cordata processed leaves (30%–64%) and is formulated to contain a concentration of the sum of the four alkaloids (SG, CH, PRO and ALL) of 1.25%, with 0.5% SG (0.4%–0.7%).

Owing to the presence of the DNA intercalators SG and CH, a concern for genotoxicity is identified.

Based on the results of the tolerance trial in weaned piglets, the FEEDAP Panel has no safety concerns for the target species when the additive Sangrovit® Extra is used up to the maximum recommended level of 0.750 mg SG/kg complete feed for weaned piglets. This conclusion was extended to suckling piglets and extrapolated to other growing Suidae. The Panel notes that these conclusions do not cover pigs and minor porcine species reared for reproduction, which are considered long‐living/reproductive animals.

Since in all consumer categories the exposure to SG and CH exceeds the threshold of toxicological concern of 0.0025 μg/kg bw per day for DNA reactive mutagens and/or carcinogens, the FEEDAP Panel cannot conclude on the safety of Sangrovit® Extra for the consumers.

The additive is irritant to the eyes but not irritant to skin or a skin sensitiser. The FEEDAP Panel cannot not exclude the potential of the additive to be a respiratory sensitiser. When handling the additive, exposure of unprotected users to SG and CH may occur. Therefore, to reduce the risk, the exposure of users should be reduced.

The use of Sangrovit® Extra as a feed additive under the proposed conditions of use is considered safe for the environment.

The additive Sangrovit® Extra has the potential to be efficacious in improving performance of weaned piglets at 0.600 mg SG/kg complete feed. This conclusion is extended to sucking piglets for the period in which solid feed is given and other growing Suidae up to the age of 120 days or a body weight of 35 kg.

RECOMMENDATIONS

5

The specification for SG in Sangrovit® Extra should not exceed 0.7%. The specifications for the sum of the four alkaloids should not exceed the highest analysed concentration (1.4%).ABBREVIATIONSALLallocryptopineAUCarea under the curvebwbody weightCHchelerythrineDHSGdihydrosanguinarineEURLEuropean Union Reference LaboratoryFEEDAPEFSA Scientific Panel on Additives and Products or Substances used in Animal FeedHGBhaemoglobinHPLChigh‐performance liquid chromatographyLC‐IT/TOFMSliquid chromatography ion trap/time of flight mass spectrometryLODlimit of detectionLOQlimit of quantificationMCHmean corpuscular haemoglobinMCHCmean corpuscular haemoglobin concentrationMCVmean corpuscular volumeNADHnicotinamide adenine dinucleotideNADPHnicotinamide adenine dinucleotide phosphateNOAELno observed adverse effect levelPROprotopineRBCred blood cell countSGsanguinarineTTCthreshold of toxicological concernUPLC–MS/MSultraperformance liquid chromatography tandem mass spectrometry

ACKNOWLEDEGMENTS

The Panel wishes to thank the following for the support provided to this scientific output (in alphabetical order of the last name): Jaume Galobart, Matteo Lorenzo Innocenti, Fabiola Pizzo and Maria Vittoria Vettori.

CONFLICT OF INTEREST

If you wish to access the declaration of interests of any expert contributing to an EFSA scientific assessment, please contact interestmanagement@efsa.europa.eu.

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2022‐00357

COPYRIGHT FOR NON‐EFSA CONTENT

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PANEL MEMBERS

Vasileios Bampidis, Giovanna Azimonti, Maria de Lourdes Bastos, Henrik Christensen, Mojca Durjava, Birgit Dusemund, Maryline Kouba, Marta López‐Alonso, Secundino López Puente, Francesca Marcon, Baltasar Mayo, Alena Pechová, Mariana Petkova, Fernando Ramos, Roberto Edoardo Villa, and Ruud Woutersen.

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 EFSA. The full output has been shared with the European Commission, EU Member States (if applicable) and the applicant. The blackening may be subject to review once the decision on the confidentiality requests is adopted by EFSA and in case it rejects some of the confidentiality requests