Assessment of the feed additive consisting of red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017 for salmon and trout for the renewal of its authorisation (ENEOS Techno Materials Corporation)

TL;DR

This paper evaluates the safety of a red carotenoid additive for salmon and trout, concluding it is safe under authorized conditions but raises concerns about consumer exposure.

Contribution

The paper provides a scientific assessment for the renewal of authorization of a specific carotenoid-rich additive in aquaculture.

Findings

The additive is safe for target species, consumers, and the environment under authorized conditions.

Consumer exposure to canthaxanthin and adonirubin exceeds acceptable levels in certain population groups.

The additive is a dermal and respiratory sensitiser, posing risks via skin or respiratory exposure.

Abstract

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017 for salmon and trout (category: sensory additives; functional group: colourants; substances which when fed to animals add colours to food of animal origin) for the renewal of its authorisation. The applicant provided evidence that the additive complies with the conditions of the authorisation. The Panel concludes that the use of the additive in salmon and trout remains safe for the target species, the consumer and the environment under the authorised conditions of use. When assessing consumer exposure to canthaxanthin and adonirubin at the level of the existing maximum residue limits (MRL) for poultry and the proposed MRL for trout/salmon (5 mg/kg muscle), the exposure of consumers exceeds the acceptable daily intake (ADI) in the population classes toddlers and other children. The Panel considers that there is no need to restrict the use of the additive to fish older than 6 months or of more than 50 g. Red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017 is not irritant to the skin, but is irritant to the eyes. It is considered a dermal and respiratory sensitiser and any exposure via skin or the respiratory tract is a risk.

Tables

TABLE 1. Carotenoid profile (g/kg) of red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017.

	Specification	Average	Range
Astaxanthin	20–23	21.68	21.2–22.0
Adonirubin	7–15	8.5	7.6–9.4
Canthaxanthin	1–5	2.04	1.8–2.3
Adonixanthin		2.74	2.2–3.5
Echinenone		0.68	0.5–0.9
beta‐Carotene		0.44	0.2–0.6
Asteroidenone		0.42	0.3–0.5
3‐Hydroxyechinenone		0.24	0.1–0.4
Others		0.1	< 0.1–0.1
Total carotenoids (TC)		36.8	35.1–38.0

TABLE 2. Proximate composition (% as is) of red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017.

	Average	Range
Crude protein	41.1	37.1–43.4
Crude ash	21.9	20.1–24.5
Crude fat	2.9	2.7–3.2
Crude fibre	< 0.1	< 0.1
Carbohydrates	5.6	5.0–5.9
Moisture	2.8	2.5–2.9

TABLE 3. Description of the main carotenoids in red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017.

	Chemical abstracts service (CAS) number	Molecular formula	Molecular weight (g/mol)
Astaxanthin (3,3′‐dihydroxy‐β,β‐carotene‐4,4′‐dione)	472–61‐7	C40H52O4	597
Canthaxanthin (β‐carotene‐4,4′‐dione)	514–78‐3	C40H52O2	565
Adonirubin (3‐hydroxy‐ββ‐carotene‐4,4′‐dione)	4418‐72‐8	C40H52O3	581

TABLE 4. Concentration of astaxanthin, canthaxanthin and adonirubin in muscle of rainbow trout fed the additive for 12 weeks.

	Astaxanthin	Canthaxanthin	Adonirubin
Average (mg/kg) ± SD	3.30 ± 1.95	0.43 ± 0.21	1.87 ± 1.02
Average + 2SD	7.20	0.85	3.91

TABLE 5. Input values used to calculate consumer exposure to carotenoid residues from red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017.

	Astaxanthin (mg/kg tissue)	Canthaxanthin + adonirubin (mg/kg tissue)
Fish (meat)	7.20	4.76

TABLE 6. Chronic dietary exposure to carotenoid residues from red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017 calculated based on residue data in trout.

Population class	Astaxanthin		Canthaxanthin + adonirubin
	Highest exposure estimate a (mg/kg bw per day)	% ADI b	Highest exposure estimate a (mg/kg bw per day)	% ADI c
Infants	0.0153	8	0.0101	34
Toddlers	0.0413	21	0.0273	91
Other children	0.0286	14	0.0189	63
Adolescents	0.0203	10	0.0134	45
Adults	0.0170	9	0.0112	37
Elderly	0.0158	8	0.0104	35
Very elderly	0.0120	6	0.0079	26

TABLE 7. Chronic dietary exposure to canthaxanthin + adonirubin residues from red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017 calculated based on existing MRLs in trout (8 mg/kg muscle) and salmon (10 mg/kg muscle).

Population class	Input value: 8 mg/kg muscle		Input value: 10 mg/kg muscle
	Highest exposure estimate a (mg/kg bw per day)	% ADI b	Highest exposure estimate a (mg/kg bw per day)	% ADI b
Infants	0.0170	56.7	0.0213	70.9
Toddlers	0.0459	152.9	0.0573	191.1
Other children	0.0318	106.1	0.0398	132.6
Adolescents	0.0226	75.2	0.0282	94.0
Adults	0.0188	62.8	0.0235	78.5
Elderly	0.0175	58.4	0.0219	73.0
Very elderly	0.0133	44.4	0.0166	55.4

TABLE 8. Chronic dietary exposure to canthaxanthin + adonirubin residues from red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017 calculated based on proposed MRL for trout/salmon.

Population class	Input value: 5 mg/kg muscle
	Highest exposure estimate a (mg/kg bw per day)	% ADI b
Infants	0.0106	35.5
Toddlers	0.0287	95.5
Other children	0.0199	66.3
Adolescents	0.0141	47.0
Adults	0.0118	39.2
Elderly	0.0109	36.5
Very elderly	0.0083	27.7

TABLE 9. Input values used to calculate consumer exposure to canthaxanthin + adonirubin from all sources (fish and poultry).

	Canthaxanthin + adonirubin (mg/kg tissue)
Fish (meat)	5
Birds fat/skin tissue	2.5
Birds liver	15
Whole eggs	8.1

TABLE 10. Chronic dietary exposure to canthaxanthin + adonirubin residues calculated based on proposed MRL in trout/salmon and existing MRLs in poultry.

Population class	Highest exposure estimate a (mg/kg bw per day)	% ADI b
Infants	0.0295	98
Toddlers	0.0385	128
Other children	0.0426	142
Adolescents	0.0248	83
Adults	0.0176	59
Elderly	0.0175	58
Very elderly	0.0186	62

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 14(1) of that Regulation lays down that an application for renewal shall be sent to the Commission at the latest 1 year before the expiry date of the authorisation.

The European Commission received a request from JX Nippon ANCI SAS (Europe) on behalf of JXTG Nippon Oil & Energy Corporation2 for renewal of the authorisation of the product red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017 for salmon and trout (category: sensory additives; functional group: colourants; substances which when fed to animals add colours to food of animal origin).

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 14(1) (renewal of the authorisation). The dossier was received on 22 August 2017 and the general information and supporting documentation are available at https://open.efsa.europa.eu/questions/EFSA‐Q‐2017‐00694. The particulars and documents in support of the application were considered valid by EFSA as of 20 November 2017.

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 product red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017, when used under the proposed conditions of use (see Section 3.1.5).

Additional information

1.2

The additive, a natural source of red carotenoids containing as main colouring principle astaxanthin, adonirubin and canthaxanthin, is currently authorised for salmon and trout (2a(ii)167).3 The FEEDAP Panel adopted two opinions on the safety and efficacy of Panaferd®‐AX (red carotenoid‐rich bacterium Paracoccus carotinifaciens NITE SD 00017) (EFSA, 2007; EFSA FEEDAP Panel, 2010).

DATA AND METHODOLOGIES

2

Data

2.1

The present assessment is based on data submitted by the applicant in the form of a technical dossier4 in support of the authorisation request for the use of red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017 (Panaferd®‐AX) as a feed additive.

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 and other scientific reports 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 astaxanthin, adonirubin and canthaxanthin in animal feed and marker residue in muscle tissue of salmon and trout.5

Methodologies

2.2

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of red carotenoid‐rich Paracoccus carotinifaciens (Panaferd®‐AX) is in line with the principles laid down in Regulation (EC) No 429/20086 and the relevant guidance documents: Guidance on the renewal of the authorisation of feed additives (EFSA FEEDAP Panel, 2021).

ASSESSMENT

3

The additive red carotenoid‐rich Paracoccus carotinifaciens is currently authorised as a sensory additive (functional group: colourants; substances which when fed to animals add colours to food of animal origin) for use in feed for salmon and trout. The present application is for the renewal of the current authorisation. The additive will be referred to in this opinion with its trade name Panaferd®‐AX.

Characterisation

3.1

Manufacturing process

3.1.1

The additive is obtained via fermentation with the strain Paracoccus carotinifaciens NITE SD 00017. The information submitted regarding the manufacturing process lists some modifications applied which have been developed since the first authorisation was granted.7 ^,^ 8 ■■■■■9 The applicant declared that no antimicrobials are used in the manufacturing process.10

Characterisation of the strain

3.1.2

The strain under assessment was isolated from soil and is deposited in the National Institute of Technology and Evaluation (NITE) of Japan with accession number NITE SD 00017.11 The strain is not genetically modified.

The taxonomic identification of the strain NITE SD 00017 was performed using ■■■■■12 ■■■■■13 the FEEDAP Panel considers that the identification of NITE SD 00017 as P. carotinifaciens is confirmed.

The susceptibility of the strain to the antimicrobials recommended by the FEEDAP Panel (EFSA FEEDAP Panel, 2018) was tested by broth microdilution following the Clinical and Laboratory Standards Institute (CLSI) performance standards ■■■■■ All the minimum inhibitory concentration (MIC) values determined were equal or fell below the corresponding cut‐off values for Enterobacteriaceae, except for fosfomycin which was exceeded by five dilutions (256 mg/L vs. 8 mg/L).14 Therefore, P. carotinifaciens NITE SD 00017 is considered susceptible to all relevant antibiotics except for fosfomycin.

The WGS data of the production strain were queried for the presence of genes coding for antimicrobial resistance (AMR) ■■■■■15 No hits of concern were identified. Although the strain was resistant to fosfomycin, since no acquired AMR genes were found in the genome, this resistance does not raise safety concerns.

Antimicrobial activity was measured in culture supernatants of the strain by a broth microdilution method.16 No inhibitory activity was detected against the 18 reference strains used, Acinetobacter baumannii NCTC 12156, A. baumannii ATCC 17978, Bacillus subtilis NC08236, B. subtilis ATCC 6633, Escherichia coli ATCC 25922, E. coli ATCC 35218, Enterococcus faecalis NCTC 12697, E. faecalis NCTC 12203, Klebsiella pneumoniae NCTC 13368, K. pneumoniae NCTC 13439, Pseudomonas aeruginosa BAA 2110, P. aeruginosa ATCC 27853, Proteus vulgaris ATCC 6380, P. mirabilis ATCC 7002, Staphylococcus aureus NCTC 12981, S. aureus ATCC 29213, S. aureus BAA 2312 and Streptococcus pneumoniae ATCC 49619.

Characterisation of the additive

3.1.3

The additive is a preparation containing dried killed cells of Paracoccus carotinifaciens NITE SD 00017 and calcium carbonate. The additive is specified by the authorising regulation to contain 20–23 g/kg astaxanthin, 7–15 g/kg adonirubin and 1–5 g/kg canthaxanthin. The analysed values from five batches (Table 1) showed compliance with the specifications.

Three of the above batches were analysed to provide information on the proximate composition (Table 2).

In the same three batches, lead concentration ranged from 0.08 to 0.17 mg/kg, cadmium ranged from 0.02 to 0.03 mg/kg while arsenic and mercury were below their limit of quantification (LOQ)17 as well as mycotoxins18 (aflatoxins B1, B2, G1 and G2, fumonisins B1 and B2, nivalenol, deoxynivalenol, zearalenone, patulin, ochratoxin A, citrinin, sterigmatocystin, penicillic acid).19

Polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and coplanar dioxin‐like polychlorinated biphenyls (Co‐planar PCBs) were analysed in three other batches. The calculated (lower bound) levels of the sum of dioxins and dioxin‐like‐PCBs ranged from 0.028 to 0.062 pg WHO‐PCDD/F‐PCB‐TEQ/kg.20

The FEEDAP Panel considers that the amounts of the detected impurities do not raise safety concerns.

Five other batches were assessed for microbiological contamination. Total aerobic count was between 2.40 × 10^4^ and 2.90 × 10^5^ CFU/g, coliform bacteria was absent in 10 g samples and Salmonella spp. was not detected in 25 g samples.21 The FEEDAP Panel notes that the values found regarding the total aerobic count are high and may deserve attention/monitoring during the production process.

Lipopolysaccharides (LPS) are present in most Gram‐negative bacteria's outer membrane and are typically recognised by Toll‐like receptor 4 (TLR4) present on the surface of phagocytic cells. The applicant provided analytical data for the determination of LPS content in five batches of the additive.22 The results of the chromogenic Limulus Amebocyte Lysate (LAL) assay averaged 1.50 × 10^6^ EU/g23 (range 0.76 × 10^6^–2.90 × 10^6^ EU/g). Moreover, the LPS of P. carotinifaciens NITE SD 00017 pure culture were extracted, purified and characterised (in terms of composition and potential for cytokines induction).24 Based on the results of silver‐staining SDS‐PAGE, GS‐MS and MALDI‐TOF‐MS, it was determined that the strain's LPS have low molecular weight and that its Lipid A is penta‐acylated.25 The strain's LPS and their Lipid A potential to induce inflammatory cytokines was investigated in two different cell lines and the results showed lower cytokine induction activity compared with the Escherichia coli O55 LPS and comparable to a TLR4 antagonist (ultrapure commercial extract of LPS from Rhodobacter sphaeroides).26 ^,^ 27 Based on the biological activity data, it is concluded that the LPS of the P. carotinifaciens NITE SD 00017 LPS trigger low TLR4‐mediated cytokine release.

The presence of viable cells of the production strain was investigated in three batches, in triplicate. One gram of product per batch was diluted in 50 mL saline and 50 μL of this suspension was spread in solid media and incubated at 28°C for 10 days (positive controls were incubated for 5 days). The production strain was not detected.28 Due to the low amount of the additive tested, the presence of viable cells cannot be excluded. However, the FEEDAP Panel considers that based on the inactivation steps in the manufacturing process, the presence of P. carotinifaciens NITE SD 00017 is unlikely. Based on the data provided to characterise P. carotinifaciens NITE SD 00017, the potential presence of viable cells in the additive would not represent a safety concern.

The Chemical Abstracts Service (CAS) number, molecular formula and molecular weight of astaxanthin, canthaxanthin and adonirubin are reported in Table 3.

Due to the presence of the long hydrocarbon chain, these substances are insoluble in water but soluble in fats and most organic solvents.

Physico‐chemical and technological properties of the additive

3.1.4

The additive is a dark red granular powder with an average bulk density of 473 kg/m^3^ and tapped density of 623 kg/m^3^.

In order to establish the impact of the changes implemented in the manufacturing process on the physico‐chemical properties of the additive, the applicant provided new data regarding the dusting potential and particle size, which are described below.

The particle size distribution was determined by laser diffraction analysis in three batches of the additive. The fractions of particles below 10, 50 and 100 μm were in the range of 3–4, 12–15 and 29–35%(V/V), respectively. Dusting potential, measured in the same batches (Stauber‐Heubach method) showed values of 1.9, 3.1 and 2.6 g/m^3^. The dusting potential was also measured in five additional batches (Stauber‐Heubach method) showing values of 2.6, 2.3, 3.1, 4.8 and 5.1 g/m^3^.29 In this experiment, the concentration of astaxanthin, adonirubin and cantaxanthin was also determined. The following mean concentrations were measured before and after dust generation, respectively: 21.7 and 20.1 mg/g astaxanthin; 9.0 and 8.4 mg/g adonirubin; 2.2 and 1.9 mg/g canthaxanthin. Particle size distribution of the dust was not analysed.

No new data were submitted on stability and homogeneity. The Panel considers that the small changes introduced in the manufacturing process would not have an impact on the stability and the homogeneous distribution of the additive in feed, and therefore, the previous assessment is still considered valid (EFSA, 2007).

The applicant reported the potential incompatibility between carotenoids and blood meal based on customer feedback and scientific literature data. Hatlen et al. (2012) observed lower muscle colour score and lower astaxanthin concentrations when the diet of salmons was supplemented with porcine blood meal, possibly due to degradation of astaxanthin catalysed by iron from haemoglobin present in blood meal. The FEEDAP Panel recommends avoiding the simultaneous use of the additive and blood meal in the diet of salmon and trout.

Conditions of use

3.1.5

The additive is currently authorised for use in feed for salmon and trout with a maximum content of 100 mg/kg complete feed where the maximum content is expressed as the sum of astaxanthin, adonirubin and canthaxanthin.

The authorisation under other provisions foresees:

1. The maximum content is expressed as the sum of astaxanthin, adonirubin and canthaxanthin.
2. Use permitted from the age of 6 months onwards or weight of 50 g.
3. The mixture of the additive with astaxanthin or canthaxanthin is allowed provided that the total concentration of the sum of astaxanthin, adonirubin and canthaxanthin from other sources does not exceed 100 mg/kg in complete feedingstuff.

Maximum residue limits are also set in the authorising regulation as follows:

• For salmon: 10 mg/kg for the sum of adonirubin and canthaxanthin/kg muscle (wet tissue).
• For trout: 8 mg/kg for the sum of adonirubin and canthaxanthin/kg muscle (wet tissue).

The applicant is requesting to delete the provision limiting the use from the age of 6 months onwards or weight of 50 g (point 2 under other provisions).

The applicant is requesting the modification of provision 3 as follows: ‘The mixture of the additive with astaxanthin is allowed provided that the total concentration of the sum of astaxanthin, adonirubin and canthaxanthin from other sources does not exceed 100 mg/kg in complete feedingstuff’ considering that as of today, there is no authorisation in force for canthaxanthin in feed for salmonids.

Lastly, the applicant is requesting to lower the MRLs for salmon to 5 mg for the sum of adonirubin and canthaxanthin/kg muscle (wet tissue).

In addition, the applicant is asking to add the following provisions:

• –For safety: breathing protection, safety glasses and gloves shall be worn during handling.
• –Red carotenoid‐rich Paracoccus carotinifaciens may be placed on the market and used as an additive consisting of a preparation.

Safety

3.2

The safety of the additive red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017 for the target species, consumer, user and environment was established in previous FEEDAP opinions (EFSA, 2007; EFSA FEEDAP Panel, 2010).

The applicant provided the following information to support that, under the approved conditions of use, the additive remains safe for the target species, consumer, user and environment: (i) a company report on adverse events (see Section 3.2.4), (ii) an extensive literature search and (iii) a report on residue monitoring.

The extensive literature search30 ^,^ 31 (ELS) covered the period June 2005 until March 2024 and the search terms and search strategy were provided.32 The main search terms regarded the additive, the active substances and included terms concerning the safety and the toxicity for the target species, consumers, users and the environment. A total of 78 publications were considered relevant by the applicant. The FEEDAP Panel reviewed the papers and mentioned them in the assessment when considered relevant.

Absorption, distribution, metabolism, excretion and residues

3.2.1

The metabolism of the three main carotenoids (astaxanthin, adonirubin and canthaxanthin) was described in the former opinion of the FEEDAP Panel (EFSA, 2007). No new studies have been submitted in the current application. No relevant publications were identified in the ELS by the Panel for the assessment of the renewal of the authorisation.

The applicant submitted a report on residue monitoring.33 Samples of salmon and trout were collected at the end of the production cycle of fish fed red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017 in one European country. The samples originated from three fish producers using the additive under assessment. The inclusion rate of the additive was variable depending on the growth rate of the fish and was not always reaching the highest authorised use level. In all three cases, the highest supplementation level was calculated to be ~ 100 mg of the sum of astaxanthin, canthaxanthin and adonirubin. The carotenoids were extracted from the fish flesh, separated and quantified using a validated HPLC method. The results (125 samples analysed) indicated 0.3 ± 0.1 mg canthaxanthin/kg muscle, 1.3 ± 0.2 mg adonirubin/kg muscle and 3.8 ± 0.6 mg astaxanthin/kg muscle. A similar analysis was performed in trout from four producers. Also in this case, the inclusion level varied based on the growing stage of the trout; the highest inclusion level was calculated to be ~ 100 mg of the sum of astaxanthin, canthaxanthin and adonirubin. The results (32 samples analysed) indicated 0.3 ± 0.1 mg canthaxanthin/kg muscle, 1.7 ± 0.8 mg adonirubin/kg muscle and 5.2 ± 2.3 mg astaxanthin/kg muscle.

The FEEDAP Panel considers that the data provided might reflect current fish farming practices (adapting use levels according to the growth phase of the fish). However, considering that the additive was not administered up to the highest authorised use level continuously during the production cycle of the fish, the data cannot be used for the calculation of consumer exposure.

Residue data derived from studies with animals exposed to the highest proposed use level of the additive were assessed in the former opinion (EFSA, 2007). The FEEDAP Panel reviewed the studies in salmon and trout and considered that the dose–response study performed with the additive under assessment in rainbow trout (Oncorhynchus mykiss) can be retained for the consumer safety assessment. The treatment group with 100 mg of the sum of astaxanthin, adonirubin and canthaxanthin will be considered to derive residue data. The residue values are reported in Table 4.

Toxicology

3.2.2

The toxicity of red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017 was assessed by the FEEDAP Panel in 2007 and it was concluded that ‘Panaferd®‐AX is non genotoxic and exhibits very low acute and sub‐chronic toxicity. Therefore, the FEEDAP Panel considers that no specific risk for the consumer related to compounds arising from the fermentation process (other than red carotenoids) is likely to occur’ (EFSA, 2007).

No new studies were submitted. The toxicology of ATX was re‐evaluated by the FEEDAP Panel in 2019 (EFSA FEEDAP Panel, 2019a). In this opinion, an acceptable daily intake (ADI) of 0.2 mg astaxanthin/kg body weight (bw) per day was established based on a lowest observed adverse effect level (LOAEL) of 40 mg ATX/kg bw per day (end point: increased incidence of multinucleated hepatocytes in a 2‐year rat study). Similarly, the toxicology of CTX was re‐evaluated by the FEEDAP Panel in 2013 (EFSA FEEDAP Panel, 2013). In this opinion, the ADI of 0.03 mg CTX/kg bw, already agreed by JECFA (JECFA, 1995), the EU Scientific Committee for Food (European Commission, 2000), and by EFSA ANS Panel (EFSA ANS Panel, 2010), was confirmed.34 The ELS did not identify relevant studies that would lead the Panel to modify these conclusions.

In its previous opinion (EFSA, 2007), the FEEDAP Panel noted that the safety of adonirubin was not established. However, it was stated that ‘adonirubin has an intermediary chemical structure between canthaxanthin and astaxanthin, which suggests that the toxicological profiles of the three compounds are similar.’ Therefore, the Panel stated that the ADI for canthaxanthin should be applied to the sum of canthaxanthin plus adonirubin on a precautionary basis. In the absence of any new evidence, the FEEDAP Panel considers that the same conclusions should be retained for the present assessment.

Conclusions on the absence of a genotoxic potential of the additive were reached in the previous opinion (EFSA, 2007) based on a negative Ames test, a negative mouse lymphoma assay and a negative in vivo micronucleus test in bone marrow erythrocytes. However, the FEEDAP Panel noted that the available data set did not comply with the current requirements on genotoxicity testing strategy (EFSA FEEDAP Panel, 2017a; EFSA Scientific Committee, 2017) since the negative results observed in the in vivo micronucleus test were not associated with evidence of target tissue exposure and did not allow to evaluate the aneugenic potential of the test item. To this aim, the applicant provided an in vitro micronucleus test performed in Chinese hamster ovary (CHO) cells following the OECD Guideline 487 and claimed to be GLP‐compliant. Based on the results of a preliminary cytotoxicity test, four concentrations were selected for the analysis of micronucleus frequency in binucleated cells. Panaferd®‐AX was tested at (i) 100, 300, 600, 1250 μg/mL applying a short treatment (3 + 21 h of recovery) in the presence of metabolic activation; (ii) 100, 200, 700, 1250 μg/mL after a short treatment in the absence of metabolic activation; (iii) 40, 60, 80, 100 μg/mL following a continuous treatment (24 + 0 h of recovery) without metabolic activation. Cytotoxicity up to 54% was recorded at the highest concentrations tested. The frequencies of micronuclei observed after treatment with Panaferd®‐AX were comparable to the values detected in the vehicle control cultures. Therefore, the FEEDAP Panel concluded that Panaferd®‐AX did not induce structural and numerical chromosomal damage in cultured Chinese Hamster Ovary (CHO) cells under the experimental conditions applied in this study.

Conclusions on toxicology

The FEEDAP Panel concludes that Panaferd®‐AX does not raise safety concern for genotoxicity.

The ADI of 0.2 and 0.03 mg/kg bw can be retained for the consumer safety assessment of astaxanthin and of the sum of canthaxanthin and adonirubin, respectively.

Safety for the target species

3.2.3

In its previous opinion (EFSA, 2007), the FEEDAP Panel concluded that the additive is safe for salmonids at the inclusion level of 0.4% based on a tolerance study in rainbow trout in which Panaferd®‐AX was tolerated at dietary incorporation rate of 12.5‐fold greater than the maximum incorporation rate of 0.4%. Considering the carotenoid levels in the additive, the Panel recommended to set the maximum use level for the sum of astaxanthin, canthaxanthin and adonirubin and to apply 100 mg/kg feed for this sum.

No new studies have been provided in the present application. No relevant publications were identified by the Panel in the ELS.

The measured levels of endotoxins in the additive 2.9 × 10^6^ EU/g are of the same order of magnitude as those commonly found in feedingstuffs (ca. 1,000,000 IU/g, Cort et al., 1990). Therefore, at the usual conditions of use of the additive in feed, the endotoxins potentially added by the additive would be around 7300 EU/kg complete feed. The Panel also notes that the characterisation of the LPS showed that those originating from the production strain under assessment are of low concern.

Considering that no adverse effects have been reported, and no relevant information was found in the ELS with regard to the safety of the product and taking into account that the manufacturing/composition of the additive has not been substantially changed, the Panel considers that the conclusions reached in the previous opinion are still valid. The FEEDAP Panel concludes that the use of the additive remains safe for the target species under the conditions of the authorisation.

The applicant requested to remove the restriction of use of the additive from the age of 6 months onwards or weight of 50 g. As already expressed by the FEEDAP Panel in 2005 (EFSA, 2005), there is no reason to restrict the use of ATX to any particular physiological developmental stage of fish. In addition, the Panel notes that the tolerance studies in rainbow trout considered in the original opinion was done in juvenile animals (6–7.5 g). Therefore, the Panel concludes that there is no need to restrict the use of the additive from the age of 6 months onwards or weight of 50 g.

Safety for the consumer

3.2.4

In its former opinion, the FEEDAP Panel concluded that ‘As consumer exposure to astaxanthin and canthaxanthin after administration of Panaferd‐AX at the maximum dose proposed would be at the most equal or less than that resulting from the use of other astaxanthin or canthaxanthin sources, there is no additional risk for the consumer. […] The calculated consumer exposure to adonirubin plus canthaxanthin derived from the use of Panaferd‐AX in salmon and trout complies with the ADI for canthaxanthin (37 %). Therefore, no additional risk due to adonirubin exposure resulting from the use of Panaferd‐AX is likely to occur.’ (EFSA, 2007).

The applicant provided a company report on adverse events35 ■■■■■ could not be related to the use of the additive and are not considered relevant for the current assessment.

The consumer exposure to residues of astaxanthin, canthaxanthin and adonirubin has been assessed following the methodology detailed in the guidance on consumer safety (EFSA FEEDAP Panel, 2017b). The input values used in the calculation are included in Table 5. The outcome of the calculation is shown in Table 6 (for detailed results per age class, country and survey, see Appendix A, Table A.1).

Following the exposure calculation, it appears that the exposure to astaxanthin is well below the ADI (6%–21%), and that of canthaxanthin plus adonirubin ranges between 26% and 91% of the respective ADI.

MRLs exists for the sum of canthaxanthin and adonirubin for salmon (10 mg/kg muscle (wet tissue)) and trout (8 mg/kg muscle (wet tissue)). For astaxanthin, the setting of MRLs was not considered necessary (EFSA FEEDAP Panel, 2014).

When assessing consumer exposure to canthaxanthin and adonirubin at the level of the existing MRLs, the exposure of consumers exceeds the ADI in the population classes toddlers and other children for both salmon and trout (Table 7) (for detailed results per age class, country and survey, see Appendix A, Table A.2 and A.3).

The applicant proposes to reduce the MRLs for salmon from 10 mg to 5 mg/kg muscle while keeping that of trout at 8 mg/kg muscle. When calculating the consumer exposure at the MRL of 5 mg/kg muscle, the exposure is below the ADI for all population classes (Table 8) (for detailed results per age class, country and survey, see Appendix A, Table A.4).

The FEEDAP Panel considers that the MRL of 5 mg/kg muscle would ensure compliance with the ADI for the sum of canthaxanthin and adonirubin, and could be applied to both salmon and trout. The existing data on residues (see Table 4), also confirmed by the data from the recent residue monitoring performed by the applicant, show that the residues would comply with this proposed MRL of 5 mg/kg for muscle of salmon and trout.

Consumer exposure needs to take into consideration all potential dietary sources. Astaxanthin is currently authorised only in salmon and trout, while canthaxanthin is also authorised for use in chickens for fattening, minor poultry species for fattening, laying poultry, poultry reared for laying and the following MRLs are in force: 2.5 mg/kg skin/fat, 15 mg/kg liver, 30 mg/kg egg yolk (8.1 mg/kg whole egg containing 27% of yolk).36 Consumer exposure considering the combined exposure from salmonids and poultry was done using the proposed MRL of 5 mg/kg muscle as input data together with existing MRLs in poultry (Table 9). The outcome of the calculation is shown in Table 10 (for detailed results per age class, country and survey, see Appendix A, Table A.5).

When assessing consumer exposure to canthaxanthin and adonirubin at the level of the existing MRLs for poultry and the proposed MRL for trout/salmon, the exposure of consumers exceeds the ADI in the population classes toddlers and other children. The main contributors to the consumer exposure are eggs and fish.

Safety for user

3.2.5

In 2007, based on the results of an acute inhalation study in rats and a skin and eye irritation study in rabbits, the FEEDAP Panel concluded that: ‘Panaferd‐AX shows very low acute inhalation toxicity and no potential for skin irritation. However, Panaferd‐AX is considered an eye irritant. Considering the nature of the product, it should also be considered a respiratory sensitiser’ (EFSA, 2007).

No additional studies were submitted in the present application, and no relevant data were found in the ELS performed by the applicant.

The highest dusting potential measured was 5.1 g/m^3^, indicating that exposure of users by inhalation is likely.

The additive consists of inactivated cells of P. carotinifaciens, and therefore, it should be considered as a skin and respiratory sensitiser. In addition, considering the high content of LPS, albeit its low TLR4 mediated cytokine release, the Panel considers that any exposure via inhalation is a risk.

Therefore, considering the above, the Panel concludes that the additive is not irritant to the skin, but is irritant to the eyes. It is considered a dermal and respiratory sensitiser and any exposure via skin or the respiratory tract is a risk.

Safety for the environment

3.2.6

In 2007, the FEEDAP Panel concluded that astaxanthin, adonirubin and canthaxanthin, the major red carotenoids contained in the additive, are not expected to pose a risk for the environment (EFSA, 2007).

In the current dossier, no new studies were provided, and the ELS did not identify any relevant papers on the safety of the additive or its main components for the environment.

Considering that the major carotenoids contained in the additive are naturally occurring in the marine environment (Mapelli‐Brahm et al., 2023), in line with the provisions of the guidance on the assessment of feed additive for the environment (EFSA FEEDAP Panel, 2019b), the FEEDAP Panel concludes that the use of red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017 as source of astaxanthin, canthaxanthin and adonirubin for salmon and trout is not expected to pose additional risk to the environment in comparison with their naturally occurring forms.

Therefore, the FEEDAP Panel concludes that the use of the additive in salmon and trout remains safe for the environment under the conditions of the authorisation.

Efficacy

3.3

The present application for renewal of the authorisation does not include a proposal for amending or supplementing the conditions of the authorisation that would have an impact on the efficacy of the additive. Therefore, there is no need for assessing the efficacy of the additive in the context of the renewal of the authorisation.

CONCLUSIONS

4

The applicant provided evidence that the additive currently in the market complies with the existing conditions of the authorisation.

The Panel concludes that the use of the additive in salmon and trout remains safe for the target species, the consumer and the environment under the authorised conditions. The Panel considers that there is no need to restrict the use of the additive to fish older than 6 months or of more than 50 g.

When assessing consumer exposure to canthaxanthin and adonirubin at the level of the existing MRLs for poultry and the proposed MRL for trout/salmon (5 mg/kg muscle), the exposure of consumers exceeds the ADI in the population classes toddlers and other children. The main contributors to the consumer exposure are eggs and fish.

Red carotenoid‐rich Paracoccus carotinifaciens NITE SD 00017 is not irritant to the skin but is considered an eye irritant, and a skin and respiratory sensitiser. Any exposure though skin and inhalation is considered a risk for the user.

There is no need for assessing the efficacy of the additive in the context of the renewal of the authorisation.

RECOMMENDATIONS

5

The Panel recommends reducing the MRLs for the sum of canthaxanthin and adonirubin for both salmon and trout to 5 mg/kg muscle. The Panel also notes that a revision of the MRL for canthaxanthin when used in poultry might be needed.

The applicant is requesting the modification of provision 3 as follows: ‘The mixture of the additive with astaxanthin is allowed provided that the total concentration of the sum of astaxanthin, adonirubin and canthaxanthin from other sources does not exceed 100 mg/kg in complete feedingstuff’ considering that as of today, there is no authorisation in force for canthaxanthin in feed for salmonids. However, the Panel notes that such a provision should be independent from other authorisations. Therefore, the Panel recommends the following wording for provision 3: ‘the mixture of the additive with other authorised feed additives containing astaxanthin, adonirubin and/or canthaxanthin is allowed provided that the total concentration of the sum of astaxanthin, adonirubin and canthaxanthin in the diet does not exceed 100 mg/kg complete feed’.

The FEEDAP Panel notes that the values found regarding the total aerobic count are high and may deserve attention/monitoring during the production process.

The FEEDAP Panel recommends avoiding the simultaneous use of the additive and blood meal in the feed of salmon and trout.

ABBREVIATIONSADIaverage daily intakeANSEFSA Scientific Panel on Additives and Nutrient Sources added to FoodBWbody weightCASChemical Abstracts ServiceCFUcolony forming unitECHAEuropean Chemicals AgencyEURLEuropean Union Reference LaboratoryFAOFood Agricultural OrganisationJECFAThe Joint FAO/WHO Expert Committee on Food AdditivesLODlimit of detectionLOQlimit of quantificationMICminimum inhibitory concentrationMRLmaximum residue limitNOAELno observed adverse effect levelTLR4Toll‐like receptor 4WHOWorld Health Organization

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‐2017‐00694

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 the European Commission. The full output has been shared with the European Commission, EU Member States and the applicant. The blackening will be subject to review once the decision on the confidentiality requests is adopted by the European Commission.