Safety and efficacy of a feed additive consisting of 25‐hydroxycholecalciferol produced with Saccharomyces cerevisiae CBS 146008 for salmonids, other fish species and all other animal species (except poultry, pigs and all ruminants) (DSM Nutritional Products Ltd.)

TL;DR

This study evaluates the safety and effectiveness of a vitamin D3 additive in animal feed for fish and other animals, excluding poultry, pigs, and ruminants.

Contribution

The study confirms the safety and efficacy of 25-hydroxycholecalciferol in feed for salmonids and other non-ruminant animals.

Findings

The additive is safe for salmonids, other fish species, and non-ruminant animals at specified levels.

The additive is considered safe for consumers and the environment.

The additive is not irritating to skin or eyes but lacks data on skin sensitization and respiratory effects.

Abstract

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of 25‐hydroxycholecalciferol as a nutritional feed additive for salmonids, other fish species and all other animal species (except poultry, pigs and all ruminants). The additive is currently authorised for use in pigs, poultry and ruminants. The additive is safe for salmonids, other fish species and all other animal species (other than pigs, poultry and ruminants) at the maximum recommended use levels (in mg 25‐OH‐D3/kg complete feed): 0.8 for salmonids, 0.075 in other fish species and 0.05 for all other animal species (other than poultry, pigs and ruminants). The use of 25‐OH‐D3 in feed for all animal species under the proposed conditions of use is considered safe for the consumers and the environment. With regard to the safety of users, the additive is not irritant to the skin or eyes. No conclusion on its potential to be a skin sensitiser or on its effects on the respiratory system can be reached due to the absence of data. The FEEDAP Panel concludes that 25‐OH‐D3, when used as a nutritional additive in feed, is efficacious for all animal species.

Tables

TABLE 1. Trial design of the tolerance study in rainbow trout.

Species total number of animals (fish × tank) replicate tanks per treatment	Group (mg 25‐OH‐D3/kg complete feed)	Analysed
		25‐OH‐D3 (mg/kg complete feed)		Vitamin D3 (mg/kg complete feed)*
		0–44 days	45–91 days	0–44 days	45–91 days
Rainbow trout (O. mykiss) 640 (40) 4	0	< LOD**	< LOD	0.131	0.137
	0.08	0.060	0.079	0.131	0.137
	0.80	0.680	0.693	0.133	0.144
	8.00	7.190	6.540	0.134	0.135

TABLE 2. Performance parameters for the rainbow trout over the 91‐day tolerance trial.

Group (mg 25‐OH‐D3/kg complete feed)	Total feed intake (g/fish)	Final body weight (g)	Body weight gain (g/fish per day)	Specific growth rate (%BW/day)	Feed to gain ratio	Mortality (%)
0	197a	323.5a	2.92a	1.90a	0.74b	0
0.08	199ab	326.5a	2.96a	1.91a	0.74b	0
0.80	203b	341.0c	3.11b	1.95b	0.72a	0
8.00	201ab	336.0b	3.06b	1.94b	0.72a	0

TABLE 3. Chronic human dietary exposure to 25‐OH‐D3 originating from fish consumption, expressed as vitamin D3 equivalents.

Population class	Maximum HRP 1 (μg/kg BW)	Default body weight (kg)	Exposure (μg/day)	UL 3 (μg/day)	% UL
Infants	0.0277	5	0.138	25	0.55
Toddlers	0.0745	12	0.894	50	1.79
Other children	0.0517	23	1.189	50	2.38
Adolescents	0.0366	52.4 2	1.920	100	1.92
Adults	0.0306	70	2.143	100	2.14
Elderly	0.0285	70	1.992	100	1.99
Very elderly	0.0216	70	1.514	100	1.51

TABLE 4. Concentrations of 25‐OH‐D3, vitamin D3 and metabolites in plasma, muscle and liver (average and standard deviation).

Treatment group	Intended inclusion level (mg/kg)	Plasma D3 (ng/mL)	Plasma 25‐OH‐D3 (ng/mL)	Plasma 1,25‐OH‐D3 (pg/mL)	Muscle D3 (ng/g)	Muscle 25‐OH‐D3 (ng/g)	Liver D3 (ng/g)	Liver 25‐OH‐D3 (ng/g)
D3	0.200	6.2 (1.7)	0.5 (0.0)	551 (183)	1.7 (0.5)	0.5 (0.7)	49 (10)	2.6 (2.3)
	0.400	15.1 (3.8)	0.5 (0.0)	534 (222)	4.4 (1.3)	0.4 (0.7)	104 (21)	1.5 (0.7)
	0.800	38.6 (8.6)	0.6 (0.4)	447 (175)	17.6 (5.5)	0.4 (0.5)	229 (46)	1.5 (0.5)
25‐OH‐D3	0.200	< LOQ	0.6 (0.4)	571 (167)	< LOQ	0.8 (1.0)	5 (1)	1.2 (0.5)
	0.400	< LOQ	1.6 (1.2)	515 (214)	< LOQ	0.8 (0.5)	4 (1)	2.2 (0.7)
	0.800	< LOQ	4.0 (1.6)	543 (217)	< LOQ	1.6 (0.7)	4 (1)	4.0 (1.7)
	2‐way ANOVA p values
Metabolite		< 0.0001	< 0.0001	0.4955	< 0.0001	0.0016	< 0.0001	< 0.0001
Inclusion level		< 0.0001	< 0.0001	0.1629	< 0.0001	0.0531	< 0.0001	< 0.0001
Interaction		< 0.0001	< 0.0001	0.3662	< 0.0001	0.0305	< 0.0001	< 0.0001
	Linear regression p values
D3 diets		< 0.0001	0.0571	0.0129	< 0.0001	0.7192	< 0.0001	0.0506
25‐OH‐D3 diets		1.0000	< 0.0001	0.7022	1.0000	0.0004	0.0037	< 0.0001

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 a feed additive shall submit an application in accordance with Article 7.

The European Commission received a request from DSM Nutritional Products Ltd.2 for the authorisation of the additive consisting of the product 25‐hydroxycholecalciferol, when used as a feed additive for salmonids, other fish species and all other animal species (except poultry, pigs and all ruminants) (category: nutritional additive; functional group: vitamins, pro‐vitamins and chemically well‐defined substances having similar effect).

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). The dossier was received on 26th April 2024 and the general information and supporting documentation are available at https://open.efsa.europa.eu/questions/EFSA‐Q‐2024‐00273. The particulars and documents in support of the application were considered valid by EFSA as of 11th July 2024.

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 the product 25‐hydroxycholecalciferol, when used under the proposed conditions of use (see Section 3.1.3).

Additional information

1.2

An additive containing 25‐hydroxycholecalciferol produced with Saccharomyces cerevisiae CBS 146008 is currently authorised in the EU as a feed additive for poultry, pigs and ruminants (3a670a).

The FEEDAP Panel of EFSA adopted several opinions on the safety and efficacy of this additive for different target species (EFSA, 2005, 2009; EFSA FEEDAP Panel, 2023a, 2023b 2023c).

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 the product 25‐hydroxycholecalciferol as a feed additive.

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 18 November to 9 December 2024, for which no comments were received.

The confidential version of the technical dossier was subject to a target consultation of the interested Member States from 16 July 2024 to 16 October 2024; the comments received were considered for the assessment.

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.

The European Union Reference Laboratory (EURL) considered that the conclusions and recommendations reached in the previous assessment regarding the methods used for the control of the 25‐hydroxycholecalciferol in animal feed are valid and applicable for the current application.6

Methodologies

2.2

The approach followed by the FEEDAP Panel to assess the safety and efficacy of 25‐hydroxycholecalciferol 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 characterisation of microorganisms used as feed additives or as production organisms (EFSA FEEDAP Panel, 2018b), 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, 2023d) and Guidance on the assessment of the efficacy of feed additives (EFSA FEEDAP Panel, 2024).

ASSESSMENT

3

The subject of the assessment is 25‐hydroxycholecalciferol monohydrate (25‐OH‐D_3_) produced with a genetically modified Saccharomyces cerevisiae strain (S. cerevisiae CBS 146008). The additive is currently authorised for pigs, poultry and ruminants as a preparation of the active substance 25‐OH‐D_3_ as a nutritional additive, functional group vitamins, pro‐vitamins and chemically well‐defined substances having a similar effect (EU register code 3a670a).8 ^,^ 9

The applicant requested the authorisation for the use of the additive in feed for salmonids, other fish species and all other animal species (except poultry, pigs and all ruminants).

Characterisation

3.1

The precursor of 25‐OH‐D_3_, 5,7,24‐cholestatrienol, is produced by fermentation with a genetically modified strain of S. cerevisiae which has been deposited in the Westerdijk Fungal Biodiversity Institute under the accession number CBS 146008. The production strain was fully characterised, and the manufacturing process, physico‐chemical characteristics, shelf‐life, stability and capacity of the additive to distribute homogeneously in feed were described in a previous opinion of the FEEDAP Panel (EFSA FEEDAP Panel, 2023b). The FEEDAP Panel considers that such information is applicable to the current application.

Characterisation of the active substance

3.1.1

The current authorisation sets specifications for the active substance with 25‐hydroxycholecalciferol > 94%, other sterol derivatives ≤ 1% each and erythrosine < 5 mg/kg.

The applicant is proposing to keep the same specifications with regard to the minimum content of the active substance, the limit for erythrosine and for other sterol derivatives.

The applicant provided information on the batch‐to‐batch variation and the impurities listed in the authorisation. Data on batch‐to‐batch variation of five analysed batches showed average concentrations of 100% 25‐hydroxycholecalciferol (range: 99.7%–100%) on as is basis (anhydrous substance), 4% moisture, 0.9% total impurities (range: 0.5%–1.1%) and erythrosine was not detected in any of the batches.10

Apart from erythrosine and other sterol derivatives, the applicant proposed limits/specifications for aluminium and lead (≤ 20 mg/kg), for moisture ≤ 5%, for total impurities ≤ 6% and for the single largest impurity (≤ 1%).11 Data from five batches showed compliance with these specifications.12

Characterisation of the additive

3.1.2

Since 25‐OH‐D_3_ is sensitive to light, heat and oxidation, the current authorisation for use in pigs, poultry and ruminants foresees that it is placed in the market as a preparation with a minimum content of 1.25% of 25‐hydroxycholecalciferol. The applicant proposed to keep the same specification for the additive under assessment, with a maximum content of moisture of 5%. The example preparation proposed by the applicant was fully characterised in the previous opinion of the Panel (EFSA FEEDAP Panel, 2023b). New data were provided by the applicant on the composition of the additive, which are described below.

Five batches analysed for batch‐to‐batch variation showed average concentrations of 25‐OH‐D_3_ of 1.34% (1.32%–1.37%), and 2.8% moisture (2%–3%).13

As regards the three batches analysed for impurities, arsenic, cadmium, lead and mercury were below the limit of quantification (LOQ) except for lead in one batch showing concentrations of 0.09 mg/kg additive.14 Aflatoxins (B1, B2, G1, G2) were also below the LOQ. Methanol ranged 124–130 mg/kg additive while ethanol and isopropanol were below the LOQ. Other residual solvents (class 1, 2a, 2b and 3) were below the LOQ.15

Conditions of use

3.1.3

The additive is intended to be used in feed for salmonids, other fish and all other animal species (excluding ruminants, pigs and poultry) at a maximum use level in complete feed of 0.8 mg 25‐OH‐D_3_/kg complete feed for salmonids, 0.075 mg/kg in other fish and 0.05 mg/kg for all other animal species. The additive is intended to be incorporated in feed via the use of premixtures.16

Safety

3.2

Safety of the production microorganism

3.2.1

In a previous opinion (EFSA FEEDAP Panel, 2023b), the safety of the production organism S. cerevisiae CBS 146008 was assessed; it was found to be suitable for the qualified presumption of safety (QPS) approach and its genetic modification was considered to be of no concern. No viable cells and recombinant DNA of the production strain were detected in three batches of the additive or of an intermediate product used to formulate the additive. Therefore, the additive does not give rise to any safety concern regarding the genetically modified production strain.

Safety for the target species

3.2.2

Safety for rainbow trout

3.2.2.1

The applicant provided a tolerance study in rainbow trout (that included an evaluation of residue levels in fish flesh) to support the safety of fish species17 with the additive under assessment.

The trial involved 640 juvenile rainbow trout (Oncorhynchus mykiss) with an initial average body weight of 57.6 g. They were randomly allocated into 16 tanks (4 replicate tanks/treatment, 40 fish/replicate tank) of a 500‐L recirculating aquaculture system.

A basal diet consisting of fish meal, soybean meal, soy protein concentrate, rapeseed meal, wheat, wheat gluten and rapeseed oil, and supplemented with 1500 IU vitamin D_3_/kg diet (the basal diet contained on average 43.0% crude protein and 23.8% lipids), was either left unsupplemented (control group) or supplemented with 0.08 (0.1× maximum use level), 0.80 (1×) or 8.00 (10×) mg 25‐OH‐D_3_/kg feed. The dietary treatments were offered ad libitum in dry extruded oil‐coated pellets for 91 days. The diameter of the pellets was 3 mm for 44 days and 4 mm for the remaining 47 days (see the Table 1 below).

Monitoring of survival, health status and growth performance parameters, such as initial and final body weight, body weight gain, feed intake, specific growth rate and feed to gain ratio (F:G), was conducted. At the end of the study, 40 fish/treatment group (10 fish per replicate tank) were used to collect blood samples for haematological18 and blood chemistry analyses,19 and to determine body weight and length, as well as external malformities and lesions, gross pathology, and to collect organ samples (liver, spleen, kidney and intestine) for histological analyses. Fish condition factor (CF) and liver somatic index (LSI) were calculated.20 In addition, plasma (from 10 trout/tank) and about 5 g of fish flesh (from 5 trout/tank) were collected to determine plasma vitamin D_3_ and 25‐OH‐D_3_ content and muscle 25‐OH‐D_3_ deposition. These results are reported and described in Section 3.2.3.2.

The zootechnical data final body weight and specific growth rate were subjected to a non‐inferiority test (prespecified non‐inferiority margin of 5%) to the control group. Subsequently, the zootechnical parameters were submitted to an analysis of variance (ANOVA) with the diet as a fixed effect and the tank as the experimental unit; significant differences between means were determined using the Tukey HSD test, with a significance level set at 0.05.

Statistical analyses included non‐inferiority assessments of performance‐related data and equivalence or non‐parametric evaluations of other parameters, such as liver weight, CF, LSI and blood parameters. The individual fish served as the experimental unit, with tank location considered within the statistical model.

No fish died during the trial, with only one fish from the control group culled due to poor growth.21 No adverse effects were observed on any of the performance parameters assessed (Table 2).

All haematological and blood chemistry parameters fell within the normal range for rainbow trout, and no statistically significant differences among treatment groups were observed. Regarding gross pathology, some liver samples showed yellowish and lighter areas due to increased fat deposition, as well as congestion and dilated gall bladders. These findings, however, were isolated and not dose‐related, thus considered clinically irrelevant.

The results of the tolerance trial with rainbow trout showed that 8.00 mg 25‐OH‐D_3_/kg feed is tolerated. Therefore, the FEEDAP Panel concludes that 0.8 mg 25‐OH‐D_3_/kg feed is safe for salmonids.

Safety for other fish and for all animal species other than poultry, pigs, ruminants and salmonids

3.2.2.2

In previous opinions, the FEEDAP Panel assessed a series of tolerance/published studies which showed that the additive was safe in chickens and turkeys at the maximum proposed use level of 0.10 mg/kg, weaned piglets at 0.05 mg/kg and cattle for fattening at 0.10 mg/kg, with a margin of safety of at least 2.5 (up to 10) (EFSA FEEDAP Panel, 2009, 2023c). From these studies, the Panel concluded that the additive is safe for all poultry, pigs and ruminants. The new tolerance study in rainbow trout showed that the additive is safe for trout at 0.8 mg/kg, with a margin of safety of 10. Considering that the safety of the additive has been demonstrated in four major species with a sufficient margin of safety, the Panel considers that it can be considered safe for fish other than salmonids at 0.075 mg/kg feed and all other animal species (other than poultry, pigs and ruminant) at 0.05 mg/kg complete feed.

Conclusions on safety for the target species

3.2.2.3

The FEEDAP Panel concludes that 25‐OH‐D_3_ is safe for salmonids at a maximum concentration of 0.8 mg/kg feed, for fish other than salmonids at 0.075 mg/kg feed, and for all other animal species (other than poultry, pigs and ruminants) at 0.05 mg/kg complete feed.

Safety for the consumer

3.2.3

Absorption, distribution, metabolism, excretion (ADME)

3.2.3.1

The ADME of the additive was assessed in the FEEDAP Panel opinion of 2005. No ADME studies were submitted in the current application.

In vertebrates including fish, vitamin D_3_ is hydroxylated in the liver to 25‐OH‐D_3_ which, by a second hydroxylation, results in 1,25‐OH‐D_3_. Data from the literature shows that, contrary to terrestrial species, in salmonids, the second hydroxylation also takes place primarily in the liver (Lock et al., 2010). A local extra‐hepatic tissue‐specific hydroxylation of 25‐OH‐D_3_ to 1,25‐OH‐D_3_ likely also takes place in some salmonid tissues (Graff, Lie, and Aksnes, 1999).22

Residue studies

3.2.3.2

Residues of 25‐OH‐D_3_ were measured in edible tissues of salmonids (20 animals per group) fed 0, 0.080 and 0.800 mg 25‐OH‐D_3_/kg complete feed in the tolerance study (Section 3.2.2). Residues in fish flesh were 0.38 and 3.12 μg/kg in the 0.080 and 0.800 mg 25‐OH‐D_3_/kg feed groups, respectively.

Residue of 25‐OH‐D_3_ was also measured in an efficacy study (Section 3.3), where salmonids (20 animals per group) were fed at 0.20, 0.40 and 0.80 mg 25‐OH‐D_3_/kg feed. Concentrations of 25‐OH‐D_3_ in fish flesh were 0.65, 0.61 and 1.03 μg/kg, respectively.

The FEEDAP Panel notes that the difference in concentration of 25‐OH‐D_3_ in fish flesh observed between the two studies is probably due to the different concentrations of vitamin D_3_ supplementation in the diets: 0.131–0.144 mg/kg in the tolerance study versus 0.006 mg/kg in the efficacy study.23

The FEEDAP Panel considers that the deposition data derived from the tolerance study represent a worst‐case scenario, with the result of higher concentration in fish flesh compared to the ones measured in the efficacy study.

Therefore, to estimate consumer exposure via fish, the FEEDAP Panel used the results of 25‐OH‐D_3_ deposition in fish flesh at the maximum proposed use level (0.80 mg 25‐OH‐D_3_ /kg feed) from the tolerance study (3.12 μg/kg) plus two times the standard deviation (SD) (1.04) as indicated in the FEEDAP Panel Guidance on the safety of the feed additives for the consumer (EFSA FEEDAP Panel, 2017a, 2017b, 2017c). The resulting deposition value is 5.2 μg/kg flesh.

Assessment of consumer exposure and consumer safety assessment

3.2.3.3

The FEEDAP Panel notes that the EFSA NDA Panel has recently revised the tolerable upper intake level (UL) levels for vitamin D_3_ for all age groups as follows: 25 μg/day for children up to 6 months, 35 μg/day for children 6–12 months, 50 μg/day for children 1–10 years and 100 μg/day for adolescents (11–17 years) and adults, including pregnant and lactating women. The NDA Panel also proposed a biopotency factor of 2.5 to convert calcidiol monohydrate (25‐OH‐D_3_) into vitamin D_3_ (EFSA NDA Panel, 2023).

In previous assessments (EFSA FEEDAP Panel, 2023a, 2023b, 2023c), the FEEDAP Panel calculated the consumer exposure to vitamin D_3_ using deposition data of 25‐OH‐D_3_ in tissues from poultry and mammals, milk and eggs and applying a biopotency factor of 5. This was done because, at the time of the previous evaluations, the above NDA Panel opinion was not adopted. In addition, the FEEDAP Panel considered that the consumer exposure assessment should cover the uses of the ongoing applications (EFSA FEEDAP Panel, 2023a, 2023b, 2023c) and the already authorised uses at that time (pigs and poultry). In that opinion, the Panel noted that the combined exposure resulting from the use of the additive in feed for poultry, pigs and ruminants would be at levels of 11.2%–38.6% of the updated ULs for vitamin D_3_ (EFSA FEEDAP Panel, 2023a). The Panel notes that considering the new biopotency factor for 25‐OH‐D_3_ (2.5), the resulting consumer exposure is halved, and therefore would account for 5.6%–19.3% of the UL.

In the framework of the current assessment, the additional exposure of consumers via fish products needs to be considered. To that effect, using the deposition value of fish flesh mentioned above (5.2 μg/kg), and considering the biopotency factor of 2.5 (input value 13 μg/kg), the exposure of consumers to 25‐OH‐D_3_ via fish products was estimated following the methodology described in the FEEDAP guidance (EFSA FEEDAP Panel, 2017a). The results of the dietary exposure to residues of 25‐OH‐D_3_ calculated as vitamin D_3_ equivalents for the different population categories are reported in Table 3. The detailed results are given in Appendix A.

The data show that exposure via fish flesh does not significantly add to the exposure of consumers to 25‐OH‐D_3_. Even considering the combined exposure from all sources, the exposure of consumers would not exceed 22% of the UL for vitamin D_3_.

Conclusions on safety for the consumer

3.2.3.4

The FEEDAP Panel considers that the use of additives in feed for all animal species at the proposed conditions of use is safe for the consumer.

Safety for the user

3.2.4

The safety for the user has been previously evaluated by the FEEDAP Panel in 2023. In that opinion, it was concluded that the additive is not irritant to the skin or eyes. However, due to lack of information, no conclusion on its potential to be a skin sensitiser or on its effects on the respiratory system could be reached (EFSA FEEDAP Panel, 2023).

No information is provided to support the safety for the user in the current dossier. Therefore, the FEEDAP Panel considers that the conclusions previously reached still apply.

Safety for the environment

3.2.5

No information was provided to support the safety of the additive for the environment.

The production strain is genetically modified. No cells and no DNA of the production strain are present in the additive (EFSA FEEDAP Panel, 2023). Therefore, no safety concerns for the environment are expected from the use of the production strain for production purposes.

The 25‐OH‐D_3_ is a physiological metabolite in animals. It derives from vitamin D_3_, either from endogenous synthesis or exogenous supply. It is excreted only in low amounts. The use of 25‐OH‐D_3_ in animal nutrition up to the maximum recommended use level does not represent a risk for the environment.

Efficacy

3.3

Since 25‐OH‐D_3_ is already authorised for poultry, pigs and ruminants,24 and EFSA concluded that its use is efficacious as a substitute for vitamin D_3_ (EFSA, 2005, 2009), no new demonstration of efficacy for salmonids, other fish species and all other animal species would be necessary.

The applicant submitted a trial with rainbow trout to demonstrate the bioavailability of the additive (25‐OH‐D_3_) when supplemented at the proposed conditions of use. For that purpose, 480 juvenile (168‐day‐old) rainbow trout (O. mykiss) (average body weight = 50.9 g) were distributed in 24 tanks (240 L; 15 ± 2°C) with 20 fish per tank and randomly allocated to six experimental groups (4 replicates per group). All groups received the same basal diet consisting of soy protein concentrate, fish meal and wheat, which was supplemented either with 0.200, 0.400 and 0.800 mg 25‐OH‐D3/kg complete feed or with 0.200, 0.400 and 0.800 mg vitamin D_3_/kg complete feed (corresponding to 8000, 16,000 and 32,000 IU/kg feed, respectively). The content of both additives in feed was analytically confirmed.25 The experimental diets were offered ad libitum in extruded form for 84 days.

Mortality and other zootechnical performance parameters were recorded during the trial. At the end of the trial (day 84), 10 fish per tank were randomly selected and blood sampled. From five of these 10 fish, muscle and livers were sampled. Plasma, muscle and liver samples were analysed for the content of vitamin D_3_, 25‐OH‐D_3_ and their metabolites.

The experimental data were analysed with a mixed model, including the additive, the supplemented level and the interaction between them as fixed factors, and the tank as random effect. Linear regression effects were assessed. The tank was considered the experimental unit. The significance level was set at 0.05. The main results are shown in Table 4.

The mortality was low and within commercial standards, and the health status was fine throughout the trial, with no detrimental effect of the additive supplementation. No effects were observed in the main zootechnical parameters.

There was a significant, linear, positive dose–response relationship in the levels of 25‐OH‐D_3_ in plasma, liver and muscle, corresponding to increasing inclusion levels of the additive in the feed. A similar trend was observed for vitamin D_3_ levels when vitamin D_3_ was supplemented.

The data on tissue deposition measured in the tolerance study and reported in Section 3.2.3.2 also showed a linear increase of 25‐OH‐D_3_ concentrations in fish flesh, with higher inclusion levels of the additive in feed.

The results of these two trials support the efficacy of 25‐OH‐D_3_ in salmonids.

Conclusions on efficacy

3.3.1

The FEEDAP Panel concludes that 25‐OH‐D_3_ when used as a nutritional additive in feed is efficacious for all animal species.

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 Regulation26 and good manufacturing practice.

CONCLUSIONS

4

The FEEDAP Panel concludes that the additive is safe for salmonids, other fish species and all other animal species (other than pigs, poultry and ruminants) at the maximum recommended use levels (in mg 25‐OH‐D_3_/kg complete feed): 0.8 for salmonids, 0.075 for other fish species and 0.05 for all other animal species (other than poultry, pigs and ruminants).

The use of 25‐OH‐D_3_ in feed for all animal species under the proposed conditions of use is considered safe for the consumers and the environment.

With regard to the safety of users, the additive is not irritant to the skin or eyes. No conclusion on its potential to be a skin sensitiser or on its effects on the respiratory system can be reached due to the absence of data.

The FEEDAP Panel concludes that 25‐OH‐D_3_, when used as a nutritional additive in feed, is efficacious for all animal species.

RECOMMENDATIONS AND/OR REMARKS

5

The FEEDAP Panel recommends that the maximum content in feed should also apply to the combinations of 25‐OH‐D_3_ with vitamin D_3_.

ABBREVIATIONSANSEFSA Scientific Panel on Additives and Nutrient Sources added to FoodBWbody weightCASChemical Abstracts ServiceCFUcolony‐forming unitDMdry matterEURLEuropean Union Reference LaboratoryF:Gfeed to gain ratioFEEDAPEFSA Scientific Panel on Additives and Products or Substances used in Animal FeedLODlimit of detectionLOQlimit of quantificationMCVmean corpuscular volumeMWmolecular weight

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00273

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PANEL MEMBERS

Roberto Edoardo Villa, Giovanna Azimonti, Eleftherios Bonos, Henrik Christensen, Mojca Durjava, Birgit Dusemund, Ronette Gehring, Boet Glandorf, Maryline Kouba, Marta López‐Alonso, Francesca Marcon, Carlo Nebbia, Alena Pechová, Miguel Prieto‐Maradona, Ilen Röhe, and Katerina Theodoridou.