This is a joint FSA and FSS publication.
1. Introduction
The FSA and FSS have undertaken an assessment of a feed additive containing 25-hydroxycholecalciferol (25-OH-D3) produced by a genetically modified strain of Saccharomyces cerevisiae SC0639 (DSM Nutritional Products Ltd in Switzerland, Wurmisweg 576, 4303, Kaiseraugst, Switzerland) under Assimilated Regulation (EC) No 1831/2003 (EC 2003). The application sought a new use (extension of use from chickens for fattening, turkeys for fattening, other poultry and pigs to include all ruminants). The additive falls under the category ‘nutritional additive’, functional group ‘vitamins, pro-vitamins and chemically well-defined substances having a similar effect’.
To support the safety assessment by FSA and FSS, the ACAF provided advice to the FSA and FSS outlined in this document.
In line with Article 8 of 1831/2003, the assessment has considered and concluded that the feed additive complies with the conditions laid down in Article 5, including: safety considerations for human, animal and environmental health; efficacy of the additive for its intended effect; potential impairment of the distinctive features of animal products. This, and the guidance put in place by EFSA for the evaluation of feed additive applications, has formed the basis and structure for the assessment.
The dossier was evaluated by the Advisory Committee on Animal Feedingstuffs (ACAF) at its December 2023 meeting, after which a request for further information was communicated to the applicant. The applicant’s response to this request was evaluated by ACAF at its June 2024 meeting.
The views of ACAF have been taken into account in this safety assessment which represents the opinion of the FSA/FSS.
2. Assessment
2.1. Section II: Identity, characterisation and conditions of use
25-OH-D3 is made from the raw material 5,7,24-cholestatrienol, which is produced by a fermentation process using a genetically modified strain of Saccharomyces cerevisiae (SC0639). The additive is an off-white to yellowish powder consisting of > 94% 25-OH-D3, ≤ 1% each of other related sterols and < 5 mg/kg erythrosine.
One example of a formulated product containing 25-OH-D3 is ROVIMIX® Hy-D® 1.25%, which is manufactured by the applicant to contain a minimum of 12.5 g/kg of the active substance. The composition of Hy-D® 1.25% is given in Table 2.
The applicant provided data from five batches for the formulated product ROVIMIX® Hy-D® 1.25% supporting the identification values outlined below (Table 3):
The Committee evaluated the identity and characterisation of the additive, requesting information relating to microbiological contamination and data to quantify the growth media within the final additive. In their response, the applicant stated that the product placed on the market is spray-dried and is therefore subjected to very high temperatures, so microbial testing is not necessary. The Committee commented that these temperatures are insufficient for sterilisation and hence the applicant provided microbial testing. Members agreed that the strain had been fully characterised using whole genome sequencing and were therefore able to conclude on the identity and characterisation of the additive.
Members noted that both HACCP (Hazard Analysis and Critical Control Points) and relevant assurance certification were not provided by the applicant, as well as only a single control point. Following a response from the applicant in addition to an updated manufacturing process and composition list, the Committee agreed that the HACCP documentation and relevant processing information provided were satisfactory.
The formulation prepared with butylated hydroxytoluene (BHT) was shown to be stable for 24 months when stored at 15 ± 2°C, 18 months when stored at 25 ± 2°C and 60% relative humidity (RH), and 6 months when stored at 40°C and 75% RH. Stability of the additive depends on the manufacturer and the composition of the stabilised form, such as the inclusion of antioxidants, e.g. BHT. Regarding stability in premixtures and feedingstuffs, the applicant demonstrated that the formulation ROVIMIX® Hy-D® 1.25% is stable during the manufacture of premixtures and can be stored up to 6 months in premixtures not containing minerals. The formulation is stable for 3 months in complete premixtures when stored at room temperature. Stability during pelleting at 70 - 90°C for 2.5 mins was demonstrated with the formulation remaining stable for 3 months after pelleting. Homogeneity of the additive in complete feed for turkeys was previously assessed.
The proposed conditions of use of the additive are described in Table 4.
The ACAF accept the applicant’s proposed conditions of use for the additive.
2.1.1. Conclusions on Section II
The ACAF concluded that the additive was fully identified and characterised using whole genome sequencing. Upon receipt of further information, members were satisfied with the manufacturing process. Members were able to conclude positively on the stability of the formulation provided and homogeneity of the additive. No further concerns were raised for Section II of the dossier.
2.2. Section III: Safety
2.2.1. Safety for the target species
The Committee reviewed a literature search conducted by the applicant to identify information relating to the safety of 25-OH-D3 for ruminants. No negative effects of dietary supplementation with the additive were reported; therefore the Committee concluded that the additive can be considered safe for the target species.
2.2.2. Safety for the consumer
A number of toxicological tests were available for 25-OH-D3 and considered previously by the EFSA (European Food Safety Authority) FEEDAP Panel (The Panel on Additives and Products or Substances used in Animal Feed) in 2005 (EFSA, 2005). These studies included: acute toxicity in mice and rats, repeated dose sub-chronic toxicity in rats, two mutagenicity studies (a bacterial reverse mutation test and an in vitro mammalian cell micronucleus test) and reproduction studies in both rats and rabbits. A further chromosome aberration test in 2009 (EFSA, 2009) confirmed that the additive is not considered to be genotoxic, as the results did not show any clastogenic activity of the additive when tested up to cytotoxic concentrations. Taking account of the results of all the mutagenicity tests, the ACAF concluded that the additive is not genotoxic.
The ACAF agreed that the dietary exposure assessment performed using the FACE (Feed Additive Consumer Exposure) model was appropriate and the tolerable upper intake levels (ULs) used in the consumer risk assessment were in line with EFSA’s previous evaluations of vitamin D3 and 25-OH-D3. The Committee concluded the additive can be considered safe for the consumer.
2.2.3. Safety for the user
The applicant had originally not provided any study data for safety for the user, hence the Committee requested reports relating to skin and eye irritancy. The following tests were presented, wherein the substance tested was ROVIMIX® Hy-D® 1.25%:
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Eye irritancy, following OECD protocol 405;
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Skin irritancy/corrosion, following OECD protocol 404.
For eye irritancy, treatment caused reversible redness and discharge which had resolved at 24 hours post-treatment; therefore the additive is not considered an eye irritant. For skin irritancy and corrosion, treatment caused mild erythema and oedema which resolved after 7 days, therefore the material is not corrosive or irritant to skin.
Dusting potential and particle size distribution data indicate that normal handling of 25-OH-D3 could generate a respirable dust. Studies assessing inhalation toxicity and dermal sensitisation were not provided; therefore the additive should be considered potentially harmful if inhaled and as a potential skin sensitiser.
2.2.4. Safety for the environment
25-OH-D3 is a natural substance that is extensively metabolised in the target animal and excreted in small amounts. Additionally, the additive predominantly substitutes for supplemental vitamin D3 at the relevant maximum content; hence the Committee concluded that environmental studies are not necessary as the use of the additive in feedingstuffs does not represent a risk for the environment.
2.2.5. Conclusions on safety
The ACAF concluded that the additive is safe for the target animals, consumers, and the environment.
The additive is not irritant to skin or eyes but should be treated as potentially harmful if inhaled and as a potential skin sensitiser.
2.3. Section IV: Efficacy
The applicant provided four published studies evaluating the additive as a source of vitamin D for ruminants, focusing on the equivalence of dietary 25-OH-D3 to vitamin D3. Members raised concerns relating to the inclusion rate for the use of the additive in ruminants as over-supplementation was a potential risk. Upon receiving this additional information, the Committee concluded that the inclusion of the additive in ruminant diets has similar effects on performance parameters as vitamin D3 and could therefore conclude the additive is an efficient source of vitamin D3.
2.3.1. Conclusions on efficacy
The ACAF concluded that the inclusion of the additive has similar effects on performance parameters as vitamin D3 when added to ruminant diets. The Committee, therefore, concluded that the additive is an efficient source of vitamin D3 for ruminants when used according to the proposed conditions of use.
3. Analytical methods evaluation
The FSA/FSS consider the EURL to be a competent and reliable body for the evaluation of analytical methods for the authorisation of feed additives. The EURL evaluation standards were agreed when the UK was part of the EU and they have remained relevant for the standards expected at UK level. Based on these premises, the conclusions on the evaluation of the analytical methods for this application have been received and accepted to be applicable to the UK authorisation process.
Conclusions on the analytical methods are presented here as an extract from the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of the Analysis for 25-hydroxycholecalciferol (25-OH-D3) (EURL, 2009):
"For the determination of 25-OH-D3 in stabilised forms of the feed additive the applicant proposed a single-laboratory validated and further verified method based on reversed-phase Ultra Performance Liquid Chromatography (UPLC) coupled to spectrophotometric (UV) detection. For the determination of 25-OH-D3 in premixtures the applicant proposed a single-laboratory validated and further verified method based on reverse-phased High Performance Liquid Chromatography (HPLC) coupled to spectrophotometric (UV) detection. The method is applicable for samples containing 25-OH-D3 at a minimum of 1500 µg/kg of the feed additive. For the determination of 25-OH-D3 in low concentrated premixtures and feedingstuffs the applicant proposed a single-laboratory validated and further verified method based on HPLC coupled to tandem mass spectrometry (MS/MS). The method is applicable for samples containing 25-OH-D3 between 10 and 3000 µg/kg of the feed additive. The following performance characteristics were reported by the applicant from the validation and verification studies for the quantification of 25-OH-D3 in the feed additive (stabilised products), premixtures, low concentrated premixtures and feedingstuffs:
In the feed additive: a relative standard deviation for repeatability (RSDr) and a relative standard deviation for intermediate precision (RSDip) of 1.2%, a recovery rate (Rrec) of 101%;
In premixtures (minimum 1500 µg/kg): a RSDr ranging from 1.5 to 4.4%; a RSDip ranging from 1.6 to 5.0%; a Rrec of 102%; and
In low concentrated premixtures and feedingstuffs (10 to 3000 µg/kg): a RSDr ranging from 3.0 to 5.3%; a RSDip ranging from 3.1 to 5.9%; a Rrec of 92%; and a limit of quantification (LOQ) of 2 µg of 25-OH-D3/kg feedingstuffs.
Based on the performance characteristics presented the EURL recommends for the official control the single-laboratory validated and further verified analytical method mentioned above based on UPLC-UV for the determination of 25-OH-D3 in stabilised forms of the feed additive; the single-laboratory validated and further verified analytical method based on HPLC-UV for the determination of 25-OH-D3 in premixtures: and the single-laboratory validated and further verified analytical method based on HPLC-MS/MS for the determination of 25-OH-D3 in low concentrated premixtures and feedingstuffs.
Further testing or validation of the methods to be performed through the consortium of National Reference Laboratories as specified by Article 10 (Commission Regulation (EC) No 378/2005, as last amended by Regulation (EU) 2015/1761) is not considered necessary."
FSA/FSS accepts the EURL analytical method evaluation reports. FSA/FSS determined the analytical methods proposed as appropriate for official controls for this feed additive.
4. Conclusions
The ACAF concluded that the additive was fully identified and characterised using whole genome sequencing. Members concluded positively on the stability of the formulation tested, noting that stability will depend on the manufacturer and composition of the stabilised form. Homogeneity of the additive was also demonstrated.
The additive is presumed safe for the target animals, the consumer and the environment. It is not irritant to skin or eyes but should be treated as potentially harmful if inhaled and as a potential skin sensitiser.
The ACAF concluded that the inclusion of the additive in ruminant diets has similar performance effects as vitamin D3. The Committee therefore concluded that the additive is an efficient source of vitamin D3 for ruminants when used according to the proposed conditions of use.
Abbreviations
Acknowledgements
With thanks to the members of the ACAF during the course of the assessment, who were: Professor Nicholas Jonsson, Martin Briggs, Professor Emily Burton, Professor Katrina Campbell, Professor Matthew Fisher, Hannah Kane, Susan MacDonald, Dr. Oonagh Markey, Christine McAlinden, Dr. Donald Morrison, Derek Renshaw, Dr. Michael Salter, Dr. Adam Smith, Christel Wake, Dr. Helen Warren and Professor Nick Wheelhouse. Dr. Adam Smith declared a direct conflict of interest and left the meetings for discussion.