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EFSA J. 2023 Jun; 21(6): e08050.
Published online 2023 Jun 9. doi: 10.2903/j.efsa.2023.8050
PMCID: PMC10251257
PMID: 37304346

Safety and efficacy of a feed additive consisting of 25‐hydroxycholecalciferol (produced by Pseudonocardia autotrophica DSM 32858) for all pigs, all poultry for fattening and ornamental birds and other poultry species (Huvepharma NV)

EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP),corresponding author Vasileios Bampidis, Giovanna Azimonti, Maria de Lourdes Bastos, Henrik Christensen, Birgit Dusemund, Mojca Durjava, Maryline Kouba, Marta López‐Alonso, Secundino López Puente, Francesca Marcon, Baltasar Mayo, Alena Pechová, Mariana Petkova, Fernando Ramos, Yolanda Sanz, Roberto Edoardo Villa, Ruud Woutersen, Georges Bories, Jürgen Gropp, Giovanna Martelli, Kettil Svensson, Davor Zeljezic, Montserrat Anguita, Jordi Ortuño Casanova, Jaume Galobart, Orsolya Holczknecht, Matteo Lorenzo Innocenti, Paola Manini, Elisa Pettenati, Maria Vittoria Vettori, and Fabiola Pizzo
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Abstract

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of a feed additive consisting of 25‐hydroxycholecalciferol (produced by Pseudonocardia autotrophica DSM 32858) for all pigs, all poultry for fattening and ornamental birds and other poultry species. The production strain P. autotrophica DSM 32858 is not genetically modified however, uncertainties remain on the possible presence of its viable cells in the final product. Due to the lack of adequate safety data and uncertainty on the presence of nano particles, the FEEDAP Panel cannot conclude on the safety of the additive for the target species and the consumer. The additive was shown not to be irritant to skin or eyes and it is not a skin sensitiser. Considering the low dusting potential of the additive, the FEEDAP Panel concluded that the exposure through inhalation is unlikely. However, the FEEDAP Panel considered that uncertainties remain on genotoxicity and on the possible presence of viable cells of P. autotrophica DSM 32858 in the final product which might have an impact on the safety for the users. The use of the feed additive is considered safe for the environment. The Panel concluded that the additive has a potential to be efficacious under the proposed conditions of use.

Keywords: nutritional additives, vitamins, Pseudonocardia autotrophica, safety, efficacy, vitamin D3 , 25‐hydroxycholecalciferol

1. Introduction

1.1. Background and Terms of Reference

Regulation (EC) No 1831/2003 1 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 Huvepharma NV 2 for the authorisation of the additive consisting of 25‐hydroxycholecalciferol produced by Pseudonocardia autotrophica DSM 32858, when used as a feed additive for all pigs, all poultry for fattening and ornamental birds and other poultry species (category: nutritional additives; 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 particulars and documents in support of the application were considered valid by EFSA as of 25 November 2021.

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 25‐hydroxycholecalciferol when used under the proposed conditions of use (see Section 3.1.7).

1.2. Additional information

The feed additive consisting of 25‐hydroxycholecalciferol (25‐OH‐D3) produced by Pseudonocardia autotrophica DSM 32858 is not currently authorised for use in animal feed.

The FEEDAP Panel issued two opinions on the use of 25‐OH‐D3 as a feed additive: one for chickens for fattening, turkeys and laying hens (EFSA, 2005) and the other one for poultry and pigs (EFSA, 2009).

Another opinion was issued on the safety of calcidiol monohydrate (25‐hydroxycholecalciferol monohydrate) produced by chemical synthesis as a novel food (EFSA NDA Panel, 2021).

25‐OH‐D3 is currently authorised for use in feed for chickens for fattening, turkeys for fattening, other poultry and pigs under the identification number 3a670a. 3

2. Data and methodologies

2.1. Data

The present assessment is based on data submitted by the applicant in the form of a technical dossier 4 in support of the authorisation request for the use of 25‐OH‐D3 produced by P. autotrophica DSM 32858 as a feed additive.

The dossier was received on 1/10/2021 and the general information and supporting documentation is available at https://open.efsa.europa.eu/questions/EFSA-Q-2021-00641.

The confidential version of the technical dossier was subject to a target consultation of the interested Member States from 9 March 2022 to 9 June 2022 for which the received comments 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' (elicitation) 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 active substance in animal feed. 5

2.2. Methodologies

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of 25‐OH‐D3 is in line with the principles laid down in Regulation (EC) No 429/2008 6 and the relevant guidance documents: Guidance on studies concerning the safety of use of the additive for users/workers (EFSA FEEDAP Panel, 2012), 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 FEEEDAP 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, 2018a), 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).

3. Assessment

25‐OH‐D3 produced with P. autotrophica DSM 32858 is intended to be used as a nutritional additive (functional group: vitamins, pro‐vitamins and chemically well‐defined substances having similar effect) in feed and water for all pigs, all poultry for fattening and ornamental birds and other poultry species as a source of Vitamin D3.

3.1. Characterisation

3.1.1. Characterisation of Pseudonocardia autotrophica DSM 32858

The active substance is produced using a non‐genetically modified strain of P. autotrophica which is deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession number DSM 32858. 7

■■■■■

■■■■■ 8

The minimum inhibitory concentration (MIC) of P. autotrophica DSM 32858 to the list of antibiotics for ■■■■■ Consequently, the production strain is considered to be resistant to these antibiotics. 9

The WGS data of the production strain was interrogated for the presence of antimicrobial resistance (AMR) genes against ■■■■■ 10 No genes of concern were identified. ■■■■■ these resistances raise no safety concerns since no acquired AMR genes were found in the WGS.

The WGS data of P. autotrophica DSM 32858 was also examined for virulence factors ■■■■■ No genes of concern were identified. 10 , 11

■■■■■ No antimicrobial activity was detected. 12

3.1.2. Manufacturing process

■■■■■

3.1.3. Characterisation of the active substance

The active substance is 25‐OH‐D3.

The Chemical Abstracts Service (CAS) number is 19356–17‐3, the European Inventory of Existing Commercial Chemical Substances (EINECS) number is 242–990‐9, molecular formula C27H44O2 and the molecular weight 400.64 g/mol. It has a melting point of 107–109°C and a boiling point of 592.25°C. It is insoluble in water. 13

The structural formula of the active substance is presented in Figure 1.

An external file that holds a picture, illustration, etc. Object name is EFS2-21-e08050-g001.jpg

Structural formula of 25‐hydroxycholecalciferol (25‐ OH‐D3)

No purity data on the active substance were provided by the applicant.

3.1.4. Characterisation of the additive

The final formulation of the additive contains a stabilised form of the active substance (25‐OH‐D3) at a minimum of 1.25%. Maltodextrin is used as a carrier. In addition to 25‐OH‐D3, the final formulation of the additive also contains other sterol derivatives, including residues of cholecalciferol (the starting substrate) and other sterols produced in minor amounts during the manufacturing process.

The applicant proposed specifications of the additive as follows: content of 25‐OH‐D3 not less than (NLT) 1.25%, 1α,25‐dihydroxycholecalciferol (1α,25‐(OH)2‐D3) not more than (NMT) 0.03%, cholecalciferol NMT 0.5%, loss of drying NMT 7%. Other sterol derivatives might be present and should be NMT 40% (of 1.25% of 25‐OH‐D3) expressed as the area per cent of the corresponding chromatographic peak (% HPLC area), assuming the sum of chromatographic areas of all detected sterols as 100%, any individual sterol derivative NMT 10% expressed as % HPLC area.

Analytical data to confirm the specifications were provided for five batches of the additive, showing the following average values: 25‐hydroxycholecalciferol mean 1.3% (range 1.28–1.32%), 1α,25‐dihydroxycholecalciferol mean 0.015% (range 0.013–0.017%), cholecalciferol mean 0.34% (range 0.3–0.4%), total related sterols (expressed as % LC area) mean 18.6% (range 16–21%), any individual sterol (expressed as % HPLC area) mean 5% (range 4–6%). 14

In addition, the applicant sent certificates of analysis for five pilot scale batches to confirm compliance with specifications. 15

Three pilot scale batches of the additive were analysed for the content of crude protein (mean: 7.34%; range: 6.52–8.33%), total of fat and crude fibre (mean: < 10%), total ash (mean: 6.62%; range: 3.11–9.97%) and moisture (mean: 4.02%; range: 3.78–4.23%). 16

Three batches of the additive were analysed for cadmium < 0.01 mg/kg, lead < 0.05 mg/kg, mercury < 0.005 mg/kg and arsenic < 0.04 mg/kg. Individual aflatoxin (B1, B2, G1, G2) levels were < 1 μg/kg and the total aflatoxin level was < 1.5 μg/kg. Polychlorinated dibenzo‐p‐dioxins and dibenzofurans (PCDD/PCDF) were 0.129 (0.128–0.131) ng TEQ‐WHO/kg and dioxin‐like polychlorinated biphenyls (PCBs) were 0.123 ng TEQ‐WHO/kg resulting in a total of 0.252 (0.251–0.254) ng TEQ‐WHO/kg PCDD/F/PCB (expressed as 88% dry matter) in all batches. 17

Microbiological contamination was analysed in six batches of the additive by determination of total aerobic counts (1.06 × 103 CFU/g; range 3.5 × 102–2.4 × 103 CFU/g), coliforms (< 30 CFU/g) and yeasts and mould counts (< 102 CFU/g). Escherichia coli and Salmonella spp. were not detected in 25 g. 18

■■■■■ 19

The Panel considered that the microbial contamination and the amounts of the detected impurities do not raise safety concerns.

The presence of viable cells of P. autotrophica DSM 32858 was investigated in three batches of the product tested in triplicate with a total of nine samples. 20 , 21 For each sample, 10 g of the product was suspended in sterile water to reach a final volume of 100 mL. Then 10 mL (corresponding to 1 g of the product) were centrifuged, the pellet suspended in 0.2 mL, and used to inoculate nutrient agar plates. Incubation was done at 28°C for 8 days. A proper positive control was included in the analysis. Colonies were found in the non‐spiked samples analysed and, according to the applicant, they were identified and excluded to belong to P. autotrophica species. However, the FEEDAP Panel notes that the identification of the colonies was not performed using molecular methods, but by morphology, microscopic analysis and using the semi‐automatic identification system BD BBL Crystal Identification system. Those methods are considered not conclusive and would not allow to unequivocally exclude the presence of viable cells of the production strain in the additive. Therefore, uncertainty remains on the possible presence of viable cells of P. autotrophica DSM 32858 in the final product.

The applicant also investigated the presence of DNA of P. autotrophica DSM 32858 in three batches of the product tested in triplicate. The extraction procedure included a lysis step using glass beads. The primers amplified a specific segment of the Pseudonocardia spp. 16S rRNA gene of 645 bp with a limit of detection of 10 ng of genomic DNA/mL. Relevant controls were included in the analysis. DNA of P. autotrophica DSM 32858 was not detected. 22 , 23

3.1.5. Physical properties of the additive

The bulk and tapped density were measured on five pilot scale batches of the additive and showed on average 351 kg/m3 (332–386 kg/m3) and 403 kg/m3 (377–438 kg/m3), respectively.

The dusting potential of three pilot scale batches (four replicates per batch) of the additive was determined using the Stauber‐Heubach method and showed values on average of 42 mg/m3 (range 30–60 mg/m3) (mg airborne dust per m3 of air). 24 The dust of the same three batches (four replicates per batch) of the additive was tested to evaluate the concentration of 25‐OH‐D3 and showed values on average of 2.62 mg 25‐OH‐D3/g (range 1.85–3.26 mg/g). 25

The particle size of the additive was analysed by laser‐diffraction method: 10% of the particles are smaller than 13.21 μm, 50% of the particles are smaller than 58.72 μm, 90% of the particles are smaller than 214 μm; the average particle size was 92.72 μm. 26

The applicant analysed the final formulation of the additive (containing 25‐OH‐D3 at a minimum of 1.25% and maltodextrin) by Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) 27 The analysis showed that the sample contained predominantly amorphous material as well as a low level of small particles which are classified as nanoparticles as they have one or more external dimension in the nano size range. However, it was not possible to differentiate the 25‐OH‐D3 particles (insoluble in water) from the particles of the maltodextrin (very soluble in water) used as carrier. Therefore, data does not allow to conclude whether 25‐OH‐D3 contains a fraction of small particles including nanoparticles.

3.1.6. Stability and homogeneity

3.1.6.1. Shelf life of the additive

The shelf life of the additive was studied on three batches when stored at 25°C (60% relative humidity ‐ RH) for 24 months and at 40°C (75% RH) for 6 months. 28 The stability in terms of loss of drying (< 7%, ranging 3.6–5.5%), content of 25‐OH‐D3 (> 1.25%, ranging 1.32–1.43%), cholecalciferol (< 0.5, ranging 0.13–0.16%), related sterols (< 40%, ranging 19–22%) and content of 1α,25‐dihydroxycholecalciferol (< 0.03, ranging 0.017–0,021%) was demonstrated under the two conditions studied.

3.1.6.2. Stability of the additive in premixtures and feedingstuffs

Stability in premixtures

The stability of the additive in premixtures for pigs and poultry was studied in three batches when supplemented at 627.3 mg 25‐OH‐D3/kg and stored at 25°C (60% RH) for 24 months and at 40°C (75% RH) for 6 months in standard packaging. 29 Losses in terms of content of 25‐OH‐D3 ranged 3.2–11.3% after 24 months storage at 25°C (60% RH) and 11.9–13.9% after 6 months storage at 40°C (75% RH).

Stability in feeds

The stability of three batches of the additive in mash and pelleted feed for poultry was investigated at two different inclusion levels of 50 or 100 μg 25‐OH‐D3/kg and when stored at 25°C (60% RH) for 3 months and at 40°C (75% RH) for 4 weeks. 30 At the lowest inclusion level (50 μg/kg) the losses of 25‐OH‐D3 ranged from 0% to 8.4% after 3 months at ambient temperature, while when stored at 40°C, after 4 weeks the loss ranged from 7.7% to 23.2%, for mash feed. For pelleted feed, the loss after 3 months at ambient temperature ranged from 11.6% to 14.7% and after 4 weeks at 40°C ranged from 15.8% to 22.9%. At the highest inclusion level (100 μg/kg) the loss of 25‐OH‐D3 ranged from 3.6% to 4.4% after 3 months at ambient temperature, while when stored at 40°C, after 4 weeks the loss ranged from 17.2% to 18.1%, for mash feed. For pelleted feed, the loss after 3 months at ambient temperature ranged from 12.2% to 21.1% and after 4 weeks at 40°C ranged from 15.4% to 18.4%.

The same conditions as described above were also applied to mash and pelleted feed for pigs but with different inclusion levels (25 or 50 μg 25‐OH‐D3/kg). At the lowest inclusion level (25 μg/kg) the losses of 25‐OH‐D3 ranged from 2.4% to 18% after 3 months at ambient temperature, while when stored at 40°C, after 4 weeks the loss ranged from 7.3% to 20%, for mash feed. For pelleted feed, the loss after 3 months at ambient temperature ranged from 0% to 2.5% and after 4 weeks at 40°C ranged from 2.7% to 12.8%. At the highest inclusion level (50 μg/kg) the losses in terms of content of 25‐OH‐D3 ranged from 11.6 to 14.4% after 3 months at ambient temperature, while when stored at 40°C, after 4 weeks the loss ranged from 16.8% to 20.4%, for mash feed. For pelleted feed, the loss after 3 months at ambient temperature ranged from 0% to 4.9% and after 4 weeks at 40°C ranged from 7.4% to 12.5%.

The applicant has also provided data on the effects on stability of the pelleting process (at 80°C) in feed for poultry and pigs. The loss of 25‐OH‐D3 ranged between 0% and 14.7% and from 0% to 11.5% for poultry and pigs feeds, respectively.

3.1.6.3. Stability of the additive in water for drinking

The stability of the additive in water for drinking was studied in two types of water: soft water (pH between 5–7 and ≤ 60 mg/mL of calcium carbonate) and hard water (pH 8–9 and 180–350 mg/L of calcium carbonate) supplemented with 25‐OH‐D3 at 0.01%. Samples were stored at ambient temperature and refrigerated (between 3 and 8°C) for 48 h. The losses in terms of content of 25‐OH‐D3 in the soft water was 9.7% at ambient temperature after 48 h and 10.7% and 21% in the refrigerated sample after 24 and 48 h, respectively. In the hard water samples, there were no losses at ambient temperature or after 24 h refrigerated but after 48 h refrigerated the loss was 16.5%.

3.1.6.4. Homogeneity

The capacity for homogeneous distribution of the additive, was evaluated in feeds for poultry and pig in two presentations each (mashed and pelleted) and supplemented with two levels of inclusion. Poultry feed was supplemented with 74–97 μg/kg or 161–205 μg/kg and pigs feed with 45–52 μg/kg or 83–92 μg/kg. The coefficient of variation (CV) was calculated in 10 subsamples of each feed. At the lowest inclusion level (74–97 μg/kg), the CV ranged from 3 to 5.9 and from 1.8 to 4.3 for mashed and pellet poultry feed respectively and from 3.5 to 7.4 and from 3.4 to 4.8 for mashed and pellet pigs feed respectively. At the highest inclusion level (161–205 μg/kg), the CV ranged from 5 to 6.1 and from 2 to 6.2 for mashed and pellet poultry feed, respectively and from 3.5 to 4.5 and from 2.3 to 3.7 for mashed and pellet pigs feed, respectively. 30

3.1.7. Conditions of use

The additive is intended for use in feed for the following animal species/categories:

  • —
    Poultry for fattening and ornamental birds: the additive is intended to be used to provide a typical content of 34.85 μg of 25‐OH‐D3/kg feed and a maximum content of 100 μg of 25‐OH‐D3/kg feed. Considering that water intake would be 2–3 times higher than feed intake (DM), the typical inclusion level in water for drinking would correspond to 11.6–17.5 μg of 25‐OH‐D3/L and the maximum content to 33.3–50 μg of 25‐OH‐D3/L in water for drinking.
  • —
    Poultry for laying and breeding: the additive is intended to be used to provide a typical content of 34.85 μg of 25‐OH‐D3/kg feed and a maximum content of 80 μg of 25‐OH‐D3/kg feed. Considering that water intake would be 2–3 times higher than feed intake (DM), the typical inclusion level in water for drinking would correspond to 11.6–17.5 μg of 25‐OH‐D3/L and the maximum content to 26.6–40 μg of 25‐OH‐D3/L in water for drinking.
  • —
    Pigs: the additive is intended to be used to provide a typical content of 25 μg of 25‐OH‐D3/kg feed and a maximum content of 50 μg of 25‐OH‐D3/kg feed. Considering that water intake would be 2–3 times higher than feed intake (DM), the typical inclusion level in water for drinking would correspond to 8.4–12.5 μg of 25‐OH‐D3/L and the maximum content to 16.6–25 μg of 25‐OH‐D3/L in water for drinking.

The maximum amount of the combination of 25‐OH‐D3 with vitamin D3 31 should be:

  1. ≤ 125 μg (equivalent to 5,000 IU of vitamin D3) for chickens for fattening and turkeys for fattening.
  2. ≤ 80 μg (equivalent to 3,200 IU of vitamin D3) for poultry for laying and breeding.
  3. ≤ 50 μg (equivalent to 2,000 IU of vitamin D3) for pigs.

3.2. Safety

3.2.1. Toxicological studies

Genotoxicity studies

The applicant has provided two Ames tests 32 , 33 and an in vitro micronucleus test. 32 One of the two Ames tests was performed with the active substance produced by the strain under assessment (P. authotrophica DSM 32858). The test item used in the other studies was the final formulation of the additive containing 25‐OH‐D3, produced enzymatically during fermentation of ■■■■■.

Due to the lack of confirmation of the toxicological equivalence between the ■■■■■, used in some of the genotoxicity studies, and the strain under assessment, P. autotrophica DSM 32858, the FEEDAP Panel considered that only the Ames test conducted with 25‐OH‐D3 produced by the strain under assessment can be further considered for the risk assessment.

In addition, the in vitro micronucleus test provided by the applicant, beside the limitation of the test item used, showed: ■■■■■ The Panel noted that these results did not fulfil the criteria of OECD TG 487 for a clearly positive or a clearly negative result, and therefore judged the study as equivocal.

Bacterial reverse mutation assay

25‐OH‐D3, produced by fermentation of P. autotrophica DSM 32858, was tested for the induction of reverse mutations ■■■■■ 33 ■■■■■

■■■■■

Repeated dose toxicity studies

The applicant submitted a 28‐day 34 and a 90‐day 35 toxicity studies to support the safety of the additive for the consumers.

The FEEDAP Panel noted that both studies were performed with ■■■■■ The FEEDAP Panel considered the lack of confirmation of the toxicological equivalence between the ■■■■■ and the strain under assessment, P. autotrophica DSM 32858. Moreover, this test item is not representative of the additive under assessment and does not reflect the realistic manufacturing conditions. Therefore, the studies submitted cannot be further considered for risk assessment.

3.2.2.

3.2.1.1. Conclusion on Toxicology

Based on the data available, the FEEDAP Panel concluded that 25‐OH‐D3 did not induce gene mutations, provided that it is not in the nano form accordingly to the criteria described in the relevant EFSA Guidance (see Section 4.2.1 of the Guidance on technical requirements for regulated food and feed product applications to establish the presence of small particles including nanoparticles; EFSA Scientific Committee, 2021). Due to the lack of adequate data, the FEEDAP Panel cannot conclude on the potential of 25‐OH‐D3 to induce chromosomal damage. The toxicological dataset provided does not allow to conclude on possible adverse effects.

3.2.3. Safety for the target species

In support of the safety for the target species, the applicant provided a combined tolerance/efficacy study 36 , 37 conducted in chickens for fattening and results of a literature search. The test item used in the study was the final formulation of the additive containing 25‐OH‐D3, ■■■■■

Due to the lack of confirmation of the toxicological equivalence between the ■■■■■ and the strain under assessment, P. autotrophica DSM 32858, the FEEDAP Panel did not further consider this study for the risk assessment.

The results of the literature search performed did not show any negative effect of 25‐OH‐D3 when given to chickens for fattening up to 5,520 IU/kg feed (138 μg/kg), 31 to laying hens up to 9,000 IU/kg (225 μg/kg) and to piglets up to 40,000 IU/kg feed (1,000 μg/kg). However, the studies provided present some limitations (e.g. limited number of replicates, the source of 25‐OH‐D3 used in the studies is not clearly reported or described, not clear study design), which prevents them to be used to support the safety of the additive under assessment for the target species.

3.2.3.1. Conclusions on safety for the target species

Due to the lack of adequate data, the FEEDAP Panel cannot conclude on the safety of the additive for the target species. Moreover, the FEEDAP Panel considered that uncertainties remain on genotoxicity and on the possible presence of viable cells of P. autotrophica DSM 32858 in the final product.

3.2.4. Safety for the consumer

3.2.4.1. Absorption, distribution, metabolism and excretion

No ADME studies performed with the additive under assessment were carried out. The applicant submitted reviews and experimental studies retrieved from the literature that are herein described.

Humans

In humans and other species, the intestinal absorption of 25‐OH‐D3 is consistently higher than that of vitamin D3. 25‐OH‐D3 is absorbed via the vena porta and vitamin D3 via the lymph pathway and this can in part explain the greater bioavailability of 25‐OH‐D3. The mean increase of 25‐OH‐D3 in human serum (considering nine randomised control studies reviewed) after intake of 1 μg vitamin D3 per day was 1.53 ± 0.89 nmol/L, whereas the mean increase after oral ingestion of 1 μg 25‐OH‐D3 per day was 4.76 ± 1.17 nmol/L. Based on this calculation, the overall relative potency of oral 25‐OH‐D3 was 3.1 fold greater when compared with oral vitamin D3 (Quesada‐Gomez and Bouillon, 2018).

25‐OH‐D3 is the major circulating form of vitamin D3, formed mainly in the liver from vitamin D3 by hydroxylation catalysed by the cytochrome P450 enzyme, the vitamin D3 25‐hydroxylase (CYP2R1). This reaction can also occur in intestine and kidney at a lower extension. From the liver, 25‐OH‐D3 enters the circulatory system transported by the vitamin D‐binding protein to the kidney where a second hydroxylation at the C‐1 position occurs by the 25‐OH‐D3‐1‐α‐hydroxylase (CYP27B1), originating 1α,25‐(OH)2‐D3. In kidney, another hydroxylated vitamin D metabolite is produced, 24R,25‐(OH)2D3, catalysed by the 25‐hydroxyvitamin D3‐24R‐hydroxylase. CYP24A1 can also metabolise 25‐OH‐D3 and 1α,25‐(OH)2‐D3 into 24‐hydroxylated metabolites. In vertebrates, 1α,25‐(OH)2‐D3 and 24R,25‐dihydroxyvitamin D3 (24R,25‐(OH)2D3) are secreted from kidney into blood. Many other metabolites are formed, being the structure of 37 compounds already elucidated (Norman and Henry, 2007). Later, more than 50 metabolites were referred to be already identified, being calcitroic acid and lactones the final metabolites of this complex biotransformation pathways (Quesada‐Gomez and Bouillon, 2018).

Vitamin D and its metabolites are excreted mainly in the faeces with the aid of bile salts, only a small fraction being excreted in the urine.

In humans, the blood half‐life of 25‐OH‐D3 is of 15 days while it is of 4–6 h for vitamin D (Jones, 2008).

Besides the reviews above referred, the applicant also submitted some experimental studies, mainly comparing the absorption of vitamin D3 with 25‐OH‐D3.

Rats

In the rat, Maislos et al. (1981) studied the absorption of vitamin D3 and of two hydroxylated metabolites (25‐OH‐D3, and 1,25‐(OH)2‐D3) from intestine into the mesenteric lymphatic and portal venous systems. With this aim, rats with mesenteric lymph fistula and portal vein cannulation were intraduodenally given the same dose of the labelled compounds. Mesenteric lymph was collected 30 min before the administration of the compounds, every 15 min in the first hour and every 30 min thereafter up to 4 h after administration. Also, blood samples were taken from both the portal and iliac veins at 0, 15, 30, 45, 60 min and every 30 min thereafter. After 4 h, the rats were killed and organs exsanguinated and removed (heart, lung, liver, spleen, kidney, duodenum, jejunum, ileum, epididymal fat and skeletal muscle) for analysis. Radioactivity was measured in lymph, serum, and dried chloroform extract of tissue homogenates. In the lymph, the peak concentration of compounds was 66, 110, and 190 pmol/h, attained at 15–30, 45–60, and 150–160 min for 1,25‐OH‐D3, 25‐OH‐D3, and vitamin D3, respectively. The data show that vitamin D3 was slowly absorbed as compared with its hydroxy metabolites. In tissues, the largest amount of vitamin D and 25‐OH‐D3 was present in the intestinal wall, mainly in jejunum. Both hydroxy compounds were distributed in liver and kidney, being the levels very low in the other tissues. The kidney extract showed an additional, more polar metabolite of 25‐OH‐D3 that was not identified. The sum of the concentrations of 25‐OH‐D3 measured in blood, liver, kidney and heart 4 h after intraduodenal administration was approximately the double of vitamin D3 (10.4% and 5.4% of the administered dose, respectively). Concluding, 25‐OH‐D3 and 1,25‐(OH)2‐D3, are mainly absorbed directly into portal blood and in a higher extension as compared with vitamin D3.

Target species

Studies in target species were also submitted, two in pigs with performance evaluation purposes (Lauridsen et al., 2010; Witschi et al., 2014) and one in birds (Bar et al., 1980).

Lauridsen et al. (2010) carried out a study in gilts from the first oestrus until day 28 of gestation and in pregnant multiparous sows from the first day of mating until weaning by giving in the diet several levels of vitamin D3 or 25‐OH‐D3 (200, 800, 1,400, 2,000 IU each). Blood samples and uterine fluids were collected from gilts at day 28 and blood at several days from sows, and also from piglets at days 4, 16 and 28 of age. Both vitamins were analysed in plasma and uterine fluids of gilts by an isotope dilution assay with HPLC‐MS (LOQ: 5 ng/mL). The levels of 25‐OH‐D3 increased linearly in plasma of gilts with increasing dietary levels of the two compounds, although in gilts supplemented with 25‐OH‐D3, the plasma level of 25‐OH‐D3 was significantly higher than in gilts supplemented with vitamin D3. No detectable 25‐OH‐D3 was found in uterine fluid samples.

25‐OH‐D3 in plasma of sows and piglets was measured by HPLC after saponification and extraction into heptane. In plasma of sows the levels of 25‐OH‐D3 were significantly higher in animals given 25‐OH‐D3 than in those given vitamin D3 in diet at 800 IU and higher. In plasma of piglets, 25‐OH‐D3 was only detected in 154 of 576 samples analysed, being the concentrations very low, although higher in piglets born of sows fed with 25‐OH‐D3 (mean 4.3 ng/mL). These data show that very low levels of the compounds were transferred to the progeny.

Concluding, at concentrations higher than 200 IU, 25‐OH‐D3 showed to be more bioavailable than vitamin D3 both in gilts and in sows, being the plasma levels of 25‐OH‐D3 increased by a factor of 2 to 3 when the 25‐OH‐D3 was included in the diet.

Witschi et al. (2014) carried out a study in piglets by comparing the effects of vitamin D3 with 25‐OH‐D3 on growth, blood status and various bone traits of pigs from birth to 77 days of age. With this aim, primiparous and multiparous sows (13 in each treatment) were given diets supplemented with either 5 or 50 μg of vitamin D3 or 50 μg of 25‐OH‐D3 per kg feed. By week 3 of lactation, piglets had access to a creep diet containing the same levels of the two vitamins as the corresponding dams and after weaning up to reaching a BW of approximately 20 kg. Blood was collected from randomly selected piglets of each group treatment on days 21, 33 (just before weaning) and 77 of age. 25‐OH‐D3 was measured in serum by an isotope dilution assay using a reversed‐phase HPLC‐mass spectrometry. Serum levels of 25‐OH‐D3 of piglets from sows given 25‐OH‐D3 were numerically higher as early as day 21, and significantly higher in the following days 33 and 77, as compared with the two groups given vitamin D3. At day 77, serum levels of piglets given diet supplemented with 25‐OH‐D3 or vitamin D3 were ~ 50 ng/mL and 15 ng/mL, respectively. Serum concentrations of 25‐OH‐D3 decreased numerically between day 21 and day 33 in both groups given diets with vitamin D3 but not in the group given 25‐OH‐D3.

The absorption and excretion of vitamin D3 and 25‐OH‐D3 was studied in chicks and turkeys and their metabolism was tentatively evaluated (Bar et al., 1980). Animals were fed diets deficient in vitamin D3 from day 1 to day 14. In the following 6 days, they received the same diet supplemented with labelled vitamin D3 at 15 or 50 μg/kg feed or labelled 25‐OH‐D3 at 50 μg/kg feed. At the end of day 6, animals were killed, intestine removed and divided in five segments, the contents collected for radioactivity measurement.

The absorption of 25‐OH‐D3 was significantly higher than vitamin D3 in chicks (84% vs. 67%) and (84% vs. 75%) in turkeys. The absorption of both compounds was almost complete in the upper jejunum in both species. 25‐OH‐D3, polar metabolites and non‐polar metabolites and esters were secreted in duodenum, in significantly greater levels in turkeys than in chicks. Some of these metabolites were reabsorbed, mainly in the upper jejunum. These metabolites were analysed by thin layer chromatography (TLC), thus their identity was not clarified. The overall daily excretion of vitamin D3 metabolites was significantly higher (20% and 14% of the daily intake in turkeys and chicks, respectively) as compared with 7% excretion of 25‐OH‐D3 metabolites.

Conclusions on ADME

Overall, the limited data available on ADME of 25‐OH‐D3 in animals show that it is more rapidly and extensively absorbed than vitamin D3, is metabolised to several hydroxylated metabolites, has a longer blood half‐life, being mainly excreted in faeces as calcitroic acid and lactones. The same pattern of disposition seems to happen in humans.

If nanoparticles of 25‐OH‐D3 are present, they are expected to partition and quickly solubilise into the lipophilic cell compartments, suggesting that systemic distribution of particles is unlikely to occur.

3.2.4.2. Residue studies

The applicant has provided information on residues of 25‐OH‐D3 derived from a combined tolerance/efficacy study conducted in chickens for fattening. Publications were retrieved through a literature search reporting data on residues in tissues and organs of pigs, birds and ruminants, as well as residues of 25‐OH‐D3 in milk. Reference was made to the previous EFSA FEEDAP Panel Opinions (EFSA, 2005, 2009) where, residue data for eggs and tissue and organs of pigs could be identified.

When evaluating the studies, the FEEDAP Panel considered that (i) 25‐OH‐D3 contributes to the exposure of the consumer to vitamin D3, for which a UL is available, (ii) other forms of vitamin D3 are available on the market and (iii) residues of vitamin D3 in tissues and products from target species other than poultry and pigs contribute to the exposure of the consumer; therefore, studies reporting residue data in target species different from pigs and poultry were also considered. Among the studies submitted, the FEEDAP Panel selected the studies with the more relevant design (e.g. supplementation level) and among those, the studies which showed the highest values for the residues of 25‐OH‐D3 in tissues, organs and products for each class (mammals, birds).

The studies considered by the FEEDAP Panel as relevant for the estimate of the consumer exposure are herein described.

Regarding poultry, recent residue data were available from the combined tolerance‐efficacy study performed with the additive containing 25‐OH‐D3 produced by ■■■■■ (Schothorst, 2019; 38 Section 3.2.2). ■■■■■

Celi et al. (2018) 39 fed growing cattle (106 kg bw, 10 animals/group) for 90 days with increasing doses of 25‐OH‐D3: 1.7 μg, 5.1 μg and 8.5 μg/kg bw (corresponding to 56.4, 168.7 and 249.1 μg/kg feed, respectively). The control group received 0.75 μg vitamin D3/kg bw. Serum 25‐OH‐D3 increased from 46 (control group without calcidiol) to 107, 188 and 217 ng/mL for the 25‐OH‐D3 supplemented groups, respectively. All calves in the four groups gained weight continually: no growth depression was observed. No adverse effects of 25‐OH‐D3 were observed for any of the haematology 40 and serum chemistry parameters41 measured monthly or during the routine clinical examinations. In the post‐mortem evaluation, no adverse effects of the different 25‐OH‐D3 doses were observed, neither during the gross pathology nor in the histopathological examination. The data allow the conclusion that about 10,000 IU vitamin D from 25‐OH‐D3 was well tolerated by growing cattle. Serum, fat, muscle, kidney and liver samples (10 per treatment group) were collected to evaluate the concentration of 25‐OH‐D3. The concentrations of 25‐OH‐D3 at day 90 of treatment in liver, muscle, kidney and fat were significantly increased in comparison to the control group (Table 2).

Table 2

Tissue content of 25‐OH‐D3 (μg/kg), 10 samples per treatment

TissueControl56.4 μg 25‐OH‐D3/kg feed168.7 μg 25‐OH‐D3/kg feed249.1 μg 25‐OH‐D3/kg feed
Liver 4.5c 14.5b 27.4a 31.2a
Kidney 7.2c 23.1b 44.0a 39.7a
Muscle 1.8c 5.7b 10.8a 12.3a
Fat 4.1c 13.2b 20.7a 26.4a

Different letters within a row indicate statistical significance (p < 0.05).

For calculating consumer exposure, an average between the groups with 56.4 and 168.7 μg 25‐OH‐D3/kg feed was used (liver: 21.0, kidney: 33.6, muscle 8.3, fat 17.0 μg/kg) as representative of the value expected from the use of 25‐OH‐D3 at the concentration of 100 μg/kg complete feed which correspond to the maximum authorised concentration for vitamin D3 of 4,000 IU/kg complete feed. 42 Since these values were higher than those reported in studies available with pigs (Jacobsen et al., 2007; Höller et al., 2010; Burild et al., 2016; von Rosenberg et al., 2016; Duffy et al., 2018a,b), values from cattle study were taken for the consumer exposure assessment.

In the study by Rodney et al. (2018), 43 a total of 25 mid‐lactation Holstein dairy cows (blocked by age and milk production) were randomly distributed to five treatment groups. The diet of the control group was left unsupplemented while the diet of the other four was supplemented with 0.5, 1, 2 or 4 mg 25‐OH‐D3/cow per day for 30 days. Data on feed intake were not provided. Corresponding levels in a complete feed could be calculated based on the default values for daily feed intake of dairy cows of DM content of 88% and 20 kg DM/day (EFSA FEEDAP Panel, 2017a,b,c) and were 22, 44, 88, and 176 μg/kg complete feed. Milk volume was measured, and milk samples taken every 2 weeks to analyse milk levels of protein and fat, as well as protein and fat yield, somatic cell count and 25‐OH‐D3 concentrations. The 25‐OH‐D3 concentration in milk of the group fed with 88 μg 25‐OH‐D3/kg complete feed was taken as representative of the value expected from the use of 25‐OH‐D3 at the concentration of 100 μg/kg complete feed which correspond to the maximum authorised concentration for vitamin D3 of 4,000 IU/kg complete feed. Following the guidance, two times the SD was added to the mean resulting in 0.465 (0.4145 + 2 × 0.0254) μg 25‐OH‐D3/kg milk.

3.2.4.3. Assessment of consumer exposure

In its first opinion (EFSA, 2005), the FEEDAP Panel proposed a provisional upper tolerable limit (UL) for 25‐OH‐D3 of 10 μg/day in adults and adolescents (11–17 years) and 5 μg/day in children (0–10 years). This was based on the UL for vitamin D3 (50 μg/day in adults and 25 μg/day in children up to 11 years) and a relative biological activity factor of 5.

In the opinion of 2009, the FEEDAP Panel conducted a ‘worst‐case scenario’ exposure assessment for the consumer, based on the consumption model described in Regulation (EC) No 429/2008 and on data from studies done with the additive at the maximum use levels for pigs and poultry species. The results indicated that exposure of adults was below the provisional UL for 25‐OH‐D3 set for adults (69%) but that of children would be exceeded (138%). A refined calculation of exposure based on more realistic data (SCOOP) indicated that exposure for both adults and children would be below the provisional UL (24% and 49%, respectively). Based on that, the Panel concluded that the total exposure resulting from the use of 25‐OH‐D3 in all poultry and pig categories at the maximum levels would not represent a risk for the consumer.

The FEEDAP Panel notes that the NDA Panel of EFSA has revised the UL levels for vitamin D3 for all age groups (EFSA NDA Panel, 2012, 2018): 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 women.

The FEEDAP Panel would like to withdraw the provisional UL for 25‐OH‐D3, since also cholecalciferol from other food and body's own synthesis would enter the body's store of 25‐OH‐D3. It is therefore considered reasonable to apply the UL for vitamin D3 and to introduce the 25‐OH‐D3 intake multiplied with a biopotency factor of 5.

In reassessing consumer exposure, the FEEDAP Panel is aware of the ongoing evaluation by the NDA Panel of setting a conversion factor for 25‐OH‐D3 into vitamin D3. At the time of the adoption of the current FEEDAP Panel's scientific opinion, the work of the NDA Panel has not been completed. Therefore, as a pragmatic approach, the Panel considered that the residues of 25‐OH‐D3 deposited in edible tissues and products should be expressed in terms of vitamin D3 activity, and therefore, multiplied by 5 to consider the relative biological activity of the different compounds (Table 1). This was then compared with the UL established by the NDA Panel for vitamin D3.

Table 1

■■■■■

■■■■■ ■■■■■ ■■■■■ ■■■■■ ■■■■■
■■■■■ ■■■■■ ■■■■■ ■■■■■ ■■■■■ ■■■■■
■■■■■ ■■■■■ ■■■■■ ■■■■■ ■■■■■ ■■■■■
■■■■■ ■■■■■ ■■■■■ ■■■■■ ■■■■■ ■■■■■
■■■■■ ■■■■■

New data, from literature search and a tolerance trial performed in chickens, have been made available with regard to the deposition of 25‐OH‐D3 in tissues or products of pigs, poultry and ruminants not considered in the past (see 3.2.3.2). Therefore, the Panel used the new residue data to perform an exposure assessment following the methodology described in the Guidance on consumer safety (EFSA FEEDAP Panel, 2017a). The input data are reported in Table 3.

Table 3

Input data on 25‐OH‐D3 and vitamin D3 content in food of animal origin used for the consumer exposure assessment

Tissue/productμg 25‐OH‐D3/kg wet tissue/productμg Vitamin D3 equivalent/kg wet tissue/product* Reference
Birds fat tissue24.12§ 120.6Schothorst (2019) 38
Birds liver29.36§ 146.8Schothorst (2019) 38
Birds meat** 9.2§ 46Schothorst (2019) 38
Birds offals and slaughtering products (other than liver)25.42§ 127.1Schothorst (2019) 38
Mammals fat tissue1785Celi et al. (2018)
Mammals liver21105Celi et al. (2018)
Mammals meat† 1050Celi et al. (2018)
Mammals offals and slaughtering products (other than liver)33.6168Celi et al. (2018)
Whole eggs13.366.5EFSA (2005)
Milk0.472.35Rodney et al. (2018)
*Calculated from the residues of 25‐OH‐D3 by multiplying with the relative biological activity factor of 5.
**90% breast muscle + 10% skin/fat.
†80% muscle +20% fat.
§Data are the mean + 2 SD.

The results of the dietary exposure to residues of 25‐OH‐D3 calculated as vitamin D3 equivalents for the different population categories are reported in Table 4. The detailed results are given in Appendix A.

Table 4

Chronic human dietary exposure to 25‐OH‐D3 expressed as vitamin D3 equivalents

Population classMaximum HRP* (μg/kg body weight per day)Default body weight† (kg)Exposure (μg/day)Upper tolerable level (μg/day)% UL
Infants0.745052.82511.2
Toddlers0.87591210.515021.05
Other children0.83902319.295038.58
Adolescents0.561752.4** 29.4310029.43
Adults0.42437029.710029.7
Elderly0.32177022.410022.4
Very elderly0.33357023.3410023.34
*HRP: highest reliable percentile
**Average of 43.4 and 61.3 kg.
†EFSA Scientific committee (2012).

The FEEDAP Panel noted that some uncertainties exist in relation to the exposure calculation (e.g. the standard deviations and the levels of 1,25‐(OH)2‐D3, an active metabolite of 25‐OH‐D3, were not available). However, the FEEDAP Panel considered these limitations not having a significant impact on the exposure calculation.

To compare the exposure to residues of 25‐OH‐D3 transformed to vitamin D3 to the UL of vitamin D3, the FEEDAP Panel used the highest reliable percentile (HRP) for the different population categories and converted it from μg/kg body weight (bw) to μg/person and day using the default values for body weight (EFSA Scientific Committee, 2012). For the population group infants, the lowest UL of 5 μg/day has been used. The contribution to consumer exposure to Vitamin D3 from products of animals fed with the additive ranged from 11.2% to 38.58% of the UL (Table 4). 38

The FEEDAP Panel concludes that there is no safety concern for the consumer resulting from the intake of food from pigs, poultry and ruminants fed with 25‐OH‐D3.

3.2.4.4. Conclusions on safety for the consumer

The FEEDAP Panel concluded that the exposure calculation does not indicate any risk for the consumer resulting from the use of food originating from animals fed with 25‐OH‐D3. However, considering the lack of adequate toxicological data and the uncertainties on the possible presence of viable cells of P. autotrophica DSM 32858 in the final product, the FEEDAP Panel cannot conclude on the safety for the consumers.

3.2.5. Safety for the user

No data on respiratory toxicity was provided. Considering the low dusting potential of the additive (up to 60 mg/m3), the FEEDAP Panel considered that exposure through inhalation is unlikely.

The skin irritation potential of the additive was tested in vitro according to OECD TG 439. 44 The results of the study indicated that the formulated additive is not irritant to the skin and is, therefore, classified according to the UN GHS as ’No Category’.

The eye irritation potential of the additive was investigated in vitro according to OECD TG 438. 45 The results of the study showed that the formulated additive is not irritant to eyes and is, therefore, classified according to the UN GHS as ‘No Category’.

The skin sensitisation potential of the additive was investigated in a study conducted in compliance with the OECD TG 429. 30 The results of the study indicated that the formulated additive is not a skin sensitiser.

3.2.5.1. Conclusions on safety for the user

Based on the studies submitted, the additive was shown not be irritant to skin or eyes. It is not a skin sensitiser. Considering the low dusting potential of the additive, the FEEDAP Panel considered that the exposure through inhalation is unlikely.

However, the FEEDAP Panel considered that uncertainties remain on genotoxicity and on the possible presence of viable cells of P. autotrophica DSM 32858 in the final product.

3.2.6. Safety for the environment

The active substance present in the additive occurs in nature, and its use in animal nutrition is not expected to substantially increase the concentration in the environment. The strain P. autotrophica DSM 32858 is non‐genetically modified, and no DNA of the production strain was detected in the additive using a test with a sensitivity of 10 ng of genomic DNA/mL. Although, uncertainty remains on the possible presence of viable cells of P. autotrophica DSM 32858 in the final product, a risk for the environment resulting from the use of the additive under assessment in animal nutrition is not foreseen.

3.3. Efficacy

According to Regulation (EC) No 429/2008, efficacy studies are not required for vitamins, pro‐vitamins and chemically defined substances having similar effects that are already authorised as feed additives under Directive 70/524/EEC.

Since 25‐OH‐D3 is already authorised for chickens for fattening, turkeys for fattening, other poultry and pigs, 46 and EFSA concluded that its use is efficacious as a substitute for vitamin D3 (EFSA, 2005, 2009), no new demonstration of efficacy would be necessary.

The applicant has provided three studies in chickens for fattening to support the efficacy of 25‐OH‐D3. Two of them are available in publications (Goodgame et al., 2011; Leyva‐Jimenez et al., 2019), and the third one is the combined tolerance‐efficacy study 37 already mentioned under Section 3.2.2. The test article used in the three studies in chickens for fattening was a fermentation product containing the active substance 25‐OH‐D3 produced by ■■■■■ (the production strain of the additive under assessment). The Panel considers that these studies could be used to support the efficacy of the additive, as the effects of the active substance would be the same.

In the study included in Leyva‐Jimenez et al. (2019), a challenge was induced to the birds with live coccidiosis vaccine intended to induce intestinal damage. This approach was not considered adequate by the Panel as evidence of the efficacy. Therefore, the study was not considered further for the assessment.

Goodgame et al. (2011) described one bioequivalence trial in which the dietary supplementation of increasing levels of the test item (25‐OH‐D3 produced by P. autotrophica 10 M213) was compared with 25‐OH‐D3 from a different source. One old‐day chicks received a basal diet with no vitamin D containing marginal levels of calcium and phosphorus for 7 days. Thereafter, the basal diet was supplemented with the two 25‐OH‐D3 sources at increasing levels from 2.5 to 80 μg/kg complete feed and offered to the chickens for 14 days. The study assessed the effect of both sources of 25‐OH‐D3 on the zootechnical performance and tibia diameter and breaking force of chickens for fattening. The results showed increases in the tibia ash content with increasing levels of 25‐OH‐D3; no statistical differences were found between the two sources of 25‐OH‐D3.

The tolerance/efficacy trial assessed the effect of the dietary supplementation of chickens for fattening ■■■■■

3.3.1. Conclusions on the efficacy

The FEEDAP Panel concludes that 25‐OH‐D3 is an efficient source of vitamin D3 under the proposed conditions of use.

3.4. Post‐market monitoring

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 Regulation 47 and Good Manufacturing Practice.

4. Conclusions

The production strain P. autotrophica DSM 32858 is not genetically modified however, uncertainties remain on the possible presence of its viable cells in the final product.

Due to the lack of adequate safety data and uncertainty on the presence of nano particles, the FEEDAP Panel cannot conclude on the safety of the additive for the target species and the consumer.

The additive is not irritant to skin or eyes and is not a skin sensitiser and the exposure through inhalation is unlikely. However, the FEEDAP Panel considers that uncertainties remain on genotoxicity and on the possible presence of viable cells of P. autotrophica DSM 32858 in the final product which might have an impact on the safety for the users.

The use of the feed additive is considered safe for the environment.

The additive is regarded as an effective dietary source of the vitamin D3 under the proposed conditions of use.

Abbreviations

AMR
Antimicrobial resistance
BW
body weight
CAS
Chemical Abstracts Service
CFU
colony forming unit
CV
coefficient of variation
dDDH
DNA–DNA hybridization
DM
dry matter
DSMZ
German Collection of Microorganisms and Cell Cultures
EINECS
European Inventory of Existing Chemical Substances
EURL
European Union Reference Laboratory
LOD
limit of detection
MW
molecular weight
NLT
Not less than
NMT
Not more than
OECD
Organisation for Economic Co‐operation and Development
RH
relative humidity
TEM
transmission electron microscopy
TLC
thin layer chromatography
SEM
scanning electron microscopy
VFDB
Virulence Factor Database

Appendix A – Calculation of the consumer exposure with FACE model

A.1. Methodology

As described in the Guidance on the safety of feed additives for consumers (EFSA FEEDAP Panel, 2017a,b,c), consumption data of edible tissues and products as derived from the EFSA Comprehensive European Food Consumption Database (Comprehensive Database) will be used to assess exposure to residues from the use of feed additives in different EU countries, age classes 35 and special population groups. For each EU country and age class, only the latest survey available in the Comprehensive Database will be used.

While the residue data reported for feed additives refer to organs and tissues (raw agricultural commodities (RAC)), the Comprehensive Database includes consumption data for foods as consumed.

In order to match those consumption data with the available residue data for feed additives, the consumption data reported in the Comprehensive Database have been converted into RAC equivalents. For assessing the exposure to coccidiostats from their use in (non‐reproductive) poultry, the following list of commodities is considered: meat, fat, liver, other offals (including kidney).

Depending on the nature of the health‐based guidance derived, either a chronic or acute exposure assessment may be required.

For chronic exposure assessments, the total relevant residues will be combi ned for each individual with the average daily consumptions of the corresponding food commodities, and the resulting exposures per food will be summed in order to obtain total chronic exposure at individual level (standardised by using the individual body weight). The mean and the higher percentile (usually the 95th percentile) of the individual exposures will be subsequently calculated for each dietary survey(country) and each age class separately.

As opposed to the chronic exposure assessments, acute exposure calculation will be carried out foreach RAC value separately. The higher percentile (usually the 95th percentile) exposures based on the consuming days only will be calculated for each food commodity, dietary survey and age class separately.

As described in the Guidance on the safety of feed additives for consumers (EFSA FEEDAP Panel, 2017a,b,c), consumption data of edible tissues and products as derived from the EFSA Comprehensive European Food Consumption Database (Comprehensive Database) will be used to assess exposure to residues from the use of feed additives in different EU countries, age classes 48 and special population groups. For each EU country and age class, only the latest survey available in the Comprehensive Database will be used.

While the residue data reported for feed additives refer to organs and tissues (raw agricultural commodities (RAC)), the Comprehensive Database includes consumption data for foods as consumed. In order to match those consumption data with the available residue data for feed additives, the consumption data reported in the Comprehensive Database have been converted into RAC equivalents. For assessing the exposure to vitamin E from their use in (non‐reproductive) poultry, the following list of commodities is considered: meat, fat, liver, other offals (including kidney).

Depending on the nature of the health‐based guidance derived, either a chronic or acute exposure assessment may be required.

For chronic exposure assessments, the total relevant residues will be combined for each individual with the average daily consumptions of the corresponding food commodities, and the resulting exposures per food will be summed in order to obtain total chronic exposure at individual level(standardised by using the individual body weight). The mean and the higher percentile (usually the 95th percentile) of the individual exposures will be subsequently calculated for each dietary survey(country) and each age class separately.

As opposed to the chronic exposure assessments, acute exposure calculation will be carried out foreach RAC value separately. The higher percentile (usually the 95th percentile) exposures based on the consuming days only will be calculated for each food commodity, dietary survey and age class separately.

Appendix B – Detailed results on chronic exposure calculation

1.

Chronic dietary exposure per population class, country and survey (μg/kg bw per day) of consumers to 25‐OH‐D3 expressed as Vitamin D3 equivalents based on residue data in birds, mammals, eggs and milk (Table B.1).

Table B.1

Chronic dietary exposure per population class, country and survey (μg/kg bw per day) of consumers to 25‐OH‐D3 expressed as Vitamin D3 equivalents based on residue data

Population classSurvey's countryNumber of subjectsHRP valueHRP description
InfantsBulgaria5230.745002231795th
InfantsGermany1420.385080301695th
InfantsDenmark7990.549198487595th
InfantsFinland4270.367825262195th
InfantsItaly90.150955538050th
InfantsUnited Kingdom1,2510.456387179195th
ToddlersBelgium360.652259848990th
ToddlersBulgaria4280.876077225395th
ToddlersGermany3480.644567980695th
ToddlersDenmark9170.672452652195th
ToddlersSpain170.703921278075th
ToddlersFinland5000.672510958195th
ToddlersItaly360.586411817090th
ToddlersNetherlands3220.644617556695th
ToddlersUnited Kingdom1,3140.629804901295th
ToddlersUnited Kingdom1850.582367069295th
Other childrenAustria1280.744438791195th
Other childrenBelgium6250.707768423595th
Other childrenBulgaria4330.839061172695th
Other childrenGermany2930.610311059795th
Other childrenGermany8350.525976244895th
Other childrenDenmark2980.586714776195th
Other childrenSpain3990.657417321395th
Other childrenSpain1560.828702567395th
Other childrenFinland7500.694946603795th
Other childrenFrance4820.689714147695th
Other childrenGreece8380.647068044195th
Other childrenItaly1930.645361128895th
Other childrenLatvia1870.579606900695th
Other childrenNetherlands9570.525847609995th
Other childrenNetherlands4470.533305533595th
Other childrenSweden1,4730.603280231795th
Other childrenCzechia3890.705345139895th
Other childrenUnited Kingdom6510.525352724995th
AdolescentsAustria2370.402353262295th
AdolescentsBelgium5760.308788792395th
AdolescentsCyprus3030.279142724495th
AdolescentsGermany3930.416651587795th
AdolescentsGermany1,0110.300575895495th
AdolescentsDenmark3770.342863301695th
AdolescentsSpain6510.433284847295th
AdolescentsSpain2090.561663952495th
AdolescentsSpain860.417935890595th
AdolescentsFinland3060.328049513795th
AdolescentsFrance9730.401028841295th
AdolescentsItaly2470.412589277395th
AdolescentsLatvia4530.424166062095th
AdolescentsNetherlands1,1420.387276704395th
AdolescentsSweden1,0180.384051249495th
AdolescentsCzechia2980.549098430495th
AdolescentsUnited Kingdom6660.305296090995th
AdultsAustria3080.321567784495th
AdultsBelgium1,2920.281633989095th
AdultsGermany10,4190.286911174095th
AdultsDenmark1,7390.240467161995th
AdultsSpain9810.363767229395th
AdultsSpain4100.342428661095th
AdultsFinland1,2950.293771870695th
AdultsFrance2,2760.291742946195th
AdultsHungary1,0740.402340049395th
AdultsIreland1,2740.288644384695th
AdultsItaly2,3130.258014649195th
AdultsLatvia1,2710.353902082395th
AdultsNetherlands2,0550.290835598095th
AdultsRomania1,2540.411564283095th
AdultsSweden1,4300.324026741895th
AdultsCzechia1,6660.424291013195th
AdultsUnited Kingdom1,2650.237159730095th
ElderlyAustria670.293036898495th
ElderlyBelgium5110.253898407995th
ElderlyGermany2,0060.259249761995th
ElderlyDenmark2740.220505034895th
ElderlyFinland4130.233354179095th
ElderlyFrance2640.249721043295th
ElderlyHungary2060.308613294595th
ElderlyIreland1490.289818606195th
ElderlyItaly2890.230105279895th
ElderlyNetherlands1730.248538853195th
ElderlyNetherlands2890.222636562595th
ElderlyRomania830.321682978895th
ElderlySweden2950.288472342095th
ElderlyUnited Kingdom1660.198291267995th
Very elderlyAustria250.212149053475th
Very elderlyBelgium7040.268428221195th
Very elderlyGermany4900.250959173995th
Very elderlyDenmark120.166783498875th
Very elderlyFrance840.256520086895th
Very elderlyHungary800.311711006895th
Very elderlyIreland770.289867853695th
Very elderlyItaly2280.194719564295th
Very elderlyNetherlands4500.232089390895th
Very elderlyRomania450.333535035790th
Very elderlySweden720.323039257995th
Very elderlyUnited Kingdom1390.211968975095th

Notes

Suggested citation: EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed) , Bampidis V, Azimonti G, Bastos ML, Christensen H, Dusemund B, Durjava M, Kouba M, López‐Alonso M, López Puente S, Marcon F, Mayo B, Pechová A, Petkova M, Ramos F, Sanz Y, Villa RE, Woutersen R, Bories G, Gropp J, Martelli G, Svensson K, Zeljezic D, Anguita M, Casanova JO, Galobart J, Holczknecht O, Innocenti ML, Manini P, Pettenati E, Vettori MV and Pizzo F, 2023. Scientific Opinion on the safety and efficacy of a feed additive consisting of 25‐hydroxycholecalciferol (produced by Pseudonocardia autotrophica DSM 32858) for all pigs, all poultry for fattening and ornamental birds and other poultry species (Huvepharma NV). EFSA Journal 2023;21(6):8050, 24 pp. 10.2903/j.efsa.2023.8050 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

Requestor: European Commission

Question number: EFSA‐Q‐2021‐00641

Panel members: Vasileios Bampidis, Giovanna Azimonti, Maria de Lourdes Bastos, Henrik Christensen, Birgit Dusemund, Mojca Durjava, Maryline Kouba, Marta López‐Alonso, Secundino López Puente, Francesca Marcon, Baltasar Mayo, Alena Pechová, Mariana Petkova, Fernando Ramos, Yolanda Sanz, 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.

Declarations of interest: If you wish to access the declaration of interests of any expert contributing to an EFSA scientific assessment, please contact ue.aporue.asfe@tnemeganamtseretni.

Acknowledgements: The Panel wishes to thank the following for the support provided to this scientific output: Daniel Pagès Plaza.

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

Adopted: 12 May 2023

Notes

1 Regulation (EC) No 1831/2003 of the European Parliament and of the council of 22 September 2003 on the additives for use in animal nutrition. OJ L 268, 18.10.2003, p. 29.

2 Huvepharma NV, Uitbreidingstraat 80, Berchem (Belgium).

3 Commission Regulation (EC) No 887/2009 of 25 September 2009 concerning the authorisation of a stabilised form of 25‐hydroxycholecalciferol as a feed additive for chickens for fattening, turkeys for fattening, other poultry and pigs. OJ 254/68, p. 3.

4 FEED dossier reference: FAD‐2021‐0057.

5 The full report is available on the EURL website: https://joint-research-centre.ec.europa.eu/publications/fad-2021-0057_en

6 Commission Regulation (EC) No 429/2008 of 25 April 2008 on detailed rules for the implementation of Regulation (EC) No 1831/2003 of the European Parliament and of the Council as regards the preparation and the presentation of applications and the assessment and the authorisation of feed additives. OJ L 133, 22.5.2008, p. 1.

7 Technical dossier/Section II/Annex_II.2.2.

8 Technical dossier/Supplementary Information (June 2022)/Section II/Appendix 1.

9 Technical dossier/Section II/Annex_II.2.18.

10 Technical dossier/Supplementary Information (June 2022)/Section II/Appendix 2.

11 Technical dossier/Section II/Annex_II.2.30.

12 Technical dossier/Section II/Annex_II.2.28.

13 https://pubchem.ncbi.nlm.nih.gov/compound/Calcifediol#section=Computed-Properties

14 Technical dossier/Section II/Annex_II.1.1.

15 Technical dossier/Section II/Annex_II.1.2.

16 Technical dossier/Section II/Annex_II.1.4.

17 Technical dossier/Supplementary information (June 2022)/Appendices/Appendix 5.6 and 7.

18 Technical dossier/Supplementary information (June 2022)/Appendices/Appendix 8.

19 Technical dossier/Section II/Annex_II.1.12.

20 Technical dossier/Section II/Annex_II.2.29.

21 Technical dossier/Supplementary Information (June 2022)/Section II/Appendix 4.

22 Technical dossier/Section II/Annex_II.2.19.

23 Technical dossier/Supplementary Information (June 2022)/Section II/Appendix 3.

24 Technical dossier/Section II/Annex_II.1.9.

25 Technical dossier/Section II/Annex_II.1.10.

26 Technical dossier/Section II/Annex_II.1.11.

27 Technical dossier/Supplementary Information (January 2023)/Section II/Appendix 1.

28 Technical dossier/Section II/Annex_II.4.1.

29 Technical dossier/Section II/Annex_II.4.2.

30 Technical dossier/Section II/Annex_II.4.3.

31 40 IU 25‐OH‐D3 = 1 μg 25‐OH‐D3.

32 Technical dossier/Section III/Annex_III_2_9.

33 Technical dossier/Supplementary information (June 2022)/Appendices/Appendix 9.

34 Technical dossier/Section III/Annex_III.2.10.

35 Technical dossier/Section III/AnnexIII.2.11.

36 Technical dossier/Section III/Annex III_1_1.

37 Technical dossier/Section IV/Annex_IV_1_3.

38 Technical dossier/Section III/Annex_III_1_1_Schothorst_2019_CONF.pdf

39 Technical dossier/Section III/Spontaneous submission (February 2023)/Celi et al.pdf

40 WBC, RBC, Haemoglobin, HTC, MCV, MCH, MCHC, platelets, neutrophils, Neu % of WBC, Lymphocytes, Lym % of WBC, monocytes, Mon % of WBC, eosinophils, Eos % of WBC, basophils, Bas % of WBC, large unstained cells, Luc % of WBC, protrombine time, activated partial thromboplastine time.

41 Urea, creatinine, urea/creatinine, glucose, sodium, potassium, chloride, ALP, ALT, AST, LDH, CPK, CGT, total protein, albumin, globulin, A/G, calcium, phosphate, magnesium, cholesterol, amylase, total bile acids.

42 Commission Implementing Regulation (EU) 2017/1492 of 21 August 2017 concerning the authorisation of cholecalciferol as a feed additive for all animal species. OJ L 216, 22.8.2017, p. 19–22.

43 Technical dossier/Section III/Spontaneous submission (February 2023)/Rodney et al., 2018.pdf

44 Technical dossier/Section III/Annex_III.3.3.

45 Technical dossier/Section III/Annex_III.3.1.

46 Commission Regulation (EC) No 887/2009 of 25 September 2009.

47 Regulation (EC) No 183/2005 of the European Parliament and of the Council of 12 January 2005 laying down requirements for feed hygiene. OJ L 35, 8.2.2005, p. 1.

48 Infants: < 12 months old, toddlers: ≥ 12 months to< 36 months old, other children: ≥ 36 months to< 10 years old, adolescents: ≥ 10 years to< 18 years old, adults: ≥ 18 years to< 65 years old, elderly: ≥ 65 years to< 75 years old, and very elderly: ≥ 75 years old.

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