Safety and efficacy of feed additives consisting of vitamin B2 (98%) and vitamin B2 (80%) produced with Bacillus subtilis CGMCC 7.449 for all animal species (Chifeng Pharmaceutical Co., Ltd.)

TL;DR

This study evaluates the safety and effectiveness of vitamin B2 produced using a genetically modified Bacillus subtilis strain for use in animal feed.

Contribution

The study confirms the safety and efficacy of vitamin B2 produced via a genetically modified Bacillus subtilis strain for all animal species.

Findings

The final products do not contain viable cells or DNA of the production strain, posing no safety concerns.

The additives are effective in meeting animals' vitamin B2 requirements when administered via feed.

The additives are dermal and respiratory sensitisers, with inhalation and dermal exposure considered a risk.

Abstract

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) of EFSA was asked to deliver a scientific opinion on the safety and efficacy of vitamin B2 98% and vitamin B2 80% in the form of riboflavin produced by fermentation with a genetically modified strain of Bacillus subtilis (CGMCC 7.449) as nutritional feed additives for all animal species. Viable cells and DNA of the production strain were not detected in the final products and therefore, the use of B. subtilis CGMCC 7.449 to produce vitamin B2 does not raise safety concerns. The use of vitamin 98% and 80% produced with B. subtilis CGMCC 7.449 in animal nutrition does not represent a safety concern for the target species, consumers and for the environment. The additives are not dermal nor eye irritants but are dermal and respiratory sensitisers. Inhalation and dermal exposure are considered a risk. The additives under assessment are effective in covering the animals' requirements of vitamin B2 when administered via feed.

Figures

Structural formula of riboflavin.

Full text

INTRODUCTION

1

Background and Terms of Reference

1.1

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

The European Commission received a request from Chifeng Pharmaceutical Co., Ltd. represented in the EU by Acies Bio d.o.o.,2 for the authorisation of the additives consisting of vitamin B_2_ (riboflavin 98% and 80%) produced with Bacillus subtilis CGMCC 7.449, when used as feed additives for all animal 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 dossier was received on 20 December 2023 and the general information and supporting documentation are available at https://open.efsa.europa.eu/questions/EFSA‐Q‐2023‐00898. The particulars and documents in support of the application were considered valid by EFSA as of 13 May 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 additives comply 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 vitamin B_2_ (riboflavin 98% and 80%) produced with Bacillus subtilis CGMCC 7.449, when used under the proposed conditions of use (see Section 3.1.6).

Additional information

1.2

The additives are preparations containing vitamin B_2_ (98% and 80%) produced with B. subtilis CGMCC 7.449. They have not been previously authorised as feed additives in the European Union.

DATA AND METHODOLOGIES

2

Data

2.1

The present assessment is based on data submitted by the applicant in the form of a technical dossier3 in support of the authorisation request for the use of vitamin B_2_ (98% and 80%) produced with B. subtilis CGMCC 7.449, as feed additives.

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

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

According to Article 32c(2) of Regulation (EC) No 178/2002 and to the Decision of EFSA's Executive Director laying down the practical arrangements on pre‐submission phase and public consultations, EFSA carried out a public consultation on the non‐confidential version of the technical dossier from 20 August to 10 September 2024 for which no comments were received.

The FEEDAP Panel used the data provided by the applicant together with data from other sources, such as previous risk assessments by EFSA or other expert bodies, peer‐reviewed scientific papers, other scientific reports and experts' (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.6

Methodologies

2.2

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of vitamin B_2_ (98% and 80%) produced with B. subtilis CGMCC 7.449 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, 2018a), Guidance on the assessment of the safety of feed additives for the environment (EFSA FEEDAP Panel, 2019), EFSA statement on the requirements for whole genome sequence analysis of microorganisms intentionally used in the food chain (EFSA, 2021), Guidance on the assessment of the safety of feed additives for the users (EFSA FEEDAP Panel, 2023), Guidance on the assessment of the efficacy of feed additives (EFSA FEEDAP Panel, 2024).

ASSESSMENT

3

Vitamin B_2_ (98% and 80%) produced with a genetically modified strain of B. subtilis (CGMCC 7.449) are intended to be used as nutritional additives (functional group: vitamins, pro‐vitamins and chemically well‐defined substances having a similar effect) in feed and water for drinking for all animal species. They will be referred to in this opinion as vitamin B_2_ 98% and vitamin B_2_ 80%.

Characterisation

3.1

Characterisation of the production organism

3.1.1

Vitamin B_2_ is produced with a genetically modified strain of B. subtilis which is deposited in the China General Microbiological Culture Collection Center (CGMCC) with accession number 7.449.8

The whole genome sequence (WGS) data were used for identification of the production strain. An average nucleotide identity (ANI) of 99.97% of the CGMCC 7.449 strain with B. subtilis 168 was obtained. The same identification results were inferred from genome‐wide alignment‐free phylogenetic distance estimation of the strain under assessment and B. subtilis 168 and phylogenomic analyses using either 1427 or 3949 core genes.9

The susceptibility of B. subtilis CGMCC 7.449 was tested ■■■■■ against the set of antimicrobials required by EFSA (EFSA FEEDAP Panel, 2018a, 2018b). ■■■■■ Therefore, the CGMCC 7.449 strain is considered to be susceptible to all the relevant antimicrobials, but resistant to ■■■■■.10

The WGS data of the production strain were interrogated for the presence of antimicrobial resistance (AMR) genes ■■■■■■■■■■■■■■■■■■■■■■■■■ ■■■■■11 ■■■■■.12

The possible cytotoxicity of the supernatant of B. subtilis CGMCC 7.449 was screened with Vero‐cells (LDH assay) according to the FEEDAP Guidance (EFSA FEEDAP Panel, 2018a, 2018b) and using positive and negative controls.13 No cytotoxic activity was detected.

Information related to the genetically modified microorganism

3.1.1.1

■■■■■.

■■■■■.

■■■■■.14

Manufacturing process

3.1.2

Vitamin B_2_ (98% and 80%) is produced by fermentation with B. subtilis CGMCC 7.449. ■■■■■ ■■■■■.

Characterisation of the active substance

3.1.3

Riboflavin (International Union of Pure and Applied Chemistry (IUPAC) name: 7,8‐dimethyl‐10‐[(2S,3S,4R)‐2,3,4,5,‐tetrahydroxypentyl]benzo[g]pteridine‐2,4(3H,10H)‐dione, synonyms: vitamin B2,7,8,‐dimethyl‐10‐(1’‐D‐ribityl)isoalloxazine; lactoflavin,1‐deoxy‐1‐(7,8,dimethyl‐2,4‐dioxo3,4dihydrobenzo[g]pteridin‐10(2H)‐yl)‐D‐ribitol) is identified by the CAS (Chemical Abstracts Service) number 83‐88‐5 and the EINECS (European Inventory of Existing Chemical Substances) number 201‐507‐1. The molecular formula of riboflavin is C_17_H_20_N_4_O_6_ and its molecular weight is 376.37 g/mol. The structural formula of riboflavin is shown in Figure 1.

Structural formula of riboflavin.

The applicant proposed the following specifications: purity of 97–103.0% in the dry matter (DM), ≤ 0.5% total impurities, ≤ 0.025% impurity A (lumiflavin), ≤ 0.2% impurity B (7,8‐dimethylbenzo[g]pteridine‐2,4(1H,3H)‐dione), C (6,7‐dimethyl‐8‐[(2S,3S,4R)‐2,3,4,5‐etrahydroxypentyl]pteridine‐2,4(3H,8H)‐dione) and D (8‐(hydroxymethyl)‐7‐methyl‐10‐[(2S,3S,4R)‐2,3,4,5‐tetrahydroxypentyl]benzo[g]pteridine2,4(3H,10H)‐dione), ≤ 0.1% for each unspecified impurity and ≤ 0.5% for total impurities. Specifications are also set for loss on drying ≤ 1.5%, sulphated ash ≤ 0.1%, specific optical rotation between −115° and −135° and absorbance ratios A_373_/A_267_ between 0.31–0.33 and A_444_/A_267_ between 0.36–0.39. The FEEDAP Panel noted that the proposed specifications comply with those in the European Pharmacopoeia Monograph for Riboflavin (0292, 6th Edition).

Analysis of six batches showed compliance with the proposed specifications, resulting in an average content of 98.8% riboflavin (range: 98.6%–98.8%) in the DM, loss on drying 0.23% (0.18%–0.33%), sulphated ash 0.1% (in all batches tested).15

In the same batches impurity A was on average 0.0041% (range: 0.0025%–0.0084%), impurity B 0.081% (range: 0.07%–0.1%), impurity C was not detected in any batches tested, impurity D 0.09% (range: 0.08%–0.1%), unspecified impurities 0.067% (range: 0.06%–0.072%, not detected in three batches) and total impurities 0.26% (range: 0.2%–0.4%).

The specific optical rotation measured in six batches ranged from −128° to −131°, absorbance ratio A_373_/A_267_ 0.32 (range: 0.32–0.33) and absorbance ratios A_444_/A_267_ 0.38 (in all batches).

Three batches of vitamin B_2_ 98% were analysed for chemical and microbiological contamination. Mycotoxins (fumonisins B_1_ and B_2_, total fumonisins, deoxynivalenol, T‐2 and HT‐2 toxins, ochratoxin A, zearalenone),16 mercury, lead, cadmium and arsenic17 were all below their corresponding limits of quantification (LOQs).18 Salmonella spp. was not detected in 25 g; total aerobic microbial counts, total yeasts, moulds and Enterobacteriaceae were < 100 colony forming units (CFU)/g and presumptive Bacillus cereus was < 10 CFU/g in all the batches.19

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

The presence of viable vegetative cells and spores of the production strain in the active substance was investigated ■■■■■.20 ■■■■■ No viable cells or spores of the production strain were detected in the final product.

The presence of DNA from the production strain was tested ■■■■■21 ■■■■■ No DNA of the production strain was detected.

The active substance is a yellow to orange–yellow crystalline powder. The tapped bulk density was on average 370 kg/m^3^ (340–380 kg/m^3^).22 The dusting potential was measured in three batches of the active substance according to Stauber–Heubach and the values ranged between 451 and 507 mg/m^3^.23 The same three batches were analysed (in duplicate) for particle size distribution by laser diffraction. No particles were found below 0.5 μm. On average 0.33% (range: 0.1%–0.8%) of the particles were below 1 μm, 29.2% (range: 22.2%–42.2%) were below 5 μm, 7% (range: 4.9%–10.7%) were below 10 μm, 7% (range: 4.9%–10.7%) were below 50 μm, 15.1% (range: 10.9%–22.7%) were below 100 μm.24

Characterisation of riboflavin containing additives

3.1.4

The applicant intends to place in the market two products containing vitamin B_2_, one at 98% and one at 80%.

Vitamin B2

98%

3.1.4.1

The formulation of the additive vitamin B_2_ 98% consists only of the active substance and has been characterised in Section 3.1.3.

Vitamin B2

80%

3.1.4.2

The preparation vitamin B_2_ 80% is formulated by mixing vitamin B_2_ produced with B. subtillis CGMCC 7.449 and an appropriate amount of maltodextrin.

Vitamin B_2_ 80% is specified to contain not less than 80% riboflavin, not more than 3% loss on drying and not more than 4% sulphated ash. Analysis of six batches showed compliance with the proposed specifications, resulting in an average content of 84.06% riboflavin (83.8%–84.2%), loss on drying 0.89% (0.83%–0.95%), sulphated ash 0.09% (0.07%–0.14%).25

Three batches of vitamin B_2_ 80% were analysed for chemical and microbiological contamination. Mycotoxins (fumonisins B_1_ and B_2,_ deoxynivalenol, T‐2 and HT‐2 toxins, ochratoxin A, zearalenone), mercury, lead, cadmium and arsenic were all below their corresponding limits of quantification (LOQs).26 Yeasts, moulds, Enterobacteriaceae, E. coli and presumptive Bacillus cereus were < 10 colony forming units (CFU)/g and Salmonella spp. was not detected in 25 g.

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

The presence of viable vegetative cells and spores of the production strain was investigated ■■■■■.27 ■■■■■ Therefore, neither viable cells nor spores of the production strain were detected in the final product.

No information on possible presence of DNA of the production strain in the additive vitamin B_2_ 80% was provided.

The additive vitamin B_2_ 80% is a yellow to orange–yellow crystalline powder. The bulk density was on average 490 kg/m^3^.28 The dusting potential was measured in three batches of the additive according to Stauber–Heubach and the values ranged between 22 and 42 mg/m^3^. The same three batches were analysed (in duplicate) for particle size distribution by laser diffraction. No particles were found below 100 μm.29

Stability and homogeneity

3.1.5

Three batches of vitamin B_2_ 98% and vitamin B_2_ 80% were stored in polyethylene bags at 25°C or 30°C for 36 months or at 40°C for 6 months. No significant losses were observed under any of the conditions tested.30

The stability of one batch of vitamin B_2_ 80% was evaluated in two premixture formulations (one containing a mixture of trace elements, vitamins and choline chloride and the other one with vitamins only) when added at 600 mg/kg and stored at 25°C for 6 months (samples were packed in heat sealed PE‐bags, protected from light).31 No significant losses were observed.

The stability of one batch of vitamin B_2_ 80% was evaluated in three mash feeds (chickens for fattening, laying hens and pigs for fattening) when added at 1.75 mg/kg feed and stored at 25°C for 3 months or at 40°C for 1 month (samples were packed in heat sealed PE‐bags, protected from light).32 No significant losses were observed at the end of the storage period.

The stability of the additive in water for drinking of three batches of vitamin B_2_ 98% was tested.33 A total of 10 mg of vitamin B_2_ 98% was suspended in 1 L of tap water and stored at 25°C. The content of riboflavin was measured in a subsample of 100 g after 0, 24 and 48 h. No significant losses were observed after 48 h.

The capacity of one batch of vitamin B_2_ 80% to distribute homogeneously in feed was studied in 10 sub‐samples in compound feed for laying hens (1.75 mg riboflavin 80%/kg feed).34 The coefficient of variation (CV) was 10%.

The capacity of one batch (tested in duplicate) of vitamin B_2_ 98% and vitamin B_2_ 80% to distribute homogeneously in water for drinking was studied in 10 sub‐samples.35 The CVs were 8.7% and 8.5%, respectively.

Conditions of use

3.1.6

Vitamin B_2_ 98% and vitamin B_2_ 80% are intended for all animal species without any time limit or withdrawal period. They may be added directly in compound feed or complementary feed, or via premixtures. The additives are also intended for use in water for drinking. No inclusion levels are proposed by the applicant as they will depend on the dietary requirements of the different species.

Safety

3.2

Safety for the production strain

3.2.1

The strain B. subtilis CGMCC 7.449 belongs to a species for which the qualified presumption of safety (QPS) approach to safety assessment applies (EFSA BIOHAZ Panel, 2023) if the requirements are met. The production strain was identified as B. subtilis and lacks toxigenic potential. However, B. subtilis CGMCC 7.449 is resistant to ■■■■■ and contains several copies of two acquired AMR genes. No viable cells (including vegetative cells and spores) were detected in the final products (vitamin B_2_ 98% and vitamin B_2_ 80%). No DNA of the production strain was detected in the product vitamin B_2_ 98% and the FEEDAP Panel considered that this conclusion can be extended to vitamin B_2_ 80% for which no data were provided. Therefore, the use of B. subtilis CGMCC 7.449 to produce vitamin B_2_ does not raise safety concerns.

Safety for the target species, consumer and environment

3.2.2

Safety concerns from the additive may derive either from riboflavin or from the residues of the fermentation process/production strain remaining in the final products. The active substance is produced with a genetically modified microorganism identified as B. subtilis, showing no toxigenic potential and whose recipient strain is considered by EFSA to qualify for the QPS approach. In addition, the genetic modification raises no toxicological concerns.

The nutrient requirements/recommendations of the target species for vitamin B_2_, the background levels of vitamin B_2_ in feed materials and the tolerance to overdoses of vitamin B_2_ were reviewed by the FEEDAP Panel in previous opinions (EFSA FEEDAP Panel, 2014, 2016, 2018b). The Panel concluded that the use levels based on the requirement/background levels would pose no safety concerns to the target species. The Panel is not aware of any more recent findings which would modify the above conclusion. The active substance used to formulate the additives is of high purity. Moreover, the production strain is considered safe from the toxicological point of view. Therefore, it can be concluded that no safety concerns for the target animal would rise from the fermentation residues that may be present in the final additive and the product used to formulate it (i.e. maltodextrin). The FEEDAP Panel concludes that vitamin B_2_ produced with B. subtilis CGMCC 7.449 is considered safe for the target species when used in feed to satisfy the nutritional requirements of the different target species.

The consumer exposure and safety of riboflavin were reviewed in previous opinions (EFSA ANS Panel, 2014, 2013, 2016). The FEEDAP Panel concluded that the supplementation of feed with riboflavin would not raise safety concern for the consumers. The Panel is not aware of any more recent findings which would modify the above conclusion.

The active substance riboflavin occurs in nature. Its use in animal nutrition is not expected to substantially increase the concentration in the environment. Considering that viable cells and DNA of the production strain B. subtilis CGMCC 7.449 were not detected in the final products, a risk for the environment resulting from the use of the additive under assessment in animal nutrition is not foreseen.

The FEEDAP Panel concludes that the use of vitamin B_2_ 98% and vitamin B_2_ 80%, produced with B. subtilis CGMCC 7.449 is safe for the target species, for the consumer and for the environment.

Safety for the user

3.2.3

The highest measured dusting potential of the additives was 507 mg/m^3^ for vitamin B_2_ 98% and 42 mg/m^3^ for vitamin B_2_ 80%. Therefore, the exposure by inhalation is considered likely.

Effect on eyes and skin

3.2.3.1

To support the safety of the additive for the users, the applicant provided studies performed with vitamin B_2_ 98% only.

The skin irritation potential of the additive was investigated in an in vitro study performed according to OECD Testing Guideline (TG) 439.36 The results of the study indicated that the additive is not irritant to the skin (UN GHS ‘No Category’).

The eye irritation potential of the additive was investigated in an in vitro study performed according to OECD TG 492.37 Based on the results of the study the additive is not irritant to the eyes (UN GHS ‘No Category’).

The skin sensitisation potential of the additive was evaluated in an in vitro study performed accordingly to OECD TG 442E.38 The results obtained showed that the additive is a dermal sensitiser.

The skin sensitisation potential of riboflavin 98% was further evaluated in an in vitro study using the adapted OECD TG 442D.39 The obtained results confirmed that the additive is a dermal sensitiser.

Conclusions on safety for the user

3.2.3.2

The results of the studies showed that the test item vitamin B_2_ 98% is neither irritant to eyes nor to the skin, but it is a dermal sensitiser. Therefore, the additive is also considered a respiratory sensitiser. Inhalation and dermal exposure are considered a risk. These conclusions are extended to vitamin B_2_ 80%.

Efficacy

3.3

Data on requirement, allowances and recommendations for feed supplementation are available in the standard literature on animal nutrition. Dietary requirements are set for domestic animals except for ruminants, owing to microbial synthesis of riboflavin in the rumen (GfE, 1995, 2001, 2003, 2007, 2021; NRC, 1996).

Riboflavin (vitamin B_2_) has been used world‐wide in animal nutrition for decades. Owing to the long history of use and its established nutritional role in domestic animals, riboflavin when administered orally is regarded as effective in covering the animal's requirement for vitamin B_2_.

The FEEDAP Panel considers that vitamin B_2_ produced with B. subtilis CGMCC 7.449 is an effective source in covering the animals' requirements when administered via feed or water.

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 Regulation40 and Good Manufacturing Practice.

CONCLUSIONS

4

The production strain harbours acquired genes for resistance to antimicrobials. However, no viable cells of the production strain or its DNA were detected in the final products. Therefore, the additives do not pose any safety concern associated with the production strain.

The use of riboflavin 98% and 80% produced with B. subtilis CGMCC 7.449 in animal nutrition is considered safe for the target species, consumers and the environment.

The additives are not dermal nor eye irritants but are dermal and respiratory sensitisers. Inhalation and dermal exposure are considered a risk.

The additives under assessment are effective in covering the animals' requirements when administered via feed and water for drinking.ABBREVIATIONSAMRAntimicrobial resistanceANIAverage nucleotide identityCASChemical Abstracts ServiceCFUcolony forming unitCGMCCChina General Microbiological Culture Collection CenterCVcoefficient of variationEINECSEuropean Inventory of Existing Chemical SubstancesEURLEuropean Union Reference LaboratoryFEEDAPEFSA Scientific Panel on Additives and Products or Substances used in Animal FeedIUPACInternational Union of Pure and Applied ChemistryLODlimit of detectionMICminimum inhibitory concentrationOECDOrganisation for Economic Co‐operation and DevelopmentQPSqualified presumption of safetyWGSwhole genome sequence

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00898

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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.

LEGAL NOTICE

Relevant information or parts of this scientific output have been blackened in accordance with the European Commission decision on the confidentiality requests formulated by the applicant and further confidentiality requests formulated by the applicant for which a decision by the European Commission is pending. The blackened text will be subject to review once the full decision on the confidentiality requests is adopted by the European Commission. The full output was shared with the European Commission, EU Member States and the applicant.