Abstract
Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the efficacy of the preparation consisting of viable cells of Bacillus subtilis CNCM I‐4606, B. subtilis CNCM I‐5043, B. subtilis CNCM I‐4607 and Lactococcus lactis CNCM I‐4609 when used as a technological additive (hygiene condition enhancer) for all animal species. In a previous opinion, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the additive is safe for the target species, consumers and the environment. The Panel also considered the additive to be non‐irritant to skin and eyes, nor a dermal sensitiser, but a respiratory sensitiser. Moreover, the data provided were not sufficient to conclude on the efficacy of the additive to significantly reduce the growth of Salmonella Typhimurium or Escherichia coli in feed. In the current assessment, the applicant provided supplementary information to address these flaws and limited the claimed effect to ‘prevent (re)contamination by Salmonella Typhimurium’. Based on the new studies, the Panel concluded that the additive at a minimum proposed inclusion level of 1 × 109 colony forming units (CFU) B. subtilis and 1 × 109 CFU L. lactis per litre showed potential to reduce Salmonella Typhimurium growth in feeds with high moisture content (60–90% moisture).
Keywords: technological additive, hygiene condition enhancer, Bacillus subtilis CNCM I‐4606, Bacillus subtilis CNCM I‐5043, Bacillus subtilis CNCM I‐4607, Lactococcus lactis CNCM I‐4609, efficacy
1. Introduction
1.1. Background and Terms of Reference as provided by the requestor
Regulation (EC) No 1831/2003 1 establishes the rules governing the Community authorisation of additives for use in animal nutrition and, in particular, Article 9 defines the terms of the authorisation by the Commission.
The applicant, MiXscience, 2 is seeking a Community authorisation of Preparation of Bacillus subtilis CNCM I‐4606, Bacillus subtilis CNCM I‐5043, Bacillus subtilis CNCM I‐4607 and Lactococcus lactis CNCM I‐4609 as a feed additive to be used as hygiene condition enhancers: substances or, when applicable, microorganisms which favourably affect the hygienic characteristics of feed by reducing a specific microbiological contamination for all animal species. (Table 1).
Table 1.
Description of the substances
| Category of additive | Technological additive |
|---|---|
| Functional group of additive | Hygiene condition enhancers |
| Description | Preparation of Bacillus subtilis CNCM I‐4606, Bacillus subtilis CNCM I‐5043, Bacillus subtilis CNCM I‐4607 and Lactococcus lactis CNCM I‐4609 |
| Target animal category | All animal species |
| Applicant | MiXscience |
| Type of request | New opinion |
On 30 September 2021, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) of the European Food Safety Authority (EFSA), in its opinion on the safety and efficacy of the product, could not conclude on the efficacy of the preparation of Bacillus subtilis CNCM I‐4606, Bacillus subtilis CNCM I‐5043, Bacillus subtilis CNCM I‐4607 and Lactococcus lactis CNCM I‐4609 in all animal species.
The Commission gave the possibility to the applicant to submit supplementary information and data in order to complete the assessment and to allow a revision of the EFSA's opinion. The new data have been received on 16 March 2022 and the applicant has been requested to transmit them to EFSA as well.
In view of the above, the Commission asks the Authority to deliver a new opinion on Preparation of Bacillus subtilis CNCM 1–4,606, Bacillus subtilis CNCM 1–5,043, Bacillus subtilis CNCM 1–4,607 and Lactococcus lactis CNCM 1–4,609 as a feed additive for all animal species based on the additional data submitted by the applicant, in accordance with Article 29(1)(a) of Regulation (EC) No 178/2002.
1.2. Additional information
This additive is a preparation containing viable cells of three strains of Bacillus subtilis (CNCM I‐4606, CNCM I‐5043 and CNCM I‐4607) and one strain of Lactococcus lactis (CNCM I‐4609).
EFSA has issued an opinion on the safety and efficacy of this additive for all animal species (EFSA FEEDAP Panel, 2021).
The additive is not currently authorised as a feed or food additive in the European Union.
2. Data and methodologies
2.1. Data
The present assessment is based on data submitted by the applicant in the form of supplementary information 3 to a previous application on the same product. 4
In accordance with Article 38 of the Regulation (EC) No 178/2002 5 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, 6 a non‐confidential version of the supplementary information has been published on Open.EFSA. 7
2.2. Methodologies
The approach followed by the FEEDAP Panel to assess the safety and the efficacy of active substance is in line with the principles laid down in Regulation (EC) No 429/2008 8 and the relevant guidance documents: Guidance on the assessment of the efficacy of feed additives (EFSA FEEDAP Panel, 2018).
3. Assessment
The additive under assessment is a preparation containing viable cells of three strains of Bacillus subtilis (CNCM I‐4606, CNCM I‐5043 and CNCM I‐4607) and one strain of Lactococcus lactis (CNCM I‐4609) with a total minimum content of 6.7 × 108 colony forming units (CFU)/g for each individual B. subtilis strain (2 × 109 CFU/g in total for the three B. subtilis strains) and 2 × 109 CFU/g for the L. lactis strain. The additive is intended to be used as a technological additive (functional group: hygiene condition enhancers) in dry and liquid compound feeds and feed materials for all animal species at a minimum inclusion level of 1 × 109 CFU total B. subtilis and 1 × 109 CFU L. lactis per kg or litre. The applicant states that in case of dry feed, the additive is to be added to feed materials or compound feeds during the manufacturing process and/or when there is a risk of high moisture content and/or of contamination. In case of liquid feed, the additive should be added and mixed to dry feed just before the liquid preparation or directly in the liquid feed. The additive is not intended to be used in commercial premixtures containing trace elements or preservatives.
In the first application dossier, the claimed effect was ‘to reduce the microbiological level of enteropathogens such as Salmonella spp. or E. coli’. In the concerned opinion, the FEEDAP Panel assessed the safety and the efficacy of the product when used in feed for all animal species and concluded that the additive is safe for the target species, consumers and the environment (EFSA FEEDAP Panel, 2021). The Panel also considered the additive to be non‐irritant to skin and eyes, not a dermal sensitiser, but, owing to the proteinaceous nature of the active agents, the additive was considered a respiratory sensitiser. Moreover, the data provided were not sufficient to conclude on the efficacy of the additive to significantly reduce the growth of Salmonella Typhimurium or Escherichia coli in feed.
In the current application, the claimed effect has been changed to ‘prevent (re)contamination by Salmonella Typhimurium’. To support this effect, the applicant has provided new data that are described below.
3.1. Efficacy
The applicant has requested to reconsider three of the seven studies assessed by the FEEDAP Panel in the previous opinion. 9 The FEEDAP Panel reconsidered these three studies, and, according to the dossier submitted initially, in one study, 10 the additive showed the potential to limit the growth of Salmonella Typhimurium in ■■■■■, but the Panel expressed reservations on the relevance of the effects observed under practical use conditions due to the duration of the trial. In the second study, 11 the addition of the additive showed the potential to decrease Salmonella Typhimurium counts in ■■■■■, but the persistence of the effect observed was not maintained over time. In the third study, 12 the addition of the additive showed the potential to limit the growth of Salmonella Typhimurium in ■■■■■ In the current submission, the applicant requested ■■■■■ The Panel reiterates its previous reservations and highlights the absence of data to assess the microbiological quality of the diets at the end of the experiments for the three studies. Therefore, these trials will not be further considered for the current assessment.
Additionally, the applicant has provided 10 new in vitro studies (studies 1–10). ■■■■■ (Table 2). 13 The feed matrices ■■■■■ were either left untreated (control) or ■■■■■ the additive ■■■■■
Table 2.
Summary of the in vitro trials of different feed matrices inoculated with S. Typhimurium ■■■■■ and treated with the additive
| Study | Feed matrix (% dry matter) Inoculant Salmonella Typhimurium strain | Treatment Additive (CFU Bacillus subtilis/L and Lactococcus lactis/L) | Salmonella spp. counts (log10 CFU/mL) at hours of the experiment | |||||
|---|---|---|---|---|---|---|---|---|
| 0 | 3 | 6 | 9 | 18 | 24 | |||
| 1 18 |
Soup for pigs (24%) ■■■■■ |
Control | 2.52 | – | – | – | – | 8.21a |
|
■■■■■ ■■■■■ |
2.25 | – | – | – | – | 6.46b | ||
| Δ | 0.27 | – | – | – | – | 1.75 | ||
| 2 18 |
Soup for pigs (19%) ■■■■■ |
Control | 4.04 | – | – | – | – | 8.75a |
|
■■■■■ ■■■■■ |
4.11 | – | – | – | – | 7.72b | ||
| Δ | 0.07 | – | – | – | – | 1.03 | ||
| 3 19 |
Soup for pigs (37%) ■■■■■ |
Control | 2.53 | – | – | – | 8.66a | 8.57a |
|
■■■■■ ■■■■■ |
2.54 | – | – | – | 7.50b | 7.02b | ||
| Δ | 0.01 | – | – | – | 1.15 | 1.55 | ||
| 420 |
Soup for pigs (37%) ■■■■■ |
Control | 2.62 | – | – | – | 8.41a | 8.80a |
|
■■■■■ ■■■■■ |
2.77 | – | – | – | 7.27b | 7.24b | ||
| Δ | 0.15 | – | – | – | 1.14 | 1.56 | ||
| 521 |
Soup for pigs (37%) ■■■■■ |
Control | 2.35 | – | – | – | 8.24a | 8.42a |
|
■■■■■ ■■■■■ |
2.40 | – | – | – | 6.93b | 6.77b | ||
| Δ | 0.05 | – | – | – | 1.31 | 1.64 | ||
| 6 22 |
Milk for cats (13%) ■■■■■ |
Control | 1.69 | – | 3.46a | – | – | – |
|
■■■■■ ■■■■■ |
1.69 | – | 2.00b | – | – | – | ||
| Δ | 0 | – | 1.46 | – | – | – | ||
| 722 |
Milk for cats (13%) ■■■■■ |
Control | 1.90 | – | 3.32a | – | – | – |
|
■■■■■ ■■■■■ |
1.96 | – | 2.27b | – | – | – | ||
| Δ | 0.06 | – | 1.05 | – | – | – | ||
| 823 |
Milk for cats (13%) ■■■■■ |
Control | 1.86 | 2.58a | 3.71a | 3.91a | – | – |
|
■■■■■ ■■■■■ |
1.80 | 2.13b | 2.55b | 2.85b | – | – | ||
| Δ | 0.05 | 0.45 | 1.16 | 1.06 | – | – | ||
| 924 |
Milk for cats (13%) ■■■■■ |
Control | 1.92 | 2.63a | 3.90a | 4.54a | – | – |
|
■■■■■ ■■■■■ |
1.86 | 1.94b | 2.80b | 3.35b | – | – | ||
| Δ | 0.06 | 0.69 | 1.11 | 1.18 | – | – | ||
| 1025 |
Milk for cats (13%) ■■■■■ |
Control | 1.84 | 2.69a | 3.61a | 4.25a | – | – |
|
■■■■■ ■■■■■ |
1.75 | 1.95b | 2.47b | 2.93b | – | – | ||
| Δ | 0.09 | 0.74 | 1.14 | 1.32 | – | – | ||
CFU, colony forming unit.
: Means in the same column within study are significantly different compared to control with p ≤ 0.05.
Δ: Salmonella spp. counts (log10 CFU/mL) between control and treatment.
–: not measured.
The target organisms used were ■■■■■ Salmonella Typhimurium strains. 14 These included ■■■■■ 15 ■■■■■ 16 ■■■■■ 17 ■■■■■ ■■■■■
The Salmonella Typhimurium strains were enumerated in both experimental groups at the beginning and end of the experiment, and at an intermediate timepoint (when applicable). Additionally, enumeration of ■■■■■ was performed to assess the microbiological quality of the feed at the beginning and at the end of the experiment for all the studies. Water activity (aW) and dry matter (DM) content of the feed matrices were also analysed. The experimental design and results are described in Table 2. ■■■■■
The feed matrices were incubated ■■■■■ (Table 2). The studies included ■■■■■ replicates per group. The results were assessed by the ■■■■■ test. Statistical significance was declared at p ≤ 0.05.
In all the trials, a statistically significant and biologically relevant reduction of Salmonella Typhimurium growth in treated feeds compared to control was observed in all the time points monitored after an incubation period of 6 h. The Panel notes that the additive has been tested against ■■■■■ independent strains, which may be considered as representative of the Salmonella Typhimurium serovar. Thus, conclusions can be drawn on the efficacy of the additive against Salmonella Typhimurium. However, the Panel is not on the position to evaluate the effect of the additive to prevent re‐contamination of the matrices as claimed by the applicant. The Panel also notes that the absence of an intermediate time point does not allow to evaluate the maintenance/persistence of the effect over time for the studies 1, 2, 6 and 7. 20 , 21 , 22 , 23 , 24 , 25
Considering the newly submitted studies, the results obtained allow to conclude that the additive did not show a potential to inhibit the growth of Salmonella Typhimurium, but it showed a potential to reduce Salmonella Typhimurium growth in feeds with high moisture content (60–90% moisture).
The Panel notes that the use of this additive cannot be considered as a replacement for the standard hygiene farming conditions.
4. Conclusions
The Panel concludes that the additive under assessment at a minimum inclusion level of 1 × 109 CFU B. subtilis (total CNCM I‐4606, CNCM I‐5043 and CNCM I‐4607) and 1 × 109 CFU L. lactis (CNCM I‐4609) per litre showed a potential to reduce Salmonella Typhimurium growth in feeds with high moisture content (60–90% moisture).
Abbreviations
- CFU
colony forming unit
- DM
dry matter
- EFSA
European Food Safety Authority
- FEEDAP
EFSA Scientific Panel on Additives and Products or Substances used in Animal Feed
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, Fašmon 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, Anguita M, Brozzi R, Innocenti M, Ortuño J and García‐Cazorla Y, 2023. Scientific Opinion on the efficacy of a feed additive consisting of Bacillus subtilis strains CNCM I‐4606, CNCM I‐5043 and CNCM I‐4607 and Lactococcus lactis CNCM I‐4609 for all animal species (MiXscience). EFSA Journal 2023;21(3):7871, 8 pp. 10.2903/j.efsa.2023.7871
Requestor European Commission
Question number EFSA‐Q‐2022‐00234
Panel members Vasileios Bampidis, Giovanna Azimonti, Maria de Lourdes Bastos, Henrik Christensen, Birgit Dusemund, Mojca Fašmon 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 EFSA. The full output has been shared with the European Commission, EU Member States (if applicable) and the applicant. The blackening may be subject to review once the decision on the confidentiality requests is adopted by EFSA and in case it rejects some of the confidentiality requests.
Declarations of interest If you wish to access the declaration of interests of any expert contributing to an EFSA scientific assessment, please contact interestmanagement@efsa.europa.eu.
Acknowledgements The Panel wishes to thank the following for the support provided to this scientific output: FEEDAP Working Group of Microbiology and Jordi Tarrés‐Call.
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: 1 February 2023
Notes
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.
On 08/09/2022, EFSA was informed by the applicant that Nolivade merged with MIXscience in April 2022. The applicant for this feed additive is now MIXscience (2/4 avenue de Ker Lann CS 17228, PC 35172, Bruz, France).
FEED dossier reference: EFSA‐Q‐2022‐00234.
FEED dossier reference: FAD‐2019‐0090.
Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety. OJ L 31, 1.2.2002, p. 1–48.
Decision available at: https://www.efsa.europa.eu/en/corporate-pubs/transparency-regulation-practical-arrangements
Available at: https://open.efsa.europa.eu/questions/EFSA-Q-2022-00234
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.
Followup EFSA‐Q‐2020‐00202_FAD‐2019‐0090.
FAD‐2019‐0090: Annex_SII_6; Annex_SIII_7, Annex_SIII_8 and Annex SIII_9.
FAD‐2019‐0090: Annex_SII_2; Annex_SIII_3; Annex_SIII_8 and Annex SIII_9.
FAD‐2019‐0090: Annex_SII_6; Annex_SIII_6; Annex_SIII_8 and Annex SIII_9.
Annex_SI_33_Protocol INVT‐SPS04 MIS02 and EFSA‐Q‐2022‐00234_SIn_11_05_22.
Annex_SI_2_■■■■■.
Annex_SI_10_■■■■■.
Annex_SI_1_■■■■■.
Annex_SI_8_■■■■■.
Annex 3_EFFiccy report INVT‐ SPS04.
Annex_SI_39_Efficacy report SPS05.
Annex_SI_42_Efficacy report SPS06.
Annex_SI_43_Efficacy report SPS07.
Annex 2_EFFiccy report INVT‐ MIS02.
Annex_SI_44_Efficacy report MIS03.
Annex_SI_45_Efficacy report MIS04.
Annex_SI_46_Efficacy report MIS05.
References
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