This is a joint FSA and FSS publication.
1. Introduction
The FSA and FSS have undertaken an assessment for a feed additive containing 6-phytase (Ronozyme® HiPhos, DSM Nutritional Products Ltd in Switzerland, Wurmigsweg 576, 4303, Kaiseraugst, Switzerland) under assimilated Regulation (EC) No 1831/2003 (EC, 2003). The application sought a renewal authorisation in the category ‘zootechnical additive’, functional group ‘digestibility enhancer’, for its use in poultry, weaned piglets, pigs for fattening and sows. The applicant also sought a modification of the terms of the existing authorization, to include a change in production organism to an optimized strain from the same safe strain line but with a higher yield.
To support the safety assessment by FSA and FSS, the ACAF provided advice to the FSA and FSS outlined in this document.
In line with Article 8 of 1831/2003, the assessment has considered and concluded that the feed additive complies with the conditions laid down in Article 5, including: safety considerations for human, animal and environmental health; efficacy of the additive for its intended effect; potential impairment of the distinctive features of animal products. This, and the guidance put in place by EFSA for the evaluation of feed additive applications, has formed the basis and structure for the assessment.
The dossier was evaluated by the Advisory Committee on Animal Feedingstuffs (ACAF) at its October 2023 meeting, after which a request for further information was communicated to the applicant. The applicant’s response to this request and subsequent requests were evaluated by ACAF at its July 2024 meeting.
The views of ACAF have been taken into account in this safety assessment which represents the opinion of the FSA/FSS.
2. Assessment
2.1. Section II: Identity, characterisation and conditions of use
The active substance of the additive Ronozyme® HiPhos is a 6-phytase enzyme derived from the genetically modified fungus Aspergillus oryzae DSMZ 33699. The additive is formulated as both a liquid (Ronozyme® HiPhos (L)) and a solid (Ronozyme® HiPhos (GT)). The applicant provided data from five batches supporting the identification values outlined below (Table 2):
During the first evaluation, the ACAF identified several items of information that were necessary to inform assessment of the additive’s identity. This included: yeast, filamentous fungi and Bacillus cereus microbial data, clarification on the relationship between the original production strain (DSMZ 22594) and the new ‘optimised’ strain (DSMZ 33699), and the 24-month stability data. This information was requested from the applicant, and all aspects were satisfactorily addressed.
The additive was shown to be stable for 24 months with residual activity of the granular and liquid product at 25°C being 45 % and 63 %, respectively. The applicant stated that the loss of activity will be compensated by overdosing to a level necessary to meet the declared minimum activity until the product expiry date.
The proposed conditions of use of the additive are described in Table 3:
2.1.1. Conclusions on Section II
The ACAF concluded that both the liquid and granular form of the additive were correctly characterised. Both forms of the additive are stable for 24 months at 25°C provided overdosing of the additive to ensure minimum activity levels are met until the product’s expiry date. No further concerns were raised for Section II of the dossier.
2.2. Section III: Safety
The Committee were satisfied that the extensive literature review identified no relevant papers that described adverse effects of the additive on target species, consumers or the environment. Furthermore, the applicant provided a signed quality statement by manufacturers and distributors of Ronozyme® HiPhos noting the absence of reports of adverse effects on target animals, users or the environment.
The ACAF were satisfied with the safety of the ‘optimised’ production strain termed 33693. The applicant provided a history of safe use of the A. oryzae strains and demonstrated an absence of known toxins.
2.2.1. Safety for the target species
The applicant provided new calculations to establish the maximum safe concentration of the additive in feed for the target species. This was derived by applying a 100-fold uncertainty factor to the NOAEL from the 90-day repeat-dose oral toxicity study performed in rats. The ACAF agree with the applicant that the calculations confirm the absence of safety concerns when supplementing with the additive at the maximum recommended dose of 4,000 FYT/kg feed. The calculated values are reported in Table 4, which shows that for all classes of target species tested the estimated maximum safe concentration (as enzyme activity) of the additive was much greater than the recommended dose.
2.2.2. Safety for the consumer
The applicant presented the following tests on the active ingredient to evaluate the safety of the additive for the consumer:
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Sub-chronic oral toxicity study (OECD 408)
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Bacterial reverse mutation (Ames) test (OECD 471)
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In vitro micronucleus test in human lymphocytes (OECD 487)
No adverse effects were found in the toxicity study up to the highest dose tested, 523710 FYT/kg body weight/day, which was taken as the NOAEL. The negative results from the two genotoxicity studies indicated that the active ingredient was not mutagenic. Ingredients other than the active ingredient were commonly used substances that are known to be safe at the levels present in the additive.
The Committee were satisfied with the evidence provided and concluded that the additive showed no adverse effect which could give rise to concern of the additive for the consumer.
2.2.3. Safety for the user
Two irritation studies performed with the active ingredient (optimised production strain DSMZ 33699) were presented to evaluate the safety of the additive for the user/worker:
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Bovine Corneal Opacity Permeability (BCOP) test performed following OECD 437 to assess eye irritation
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In vitro EpiSkin™ reconstructed human epidermis model test performed following OECD 439 to assess skin irritation
The Committee concluded that the active substance is non-irritant to the skin and eyes; however, because the additive is an enzyme it is considered to be a respiratory sensitiser, and in the absence of skin sensitisation tests, a potential skin sensitiser.
2.2.4. Safety for the environment
No studies were provided as the active substance is an enzyme and the other ingredients are commonly used substances that are not expected to harm the environment.
2.2.5. Conclusions on safety
The ACAF concluded that there were no safety concerns to the target species when supplementing with the additive at the maximum recommended dose of 4,000 FYT/kg feed.
The additive showed no adverse effect which could give rise to concern of the additive for target animals or the consumer.
The active substance is non-irritant to the skin and eyes; however, because the additive is an enzyme it is considered to be a respiratory sensitiser, and in the absence of skin sensitisation tests, a potential skin sensitiser.
The additive poses no risk to the environment.
2.3. Section IV: Efficacy
The Committee questioned the potential difference between the original production strain and the new ‘optimised’ strain. Upon accepting the similarities between strains, the ACAF noted that the phytase product obtained from the ‘optimised’ strain shares the same composition, manufacturing process and phytase. The ACAF deemed the efficacy studies submitted to be acceptable for the application. The applicant submitted the efficacy trials previously submitted to FEEDAP Panel (FEEDAP, 2012b, 2012a, 2016). The Committee concluded that the additive demonstrated efficacy in poultry, weaned piglets, pigs for fattening and sows at the minimum recommended dose of 500 FYT/kg feed.
2.3.1. Conclusions on efficacy
The ACAF concluded that the additive was demonstrated to be efficacious.
3. Analytical methods evaluation
Conclusions on the analytical methods are presented here as an extract from the Evaluation Report of the European Union Reference Laboratory (EURL) for Feed Additives on the Method(s) of the Analysis for Ronozyme HiPhos (EURL, 2018):
"For the quantification of the phytase activity in feedingstuffs the Applicant submitted the ring-trial validated colorimetric standard method EN ISO 30024 and demonstrated the suitability of the method at 200 FYT/kg feedingstuffs. In addition, the Applicant applied this method with minor experimental modifications to analyse the feed additive (Ronozyme® HiPhos) and premixtures and obtained similar method performance characteristics.
Based on the performance characteristics available the EURL recommends for official control the colorimetric methods mentioned above for the quantification of the phytase activity in the feed additive, premixtures and feedingstuffs".
FSA/FSS accepts the EURL analytical method evaluation reports. FSA/FSS determined the analytical methods proposed as appropriate for official controls for this feed additive. The analytical methods were approved in 2018, at the time in which the UK was still an EU member.
4. Conclusions
The ACAF concluded the additive was correctly characterised, and that it is stable for 24 months at 25°C, with overfilling of the additive to compensate for residual loss of activity. No further concerns were raised for Section II of the dossier.
The active substance is non-irritant to the skin or eyes; however, because the additive is an enzyme it is considered a respiratory sensitiser, and in the absence of skin sensitisation tests, a potential skin sensitiser.
The additive poses no risk to the environment.
The ACAF concluded that the additive is efficacious in poultry, weaned piglets, pigs for fattening and sows at the dose range of 500 – 4000 FYT/kg feed.
Abbreviations
Acknowledgements
With thanks to the members of the ACAF during the course of the assessment, who were: Professor Nicholas Jonsson, Martin Briggs, Professor Emily Burton, Professor Katrina Campbell, Professor Matthew Fisher, Hannah Kane, Susan MacDonald, Dr. Oonagh Markey, Christine McAlinden, Dr. Donald Morrison, Derek Renshaw, Dr. Michael Salter, Dr. Adam Smith, Christel Wake, Dr. Helen Warren and Professor Nick Wheelhouse. Dr. Adam Smith declared a direct conflict of interest and left the meetings for discussion.