Safety evaluation of the food enzyme ribonuclease P from the non‐genetically modified Penicillium citrinum strain AE‐RPE

TL;DR

This study evaluates the safety of a food enzyme produced by a non-genetically modified fungus and concludes it is safe for use in yeast processing.

Contribution

The study provides a comprehensive safety assessment of a novel food enzyme from Penicillium citrinum.

Findings

Genotoxicity tests showed no safety concerns for the food enzyme.

The no observed adverse effect level was 984 mg TOS/kg bw per day.

The likelihood of allergic reactions is considered low.

Abstract

The food enzyme ribonuclease P (EC 3.1.26.5) is produced with the non‐genetically modified Penicillium citrinum strain AE‐RPE by Amano Enzyme Inc. It was considered free from viable cells of the production organism. It is intended to be used in the processing of yeast and yeast products. Dietary exposure was estimated to be up to 0.0014 mg total organic solids (TOS)/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 984 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 702,857. A search for the homology of the amino acid sequence of the ribonuclease P to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure cannot be excluded, but that the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Tables

TABLE 1. Composition of the food enzyme preparation.

Parameters	Unit	Batches
		1	2	3	4 a	5 b	6 c
Ribonuclease P activity	U/g d	84,800	126,000	127,000	58,700	97,100	72,600
Protein	%	12.8	10.9	10.8	NA e	7.6	NA e
Ash	%	1.8	1.7	2.1	3.0	2.2	2.7
Water	%	7.6	5.7	6.1	4.5	3.2	4.0
■■■■■ (excipient)	%	38.7	42.3	42.4	50.0	42.7	44.1
Total organic solids (TOS) f	%	51.9	50.3	49.4	42.5	51.9	49.2
Activity/TOS ratio	U/mg TOS	163.4	250.5	257.1	138.1	187.1	147.6

TABLE 2. Intended use and recommended use level of the food enzyme provided by the applicant. 31

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM) b
Processing of yeast and yeast products	Yeast	1.16–23.2

TABLE 3. Summary of the estimated dietary exposure to the food enzyme–TOS in six population groups.

Population group	Estimated exposure (mg TOS/kg body weight per day)
	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	4–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0–0.0002 (14)	0–0.0006 (17)	0–0.0005 (21)	0–0.0003 (23)	0–0.0001 (23)	0–0.0002 (25)
Min–max 95th percentile (number of surveys)	0–0.0010 (13)	0.0001–0.0007 (16)	0.0001–0.0014 (21)	0.0001–0.0007 (22)	0–0.0004 (23)	0–0.0006 (24)

TABLE 4. Qualitative evaluation of the influence of uncertainties on the dietary exposure estimate.

Sources of uncertainties	Direction of impact
Model input data
Consumption data: different methodologies/representativeness/ underreporting/misreporting/no portion size standard	+/−
Use of data from food consumption surveys of a few days to estimate long‐term (chronic) exposure for high percentiles (95th percentile)	+
Possible national differences in categorisation and classification of food	+/−
Model assumptions and factors
Selection of broad FoodEx categories for the exposure assessment	+
For yeast processing, the food categories chosen for calculation are not only those containing yeast extract as indicated by the applicant, 35 but also those containing mannoproteins resulted from the treatment of yeast cell walls	+
Exposure to food enzyme–TOS always calculated based on the recommended maximum use level	+
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−

Full text

INTRODUCTION

1

Article 3 of the Regulation (EC) No 1332/20081 provides definition for ‘food enzyme’ and ‘food enzyme preparation’.

‘Food enzyme’ means a product obtained from plants, animals or micro‐organisms or products thereof including a product obtained by a fermentation process using micro‐organisms: (i) containing one or more enzymes capable of catalysing a specific biochemical reaction; and (ii) added to food for a technological purpose at any stage of the manufacturing, processing, preparation, treatment, packaging, transport or storage of foods.

‘Food enzyme preparation’ means a formulation consisting of one or more food enzymes in which substances such as food additives and/or other food ingredients are incorporated to facilitate their storage, sale, standardisation, dilution or dissolution.

Before January 2009, food enzymes other than those used as food additives were not regulated or were regulated as processing aids under the legislation of the Member States. On 20 January 2009, Regulation (EC) No 1332/2008 on food enzymes came into force. This Regulation applies to enzymes that are added to food to perform a technological function in the manufacture, processing, preparation, treatment, packaging, transport or storage of such food, including enzymes used as processing aids. Regulation (EC) No 1331/20082 established the European Union (EU) procedures for the safety assessment and the authorisation procedure of food additives, food enzymes and food flavourings. The use of a food enzyme shall be authorised only if it is demonstrated that:

• it does not pose a safety concern to the health of the consumer at the level of use proposed;
• there is a reasonable technological need;
• its use does not mislead the consumer.

All food enzymes currently on the EU market and intended to remain on that market, as well as all new food enzymes, shall be subjected to a safety evaluation by the European Food Safety Authority (EFSA) and approval via an EU Community list.

Background and Terms of Reference as provided by the requestor

1.1

Background as provided by the European Commission

1.1.1

Only food enzymes included in the European Union (EU) Community list may be placed on the market as such and used in foods, in accordance with the specifications and conditions of use provided for in Article 7 (2) of Regulation (EC) No 1332/2008 on food enzymes.

On 19 May 2023, a new application has been introduced by the applicant “Amano Enzyme Inc.” for the authorization of the food enzyme Ribonuclease P from a non‐genetically modified strain of Penicillium citrinum (strain AE‐RPE).

Terms of Reference

1.1.2

The European Commission requests the European Food Safety Authority to carry out the safety assessment and the assessment of possible confidentiality requests of the following food enzyme: Ribonuclease P from Penicillium citrinum (strain AE‐RP), in accordance with Regulation (EC) No 1332/2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings.^1^

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme Ribonuclease P from a non‐genetically modified P. citrinum strain AE‐RPE.

Additional information was requested from the applicant during the assessment phase on 21 October 2024 and on 10 June 2025, and they were received on 30 May 2025 and on 16 June 2025, respectively (see ‘Documentation provided to EFSA’).

Methodologies

2.2

The assessment was conducted in line with the principles described in the EFSA ‘Guidance on transparency in the scientific aspects of risk assessment’ (EFSA, 2009) and following the relevant guidance documents of the EFSA Scientific Committee.

The ‘Scientific Guidance for the submission of dossiers on food enzymes’ (EFSA CEP Panel, 2021) and the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023) have been followed for the evaluation of the application.

Public consultation

2.3

According to Article 32c(2) of Regulation (EC) No 178/20023 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 29 October to 19 November 2024 for which no comments were received.

ASSESSMENT

3

IUBMB nomenclatureRibonuclease PSystematic name–SynonymsRNase PIUBMB NoEC 3.1.26.5CAS No71427‐00‐4EINECS NoNA

Ribonucleases P hydrolyse the phosphodiester bonds of RNA to release nucleotides. The food enzyme under assessment is intended to be used in the processing of yeast and yeast products as defined in the EFSA guidance (EFSA CEP Panel, 2023).

Source of the food enzyme

3.1

The ribonuclease P is produced with the filamentous fungus P. citrinum strain AE‐RPE, which is deposited ■■■■■ with the deposition number ■■■■■.4 The production strain was identified as P. citrinum ■■■■■.5

Production of the food enzyme

3.2

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,6 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with Good Manufacturing Practice.7

The production strain is grown as a pure culture using a typical industrial medium in a submerged, batch or fed‐batch fermentation system with conventional process controls in place. After completion of the fermentation, the solid biomass is removed from the fermentation broth by filtration. The filtrate containing the enzyme is then further purified and concentrated, including an ultrafiltration step in which enzyme protein is retained, while most of the low molecular mass material passes the filtration membrane and is discarded.8 The applicant provided information on the identity of the substances used to control the fermentation and in the subsequent downstream processing of the food enzyme.9

The Panel considered that sufficient information has been provided on the manufacturing process and the quality assurance system implemented by the applicant to exclude issues of concern.

Characteristics of the food enzyme

3.3

Properties of the food enzyme

3.3.1

The ribonuclease P is a single polypeptide chain of ■■■■■ amino acids.10 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa.11 The food enzyme was analysed by size exclusion chromatography.12 The chromatograms of the three batches showed similar profiles.

No other enzyme activities were reported.13

The applicant's in‐house determination of ribonuclease P activity is based on ■■■■■.14

The food enzyme has a temperature optimum around 70°C (pH 7.0) and a pH optimum around pH 5.0 (37°C).15 Thermostability was tested by pre‐incubation of the food enzyme for 15 min at different temperatures (pH 5.0). Enzyme activity decreased above 60°C showing 6% residual activity at 80°C.16

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme preparation were provided for three batches intended for commercialisation and three batches produced for the toxicological tests (Table 1).17 The mean total organic solids (TOS) of the three batches intended for commercialisation was 50.5% and the mean enzyme activity/TOS ratio was 223.7 U/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches was below 0.03 mg/kg18 ^,^ 19 which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).

The food enzyme preparation complies with the microbiological criteria for total coliforms, Escherichia coli and Salmonella, as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).20 No antimicrobial activity was detected in any of the tested batches.21

P. citrinum is known to produce the toxin citrinin (Park et al., 2008), which was analysed in the three commercial batches and was below LoQ of the applied method.22 ^,^ 23 In addition, the presence of aflatoxins (B1, B2, G1, G2), HT‐2 toxin, T‐2 toxin, zearalenone, ochratoxin A and sterigmatocystin was examined in the three food enzyme batches and all were below the limits of quantification (LoQs) of the applied analytical methods.24 Adverse effects caused by the possible presence of other secondary metabolites are addressed by the toxicological examination of the food enzyme.

The Panel considered that the information provided on the purity of the food enzyme was sufficient.

Viable cells of the production strain

3.3.4

The absence of viable cells of the production strain in the food enzyme was demonstrated in three independent batches analysed in triplicate. ■■■■■. No colonies of the production strain were recovered. A positive control was included.

Toxicological data

3.4

A battery of toxicological tests including a bacterial reverse mutation test (Ames test), an in vitro mammalian chromosomal aberration test, an in vitro mammalian cell micronucleus test, and a repeated dose 90‐day oral toxicity study in rats has been provided.

The batches 4, 5 and 6 (Table 1) used in these studies have similar or lower activity/TOS ratio as the batches intended for commercialisation, and thus are considered suitable as test items.

Genotoxicity

3.4.1

Bacterial reverse mutation test

3.4.1.1

A bacterial reverse mutation test (Ames test) was performed according to the Organisation for Economic Co‐operation and Development (OECD) Test Guideline 471 (OECD, 1997a) and following Good Laboratory Practice (GLP).25

Four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2uvrA(pKM101) were used with or without metabolic activation (S9‐mix), applying the pre‐incubation method.

Based on the results of a range‐finding test, the main experiment was carried out in triplicate, using 5 concentrations of the food enzyme of 132.6, 265.2, 530.4, 1060.8 and 2125 μg TOS/plate.

No cytotoxicity was observed at any concentration of the test substance. Upon treatment with the food enzyme there was no biologically relevant increase in the number of revertant colonies above the control values, in any strain tested, with or without S9‐mix.

The study was considered reliable without restrictions and the results of high relevance.

The Panel concluded that the food enzyme ribonuclease P did not induce gene mutations under the test conditions applied in this study.

In vitro mammalian cell micronucleus test

3.4.1.2

The in vitro mammalian cell micronucleus test was carried out according to OECD Test Guideline 487 (OECD, 2016) and following GLP.26

A range finding test and two main experiments were performed with duplicate cultures of human lymphoblastoid cell line TK6. The cell cultures were treated with the food enzyme with or without metabolic activation (S9‐mix).

Based on the results of a range finding test, in the first experiment, cells were exposed to the food enzyme and scored for the frequency of micronucleated mononuclear cells (MNMC) at concentrations of 8195, 12,292 and 15,570 μg TOS/mL in a short‐term treatment (3‐h exposure and 21‐h recovery period) either with or without S9‐mix.

In the second experiment, cells were exposed to the food enzyme and scored for MNMC at concentrations of 5463, 6146 and 7512 μg TOS/mL in a long‐term treatment (24‐h exposure without recovery period) without S9‐mix.

In the short‐term treatment without S9‐mix and in the long‐term treatment, cytotoxicity of 50.6% and 57.9% evaluated as relative population doubling were observed at the highest concentration tested, respectively.

The frequency of MNMC was not statistically significantly different to the negative controls at all concentrations tested.

The study was considered reliable without restrictions and the results of high relevance.

The Panel concluded that the food enzyme ribonuclease P did not induce an increase in the frequency of MNMC under the test conditions applied in this study.

In vitro mammalian chromosomal aberration test

3.4.1.3

The in vitro mammalian chromosomal aberration test was carried out according to OECD Test Guideline 473 (OECD, 1997b) and following GLP.27

A range finding test and two main experiments were performed with duplicate cultures of human peripheral whole blood lymphocytes. The cell cultures were treated with the food enzyme either with or without metabolic activation (S9‐mix).

The range finding test was carried out at eight concentrations of food enzyme up to 2125 μg TOS/mL for 6 h (a short‐term treatment) with or without S9‐mix and for 24 h (a long‐term treatment) without S9‐mix. A cytotoxicity reaching 50%, as relative mitotic index, was estimated to be 1530 μg TOS/mL, for the short‐term treatment with S9‐mix and 1658 μg TOS/mL for the 24‐h continuous treatment. No cytotoxicity above 50% was seen at any concentration tested in the short‐term treatment without S9‐mix.

In the first experiment, cells were exposed to the food enzyme and scored for chromosomal aberrations at concentrations of 531, 1063, 2125 μg TOS/mL, in a short‐term treatment (3‐h exposure and 21‐h recovery period) either with or without S9‐mix.

In the second experiment, cells were exposed to the food enzyme and scored for chromosomal aberrations at concentrations of 531, 1063, 2125 μg TOS/mL, in a long‐term treatment (24‐h exposure without recovery period) without S9‐mix.

No cytotoxicity was seen either in the short‐term treatment (with or without S9‐mix) or in long‐term treatment. The frequency of structural and numerical aberrations was not statistically significantly different to the negative controls at all concentrations tested.

The study was considered reliable without restrictions and the results of high relevance.

The Panel concluded that the food enzyme ribonuclease P did not induce an increase in the frequency of structural and numerical aberrations under the test conditions applied in this study.

Repeated dose 90‐day oral toxicity study in rodents

3.4.2

The repeated dose 90‐day oral toxicity study was performed following GLP and in accordance with guidelines of the Japanese Ministry of Health and Welfare (1996 and 1999). The study is in accordance with OECD Test Guideline 408 (OECD, 1998) with the following deviations: functional observations were not performed; urea was not measured in clinical chemistry (blood urea nitrogen was included); the medulla/pons was not included and only one level of the spinal cord was included in the histological examination. The Panel considered that these deviations are minor and do not impact on the evaluation of the study.

Groups of 12 male and 12 female Sprague–Dawley (Crl:CD(SD)) rats received the food enzyme by gavage in doses of 246, 492 or 984 mg TOS/kg body weight (bw) per day. Controls received the vehicle (water for injection).

One mid‐dose female was found dead on day 17 of administration. The Panel considered the death as incidental because no relation to the treatment could be established based on the clinical signs prior to the death and the gross and histopathological findings.

Haematological investigations revealed a statistically significant increase in reticulocyte percentage in mid‐dose males (+29%). The Panel considered the change as not toxicologically relevant, as it was only observed in one sex and there was no dose–response relationship.

Statistically significant changes detected in organ weights were an increase in absolute and relative adrenal weight in mid‐dose females (+22% and +14%), and an increase in relative spleen weight in mid‐dose males (+7%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all parameters), there was no dose–response relationship (all parameters) and there were no histopathological changes in the relevant organs.

No other statistically significant or toxicologically relevant differences from controls were reported.

The Panel identified a no observed adverse effect level (NOAEL) of 984 mg TOS/kg bw per day, the highest dose tested.

Allergenicity

3.4.3

The allergenicity assessment considered only the food enzyme and not additives, carriers or other excipients that may be used in the final formulation.

The potential allergenicity of the ribonuclease P produced with the P. citrinum strain AE‐RPE was assessed by comparing its amino acid sequence with those of known allergens as described in the EFSA GMO Scientific Opinion (EFSA GMO Panel, 2010). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, no match was found in the Allermatch and AllergenOnline databases.28

No reports on oral or respiratory sensitisation or elicitation reactions of the ribonuclease P under assessment have been published. No allergic reactions upon dietary exposure to any ribonuclease P have been reported in the literature.29

The Panel considered that the results of the sequence homology search and the available literature do not indicate a risk of allergic reactions upon dietary exposure to the ribonuclease P under assessment.

The production strain belongs to the Penicillium genus, which is known to cause respiratory allergy (Kurup, 2003; Shen & Han, 1998). Allergic reactions upon dietary exposure have been observed, but are rare (Xing et al., 2022). The biomass is removed during the production process; however, allergenic proteins of the production strain can be released into the culture medium from which the food enzyme is obtained.

■■■■■ that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/201130) is used as raw material. In addition, ■■■■■, a known source of allergens, is present in the culture medium. During the fermentation process, these products will mostly be degraded and utilised by the production strain.

Taken together, concerning the potential allergic reactions due to the production strain and the raw material in the culture medium, the Panel considered that residual amounts of allergenic proteins could be present in the food enzyme. Taking into account the level of dietary exposure (see Section 3.5), this would result in minute amounts in the final foods, from which allergic reactions are usually not expected.

In conclusion, the Panel considered that under the intended conditions of use, a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but that the likelihood is low.

Dietary exposure

3.5

Intended use of the food enzyme

3.5.1

The food enzyme is intended to be used in one food manufacturing process at the recommended use level summarised in Table 2.

TABLE 2: Intended use and recommended use level of the food enzyme provided by the applicant. 31

In the processing of yeast and yeast products, the food enzyme is added to yeast cultures during autolysis or during the incubation32 to release nucleotides that enhance the umami taste of the yeast extracts33 in which the food enzyme–TOS remain.

Based on data provided on temperature profile, thermostability (see Section 3.3.1) and the downstream processing within the food manufacturing process, the Panel considered that the food enzyme is inactivated during the processing of yeast and yeast products.

Dietary exposure estimation

3.5.2

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool34 by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021). The estimation involved selection of relevant food categories and application of technical conversion factors (EFSA CEP Panel, 2023).

Table 3 provides an overview of the derived exposure estimates across all surveys. Detailed mean and 95th percentile exposure to the food enzyme–TOS per age class, country and survey, as well as contribution from each FoodEx category to the total dietary exposure are reported in Appendix A – Tables 1 and 2. For the present assessment, food consumption data were available from 51 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 27 European countries (Appendix A). The highest dietary exposure was estimated to be 0.0014 mg TOS/kg bw per day in children at the 95th percentile.

Uncertainty analysis

3.5.3

In accordance with the guidance provided in the EFSA opinion related to uncertainties in dietary exposure assessment (EFSA, 2006), the following sources of uncertainties have been considered and are summarised in Table 4.

The conservative approach applied to estimate the dietary exposure to the food enzyme–TOS, in particular assumptions made on the occurrence and use levels of this specific food enzyme, is likely to have led to an overestimation of the exposure.

Margin of exposure

3.6

A comparison of the NOAEL (984 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0–0.0006 mg TOS/kg bw per day at the mean and from 0 to 0.0014 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 702,857.

CONCLUSIONS

4

Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme ribonuclease P produced with the non‐genetically modified P. citrinum strain AE‐RPE does not give rise to safety concerns under the intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for the authorisation of ribonuclease P from a non‐genetically modified Penicillium citrinum strain AE‐RPE as a new food enzyme. December 2023. Submitted by Amano Enzyme Inc.

Additional information. May 2025. Submitted by Amano Enzyme Inc.

Additional information. June 2025. Submitted by Amano Enzyme Inc.

ABBREVIATIONSbwbody weightCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFAOFood and Agricultural Organization of the United NationsGLPGood Laboratory PracticeGMOgenetically modified organismITSinternal transcribed spacerIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonLOQlimit of quantificationMNMCmicronucleated mononuclear cellsMOEmargin of exposureOECDOrganisation for Economic Cooperation and DevelopmentWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00877

COPYRIGHT FOR NON‐EFSA CONTENT

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PANEL MEMBERS

José Manuel Barat Baviera, Claudia Bolognesi, Francesco Catania, Gabriele Gadermaier, Ralf Greiner, Baltasar Mayo, Alicja Mortensen, Yrjö Henrik Roos, Marize de Lourdes Marzo Solano, Henk Van Loveren, Laurence Vernis, and Holger Zorn.

NOTE

The scientific output published implements EFSA's decision on the confidentiality requests submitted on specific items. As certain items have been awarded confidential status by EFSA they are consequently withheld from public disclosure by redaction.

Supporting information

APPENDIX A Dietary exposure estimates to the food enzyme–TOS in details