Safety evaluation of the food enzyme papain from the latex of Carica papaya L

TL;DR

This study evaluates the safety of papain, an enzyme from unripe papaya latex, used in food manufacturing and concludes it is safe under intended conditions.

Contribution

The study provides a safety evaluation of papain as a food enzyme, including allergenicity and dietary exposure assessments.

Findings

Dietary exposure to papain is up to 1.112 mg TOS/kg body weight per day.

Papain and chymopapain are known food allergens, and sequence homology suggests potential allergenicity.

The enzyme is considered safe for use in food manufacturing under the evaluated conditions.

Abstract

The food enzyme papain (EC 3.4.22.2) is extracted from the latex of unripe Carica papaya L. by Nagase (Europe) GmbH. It is intended to be used in six food manufacturing processes. The dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 1.112 mg TOS/kg body weight per day. This exposure is up to one order of magnitude lower than the intake of the corresponding fraction from unripe C. papaya L. latex. The toxicological studies provided were not required according to the current guidance, nevertheless, were evaluated as supporting evidence. For the allergenicity assessment, the Panel considered the papain as well as three other cysteine endopeptidases known to be present in the food enzyme. Papain and chymopapain are known food allergens. In addition, homology searches of the amino acid sequences of the four proteins to known allergens identified matches with 6 food and 8 respiratory allergens. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded. Based on the data provided, the origin of the food enzyme being an edible plant source and the estimated dietary exposure, the Panel concluded that the food enzyme does not give rise to safety concerns under the intended conditions of use.

Figures

Tables

TABLE 1. Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3	4 a
Papain activity	TU/mg b	990.8	1119.9	1068.2	1048
Protein	%	86.7	88.1	88.0	–
Ash	%	7.6	7.6	7.2	8.5
Water	%	3.7	2.4	2.7	3.8
Total organic solids (TOS) c	%	88.7	90.0	90.1	87.7
Activity/TOS ratio	TU/mg TOS	1117	1244	1186	1195

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 35

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM) b
Processing of meat and fish products
Production of modified meat and fish products	Meat and fish	89.6
Production of protein hydrolysates from meat and fish proteins	Meat and fish proteins	179.2
Processing of cereals and other grains
Production of baked products	Flour	22.4
Production of rice‐based meals c , 36	Rice	67.2
Processing of plant‐ and fungal‐derived products
Production of protein hydrolysates from plants and fungi	Plant proteins	179.2
Processing of yeast and yeast products	Yeast	179.2

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

Consumer 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
Actual consumers of the relevant food categories
Min–max mean (number of surveys)	0.056–0.306 (14)	0.198–0.501 (17)	0.222–0.477 (21)	0.125–0.258 (23)	0.086–0.125 (23)	0.067–0.150 (25)
Min–max 95th percentile (number of surveys)	0.250–0.946 (13)	0.540–1.112 (16)	0.461–0.975 (21)	0.277–0.593 (22)	0.198–0.435 (23)	0.149–0.331 (24)

TABLE 4. Summary of estimated dietary exposure to the SMT–Equivalent resulting from eating raw and cooked unripe papaya.

TABLE 5. Qualitative evaluation of the influence of uncertainties on the dietary exposure estimate.

Sources of uncertainties	Direction of impact
	Exposure to FE–TOS	Exposure to SMT–equivalent
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)	+	NA
Possible national differences in categorisation and classification of food	+/−	+/−
Use of the consumption data of unripe C. papaya L. from Thailand and Philippines to calculate the exposure to the SMT–Equivalent	NA	+/−
Model assumptions and factors
Selection of broad FoodEx categories to calculate the exposure to FE–TOS	+	NA
Only green papaya salad and soup are included to calculate the intake of SMT–Equivalent	NA	−
Use of recipe fractions to disaggregate FoodEx categories	+/−	NA
Use of technical factors in the exposure model	+/−	NA
The applied food enzyme yield factor was the mean value	NA	+/−

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 microorganisms or products thereof including a product obtained by a fermentation process using microorganisms: (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 Union 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.

Five applications have been introduced by the Association of Manufacturers and Formulators of Enzyme Products (AMFEP) for the authorisation of the food enzyme Papain from Carica papaya, and the companies ‘Qualifica’ for the authorisation of the food enzyme Plant coagulant from Cardoon flower (Cynara cardunculus L.), ‘Advanced Enzyme Technologies Ltd.’ for the authorisation of the food enzyme Triacylglycerol lipase from a genetically modified strain of Aspergillys niger agg. (strain FL105SC); ‘Kerry Ingredients & Flavours’ for the authorisation of the food enzyme Endo‐1,3(4)‐beta‐glucanase from a genetically modified strain of Bacillus subtilis (strain CBS 613.94) and ‘AB Enzymes GmbH.’ for the authorisation of the food enzyme Endo‐1,4‐beta‐glucanase from a genetically modified strain of Trichoderma reesei (RF5261).

Following the requirements of Article 12.1 of Regulation (EC) No 234/20113 implementing Regulation (EC) No 1331/2008, the Commission has verified that the five applications fall within the scope of the food enzyme Regulation and contain all the elements required under Chapter II of that Regulation.

Terms of Reference

1.1.2

The European Commission requests the European Food Safety Authority to carry out the safety assessments on the food enzymes Papain from Carica papaya, Plant coagulant from Cardoon flower (Cynara cardunculus L.), Triacylglycerol lipase from a genetically modified strain of Aspergillus niger agg. (strain FL105SC); Endo‐1,3(4)‐beta‐glucanase from a genetically modified strain of Bacillus subtilis (strain CBS 613.94) and Endo‐1,4‐beta‐glucanase from a genetically modified strain of Trichoderma reesei (RF5261) in accordance with Article 17.3 of Regulation (EC) No 1332/2008 on food enzymes.

Interpretation of the Terms of Reference

1.2

The present scientific opinion addresses the European Commission's request to carry out the safety assessment of food enzyme Papain from C. papaya submitted by AMFEP.

The application was submitted initially as a joint dossier4 and identified as the EFSA‐Q‐2015‐00559. During the risk assessment, a meeting between EFSA, the European Commission and the AMFEP,5 it was agreed that joint dossiers will be split into individual data packages.

The current opinion addresses one data package originating from the former joint dossier. This data package is identified as EFSA‐Q‐2023‐00226 and concerns the food enzyme papain produced from the latex of the unripe C. papaya L. submitted by Nagase (Europe) GmbH.

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme papain from the latex of the unripe fruit of C. papaya L.

Additional information was spontaneously submitted on 15 February 2024 (see ‘Section 5’).

Additional information, requested from the applicant during the assessment process on 1 March 2024 and 22 September 2025, was received on 5 April 2024 and on 25 September 2025, respectively (see Section 5).

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 ‘Guidance on the submission of a dossier on food enzymes for safety evaluation’ (EFSA CEF Panel, 2009) have been followed for the evaluation of the application. Additional information was requested in accordance with the updated ‘Scientific Guidance for the submission of dossiers on food enzymes’ (EFSA CEP Panel, 2021) and the guidance on the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023).

ASSESSMENT

3

The food enzyme under application has one declared activity. The term ‘papain’ covers a number of cysteine endopeptidases present in papaya.IUBMB nomenclaturePapainSystematic name–SynonymsPapayotin; papaya peptidase IIUBMB NoEC. 3.4.22.2CAS No9001‐73‐4EINECS No232‐627‐2

Papains catalyse the hydrolysis of proteins with broad specificity for peptide bonds, with preference for amino acids with large hydrophobic side chains at the P2 position, resulting in the generation of peptides and amino acids.

The food enzyme under assessment is intended to be used in six food manufacturing processes: processing of meat and fish products for the production of (1) modified meat and fish products and (2) protein hydrolysates from meat and fish proteins; processing of cereals and other grains for the production of (3) baked products and (4) rice‐based meals; (5) processing of plant‐ and fungal‐derived products for the production of protein hydrolysates from plants and fungi and (6) processing of yeast and yeast products.

Source of the food enzyme

3.1

The food enzyme papain is obtained from the latex harvested from the unripe fruit of non‐genetically modified C. papaya L.,6 a species belonging to the Caricaceae family (OECD, 2010) and a perennial tree native to Mexico and South America (Garrett, 1995; Siar et al., 1998).

The milky latex is stored in secretory structures known as laticifers, which are distributed throughout various parts of the papaya plant. The highest concentration of latex is present in the peels of unripe papaya fruit. During the ripening of the papaya fruit, the production of the latex by the laticifers gradually declines. Mechanical injury, such as longitudinal incisions on the surface of the unripe fruit induce latex exudation (El Moussaoui et al., 2001).

The ripe papaya fruit is commonly consumed worldwide. Unripe papaya is also used as vegetable in south Asian countries such as Thailand, where it is cooked in soup or consumed raw as a salad ingredient (Sone et al., 1998) or canned in sugar syrup in Puerto Rico (Morton, 1987).

A literature search was conducted by EFSA to identify compounds in papaya peels or latex or extracts thereof that could be hazardous to human health upon oral exposure. The Panel did not identify any study both reliable and relevant to the source of the food enzyme, except for allergenicity.

No issues of concern were identified by the Panel from the source material.

Production of the food enzyme

3.2

The food enzyme is produced by a third‐party supplier. The applicant re‐sieves and tests the quality of the food enzyme to confirm specifications.7 The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,8 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with Good Manufacturing Practice.9

The food enzyme is obtained by ■■■■■ from the papaya latex. After a flocculation step, the liquid containing the enzyme is centrifuged and filtrated. The filtrate containing the enzyme is further purified and concentrated, including an ultrafiltration step in which the enzyme protein is retained, while most of the low molecular mass material passes the filtration membrane and is discarded. Finally, the food enzyme is dried.10 Around ■■■■■ kg of fresh latex is needed to produce 1 kg of papain,11 corresponding to a yield factor of ■■■■■. The applicant provided information on the identity of the substances used in the extraction and in the subsequent downstream processing of the food enzyme.

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 papain is a single polypeptide chain of 345 amino acids,12 which includes the signal peptide, propeptide and the mature protein. The molecular mass of the mature protein, calculated from the amino acid sequence, is 23.3 kDa.13 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.14 A consistent protein pattern was observed across all batches. The gel showed the target protein migrating between the marker proteins of 20 and 25 kDa in all batches, consistent with the expected mass of the enzyme. The food enzyme was tested for amylase and lipase activities, and none were detected.15

No other enzyme activities were reported.

The applicant's in‐house determination of papain activity is based on the hydrolysis of bovine casein (reaction conditions: ■■■■■ ■■■■■, ■■■■■).16 The release of peptides containing L‐tyrosine is measured spectrophotometrically at 280 nm. The enzyme activity is quantified relative to an internal enzyme standard and expressed in Tyrosine Unit (TU)/g. One TU is defined as the amount of enzyme which release one microgram of L‐tyrosine equivalents from casein per minute under the conditions of the assay.17

The food enzyme has a temperature optimum around 80°C (pH 7.5, 10 min), the maximum temperature tested and a pH optimum around 6–7 (40°C).^9^ Thermostability was tested by pre‐incubation of the food enzyme for 30 min at different temperatures (pH 7). Enzyme activity decreased above 40°C showing about 10% of residual activity at 80°C.18

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation and one batch produced for the toxicological tests (Table 1).19 ^,^ 20 The mean total organic solids (TOS) of the three batches for commercialisation was 89.6% and the mean enzyme activity/TOS ratio was 1182 TU/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches was below 1 mg/kg,21 which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). In addition, mercury and arsenic contents were below the limits of quantification (LoQs) of the employed methods.22 ^,^ 23 Cadmium was detected only in one of the three batches with a concentration of 0.07 mg/kg. The Panel considered this concentration as not of concern.

The food enzyme 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).24 A total viable bacteria count of 100 cfu/g was detected in two of the three tested batches.25 Additionally, the presence of Bacillus cereus was examined in four food enzyme batches and it was found to be below the limit of detection (100 cfu/g).26

The presence of aflatoxins (B1, B2, G1 and G2), fumonisin B1 and B2, deoxynivalenol, T‐2 toxin, zearalenone and ochratoxin A was examined in three food enzyme batches and were below the LoQ.27 ^,^ 28

More than 600 pesticides were tested in three food enzyme batches and no pesticide residues were detected.29

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

Toxicological data

3.4

Toxicological tests are not required (EFSA CEP Panel, 2021), because: (i) the food enzyme is obtained from an edible plant source, (ii) no issues of concern arise from the food enzyme production process, (iii) the dietary exposure to the food enzyme–TOS under the intended conditions of use is within the same magnitude compared to the intake of the fraction from C. papaya L. (see Section 3.5). However, the applicant, following the guidance available at the time of the submission of the application (EFSA CEF Panel, 2009), provided a battery of toxicological tests performed with the food enzyme under assessment (batch 4, Table 1). These studies were assessed and reported as supporting evidence for this evaluation.

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

The batch 4 (Table 1) used in these studies has similar enzyme activity/TOS ratio as the batches used for commercialisation, and is thus considered suitable as a test item.

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, 2020) and following Good Laboratory Practice (GLP).30 Four strains of S. Typhimurium (TA98, TA100, TA1535 and TA1537) and E. coli WP2uvrA (pKM101) were used with or without metabolic activation (S9‐mix). A dose range‐finding test, and a main experiment were carried out in triplicate applying the pre‐incubation method.

The dose range‐finding test was carried out with eight concentrations of food enzyme, namely 8, 21, 51, 128, 320, 800, 2000 and 5000 μg TOS/plate, with and without S9‐mix. Dose‐dependent growth inhibition was observed in all tester strains without S9‐mix (TA1537 at ≥ 20 μg TOS/plate, TA100 at ≥ 51 μg TOS/plate, TA98 at ≥ 128 μg TOS/plate, TA1535 and WP2uvrA at ≥ 320 μg TOS/plate) and with S9‐mix in TA100 and TA98 strains at ≥ 2000 μg TOS/plate; moreover, a reduction in the number of revertant colonies occurred in TA1535 and TA1537 strains at 5000 μg TOS/plate with S9‐mix. To identify a minimum of four concentration levels with no bacterial growth inhibition, an additional dose range‐finding test was carried out with seven concentrations of food enzyme without S9‐mix in strains TA100 (ranging from 0.33 to 80 μg TOS/plate), TA98 (0.82 to 200 μg TOS/plate) and TA1537 (0.13 to 32 μg TOS/plate). Growth inhibition was observed in strain TA1537 at ≥ 13 μg TOS/plate, in TA100 at ≥ 32 μg TOS/plate and in TA98 at ≥ 80 μg TOS/plate.

The main experiment was carried out using seven concentrations of the food enzyme ranging from 0.6 to 625 μg TOS/plate without S9‐mix, or from 39 to 5000 μg TOS/plate with S9‐mix. Growth inhibition was observed in all tester strains without S9‐mix (TA1537 at ≥ 19 μg TOS/plate, TA100 at ≥ 39 μg TOS/plate, TA98 at ≥ 78 μg TOS/plate, TA1535 at ≥ 156 μg TOS/plate and WP2uvrA at ≥ 313 μg TOS/plate) and in TA100 at ≥ 1250 μg TOS/plate and TA98 at ≥ 2500 μg TOS/plate, with S9‐mix. 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 papain 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, 2016a) and following GLP.31 A dose range‐finding test and two main experiments were performed with duplicate cultures of a human lymphoblastoid cell line (TK6). The cell cultures were treated with the food enzyme with or without metabolic activation (S9‐mix).

In the range‐finding test cytotoxicity ≥ 59.5% was seen at concentrations of ≥ 350 μg TOS/mL in the short‐term treatment without S9‐mix, ≥ 58.3% at concentrations of ≥ 650 μg TOS/mL in the short‐term treatment with S9‐mix and ≥ 55% at concentrations of ≥ 150 μg TOS/mL in the long‐term treatment without S9‐mix.

Based on these results, in the first experiment, cells were exposed to the food enzyme and scored for the frequency of bi‐nucleated cells with micronuclei (MNBN) in a short‐term treatment (3‐h exposure and 24‐h recovery period) at concentrations of 100, 200 and 350 μg TOS/mL without S9‐mix and 300, 500 and 600 μg TOS/mL with S9‐mix.

In the second experiment, cells were exposed to the food enzyme and scored for MNBN at concentrations of 25, 75, 125 and 150 μg TOS/mL in a long‐term treatment (24‐h exposure without recovery period) without S9‐mix.

In the short‐term treatment, cytotoxicity of 51.2% and 52.7% based on replication index was observed at 350 μg TOS/mL without S9‐mix and at 600 μg TOS/mL with S9‐mix. In the long‐term treatment, cytotoxicity of 55% was observed at a concentration of 150 μg TOS/mL. The frequency of MNBN was statistically significantly different to the negative controls at concentrations of 125 and 150 μg TOS/mL in the long‐term treatment without S9‐mix. A concentration response relationship was also observed, and the values were outside the historical control range.

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

The Panel concluded that the food enzyme papain induced an increase in the frequency of MNBN under the test conditions applied in this study.

In vivo combined mammalian erythrocyte micronucleus and comet test

3.4.1.3

The combined in vivo mammalian erythrocyte micronucleus and Comet test was carried out according to OECD Test Guidelines 474 (OECD, 1997) and 489 (OECD, 2016b) respectively, and following GLP.32

In the repeated dose 90‐day oral toxicity study, 2000 mg/kg bw per day was not tolerated and resulted in mortality of all treated animals, while no test item‐related changes were observed at 500 mg/kg bw per day. Based on these results, 1000 mg/kg bw per day was considered the maximum tolerated dose and the food enzyme was given by gavage for three consecutive days to groups of six male Sprague–Dawley rats (Crl:CD(SD)) at 125, 250, 500 and 1000 mg TOS/kg bw per day. The rats were euthanised 3 h after dosing. Liver, kidney and duodenum samples were taken for the Comet assay and the bone marrow for the micronucleus test.

One high‐dose male was found dead after the first administration and decreased in locomotor activity, irregular respiration and ptosis was observed in another rat in the same group.

A significant decrease in the ratio of polychromatic erythrocytes (% PCE) was observed at ≥ 500 mg TOS/kg bw per day. No statistically significant increase in mean frequencies of micronucleated polychromatic erythrocytes (MNPCE) was observed in animals treated with food enzyme compared to the concurrent vehicle control group. The toxicity to the bone marrow, evaluated as decrease in %PCE is a sufficient evidence of bone marrow exposure.

No statistically significant increase in percentage of tail DNA values was observed in liver, kidney and duodenum of animals treated with the food enzyme compared to the concurrent vehicle control group.

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

The Panel concluded that the food enzyme papain did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in rat bone marrow or DNA damage in the rat liver, kidney and duodenum.

Conclusion on genotoxicity

3.4.1.4

The food enzyme papain was evaluated in a battery of reliable in vitro genotoxicity studies. In the presence or absence of S9‐mix, it did not induce gene mutations in bacteria in the Ames test. Positive results were obtained in the absence of metabolic activation in an in vitro micronucleus test. An in vivo follow‐up with a combined mammalian erythrocyte micronucleus and comet assay did not show any increase of MNPCE in the bone marrow at toxic doses, or of DNA damage in liver, kidney and duodenum. The Panel concluded that there is no concern for genotoxicity of the food enzyme papain.

Repeated dose 90‐day oral toxicity study in rodents

3.4.2

The repeated dose 90‐day oral toxicity study was performed under GLP and according to OECD Test Guideline 408 (OECD, 2018)33 with the following deviation: plasmatic urea was not determined. The Panel considered that this deviation is minor and does not impact the evaluation of the study.

Groups of 10 male and 10 female Sprague–Dawley (Crl:CD(SD)) rats received the food enzyme by gavage in doses of 125, 500 and 2000 mg TOS/kg body weight (bw) per day. Controls received the vehicle (water for injection).

All high‐dose males and females were found dead between days 2 and 12 of administration. At microscopic examination, severe and extensive degenerative findings were observed in all rats affecting the entire gastrointestinal tract, including autolysis/melting, atrophy and haemorrhage. These findings variably accounted for the macroscopic observations of reddish contents and red/black patches seen at necropsy in the stomach and intestine. The Panel considered these findings as the result of direct local irritation of the gastrointestinal mucosa and the cause of death in high‐dose rats.

Clinical chemistry investigations in rats euthanised at the end of the treatment period revealed a statistically significant decrease in glucose in low‐ and mid‐dose males (−12% both), a decrease in blood urea nitrogen (BUN) in mid‐dose males (−18%) and an increase in aspartate aminotransferatse (AST) in low‐dose females (+77%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all), there was no dose–response relationship (all), there were no changes in other relevant parameters (creatinine for urea, biomarkers of liver damage for AST), there were no histopathological changes in kidney or liver, and the changes were within the historical control values (glucose and BUN).

The urinalysis in rats euthanised at the end of the treatment period revealed a statistically significant increase in sodium and chloride excretion in mid‐dose females (+35%, +31%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex, there were no changes in other relevant urinalysis parameters, there were no histopathological changes in the kidney and the changes were within the historical control values.

Statistically significant changes detected in organ weights in rats euthanised at the end of the treatment period were a decrease in relative thymus weight in mid‐dose females (−22%). The Panel considered the change as not toxicologically relevant, as it was only observed in one sex, there were no changes in circulating white blood cells, there were no histopathological changes in the thymus and the change was within the historical control values.

All low‐ and mid‐dose rats survived until the end of the treatment period and no test item‐related macro‐ nor microscopic changes were observed. No other statistically significant or toxicologically relevant differences from controls were reported.

The Panel identified a no observed adverse effect level (NOAEL) of 500 mg TOS/kg bw per day, the mid‐dose tested, based on the mortality observed in both sexes at 2000 mg TOS/kg bw per day.

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.

Papain is one of the endopeptidases within the cysteine endopeptidase complex, which contains four proteolytic activities: papain, chymopapain, caricain and glycyl endopeptidase. The amino acid sequences of these four proteins present a high degree of homology (from 62% to 81% sequence identity). The Panel considered all four proteins in the allergenicity assessment.

The potential allergenicity of these four proteins was assessed separately by the Panel by comparing each 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, matches with six food and eight respiratory allergens were found using the AllergenOnline and COMPARE databases.34

Papain and chymopapain (Cari p 2) are food allergens from papaya (C. papaya L.). Cari p 2 was detected in pollen and pulp of raw and ripe papaya fruits (Bhowmik et al., 2021). Several studies reported occupational rhinitis and asthma in workers of industries where papain is handled (Baur et al., 1982; Baur & Fruhmann, 1979; Niinimaki et al., 1993; Soto‐Mera et al., 2000; Van Kampen et al., 2005). Administration of chymopapain for chemonucleolysis resulted in sensitisation in some patients (Garcia‐Ortega et al., 1991). Severe systemic reactions mediated by papain‐specific IgE were observed in some individuals that ingested papain‐containing meat tenderiser (Mansfield & Bowers, 1983). Caricain and glycyl endopeptidase showed 82.5%–87.5% sequence identity to papain and chymopapain, and IgE binding to caricain and glycyl endopeptidase was reported (Dando et al., 1995).

Matching food allergens were Ana c 2 (52.5%–62.5% sequence identity), a bromelain from pineapple (Ananas comosus); actinidins (56.3%–61.3% sequence identity) from kiwi fruits (Actinidia deliciosa, A. chinensis) and a cysteine protease (47.5%–50% sequence identity) from soybean (Glycine max).

Bromelain from pineapple was described as occupational allergen eliciting allergic reactions after inhalation or dietary exposure (Gailhofer et al., 1988; Nettis et al., 2001).

Actinidins are major kiwi allergens (Grozdanovic et al., 2014). Kiwi allergic individuals also demonstrate IgE reactivity to bromelain and papain. In addition, the presence of cross‐reactive allergens in papaya and fig was shown by cross‐inhibition experiments (Hemmer et al., 2004).

IgE binding to the soybean cysteine protease was described in soybean allergic individuals with skin‐related symptoms (Morita et al., 2012; Ogawa et al., 1993).

The matching pollen allergen was Amb a 11 (53.8%–58.8% sequence identity), a cysteine protease from ragweed (Ambrosia artemisiifolia). Ragweed is associated with the pollen‐food allergy syndrome. Reactions within this syndrome are usually restricted to the buccal cavity and seldomly lead to anaphylaxis (Sarkar et al., 2018).

The matching respiratory allergens were group 1 mite allergens (36.3%–48.8% sequence identity), cysteine proteases from Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis, Euroglyphus maynei, Tyrophagus putrescentiae and Sarcoptes scabiei. Der p 1 and Der f 1 are major mite allergens associated with rhinitis and asthma (Xu et al., 2025). No evidence of papain‐related allergic reactions upon dietary exposure in individuals sensitised to mites is available (Giangrieco et al., 2023).

The Panel considered that the results of the sequence homology search and the available literature indicate a risk of allergic reactions for papaya, ananas, kiwi, soy, fig and pollenallergic individuals upon dietary exposure to the papain under assessment.

The food enzyme is obtained from the latex of unripe papaya. Papaya also contains other food allergens, for example endo‐polygalacturonase (Cari p 1) and endochitinase, involved in the latex‐fruit syndrome (Rojas‐Mandujano et al., 2018). Cari p 1 was detected in papaya peel, pulp and pollen (Sarkar et al., 2018). The Panel considered that these allergenic proteins could also be present in the food enzyme.

In conclusion, the Panel considered that under the intended conditions of use, a risk of allergic reactions upon dietary exposure to this food enzyme, particularly for papaya, ananas, kiwi, soy, fig and pollen allergic individuals, cannot be excluded. However, the likelihood of such reactions will not exceed the risk of reactions after consumption of papaya, ananas, kiwi, soy and fig.

Dietary exposure

3.5

Intended use of the food enzyme

3.5.1

The food enzyme is intended to be used in six food manufacturing processes at the recommended use levels summarised in Table 2.

TABLE 2: Intended uses and recommended use levels of the food enzyme as provided by the applicant. 35

In the production of modified meat and fish products, the food enzyme is added to meat or fish37 to hydrolyse the fibrous proteins for tenderising purposes. The food enzyme–TOS remain in the final processed foods.

In the production of protein hydrolysates, the food enzyme is added to a variety of protein‐rich materials from animal (e.g. meat, fish, gelatine, collagen) or plant sources (e.g. wheat, corn)38 to achieve the desired degree of hydrolysis. The food enzyme–TOS remain in these protein hydrolysates.

In the production of baked products, the food enzyme is added to flour during the preparation of the dough39 to hydrolyse gluten proteins, facilitating the handling of the dough. The food enzyme–TOS remain in the baked products.

In the production of rice‐based meals,40 the food enzyme is added to rice prior to cooking41 to increase the water absorption of rice.42 The food enzyme–TOS remain in the cooked rice.

In the processing of yeast and yeast products, the food enzyme is added to yeast before autolysis or directly to autolysed yeast or to yeast extracts43 to increase the yield and to enrich the savoury taste of the yeast products, in which the food enzyme–TOS remain.

Based on data provided on thermostability (see Section 3.3.1) and the downstream processing steps applied in the respective food manufacturing processes, the Panel considered that the food enzyme may remain in its active form in all food manufacturing processes, depending on the processing conditions.

Dietary exposure estimation

3.5.2

Following the EFSA Guidance Document on food enzymes (EFSA CEP Panel, 2021), a comparison was made between the chronic exposures:

• dietary exposure to the food enzyme–TOS, resulting from the intended uses as proposed by the applicant (herein referred as ‘FE–TOS’) and
• dietary exposure to a fraction of C. papaya L. unripe fruit comparable to the food enzyme–TOS, resulting from the consumption of raw or cooked unripe unripe C. papaya L. (herein referred to as source material TOS equivalent,‘SMT–Equivalent’)

Estimated dietary exposure to the food enzyme–TOS

3.5.2.1

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool44 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) together with the information provided in Appendix C.

Table 3 provides an overview of the derived exposure estimates across all surveys from actual consumers of the relevant food categories. 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 B). The highest dietary exposure was estimated to be 1.112 mg TOS/kg bw per day in toddlers at the 95th percentile. As the food categories relevant to the intended uses of this food enzyme are commonly consumed by Europeans, the estimates calculated for survey respondents are the same as those calculated for actual consumers.

Estimated dietary exposure to the SMT–Equivalent

3.5.2.2

Unripe papaya (also known as green papaya) is rich of papain, especially in the latex. The food enzyme is obtained from the papaya latex. In Europe, papaya is consumed mainly as ripe fruit, while the consumption of unripe papaya is limited and consumption data are not available. In Southeast Asia, unripe papaya is commonly eaten raw as salad or cooked in soup.45 For the estimation of the SMT–Equivalent, consumption data from actual consumers of green papaya salad or soupin the Philippines46 and Thailand47 were used.

The chronic dietary exposure to the SMT–Equivalent was calculated in two steps: firstly, the intake of green papaya was converted to the intake of latex, using a factor of 0.016,48 as provided by the applicant, to account for the amount of latex in unripe C. papaya L. Secondly, the intake of latex was converted into a fraction comparable to the food enzyme–TOS, by applying a yield factor (■■■■■)49 to take into account the yield of the food enzyme–TOS from fresh latex (Section 3.2).

Table 4 provides an overview of the estimated exposure to the SMT–Equivalent for the actual consumers of raw and cooked unripe papaya. The age ranges are presented as provided in the surveys from the Philippines and Thailand. The highest dietary exposure was estimated to be 4.212 mg/kg bw per day in children at the 95th percentile.

Comparison of the two exposure estimates

3.5.2.3

The intakes of the SMT–Equivalent by consumers from the Philippines and Thailand (Table 4) are up to one order of magnitude greater than the dietary exposure to the food enzyme‐TOS for European consumers (Table 3).

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 5.

The estimation of the SMT–Equivalent is based on a realistic scenario, while the estimation of the dietary exposure to the food enzyme–TOS is based on a conservative approach. In particular assumptions made on the occurrence and use levels of this specific food enzyme have likely led to an overestimation of the dietary exposure to the food enzyme–TOS.

Margin of exposure

3.6

Since toxicological tests were not required for this food enzyme (see Section 3.4), the margin of exposure was not calculated.

CONCLUSIONS

4

Based on the data provided, the origin of the food enzyme being an edible plant source and the estimated dietary exposure, the Panel concluded that the food enzyme papain extracted from the latex of the unripe C. papaya L. does not give rise to safety concerns under the intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for authorisation of papain from Carica papaya in accordance with Regulation (EC) No.1331/2008. March 2023. Submitted by Nagase (Europe) GmbH.

Additional information: spontaneous data. February 2024. Submitted by Nagase (Europe) GmbH.

Additional information. April 2024. Submitted by Nagase (Europe) GmbH.

Additional information. September 2025. Submitted by Nagase (Europe) GmbH.

ABBREVIATIONSAMFEPAssociation of Manufacturers and Formulators of Enzyme ProductsASTaspartate aminotransferaseBUNblood urea nitrogenbwbody weightCASChemical Abstracts ServiceCEFEFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing AidsCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsCFUcolony forming unitsDRFdose range findingEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFAOFood and Agricultural Organization of the United NationsFEIMFood Enzyme Intake ModelFEZEFSA Panel on Food EnzymesGLPGood Laboratory PracticeGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonLODlimit of detectionLOQlimit of quantificationMNBNbi‐nucleated cells with micronucleiMNPCEmicronucleated polychromatic erythrocytesNOAELno observed adverse effect levelOECDOrganisation for Economic Co‐operation and DevelopmentPCEpolychromatic erythrocytesRMRaw MaterialSDS‐PAGEsodium dodecyl sulfate‐polyacrylamide gel electrophoresisTOStotal organic solidsTUtyrosine UnitWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00226

COPYRIGHT FOR NON‐EFSA CONTENT

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.

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 full opinion will be published in accordance with Article 12 of Regulation (EC) No 1331/2008 once the decision on confidentiality will be received from the European Commission.

Supporting information

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