Safety evaluation of the food enzyme cyclomaltodextrin glucanotransferase from the non‐genetically modified Anoxybacillus caldiproteolyticus strain AE‐KCGT

TL;DR

This study evaluates the safety of a food enzyme produced by a non-genetically modified bacteria, finding it safe for most age groups but raising concerns for infants and children.

Contribution

The study provides a safety evaluation of cyclomaltodextrin glucanotransferase from Anoxybacillus caldiproteolyticus for food use.

Findings

The enzyme is safe for adolescents, adults, and the elderly with a margin of exposure above 362.

Infants, toddlers, and children have insufficient margins of exposure to ensure safety.

Potential allergenic risk exists due to homology with three respiratory allergens.

Abstract

The food enzyme cyclomaltodextrin glucanotransferase ((1–4)‐α‐d‐glucan 4‐α‐d‐[(1–4)‐ α‐d‐glucano]‐transferase; EC 2.4.1.19) is produced with the non‐genetically modified Anoxybacillus caldiproteolyticus strain AE‐KCGT by Amano Enzyme Inc. The food enzyme is free from viable cells of the production organism. It is intended to be used in two food manufacturing processes. The dietary exposure was estimated to be up to 20.27 mg total organic solids (TOS)/kg bw per day in European populations. Genotoxicity tests did not raise safety concerns. 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 2246 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure (MoE) of at least 141 for infants, 111 for toddlers, 154 for children, 362 for adolescents, 508 for adults and 713 for the elderly. A search for the homology of the amino acid sequence of the cyclomaltodextrin glucanotransferase to known allergens was made and matches with three respiratory allergens were found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but 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 for adolescents, adults and the elderly. The MoE is insufficient to exclude safety concerns for infants, toddlers and children.

This publication is linked to the following EFSA scientific opinion: https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9222

Tables

TABLE 1. Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3	4 a
Cyclomaltodextrin glucanotransferase activity	U/g b	719	871	649	865
Protein	%	2.5	2.2	2.1	NA c
Ash	%	9.5	9.6	9.8	0.8
Water	%	68.1	69.7	68.4	88.4
Total organic solids (TOS) d	%	22.4	20.7	21.8	10.8
Activity/TOS ratio	U/mg TOS	3.2	4.2	3.0	8.0

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 31

Food manufacturing process a	Raw material	Recommended dosage of the food enzyme (mg TOS/kg RM) b
Processing of plant‐ and fungal‐derived products
Production of plant extracts	Leaf (herb, tea)	1400–70,000
	Leaf extracts rich in steviol glycosides
	Fruit peels rich in glycosides such as hesperidin 32
Processing of sugars
Production of specialty carbohydrates (cyclodextrins)	Starch	140–2808

TABLE 3. Summary of the estimated dietary exposure to 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	3–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0.046–2.9 (12)	0.73–6.01 (15)	1.04–5.19 (19)	0.34–2.19 (21)	0.17–1.38 (22)	0.064–0.95 (23)
Min–max 95th percentile (number of surveys)	0.092–15.91 (11)	4.12–20.27 (14)	4.07–14.58 (19)	1.53–6.2 (20)	0.7–4.42 (22)	0.35–3.15 (22)

TABLE 4. Summary of dietary exposure to β‐cyclodextrin (E 459) from their use as food additives in the maximum level exposure assessment scenario and subsequent exposure to the food enzyme, in six population groups (minimum–maximum across the dietary surveys in mg/kg bw per day).

Population group	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	3–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Regulatory maximum level exposure scenario (mg β‐cyclodextrin (E 459)/kg bw per day)
Min–max mean (number of surveys)	0.7–4.5 (6)	1.9–19.1 (10)	2–24.4 (18)	1.7–16.2 (17)	1–7.7 (17)	0.9–6.8 (14)
Min–max 95th percentile (number of surveys)	2.6–16.2 (5)	8.7–56.6 (7)	8–73.1 (18)	6.3–56.8 (17)	4–26.3 (17)	3.4–15.2 (14)
Estimated exposure to the food enzyme–TOS (mg TOS/kg bw per day)
Min–max mean (number of surveys)	0.002–0.013 (6)	0.005–0.054 (10)	0.006–0.069 (18)	0.005–0.045 (17)	0.003–0.022 (17)	0.003–0.019 (14)
Min–max 95th percentile (number of surveys)	0.007–0.045 (5)	0.024–0.159 (7)	0.022–0.205 (18)	0.018–0.159 (17)	0.011–0.074 (17)	0.01–0.043 (14)

TABLE 5. 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
Exposure to food enzyme–TOS always calculated based on the recommended maximum use level	+
Selection of broad FoodEx categories for the exposure assessment	+
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−
Assumption that 100% of TOS remains in the final foods	+
Overall intake via all sources of dietary exposure
Only the highest intake at the P95 percentile shown in Table 3 is used to derive the lowest margin of exposure	–

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 European Union 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.

The ‘Guidance on submission of a dossier on food enzymes for safety evaluation’ (EFSA, 2009a) lays down the administrative, technical and toxicological data required.

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 company “Amano Enzyme Inc.” for the authorization of the food enzymes Cyclomaltodextrin glucanotransferase from Geobacillus stearothermophilus (strain AE‐KCGT), Cyclomaltodextrin glucanotransferase from Paenibacillus macerans (strain AE‐CGT) and Thermolysin from Geobacillus stearothermophilus (strain AE‐TP), “Sanyo Fine Co., Ltd.” for the authorization of the food enzyme Phospholipase A_2_ from porcine pancreas, and “Nagase (Europa) GmbH” for the authorization of the food enzyme Beta amylase form soybean (Glycine max).

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 three 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 a safety assessments of the food enzymes Cyclomaltodextrin glucanotransferase from Geobacillus stearothermophilus (strain AE‐KCGT), Cyclomaltodextrin glucanotransferase from Paenibacillus macerans (strain AE‐CGT), Thermolysin from Geobacillus stearothermophilus (strain AE‐TP), Phospholipase A_2_ from porcine pancreas and Beta‐amylase from soybean (Glycine max) 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 request to carry out of the safety assessment of the food enzyme cyclomaltodextrin glucanotransferase from Geobacillus stearothermophilus (strain AE‐KCGT).

Recent data identified the production microorganism as Anoxybacillus caldiproteolyticus (Section 3.1). Therefore, this name will be used in this opinion instead of G. stearothermophilus.

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme cyclomaltodextrin glucanotransferase from Paenibacillus macerans (strain AE‐CGT). The dossier was updated on 26 February 2015.

Additional information was requested from the applicant during the assessment process on 20 January 2021 and on 24 September 2024, and received on 23 February 2022 and on 15 October 2024, 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, 2009b) and following the relevant existing guidance's of EFSA Scientific Committees.

The ‘Guidance on the submission of a dossier on food enzymes for safety evaluation’ (EFSA, 2009a) has 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

IUBMB nomenclatureCyclomaltodextrin glucanotransferaseSystematic name(1–4)‐α‐d‐glucan 4‐α‐d‐[(1–4)‐α‐d‐glucano]‐transferase (cycling)SynonymsCyclodextrin glycosyltransferase; α‐cyclodextrin glucanotransferaseIUBMB NoEC 2.4.1.19CAS No9030‐09‐5EINECS No618‐522‐8

Cyclomaltodextrin glucanotransferases catalyse the transglycosylation of glucans by formation of (1–4)‐α‐d‐glucosidic bonds, resulting in the generation of α‐, β‐ and γ‐cyclodextrins and transglycosylated glucans.

The food enzyme under assessment is intended to be used in two food manufacturing processes: (1) processing of plant‐ and fungal‐derived products for the production of plant extracts; (2) processing of sugars for the production of specialty carbohydrates (cyclodextrins).

Source of the food enzyme

4

3.1

The cyclomaltodextrin glucanotransferase is produced with the non‐genetically modified bacterium A. caldiproteolyticus strain AE‐KCGT which is deposited at the National Institute of Technology and Evaluation (NITE) Biological Resource Center (Japan) with the deposition number ■■■■■.5 The production strain was obtained from the parental strain ■■■■■.6

The production strain was identified as A. caldiproteolyticus ■■■■■.7

Whole genome sequencing (WGS) analysis did not detect antibiotic resistance (AMR) genes of concern in the production strain.

Production of the food enzyme

3.2

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 current Good Manufacturing Practice.9

The production strain is grown as a pure culture using a typical industrial medium in a submerged, 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 leaving a filtrate containing the food enzyme. 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.10 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.11

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 cyclomaltodextrin glucanotransferase is a single polypeptide chain of ■■■■■ amino acids including a signal peptide.12 The molecular mass of the protein, calculated from the amino acid sequence, is ■■■■■ kDa.13 The food enzyme was analysed by size exclusion chromatography. The chromatograms for the three food enzyme batches for commercialisation showed similar patterns.14

No other enzymatic activities were reported.15

The applicant's in‐house determination of cyclomaltodextrin glucanotransferase activity is based on the reaction with starch (reaction conditions: ■■■■■°C, ■■■■■ min). The enzyme activity is measured by quantifying the degradation of starch which is coloured with iodine and detected spectrophotometrically at 660 nm. The enzyme activity is expressed in Units (U)/g. One unit of activity is defined as the amount of enzyme causing a decrease in extinction of 1% per minute under the conditions of the assay.16

The food enzyme has a temperature optimum around 70°C (pH 7.0) and a pH optimum between pH 5.0 and 6.0 (37°C). Thermostability was tested after a pre‐incubation of the food enzyme for 10 min at different temperatures (pH 7.0). Enzyme activity decreased above 60°C showing no residual activity after pre‐incubation at 70°C.17

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches used for commercialisation and one batch produced for the toxicological studies (Table 1).18 The mean total organic solids (TOS) of the three food enzyme batches was 21.6% and the mean enzyme activity/TOS ratio was 3.4 U/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches was below 0.01 mg/kg19 ^,^ 20 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 complies with the microbiological criteria for total coliforms, Escherichia coli and Salmonella and no antimicrobial activity was detected in any of the tested batches (FAO/WHO, 2006).21

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 the production strain in the food enzyme was demonstrated ■■■■■. No colonies were produced. ■■■■■.22

Toxicological data

3.4

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

The Panel considered that batch 4 (Table 1), despite having higher activity per unit TOS than the commercial batches, was suitable for toxicological studies.

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, 1997) and following Good Laboratory Practice (GLP).23

Four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2uvrA were used with or without metabolic activation (S9‐mix), applying the pre‐incubation assay with all S. Typhimurium strains and plate incorporation method with E. coli WP2uvrA. Two separate experiments were carried out in triplicate applying eight concentrations of the food enzyme of 87.8, 175.5, 351, 702, 1404, 2808, 5616, 11,232 μg TOS/plate.24

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 Panel concluded that the food enzyme cyclomaltodextrin glucanotransferase did not induce gene mutations under the test conditions applied in this study.

In vitro mammalian chromosomal aberration test

3.4.1.2

The in vitro mammalian chromosomal aberration test was carried out according to OECD Test Guideline 473 (OECD, 2016) and following GLP.25

An experiment was performed with duplicate cultures of Chinese hamster lung‐derived fibroblasts (CHL/IU). The cell cultures were treated with the food enzyme either with or without metabolic activation (S9‐mix).

Based on the results of two preliminary tests, the concentrations giving 50% inhibition of cell growth were applied.

In the main experiment, cells were exposed to the food enzyme and scored for chromosomal aberrations at 337, 449 and 562 μg TOS/mL in the short‐term treatment (6 h exposure and 18 h recovery period) without S9‐mix, and at 112, 169 and 225 μg TOS/mL in the short‐term treatment with S9‐mix and at 225, 281 and 337 μg TOS/mL in the long‐term treatment (24 h exposure) without S9‐mix.26

A cytotoxicity of 50%, 55% and 57% was reported at the highest concentration tested in the short‐term treatment without S9‐mix, in the short‐term treatment with S9‐mix and in the long‐term treatment without S9‐mix, respectively. The frequency of structural and numerical aberrations was not statistically significantly different to the negative controls at all concentrations tested.

The Panel concluded that the food enzyme cyclomaltodextrin glucanotransferase 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 in accordance with guidelines of the Japanese Ministry of Health and Welfare (1999) and following GLP.27 The study is in accordance with OECD Test Guideline 408 (OECD TG, 1998) with the following deviations: only one section of the brain and of the spinal cord (cervical level) were examined histologically. The Panel considered that these deviations are minor and do 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 5200, 10,400 or 20,800 mg/kg body weight (bw) per day, corresponding to 562, 1123 or 2246 mg TOS/kg bw per day.28 Controls received the vehicle (distilled water).

No mortality was observed.

Haematological investigations revealed a statistically significant decrease in activated partial thromboplastin time (APTT, −12%) in high‐dose males. The Panel considered the change as not toxicologically relevant as it was only observed in one sex and there were no changes in other relevant parameters (prothrombin time and platelet count).

Clinical chemistry investigations revealed a statistically significant increase in γ‐globulin (γ‐G, +26%) in high‐dose males, a decrease in γ‐glutamyl transpeptidase (γ‐GTP, −42%) in high‐dose females and an increase in calcium concentration (Ca, +5%) in mid‐dose females. The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (all parameters), the change was small (γ‐GTP), there was no dose–response relationship (Ca) and there were no changes in other relevant parameters (for γ‐GTP in other liver enzymes, for γ‐G in total protein, albumin/globulin ratio and other fractions of globulin).

The urinalysis revealed a statistically significant increase in sodium concentration (U‐Na, +44%) in high‐dose males and a decrease in potassium concentration (U‐K, −19%) in mid‐dose males. The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (both parameters), there was no dose–response relationship (U‐K) and there were no histopathological changes in the kidneys.

Statistically significant changes in organ weights detected were a decrease in the absolute brain weight in mid‐dose females (−5%) and an increase in the absolute thyroid weight in low‐dose females (+19%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (both organs), there was no dose–response relationship (both organs), the change was small (brain) and there were no histopathological changes in the thyroid and brain.

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

The Panel identified a no observed adverse effect level (NOAEL) of 2246 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 cyclomaltodextrin glucanotransferase produced with the genetically modified A. caldiproteolyticus strain AE‐KCGT 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, matches with 3 respiratory allergens were found in the AllergenOnline and Allermatch databases.29 The matching allergens were Asp o 21 (46.2% sequence identity), an α‐amylase from Aspergillus oryzae; Sch c 1 (43.8% sequence identity), a glucoamylase from Schizophyllum commune and Aed a 4 (36.3% sequence identity), an α‐glucosidase from Yellow fever mosquito.

No reports on oral or respiratory sensitisation or elicitation reactions of the cyclomaltodextrin glucanotransferase under assessment have been published.30

α‐Amylase from A. oryzae (Brisman, 2002; Quirce et al., 1992, 2002; Sander et al., 1998) and glucoamylase from S. commune (Toyotome et al., 2014) are known as occupational respiratory allergens associated with asthma. However, several studies have shown that individuals respiratorily sensitised to a food enzyme (as described for α‐amylase from A. oryzae) are usually able to ingest the corresponding enzyme without acquiring clinical symptoms of food allergy (Armentia et al., 2009; Cullinan et al., 1997; Poulsen, 2004). Taking into account the wide use of α‐amylases as food enzyme, only a low number of allergic reactions upon oral exposure in individuals respiratorily sensitised to α‐amylases have been described (Baur & Czuppon, 1995; Kanny & Moneret‐Vautrin, 1995; Losada et al., 1992; Moreno‐Ancillo et al., 2004; Quirce et al., 1992). α‐Glucosidase has been associated with allergic reactions to yellow fever mosquito bites, but allergic reactions upon oral exposure to the α‐glucosidase from the yellow fever mosquito have not been reported. No allergic reactions upon dietary exposure to any α‐glucosidase have been reported in the literature.

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

■■■■■ and ■■■■■, known sources of allergens, are present in the medium fed to the production strain. However, during the fermentation process, these products will mostly be degraded and utilised by the production strain. 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 3.5.2), 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 two 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. 31

In the production of plant extracts, the food enzyme is added to herbal or tea leaves (e.g. Stevia or a mixture containing Stevia) or leaf extracts that are rich in steviol glycosides or di−/triterpene glycosides.33 The cyclodextrin glucanotransferase glycosylates steviol glycosides with soluble starch serving as a glycosyl donor. Glucosylated steviol glycosides are sweeter than their non‐glycosylated precursors. The food enzyme–TOS remain in the glucosylated leave extracts, a flavouring agent used in a wide variety of final foods (e.g. herbal and fruit infusions, breath refreshing micro‐sweets).

For the production of cyclodextrins, the food enzyme is added to the liquefied starch together with α‐amylase, where the cyclodextrin glucanotransferase catalyses a transglycosylation reaction to degrade the amylose in the starch and form a mixture of α‐, β‐, γ‐cyclodextrins. After the enzymatic reaction, 1‐decanol is used to recover cyclodextrins. The cyclodextrin/1‐decanol complex is separated from the reaction mixture and purified in multiple steps, including centrifugation, dissolution of the solid phase in water and re‐precipitation at temperature > 90°C to remove the 1‐decanol by steam distillation. At the end, the recovered cyclodextrins are dried into powdered products.34

The applicant clarified that no specific step is included to remove proteins in the manufacturing process of cyclodextrins.35 Less amounts of total protein were found in three batches of cyclodextrin powder by polyacrylamide gel electrophoresis.36 The Panel considered that the technical information about the purification process and the experimental data were insufficient to establish the residual amounts of the food enzyme–TOS in the cyclodextrin products being negligible.

Based on the data provided on thermostability (see Section 3.3.1) and the downstream processing steps applied in the food manufacturing processes, it is expected that the food enzyme is inactivated in all the relevant final foods.

Dietary exposure estimation

3.5.2

α‐Cyclodextrins37 and γ‐cyclodextrins38 are authorised novel food ingredients in the EU under Regulation (EC) No 278/97. β‐Cyclodextrin (E 459) is an authorised food additive in the EU according to Annex II and Annex III to Regulation (EC) No 1333/2008 on food additives. Dietary exposure assessment has already been evaluated by two scientific panels of EFSA for respective uses (EFSA NDA Panel, 2007; EFSA ANS Panel, 2016). Therefore, in this assessment, two sets of calculation were made to estimate the dietary exposure of the food enzyme–TOS via the production of plant extracts, and via the use of cyclodextrins as a food additive and novel foods.

Dietary exposure via the production of plant extracts

3.5.2.1

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool39 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). Exposure from all FoodEx categories was subsequently summed up, averaged over the total survey period (days) and normalised for body weight. This was done for all individuals across all surveys, resulting in distributions of individual average exposure. Based on these distributions, the mean and 95th percentile exposures were calculated per survey for the total population and per age class. Surveys with only one day per subject were excluded and high‐level exposure/intake was calculated for only those population groups in which the sample size was sufficiently large to allow calculation of the 95th percentile (EFSA, 2011).

Table 3 provides an overview of the derived exposure estimates across all surveys. They ranged from, with the highest dietary exposure being 20.27 mg TOS/kg bw per day in toddlers at the 95th percentile. 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 48 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 26 European countries (Appendix B).

Dietary exposure via cyclodextrins as a food additive and novel foods

3.5.2.2

The safety of α‐cyclodextrins has also been evaluated by the EFSA NDA panel as a novel food ingredient to be added as dietary fibre to a variety of foods at the inclusion rate of 1%–10%, reaching a total intake of 65 g/person/day from both additive and ingredient use. This estimation made by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) for European consumers was based on food balance sheets (EFSA NDA Panel, 2007).

In 2016, using actual consumption data collected from European population and use levels provided by industries, EFSA carried out another exposure assessment to β‐cyclodextrin (E 459) as a food additive as part of the food additive re‐evaluation program (EFSA ANS Panel, 2016).

The three types of cyclodextrins are not differentiated on food labels. Therefore, the EFSA FEZ Panel decided to combine the intake estimate for β‐cyclodextrin (E 459) in 2016 with the food enzyme use levels provided by the applicant, in order to derive exposure levels to the food enzyme–TOS via the use as a food additive and novel food ingredients. Table 4 provides an overview of the derived exposure estimates across all surveys. The highest dietary exposure at the 95th percentile was estimated to be 0.205 mg TOS/kg bw per day in children.

Overall dietary exposure

3.5.2.3

The sources of the food consumption data behind these two sets of exposure estimation are different. The estimates reported in Tables 3 and 4 should not be summed to derive the highest overall intake. Therefore, the results are kept separately. Since the estimates shown in Table 3 exceed those in Table 4 by 1–2 orders of magnitude, to avoid excessive overestimation, the Panel chose only the estimates shown in Table 3 to derive the margin of exposure.

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.

For figures reported in Tables 3 and 4, the conservative approach applied to the exposure estimate to food enzyme–TOS, in particular assumptions made on the occurrence and use levels of this specific food enzyme, is likely to have led to overestimation of the exposure.

The choice of using only figures reported in Table 3 to derive the margin of exposure (MoE) may lead to underestimation of the overall intake. However, estimates in Table 3 exceed greatly those in Table 4, thus, the underestimation, if it occurs, would be minimal.

Margin of exposure

3.6

A comparison of the NOAEL (2246 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0.046–6.01 mg TOS/kg bw per day at the mean and from 0.092–20.27 mg TOS/kg bw per day at the 95th percentile, resulted in MoE of at least 141 for infants, 111 for toddlers, 154 for children, 362 for adolescents, 508 for adults, 713 for the elderly.

CONCLUSION

4

Based on the data provided and the derived margin of exposure for food manufacturing processes, the Panel concluded that the food enzyme cyclomaltodextrin glucanotransferase produced with the non‐genetically modified A. caldiproteolyticus strain AE‐KCGT does not give rise to safety concerns under the intended conditions of use for adolescents, adults and the elderly. However, the MoE is insufficient to exclude safety concerns for infants, toddlers and children.

REMARK

5

The production of the food additive (E 960d glucosylated steviol glycosides) as defined in Regulation (EU) No 231/2012 laying down specifications for food additives, is not covered by this assessment.

This food enzyme is intended to be used in the manufacturing of cyclodextrins. The safety evaluation of cyclodextrins as food additives and novel food ingredients is outside the remit of the EFSA FEZ Panel.

DOCUMENTATION AS PROVIDED TO EFSA

6

Dossier for cyclomaltodextrin glucanotransferase from Paenibacillus macerans AE‐CGT. February 2015. Submitted by Amano Enzyme Inc.

Additional information. February 2022. Submitted by Amano Enzyme Inc.

Additional information. October 2024. Submitted by Amano Enzyme Inc.

ABBREVIATIONSbwbody weightCASChemical Abstracts ServiceCEFEFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing AidsCEPEFSA 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 organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonLoDlimit of detectionMoEmargin of exposureOECDOrganisation for Economic Co‐operation and DevelopmentPCRpolymerase chain reactionTOStotal organic solidsWGSWhole genome sequencingAMRantibiotic resistanceWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2016‐00081

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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, Monika Sramkova, Henk Van Loveren, Laurence Vernis, and Holger Zorn.

LEGAL NOTICE

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

Dietary exposure estimates to the food enzyme–TOS in details