Safety evaluation of the food enzyme α‐amylase from the Parageobacillus thermoglucosidasius strain DP‐Gzb47

Abstract

The food enzyme α‐amylase (1,4‐α‐d ‐glucan glucanohydrolase; EC 3.2.1.1) is produced with a non‐genetically modified Parageobacillus thermoglucosidasius strain DP ‐Gzb47 by Danisco US Inc. The α‐amylase food enzyme is intended to be used in brewing processes and distilled alcohol production. Since residual amounts of the food enzyme are removed by distillation, dietary exposure was only calculated for brewing processes. Based on the maximum use levels recommended for brewing processes, and individual data from the EFSA Comprehensive European Food Database, dietary exposure to the food enzyme–Total Organic Solids (TOS ) was estimated to be up to 0.880 mg TOS /kg body weight (bw) per day in European populations. As the production strain of P. thermoglucosidasius meets the requirements for a Qualified Presumption of Safety (QPS ) approach, no toxicological data are required. Similarity of the amino acid sequence to those of known allergens was searched for and no match was found. The Panel considered that under the intended conditions of use, other than distilled alcohol production, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be 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.

1 Introduction

Article 3 of the Regulation (EC) No 1332/200811 Regulation (EC) No 1332/2008 of the European Parliament and of the Council of 16 December 2008 on Food Enzymes and Amending Council Directive 83/417/EEC, Council Regulation (EC) No 1493/1999, Directive 2000/13/EC, Council Directive 2001/112/EC and Regulation (EC) No 258/97. OJ L 354, 31.12.2008, pp. 7–15.
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 1331/200822 Regulation (EC) No 1331/2008 of the European Parliament and of the Council of 16 December 2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings. OJ L 354, 31.12.2008, pp. 1–6.
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:

1. it does not pose a safety concern to the health of the consumer at the level of use proposed;
2. there is a reasonable technological need;
3. 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.

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

1.1 Background and Terms of Reference as provided by the requestor

1.1.1 Background as provided by the European Commission

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.

Five applications have been introduced by the company “Danisco US Inc.” for the authorisation of the food enzymes Beta‐galactosidase from a genetically modified strain of Aspergillus oryzae (DP‐Bzg59), Alpha, alpha trehalase from a genetically modified strain of Trichoderma reesei (DP‐Nzs51), Alpha‐amylase from a genetically modified strain of Bacillus licheniformis (DP‐Dzb45), Glucose oxidase from a genetically modified strain of Aspergillus niger (DP‐Aze23), and Alpha‐amylase from Geobacillus stearothermophilus (DP‐Gzb47).

Following the requirements of Article 12.1 of Regulation (EC) No 234/201133 Commission Regulation (EU) No 234/2011 of 10 March 2011 implementing Regulation (EC) No 1331/2008 of the European Parliament and of the Council establishing a common authorisation procedure for food additives, food enzymes and food flavourings. OJ L 64, 11.3.2011, p. 15–24.
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.

1.1.2 Terms of Reference

The European Commission requests the European Food Safety Authority to carry out the safety assessments on the food enzymes Beta‐galactosidase from a genetically modified strain of Aspergillus oryzae (DP‐Bzg59), Alpha, alpha trehalase from a genetically modified strain of Trichoderma reesei (DP‐Nzs51), Αlpha‐amylase from a genetically modified strain of Bacillus licheniforrnis (DP‐Dzb45), Glucose oxidase from a genetically modified strain of Aspergillus niger (DP‐Aze23), and Αlpha‐amylase from Geobacillus stearothermophilus (DP‐Gzb47) in accordance with Article 17.3 of Regulation (EC) No 1332/2008 on food enzymes.

1.2 Interpretation of the Terms of Reference

The present scientific opinion addresses the European Commission request to carry out the safety assessment of food enzyme α‐amylase from P. thermoglucosidasius (initially indicated as G. stearothermophilus ; strain DP‐Gzb47).

2 Data and methodologies

2.1 Data

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme α‐amylase from P. thermoglucosidasius (strain DP‐Gzb47).

Additional information was sought from the applicant during the assessment process in response to a request from EFSA sent on 19 April 2018 and was consequently provided (see ‘Documentation provided to EFSA’).

Following the reception of additional data by EFSA on 1 July 2019, EFSA requested a clarification teleconference, which was held on 11 July 2019, after which the applicant provided additional data on 26 March 2020.

2.2 Methodologies

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 of EFSA Scientific Committees.

The current ‘Guidance on the submission of a dossier on food enzymes for safety evaluation’ (EFSA, 2009a) has been followed for the evaluation of the application with the exception of the exposure assessment, which was carried out in accordance to the methodology described in the CEF Panel statement on the exposure assessment of food enzymes (EFSA CEF Panel, 2016).

3 Assessment

IUBMB nomenclature: α‐amylase

Systematic name: 1,4‐α‐d ‐glucan glucanohydrolase

Synonyms: glycogenase

IUBMB No.: EC 3.2.1.1

CAS No.: 9000‐90‐2

EINECS No.: 232‐565‐6.

The α‐amylase catalyses the hydrolysis of 1,4‐α‐glucosidic linkages in starch (amylose and amylopectin), glycogen and related polysaccharides and oligosaccharides, resulting in the generation of soluble dextrins and other malto‐oligosaccharides. It is intended to be used in brewing processes and distilled alcohol production.

3.1 Source of the food enzyme

The α‐amylase is produced with the non‐genetically modified bacterium P. thermoglucosidasius strain DP‐Gzb47. Strain DP‐Gzb47, initially indicated in the dossier as G. stearothermophilus was identified as P. thermoglucosidasius ■■■■■ in agreement with the renaming to P. thermoglucosidasius proposed by Aliyu et al. (2016, 2018).44 Technical dossier/Additional data July 2019/Annex U_SI.
The production strain is deposited as G. stearothermophilus at the ■■■■■ with deposit number ■■■■■.55 Technical dossier/Additional data July 2019/Annex T_SI.

P. thermoglucosidasius is included in the list of microorganisms considered suitable for the Qualified Presumption of Safety (QPS) approach (EFSA BIOHAZ Panel, 2020) with the qualification that toxigenic potential and antimicrobial resistance are absent.

Cytotoxicity test was performed with the culture supernatant of the production strain in VERO cells applying the lactate dehydrogenase assay. No evidence of cytotoxicity was detected.66 Technical dossier/Additional data July 2019/Annex X_SI.
No antimicrobial resistance genes were found in the production strain by whole genome sequence analysis using the CARD database.77 Technical dossier/Additional data July 2019/Annex W.

Consequently, the production strain qualifies for the QPS approach for further assessment.

3.2 Production of the food enzyme

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/200488 Regulation (EC) No 852/2004 of the European Parliament and of the Council of 29 April 2004 on the hygiene of food additives. OJ L 226, 25.6.2004, pp. 3–21.
, with food safety procedures based on hazard analysis and critical control points, and in accordance with current Good Manufacturing Practice.

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 and release of the intracellular enzyme, the solid biomass is removed from the fermentation broth by filtration, leaving a supernatant 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 weight material passes the filtration membrane and is discarded.99 Technical dossier/Additional data July 2019/Annex S_SI.
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.1010 Technical dossier/Additional data March 2020/Annex Z_SI.

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.

3.3 Characteristics of the food enzyme

3.3.1 Properties of the food enzyme

The α‐amylase is a single polypeptide chain of ■■■■■ amino acids.1111 Technical dossier/1st submission/Annex I.
The molecular mass of the mature protein, derived from the amino acid sequence, was calculated to be ■■■■■ kDa. The food enzyme was analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis. A consistent protein pattern was observed across all batches. The gels showed a single major protein band corresponding to an apparent molecular mass of about ■■■■■ kDa.1212 Technical dossier/2nd submission/p 31–32.
No other enzymatic side activities were reported.

The in‐house determination of α‐amylase activity is based on hydrolysis of starch (reaction conditions: pH 6.0, 60°C, 10 min). An iodine solution is added and, after 3 min, the iodine‐staining capacity is determined spectrophotometrically at 620 nm. The enzyme activity is expressed in α‐amylase units (U)/g. One U is defined as the amount of enzyme required to hydrolyse 10 mg of starch per minute under the specified conditions.1313 Technical dossier/1st submission/Annex D.

The food enzyme has a temperature optimum around 80°C (pH 5.6) and a pH optimum around pH 6.0 (90°C). Thermostability was tested after a pre‐incubation of the food enzyme for 20 min at 75°C. Under the conditions (pH 5.6) of the applied temperature stability assay, α‐amylase activity decreased rapidly, showing no residual activity after 20 min.1414 Technical dossier/2nd submission/p. 35–36.

3.3.2 Chemical parameters

Data on the chemical parameters of the food enzyme were provided for, three batches used for commercialisation (Table 1).1515 Technical dossier/Additional data March 2020/Annex Y_SI.
The average Total Organic Solids (TOS) of the food enzyme batches for commercialisation was 5.63%. The average enzyme activity/TOS ratio of the three food enzyme batches for commercialisation is 89.06 U/mg TOS.

Table 1. Compositional data of the food enzyme

Parameter	Unit	Batches
		1	2	3
α‐Amylase activity	U/g batcha^a U: α‐Amylase units(see Section 3.3.1).	8,730	2,863	3,230
Protein	%	6.46	1.49	1.34
Ash	%	0.98	0.56	0.94
Water	%	88,87	95.99	95.77
Total Organic Solids (TOS)b^b TOS calculated as 100% – % water – % ash.	%	10.15	3.45	3.29
α‐Amylase activity/mg TOS	U/mg TOS	86.01	82.99	98.18

• ^a U: α‐Amylase units(see Section 3.3.1).
• ^b TOS calculated as 100% – % water – % ash.

3.3.3 Purity

The lead content in the three commercial batches was below 0.05 mg/kg which complies with the specification for lead (≤ 5 mg/kg) as laid down in the general specifications and considerations for enzymes used in food processing (FAO/WHO, 2006).1515 Technical dossier/Additional data March 2020/Annex Y_SI.
^,1616 LODs: Pb = 0.05 mg/kg.

The food enzyme complies with the microbiological criteria as laid down in the general specifications and considerations for enzymes used in food processing (FAO/WHO, 2006), which stipulate that Escherichia coli and Salmonella species are absent in 25 g of sample and total coliforms should not exceed 30 colony forming units per gram. No antimicrobial activity was detected in any of these batches (FAO/WHO, 2006).1515 Technical dossier/Additional data March 2020/Annex Y_SI.

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

3.3.4 Viable cells of the production strain

The absence of the production strain in the product was demonstrated using non‐selective medium at 36°C for 48 h to facilitate resuscitation. Three independent batches were analysed.1717 Technical dossier/2nd submission/Annex G and Additional data March 2020/Annex Y_SI

3.4 Toxicological data

As the production strain qualifies for the QPS approach of safety assessment and as no issue of concern arising from the production process of the food enzyme were identified (see Sections 3.1, 3.2 and 3.3), the Panel considers that no toxicological studies other than assessment of allergenicity are necessary.1818 Article 1.2 of the Regulation (EU) No 562/2012 of 27 June 2012 amending Commission Regulation (EU) No 234/2011.

3.4.1 Allergenicity

The allergenicity assessment considers only the food enzyme and not any carrier or other excipient, which may be used in the final formulation.

The potential allergenicity of the α‐amylase produced with the P. thermoglucosidasius strain DP‐Gzb47 was assessed by comparing its amino acid sequence with those of known allergens according to the ‘Scientific opinion on the assessment of allergenicity of GM plants and microorganisms and derived food and feed of the Scientific Panel on Genetically Modified Organisms’ (EFSA GMO Panel, 2010). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, no match was found.

No information is available on oral and respiratory sensitisation or elicitation reactions of this α‐amylase from P. thermoglucosidasius .

Several studies have shown that adults with occupational asthma to a food enzyme (as described for α‐amylase from A. oryzae ) may be able to ingest the corresponding allergen without acquiring clinical symptoms of food allergy (Cullinan et al., 1997; Poulsen, 2004; Armentia et al., 2009). Taking into account the wide use of α‐amylase as food enzyme only a low number of case reports of allergic reactions upon oral exposure to α‐amylase in individuals respiratory sensitised to α‐amylase have been described in literature (Losada et al., 1992; Quirce et al., 1992; Baur and Czuppon, 1995; Kanny and Moneret‐Vautrin, 1995; Moreno‐Ancillo et al., 2004). Therefore, it can be concluded that an allergic reaction upon oral ingestion of α‐amylase from P. thermoglucosidasius in individuals sensitised to α‐amylase or other food enzymes by inhalation cannot be ruled out, but the likelihood of such reaction to occur is considered to be low.

According to the information provided, substances or products that may cause allergies or intolerances are used as raw materials (■■■■■) in the media fed to the microorganisms. However, during the fermentation process, these products will be degraded and utilised by the microorganisms for cell growth, cell maintenance and production of enzyme protein. In addition, the microbial biomass and fermentation solids are removed. Taking into account the fermentation process and downstream processing, the Panel considered that potentially allergenic residues of these foods employed as protein sources are not expected to be present.

The Panel considers that under the intended condition of use the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded but the likelihood of such reactions to occur is considered to be low.

3.5 Dietary exposure

3.5.1 Intended use of the food enzyme

The food enzyme is intended to be used in two food processes. Intended uses and the recommended use levels are summarised in Table 2.1919 Technical dossier/p. 55.

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

Food manufacturing processa^a The description provided by the applicant has been harmonised by EFSA according to the ‘EC working document describing the food processes in which food enzymes are intended to be used’ – not yet published at the time of adoption of this opinion.	Raw material	Recommended dosage of the food enzyme
Brewing processes	Cereals	Up to 192 mg TOS/kg cereal
Distilled alcohol production	Cereals	Up to 360 mg TOS/kg cereal

• TOS: Total Organic Solids.
• ^a The description provided by the applicant has been harmonised by EFSA according to the ‘EC working document describing the food processes in which food enzymes are intended to be used’ – not yet published at the time of adoption of this opinion.

In brewing processes, the food enzyme is added during the mashing step and/or cereal cooking step. The α‐amylase is used to convert liquefied starch into a maltose‐rich solution,2020 Technical dossier/p. 70.
improving the amounts of fermentable sugars and thus increasing brewing yield, decrease mash viscosity and remove beer haze.

The food enzyme remains in the final brewing product. Based on data provided on thermostability (see Section 3.3.1), it is anticipated that the α‐amylase is inactivated during brewing processes.

In distilled alcohol production, the food enzyme is added during the slurry mixing step, in the liquefaction step and, if needed, in the pre‐saccharification step.2121 Technical dossier/p. 71.
The ɑ‐amylase is intended to convert liquefied starch into a maltose‐rich solution, to increase the amounts of fermentable sugars, which results in higher alcohol yields.

Concerning distilled alcohol production, technical information and experimental data provided on the removal of food enzyme–TOS was considered by the Panel as sufficient to exclude this process from the exposure assessment (Annex B in EFSA CEF Panel, 2016).

3.5.2 Dietary exposure estimation

As residual amounts of TOS are removed by distillation, foods/ingredients derived through this process, i.e. distilled alcohol, were excluded from the estimation.

For the brewing processes, chronic exposure was calculated using the methodology described in the CEF Panel statement on the exposure assessment of food enzymes (EFSA CEF Panel, 2016). The assessment involved selection of relevant food categories from the EFSA Comprehensive European Food Consumption Database and application of process and technical conversion factors (Annex B in EFSA CEF Panel, 2016).

Chronic exposure was calculated by combining the maximum recommended use level provided by the applicant (see Table 2) with the relevant FoodEx categories (Annex B in EFSA CEF Panel, 2016), based on individual consumption data. Exposure from individual FoodEx categories was subsequently summed up, averaged over the total survey period 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 average 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. Detailed average 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 35 different dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 22 European countries (Appendix B).

Table 3. Summary of 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 (10)	0–0.002 (14)	0–0.005 (19)	0–0.037 (18)	0.015–0.195 (19)	0.004–0.096 (18)
Min–max 95th percentile (number of surveys)	0 (8)	0 (12)	0 (19)	0–0.232 (17)	0.109–0.880 (19)	0.024–0.402 (18)

• TOS: Total Organic Solids.

3.5.3 Uncertainty analysis

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.

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
FoodEx categories included in the exposure assessment were assumed to always contain the food enzyme–TOS	+
Exposure to food enzyme–TOS was always calculated based on the recommended maximum use level	+
Selection of broad FoodEx categories for the exposure assessment	+
Use of recipe fractions in disaggregation FoodEx categories	+/−
Use of technical factors in the exposure model	+/−
Exclusion of other processes from the exposure estimate: – distilled alcohol production	−

• TOS: Total Organic Solids.
• + uncertainty with potential to cause overestimation of exposure; –: uncertainty with potential to cause underestimation of exposure.

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 a considerable overestimation of the exposure.

The exclusion of one food manufacturing processes (distilled alcohol production) from the exposure assessment was based on > 99% of TOS removal during these processes and is not expected to have an impact on the overall estimate derived.

3.6 Margin of exposure

Since no toxicological assessment was considered necessary by the Panel, the margin of exposure was not calculated.

4 Conclusions

Based on the data provided, the Panel concluded that the food enzyme α‐amylase produced with the P. thermoglucosidasius strain DP‐Gbz47 does not give rise to safety concerns under the intended conditions of use.

Documentation provided to EFSA

1. Technical dossier ‘Application for authorisation of Alpha‐amylase from Geobacillus stearothermophilus DP‐Gzb47′. February 2016. Submitted by Danisco US Inc.
2. Additional information. July 2019. Submitted by Danisco US Inc.
3. Additional information. March 2020. Submitted by Danisco US Inc.

Abbreviations

bw

body weight

CAS

Chemical Abstracts Service

CEP

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EC

Enzyme Commission

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

GMO

Genetically Modified Organism

IUBMB

International Union of Biochemistry and Molecular Biology

JECFA

Joint FAO/WHO Expert Committee on Food Additives

LOD

Limit of Detection

QPS

Qualified Presumption of Safety

SDS–PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

TOS

Total Organic Solids

WHO

World Health Organization

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

Information provided in this appendix is shown in an excel file (downloadable https://doi.org/10.2903/j.efsa.2020.6129).

The file contains two sheets, corresponding to two tables.

Table 1: Average and 95th percentile exposure to the food enzyme–TOS per age class, country and survey.

Table 2: Contribution of food categories to the dietary exposure to the food enzyme–TOS per age class, country and survey.

Appendix B – Population groups considered for the exposure assessment

Population	Age range	Countries with food consumption surveys covering more than one day
Infants	From 12 weeks on up to and including 11 months of age	Bulgaria, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Portugal, United Kingdom
Toddlers	From 12 months up to and including 35 months of age	Belgium, Bulgaria, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Netherlands, Portugal, Spain, United Kingdom
Childrena^a The terms ‘children’ and ‘the elderly’ correspond, respectively, to ‘other children’ and the merge of ‘elderly’ and ‘very elderly’ in the Guidance of EFSA on the ‘Use of the EFSA Comprehensive European Food Consumption Database in Exposure Assessment’ (EFSA, 2011).	From 36 months up to and including 9 years of age	Austria, Belgium, Bulgaria, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Italy, Latvia, Netherlands, Portugal, Spain, Sweden, United Kingdom
Adolescents	From 10 years up to and including 17 years of age	Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Netherlands, Portugal, Spain, Sweden, United Kingdom
Adults	From 18 years up to and including 64 years of age	Austria, Belgium, Croatia, Czech Republic, Denmark, Estonia, Finland, France, Germany, Hungary, Ireland, Italy, Latvia, Netherlands, Portugal, Romania, Spain, Sweden, United Kingdom
The elderlya^a The terms ‘children’ and ‘the elderly’ correspond, respectively, to ‘other children’ and the merge of ‘elderly’ and ‘very elderly’ in the Guidance of EFSA on the ‘Use of the EFSA Comprehensive European Food Consumption Database in Exposure Assessment’ (EFSA, 2011).	From 65 years of age and older	Austria, Belgium, Denmark, Estonia, Finland, France, Germany, Hungary, Ireland, Italy, Latvia, Netherlands, Portugal, Romania, Spain, Sweden, United Kingdom

• ^a The terms ‘children’ and ‘the elderly’ correspond, respectively, to ‘other children’ and the merge of ‘elderly’ and ‘very elderly’ in the Guidance of EFSA on the ‘Use of the EFSA Comprehensive European Food Consumption Database in Exposure Assessment’ (EFSA, 2011).