Evaluation of certain food additives: ninety-second report of the Joint FAO/WHO Expert Committee on Food Additives

W H O T e c h n i c a l R e p o r t S e r i e s Evaluation of certain food additives Ninety-second report of the Joint FAO/WHO Expert Committee on Food Additives 1037 The World Health Organization (WHO) was established in 1948 as a specialized agency of the United Nations serving as the directing and coordinating authority for international health matters and public health. One of WHO’s constitutional functions is to provide objective and reliable information and advice in the field of human health, a responsibility that it fulfils in part through its extensive programme of publications. The Organization seeks through its publications to support national health strategies and address the most pressing public health concerns of populations around the world. 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To purchase WHO publications, please contact: WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland; email: bookorders@who.int; order online: http://apps.who.int/bookorders. W H O T e c h n i c a l R e p o r t S e r i e s 1 0 3 7 Evaluation of certain food additives Ninety-second report of the Joint FAO/WHO Expert Committee on Food Additives This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the World Health Organization Evaluation of certain food additives: ninety-second report of the Joint FAO/WHO Expert Committee on Food Additives (WHO Technical Report Series, No. 1037) ISBN (WHO) 978-92-4-005464-6 (electronic version) ISBN (WHO) 978-92-4-005465-3 (print version) ISBN (FAO) 978-92-5-137190-9 ISSN 0512-3054 © World Health Organization and Food and Agriculture Organization of the United Nations, 2022 Some rights reserved. 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This publication contains the collective views of an international group of experts and does not necessarily represent the decisions or the policies of WHO or FAO. iii Contents Acknowledgements v List of participants vi List of abbreviations and acronyms viii 1. Introduction 1 1.1 Procedural matters 1 1.2 Declarations of interests 2 1.3 Adoption of the agenda 2 2. Toxicological evaluations and exposure assessments 5 2.1 Benzoic acid and its salts 5 2.2 Collagenase 14 2.3 β-Glucanase 19 2.4 Phospholipase A2 24 2.5 Riboflavin from Ashbya gossypii 28 2.6 Ribonuclease P from Penicillium citrinum 37 3. Revision of specifications and analytical methods 43 3.1 Modified starches 43 Corrigenda 45 Annex 1 Reports and other documents resulting from previous meetings of the Joint FAO/WHO Expert Committee on Food Additives 47 Annex 2 Toxicological and dietary exposure information and information on specifications 61 Annex 3 Meeting agenda 63 iv vAcknowledgements FAO and WHO acknowledge the significant contributions of the experts, as well as their institutions (where relevant), to the work of the ninety-second meeting of JECFA. The Committee thanks Ms Elisabeth Heseltine, Saint Léon-sur-Vézère, France, for her assistance in the preparation of the report. vi List of participants Ninety-second meeting of the Joint FAO/WHO Expert Committee on Food Additives Virtual meeting, 7–18 June 2021 Members Dr S. Barlow, Brighton, East Sussex, United Kingdom Dr J. Bend, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada Dr D. Benford (Co-Chairperson), Cheddington, United Kingdom Dr P.E. Boon, Department for Food Safety, Centre for Nutrition, Prevention and Health, National Institute for Public Health and the Environment, Bilthoven, Netherlands Dr R. Cantrill (Co-Chairperson), Bedford, Nova Scotia, Canada Dr E. Dessipri, General Chemical State Laboratory, Athens, Greece Ms T. Hambridge, Food Standards Australia New Zealand, Kingston, Australian Capital Territory, Australia Ms K. Laurvick, Food Standards, United States Pharmacopeia, Rockville (MD), United States of America (Co-rapporteur) Dr U. Mueller, Australian Pesticide and Veterinary Medicines Authority, Armidale, New South Wales, Australia (Co-rapporteur) Dr J. Schlatter, Zürich, Switzerland Dr J. Smith, Executive Director Bio|Food|Tech, Charlottetown, Prince Edward Island, Canada Dr J.R Srinivasan, Food and Drug Administration, College Park (MD), United States of America Dr N. Sugimoto, Section 2, Division of Food Additives, National Institute of Health Sciences, Kanagawa, Japan Secretariat Dr F. Aguilar Morales, Agency for Food, Environmental and Occupational Health and Safety, Paris, France (WHO temporary adviser) vii Dr M. DiNovi, Food and Drug Administration, College Park (MD), United States of America (WHO temporary adviser) Ms E. Heseltine, Saint Léon-sur-Vézère, France (WHO technical editor) Dr S.M.F. Jeurissen, Department for Food Safety, Centre for Nutrition, Prevention and Health, National Institute for Public Health and the Environment, Netherlands (WHO temporary adviser) Dr M. Lipp, Agriculture and Consumer Protection Department, Food and Agriculture Organization of the United Nations, Rome, Italy (FAO Joint Secretary) Dr O.E. Orisakwe, University of Port Harcourt, Port Harcourt, Nigeria (WHO temporary adviser) Mr K. Petersen, Department of Nutrition and Food Safety, World Health Organization, Geneva, Switzerland (WHO Joint Secretary) Dr J. Rotstein, Pre-market Toxicology Assessment Section, Chemical Health Hazard Assessment Division, Bureau of Chemical Safety, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada (WHO temporary adviser) Dr S.G. Walch, Executive Director, Chemisches und Veterinäruntersuchungsamt, Karlsruhe, Germany (FAO expert) Dr X. Yang, School of Public Health, Southern Medical University, China (WHO temporary adviser) Dr H.J. Yoon, Korea Food and Drug Administration, Seoul, Republic of Korea (WHO temporary adviser) viii List of abbreviations and acronyms ADI acceptable daily intake bw body weight CCFA Codex Committee on Food Additives CSAF chemical-specific adjustment factor EFSA European Food Safety Authority FAD flavin-adenine dinucleotide FAO Food and Agricultural Organization of the United Nations GSFA General Standard for Food Additives INS International Numbering System JECFA Joint Expert Committee on Food Additives LD50 median lethal dose MOE margin of error MPL maximum permitted level MRUL maximum reported use level NOAEL no-observed-adverse-effect level TMDI theoretical maximum daily intake TOS total organic solids WHO World Health Organization 11. Introduction The ninety-second meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) was convened by video conference from 7 to 18 June 2021. The meeting was opened on behalf of the Director-General of the Food and Agriculture Organization of the United Nations (FAO) by Dr Markus Lipp (Food Systems and Food Safety Division, FAO) and on behalf of the Director-General of the World Health Organization (WHO) by Mr Kim Petersen (Programme Manager, Department of Nutrition and Food Safety, WHO). Dr Lipp welcomed all meeting participants and stressed that, despite the challenges of the ongoing COVID-19 pandemic, the work of JECFA had progressed, and the Committee continued to provide sound scientific advice to the Codex Alimentarius Commission and Member States. He reminded the participants of their responsibility to impart the least biased, best scientific advice possible and that they had been invited to serve solely in their capacity as experts to provide sound scientific advice and not as representatives of their employer or country. He closed by reiterating his sincere gratitude to all participants for providing their time and expertise to the present JECFA meeting. Mr Petersen welcomed all meeting participants on behalf of WHO and thanked all experts for their commitment and dedication to the work of JECFA. He underlined the importance of their work in relation to the work of the Codex Alimentarius Commission in developing international food safety standards. 1.1 Procedural matters Owing to travel restrictions and lockdowns due to the response to the COVID-19 pandemic in many countries, it was not possible to convene a physical meeting and it was instead decided to hold it online by video-conferencing. In view of the time differences in the countries of the invited experts, the only possible time for a video-conference was restricted to a 4-h time slot (12:00–16:00 CET) each day. This allowed only 40% of the usual daily length (8–10 h) of a typical JECFA meeting. Although the experts participated fully, they noted that online meetings do not permit the necessary in-depth, robust scientific discussions that are characteristic of JECFA meetings and are therefore not a suitable substitute for face-to-face meetings. In particular, the experts felt that the online format did not foster the atmosphere of trust, inclusiveness and openness that has marked all physical JECFA meetings. The experts considered that the success of the ninety- second meeting was due mainly to the cohesion among them stemming from the trust built on the relationships they had formed during previous face-to- face meetings. The experts also decried the significant difficulty of holding any 2W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report informal meetings outside the scheduled meeting times because of the widely differing time zones. Perhaps the greatest loss due to the virtual meeting format rather than in-person meetings is in efficiency in solving issues that arise shortly before or during the meeting that require immediate input from individuals or small groups of both FAO and WHO representatives. Indeed, this deficiency means that fewer food additives can be evaluated within a two-week meeting. The experts emphasized further that an invitation to a physical JECFA meeting at FAO or WHO headquarters gives rise to more significant recognition by the expert’s employer of the weight, reach, responsibility and workload required for full participation in a JECFA meeting. The same degree of acknowledgement is not granted by employers for online meetings, as the experts remain available locally. This lack of recognition of the workload and significance of participation in a JECFA meeting led to an increase in other demands on the experts, resulting in more distractions and more frequent scheduling conflicts. The experts concluded that, cumulatively, such factors would be counterproductive for participation in future JECFA meetings if FAO and WHO maintained the online-only format. In recognition of the difficulties and the tremendous efforts made, the Joint FAO/WHO Secretariat expressed its deep gratitude to all the experts for their commitment and flexibility, not least as the scheduled meeting times were exceedingly inconvenient for many. The meeting report was adopted on 18 June 2021. 1.2 Declarations of interests The Secretariat informed the Committee that all experts participating in the ninety-second JECFA meeting had completed a declaration of interests form. The declarations were assessed to determine the extent to which any interest could be reasonably expected to influence the experts’ judgement. The declared interests were considered unlikely to impair any individual’s objectivity or have any significant influence on the impartiality, neutrality and integrity of their work. Neither FAO nor WHO received any public comments in response to the online posting of the names and brief biographies of the individuals considered for participation in the expert meeting. The interests of all participants were disclosed at the beginning of the meeting to all attendees. 1.3 Adoption of the agenda After discussion between the experts and the sponsor, the name of the enzyme listed on the agenda as “phosphodiesterase” was changed to “ribonuclease P”, and 3Introduction this change was reflected on the agenda and in the subsequent monograph and specifications. Spirulina was removed from the agenda as the sponsor could not provide the information necessary for establishing full specifications before the meeting. The meeting agenda was adopted with no further modification.  4 52. Toxicological evaluations and exposure assessments 2.1 Benzoic acid, its salts and derivatives 2.1.1 Explanation The Committee first evaluated benzoic acid and its salt, sodium benzoate, at its sixth meeting (Annex 1, reference 6) in 1962. A group acceptable daily intake (ADI) of 0–5 mg/kg body weight (bw) for benzoic acid and sodium benzoate (expressed as benzoic acid) was established at that meeting. This group ADI was based on the absence of any observed adverse effects in rats over four successive generations, two of which involved lifetime dietary exposure to benzoic acid at a maximal concentration of 1% (equivalent to 500 mg/kg bw per day). The potassium and calcium salts were subsequently included in the group ADI for benzoic acid at the ninth, seventeenth, twenty-seventh and forty-sixth meetings (Annex 1, references 11, 32, 62 and 122). Dietary exposure to benzoic acid and its salts (benzoates) was evaluated by the Committee at its fifty-first and eightieth meetings (Annex 1, references 137, 138, 223 and 224). At its eightieth meeting, in 2015, the Committee estimated dietary exposure to benzoates through consumption of water-based flavoured drinks on the basis of reported average typical use levels up to 209 mg/L. The highest high dietary exposure estimates were for consumers-only of these drinks, which was up to 10.9 mg/kg bw per day for toddlers and young children. After a literature review, the Committee concluded that, in most countries, water-based flavoured drinks contribute most to dietary exposure to benzoates. Benzoic acid and its salts are used as food preservatives, whereas derivatives such as benzaldehyde, benzyl acetate, benzyl alcohol and benzyl benzoate are used as flavouring agents. Benzyl alcohol and benzyl benzoate are also used as carrier solvents in foods. The Committee has evaluated benzyl derivatives when used as flavouring agents, most recently at its fifty-seventh meeting (Annex 1, references 154 and 155). Benzyl acetate was evaluated at the eleventh, twenty-seventh, twenty-ninth, thirty-first, thirty-fifth, forty-first and forty-sixth meetings (Annex 1, references 14, 62, 70, 77, 88, 107 and 122). Benzaldehyde and benzyl alcohol were evaluated at the eleventh, twenty-third and forty-sixth meetings (Annex 1, references 14, 50 and 122). As all these structurally related compounds are metabolized along common pathways to benzoate in both rodents and humans, the Committee at its forty-sixth meeting evaluated benzyl acetate, benzyl alcohol, benzaldehyde, benzoic acid and the benzoate salts (calcium, potassium and sodium) together and re-affirmed the group ADI of 0–5 mg/kg bw expressed as benzoic acid equivalents (Annex 1, reference 122). 6W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report The current request to re-evaluate benzoic acid and its salts was made by the Codex Committee on Food Additives at its Forty-ninth Session (1). The sponsor provided a report of an extended one-generation reproductive toxicity study according to OECD 443 and two published reports on the use of default uncertainty factors for benzoic acid. In addition, the sponsor provided dietary exposure estimates for benzoates from water-based flavoured drinks for Brazil, Canada, Mexico and the USA based on maximum use levels and market volume- weighted average use levels of benzoates (expressed as benzoic acid). A comprehensive literature search for toxicological and biochemical data from January 2002 to March 2021 was conducted in PubMed/TOXLINE; 49 publications were considered relevant and further evaluated. As benzoic acid and its salts were evaluated by the European Food Safety Authority (EFSA) from the literature available up to 2016, the Committee decided to use only 20 studies published after 2016 and those published since 2002 that the Committee relevant for this evaluation. At its eightieth meeting, the Committee reviewed publications on dietary exposure to benzoates from all foods between 2000 and 2015. Therefore, the Committee performed a literature search from January 2015 to March 2021 in PubMed and found four publications that were considered relevant for the evaluation. 2.1.2 Chemical and technical considerations Benzoic acid (C7H6O2; CAS No. 65-85-1; INS 210) occurs naturally in organic tissues and can be generated in fermented products. As benzoic acid has antibacterial and antifungal activities, it has applications in food manufacture. Benzoic acid is synthesized by liquid-phase oxidation of toluene with oxygen in the presence of a cobalt-containing catalyst (2). During oxidation, several by-products are formed, such as benzaldehyde, benzyl alcohol and benzyl benzoate; small amounts of benzyl formate, benzyl acetate, biphenyl and methyl biphenyls and phthalic acid may also be formed. For food and pharmaceutical uses, benzoic acid is purified by further processing, including sublimation, recrystallization and neutralization. Treatment with amines and rinsing are required to remove phthalic acid. Benzoic acid has been used as a preservative or flavouring agent in food, cosmetic, hygiene and pharmaceutical products. To extend its application in foods, the following water- soluble salts have been produced by neutralization: sodium benzoate (C7H5NaO2; CAS No. 532-32-1; INS 211), potassium benzoate (C7H5KO2; CAS No. 582-25-2; INS 212) and calcium benzoate (C14H10CaO4; CAS No. 2090-05-3; INS 213). 7Toxicological evaluations and exposure assessments 2.1.3 Biochemical aspects The Committee noted previously that benzoic acid is absorbed, primarily metabolized in the liver and completely excreted in the urine as hippuric acid (major metabolite) and benzoyl-glucuronide. Two pharmacokinetics approaches have been proposed in order to reduce the default uncertainty (safety) factor used to set the current ADI for benzoic acid, its salts and derivatives (e.g., benzyl acetate, benzyl alcohol and benzaldehyde). Zu et al. (3) applied the procedures described in the IPCS Guidance document for use of data in dose/concentration–response assessment (4) to derive a chemical- specific adjustment factor (CSAF) from data on rat and human pharmacokinetics. A reduction of four to two times was proposed for the pharmacokinetics subfactor usually applied to account for inter-species differences. This reduction resulted in an overall uncertainty factor of 50 (i.e., 2 × 2.5 × 10), rather than the default 100 (i.e. 4 × 2.5 × 10). The Committee noted that a CSAF can be set for a food additive only when suitable pharmacokinetics data are available for both the relevant experimental animal species and humans. For deriving a CSAF for benzoic acid, data are available from the use of sodium benzoate at doses as high as 500 mg/kg bw per day in the long-term treatment of patients with inborn errors of urea cycle enzymes that result in hyperammonaemia. Using a physiologically based pharmacokinetics modelling approach to simulate benzoic acid concentrations in the plasma of rats and humans, Hoffman and Hanneman (5) incorporated a seven-compartment model (i.e., blood, liver, brain, adipose, testes/ovaries and rapidly and poorly perfused tissues) with input from three compartments (blood, liver and the remaining body) for some metabolic precursors (i.e., benzyl acetate, benzyl alcohol and benzaldehyde). After a comparison of simulated benzoic acid concentrations in rat and human plasma, the authors concluded that the pharmacokinetics subfactor used to account for inter-species differences was in the range of 0.3–0.4. Hoffman and Hanneman suggested that this fractional value could be used instead of the conventional interspecies pharmacokinetics factor of 4 to yield an overall uncertainty factor of 7.5–10 (i.e., 0.3–0.4 × 2.5 × 10). The Committee noted that, although the absorption and disposition of benzoic acid and its precursors is essentially the same, Hoffman and Hanneman provided no comparison of goodness- of-fit to experimental results with fewer compartments, which increases the model uncertainty. Furthermore, as the interspecies pharmacokinetics factor is essentially determined by hepatic clearance in both rats and humans, the low hepatic clearance in rats (i.e., the Michaelean constant for conversion of benzoic acid to hippuric acid) used in this model may be an underestimate, as it is based on the data after a single intravenous dose of 122 mg/kg bw. 8W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report 2.1.4 Toxicological studies In studies previously evaluated by the Committee, the oral acute toxicity (median lethal dose, LD50) of benzoic acid ranged from 200 to 1200 mg/kg bw in mice to 2700 mg/kg bw for sodium benzoate in rats and 2000 mg/kg bw in rabbits and dogs. A large number of short-term studies on benzoic acid, sodium benzoate and its benzyl derivatives have been evaluated previously by the Committee, none of which showed effects at doses up to 1000 mg/kg bw per day. No new long-term studies were found. The Committee previously reviewed long-term studies in mice and rats on benzyl derivatives, benzyl alcohol, benzaldehyde, benzyl acetate, benzoic acid and sodium benzoate and concluded that the data did not indicate carcinogenic potential. The Committee previously reviewed studies on genotoxicity, and, although positive results were seen in some in vitro studies, the results of in vivo studies were consistently negative. The Committee concluded that there was no concern about the genotoxicity of benzoic acid, its salts and its derivatives. The Committee previously evaluated a four-generation reproductive toxicity study in rats, in which the highest dose tested, 10 000 ppm (1%) in the diet equivalent to 500 mg/kg bw day, was not associated with any toxicological effect (6). On the basis of these results, the Committee established a group ADI of 0–5 mg/kg bw for benzoic acid, the benzoate salts (calcium, potassium and sodium), benzaldehyde, benzyl acetate, benzyl alcohol and benzyl benzoate, expressed as benzoic acid equivalents, applying a default uncertainty factor of 100. In an extended one-generation reproductive toxicity study, conducted according to OECD 443 extended one-generation reproductive toxicity test guideline, doses of 0, 500, 750 or 1000 mg benzoic acid/kg bw per day were given in the diet to rats through F0, F1 and F2 generations (7, 8). The study included offspring cohorts that were assessed for potential developmental immunotoxicity and developmental neurotoxicity. No treatment-related adverse effects were observed on reproductive performance, estrous cycles, parturition, litter viability or survival, pre- or post-weaning developmental landmarks, neurobehaviour, thyroid hormones, clinical pathology, gross necropsy, organ weights, histopathology or sperm parameters. Immunophenotyping and T-cell- dependent antibody responses, organ weights, histopathological examination, neuropathology and brain morphometry in the offspring were not affected by the treatment. The Committee identified a no-observed-adverse effect level (NOAEL) of 1000 mg/kg bw per day, the highest dose tested, for reproductive and developmental toxicity. 9Toxicological evaluations and exposure assessments 2.1.5 Allergenicity The available human data indicate that benzoic acid and its sodium salt can trigger intolerance and allergic reactions in some individuals when ingested in food. 2.1.6 Assessment of dietary exposure Benzoic acid and its salts are endorsed for use in 59 food categories at maximum permitted levels (MPLs) ranging from 200 mg/kg up to 5000 mg/kg, as specified in the Codex General Standard for Food Additives (GSFA), all expressed as benzoic acid. At the current meeting, the Committee evaluated estimates of dietary exposure to benzoates from water-based flavoured drinks submitted by the sponsor for Brazil, Canada, Mexico and the USA (9, 10), based on maximum use levels of benzoates (expressed as benzoic acid) of up to 438 mg/kg in regular carbonated soft drinks and market volume-weighted average use levels ranging from 39 to 197 mg/kg. In addition, dietary exposure estimates from the literature were assessed for Europe (11), India (12), the Islamic Republic of Iran (13, 14) and various other countries, as reviewed at the eightieth meeting of the Committee (Annex 1, references 223 and 224). Table 1 gives an overview of the dietary exposure estimates, all expressed as benzoic acid. The estimates of dietary exposure for Brazil, Canada, Mexico and the USA and for Europe are “brand- loyal estimates”, which account for brand loyalty by mapping the consumption of such foods at a maximum reported use level and that of other foods that may contain benzoates at a typical use level or at a market volume-weighted average use level. The estimates of dietary exposure to benzoates for Europe covered 26 of the 32 food categories for which the use of benzoates is authorized in the European Union according to Annex II of Regulation (EC) No. 1333/2008. Benzoic acid may also be present naturally in foods, such as in berries, but the concentrations are usually not high. In Europe, benzoates may also be present in food due to their use as preservatives in food additives, food enzymes and flavouring preparations according to Annex III to Regulation (EC) No 1333/2008; however, the concentrations of benzoic acid in food are not expected to be high. In view of the wide range of foods in which benzoic acid may occur, naturally and from use of benzoates in food additives, food enzymes and flavouring preparations, however, dietary exposure to benzoic acid may not be negligible. The most complete assessment of dietary exposure to benzoates from their use as food additives was performed for the European population. Dietary exposure in a brand-loyal scenario could be as high as 7.1 mg/kg bw per day for children aged 3–9 years (Table 1). The Committee considered that this high estimate of dietary exposure was the most suitable estimate currently available for 10 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report Table 1 Overview of estimated dietary exposure of the total population to benzoates (as benzoic acid)a from their use as food additives Country/ source Foods included Source concentrations Consumption Dietary exposure (mg/kg bw per day)b,c Childrend General populatione Mean High Mean High Brazil, Canada, Mexico, USA Water-based flavoured drinks Maximum and market volume- weighted use levels Individual food consumption data 0.63 0.15–0.48 1.5–1.6 0.44–0.83 2.7 0.89-2.4 4.8-5.1 2.7-3.2 0.41 0.33 1.2 0.74 1.8 1.7 4.3 2.9 Europef Whole diet Maximum and average use; analytical concentrations Individual food consumption data 0.07–3.2 0.4–7.1 0.07–3.2 0.4–7.1 India Pickles, sauces, soft and fruit drinks, jellies, jams Analytical concentrations Individual food consumption data 0.9–1.3 – – 4.3 Iran, Islamic Republic of Orange juice Cake, toast bread, tomato paste, mayonnaise, carbonated soft drinks, olovieh salad Analytical concentrations Per capita Mean consumption – 0.94g 0.14 – 2.9 Eightieth meeting literatureh Whole diet Use / analytical concentrations Individual food consumption datai 0.1–1.5 0.4–3.9j 0.01–1.5 0.2–3.1 Source: references 9–14, Annex I, reference 244. a Exposure estimates for India and the Islamic Republic of Iran refer to dietary exposure to sodium benzoate but converted to benzoic acid according to molecular weight. As no information was provided for the estimates from the literature summarized by the Committee at its eightieth meeting, they were assumed to refer to benzoic acid. b High exposure: 95th percentile c Dietary exposure for Brazil, Canada, Mexico and the USA and for Europe were calculated for a “brand-loyal” scenario. For more details see the text. d Children aged 10–17 years in Brazil, 2–17 years in Canada, 1–17 years in Europe, Mexico, the USA and literature from the eightieth meeting, and 2–19 years for India. e The general population of Europe covers people aged from 12 weeks up to > 65 years; for Mexico, the USA and estimates from the literature, people aged ≥ 1 year; for Brazil, ≥ 10 years and for Canada ≥ 2 years. The ages for the general population in the Islamic Republic of Iran were not specified. f The European countries were Austria, Belgium, Bulgaria, Czechia, Cyprus, Denmark, France, Germany, Greece, Finland, Hungary, Ireland, Italy, Latvia, Netherlands, Romania, Spain, Sweden and the United Kingdom. g The Committee noted that no information was provided on the consumption level of orange juice, body weight and the actual sodium benzoate concentration used to obtain this dietary exposure estimate. h Refers to literature-derived dietary exposure estimates for exposure to benzoates in all foods for the total population as published between 2000 and 2015 and summarized by the Committee at its eightieth meeting. These estimates are for Australia, Austria, Belgium, Brazil, China, Denmark, France, Ireland, Italy, Lebanon, New Zealand, Republic of Korea, Saudi Arabia, Serbia and the United Kingdom. i Exposure estimates for Brazil were based on per capita estimates of consumption. j High exposure in children is the 97.5th percentile. evaluating dietary exposure to benzoates expressed as benzoic acid, as it accounts for people who are loyal to brands of foods, such as water-based flavoured drinks, over a long period. In addition, this estimate applies to the majority of foods to which benzoates may be added as additives in the European Union. The estimate was also considered conservative enough to include dietary exposure to benzoic acid from natural sources and from authorized use of benzoates as preservatives in food additives, food enzymes and flavouring preparations in the European 11 Toxicological evaluations and exposure assessments Union. The Committee further noted that this high exposure estimate exceeds the high dietary exposure estimates for consumers only reported by the sponsor. 2.1.7 Benzene as a reaction product in benzoic acid-containing beverages Benzene is a known human carcinogen after chronic inhalation (15). During the early 2000s, it was found in trace quantities in some soft drinks and other beverages, where it might have been formed during storage by radical-initiated decarboxylation of benzoic acid (16). Studies have indicated higher concentrations of benzene in beverages that contain benzoate and ascorbic acid (17, 18). Following investigations by a number of national regulatory agencies and the development of mitigation strategies, analyses of reformulated products demonstrated that benzene could not be detected in some samples and that benzene levels were commonly < 5 ng/mL in all others (17–20). The present Committee considered these findings in its assessment of the safety of foods containing benzoic acid and related compounds. Estimated dietary exposure to benzene from beverages and foods is low. An assessment made as part of an examination of the use of the concept of “margin of exposure” (MOE) presented estimates of dietary exposure to benzene from beverages of 8 ng/kg bw per day and from food of 3–50 ng/kg bw per day. Based on a benchmark dose level with a 10% extra risk of adverse effects (BMDL10) of 17.6 mg/kg bw per day from dose–response modelling of data on experimental animals, the MOEs were calculated to be 2 × 106 for beverages and between 6 × 106 and 0.4 × 106 for food (21). The Committee noted that food and beverages make a much smaller contribution to human exposure than other sources, such as inhalation, vehicle fuel fumes and cigarette smoking (20–22). WHO has set a guideline for benzene in drinking-water at 10 µg/L (23). 2.1.8 Evaluation The Committee previously established a group ADI of 0–5 mg/kg bw for benzoic acid, its salts and derivatives expressed as benzoic acid on the basis of a four- generation reproductive toxicity study in rats that showed no toxicological effects at the highest dose tested, 10 000 ppm (1%) in the diet equivalent to 500 mg/kg bw per day. The previous Committee applied a default uncertainty factor of 100 to the dose of 500 mg/kg bw per day. At its present meeting, the Committee evaluated a new extended one- generation reproductive toxicity study on benzoic acids. This study showed no treatment-related adverse effects, indicating a NOAEL of 1000 mg/kg bw per day, the highest dose tested. 12 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report The Committee evaluated two approaches to refining the uncertainty factor to be used in establishing an ADI and concluded that the CSAF approach of Zu et al. (3) was the most appropriate. The Committee therefore applied a CSAF of 2 for interspecies toxicokinetics variation instead of the default factor of 4.0. An overall uncertainty factor of 50 (2 for interspecies toxicokinetic variation × 2.5 for interspecies toxicodynamic variation × 10 for interindividual variation) was applied to the NOAEL of 1000 mg/kg bw per day identified in the new one-generation reproductive toxicity study in rats. The Committee established a group ADI of 0–20 mg/kg bw. This group ADI applies to benzoic acid, the benzoate salts (calcium, potassium and sodium), benzaldehyde, benzyl acetate, benzyl alcohol and benzyl benzoate, expressed as benzoic acid equivalents. The Committee withdrew the previous group ADI of 0–5 mg/kg bw. The Committee noted that the high dietary exposure estimate, expressed as benzoic acid, of 7.1 mg/kg bw per day for children aged 3–9 years does not exceed the group ADI of 0–20 mg/kg bw. The Committee considered this dietary exposure to be of no concern. In addition, the Committee noted that the highest estimates of dietary exposure, expressed as benzoic acid, from water-based flavoured drinks evaluated by the Committee at its eightieth meeting also did not exceed the ADI of 0–20 mg/kg bw (Annex 1, references 223 and 224). These dietary exposure estimates do not include contributions from benzaldehyde, benzyl acetate, benzyl alcohol and benzyl benzoate due to their use as flavouring agents; however, dietary exposure to these derivatives is expected to be low and would be covered by the conservative dietary exposure estimates to benzoic acid and its salts as food additives. At its previous meeting, the Committee identified reports of human idiosyncratic intolerance and of allergy to benzoate. The Committee noted that intolerance and allergenicity to benzoate may pose a health concern to sensitive individuals. The Committee noted that benzene can be formed as a reaction product in benzoic acid-containing beverages. On the basis of the available information, the Committee concluded that exposure to benzene from soft drinks and other foods formulated with benzoic acid or its salts is of little concern from a public health perspective. An addendum to the toxicology and dietary exposure monograph was prepared. The specifications were revised, and a chemical and technical assessment was prepared. 13 Toxicological evaluations and exposure assessments References 1. Forty-ninth Session of the Codex Committee on Food Additives. Rome: Food and Agriculture Organization of the United Nations; Geneva: World Health Organization; 2016. 2. Maki T, Takeda K. Benzoic acid and derivatives. In: Ullmann's Encyclopedia of industrial Chemistry. Weinheim: Wiley-VCH Verlag GmbH; 2012:329–42 (https://doi.org/10.1002/14356007.a03_555). 3. Zu K, Pizzurro DM, Goodman JE, Lewandowski TA. Pharmacokinetic data reduce uncertainty in the acceptable daily intake for benzoic acid and its salts. Regul Toxicol Pharmacol. 2017;89:83–94. 4. Chemical-specific adjustment factors for interspecies differences and human variability: guidance document for use of data in dose/concentration–response assessment (IPCS Harmonization Project Document No. 2). Geneva: World Health Organization; International Labour Organisation; United Nations Environment Programme; 2005 (http://www.who.int/ipcs/methods/harmonization/areas/ uncertainty/en). 5. Hoffman TE, Hanneman WH. Physiologically-based pharmacokinetic analysis of benzoic acid in rats, guinea pigs and humans: Implications for dietary exposures and interspecies uncertainty. Comput Toxicol. 2017;3:19–32. 6. Kieckebusch W Lang K. Die Verträglichkeit der Benzoesäure im chronischen Fütterungsversuch. [The tolerability of benzoic acid in chronic feeding experiments]. Arzneimittel Forsch. 1960;10:1001–3. 7. Coder PS. An oral (dietary) extended one-generation reproductive toxicity study of benzoic acid in rats. Final report. Laboratory Project ID 00865014. Ashland (OH): Charles River Laboratories; 2020. 8. Turnbull D, Jack MM, Coder PS, Rodricks JV. Extended one-generation reproductive toxicity study (EOGRTS) of benzoic acid in Sprague-Dawley rats. Regul Toxicol Pharmacol. 2021;122:104897 (https:// doi.org/10.1016/j.yrtph.2021.104897). 9. Darch M, Martyn D, Ngo K, Jack MM. An updated estimate of benzoate intakes from non- alcoholic beverages in Canada and the United States. Food Addit Contam Part A. 2021. doi: 10.1080/19440049.2020.1859624. 10. Martyn D, Lau A, Darch M, Roberts A. Benzoate intakes from non-alcoholic beverages in Brazil, Canada, Mexico and the United States. Food Addi Contam Part A. 2017;34:9:1485–99. doi: 10.1080/19440049.2017.1338836. 11. Scientific opinion on the re-evaluation of benzoic acid (E 210), sodium benzoate (E 211), potassium benzoate (E 212) and calcium benzoate (E 213) as food additives. EFSA J. 2016;14(3):4433. doi: 10.2903/j.efsa.2016.4433. 12. Vivek Reddy M, Aruna G, Angala Parameswari S, Haseena Banu B, Jayachandra Reddy P. Estimated daily intake and exposure of sodium benzoate and potassium sorbate through food products in school children of Tirupati, India. Int J Pharmacy Pharmaceut Sci. 2015;7:129–33. 13. Akbari-Adergani B, Poorasad M, Esfandiari Z. Sunset yellow, tartrazine and sodium benzoate in orange juice distributed in Iranian market and subsequent exposure assessment. Int Food Res J. 2018;25: 975–81 14. Chaleshtori FS, Arian A, Chaleshtori RS. Assessment of sodium benzoate and potassium sorbate preservatives in some products in Kashan, Iran with estimation of human health risk. Food Chem Toxicol. 2018;120:634–8. doi: 10.1016/j.fct.2018.08.010. 14 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report 15. Snyder R, Witz G, Goldstein BD. The toxicology of benzene. Environ Health Perspect. 1993;100:293– 306. doi: 10.1289/ehp.93100293. 16. Gardner LK, Lawrence GD. Benzene production from decarboxylation of benzoic acid in the presence of ascorbic acid and a transition-metal catalyst. J Agric Food Chem. 1993;41:693–5. https://doi. org/10.1021/jf00029a001. 17. Nyman PJ, Diachenko GW, Perfetti GA, McNeal TP, Hiatt MH, Morehouse KM. Survey results of benzene in soft drinks and other beverages by headspace gas chromatography/mass spectrometry. J Agric Food Chem. 2008;56:571–6. doi: 10.1021/jf072479l. 18. Bonaccorsi G, Perico A, Colzi A, Bavazzano P, Di Giusto M, Lamberti I et al. Benzene in soft drinks: a study in Florence (Italy). Ig Sanita Pubbl. 2012;68(4):523–32. 19. Cao XL, Casey V, Seaman S, Tague B, Becalski A. Determination of benzene in soft drinks and other beverages by isotope dilution headspace gas chromatography/mass spectrometry in humans. J AOAC Int. 2007;90:479–84. 20. Benzene in flavoured beverages. Canberra: Food Standards Australia New Zealand; 2013 (https:// www.foodstandards.gov.au/consumer/chemicals/benzene/Pages/default.aspx). 21. Smith B, Cadby P, DiNovi M, Setzer RW. Application of the margin of exposure (MoE) approach to substances in food that are genotoxic and carcinogenic example: benzene, CAS: 71-43-2. Food Chem Toxicol. 2010;48:S49–56. 22. Salviano dos Santos VP, Salgado AM, Torres AG, Pereira KS. Review article. Benzene as a chemical hazard in processed foods. Int J Food Sci. 2015;2015: 545640.doi: 10.1155/2015/545640. 23. Preventing disease through healthy environments. Exposure to benzene: a major public health concern. Geneva: World Health Organization; 2019. 2.2 Collagenase from Streptomyces violaceoruber expressed in S. violaceoruber 2.2.1 Explanation At the request of the Codex Committee on Food Additives (CCFA) at its Fifty- first Session (1), the Committee evaluated the safety of collagenase (microbial collagenase; IUBMB EC No. 3.4.24.3) from Streptomyces violaceoruber pCol, which it had not previously considered. In this report, the term “collagenase” refers to the collagenase enzyme and its amino acid sequence, the term “enzyme concentrate” to the test material used in the toxicity studies and the term “enzyme preparation” to the formulated product for commercial use. At its present meeting, the Committee considered the submitted data and also conducted a literature search in Google Scholar with the linked search terms “collagenase” and “Streptomyces violaceoruber”, which identified 27 references. One reference (2) was relevant to this toxicological evaluation; however, it was based entirely on the studies in the submitted dossier. 15 Toxicological evaluations and exposure assessments 2.2.2 Genetic background The production organism, S. violaceoreuber, also referred to as S. lividans or S. coelicolor, belongs to the genus Streptomyces. S. violaceoreuber is non-pathogenic, non-toxigenic, occurs in nature as a component of soil (3) and has a history in the production of enzymes intended for use in food processing (4). The S. violaceoreuber pCol production strain was obtained by transforming a plasmid containing a promoter sequence obtained from S. avermitilis ATCC 31267, the collagenase gene obtained from S. violaceoruber NBRC 15146, a terminator sequence obtained from S. cinnamoneus NBRC 12852 and a selectable marker. The resulting plasmid was incorporated into the host organism,  S. violaceoruber  1326, by electroporation. The stability of the introduced sequences was confirmed by cultivating the production strain over three generations and measuring collagenase activity each time. The final enzyme preparations were tested for the absence of an antibiotic resistance gene by polymerase chain reaction. The production strain has been deposited at National Institute of Technological Evaluation in Japan. 2.2.3 Chemical and technical considerations Collagenase is produced by controlled fermentation of a pure culture of the S. violaceoruber production strain. Manufacture of the collagenase enzyme preparation includes fermentation (pre-, seed and main fermentation), recovery and formulation. After fermentation, the broth containing the collagenase enzyme is separated from the biomass by sedimentation; this is followed by three filtration steps. The resulting liquid preparation is formulated with water and glycerol. Two powdered enzyme preparations are produced by further filtering and freeze-drying the liquid filtrate, followed by formulation with dextrin. The entire process is performed in accordance with current good manufacturing practices and with raw materials that are food-grade. The primary sequence of collagenase produced by S. violaceoruber consists of 865 amino acids; its molecular weight calculated from the determined amino acid sequence is 92.4 kDa. The enzyme concentrate is tested to ensure that it is free from the production organism and any antibiotic activity. The activity of collagenase is determined spectrophotometrically by measuring the hydrolysis of a defined peptide substrate at 570 nm; one unit of activity is defined as the quantity of enzyme required to liberate one µmol/min of glycine under the conditions of the assay. The mean activities of collagenase from three batches each of the liquid and the two powder enzyme preparations were 477 U/g, 122 U/g and 2690 U/g, respectively. The enzyme catalyses the hydrolysis of peptide bonds in collagen. The collagenase enzyme preparation is intended for use as a processing aid in the 16 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report production of meat and sausage casings and in the production of collagen hydrolysates used as ingredients in foods, such as those for special nutritional purposes, sports foods and health foods and in dietary supplements. The collagenase enzyme preparations are used at maximum levels of 1188 mg total organic solids (TOS)/kg raw material (as a liquid) or 36 mg TOS/kg raw material (as a powder) and 1566 mg TOS/kg raw material (as a powder). The TOS includes the enzyme of interest and residues of organic materials, such as proteins, peptides and carbohydrates, derived from the production organism during manufacture. The collagenase enzyme is inactivated by heat treatment before use of the final foods. If it were present in the finished food it would probably be digested, like most other proteins, although no data were available on its digestibility. 2.2.4 Biological data Biotransformation No information was available. 2.2.5 Allergenicity The enzyme collagenase from S. violaceoruber was assessed as a potential allergen according to bioinformatics, consistent with the criteria recommend by FAO/ WHO and others (5–7). The amino acid sequence of the enzyme was compared with those of known allergens in two online databases of known allergens. No statistically significant matches were found in either database. The Committee concluded that the enzyme was not anticipated to pose an allergenic risk. 2.2.6 Toxicological studies A study of acute oral toxicity in rats (8) was conducted with the enzyme concentrate (TOS: 93.6%), which was mixed in water and administered by gavage. The oral LD50 of the enzyme concentrate was > 2000 mg/kg bw enzyme concentrate, equal to 1879.2 mg TOS/kg bw. In a 13-week study of oral toxicity in rats (9), a powdered enzyme concentrate (TOS: 93.96 %) was mixed in water and administered by gavage at 1000 mg /kg bw per day, equal to 939.6 mg TOS/kg bw per day, for 90 days. Adverse effects included several anomalies in blood chemistry, but these were observed only in one high-dose female. The cause of these changes could not be established; however, the Committee noted that the changes were extreme. The findings were considered not to be due to treatment, as only one animal was affected, and there was no indication of similar effects in any other animal. The results for the one animal were therefore excluded. The Committee identified a NOAEL of 940 mg TOS/kg bw per day (rounded from 939.6), the highest dose tested. 17 Toxicological evaluations and exposure assessments A powdered enzyme concentrate (TOS content, 93.96%) was tested for genotoxicity in a bacterial reverse mutation test, an in vitro mammalian cell gene mutation assay (mouse lymphoma TK assay) and an in vivo micronucleus induction assay in rats (10–13). The results of the bacterial reverse mutation assay were negative, those of the mammalian cell gene mutation assay were equivocal, and those of the in vivo micronucleus induction assay were negative. The Committee had no concern about the genotoxicity of the collagenase enzyme concentrate. 2.2.7 Observations in humans No information was available. 2.2.8 Assessment of dietary exposure The Committee evaluated an estimate of the theoretical maximum daily intake (TMDI) of the collagenase enzyme preparation conducted with the budget method. The TMDI was based on the level of TOS in the collagenase enzyme preparation and its maximum proposed use levels (equivalent to ≤ 36.36 mg TOS/ kg in solid foods and ≤ 7 mg TOS/kg in non-milk beverages) and an assumption that 12.5% of solid foods and 25% of the non-milk beverages contain the enzyme preparation. The TMDI also included exposure to dietary supplements based on maximum proposed use levels (70 mg TOS/kg) and a daily dose of 24 g/day. The resulting TMDI was 0.43 mg TOS/kg bw per day from solid food, non-milk beverages and dietary supplements. For the dietary exposure assessment, it was assumed that 100% of the enzyme remains in the final food. The Committee noted that the enzyme is inactivated during the processing of food ingredients and will have no function in the final food. 2.2.9 Evaluation The Committee identified a NOAEL of 940 mg TOS/kg bw per day, the highest dose tested in a 13-week study of oral toxicity in rats. When this NOAEL was compared with the estimated dietary exposure of 0.43 mg TOS/kg bw per day, the MOE was > 2100. In view of this MOE and the lack of concern about genotoxicity, the Committee established an ADI “not specified”1 for collagenase from S. violaceoruber, when used in the applications specified and in accordance with good manufacturing practice. A toxicology and dietary exposure monograph was prepared. New specifications and a chemical and technical assessment were prepared. 1 The reader is referred to the Technical Report of the 87th JECFA meeting (Annex 1, reference 243) for clarification of the term “ADI not specified”. 18 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report References 1. Collagenase from Streptomyces violaceoruber expressed in S. violaceoruber. In: List of substances scheduled for evaluation and request for data, addendum [to be presented at]: Joint FAO/WHO Expert Committee on Food Additives (JECFA), Eighty-ninth meeting, 2–11 June, 2020. Rome: Food and Agriculture Organization of the United Nations; Geneva: World Health Organization; 2019:4 (http:// www.fao.org/3/ca6809en/ca6809en.pdf). 2. Harazono K, Atsumi Y, Shirasaka N. Safety evaluation of collagenase from Streptomyces violaceoruber. Regul Toxicol Pharmacol. 2020;113:104665. 3. Duangmal K, Ward AC, Goodfellow M. Selective isolation of members of the Streptomyces violaceoruber clade from soil. FEMS Microbiol Lett. 2005;245(2):321–7. doi:10.1016/j.femsle.2005.03.028. 4. Pariza MW, Johnson EA. Evaluating the safety of microbial enzyme preparations used in food processing: update for a new century. Regul Toxicol Pharmacol. 2001;33(2):173–86. 5. Evaluation of allergenicity of genetically modified foods. Report of a Joint FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnology, 22–25 January 2001. Rome: Food and Agriculture Organization of the United Nations; Geneva: World Health Organization; 2001 (http:// www.who.int/foodsafety/publications/gmo-allergenicity/en). 6. Codex Alimentarius. Foods derived from modern biotechnology, second edition. Geneva: World Health Organization; Rome: Food and Agriculture Organization of the United Nations; 2009 (http://www.fao. org/3/a-a1554e.pdf). 7. 2.4.1 Potential allergenicity of enzymes: change to the number of amino acids in segments used in allergen database searches. In: Evaluation of certain food additives and contaminants. Eightieth report of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), 1–25 June 2015. Rome: Food and Agriculture Organization of the United Nations; Geneva: World Health Organization (WHO Technical Report Series, No. 995); 2016 (https://www.who.int/docs/default-source/food-safety/food- genetically-modified/9789240695405-eng.pdf?sfvrsn=6eeac771_2). 8. Katsumata T. Acute oral toxicity study of pCol protease bulk powder in rats. Study No. B-7208. Bozo Research Center Inc., Gotemba-shi, Shizuoka, Japan; 2012. Submitted to WHO by Nagase ChemteX Corporation, Tokyo, Japan. 9. Katsumata T. A 13-week oral gavage toxicity study of pCol protease bulk powder in rats. Study No. B-7209. Unpublished study. Gotemba Laboratory, Bozo Research Center Inc., Gotemab-shi, Shizuoka, Japan; 2012. Submitted to WHO by Nagase ChemteX Corporation, Tokyo, Japan. 10. Bozo Research Center Inc. A bacterial reverse mutation test of pCol-protease bulk powder (Study No. T-0980). Final report [English translation]. Prepared by Bozo Research Center Inc. for Hyogo, Japan; 2012. Nagase ChemteX Corporation, Tokyo, Japan. 11. Bozo Research Center Inc. A micronucleus test of pCcl protease bulk powder in rats (Study No. M-1498). Final report [English translation]; 2013. Prepared by Bozo Research Center Inc., Gotemba Laboratory, Shizuoka, Japan, for Nagase ChemteX Corporation, Tokyo, Japan. 12. Bozo Research Center Inc. A bacterial reverse mutation test of pCol-protease bulk powder (Study No. N-T3112). Final report [English translation]; 2014. Prepared by Bozo Research Center Inc., Tokyo, Japan for Nagase ChemteX Corporation, Kyoto, Japan. 19 Toxicological evaluations and exposure assessments 13. Bozo Research Center Inc. A mouse lymphoma tk study of pCol protease. Final report [English translation] (Study No. G-042); 2014. Prepared by Bozo Research Center Inc., Tokyo, Japan, for Nagase ChemteX Corporation, Research & Development Section, Enzyme Division, Bio & Fine Chemicals Department, Hyogo, Japan. 2.3 β-Glucanase from Streptomyces violaceoruber expressed in S. violaceoruber 2.3.1 Explanation At the request of the Codex Committee on Food Additives at its fifty-first session (1), the Committee evaluated the safety of β-glucanase (Enzyme Commission No. 3.2.1.39; Chemical Abstract Services No. 9025-37-0) from Streptomyces violaceoruber pGlu. The Committee had not evaluated this enzyme preparation previously. In this report, the term “β-glucanase” refers to the β-glucanase enzyme and its amino acid sequence, the term “enzyme concentrate” to the test material used in the toxicity studies and the term “enzyme preparation” to the formulated product for commercial use. At the present meeting, the Committee considered the submitted data and also conducted a literature search in Google Scholar with the linked search terms “β-glucanase” and “Streptomyces violaceoruber”, which identified 25 references; however, none was considered relevant for the toxicological evaluation. 2.3.2 Genetic background The production organism, S. violaceoruber, also referred to as S. lividans or S. coelicolor, belongs to the genus Streptomyces. S. violaceoruber is non-pathogenic, non-toxigenic, occurs in nature as a component of soil (2) and has a history of use in the production of enzymes intended for use in food processing (3). The S. violaceoruber pGlu production strain was obtained by transforming a plasmid containing a promoter sequence obtained from S. cinnamoneus TH 2, the β-glucanase gene obtained from S. violaceoruber NBRC 15146 and a terminator sequence obtained from S. cinnamoneus NRBC 12852. The stability of the introduced sequences was confirmed by cultivating the production strain for multiple generations and measuring β-glucanase activity each time. The final enzyme preparations were tested for the absence of antibiotic resistance genes by PCR. The production strain has been deposited at the National Institute of Technology and Evaluation in Japan. 20 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report 2.3.3 Chemical and technical considerations β-Glucanase is produced by controlled fermentation of a pure culture of the S. violaceoruber production strain. The manufacture of the β-glucanase enzyme preparation includes fermentation (seed, pre- and main culture), recovery and formulation. After fermentation, the broth containing the β-glucanase enzyme is separated from the biomass; this is followed by multiple filtration steps and dispersion at controlled temperature, pressure and pH. The resulting precipitate is formulated with glycerol to the final β-glucanase enzyme preparation. A powdered enzyme preparation is produced by further filtering and freeze-drying of the liquid formulation, followed by standardization with sodium chloride. The entire process is performed in accordance with current good manufacturing practices and with food-grade raw materials. The primary sequence of β-glucanase produced by S. violaceoruber consists of 453 amino acids; its molecular weight calculated from the determined amino acid sequence is 42.7 kDa. The enzyme concentrate is tested to ensure that it is free of the production organism and any antibiotic activity. The activity of β-glucanase is determined spectrophotometrically by measuring the hydrolysis of 1,3-β-d-glucan substrate by the enzyme at 490 nm; one unit of activity is defined as the quantity of enzyme required to catalyse the formation of 1 µmol/min of glucose under the conditions of the assay. The mean activity of β-glucanase from three batches of the liquid and powder enzyme concentrates were 12 897 U/g and 23 041 U/g, respectively. β-Glucanase catalyses the hydrolysis of the (1→3)-β-d-glucosidic linkages in (1→3)-β-d-glucans to produce d-glucose and β-glucans. The enzyme preparation is intended for use as a processing aid in the manufacture of yeast and mushroom extracts for use as ingredients in seasonings and in the production of beer. The enzyme preparation is added to disrupt the cell walls of mushroom and yeast raw material to improve yield, with residual filtration of the extract products; it is used as a clarifying and filtration aid in the production of beer. The β-glucanase enzyme preparation is intended to be used at a maximum level of 151 mg TOS of powdered β-glucanase/kg raw material and 202 mg TOS of liquid β-glucanase/kg raw material. The TOS includes the enzyme of interest and residues of organic materials, such as proteins, peptides and carbohydrates, derived from the production organism during manufacture. The β-glucanase enzyme is inactivated by heat treatment during processing. It is not expected to have any technological function in the finished foods. If it is present in finished foods, it would probably be digested, like most other proteins, although no data were available on its digestibility. 21 Toxicological evaluations and exposure assessments 2.3.4 Biological data Biotransformation No information was available. 2.3.5 Assessment of potential allergenicity β-Glucanase was assessed as a potential allergen according to bioinformatics consistent with the criteria recommend by FAO/WHO (4), Codex Alimentarius (5) and JECFA (6). The amino acid sequence of the enzyme was compared with those of known allergens in two publicly available databases. A search for matches with > 35% identity over a sliding window of 80 amino acids and a search for sequence identity of eight contiguous amino acids produced a small number of matches. Upon examination, however, these matches were considered not significant. In view of the intended use and available information, the Committee did not anticipate that β-glucanase would pose an allergenic risk. 2.3.6 Toxicological studies A study of acute oral toxicity in rats (7) was conducted with the enzyme concentrate mixed in water and administered as a single gavage dose. The oral LD50 was > 2000 mg/kg bw of the enzyme concentrate, equal to 1906.6 mg TOS/ kg bw. In a 2-week dose range-finding study in rats (8), no significant toxicity was observed when the enzyme concentrate was mixed in water and administered by gavage at doses ≤ 1000 mg/kg bw, equal to 953.3 mg TOS/kg bw. In a 13-week study of oral toxicity in rats (9), the enzyme concentrate was mixed in water and administered at doses ≤ 1000 mg/kg bw, equal to 953.3 mg TOS/kg bw, to groups of rats by gavage. The only treatment-related observation was hyperplasia of the forestomach in male and female rats at the high dose. This was considered not to be related to systemic toxicity but rather an artefact of gavage with increasing concentrations of an acidic substance, which resulted in local irritation. The Committee identified a NOAEL of 950 mg TOS/kg bw per day (rounded by the Committee from 953.3), the highest dose tested. The enzyme concentrate gave negative results in a bacterial reverse mutation assay, an in vitro micronucleus assay and an in vivo micronucleus assay (10–12). The enzyme concentrate gave negative results in an in vitro chromosomal aberration assay without metabolic activation and positive results with metabolic activation, after 6 h of exposure. Under the conditions of the in vitro chromosomal aberration assay with metabolic activation, the enzyme concentrate caused structural aberrations, but it did not induce polyploidy aberrations (13). The Committee had no concern about the genotoxicity of the enzyme concentrate. 22 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report 2.3.7 Observations in humans No information was available. 2.3.8 Assessment of dietary exposure The Committee evaluated an estimate of the TMDI of the β-glucanase enzyme preparation derived with the budget method. The TMDI was based on the level of TOS in the β-glucanase enzyme preparation and its maximum proposed use levels (equivalent to ≤ 8.08 mg TOS/kg in solid foods and ≤ 1.9 mg TOS/kg in non-milk beverages) and on the assumption that 25% of the food supply contains the enzyme preparation. The resulting TMDI was 0.15 mg TOS/kg bw per day (rounded by the Committee from 0.149) from both solid food and non-milk beverages. For the dietary exposure assessment, it was assumed that 100% of the enzyme remains in the final food. The Committee noted that the enzyme will be inactivated during the processing of food ingredients and will have no technical function in the final food. 2.3.9 Evaluation The Committee identified an NOAEL of 950 mg TOS/kg bw per day, the highest dose tested in the 13-week study of oral toxicity in rats. Comparison of this NOAEL with the estimated dietary exposure of 0.15 mg TOS/kg bw per day gave an MOE > 6300. On the basis of this MOE and the lack of concern about genotoxicity, the Committee established an ADI “not specified”1 for β-glucanase from S. violaceoruber, for the proposed uses and in accordance with good manufacturing practice. A toxicology and dietary exposure monograph was prepared. New specifications and a chemical and technical assessment were prepared. References 1. Beta-glucanase from Streptomyces violaceoruber expressed in S. violaceoruber. In: List of substances scheduled for evaluation and request for data, addendum [to be presented at]: Joint FAO/WHO Expert Committee on Food Additives (JECFA), Eighty-ninth meeting, June 2–11, 2020. Rome: Food and Agriculture Organization of the United Nations; Geneva: World Health Organization; 2019:4 (http:// www.fao.org/3/ca6809en/ca6809en.pdf). 2. Duangmal K, Ward AC, Goodfellow M. Selective isolation of members of the Streptomyces violaceoruber clade from soil. FEMS Microbiol Lett. 2005;245(2):321–7. doi: 10.1016/j.femsle.2005.03.028. 1 The reader is referred to the Technical Report of the 87th JECFA meeting (Annex 1, reference 243) for clarification of the term “ADI not specified”. 23 Toxicological evaluations and exposure assessments 3. Pariza MW, Johnson EA. Evaluating the safety of microbial enzyme preparations used in food processing: update for a new century. Regul Toxicol Pharmacol. 2001;33(2):173–86. 4. Evaluation of allergenicity of genetically modified foods. Report of a Joint FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnology 22–25 January 2001. Rome: Food and Agriculture Organization of the United Nations; Geneva: World Health Organization; 2001 (http:// www.who.int/foodsafety/publications/gmo-allergenicity/en). 5. Foods derived from modern biotechnology, second edition. Geneva: World Health Organization; Rome: Food and Agriculture Organization of the United Nations, Codex Alimentarius; 2009 (http://www.fao. org/3/a-a1554e.pdf). 6. 2.4.1 Potential allergenicity of enzymes: change to the number of amino acids in segments used in allergen database searches. In: Evaluation of certain food additives and contaminants. Eightieth report of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), 1–25 June 2015 (WHO Technical Report Series, No. 995). Geneva: World Health Organization; 2016:9 (https://www. who.int/docs/default-source/food-safety/food-genetically-modified/9789240695405-eng. pdf?sfvrsn=6eeac771_2). 7. Katsumata T. Acute oral toxicity study of pglu glucanse bulk powder in rats. Study number: B-7067. Unpublished study conducted by Bozo Research Center Inc., Tokyo, Japan; 2011. Submitted to WHO by Nagase CehmteX Corporation, Osaka, Japan. 8. Serisawa H. A 2 week oral toxicity study of pglu glucanase in rats (dose range finding study). Study No.: C-B 584. Unpublished study conducted by Bozo Research Center Inc., Shizuoka, Japan; 2011. Submitted to WHO by Nagase ChemteX Corporation, Osaka, Japan. 9. Katsumata T. A 13-week oral gavage toxicity study of pglu glucanase bulk powder in rats. Study No. B-7068. Unpublished study conducted by Bozo Research Center Inc., Tokyo, Japan; 2012. Submitted to WHO by Nagase ChemteX Corporation, Osaka, Japan. 10. Bozo Research Center Inc. A bacterial reverse mutation test of pGlu-glucanase powder. Final report [English translation]. Prepared by Bozo Research Center Inc., Tokyo, Japan; Gotemba Laboratory for Kobe-shi, Japan; Nagase Chemtex Corporation, Osaka, Japan (Study No. T-0796); 2011. 11. In vitro assessment of pGLU glucanase in the micronucleus test in cultured human peripheral lymphocytes. Hamburg: Laboratory of Pharmacology and Toxicology GmbH & Co., 2019. 12. Bozo Research Center Inc. Micronucleus test of pGlu glucanase bulk powder in rats. Final report [English translation]. Prepared by Bozo Research Center Inc., Tokyo, Japan; Gotemba Laboratory for Kobe-shi, Japan; Nagase Chemtex Corporation, Osaka, Japan (Study No. M-1497); 2013. 13. Bozo Research Center Inc. Chromosome aberration test in cultured Chinese hamster cells treated with pGlu-glucanase. Final report [English translation]. Prepared by Bozo Research Center Inc., Tokyo, Japan; Gotemba Laboratory for Kobe-shi, Japan; Nagase Chemtex Corporation, Osaka, Japan (Study No. T-G011); 2011. 24 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report 2.4 Phospholipase A2 from Streptomyces violaceoruber expressed in S. violaceoruber 2.4.1 Explanation At the request of the CCFA at its fifty-first Session (1), the Committee evaluated the safety of phospholipase A2 (Enzyme Commission No. 3.1.1.4) from Streptomyces violaceoruber for the first time. In this report, the term “phospholipase A2” refers to the phospholipase A2 enzyme and its amino acid sequence, the term “enzyme concentrate” refers to the test material used in the toxicity studies, and the term “enzyme preparation” refers to the product formulated for commercial use. The Committee at its present meeting considered the submitted data and conducted a literature search in the PubMed (all fields), Scopus (title, abstract, keywords) and Embase (title, abstract, keywords) with the linked search terms “phospholipase A2” and “streptomyces” or “violaceoruber”. The search yielded 118 unique references, none of which reported biochemical and/or toxicological studies on phospholipase A2 from S. violaceoruber. 2.4.2 Genetic background The production organism, S. violaceoruber, also referred to as S. lividans or S. coelicolor, belongs to the genus Streptomyces. S. violaceoruber is non-pathogenic and non-toxigenic and occurs in nature as a component of soil (2). It has a history of use in the production of enzymes intended for use in food processing (3). The S. violaceoruber AS-10 production strain was obtained by transforming a plasmid containing an expression cassette with the phospholipase A2 encoding gene from S. violaceoruber NBRC 15146 donor, a suitable promoter and terminator encoding phospholipase D from S. cinnamoneum and a selectable marker, ligated with a plasmid obtained from S. violaceoruber ATCC 35287. The resulting plasmid was incorporated into the host organism, S. violaceoruber 1326, by electroporation. The stability of the introduced sequences was confirmed by cultivating the production strain over three generations and by measuring phospholipase A2 activity each time. The final enzyme preparations were tested for the absence of an antibiotic resistance gene by PCR. The production strain has been deposited at the National Institute of Technology and Evaluation in Japan. 2.4.3 Chemical and technical considerations Phospholipase A2 is produced by controlled submerged fermentation of a pure culture of the S. violaceoruber production strain. Manufacture of the phospholipase A2 enzyme preparation includes fermentation (pre-, seed and main fermentation), recovery and formulation. After fermentation, the broth containing phospholipase A2 enzyme is separated from the biomass by 25 Toxicological evaluations and exposure assessments sedimentation; this is followed by several filtration steps. The resulting liquid filtrate is formulated with water, sorbitol, potassium sorbate and sodium chloride to obtain the liquid phospholipase A2 enzyme preparation. A powdered enzyme preparation is produced by further filtering and freeze-drying the liquid filtrate, followed by formulation with sodium chloride. The entire process is performed in accordance with current good manufacturing practices and with food-grade raw materials. The primary amino acid sequence of phospholipase A2 produced by S. violaceoruber consists of 151 amino acids; its molecular weight, calculated from the determined amino acid sequence, is 16.4 kDa. The enzyme preparation is tested for the absence of any of the major food allergens that are present in the fermentation medium. The enzyme concentrate is tested to ensure that it contains neither the production organism nor any antibiotic activity. The activity of phospholipase A2 is determined spectrophotometrically by measuring the hydrolysis of a phosphatidylcholine substrate by the enzyme at 550 nm; one unit of activity is defined as the quantity of enzyme required to liberate 1 µmol/min of fatty acid from l-α-phosphatidylcholine under the conditions of the assay. The mean activities of phospholipase A2 from three batches of the liquid and the powder enzyme concentrates are 10 400 U/g and 114 200 U/g, respectively. Phospholipase A2 catalyses the hydrolysis of the sn-2 ester bonds of diacylphospholipids to form 1-acyl-2-lysophospholipids and free fatty acids; when added to food, this improves emulsification. The enzyme preparation is intended for use as a processing aid in the manufacture of enzyme-modified egg yolk, lecithin, cereal flour, dairy products and vegetable oil. The phospholipase A2 enzyme preparation is intended to be used as a processing aid at a maximum level of 105 mg total organic solids (TOS) of powdered phospholipase A2/kg raw material and 459 mg TOS of liquid phospholipase A2/kg raw material. The TOS includes the enzyme of interest and residues of organic materials, such as proteins, peptides and carbohydrates, derived from the production organism during the manufacturing process. The phospholipase A2 enzyme is inactivated by heat treatment before use of the final foods. If present, it is expected that phospholipase A2 will be digested, as would most other protein occurring in food, but no data were available on its digestibility. 2.4.4 Biotransformation No information was available. 26 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report 2.4.5 Assessment of potential allergenicity Phospholipase A2 from S. violaceoruber was evaluated for allergenicity according to the bioinformatics criteria recommended by FAO/WHO (4, 5) and modified at the eightieth meeting of the Committee (Annex 1, reference 223). The amino acid sequence of phospholipase A2 from S. violaceoruber was compared with those of known allergens in publicly available databases. A search for matches with > 35% identity in a sliding window of 80 amino acids, a search for sequence identity of 8 contiguous amino acids and a full-length FASTA sequence search produced no matches. Therefore, the Committee considered that dietary exposure to phospholipase A2 from S. violaceoruber is not anticipated to pose a risk of allergenicity. 2.4.6 Toxicological data In a study of oral acute toxicity in rats with powdered phospholipase A2 concentrate, the LD50 was estimated to be > 1912 mg TOS/kg (6). No treatment-related effects were observed in a range-finding study in which rats were given powdered enzyme concentrate at doses up to 956 mg TOS/ kg bw per day by oral gavage for 2 weeks (7). In a 13-week study of oral toxicity in rats, treatment-related effects were observed on the caecum and the stomach when the powdered phosphodiesterase enzyme concentrate was administered by gavage (8). In the stomach, hyperplasia of the limiting ridge was observed in all animals at the high dose, one female at the mid dose, one male at the low dose and two males in the control group. Diffuse mucosal hyperplasia of the stomach was observed in four males and two females at the high dose, and globule leukocyte infiltration was observed in the stomachs of two males and five females at the high dose, one female at the mid dose and one male in the control group. The effects were classified as minimal or mild. The Committee considered that the effects in the stomach were probably local irritation due to administration of the enzyme concentrate by gavage and were not relevant to the human situation. In the caecum, minimal diffuse mucosal hyperplasia was observed in three males at the high dose, one female at the mid dose and one female at the high dose. The Committee considered that the effects in the caecum were treatment-related adverse effects. In addition, two males at the high dose had extramedullary haematopoiesis in the spleen, although no accompanying effects on haematological parameters were seen. Minimal mineralization of the arterial wall of the lungs occurred in two males and two females at the high dose and in one male in the control group. A statistically significant decrease in grip strength and a statistically significant increase in relative, but not absolute, liver weight were also observed in males at the high dose. Although the effects at the high dose of 956 mg/kg bw per day were small 27 Toxicological evaluations and exposure assessments or occurred at a low incidence, they might have been related to treatment. On this basis, the Committee identified a no-observed-adverse-effect level (NOAEL) of 190 mg/kg per day (rounded by the Committee from 191 mg/kg bw per day). The powdered enzyme concentrate was not genotoxic in a bacterial reverse mutation assay (9) or in an in-vitro chromosomal aberration assay (10). The Committee had no concerns with respect to the genotoxicity of the phospholipase A2 enzyme preparation. 2.4.7 Assessment of dietary exposure The Committee evaluated one estimate of dietary exposure for phospholipase A2 from S. violaceoruber submitted by the sponsor. The enzyme is used in a broad spectrum of food and beverages, including milk. The estimate was derived with the budget method and was based on maximum use levels of 6.42 mg TOS/kg for solid foods, 4.59 mg TOS/kg for non-milk beverages and 9.17 mg TOS/kg for milk, and on the assumption that 25% of the food supply would contain the enzyme preparation. The theoretical maximum daily intake was estimated to be 0.25 mg TOS/kg bw per day (rounded by the Committee from 0.252 mg TOS/kg bw per day). For the dietary exposure assessment, it was assumed that the enzyme is not removed during final processing of ingredients or foods and that 100% of the enzyme remains in the ingredient and final food. The Committee noted that the enzyme will be inactivated during the processing of food ingredients and has no function in the final food. 2.4.8 Evaluation The Committee identified a NOAEL of 190 mg TOS/kg bw per day in a 13-week study in rats. A comparison of the estimated dietary exposure of 0.25 mg TOS/ kg bw per day with the NOAEL of 190 mg TOS/kg bw per day from the oral toxicity study gives a MOE of 760. On this basis and in the absence of concern about genotoxicity, the Committee established an ADI “not specified”1 for the phospholipase A2 enzyme preparation from S. violaceoruber when used in the applications specified and in accordance with good manufacturing practice. A toxicology and dietary exposure monograph was prepared. New specifications and a chemical and technical assessment were prepared. 1 The reader is referred to the Technical Report of the 87th JECFA meeting (Annex 1, reference 243) for clarification of the term “ADI not specified”. 28 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report References 1. Report of the 51th Session of the Codex Committee on Food Additives, Jinan, China, 25–29 March 2019. Rome: Food and Agriculture Organization of the United Nations; Geneva: World Health Organization, Joint FAO/WHO Food Standards Programme, Codex Alimentarius Commission; 2019 (REP19/FA; http://www.fao.org/fao-who-codexalimentarius/sh-proxy/ en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FMeetin gs%252FCX-711-51%252FReport%252FREP19_FAe.pdf). 2. Duangmal K, Ward AC, Goodfellow M. Selective isolation of members of the Streptomyces violaceoruber clade from soil. FEMS Microbiol Lett. 2005;245(2):321–7. doi: 10.1016/j.femsle.2005.03.028. 3. Pariza MW, Johnson EA. Evaluating the safety of microbial enzyme preparations used in food processing: update for a new century. Regul Toxicol Pharmacol. 2001;33(2):173–86. 4. Evaluation of allergenicity of genetically modified foods. Report of a Joint FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnology, 22–25 January 2001. Rome: Food and Agriculture Organization of the United Nations; Geneva: World Health Organization; 2001 (http:// www.who.int/foodsafety/publications/biotech/en/ec_jan2001.pdf). 5. Foods derived from modern biotechnology. Annex 1. Assessment of possible allergenicity. Rome: Food and Agriculture Organization of the United Nations; Geneva: World Health Organization, Joint FAO/WHO Food Standards Programme, Codex Alimentarius Commission; 2009 (http://www.fao.org/ docrep/011/a1554e/a1554e00.htm). 6. Oda S. Acute oral toxicity study of PLA2 Nagase concentrate in rats [English translation]. Unpublished report (Study No. B-5474) from Bozo Research Center Inc., Tokyo, Japan; 2005. Submitted to WHO by Nagase ChemteX Corporation, Osaka, Japan. 7. Oda S. A 2-week oral toxicity study of PLA2 Nagase concentrate in rats (dose range finding study) [English version]. Unpublished report (Study No. C-B213) from Bozo Research Center Inc., Tokyo, Japan; 2005. Submitted to WHO by Nagase ChemteX Corporation, Osaka, Japan. 8. Oda S. A 90-day oral toxicity study of PLA2 Nagase concentrate in rats [English translation]. Unpublished report (Study No. B-5475) from Bozo Research Center Inc., Tokyo, Japan; 2006. Submitted to WHO by Nagase ChemteX Corporation, Osaka, Japan. 9. Yokota F. A reverse mutation test of PLA2 Nagase concentrate in bacteria [English translation]. Unpublished report (Study No. 10057) from Bozo Research Center Inc., Tokyo, Japan; 2005. Submitted to WHO by Nagase ChemteX Corporation, Osaka, Japan. 10. Sono A. Chromosome aberration test in CHL/IU cells treated with PLA2 Nagase concentrate [English translation]. Unpublished report (Study No. M-1193) from Bozo Research Center Inc., Tokyo, Japan; 2005. Submitted to WHO by Nagase ChemteX Corporation, Osaka, Japan. 2.5 Riboflavin from Ashbya gossypii 2.5.1 Explanation  Riboflavin, or 7,8-dimethyl-10-(1´-d-ribityl)isoalloxazine, (7,8-dimethyl-10-[(2S, 3S,4R)-2,3,4,5-tetrahydroxypentyl]benzo[g]pteridine-2,4-dione),  is commonly 29 Toxicological evaluations and exposure assessments known as vitamin B2. It can be obtained by chemical synthesis or by fermentation. Riboflavin and riboflavin-5´-phosphate are used as food colours.  At its thirteenth meeting, the Committee established an ADI of 0–0.5 mg/kg  bw  for  riboflavin on the basis of the absence of any adverse effects at the only dose tested of 50 mg/kg bw per day in a three-generation study of reproductive  toxicity  in rats (1). At its twenty-fifth meeting, the Committee included riboflavin and riboflavin-5´-phosphate, expressed as riboflavin, in a newly established group ADI of 0–0.5 mg/kg bw (2). At its fifty-first meeting, the Committee evaluated riboflavin produced by fermentation from Bacillus subtilis and included it in the group ADI of 0–0.5 mg/kg bw for riboflavin and riboflavin- 5´-phosphate, on the basis of its equivalence to riboflavin (3).  The CCFA at its Fifty-first Session requested the Committee to evaluate  riboflavin from  Ashbya  gossypii  as an alternative source of riboflavin for colouring purposes and as a nutrient source (4). At its present meeting, the Committee evaluated riboflavin from A. gossypii for use as a food colour for the first time. It did not review the nutrient properties of riboflavin but took  into account dietary exposure from all sources of riboflavin, including as a nutrient. A toxicological dossier was  received, with relevant study reports and publications. A comprehensive literature search on riboflavin from A. gossypii conducted on eight databases identified two additional studies for evaluation, and an additional search on riboflavin from other sources identified four additional studies for evaluation.    2.5.2 Genetic background  The Committee at its present meeting evaluated the information provided by the sponsor on use of the filamentous fungus A. gossypii (Eremothecium gossypii) in the production of commercial riboflavin. A. gossypii is a naturally occurring phenotypic riboflavin-overproducing organism  (5),  which possibly provides  protection against ultraviolet radiation (6).  Early development of  A. gossypii  strains for commercial production of riboflavin involved classical mutagenesis and strain selection to obtain a high riboflavin  titre. The  A. gossypii  strain LU8907  was continuously developed into several commercial strains to further increase riboflavin production. The present production strain,  A. gossypii LU11439, was  constructed  from  the recipient strain A. gossypii LU8907. The recipient was modified by the addition of several genes from the wild-type A. gossypii strain under the control of translation elongation factor promoters and antibiotic resistance marker genes. The DNA sequences for transformation were prepared as linear, vector-free fragments and inserted by electroporation, followed by homologous recombination and targeted 30 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report integration. The production strain was confirmed to be genetically stable and not to contain any transferable marker genes or sequences derived from vector DNA. The complete sequence and annotation of the A. gossypii  genome was published in 2004. It shows a 95% homology and gene synteny to the genome of budding yeast,  Saccharomyces cerevisiae  (7, 8). The complete genome sequences of the sponsor's first self-cloned production strain and of the published A. gossypii wild-type strain (ATCC 10895) were analysed for the presence of gene clusters encoding secondary metabolites. No gene clusters of polyketide synthases or non-ribosomal peptide synthases were identified in the genome. The genome of A. gossypii has no potential for production of secondary metabolites.    2.5.3 Chemical and technical considerations  Riboflavin is obtained from A.  gossypii by  fermentation  under controlled conditions. Several filtration and precipitation or crystallization steps result in a highly purified food-grade dry powder containing not less than 98% riboflavin, free of fermentation medium components and the production organism.  The entire process is carried out in accordance with current good manufacturing practices; all raw materials used in the manufacture are food-grade.  Riboflavin was evaluated previously by JECFA (9) as a synthetic product (1987) and as a product of fermentation from B. subtilis (1999). Independent of the source, these additives contain not less than 98% and not more than 101% of riboflavin (on a dried basis). Riboflavin is relatively stable during food processing and storage but is very sensitive to light.   2.5.4 Biochemical aspects  Riboflavin is absorbed actively and passively mainly in the proximal small intestine, partly in the large intestine and also in the colon (10–12). Riboflavin is absorbed by two mechanisms – a saturable active component that dominates at near-physiological vitamin concentrations and a passive component that is revealed under conditions of high levels of supplementation with riboflavin. In plasma, some riboflavin is bound to albumin; however, a large portion of riboflavin is associated with immunoglobulins (A or G) for transport (13). When riboflavin is absorbed in high concentrations, little is stored in the body tissues, and the excess is excreted, primarily in the urine (14–16).  The metabolism of riboflavin begins with ATP-dependent phosphorylation to flavin mononucleotide, catalysed by the enzyme flavokinase under hormonal control. Flavin mononucleotide is then complexed with specific apoenzymes to form a variety of flavoproteins or is mainly converted to flavin-adenine dinucleotide (FAD) by FAD synthetase (17). Although FAD was the major 31 Toxicological evaluations and exposure assessments form in plasma, plasma riboflavin and erythrocytes flavin mononucleotide were suggested to represent riboflavin status in humans (18). Lumichrome and lumiflavin have been identified as metabolites of riboflavin in the rat, while  hydroxyriboflavin  and  formylmethylflavin  have been identified as metabolites in human plasma (19, 20).   2.5.5 Toxicological studies  The acute oral toxicity of riboflavin from A. gossypii is low, with an LD50 of > 2500 mg/kg bw (21, 22).  In a 90-day repeated-dose oral toxicity study in rats (23), riboflavin from A. gossypii (purity, 82.3%, feed grade) was fed in the diet at a concentration of 0, 500, 5000 or 50 000 mg/kg diet, equal to 0, 35, 362 or 3659 mg/kg bw in males and 0, 41, 410 or 4325 mg/kg bw in females. Treatment had no effects on bw, bw gain, feed or drinking-water consumption. Foci were detected in the kidneys of two female rats at the highest dose, but were considered not to be toxicologically relevant. When accounting for the purity of the preparation used, the Committee identified a NOAEL of 3011 mg/kg bw per day for males and 3559 mg/kg bw per day for females, the highest doses tested.  In another 90-day oral toxicity study in rats (24), reviewed previously by the Committee (3), riboflavin from B.  subtilis with a purity of 98% or 96% was fed in the diet at concentrations providing 0, 20, 50, or 200 mg/kg bw per day. The previous Committee identified a NOAEL of 200 mg/kg bw per day (3).  The European Food Safety Authority (EFSA) (25) described a further 90- day oral toxicity study in rats (Bachmann et al., 2005) performed with riboflavin from B. subtilis (containing 80.1 % riboflavin, feed grade). The test material was administered in the diet to provide doses of 0, 50, 100 and 200 mg/kg bw per day. Additional groups of rats were treated at the same doses for 13 weeks and then observed for a 4-week recovery period. Eosinophilic granules were observed in the renal tubules of male rats receiving 100 or 200 mg/kg bw per day at the end of the treatment period, but renal morphology returned to normal after the 4-week recovery period. The study authors pointed out that accumulation of hyaline droplets is associated with α-2μ-globulin and is considered to be a response specific to male rats and therefore not relevant to humans. EFSA (25) concurred with this consideration and concluded that the NOAEL for the test material in this study was 200 mg/kg  bw  per day, the highest dose tested, corresponding to 160 mg/kg bw per day expressed as riboflavin. The Committee at its present meeting agreed with this evaluation.  No studies of chronic toxicity or carcinogenicity with riboflavin from A. gossypii or riboflavin from any other source were available. 32 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report Riboflavin from A. gossypii  (purity, 99% and 80.8%) was tested in two bacterial mutagenicity assays and in an in vitro micronucleus induction assay in human lymphocytes (26–28). In spite of minor limitations, the combination of these tests fulfilled the basic requirements for an assessment of genotoxic potential, and the Committee concluded that there is no concern with respect to the genotoxicity of riboflavin from A. gossypii.  No reproductive or developmental toxicity was observed in a multigeneration study in which rats received riboflavin at a daily dose of 0 or 10 mg per rat (equivalent to 0 or approximately 50 mg/kg bw per day) from weaning for three generations (29). The dose of 50 mg/kg bw per day was used as the basis for the ADI of 0–0.5 mg/kg bw per day established by the Committee in 1969 (1). The present Committee noted that the report of the study provided limited experimental data, poor reporting and only one dose level was used.  In a series of intervention studies with oral administration of riboflavin, no adverse effects were reported in populations of children and adults, including healthy individuals and patients suffering from migraine, cardiovascular diseases, colorectal polyp or anaemia (30–40).   2.5.6 Assessment of dietary exposure  The Committee noted that riboflavin is endorsed for use in 71 food categories in the Codex GSFA at MPLs of 30–1000 mg/kg, while riboflavin may be used in amounts consistent with national good manufacturing practice in Australia, New Zealand, the Republic of Korea and the USA and in the European Union. The sponsor provided maximum reported use levels (MRULs) for riboflavin of 10–400 mg/kg as a food colour for the 29 food categories in which it is authorized in the European Union according to Annex II to Regulation (EC) No. 1333/2008.  Estimates of dietary exposure to riboflavin from GSFA MPLs and MRULs, in combination with food consumption data from the Food Additive Intake Model 2.0, by the sponsor were reviewed by the Committee. This model includes food consumption data from  various European countries for six age groups. High-level dietary exposure estimates are calculated by adding the 95th percentile of dietary exposure to one food category at the highest dietary exposure to the mean  dietary  exposure resulting from consumption of all other food categories. Estimated mean and high-level dietary exposure to riboflavin of the six age groups were 2.8–18.3. mg/kg bw per day and 4.8–25.5 mg/kg bw per day with GSFA MPLs and 0.2–2.4. mg/kg bw per day and 0.4–3.6 mg/kg bw per day with MRULs. The Committee noted that the MRULs for riboflavin as a food colour were well below the GSFA MPLs for most food categories.  Estimates of dietary exposure to riboflavin from all sources, including from its use as a food additive, are available from many national dietary surveys. 33 Toxicological evaluations and exposure assessments EFSA (25) estimated dietary exposure to be in the range of 0.05–0.09 mg/kg bw per day for children and 0.02–0.04 mg/kg bw per day for adults. The Committee also noted estimates of dietary exposure to riboflavin from Australia (0.03 mg/kg bw per day for adults and 0.06 mg/kg bw per day for children; 41), New Zealand (0.03 mg/kg bw per day for adults and 0.05 mg/kg bw per day for children; 41), the Republic of Korea (0.02–0.03 mg/kg bw per day for adults; 42) and the USA (from 0.05 mg/kg bw per day for men to 0.14 mg/kg bw per day for children; 43). The group pf milk and dairy products group was the main contributor to dietary exposure to riboflavin in Spain at 32.3% (44), Australia at 27–28% (45) and New Zealand at 23% (46, 47). The Committee concluded that the highest estimate of high-level dietary exposure to riboflavin of 3.6  mg/kg bw per day for  children aged 3–9 years, calculated with the Food Additive Intake Model 2.0 with MRULs, should be considered in the safety assessment of riboflavin.   2.5.7 Evaluation  In its present evaluation of riboflavin from A. gossypii, the Committee noted that it has low acute toxicity and did not raise concern for genotoxicity. The NOAEL from a 90-day oral toxicity study in rats on riboflavin from A. gossypii was 3000 mg/kg bw per day (rounded by the Committee from 3011 mg/kg bw per day), the highest dose tested. Comparison of the NOAEL of 3000 mg/kg bw per day with the estimate of dietary exposure of 3.6 mg/kg bw per day, based on maximum reported use levels, resulted in an MOE > 800. The Committee concluded that exposure  to riboflavin from all sources does not represent a safety concern.  The NOAEL  of 3000 mg/kg bw per day in the present evaluation of riboflavin from A. gossypii is considerably higher than the 50 mg/kg bw per day in the multigeneration study with a single dose level that was used by the previous Committee to establish an ADI of 0–0.5 mg/kg bw. The Committee at its present meeting noted that the toxicity database on riboflavin from various sources reviewed previously by the Committee does not indicate any adverse effects. The Committee at its present meeting established a group ADI “not specified”1  for riboflavin, riboflavin-  5´-phosphate, riboflavin from B.  subtilis  and riboflavin from A. gossypii and withdrew the previous group ADI of 0–0.5 mg/kg bw.  A toxicology and a dietary exposure monograph was prepared.  New specifications and a chemical and technical assessment were prepared.  34 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report 2.5.8 Future work  Regarding the previously established specifications for riboflavin and riboflavin from B. subtilis, the Committee proposes to:  ■ rename “riboflavin” as “riboflavin, synthetic”;  ■ replace the existing method for determination of lumiflavin in both specifications to avoid the use of chloroform; and ■ delete the functional use of “nutrient supplement” from the specifications monograph on riboflavin from B. subtilis, as the Codex food additive definition does not include nutrients.  2.5.9 Recommendation  In view of  information received at the current meeting which implies  that riboflavin is no longer produced synthetically for use  as a  food additive, the Committee  recommends that the CCFA reconsider the requirement for specifications for synthetically produced riboflavin.  References 1. Toxicological evaluation of some food colours, emulsifiers, stabilizers, anticaking agents, and certain other substances (FAO Nutrition Meetings Report Series, No. 46a). Rome: Food and Agricultural Organization of the United Nations; Geneva: World Health Organization; 1969.  2. Safety evaluation of certain food additives (Twenty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives; WHO Food Additives Series, No.16). Rome: Food and Agricultural Organization of the United Nations; Geneva: World Health Organization; 1981.  3. Safety evaluation of certain food additives (Fifty-first report of the Joint FAO/WHO Expert Committee on Food Additives; WHO Food Additives Series, No. 42). Rome: Food and Agricultural Organization of the United Nations; Geneva: World Health Organization; 1988. 4. Report of the 51st session of the Codex Committee on Food Additives. Joint FAO/WHO Food Standards Programme. Codex Alimentarius Commission, Forty-second Session; 2019.  5. Demain AL. Riboflavin oversynthesis. Annu Rev Microbiol. 1972;26(1):369–88.  6. Stahmann KP, Arst HN, Althöfer H, Revuelta J L, Monschau N, Schlüpen C et al. Riboflavin, overproduced during sporulation of  Ashbya  gossypii, protects its hyaline spores against ultraviolet light. Environ Microbiol. 2001;3(9):545–50. Doi: 10.1046/j.1462-2920.2001.00225.x  7. Dietrich FS, Voegeli S, Brachat S, Lerch A, Gates K, Steiner S et al. The Ashbya gossypii genome as a tool for mapping the ancient Saccharomyces cerevisiae genome. Science. 2004;304(5668):304–7. doi: 10.1126/science.1095781.  35 Toxicological evaluations and exposure assessments 8. Gattiker  A,  Rischatsch  R,  Demougin  P,  Voegeli  S, Dietrich FS,  Philippsen  P et al. Ashbya  genome database 3.0: a cross-species genome and transcriptome browser for yeast biologists. BMC Genomics. 2007;8:9. doi: 10.1186/1471- 2164-8-9.  9. Compendium of food additive specifications, JECFA Monographs 1 vol. 3/243. Rome: Food and Agricultural organization of the United Nations; Geneva: World Health Organization; 2006.  10. Jusko  WJ, Levy G. Absorption, metabolism, and excretion of riboflavin-5’-phosphate in man. J Pharmaceut Sci. 1967;56:58–62.  11. Merrill AH Jr, Lambeth JD, Edmondson DE, McCormick DB. Formation and mode of action of flavoproteins. Annu Rev Nutr. 1981;1:281–317.  12. Yuasa H, Hirobe M, Tomei S. Carrier-mediated transport of riboflavin in the rat colon. Biopharmaceutics Drug Disposition. 2000;21:77–82.  13. Innis WS, McCormick DB, Merrill AH Jr. Variations in riboflavin binding by human plasma: identification of immunoglobulins as the major proteins responsible. Biochem Med. 1985;34:151–65.  14. McCormick DB. Two interconnected B vitamins: riboflavin and pyridoxine. Physiol Rev. 1989;69:1170– 98. 15. Sauberlich HE. Laboratory tests for the assessment of nutritional status. Boca Raton (FL): CRC Press; 1999:55–69 (cited in reference 25).  16. Said HM, Ross AC. Riboflavin. In: Modern Nutrition in Health and Disease. Philadelphia (PA): Lippincott Williams & Wilkins; 2012 (cited in reference 25).  17. Yamada Y, Merrill AH Jr, McCormick DB. Probable reaction mechanisms of  flavokinase  and FAD synthetase from rat liver. Arch Biochem Biophys. 1990;278:125–30.  18. Hustad  S, McKinley MC, McNulty H,  Schneede  J, Strain JJ, Scott JM et al. Riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in human plasma and erythrocytes at baseline and after low-dose riboflavin supplementation. Clin Chem. 2002;48:1571–7.  19. Yang CS, McCormick DB. Degradation and excretion of riboflavin in rats. J Nutr. 1967;93:445–53.  20. West DW, Owen EC. The urinary excretion of metabolites of riboflavin by man. Br J Nutr. 1969;23:889– 98.  21. Kuehlem. Study on the acute oral toxicity of riboflavin CF in rats. Report, Project No. 10A0346/961143, 22 June 1998.  22. Wiemann C. Report Lutavit B2 SG 80. Acute oral toxicity in rats. Report, Project No. 10A0001/981002, 9 July 1998.  23. Mellert W. Report Lutavit® B2 SG 80. Subchronic oral toxicity study in Wistar rats. Administration in the diet for 3 months. Report, Project No 50S0001/98006, 14 September 1998.  24. Buser S, Hofmann P, Lina B, Forster S,  Zabka S. Subchronic  oral toxicity study with three different qualities of riboflavin (Ro 01-3131/055 96% ex fermentation, Ro 01-3131/054 98% ex fermentation and Ro 01-3131/000 98% ex synthesis) in rats (Project No.920V94), parts I to III. Unpublished study. TNO Nutrition and Food Institute, Zeist, Netherlands, 14 November 1995. Submitted to WHO by F. Hoffmann La Roche Ltd, Basel, Switzerland (cited in reference 3).  36 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report 25. Scientific opinion on the re-evaluation of riboflavin (E 101(i)) and riboflavin-5’-phosphate sodium (E 101(ii)) as food additives (EFS2 11 (10), S. 209). Parma: European Food Safety Authority; 2013. doi: 10.2903/j.efsa.2013.3357. 26. Engelhardt G. Report on the study of Bio B2 Pharmaqual in the Ames test (Salmonella/mammalian- microsome mutagenicity test – standard plate test and preincubation  test). Report, Project No. 40M0323/94239, 1 September 1994.  27. Naumann S. Lutavit B2 SG 80: Micronucleus test in human lymphocytes in vitro. Report, Project No. 31M0115/09X203, 20 July 2017.  28. Woitkowiak  C.  Lutavit  B2 SG 80 Salmonella typhimurium / Escherichia coli reverse mutation assay. Report, Project No 40M0115/09M059, 10 May 2017.  29. Unna K, Greslin JG. Studies on the toxicity and pharmacology of riboflavin. J Pharmacol Exp Therapeut. 1942;76(1):75–80.  30. Madigan SM, Tracey F, McNulty H, Eaton-Evans J, Coulter J, McCartney H et al Riboflavin and vitamin B-6 intakes and status and biochemical response to riboflavin supplementation in free-living elderly people. Am J Clin Nutr. 1998;2:389.  31. Boehnke C, Reuter U, Flach U, Schuh-Hofer S, Arnold G. High-dose riboflavin treatment is efficacious in migraine prophylaxis: an open study in a tertiary care centre. Eur J Neurol. 2004;11(7):475–7.  32. MacLennan SC, Wade FM, Forrest KML,  Ratanayake  PD, Fagan E, Antony J. High-dose riboflavin for migraine prophylaxis in children: a double-blind, randomised, placebo-controlled trial. J Child Neurol. 2008;23:1300–4.  33. Powers HJ. Riboflavin (vitamin B-2) and health. Am J Clin Nutr. 2003;77:1352–60.  34. Condò  M,  Posar  A,  Arbizzani  A,  Parmeggiani  A. Riboflavin prophylaxis in  paediatric  and adolescent migraine. J Headache Pain. 2009;10:361–5.  35. Tavares NR, Moreira PA, Amaral  TF. Riboflavin supplementation and biomarkers of cardiovascular disease in the elderly. J Nutr Health Aging. 2009;13(5):441–6.  36. Bruijn  J, Duivenvoorden  H, Passchier  J, Locher  H, Dijkstra N, Arts WF. Medium-dose riboflavin as a prophylactic agent in children with migraine: a preliminary placebo-controlled, randomised, double- blind, cross-over trial. Cephalalgia. 2010;30(12):1426–34.  37. Ma  AG,  Schouten  EG, Sun YY,  Fang Y, Kok  FJ. Supplementation of iron alone and combined with vitamins improves  haematological  status, erythrocyte membrane fluidity and oxidative stress in anaemic pregnant women. Br J Nutr. 2010;104(11):1655–61.  38. Gaul C, Diener HC, Danesch U. Improvement of migraine symptoms with a proprietary supplement containing riboflavin, magnesium and Q10: a randomized, placebo-controlled, double-blind, multicenter trial. J Headache Pain. 2015;16:516. 39. Rahimdel A, Zeinali A, Yazdian-Anari P, Hajizadeh R, Arefnia E. Effectiveness of vitamin B2 versus sodium valproate in migraine prophylaxis: a randomized clinical trial. Electronic Physician. 2015;7(6):1344–8.  40. Yamanaka G, Suzuki S, Takeshita M, Go S, Morishita N, Takamatsu T et al. Effectiveness of low-dose riboflavin as a prophylactic agent in pediatric migraine. Brain Dev. 2020;42(7):523–8.  41. Estimated dietary intakes of riboflavin for nutrition survey population groups as derived using the Harvest dietary exposure assessment program. Canberra: Food Standards Australia New Zealand (personal communication, 2020).  37 Toxicological evaluations and exposure assessments 42. Shin WY, Kim JH. Low riboflavin intake is associated with cardiometabolic risks in Korean woman. Asia Pac J Clin Nutr. 2019;28(2):285–99. 43. What we eat in America, 2009–2010. Beltsville (MD): Department of Agriculture, Agricultural Research Service; 2012 (http://www/ars/ussda.gov/Services/docs.htm?Docid=18349). 44. Miego-Ayuso J, Aparicio-Ugarriza R, Olza J, Aranceta-Bartrina J, Gil A, Ortega R et al. Dietary intake and food sources of niacin, riboflavin, thiamin and vitamin B6 in a representative sample of the Spanish population. The anthropometry, intake, and energy balance in Spain (ANIBES) study. Nutrients. 2018;10(7):846.  45. Australian Health Survey: Nutrition first results – foods and nutrients (Catalogue no. 4364.0.55.007). Canberra: Australian Bureau of Statistics; 2014.  46. A focus on nutrition: Key findings of the 2008/09 New Zealand adult nutrition survey. Wellington: University of Otago and Ministry of Health; 2011. 47. NZ Food NZ Children: Key results of the 2002 national children’s nutrition survey. Wellington: Ministry of Health; 2003.  2.6 Ribonuclease P from Penicillium citrinum 2.6.1 Explanation At the request of the CCFA at its Fifty-first Session (1), the Committee evaluated the safety of ribonuclease P (IUBMB EC No. 3.1.26.5) from Penicillium citrinum, which has not previously been evaluated. In this report, the term “ribonuclease P” refers to the ribonuclease P enzyme and its amino acid sequence, the term “powdered enzyme concentrate” to the test material used in the toxicity studies submitted and the term “enzyme preparation” to the product formulated for commercial use. The Committee at its present meeting considered the submitted data and searched the literature in the PubMed database (all fields), Scopus and Embase (title, abstract, keywords) with the linked search terms “ribonuclease” and (“penicillium” or “citrinum”). In total, 188 unique references were found, of which only two described biochemical and/or toxicological studies with ribonuclease P from P. citrinum. Most of the toxicological studies with ribonuclease P from P. citrinum described below were therefore submitted by the sponsor. 2.6.2 Genetic background P. citrinum is a filamentous fungus that is ubiquitous in the environment. It occurs on various plants, including citrus fruits and wheat and other cereal grains (2). Penicillium species are recognized for use in food applications (3), including as a source organism in the production of ribonuclease P for use in food processing (4). The taxonomy of the source organism was confirmed from its macroscopic and microscopic characteristics. The P. citrinum production strain used in the 38 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report manufacture of ribonuclease P was P. citrinum AE-RP. The strain was verified as from P. citrinum by phylogenetic analysis of the rDNA sequence from the results of a BLAST homology search in the APOLLON DB-FU ver.1.0 database, which includes all sequences in the International Nucleotide Sequence Database (GenBank/DBK/EMBL). The P. citrinum production strain was obtained by conventional mutation with N-methyl-N´-nitrosoguanidine, ultraviolet light and monospore isolation of the parent strain, P. citrinum IAM 7003. The parent strain was originally housed at the Institute of Applied Microbiology Culture Collection; it is presently held at the Japan Collection of Microorganisms under P. citrinum JCM 22500. P. citrinum is known to produce citrinin, a mycotoxin (5); however, citrinin is not produced in the manufacture of ribonuclease P by fermentation of P. citrinum AE-RP. P. citrinum is an occasional opportunistic human pathogen and has been identified rarely as a cause of pneumonia in immunocompromised individuals (6, 7). No viable P. citrinum organisms are present in the enzyme preparation. 2.6.3 Chemical and technical considerations Ribonuclease P is produced by controlled aerobic submerged batch fermentation of a pure culture of a selected strain of P. citrinum AE-RP. Ribonuclease P can also be produced by P. citrinum RP-4, but insufficient information was available on the enzyme concentrate produced from this strain, and enzyme preparations manufactured with the RP-4 strain were not included in this evaluation. During fermentation, the enzyme is secreted into the fermentation broth by the microbial cells. Fermentation continues for a predetermined time or until the enzyme production rate decreases below a defined threshold. The enzyme is separated from the fermentation medium in a series of filtration steps. The biomass is pre- treated with flocculants and filtration aids to facilitate removal of cell material. Germ and polish filtration are performed as part of the recovery process to prevent microbial contamination. The liquid enzyme concentrate is spray-dried, and the activity is standardized with dextrin in production of the final powdered enzyme preparation. The entire process is performed in accordance with current good manufacturing practice with food-grade raw materials. The final ribonuclease P enzyme preparation does not contain the production strain. The enzyme preparation conforms to the General Specifications and Considerations for Enzyme Preparations Used in Food Processing. The primary sequence of ribonuclease P produced by P. citrinum consists of 342 amino acids; its molecular weight by calculation from the determined amino acid sequence is 35 kDa. Ribonuclease P catalyses the hydrolysis of RNA to monophosphate nucleotides. Ribonuclease P enzyme preparation is intended for use in processing 39 Toxicological evaluations and exposure assessments yeast products and flavouring substances and preparations with naturally occurring RNA. The degradation of the RNA substrate in raw materials to produce free phosphonucleotides, specifically guanine and adenine, enhances the consistency and organoleptic (flavour) properties of the final food or food ingredient. Ribonuclease P activity is measured spectrophotometrically as the release of phosphate from adenosine 3´-phosphate. One unit is defined as the amount of enzyme that liberates one μmol/min of phosphate under the assay conditions. The mean activity of ribonuclease P from three batches of the powdered enzyme concentrate was 112 600 U/g. The mean TOS content of the enzyme concentrate is 444 mg/g. The TOS includes the enzyme of interest and residues of organic materials, such as proteins, peptides and carbohydrates, derived from the production organism during manufacture. Ribonuclease P enzyme preparation is used at concentrations up to 1000 mg TOS/kg raw material. Ribonuclease P is denatured and inactivated by high temperatures (> 80 °C) during the production of processed yeast and has no technological effect in the final food. When used in the production of flavouring substances or flavouring preparations, the enzyme is either denatured or removed from the final product. Any carry-over of active ribonuclease P to food is negligible. If present, it is expected that ribonuclease P will be digested, as are most other proteins occurring in food, but no data were available on its digestibility. 2.6.4 Biotransformation No data were available. 2.6.5 Assessment of allergenicity Ribonuclease P from P. citrinum was evaluated for potential allergenicity according to the bioinformatics criteria recommended by FAO/WHO (8, 9) and modified at the eightieth meeting of the Committee (Annex 1, reference 223). The amino acid sequence of ribonuclease P from P. citrinum was compared with those of known allergens in publicly available databases. A search for matches with > 35% identity in a sliding window of 80 amino acids, a search for sequence identity of eight contiguous amino acids and a full-length FASTA sequence search produced no matches. Therefore, the Committee concluded that dietary exposure to ribonuclease P from P. citrinum would not be anticipated to pose a risk of allergenicity. 2.6.6 Toxicological studies In addition to the studies submitted on ribonuclease P from P. citrinum AE- RP, the literature search resulted in toxicity studies with ribonuclease P from P. 40 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report citrinum RP-4 (10, 11). Manufacture of ribonuclease P from P. citrinum RP-4 includes a precipitation step with ethanol. Therefore, the composition of the enzyme concentrates obtained with P. citrinum AE-RP is different from those obtained with P. citrinum RP-4. The Committee concluded that the studies with ribonuclease from P. citrinum RP-4 were not relevant for the current evaluation of ribonuclease P from P. citrinum AE-RP and did not include them in their evaluation. In a 2-week dose range-finding study and a 13-week study of oral toxicity in rats with ribonuclease P from P. citrinum AE-RP, no treatment-related adverse effects were seen when the powdered enzyme concentrate was administered by gavage at doses up to 984 mg TOS/kg bw per day, the highest dose tested (12, 13). The Committee identified a NOAEL of 980 mg TOS/kg bw per day (rounded by the Committee from 984 mg TOS/kg bw per day), the highest dose tested. Powdered ribonuclease P concentrate from P. citrinum AE-RP was not genotoxic in a bacterial reverse mutation assay or in an in vitro chromosomal aberration assay (14, 15). The Committee had no concern with respect to the genotoxicity of the preparation of ribonuclease P from P. citrinum AE-RP. 2.6.7 Observations in humans No data were available. 2.6.8 Assessment of dietary exposure The Committee evaluated an estimate of dietary exposure to ribonuclease P from P. citrinum submitted by the sponsor. The estimate was derived with the budget method and was based on maximum use levels of 20 mg TOS/kg for solid foods and for non-milk beverages and 1000 mg TOS/kg for dietary supplements and the assumption that 25% of the food supply would contain the enzyme preparation. It was assumed that the maximum consumption of dietary supplements would be 30 g/day. The theoretical maximum daily intake was estimated to be 1.3 mg TOS/ kg bw per day (rounded by the Committee from 1.25 mg TOS/kg bw per day). For the dietary exposure assessment, it was assumed that 100% of the enzyme remains in the ingredient and final food. The enzyme is removed or inactivated by high temperatures during processing of food ingredients and would have no technological function in the final food. 2.6.9 Evaluation The Committee identified a NOAEL of 980 mg TOS/kg bw per day (the highest dose tested) in a 13-week study in which rats were treated with ribonuclease P concentrate from P. citrinum AE-RP by gavage. A comparison of the estimated dietary exposure of 1.3 mg TOS/kg bw per day with the NOAEL of 980 mg TOS/ 41 Toxicological evaluations and exposure assessments kg bw per day gives an MOE > 750. On the basis of this MOE and the lack of concern for genotoxicity, the Committee established an ADI “not specified”1 for the ribonuclease P enzyme preparation from P. citrinum AE-RP, used in the applications specified and in accordance with good manufacturing practice. A toxicology and dietary exposure monograph was prepared. New specifications and a chemical and technical assessment were prepared. 2.6.10 Recommendations Ribonuclease P can also be produced by P. citrinum RP-4, but insufficient information was available on the enzyme concentrate produced from this strain. To evaluate the safety of ribonuclease P from P. citrinum RP-4, toxicological studies with well-characterized enzyme concentrate are required. References 1. Report of the Fifty-first Session of the Codex Committee on Food Additives, Jinan, China, 25–29 March 2019. Rome: Food and Agriculture Organization of the United Nations; Geneva: World Health Organization, Joint FAO/WHO Food Standards Programme, Codex Alimentarius Commission; 2019 (REP19/FA; http://www.fao.org/fao-who-codexalimentarius/sh-proxy/ en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FMeetin gs%252FCX-711-51%252FReport%252FREP19_FAe.pdf). 2. Schmidt-Heydt M, Stoll D, Geisen R. Whole-genome sequencing of the fungus Penicillium citrinum reveals the biosynthesis gene cluster for the mycotoxin citrinin. Microbiol Resour Announc. 2019;8:e01419-18 (https://doi.org/10.1128/MRA.01419-18). 3. Bourdichon F, Casaregola S, Farrokh C, Frisvad JC, Gerds ML, Hammes WP et al. Food fermentations: microorganisms with technological beneficial use. Int J Food Microbiol. 2012;154(3):87–97. 4. Pariza MW, Johnson EA. Evaluating the safety of microbial enzyme preparations used in food processing: update for a new century. Regul Toxicol Pharmacol. 2001;33(2):173–86. 5. Park SY, Kim R, Ryu CM, Choi SK, Lee CH, Kim JG et al. Citrinin, a mycotoxin from Penicillium citrinum, plays a role in inducing motility of Paenibacillus polymyxa. FEMS Microbiol Ecol. 2008;65:229–37. 6. Mok T, Koehler AP, Yu MY, Ellis DH, Johnson PJ, Wickham NW. Fatal Penicillium citrinum pneumonia with pericarditis in a patient with acute leukemia. J Clin Microbiol. 1997;35(10):2654–6 (https://doi. org/10.1128/JCM.35.10.2654-2656.1997). 7. Hesse SE, Luethy PM, Beigel JH, Zelazny AM. Penicillium citrinum: opportunistic pathogen or idle bystander? A case analysis with demonstration of galactomannan cross-reactivity. Med Mycol Case Rep. 2017;17, 8–10 (https://doi.org/10.1016/j.mmcr.2017.05.003). 1 The reader is referred to the Technical Report of the 87th JECFA meeting (Annex 1, reference 243) for clarification of the term “ADI not specified”. 42 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report 8. Evaluation of allergenicity of genetically modified foods. Report of a Joint FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnology, 22–25 January 2001. Rome: Food and Agriculture Organization of the United Nations; Geneva: World Health Organization; 2001 (http:// www.who.int/foodsafety/publications/biotech/en/ec_jan2001.pdf). 9. Foods derived from modern biotechnology. Annex 1. Assessment of possible allergenicity. Rome: Food and Agriculture Organization of the United Nations; Geneva: World Health Organization, Joint FAO/WHO Food Standards Programme, Codex Alimentarius Commission; 2009 (http://www.fao.org/ docrep/011/a1554e/a1554e00.htm). 10. Burdock GA, Flamm WG, Carabin G. Toxicity and mutagenicity studies of DN-500001 and RP-11 Enzymes. Food Chem. Toxicol. 2000;38:429–42. 11. Kondo M, Nishimura S, Tanaka N, Flood M. Safety evaluation of phosphodiesterase produced from Penicillium citrinum: summary of toxicological data. Regul Toxicol Pharmacol. 2001;33:2–11. 12. Katsumata T. 2-week repeated oral dose toxicity study of enzyme RP-1 (DRP-SD) in rats (preliminary study). Unpublished report No. C-B313 from Bozo Research Center Inc., Tokyo, Japan; 2007. Submitted to WHO by Amano Enzyme Inc., Nagoya, Japan. 13. Katsumata T. A 13-week oral toxicity study of enzyme RP-1 (DRP-SD) in rats. Unpublished report No. B-6067 from Bozo Research Center Inc., Tokyo, Japan; 2008. Submitted to WHO by Amano Enzyme Inc., Nagoya, Japan. 14. Kawakami K. Safety studies of enzyme RP-1 (DRP-SD) produced by Penicillium citrinum – reverse mutation test in bacteria. Unpublished report (study no. M-06-113) from Hatano Research Institute, Food and Drug Safety Center, Ochiai, Japan; 2007. Submitted to WHO by Amano Enzyme Inc., Nagoya, Japan. 15. Yamakage K. Safety studies of enzyme RP-1 (DRP-SD) produced by Penicillium citrinum – chromosomal aberration test using human lymphocytes. Unpublished report (study no. G-06-057) from Hatano Research Institute, Food and Drug Safety Center, Ochiai, Japan; 2007. Submitted to WHO by Amano Enzyme Inc., Nagoya, Japan. 43 3. Revision of specifications and analytical methods 3.1 Modified starches 3.1.1 Explanation The Committee at its eighty-sixth meeting reviewed full specifications for three modified starches, International Numbering System (INS) 1404, 1420 and 1451, tentative specifications for the remaining 13 modified starches (INS 1400, 1401, 1402, 1403, 1405, 1410, 1412, 1413, 1414, 1422, 1440, 1442 and 1450) and data on the method of manufacture, identity and purity of all 16 modified starches. At the same meeting, the Committee drafted a modular specifications monograph entitled “Modified starches”, consisting of an explanatory introduction, “General specifications for modified starches”, applying to all 16 modified starches, and eight annexes with specifications applicable to individual modified starches according to their treatment(s) (Table 2). The general specifications and annexes 1, 2, 3, 5, 7 and 8 were made tentative. Data and the information necessary to remove the tentative status and revise the modular specifications monograph were requested. At its current meeting, the Committee reviewed the information and data received, revised the modular specifications monograph and removed the tentative status of the “General specifications” and annexes 1, 2, 3, 5, 7 and 8. Each modified starch should fulfil the specification requirements of the “General specifications” and in the applicable annexes. Table 2 Modified starches considered and applicable annexes Modified starch INS Annex Dextrin roasted starch 1400 1 Acid treated starch 1401 1 Alkaline treated starch 1402 1 Bleached starch 1403 2 Oxidized starch 1404 5 Enzyme-treated starch 1405 1 Monostarch phosphate 1410 3 Distarch phosphate 1412 3 Phosphated distarch phosphate 1413 3 Acetylated distarch phosphate 1414 3, 4 Starch acetate 1420 4 Acetylated distarch adipate 1422 4, 8 Hydroxypropyl starch 1440 7 Hydroxypropyldistarch phosphate 1442 3, 7 44 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report Table 2 (continued) Modified starch INS Annex Starch sodium octenylsuccinate 1450 6 Acetylated oxidized starch 1451 4, 5 45 Corrigenda The following requests for corrections, reported to the JECFA Secretariat, were evaluated by the  92nd  JECFA meeting and found to be necessary. These corrections will be made, however, only in the electronic versions and in the online database of specifications.   Food additive Original text Revised text Additional information Riboflavin INS 101(i) % Riboflavin = A × 5000328 × W × 1.367% riboflavin = A × 5000328 × W × 1.367 % Riboflavin = A × 5000328 × W% riboflavin = A × 5000328 × W Correction to calculation in the method of assay; removal of a wrongly assigned factor Riboflavin from Bacillus subtilis INS 101(iii) % Riboflavin = A × 5000328 × W × 1.367% riboflavin = A × 5000328 × W × 1.367 % Riboflavin = A × 5000328 × W% riboflavin = A × 5000328 × W Correction to calculation in the method of assay; removal of a wrongly assigned factor Riboflavin 5´-phosphate sodium INS 101(ii) CAS number 130-40-5 CAS number 130-40-5 (anhydrous) CAS number 6184-17-4 (dihydrate) Current specifications provide the formula for the dihydrate but no applicable CAS number Potassium polyaspartate Missing “Method of assay” Add “Method of assay” under “Purity tests” after the test entitled “Molecular weight and molecular weight distribution”. Delete the bold text “Potassium polyaspartate”, which appears in the test for “Molecular weight and molecular weight distribution”, and replace with “Principle” (as the method of assay). Correct errors in format of specifications monograph Vol. 4 procedure Unsulfonated primary aromatic amines See printed version of Vol. 4 See revised text below; modified text is in bold. Correction to the range of the standard curve Revised text:  Procedure  Preparation of standard aniline solution Weigh 100 mg of redistilled aniline into a small beaker, and transfer to a 100-mL volumetric flask, rinsing the beaker several times with water. Add 30 mL of 3 N hydrochloric acid, and dilute to the mark with water at room temperature. Dilute 10.0 m of this solution to 100 mL with water, and mix well. Dilute 20.0 mL of this solution to 100 mL with water, and mix well (1 mL of this standard solution is equivalent to 20 µg of aniline). Measure the following volumes of the standard aniline solution into a series of 100-mL volumetric flasks: 5 mL, 10 mL, 15 mL, 20 mL and 25 mL. Dilute to 100 mL with 1 N hydrochloric acid, and mix well (100 mL of the resulting working standard solutions contains 100, 200, 300, 400 and 500 µg of aniline, respectively). Prepare all standard solutions freshly.  46 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report Construction of standard curve Pipette 10 mL of each working standard solution into clean, dry test tubes; cool them for 10 min by immersion in a beaker of ice water. To each tube, add 1 mL of the potassium bromide solution and 0.05 ml of the sodium nitrite solution. Mix, and allow the tubes to stand for 10 min in the ice-water bath while the aniline is diazotized. Into each of five 25-mL volumetric flasks, measure 1 mL of the R salt solution and 10 ml of the sodium carbonate solution. Pour each diazotized aniline  solution into a separate flask containing R salt solution and sodium carbonate solution; rinse each test tube with a few drops of water. Dilute to the mark with water, stopper the flasks, mix the contents well, and allow them to stand for 15 min in the dark. Measure the absorbance of each coupled solution at 510 nm in 40-mm cells. As a reference solution, use a mixture of 10.0 mL of 1 N hydrochloric acid, 10.0 mL of the sodium carbonate solution and 2.0 mL of the R salt solution, diluted to 25.0 mL with water. Construct a standard curve of the absorbance versus the weight (g) of aniline in each 100 mL of working standard solution.  Preparation and evaluation of a test solution  Weigh, to the nearest 0.01 g, about 2.0 g of the colouring matter sample (W) into a separatory funnel containing 100 mL of water, rinse the sides of the funnel with a further 50 mL of water, swirling to dissolve the sample, and add 5 mL of 1 N sodium hydroxide. Extract with two 50-mL portions of toluene, and wash the combined toluene extracts with 10-mL portions of 0.1 N sodium hydroxide to remove traces of colour. Extract the washed toluene with three 10-mL portions of 3 N hydrochloric acid, and dilute the combined extract to 100 mL with water. Mix well. Call this “solution T”. Pipette 10.0 mL of solution T into a clean, dry test tube, cool for 10 min by immersion in a beaker of iced water, add 1 mL of the potassium bromide solution, and proceed as described above for preparation of the standard curve, starting with addition of 0.05 mL of the sodium nitrite solution. Measure the absorbance of the coupled test solution at 510 nm in a 40-mm cell. Use a reference solution prepared from 10.0 mL of solution T, 10 mL of the sodium carbonate solution and 2.0 mL of the R salt solution diluted to 25.0 mL with water. From the standard curve, read the weight of aniline (WA) corresponding to the observed absorbance of the test solution.   Calculation: % unsulfonated primary aromatic amine (as aniline) = 100 × WA/W 47 Annex 1 Reports and other documents resulting from previous meetings of the Joint FAO/WHO Expert Committee on Food Additives 1. General principles governing the use of food additives (First report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Report Series, No. 15, 1957; WHO Technical Report Series, No. 129, 1957 (out of print). 2. Procedures for the testing of intentional food additives to establish their safety for use (Second report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Report Series, No. 17, 1958; WHO Technical Report Series, No. 144, 1958 (out of print). 3. Specifications for identity and purity of food additives (antimicrobial preservatives and antioxidants) (Third report of the Joint FAO/WHO Expert Committee on Food Additives). These specifications were subsequently revised and published as Specifications for identity and purity of food additives, Vol. I. Antimicrobial preservatives and antioxidants, Rome, Food and Agriculture Organization of the United Nations, 1962 (out of print). 4. Specifications for identity and purity of food additives (food colours) (Fourth report of the Joint FAO/ WHO Expert Committee on Food Additives). These specifications were subsequently revised and published as Specifications for identity and purity of food additives, Vol. II. Food colours, Rome, Food and Agriculture Organization of the United Nations, 1963 (out of print). 5. Evaluation of the carcinogenic hazards of food additives (Fifth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Report Series, No. 29, 1961; WHO Technical Report Series, No. 220, 1961 (out of print). 6. Evaluation of the toxicity of a number of antimicrobials and antioxidants (Sixth report of the Joint FAO/ WHO Expert Committee on Food Additives). FAO Nutrition Meetings Report Series, No. 31, 1962; WHO Technical Report Series, No. 228, 1962 (out of print). 7. Specifications for the identity and purity of food additives and their toxicological evaluation: emulsifiers, stabilizers, bleaching and maturing agents (Seventh report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 35, 1964; WHO Technical Report Series, No. 281, 1964 (out of print). 8. Specifications for the identity and purity of food additives and their toxicological evaluation: food colours and some antimicrobials and antioxidants (Eighth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 38, 1965; WHO Technical Report Series, No. 309, 1965 (out of print). 9. Specifications for identity and purity and toxicological evaluation of some antimicrobials and antioxidants. FAO Nutrition Meetings Report Series, No. 38A, 1965; WHO/Food Add/24.65 (out of print). 10. Specifications for identity and purity and toxicological evaluation of food colours. FAO Nutrition Meetings Report Series, No. 38B, 1966; WHO/Food Add/66.25. 11. Specifications for the identity and purity of food additives and their toxicological evaluation: some antimicrobials, antioxidants, emulsifiers, stabilizers, flour treatment agents, acids, and bases (Ninth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 40, 1966; WHO Technical Report Series, No. 339, 1966 (out of print). 48 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report 12. Toxicological evaluation of some antimicrobials, antioxidants, emulsifiers, stabilizers, flour treatment agents, acids, and bases. FAO Nutrition Meetings Report Series, No. 40A, B, C; WHO/Food Add/67.29. 13. Specifications for the identity and purity of food additives and their toxicological evaluation: some emulsifiers and stabilizers and certain other substances (Tenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 43, 1967; WHO Technical Report Series, No. 373, 1967. 14. Specifications for the identity and purity of food additives and their toxicological evaluation: some flavouring substances and non-nutritive sweetening agents (Eleventh report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 44, 1968; WHO Technical Report Series, No. 383, 1968. 15. Toxicological evaluation of some flavouring substances and non-nutritive sweetening agents. FAO Nutrition Meetings Report Series, No. 44A, 1968; WHO/Food Add/68.33. 16. Specifications and criteria for identity and purity of some flavouring substances and non-nutritive sweetening agents. FAO Nutrition Meetings Report Series, No. 44B, 1969; WHO/Food Add/69.31. 17. Specifications for the identity and purity of food additives and their toxicological evaluation: some antibiotics (Twelfth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 45, 1969; WHO Technical Report Series, No. 430, 1969. 18. Specifications for the identity and purity of some antibiotics. FAO Nutrition Meetings Series, No. 45A, 1969; WHO/Food Add/69.34. 19. Specifications for the identity and purity of food additives and their toxicological evaluation: some food colours, emulsifiers, stabilizers, anticaking agents, and certain other substances (Thirteenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 46, 1970; WHO Technical Report Series, No. 445, 1970. 20. Toxicological evaluation of some food colours, emulsifiers, stabilizers, anticaking agents, and certain other substances. FAO Nutrition Meetings Report Series, No. 46A, 1970; WHO/Food Add/70.36. 21. Specifications for the identity and purity of some food colours, emulsifiers, stabilizers, anticaking agents, and certain other food additives. FAO Nutrition Meetings Report Series, No. 46B, 1970; WHO/ Food Add/70.37. 22. Evaluation of food additives: specifications for the identity and purity of food additives and their toxicological evaluation: some extraction solvents and certain other substances; and a review of the technological efficacy of some antimicrobial agents (Fourteenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 48, 1971; WHO Technical Report Series, No. 462, 1971. 23. Toxicological evaluation of some extraction solvents and certain other substances. FAO Nutrition Meetings Report Series, No. 48A, 1971; WHO/Food Add/70.39. 24. Specifications for the identity and purity of some extraction solvents and certain other substances. FAO Nutrition Meetings Report Series, No. 48B, 1971; WHO/Food Add/70.40. 25. A review of the technological efficacy of some antimicrobial agents. FAO Nutrition Meetings Report Series, No. 48C, 1971; WHO/Food Add/70.41. 26. Evaluation of food additives: some enzymes, modified starches, and certain other substances: Toxicological evaluations and specifications and a review of the technological efficacy of some 49 Annex 1 antioxidants (Fifteenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 50, 1972; WHO Technical Report Series, No. 488, 1972. 27. Toxicological evaluation of some enzymes, modified starches, and certain other substances. FAO Nutrition Meetings Report Series, No. 50A, 1972; WHO Food Additives Series, No. 1, 1972. 28. Specifications for the identity and purity of some enzymes and certain other substances. FAO Nutrition Meetings Report Series, No. 50B, 1972; WHO Food Additives Series, No. 2, 1972. 29. A review of the technological efficacy of some antioxidants and synergists. FAO Nutrition Meetings Report Series, No. 50C, 1972; WHO Food Additives Series, No. 3, 1972. 30. Evaluation of certain food additives and the contaminants mercury, lead, and cadmium (Sixteenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 51, 1972; WHO Technical Report Series, No. 505, 1972, and corrigendum. 31. Evaluation of mercury, lead, cadmium and the food additives amaranth, diethylpyrocarbamate, and octyl gallate. FAO Nutrition Meetings Report Series, No. 51A, 1972; WHO Food Additives Series, No. 4, 1972. 32. Toxicological evaluation of certain food additives with a review of general principles and of specifications (Seventeenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 53, 1974; WHO Technical Report Series, No. 539, 1974, and corrigendum (out of print). 33. Toxicological evaluation of some food additives including anticaking agents, antimicrobials, antioxidants, emulsifiers, and thickening agents. FAO Nutrition Meetings Report Series, No. 53A, 1974; WHO Food Additives Series, No. 5, 1974. 34. Specifications for identity and purity of thickening agents, anticaking agents, antimicrobials, antioxidants and emulsifiers. FAO Food and Nutrition Paper, No. 4, 1978. 35. Evaluation of certain food additives (Eighteenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 54, 1974; WHO Technical Report Series, No. 557, 1974, and corrigendum. 36. Toxicological evaluation of some food colours, enzymes, flavour enhancers, thickening agents, and certain other food additives. FAO Nutrition Meetings Report Series, No. 54A, 1975; WHO Food Additives Series, No. 6, 1975. 37. Specifications for the identity and purity of some food colours, enhancers, thickening agents, and certain food additives. FAO Nutrition Meetings Report Series, No. 54B, 1975; WHO Food Additives Series, No. 7, 1975. 38. Evaluation of certain food additives: some food colours, thickening agents, smoke condensates, and certain other substances (Nineteenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 55, 1975; WHO Technical Report Series, No. 576, 1975. 39. Toxicological evaluation of some food colours, thickening agents, and certain other substances. FAO Nutrition Meetings Report Series, No. 55A, 1975; WHO Food Additives Series, No. 8, 1975. 40. Specifications for the identity and purity of certain food additives. FAO Nutrition Meetings Report Series, No. 55B, 1976; WHO Food Additives Series, No. 9, 1976. 50 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report 41. Evaluation of certain food additives (Twentieth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Food and Nutrition Meetings Series, No. 1, 1976; WHO Technical Report Series, No. 599, 1976. 42. Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 10, 1976. 43. Specifications for the identity and purity of some food additives. FAO Food and Nutrition Series, No. 1B, 1977; WHO Food Additives Series, No. 11, 1977. 44. Evaluation of certain food additives (Twenty-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 617, 1978. 45. Summary of toxicological data of certain food additives. WHO Food Additives Series, No. 12, 1977. 46. Specifications for identity and purity of some food additives, including antioxidants, food colours, thickeners, and others. FAO Nutrition Meetings Report Series, No. 57, 1977. 47. Evaluation of certain food additives and contaminants (Twenty-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 631, 1978. 48. Summary of toxicological data of certain food additives and contaminants. WHO Food Additives Series, No. 13, 1978. 49. Specifications for the identity and purity of certain food additives. FAO Food and Nutrition Paper, No. 7, 1978. 50. Evaluation of certain food additives (Twenty-third report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 648, 1980, and corrigenda. 51. Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 14, 1980. 52. Specifications for identity and purity of food colours, flavouring agents, and other food additives. FAO Food and Nutrition Paper, No. 12, 1979. 53. Evaluation of certain food additives (Twenty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 653, 1980. 54. Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 15, 1980. 55. Specifications for identity and purity of food additives (sweetening agents, emulsifying agents, and other food additives). FAO Food and Nutrition Paper, No. 17, 1980. 56. Evaluation of certain food additives (Twenty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 669, 1981. 57. Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 16, 1981. 58. Specifications for identity and purity of food additives (carrier solvents, emulsifiers and stabilizers, enzyme preparations, flavouring agents, food colours, sweetening agents, and other food additives). FAO Food and Nutrition Paper, No. 19, 1981. 59. Evaluation of certain food additives and contaminants (Twenty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 683, 1982. 60. Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 17, 1982. 61. Specifications for the identity and purity of certain food additives. FAO Food and Nutrition Paper, No. 25, 1982. 51 Annex 1 62. Evaluation of certain food additives and contaminants (Twenty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 696, 1983, and corrigenda. 63. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 18, 1983. 64. Specifications for the identity and purity of certain food additives. FAO Food and Nutrition Paper, No. 28, 1983. 65. Guide to specifications – General notices, general methods, identification tests, test solutions, and other reference materials. FAO Food and Nutrition Paper, No. 5, Rev. 1, 1983. 66. Evaluation of certain food additives and contaminants (Twenty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 710, 1984, and corrigendum. 67. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 19, 1984. 68. Specifications for the identity and purity of food colours. FAO Food and Nutrition Paper, No. 31/1, 1984. 69. Specifications for the identity and purity of food additives. FAO Food and Nutrition Paper, No. 31/2, 1984. 70. Evaluation of certain food additives and contaminants (Twenty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 733, 1986, and corrigendum. 71. Specifications for the identity and purity of certain food additives. FAO Food and Nutrition Paper, No. 34, 1986. 72. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 20. Cambridge University Press, 1987. 73. Evaluation of certain food additives and contaminants (Thirtieth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 751, 1987. 74. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 21. Cambridge University Press, 1987. 75. Specifications for the identity and purity of certain food additives. FAO Food and Nutrition Paper, No. 37, 1986. 76. Principles for the safety assessment of food additives and contaminants in food. WHO Environmental Health Criteria, No. 70. Geneva, World Health Organization, 1987 (out of print). The full text is available electronically at www.who.int/pcs. 77. Evaluation of certain food additives and contaminants (Thirty-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 759, 1987, and corrigendum. 78. Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 22. Cambridge University Press, 1988. 79. Specifications for the identity and purity of certain food additives. FAO Food and Nutrition Paper, No. 38, 1988. 80. 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Safety evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 40, 1998. 133. Compendium of food additive specifications: addendum 5. FAO Food and Nutrition Paper, No. 52, Add. 5, 1997. 134. Evaluation of certain veterinary drug residues in food (Fiftieth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 888, 1999. 135. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 41, 1998. 136. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/11, 1999. 137. Evaluation of certain food additives (Fifty-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 891, 2000. 138. Safety evaluation of certain food additives. WHO Food Additives Series, No. 42, 1999. 139. Compendium of food additive specifications, addendum 6. FAO Food and Nutrition Paper, No. 52, Add. 6, 1998. 140. Evaluation of certain veterinary drug residues in food (Fifty-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 893, 2000. 141. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 43, 2000. 142. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/12, 2000. 143. Evaluation of certain food additives and contaminants (Fifty-third report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 896, 2000. 144. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 44, 2000. 55 Annex 1 145. Compendium of food additive specifications, addendum 7. FAO Food and Nutrition Paper, No. 52, Add. 7, 1999. 146. Evaluation of certain veterinary drug residues in food (Fifty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 900, 2001. 147. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 45, 2000. 148. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/13, 2000. 149. Evaluation of certain food additives and contaminants (Fifty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 901, 2001. 150. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 46, 2001. 151. Compendium of food additive specifications: addendum 8. FAO Food and Nutrition Paper, No. 52, Add. 8, 2000. 152. Evaluation of certain mycotoxins in food (Fifty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 906, 2002. 153. Safety evaluation of certain mycotoxins in food. WHO Food Additives Series, No. 47/FAO Food and Nutrition Paper, No. 74, 2001. 154. Evaluation of certain food additives and contaminants (Fifty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 909, 2002. 155. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 48, 2002. 156. Compendium of food additive specifications: addendum 9. FAO Food and Nutrition Paper, No. 52, Add. 9, 2001. 157. Evaluation of certain veterinary drug residues in food (Fifty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 911, 2002. 158. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 49, 2002. 159. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/14, 2002. 160. Evaluation of certain food additives and contaminants (Fifty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 913, 2002. 161. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 50, 2003. 162. Compendium of food additive specifications: addendum 10. FAO Food and Nutrition Paper, No. 52, Add. 10, 2002. 163. Evaluation of certain veterinary drug residues in food (Sixtieth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 918, 2003. 164. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 51, 2003. 165. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/15, 2003. 56 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report 166. Evaluation of certain food additives and contaminants (Sixty-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 922, 2004. 167. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 52, 2004. 168. Compendium of food additive specifications: addendum 11. FAO Food and Nutrition Paper, No. 52, Add. 11, 2003. 169. Evaluation of certain veterinary drug residues in food (Sixty-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 925, 2004. 170. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/16, 2004. 171. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 53, 2005. 172. Compendium of food additive specifications: addendum 12. FAO Food and Nutrition Paper, No. 52, Add. 12, 2004. 173. Evaluation of certain food additives (Sixty-third report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 928, 2005. 174. Safety evaluation of certain food additives. WHO Food Additives Series, No. 54, 2005. 175. Compendium of food additive specifications: addendum 13. FAO Food and Nutrition Paper, No. 52, Add. 13 (with Errata), 2005. 176. Evaluation of certain food contaminants (Sixty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 930, 2005. 177. Safety evaluation of certain contaminants in food. WHO Food Additives Series, No. 55/FAO Food and Nutrition Paper, No. 82, 2006. 178. Evaluation of certain food additives (Sixty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 934, 2006. 179. Safety evaluation of certain food additives. WHO Food Additives Series, No. 56, 2006. 180. Combined compendium of food additive specifications. FAO JECFA Monographs 1, Volumes 1–4, 2005, 2006. 181. Evaluation of certain veterinary drug residues in food (Sixty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 939, 2006. 182. Residue evaluation of certain veterinary drugs. FAO JECFA Monographs 2, 2006. 183. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 57, 2006. 184. Evaluation of certain food additives and contaminants (Sixty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 940, 2007. 185. Compendium of food additive specifications. FAO JECFA Monographs 3, 2006. 186. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 58, 2007. 187. Evaluation of certain food additives and contaminants (Sixty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 947, 2007. 57 Annex 1 188. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 59, 2008. 189. Compendium of food additive specifications. FAO JECFA Monographs 4, 2007. 190. Evaluation of certain food additives (Sixty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 952, 2009. 191. Safety evaluation of certain food additives. WHO Food Additives Series, No. 60, 2009. 192. Compendium of food additive specifications. FAO JECFA Monographs 5, 2009. 193. Evaluation of certain veterinary drug residues in food (Seventieth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 954, 2009. 194. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 61, 2009. 195. Residue evaluation of certain veterinary drugs. FAO JECFA Monographs 6, 2009. 196. Evaluation of certain food additives (Seventy-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 956, 2010. 197. Safety evaluation of certain food additives. WHO Food Additives Series, No. 62, 2010. 198. Compendium of food additive specifications. FAO JECFA Monographs 7, 2009. 199. Evaluation of certain contaminants in food (Seventy-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 959, 2011. 200. Safety evaluation of certain contaminants in food. WHO Food Additives Series, No. 63/FAO JECFA Monographs 8, 2011. 201. Residue evaluation of certain veterinary drugs. FAO JECFA Monographs 9, 2010. 202. Evaluation of certain food additives and contaminants (Seventy-third report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 960, 2011. 203. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 64, 2011. 204. Compendium of food additive specifications. FAO JECFA Monographs 10, 2010. 205. Evaluation of certain food additives and contaminants (Seventy-fourth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 966, 2011. 206. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 65, 2011. 207. Compendium of food additive specifications. FAO JECFA Monographs 11, 2011. 208. Evaluation of certain veterinary drug residues in food (Seventy-fifth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 969, 2012. 209. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 66, 2012. 210. Residue evaluation of certain veterinary drugs. FAO JECFA Monographs 12, 2012. 211. Evaluation of certain food additives (Seventy-sixth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 974, 2012. 212. Safety evaluation of certain food additives. WHO Food Additives Series, No. 67, 2012. 58 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report 213. Compendium of food additive specifications. FAO JECFA Monographs 13, 2012. 214. Evaluation of certain food additives and contaminants (Seventy-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 983, 2013. 215. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 68, 2013. 216. Compendium of food additive specifications. FAO JECFA Monographs 14, 2013. 217. Evaluation of certain veterinary drug residues in food (Seventy-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 988, 2014. 218. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 69, 2014. 219. Residue evaluation of certain veterinary drugs. FAO JECFA Monographs 15, 2014. 220. Evaluation of certain food additives (Seventy-ninth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 990, 2015. 221. Safety evaluation of certain food additives. WHO Food Additives Series, No. 70, 2015. 222. Compendium of food additive specifications. FAO JECFA Monographs 16, 2014. 223. Evaluation of certain food additives and contaminants (Eightieth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 995, 2016. 224. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 71, 2015. 225. Compendium of food additive specifications. FAO JECFA Monographs 17, 2015. 226. Evaluation of certain veterinary drug residues in food (Eighty-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 997, 2016. 227. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 72, 2016. 228. Residue evaluation of certain veterinary drugs. FAO JECFA Monographs 18, 2016. 229. Safety evaluation of certain food additives and contaminants. Supplement 1: Non-dioxin-like polychlorinated biphenyls. WHO Food Additives Series, No. 71-1, 2016. 230. Evaluation of certain food additives (Eighty-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 1000, 2016. 231. Compendium of food additive specifications. FAO JECFA Monographs 19, 2016. 232. Safety evaluation of certain food additives. WHO Food Additives Series, No. 73, 2017. 233. Evaluation of certain contaminants in food (Eighty-third report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 1002, 2017. 234. Evaluation of certain food additives (Eighty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 1007, 2017. 235. Safety evaluation of certain contaminants in food. WHO Food Additives Series, No. 74, FAO JECFA Monographs 19 bis, 2018. 236. Compendium of food additive specifications. FAO JECFA Monographs 20, 2017. 59 Annex 1 237. Safety evaluation of certain food additives. WHO Food Additives Series, No. 75, 2019. 238. Evaluation of certain veterinary drug residues in food (Eighty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 1008, 2018. 239. Residue evaluation of certain veterinary drugs. FAO JECFA Monographs 21, 2018. 240. Compendium of food additive specifications. FAO JECFA Monographs 22, 2018. 241. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 76, 2019. 242. Evaluation of certain food additives (Eighty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 1014, 2019. 243. Evaluation of certain food additives (Eighty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives) WHO Technical Report Series, No. 1020, 2019. 244. Compendium of food additive specifications. FAO JECFA Monographs 23, 2019. 245. Evaluation of veterinary drug residues in food (Eighty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 1023, 2020. 246. Evaluation of certain food additives (Eighty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 1027, 2021. 247. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 79, 2021. 60 61 Annex 2 Toxicological and dietary exposure information and information on specifications Food additives evaluated toxicologically and assessed for dietary exposure Food additive Specifications Acceptable daily intakes (ADIs) and other conclusions on toxicology and dietary exposure Benzoic acid, its salts and derivatives N The Committee evaluated a new extended one-generation reproductive toxicity study on benzoic acid. This study showed no treatment-related adverse effects, indicating a NOAEL of 1000 mg/kg bw per day, the highest dose tested. Applying a chemical specific adjustment factor of 2 for interspecies toxicokinetics variation instead of the default factor of 4.0, the Committee established a group ADI of 0–20 mg/kg bw, which applies to benzoic acid, the benzoate salts (calcium, potassium and sodium), benzaldehyde, benzyl acetate, benzyl alcohol and benzyl benzoate, expressed as benzoic acid equivalents. The Committee withdrew the previous group ADI of 0–5 mg/kg bw. The Committee noted that the high dietary exposure estimate, expressed as benzoic acid, of 7.1 mg/kg bw per day for children aged 3–9 years does not exceed the group ADI of 0–20 mg/kg bw. Collagenase from Streptomyces violaceoruber expressed in S. violaceoruber N Negative results were observed in genotoxicity studies with a powdered enzyme concentrate. The Committee identified a NOAEL of 940 mg TOS/kg bw per day (rounded from 939.6), the highest dose tested in a 13-week study of oral toxicity in rats. The Committee identified a NOAEL of 940 mg TOS/kg bw per day, the highest dose tested in a 13-week study of oral toxicity in rats. Comparison of this NOAEL with the estimated dietary exposure of 0.43 mg TOS/kg bw per day gave a margin of exposure (MOE) of > 2100. In view of this MOE and the lack of concern about genotoxicity, the Committee established an ADI “not specified”1 for collagenase from S. violaceoruber, when used in the applications specified and in accordance with good manufacturing practice. β-Glucanase from Streptomyces violaceoruber expressed in S. violaceoruber N The Committee noted negative results in studies of genotoxicity and in studies of oral toxicity in rats. The Committee identified a NOAEL of 950 mg TOS/kg bw per day (rounded by the Committee from 953.3), the highest dose tested. Comparison of this NOAEL with the estimated dietary exposure of 0.15 mg TOS/kg bw per day gave an MOE > 6300. On the basis of this MOE and the lack of concern about genotoxicity, the Committee established an ADI “not specified”1 for β-glucanase from S. violaceoruber, for the proposed uses and in accordance with good manufacturing practice. 1 The reader is referred to the Technical Report of the 87th JECFA meeting for clarification of the term “ADI not specified”. 62 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report Food additive Specifications Acceptable daily intakes (ADIs) and other conclusions on toxicology and dietary exposure Phospholipase A2 from Streptomyces violaceoruber expressed in S. violaceoruber R Negative results were obtained in genotoxicity tests. In a 13-week study of oral toxicity in rats, small effects were seen at low incidence at the high dose of 956 mg TOS/kg bw per day, which might have been related to treatment. The Committee therefore identified a NOAEL of 190 mg TOS/kg per day (rounded by the Committee from 191 mg TOS/kg bw per day). A comparison of the estimated dietary exposure of 0.25 mg TOS/kg bw per day with the NOAEL of 190 mg TOS/kg bw per day from the oral toxicity study gives an MOE of 760. On this basis and in the absence of concern about genotoxicity, the Committee established an ADI “not specified”1 for the phospholipase A2 enzyme preparation from S. violaceoruber when used in the applications specified and in accordance with good manufacturing practice. Riboflavin from Ashbya gossypii N The Committee noted that riboflavin from A. gossypii has low acute toxicity and does not raise concern for genotoxicity. The NOAEL from a 90-day oral toxicity study in rats was 3000 mg/kg bw per day, the highest dose tested. Comparison of this NOAEL with the estimated dietary exposure of 3.6 mg/kg bw per day, based on maximum reported use levels, resulted in an MOE > 800. The Committee established a group ADI “not specified”1 for riboflavin, riboflavin- 5´-phosphate, riboflavin from B. subtilis and riboflavin from A. gossypii, expressed as riboflavin. The Committee withdrew the previous group ADI of 0–0.5 mg/kg bw. Ribonuclease P from Penicillium citrinum N The Committee identified a NOAEL of 980 mg TOS/kg bw per day (the highest dose tested) in a 13-week study in which rats were treated with ribonuclease P concentrate from P. citrinum AE-RP by gavage. A comparison of the estimated dietary exposure of 1.3 mg TOS/kg bw per day with the NOAEL of 980 mg TOS/kg bw per day gives an MOE > 750. On the basis of this MOE and the lack of concern for genotoxicity, the Committee established an ADI “not specified”1 for the ribonuclease P enzyme preparation from P. citrinum AE-RP, used in the applications specified and in accordance with good manufacturing practice. 1 The reader is referred to the Technical Report of the 87th JECFA meeting for clarification of the term “ADI not specified”. N: new specifications, R: revised specifications R: revised specifications Food additive Specifications Modified starches R Food additives considered for specifications only 63 Annex 3 Meeting agenda 92nd JOINT FAO/WHO EXPERT COMMITTEE ON FOOD ADDITIVES (JECFA) 7–18 June 2021 Virtual meeting: 12:00–16:00 (CET) 1. Opening 2. Declarations of Interests (information by the Secretariat on any declared interests and discussion, update by experts) 3. Election of Chairperson and Vice-Chairperson, appointment of Rapporteurs 4. Adoption of the agenda 5. Matters of interest arising from previous Sessions of the Codex Committee on Food Additives 6. Critical issues and questions from Working Papers (first brief round of discussion on all subjects to inform the full Committee) 7. Evaluations 7.1. Food additives • Benzoic acid and its salts • Riboflavin from Ashbya gossypii 7.2. Enzymes • β-Glucanase • Collagenase • Phosphodiesterase • Phospholipase A2 8. Other matters to be considered (general considerations). 64 W H O T ec hn ic al R ep or t S er ie s, N o. 1 03 7, 2 02 2 Joint FAO/WHO Expert Committee on Food Additives Ninety-second report 9. Other matters as may be brought forth by the Committee during discussions at the meeting. 10. Adoption of the report. SELECTED WHO PUBLICATIONS OF RELATED INTEREST Evaluation of certain food additives Eighty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives WHO Technical Report Series, No. 1027, 2020 (106 pages) Evaluation of certain veterinary drug residues in food Eighty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives WHO Technical Report Series, No. 1023, 2020 (116 pages) Evaluation of certain food additives Eighty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives WHO Technical Report Series, No. 1020, 2019 (109 pages) Evaluation of certain food additives Eighty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives WHO Technical Report Series, No. 1014, 2019 (156 pages) Evaluation of certain veterinary drug residues in food Eighty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives WHO Technical Report Series, No. 1008, 2017 (150 pages) Safety evaluation of certain food additives Eighty-fourth meeting of the Joint FAO/WHO Expert Committee on Food Additives WHO Food Additives Series, No. 75, 2018 (244 pages) Evaluation of certain food additives Eighty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives WHO Technical Report Series, No. 1007, 2017 (92 pages) Safety evaluation of certain contaminants in food Eighty-third meeting of the Joint FAO/WHO Expert Committee on Food Additives WHO Food Additives Series, No. 74, 2018 (897 pages) Evaluation of certain contaminants in food Eighty-third report of the Joint FAO/WHO Expert Committee on Food Additives WHO Technical Report Series, No. 1002, 2017 (166 pages) Further information on these and other WHO publications can be obtained from WHO Press, World Health Organization 1211 Geneva 27, Switzerland www.who.int/bookorders tel.: +41 22 791 3264; fax: +41 22 791 4857; email: bookorders@who.int Evaluation of certain food additives This report presents the conclusions of a Joint FAO/WHO Expert Committee on Food Additives (JECFA), which met virtually from 7 to 18 June 2021. The toxicological and dietary exposure monographs in this volume summarize data on the safety of and dietary exposure to specific food additives: benzoic acid, its salts and derivatives; collagenase from Streptomyces violaceoruber expressed in S. violaceoruber; β-glucanase from Streptomyces violaceoruber expressed in S. violaceoruber; phospholipase A2 from Stretomyces violaceoruber expressed in S. violaceoruber; riboflavin from Ashbya gossypii; and ribonuclease P from Penicillium citrinum. This volume and others in the WHO Food Additives series contain information that is useful to those who produce and use food additives and veterinary drugs and those involved in controlling contaminants in food, government and food regulatory officers, industrial testing laboratories, toxicological laboratories and universities. CC2907EN/1/11.22