Scientific Opinion on the safety of polyvinylpyrrolidone-vinyl acetate copolymer for the proposed uses as a food additive 1

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1 SCIENTIFIC OPINION Scientific Opinion on the safety of polyvinylpyrrolidone-vinyl acetate copolymer for the proposed uses as a food additive 1 EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) 2, 3 European Food Safety Authority (EFSA), Parma, Italy ABSTRACT The Panel on Food Additives and Nutrient Sources added to Food provides a scientific opinion on the use of polyvinylpyrrolidone-vinyl acetate (PVP/VA) copolymer in food supplements. PVP/VA copolymer is poorly absorbed orally and largely excreted intact in the faeces. No genotoxicity data are available. Both a 28-day and a 90-day feeding study with PVP/VA copolymer in the rat, revealed a NOAEL of 1000 mg/kg bw/day (highest dose tested). Oral administration of PVP/VA copolymer to male/female rats for 24 months and to male/female dogs for 52 weeks revealed NOAELs of 2800 mg/kg bw/day and 2500 mg/kg bw/day respectively (highest doses tested). No reproductive or developmental toxicity data are available. The exposure to PVP/VA copolymer from its use in food supplements and in pharmaceuticals is 23.4 mg/kg bw/day (adult high consumers) and 16 mg/kg bw/day (child high consumers). The toxicological database was too limited to derive an ADI. From the given range of NOAELs and the intake of PVP/VA copolymer, a Margin of Safety (MOS) varying from 43 to 120 for adults and from 63 to 175 for children can be calculated. These MOS are considered sufficient given the lack of absorption of PVP/VA, the fact that the NOAELs were the highest doses tested and that exposure estimates are based on worst case assumptions. From the maximum residual levels of VA, Margins of Exposure (MOE) of > 10 6 were calculated. The MOE for hydrazine were above The Panel concluded that the residual levels of hydrazine (up to a maximum of 1.0 mg/kg in the final product) are unlikely to be of safety concern, but considered it would be prudent to lower the level of hydrazine as far as reasonably achievable. The Panel concluded that the use of PVP/VA copolymer in solid food supplements as a binding/coating agent is unlikely to be of safety concern at the proposed uses. European Food Safety Authority, 2010 KEY WORDS Polyvinylpyrrolidone-vinyl acetate copolymer, CAS Registry Number , vinyl acetate; polyvinylpyrrolidone; copolyvidon; copovidone; 1-vinyl-2-pyrrolidone-vinyl acetate copolymer; 2- pyrrolidinone, 1-ethenyl-, polymer with ethenyl acetate. 1 On request from the European Commission, Question No EFSA-Q , adopted on 9 December Panel members: F. Aguilar, B. Dusemund, P. Galtier, J. Gilbert, D.M. Gott, S. Grilli, R. Gürtler, J. König, C. Lambré, J-C. Larsen, J-C. Leblanc, A. Mortensen, D. Parent-Massin, I. Pratt, I.M.C.M. Rietjens, I. Stankovic, P. Tobback, T. Verguieva, R.A. Woutersen. Correspondence: ans@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the ANS Working Group B on Food Additives and Nutrient Sources for the preparatory work on this scientific opinion: M. Bakker, D. Boskou, B. Dusemund, D. Gott, T. Hallas- Møller, A. Hearty, J. König, D. Marzin, D. Parent-Massin, I.M.C.M. Rietjens, G.J.A. Speijers, P. Tobback, T. Verguieva, R.A. Woutersen. Suggested citation: EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS); Scientific Opinion on the safety of polyvinylpyrrolidone-vinyl acetate copolymer for the proposed uses as a food additive. EFSA Journal 2010;8(12):1948. [28 pp.] doi: /j.efsa Available online: European Food Safety Authority,

2 SUMMARY Following a request from the European Commission, the Panel on Food Additives and Nutrient Sources added to Food (ANS) was asked to deliver a scientific opinion on polyvinylpyrrolidone-vinyl acetate (PVP/VA) copolymer when used as a food additive. The present opinion deals with the safety of PVP/VA copolymer for use in food supplements in tablet form as a binding/coating agent in an amount of up to 10% of weight per tablet, for a tablet weight of 1000 mg. PVP/VA copolymer is a copolymer produced by polymerisation of the monomers N-vinyl-2- pyrrolidone (NVP) and vinyl acetate (VA). The substance will be used in food supplement tablets as a binding agent and as a coating agent. No absorption, distribution, metabolism and excretion (ADME) studies were provided by the petitioner. It is reported in literature that PVP/VA copolymer, given its high weight average molecular weight of about to g/mol, is only minimally absorbed after oral administration (approximately 2% of the administered dose), and that it is largely excreted intact in the faeces. Data from a 28-day oral feeding study and from a 90-day oral feeding study, both in the rat, demonstrated that there were no effects on body weight gain, feed consumption, haematology parameters, serum chemistry and urinalyses. There were also no test-article-related effects on organ weights, macroscopic and microscopic evaluations. The No-Observed-Adverse-Effect Level (NOAEL) in both studies was determined to be 1000 mg/kg bw/day, the highest dose level tested. The Panel agreed with these NOAELs. PVP/VA copolymer was administered in the diet to male and female Wistar rats for 24 months and to male and female pure-bred Beagle dogs for 52 weeks. No significant toxicological findings resulting from high levels of PVP/VA copolymer in the diet of both animal species were observed. From these studies, the NOAEL was estimated to be 2800 mg PVP/VA/kg bw/day for the rat, and 2500 mg PVP/VA/kg bw/day for the dog, being the highest dose levels tested. The Panel agreed with these NOAELs. The petitioner also referred to studies in mice and rats as summarised in a review paper. However, the Panel noted that no details of the studies were provided. It was only stated that the animals were given daily an aqueous solution of 10.2 mg PVP/VA/l (equivalent to about 2.7 mg PVP/VA/kg bw/day for the mice, and to about 1.6 mg PVP/VA/kg bw/day for the rats) for a period of one year. There were no changes attributable to the copolymer in either mice or rats, including no histological changes in the internal organs. Data from a 2-year feeding study in the rat with PVP/VA copolymer at a dose level of 450 mg PVP/VA/kg bw/day showed that no treatment-related clinical chemistry changes were observed. No treatment-related tumours or other gross pathologically detectable lesions were induced. Histopathological examination showed an increased incidence of liver congestion and fatty degeneration in the test group compared with the cellulose control group. There were no other histopathologically detectable organ changes observed. The test and control diets were tolerated without signs of toxicity. It was however noted, that the survival in both the control and test groups was poor (8-14%), reportedly due to inflammatory diseases of the respiratory tract and from otitis. There were no reproductive or developmental toxicity data available for PVP/VA copolymer. The petitioner considers that the copolymer is not expected to have reproductive or developmental effects considering the low oral bioavailability of PVP/VA copolymer and given the presence of only low levels (<5 mg/kg) of residual monomers (VP and VA) in the copolymer. In support of this, the Panel noted that a similar polymer produced from VA, (i.e. polyvinyl alcohol) did not show reproductive or developmental effects at doses up to 5000 mg/kg bw/day. 2

3 Data from the UK Food Standards Agency on the consumption of food supplements indicate that the use of food supplements among high consumers (97.5 th percentile) ranged from 2 tablets/capsules per day in children (4-18 years old) to 7 tablets/capsules per day in adults. Based on a coating level of 100 mg PVP/VA copolymer/tablet for high consumers, an anticipated exposure of 200 mg PVP/VA copolymer/day for children and 700 mg PVP/VA copolymer/day for adults, corresponding to 8 mg/kg bw/day for children and to 11.7 mg/kg bw/day for adults, can be calculated. Assuming similar levels of use and intake data of PVP/VA copolymer from pharmaceuticals, the Panel calculated the combined intake from food supplements and pharmaceutical products to be, respectively, 16 mg/kg bw/day for children, and 23.4 mg/kg bw/day for adults. The Panel considered that the safety of the residual levels of VA and hydrazine should also be assessed. The petitioner stated that no data on the genotoxicity of the PVP/VA copolymer were available and provided some data on the genotoxicity of the monomers (VP and VA) from which the PVP/VA copolymer is derived. The Panel noted that the safety of VP residues in certain food additives has been evaluated by the former European Commission Scientific Committee on Food (SCF) and that the mutagenicity of both monomers has already been evaluated in the course of the European chemicals evaluation. Based on these evaluations, the Panel considered that there is no concern with respect to genotoxicity of the monomers VP and VA from which the PVP/VA copolymer is derived, nor for PVP/VA copolymer itself. Four chronic toxicity and carcinogenicity studies with the monomer VA supplied in drinking water of rats and mice for periods up to 104 weeks were also provided. From these studies the different authors concluded that VA should be considered a multipotential carcinogen for rodents. The Panel noted that VA is present in PVP/VA copolymer at a level of maximum 5 mg/kg. The Panel calculated the exposure to VA, for the highest exposure levels, based on the proposed uses to be 0.12 µg/kg bw/day for adults and to 0.08 µg/kg bw/day for children. The Panel noted that the studies provide evidence for a VA-induced animal carcinogenicity. The Panel also noted that according to the IARC evaluation, VA is considered as a possibly human carcinogen of Group 2B (IARC, 1995). The Panel derived a lower confidence limit on the benchmark dose (BMDL 10 ) of 440 mg/kg bw/day from the combined incidence of squamous cell carcinomas and papillomas in mice and rats induced by VA in the oral cavity as reported by Umeda et al. in Taking the total combined exposure to VA at 0.12 µg/kg bw/day for adults and 0.08 µg/kg bw/day for children, this would lead to a Margin of Exposure (MOE) of respectively 3.7 x 10 6 for adults and 0.6 x 10 7 for children, and therefore the Panel concluded that the presence of VA at levels up to 5 mg/kg is unlikely to be of safety concern. The petitioner did not provide data on chronic toxicity and carcinogenicity of hydrazine, present in PVP/VA copolymer at levels up to a maximum of 1.0 mg/kg. IARC evaluated hydrazine and hydrazine sulphate in Hydrazine was found to be genotoxic in vivo and a genotoxic mechanism of the carcinogenicity cannot be excluded. IARC considered that hydrazine and hydrazine sulphate are reasonably anticipated to be human carcinogens based on sufficient evidence of carcinogenicity in experimental animals. IARC however, considered that there is inadequate evidence in humans for the carcinogenicity of hydrazine and concluded that hydrazine is possibly carcinogenic to humans (Group 2B) (IARC, 1999). Based on the combined exposure for the highest exposure levels, the Panel calculated the exposure to hydrazine for adults to be μg/kg bw/day whereas the estimated potential exposure for children would be μg/kg bw/day. From the available data on the incidence of hepatocellular tumours induced in male CBA/Cb/Se mice exposed daily to hydrazine for 25 weeks (Biancifiori, 1970) the Panel derived a BMDL 10 for hydrazine sulphate of 2.3 mg/kg bw/day and for hydrazine anhydrous of 0.57 mg/kg bw/day. Comparison of the 3

4 BMDL 10 of 0.57 mg kg bw/day to the estimated exposure to hydrazine resulting from its residual presence in the final product amounting to μg/kg bw/day for adults and μg/kg bw/day for children the Panel calculated a MOE for hydrazine sulphate of 9.6 x10 4 for adults and of 14 x 10 4 to for children. For hydrazine anhydrous these values become 2.3 x 10 4 for adults and 3.6 x 10 4 for children. Given that these Margins of Exposure are above and considering the opinion of the EFSA Scientific Committee (EFSA, 2005), the Panel concluded that the residual levels of hydrazine, proposed to be up to a maximum of 1.0 mg/kg in the final product, are unlikely to be of safety concern. The Panel noted that no data on reproductive and developmental toxicity of PVP/VA were provided and that the overall toxicological database on PVP/VA is too limited to derive an acceptable daily intake (ADI). However, the Panel noted the limited absorption and the lack of toxicity observed at the highest doses tested in the available repeated-dose toxicity studies. The NOAELs derived from the available studies were: 1000 mg/kg bw/day from a 28-day oral feeding study in the rat, 1000 mg/kg bw/day from a 90-day oral feeding study in the rat, 2800 mg/kg bw/day from a 24-month feeding study in the rat and 2500 mg/kg bw/day from a 52-week feeding study in the dog. All the NOAELs in the studies were the highest doses tested. Using this range of NOAELs and taking into account the combined intake of PVP/VA copolymer from its use in food supplements and pharmaceuticals, the Panel calculated a Margin of Safety (MOS) varying from at least 43 to 120 for adults, and 63 to 175 for children. Given the lack of absorption of PVP/VA, the fact that the MOS values are based on NOAELs that were the highest dose levels tested and that the exposures estimates were worst case assumptions, the Panel considered these MOS values sufficient. In conclusion the Panel noted that, in the absence of data on reproductive and developmental toxicity, chronic effects in the gastrointestinal tract following oral administration cannot be excluded. Therefore, the Panel considered that an ADI should not be established and that a MOS approach is appropriate. The Panel considered the calculated Margins of Safety for PVP/VA copolymer sufficient. The Panel concluded that the residual level of hydrazine, proposed to be up to a maximum of 1.0 mg/kg in the final product, is unlikely to be of safety concern. Overall, the Panel concluded that the use of PVP/VA copolymer in solid food supplements as a binding/coating agent is unlikely to be of safety concern at the proposed uses and use levels provided. The Panel however, considered that it would be prudent to lower the level of hydrazine as far as reasonably achievable. 4

5 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 5 Background as provided by the European Commission... 6 Terms of reference as provided by the European Commission... 6 Assessment Introduction Technical data Identity of the substance Specifications Manufacturing process Methods of analysis in food Reaction and fate in food Case of need and proposed uses Information on existing authorisations and evaluations Exposure Biological and toxicological data Absorption, distribution, metabolism and excretion Toxicological data Acute oral toxicity Short-term and subchronic toxicity Genotoxicity Chronic toxicity and carcinogenicity Reproductive and developmental toxicity Discussion Documentation provided to EFSA References Glossary/Abbreviations

6 BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION Food supplements, like pharmaceuticals need to be coated to provide taste and odour masking, to modify the release of the active ingredient and to increase the stability of the product. Cellulose esters have been used for this purpose. Polyvinylpyrrolidone-vinyl acetate copolymer is a synthetic, 60:40 random copolymer of N-vinyl-2- pyrrolidone and vinyl acetate. The copolymer has a long history of use as excipient in pharmaceutical tablets. There is a monograph of the substance under the name Copovidone in the European Pharmacopoeia. The addition of the polyvinylpyrrolidone-vinyl acetate copolymer to a cellulosic formulation in food supplements would improve film toughness, increase coating application rates and promote better film adhesion, with a film which is stronger and more flexible. In addition, the use of the copolymer would also enable a continuous coating process resulting in reduction in coating process time. TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION In accordance with Article 29 (1) (a) of Regulation (EC) No 178/2002 4, the European Commission asks the European Food Safety Authority to provide a scientific opinion on the safety of polyvinylpyrrolidone-vinyl acetate copolymer in food supplement tablets as a binding agent and in tablet coatings. 4 Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety. OJ L 31, , p

7 ASSESSMENT 1. Introduction The present opinion deals with the safety of polyvinylpyrrolidone-vinyl acetate copolymer (PVP/VA copolymer) for use in food supplement tablets as a binding agent and as a coating agent. 2. Technical data 2.1. Identity of the substance PVP/VA copolymer has the chemical name: Acetic acid, ethenyl ester, polymer with 1-ethenyl-2- pyrrolidinone and CAS Registry Number According to the petitioner, the weight average molecular weight (Mw) is between and g/mol. The molecular formula as given by the petitioner is: (C 6 H 9 NO) n. (C 4 H 6 O 2 ) m.. The representative structural formula of PVP/VA copolymer as provided by the petitioner is shown in Figure 1. Figure 1: The structural formula of PVP/VA copolymer as provided by the petitioner (n = 1.2 m, on molar basis); n:m = 60:40 (w/w) Synonyms: There are more than 50 synonyms listed (Internet ChemIDplus Advanced) for PVP/VA copolymer (CAS No ), e.g. vinyl acetate; polyvinylpyrrolidone; copolyvidon; copovidone; 1-vinyl-2-pyrrolidone-vinyl acetate copolymer; 2-pyrrolidinone, 1-ethenyl-, polymer with ethenyl acetate. Physical state The physical state is described as a white to yellowish-white powder or flakes with an average particle size of µm. Solubility The substance is freely soluble in water, alcohol, ethylene chloride and ether Specifications The specifications for PVP/VA as proposed by the petitioner are given in Table 1. 7

8 Table 1: Specifications of PVP/VA copolymer as proposed by the petitioner Characteristics Proposed specifications Identification IR spectroscopy European Colour Test (BY Colour) Minimum BY5 K-value* (1% solids in aqueous solution) ph-value (10% w/w in distilled water) Vinyl acetate component in copolymer Maximum % Nitrogen content % Loss on drying Maximum 5.0% Residuals Aldehydes (as acetaldehyde) 0.2% Vinyl Acetate (by GC) Maximum 5 mg/kg Vinyl pyrrolidone (by HPLC) Maximum 5 mg/kg Hydrazine Maximum 1.0 mg/kg Peroxide content (Titanyl sulphate method) Isopropanol Maximum 400 mg/kg Maximum 150 mg/kg Heavy metals Arsenic: max. 3 mg/kg Lead: max. 2 mg/kg Mercury: max. 1 mg/kg Cadmium: max. 1 mg/kg Ash (residue on ignition/sulphated) Max. 0.1% *K-value: dimensionless index, calculated from kinematic viscosity measurements of dilute solutions, used to indicate the likely degree of polymerization or molecular size of a polymer (Kline, 1945). The Panel noted that the proposed limitation for aldehydes is above the maximum level of 0.05% in the European Pharmacopeia (Ph. Eur, 2008). Furthermore, the Panel noted that according to the evaluation by IARC, vinyl acetate is possibly carcinogenic to humans (Group 2B) (IARC, 1995), and that according to the evaluation by IARC also hydrazine is possibly carcinogenic to humans (Group 2B) (IARC, 1999) Manufacturing process The manufacturing process is described in detail by the petitioner and can be summarised as follows. PVP/VA copolymer is produced by free radical copolymerisation of N-vinyl-2-pyrrolidone (NVP; CAS No ) and vinyl acetate (VA; CAS No ) in solution in isopropanol (iproh; CAS No ), in the presence of initiators. The process is continuous and temperature controlled. Hydrazine is formed from amines present in this reaction mixture. Sodium bisulphite is added to the batch for colour stability. Isopropanol is exchanged for de-ionised water by adding de-ionised water to the reactor and performing a solvent exchange via vacuum distillation. Sodium acetate (for ph stabilisation) and a microbiological preservative (not specified) are added. The batch is then heated, sampled, and adjusted for solids content. The product is isolated as an aqueous solution, or as a spray-dried solid. The product is stored at ambient temperature protected from moisture. The Panel noted that sodium bisulphite (E 222) is an authorised food additive. However, the Panel also noted that the specifications do not indicate whether (or at what level) sodium bisulphite is present in the product of commerce. 8

9 2.4. Methods of analysis in food The safety of polyvinylpyrrolidone-vinyl acetate copolymer used as a food additive The petitioner states that PVP/VA copolymer can be determined by High Performance Liquid Chromatography (HPLC) on Phenomenex Poly-Sep GFC-P 3000 at 40 C Reaction and fate in food No data on the reaction and fate of PVP/VA copolymer in food have been provided. Instead, the petitioner provided data on bulk stability studies with PVP/VA copolymer. In these studies the copolymer was packaged in closed containers and stored at room temperature for a minimum of 24 months. Stability attributes including physical appearance, solution appearance, K-value, aldehyde content (as formaldehyde), hydrogen peroxide content, ph in the range of 3.0 to 4.0, and loss on drying, were evaluated at various time intervals up to 48 months. From the data provided it is shown that there were no consistent changes observed over time in any of the stability-indicating assays. From these data, the petitioner concluded that PVP/VA copolymer has a minimum shelf-life of 24 months under the given conditions Case of need and proposed uses PVP/VA copolymer is intended to be used as a binding agent and as a coating agent in food supplements at levels up to 10% of the final dietary supplement product. According to the petitioner, the insertion of vinyl acetate (VA) into the vinylpyrrolidone polymer chain reduces hydrophilicity and glass transition temperature (Tg) of the polymer Information on existing authorisations and evaluations PVP/VA copolymer is currently approved by the US FDA as an inactive ingredient in pharmaceuticals for use in oral tablets ( PVP/VA copolymer is also listed for use as an indirect food additive in food contact materials: - food contact adhesives (21 CFR Part ) components of paper and paperboard in contact with aqueous and fatty foods (21 CFR Part ); - components of paper and paperboard in contact with dry food (21 CFR Part ). According to the petitioner, PVP/VA copolymer is permitted for pharmaceutical use. It conforms to USP/NF and Ph. Eur. monographs. It is also used in pharmaceutical products and personal care products in the EU. In 2002, the Scientific Committee on Food (SCF) evaluated the safety of the residues of N-vinyl-2- pyrrolidone (NVP) monomer, proposed at the use level of 10 mg/kg in the specifications for the food additives polyvinylpyrrolidone (PVP) and polyvinylpolypyrrolidone (PVPP). The SCF concluded that the intakes of NVP from food additive uses of PVP and PVPP do not give cause for concern (SCF, 2002). In 2004, the Joint FAO/WHO Expert Committee on Food additives (JECFA) evaluated Polyvinyl alcohol (PVA) used as a coating, binder, sealing and surface finishing agent in food products. JECFA established an Acceptable Daily Intake (ADI) for polyvinyl alcohol of 50 mg/kg bw/day (JECFA, 2004). In 2005, the European Food safety Authority (EFSA) evaluated the use of PVA as a film coating agent for food supplements. The Panel concluded that the consumption of the PVA is not of safety concern (EFSA, 2005a). 9

10 In 1983, the Joint FAO/WHO Expert Committee on Food additives (JECFA) evaluated insoluble polyvinylpolypyrrolidone (PVPP) and established an ADI not specified for this substance (JECFA, 1983). JECFA evaluated polyvinylpyrrolidone (PVP) on several occasions, and in 1986 established an ADI of 0-50 mg/kg bw/day (JECFA, 1986). According to Annex I of Directive 67/548/EEC on Dangerous Substances, vinylacetate is classified as F; R11 and hydrazine as R10, Carc. Cat. 2; R45, T; R23/24/25, C; R34, R43, N; R ( IARC (International Agency for Research on Cancer) in 1995 and JSOH (the Japanese Society for Occupational Health) in 2003 evaluated vinyl acetate, which, according to the proposed specifications may be present at a maximum level of 5 mg/kg, and concluded that the substance is possibly carcinogenic to humans (Group 2B) (IARC, 1995; JSOH, 2003). IARC also evaluated hydrazine, which, according to the proposed specifications may be present at a maximum level of 1 mg/kg, and concluded that hydrazine is possibly carcinogenic to humans (Group 2B) (IARC, 1999). In the EU Risk Assessment Report on N-vinyl-2-pyrrolidone (EU RAR, 2003) it is stated that no concerns were identified for workers whose only form of exposure to N-VP was as a residue in N-VP based polymers owing to the very low level of N-VP to which these workers would be exposed Exposure PVP/VA copolymer is intended to be used as a binding/coating agent in an amount of up to 10% of weight per tablet. The petitioner provided information on the use of PVP in dietary supplements and the anticipated exposure. The petitioner stated that the average daily serving for dietary supplement products was recently reported to be two 600 mg supplement tablets per day, or a total of 1200 mg/day; however, no reference was provided for these figures. The petitioner also quoted another research survey, which found that in a diverse population an average of 5.5 dietary supplement tablets were consumed each day (Kemper et al., 2006). Therefore, the petitioner assumed a maximum daily dietary supplement intake of twice the average to cover high percentage of consumption, or 11 tablets of 600 mg or a total of 6600 mg/person/day. Based on this assumption and the intended use level of the food additive of 10% of the weight per tablet, the petitioner calculated the estimated maximum daily exposure from its use in dietary supplements for adults with a body weight (bw) of 60 kg to be 11 mg/kg bw/day PVP/VA copolymer. The Panel noted that the estimates provided by the petitioner for supplement consumption are higher than the estimates made on the assumptions used by the Panel in previous evaluations (EFSA, 2004; EFSA, 2005b), but the tablet weight used for the calculations by the petitioner was lower than those used in previous EFSA evaluations. Data from the UK Food Standards Agency on the consumption of food supplements indicate that the use among high consumers (97.5 th percentile) ranged from 2 tablets/capsules per day in children (4-18 year olds) (Gregory, 2000), to 7 tablets/capsules per day in adults (Henderson et al., 2002). Based on these data, a tablet weight of 1000 mg and using a coating level of 100 mg PVP/VA copolymer per tablet (10% on a 1000 mg tablet) for high consumers, the anticipated exposure estimate made by the Panel would be 200 mg PVP/VA copolymer/day for children, and 700 mg PVP/VA 5 Nomenclature according to 10

11 copolymer/day for adults, corresponding to 8 mg/kg bw/day for children (4-18 years, using 25 kg bw typical for a 4-year old child) and to 11.7 mg/kg bw/day for adults. The Panel assumed similar levels of use and intake of pharmaceutical products and food supplements per day (EFSA, 2005b). Given this assumption, the Panel estimated that the combined intake from food supplements and pharmaceutical products would be twice as high as the estimated intake for supplements only and would amount respectively for high consumers (97.5 th percentile) to 16 mg/kg bw/day for children and to 23.4 mg/kg bw/day for adults for a coating level of 100 mg PVP/VA copolymer/tablet, and to 4.8 mg/kg bw/day for child high consumers and 7.0 mg/kg bw/day for adults high consumers for a coating level of 30 mg PVP/VA copolymer/tablet. The estimated exposure data for a coating level of 100 mg PVP/VA per supplement are summarised in Table 2. Table 2: Estimated potential exposure to PVP/VA copolymer for a coating level of 100 mg PVP/VA per supplement based on assumptions of the Panel for high consumers Type of user Estimated daily number of tablets Estimated exposure (mg/kg bw/day) to PVP/VA copolymer based on 100 mg coating per supplement Supplements Pharmaceuticals Total combined exposure Adult Children 4 18 years Based on the measurement of the residual levels of 5 mg/kg each for both VA and vinyl pyrrolidone (VP), the petitioner calculated the estimated maximum daily exposure for these residuals (based on the same assumptions as for the exposure calculations for the PVP/VA copolymer) for adults with body weights of 60 kg to be 0.06 µg/kg bw/day. The petitioner did not provide an exposure estimate for children. Based on the total anticipated combined exposure calculations made by the Panel (Table 2) for the highest exposure levels, the calculated estimated potential exposure to those residuals (VA and VP separately) in the product for adults would be 0.12 µg/kg bw/day each, whereas the estimated potential exposure for children would be 0.08 µg/kg bw/day. Based on the combined exposure calculations made by the Panel for the highest exposure levels, the calculated estimated potential exposure to hydrazine residuals (maximum 1 μg/kg) in the product for adults would be μg/kg bw/day, whereas the estimated potential exposure for children would be μg/kg bw/day. 3. Biological and toxicological data 3.1. Absorption, distribution, metabolism and excretion No absorption, distribution, metabolism and excretion (ADME) studies were provided by the petitioner. The petitioner only referred to papers by the International Specialty Products (ISP, 2005) and by the US Environmental Protection Agency (US EPA, 2006) where it is considered that PVP/VA having a weight average molecular weight of about to g/mol, can be expected to be poorly absorbed from the gastrointestinal tract. This assessment is consistent with unpublished data cited by Mellert et al. (2004), that only approximately 2% of an orally administered dose of PVP/VA is absorbed and the copolymer is mainly excreted intact in the faeces. 11

12 3.2. Toxicological data The safety of polyvinylpyrrolidone-vinyl acetate copolymer used as a food additive Acute oral toxicity In five studies, PVP/VA copolymer was administered by gavage at doses of either 0.63 or 2.39 g PVP- VA/kg bw, to albino rats (10 and 6 animals/group respectively) (gender not specified). At the high dose level, piloerection was observed in some of the 6 rats, none of the animals died, and there were no remarkable observations at necropsy. At the 0.63 g/kg bw dose level, and during the 14 days following dosing the animals showed decreased activity and ataxia for an unspecified length of time, but none died. It was concluded that the solution is practically non-toxic (CIR, 1983) Short-term and subchronic toxicity PVP/VA copolymer was administered in the diet of 3 groups of male and female Sprague-Dawley rats for 28 days (5 animals/sex/group; control group 5 animals/sex) and for 90 days (10 animals/sex/group; control group 10 animals/sex), respectively. The control groups received a basal diet on a comparable regimen. The studies were conducted according to GLP and to the OECD Testing Guidelines 407 or 408 (OECD, 1995 and 1998). In both studies PVP/VA copolymer was administered at doses of 0 (control), 100, 300, and 1000 mg/kg bw/day. All animals survived to scheduled necropsy. In the 28 days study there were no clinical signs of toxicity, and there were no effects on body weight gain, feed consumption, haematology parameters, serum chemistry and urinalyses. There were no test-articlerelated effects on organ weights, macroscopic and microscopic evaluations. The authors concluded that the No-Observed-Adverse-Effect Level (NOAEL) was 1000 mg/kg bw/day PVP/VA, the highest dose level tested (ISP, 2005). The Panel agreed with this NOAEL. In the 90-day study there were no clinical signs of toxicity, and there were no effects on body weight gain, feed consumption, functional observational battery and locomotor activity evaluations. There were no ophthalmic lesions indicative of toxicity, and no test-substance-related effects on haematology parameters, serum chemistries and urinalyses. There were no test-article-related effects on organ weights, macroscopic and microscopic evaluations. The authors concluded that the NOAEL was 1000 mg/kg bw/day, the highest dose level tested (ISP, 2006). The Panel agreed with this NOAEL Genotoxicity Genotoxic potential of PVP/VA The petitioner stated that no data on the genotoxicity of the PVP/VA copolymer were available and provided some data on the genotoxicity of the monomers (VP and VA) from which the PVP/VA copolymer is derived. The Panel noted that the safety of VP residues in certain food additives has been evaluated by the former European Commission Scientific Committee on Food (SCF) and that the mutagenicity of both monomers has already been evaluated in the course of the European chemicals evaluation. The results of these evaluations are described below Genotoxic potential of vinylpyrrolidone (VP) VP residues in polyvinylpyrrolidone and polyvinylpolypyrrolidone when used as food additives have been evaluated by the SCF (SCF 2002). The Committee concluded that the intakes of VP from food additive uses of PVP and PVPP do not give cause for concern. The genotoxicity of VP was described by the SCF as follows: 12

13 The genotoxicity of NVP was examined in several in vitro studies in bacterial systems. In these tests NVP was found to be not mutagenic (HRC, 1978; BASF, 1978b; Simmon and Baden, 1980; Knaap et al., 1985). Various in vitro tests in cultured mammalian cells, including the use of closed systems, also indicated the absence of any mutagenic potential. These comprised assays for chromosomal aberrations in human lymphocytes (BASF, 1987), assays for gene mutations at the HPRT and TK locus in mouse lymphoma L5178Y cells (Litton Bionetics, 1980a; Knaap et al., 1985), an assay for unscheduled DNA synthesis in rat hepatocytes (Litton Bionetics, 1980b), assays for the induction of sister chromatid exchanges in cultured lymphocytes (Norppa & Tursi, 1984) and for cell transformation in BALB/3T3 cells (Litton Bionetics, 1980c). In vivo tests with negative outcome included a sex-linked recessive lethal test in Drosophila melanogaster (Knaap et al., 1985) and a micronucleus test in NMRI mice (BASF, 1993b). NVP was thus found to be non-genotoxic. In addition, there is an EU risk assessment report on VP (EU RAR, 2003) in which the evaluation of mutagenicity has been summarized as follows: N-VP has yielded consistently negative results in genotoxicity tests in a wide variety of in vitro systems, and one well conducted in vivo test. On this basis, it can be concluded that N-VP is not a genotoxicant Genotoxic potential of vinylacetate (VA) There is an EU Risk Assessment Report on VA (EU RAR, 2008) in which the evaluation of mutagenicity is described as follows: Vinyl acetate is not mutagenic to bacteria, but it induces chromosomal aberrations, gene mutations and SCE in several tests with mammalian cells from different sources in culture. Furthermore, at high concentrations the formation of DNA-protein-cross links and DNA-DNA-cross links is shown with mammalian cells. The in vivo genotoxicity of vinyl acetate appears to be limited to toxic doses thus it may be reasonable to assume a threshold mechanism of action for germ cell mutagenicity. Taking into account the exposure estimates resulting for a point source from a worst case calculation and from regional background concentrations it may be concluded that there is presently no concern regarding in vivo mutagenicity. Based on these evaluations, the Panel considered that there is no concern with respect to genotoxicity of the monomers VP and VA from which the PVP/VA copolymer is derived for PVP/VA copolymer itself Genotoxic potential of hydrazine The International Agency for Research on Cancer (IARC) published a review on the genotoxic potential of hydrazine (IARC, 1999). The results of the IARC analysis is cited below. Humans studies No data were available. Studies in experimental systems Hydrazine was mutagenic to yeast and bacteria and induced DNA damage in bacteria. Hydrazine induced somatic mutations in Drosophila. It induced DNA strand breaks in rat hepatocytes and unscheduled DNA synthesis in mouse hepatocytes in vitro. Conflicting results were obtained for induction of gene mutations in mouse lymphoma L5178Y cells. It did not induce chromosomal aberrations in rat liver cell line in vitro but did induce sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells. In single studies in vivo in mice, hydrazine induced DNA strand breaks in liver and lungs. It did not induce sister chromatid 13

14 exchanges in bone marrow or liver of mouse treated in vivo in one study. It weakly induced micronuclei in bone-marrow cells of mice treated in vivo in one of three studies. Hydrazine induced the formation of DNA adducts in vitro and of N 7 -methylguanine and O 6 -methylguanine in liver of mice, rats and hamsters treated in vivo. In in vivo studies with mice, hydrazine did not induce dominant lethal mutations in a single study or sperm abnormalities in two studies. In the experiment described in Section 3.1.3, in which Syrian hamsters were given average doses of 4.6, 8.3 and 10 mg/kg bw hydrazine free base as sulphate salt in the drinking-water for two years, the levels of methylation of DNA guanine in liver, kidney and lung were measured. Both N 7 - and O 6 -methylguanine were readily detectable at six months of exposure, but only trace amounts of these bases were detected after 12 months of exposure; these bases were again detected in liver DNA at exposure times of 18 and 24 months (Bosan et al., 1987). Rats exposed to single doses (gavage) of hydrazine in the range of mg/kg bw showed an increase in N 7 - and O 6 -methylguanine above background levels in liver DNA only after doses greater than mg/kg bw, doses which were still non-toxic or only weakly toxic (van Delft et al., 1997). Since the IARC monograph was published further data based on new methodologies became available. These data are summarised below. Comet assay Sasaki et al. (1998) used the Comet assay to test the in vivo genotoxicity of hydrazine in mouse liver, lung, kidney, brain, and bone marrow, and also in the mucosa of stomach, colon, and bladder. Mice were sacrificed 3 and 24 hours after intra-peritoneal (i.p.) and oral (p.o.) administration. Hydrazine at 100 mg/kg yielded DNA damage in the stomach, liver, and lung when given i.p. and in the brain after oral administration. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured in vitro by Robbiano et al. (2006) using the single-cell gel electrophoresis (Comet) assay, were confirmed in primary lung cells from male rats with hydrazine (0.5-4 mm). Similar degrees of DNA fragmentation were obtained in vitro in primary human lung cells. The DNA-damaging potency of hydrazine was higher in lung cells in rats than in humans. In agreement with these findings, statistically significant increases in the average frequency of DNA breaks was obtained in the lung of rats given a single oral dose (1/2 LD50) of hydrazine. According to Robbiano et al. (2006), these findings give evidence that hydrazine is a genotoxic lung carcinogen that produces DNA-damaging effects in primary cultures of lung cells from human donors substantially similar to those observed in rats. Transgenic mice Transgenic mice from strain 40.6 (Mutamouse) were administered single oral doses of hydrazine sulphate up to a toxic concentration (400 mg/kg bw) (Douglas et al., 1995). None of the doses induced lacz mutations in lung, liver or bone marrow. Since the highest single dose used was higher than the cumulative dose that induced tumours in carcinogenicity studies, the authors concluded that it may be that either hydrazine sulphate is genotoxic in target tissues in vivo only when given in multiple doses or that it is a non-mutagenic carcinogen. Cell transformation assay An in vitro test was used by Kowalski et al. (2000) in order to predict rodent carcinogenicity of chemicals. The assay uses focus formation in a stable, bovine papilloma virus type 1 (BPV-1) DNA carrying C3H/10T1 2 mouse embryo fibroblast cell line (T1) that does not require transfection, infection with virus, isolation of primary cells from animals, or addition of a microsomal fraction. Of a total database of 64 compounds, 92% of the carcinogens, promoters, or non-carcinogens were correctly predicted. Hydrazine was found positive in a range of concentrations from 0.01 to 5 mg/ml. 14

15 From the above studies it can be concluded that hydrazine induces gene mutations in bacteria, yeast and Drosophila and in-vivo treatment of mice, rats and Syrian hamsters results in the formation of N7- methylguanine and O 6 -methylguanine in liver DNA. Hydrazine increases the frequency of DNA single-strand breaks and alkali-labile sites in several organs including the lung in mice and rats that is one of the carcinogenicity target organs in both species. It induces no gene mutations in an in vivo mutamouse strain after a single treatment at high dose level. Hydrazine induces cell transformation in a C3H/10T1 2 mouse embryo fibroblast cell line Chronic toxicity and carcinogenicity PVP/VA copolymer PVP/VA copolymer (CAS No ) was administered in the diet for 24 months to male and female Wistar rats (50 animals/sex/dose; 10 animals/sex/dose as satellite group for haematological evaluation) at doses of 0, 700, 1400, and 2800 mg/kg bw/day (Mellert et al., 2004). The study was performed following OECD Testing Guideline Nos. 451 (OECD, 1981a). The study was conducted in compliance with GLP. In a parallel study, PVP/VA copolymer was also administered in the diet to male and female pure-bred Beagle dogs over a period of 52 weeks. The study was performed following OECD Testing Guideline No. 452 (OECD, 1981b) and was also conducted in compliance with GLP. The dogs were allocated to 4 treatment groups: Group I: 0 mg/kg bw/day (control) (6 animals/sex); Group II: 500 mg/kg bw/day (4 animals/sex); Group III: 1500 mg/kg bw/day (4 animals/sex); and Group IV: 2500 mg/kg bw/day (6 animals/sex). Additionally, at the end of the 52-week treatment period, two males and females each, from Groups I and IV (control and high-dose) were maintained treatment-free for an additional 6-week recovery period. Clinical signs, body weight, food consumption, haematology, and gross and histopathological evaluations were conducted on both rats and dogs; dogs also underwent hearing tests, ophthalmoscopic examinations, electrocardiograms, blood pressure measurements, and clinical chemistry and urinalysis evaluations. No adverse in-life observations related to treatment were observed in either species. The rats exhibited dark discoloration of the faeces that was attributed to the intake and excretion of large amounts of PVP/VA copolymer and this was not considered to be an adverse effect by the authors. No treatment-related haematological changes or gross or histopathological lesions were observed in either species that could be considered clinically relevant. The authors stated that vacuolated histiocytosis in the mesenteric lymph nodes of 4 dogs that was not accompanied by inflammation or degenerative changes reflected histiocytic removal and degradation of the test article rather than a toxic effect. The authors concluded that the results of the studies demonstrated the absence of any significant toxicological findings in rats and dogs resulting from high dietary levels of PVP/VA copolymer, including the absence of any treatment-related increase in the incidence of neoplasms in the 24 month rat study. The authors estimated the NOAEL to be 2800 mg PVP/VA/kg bw/day in the rat and 2500 mg PVP/VA/kg bw/day in the dog, the highest dose levels tested. The Panel agreed with these NOAELs. The petitioner also refers to 1-year drinking water studies in mice and rats as summarised in a review paper (CIR, 1983). It is stated that the animals were given daily an aqueous solution of 10.2 mg PVP/VA/l (equivalent to about 2.7 mg PVP/VA/kg bw/day for the mice, and to about 1.6 mg PVP/VA/kg bw/day for the rats) for a period of one year. There were no changes attributable to the copolymer in either mice or rats, including no histological changes in the internal organs. The petitioner also provided data from a 2-year feeding study in the rat with PVP/VA copolymer (CAS No ) consisting of a copolymer of 60% VP and 40% VA. The study was conducted 15

16 using 51 male and 51 female Sprague-Dawley rats in both the test and control groups. Test animals were given feed containing 5% test substance, and control animals were given feed containing 5% cellulose. Based on the consumption of the entire diet, test animals were fed about 0.67 g of the test substance per day (equivalent to about 450 mg PVP/VA/kg bw/day) for 24 months. Haemoglobin content and leukocyte count were determined in 5 rats of each sex in test and control groups after 24, 100, 175, 245, and 364 days of the study. Haematology, blood chemistries and urinalysis were determined after about 500 days of the study in 10 test and control rats of each sex, as well as in all test (20) and control (11) rats still alive after 675 days. It was noted however, that the survival in both the control and test groups was poor (8-14%), reportedly due to inflammatory diseases of the respiratory tract and from otitis. The test and control diets were tolerated without signs of toxicity. No treatment-related clinical chemistry changes were observed. Liver and kidneys as well as all pathological organ changes were examined histopathologically. No treatment related tumours or other gross pathologically detectable lesions were induced. Histopathological examination showed an increased incidence of liver congestion and fatty degeneration in the test group compared with the cellulose control group. There were no other histopathologically detectable organ changes observed (Oettel and Frohberg, 1965) VA In literature, studies on chronic toxicity and carcinogenicity are available regarding VA administrated via the drinking water to rats and mice at dose levels up to 5000 mg/l (equivalent to 1360 mg VA/kg bw/day for the mouse and 760 mg VA/kg bw/day for the rat) (Lijinsky and Reuber, 1983; Maltoni et al., 1997; Minardi et al., 2002). All these studies lead to the general conclusion that VA should be considered as a multipotential carcinogen. In a study by Umeda et al. (2004), the carcinogenicity and chronic toxicity of VA were examined in male and female Crj:BDF 1 mice and F344/DuCrj rats. Groups of 50 mice and 50 rats of each sex were orally administered VA in drinking water containing 0, 400, 2000 or mg/l over 104 weeks. The authors of this study reported that the corresponding average VA intakes (based on the recorded water consumption) were 0, 21, 98, 442 mg/kg bw/day for male rats and 0, 31, 146, 575 mg/kg bw/day for female rats. In the mouse, the corresponding intakes were 0, 42, 202, 989 mg/kg bw/day for male mice and 0, 63, 301, 1418 mg/kg bw/day for female mice. Squamous cell tumours were clearly evident in the upper digestive tract of treated mice and rats, and in the larynx of treated mice of both sexes. In mice, squamous cell carcinomas and papillomas were observed in the oral cavity, oesophagus, forestomach and larynx of the mg/l group, together with basal cell hyperplasia, squamous cell hyperplasia and epithelial dysplasia. In rats, incidences of squamous cell carcinomas and papillomas were increased in the oral cavity of the mg/l group of both sexes, and an oesophagus squamous cell carcinoma was observed in a mg/l female. Pre-neoplastic hyperplasias were also noted. The neoplastic and pre-neoplastic lesions in the oral cavity of the mg/l groups revealed that both lesions occurred predominantly in the mandible area of the oral cavity of the animals. A lower confidence limit of a benchmark dose (BMDL 10 ) of 477 mg/kg bw/day was obtained by the authors from a dose-response relationship between combined incidence of squamous cell carcinomas and papillomas in the oral cavity of mice and rats and the estimated daily VA intakes per body weight. Table 3 presents the data on the combined incidence of squamous cell carcinomas and papillomas in mice and rats induced by VA in the oral cavity as reported by Umeda et al. (2004). Table 4 presents the results of a BMD analysis performed by the Panel using these data. Table 3: Incidence of squamous cell carcinomas and papillomas in mice and rats induced by VA in the oral cavity as reported by Umeda et al. (2004). Dose Species Gender Dose No Combined incidence of 16