Scientific Opinion on the safety of neutral methacrylate copolymer for the proposed uses as a food additive 1

Transcription

1 SCIENTIFIC OPINION Scientific Opinion on the safety of neutral methacrylate 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 neutral methacrylate copolymer (NMC, a 30% dispersion of the dry copolymer in water) as a glazing agent in solid food supplements and solid foods for special medical purposes (FSMPs). The dispersion contains 0.7% of polyethylene glycol monostearyl ether, which is not a authorised food additive; the opinion does not include a safety evaluation of this substance. From studies on metabolism/toxicokinetics, acute and subchronic oral toxicity, genotoxicity, and developmental toxicity it is concluded that NMC is essentially not absorbed and that any absorbed material is not retained in the tissues. No data on reproductive toxicity, chronic toxicity and carcinogenicity are provided. In the absence of such data, chronic effects on the gastrointestinal tract following oral administration cannot be excluded. Therefore, the Panel considers that an ADI should not be established and that a margin of safety (MOS) approach is appropriate. Data from in vitro Ames and mammalian cell mutation assays and an in vivo micronucleus assay show that NMC does not raise concern with respect to genotoxicity. The calculated anticipated exposure (coating level 200 mg/tablet) to NMC from both its proposed use in food supplements and its approved use in pharmaceuticals is 46.7 mg/kg bw/day for high consumer adults, and 32 mg/kg bw/day for children. Two subchronic toxicity studies in rats and a developmental study in rabbits revealed NOAELs of 2000 mg/kg/bw/day (highest dose level tested) resulting in a MOS of at least 43 for adults and at least 63 for children. The Panel considers these margins of safety sufficient given the lack of absorption and that the exposure estimates are based on worst case assumptions. The Panel concludes that the use of NMC in solid food supplements at the proposed use and use levels is not of safety concern. The Panel could not assess the safety of neutral methacrylate copolymer for uses in solid foods for special medical purposes. KEY WORDS Neutral methacrylate copolymer; poly(ethyl acrylate-co-methyl methacrylate) 2:1; methyl methacrylate (CAS-No: ); ethyl acrylate (CAS-No: ); ethyl acrylate methyl methacrylate polymer; ethyl acrylate, methyl 1 On request from the European Commission, Question No EFSA-Q , adopted on 24 June 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: uni-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 preparation of this 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. EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS); Scientific Opinion on the safety of neutral methacrylate copolymer for the proposed uses as a food additive. 24 June [24 pp.]. doi: /j.efsa Available online: European Food Safety Authority,

2 methacrylate polymer; ethyl acrylate, polymer with methyl methacrylate; methyl methacrylate, ethyl acrylate polymer; methyl methacrylate, polymer with ethyl acrylate. 2

3 SUMMARY Following a request from the European Commission to the European Food Safety Authority (EFSA), the Scientific Panel on Additives and Nutrient Sources added to Food (ANS) was asked to provide a scientific opinion on the use of neutral methacrylate copolymer as a glazing agent for sustained release formulations of solid food supplements as defined in the European Directive 2002/46/EC and in solid foods for special medical purposes as defined in the European Directive 1999/21/EC. Neutral methacrylate copolymer is a fully polymerised copolymer of methyl methacrylate and ethyl acrylate. The commercial form is a 30% dispersion of the dry substance in water together with 0.7% of the emulsifier polyethylene glycol monostearyl ether. The Panel noted that polyethylene glycol monostearyl ether is not an authorised food additive; the present opinion does not include a safety evaluation of this substance. The Panel considered the identity of the test substances used in the toxicological studies submitted by the petitioner as compared to the commercial substance. From the information provided by the petitioner it is deduced that the proportion of monomers in the copolymer are either identical or very similar to the substances used in the ADME study and toxicity studies. The petitioner provided toxicokinetics, acute and subchronic oral toxicity, genotoxicity, and reproductive toxicity studies on neutral methacrylate copolymer. From the data using radiolabelled neutral methacrylate copolymer it can be concluded that the polymer is essentially not absorbed and that the very low amounts of absorbed radioactivity are not retained in the tissues. From the in vitro Ames and mammalian cell mutation assays and the micronucleus assay in vivo, the Panel considers that neutral methacrylate copolymer does not raise concern with respect to genotoxicity. From a 35 day oral feeding study in the rat a NOAEL of 2000 mg neutral methacrylate copolymer/kg bw/day was derived, the highest dose tested. From a 28 day oral (gavage) study with minipigs a NOAEL of 454 mg/kg bw/day was derived. In a 26-week feeding study with rats no treatment related effects were noted on body weight, body weight gain, and food or water consumption. No clinical signs were apparent to indicate a treatment related effect. Haematology and clinical chemistry parameters were not remarkable. No macroscopic or microscopic abnormalities were noted that were deemed to be a result of treatment. From the study a NOAEL of 2000 mg neutral methacrylate copolymer /kg bw/day was derived, the highest dose tested. In a 26-week feeding study with dogs no toxicologically relevant changes in water consumption, clinical observations (reaction to treatment and clinical signs) were reported. There were also no changes noted in clinical pathology investigations or organ weights. Histopathological examination did not reveal any evident differences in the incidence of findings observed in treated and control animals which could be considered treatment-related. From this study a NOAEL of 250 mg neutral methacrylate copolymer/kg bw/day was derived. From developmental toxicity studies in rats and rabbits no evidence of an effect of treatment on maternal animals was found. Fetal survival and fetal and placental weights were unaffected by the treatment and no malformations were seen. From both studies a NOAEL for both dams and fetuses of 2000 mg/kg bw/day was derived. 3

4 Based on data from the UK Food Standard Agency on the consumption of food supplements the Panel calculated an anticipated exposure (assuming coating levels of respectively 200 mg/tablet as proposed by the petitioner) to neutral methacrylate copolymer from both its proposed use in food supplements and from its approved use in pharmaceuticals to be equal to 46.7 mg neutral methacrylate copolymer /kg bw/day for high adult consumers, and 32 mg/kg bw/day for children (4-17 years; 25 kg bw). Using the worst case scenario, the calculated total exposure to the residual monomers (MMA and EA) present in the substance would be 0.17 µg kg bw/day MMA and 2.9 µg kg bw/day EA for adults and 1.2 µg/kg bw/day MMA and 2.0 µg kg bw/day EA for children. The SCF allocated a group TDI of 0.1 mg/kg bw/day for these monomers. Considering the toxicological profile of these compounds, the Panel considers the calculated total exposure as insignificant. The NOAELs derived from the studies available are: 2000 mg/kg bw/day from a 35 day oral feeding study in the rat, 2000 mg/kg bw/day from a 26-week feeding study in the rat, 2000 mg/kg bw/day from a developmental study in rabbits, 454 mg/kg bw/day from a 28-day (gavage) study in minipigs and 250 mg/kg bw/day from a 26-week feeding study in the dog (including a 3 week recovery period. The NOAELs in the studies are all the highest doses tested. Using this range of NOAELs and the estimated total intakes from both the proposed use in food supplements and from the approved use in pharmaceuticals, of 46.7 mg neutral methacrylate copolymer /kg bw/day for high adult consumers and 32 mg/kg bw/day for children, the Panel calculates a MOS varying from at least 5 to 43 for adults and 8 to 63 for children. The Panel considers these margins of safety sufficient taking into account that the NOAEL values were all the highest dose levels tested in the respective studies, that the two sub chronic toxicity studies in rats and a developmental study in rabbits all reveal NOAELs of 2000 mg/kg/bw/day and given the lack of absorption of neutral methacrylate copolymer and the fact that the estimated intakes were all based on worst case scenarios. The Panel noted that the petitioner did not provide use levels for neutral methacrylate copolymer for uses in solid foods for special medical purposes (FSMPs). Therefore, the safety of this usage could not be evaluated. The Panel concludes that in the absence of data on reproductive toxicity, chronic toxicity and carcinogenicity, chronic effects in local tissues cannot be excluded. Therefore, the Panel considers that an ADI should not be established and that a margin of safety (MOS) approach is appropriate. The Panel concludes that the use of neutral methacrylate copolymer in solid food supplements as glazing agent/coating agent for sustained release is not of safety concern at the proposed uses and use levels. The Panel could not assess the safety of neutral methacrylate copolymer for uses in solid foods for special medical purposes. 4

5 TABLE OF CONTENTS Abstract... 1 Summary... 3 Table of Contents... 5 Background as provided by the European Commission... 6 Terms of reference as provided by the European Commission... 6 Assessment Identity of substance Specifications Manufacturing process Methods of analysis in food Reaction and fate in food Case of need and proposed uses Technological need and intended use Proposed use levels Information on existing authorisations and evaluations Exposure Absorption, distribution, metabolism and excretion (ADME) Toxicological data Acute toxicity Short-term toxicity and subchronic toxicity Short-term toxicity Subchronic toxicity Genotoxicity studies Chronic toxicity and carcinogenicity Reproductive and developmental toxicity Human studies Conclusions Documentation provided to EFSA References Glossary / Abbreviations

6 BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION Glazing agents are a functional class of food additives which are regulated under Directive 95/2/EC 4 of the European Parliament and of the Council on food additives other than colours and sweeteners. A manufacturer has requested the authorisation of neutral methacrylate copolymer under Directive 95/2/EC. According to the applicant the substance is a fully polymerised neutral copolymer based on methyl methacrylate and ethyl acrylate. Neutral methacrylate copolymer is proposed to be used as a glazing/coating agent in solid food supplements of sustained release formulations as defined by European Parliament and Council Directive 2002/46/EC 5 and in solid foods for special medical purposes as defined by Commission Directive 1999/21/EC 6. The substance is proposed to be used to permit a sustained-release that appears to contribute to successful supplementation therapy. Additionally to this, fewer polymer is needed for sustained release formulation as compared with authorised glazing agents already available on the market. The company states that the substance has been used for similar purposes in pharmaceutical products for decades. TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION In accordance with Article 29 (1) (a) of Regulation (EC) No 178/2002, the European Commission asks the European Food Safety Authority to provide a scientific opinion on the safety of neutral methacrylate copolymer as a glazing agent/coating agent for solid food supplements and in solid foods for special medical purposes. 4 Directive 95/2/EC of the European Parliament and of the Council of 20 February 1995on food additives other than colours and sweeteners. OJ L 61, , p.1. 5 Directive 2002/46/EC of the European Parliament and of the Council of 10 June 2002 on the approximation of the laws of the Member States relating to food supplements. OL L 183, , p Commission Directive 1999/21/EC of 25 March 1999 on dietary foods for special medical purposes. OJ L 91/29,

7 ASSESSMENT 1. Introduction The present opinion deals with the safety of neutral methacrylate copolymer intended to be used as glazing agent/coating agent for sustained release of nutrients in solid food supplements and solid foods for special medical purposes (FSMPs). 2. Technical data 2.1. Identity of substance Neutral methacrylate copolymer is a fully polymerized copolymer of methyl methacrylate (CAS Registry Number: ) and ethyl acrylate (CAS Registry Number: ), the monomers being randomly distributed. The commercial form is a 30% dispersion of the dry substance in water together with 0.7% of the emulsifier polyethylene glycol monostearyl ether [macrogol stearyl ether 20 (CAS Registry Number: )]. The Panel noted that polyethylene glycol monostearyl ether is not an authorised food additive. The International Union of Pure and Applied Chemistry (IUPAC) designation is as follows: Poly(ethyl acrylate-co-methyl methacrylate) 2:1. The ratio indicates the molar proportion of the monomer units. The CAS Registry Number is The petitioner states that based on the analysis by gel permeation chromatography (GPC) of 10 lots of neutral methacrylate copolymer manufactured in 2007/2008, the molecular weight was determined to be as follows: weight average molecular weight (Mw), approximately 600x10 3 g/mol; number average molecular weight (Mn), approximately 220x10 3 g/mol. Molecular formula The representative structural formula of neutral methacrylate copolymer as given by the petitioner is shown in Figure 1. Figure 1: The representative structural formula of neutral methacrylate copolymer 7

8 Neutral methacrylate copolymer is synthesised from defined amounts of two synthetic monomers [ethyl acrylate (EA) and methyl methacrylate (MMA)] in the presence of methacrylic acid (MAA) as stabiliser. The molar ratio of these monomers is 69.2:29.1:1.2. Synonyms: Ethyl acrylate methyl methacrylate polymer; Ethyl acrylate, methyl methacrylate polymer; Ethyl acrylate, polymer with methyl methacrylate; Methyl methacrylate, ethyl acrylate polymer; Methyl methacrylate, polymer with ethyl acrylate (Internet ChemIDplus Advanced). Physical state Neutral methacrylate copolymer is described as a milky-white dispersion of low viscosity with a faint characteristic odour. The odour is nonexistent on the final product (polymer film on tablets or granules). Solubility Neutral methacrylate copolymer dispersion is miscible with water in any proportion, the milky-white appearance being retained. When one part of neutral methacrylate copolymer is mixed with 5 parts acetone, a clear to slightly cloudy, viscous dispersion is obtained. The same occurs when mixed with ethanol or isopropyl alcohol; initially the polymer is precipitated, but then dissolves again in the excess organic solvent. When mixed with 1 N sodium hydroxide in a ratio of 1:2, the dispersion does not dissolve. The milkywhite appearance is retained Specifications The specifications for neutral methacrylate copolymer (30% dispersion) as proposed by the petitioner are given in Table 1. Table 1: Specifications of neutral methacrylate copolymer (30% dispersion) as proposed by the petitioner Characteristics Proposed specifications % (Ph Eur. 6.0, Volume 3, method ) Assay/Residue on evaporation 1 g dispersion is dried in an oven for 3 hours at 110 C The dispersion must form a clear film after drying. Viscosity Max. 50 mpa.s (Brookfield viscosimetry) ph-value (Ph Eur. 6.0, Volume 2, method ) Relative density(at 20 C) (Ph Eur. 6.0, Volume 2, method ) Residual monomers Total of monomers (sum of methyl methacrylate and ethyl acrylate): < 100 mg/kg (Ph. Eur. 6.0, Volume 3, method ) Residual solvents Ethanol Methanol Residual emulsifier polyethylene glycol monostearyl ether (macrogol stearyl ether 20) Heavy metals < 0.5% < 0.1% 0.7% Arsenic: < 2 mg/kg; lead: < 2 mg/kg; mercury: < 2 mg/kg; zinc: < 10 mg/kg; copper: < 10 mg/kg (Ph. Eur method C) 8

9 Sulphated ash Max. 0.4% (Ph Eur. 6.0 method ) Microbial count Total aerobic count Max cfu/g (Ph Eur. 6.0, Volume 3, method ) Total combined yeasts and moulds Max. 100 cfu/g (Ph Eur. 6.0, Volume 3, method ) 2.3. Manufacturing process Neutral methacrylate copolymer is produced using a process of emulsion polymerisation. The principles of emulsion polymerisation have been described by Lehmann et al. (1973), Cowie (1976), Houben-Weyl (1987) and Elias (1990). The process used by the petitioner is described in detail and can be summarised as follows. Neutral methacrylate copolymer is manufactured by redox initiated polymerisation of the monomers ethyl acrylate (EA), methyl methacrylate (MMA) by using a free radical donor redox initiator system stabilised with polyethylene glycol monostearyl ether and vinylic acid/sodium hydroxide. An alkyl mercaptane is used as a chain modifying agent. At the end of the polymerisation an emulsifier is added, to reduce foaming, and the residual monomers and excess water are removed by distillation. The dispersion is cooled to room temperature and the ph of the reaction mixture is adjusted. Sodium hypochlorite is added to prevent microbial contamination during the filtration and filling process. The petitioner states that polyethylene glycol monostearyl ether is used as an emulsifier in the process. The emulsifier is not removed after manufacturing. It is present at a concentration of 0.7% in the aqueous dispersion containing 30% copolymer (commercial form). The petitioner further indicates that since the aqueous dispersion concentrates during processing of the copolymer in its application as a coating material, residual levels polyethylene glycol monostearyl ether in the product will be 2.4% (24000 ppm) Methods of analysis in food Neutral methacrylate copolymer is a coating for solid dosage forms such as tablets and fine particles. For the quantitative analysis the dosage forms are extracted with tetrahydrofuran. The insoluble substances are isolated by centrifugation and are further extracted with tetrahydrofuran. The clear filtrate is concentrated by boiling and further drying is performed by oven drying. The residue is identified by NMR spectroscopy Reaction and fate in food The petitioner states that due to the polyethylene carbon backbone, degradation of the polymer chains, e.g. by hydrolysis, can be excluded and has never been observed under normal storage and handling conditions. Storage stability of the substance has been studied by the petitioner. From the data obtained it can be concluded that the substance is stable for at least 24 months at temperatures between 8 C and 25 C in well closed containers. As regards the stability during processing, the petitioner states that when using the conventional methods for coating solid dosage forms (pan-coating/fluid-bed coating) the polymer shows high thermal stability (up to 300 C). Thermogravimetric analysis shows that depolymerisation only occurs at temperatures above 300 C. 9

10 The petitioner further states that possible interactions between nutrients and the polymer can be excluded, because neutral methacrylate polymer does not contain reactive functional groups Case of need and proposed uses Technological need and intended use Neutral methacrylate copolymer is intended to be used as a sustained-release glazing agent/coating agent in solid food supplements. Sustained-release formulations allows the continuous dissolution of a nutrient over a defined time. Neutral methacrylate copolymer is insoluble in aqueous media over the complete ph range. The sustainedrelease functionality is obtained via the permeability of the coating for the nutrients in aqueous media. The addition of organic solvents is not necessary. The petitioner indicates that coating is done at temperatures in the range of 25 C - 35 C Proposed use levels The petitioner indicates that the amount of neutral methacrylate copolymer as glazing agent can be adjusted to the formulation to achieve sustained-release functionality. The sustained-release functionality can be achieved with a polymer amount of 10%. By increasing the polymer amount of the coating, the release profile can be modified to provide lower or higher release rates. It is indicated that the conventional polymer amount for a sustained-release profile is in the range of 10% to 20% ( mg/cm²), equivalent to approximately mg/tablet (for a tablet weight of 1000 mg). No uses and use levels were provided by the petitioner for solid foods for special medical purposes (FSMPs) as defined in the European Directive 1999/21/EC Information on existing authorisations and evaluations No specific information on existing authorisations and evaluations is provided. As regards Europe, the petitioner indicates that acrylic polymers, including neutral methacrylate copolymer (30% dispersion in water), are used as excipients in a large number of pharmaceutical preparations registered with the national authorities within the EU (Ph. Eur., 2009). The petitioner further indicates that pharmaceutical preparations containing neutral methacrylate copolymer are used worldwide and are listed in the German Rote Liste ( in the French Vidal ( and the British Electronic Medicines Compendium ( As regards the USA neutral methacrylate copolymer is also listed in the US FDA s Inactive Ingredients Guide ( In addition, the US Physician's Desk Reference lists preparations containing neutral methacrylate copolymer. As regards Japan, the whole range of acrylic polymers including neutral methacrylate copolymer is used on a large scale in pharmaceutical specialties registered with Japan's Ministry of Health, Labour and Welfare (MHLW) ( 10

11 The Scientific Committee on Food (SCF) did evaluate methyl methacrylate (MMA, Ref. No 21130) for which a group Tolerable Daily Intake (TDI) of 0.1 mg/kg bw/day (either as methacrylic acid or acrylic acid) was allocated (SCF, 1999). Methyl methacrylate is listed in Directive 2002/72/EC 7 and its amendments, for use in food contact materials Exposure Neutral methacrylate copolymer is intended to be used as a coating agent in an amount of up to 10% to 20% of weight per tablet. Due to the greater ratio of surface area to volume, smaller tablets will contain relatively more substance than the larger tablets. For estimating the exposure to neutral methacrylate copolymer, an absolute weight per tablet of 1000 mg will be used as it represents the bulk of supplements and pharmaceutical tablets. The Dutch National Food Consumption Survey (DNFCS) and several monitoring and cohort surveys performed after 1998 report that about half of the supplement users limit their intake to one supplement per day. About one third of users take two specific supplements/day, about 10% take three specific supplements/day and about 6% take four or more specific supplements/day. A maximum of 6% of users take eight tablets via four different supplements twice a day (Ocké et al., 2005). Based on this data, the petitioner provided an exposure assessment for (200 mg/tablet) using 2 supplements and 3 pharmaceutical pills per day for average user adults (3 supplements/day for heavy users) and 1 supplement and 1 pharmaceutical pill per day for children above 6 years. The total anticipated exposure from its combined use in solid food supplements and pharmaceutical pills of 200 mg/tablet would amount to either 16.7 mg/kg bw/day of neutral methacrylate copolymer for average adult users (i.e. 200 mg x 5 tablets), to 20 mg/kg bw/day for heavy adult users (i.e. 200 mg x 6 tablets), and to 16 mg/kg bw/day for children (i.e. 200 mg x 2 tablets), respectively. The Panel noted that the estimates provided by the petitioner are (slightly) lower than the estimates made on assumptions used by the Panel in previous evaluations (EFSA, 2004; EFSA, 2005). Data from the UK Food Standard Agency on the consumption of food supplements indicate that the use among high consumers (97.5th percentile) ranged from 2 tablets/capsules per day in young people (4-18 years) (Gregory, 2000) to 7 tablets/capsules per day in adults (Henderson et al., 2002). Therefore, the Panel calculated an anticipated exposure to neutral methacrylate copolymer (see Table 2) from its combined use in solid food supplements and pharmaceutical pills of 46.7 mg/kg bw/day of neutral methacrylate copolymer for high adult consumers (based on a coating of 200 mg/tablet) and of 32 mg/kg bw/day for children, respectively. Assuming a coating level of 200 mg methacrylate copolymer per tablet, the anticipated total exposure for high consumers resulted in 2800 mg/day (200 mg x 14 tablets) and 800 mg/day (200 mg x 4 tablets) of neutral methacrylate copolymer for adults and children, respectively. 7 Commission Directive 2002/72/EC of 6 August 2002 relating to plastic materials and articles intended to come into contact with foodstuffs. OJ L 220, , p

12 Table 2: Estimated potential exposures to neutral methacrylate copolymer (NMC) based on assumptions of the Panel Adults Type of user Children 4 17 years (25 kg body weight) Number of tablets* 7 supplements and 7 pharmaceutical pills 2 supplements and 2 pharmaceutical pills Estimated exposure (mg/kg bw/day) to NMC, based on 200 mg coating per supplement Supplements Pharmaceuticals Total exposure *These data are derived from the use of food supplements among high consumers in young people (4-18 years) (Gregory, 2000) and in adults (Henderson et al., 2002) in the UK. Based on these intakes the Panel assumed similar levels of use and intake of pharmaceutical products by children and adults (EFSA, 2005). Exposure to the residual monomers in the copolymer product has also been considered by the petitioner. Residual monomers ethyl acrylate (EA) and methyl methacrylate (MMA) are, according to SCF (1998), expected to be hydrolysed to either methacrylic acid or acrylic acid and the corresponding alcohol. It is also expected that these will be further metabolised via endogenous pathways. Therefore, the potential residual levels of the monomers are expressed in terms of methacrylic acid. Exposure to these residual monomers was estimated by the Panel for adults as mg/kg bw/day MMA and mg/kg bw/day EA (based on total exposure of 46.7 mg/kg bw/day neutral methacrylate copolymer) and for children mg/kg bw/day MMA and mg/kg bw/day EA (based on total exposure 32 mg/kg bw/day neutral methacrylate copolymer) 8. The exposure for solid foods for special medical purposes could not be calculated as uses and use levels were not provided by the petitioner. 3. Biological and toxicological data The petitioner states that the identity of the substances used in the toxicity studies are similar to neutral methacrylate copolymer Absorption, distribution, metabolism and excretion (ADME) An ADME study was performed with adult male rats (Charles River CD) in two phases. In the first experiment 14 C-labelled neutral methacrylate copolymer film (place of the label not specified) was administered, via a cannula into the stomach of the animal, at a level of mg/animal to 5 adult male rats. Urine and faeces were collected at 24 hour intervals until only background levels of radioactivity were present in these samples. The animals were killed 7 days after dosing and liver, lungs, spleen, kidneys, mesenteric lymph nodes, small intestine and large intestine were removed for the assessment of radioactivity (LSC Report, 1974). 8 Example of calculation for adults: 46.7 mg/kg bw/day copolymer intake x 0.01% total monomer content (100 ppm) = mg/kg/day. Assuming equal residual amounts of monomer in relation to their molar ratio on the copolymer this equates to mg/kg/day methyl methacrylate and mg/kg/day ethyl acrylate. 12

13 In the second experiment, to investigate the rate of elimination of the radioactivity from the tissues, a single dose (55-75 mg/animal) of 14 C-labelled neutral methacrylate copolymer film was administered orally to 9 adult male rats. Groups of 3 animals were killed 1, 3, and 14 days following dosing. The liver, kidneys, spleen, mesenteric lymph nodes, small intestine and large intestine and cardiac blood were removed for the assessment of radioactivity. The major route of excretion was via the faeces. A mean total of 97.2% (range: %) of the dose was eliminated via this route, mostly occurring within the 48 hours following dosing. Urine showed very low levels of elimination (0.009% of the administered dose). Extremely low levels of radioactivity were recovered from the blood and a number of tissues, resulting in the conclusion that absorption from the gastro-intestinal tract was less than 0.02% of the administered dose. Analysis of blood and tissues did not indicate that significant amounts of any absorbed material were retained. From the data it can be concluded that the polymer is essentially not absorbed and that any absorbed material is not retained in the tissues Toxicological data Acute toxicity The petitioner provided data from studies on acute toxicity in a number of laboratory species and by a number of routes of administration showing low acute toxicity (oral, rat: LD 50 > mg/kg bw; oral, dog: LD 50 > 7950 mg/kg bw (Leuschner, 1971a; Leuschner, 1971b) Short-term toxicity and subchronic toxicity Short-term toxicity The petitioner provided data on a 35 day oral feeding study in the rat. The Panel noted that the study was performed in 1981 and therefore, probably not conducted following current test guidelines and in accordance with GLP. However, according to the petitioner the methods used in the study are generally in accordance with the requirements of OECD Test Guideline No.408. In the study a group of 15 male (5 weeks of age, weight range g) and 15 female (5 weeks of age, weight range g) animals (Sprague-Dawley; SD-CRJ) received neutral methacrylate copolymer, by gavage, at a dose level of 2000 mg/kg bw/day. An additional group of 15 males and 15 females acted as controls. Two additional groups, each of 10 male and 10 female rats, received intermediate dose levels of 500 and 1000 mg neutral methacrylate copolymer/kg bw. A group of 10 male and 10 female animals were treated with silicon dioxide alone at a dose level of 70 mg/kg bw/day, being the silicon dioxide exposure of animals treated with neutral methacrylate copolymer at the highest dose level. A second group of control animals received distilled water alone. At the end of the treatment period, 5 males and 5 females from the high-dose and control groups were maintained without treatment for a recovery period of 2 weeks. The animals were checked daily (prior to and following dosing) for clinical signs and mortality. Body weight and food and water consumption were recorded 3 times weekly during the first week of the study and twice weekly thereafter. Blood and urine samples were collected from animals at the end of the treatment and recovery periods to investigate selected hematology, blood biochemistry and urinalysis parameters. At the end of the treatment period or recovery period the animals were killed and a full macroscopic post mortem examination was performed. Selected major organs were weighed. Selected tissue specimens were preserved and submitted to a microscopic examination. The authors concluded that no specific changes 13

14 occurred during the study that were attributed to treatment and that the No-Observed-Adverse-Effect Level (NOAEL) identified in this study may be regarded as 2000 mg/kg bw/day, the highest dose tested (Hirakawa, 1981). The Panel agrees with this NOAEL. In a study with minipigs, conducted in accordance with to EEC Council Directives 65/65 and 75/318 and subsequent amendments, as detailed in the Rules Governing Medicinal Products in the European Union, Volume 3B (1998), three groups of 3 male and 3 female animals (age 6-7 months; weight kg/animal) received by gavage, over a period of 4 consecutive weeks (28 days), Neutral methacrylate copolymer coated onto cellulose pellets at dose levels of 500, 1000 and 2000 mg/kg bw/day, equivalent to 113, 227 and 454 mg/kg bw/day expressed as Neutral methacrylate copolymer dry substance. An additional group of 3 males and 3 females acted as a control group. Following the 4 weeks of treatment, no toxicologically relevant changes were observed in body weight, food consumption, clinical observations, and clinical pathology investigations and in the organ weights. Histopathological examination revealed sporadic instances of mucosal/submucosal oedema in the caecum and colon of one male receiving 454 mg/kg bw/day and in the caecum of one male dosed at 227 mg/kg bw/day. According to the authors of the study, the effect of the high doses is unclear and the finding may be a physiological reaction of the intestine to the high amounts of non-soluble or non-degradable particles resulting in osmotic imbalance in this part of the intestine. According to the authors, no toxicological relevance could be attributed to this observation (Rossiello, 2006). The Panel agrees with this conclusion and identified a NOAEL of 454 mg/kg bw/day Subchronic toxicity The petitioner provided data of a 26 week dietary study in the rat. The Panel noted that the study was carried out in 1973 when OECD and GLP guidelines were not yet implemented. However, the Panel also noted the study was undertaken by a recognised research organisation and that the original report was amended by the research organisation in In this study two groups of 20 male and 20 female Sprague-Dawley rats (males approximately 39 days old and females 43 days old; body weight range g) received a dry lacquer preparation (diet coated with test substance) of neutral methacrylate copolymer, by dietary admixture at dose levels of 500 and 2000 mg/kg bw/day. An additional group of 20 males and 20 females received an untreated diet ad libitum and acted as a control group. The petitioner points out that the test substance used was obtained by spraying the aqueous polymer dispersion of neutral methacrylate copolymer onto the powdered diet at a ratio of 1:10, in order to coat the diet in an analogous manner to its proposed use in the commercial supplements tablet coating. The animals were checked at least daily for clinical signs and mortality and were also examined weekly. Body weight was recorded weekly throughout the treatment period. Food consumption was measured daily and the achieved dosage calculated and adjusted weekly. Blood and urine samples were taken and collected respectively from 10 animals/sex/group during weeks 6, 13, 18 and 26 to allow selected haematology, blood biochemistry and urinalysis parameters to be investigated. At the end of the treatment period, the animals were killed, and selected major organs were weighed. All animals were submitted to a full macroscopic post mortem examination. Selected tissue specimens were preserved and those from 10 animals/sex of the high-dose groups and control groups were submitted to a microscopic examination. The results of the study show that the test substance, administered in the diet at dose levels up to 2000 mg/kg bw/day for 26 weeks was generally well tolerated. No treatment-related effects were noted on body weight, body weight gain, and food or water consumption. No clinical signs were apparent to indicate a treatment-related effect. Haematology and clinical chemistry parameters were not affected. No macroscopic or microscopic abnormalities were noted that were deemed to be a result of treatment. From the study the authors derived a NOAEL of 2000 mg/kg bw/day, the highest dose tested. (Leuschner, 1972). The Panel agrees with this NOAEL. 14

15 The petitioner provided also data of another 26 weeks study with Beagle dogs (age, 24 weeks; weight, kg/animal) in which three groups of 4 male and 4 female animals received orally (via gelatin capsules) neutral methacrylate copolymer coated onto cellulose pellets at dose levels of 200, 500 and 1000 mg/kg bw/day, equivalent to 50, 125 and 250 mg/kg bw/day expressed as neutral methacrylate copolymer. A group of 4 males and 4 females acted as a control group. An additional 3 males and 3 females in the control group and in the high-dose group were treated for 26 weeks and then permitted a 3 week recovery period without treatment. The petitioner states that the study was conducted in accordance with the Rules Governing Medicinal Products in the European Community, Volume 3B (1998) and in compliance with the EU, OECD, US FDA and Italian GLP Guidelines. From the data of this study it was concluded that there were no toxicologically relevant changes in water consumption and clinical observations (reaction to treatment and clinical signs). There were also no changes noted in clinical pathology investigations or organ weights. The slightly lower body weights correlated with reduced food consumption in males treated at 250 mg/kg bw/day and in all treated females was thought, by the authors of the study, to be a characteristic of the physical properties of the test substance, being insoluble but swelling in water. It was considered, as a result, that long-term administration may influence food consumption and food absorption. Histopathological examination did not reveal any evident differences in the incidence of findings observed in treated and control animals, which could be considered treatment-related. According to the authors, the lower body weights and the reduced food consumption observed at the highest dose tested, could be explained by the characteristics of the test substance and were not accompanied by any signs of toxicity to organs or tissues, a NOAEL of 250 mg neutral methacrylate copolymer/kg bw/day, the highest dose tested (Rossiello, 2007). The Panel agrees with this NOAEL Genotoxicity studies Neutral methacrylate copolymer was tested in the Ames assay on Salmonella typhimurium TA98, TA100, TA1535 and TA1537, in the presence and absence of metabolic activation (S-9 mix). The test substance was added to the cultures at 5 concentrations in the range between to 5000 µg/plate. No information is provided on whether the study was carried out according to GLP. No substantial increase in revertant colony numbers was observed either in the presence or absence of metabolic activation. It was concluded that neutral methacrylate copolymer did not show mutagenic activity under the test conditions used (Bootman, 1977). The petitioner provided also data of a second reverse mutation assay with neutral methacrylate copolymer, performed according to OECD guideline 471, on S. typhimurium TA98, TA100, TA102, TA1535 and TA1537, both in the presence and absence of metabolic activation (S9 mix). The assay was performed in two independent experiments: in a pre-experiment/experiment I neutral methacrylate copolymer was tested in a concentration range between 3 and 5000 µg/plate and in experiment II in a concentration range between 33 and 5000 µg/plate. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S-9 mix in all the strains used. No toxic effects occurred in the test groups with or without metabolic activation. No substantial increase in revertant colony numbers was observed either in the presence of absence of S-9 mix. It was concluded that the test substance did not show mutagenic activity under the conditions used. (Sokolowski, 2005). Neutral methacrylate copolymer was also tested for genotoxic activity in L5178Y TK +/- mouse lymphoma cells in the absence and presence of metabolic activation. Tests were conducted in compliance with OECD guideline No 476 and in accordance with GLP. Two independent assays were performed in two independent experiments: in the first assay following a 4-hour exposure both in the presence and the absence of S9 the test article concentrations were 390.6, 781.3, , 3125 and 6250 μg/ml; in the second assay following a 24-hour exposure in absence of S9 the concentrations were 390.6, 781.3, , 15

16 3125 and 6250 μg/ml. Precipitation of the test substance was visible at the two highest concentrations 3125 and 6250 μg/ml in both experiments. In both experiments no relevant toxicity was detected. No relevant and reproducible increase of the mutation frequency was observed in the main experiments with and without metabolic activation. The total numbers of mutant colonies per 10 6 cells remained well within the range of historical solvent controls. It was concluded that neutral methacrylate copolymer did not demonstrate mutagenic potential in this in vitro cell mutation assay. (Wollny, 2005). Neutral methacrylate copolymer was further tested in an in vivo micronucleus assay in mice in compliance with OECD Guideline 474 and accordance with GLP. Based on a preliminary toxicity assay, the doselevels selected were 500, 1000 and 2000 mg dry neutral methacrylate copolymer/kg bw for male and female animals. Groups of Hsd:ICR (CD-1) mice (age range 5-6 weeks; weight range g) were dosed, by gavage, once only, with sterile distilled water (vehicle of the test substance), neutral methacrylate copolymer at the selected dose-levels or with the positive control substance (mitomycin-c, 3mg/kg bw). Each group consisted of five male and five female animals with the exception of the control and high-dose group, which included an additional five animals for each sex per group. Five animals per sex from each group were sacrificed at the 24 hour sampling time. The additional animals were sacrificed at the 48 hour sampling time. In the preliminary assay, no mortality occurred and no clinical signs were observed at any dose level. Analysis of the bone marrow smears obtained from the animals treated at a dose level of 2000 mg/kg (expressed as dry substance) revealed no adverse effect on the proliferation of bone-marrow erythropoietic cells. In the main study, one male animal from the positive control group was found dead on the day after dosing. Necropsy examination revealed a red fluid in the thoracic cavity together with a ruptured oesophagus. According to the petitioner these findings were considered to be indicative of misdosing. The femurs of this animal were removed and slides were prepared with the extracted bone marrow cells. On the day after dosing with the test substance one female treated at 500 mg/kg (expressed as dry substance) exhibited a pale aspect, hunched posture and piloerection. Hunched posture, piloerection, hypoactivity, semi-closed eyes and loss of hair were observed in one male treated at 1000 mg/kg (expressed as dry substance). A slight reduction in body weight was observed in one male animal treated at 2000 mg/kg (expressed as dry substance) and similar findings were noted in female animals from the positive control group. Following treatment with neutral methacrylate copolymer, minimal increases in the incidences of micronucleated polychromatic erythrocytes (PCE s) over the control values, reaching statistical significance, were observed in the intermediate and high dose groups (treated at 1000 and 2000 mg/kg bw, expressed as dry substance) at the 24 hour sampling time. These increases were not considered biologically relevant since the incidences of micronucleated cells were within the range of historical values observed for negative controls by the testing laboratory and no dose response was observed. A statistically significant increase in the frequency of micronucleated PCE's was observed in the positive control group at the 24 hour sampling time, indicating the correct functioning of the test system. No induction of micronucleated PCE's was observed in the animal that died due to misdosing during the administration of mitomycin-c. The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate bone marrow cell toxicity. Based on these results, no remarkable inhibitory effect on erythropoietic cell division was observed at any sampling time, at any dose-level. It was concluded that when neutral methacrylate copolymer was administered by oral gavage at dose levels up to 2000 mg/kg body weight (expressed as dry substance), there was no biologically significant induction of micronuclei in polychromatic erythrocytes under the reported experimental conditions (Ciliutti, 2007) Chronic toxicity and carcinogenicity No data are provided. 16

17 Reproductive and developmental toxicity The petitioner did not provide data from one or two generation reproductive toxicity studies. The petitioner provided data of two studies focussing on prenatal developmental toxicity. The Panel noted that the studies were carried out in The methods used were those described by the guidelines for reproductive studies for safety evaluation of drugs for human use of the FDA (1966) and the principles for the testing of drugs for teratogenicity, WHO, There were no indications that the studies were conducted in compliance with GLP. In a first study the effects of neutral methacrylate copolymer on prenatal development of rats was assessed. Two test groups and a control group of 20 female rats (Sprague Dawley, body weight 202 to 258 g) were paired overnight with males of the same strain. The rats of the test groups received the daily dose of 500 or 2000 mg neutral methacrylate copolymer/kg bw/day in the diet from day 6 to day 15 of gestation inclusive (period of organogenesis). The test substance was prepared by spraying the aqueous polymer dispersion of neutral methacrylate copolymer onto the powdered diet at a ratio of 1:10. Final concentrations were achieved by diluting aliquots of the premix with an appropriate amount of basal diet and mixing. Control diet was prepared by spraying with water, allowing drying, and furthering mixing with additional diet. The petitioner stated that examination of the analytical data of the batch and manufacturing formula for the lot used indicated the substance to be comparable to current commercial lots of neutral methacrylate copolymer. There was no evidence of an effect of treatment on maternal animals with the exception of a possible increase in urinary excretion during the treatment period only in those dosed at 2000 mg/kg bw/day. Behaviour, external appearance, faeces, body weight gain and food intake were regarded as normal. All animals were pregnant with live fetuses at termination. There were no maternal necropsy findings which were considered to be related to treatment at sacrifice on GD 19. No adverse effect on prenatal development of fetuses was reported. The number and weight of alive fetuses, variations, resorption rates and post-implantation losses were within control values. Pre-implantation losses were 2.2% in control animals and 1.5% and 1.8% in animals treated at 500 and 2000 mg/kg bw/day respectively and post-implantation losses were respectively 11.4%, 12.2% and 9.5%. Mean litter size was 11.7 in the control group, 11.9 in the group treated with 500 mg/kg/day and 12.4 in the group treated with 2000 mg/kg/day. No fetuses were found with external malformations, malformations of the internal organs (as examined after opening of abdominal and thoracic cavity, but not by Wilson s section to reveal brain abnormalities) and such of skeleton (after staining with alizarin red and evaluated according to Dawson). The rate of fetuses with variants or delay in ossification of sternebrae, cranial bones, phalanges and 12/13 ribs, as presented for each type and as a number of their sum or the number of affected fetuses, was similar in the test groups and in controls. The authors concluded the NOAEL for both dams and fetuses to be 2000 mg/kg bw/day (Leuschner, 1974a). The Panel agrees with this NOAEL. In a second study with female rabbits (Strain New Zealand White, body weight 3.6 to 4.4 g), two test groups of 10 animals each and a control group of 10 female rats were paired with males of the same strain. Day 0 of gestation was designated when a successful mating was observed. The test substance (neutral methacrylate copolymer) was prepared and applied to feedstuff in exactly the same way as in the study with rats described above. The rabbits of the test groups received the daily dose of the test substance in the diet from day 6 to day 18 of gestation inclusive. The concentration of the test substance in the diet was adjusted daily to give dose levels of 500 and 2000 mg neutral methacrylate copolymer/kg bw/day. Control animals received the water treated and subsequently dried diet over the same period. There was no evidence of an effect of treatment on maternal animals. Behaviour, external appearance, faeces, body weight gain, food and water intake were regarded as normal. At autopsy performed on GD 29 there were no maternal necropsy findings which were considered to be related to treatment. There were also no indications of an adverse effect on embryo/fetal development. All the fetal parameters assessed (mean number of corpora lutea, of resorptions, of implantation sites, of alive fetuses and their viability as well as 17