The EFSA Journal (2005) 274, 1-10

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1 The EFSA Journal (2005) 274, 1-10 Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in ontact with Food on a request from the ommission related to Epoxy Phenolic Novolac Resins (NOGE) Question N EFSA-Q Adopted on 6 October 2005 SUMMARY EFSA is asked to carry out risk assessments for substances intended for use in materials in contact with food, according to Regulation (E) No. 1935/2004 of the European Parliament and of the ouncil of 27 October 2004 on materials and articles intended to come into contact with food. In particular, EFSA is asked to advise the ommission on the implications for human health of the use of Epoxy Phenolic Novolac Resins (Novolac Glycidyl Ether, NOGE) as a starting substance in epoxy resins used for heavy duty coatings. The Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in ontact with Food noted that the Scientific ommittee on Food (SF) had issued a statement on the use of NOGE as an additive in food contact materials in December Based on the presence of structural alerts for toxic effects, and in the absence of information on exposure and toxicity profile, the SF evaluated as not appropriate the use of NOGE as an additive in organosols for food contact materials (SF, 1999). In a subsequent opinion on bisphenol-f-diglycidyl ether (BFDGE), which can be considered as the lowest molecular weight component of NOGE, the SF noted that NOGE, may give rise to a broader scope of cross-linking reactions compared to BFDGE because NOGE contains oligomers bearing more than 2 epoxy groups. Therefore BFDGE cannot be regarded as a worst case test compound for evaluating the toxicity of NOGE. In 2000, the SF therefore requested mutagenicity studies with the oligomeric fraction of NOGE having a molecular weight lower than 1000 D. onsidering the available evidence of mutagenicity in vitro of BFDGE, and the presence of epoxy groups on NOGE oligomers, the SF concluded that a positive response in the standard genotoxicity tests in vitro could be anticipated. Therefore a package of in vivo assays addressing various end-points and tissues was recommended by the SF in In this opinion the Panel considered three recent in vivo mutagenicity studies carried out with a low molecular weight commercial Epoxy Phenolic Novolac Resin containing high percentages of oligomers with molecular weight below 1000 D and BFDGE. The material

2 was tested in a transgenic rodent system with the analysis of mutations in the liver and the forestomach, in the rat liver unscheduled DNA synthesis (UDS) assay and in the mouse bone marrow micronucleus test. Negative results were obtained in all three studies following the administration of NOGE oligomers and BFDGE up to the highest recommended dose level. Based on the examined data, the Panel concluded that the oligomeric fraction of NOGE with molecular weight below 1000 D and BFDGE are not genotoxic in vivo. onsidering the lack of genotoxicity and the information on specific migration from heavy duty coatings provided by the applicant showing that migration is 50 g/kg food, the Panel concluded that the use of NOGE as a starting substance in heavy duty epoxy coatings is not of concern from a safety point of view. KEY WORDS Epoxy Phenolic Novolac Resin, Novolac Glycidyl Ether, NOGE, AS No and , Bisphenol-F-diglycidyl ether (BFDGE), AS No , PM REF 12976, food contact materials, coatings, genotoxicity. BAKGROUND According to ommission Directive 2002/16/E on the use of certain epoxy derivatives in materials and articles intended to come into contact with foodstuffs NOGE may not be used in materials and articles made of plastics, articles covered by surface coatings or adhesives except for containers and storage tanks having a capacity greater than liters and for pipelines belonging to or connected with them. At the time of the adoption of the Directive information on the toxicological profile and potential exposure to NOGE was not available. Previous evaluations NOGE was first evaluated by the SF in November 1999 during its 119 th meeting. The ommittee had been informed that NOGE was used as alternative to bisphenol A based glycidyl ether (BADGE) as additive in organosols. The ommittee noted that NOGE had structural alerts for toxic effects (i.e. the presence of epoxy groups and the potential for chlorohydrin formation) and that analytical methods for identification and estimation of NOGE and its reaction products were not available. Therefore the ommittee concluded that, in the absence of information on potential exposure and toxicity of NOGE and its reaction products, the use of NOGE as an additive in organosols in food contact materials was not appropriate (SF, 1999). NOGE was again evaluated by the SF during its 123 rd meeting in October 2000, together with bisphenol-f-diglycidyl ether (BFDGE), the lowest molecular weight component of NOGE (SF, 2000). The ommittee noted that NOGE contains oligomers bearing more than 2 epoxy groups which may give rise to a broader scope of cross-linking reactions than 2

3 bis-epoxides like BFDGE, BADGE and BADGE oligomers. Therefore these bisepoxides cannot be considered as worst case test compounds. For this reason, in addition to nontoxicity data according to guidelines, the results of 3 mutagenicity studies on the more representative NOGE fraction having a MW less than 1000 D were requested to be provided within one year. oncerning BFDGE, based on limited non-toxicity data and mutagenicity studies showing positive results in vitro but not in vivo, the compound was provisionally classified by the ommittee in List 7 with the request for absorption, distribution, metabolism and excretion (ADME) and DNA binding studies to be provided within three years (SF, 2000). Industry proposal for the toxicological testing of NOGE Following the adoption of the above mentioned SF opinion, an alternative proposal for the toxicological testing of NOGE was put forward by industry. According to the proposal, standard in vitro mutagenicity tests on NOGE were regarded as of limited utility, because a positive response was anticipated in view of the presence of the in vitro mutagens BFDGE in NOGE. Therefore a transgenic rat mutagenicity study, coupled with a rat micronucleus test for clastogenic and aneugenic effects was proposed (APME, 2002). The proposal was discussed during a hearing with industry held on the occasion of the 93 rd meeting of the SF working group Food ontact Materials on 13 October It was agreed that a low viscosity commercial product (with a ratio 1:1 of BFDGE:oligomeric fraction <1000 D and < 20% with MW above 1000 D) was the preferred test material because it was likely to reflect more the substances which will migrate from the coating. It was recommended by the working group to optimise the dosing to ensure that the fraction of interest reached the doses recommended in guidelines. The working group recommended that all the tests outlined by industry, i.e. a transgenic rodent study, an UDS in vivo/in vitro study, and a micronucleus in vivo study be performed. The WG agreed that ADME and DNA-binding studies were not warranted for NOGE, and could be postponed for BFDGE awaiting the results of the in vivo genotoxicity studies on the mixture (SF, 2002). The above mentioned studies have been performed and forwarded to EFSA for evaluation. TERMS OF REFERENE In accordance with Article 29 (1) (a) of Regulation (E) No. 178/2002 the European Food Safety Authority is asked to carry out risk assessments on substances intended for use in materials in contact with food. In particular, EFSA is asked to advise the ommission on the implications for human health of the use of Epoxy Phenolic Novolac Resins (Novolac Glycidyl Ether, NOGE) as starting substance only for special coatings, called heavy duty coatings, applied to containers having a capacity of more than liters and to pipelines connected to them. 3

4 ASSESSMENT The Panel has given consideration to the data already available on BFDGE and evaluated by the SF, as well as to the new mutagenicity studies with low molecular weight (MW) commercial Epoxy Novolac Resin. hemistry Epoxy Phenolic Novolac Resins (NOGE) are polymeric reaction products of phenol, formaldehyde and epichlorohydrin described by the AS No (Phenolformaldehyde-glycidyl ether) or AS No (phenol-formaldehydeepichlorohydrin polymer). Epoxy Phenolic Novolac Resins are manufactured in two stages: I. reaction of phenol with formaldehyde to yield phenolic novolac resins (mixture of the 2-ring bisphenol F plus components with 3 or more rings) II. reaction of phenolic novolac resins with epichlorohydrin, to form Epoxy Phenolic Novolac Resins or NOGE (mixture of the 2-ring bisphenol-f-diglycidyl ether (BFDGE) plus epoxides with 3 or more aromatic rings and epoxy groups) The lowest molecular weight oligomeric fraction in commercial Epoxy Phenolic Novolac Resins is bisphenol-f diglycidylether. BFDGE exists as a mixture of three isomers with MW of 312 and is present in NOGE at levels of 20 45%. NOGEs have the following general structure: H 2 O H H 2 O O O H 2 H H 2 O H 2 H2 O H2 H H 2 n BFDGE can be considered as an Epoxy Phenolic Novolac Resin with n = 0. If n = 1, three epoxy groups are available, and the MW is 474. With n = 4, there are 6 epoxy groups and the MW is 964. A typical commercial Epoxy Phenolic Novolac Resin has the following oligomeric distribution: n = 0 : ~45% (BFDGE) n = 1: ~20 % n = 2 : ~10 % n = 3 : ~7 % n = 4 : ~5 % n > 4 : ~13 % ( MW > 1000 D) 4

5 Uses in food contact materials Epoxy Phenolic Novolac Resins are used as starting substance in heavy duty epoxy coatings, with the function of crosslinker and polymeric epoxy binder, and in some countries outside the EU as polymeric additive in PV organosol can coatings, with the function of heat stabilizer and Hl scavenger. According to the information provided by industry, the presence of NOGE as an additive in PV organosols has been phased out in the EU since the end of year 2000 (EPE, 2002). Exposure Both Novolacs and BADGE-based resins have been on the market for a long time. From 1997, Novolacs coatings largely replaced those based on BADGE in vinylic organosols (JIG, 1998). The use of epoxy resins as stabilizers in PV-organosols is associated with higher migration levels compared to heavy duty epoxy coatings. In 1997 a survey of Swiss samples of canned oily foods showed levels of 3- and 4-ring NOGE from < 50 g/kg up to 3330 g/kg food (Bronz et al., 1997). As mentioned above, the use of NOGE in PV organosols has been discontinued in the EU, and restricted to heavy duty coatings for containers bigger than liters (EPE, 2002). onsidering a surface to volume ratio of 0.4 dm 2 per kg of food as appropriate for the specific application, migration levels lower or equal to 50 g/kg have been reported in food simulants (EPE, 2001). New toxicological studies (not considered in previous SF evaluations) Three in vivo mutagenicity studies were performed with a sample of commercial Epoxy Phenolic Novolac Resin. The analytical characterisation of the material showed the presence of approximately 45-50% of BFDGE (n=0), % of oligomers with MW (n=1-4), and 1-5% of polymers with MW >1000 D (n>4) (depending on the method of analysis) (Huntsman, 2003). The studies performed were in compliance with Good Laboratory Practice and OED Guidelines; the main findings obtained are summarized below. Epoxy Phenolic Novolac Resin was assayed in the bone marrow micronucleus test in D-1 mice (Whitwell, 2003). The substance was formulated in 1% (w/v) methyl cellulose in water and administered by gavage at the dose of 4000 mg/kg body weight/day once daily on two consecutive days. The administered dose was equivalent to about 2000 mg/kg body weight (maximum recommended dose) of the oligomeric fraction with MW Based on the results of a preliminary toxicity test showing no clinical signs both in males and females, only males (six per group) were treated in the main test. The negative control group received the vehicle alone according to the same treatment schedule. yclophosphamide, dissolved in saline, was given to the positive control group by single intragastric administration. Animals were sacrificed 24 hours after the second (or for positive controls unique) treatment. The incidence of micronuclei was determined by scoring 2000 polychromatic erythrocytes (PEs) per animals. The ratio between poly- and normochromatic erythrocytes (PE/NE) was calculated during the scoring of the first 1000 PEs. The results obtained indicate that NOGE administration was well tolerated, with no animals dying or showing signs of overt toxicity prior to the scheduled sampling time. The 5

6 incidence of micronucleated PEs was similar in mice receiving NOGE or the vehicle alone, whereas a distinct increase of micronucleated PEs was detected in animals belonging to the positive control group. No significant difference in the PE/NE ratio was observed among treatment groups. Epoxy Phenolic Novolac Resin was tested for its ability to induce unscheduled DNA synthesis (UDS) in the livers of male rats dosed via oral gavage using an in vivo/in vitro procedure (Beevers, 2003). In an initial range-finding study, outbred male Han Wistar rl:wi (Glx/BRL/Han) IGS BR rats were dosed once with 4000 mg/kg body weight Epoxy Phenolic Novolac Resin formulated in 1% (w/v) methylcellulose (equivalent to the maximum recommended dose of 2000 mg/kg b.w. for the d oligomeric fraction). During a 2 day post-dose observation period, no significant clinical signs were observed. In the main experiment, groups of three male rats were treated once by gavage with the vehicle or with mg/kg body weight of Epoxy Phenolic Novolac Resin (equivalent to approximately mg/kg body weight of D oligomers). The following positive control substances were administered by the same route to groups of three rats: 75 mg/kg body weight of 2-acetamidofluorene (2-AAF) suspended in corn oil (12-14 hour experiment), and 10 mg/kg body weight of dimethylnitrosamine (DMN) dissolved in purified water (2-4 hour experiment). Approximately hours (Experiment 1) or 2-4 hours (Experiment 2) after dosing, animals were sacrificed and their livers perfused with collagenase to provide a primary culture of hepatocytes. ultures were made from at least three animals in each dose group and were treated with [ 3 H] thymidine. Six slides from each animal were prepared with fixed hepatocytes and examined microscopically after autoradiography. For each animal the net nuclear grain count (NNG), i.e. the number of grains present in the nucleus minus the mean number of grains in an equivalent area of cytoplasm, was determined in one hundred cells from 1-3 slides. Treatments did not produce clinical signs at any time. Mean NNG value in the negative control group was less than zero with no cells in repair. Similarly, negative mean NNG values were observed in animals treated with NOGE, with less than 1% of cells in repair. A distinct increase in NNG (above 10) was observed in animals treated with positive control substances. Epoxy Phenolic Novolac Resin was evaluated for the ability to induce gene mutation in the lacz transgene in tissues from Muta TM Mice (D 2 -lacz80/hazfbrstrain) (Ballantyne, 2004). The Panel noted that this study was conducted in accordance with the newly agreed OED guidelines (OED, 2005). The test substance was formulated in 1% (w/v) methylcellulose (M) in water and administered by gavage at 20 ml/kg body weight. In a preliminary range finding study, three male Muta TM Mice were treated for 7 consecutive days with 4000 mg/kg body weight/day of Epoxy Phenolic Novolac Resin, equivalent to about 2000 mg/kg body weight/day of oligomers with MW No clinical signs of toxicity, and no abnormal organ morphology was observed in any animal at sacrifice. Based on these data, 4000 mg NOGE/kg body weight/day (or 2000 mg/kg body weight/day of oligomers D) was selected as maximum recommended dose for the main study where groups of seven male mice were treated once a day for 28 consecutive days. A lower dose of 2680 mg NOGE/kg body weight/day (approximately 2/3 the maximum dose) was also included in the main study, together with a vehicle control group, dosed with 1% M alone. oncurrent positive controls are not normally necessary in this type of study, but it is recommended that positive control DNA be included to confirm the success of the 6

7 laboratory procedures (Heddle et al., 2000). In this work DNA samples obtained from the liver of mice treated orally with 4-nitroquinoline-1-oxide (200 mg/kg body weight, 24 hours before sacrifice) were processed together with other DNA samples. No compound related clinical signs of toxicity occurred during the course of the treatment period. One animal from each of the high and low dose groups was incorrectly dosed and killed in extremis. All surviving animals were sacrificed and necropsied 3 days after the final administration (Day 31). DNA was extracted from forestomach and liver, aliquots of isolated DNA were then introduced into competent E. coli lac - gale - Kan r (gale - Amp r ) bacteria using a bacteriophage lambda vector. Packaged DNA was absorbed to competent bacteria for approximately 20 minutes, and then plated on titration plates to determine the total number of plaque-forming units (pfu), and plates containing phenylgalactose (P-gal, 0.3% w/v) for the scoring of lacz - mutants, which produce clear plaques on this medium. Mutation frequencies per million (MF) were calculated scoring at least 200,000 plaqueforming units (pfu) per animal, in at least five animals per group, giving a total yield of more than one million pfu analysed per group. MF in liver and forestomach of treated animals were as follows: Treatment Liver a MF x 10-6 Group MF 10-6 (SD) Forestomach a MF x 10-6 Group MF x 10-6 (SD) 1% M Vehicle (19.20) (23.56) Epoxy Phenolic Novolac Resin (fraction D) 1340 mg/kg Epoxy Phenolic Novolac Resin (fraction D) 2000 mg/kg (62.54) (18.68) (35.34) (18.97) a mutation data could not be obtained from some animals due to poor packaging efficiencies, which led to too few analysable pfu; in all cases, however, data from at least five animals per group were available, as indicated in the study protocol. 7

8 Liver and forestomach mean MF (± standard deviation) for the vehicle group (62.83 ± 19.2 and ± 23.6 respectively) were comparable to historical control data (64.25 ± 26.4 and ± 25.9 for liver and stomach, respectively; no data were available for forestomach). MF from positive control DNA was (± , 10 samples). A slight increase in mean mutant frequency was observed in liver samples from animals treated with the low dose of NOGE, and from forestomach samples of animals treated with the high dose. These increases were not statistically significant compared to vehicle control groups (ANOVA and Dunnett s tests on untransformed and rank transformed data) and can be attributed to the variability of data which is typically observed with transgenic mutation assays. Moreover, the larger increase in mutant frequency (observed in liver samples) did not satisfy any of the conditions required for a positive response, i.e. it was not dose related and did not exceed twice the mean historical negative control frequency, nor two standard deviations above the mean historical control(heddle et al., 2000). DISUSSION The newly provided genotoxicity studies on NOGE show that commercial Epoxy Phenolic Novolac Resin (~ 50 % of BFDGE, ~ 50 % oligomers with MW ) did not induce clastogenic or aneugenic effects in the mouse bone marrow micronucleus test, nor unscheduled DNA synthesis, an hallmark of DNA damage and repair, in the liver of rats in vivo. Even though no alteration in the ratio polychromatic/normochromatic erythrocytes was observed in the mouse micronucleus test, the Panel noted that myeologram changes, indicative of toxicity to bone marrow, were observed in a previous general toxicity study in rats with BFGDE, suggesting that the target tissue was exposed to the test material. After repeated oral administration of the same commercial Epoxy Phenolic Novolac Resin, only non-statistically significant increases in mutant frequencies were observed in liver and forestomach of lacz transgenic mice. These results are attributed to the variability of data which is typically observed with transgenic mutation assays, as no one of the conditions required for a positive response by current evaluation criteria, i.e. a relationship with the dose or an increase exceeding twice the mean historical negative control frequency or two standard deviations above the mean historical control, was satisfied. Based on these results, it is concluded NOGE does not induce gene mutations in vivo in the liver nor at the site-ofcontact following oral administration to mice. Overall, the experimental data provided by the studies considered in this opinion show that commercial Epoxy Phenolic Novolac Resin (~ 50 % BFDGE, ~ 50 % of oligomers with MW ) is not genotoxic in vivo. The SF classified BFDGE in List 7, with the request for ADME and DNA binding studies to clarify the systemic availability of orally dosed BFDGE and its potential genotoxicity in vivo. The new studies with commercial Epoxy Phenolic Novolac Resin, which contained ~ 50 % of BFDGE in addition to NOGE oligomers, demonstrate that also BFDGE does not induce genotoxic effects in liver and bone marrow, nor at the site of contact. On this basis, and also considering the evidence of systemic exposure provided by 8

9 the rat sub-chronic toxicity study on BFGDE, it is concluded that BFDGE is not genotoxic in vivo. Therefore the additional studies required by the SF to clarify the systemic availability and in vivo genotoxic potential of BFDGE (i.e. ADME and DNA binding studies) are no longer considered necessary. ONLUSIONS The studies evaluated by the Panel address the reservation expressed by the SF on the genotoxic potential of oligomers of bisphenol F diglycidylether with n 1, raised by the presence of multiple ( 3) epoxy groups. The studies were performed on a commercial, low molecular weight Epoxy Phenolic Novolac Resin (NOGE) containing BFDGE and the oligomeric fraction <1000 D in 1:1 ratio and < 20% with MW above 1000 D. Dosages were adjusted in order to administer the highest recommended dose of both the oligomeric fraction with molecular weight (corresponding to 3- to 6-ring oligomers), and BFGDE (the 2-ring lowest molecular weight component of NOGE). The studies, addressing various end-points and target tissues, show that NOGE is not genotoxic in vivo. onsidering the lack of genotoxicity and the information provided by the applicant on specific migration from heavy duty coatings ( 50 µg/kg), the Panel concluded that the use of NOGE as a starting substance in heavy duty epoxy coatings is not of concern from a safety point of view. The new studies on NOGE evaluated by the Panel provided additional confirmation that BFDGE is not genotoxic in vivo. Therefore the additional studies previously requested by the SF to clarify the genotoxic potential of BFDGE in vivo (ADME and DNA binding) are no longer considered necessary. REFERENES APME, Association of Plastics Manufacturers in Europe (2002), Proposal for the Toxicological Testing of NOGE. APME Epoxy Resins ommittee, March 5, 2002 (S/PM/4005) Ballantyne M. (2004), Epoxy Phenolic Novolac Resin: Induction of lacz mutations in tissues of treated Muta Mice. ovance report n. 1436/71-D6171, April Beevers. (2003), Epoxy Phenolic Novolac Resin: measurement of unscheduled DNA synthesis in rat liver using an in vivo/in vitro procedure. ovance report n 1436/70-D6173, December Bronz M., et al (1997), Diglycidyl ethers of bisphenol-f and Novolac in canned oily foods. Mitt. Giebiet Lebens. Hyg. 88, EPE (2001), Specific migration studies from heavy duty coatings. February EPE (2002). presentation in the course of the hearing with the SF Food contact materials working group, 13 Novembre

10 Heddle, J.A., Dean, S., Nohmi, T., Boerringter, M., asciano, D., Douglas, G.R., Glickman, B., Gorelick, N., Mirsalis, J., Martus, H.J., Skopek, T., Thybaud, V., Tindall, K., Yajima, I. (2000), In vivo transgenic mutation assays. Environ. Mol. Mutagen. 35, Huntsman Advanced Material (2003), Epoxy Novolac Resin (NOGE), Batch n. 0421: Analytical characterisation. Request n. 194/2003, report date JIG, Joint Industry Group Metal Packaging (1998), meeting with SF delegates, Birmingham UK, July OED (2005), Draft Detailed Review Paper on Transgenic Rodent Mutation Assays, Paris, February SF (1999), Minutes statement of the 119th meeting of the Scientific ommittee on Food (1-2 December 1999). Statement on the use of novolac glycidyl ethers (NOGE) as additives in food contact materials, SF (2000), Opinion of the Scientific ommittee on Food on the 11th additional list of monomers and additives for food contact materials (expressed on 19 October 2000), SF (2002), Minutes of the 93 rd meeting of the SF working group Food contact materials, November Whitwell J. (2003), Epoxy Phenolic Novolac Resins: induction of micronuclei in the bone marrow of treated mice. ovance report n 1436/69-D6172. December SIENTIFI PANEL MEMBERS Robert Anton, Sue Barlow, Dimitrios Boskou, Laurence astle, Riccardo rebelli, Wolfgang Dekant, Karl-Heinz Engel, Stephen Forsythe, Werner Grunow, Marina Heinonen, John hr. Larsen, atherine Leclercq, Wim Mennes, Maria Rosaria Milana, Iona Pratt, Ivonne Rietjens, Kettil Svensson, Paul Tobback, Fidel Toldrá. 10