Title: Safety assessment of the substance poly((R)‐3‐hydroxybutyrate‐co‐(R)‐3‐hydroxyhexanoate) for use in food contact materials
Abstract: EFSA JournalVolume 17, Issue 1 e05551 Scientific OpinionOpen Access Safety assessment of the substance poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) for use in food contact materials EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP), EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP)Search for more papers by this authorVittorio Silano, Vittorio SilanoSearch for more papers by this authorJosé Manuel Barat Baviera, José Manuel Barat BavieraSearch for more papers by this authorClaudia Bolognesi, Claudia BolognesiSearch for more papers by this authorBeat Johannes Brüschweiler, Beat Johannes BrüschweilerSearch for more papers by this authorAndrew Chesson, Andrew ChessonSearch for more papers by this authorPier Sandro Cocconcelli, Pier Sandro CocconcelliSearch for more papers by this authorRiccardo Crebelli, Riccardo CrebelliSearch for more papers by this authorDavid Michael Gott, David Michael GottSearch for more papers by this authorKonrad Grob, Konrad GrobSearch for more papers by this authorEvgenia Lampi, Evgenia LampiSearch for more papers by this authorAlicja Mortensen, Alicja MortensenSearch for more papers by this authorInger-Lise Steffensen, Inger-Lise SteffensenSearch for more papers by this authorChristina Tlustos, Christina TlustosSearch for more papers by this authorHenk Van Loveren, Henk Van LoverenSearch for more papers by this authorLaurence Vernis, Laurence VernisSearch for more papers by this authorHolger Zorn, Holger ZornSearch for more papers by this authorLaurence Castle, Laurence CastleSearch for more papers by this authorJean-Pierre Cravedi, Jean-Pierre CravediMember of the former Working Group on "Food Contact Materials" of the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF).Search for more papers by this authorRoland Franz, Roland FranzMember of the former Working Group on "Food Contact Materials" of the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF).Search for more papers by this authorMartine Kolf-Clauw, Martine Kolf-ClauwMember of the former Working Group on "Food Contact Materials" of the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF).Search for more papers by this authorMaria Rosaria Milana, Maria Rosaria MilanaSearch for more papers by this authorKarla Pfaff, Karla PfaffSearch for more papers by this authorKettil Svensson, Kettil SvenssonMember of the former Working Group on "Food Contact Materials" of the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF).Search for more papers by this authorMaria de Fatima Tavares Poças, Maria de Fatima Tavares PoçasMember of the former Working Group on "Food Contact Materials" of the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF).Search for more papers by this authorDetlef Wölfle, Detlef WölfleMember of the former Working Group on "Food Contact Materials" of the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF).Search for more papers by this authorKatharina Volk, Katharina VolkSearch for more papers by this authorGilles Rivière, Gilles RivièreSearch for more papers by this author EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP), EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP)Search for more papers by this authorVittorio Silano, Vittorio SilanoSearch for more papers by this authorJosé Manuel Barat Baviera, José Manuel Barat BavieraSearch for more papers by this authorClaudia Bolognesi, Claudia BolognesiSearch for more papers by this authorBeat Johannes Brüschweiler, Beat Johannes BrüschweilerSearch for more papers by this authorAndrew Chesson, Andrew ChessonSearch for more papers by this authorPier Sandro Cocconcelli, Pier Sandro CocconcelliSearch for more papers by this authorRiccardo Crebelli, Riccardo CrebelliSearch for more papers by this authorDavid Michael Gott, David Michael GottSearch for more papers by this authorKonrad Grob, Konrad GrobSearch for more papers by this authorEvgenia Lampi, Evgenia LampiSearch for more papers by this authorAlicja Mortensen, Alicja MortensenSearch for more papers by this authorInger-Lise Steffensen, Inger-Lise SteffensenSearch for more papers by this authorChristina Tlustos, Christina TlustosSearch for more papers by this authorHenk Van Loveren, Henk Van LoverenSearch for more papers by this authorLaurence Vernis, Laurence VernisSearch for more papers by this authorHolger Zorn, Holger ZornSearch for more papers by this authorLaurence Castle, Laurence CastleSearch for more papers by this authorJean-Pierre Cravedi, Jean-Pierre CravediMember of the former Working Group on "Food Contact Materials" of the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF).Search for more papers by this authorRoland Franz, Roland FranzMember of the former Working Group on "Food Contact Materials" of the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF).Search for more papers by this authorMartine Kolf-Clauw, Martine Kolf-ClauwMember of the former Working Group on "Food Contact Materials" of the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF).Search for more papers by this authorMaria Rosaria Milana, Maria Rosaria MilanaSearch for more papers by this authorKarla Pfaff, Karla PfaffSearch for more papers by this authorKettil Svensson, Kettil SvenssonMember of the former Working Group on "Food Contact Materials" of the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF).Search for more papers by this authorMaria de Fatima Tavares Poças, Maria de Fatima Tavares PoçasMember of the former Working Group on "Food Contact Materials" of the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF).Search for more papers by this authorDetlef Wölfle, Detlef WölfleMember of the former Working Group on "Food Contact Materials" of the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF).Search for more papers by this authorKatharina Volk, Katharina VolkSearch for more papers by this authorGilles Rivière, Gilles RivièreSearch for more papers by this author First published: 25 January 2019 https://doi.org/10.2903/j.efsa.2019.5551 Correspondence: [email protected] Requestor: Ministry of Health, Welfare and Sports; The Netherlands Question number: EFSA-Q-2017-00412 Panel members: José Manuel Barat Baviera, Claudia Bolognesi, Beat Johannes Brüschweiler, Andrew Chesson, Pier Sandro Cocconcelli, Riccardo Crebelli, David Michael Gott, Konrad Grob, Evgenia Lampi, Alicja Mortensen, Gilles Rivière, Vittorio Silano, Inger-Lise Steffensen, Christina Tlustos, Henk Van Loveren, Laurence Vernis, Holger Zorn. Acknowledgements: The CEP Panel wishes to thank the WG on Food Contact Materials of the former EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) for the preparatory work on this scientific output. Note: The full opinion will be published in accordance with Article 10(6) of Regulation (EC) No 1935/2004 once the decision on confidentiality, in line with Article 20(3) of the Regulation, will be received from the European Commission. The following information has been provided under confidentiality and it is redacted awaiting the decision of the Commission: results of migration testing (food simulant E) of reaction products and/or residuals from the starting substance; report on fluid tests simulating gastric and intestinal pH conditions. Adopted: 5 December 2018 This output supersedes two previous scientific opinions on the Safety assessment of the substance Poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) for use in food contact materials (https://doi.org/10.2903/j.efsa.2016.4464 and https://doi.org/10.2903/j.efsa.2018.5326). AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract The EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP Panel) assessed the safety of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) (PHBH), CAS No 147398-31-0 and food contact material (FCM) substance No 1059. This biodegradable copolymer is produced by fermentation of palm oil using a genetically modified microorganism (Cupriavidus necator). Overall migration was up to 5.4 mg/kg. Oligomers are hydroxyl-terminated or with crotyl- and hexenyl end-groups from dehydration of hydroxyl end-groups. In the absence of calibration standards, the total oligomer migration was set at the overall migration values. Other degradation products are crotonic acid and (E)-2-hexenoic acid. Crotonic acid is authorised for use in FCMs with a specific migration limit (SML) of 0.05 mg/kg food. For (E)-2-hexenoic acid, no indication for genotoxicity was identified by the EFSA CEF Panel in its group evaluation of flavouring substances in FGE.05Rev2 (EFSA CEF Panel, 2010b). The other migrating substances detected, ■■■■■, are from the authorised substance 'palm oil and/or palm fatty acid distillate' (FCM substance No 9) used as a carbon source for the fermentation and do not give rise to safety concern. A PHBH oligomer mixture was synthesized to simulate that migrating. It did not give rise to concern for genotoxicity. From the repeated dose 90-day oral toxicity study in rats, the Panel identified the no-observed-adverse-effect level (NOAEL) at the highest dose tested in males, 1,364 mg/kg body weight (bw) per day. The Panel concluded that the potential for bioaccumulation of oligomers is low. Overall, the CEP Panel concluded that the substance PHBH is not of safety concern for the consumer if it is used alone or blended with other polymers in contact with all kinds of food during more than 6 months at room temperature or below, including hot-fill or a short heating up phase. The specific migration of all oligomers < 1,000 Da should not exceed 5 mg/kg food. The migration of crotonic acid should not exceed the SML of 0.05 mg/kg food. As the migration of (E)-2-hexenoic acid can be expected to be always lower than that of crotonic acid, no individual restriction is necessary. 1 Introduction 1.1 Background and Terms of Reference as provided by the requestor Before a substance is authorised to be used in food contact materials (FCM) and is included in a positive list EFSA's opinion on its safety is required. This procedure has been established in Articles 8, 9 and 10 of Regulation (EC) No 1935/20041 of the European Parliament and of the Council of 27 October 2004 on materials and articles intended to come into contact with food. According to this procedure, the industry submits applications to the Member States' competent authorities which transmit the applications to EFSA for their evaluation. In this case, EFSA received an application from the Ministry of Health, Welfare and Sport, the Netherlands, requesting the evaluation of the substance poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate), with the Chemical Abstracts Service (CAS) number 147398-31-0, and the FCM substance No 1059. The dossier was submitted by TNO Triskelion BV on behalf of Kaneka Belgium N.V. According to Regulation (EC) No 1935/2004 of the European Parliament and of the Council on materials and articles intended to come into contact with food, EFSA is asked to carry out an assessment of the risks related to the intended use of the substance and to deliver a scientific opinion. 2 Data and methodologies 2.1 Data The applicant has submitted a dossier in support of his application for the authorisation of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) (PHBH) to be used in FCM in contact with all kinds of food. Information provided in this dossier was already taken into consideration for a previous evaluation of the substance for use in contact with dry foods (EFSA CEF Panel, 2018), as requested by the European Commission in June 2017 (EFSA-Q-2017-00495). The current evaluation reflects, as requested by the applicant, the use of the substance with all foods, therefore including the previously assessed use with dry foods. Additional information was provided by the applicant during the assessment process in response to requests from EFSA sent on 12 July 20172, 20 October 20173, 20 December 20174 and 4 June 20184 (see 'Documentation provided to EFSA'). Following the requests for additional data sent by EFSA on 20 October 2017, 20 December 2017 and 04 June 2018, the applicant requested clarification teleconferences, which were held on 10 November 2017, 09 March 2018 and 19 June 2018. Data submitted and used for the evaluation are: Non-toxicological data and information Chemical identity Description of manufacturing process of substance/FCM Physical and chemical properties Intended use Existing authorisation(s) Identification, quantification and migration of oligomers, reaction products and impurities. Toxicological data Bacterial gene mutation tests In vitro mammalian cell gene mutation test In vivo mouse bone marrow micronucleus test repeated dose 90-day oral toxicity study in rats Fluid tests simulating gastric and intestinal pH conditions Information on accumulation in rats. 2.2 Methodologies The assessment was conducted in line with the principles laid down in Regulation (EC) No 1935/2004 on materials and articles intended to come into contact with food. This Regulation underlines that applicants may consult the Guidelines of the Scientific Committee on Food (SCF) for the presentation of an application for safety assessment of a substance to be used in FCM prior to its authorisation (European Commission, 2001), including the corresponding data requirements. The dossier that the applicant submitted for evaluation was in line with the SCF guidelines (European Commission, 2001). The methodology is based on the characterisation of the substance that is the subject of the request for safety assessment prior to authorisation, its impurities and reaction and degradation products, the evaluation of the exposure to those substances through migration and the definition of minimum sets of toxicity data required for safety assessment. To establish the safety from ingestion of migrating substances, the toxicological data indicating the potential hazard and the likely human exposure data need to be combined. Exposure is estimated from studies on migration into food or food simulants and considering that a person may consume daily up to 1 kg of food in contact with the relevant FCM. As a general rule, the greater the exposure through migration, the more toxicological data is required for the safety assessment of a substance. Currently, there are three tiers with different thresholds triggering the need for more toxicological information as follows: In case of high migration (i.e. 5–60 mg/kg food), an extensive data set is needed. In case of migration between 0.05 and 5 mg/kg food, a reduced data set may suffice. In case of low migration (i.e. < 0.05 mg/kg food), only a limited data set is needed. More detailed information on the required data is available in the SCF guidelines (European Commission, 2001). The assessment was conducted in line with the principles described in the EFSA Guidance on transparency in the scientific aspects of risk assessment (EFSA, 2009) and considering the relevant guidance from the EFSA Scientific Committee. 3 Assessment According to the applicant, the substance PHBH, CAS No 147398-31-0, is a biodegradable copolymer. It is intended to be used as such or compounded with other bio-based/biodegradable materials, such as starch, polylactic acid, polybutyrate adipate terephthalate and polybutylene succinate, to produce packaging articles like bags and trays at a maximum process temperature of 200°C. The articles are intended to be in contact with all kinds of food for long-term storage, i.e. 6 months or more at room temperature or below, including possible hot-fill or short heating up conditions. 3.1 Non-toxicological data 3.1.1 Physical and chemical properties Chemical formula: H[C4H6O2]x[C6H10O2]yOH (Figure 1) Figure 1Open in figure viewerPowerPoint Chemical structure of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) The substance PHBH is produced by fermentation of palm oil and/or palm oil fatty acid distillate as carbon source using a genetically modified microorganism (Cupriavidus necator). It consists of 80–99% 3-hydroxybutyrate (3HB) and 1–20% 3-hydroxyhexanoate (3HH). Before isolating the copolymer, the microorganisms are inactivated by heat treatment. The purity is higher than 98%. The molecular weight (Mw) ranges from 10,000 to 1,000,000 Da with Mw = 518,000 Da, number average molecular weight (Mn) = 182,000 Da. The fraction below 1,000 Da amounts to 0.5%. The substance has a melting point ranging from 120 to 150°C, depending on the monomeric composition. It is virtually insoluble in water and ethanol and is sparingly soluble in toluene, chloroform and tetrahydrofuran (THF). It is prone to dehydration at high temperature as well as to both dehydration and partial hydrolysis in aqueous media. 3.1.2 Migration studies Since the carbon source for the fermentation is palm oil, which is listed as FCM substance No 9 under Regulation (EU) No 10/2011 without a specific restriction, specific migration of this starting substance was not tested. In accord with the intended use in contact with all food types, migration tests were conducted with simulant E (modified polyphenylene oxide, for dry foods), 10% ethanol (for water and aqueous foods), 3% acetic acid (for acidic foods) as well as olive oil, isooctane and 95% ethanol (for fatty foods). A range of time and temperature conditions were used, up to and including 10 days contact at 60°C. All migration tests used a polymer containing 89 mol% of 3HB and 11 mol% of 3HH. Overall migration into 10% ethanol, 3% acetic acid and olive oil (each for 10 days/60°C) was respectively 5.4, 3.0 and not detectable (< 6 mg/kg). The overall migration into isooctane (10 days/60°C) and 95% ethanol (10 days/20°C) was much higher at 41 and 17 mg/kg. It was concluded that these alternative food simulants, isooctane and 95% ethanol, are not appropriate for this type of polar polyester and that the intended food contact uses are adequately covered by the other simulants used. Of all the liquid food simulants used, the 10% ethanol simulant was considered to be the most representative. Overall migration was not determined using the dry powder simulant E, but it is expected to be low. This simulant is especially diagnostic for testing the migration of volatile and semi-volatile substances. The migration extracts from the simulant E test (10 days/40°C) were analysed by gas chromatography–mass spectrometry (GC–MS) in order to investigate the presence and identity of other migrants, such as residuals from the starting substance or reaction products. Assuming uniform response factors for their semi-quantification, the following migrants and migration levels were found: ■■■■■ These substances were concluded to originate from or to be likely related to the source material 'palm oil and/or palm fatty acid distillate' already authorised under Regulation (EU) No 10/2011. The polymer end-groups can be dehydrated by heating and/or acid/base-catalysed reaction when the polymer is heated for processing into films and sheets or exposed to aqueous foods/simulants. A minor reaction pathway for dehydration is the formation of free crotonic acid ((E)-2-butenoic acid) and (E)-2-hexenoic acid. Crotonic acid is listed in Regulation (EU) No 10/2011 under FCM substance No 467 with a specific migration limit (SML) of 0.05 mg/kg food; (E)-2-hexenoic acid is listed as flavouring in Regulation (EC) No 872/2012 under [FL-no: 08.119]. In all migration tests conducted (simulant E, 3% acetic acid, 10% ethanol and olive oil), the migration of crotonic acid was below its SML. From the ratio of C4 to C6 units in the copolymer and assuming equal susceptibility to dehydration, the hexenoic acid should migrate at lower concentrations than crotonic acid. The 10% ethanol migration solutions, analysed using liquid chromatography–mass spectrometry (LC–MS) analysis, revealed a range of hydroxyl (OH-)-terminated (i.e. unchanged) and crotyl-terminated (i.e. dehydrated) oligomers from the dimer (n = 2) up to the limit of the MS scan range used (n = 12, Mw 1,050 for OH-terminated, Mw 1,032 for crotyl-terminated). The smaller oligomers (dimers, trimers, tetramers and pentamers) dominated in the 10% ethanol migration solution, particularly on the molar basis. Assuming a uniform molar MS response and correcting the MS response for the Mw, the OH-terminated oligomers were in the following proportions on a w/w basis: n = (1–4) at 25.8%, n = (1–5) at 43.0%, n = (1–6) at 50.9%. The crotyl-terminated oligomers were in the following proportions: n = (1–4) at 21.6%, n = (1–5) at 31.4% and n = (1–6) at 36.1%. The migration of all oligomers ≤ 1,000 Da, n = (1–11), was 59.8% hydroxyl- and 40.0% crotyl-terminated. The pentamers (Mw of 448 (OH-) or 430 (crotyl-)) were used as cut-off for characterising the 10% ethanol migrate, with 43.0% (OH-) and 31.4% (crotyl-) meaning that 74.4% of the total oligomer migration were oligomers < 500 Da. The applicant attempted to quantify the LC–MS results using 3-hydroxybutyric acid as a calibrant and assuming that the oligomers had the same response. This was unsuccessful because the sum of oligomers resulted about 10 times the overall migration value, which is not possible. The Panel decided, therefore, to scale the oligomer results using the overall migration value. For the 10% ethanol simulant the overall migration was 5.4 mg/kg. On this basis, the migration into 10% ethanol of oligomers up to and including the pentamer (i.e. Mw < 500 Da) is estimated to be 2.3 mg/kg of OH-terminated and 1.7 mg/kg of crotyl-terminated oligomers. In reality, a substantial part of the oligomers contains at least one 3HH unit and 11% of the end groups were probably 3HH and hexenoyl units in the same proportion as crotyls among the 3HB terminal units. As this has no effect on the comparison with the synthetic mixture used for toxicity testing (see Section 3.1.3), this aspect was not elaborated further. 3.1.3 Preparation and characterisation of the oligomer mix used in toxicity studies The substance (containing 11 mol% 3HH) was dissolved in hot aqueous acetonitrile and hydrolysed with sulfuric acid. After neutralisation and evaporation of the solvent, an oligomer mixture was obtained as a white precipitate. This mixture was analysed by 1H-nuclear magnetic resonance (1H-NMR) and 13C-NMR spectroscopy. It revealed OH-terminated oligomers with a varying number of 3HB and 3HH repeated units. No alkene groups, i.e. dehydrated end groups, were detected. The 3HH content was 11.9 mol%. The molecular weight estimated by gel permeation chromatography (GPC) was Mw = 2,000 Da; Mn: 1,050 Da, the fraction ≤ 1,000 Da 30%. LC–MS analysis found 15 PHBH oligomer peaks, of which 11 being < 1,000 Da. The applicant noted that LC–MS (using the atmospheric pressure chemical ionisation (APCI) technique) is not generally capable of detecting oligomers above ~ 1,000 Da. The smallest oligomer was a hexamer with a molecular weight of 535 Da. As described above, in the 10% ethanol migration solutions, substantial amounts of crotyl-terminated oligomers were present and the migrating oligomers were predominantly dimers, trimers and tetramers, i.e. with molecular weights considerably lower than the smallest oligomer identified in the oligomer mixture used for the toxicity studies. Therefore, its representativeness was questionable and so the applicant provided data on simulated gastric and intestinal stability tests. ■■■■■ The LC–MS data were treated using the same assumptions as described above for the migration studies and corrected for the fact that 70% of the original oligomer mix was > 1,000 Da, i.e. outside of the range covered by the LC–MS analysis. Then, the oligomer mix after simulated gastric treatment had the following composition on a w/w basis: the OH-terminated oligomers were ■■■■■; the crotyl-terminated oligomers were ■■■■■. Hence, the mixture contained ■■■■■ OH-terminated and ■■■■■ crotyl-terminated oligomers ≤ 1,000 Da (n = (1–11). As discussed above, the oligomers up to the pentamers were considered characteristic for the 10% ethanol migrates. The gastric-treated oligomers had a ■■■■■ content of OH-terminated oligomers and ■■■■■ crotyl-terminated oligomers than the migrate into 10% ethanol. If the oligomers ≤ 1,000 Da, n = (1–11), were used for the comparison, the corresponding figures would be ■■■■■ and ■■■■■ for the OH- and crotyl-terminated oligomers, respectively. The specific masses for the 3HH-containing oligomers were not looked for in this LC–MS analysis. As was done above for the 10% ethanol migrate, the Panel assumes that the oligomer mix after simulated gastric treatment, contains 3HH-units pro-rata to the polymer composition, being 11 mol% for the sample prepared and confirmed (at 11.9 mol%) by the NMR analysis. 3.2 Microbiological information Given the heat treatment and the applied purification steps at the end of the production process and the heat treatment during manufacturing of the final FCM, the use of Cupriavidus necator as a producing organism is considered to be of no safety concern. 3.3 Toxicological data 3.3.1 Evaluation of the substance itself No genotoxicity data are required for PHBH as the substance is a large polymer with a molecular weight ranging between 10,000 and 1,000,000 Da, which are unlikely to be absorbed in the gastrointestinal tract. Nevertheless, the applicant provided a complete set of genotoxicity tests, i.e. a bacterial reverse mutation test, an in vitro mammalian chromosome aberration test and an in vivo micronucleus bone marrow test. The in vitro tests were negative but limited due to the precipitation of the compound in the culture medium. No increase in micronuclei frequency was observed in the in vivo test in mice, but no evidence was provided for the exposure of the bone marrow. The fraction below 1,000 Da amounts to 0.5%, with the major part being above 500 Da. Nevertheless, the Panel noted that the major unit of this polymer, 3-hydroxybutyric acid, is an intermediate in fatty acid metabolism. For the minor unit of this polymer, 3-hydroxyhexanoic acid, a bacterial reverse mutation test according to OECD TG471 was provided. It was tested for gene mutation in four histidine-requiring strains TA98, TA100, TA1535 and TA1537 of Salmonella Typhimurium, and in Escherichia coli strain WP2 uvrA both in the absence and in the presence of metabolic activation up to 5,000 μg/plate. The results of the study were considered negative. 3.3.2 Evaluation of the migrating oligomers The PHBH oligomer mixture, synthesized as described above to simulate the migration mixture into 10% ethanol, was tested in a basic battery of genotoxicity tests, i.e. a bacterial reverse mutation test, an in vitro mammalian cell gene mutation test, and an in vitro mammalian chromosome aberration test, and in a repeated dose 90-day oral toxicity study. As derived above, even after simulated gastric treatment, the composition of the synthesized oligomers deviated substantially from that of the oligomers in the 10% ethanol migrate, both in terms of molecular mass distribution and proportion of crotyl-terminated species. The data of the toxicological experiments were nonetheless considered and evaluated together with other facts. A bacterial reverse mutation (Ames) test (plate incorporation method) with the PHBH oligomer was conducted according to OECD guideline 471 and in compliance with Good laboratory practice (GLP) guidelines. S. Typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli strain WP2 uvrA were used in the absence and presence of metabolic activation (S9-mix). The test substance PHBH oligomer was dissolved in dimethyl sulfoxide (DMSO). A single experiment was performed in triplicate at the concentrations: 62, 185, 556, 1,667 and 5,000 μg/plate. No cytotoxicity was observed at any bacterial strain tested up to 5,000 μg/plate. A dose-related precipitation was observed at and above a concentration of 556 μg/plate. The test substance did not induce statistically significant increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control in any bacterial strain tested both in the absence and presence of S9-mix. An in vitro mammalian cell gene mutation test at the TK-locus of cultured mouse lymphoma L5178Y cells in the absence and presence of metabolic activation (S9-mix) was carried out according to OECD guideline 490 and in compliance with GLP. The PHBH oligomer mixture was dissolved in DMSO. The maximum final concentration of PHBH oligomer (800 μg/mL) was limited by solubility of the test substance in culture medium. No cytotoxicity was observed after 4 and 24 h treatment in the presence of S9-mix and 24 h in the absence of S9-mix. Following 4 h treatment in the absence of S9-mix, the test substance PHBH oligomer was found to be cytotoxic at the highest concentration tested. No increase in mutant frequency