Safety assessment of the substance amines, di-C14-C20-alkyl, oxidised, from hydrogenated vegetable oil, for use in food contact materials

Abstract

The EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) assessed the safety of amines, di-C14-C18-alkyl, oxidised, renamed by the Panel as amines, di-C14-C20-alkyl, oxidised, from hydrogenated vegetable oil. The substance amines, bis(hydrogenated tallow alkyl) oxidised, consisting of the same components, but originating from tallow, is currently authorised as FCM substance No 768. The vegetable-sourced substance is intended to be used at up to 0.1% w/w as antioxidant and/or stabiliser in the manufacture of polyolefin food contact materials (FCM) and articles intended for contact with dry, aqueous and acidic foods. The substance is a mixture consisting of linear N,N-dialkyl hydroxylamines and their corresponding amine, nitrone and oxime derivatives, as well as further components: tert-N-oxides, secondary amides and carboxylic acids. Specific migration was tested from polyethylene samples in 10% ethanol and 3% acetic acid for 2 h at 100°C followed by 10 days at 60°C. None of the non-authorised components were detected to migrate at detection limits (LoD) in the range 0.003–0.029 mg/kg. The LoD of authorised carboxylic acids was 0.35 mg/kg. The Panel reassessed the genotoxicity studies carried out on FCM No 768 and evaluated two new bacterial reverse mutation tests on the nitrone and oxime derivatives as well as new (qualitative/quantitative) structure–activity relationship (Q)SAR analyses on other components. The Panel concluded that the substance did not raise a concern for genotoxicity. The Panel concluded that the substance is not of safety concern for the consumers if it is used as an additive at 0.1% w/w in the manufacture of polyolefin FCM intended to be in contact with foods simulated by food simulants A, B, C and E, except for infant formula and human milk, for storage above 6 months at room temperature and below, including hot-fill conditions and heating up to 100°C for 2 h.

1 INTRODUCTION

1.1 Background and Terms of Reference

Before a substance is authorised to be used in food contact materials (FCM) and is included in a positive list, the European Food Safety Authority (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 competent authorities of Member States, which transmit the applications to the (EFSA) for evaluation.

In this case, EFSA received an application from the German competent authority (Bundesamt für Verbraucherschutz und Lebensmittelsicherheit, BVL), requesting the safety evaluation of the substance amines, di-C14-C18-alkyl, oxidised, FCM substance No 1092, with the CAS number 1801863-42-2. The request has been registered in EFSA's register of received questions under the number EFSA-Q-2021-00555. The dossier was submitted by BASF SE.

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 confidential and a non-confidential version of a dossier following the ‘EFSA Note for Guidance for the preparation of an application for the Safety Assessment of a Substance to be used in Plastic Food Contact Materials (EFSA CEF Panel, 2008) and the ‘Administrative guidance for the preparation of applications on substances to be used in plastic food contact materials' (EFSA, 2021).

In accordance with Art. 38 of the Commission Regulation (EC) No 178/2002² and taking into account the protection of confidential information and of personal data in accordance with Articles 39 to 39e of the same Regulation and of the Decision of the EFSA's Executive Director laying down practical arrangements concerning transparency and confidentiality,³ the non-confidential version of the dossier is published on Open.EFSA.⁴

According to Art. 32c(2) of Regulation (EC) No 178/2002 and to the Decision of EFSA's Executive Director laying down the practical arrangements on pre-submission phase and public consultations,³ EFSA carried out a public consultation on the non-confidential version of the application from 5 July to 26 July 2023, for which no comments were received.

Additional information was provided by the applicant during the assessment process in response to requests from EFSA sent on 21 April 2022 (see ‘Documentation provided to EFSA’).

Data submitted and used for the evaluation are:

Non-toxicological data and information

• Chemical identity
• Description of manufacturing process of the substance and of the FCM
• Physical and chemical properties
• Intended application
• Migration of the substance
• Residual content of the substance in the FCM
• Identification, quantification and migration of reaction products

Toxicological data

Amines, bis(hydrogenated tallow alkyl) oxidised:

• Bacterial gene mutation test
• In vitro mammalian cell gene mutation test
• In vitro mammalian chromosome aberration test
• 90-day oral toxicity study in rats
• 90-day oral toxicity study in dogs
• One-generation reproduction toxicity study⁵

Amines, di-C14-C18-alkyl:

• Bacterial gene mutation test
• In vitro mammalian chromosome aberration test

Nitrones, di-C14-C18-alkyl:

• Bacterial gene mutation test⁵
• In vitro mammalian chromosome aberration test

Oximes, C14-C18-alkyl:

• Bacterial gene mutation test⁵
• In vitro mammalian chromosome aberration test

Tertiary N-oxides, tri-C14-C18-alkyl and aldehydes, C14-C18-alkyl and secondary amides, C14-C18-alkyl and carboxylic acids, C14-C18-alkyl:

• In silico data⁵

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 are 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:

1. In case of high migration (i.e. 5–60 mg/kg food), an extensive data set is needed.
2. In case of migration between 0.05 and 5 mg/kg food, a reduced data set may suffice.
3. 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 ‘amines, di-C14-C18-alkyl, oxidised’, renamed by the Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) as ‘amines, di-C14-C20-alkyl, oxidised, from hydrogenated vegetable oil’, is a non-defined process mixture. It is called ‘the substance’ hereafter. It is intended to be used in polyolefins at up to 0.1% w/w as antioxidant and/or stabiliser to reduce the degradation of polyolefins during their manufacture. Final articles are intended for contact with dry, aqueous and acidic foods for long-term storage above 6 months at room temperature and below, including hot-fill conditions and/or heating up to between 70°C and 100°C for a maximum period calculated as t = 120/2^((T – 70)/10) min.⁶ The uses requested by the applicant do not specify contact with infant formula and human milk.

The substance ‘amines, bis(hydrogenated tallow alkyl) oxidised’ consisting of the same components but originating from a different source material (tallow), was evaluated by the SCF in 2003 (European Commission, 2003). It is currently authorised according to Annex I of Regulation (EU) No 10/2011⁷ as FCM substance No 768 with the following restrictions: ‘not to be used for articles in contact with fatty foods for which simulant D1 and/or D2 is laid down, only to be used in: polyolefins at 0.1% (w/w) concentration and in PET at 0.25% (w/w) concentration, and verification of compliance by residual content per food contact surface area (QMA) pending the availability of an analytical method’. Its synthesis uses tallow as starting material, while the substance subject of this application uses vegetable oil.

3.1 Non-toxicological data

3.1.1 Identity of the substance⁸

The substance is a mixture consisting of linear N,N-dialkyl hydroxylamines and their corresponding amine, nitrone and oxime derivatives, as well as further components: tert-N-oxides, secondary amides and carboxylic acids. The composition as reported by the applicant is shown in Table 1. N,N-dialkyl hydroxylamines and the nitrones are the main functional components.

TABLE 1. Composition, average molecular weight (MW), chemical formula and structure of the individual components of the substance, as reported by the applicant.

Component	% (w/w)	Average MW	Chemical formula of the C18 component	Chemical structure^9
Hydroxylamines, di-C14-C18-alkyl	■■■■■	516	C36H75NO
Amines, di-C14-C18-alkyl	■■■■■	500	C36H75N
Nitrones, di-C14-C18-alkyl	■■■■■	514	C36H73NO
Oximes, C14-C18-alkyl	■■■■■	272	C18H37NO
Tert-N-oxides, tri-C14-C18-alkyl	■■■■■	756	C54H111NO
Aldehydes, C14-C18-alkyl	■■■■■	257	C18H36O
Secondary amides, di-C14-C18-alkyl	■■■■■	514	C36H73NO
Carboxylic acids, C14-C18-alkyl	■■■■■	273	C18H36O2

The substance subject of this application was synthesised from a vegetable oil (of not disclosed identity) with 32.9% C16- and 65.5% C18- alkyl chains. C14- and C20- alkyl chains were also present in low percentages (1%–2%). The Panel noted that the composition of the fatty acids within a given vegetable oil varies and even more between different vegetable oils. Therefore, the Panel considered that C14 and C20 should be included in the name of the substance and renamed it as ‘amines, di-C14-C20-alkyl, oxidised, from hydrogenated vegetable oil’.

The carboxylic acids myristic- (C14), palmitic- (C16), stearic- (C18) and arachidic- (C20) acids are authorised to be used in plastic FCM according to Annex I of Regulation (EU) 10/2011 (FCM No 348, 105, 106 and 345, respectively) and as acids, fatty, from animal or vegetable food fats and oils under FCM No 12, all without a specific migration limit (SML).¹⁰

The purity of the substance is specified as minimum ■■■■■% of ■■■■■. For nine provided tested batches, the compositions were within the ranges given in Table 1. Besides the above-mentioned components, two impurities, both authorised in accordance with Reg. (EU) 10/2011 without specific restriction, ■■■■■ (FCM No ■■■■■) and ■■■■■ (FCM No ■■■■■), are specified at levels < ■■■■■% and < ■■■■■%, respectively.

The tallow-sourced substance, the amines, bis(hydrogenated tallow alkyl) oxidised, the composition of which is given in the SCF Opinion (European Commission, 2003) and reported in Table 2, is composed of the same components as the substance under assessment, and similarly to it, the composition varies depending on the source. Also tallow may include low percentages of C14 and C20 acids. The chain lengths of the main fatty acids in tallow are within the range of the fatty acids of vegetable oils.

TABLE 2. Composition of FCM No 768 as given in the application assessed by the SCF (European Commission, 2003).

Component	% (w/w)
Bis(hydrogenated tallow, C16-C18, alkyl) hydroxylamines	63–80
Bis(hydrogenated tallow, C16-C18, alkyl) amines	12–20
Bis(hydrogenated tallow, C16-C18, alkyl) nitrones	2–10
(Hydrogenated tallow, C16-C18, alkyl) oximes	2–10
Fatty acids (C16-C18)	0–5
(C16-C18) secondary amides	0–7

3.1.2 Physical and chemical properties¹¹

All components of the substance are highly lipophilic; the log Po/w of the mixture was not measurable and, hence, was calculated for the main component to be 14.15. The solubilities in water of all components were measured at 25°C and found to be below the limit of detection (LoD) of 20 μg/L. The solubility can be anticipated to be marginally higher in real circumstances: (i) at higher temperatures of use of the FCM; (ii) in aqueous food simulants containing an organic component (e.g. in simulants A and B); and (iii) in aqueous foods and beverages.

Thermogravimetric analysis (TGA) provided by the applicant showed that thermal decomposition starts at around 150°C, with a 2% weight loss at 200°C, which is due to loss of volatiles. Therefore, no relevant thermal degradation of the substance is expected at the provided specification of the maximum temperature of manufacture of polyolefins (200°C). The Panel noted that tert-alkyl-N-oxides are thermally unstable under the polymer processing conditions; but they decompose by the Cope elimination into the N,N-dialkyl hydroxylamines with the formation of alpha olefins (Cope et al., 1949), one of them being the C14 olefin which is authorised as 1-tetradecene, FCM No 388, with an SML of 0.05 mg/kg food. Part of the N,N-dialkyl hydroxylamines is consumed by grafting onto the polymer chains.

3.1.3 Residual content and specific migration of the substance

The residual contents¹² of all the components in the test samples, except for the tert-alkyl-N-oxides, were determined by extractive solvent dissolution/precipitation of the polymer and by exhaustive solvent extraction, followed by applying the same analytical methods as for the specific migration tests below. In agreement with the technological function of the substance, the N,N-dialkyl hydroxylamines were no longer the main component or not detectable in the polymer at the LoD of 10 mg/kg polymer. For all the other components, the individual residual amounts in the polymer samples were in accordance with the transformation chemistry of the substance. The total nitrogen determinations in the test samples confirmed that they contained the substance at the intended use level of approximately 0.1% w/w.

The specific migration¹³ of all substance components, again except the tert-alkyl-N-oxides, has been tested from low-density polyethylene (LDPE), high-density polyethylene and polypropylene samples in contact with 10% ethanol (food simulant A) and 3% acetic acid (food simulant B) for 2 h at 100°C followed by 10 days at 40°C. It was below the limits of quantification (LoQ) ranging from 0.14 to 0.73 mg/kg.

Considering such high LoQ, the Panel requested second migration tests, which were carried out using an analytical method with an improved sensitivity. Hydroxylamine, amine, nitrone, secondary amide and oxime derivatives were determined by ultra-performance convergence chromatography (UPC²) with photo diode array (PDA) detection and quadrupole mass analyser. The aldehyde derivatives were detected by ultra-performance liquid chromatography (UPLC) with tunable ultraviolet (UV) detector; stearic acid by high-pressure liquid chromatography coupled with mass spectrometry (HPLC/MS). The migration tests were performed using the maximum intended use level of 0.1% w/w of the substance in LDPE samples, again using simulants A and B. More severe contact conditions were applied: 2 h at 100°C followed by 10 days at 60°C. Considering these testing conditions and that the migrants are not volatile, the Panel agreed that no testing for dry foods was required (additionally, Annex V, Chapter 2 of Reg. 10/2011 does not require simulant E tests below 100°C). None of the components were detected to migrate at LoD of 0.008 mg/kg for hydroxylamines, 0.025 mg/kg for the amines, 0.003 mg/kg for the nitrones and 0.029 mg/kg for the oximes, as well as 0.004, 0.007 and 0.35 mg/kg for the secondary amides, aldehydes and stearic acid, respectively. The Panel noted that the alpha olefins C14-C20 are lipophilic with very low solubilities in aqueous simulants (e.g. around 12 μg/L for 1-tetradecene), hence, they are not expected to exceed a migration of 0.05 mg/kg food.

The Panel noted that the results from the second test can be considered representative for polyolefins and overrule the data from the first test. For the reasons explained in Section 3.1.2, the tert-alkyl-N-oxides were not included in the migration test. Furthermore, even if they were still present in the polymer, due to their higher lipophilicity, their migration would be expected to be orders of magnitude lower than those measured for the other components.

As specific migration tests were carried out into aqueous simulants A and B and the components are lipophilic, migration is expected to be controlled by partitioning, rapidly reaching equilibrium. Therefore, migration into the aqueous simulant C (20% ethanol) is expected to be comparable to that into simulant A.

3.2 Toxicological data

No toxicological data were submitted on the substance, ‘amines, di-C14-C20-alkyl, oxidised, from hydrogenated vegetable oil’, but instead on the FCM No 768, the ‘oxidised bis(hydrogenated tallow alkyl) amines’, and on the corresponding amine, nitrone and oxime derivatives. The Panel considered the studies on the tallow-sourced substance appropriate for the assessment of the substance (vegetable-sourced), because, as explained in Section 3.1.1, both substances consist of essentially the same components.

C14-, C16-, C18-, C20- carboxylic acids and, more generally, acids, fatty, from animal or vegetable food fats and oils are authorised in accordance with Annex I of Reg. (EU) 10/2011 (respectively as FCM No 348, 105, 106, 345 and 12) without any specific migration limits, and therefore they are not addressed further. Also, C14-alpha olefin (1-tetradecene) is authorised according to Reg. (EU) 10/2011 as FCM No 388 with an SML of 0.05 mg/kg food and therefore is not addressed further.

The migration of the components of the substance other than carboxylic acids was not detected at the LoD (in the range 3–29 μg/kg food simulant). Therefore, according to the ‘EFSA Note for guidance’ (EFSA CEF Panel, 2008), only their genotoxicity potential should be addressed (migration below 0.05 mg/kg food).

In addition to genotoxicity studies, two 90-day studies in rats and dogs, which were already evaluated by the SCF (European Commission, 2003), and a new one-generation reproduction toxicity study (OGRTS) were submitted, even if, in principle, not required. The 90-day study in rats followed an old Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) (No 408, 1981) without addressing endpoints that would be considered by the current one (OECD, 2018). In addition, no certificate of analysis was provided for the OGRTS, and no concern that would have affected the outcome of the assessment and its restriction was identified from the 90-day and the OGRT studies. Consequently, and since those studies were not required, they were not reported in the opinion.

With regards to the genotoxicity studies, those already evaluated by SCF (2003) plus two bacterial reverse mutation tests on the nitrone and oxime derivatives, respectively, were provided. Even though the genotoxicity studies evaluated by SCF were carried out in accordance with the standards and guidelines relevant at that time, considering the publication of several EFSA's guidance documents for assessing genotoxicity over the last 20 years, the Panel considered appropriate to reassess them.

3.2.1 Genotoxicity¹⁴

The genotoxic potential of the substance was assessed with an Ames test, a mouse lymphoma test and an in vitro chromosome aberration test in Chinese hamster lung (CHL) cells carried out with the whole mixture ‘bis(hydrogenated tallow alkyl) amines, oxidised’ (FCM No 768) which was considered acceptable to cover the main components, the hydroxylamine and the amine derivatives. Furthermore, the nitrone and the oxime derivatives were assessed with a battery of reliable genotoxicity tests, that is, Ames test and in vitro chromosomal aberration test. The two in vitro micronucleus tests foreseen in the ‘EFSA Note for Guidance’ (EFSA CEF Panel, 2008) were not submitted, but both the in vitro chromosomal aberration tests reported the numerical chromosomal aberrations, hence covering the aneugenicity end-point. Secondary amides and aldehydes were assessed with in silico data.

As explained in Section 3.1.2, tert-alky-N-oxides decompose into the component N,N-dialkyl hydroxylamines, being no longer present in the polymer, and can be expected to migrate, if at all, below 0.15 μg/kg food (corresponding to the threshold of toxicological concern of 0.0025 μg/kg body weight per day, EFSA Scientific Committee, 2012).

Neither the C16-, C18- and C20- alpha olefins were assessed individually, as they are structurally related to the C14 one which is authorised as FCM No 388 with a SML of 0.05 mg/kg food based on the lack of genotoxicity potential, and all together are not expected to exceed a migration of 0.05 mg/kg food (see Section 3.1.3).

The Panel acknowledged that percentages of C14- and C20- components are low for being covered by the genotoxicity tests. However, considering that C14 and C20 are structurally related to C16 and C18, the Panel accepted read across from C16-C18.

3.2.1.1 Oxidised bis(hydrogenated tallow alkyl) amines

3.2.1.1.1 Bacterial reverse mutation test

‘Oxidized bis(hydrogenated tallow alkyl) amines’ was tested in a bacterial reverse mutation test (Ames test) according to the OECD Test Guideline 471 (OECD, 2020) and following Good Laboratory Practice (GLP). Four Salmonella Typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA/pKM101 were used. Two separate experiments in triplicate were performed both in the absence and in the presence of metabolic activation by Aroclor 1254-induced rat liver S9 fraction (S9-mix) applying the preincubation method. The substance was dissolved in acetone and tested at five concentrations from 312.5 to 5000 μg/plate, determined in a preliminary toxicity test. Precipitation in the plates was observed at all the concentrations tested. No signs of toxicity and no increase in the number of revertant colonies were observed. Based on the ‘Harmonised approach for reporting reliability and relevance of genotoxicity studies’ (EFSA, 2023), the Panel considered the study reliable without restrictions and the negative results to be of high relevance.

3.2.1.1.2 In vitro mammalian cell gene mutation test

‘Oxidized bis(hydrogenated tallow alkyl) amines’ was tested in the in vitro mammalian cell gene mutation test using the thymidine kinase gene according to OECD TG 476 (1984) and following GLP. Two separate experiments were carried out in duplicate cultures of L5178Y mouse lymphoma cells in presence and absence of metabolic activation from Aroclor 1254-induced rat liver S9 fraction. The substance was dissolved in DMSO and tested up to the limit of solubility, at concentrations of 10, 33, 100 and 333 μg/mL for 3 h in the presence of S9-mix and for 24 h in the absence of S9-mix. The 24-h extensive treatment was considered because, based on the results of the range finding test, the selected dose range did not include a clearly toxic concentration. A slight precipitation was reported at 333 μg/mL. Both in the absence and presence of S9-mix, no reduction was observed in the cell count or in the cloning efficiency. In the absence of S9-mix, the test substance did not induce any significant increase in the mutant frequency. In the presence of S9-mix, a twofold increase in mutant frequency was reported only at 100 μg/mL in the second experiment. This increase was not reproduced in the first experiment and no dose response was observed; therefore, this result was considered not biologically relevant. The Panel considered the study reliable with restrictions because the experimental protocol did not comply with the most recent OECD TG 490 (OECD, 2016a, 2016b) (the range of concentrations applied did not reach the level of cytotoxicity recommended) and the negative results to be of limited relevance.

3.2.1.1.3 In vitro mammalian chromosomal aberration test

Oxidised bis(hydrogenated tallow alkyl) amines was tested in a chromosomal aberration test in Chinese Hamster lung cells, with and without metabolic activation from Aroclor 1254-induced rat liver S9 fraction according to OECD TG 473 (2020) and in compliance with GLP. Two separate experiments were carried out in duplicate. The substance was dissolved in DMSO and tested up to the limit of solubility, at concentrations of 10, 33, 100 and 333 μg/mL.

In the first experiment, in the presence of S9-mix, cells were exposed for 3 hours followed by 24 and 48 hours recovery periods. In the absence of S9-mix, cell cultures were treated for 3 hours with 24 hours recovery period and for 24 and 48 hours continuous treatment. In the second experiment, cells were treated for 3 hours followed by 24 h recovery period in the presence of S9-mix and for 24 h continuous treatment in the absence of S9-mix.

At 333 μg/mL, some precipitation was reported. The mitotic index was not reduced significantly. No statistically significant increase in structural or numerical chromosomal aberrations was reported at any condition of treatment. The Panel considered the study reliable with restrictions because the experimental protocol did not comply with the most recent OECD TG 473 (OECD, 2016a, 2016b) (100 metaphases chromosome spreads per culture instead of 300 were examined. No historical controls were provided). The negative results were considered to be of limited relevance.

3.2.1.2 Amines, di-C14-C20-alkyl

In addition to the above tests provided on the mixture that were considered sufficient to rule out the concern of genotoxicity for the amine derivatives (present as major component of the mixture), they were also tested in an in vitro chromosomal aberration test.

3.2.1.2.1 In vitro mammalian chromosomal aberration test

The chromosomal aberration test was carried out in Chinese Hamster lung cells, with and without metabolic activation from phenobarbital/β-naphthoflavone-induced rat livers (S9-mix) according to OECD TG 473 (2020) and in compliance with GLP. The substance was suspended in ethanol. A range finding test was performed from 9.8 to 1250 μg/mL with 4 and 24 h treatment. Precipitation was observed at 156 μg/mL and above in the absence of S9-mix and at 312 μg/mL and above in the presence of S9-mix. Cytotoxicity, detected as reduced cell numbers below 50% of control, was observed after 4 hours treatment at 78.1 μg/mL and above in the absence of S9-mix and after treatment with 156 μg/mL the presence of S9-mix. In the first experiment, cells were exposed for 4 hours followed by 14 h recovery period at the concentration of 12.5, 25 and 50 μg/mL in the absence of S9-mix and at 100, 200 and 300 μg/mL in the presence of S9-mix. In the second experiment, cells were treated for 4 h with 24 h recovery at 100, 200 and 300 μg/mL in the presence of S9-mix and for 18 h continuous treatment at 5, 10, 20 and 40 μg/mL in the absence of S9-mix. Due to the high cytotoxic effects observed at 40 μg/mL (72%), a further experiment was carried out with 18 hours continuous treatment at a single concentration of 40 μg/mL. Cytotoxicity, evaluated as mitotic indices reduced by 50% or more, were observed at the highest concentrations in the absence and presence of S9-mix. No biologically relevant increase in cells with structural or numerical chromosomal aberrations was reported. The Panel considered the study reliable with restrictions because the experimental protocol did not comply with the most recent OECD TG 473 (OECD, 2016a, 2016b) (100 metaphases chromosome spreads per culture instead of 300 were examined. No historical controls were provided). The negative results were considered to be of limited relevance.

The experimental data provided on the substance, oxidised bis(hydrogenated tallow alkyl) amines were considered sufficient for the assessment of the N,N-dialkyl hydroxylamines, because it is present at high percentage (min. ■■■■■%) in the substance.

3.2.1.3 Nitrones, di-C14-C20-alkyl

3.2.1.3.1 Bacterial reverse mutation test

Nitrone derivatives (batch KOLAMBP-L-00594, purity > 90%) were tested in a bacterial reverse mutation test (Ames test) according to the OECD TG 471 (2020) and following GLP. Four Salmonella Typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA/pKM101 were used. Two separate experiments in triplicate were performed both in the absence and in the presence of metabolic activation by phenobarbital/ β-naphthoflavone -induced rat liver S9 fraction (S9-mix). The test substance was suspended in ethanol and then treated with ultrasonic waves. The test concentrations were adjusted considering the purity of the substance. In a first experiment, the substance was tested at six concentrations 33, 100, 333, 1000, 2500 and 5000 μg/plate applying the standard plate incorporation method. A second experiment was performed at the same concentrations applying the preincubation method. Some precipitation was reported starting from 100 μg/plate. A bacteriotoxic effect was observed in the standard plate incorporation test and in the preincubation assay depending on the strain and test conditions, at and above 333 μg/plate in the absence of S9-mix and at and above 100 μg/plate in the presence of S9-mix. The test substance did not induce a biologically relevant increase in the number of revertant colonies with or without S9-mix.

The Panel considered the study reliable without restrictions and the negative results to be of high relevance.

3.2.1.3.2 In vitro mammalian chromosomal aberration test

Nitrone derivatives were tested in a chromosomal aberration test in Chinese Hamster lung cells, with and without metabolic activation from phenobarbital/β-naphthoflavone-induced rat livers (S9-mix) according to OECD TG 473 (2020) and in compliance with GLP. Two separate experiments were carried out in duplicate. The substance was suspended in ethanol and tested up to the limit of solubility, at concentrations of 1.25, 2.5, 5.0 and 10.0 μg/mL. In the first experiment, cells were exposed without and with S9-mix for 4 h followed by 14 h recovery period. In the second experiment, cells were treated for 4 h in the presence of S9-mix with 24 h recovery, and for 18 and 28 h continuous treatment in the absence of S9-mix. Only two concentrations (5.0 and 10.0 μg/mL) were applied in the second experiment with the 28 h continuous treatment. Precipitation was reported at 5 μg/mL and above in all experiments, except in the first experiment without S9-mix. No toxicity was observed. No biologically relevant increase in cells with structural or numerical chromosomal aberrations was reported. The Panel considered the study reliable with restrictions because the experimental protocol did not comply with the most recent OECD TG 473 (OECD, 2016a, 2016b) (100 metaphases chromosome spreads per culture instead of 300 were examined. No historical controls were provided). The negative results were considered to be of limited relevance.

3.2.1.4 Oximes, C14-C20-alkyl

3.2.1.4.1 Bacterial reverse mutation test

Oxime derivatives (batch KOLAMBP-L-00592, purity > 90%) was tested in a bacterial reverse mutation test (Ames test) according to the OECD TG 471 (2020) and following GLP. Four Salmonella Typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA/pKM101 were used. Two separate experiments in triplicate were performed both in the absence and in the presence of metabolic activation by phenobarbital/beta-naphthoflavone-induced rat liver S9 fraction (S9-mix). The test substance was finely ground in a mortar and then suspended in ethanol. The test concentrations were adjusted considering the purity of the substance. In a first experiment the substance was tested at six concentrations 33, 100, 333, 1000, 2500 and 5000 applying the standard plate incorporation method. A second experiment was performed at the same concentrations applying the preincubation method. A bacteriotoxic effect was observed in the standard plate incorporation test and in the preincubation assay depending on the strain and test conditions at and above 100 μg/plate. In strain TA1535, the bacteriostatic effect was reported starting from the lowest concentration tested. A third experiment was carried out in this strain with adjusted concentrations 3.3, 10, 33, 100, 333 and 1000 μg/plate using the preincubation test. Bacteriostatic effect was recorded at and above 333 μg/plate. No relevant increase in the revertant colony number was reported in any strain at any condition of treatment.

The Panel considered the study reliable without restrictions and the negative results to be of high relevance.

3.2.1.4.2 In vitro mammalian chromosomal aberration test

Oxime derivatives were tested in a chromosomal aberration test in Chinese Hamster lung cells, with and without metabolic activation from phenobarbital/β-naphthoflavone induced rat livers (S9-mix) according to OECD TG 473 (2020) and in compliance with GLP. Two separate experiments were carried out in duplicate. The substance was suspended in ethanol and tested up to the limit of solubility, at concentrations of 12.5, 25, 50 and 100 μg/mL. In the first experiment, cells were exposed to the test item without and with S9-mix for 4 h followed by 14 hours recovery period. In the second experiment, cells were treated for 4 h in the presence of S9-mix with 24 h recovery, and for 18 and 28 h continuous treatment in the absence of S9-mix. Three concentrations (25, 50 and 100 μg/mL) were applied in the second experiment with 28 h continuous treatment. Precipitation was reported at 50 μg/mL and above in all experiments. No toxicity was observed. No biologically relevant increase in cells with structural or numerical chromosomal aberrations was reported. The Panel considered the study reliable with restrictions because the experimental protocol did not comply with the most recent OECD TG 473 (OECD, 2016a, 2016b) (100 metaphases chromosome spreads per culture instead of 300 were examined. No historical control data were provided). The negative results were considered to be of limited relevance.

3.2.1.5 Carboxylic C14-C20 amides

No experimental data have been provided for the amide derivatives. The genotoxicity of the compound was assessed with in silico tools. Structural alerts were investigated with profilers available in the OECD QSAR Toolbox and the outcome of an Ames and a chromosomal aberration study, as well as an in vivo micronucleus test were predicted using OASIS Times and DEREK. No alerts were triggered using the OECD toolbox regarding DNA binding and in vitro and in vivo mutagenicity. OASIS Times predicted the carboxylic amide derivatives to be non-mutagenic and non-clastogenic. Negative results were also obtained with an in vivo micronucleus model. The model classified the substance as inside the model's parametric domain as well as structural domain. DEREK predicted the carboxylic amide derivatives to be not mutagenic in an Ames test. No alerts were identified for chromosomal aberrations.

3.2.1.6 C14-C20 alkyl aldehydes

The genotoxicity of the compound was assessed with in silico tools. Structural alerts were investigated with profilers available in the OECD QSAR Toolbox and the outcome of an Ames and a chromosomal aberration study as well as an in vivo micronucleus test were predicted using OASIS Times and DEREK. Additionally, prediction of mutagenicity in bacteria and an in vivo micronucleus test were conducted using CASE Ultra. No alert for genotoxicity were identified.

3.2.1.7 Conclusions on genotoxicity

Oxidised bis(hydrogenated tallow alkyl) amines was evaluated in a battery of reliable in vitro genotoxicity studies. In the presence or absence of S9-mix, the tallow-sourced substance did not induce gene mutations in bacteria (four strains of Salmonella Typhimurium, TA1535, TA1537, TA98 and TA100, and one strain of E. coli WP2 uvrA) and in mammalian cells (mouse lymphoma L5178Y cells). The tallow-sourced substance did not induce structural and numerical chromosomal aberrations in Chinese Hamster lung cells in the presence and absence of S9-mix.

The negative results of a chromosomal aberration study on the amine derivatives and of the battery of in vitro tests on the tallow-sourced substance were considered sufficient to rule out the concern for genotoxicity of the hydroxylamine and amine derivatives, present as major components (■■■■■) in the substance.

Nitrone and oxime derivatives were evaluated in a basic battery of reliable genotoxicity tests: an Ames test and a chromosomal aberration assay in Chinese Hamster lung cells. Neither component induced gene mutations in bacteria nor chromosomal aberrations in mammalian cells in the presence and absence of S9-mix.

The genotoxicity of carboxylic C14/C20 amides and alkyl aldehydes were assessed with in silico tools and no alerts were identified.

Based on the results of experimental studies and (Q)SAR analyses on the tallow-sourced substance and on the identified components which are present in the polymer and could migrate into food, the Panel concluded that the substance ‘amines, di-C14-C20-alkyl, oxidised, from hydrogenated vegetable oil’ did not raise a concern for genotoxicity.

3.3 Discussion

The solubility in water of all the substance components was below the LoD of 20 μg/L at room temperature. No migration of the substance components other than carboxylic acids was detected at the LoD (3–29 μg/kg). C14-, C16-, C18-, C20- carboxylic acids and, more generally, acids, fatty, from animal or vegetable food fats and oils are authorised in accordance with Annex I of Reg. (EU) 10/2011 (respectively as FCM No 348, 105, 106, 345 and 12) without any specific migration limits.

The Panel considered the submitted toxicological studies on the tallow-sourced substance FCM No 768 appropriate for the assessment of the substance subject of the application, that is, ‘amines, di-C14-C20-alkyl, oxidised, from hydrogenated vegetable oil’, because the two substances consist of the same components and the main fatty acids in tallow are within the range of the C14-C20 vegetable fatty acids.

Based on the results of the experimental studies and (Q)SAR analyses on the components, the Panel concluded that the substance did not raise a concern for genotoxicity. The current evaluation confirms the assessment carried out by the SCF for FCM No 768 (European Commission, 2003). The reaction products C14-20 alpha olefins are not of concern as they are not expected to exceed a migration of 0.05 mg/kg food.

The two 90-day studies in rats and dogs, which were already evaluated by the SCF (European Commission, 2003), and a new one-generation reproduction toxicity study were submitted, even if, in principle, they were not required because of the low migration potential of the components. The 90-day study in rats followed an old OECD Guideline (No 408, 1981) without addressing endpoints that would be considered by the current one (OECD, 2018). In addition, no certificate of analysis was provided for the OGRTS and no concern that would have affected the outcome of the assessment and its restriction was identified from the 90-day and the OGRT studies. Consequently, and since those studies were not required, they were not reported in the opinion.

The tallow-sourced substance FCM No 768 as well as the substance under assessment starts degrading at around 150°C with no relevant thermal degradation of the substance at the maximum temperature of manufacture of polyolefins (200°C). The Panel noted that the substance FCM No 768 is currently authorised to be used in PET at 0.25% w/w (not for articles in contact with fatty foods) for which the manufacturing process temperature is in the range of 280–300°C, hence, above the thermal degradation of the substance. The Panel considered the previous assessment on FCM No 768 (application submitted by the same applicant) by the SCF in 2003, but could not retrieve the assessment of the thermal degradation products under the high temperature processing conditions of PET. Due to the missing information on the working mechanism, chemical reactions and fate of the substance when used to manufacture PET, the Panel could not corroborate the safe use of the substance FCM No 768 for such application.

The Panel proposed to rename the substance under assessment as ‘amines, di-C14-C20-alkyl, oxidised, from hydrogenated vegetable oil’ and the substance FCM No 768 as ‘amines, di-C14-C20-alkyl, oxidised, from hydrogenated tallow’.

4 CONCLUSIONS

The CEP Panel concluded that the substance ‘amines, di-C14-C20-alkyl, oxidised, from hydrogenated vegetable oil’ is not of safety concern for the consumer if it is used as an additive at 0.1% w/w in the manufacture of polyolefin materials and articles intended to be in contact with foods simulated by food simulants A, B, C and E (as defined in the Regulation (EU) 10/2011), except for infant formula and human milk, for storage above 6 months at room temperature and below, including hot-fill conditions and heating up to 100°C for 2 hours.

5 RECOMMENDATIONS TO THE EC

Regulation (EU) No 10/2011 specifies for FCM No 768, under the entry for ‘Notes on verification of compliance’, that ‘(1) Verification of compliance by residual content per food contact surface area (QMA) pending the availability of an analytical method.’ The Panel noted that no value for QMA is given in that Regulation. The Panel further notes that the migration of this lipophilic substance is solubility controlled for the intended food contact uses and the migration will be low, if any. This would be ensured by the maximum use level which would serve as a reference value for control purposes, for example by measurement of total nitrogen. The Panel recommends therefore to delete the footnote entry (1) for FCM No 768.

DOCUMENTATION AS PROVIDED TO EFSA

Dossier ‘amines, di-C14-C18-alkyl, oxidised’. October 2021. Submitted by BASF SE.

Additional information, January 2024. Submitted by BASF SE.

ABBREVIATIONS

CAS

Chemical Abstracts Service

CEF Panel

EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids

CEP Panel

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

FCM

food contact materials

GLP

Good Laboratory Practice

LDPE

low-density polyethylene

LoD

limit of detection

LoQ

limit of quantification

OECD

Organisation for Economic Co-operation and Development

OGRTS

one-generation reproduction toxicity study

QMA

residual content per food contact surface area

(Q)SAR

(qualitative/quantitative) structure–activity relationship

SCF

Scientific Committee on Food

SML

specific migration limit

CONFLICT OF INTEREST

If you wish to access the declaration of interests of any expert contributing to an EFSA scientific assessment, please contact [email protected].

REQUESTOR

German Competent Authority (Bundesamt für Verbraucherschutz und Lebensmittelsicherheit - BVL)

QUESTION NUMBER

EFSA-Q-2021-00555

COPYRIGHT FOR NON-EFSA CONTENT

EFSA may include images or other content for which it does not hold copyright. In such cases, EFSA indicates the copyright holder and users should seek permission to reproduce the content from the original source.

PANEL MEMBERS

José Manuel Barat Baviera, Claudia Bolognesi, Andrew Chesson, Pier Sandro Cocconcelli, Riccardo Crebelli, David Michael Gott, Konrad Grob, Claude Lambré, Evgenia Lampi, Marcel Mengelers, Alicja Mortensen, Gilles Rivière, Vittorio Silano (until 21 December 2020 †), Inger-Lise Steffensen, Christina Tlustos, Henk Van Loveren, Laurence Vernis and Holger Zorn.

LEGAL NOTICE

Relevant information or parts of this scientific output have been blackened in accordance with the confidentiality requests formulated by the applicant pending a decision thereon by EFSA. The full output has been shared with the European Commission, EU Member States (if applicable) and the applicant. The blackening may be subject to review once the decision on the confidentiality requests is adopted by EFSA and in case it rejects some of the confidentiality requests.

Notes

¹ Regulation (EC) No 1935/2004 of the European parliament and of the council of 27 October 2004 on materials and articles intended to come into contact with food and repealing Directives 80/590/EEC and 89/109/EEC. OJ L 338, 13.11.2004, p. 4–17.

² Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety. OJ L 31, 1.2.2002, p. 1–48.

³ Decision available at https://www.efsa.europa.eu/en/corporate-pubs/transparency-regulation-practical-arrangements.

⁴ The non-confidential version of the dossier, following EFSA's assessment of the applicant's confidentiality requests, is published on Open.EFSA and is available at the following link: https://open.efsa.europa.eu/dossier/FCM-2021-1188.

⁵ New data, that is, not submitted for the assessment by the EC SCF of the amines, bis(hydrogenated tallow alkyl) oxidised, FCM No 768 (European Commission, 2003).

⁶ Technical dossier, section ‘Intended application of substance’.

⁷ Commission Regulation (EU) No 10/2011 of 14 January 2011 on plastic materials and articles intended to come into contact with food. Text with EEA relevance. OJ L 12, 15.1.2011, p. 1–89.

⁸ Technical dossier, section ‘Intended application of substance’.

⁹ In several certificates of analysis submitted by the applicant, the components were reported with Rn as C16–C18.

¹⁰ Fatty acids (E 570) are also authorised food additives in accordance with Regulation (EC) No 1333/2008. The latest EFSA's Re-evaluation of fatty acids (E 570) as a food additive (https://www.efsa.europa.eu/en/efsajournal/pub/4785) included, among others, myristic-, palmitic- and stearic- acids.

¹¹ Technical dossier, section ‘Physical and chemical properties of the substance’.

¹² Technical dossier, section ‘Data on residual content of substance in the food contact material’.

¹³ Technical dossier, section ‘Data on migration of substance’.

¹⁴ Technical dossier, section ‘Genotoxicity’.