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Administrative data

Key value for chemical safety assessment

Additional information

Justification for grouping of substances and read-across

The Polyfunctional acid ester (PFAE) aromatic category covers fatty alcohol esters of Benzene-1,2,4-tricarboxylic acid. The category contains both mono constituent and UVCB substances with fatty alcohol carbon chain lengths from C8-C13 (linear and iso-alcohols) building tri-esters with Benzene-1,2,4-tricarboxylic acid in variable proportions. A further surrogate substance of similar structure is included, namely a triester of Benzene-1,2,4-tricarboxylic acid with a C8 alcohol, but the alcohol moiety is branched (2-ethylhexyl).

The available data allows for an accurate hazard and risk assessment of the category and the category concept is applied for the assessment of environmental fate, environmental and human health hazards. Thus, where applicable, environmental and human health effects are predicted from adequate and reliable data for source substance(s) within the group by interpolation to the target substances in the group (read-across approach) applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006. In particular, for each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across.

A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Sections 7.1 and 13) and within Chapter 5.1 of the CSR.

Endpoint specific data matrix:

Table: Genetic toxicity.

ID No.

CAS

Genetic Toxicity in vitro: Gene mutation in bacteria

Genetic Toxicity in vitro: cytogenicity in mammalian cells

Genetic Toxicity in vitro: gene mutation in mammalian cells

Genetic toxicity in vivo

#1

3319-31-1 (c)

Negative

Negative

Negative

--

#2

90218-76-1 (b) (former CAS: 67989-23-5)

Negative

Negative

Negative

--

#4

94279-36-4 (a)

RA: CAS 90218-76-1

RA: CAS 3319-31-1

RA: CAS 90218-76-1

RA: CAS 3319-31-1

RA: CAS 90218-76-1

RA: CAS 3319-31-1

Not required

#5

72361-35-4

RA: CAS 90218-76-1

RA: CAS 3319-31-1

RA: CAS 90218-76-1

RA: CAS 3319-31-1

RA: CAS 90218-76-1

RA: CAS 3319-31-1

Not required

(a) Category members subject to the REACh Phase-in registration deadline of 31 May 2013 are indicated in bold font.

(b) Substances that are either already registered under REACh or not subject to the REACh Phase-in registration deadline of 31 May 2013 are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

(c) Surrogate substances are either chemicals forming part of a related category of structurally similar fatty acid esters or precursors/breakdown products of category members (i.e. alcohol and fatty acid moieties). Available data on these substances are used for assessment of toxicological properties by read-across on the same basis of structural similarity and/or mechanistic reasoning as described below for the present category.

 

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 90218-76-1

The potential mutagenicity of 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters was tested in three histidine requiring strains (TA97, TA98 and TA100) of Salmonella typhimurium both in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S9 mix) similar to OECD guideline 471 under GLP conditions (Kenelly, 1987). An initial toxicity range-finder experiment was carried out in TA100 only, using final concentrations of the test substance at 8, 40, 200, 1000 and 5000 µg/plate plus a solvent (DMSO) and positive control. From these results the highest test dose were chosen for the main mutation assay. In the mutation assay bacteria were tested with 8, 40, 200, 1000 and 50000 µg/plate of the test compound. Negative (DMSO) and positive control (2-aminoanthracene) treatments were included for all strains. Precipitation was observed at 5000 µg/plate. The mean numbers of revertant colonies were within acceptable ranges. The positive controls gave the expected results. No cytotoxicity was observed for all concentrations tested. 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters failed to induce a two-fold increase in revertant numbers with any tester strain either in the absence or presence of S9 mix, so was considered non-mutagenic in this assy.

CAS 3319-31-1

The potential mutagenicity of Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate (> 99% pure) was investigated in a bacterial mutation assay (Ames test) according to OECD guideline 471 under GLP conditions (Shibuya, 1996). S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 were tested in the presence or absence of metabolic activation (S9 mix) at test substance concentrations of 313, 625, 1250, 2500 and 5000 μg/plate. Acetone was used as a vehicle. Two independent experiments were conducted, each with triplicates. The exposure duration was 48 h and 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 9-aminooacridine (without S9) and 2-aminoanthracene (with S9) were used as positive controls. Precipitation was observed from 1250 and 313 µg/plate with and without metabolic activation, respectively. No cytotoxicity was seen up to the limit concentration of 5000 µg/plate. The test substance did not induce an increase in the number of revertants. Thus, Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate is considered non-mutagenic in bacteria under the experimental conditions chosen in this study.

 

Genetic toxicity (cytogenicity) in mammalian cells in vitro

CAS 90218-76-1

The test item, 1,2,4-Benzenetricarboxylic acid, decyl octyl ester (≥ 97.5 % pure), was assayed for the ability to cause chromosomal damage in cultured human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation (S9 mix) according to OECD guideline 473 under GLP conditions. Two independent experiments for chromosomal damage were performed. In the first experiment, the cells were treated 3 hours in the presence and absence of S9 mix. The harvest time of 24 hours corresponding to approx. 1.5 cell cycles was used. As negative results were obtained, a second experiment was performed using the same harvest time (24 hours). A continuous treatment until harvest was used. Both for the first and second experiments, dose levels of 2500, 1250, 625, 313, 156, 78.1, 39.1, 19.5 and 9.77 µg/mL were used in the absence or presence of S9 mix. Each experiment included appropriate negative (ethanol) and positivecontrols (mitomycin C without S9 mix, cyclophosphamide with S9 mix). Two cell cultures were prepared at each test point. For both experiments, the dose levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity determined by the reduction in mitotic index. Where no cytotoxicity occurred, the highest treatment level was selected as the maximum dose level for scoring. The following dose levels were selected for scoring (100 metaphase spreads were scored for chromosomal aberrations from each culture, 200 for each experimental point): Experiment 1: 1250, 625 and 313 µg/mL with and without S9 metabolism (3 hours treatment time, 24 hours harvest time); Experiment 2: 2500, 1250 and 625 µg/mL without S9 metabolism (24 hours treatment and harvest time). Following treatment with the test item, no statistically significant increase in the incidence of cells bearing aberrations, including or excluding gaps, was observed at any dose level of any sampling time. The positive controls gave the expected results. It was concluded that 1,2,4-Benzenetricarboxylic acid, decyl octyl ester did not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.

CAS 3319-31-1

The potential for chromosomal damage of Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate has been investigated in a chromosomal aberration assay performed according to OECD guideline 473 under GLP conditions. The assay was conducted with Chinese hamster lung (CHL/IU) cells with and without metabolic activation. Test substance concentrations were 1.3, 2.5, and 5.0 mg/mL with and without metabolic activation, in short term and continuous treatment. Acetone was used as vehicle and mitomycin C (continuous exposure) and cyclophosphamide (short-term treatment) as positive controls. The cells were treated with test substance for 6, 24, and 48 hours. 200 cells per concentration were evaluated in each of two independent experiments. Cytotoxicity was determined based on the relative total growth (mitotic index). The relative (compared to vehicle control) number of aberrant cells with and without gaps was reported, as well as the mitotic index. Polyploidy was also determined. No reduction in mitotic index was seen and no increase in the number of aberrant cells (with or without gaps) was observed in the test substance treated cells. The positive controls gave the expected results. Thus, based on the result of this assay, Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate is not considered to cause chromosomal damage under the conditions in this study.

 

Genetic Toxicity in vitro: gene mutation in mammalian cells

CAS 90218-76-1

The test item 1,2,4-Benzenetricarboxylic acid, decyl octyl ester (≥ 97.5% pure) was examined for mutagenic activity by assaying for the induction of 5-trifluorothymidine resistant mutants at the TK locus in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation (S9 mix), using a fluctuation method. The study was performed according to OECD guideline 476 under GLP conditions (Salvador, 2009).

A solubility trial indicated that the maximum practicable concentration of the test item in the final test medium was 2500 µg/mL using ethanol as the solvent. On the basis of this result a preliminary cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 2500 µg/mL and at a wide range of lower dose levels: 1250, 625, 313, 78.1, 39.1, 19.5 and 9.77 µg/mL. No relevant toxicity was observed at any dose level at any sampling time, in the absence and presence of S9 metabolism. Based on the toxicity results obtained in the preliminary assay, two independent assays for mutation to 5-trifluorothymidine resistance were performed using the following dose levels and treatment times: Experiment 1: 156, 313, 626, 1250 and 2500 µg/mL, 3 hours treatment with and without metabolic activation; Experiment 2: 156, 313, 625, 1250 and 2500 µg/mL (24 hours treatment, -S9 mix) and 875, 1138, 1479, 1923 and 2500 µg/mL (3 hours treatment, + S9 mix). No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 mix. Negative (ethanol) and positive (Benzo(a)pyrene with S9 mix, methyl methanesulphonate without S9 mix) control treatments were included in each mutation experiment. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

It was concluded that 1,2,4-Benzenetricarboxylic acid, decyl octyl ester does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

CAS 3319-31-1

The potential for gene mutation of Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate has been evaluated in a CHO/HGPRT forward mutation assay performed according to OECD 476 under GLP conditions (Young, 1985). The test substance (98.95% pure) was applied to Chinese hamster ovary (CHO-K1-BH4) cells at concentrations of 5, 10, 20, 50, 100 and 200 nL/mL, corresponding to approx. 4.95, 9.9, 19.8, 49.5, 99, 198, 495, 990, 1980 and 4950 µg/mL based on a density of 0.99 g/mL, (4 h treatment) in the first experiment with and without metabolic activation (S9 mix) and at concentrations of 10, 100 and 200 nL/mL corresponding to approx. 4.95, 9.9, 19.8, 49.5, 99, 198 µg/mL based on a density of 0.99 g/mL (4 h treatment) with S9 mix. The concentrations were chosen based on a preliminary cytotoxicity test. 1% ethanol was used as vehicle and 5-bromo-2'-deoxyuridine and 3-methylcholanthrene were used as positive controls. 7 days after the end of treatment, cells were plated for determination of cloning efficiency and mutation frequency in mutant selection medium containing 10 µg/mL of 6-thioguanine (TG). The plates were incubated for 7-10 days. After harvest, the cells were stained with Giemsa. Duplicates of each condition were evaluated. Cytotoxicity was determined as cloning efficiency, relative population growth and relative survival.

The test substance remained in solution in culture medium from 4.95 to 9.9 µg/mL. In both the preliminary cytotoxicity test and the mutation assays, very slight cloudiness was observed at 19.8 and 49.5 µg/mL. The test material was insoluble at 99.9 µg/mL and higher, producing a cloudy white suspension in F12 culture medium. At 4950 µg/mL, an oily suspension was formed with droplets of test material that floated to the surface upon standing. Because of the cloudiness of the S9 mix used in the metabolic activation assays, accurate microscopic observations of the activation test media could not be made. Very slight cytotoxicity at 4.95 to 4950 µg/mL in the preliminary test and at 9.9 to 198 µg/mL in the main assay without metabolic activation.

In the absence of S9 mix, no significant changes in the mutant frequency were observed with the exception of one culture exposed to 49.5 µg/mL. As this was an isolated, not dose-related increase, the mutant frequency was typical of vehicle controls in other assays and the duplicate treated culture showed no increase in mutant frequency, this increase was not considered a positive response.

In the presence of S9 mix, the mutant frequencies associated with the duplicate treatments between 9.9 and 99.9 µg/mL fluctuated randomly in a range that was typical of vehicle control variation between trials (1E-06 to 15E-06). None of the treatments had a mutant frequency significantly higher than that obtained for the concurrent vehicle controls. However, the mutant frequencies in the two 198 µg/mL cultures were statistically elevated over the concurrent vehicle controls. This response was small and was not expected in view of the lack of any increase in the toxicity of treatment. Therefore, another trial was initiated to determine whether this response was repeatable or represented an assay fluctuation.

The second activation trial employed duplicate treatments with 9.9, 99 and 198 µg/mL of test material. Similar patterns of toxicity and solubility were seen in both the first and second activation trials. No significant or dose-related increases in mutant frequency were observed in the repeat trial. Based on these results, Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate is not considered to induce gene mutations under the conditions chosen in this assay.

Conclusion for genetic toxicity

In summary, 2 studies investigating the genetic mutation in bacteria in vitro are available within the PFAE aromatic category for 1,2,4-Benzenetricarboxylic acid, decyl octyl ester (CAS 90218-76-1) and Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate (3319-31-1), both providing negative results. Furthermore, neither cytogenicity nor gene mutation in mammalian cells in vitro was observed for these category members.

Therefore, no properties for genetic toxicity were observed within the PFAE aromatic group for any member.

 

References

Gubicza, L. et al. (2000). Large-scale enzymatic production of natural flavour esters in organic solvent with continuous water removal. Journal of Biotechnology 84(2): 193-196.

Lilja, J. et al. (2005). Esterification of propanoic acid with ethanol, 1-propanol and butanol over a heterogeneous fiber catalyst. Chemical Engineering Journal, 115(1-2): 1-12.

Liu, Y. et al. (2006). A comparison of the esterification of acetic acid with methanol using heterogeneous versus homogeneous acid catalysis. Journal of Catalysis 242: 278-286.

Radzi, S.M. et al. (2005). High performance enzymatic synthesis of oleyl oleate using immobilised lipase from Candida antartica. Electronic Journal of Biotechnology 8: 292-298.

Zhao, Z. (2000). Synthesis of butyl propionate using novel aluminophosphate molecular sieve as catalyst. Journal of Molecular Catalysis 154(1-2): 131-135.


Short description of key information:
No indication for genetic toxicity were observed within the PFAE aromatic group for any member. All available studies had a negative outcome.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the group concept is applied to the members of the PFAE aromatic Category, data will be generated from representative reference substance(s) within the category to avoid unnecessary animal testing. Additionally, once the group concept is applied, substances will be classified and labelled on this basis.

Based on the group concept, all available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.