Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane

Endpoint summary

Additional information

Justification for grouping of substances and read-across

There are no data available for the assessment of mutagenicity in mammalian cells of Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane (CAS 147256-33-5). In order to fulfil the standard information requirements set out in Annex VII and VIII, 8.4, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from structurally related substances was conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

Overview of genetic toxicity

CAS	147256-33-5 (a)	61788-89-4 (b)	77-99-6
Chemical Name	Fatty acids,C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane	Fatty acids, C18-unsatd., dimers	Propylidynetrimethanol
MW	624.97 1115.78; 1606.58; 677.05; 1219.92; 1762.80	556.87; 564.93	134.18
Genetic Toxicity in vitro: Gene mutation in bacteria	Experimental result: Negative	Experimental result: Negative	Experimental result: Negative
Genetic Toxicity in vitro: cytogenicity in mammalian cells	Experimental result: Negative	Experimental result: Negative	Experimental result: Negative
Genetic Toxicity in vitro: gene mutation in mammalian cells	RA: CAS 61788-89-4 RA: CAS 77-99-6	Experimental result: Negative	Experimental result: Negative

(a) Substances 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 “--“.

The available endpoint information is used to predict the same endpoints for Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane (CAS 147256-33-5).

A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

 

Discussion

Genetic toxicity

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 147256-33-5

A bacterial gene mutation assay (Ames test) with Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane was performed according to OECD Guideline 471 and under GLP conditions (Sokolowski, 2012). The study included two independent experiments both in the absence and in the presence of liver microsomal activation system (S9 mix). In the first experiment, the direct plate incorporation procedure was conducted with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and the Escherichia coli strain WP2 uvrA at concentrations ranging from 3 to 5000 µg/plate for a period of 48 h. In the second experiment, the same concentrations and exposure duration were applied for treatment of bacteria, but an additional pre-incubation period of 60 min was included in the test. In both experiments, no cytotoxicity was evident up to the limit concentration of 5000 µg/plate in all bacterial strains. In the first experiment, precipitation was observed at concentrations ≥ 333 µg/plate or ≥ 1000 µg/plate with or without metabolic activation, respectively. In the second experiment precipitation was evident at ≥ 1000 µg/plate in the presence or absence of S9 mix. The mean number of revertant colonies was not increased in the bacteria at any concentrations tested. The positive and negative controls included showed the expected results in each experiment.

Under the conditions of this assay, Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane did not induce mutagenicity in the selected strains of S. typhimurium (TA 98, TA 100, TA 1535, TA 1537) and E. coli WP2 uvrA in the absence and presence of metabolic activation.

 

Genetic toxicity (cytogenicity) in mammalian cells in vitro

CAS 147256-33-5

The cytogenicity of Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane in mammalian cells in vitro has been investigated in two studies (CAS 147256-33-5).

An in vitro mammalian chromosome aberration test according to OECD Guideline 473 was performed with Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane in Chinese hamster lung fibroblasts (V79) (Flügge, 2009). In this GLP-conform study, duplicate cell cultures were treated with the test substance at concentrations ranging from 312.5 to 5000 µg/mL for a period of 4 and 20 h (without metabolic activation) or 4 h only (with metabolic activation). These concentrations were selected based on a preliminary cytotoxicity test, in which no toxicity was observed up to the highest concentration of 5000 µg/mL. Consistent with this, treatment of cells in the main study did not induce cytotoxicity in V79 cells up to the maximum concentration of 5000 µg/mL. Based on these results, the analysis of chromosome aberrations was performed at concentrations of 625, 1250, 2500, 5000 µg/mL, both in the presence or absence of metabolic activation. No significant increase in the number of phases with aberrations at any preparation time and concentration was observed. The positive controls significantly increased the rate of chromosome aberrations, thus indicating the sensitivity of the assay. In conclusion, the test substance was not clastogenic in Chinese hamster lung fibroblasts (V79), neither in the presence nor in the absence of a metabolic activation system, under the chosen experimental conditions.

In a further in vitro chromosome aberration test according to OECD Guideline 473 and under conditions of GLP, cultured peripheral human lymphocytes were treated with the test substance at concentrations ranging from 0.01 to 5 µL/mL in the presence and absence of metabolic activation (S9 mix) in two independent experiments (Bohnenberger, 2012). These concentrations were selected based on a preliminary cytotoxicity test, in which no toxicity was observed up to the maximum concentration. In the first experiment, cells were exposed to the test substance for 4 h with and without S9 mix, whereas exposure periods of 22 h (without S9) and 4 h (with S9) were chosen in the second experiment. No cytotoxicity was observed in both experiments up to the maximum concentration of 5 µL/mL with and without S9 mix. Based on these results, concentrations of 0.01, 2.86 and 5 µL/mL were selected for chromosome analysis in the absence and presence of metabolic activation in the first and second experiment. No increase in the number of cells with chromosomal aberrations was observed compared to controls in any of the experiments performed. At the end of treatment, phase separation was observed in the first experiment in the absence and presence of S9 mix and in the second experiment in the absence of S9 mix at 0.02 μL/mL and above. In the presence of S9 mix, phase separation was observed at 0.03 μL/mL and above in the second experiment. The positive controls included in both experiments showed the expected results and thus verified the sensitivity of the assay. Based on the negative results obtained in this chromosome aberration assay, it was concluded that the test substance did not show clastogenic activity in cultured peripheral human lymphocytes in the presence or absence of metabolic activation.

In summary, Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane is not clastogenic to mammalian cells in vitro.

 

Gene mutation in mammalian cells in vitro

CAS 61788-89-4

Fatty acids, C18-unsaturated, dimers were tested for its mutagenic potential in a vitro mammalian cell mutation assay following OECD Guideline 476 and in compliance with GLP (Adams et al., 1993). In two independent experiments, mouse lymphoma L5178Y cells were treated with the test substance at concentrations up to 300 µg/mL (limit of solubility) with and without metabolic activation (S9 mix) for a period of 3 h. After an expression time of 48 h in growth medium, cells were incubated for 12 days with trifluorothymidine as selection agent for forward mutation at the thymidine kinase locus. The test material was cytotoxic in both the absence and presence of S9 mix (at 300 and 150 µg/mL, respectively). A statistically significant increase in mutant frequency was observed at 250 µg/mL in one of the two experiments without S9 mix. However, this increase was small and not considered to be biologically significant. In the presence S9 mix, no statistically significant increases in mutant frequency were observed in any of the two experiments performed. The corresponding positive and negative control substances showed the expected results and thus verified the sensitivity of the assay.

Based on the results of this study, it was concluded that Fatty acids, C18-unsaturated, dimers does not have a mutagenic potential in mammalian cells in vitro.

CAS 77-99-6

The genotoxic potential of the propylidynetrimethanol was assessed using a gene mutation assay in cultured mammalian cells (Chinese hamster lung fibroblasts (V79)) according to OECD Guideline 476 and under conditions of GLP (Harlan CCR, 2010). Based on a preliminary cytotoxicity test, gene mutations in the HPRT locus were investigated in the presence and absence of metabolic activation (rat liver S9-mix) with test substance at concentrations ranging from 43.8 to1400 µg/mL. In the first experiment, cells were exposed to the test substance for a period of 4 h, either in the presence or absence of S9 mix. The second experiment was performed in a similar manner, but the exposure duration for the treatment in the absence of S9 mix was extended to a period of 24 h. No significant cytotoxicity was reported. An increase in mutant frequency was not observed at any concentration tested, neither with nor without metabolic activation. The positive controls significantly increased the mutant frequency and thus confirmed the sensitivity of the assay.

In summary, the propylidynetrimethanol was not mutagenic at the HPRT locus of Chinese hamster lung fibroblasts in the absence and presence of metabolic activation.

Conclusion for genetic toxicity

In summary, studies on the in vitro bacterial mutagenicity and cytogenicity in mammalian cells are available for Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane (CAS 147256-33-5), all showing negative results. No information is available for the potential of Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane to cause gene mutation in mammalian cells, thus read across from possible breakdown products was conducted. Although oral, dermal or inhalative absorption of the registered substance is deemed unlikely due to its very high MW and physico-chemical properties, and cleavage of the substance is not expected to occur in significant amounts, the hydrolysis products can be used as a marker for toxicological behaviour of the target substance following a worst case approach.

Both surrogate substances, Fatty acids, C18-unsatd., dimers (CAS 61788-89-4) and propylidynetrimethanol (CAS 77-99-6), which would result from enzymatic ester hydrolysis of the target substance, were negative in studies investigating mutagenicity in mammalian cells.

Therefore, no genetic toxicity is expected for Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane.

Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted by means of read-across from a structural analogue. No study was selected, since all available in vitro genetic toxicity studies were negative. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substance and overall quality assessment (refer to the endpoint discussion for further details).

Short description of key information:
In vitro:
Mutagenicity in bacteria (OECD TG 471) using S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA: negative
Cytogenicity in mammalian cells in a chromosome aberration test (OECD TG 473) using Chinese hamster lung fibroblasts (V79): negative
Mutagenicity in mammalian cells in a MLA and HPRT assay (OECD TG 476): negative (based on read-across from the hydrolysis products CAS 61788-89-4 and CAS 77-99-6)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the target and structurally similar substances, the 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.