Bis(16-methylheptadecyl) malate

Endpoint summary

Additional information

Justification for grouping of substances and read-across

There are only limited data available on genetic toxicity of Diisooctadecyl malate (CAS 67763-18-2). 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	Chemical name	Molecular weight [g/mol]	Gene mutation in vitro (Ames)	Gene mutation in vitro (MLA)	Chromosome aberration in vivo (MN)
67763-18-2 (a)	Diisooctadecyl malate	639.06	Experimental result: negative	RA: CAS 103-23 -1	RA: CAS 149144-85-4
149144-85-4 (b)	Di C12-13 Alkyl Malate	470.73/498.78	Experimental result: negative	--	Experimental result: negative
103-23-1 (b)	Bis(2-ethylhexyl) adipate	370.57	--	Experimental result: negative	--

(a) The substance subject to registration is indicated in bold font.

(b) Reference (read-across) substances are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

The above mentioned substances are considered to be similar on the basis of the structural similar properties and/or activities. The available endpoint information is used to predict the same endpoints for Diisooctadecyl malate (CAS 67763-18-2). A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

Discussion

Only limited data on genetic toxicity is available with Diisooctadecyl malate (CAS 67763-18-2). Therefore, read across from the structurally analogue substances Di C12-13 Alkyl Malate (CAS 149144-85-4) and Bis(2-ethylhexyl) adipate (CAS 103-23-1) was applied.

 

Genetic toxicity (mutagenicity) in bacteria in vitro

A reliable bacterial gene mutation study (Ames test) performed according to OECD TG 471 and in compliance with GLP with Diisooctadecyl malate (CAS 67763-18-2) is available (Sire, 2005). The strains Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were tested according to the plate incorporation (experiment I, II (without S9-mix) and III) and preincubation (experiment II (with S9-mix)) procedure in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). Two independent experiments were conducted in three repetitions at each concentration from 312.5 to 5000 µg/plate (experiment I and II). A third confirmatory experiment was performed with tester strain TA 102 at concentrations ranging from 1250 to 5000 µg/plate in the presence of metabolic activation. No cytotoxicity was observed in a preliminary toxicity test performed with tester strains TA 98, TA 100 and TA 102 after treatment with the test material up to 5000 µg/plate in the absence and presence of metabolic activation. No precipitation was recorded at any test concentration. Appropriate solvent (ethanol) and positive controls were included and gave the expected results. No significant increase in the number of revertants was observed in any of the tester strains with and without metabolic activation. Therefore, the test material was considered to be non-mutagenic under the conditions of the test.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

An in vitro mammalian cell gene mutation assay with Bis(2-ethylhexyl) adipate (CAS 103-23-1) was performed equivalent or similar to OECD TG 476 (McGregor, 1988). Mutations at the TK locus of mouse-lymphoma L5178Y cells in the presence of metabolic activation (S9 mix) were investigated in two independent experiments at test substance concentrations ranging from 312.5 to 5000 µg/mL. In two further experiments, cells were exposed to the test substance at concentrations ranging from 1000 to 5000 µg/mL in the absence of metabolic activation. The treatment of cells in all experiments included an exposure period of 4 h, followed by an expression period of 48 h and a selection period of 11-14 days in the presence of 5-trifluorothymidine (TFT). The test substance did not induce a significant increase in the mutant frequency at any test substance concentration in the absence of S9-mix. No significant increase in mutant frequency was observed in the first experiment in the presence of S9-mix. In contrast, a statistically significant increase in the mutant frequency was observed in cells treated at concentrations ≥2000 µg/mL in the second experiment with S9-mix. However, since no dose-response relationship was observed and precipitation was evident at concentrations of 1000 µg/mL, the slight significant increase in mutant frequencies in the presence of S9-mix was not considered to be of biological relevance. In addition, significant cytotoxicity, as indicated by a reduction in relative total growth, was noted in all experiments at concentrations ≥1000 µg/mL, both with and without S9-mix. The positive controls significantly increased mutant frequency, demonstrating the sensitivity of the test system and the efficacy of the metabolic activation system. In conclusion, the test substance did not induce the mutant frequency in mouse-lymphoma L5178Y cells in the presence and absence of metabolic activation.

Genetic toxicity (cytogenicity) in mammals in vivo

An in vivo chromosome aberration test (micronucleus assay) with Di C12-13 Alkyl Malate (CAS 149144-85-4) was performed according to EU Method B.12 and in compliance with GLP (Biffi, 1994). The test material was administered intraperitoneal to 15 C57BL mice of each sex at a single dose of 12500 mg/kg bw. Additionally, 15 mice of each sex were either treated with a positive control substance (75 mg/kg bw cyclophosphamide) or vehicle alone (50 mL/kg bw sesame seed oil) as negative control, respectively. 24, 48 and 72 hours post-treatment 10 mice of each group were sacrificed, and the bone marrow of each animal was extracted, smeared on a slide, stained and observed with a microscope. For each animal 1000 polychromatic erythrocytes were counted and the frequency of normochromatic and micronucleated polychromatic erythrocytes was recorded. The data obtained from the proportional relationship between the polychromatic erythrocytes and the normochromatic erythrocytes was statistically evaluated by means of Student's t-test. No inhibitory effect on erythropoetic cell division was observed for both sexes 24, 48 and 72 hours after intraperitoneal application of the test material. Positive and negative controls gave the expected results. On the slides prepared with the bone marrow of the animals treated with cyclophosphamide and sacrificed 72 hours after the beginning of the treatment, only platelets and blasts were observed and could not be evaluated. Based on the results of the study, it was concluded that the test substance did not induce clastogenic effects on the tested mitotic system under the conditions of the study.

Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted by means of read-across based on an analogue approach. No study was selected since all available in vitro and in vivo 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.

Short description of key information:
In vitro gene mutation:
Bacterial reverse mutation assay (Ames test / OECD 471): negative
In vitro gene mutation test in mouse lymphoma cells (MLA / OECD 476): negative (RA from CAS 103-23-1)

In vivo gene mutation:
In vivo chromosome aberration test in mice (MN / EU Method B.12): negative (RA from CAS 149144-85-4)

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 analogue concept is applied to Diisooctadecyl malate (CAS 67763-18-2), data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.