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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471, GLP): negative in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA with and without metabolic activation

MLA (OECD 490, GLP): negative with and without metabolic activation (4 h exposure duration) and positive without metabolic activation (24 h exposure duration) in mouse lymphoma 5178Y cells

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

MNT (OECD 474, non-GLP, source substance): negative in rats

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for read-across

There are data available on mutagenicity in bacterial and mammalian cells for the target substance Hexanedioic acid, di-C16-18 (even numbered)-alkyl esters (CAS 92969-90-9). In addition, read-across from an appropriate source substance is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. in order to fulfil the standard data requirements, defined in Regulation (EC) No 1907/2006, Annex VIII, 8.4. 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 analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).


In vitro


Gene mutation in bacteria

The potential mutagenicity in bacterial cells of

Hexanedioic acid, di-C16-18 (even numbered)-alkyl esters (CAS 92969-90-9) was tested in a GLP-compliant bacterial reverse mutation assay performed according to OECD TG 471 (Ames, 2017). The assay was performed with a standard battery of Salmonella typhimurium tester strains TA 1535, TA 1537, TA 98 and TA 100, and Escherichia coli tester strain WP2uvrA. In a preliminary cytotoxicity test the highest concentration of 5000 µg/plate was tested in TA 100 and WP2uvrA. The test substance precipitated at dose levels of 1600 and 5000 µg/plate, but cytotoxicity was not observed. Based on these results, the concentration range 52 to 5000 µg/plate was selected in the first mutation experiment and the concentration range 17 to 5000 µg/plate was selected in the second mutation experiment. Hexanedioic acid, di-C16-18 (even numbered)-alkyl esters (CAS 92969-90-9) did not exhibit mutagenic properties in the absence or presence of metabolic activation. No cytotoxicity was observed in any strains up to and including the limit dose of 5000 µg/plate. The test substance precipitated at concentrations equal to or greater than 1600 µg/plate. The positive control substances induced a distinct increase in the number of revertants in all strains with and without metabolic activation, indicating the validity of the assay. Based on the results of the conducted study, Hexanedioic acid, di-C16-18 (even numbered)-alkyl esters (CAS 92969-90-9) is not considered to exhibit mutagenic properties in bacterial cells.


In vitro gene mutation in mammalian cells

An in vitro mammalian cell gene mutation assay was performed in mouse lymphoma L5178Y cells according to OECD guideline 490 and under GLP conditions with Hexanedioic acid, di-C16-18 (even numbered)-alkyl esters (CAS 92969-90-9) (MLA, 2017). The cells were treated for 3 h with and without metabolic activation (cofactor-supplemented post-mitochondrial fraction; S9 mix), and for 24 h without metabolic activation. The concentrations tested were 0.54, 1.7, 5.4, 17, 52, 164, 512 and 1600 µg/mL in Experiment I (with and without metabolic activation, 3 h treatment) and in Experiment II (without metabolic activation, 24 h treatment), and precipitation was observed at the highest concentration. Cyclophosphamide and methylmethanesulfonate were used as positive controls with and without S9 mix, respectively. No decrease in the relative growth factor (RTG) was observed in any of the tested concentrations in Experiment I and II, respectively. The positive controls induced distinct increases in mutation frequencies, indicating the validity of the test and of the activity of the metabolizing system.

In the first mutation experiment (3 h treatment), no significant increase in the mutation frequency (MF) at the TK locus was observed in the absence or in the presence of metabolic activation. An increase above the 95% control limit was observed at the mid-dose of 5.4μg/mL without metabolic activation. Although this increase is above the historical control data range, no dose-related response was observed, the increase in the mutation frequency (168 per 10E6 survivors) was below the MF (controls) + 126 (mutation frequency of the control, in this study 248 per 10E6 survivors) and not more than a 1.4-fold increase was observed. Therefore the increase observed after the 3-h treatment without metabolic activation was considered to be not biologically relevant.

In the second mutation experiment (24 h treatment), the test substance induced an up to 2.3-fold increase in the mutation frequency at the TK locus at the non-precipitating dose level of 52μg/mL. This result was above the global evaluation factor (GEF) of (245 x 10E6), and therefore indicative of a positive result. The test item showed up to 2.6- and 1.6-fold increases in the mutation frequency of the small and large colonies, respectively, compared with the mean mutation frequency of the small and large colonies of the respective solvent controls. At the precipitating dose levels from 164μg/mL, an increase in the mutation frequency was observed. However, the MF was 176 x 10E6 at 164μg/mL, 194 x 10E6 at 512 µg/mL and 192 x 10E6 at 1600 µg/mL, which is below the GEF. A statistical significant dose related trend (p<0.001) was observed following the 24-h treatment, and considered to be biologically relevant (the concentrations for which this trend was observed were not given in the study report). Hexanedioic acid, di-C16-18 (even numbered)-alkyl esters (CAS 92969-90-9) was mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this study in the absence of metabolic activation after 24 h treatment.


In vivo cytogenicity


CAS 16958-92-2

An in vivo mammalian cell gene cytogenicity assay was performed following a protocol similar to OECD guideline 474, with the source substance ditridecyl adipate (CAS 16958-92-2) (MNT, 1985). The study was part of a 13-weeks dermal repeated dose toxicity study in male and female Sprague-Dawley rats. The animals were exposed to doses of 800 and 2000 mg/kg bw; applied to the back of the animals for 24 h without a dressing; 5 days/week for a period of 13 week. Peripheral blood smears and bone marrow were taken from the femurs of 5 animals/sex/dose and 2 slides per animal were prepared and stained according to the ‘Wright-stain-procedure’. A negative control was included in the study. A positive control was not run in parallel, as the animals were part of the 13-weeks repeated dose toxicity study. No clear signs of systemic toxicity were observed in male and female rats in any of the dose groups. The treatment groups did not show a decrease in the ratio of polychromatic to normochromatic erythrocytes, compared with the control group. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes isolated from bone marrow and peripheral blood, respectively, for any treatment group compared with the control group. Under the conditions of the study, the test substance was not clastogenic in Sprague-Dawley rats.

Justification for classification or non-classification

The in vitro data for gene mutation revealed a positive result, however there is no positive result determined in vivo, thus there is no need to classifiy according to the CLP Regulation 1272/2008 based on the currently available data. The classification/non-classification of the target substance will be reassessed when additional in vivo data has been generated.