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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation assays in bacteria with the submission substance and surrogates of the submission substance gave negative results. A gene mutation study in mammalian cells as well as a chromosome aberration test performed with the submission substance and a surrogate of the submission substance reported negative findings too.

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

Additional information

All available in vitro studies were conducted according to a respective OECD testing guideline and GLP conditions applied.

Bacterial reverse mutation assay:

There is one reliable reverse gene mutation study in bacteria available for a surrogate of the submission substance (i.e. 3,5,5 -trimethylhexanoic acid). This test substance was not mutagenic in an Ames-test with the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 and Escherichia coli WP2uvrA in concentrations up to 10000 µg/plate, both with or without metabolic activation.

Mutagenic activity of the submission substance was also investigated in Salmonella typhimurium strains TA 97a, TA 98, TA 100 and TA 1535 with (induced rat liver S9 mix) and without metabolic activation at concentrations of either 0, 5, 50, and 500 µg/plate (experiment I) or 0, 50, 160, 500, 1600 and 5000 µg/plate (experiment II) using the plate incorporation assay. The test was performed according to GLP and OECD testing guideline 471 (adopted May 26, 1983), therefore missing the now required strains S. typhimurium 102 or Escherichia coli WP2uvrA of today standard method (thus only RL3).

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.

There is one reliable reverse gene mutation study in bacteria available for the a surrogate of the submission substance:6-(Isononanoylamino)hexanoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1). The test item was assessed for its mutagenic effects usingSalmonella typhimuriumstrains: TA98, TA100, TA1535, TA1537 andEscherichiacoliWP2 uvrA (pKM101).  The test item was tested at the concentrations of0.05, 0.16, 0.5, 1.6and 5 mg/platefor plate incorporation method and preincubation method using Distilled water as vehicle based on the results of solubility, precipitation and initial cytotoxicity test.From the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both the trials, in the presence and absence of metabolic activation. There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both the trials. The number of revertant colonies in the positive controls resulted in 2.2 to 20.5 fold increase underidenticalconditions. Based on the results of the study, it can be concluded that the test itemwas “non-mutagenic” in the Bacterial Reverse Mutation Testup to the highest tested concentration of5mg/plate under the test conditions.

Gene mutation in mammalian cells:

An in vitro mammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at the HPRT locus to test the potential of the submission substance to cause gene mutation. Treatments were carried out for 5 hours with and without metabolic activation (+/- S9-mix) and for 24 hours without metabolic activation (-S9-mix) and the test was performed according to OECD testing guideline 476 (GLP).

 Dimethyl sulfoxide was used as the solvent of the test item in this study. Treatment concentrations for the mutation assay were selected based on the results of a preliminary toxicity test. The following concentrations of test item were selected for the main assays:

Assay 1: 5-hour treatment in the presence of S9-mix: 3000; 1500; 1000; 750; 500; 250; 125; 62.5 and 31.25 μg/mL

5-hour treatment in the absence of S9-mix: 3000; 2000; 1500; 1000; 500; 250; 125 and 62.5 μg/mL

Assay 2: 5-hour treatment in the presence of S9-mix and 24-hour treatment in the absence of S9-mix: 3000; 1500; 1000; 750; 500; 250; 125; 62.5 and 31.25 μg/mL

 In the main assays cytotoxicity was obvious starting from concentrations of 750 µg/mL and above (i.e. result from Assay 2 in the presence of S9-mix; in the other assays cytotoxicity was observed at higher concentrations tested and evaluation of mutation frequency was adapted to the respctive circumstances).

Overall there was no statistically significant increase in the mutant frequency compared to the solvent control observed in these experiments. There was no dose response to the treatment; a trend analysis showed no effect of treatment.

  The spontaneous mutation frequency of the negative (solvent) control was in accordance with the historical control range in all assays. The positive controls gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays. At least four evaluated concentrations were presented in all assays.

 The cloning efficiencies for the negative controls were within the target range in all assays.

The study was considered to be valid.

 In conclusion, no mutagenic effect of the submission substance was observed either in the presence or absence of metabolic activation system under the conditions of this HPRT assay.

In vitro cytogenicity study:

The submission substance was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells according to OECD guideline 473. In the performed independent Chromosome Aberration Assays using duplicate cultures at least 200 well-spread metaphase cells were analysed for each test item treated, negative (solvent) control or positive control sample where possible.

In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item in Assay 1 with and without metabolic activation were 5000, 2500, 1250, 625 and 312.5 µg/mL.

In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases. The examined concentrations of the test item in Assay 2 were 5000, 2500, 1250, 625, 312.5 and 156.25 µg/mL.

Both experiments were carried out up to cytotoxic concentrations.

In both assays none of the treatment doses caused a significant increase in the number of cells with structural chromosome aberrations either in the absence or in the presence of metabolic activation. Both assays were considered as negative.

The negative (solvent) control data were within the laboratory’s normal range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system; therefore, the tests were considered to be valid.

In conclusion, no induction of chromosome aberrations in Chinese hamster V79 cells was observed following treatment with the submission substance up to the cytotoxicity/solubility limit; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study. Therefore, the submission substance is considered non-clastogenic in this test system.

A surrogate of the submission substance 6-(Isononanoylamino)hexanoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) was testedon CHO-K1 cells in duplicate culturalflasks.The test item resulted in mean percentage reduction in RICC of 19.18% in the presence of metabolic activation, 19.12% and 23.19% in the absence of metabolic activation for short and long term treatments, respectively at 2 mg/mL, the highest dose concentration tested. Polyploidy and endoreduplication was not observed at any of the test item doses tested. There was no statistically significant increase in the number of chromosomal aberrations observed when compared with vehicle control at any of the dose levels of 6-(Isononanoylamino)hexanoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) tested.Based on the results obtained, the test item,6-(Isononanoylamino)hexanoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1)is considered as non-clastogenic at and up to the concentration of 2 mg/mL both in the presence and absence of metabolic activation, under the test conditions.


Justification for selection of genetic toxicity endpoint
No study was selected, since all available studies showed negative findings.

Short description of key information:
Gene mutation assays in bacteria with the submission substance and a surrogate of the submission substance gave negative results. A gene mutation study in mammalian cells as well as a chromosome aberration test performed with the submission substance reported negative findings too.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In a reliable set of in vitro gene mutation studies in bacteria and mammalian cells, as well as a chromosome aberration test no mutagenic potential was found for the submission substance or a surrogate substance.

Based on the available data no classification according to Regulation (EC) No. 1272/2008 and Council Directive 67/548/EEC on mutagenicity is warranted.