Cyclohexane, 1,4-bis(ethoxymethyl)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames assay

Key

The test substance 1,4-bis(ethoxymethyl)-Cyclohexane was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay according to OECD 471 (BASF SE, 2013, 40M0766/12M359). The strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA were tested at a dose range of 33 μg - 5 000 μg/plate (SPT), 33 μg - 5 000 μg/plate (PIT, TA strains without S9, E. coli) and 3.3 μg - 5 000 μg/plate (PIT; TA strains with S9). Standard plate test (SPT) and preincubation test (PIT) were performed both with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test substance was found neither with nor without S9 mix. A strong bacteriotoxic effect was observed depending on the strain and test conditions from about 100 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

Thus, under the experimental conditions of this study, the test substance 1,4- bis(ethoxymethyl)-Cyclohexane is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

 

HPRT assay

Key

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro according to OECD guideline 476 and EU method B.17 (BASF SE, 2016, 50M0261/14M306). Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested.

 

1st Experiment

without S9 mix

0; 9.4; 18.8; 37.5; 75.0; 150.0; 300.0; 600.0 μg/mL

(0; 9.4; 18.8; 37.5; 75; 150.0 µg/mL were evaluated for gene mutations)

 

with S9 mix

0; 18.8; 37.5; 75.0; 150.0; 300.0; 600.0; 1200.0 μg/mL

(0; 18.8; 37.5; 75; 150.0; 300.0 μg/mL were evaluated for gene mutations)

 

2nd Experiment

without S9 mix

0; 6.3; 12.5; 25.0; 50.0; 100.0; 200.0; 400.0 μg/mL

(0; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL were evaluated for gene mutations)

 

with S9 mix

0; 12.5; 25.0; 50.0; 100.0; 200.0; 400.0; 800.0 μg/mL

(0; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL were evaluated for gene mutations)

 

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]anthracene (DMBA), led to the expected increase in the frequencies of forward mutations. In this study in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest required concentration evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

 

Micronucleus assay

Key

The test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) according to OECD guideline 487 and EU method B.49 (BASF SE, 2016, 33M0261/14M307). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). Based on the pretests for dose selection, the following concentrations were tested.

 

1st Experiment

4 hours exposure, 24 hours harvest time, without S9 mix

0; 15.6; 31.3; 62.5; 125.0; 250.0; 500.0 μg/mL

(0; 62.5; 125.0 μg/mL were evaluated)

 

4 hours exposure, 24 hours harvest time, with S9 mix

0; 15.6; 31.3; 62.5; 125.0; 250.0; 500.0 μg/mL

(0; 62.5; 125.0; 250.0 μg/mL were evaluated)

 

2nd Experiment

24 hours exposure, 24 hours harvest time, without S9 mix

0; 15.6; 31.3; 62.5; 125.0; 250.0; 500.0 μg/mL

(0; 31.3; 62.5; 125.0; 250.0 μg/mL were evaluated)

 

4 hours exposure, 44 hours harvest time, with S9 mix

0; 15.6; 31.3; 62.5; 125.0; 250.0; 500.0 μg/mL

(0; 62.5; 125.0; 250.0 μg/mL were evaluated)

 

3rd Experiment

4 hours exposure, 24 hours harvest time, with S9 mix

0; 50.0; 75.0; 100.0; 150.0; 200.0; 300.0 μg/mL

(0; 75.0; 100.0; 150.0 μg/mL were evaluated)

 

4th Experiment

4 hours exposure, 44 hours harvest time, with S9 mix

0; 50.0; 75.0; 100.0; 150.0; 200.0; 300.0 μg/mL

(0; 50; 75.0; 100.0; 150.0; 200 μg/mL were evaluated)

 

A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. The negative controls gave frequencies of micronucleated cells within the historical negative control data range for V79 cells. Both positive control substances, ethyl methanesulfonate (EMS) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei. In this study, no cytotoxicity indicated by reduced cell count (indicated by relative population doubling) or proliferation index (CBPI) was observed up to the highest applied test substance concentration. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Thus, under the experimental conditions described, potassium N,N-dimethylglycinate is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

 

Justification for selection of genetic toxicity endpoint
GLP and guideline compliant study reports (Ames, HPRT, and MN) are available to assess the genetic toxicity of the test substance.

Short description of key information:
In a reverse gene mutation assay in bacteria (Ames Test), no mutagenicity was observed with or without metabolic activation. (BASF SE, 2013).
No mutant colonies were detected in the in vitro mammalian cell gene mutation test examining the HPRT locus of Chinese hamster ovary cells (BASF SE, 2016).
The test substance did not show any potential to induce micronuclei under in vitro conditions in a micronucleus assay using the cytokinesis block method (BASF SE, 2016).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance does not need to be classified and labelled for genetic toxicity under Regulation (EC) No 1272/2008.