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Key value for chemical safety assessment

Additional information

Ames test

In a reverse gene mutation assay in bacteria according to OECD guideline 471 and EU method B. 13/14, strains of S. typhimurium (TA98, TA100, TA1535, TA1537) and E. coli (WP2 uvr A) were exposed to the test substance in water at concentrations of 33, 100, 333, 1000, 2500 and 5000 µg/plate. A standard plate test (SPT) and a preincubation test (PIT) both with and without metabolic activation were performed. Each concentration was tested in triplicate.

No precipitation of the test substance was found with and without S9 mix. No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate and in the preincubation test up to the highest required concentration.

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data.

Thus, under the experimental conditions chosen here, it is concluded that the test substance is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

 

Ames II test

In a reverse gene mutation assay in bacteria following the experimental design according to Gee, P . et al. (1998), strains of S. typhimurium (TA 98, TA 7001 -TA 7006) were examined in a modified version of the traditional Ames test, i.e. a microtiter version. The test substance was tested up to a concentration of 5000 µg/mL in a liquid fluctuation test both with and without metabolic activation. Each concentration was tested in triplicate.

No precipitation of the test substance was found. Bacteriotoxicity was not observed at any of the tested doses. The positive controls induced the appropriate responses in the corresponding strains. An increase in the number of positive wells (his+ revertants) was not observed either with or without the addition of a metabolizing system. According to the results of the present study, the test substance is not mutagenic in the Ames II Assay (Salmonella typhimurium reverse mutation assay) under the experimental conditions chosen here.

 

HPRT test

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. 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 (4-hour exposure period)

0; 312.5; 625.0; 1250.0; 2500.0 μg/mL

with S9 mix (4-hour exposure period)

0; 312.5; 625.0; 1250.0; 2500.0 μg/mL

2nd Experiment

without S9 mix (4-hour exposure period)

0; 375; 750; 1500; 2500 μg/mL

with S9 mix (4-hour exposure period)

0; 375; 750; 1500; 2500 μg/mL

 

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, EMS and 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

The substance tris(2-hydroxyethyl)methylammonium methyl sulphate 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. Two independent experiments were carried out, both with and without the addition of liver S9 mix from 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

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

0; 312.5; 625.0; 1250.0; 2500.0 μg/mL

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

0; 312.5; 625.0; 1250.0; 2500.0 μg/mL

2nd Experiment

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

0; 312.5; 625.0; 1250.0; 2500.0 μg/mL

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

0; 312.5; 625.0; 1250.0; 2500.0 μg/mL

A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group.

 

The negative controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei. In this study, no cytotoxicity indicated by reduced cell count or proliferation index (CBPI) was observed up to the highest required 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, tris(2-hydroxyethyl)methylammonium methyl sulphate 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.


Short description of key information:
Ames test (BASF SE, 2014): negative
Ames II Test (BASF, 1999): negative
HPRT assay (BASF SE, 2014): negative
Micronucleus assay (BASF SE, 2015): negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies on mutagenicity are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance does not need to be classified and labelled under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EC.

 

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance does not need to be classified and labelled for mutagenicity under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation EC No 605/2014.