Potassium N,N-dimethylglycinate

Endpoint summary

Additional information

Ames assay
Key
In a reverse mutation assay in bacteria, strains of Salmonella typhimurium ( TA 1535, TA 100, TA 1537, TA 98) and Escherichia coli ( WP2 uvrA) were exposed to the test substance at concentrations of 0; 33; 100; 333; 1000; 2650 and 5300 μg/plate (BASF SE, 2015). A standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats) were conducted.
No precipitation of the test substance was found with and without S9 mix. A bacteriotoxic effect was occasionally observed in the preincubation test at a concentration of 5300 μg/plate. 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, marginally below or above the range of the historical positive control data.
Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Supporting
Two publications further support the key study on bacterial reverse mutation. Hoorn et al. (1989) used the read across substance N, N-dimethylglycine hydrochloride (CAS# 2491-06-7) and tested the S. typhimurium strains TA100 and TA1535 with and without metabolic activation. Colman et al. tested the S. typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 with the above mentioned read across substance with and without metabolic activation. No mutagenic properties were observed with or without S9 mix in both publications.

HPRT assay
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:
0; 100.0; 200.0; 400.0; 800.0; 1600.0 μg/mL

[0; 200.0; 400.0; 800.0; 1600.0 μg/mL were evaluated for gene mutations]

with S9 mix:
0; 100.0; 200.0; 400.0; 800.0; 1600.0 μg/mL

[0; 200.0; 400.0; 800.0; 1600.0 μg/mL were evaluated for gene mutations]

2nd Experiment
without S9 mix:
0; 87.5; 175.0; 350.0; 700.0; 1600.0 μg/mL

[0; 175.0; 350.0; 700.0; 1600.0 μg/mL were evaluated for gene mutations]

with S9 mix:
0; 87.5; 175.0; 350.0; 700.0; 1600.0 μg/mL

[0; 175.0; 350.0; 700.0; 1600.0 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
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. 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 observations and the toxicity data of a previously performed pretest for a HPRT study (BASF project No. 50M0456/01M022) the following concentrations were tested.

1st Experiment
4 hours exposure, 24 hours harvest time, without S9 mix:
0; 50.0; 100.0; 200.0; 400.0; 800.0; 1600.0 μg/mL

[0; 400.0; 800.0; 1600.0 μg/mL were evaluated]

4 hours exposure, 24 hours harvest time, with S9 mix:
0; 50.0; 100.0; 200.0; 400.0; 800.0; 1600.0 μg/mL

[0; 400.0; 800.0; 1600.0 μg/mL were evaluated]

2nd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix:
0; 200.0; 400.0; 800.0; 1600.0 μg/mL

[0; 400.0; 800.0; 1600.0 μg/mL were evaluated]

4 hours exposure, 44 hours harvest time, with S9 mix:
0; 200.0; 400.0; 800.0; 1600.0 μg/mL

[0; 400.0; 800.0; 1600.0 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 our 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 are available to assess the mutagenic acitivity 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, 2015). 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, 2015). The test substancde 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 1272/2008. As a result the substance does not need to be classified and labelled for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the seventh time in Regulation (EU) No 2015/1221.