Tetramethylammonium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro studies are available, one (WIL2012) was conducted with TMAC, the other two (WIL2013 and MHLW2001) were conducted with the Read Across substance TMAH. All three studies were conducted according to OECD guidelines and GLP principles.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11OCT2012-25OCT2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

- Solvent used: Milli-Q water
The stock solution was filter (0.22 μm)-sterilized. Test substance concentrations were used within 4.5 hours after preparation. The performance of the plating procedure exceeded the 4 hours, which was stated for the test solutions. However since this was only half hour and no indication has been given by the sponsor that the test substance is unstable in water, this deviation had no influence on the study result.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

with S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Remarks:

without S9; Sodium azide: 1535; ICR-191: 1537; 2-nitrofluorene: TA98; methylmethanesulfonate: TA100; 4-nitroquinoline-N-oxide: WP2uvrA.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted (with 50 or 10% S9 mix).

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Conclusions:

Interpretation of results (migrated information):
negative

In an AMES test, performed according to OECD guideline and GLP principles, TMAC was found not to be mutagenic with or without metabolic activation.

Executive summary:

An AMES test was performed according to OECD guideline and GLP principles. All bacterial strains showed negative responses up to 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that TMAC is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In an AMES test performed with TMAC, all bacterial strains showed negative responses up to 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that TMAC is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation.

A chromosomal aberration test was conducted with TMAH. Chinese hamster lung (CHL/IU) cells were exposed to 228, 455 or 910 ug/ml with and without metabolic activation.No considerable inhibition of cell growth was observed up to the highest dose.No increase in the number of polyploid cells or cellswith structural genetic aberrations were found after treatmentwith the test substance for 24 hr in the absence of metabolic activation or shortly for 6 hr with the test substance in the presence or absence of metabolic activation.Based on these findings, tetramethylammonium hydroxide was considered negative in the induction of chromosomal aberrations.

A mouse lymphoma assay was conducted with TMAH. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. In the absence of S9-mix, TMAH did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of 8% v/v S9-mix, TMAH did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with 12% v/v S9 for metabolic activation. It is concluded that TMAH is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Justification for using the two studies performed with TMAH for determination of the genotoxicity of TMAC is given in the attached Read Across document.

Justification for selection of genetic toxicity endpoint
One study available conducted with TMAC.

Justification for classification or non-classification

Based on the available data, TMAC is not classified for genotoxicity according to CLP Regulation (EC) No. 1272/2008.