Docosyltrimethylammonium methyl sulphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A structural analogue substance was not mutagenic in a guideline-compliant Salmonella typhimurium reverse mutation assay using strains TA 100, TA 1535, TA 1537 and TA 98 in either the absence or presence of rat liver S9 metabolic activation. This supporting substance was not genotoxic in a guideline-compliant mammalian cell gene (HPRT) mutation test in V79 Chinese Hamster cells and did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and the presence of metabolic activation in a guideline-compliant study.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study (OECD TG 471) with acceptable restrictions. For justification for read-across see chapter 1 of the chemical safety report.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Study was performed according to OECD guideline published at that time and according to GLP. E. coli WP2 strain was not used. The second experiment was performed as a plate incorporation assay, but not as a preincubation assay. Since the effects were clearly negative, these limitations are not considered to decisively limit the reliability of the study.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Pre-Experiment and Experiment I: 0 (control), 4, 20, 100, 500, 2500, 5000 µg/plate
Experiment II: 0 (control), 4, 20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below for additional information

Details on test system and experimental conditions:

The assay was performed in two independent experiments:

experiment I: plate incorporation assay with and without induced rat liver S9 mix
experiment II: plate incorporation assay with and without induced rat liver S9 mix

Rat liver S9 mix from Aroclor 1254 induced rats was used.

Top agar was prepared for the Salmonella strains by mixing 100 ml agar (0.6% (w/v) agar, 0.5% (w/v) NaCI) with 10 ml of a 0.5 mM histidine-biotin solution. After mixing, the liquid was poured into a petridish with minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2 % (wlv) glucose). After incubation for approximately 48 hours at approx. 37 °C in the dark, colonies (hi? revertants) were counted.

DURATION
- Exposure duration: after solidification the plates were incubated upside down for approx. 48 hours at 37°C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: The first experiment was performed with all tester strains using three plates per dose to get information on mutagenicity and toxicity for calculating an appropriate dose range. A reduced rate of spontaneously occuring colonies and visible thinning of the bacterial lawn were used as toxicity indicators. Thinning of the bacterial lawn was evaluated microscopically.
In combination with the second experiment, toxicity testing was performed as follows: 0.1 ml of the different dilutions of the test compound were thoroughly mixed with 0.1 ml of 108 dilution of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control is compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (= surviving fraction).

POSITIVE CONTROL SUBSTANCES:
without metabolic activation: sodium azide (TA 1535, TA 100), 9-aminoacridine (TA 1537), 2-nitrofluorene (TA 98);
with metabolic activation: 2-aminoanthracene (all strains)

Evaluation criteria:

A test article is classified as mutagenic if it has either of the following effects:
a) a test article produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) a test article induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test article at complete bacterial background lawn.
The test results must be reproducible.

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:

cytotoxicity

Remarks:

see below for additional information

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test compound was tested at doses of 4 to 5000 microgram/plate and proved to be toxic to most of the bacterial strains at doses of 2500 microgram/plate and above. Thinning of the bacterial lawn and a reduction in the number of colonies were observed at this dose.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Mean mutant number

Exp. I: plate incorporation method without S9 mix

Concentrations given in µg/plate

Strain -- 0 -- 4 -- 20 -- 100 -- 500 -- 2500 -- 5000

TA100 -- 176.7 -- 168.0 -- 201.0 -- 217.0 -- 25.0 -- 1.3 -- 1.0

TA1535 -- 10.7 -- 6.3 -- 8.3 -- 8.0 -- 1.3 -- 1.0 -- 0.3

TA1537 -- 7.0 -- 10.7 -- 9.7 -- 11.3 -- 5.0 -- 0.0 -- 0.0

TA98 -- 27.7 -- 24.0 -- 31.3 -- 23.0 -- 7.3 -- 1.0 -- 0.0

Exp. I: plate incorporation method with rat S9 mix

Concentrations given in µg/plate

Strain -- 0 -- 4 -- 20 -- 100 -- 500 -- 2500 -- 5000

TA100 -- 137.7 -- 207.0 -- 182.7 -- 185.0 -- 165.7 -- 43.0 -- 6.0

TA1535 -- 9.3 -- 11.7 -- 14.7 -- 12.3 -- 10.0 -- 5.0 -- 1.0

TA1537 -- 11.7 -- 8.7 -- 10.0 -- 8.3 -- 6.3 -- 2.3 -- 0.7

TA98 -- 36.3 -- 34.7 -- 36.7 -- 41.7 -- 37.0 -- 15.0 -- 2.0

Exp. II: plate incorporation method without S9 mix

Concentrations given in µg/plate

Strain -- 0 -- 4 -- 20 -- 100 -- 500 -- 2500 -- 5000

TA100 -- 141.7 -- 184.3 -- 146.3 -- 145.7 -- 2.0 -- 1.0 -- 1.0

TA1535 -- 16.0 -- 12.7 -- 7.7 -- 8.7 -- 3.3 -- 0.0 -- 0.0

TA1537 -- 7.7 -- 7.7 -- 8.7 -- 8.3 -- 4.3 -- 0.0 -- 0.0

TA98 -- 26.3 -- 24.3 -- 22.0 -- 31.0 -- 9.3 -- 1.3 -- 0.0

Exp. II:

plate incorporation method with rat liver S9 mix

Concentrations given in µg/plate

Strain -- 0 -- 4 -- 20 -- 100 -- 500 -- 2500 -- 5000

TA100 -- 176.3 -- 172.7 -- 170.3 -- 170.3 -- 123.3 -- 98.7 -- 24.0

TA1535 -- 19.3 -- 13.0 -- 16.7 -- 13.3 -- 15.0 -- 1.3 -- 1.0

TA1537 -- 8.0 -- 7.0 -- 9.7 -- 10.3 -- 6.0 -- 9.3 -- 0.3

TA98 -- 36.7 -- 33.3 -- 36.3 -- 29.3 -- 30.7 -- 8.3 -- 1.3

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

The test substance was not mutagenic in a guideline-compliant Salmonella typhimurium reverse mutation assay using strains TA 100, TA 1535, TA 1537 and TA 98 in either the absence or presence of rat liver S9 metabolic activation.

Executive summary:

A similar supporting substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. The test substance was dissolved in ethanol and a dose range of 6 different doses from 4 microgram/plate to 5000 microgram/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to

that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: The test compound proved to be toxic of the bacterial strains at doses of 2500 microgram/plate and above.

5000 microgram/plate was chosen as top dose level for the mutagenicity study.

Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacteria) strains. Also in the presence of a metabolic activation system, treatment of the cells with the test substance did not result in relevant increases in the number of revertant colonies.

Summarizing, it can be stated that the supporting substance is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The structural analogue substance Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides, which differs only in the anion, showed negative results in a study for the induction of gene mutations (bacterial reverse mutation assays) by frameshift or base-pair substitutions with and without metabolic activation. Studies were performed with the test strains S. typhimurium TA 98, TA 100, TA 1535, and TA 1537. Test concentrations up to the limit concentration of 5000 µg/plate were tested; cytotoxic effects were observed at 2500 µg/plate and higher. This study was selected as key study.

The supporting substance also yielded negative results in an in vitro gene (HPRT) mutation study in mammalian cells V79 cells (Chinese hamster cell line) at up to cytotoxic concentrations in the presence and absence of metabolic activation. Relevant toxic effects indicated by a relative cloning efficiency I (CE I) below 50% occurred in the first experiment at 1.3 microg/ml and above without metabolic activation and at 9.9 (culture I only) and 19.7 microg/ml and above in the presence of metabolic activation. In the second experiment such toxic effects occurred at 4.7 microg/ml and above. This study was selected as key study.

The supporting substance did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and the presence of metabolic activation under the experimental conditions used when test up to precipitating/cytotoxic concentrations. The highest applied concentration in Experiment l (400 µg/ml) was chosen with regard to the ability to formulate a homogeneous suspension in an appropriate solvent following the current OECD Guideline 473. In the absence and presence of S9 mix, clear cytotoxicity with relative cell numbers of below 40 % of control were observed after treatment with the highest scored concentrations, except in Experiment I in the presence of S9 mix. In this part of the study concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment I (highest evaluated concentration 12.5 µg/ml), in the absence and the presence of S9 mix and in Experiment IIA (highest evaluated concentration 12.5 µg/ml), in the presence of S9 mix, no statistically significant or biologically relevant increase in the number of micronucleated cells was observed up to the highest evaluated concentrations. In Experiment IIA, in the absence of S9 mix, dose-related and statistically significant increases in the percentage of micronucleated cells were observed at the three tested concentrations (3.1, 6.3 and 12.5 µg/ml). However, the only concentration that produced a frequency of micronucleated cells outside the historical control range was also associated with high cytotoxicity. To verify the observation in Experiment IIA, a repeat experiment in the absence of S9 mix within a narrow concentration range (5.0, 6.0 and 7.0 µg/ml), designated Experiment IIB, was performed. In Experiment IIB, no clastogenicity was observed up to the highest scorable concentration, which was associated with appropriate cytotoxicity. Accordingly, the increased incidence of micronucleated cells observed at a concentration associated with high cytotoxicity (Experiment IIA in the absence of S9 mix) could not be reproduced, and this finding was considered to bear no biological significance. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei in V79 cells in vitro in the absence and the presence of metabolic activation. This study was selected as key study.

As a representative of the anion of the submission substance, literature data about the related substance monomethyl sulfate (CAS-No. 75-93-4) was also considered. Tan et al. (1983) tested the mutagenicity of methyl sulfate in the CHO/HGPRT System. This test was suitable to detect direct-acting methylating agents (such as dimethylsulfate). The test substance did not increase the mutant frequency at the HGPRT locus in Chinese hamster CHO cells and is therefore considered to be non-mutagenic under the conditions of the test.

In conclusion, the submission substance is found to be not mutagenic in the bacterial reverse mutation assay, the mammalian gene (HPRT) mutation assay and in the in vitro micronucleus assay.

Justification for selection of genetic toxicity endpoint
Several key studies.

Justification for classification or non-classification

The structural analogue substance Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

and thus the submission substance do not have to be classified for mutagenicity according to the criteria laid down in the EU Dangerous Substances Directive (67/548/EEC) and in the EU Classification Labelling and Packaging Regulation (1272/2008/EC) because the similar supporting substance did not reveal any mutagenic effect in the bacterial reverse mutation assay in the presence or absence of metabolic activation , in the mammalian cell gene (HPRT) mutation test in V79 cells and in the in vitro micronucleus test in V79 cells.