Docosyltrimethylammonium methyl sulphate

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1955

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

EC Number:

271-756-9

EC Name:

Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

Cas Number:

68607-24-9

IUPAC Name:

68607-24-9

Method

Species / strain

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

Controls

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.