Quaternary ammonium compounds, C12-14-alkyltrimethyl, Me sulfates

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 05 December, 2002 to 13 January, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, C12-14 (even-numbered)-alkylethyldimethyl, ethyl sulphates

EC Number:

939-607-9

Molecular formula:

C18 H41 N1 O4 S1

IUPAC Name:

Quaternary ammonium compounds, C12-14 (even-numbered)-alkylethyldimethyl, ethyl sulphates

Test material form:

other: solid

Specific details on test material used for the study:

- Physical state: whitish pieces
- Lot/batch No.: LC0780
- Storage conditions: at room temperature
- Date of manufacture: 27, September, 2002
- Date of analysis: 27, September, 2002
- Date of receipt: 27, November, 2002
- Description: Refer to appendex 1 of the attached report for more details (page 22)

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other:

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route. The S9 fraction was preserved in sterile tubes at -80°C, until use)

Test concentrations with justification for top dose:

Preliminary test concentrations:10, 100, 500, 1000, 2500 and 5000 μg/plate for both with and without S9-mix

Main test concentrations: Experiments without S9 mix:
• 1.23, 3.7, 11.1, 33.3 and 100 μg/plate, for all tester strains in the first experiment,
• 0.41, 1.23, 3.7, 11.1 and 33.3 μg/plate, for all tester strains in the second experiment.

Experiments with S9 mix:
• 1.23, 3.7, 11.1, 33.3 and 100 μg/plate, for all tester strains in the first experiment,
• 3.7, 11.1, 33.3, 100 and 200 μg/plate, for all tester strains in the second experiment, except for the TA 1537 strain,
• 1.23, 3.7, 11.1, 33.3 and 100 μg/plate, for the TA 1537 strain in the second experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO), batch No. K30379650 214 (Merck Eurolab, Fontenay-Sous-Bois, France).

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

1 μg/plate: for TA 1535 and TA 100 in the absence of S-9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 μg/plate: for TA 1537 in the absence of S-9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

0.5 μg/plate: for TA 98 in the absence of S-9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.5 μg/plate: for TA 102 in the absence of S-9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-anthramine

Remarks:

2 μg/plate: for TA 1535, TA1537, TA 98 and TA 100 in the presence of S9 mix and 10 μg/plate: TA 102 in the presence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation method) as well as preincubation method

DURATION
- Incubation period: 48 to 72 h at approximately 37°C

NUMBER OF REPLICATIONS: Three/dose

DETERMINATION OF CYTOTOXICITY
- Method: Number of revertant colonies were counted, with an automatic counter (Artek counter, model 880, OSi, 75015 Paris, France or cardinal counter, Perceptive instruments, Suffolk CB9 7 BN, UK)

Evaluation criteria:

- Treatment of results
In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test item/mutants obtained in the presence of the vehicle), are presented in tabular form.

- Acceptance criteria
This study is considered valid if the following criteria are fully met:
• the number of revertants in the vehicle controls is consistent with the historical data of the testing facility (appendix 1),
• the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data of the testing facility.

- Evaluation criteria
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Results and discussion

Test results

Key result

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:

A moderate to strong toxicity was noted in all tester strains mainly at concentration-levels ≥ 33.3 μg/plate (experiments without S9). A moderate to strong toxicity was noted in all tester strains mainly at concentration-levels ≥ 100 μg/plate (with S9).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test: Yes

Any other information on results incl. tables

Refer to the attached report (pages 15 to 19) for full details

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance is not considered to be mutagenic in the presence and absence of exogenous metabolic activation.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD 471 Guideline and EU Method B.13/14, in compliance with GLP. The test substance was examined for mutagenic activity in five strains of Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102, using the plate incorporation and pre-incubation methods. The studies were performed in the absence and presence of metabolic activation (S9-mix). The concentrations ranged from 1.23 to 100 µg/plate in first experiment and 0.41 to 200 µg/plate in second experiment. No increase in reversion to prototrophy was obtained with any of the bacterial strain at any concentration either in the presence or absence of S9-mix. Further, inhibition of growth, observed as thinning of the background lawn of non-revertant cells, occurred in all strains following exposure to the test substance at ≥100 µg/plate. Under the study conditions, the test substance was not considered to be mutagenic in the presence and absence of exogenous metabolic activation (Haddouck, 2003).