Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 21 August, 2006 to 01 November, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted according to the OECD guideline 471 and Commission Directive 2000/32/EC, Annex-4D as well as in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4D
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Cetrimonium chloride
EC Number:
203-928-6
EC Name:
Cetrimonium chloride
Cas Number:
112-02-7
Molecular formula:
C19H42N.Cl
IUPAC Name:
N,N,N-trimethylhexadecan-1-aminium chloride
additive 1
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
water
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment I (with and without S9 mix): 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/plate
Experiment II (with and without S9 mix): 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: Deionised water was chosen because of its solubility properties and its low toxicity to the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
10 µg/plate for TA 1535 and TA 100 (Without metabolic activation)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water
Positive controls:
other:
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 10 µg/plate for TA 98, 50 µg/plate for TA 1537 (Without metabolic activation)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
3 µL/plate for WP2 uvrA (Without metabolic activation)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 2.5 µg/plate for TA 1535, TA 1537, TA 98, TA 100 and 10 µg/plate for WP2 uvrA (With metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Triplicates

Evaluation criteria:
A test item is considered as a mutagen if there is a biologically relevant increase in the number of revertants exceeding the threshold by twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control.

A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Reduction in the number of revertants observed in the test groups (i.e., at 100 – 333, 3 – 333 µg/plate with and without S9 mix respectively in experiment I; 100 – 333, 3 – 333 µg/plate respectively with and without S9 mix in experiment II)
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the test substance was non-mutagenic in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2 uvrA in the reverse mutation assay with and without metabolic activation.
Executive summary:

An in vitro study was conducted to investigate the potential of test substance, C16 TMAC (25% active in water) to induce gene mutations Salmonella typhimurium strains, according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA were treated with the test substance using the Ames plate incorporation (pre-experiment and experiment I) and pre-incubation methods (experiment II). The assay was performed in three independent experiments both with and without metabolic activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The concentrations of the test substance ranged from 3 to 5000 µg/plate in the pre-experiment, from 0.1 to 333 µg/plate in experiment I and from 0.01 to 333 µg/plate in experiment II. The plates incubated with the test substance showed reduced background growth in all strains with and without metabolic activation in all experiments. Strong cytotoxic effects, evidenced by a reduction in the number of revertants, occurred in the test groups with and without S9 mix. No substantial increase in revertant colony numbers in any of the five tester strains was observed following treatment with the test substance either in the presence or absence of S9 mix. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. All positive control showed a distinct increase in the number of revertant colonies. Under the study conditions, the substance was determined to be non-mutagenic with and without metabolic activation in the reverse mutation assay (Sokolowski, 2006).