N-(1,3-dimethylbutylidene)-5-[(1,3-dimethylbutylidene)amino]-1,3,3-trimethylcyclohexanemethylamine

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:

28 July 2014 - 15 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Specific details on test material used for the study:

Identification: CAS#71077-09-3
Batch: 76200
Purity: 98,70%
Physical state: Pale yellow liquid
Expiry date 04 March 2015
Storage conditions: room temperature in dark

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and the WP2uvrA, both with and without S9-mix. Seven concentrations 0, 1.5, 5, 15, 50,150, 500, 1500 and 5000 μg/plate were tested in triplicate.

Vehicle / solvent:

The vehicle of the test item was dimethyl sulfoxide.

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation methods at five dose concentrations

DURATION
- Preincubation period: 20 minutes
- Exposure duration:48 h

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

There are several criteria determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study.
1. A dose related increase in mutant frequency over the dose range tested
2. A reproducible increase at one or more concentrations
3. Biological relevance against in-house historical control ranges
4. Statistical analysis of data determined by UKEMS
5. Fold increase greater than two times the concurrent solvent control for any tester strain
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. If no conclusion on mutagenicity can be made the result will be reported as equivivocal

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed.

RANGE-FINDING/SCREENING STUDIES: The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA) in all concentrations tested (0 - 5000ug/plate).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: All positive control chemicals used in the test induced marked increase in the frequency of revertant colonies thus confriming the activity of the S9 mix and the sensititivity of the bacterrial strains.
- Negative (solvent/vehicle) historical control data: Results for the negative control (spontaneous mutation rates) were considered to be acceptable.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was observed in TA 100, TA1535 and TA1537 tester strains at the maximum dose level of 5000ug/plate. No toxicity was observed in TA98 and E.coli strains.

Applicant's summary and conclusion

Conclusions:

All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. 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. The test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The objective of this study was to determine the potential of the test substance and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli(E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. The study procedures described in this report were based on the most recent OECD and EC guidelines. The test item was dissolved in dimethyl sulfoxide.

In the dose-range finding study, the test item was initially tested up to concentration of 5000μg/plate in all tester strains in the direct plate assay. The test item reduced the growth of the bacterial background lawns of TA100, TA1535 and 1537 at 5000ug/plate in both the absence and precence of S9 -mix. No toxicity was observed at was noted in TA98 and WP2uvrA strains at 5000ug/plate.

Consequently, thye same maximum dose level was used in the second mutation test. In the second mutation test (pre-incubation method) the test item again induced a toxic response with visible reductions in the growth of the bacterial background lawns of Salmonella tester strains TA100, TA1535 and TA 1537 noted at 5000ug/plate in the absence of S9 -mix only. No toxicity was noted to any of the tester strains dosed in the presence of S9 -mix and TA98 or WP2uvrA in the presence or absence of S9 -mix.

No the test item precipitate was noted at any doses tested.

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.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation.

In conclusion, based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.