Reaction Mass of 2,6-dimethyloct-7-en-2-ol and 2,6-dimethyloct-7-en-2-yl formate

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:

20 February 2019 to 08 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labor and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries, 24 November 2000

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of 2,6-dimethyloct-7-en-2-ol and 2,6-dimethyloct-7-en-2-yl formate

EC Number:

915-335-6

Molecular formula:

C11H20O2 C10H20O

IUPAC Name:

Reaction Mass of 2,6-dimethyloct-7-en-2-ol and 2,6-dimethyloct-7-en-2-yl formate

Test material form:

liquid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Naphtha flavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Experiment 2: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate (TA100, TA98); 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate (TA1535); 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate (WP2uvrA, TA1537)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

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:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: preincubation

DURATION
- Preincubation period: 20 minutes (Experiment 2)
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning

Evaluation criteria:

There are several criteria for 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 (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnett’s Regression Analysis (p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Key result

Species / strain:

bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Controls:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
- Results for the negative controls (spontaneous mutation rates) are considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
- The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Experiment 1:
- The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of metabolic activation (S9-mix), initially from 500 μg/plate.
- No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
- There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix). Two statistically significant values were noted (TA1535 at 1.5 μg/plate and TA1537 at 500 μg/plate in the presence of metabolic activation (S9-mix). These responses were within the in-house historical vehicle/untreated control range for the strains and were, therefore considered of no biological relevance.

Experiment 2:
- Results from the second mutation test indicated that the test item induced a stronger toxic response employing the pre-incubation modification with weakened bacterial background lawns initially noted in the absence of metabolic activation (S9-mix) from 150 μg/plate (TA98 and TA1535) and 500 μg/plate (TA100, WP2uvrA and TA1537). In the presence of metabolic activation (S9-mix), weakened bacterial background lawns were initially noted from 500 μg/plate (TA100, TA1535 and TA98) and 1500 μg/plate (WP2uvrA and TA1537).
- No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
- No biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix). One statistically significant value was noted (TA1535 at 5 μg/plate in the presence of metabolic activation (S9-mix). However, as in Experiment 1 also, this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.

Applicant's summary and conclusion

Conclusions:

The test material was considered non-mutagenic under the conditions of this test.

Executive summary:

The test substance was evaluated in a bacterial reverse mutation assay performed according to OECD 471 and following GLP using Salmonella typhimurium strains TA98, TA100, TA 1535 and TA1537 and Escherichia coli strain WP2 uvrA both in the presence and absence of an exogenous metabolic activation system (S9-mix). The test substance was evaluated using an initial plate incorporation assay and a confirmatory preincubation procedure in triplicate with and without metabolic activation. In the first experiment, test substance concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate were assessed. In the second experiment, test substance concentrations of 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate (TA100, TA98), 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate (TA1535), and 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate (WP2uvrA, TA1537) were assessed. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. In the first mutation test (plate incorporation method), the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 and 500 μg/plate in the absence and presence of metabolic activation, respectively. In the second experiment, the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 and 500 μg/plate in the absence and presence of metabolic activation, respectively. No test item precipitate was observed on any of the plates. There were no significant biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. Therefore, the test material was considered to be non-mutagenic under the conditions of this test.