Reaction mass of 3,5,6,6-tetramethyl-4-methyleneheptan-2-one and (E)-3,4,5,6,6-pentamethylhept-3-en-2-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Two instead of three replicates was used for the substance treatment.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Standards for Toxicity Investigations, Ministry of Labour, Japan

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Procedures of Mutagenicity Test Using Microorganisms and Evaluation of Test Results, Ministry of Labor, Japan

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (E)-3,4,5,6,6-pentamethylhept-3-en-2-one and 3,5,6,6-tetramethyl-4-methyleneheptan-2-one

EC Number:

946-245-5

Molecular formula:

C12H22O

IUPAC Name:

Reaction mass of (E)-3,4,5,6,6-pentamethylhept-3-en-2-one and 3,5,6,6-tetramethyl-4-methyleneheptan-2-one

Test material form:

liquid

Details on test material:

According to ECHA communication with reference to Annotation number: SUB-C-2114629169-42-01/F, the substance ID for Koavone has been changed with EC# from 939-627-8 to 946-245-5, and the IUPAC name from Reaction mass of (3R,5R)-3,5,6,6-tetramethyl-4-methylideneheptan-2-one and (3R,5S)-3,5,6,6-tetramethyl-4-methylideneheptan-2-one and (E)-3,4,5,6,6-pentamethylhept-3-en-2-one to Reaction mass of (E)-3,4,5,6,6-pentamethylhept-3-en-2-one and 3,5,6,6-tetramethyl-4-methyleneheptan-2-one.

Method

Target gene:

S. typhimurium TA1535: hisG46 rfa uvrB; S. typhimurium TA1537: hisC3076 rfa uvrB; S. typhimurium TA98: hisD3052 rfa uvrB pKMl01; S. typhimurium TA100: hisG46 rfa uvrB pKM101; E. coli WP2 uvrA: trp

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:

Rat liver S9

Test concentrations with justification for top dose:

Dose-range finding test 1: with and without S9: 0, 4.88, 19.5, 78.1, 313, 1250 and 5000 μg/plate
Dose-range finding test 2: all strains without S9 mix and TA100 and TA1535 with S9 mix: 0, 2.44, 4.88, 9.77 ,19.5, 39.1 and 78.1 μg/plate; WP2 uvrA, TA98 and TA1537 with S9 mix: 0, 9.77, 19.5, 39.1, 78.1, 156 and 313 μg/plate
Main test: without S9 mix: all strains without S9 mix and TA100 and TA1535 with S9 mix: 0, 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 μg/plate; WP2 uvrA, TA98 and TA1537 with S9 mix: 0, 9.77, 19.5, 39.1, 78.1, 156 and 313 μg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: the substance was insoluble in distilled water at 50 mg/mL, but was soluble in DMSO at 50 mg/mL. The test substance solution of 50 mg/mL prepared with DMSO was considered to be stable, as there was no change in colour nor heat generation at room temperature within 2 hours after preparation.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: AF-2 (2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide); ICR-191 (2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine x 2 HCl), 2-aminoanthracene

Details on test system and experimental conditions:

Two dose-finding tests and one main test were performed.

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate plates for the negative control group and duplicate plates per dose for the substance treatment groups and the positive control groups

METHODS FOR MEASUREMENT OF CYTOTOXICITY: bacterial growth inhibition

Evaluation criteria:

The test substance was judged positive when the number of revertant colonies increased to two times or more than that in the negative control in a concentration-dependent manner and also reproducibility of the results was obtained. In all other cases, the results were judged negative.

Statistics:

Statistical analysis was not performed.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation: Precipitation of the substance was not observed at any doses.

RANGE-FINDING/SCREENING STUDIES: The bacterial growth inhibition was observed at 78.1 μg/plate or higher in all test strains without S9 mix and in TA100 and TA1535 with S9 mix and at 313 μg/plate or higher in WP2 uvrA, TA98 and TA1537 with S9 mix in dose-finding test 1. In dose-finding test 2, the bacterial growth inhibition was observed at 78.1 μg/plate or higher in all test strains without S9 mix, at 78.1 μg/plate in TA100 and TA1535 with S9 mix, at 313 μg/plate in WP2 uvrA with S9 mix and 156 μg/plate or higher in TA98 or TA1535 with S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the main test, the bacterial growth inhibition was observed at 78.1 μg/plate in all test strains without S9 mix, at 78.1 μg/plate in TA100 and TA1535 with S9 mix, at 313 μg/plate in WP2 uvrA with S9 mix and 156 μg/plate or higher in TA98 and TA1537 with S9 mix.

Applicant's summary and conclusion

Conclusions:

The substance gave negative results in an Ames test wih S. typhimurium TA98, TA100, TA1535 and TA1537 and E. coli WP2 uvr A, both with and without metabolic activation (OECD 471).

Executive summary:

An in vitro mutagenicity test using bacteria was performed according to OECD TG 471, and in compliance with GLP. The ability of the substance to induce mutations was investigated using Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and E. coli strain WP2 uvr A using a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix). In the absence of the S9 and in case of TA100 and TA1535 in the presence of S9, the highest dose tested was 78.1 µg/plate, while for TA98, TA1537 and WP2 uvr A in the presence of S9 mix it was 313 µg/plate based on two dose-range finding tests.

No precipitation was observed at any dose level with and without S9 mix. Bacterial growth inhibition was observed at 78.1 μg/plate in all test strains without S9 mix, at 78.1 μg/plate in TA100 and TA1535 with S9 mix, at 313 μg/plate in WP2 uvrA with S9 mix and 156 μg/plate or higher in TA98 and TA1537 with S9 mix. The numbers of revertant colonies in the substance treatment groups were less than two times that observed in each negative control in all test strains and therefore, the mutagenicity of the substance was judged negative.

As such, it is concluded that the substance has no ability to induce mutations under the present test conditions.