reaction mass of 1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one and 1-(1,2,3,4,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one and 1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 November 1994 - 29 November 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Remarks:

Statement of Compliance

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Reaction Mass of 1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one and 1-(1,2,3,4,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one and 1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

EC Number:

915-730-3

Molecular formula:

C16H26O

IUPAC Name:

Reaction Mass of 1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one and 1-(1,2,3,4,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one and 1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

Constituent 1

Reference substance name:

1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

EC Number:

259-174-3

EC Name:

1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

Cas Number:

54464-57-2

Molecular formula:

C16H26O

IUPAC Name:

1-(2,3,8,8-tetramethyl-1,2,3,4,5,6,7,8-octahydronaphthalen-2-yl)ethanone

Constituent 2

Reference substance name:

1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

EC Number:

268-978-3

EC Name:

1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

Cas Number:

68155-66-8

Molecular formula:

C16H26O

IUPAC Name:

1-(2,3,8,8-tetramethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen-2-yl)ethanone

Constituent 3

Reference substance name:

1-(1,2,3,4,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

EC Number:

268-979-9

EC Name:

1-(1,2,3,4,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one

Cas Number:

68155-67-9

Molecular formula:

C16H26O

IUPAC Name:

1-(2,3,8,8-tetramethyl-1,2,3,4,6,7,8,8a-octahydronaphthalen-2-yl)ethanone

Test material form:

liquid

Method

Target gene:

His-gene: Amino acid histidine, and Trp-gene: amino acid tryptophan

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 , TA 100 and E. coli WP2 uvrA

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: S. typhimurium TA1535, TA1537, TA1538: rfa, uvrB; S. typhimurium TA98, TA100: rfa, uvrB, pKM101; E. coli WP2: uvrA

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 induced by Aroclor 1254

Test concentrations with justification for top dose:

5000, 2500, 1250, 625, and 312.5 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was virtually insoluble in water.

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: S. typhimurium TA 1535, TA 100, E. coli WP2 uvrA: N-Ethyl-N'-nitro-N-nitrosoguanidine; S. typhimurium TA 1537: 9-Aminoacridine; S. typhimurium TA 1538, TA 98: 2-Nitrofluorene. +S9: all strains: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Selection time (if incubation with a selection agent): 3 days

SELECTION AGENT (mutation assays): agar containing Histidine or Tryptophan

NUMBER OF REPLICATIONS: 3 plates per concentration

NUMBER OF CELLS EVALUATED: 10*9 viable cells

DETERMINATION OF CYTOTOXICITY
- Method: determination of a concentration related reduction in the mean number of revertants per plate and/or the reduction of the microcolony background lawns

OTHER EXAMINATIONS: Preliminary toxicity/Dose range finding test at 5, 50, 500, and 5000 ug/plate.

Evaluation criteria:

If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, the following approach is taken in order to resolve the issue of the substance's mutagenic activity in this test system.
- Repeat tests may be performed using modifications of the experimental method.
- If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers.

Statistics:

Mean and Standard Deviation

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: OTNE was not toxic towards the tester strains in the preliminary toxicity test. Therefore 5000 ug/plate was chosen as the top dose level in the mutation tests.

Applicant's summary and conclusion

Conclusions:

No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with OTNE at any dose level, in the presence or absence of S-9 mix, in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that OTNE tested in DMSO shows no evidence of mutagenic activity in this bacterial system.

Executive summary:

In this in vitro assessment of the mutagenic potential of OTNE, histidine dependent auxotrophic mutants of S. typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a tryptophan dependent mutant of E. Coli (WP2uvrA) were exposed to the test substance, diluted in DMSO which was also used as a negative control. Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats. In the preliminary dose range finding study with dose levels of up to 5000 ug/plate, no toxicity was observed. A top dose level of 5000 ug/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 2500, 1250, 625, and 312.5 ug/plate. No evidence of mutagenic activity was seen at any dose level of OTNE in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that OTNE tested in DMSO was not mutagenic in this bacterial system.