Octahydro-4,7-methano-1H-inden-5-yl acetate

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:

2-23-2007 - 3-26-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test was conducted according to OECD Test Guideline No. 471, 1997, under GLP Standards and QA.

Justification for type of information:

Information used for read across to Cyclacet Dihydro

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The information is based on read across

Justification for type of information:

Information used for read across to Cyclacet Dihydro. The full read across is documented in the Endpoint summary. The accompanying files are attached below.
Executive summary
Cyclacet Dihydro will have the same result in the Ames test as Cyclacet in view of high similarity in structure: backbone and functional group, which results in a negative result in the Ames test.
Structural similarities and differences: Cyclacet Dihydro, the target, and the source chemical Cyclacet have identical structural features consisting of a tricyclodecan-e/yl fused ring structure and an acetic ester attached at the bridged hexyl ring. The difference is that Cyclacet Dihydro (as presented in the name) does not have a double bond in the pentyl -ring which Cyclacet has.
Toxico-kinetics: The molecular weights, appearance, physic-chemical properties all indicate a similar bioavailability. Metabolism: In both Cyclacet Dihydro and Cyclacet the ester bond will be cleaved by bacteria in the gut and/or enzymes in the liver (Toxicological Handbook). Acetic acid will be one product and the Cyclacet Dihydro- and Cyclacet-alcohol the other metabolite. The parent and the metabolites are very similar and are not expected to have a different toxico-kinetic profile.
Toxico-dynamic: Cyclacet Dihydro and Cyclacet are anticipated to have similar genotoxicity based on their similarity in structure and similar metabolites. The absence of the double bond in Cyclacet Dihydro indicates a lower electrophilicity compared to Cyclacet. In view of this the read across from Cyclacet may be considered conservative.
Uncertainty: There is no uncertainty in the prediction based on the same functional group and very similar backbone.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: S. typhimurium TA97a, TA98, TA100, TA 102, TA1535 and E. coli WP2 uvrA (328)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Reaction mass of 3a,4,5,6,7,7a-hexahydro-4,7-methanoinden-5-yl acetate and 3a,4,5,6,7,7a-hexahydro-4,7-methanoinden-6-yl acetate

EC Number:

911-369-0

Molecular formula:

C12H16O2

IUPAC Name:

Reaction mass of 3a,4,5,6,7,7a-hexahydro-4,7-methanoinden-5-yl acetate and 3a,4,5,6,7,7a-hexahydro-4,7-methanoinden-6-yl acetate

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 TA97a, TA98, TA100, TA 102, TA1535 and E. coli WP2 uvrA (328)

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: S. typhimurium TA97a, TA98, TA100: rfa, ∆uvrB, pKM101 S. typhimurium TA1535: rfa, ∆uvrB S. typhimurium TA102: rfa, pAQ1 Plasmid, pKM101 E. coli WP2 uvrA (328): ∆uvrB

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 induced by Aroclor 1254

Test concentrations with justification for top dose:

Plate incorporation assay: 5, 10, 50, 100, 500, 1000, 2500, and 5000 μg/plate
Preincubation assay: 5, 10, 50, 100, and 250 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Selection of the appropriate solvent and diluent for the test substance was based on solubility information provided by the Sponsor and on a solubility assessment at the testing facility.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: S. typhimurium TA97a: ICRI91 Acridine mutagen; S. typhimurium TA98: 2-Nitrofluorene; S. typhimurium TA100 and TA1535: Sodium azide; E. coli WP2 uvrA (328): N-Methyl-N'-nitro-N-nitroguanidine +S9: S. typhimurium TA97a: 9,10-Dimethy1-1,2-benzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 minutes
- Selection time (if incubation with a selection agent): 48-72 hours

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

NUMBER OF REPLICATIONS: 3 plates per concentration

NUMBER OF CELLS EVALUATED: 1 x 10*8 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:
- Other: The dosage regimen was determined from a solubility study conducted prior to the experimental start.

Evaluation criteria:

A test substance was classified as positive if either of the first two conditions and the third condition were met:
1) The mean number of revertants in Salmonella strains TA97a, TA98, TA100, TA102 or Escherichia coli WP2 uvrA (328) at any test substance concentration was at least two times greater than the mean in the concurrent vehicle control.
2) The mean number of revertants in Salmonella strains TA1535 or TA1537 at any test substance concentration was at least three times greater than the mean in the concurrent vehicle control.
3) For a positive classification in any strain, there must have been a concentration-related increase in the mean revertants per plate in that same strain.

A test substance was classified as negative (e.g., nonmutagenic in bacteria) if the following conditions were met:
1) There were no test substance concentrations in Salmonella strains TA97a, TA98, TA100, TA102 or Escherichia coli WP2 uvrA (328) with a mean number of revertants that were at least two times greater than the mean in the concurrent vehicle control.
2) There were no test substance concentrations in Salmonella strains TAl535 or TAl537 with a mean number of revertants that were at least three times greater than the mean in the concurrent vehicle control.
3) For a negative classification in any strain, there could not be a concentration related increase in the mean revertants per plate in that same strain. In consultation with the sponsor, negative results may be confirmed as needed.

Results not meeting criteria for positive or negative classification were evaluated using scientific judgment and may have been reported as equivocal. In a two trial study, if one trial is positive and the other is negative, additional trials may be conducted.

Statistics:

Trials were evaluated independently. For each selected test strain, the average number of revertants and the standard deviation at each concentration level with and without the exogenous metabolic activation system was calculated.

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA97a, TA98, TA100, TA 102, TA1535 and E. coli WP2 uvrA (328)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Due to test substance associated toxicity in the plate incorporation assay, test substance concentrations were lowered for the confirmatory assay.

COMPARISON WITH HISTORICAL CONTROL DATA: The mean number of revertants observed in the negative controls (DMSO) for each of the test strains was within acceptable historical negative control ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Plate incorporation assay: S. typhimurium TA97a, TA98, TA100 and TA1535 (± S9), and E. coli WP2 uvrA (–S9): ≥ 500 ug/plate; E. coli WP2 uvrA (+S9): ≥ 1000 ug/plate.
Preincubation assay (–S9): S. typhimurium TA98: ≥ 100 ug/plate; S. typhimurium TA97a, TA100, TA1535, and E. coli WP2 uvrA (328): 250 ug/plate.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, no evidence of mutagenic activity was detected for the test substance, Cyclacet, in Salmonella typhimurium test strains TA97a, TA98, TAI00, and TA1535 along with Escherichia coli strain WP2 uvrA (328). The findings of this study show the test substance to be negative for the induction of mutagenicity in the bacterial reverse mutation test.

Executive summary:

The test substance, Cyclacet, was evaluated in a bacterial reverse mutation assay employing Salmonella typhimurium strains TA97a, TA98, TA100, and TA1535 along with Escherichia coli strain WP2 uvrA (328) both in the presence and absence of an exogenous metabolic activation system. The test substance was evaluated using an initial plate incorporation assay and a confirmatory preincubation procedure.

The solvent, diluent and negative control used in this assay was dimethyl sulfoxide (DMSO). Test substance concentrations of 5, 10, 50, 100, 500, 1000, 2500, and 5000 ug per plate were assessed with respect to negative (solvent) controls in the plate incorporation assay. Due to test article associated toxicity, test substance concentrations were lowered for the confirmatory assay. Concentrations of 5, 10, 50, 100, and 250 ug per plate were assessed with respect to negative (solvent) controls in the confirmatory preincubation assay. In the plate incorporation assay, test substance associated toxicity, as evidenced by a concentration related reduction in the mean number of revertants per plate and/or the reduction of the microcolony background lawns, was observed at 500 ug per plate and above in S. typhimurium tester strains TA97a, TA98, TA100, and TA1535 with and without exogenous metabolic activation along with E. coli strain WP2 uvrA (328) without exogenous metabolic activation. Toxicity was observed at 1000 ug per plate and above in E. coli strain WP2 uvrA (328) with exogenous metabolic activation. Test substance associated precipitate was not observed at any concentration level.

In the preincubation assay, toxicity was observed at 100 ug per plate and above in S. typhimurium test strain TA98 without exogenous metabolic activation and at 250 ug per plate in S. typhimurium strains TA97a, TA100, and TA1535 along with E. coli strain WP2 uvrA (328) without exogenous metabolic activation. No toxicity was observed at the tested concentrations for any strain with exogenous metabolic activation.

The mean number of revertants observed in the negative controls (DMSO) for each of the test strains was within acceptable historical negative control ranges. All test strains demonstrated appropriate phenotypic characteristics.

Under the conditions of this study, no evidence of mutagenic activity was detected for the test substance, Cyclacet, in S. typhimurium test strains TA97a, TA98, TAI00, and TA1535 along with E. coli strain WP2 uvrA (328). The findings of this study show the test substance to be negative for the induction of mutagenicity in the bacterial reverse mutation test.