Decahydro-1,1,7-trimethyl-3a,7-methano-3aH-cyclopentacyclooct-3-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:

20.03.2018 to 27.03.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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

Test material information

Constituent 1

Reference substance name:

Decahydro-1,1,7-trimethyl-3a,7-methano-3aH-cyclopentacyclooct-3-yl formate

EC Number:

261-118-8

EC Name:

Decahydro-1,1,7-trimethyl-3a,7-methano-3aH-cyclopentacyclooct-3-yl formate

Cas Number:

58096-47-2

Molecular formula:

C16H26O2

IUPAC Name:

decahydro-1,1,7-trimethyl-3a,7-methano-3aH-cyclopentacyclooct-3-yl formate

Test material form:

liquid

Method

Target gene:

Following strains of bacteria accepted under OECD guideline for the assessment of point gene mutation were used:
Histidine auxotrophic strains of Salmonella typhimurium: TA98, TA100, TA1535 and TA1537.
Tryptophan auxotrophic strain of Escherichia coli: WP2uvrA (pKM101).

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 homogenate was used as the metabolic activation system.

Test concentrations with justification for top dose:

The test item was not toxic to the tester strain at any of the tested doses as the intensity of the bacterial background lawn and the mean number of revertant colonies were comparable to the THF control.

Based on these observations, the highest OECD 471-recommended dose of 5000 g/plate was tested in the mutation assay.

Vehicle / solvent:

Tetrahydrofuran (THF)

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

Test System
Following strains of bacteria accepted under OECD guideline for the assessment of point gene mutation were used:
Histidine auxotrophic strains of Salmonella typhimurium: TA98, TA100, TA1535 and TA1537.
Tryptophan auxotrophic strain of Escherichia coli: WP2uvrA (pKM101).

Each S. typhimurium tester strain contains, in addition to a mutation in the histidine operon, additional mutations that enhance sensitivity to some mutagens. The rfa mutation results in a cell wall deficiency that increases the permeability of the cell to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA excision repair system. Tester strains TA98 and TA100 also contain the pKM101 plasmid (carrying the R factor). It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch repair process.

TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both frameshift and base substitution mutagens and TA1535 is reverted only by mutagens that cause base substitutions.

The preliminary toxicity test was conducted to check toxicity of the test item to the Salmonella typhimurium tester strain TA100 at 50, 100, 200, 400, 800, 1600, 3200, and 5000 µg/plate test concentrations along with the THF control.

The following five test doses were selected (with approximately half-log dose interval) for testing in the mutation assay:

(a) 50, (b) 158, (c) 500, (d) 1581, and (e) 5000 µg/plate.


The E. coli tester strain has an AT base pair at the critical mutation site within the trpE gene (Wilcox et al., 1990). Tester strain WP2uvrA (pKM101) has a deletion in the uvrA gene resulting in a deficient DNA excision repair system. Tryptophan revertants can arise due to a base change at the originally mutated site or by a base change elsewhere in the chromosome causing the original mutation to be suppressed. Thus, the specificity of the reversion mechanism is sensitive to base pair substitution mutations (Green and Muriel, 1976).

Evaluation criteria:

To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system.

The test will be judged positive, if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value for strains TA98, TA100 and WP2uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value for strains TA1535 and TA1537.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Results and discussion

Test results

Test results 1

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

Positive controls validity:

valid

Test results 2

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

Positive controls validity:

valid

Test results 3

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

Positive controls validity:

valid

Test results 4

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

Positive controls validity:

valid

Any other information on results incl. tables

Results of Preliminary Toxicity Test

Treatment (mg/plate)	TA 100 revertant colonies/plate^*
	Presence of Metabolic Activation			Absence of Metabolic Activation
	Mean	Background lawn intensity	Precipitation	Mean	Background lawn intensity	Precipitation
THF (100mL)	108	4+	NPO	107	4+	NPO
50	106	4+	NPO	108	4+	NPO
100	101	4+	NPO	104	4+	NPO
200	104	4+	NPO	106	4+	NPO
400	104	4+	NPO	101	4+	NPO
800	106	4+	NPO	102	4+	NPO
1600	101	4+	NPO	102	4+	NPO
3200	101	4+	NPO	100	4+	NPO
5000	102	4+	NPO	104	4+	NPO

*: Mean of two replicates and rounded off to the nearest whole number

4+ - Normal                            3+ - Slightly reduced               2+ - Moderately reduced

1+ - Severely reduced             0- Absent                                NPO – No precipitation observed

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item, Decahydro-1,1,7-trimethyl-3a,7-methano-3aH-cyclopentacyclooct-3-yl formate [Common name: Clovanyl-3-Formate (CARYOLAN), was not mutagenic in this bacterial reverse mutation test up to the highest OECD 471-recommended dose of 5000 µg/plate under the conditions of testing employed.

Executive summary:

Clovanyl-3-Formate (CARYOLAN) was tested for its mutagenic potential in the bacterial reverse mutation assay. The study was conducted using TA98, TA100, TA1535 and TA1537 strains ofSalmonella typhimuriumand WP2uvrA (pKM101) strain ofEscherichia coli.The study consisted of a preliminary toxicity test and two mutation assays comprising four independent experiments. The bacterial tester strains were exposed to the test item in the presence and absence of a metabolic activation system
(S9 fraction prepared from Aroclor 1254 induced rat liver).

Clovanyl-3-Formate (CARYOLAN) was soluble in Tetrahydrofuran (THF) at 50 mg/mL. The test item was found to be stable in THF for 24 hours at room temperature at the dose concentrations of 25 and 50,000 µg/mL.

In a preliminary toxicity test to select test doses for the mutation assay, the test item did not precipitate on the basal agar plates at any of the tested doses, The test item did not show toxicity to the tester strain at any of the tested doses as the intensity of the bacterial background lawn and the mean number of revertant colonies were comparable to the vehicle control plates. Based on these observations, Clovanyl-3-Formate (CARYOLAN)was testedup to the highest OECD 471-recommended dose of 5000 µg/plate in the mutation assay.

The bacterial tester strains were exposed to Clovanyl-3-Formate (CARYOLAN)in triplicate at 50, 158, 500, 1581, and 5000 µg/plate using the direct plate incorporation mode of exposure in the initial mutation assay and using the pre-incubation mode of exposure in the confirmatory mutation assay. The vehicle control (THF) and the appropriate positive controls (2-aminoanthracene, 2-nitrofluorene, sodium azide, 9-aminoacridine, and 4-nitroquinoline-1-oxide) were tested simultaneously. The mean and standard deviation of numbers of revertant colonies were calculated for each test dose and the controls for all the tester strains.

The results from the initial as well as from the confirmatory assays,indicated that the tested dosesshowed no positive mutagenic increase in the mean numbers of revertant colonies for all tester strains when compared to the respective vehicle control plates, either in the presence or absence of metabolic activation.Under identical test conditions, there was a more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay procedure used.

The results of the concentration analysis of the dose formulation samples of the mutation assay confirmed that thetop dose level of 5000µg/plate wasachieved in the initial as well as in the confirmatory mutation assays and the results support the validity of the study conclusion.

It is concluded that the test item, Clovanyl-3-Formate (CARYOLAN) was not mutagenic in this Bacterial Reverse Mutation Assay up to the highest OECD 471-recommended dose of 5000 µg/plate, under the conditions of testing employed.