1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol

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:

11 May 2020 -28 may 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”. Official Journal of the European Union No. L142

Version / remarks:

31 May 2008

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol

EC Number:

255-256-8

EC Name:

1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol

Cas Number:

41199-19-3

Molecular formula:

C13H22O

IUPAC Name:

2,5,5-trimethyl-1,2,3,4,4a,5,6,7-octahydronaphthalen-2-ol

Test material form:

liquid

Method

Target gene:

S. typhimurium: Histidine gene
E. coli: Tryptophan gene

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:

Type and composition of metabolic activation system:
- source of S9 : rat liver S9-mix induced by Aroclor 1254 (500 mg/kg bw)
- method of preparation of S9 mix: S9-mix per 10 mL: 30 mg NADP, 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The solution was filter (0.22 μm)-sterilized and 0.5 mL S9-fraction was added
- concentration of S9 in S9 mix: 5%
- quality controls of S9: Each S9 batch was characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively

Test concentrations with justification for top dose:

Justification for top dose: 5 mg is the recommended maximum test concentration according to the guideline.

Dose-range finding test (without and with S9-mix; tester strains TA100 and WP2uvrA): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (reported as part of experiment 1)

First experiment (Direct plate assay) (without and with S9; tester strains TA1535, TA1537 and TA98): 5.4, 17, 52, 164, 512, 1600 μg/plate

Second experiment (pre-incubation assay) (without and with S9-mix, tester strains WP2uvrA, TA1535, TA1537, TA 98 and TA100): 5.4, 17, 52, 164, 512, 1600, 5000 μg/plate

Vehicle / solvent:

The vehicle of the test item was DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene; ICR-191

Remarks:

For details on positive control substances, see Table 1 and Table 2 under 'any other information'

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Doses levels were tested in triplicate in each strain.

METHODS:
Direct plating assay:
The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (1E9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in Milli-Q water and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark.
Pre-incubaton assay:
The solution in the direct plating assay were pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°C before added to 3 mL molten top agar

DURATION
- Exposure duration: 48 ± 4 h (in the dark at 37.0 ± 1.0 °C) (actual temperature range 35.5-38.6)

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.
- Other: precipitation of the test item was recorded.

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:

a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Rationale for test conditions:

According to guideline

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

In addition to these criteria, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

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

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

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

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

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test item on the plates was not observed in any tester strain.

RANGE-FINDING/SCREENING STUDIES:
- In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both tester strains in the absence and presence of S9-mix.

STUDY RESULTS
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
- In the first mutation experiment, the test item was tested up to concentrations of 1600 μg/plate in the strains TA1535, TA1537 and TA98. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, in the absence and presence of S9-mix at dose levels of 1600 and/or 5000 μg/plate.
- In the second mutation experiment, the test item was tested in the pre-incubation assay up to the concentration of 1600 μg/plate in tester strains TA1535, TA1537, TA98 and TA100 and up to 5000 μg/plate in tester strain WP2uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, due to severe cytotoxicity, five analyzable dose levels have not been achieved for the determination of the mutagenicity of the test item. However, at least 3 analysable dose levels without toxicity could be evaluated and clear negative results were obtained. Thus, the testing of lower dose levels were not considered to provide additional information.
- The test item did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

HISTORICAL DATA
Historical control data and detailes results: See attached background material.

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

A bacterial reverse mutation test was performed according to OECD TG 471 and in accordance with GLP principles. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. No precipitation was observed in any tester strain. In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both tester strains in the absence and presence of S9-mix. In the first mutation experiment, the test item was tested up to concentrations of 1600 μg/plate in the strains TA1535, TA1537 and TA98.  Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix. In the second mutation experiment, the test item was tested in the pre-incubation assay up to the concentration of 1600 μg/plate in tester strains TA1535, TA1537, TA98 and TA100 and up to 5000 μg/plate in tester strain WP2uvrA. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9 -metabolic activation when tested up to and including the recommended top concentration. These results were confirmed in a follow-up experiment. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.