Cyclooctane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test (reference 7.6.1-1).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-05-25 to 2021-06-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium TA98, TA100, TA1535, TA1537: mutations in the histidine operon
Escherichia coli WP2 uvrA: defect in one of the genes for tryptophan biosynthesis

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system
- source of S9: post-mitochondrial S9 fraction derived from rat liver homogenate; from male Wistar rats, Crl:WI (HAN) (Charles River, Germany), age 6-8 weeks, pretreated with ß-Naphthoflavone (100 mg/kg bw) and Phenobarbital (80 mg/kg bw)
- method of preparation of S9 mix: Please refer to “Any other information on materials”.
- concentration or volume of S9 mix and S9 in the final culture medium: 10 % and 20 % S9 in the S9 mix were used in the first and second test series, respectively.
- quality controls of S9: Each S9 batch was tested for its metabolic activity using specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for each S9 batch.

Test concentrations with justification for top dose:

1st series: 5, 15.8, 50, 158, 500, 1580, 5000 µg/plate
2nd series: 88.9, 281, 500, 1580, 5000 µg/plate

5000 µg/plate was chosen as the appropriate maximum test material concentration. The test material concentrations used were selected according to the EEC, OECD and Japanese guidelines for this test system.

Vehicle / solvent:

- Solvent used: ethanol

- Justification for choice of solvent and amount in the final culture medium: The selection of the solvent for this assay was based on the available information from a prelimi­nary solubility test. Ethanol showed best performance and was thus used for this experiment at a maximum concentration of 10 µL/plate.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

other:

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate for test item and positive control, 6 plates for solvent controls
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- increase of revertant colonies

Rationale for test conditions:

Test conditions in line with respective test guidelines were used.

Evaluation criteria:

A test material was to be defined as positive or mutagenic in this assay if
• the assay is considered valid and
• a biologically relevant increase in the mean number of revertants above a threshold of 2- fold (TA98, TA100, WP2 uvrA) or 3-fold (TA1535, TA1537) as compared to the concurrent negative controls is observed
• an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
• a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration

A test material is defined as negative or non-mutagenic in this assay if
• the assay is considered valid and
• none of the above-mentioned criteria are met

Whenever colony counts remain within the historical range of negative controls, such increases are considered as biologically not meaningful. In general, two series of experiments must be performed. However, there is no requirement for verification of a clear positive response (OECD TG 471, 1997).

Statistics:

Not specified

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

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

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

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

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

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

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

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

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Confounding effects: No confounding effects were described.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Please refer to attached document under "Overall remarks, attachments".

Ames test:
- Signs of toxicity: No toxicity was observed.
- Individual plate counts: Please refer to attached document under "Overall remarks, attachments".
- Mean number of revertant colonies per plate and standard deviation: Please refer to attached document under "Overall remarks, attachments".

HISTORICAL CONTROL DATA
Please refer to attached document under "Overall remarks, attachments".

Conclusions:

The test item was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation test.

Executive summary:

The present study according to OECD TG 471 was conducted to investigate the test material for its mutagenic potential in a bacterial reverse mutation test in the absence and presence of a rat liver metabolizing system (S9 mix). The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pretreated with ß­ Naphthoflavone/Phenobarbital was used. In this study, two experimental series were performed. The S9 mix used contained 10 % S9 in the first and 20 % S9 in the second series, respectively. Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following treatment of all bacteria tester strains with the test item in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed. Under the experimental conditions reported here, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The present study according to OECD TG 471 was conducted to investigate the test material for its mutagenic potential in a bacterial reverse mutation test in the absence and presence of a rat liver metabolizing system (S9 mix). The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pretreated with ß­ Naphthoflavone/Phenobarbital was used. In this study, two experimental series were performed. The S9 mix used contained 10 % S9 in the first and 20 % S9 in the second series, respectively. Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following treatment of all bacteria tester strains with the test item in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed. Under the experimental conditions reported here, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data the test item does not require classification for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP).