Reaction mass of 5-[(2R)-butan-2-yl]-2-[(1R,2R)-2,4-dimethylcyclohex-3-en-1-yl]-5-methyl-1,3-dioxane and 5-[(2R)-butan-2-yl]-2-[(1R,6R)-4,6-dimethylcyclohex-3-en-1-yl]-5-methyl-1,3-dioxane and 5-[(2S)-butan-2-yl]-2-[(1R,2R)-2,4-dimethylcyclohex-3-en-1-yl]-5-methyl-1,3-dioxane and 5-[(2S)-butan-2-yl]-2-[(1S,2R)-2,4-dimethylcyclohex-3-en-1-yl]-5-methyl-1,3-dioxane and 5-[(2S)-butan-2-yl]-2-[(1S,6R)-4,6-dimethylcyclohex-3-en-1-yl]-5-methyl-1,3-dioxane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was tested in the Salmonella typhimurium assay with strains TA1535, TA1537, TA98 and TA100 equivalent or similar to OECD 471 guideline. In a preliminary toxicity test all four strains were tested up to 5000 ug/plate with and without metabolic activation. In the mutagenicity assay concentrations used were chosen on data obtained in the preliminary toxicity assay. In the mutagenicity assay no toxicity and no increase in the number of revertant colonies was observed in strain TA98 up to and including the top dose of 5000 µg/plate, with and without S9 -mix. No toxicity and no increase in the number of revertant colonies was observed up to and including the top dose of 500 µg/plate in strain TA1535 (with and without S9-mix), TA1537 (without S9-mix), and no toxicity and no increase in the number of revertant colonies was observed up to and including the top dose of 1500 µg/plate in strain TA1537 (with S9 -mix) and TA100. From this it can be concluded that the substance is not mutagenic in strains TA1535, TA1537, TA98 and TA100 with and without S9-mix in two main experiments.

Link to relevant study records

Reference

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:

September 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed equivalent or similar to OECD 471 guideline, but predating GLP. A fifth strain to detect certain oxidising mutagens, cross-linking agents and hydrazines was not included.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

No fifth strain was tested (E. coli or TA102).

GLP compliance:

no

Remarks:

Study performed around/just before the time GLP was introduced in Europe, but internal QA statement.

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary toxicity assay (all strains):
Without and with S9-mix: 0, 0.5, 5, 50, 500 and 5000 µg/plate

Main study 1 and 2:
TA1535 (without and with S9-mix) and TA1537 (without S9-mix): 0, 5, 15, 50, 150 and 500 µg/plate
TA1537 (with S9-mix) and TA100 (without and with S9-mix): 0, 15, 50, 150, 500 and 1500 µg/plate
TA98 (without and with S9-mix): 0, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO is accepted and approved by authorities and international guidelines.

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9-mix: 10 µg/plate in water for TA1535 and TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9-mix: 40 µg/plate in DMSO for TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9-mix: 10 µg/plate in DMSO for TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene 2 µg/plate in DMSO for all tester strains

Remarks:

With S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 or 72 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the revertant colonies.

Evaluation criteria:

The average number of mutant colonies per plate is compared with the average number of spontaneous revertants in the control. A dose-related
increase in the number of colonies which reaches at least a doubling of the control values is usually considered to be a positive response.

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

Remarks:

of 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA1535, TA1537 and TA100

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No information on precipitation in doses tested.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at the top dose of 5000 µg/plate in strain TA1535 (with and without S9-mix), TA1537 (without S9-mix) and TA100 (without S9-mix)

COMPARISON WITH HISTORICAL CONTROL DATA:
- The positive control substances produced the expected mutagenic results.

ADDITIONAL INFORMATION ON CYTOTOXICITY MAIN STUDY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate in strain TA98.
- No toxicity or mutagenicity was observed up to and including the top dose of 500 µg/plate in strain TA1535 (with and without S9-mix), TA1537 (without S9-mix).
- No toxicity or mutagenicity was observed up to and including the top dose of 1500 µg/plate in strain TA1537 (with S9-mix) and TA100 (with and without S9-mix).

Conclusions:

Interpretation of results (migrated information):
negative

The substance was tested in the Salmonella typhimurium assay with strains TA1535, TA1537, TA98 and TA100 equivalent or similar to OECD 471 guideline. The substance is not mutagenic in strain TA1535, TA1537, TA98 and TA100 with and without S9-mix.

Executive summary:

The substance was tested in the Salmonella typhimurium assay with strains TA1535, TA1537, TA98 and TA100 equivalent or similar to OECD 471 guideline. In a preliminary toxicity test all four strains were tested up to 5000 ug/plate with and without metabolic activation. In the mutagenicity assay concentrations used were chosen on data obtained in the preliminary toxicity assay. In the mutagenicity assay no toxicity and no increase in the number of revertant colonies was observed in strain TA98 up to and including the top dose of 5000 µg/plate, with and without S9 -mix. No toxicity and no increase in the number of revertant colonies was observed up to and including the top dose of 500 µg/plate in strain TA1535 (with and without S9-mix), TA1537 (without S9-mix), and no toxicity and no increase in the number of revertant colonies was observed up to and including the top dose of 1500 µg/plate in strain TA1537 (with S9 -mix) and TA100. From this it can be concluded that the substance is not mutagenic in strains TA1535, TA1537, TA98 and TA100 with and without S9-mix in two main experiments.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Justification for selection of genetic toxicity endpoint
Only study available and equivalent or similar to OECD471 because the non-testing of e.g. E.coli is not critical because the substance does not have oxidising or cross-linking functional groups.

Justification for classification or non-classification

Based on the results available, i.e. only for in vitro mutagenicity, the substance does not need to be classified for genetic toxicity under DSD 67/548/EC or CLP 1272/2008/EC.