Reaction mass of stereoisomers of 4-METHYLENE-2-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-TETRAHYDRO-PYRAN and; 4-METHYL-6-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-3,6-DIHYDRO-2H-PYRAN and; 4-METHYL-2-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-3,6-DIHYDRO-2H-PYRAN

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:

11-25 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 471 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Directive 2000/32/EC

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 2009-07-13 and 2009-07-22 / Signed on 2009-08-3 / Valid until 2011-08-003

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-METHYLENE-2-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-TETRAHYDRO-PYRAN

Cas Number:

947237-93-6

Molecular formula:

C15 H24 O

IUPAC Name:

4-METHYLENE-2-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-TETRAHYDRO-PYRAN

Constituent 2

Reference substance name:

4-METHYL-6-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-3,6-DIHYDRO-2H-PYRAN

Cas Number:

947237-84-5

Molecular formula:

C15 H24 O

IUPAC Name:

4-METHYL-6-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-3,6-DIHYDRO-2H-PYRAN

Constituent 3

Reference substance name:

4-METHYL-2-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-3,6-DIHYDRO-2H-PYRAN

Cas Number:

947237-75-4

Molecular formula:

C15 H24 O

IUPAC Name:

4-METHYL-2-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-3,6-DIHYDRO-2H-PYRAN

Test material form:

liquid

Details on test material:

- Physical state: Colourless to pale yellow liquid

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH
- method of preparation of S9 mix : not detailed
- concentration of S9 in the final culture medium: S9-fraction 10% v/v
- quality controls of S9: enzymatic activity, sterility, metabolic capability (certificate included in the study report)

Test concentrations with justification for top dose:

Cytotoxicity test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 100 without S9 under the direct plate incorporation method.

Mutagenicity test:
Main test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.
Confirmation test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2 (pKM101) with and without S9 under the pre-incubation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol 96%
- Appearance in solvent: Colourless liquid solution
- Basis for dose calculation: The test item was used as provided
- Formulation conditions: Room temperature
- Formulation frequency: Daily
- Formulation preparation: The highest exposure concentration was 5 μL/plate. Further lower concentrations were prepared by 1:3 serial dilutions in the selected solvent from the highest concentration.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol (95%)

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol (95%)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

SOURCE OF TESTER STRAINS: Strains of salmonella typhimurium and E. Coli were obtained from Moltox. Bacterial strains used for the study were grown from controlled Working Banks obtained from Master Banks (generated in Vivotecnia).

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

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 37 °C for 48 h

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity evaluation of the test item was based on the decrease in the number of revertant colonies, or a clearing or diminution of the background lawn.

OTHER:
- After an incubation of about 48 hours at about 37 °C, the number of colonies per plate was counted.
- The number of revertant colonies per plate was counted and recorded by an automatic colony counter. Average plate counts was presented with the mean and the standard deviation for each set of triplicates per test item concentration and was used to calculate the ratio of colonies per exposed plate compared to the corresponding negative control.
- Test item solubility: The solubility of the test item was evaluated in a standard solvent panel (milliQ water, ethanol 96%, DMSO and corn oil). Observation of precipitation by the unaided eye indicated that the test item is not soluble. The test item was found to be soluble when diluted 1/3 in ethanol (96%).
- Test item sterility assay: The sterility of the test item was assayed by adding of 5 μL/plate to a minimal agar plate and incubating at 37 °C for 48 h. No growth was observed in the minimal agar plate after incubation with the test item.

Evaluation criteria:

The criteria used for determining a positive result take into account a dose-response effect in the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
A result is considered positive whenever the number of revertants of the test item treated plates is increased when compared to the solvent treated plates according to the following criteria: 2 fold for S.typhimurium TA98, TA 100 and E.coli WP2(pKM101); 3 fold for S.typhimurium TA1535 and TA1537.
Biological relevance of the results was also considered.

Statistics:

None

Results and discussion

Test results

Key result

Species / strain:

bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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
- Water solubility: Test item was found to be soluble when diluted 1/3 in ethanol (96%).
- Precipitation: None
- Other confounding effects: None

CYTOTOXICITY TEST:
No cytotoxic activity was observed by the test item in the bacterial system at a concentration of 5 μL/plate.

MUTAGENICITY TEST:
- None of the concentrations assayed for the test item showed an increase in the R value either with or without S9 metabolic activation regardless of the procedure.
- No dose response for the test item was observed in any of the tested bacterial strains.

HISTORICAL CONTROL DATA
- Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data of the test facility.

Any other information on results incl. tables

Ames test acceptance criteria:

The bacterial reverse mutation test for the test item was considered valid as the following criteria were met:

The mean solvent control counts complied with Vivotecnia historical data for each strain.

The mean reference item control counts complied with Vivotecnia historical data for each strain.

Applicant's summary and conclusion

Conclusions:

Under the test condition, test substance is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2(pKM101) were exposed to the test substance diluted in ethanol (95%) at the following concentrations both in the presence and absence of metabolic activation system (10% v/v S9).

Cytotoxicity test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 100 without S9 under the direct plate incorporation method. 

Mutagenicity test:

Main test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.

Confirmation test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2 (pKM101) with and without S9 under the pre-incubation method.

Vehicle and positive control groups were also included in mutagenicity tests.

 

Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data.

 

No cytotoxic effect was observed. None of the concentrations assayed for the test item showed an increase in the R value either with or without S9 metabolic activation regardless of the procedure. No dose response for the test item was observed in any of the tested bacterial strains.

 

Under the test condition, test substance is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.