Reaction mass of [1S-(1α,3aβ,4α,8aβ)]-decahydro-4,8,8-trimethyl-9-methylene-1,4-methanoazulene and caryophyllene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): negative

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:

30 May 2001 - 18 June 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

- Dose range finding test (direct plate and pre-incubation):
TA 98 and TA 100 (without and with S9): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

- Experiment 1:
TA 1535, TA 1537, TA 98, TA 100 and TA 102 (without and with S9): 33, 100, 333, 1000, 2500 and 5000 µg/plate

- Experiment 2:
Due to toxicity in the pre-incubation dose range finding test the following dose levels were used:
TA 1535, TA 1537 and TA 102 (without and with S9): 3, 10, 33, 100, 333 and 1000 µg/plate
TA 98 and TA 100 (without and with S9): 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Solvent used: Ethanol
- Justification for choice of solvent: The test substance was dissolved in ethanol. The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(100 µL/plate ethanol)

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours

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

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

Evaluation criteria:

A test item is considered as positive if a biologically relevant and dose related increase in the number of revertants is induced.
A test item producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any one of the test points is considered non mutagenic in this system.

A biologically relevant response is described as follows:
A test item is considered mutagenic if in strains TA 98, TA 100, and TA 102 the number of reversions will be at least twice as high and in strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose will induce the above described enhancement factors or not.

Statistics:

A statistical analysis of the data is not required.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed in both TA 98 and TA 100 in the pre-incubation assay up to and including 5000 µg/plate, both in the absence and presence of S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I, toxic effects, evident as a reduction in the number of revertants, were observed in strain TA 1537 with 59 mix and in strain TA 100 with and without 59 mix. In experiment II, toxic effects were observed in strains TA 1535, 1537, 98, and 100 with and without 59 mix.
Irregular background growth was observed in strains TA 1537 and TA 98 in the second experiment at 333 µg/plate and above with metabolic activation and at 1000 µg/plate and above in strain TA 100 without metabolic activation.

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay performed according to OECD 471 guideline and GLP principles.

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent experiments, the first in agar (plate incorporation) and the second a pre-incubation experiment, both in the absence and presence of S9-mix up to and including 5000 μg/plate. Adequate negative and positive controls were included.

In experiment I, toxic effects, evident as a reduction in the number of revertants, were observed in strain TA 1537 with S9 mix and in strain TA 100 with and without S9 mix. In experiment II, toxic effects were observed in strains TA 1535, 1537, 98, and 100 with and without S9 mix. Irregular background growth was observed in strains TA 1537 and TA 98 in the second experiment at 333 µg/plate and above with metabolic activation and at 1000 µg/plate and above in strain TA 100 without metabolic activation.

The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100 and TA102), both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test (OECD TG 471):

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent experiments, the first in agar (plate incorporation) and the second a pre-incubation experiment, both in the absence and presence of S9-mix up to and including 5000 μg/plate. Adequate negative and positive controls were included.

In experiment I, toxic effects, evident as a reduction in the number of revertants, were observed in strain TA 1537 with S9 mix and in strain TA 100 with and without S9 mix. In experiment II, toxic effects were observed in strains TA 1535, 1537, 98, and 100 with and without S9 mix. Irregular background growth was observed in strains TA 1537 and TA 98 in the second experiment at 333 µg/plate and above with metabolic activation and at 1000 µg/plate and above in strain TA 100 without metabolic activation.

The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100 and TA102), both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Justification for classification or non-classification

Based on the results of the Ames test, the substance does not have to be classified for genotoxicity in accordance with Regulation (EC) No 1272/2008 and its updates.