Reaction mass of (4R)-isopropenyl-1-methylcyclohexene and (4S)-isopropenyl-1-methylcyclohexene and 1-isopropyl-4-methylcyclohexa-1,3-diene and 1-isopropyl-4-methylcyclohexa-1,4-diene and 4-isopropylidene-1-methylcyclohexene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January- February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

17 June 2015

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(R)-p-mentha-1,8-diene

EC Number:

227-813-5

EC Name:

(R)-p-mentha-1,8-diene

Cas Number:

5989-27-5

Molecular formula:

C10H16

IUPAC Name:

(4R)-isopropenyl-1-methylcyclohexene

Constituent 2

Reference substance name:

(S)-p-mentha-1,8-diene

EC Number:

227-815-6

EC Name:

(S)-p-mentha-1,8-diene

Cas Number:

5989-54-8

Molecular formula:

C10H16

IUPAC Name:

(4S)-isopropenyl-1-methylcyclohexene

Constituent 3

Reference substance name:

p-mentha-1,3-diene

EC Number:

202-795-1

EC Name:

p-mentha-1,3-diene

Cas Number:

99-86-5

Molecular formula:

C10H16

IUPAC Name:

1-isopropyl-4-methylcyclohexa-1,3-diene

Constituent 4

Reference substance name:

p-mentha-1,4(8)-diene

EC Number:

209-578-0

EC Name:

p-mentha-1,4(8)-diene

Cas Number:

586-62-9

Molecular formula:

C10H16

IUPAC Name:

4-isopropylidene-1-methylcyclohexene

Constituent 5

Reference substance name:

p-mentha-1,4-diene

EC Number:

202-794-6

EC Name:

p-mentha-1,4-diene

Cas Number:

99-85-4

Molecular formula:

C10H16

IUPAC Name:

1-isopropyl-4-methylcyclohexa-1,4-diene

impurity 1

Reference substance name:

(3R)-isopropylidene-6-methylcyclohexene

Cas Number:

43124-56-7

Molecular formula:

C10H16

IUPAC Name:

(3R)-isopropylidene-6-methylcyclohexene

impurity 2

Reference substance name:

(3S)-isopropylidene-6-methylcyclohexene

Cas Number:

62504-16-9

Molecular formula:

C10H16

IUPAC Name:

(3S)-isopropylidene-6-methylcyclohexene

impurity 3

Reference substance name:

p-cymene

EC Number:

202-796-7

EC Name:

p-cymene

Cas Number:

99-87-6

Molecular formula:

C10H14

IUPAC Name:

1-isopropyl-4-methylbenzene

impurity 4

Reference substance name:

(1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

EC Number:

227-336-2

EC Name:

(1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

Cas Number:

5794-03-6

Molecular formula:

C10H16

IUPAC Name:

(1R,4S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

impurity 5

Reference substance name:

(1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

EC Number:

227-337-8

EC Name:

(1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

Cas Number:

5794-04-7

Molecular formula:

C10H16

IUPAC Name:

(1S,4R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

impurity 6

Reference substance name:

(5S)-isopropyl-2-methylcyclohexa-1,3-diene

Cas Number:

2243-33-6

Molecular formula:

C10H16

IUPAC Name:

(5S)-isopropyl-2-methylcyclohexa-1,3-diene

impurity 7

Reference substance name:

(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

EC Number:

224-167-6

EC Name:

(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

Cas Number:

4221-98-1

Molecular formula:

C10H16

IUPAC Name:

(5R)-isopropyl-2-methylcyclohexa-1,3-diene

Test material form:

liquid

Details on test material:

Batch No.: 173391
Purity: 85.8% (sum of the five main constituents)
Name of test material (as cited in study report): Reaction mass of dl-limonene, alpha- gamma-terpinenes, terpinolene
Physical state: colourless - slightly yellow liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 1 October 2016

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item
(1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

The solvent (vehicle) control used was dimethyl sulphoxide

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM:
All the strains were originally sourced from the University of California, Berkley, Syngenta CTL Ltd and the British Industrial Biological Research Association (BIBRA)

METHOD OF APPLICATION: In agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes at 37 ± 3 °C, with shaking
- Incubation period: Plates were incubated at 37 ± 3 °C for approximately 48 hours.

NUMBER OF REPLICATIONS:
- Vehicle and positive controls were included in triplicate plates.
- Treatment (test item) groups were included in triplicate plates

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

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

Remarks:

Sparse bacterial background lawn at 5000µl/plate and In the pre-incubation plate method Sparse bacterial background lawn at concentration from 500 µk/plate

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

Remarks:

In the pre-incubation plate method only Sparse bacterial background lawn at concentration from 500 µk/plate

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

Remarks:

In the pre-incubation plate method only Sparse bacterial background lawn at concentration from 500 µk/plate

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

Remarks:

In the pre-incubation plate method only Sparse bacterial background lawn at concentration from 500 µk/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE was considered to be non-mutagenic in a Ames test.

Executive summary:

REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE was tested for mutagenicity purpose according to OECD Guideline 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. An interim (confirmatory) experiment was performed between the first and second mutation tests employing one strain only (TA1535) following the observation of statistically significant increases in Experiment 1. The experiment employed a tightened test item dose range to potentially enhance any response. The response was not repeated in the interim experiment; therefore a second mutation test was performed on all of the bacterial strains employing the pre-incubation method using fresh cultures of the bacterial strains and fresh test item formulations. The dose range for the second mutation test was amended slightly and was 5 to 5000 μg/plate. Seven test item concentrations were selected in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test, although weakened bacterial background lawns were noted to TA1535 at 5000 μg/plate in both the absence and presence of S9-mix in Experiment 1 (confirmatory test). These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 μg/plate in the second mutation test. The pre-incubation modification was employed for the second mutation test and this change in methodology caused the test item to exhibit a toxic response in the form of weakened bacterial background lawns to all of the Salmonella strains at and above 500 μg/plate in both the presence and absence of metabolic activation (S9-mix). No significant weakening of the bacterial background lawns was noted for Escherichia coli strain WP2uvrA at any test item dose level. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). In the first mutation test, statistically significant increases in TA1535 revertant colony frequency were noted from 1500 μg/plate in both the presence and absence of S9-mix. These results were investigated in a confirmatory experiment employing a tightened concentration range at the upper dose levels. However, there were no toxicologically significant increases in the frequency of TA1535 revertant colonies recorded after the confirmatory test but weakened bacterial background lawns were noted at 5000 μg/plate. Therefore, the responses noted for TA1535 in the original test were considered due to the fact that the test item exhibited toxicity under certain circumstances and exposure conditions (a much stronger toxic response was noted after performing the pre-incubation modification) and the increases in revertant colony frequency noted may have been an artefact resulting from a modest level of toxicity to the tester strain at the upper test item dose levels. Even though weakened background lawns were not noted in the original test there may have been enough weakening (toxicity) to induce a ‘false’ response. A possible mechanism may be that low level toxicity has caused a selective effect on the number of bacterial cells plated, resulting in an increase in non-revertant histidine-dependent bacteria, which are able to manifest as false ‘colonies’.

Therefore, REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE was considered to be non-mutagenic in a Ames test.