p-mentha-1,4-diene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

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:

p-mentha-1,4-diene

Specific details on test material used for the study:

Test Item
Designation in Test Facility: 17022802G
Date of Receipt: 28. Feb. 2017
Condition at Receipt Room temperature, in proper conditions
Specification
The following information concerning identity and composition of the test item was pro-vided by the sponsor.
Name Gamma Terpinene
Batch no. 161215
Appearance Clear liquid
Composition p-mentha-1,4-diene
Purity 98%
Homogeneity homogeneous
Expiry date 14. Dec. 2018
Storage Room Temperature (20 ± 5 °C), keep away from light

The following additional information is relevant to the conduct of the study, according to
OECD 471:

CAS No. 99-85-4
EINECS-No. 202-794-6
Stability H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility H2O: < 0.1 g/L; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown

It is provided by the sponsor as well.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA97a

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Test concentrations with justification for top dose:

5 µL/plate

Vehicle / solvent:

DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene Diamine

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Amino-Anthracene

Details on test system and experimental conditions:

Specification
Species: Salmonella typhimurium LT2
Strains: TA97a, TA98, TA100, TA102 and TA1535
Origin and Culture
All Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D) and were stored as lyophilizates in the refrigerator at 2-8 °C.
The lyophilizates were used to prepare permanent cultures which were filled into vials and stored at < - 75 °C.

Eight hours before the start of each experiment, an aliquot of a permanent culture per strain to be used was taken from the deep freezer to inoculate a culture vessel containing nutrient broth. After incubation overnight for eight hours at 37 ± 1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Three valid experiments and one invalid experiment were performed.
The experiment 2a was not valid, because the bacteria strain TA98 showed a contamina-tion and the stock solution contained 50 mL/L, instead of 15 mL/L.
This experiment is not reported in this report, but the raw data are kept in the test facility in the GLP- archive. The other experiments were valid and the results are reported here.


The study procedures described in this report were based on the most recent OECD and EC guidelines.
The test item Gamma Terpinene was tested in the Salmonella typhimurium reverse muta-tion assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535).
The test was performed in three experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

Experiment 1a:
In experiment 1a, the test item (dissolved in DMSO) was tested up to concentrations of 5 µL/plate (five concentrations) in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.
The test item showed no precipitates on the plates at any of the concentrations.
The test item showed signs of toxicity towards all bacteria strains in both the absence and presence of metabolic activation in the highest concentration (5 µL/plate).
In this concentration, no bacterial background lawn and no bacteria growth was observed.
In the lower four concentrations, the bacterial background lawn was not reduced and no relevant decrease in the number of revertants was observed in all bacteria strains.
The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Experiment 1b:
Based on the results of the experiment 1a, the test item was also tested up to concentra-tions of 5 µL/plate (six concentrations), in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.
The test item showed no precipitates on the plates at any of the concentrations.
The test item showed signs of toxicity towards all bacteria strains in both the absence and presence of metabolic activation in the highest concentration (5 µL/plate).
In this concentration, no bacterial background lawn and no bacteria growth was observed.
In the lower five concentrations, the bacterial background lawn was not reduced and no relevant decrease in the number of revertants was observed in all bacteria strains.
The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Experiment 2b:
Based on the results of the plate incorporation method, the test item was tested up to con-centrations of 1.5 µL/plate (seven concentrations) in the absence and presence of S9-mix in all bacteria strain using the pre-incubation method.
The test item showed no precipitates on the plates at any of the concentrations.
The test item showed signs of toxicity towards all bacteria strains in both the absence and presence of metabolic activation in the highest concentration (1.5 µL/plate). No bacterial background was observed and only the bacteria strains TA102 and TA1535 showed a low bacteria growth.
In the next lower concentration (0.75 µL/plate) signs of toxicity were observed towards the bacteria strains TA100 and TA102.
In the lower concentrations, the bacterial background lawn was not reduced and no rele-vant decrease in the number of revertants was observed in all bacteria strains.
The results of this experiments showed that the test item caused no increase in the num-ber of revertants in all bacteria strains compared to the solvent control, in both the ab-sence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

Based on the results of this study it is concluded that Gamma Terpinene is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.