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

Genetic toxicity: in vitro

Administrative data

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:

06 December 2000 - 12 January 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD Guideline No 471 without any deviation

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:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

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

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

Name of test material (as cited in study report): Terpinolene
Colour: colourless to yellowish
Analytical purity: 91.2 %
Lot/batch No.: 290600015
Storage Conditions: Refrigerator 7 ± 2 °C, protected from light and moisture
Expiration Date: 31 January 2001

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: the genotypes of the tested strains were checked at each study by histidine auxotrophy, ampicillin resistance, tetracycline resistance (only TA 102), UV-sensitivity (except TA 102) and growth inhibition with crystal violet (rfa-mutation)

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction prepared from liver homogenates of male Wistar rats induced with phenobarbital (intraperitoneal route) and ß-Naphthoflavone (oral route)

Test concentrations with justification for top dose:

Study 1:
- TA 97a, TA 100 and TA 1535 (- S9): 0.5, 1.6, 5, 16 and 50 µg/plate
- TA 98 (± S9), TA 97a and TA 100 (+ S9): 5, 16, 50, 160 and 500 µg/plate
- TA 102 (± S9) and TA 1535 (+ S9): 50, 160, 500, 1600 and 5000 µg/plate

Study 2:
- TA 97a, TA 100 and TA 1535 (- S9): 0.5, 1.6, 5, 16 and 50 µg/plate
- TA 100 (+ S9) and TA 98 (± S9): 16, 50, 160, 500 and 1600 µg/plate
- TA 102 (± S9), TA 97a and TA 1535 (+ S9): 50, 160, 500, 1600 and 5000 µg/plate

Vehicle / solvent:

Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR 191 (0.5 µg/plate in TA 97a); 4-Nitro-1,2-phenylenediamine (0.5 µg/plate in TA 98); Nitrofurantoine (0.2 µg/plate in TA 100); sodium azide (0.25 µg/plate in TA 1535); cumene hydroperoxide (100 µg/plate in TA 102)

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2 µg/plate in TA 97a, TA 98, TA 100 and TA 1535); danthron (30 µg/plate in TA 102)

Remarks:

with metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: University of California, Berkeley Division of Biochemistry and Molecular Biology, USA

METHOD OF APPLICATION: In agar (plate incorporation method)

DURATION
Incubation period: Plates for confirmation of the genotypes were incubated for 24 h and plates of the mutagenicity test for 48 h, respectively, at 37± 1 °C.

NUMBER OF REPLICATIONS: 3 plates/dose (3 replicates per concentration level and control)

DETERMINATION OF CYTOTOXICITY
Method: Evaluation of the toxicity was performed on the basis of the observation of a reduced background lawn.

OTHER: Revertants were scored with colony counter (manual counting).

Evaluation criteria:

Test item is to be interpretated mutagenic if there is a concentration effect relationship and the induction rate is ≥ 2.

Statistics:

none

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY:
Study 1:
- Reduced background lawn was observed in TA 97a, TA 100 and TA 1535 strains at 50 µg/plate (- S9)
- Reduced background lawn was observed in TA 1535 strain at 5000 µg/plate (+ S9)
Study 2:
- Reduced background lawn was observed in TA 98 strain at 1600 µg/plate (± S9)
- Reduced background lawn was observed in TA 100 strain at 1600 µg/plate (+ S9) and 50 µg/plate (- S9)
- Reduced background lawn was observed in TA 1535 strain at 50 µg/plate (- S9)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Terpinolene monoconstituent is not considered as mutagenic in S. typhimurium strains (TA 97a, TA 98, TA 100, TA 102 and TA 1535).

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline No 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to terpinolene monoconstituent at the following concentrations:

Study 1:

- TA 97a, TA 100 and TA 1535 (- S9): 0.5, 1.6, 5, 16 and 50 µg/plate

- TA 98 (± S9), TA 97a and TA 100 (+ S9): 5, 16, 50, 160 and 500 µg/plate

- TA 102 (± S9) and TA 1535 (+ S9): 50, 160, 500, 1600 and 5000 µg/plate

Study 2:

- TA 97a, TA 100 and TA 1535 (- S9): 0.5, 1.6, 5, 16 and 50 µg/plate

- TA 100 (+ S9) and TA 98 (± S9): 16, 50, 160, 500 and 1600 µg/plate

- TA 102 (± S9), TA 97a and TA 1535 (+ S9): 50, 160, 500, 1600 and 5000 µg/plate

S9 mix was used in the experiments conducted with metabolic activation. S9 fraction was prepared from liver homogenates of male Wistar rats induced with phenobarbital (intraperitoneal route) and ß-Naphthoflavone (oral route). Vehicle and positive control groups were also included in these experiments.

Test substance induced cytotoxicity in the form of background lawn inhibition in TA 97a, TA 100 and TA 1535 strains at 50 µg/plate (- S9); TA 1535 strain at 5000 µg/plate (+ S9) in study 1. In study 2, cytotoxicity was observed in TA 98 strain at 1600 µg/plate (± S9); TA 100 strain at 1600 µg/plate (+ S9) and 50 µg/plate (- S9); TA 1535 strain at 50 µg/plate (- S9). The positive and negative (vehicle) controls induced the appropriate responses in the corresponding strains. Terpinolene monoconstituent showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S9 mix.

Therefore, terpinolene monoconstituent is not considered as mutagenic in this bacterial system.