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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study performed similarly to OECD Guideline 476 with minor deviations: no data on maintenance of cell cultures and absence of mycoplasma

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:

publication

Title:

Unnamed

Year:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

no data on maintenance of cell cultures and absence of mycoplasma

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of assay:

mammalian cell gene 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:

4-isopropenyl-1-methylcyclohexene

Test material form:

liquid

Method

Target gene:

Thymidine kinase, TK +/- locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Source of cells: National Toxicology Program's (NTP) chemical repository (Radian Corporation, Austin, USA)
- Type and identity of media: Fischer’s medium used for expression and cloning; horse serum used at 20% v/v for soft agar cloning

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of male Fischer 344 rat liver induced with Aroclor-1254

Test concentrations with justification for top dose:

Without S9:
- Trail 1: 0, 10, 20, 30, 40, 50 and 60 nL/mL
- Trail 2: 0, 30, 40, 50, 60, 80 and 100 nL/mL
- Trail 3: 0, 5, 10, 20, 30, 40 and 50 nL/mL
- Trail 4: 0, 5, 10, 20, 30, 40, 50 and 60 nL/mL

With S9:
- Trail 1: 0, 10, 20, 30, 40, 50, 60 and 80 nL/mL
- Trail 2: 0, 10, 20, 30, 40, 50, 60 and 80 nL/mL
- Trail 3: 0, 30, 40, 50, 60, 80 and 100 nL/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 1% ethanol

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation Migrated to IUCLID6: 5 nL/mL

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation Migrated to IUCLID6: 2.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 4 hours at 37 °C
- Expression time (cells in growth medium): 48 hours at 37 °C
- Selection time (if incubation with a selection agent): 9-12 days at 37 °C

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Duplicate (at least)

NUMBER OF CELLS EVALUATED: 6 x 10^6 cells exposed to the test item, 3 x 10^6 cells to select mutant cells; 600 cells to determine cloning efficiency

DETERMINATION OF CYTOTOXICITY
- Method: Relative growth on Days 1 and 2, cloning efficiency and relative total growth

OTHER EXAMINATIONS:
- Colony sizing: Number of small and large mutant colonies were determined by recording TFT colony counts for increments of 0.2 on the colony size discriminator.

OTHER: Colonies were counted on an Artek 880 colony counter fitted with a 10-turn size discriminator.

Evaluation criteria:

Positive (+):
- Significant response for at least one of the three highest dose sets and a significant trend (P ≤ 0.05)

Questionable (?):
- Significant response for one of the three highest dose sets but no significant trend
- Significant trend but no significant dose set

Inconclusive (i):
- Significant response for a dose set other than one of the three highest but no significant trend
- No significant responses or trend, but the relative total growth is greater than 30% and higher toxicity can be attained

No response (=):
- No significant responses or trend, and the relative total growth is greater than 30% under conditions where a 1.5-fold increase in dose cause precipitation or where the 5 mg/mL (or 5 µL/mL) concentration limit is attained.

Negative (-):
- No significant responses or trend, and either the relative total growth is less than 30% or excessive toxicity occurs for a 1.5-fold higher dose.

Statistics:

Consistency among the mutant frequencies was analysed using chi-square test; acceptable cultures must be significant at P ≤ 0.05

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at or above 50 and 60 nL/mL (with and without S9, respectively)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Response in trials without S9:
- Trail 1: Inconclusive (i)
- Trail 2: Questionable (?)
- Trail 3 and 4: Negative (-)
- Overall response: Negative (-)

Response in trials with S9:
- Trail 1 and 2: Negative (-)
- Trail 3: Inconclusive (i)
- Overall response: Negative (-)


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Without S9: Cytotoxicity was observed in one or more replicates tested at concentration of 50 nL/mL or above
- With S9: Cytotoxicity was observed in one or more replicates tested at concentration of 60 nL/mL or above

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Chemical-induced changes in the large and small classes of mutant colonies

 

Chemical treatment	Trial	Mutant colony count and CE						Mutant frequency				Mutant frequency change
		Treatment			Solvent control			Treatment		Solvent control		Difference
		L	S	CE	L	S	CE	L	S	L	S	L	S
50 nL/mL	WO 2	48	119	98	44	53	85	16	40	17	21	-1	19
50 nL/mL	S9 2	98	143	79	72	109	115	41	60	21	32	20	28

Applicant's summary and conclusion

Conclusions:

d-Limonene was not considered as mutagenic in mouse lymphoma L5178Y cells and does not need to be classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Executive summary:

In an in vitro mammalian cell gene mutation test performed similarly to OECD Guideline 476, mouse lymphoma L5178Y TK+/- cells were exposed to d-limonene in 1% ethanol in Fischer’s medium with and without metabolic activation (S9 fraction of male Fischer 344 rat liver induced with Aroclor-1254) at the concentrations below

Without S9:

- 0, 10, 20, 30, 40, 50 and 60 nL/mL (trial 1)

- 0, 30, 40, 50, 60, 80 and 100 nL/mL (trial 2)

- 0, 5, 10, 20, 30, 40 and 50 nL/mL (trial 3)

- 0, 5, 10, 20, 30, 40, 50 and 60 nL/mL (trial 4)

With S9:

- 0, 10, 20, 30, 40, 50, 60 and 80 nL/mL (trial 1)

- 0, 10, 20, 30, 40, 50, 60 and 80 nL/mL (trial 2)

- 0, 30, 40, 50, 60, 80 and 100 nL/mL (trial 3)

 

Positive controls (methyl methanesulphonate at 5 nL/mL without S9 and 3-methylcholanthrene at 2.5 µg/mL with S9) induced the appropriate response. In experiment without S9, mutagenic responses in trials 1, 2, 3 and 4 were inconclusive, questionable, negative and negative, respectively. In experiment with S9, mutagenic responses in trials 1, 2 and 3 were negative, negative and inconclusive, respectively. Overall, d-limonene was not considered as mutagenic in either presence or absence of S9 mix. Cytotoxicity was observed in one or more replicates tested at or above 50 nL/mL.

 

Therefore, d-limonene was not considered as mutagenic in mouse lymphoma L5178Y cells and does not need to be classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.