Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 June - 22 July 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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)-4-isopropenyl-1-methylcyclohexene

Constituent 2

Reference substance name:

(4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene

Cas Number:

30199-25-8

Molecular formula:

C11H20O

IUPAC Name:

(4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene

Test material form:

liquid: viscous

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ca. 4 ºC in the dark and under nitrogen
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Fully miscible in DMSO (dimethyl sulfoxide) and assumed stable.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (27.6%) of the test item.
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: Concentration of stock solution = 50 mg/mL.
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: No

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Salmonella strains (presence of S9-mix) - 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate
E. coli (presence of S9-mix) - 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
All bacterial strain (absence of S9-mix) - 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: guideline recommended

Controls

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:

METHOD OF APPLICATION: preincubation; in suspension (main test)
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): n/a
- Fixation time (start of exposure up to fixation or harvest of cells): n/a

SELECTION AGENT (mutation assays): n/a

SPINDLE INHIBITOR (cytogenetic assays): n/a

STAIN (for cytogenetic assays): n/a

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: n/a

NUMBER OF CELLS EVALUATED: n/a

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): n/a

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: n/a

DETERMINATION OF CYTOTOXICITY
- Method: presence of bacterial lawn
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:n/a
- Determination of endoreplication: n/a
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): n/a

- OTHER:

Rationale for test conditions:

The study was based on the in vitro technique described by Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence (Green and Muriel (1976)) is used to complement the Salmonella strains.

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).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not examined
- Effects of osmolality: not examined
- Evaporation from medium: not examined
- Water solubility: not examined
- Precipitation: none
- Definition of acceptable cells for analysis: All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: In the range-finding test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 1500 μg/plate in the absence and presence of S9-mix.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: n/a

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: n/a
- Indication whether binucleate or mononucleate where appropriate: n/a

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No
- Negative (solvent/vehicle) historical control data: Yes (2009 - 2010 data)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: presence of bacterial lawn
- Other observations when applicable: no

Any other information on results incl. tables

Table 1       Main test results

Base-pair substitution and frameshift type	Concentration [µg/plate]	No. of revertants per plate*
		— MA	Cytotoxicity	+ MA	Cytotoxicity
TA100 (Base pair substitution)	0	110	N	123	N
	0.15	104	N	-	-
	0.5	113	N	-	-
	1.5	98	N	103	N
	5	110	N	109	N
	15	95	Y	100	N
	50	80	Y	93	N
	150	0	Y	78	Y
	500	-	-	0	Y
	1500	-	-	0	Y
	5000	-	-	-	-
	Positive control	711	N	647	N
TA1535 (Base pair substitution)	0	18	N	11	N
	0.15	17	N	-	-
	0.5	16	N	-	-
	1.5	17	N	10	N
	5	15	N	11	N
	15	18	N	10	N
	50	12	Y	12	N
	150	10	Y	8	N
	500	-	-	0	Y
	1500	-	-	0	Y
	5000	-	-	-	-
	Positive control	110	N	289	N
WP2uvrA (Base pair substitution)	0	19	N	39	N
	0.15	26	N	-	-
	0.5	27	N	-	-
	1.5	23	N	-	-
	5	26	N	38	N
	15	26	Y	37	N
	50	21	Y	33	N
	150	24	Y	34	N
	500	-	-	32	N
	1500	-	-	25	Y
	5000	-	-	0	Y
	Positive control	627	N	250	N
TA98 (Frameshift)	0	18	N	20	N
	0.15	19	N	-	-
	0.5	13	N	-	-
	1.5	17	N	18	N
	5	14	N	16	N
	15	12	N	22	N
	50	14	Y	22	N
	150	12	Y	16	N
	500	-	-	6	Y
	1500	-	-	0	Y
	5000	-	-	-	-
	Positive control	112	N	190	N
TA1537 (Frameshift)	0	9	N	13	N
	0.15	9	N	-	-
	0.5	11	N	-	-
	1.5	10	N	13	N
	5	13	N	15	N
	15	9	N	13	N
	50	6	Y	14	N
	150	2	Y	9	N
	500	-	-	0	Y
	1500	-	-	0	Y
	5000	-	-	-	-
	Positive control	617	N	98	N

- not conducted

MA: metabolic activation (S9 mix)

* mean of 3 replicates

Applicant's summary and conclusion

Conclusions:

Te test item was considered to be non-mutagenic under the conditions of the test.

Executive summary:

OECD 471 (2011) - In a reverse gene mutation assay in bacteria, strains TA100, TA1535, TA98 and TA1537 of S. typhimuriumand WP2uvrA of E. coliwere exposed to Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene (limonene) and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene (ether MT) in DMSO at concentrations of 0.15, 0.5, 1.5, 5, 1550 and 150 µg/plate without metabolic activation and 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation, in a pre-incubation test system.

 

Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene (limonene) and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene (ether MT)was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains.

 

There was no evidence of induced mutant colonies over background in the test item treated colonies.

 

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.