Reaction mass of cis-1-methyl-1-(4-methylcyclohexyl) ethyl acetate and trans-1-methyl-1-(4-methylcyclohexyl) ethyl acetate and cis- 4-isopropyl-1-methylcyclohexyl acetate and trans-4-isopropyl-1-methylcyclohexyl acetate

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:

12 April - 6 June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD Guideline 471 with minor deviation: temperature of the incubator rose to 38.1 °C overnight, just above the 37 ± 1 °C limit

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:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

temperature of the incubator rose to 38.1 °C overnight, just above the 37 ± 1 °C limit

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:

(1r,4r)-1-methyl-4-(propan-2-yl)cyclohexyl acetate; (1s,4s)-1-methyl-4-(propan-2-yl)cyclohexyl acetate; 2-[(1r,4r)-4-methylcyclohexyl]propan-2-yl acetate; 2-[(1s,4s)-4-methylcyclohexyl]propan-2-yl acetate

EC Number:

939-728-7

Molecular formula:

C12H22O2

IUPAC Name:

(1r,4r)-1-methyl-4-(propan-2-yl)cyclohexyl acetate; (1s,4s)-1-methyl-4-(propan-2-yl)cyclohexyl acetate; 2-[(1r,4r)-4-methylcyclohexyl]propan-2-yl acetate; 2-[(1s,4s)-4-methylcyclohexyl]propan-2-yl acetate

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Name of test material (as cited in study report): Menthanyl acetate multiconstituent
- Physical state: Colourless to slightly amber liquid
- Analytical purity: 98.3 %
- Lot/batch No.: 110996
- Date of receipt: 21 February 2012
- Expiration date of the lot/batch: 9 September 2012
- Storage condition of test material: Stored at 2-8 °C, protected from light and under nitrogen

Method

Target gene:

Histidine gene

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: All strains were checked for characteristics (histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline).

Metabolic activation:

with and without

Metabolic activation system:

10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Mutagenicity tests:
- Experiment 1: 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate, with and without S9 mix in all 5 strains (plate incorporation method)
- Experiment 2a: 1.563, 3.125, 6.25, 12.5, 25, 50 and 100 μg/plate, without S9 mix in strains TA100 and TA1535 (plate incorporation method)
- Experiment 2b: 4.096, 10.24, 25.6, 64, 160 and 400 μg/plate; strains TA 98, TA 1537 and TA 102 in the absence (plate incorporation method) and presence of S9 (preincubation method) and strains TA 100 and TA 1535 in the presence of S9 (preincubation method) [1.638 μg/plate used in strains TA98 and TA1537 in the absence of S9 and strain TA102 in the presence of S9 only; 1000 μg/plate used in strain TA 102 in the absence of S9 and strains TA 98, TA 100, TA 1535 and TA 1537 in the presence of S9 only]
- Experiment 3: 1.563, 3.125, 6.25, 12.5, 25, 50 and 100 μg/plate, with S9 mix in strain TA1537 (preincubation method)
- Experiment 4: 1.563, 3.125, 6.25, 12.5, 25 and 50 μg/plate, with S9 mix in strains TA 1535, TA 98, TA 100 and TA 102 (preincubation method) [0.7813 μg/plate used in strain TA 102; 100 μg/plate used in strains TA 98, TA 100 and TA 1535]

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
Formulation procedure:
- Test article stock solutions were prepared by formulating Menthanyl acetate multiconstituent in ethanol under subdued lighting conditions with the aid of vortex mixing and warming to 30 °C as required, immediately prior to assay to give the maximum required treatment solution concentration. Subsequent dilutions were made using ethanol. The test article solutions were protected from light and used within approximately 5.5 h of initial formulation.

Volume addition: 0.1 mL volume additions of vehicle/test article solution were used for all plate-incorporation treatments, 0.05 mL volume additions of vehicle/test article solution were used for pre-incubation treatments.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2- Aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Strains TA 98, TA 1535 and TA 1537 were originally obtained from the UK NCTC. Strains TA 100 and TA 102 were derived from cultures originally obtained from Covance Laboratories Inc., USA.

METHOD OF APPLICATION: In agar (plate incorporation and preincubation method)

DURATION
- Preincubation period: 20 minutes at 37 ± 1 °C
- Incubation period: Plates were inverted and incubated at 37 ± 1 °C in the dark for 3 days.

NUMBER OF REPLICATIONS:
- Treatment and positive control groups: 3 plates/dose
- Vehicle control group: 5 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn was inspected for signs of toxicity.

OTHER:
Colony counting: Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles or splits in the agar affected the accuracy of the automated counter.

Evaluation criteria:

- For valid data, the test article was considered to be mutagenic if:
1. When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p ≤ 0.01) which was concentration related.
2. The positive trend/effects described above were reproducible.
- Test article was considered positive in this assay if all of the above criteria were met.
- Test article was considered negative in this assay if none of the above criteria were met.

- Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

- Dunnett's test was used to compare the counts at each concentration with the control. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate).

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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:

COMPARISON WITH HISTORICAL CONTROL DATA: Mean vehicle control counts fell within the normal historical ranges (historical control data of February 2008 - July 2009).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment 1: Toxicity was observed at concentrations of 50 μg/plate and above in strains TA 100 and TA 1535 in the absence of S9; 158.1 μg/plate and above in strains TA 98 and TA 1537 in the absence of S9 and strain TA 102 in the presence of S9; and 500 μg/plate and above in strains TA 98, TA 100, TA 1535 and TA 1537 in the presence of S9 and strain TA 102 in the absence of S9.
- Experiment 2: Toxicity was observed at 1000 μg/plate in strains TA 98, TA 100, TA 1535 and TA 1537 in the presence of S9 and TA 102 in the absence of S9; 400 μg/plate in strains TA 98 and TA 1537 in the absence of S9 and strain TA 102 in the presence of S9; and 100 μg/plate in strains TA 100 and TA 1535 in the absence of S9. Toxicity was observed at concentrations of 10.24 and/or 25.6 μg/plate and above in all strains in the presence of S9; 50 μg/plate and above in strain TA 100 in the absence of S9, 64 μg/plate and above in strain TA 98 in the absence of S9, 100 μg/plate and above in strain TA 1535 in the absence of S9, and 160 μg/plate and above in strains TA 1537 and TA 102 in the absence of S9.
- Experiment 3 and 4: Toxicity was observed at concentrations of 25 μg/plate and above in all strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/2: Results of mutagenicity

Group	Concentration (µg/plate)	Mean revertant numbers/plate	Concentration (µg/plate)	Mean revertant numbers/plate
Experiment 1			Experiment 2
TA 98 -S-9
Ethanol		27.6		26.8
Test item	5	26.7	1.638	18.7
	15.81	27.3	4.096	28.7
	50	28.3	10.24	27.7
	158.1	27.7	25.6	22.3
	500	30.0	64	33.0
	1581	25.0	160	22.7
	5000	16.3	400	28.7
2NF	5	1070.3	5	1134.3
TA 98 +S-9
Ethanol		37.8		32.0
Test item	5	29.0	4.096	30.7
	15.81	32.3	10.24	25.0
	50	32.0	25.6	31.0
	158.1	36.7	64	17.0
	500	34.0	160	30.7
	1581	32.7	400	25.7
	5000	19.3	1000	23.7
B[a]P	10	440.3	10	548.7
TA 100 -S-9
Ethanol		93.2		87.2
Test item	5	82.0	1.563	94.3
	15.81	111.7*	3.125	93.7
	50	92.7	6.25	72.7
	158.1	76.0	12.5	85.7
	500	69.3	25	77.0
	1581	49.0	50	92.7
	5000	26.0	100	53.7
NaN3	2	690.3	2	789.0
TA 100 +S-9
Ethanol		105.0		98.4
Test item	5	119.7	4.096	91.0
	15.81	106.0	10.24	80.7
	50	86.0	25.6	75.3
	158.1	100.3	64	58.0
	500	85.3	160	68.0
	1581	27.0	400	72.7
	5000	T	1000	56.7
AAN	5	1443.0	5	1139.7
TA 1535 -S-9
Ethanol		21.8		17.8
Test item	5	22.0	1.563	15.3
	15.81	20.3	3.125	11.3
	50	21.0	6.25	15.0
	158.1	12.0	12.5	14.7
	500	10.0	25	15.3
	1581	9.0	50	15.7
	5000	8.0	100	15.0
NaN3	2	690.7	2	653.0
TA 1535 +S-9
Ethanol		22.6		17.4
Test item	5	22.0	4.096	15.3
	15.81	27.3	10.24	20.7
	50	25.0	25.6	18.0
	158.1	36.7	64	13.0
	500	21.3	160	11.7
	1581	17.7	400	8.3
	5000	15.0	1000	9.3
AAN	5	251.3	5	193.3
TA 1537 -S-9
Ethanol		8.2		8.4
Test item	5	8.3	1.638	5.3
	15.81	11.0	4.096	6.3
	50	8.0	10.24	6.3
	158.1	8.0	25.6	6.7
	500	6.3	64	5.7
	1581	T	160	7.3
	5000	T	400	3.3
AAC	50	183.3	50	126.7
TA 1537 +S-9
Ethanol		19.8		14.2
Test item	5	17.7	4.096	20.7
	15.81	20.0	10.24	6.0
	50	15.7	25.6	12.3
	158.1	17.7	64	T
	500	14.7	160	T
	1581	T	400	T
	5000	T	1000	T
AAN	5	294.3	5	120.3
TA 102 -S-9
Ethanol		246.2		246.8
Test item	5	267.7	4.096	216.0
	15.81	261.7	10.24	220.3
	50	217.0	25.6	231.3
	158.1	196.0	64	232.0
	500	198.0	160	196.3
	1581	200.0	400	155.0
	5000	200.3	1000	107.3
MMC	0.2	875.3	0.2	962.3
TA 102 +S-9
Ethanol		251.0		215.0
Test item	5	264.3	1.638	250.0
	15.81	248.0	4.096	256.7
	50	244.3	10.24	190.3
	158.1	199.7	25.6	182.0
	500	195.7	64	92.3
	1581	264.0	160	105.7
	5000	243.7	400	131.3
AAN	20	1751.0	20	1134.3
Experiment 3 (TA 1537 +S-9)			Experiment 4 (TA 98 +S-9)
Ethanol		16.4		36.0
Test item	1.563	14.7	1.563	38.0
	3.125	7.7	3.125	30.7
	6.25	15.3	6.25	25.7
	12.5	9.3	12.5	29.3
	25	11.3	25	22.3
	50	T	50	27.3
	100	T	100	T
AAN	5	178.0	B[a]P 10	455.7
Experiment 4 (TA 100 +S-9)			Experiment 4 (TA 1535 +S-9)
Ethanol		108.2		16.6
Test item	1.563	96.7	1.563	16.3
	3.125	93.7	3.125	16.0
	6.25	99.7	6.25	20.7
	12.5	115.7	12.5	14.0
	25	91.7	25	7.7
	50	77.0	50	12.0
	100	T	100	12.0
AAN	5	901.0	AAN 5	197.0
Experiment 4 (TA 102 +S-9)
Ethanol		195.4	NA
Test item	0.7813	162.7
	1.563	154.3
	3.125	148.3
	6.25	129.7
	12.5	140.3
	25	128.7
	50	112.3
AAN	20	901.0

T: Toxic, no revertant colonies; * p ≤ 0.05

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the test conditions, Menthanyl acetate multiconstituent is not considered as mutagenic in S. typhimurium strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102).

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were exposed to Menthanyl acetate multiconstituent at the following concentrations:

- Experiment 1: 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate, with and without S9 mix in all 5 strains (plate incorporation method)

- Experiment 2a: 1.563, 3.125, 6.25, 12.5, 25, 50 and 100 μg/plate, without S9 mix in strains TA100 and TA1535 (plate incorporation method)

- Experiment 2b: 4.096, 10.24, 25.6, 64, 160 and 400 μg/plate; strains TA 98, TA 1537 and TA 102 in the absence (plate incorporation method) and presence of S9 (preincubation method) and strains TA 100 and TA 1535 in the presence of S9 (preincubation method) [1.638 μg/plate used in strains TA 98 and TA 1537 in the absence of S9 and strain TA102 in the presence of S9 only; 1000 μg/plate used in strain TA 102 in the absence of S9 and strains TA 98, TA 100, TA 1535 and TA 1537 in the presence of S9 only]

- Experiment 3: 1.563, 3.125, 6.25, 12.5, 25, 50 and 100 μg/plate, with S9 mix in strain TA1537 (preincubation method)

- Experiment 4: 1.563, 3.125, 6.25, 12.5, 25 and 50 μg/plate, with S9 mix in strains TA 1535, TA 98, TA 100 and TA 102 (preincubation method) [0.7813 μg/plate used in strain TA 102; 100 μg/plate used in strains TA 98, TA 100 and TA 1535]

Metabolic activation system used in this test was10 % S9 mix. S9 fraction was prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254. Vehicle control and positive control groups were also included in mutagenicity tests.

In experiment 1, toxicity was observed at concentrations of 50 μg/plate and above in strains TA 100 and TA 1535 in the absence of S9; 158.1 μg/plate and above in strains TA 98 and TA 1537 in the absence of S9 and strain TA 102 in the presence of S9; and 500 μg/plate and above in strains TA 98, TA 100, TA 1535 and TA 1537 in the presence of S9 and strain TA 102 in the absence of S9. In Experiment 2, toxicity was observed at 1000 μg/plate in strains TA 98, TA 100, TA 1535 and TA 1537 in the presence of S9 and TA 102 in the absence of S9; 400 μg/plate in strains TA 98 and TA 1537 in the absence of S9 and strain TA 102 in the presence of S9; and 100 μg/plate in strains TA 100 and TA 1535 in the absence of S9. Toxicity was observed at concentrations of 10.24 and/or 25.6 μg/plate and above in all strains in the presence of S9; 50 μg/plate and above in strain TA 100 in the absence of S9, 64 μg/plate and above in strain TA 98 in the absence of S9, 100 μg/plate and above in strain TA 1535 in the absence of S9, and 160 μg/plate and above in strains TA 1537 and TA 102 in the absence of S9. In Experiment 3 and 4, toxicity was observed at concentrations of 25 μg/plate and above in all strains. The positive and vehicle controls induced the appropriate responses in the corresponding strains. Test item did not induce statistically significant increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of metabolic activation.

Under the test conditions, Menthanyl acetate multiconstituent is not considered as mutagenic in this bacterial system.