reaction mass of [(1R*,5S*)-3,3,5-trimethylcyclohexyl] acetate and [(1S*,5S*)-3,3,5-trimethylcyclohexyl] acetate

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:

21 June - 04 July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

reaction mass of [(1R*,5S*)-3,3,5-trimethylcyclohexyl] acetate and [(1S*,5S*)-3,3,5-trimethylcyclohexyl] acetate

EC Number:

946-282-7

Molecular formula:

C11H20O2

IUPAC Name:

reaction mass of [(1R*,5S*)-3,3,5-trimethylcyclohexyl] acetate and [(1S*,5S*)-3,3,5-trimethylcyclohexyl] acetate

Method

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Experiment I and II: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative low toxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA), methylmethanesulfonate (MMS)

Remarks:

+S9: 2-AA (2.5 µg/plate, TA1535, TA1537, TA98, TA100; 10 µg/plate, WP2 uvrA); -S9: NaN3 (10 µg/plate, TA1535, TA100); 4-NOPD (10 µg/plate, TA98; 50 µg/plate, TA1537); MMS (2 µL/plate, WP2 uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (Experiment I); preincubation (Experiment II)

DURATION
- Preincubation period: 1 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Evaluation criteria:

A test substance is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Mean values and standard deviation were calculated.

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

Remarks:

exp. 1: -S9: starting at 1000 µg/plate, +S9: starting at 2500 µg/plate; exp. 2: -S9: starting at 333 µg/plate, +S9: starting at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Remarks:

exp. 1: -S9 and +S9: starting at 2500 µg/plate; exp. 2: -S9: starting at 333 µg/plate, +S9: starting at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Remarks:

exp. 1: -S9: starting at 1000 µg/plate, +S9: starting at 2500 µg/plate; exp. 2: -S9 and +S9: starting at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Remarks:

exp. 1: -S9: starting at 1000 µg/plate, +S9: starting at 2500 µg/plate; exp. 2: -S9: starting at 100 µg/plate, +S9: starting at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Remarks:

exp. 2: +S9: at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to the highest investigated dose.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as experiment I.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Reduced background growth was observed at the higher concentrations with and without metabolic activation in both experiments. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations with and without metabolic activation in both experiments.

Any other information on results incl. tables

Table 1. Test results of main test 1 (plate incorporation).

With or without S9 mix	Test substance concentration [μg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvrA	TA98	TA1537
-	0	134 ± 6	13 ± 1	46 ± 9	31 ± 4^B	14 ± 5
-	0 (DMSO)	117 ± 22	16 ± 3	50 ± 5	22 ± 3^B	14 ± 4
-	3	126 ± 10	17 ± 5	44 ± 6	25 ± 3^B	13 ± 3
-	10	123 ± 25	17 ± 3	47 ± 2	22 ± 3^B	13 ± 4
-	33	120 ± 20	19 ± 5	50 ± 2	21 ± 2^B	10 ± 4
-	100	130 ± 6	18 ± 2	48 ± 3	22 ± 4^B	11 ± 4
-	333	107 ± 18	10 ± 4	45 ± 10	20 ± 4^B	11 ± 4
-	1000	26 ± 7^R	9 ± 3^R	49 ± 5	16 ± 3^B R	8 ± 1
-	2500	9 ± 2^R	5 ± 1^R	54 ± 8	10 ± 2^B R	11 ± 2^R
-	5000	5 ± 1^R	5 ± 2^R	50 ± 17	7 ± 2^B R	3 ± 1^R
Positive controls, –S9 mix	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentrations [μg/plate]	10	10	2.0 μl	10	50
	Mean No. of colonies/plate (average of 3 ± SD)	2232 ± 43	2026 ± 7	956 ± 36	341 ± 14	81 ± 2
+	0	196 ± 17	15 ± 7	58 ± 2	39 ± 4^B	19 ± 6
+	0 (DMSO)	201 ± 4	18 ± 6	59 ± 8	44 ± 7^B	17 ± 2
+	3	202 ± 24	20 ± 1	50 ± 3	41 ± 6^B	19 ± 8
+	10	199 ± 25	18 ± 6	50 ± 7	43 ± 5^B	20 ± 8
+	33	202 ± 24	24 ± 2	64 ± 9	41 ± 5^B	16 ± 4
+	100	228 ± 13	21 ± 1	57 ± 7	42 ± 5^B	21 ± 8
+	333	223 ± 30	17 ± 5	53 ± 9	32 ± 10^B	17 ± 6
+	1000	189 ± 10	15 ± 4	57 ± 13	23 ± 5^B	20 ± 3
+	2500	7 ± 2^R	7 ± 1^R	30 ± 6	4 ± 2^B	2 ± 1^R
+	5000	1 ± 1^R	1 ± 1^R	32 ± 7	1 ± 1^B	1 ± 1^R
Positive controls, +S9 mix	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations [μg/plate]	2.5	2.5	10	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	3418 ± 159	472 ± 45	431 ± 29	2905 ± 19	349 ± 101

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

B: extensive bacterial growth

R: reduced background growth

Table 2. Test results of main test 2 (preincubation).

With or without S9 mix	Test substance concentration [μg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvrA	TA98	TA1537
-	0	124 ± 12	13 ± 1	66 ± 4	31 ± 5	13 ± 6
-	0 (DMSO)	114 ± 3	14 ± 1	64 ± 7	27 ± 3	10 ± 3
-	3	98 ± 2	14 ± 6	63 ± 4	26 ± 7	12 ± 4
-	10	104 ± 11	15 ± 2	58 ± 1	28 ± 10	8 ± 2
-	33	98 ± 10	12 ± 4	59 ± 4	28 ± 7	13 ± 4
-	100	38 ± 4^R	14 ± 3	57 ± 7	29 ± 7	8 ± 1
-	333	20 ± 4^R	9 ± 2^R	46 ± 6	26 ± 2	6 ± 0^R
-	1000	10 ± 3^R	8 ± 0^R	47 ± 9	6 ± 2^R	2 ± 2^R
-	2500	7 ± 2^R	0 ± 0^R	40 ± 5	5 ± 1^R	2 ± 1^R
-	5000	3 ± 1^R	0 ± 0^R	49 ± 5	2 ± 1^R	2 ± 1^R
Positive controls, –S9 mix	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentrations [μg/plate]	10	10	2.0 μl	10	50
	Mean No. of colonies/plate (average of 3 ± SD)	2128 ± 51	2106 ± 74	674 ± 19	325 ± 48	75 ± 2
+	0	167 ± 5	17 ± 7	62 ± 6	38 ± 9^B	19 ± 3
+	0 (DMSO)	154 ± 14	21 ± 7	62 ± 6	28 ± 9^B	20 ± 3
+	3	169 ± 13	19 ± 3	54 ± 9	28 ± 9^B	23 ± 1
+	10	178 ± 16	23 ± 1	65 ± 9	27 ± 7^B	16 ± 3
+	33	171 ± 9	19 ± 4	67 ± 10	29 ± 4^B	21 ± 1
+	100	169 ± 3	19 ± 7	67 ± 4	26 ± 4^B	17 ± 6
+	333	176 ± 7	15 ± 4	56 ± 4	27 ± 4^B	18 ± 4
+	1000	13 ± 3^R	12 ± 2^R	35 ± 9	7 ± 2^B R	10 ± 2^R
+	2500	0 ± 0^R	0 ± 0^R	41 ± 6	0 ± 0^B R	0 ± 0^R
+	5000	0 ± 0^R	0 ± 0^R	7 ± 2^R	0 ± 0^B R	0 ± 0^R
Positive controls, +S9 mix	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations [μg/plate]	2.5	2.5	10	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	3259 ± 99	231 ± 17	389 ± 25	405 ± 31	310 ± 23

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

B: extensive bacterial growth

R: reduced background growth

Applicant's summary and conclusion

Conclusions:

Under the conditions of the Ames test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation up to 5000 µg/plate.

Executive summary:

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2013). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and the Escherichia coli strain WP2 uvrA were exposed to the test substance using either the plate incorporation or the preincubation method. Test substance concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate were selected for the first and second experiment (plate incorporation and preincubation method, respectively) with and without metabolic activation. No substantial increase in the mean number of revertants per plate was observed in any of the test strains compared to the control, neither in the presence nor absence of metabolic activation. Reduced background growth was observed at the higher concentrations with and without metabolic activation in both experiments. Furthermore, toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations with and without metabolic activation in both experiments. All positive and negative control values were found to be within the respective historical control ranges. Under the conditions of this experiment, the test substance did not show mutagenicity in the selected S. typhimurium strains and in the E. coli strain in the presence and absence of metabolic activation.