(1R,2R,4S)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-ol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 May 2013 - 15 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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(1R,2R,4S)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl acetate

EC Number:

807-751-0

Cas Number:

4057-31-2

Molecular formula:

C12H20O2

IUPAC Name:

(1R,2R,4S)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl acetate

Test material form:

liquid

Method

Target gene:

Mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver)

Test concentrations with justification for top dose:

3h, ± S9 mix: 140; 130; 120; 110; 100; 90; 80; 60; 40; 20; 10 and 5 µg/mL
3h, + S9 mix: 110; 100; 95; 90; 85; 80; 70; 60; 40; 20; 10 and 5 µg/mL
24h, - S9 mix. 80; 75; 70; 65; 60; 55; 50; 45; 40; 30; 20; 10 and 5 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test item was non-soluble in Distilled water or Dimethyl sulfoxide at 500 mg/mL concentration. However, a proper formulation could be made at the same concentration using Acetone or Ethanol (96%) as vehicle. Due to the better biocompatibility to the test system, Ethanol (96%) was selected for vehicle of the study.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

(Untreated)

Negative solvent / vehicle controls:

yes

Remarks:

(Ethanol 96%, DMSO)

Remarks:

Test item vehicle: Ethanol (10 µL/mL), positive control vehicle: DMSO (10 µL/mL)

Controls 2

Positive controls:

yes

Remarks:

(Without metabolic activation)

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.15 µg/mL (3h treatment), 0.10 µg/mL (24h treatment), in DMSO

Controls 3

Positive controls:

yes

Remarks:

(With metabolic activation)

Positive control substance:

cyclophosphamide

Remarks:

4 µg/mL in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (RPMI-5 medium)

DURATION
- Exposure duration: 3h (with and without S9-mix), 24h (without S9-mix)
- Expression time (cells in growth medium, to allow expression of the TK- mutation ): 3 days
- Selection time (plating for -trifluorothymidine (TFT) resistance): Two weeks

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Duplicate cultures were used for each treatment.

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was measured by relative survival

OTHER EXAMINATIONS:
- Determination of Survival or Viability:
After the exposure period and the expression time, cells were also diluted to be plated for survival and viability respectively. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

Parameter calculated:
- Relative survival
- Mutant Frequency
- Small and large colony mutant frequencies

Others:
The number of viable cells in the individual samples was counted manually using a haemocytometer.
Measurement of pH and osmolality was performed after the treatment period.

Evaluation criteria:

The test item was considered to be mutagenic in this assay if all the following criteria were met (based on M. Moore 2006):
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency are observed in treated cultures compared to the corresponding negative (vehicle) control values at one or more concentrations.
3. The increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. There is a significant concentration-relationship as indicated by the linear trend analysis (p < 0.05).
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle) control value.

Statistics:

Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel software. The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

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:

(Assay 1: ±S9mix, 3h: >= 100 µg/L; Assay 3: +S9mix, 3h: >= 95 µg/L; -S9mix, 24h: >= 55 µg/L)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There were no large changes in pH after treatment.
- Effects of osmolality: There were no large changes in osmolality after treatment.
- Water solubility: No insolubility was detected in the final treatment medium at the end of the treatment (although discoloured medium was detected at some concentrations)

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed to select dose levels for the main assays. During the preliminary test, a 3 hour treatment in the presence and absence of S9-mix and a 24 hour treatment in the absence of S9-mix were performed with a range of test item concentration to determine toxicity. The highest concentration tested in the preliminary experiment was 2500 μg/mL. Insolubility and cytotoxicity were observed in the preliminary experiment. Concentrations for the main experiments were selected to cover the range from cytotoxicity to little or no cytotoxicity according to the instructions of the relevant OECD guideline.

ADDITIONAL INFORMATION:
No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations.
No significant dose-response to the treatment was indicated by the linear trend analysis.

OBSERVATION:
During Assay 2, a contamination was detected in the samples, therefore it was considered invalid and terminated. An additional experiment (Assay 3) was performed using the same experimental conditions as in Assay 2 to provide valid and interpretable data.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Mutagenicity Results of Assay 1

S9 mix	Treatment period (hours)	Test item or control concentration	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/ total number of wells	Dn^2/var(Dn)♦	Mutation frequency
+	3	140 µg/mL	ND	ND	ND	ND	ND
		130 µg/mL	ND	ND	ND	ND	ND
		120 µg/mL	ND	ND	ND	ND	ND
		110 µg/mL	ND	ND	ND	ND	ND
		100 µg/mL	NE	NE	NE	NE	NE
		90 μg/mL	642/768	64/768	62/768	0.047	125.0
		80 µg/mL	640/768	71/768	57/768	0.056	124.5
		60 µg/mL	640/768	74/768	54/768	0.087	122.7
		40 µg/mL	641/768	74/768	53/768	0.009	128.8
		20 µg/mL	652/768	68/768	48/768	0.108	143.1
		10 µg/mL	654/768	65/768	49/768	0.785	105.8
		5 µg/mL	636/768	75/768	57/768	0.003	133.5
		Vehicle control	640/768	71/768	57/768	--	131.8
		Vehicle control for CP	645/768	64/768	59/768	--	118.3
		Untreated control	642/768	64/768	62/768	--	135.2
		Positive control (CP: 4 μg/mL)	277/768	204/768	287/768	♦♦ 3.55E-13	1076.7*

In linear trend analysis β²/var (β) = 0.002, not significant.

S9 mix	Treatment period (hours)	Test item or control concentration	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/ total number of wells	Dn^2/var(Dn) ♦	Mutation frequency
-	3	140 µg/mL	ND	ND	ND	ND	ND
		130 µg/mL	ND	ND	ND	ND	ND
		120 µg/mL	ND	ND	ND	ND	ND
		110 µg/mL	ND	ND	ND	ND	ND
		100 µg/mL	ND	ND	ND	ND	ND
		90 μg/mL	671/768	38/768	59/768	0.710	111.7
		80 µg/mL	672/768	42/768	54/768	0.229	100.8
		60 µg/mL	652/768	28/768	88/768	0.818	112.6
		40 µg/mL	683/768	38/768	47/768	0.139	79.0
		20 µg/mL	673/768	39/768	56/768	<0.001	87.4
		10 µg/mL	679/768	47/768	42/768	0.094	80.6
		5 µg/mL	672/768	36/768	60/768	0.254	101.5
		Vehicle control	672/768	52/768	44/768	--	88.0
		Vehicle control for NQO	670/768	39/768	59/768	--	100.8
		Untreated control	653/768	40/768	75/768	--	119.8
		Positive control (NQO: 0.15 μg/mL)	407/768	199/768	162/768	♦♦ 2.54E-12	749.6*

In linear trend analysis β²/var (β) =1.199, not significant.

Mutagenicity Results of Assay 3

S9-mix	Treatment period (hours)	Test item or control concentration	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/ total number of wells	Dn2/var(Dn)♦	Mutation frequency
+	3	110 µg/mL	ND	ND	ND	ND	ND
		100 µg/mL	NE	NE	NE	NE	NE
		95 µg/mL	NE	NE	NE	NE	NE
		90 μg/mL	NE	NE	NE	NE	NE
		85 µg/mL	646/768	67/768	55/768	0.014	120.7
		80 µg/mL	655/768	52/768	61/768	<0.001	123.5
		70 µg/mL	646/768	59/768	63/768	<0.001	124.2
		60 µg/mL	646/768	60/768	62/768	0.276	109.1
		40 µg/mL	661/768	50/768	57/768	0.464	103.9
		20 µg/mL	661/768	51/768	56/768	0.531	103.2
		10 µg/mL	667/768	51/768	50/768	1.148	94.2
		62.5 µg/mL	671/768	51/768	46/768	1.564	88.9
		Vehicle control	649/768	57/768	62/768	--	124.4
		Vehicle control for CP	654/768	66/768	48/768	--	105.1
		Untreated control	658/768	57/768	53/768	--	100.4
		Positive control (CP: 4 μg/mL)	183/768	240/768	345/768	♦♦ 1.58E-15	1571.2*

In linear trend analysis β²/var (β) = 1.619, not significant.

S9-mix	Treatment period (hours)	Test item or control concentration	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/ total number of wells	Dn^2/var(Dn) ♦	Mutation frequency
-	24	80 µg/mL	ND	ND	ND	ND	ND
		75 µg/mL	ND	ND	ND	ND	ND
		70 µg/mL	ND	ND	ND	ND	ND
		65 µg/mL	ND	ND	ND	ND	ND
		60 µg/mL	ND	ND	ND	ND	ND
		55 µg/mL	NE	NE	NE	NE	NE
		50 μg/mL	684/768	44/768	40/768	0.270	80.2
		45 μg/mL	678/768	51/768	39/768	0.245	80.9
		40 μg/mL	663/768	51/768	54/768	0.043	97.5
		30 μg/mL	672/768	68/768	28/768	0.058	86.7
		20 μg/mL	683/768	47/768	38/768	0.049	87.0
		10 μg/mL	670/768	49/768	49/768	0.001	93.2
		5 μg/mL	668/768	65/768	35/768	<0.001	91.9
		Vehicle control	674/768	55/768	39/768	--	92.4
		Vehicle control for NQO	678/768	65/768	25/768	--	87.0
		Untreated control	681/768	53/768	34/768	--	82.1
		Positive control (NQO: 0.1 μg/mL)	384/768	269/768	115/768	♦♦ 2.27E-13	830.8*

In linear trend analysis β²/var (β) =0.359, not significant.

* = Statistically significant.

♦ = Evaluated by Dunnett’s test for multiple comparisons. Significant if D_n²/var(D_n) >5.48(at p<0.05).

♦♦ = Evaluated by T-test for independent samples. Significant at p<0.05.

D_n= Difference of log mutant frequency of dose “n” and that of the vehicle control

var(D_n) = variance of D_n; β = slope of the curve; var(β) = variance of the slope

+ = in the presence of S9-mix

- = in the absence of S9-mix

Vehicle control = Ethanol

Vehicle control for CP = DMSO

CP = Cyclophosphamide

Vehicle control for NQO = DMSO

NQO = 4-Nitroquinoline-N-oxide

DMSO = Dimethyl sulfoxide

ND = no data (No cells were plated for colony growing due to the observed cytotoxicity during treatment or in the expression period.)

NE = not evaluated (Due to the high level of cytotoxicity)

Note: Mutation frequency refers to 10⁶viable cells

The overall study was considered to be valid:

The spontaneous mutation frequency of the negative (vehicle) control was in the recommended range (50-170 mutants per 106 viable cells) in each experiment.

The positive controls (Cyclophosphamide in the presence of metabolic activation and 4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays.

The plating efficiencies for the negative (vehicle) control at the end of the expression period (PEviability) were within the acceptable range (65-120 %) in all assays.

The number of test concentrations evaluated for each treatment was at least seven in each case,.

The tested concentration range in the study was considered to be adequate as the highest examined concentration produced approximately 80-90% toxicity (i.e. approximately 10-20% relative survival or relative total growth) and lower test concentrations were evenly spaced by a factor of no more than two. For determination of the cytotoxicity, the relative total growth was taken into consideration, as the test item showed late phase cytotoxicity during the expression period.

Applicant's summary and conclusion

Conclusions:

No mutagenic effect was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus (OECD 476) to test the potential to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix). Ethanol was used as vehicle of the test item in this study. The test item was examined up to 2500 μg/mL in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:

Assay 1, 3-hour treatment with metabolic activation: 140; 130; 120; 110; 100; 90; 80; 60; 40; 20; 10 and 5 μg/mL

Assay 1, 3-hour treatment without metabolic activation: 140; 130; 120; 110; 100; 90; 80; 60; 40; 20; 10 and 5 μg/mL

Assay 3, 3-hour treatment with metabolic activation: 110; 100; 95; 90; 85; 80; 70; 60; 40; 20; 10 and 5 μg/mL

Assay 3, 24-hour treatment without metabolic activation: 80; 75; 70; 65; 60; 55; 50; 45; 40; 30; 20; 10 and 5 μg/mL

No insolubility was detected in the final treatment medium at the end of the treatment. There were no large changes in pH or osmolality after treatment. Cytotoxicity was observed in both assays (Assay 1: ± S9mix, 3h: >= 100 µg/L; Assay 3: + S9mix, 3h: >= 95 µg/L; - S9mix, 24h: >= 55 µg/L). No biologically relevant or statistically significant increase in the mutation frequency was observed. No dose response to the treatment was indicated by the linear trend analysis. The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases and the study was considered to be valid. In conclusion, no mutagenic effect was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.