1,2,3,4,4a,7,8,8a-octahydro-2,4a,5,8a-tetramethyl-1-naphthyl formate

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:

The study was conducted between 09 May 2014 and 25 June 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Kanpoan No. 287 - Japanese Environment Protection Agency; Eisei No. 127 - Japanese Ministry of Health and Welfare and Heisei 09/10/31 Kikyoku No. 2 - Japanese Ministry of International Trade & Industry.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3,4,4a,7,8,8a-octahydro-2,4a,5,8a-tetramethyl-1-naphthyl formate

EC Number:

265-742-1

EC Name:

1,2,3,4,4a,7,8,8a-octahydro-2,4a,5,8a-tetramethyl-1-naphthyl formate

Cas Number:

65405-72-3

Molecular formula:

C15H24O2

IUPAC Name:

1,2,3,4,4a,7,8,8a-octahydro-2,4a,5,8a-tetramethyl-1-naphthyl formate

Test material form:

liquid

Specific details on test material used for the study:

Batch: SC00010054
Purity: 96.5%
Physical state/Appearance: Clear colourless liquid

Method

Target gene:

Thymidine kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Experiment 1
4-hour without S9: 2.5, 5, 10, 20 µg/mL
4-hour with S9 (2%): 2.5, 5, 10, 20, 40, 50 µg/mL

Experiment 2
24-hour without S9: 2, 4, 8, 16, 24, 32 µg/mL
4-hour with S9 (2%): 8, 16, 32, 40, 48, 56 µg/mL

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Controls

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Mutagenicity Test
Experiment 1
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1E+06 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (2.5 to 40 µg/mL in the absence of metabolic activation and 2.5 to 70 µg/mL in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 0.2 mL of the treatment dilutions, (0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL.

The treatment vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

Experiment 2
As in Experiment 1, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL cultures in R10 medium for the 4 hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours therefore 0.3E+06 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks. The treatments were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (2 to 48 µg/mL in the absence of metabolic activation, and 2 to 56 µg/mL in the presence of metabolic activation), vehicle and positive controls. To each culture vessel was added 2 mL of S9 mix if required, 0.2 mL of the treatment dilutions, (0.2 mL for the positive controls) and sufficient R0 medium to give a final volume of 20 mL (R10 was used for the 24 hour exposure group).

The treatment vessels were incubated at 37 °C with continuous shaking using an orbital shaker within an incubated hood for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

Measurement of Survival, Viability and Mutant Frequency
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2E+05 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2E+05 cells/mL, unless the mean cell count was less than 3E+05 cells/mL in which case all the cells were maintained.

On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.

The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Plate Scoring
Microtitre plates were scored using a magnifying mirror box after ten to fourteen days’ incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al, 1990). Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolized to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups in the preliminary toxicity test.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Cytotoxicity Test
The dose range of the test item used in the preliminary toxicity test was 9.2 to 2363 µg/mL in all three exposure groups.

There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups. The onset of test item-induced toxicity was very sharp in all three of the exposure groups. A precipitate of the test item was observed at and above 295.4 µg/mL in the absence of metabolic activation (4-hour and 24-hour exposure). A precipitate of the test item was observed at and above 590.8 µg/mL in the presence of metabolic activation (4-hour exposure). Based on the %RSG values observed, the maximum dose level in the subsequent mutagenicity experiment was limited by test item induced toxicity.

Experiment 1
There was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of reductions in viability (%V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Based on the %RSG and RTG values observed, optimum levels of toxicity were achieved in the absence of metabolic activation. Optimum levels of toxicity were not achieved in the presence absence of metabolic activation, despite using a narrow dose interval, due to the very steep toxicity curve of the test item. Whilst optimum levels of toxicity were not achieved a dose level, in the presence of metabolic activation, that exceeded the upper limit of toxicity was plated for viability and 5-TFT resistance as sufficient cells were available at the time of plating. The excessive toxicity observed at and above 25 µg/mL in the absence of metabolic activation, and at and above 60 µg/mL in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 50 µg/mL in the in the presence of metabolic activation, exceeded the upper acceptable limit of 90%, therefore, this dose was excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances.

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell, at any of the dose levels (including the dose levels that exceeded the upper limit of acceptable toxicity), in either the absence or presence of metabolic activation. With no evidence of any toxicologically significant increases in mutant frequency in either the absence or presence of metabolic activation in this Experiment, the test item was considered to have been adequately tested. Precipitate of the test item was not observed at any of the dose levels.

Experiment 2
As was seen previously, there was evidence of marked toxicity in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of a reduction in viability (%V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Based on the RTG and / or %RSG values observed, optimum levels of toxicity were considered to have been achieved in the absence metabolic activation only. Despite using a narrow dose range, optimum levels of toxicity could not be achieved in the presence of metabolic activation due to shift in toxicity. The excessive toxicity observed at and above 40 µg/mL in the absence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 56 µg/mL in the presence of metabolic activation exceeded the upper acceptable limit of 90%. Therefore, this dose level was excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances.

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a modest effect on the toxicity of the test item.

The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 1E-06 per viable cell, at any of the dose levels (including the dose level that exceeded the upper limit of acceptable toxicity), in either the absence or presence of metabolic activation. Precipitate of test item was not observed at any of the dose levels.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (mg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
9.2	106	10	110
18.5	76	88	73
36.9	0	85	8
73.8	0	0	0
147.7	0	0	0
295.4	0	0	0
590.8	0	0	0
1181.5	0	0	0
2363	0	0	0

 

Summary of results

Experiment 1

Treatment (µg/ml)		4-hours-S-9				Treatment (µg/ml)		4-hours+S-9
		%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	137.59		0		100	1.00	128.61
2.5		94	1.03	115.89		2.5		98	1.00	117.24
5		94	0.99	128.48		5		97	1.04	108.62
10		86	0.98	94.03		10		94	0.88	115.31
20		22	0.27	104.22		20		80	0.97	108.70
25	Ø	1				40		37	0.36	117.40
30	Ø	0				50	X	8	0.07	149.70
35	Ø	0				60	Ø	1
40	Ø	0				70	Ø	0
Linear trend				NS		Linear trend				NS
EMS						CP
400		57	0.48	839.87		2		37	0.19	1036.19

Experiment 2

Treatment (µg/ml)		24-hours-S-9				Treatment (µg/ml)		4-hours+S-9
		%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	126.54		0		100	1.00	146.88
2		116	0.95	145.67		2	Ø	92
4		108	0.93	130.01		4	Ø	93
8		93	1.01	111.55		8		90	0.98	107.82
16		64	0.83	113.49		16		94	1.00	142.45
24		32	0.45	122.83		32		90	1.01	127.00
32		7	0.14	143.07		40		87	0.90	132.42
40	Ø	1				48		39	0.46	150.35
48	Ø	0				56	X	4	0.05	182.95
Linear trend				NS		Linear trend				NS
EMS						CP
150		53	0.42	1075.60		2		56	0.35	1389.62

Cell and 96-Well Plate Counts: Experiment 1 (-S9) 4-Hour Exposure

Treatment (µg/ml)		Cell counts $			Viability § after day 2 2 cells/well				Resistant mutants § after day 2 2000 cells/well
		0h	24h	48h
0	A B	8.81 8.83	7.65 7.99	7.24 6.34	72 74	73 75	85 76	77 85	17 18	25 18	18 14	19 25
2.5	A B	8.28 8.36	8.65 8.27	6.03 6.48	80 76	87 77			21 15	19 17
5	A B	8.62 8.85	8.17 8.32	6.48 5.71	76 82	74 83			22 13	26 15
10	A B	8.39 8.20	8.94 8.39	5.34 5.90	83 82	80 78			12 18	15 16
20	A B	6.80 4.99	3.69 3.85	4.66 4.82	82 81	87 80			22 14	18 17
25	A B	2.32 2.28	1.56(2.32) 1.74(2.28)	1.25(1.56) 1.52(1.74)	NP NP	NP NP			NP NP	NP NP
30	A B	1.74 1.55	1.18(1.74) 1.05(1.55)	0.81(1.18) 0.73(1.05)	NP NP	NP NP			NP NP	NP NP
35	A B	1.40 1.66	0.85(1.40) 0.94(1.66)	0.49(0.85) 0.48(0.94)	NP NP	NP NP			NP NP	NP NP
40	A B	1.19 0.90	0.68(1.19) 0.46(0.90)	0.42(0.68) 0.25(0.46)	NP NP	NP NP			NP NP	NP NP
Positive Control EMS (µg/ml)
400	A B	7.93 7.88	6.63 6.59	4.27 5.99	70 73	72 70			77 59	70 55

Statistical Analysis: Experiment 1 (-S9) 4-Hour Exposure

Treatment (µg/ml)		SG	%RSG	%V	RTG	MF§
0		13.27	100	81.33	1.00	137.59
2.5		13.23	94	89.59	1.03	115.89
5		12.56	94	85.83	0.99	128.48
10		12.17	86	91.99	0.98	94.03
20		4.47	22	98.08	0.27	104.22
25	Ø	0.57	1
30	Ø	0.21	0
35	Ø	0.11	0
40	Ø	0.05	0
Positive Control EMS
Treatment (µg/ml)		SG	%RSG	%V	RTG	MF§
400		8.48	57	67.78	0.48	839.87

Test for linear trend

Slope	‑1.664E‑006
Variance	7.174E‑013
b²/Sb	3.861

Applicant's summary and conclusion

Conclusions:

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In VitroMammalian Cell Gene Mutation Tests" adopted 21 July 1997, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

 

Methods…….

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle, dimethyl sulfoxide (DMSO), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4‑hour exposure group in the presence of metabolic activation (2% S9) and a 24-hour exposure group in the absence of metabolic activation.

 

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for viability and expression of mutant colonies were as follows:

 

Experiment 1

Group	Concentration of OxyoctalineFormate (µg/mL) plated for mutant frequency
4-hour without S9	2.5, 5, 10, 20,
4-hour with S9 (2%)	2.5, 5, 10, 20, 40, 50

 

Experiment 2

Group	Concentration of OxyoctalineFormate (µg/mL) plated for mutant frequency
24-hour without S9	2, 4, 8, 16, 24, 32
4-hour with S9 (2%)	8, 16, 32, 40, 48, 56

 

Results……..

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate of the test item was not observed at any of the dose levels in the Mutagenicity Test. The vehicle controls (DMSO) had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

 

The test item did not induce any toxicologically significant dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.

 

Conclusion

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.