3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)pent-4-en-2-ol

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:

Mar - May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)pent-4-en-2-ol

EC Number:

267-140-4

EC Name:

3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)pent-4-en-2-ol

Cas Number:

67801-20-1

Molecular formula:

C14H24O

IUPAC Name:

3-methyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pent-4-en-2-ol

Details on test material:

IUPAC name : 3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)-4-penten-2-ol
Molecular formulae : C14H24O
CAS number : 67801-20-1
EINECS number : 267-140-4
Description : Pale yellow liquid
Batch number : VE00071661
Purity : 96% (sum of isomers)
Date received : 29 January 2010
Expiry date : 10 June 2011
Storage conditions : Room temperature in the dark

Method

Target gene:

The thymidine kinase heterozygote system, TK +/- to TK -/-.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

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.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Male Sprague-Dawley rats: Three consecutive daily doses of phenobarbital/Beta-naphthoflavone (80/100 mg per kg per day). S9 was stored at approximately 196°C in a liquid nitrogen. Final concentration of S9 was 2% throughout the study.

Test concentrations with justification for top dose:

Concentrations assessed for mutation Frequency (µg/mL)

Test 1
4 hour -S9 (2.5, 5, 10, 20, 30)
4 Hour +S9 ( 5, 10, 20, 30, 40, 50)
24 Hour -S9 ( 2.5, 5, 10, 20, 30)

Test 2
4 Hour +S9 (10, 20, 30, 40, 45, 50, 55)

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

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.

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 1 x 106 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 (S9-mix) at eight dose levels of the test material (2.5 to 60 µg/ml), 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.

At the end of the treatment period the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/ml. The cultures were incubated and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 105 cells/ml.
On Day 2 of each 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 Percentage 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.

Microtitre plates were scored using a magnifying mirror box after ten to fourteen days incubation. 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. Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately to 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 MTT solution (2.5 mg/ml in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

Evaluation criteria:

The normal range for mutant frequency per survivor is 50-200 x 10-6 for the TK+/- locus in L5178Y cells at this laboratory. Vehicle controls results should ideally be within this range.
Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.
Dose levels that have survival values (RTG) less than 10% are usually excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.
For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 will be considered positive.
However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Statistics:

The International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) stated that the IMF must exceed some value based on the global background MF. This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 will be considered positive.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(4 hour -S9 at 40ug/ml, 4 hour +S9 at 60ug/ml, 24 hour _S9 at 40

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: L5178Y mouse lymphoma cell line

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table1              Summary of Results

 

Experiment 1

Treatment (µg/ml)		4-Hours-S-9				Treatment (µg/ml)		4-Hours+S-9
		%	RTG	MF§				%	RTG	MF§
0		100	1.00	64.39		0		100	1.00	75.58
2.5		111	1.15	71.60		2.5	Ø	107
5		107	1.07	60.82		5		101	0.96	106.08
10		102	1.00	65.22		10		93	1.08	79.57
20		84	0.92	71.56		20		107	1.00	82.89
30		25	0.26	73.99		30		99	0.89	101.72
40	Ø	0				40		85	0.78	108.88
50	Ø	0				50		43	0.50	67.74
60	Ø	0				60	Ø	0
Linear trend				NS		Linear trend				NS
EMS						CP
400		85	0.60	812.98		2		66	0.39	1000.00

Experiment 2

Treatment (µg/ml)		24-Hours-S-9				Treatment (µg/ml)		4-Hours+S-9
		%	RTG	MF§				%	RTG	MF§
0		100	1.00	65.51		0		100	1.00	79.90
2.5		97	1.15	84.15		2.5	Ø	91
5		92	0.96	88.37		5	Ø	86
10		96	0.94	101.71		10		82	0.89	85.01
20		56	0.53	71.39		20		83	0.84	87.03
30		33	0.27	76.94		30		77	0.99	56.41
40	Ø					40		68	0.70	77.86
50	Ø					45		53	0.58	95.58
60	Ø					50		41	0.35	66.56
						55		8	0.12	108.62
						60	Ø	1
Linear trend				NS		Linear trend				NS
EMS						CP
150		59	0.49	1421.09		2		61	0.30	821.39

Applicant's summary and conclusion

Conclusions:

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.