Cyclohexanemethanol, 4-(1-methylethyl)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance tested uniformly negative in 3 in vitro assays, including a bacterial reverse mutation assay that was consistent with the current OECD Test Guideline 471 (OECD, 1997a), a chromosome aberration assay in human lymphocytes that was consistent with the current OECD Test Guideline 473 (OECD, 1997b), and a gene mutation assay in mouse lymphoma L5178Y TK+/- cells that was consistent with the current OECD Test Guideline 476 (OECD, 1997c).  The conclusion is that the substance is not considered to be a genotoxic compound.  

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study in compliance with guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Acceptable to the Japanese New Chemical Substance Law (METI)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at 37ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Microsomal fraction prepared from livers of male Sprague-Dawley rats

Test concentrations with justification for top dose:

EXPERIMENT 1:
without S9 (μg/ml): 0, 12.5, 25, 50, 100, 150 and 200
with S9 (2%) (μg/ml): 0, 12.5, 25, 50, 100, 150 and 200
EXPERIMENT 2:
without S9 (μg/ml): 0, 3.13, 6.25, 12.5, 25, 50 and 100
with S9 (1%) (μg/ml): 0, 12.5, 25, 50, 100, 150 and 200

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DSMO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: cyclophosphamide (+S9)

Details on test system and experimental conditions:

DURATION
- Exposure duration: 4 hours (± S9) in Experiment 1, 4 hours (+S9) and 24 hours (-S9) in Experiment 2
- Expression time (cells in growth medium): 20 hours (± S9) in Experiment 1, 20 hours (+S9) in Experiment 2

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 consecutive well-spread metaphases from each culture were counted

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: If greater than 44 chromosomes are scored and the number is a multiple of the haploid count then the cell is classified as a polyploid cell.
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, i.e., 4 chromatids instead of 2, the cell is scored as endoreduplicated (E).

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells were compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Key result

Species / strain:

mammalian cell line, other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: >100 μg/ml (-S9) and >150 μg/ml (+S9); Experiment 2: >50 μg/ml (-S9) and >100 μg/ml (+S9).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test item showed marked evidence of toxicity in all three exposure groups. Haemolysis was seen in the blood cultures at the end of the exposure period at and above 24.42 μg/ml in the 4(20)-hour exposure group in the absence of S9, and at and above 48.84 μg/ml in the 4(20)-hour exposure group in the presence of S9 and in the 24-hour continuous exposure group.

Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 97.69 μg/ml in the 4(20)-hour treatment in the absence of metabolic activation (S9) and in the 24-hour continuous exposure group in the absence of S9. The maximum dose with metaphases present in the 4(20)-hour exposure in the presence of S9 was 195.38 μg/ml.

EXPERIMENT 1:
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the test item dose level of 150 μg/ml in both the presence and absence of metabolic activation (S9). The toxicity curve was relatively sharp with no observed metaphases present at the higher dose level of 200 μg/ml in either exposure group. The ‘A’ replicate of the 150 μg/ml dose level in the presence of S9 was lost due to a technical error during the harvesting process and therefore extra scoring was performed on the ‘B’ culture to compensate. This is acceptable under the test guidelines.

The results of the mitotic indices (MI) from the cultures after their respective treatments show 69% and 31% growth inhibition was achieved at 150 μg/ml μg/ml in the absence and presence of S9 respectively.

No precipitate of the test item was observed at the end of the treatment period in either exposure group. Haemolysis was observed at the end of the exposure period in both exposure groups at and above 50 μg/ml.

Thus, the selection of maximum dose level selected for metaphase analysis was 150 μg/ml for both exposure groups and was the highest dose level with metaphases suitable for scoring in both exposure groups.

EXPERIMENT 2:
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test item dose level of 100 μg/ml in both the presence and absence of S9.

The results of the mitotic indices (MI) from the cultures after their respective treatments show a dose related increase in toxicity in the 24-hour exposure group and 36% and 65% mitotic inhibition was achieved at 50 and 100 μg/ml respectively. In the presence of S9 the toxicity curve was very sharp with no toxicity being demonstrated up to the test item dose level of 100 μg/ml and no observed metaphases present at 150 μg/ml. The increase in toxicity in the 4(20)-hour exposure group in the presence of S9 when compared to the same Experiment 1 exposure group is considered to be due to the reduction in the S9 concentration and its protective effects.

No precipitate of the test item was observed at the end of the treatment period in either exposure group. Haemolysis was seen at the end of exposure at and above 50 μg/ml in both exposure groups.

Thus, the maximum dose level selected for metaphase analysis was the highest dose level with metaphases suitable for scoring and was 100 μg/ml for both exposure groups. Although optimum toxicity was not achieved in the 4(20)-hour exposure group in the presence of S9, due to the relative steepness of the toxicity curve, the test item was considered to have been adequately tested.

Experiment 1

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the test item dose level of 150 µg/ml in both the presence and absence of metabolic activation (S9). 

The results of the mitotic indices (MI) from the cultures after their respective treatments show 69% and 31% growth inhibition was achieved at 150 µg/ml in the absence and presence of S9, respectively. 

Thus, the selection of maximum dose level selected for metaphase analysis was 150 µg/ml for both exposure groups and was the highest dose level with metaphases suitable for scoring in both exposure groups. 

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

 

Experiment 2

The results of the mitotic indices (MI) from the cultures after their respective treatments show a dose related increase in toxicity in the 24-hour exposure group and 36% and 65% mitotic inhibition was achieved at 50 and 100 µg/ml respectively. 

Thus, the maximum dose level selected for metaphase analysis was the highest dose level with metaphases suitable for scoring and was 100 µg/ml for both exposure groups. 

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.

The test item did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Conclusions:

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

The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. Therefore the substance was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

The chromosome aberration test for the substance was conducted according to OECD Guideline No. 473 and under GLP conditions. The test substance did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments at test concentrations of either 100 or 200 µg/mL, depending on the test. Consequently, the substance was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 2

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:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

guideline study under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Storage condition of test material: Approximately 4ºC in the dark under nitrogen

Target gene:

Salmonella typhimurium strains: TA1535, TA1537, TA98 and TA100 primarily detected G/C base pair and frameshift mutations. Escherichia coli strain WP2uvrA is primarily detected A/T base pair damage.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 from the livers of SD rats

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, or 1500 µg/plate ( 5000 µg/plate included in the preliminary / dose range-finding test)

Vehicle / solvent:

dimethyl sulphoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without s9

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

2 µg/plate for WP2uvrA- , 3 µg/plate for TA100 and 5 µg/plate for TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without s9

Positive control substance:

9-aminoacridine

Remarks:

80 µg/plate for TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without s9

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2 µg/plate for TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with s9

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

1 µg/plate for TA100; 2 µg/plate for TA1535 and TA1537; 10 µg/plate for WP2uvrA-

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with s9

Positive control substance:

benzo(a)pyrene

Remarks:

5 µg/plate for TA98

Details on test system and experimental conditions:

Incubation period: 24 hrs for preliminary toxicity and 48 hrs for main test at 37ºC

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 24 hours
- Period of pre-culture: 19 hours

NUMBER OF REPLICATIONS: triplicate

Evaluation criteria:

Dose response effect and statistically significant increase were observed at least one strain, results can be reproduced.

Statistics:

Dunnett's method of linear regression

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1 Test Results: Main Test– Without Metabolic Activation
Test Period		From:30 July 2004 To: 02 August 2004
Without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		TA100	TA1535	WP2uvrA-	TA98	TA1537
	0	103	26	29	24	15
		102	32	29	18	15
		101	26	32	29	10
	5	93	35	34	23	7
		94	30	24	22	15
		101	24	21	26	15
	15	118	32	23	30	11
		86	35	22	18	16
		114	23	26	23	16
	50	107	27	31	18	18
		105	27	24	20	12
		106	27	22	22	12
	150	101	22	31	21	9
		112	27	21	24	18
		90	27	27	22	15
	500	123 *	34 *	11	16 *	3 *
		84 *	21 *	20	23 *	4 *
		119 *	22 *	15	17 *	4 *
	1500	0 *	0 *	0 *	0 *	0 *
		0 *	0 *	0 *	0 *	0 *
		0 *	0 *	0 *	0 *	0 *
Positive controls	Name Concentration (µg/plate) No. colonies per plate	ENNG	ENNG	ENNG	4NQO	9AA
		3	5	2	0.2	80
		554	953	1016	195	1161
		590	733	905	192	1391
		552	1063	879	205	1405
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine 4NQO 4-Nitroquinoline-1-oxide 9AA 9-Aminoacridine * Partial or complete absence of bacterial background lawn

Table 2 Test Results: Main Test– With Metabolic Activation
Test Period		From:30 July 2004 To: 02 August 2004
With S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		TA100	TA1535	WP2uvrA-	TA98	TA1537
	0	97	11	25	27	19
		95	13	24	21	20
		100	13	26	25	15
	5	105	20	25	31	10
		110	11	24	23	13
		102	10	17	17	23
	15	83	8	24	20	10
		80	9	21	24	17
		100	16	26	22	16
	50	104	6	27	22	16
		76	6	18	34	12
		83	12	18	17	13
	150	78	7	13	24	15
		90	9	16	20	10
		100	6	15	28	19
	500	57 *	5	18	15	15
		55 *	5	16	21	13
		63 *	6	16	19	24
	1500	0 *	0 *	0 *	0 *	0 *
		0 *	0 *	0 *	0 *	0 *
		0 *	0 *	0 *	0 *	0 *
Positive controls	Name Concentration (µg/plate) No. colonies per plate	2AA	2AA	2AA	BP	2AA
		1	2	10	5	2
		482	266	310	264	119
		807	263	309	237	117
		828	218	278	245	332
BP Benzo(a)pyrene 2AA 2-Aminoanthracene * Partial or complete absence of bacterial background lawn

Conclusions:

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

No significant increase in the frequency of revertant colonies and dose-related effects were recorded for any bacterial strain used at any dose of the test material in two separate experiments either with or without metabolic activation.

Executive summary:

The Ames test was conducted well and in accordance with the OECD Guideline No. 471 under GLP conditions. The substance at test concentrations up to 1,500 µg/plate did not increase the reverse mutation rate when tested in various bacterial strains (Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA100, and Escherichia coli strain WP2uvrA) either in the presence and/or absence of an externally-added metabolic activating system (S9). Consequently, the test substance was considered non-mutagenic under the conditions of this study.

Reference 3

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:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study with no deviations.

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

Target gene:

TK (thymidine kinase) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

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% carbon dioxide 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.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction prepared from the livers of male Sprague Dawley rats induced with phenobarbital and beta-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1 (±S9, 4 hours): 0, 2.5, 5, 10, 20, 40, 60, 80, or 100 µg/mL
Experiment 2 (-S9, 24 hours): 0, 10, 20, 30, 40, 50, 60, 70, or 80 µg/mL
Experiment 2 (+S9, 4 hours): 0, 30, 40, 50, 60, 70, 80, 90, or 100 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Cyclophosphamide was the positive control compound used in the presence of S9 while ethylmethylsulfonate was the positive control compound used in the absence of S9.

Details on test system and experimental conditions:

Preliminary Toxicity Test: Conducted on cell cultures [5x10^5 cells/mL for 4 hours (± S9) and 1.5x10^5 cells/mL for 24 h (-S9)] at concentrations of 3.13-400 µg/mL in order to select dose levels for the Experiments.

Experiment 1: Cell cultures: 1 x 10^6 cells/ml. Treatment vessels were incubated at 37°C for 4 hours (± S9) with continuous shaking using an orbital shaker within an incubated hood.

Experiment 2: Cell cultures: 1 x 10^6 cells/ml (4-hour incubations) and 0.3 x 10^6 cells/ml (24-hour incubations). Treatment vessels were incubated at 37°C with continuous shaking using an orbital shaker within an incubated hood for 24 hours (-S9) and 4 hours (+S9).

At the end of the treatment period, for each experiment, the cells were washed, resuspended in medium, and incubated at 37°C for the expression period of two days.

Evaluation criteria:

The normal range for mutant frequency per survivor is 50-170 x 10^-6 for the TK+/- locus in L5178Y cells at this laboratory. Vehicle control results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200 x 10-6 mutant frequency per survivor are not normally acceptable and will be repeated.

Positive control chemicals should induce at least 3 to 5-fold increases in mutant frequency greater than the corresponding vehicle control.

Statistics:

Not required.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Marked dose-related toxicity as indicated by the RTG and %RSG values at concentrations of 60 µg/mL and over (Experiment 1; -S9), 80 µg/mL and over (Experiment 1, +S9), 30 µg/mL and over (Experiment 2; -S9), and 90 µg/mL (Experiment 2; +S9).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

See tables below (Summary of Results). In addition, there were no unusual findings in the numbers of small and large colonies and their analysis.

Tabulated Summary of Results

[Ø = not plated for viability or 5-TFT resistance; X = treatment excluded from test statistics due to toxicity; CP = cyclophosphamide; EMS=ethylmethanesulphonate; MF§ = 5 -TFT--resistant mutants/10^6 viable cells after 2 days of treatment; %RSG = relative suspension growth; RTC = relative total growth]

Experiment 1

Treatment (µg/ml)			4-Hours-S9					Treatment (µg/ml)			4-Hours+S9
			%RSG	RTG	MF§						%RSG	RTG	MF§
0			100	1.00	134.57			0			100	1.00	130.89
2.5		Ø	97					2.5		Ø	95
5			102	1.01	115.39			5		Ø	98
10			100	1.14	104.85			10			94	0.99	130.05
20			86	0.85	101.63			20			94	0.94	125.07
40			76	0.85	98.67			40			86	0.98	111.55
60			45	0.48	109.39			60			74	0.78	130.80
80		X	3	0.02	111.12			80			48	0.55	119.03
100		Ø	1					100		X	10	0.07	71.82
Linear trend					NS			Linear trend					NS
EMS								CP
400			70	0.46	1122.41			2			66	0.41	1345.65

Experiment 2

Treatment (µg/ml)		24-Hours-S9				Treatment (µg/ml)		4-Hours+S9
		%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	119.82		0		100	1.00	126.77
10		76	0.85	96.20		30	Ø	88
20		53	0.68	117.24		40	Ø	92
30		31	0.45	89.82		50		87	0.94	100.78
40		20	0.37	101.76		60		76	0.76	112.43
50		13	0.21	110.67		70		69	0.76	113.45
60		8	0.15	121.07		80		53	0.52	117.91
70	Ø	5				90		33	0.37	86.06
80	Ø	2				100	X	7	0.06	90.45
Linear trend				NS		Linear trend				NS
EMS						CP
150		45	0.36	1354.61		2		63	0.35	1558.98

 

Conclusions:

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

The test item 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 mouse lymphoma assay.

Executive summary:

The ability of the substance to induce genetic mutations in a mammalian cell assay was evaluated in a mouse lymphoma assay at the TK +/- locus. Test item concentrations were selected based on preliminary tests and the maximum dose levels used in the mutagenicity tests (2 experiments) were limited by test item-induced toxicity. Overall, greasy / oily precipitate of the test item was observed at and above 80 µg/ml in the Mutagenicity Test. The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency confirming the validity of the test and the activity of the metabolising system.

 

The test substance did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, in either of the two separate experiments, in the presence or absence of metabolic activation. It was concluded that the substance did not show potential to induce significant increases in mutation frequency in mammalian cells and is therefore considered to be non-mutagenic under the conditions of the mouse lymphoma test at the TK locus.

Additional information

In the bacterial reverse mutation assay (Safepharm Laboratories Limited, 2004), which was consistent with OECD Test Guideline No. 471 and conducted according to Good Laboratory Practices (GLP), the substance was evaluated in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and in Escherichia coli WP2 uvrA. The substance was evaluated in each bacterial strain at concentrations of 0, 5, 15, 50, 150, 500, or 1,500 µg/plate in both the presence and absence of external metabolic activation (S9). The reported purity of the substance was 99.7%, and dimethylsulfoxide (DMSO) was used as the solvent control. No genotoxicity was observed in any bacterial tester strain incubated with the test substance in the presence or absence of S9. Cytotoxicity was reported the substance at concentrations of 500 µg/plate and higher. Positive control substances evaluated both in the absence and presence of S9 demonstrated the sensitivity of the assay.

 

In the chromosome aberration study, the substance was evaluated in an assay that was conducted in human lymphocytes (Harlan Laboratories Ltd., 2012). The assay was performed according to OECD Test Guideline No. 473 (OECD, 1997b) and was conducted according to GLP. In this study, the test substance was evaluated in 2 individual experiments at concentrations of 0, 12.5, 25, 50, 100, 150, or 200 μg/mL (Experiment 1; ± S9, 4 hours), 0, 3.13, 6.25, 12.5, 25, 50, or 100 µg/mL (Experiment 2; -S9, 24 hours), or 0, 12.5, 25, 50, 100, 150, or 200 µg/mL (Experiment 2; + S9, 4 hours). The purity of the substance was reported to be 99.8% and DMSO was used as the solvent control. No effects on chromosome aberrations were observed at any test substance concentration either in the presence or absence of S9. The positive controls cyclophosphamide (CP) and mitomycin C demonstrated the sensitivity of the test system in the presence and absence of S9, respectively.

 

In the mammalian gene mutation assay, the substance was evaluated in mouse lymphoma L5178Y TK+/-cells (Harlan Laboratories Ltd., 2013). The assay was performed according to OECD Test Guideline No. 476 (OECD, 1997c) and was conducted according to GLP. In this study, the substance was evaluated at concentrations ranging from 2.5 to 100 µg/mL both in the absence and presence of S9. The test material was reported to have a purity of 99.8% and DMSO was used as the solvent control. Cytotoxicity was noted at test substance concentrations of ≥60 µg/mL (Experiment 1; -S9), ≥80 µg/mL (Experiment 1; +S9), ≥30 µg/mL (Experiment 2; -S9), and ≥90 µg/mL (Experiment 2; +S9); however, no significant increase was observed in the mutant frequency at the TK locus either in the presence or absence of S9. The positive controls ethylmethane sulfonate and CP demonstrated the sensitivity of the test system in the absence and presence of S9, respectively. All three of the in vitro genetic toxicity assays have demonstrated that the substance shows no genotoxic potential. As such, the substance is not considered to be a genotoxic compound.

Justification for classification or non-classification

The substance tested uniformly negative in 3 in vitro assays, including a bacterial reverse mutation assay that was consistent with the current OECD Test Guideline 471, a chromosome aberration assay in human lymphocytes that was consistent with the current OECD Test Guideline 473, and a gene mutation assay in mouse lymphoma L5178Y TK+/- cells that was consistent with the current OECD Test Guideline 476. The conclusion is that the substance is not considered to be a genotoxic compound. As a result, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.5.