Oxirane, 2,2'-[methylenebis[(2,6-dimethyl-4,1-phenylene)oxymethylene]]bis-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test, Inai (1994)

Under the conditions of this study, the test material was judged to have reverse mutagenic potential.

Chromosomal Aberration Test with Cultured Mammalian Cells, Ogura (1995)

Under the conditions of the study the test material induced chromosomal aberration in Chinese hamster lung fibroblasts.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 1994 to 28 February 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: The Notification on Partial Revision of Testing Methods Relating to New Chemical Substances

Version / remarks:

Notification No. 700 of Kanpogyo, No.1039 of Yakuhatsu and No.1014 of 61 Kikyoku, December 5, 1986

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: National Institute of Hygienic Sciences. Cells of passage number 18 were supplied.
- Suitability of cells: Chinese hamster lung fibroblasts (CHL cells, clone No. 11) were used in this test because they are widely employed, for in vitro chromosomal aberration tests, show quite high sensitivity to chemical mutagens, and a large amount of data is available about their chromosomal aberrations.
- The modal number of chromosomes is 25 per cell. The time required for doubling of cell number is about 15 hours.
- CHL cells preserved by freezing were defrosted and cultured. Two or three days after incubation, a subculture was made, and the cells in logarithmic growth phase were used for the test.
- The passage number of cells used in the chromosomal aberration test was 40 for 24 hours treatment, 40 for 48 hours treatment by the direct method, and 23 by the metabolic activation method.

MEDIA USED
- Eagle's minimum essential medium was supplemented with new-born calf serum at a rate of 10 v/v %. This medium is referred to hereafter as 10 % NCS/MEM.
- Purified water for preparation of the medium was obtained from a water ultra purifier, through which distilled water prepared with a glass still was passed.

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

24 h treatment by the direct method: 20, 25 and 30 μg/mL
48 h treatment by the direct method: 20, 25 and 30 μg/mL
Metabolic activation method: 200, 215 and 230 μg/mL
The highest concentration decided in the dose determination test was employed as the maximum dose for the chromosomal aberration test. The test was carried out at three dose levels prepared by diluting the maximum dose using a arithmetical progression.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

CELL CULTURE
- Five mL of 10 % NCS/MEM containing 1.5 x 10^4 or 0.5 x 10^4 cells/mL was seeded in a 60 mm diameter Petri dish and cultured for two or three days, respectively. Monolayer cells in logarithmic growth phase were exposed to the test material two or three days after culturing.
- Throughout the study, the incubation of the cells was conducted in a CO2 incubator controlled at 37+0.5°C, 5% of CO2 and nearly 100% relative humidity.

PREPARATION OF THE TEST MATERIAL AND THE POSITIVE CONTROL MATERIAL SOLUTIONS
- Test Material: The test material was dissolved in dimethylsulfoxide. The original solution was diluted with the same solvent to make the required concentrations. Test material solutions were prepared immediately before use and used within 2 hours. To the medium, 1% solution was added.
- Positive Control Material: Positive control materials were dissolved in pure water, and diluted with the same solvent to make the required concentrations. Positive control material solutions were prepared immediately before use and used within 2 hours.

PREPARATION OF MICROSCOPE SLIDES
- Cells were removed from each dish with 0.25% trypsin, and collected by centrifugation in a tube. Then, 0.075 M KCl was added to the tube, and hypotonic treatment was carried out at 37°C for 15 minutes. Methanol-acetic acid (3:1) solution was then poured into the tube to fix the cells and make a slightly cloudy cell suspension, which is then plated onto microscope slides and spread. The cells on each slide were finally dried and stained with 2% Giemsa solution. Two slides per dish were prepared.

DOSE DETERMINATION TEST
- Preliminary tests for cell growth inhibition and cell division inhibition were performed to determine the dose levels for the chromosomal aberration test. In the former test, the 50% growth inhibition concentrations of the test material were determined. In the latter test, the highest concentration at which metaphase cells were practically scorable was determined as the maximum dose for the chromosomal aberration test.
- Cell growth inhibition test: In the test without metabolic activation (direct method), 5 mL of the medium including the test material was added to each dish after removal of the medium from two- or three-day-old cultures, followed by culture for 24 or 48 hours. In the test with metabolic activation, 3 mL of S9 Mix/MEM including the test material was added to each dish after removal of the medium, and treated for 6 hours. After S9 Mix/MEM including the test material had been removed, the dishes were rinsed three times with 2 mL of Ca2+, Mg2+ -free phosphate-buffered saline [PBS(--)]. Then, 5 mL of 10% NCS/MEM was added to each dish, and the cells were cultured for 18 hours. After removal of the medium at the end of incubation, the cells were detached from each dish with 2 mL of 0.25% trypsin, and counted using a Microcell Counter F-500. Two dishes were used for each dose, and the cells were counted in each respective dish.
- Cell division inhibition test: Cell cultures and treatments with the test material for both the direct and metabolic activation methods were done using the same procedure as that for the cell growth inhibition test. Two hours before the end of incubation, colcemid was added to the medium to give a concentration of 0.1 μg/mL. The chromosome preparations were made and observed microscopically for the incidence of mitotic cells.

CHROMOSOMAL ABERRATION TEST
- The highest concentration decided in the dose determination test was employed as the maximum dose for the chromosomal aberration test. The test was carried out at three dose levels prepared by diluting the maximum dose using an arithmetical progression.
- The following concentrations were used in the positive controls, based on background data: Direct method 24 and 48 hour treatment: MMC 0.05 µg/mL. Metabolic activation method: CPA 10 μg/mL.
- Direct Method: After removal of the medium from three-day-old cultures, 5 mL of the medium including the test material was added and incubated for 24 or 48 hours. Two hours before the end of incubation, colcemid was added to the medium to give a concentration of 0.1 μg/mL. Then chromosome preparations were made. A solvent-treated group and a MMC-treated group were used as a negative and a positive control, respectively.
- Metabolic activation method: After removal of the medium from three-day-old cultures, 3 mL of S9 Mix/MEM including the test material was added, followed by incubation for 6 hours. After removal of the S9 Mix/MEM, the dishes were rinsed three times with 2 mL of PBS(-), and 5 mL of 10% NCS/MEM was added. The cells were cultured for 18 hours. Two hours before the end of incubation, colcemid was added to give a concentration of 0.1 μg/mL. Then, chromosome preparations were made. A solvent-treated group and a CPA-treated group were used as a negative and a positive control, respectively. To clarify the effect of metabolic activation, a culture without S9 Mix was treated with the test material at the same dose levels and the same treatment time as those for the control of this test.
- Two dishes were chosen at random to minimise any variation in results, and used for each dose. Preparation was conducted in each respective dish. The test code number, treatment method and dose level were noted on the dishes for identification of each culture.

OBSERVATION AND SCORING OF CHROMOSOMAL ABERRATIONS
- The numbers of cells with chromatid or chromosome-type structural aberrations (such as gaps, breaks, exchanges, etc.) and with numerical aberrations (polyploid, endo-reduplication) were checked by observing 100 metaphases for each dish. The incidences of cells with numerical and/or structural aberrations were obtained by observing 200 metaphases for each group. The incidence of structural aberration was calculated with and without gaps, respectively.
- A gap was scored when a clear discontinuity (larger than a chromatid width) was evident, and when the distal part of the chromatid or chromosome showed no dislocation. Slides were all coded and blind-tested by microscopy.

Evaluation criteria:

EVALUATION OF RESULTS
- The results obtained were assessed as follows. The incidence of cells (mean value for two dishes) with aberrations including gaps was:
less than 5 % = negative ( - )
5% or more and less than 10% = suspect positive (±)
10% or more and less than 20% = positive (+)
20% or more and less than 50% positive (++)
50% or more = positive (+++)
- The test material is judged to be positive for induction of chromosomal aberration when the incidence of 10% or more was dose related or reproducible. If it was judged to be positive, the D20 value, which meant the dose being inducible the chromosomal aberration of 20%, was calculated. Re-examination was performed whenever the result was suspect positive, positive at only one dose level, or not evidently dose-related.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

VALIDITY OF THE TEST
- The values for two dishes were not markedly different, the incidence of cells with any aberration did not exceed 5% in the negative control, the incidence of cells with structural aberrations excluding gap was 10% or more in the positive control. There were no fluctuations in the test conditions and no contamination with microorganisms in the cultures which might have affected the results. The test results were therefore considered to be valid.

CELL GROWTH INHIBITION TEST AND CELL DIVISION INHIBITION
- The growth rate in the solvent control was considered to be 100%. Fifty percent growth inhibition concentrations of the test material were about 28 μg/mL in the 24 hour treatment, about 16 μg/mL in the 48 hour treatment by the direct method, and about 205 μg/mL by the metabolic activation method with S9 Mix. Mitotic metaphases of chromosomes sufficient for assessing chromosomal aberration were observed at doses of 30 μg/mL or less in 24 and 48 hour treatment by the direct method, and 230 μg/mL or less by the metabolic activation method with S9 Mix.
- Cells and their induction of chromosomal aberration were observed microscopically. Based upon the results, the following 3 dose levels were employed in each method of chromosomal aberration test, in order to examine dose-dependent reaction of chromosomal aberration:
24 h treatment by the direct method: 20, 25 and 30 μg/mL
48 h treatment by the direct method: 20, 25 and 30 μg/mL
Metabolic activation method: 200, 215 and 230 μg/mL

CHROMOSOMAL ABERRATION TEST
Direct method
- 24 hour treatment: The incidences of cells with structural aberrations including gap were 1.5% at 20 μg/mL, 9.5% at 25 μg/mL and 18.5% at 30 μg/mL. The incidence in the solvent control was 1.5%, i.e. within the normal range. The positive control treated with MMC showed the structural aberrations of 39.5%. The incidences of polyploid cells at any doses were less than 5%.
- 48 hour treatment: The incidences of cells with structural aberrations including gap were 1.5% at 20 μg/mL, 3.5% at 25 μg/mL, and 3.5% at 30 μg/mL. The incidence in the solvent control was 0.5%, i.e. within the normal range. The positive control treated with MMC showed the structural aberration of 51. 5%. The incidences of polyploid cells at any doses were less than 5%.

Metabolic activation method
- With S9 Mix: The incidences of cells with structural aberrations including gap were 2.5% at 200 μg/mL, 6.5% at 215 μg/mL and 11.0% at 230 μg/mL. The incidence in the solvent control was 1.0%, i.e. within the normal range. The positive control treated with CPA showed the structural aberration of 27.5%. The incidences of polyploid cells at any doses were less than 5%.
- Without S9 Mix: The incidences of cells with structural aberrations including gap at 200, 215 and 230 μg/mL of the test material were unable to be observed due to cytotoxicity. On the other hand, the appearance rates in the solvent and positive control groups were 0 and 1.5%, respectively, which were within a normal range. The incidences of polyploid cells at any doses were less than 5%.

Table 1: Results of Chromosomal Aberration Test (Without Metabolic Activation)

Treatment	Time (h)	Concentration (µg/mL)	Observed	No. of Cells		No. and % of cells showing structural chromosomal aberrations
				No. of Polyploids	Judgement	Gap (g)	Chromatid type		Chromosome type		Others	Total		Evaluation
							ctb	cta	csb	cse		-g	+g
Solvent (DMSO)	24	0	100	1		0	0	0	0	2	0	2	2
			100	2		0	0	0	0	1	0	1	1
			200	3 (1.5)		0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	3 (1.5)	0 (0.0)	3 (1.5)	3 (1.5)
	48	0	100	1		0	0	0	0	0	0	0	0
			100	0		0	0	1	0	0	0	1	1
			200	1 (0.5)		0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	0 (0.0)	(0.0)	1 (0.5)	1 (0.5)
Test Material	24	20	100	0	-	0	1	0	0	0	0	1	1	-
			100	1		0	0	2	0	0	0	2	2
			200	1 (0.5)		0 (0.0)	1 (0.5)	2 (1.0)	0 (0.0)	0 (0.0)	0 (0.0)	3 (1.5)	3 (1.5)
		25	100	0	-	1	4	3	1	2	0	9	10	±
			100	1		3	4	3	1	0	0	6	9
			200	1 (0.5)		4 (2.0)	8 (4.0)	6 (3.0)	2 (1.0)	2 (1.0)	0 (0.0)	15 (7.5)	19 (9.5)
		30	100	0	-	2	7	5	1	0	0	12	14	+
			100	0		3	13	11	0	0	0	22	23
			200	0 (0.0)		5 (2.5)	20 (10.0)	16 (8.0)	1 (0.5)	0 (0.0)	0 (0.0)	34 (17.0)	37 (18.5)
	48	20	100	0	-	0	0	1	0	0	0	1	1	-
			100	0		0	0	1	0	1	0	2	2
			200	0 (0.0)		0 (0.0)	0 (0.0)	2 (1.0)	0 (0.0)	1 (0.5)	0 (0.0)	3 (1.5)	3 (1.5)
		25	100	1	-	1	0	0	0	1	0	1	2	-
			100	0		2	0	2	1	0	0	3	5
			200	1 (0.5)		3 (1.5)	0 (0.0)	2 (1.0)	1 (0.5)	1 (0.5)	0 (0.0)	4 (2.0)	7 (3.5)
		30	100	1	-	1	2	1	0	0	0	2	3	-
			100	0		1	0	2	0	1	0	3	4
			200	1 (0.5)		2 (1.0)	2 (1.0)	3 (1.5)	0 (0.0)	1 (0.5)	0 (0.0)	5 (2.5)	7 (3.5)
Positive Control (MMC)	24	0.05	100	0	-	0	9	33	1	0	0	40	40	++
			100	0		0	5	36	0	0	0	39	39
			200	0 (0.0)		0 (0.0)	14 (7.0)	69 (34.5)	1 (0.5)	0 (0.0)	0 (0.0)	79 (39.5)	79 (39.5)
	48	0.05	100	1	-	3	9	44	0	2	0	50	53	+++
			100	0		3	14	37	0	4	0	49	50
			200	1 (0.5)		6 (3.0)	23 (11.5)	81 (40.5)	0 (0.0)	6 (3.0)	0 (0.0)	99 (49.5)	103 (51.5)

 

 

Table 2: Results of Chromosomal Aberration Test (With Metabolic Activation)

Treatment	S9 Mix	Concentration (µg/mL)	Observed	No. of Cells		No. and % of cells showing structural chromosomal aberrations
				No. of Polyploids	Judgement	Gap (g)	Chromatid type		Chromosome type		Others	Total		Evaluation
							ctb	cta	csb	cse		-g	+g
Solvent (DMSO)	-	0	99	1		0	0	0	0	0	0	0	0
			100	0		0	0	0	0	0	0	0	0
			199	1 (0.5)		0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
	+	0	100	2		0	0	0	0	0	0	0	0
			100	0		1	0	1	0	0	0	1	2
			200	2 (1.0)		1 (0.5)	0 (0.0)	1 (0.5)	0 (0.0)	0 (0.0)	(0.0)	1 (0.5)	2 (1.0)
Test Material	-	200	Mitotic metaphases were not obtained because of cytotoxicity
		215	Mitotic metaphases were not obtained because of cytotoxicity
		230	Mitotic metaphases were not obtained because of cytotoxicity
	+	200*	100	3	-	2	1	0	0	0	0	1	3	-
			100	2		0	1	0	0	1	0	2	2
			200	5 (2.5)		2 (1.0)	2 (1.0)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	3 (1.5)	5 (2.5)
		215*	100	1	-	0	3	1	0	0	0	4	4	±
			100	3		0	4	6	0	1	0	9	9
			200	4 (2.0		0 (0.0)	7 (3.5)	7 (3.5)	0 (0.0)	1 (0.5)	0 (0.0)	13 (6.5)	13 (6.5)
		230*	100	1	-	4	1	13	0	0	0	13	14	+
			100	0		1	0	6	0	1	0	7	8
			200	1 (0.5)		5 (2.5)	1 (0.5)	19 (9.5)	0 (0.0)	1 (0.5)	0 (0.0)	20 (10.0)	22 (11.0)
Positive Control (CPA)	-	10	100	1	-	0	0	0	0	3	0	3	3	-
			100	0		0	0	0	0	0	0	0	0
			200	1 (0.5)		0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	3 (1.5)	0 (0.0)	3 (1.5)	3 (1.5)
	+	10	100	1	-	0	11	19	2	1	0	30	30	++
			100	1		0	2	24	0	0	0	25	25
			200	2 (1.0)		0 (0.0)	13 (6.5)	43 (21.5)	2 (1.0)	1 (0.5)	0 (0.0)	55 (27.5)	55 (27.5)

* Precipitation of the test material was observed in culture medium.

Conclusions:

Under the conditions of this study the test material induced chromosomal aberrations in Chinese hamster lung fibroblasts.

Executive summary:

The genetic toxicity of the test material was investigated in a chromosomal aberration test using Chinese hamster lung fibroblasts (CHL cells). The test was performed in accordance with ‘The Notification on Partial Revision of Testing Methods Relating to New Chemical Substances’ (Notification No. 700 of Kanpogyo, No.1039 ofYakuhatsu and No.1014 of61 Kikyoku, December 5, 1986). The testing was performed under GLP conditions.

Mitomycin C (MMC) and cyclophosphamide (CPA) were employed as the positive controls for the tests without and with metabolic activation, respectively. Metabolic activation was provided in the form of S9-mix.

A cell growth inhibition test and cell division inhibition test were carried out to determine the appropriate dose levels of the test material. From the results of these tests, chromosomal aberration tests were carried out using 20, 25 and 30 µg/mL of the test material for 24 and 48 hour treatment by the direct method (without metabolic activation), and 200, 215 and 230 µg/mL by the metabolic activation method. As the positive control, MMC was employed at 0.05 µg/mL for 24 and 48 hour treatment by the direct method, and CPA at 10 µg/mL by the metabolic activation method.

The test material induced structural aberrations dose-dependently within the dose range of 25 - 30 μg/mL in the 24 hour treatment, and within the dose range of 215 - 230 μg/mL by the metabolic activation method. D20 value of 24 hour treatment group in the direct method and with S9 Mix treatment group by the metabolic activation method were 34, and 280 μg/mL, respectively. MMC and CPA induced evident chromosomal aberrations.

Under the conditions of this study the test material induced chromosomal aberration in Chinese hamster lung fibroblasts.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 June 1994 to 07 July 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Standards for Toxicity Investigations

Version / remarks:

Japan's ML, No.77, September 1, 1988

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Notification on Partial Revision of Testing Methods Relating to New Chemical Substances

Version / remarks:

Notification No.700 of Kanpogyo, No.1039 of Yakuhatsu and No.1014 of 61 Kikyoku, December 5, 1986).

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- The test material was dissolved in DMSO to make 5 w/v % concentration and diluted with the same solvent to give lower concentrations.

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Salmonella typhimurium TA100, TA98, TA1535 and TA1537 obtained from Dr. B.N. Ames, University of California, U.S.A. , on June 20, 1990.
- The test strains were stored as frozen stock cultures (0.045 mL of DMSO/ 0.5 mL of broth culture) at -80 °C.
- The amino acid requirement for growth was demonstrated by using histidine for S. typhymurium strains. The presence of R-factor, membrane mutation and mutation on the ability to repair DNA lesions were confirmed by ampicillin resistance, sensitivity to crystal violet and UV sensitivity, respectively.
- From the stock cultures, 20 μL of bacterial suspension was inoculated to L-tube containing 10 mL nutrient broth No.2, the bacterial culture was incubated at 37 ± 0.5 °C for 8 h with shaking at 50 times/min by the Monad shaker. The viable cell counts calculated from the values determined by spectrophotometry at 660 nm at the end of incubation.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Escherichia coli WP2 uvrA obtained from Dr. T.Matsushima, Institute of Medical Science, University of Tokyo, on June 13, 1987.
- The test strains were stored as frozen stock cultures (0.045 mL of DMSO/ 0.5 mL of broth culture) at -80 °C.
- The amino acid requirement for growth was demonstrated by using tryptophan for E. coli strain. The presence of R-factor, membrane mutation and mutation on the ability to repair DNA lesions were confirmed by ampicillin resistance, sensitivity to crystal violet and UV sensitivity, respectively.
- From the stock cultures, 20 μL of bacterial suspension was inoculated to L-tube containing 10 mL nutrient broth No.2, the bacterial culture was incubated at 37 ± 0.5 °C for 8 h with shaking at 50 times/min by the Monad shaker. The viable cell counts calculated from the values determined by spectrophotometry at 660 nm at the end of incubation.

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

- Dose range-finding test: 5000, 2000, 1000, 500, 200, 100 and 50 μg/plate.
- Main test: 5000, 2500, 1250, 625, and 313 µg/plate (without S9 mix) and 5000, 2500, 1250, 625, 313 and 156 µg/plate (with S9 mix).
- Based on the results of the dose range finding test, the main test was performed at the highest dose of 5,000 μg/plate and 4 or 5 more doses were set by dilution with a geometric progression of 2.

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:

sodium azide

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino] acridine·2HCl (ICR-191) and 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
- The test was carried out using the pre-incubation method both with and without metabolic activation system, represented S9 Mix(+) and S9 Mix(-), respectively.
- Three plates were used for the negative control, and two plates for the test material and positive controls.

PROCEDURES
- After 0.1 mL of the test material solution, 0.5 mL of 0.1 M sodium phosphate buffer (pH 7.4) or S9 Mix, and 0.1 ml of the bacterial culture was added to a test tube, the mixture was incubated for 20 min at 37 ± 0.5 °C. Two mL of the soft agar was added to each test tube and poured onto a minimal glucose agar plate.
- After incubation for 48 hours at 37 ± 0.5 °C, the number of revertant colonies were counted.
- For the sterility test, 0.1 mL of the test material solution, the S9 Mix and 0.1 M sodium phosphate buffer (pH 7.4) were smeared on a minimal glucose agar plate and incubated at 37 ± 0.5 °C for 48 hours.

MICROSCOPIC OBSERVATION AND COLONY COUNTING
- Microscopic observation: The state of revertant colonies (size and number of colonies), deposition of the test material and the growth inhibition were examined with a stereo microscope.
- Colony counting: The number of colonies was counted using the colony analyser three times per plate with correction for number, and a mean value per plate was calculated. A mean value on each dose was calculated from each individual mean value of plates set with rounding off a decimal point. When precipitation of the test material or inhibition of growth might disturb measurement by the colony analyser or the number of revertant colonies was easily counted, the number of colonies was counted by a manual counter.

Evaluation criteria:

INTERPRETATION OF RESULTS
- The test material was judged to be positive, when the number of revertant colonies increased more than twice that of the negative control in a dose dependent manner, and the reproducibility was also observed.
- In the case of a positive result, specific activities (number of revertant colonies/mg) were calculated in the dose levels in which the number of revertant colonies was twice or more than that in the negative control, and the highest value was designated as the specific activity of the test material:

Specific activity = ( M – M0) / D
M = the number of revertant colonies for the test material
M0 = the number of revertant colonies for the negative control
D = dose (mg/plate)

Statistics:

Any statistical procedure was not adopted.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 1537 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- In the dose range finding test, the number of revertant colonies for TA1535 with S9 Mix, was more than twice that of the negative control.
- In the main test, the number of revertant colonies for TA1535 with and without S9 Mix and TA100 with S9 Mix, were more than twice that of the negative control.
- White needle-like deposition of the test material without S9 Mix was observed at more than 1,000 μg/plate.
- The positive control substances significantly induced the revertant colonies, and the number of revertant colonies was, together with that of the negative control, within a range of the background data in the laboratory.
- There were no fluctuations which affected the test results, since no contamination of micro-organisms was confirmed in the sterility test.

Table 1: Summary of Main Test

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 313 625 1250 2500 5000	98 116 111 129 148 141	8 9 10 4 10 19	42 47 57 48 48 50	18 23 19 19 15 27	8 7 7 7 7 8
+	Solvent 156 313 625 1250 2500 5000	120 137 134 137 242 210 150	8 11 13 32 92 95 96	62 - 66 76 75 68 66	28 - 26 29 23 31 18	11 - 12 11 10 11 9
Positive Controls
-	Name	AF-2	SA	AF-2	AF-2	ICR-191
	Concentration (µg/plate)	0.01	0.5	0.01	0.1	1
	Mean no. colonies/plate	349	240	571	445	1824
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	1	2	10	0.5	2
	Mean no. colonies/plate	530	160	438	140	112

2AA = 2-aminoanthracene

SA = Sodium azide

AF-2 =2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

ICR-191 = 2-Methoxy-6-chloro-9-(3-(2-chloroethyl)-aminopropylamino) acridine·2HCl

Conclusions:

Under the conditions of this study, the test material was judged to have reverse mutagenic potential.

Executive summary:

The genetic toxicity of the test material was investigated in a reverse mutation test, performed under GLP conditions.

The reverse mutation test was performed on Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and Escherichia coli strain WP2 uvrA using the pre-incubation method with and without metabolic activation system. Metabolic activation was provided in the form of S9 Mix.

In the dose range finding test, the number of revertant colonies for TA1535 with S9 Mix, was more than twice that of the negative control. In the main test, the number of revertant colonies for TA1535 with and without S9 Mix and TA100 with S9 Mix, were more than twice that of the negative control.

The negative control and positive controls were within background data for the laboratory.

Under the conditions of this study, the test material was judged to have reverse mutagenic potential.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Mouse Micronucleus Study, Durward (2002)

Under the conditions of the study the test material was considered to be non-genotoxic.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 July 2002 to 19 July 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: USA EPA, TSCA and FIFRA guidelines

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese METl/MHLW guidelines

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vivo mouse micronucleus

Species:

mouse

Strain:

CD-1

Remarks:

Crl:CD-1™(ICR)BR

Details on species / strain selection:

Using existing toxicity data there was no marked difference in test material toxicity between the sexes, therefore the study was performed using only male mice.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: five to eight weeks old
- Weight at study initiation: 25 to 30 g
- Assigned to test groups randomly: the animals were selected at random and given a number unique within the study by ear punching and a number written on a colour coded cage card
- Housing: The animals were housed in groups of up to seven in solid-floor polypropylene cages with wood-flake bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: a minimum of seven days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25°C
- Humidity: 30 - 70%
- Air changes: approximately fifteen changes per hour
- Photoperiod: the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: arachis oil

Duration of treatment / exposure:

A single dose administration

Frequency of treatment:

Once

Post exposure period:

One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with 2000 mg/kg was killed after 48 hours.

Dose / conc.:

2 000 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

500 mg/kg bw (total dose)

No. of animals per sex per dose:

Seven maless per dose. An additional seven males were treated at 2000 mg/kg for termination after 48 hours.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Cyclophosphamide
- For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water.

Tissues and cell types examined:

Slides were prepared from the bone marrow of the femora and stained. The numbers of micronucleated polychromatic erythrocytes and normochromatic erythrocytes were recorded.

Details of tissue and slide preparation:

RANGE-FINDING STUDY
- A range-finding toxicity study was performed to determine a suitable dose level and route of administration for the micronucleus study. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg. Due to limitations of the test material formulation it was not possible to achieve 2000 mg/kg via the intraperitoneal route. Using existing toxicology data it was considered to be acceptable to only use male animals in the study.
- Two males were dosed at 2000 mg/kg (oral) and two males were dosed at 1000 mg/kg (i.p.). All animals were dosed once only at the appropriate dose level by gavage using a metal cannula or with a hypodermic needle attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of dosing.
- Animals were observed one hour after dosing and subsequently once daily for two days. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.

MICRONUCLEUS STUDY
- Groups, each of seven mice, were dosed once only via the oral route with the test material at 500, 1000 or 2000 mg/kg. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with 2000 mg/kg was killed after 48 hours. In addition, three further groups of mice were included in the study; two groups (seven mice) were dosed via the oral route with the vehicle alone (arachis oil) and a third group (five mice) was dosed orally with cyclophosphamide, a positive control material known to produce micronuclei under the conditions of the test. The vehicle controls were killed 24 or 48 hours following dosing and positive control group animals were killed 24 hours following dosing.
- All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.

SLIDE PREPARATION
- Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grünwald/Giemsa, allowed to air-dry and cover-slipped using mounting medium.

SLIDE EVALUATION
- Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes were counted; these cells were also scored for incidence of micronuclei.
- The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Evaluation criteria:

INTERPRETATION OF RESULTS
- A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.
- A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group.
- If these criteria were not demonstrated, then the test material was considered to be non-genotoxic under the conditions of the test.
- A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.
- All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a √(x + 1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING TOXICITY STUDY
- In animals dosed with test material via the intraperitoneal route there were no premature deaths but clinical signs were observed at 1000 mg/kg as follows: hunched posture and lethargy.
- In animals dosed with the test material via the oral route there were no premature deaths, however, clinical signs were observed at 2000 mg/kg as follows: hunched posture.
- There was little difference between the dose routes in the toxic response and the test material suspension was more suited to dosing via the oral route. Results from the 28-day oral toxicity study indicated clinical effects were induced following oral administration. It was, therefore, considered that adequate exposure would be achieved via the oral route. Therefore, the oral route was selected for use in the main study. The maximum recommended dose (MRD) of the test material, 2000 mg/kg, was selected for use in the main study, with 1000 and 500 mg/kg as the lower dose levels.

MICRONUCLEUS STUDY
Mortality Data and Clinical Observations
- There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at 2000 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: hunched posture.

Evaluation of Bone Marrow Slides
- There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hour test material groups when compared to their concurrent vehicle control groups.
- There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.
- The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.
- The test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Conclusions:

Under the conditions of this study the test material was considered to be non-genotoxic.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 474, EU Method B.12., the USA EPA, TSCA and FIFRA guidelines and the Japanese METl/MHLW guidelines for testing of new chemical substances. The method was designed to comply with the UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report, Part 1 revised (Basic Mutagenicity Tests: UKEMS recommended procedures, 1990). The testing was conducted under GLP conditions.

A range-finding study was performed to find suitable dose levels of the test material and route of administration. Using existing toxicity data there was no marked difference in test material toxicity between the sexes, therefore the study was performed using only male mice. The micronucleus study was conducted using the oral route in groups of seven mice at the maximum recommended dose (2000 mg/kg) with 1000 and 500 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Further groups of mice were given a single oral dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.

Under the conditions of the study no statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups.

The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes.

The test material was therefore considered to be non-genotoxic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

IN VITRO

Ames Test, Inai (1994)

The genetic toxicity of the test material was investigated in a reverse mutation test, performed under GLP conditions. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The reverse mutation test was performed on Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and Escherichia coli strain WP2 uvrA using the pre-incubation method with and without metabolic activation system. Metabolic activation was provided in the form of S9 Mix.

In the dose range finding test, the number of revertant colonies for TA1535 with S9 Mix, was more than twice that of the negative control. In the main test, the number of revertant colonies for TA1535 with and without S9 Mix and TA100 with S9 Mix, were more than twice that of the negative control.

The negative control and positive controls were within background data for the laboratory.

Under the conditions of this study, the test material was judged to have reverse mutagenic potential.

Chromosomal Aberration Test with Cultured Mammalian Cells, Ogura (1995)

The genetic toxicity of the test material was investigated in a chromosomal aberration test using Chinese hamster lung fibroblasts (CHL cells). The test was performed in accordance with ‘The Notification on Partial Revision of Testing Methods Relating to New Chemical Substances’ (Notification No. 700 of Kanpogyo, No.1039 ofYakuhatsu and No.1014 of61 Kikyoku, December 5, 1986). The testing was performed under GLP conditions. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Mitomycin C (MMC) and cyclophosphamide (CPA) were employed as the positive controls for the tests without and with metabolic activation, respectively. Metabolic activation was provided in the form of S9-mix.

A cell growth inhibition test and cell division inhibition test were carried out to determine the appropriate dose levels of the test material. From the results of these tests, chromosomal aberration tests were carried out using 20, 25 and 30 µg/mL of the test material for 24 and 48 hour treatment by the direct method (without metabolic activation), and 200, 215 and 230 µg/mL by the metabolic activation method. As the positive control, MMC was employed at 0.05 µg/mL for 24 and 48 hour treatment by the direct method, and CPA at 10 µg/mL by the metabolic activation method.

The test material induced structural aberrations dose-dependently within the dose range of 25 - 30 μg/mL in the 24 hour treatment, and within the dose range of 215 - 230 μg/mL by the metabolic activation method. D20 value of 24 hour treatment group in the direct method and with S9 Mix treatment group by the metabolic activation method were 34, and 280 μg/mL, respectively. MMC and CPA induced evident chromosomal aberrations.

Under the conditions of the study the test material induced chromosomal aberration in Chinese hamster lung fibroblasts.

IN VIVO

Mouse Micronucleus Study, Durward (2002)

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 474, EU Method B.12., the USA EPA, TSCA and FIFRA guidelines and the Japanese METl/MHLW guidelines for testing of new chemical substances. The method was designed to comply with the UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report, Part 1 revised (Basic Mutagenicity Tests: UKEMS recommended procedures, 1990). The testing was conducted under GLP conditions. The testing was conducted under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

A range-finding study was performed to find suitable dose levels of the test material and route of administration. Using existing toxicity data there was no marked difference in test material toxicity between the sexes, therefore the study was performed using only male mice. The micronucleus study was conducted using the oral route in groups of seven mice at the maximum recommended dose (2000 mg/kg) with 1000 and 500 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Further groups of mice were given a single oral dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.

Under the conditions of the study no statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups.

The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes.

The test material was therefore considered to be non-genotoxic.

Justification for classification or non-classification

In consideration of the positive result obtained in the in vitro reverse mutation and chromosome aberation tests and bearing in mind the substance is a reactive di-epoxide the substance requires classification with respect to genetic toxicity (category 2) and is assigned the hazard statement H341: Suspected of causing genetic defects (ref Regulation (EC) No 1272/2008, Table 3.5.1)