4-fluoro-1,3-dioxolan-2-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two in vitro studies are available on the substance conducted in accordance with OECD 471 (Ames test) and 473 (chromosome aberration assay) guidelines.

 

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:

From 8 December 2003 to 30 March 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP cmpliance

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Korea National lnstitute of Environmental Research, Test Guidelines of Chemicals, Notification 1998-41, 23 December 1998

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Chromosome aberration (clastogenic potential)

Species / strain / cell type:

mammalian cell line, other: Chinese Hamster Lung cells

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver post-mitochondrial fraction (S9) obtained from Molecular Toxicology lncorporated and prepared from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Dose-levels were:
- 5, 10, 20, 50, 100, 200, 500,1000, 2000 and 5000 µg/mL for the first experiment with and without S9,
- 30, 40, 50, 60, 70 and 80 µg/mL for the confirmation experiment with and without S9.

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

CELLS:
The cells were a line of Chinese Hamster Lung (CHL) cells, obtained from American Type Culture Collection (ATCC). The CHL cell line was selected because it is recommended for the chromosome aberration test by other various regulatory authorities and background data are available. The modal chromosome number is 25 and the average generation time is 15-17 hours.

CULTURE ESTABLISHMENT
CHL cells in logarithmic growth were trypsinized and subcultured at low density into tissue culture flasks. After 2 or 3 days incubation at 37 °C in an atmosphere of 5% (v/v) C02 100% humidity, cultured cells at suitable confluence were selected for treatment.

CULTURE TREATMENT CONDITIONS AND HARVEST TIME
The test system was suitably labeled (using a color-coded procedure) to clearly identify the study number, test article, positive and negative control groups, absence or presence of metabolic activation system, dose levels and treatment conditions. ln each experiment duplicated cultures were employed for test article treatment, also negative (vehicle) and positive control articles were included in duplicate.
A series of more than six dose levels of test article spaced at two-fold intervals were employed in chromosome aberration experiment both in the absence and in the presence of metabolic activation system. Duplicate cultures were treated with the test article, the negative (vehicle) and positive controls both in the absence and in the presence of metabolic activation system. The final concentration of 10% S9 mix in the test system was 10% (v/v). Cultures treated in the absence of S9 mix were received an equal volume of 0.2 M Phosphate buffer. See "Any other information on materials and methods incl. tables" for more details.

PREPARATION OF METAPHASE SPREADS
Six harvesting dose levels were selected from a series of more than six dose levels of test article treatment. Approximately 2 hours prior to harvest, colcemid® was added to give a final concentration of approximately 0.2 μg/mL to arrest dividing cells in metaphase. The monolayer of these cultures were then harvested. The suspension from each flask was transferred to a plastic centrifuge tube and the cells were pelleted by centrifuging at 100 x g for 5 minutes. The supernatant was removed and cells were resuspended in 5 mL (hypotonic) of 0.075 M KCI at 37°C for 15 minutes to allow cell swelling to occur. 1 mL of fresh, ice-cold fixer solution was added for pre-fixation. The cells were pelleted by centrifuging at 200 x g for 5 minutes. Cells were resuspended in 5 mL of fresh, ice-cold fixer solution and the fixative was changed by centrifugation at 200 x g for 5 minutes. This procedure was repeated several times until the supernatants become clean. Cells were kept in fixative in the refrigerator before slides are made, but slides were not be made on the day of harvest in order to ensure that cells were adequately fixed. Cells were pelleted and resuspended in a minimal amount of fresh fixative so as to give a milky suspension. Two drops of suspension were transferred on to clean microscope slides. After the slides had dried the cells were stained for 10 minutes in 3% (v/v) Giemsa in pH 6.8 Sörenson buffer. The slides were then rinsed and dried.

SELECTION OF DOSES FOR CHROMOSOME ANALYSIS
At least 1000 cells were scored from slides prepared from each culture to assess the mitotic indices. The mitotic index (Ml) of every slide scored was taken from the number of mitotic cells seen in a total of at least 1000 cells.
ln the absence and in the presence of metabolic activation system, metaphases were not observed at above 100 μg/mL dose level, so 50 μg/mL dose level was selected for highest dose level.
ln the absence and in the presence of metabolic activation system of confirmation experiment, about 50% reduction in Ml was observed at 70 μg/mL dose level, so 70 µg/ml dose level was selected for highest dose level.
This was the maximum dose and next two lower doses were taken for scoring.

SCORING OF CHROMOSOME ABERRATIONS
Slides from the test article treatments and controls were coded using randomly generated letters by a person not involved with slide scoring. Labels bearing only the study reference number and the assigned code were used to cover treatment details on the slides. 100 metaphases from each coded slide were analyzed for chromosome aberrations. Only cells with the modal number of chromosome 25±2 were considered acceptable for analysis. Cells with greater numbers of chromosomes observed during this evaluation were noted and recorded separately. Observations were recorded on raw data sheets by following criteria with the microscope stage coordinates of any aberrant cell.
- Structural aberration:
* Gap (g)
* Chromatid break ( chtb)
* Chromatid exchange (chte)
* Chromosome break (chrb)
* Chromosome exchange (chre)
* Other
- Numerical aberration:
* Hyperploid
* Polyploid
* Endoreduplication (end)

Evaluation criteria:

TREATMENT OF DATA
After completion of microscopic analysis, data were decoded. The aberrant cells in each culture were categorized as follows:
1) Cells with structural aberrations including gaps
2) Cells with structural aberrations excluding gaps
3) Cells with numerical aberrations
4) Cells with numerical aberrations excluding hyperploid

ACCEPTANCE CRITERIA
The assay was considered valid if the following criteria are met:
1) The proportion of cells with structural aberrations (excluding gaps) in negative control cultures falls within the normal range
2) The proportion of cells with numerical aberrations in negative control cultures falls within the normal range
3) The positive control chemicals induce statistically significant increase in the proportion of cells with structural aberrations
4) At least 160 cells are analyzable at each dose level.

EVALUATION CRITERIA
The test article was considered as positive in this assay if:
1) The assay is valid (see acceptance criteria)
2) Statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurs at one or more dose level
3) 2) exceed the normal range
4) If the result of 2) is reproducible, the test article will be considered as positive on structural aberrations
5) Statistically significant increase in the proportion of cells with numerical aberrations (excluding hyperploid) occurs at one or more dose level
6) 5) exceed the normal range
7) If the result of 5) is reproducible, test article will be considered as positive on numerical aberrations

Statistics:

Normally, the proportion of cells in category 2) and 4) (see "TREATMENT OF DATA") for each treatment condition were compared with the proportion in concurrent negative controls by using Fisher's exact test. Probability values of p ≤ 0.05 were accepted as significant. A further statistical test (for linear trend) used to evaluate possible dose-response relationships.

Key result

Species / strain:

mammalian cell line, other: Chinese Hamster Lung

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ln the chromosome aberration experiment, the dose levels for chromosome analysis were selected 10, 20, 50 μg/mL dose levels in the absence and in the presence of metabolic activation system (-S9 mix 6+18, +S9 mix, 6+18).
Significant increases in the proportion of cells with structural aberrations were observed in cultures at 50 μg/mL dose level in the absence of metabolic activation system (p ≤ 0.05). Significant increases in the proportion of cells with numerical aberrations were not observed in cultures at all dose levels.
Significant increases in the proportion of cells with structural aberrations were observed in cultures at 50 μg/mL dose level in the presence of metabolic activation system (p ≤ 0.05). Significant increases in the proportion of cells with numerical aberrations were not observed in cultures at all dose levels.
Since a positive result was obtained, confirmation experiment was performed under the same treatment condition with modified dose levels. Treatment was applied for 6 hours followed by 18 hours recovery period prior to harvest both in the absence and in the presence of metabolic activation system. The confirmation experiment dose levels for chromosome analysis were selected 50, 60, 70 μg/mL dose levels both in the absence and in the presence of metabolic activation system.
Significant increases in the proportion of cells with structural aberrations were observed in cultures at all dose levels in the absence of metabolic activation system (p ≤ 0.05). Significant increases in the proportion of cells with numerical aberrations were not observed in cultures at all dose levels.
Significant increases in the proportion of cells with structural aberrations were observed in cultures at all dose levels in the presence of metabolic activation system (p ≤ 0.05). Significant increases in the proportion of cells with numerical aberrations were not observed in cultures at all dose levels.

Negative (vehicle) and positive control treatments were included in each treatment condition. The proportion of cells with structural aberrations on the negative control treatments fell within acceptable ranges, while the positive control treatments induced clear increases in the proportion of cells with structural aberrations.

The following table summarizes the mitotic indices for all treatment conditions and selecting dose levels:

Experiment	Treatment condition	Treatment concentration (µg/mL)	Mitotic index (%)
			A	B
Chromosome aberration experiment	- S9 6 + 18	0 5 10 20 50 100 200	8.6 8.5 8.2 6.1 5.3 0.0 0.0	8.3 8.1 8.4 5.9 5.7 0.0 0.0	C NC C C C NC NC
	+ S9 6 + 18	0 5 10 20 50 100 200	8.7 8.5 8.4 6.2 5.7 0.0 0.0	9.0 8.5 8.0 6.9 5.5 0.2 0.0	C NC C C C NC NC
Confirmation experiment	- S9 6 + 18	0 30 40 50 60 70 80	8.9 6.1 5.5 5.8 5.1 3.6 1.8	8.7 6.3 6.0 4.5 4.8 4.5 2.6	C NC NC C C C NC
	+ S9 6 + 18	0 30 40 50 60 70 80	8.8 6.3 5.0 5.8 6.2 3.8 2.0	9.2 6.0 5.1 5.2 5.7 4.9 2.8	C NC NC C C C NC

Table 1: Summary of numbers and types of structural aberrations, - S9 mix, 6 hour treatment 18 hour recovery (6 +18)

Treatment (µg/mL)		Replicate	Cells counted	Cells with strutural aberrations
				chtb	chte	chrb	chre	g	Other	Total(+g)	Total(-g)
- S9 6 + 18	Dimethylsulfoxide	A B	100 100	1 1	0 0	0 0	0 0	2 1	0 0	3 2	1 1
		Total	200	2	0	0	0	3	0	5	2
	10	A B	100 100	0 0	0 0	0 2	0 0	1 2	0 0	1 4	0 2
		Total	200	0	0	2	0	3	0	5	2
	20	A B	100 100	0 2	0 3	0 0	0 0	0 2	0 0	0 7	0 5
		Total	200	2	3	0	0	2	0	7	5
	50	A B	100 100	3 14	12 22	0 2	0 0	3 6	0 0	15 30	14 28
		Total	200	17	34	2	0	9	0	45	42
	MMC 0.1 µg/mL	A B	100 100	5 10	9 11	0 0	0 0	3 2	0 0	14 19	11 17
		Total	200	15	20	0	0	5	0	33	28

Table 2: Summary of numbers and types of structural aberrations, + S9 mix, 6 hour treatment 18 hour recovery (6 +18)

Treatment (µg/mL)		Replicate	Cells counted	Cells with strutural aberrations
				chtb	chte	chrb	chre	g	Other	Total(+g)	Total(-g)
+ S9 6 + 18	Dimethylsulfoxide	A B	100 100	0 2	0 0	0 0	0 0	2 2	0 0	2 4	0 2
		Total	200	2	0	0	0	4	0	6	2
	10	A B	100 100	0 2	0 0	0 0	0 0	1 1	0 0	1 3	0 2
		Total	200	2	0	0	0	2	0	4	2
	20	A B	100 100	0 4	0 2	0 2	0 0	2 1	0 0	2 8	0 7
		Total	200	4	2	2	0	3	0	10	7
	50	A B	100 100	5 3	10 5	0 2	0 0	7 4	0 0	20 12	16 10
		Total	200	8	15	2	0	11	0	32	26
	B(a)P 5 µg/mL	A B	100 100	1 9	15 15	0 0	0 0	2 3	0 0	17 20	15 19
		Total	200	10	30	0	0	5	0	37	34

Table 3: Summary of numbers and types of structural aberrations, - S9 mix, 6 hour treatment 18 hour recovery (6 +18), confirmation experiment

Treatment (µg/mL)		Replicate	Cells counted	Cells with strutural aberrations
				chtb	chte	chrb	chre	g	Other	Total(+g)	Total(-g)
- S9 6 + 18	Dimethylsulfoxide	A B	100 100	0 0	0 0	0 0	0 0	0 1	0 0	0 1	0 0
		Total	200	0	0	0	0	1	0	1	0
	50	A B	100 100	7 14	18 21	0 1	0 0	3 2	0 0	25 31	23 30
		Total	200	21	39	1	0	5	0	56	53
	60	A B	100 100	34 21	32 36	3 2	0 0	7 2	0 0	61 47	59 47
		Total	200	55	68	5	0	9	0	108	106
	70	A B	100 100	35 65	25 67	1 7	0 0	6 12	0 0	59 83	58 83
		Total	200	100	92	8	0	18	0	142	141
	MMC 0.1 µg/mL	A B	100 100	10 3	5 12	0 0	0 0	2 1	0 0	17 15	15 14
		Total	200	13	17	0	0	3	0	32	29

Table 4: Summary of numbers and types of structural aberrations, + S9 mix, 6 hour treatment 18 hour recovery (6 +18), confirmation experiment

Treatment (µg/mL)		Replicate	Cells counted	Cells with strutural aberrations
				chtb	chte	chrb	chre	g	Other	Total(+g)	Total(-g)
+ S9 6 + 18	Dimethylsulfoxide	A B	100 100	0 0	0 0	0 0	0 0	2 1	0 0	2 1	0 0
		Total	200	0	0	0	0	3	0	3	0
	50	A B	100 100	12 9	26 24	2 2	0 0	6 3	0 0	41 27	38 26
		Total	200	21	50	4	0	9	0	68	64
	60	A B	100 100	2 3	6 12	1 2	0 0	4 3	0 0	11 15	9 15
		Total	200	5	18	3	0	7	0	26	24
	70	A B	100 100	25 52	39 53	2 7	0 0	9 10	0 0	63 68	58 68
		Total	200	77	92	9	0	19	0	131	126
	B(a)P 5 µg/mL	A B	100 100	6 10	22 19	0 0	0 0	2 1	0 0	27 21	26 20
		Total	200	16	41	0	0	3	0	48	46

Conclusions:

Based on the results that the treatment of Monofluoroethylene carbonate induced increases in the proportion of cells with structural aberrations both in the absence and in the presence of metabolic activation system.
lt is concluded that Monofluoroethylene carbonate exhibit clastogenic activity in cultured Chinese Hamster Lung cells when tested under the conditions employed for this test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 8 december 2003 to 26 February 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliance

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Kcirea National lnstitute of Environmental Research Test Guidelines of Chemicals, Notification 1998-41 , 23 December 1998

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine dependence, rfa mutation, uvrB mutation, R-factor and spontaneous revertant numbers have been checked for strain genotypes of Salmonella typhimurium. The uvrA mutation and spontaneous revertant numbers have been checked for strain genotypes of Escherichia co/i.

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:

Mammalian liver post-mitochondrial fraction (S9) obtained from Molecular Toxicology lncorporated and prepared from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

0, 33.3, 100, 300, 1000, 3000 and 5000 µg/plate for all mutagenicity experiments with and without S9 mix

Justification of top dose (Result of dose range finding experiment): Significant increases of revertant colonies were observed at 1000 μg/plate dose level in Salmonella typhimurium TA100 in the absence and presence of metabolic activation system.
Bacterial growth inhibition was observed at 1000, 5000 μg/plate dose level in Salmonella typhimurium TA98 and 5000 μg/plate dose level in Salmonella typhimurium TA100, TA1535 and TA 1537 in the absence of metabolic activation system. Therefore, 5000 μg/plate was chosen as the highest dose level for mutation experiment.

Vehicle / solvent:

- Vehicle/solvent used: Dimethylsulfoxide (lot number: 122K0027)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: All experiments were performed according to the preincubation method.

DURATION
- Preincubation period: 0.1 mL of bacterial culture, 0.5 mL of 5% S9 mix or 0.2 M phosphate buffer solution (pH 7.4) and 0.1 mL of test article or controls were placed in tubes. 2 mL of molten top agar at 46°C was added followed by rapid mixing and pouring on to minimal glucose agar plates.
- Exposure duration: 48 hours
- Incubation: 37°C
- Expression time (cells in growth medium): The number of revertants is determined at the end of the exposure time.

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: The revertant colonies per plates are counted

Evaluation criteria:

ACCEPTANCE CRITERIA:
The assay was considered valid if the following criteria were met.
1) The mean negative control counts fell within the normal ranges.
2) The positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active metabolic activation system.
3) The plates from each bacterial strain at least 4 concentrations were scored the colonies.

EVALUATION CRITERIA:
The test article was considered to be mutagenic in this assay if.
1) The assay was valid (see acceptance criteria).
2) The number of revertant colonies increased significantly in one strain at least one or more concentrations or the data set(s) showed a dose related correlation.
3) The mutagenic response in 2) was reproducible.

Statistics:

For evaluation of test article data the m-statistics was calculated to check the data was Poisson-distributed and Dunnett's test was used to compare the counts at each dose level with the control. Probability value of p ≤ 0.01 was accepted as significant.

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

not applicable

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:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Mutation experiment: Significant increases of revertant colonies were observed at 3000 μg/plate dose level in the presence of metabolic activation system in Salmonella typhimurium TA98 and 1000, 3000 μg/plate dose level in the absence of metabolic activation system and 1000, 3000, 5000 μg/plate dose level in the presence of metabolic activation system in Salmonella typhimurium TA100. Bacterial growth inhibition was observed at 5000 μg/plate in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 in the absence of metabolic activation system.

Confirmed experiment: Significant increases of revertant colonies were observed at 3000 μg/plate dose level in the presence of metabolic activation system in Salmonella typhimurium TA98 and 1000, 3000 μg/plate dose level in the absence of metabolic activation system and 300, 1000, 3000, 5000 μg/plate dose level in the presence of metabolic activation system in Salmonella typhimurium TA100.

All mean values of revertant colony counts on negative control treatments fell within acceptable ranges, while positive control treatments induced clear increases in the mean values of revertant colony counts.

Table 1: Summary of revertant colony numbers obtained per plate in the first experiment

Dose (µg/plate)	S9 mix (5%)	Number of revertant colony per plate (mean ± S.D.)
		Salmonella typhimurium				Escherichia coli
		TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
0   33.3 100 300 1000 3000 5000	-   - - - - - -	37± 4   26± 5 31 ± 3 34 ± 5 48 ± 5 15 ± 11 8 ± 3^5	117± 5   113 ± 7 119 ± 14 151 ± 27 257 ± 26^SS 370 ± 21^SS 86 ± 4^1	23± 2   17 ± 5 27 ± 4 22 ± 4 24 ± 5 30 ± 14 7 ± 2^1	12± 3   9 ± 2 12 ± 4 11 ± 5 18 ± 5 17 ± 4 6 ± 5^1	13± 3   8 ± 4 12 ± 2 14 ± 5 18 ± 7 19 ± 4 17 ± 3
0   33.3 100 300 1000 3000 5000	+   + + + + + +	32± 9   28 ± 6 35 ± 3 39 ± 2 46 ± 8 59 ± 7^SS 34 ± 6	106± 15   141 ± 20 119 ± 13 122 ± 8 216 ± 16^SS 435 ± 18^SS 324 ± 70^SS	15± 3   11 ± 4 15 ± 3 16 ± 2 16 ± 2 22 ± 3 19 ± 9	8± 4   10 ± 1 8 ± 2 10 ± 7 16 ± 3 14 ± 2 15 ± 2	14± 2   10 ± 1 12 ± 2 14 ± 5 16 ± 1 19 ± 4 21 ± 7
Positive control	-	253± 11^SS	481± 26^SS	352± 58^SS	341± 19^SS	513± 43^SS
Positive control	+	575± 34^SS	952± 16^SS	261± 14^SS	404± 17^SS	279± 31^SS

^SS: Statistical significance was observed (p ≤ 0.01)

¹: Colony count and slight thin lawn, some very slight toxicity

⁵: Complete killing

Table 2: Summary of revertant colony numbers obtained per plate in the confirmed experiment

Dose (µg/plate)	S9 mix (5%)	Number of revertant colony per plate (mean ± S.D.)
		Salmonella typhimurium
		TA 98	TA 100
0   33.3 100 300 1000 3000 5000	-   - - - - - -	29 ± 5   36 ± 6 36 ± 8 47 ± 2 42 ± 2 15 ± 7 -      ^5	105 ± 1   93 ± 6 104 ± 6 117 ± 9 213 ± 11^SS 368 ± 20^SS -      ^5
0   33.3 100 300 1000 3000 5000	+   + + + + + +	40 ± 5   37 ± 12 40 ± 4 37 ± 4 51 ± 8 76 ± 6^SS 31 ± 7	95 ± 13   139 ± 25 147 ± 8 183 ± 13^SS 198 ± 27^SS 460 ± 57^SS 247 ± 45^SS
Positive control	-	269 ± 24^SS	407 ± 12^SS
Positive control	+	456 ± 35^SS	835 ± 50^SS

^SS: Statistical significance was observed (p ≤ 0.01)

⁵: Complete killing

Conclusions:

From these data, significant increases of revertant colonies were observed in Salmonella typhimurium TA98 in the presence of metabolic activation system and Salmonella typhimurium TA 100 in the absence and presence of metabolic activation system.
lt is concluded that Monofluoroethylene carbonate exhibited mutagenic activity in Salmonella typhimurium TA98, TA 100 under the conditions employed for this test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

An in vivo rat Comet assay conducted in accordance with OECD 489 is available. This was conducted to ascertain whether the positive effect observed in the in vitro Ames assay would be replicated in vivo. An in vivo micronucleus assay is also available, conducted in accordance with OECD 474 guidance and GLP.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 6 July 2007 to 19 October 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliance

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(19 December 2006)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

This strain was used because it is routinely used at the testing facility for this type of studies.

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: young adults
- Weight at study initiation: 27.4 to 28.0 g
- Assigned to test groups randomly: yes. Computer randomization
- Fasting period before study: no
- Housing: The mice were housed with five per cage, under conventional conditions in one room, in sterilised macrolon cages (type III), fitted with a grid cover of stainless steel with a bedding of wood shavings (Lignocel) and shreds of paper as environmental enrichment (Enviro-dri).
- Diet: ad libitum. The animals received a commercial rodent diet (Rat and Mouse No. 3 Breeding Diet, RM3); Batch 5650 (expiry date 8 October 2007); Supplier: SDS Special Diets Services, Witham, England.
- Water: ad libitum. Each cage was supplied with domestic mains tap-water suitable for human consumption. The tap-water was supplied by N.V. Hydron Midden-Nederland and given in polypropylene bottles.
- Acclimation period: yes, 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 40-70%
- Air changes: 10 air changes per hour
- Photoperiod: 12 hours light and 12 hours dark

IN-LIFE DATES: From: 4 July 2007 To: 12 July 2007

Route of administration:

intraperitoneal

Vehicle:

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

Details on exposure:

On day 0 and day 1 (prior to dosing), the test substance was freshly dissolved in corn oil, at concentrations of 10, 5 and 2.5 mg/mL. Prior to each dosing, the animals were weighed and the given dosing volume was 10 mL/kg-bw.

Frequency of treatment:

The test substance was dosed twice intraperitoneally, on two successive days, with an interval of ca. 24 hours.

Post exposure period:

24 hours after treatment

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Negative control (corn oil)

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Treated group

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Treated group

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Treated group

Dose / conc.:

0.75 mg/kg bw/day (nominal)

Remarks:

Mitomycin C (positive control)

No. of animals per sex per dose:

5 females per groups except the group at the dose-level 100 mg/kg bw/day: 7 females (two extra mouse were treated to replace mortality)

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C
The mice were treated once intraperitoneally (0.075 mg/mL; 10 mL/kg) with the mutagen mitomycin C.

Tissues and cell types examined:

From each mouse, the bone marrow cells of both femurs were immediately collected into foetal calf serum and processed into glassdrawn smears according to the method described by Schmid (1976). Two bone marrow smears per animal were prepared, air-dried and fixed in methanol. One smear per animal was stained with a May-Grünwald Giemsa solution. The other fixed but unstained smear was kept in reserve and discarded after analysis of the stained smear.

Details of tissue and slide preparation:

The slides were randomly coded by a person not involved in the scoring of slides. The slides (one slide per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines, taking care that areas selected for evaluation were evenly distributed over the whole smear.The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 200 erythrocytes (E) per animal; if micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 200 E (PE + NE) had been scored, an additional number of PE was scored for the presence of rnicronuclei until a total number of 2000 PE had been scored. Thus the incidence of MPE was recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.

Evaluation criteria:

The study was considered valid because the positive controls give a statistically significant increase in the mean number of MPE/2000 PE and the negative controls were within the historical range.
A test substance is considered to cause chromosomal damage and/or damage to the mitotic apparatus, if the mean number of MPE/2000 PE is statistically significantly higher, when compared to the mean number of the vehicle controls.
A test substance is considered to be negative in the micronucleus test if it produces no positive response at the used dose level.
Both statistical significance and biological relevance are considered together in the evaluation.

Statistics:

The statistical procedures used in the evaluation of data are generally as follows: Data were analysed by Analysis of Variance (ANOVA); if necessary, data were analysed after square root transformation (sqrt(x+ 1)) to 'normalise' the counts (Lovell et al. 1989). If the ANOVA yields significant results, pairwise comparisons between treated and control groups were made.

All statistical tests were performed using BMDP statistical software (W.J. Dixon, BMDP Statistical Software Manual, University of Califomia Press, Berkeley, 1992).

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Cytotoxic to the bone marrow cells

Additional information on results:

STATISTICAL ANALYSIS OF THE TEST RESULTS
* Group 4 (100 mg/kg bw/day) for PE: Statistical analysis (two-way Anova t-test and Dunnett test) of the results indicated there was a marginal statistically significantly (*p<0.05) decrease in the mean number of PE, when compared to the mean number of PE found in the negative control group (group 1; corn oil). This indicates that the test substance reached the bone marrow and induced cytotoxicity to the bone marrow cells. Furthermore, the trend analysis showed a significant linear decrease in the mean numbers of PE.

* Groups 2, 3 and 4 (25, 50 and 100 mg/kg bw/day, respectively) for MPE: Statistical analysis (two-way Anova) of the results indicated there were no statistically significant differences in the mean numbers of MPE, at any of the dose levels used, when compared to the mean number of MPE found in the negative control group (group 1; corn oil). This indicates that treatment with Monofluoroethylene carbonate, up to 100 mg/kg bw/day, did not result in genotoxicity to bone marrow cells.

* Positive control for MPE: Statistical analysis with both Anova (t-test) and the Kruskal-Wallis test indicated there was a statistically significant (***p<0.001) increase in the number of MPE, when compared to the mean number of MPE found in the negative control group (groupl; corn oil). This indicates that the positive control substance reached the bone marrow and induced genotoxicity to the bone marrow cells. The latter results demonstrate the validity of the test system.

CLINICAL SIGNS
* Group 4 (100 mg/kg bw/day) - 4 hours after the first administration: All animals (including reserve animals) showed blepharospasm (bilateral) and lethargy. All animals of treatment group 2 (25 mg/kg bw) and treatment group 3 (50 mg/kg bw), did not show any clinical signs.

* Group 4 (100 mg/kg bw/day) - 4 hours after the second administration: One reserve animal and two animals (31 and 33) were found dead. Two another animals (35 and 39) showed blepharospasm (bilateral), piloerection, hunched posture and lethargy. Another animal (37) showed no clinical signs.

* Groups 2 and 3 (25 and 50 mg/kg bw/day, respectively) - 4 hours after the second administration: All animals showed piloerection.

* Group 3 (50 mg/kg bw/day) - prior to sacrifice: One animal (27) showed blepharospasm (bilateral), piloerection, hunched posture and lethargy.

Table 1: Group numbers (mean ± S.D.) of MPE per 2000 PE, 24 hours after the final administration of Monofluoroethylene carbonate 

Group number:		1: Negative control (corn oil)	Monofluoroethylene carbonate (dose-level/day)			5: Positive control mitomycin C (0.75 mg/kg)
Sex	N		2: 25 mg/kg	3: 50 mg/kg	4: 100 mg/kg
Female	5 4^1)	2.0 ± 1.2	2.2 ± 1.3	2.0 ± 1.6	2.8 ± 0.5	34.6 ± 10.1***

¹⁾ As a result of treatment with the test substance, three out of seven mice died prior to scheduled sacrifice.

N: number of animals per treatment group

*** p<0.001 (statistical analysis: Anova t-test, confirmed by Krushal-Wallis test)

 

Table 2: Group numbers (mean ± S.D.) of PE per 2000 E, 24 hours after the final administration of Monofluoroethylene carbonate

Group number:		1: Negative control (corn oil)	Monofluoroethylene carbonate (dose-level/day)			5: Positive control mitomycin C (0.75 mg/kg)
Sex	N		2: 25 mg/kg	3: 50 mg/kg	4: 100 mg/kg
Female	5 4^1)	107.8 ± 6.83	112.6 ± 14.3	98.6 ± 13.9	84.3 ± 8.1*	91.4 ± 20.5

¹⁾ As a result of treatment with the test substance, three out of seven mice died prior to scheduled sacrifice.

N: number of animals per treatment group

* p<0.05 (statistical analysis: Anova t-test, confirmed by Krushal-Wallis test)

 

Table 3: Individual data of the microscopic evaluation of the bone marrow smears

Group	Mouse	SD	bwd(0)	bwd(1)	PE	NE	MPE	MNE
1 1 1 1 1	1 3 5 7 9	24 24 24 24 24	27.3 28.1 26.3 27.4 29.0	27.7 27.9 27.0 28.1 28.0	109 96 112 113 109	91 104 88 87 91	2 1 4 1 2	0 0 1 0 0
2 2 2 2 2	11 13 15 17 19	24 24 24 24 24	27.1 28.9 27.0 25.1 28.9	26.0 29.6 27.4 26.3 29.1	113 128 111 121 90	87 72 89 79 110	1 4 1 3 2	0 0 0 0 0
3 3 3 3 3	21 23 25 27 29	24 24 24 24 24	30.0 29.7 25.4 28.9 26.1	30.1 29.6 26.8 28.1 26.1	110 106 110 82 85	90 94 90 118 115	3 1 4 0 2	0 0 0 0 0
4 4 4 4 4 4 4	31* 33* R2016 R2017* 35 37 39	-- -- 24 -- 24 24 24	26.3 28.4 31.3 31.7 26.2 28.1 28.4	26.2 27.1 29.3 30.0 26.1 25.8 27.9	-- -- 83 -- 79 96 79	-- -- 117 -- 121 104 121	-- -- 3 -- 3 3 2	-- -- 0 -- 1 0 0
5 5 5 5 5	41 43 45 47 49	24 24 24 24 24	26.0 27.1 28.9 28.3 29.8	25.7 27.3 29.5 28.8 29.4	103 91 88 115 60	97 109 112 85 140	29 40 49 23 32	1 0 0 0 0

Group:

1 = Negative control (corn oil)

2 = Monofluoroethylene carbonate (25 mg/kg bw/day)

3 = Monofluoroethylene carbonate (50 mg/kg bw/day)

4 = Monofluoroethylene carbonate (100 mg/kg bw/day)

5 = Mitomycin C (positive control; 0.75 mg/kg bw)

 

Mouse: mouse number

SD: scheduled dead after all treatments (h)

bwd(0): body weight (g) prior to the first administration

bwd(1): body weight (g) prior to the second administration

PE: number of PE scored per 200 E scored

NE: number of NE scored per 200 E scored

MPE: number of MPE scored per 2000 PE scored

MNE: number of MNE scored per number of NE scored

* As a result of treatment with the test substance, this animal died prior to scheduled sacrifice.

Conclusions:

Under the conditions used in this study it is concluded that, the test substance Monofluoroethylene carbonate was cytotoxic to the bone marrow cells but did not show any indication of chromosomal damage and/or damage to the mitotic apparatus of the bone marrow target cells in female mice, treated intraperitoneally with Monofluoroethylene carbonate, up to 100 mg/kg bw.

Executive summary:

The test substance Monofluoroethylene carbonate was examined for its mutagenic potential in a bone marrow micronucleus test, performed with female mice. The dose levels administered (100, 50 and 25 mg/kg bw/day) were based on treatment related toxicity, observed in a micronucleus test performed with male mice and the same dose levels (information provided by the sponsor).

Animals were treated twice intraperitoneally, on two successive days with an interval of 24 hours, with three graded dose levels of the test substance Monofluoroethylene carbonate. The high dose level group (4), consisted of 7 animals, and each animal received a dose of 100 mg/kg bw/day. The mid dose level group (3), consisted of 5 animals, and each animal received a dose of 50 mg/kg bw/day. The low dose level group (2), consisted of 5 animals, and each animal received a dose of 25 mg/kg bw/day. The vehicle control group (1) consisted of 5 animals, and each animal was dosed in a similar way with the vehicle corn oil. A positive control group (5) consisted of 5 animals, and each animal was given a single intraperitoneal dose of mitomycin C at 0.75 mg/kg bw. At 24 hours after all treatments, all surviving mice were euthanized, bone marrow cells were collected and pooled from both femurs and processed into smears for microscopic examination.

The number of polychromatic erythrocytes (PE) per 200 erythrocytes (E) and the number of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) were counted for each mouse.

The group mean number of polychromatic erythrocytes (PE) per 200 erythrocytes (E) was statistically significantly lower in the animals treated with the highest dose level (100 mg/kg bw/day) of Monofluoroethylene carbonate, compared to the mean number found in the vehicle control animals, treated with corn oil. This result is a confirmation that Monofluoroethylene carbonate reached the target cells in the bone marrow and induced cytotoxicity to the bone marrow cells.

The mean numbers of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) in the animals, treated with dose levels up to 100 mg/kg bw/day, were not statistically significantly higher than the mean number found in the vehicle control animals, treated with corn oil. Therefore, Monofluoroethylene carbonate is not genotoxic to bone marrow cells. For the mice of the positive control group, the mean number of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) was statistically significantly higher than the mean number found in the vehicle control mice. The latter results demonstrate the validity of the test system.

Under the conditions used in this study it is concluded that Monofluoroethylene carbonate was cytotoxic to the bone marrow cells, but did not show any indication of chromosomal damage and/or damage to the mitotic apparatus of the bone marrow target cells in female mice, treated intraperitoneally with Monofluoroethylene carbonate, up to 100 mg/kg bw.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl: WI(Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland
- Age at study initiation: 6 - 10 weeks
- Weight at study initiation: The mean body weights were for males 187.5 ± 8.2 g and the range for males 171 – 203 g.
- Assigned to test groups randomly: The animals were allocated at random to treatment groups.
- Fasting period before study: No
- Housing: Polycarbonate cages (Makrolon MIV type; height 18 cm.)
- Diet (e.g. ad libitum): SM R/M-Z from SSNIFF pellets fed ad libitum except during designated procedures.
- Water (e.g. ad libitum): Municipal tap water freely available to each animal via water bottles.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9 to 20.9°C
- Humidity (%): 47 to 67%
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: Standard vehicle
- Concentration of test material in vehicle: 10, 20 and 40 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/Kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

A solubility test was performed based on visual assessment. The test item was suspended (yellow homogenic suspension) in corn oil. The specific gravity of corn oil is 0.9 g/mL. Test item concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension. Test item concentrations were dosed within 3 hours after preparation. During the dose-range finding phase the test item solidified (melting point is 22°C) and was placed in a waterbath at 30°C to completely liquefy. Thereafter, the test item was a liquid during the course of testing.

Duration of treatment / exposure:

Two doses were administered at 24 intervals

Frequency of treatment:

Twice

Post exposure period:

Approximately 3 to 4 hours after the last dose.

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

MTD based on results from a dose range finder

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males per group

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control substance: Ethyl methanesulfonate
- Justification for choice of positive control(s): Standard as per test guidelines
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg/day

Tissues and cell types examined:

Liver, Glandular stomach and Duodenum were examined. 150 cells were examined per animal.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In the dose-range finding test, male and female animals dosed with 300, 400 and 500 mg test item per kilogram body weight showed treatment related clinical signs or mortality. Based on the results of the dose-range finding study dose levels of 100, 200 and 400 mg/kg body weight were selected as appropriate doses for the main test. There were no sex differences observed in the dose-range finder and accordingly males were use in the main test.

DETAILS OF SLIDE PREPARATION:
Isolation of cells: Approximately 3-4 hours after the last treatment with the test item, vehicle or EMS, Liver, Duodenum and glandular stomach were collected/isolated and examined for DNA damage with the alkaline Comet assay.

Liver:
A portion of 0.6-0.7 gram from the liver was removed and minced thoroughly on aluminium foil in ice. The minced liver tissue was added to 10 mL of collagenase (20 Units/mL) dissolved in HBSS (Ca2+- and Mg2+-free) and incubated in a shaking water bath at 37 °C for 20 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris (40 g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359 g for 10 min). The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+- and Mg2+-free) and kept on ice.

Isolation of glandular stomach cells:
The stomach was cut open and washed free from food using cold Hank’s Balanced Salt Solution (HBSS; Ca++, Mg++ free). The forestomach was removed and discarded. The glandular stomach was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA). The glandular stomach was then transferred to a petri-dish on ice containing 10 mL mincing
buffer incomplete. The surface epithelia of the glandular epithelia were gently scraped with a cell scraper. This layer was discarded since the lifetime of these cells is very short in the body with a maximum of 3 days. Therefore, this layer contains a high amount of apoptotic cells which disturb the interpretation in the Comet assay. Moreover, since the lifetime of these cells is very short it is unlikely that these cells play a role in carcinogenesis. The glandular stomach was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The stomach was then scraped multiple times
with a cell scraper and the cells were collected in the mincing buffer present in the petri-dish. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution, pH 7.5 (DMSO) was added immediately before use). The cell suspension was filtered through a 100 μm Cell Strainer to purify the cell suspension and collected in a tube and stored on ice.

Isolation of duodenum
The duodenum was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA). The duodenum was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The duodenum was cut open and the surface epithelia of the glandular epithelia were gently scraped with a cell scraper to remove apoptotic cells in the upper cell layer. This layer was discarded. The duodenum was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The duodenum was then scraped multiple times with a cell scraper and the cells are collected in the mincing buffer present in the petri-dish. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca++, Mg++ free, and phenol red free if available), pH 7.5 (DMSO was added immediately before use). The cell suspension was filtered through a 100 μm Cell Strainer to purify the cell suspension and collected in a tube and stored on ice.

Preparation of slides:
To the cell suspension, melted low melting point agarose was added (ratio 10:140). The cells were mixed with the LMAgarose and 50 μL was layered on a pre-coated Comet slide (Trevigen) in duplicate. Three slides per tissue per animal were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 15-19 minutes in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area.

Lysis, Electrophoresis and Staining of the Slides:
The cells on the slides were overnight (approximately 16-17 h) immersed in pre-chilled lysis
solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 30 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 0.7 Volt/cm. The electrophoresis was performed for 20 (liver) or 30 minutes (glandular
stomach and duodenum) under constant cooling (actual temperature 4.0°C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5 minutes. The slides were subsequently immersed for 5 minutes in Absolut ethanol (99.6%) and allowed to dry at room temperature. The slides were stained for approximately 10 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark and fixed with a coverslip.

Sampling, fixation and storage of tissue for histotechnology and histopathology:
Part of the liver, stomach and duodenum from the animals (with exception of the positive control) used (after isolation of a part for the comet assay) was collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No histotechnology and histopathology was needed.

Comet scoring:
To prevent bias, slides were randomly coded (per tissue) before examination of the Comets. An adhesive label with study identification number and code were placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system. One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample.

The following criteria for scoring of Comets were used:
• Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
• Cells that showed overlap or were not sharp were not scored.
In addition, the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal in the repeat experiment. The occurrence of hedgehogs was scored in all treatment groups and the control. Since there was no effect of the test item Hedgehogs data was not reported and maintained in the raw data.

Evaluation criteria:

The in vivo comet is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.

b) The positive control EMS should produce at least a statistically significant increase in the percentage Tail Intensity compared to the vehicle treated animals. The response should be compatible with the data in the historical control database. The positive control data was analyzed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances (duodenum and glandular stomach) or by the Welch t test in case of inhomogeneous variances (liver) (one-sided, p < 0.05).

c) Adequate numbers of cells and doses have been analysed

d) The highest test dose is the MTD or 2000 mg/kg/day

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the comet assay data .
A test item is considered positive in the Comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative in the Comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose range-finding study: In the dose-range finding test, male and female animals dosed with 300, 400 and 500 mg test item per kilogram body weight showed treatment related clinical signs or mortality.

Main study:
Mortality and clinical signs:
The animals of the groups treated with 100 mg test item/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality. Positive control animals did not completely eat the food provided. Clinical observations were made in the groups treated with 200 and 400 mg test item /kg body weight. One of the high dose animals (no.18) showed pathology of the stomach during necropsy. One of the animals (no. 20) died just before necropsy and was replaced (no. 23).

Comet slide analysis:
Comet slides were prepared and analyzed. No statistically significant increase in the mean Tail Intensity (%) was observed in Liver, Duodenum and Stomach cells of test item treated male animals compared to the vehicle treated animals.

The mean Tail Intensity in Liver, Duodenum and Stomach cells of vehicle-treated rats was 0.97 ± 0.39% (mean ± SD), 5.59 ± 3.17% (mean ± SD) and 4.42 ± 2.62% (mean ± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control. The positive control EMS induced a significant increase and showed a mean Tail Intensity of 69.06 ± 5.30% (mean ± SD; p<0.001 Students t test) and 51.81 ± 3.26% (mean ± SD; p<0.001 Students t test) and 49.87 ± 3.03% (mean ± SD; p<0.001 Students t test) in male animals in Liver, Duodenum and Stomach cells,
respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database, except in the liver cells. Since the mean Tail Intensity was just below the lower limit of the acceptability criteria range and the positive control EMS induced in Liver cells a significant 71-fold increase, this has no effect on the validity of the results.

Adequate numbers of cells (150 cells per animal) and doses were analyzed and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.

Mean Body Weight Immediately Prior to Dosing

Group	Dose (mg/kg)	Day 1 Bodyweight gram (Mean ± S.D.)	Day 2 Bodyweight gram (Mean ± S.D.)
1	0	192.6 ± 9.3	189.6 ± 9.1
2	100	184.4 ± 9.1	185.0 ± 9.2
3	200	188.0 ± 2.9	190.2 ± 3.3
4	400	181.6 ± 5.1	174.4 ± 6.5
4 Additional	400	192.0 ± 5.6	181.3 ± 7.6
5	200 (EMS)	187.8 ± 11.6	181.0 ± 13.1

 

Overview tail intensity in liver cells in male rats

Dose	Tail intensity (%)	S.D.
Vehicle Control	0.97	0.39
Test item 100 mg/kg	0.77	0.18
Test item 200 mg/kg	1.29	0.56
Test item 400 mg/kg	1.41	0.66
EMS 200 mg/kg	69.06	5.30

 

Overview tail intensity in duodenum cells in male rats

Dose	Tail intensity (%)	S.D.
Vehicle Control	5.59	2.62
Test item 100 mg/kg	6.18	1.71
Test item 200 mg/kg	2.95	3.95
Test item 400 mg/kg	4.53	3.17
EMS 200 mg/kg	51.81	3.03

 

Overview tail intensity in stomach cells in male rats

Dose	Tail intensity (%)	S.D.
Vehicle Control	4.42	2.62
Test item 100 mg/kg	3.84	1.71
Test item 200 mg/kg	5.60	3.95
Test item 400 mg/kg	6.55	3.17
EMS 200 mg/kg	49.87	3.03

 

Historical data Comet assay Negative control

	Duodenum Tail intensity (%) Males and Females	Liver Tail intensity (%) Males and Females	Glandular stomach Tail intensity (%) Males and Females
Mean	4.4	2.6	3.7
SD	1.9	1.8	1.7
N	23	40	26
Lower control limit (95% control limits)	0.6	-0.9	0.3
Upper control limit (95% control limits)	8.2	6.1	7.1

 

Historical data Comet assay (200 mg/kg EMS orally dosed for two consecutive days)

	Duodenum Tail intensity (%) Males and Females	Liver Tail intensity (%) Males and Females	Glandular stomach Tail intensity (%) Males and Females
Mean	45.3	87.6	54.8
SD	11.3	6.7	10.9
N	23	39	26
Lower control limit (95% control limits)	23.1	74.5	33.4
Upper control limit (95% control limits)	67.5	100.8	76.2

Conclusions:

The comet assay is valid and the susbtance is not genotoxic in the Comet assay in Liver, Duodenum and Stomach cells when sampled approximately 3-4 hours post dosing, of male rats that were dosed via oral gavage for two consecutive days
up to a dose of 400 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The available in vitro chromsome aberration assay provided a positive response indicating the substance has the potential to lead to chromosome aberrations. However, an in vivo micronucleus assay is also available which provided a negative response with evidence of bone marrow exposure. As such the substance is not deemed to be clastogenic/aneugenic.

The substance provided a positive response in an Ames test and accordingly a follow up in vivo test (OECD 489; comet assay) was conducted. The results of this were also negative and accordingly the substance is not expected to cause gene mutation in vivo.

Two follow up in vivo studies have been conducted to investigate positive in vitro findings and as all results were negative, the substance is not expected to be a germ cell mutagen. No further testing is deemed necessary.