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Administrative data

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

Referenceopen allclose all

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.
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, other: TA 1535, 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
Key result
Species / strain:
S. typhimurium, other: TA 1535, 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:
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 ± 35

117± 5

 

113 ± 7

119 ± 14

151 ± 27

257 ± 26SS

370 ± 21SS

86 ± 41

23± 2

 

17 ± 5

27 ± 4

22 ± 4

24 ± 5

30 ± 14

7 ± 21

12± 3

 

9 ± 2

12 ± 4

11 ± 5

18 ± 5

17 ± 4

6 ± 51

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 ± 7SS

34 ± 6

106± 15

 

141 ± 20

119 ± 13

122 ± 8

216 ± 16SS

435 ± 18SS

324 ± 70SS

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± 11SS

481± 26SS

352± 58SS

341± 19SS

513± 43SS

Positive control

+

575± 34SS

952± 16SS

261± 14SS

404± 17SS

279± 31SS

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 ± 11SS

368 ± 20SS

-      5

0

 

33.3

100

300

1000

3000

5000

+

 

+

+

+

+

+

+

40 ± 5

 

37 ± 12

40 ± 4

37 ± 4

51 ± 8

76 ± 6SS

31 ± 7

95 ± 13

 

139 ± 25

147 ± 8

183 ± 13SS

198 ± 27SS

460 ± 57SS

247 ± 45SS

Positive control

-

269 ± 24SS

407 ± 12SS

Positive control

+

456 ± 35SS

835 ± 50SS

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:
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.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

An in vivo micronucleus assay is available conducted in accordance with OECD 474 guidance and GLP.

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:
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

41)

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

41)

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

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) has been proposed to assess this finding in vivo. Once the results are available, the considerations for classification and labelling of the substance will be reviewed.