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Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
483-360-5
EC Name:
-
Cas Number:
114435-02-8
Molecular formula:
C3H3FO3
IUPAC Name:
4-fluoro-1,3-dioxolan-2-one
Test material form:
other: Solid with a low melting point (i.e. liquefied solid)
Details on test material:
Name of the test susbtance: Monofluoroethylene carbonate

Method

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 / strainopen allclose all
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)
Controls
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.

Results and discussion

Test resultsopen allclose all
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.