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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: - Guideline study (OECD, etc.) - number of replicates to low for some concentrations

Data source

Reference
Reference Type:
publication
Title:
Mutagenicity of carbon tetrachloride and chloroform in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA/pKM101 and WP2/pKM101, using a gas exposure method.
Author:
Araki A, Kamigaito N, Sasaki T, Matsushima T,
Year:
2004
Bibliographic source:
Environmental and molecular mutagenesis, vol. 43, no 2, p. 128-133

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
: gas exposure method
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): carbon tetrachloride
- Physical state: liquid
- Analytical purity: sample No 1: 99.5 %, sample no. 2: 99.9 %
- Supplier: Wako Pure Chemicals Industries
- Lot/batch No.: sample No 1: APE7331, sample no. 2: KSG7670
- Stability under test conditions: not reported, expected to be stable
- Storage condition of test material: not reported

Method

Target gene:
no data
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 pKM 101
Species / strain / cell type:
E. coli WP2
Additional strain / cell type characteristics:
other: carrying the plasmid pKM101
Metabolic activation:
with and without
Metabolic activation system:
S9 extract from phenobarbitone + 5,6-benzoflavone treated male Sprague-Dawley rats
Test concentrations with justification for top dose:
100, 500, 1000, 2000, 5000, 10'000, 20'000, 50'000 ppm (= 640, 3200, 6400, 12'800, 32'000, 64'000, 128'000, 320'000 mg/m³)
assuming 500 mL air volume/plate: 0.8 - 160 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none
Controls
Untreated negative controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamidMe, MMC: mitomycin C, 2AA: 2-aminoanthracen, see table 1 for details
Remarks:
with the gas exposure, sham exposed controls are both negative and true negative controls
Details on test system and experimental conditions:
METHOD OF APPLICATION: gas exposure method


DURATION
- Preincubation period: 0 min, no preincubation
- Exposure duration: 24 h
- Expression time (cells in growth medium): 0 h
- Selection time (if incubation with a selection agent): 24 h (+24 h during exposure)



SELECTION AGENT (mutation assays): Salmonella strains: L-histidine, Ecoli strains: L-trytophane




DETERMINATION OF CYTOTOXICITY
- Method: not reported

Evaluation criteria:
- a factor of 2 between the average no of revertants on exposed plates compared to the everage no of revertants on unexposed plates
Statistics:
- Bartlett's test for evaluation of homogenity of variance,
- one way ANOVA in the case of variance homogenity
- Kruskal-Wallis- rank sum test for non homogenous variance

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
only at concentrations > 5000 ppm
Cytotoxicity / choice of top concentrations:
other: only at the highest concentration
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
only at concentrations > 5000 ppm
Cytotoxicity / choice of top concentrations:
other: only at the highest concentration
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
without activation: only at concentrations > 500 ppm, with activation: only at concentrations > 2000 ppm
Cytotoxicity / choice of top concentrations:
other: only at the highest concentration
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not expected as CTC is apolarm and stable
- Effects of osmolality: not expected as CTC is apolarm and stable
- Evaporation from medium: not applicable due to gas exposure method
- Water solubility: not applicable due to gas exposure method
- Precipitation: not applicable due to gas exposure method
- Other confounding effects: not expected


RANGE-FINDING/SCREENING STUDIES: No


COMPARISON WITH HISTORICAL CONTROL DATA: No


ADDITIONAL INFORMATION ON CYTOTOXICITY:
The maximum exposure concentration was 2% or 5%; carbon tetrachloride showed toxicity for all strains in tests conducted with 5% of the agents

see tables I - II for detailed results
Remarks on result:
other: strain/cell type: TA 98
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

- Table Ia Mutagenicity Testing of Carbon Tetrachloride in S.typhimurium TA98

TA98

Lot. Nr. APE7331

Lot No. KSG7670

Dose %

Test 1 (Mean)

Test 2 (Mean)

Test 3 (Mean

Test 1-3 Mean + SD

Negative control (Air)

9

16

13

18

8

15

13

16

(14)

17

13

(15)

9

13

(11)

13+3.3

Without S 9 mix

0.005

NT

NT

13

23

(18)

NT

NT

18a

0.01

NT

NT

16

20

(18)

NT

NT

18a

0.05

NT

NT

28

16

(22)

11

16

(14)

18+7.2

0.1

13

20

(17)

22

23

(23)

11

13

(12)

17+5.3

0.2

NT

NT

NT

NT

15

17

(16)

16a

0.5

29

20

(25)

28

31

(30)

10

22

(16)

23+7.8*

1

40

24

(32)

21

21

(21)

26

21

(24)

26+7.4**

2

NT

NT

NT

NT

32

36

(34)

34a

5

36

38

(37)

34

30

(32)

31

17

(24)

31+7.5

AF2 0.1 μg/plate

719

680

(700)

627

614

(621)

584

596

(590)

AF2 0.005 μg/plate

TA98

Lot. Nr. APE7331

Lot No. KSG7670

Dose %

Test 1 (Mean)

Test 2 (Mean)

Test 3 (Mean

Test 1-3 Mean + SD

Negative control (Air)

22

28

26

39

14

28

24

26

(25)

36

32

(33)

24

23

(22)

27+6.6

With S 9 mix

0.005

NT

NT

37

29

(33)

NT

NT

33a

0.01

NT

NT

32

26

(29)

NT

NT

29a

0.05

NT

NT

22

32

(27)

11

23

(17)

22+8.6

0.1

29

30

(30)

37

22

(30)

29

23

(26)

28+5.4

0.2

NT

NT

NT

NT

21

26

(24)

24a

0.5

25

26

(26)

25

28

(25)

28

28

(28)

26+1.5

1

33

36

(35)

31

38

(35)

31

24

(28)

32+4.9

2

NT

NT

NT

NT

36

32

(34)

34a

5

52

38

(45)

30

36

(33)

24

28

(26)

35+9.9

2AA 0.5 μg/plate

332

355

(344)

343

450

(397)

444

436

(440)

2AA 2.0 μg/plate

* P<0.05

** P<0.01, compared with results of negative control group in each strain

NT not ested

AF2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

2AA 2-aminoanthracene

a Statistical analysis was not applied because number of measurements was less than three

- Table Ib Mutagenicity Testing of Carbon Tetrachloride in E.coli WP2uvrA/pKM101

TA98

Lot. Nr. APE7331

Lot No. KSG7670

Dose %

Test 1 (Mean)

Test 2 (Mean)

Test 3 (Mean

Test 1-3 Mean + SD

Negative control (Air)

9

16

13

18

8

15

13

16

(14)

17

13

(15)

9

13

(11)

13+3.3

Without S 9 mix

0.005

NT

NT

13

23

(18)

NT

NT

18a

0.01

NT

NT

16

20

(18)

NT

NT

18a

0.05

NT

NT

28

16

(22)

11

16

(14)

18+7.2

0.1

13

20

(17)

22

23

(23)

11

13

(12)

17+5.3

0.2

NT

NT

NT

NT

15

17

(16)

16a

0.5

29

20

(25)

28

31

(30)

10

22

(16)

23+7.8*

1

40

24

(32)

21

21

(21)

26

21

(24)

26+7.4**

2

NT

NT

NT

NT

32

36

(34)

34a

5

36

38

(37)

34

30

(32)

31

17

(24)

31+7.5

AF2 0.1 μg/plate

719

680

(700)

627

614

(621)

584

596

(590)

AF2 0.005 μg/plate

TA98

Lot. Nr. APE7331

Lot No. KSG7670

Dose %

Test 1 (Mean)

Test 2 (Mean)

Test 3 (Mean

Test 1-3 Mean + SD

Negative control (Air)

22

28

26

39

14

28

24

26

(25)

36

32

(33)

24

23

(22)

27+6.6

With S 9 mix

0.005

NT

NT

37

29

(33)

NT

NT

33a

0.01

NT

NT

32

26

(29)

NT

NT

29a

0.05

NT

NT

22

32

(27)

11

23

(17)

22+8.6

0.1

29

30

(30)

37

22

(30)

29

23

(26)

28+5.4

0.2

NT

NT

NT

NT

21

26

(24)

24a

0.5

25

26

(26)

25

28

(25)

28

28

(28)

26+1.5

1

33

36

(35)

31

38

(35)

31

24

(28)

32+4.9

2

NT

NT

NT

NT

36

32

(34)

34a

5

52

38

(45)

30

36

(33)

24

28

(26)

35+9.9

2AA 0.5 μg/plate

332

355

(344)

343

450

(397)

444

436

(440)

2AA 2.0 μg/plate

* P<0.05

** P<0.01, compared with results of negative control group in each strain

NT not ested

AF2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

2AA 2-aminoanthracene

a Statistical analysis was not applied because number of measurements was less than three

- Table II Mutagenicity Testing of Carbon Tetrachloride and chloroform in E.coli WP2/pKM101

Carbon Tetrachloride Lot No. APE7331

Dose (%)

Mean

Negative control (Air)

20

37

29

38

(30)

Without S9 mix

0.01

44

43

(44)*

0.05

54

61

(58)*

0.1

74

83

(79)*

0.2

NT

NT

0.5

138

155

(147)*

1

243

230

(237)*

2

320

367

(344)*

5

232

208

(12)*

AF2 0.1μg/plate 0.1μg/plate

460

456

(220)*

Negative Control (Air)

48

71

53

51

(56)

With S9 mix

0.01

55

59

(57)*

0.05

70

39

(55)*

0.1

91

85

(88)*

0.2

NT

NT

0.5

150

180

(165)*

1

288

284

(286)*

2

445

395

(420)*

5

101

71

(86)*

2AA 10μg/plate
2AA 25μg/plate

272

358

(315)

* P<0.05

** P<0.01, compared with results of negative control group in each strain

NT not ested

AF2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

2AA 2-aminoanthracene

a Statistical analysis was not applied because number of measurements was less than three

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
ambiguous

- A CTC concentration dependent rise of the no. of revertants was found in the strains TA98, E. coli WP2 uvr A pKM 101 and E. coli WP2 pKM 101
- The used concentrations are very high
- Ambiguous results
Executive summary:

The study Araki (2004) presents an ambigous result concerning genotoxicity of CTC in the bacterial reverse mutation assay.

Salmonella strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli strains E. coli WP2 uvr A pKM 101 and E. coli WP2 pKM 101 were exposed to CTC using the gas exposure method.

A CTC concentration dependent rise of the no. of revertants was found in the strains TA98, E. coli WP2 uvr A pKM 101 and E. coli WP2 pKM 101. But for TA98, E. coli WP2 uvr A pKM 101 the concentrations where the criterion of 2 fold increase of no. of revertants compared to controls were reached were 10´000 ppm. And the results for E. coli WP2 pKM 101 (criterion reached at 1000 ppm) were not reproduced and based only on 2 replicate plates per concentration.