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Toxicological information

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:
13 Aug - 17 Nov 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Directive 92/69/EEC
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1 and 2: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation


Vehicle / solvent:
- Vehicle/solvent used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene (2AA); 1,8-dihydroxyanthraquinone (Danthron)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: approx. 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn

Evaluation criteria:
The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett´s method of linear regression) significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Mean values and standard deviation were calculated.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In order to select appropriate dose levels for the main study, a preliminary test was carried out in S. typhimurium strain TA 100 to determine the toxicity of the test substance. The dose range for the test substance used was 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate wiht and without metabolic activation. The test substance was non-toxic to strain TA 100 at the tested concentrations.

Any other information on results incl. tables

Table 1: Test Results of Experiment 1

Experiment 1

S9-Mix

Without

Test item (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

Solvent control (DMSO)

30 ± 4

124 ± 18

299 ± 20

28 ± 7

18 ± 4

50

30 ± 5

123 ± 7

286 ± 4

20 ± 6

11 ± 0

150

26 ± 8

122 ± 10

301 ± 14

23 ± 6

15 ± 3

500

31 ± 7

145 ± 13

297 ± 27

27 ± 9

19 ± 3

1500

25 ± 4

110 ± 14

287 ± 26

30 ± 6

16 ± 1

5000

20 ± 6

128 ± 5

307 ± 35

33 ± 2

9 ± 1

ENNG (3)

-

539 ± 64

-

-

-

ENNG (5)

-

-

-

222 ± 39

-

9-AA (80)

-

-

-

-

784 ± 251

4NQO (0.2)

160 ± 2

-

-

-

-

MMC (0.5)

-

-

751 ± 185

-

-

S9-Mix

With

Test item (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

Solvent control (DMSO)

44 ± 6

143 ± 21

330 ± 24

24 ± 4

22 ± 2

50

50 ± 17

129 ± 16

313 ± 26

13 ± 5

22 ± 1

150

48 ± 11

138 ± 8

321 ± 9

18 ± 7

23 ± 3

500

56 ± 14

144 ± 5

331 ± 22

20 ± 1

24 ± 1

1500

37 ± 5

131 ± 17

316 ± 19

16 ± 5

22 ± 3

5000

38 ± 5

127 ± 3

289 ± 12

17 ± 5

20 ± 1

2-AA (1)

-

2057 ± 428

-

-

-

2-AA (2)

-

-

-

274 ± 43

814 ± 11

DAN (10)

-

-

813 ± 53

-

-

BP (5)

814 ± 11

-

-

-

-

EENG: N-ethyl-N-nitro-N-nitrosoguanidin

4NQO: 4-Nitroquinoline-1-oxide

9-AA: 9-Aminoacridine

MMC: Mitomycin C

2-AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

DAN: 1,8-Dihydroxyanthraquinone

Table 2: Test Results of Experiment 2

Experiment 2

S9-Mix

Without

Test item (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

Solvent control (DMSO)

27 ± 4

119 ± 11

352 ± 4

25 ± 2

16 ± 4

50

29 ± 3

123 ± 4

338 ± 5

20 ± 3

16 ± 5

150

29 ± 7

137 ± 15

331 ± 15

25 ± 7

17 ± 3

500

23 ± 6

135 ± 6

350 ± 12

23 ± 1

14 ± 2

1500

27 ± 7

121 ± 7

323 ± 56

24 ± 2

11 ± 3

5000

22 ± 8

124 ± 16

324 ± 17

32 ± 3

15 ± 5

ENNG (3)

-

481 ± 25

-

-

-

ENNG (5)

-

-

-

167 ± 15

-

9-AA (80)

-

-

-

-

926 ± 324

4NQO (0.2)

168 ± 14

-

-

-

-

MMC (0.5)

-

-

955 ± 75

-

-

S9-Mix

With

Test item (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

Solvent control (DMSO)

39 ± 2

127 ± 15

311 ± 20

15 ± 7

20 ± 1

50

34 ± 3

112 ± 11

308 ± 17

14 ± 1

19 ± 5

150

40 ± 8

157 ± 18

318 ± 25

18 ± 1

22 ± 3

500

31 ± 4

132 ± 29

317 ± 3

16 ± 3

17 ± 4

1500

29 ± 7

119 ± 11

317 ± 3

17 ± 4

14 ± 2

5000

38 ± 7

138 ± 19

269 ± 18

14 ± 1

12 ± 1

2-AA (1)

-

1484 ± 94

-

-

-

2-AA (2)

-

-

-

279 ± 41

551 ± 35

DAN (10)

-

-

875 ± 204

-

-

BP (5)

257 ± 6

-

-

-

-

EENG: N-ethyl-N-nitro-N-nitrosoguanidin

4NQO: 4-Nitroquinoline-1-oxide

9-AA: 9-Aminoacridine

MMC: Mitomycin C

2-AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

DAN: 1,8-Dihydroxyanthraquinone

Applicant's summary and conclusion

Conclusions:
Under the conditions of the Ames Assay the substance was not mutagenic in any of the five strains (TA 100, TA 1535, TA 102, TA 98 and TA 1537) tested with and without metabolic activation up to 5000 µg/plate.