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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:
26 July - 09 Aug 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(adopted July 1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(2000/32/EC)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Comunidad de Madrid
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon (S. typhimurium) and trp operon (E. coli)
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
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
0.06, 0.19, 0.56, 1.67 and 5 µL/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: corn oil
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-amino-anthracene (2AA; 1.5 µg/plate in DMSO, +S9, TA98, 2.5 µg/plate in DMSO, +S9, TA100 and TA1537, 30 µg/plate in DMSO, +S9, TA1535 and WP2)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation (main study) ; preincubation (confirmatory study)
DURATION
- Preincubation period: 20 min
- Exposure duration: 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 positive in this test system if the following criteria are met: For the test item to be considered mutagenic, two-fold (TA98, TA100, WP2) or three-fold (TA1535, TA1537) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
Statistics:
Mean values and standard deviation were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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:
not specified
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
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:
not specified
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Test Results of Experiment 1 (plate incorporation)

With or without S9-Mix

Test substance concentration

(μL/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2

TA98

TA1537

0

103

23

184

22

6

0.06

110

25

188

17

4

0.19

94

30

222

20

5

0.56

91

24

212

18

5

1.67

95

19

207

18

4

5

93

17

232

18

5

Positive controls, –S9

Name

SA

SA

4NQO

2NF

9AA

Concentrations

(μg/plate)

2.5

3.5

0.4

5

45

Mean No. of colonies/plate

(average of 3)

762

798

1776

418

188

+

0

92

23

234

22

4

+

0.06

87

21

208

20

5

+

0.19

90

17

203

17

6

+

0.56

95

18

207

20

6

+

1.67

92

21

212

20

7

+

5

97

23

213

22

8

Positive controls, + S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

30

30

1.5

2.5

Mean No. of colonies/plate

(average of 3)

1677

419

1605

1012

190

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

2NF = 2-nitroflurene

SA = sodium azide

 

 

Table 2: Test Results of Experiment 2 (pre-incubation)

With or without S9-Mix

Test substance concentration

(μL/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2

TA98

TA1537

0

83

24

138

23

5

0.06

87

19

187

18

7

0.19

79

27

194

17

7

0.56

85

24

170

19

4

1.67

89

22

181

23

5

5

91

19

193

20

5

Positive controls, –S9

Name

SA

SA

4NQO

2NF

9AA

Concentrations

(μg/plate)

2.5

3.5

0.4

5

45

Mean No. of colonies/plate

(average of 3)

792

1036

2482

409

161

+

0

82

33

163

26

9

+

0.06

91

23

199

21

6

+

0.19

96

26

219

20

7

+

0.56

95

27

182

19

9

+

1.67

83

26

208

22

6

+

5

91

23

208

20

8

Positive controls, + S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

30

30

1.5

2.5

Mean No. of colonies/plate

(average of 3)

1007

320

2118

1049

189

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

2NF = 2-nitroflurene

SA = sodium azide

Applicant's summary and conclusion

Conclusions:
Under the tested experimental conditions the test substance did not induce gene mutations in E. coli and and S. typhimurium strains up to precipitating concentrations.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.