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Toxicological information

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
11 Nov 1999 - 13 Mar 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study, tested with the source substance CAS 2306-88-9. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 2-AA used as positive control in the presence of S9-mix
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
certified by the Hessische Ministerium für Umwelt, Energie, Jugend, Familie und Gesundheit
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): only trade name given
- Physical state: light yellow liquid
- Analytical purity: 100%
- Expiration date of the lot/batch: December 31, 2000
- Storage condition of test material: room temperature

Method

Target gene:
his opreon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: rfa-; uvrB- (R+ for TA 98 and TA 100)
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: rfa-; uvrB+; R+
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and β-naphthoflavone.
Test concentrations with justification for top dose:
Experiment 1 and 2: 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment 2: 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (MERCK, D-64293 Darmstadt; purity > 99%)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 10 µg/plate sodium azide (TA 1535 and TA 100; -S9); 4-nitro-o-phenylene-diamine (10 µg/plate in TA 98 and 50 µg/plate in TA 1537; -S9); 5.0 µl/plate methylmethanesulfonate (TA 102; -S9); 2.5 µg/plate 2-aminoanthracene (10.0 µg/plate in TA 102) (+S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (first experiment); preincubation (second and third experiment)

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in three independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn
Evaluation criteria:
A test item is considered positve if either a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration is induced. A test item producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any one of the test points is considered non-mutagenic in the test system.
A biologically relevant response is described as follows:
A test item is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, TA 100 and TA 102 or thrice in strains TA 1535 and TA 1537. Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose fulfilled the criteria described above or not.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and 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:
COMPARISON WITH HISTORICAL CONTROL DATA: yes

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test results without S9-mix

Without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates; 2 or 3 independent experiments)

Base-pair substitution type

Frameshift type

TA 100

TA1535

TA102

TA98

TA1537

Experiment

I

II

I

II

III

I

II

I

II

I

II

 

0

132

58

19

11

26

184

208

27

15

24

18

Solvent control

114

62

16

13

26

152

201

26

14

24

22

33

104

69

11

12

-

173

198

25

14

28

24

100

123

71

17

12

-

167

191

25

10

27

22

333

127

68

23

12

25

178

280

20

13

30

23

1000

117

58

14

26

25

169

183

23

13

31

21

2500

115

68

16

35

23

119

266

20

7

23

25

5000

122

63

16

32

25

176

270

22

11

23

20

Positive controls, –S9

Name

SA

SA

MMS

4NOPD

4NOPD

Concentrations

(μg/plate)

10.0

10.0

5 µL/plate

10.0

50.0

Mean No. of colonies/plate

(average of 3)

1490

1150

1075

1210

760

1387

1270

389

250

86

95

Table 2. Test results with S9-mix

With S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates; 2 independent experiments)

Base-pair substitution type

Frameshift type

TA 100

TA1535

TA102

TA98

TA1537

Experiment

I

II

I

II

III

I

II

I

II

I

II

+

0

143

96

8

11

-

192

227

45

26

26

20

+

Solvent control

129

70

10

10

-

154

215

36

19

31

22

+

33

118

72

9

8

-

144

201

46

20

28

24

+

100

118

76

8

9

-

166

219

46

24

34

24

+

333

131

80

6

10

-

170

220

41

17

32

26

+

1000

125

50

9

6

-

186

211

49

17

26

17

+

2500

122

75

5

8

-

154

172

55

17

23

28

+

5000

128

77

11

6

-

153

217

45

22

33

27

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

2.5

2.5

10.0

2.5

2.5

Mean No. of colonies/plate

(average of 3)

1803

536

231

151

-

726

1497

892

536

117

169

SA = Sodium azide

4NOPD = 4-nitro-o-phenylene-diamine

MMS = Methyl methane sulfonate

2-AA = 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative