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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:
experimental study
Adequacy of study:
key study
Study period:
22 Feb - 23 Mar 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 July 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Umwelt und Verkehr, Baden-Württemberg, Stuttgart, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon (for S. typhimurium strains)
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:
First experiment:
5, 15, 50, 150 and 500 µg/plate without metabolic activation (TA1537)
15, 50, 150, 500 and 1500 µg/plate without metabolic activation (TA98, TA100 and TA 102)
50, 150, 500, 1500 and 5000 µg/plate without metabolic activation (TA1535)
50, 150, 500, 1500 and 5000 µg/plate with metabolic activation (all strains)

Second experiment:
15, 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation (TA1535)
15, 50, 150, 500 and 1500 µg/plate without metabolic activation (TA102)
50, 150, 500, 1500 and 5000 µg/plate without metabolic activation (TA98, TA100)
500, 1000, 1500, 2000 and 3000 µg/plate without metabolic activation (TA1535)
15, 50, 150, 500 and 1500 µg/plate with metabolic activation (TA100, TA102, TA1537)
50, 150, 500, 1500 and 5000 µg/plate with metabolic activation (TA98, TA1535)
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
other: sodium azide (NaN3), 2-nitrofluorene (2-NF), 9-aminoacridine (9-AA), 2-aminoanthracene (2-AA)
Remarks:
+S9: 2-AA (0.8 µg/plate TA98, TA100; 0.9 µg/plate TA102, TA1535; 1.7 µg/plate, TA1537); -S9: NaN3 (0.7 µg/plate, TA100, TA1535); 2-NF (2.5 µg/plate, TA98); 9-AA (50 µg/plate, TA1537); Mitomycin C (0.15 µg/plate, TA102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies and/or diminution of the background lawn

Statistics:
X²-test was used to estimate the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level. Mean values and standard deviation were calculated.

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:
cytotoxicity
Remarks:
at 1500 µg/plate (-S9) in TA102 and at 5000 µg/plate (-S9) in TA1537 and TA100; at 5000 µg/plate (+S9) in TA1535, TA1537, TA100 and TA102
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation occurred up to the highest investigated dose.

RANGE-FINDING/SCREENING STUDIES:
An initial toxicity test was conducted. No further details were given in the study report.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and positive control data were between the minimum and maximum value of the historical control data of the laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9 mix the test substance was bacteriotoxic towards the strains TA102 at 1500 µg/plate and towards the strains TA1537 and TA100 at 5000 µg/plate. In the presence of S9 mix the test substance was bacteriotoxic towards the strains TA1535, TA1537, TA100, and TA102 at 5000 µg/plate.
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 of Experiment 1

EXPERIMENT 1

S9-Mix

Without

 

Test item (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

NC

34 ± 6

99 ± 8

216 ± 26

19 ± 3

10 ± 3

Solvent control (DMSO)

30 ± 3

86 ± 2

235 ± 11

17 ± 3

10 ± 0

5

-

-

-

-

8 ± 2

15

33 ± 6

86 ± 5

216 ± 22

-

9 ± 2

50

33 ± 8

94 ± 14

240 ± 19

19 ± 2

8 ± 3

150

33 ± 7

98 ± 6

211 ± 19

24 ± 4

6 ± 3

500

36 ± 5

100 ± 2

253 ± 7

29 ± 1

7 ± 3

1500

32 ± 4

106 ± 6

172 ± 6 (T)

34 ± 8

-

5000

-

-

-

25 ± 5

-

NaN3 (0.7)

-

503 ± 58

-

885 ± 104

-

2-NF (2.5)

812 ± 39

-

-

-

-

9-AA (50)

-

-

-

-

272 ± 43

Mitomycin C (0.15)

-

-

723 ± 53

-

-

S9-Mix

With

 

Test item (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

 

 

 

 

 

 

NC

32 ± 8

97 ± 10

174 ± 29

7 ± 3

10 ± 4

Solvent control (DMSO)

25 ± 2

91 ± 7

208 ± 3

9 ± 2

14 ± 2

50

22 ± 3

86 ± 2

204 ± 29

10 ± 2

11 ± 4

150

25 ± 4

86 ± 3

229 ± 7

9 ± 2

13 ± 4

500

25 ± 3

83 ± 4

239 ± 12

6 ± 3

13 ± 3

1500

24 ± 1

81 ± 11

187 ± 27

6 ± 1

9 ± 4

5000

25 ± 7 (T)

58 ± 4 (T)

38 ± 10 (T)

6 ± 1

3 ± 3 (T)

2-AA (0.8)

926 ± 31

627 ± 90

-

-

-

2-AA (0.9)

-

-

308 ± 62

135 ± 20

-

2-AA (1.7)

-

-

-

-

362 ± 58

NC = Negative Control

T = bacteriotoxic

2-NF: 2-nitrofluorene; 9-AA: 9-aminoacridine; 2-AA: 2-aminoanthracene; NaN3: sodium azide

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

NC

18 ± 4

201 ± 16

328 ± 28

22 ± 4

8 ± 2

Solvent control (DMSO)

21 ± 3

158 ± 6

300 ± 13

24 ± 1

6 ± 2

15

-

-

282 ± 34

-

7 ± 2

50

19 ± 3

138 ± 12

273 ± 14

-

8 ± 4

150

18 ± 2

177 ± 10

282 ± 12

-

7 ± 3

500

17 ± 4

166 ± 29

250 ± 18

35 ± 7

7 ± 5

1000

-

-

-

38 ± 9

-

1500

16 ± 2

130 ± 28

199 ± 12 (T)

38 ± 3

7 ± 3

2000

-

-

-

33 ± 15

-

3000

-

-

-

41 ± 3

-

5000

16 ± 2

79 ± 23 (T)

-

-

0 ± 0 (T)

NaN3 (0.7)

-

471 ± 91

-

841 ± 127

-

2-NF (2.5)

607 ± 67

-

-

-

-

9-AA (50)

-

-

-

-

381 ± 54

Mitomycin C (0.15)

-

-

967 ± 67

-

-

S9-Mix

With

 

Test item (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

 

 

 

 

 

 

NC

23 ± 5

171 ± 23

228 ± 38

12 ± 3

9 ± 3

Solvent control (DMSO)

22 ± 4

174 ± 6

272 ± 16

13 ± 3

11 ± 2

15

-

170 ± 12

223 ± 12

-

12 ± 5

50

22 ± 3

179 ± 14

263 ± 15

11 ± 1

13 ± 4

150

20 ± 6

180 ± 13

227 ± 17

14 ± 4

14 ± 4

500

21 ± 5

144 ± 23

252 ± 19

13 ± 5

10 ± 2

1500

24 ± 3

140 ± 12

209 ± 45

13 ± 3

12 ± 4

5000

20 ± 4

-

-

6 ± 4 (T)

-

2-AA (0.8)

420 ± 19

647 ± 113

-

-

-

2-AA (0.9)

-

-

477 ± 19

308 ± 51

-

2-AA (1.7)

-

-

-

-

321 ± 21

NC = Negative Control

T = bacteriotoxic

2-NF: 2-nitrofluorene; 9-AA: 9-aminoacridine; 2-AA: 2-aminoanthracene; NaN3: sodium azide

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of the Ames test the test substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) tested with and without metabolic activation.