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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 - 03 Mar 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(adopted 26 May 1983)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Version / remarks:
(adopted 26 May 1983)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Version / remarks:
(adopted Sep 1985)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
(adopted Sep 1995)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
His operon
Trp operon
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
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:
4, 20, 100, 500, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9:sodium azide (1 μg/plate) for TA100 and TA1535; 9-aminoacridine (50 μg/plate) for TA1537; 2-nitrofluorene (2,5 μg/plate) for TA98 ; N-methyl-N-nitro-N-nitrosoguanidine (2.5 µg/plate) for WP2uvrA; +S9: 2 aminoanthracene (0.5 to 10 μg/plate) all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, reduction of bacterial background lawn
Evaluation criteria:
A test article is classified mutagenic if either of the following conditions under a) and b) is achieved:
a) a test article produces at least a 2-fold increase in the mean number of revertants per plate of at least
one of the tester strains over the mean number of revertants per plate of the appropriate vehicle
control at complete bacterial background lawn
b) a test article induces a dose-related increase in the mean number of revertants per plate of at least one
of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in
at least two to three concentrations of the test article at complete bacterial background lawn.
The test results must be reproducible.
Statistics:
Mean values and standard deviation were calculated.

Results and discussion

Test resultsopen allclose all
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: visible precipitation of the test compound on the plates has been observed at 2500 µg/plate and above

RANGE-FINDING/SCREENING STUDIES: the test compound was tested at doses of 4 to 5000 µg/plate in TA 100 and proved to be not toxic to the
bacterial strain, therefore 5000 µg/plate was selected as the highest concentration tested.
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 first experiment (plate incorporation)           

With or without

S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

Cross-linking type

Frameshift type

 

TA 100

TA 1535

WP2 uvr A

TA 98

TA 1537

0

173.0 ± 16.1

10.0 ± 2.0

40.7 ± 6.8

36.0 ± 5.2

17.7 ± 2.5

0 (DMSO)

162.3 ± 17.2

14.0 ± 6.1

37.0 ± 6.2

32.0 ± 5.3

18.0 ± 4.6

4

176.0 ± 7.5

14.0 ± 4.4

37.3 ± 9.6

27.3 ± 4.0

15.0 ± 2.6

20

175.7 ± 1.5

13.0 ± 3.6

35.3 ± 3.5

34.0 ± 5.3

20.7 ± 2.5

100

173.3 ± 7.5

15.3 ± 2.3

33.0 ± 0.0

30.0 ± 7.2

12.3 ± 1.5

500

187.3 ± 22.1

18.3 ± 6.0

34.3 ± 2.1

30.3 ± 8.5

17.0 ± 1.7

2500

190.7 ± 11.0 P

14.0 ± 3.5 P

36.7 ± 1.5 P

29.7 ± 2.1 P

12.7 ± 3.5 P

5000

206.7 ± 13.1 P

12.0 ± 4.0 P

38.7 ± 3.5 P

30.7 ± 3.8 P

14.0 ± 3.0 P

Positive controls,

-S9

Name

SA

SA

MNNG

2NF

9AA

Concentrations (μg/plate)

1

1

2.5

2.5

50

Mean No. of colonies/plate (average of 3 ± SD)

762.7 ± 29.9

537.0 ± 32.1

225.3 ± 12.6

643.0 ± 83.0

114.3 ± 4.6

+

0

170.7 ± 26.6

13.7 ± 5.9

43.3 ± 6.4

36.3 ± 9.1

10.3 ± 0.6

+

0 (DMSO)

159.0 ± 12.1

11.7 ± 0.6

42.3 ± 4.7

41.3 ± 6.1

11.7 ± 3.2

+

4

158.7 ± 15.6

12.0 ± 1.0

37.3 ± 7.4

41.0 ± 2.6

9.7 ± 4.0

+

20

166.0 ± 17.1

14.3 ± 1.2

48.0 ± 7.5

34.7 ± 2.3

9.3 ± 4.5

+

100

167.3 ± 18.6

12.0 ± 4.6

40.0 ± 7.2

43.0 ± 7.8

10.0 ± 1.0

+

500

180.3 ± 11.5

17.3 ± 4.5

42.0 ± 5.2

40.3 ± 11.6

12.0 ± 1.0

 

2500

168.7 ± 6.7 P

16.3 ± 6.4 P

39.7 ± 1.5 P

37.3 ± 10.3 P

10.7 ± 1.5 P

+

5000

192.0 ± 12.8 P

12.0 ± 5.3 P

36.0 ± 2.6 P

33.3 ± 4.5 P

13.0 ± 5.0 P

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations (μg/plate)

0.5

1

10

0.5

1

Mean No. of colonies/plate (average of 3 ± SD)

1194.7 ± 49.4

137.7 ± 10.1

273.7 ± 6.7

996.0 ± 82.3

244.0 ± 12.5

2NF = 2-nitrofluorene                            

SA = sodium azide                              

MNNG= N-methyl-N-nitro-N-nitrosoguanidine                             

9AA = 9-aminoacridine                         

2AA = 2-Aminoanthracene                               

P = Precipitate                        

 

Table 2. Test results of second experiment (plate incorporation)                                  

With or without

S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

Cross-linking type

Frameshift type

 

TA 100

TA 1535

WP2 uvr A

TA 98

TA 1537

0

222.3 ± 23.4

15.3 ± 2.1

33.0 ± 7.0

36.0 ± 2.6

20.3 ± 2.1

0 (DMSO)

190.7 ± 16.8

12.3 ± 4.0

32.3 ± 5.5

37.0 ± 2.6

24.0 ± 6.6

4

188.3 ± 11.6

19.3 ± 5.1

30.0 ± 4.4

35.3 ± 4.0

16.0 ± 9.2

20

186.3 ± 14.5

10.7 ± 5.5

32.7 ± 9.0

32.7 ± 10.1

17.7 ± 4.6

100

175.0 ± 18.5

12.0 ± 4.6

36.3 ± 3.5

37.7 ± 2.3

17.3 ± 4.5

500

187.3 ± 1.2

15.7 ± 7.5

37.0 ± 8.5

32.3 ± 7.0

16.3 ± 4.2

2500

199.7 ± 30.0 P

18.3 ± 2.5 P

34.3 ± 8.5 P

27.7 ± 2.9 P

20.3 ± 3.5 P

5000

233.7 ± 15.8 P

15.7 ± 1.2 P

34.7 ± 3.2 P

26.7 ± 3.8 P

19.7 ± 3.5 P

Positive controls,

-S9

Name

SA

SA

MNNG

2NF

9AA

Concentrations (μg/plate)

1

1

2.5

2.5

50

Mean No. of colonies/plate (average of 3 ± SD)

949.7 ± 64.5

731.7 ± 16.8

242.7 ± 62.5

1334.7 ± 82.7

155.7 ± 27.0

+

0

186.0 ± 10.1

16.7 ± 6.0

40.7 ± 6.8

39.0 ± 8.2

21.0 ± 3.0

+

0 (DMSO)

163.0 ± 6.0

16.7 ± 1.5

38.0 ± 4.6

40.0 ± 4.4

19.0 ± 2.6

+

4

179.3 ± 17.2

13.0 ± 1.0

36.3 ± 11.2

44.7 ± 3.8

15.7 ± 4.9

+

20

199.3 ± 4.2

14.3 ± 1.5

36.0 ± 5.3

41.0 ± 9.2

14.3 ± 1.2

+

100

199.0 ± 8.2

18.3 ± 2.5

38.7 ± 1.5

42.7 ± 4.9

14.7 ± 0.6

+

500

198.0 ± 7.8

13.3 ± 1.2

39.3 ± 2.3

46.0 ± 7.2

19.0 ± 2.6

 

2500

199.0 ± 24.3 P

14.7 ± 3.5 P

39.7 ± 9.0 P

40.0 ± 7.8 P

18.3 ± 3.5 P

+

5000

207.7 ± 4.5 P

16.3 ± 2.9 P

34.0 ± 6.1 P

28.0 ± 7.5 P

10.7 ± 3.1 P

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations (μg/plate)

0.5

1

10

0.5

1

Mean No. of colonies/plate (average of 3 ± SD)

2041.7 ± 80.7

167.7 ± 7.6

275.3 ± 57.5

1712.3 ± 42.2

289.7 ± 0.6

2NF = 2-nitrofluorene                            

SA = sodium azide                              

MNNG= N-methyl-N-nitro-N-nitrosoguanidine                             

9AA = 9-aminoacridine                         

2AA = 2-Aminoanthracene                               

P = Precipitate               

Precipitation occurred at concentrations of 2500 µg/plate and above in all assays.

No indications of a mutagenic effect of the test substance occurred at assessable doses up to 500 µg/plate in any of the strains tested.

Conclusion:

Under the test conditions chosen in this study the test substance has to be regarded as non-mutagenic.

         

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative