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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(E.coli was not included, only few details on experimental procedure, in experiments with metabolic activation a positive control was only investigated in TA1535)
GLP compliance:
no
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 and TA 100
Additional strain / cell type characteristics:
other: histidine-auxotrophic
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of liver from rats induced with Aroclor 1254 and a solution of co-factors
Test concentrations with justification for top dose:
25, 75, 225, 675, and 2025 µg/0.1 ml
Vehicle / solvent:
- Solvent used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below for details
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Exposure duration: 48 h at 37 °C in darkness

NUMBER OF REPLICATIONS: 2 independent experiments, each with 3 plates each concentration or control group

POSITIVE CONTROLS:
no metabolic activation:
- Strain TA 98: Daunorubicin-HCl (5 and 10 µg/0.1 ml phosphate buffer)
- Strain TA 100: 4-nitroquinoline-N-oxide (0.125 and 0.25 µg/0.1 ml phosphate buffer)
- Strain TA 1535: N-methyl-N'-nitro-N-nitrosoguanidine (3 and 5 µg/0.1 ml phospahte buffer)
- Strain TA 1537: 9(5)aminoacridine hydrochloride monohydrate (50 and 100 µg/0.1 ml DMSO)
with metabolic activation:
- Starin TA 1537: Cyclophophamide (250 µg/0.1 ml phosphate buffer)
Evaluation criteria:
A test substance was generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.
Statistics:
When the colonies had been counted, the arithmetic mean was calculated.

Results and discussion

Test results
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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 225 µg/0.1 ml and above the substance precipitated in soft agar.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Tab. 1: Salmonella/Mammalian-Microsome Mutagenicity Test: First experiment without microsomal activation. Number of Back-Mutant Colonies per Plate (Arithmetic mean) (1981 -06 -25)

Compound

Concentration

TA 98

TA 100

TA 1535

TA 1537

Test substance

Control

20

152

8

2

 

25 µg/0.1 ml

16

126

11

3

 

75 µg/0.1 ml

18

131

5

2

 

225 µg/0.1 ml

13

134

10

4

 

675 µg/0.1 ml

14

123

7

2

 

2025 µg/0.1 ml

10

142

5

4

 Daunorubicin-HCl

Control

21

 

 

 

 

5 µg/0.1 ml

675

 

 

 

 

10 µg/0.1 ml

1110

 

 

 

4-Nitroquinoline-N-oxide

Control

 

147

 

 

 

0.125 µg/0.1 ml

 

661

 

 

 

0.25 µg/0.1 ml

 

1083

 

 

N-Methyl-N'-nitro- N-nitrosoguanidine

Control

 

 

10

 

 

3 µg/0.1 ml

 

 

350

 

 

5 µg/0.1 ml

 

 

1352

 

9(5)Aminoacridine hydrochloride

Control

 

 

 

3

 

50 µg/0.1 ml

 

 

 

254

 

100 µg/0.1 ml

 

 

 

866

Tab. 2: Salmonella/Mammalian-Microsome Mutagenicity Test: First experiment with microsomal activation. Number of Back-Mutant Colonies per Plate (Arithmetic mean) (1981 -06 -25)

Compound

Concentration

TA 98

TA 100

TA 1535

TA 1537

Test substance

Control

28

154

6

7

 

25 µg/0.1 ml

31

145

9

5

 

75 µg/0.1 ml

33

153

17

6

 

225 µg/0.1 ml

37

137

9

3

 

675 µg/0.1 ml

23

152

9

10

 

2025 µg/0.1 ml

26

155

14

6

Cyclophosphamide

Control

 

 

6

 

 

250 µg/0.1 ml

 

 

384

 

Tab. 3: Salmonella/Mammalian-Microsome Mutagenicity Test: Second experiment without microsomal activation. Number of Back-Mutant Colonies per Plate (Arithmetic mean) (1981 -07-09)

Compound

Concentration

TA 98

TA 100

TA 1535

TA 1537

Test substance

Control

21

166

11

5

 

25 µg/0.1 ml

20

147

10

3

 

75 µg/0.1 ml

26

151

12

6

 

225 µg/0.1 ml

22

152

12

7

 

675 µg/0.1 ml

20

151

14

4

 

2025 µg/0.1 ml

21

135

17

4

 Daunorubicin-HCl

Control

31

 

 

 

 

5 µg/0.1 ml

629

 

 

 

 

10 µg/0.1 ml

1025

 

 

 

4-Nitroquinoline-N-oxide

Control

 

160

 

 

 

0.125 µg/0.1 ml

 

693

 

 

 

0.25 µg/0.1 ml

 

1176

 

 

N-Methyl-N'-nitro- N-nitrosoguanidine

Control

 

 

14

 

 

3 µg/0.1 ml

 

 

489

 

 

5 µg/0.1 ml

 

 

1820

 

9(5)Aminoacridine hydrochloride

Control

 

 

 

5

 

50 µg/0.1 ml

 

 

 

164

 

100 µg/0.1 ml

 

 

 

985

Tab. 4: Salmonella/Mammalian-Microsome Mutagenicity Test: Second experiment with microsomal activation. Number of Back-Mutant Colonies per Plate (Arithmetic mean) (1981 -07-09)

Compound

Concentration

TA 98

TA 100

TA 1535

TA 1537

Test substance

Control

45

159

13

11

 

25 µg/0.1 ml

39

160

13

7

 

75 µg/0.1 ml

42

163

13

13

 

225 µg/0.1 ml

43

156

14

10

 

675 µg/0.1 ml

41

144

16

14

 

2025 µg/0.1 ml

35

165

9

9

Cyclophosphamide

Control

 

 

9

 

 

250 µg/0.1 ml

 

 

502

 

Applicant's summary and conclusion

Conclusions:
No evidence of the induction of point mutations by the test article or by the metabolites of the substance formed as a result of microsomal activation was detectable.
Executive summary:

In a bacterial reverse mutation assay conducted similar to OECD Guideline 471 the test substance was analysed for mutagenicity by the detection of point mutations in bacteria (histidine-auxotrophic mutants of Salmonella typhimurium). To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were also performed with the addition of an activation mixture (rat liver microsomes and co-factors). The strains TA 98, TA 100, TA 1535, and TA 1537 were treated with the test substance dissolved in DMSO in doses of 25, 75, 225, 675, and 2025 µg/0.1 ml (plate incorporation test). A negative control (vehicle) and appropriate positive control substances were also evaluated. At concentrations of 225 µg/0.1 ml and higher the test compound precipitated in soft agar. No dose-related biologically relevant increase or doubling in the number of his+ revertants was observed in plate incorporation test in any of the tested strains with or without metabolic activation. Effects on cytotoxicity were not given. According to the results of the present study, the test substance is not mutagenic in the bacterial reverse mutation assay under the experimental conditions chosen. The positive controls yielded the expected results.