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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restriction - No data on the purity of the tested article. - Neither TA 102 nor E. Coli is included in the panel of cells tested. - No data on GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(neither TA 102 nor E. coli is included in the panel of cells tested; no statistical analysis, no data on purity of the tested article;)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: Lab sample
- Storage: room temperature
- Physical state: solid, insoluble in water
- Stability: guaranteed by sponsor

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 fraction mixed with a series of cofactors
Test concentrations with justification for top dose:
20, 78, 313, 1,250 and 5000 µg/0.1 mL
Vehicle / solvent:
- Vehicle: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without metabolic activation: Daunorubincin-HCl, 4-Nitroquinoline-N-Oxide, Sodium azide, 9(5) aminoacridine HCl; with metabolic activation: 2 aminoanthracene and cyclophosphamide
Remarks:
further information of positive control substances; see "any other information on materials and methods"
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation test)

DURATION
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: 2 experiments; 3 cultures / experiment

DETERMINATION OF CYTOTOXICITY
- Method: Reduction of revertants or reduction or clearing of background lawn
Evaluation criteria:
Criteria for a positive response:
The test substance is considered to be positive in this test system if one or both of the following conditions are met:
- a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration level for one or more of the following strains: TA 98, TA 1535 and TA 1537,
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by a factor of 1.5 for strain TA 100. Generally a concentration-related effect should be demonstrable.

Assay acceptance criteria:
A test is considered acceptable if the mean colony counts of the control values of all strains are within the historical control ranges and if the results of the positive controls meet the criteria for a positive response.

Acceptable range for negative control (without metabolic activation).
Revertants/plate
TA 98: 12-50
TA 100: 80-220
TA 1535: 7-30
TA 1537: 3-20

Acceptable range for negative control (with metabolic activation).
Revertants/plate
TA 98: 20-70
TA 100: 70-220
TA 1535: 7-35
TA 1537: 5-30
Statistics:
Arithmetical mean was calculated of the number of revertants.

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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item in soft agar was observed at concentrations of 313 µg/0.1 mL and above.

RANGE-FINDING/SCREENING STUDIES:
- Strain tested: TA 100
- Metabolic activation: without
- Concentrations: 0.08, 0.31, 1.2, 4.9, 19.5, 78.1, 312.5, 1250.0, 5000 µg/0.1 mL
- Repetitions: 2 cultures/concentration
- Method: the protocol used was the same as in the mutagenicity test
- Results: 5000 µg/0.1 mL of the test substance did not lead to a growth-inhibiting effect. This concentration was therefore selected as the highest in the mutagenicity tests. Number of revertants at 5000 µg/0.1 mL = 132 vs. 99 in control.

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

Any other information on results incl. tables

Table 2: Experiment 1: Mean values (without S9 mix)

 

 

 

TA 98

TA 100

TA 1535

TA 1537

 

Concentration (µg/0.1 ml)

S9

X (n=3)

revertant factor

X (n=3)

revertant factor

X (n=3)

revertant factor

X (n=3)

revertant factor

Test substance

control

no

27

 

144

 

12

 

10

 

20

no

18

0.7

127

0.9

11

0.9

6

0.6

78

no

21

0.8

140

1.0

11

0.9

7

0.7

313*

no

21

0.8

139

1.0

13

1.1

8

0.8

1250*

no

22

0.8

145

1.0

12

1.0

11

1.1

5000*

no

30

1.1

144

1.0

11

0.9

11

1.1

positive control

control

no

19

 

131

 

10

 

6

 

Low conc.

no

681

35.8

837

6.4

776

7.8

105

17.8

 

High conc.

no

881

46.4

1447

11.0

1155

115.5

1155

192.5

 

Table 3: Experiment 1: Mean values (with S9 mix)

 

 

 

TA 98

TA 100

TA 1535

TA 1537

 

Concentration (µg/0.1 ml)

S9

X (n=3)

revertant factor

X (n=3)

revertant factor

X (n=3)

revertant factor

X (n=3)

revertant factor

Test substance

control

yes

31

 

125

 

16

 

15

 

20

yes

36

1.2

128

1.0

24

1.5

9

0.6

78

yes

31

1.0

139

1.1

16

1.0

13

0.9

313*

yes

36

1.2

149

1.2

17

1.1

8

0.5

1250*

yes

51

1.6

129

1.0

9

0.6

13

0.9

5000*

yes

37

1.2

143

1.1

8

0.5

20

1.3

positive control

control

yes

32

 

163

 

14

 

18

 

Test conc.

yes

1246

38.9

1611

9.9

530

37.9

291

16.2

 

Table 4: Experiment 2: Mean values (without S9 mix)

 

 

 

TA 98

TA 100

TA 1535

TA 1537

 

Concentration (µg/plate)

S9

X (n=4)

revertant factor

X (n=4)

revertant factor

X (n=4)

revertant factor

X (n=4)

revertant factor

Test substance

control

no

17

 

146

 

12

 

7

 

20

no

18

1.0

179

1.2

10

0.9

7

1.0

78

no

17

1.0

191

1.3

7

0.6

6

0.9

313*

no

19

1.1

165

1.1

8

0.7

9

1.4

1250*

no

21

1.2

147

1.0

13

1.1

7

1.0

5000*

no

21

1.2

161

1.1

10

0.8

9

1.3

Positive control

Control

no

11

 

138

 

9

 

5

 

Low conc.

no

295

17.7

263

1.9

523

58.1

49

9.8

High conc.

no

752

68.4

496

3.4

911

101.2

1048

209.6

 

Table 5: Experiment 2: Mean values (with S9 mix)

 

 

 

TA 98

TA 100

TA 1535

TA 1537

 

Concentration (µg/plate)

S9

X (n=4)

revertant factor

X (n=4)

revertant factor

X (n=4)

revertant factor

X (n=4)

revertant factor

Test substance

control

yes

29

 

136

 

13

 

10

 

20

yes

23

0.8

137

1.0

15

1.1

10

1.0

78

yes

29

1.0

164

1.2

9

0.7

9

0.9

313*

yes

30

1.0

164

1.2

10

0.8

11

1.1

1250*

yes

29

1.0

169

1.2

9

0.7

11

1.1

5000*

yes

36

1.3

180

1.3

10

0.8

9

0.9

Positive control

Control

no

29

 

127

 

11

 

6

 

 

Test conc.

no

1288

44.4

965

7.6

502

45.6

109

18.2

 

  * Precipitation on test plate

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative