Registration Dossier

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:
April 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
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 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Histidine gene locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced male rat liver S9 mix
Test concentrations with justification for top dose:
0, 50, 158, 500, 1581, 5000 µg/plate (first and repeat test)








Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Remarks:
No solvent control was used since sufficient evidence was available in the literature and from testing laboratory experience, indicating that the solvents used had no influence on the spontaneous mutant counts of the used strains.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (only TA 1535), nitrofurantoin (only TA 100), 4-nitro-1,2-phenylene diamine (TA 1537 and TA 98), cumene hydroperoxide (only TA 102), 2-aminoanthracene.
Remarks:
The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and cumene hydroperoxide were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.
Details on test system and experimental conditions:
METHOD: Standard plate test and preincubation test; each concentration including the controls was tested in triplicate.
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 1537, TA 100 and TA 98 this increase should be about twice that of negative controls. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
not specified

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:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of results from the Salmonella mutagenicity assay (first test) with the test substance (mean values of revertants per plate)

 Dose (µg per plate)

Without metabolic activation

 

 TA 1535

 TA 100

 TA 1537

 TA 98

 TA 102

0

9

80

12

14

156

50

8

93

10

15

153

158

10

69

7

15

144

500

13

61

8

15

134

1581

14

76

8

16

136

5000

12

75

8

16

152

 Positive control

793

241

104

146

291 

 Dose ( µg per plate )

With metabolic activation (liver S9 mix)

 

 TA 1535

 TA 100

 TA 1537

 TA 98

TA 102

0

12

86

11

20

206

50

11

92

7

21

218

158

8

89 

9

19

197

500

10

88

11

17

253

1581

11

107

11

26

263

5000

10

102

9

27

276

 Positive control

351

1797

134

1512

390

Table 2: Summary of results from the Salmonella mutagenicity assay (repeat test) with the test substance (mean values of revertants per plate)

 Dose (µg per tube)

Without metabolic activation

 

 TA 1535

 TA 100

 TA 1537

 TA 98

 TA 102

0

8

82

8

13

244

50

6

78

9

13

242

158

8

76

8

12

266

500

6

71

7

14

255

1581

11

73

8

15

265

5000

8

77

8

12

240

 Positive control

895

258

126

157

671 

 Dose ( µg per tube )

With metabolic activation (liver S9 mix)

 

 TA 1535

 TA 100

 TA 1537

 TA 98

TA 102

0

11

117

11

21

303

50

13

82

13

21

341

158

10

100 

9

18

350

500

9

103

10

18

284

1581

10

100

12

20

308

5000

12

112

9

24

333

 Positive control

143

990

96

866

474

Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed.

Evidence of mutagenic activity of the test substance was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Applicant's summary and conclusion

Executive summary:

The mutagenic potential of the test substance was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Evidence of mutagenic activity was not seen up to the maximum recommended dose level of 5000 µg/plate. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. The positive controls had a marked mutagenic effect. Therefore the test substance was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.