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 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

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 test, +/-S9 mix, all strains)
0, 50, 100, 200, 400, 800, 1600 µg/plate (repeat test, +/-S9 mix, all strains)








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 resultsopen allclose all
Species / strain:
S. typhimurium, other: TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
strong, strain-specific bacteriotoxic effect at 800 µg/plate and above
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
strong, strain-specific bacteriotoxic effect at 800 µg/plate and above
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 1537, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
strong, strain-specific bacteriotoxic effect at 800 µg/plate and above
Vehicle controls validity:
not examined
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 Ethoxybenzonitril (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

73

12

18

170

50

7

66

9

15

185

158

4

90

8

24

172

500

4

55

7

18

128

1581

4

59

2

20

48

5000

-

0

0

-

-

 Positive control

634

229

95

225

280 

 Dose ( µg per plate )

With metabolic activation (liver S9 mix)

 

TA 1535

 TA 100

 TA 1537

 TA 98

TA 102

0

10

78

10

23

230

50

12

75

10

27

236

158

20

91 

9

26

253

500

23

85

11

23

233

1581

13

64

6

18

155

5000

-

-

-

-

6

 Positive control

225

1170

191

1058

429

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

Dose (µg per plate)

Without metabolic activation

 

TA 1535

 TA 100

 TA 1537

 TA 98

 TA 102

0

6

81

7

17

226

50

5

80

6

16

218

100

7

78

6

17

239

200

8

82

6

17

226

400

9

61

5

14

183

800

6

44

4

5

131

1600

 -

18

 Positive control

536

263

125

188

419 

 Dose ( µg per plate )

With metabolic activation (liver S9 mix)

 

TA 1535

 TA 100

 TA 1537

 TA 98

TA 102

0

10

69

8

35

284

50

12

72

7

28

295

100

15

86 

7

19

270

200

22

92

6

17

227

400

21

100

5

20

244

800

21

88

4

18

210

1600

10 49 - 10 94

 Positive control

114

1019 

117 

1172 

491 

 

 

Table 3: Summary of results from the Salmonella mutagenicity assay (repeated plate incorporation and pre-incubation test with TA 1535 and metabolic activation) with Ethoxybenzonitril (mean values of revertants per plate)

 

 Dose (µg per plate )

With metabolic activation (liver S9 mix)

 

 TA 1535

 TA 1535

plate incorporation

pre-incubation

 0

9

9

 50

12

 12 

 100

20

19

 200

25

27

 400

31

26

 800

27

18

1600

11

5

 Positive control

184

259

Doses up to and including 500 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a limited extent up to and including 1600 µg per plate for assessment purposes.

Evidence of mutagenic activity of Ethoxybenzonitril was seen. On Salmonella typhimurium TA 1535, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Positive response was found only with S9 mix. The lowest reproducible effective dose was 100µg per plate. The Salmonella/microsome test thus showed Ethoxybenzonitril to have a mutagenic effect. However, it can be assumed that this result may be based on the effect of an impurity.

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

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation
Executive summary:

The mutagenic potential of Ethoxybenzonitril 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. Doses up to and including 500 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a limited extent up to and including 1600 µg per plate for assessment purposes. Evidence of mutagenic activity of Ethoxybenzonitril was seen. On Salmonella typhimurium TA 1535, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Positive response was found only with S9 mix. The lowest reproducible effective dose was 100 µg per plate. The Salmonella/microsome test thus showed Ethoxybenzonitril to have a mutagenic effect. However, it can be assumed that this result may be based on the effect of an impurity.