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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxybenzamide
- Analytical purity: 97.4%
- Lot/batch No.: 08466/0013

Method

Target gene:
his operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: nutrient broth supplemented with ampicillin for the TA 100, TA98 strains
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
- Type and identity of media: nutrient broth supplemented with tetracycline
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:
0, 16, 50, 158, 500, 1581, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility of test compound
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (10 µg/plate, -S9, TA1535); nitrofurantoin (0.2 µg/plate, -S9, TA100); 4-nitro-1,2-phenylene diamine (0.5 and 10 µg/plate, -S9, TA98 and TA1537); mitomycin C (0.2 µg/plate, -S9, TA102); cumene hydroperoxide (50 µg/plate, -S9, TA102)
Positive control substance:
other: 2-aminoanthracene (3 µg/plate, +S9, all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation (served as independent repeat)

DURATION
- Preincubation period: 20 minutes (only preincubation assay) at 37 °C
- Exposure duration: 48 hours (both plate incorporation and preincubation) at 37 °C

SELECTION CRITERIA (mutation assays): reverse mutation to histidine prototrophy

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: reduction in background growth; reduction in mutant count per plate; titer
Evaluation criteria:
Acceptance of an assay:
1. The negative controls had to be within the expected range, as defined by published data (e.g. Maron and Ames, 1983) and/ or the laboratories' own historical data.
2. The positive controls had to show sufficient effects, as defined by the laboratories' experience.
3. Titer determinations had to demonstrate sufficient bacterial density in the suspension.
Even if the criteria for points 1 and 2 were not met, a trial was accepted if it showed mutagenic activity of the test compound.

Criteria for a positive result:
A reproducible and dose-related increase in mutant counts of at least one strain (For TA1535, TA100 and TA98 about twice that of negative controls, for TA1537 at least threefold; for TA102 an increase of about 100 mutants should be reached). Otherwise the result is evaluated as negative.
Statistics:
Means and standard deviation of triplicate plates 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:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/per plate, the substance had a weak, bacteriotoxic effect in the first trial
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The doses for the first trial (plate incorporation assay) were determined on the basis of a standard protocol: if not limited by solubility 5000 µg or 5 µl per plate were used as the highest dose. At least five additional doses were routinely used. The results of the first experiment were then considered as a pre-test for toxicity. If at least three doses could be used for assessment, the first trial was included in the overall assessment. If the second test (preincubation assay) confirmed the results of the first, no additional repeat was performed. Doses of repeats were chosen on the basis of the results obtained in the first experiment.

COMPARISON WITH HISTORICAL CONTROL DATA:
The experimental data is well comparable with the provided historical control data.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Maximum number of revertants (Mean ± SD):

Plate incorporation assay

 

-S9

+S9

Strain

control

test compound (µg/plate)

pos. Control

control

test compound (µg/plate)

pos. control

TA1535

21 ± 4

20 ± 4 (16)

467 ± 51

11 ± 2

8 ± 1 (16)

132 ± 9

 

 

20 ± 5 (50)

 

 

10 ± 3 (50)

 

 

 

16 ± 3 (158)

 

 

8 ± 1 (158)

 

 

 

20 ± 3 (500)

 

 

8 ± 1 (500)

 

 

 

20 ± 6 (1581)

 

 

9 ± 2 (1581)

 

 

 

11 ± 6 (5000)

 

 

7 ± 1 (5000)

 

TA 100

128 ± 6

141 ± 16 (16)

320 ± 2

133 ± 25

134 ± 3 (16)

1366 ± 102

 

 

129 ± 21 (50)

 

 

136 ± 14 (50)

 

 

 

144 ± 20 (158)

 

 

136 ± 22 (158)

 

 

 

128 ± 5 (500)

 

 

138 ± 17 (500)

 

 

 

131 ± 8 (1581)

 

 

125 ± 2 (1581)

 

 

 

138 ± 3 (5000)

 

 

124 ± 6 (5000)

 

TA 1537

6 ± 1

5 ± 1 (16)

87 ± 13

9 ± 4

6 ± 1 (16)

248 ± 9

 

 

6 ± 2 (50)

 

 

6 ± 1 (50)

 

 

 

7 ± 3 (158)

 

 

7 ± 2 (158)

 

 

 

5 ± 2 (500)

 

 

10 ± 3 (500)

 

 

 

4 ± 1 (1581)

 

 

6 ± 2 (1581)

 

 

 

2 ± 1 (5000)

 

 

6 ± 1 (5000)

 

TA 98

44 ± 16

35 ± 5 (16)

179 ± 14

55 ± 10

43 ± 2 (16)

1326 ± 48

 

 

38 ± 2 (50)

 

 

50 ± 7 (50)

 

 

 

42 ± 10 (158)

 

 

45 ± 6 (158)

 

 

 

38 ± 2 (500)

 

 

55 ± 9 (500)

 

 

 

42 ± 10 (1581)

 

 

43 ± 8 (1581)

 

 

 

36 ± 3 (5000)

 

 

43 ± 5 (5000)

 

TA102

205 ± 12

174 ± 28 (16)

577 ± 95

291 ± 4

246 ± 6 (16)

586 ± 47

 

 

224 ± 7 (50)

 

 

286 ± 39 (50)

 

 

 

214 ± 8 (158)

 

 

286 ± 21 (158)

 

 

 

205 ± 17 (500)

 

 

250 ± 22 (500)

 

 

 

208 ± 6 (1581)

 

 

236 ± 28 (1581)

 

 

 

186 ± 15 (5000)

 

 

252 ± 32 (5000)

 

Preincubation assay

 

-S9

+S9

Strain

control

test compound (µg/plate)

pos. control

control

test compound (µg/plate)

pos. control

TA1535

16 ± 2

17 ± 3 (16)

580 ± 123

13 ± 3

13 ± 4 (16)

150 ± 19

 

 

13 ± 5 (50)

 

 

14 ± 4 (50)

 

 

 

16 ± 4 (158)

 

 

11 ± 3 (158)

 

 

 

14 ± 3 (500)

 

 

16 ± 2 (500)

 

 

 

16 ± 6 (1581)

 

 

10 ± 2 (1581)

 

 

 

18 ± 4 (5000)

 

 

14 ± 3 (5000)

 

TA 100

138 ± 4

132 ± 7 (16)

540 ± 27

 167 ± 5

166 ± 4 (16)

1538 ± 75

 

 

141 ± 6 (50)

 

 

154 ± 19 (50)

 

 

 

146 ± 30 (158)

 

 

167 ± 19 (158)

 

 

 

154 ± 10 (500)

 

 

161 ± 6 (500)

 

 

 

152 ± 19 (1581)

 

 

152 ± 18 (1581)

 

 

 

137 ± 15 (5000)

 

 

154 ± 16 (5000)

 

TA 1537

7 ± 1

9 ± 2 (16)

132 ± 17

11 ± 3

10 ± 2 (16)

214 ± 11

 

 

9 ± 1 (50)

 

 

9 ± 4 (50)

 

 

 

9 ± 3 (158)

 

 

12 ± 2 (158)

 

 

 

9 ± 0 (500)

 

 

11 ± 4 (500)

 

 

 

6 ± 1 (1581)

 

 

8 ± 1 (1581)

 

 

 

5 ± 1 (5000)

 

 

10 ± 5 (5000)

 

TA 98

26 ± 1

25 ± 5 (16)

141 ± 40

34 ± 3

34 ± 5 (16)

1265 ± 105

 

 

25 ± 8 (50)

 

 

37 ± 2 (50)

 

 

 

27 ± 4 (158)

 

 

33 ± 4 (158)

 

 

 

21 ± 7 (500)

 

 

27 ± 1 (500)

 

 

 

20 ± 4 (1581)

 

 

32 ± 5 (1581)

 

 

 

19 ± 1 (5000)

 

 

29 ± 3 (5000)

 

TA102

236 ± 14

237 ± 23 (16)

497 ± 28

277 ± 4

244 ± 25 (16)

665 ± 12

 

 

259 ± 9 (50)

 

 

240 ± 32 (50)

 

 

 

254 ± 11 (158)

 

 

239 ± 37 (158)

 

 

 

240 ± 11 (500)

 

 

307 ± 11 (500)

 

 

 

230 ± 22 (1581)

 

 

281 ± 16 (1581)

 

 

 

228 ± 17 (5000)

 

 

286 ± 40 (5000)

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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