Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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: well performed GLP and OECD guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
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): p-Nitrobenzoylaminobenzamid trocken

Method

Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
Experiment I (plate incorporation test): 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II (pre-incubation test): 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment I: plate incorporation test
Experiment II: pre-incubation test

NUMBER OF REPLICATIONS:
3 plates

DETERMINATION OF CYTOTOXICITY:
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see additional information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Precipitation of the test item was observed in the test tubes at 2500 and 5000 µg/plate in both experiments. Precipitation of the test item was also observed at 2500 and 5000 µg/plate with S9 mix in experiment I, and from 1000 - 5000 µg/plate without S9 mix in experiment I and II, and with S9 mix in experiment II.
ADDITIONAL INFORMATION ON CYTOTOXICITY
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.
Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed without metabolic activation in strain TA 1535 at 2500 µg/plate in experiment I, and in strain TA 1537 at 5000 µg/plate without metabolic activation in experiment I and II.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Tables

   Summary of Results Pre-Experiment and Experiment I

Study Name: 1261901

Study Code: Harlan-CCR 1261901

Experiment: 1261901 VV Plate

Date Plated: 21/04/2009

Assay Conditions:

Date Counted: 24/04/2009

Metabolic

Activation

Test

Group

Dose Level

(per plate)

Revertant Colony Counts (Mean ±SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without Activation

DMSO

21 ± 2

14 ± 2

31 ± 8

182 ± 11

56 ± 2

Untreated

14 ± 2

13 ± 1

36 ± 3

168 ± 21

50 ± 13

p-Nitrobenzoylaminobenzamid

3 µg

21 ± 5

10 ± 3

35 ± 2

150 ± 14

52 ± 6

trocken

10 µg

12 ± 2

8 ± 2

37 ± 5

156 ± 12

59 ± 7

33 µg

16 ± 1

8 ± 1

36 ± 8

172 ± 32

59 ± 4

100 µg

17 ± 2

9 ± 1

38 ± 4

174 ± 9

81 ± 8

333 µg

18 ± 4

8 ± 1

47 ± 6

161 ± 16

59 ± 10

1000 µg

10 ± 1P

8 ± 2P

34 ± 3P

178 ± 9P

58 ± 8P

2500 µg

9 ± 2P M

8 ± 1P M

30 ± 3P M

171 ± 23P M

48 ± 4P M

5000 µg

12 ± 2P M

5 ± 2P M

33 ± 4P M

168 ± 15P M

41 ± 3P M

NaN3

10 µg

2272 ± 76

2131 ± 47

4-NOPD

10 µg

576 ± 110

4-NOPD

50 µg

239 ± 59

MMS

3.0 µL

1383 ± 41

With Activation

DMSO

19 ± 3

11 ± 2

46 ± 9

169 ± 11

77 ± 5

Untreated

12 ± 1

11 ± 2

51 ± 13

179 ± 13

76 ± 5

p-Nitrobenzoylaminobenzamid

3 µg

14 ± 1

10 ± 3

49 ± 1

142 ± 8

71 ± 18

trocken

10 µg

11 ± 5

12 ± 7

40 ± 3

154 ± 9

60 ± 6

33 µg

22 ± 7

11 ± 3

43 ± 4

184 ± 13

57 ± 2

100 µg

21 ± 7

12 ± 2

40 ± 6

183 ± 14

74 ± 16

333 µg

14 ± 1

12 ± 3

40 ± 6

161 ± 22

69 ± 4

1000 µg

18 ± 3

12 ± 3

41 ± 4

188 ± 15

63 ± 4

2500 µg

16 ± 2P M

9 ± 2P M

44 ± 7P M

169 ± 21P M

77 ± 12P M

5000 µg

15 ± 1P M

8 ± 1P M

39 ± 5P M

167 ± 12P M

45 ± 11P M

2-AA

2.5 µg

288 ± 9

256 ± 10

1632 ± 75

1830 ± 55

2-AA

10.0 µg

337 ± 13

Key to Positive Controls

Key to Plate Postfix Codes

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

P

M

Precipitate

Manual count

  Summary of Results Experiment II

Study Name: 1261901

Study Code: Harlan-CCR 1261901

Experiment: 1261901 HV2 pre

Date Plated: 07/05/2009

Assay Conditions:

Date Counted: 13/05/2009

Metabolic

Activation

Test

Group

Dose Level

(per plate)

Revertant Colony Counts (Mean ±SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without Activation

DMSO

14 ± 1

13 ± 3

32 ± 9

126 ± 29

55 ± 5

Untreated

15 ± 7

10 ± 2

31 ± 9

148 ± 10

61 ± 9

p-Nitrobenzoylaminobenzamid

3 µg

13 ± 9

11 ± 2

25 ± 4

112 ± 13

58 ± 9

trocken

10 µg

18 ± 4

19 ± 9

27 ± 3

129 ± 11

59 ± 17

33 µg

16 ± 3

9 ± 2

28 ± 5

128 ± 12

55 ± 4

100 µg

14 ± 1

11 ± 3

30 ± 6

129 ± 8

59 ± 4

333 µg

21 ± 5

13 ± 5

25 ± 4

129 ± 27

60 ± 4

1000 µg

18 ± 3P

16 ± 4P

29 ± 3P

120 ± 8P

48 ± 9P

2500 µg

18 ± 5P M

8 ± 3P M

34 ± 3P M

118 ± 11P M

54 ± 5P M

5000 µg

22 ± 4P M

6 ± 1P M

28 ± 5P M

108 ± 4P M

41 ± 2P M

NaN3

10 µg

1784 ± 70

1846 ± 166

4-NOPD

10 µg

423 ± 30

4-NOPD

50 µg

104 ± 14

MMS

3.0 µL

276 ± 17

With Activation

DMSO

24 ± 2

14 ± 1

37 ± 3

130 ± 15

68 ± 9

Untreated

28 ± 14

19 ± 5

44 ± 4

168 ± 13

73 ± 6

p-Nitrobenzoylaminobenzamid

3 µg

21 ± 7

17 ± 3

44 ± 12

149 ± 16

67 ± 6

trocken

10 µg

24 ± 5

13 ± 1

42 ± 12

123 ± 6

69 ± 16

33 µg

26 ± 2

15 ± 2

39 ± 12

122 ± 4

71 ± 18

100 µg

26 ± 4

23 ± 2

42 ± 4

149 ± 6

76 ± 8

333 µg

11 ± 4

19 ± 8

37 ± 4

138 ± 18

62 ± 4

1000 µg

26 ± 9P

17 ± 8P

43 ± 5P

133 ± 5P

71 ± 28P

2500 µg

17 ± 3P M

14 ± 5P M

40 ± 7P M

132 ± 8P M

61 ± 10P M

5000 µg

23 ± 5P M

11 ± 1P M

38 ± 3P M

129 ± 18P M

52 ± 10P M

2-AA

2.5 µg

243 ± 6

183 ± 19

1033 ± 137

2019 ± 79

2-AA

10.0 µg

381 ± 16

Key to Positive Controls

Key to Plate Postfix Codes

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

P

M

Precipitate

Manual count

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, p-Nitrobenzoylaminobenzamid trocken is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

The test item p-Nitrobenzoylaminobenzamid trocken was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA according to OECD guideline 471.

The assay was performed in two independent experiments both with and without metabolic activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed without metabolic activation in strain TA 1535 at 2500 µg/plate in experiment I, and in strain TA 1537 at 5000 µg/plate without metabolic activation in experiment I and II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with p-Nitrobenzoylaminobenzamid trocken at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, p-Nitrobenzoylaminobenzamid trocken is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.