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:
2011 -08-01 till 2011-08-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
614-960-9
EC Number:
614-960-9
Cas Number:
69399-57-1
Molecular formula:
C9H7N3O5
IUPAC Name:
614-960-9
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Nitro-C-Dion trocken (NMQD)
- Physical state: Yellow powder
- Analytical purity: 91.0 % (w/w); (dose calculation not adjusted to purity)
- Impurities (identity and concentrations):
- Stability under test conditions: > 72 h in DMSO at room temperature
- Storage condition of test material: At room temperature

Method

Species / strain
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I and II
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: better than other
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: plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


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: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
in strain TA 1537 and TA 98 without S9 mix
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation:
The test item precipitated in the overlay agar in the test tubes and on the incubated agar plates from 1000 to 5000 µg/plate. The undissolved particles had no influence on the data recording.
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxic effects
Remarks on result:
other: other: reverse mutation assay
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

     Summary of Results Pre-Experiment and Experiment I

 

Study Name: 1431901

Study Code: Harlan CCR 1431901

Experiment: 1431901 VV Plate

Date Plated: 01/08/2011

Assay Conditions:

Date Counted: 04/08/2011

 

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 ± 5

13 ± 4

33 ± 11

138 ± 9

48 ± 6

Untreated

 

 

18 ± 4

19 ± 2

36 ± 4

134 ± 9

50 ± 6

Nitro-C-Dion

3 µg

 

14 ± 7

17 ± 5

33 ± 5

133 ± 9

46 ± 5

trocken (NMQD)

10 µg

 

12 ± 4

14 ± 2

33 ± 8

132 ± 3

52 ± 3

 

33 µg

 

14 ± 2

13 ± 2

46 ± 3

130 ± 13

54 ± 3

 

100 µg

 

13 ± 3

19 ± 3

56 ± 14

146 ± 12

53 ± 10

 

333 µg

 

16 ± 3

22 ± 1

55 ± 12

123 ± 7

41 ± 11

 

1000 µg

 

16 ± 3P

27 ± 1P

60 ± 11P M

145 ± 10P

40 ± 8P

 

2500 µg

 

7 ± 1P M

31 ± 4P M

52 ± 10P M

141 ± 28P

46 ± 6P

 

5000 µg

 

12 ± 2P M

41 ± 4P M

62 ± 8P M

150 ± 3P M

38 ± 4P M

NaN3

10 µg

 

1892 ± 120

 

 

2097 ± 22

 

4-NOPD

10 µg

 

 

 

338 ± 34

 

 

4-NOPD

50 µg

 

 

94 ± 14

 

 

 

MMS

3.0 µL

 

 

 

 

 

1021 ± 41

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

18 ± 5

18 ± 3

37 ± 7

140 ± 6

61 ± 5

Untreated

 

 

16 ± 5

19 ± 9

45 ± 6

156 ± 3

56 ± 13

Nitro-C-Dion

3 µg

 

18 ± 5

13 ± 2

42 ± 14

158 ± 19

58 ± 5

trocken (NMQD)

10 µg

 

21 ± 4

15 ± 5

38 ± 4

137 ± 7

58 ± 12

 

33 µg

 

16 ± 1

17 ± 2

43 ± 7

144 ± 14

53 ± 3

 

100 µg

 

19 ± 4

16 ± 6

45 ± 3

144 ± 19

62 ± 3

 

333 µg

 

20 ± 6

20 ± 8

57 ± 1

151 ± 12

50 ± 2

 

1000 µg

 

18 ± 8P

21 ± 6P

44 ± 4P M

169 ± 16P

53 ± 2P

 

2500 µg

 

19 ± 6P M

18 ± 5P M

44 ± 6P M

168 ± 15P M

40 ± 5P M

 

5000 µg

 

11 ± 4P M

18 ± 5P M

43 ± 6P M

153 ± 13P M

48 ± 4P M

2-AA

2.5 µg

 

358 ± 39

283 ± 12

2027 ± 170

2284 ± 166

 

2-AA

10.0 µg

 

 

 

 

 

362 ± 18

 

 

 

 

 

 

 

 

 

 

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: 1431901

Study Code: Harlan CCR 1431901

Experiment: 1431901 HV2 Pre

Date Plated: 09/08/2011

Assay Conditions:

Date Counted: 12/08/2011

 

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

 

 

13 ± 1

14 ± 1

25 ± 3

99 ± 1

45 ± 8

Untreated

 

 

10 ± 5

19 ± 6

35 ± 1

131 ± 12

48 ± 6

Nitro-C-Dion

3 µg

 

15 ± 6

16 ± 1

31 ± 7

99 ± 7

58 ± 8

trocken (NMQD)

10 µg

 

10 ± 3

17 ± 6

40 ± 4

102 ± 12

52 ± 6

 

33 µg

 

15 ± 2

24 ± 5

46 ± 4

112 ± 12

59 ± 9

 

100 µg

 

15 ± 1

22 ± 7

70 ± 1

109 ± 14

52 ± 8

 

333 µg

 

17 ± 3

17 ± 5

70 ± 4

115 ± 11

58 ± 6

 

1000 µg

 

12 ± 4P

25 ± 4P

63 ± 10P

113 ± 1P

48 ± 11P

 

2500 µg

 

10 ± 2P M

25 ± 2P M

53 ± 7P M

124 ± 4P M

46 ± 6P M

 

5000 µg

 

13 ± 1P M

47 ± 6P M

70 ± 7P M

121 ± 6P M

52 ± 5P M

NaN3

10 µg

 

1886 ± 174

 

 

2037 ± 110

 

4-NOPD

10 µg

 

 

 

300 ± 2

 

 

4-NOPD

50 µg

 

 

89 ± 15

 

 

 

MMS

3.0 µL

 

 

 

 

 

539 ± 59

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

18 ± 5

21 ± 8

41 ± 9

142 ± 9

65 ± 6

Untreated

 

 

18 ± 3

22 ± 5

44 ± 6

150 ± 13

60 ± 11

Nitro-C-Dion

3 µg

 

22 ± 5

23 ± 2

38 ± 11

141 ± 21

66 ± 7

trocken (NMQD)

10 µg

 

18 ± 5

19 ± 4

45 ± 14

126 ± 17

61 ± 3

 

33 µg

 

21 ± 9

22 ± 4

44 ± 9

135 ± 14

64 ± 7

 

100 µg

 

20 ± 4

22 ± 9

62 ± 4

118 ± 21

60 ± 3

 

333 µg

 

18 ± 2

24 ± 3

55 ± 1

130 ± 15

46 ± 10

 

1000 µg

 

23 ± 7P

23 ± 4P

63 ± 6P

143 ± 11P

67 ± 18P

 

2500 µg

 

18 ± 3P M

19 ± 4P M

59 ± 2P M

141 ± 6P M

67 ± 7P M

 

5000 µg

 

16 ± 6P M

20 ± 5P M

63 ± 21P M

135 ± 26P M

65 ± 14P M

2-AA

2.5 µg

 

311 ± 8

230 ± 6

1639 ± 86

1356 ± 122

 

2-AA

10.0 µg

 

 

 

 

 

399 ± 99

 

 

 

 

 

 

 

 

 

 

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):
positive without S9 mix in strains TA 1537 and TA 98

In conclusion, it can be stated that during the described mutage¬nicity test and under the experimental conditions reported, the test item did induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

The test item Nitro-C-Dion trocken (NMQD) was assessed for its potential to induce gene mutations in the plate incorporation test (experi­ment I) and the pre-incubation test (experiment II) usingSalmo­nella typhimuriumstrains TA 1535, TA 1537, TA 98, and TA 100, and theEscheri­chia colistrain WP2 uvrA.

The assay was performed with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I and II: 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 S9 mix in both experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabol­ic activation.

Substantial and dose dependent increases in revertant colony numbers were observed following treatment with Nitro-C-Dion trocken (NMQD) in strains TA 98 and TA 1537 in the absence of metabolic activation (S9 mix). The number of colonies exceeded the threshold of trice the number of the corresponding solvent control in strain TA 1537 at 5000 µg/plate. A slight and dose dependent increase in the number of revertants was also detected in strain TA 98 without metabolic activation but the threshold of two was not quite reached. To verify the weak positive response of the first experiment, a second experiment was performed using the more sensitive pre-incubation method. In experiment II without S9 mix the number of colonies exceeded the threshold of trice the number of the corresponding solvent control in strain TA 1537 at 5000 µg/plate and of twice the number of the corresponding solvent control in strain TA 98 from 100 µg/plate up to 5000 µg/plate.

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