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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform OECD 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:
2,3-Quinoxalinedione, 6-amino-1,4-dihydro-7-methoxy-
EC Number:
615-027-9
Cas Number:
69904-10-5
Molecular formula:
C9H9N3O3
IUPAC Name:
2,3-Quinoxalinedione, 6-amino-1,4-dihydro-7-methoxy-
Details on test material:
- Name of test material (as cited in study report): Amino-C-Dion trocken
- Physical state: solid, yellow
- Analytical purity: 93.8 % (w/w) comp.1
- Stability under test conditions: at least 72 h in DMSO at room temperature
- Storage condition of test material: At room temperature
- Other:
CAS No: 69904-10-5
Correction factor: 1.0

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
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: better than others (Wter and ethanol)
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


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 98 with 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
- Effects of pH: none
- Water solubility: not soluble
- Precipitation:
The test item precipitated in the overlay agar in the test tubes and on the incubated agar from 2500 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:
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 metabol¬ic activation.
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: 1423202

Study Code: Harlan CCR 1423202

Experiment: 1423202 VV Plate

Date Plated: 18/07/2011

Assay Conditions:

Date Counted: 21/07/2011

 

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 ± 4

9 ± 1

33 ± 3

122 ± 18

51 ± 6

Untreated

 

 

14 ± 2

9 ± 2

36 ± 10

121 ± 15

49 ± 6

Amino-C-Dion

3 µg

 

16 ± 6

9 ± 1

31 ± 10

119 ± 2

48 ± 14

trocken

10 µg

 

14 ± 1

9 ± 1

36 ± 5

130 ± 24

49 ± 12

 

33 µg

 

16 ± 4

12 ± 0

29 ± 6

124 ± 11

58 ± 7

 

100 µg

 

17 ± 5

9 ± 3

34 ± 6

121 ± 25

49 ± 7

 

333 µg

 

17 ± 10

12 ± 3

31 ± 3

129 ± 3

45 ± 8

 

1000 µg

 

15 ± 4

11 ± 2

29 ± 4

112 ± 12

51 ± 14

 

2500 µg

 

16 ± 4P

9 ± 3P

24 ± 3P

107 ± 2P

41 ± 3P

 

5000 µg

 

18 ± 2P

10 ± 2P

30 ± 6P

123 ± 7P

48 ± 6P

NaN3

10 µg

 

1901 ± 95

 

 

2245 ± 49

 

4-NOPD

10 µg

 

 

 

345 ± 16

 

 

4-NOPD

50 µg

 

 

87 ± 11

 

 

 

MMS

3.0 µL

 

 

 

 

 

886 ± 26

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

20 ± 4

15 ± 1

43 ± 5

140 ± 8

58 ± 4

Untreated

 

 

18 ± 2

9 ± 6

44 ± 4

143 ± 7

59 ± 7

Amino-C-Dion

3 µg

 

22 ± 5

13 ± 5

40 ± 7

134 ± 17

51 ± 5

trocken

10 µg

 

18 ± 6

16 ± 5

35 ± 2

127 ± 9

56 ± 9

 

33 µg

 

19 ± 4

15 ± 7

37 ± 3

129 ± 14

60 ± 7

 

100 µg

 

15 ± 1

16 ± 6

48 ± 3

135 ± 8

62 ± 23

 

333 µg

 

20 ± 4

18 ± 3

68 ± 10

156 ± 5

54 ± 2

 

1000 µg

 

15 ± 2

19 ± 5

134 ± 12

138 ± 15

54 ± 8

 

2500 µg

 

17 ± 10P

34 ± 4P

219 ± 6P

121 ± 11P

53 ± 4P

 

5000 µg

 

15 ± 6P

36 ± 6P

237 ± 31P

134 ± 22P

47 ± 9P

2-AA

2.5 µg

 

377 ± 11

319 ± 24

2470 ± 66

2353 ± 113

 

2-AA

10.0 µg

 

 

 

 

 

288 ± 41

 

 

 

 

 

 

 

 

 

 

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

Precipitate

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation in strain 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 frameshifts in the genome of the strain TA 98 with S9 mix.
Executive summary:

The test item Amino-C-Dion trocken was assessed for its potential to induce gene mutations in the plate incorporation test (experi­ment I) 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:          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 Amino-C-Dion trocken in strains TA 98 and TA 1537 in the presence of metabolic activation (S9 mix). The number of colonies exceeded the threshold of twice in strain TA 98 the number of the corresponding solvent control at concentrations from 1000 up to 5000 µg/plate. A slight and dose dependent increase in the number of revertants was also detected in strain TA 1537 with metabolic activation but the threshold of three was not quite reached.

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