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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
Study period:
2010 -09-20 till 2010-10-22
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:
2010
Report date:
2010

Materials and methods

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

Test material

Constituent 1
Reference substance name:
Automatically generated during migration to IUCLID 6, no data available
IUPAC Name:
Automatically generated during migration to IUCLID 6, no data available
Details on test material:
- Name of test material (as cited in study report): Acid Yellow RN 2903

- Physical state: Solid
- Purity test date: Approx. 65 %
- Lot/batch No.: Vers. Kilo 6,26.05.2009
- Expiration date of the lot/batch: 2015-08-01
- Stability under test conditions: 4 hours in water, polyethylene glycol, CMC at room temperature
- Storage condition of test material: Room temperature
- Other:

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 / non induced hamster liver S9
Test concentrations with justification for top dose:
3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.
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, congo red
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar, 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:
negative
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 at 2500 and 5000 µg/plate and on the incubated agar plates from 1000 - 5000 µg/plates. 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 metabolic activation in both independent experiments.
No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation with the exception of strain TA 98 with S9 mix where a minor toxic effect was observed at 5000 µg/plate in experiment II.
Remarks on result:
other: other: reverse mutation assay
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Tabellen

Pre-Experiment/Experiment I

Study Name: 1368902

Study Code: Harlan CCR 1368902

Experiment: 1368902 VV Plate

Date Plated: 20/09/2010

Assay Conditions:

Date Counted: 23/09/2010

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

Deionised water

12 ± 3

10 ± 1

39 ± 3

189 ± 9

50 ± 4

Untreated

11 ± 2

13 ± 0

39 ± 7

198 ± 4

61 ± 2

Acid Yellow RN

3 µg

13 ± 1

9 ± 3

44 ± 8

181 ± 13

50 ± 2

2903

10 µg

12 ± 2

13 ± 2

38 ± 10

193 ± 7

57 ± 1

33 µg

12 ± 2

10 ± 3

30 ± 3

206 ± 8

54 ± 9

100 µg

18 ± 4

13 ± 1

40 ± 5

202 ± 5

50 ± 12

333 µg

13 ± 7P

11 ± 3P

34 ± 2P

201 ± 10P

51 ± 4P

1000 µg

15 ± 4P

11 ± 4P

33 ± 1P

198 ± 9P

52 ± 5P

2500 µg

13 ± 4P

9 ± 1P

33 ± 5P

200 ± 10P

54 ± 4P

5000 µg

10 ± 4P M

6 ± 3P

26 ± 2P

133 ± 12P

43 ± 7P

NaN3

10 µg

1661 ± 22

1802 ± 57

4-NOPD

10 µg

342 ± 14

4-NOPD

50 µg

99 ± 12

MMS

3.0 µL

1350 ± 21

With Activation

Deionised water

14 ± 1

12 ± 0

42 ± 6

185 ± 25

66 ± 19

Untreated

18 ± 9

15 ± 4

50 ± 15

175 ± 9

62 ± 5

Acid Yellow RN

3 µg

15 ± 4

16 ± 2

41 ± 7

186 ± 19

64 ± 9

2903

10 µg

18 ± 2

12 ± 3

46 ± 7

190 ± 5

51 ± 3

33 µg

16 ± 4

14 ± 1

37 ± 2

201 ± 18

63 ± 11

100 µg

23 ± 7

10 ± 3

37 ± 3

194 ± 2

59 ± 10

333 µg

16 ± 4P

9 ± 3P

37 ± 5P

190 ± 6P

62 ± 5P

1000 µg

20 ± 4P

14 ± 6P

35 ± 8P

212 ± 13P

58 ± 6P

2500 µg

17 ± 1P

11 ± 4P

41 ± 3P

216 ± 11P

64 ± 4P

5000 µg

17 ± 4P M

7 ± 1P M

19 ± 2P M

153 ± 11P M

45 ± 7P M

2-AA

2.5 µg

433 ± 25

309 ± 22

2471 ± 212

2673 ± 31

2-AA

10.0 µg

283 ± 19

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

Experiment II

Study Name: 1368902

Study Code: Harlan CCR 1368902

Experiment: 1368902 HV2 Pre

Date Plated: 05/10/2010 / 19/10/2010*

Assay Conditions:

Date Counted: 08/10/2010 / 22/10/2010*

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

Deionised water

15 ± 2

9 ± 4

20 ± 2

177 ± 7

69 ± 6

Untreated

14 ± 2

7 ± 3

24 ± 2

200 ± 12

68 ± 7

Acid Yellow RN

3 µg

14 ± 2

10 ± 2

26 ± 3

185 ± 15

71 ± 14

2903

10 µg

16 ± 3

11 ± 7

27 ± 7

183 ± 6

62 ± 7

33 µg

13 ± 3

11 ± 3

17 ± 2

185 ± 13

85 ± 11

100 µg

22 ± 3

8 ± 2

20 ± 3

187 ± 6

76 ± 13

333 µg

14 ± 2

10 ± 1

26 ± 4

192 ± 8

62 ± 7

1000 µg

16 ± 3P

9 ± 1P

28 ± 7P

193 ± 8P

65 ± 7P

2500 µg

17 ± 5P

9 ± 4P

25 ± 4P

185 ± 7P

72 ± 1P

5000 µg

13 ± 1P

7 ± 2P

25 ± 4P

160 ± 17P

74 ± 10P

NaN3

10 µg

1743 ± 48

1598 ± 57

4-NOPD

10 µg

529 ± 115

4-NOPD

50 µg

73 ± 21

MMS

3 µL

1048 ± 16

With Activation

Deionised water

11 ± 2

7 ± 2

31 ± 9

157 ± 3

52 ± 7

Untreated

17 ± 4

6 ± 2

37 ± 6

143 ± 14

57 ± 5

Acid Yellow RN

3 µg

11 ± 2

7 ± 1

40 ± 6

152 ± 9

55 ± 9

2903

10 µg

10 ± 8

7 ± 3

34 ± 4

166 ± 12

57 ± 8

33 µg

12 ± 4

8 ± 2

26 ± 3

155 ± 10

50 ± 15

100 µg

12 ± 3

9 ± 4

31 ± 4

167 ± 11

60 ± 6

333 µg

17 ± 2

6 ± 1

45 ± 5

172 ± 14

63 ± 8

1000 µg

15 ± 4P

6 ± 2P

40 ± 1P

231 ± 6P

70 ± 7P

2500 µg

14 ± 5P

11 ± 4P

23 ± 4P M

203 ± 17P

64 ± 8P

5000 µg

12 ± 2P M

6 ± 2P M

13 ± 2P M

135 ± 20P M

53 ± 7P M

2-AA

2.5 µg

213 ± 28

151 ± 29

3189 ± 55

2-AA

2.5 µg

2-AA

10 µg

715 ± 73

Congo red

500 µg

249 ± 2

Key to Positive Controls

Key to Plate Postfix Codes

NaN3

2-AA

4-NOPD

Congo red

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

Congo red

methyl methane sulfonate

P

M

N

Precipitate

Manual count

Analysis not possible

* = repeated experiment

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutage¬nicity 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.
Executive summary:

This study was performed to investigate the potential of Acid Yellow RN 2903 to induce gene mutations according to the plate incorporation assay with rat liver S9 (experiment I), and the pre-incubation test with hamster liver S9 (experiment II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98 and TA 100 and theEscherichia colistrain WP2uvrA.

The assay was performed in two independent experiments with and without liver microsomal activation. Due to contamination, analysis of strain TA 98 was not possible in experiment II. This part of the experiment was repeated under identical conditions. The repeated part is reported as experiment II. Each concentration, including the controls, was 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.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation with the exception of strain TA 98 with S9 mix where a minor toxic effect was observed at 5000 µg/plate in experiment II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Acid Yellow RN 2903 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.