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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:
2014-08-26 till 2014-08-29
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:
2014
Report Date:
2014

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity: 99.2 % (w/w)
Expiry Date: 25 June 2024

Method

Species / strain
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Additional strain 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:
- Vehicle(s)/solvent(s) used: suspended in DMSO
- Justification for choice of solvent/vehicle: Better than others
Controls
Negative controls:
yes
Solvent 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 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 metabolic activation
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Water solubility: unsoluble
- Precipitation:
The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. No precipitation of the test item was observed on the incubated agar plates. The undissolved particles had no influence on the data recording.

- Other confounding effects: none
COMPARISON WITH HISTORICAL CONTROL DATA: performed
The data in the untreated control of strain TA 1535 were slightly above our historical control range in the absence of metabolic activation. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal back¬ground growth up to 5000 µg/plate with and without S9 mix in all strains used.
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.

Any other information on results incl. tables

Study Name: 1652800

Study Code: Harlan CCR 1652800

Experiment: 1652800 VV Plate

Date Plated: 26/08/2014

Assay Conditions:

Date Counted: 29/08/2014

 

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

 

 

26 ± 4B M

7 ± 2

21 ± 7

174 ± 10

35 ± 13

Untreated

 

 

28 ± 3B M

10 ± 3

22 ± 6

162 ± 30

48 ± 5

Fat Blue B 01

3 µg

 

27 ± 4B M

10 ± 4

28 ± 1

177 ± 2

42 ± 0

 

10 µg

 

26 ± 2B M

8 ± 2

19 ± 4

166 ± 9

39 ± 5

 

33 µg

 

30 ± 2B M

8 ± 1

22 ± 3

191 ± 2

44 ± 6

 

100 µg

 

26 ± 1B M

7 ± 1

17 ± 2

216 ± 16

41 ± 4

 

333 µg

 

28 ± 2B M

9 ± 1

19 ± 9

185 ± 16

54 ± 9

 

1000 µg

 

28 ± 1B D M

7 ± 1D

21 ± 1D

173 ± 10D

44 ± 9D

 

2500 µg

 

26 ± 5B D M

6 ± 2D M

21 ± 4D M

174 ± 11D M

32 ± 4D M

 

5000 µg

 

28 ± 2B D M

6 ± 1D M

17 ± 2D M

176 ± 13D M

27 ± 4D M

NaN3

10 µg

 

2459 ± 235

 

 

1835 ± 57

 

4-NOPD

10 µg

 

 

 

395 ± 13

 

 

4-NOPD

50 µg

 

 

80 ± 8

 

 

 

MMS

2.0 µL

 

 

 

 

 

1107 ± 49

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

13 ± 6

15 ± 2

30 ± 10

123 ± 23

55 ± 4

Untreated

 

 

14 ± 7

12 ± 5

38 ± 6

175 ± 4

57 ± 14

Fat Blue B 01

3 µg

 

19 ± 5

20 ± 1

41 ± 1

155 ± 7

41 ± 4

 

10 µg

 

18 ± 2

18 ± 5

32 ± 3

146 ± 5

54 ± 6

 

33 µg

 

17 ± 0

19 ± 4

37 ± 13

155 ± 4

53 ± 2

 

100 µg

 

16 ± 4

16 ± 1

42 ± 2

163 ± 16

51 ± 5

 

333 µg

 

13 ± 5

22 ± 4

73 ± 3

148 ± 16

50 ± 2

 

1000 µg

 

14 ± 3D

20 ± 5D

117 ± 13D

151 ± 18D

46 ± 5D

 

2500 µg

 

13 ± 2D M

21 ± 2D M

172 ± 33D M

168 ± 6D M

49 ± 10D M

 

5000 µg

 

13 ± 3D M

18 ± 2D M

192 ± 7D M

179 ± 5D M

34 ± 3D M

2-AA

2.5 µg

 

454 ± 13

310 ± 18

2167 ± 35

4272 ± 129

 

2-AA

10.0 µg

 

 

 

 

 

212 ± 10

 

 

 

 

 

 

 

 

 

 

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

D

M

B

Densely coloured plate

Manual count

Extensive bacterial growth

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive in strain TA 98 with metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strain TA 98 with S9 mix.
Executive summary:

The test item Fat Blue B 01 was assessed for its potential to induce gene mutations according to the plate incorporation test using Salmo­nella typhimurium strains TA 1535, TA 1537, TA 98, 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:

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

The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. No precipitation of the test item was observed on the incubated agar plates. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without S9 mix in all strains used.

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.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with Fat Blue B 01 at any dose level in the absence of metabolic activation (S9 mix).

In the presence of metabolic activation a substantial and dose dependent increase was observed in strain TA 98.The number of colonies exceeded the threshold of twice the number of the corresponding solvent control at concentrations from 333 µg/plate to 5000 µg/plate

 

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

The data in the untreated control of strain TA 1535 were slightly above our historical control range in the absence of metabolic activation. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.