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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:
2015 -04-01 till 2015-05-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

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

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
33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dissolved in deionised water[
- Justification for choice of solvent/vehicle: best suitable solvent
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: 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
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
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
Water solubility: soluble
Precipitation:
The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 µg/plate with and without S9 mix in experiment I and from 2500 to 5000 µg/plate with S9 mix in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 2500 to 5000 µg/plate. The undissolved particles had no influence on the data recording.
Other confounding effects: none
COMPARISON WITH HISTORICAL CONTROL DATA: performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY: 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 Tabellen

Table1     Summary of Experiment I

Study Name: 1684702

Study Code: Harlan CCR 1684702

Experiment: 1684702 VV Plate

Date Plated: 01/04/2015

Assay Conditions:

Date Counted: 08/04/2015

 

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

 

 

9 ± 1

9 ± 3

27 ± 6

156 ± 19

41 ± 6

Untreated

 

 

8 ± 3

9 ± 1

23 ± 7

176 ± 21

37 ± 4

3 µg

 

9 ± 3

10 ± 1

25 ± 7

158 ± 14

40 ± 8

4N trocken

10 µg

 

9 ± 3

10 ± 2

30 ± 6

176 ± 9

49 ± 1

 

33 µg

 

9 ± 2

10 ± 2

25 ± 8

173 ± 10

46 ± 10

 

100 µg

 

10 ± 2

9 ± 3

25 ± 4

184 ± 9

41 ± 10

 

333 µg

 

11 ± 3

10 ± 3

27 ± 4

178 ± 19

45 ± 11

 

1000 µg

 

9 ± 3

6 ± 2

24 ± 6

161 ± 6

46 ± 7

 

2500 µg

 

8 ± 2P

8 ± 3P

24 ± 6P

166 ± 4P

44 ± 2P

 

5000 µg

 

8 ± 2P

9 ± 1P

23 ± 2P

169 ± 18P

42 ± 5P

NaN3

10 µg

 

1175 ± 97

 

 

2116 ± 124

 

4-NOPD

10 µg

 

 

 

325 ± 21

 

 

4-NOPD

50 µg

 

 

68 ± 9

 

 

 

MMS

2.0 µL

 

 

 

 

 

850 ± 137

 

 

 

 

 

 

 

 

 

With Activation

Deionised water

 

 

11 ± 3

14 ± 3

38 ± 1

121 ± 16

53 ± 5

Untreated

 

 

9 ± 4

14 ± 2

39 ± 10

122 ± 3

50 ± 7

Sanodal Gold

3 µg

 

11 ± 2

13 ± 1

38 ± 10

122 ± 8

45 ± 5

4N trocken

10 µg

 

10 ± 3

12 ± 3

35 ± 7

121 ± 6

50 ± 6

 

33 µg

 

13 ± 1

13 ± 2

32 ± 9

130 ± 26

54 ± 5

 

100 µg

 

10 ± 3

15 ± 5

35 ± 11

122 ± 2

47 ± 7

 

333 µg

 

9 ± 2

14 ± 5

37 ± 5

140 ± 17

66 ± 4

 

1000 µg

 

11 ± 2

12 ± 3

33 ± 6

151 ± 7

52 ± 10

 

2500 µg

 

10 ± 3P

13 ± 2M P

35 ± 2P

136 ± 5P

43 ± 8P

 

5000 µg

 

10 ± 3P

14 ± 2M P

38 ± 15P

165 ± 15P

54 ± 16P

2-AA

2.5 µg

 

454 ± 30

165 ± 20

4724 ± 512

3255 ± 160

 

2-AA

10.0 µg

 

 

 

 

 

379 ± 34

 

 

 

 

 

 

 

 

 

 

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

 

 


Table2     Summary of Experiment II

Study Name: 1684702

Study Code: Harlan CCR 1684702

Experiment: 1684702 HV2 pre

Date Plated: 28/04/2015

Assay Conditions:

Date Counted: 04/05/2015

 

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

 

 

8 ± 1

11 ± 3

23 ± 8

179 ± 11

41 ± 8

Untreated

 

 

10 ± 2

8 ± 2

32 ± 9

189 ± 17

39 ± 3

Sanodal Gold

33 µg

 

8 ± 2

13 ± 2

24 ± 3

202 ± 24

43 ± 7

4N trocken

100 µg

 

6 ± 5

8 ± 5

23 ± 7

197 ± 19

45 ± 9

 

333 µg

 

9 ± 3

8 ± 1

25 ± 10

158 ± 0

38 ± 9

 

1000 µg

 

13 ± 4

11 ± 5

29 ± 6

204 ± 15

39 ± 11

 

2500 µg

 

12 ± 2

9 ± 4

22 ± 2

183 ± 7

41 ± 5

 

5000 µg

 

14 ± 1

8 ± 2

27 ± 8

182 ± 13

36 ± 1

NaN3

10 µg

 

890 ± 39

 

 

1670 ± 149

 

4-NOPD

10 µg

 

 

 

334 ± 38

 

 

4-NOPD

50 µg

 

 

74 ± 3

 

 

 

MMS

2.0 µL

 

 

 

 

 

661 ± 69

 

 

 

 

 

 

 

 

 

With Activation

Deionised water

 

 

11 ± 2

17 ± 0

30 ± 7

204 ± 6

42 ± 7

Untreated

 

 

10 ± 3

21 ± 4

30 ± 6

189 ± 7

48 ± 7

Sanodal Gold

33 µg

 

6 ± 1

12 ± 3

32 ± 5

202 ± 19

40 ± 8

4N trocken

100 µg

 

18 ± 4

17 ± 6

38 ± 2

193 ± 29

55 ± 8

 

333 µg

 

9 ± 1

10 ± 3

39 ± 9

144 ± 7

50 ± 9

 

1000 µg

 

12 ± 3

14 ± 1

34 ± 6

192 ± 23

53 ± 3

 

2500 µg

 

10 ± 5

14 ± 3

33 ± 3

228 ± 129

40 ± 11

 

5000 µg

 

9 ± 2

16 ± 4

36 ± 4

154 ± 18

39 ± 11

2-AA

2.5 µg

 

176 ± 14

155 ± 29

3415 ± 381

3108 ± 509

 

2-AA

10.0 µg

 

 

 

 

 

333 ± 121

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

 

 

 

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

 

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative

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, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was 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

Experiment II:                                33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 µg/plate with and without S9 mix in experiment I and from 2500 to 5000 µg/plate with S9 mix in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 2500 to 5000 µg/plate. 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 five tester strains was observed following treatment with the test item 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 and showed a distinct in­crease of induced revertant colonies.