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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:
2011 -01-26 till 2011-01-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conforming study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
from May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
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:
- Physical state: solid, brown
- Storage condition of test material: at room temperature < 30° C, light protected

Method

Species / strain
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA pKM101, WP2 pKM101
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; experiment II
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubilisation properties and its relative non-toxicity to the bacteria.
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 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 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 pKM101, WP2 pKM101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Precipitation of the test item was observed in the overlay agar in the test tubes from 1000 - 5000 µg/plate. Precipitation was also observed on the incubated agar plates without S9 mix from 333 - 5000 µg/plate in experiment I and at 2500 and 5000 µg/plate in experiment II, and with S9 mix from 1000 - 5000 µg/plate in both experiments. 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.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations in strain WP2 pKM101 without S9 mix and in strains TA1535, TA98, WP2 pKM101, and WP2 uvrA pKM101 with S9 mix in experiment I. In experiment II, toxic effects were observed without S9 mix in all strains and with S9 mix in strains TA 1537, TA 97, and WP2 pKM101.

Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

 Summary of Results Pre-Experiment/Experiment

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA1535

TA1537

TA98

TA100

WP2 pKM101

WP2 uvrA pKM101

 

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

 

15 ± 4

9 ± 3

25 ± 2

138 ± 4

170 ± 5

340 ± 10

Untreated

 

 

19 ± 3

10 ± 4

30 ± 5

156 ± 2

224 ± 10

383 ± 34

Test substance

3 µg

 

15 ± 4

8 ± 1

23 ± 5

142 ± 20

187 ± 43

417 ± 18

 

10 µg

 

15 ± 5

8 ± 1

25 ± 4

117 ± 13

180 ± 25

334 ± 25

 

33 µg

 

13 ± 4

8 ± 1

26 ± 3

132 ± 21

147 ± 13

322 ± 18

 

100 µg

 

13 ± 2

7 ± 2

20 ± 2

134 ± 18

151 ± 22

301 ± 20

 

333 µg

 

19 ± 2P

9 ± 1P

28 ± 6P

143 ± 12P

162 ± 21P

397 ± 25P

 

1000 µg

 

13 ± 3P

7 ± 1P

25 ± 8P

158 ± 3P

101 ± 1P

347 ± 43P

 

2500 µg

 

9 ± 1P M

6 ± 1P

21 ± 5P M

106 ± 1P

100 ± 2P

259 ± 7P

 

5000 µg

 

8 ± 2P M

5 ± 1P M

22 ± 4P M

86 ± 10P M

42 ± 9P M

258 ± 25P M

NaN3

10 µg

 

2129 ± 83

 

 

2025 ± 101

 

 

4-NOPD

10 µg

 

 

 

277 ± 20

 

 

 

4-NOPD

50 µg

 

 

76 ± 3

 

 

 

 

MMS

3.0 µL

 

 

 

 

 

2531 ± 197

3035 ± 329

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

18 ± 2

13 ± 3

32 ± 5

126 ± 9

174 ± 6

404 ± 24

Untreated

 

 

24 ± 5

22 ± 2

33 ± 10

145 ± 6

241 ± 29

497 ± 4

Test substance

3 µg

 

20 ± 4

14 ± 7

32 ± 5

124 ± 19

199 ± 23

395 ± 51

 

10 µg

 

20 ± 2

11 ± 4

34 ± 7

134 ± 14

180 ± 35

353 ± 42

 

33 µg

 

14 ± 5

13 ± 2

33 ± 9

113 ± 7

188 ± 20

344 ± 36

 

100 µg

 

16 ± 1

13 ± 3

29 ± 5

132 ± 26

137 ± 4

316 ± 35

 

333 µg

 

16 ± 7

18 ± 2

29 ± 4

147 ± 8

151 ± 3

344 ± 19

 

1000 µg

 

8 ± 2P M

8 ± 2P M

30 ± 6P

121 ± 11P

113 ± 12P

294 ± 32P

 

2500 µg

 

8 ± 2P M

8 ± 2P M

13 ± 2P M

117 ± 3P

105 ± 12P

233 ± 9P

 

5000 µg

 

8 ± 1P M

7 ± 3P M

12 ± 2P M

96 ± 8P M

41 ± 8P M

168 ± 34P

2-AA

2.5 µg

 

392 ± 36

269 ± 40

1731 ± 173

1984 ± 352

 

 

2-AA

10.0 µg

 

 

 

 

 

1412 ± 60

1443 ± 27

 

 

 

 

 

 

 

 

 

 

 

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

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA1535

TA1537

TA98

TA100

WP2 pKM101

WP2 uvrA pKM101

 

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

 

16 ± 1

10 ± 3

22 ± 6

124 ± 10

183 ± 21

365 ± 20

Untreated

 

 

20 ± 1

13 ± 2

30 ± 1

160 ± 5

202 ± 40

372 ± 18

Test substance

3 µg

 

16 ± 4

11 ± 4

21 ± 1

130 ± 5

199 ± 5

331 ± 30

 

10 µg

 

14 ± 2

11 ± 4

24 ± 3

115 ± 15

185 ± 13

312 ± 10

 

33 µg

 

17 ± 3

10 ± 2

24 ± 2

104 ± 3

178 ± 13

324 ± 25

 

100 µg

 

17 ± 2

13 ± 1

23 ± 4

111 ± 11

177 ± 7

336 ± 19

 

333 µg

 

15 ± 2

14 ± 1

21 ± 1

89 ± 12

173 ± 19

334 ± 21

 

1000 µg

 

10 ± 2

12 ± 4

23 ± 2

78 ± 22

110 ± 4

288 ± 50

 

2500 µg

 

8 ± 1P

7 ± 2P

21 ± 2P

54 ± 8P

93 ± 4P

195 ± 11P

 

5000 µg

 

2 ± 0P M

1 ± 1P M

5 ± 2P M

21 ± 5P M

50 ± 6P M

149 ± 17P M

NaN3

10 µg

 

1735 ± 114

 

 

2196 ± 108

 

 

4-NOPD

10 µg

 

 

 

374 ± 14

 

 

 

4-NOPD

50 µg

 

 

74 ± 9

 

 

 

 

MMS

3.0 µL

 

 

 

 

 

3224 ± 165

2523 ± 147

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

15 ± 7

21 ± 1

34 ± 6

133 ± 22

200 ± 6

397 ± 11

Untreated

 

 

20 ± 6

26 ± 5

44 ± 7

154 ± 12

249 ± 39

412 ± 47

Test substance

3 µg

 

17 ± 7

22 ± 2

37 ± 6

136 ± 10

196 ± 27

373 ± 18

 

10 µg

 

16 ± 6

19 ± 3

29 ± 7

130 ± 7

186 ± 38

379 ± 9

 

33 µg

 

11 ± 3

19 ± 7

31 ± 5

129 ± 18

219 ± 22

368 ± 18

 

100 µg

 

14 ± 1

19 ± 4

36 ± 1

116 ± 12

221 ± 17

369 ± 15

 

333 µg

 

18 ± 2

19 ± 3

33 ± 9

125 ± 15

198 ± 26

358 ± 19

 

1000 µg

 

15 ± 2P

15 ± 5P

35 ± 2P

104 ± 13P

122 ± 4P

332 ± 26P

 

2500 µg

 

12 ± 2P M

11 ± 4P M

22 ± 2P M

84 ± 12P M

77 ± 6P M

233 ± 14P M

 

5000 µg

 

9 ± 3P M

6 ± 2P M

14 ± 1P M

85 ± 6P M

65 ± 11P M

201 ± 22P M

2-AA

2.5 µg

 

296 ± 16

199 ± 1

1447 ± 136

2103 ± 137

 

 

2-AA

10.0 µg

 

 

 

 

 

1459 ± 66

1664 ± 73

 

 

 

 

 

 

 

 

 

 

 

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

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at the following concentrations (μg/plate):

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

 without S9 mix

 with S9 mix

TA1535

/

2500

5000

/

TA1537

/

/

5000

5000

TA98

/

2500, 5000

5000

5000

TA100

/

/

2500, 5000

/

WP2 pKM101

5000

5000

5000

2500, 5000

WP2 uvrA pKM101

/

5000

5000

/

/ = no toxic effects observed

 

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 substance 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 substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strains WP2 uvrA pKM101 and WP2 pKM101.

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.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5),

were observed at higher concentrations in strain WP2 pKM101 without S9 mix and in strains TA1535, TA98, WP2 pKM101, and WP2 uvrA pKM101 with S9 mix in experiment I. In experiment II, toxic effects were observed without S9 mix in all strains and with S9 mix in strains TA 1537, TA97, and WP2 pKM101.

No substantial increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance 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.