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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: other: bacterial reverse mutation test, gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010 -01-27 till 2010-02-22
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:
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)
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
- Stability under test conditions: not reported
- Storage condition of test material: At room temperature at about 20 °C

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 pKM 101, WP2 pKM101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
minor toxic effect in strain TA98
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 and on the incubated agar plates from 1000 - 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 metabolic activation in both independent experiments.

In both experiments, no toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed with the exception of strain TA 98 where a minor toxic effect was observed at 5000 µg/plate with S9 mix in experiment I.
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

         Summary of Results Pre-Experiment/Experiment I

 

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 pKM101

S uvrA pKM101

 

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

 

13 ± 1

12 ± 3

25 ± 4

117 ± 5

191 ± 5

363 ± 8

Untreated

 

 

10 ± 4

11 ± 5

31 ± 3

128 ± 19

202 ± 6

372 ± 13

Test Substance

3 µg

 

9 ± 2

11 ± 3

25 ± 4

117 ± 7

215 ± 35

327 ± 17

 

10 µg

 

10 ± 2

12 ± 3

27 ± 4

114 ± 7

200 ± 36

322 ± 14

 

33 µg

 

12 ± 3

11 ± 2

25 ± 4

120 ± 21

195 ± 14

334 ± 34

 

100 µg

 

11 ± 4

9 ± 1

25 ± 8

119 ± 4

194 ± 22

321 ± 35

 

333 µg

 

13 ± 4

14 ± 1

28 ± 7

131 ± 10

203 ± 4

320 ± 23

 

1000 µg

 

12 ± 2P M

8 ± 3P M

23 ± 6P

115 ± 2P

181 ± 9P

321 ± 52P

 

2500 µg

 

11 ± 2P M

6 ± 1P M

14 ± 2P M

107 ± 5P M

176 ± 17P

335 ± 23P M

 

5000 µg

 

8 ± 1P M

6 ± 2P M

12 ± 3P M

104 ± 10P M

93 ± 14P M

309 ± 11P M

NaN3

10 µg

 

1620 ± 133

 

 

2072 ± 214

 

 

4-NOPD

10 µg

 

 

 

345 ± 18

 

 

 

4-NOPD

50 µg

 

 

75 ± 14

 

 

 

 

MMS

3.0 µL

 

 

 

 

 

3381 ± 703

2653 ± 127

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

20 ± 6

17 ± 2

33 ± 6

108 ± 6

211 ± 30

414 ± 31

Untreated

 

 

15 ± 6

22 ± 1

45 ± 4

122 ± 1

257 ± 11

435 ± 30

Test Substance

3 µg

 

18 ± 4

18 ± 5

35 ± 4

115 ± 1

221 ± 39

351 ± 79

 

10 µg

 

17 ± 3

19 ± 2

38 ± 9

113 ± 10

217 ± 2

446 ± 44

 

33 µg

 

15 ± 2

16 ± 3

34 ± 6

115 ± 11

205 ± 22

383 ± 41

 

100 µg

 

19 ± 6

20 ± 1

35 ± 2

117 ± 17

193 ± 15

420 ± 13

 

333 µg

 

13 ± 3

18 ± 4

36 ± 4

102 ± 9

212 ± 54

373 ± 23

 

1000 µg

 

15 ± 2P M

13 ± 5P M

35 ± 2P

106 ± 10P

157 ± 14P

396 ± 9P M

 

2500 µg

 

11 ± 1P M

10 ± 1P M

27 ± 1P M

96 ± 13P M

133 ± 11P M

408 ± 5P M

 

5000 µg

 

14 ± 2P M

8 ± 2P M

13 ± 2P M

102 ± 4P M

106 ± 11P M

382 ± 12P M

2-AA

2.5 µg

 

249 ± 6

207 ± 37

1719 ± 236

1585 ± 66

 

 

2-AA

10.0 µg

 

 

 

 

 

1261 ± 18

1556 ± 6

 

 

 

 

 

 

 

 

 

 

 

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

 

Study Name: 1389001

Study Code: Harlan CCR 1389001

Experiment: 1389001 HV2 Pre

Date Plated: 16/02/2011

Assay Conditions:

Date Counted: 22/02/2011

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 pKM101

WP2 uvrA pKM101

 

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

 

12 ± 2

11 ± 4

23 ± 3

122 ± 6

178 ± 4

343 ± 13

Untreated

 

 

12 ± 8

12 ± 5

33 ± 8

136 ± 3

206 ± 19

363 ± 35

Test Substance

3 µg

 

12 ± 5

9 ± 1

25 ± 4

112 ± 4

192 ± 13

347 ± 29

 

10 µg

 

10 ± 2

12 ± 3

20 ± 4

118 ± 16

177 ± 11

320 ± 12

 

33 µg

 

15 ± 6

12 ± 2

25 ± 2

115 ± 12

168 ± 4

274 ± 21

 

100 µg

 

15 ± 2

8 ± 3

22 ± 5

109 ± 9

173 ± 10

275 ± 28

 

333 µg

 

14 ± 5

11 ± 4

22 ± 5

113 ± 23

179 ± 25

335 ± 14

 

1000 µg

 

10 ± 3P M

12 ± 1P M

22 ± 5P M

108 ± 8P

179 ± 1P

308 ± 23P

 

2500 µg

 

13 ± 2P M

13 ± 2P M

23 ± 4P M

112 ± 8P M

124 ± 9P M

312 ± 40P M

 

5000 µg

 

10 ± 2P M

12 ± 4P M

17 ± 3P M

112 ± 8P M

106 ± 15P M

244 ± 4P M

NaN3

10 µg

 

2047 ± 55

 

 

2262 ± 79

 

 

4-NOPD

10 µg

 

 

 

344 ± 25

 

 

 

4-NOPD

50 µg

 

 

63 ± 4

 

 

 

 

MMS

3.0 µL

 

 

 

 

 

2911 ± 21

2536 ± 47

 

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

19 ± 3

18 ± 2

37 ± 10

145 ± 8

207 ± 9

396 ± 19

Untreated

 

 

23 ± 7

27 ± 4

44 ± 8

163 ± 16

263 ± 15

501 ± 26

Test Substance

3 µg

 

21 ± 6

21 ± 6

38 ± 6

140 ± 10

205 ± 14

339 ± 20

 

10 µg

 

21 ± 3

17 ± 2

37 ± 3

153 ± 12

202 ± 15

382 ± 73

 

33 µg

 

20 ± 6

17 ± 4

33 ± 8

141 ± 8

172 ± 11

359 ± 60

 

100 µg

 

16 ± 4

16 ± 6

36 ± 10

144 ± 3

173 ± 23

365 ± 38

 

333 µg

 

14 ± 2

21 ± 3

40 ± 6

147 ± 27

194 ± 33

368 ± 57

 

1000 µg

 

14 ± 0P M

14 ± 2P M

26 ± 3P M

143 ± 13P

176 ± 2P

319 ± 30P

 

2500 µg

 

10 ± 4P M

11 ± 3P M

31 ± 1P M

113 ± 6P M

146 ± 8P M

303 ± 40P M

 

5000 µg

 

11 ± 3P M

9 ± 3P M

29 ± 1P M

116 ± 6P M

128 ± 6P M

292 ± 21P M

2-AA

2.5 µg

 

307 ± 15

209 ± 26

1323 ± 97

1540 ± 56

 

 

2-AA

10.0 µg

 

 

 

 

 

1721 ± 77

1322 ± 74

 

 

 

 

 

 

 

 

 

 

 

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

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 theSalmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strains WP2uvrApKM101 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.

 

In both experiments, no toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed with the exception of strain TA 98 where a minor toxic effect was observed at 5000 µg/plate with S9 mix in experiment I.

 

No substantial increase in revertant colony numbers of any of the five 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.

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.