Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
From 22 April to 08 May, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, guidline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

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
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strainopen allclose all
Species / strain:
other: TA 1535, TA 100, TA 1537, TA 98
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Napthoflavone induced rat liver S9
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Napthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Doses adjusted for content of the main constituent
Experiment 1: 3; 10; 33; 100; 333; 1000; 2500; and 5000; µg/plate
Experiment 2: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: acceptable solubility of test substance
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535; TA 100 without metabolic activation
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Remarks:
TA 1537; TA 98 without metabolic activation
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA without metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535; TA 1537; TA 98, TA100, Wp2 uvrA with metabolic activation
Details on test system and conditions:
METHOD OF APPLICATION: experiment 1: plate incorporation; experiment 2: preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
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.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

There were no toxic effects. Plates incubated with the test item showed normal background growth up to 5000µg/plate with and without S9 mix in both experiments.

No substantial increase in revertant colony numbers of any of the 5 tester strains was observed following treatment with TRIQUAT MONOMER 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 these showed a distinct increase of induced revertant colonies.

Results of Gene Mutation Assay: Pre-experiment and Experiment 1:

Metabolic

Activation

Test

Group

Dose Level

(µg/plate)

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without Activation

Deionised water

14 ± 2

15 ± 4

37 ± 3

150 ± 12

49 ± 10

Untreated

14 ± 4

16 ± 2

39 ± 5

129 ± 20

50 ± 1

Triquat

3 µg

14 ± 3

18 ± 3

33 ± 5

153 ± 18

35 ± 4

Monomer

10 µg

17 ± 2

17 ± 2

35 ± 2

157 ± 8

48 ± 4

33 µg

15 ± 1

15 ± 3

34 ± 1

143 ± 9

46 ± 3

100 µg

11 ± 2

15 ± 2

36 ± 1

150 ± 8

43 ± 6

333 µg

13 ± 1

18 ± 4

28 ± 2

148 ± 10

37 ± 6

1000 µg

14 ± 5

15 ± 5

29 ± 3

145 ± 6

44 ± 8

2500 µg

14 ± 3

18 ± 3

33 ± 7

167 ± 11

50 ± 2

5000 µg

19 ± 4

19 ± 4

30 ± 3

187 ± 10

54 ± 2

NaN3

10 µg

2121 ± 70

2568 ± 110

4-NOPD

10 µg

543 ± 23

4-NOPD

50 µg

106 ± 13

MMS

3.0 µL

1283 ± 21

With Activation

Deionised water

17 ± 2

24 ± 5

45 ± 5

171 ± 5

70 ± 7

Untreated

17 ± 3

21 ± 8

46 ± 4

160 ± 7

66 ± 3

Triquat

3 µg

17 ± 8

24 ± 4

43 ± 8

142 ± 11

56 ± 7

Monomer

10 µg

18 ± 1

23 ± 3

39 ± 5

156 ± 16

67 ± 5

33 µg

21 ± 4

20 ± 1

34 ± 7

177 ± 9

66 ± 9

100 µg

17 ± 5

21 ± 1

41 ± 2

159 ± 19

49 ± 13

333 µg

17 ± 3

21 ± 1

36 ± 1

157 ± 7

59 ± 12

1000 µg

14 ± 2

25 ± 2

46 ± 5

152 ± 6

65 ± 2

2500 µg

14 ± 1

26 ± 3

44 ± 8

147 ± 8

65 ± 5

5000 µg

24 ± 3

22 ± 4

39 ± 5

138 ± 14

60 ± 8

2-AA

2.5 µg

403 ± 10

297 ± 13

2145 ± 42

2447 ± 104

2-AA

10.0 µg

297 ± 5

Key to Positive Controls

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

Results of Gene Mutation Assay: Experiment 2:

Metabolic

Activation

Test

Group

Dose Level

(µg/plate)

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without Activation

Deionised water

16 ± 4

12 ± 4

32 ± 9

137 ± 12

58 ± 5

Untreated

17 ± 0

17 ± 7

27 ± 4

134 ± 6

55 ± 7

Triquat

33 µg

16 ± 6

9 ± 1

30 ± 7

135 ± 12

49 ± 2

Monomer

100 µg

17 ± 3

12 ± 5

30 ± 9

136 ± 11

53 ± 3

333 µg

17 ± 3

13 ± 3

30 ± 10

128 ± 11

58 ± 10

1000 µg

13 ± 4

10 ± 3

29 ± 7

134 ± 7

55 ± 6

2500 µg

17 ± 2

11 ± 2

34 ± 3

132 ± 12

60 ± 3

5000 µg

16 ± 8

15 ± 1

31 ± 4

131 ± 8

60 ± 9

NaN3

10 µg

2040 ± 40

2152 ± 55

4-NOPD

10 µg

491 ± 55

4-NOPD

50 µg

124 ± 5

MMS

3.0 µL

635 ± 85

With Activation

Deionised water

21 ± 6

16 ± 4

39 ± 3

172 ± 12

59 ± 8

Untreated

16 ± 3

14 ± 1

46 ± 11

161 ± 25

58 ± 9

Triquat

33 µg

22 ± 2

16 ± 4

35 ± 4

176 ± 13

57 ± 8

Monomer

100 µg

23 ± 6

16 ± 3

39 ± 8

174 ± 12

61 ± 9

333 µg

22 ± 4

18 ± 3

43 ± 11

176 ± 12

66 ± 5

1000 µg

18 ± 5

19 ± 0

38 ± 5

159 ± 12

64 ± 6

2500 µg

24 ± 4

15 ± 7

35 ± 10

159 ± 14

61 ± 4

5000 µg

23 ± 1

15 ± 6

36 ± 12

163 ± 11

63 ± 10

2-AA

2.5 µg

245 ± 7

125 ± 10

1041 ± 58

977 ± 93

2-AA

10.0 µg

227 ± 25

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

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.
Executive summary:

This study was performed to investigate the potential of TRIQUAT MONOMER to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and 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 of the active ingredient (main constituent):

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 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 (below the induction factor of 0.5), occurred in the test groups with or without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with TRIQUAT MONOMER at any dose level, either in the presence or 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.

In conclusion, in 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, TRIQUAT MONOMER is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.