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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Jan 2018 - 07 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon for S. typhimurium strains
trp operon for E. coli strains

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Arocolor 1254.
Test concentrations with justification for top dose:
Pre-experiment (dose-range finding test, Direct Plate Assay): 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 µg/plate
Main-experiment (Pre-incubation Assay): 17, 52, 164, 512, 1600, 5000 µg/plate (Based on the results of the dose-range finding test)
Vehicle / solvent:
- Vehicle/solvent used: water (Milli-Q-water, Millipore Corp., Bedford, MA., USA).
- Justification for choice of solvent/vehicle: The test item formed a clear colorless solution in Milli-Q water.

- Vehicle/solvent used for positive control items: Saline = physiological saline (Eurovet Animal Health, Bladel, The Netherlands)
DMSO = dimethyl sulfoxide (Merck, Darmstadt, Germany)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar, Direct Plate Assay, Pre-incubation assay

DURATION
- Preincubation period: 30 ± 2 min
- Exposure duration: at least 48 ± 4 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.
Evaluation criteria:
ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

EVALUATION CRITERIA
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment. A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:
Mean values and standard deviations were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
2.3-fold dose-related increase in the number of revertant colonies in the absence of S9-mix.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
1.9-fold dose-related increase in the number of revertant colonies in the presence of S9-mix. The increase observed was within the laboratory historical control data ranges and less than two-fold the concurrent solvent control
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Direct Plate Assay
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in all tester strains. Except for tester strain WP2uvrA, where no toxicity was observed in the absence or presence of S9-mix.

Pre-Incubation Assay
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in all tester strains from 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation. Except for tester strain WP2uvrA in the presence of S9-mix, where no toxicity was observed

Any other information on results incl. tables

  Table 3: Dose-Range Finding Test (Direct-Plate assay)

 

EXPERIMENT 1 (Revertant colonies per plate ± SD)

 

S9-Mix

Without

 

 

Test item (µg/plate)

TA 100

 

WP2uvrA

 

Vehicle Control

 

113 ± 11

33 ± 11

Test Substance

1.7

121 ± 15

25 ± 5

5.4

123 ± 19

37 ± 2

17

115 ± 12

21 ± 1

52

134 ± 12

33 ± 6

164

141 ± 13

37 ± 6

512

166 ± 16

35 ± 0

1600

292 ± 22n 

41 ± 9

5000

0 ± 0aNP 

51 ± 4nNP

Positive Control

 

744 ± 65

1602 ± 77

 

S9-Mix

With

 

 

Test item (µg/plate)

TA 100

WP2uvrA

Vehicle Control

-

118 ± 6

43 ± 7

Test Substance

1.7

100 ± 8

50 ± 6

5.4

106 ± 13

40 ± 3

17

116 ± 13

35 ± 9

52

122 ± 6

38 ± 8

164

135 ± 18

43 ± 4

512

169 ± 21

58 ± 5

1600

310 ± 19

75 ± 4

5000

106 ± 941NP

52 ± 5nNP

Positive Control

 

2156 ± 227

558 ± 54

 

1=Revertants were only observed on a small part of the plate with normal background, no bacterial growth or background was observed on a large part of the plate

NP= No precipitate

a = Bacterial background lawn absent

n = Normal bacterial background lawn

s = Bacterial background lawn slightly reduced

 

Table 4: Direct-Plate assay

 

EXPERIMENT 1 (Revertant colonies per plate ± SD)

 

S9-Mix

Without

 

 

Test item (µg/plate)

TA 1535

TA 1537

TA 98

Vehicle Control

 

12 ± 2

3 ± 2

11 ± 1

Test Substance

17

12 ± 2

3 ± 0

17 ± 5

52

13 ± 5

4 ± 3

14 ± 3

164

14 ± 4

2 ± 1

11 ± 3

512

13 ± 2

2 ± 1

10 ± 6

1600

11 ± 3n

2 ± 1n

12 ± 6n

5000

0 ± 0aNP

2 ± 2eNP

0 ± 0aNP

Positive Control

 

1091 ± 65

834 ± 49

1141± 30

 

S9-Mix

With

 

 

Test item (µg/plate)

TA 1535

TA 1537

TA 98

Vehicle Control

-

11 ± 6

3 ± 4

16 ± 2

Test Substance

17

12 ± 5

4 ± 3

16 ± 3

52

10 ± 2

7 ± 4

22 ± 6

164

13 ± 5

4 ± 1

16 ± 4

512

17 ± 8

3 ± 2

17 ± 4

1600

18 ± 3

3 ± 2

19 ± 7

5000

5 ± 81NP

1 ± 21NP

3 ± 21NP

Positive Control

 

408 ± 32

374± 108

1298± 195

 

1=Revertants were only observed on a small part of the plate with normal background, no bacterial growth or background was observed on a large part of the plate

NP= No precipitate

a = Bacterial background lawn absent

n = Normal bacterial background lawn

s = Bacterial background lawn slightly reduced

 

Table 5: Plate-Incubation Test

 

EXPERIMENT 2 (Revertant colonies per plate ± SD)

 

S9-Mix

Without

 

 

Test item (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

(fold)

WP2uvrA

(fold)

Vehicle Control

 

10 ± 2

3 ± 4

18 ± 3

106 ± 14

19 ± 5

Test Substance

17

13 ± 5

3 ± 1

17 ± 6

122 ± 15

28 ± 2

52

12 ± 2

3 ± 2

13 ± 5

130 ± 5

29 ± 15 (1.5)

164

19 ± 1

3 ± 2

14 ± 3

134 ± 16 (1.3)

35 ± 7 (1.8)

512

13± 3n

8 ± 2n

17 ± 3n

195 ± 5n (1.8)

44 ± 8n(2.3)

1600

0 ± 0a

0± 0a

0± 0a

0± 0a

1± 01n

5000

0 ± 0aNP

0 ± 0aNP

0 ± 0aNP

0 ± 0aNP

0 ± 0aNP

Positive Control

 

1036 ± 33

138 ± 14

1146 ± 68

781 ± 11

152 ± 85

 

S9-Mix

With

 

 

Test item (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

Vehicle Control

-

11 ± 2

5 ± 3

19 ± 6

102 ± 2

30 ± 7

Test Substance

17

13 ± 2

4 ± 2

15 ± 6

166 ± 18

36 ± 6

52

10 ± 5

7 ± 2

13 ± 2

180 ± 20

42 ± 6

164

14 ± 3

7 ± 2

19 ± 4

139 ± 7

39 ± 8

512

10 ± 0

5 ± 3n

13 ± 2

190 ± 26 (1.9)

42 ± 4 (1.4)

1600

14 ± 6

2 ± 1s

15 ± 3n

304 ± 22n(3.0)

56 ± 13 (1.9)

5000

0 ± 11nNP

0 ± 0aNP

0 ± 0aNP

0 ± 0aNP

57 ± 11nNP(1.9)

Positive Control

 

247 ± 22

230 ± 32

762 ± 14

1586 ± 89

587 ± 30

 

1=Revertants were only observed on a small part of the plate with normal background, no bacterial growth or background was observed on a large part of the plate

NP= No precipitate

a = Bacterial background lawn absent

n = Normal bacterial background lawn

s = Bacterial background lawn slightly reduced

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of the conducted test the substance induced significant dose-related increases in the number of revertant colonies in the tester strain TA100 and WP2uvrA both in the absence and presence of S9-mix. In the latter, only in the absence of S-9 mix in experiment 2, these increases exceeded the two-fold threshold (2.3-fold). Based on these results, the test substance is regarded to be mutagenic in bacteria.