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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:
02 Jan - 26 Jan 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

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

Test material

1
Chemical structure
Reference substance name:
Reaction mass of 2-ethyl-4-methyl-1H-imidazole-1-propiononitrile and 2-ethyl-5-methyl-1H-imidazole-1-propiononitrile
EC Number:
947-727-8
Cas Number:
568591-00-4
Molecular formula:
C9H13N3
IUPAC Name:
Reaction mass of 2-ethyl-4-methyl-1H-imidazole-1-propiononitrile and 2-ethyl-5-methyl-1H-imidazole-1-propiononitrile

Method

Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strain)
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 pKM 101
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital (PB) and 5,6-benzoflavone (BF)
Test concentrations with justification for top dose:
Dose range finding experiment: 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate
Main experiment: 313, 625,1250, 2500 and 5000 µg/plate
As a result of the dose range finding study, the growth inhibition and precipitation of the test substance were not evident at any dose level of the test substance in any strain in the presence or absence of metabolic activation. Therefore, the high dose in the main study was selected at 5,000 μg/plate and sequentially diluted by applying a geometric ratio of 2 to produce lower dose levels.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water for injection
- Justification for choice of solvent/vehicle: In order to produce the high dose level (5000 μg/plate) of the dose range finding study, a preliminary solubility test was conducted based on the information provided by the sponsor. As a result, the test substance was dissolved in water for injection at a dose level of 50.0 mg/mL. Therefore, water for injection was selected as the vehicle for this study.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-Aminoanthracene (2-AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Dose-range-finding and main experiment (pre-incubation method), in agar

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

NUMBER OF REPLICATIONS:
3 replications each in 2 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: Growth inhibition was detected by a reduction in the number of revertant colonies, or by diminution or clearing of background lawn compared to the negative control group.

Evaluation criteria:
Acceptance Criteria

Evaluation of the validity of the study results was conducted based on the following criteria:
- The mean number of revertant colonies for the positive and negative control groups is within the range of the historical control data or the mean number of revertant colonies in the positive control groups is increased at least twice as compared to the negative control group.
- No plate shows any evidence of contamination.

Evaluation Criteria
The results of the study were considered to be positive when the following conditions were met.
- The number of revertant colonies in any strain at one or more doses is increased at least two times when compared to the negative control group. There should be dose dependency or reproducibility as dose increases.
Statistics:
Individual plates were counted for revertant colonies. The average and standard deviation of the number of revertant colonies were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 100
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
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
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
- Precipitation: No precipitation of the test substance occurred up to the highest investigated dose.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Growth inhibition of the test substance were not evident at any dose level of the test substance in any strain in the presence or absence of metabolic activation.

Any other information on results incl. tables

Table 2: Main Experiment 1 

 

EXPERIMENT 1 (Revertant colonies per plate ± SD)

 

S9-Mix

Without

 

 

Test item (µg/plate)

TA98

TA 100

TA 1535

TA 1537

WP2 uvrA pKM101

water

0

23 ± 1

103 ± 3

16 ± 1

9 ± 1

110 ± 4

Test Substance

313

24 ± 1

108 ± 6

14 ± 1

9 ± 1

106 ± 3

625

29 ± 1

113 ± 4

13 ± 2

10 ± 1

115 ± 2

1250

22 ± 1

108 ± 4

17 ± 1

9 ± 1

125 ± 3

2500

22 ± 1

138 ± 5

15 ± 1

9 ± 1

118 ± 4

5000

28 ± 2

130 ± 5

14 ± 1

8 ± 1

121 ± 3

2-NF

5

731 ± 10

-

-

 

-

SA

1.5

-

732 ± 11

589 ± 12

-

-

9-AA

80

 

 

 

602 ± 6

-

4-NQO

0.1

 

 

 

 

715 ± 11

 

S9-Mix

With

 

 

Test item (µg/plate)

TA98

TA 100

TA 1535

TA 1537

WP2 uvrA pKM101

water

0

35 ± 2

94 ± 1

10 ± 1

21 ± 1

147 ± 4

Test Substance

313

38 ± 1

104 ± 4

10 ± 1

16 ± 1

159 ± 4

625

35 ± 1

109 ± 2

11 ± 1

16 ± 1

148 ± 3

1250

40 ± 1

116 ± 4

9 ± 2

16 ± 1

167 ± 3

2500

42 ± 2

138 ± 4

11 ± 1

17 ± 1

171 ± 4

5000

38 ± 3

141 ± 4

12 ± 1

18 ± 1

149 ± 4

2-AA

1.0 (TA 98), 2.0 (TA 100, WP2 uvr pKM101), 3.0 (TA 1535, TA 1537)

453 ± 6

971 ± 32

180 ± 1

216 ± 2

601 ± 9

 

NC = Negative/Vehicle Control

PC = Respective positive control substances (for details see method description)

SD = Standard Deviation

 

Table 3: Main Experiment 2

 

EXPERIMENT 2 (Revertant colonies per plate ± SD)

 

S9-Mix

Without

 

 

Test item (µg/plate)

TA98

TA 100

TA 1535

TA 1537

WP2 uvrA pKM101

water

0

25 ± 1

95 ± 3

13 ± 1

9 ± 1

121 ± 5

Test Substance

313

26 ± 1

104 ± 5

14 ± 1

12 ± 1

127 ± 3

625

22 ± 1

105 ± 5

15 ± 1

11 ± 1

116 ± 5

1250

26 ± 1

112 ± 4

14 ± 1

12 ± 1

117 ± 3

2500

26 ± 1

115 ± 3

16 ± 1

11 ± 1

126 ± 3

5000

24 ± 1

128 ± 5

14 ± 1

8 ± 1

132 ± 6

2-NF

5

717 ± 15

-

-

 

-

SA

1.5

-

722 ± 11

521 ± 14

-

-

9-AA

80

-

-

-

563 ± 3

-

4-NQO

0.1

 

 

 

 

735 ± 13

 

S9-Mix

With

 

 

Test item (µg/plate)

TA98

TA 100

TA 1535

TA 1537

WP2 uvrA pKM101

water

0

35 ± 1

105 ± 3

13 ± 1

17 ± 1

157 ± 3

Test Substance

313

38 ± 1

142 ± 5

10 ± 1

20 ± 1

157 ± 5

625

42 ± 1

130 ± 6

11 ± 1

23 ± 1

166 ± 5

1250

40 ± 1

114 ± 5

9 ± 1

19 ± 1

175 ± 5

2500

44 ± 1

142 ± 5

12 ± 1

23 ± 1

175 ± 5

5000

39 ± 1

146 ± 5

11 ± 1

20 ± 1

160 ± 6

2-AA

1.0 (TA 98), 2.0 (TA 100, WP2 uvr pKM101), 3.0 (TA 1535, TA 1537)

457 ± 18

961 ± 20

175 ± 4

228 ± 3

589 ± 6

 

NC = Negative/Vehicle Control

PC = Respective positive control substances (for details see method description)

SD = Standard Deviation

Applicant's summary and conclusion

Conclusions:
Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA pKM101) tested with and without metabolic activation up to 5000 µg/plate.

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