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EC number: 947-727-8 | CAS number: 568591-00-4
Genetic toxicity: in vitro
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:
- 2 018
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
- 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
1
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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