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EC number: 608-818-5 | CAS number: 32997-86-7
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- Short-term toxicity to fish
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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:
- 2018-01-11 to 2018-03-20
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- sodium 2-chloroprop-2-enoate
- EC Number:
- 608-818-5
- Cas Number:
- 32997-86-7
- Molecular formula:
- C3H2ClO2.Na
- IUPAC Name:
- sodium 2-chloroprop-2-enoate
- Test material form:
- liquid
Constituent 1
Method
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- induced rat liver S9 mix
- Test concentrations with justification for top dose:
- Both trials (plate incorporation and pre-incubation) were performed at concentrations of 0.05, 0.16, 0.5, 1.6 and 5 mg/plate.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: recommended by guideline
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- mitomycin C
- other: 2-Aminoanthracene: 4 µg/plate, all strains with S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding: 10^8 viable cells per plate
DURATION
- Preincubation period: 30 min
- Exposure duration: 48 hrs +/- 30 min (plate incorporation); 66.75 hrs (pre-incubation)
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: background lawn - Rationale for test conditions:
- According to guideline and based on preliminary dose-range finding study.
- Evaluation criteria:
- Evaluation:
A substance is considered positive if the following criteria apply:
There should be a dose related increase over the range tested and/or a reproducible increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test item either in the presence or absence of the metabolic activation system.
The test will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and TA102 or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.
The mutation test is considered acceptable as it met the following criteria:
- All tester strains confirmed to their genetic characteristics.
- The positive controls showed increase in revertant colony numbers of at least twice or thrice the concurrent vehicle control levels with the appropriate bacterial strain.
Results and discussion
Test resultsopen allclose all
- 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:
- not examined
- 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:
- not examined
- Positive controls validity:
- valid
- 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:
- not examined
- 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:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no
- Water solubility: well soluble
- Precipitation: not observed
RANGE-FINDING/SCREENING STUDIES: yes
Any other information on results incl. tables
Table 1 SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-I: Plate Incorporation Method
Treatment |
Test Concentration (µL/plate) |
No. of Revertants (Mean of 3 Plates) |
|||||||||||
With S9 |
|
Without S9 |
|||||||||||
Salmonella typhimurium |
Salmonella typhimurium |
||||||||||||
TA 98 |
TA 100 |
TA 102 |
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
TA 102 |
TA 1535 |
TA 1537 |
||||
Vehicle Control |
100 µL of Distilled water |
Mean |
27.0 |
102.0 |
267.7 |
20.0 |
10.3 |
23.0 |
98.3 |
271.3 |
18.3 |
8.3 |
|
±SD |
2.6 |
4.4 |
13.1 |
1.7 |
3.5 |
3.0 |
3.5 |
13.6 |
2.5 |
2.5 |
|||
Lawn Intensity |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
|||
2-Chloroacrylic acid sodium salt |
0.05 |
Mean |
24.3 |
100.3 |
262.0 |
17.7 |
10.7 |
22.7 |
97.3 |
265.3 |
19.7 |
7.7 |
|
±SD |
2.1 |
6.0 |
12.5 |
2.5 |
2.1 |
2.5 |
6.1 |
13.1 |
1.5 |
0.6 |
|||
Fold Increase |
0.9 |
1.0 |
1.0 |
0.9 |
1.0 |
1.0 |
1.0 |
1.0 |
1.1 |
0.9 |
|||
Lawn Intensity |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
|||
0.16 |
Mean |
27.7 |
97.3 |
264.0 |
17.3 |
8.7 |
20.0 |
97.7 |
270.7 |
16.3 |
8.0 |
||
±SD |
3.2 |
7.5 |
9.8 |
2.5 |
2.9 |
3.6 |
4.0 |
7.8 |
2.1 |
2.6 |
|||
Fold Increase |
1.0 |
1.0 |
1.0 |
0.9 |
0.8 |
0.9 |
1.0 |
1.0 |
0.9 |
1.0 |
|||
Lawn Intensity |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
|||
0.5 |
Mean |
25.0 |
97.0 |
263.0 |
18.7 |
8.0 |
19.7 |
94.0 |
265.3 |
15.3 |
9.0 |
||
±SD |
5.0 |
6.2 |
7.5 |
2.5 |
2.6 |
3.2 |
7.0 |
9.7 |
1.5 |
2.6 |
|||
Fold Increase |
0.9 |
1.0 |
1.0 |
0.9 |
0.8 |
0.9 |
1.0 |
1.0 |
0.8 |
1.1 |
|||
Lawn Intensity |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
|||
1.6 |
Mean |
25.7 |
98.0 |
261.7 |
18.7 |
9.0 |
21.7 |
102.3 |
262.0 |
16.0 |
7.3 |
||
±SD |
2.5 |
8.2 |
8.5 |
2.1 |
2.6 |
2.5 |
3.8 |
12.1 |
1.0 |
2.1 |
|||
Fold Increase |
1.0 |
1.0 |
1.0 |
0.9 |
0.9 |
0.9 |
1.0 |
1.0 |
0.9 |
0.9 |
|||
Lawn Intensity |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
|||
5 |
Mean |
24.3 |
95.7 |
279.0 |
17.0 |
9.3 |
20.0 |
101.0 |
265.7 |
17.0 |
9.3 |
||
±SD |
3.5 |
4.7 |
6.6 |
3.0 |
2.3 |
2.0 |
6.2 |
7.5 |
2.6 |
2.1 |
|||
Fold Increase |
0.9 |
0.9 |
1.0 |
0.9 |
0.9 |
0.9 |
1.0 |
1.0 |
0.9 |
1.1 |
|||
Lawn Intensity |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
|||
Positive Control |
100 µL of respective Positive Control |
Mean |
354.7 |
406.7 |
605.0 |
144.0 |
112.0 |
361.3 |
390.7 |
596.3 |
136.7 |
108.0 |
|
±SD |
23.9 |
7.5 |
13.5 |
6.2 |
11.0 |
9.6 |
14.0 |
7.8 |
8.6 |
9.5 |
|||
Fold Increase |
13.1 |
4.0 |
2.3 |
7.2 |
10.8 |
15.7 |
4.0 |
2.2 |
7.5 |
13.0 |
|||
Lawn Intensity |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
Table 2 SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-II: Preincubation Method
Treatment |
Test Concentration (µL/plate) |
No. of Revertants (Mean of 3 Plates) |
|||||||||||
With S9 |
|
Without S9 |
|||||||||||
Salmonella typhimurium |
Salmonella typhimurium |
||||||||||||
TA 98 |
TA 100 |
TA 102 |
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
TA 102 |
TA 1535 |
TA 1537 |
||||
Vehicle Control |
100 µL of Distilled water |
Mean |
28.7 |
99.0 |
263.7 |
19.3 |
9.3 |
24.7 |
96.7 |
259.0 |
21.0 |
10.0 |
|
±SD |
1.5 |
5.6 |
7.6 |
2.5 |
3.2 |
3.5 |
6.1 |
5.6 |
3.0 |
1.0 |
|||
Lawn Intensity |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
|||
2-Chloroacrylic acid sodium salt |
0.05 |
Mean |
22.3 |
92.7 |
255.7 |
17.7 |
11.0 |
22.0 |
91.7 |
264.0 |
18.0 |
7.7 |
|
±SD |
3.1 |
2.5 |
6.7 |
2.3 |
2.6 |
4.0 |
4.7 |
7.0 |
2.0 |
3.1 |
|||
Fold Increase |
0.8 |
0.9 |
1.0 |
0.9 |
1.2 |
0.9 |
0.9 |
1.0 |
0.9 |
0.8 |
|||
Lawn Intensity |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
|||
0.16 |
Mean |
25.3 |
105.0 |
263.0 |
18.7 |
10.0 |
20.7 |
101.3 |
267.7 |
18.0 |
8.3 |
||
±SD |
1.5 |
7.5 |
10.5 |
3.2 |
2.0 |
2.5 |
6.7 |
9.3 |
2.6 |
4.9 |
|||
Fold Increase |
0.9 |
1.1 |
1.0 |
1.0 |
1.1 |
0.8 |
1.0 |
1.0 |
0.9 |
0.8 |
|||
Lawn Intensity |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
|||
0.5 |
Mean |
25.0 |
108.0 |
259.0 |
19.7 |
9.0 |
21.7 |
102.3 |
260.3 |
18.3 |
8.7 |
||
±SD |
3.6 |
6.2 |
9.2 |
2.1 |
2.0 |
4.5 |
7.6 |
6.7 |
3.8 |
1.5 |
|||
Fold Increase |
0.9 |
1.1 |
1.0 |
1.0 |
1.0 |
0.9 |
1.1 |
1.0 |
0.9 |
0.9 |
|||
Lawn Intensity |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
|||
1.6 |
Mean |
26.0 |
106.0 |
268.0 |
19.3 |
10.0 |
21.7 |
102.7 |
258.7 |
18.3 |
8.0 |
||
±SD |
3.0 |
9.6 |
13.7 |
4.5 |
3.6 |
4.2 |
8.3 |
7.8 |
2.5 |
3.6 |
|||
Fold Increase |
0.9 |
1.1 |
1.0 |
1.0 |
1.1 |
0.9 |
1.1 |
1.0 |
0.9 |
0.8 |
|||
Lawn Intensity |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
|||
5 |
Mean |
31.7 |
99.7 |
274.3 |
19.0 |
7.7 |
30.3 |
91.0 |
262.7 |
19.3 |
7.0 |
||
±SD |
2.1 |
1.5 |
12.7 |
3.6 |
2.9 |
3.8 |
2.0 |
6.4 |
2.1 |
3.0 |
|||
Fold Increase |
1.1 |
1.0 |
1.0 |
1.0 |
0.8 |
1.2 |
0.9 |
1.0 |
0.9 |
0.7 |
|||
Lawn Intensity |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
|||
Positive Control |
100 µL of respective Positive Control |
Mean |
373.0 |
397.3 |
605.7 |
136.7 |
118.7 |
357.0 |
393.0 |
594.7 |
141.3 |
114.0 |
|
±SD |
8.2 |
11.1 |
8.5 |
5.5 |
6.7 |
16.0 |
9.8 |
13.9 |
10.6 |
6.0 |
|||
Fold Increase |
13.0 |
4.0 |
2.3 |
7.1 |
12.7 |
14.5 |
4.1 |
2.3 |
6.7 |
11.4 |
|||
Lawn Intensity |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
4+ |
Positive controls:
With S9:
For Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene
Without S9:
For TA98: 2 µg/plate of 2-Nitrofluorene
For TA100 and TA1535: 1 µg/plate of Sodium azide.
For TA102: 0.5 µg/plate of Mitomycin C
For TA1537: 50 µg/plate of 9-Aminoacridine
Applicant's summary and conclusion
- Conclusions:
- Based on the results obtained from this study, it is concluded that the test item is non-mutagenic in the Bacterial Reverse Mutation Test up to the highest tested concentration of 5 µL/plate under the test conditions chosen.
- Executive summary:
The test item was evaluated for mutagenicity in a Bacterial Reverse Mutation Test according to OECD guideline for testing of chemicals No. 471, “Bacterial Reverse Mutation Test”, adopted on 21st July 1997.
The test concentrations used in the mutation assay were selected based on the results obtained in the pre-tests. The mutagenic potential of the test item was investigated in two independent experiments, in plate incorporation test (trial 1) and pre-incubation test (trial 2). Each assay was conducted with and without metabolic activation (± S9 mix). Both trials were performed at concentrations of 0.05, 0.16, 0.5, 1.6 and 5 µL/plate. All concentrations, including the controls, were tested in triplicate. Vehicle control (distilled water) and appropriate positive controls (2-nitrofluorene, sodium azide and 9-Aminoacridine, Mitomycin C for trials without metabolic activation and 2-Aminoanthracene for trials with metabolic activation) were tested simultaneously.
The tester strains used in the mutation assay were Salmonella typhimurium TA98, TA100, TA 102, TA1535 and TA1537.
In the performed trials all of the validity criteria regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both trials in the presence and absence of metabolic activation. There was no substantial increase in number of revertant colonies at any of the tested concentrations in both trials. In the performed experiments an inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case.
The number of revertant colonies in the positive controls resulted in 2.2 to 16.1 fold increase under identical conditions.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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