Registration Dossier
Registration Dossier
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EC number: 201-161-1 | CAS number: 78-95-5
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:
- 2021-01-22 to 2021-02-01
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 021
- Report date:
- 2021
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 26 June 2020
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Chloroacetone
- EC Number:
- 201-161-1
- EC Name:
- Chloroacetone
- Cas Number:
- 78-95-5
- Molecular formula:
- C3H5ClO
- IUPAC Name:
- chloroacetone
Constituent 1
Method
- Target gene:
- S. typhimurium: histidine
E. coli: tryptophan
Species / strainopen allclose all
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: S9 liver microsomal fraction from male Sprague Dawley rats, induced with phenobarbital/β-naphthoflavone (obtained from Trinova Biochem GmbH, Gießen, Germany), protein concentrations in the S9 preparations were adjusted to 30 mg/mL
- method of preparation of S9 mix: The S9 mix preparation was performed according to Ames et al.. 100 mM of ice-cold sodium-ortho-phosphate-buffer, pH 7.4, was added to the following pre-weighed sterilised reagents to give final concentrations in the S9 mix of:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion:
co-factor solution 9.5 parts
liver preparation 0.5 parts
- volume of S9 mix in the final culture medium: 500 μL S9 mix/plate
- quality controls of S9: Alkoxyresorufin-O-dealkylase activities, Test for the presence of adventitious agents, Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene) (performed by Trinova Biochem GmbH) - Test concentrations with justification for top dose:
- pre-experiment:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
Experiment I:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
Experiment II:
0.000158, 0.00050, 0.00158, 0.0050, 0.0158, 0.050, 0.158 and 0.5 μL/plate
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. - Vehicle / solvent:
- - Solvent used: water (aqua dest.)
- Justification for choice of solvent: The solvent was compatible with
the survival of the bacteria and the S9 activity.
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition; reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control
METHODS FOR MEASUREMENTS OF GENOTOXICIY
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and E. coli WP2 uvrA (pKM101) the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic
in this system. - Rationale for test conditions:
- The OECD Guideline for Testing of Chemicals, Section 4, No. 471 – Bacterial Reverse Mutation Test - recommends using a combination of S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM101).
- Evaluation criteria:
- A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, E. coli WP2 uvrA (pKM101))
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean
values of the spontaneous reversion frequency are within the historical control data range (for values please refer to “Any other information on results”)
- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable. - Statistics:
- According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- starting at 0.050 μg/plate (with and without metabolic activation)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True 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:
- starting at 0.050 μg/plate (with and without metabolic activation)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True 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:
- starting at 0.100 μg/plate (without metabolic activation) and 0.050 μg/plate (with metabolic activation)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- starting at 0.050 μg/plate (without metabolic activation) and 0.100 μg/plate (with metabolic activation)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True 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:
- starting at 0.158 μg/plate (with and without metabolic activation)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not specified
- Data on osmolality: not specified
- Possibility of evaporation from medium: not specified
- Water solubility: The test item was water soluble.
- Precipitation and time of the determination: No precipitation of the test item was observed in any tester strain used in experiment I and II.
- Other confounding effects: None reported.
RANGE-FINDING/SCREENING STUDIES : A pre-experiment was conducted to determine the concentrations for the main experiment.
STUDY RESULTS
- Concurrent vehicle negative and positive control data: Please refer to Any other information on results.
Ames test:
- Signs of toxicity: Toxic effects of the test item were noted in all tester strains evaluated in experiment I and II.
- Individual plate counts: Please refer to Any other information on results.
- Mean number of revertant colonies per plate and standard deviation: Please refer to Any other information on results.
- Others: Due to the high toxicity of the test item, in experiment I only four concentrations out of eight in each tester strain were analysable, which is below the threshold limit of five analysable concentrations set by the OECD 471. However, as the concentrations used in experiment II were lower than those of experiment I, a total of six to seven concentrations used in each tester strain were analysable.
HISTORICAL CONTROL DATA
Please refer to Any other information on results.
Any other information on results incl. tables
Experiment 1
Table 1: Tester Strain: TA98 Experiment 1
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||||
Without activation (-S9) |
With activation (+S9) |
||||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||||
A. dest |
|
20 36 38 |
C |
31 |
9.9 |
20 21 51 |
|
31 |
17.6 |
1.0 |
1.0 |
Test Item |
0.00316 µL |
35 40 31 |
|
35 |
4.5 |
48 35 31 |
|
38 |
8.9 |
1.1 |
1.2 |
Test Item |
0.0100 µL |
28 25 23 |
|
25 |
2.5 |
43 53 36 |
|
44 |
8.5 |
0.8 |
1.4 |
Test Item |
0.0316 µL |
20 32 33 |
|
28 |
7.2 |
35 14 21 |
|
23 |
10.7 |
0.9 |
0.8 |
Test Item |
0.100 µL |
18 24 30 |
|
24 |
6.0 |
22 13 18 |
|
18
|
4.5 |
0.8 |
0.6 |
Test Item |
0.316 µL |
0 0 0 |
B B B |
0 |
0.0 |
0 0 0 |
B B B |
0 |
0.0 |
0.0 |
0.0 |
Test Item |
1.0 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
Test Item |
2.5 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
Test Item |
5.0 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
4-NOPD |
10 µg |
456 402 454 |
|
437 |
30.6 |
/ / / |
|
/ |
/ |
14.0 |
/ |
2-AA |
2.5 µg |
/ / / |
|
/ |
/ |
1584 1291 1726 |
|
1534 |
221.8 |
/ |
50.0 |
SD:
Standard-deviation P: Precipitation
B: Background lawn reduced C:
Contamination
N: No background lawn
mean revertants (test item)
Mutation factor =
mean revertants (vehicle
control)
Table 2: Tester Strain. TA100 Experiment: 1
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||||
Without activation (-S9) |
With activation (+S9) |
||||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||||
A. dest |
|
127 89 73 |
|
96 |
27.7 |
108 107 91 |
|
102 |
9.5 |
1.0 |
1.0 |
Test Item |
0.00316 µL |
95 52 67 |
|
71 |
21.8 |
123 108 108 |
|
113 |
8.7 |
0.7 |
1.1 |
Test Item |
0.0100 µL |
84 67 82 |
|
78 |
9.3 |
127 76 121 |
|
108 |
27.9 |
0.8 |
1.1 |
Test Item |
0.0316 µL |
49 37 43 |
|
43 |
6.0 |
86 88 70 |
|
81 |
9.9 |
0.4 |
0.8 |
Test Item |
0.100 µL |
3 15 21 |
|
13 |
9.2 |
31 24 19 |
C |
25 |
6.0 |
0.1 |
0.2 |
Test Item |
0.316 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
Test Item |
1.0 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
Test Item |
2.5 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
Test Item |
5.0 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
NaN3 |
10 µg |
199 238 262 |
|
233 |
31.8 |
/ / / |
|
/ |
/ |
2.4 |
/ |
2-AA |
2.5 µg |
/ / / |
|
/ |
/ |
623 669 739 |
|
677 |
58.4 |
/ |
6.6 |
SD:
Standard-deviation P: Precipitation
B: Background lawn reduced C:
Contamination
N: No background lawn
mean revertants (test item)
Mutation factor =
mean revertants (vehicle
control)
Table 3: Tester Strain: TA1535 Experiment: 1
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||||
Without activation (-S9) |
With activation (+S9) |
||||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||||
A. dest |
|
18 14 11 |
|
14 |
3.5 |
6 9 6 |
|
7 |
1.7 |
1.0 |
1.0 |
Test Item |
0.00316 µL |
11 6 11 |
|
9 |
2.9 |
11 9 11 |
|
10 |
1.2 |
0.7 |
1.5 |
Test Item |
0.0100 µL |
14 8 7 |
|
10 |
3.8 |
12 5 6 |
|
8 |
3.8 |
0.7 |
1.1 |
Test Item |
0.0316 µL |
9 6 12 |
|
9 |
3.0 |
8 6 6 |
|
7 |
1.2 |
0.6 |
1.0 |
Test Item |
0.100 µL |
10 4 4 |
|
6 |
3.5 |
9 10 4 |
|
8 |
3.2 |
0.4 |
1.1 |
Test Item |
0.316 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
Test Item |
1.0 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
Test Item |
2.5 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
Test Item |
5.0 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
NaN3 |
10 µg |
226 349 348 |
|
308 |
70.7 |
/ / / |
|
/ |
/ |
21.5 |
/ |
2-AA |
2.5 µg |
/ / / |
|
/ |
/ |
138 124 107 |
|
123 |
15.5 |
/ |
17.6 |
SD:
Standard-deviation P: Precipitation
B: Background lawn reduced C:
Contamination
N: No background lawn
mean revertants (test item)
Mutation factor =
mean revertants (vehicle
control)
Table 4: Tester Strain: TA1537 Experiment 1
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||||
Without activation (-S9) |
With activation (+S9) |
||||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||||
A. dest |
|
8 13 11 |
|
11 |
2.5 |
15 1011 |
|
12 |
2.6 |
1.0 |
1.0 |
Test Item |
0.00316 µL |
8 10 8 |
|
9 |
1.2 |
7 10 5 |
|
7 |
2.5 |
0.8 |
0.6 |
Test Item |
0.0100 µL |
6 7 6 |
|
6 |
0.6 |
12 6 7 |
|
8 |
3.2 |
0.6 |
0.7 |
Test Item |
0.0316 µL |
3 11 9 |
|
8 |
4.2 |
7 10 6 |
|
8 |
2.1 |
0.7 |
0.6 |
Test Item |
0.100 µL |
4 6 4 |
B B B |
5 |
1.2 |
3 5 4 |
B B B |
4 |
1.0 |
0.4 |
0.3 |
Test Item |
0.316 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
Test Item |
1.0 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
Test Item |
2.5 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
Test Item |
5.0 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
4-NOPD |
40 µg |
29 34 61 |
|
41 |
17.2 |
/ / / |
|
/ |
/ |
3.9 |
/ |
2-AA |
2.5 µg |
/ / / |
|
/ |
/ |
155 163 162 |
|
160 |
4.4 |
/ |
13.3 |
SD:
Standard-deviation P: Precipitation
B: Background lawn reduced C:
Contamination
N: No background lawn
mean revertants (test item)
Mutation factor =
mean revertants (vehicle
control)
Table 5: Tester Strain: WP2 uvra (pKM101) Experiment 1
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||||
Without activation (-S9) |
With activation (+S9) |
||||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||||
A. dest |
|
314 300 258 |
|
291 |
29.1 |
258 355 313 |
|
309 |
48.6 |
1.0 |
1.0 |
Test Item |
0.00316 µL |
274 327 327 |
|
309 |
30.6 |
349 380 349 |
|
359 |
17.9 |
1.1 |
1.2 |
Test Item |
0.0100 µL |
358 304 276 |
|
313 |
41.7 |
3372 411 368 |
|
384 |
23.8 |
1.1 |
1.2 |
Test Item |
0.0316 µL |
300 290 249 |
|
280 |
27.0 |
287 319 329 |
|
312 |
21.9 |
1.0 |
1.0 |
Test Item |
0.100 µL |
106 116 152 |
|
125 |
24.2 |
148 160 162 |
|
157 |
7.6 |
0.4 |
0.5 |
Test Item |
0.316 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
Test Item |
1.0 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
Test Item |
2.5 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
Test Item |
5.0 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
MMS |
1.0 µL |
1261 1547 1556 |
|
1455 |
167.8 |
/ / / |
|
/ |
/ |
5.0 |
/ |
2-AA |
10 µg |
/ / / |
|
/ |
/ |
865 739 953 |
|
852 |
107.6 |
/ |
2.8 |
SD:
Standard-deviation P: Precipitation
B: Background lawn reduced C:
Contamination
N: No background lawn
mean revertants (test item)
Mutation factor =
mean revertants (vehicle
control)
Experiment 2
Table 6: Tester Strain: TA98 Experiment 2
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||||
Without activation (-S9) |
With activation (+S9) |
||||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||||
A. dest |
|
14 46 20 |
|
27 |
17.0 |
16 18 26 |
|
20 |
53. |
1.0 |
1.0 |
Test Item |
0.000158 µL |
25 25 27 |
|
26 |
1.2 |
21 11 10 |
|
14 |
6.1 |
1.0 |
0.7 |
Test Item |
0.00050 µL |
23 27 22 |
|
24 |
2.6 |
29 15 18 |
|
21 |
7.4 |
0.9 |
1.0 |
Test Item |
0.00158 µL |
14 21 19 |
|
18 |
3.6 |
19 19 28 |
|
22 |
5.2 |
0.7 |
1.1 |
Test Item |
0.0050 µL |
27 18 13 |
|
19 |
7.1 |
12 13 10 |
|
12 |
1.5 |
0.7 |
0.6 |
Test Item |
0.0158 µL |
25 23 18 |
|
22 |
3.6 |
15 16 9 |
|
13 |
3.8 |
0.8 |
0.7 |
Test Item |
0.050 µL |
11 24 11 |
|
15 |
7.5 |
12 13 13 |
|
13 |
0.6 |
0.6 |
0.6 |
Test Item |
0.158 µL |
0 0 5 |
B B B |
2 |
2.9 |
2 6 3 |
B B B |
4 |
2.1 |
0.1 |
0.2 |
Test Item |
0.5 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
4-NOPD |
10 µg |
331 337 292 |
|
320 |
24.4 |
/ / / |
|
/ |
/ |
12.0 |
/ |
2-AA |
2.5 µg |
/ / / |
|
/ |
/ |
947 737 754 |
|
813 |
116.6 |
/ |
40.6 |
SD:
Standard-deviation P: Precipitation
B: Background lawn reduced C:
Contamination
N: No background lawn
mean revertants (test item)
Mutation factor =
mean revertants (vehicle
control)
Table 7: Tester Strain: TA100 Experiment 2
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||||
Without activation (-S9) |
With activation (+S9) |
||||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||||
A. dest |
|
88 1 94 |
|
88 |
6.5 |
82 102 89 |
|
91 |
10.1 |
1.0 |
1.0 |
Test Item |
0.000158 µL |
65 73 87 |
|
75 |
11.1 |
95 87 93 |
|
92 |
4.2 |
0.9 |
1.0 |
Test Item |
0.00050 µL |
107 79 85 |
|
90 |
14.7 |
73 89 93 |
|
85 |
10.6 |
1.0 |
0.9 |
Test Item |
0.00158 µL |
86 80 62 |
|
76 |
12.5 |
87 59 85 |
|
77 |
5.6 |
0.9 |
0.8 |
Test Item |
0.0050 µL |
57 78 94 |
|
76 |
18.6 |
90 855 61 |
|
79 |
15.5 |
0.9 |
0.8 |
Test Item |
0.0158 µL |
46 56 66 |
|
56 |
10.0 |
62 75 41 |
|
59 |
17.2 |
0.6 |
0.7 |
Test Item |
0.050 µL |
32 19 27 |
B B B |
26 |
6.6 |
40 29 45 |
|
38 |
8.2 |
0.3 |
0.4 |
Test Item |
0.158 µL |
5 11 15 |
B B B |
10 |
5.0 |
2 4 3 |
B B B |
3 |
1.0 |
0.1 |
0.0 |
Test Item |
0.5 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
NaN3 |
10 µg |
315 348 320 |
|
328 |
17.8 |
/ / / |
|
/ |
/ |
3.7 |
/ |
2-AA |
2.5 µg |
/ / / |
|
/ |
/ |
875 707 893 |
|
825 |
102.6 |
/ |
9.1 |
SD:
Standard-deviation P: Precipitation
B: Background lawn reduced C:
Contamination
N: No background lawn
mean revertants (test item)
Mutation factor =
mean revertants (vehicle
control)
Table 8: Tester Strain: TA1535 Experiment 2
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||||
Without activation (-S9) |
With activation (+S9) |
||||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||||
A. dest |
|
9 15 15 |
|
13 |
3.5 |
10 12 5 |
|
9 |
3.6 |
1.0 |
1.0 |
Test Item |
0.000158 µL |
7 15 5 |
|
9 |
5.3 |
10 5 11 |
|
9 |
3.2 |
0.7 |
1.0 |
Test Item |
0.00050 µL |
7 11 9 |
|
9 |
2.0 |
7 6 11 |
|
8 |
2.6 |
0.7 |
0.9 |
Test Item |
0.00158 µL |
5 10 12 |
|
9 |
3.6 |
10 6 5 |
|
7 |
2.6 |
0..7 |
0.8 |
Test Item |
0.0050 µL |
7 10 7 |
|
8 |
1.7 |
13 6 14 |
|
11 |
4.4 |
0.6 |
1.2 |
Test Item |
0.0158 µL |
9 7 8 |
|
8 |
1.0 |
3 7 7 |
|
6 |
2.3 |
0.6 |
0.6 |
Test Item |
0.050 µL |
8 9 5 |
|
7 |
2.1 |
3 9 2 |
|
5 |
3.8 |
0.6 |
0.5 |
Test Item |
0.158 µL |
2 2 3 |
B B B |
2 |
0.6 |
2 1 1 |
B B |
1 |
0.6 |
0.2 |
0.1 |
Test Item |
0.5 µL |
0 0 0 |
N N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
NaN3 |
10 µg |
339 342 355 |
|
345 |
8.5 |
/ / / |
|
/ |
/ |
26.6 |
/ |
2-AA |
2.5 µg |
/ / / |
|
/ |
/ |
176 121 125 |
|
141 |
30.7 |
/ |
15.6 |
SD:
Standard-deviation P: Precipitation
B: Background lawn reduced C:
Contamination
N: No background lawn
mean revertants (test item)
Mutation factor =
mean revertants (vehicle
control)
Table 8: Tester Strain: TA153 Experiment 2
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||||
Without activation (-S9) |
With activation (+S9) |
||||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||||
A. dest |
|
5 4 8 |
|
6 |
2.1 |
11 8 7 |
|
9 |
2.1 |
1.0 |
1.0 |
Test Item |
0.000158 µL |
5 6 5 |
|
5 |
0.6 |
8 8 10 |
|
9 |
1.2 |
0.9 |
1.0 |
Test Item |
0.00050 µL |
8 4 5 |
|
6 |
2.1 |
7 8 10 |
|
8 |
1.5 |
1.0 |
1.0 |
Test Item |
0.00158 µL |
6 6 7 |
|
6 |
0.6 |
8 4 9 |
|
7 |
2.6 |
1.1 |
0.8 |
Test Item |
0.0050 µL |
4 9 9 |
|
7 |
2.9 |
8 8 13 |
|
10 |
2.9 |
1.3 |
1.1 |
Test Item |
0.0158 µL |
3 13 3 |
|
6 |
5.8 |
6 7 5 |
|
6 |
1.0 |
1.1 |
0.7 |
Test Item |
0.050 µL |
4 2 2 |
B B B |
3 |
1.2 |
4 4 5 |
|
4 |
0.6 |
0.5 |
0.5 |
Test Item |
0.158 µL |
1 0 1 |
B B B |
1 |
0.6 |
3 1 1 |
B B B |
2 |
1.2 |
0.1 |
0.2 |
Test Item |
0.5 µL |
0 0 0 |
N N |
0 |
0.0 |
0 0 0 |
N N |
0 |
0.0 |
0.0 |
0.0 |
4-NOPD |
40 µg |
39 51 50 |
|
47 |
6.7 |
/ / / |
|
/ |
/ |
8.22 |
/ |
2-AA |
2.5 µg |
/ / / |
|
/ |
/ |
105 83 75 |
|
88 |
15.5 |
/ |
10.1 |
SD:
Standard-deviation P: Precipitation
B: Background lawn reduced C:
Contamination
N: No background lawn
mean revertants (test item)
Mutation factor =
mean revertants (vehicle
control)
Table 9: Tester Strain: WP2 uvrA (pKM101) Experiment 2
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||||
Without activation (-S9) |
With activation (+S9) |
||||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||||
A. dest |
|
267 188 205 |
|
220 |
41.6 |
251 232 181 |
|
221 |
36.2 |
1.0 |
1.0 |
Test Item |
0.000158 µL |
207 245 188 |
|
213 |
29.0 |
338 240 346 |
|
308 |
59.0 |
1.0 |
1.4 |
Test Item |
0.00050 µL |
254 210 235 |
|
233 |
22.1 |
301 299 285 |
|
295 |
8.7 |
1.1 |
1.3 |
Test Item |
0.00158 µL |
179 238 200 |
|
206 |
29.9 |
298 254 250 |
|
267 |
26.6 |
0.9 |
1.2 |
Test Item |
0.0050 µL |
292 233 247 |
|
257 |
30.8 |
349 226 238 |
|
271 |
67.8 |
1.2 |
1.2 |
Test Item |
0.0158 µL |
159 152 113 |
|
141 |
24.8 |
133 124 140 |
|
132 |
8.0 |
0.6 |
0.6 |
Test Item |
0.050 µL |
60 68 67 |
|
65 |
4.4 |
97 119 131 |
|
116 |
17.2 |
0.3 |
0.5 |
Test Item |
0.158 µL |
8 30 6 |
B B B |
15 |
13.3 |
30 14 12 |
B B B |
19 |
9.9 |
0.1 |
0.1 |
Test Item |
0.5 µL |
0 0 0 |
N N |
0 |
0.0 |
0 0 0 |
N N N |
0 |
0.0 |
0.0 |
0.0 |
MMS |
1.0 µg |
584 556 590 |
|
577 |
18.1 |
/ / / |
|
/ |
/ |
2.6 |
/ |
2-AA |
10 µg |
/ / / |
|
/ |
/ |
421 / 451 |
|
436 |
21.2 |
/ |
2.0 |
SD:
Standard-deviation P: Precipitation
B: Background lawn reduced C:
Contamination
N: No background lawn
mean revertants (test item)
Mutation factor =
mean revertants (vehicle
control)
Historical Laboratory Control Data
Table 10: Historical Laboratory Control Data of the Negative Control without S9 (-S9)
(Data from 2017 – 2019 for all tester strains, except for E. coli WP2 uvrA (pKM101). For this tester strain the period was December 2019 to May 2020))
|
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2 uvrA (pKM101) |
Mean |
28,4 |
95,4 |
15,6 |
15,6 |
181,5 |
SD |
8,0 |
16,3 |
5,9 |
6,1 |
42,7 |
Min |
14 |
44 |
5 |
3 |
110 |
Max |
61 |
143 |
35 |
35 |
315 |
RSD [%] |
28,2 |
17,0 |
37,8 |
39,2 |
23,5 |
n |
976 |
1023 |
926 |
927 |
87 |
S9: metabolic activation
Mean: mean of revertants/plate
Min.: minimum of revertnts/plate
Max.: maximum of revertants/plate
SD: Standard Deviation
RSD: Relative Standard Deviation
n: Number of control values
Table 10: Historical Laboratory Control Data of the Positive Control without S9 (-S9)
(Data from 2017 – 2019 for all tester strains, except for E. coli WP2 uvrA (pKM101). For this tester strain the period was December 2019 to May 2020))
|
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2 uvrA (pKM101) |
Substance Conc./plate |
4-NOPD 10 µg |
NaN3 10 µg |
NaN3 10 µg |
4-NOPD 40 µg |
MMS 1 µL |
Mean |
443,2 |
614,2 |
826,2 |
123,3 |
1702,7 |
SD |
168,7 |
208,2 |
362,2 |
49,4 |
551,4 |
Min |
89 |
166 |
28 |
33 |
831 |
Max |
2013 |
2493 |
1863 |
57 |
3391 |
RSD [%] |
38,1 |
33,9 |
43,8 |
40,0 |
32,4 |
n |
985 |
1034 |
935 |
934 |
89 |
S9: metabolic activation
Mean: mean of revertants/plate
Min.: minimum of revertnts/plate
Max.: maximum of revertants/plate
SD: Standard Deviation
RSD: Relative Standard Deviation
n: Number of control values
Table 12: Historical Laboratory Control Data of the Negative Control with S9 (+S9)
(Data from 2017 – 2019 for all tester strains, except for E. coli WP2 uvrA (pKM101). For this tester strain the period was December 2019 to May 2020))
|
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2 uvrA (pKM101) |
Mean |
29,9 |
92,7 |
14,1 |
16,2 |
219,4 |
SD |
7,3 |
14,0 |
5,3 |
6,3 |
48,6 |
Min |
15 |
60 |
4 |
5 |
142 |
Max |
60 |
154 |
37 |
41 |
381 |
RSD [%] |
245 |
15,1 |
37,5 |
38,8 |
22,2 |
n |
974 |
1020 |
923 |
924 |
87 |
S9: metabolic activation
Mean: mean of revertants/plate
Min.: minimum of revertnts/plate
Max.: maximum of revertants/plate
SD: Standard Deviation
RSD: Relative Standard Deviation
n: Number of control values
Table 13: Historical Laboratory Control Data of the Positive Control without S9 (+S9)
(Data from 2017 – 2019 for all tester strains, except for E. coli WP2 uvrA (pKM101). For this tester strain the period was December 2019 to May 2020))
|
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2 uvrA (pKM101) |
Substance Conc./plate |
2-AA 2,5 µg |
2-AA 2,5 µg |
2-AA 2,5 µg |
2-AA 2,5 µg |
2-AA 10 µg |
Mean |
1305,9 |
1054,5 |
185,7 |
187,3 |
708,4 |
SD |
792,7 |
611,0 |
130,1 |
104,0 |
153,5 |
Min |
70 |
119 |
19 |
23 |
424 |
Max |
3609 |
2920 |
1856 |
1476 |
1138 |
RSD [%] |
60,7 |
57,9 |
70,1 |
55,5 |
21,7 |
n |
976 |
1026 |
929 |
929 |
89 |
S9: metabolic activation
Mean: mean of revertants/plate
Min.: minimum of revertnts/plate
Max.: maximum of revertants/plate
SD: Standard Deviation
RSD: Relative Standard Deviation
n: Number of control values
Applicant's summary and conclusion
- Conclusions:
- The test item is considered to be non-mutagenic in the bacterial reverse mutation assay.
- Executive summary:
The test item was investigated for its potential to induce gene mutations according to OECD 471 in the plate incorporation test (experiment I and experiment II) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101). In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation using S9. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:
Experiment I:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
Experiment II:
0.000158, 0.00050, 0.00158, 0.0050, 0.0158, 0.050, 0.158 and 0.5 μL/plate
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). Toxic effects of the test item were noted in all tester strains used in experiment I and II. In experiment I toxic effects of the test item were observed at concentrations of 0.100 μg/plate and higher (with and without metabolic activation) depending on the particular tester strain. In experiment II toxic effects of the test item were noted at concentrations of 0.050 μg/plate and higher (with and without metabolic activation) depending on the particular tester strain. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation. All criteria of validity were met. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test itenm did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.
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