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EC number: 202-077-8 | CAS number: 91-56-5
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From March 10 to May 22, 2015.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Test method according to OECD 471. GLP study.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 015
- Report date:
- 2015
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Indoline-2,3-dione
- EC Number:
- 202-077-8
- EC Name:
- Indoline-2,3-dione
- Cas Number:
- 91-56-5
- Molecular formula:
- C8H5NO2
- IUPAC Name:
- 2,3-dihydro-1H-indole-2,3-dione
- Test material form:
- solid: particulate/powder
- Remarks:
- migrated information: powder
- Details on test material:
- - Name of test material (as cited in study report): Indoline-2,3-dione
- Synonym: Isatin.
- Physical state: Red-orange crystalline powder.
- Analytical purity: 98%
- Lot/batch No.: MP1076.3
- Expiration date of the lot/batch: 30 November 2015
Constituent 1
Method
- Target gene:
- Histidine-requiring gene in Salmonella typhimurium and Tryptophan-requiring gene in the strain of Escherichia coli.
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix from rat liver
- Test concentrations with justification for top dose:
- 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: Based on the results of a solubility test, DMSO was selected for vehicle (solvent) of the study. The formulation at 100 mg/mL concentration using this vehicle was achievable and considered to be suitable for the test.
Controlsopen allclose all
- Negative solvent / vehicle controls:
- yes
- Remarks:
- (DMSO and distilled water)
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-1,2-phenylene-diamine
- Remarks:
- TA98, -S9 (4 µg/plate in DMSO)
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- TA100, TA1535, -S9 (2 µg/plate in distilles water)
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- TA1537, -S9 (50 µg/plate in DMSO)
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- E.coli WP2 uvrA, -S9 (2 µg/plate in distilled water)
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoantracene
- Remarks:
- all strains, +S9 (2 µg/plate Salmonella strains; 50 µg/plate E.coli; in DMSO)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Plate incorporation (initial mutation test):
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level).
The equivalent number of minimal glucose agar plates was properly labelled. The test item (or controls) and other components (top agar, overnight culture of test strain, phosphate buffer (pH 7.4) or S9 mix) were prepared freshly and added to the overlay (45°C). This solution was mixed and poured on the surface of minimal agar plates. After preparation, the plates were incubated at 37°C for 48 hours.
For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube.
Pre-incubation (confirmatory mutation test):
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control or concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes; the content was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. After preparation, the plates were incubated at 37°C for 48 hours.
NUMBER OF REPLICATIONS: 3 replicates per dose and controls.
EVALUATION OF EXPERIMENTAL DATA:
The colony numbers were determined by manual counting.
Visual examination of the plates was performed, precipitation or signs of growth inhibition (if any) were recorded.
The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated. - Evaluation criteria:
- Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
Criteria for a Negative Response:
A test article was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation. - Statistics:
- The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
Results and discussion
Test results
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- 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
RANGE-FINDING/SCREENING STUDIES: The numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system). No precipitate or signs of inhibitory, cytotoxic effect were detected on the plates in the preliminary experiment.
COMPARISON WITH HISTORICAL CONTROL DATA: The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains.
ADDITIONAL INFORMATION ON CYTOTOXICITY: No inhibitory, cytotoxic effect of the test item was seen in the main tests in any examined strains with and without metabolic activation.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Table 1. Summary Table of the Confirmatory Mutation Test
Concentrations (µg/plate) |
Mean values of revertants / Mutation factor (MF) |
Salmonella typhimurium |
Escherichia coli |
||||||||
TA98
|
TA100 |
TA1535 |
TA1537 |
WP2uvrA |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||
Untreated control |
Mean |
23.3 |
28.0 |
114.7 |
116.7 |
9.7 |
9.3 |
5.7 |
9.0 |
36.0 |
42.0 |
MF |
1.17 |
0.83 |
1.10 |
1.13 |
1.21 |
1.00 |
0.77 |
1.59 |
0.84 |
0.98 |
|
DMSO control |
Mean |
20.0 |
33.7 |
104.3 |
103.0 |
8.0 |
9.3 |
7.3 |
5.7 |
43.0 |
42.7 |
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|
Distilled water control |
Mean |
-- |
-- |
107.3 |
-- |
10.3 |
-- |
-- |
-- |
43.7 |
-- |
MF |
-- |
-- |
1.03 |
-- |
1.29 |
-- |
-- |
-- |
1.02 |
-- |
|
5000 |
Mean |
26.3 |
28.7 |
132.7 |
123.3 |
10.0 |
13.0 |
11.3 |
10.3 |
55.0 |
58.0 |
MF |
1.32 |
0.85 |
1.27 |
1.20 |
1.25 |
1.39 |
1.55 |
1.82 |
1.28 |
1.36 |
|
1581 |
Mean |
22.3 |
33.3 |
124.3 |
117.7 |
8.7 |
11.7 |
7.3 |
8.7 |
47.0 |
49.7 |
MF |
1.12 |
0.99 |
1.19 |
1.14 |
1.08 |
1.25 |
1.00 |
1.53 |
1.09 |
1.16 |
|
500 |
Mean |
28.3 |
25.7 |
119.7 |
88.7 |
7.0 |
8.7 |
7.3 |
8.7 |
45.3 |
44.0 |
MF |
1.42 |
0.76 |
1.15 |
0.86 |
0.88 |
0.93 |
1.00 |
1.53 |
1.05 |
1.03 |
|
158.1 |
Mean |
18.0 |
32.7 |
110.3 |
100.0 |
11.0 |
11.7 |
6.3 |
8.0 |
46.0 |
48.7 |
MF |
0.90 |
0.97 |
1.06 |
0.97 |
1.38 |
1.25 |
0.86 |
1.41 |
1.07 |
1.14 |
|
50 |
Mean |
18.7 |
30.7 |
98.7 |
93.0 |
7.3 |
5.7 |
9.0 |
9.3 |
42.0 |
49.7 |
MF |
0.93 |
0.91 |
0.95 |
0.9 |
0.92 |
0.61 |
1.23 |
1.65 |
0.98 |
1.16 |
|
15.81 |
Mean |
24.7 |
27.3 |
100.7 |
96.0 |
6.3 |
11.0 |
9.3 |
6.7 |
45.7 |
47.3 |
MF |
1.23 |
0.81 |
0.96 |
0.93 |
0.79 |
1.18 |
1.27 |
1.18 |
1.06 |
1.11 |
|
5 |
Mean |
21.7 |
32.0 |
100.0 |
93.7 |
8.0 |
8.0 |
7.0 |
7.0 |
44.0 |
51.7 |
MF |
1.08 |
0.95 |
0.96 |
0.91 |
1.00 |
0.86 |
0.95 |
1.24 |
1.02 |
1.21 |
|
NPD (4 µg) |
Mean |
361.3 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
MF |
18.07 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
2AA (2 µg) |
Mean |
-- |
2421.3 |
-- |
2255.3 |
-- |
284.7 |
-- |
192.7 |
-- |
-- |
MF |
-- |
71.92 |
-- |
21.90 |
-- |
30.5 |
-- |
34.00 |
-- |
-- |
|
2AA (50 µg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
184 |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
4.31 |
|
SAZ (2 µg) |
Mean |
-- |
-- |
1084 |
-- |
1169.3 |
-- |
-- |
-- |
-- |
-- |
MF |
-- |
-- |
10.1 |
-- |
113.16 |
-- |
-- |
-- |
-- |
-- |
|
9AA (50 µg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
394.7 |
-- |
-- |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
53.82 |
-- |
-- |
-- |
|
MMS (2 µL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
985.3 |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
22.56 |
-- |
|
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative (with and without metabolic activation)
The test item had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study. - Executive summary:
The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Guideline 471, EU Method B.13/14 and EPA OPPTS 870.5100. GLP study.
The experiments were carried out using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA in the presence and absence of metabolic activation (S9 fraction prepared from the livers of rats). Based on the results of the Solubility Test, the test item was dissolved in DMSO. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the preliminary experiment, the test item concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.
In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the vehicle control and were within the normal biological variability of the test system.
No precipitate was detected on the plates in the main tests in any examined bacterial strains with and without metabolic activation.
No inhibitory, cytotoxic effect of the test item was observed in the main tests in all examined strains with and without metabolic activation, and the controls were valid.
The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item Indoline-2,3-dione had no mutagenic activity in the examined bacterial strains under the test conditions of this study.
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