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Genetic toxicity: in vitro

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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:
29 September - 16 October, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is performed according to OECD Guideline 471 under GLP conditions. No deviations reported.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 4, 4'-methylene bis (2-chlorobenzen amine)
- Substance type: white, needlelike crystalline powder
- Physical state: solid
- Analytical purity: 99.76%
- Impurities (identity and concentrations): not mentioned
- Purity test date: 2002-12-13
- Lot/batch No.: FG-3002
- Expiration date of the lot/batch: not mentioned
- Stability under test conditions: stable during study period; purity was 99.11% at 2003-12-15
- Storage condition of test material: protected from air and mosture at 3-6 °C

Method

Target gene:
Histidine gene in S. Typhimurium
Tryptophan gene in E. Coli
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
Metabolic activation:
with and without
Metabolic activation system:
S9 (rat)
Test concentrations with justification for top dose:
Dose-range finding: 0, 0.0191, 0.0763, 0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250 µg/plate.
Main test: 0, 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: evaluated before start of the study, dissolution observable, without foaming, heat generation/absorption, discoloration.
Controls
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: 2-aminoanthracene, AF-2, sodium azide, 2-methoxy-6-chloro-9-[3-2(-chloroethyl)-aminopropylamino]acridine-2HCl
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 20 min.
- Exposure duration: 48 hours
Evaluation criteria:
(1) In the positive control groups, the number of revertant colonies in each bacterial strain increased to more than double the number in the negative control.
(2) In all dosage groups there was no marked difference in number of revertant colonies between the three plates.
(3) The number of revertant colonies in each bacterial tester strain, with or without metabolic activation and in both negative and positive controls, was within the range of the test facility’s background data values. The test system was deemed valid as the above criteria were met.
Statistics:
No statistical methods were used in the evaluation.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
at 19.5 and 78.1 µg/plate (+S9) not without S9
Cytotoxicity / choice of top concentrations:
other: white deposition observed from 19.5 µg/plate onwards
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
at 19.5, 39.1, 78.1 and 156 µg/plate (+S9) not without S9
Cytotoxicity / choice of top concentrations:
other: white deposition observed from 19.5 µg/plate onwards
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: white deposition observed at concentrations considered genotoxic

RANGE-FINDING/SCREENING STUDIES:
Range-finding:
1) Results of observation following completion of culturing:
Observation by stereoscopic microscope following completion of culturing revealed the presence of a white precipitate in the test article treatment groups at 313 μg/plate and above without metabolic activation and at 19.5 μg/plate and above with metabolic activation. Inhibition of growth in tester strains was seen in test article treatment groups at 313μg/plate and above irrespective of tester strains or conditions with/without metabolic activation.
2) Number of revertant colonies
In test article treatment groups at 19.5 μg/plate and above with metabolic activation, the number of S. typhimurium TA100 and TA98 revertant colonies increased to at least 2 folds more than the number in the negative (vehicle) control. The highest number of revertant colonies occurred under metabolic activation at 78.1 μg/plate, with S. typhimurium TA100 having 4.81 times more revertant colonies and TA98 having 3.58 times more revertant colonies than the negative control. However, under metabolic activation in all test article treatment groups (0.0191-5000 μg/plate), the number of revertant colonies of S. typhimurium TA1535, TA1537 and E.coli WP2 uvrA did not increase to double the number in the negative control. Similarly, the number of revertant colonies did not increase to double the number in the negative control in any test article treatment group (0.0191-5000 μg/plate) without metabolic activation, irrespective of tester strains.

Main test:
Results of observation following completion of culturing:
1) Observation by stereoscopic microscope following completion of culturing revealed the presence of a white precipitate in the test article treatment groups at the highest dosage of 313 μg/plate without metabolic activation and at 19.5 μg/plate and above with metabolic activation, irrespective of bacterial tester strain. However, inhibition of growth in tester strains was seen in the test article treatment group with the highest dosage of 313 μg/plate, irrespective of tester strains or conditions with/without metabolic activation.
2) Number of revertant colonies
In the main test, in test article treatment groups at 19.5 μg/plate and above with metabolic activation, the number of revertant colonies of S. typhimurium TA100 and TA98 increased to at least double the number in the negative control. The highest number of revertant colonies occurred at 156 μg/plate under metabolic activation, with S. typhimurium TA100 having 5.65 times more revertant colonies and TA98 having 3.58 times more revertant colonies than the negative control. However, under metabolic activation in all test article treatment groups (2.44-313 μg/plate), the number of revertant colonies of S. typhimurium TA1535, TA1537 and E.coli WP2 uvrA did not increase to double the number in the negative control. Similarly, an increase in number of revertant colonies to double that of the negative control was not seen in any test article treatment group (2.44-313 μg/plate) without metabolic activation, irrespective of tester strains.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation in TA 98 and TA 100

The gene reverse mutation test was performed using Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA. Both in the dose–range finding test and in main test , with metabolic activation (rat S9) for S. typhimurium TA100 and TA98, the number of revertant colonies increased more than 2-folds that of the negative control in the groups treated with the test article of 19.5 μg/plate or over. Meanwhile, in S. typhimurium TA1535, TA1537 and E. coli WP2 uvrA with metabolic activation (rat S9), the number of revertant colonies did not increase more than 2-folds that of the negative control in all the groups treated with the test article. Without metabolic activation, regardless of types of the tester strains, the number of revertant colonies did not increase more than 2-folds that of the negative control in all the groups treated with the test article. Based on the described results, 4,4'-methylene bis (2-chlorobenzene amine) was considered to express reverse mutagenicity under the present study conditions.