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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 March 2017 - 26 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Willing New Material Technology Co.,Ltd.; 23161211201
- Expiration date of the lot/batch: 10 December 2017
- Purity: 97%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 ºC)

Method

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:
Phenobarbital/β-naphthoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Range Finding Test: 5000; 2500; 1000; 316; 100; 31.6 ,10 μg/plate
Main Test 1: 5000, 1581, 500, 158.1, 50, 15.81 μg/plate
Main Test 2: 5000, 1581, 500, 158.1, 50, 15.81, 5 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: N,N-Dimethylformamide (DMF)
- Justification for choice of solvent/vehicle:
The solubility of the test item was examined using Distilled water, N,N-Dimethylformamide (DMF) and Dimethyl sulfoxide (DMSO). Test item was insoluble at 100 mg/mL concentration using Distilled water. Partial dissolution was observed at the same concentration using DMSO. The test item was soluble at this concentration using DMF. DMF was selected as vehicle (solvent) for the study
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMF
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
Remarks:
TA98: 4-nitro-1,2-phenylenediamine (NPD) without activation; All strains: 2-aminoanthracene (2AA) with activation
Details on test system and experimental conditions:
ETHOD OF APPLICATION: Preliminary Concentration Range Finding Test as well as in the first Main Test, the plate incorporation method was used. In the second main test, the pre-incubation method was used.

DURATION
- Preincubation period: 20min at 37ºC
- Exposure duration: 48±1 hours

NUMBER OF REPLICATIONS: Triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in background lawn
Evaluation criteria:
Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

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 positiveresponse 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 was 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 was more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and
TA1537 bacterial strains.

Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produces 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:
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.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: All stains
Remarks:
Main test 1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: All strains
Remarks:
Main test 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate/slight precipitate was detected on the plates in the main tests in all examined strains with and without metabolic activation at higher concentrations


RANGE-FINDING/SCREENING STUDIES:
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.

Following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
In the preliminary experiment, 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). Precipitate/slight precipitate was observed in the Preliminary Concentration Range Finding Test in both bacterial strains with and without metabolic activation at 5000, 2500 and 1000 μg/plate concentrations. Inhibitory or toxic effects of the test item were not detected in the preliminary Experiment. Based on the results of the Range Finding Test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 μg/plate. The experimental results (revertant colony numbers per plate, mutation factors and standard deviations) are detailed in Table 7 (Appendix 2) and in Appendix 3.


HISTORICAL CONTROL DATA
- Positive historical control data: Yes with ranges, means and standard deviation (Period of 2011-2015) Appendix 6
- Negative (solvent/vehicle) historical control data: Yes with ranges, means and standard deviation (Period of 2011-2015) Appendix 6

Applicant's summary and conclusion

Conclusions:
TiBTD has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.
Executive summary:

In a reverse gene mutation assay in bacteria (17_053-007M), strains of S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A were exposed to TiBTD (97%) in DMF at concentrations of 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.58 μg/plate (direct plate incorporation; experiment 1) and 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate (20 minute pre-incubation; experiment 2) in the presence and absence of mammalian metabolic activation (Phenobarbital/β- naphthoflavone-induced rat liver S9).

TiBTD was tested up to the limit concentration (5000 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.