Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09-10-2016 - 31-10-2016 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test". (adopted July 21, 1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 31 May 2008.
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
his (S. typhimurium)
trp (E. coli)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535: 2006, TA1537: 2016, TA98: 2015, TA100: 2015; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA: 2008)]
- Suitability of cells:Recommended test system in international guidelines (e.g. OECD, EC).

MEDIA USED
- Periodically 'cleansed' against high spontaneous background: yes
Additional strain / cell type characteristics:
other: see "Any other information on materials and methods"
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced male Sprague Dawley rat S9
Test concentrations with justification for top dose:
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and the WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate.
The highest concentration of the test item used in the subsequent mutation assays was 1600 µg/plate. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix.
Plate incorporation: The test item was initially tested in the tester strains TA100 and WP2uvrA as a dose range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 17, 52, 164, 512 and 1600 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Remarks:
solvent controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (for test item, positive control); physiological saline (for positive control)
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191; 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, 1st experiment); preincubation (2nd experiment)
- Cell density at seeding (if applicable): 0.1 ml of a fresh bacterial culture (10exp9 cells/ml)

DURATION
- Preincubation period: 30 minutes by 70 rpm at 37°C,
- Exposure duration: 48 ± 4 h

SELECTION AGENT (mutation assays): his/trp minimal agar

NUMBER OF REPLICATIONS: triplicates, two experiments

DETERMINATION OF CYTOTOXICITY
- Method: evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn
Rationale for test conditions:
Recommended test system in international guidelines (e.g. OECD, EC).
Evaluation criteria:
Data evaluation and statistical procedures
No formal hypothesis testing was done.
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment. A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated 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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated 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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated 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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none stated
- Effects of osmolality: none stated
- Precipitation:
Direct plate assay: Precipitation of the test item on the plates was not observed at the start of the incubation period. At the end of the incubation period the test item precipitated at 512 μg/plate and above in the tester strains TA100 (absence of S9-mix) and WP2uvrA (absence and presence of S9-mix), and at 1600 μg/plate and above in the tester strains TA100 (presence of S9-mix) and TA1537 and TA98 (absence and presence of S9-mix). No precipitation was observed in tester strain TA1535 in the absence and presence of S9-mix.
Pre-incubation assay: Precipitation of the test item on the plates was not observed at the start of the incubation period. At the end of the incubation period, the test item precipitated at 1600 μg/plate.

RANGE-FINDING/SCREENING STUDIES:
Direct plate assay: The test item was initially tested in the tester strains TA100 and WP2uvrA as a dose range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 17, 52, 164, 512 and 1600 μg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used:
Direct plate assay: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in the tester strains TA100 and WP2uvrA in the absence and presence of S9-mix. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in the tester strains TA1535, TA1537 and TA98 in the absence and presence of S9-mix.
Pre-incubation assay: Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA98 in the absence of S9-mix at 512 and 1600 μg/plate. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all other tester strains in the absence and presence of S9-mix.

Applicant's summary and conclusion

Conclusions:
The study was conducted according to OECD guideline 471 under GLP on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of Isobutyl 4-chloro-3,5-diaminobenzoate to induce gene mutations in bacteria.
All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Evaluation of the mutagenic activity of (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)benzenecarbothioate was performed according to OECD 471 under GLP in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (plate incorporation and pre-incubation methods)

(S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay both in the absence and presence of S9-mix (rat liver S9-mix induced Aroclor 1254).

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The vehicle of the test item was dimethyl sulfoxide.

In the dose range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item precipitated on the plates at dose levels of 512 or 1600 µg/plate and upwards. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in both tester strains in the absence and presence of S9-mix.

In the first mutation experiment, the test item was tested up to concentrations of 1600 µg/plate in the strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the dose level of 1600 µg/plate, except in tester strain TA1535. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation experiment, the test item was tested up to concentrations of 1600 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item precipitated on the plates at the dose level of 1600 µg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA98 in the absence of S9-mix.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.