Registration Dossier

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:
26 February to 06 March 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to OECD 471 and GLP guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Thiazol Blau

Method

Target gene:
Salmonella typhimurium:
TA98 hisD3052 Frameshift
TA100 hisG46 Base pair substitution
TA1535 hisG46 Base pair substitution
TA1537 hisC3076 Frameshift

Escherichia coli:
WP2uvrA trpE Base pair substitution
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine dependent
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: tryptophan dependent
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
Initial Mutation Test and Confirmatory Mutation Test: 5000; 1581; 500; 158.1; 50; 15.81; 5 and 1.581 μg/plate
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
w/o S9

Migrated to IUCLID6: TA100, TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
w/o S9

Migrated to IUCLID6: TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine
Remarks:
TA98: w/o S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
w/o S9

Migrated to IUCLID6: WP2uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9: TA98, TA100, TA1535, TA1537, WP2uvrA
Details on test system and experimental conditions:
A standard plate incorporation procedure was performed, as an Initial Mutation Test. Bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system (rat liver S9 mix).
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 labeled. The test item and other components were prepared fresh and added to the overlay (45°C).

The content of the tubes:
top agar 2000 µL
solvent or test item solution or (reference controls) 100 (50) µL
overnight culture of test strain 100 µL
phosphate buffer (pH: 7.4) or S9 mix 500 µL

This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of negative and positive controls. The plates were incubated at 37°C for 48 hours.


A pre-incubation procedure was performed as a Confirmatory Mutation Test since the result of the Initial Mutation Test was negative.
Before the overlaying of the test item, the bacterial culture and the hamster S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its solvent). These tubes were gently mixed and incubated for 30 min at 30ºC 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. The entire test consisted of non-activated and activated test conditions, with the addition of negative and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.
Evaluation criteria:
The colony numbers on the untreated / negative / positive control and test plates were determined. 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 Excel software.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control

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 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.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
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:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Spontaneous Reversion of Tester Strain: Historical control values for untreated control plates without metabolic activationin the period of 1999 to 2008 are (as guide) as follows: (-S9)Salmonella typhimuriumTA98: 9-54, TA100: 58-211, TA1535: 4-31, TA1537: 1-24,Escherichia coliWP2uvrA: 9-66revertants/plate.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item Thiazol Blau was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from induced (phenobarbital/-naphthoflavone) rat liver in the experiment using the plate incorporation method and a cofactor-supplemented post-mitochondrial S9 fraction prepared from uninduced hamster liver in the experiments using the pre-incubation method.

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test, an Initial Mutation Test and a Confirmatory Mutation Test. In the Range Finding Test and Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.

Based on the results of the Solubility Test, the test item was dissolved in DMF
(N,N-Dimethylformamide) at a concentration of 50 mg/mL (stock solution). Based on the results of the preliminary Range Finding Test the following concentrations of the test item were used in the Initial Mutation Test and Confirmatory Mutation Test: 5000; 1581; 500; 158.1; 50; 15.81; 5 and 1.581 µg/plate. The test item concentrations, including the controls (untreated, solvent and positive reference) were tested in triplicate.

None of the observed revertant colony numbers were above the respective biological threshold value in the experiments. There were no dose-related trends and no indication of any treatment-related effect in any of the five bacterial strains either in the presence or absence of metabolic activation (±S9-mix).

Sporadically, higher revertant colony numbers compared to the solvent control plates were observed in the two independently performed main experiments. However, those values did not follow a dose-response relationship and they were below the biologically relevant threshold value. In conclusion, the results were considered to reflect the biological variability of the test in the performed experiments.

Reduced number of revertant colonies compared to the solvent control plates was detected in the Confirmatory Mutation Test in E. coli WP2 uvrA bacterial strain at 5000 g/plate concentration with metabolic activation, but no effect of the test item was observed on the background lawn development.

Precipitate was observed in the Initial Mutation Test at 5000 µg/plate concentration in all of the tester strains without and with metabolic activation (±S9 Mix) and in the Confirmatory Mutation Test at 5000 and 1581 µg/plate concentrations in all of the tester strains without and with metabolic activation (±S9 Mix).

The mean values of revertant colonies of the untreated and solvent control plates were within the historical control data range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied 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 Thiazol Blau had no mutagenic activity on the growth of the bacterium tester strains under the test conditions used in this study.
Executive summary:

The test item Thiazol Blau was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains ofSalmonella typhimurium(Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain ofEscherichia coli(Escherichia coliWP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from induced (phenobarbital/b-naphthoflavone) rat liver in the experiment using the plate incorporation method and a cofactor-supplemented post-mitochondrial S9 fraction prepared from uninduced hamster liver in the experiments using the pre-incubation method.

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test, an Initial Mutation Test and a Confirmatory Mutation Test. In the Range Finding Test and Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.

Based on the results of the Solubility Test, the test item was dissolved in DMF
(N,N-Dimethylformamide) at a concentration of 50 mg/mL (stock solution). Based on the results of the preliminary Range Finding Test the following concentrations of the test item were used in the Initial Mutation Test and Confirmatory Mutation Test:5000; 1581; 500; 158.1; 50; 15.81; 5 and 1.581 µg/plate. The test item concentrations, including the controls (untreated, solvent and positive reference) were tested in triplicate.

None of the observed revertant colony numbers were above therespective biological threshold value in the experiments. There were no dose-related trends and no indication of any treatment-related effect in any of the five bacterial strains either inthe presence or absence of metabolic activation (±S9-mix).

Sporadically, higher revertant colony numbers compared to the solvent control plates were observed in the two independently performed main experiments. However, those valuesdid not follow a dose-response relationship and they were below the biologically relevant threshold value. In conclusion, the results wereconsidered to reflect the biological variability of the test in the performed experiments.

Reduced number of revertant colonies compared to the solvent control plates was detected in the Confirmatory Mutation Test inE. coliWP2uvrAbacterial strain at 5000 mg/plate concentration with metabolic activation, but no effect of the test item was observed on the background lawn development.

Precipitate was observed in the Initial Mutation Test at 5000 µg/plate concentration in all of the tester strains without and with metabolic activation (±S9 Mix) and in the Confirmatory Mutation Test at 5000and 1581 µg/plate concentrations in all of the tester strains without and with metabolic activation (±S9 Mix).

The mean values of revertant colonies of the untreated and solvent control plates were within the historical control data range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test itemdid not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. 

In conclusion, the test itemThiazol Blau had no mutagenic activity on the growth of the bacterium tester strainsunder the test conditions used in this study.