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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:
Experimental Period I 26-28 June 2019, Experimental Perios II 01 - 04 July 2019.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
3-tert-butyl-1-(3,5-dicarboxyphenyl)-1H-pyrazol-5-aminium chloride
EC Number:
941-661-3
Molecular formula:
C15H18ClN3O4
IUPAC Name:
3-tert-butyl-1-(3,5-dicarboxyphenyl)-1H-pyrazol-5-aminium chloride
impurity 1
Reference substance name:
Unidentified impurity
IUPAC Name:
Unidentified impurity

Method

Target gene:
Histidine for Salmonella. Trytophan for E.coli.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: MOLTOX - Molecular Toxicology Inc, Boone, North Carolina, USA
Additional strain / cell type characteristics:
other: histidine negative
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
Additional strain / cell type characteristics:
other: trytophan negative
Metabolic activation:
with and without
Metabolic activation system:
cofactor-supplemented post-mitochondrial S9 fraction
Test concentrations with justification for top dose:
Concentrations for the main tests were selected on the basis of the Preliminary Compatibility Test and Preliminary Range Finding Test (Informatory Toxicity Test). In the Assay 1 and Assay 2, the same concentrations were used.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO; distilled water
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO), N,N-Dimethylformamide (DMF), Acetone and Ethanol. At the concentration level of 100 mg/mL, partial solubility was detected using Distilled water, Acetone and Ethanol. After approximately 5 minutes of vortex no changes in the formulations were observed. However, at the same concentration level after approximately 5 minutes of vortex, complete solubility was detected using DMSO and DMF. Due to the better biocompatibility, DMSO was selected as vehicle for the study.
Controls
Untreated negative controls:
yes
Remarks:
solvent
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylene-diamine, methyl-methanesulfonate; 2-aminoanthracene
Details on test system and experimental conditions:
Assay 1 followed the standard plate incorporation procedure. Bacteria (cultured in Nutrient Broth No.2 as described in Section 5.3.6.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
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 and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes:
top agar 2000 μL
vehicle or test item formulation (or reference controls) 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 untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48(±1) hours.

Assay 2 followed the standard pre-incubation procedure [1][2][5][6][7][8] since no biologically relevant increase in the number of revertant colonies was observed in the Assay 1 (see details in Section 6.).
Bacteria (cultured in Nutrient Broth No.2. as described in section 5.3.6.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture (Section 5.3.6.) 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 same volumes were used as in Assay 1. The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48(±1) hours.

The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. 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.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.
Rationale for test conditions:
Based on the results of the preliminary experiment, the examined test concentrations in the assays were: 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.
Evaluation criteria:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analysable concentrations are presented in all strains of the main tests

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a concentration-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 is considered biologically relevant if:
- in all strains: the number of reversions is more than two times higher than the reversion rate of the negative (solvent) 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 is considered non-mutagenic if it produces neither a concentration-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the concentration groups, with or without metabolic activation.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: slightly reduced background lawn develpoment in assay 2 at 5000 ug/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: slightly reduced background lawn develpoment in assay 2 at 5000 ug/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: slightly reduced background lawn develpoment in assay 2 at 5000 ug/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: slightly reduced background lawn develpoment in assay 2 at 5000 ug/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
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:
not applicable
Positive controls validity:
valid
Additional information on results:
Slight precipitate was detected on the plates in Assay 2 in all examined bacterial strains with and/or without metabolic activation at 5000 μg/plate concentration.
Inhibitory, cytotoxic effect of the test item (reduced / slightly reduced background lawn development) was observed in Assay 2 in all Salmonella typhimurium bacterial strains with metabolic activation at 5000 μg/plate concentration.
In Assay 1 (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 strain at 15.81 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.23). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.
In Assay 2 (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 1581 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.44). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

Applicant's summary and conclusion

Conclusions:
The test item had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.