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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:
From April 18, 2017 to May 18, 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 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
Test material form:
solid
Details on test material:
CAS No.: 23054-60-6 + 23054-61-7; Batch No.: 7723329-R802; Purity: 100 %; Appearance: colourless/pale straw clear liquid (white solid was confirmed by visual inspection upon arrival at test facility at room temperature. Test substance was clear liquid after incubation at 37 °C).
Specific details on test material used for the study:
CAS No.: 23054-60-6 + 23054-61-7; Batch/Lot No.: 7723329-R802; Purity: 100 %; Appearance: colourless/pale straw clear liquid (white solid was confirmed by visual inspection upon arrival at test facility at room temperature. Test substance was clear liquid after incubation at 37 °C).

Method

Target gene:
In addition to histidine or tryptophan mutation, each strain has additional mutations, which enhances its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair, making them more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The presence of rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2 uvrA) is also defective in DNA excision repair.
Genotypes of the used strains: S.typhimurium TA98 (hisD3052), S.typhimurium TA100 (hisG46), S.typhimurium TA1535 (hisG46), S.typhimurium TA1537 (hisC3076), WP2 (uvrA trpE).
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Range finding test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation: 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate;

Salmonella typhimurium strains with and without metabolic activation:
Initial mutation test: 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate
Confirmatory mutation test: 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate

Escherichia coli WP2 uvrA strain with and without metabolic activation:
Initial mutation test and in the confirmatory mutation test: 5000, 1581, 500, 158.1, 50 15.81, 5 and 1.581 μg/plate
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, distilled water
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene; 4-nitro-1,2-phenylenediamine
Details on test system and experimental conditions:
The study included a preliminary compatibility test, a preliminary concentration range finding test (informatory toxicity test), an initial mutation test and a confirmatory mutation test. In the preliminary concentration range finding test as well as in the initial mutation test, the plate incorporation method was used. In the confirmatory mutation test, the pre-incubation method was used.

Preliminary compatibility test:

The appropriate vehicle (solvent) and the behaviour of the test substance formulations with the solution of top agar and phosphate buffer were examined in a preliminary compatibility test. Distilled water was used as solvent to prepare the stock solution of the test substance. Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent and were used within 4 hours after preparation.

Preliminary concentration range finding test:

Based on the solubility test, a 100 mg/mL stock solution was prepared in distilled water. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test substance, in the absence and presence of metabolic activation.
Test substance concentrations in the mutagenicity tests (initial mutation test and confirmatory mutation test).
Based on the results of the preliminary tests, a 100 mg/mL stock solution was prepared in distilled water. Maximum eight test concentrations were prepared by successive dilutions of the stock solution, to obtain lower doses. The maximum test concentration was 5000 μg test substance/plate.

Procedure for exposure in the initial mutation test:

The initial mutation test followed the standard plate incorporation procedure. Bacteria (cultured in nutrient broth No.2 were exposed to the test substance 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 substance 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 substance 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.

Procedure for exposure in the confirmatory mutation test:
The confirmatory mutation test followed the standard pre-incubation procedure since no biologically relevant increase in the number of revertant colonies was observed in the initial mutation test. Bacteria (cultured in nutrient broth No.2.) were exposed to the test substance 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 substance formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test substance (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37 ºC 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.
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 substance 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 was considered biologically relevant if:
- the number of reversions is 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 is 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:
A test article was considered non-mutagenic if:
- the total number of revertants in tester strain Salmonella typhimurium TA98, TA100 or Escherichia coli WP2 uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strain Salmonella typhimurium TA1535 or TA1537 is not greater than three times the concurrent vehicle control;
- the negative response should be reproducible in at least one follow up experiment.
Statistics:
The colony numbers on the untreated / negative (solvent) / positive control and test substance 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 substance and for the controls using Microsoft ExcelTM software.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary experiment:
- 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). No precipitate was observed in the preliminary concentration range finding test in both bacterial strains with and without metabolic activation.
- Slightly reduced background lawn was detected in the preliminary range finding test in Salmonella typhimurium TA98 strain on the plates at 5000 μg/plate without metabolic activation, in Salmonella typhimurium TA100 strain absent/reduced background lawn was observed on the plates at 5000, 2500 μg/plate without metabolic activation and at 5000 μg/plate concentration with metabolic activation.

The initial mutation test and confirmatory mutation test:

- No precipitate was observed in the main tests in all examined strains with and without metabolic activation.

- In the initial mutation test absent/reduced background lawn was detected in all Salmonella typhimurium strains on the plates at 5000 μg/plate concentration with and without metabolic activation.
- In the initial mutation test and confirmatory mutation test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.
- In the initial mutation test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 strain at 500 and 5 μg/plate concentrations without metabolic activation (the observed mutation factor value was: MF: 1.47). 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 the confirmatory mutation test absent/reduced/slightly reduced background lawn was observed in all Salmonella typhimurium TA98 strain and in Escherichia coli WP2 uvrA strain on the plates at 5000 and 1581 μg/plate concentrations with and without metabolic activation. The same effect was detected in Salmonella typhimurium TA100, TA1535, TA1537 strains at 5000, 1581 μg/plate concentrations with metabolic activation and 5000, 1581, 500 μg/plate concentrations without metabolic activation.
- In the confirmatory mutation test (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 15.81 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.39). 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.

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the reported data of this mutagenicity assay show that the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test substance has no mutagenic activity on the growth of the bacterial strains.
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471, EU Method B.13/14 and OPPTS 870.5100, in compliance with GLP. The experiments were carried out using strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and WP2 uvrA strain of Escherichia coli in the presence and absence of a post mitochondrial supernatant (S9 fraction). The test substance was dissolved in distilled water at a concentration of 100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the range finding test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of this test, the test substance concentrations in the initial mutation test were within range of 5000 - 5 μg/plate and in the confirmatory mutation test were in the range of 5000 - 1.581 μg/plate at the Salmonella typhimurium strains with and without metabolic activation. In the initial mutation test and in the confirmatory mutation test tested concentrations in Escherichia coli WP2 uvrA strain with and without metabolic activation were in between 5000 and 1.581 μg/plate. No precipitate was observed in the main tests in all examined strains with and without metabolic activation. Inhibitory, cytotoxic effect of the test substance (absent / reduced / slightly reduced background lawn development) was observed in the main tests in all examined strains with and without metabolic activation at several concentrations (expect in the initial mutation test at Escherichia coli WP2 uvrA strain). In the initial mutation test and confirmatory mutation test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect. The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control 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. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid. Under the study conditions, the reported data of this mutagenicity assay show that the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test substance has no mutagenic activity on the growth of the bacterial strains (Orovecz, 2017).