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Genetic toxicity: in vitro

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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:
14 April 2010 - 6 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
S. typhimurium: Histidine locus
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):
Not applicable
Additional strain / cell type characteristics:
other: the Salmonella typhimurium strains are constructed to differentiate between base-pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: The Escherichia coli WP2 uvrA detects mutagens that cause base-pair substitutions (AT to GC).
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 5000; 1581; 500; 158.1; 50; 15.81; 5 and 1.581 μg/plate
Concentration range in the main test (without metabolic activation): 5000; 1581; 500; 158.1; 50; 15.81; 5 and 1.581 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: N,N-Dimethylformamide
- Justification for choice of solvent/vehicle: The test item could not be dissolved in Dimethyl sulfoxide at 100, 50 or 25 mg/mL concentration. However, it was soluble in Tetrahydrofuran (THF) and N,N-Dimethylformamide (DMF) at 100 mg/mL concentration. As DMF has better biocompatibility with the test system, it was chosen as a solvent for the study.

Concentration Range Finding Test (Informatory Toxicity Test)
Based on the solubility test, 100 mg/mL stock solution was prepared in DMF, which was diluted in 6 steps 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 the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item.

Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test and Confirmatory Mutation Test)
Based on the results of the preliminary tests, 100 mg/mL stock solution was prepared from the test item with DMF, which was diluted by serial dilutions in seven steps to obtain eight dosing solutions for lower doses. The maximum test concentration was 5000 μg test item/plate. Examined test material concentrations were: 5000; 1581; 500; 158.1; 50; 15.81; 5 and 1.581 μg/plate.

Strain-specific positive and negative (solvent) controls, both with and without metabolic activation were included in each test. In addition, untreated control was used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See "Any other information on materials and methods incl. tables" section below
Details on test system and experimental conditions:
Procedure for Exposure in the Initial Mutation Test
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. 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
solvent or test item solution 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 and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.

Procedure for Exposure in the Confirmatory Mutation Test
A pre-incubation procedure was performed as a Confirmatory Mutation Test since the result of the Initial Mutation Test was negative. Before the overlaying, the test item solution (or 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 item (in its solvent). The tubes were gently mixed and incubated for 20 min at 37ºC 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 untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.
Evaluation criteria:
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:
- the number of reversions is at least twice as high as the reversion rate of the solvent control in all strains
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.
Statistics:
The colony numbers on the untreated / negative / positive control and test item treated plates were determined by manual counting. 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.

Results and discussion

Test resultsopen allclose all
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:
cytotoxicity
Remarks:
Slightly reduced background lawn development was observed in S. typhimurium TA98 strain at 5000 μg/plate without metabolic activation and in S. typhimurium TA1537 bacterial strain at 5000 μg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)

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. In the test each samples (including the controls) were tested in triplicate.
In the Range Finding Test the concentrations examined were: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
Sporadically, higher numbers of revertant colonies compared to the solvent control plates were observed in Salmonella typhimurium TA98 bacterial strain without metabolic activation. However, no dose-dependence was observed, the observed mutation factor values were below the biologically relevant threshold value and the numbers of revertant colonies were within the historical control range in each case.
Sporadically, slightly lower numbers of revertant colonies compared to the solvent control plates were observed in Salmonella typhimurium TA98 bacterial strain with metabolic activation. However, the numbers of revertant colonies were within the historical control range in each case.
Note: No revertant colonies were observed in in Salmonella typhimurium TA98 bacterial strain at 5000 μg/plate with and without metabolic activation, however no effect on background lawn development was detected.
The observed revertant rates in Salmonella typhimurium TA100 bacterial strain with and without metabolic activation were in the normal range.
Microdrops were observed in both tester strains at 5000 and 2500 μg/plate with and without metabolic activation in the Preliminary Range Finding Test.

INITIAL AND CONFIRMATORY MUTATION TESTS

In the Initial Mutation Test, the plate incorporation method; in the Confirmatory Mutation Test, the pre-incubation method was used. The Initial Mutation Test and Confirmatory Mutation Test were carried out using Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strain. Each test was performed in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. The test item concentrations (including the controls) were tested in triplicate.
The examined test item concentrations in the main tests were: 5000; 1581; 500; 158.1; 50; 15.81; 5 and 1.581 μg/plate.
In the Initial Mutation Test and Confirmatory Mutation Tests, none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect.
Using the plate incorporation method, the highest revertant rate was observed in the Initial Mutation Test in Salmonella typhimurium TA98 tester strain without metabolic activation at the concentration of 15.81 μg/plate. The mutation factor value was 1.95. Higher numbers of revertant colonies compared to the solvent control were observed at other tested concentration also in this experiment. However, no dose-dependence was observed, the observed mutation factor values did not reach the biologically relevant threshold value and the numbers of revertant colonies were within the historical control range. Furthermore, higher revertant rate compared to the solvent control was detected for DMSO control (MF=1.33) also.
Using the pre-incubation method, highest revertant rate was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA1535 tester strain without metabolic activation at the concentration of 5000 μg/plate (the mutation factor value was 1.57). However, no dose-dependence was observed, this value was below the biologically relevant threshold value and the numbers of revertant colonies were within the historical control range.
Sporadically, higher numbers of revertant colonies compared to the solvent control were detected in the Initial Mutation Test and Confirmatory Mutation Test in some cases. However, no dose-dependence was observed and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in all cases, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the solvent control were observed in the Initial Mutation Test and Confirmatory Mutation Test at some other cases. However, the mean numbers of revertant colonies were in the historical control range in all cases and were considered as biological variability of the test system.
A weak inhibitory effect of the test item (slightly reduced background lawn development) was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA98 strain at 5000 μg/plate concentration without metabolic activation and in Salmonella typhimurium TA1537 bacterial strain at 5000 μg/plate concentration with and without metabolic activation.
Microdrops were observed in all of the five tester strains at 5000 μg/plate with and without metabolic activation in the Initial Mutation Test and Confirmatory Mutation Test.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

VALIDITY OF THE TESTS

Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated and solvent control plates were within the historical control data range*.

*Note: In the Initial Mutation Test, in Salmonella typhimurium TA100 strain without metabolic activation, the mean values of revertant colony numbers of DMF control plates was 67.3. This value was lower than the lower limit of the historical control range (70-195) of this solvent. However, the observed lower counts were compatible with the much broader historical control range of untreated control (54-200), DMSO control (58-211) or Distilled water control (51-208) samples. As there was no indication of any cytotoxic effect of the DMF solvent, furthermore the values observed for untreated and DMSO control samples were within their historical control range in these experiments, the results of the DMF control were considered to reflect the biological variability of the test system.

The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

In conclusion, the test item had no mutagenic activity on the growth of the applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to EU Test Method B.13/14.

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 post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Preliminary Solubility Test, the test item was dissolved in N,N-Dimethylformamide. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Preliminary Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the two independently performed main experiments (Initial Mutation Test and Confirmatory Mutation Test) were: 5000; 1581; 500; 158.1; 50; 15.81; 5 and 1.581 μg/plate.

In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends and no indication of any treatment effect.

In all test item treated groups, the numbers of revertant colonies were below the biological relevance when compared with the solvent controls and were within the historical control range and were within the normal biological variability of the test system.

A weak inhibitory effect of the test item (slightly reduced background lawn development) was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA98 strain at 5000 μg/plate concentration without metabolic activation and in Salmonella typhimurium TA1537 bacterial strain at 5000 μg/plate concentration with and without metabolic activation.

Microdrops were observed in all of the five tester strains at 5000 μg/plate with and without metabolic activation in the Initial Mutation Test and Confirmatory Mutation Test.

The mean values of revertant colonies of the solvent control plates were within the acceptable 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 had no mutagenic activity on the growth of the bacterium tester strains under the test conditions used in this study.