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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 29th to 31st October, 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)
Version / remarks:
1997
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Acid Brown 303
IUPAC Name:
Acid Brown 303
Test material form:
solid: particulate/powder

Method

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
Metabolic activation system:
The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).

Rat liver S9 fraction
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was used.

S9 mix (with rat liver S9)
Salt solution for S9 mix Final concentration in S9 mix
NADP Na 7.66 g 4 mM
D-glucose-6 phosphate Na 3.53 g 5 mM
MgCl2 1.90 g 8 mM
KCl 6.15 g 33 mM
Ultrapure water ad 1000 ml
Sterilized by filtration through a 0.22 µm membrane filter.

The complete S9 mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 ml
Rat liver homogenate (S9) 100 ml
Salt solution for S9 mix 400 ml
The S9 mix (containing 10 % S9) was kept in an ice bath before it was added to the culture medium.

The present test item contains azo-bonds. Therefore, the modified protocol proposed by Prival and Mitchell [10] (e.g.: using of hamster liver S9 Mix that contains 30 % hamster liver extract etc.) was originally planned for the confirmatory mutation test.
However, in this study unequivocal, demonstrative positive results were obtained in the initial mutation test (plate incorporation test); therefore, further investigation, following the modified protocol proposed by Prival and Mitchell was considered as not necessary for the final conclusion of the study.

Sodium phosphate buffer (0.2 M, pH 7.4)
Solution A:
Na2HPO4 × 12H2O 71.63 g
Ultrapure water ad 1000 ml
Solution B:
NaH2PO4 × H2O 27.6 g
Ultrapure water ad 1000 ml
Solution A 880 ml
Solution B 120 ml*
* The components were mixed in the above ratio; thereafter the pH was checked and corrected. The correction was performed with admixture of the solution A or B.
After the pH setting the sterilization was performed by filtration through a 0.22 µm membrane filter.
Test concentrations with justification for top dose:
Concentration tested in µg/plate: 5000, 1600, 500, 160, 50, 16, 5
Justification of concentrations: selection of the concentrations for the initial mutation test was based on the results of the solubility trials and the concentration range finding test (informatory toxicity test).
The maximum test concentration was 5000 µg/plate (±S9), as recommended in the guideline. For the dose selection the cytotoxicity, the solubility and a possible positive effect of the test item were taken into consideration.
In the main test, at the preparation of the test item stock solution a correction (multiplier) factor of 1.25 (1/0.80=1.25) based on its purity of 80 % was taken into consideration.
For facilitating the test item dissolution, in the main test, for the preparation of test item stock solution 2-3 min. ultrasonic treatment was applied.
The test solutions were freshly prepared at the beginning of the experiment.
Vehicle / solvent:
- DMSO for test item
- DMSO or ultrapure water for positive controls
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylenediamine
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
Genotypes of the strains used for mutagenicity testing
In addition to mutations in genes involved in histidine or tryptophan synthesis, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. 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 (WP2uvrA) is also defective in DNA excision repair.

Storage of tester strains
The strains are stored at -80 ± 10 °C. in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO (8 % (v/v)) is added as a cryoprotective agent.

Confirmation of phenotypes of tester strains
The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al..

Spontaneous reversion of tester strains
Each tester strain reverts spontaneously at a frequency that strain specific. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs are measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate. The historical control values for spontaneous revertants are available.

Procedure for bacterial cultures
The frozen bacterial cultures were thawed at room temperature and 200 µl inoculum was used to inoculate each 50 ml of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 10-12 hours in a 37 °C Benchtop Incubator Shaker.

Viability and the cell count of the testing bacterial cultures
Fresh cultures of bacteria were grown up to the late exponential or early stationary phase of growth (approximately 10^9 cells per ml). Cultures in late stationary phase were not used. The titer was demonstrated in each assay through the determination of viable cell numbers by a plating experiment.
The viability of each testing culture was determined by plating 0.1 ml of the 10^-5, 10^-6, 10^-7 and 10^-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by manual colony counting.

Media
An appropriate minimal agar and an overlay agar containing histidine and biotin or tryptophan, to allow for a few cell divisions were used.

The Minimal Glucose Agar (MGA) plates
Ready-to-use minimal glucose agar (MGA) plates were used in the study.
Typical composition (g/1000 ml) of MGA plates:
Glucose 20.0 g
Magnesium sulfate 0.2 g
Citric acid 2.0 g
di-Potassium hydrogenphosphate 10.0 g
Sodium ammonium hydrogenphosphate 3.5 g
Agar agar 13.0 g

Nutrient broth no. 2
Nutrient broth no. 2 25.0 g
Ultrapure water ad 1000.0 ml
Sterilization for 20 minutes was performed at 121˚C in an autoclave.

Nutrient agar
Nutrient agar 20.0 g
Ultrapure water ad 1000.0 ml
Sterilization for 20 minutes was performed at 121˚C in an autoclave.

Top agar for Salmonella typhimurium strains
Agar solution:
Agar bacteriological 4.0 g
NaCl 5.0 g
Ultrapure water ad 1000.0 ml
Sterilization for 20 minutes was performed at 121˚C in an autoclave.

Histidine – biotin solution (0.5 mM):
D-Biotin 122.2 mg
L-Histidine•HCl H2O 104.8 mg
Ultrapure water ad 1000.0 ml
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete top agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100.0 ml
Agar solution 900.0 ml

Top agar for Escherichia coli strain
Tryptophan solution (2 mg/ml):
L-Tryptophan 2000.0 mg
Ultrapure water ad 1000.0 ml
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete top agar for Escherichia coli strain:
Nutrient broth 50.0 ml
Tryptophan solution (2 mg/ml) 2.5 ml
Agar solution 947.5 ml

Description of test procedure
The study included a preliminary solubility test, a preliminary concentration range finding test (informatory toxicity test) and an initial mutation test (plate incorporation test).
In the preliminary concentration range finding test as well as in the initial mutation test the plate incorporation method was used. Because of the unequivocal, positive results of the initial mutation test further testing for the final conclusion was considered as not necessary.

Solubility test
In the solubility trials the test item behavior was investigated in the applied test system when formulated in dimethyl sulfoxide (DMSO). The test item was dissolved in dimethyl sulfoxide (DMSO). The obtained solutions with the solution of top agar and phosphate buffer were examined in a test tube without test bacterium suspension.

Concentration of test item in the solvent (mg/ml): 50
Solubility, visible behavior in the solvent: clear solution
Solubility in the top solution (final treatment mixture) (test item solution 100 µl + phosphate buffer 500 µl + top agar 2 ml): clear solution
Test item concentration in the test tube µg/tube: 5000

In the solubility test any correction factor, based on the purity of the test item (80 %) was not taken into consideration; therefore the 50 mg/ml solution concentrations corresponded to 40 mg active component/ml.

Concentration range finding tests (informatory toxicity tests)
Based on the solubility trials, the stock solution with a concentration of 50 mg/ml (40 mg/ml active ingredient content) was prepared in DMSO and diluted in 6 steps by factor of 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, TA100) were determined at the concentrations of 4000, 1280, 400, 128, 40, 13 and 4 µg active ingredient content/plate, (planned concentrations: 5000, 1600, 500, 160, 50, 16 and 5 µg test item/plate).
In the informatory toxicity test a correction factor, based on the purity of the test item (80 %) was not taken into consideration.
In the performed informatory toxicity test inhibitory effect of the test item was not observed, the colony and background lawn development was not affected in any case. All of the obtained slight revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.
Biologically relevant dose-related changed revertant colony number increases, high revertant colony numbers above the corresponding genotoxicological thresholds for being positive were obtained in both strains at 4000-400 µg/plate in the absence of exogenous metabolic activation ( S9); in S. typhimurium TA98 down to and including the concentration of 40 µg/plate in the presence of exogenous metabolic activation (+S9), in TA100 at 4000, 1280 and 128 µg/plate (+S9).
The high revertant colony numbers were above the corresponding historical control data range in S. typhimurium TA98 at 13 µg/plate (+S9), in TA100 at 400, 40 and 13 µg/plate (+S9).
No precipitation of the test item was observed on the plates in the above bacterial strains at any examined concentration level (±S9).

Procedure for the initial mutation test
A standard plate incorporation procedure was performed as an initial mutation test. Bacteria were exposed to the test item both in the presence and absence of rat liver S9 as metabolic activation system. Molten top agar was prepared and kept at 45 °C. Two ml of top agar was aliquoted into individual test tubes (3 tubes per both control and each concentration level). The equivalent number of minimal glucose agar plates was properly labelled. Conditions were investigated in triplicate. 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 solution of test item or positive controls 100 µl
overnight culture of test strain* (containing approximately 10^8 viable cells) 100 µl
phosphate buffer (pH: 7.4) or S9 mix 500 µl
* : Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA.
This solution was mixed and poured on the surface of the properly labeled minimal agar plates (3 plates per both control and each concentration level). For incubations with metabolic activation, 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 and each with the addition of negative and positive controls. The plates were incubated at 37 °C for about 48 hours.
Unequivocal, demonstrative positive results were obtained in the initial mutation test (plate incorporation test) in the Salmonella typhimurium TA98, TA100 and TA1537 strains (indicator strains for base-pair substitution and frameshift mutations), in the absence and also in the presence of exogenous metabolic activation (±S9); therefore, the performance of an additional confirmatory experiment was considered as not necessary for the final conclusion of the study.
Evaluation criteria:
The evaluation of revertant colony count, background lawn development and test item precipitate were performed manually by unaided eye.
The colony numbers on the untreated, solvent control, positive control and the test item treated plates were determined, the mean values, standard deviations and the mutation rates were calculated.
Mutation Rate (MR) = Mean number of revertants at the test item (or control*) treatments / mean number of revertants of solvent control
* : untreated, solvent or positive control

Evaluation and interpretation of results
A test item is 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 is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the solvent control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the solvent control.
Statistics:
According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a negative response: a test article is 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.

Conditions for the validity of the test
The tests are considered valid if:
- all of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- S. typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- E. coli WP2 uvrA culture demonstrate the deletion in the uvrA gene.
- bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the solvent controls.
- tester strain culture titers are in the 10^9 cells/ml order.
- batch of S9 used in this study shows the appropriate biological activity.
- positive control reference items (known mutagens) show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective solvent control, and the number of induced revertant colonies are in accordance with the corresponding historical control ranges.
- there are at least five analyzable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).

A dose level is considered toxic if
- reduced revertant colony numbers are observed as compared to the mean solvent control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range, and / or
- pinpoint colonies appear, and / or
- reduced background lawn development occurs.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA100, TA98, TA1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA 1535 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Validity of the performed experiments
Tester strains used in this study demonstrated the specific phenotype characteristics, were in line with the corresponding historical control data ranges and showed the adequate strain culture titer.
Each batch of the S9 fraction used in this test had the appropriate biological activity and was active in the applied system.
The positive control reference items (diagnostic mutagens) induced the expected, biological relevant increases (more than 3-fold increase) in revertant colonies and most of the number of revertant colonies was within the historical control data range per strain, thereby meeting the criteria for a valid positive control in all experimental phases and all tester strains.
The numbers of revertant colonies in the solvent control (dimethyl sulfoxide) showed characteristic mean numbers agreed with the actual historical control data ranges in all strains in all experimental phases.
Seven concentration levels were investigated in the informatory toxicity test and in the main mutation experiment (initial mutation test).
In the performed experimental phases there were at least five analyzable concentrations and a minimum of three non-toxic and non-precipitated dose levels at each tester strain.
All criteria for the validity of the performed experiments have thus been met.

Controls
In the performed initial mutation test multiple test items were tested with reference values from the common parallel controls.
The spontaneous revertant colony numbers of the dimethyl sulfoxide (DMSO) solvent control plates showed characteristic mean numbers in line with the actual historical control data ranges in all strains.
Each of the investigated reference mutagens (positive controls) showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective solvent control in the initial mutation test and additionally the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all tester strains.
The revertant colony numbers of the untreated and ultrapure water control plates in the initial mutation test were slightly higher or lower than the dimethyl sulfoxide (DMSO) control plates. The higher or lower revertant counts of these controls remained in the corresponding historical control data ranges.
In summary, the actual values of untreated, solvent and positive controls were in line with the criteria for validity of the assay.

Initial mutation test (plate incorporation test)
In the initial mutation test biologically relevant increases were observed in revertant colony numbers of Salmonella typhimurium TA98, TA100 and TA1537 strains (indicator strains for base-pair substitution and frameshift mutations), in the absence and also in the presence of exogenous metabolic activation (±S9).
The relevant revertant colony number increases, high revertant colony numbers above the corresponding genotoxicological thresholds for being positive were obtained in S. typhimurium TA98 in the concentration range of 5000-500 µg/plate, in the absence and also in presence of exogenous metabolic activation (±S9); in TA100 at the concentration range of 5000-160 µg/plate ( S9) and at 5000 and 1600 µg/plate (+S9); in TA1537 at the concentration range of 5000-160 µg/plate (±S9). In these strains the highest MR values were obtained at the highest examined concentration of 5000 µg/plate and the further revertant colony number increases followed clear dose-relationship (±S9).
Furthermore, the obtained revertant colony numbers were above the corresponding historical control data range, but remained below the genotoxicological threshold for being positive in TA98 at 160 µg/plate (±S9), at 50 µg/plate (+S9) and in TA100 at 160 µg/plate (+S9).
Additionally, the obtained increased revertant colony numbers showed clear dose related tendency, and at 5000 µg/plate (at the highest examined concentration level) were above the corresponding historical control data range, but remained below the genotoxicological threshold for being positive in Salmonella typhimurium TA1535, in the absence of exogenous metabolic activation ( S9).
In Escherichia coli WP2 uvrA strain equivocal, borderline positive results (revertant colony counts above or just above the genotoxicological threshold for being positive and above the corresponding historical control data range) were obtained at the concentrations of 160 and 50 µg/plate, with addition of S9 (+S9), and the further increased revertant colony numbers followed the clear dose relationship, were above the corresponding historical control data range; however remained below the threshold for being positive at 16 and 5 µg/plate (+S9). Supposedly a genotoxic, but in higher concentrations slightly cytotoxic metabolite of the test item is responsible for these latter changes.
In the performed mutation test, inhibitory effect of the test item on bacterial growth was not observed. All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding solvent control) remained in the range of the biological variability of the applied test system and the background lawn development was not affected in any case.
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9).

Applicant's summary and conclusion

Conclusions:
Test item induced gene mutations by base-pair substitution in the genome of the strains of Salmonella typhimurium TA100 and by frameshifs in Salmonella typhimurium TA98 and TA1537.
In conclusion, test item has mutagenic activity on Salmonella typhimurium TA98, TA100 and TA1537 carrying base-pair substitution and frameshift mutation in the absence and presence of exogenous metabolic activation, under test conditions used in this study.
Executive summary:

Test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

Bacterial strains, exogenous metabolic activation

Experiments were carried out using S. typhimurium TA98, TA100, TA1535 and TA1537, and E. coli WP2 uvrA in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats.

Experimental phases

The study included preliminary solubility trials, a preliminary concentration range finding test (informatory toxicity test applying the plate incorporation method), an initial mutation test (plate incorporation test).

The present test item contains azo-bonds. Therefore, the modified protocol proposed by Prival and Mitchell [10] (e.g.: using of hamster liver S9 Mix that contains 30 % hamster liver extract etc.) was originally planned for the confirmatory mutation test.

However, in this study unequivocal, demonstrative positive results were obtained in the initial mutation test (plate incorporation test); therefore, further investigation, was not necessary for the final conclusion of the study.

Solvent, test item concentrations, rationale for dose selection

Based on the results of the solubility test and the concentration range finding test, the test item was dissolved in dimethyl sulfoxide (DMSO) and the following concentrations were prepared and investigated in the initial mutation testin the absence and presence of exogenous metabolic activation: ±S9: 5000; 1600; 500; 160; 50; 16 and 5 µg/plate.

The selection of the concentration range was in accordance with the OECD 471 guideline recommendations, accordingly, the cytotoxicity (absent), the solubility and a possible positive effect of the test item were taken into consideration and the maximum test concentration was in all strains 5000 µg/plate (the recommended maximum test item concentration for soluble non-cytotoxic test items).

At the preparation of the test item suspensions, solutions a correction (multiplier) factor of 1.25 (1/0.8=1.25) based on its purity of 80 % was taken into consideration.

Solubility, precipitation

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9).

Cytotoxicity results

In the performed experiments inhibitory effect of the test item on bacterial growth was not observed.All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding solvent control) remained in the range of the biological variability of the applied test system and the background lawn development was not affected in any case.

Mutagenicity results

The revertant colony numbers of solvent control (dimethyl sulfoxide) plates with and without S9 Mix demonstratedthe characteristic mean number of spontaneous revertantsthat was in line with the corresponding historical control data ranges.The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase)in induced revertant coloniesand the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive controlin all experimental phases, in all tester strains.

In the performed initial mutation test biologically relevant, dose dependently changed revertant colony number increases, positive results were observed in revertant colony numbers of in case of S. typhimurium TA98, TA100 and TA1537 without and with addition of exogenous metabolic activation (±S9) and equivocal borderline positive results (revertant colony counts above or just above the genotoxicological threshold for being positive and above the corresponding historical control data ranges) were obtained in E. coli WP2 uvrA strain in the presence of exogenous metabolic activation (+S9).

The results of the main experiment confirmed well the concentration range finding test results in TA98 and TA100 strains in the absence and the presence of exogenous metabolic activation (±S9). The obtained slight differences (in mutation rates at different concentration levels at these two strains) remained within the biological variability range of the applied test system.

Additionally, in the case of S. typhimurium TA1537 unequivocal, demonstrative positive results were gained in the absence and presence of exogenous metabolic activation (±S9) and equivocal positive results were obtained in E. coli WP2 uvrA, in the presence of exogenous metabolic activation (+S9).

These obtained positive results were demonstrative and enough for the final conclusion of the study.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test iteminduced gene mutations by base-pair substitution and frameshift mutation in the genome of the strains of S. typhimurium TA98, TA100 and TA1537. Equivocal positive results were obtained in E. coli WP2 uvrA.

Conclusion

The reported data of this mutagenicity assay show that under the experimental conditions applied, test item induced gene mutations by base-pair substitution in the genome of the strains of S. typhimurium TA100 and by frameshifts in S. typhimurium TA98 and TA1537.

In conclusion, test item has mutagenic activity on S. typhimurium TA98, TA100 and TA1537 carrying base-pair substitution and frameshift mutation in the absence and presence of exogenous metabolic activation, under the test conditions used in this study.