Methyl 4-[2-[4-(5-methyl-2-benzoxazolyl)phenyl]vinyl]benzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November from 15th to 24th, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July, 1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fluorescent Brightener 371

IUPAC Name:

Fluorescent Brightener 371

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Trinova Biochem GmbH
- Storage: the strains were stored at -80 ± 10 ºC in the testing facility in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.

BACTERIA 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 10-13 hours in a 37 °C Benchtop Incubator Shaker

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 were checked regularly according to Ames et al.

SPONTANEOUS REVERSION OF TESTER STRAINS
Each tester strain reverts spontaneously at a frequency that is characteristic for the strain. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs were measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.

Metabolic activation:

with and without

Metabolic activation system:

Rat Liver S9 Fraction

Test concentrations with justification for top dose:

MAIN EXPERIMENTS: 5, 16, 50, 160, 500, 1600 and 5000 µg/plate
CONCENTRATION RANGE FINDING 1: 4900, 1568, 490, 157, 49, 16 and 5 µg/plate
CONCENTRATION RANGE FINDING 2: 4900, 2450, 1960, 1568, 980 and 490 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent/vehicle: in the preliminary Concentration Range Finding Tests dimethyl sulfoxide (DMSO) was found as appropriate vehicle for preparing the test item solutions. The vehicle is compatible with the survival of the bacteria and the S9 activity.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine // 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION
EXPERIMENT 1 - in agar (plate incorporation)
- Culturing: bacteria were cultured in Nutrient Broth No.2
- Medium: molten top agar was prepared and kept at 45 °C. 2ml of top agar was aliquoted into individual test tubes.
- 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.
- Incubation: the plates were incubated at 37 °C for about 48 hours.
- Replicates: 3 tubes per both control and each concentration level.

EXPERIMENT 2 - pre-incubation test
- Medium: before the overlaying of the test item, the bacterial culture (0.1 ml, containing approximately 10^8 viable cells) and the S9 mix or phosphate buffer (0.5 ml) was added into appropriate tubes to allow direct contact between bacteria and the test item (in its solvent).
- Preincubation: the tubes were gently mixed and incubated for 20 min at 37 ºC in a shaking incubator.
- Content of the tubes: after the incubation period, 2 ml of molten top agar was added to the tubes and the content was mixed and poured onto minimal glucose agar plates as described for the standard plate incorporation method.
- Aeration: tubes were aerated during pre-incubation by using a shaker.
- Incubation: after preparation the plates were incubated at 37 °C for about 48 hours.
- Replicates: triplicate plating was used at each dose level.

PRELIMINARY CONCENTRATION RANGE FINDING
- Method of application: plate incorporation.
- Number of experiments: 2: first informatory toxicity test conducte with and without S9; equivocal positive findings obtained in the first informatory toxicity test (at a precipitated concentration) were further investigated in the second informatory toxicity test conducted in the absence of exogenous metabolic activation (S9 Mix).
- Strains: TA 98 and TA 100 in the first informatory toxicity test; TA 100 in the second informatory toxicity test.
- Concentrations: 4900, 1568, 490, 157, 49, 16 and 5 µg/plate, both with and withoud S9 mix, first informatory toxicity test; 4900, 2450, 1960, 1568, 980 and 490 µg/plate, withoud S9 mix, second informatory toxicity test.

VIABILITY AND CELL COUNT
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 titre 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.

METABOLIC ACTIVATION
Rat Liver S9 Fraction
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH.

VALIDITY OF THE TEST
The tests (initial and confirmatory mutation 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
- the Salmonella typhimurium TA 98 and TA 100 tester strains demonstrate the presence of the pKM101 plasmid R-factor
- the Escherichia coli WP2 uvrA culture demonstrate the deletion in the uvrA gene
- the bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the solvent controls
- the tester strain culture titers are in the 10^9 cells/ml order
- the batch of S9 used in this study shows the appropriate biological activity
- the 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.

Evaluation criteria:

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 TA 100 the number of reversions is at least twice as high as the reversion rate of the solvent control,
- in strain Salmonella typhimurium TA 98, TA 1535, TA 1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the solvent control.

Criteria for a negative response: a test item 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.

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.
In the performed experiments, sporadically increased revertant colony numbers were observed. These increases did not show a clear dose-response relationship, were mostly of minor intensity and all of the increases remained below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest revertant colony number increase was observed in the confirmatory mutation test (pre-incubation test) in S. typhimurium TA1537 at the concentration of 1600 µg/plate, in the absence of an exogenous metabolic activation (S9 mix). The increased revertant colony numbers at this treatment remained within the corresponding solvent historical control data range and the appearance of this value was considered to be unique without concentration related increases. The mutation rate was 2.05, which was far below the genotoxicological threshold for being positive.
In the initial and confirmatory mutation tests unequivocal 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.
When evaluated by naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains in the concentration rage of 5000-500 µg/plate in the absence and presence (±S9 mix) of exogenous metabolic activation following the plate incorporation procedure, and at 5000 and 1600 µg/plate in the absence and at 5000 µg/plate in the presence of exogenous metabolic activation following the pre-incubation procedure.

PRELIMINARY CONCENTRATION RANGE FINDING
In the first informatory toxicity test the revertant colony numbers of solvent control plates with and without S9 mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected biological relevant increases in induced revertant colonies in both tester strains.
In the first informatory toxicity test cytotoxic effect of the test item on the bacterial strains was not observed. The colony and background lawn development was not affected in any case. All of the observed slight revertant colony number decreases (compared to the revertant colony numbers of the solvent control) remained within the biological variability range of the applied test system.
In this experimental phase significant, biologically relevant revertant colony number increases, revertant colony numbers above the solvent control data and above the historical control data ranges were obtained in S. typhimurium TA100 at the concentrations of 4900 and 1568 µg/plate in the absence of exogenous metabolic activation (S9 mix).
The increased revertant colony numbers were above the threshold for being positive in S. typhimurium TA100 at the concentration of 4900 µg/plate (S9 mix).
The further obtained revertant colony numbers were sporadically higher than the revertant colony numbers of the solvent control; however all of the observed higher colony numbers remained within the biological variability range of the applied test system and far below the biologically relevant threshold for being positive.
In the first informatory toxicity test non-interfering, not disturbing precipitate was observed by the unaided eye on the plates at the concentrations of 4900, 1568 and 490 µg/plate in the absence and presence of an exogenous metabolic activation (±S9 mix).
The equivocal positive findings obtained in the first informatory toxicity test (at a precipitated concentration) were further investigated in the second informatory toxicity test.
In the second informatory toxicity test the revertant colony numbers of solvent control plates were in line with the corresponding historical control data ranges. The positive control treatment showed the expected biological relevant increase in induced revertant colonies.
Similarly to the first informatory toxicity test cytotoxic effect of the test item was not observed and the appearance of precipitate was the same as described in the former experiment.
In the second informatory toxicity test all of the obtained higher revertant colony numbers remained within the biological variability range of the applied test system and far below the biologically relevant threshold for being positive.
The positive findings, significant increases of the first preliminary experiment were obtained at precipitated plates, were not confirmed in the subsequent preliminary experiment.

VALIDITY OF THE PERFROMED EXPERIMENTS
The tester strains used in the 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 had the appropriate biological activity and was active in the applied system.
Each of the investigated reference mutagens showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective solvent control in all main experimental phases and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in the main experimental phases, in the tester strains.
The spontaneous revertant colony numbers of the dimethyl sulfoxide (DMSO) solvent control plates showed characteristic mean numbers agreed with the actual historical control data ranges in all strains in both main experimental phases.
In the performed main 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 therefore been met.

CONTROLS
In the initial and confirmatory mutation tests the revertant colony numbers of the dimethyl sulfoxide (DMSO) solvent control plates with and without S9 mix were in line with the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.
The revertant colony numbers of the untreated and ultrapure water control plates in different experimental phases 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.

Applicant's summary and conclusion

Conclusions:

The test item has no mutagenic activity on the applied bacterium tester strains under the tested conditions.

Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay, in accordance with the testing method and procedures outlined into the OECD guideline 471.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (strains TA 98, TA 100, TA 1535 and TA 1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (strain WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats.

The study included a preliminary solubility tests, a preliminary concentration range finding tests (informatory toxicity tests), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test).

Based on the results of the solubility test and the concentration range finding tests the test item was dissolved in dimethyl sulfoxide (DMSO). At the formulation of test item solutions a correction of the concentrations for the purity (98 %) of test item was made in the experiments.

Based on the cytotoxicity and solubility results of the preliminary concentration range finding tests (informatory toxicity tests) and based on the recommendations in OECD 471 guideline [6], the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 5000, 1600, 500, 160, 50 and 16 µg/plate.

The results of the preliminary concentration range finding tests allowed the applying of the recommended maximum test concentration of 5000 µg/plate.

When evaluated by naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains in the concentration rage of 5000-500 µg/plate in the absence and presence (±S9 mix) of exogenous metabolic activation following the plate incorporation procedure, and at 5000 and 1600 µg/plate in the absence and at 5000 µg/plate in the presence of exogenous metabolic activation following the pre-incubation procedure.

The OECD 471 permits the use of just one or two dose levels showing precipitate for non-cytotoxic test items. In the present case, because of the low water solubility of the test item and appearance of non-disturbing precipitate, it was decided to test up to the maximum recommended dose level of 5000 µg/plate in order to confirm the absence of its mutagenic potential.

The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the solvent control) remained within the biological variability range of the applied test system.

The revertant colony numbers of solvent control dimethyl sulfoxide (DMSO) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that 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 colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

The reported data 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.

Conclusion

In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the tested conditions.