4-[(2E)-3-(3,5-dichloro-4-fluorophenyl)-4,4,4-trifluorobut-2-enoyl]-N-[(4R)-2-ethyl-3-oxo-1,2-oxazolidin-4-yl]-2-methylbenzamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro studies

Negative, with and without metabolic activation (S9-mix): Bacterial reverse mutation assay (Ames test), OECD TG 471, S. typhimurium TA98, TA100, TA1535, TA1537, E. coli WP2 uvrA pKM101

Negative, with and without metabolic activation (S9-mix): In vitro micronucleus assay, OECD TG 487, human lymphocytes

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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 S9 Microsomal fraction (Sprague-Dawley) was purchased from Moltox and stored at approximately -196 °C in a liquid nitrogen freezer; Lot No. 4123 was used in this study and the protein level was adjusted to 20 mg/mL.

Test concentrations with justification for top dose:

Experiment 1 (plate incorporation test): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (highest concentration is the maximum recommended concentration level)
Experiment 2 (pre-incubation test): 15, 50, 150, 1500 and 5000 μg/plate

Vehicle / solvent:

Dimethyl sulphoxide (supplied by ThermoFisher Scientific, >99.7% purity)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

Without S9-mix: N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, 4-nitroquinoline-1-oxide
With S9-mix: 2-aminoanthracene, benzo[a]pyrene

Details on test system and experimental conditions:

Media: Top agar was prepared using 0.6% w/v Bacto agar (lot number 8255817 07/2023) and 0.5% w/v sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 51673 09/2019 and 51956 10/2019).
Precultures: A culture of each of the bacterial strains was prepared by inoculating nutrient broth with the appropriate coded stock culture and incubated, with shaking, for approximately 10 hours at 37 ± 3 °C. The bacterial cell count for each culture was determined by viable count analysis on nutrient agar plates on the day of test. The cell number after the pre-culture period in experiments 1 and 2 was 3.1E09 and 1.9E09 for TA100, 1.3E09 and 3.6E09 for TA1535, 2.4E09 and 3.3E09 for WP2uvrA pKM101, 2.2E09 and 1.4E09 for TA98 and 1.7E09 and 2.1E09 for TA1537, respectively.
Experiment 1: plate incorporation test
Eight concentrations of the test item were assayed in triplicate against each tester strain, using the direct plate incorporation method. For the study without metabolic activation, 0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlaid onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates. For the study with metabolic activation, the same procedure was used except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer. All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at 5000 μg/plate because of test item precipitation.
Experiment 2: pre-incubation method
Six test item concentration levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic concentration levels and the maximum recommended concentration following the change in test methodology from plate incorporation to pre-incubation. For the test without metabolic activation, 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 30 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate. The procedure for the study with metabolic activation was the same except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 30 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate. All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed because of test item precipitation from 150 and 500 μg/plate in the absence and presence of S9, respectively.

The sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix; Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).

Rationale for test conditions:

The maximum concentration of the test item in the first experiment was selected as the maximum recommended concentration of 5000 μg/plate.

Evaluation criteria:

If exposure to a test item produces a reproducible increase, in one or more concentration, in mean revertant colony numbers of at least twice that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship in at least one strain with or without metabolic activation system, it will be considered to exhibit mutagenic activity in this test system (Mortelmans and Zeiger 2000). No statistical analysis was performed.
If exposure to a test item does not produce an increase in mean revertant colony numbers, it will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis was performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used will usually be Dunnett’s test followed, if appropriate, by trend analysis (Mahon et al, 1989). Biological significance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below twice those of the concurrent vehicle controls (as described above) will not be considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director will use his/her scientific judgment.

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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

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

True negative controls validity:

not applicable

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

True negative controls validity:

not applicable

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

True negative controls validity:

not applicable

Positive controls validity:

valid

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There was no toxicity, evident as a reduction in the number of revertants (below an induction factor of 0.5) or a reduction in the background lawn in any of the five tester strains either with or without S9 mix following exposure to the test substance at any test concentration in both experiments. A test substance precipitate was noted from 500 and 150 μg/plate in both the presence and absence of S9-mix in experiments 1 and 2.
There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any concentration of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) or Experiment 2 (pre-incubation method). A minor increase was noted in Experiment 2 (TA1535 at 15 μg/plate in the absence of S9-mix), however this response was within the in-house historical vehicle/untreated control range for the relevant strain, did not exceed the threshold of twice the concurrent control and was, therefore considered of no biological relevance.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All counts were within the min-max range of historical control data. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, consistent with the laboratory’s positive historical control data, thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Conclusions:

Under the experimental conditions, the substance did not induce gene mutations by base pair changes or frameshifts in the genome of the Salmonella typhimurium and Escherichia coli strains used.

Executive summary:

The ability of the substance to induce reverse mutations in bacteria, either directly or after metabolic activation, in the plate incorporation test (Experiment 1) and the pre-incubation test (Experiment 2), using the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strain WP2uvrApKM101 was tested under GLP to OECD TG 471 (1997).
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range.
The maximum concentration of the test item in the first experiment was selected as the maximum recommended concentration of 5000 μg/plate.
In the first mutation test (plate incorporation method), there was no toxicity, evident as a reduction in the number of revertants (below an induction factor of 0.5) or a reduction in the background lawn, in any of the five tester strains either in the presence or absence of metabolic activation (S9 mix) following exposure to the substance. Consequently, the same maximum concentration of 5000 μg/plate was used as the maximum concentration in the second mutation test. Similarly, there was no toxicity in either the presence or absence of metabolic activation (S9-mix), at any test item concentration in the second mutation test (pre-incubation method).
A test item precipitate (particulate in appearance) was noted from 500 and 150 μg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2, respectively. This observation did not prevent the scoring of revertant colonies.
There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any concentration of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) or Experiment 2 (pre-incubation method).
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies which were consistent with the laboratory’s historical positive control data, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
Based on the results it was found that the substance did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterial tester strains used.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (18-35). The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. All blood donors in the experiment were female aged 24 to 35 years.

Cytokinesis block (if used):

Cytochalasin B was formulated in DMSO and added to all cultures, after washing, at the end of the exposure period at a final concentration of 4.5 µg/mL for a period of 24 hours.

Metabolic activation:

with and without

Metabolic activation system:

The Rat S9 Microsomal fraction was purchased from Moltox, Lot No. 4061 and 4127, Expiry 14 February 2021 and 25 July 2021, respectively. The S9 was adjusted to a final protein content of 20 mg/mL before use. The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Test concentrations with justification for top dose:

Preliminary test: 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL (top dose selected as highest recommended dose)
Main experiment (top dose selected due to cell toxicity)
4-hour exposure without S9: 0, 2, 4, 8, 16, 24, 32, 40 and 48 µg/mL
4-hour exposure with S9: 0, 2, 4, 8, 16, 32, 40, 48 and 64 µg/mL
24-hour exposure without S9: 0, 2, 4, 8, 16, 24, 32, 40 and 48 µg/mL
Main experiment repeat
4-hour exposure without S9: 0, 2, 4, 8, 16, 24, 32, 40 and 48 µg/mL
24-hour exposure without S9: 0, 2, 4, 8, 16, 24, 32, 40 and 48 µg/mL
Main experiment repeat II
4-hour exposure without S9: 0, 8, 16, 24, 28, 32 and 40 µg/mL
4-hour exposure with S9: 0, 16, 32, 40, 48, 56 and 64 µg/mL
24-hour exposure without S9: 0, 8, 16, 24, 28, 32 and 40 µg/mL
Main experiment repeat III
4-hour exposure with S9: 0, 16, 32, 40, 44, 48, 52 and 56 µg/mL

Vehicle / solvent:

Dimethyl sulphoxide (0.1%)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine

Remarks:

Without S9-mix: mitomycin C, demecolcine
With S9-mix: cyclophosphamide

Details on test system and experimental conditions:

Cell culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 °C with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05-9.10 mL MEM, 10% (FBS) 0.1 mL Li-heparin 0.1 mL phytohaemagglutinin 0.70-0.75 mL heparinized whole blood
4-hour exposure with S9-mix
After approximately 48 hours incubation at approximately 37 °C, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9¯mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment. After 4 hours at approximately 37 °C, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.
4-hour exposure without S9-mix
After approximately 48 hours incubation at approximately 37 °C, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL. After 4 hours at approximately 37 °C, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.
24-hour exposure without S9-mix
The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After 44 - 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 24 hours. The extended exposure detailed above does not follow the suggested cell treatment schedule in the Guideline. The OECD guideline permits modified treatment times where justified.. This design avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response (Whitwell et al., 2019). Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format. The Preliminary Toxicity Test was performed using the exposure conditions as described for the Main Experiment but using single cultures for the test item dose levels and duplicate cultures for the vehicle controls, whereas the Main Experiment used duplicate cultures and quadruplicate cultures for the vehicle controls.
Preliminary toxicity test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate concentrations were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the concentrations for the exposure groups of the main test.
Main experiment
Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Main experiment repeat
Due to a technical issue, both exposure groups in the absence of metabolic activation (S9) were dosed in the Main Experiment Repeat:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Main experiment repeat II
Three exposure groups were used for the Main Experiment Repeat II were dosed due to the Main repeat Experiment exposures not meeting the toxicity range as stated in the OECD 487 Guideline and a small but statistically significant increase in the 4-hour in the presence of S9:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Main experiment repeat III
One exposure group was used for the Main Experiment Repeat III because optimum toxicity was not achieved in the 4-hour exposure group dose in Main Experiment Repeat II:
i) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Cell harvest
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 °C prior to slide making.
Preparation of microscope slides
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry with gentle warming. Each slide was permanently labeled with the appropriate identification data.
Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Rationale for test conditions:

The study underwent multiple Main Experiment repeats due to technical issues so the test guideline could be met. Consequently, each exposure group was performed more than once and, where possible, all data reported.

Evaluation criteria:

Providing that all of the acceptance criteria have been met, the test item is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control.
- The increase in the frequency of micronucleated cells is dose-related when evaluated with an appropriate trend test.
- Any of the results are outside the distribution of the historical vehicle control data (above the upper 95% control limit).
- If all of these criteria are met, the test item will be considered able to induce chromosome breaks and/or gain or loss in the test system.
Providing that all of the acceptance criteria have been met, a clearly negative response will be claimed if, in all of the experimental conditions examined:
- None of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control.
- There is no concentration-related increase when evaluated with an appropriate trend test.
- All results are inside the distribution of the historical vehicle control data (within the 95% control limits).
- If all of these criteria are met, the test item will be considered unable to induce chromosome breaks and/or gain or loss in the test system.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. The Study Director may make a judgement based on experience and the biological relevance of the data and any justification for acceptance of the data will be included in the report. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic a

Statistics:

When there is no indication of any increase at all concentrations tested then statistical analysis may not be necessary. In all other circumstances comparisons were made between the appropriate vehicle control and each individual concentration, using Chi-squared Test on observed numbers of cells with micronuclei. A statistically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 when compared to its concurrent control. The concentration-relationship was assessed using a linear regression model. An arcsin square root transformation was applied to the percentage of binucleated cells containing micronuclei (excluding positive control(s)). A linear regression model was applied to these transformed values with concentration values fitted as the explanatory variable. The F-value from the model was assessed at the 5% statistical significance level.

Key result

Species / strain:

lymphocytes: cells derived from human blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was seen in all exposure groups, and maximum dose levels in the main experiment were selected based on toxcity.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Main experiment: Due to a technical issue, there were no binucleate cells present on the slides for the 4-hour exposure group in the absence of S9. Although the 24-hour exposure group did have binucleate cells present, there were many more multinucleate cells than expected and the exposure was not considered suitable for micronucleus analysis. Therefore, no binucleate cells were analysed for micronuclei in the exposure groups in the absence of S9 and these conditions were repeated.
Main experiment repeat: As both exposure groups did not meet the optimum toxicity requirements of the OECD 487 Test Guideline the experiment was terminated and the exposures repeated using modified concentration ranges.
Main experiment repeat II: In the presence of S9-mix, 29%, 65% and 83% cytostasis was achieved at 40, 48 and 56 μg/mL, respectively. Therefore, optimum toxicity was not achieved and a further experiment was necessary.

Conclusions:

The substance did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes under the conditions of the experiment and thus was found to be non-clastogenic and non-aneugenic.

Executive summary:

The clastogenic and aneugenic potential of the substance on the nuclei of normal cultured human lymphocytes was studied in vitro under GLP to OECD TG 487. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells with at least three concentrations, together with vehicle and positive controls. Three exposure conditions were used for the study; a 4 hour exposure in the presence and absence of a standard metabolizing system (S9-mix) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B. The study underwent multiple Main Experiment repeats due to technical issues so the test guideline could be met. Consequently, each exposure group was performed more than once. All vehicle (dimethyl sulphoxide) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes in all experiments conducted. The positive control items induced statistically significant increases in the frequency of cells with micronuclei compatible with the historical positive control data. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated for all experiments conducted.
In the 4 hour exposure in the presence of S9-mix, the substance was tested to a precipitating concentration resulting in 29% cytostasis. At the highest concentration tested there was a statistically significant increase in the percentage of micronucleated binucleated cells. However, all micronucleus frequencies remained within the 95% control limits (CL) of the historical vehicle control data and there was no concentration related response as judged by an appropriate trend test. As the exposure did not meet the criteria for a clear negative response this exposure was repeated. In the repeat experiment, the substance was tested up to 48 µg/mL resulting in 55% cytostasis. All micronucleus frequencies remained within the 95% CL of the historical vehicle control data and there were no statistically significant increases or concentration related increase in micronucleated binucleate cells. The criteria for a clear negative experiment were therefore met in this experiment. Overall, exposure of the substance for 4 hours in the presence of S9-mix was concluded to be negative. In the 4 hour exposure in the absence of S9-mix the substance was tested up to a precipitating concentration of 32 µg/mL which induced 49% cytostasis. There was a statistically significant increase in the percentage of micronucleated binucleate cells. However, the micronucleus frequencies remained within the 95% CL of the historical vehicle control data at all concentrations and there was no concentration related relationship, as judged by an appropriate trend test. Overall, this treatment was concluded to be negative. In the 24 hour exposure in the absence of S9-mix, the substance was tested up to a precipitating concentration of 32 µg/mL which induced 48% cytostasis. All micronucleus frequencies remained within the 95% CL of the historical vehicle control data and there were no statistically significant increases and no concentration related increase in micronucleated binucleate cells. The criteria for a clear negative experiment were therefore met in this experiment. Overall, treatment with the substance in the absence of S9 was concluded to be negative.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No data available

Additional information

The substance was found to be non-mutagenic in bacterial cells of commonly used S. typhimurium and E. coli strains in a valid and reliable Ames test conducted under GLP to OECD TG 471. Furthermore, the substance was non-clastogenic and non-aneugenic to the nuclei of normal human lymphocytes in a valid and reliable in vitro micronucleus study performed under GLP to OECD TG 487.

Justification for classification or non-classification

The substance does not meet the criteria for classification for mutagenicity under Regulation (EC) No 1272/2008.