Boron, (benzenemethanamine)trifluoro-, (T-4)-, reaction products with Bu glycidyl ether

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames test (OECD 471) Boron, (benzenemethanamine)trifluoro-,(t-4)-, reaction products with bu glycidyl ether was tested using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system. The dose range was 1.5 to 5000 μg/plate. Boron, (benzenemethanamine)trifluoro-,(t-4)-, reaction products with bu glycidyl ether was considered to be non-mutagenic under the conditions of this test (Envigo 2017).

Test samples of the test item were assayed in an in vitro micronucleus test (OECD 487)using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals. Under the present test conditions, the test item tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times without S9 mix and one exposure time with S9 mix revealed no indications of chromosomal damage in the in vitro micronucleus test (LPT 2020).

The test item Boron, (benzenemethanamine)trifluoro-, (T-4)-, reaction products with Bu glycidyl ether was tested in an OECD 476 test for its mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals. The duration of the exposure with the test item was 4 hours in the experiments without and with S9 mix.

The test item was completely dissolved in dimethyl sulfoxide (DMSO). The vehicle DMSO was employed as the negative control.

Under the present test conditions, the test item, tested up to the concentration of 600 µg /mL medium, in the absence of metabolic activation, is considered to be clearly positive. I shows a concentration-related increase and a statistically significant increase compared to the concurrent negative control as well as a value, which is outside the distribution of the historical negative control data in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects, was observed. In the presence of metabolic activation no mutagenic effects were observed up to the top concentration of 800 µg test item/mL medium (LPT 2020).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2019-06-03 until 2019-08-23

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:

adopted July 29, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: -EC method B.49:

Version / remarks:

-EC method B.49: In vitro Mammalian Cell Micronucleus Test, Commission Regulation (EU) No 2017/735 adopted February 14, 2017, published in the Official Journal of the European Union L 112/1, dated April 28, 2017.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

The test item was completely dissolved in dimethylsulfoxide (DMSO)

Species / strain / cell type:

lymphocytes:

Remarks:

CULTURED HUMAN PERIPHERAL LYMPHOCYTES

Details on mammalian cell type (if applicable):

Human peripheral blood was obtained by venipuncture from young, healthy, non-smoking individuals with no known recent exposures to genotoxic chemicals or radiation, and collected in heparinised vessels. Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin . The tubes were sealed and incubated at 37°C, and shaken occasionally to prevent clumping

Cytokinesis block (if used):

The appropriate concentration of CytoB was determined for human lymphocytes to achieve the optimal frequency of binucleated cells in the vehicle control cultures. The appropriate concentration of CytoB is usually between 3 and 6 µg/mL. The concentration used for this assay was 5 µg/mL.

Metabolic activation:

with and without

Metabolic activation system:

Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254 (Monsanto KL615, 500 mg/kg i.p.) and prepared according to MARON and AMES was obtained from Trinova Biochem . S9 was collected from male rats.

Test concentrations with justification for top dose:

0; 50; 100; 200; 400; 600 µg/mL medium in the experiments with 4 hours with and without metabolic activation
0; 12.5; 25; 50; 100; 200 µg/mL medium in the experiment with 24 hours without metabolic activation

At least three analysable test concentrations were evaluated. In order to achieve this, a preliminary cytotoxicity test was performed to narrow the range of concentrations used for the definitive test. In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg Boron,…/mL medium were employed. Complete cytotoxicity was noted starting at a concentration of 316 µg/mL medium in the experiment without metabolic activation and at concentrations of 1000 or 2000 µg/mL medium in the presence of metabolic activation (24- or 4-hour exposure, respectively). Test item precipitation was noted at concentrations of 1000 and 2000 µg/mL medium in the absence and presence of metabolic activation (24-hour or 4-hour exposure)

Vehicle / solvent:

The test item was completely dissolved in dimethyl sulfoxide (DMSO) . The vehicle DMSO was employed as the negative control. Fresh preparations of the test item were made on the day of the experiment and used for the treatment in all experimental parts.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

SYSTEM OF TESTING
- Species/cell type: Human peripheral blood was obtained by venipuncture from young (approximately 18 – 35 years of age), healthy, non-smoking male or female individuals with no known recent exposures to genotoxic chemicals or radiation, and collected in heparinised vessels.
- Metabolic activation system: male rat liver S9 from Aroclor 1254 induced animals
ADMINISTRATION:
- Solubility: The test item was completely dissolved in dimethylsulfoxide (DMSO). The vehicle DMSO served as the negative control. Fresh preparations of the test item were prepared on the day of the experiment and used for the treatment in all experimental parts.
- Preliminary experiment: without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg test item/mL medium were employed. Complete cytotoxicity was noted starting at a concentration of 316 µg/mL medium in the experiment without metabolic activation and at concentrations of 1000 or 2000 µg/mL medium in the presence of metabolic activation (24- or 4-hour exposure, respectively). Test item precipitation was noted at concentrations of 1000 and 2000 µg/mL medium in the absence and presence of metabolic activation (24-hour or 4-hour exposure). No relevant changes in pH or osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 2000 µg/mL medium.
Hence, 600 µg/mL medium were employed as the top concentration for the genotoxicity tests without and with metabolic activation with a 4-hour exposure and 200 µg/mL medium for the 24-hour exposure experiment without S9 mix

- Dosing:
Without metabolic activation:
4-h exposure 50, 100, 200, 400 and 600 µg/mL medium;
24-h exposure: 12.5, 25, 50, 100 and 200 µg/mL medium;
With metabolic activation:
4-h exposure: 50, 100, 200, 400 and 600 µg/mL medium.
- Positive and negative control groups and treatment:
negative/solvent: DMSO
positive, clastogen (+S9): cyclophosphamide in highly purified water, c = 10 µg/mL and c = 20 µg/mL
positive, clastogen (-S9): mitomycin C in highly purified water, c = 0.1 µg/mL and c = 0.2 µg/mL
positive, aneugen (-S9): colchicine in highly purified water, c = 0.01 µg/mL and c = 0.020 µg/mL

TREATMENT SCHEDULE:
- 0.5 mL of freshly prepared blood lymphocytes were seeded with 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin. The tubes are sealed and incubated at 37°C, and shaken occasionally to prevent clumping.
- After initiation appropriate concentration of the test item in the vehicle were added to the cell cultures for each target concentration of the test item in the test medium and each experiment.
- Precipitation of the test item was checked before and after each experiment. Evaluation of precipitation was done by light microscopy at the beginning and end of treatment.
- Theoretical considerations, together with published data, indicate that most aneugens and clastogens are detected by a short term treatment period of 4 hours in the presence and absence of S9, followed by removal of the test item and a growth period of 1.5 cell cycles.
- Cells were sampled at a time equivalent to about 1.5 times the normal (i.e. untreated) cell cycle length either after the beginning or at the end of treatment.
- Sampling or recovery times would have been extended if it is known or suspected that the test item affects the cell cycling time (e.g. when testing nucleoside analogues). Because of the potential cytotoxicity of S9 preparations for cultured mammalian cells, an extended exposure treatment was used only in the absence of S9.
- All treatments were conducted while the cells were growing exponentially.

DURATION:
Cell treatment and harvest times for the used human lymphocytes line:
Without S9 mix: 4-hour exposure
- 0.5 mL of freshly prepared blood lymphocytes were seeded with 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin. The tubes are sealed and incubated at 37°C, and shaken occasionally to prevent clumping.
- After 48 hours the cultures were centrifuged (10 minutes at 800 – 900 rpm) and the medium was replaced by 4.95 mL of fresh Ham’s F10 medium with fetal calf serum (FCS).
- Five concentrations of 50, 100, 200, 400 and 600 µg test item/mL were employed. The test item treatments and the controls were added at a volume of 50 µL to obtain the corresponding target concentrations.
- The cultures were then incubated for 4 hours at +37°C.
- Afterwards the medium was removed and the cultures were washed twice with Ham’s F10 medium.
- After addition of 5 mL Chromosome medium containing 5 µg/mL Cytochalasin B the cultures were incubated for further 20 hours at 37°C.

Without S9 mix: 24-hour exposure
- 0.5 mL of freshly prepared blood lymphocytes were seeded with 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin. The tubes are sealed and incubated at 37°C, and shaken occasionally to prevent clumping.
- After 48 hours the cultures were centrifuged (10 minutes at 800 – 900 rpm) and the medium was replaced by 4.95 mL of fresh Ham’s F10 medium with fetal calf serum (FCS).
- Five concentrations of 12.5, 25, 50, 100 and 200 µg test item/mL were employed. The test item treatments and the controls were added at a volume of 50 µL to obtain the corresponding target concentrations.
- The cultures were then incubated for 24 hours at +37°C.
- Afterwards the medium was removed and the cultures were washed twice with Ham’s F10 medium.
- After addition of 5 mL Chromosome medium containing 5 µg/mL Cytochalasin B the cultures were incubated for further 20 hours at 37°C.

With S9 mix: 4-hour exposure
- 0.5 mL of freshly prepared blood lymphocytes were seeded with 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin. The tubes are sealed and incubated at 37°C, and shaken occasionally to prevent clumping.
- After 48 hours the cultures were centrifuged (10 minutes at 800 – 900 rpm) and the medium was carefully removed and replaced by 4.45 mL Ham’s F10 medium with FCS and 0.5 mL S9 Mix.
- Five concentrations of 50, 100, 200, 400 and 600 µg test item/mL were employed. The test item treatments and the controls were added at a volume of 50 µL to obtain the corresponding target concentrations.
- The cultures were then incubated for 4 hours at +37°C. Afterwards the medium was removed and the cultures were washed twice with Ham’s F10 medium.
- After addition of 5 mL Chromosome medium containing 5 µg/mL Cytochalasin B the cultures were incubated for further 20 hours at 37°C.


STAIN (for cytogenetic assays):
- Each culture was harvested and processed separately.
- After the test item incubation, mitotic activity was arrested by the addition of CytoB to each culture at a final concentration of 5 µg/mL.
- After an additional incubation of 20 hours the cultures were centrifuged for 10 minutes at 800 rpm, the supernatant was discarded and the cells resuspended in KCl (0.56%).
- After incubation for 17 minutes at 37°C, the cell suspensions were centrifuged for 10 minutes at 800 rpm. The supernatant was discarded and 5 mL of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid v/v) added.
- The cells were left in fixative for 30 minutes followed by centrifugation at 800 rpm.
- The supernatant was carefully removed and discarded, and the cell pellet was resuspended in about 0.5 mL of fresh fixative and 30% glacial acetic acid by repeated aspiration through a Pasteur pipette.
- Two drops of this cell suspension were dropped onto a prewarmed, pre-cleaned microscope slide and left to air-dry at room temperature.
- The slides were then stained using 10% Giemsa and left to air-dry at room temperature.


NUMBER OF REPLICATIONS: Two replicate cultures were used for each test item concentration and for the vehicle and positive control cultures

NUMBER OF CELLS EVALUATED: The micronucleus frequencies were analysed in at least 2000 binucleated cells per concentration (at least 1000
binucleated cells per culture; two cultures per concentration).

DETERMINATION OF CYTOTOXICITY
- Method: evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).
The CBPI indicates the average number of cell cycles per cell during the period of exposure to cytoB, and is used to calculate cell proliferation.
The RI indicates the relative number of nuclei in treated cultures compared to control cultures and can be used to calculate the % cytostasis:
At least 500 cells per replicate cell culture (two cultures per concentration in the main study, one culture per concentration in the preliminary test) were scored and classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of toxicity.
Thus, an RI of 53% means that, compared to the numbers of cells that have divided to form binucleate and multinucleate cells in the control culture, only 53% of this number divided in the treated culture, i.e. 47% cytostasis

OTHER EXAMINATIONS:
- The micronucleus frequencies were analysed in at least 2000 binucleated cells per concentration (at least 1000 binucleated cells per culture; two cultures per concentration). If substantially fewer than 1000 binucleate cells per culture are available for scoring at each concentration, and if a significant increase in micronuclei is not detected, the test would be repeated using more cells, or at less toxic concentrations, whichever is appropriate. Care was taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size; neither would binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei were not analysed for micronuclei, as the baseline micronucleus frequency might be higher in these cells. Scoring of mononucleate cells is acceptable if the test item is shown to interfere with CytoB activity.

- pH values and osmolality measurements
The pH and osmolality of the negative control and all test item formulations in the medium of the preliminary experiment were determined employing the methods given below:
pH values: using a digital pH meter type SevenCompact s’210 .
Osmolality: with a Semi-micro osmometer Typ ML A0299.

Rationale for test conditions:

For relatively non-cytotoxic items the maximum concentration should be 2 mg/mL, 2 µL/mL or 0.01 M, whichever is the lowest, when not limited by solubility in the solvent or culture medium, or cytotoxicity.
Where there is cytotoxicity (tested in the pre-test), these concentrations should cover a range from the maximum to little or no toxicity; this will usually mean that the concentration levels should be separated by no more than a factor between 2 and √10. If the maximum concentration is based on cytotoxicity then it should result in approximately 10 - 20% (but not less than 10%) relative survival (relative cloning efficiency) or relative total growth. Relatively insoluble items will be tested up to or beyond their limit of solubility under culture conditions.
The concentrations employed in the main experiment were chosen based on the results of a preliminary cytotoxicity test with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL. In this preliminary test pronounced cytotoxicity in form of decreased plating efficiency was noted at concentrations of 100 µg/mL and higher in the experiment without metabolic activation and at 1000 and 2000 µg/mL in the experiment with metabolic activation. Test item precipitation was noted at concentrations of 1000 and 2000 µg/mL medium in both experiments.
Test item was completely dissolved in dimethylsulfoxide (DMSO)

Evaluation criteria:

The assay demonstrates its ability to reliably and accurately detect substances of known aneugenic and clastogenic activity, with and without
metabolic activation.
Acceptance of a test is based on the following criteria:
• The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database (Poisson-based 95% control limits). Where concurrent negative control data fall outside the 95% control limits, they may be acceptable for inclusion in the historical control data as long these data are not extreme outliers.
• Concurrent positive controls induce responses that are compatible with those generated in the laboratory’s historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
• Adequate number of cells, cell proliferation criteria and concentrations are analysable and are consistent with those described in Exosure Concentrations

Vehicle control and untreated cultures give reproducibly low and consistent micronucleus frequencies. Data from vehicle and positive controls are used to establish historical control ranges. These values are used in deciding the adequacy of the concurrent vehicle controls or positive controls for an experiment

Statistics:

Only the frequencies of binucleate cells with micronuclei (independent of the number of micronuclei per cell) were used in the evaluation of micronucleus induction. Concurrent measures of cytotoxicity and/or cytostasis for all treated and vehicle control cultures were determined. Individual culture data were provided.
Providing that all acceptability criteria are fulfilled, a test chemical 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 compared with the concurrent negative control
• the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test
• any of the results are outside the distribution of the historical negative control data (Poisson-based 95% control limits)
When all of these criteria are met, the test chemical is then considered able to induce chromosome breaks and/or gain or loss in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test,
• all results are inside the distribution of the historical negative control data (Poisson-based 95% control limits).
The test chemical is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Key result

Species / strain:

lymphocytes: CULTURED HUMAN PERIPHERAL LYMPHOCYTES

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The results for the vehicle controls were within the historical control range.

Text table 9-1: pH values and osmolality

Concentrationof Boron,… [µg/mL medium]	pH value	osmolality [mOsmol/kg]
Medium	7.57	295
0, vehicle control	7.70	430
3.16	7.64	440
10.0	7.64	450
31.6	7.61	450
100	7.57	440
316	7.46	440
1000	7.20	440
2000	6.96	430

Conclusions:

Under the present test conditions, the test item tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times without S9 mix and one exposure time with S9 mix revealed no indications of chromosomal damage in the in vitro micronucleus test.
The results for the vehicle controls were within the historical control range.
In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

Executive summary:

Test samples of the test item were assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied.

The test item was completely dissolved in dimethyl sulfoxide (DMSO). The vehicle DMSO was employed as the negative control.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg test item/mL medium were employed. Complete cytotoxicity was noted starting at a concentration of 316 µg/mL medium in the experiment without metabolic activation and at concentrations of 1000 or 2000 µg/mL medium in the presence of metabolic activation (24- or 4-hour exposure, respectively). Test item precipitation was noted at concentrations of 1000 and 2000 µg/mL medium in the absence and presence of metabolic activation (24-hour or 4-hour exposure).No relevant changes in pH or osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 2000 µg/mL medium.

Hence, 600 µg test item /mL medium were employed as the top concentration for the genotoxicity tests without and with metabolic activation with a 4-hour exposure and 200 µg/mL medium for the 24-hour exposure experiment without S9 mix.

In the main study cytotoxicity was noted at the top concentration of 600 µg test item /mL medium in the 4-hour exposure experiments without and with metabolic activation. Thelong-term treatment(24-hour exposurewithout S9)caused cytotoxicity at the top concentration of 200 µg/mL medium.

Mitomycin C (at 0.2 µg/mL) and colchicine (at 0.02 µg/mL) were employed as positive controls in the absence and cyclophosphamide (at 20 µg/mL) in the presence of metabolic activation.

Tests without metabolic activation (4- and 24-hour exposure)

The mean micronucleus frequencies of cultures treated with the concentrations of 100, 200, 400 and 600 or 25, 50, 100 and 200 µgtest item/mL medium in the absence of metabolic activation (4- and 24-hour exposure, respectively) ranged from 2.0 to 4.0 micronucleate cells per 1000 binucleate cells. There was no dose-related increase in micronuclei up to the top concentrations of 600 or 200 µg/mL medium (4- and 24‑hour exposure, respectively). The frequency of micronucleate cells was within the historical control range of the untreated and vehicle controls.

Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test mean frequencies of 4.0 or 4.5 micronucleate cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively, were observed. The vehicle result was within the historical control ranges.

In the positive control cultures the mean micronucleus frequencies were increased to 13.5 or 19.5 micronucleate cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively.This demonstrated that Mitomycin C induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus.

Test with metabolic activation (4-hour exposure)

The mean micronucleus frequencies of cultures treated with the concentrations of100, 200, 400 and 600 µgtest item/mL medium(4-h exposure) in the presence of metabolic activation ranged from 2.5 to 4.5 micronucleate cells per 1000 binucleate cells.There was no dose-related increase in micronuclei upto the top concentration of 600 µg/mL medium. The frequency of micronucleate cells was within the historical control range of the untreated and vehicle controls.

Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test a mean frequency of 4.0 micronucleate cells per 1000 binucleate cells was observed. The vehicle result was within the historical control ranges.

In the positive control culture the mean micronucleus frequency was increased to 17.5 micronucleate cells per 1000 binucleate cells for the 4-hour exposurewith metabolic activation.This demonstratedthat cyclophosphamideinduced significant chromosomal damage.

Conclusion

Under the present test conditions, the test item tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times without S9 mix and one exposure time with S9 mix revealed no indications of chromosomal damage in the in vitro micronucleus test.

The results for the vehicle controls were within the historical control range.

In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2019-06-19 untill 2019-12-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

adopted 29 July 2016

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

Council Regulation (EC) No. 440/2008 of 30 May 2008;

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

The test item was completely dissolved in dimethylsulfoxide (DMSO)

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The cells were periodically checked for the absence of mycoplasma contamination. The spontaneous mutation rate was continuously monitored.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254 (Monsanto KL615, 500 mg/kg i.p.) and prepared according to MARON and AMES was obtained from Trinova Biochem . S9 was collected from male rats.

Test concentrations with justification for top dose:

Concentrations of 50, 100, 200, 400 and 600 and 100, 200, 400, 600 and 800 µg test item /mL medium were selected for experiments without and with metabolic activation, respectively.

In a preliminary test cytotoxicity in form of decreased relative survival compared to the control was noted starting at a concentration of 316 µg/mL and 1000 µg/mL test item /mL medium in the absence and presence of metabolic activation, respectively. Test item precipitation was noted macroscopically at the top concentration of 2000 µg /mL medium in both experiments. No relevant changes in pH or osmolality were noted in the test cultures compared to the negative control treated with DMSO. Hence, 600 or 800 µg test item / mL medium was employed as highest concentration for the genotoxicity tests without and with metabolic activation, respectively.

Vehicle / solvent:

The test item was completely dissolved in dimethyl sulfoxide (DMSO) . The vehicle DMSO was employed as the negative control. Fresh preparations of the test item were made on the day of the experiment and used for the treatment in all experimental parts.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Each experiment (in the absence and presence of S9 mix) was conducted in duplicates
- Both experiments (in the absence and presence of S9 mix) were performed in independent replicates.


METHOD OF TREATMENT/ EXPOSURE:
- Approximately 1500000 cells were placed in 15 mL growth medium per 75 cm2 culture flask
- On the following day, the growth medium was removed and the cells were resuspended in treatment medium (Dulbecco's modified Eagle-Medium) supplemented with 5% foetal calf serum and 1% penicillin/streptomycin solution


CELLS AND TISSUE CULTURE MEDIA
- V79 cells were maintained in Dulbecco's modified Eagle-Medium supplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin
(100 µg/mL) called DMEM-FCS
- Incubation of cultures: at 37°C in a humidified atmosphere (90%) containing 10% CO2
- For subculturing, a trypsin (0.05%)-EDTA (ethylenediaminetetraacetic acid, 0.02%) solution in modified Puck's salt solution A was used.


TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 1 day (in 15 mL DMEM-FCS), on day 1 of the experiment, approximately 1 500 000 cells were seeded in 15 mL DMEM-FCS per 60 mm diameter dish.
- Exposure duration:
* 4 hours, in the absence and presence of S9 mix, the cells were exposed to the test item in DMEM-FCS for 4 hours.

For experiments without metabolic activation 0.15 mL test item solution, negative or positive controls were added to 14.85 mL treatment medium.
For experiments with metabolic activation 3 mL of the S9 mix were added to 11.85 mL treatment medium before treatment.
Concurrent positive and negative (vehicle) controls, both with and without metabolic activation were included in each experiment


At the end of the exposure period and removal of the test item the cells were washed with PBS and the cells were trypsinised and then suspended in 9 mL growth medium. The cells were pelleted by centrifugation (250 x g for 5 minutes), the supernatant was removed, and the cells were resuspended in 3 mL growth medium.
*one part of the cells was used for the determination of the relative plating efficiency. Therefore, three replicate plates (60 mm diameter dishes) were used with 150 cells per plate in 5 mL growth medium.
*After about 8 days, the cells were fixed and stained with methylene blue in ethanol. The colonies were then counted for plating efficiency (PE1).

Another part of the cells was used for the determination of the mutant frequency. The cells were further incubated for 6 days including one cell passage in between. This period was required for expression of the new genotype, i.e. for sufficient dilution and catabolism of the previously expressed HPRT.
After the expression period the cells were harvested by trypsinisation and replated at a density of 500000 cells per 100 mm diameter dish in DMEM-FCS containing 6-thioguanine (10 µg/mL) for selection of mutants (4 replicate plates), and 150 cells per 60 mm diameter dish in medium without 6-thioguanine for the estimation of plating efficiencies (PE2), (3 replicate plates).
After about 8 days (PE2) or about 12 days (mutant plates), the cells were fixed and stained and the colonies were then counted.



DETERMINATION OF CYTOTOXICITY (same procedure was used as employed for the mutagenicity experiments, except that no mutant selection was carried out)
- Method: survival
- A concentration of the test item which produces a low level of survival (10 to 20%) would be used as highest concentration and the survival in the
lowest concentration being approximately the same as that in the negative control.
- Five adequately spaced concentrations are employed
- The concentrations employed in the main experiment were chosen based on the results of a preliminary cytotoxicity test with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL.
- In this preliminary test pronounced cytotoxicity in form of decreased plating efficiency was noted at concentrations of 316 µg/mL and higher in the experiment without metabolic activation and at 1000 µg/mL in the experiment with metabolic activation.
- Test item precipitation was noted at concentrations of 2000 µg/mL medium in both experiments.
- No changes in pH or osmolality were noted in the test cultures compared to the negative control treated with DMSO.
- Hence, the highest concentrations employed in the main study were 600 µg test item/mL medium in the absence and 800 µg/mL medium in the presence of metabolic activation.

DETERMINATION OF GENE MUTATION:

Another part of the cells was used for the determination of the mutant frequency. The cells were further incubated for 6 days including one cell passage in between. This period was required for expression of the new genotype, i.e. for sufficient dilution and catabolism of the previously expressed HPRT.
After the expression period the cells were harvested by trypsinisation and replated at a density of 500000 cells per 100 mm diameter dish in DMEM-FCS containing 6-thioguanine (10 µg/mL) for selection of mutants (4 replicate plates), and 150 cells per 60 mm diameter dish in medium without 6-thioguanine for the estimation of plating efficiencies (PE2), (3 replicate plates).
After about 8 days (PE2) or about 12 days (mutant plates), the cells were fixed and stained and the colonies were then counted.


POSITIVE CONTROL:
- ethyl methanesulphonate (EMS) in mutagenicity experiments in the absence of exogenous metabolic activation;
- 9,10-dimethyl-1,2-benzanthracene (DMBA) in the S9 mix supplemented assays. This compound is mutagenic in V79 cells in the presence, but not in the absence of S9 mix.
Both EMS and DMBA were dissolved in DMSO. The applied concentrations were 600 or 700 µg EMS/mL medium or 20 or 30 µg DMBA/mL

Rationale for test conditions:

For relatively non-cytotoxic items the maximum concentration should be 2 mg/mL, 2 µL/mL or 0.01 M, whichever is the lowest, when not limited by solubility in the solvent or culture medium, or cytotoxicity.
Where there is cytotoxicity (tested in the pre-test), these concentrations should cover a range from the maximum to little or no toxicity; this will usually mean that the concentration levels should be separated by no more than a factor between 2 and √10. If the maximum concentration is based on cytotoxicity then it should result in approximately 10 - 20% (but not less than 10%) relative survival (relative cloning efficiency) or relative total growth. Relatively insoluble items will be tested up to or beyond their limit of solubility under culture conditions.
The concentrations employed in the main experiment were chosen based on the results of a preliminary cytotoxicity test with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL. In this preliminary test pronounced cytotoxicity in form of decreased plating efficiency was noted at concentrations of 100 µg/mL and higher in the experiment without metabolic activation and at 1000 and 2000 µg/mL in the experiment with metabolic activation. Test item precipitation was noted at concentrations of 1000 and 2000 µg/mL medium in both experiments.
Test item was completely dissolved in dimethylsulfoxide (DMSO)

Evaluation criteria:

Individual plate counts for the test item and controls are presented for both mutation induction and survival. Mutation frequency is expressed as number of mutants per number of surviving cells. Plating efficiencies (PE1 and PE2) and relative survival (RS) are presented in the tables (Tables 2 - 5).
The methods of calculation of the analysed data are listed below:
Results of determination of survival following exposure to the test item, expressed as Relative Survival were calculated using the following equations:

Relative Survival (RS) [%] = (PE1 (treated culture) / PE1 (control culture)) *100

Based on the plating efficiency (PE1) = (Mean of the number of clones/culture dish / number of cells plated/culture dish


Results of determination of mutant frequency per 106 cells (MF/106) were calculated with the following equation:

Mutant Frequency (MF/106) = Cloning efficiency (CE) / PE2


Based on the cloning efficiency (CE) of mutant cells in selective media:

Cloning efficiency (CE) = Sum of the number of mutant clones / Sum of the number of cells plated


and based on the plating efficiency (PE2), the determination of survival following incubation for selection of mutants:

Plating Efficiency step 2 (PE2) = Mean of the number of clones/culture dish / number of cells plated/culture dish

Statistics:

No satisfactory mathematical methods are available for the statistical analysis of mammalian cell mutagenicity experiments.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity in form of decreased plating efficiency was noted at 400 and 600 μg/mL medium in the absence of metabolic activation and at 600 (second experiment) and 800 μg/mL medium (first and second experiment) in the presence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The concentrations to be employed in the main experiment were chosen based on the results of a preliminary cytotoxicity study without and with metabolic activation with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg test item /ml medium. In this preliminary test cytotoxicity in form of decreased relative survival compared to the control was noted starting at a concentration of 316 µg and 1000 µg test item /mL medium in the absence and presence of metabolic activation, respectively. Test item precipitation was noted macroscopically at the top concentration of 2000 µg test item /mL medium in both experiments. No relevant changes in pH or osmolality were noted in the test cultures compared to the negative control treated with DMSO. Hence, 600 or 800 µg test item / mL medium was employed as highest concentration for the genotoxicity tests without and with metabolic activation, respectively.

Remarks on result:

other:

Criteria for assay acceptance

Acceptance of a test is based on the following criteria:

-    The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database.

-    Concurrent positive controls induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.

-    Four tested concentrations are analysable.

 

As the total number of colonies is normally low and as a single mutation may cause several colonies due to cell division during the expression period, a relatively large variation of mutation frequency may result. The background data obtained at LPTare given at the end of this chapter (Text table 4-2). The spontaneous mutation frequency may vary from experiment to experiment, but should normally lie within the above-mentioned range.

The mutation frequencies of the solvent controls and the positive controlfor the last experiments of the years 2016 to 2018 (background data, not audited by the QAU-department) are given as follows:

Text table 4-2Historical background data ofmutation frequencies (n = 25)

Mutation frequency: mutant colonies per 10^6cells
	Without metabolic activation (4-hour exposure)	With metabolic activation (4-hour exposure)
Solvent control
mean	16.1	16.3
SD	9.5	12.0
range	1.6 - 44.3	2.3 - 40.7
Positive control (µg/mL)
	EMS	DMBA
mean	414.7	386.1
SD	164.7	136.7
range	159.4 - 703.4	155.7 - 711.8

SD       = standard deviation

EMS    = ethyl methanesulfonate

DMBA  = 9,10-dimethyl-1,2-benzanthracene

Conclusions:

Under the present test conditions, the test item tested up to the concentration of 600 µg /mL medium, in the absence of metabolic activation, is considered to be clearly positive according to the OECD guideline 476 as a concentration-related increase, statistically significant increase compared to the concurrent negative control as well as a value, which is outside the distribution of the historical negative control data in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects, was observed. In the presence of metabolic activation no mutagenic effects were observed up to the top concentration of 800 µg /mL medium.

Executive summary:

The test item Boron, (benzenemethanamine)trifluoro-, (T-4)-, reaction products with Bu glycidyl ether was tested for its mutagenic potential in a gene mutation assay in cultured mammalian cells(V79, genetic marker HPRT) both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals. The duration of the exposure with the test item was 4 hours in the experiments without and with S9 mix.

The test item was completely dissolved in dimethyl sulfoxide (DMSO). The vehicle DMSO was employed as the negative control.

 

Preliminary cytotoxicity test

The concentrations to be employed in the main experiment were chosen based on the results of a preliminary cytotoxicity study without and with metabolic activation with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg test item / ml medium. In this preliminary test cytotoxicityin form of decreased relative survival compared to the controlwas noted starting at a concentration of 316 µg and 1000 µg test item/

mL medium in the absence and presence of metabolic activation, respectively.Test item precipitation was noted macroscopically at the top concentration of 2000 µg /mL medium in both experiments. No relevantchanges in pH or osmolality werenoted in the test cultures compared to the negative control treated with DMSO.Hence, 600 or 800 µg test item /mL medium was employed as highest concentrationfor the genotoxicity tests without and with metabolic activation, respectively.

Main study

Concentrations of50, 100, 200, 400 and 600and 100, 200, 400, 600 and 800 µg test item/mL medium were selected for themutagenicity experiments without or with metabolic activation, respectively. The experiments without and with metabolic activation were conducted in duplicates.

Cytotoxicity

Cytotoxicityin form of decreased relative survival compared to the controlwas noted in both experiments without metabolic activation at the concentrations of 400 and 600 µg test item/mL medium as well asin the experiments with metabolic activation at the concentrations of 800 µg (first experiment) and 600 and 800 µg (second experiment) test item/mL medium. No test item precipitation was noted macroscopically at all concentrationsin both experiments each carried out without and with metabolic activation.

Mutagenicity

Experiments without metabolic activation

The mutation frequencies of the solvent controlDMSO were 6.80 and 11.87 mutant colonies per 10⁶cells, for the 1st and the 2nd experiment, respectively. Hence, the solvent controls were well within the expected range.

The mutation frequencies of the cultures treated with concentrations of 50, 100 or 200 µg test item/mL culture medium (non-cytotoxic concentrations) were 10.18, 15.70, 24.51 (first experiment) and 10.89, 27.98, 45.14 (second experiment) mutant colonies per 10⁶cells. Hence, a concentration-related increase in the mutation frequency was noted in both experiments. The regression analysis for the data showed a significant concentration (log value)-related effect (Spearman’s rank correlation coefficient, p≤0.05), a statistically significant increased value for the test concentration of 100 µg test item/mL (Wilcoxon-Mann-Whitney test, p≤0.05) and a value, which is outside the distribution of the historical negative control data for 200 µg test item/mL. Hence, the test item is considered able to induce gene mutations in cultured mammalian cells in this test system.

Experiments with metabolic activation

The mutation frequencies of the solvent controlDMSO were 4.28 and 11.81 mutant colonies per 10⁶cells, for the 1st and the 2nd experiment, respectively. Hence, the solvent controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 100, 200, 400, 600 and 800 µg test item/mL medium ranged from1.61to12.07mutant colonies per 10⁶cells. These results are within the normal range of the solvent controls.

The positive controls in the direct test EMS (ethyl methanesulfonate) and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 362.30 to 605.71 mutant colonies per 10⁶cells in the case of EMS and ranging from 244.96 to 617.76 mutant colonies per 10⁶cells in the case of DMBA, indicating the validity of this test system.

Conclusion

Under the present test conditions, the test item, tested up to the concentration of 600 µg /mL medium, in the absence of metabolic activation, is considered to be clearly positive according to the OECD guideline 476 as a concentration-related increase, statistically significant increase compared to the concurrent negative control as well as a value, which is outside the distribution of the historical negative control data in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects, was observed. In the presence of metabolic activation no mutagenic effects were observed up to the top concentration of 800 µg test item/mL medium.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Ames test

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

uvrB-

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

Dimethyl sulphoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

The five strains of bacteria used, and their mutations, are defined above.
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine
operon and are derived from S. typhimurium strain LT2 through mutations in the
histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty
lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to
larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an
inactivation of the excision repair system and a dependence on exogenous biotin. In the
strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced
mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers
ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In
addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA
repair deficiency which enhances its sensitivity to some mutagenic compounds. This
deficiency allows the strain to show enhanced mutability as the uvrA repair system would
normally act to remove and repair the damaged section of the DNA molecule (Green and
Muriel, 1976 and Mortelmans and Riccio, 2000).
The bacteria used in the test were obtained from:
• University of California, Berkeley, on culture discs, on 04 August 1995.
• British Industrial Biological Research Association, on a nutrient agar plate, on
All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen
freezer, model SXR 34.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in
nutrient broth (Oxoid Limited; lot number 1758279 10/20) and incubated at 37 °C for
approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity
with titres determined by viable count analysis on nutrient agar plates.

Experimental Design and Study Conduct
Test Item Preparation and Analysis
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house.
Dimethyl sulphoxide was therefore selected as the vehicle.
The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. No
correction was made for purity. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined.
This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method

Dose selection
The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
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 overlayed 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.

With Metabolic Activation
The procedure was the same as described previously 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. 3.3.2.4 Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 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). Several manual counts were
required, predominantly due to revertant colonies spreading slightly, thus distorting the actual
plate count.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 μg/plate.
Six test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

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 20 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.

With Metabolic Activation
The procedure was the same as described previously (see 3.3.3.2) 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 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 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).

Acceptability Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60
Combined historical negative and solvent control ranges for 2014 and 2015 are presented in Appendix 1.
All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix.
All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. The historical ranges of the positive control reference items for 2014 and 2015 are presented in Appendix 1. There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.

Evaluation criteria:

Evaluation Criteria
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and
Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester
strain (especially if accompanied by an out-of-historical range response (Cariello and
Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. In the first mutation test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 500 μg/plate in the absence of S9-mix and 1500 μg/plate in the presence of S9-mix. Consequently the same maximum dose level of 5000 μg/plate was employed in the second mutation test. In the second mutation test (pre-incubation method) the test item induced a stronger toxic response with weakened bacterial background lawns initially noted in the absence of S9-mix from 150 μg/plate (TA1535), 500 μg/plate (TA100, TA98 and TA1537) and 1500 μg/plate (WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were initially noted from 1500 μg/plate (all Salmonella strains) and at 5000 μg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). A small, statistically significant increase in TA1535 revertant colony frequency was observed in the second mutation test at 1500 μg/plate in the presence of S9-mix. This increase was considered to be of no biological relevance because weakened bacterial background lawns were also noted. Therefore the response would be due to additional cell divisions and presenting as non-revertant colonies.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies generally within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Conclusions:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with Boron, (benzenemethanamine)trifluoro-,(t-4)-, reaction products with bu glycidyl ether using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system. The dose range was 1.5 to 5000 μg/plate. Boron, (benzenemethanamine)trifluoro-,(t-4)-, reaction products with bu glycidyl ether was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with Boron, (benzenemethanamine)trifluoro-,(t-4)-, reaction products with bu glycidyl ether using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system. The dose range was 1.5 to 5000 μg/plate. Boron, (benzenemethanamine)trifluoro-,(t-4)-, reaction products with bu glycidyl ether was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

study proposal for Comet Assay

Type of information:

experimental study planned

Justification for type of information:

For the test item three in vitro tests were performed from 2017 to 2020. The Ames test according to OECD 471 and the micronucleus test according to OECD 487 have a negative outcome for genotoxicity. However, the HPRT test, which was performed 2018 at LPT showed clear positive genotoxic effects in the absence of metabolic activation. According to the REACH regulation Annex VIII an in vivo genotoxicity study has to be performed if at least one in vitro genotoxicity test showes a positive outcome.
Based on these data, we recommend to perform the combined in vivo mammalian erythrocyte micronucleus test according to OECD 474) and the in vivo mammalian alkaline Comet assay according to OECD 489 (in the liver and the stomach) as the most suitable in vivo genotoxicity tests for the test item.
Literature search showed no other available GLP and or no GLP studies for this endpoint nor any historical human data for the substance. As there are no other similar substances available grouping and read across can not be performed. As the substance is an UVCB substance application of QSAR tools/methods is not applicable.
Other in vitro methods are not relevant as all three endpoints (gene mutation, chromosome aberration, and micronucleus test) are already covered and an in vivo test is necessary after one positive in vitro test according to REACH regulation Annex VIII: According to REACH regulation Annex VIII ….”an in vivo method should be performed if there is one or more positive in vitro test in annex VII or VIII”.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)