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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted July 29, 2016

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

270-821-9

EC Name:

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

Cas Number:

68478-46-6

Molecular formula:

C14H23BF3NO2

IUPAC Name:

1-phenylmethanamine; 7-[(benzylamino)methyl]-10-(butoxymethyl)-5,8,11,15-tetraoxaicosan-13-ol; benzyl(3-butoxy-2-hydroxypropyl)amine; benzyl[3-butoxy-2-(3-butoxy-2-hydroxypropoxy)propyl]amine; dibenzyl(3-butoxy-2-hydroxypropyl)amine

Test material form:

liquid: viscous

Details on test material:

Boron, (benzenemethanamine)trifluoro-, (T-4)-, reaction products with Bu glycidyl ether, Batch: NC18201915

Specific details on test material used for the study:

The test item was completely dissolved in dimethylsulfoxide (DMSO)

Method

Species / strain

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.

Controls

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.