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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 June 2014 to 28 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Trichloro(N,N-dimethyloctylamine)boron
EC Number:
252-200-4
EC Name:
Trichloro(N,N-dimethyloctylamine)boron
Cas Number:
34762-90-8
Molecular formula:
C10H23BCl3N
IUPAC Name:
trichloro(N,N-dimethyloctylamine)boron
Test material form:
other: solid
Details on test material:
- Test Substance I.D.: (TK 12146)
- Test Substance Lot Number: AEC0123100
- Test Substance Purity: 99% (provided by Sponsor)
- Test Substance Description: Amber solid
- Storage Conditions: Room temperature, protected from light
- Test Substance Receipt/Login Date: 28 May 2014

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese hamster ovary (CHO-K1) cell line is a proline auxotroph with a modal chromosome number of 20 and a population doubling time of 10-14 hours. CHO-K1 cells were obtained from the American Type Culture Collection (repository number CCL 61), Manassas, VA. The stock cell line was checked for stability of the modal chromosome number and was determined to be free from mycoplasma contamination. This system has been demonstrated to be sensitive to the clastogenic activity of a variety of chemicals.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
- Preliminary toxicity test with and without metabolic activation (4 hours treatment, 16-hour recovery period):
0.274/0.822/2.74/8.22/27.4/82.2p/274p/812p/2740p μg/mL

- Preliminary toxicity test without metabolic activation (20 hour continuous treatment):
0.274/0.822/2.74/8.22/27.4/82.2p/274p/812p/2740p μg/mL
p:Visible precipitate was observed in the treatment medium at the conclusion of the treatment period.

Based upon the results of the toxicity study, the dose levels selected for testing in the chromosome aberration were as follow:
- 5, 10, 25, 50, 60, 70, 80, 90 μg/mL (without metabolic activation, 4 hours treatment and 16 hours recovery time)
- 2.5, 5, 10, 15, 17.5, 20, 22.5, 25 μg/mL (without metabolic activation, 20 hours treatment and 0 hours recovery time)
- 5, 10, 25, 50, 60, 70, 80, 90 μg/mL (with metabolic activation, 4 hours treatment and 16 hours recovery time)
Vehicle / solvent:
The vehicle used to deliver Trichloro(N,N-dimethyloctylamine)boron (TK 12146) to the test system was DMSO (CAS No. 67-68-5, Lot No. 52193349, Exp. Date January 2017) obtained from EMD Chemicals. Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under yellow light.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
Cyclophosphamide (with metabolic activation); Mitomycin C (without metabolic activation)
Details on test system and experimental conditions:
Preparation of Target Cells:
Exponentially growing CHO-K1 cells were seeded in complete medium (McCoy's 5A medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 2.5 μg/mL Amphotericin B) for each treatment condition at a target of 5 x 105 cells/culture. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 16-24 hours.

Scoring for Metaphase Chromosome Aberrations (Definitive Assay)
To ensure that a sufficient number of metaphase cells are present on the slides, the percentage of cells in mitosis per 500 cells scored (mitotic index) was determined and recorded for each coded treatment group selected for scoring chromosome aberrations. Slides were coded using random numbers by an individual not involved with the scoring process. Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 200 metaphase spreads from each dose level (100 per duplicate culture) were examined and scored for chromatid-type and chromosome-type aberrations (Scott et al., 1990). The number of metaphase spreads that were examined and scored per duplicate culture may be reduced if the percentage of aberrant cells reaches a significant level (at least 10% determined based on historical positive control data) before 100 cells are scored. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but were considered part of the incomplete exchange. Pulverized cells and severely damaged cells (counted as 10 aberrations) were also recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY vernier for each cell with a structural aberration was recorded. The percentage of cells with numerical aberrations (polyploid and endoreduplicated cells) was evaluated for 100 cells per culture (a total of 200 cells per dose level).
Evaluation criteria:
Toxicity induced by treatment was based upon cell growth inhibition relative to the vehicle control and was reported for the preliminary toxicity and chromosome aberration portions of the study. The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group. Chromatid and isochromatid gaps were presented in the data but were not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.
Statistical analysis of the percentage of aberrant cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the vehicle control. The Cochran-Armitage test was used to measure dose-responsiveness.
The test substance was considered to have induced a positive response if it induced a statistically significant and dose-dependent increase the frequency of aberrant metaphases (p ≤ 0.05). If only one criterion was met (statistically significant OR dose-dependent increase), the result may be considered equivocal. If neither criterion was met, the results were considered to be negative.
Statistics:
Electronic systems used for the collection or analysis of data included but not be limited to the following (version numbers are maintained in the system documentation):
- LIMS Labware System. Test Substance Tracking
- Excel (Microsoft Corporation): Calculations
- Kaye Lab Watch Monitoring system (Kaye GE): Environmental Monitoring
- BRIQS: Deviation and audit reporting

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Solubility Test

Dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested for solubility.

Preliminary Toxicity Assay

A preliminary toxicity assay was conducted to observe the cytotoxicity profile of the test substance and to select suitable dose levels for the definitive chromosome aberration assay. CHO cells were exposed to vehicle alone and to nine dose levels of test substance, ranging from 0.274 to 2740 μg/mL in the absence and presence of an S9 reaction mixture. The test substance was soluble in DMSO at all concentrations tested. Visible precipitate was observed in treatment medium at dose levels ≥ 82.2 μg/mL, while dose levels ≤ 27.4 μg/mL were soluble in treatment medium at the beginning and conclusion of the treatment period.

The osmolality in treatment medium of the highest dose level tested, 2740 μg/mL, was 396 mmol/kg. The osmolality in treatment medium of the lowest precipitating dose level, 82.2 μg/mL, was 422 mmol/kg. The osmolality in the treatment medium of the highest soluble dose level, 27.4 μg/mL, was 425 mmol/kg. The osmolality of the solvent (DMSO) in the treatment medium was 421 mmol/kg. The osmolality of the test substance dose levels in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 20%. The pH of the highest dose level of test substance in treatment medium was 7.5.

Substantial toxicity (≥ 50% cell growth inhibition, relative to the vehicle control) was observed at dose levels ≥ 82.2 μg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 274 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 27.4 μg/mL in the non-activated 20-hour exposure group. Based upon the results of the toxicity study, the dose levels selected for testing in the chromosome aberration assay were as follows:

- 5, 10, 25, 50, 60, 70, 80, 90 μg/mL (without metabolic activation, 4 hours treatment and 16 hours recovery time)

- 2.5, 5, 10, 15, 17.5, 20, 22.5, 25 μg/mL (without metabolic activation, 20 hours treatment and 0 hours recovery time)

- 5, 10, 25, 50, 60, 70, 80, 90 μg/mL (with metabolic activation, 4 hours treatment and 16 hours recovery time)

Chromosome Aberration Assay

In the chromosome aberration assay, the test substance was soluble in DMSO at all concentrations tested. Visible precipitate was observed in treatment medium at dose levels ≥ 50 μg/mL, while dose levels ≤ 25 μg/mL were soluble in treatment medium at the beginning of the treatment period. At the conclusion of the treatment period, in the non-activated and S9-activated 4-hour exposure groups, visible precipitate was observed in treatment medium at dose levels ≥ 70 μg/mL, while dose levels ≤ 60 μg/mL were soluble in treatment medium. In the non-activated 20-hour exposure group, all dose levels were soluble in the treatment medium at the conclusion of the treatment period. The pH of the highest dose level of test substance in treatment medium was 7.5.

Toxicity of Trichloro(N,N-dimethyloctylamine)boron (TK 12146) (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the absence of S9 activation was 53% at 50 μg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 50 μg/mL, was not reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 10, 25, and 50 μg/mL. The percentage of cells with structural aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of cells with numerical aberrations in the test substance-treated group was statistically increased relative to vehicle control at 50 μg/mL (p ≤ 0.05, Fisher's Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). In addition, the percent increase observed (3.5%) was within the historical control data of 0.0% to 5.5%. Therefore the statically significant increase in numerical aberrations was considered biologically irrelevant. The percentage of structurally aberrant cells in the MMC (positive control) treatment group (12.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test).

Toxicity of Trichloro(N,N-dimethyloctylamine)boron (TK 12146) (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the presence of S9 activation was 50% at 70 μg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 70 μg/mL, was 16% reduced relative to the vehicle control. The dose levels initially selected for microscopic analysis were 10, 25, and 70 μg/mL. Due to scoring error, a statistically significant and dose-dependent increase in structural aberrations was observed at 70 μg/mL (p ≤ 0.05; Fisher’s Exact and Cochran-Armitage tests). Therefore, two additional lower doses (50 and 60 μg/mL) were included for microscopic evaluation. Upon re-evaluation of dose level 70 μg/mL, no statistically significant increase in structural aberrations was observed at any of the doses scored. In addition, the Cochran-Armitage test was negative for a dose-response. The percentage of cells with numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p >0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the CP (positive control) treatment group (22.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test).

Toxicity of Trichloro(N,N-dimethyloctylamine)boron (TK 12146) (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 20 hours in the absence of S9 activation was 59% at 15 μg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 15 μg/mL, was 11% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 5, 10, and 15 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) treatment group (22.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test).

The results for the positive and negative controls indicate that all criteria for valid assay were met. Based on these criteria, the results are justified and do not require a repeat of any portions of the study.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative With and without metabolic activation

The positive and vehicle controls fulfilled the requirements for a valid test.
Under the conditions of the assay described in this report, Trichloro(N,N-dimethyloctylamine) boron (TK 12146) was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells in both the non-activated and the S9-activated test systems.
Executive summary:

The purpose of this study was to evaluate the potential of the test substance and/or its metabolites to induce structural chromosomal aberrations in CHO cells in the presence and absence of an exogenous metabolic activation system.

The study was conducted in conformance with the testing guidelines of the ICH (2011) and OECD 473 (1997).

The test substance, Trichloro(N,N-dimethyloctylamine)boron (TK 12146), was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance. In both phases, CHO cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation.

Dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested for solubility. Cyclophosphamide and mitomycin C were evaluated as the concurrent positive controls for treatments with and without S9, respectively.

In the preliminary toxicity assay, the dose levels tested ranged from 0.274 to 2740 μg/mL (10 mM). Substantial toxicity (≥ 50% cell growth inhibition, relative to the vehicle control) was observed at dose levels ≥ 82.2 μg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 274 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 27.4 μg/mL in the non-activated 20-hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 5 to 90 μg/mL for the non-activated and the S9-activated 4-hour exposure groups, and from 2.5 to 25 μg/mL for the non-activated 20-hour continuous exposure group.

In the chromosome aberration assay, substantial toxicity was observed at dose levels ≥ 50 μg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 70 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 15 μg/mL in the non-activated 20-hour exposure group. The highest dose analyzed under each treatment condition produced an approximately 50% reduction in cell growth index relative to the vehicle control, which met the dose limit as recommended by testing guidelines for this assay.

In the non-activated 4-hour exposure group, no significant or dose-dependent increases in structural aberrations were observed at any dose level (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). A statistically significant increase in numerical aberrations (polyploid or endoreduplicated cells) was observed at 50 μg/mL in the non-activated 4-hour exposure group (p ≤ 0.05; Fisher’s Exact test). However, the Cochran-Armitage test was negative for a dose-response (p > 0.05). In addition, the percent increase observed (3.5%) was within the historical control data of 0.0% to 5.5%. Therefore the statically significant increase in numerical aberrations was considered biologically irrelevant.

In the S9-activated 4-hour and the non-activated 20-hour exposure group, no significant or dose-dependent increases in structural or numerical aberrations were observed at any dose level (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ≤ 0.01). Thus, all criteria for a valid study were met.

These results indicate Trichloro(N,N-dimethyloctylamine)boron (TK 12146) was negative in the in vitro chromosome aberration assay in CHO cells under the conditions, and according to the criteria of the study protocol.