Trichloro(N,N-dimethyloctylamine)boron

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 June 2014 to 14 July 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Evaluation the test substance for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (1-IPRT) locus (hprt) of Chinese Hamster Ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TG').

Species / strain / cell type:

Chinese hamster Ovary (CHO)

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:

TK 12146 was prepared in DMSO and evaluated in a preliminary toxicity assay at concentrations of 10.7, 21.4, 42.8, 85.6, 171, 343, 685, 1370 and 2740 microg/mL with and without S9 (the maximum concentration evaluated approximated the I0 mM limit dose for this assay).

Based on these results, TK 12146 was evaluated in the definitive mutagenicity assay at concentrations of 6.25, 12.5, 25.0, 50.0, 100 and 200 microg/mL with and without S9.

Vehicle / solvent:

The vehicle used to deliver TK 12146 to the test system was DMSO (CAS No. 67-68-5; Lot No. Sl-IBC3749V, Pmity: 99.92%, Expiration Date: April
2016), obtained fi·om Sigma-Aldrich.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (Dimethylsulfoxide)

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: Benzo(a)pyrene

Remarks:

Both were prepared at the appropriate concentration in DMSO

Evaluation criteria:

A test substance was considered to have induced a positive response if there was a concentration-related increase in mutant fi·equency with at least two consecutive doses showing mutant frequencies of>40 mutants per 10E6 clonable cells. If a single point above 40 mutants per I 06 clonable cells was observed at the highest dose, the results were considered equivocal. If no culture exhibited a mutant frequency of >40 mutants per 10E6 clonable cells, the test
substance was considered negative.

Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.

Statistics:

The primary computer or electronic systems used for the collection of data or analysis included but were not limited to the following:
- LIMS Labware System: Test Substance tracking
- Excel (Microsoft Corporation): Calculations
- Kaye Lab Watch Monitoring system (Kaye GE): Environmental monitoring
- BRIQS: Deviation and audit repmting
- ProtoCOL Colony Counter: Data collection

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'.

Solubility Test

DMSO was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approx.500 mg/mL, the maximum tested.

Preliminary Toxicity Assay

Results of the preliminary toxicity assay are presented in Table I. TK 12146 was prepared in DMSO and evaluated in a preliminary toxicity assay at concentrations of I 0.7, 21.4, 42.8, 85.6, 171, 343, 685, 1370 and 2740 microg/mL with and without S9 (the maximum concentration evaluated approximated the 10 mM limit dose for this assay). Visible precipitate was observed at concentrations >=85.6 microg/mL at the beginning and end of treatment. The test substance did not have an adverse impact on the pH or osmolality of the cultures (437 and 431 mmol/kg for the solvent control and 42.8 f!g/mL, the highest soluble concentration at the

beginning of treatment, respectively). Relative survival was 112.10 and 84.58% at a concentration of 2740 microg/mL with and without S9, respectively.

Definitive Mutagenicity Assay

Results of the mutagenicity assay are presented in Table 2. Based on the results of the preliminary toxicity assay, TK 12146 was evaluated in the definitive mutagenicity assay at concentrations of 6.25, 12.5, 25.0, 50.0, 100 and 200 microg/mL with and without S9. Visible precipitate was observed at concentrations 2:50.0 microg/mL at the beginning and end of treatment.Cultures treated at concentrations of6.25, 12.5, 25.0, 50.0 and 100 microg/mL with and without S9 were chosen for mutant selection (cultures treated at 200 pg/mL were discarded prior to selection because the limit of solubility was reached). The average relative survival was 85.47

and I 03.79% at a concentration of I 00 pg/mL with and without S9, respectively. No increases in average mutant frequency >40 mutants per I10E6 were observed at any concentration evaluated with or without S9.

All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.

Conclusions:

Under study conditions, test substance was negative in the In vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay.

Executive summary:

An in vitro study was conducted to investigate the potential of test substance, trichloro(N,N-dimethyloctylamine) boron for its ability to induce forward mutations at the hypoxanthineguanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, according to OECD Guideline 476, in compliance with GLP in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of6-thioguanine (TG resistance, TG'). Test substance was prepared in DMSO and evaluated in a preliminary toxicity assay at concentrations of 10.7, 21.4, 42.8, 85.6, 171, 343, 685, 1370 and 2740 micromL with and without S9 (the maximum concentration evaluated approximated the I0 mM limit dose for this assay). Visible precipitate was observed at concentrations 2:85.6 microg/mL at the beginning and end of treatment. The test substance did not have an adverse impact on the pH or osmolality of the cultures. Relative survival was 112.10 and 84.58% at a concentration of 2740 microg/mL with and without S9, respectively. Based on these results, test substance was evaluated in the definitive mutagenicity assay at concentrations of 6.25, 12.5, 25.0, 50.0, 100 and 200 microg/mL with and without S9. Visible precipitate was observed at concentrations >=50.0 microg/mL at the beginning and end of treatment. Cultures treated at concentrations of 6.25, 12.5, 25.0, 50.0 and 100 microg/mL with and without S9 were chosen for mutant selection (cultures treated at 200 microg/mL were discarded prior to selection because the limit of solubility was reached). The average relative survival was 85.47 and 103.79% at a concentration of 100 microg/mL with and without S9, respectively. Under study conditions, test substance was negative in the In vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay (Stankowski, 2014).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 June 2014 to 24 June 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Loci of several strains of Salmonel!a typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA.
Tester strains TA98 and TAI537 are revetted fi·om histidine dependence (auxotrophy) to histidine independence (prototrophy) by fi·ameshift mutagens. Tester strain TA 1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA I 00 is revetted by mutagens that cause both fi·ameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel. 1976).

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

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:

- In the initial toxicity-mutation assay, the dose levels tested were: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate.

- In the confirmatory mutagenicity assay, the dose levels tested were: 50, 150, 500, 1500 and 5000 μg per plate.

Vehicle / solvent:

The vehicle used to deliver TK 12146 to the test system was Dimethyl sulfoxide (DMSO) and was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells.
(CAS No. 67-68-5, Lot No. SHBD1324V, Purity: 99.98%, Exp. Date: May 2017), obtained fi·om Sigma-Aldrich. Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under yellow light.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (DIMETHYL sulfoxide)

True negative controls:

no

Positive controls:

yes

Remarks:

At 1 (microg/plate) for TA98 and TA 1535, 2.0 microg/plate for TA100 and TA 1537 and 15 microgr/plate for WP2 uvrA.

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (DIMETHYL sulfoxide)

True negative controls:

no

Positive controls:

yes

Remarks:

At 1 (microg/plate) for TA98

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (DIMETHYL sulfoxide)

True negative controls:

no

Positive controls:

yes

Remarks:

At 1 (microg/plate) for TA100 and TA 1535

Positive control substance:

sodium azide

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (DIMETHYL sulfoxide)

True negative controls:

no

Positive controls:

yes

Remarks:

At 75 (microg/plate) for TA1537

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (DIMETHYL sulfoxide)

True negative controls:

no

Positive controls:

yes

Remarks:

At 1 000 (microg/plate) for WP2 uvrA

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS: All dose levels oftest substance, vehicle control and positive controls were plated in triplicate.

METHOD OF APPLICATION: In agar (plate incorporation).

Overnight cultures were prepared by inoculating fi·om the appropriate frozen permanent stock
into a vessel, containing 30 to 50 mL of culture medium. To assure that cultures were harvested
in late log phase, the length of incubation was controlled and monitored. Following inoculation,
each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and
incubating at 37±2°C for approximately 12 hours before the anticipated time of harvest. Each
culture was monitored spectrophotometrically for turbidity and was harvested at a percent
transmittance yielding a titer of greater than or equal to 0.3x I 09 cells per milliliter. The actual
titers were determined by viable count assays on nutrient agar plates.

Evaluation criteria:

Evaluation of Results
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are repmted.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean reve11ants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance.

Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA I 00 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.

An equivocal response is a biologically relevant increase in a reve1tant count that pmtially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the
respective threshold cited. A response was evaluated as negative, if it was neither positive nor equivocal.

Statistics:

The primary computer or electronic systems used for the collection of data or analysis included but were not limited to the following:
Sorcerer Colony Counter and Ames Study Manager (Perceptive Instruments), LIMS System (BioReliance), Excel2007 (Microsoft Corporation), BRIQS
(BioReliance) and Kaye Lab Watch Monitoring System (Kaye GE).

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle 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:

no cytotoxicity

Vehicle 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:

no cytotoxicity

Vehicle 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:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Solubility Test

DMSO was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test conducted at BioReliance.

Sterility Results

No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

Tester Strain Titer Results

Experiment	Tester starin
Experiment	TA98	TA100	TA1535	TA1537	WP2 uvrA
Experiment	Titer Value (x 10 9 cells per mL)
B1	1.1	1.3	2.0	2.1	2.3
B2	2.3	1.	2.9	2.9	4.0

Initial Toxicity-Mutation Assay

In Experiment B 1 (Initial Toxicity-Mutation Assay), the maximum dose tested was 5000 microg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 microL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 microg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 500 or 1500 microg per plate. No toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 microg per plate.

Confirmatory Mutagenicity Assay

In Experiment B2 (Confirmatory Mutagenicity Assay), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 microg per plate. Precipitate was observed beginning at 500 microg per plate. No toxicity was observed.

Conclusions:

Under the conditions of this study, test substance, trichloro(N,N-dimethyloctylamine) boron was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay (Valentine et al, 2014).

Executive summary:

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, trichloro(N,N-dimethyloctylamine)boron according to OECD Guideline 471, in compliance with GLP. The test substance was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA I00, TA 1535 and TA 1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance. Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test conducted at Bio Reliance. In the initial toxicity-mutation assay, the maximum dose tested was 5000 microg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 microL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 microg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 500 or 1 500 microg per plate. No toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 microg per plate. In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 50, 150, 500, 1 500 and 5000 microg per plate. Precipitate was observed beginning at 500 microg per plate. No toxicity was observed. Under the conditions of this study, test substance trichloro(N,N-dimethyloctylamine) boron was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay (Valentine et al, 2014).

Reference 3

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.

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

Target gene:

Not applicable

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.

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

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'.

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.

Conclusions:

Under study conditions Trichloro(N,N-dimethyloctylamine)boron was negative in the in vitro chromosome aberration assay in CHO cells.

Executive summary:

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, trichloro(N,N-dimethyloctylamine)boron, according to OECD Guideline 473, in compliance with GLP.The test substance 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. Based preliminary 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 non-activated 4-hour exposure group, no significant or dose-dependent increases in structural aberrations were observed at any dose level . 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 .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. Under study conditions Trichloro(N,N-dimethyloctylamine)boron was negative in the in vitro chromosome aberration assay in CHO cells (Shambhu Roy, 2014).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Chromosome aberration test

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, trichloro(N,N-dimethyloctylamine)boron, according to OECD Guideline 473, in compliance with GLP.The test substance 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. Based preliminary 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 non-activated 4-hour exposure group, no significant or dose-dependent increases in structural aberrations were observed at any dose level . 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 .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. Under study conditions Trichloro(N,N-dimethyloctylamine)boron was negative in the in vitro chromosome aberration assay in CHO cells (Shambhu Roy, 2014).

 

Bacterial Reverse Mutation Assay

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, trichloro(N,N-dimethyloctylamine)boron according to OECD Guideline 471, in compliance with GLP. The test substance was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA I00, TA 1535 and TA 1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance. Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test conducted at Bio Reliance. In the initial toxicity-mutation assay, the maximum dose tested was 5000 microg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 microL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 microg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 500 or 1 500 microg per plate. No toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 microg per plate. In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 50, 150, 500, 1 500 and 5000 microg per plate. Precipitate was observed beginning at 500 microg per plate. No toxicity was observed. Under the conditions of this study, test substance trichloro(N,N-dimethyloctylamine) boron was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay (Valentine et al, 2014).

 

Mammalian Cell Forward Gene Mutation Assay

 

An in vitro study was conducted to investigate the potential of test substance, trichloro(N,N-dimethyloctylamine) boron for its ability to induce forward mutations at the hypoxanthineguanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, according to OECD Guideline 476, in compliance with GLP in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of6-thioguanine (TG resistance, TG'). Test substance was prepared in DMSO and evaluated in a preliminary toxicity assay at concentrations of 10.7, 21.4, 42.8, 85.6, 171, 343, 685, 1370 and 2740 micromL with and without S9 (the maximum concentration evaluated approximated the I0 mM limit dose for this assay). Visible precipitate was observed at concentrations 2:85.6 microg/mL at the beginning and end of treatment. The test substance did not have an adverse impact on the pH or osmolality of the cultures. Relative survival was 112.10 and 84.58% at a concentration of 2740 microg/mL with and without S9, respectively. Based on these results, test substance was evaluated in the definitive mutagenicity assay at concentrations of 6.25, 12.5, 25.0, 50.0, 100 and 200 microg/mL with and without S9. Visible precipitate was observed at concentrations >=50.0 microg/mL at the beginning and end of treatment. Cultures treated at concentrations of 6.25, 12.5, 25.0, 50.0 and 100 microg/mL with and without S9 were chosen for mutant selection (cultures treated at 200 microg/mL were discarded prior to selection because the limit of solubility was reached). The average relative survival was 85.47 and 103.79% at a concentration of 100 microg/mL with and without S9, respectively. Under study conditions, test substance was negative in the In vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay (Stankowski, 2014).

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to EU CLP regulation (EC) 1272/2008).