Trichloro(N,N-dimethyloctylamine)boron

Genetic toxicity: in vitro

Administrative data

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.

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

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:

dimethyl(octyl)(trichloro-λ⁵-boranylidene)amine

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:

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

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.

Controls

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

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

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.

Applicant's summary and conclusion

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).