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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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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:
weight of evidence
Study period:
03-06-2014 to 12-03-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
In vitro mammalian cell gene mutation toxicity study was peformed to determine the mutagenic nature of Tetrabutylammonium bromide to Chinese hamster ovary cell line with S9 metabolic activation system
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrabutylammonium bromide
EC Number:
216-699-2
EC Name:
Tetrabutylammonium bromide
Cas Number:
1643-19-2
Molecular formula:
C16H36N.Br
IUPAC Name:
N,N,N-tributylbutan-1-aminium bromide
Details on test material:
- Name of the test material: Tetrabutylammonium bromide
- Molecular Formula: C16H36NBr
- Molecular Weight: 322.371 g/mol
- Substance type: Organic
Specific details on test material used for the study:
- Name of the test material: Tetrabutylammonium bromide
- IUPAC name: N,N,N-tributylbutan-1-aminium bromide
- Molecular formula: C16H36N.Br
- Molecular weight: 322.371 g/mol
- Substance type: Organic

Method

Target gene:
Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable
Additional strain / cell type characteristics:
other: Hypodiploid, modal No. 20
Metabolic activation:
with
Metabolic activation system:
S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
Test concentrations with justification for top dose:
0, 0.625, 1.25, 2.5 or 5 uM
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Phosphate-buffered saline (PBS)
Justification for choice of solvent/ vehicle: Tetrabutylammonium bromide was easily dissolved in PBS.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium with pre-incubation

DURATION
Pre-incubation
One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.

Exposure duration
3 hours

Expression time
7 days

Selection time
14 days

Fixation time
7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)

SPINDLE INHIBITOR (cytogenetic assays): Not applicable

STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.
Rationale for test conditions:
No data
Evaluation criteria:
The plates were scored for an increase in the number of colonies
Statistics:
No data

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No data

Applicant's summary and conclusion

Conclusions:
Tetrabutylammonium bromide in the concentration of 0, 0.625, 1.25, 2.5 or 5 micromolar did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical in the presence of S9 metabolic activation system.
Executive summary:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to Tetrabutylammonium bromide (IUPAC name: N,N,N-tributylbutan-1-aminium bromide ) in the concentration of 0, 0.625, 1.25, 2.5 or 5 micromolar and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment. Independently of tested Tetrabutylammonium bromide concentration, the results showed no evidence of gene toxicity. Therefore, it is considered that Tetrabutylammonium bromide in the concentration of 0, 0.625, 1.25, 2.5 or 5 micromolar does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.