Methyltrioctylammonium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro transformation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Gene mutation toxicity study of the test chemical

Author:

Inoue et al

Year:

1980

Bibliographic source:

Food and chemical toxicology

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

In vitro mammalian cell transformation assay was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cetalkonium chloride

EC Number:

204-526-3

EC Name:

Cetalkonium chloride

Cas Number:

122-18-9

Molecular formula:

C25H46NCl

IUPAC Name:

Cetyldimethylbenzylammonium chloride

Details on test material:

- Name of test material: cetyldimethylbenzylammonium chloride (CDBAC)
- IUPAC name: Benzylcetyldimethylammonium Chloride Hydrate
- Molecular formula: C25H46NCl
- Molecular weight: 396.098 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: 98.4%
- Impurities (identity and concentrations): 1.6%

Method

Target gene:

No data

Species / strain

Species / strain / cell type:

mammalian cell line, other: In vitro mammalian cell transformation assay

Details on mammalian cell type (if applicable):

- Type and identity of media: The standard complete culture medium used was Dulbecco’s modified Eagle medium supplemented with 2 mM+glutamine and 20% foetal bovine serum without any antibiotics. For dissociation of embryos and preparation of subcultures, 0.25% trypsin solution was used; i.e. 10 ml trypsin- EDTA solution (10 x) and 90 ml Ca2+- and Mg 2+-free solution (PBS).
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

not specified

Metabolic activation system:

No data

Test concentrations with justification for top dose:

- Type and identity of media: The standard complete culture medium used was Dulbecco’s modified Eagle medium supplemented with 2 mM+glutamine and 20% foetal bovine serum without any antibiotics. For dissociation of embryos and preparation of subcultures, 0.25% trypsin solution was used; i.e. 10 ml trypsin- EDTA solution (10 x) and 90 ml Ca2+- and Mg 2+-free solution (PBS).
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: No data

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Controls

Untreated negative controls:

yes

Remarks:

Medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 3-methylcholanthrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20 mins
- Exposure duration: 9 days
- Expression time (cells in growth medium): 9 days
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): 10 mins

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): Giemsa stain

NUMBER OF REPLICATIONS: Nine dishes were used for each dose level in all the experiments (in a few cases only eight or seven dishes were used for controls).

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

Criteria for transformation: Randomly oriented three-dimensional growth with extensive crossing over of the cells at the periphery of the colony was considered to be the endpoint of morphological transformation. The centres of transformed colonies usually exhibit dense piling-up of cells. Moreover, these cells usually have an increased ratio of nucleus to cytoplasm, are more basophilic, and are variable in size.

Statistics:

No data

Results and discussion

Test results

Species / strain:

mammalian cell line, other: Pregnant Syrian golden hamsters embryo cell line

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No data

Any other information on results incl. tables

Table 1. In vitro cell transformation of the test compound and the respective control chemicals

 

Compound	No. of transformed colonies/No. of surviving colonies
	0.2% DMSO	3MC (µg/mL)			Dose of the test chemical (µg/mL)
		0.1	0.5	1.0	0	0.01	0.05	0.1.	0.5	1	5	10	20	50	100	300
Test chemical	0/603	0/538	1/527	0/500	0/620	0/583	0/606	0/576	0/496	-	-	-	-	-	-	-

DMSO = Dimethylsulphoxide

MC = 3-Methylcholanthrene

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce cell transformation in the Pregnant Syrian golden hamsters embryo cell line used and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

In vitro mammalian cell transformation assay was performed to determine the mutagenic nature of the test chemical. The test chemical was dissolved in DMSO as the solvent and used at dose level of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 50, 100 or 300µg/mL. Pregnant Syrian golden hamsters were killed on days 13 and 14 of gestation for preparation of target cells and feeder-layer cells respectively. Embryos without heads and viscera were minced with scissors,a nd trypsinized with 0.25% trypsin. Inocula of 10000000 embryo cells per 75-cm’ flask were incubated at 37°C in a humidified atmosphere of 10% CO2 in air. When they became confluent primary cultures were trypsinized, dispensed in lots of 5000000 cells in glass ampules, and stored in liquid nitrogen for use as target and feeder-layer cells. The assay takes 15 days from start to finish. On Day 0, an ampule of cryopreserved primary cells prepared as feeder-layer cells was rapidly thawed and plated in a 75-cm2 flask containing 20 ml of the culture medium. The medium was changed every day. On day 3, an ampule of cryopreserved primary cells prepared as target cells was also rapidly thawed and plated in a 75-cm2 flask. On day 4, the feeder cells which were shifting from a stage of logarithmic growth to a stationary phase were irradiated with 5000 R from a linear accelerator, trypsinized, and then plated at 6 x lo4 tells/60-mm dish in 2 ml of complete medium. On day 5, the target cells which were approximately 80 -90% confluent were trypsinized, and a suspension of 500 target cells in 2 ml of complete medium was then added to each of the dishes plated the day before with irradiated feeder-layer cells. On day 6, an appropriate dose of the test chemical in a volume of 4 ml was added, giving a total volume of 8 ml of medium in each dish. Nine dishes were used for each dose level in all the experiments (in a few cases only eight or seven dishes were used for controls). On day 14, the cultures werefixed with absolute methanol for 10min and stained with Giemsa solution for 45 min or more. The stained dishes were examined with a stereoscopic dissection microscope to count normal and transformed colonies. The exposure duration was 8 days. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did not induce cell transformation in the Pregnant Syrian golden hamsters embryo cell line used and hence it is not likely to classify as a gene mutant in vitro.