p-phenylenebis(methylamine)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Data source

Reference

Reference Type:

publication

Title:

Chromosomal Aberration Test in CHL/IU Cell

Year:

2000

Bibliographic source:

JAPAN CHEMICAL INDUSTRY ECOLOGY- TOXICOLOGY AND INFORMATION CENTER, JAPAN; MUTAGENICITY TEST DATA OF EXISTING CHEMICAL SUBSTANCES BASED ON THE TOXICITY INVESTIGATION OF THE INDUSTRIAL SAFETY AND HEALTH LAW; (SUPPL. 2), 2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: "Standard for Chromosomal Aberration Test in Cultured Mammalian Cells" of the Notification No.652 of the Labour Standards Bureau, Ministry of Labour, Japan

Version / remarks:

September 29, 1997

Deviations:

no

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

p-phenylenebis(methylamine)

EC Number:

208-719-3

EC Name:

p-phenylenebis(methylamine)

Cas Number:

539-48-0

Molecular formula:

C8H12N2

IUPAC Name:

1,4-Bis(aminomethyl)benzene

Test material form:

solid

Specific details on test material used for the study:

Lot No.: FBX01
Purity: 99.5%

Method

Species / strain

Species / strain / cell type:

other: Chinese hamster lung cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: supplied by Dr. M. Ishidate, Jr. in 1985
- Modal number of chromosomes: 25
- Doubling time: approximately 15 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagle's MEM supplemented with 10% heat-inactivated (56°C, 30 min) calf serum

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

24 and 48 h treatment: 0.015, 0.03, 0.06, 0.09, 0.12, 0.15 mg/mL
6h treatment: 0.05, 0.1, 0.2, 0.3, 0.4 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was soluble in DMSO and insoluble in water.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with and without S9

Details on test system and experimental conditions:

- Short-period treatment with and without Metabolic Activation (6 hours treatment):
Twenty thousands cells were seeded in culture vessel (with 5 mL of culture medium) and cultured for three days, then culture medium was changed. Cells were simultaneously treated with and without S9 mix and the test substance solution for 6 hours. Then, the test substance mixture was changed to fresh culture medium. After 18 hours further culture, rates of cell growth inhibition were determined.
- Continuous Treatment without Metabolic Activation (24 and 48 hours treatment):
Twenty thousands cells were seeded in a culture vessel (with 5 mL of culture medium), and cultured for three days. Then culture medium was changed and the test substance solution was added to a culture medium. After 24 or 48 hours treatment, rates of cell growth inhibition were determined.

- Determination of Cell Growth Inhibition Rate
The medium was discarded and the cells were washed with physiological saline. Ethanol was added to fix the cells which were then stained with 0.1% crystal violet. After washing and drying, each dish was placed under a cell densitometer to measure the color absorption value (550nm). The cell growth index was calculated with the negative control being 100% and Petri dish without cells being 0%.
- Slide preparation:
Two hours before the end of culture, the cells were treated with 0.2μg/mL Colcemid. After finishing culture, they were dissociated with trypsin (0.1% trypsin + 0.036% EDTA) and centrifuged (1000 rpm, 5 min) to collect cells. After removal of the supernatant, the cells were incubated in a 75 mM hypotonic KCl solution for 20 minutes at 37 °C. The cells were then fixed three times with acetic acid-ethanol (1:3) and spread onto clean glass slides. Each slide was stained with Giemsa solution (2.5% at pH 6.8) for 12 minutes after air-drying.
- Observation:
The number of cells with structural aberrations in 100 well-spread metaphase cells were counted per each culture vessel. The incidence of polyploid cells were recorded simultaneously.

Evaluation criteria:

Negative: frequencies of structural aberrations and of polyploidy less than 5%
Equivocal: frequencies of structural aberrations and of polyploidy from 5% to less than 10%
Positive: frequencies of structural aberrations and of polyploidy 10% or more

Results and discussion

Test results

Key result

Species / strain:

other: Chinese hamster lung cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Negative result got for test item in chromosomal aberration test in CHL.

Executive summary:

In vitro mammalian chromosome aberration test for test item was performed using lung cells of a newborn Chinese hamster according to "Standard for Chromosomal Aberration Test in Cultured Mammalian Cells" of the Notification No.652 of the Labour Standards Bureau, Ministry of Labour, Japan. The dose level were 0.015, 0.03, 0.06, 0.09, 0.12, 0.15 mg/mL for 24 and 48 h treatment without S9 mix activation, 0.05, 0.1, 0.2, 0.3, 0.4 mg/mL for 6h treatment with and without S9 mix activation. The recovery time forshort-period treatment (6h) was 18 h.

Negative result was given under the test condition.