1,3-bis(isocyanatomethyl)benzene

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

Study period:

September 12, 2008 - March 18, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

1,3-bis(isocyanatomethyl)benzene

EC Number:

222-852-4

EC Name:

1,3-bis(isocyanatomethyl)benzene

Cas Number:

3634-83-1

Molecular formula:

C10H8N2O2

IUPAC Name:

1,3-bis(isocyanatomethyl)benzene

Details on test material:

- Name of test material (as cited in study report): m-Xylylene diisocyanate (XDI)
- Appearance: colourless transparent liquid

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

other: Chinese hamster lung fibroblasts (CHL/IU cells)

Details on mammalian cell type (if applicable):

Cells:
CHL/IU cells were supplied by Health Science Research Resources Bank, Japan Health Sciences Foundation on April 17, 2002. The modal number of chromosomes was 25 per cell. The doubling time was about 15 hours. It was confirmed in the testing facility that the cells were mycoplasma free and the spontaneous frequencies of cells with structural aberration and the numerical aberration were below 5%.

Storage of cells:
Cells were suspended in medium (Eagle's minimum essential medium, Nissui Pharmaceutical Co., Ltd.) and 10 vol% heat-inactivated newborn calf serum (NBCS, Sanko Junyaku Co., Ltd.) including 10 vol% DMSO and were frozen and stored in liquid nitrogen.

Culture condition:
Cells were cultured in a CO2 incubator (MCO-18AIC, SANYO Electric Co.), which was set at 37 degrees Celsius and 5% CO2 under humid condition

Subculture:
Cells were subcultured in a 90 mm diameter Petri dishes twice a week. Passage number of cells was at 11 for the cell growth inhibition test (dose range finder) and 17 for the chromosomal aberration test after the receipt.

Medium:
Basal medium (MEM) was prepared by adding L-Glutamine (final concentration: 0.292 g/L) and sodium hydrogen carbonate (final concentration: approximately 1.85 g/L) to Eagle's minimum essential medium (Lot no. 588803, Nissui Pharmaceutical Co., Ltd.) This medium was then supplemented with 10 vol% heat-inactivated newborn calf serum (lot. no. ASC29136, HyClone) and used for culture

Cell pre-culture:
A 60-mm diameter plastic dish (Asahi Glass Corp.) was used for cell culture. Five mL of a cell suspension of 1.5x10^4 cells/mL were seeded into a dish and were cultured continuously for 2 days in the cell growth inhibition test. Five mL of a cell suspension of 5.0x10^3 cells/mL were seeded into a dish and were cultured continuously for 3 days in the chromosomal aberration test.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without S9 mix, 6 hr/24 hr exposure; 18 hr fixation: 7.34; 14.7; 29.4; 58.8; 118; 235; 470 and 940 µg/mL (=0.01 M)
With S9 mix, 6 hr exposure; 18 hr fixation: 7.34; 14.7; 29.4; 58.8; 118; 235; 470 and 940 µg/mL (=0.01 M)
First cytogenetic test:
Without S9-mix, 6 hr exposure; 18 hr fixation: 230; 280; 330; 380; 430 and 480 µg/mL
With S9-mix, 6 hr exposure; 18 hr fixation: 230; 280; 330; 380; 430; 480; 530 and 580 µg/mL
Second cytogenetic test: not conducted as the 1st was positive
Positive controls:
MMC, without S9 mix, 0.1 µg/mL for a 6 hr exposure period
CPA, with S9 mix, 6 µg/mL for a 6 hr exposure period

Vehicle / solvent:

- Solvent used: DMSO (purity: 100%), dehydrated with molecular sieves
- Justification for choice of solvent/vehicle: water is not possible, as the substance reacts with water. The substance dissolved at 188 mg/mL in dehydrated DMSO.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

; DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Mitomycin C (MMC) and Cyclophosphamide monohydrate (CPA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period:
Cell growth inhibition test (dose range finding assay): 2 days
Chromosomal aberration test: 3 days
- Exposure duration: 6 hr (with and without S9-mix) and 24 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hr

SPINDLE INHIBITOR (cytogenetic assays): demecolcine solution
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: duplicates in 1 experiment
NUMBER OF CELLS EVALUATED: 50 metaphase chromosome spreads per culture
DETERMINATION OF CYTOTOXICITY: number of cells of each culture was measured using a Microcell counter

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

The experiment was judged positive when the frequencies of cells with structural aberrations showed 10% or more with a dose-related increase or the frequencies of cells with structural aberrations showed 5% or more both in the chromosomal aberration test. The other cases were judged to be negative. No statistical analyses were performed.

Statistics:

Not applicable

Results and discussion

Test results

Species / strain:

other: Chinese hamster lung fibroblasts (CHL/IU cells)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Precipitation: observed at the end of the treatment period at dose levels of 330 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
- Little to no toxicity was observed up to and including the highest precipitating tested dose

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

Any other information on results incl. tables

- Without S9 mix, the frequencies of cells with structural aberrations at 230, 280 and 330 µg/mL were 4.0; 11.5 and 13%, respectively (more than 10% and a dose-related increase: positive). The frequencies of numerical aberration cells at 230; 280 and 330 µg/mL were 1.0; 2.0 and 1.5%, respectively (all below 5%).

- With S9 mix, the frequencies of cells with structural aberrations at 230, 280; 330 and 380 µg/mL were 5.0; 16.0; 23.5 and 38.5%, respectively (more than 10% and a dose-related increase: positive). The frequencies of numerical aberration cells at 230; 280; 330 and 380 µg/mL were 3.0; 3.5; 3.0 and 2.5%, respectively (all below 5%).

- No effects of the substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the substance does not disturb mitotic processes and cell cycle progression.

Applicant's summary and conclusion

Conclusions:

The substance did induce a relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix. It is concluded that this test is valid and that the substance is clastogenic in Chinese hamster lung fibroblasts.

Executive summary:

The potential of the substance to induce structural chromosome aberrations in cultured mammalian somatic cells has been investigated according to OECD 473 and GLP. Chinese hamster lung fibroblasts cells were treated for 6 hr (with and without S9-mix) and for 24 hr (without S9-mix) with the substance at doses ranging from 7.34 to 940 µg/mL (=0.01 M). Precipitation was observed (at the end of the treatment period) at dose levels of 330 µg/mL and above. The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

Without S9 mix, the frequencies of cells with structural aberrations at 280 and 330 µg/mL were 11.5 and 13%, respectively and with S9 mix, the frequencies of cells with structural aberrations at 280; 330 and 380 µg/mL were 16.0; 23.5 and 38.5%, respectively. As the frequency is more than 10% and increases in a dose-related manner, it is concluded that the substance is clastogenic in Chinese hamster lung fibroblasts.