reaction product of diphenylmethanediisocyanate, toluenediisocyanate ( reaction mass of isomers: 65 % 2,4- and 35 % 2,6-diisocyanate), octylamine, oleylamine and 4-ethoxyaniline (molar ratio 3.88:1:6.38:0.47:2.91)

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:

1999-04-14 to 1999-05-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 1997-07-21

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

dated 1992-12-29

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

EPA712-C-96-223, June 1996

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

430-970-4

EC Name:

-

Cas Number:

1266545-66-7

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Reaction product of (C8 – C18) aliphatic primary amines (partly unsaturated) and p-phenetidine with a mixture of aromatic isocyanates comprising of primarily 4,4’-methylenediphenyl diisocyanate and 4-methyl-m-phenylene

Details on test material:

NA

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimum Essential Medium (MEM), supplemented with 10% foetal calf serum (FCS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (S9 liver microsomal fraction induced with beta-Naphtoflavone and Phenobarbital)

Test concentrations with justification for top dose:

Experiment I: 4 h treatment, 20 h preparation interval
with and without metabolic activation: 25, 250, 500 µg/mL

Experiment II: 20 h treatment and preparation interval:
without metabolic activation: 25, 100, 250 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (final concentration 1 %)
- Justification for choice of solvent/vehicle: The utilised final concentration of DMSO is compatible with the survival of the cells and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

, culture medium

Negative solvent / vehicle controls:

yes

Remarks:

, DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Controls 2

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

TWO INDEPENDENT EXPERIMENTS
-Experiment I: Exposure period (with and without metabolic activation): 4 hours
-Experiment II: Exposure period (without metabolic activation): 20 hours

Fixation time:
With and without S9 mix: 20 h

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: (with and without metabolic activation): 4 hours in experiment I and 20 hours in experiment II
- Fixation time (start of exposure up to fixation or harvest of cells): approx. 20 hours after start of the exposure
- SPINDLE INHIBITOR (cytogenetic assays): colcemide, 17.5 hours after the start of the treatment.
- STAIN (for cytogenetic assays): After fixation the cells were stained with Giemsa

NUMBER OF REPLICATIONS: 2 replications

NUMBER OF CELLS EVALUATED: At least 200 well spread metaphases per concentration and control were scored for cytogenetic damage.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploid: the number of polyploid cells was scored. Polyploid means a near tetraploid karyotype in the case of this aneuploid cell line.

Evaluation criteria:

There are several criteria for determining a positive result:
- dose-related increase in the number of cells with aberration
- biologically relevant positive response for at least one of the test points

Statistics:

The chi-square test was used as an aid for the interpretation. A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose level.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight cytotoxic effects at concentrations >= 250 µg/mL, only in the absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight cytotoxic effects at concentrations >= 100 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the two highest concentrations the precipitate of the test item began to stick to the cells.

RANGE-FINDING/SCREENING STUDIES: Not performed

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

In experiment I, with metabolic activation, a reduction of the mitotic index accompanied by a slight reduction of the cell density was observed at the 250 and 500 µg/mL concentrations. In exp. I, with metabolic activation, as well as in exp. II, without S9, a clear reduction of the relative cell density was seen at the two highest concentrations, but no reduction of the mitotic index. These findings, suggesting slight toxic properties of the test item, may be explained by the solubility properties of the test item. At the two highest concentrations the precipitate of the test item began to stick to the cells.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the conditions of the study, the test substance did not induce structural chromosomal in the in vitro mammalian chromosome aberration test with and without metabolic activation using V79 Chinese hamster cells. Based on the study results, the test item was considered to be non-mutagenic.

Executive summary:

An in vitro mammalian chromosome aberration test was performed with the test item according to EU Method B.10, OECD Guideline 473 and EPA OPPTS 870.5375. The test item potential to induce structural chromosome aberrations in V79 Chinese hamster cells was investigated in two independent experiments. In the two independent experiments the chromosomes were prepared 20 h after start of treatment with the test item. Experiment I was performed with and without S9 mix using a treatment interval of 4 h and a preparation interval of 20 h. Experiment II was performed only without metabolic activation with a treatment and preparation interval of 20 h. Two parallel cultures were set up per test group. Per culture 100 metaphases were scored for structural chromosomal aberrations. The following concentrations were evaluated (higher concentrations could not be evaluated due to the solubility characteristics of the test item):

Experiment I: 4 h treatment, 20 h preparation interval:

with and without metabolic activation (S9 mix) and test item concentrations: 25, 250, 500 µg/mL

Experiment II: 20 h treatment and preparation interval:

without metabolic activation (S9 mix) and test item concentrations: 25, 100, 250 µg/mL

Reference mutagens as positive controls were tested in parallel to the test item and showed an expected distinct increase in cells with structural chromosome aberrations.

Mild cytotoxic effects of the test item, indicated by a reduced relative cell density value, were only observed in experiments performed in the absence of metabolic activation starting at a concentration of 250 µg/ml in experiment II and at a concentration of 100 µg/m in experiment II.

Treatment of the cells with the test item in both experiments did not lead to a relevant decrease of the relative mitotic index or of the cell density.

When compared to the corresponding solvent control no biologically relevant increase in aberrant cells was obtained with and without metabolic activation in both independent experiments. No biologically relevant increase in the frequencies of polyploidy metaphases was found after treatment with the test item if compared to the frequencies of the controls.

It was concluded that the test item did not induce structural chromosome aberrations with and without metabolic activation. Thus, the test substance was considered non-mutagenic in this chromosome aberration test.