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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov - Feb 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(1997)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-methylenedicyclohexyl diisocyanate
EC Number:
225-863-2
EC Name:
4,4'-methylenedicyclohexyl diisocyanate
Cas Number:
5124-30-1
Molecular formula:
C15H22N2O2
IUPAC Name:
1,1'-methylenebis(4-isocyanatocyclohexane)
Details on test material:
- Content: not indicated by the sponsor
- Stability under test conditions: A stability test in the solvent did not reveal significant degradation of the active ingredient.
Specific details on test material used for the study:
- Stability under test conditions: A stability test in the solvent did not reveal significant degradation of the active ingredient.

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's minimal essential medium (MEM, Earle) with supplements
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Cytokinesis block (if used):
0.2 mL Colcemid solution (40 µg/mL water)
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from liver homogenates of Aroclor 1254 induced male Spargue Dawley rats.
Test concentrations with justification for top dose:
Experiment I (4-hours treatment, harvest time 18 hours): without S9 mix 0, 1.5, 3, 4.5, 6 and 7.5 µg/mL, with S9 mix 0, 6, 12, 20, 24 and 28 µg/mL
Experiment II (4-hours treatment, harvest time 30 hours): without S9 mix 0, 4.5, 6 and 7.5 µg/mL, with S9 mix 0, 20, 24 and 28 µg/mL
Experiment III (continuous treatment for 18 hours): without S9 mix 0, 2.5, 5, 8, 10 and 12 µg/mL
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
CP (2 µg/mL) was only used with S9-mix, MMC (4-hour treatment 0.1 µg/mL, 18-hour treatment 0.03 µg/mL) was only used without S9-mix
Details on test system and experimental conditions:
PRETESTING: Cells were exposed (in duplicate) for a 4 hour-treatment time to concentrations of 1-8 µg/mL without S9-mix and to concentrations of 10-50 µg/mL with S9-mix. Pre-test concentrations for the 18 hour-continuous treatment were 1-25 µg/mL. As indicators of cytotoxic effects mitotic indices and numbers of surviving cells (survival index) were used.

TREATMENT RPOTOCOL FOR MAIN TESTING: The general protocol was similar to published procedures (e.g. Dean and Danford, in: Mutagenicity testing - a practical approach, (ed. Venitt and Parry) IRL Press, Oxford, 1984).
Initially Chinese hamster V79 cells were exposed in the absence of S9 mix for 4 hours to concentrations of 0, 1.5, 3, 4.5, 6 and 7.5 µg/mL of test substance. Cultures of all concentrations were harvested 18 hours after the beginning of the treatment. In addition, cells treated with 0, 4.5, 6 and 7.5 µg/ml were harvested 30 hours after the beginning of the treatment. In the presence of S9 mix cells were exposed to concentrations of 0, 6, 12, 20, 24 and 28 µg/ml of test substance. Cultures of all concentrations were harvested 18 hours after the beginning of the treatment. In addition, cells treated with 0, 20, 24 and 28 µg/ml were harvested 30 hours after the beginning of the treatment. Without S9 mix an additional experiment was performed using continuous treatment for 18 hours, harvest at the same time, and test substance-concentrations of 0, 2.5, 5, 8, 10 and 12 µg/ml.
Based on their cytotoxicity, which was also determined 8 hours after the beginning of the treatment, concentrations were selected for reading of metaphases.

NUMBER OF CELLS EVALUATED: Chromosomes of approximately 200 metaphases per concentration, 100 metaphases from each of two parallel cultures, were examined. The classes of structural chromosome damage were defined and recorded by using essentially the terminology of Rieger and Michaelis (Die Chromosomenmutationen, VEB Gustav-Fischer Verlag, Jena, 1967). Both chromatid and chromosome-type aberrations were assessed.
Polyploid metaphases were recorded.

SPINDLE INHIBITOR (cytogenetic assays): 0.2 ml Colcemid-solution (40 µg/ml water) was added to each flask two hours prior to the end of the incubation period.

STAIN (for cytogenetic assays): 3% Giemsa solution

DETERMINATION OF CYTOTOXICITY: was assessed in the pre-test as well as in the main-study.
- Method: Cell survival as well as mitotic index were determined in the presence and absence of S9 mix.

OTHER: Influence on pH and osmolality was assessed.
Evaluation criteria:
- An increased incident of gaps of both types without concomitant increase of other aberration types was not considered as indication of a clastogenic effect.
- A test was considered positive, if there was a relevant and statistically significant increase in the aberration rate.
- A test was considered negative, if there was no such increase at any time interval.
- A test was also considered negative, if there were statistical significant values, which were, however, within the range of historical negative controls.
- A test was considered equivocal, if there was an increase above the range of historical negative controls which was statistically significant but not considered relevant, or if an increase occurred, which was considered relevant, but which was not statistically significant.
Statistics:
Pair-wise comparison of treated and positive control groups to the respective solvent control group.
The statistical analysis used the one-sided chi²-test for the mitotic index, the recommendations outlined by Richardson et. al. (1989) and the Fisher's exact test for assessing the numbers of metaphases with aberrations/exchanges.
A difference was considered to be significant, if the probability of error was below 5%.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Concentrations of 4.5, 6 and 7.5 µg/mL test substance (4 hours treatment) and 2.5, 5 and 8 µg/ml (18 hours treatment) were chosen for reading in the absence of S9-mix. In the presence of S9-mix 6, 12 and 20 µg/mL test substance concentrations were employed. All of these cultures harvested 18 hours after the beginning of the treatment were included. In addition, cultures treated in the absence of S9-mix with 7.5 µg/mL and harvested 30 hours after the beginning of the treatment were used. The same was true for cultures treated in the presence of S9-mix with 20 µg/mL.
None of the cultures treated with test substance in the absence and in the presence of S9-mix showed biologically relevant or statistically significant increased numbers of aberrant metaphases.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Concentrations of up to at least 500 µg/mL did not change the pH in the medium in the pre-test.
- Effects of osmolality: The osmolality in the medium of the pre-test was not changed by concentrations of up to at least 500 µg/mL.
- Precipitation: Precipitation in the medium was not observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Without S9-mix cytotoxic effects were observed at 4.5 µg/mL and above after 4 hours treatment and at 8 µg/mL and above after 18 hours treatment. With S9-mix cytotoxic effects were observed at 12 µg/mL and above.

Any other information on results incl. tables

Data from IUCLID4

RS-Freetext:
Without S9 mix cytotoxic effects were observed at 4.5 µg/ml and above after 4 hours treatment and at 8 µg/ml and above after 18 hours treatment. With S9 mix cytotoxic effects
were observed at 12 µg/ml and above. Precipitation in the medium was not observed.
Therefore, concentrations of 4.5, 6 and 7.5 µg/ml test substance (4 hours treatment) and 2.5, 5 and 8 µg/ml (18 hours treatment) were chosen for reading in the absence of S9
mix. In the presence of S9 mix 6, 12 and 20 µg/ml of test substance were employed. All of these cultures harvested 18 hours after the beginning of the treatment were included. In addition, cultures treated in the absence of S9 mix with 7.5 µg/ml and harvested 30 hours after the beginning of the treatment were used. The same was true for cultures treated in the presence of S9 mix with 20 µg/ml.
None of the cultures treated with test substance in the absence and in the presence of S9 mix showed biologically relevant or statistically significant increased numbers of aberrant metaphases.

Applicant's summary and conclusion

Executive summary:

4,4´-Methylenedicyclohexyl diisocyanate has been assessed in the OECD HPV programme, 2005.

Cited from SIAR of SIAM 20 (Paris, April 19 -22, 2005): "4,4´-Methylenedicyclohexyl diisocyanate was tested in the chromosome aberration assay with Chinese hamster V79 cells in vitro according to OECD TG 473. None of the cultures treated with 4,4´-methylenedicyclohexyl diisocyanate in the absence and in the presence of S9 mix up to cytotoxic concentrations (4.5 μg/ml without S9 mix and 12 mg/ml with S9 mix) showed biologically relevant or statistically significant increased numbers of aberrant metaphases. The positive controls induced clastogenic effects."

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