4,4'-methylenedicyclohexyl diisocyanate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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

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

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.

Target gene:

not applicable

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

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%.

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.

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.

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."

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 2006 - Feb 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

(1997)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

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.

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: PPA Ready mix PAA, Paching, Austria; medium consists of MEM with supplements; for the selection of mutant cells the complete medium was supplemented with 10 µg/mL 6-thioguanine.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from the liver of male Aroclor 1254 induced Sprague-Dawley rats.

Test concentrations with justification for top dose:

The top dose was selected based on a preliminary cytotoxicity test with concentrations up to 1000 µg/mL.
Experiment I: without S9-mix 1.5, 3, 6, 12, 24, 36, and 48 µg/mL (trials with concentrations above 48 µg/mL and up to 96 µg/mL were terminated due to excessive cytotoxicity), with S9-mix 3, 6, 12, 24, 48, 72, and 96 µg/mL
Experiment II (repeat): without S9-mix 0.75, 1.5, 3, 6, 12, 24, and 36 µg/mL, with S9-mix 6, 12, 24, 36, 48, 60, and 72 µg/mL, 2nd repeat with S9-mix: 6, 12, 24, 36, 48, 60, and 72 µg/mL

Vehicle / solvent:

DMSO, dried over molecular sieve 0.3 nm

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

EMS (900 µg/mL) was only used without metabolic activation, DMBA (20 µg/mL) was only used with metabolic activation

Details on test system and experimental conditions:

A preliminary cytotoxicity test was conducted without and with metabolic activation using concentrations ranging from 1 µg/mL to 1000 µg/mL. In this test precipitation of the test substance in the culture medium started at 250 µg/mL. Without S9 mix cytotoxic effects were observed at 10 µg/mL and above. With S9 mix cytotoxicity was evident at 50 µg/mL and above. No cells survived at concentrations where precipitation occurred.
DETERMINATION OF CYTOTOXICITY: cloning efficiency

METHOD OF APPLICATION:
Treatment protocol of main testing without metabolic activation: The method is based on Myhr and DiPaolo (Cancer Res. 38, 2539-2543, 1978). Exponentially growing V79 cells were plated in culture medium at a final volume of 20 mL in two 75 cm² flasks per concentration (4x10exp6 per flask) including all control groups. After attachment (16-24 hours later), the cells were exposed for 5 hours in 20 mL culture medium with reduced serum content (2%). The corresponding controls were incubated under the same conditions. Thereafter, cell monolayers were washed with PBS, trypsinized and replated in 20 mL culture medium using 1.5x10exp6 cells per 75 cm² flask and in 5 mL culture medium using 200 cells per Petri dish (diameter 60 mm). Per culture one flask and 3 Petri dishes were used. The Petri dishes were incubated (normally 6 days) to allow colony development and to determine the cytotoxicity associated with each test substance directly after treatment (“Survival to Treatment”).
Cells in 75 cm² flasks were incubated to permit growth and expression of induced mutations. Cells were subcultured (= count 1, normally after 3 days) by reseeding 1.5x10exp6 cells into 20 mL medium in 75 cm² flasks. At the end of the expression period (= count 2, normally a total of 6 days), cultures were reseeded in Petri dishes (diameter 100 mm) at 3x10exp5 cells per dish (8 dishes per culture) in 20 mL culture medium without hypoxanthine but containing 10µg/ml 6-TG for selection of mutants. In addition, 200 cells per dish (diameter 60 mm, 3 dishes per culture) were seeded in 5 mL culture medium to determine the absolute cloning efficiency for each concentration. After incubation for 6-8 days, the colonies were fixed, stained with Giemsa and counted to determine the number of 6-TG resistant colonies in the mutation assay dishes and the number of colonies in the cloning efficiency dishes.
At least two trials were performed. Mutant frequencies for at least four concentrations were determined in each trial.

Treatment protocol of main testing with metabolic activation: The activation assay was performed independently. The procedure was identical to the nonactivation assay except for the addition of S9-mix. In these experiments 19 instead of 20 mL culture medium and additionally 1 mL of S9.mix were added to the flasks for the treatment period, resulting in a concentration of 5% S9-mix in the cultures. The number of 6-TG resistant mutants and viability were determined as in the nonactivation assay.

Evaluation criteria:

- Mutant frequencies will only be used for assessment, if at least 5 dishes per culture were available and relative survival to treatment, relative population growth and absolute cloning efficiency were 10% or greater.
- A trial will be considered positive if a concentration-related and in parallel cultures reproducible increase in mutant frequencies is observed. To be relevant, the increase in mutant frequencies should be at least two to three times that of the highest negative or vehicle control value observed in the respective trial. If this result can be reproduced in a second trial, the test substance is considered to be mutagenic.
- Despite these criteria, a positive result will only be considered relevant, if no signifcant change in osmolality compared to the vehicle control can be observed. Otherwise, unphysiological culture conditions may be the reason for the positive result.
- A test substance will be judged as equivocal if there is no strictly concentration related increase in mutation frequencies but if one or more concentrations induce a reproducible and biologically relevant increase in mutant frequencies in all trials.
- An assay will be considered as negative if no reproductive and relevant increases of mutant frequencies were observed.

Statistics:

The statistical analysis relies on the mutant frequencies which are submitted to a weighted analysis of variance as well as to a weighted recursive regression, both with Poisson derived weights (Hsie, Mutation Res. 86, 193-214, 1981; Arlett, in Kirkland DJ (ed.), Statistical evaluation of mutagenicity test data, UKEMS sub-committee on guidelines for mutagenicity testing, Report Part Ill, Cambridge University Press, Cambridge, 66-101, 1989).

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:

valid

Positive controls validity:

valid

Additional information on results:

Without and with S9 mix there was no biologically relevant increase in mutant frequency above that of the vehicle controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Without and with S9 mix the test substance induced decreases in survival to treatment and decreases in relative population growth reaching 100% toxicity without S9 mix at 36 µg/mL and with S9 mix at 72 µg/mL. These results revealed a significant concentration-related cytotoxicity of the substance. Therefore, without S9 mix only concentrations of up to 24 µg/mL could be used for assessment. With S9 mix, only concentrations up to 60 µg/mL were assessable.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Concentrations of up to 1000 µg/mL did not change the pH in the medium of the pre-test.
- Effects of osmolality: The osmolality in the medium of the pre-test was also not changed by concentrations of up to 1000 µg/mL.
- Precipitation: Precipitation of the test substance in the culture medium was observed in the dose-range finder at 250 µg/mL and above.

Remarks on result:

other: strain/cell type: Chinese hamster lung fibroblasts (V79)

Remarks:

Migrated from field 'Test system'.

Executive summary:

According to the results of the present in vitro gene mutation study (HPRT) in Chinese hamster lung fibroblast/V79 cells (test conducted according to OECD TG 476) 4, 4'-methylenedicyclohexyl diisocyanate did not lead to a biologically relevant increase in mutant frequency either without or with the addition of a metabolizing system (S9 -mix). The mutant frequencies at any concentration were within the range of the concurrent vehicle control values. The increase in the frequencies of mutant colonies induced by the positive control substances ethylmethanesulphonate and dimethylbenzanthracene clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S9 mix employed.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from the liver of male Aroclor 1254 induced Sprague-Dawley rats.

Test concentrations with justification for top dose:

An initially conducted test used concentrations of 50 up to 5000 µg/plate. Due to the substance's toxicity the following concentrations were used for plate incorporation and independent repeat (preincubation method):
- without S9 mix 0.05, 0.16, 0.5, 1.6, 5.0, 16.0 µg/plate
- with S9 mix: 0.16, 0.5, 1.6, 5.0, 16.0, 50 µg/plate

Vehicle / solvent:

Ethylene glycol dimethylether (EGDE)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

EGDE

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium azide (NaN3), Nitrofurantoin (NF), 4-Nitro-1,2-phenylene diamine (4-NPDA), Mitomycin C (MMC), Cumene hydroperoxide (Cumene), 2-Aminoanthracene (2-AA)

Remarks:

The positive controls NaN3, NF, 4-NPDA, MMC and Cumene were used without S9 mix, the positive control 2-AA was only used with S9 mix.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for initial testing; for independent repeat the preincubation modification was performed (preincubation for 20 min. at 37 °C).
Each strain and dose, with and without S9 mix, was tested in triplicate. This applies also for solvent and positive controls.

DETERMINATION OF CYTOTOXICITY
- gross appraisal of background growth
- mutant count: A toxic effect was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 1.6 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Evidence of mutagenic activity of the substance was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Doses up to and including 0.5 µg/plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition in growth was observed. At higher doses, the substance had a strain-specific bacteriotoxic effect. Nevertheless this range could partially be used up to 50 µg/plate for assessment purposes.

Data from IUCLID4

RS-Freetext:
GENOTOXIC EFFECTS:
Evidence of mutagenic activity was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

CYTOTOXIC CONCENTRATION:
Doses up to and including 0.5 µg/plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition in growth was observed. At higher doses, the substance had a strain-specific bacteriotoxic effect. Nevertheless this range could partially be used up to 50 µg/plate for assessment purposes.

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 did not induce gene mutations in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in an Ames test conducted under GLP, and according to OECD TG 471 (...). Concentrations of up to 50 μg/plate (with S9- mix) and 16 μg/plate (without S9-mix) were used. 4,4´-Methylenedicyclohexyl diisocyanate showed a strain-specific cytotoxicity, starting at 1.6 μg/plate. Appropriate reference mutagens were used as positive controls and showed the expected results throughout all tested strains."

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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 did not induce gene mutations in bacteria (OECD TG 471)" or in mammalian cells (OECD TG 476) "and demonstrated no potential to induce chromosome aberrations in Chinese hamster V79 cells mammalian cells in vitro (OECD TG 473) either with or without metabolic activation."

Justification for classification or non-classification

According to Regulation (EC) No 1272/2008, Annex VI, not classified for genetic toxicity.