1,1'-Methylenebis(4-isocyanatobenzene) and oligomeric reaction products of 1,1'-methylenebis(4-isocyanatobenzene) and oxydipropanol

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully reported guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The study (exposure technology) was performed according to OPPTS guideline 870.1300 as far as applicable to this particular study. The OPPTS guideline 870.5.395 was used for the evaluation in the bone marrow micronucleus assay. It meets also OECD Guideline 474.

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-methylenediphenyl diisocyanate

EC Number:

202-966-0

EC Name:

4,4'-methylenediphenyl diisocyanate

Cas Number:

101-68-8

Molecular formula:

C15H10N2O2

IUPAC Name:

1,1'-methylenebis(4-isocyanatobenzene)

Constituent 2

Reference substance name:

benzene, 1,1'- methylenebis[4-isocyanato-

IUPAC Name:

benzene, 1,1'- methylenebis[4-isocyanato-

Details on test material:

99% pure;
re-analysis by Bayer AG at the end of the study: 4,4'-MDI: 99%; 2,4'-MDI: 0/98%; 2,2'-MDI:<0.01%; phenyl isocyanate: <1 ppm, stabiliser (BHT): 1060 ppm

Test animals

Species:

rat

Strain:

Brown Norway

Sex:

male

Details on test animals or test system and environmental conditions:

Age at study initiation: animals were approx. 2 months old
Weight: the variation of individual weights did not exceed +/- 10% of the mean.

Administration / exposure

Route of administration:

inhalation

Details on exposure:

MODE OF EXPOSURE: inhalation; were either exposed whole-body to conditioned air (negative control) or respirable MDI-aerosol at actual breathing zone concentrations of 9.2+/- 1.5 and 118 +/-8.6 mg/m3. One additional group of rats was exposed to 110 +/- 14.4 mg/m3 using a directed-flow nose-only mode of exposure utilizing a more refined aerosol generation technology.

AEROSOL GENERATION: In whole-body exposed rats the principles of aerosol generation used by Siegel (1999) were duplicated (MDI condensation aerosol formed by bubbling air through an impinger containing MDI monomer at 125°C) whilst in the nose-only exposed group a combined dispersion-condensation aerosol principle was devised (dispersion aerosol from heated MDI @80°C and evaporation/re-condensation under controlled conditions). In whole body exposed rats the mean mass median aerodynamic diameter (MMAD) was in the range of 2.4 - 3.1 gm (GSD 1.6) whereas in nose-only exposed rats it was 1.2 gm (GSD 1.5). In addition to gravimetric analyses, the nitro-reagent and the modified Marcali methods were used to characterize exposure atmospheres. Though both methods showed somewhat lower concentrations when compared to the gravimetric methods, it does neither appear that the principle of aerosol generation nor that the different humidity (whole-body: ab. 40%, nose-only: ab. 5%) appeared to have any impact on the chemical stability of airborne MDI.

SAMPLING TIME: Bone marrow smears were prepared from rats sacrificed 1, 2 and 7 days after cessation of exposure.

Duration of treatment / exposure:

1 hr/day, 1 exposure per week, 3 consecutive weeks

Frequency of treatment:

one hour/day, one exposure per week on three consecutive weeks

Post exposure period:

IN-LIFE OBSERVATIONS: Body weights were recorded prior to exposure weekly, clinical signs before and after each exposure. Clinical signs on exposure-free days: once/day. Rectal temperature measurements: before, directly after, and approx. 2-hrs after cessation of each exposure.

Doses / concentrations

Remarks:

Doses / Concentrations:
about 113 mg MDI/m3, 7 mg/M3-whole body. about 113 mg/MDI/m3 nose only mode of exposure
Basis:

No. of animals per sex per dose:

4 groups of young adult male Brown-Norway rats (Strain: BN/RijHsd); 6 per serial sacrifice 1, 2 and 7 days after last treatment/exposure in all but in the negative control group (day 1 only)

Positive control(s):

a) for clastogenic effects; cyclophosphamide (20 mg/kg, gavage) b) for spindle Poison effects - Colcemid (4 mg/kg, i.p.) Negative control; Conditioned, dry filtered air (whole-body exposure)

Examinations

Details of tissue and slide preparation:

SACRIFICE: 24, 48 hours and one week after last exposure (pentobarbital anesthesia).

TISSUE PREPARATION: Bone marrow from femur by aspiration with fetal bovine serum (FBS-solution); 5 replicate slides/animal were prepared and allowed to dry overnight. The slides were fixed with absolute methanol for 5 minutes. After completion of the in-live phase of study the slides were shipped to Dr. Bhaskar Gollapudi for staining and evaluation.

CELL STAINING: Acridine Orange and Wright-Giemsa (from duplicate slides) SCORING: 200 erythrocytes for PCE:NCE per animal and 2000 PCE per animal (coded and blind), Mast cells: 1000 nucleated cells per animal for determining the mast cell incidence.

FIXATION AND WEIGHT OF LUNGS: Lungs were fixed by instillation of formaldehyde fixative and stored in the same fixative for possible future histopathological examination. Prior to fixation, after exsanguination of rats the lung weights were determined.

Statistics:

One-way ANOVA with Tukey post-hoc test following arcsine-square root transformation, if necessary to achieve normal distribution of data

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Any other information on results incl. tables

- All rats exposed to 0 (air) and 9.2 mg MDI/m3 tolerated the whole-body exposure without specific signs whilst the 118 and 110 mg MDI/m3-exposed rats experienced labored and irregular breathing patterns, bradypnea and a serous nasal discharge. Mortality did not occur in any group. Apart from the 'irregular breathing pattern' both the incidence and intensity of clinical findings were almost identical in whole-body and nose-only exposed rats. The signs observed were related to respiratory tract irritation.

- Comparisons of control and MDI-exposed rats did not reveal any statistically significant effect on body weights, though there was a tendency of slightly decreased body weight gains in nose-only exposed rats.

- Statistical analysis of measurements of rectal temperatures revealed a significant hypothermia in the nose-only exposed rats.

- in nose-only exposed rats, lung weights were increased to a greater extent than in the in whole-body exposed rats. These findings are taken as indirect evidence that the nose-only mode of exposure provides a more rigorous means the expose rats to MDI. In whole-body exposed rats measures were taken to prevent their huddling together or burying their noses into the fur of neighboring rats in order to minimize their exposure to irritant particulates. Nonetheless, the irritant-related, reflexively induced hypothermia, associated with elevated lung weights , was only discernible in nose-only exposed rats.

- At no time point was there evidence of any effect on the frequency of mast cells. The frequency of mast cells was low enough not to interfere with the outcome of test. Rats treated with the positive control substances displayed increased MN-PCE on sampling days 1 and 2, but not on day 7. At no time point there was any evidence of an effect on the frequency of MN-PCE in MDI-exposed animals. -No differences in outcome existed following staining with the DANN-specific Acridine -Orange or Wright-Giemsa. These results indicate that, under the experimental conditions used, aerosolized MDI did not induce cytogenetic damage in vivo.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Of the authors: this study did not demonstrate any gentoxic (clastogenic) activity of aerosolized, inhaled MDI at concentrations as high as 118 mg/M3 air.