4,4'-methylenediphenyl diisocyanate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 1991 - April 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards, well documented and acceptable for assessment. Non-guideline exposure duration.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Chronic pulmonary effects of respirable methylene diphenyl diisocyanate (MDI) aerosol in rats: combination of findings from two bioassays

Author:

Feron VJ, Kittel B, Kuper CF, Ernst H, Rittinghausen S, Muhle H, Koch W, Gamer A, Mallett AK and Hoffmann HD

Year:

2001

Bibliographic source:

Arch Toxicol.75: 159-175.

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: Directive 87/302/EEC, Part B, p37.

Deviations:

yes

Remarks:

- females only; exposure 17 hours per day

GLP compliance:

yes

Remarks:

The study largely conformed to the German Principles of Good LaboratoryPractice.

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-isocyanato-4-[(4-isocyanatophenyl)methyl]benzene

Constituent 2

Reference substance name:

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

IUPAC Name:

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

Details on test material:

Monomeric 4,4'-methylenediphenyl diisocyanate (Desmodur 44 M Schuppen from Bayer AG, Leverkusen); 13 batches were tested (purity: >99.5 %)

Test animals

Species:

rat

Strain:

other: Crl:[WI]BR Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

-Age: at the start of the study the animals were approximately 10 weeks old
-Acclimation: approx. 2 weeks
-Origin: Charles River Wiga GmbH, Sulzfeld

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: The median mass aerodynamic diameters (in µm) were 1.03, 1.03, and 1.06, respectively.

Details on inhalation exposure:

The MDI aerosol was generated using an evaporation-condensation technique. The rats were exposed in 6 m3 stainless steel inhalation chambers (horizontal air flow, renewal rate: approx. 15-fold per hour). Since the vapour saturation of MDI at 23°C is about 0.1 mg/m3, a part of the exposure is as vapour.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Monitoring of total MDI was performed by gravimetrically calibrated, light scattering aerosol sensors. Concentrations of monomeric MDI in the inhalation chamber were measured with HPLC.

Duration of treatment / exposure:

up to 2 years

Frequency of treatment:

17 hours per day; 5 days per week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.2, 0.7, 2.1 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

80 females per dose; at each dose level there were additional 80 rats per group in satellite groups for : chronic toxicity over 12 months (20 animals); lung function over 20 months (12 animals); lung clearance over 20 months (8 animals); bronchoalveolar lavage, biochemistry over 3 months +1 week recovery (20 animals); bronchoalveolar lavage, biochemistry over 12 months + 1 week recovery (20 animals).

Control animals:

yes, sham-exposed

Examinations

Observations and examinations performed and frequency:

-Clinical signs: all animals were observed for clinical signs at least once a day; if clinical signs were present, the animals were further examined; animals in bad condition were killed and organs put in formalin.
-Body weight: in the groups for chronic toxicity and carcinogenicity testing during the first 14 days: individual weights were measured once a week; thereafter: measurements took place once per 2-weeks.
-Food consumption: for the groups of the carcinogenicity test: individual records are made for 10 animals/group weekly during the first 15 weeks; thereafter: once in 3 months till testing day 482.
- Hematology/clinical-biochemical analysis: performed after 6 and 12 months exposure (satellite groups for chronic toxicity after 12 months) and after 20 months exposure (satellite groups for lung function and clearance testing); blood for hematological and clinical/biochemical analysis are collected after 16h fasting by puncture of the retroorbital veneplexus and treated with K-EDTA (hematology) resp. Li-Heparinat (clinical chemistry); immunglobulines are gathered from the serum.
- Urinalysis: is included

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY:
- Macroscopic examination: full pathological examination is done on the surviving rats of the chronic tox test killed at 12 months exposure (satellite groups) and at 12 months resp. 24 months (animals with number 101-120 resp. 1-80) of the carcinogen test.
Following organs are preserved in 10 % neutral buffered formalin solution: all organs/tissues that are macroscopically changed, brains, pituitary, thyroid, thymus, larynx and laryngopharynx, trachea, lungs, heart, aorta, pancreas, liver, kidney, adrenals, periferal nerve, sternum, femur and knee, vertebrae, tongue, lymph nodes (submandibular and mesenteric), mediastinal lymph nodes, nose, sinus, eyes/Harderian glands; lacrimal glands
(extraorbitale), ovaries, uterus and vagina, mammary, skin, oesophgus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, urinary
bladder, muscles, pancreas, mesenterium.
Lungs (incl trachea), under +/- 20 cm water pressure, are preserved in formaline solution.
- Organ weights: are performed on the animals of the satellite group used for chronic tox test after 12 months of exposure: in 10 animals/ group: fresh weights of brain, liver, kidneys and adrenals and ovaries. Also the relative organ weights are calculated (vs. the body weight at the end of the test). This examination was not performed in rats after 24 months of testing due to increased mortality and the number of surviving animals being too limited to allow any firm conclusions to be drawn.
In the satellite groups used to examine BAL (10 animals/ group) at the end of the exposure time as well as on the remaining 10 animals/group after recovery (=after 20 months: in surviving animals of the 20 animals/group at end of the test) terminal body weights and fresh weight on lungs (incl
trachea) as well as the relative lung weight are calculated.
- Microscopic examination (light microscopy) was done for all animals of the control group and the high dose group of the carcinogenicity test and the chronic tox after 12 months, on above tissues/organs after haematoxylin-eosin staining (Lilly-Meyer).
In case of substance related pathological findings found in these groups, all corresponding organs (respiratory tract) of all other animals of low
and mid-dose groups are examined. Moreover all organs with tumor-like or similar modifications were histologically examined.
Peer review of the lung examinations (review examination by an external pathologist by Prof. Dr. D.L. Dungworth, University of California, Davis, USA.Data record and statistical treatment of the pathological findings was done using the PLACES program.

Other examinations:

- Lung function: on rats under narcosis, with non-invasive method (cfr Heinrich et al. 1989, Hoyman et al. 1994). After 6, 12 and 17 months identical tests were done on the same rats (of the satellite groups).
a) Whole-body plethysmography and parameter on spontaneous breathing.
b) Forced Expiration
c) Lung volume and elasticity
d) N-exchange test: homogenity of ventilation
e) CO-diffusion test: diffusion

Bronchoalveolar Lavage (BAL)
a) Biochemical and cytological parameter of lung lavage
b) Measurement of surface tension
Lung clearance

Investigations on MDI-metabolism: in blood and urine.

Statistics:

Differences between test and control groups are judged statistically significant at level p<0.05. Body weight and food consumption, absolute and relative organ weight and hematological/biochemical data, BAL, clearance and lungfunction data are checked for difference between groups by variance analysis. If statistical difference was found between group means, the mean of the test group was compared to the mean of the control by t-test (lung function) or adapted t-test (Dunnett-test). The Wilcoxon test was used for surfactant data. Qualitative and semi-quantitative data (histopathology) are analysed by Fisher-test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

- Mortality: decreased survival time was seen in all groups (including controls). This was due to the earlier onset of age related changes e.g. tumors of pituitary and mammary gland. The cause of this finding could not be foreseen at the start of the test nor can it be clarified.
In the carcinogenicity test:
No significant differences occurred between the test groups and the controls. After 17-18 months exposure (i.e. 19-20 months age) cumulative mortality was 50 %. Compared to internal and external historical data (1984-1988) on the same rat species, this represents a real decrease in survival time. After 17 months of exposure the weight differences from low, mid and high dose groups compared to controls were -6.7 %; -7.9 % and -11.3 %. However it should be noted that at day 0 the weights of mid and high dose group were 2.4 and 2.2 % lower.

- Body weight: since 4.5 months of testing, the mean weight of the animals in the mid- and low dose groups were significantly decreased compared to the control group.

- Food/water consumption: mean weekly food consumption data of 10 animals were calculated over the time delay 0 - 482 days. No significant difference occurred between test and controls. However slightly decreased food consumption was seen that in general was correlated with a lower body weight.

- Clinical chemistry: during the test no abnormal findings could be detected between the groups, so no substance specific effects can be derived.

- Haematology: even if isolated statistically significant differences were seen between animals of the groups, substance related changes could not be found.

- Urinalysis: cfr MDI adducts

- Organ weights: Lungs: relative fresh weights (normalised to body weight) for lungs are increased after 3, 12 and 20 months exposure. After 3 months: significantly increased weights in all test groups. After 12 and 20 months these differences are only present at the highest dose group. After 1 week recovery (clean air) following 3 months exposure, a recovery effect is seen in the low and mid dose. However, in the high dose group animals the lung weight remains sign increased. Histopathological changes corroborate with this finding. Other organs: no significant difference are seen between the test and control groups

- Gross pathology: with exception of the changes as described under histopathological changes, no substance related changes could be found

- Histopathology:
I. After 12 months of exposure (satellite-groups):

Neoplastic changes: statistically not significant increase in the number of tumor-bearing animals in the high dose group compared to control (resp. 11/15 vs 8/18). The total number of tumors was 16 in the high dose groups, 11 in the control. The number of malign tumors (n=2) was the same in both groups. Benign neoplasms in the high dose group: due to adenomas of the pituitary (9 vs 1) and fibroadenomas of the mammary gland. Other tumors occurred sporadically in different organs. In the mid and low dose groups, the frequency of tumors was lower than in the high dose group.
Non-neoplastic changes: Exposure related pathological changes were only found in the nose, lungs and lung associated lymph nodes (LALN).

Nose: Very low to low graded (multi)focal degeneration of the olfactory epithelium: in 5/15 animals of the high dose group; in 1/19 animals of the mid dose group. These changes were absent in the low and control group. Statistically different were control and high dose group. Other changes were seen but these were not statistically significant from the controls. After 12 months MDI exposure: MDA-DNA adducts were found in olfactory nose epithelium, however only in marginal amount.
Remark: The proof of MDA-DNA adducts is possibly feigned by the strong protein binding. The toxicological relevance of this finding is doubtful since MDI leads only in high concentrations to degeneration of the olfactory epithelium (Greim H (ed.) 2008, in: Occupational Toxicants - Critical data evaluation for MAK values and classification of carcinogens, Wiley-VCH, Weinheim, Vol. 14).

Lungs: Statistically significant multifocal to diffuse interstitial (septal) fibrosis in all exposure groups. Slight to moderate interstitial fibrosis in mid and high dose group: present in resp. 18/19 animals and 15/15 animals (diff. not statistically significant). In the low dose group: 6/19. Moderate (multi)focal bronchiole-alveolar hyperplasia: higher frequency in mid and high dose groups. Focal alveolar hyperplasia (Type II cells especially): only in exposed groups (1 animal in low and in mid dose; 3 in the high dose). Not significant different but presumably related to exposure. Alveolar accumulation of macrophages with inclusion of particles in low amount and dose related frequency: only present in groups exposed to the test substance (statistically different compared to control: low dose: 8/19; mid: 16/19 and high dose: 15/15 animals). Epithelium associated giant cells of Langhans: difference very significant in mid and high dose groups. Low to moderate interstitial mononuclear cell infiltration in control to high dose animals: resp. 2/18; 5/19; 18/19 and 13/15. In the BAL there were after 3 and 12 months in the highest dose; increased macrophages, lymphocytes numbers; after 20 months increased number of lymphocytes. At no point in time was there a change in the number of granulocytes.

Lung associated lymph nodes (LALN):
Exposure related multifocal accumulation of particle bearing macrophages: in the mid (16/19) and high (6/14) dose group (statistically different
from control). Slight reactive hyperplasia of the lymphoid tissue associated with macrophage accumulation: dose dependent increase in incidence.

Other organs:
Exposure related changes could not be detected.

- Histopathology:
II. After 24 months of exposure (carcinogenicity test):

Neoplastic changes: Identical number of tumor bearing animals in highest dose vs control group (both 77/80). Total number of tumors: low dose: 195; control: 183. Number of malign tumors in the high dose group is slightly higher than in the control (n=39 vs n=36). Number of benign tumors clearly lower in high dose group. The number of animals with metastasis tumors was twice high in controls than in high dose. A similar number of animals with multiple tumors were seen in both groups (61/80; 60/80).

Lungs: A dose related neoplastic effect was only seen in the lungs. In 1 animal of the high dose group: bronchiole-alveolar adenoma built of
dyspastic alveolar cells (type II pneumocytes). Further: dose dependent (multi)focal high grade dysplastic alveolar hyperplasia.

Pituitary: Single and multiple pituitary adenomas are seen in control and high dose group: 63/80 and 67/80. Carcinomas of adenohypophysis were seen in resp 69/80 and 70/80 animals. These frequencies are clearly above the historical controls of the same strain (where figures for tumor of
pituitary are: 57 % at age of 25 months; 70 % at age of 30 months). The extreme high incidence of tumors of the pituitary and the related clinical
symptoms (decreased food intake; anomalies in mobility etc) are mainly due to the high mortality in this study.

Mammary tumors: No significant difference incidence between controls and high dose group (adenocarcinoma, fibroadenoma; adenoma and
fibroma).
Significantly higher frequency as in historical control data.

Numerous other tumors observed in several organs could not be attributed to exposure related effect. Incidence and type of these tumors are
characteristic for rats of the used strain and age.

Non-neoplastic changes: Exposure related changes could only be found in the nose, larynx, lungs and lung-associated lymph nodes.

Nose (only examined in control and high dose group): (Multi)focal, in general moderate squamous metaplasia, mainly in the proximity of the olfactory epithelium (in high dose significantly higher than in control: 16/80 vs 5/80). (Multi)focal generally moderate Becker cell hyperplasia (50/80 vs 33/80) and inflammatory cell infiltration of the mucosa (29/80 vs 10/80). Other changes, non significant but obviously dose related were: metaplasia of the respiratory epithelium, degeneration, erosion, respiratory and/or olfactory epithelium.

Larynx (only examined in controls and high dose group): Slight multi(focal) squamous metaplasia significantly higher (13/79 vs 1/80). Focal hyperkeratosis (in the area of the epiglottis) and inflammatory infiltration of the mucosa (however non significant).

Lungs:
Alveolar cell hyperplasia: in frequency and severity significant difference between mid and high dose compared to controls. In the following incidences and severity are described for the 3 dose groups (number of animals with grade of the effect: very slight, slight, moderate, high; total animals displaying these changes):
Low dose: 1/80; 4/80; 2/80; 1/80; 8/80
Mid dose: 0/80; 5/80; 5/80; 2/80; 12/80
High dose: 0/80; 6/80; 8/80; 7/80; 21/80

Alveolar bronchiolisation:
(Multi)focal bronchiole-alveolar hyperplasia: is significantly higher in mid and high dose group (frequency in low; mid, high dose and control: 3/80; 14/80; 41/80; 3/80). The grading of this finding appeared to be dose related. The moderate and high grade hyperplasia only occurred in resp 5 and 2 animals of the high dose exclusively.

Interstitial and peribronchiolar fibrosis:
In all MDI exposed groups: statistically highly (p<0.001) significant compared to control (low, mid, high dose; control: 51/80; 73/80; 77/80; 4/80. Also the severity was significant difference in the different exposure groups: generally very slight (minimal) in low dose; mainly slight and slight to moderate in the high dose group.

Other statistically significant dose dependent effects in lungs:
Focal to multifocal alveolar accumulations of particle-laden (MDI?) macrophages: in very slight to moderate grade in all exposure groups: 52/80; 70/80 and 78/80 (highly sign diff with controls). Identity of the inclusion could not be defined via light microscopy. In BAL: after 3 and 12 months of exposure increased number of macrophages and lymphocytes were seen; after 20 months only increased number of lymphocytes.

Interstitial mononuclear cell infiltration (mainly low grade):
Statistically significant in all exposure groups: number of animals with this finding in resp low; mid, high dose and controls were: 24/80; 48/80; 73/80 and 11/80. Accumulation of hemosiderin pigmented macrophages: from low to high grade dose dependent significantly increased in all exposure groups compared to controls: numbers for low, mid, high dose and control: 6/80; 9/80; 14/80 and 0/80. Small focal to multifocal cholesterol granulomas: in the high dose group: 11/80 vs 0/80 in controls. In the other groups: 4/80 low dose and 1/80 in the mid dose group.

Focal osseous metaplasias:
Incidence: significantly higher in high dose group vs control (resp. 11/80 and 1/80). In the low and mid dose group resp: 6/80 and 4/80.

Lung associated lymph nodes (LALN; only examined in control and high dose group):
Accumulation of macrophages with cytoplasmatic inclusions were seen in 68/80 high dose animals (highly significant differences with control were no such changes were observed). In addition, slight to moderate reactive lymphoid hyperplasia was seen, more frequent in high dose (13/80 vs control 6/80).

Other organs: Exposure related changes could not be detected.

- Lung function tests:
1. Significant increased flow resistance in the small, peripheral air tracts in highest dose after 6 months. After 12 and 17 months also detected in the mid and low dose detected (cfr FEV0.1; FEF50 and FEF25).
2. Significantly reduced vital to total lung volume and elasticity of the lung tissue in the high dose already after 6 months (restrictive lung changes). After 12 resp 17 months increased incidence and finally also in the mid dose group and marginally in the low dose group.
3. Positive N-exchange test (indication of increased non-homogenity of the alveolar respiration) after 17 months in the mid and more expressed in the high dose group (already as a trend to be seen after 12 months).
4. Positive CO-diffusion test after 12 and 17 months : particularly in the high dose, less in the mid and marginally in the low dose group (indicating impairment of the diffusion through the alveolar-capillary membrane.

BAL findings: Changes in biochemical lavage parameters (INCREASED LACTATE DEHYDROGENASE, BETA-GLUCURONIDASE, TOTAL PROTEIN, GAMMA-GLUTAMYL TRANSFERASE, HYDROXYPROLINE CONCENTRATION, PHOSPHOLIPID CONCENTRATION; indications of damage to the cell membrane vessel endothelium, cell necrosis, increased collagen metabolism) occurred generally already after 3 months exposure and increased after 12 and 20 months. After 1 week recovery with clean air, these findings seemed partially reversible.

Increased concentration of surfactant-phospholipid were found in the mid and high dose groups. Functionally: a slight decrease in 'specific' surface activity of the phospholipid standardised surfact sample is observed in the high dose group (increased surface tension as measured by surfactometer).

Increased lymphocyte concentration was seen after 3, 12 and 20 months (partially reversible after 1 week recovery with clean air). Increased numberof macrophages after 3 months.

The increased lung weights especially in the high dose group were still increased after 1 week recovery. This indicates chronic lung changes that were confirmed by the histopathological findings.

Examination of the lung clearance (alveolar lung wash): After 6 months in the high dose group nearly doubled clearance half time compared to control. After 18 months this effect was not detectable anymore.

Examination of blood and urine:
Hemoglobin adducts and MDA urine concentrations were found in all MDI groups after 3 and 12 months exposure. A steady-state was observed after 3 months exposure.

Effect levels

Dose descriptor:

LOAEC

Effect level:

0.23 mg/m³ air

Sex:

female

Basis for effect level:

other: exposure: 17 hours/day

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Executive summary:

Of the authors:

In a long-term inhalation study over a maximum of 24 months including satellite groups with 3, 12, and 20-month exposure, the chronic toxicity and carcinogenicity of monomeric methylene diphenyl diisocyanate (MDI) were investigated. Female Wistar rats were exposed in 6 m3 inhalation chambers for 17 hours/day, 5 days/week to 0.23, 0.70 and 2.05 mg/m3 MDI in aerosol form, a control group was kept in clean air. Essentially, a dose-dependent impairment of the lung function in the sense of an obstructive-restrictive malfunction with diffusion disorder, increased lung weights, an inflammatory reaction with increased appearance of lymphocytes (but not of granulocytes) in the lung in the high dose group as a sign of specific stimulation of the immune system by MDI, an intermediately retarded lung clearance in the high dose group as well as dose-dependent interstitial and peribronchiolar fibrosis, alveolar bronchiolisations and a proliferation of the alveolar epithelium, which was classified as preneoplastic, as well as a bronchiolo-alveolar adenoma were ascertained. The LOAEC for the female rat was 0.23 mg/m3 after long-term inhalation of 4,4'-MDI aerosols.

Remark: In a chronic rat study with polymeric MDI the NOAEC was 0.2 mg/m3 after aerosol exposure over 2 years (6 hours/day, 5 days/week) (Reuzel et al. (1994), Fund. Appl. Toxicol. 22: 195-210). A comparison of the both chronic rat studies with 4,4'-MDI and polymeric MDI has shown very good consistency across the studies with respect to gradation of inhaled dose and the observed histopathological changes. In this review it turned out that a daily 17 hr-exposure of 0.23 mg/m3 4,4'-MDI corresponds approximately to a daily 6 hr-exposure of 1 mg/m3 polymeric MDI (Feron et al. (2001), Arch. Toxicol. 75: 159-175).