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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
chronic toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 1985 - June 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment.
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990
Reference Type:
publication
Title:
Unnamed
Year:
1994

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
yes
Remarks:
- some omitted exposures; clinical chemistry, urinalysis and haematological samples only taken at the end of the study
Principles of method if other than guideline:
Details of deviations:
Omitted exposures due to public holidays or to technical maintenance or repair of inhalation equipment there were no exposures during 21 days. (day 45, 100, 119, 197-198, 204, 290, 293, 323, 326, 331, 342,562-563, 569, 675, 678, 688, 692-693, 716, 727).
Clinical chemistry, urinalysis samples and haematological examination samples only taken at the end of the study (1 yr satellite group, 2 yr main group).
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
SOURCE: polymeric methylene diphenyl diisocyanate (pMDI) was supplied by Bayer AG, Leverkusen.

PURITY: 8 different batches were used, each of which was analyzed by the supplier and fulfilled the following specifications: monomeric MDI 44.8-50.2 % (w/w), NCO-content 31.0-31.7 % (w/w), hydrolysable chlorine 0.06-0.12 % (w/w), total chlorine 0.20-0.37 % (w/w), chlorobenzenes 0.0001-0.0069 % (w/w), phenyl isocyanate 0.003-0.005 % (w/w), sediment content<0.01 % (w/w). Average molecular weight ca. 400, content mol. wt <500 ca. 70%, content mol. wt <1,000 ca. 90 %. An aerosol with 95 % of the particles at < 5 micrometres. The viscosity ranged from 195 to 265 mP.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- AGE: at the start of the study the animals were approximately 6 weeks old.
- WEIGHT AT STUDY INITIATION: mean weight of male rats was 179 g and of female rats 141 g. The rats were weighted just prior to the start of the study and then weekly during the first 13 weeks and every 4 weeks afterwards. At the end of the exposure time, surviving rats were killed and weighed at the day of scheduled autopsy.
- NUMBER OF ANIMALS: 280 males and 280 females randomly allocated to four groups. Each group, composed of 70 males and 70 females. Each group was subdivided into one satellite group of 10 rats/sex and a main group of 60 rats/sex.
- STRAIN: Cpb:WU Wistar

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: The mean mass median aerodynamic particle size during the study was 0.68, 0.70 and 0.74 µm with geometric standard deviation of 2.93, 2.46 and 2.31 at the level of 0.2, 1.0 and 6.0 mg/m3, respectively. On average the aerodynamic diameter was for at least 93.5 % of the particles smaller than 4.2 µm.
Details on inhalation exposure:
- EXPOSURE CHAMBERS-EXPOSURE CONDITIONS: Animals were exposed to the test atmospheres in H1000 multitiered inhalation chambers (capacity about 2.3 m3) manufactured by Hazleton Systems, Inc.. During exposure the rats were housed individually in wire mesh stainless-steel cages. Total airflow through the chambers was on average between 35 and 45 m3/hr depending on the volume of air needed to reach the required concentration of polymeric MDI in the atmosphere. The temperature and relative humidity in the chambers were generally between 20 and 25°C and between 40 and 70 %, respectively. Except for exposure to polymeric MDI, control rats were treated similarly to test animals including housing in an H1000 multitiered inhalation chamber.
- GENERATION OF TEST ATMOSPHERES: Test atmospheres were generated by atomizing polymeric MDI liquid into droplets by using compressed air in a nebulizer designed by TNO. The nebulizer consisted of an atomizer and a glass jar. The atomizer coded DR 0 11 was purchased from Lechler (Germany). Before use, the nozzle of the atomizer was slightly modified by reducing empirically the internal diameter of the nozzle orifice. The nebulizer was operated at a pressure of approximately 2.5 bar. A baffle was fitted approximately 4 cm below the nozzle orifice to remove the larger droplets from the spray The smaller droplets followed the upward airflow and were emitted through the outlet port and passed through a cyclone with a diameter of 9 cm. The remaining particles larger than 5 µm were impinged onto the wall of the cyclone to obtain an aerosol of which 95% of the particles were smaller than 5 µm. The aerosol was then passed through a manifold pipe system constructed of polyvinyl chloride tubing, having a length of approximately 10 in and an internal diameter of 4.5 cm. Air-operated vacuum pumps (air movers, AIRNAC, Milford, CT; TD series, 110 size) adjacent to the top of the respective inhalation chambers moved polymeric MDI aerosol from the delivery system to the inlet of the inhalation chamber, where it was diluted in air from the main air supply of the inhalation chamber. By varying the operating air pressure to the vacuum pump the amount of withdrawn aerosol could be adjusted to the desired concentration of test material within the chamber.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- ANALYSIS OF POLYMERIC MDI IN THE TEST ATMOSPHERES: Gravimetry was used as primary standard for assessing the polymeric MDI aerosol concentration in the test atmospheres. Test atmosphere samples were collected using closed filter-type collectors provided with glass fiber filters (Sartorius SM 13430; diameter, 44 mm). The filters were weighed before and after sampling. From the increase in weight and the volume of test atmosphere drawn through the filter the time-weighted average concentration of polymeric MDI was calculated. Beta attenuation using attenuators from Verewa (Germany) were used in parallel with gravimetry during the first 3 months of the study, and the results were compared with those obtained from gravimetry. In view of the proven reliability of beta attenuation during this period gravimetric determinations were carried out only once every 2 weeks thereafter. The particle size distribution in each of the test atmospheres was determined at weekly intervals using a 10-stage Berkeley quartz crystal microbalance cascade impactor.
Duration of treatment / exposure:
main groups: 2 years; satellite groups: 1 year
Frequency of treatment:
6 hours/day; 5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 0.2, 1.0, 6.0 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 0.19, 0.98, 6.03 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
60 (main groups); 10 (satellite groups)
Control animals:
yes, sham-exposed

Examinations

Observations and examinations performed and frequency:
- HEMATOLOGICAL PARAMETERS (red and white blood cell counts, hemoglobin, packed cell volume, differential white blood cell count, prothrombin time) and urinary parameters (appearance, volume, density, pH, protein, occult blood, glucose, ketones, microscopy of the sediment) were measured in all rats of the satellite groups in week 52.
- BIOCHEMICAL BLOOD PARAMETERS (albumin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, urea nitrogen, total protein, creatinine, total bilirubin, Ca, K, Na, inorganic phosphate, cholesterol, triglycerides, and glucose) were measured in blood samples taken at euthanization from all rats of the satellite groups, except for glucose, which was determined in blood samples after overnight fasting in week 52.
Sacrifice and pathology:
- ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): All rats of the satellite groups were randomly killed on two successive days in week 53, and all survivors of the main groups were randomly euthanized on seven successive working days in weeks 105 and 106 by
exsanguination from the abdominal aorta under ether anesthesia, autopsied, and examined for gross pathological changes. Adrenals, brain,
heart, kidneys, Iiver, Iungs with mediastinal lymph nodes, trachea and larynx, spleen and testes of all rats of the satellite groups and of all survivors of the main groups were weighed. A wide range of organs or samples of organs or tissues were preserved in an aqueous neutral
phosphate-buffered 4% formaldehyde solution. The lungs were fixed by intratracheal infusion with the main groups, nose, lungs, mediastinal
lymph nodes, and all gross lesions fixative under 10 cm water pressure. The urinary bladder was fixed by infusion of the fixative through the
bladder wall in the neck of the bladder. The nose was fixed by infusion of the fixative through the pharyngeal duct. In the 1-year study (satellite
groups) histopathological examination was carried out of kidneys, liver, nose, larynx, trachea with bronchi, lungs, mediastinal lymph nodes, and
all gross lesions all rats. In the 2-year study (main groups) 43 different organs (**) or tissues and all grossly visible lesions were examined by light microscopy of the control and high-concentration animals and of the low- and mid-concentration decedents. Moreover, in the low- and
mid-concentration survivors of the main groups, nose, lungs, mediastinal lymph nodes, and all gross lesions were subjected to histopathologicaI examination.

For transmission electron microscopic studies from selected paraffin blocks of the 13-week study, (Reuzel el aL, 1994), and the 1- and 2-year
studies, a portion of the block containing lung tissue was trimmed away and, deparaffinized in several changes of xylene over a 24-hr period. The
tissue was then rehydrated, and postfixed with 1% osmium Tetroxide in phosphate buffer, dehydrated using a graded series of alcohol, and then
infiltrated and embedded in Epon/Araldite. After polymerization overnight in a vacuum oven, sections were cut with a diamond knife, placed in of
copper grids, and stained with lead citrate and uranyl acetate. The sections were then examined and photomicrographs were taken using a Zeiss
EM-10 electron microscope.

** (Adrenals, aorta, axillary lymph nodes, brain-brainstem, cerebrum and cerebellum, caecum, coagulating glands, colon, epididymides, exorbital lachrymal glands, eyes, femur with joint and bone marrow, Harderian glands, heart, kidneys, larynx, liver, lungs, mammary glands, mediastinal lymph nodes, mesenteric lymph nodes, nerve-peripheral, nose oeasophagus, ovaries, pancreas, parathyroids, parotid salivary glands, pharynx, pituitary, preputial/clitoral glands, prostate, rectum, seminal vesicles, skeletal muscle, skin/subcutis-flank, small intestines (duodenum, ileum, jejunum), spinal cord, spleen, sternum with bone marrow, stoamch, sublingual salivary glands, submaxillary salivary glands, testes, thymus, thyroid, trachea/bronchi, urinary bladder, uterus, Zymbal glands)
Statistics:
Body weights were analyzed by an analysis of covariance (Cochran, 1957) followed by the Dunnett's multiple comparison test (Dunnett, 1955). Analysis of variance (Steel and Torrie, 1960) followed by the Dunnett's multiple comparison test was applied to hematological, biochemical, and organ weight data. Differential white blood cell count data were analyzed by the Mann-Whitney U test (Siegel, 1956a). Incidences of histopathological changes and numbers of deaths were analyzed by the Fisher exact probability test (Siegel, 1956b).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
0.2 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEC
Effect level:
0.19 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
LOAEC
Effect level:
1 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
LOAEC
Effect level:
0.98 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Mortality incidences in males were comparable in all groups. Female rats showed negatively concentration-related mortality incidences. The number of animals with palpable masses did not differ between test and control animals. No treatment-related differences in body weights were observed between control and test groups.

Hematological examination of rats at day 357-358 revealed no exposure-related differences between the groups. Biochemical examination performed on days 360, 366 and/or 367 was essentially negative. No alterations were observed from parameters measured in urine of rats exposed to polymeric MDI aerosol for 360 days.

Lung weights were statistically significantly increased in both males and females exposed to 6.0 mg/m³ for 12 or 24 months as summarized in the following table 1:

Table 1: Mean absolute lung weights and lung-to-body weight ratios in rats exposed by inhalation to polymeric MDI aerosol for 1 year (satellite groups) or for up to 2 years (main groups)

Polymeric MDI (mg/m3)

Satellite groups (a)

Main groups (b)

Absolute (g)

Relative (g/kg)

Absolute (g)

Relative (g/kg)

Males

0

0.2

1.0

6.0

2.30 ± 0.15

2.32 ± 0.09

2.44 ± 0.10

2.60 ± 0.06

4.29 ± 0.16

4.48 ± 0.22

4.48 ± 0.16

5.06 ± 0.08**

2.90 ± 0.06

2.88 ± 0.10

2.88 ± 0.08

3.28 ± 0.07**

5.04 ± 0.14

5.00 ± 0.15

5.19 ± 0.24

5.80 ± 0.16**

Females

0

0.2

1.0

6.0

1.67 ± 0.07

1.66 ± 0.05

1.69 ± 0.06

1.93 ± 0.09*

5.66 ± 0.29

5.63 ± 0.15

5.61 ± 0.21

6.51 ± 0.22*

2.10 ± 0.06

2.09 ± 0.04

2.06 ± 0.04

2.41 ± 0.05**

6.14 ± 0.28

5.87 ± 0.09

5.68 ± 0.11

6.68 ± 0.17

(a) Number of lungs weighed/sex/group was 10
(b) Number of lungs weighed/sex/group varied from 35 to 50

* p<0.05; ** p<0.01

No treatment-related gross changes were found in animals exposed for 12 months. Gross examination of animals exposed for 24 months revealed increased incidence of lungs with spotted surface and/or discoloured appearance in male rats exposed to 6.0 mg/m3.

Histopathology (main groups exposed over 2 years):

Nose: increased incidence of rats with a higher degree of basal cell hyperplasia frequently accompanied by hyperplasia of Bowman's glands in the olfactory epithelium in the nose at levels of 1.0 and 6.0 mg/m³. At 6.0 mg/m³ basal cell hyperplasia occurred in 32/60 males and 49/60 females against 14/60 and 4/60 for the corresponding controls.

Lungs: incidences of non-neoplastic findings and tumors are summarized in the following table 2:

Table 2: Number of rats with non-neoplastic findings and tumors in lungs after 2-year exposure to polymeric MDI (main groups)

 Polymeric MDI (mg/m3)    0  0.2  1.0  6.0

Animal number

M 60   60 60 60  

60  60 60  60

Surviving animals

M 38 38 42 36
F 41 42 48 50

- Macrophages with yellow pigment

M 0 3 21** 60**
F 0 1 23** 59** 

- Localized fibrosis

M 1 0 9* 44**
0 0 4 48**

- Alveolar duct epithelialization 

M 1 0 8* 54**
F 0 0 8* 57*

- Localized alveolar bronchiolization

1 12** 
  F 14** 

- Mineralized deposits in the bronchial and alveolar region 

0 1 13** 
  24** 

- Pneumonitis 

13 13   17 28 
  F 3

- Adenoma 

0 6* 
 

- Adenocarcinoma 

  F

0

0

* p<0.05; ** p<0.01

Mediastinal lymph nodes: increased incidence of rats with an accumulation of macrophages with yellow pigment at levels of 1.0 and 6.0 mg/m³. At 6.0 mg/m³ this finding occurred in 50/60 males and 43/60 females against 0/60 for the corresponding controls.

Other organs: the incidence and distribution of other tumour types was not affected by treatment.

Histopathology (satellite groups exposed over 1 year):

Rats killed after 1 year of exposure had treatment-related histopathological changes in the nasal cavity, lungs, and mediastinal lymph nodes starting at 1.0 mg/m3, but to a lower degree of severity compared to animals exposed over 2 years. There was no microscopic evidence of lung tumors or any other tumors following exposure to polymeric MDI for 1 year.

Applicant's summary and conclusion

Executive summary:

In a combined chronic toxicity and carcinogenicity study rats were exposed for 6 hours/day, 5 days/week for 1 (satellite groups) or 2 years (main groups) to polymeric MDI aerosol concentrations of 0, 0.2, 1.0 or 6.0 mg/m3. The effect of chronic exposure of rats to respirable polymeric MDI aerosol was confined to the respiratory tract. The compound-related changes were found in the nasal cavity, the lungs and the mediastinal lymph nodes, and to some degree they were already present after 1 year of exposure. Histopathology of the organs/tissues investigated showed that exposure to 6.0 mg/m³ over 2 years was related to the occurence of pulmonary tumors in males (6 adenomas and 1 adenocarcinoma) and females (2 adenomas).

In this 2-year rat study the NOAEC was 0.2 mg/m3 for the repeated dose toxicity of polymeric MDI. The LOAEC was set on 1.0 mg/m3.