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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Remarks:
5-day range-finding study for 28-day study 40I0338/18I093
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
No testing guidelines exist for this type of study.

The conduct of inhalation exposures was performed according to the following test guidelines concerning repeated dose inhalation toxicity studies:

• Organization for Economic Cooperation and Development (OECD), OECD Guidelines on the testing of chemicals, Section 4: Health Effects, No. 412, “28-day (subacute) Inhalation Toxicity Study” adopted 25 June 2018.

• COMMISSION REGULATION (EU) No 260/2014 of 24 January 2014 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Appendix B.8.: Subacute inhalation toxicity: 28-day study; Official Journal of the European Union, No. L 81
GLP compliance:
no
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1'-Methylenebis(4-isocyanatobenzene) and oligomeric reaction products of 1,1'-methylenebis(4-isocyanatobenzene) and oxydipropanol and oligomerization reaction products of oxydipropanol
EC Number:
701-041-3
Molecular formula:
C8H6NO [C7H5NO [C3H6O]n C8H7NO ]m C7H4NO with 2<=n<20 and 0<=m<=3
IUPAC Name:
1,1'-Methylenebis(4-isocyanatobenzene) and oligomeric reaction products of 1,1'-methylenebis(4-isocyanatobenzene) and oxydipropanol and oligomerization reaction products of oxydipropanol
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: about 7 weeks
- Identification: The animals were identified individually by tattooing the respective animal number into the ears

Only animals free from clinical signs of disease were used for the study.

The rats were housed together (up to 5 animals per cage) in Typ 2000P ca. 2065 cm2 (polysulfone cages) supplied by TECNIPLAST, Germany. Bedding in the Polycarbonate cages were dust-free bedding. Dust-free wooden bedding was used in this study. For enrichment wooden gnawing blocks (Typ NGM E-022), supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria and Play Tunnel, large (Art. 14153); PLEXX b.v., Elst, Netherlands were added. Wooden gnawing blocks (SAFE® block large) J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany. The animals were kept in fully air conditioned rooms in which a temperature in the range of 20 24°C and relative humidity in the range of 45 – 65 % relative humidity was guaranteed and supervised, were ensured by means of a central air conditioning system. There were 15 air changes per hour.

A light/dark rhythm of 12 hours was maintained:

• 06.00 a.m. 06.00 p.m. light
• 06.00 p.m. 06.00 a.m. dark

Deviations from these ranges did not occur.

The room was completely disinfected using a disinfector (Geschko MLT 17 hydrogen peroxide gas generator (PEA; Germany)) before the start of the study. The floor and the walls were cleaned once a week with water containing an appropriate disinfectant.
The animals were maintained on mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland and tap water ad libitum.
During urine collection water was withdrawn.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 2.02 - <= 2.22 µm
Remarks on MMAD:
Determined by cascade impactor measurement
GSD 1.74-2.01 um
Details on inhalation exposure:
GENERATION OF THE INHALATION ATMOSPHERES:
Generator systems:

• Continuous infusion pumps PHD Ultra (Harvard Apparatus, Inc., Holliston, Massachusetts, U.S.A.)
• Two-component atomizers (stainless steel, Model 970; Düsen-Schlick GmbH, Untersiemau/Coburg, Germany)
• Glass mixing stages (BASF SE, Ludwigshafen, Germany)
• Glass dilution tube (BASF SE, Ludwigshafen, Germany)
• Glass cyclonic separators (BASF SE, Ludwigshafen, Germany)
• Silicon insulated heating tape (230 V) (Winkler AG, Heidelberg, Germany), with controller (Messner, Germany)

Generation procedure:

For each concentration, a respective constant amount of the test substance was supplied to a tempered two component atomizer by means of a metering pump and sprayed with compressed air into a glass mixing stage. The so generated aerosol was mixed with dry air and passed after appropriate dilution into the inhalation system via a cyclonic separator. In order to reduce the viscosity of the test substance, silicon insulated heating tapes are loosely wrapped around the conducting tubes and the atomizers.

Due to the low target concentrations in the test groups 1 and 2, the aerosol generation is performed with only small differences in test substance flows. The exposure concentrations are adjusted after generation by diluting the aerosols (removal of a partial flow of the aerosol and replacement by dry compressed supply air) before entering the exposure systems.

The desired concentrations in test groups 1 and 2 was achieved by substituting appropriate amounts of aerosol by conditioned supply air.

The control group was exposed to conditioned air.

Because the test substance reacts with water in the air, the relative humidity was kept as low as possible by using compressed air. The range of the relative humidity was set to be between 0 to 20 % relative humidity. The chamber temperature were from 20 to 24 °C. The surface temperature of the nozzle was kept between 20 to 40 °C. The atomizer pressure was between 1.0 and 2.0 bar.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the inhalation atmospheres were determined by gravimetric measurement of filter samples. This analytical method is judged to be valid because the polymeric test substance does not possess an appreciable vapor pressure.

Daily means were calculated based on 3 measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived.

In all test groups, the constancy of aerosol concentrations in the inhalation systems was continuously monitored using scattered light photometers.

In the control group (test group 0) no sample was taken.
Prior to the study, an analytical study (project No. 02Y0338/18X243) was performed to assess whether the gravimetric measurement of the atmospheric concentration reflect the concentration determined by chemical analysis, and whether the monomeric 4,4’-MDI fraction in the atmosphere was comparable with those in the bulk material. According to the analytical work, gravimetric measurement of the filters reflected the chemical analysis. There was evidence of a trend for slight relative enrichment of the 4,4’-MDI portion of the test atmosphere as test substance concentration was reduced. This enrichment was hypothesized to be an effect of evaporation of 4,4’-MDI from the test substance liquid lining the walls of the atmosphere generation system. Overall, it was justified to use gravimetric method to determine atmospheric concentration and cascade impactor samples.


GRAVIMETRICAL METHOD OF ANALYESES
A preweighed filter was placed into the filtration equipment. By means of a vacuum compressed air pump a defined volume of the aerosol was drawn through the filter.

The dust concentration in mg/m³ was calculated from the difference between the weight of the preweighed filter and the weight of the filter after sampling, with reference to the sample volume of the inhalation atmosphere.



SAMPLING FOR GRAVIMETRIC ANALYSES
Equipment:
• Sampling equipment with probe (Millipore Corporation, Billerica, MA 01821, USA)
• Internal probe diameter: 7 mm
• Filter: MN 85/90 BF (d = 4.7 cm)
• Vacuum pump (Millipore Corporation, Billerica, MA 01821, USA)
• Balance: MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany)

Sampling:

• Sampling velocity: 1.25 m/s
• Flow rate of sampling: 3 L/min
• Sample volumes:
• Test group 1: 180 L
• Test group 2: 90 L
• Test group 3: 45 L
• Sampling site: immediately adjacent to the animals' noses at a separate spare port
• Sampling frequency: as a rule, two samples per exposure and concentration group
REAL TIME MONITORING OF CONSTANCY OF CONCENTRATIONS:
Scattered light photometers (VisGuard (Sigrist) in test groups 1-3 were used to continuously monitor the constancy of concentrations of test substance aerosols in the inhalation systems. To this end the inhalation atmosphere was continuously sampled by the measuring devices. The measurements were transferred to the automated measuring system.

PARTICLE SIZE ANALYSIS:
The particle size analysis was carried out with a cascade impactor.

Equipment for particle size analysis:

• Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA)
• Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA)
• Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA)
• Sampling probe internal diameter 7 mm
• Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany)

Sampling for particle size analyses:

Preweighed metal collecting discs and a backup particle filter were placed into the cascade impactor and two samples were taken in each concentration at a sampling velocity of 1.25 m/sec. from the breathing zones of the animals.
Method of analysis: Gravimetrical measurement

The amount of dust deposited by each stage in mg was calculated from the difference between the weight of metal collecting disc and backup filter before and after sampling.

Evaluation:

The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39).

PARTICLE SIZE DISTRIBUTION MEASUREMENTS WITH APS:
Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321.
Frequency: On two days during the exposure period, with 3 repeats on each day.
Duration of treatment / exposure:
6 hours
Frequency of treatment:
once daily for 5 consecutive days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/m³ air
Remarks:
Air control
Dose / conc.:
20.07 mg/m³ air (analytical)
Remarks:
Test group 3
Dose / conc.:
10.15 mg/m³ air (analytical)
Remarks:
Test group 2
Dose / conc.:
3.94 mg/m³ air (analytical)
Remarks:
Test group 1
Dose / conc.:
20 mg/m³ air (nominal)
Remarks:
Test group 3
Dose / conc.:
10 mg/m³ air (nominal)
Remarks:
Test group 2
Dose / conc.:
4 mg/m³ air (nominal)
Remarks:
Test group 1
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
Acclimatization and exposure period
The animals were delivered and subjected immediately to the acclimatization period (2 weeks) in which they were adapted to the surroundings.

Prior to the pre-exposure period, the animals were distributed according to weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex. The list of randomization instructions was compiled with a computer.

EXPOSURE SYSTEMS; EXPOSURE OF THE ANIMALS
Air conditions

Conditioned air:
The central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.

Compressed air:
Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage vessel of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
Exhaust air:
The exhaust air was filtered and released into the exhaust air of the building.

Nose-only exposure systems
The inhalation atmosphere was maintained inside aerodynamic exposure systems (INA 60, volume V ca. 90 L, BASF SE) consisting of a cylindrical inhalation chamber made of stainless-steel sheeting and cone shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol.

The exposure systems were located in exhaust hoods in an air-conditioned room.

Exposures
The nose-only exposure technique was preferably selected for this aerosol/dust/ inhalation study to minimize fur contamination of the animals with the substance, which cannot be avoided during whole body exposure. Fur contamination may lead to an additional dermal and oral uptake (animals preen as their fur becomes contaminated). Thus, an estimation of a nominal dose, taken up by the animals and its correlation to a toxic effect becomes more difficult.

Furthermore, by using the dynamic mode of operation with a low volume chamber, the equilibrium characteristic of this exposure technique is favorable: t99 (the time to reach 99% of the final target concentration) was about 4 min, considerably shorter as compared to whole body chambers with a higher chamber volume.

A positive pressure was maintained inside the exposure systems by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air.

In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on two days before start of exposure (pre-exposure period). Then all test groups were exposed for 6 hours on each workday over a time period suitable to reach 5 exposures.

For adaptation to the exposure conditions the male and female animals were placed into restraining tubes and exposed to conditioned clean air on study day -3, -2 and study day -1, before start of the exposure period (details are available in the raw data). This procedure is referred to as pre-exposure period. On the first day of the pre-flow period (day -3) animals were exposed to air for 2 hours, on the second day (day -2) for 4 hours. On the third day (day -1) they were exposed to air for 6 hours.

The animals did not have access to water or food during the exposure.

Measurements of the exposure conditions
Recording of exposure parameters was performed as follows (Exposure parameters - Determination method - Recording):

Supply air 1 (compressed), test group 0 - orifice plate with differential pressure measurement - automated system
Exhaust air 1, test groups 0, 2 and 3 - orifice plate with differential pressure measurement - automated system
Chamber relative humidity - Dielectric probe (Testo) - automated system
Chamber temperature - Thermosensor - automated system
Real time concentration surveillance - Scattered light photometers - automated system
Supply air 1 (compressed), test groups 1, 2 and 3 - Rotameter - Three times per exposure
Supply air 2 (compressed), test groups 1, 2 and 3 - Rotameter - Three times per exposure
Supply air 3 (compressed), test group 1 - Rotameter - Three times per exposure
Exhaust air 1, test group 1 - Rotameter - Three times per exposure
Exhaust air 2, test groups 1 and 2 - Rotameter - Three times per exposure
Atomizer Pressure - manometer - once per exposure
Generator temperature - Thermosensor - once per exposure
Pump rate - Reading from pump display - once per exposure

No surveillance of the oxygen content in the inhalation system was performed. The air change within the inhalation systems was judged to be sufficient to prevent oxygen depletion by the breathing of the animals and the concentrations of the test substance used could not have a substantial influence on oxygen partial pressure.

Principles of recording with the automated measuring system:
Each parameter was measured at appropriate measuring points using suitable measuring equipment (sensors, orifice plates etc.). The measurements were standardized (0-20 or 4-20 mA) and transferred to instrumentation consoles. There, the measured values were displayed in an analogous way (where this is provided for) and some were used as actual value for regulating the specific parameter.

In addition, the measured values were scanned every 10 seconds, converted from analog to digital, transferred to a personal computer, displayed on its screen, and saved on hard disk. The computer checked the arriving values against preset threshold values, displayed warnings if violations of thresholds occurred and recorded the start and the end of threshold violations for each measured parameter affected. After the end of each exposure all data gathered during this exposure were backed up on optical media.

Daily protocols were prepared from the recorded values using suitable software. The protocols include start and stop times of exposure and possible threshold violations, and daily means of each parameter. The records saved on optical media and the printed daily records are considered as raw data.

Examinations

Observations and examinations performed and frequency:
CLINICAL EXAMINATIONS
Mortality
The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays

Clinical observations
The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On exposure-free weekends and post exposure observation weekends, no clinical observation was performed. Signs and findings were recorded for each animal.
During exposure only a group wise examination was possible.

Body weight data
The animals were weighed prior to the pre-exposure period, at the start of the exposure period (day 0) and one day prior to gross necropsy. The body weight change was calculated as the difference of actual body weights and the weights on study day 0.


CLINICAL PATHOLOGY
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence (the list of randomization instructions was compiled with a computer).

The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.

The results of clinical pathology examinations were expressed in International System (SI) units.

The following parameters of the animals were examined:

Hematology
The following parameters were determined in blood with EDTA K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany) --> see section "Any other information on materials and methods incl. tables"

Clinical chemistry
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters (see section "any other information on materials and methods incl. tables").

BRONCHOALVEOLAR LAVAGE FLUID (BAL):
The animals designated for lung lavage were killed by exsanguination from aorta abdominalis and vena cava under Narcoren® anesthesia. The lung was lavaged by two instillations of physiologic saline.

The following examinations were carried out:

Cytology in BAL
Total cell counts were determined using a haematology analyzer (Advia 120 Siemens Diagnostics, Fernwald, Germany). Cytocentrifuge preparations were stained according to Wright and evaluated microscopically. For details on parameters and methods of cytological examination in BAL see section "any other information on materials and methods incl. tables".
Total Protein and enzymes in BAL
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters.
For details see section "any other information on materials and methods incl. tables".
Sacrifice and pathology:
Necropsy
All animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal vessels (aorta and vena cava). Afterwards, the thorax was opened, the right lung lobes were lavaged, whereas the left lung lobe was ligated during lavage. After the lavage procedure, the left and right lung lobes were instilled with formalin for weight assessment and further histopathological assessment.
Immediately after lung lavage, the animals were necropsied and assessed by gross pathology.

Weights:
The weights of anesthetized animals and lungs were determined in all animals sacrificed on schedule .

Histopathology:
Histological examination was performed of larynx (3 levels) lungs (left lobe, right caudal lobe, right cranial lobe, right middle lobe, accessory lobe), lymph nodes (tracheobronchial, mediastinal), nasal cavity (4 levels) and trachea in both sexes.
Cell proliferation was assessed in the lungs. Paraffin sections of 3 – 5 μm thickness were prepared. Slides were stained (immunohistochemistry) with mouse antiBrdU (Zytomed, Germany, clone Bu20a) and counterstained with hematoxylin.

Labeling indices
Two lung lobes per animal were evaluated (left lobe and accessory lobe) and labeling indices determined separately in:

- Large and medium bronchi
- Terminal bronchi
- Alveoli

Cell proliferation in the lungs was determined in two steps (generation of the reference values and main evaluation).
Generally, positively stained cells are characterized by a brown reaction product covering the nuclei. Light microscopy was performed at an original magnification of 400x for alveoli and 100x for bronchi using an image analysis system.

Generation of the reference values
The reference value is an estimate of the total cell number in a given compartment (see below) and is determined before the main evaluation of the study as described below for each compartment.

Alveoli:
Separately for each sex, two animals were taken at random from each test group (control and each treated group), to determine the reference value. For each of these selected animals, all pneumocytes type 1 were counted in 15 fields of observation of a predetermined size in each evaluated lung lobe (in total 30 fields of observation). The mean number of cells was calculated for each lung lobe and then multiplied by 30 to give the reference value.
Bronchi:
Separately for each sex, two animals were taken at random from each test group (control and each treated group), to determine the reference value. For each of these selected animals, 1000 epithelial cells were counted in large/ medium bronchi and in terminal bronchi, respectively. The corresponding length of the epithelium was measured as well. For the reference value, the mean number of cells divided by the length of the corresponding epithelium (mm) was calculated.
The reference values are recorded in the raw data but not reported.

Main evaluation
Alveoli:
For the main evaluation, 15 random fields of observation of the same size as used for the determination of the reference value were evaluated in all animals for each lung lobe (left and accessory lobe; 30 field in total). The labeling index (LI) was calculated as shown below:

BrdU LI (%) = (Positively labeled alveolar cells/reference value) x100

Bronchi:
Positively labeled bronchial epithelial cells were counted and the corresponding length of the epithelium was measured in all animals. The labeling index was calculated as shown below.

BrdU LI (%) = (Positively labeled bronchial cells per epithelial length/reference value) x 100

Calculations were performed with an excel spreadsheet, they were based on unrounded values and performed with a full set of decimal places. Therefore, there may be deviations in the relative values given.

Peer review
After completion of the histopathological assessment by the study pathologist an internal peer review was performed by Dr. Silke Treumann (BASF SE, Ludwigshafen) including all evaluated tissues in all test groups.
Results presented in this “summary of results” reflect the consensus opinion of the study pathologist and the peer review pathologist.
Statistics:
Means, medians and standard deviations of each test group were calculated for several parameters (see tables).

In summary tables of part I, mean values were rounded, but deviations of means versus control means were calculated with not rounded values. Therefore, slight differences may occur when changes were re-calculated with rounded means. In these tables “deviation vs control” means x-fold of controls expressed as percentages minus 100%. In the urinalysis summary tables „deviation vs control“ is not calculated because it is undefined for semiquantitative values.

The tables in section "any other information on materials and methods incl. tables" contain the statistical analyses used in this report.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
During the exposure period one animal of each test group, including control group, showed injury (scratch) on their dorsal body side, as well as two of the five females in test group 1 and one of the five females of test group 2. This finding was considered incidental due to the missing relation to the exposure concentration.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were observed in several animals of all test groups and the control group without any concentration-response relationship.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the administration period, in males of test groups 2 and 3 (10 and 20 mg/m3) absolute reticulocyte counts were significantly decreased. The change was not dose dependent and this was the only changed red blood cell parameter within these individuals. Therefore, this change was regarded as incidental and not treatment related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the administration period in males of test groups 2 and 3 (10 and 20 mg/m3) glucose values were significantly decreased. In contrast, in females of test group 3 glucose levels were significantly increased. In males, the change was not dose dependent. Therefore, this change was regarded as incidental and not treatment related. In females, the glucose mean was slightly above the historical control range (females, glucose 4.36-6.47 mmol/L). However, this was the
only changed serum parameter and therefore, this alteration was regarded as possibly treatment related but non-adverse (ECETOC Technical Report No. 85, 2002).
In males of test group 1 (4 mg/m3) alkaline phosphatase (ALP) activities and calcium levels were significantly decreased. In females of test group 2 (10 mg/m3) chloride values were significantly decreased. However, all mentioned alterations were not dose dependent and therefore, they were regarded as incidental and not treatment related.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
When compared to the control group 0, the mean relative weight of the lungs was statistically significantly increased in test group 3 male animals (+23%, p≤0.05, Kruskal-Wallis H and Wilcoxon test, two sided).
None of the other absolute or relative weight parameters showed statistically significant differences to the control group 0.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The mediastinal lymph nodes were enlarged in one male animal of each treated test group and
two females of test group 3. The tracheobronchial lymph nodes were enlarged in two males and
one female of test group 2, and 1 male and 3 females of test group 3.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
For further details on incidences and grading of reported findings see table in section "Any other information on results incl. tables".

Treatment-related findings were observed in lungs (all lobes), trachea, larynx (level I), tracheobronchial lymph nodes and mediastinal lymph nodes in male and female animals of all test groups.
Lungs
All examined lung lobes were equally affected.
Bronchi
Large, medium and terminal bronchi showed hypertrophy/hyperplasia characterized by occasional focal slight piling up and enlargement of bronchial epithelial cells. These areas showed increased cell proliferation on the BrdU-stained slides.
Alveoli
One type of alveolar lining cells, pneumocytes type II, showed minimal to mild proliferation histopathologically characterized by multiple cuboidal cells lining the alveolar wall. The interstitial spaces between alveoli were expanded by an influx of mainly lymphocytes and frequently also by fibrosis, both findings were combined in the term “inflammation, interstitial”.
These areas tended to show increased cell proliferation on the BrdU-stained slides.
In the alveolar lumina, neutrophilic inflammation was found occasionally. Debris was sometimes seen in alveolar lumina consisting of fragments of cells.

Alveolar macrophages
Alveolar macrophage accumulation was characterized by an increased number of macrophages
per alveolar space, often these macrophages showed many, tiny, clear vacuoles in the
cytoplasm (foamy cytoplasm), which was recorded separately.
Occasionally, alveolar macrophages started to fuse, which was recorded as “macrophage aggregates”. Granulomatous inflammation was characterized by macrophages forming syncytia with ingested foreign, brown to birefringent material visible in the cytoplasm.

Trachea
The epithelium on the tip of the carina was changed from its normal cuboidal, ciliated appearance to flattened cells with loss of cilia. Beneath the epithelium, there was an increased infiltration of lymphocytes in treated animals.

Larynx, level I
Epithelial alteration in the larynx was characterized by a focal, ventrally located change of the epithelium from cuboidal to focally flattened cells and was noted with increased incidence and severity in treated animals.
Lymphocyte infiltration was seen in the submucosa in treated animals.

Mediastinal lymph nodes
Foamy macrophages with the same appearance as observed in the lungs were seen in one male of test group 3.
The term “hyperplasia, lympho-reticular” was used to describe a diffuse enlargement of the lymph nodes, which could not be attributed to a specific compartment. This correlated often with the macroscopic finding “enlarged”.
Sinus histiocytosis was seen with a possibly dose-dependent increase in incidence and severity in males. In comparison, sinus histiocytosis showed a higher incidence in control females compared to treated animals.

Tracheobronchial lymph nodes
The term “hyperplasia, lympho-reticular” was used to describe a diffuse enlargement of the lymph nodes, which could not be attributed to a specific compartment. This correlated often with the macroscopic finding “enlarged”.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
BRONCHOALVEOLAR LAVAGE FLUID (BAL):
For details on findings in BAL see section "any other information on results incl. tables". At study day 5, in the bronchoalveolar lavage (BAL) of males and females in test groups 2 and
3 (10 and 20 mg/m3) total cell and absolute macrophage counts as well as absolute and relative
lymphocyte, neutrophil and monocyte counts were significantly increased. Relative macrophage
counts were significantly decreased in the mentioned individuals. These changes were regarded
as treatment-related and adverse.
In males of test group 1 (4 mg/m3) significant increases of total BAL cell and absolute
macrophages were counted. Absolute neutrophils, lymphocyte and monocyte counts among
these individuals were also higher compared to study controls (neutrophils 2.3fold; lymphocytes
7fold; monocytes 24fold increased) although the values were not statistically significantly
changed. However, total cell counts as well as absolute neutrophil and macrophage counts were
within historical control ranges, whereas only absolute lymphocyte and monocyte counts were
above the ranges (males BAL, total cell counts 38.21-101.23 cn/μL; macrophages 37.39-89.80
cn/μL; neutrophils 0.24-5.36 cn/μL, lymphocytes 0.00-1.04 cn/μL; monocytes 0.00-0.08 cn/μL).
In combination with slight total protein and enzyme activity increases in BAL in males of test
group 1 and histopathologic findings in the lungs, at least the absolute and relative lymphocyte
and monocyte increases it cannot be excluded that these effects are treatment related and
adverse.
In the BAL of females in test group 1 (4 mg/m3) monocyte counts were significantly increased.
Absolute lymphocyte and neutrophil counts were also more than 2-fold increased. In these rats,
only the monocyte counts were clearly above the preliminary historical control* range whereas
absolute lymphocyte and neutrophil counts were within the ranges (females BAL, monoyctes
0.00-0.06 cn/μL; lymphocytes 0.22-1.65 cn/μL; neutrophils 0.69-3.81 cn/μL). However, in
combination with histopathologic findings in the lungs increased monocyte counts in females of
test group 1 a treatment related and adverse effect cannot be excluded.
Additionally, in females of test group 1 (4 mg/m3) relative macrophages were significantly
decreased, but the deviation of the mean versus the study control was so marginal (-2.3%) that
the change was regarded as incidental and not treatment related. At study day 5, in the bronchoalveolar lavage (BAL) of males and females in test groups 2 and
3 (10 and 20 mg/m3) total protein values, γ-glutamyl-transferase (GGT) and alkaline
phosphatase (ALP) activities were significantly increased. Lactate dehydrogenase (LDH)
activities in BAL were significantly higher in males of test groups 2 and 3, but in females only in
test group 3. These changes were regarded as treatment related and adverse.
In males of test group 1 (4 mg/m3), total protein levels, GGT, LDH and ALP activities were
already significantly increased. Total protein levels and GGT activities were above the historical
control range, LDH activities at the upper border of this range, and ALP activities within this
range (males BAL, total protein 22-56 mg/L; GGT 4-59 nkat/L; LDH 0.21-0.61 μkat/L; ALP 0.21-
0.53 μkat/L). Therefore, the increases of total protein levels, GGT and LDH activities in males of
test group 1 in combination with the BAL cytology changes were regarded as treatment related
and adverse. In females, these effects were not observed.

CELL PROLIFERATION IN THE LUNGS:
For detailed results see section "any other information on results incl. tables".
Statistically significantly increased cell proliferation was seen in all compartments of the lungs in all treated test groups in males. In females, statistically significantly increased cell proliferation was seen in large, medium and terminal bronchi. In alveoli, there was a trend towards increased cell proliferation in treated groups, which showed, however, neither a dose-response relationship nor statistical significance.

Effect levels

open allclose all
Key result
Dose descriptor:
LOAEC
Remarks:
local toxicity
Effect level:
4 mg/m³ air
Sex:
male/female
Basis for effect level:
other:
Key result
Dose descriptor:
NOAEC
Remarks:
systemic effects
Effect level:
>= 20 mg/m³ air
Sex:
male/female
Basis for effect level:
other:

Any other information on results incl. tables

Bronchoalveolar lavage fluid (BAL)


Changes in mean absolute cell counts in BAL (x-fold of concurrent control) of males and females on study day 5 (1 day after last exposure):



















































































Analyte



Males



Females



 



Gr. 1


4 mg/m3



Gr. 2


10 mg/m3



Gr. 3


20 mg/m3



Gr. 1


4 mg/m3



Gr. 2


10 mg/m3



Gr. 3


20 mg/m3



Total Cells



1.9*



2.5**



3.2**



1.5



1.5*



3.4**



Eosinophils



1.2



4.1



5.6



2.1



2.0



2.6



Lymphocytes



7.2



21.9**



27.1**



2.4



8.1*



16.9*



Macrophages



1.6*



2.3**



2.8**



1.6



1.5*



3.5*



Neutrophils



2.3



10.3**



36.4*



2.9



12.4*



32.6*



Monocytes



23.5



101.8**



161.6**



38.5*



82.2*



204.6*



Epithelial cells



0.4



0.5



0.0



0.0



0.0



0.0



One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01


+ increase could not be calculated because of zero activity in controls


 


Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) of males and females on study day 5 (1 day after last exposure):











































































Analyte



Males



Females



 



Gr. 1


4 mg/m3



Gr. 2


10 mg/m3



Gr. 3


20 mg/m3



Gr. 1


4 mg/m3



Gr. 2


10 mg/m3



Gr. 3


20 mg/m3



Total Protein



1.7**



2.3**



3.2**



1.6



2.3*



2.4**



GGT



2.5**



2.5**



3.3**



1.1



1.9*



2.6**



LDH



2.0*



2.3**



2.8**



1.7



1.6



2.0**



ALP



1.6*



2.0**



2.8**



1.3



1.8*



2.5**



NAG



1.8



1.2



1.5



1.1



1.2



1.3


        

GGT = g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase;


NAG = b-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p <= 0.05; ** : p <= 0.01


 


PATHOLOGY:


Incidences and grading of histological findings in lungs:






















































































































































































































































































































 



Male animals



Female animals



Test group


(mg/m³)



0


(0)



1


(4)



2


(10)



3


(20)



0


(0)



1


(4)



2


(10)



3


(20)



No. of animals



5



5



5



5



5



5



5



5



Alveolar macrophage accumulation



2



3



5



5



0



0



5



5




  • Grade 1



2



2



4



 



 



 



2



2




  • Grade 2



 



1



1



4



 



 



3



3




  • Grade 3



 



 



 



1



 



 



 



 



Debris



0



1



0



2



0



0



0



3




  • Grade 1



 



1



 



2



 



 



 



3



Foamy macrophages, alveolar



0



5



5



5



0



3



5



5




  • Grade 1



 



5



5



5



 



3



4



4




  • Grade 2



 



 



 



 



 



 



1



1



Hyperplasia, type II, (m)f



0



0



1



3



0



1



2



5




  • Grade 1



 



 



1



3



 



1



2



4




  • Grade 2



 



 



 



 



 



 



 



1



Hypertrophy/


hyperplasia bronchiolar



0



4



5



5



0



5



5



5




  • Grade 1



 



4



 



 



 



5



2



 




  • Grade 2



 



 



3



3



 



 



3



3




  • Grade 3



 



 



2



2



 



 



 



2



Inflammation, granulomatous



0



0



1



0



0



0



1



0




  • Grade 1



 



 



1



 



 



 



1



 



Inflammation, interstitial, (m)f



0



4



5



5



0



2



5



5




  • Grade 1



 



4



3



5



 



2



5



4




  • Grade 2



 



 



2



 



 



 



 



1



Inflammation, neutrophilic, alveolar



0



0



0



0



0



1



0



1




  • Grade 1



 



 



 



 



 



1



 



1



Macrophage aggregates, (m)f



0



0



1



0



0



0



2



2




  • Grade 1



 



 



1



 



 



 



2



2



 


Incidences and grading of histological findings in trachea


























































































 



Male animals



Female animals



Test group


(mg/m³)



0


(0)



1


(4)



2


(10)



3


(20)



0


(0)



1


(4)



2


(10)



3


(20)



No. of animals



5



5



5



5



5



5



5



5



Epithelium flattened, carina



0



1



2



2



0



2



2



3




  • Grade 1



 



1



2



2



 



2



2



3



Infiltration, lymphoid, (m)f



1



2



3



4



0



0



1



2




  • Grade 1



1



2



3



3



 



 



1



2




  • Grade 2



 



 



 



1



 



 



 



 



Incidences and grading of histological findings are shown in larynx:





































































































 



Male animals



Female animals



Test group


(mg/m³)



0


(0)



1


(4)



2


(10)



3


(20)



0


(0)



1


(4)



2


(10)



3


(20)



No. of animals



5



5



5



5



5



5



5



5



Epithelial alteration, (m)f



0



5



5



5



1



4



5



5




  • Grade 1



 



5



3



2



1



3



5



4




  • Grade 2



 



 



2



3



 



1



 



1



Infiltration, lymphoid, (m)f



0



5



4



4



0



5



5



4




  • Grade 1



 



3



2



3



 



3



2



4




  • Grade 2



 



2



2



1



 



2



3



 



Incidences and grading of histological findings in mediastinal lymph nodes:






































































































































 



Male animals



Female animals



Test group


(mg/m³)



0


(0)



1


(4)



2


(10)



3


(20)



0


(0)



1


(4)



2


(10)



3


(20)



No. of animals



5



5



5



5



5



5



5



5



Foamy macrophages, (m)f



0



0



0



1



0



0



0



0




  • Grade 1



 



 



 



1



 



 



 



 



Hyperplasia, lympho-reticular



0



0



1



0



0



0



1



3




  • Grade 1



 



 



 



 



 



 



1



1




  • Grade 2



 



 



1



 



 



 



 



1




  • Grade 3



 



 



 



 



 



 



 



1



Sinus histiocytosis



0



2



1



3



4



2



0



2




  • Grade 1



 



2



1



2



4



2



 



2




  • Grade 2



 



 



 



1



 



 



 



 



The incidence and grading of the histological finding in tracheobronchial lymph nodes:




































































 



Male animals



Female animals



Test group


(mg/m³)



0


(0)



1


(4)



2


(10)



3


(20)



0


(0)



1


(4)



2


(10)



3


(20)



No. of animals



5



5



5



5



5



5



5



5



Hyperplasia, lympho-reticular



0



0



2



5



0



4



4



4




  • Grade 1



 



 



 



2



 



4



2



1




  • Grade 2



 



 



2



3



 



 



2



3



 


CELL PROLIFERATION IN THE LUNGS:


Labeling indices in different compartment in the lungs of male animals:

























































 



Large + medium bronchi



Terminal bronchi


 



Alveoli


 



Test group


(mg/m³)



LI



%



LI



%



LI



%



Control


(0)



0.72



100



0.51



100



4.03



100



1


(4)



5.53**



773



13.65**



2676



5.29*



131



2


(10)



13.10**



1832



25.21**



4942



5.60*



139



3


(20)



13.68**



1913



30.60**



6000



6.27**



156



*:p ≤ 0.05, **:p ≤ 0.01 Wilcoxon-test (one-sided)


Labeling indices in different compartment in the lungs of female animals:

























































 



Large + medium bronchi



Terminal bronchi


 



Alveoli


 



Test group


(mg/m³)



LI



%



LI



%



LI



%



Control


(0)



0.91



100



0.99



100



5.20



100



1


(4)



8.74**



958



18.21**



1843



5.66



109



2


(10)



12.91**



1416



21.43**



2169



6.39



123



3


(20)



14.15**



1551



25.07**



2537



6.18



119



 


*:p ≤ 0.05, **:p ≤ 0.01 Wilcoxon-test (one-sided)


 

Applicant's summary and conclusion

Conclusions:
The inhalation exposure to 4, 10 and 20 mg/m³ 4,4-MDI/DPG/HMWP caused
concentration-related changes in lavage fluid and morphological changes in lungs, as well
as some mild reactive changes in lung-draining lymph nodes, trachea and larynx.
Moreover, significantly increased cell proliferation in large, medium and terminal bronchi
in males and females and increased cell proliferation in alveoli in males (statistically
significant) and females (not statistically significant) were observed.
No systemic effect could be observed throughout the study. Under the current study
conditions, no observed adverse effect level (NOAEL) for local toxicity could not be
determined. The NOAEC for systemic toxicity was 20 mg/m³.