m-TDI oligomers, isocyanurate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 39 (2009)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(2,4,6-trioxotriazine-1,3,5(2H,4H,6H)-triyl)tris(methyl-m-phenylene) isocyanate

EC Number:

247-840-6

EC Name:

(2,4,6-trioxotriazine-1,3,5(2H,4H,6H)-triyl)tris(methyl-m-phenylene) isocyanate

Cas Number:

26603-40-7

Molecular formula:

C27H18N6O6

IUPAC Name:

1,3,5-tris(3-isocyanato-4-methylphenyl)-1,3,5-triazinane-2,4,6-trione

Constituent 2

Reference substance name:

TDI Trimer

IUPAC Name:

TDI Trimer

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Nederland, AD Horst, Netherlands
- Strain: HsdCpb:Wu (SPF)
- Age at study initiation: approx. 2 months
- Weight at study initiation: at the study start the variation of individual weights did not exceed ± 10 per cent of the mean for each sex
- Housing: singly in conventional Makrolon® Type IIIH cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: approx. 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 40-60
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: dry conditioned air

Remarks on MMAD:

MMAD / GSD: In all exposure groups the MMAD was in the range of 1.6-2.2 µm ( GSD 2.2-2.3).

Details on inhalation exposure:

- MODE OF EXPOSURE:
Animals were exposed to the aerosolized test substance in Plexiglas exposure restrainers. Restrainers were chosen that accommodated the animals' size. These restrainers were designed so that the rat's tail remained outside the restrainer, thus restrained-induced hyperthermia can be avoided. This type of exposure principle is comparable with a directed-flow exposure design (Moss and Asgharian, 1994) and is preferable to whole-body exposure on scientific (OECD, 2008) and technical reasons (rapid attainment of steady-state concentrations, no technical problems with regard to test atmosphere inhomogeneities, better capabilities to control all inhalation chamber parameters, easier cleaning of exhaust air, and lower consumption of test substance). Moreover, contamination of the haircoat can largely be avoided and confounding effects as a result of uptake of test substance by non-inhalation routes are minimized. The chambers used are commercially available (TSE, 61348 Bad Homburg) and the performance as weil as their validation has been published (Pauluhn, 1984; Pauluhn, 1994; Pauluhn and Thiel, 2007).

- DESCRIPTION OF APPARATUS:
Dry conditioned air was used to aerosolize the test substance. The test atmosphere was then forced through openings in the inner concentric cylinder of the chamber, directly towards the rats' breathing zone. This directed-flow arrangement minimizes re-breathing of exhaled test atmosphere. Each inhalation chamber segment was suitable to accommodate 20 rats at the perimeter location. All air flows were monitored and adjusted continuously by means of calibrated and computer controlled mass-flow-controllers. A digitally controlled calibration flow meter was used to monitor the accuracy of mass-flow-controller. The ratio between supply and exhaust air was selected so that 90% of the supplied air was extracted via the exhaust air location and, if applicable, via sampling ports. Aerosol scrubbing devices were used for exhaust air clean-up. During sampling, the exhaust air was reduced in accordance with the sampling flow rate using a computerized Data Acquisition and Control System so that the total exhaust air flow rate was adjusted online and maintained at the specified 90%. The slight positive balance between the air volume supplied and extracted ensured that no passive influx of air into the exposure chamber occurred (via exposure restrainers or other apertures). The slight positive balance provides also adequate dead-space ventilation of the exposure restrainers. The pressure difference between the inner inhalation chamber and the exposure zone was 0.02 cm H20 (Pauluhn, 1994). The exposure system was accommodated in an adequately ventilated enclosure. Temperature and humidity are measured by the Data Acquisition and Control System using calibrated sensors. The sensors were located in the inhalation chamber.

- INHALATION CHAMBER:
The aluminum inhalation chamber has the following dimensions: inner diameter = 14 cm, outer diameter.= 35 cm (two-chamber system), height = 25 cm (internal volume = about 3.8 L). To be able to perform all measurements required to define exposure in a manner that is similar to the exposure of rats, 'two segment' chambers were used in all groups. Details of this nose-only exposure system, including its validation, have been published previously (Pauluhn, 1994; Pauluhn and Thiel, 2007).

- INHALATION CHAMBER EQUILIBRIUM CONCENTRATION:
The test atmosphere generation conditions provide an adequate number of air exchanges per hour [30 L/min x 60 min/(2 x 3.8 L/chamber) = 237, continuous generation of test atmosphere]. Based on OECD-GD39 the equilibrium concentration (t95) can be calculated as folIows:
t95 (mln) = 3x (chamber volume/chamber airflow)
Under the test conditions used a chamber equilibrium is attained in less than one minute of exposure (McFarland, 1976). At each exposure port a minimal air flow rate of 0.75 L/min was provided. The test atmosphere can by no means be diluted by bias-air-flows.

- CONDITIONING THE COMPRESSED AIR:
Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer. Adequate control devices were employed to control supply pressure.

- AIR FLOWS:
During the exposure period air flows were monitored continuously by flow meters and, if necessary, readjusted to the conditions required. Measured air-flows were calibrated with precision flow-meters and/or specialized flow-calibration devices (DryCal Defender 510; http://www.smglink.com/bios/drycal defender/drycal defender.html and TSI Model 4199 Mass Flowmeter; http://www.tsi.com/en-1033/models/3472/4043.aspx) and were checked for correct performance at regular intervals.

- TREATMENT OF EXHAUST AIR:
The exhaust air was purified via cotton-wool, activated char coal filter, and HEPA filters. These filters were disposed of by Bayer AG.

- INHALATION CHAMBER TEMPERATURE AND HUMIDITY:
Temperature and humidity measurements are also performed by the computerized Data Acquisition and Control System using FTF11 sensors (ELKA ELEKTRONIK, Lüdenscheid, Germany). The position of the probe was at the exposure location of rats. Measurements were performed in the exhaust air. Temperature and humidity data are integrated for 30-seconds and displayed accordingly. The humidity sensors are calibrated using saturated salt solutions according to Greenspan (1977) and Pauluhn (1994) in a two-point calibration at 33% (MgCI2) and at 75% (NaCI) relative humidity. The calibration of the temperature sensors is also checked at two temperatures using reference thermometers.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- ANALYSIS OF TEST ATMOSPHERES:
Nominal concentration: The nominal concentration was calculated from the ratio of the total quantity of test item consumed during the exposure period and the total throughput of air through the inhalation chamber.
Total mass concentration: The test substance concentration was determined by gravimetrie analysis (filter: glass-fiber filter, Sartorius, Göttingen, Germany; digital balance). This method was used to define the actual total mass concentrations.
Sampling: Chamber samples were taken in the vicinity of the breathing zone. The number of samples taken was sufficient to characterize the test atmosphere and was adjusted so as to accommodate the sampling duration and/or the need to confirm specific concentration values. Optimally, three samples per exposure day were collected from each exposure chamber. The actual concentrations reported refer to mg/m3 Desmodur RC. This means the gravimetric concentrations were not corrected for the volatile constituents.

- STABILITY OF TEST ATMOSPHERES:
To monitor the integrity and stability of the aerosol generation and exposure system either a Microdust Pro real-time aerosol photometer (Casella, USA) (4.4 and 20.6 mg/m3) or RAS-2 real-time aerosol photometer (MIE, Bedford, Massachusetts, USA) (80.7 and 318.7 mg/m3) was used. Samples were taken continuously from the vicinity of the breathing zone. The results are displayed on the computer screen and printed after cessation of exposure. For data recording and display the system integration time was 30 sec. This chamber monitoring allows for an overall survey of toxicologically relevant technical parameters (inlet and exhaust flows as well as atmosphere homogeneity, temporal stability, and generation performance). Interruptions in exposure (e.g. resulting from obstruction of nozzles or other technical mishaps) are recorded and, if applicable, a commensurate interval is added to the exposure duration for compensation.

- CHARACTERIZATION OF AERODYNAMIC PARTICLE-SIZE DISTRIBUTION:
Samples for the analysis of the particle-size distribution were also taken in the vicinity of the breathing zone. The particle-size distribution was analyzed using a BERNER-TYPE AERAS low-pressure critical orifice cascade impactor (Hauke,Gmunden, Austria). The individual impactor stages were covered by an aluminum foil and glass fiber filter which were subjected to gravimetric analysis. Gravimetric analyses were made using a digital balance.

The parameters characterizing the particle-size distribution were calculated according to the following procedure:
Mass Median Aerodynamic Diameter (MMAD): Construct a 'Cumulative Percent Found - Less Than Stated Particle Size' table, calculate the total mass of test substance collected in the cascade impactar. Start with the test substance collected on the stage that captures the smallest particle-size fraction, and divide this mass of the test substance by the total mass found above. Multiply this quotient by 100 to convert to percent. Enter this percent opposite the effective cut-off diameter of the stage above it in the impactor stack. Repeat this step for each of the remaining stages in ascending order. For each stage, add the percentage of mass found to the percentage of mass of the stages below it. Plot the percentage of mass less than the stated size versus particle size in a probability scale against a log particle-size scale, and draw a straight line best fitting the plotted points. A weighted least square regression analysis may be used to achieve the best fit. Note the particle size at which the line crosses the 50% mark. This is the estimated Mass Median Aerodynamic Diameter (MMAD).

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 5, 20, 80, 320 mg/m3
Basis:
other: target concentration

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 4.4, 20.6, 80.7, 318.7 mg/m3
Basis:
other: actual breathing zone concentrations (gravimetric)

No. of animals per sex per dose:

5 animals per sex per dose (main groups);
5 animals per sex for control and high dose group (satellite groups for 4-week recovery);
6 males per dose (satellite groups for bronchoalveolar lavage at the end of the 4-week exposure period)

Control animals:

yes, sham-exposed

Details on study design:

- DOSE SELECTION RATIONALE:
Based on the results of a 1-week pilot inhalation study in rats (Pauluhn, 2011) 5 mg/m3 is expected to be the NOAEL of the study and 20 mg/m3 is likely to be in the range where portal-of-entry related local effects start to occur. Due to the difficulty to unequivocally distinguish the physiological response to particle exposure and upregulation of clearance from early signs of particle-induced adversity - indicated by inflammation and tissue remodeling - two high-level exposure groups to 80 and 320 mg/m3 where chosen.

- POST-EXPOSURE RECOVERY PERIOD:
Five male and female rats each of the control and high dose groups were examined during a post-exposure recovery period of 4 weeks.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

- BODY WEIGHTS:
Body weights of all animals were measured on a twice per week basis during the exposure period and once weekly during the exposure-free recovery period.

- FOOD AND WATER CONSUMPTION:
Food and water consumption were determined on a per week basis.

- CLINICAL OBSERVATIONS:
The appearance and behavior of each rat was examined carefully at least twice on exposure days (before and after exposure) and once a day on exposure-free days. If considered applicable due to unequivocal signs, in nose-only exposed rats observations were also made during exposure. Following exposure, observations were made and recorded systematically; individual records were maintained for each animal, if applicable. Cage side observations included, but were not limited to changes in the skin and hair-coat, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, and sensori- as weil as somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors,
convulsions, salivation, diarrhea, lethargy, somnolence and prostration. The time of death was recorded as precisely as possible, if applicable. Since these signs can only be assessed adequately in their home cages, no specific assessment was performed during exposure while animals were restrained. During the course of study, additional clinical observations which took into account the pattern of examination consistent with a Functional Observational Battery (FOB). Measurements were made in 5 rats/sex/group. Each rat was first observed in its home cage and then individually examined. The following reflexes were tested, based on recommendations made by Irwin (1968) and Moser et. al., (1988): visual placing response and grip strength on wire mesh (wire-mesh grid-gripping resistance of the animal to pull), abdominal muscle tone, corneal and pupillary reflexes, pinnal reflex, righting reflex, tail-pinch response, startle reflex with respect to behavioral changes stimulated by sounds (e.g. finger snapping) and touch (back).

- CLINICAL PATHOLOGY AND HEMATOLOGY:
General clinical pathology was performed at the end of the exposure period on 5 animals/sex/group. For measurements at the post-exposure period endpoints were selected case-by-case, depending on the outcome after the exposure period. The terminal blood samples were obtained by cardiac puncture of· the deeply anesthetized, non-fasted rats (Narcoren®; intraperitoneal injection). As anticoagulants Li-heparin- or EDTA-coated tubes were used except for blood collected for to examine hemostasis endpoints where sodium citrate was used as anticoagulant.
1. Hematology: Hematrocit, Hemoglobin, Leukocytes, Erythrocytes, Mean corpuscular volume, Mean corpuscular hemoglobin concentration, Mean corpuscular hemoglobin, Thrombocyte count, Reticulocytes, Leukocyte differential count (Lymphocytes, Granulocytes, Segmented neutrophils, Eosinophilic neutrophils, Basophils, Monocytes, Plasma cells, miscellaneous abnormal cell types).
2. Clinical Pathology: Aspartate aminotransferase, optimized (ASAT), Alanine aminotransferase, optimized (ALAT), Glutamate dehydrogenase (GLDH), y-Glutamylaminotransferase (y-GT), Lactate dehydrogenase (LDH), Alkaline phosphatase (APh), Albumin, Bilirubin (total), Blood glucose, Calcium, Chloride, Cholesterol,Creatinine kinase, Creatinine, Magnesium, Phosphate, Potassium, Sodium, Total protein, Triglycerides, Urea, Prothrombin time (PT, Ouick value, "Hepato Quick").

- OPHTHALMIC EXAMINATION:
Ophthalmic examinations were conducted by a laboratory animal veterinarian or assistant trained in ophthalmoscopic examinations. Eye examinations were performed prior to the first exposure and towards the end of the exposure period. For examinations, an indirect ophthalmoscope was used. Five to ten minutes prior to the examination, the pupils were dilated with mydriatic (STULLN®). Routine screening examinations included an examination of the anterior segment of the eye, the posterior segment of the eye and adnexal structures. Structures examined in the anterior segment of the eye will typically include the cornea, sclera, iris, pupiI, lens, aqueous, and anterior chamber. Structures examined in the posterior segment of the eye will typically include the vitreous, retina and optic disc. Examination of adnexal structures will typically include conjunctiva, eyelids and eyelashes.

Sacrifice and pathology:

-ORGAN WEIGHTS:
The following organs were weighted at necropsy after exsanguination: Adrenal glands, Brain, Heart, Kidneys, Liver, Lung (incl. trachea), Ovaries, Spleen, Testes, Thymus.
No organ weight data were collected from animals found dead. Paired organs were weighted together.

- NECROPSY:
All surviving rats were sacrificed at the end of the exposure and post-exposure observation period using sodium pentobarbital as anaesthetic and complete exsanguination by heart puncture (Narcoren®; at least 120 mg/kg body weight, intraperitoneal injection). All rats, irrespective of the day of death, were given a gross-pathological examination. Consideration was given to performing a gross necropsy on animals as indicated by the nature of toxic effects, with particular reference to changes related to the respiratory tract. All gross pathological changes were recorded and evaluated.

- HISTOPATHOLOGY:
The following organs/tissues were collected and fixed in 10 % neutral buffered formalin or Davidson's solution: Adrenals, aorta, bone and bone marrow section (sternum), brain (cerebrum, cerebellum, pons/medulla), epididymides, esophagus, eyes with optic nerve, eyelids, extraorbital lacrimal glands, femur with knee joint, Harderian glands, head with nasal cavity, heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), kidneys including pelvis, lacrimal glands, larynx, liver, lungs and main bronchi (all lobes), lymph nodes (lung associated, mandibular, mesenterics, popliteal, mediastinal), mammary gland, muscle (biceps femoris), ovaries with oviducts, pancreas, pharynx, pituitary gland, prostate, salivary glands, sciatic nerve, seminal vesicles (incl. coagulation glands), skin (flank, nose region and facial area), spinal cord (cervical, thoracal, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, ureters, urinary bladder, uterus with cervix, vagina, Zymbal glands and tissues with macroscopic findings.
Histopathology was performed on all organs/tissue shown above at least in the control and high dose groups. The tissues of the respiratory tract were examined in all groups, including those of the recovery groups. Other groups (and/or tissues) were evaluated at the discretion of the clinical pathologist only if warranted by specific changes.

Other examinations:

- RECTAL TEMPERATURE:
The rectal (colonic) temperatures were measured at several time points shortly after cessation of exposure (within 1/2 hour of cessation of exposure) using a digital rectal probe (H. Sachs, March, Germany). Five rats/main group/sex were examined after the first exposure, midterm and the end of the exposure period.

- BRONCHOALVEOLAR LAVAGE (BAL):
Samples of bronchoalveolar lavage fluid were collected from the lungs of rats (six male rats/group) at the end of the exposure period (one day after the last exposure). In BAL-fluid (BALF), several indicators of pulmonarydamage were assessed:
Alkaline phosphatase, Soluble collagen, Lactate dehydrogenase (LDH), N-Acetylglucosaminidase (B-NAG), Phospholipids, Total protein, y-Glutamyltransferase (y-GT), Total number of lavaged cells (including the volume and diameter), Cytodifferentiation.

Statistics:

- IN-LIFE DATA: Statistical tests on body weights and weight gain as well as on absolute organ weights or relative log1O-transformed organ weights are analyzed using the Dunnett Exact Homogeneous Test. Food/water intake per animal and day are calculated and analyzed by the adjusted Mann-Whitney U-tests. Terminal body weights (TWS) serve as covariate for calculation of the organ-to-body-weight ratio (percentage). Likewise, the Dunnett Exact Homogeneous or Heterogeneous Test, the Dunnett Exact Homogeneous Test after log10-transformation or the Bonferroni/Mann-Whitney U-test are used for the statistical analysis of clinical pathology parameters. Descriptive statistics were provided per sex, dose group and time point for all parameters that were recorded with a specified unit. This included measures of general tendency (mean and median (median not given for food and water intake)) and general variability (standard deviation, minimum and maximum) as appropriate. For continuous variables, the statistical test procedure was based on prior knowledge of the respective variable derived from previous studies. For normally distributed variables with equal variances across treatment groups Dunnett's tests were performed. Heteroscedastic normally distributed variables were analysed using appropriately adjusted Dunnett's tests, using. Satterthwaite adjustments for the degrees of freedom and taking the different variances within the groups into account. For log-normally distributed variables, Dunnett's tests were performed after log transformation of the original values. If experience with historical data indicated that the assumptions for parametric analyses are violated, Bonferroni-adjusted Mann-Whitney
U-tests were employed in the analyses. For small sample sizes, the exact version of this test was used.

- RECTAL TEMPERATURE, BRONCHOALVEOLAR LAVAGE: Data were statistically evaluated using the ANOVA procedure.

Results and discussion

Results of examinations

Details on results:

- MORTALITY:
All exposures were tolerated without test substance-induced mortality (see also Table 1).

- CLINICAL SIGNS:
All rats of the dose groups 4.4, 20.6 and 80.7 mg/m3 tolerated the exposure without specific signs. In the high dose group 318.7 mg/m3 the following substance-related clinical signs were recorded in all animals (see also Table 1):
Males: Irregular breathing patterns-b, irregular breathing patterns-a, labored breathing patterns-b, labored breathing patterns-a, bradypnea-b, high-legged gait-b, high-legged gait-a, motility reduced-b, motility reduced-a, atony-b, atony-a, piloerection-b, piloerection-a, and haircoat ungroomed-b (a/b: after/before exposure).
Females: Irregular breathing patterns-b, irregular breathing patterns-a, labored breathing patterns-b, labored breathing patterns-a, high-legged gait-b, high-legged gait-a, motility reduced-b, motility reduced-a, atony-b, atony-a, piloerection-b, piloerection-a (a/b: after/before exposure).

- REFLEXES:
The examination of reflexes did not reveal any differences between control and dose groups 4.4, 20.6 and 80.7 mg/m3. At 318.7 mg/m3 the vertical grip strength was reduced.

- RECTAL TEMPERATURE:
In comparison to the concurrent air control group, there was no evidence of a conclusive, toxicologically significant effect on body (rectal) temperatures at any exposure concentration. Additionally, the temperature measurements made on control animals demonstrate clearly that the animal restrainer had no apparent effect on the body temperature (normal body temperature of the rats: 37.5°C - 38.5°C).

- BODY WEIGHTS:
There was no toxicologically consistent, i.e., concentration- or time-dependent effect on body weights up to and including 80.7 mg/m3. At 318.7 mg/m3 the body weights were statistically significant decreased in both sexes.

- FOOD AND WATER CONSUMPTION:
There was no toxicologically consistent, i.e., concentration- and/or clearly time-dependent effect on food/water consumption up to the maximum concentration tested. Nonetheless, in dose group 318.7 mg/m3 there was a trend of decreased food/water intake.

- BRONCHOALVEOLAR LAVAGE AND LUNG WEIGHTS:
Lung weights were significantly increased at 80.7 and 318.7 mg/m3. The average recovery of bronchoalveolar lavage fluid (BAL) was approximately 88 % of the instilled volume and was similar amongst the groups. The results did not show changes of adversity up to 20.6 mg/m3. At higher concentrations the effects observed appear to be related to the physiological removal and clearance of the inhaled aerosol. These effects were characterized by increased influx of phagocytic cells (alveolar macrophages with inclusions "NC-PM", neutrophilic granulocytes (PMNs) (absolute and relative), BAL-protein/-collagen, LDH, and y-GT. Evidence of increased lysosomal (ß-NAG) and Type II cell activities (AP) occurred at 318.7 mg/m3 only. At all exposure levels with the test article alveolar macrophages appeared to be "uploaded" with polymerized test material; however, in the absence of any change suggestive of injury because the endpoint "NC-RB" (phagocytosed red blood cells) was not affected in concentration-dependent manner. Collectively, the NOAEL based on BAL-analysis is 20.6 mg/m3. Macrophages with phagocytized red blood cells and translucent inclusions were attributed to the category 'non-classifiable cells'. It appears to be scientifically justified to assume that most of the NCs are morphologically alveolar macrophages containing phagocytosed material from the alveolar lining fluid.

- HEMATOLOGY AND DIFFERENTIAL BLOOD COUNT:
There were no conclusive concentration-dependent changes between control and dose groups. Isolated statistical significances are considered to be of no pathodiagnostic or prognostic significance.

- CLINICAL PATHOLOGY:
There were no conclusive concentration-dependent changes between control and dose groups. Isolated statistical significances are considered to be of no pathodiagnostic or prognostic significance.

- OPHTHALMOLOGY:
Ophthalmology performed prior to the start of the study and towards the end of the study did not reveal any conclusive evidence of test substance-induced changes in the dioptric media or in the fundus up to 318.7 mg/m3.

- ORGAN WEIGHTS:
Lung weights were statistically significantly increased at 80.7 and 318.7 mg/m3 with no reversibility at the end of the 4-week recovery period. Stress organs (thymus, spleen, and testes) showed some typical changes in organ weights at the high exposure group only, i.e., that group showing significantly decreased body weights. Hence, apart from the lung, no other significant changes in organ weights or the organ-to-body weight or organ-to-brain weight ratios considered to be of pathognostic significance occurred the remaining groups and organs.

- NECROPSY:
Necropsy findings were unremarkable at 4.4 and 20.6 mg/m3. In rats exposed at 80.7 and 318.7 mg/m3 dark-red areas were observed on lungs and lung-associated lymph nodes. After recovery during the 4-week recovery period, lung discolorations and enlarged lung-associated lymph nodes were not found. Other respiratory tract-specific of extrapulmonary macroscopic findings were not found.

- HISTOPATHOLOGY:
Histopathologically, at the end of the exposure period, deposits of the test article were detected in almost all levels of the nasal cavity. Areas with substance deposition, the epithelium showed focal atrophy and/or degeneration at 80.7 mg/m3 and above. However, in these areas no indication of any inflammatory response to deposited material was apparent. Minimal epithelial alteration of the larynx was seen in females from the 318.7 mg/m3 group and in each one male and female at 80.7 mg/m3. In the lumen of the trachea an increased incidence and/or grading of mucus and/or macrophages containing the test compound was detected at 20.6 mg/m3 and above. In the lungs, hypercellularity of the bronchiolo-alveolar junction and enlarged and/or particle-laden macrophages were the major findings. At 4.4 mg/m3 changes were characterized by macrophages with translucent inclusions without any other inflammatory and/or degenerative findings. In lung associated lymph nodes, an increased incidence of increased cellularity of the paracortex as well as test compound in macrophages occurred in males and females at 20.6 mg/m3 and above. A lymphatic activation (increased secondary follicles with germinal centers) occurred in some rats at 318.7 mg/m3. The above mentioned findings are assessed as substance-related. All other findings seen in the organs/tissues evaluated in this subacute inhalation study were equally distributed between controls and substance-exposed groups and/or are known to be spontaneous findings from previous studies.

Effect levels

1

Dose descriptor:

NOAEC

Effect level:

20.6 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

2

Dose descriptor:

LOAEC

Effect level:

80.7 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: changes of bronchoalveolar lavage, histopathological changes of the respiratory tract

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Summary of subacute inhalation toxicity (6 hrs/day for 4 weeks) of Desmodur RC (25 % in ethyl acetate)

Sex	Analytical concentration (gravimetric) (mg/m3)	Toxicological results	Onset and duration of signs	Onset and duration of mortality
male	0 (Control)	0 / 0 / 16	---	---
	4.4	0 / 0 / 11	---	---
	20.6	0 / 0 / 11	---	---
	80.7	0 / 0 / 11	---	---
	318.7	0 / 16 / 16	0d - 29d	---
female	0 (Control)	0 / 0 / 10	---	---
	4.4	0 / 0 / 5	---	---
	20.6	0 / 0 / 5	---	---
	80.7	0 / 0 / 5	---	---
	318.7	0 / 10 / 10	0d - 34d	---

Toxicological results:

number of dead animals / number of animals with signs after cessation of exposure / number of animals exposed

Applicant's summary and conclusion

Executive summary:

In a subacute inhalation toxicity study (OECD TG 412) 5 male and 5 female Wistar rats per dose group were nose-only exposed for 4 weeks (6 hours /day, 5 days/week) to a liquid aerosol of Desmodur RC (25 % in ethyl acetate). The mean actual concentrations (gravimetric) were 4.4, 20.6, 80.7 and 318.7 mg/m3. Rats exposed under otherwise identical test conditions to dry air served as concurrent control group. Additional 5 rats/sex/satellite group (control and high level exposure groups) were allowed to recover during a 4-week post-exposure period. Additional 6 male rats/group were subjected to bronchoalveolar lavage at the end of the 4-week exposure period.

The rats exposed up to 80.7 mg/m3 did not display any substance-specific clinical signs while at 318.7 mg/m3 the following clinical signs were recorded: irregular and labored breathing patterns, bradypnea, high-legged gait, reduced motility, atony, piloerection, ungroomed haircoat, hypothermia, decreased grip strength, and decreased body weights. Conclusive changes in feed/water consumption did not occur. Of note is that all findings were most pronounced during the first week and waned during the remaining exposure weeks. During this week the transiently significantly decreased body weights coincided with the significantly decreased food/water consumption. This constellation appears to suggest that non-specific sensory irritation is the cause for these findings which decreased to almost normal due to adaptation at continued exposure.

Ophthalmology was unremarkable. There was no evidence of any adverse hematological effects. Likewise, clinical pathology did not reveal any pathodiagnostically relevant effects considered to be causally related to the exposure to the test article aerosol. There were no statistically significant or conclusive changes in absolute or relative organ weights with the exception of increased lung weights at 80.7 and 318.7 mg/m3. Lung weights were still elevated at the end of the 4-week postexposure period.

Bronchoalveolar lavage did not show changes of adversity up to 20.6 mg/m3. At higher concentrations the effects observed appear to be related to the physiological removal and clearance of the inhaled aerosolized test article and its reaction products with biopolymers of the proteins contained in the fluids lining the respiratory tract. These effects were characterized by increased influx of phagocytic cells (alveolar macrophages with inclusions, neutrophilic granulocytes (PMN) (absolute and relative), BAL-protein/-collagen, LDH, and y-GT. Evidence of increased lysosomal (P-NAG) and Type II cell activities (AP) occurred at 318.7 mg/m3 only. At all exposure levels with the test article alveolar macrophages appeared to be "uploaded" with polymerized test material; however, in the absence of any change suggestive of injury because any increase in phagocytosed red blood cells was not apparent. Collectively, the NOAEL based on BAL-analysis was 20.6 mg/m3.

Histopathologically, at the end of the exposure period, deposits of the test article (or its polymerized reaction products) were detected in almost all levels of the nasal cavity. Areas with substance deposition, the epithelium showed focal atrophy and/or degeneration at 80.7 mg/m3 and above. However, in these areas no indication of any inflammatory response to deposited material was apparent. Minimal epithelial alteration of the larynx was seen in females from the 318.7 mg/m3 group and in each one male and female at 80.7 mg/m3. In the lumen of the trachea an increased incidence and/or grading of mucus and/or macrophages containing the test compound was detected at 20.6 mg/m3 and above. In the lungs, hypercellularity of the bronchiolo-alveolar junction and enlarged and/or particle-laden macrophages were the major findings. At 4.4 mg/m3 changes were characterized by macrophages with translucent inclusions without any other inflammatory and/or degenerative findings. In lung associated lymph nodes, an increased incidence of increased cellularity of the paracortex as well as test compound in macrophages occurred in males and females at 20.6 mg/m3 and above. A lymphatic activation (increased secondary follicles with germinal centers) occurred in some rats at 318.7 mg/m3. The above mentioned findings are assessed as substance-related. All other findings seen in the organs/tissues evaluated in this subacute inhalation study were equally distributed between controls and substance-exposed groups and/or are known to be spontaneous findings from previous studies. Collectively, histopathology did not reveal any adverse effects at 4.4 mg/m3.

In summary, this study did not reveal any conclusive evidence of particle-induced irritation at any exposure level. As far as epithelial changes of the respiratory epithelium occurred they followed the typical anterior-posterior gradient of deposition intensity. Tissue inflammatory responses to deposited material did not occur. Histopathology findings were essentially confined to the alveolar level and were largely complementing the changes in lung weights and bronchoalveolar lavage. Overall, the changes observed appear to be predominated by (over-) dosed alveolar macrophages by the test article itself or its polymerized reaction products. Taking all findings into account, 20.6 mg/m3 constitutes the no-observed-adverse-effect-level (NOAEL) for respiratory tract responses. In regard to extrapulmonary toxicity, no effects were found up to the maximum concentration examined.