Methylidynetri-p-phenylene triisocyanate

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:

Methylidynetri-p-phenylene triisocyanate

EC Number:

219-351-8

EC Name:

Methylidynetri-p-phenylene triisocyanate

Cas Number:

2422-91-5

Molecular formula:

C22H13N3O3

IUPAC Name:

1-[bis(4-isocyanatophenyl)methyl]-4-isocyanatobenzene

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 2.3 µm ( GSD 1.9).

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, Respiratory Drug Delivery IV pp. 197-201, 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, Journal of Applied Toxicology 13:55-62, 1994; Pauluhn and Thiel, J. Appl. Toxicol. 27:160-167, 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, Journal of Applied Toxicology 13:55-62, 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 RE. 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)or a Microdust Pro real-time aerosol photometer (MIE, Bedford, Massachusetts, USA) 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, 0.5, 4, 35 mg/m³
Basis:
other: target concentration

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 0.47, 3.9, 34.9 mg/m³
Basis:
other: gravimetric concentration

No. of animals per sex per dose:

5 animals per sex per dose (main groups);
5 animals per sex for control and 10 animals per sex for 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:

other: yes, concurrent dry air

Details on study design:

- DOSE SELECTION RATIONALE:
The exposure regimen used in the acute inhalation studies (see IUCLID chapter 7.2.2: Pauluhn, 2012a, b)supports the following exposure concentrations: 0.5, 4, and 35 mg/m³. The top level is likely to elicit unequivocal respiratory tract toxicity without causing undue irritant stress or irreversible effects. The high concentration is in the range of a fourth of the time-adjusted non-lethal threshold concentration of the acute inhalation study. Therefore, mild irritation was anticipated to occur with full recovery over the exposure-free weekends. Moreover, based on the findings of the pilot lavage study, a fourth of the 1.9 mg/m³ is expected to be the NOAEL of study. The interim concentration of 4 mg/m³ is likely to be in the range where portal-of-entry related local effects start to occur and represents the geometric mean from the lowand top concentrations.


- POST-EXPOSURE RECOVERY PERIOD:
Five male and female rats each of the control and high dose groups were allowed to recover during a post-exposure 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 (Psychopharmacologica 13, pp. 222-257, 1968) and Moser et. al., (Fundamental and Applied Toxicology, 11. 189-206, 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), Lung-associated-lymph nodes (LALNs), 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 pulmonary damage were assessed:
Alkaline phosphatase, Lactate dehydrogenase (LDH), N-Acetylglucosaminidase (B-NAG), 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 below).

- CLINICAL SIGNS:
All rats of the dose groups 0 and 0.5 mg/m³ tolerated the exposure without specific signs.
4 mg/m³/males: Nostrils: red encrustations.
35 mg/m³/males: Irregular and labored breathing patterns, motility reduced, atony, piloerection, high-legged gait, nose: red encrustations, stridor, nostrils: red encrustations, bradypnea, head (hairs): red-brownish stains, dorsal skin: areal redencrustations.
4 mg/³/females: Nostrils: red encrustations.
35 mg/m³/females: Irregular and labored breathing patterns, motility reduced, atony, piloerection, high-legged gait, nose: red encrustations, stridor, nostrils: red encrustations, head (hairs): red-brownish stains.
Collectively, the respective data presentations show that rats exposed at 35 mg/m³displayed concentration-dependent signs of respiratory distress during the exposureperiod with rapid recovery from the exposure day towards the following day before exposure (as indicated by the plateau during the exposure-free weekend).

- REFLEXES:
The examination of reflexes did not reveal any differences between control and dose groups.

- 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 low and mid dose. At the high exposure level there was evidence of a hypothermia which magnitude appears to level-off with increased study duration. 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:
The data in the respective representations show that no toxicologically consistent, i.e., clearly concentration- and/or time-dependent effect on body weights occured up to the maximum concentration tested. Accordingly, as far as significant changes were sporadically, especially at the high exposure levelobserved they are considered to be of pathodiagnostic or prognostic relevance only at 35 mg/m³. This interpretation is also supported by the analysis of the cumulative body weight changes.

- 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. Accordingly, as far as significant changes were sporadically observed, especially at the high exposure level, they are considered to be of no pathodiagnostic or prognostic relevance. This interpretation is also supported by the analysis the of cumulative changes in food/water consumptions.


- BRONCHOALVEOLAR LAVAGE AND LUNG WEIGHTS:
The results did not show changes at 35 mg/m³ reflecting adversity. As far as significant effects occurred they appear to be related to the physiological removal and clearance of the inhaled aerosol. PMNs below 2% have no pathodiagnostic significance in rats due to the low-background levels in this species. Definite changes occurred at 4 and especially at 35 mg/m³. These effects were characterized by increased total cell numbers, neutrophilic
granulocytes (PMNs) (absolute and relative) and BAL y-GT. Significant elevations of BAL-protein and -LDH, and increased Type II cell activity (AP) occurred at 35 mg/m³ only. At the 35 mg/m³ exposure level alveolar macrophages appeared to be slightly more 'foamy'. Macrophages with phagocytized red blood cells or hemosiderin (subsumed as NCRBC) and other morphological changes (macrophages with translucent granular structures, subsumed under NC-PM) were attributed to the category 'non-classifiable cells'. It appears to be scientifically justified to assume the most of the NCs are
alveolar macrophages containing phagocytosed polymeric material (test substance) and associated pulmonary inflammation.

- HEMATOLOGY AND DIFFERENTIAL BLOOD COUNT:
There were no conclusive concentration-dependent changes with the exception of a trend of increased hepatoquick at 35 mg/m³. Collectively, the statistical significances observed are considered to be of no specific 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 35 mg/m³.

- ORGAN WEIGHTS:
Lung weights were increased at 4 and 35 mg/m³ with no reversibility at the end of the 4-week recovery period. Gradually, this response was more pronounced in males than in females. No other significant changes in organ weights or the organ-to-body weight or organ-to-brain weight ratios considered to be of pathodiagnostic significance occurred the remaining groups and organs.

- NECROPSY:
Necropsy findings were unremarkable and somewhat equivocal (discolorations only) at the low and intermediate exposure levels, respectively. In rats exposed at 35 mg/m³ the lungs appears to be less collapsed and with discolorations. Also the lung-associated lymph nodes were enlarged.

- HISTOPATHOLOGY:
In brief, histopathology revealed laryngeal epithelial alteration at 4 mg/m³ and above at the end of exposure period. These findings showed evidence of reversibility in most of the rats examined. Deposits of test substance was detected at 35 mg/m³ at both time points. A similar concentration-response relationship occurred in the lung and were characterized by increased mucus and/or cells in airways, thickened epithelium lining the airways, and hypercellularity of the epithelium of bronchiolo-alveolar junction. Both hematoxylin-eosin (H&E) and Sirius red - Fast green (SR-FG) staining
demonstrated increased mesenchymal reactions typical of a beginning interstitial fibrosis. Enlarged and/or foamy macrophages, laden with phagocytosed test substance, occurred with increased severity at 35 mg/m³. BALT appeared to be increased in some rats at 35 mg/m³. In the lung-associated lymph nodes (LALN) of the hilus region lymphatic activation and/or secondary follicles were observed at 4 and 35 mg/m³. After the 4 week recovery period, the hypercellularity of epithelial at the bronchiolo-alveolar junction showed evidence of regression, whilst the increased interstitial collagen did not. Clusters of macrophages, partly with phagocytosed test substance, were still observed. 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 as spontaneous findings from previous studies. Collectively,histopathology did not reveal changes considered to be adverse at 0.5 mg/m³.

Effect levels

Dose descriptor:

NOAEL

Effect level:

0.47 mg/m³ air (analytical)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: pulmonary irritation

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 RE (25 % Triphenylmethane-4,4',4''-triisocyanatein 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	---	---
	0.5	0 / 0 / 11	---	---
	4	0 / 3 / 11	0d-1d	---
	35	0 / 21/ 21	0d-56d	---
female	0 (Control)	0 / 0 / 10	---	---
	0.5	0 / 0 / 5	---	---
	4	0 / 1/ 5	0d	---
	35	0 / 15 / 15	0d-56d	---

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 the trade product (25 % Triphenylmethane-4,4',4''-triisocyanate in ethyl acetate). The mean actual concentrations (gravimetric) were 0.47, 3.9 and 34.9 mg Triphenylmethane-4,4',4''-triisocyanate/m³. Rats exposed under otherwise identical test conditions to dry air served as concurrent control group. Additional satellite group (control and high level exposure groups) were allowed to recover during a 4-week post-exposure period. Additional male rats/group were subjected to bronchoalveolar lavage at the end of the 4-week exposure period.

The rats exposed up to 0.47 mg/m³ did not display any substance-specific clinical signs while at 3.9 mg/m³ a low incidence of minimal findings occurred. Unequivocal evidence of substance-induced effects were observed at 34.9 mg/m³ . At this exposure level the following concentration-dependent clinical signs were recorded: irregular and labored breathing patterns, motility reduced, atony, piloerection, high-legged gait, nose: red encrustations, stridor, nostrils: red encrustations, bradypnea, head (hairs): red-brownish stains, dorsal skin: areal red encrustations. The signs of respiratory distress showed rapid recovery from one exposure day to the next. Hypothermia occurred in rats exposed at 34.9 mg/m³ only. Conclusive changes in reflexes and feed/water consumption were not observed. Body weights were mildly reduced at 34.9 mg/m³ (male rats only).

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 significantly increased lung weights at 3.9 and 34.9 mg/m³. Lung weights were still significantly increased at the end of the 4-week postexposure period.

Determinations in bronchoalveolar lavage fluids resulted in effects suggestive of alveolar irritation at 3.9 and 34.9 mg/m³ and included an increase of neutrophilic granulocyte and total cell counts and y-GT. Significant elevations of BAL-protein and -LDH, and increased Type II cell activity (AP) occurred at 34.9 mg/m3 only. At this exposure level alveolar macrophages appeared to be slightly more 'foamy'.

Histopathology revealed laryngeal epithelial alteration at 3.9 mg/m³ and above at the end of exposure period. These findings showed evidence of reversibility in most of the rats examined. Deposits of Triphenylmethane-4,4',4''-triisocyanate was detected at 34.9 mg/m³ at both time points. A similar concentration-response relationship occurred in the lung and were characterized by increased mucus and/or cells in airways, thickened epithelium lining the airways, and hypercellularity of the epithelium of bronchiolo-alveolar junction. Both hematoxylin-eosin (H&E) and Sirius red - Fast green (SR-FG) staining demonstrated increased mesenchymal reactions typical of a beginning interstitial fibrosis. Enlarged and/or foamy macrophages, laden with phagocytosed Triphenylmethane-4,4',4''-triisocyanate, occurred with increased severity at 34.9 mg/m³. BALT appeared to be increased in some rats at 34.9 mg/m³. In the LALN lymphatic activation and/or secondary follicles were observed at 3.9 and 34.9 mg/m³. After the 4 week recovery period, the hypercellularity of epithelial at the bronchiolo-alveolar junction showed evidence of regression, whilst the increased interstitial collagen did not. Clusters of macrophages, partly with phagocytosed Triphenylmethane-4,4',4''-triisocyanate, were still observed.

In summary, this study reveal robust evidence of pulmonary irritation at 3.9 mg/m³ and above. Taking all findings into account, 0.47 mg/m³ constitutes the no-observed-adverse-effect-level (NOAEL) for respiratory tract irritation. In regard to extrapulmonary toxicity, no effects were found up to the maximum concentration examined.