Tris(p-isocyanatophenyl) thiophosphate

Endpoint summary

Description of key information

This 4 week inhalation study did not reveal robust evidence of pulmonary irritation and injury rather than changes suggestive of increased accumulation of polymeric material engulfed by alveolar macrophages. Overall, as far as alterations from normal were observed they appear to follow a particle-overload-like phenomenon. Taking all findings into account, 2.8 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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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 conform Guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Principles of method if other than guideline:

The procedures calles for by OECD Guidance Document No. 39 were closely observed.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Hsd Cpb:WU (SPF)
- Source: Harlan-Nederland (NL), AD Horst
- Age at study initiation: approximately 2 months old
- Weight at study initiation: At the study start the variation of individual weights did not exceed ± 20 % of the arithmetic mean for each sex.
- Housing: singly in conventional Makrolon Type II cages.
- Diet and water: ad libitum
- Acclimation period: at least 5 days

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

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: conditioned air

Remarks on MMAD:

MMAD / GSD: 2.2 - 2.5 µm (GSD appr. 2)

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, 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 filter systems. These filters were disposed of by Bayer Pharma 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:

5 days/week, 6 hours/day

Remarks:

Doses / Concentrations:
0, 3, 15, and 75 mg/m³
Basis:
other: target concentrations

Remarks:

Doses / Concentrations:
0, 2.8, 15.4, 74.0 mg/m³
Basis:
other: actual concentrations

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

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: 3, 15, and 75 mg/m³. The top level is likely to elicit unequivocal respiratory tract toxicity without causing undue irritant stress or irreversible effects. Moreover, based on the findings of the pilot study, 3 mg/m³ is expected to be the NOAEL of study because the PMN recruitment observed at 26 mg/m³ was still in a range considered to be non-adverse. The interim concentration of 15 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 low and 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. Additional 6 male rats/group were subjected to bronchoalveolar lavage at the end of the 4-week exposure period.

Positive control:

none

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 postexposure 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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

labored breathing patterns, irregular breathing patterns, bradypnea, tachypnea, piloerection, motility reduced, atony, high-legged gait, nose: encrustations (red), nose: deposits (white), muzzle: deposits (white), and head: hair-coat red-brown stains.

Mortality:

mortality observed, treatment-related

Description (incidence):

labored breathing patterns, irregular breathing patterns, bradypnea, tachypnea, piloerection, motility reduced, atony, high-legged gait, nose: encrustations (red), nose: deposits (white), muzzle: deposits (white), and head: hair-coat red-brown stains.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increased lung and lung-associated lymph node weights; at 25.8 (borderline) and 74 (significant) mg/m³

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

The rats exposed up to 2.8 mg/m³ did not display substance-specific clinical signs while at 15.4 and 74 mg/m³ the following concentration-dependent clinical signs were recorded: labored breathing patterns, irregular breathing patterns, bradypnea, tachypnea, piloerection, motility reduced, atony, high-legged gait, nose: encrustations (red), nose: deposits (white), muzzle: deposits (white), and head: hair-coat red-brown stains. The signs of respiratory distress showed rapid recovery from one exposure day to the next. Hypothermia occurred in rats exposed at 74 mg/m³. Conclusive changes in reflexes, body weights, and feed/water consumption was not observed.
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 and lung-associated- lymph node weights at 25.8 (borderline) and 74 mg/m³ (significant). Lung weights were still significantly increased at the end of the 4-week postexposure period. Determinations in broncho-alveolar lavage fluids resulted in effects suggestive of alveolar irritation at 25.8 and 74 mg/m³ and included an increase neutrophilic granulocyte and total cell counts and g-GT. As far as significant effects occurred they appear to be related to the physiological removal and clearance of the inhaled aerosol. In none of the groups significant elevations of BAL-protein and -LDH, lysosomal (b-NAG), and increased Type II cell activity (AP) occurred. At the 74 mg/m³ exposure level alveolar macrophages appeared to be slightly more ‘foamy’; however, in the absence of any change suggestive of phospholipidosis. This findings is coherent with the absence of changes indicative of surfactant dysfunction.
Histopathology revealed at the end of the exposure period minimal epithelial alteration of the larynx in some rats exposed at 74 mg/m³. After the 4 week recovery period, epithelial alteration of the larynx could not be detected anymore. In the lungs, concentration dependent minimal to slight hypercellularity of the bronchiolo-alveolar junction was observed at both 25.8 and 74 mg/m³. Especially at 74 mg/m³ enlarged and/or foamy macrophages were observed. These findings were still apparent at the end of the recovery period. In the lung-associated-lymph nodes clusters of substance-laden macrophages were still found. 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 revealed no adverse effects at 2.8 mg/m³.

Dose descriptor:

NOAEL

Effect level:

2.8 mg/m³ air

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: particle-overload phenomenon in the respiratory tract

Dose descriptor:

NOAEL

Effect level:

74 mg/m³ air

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: extrapulmonary toxicity

Critical effects observed:

not specified

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 (6-hrs/day, 5 days/week) to Desmodur RFE (atomized neat of tris(p-isocyanatophenyl)thiophosphate in ethyl acetate). The mean actual concentrations of 2.8, 15.4, and 74.0 mg/m³ closely reflect the solvent-free tris(p-isocyanatophenyl)thiophosphate. Rats exposed under otherwise identical test conditions to conditioned air served as concurrent control group. Additional 5 male rats/sex/satellite group (control and high-level exposure groups) were allowed to recover during a 4-week postexposure 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 2.8 mg/m³ did not display substance-specific clinical signs while at 15.4 and 74 mg/m³ the following concentration-dependent clinical signs were recorded: labored breathing patterns, irregular breathing patterns, bradypnea, tachypnea, piloerection, motility reduced, atony, high-legged gait, nose: encrustations (red), nose: deposits (white), muzzle: deposits (white), and head: hair-coat red-brown stains. The signs of respiratory distress showed rapid recovery from one exposure day to the next. Ophthalmology was unremarkable. Hypothermia occurred in rats exposed at 74 mg/m³. Conclusive changes in reflexes,body weights, and feed/water consumption were not observed. There were no statistically significant or conclusive changes in absolute or relative organ weights with the exception of increased lung and lung-associated- lymph node weights at 25.8 (borderline) and 74 mg/m³ (significant). 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 25.8 and 74 mg/m³ and included an increase of neutrophilic granulocyte and total cell counts and g-GT. As far as significant effects occurred they appear to be related to the physiological removal and clearance of the inhaled aerosol. In none of the groups significant elevations of BAL-protein and -LDH, lysosomal (b-NAG), and increased Type II cell activity (AP) occurred. At the 74 mg/m³ exposure level alveolar macrophages appeared to be slightly more ‘foamy’; however, in the absence of any change suggestive of phospholipidosis. This finding is coherent with the absence of changes indicative of surfactant dysfunction.

Histopathology revealed at the end of the exposure period minimal epithelial alteration of the larynx in some rats exposed at 74 mg/m³. After the 4 week recovery period, epithelial alteration of the larynx could not be detected anymore. In the lungs, concentration dependent minimal to slight hypercellularity of the bronchiolo-alveolar junction was observed at both 25.8 and 74 mg/m³. Especially at 74 mg/m³enlarged and/or foamy macrophages were observed. These findings were still apparent at the end of the recovery period. In the lung-associated-lymph nodes clusters of substance-laden macrophages were still found. 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 revealed no adverse effects at 2.8 mg/m³.

In summary, this study did not reveal robust evidence of pulmonary irritation and injury rather than changes suggestive of increased accumulation of polymeric material engulfed by alveolar macrophages. Overall, as far as alterations from normal were observed they appear to follow a particle-overload-like phenomenon. Taking all findings into account, 2.8 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

74 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is GLP compliant and of high quality (Klinisch score = 1)

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

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 conform Guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Principles of method if other than guideline:

The procedures calles for by OECD Guidance Document No. 39 were closely observed.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Hsd Cpb:WU (SPF)
- Source: Harlan-Nederland (NL), AD Horst
- Age at study initiation: approximately 2 months old
- Weight at study initiation: At the study start the variation of individual weights did not exceed ± 20 % of the arithmetic mean for each sex.
- Housing: singly in conventional Makrolon Type II cages.
- Diet and water: ad libitum
- Acclimation period: at least 5 days

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

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: conditioned air

Remarks on MMAD:

MMAD / GSD: 2.2 - 2.5 µm (GSD appr. 2)

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, 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 filter systems. These filters were disposed of by Bayer Pharma 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:

5 days/week, 6 hours/day

Remarks:

Doses / Concentrations:
0, 3, 15, and 75 mg/m³
Basis:
other: target concentrations

Remarks:

Doses / Concentrations:
0, 2.8, 15.4, 74.0 mg/m³
Basis:
other: actual concentrations

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

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: 3, 15, and 75 mg/m³. The top level is likely to elicit unequivocal respiratory tract toxicity without causing undue irritant stress or irreversible effects. Moreover, based on the findings of the pilot study, 3 mg/m³ is expected to be the NOAEL of study because the PMN recruitment observed at 26 mg/m³ was still in a range considered to be non-adverse. The interim concentration of 15 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 low and 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. Additional 6 male rats/group were subjected to bronchoalveolar lavage at the end of the 4-week exposure period.

Positive control:

none

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 postexposure 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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

labored breathing patterns, irregular breathing patterns, bradypnea, tachypnea, piloerection, motility reduced, atony, high-legged gait, nose: encrustations (red), nose: deposits (white), muzzle: deposits (white), and head: hair-coat red-brown stains.

Mortality:

mortality observed, treatment-related

Description (incidence):

labored breathing patterns, irregular breathing patterns, bradypnea, tachypnea, piloerection, motility reduced, atony, high-legged gait, nose: encrustations (red), nose: deposits (white), muzzle: deposits (white), and head: hair-coat red-brown stains.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increased lung and lung-associated lymph node weights; at 25.8 (borderline) and 74 (significant) mg/m³

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

The rats exposed up to 2.8 mg/m³ did not display substance-specific clinical signs while at 15.4 and 74 mg/m³ the following concentration-dependent clinical signs were recorded: labored breathing patterns, irregular breathing patterns, bradypnea, tachypnea, piloerection, motility reduced, atony, high-legged gait, nose: encrustations (red), nose: deposits (white), muzzle: deposits (white), and head: hair-coat red-brown stains. The signs of respiratory distress showed rapid recovery from one exposure day to the next. Hypothermia occurred in rats exposed at 74 mg/m³. Conclusive changes in reflexes, body weights, and feed/water consumption was not observed.
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 and lung-associated- lymph node weights at 25.8 (borderline) and 74 mg/m³ (significant). Lung weights were still significantly increased at the end of the 4-week postexposure period. Determinations in broncho-alveolar lavage fluids resulted in effects suggestive of alveolar irritation at 25.8 and 74 mg/m³ and included an increase neutrophilic granulocyte and total cell counts and g-GT. As far as significant effects occurred they appear to be related to the physiological removal and clearance of the inhaled aerosol. In none of the groups significant elevations of BAL-protein and -LDH, lysosomal (b-NAG), and increased Type II cell activity (AP) occurred. At the 74 mg/m³ exposure level alveolar macrophages appeared to be slightly more ‘foamy’; however, in the absence of any change suggestive of phospholipidosis. This findings is coherent with the absence of changes indicative of surfactant dysfunction.
Histopathology revealed at the end of the exposure period minimal epithelial alteration of the larynx in some rats exposed at 74 mg/m³. After the 4 week recovery period, epithelial alteration of the larynx could not be detected anymore. In the lungs, concentration dependent minimal to slight hypercellularity of the bronchiolo-alveolar junction was observed at both 25.8 and 74 mg/m³. Especially at 74 mg/m³ enlarged and/or foamy macrophages were observed. These findings were still apparent at the end of the recovery period. In the lung-associated-lymph nodes clusters of substance-laden macrophages were still found. 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 revealed no adverse effects at 2.8 mg/m³.

Dose descriptor:

NOAEL

Effect level:

2.8 mg/m³ air

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: particle-overload phenomenon in the respiratory tract

Dose descriptor:

NOAEL

Effect level:

74 mg/m³ air

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: extrapulmonary toxicity

Critical effects observed:

not specified

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 (6-hrs/day, 5 days/week) to Desmodur RFE (atomized neat of tris(p-isocyanatophenyl)thiophosphate in ethyl acetate). The mean actual concentrations of 2.8, 15.4, and 74.0 mg/m³ closely reflect the solvent-free tris(p-isocyanatophenyl)thiophosphate. Rats exposed under otherwise identical test conditions to conditioned air served as concurrent control group. Additional 5 male rats/sex/satellite group (control and high-level exposure groups) were allowed to recover during a 4-week postexposure 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 2.8 mg/m³ did not display substance-specific clinical signs while at 15.4 and 74 mg/m³ the following concentration-dependent clinical signs were recorded: labored breathing patterns, irregular breathing patterns, bradypnea, tachypnea, piloerection, motility reduced, atony, high-legged gait, nose: encrustations (red), nose: deposits (white), muzzle: deposits (white), and head: hair-coat red-brown stains. The signs of respiratory distress showed rapid recovery from one exposure day to the next. Ophthalmology was unremarkable. Hypothermia occurred in rats exposed at 74 mg/m³. Conclusive changes in reflexes,body weights, and feed/water consumption were not observed. There were no statistically significant or conclusive changes in absolute or relative organ weights with the exception of increased lung and lung-associated- lymph node weights at 25.8 (borderline) and 74 mg/m³ (significant). 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 25.8 and 74 mg/m³ and included an increase of neutrophilic granulocyte and total cell counts and g-GT. As far as significant effects occurred they appear to be related to the physiological removal and clearance of the inhaled aerosol. In none of the groups significant elevations of BAL-protein and -LDH, lysosomal (b-NAG), and increased Type II cell activity (AP) occurred. At the 74 mg/m³ exposure level alveolar macrophages appeared to be slightly more ‘foamy’; however, in the absence of any change suggestive of phospholipidosis. This finding is coherent with the absence of changes indicative of surfactant dysfunction.

Histopathology revealed at the end of the exposure period minimal epithelial alteration of the larynx in some rats exposed at 74 mg/m³. After the 4 week recovery period, epithelial alteration of the larynx could not be detected anymore. In the lungs, concentration dependent minimal to slight hypercellularity of the bronchiolo-alveolar junction was observed at both 25.8 and 74 mg/m³. Especially at 74 mg/m³enlarged and/or foamy macrophages were observed. These findings were still apparent at the end of the recovery period. In the lung-associated-lymph nodes clusters of substance-laden macrophages were still found. 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 revealed no adverse effects at 2.8 mg/m³.

In summary, this study did not reveal robust evidence of pulmonary irritation and injury rather than changes suggestive of increased accumulation of polymeric material engulfed by alveolar macrophages. Overall, as far as alterations from normal were observed they appear to follow a particle-overload-like phenomenon. Taking all findings into account, 2.8 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

2.8 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is GLP compliant and of high quality (Klinisch score = 1)

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Tris(p-isocyanatophenyl) thiophosphate is marketed and handled as solution in ethyl acetate containing approximately 27% of tris(p-isocyanatophenyl) thiophosphate. The trade name of the solution is Desmodur RFE. Removal of the solvent from Desmodur RFE does invariably lead to generation of higher molecular weight species. This is due to the inherent reactivity of the isocyanate moieties and the process can thus be monitored via the decrease of the isocyanate content (see IUCLID section 1.4: analytical material balances before/after solvent removal). Therefore, the trade product Desmodur RFE (27% active ingredient in ethyl acetate) was employed as test substance for all toxicological tests as this was believed to best represent the substance to be registered. In toxicological studies with inhalative exposure to Desmodur RFE the solvent evaporated during the exposure procedure so that the samples taken from the breathing zone were nearly solvent free. Thus, in the inhalation studies the exposure can be considered as exposure to the neat active ingredient tris(p-isocyanatophenyl)thiophosphate.

In a subacute inhalation toxicity study (OECD TG 412) 5 male and 5 female Wistar rats per dose group were nose-only exposed (6-hrs/day, 5 days/week) to Desmodur RFE (atomized neat of tris(p-isocyanatophenyl)thiophosphate in ethyl acetate). The mean actual concentrations of 2.8, 15.4, and 74.0 mg/m³ closely reflect the solvent-free tris(p-isocyanatophenyl)thiophosphate.Rats exposed under otherwise identical test conditions to conditioned air served as concurrent control group. Additional 5 male rats/sex/satellite group (control and high-level exposure groups) were allowed to recover during a 4-week postexposure 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 2.8 mg/m³ did not display substance-specific clinical signs while at 15.4 and 74 mg/m³ the following concentration-dependent clinical signs were recorded: labored breathing patterns, irregular breathing patterns, bradypnea, tachypnea, piloerection, motility reduced, atony, high-legged gait, nose: encrustations (red), nose: deposits (white), muzzle: deposits (white), and head: hair-coat red-brown stains. The signs of respiratory distress showed rapid recovery from one exposure day to the next. Hypothermia occurred in rats exposed at 74 mg/m³. Conclusive changes in reflexes,body weights, and feed/water consumption were not observed.There were no statistically significant or conclusive changes in absolute or relative organ weights with the exception of increased lung and lung-associated- lymph node weights at 25.8 (borderline) and 74 mg/m³ (significant). Lung weights were still significantly increased end of the 4-week postexposure period. Determinations in broncho­alveolar lavage fluids resulted in effects suggestive of alveolar irritation at 25.8 and 74 mg/m³ and included an increase of neutrophilic granulocyte and total cell counts and g-GT. As far as significant effects occurred they appear to be related to the physiological removal and clearance of the inhaled aerosol. In none of the groups significant elevations of BAL-protein and -LDH, lysosomal (b-NAG), and increased Type II cell activity (AP) occurred. At the 74 mg/m³ exposure level alveolar macrophages appeared to be slightly more ‘foamy’; however, in the absence of any change suggestive of phospholipidosis. This finding is coherent with the absence of changes indicative of surfactant dysfunction.

Histopathology revealed at the end of the exposure periodminimal epithelial alteration of the larynx in some rats exposed at 74 mg/m³.After the 4 week recovery period, epithelial alteration of the larynx could not be detected anymore.In the lungs, concentration dependent minimal to slight hypercellularity ofthe bronchiolo-alveolar junction was observed at both25.8 and 74mg/m³.Especially at74 mg/m³enlarged and/or foamy macrophages were observed. These findings were still apparent at the end of the recovery period. In the lung-associated-lymph nodes clusters of substance-laden macrophages were still found.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 revealed no adverse effects at 2.8 mg/m³.

In summary, this study did not reveal robust evidence of pulmonary irritation and injury rather than changes suggestive of increased accumulation of polymeric material engulfed by alveolar macrophages. Overall, as far as alterations from normal were observed they appear to follow a particle-overload-like phenomenon. Taking all findings into account, 2.8 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.

Based on the unspecific effects observed in the subacute inhalation toxicity study it is not assumed that a longer treatment duration would substantially change the hazard assessment of the substance. For DNEL derivation after long-term exposure the conservative (default) time extrapolation factor of 6 was used to take into account the exposure duration subacute to chronic. The use of this conservative factor can be considered as worst-case extrapolation of the NOAEL. For particular substances that cause lung-overload it can be expected that an extended study duration would not significantly change the outcome of the study during homeostasis and as long as the overload condition is not reached.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
only one study available

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
only one study available

Justification for classification or non-classification

Based on the study results (see "Discussion") a classification according to Regulation (EC) No. 1272/2008 (CLP) is not warranted.