1,5-naphthylene diisocyanate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic 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:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,5-naphthylene diisocyanate

EC Number:

221-641-4

EC Name:

1,5-naphthylene diisocyanate

Cas Number:

3173-72-6

Molecular formula:

C12H6N2O2

IUPAC Name:

1,5-diisocyanatonaphthalene

Constituent 2

Reference substance name:

Desmodur 15

IUPAC Name:

Desmodur 15

Details on test material:

- Name of test material (as cited in study report): 1,5-Naphthylene diisocyanate (NDI)
- Physical state: solid
- Content (micronized): 99.5 %
- Re-analysis of test article after study: 96 %; NCO-content: 39.15 % (theoretical value: 40 %)
- Lot/batch No.: P3YE591000
- Storage condition of test material: room temperature/darkness; the head-space of containers was always purged with dry nitrogen after use.
- Micronization: The test article was of low dustiness. Therefore it was micronized by the performing laboratory in order to achieve the objective of test. For micronization a Retsch Centrifugal Ball Mill S100 was used. Grinding jars contained agate balls (4 of 40 mm balls). The micronization lasted 40 min at 500 revolutions/min (load: 100 g). Heat stress to the test article was minimized by this procedure. Prior to micronization the head space was purged with dry nitrogen.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Nederland (Horst, Netherlands)
- Age at study initiation: approx. 2 months
- Weight at study initiation: males: 204-246 g; females: 173-199 g
- Housing: individual
- 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:

air

Remarks on MMAD:

MMAD / GSD: In all exposure groups the MMAD was in the range of 2.1-2.6 µm ( GSD 1.7-2.0).

Details on inhalation exposure:

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

- DESCRIPTION OF APPARATUS:
Dry conditioned air was used to generate the test substance atmospheres. The test atmosphere was conveyed 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. Gas scrubbing devices were used for exhaust air clean-up. During sampling, the exhaust air was reduced in accordance with the sampling flow rate using a computerized Data Acquisition and Control System so that the total exhaust air flow rate was adjusted online and maintained at the specified 90%. The slight positive balance between the air volume supplied and extracted ensured that no passive influx of air into the exposure chamber occurred (via exposure restrainers or other apertures). The slight positive balance provides also adequate dead-space ventilation of the exposure restrainers. The pressure difference between the inner inhalation chamber and the exposure zone was 0.02 cm H20 (Pauluhn, 1994). The exposure system was accommodated in an adequately ventilated enclosure. Temperature and humidity are measured by the Data Acquisition and Control System using calibrated sensors. The sensors were located in the inhalation chamber.

- INHALATION CHAMBER:
The aluminum inhalation chamber has the following dimensions: inner diameter = 14 cm, outer diameter.= 35 cm (two-chamber system), height = 25 cm (internal volume = about 3.8 L). To be able to perform all measurements required to define exposure in a manner that is similar to the exposure of rats, a 'four segment' chamber were used in all groups. Details of this modular chamber and 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 [60 L/min x 60 min/(4 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:
Air flows are monitored and controlled continuously by calibrated mass flow meters (Hastings HFC-C Mass Flow Controllers, Teledyne Hastings-Raydist, Hampton, VA, USA). For analytical sampling TYLAN FC-280 S mass flow controllers are used (TYLAN General, Torrance, California, USA). The proper performance of the mass flow controllers was measured using a digital precision flow-meter calibration device (DryCal DC-2 primary gas flow meter). The calibration of mass flow controllers is performed by computer under actual operating conditions. Voltage specifications exceeding or falling below the specified range are indicated by an alarm/error listing. The Data Acquisition and Control System monitors/controls up to five inhalation chambers simultaneously.

- 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: A nominal concentration was not calculated since the construction and weight of the dust generator used did not allow for a precise measurement of the powder aerosolized (without dismantling the generator).
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 concentration.
Analytical concentration: The aerosolized test material (dry powder aerosol) was adsorbed on glass powder loaded with N-4-nitrobenzyl-N-n-propylamine solution (nitro reagent). The isocyanate component reacts to form the corresponding urea derivative. After desorption with nitro reagent solution these samples were diluted with a mixture of aqueous phosphoric acid/acetonitrile (1:9), the reaction product is quantified by high-performance liquid chromatography (HPLC; UV detection). Standard solutions of the authentic test item were used for calibration. Linearity, precision, specificity, robustness and accuracy of the analytical method were evaluated apart from this study and fulfil the predefined acceptance criteria. Additionally, system suitability tests in terms of specificity, precision and linearity indicated that qualified analytical procedures were followed during the study. As back-up methodology, selected glass fiber filters were also re-analyzed with the nitroreagent/HPLC method.
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 of test substance/m3 (gravimetric concentration).

- STABILITY OF TEST ATMOSPHERES:
The integrity stability of the aerosol generation system was monitored using a Microdust Pro (Casella) digital real-time aerosol photometer. At 4 mg/m3 only, a FHG SMZ-SE (Fraunhofer, Hannover, Germany) system was used. All 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 is 30 sec. This chamber monitoring allows for an overall survey of all 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 aerodynamic particle-size distribution were taken in the vicinity of the breathing zone. Cascade impactor analyses were not feasible at 0.06 and 0.25 mg/m3 as this would had required extensive sampling periods. Therefore, a TSI-Laser Velocimeter APS 3321 was used for particle size analyses in these groups. Due to the entirely different physical principle of measurement, this analysis was also performed in the remaining exposure groups for comparison. Technical details of this system have been described (Remiarz et al., 1983). The APS 3321 was used especially for determinations at low concentrations. Analyses performed at higher concentrations were made to allow comparisons with cascade impactor analyses. Therefore, 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 which was subjected to gravimetric analysis. An adhesive stage coating (silicone spray) was used to prevent particle bounce. 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:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.065, 0.25, 1.02, 3.96 mg/m3
Basis:
other: actual breathing zone concentration (gravimetric filter analyses)

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.035, 0.21, 0.94, 3.66 mg/m3
Basis:
other: analytical concentration (glass fiber filter)

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0.061, 0.22, 0.87, 3.33 mg/m3
Basis:
other: analytical concentration (glass fiber filter processed with nitro-reagent post-sampling)

No. of animals per sex per dose:

10 animals for dose groups 0.065, 0.25 and 1.02 mg/m3;
20 animals for controls and high dose groups 3.96 mg/m3 (each containing 10 animals for recovery groups)

Control animals:

yes, sham-exposed

Details on study design:

- DOSE SELECTION RATIONALE:
Based on a 2-week pilot study in rats (Pauluhn, 2008) it appears that NDI aerosol in concentrations in the range of 0.2 mg/m3 may cause distinct, although mild respiratory tract irritation following 13 weeks repeated inhalation exposure. Therefore, in this repeated subchronic exposure study the following target concentrations were selected: 0.06, 0.25, 1, and 4 mg/m3 . With regard to respiratory tract irritation 4 mg/m3 was estimated to be in the range of the irritant threshold concentration. At this exposure level sensory effects are minimal and undue distress or pain to experimental animals are unlikely to occur. In regard to laryngeal irritation borderline effects were already apparent at 0.2 mg/m3 . Accordingly, in regard to the anticipated most sensitive endpoint (Iaryngeal), 0.06 mg/m3 is considered to be the NOAEL while 0.25 to 1 mg/m3 was anticipated to be in the LOAEL range for alveolar irritation and increased lung weights.

- POST-EXPOSURE RECOVERY PERIOD:
Ten male and female rats each of the control and high dose groups were examined during a post-exposure recovery period of 1 month.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

- BODY WEIGHTS:
Body weights of all animals were measured before exposure on a twice per week basis. During the exposure-free recovery period the body weights were determined once per week.

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

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

- CLINICAL PATHOLOGY AND HEMATOLOGY:
General clinical chemistry tests were performed at the end of the 3 months exposure period and at the end of the recovery period on all animals. The terminal blood samples were obtained by cardiac puncture of the deeply anesthetized, non-fasted rats (Narcoren®; intraperitoneal injection). The blood required for the glucose determination was obtained during the last exposure week (after urine collection) from the caudal vein of non-fasted rats. Anticoagulant-coated tubes were used except for blood collected for to examine hemostasis endpoints where sodium citrate was used as anticoagulant.
Hematology: Hematrocit, Hemoglobin, Leukocytes, Erythrocytes, Mean corpuscular volume, Mean corpuscular hemoglobin concentration, Mean corpuscular hemoglobin, Thrombocyte count, Reticulocytes, Heinz Bodies, Leukocyte differential count (Lymphocytes, Granulocytes, Segmented neutrophils, Eosinophilic neutrophils, Basophils, Monocytes, Plasma cells, miscellaneous abnormal cell types).
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, Magnesium, Phosphate, Potassium, Sodium, Total protein, Triglycerides, Urea, Prothrombin time (PT, Ouick value, "Hepato Quick").

- URINALYSIS:
General urinalysis was performed towards the end of the 3 month study and 4 week post-exposure periods on 10 animals/sex/group. The rats' urine was individually collected overnight (approximately 3 p.m.to 7 a.m.) during the last study week from animals in metabolism cages (with watering bottles, pulverized Food ad libitum). The construction of the urine collection equipment did not allow contamination of urine with water or chow. The collection containers were cooled to nominal 12 °C in order to prevent/suppress uncontrolled growth of microorganisms overnight.
The following parameters were evaluated semiquantitatively (SQ) or quantitatively (Q) in the collected urine: .
Sediment composition (SQ), Urine osmolality (Q), pH (SQ), Volume (Q), Protein (SQ), Glucose (SQ), Blood (SQ), Bilirubin (SQ), Urobilinogen (SQ), Ketone bodies (SQ).

- 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 (HEINE) 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, Epididymides, Heart, Kidneys, Liver, Lung (incl. trachea), Ovaries, Spleen, Testes, Thymus.
No organ weight data were collected from animals found dead. Paired organs were weighted together.

- NECROPSY:
All surviving rats were sacrificed at the end of the respective 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 % hour of cessation of exposure) using a digital rectal probe (H. Sachs, March, Germany). Five rats/group/sex were examined after the first exposure, midterm and the end of the exposure period.

Statistics:

- DESCRIPTIVE ANALYSIS:
All variables that are not dichotomous are described by sex, dosage group and date using appropriate measures of central tendency (mean, median) and general variability (standard deviation, in most instances also minimum, maximum).

- STATISTICAL TESTS:
For the statistical evaluation of samples drawn from continuously distributed random variates three types of statistical tests are used, the choice of the test being a function of prior knowledge obtained in former studies. Provided that the variate in question can be considered approximately normally distributed with equal variances across treatments, the Dunnett test is used, if heteroscedasticity appeared to be more likely a p value adjusted Welch test is applied. If the evidence based on experience with historical data indicates that the assumptions for a parametric analysis of variance cannot be maintained, distribution-free tests in lieu of ANOVA are carried out, i.e. the Kruskal-Wallis test followed by adjusted MWW tests (U tests) where appropriate. Global tests including more than two groups are performed by sex and date, i.e. each sex x date level defines a family of tests in the context of multiple comparison procedures (Miller (1981). Within such a family, the experiment-wise error is controlled. If not otherwise noted, all pair-wise tests are two-sided comparisons.

Results and discussion

Results of examinations

Details on results:

- MORTALITY:
All exposures were tolerated without test substance-induced mortality. One female rat of the dose group 3.96 mg/m3 was killed in a moribund state on day 15. Moribundity was possibly restraint-related.

- CLINICAL SIGNS:
All rats of the dose groups 0.065, 0.25 and 1.02 mg/m3 tolerated the exposure without specific signs. In these groups there were incidental findings in individual animals (e.g., alopecia, piloerection, ungroomed hair-coat). They are not considered to be causally related to the test substance due to the absence of any time-dependent exacerbation or concentration-dependence. In the high dose group 3.96 mg/m3 the following substance- related clinical signs were recorded in 19/20 males and all females: Bradypnea, irregular breathing patterns, labored breathing patterns, dyspnea, breathing sounds, stridor, nasal discharge (serous), nostrils: red encrustations, eye lids: red encrustations, motility reduced, limp, high-legged gait, cyanosis, piloerection, haircoat ungroomed, and bloated abdomen (onset of signs in males on day 15 and in females on day 2).

- 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 any exposure concentration. Additionally, the temperature measurements made on control animals demonstrate clearly that the animal restrainer had no apparent effect on the body temperature (normal body temperature of the rats: 37.5°C - 38.5°C).

- BODY WEIGHTS:
There was no toxicologically consistent, i.e., concentration- or time-dependent effect on body weights up to and including 3.96 mg/m3. Statistical significant changes appeared to be related to an increase rather than a decrease in body weights (vs. air control). Accordingly, as far as significant changes were observed they are considered to be of no pathodiagnostic or prognostic relevance. This interpretation is also supported by the analysis of incremental body weight changes (body weight gain).

- FOOD CONSUMPTION:
There was no consistent evidence of effected food consumption across the exposure groups.

- WATER CONSUMPTION:
There was no consistently affected water consumption throughout the exposure period considered to be of toxicological significance.

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

- CLINICAL PATHOLOGY:
There were no concentration-dependent changes in any group, except statistically and concentration-dependently increased total bilirubin at 3.96 mg/m3. The other isolated statistical significances are considered to be of no pathodiagnostic or prognostic significance.

- Urinalysis:
There were no urine effects up to 3.96 mg/m3 considered to be of pathodiagnostic relevance.

- 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 3.96 mg/m3.

- ORGAN WEIGHTS:
Lung weights were significantly increased in both sexes at 1.02 and 3.96 mg/m3 and were not reversible within the 4-week post-exposure period. In addition, in the female rats exposed at 3.96 mg/m3, heart weights were significantly increased at the end of the post-exposure period. No other significant changes in organ weights or the organ-to-body weight or organ-to-brain weight ratios are considered to be of pathodiagnostic significance.

- NECROPSY:
All necropsy findings were unobtrusive in the control and dose groups up to 3.96 mg/m3.

- HISTOPATHOLOGY:
At the end of the exposure period, minimal or slight histopathological findings were observed in the nasal cavities, pharynx, larynx, trachea and lungs of rats exposed at 1.02 and 3.96 mg/m3. In the nasal cavities findings were characterized by epithelial degeneration and/or atrophy, goblet cell hyperplasia and increased inflammatory infiltrates. As can be appreciated from the degree of responses at the level I and IV of the nasal cavities, there is an apparent equal susceptibility of regions with respiratory epithelium and olfactory epithelium. This lack of specificity supports a non-specific
irritant mechanism. Likewise, also laryngeal epithelium shows irritant-related changes ranging from epithelial alterations, focal inflammatory infiltrations to epithelial squamous metaplasia especially at the ventral aspect of the larynx. In the lungs, bronchiolo-alveolar hypercellularity, minimal septal thickening, inflammatory infiltrates and increased alveolar macrophages with foamy appearance occurred. In addition, females of the dose group 0.25 mg/m3 showed minimal focal inflammatory infiltrations and epithelial alterations in the larynx. After 4 weeks of recovery, in the nasal cavities minimal or slight degeneration and/or atrophy of the olfactory epithelium was still apparent in all rats from the high dose group 3.96 mg/m3. Additionally, unusual nerve-like structures were obvious in the epithelium and neuronal degeneration of nerve bundles was detected in the lamina propria. In the lungs, minimal hypercellularity of the bronchiolo-alveolar region, an increased amount of macrophages and an increased incidence of minimal inflammatory infiltrates was still seen. Histopathological findings of the pharynx, larynx, and trachea recovered entirely. Overall, there was a tendency that female rats were mildly more susceptible than male ones. No respiratory tract lesions were still apparent in male and female rats at 0.25 and 1.02 mg/m3 after the 4-week recovery period.

Effect levels

1

Dose descriptor:

NOAEC

Effect level:

0.25 mg/m³ air

Based on:

test mat.

Sex:

male/female

2

Dose descriptor:

LOAEC

Effect level:

1.02 mg/m³ air

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Executive summary:

In a subchronic toxicity study (OECD TG 413) 10 male and 10 female Wistar rats per dose group were nose-only exposed for 13 weeks (6 hours /day, 5 days/week) to a solid aerosol of 1,5-Naphthylene diisocyanate (NDI). The mean actual breathing zone concentrations (gravimetric) were 0.065, 0.25, 1.02 and 3.96 mg/m3. Rats exposed under otherwise identical test conditions served as concurrent control group. Additional 10 male and 10 female rats were exposed in the air control and high dose exposure groups and were allowed to recover during a 4-week post-exposure period.

The rats exposed up to and including 1.02 mg/m3 did not display any clinical effects attributable to the test substance. At 3.96 mg/m3 animals displayed responses, such as bradypnea, irregular and labored breathing patterns, dyspnea, breathing sounds, stridor, nasal discharge (serous), nostrils: red encrustations, eye lids: red encrustations, motility reduced, limp, high-legged gait, cyanosis, piloerection, haircoat ungroomed, and bloated abdomen. In regard to visible signs of respiratory tract irritation, female rats appeared to be slightly more susceptible than male rats. Conclusive changes in body temperature, reflexes, body weights or food/water consumption did not occur. Ophthalmology was unobtrusive. There was no evidence of adverse hematological effects and clinical-pathology did not provide pathodiagnostically relevant evidence of NDI-aerosol induced effects. However, an isolated increase in total bilirubin was observed at 3.96 mg/m3 (trend in males, and significant elevations in females). Urinalysis was unremarkable. There were no statistically significant or conclusive changes in absolute or relative organ weights of adrenals, brain, epididymides, kidneys, liver, ovaries, spleen, testes, and thymus. However, at 1.02 and 3.96 mg/m3 , lung weights were significantly increased. In addition, significantly increased heart weights occurred in female rats exposed at 3.96 mg/m3. The elevations in organ weights were not reversible with the 4-week post-exposure period. After 13-weeks, minimal to slight histopathological findings observed in the nasal cavities, pharynx, larynx, trachea and lungs of rats exposed at 1.02 and 3.96 mg/m3 . In the nasal cavities findings were characterized by epithelial degeneration and/or atrophy, goblet cell hyperplasia and increased inflammatory infiltrates. As can be appreciated from the degree of responses at the level I and IV of the nasal cavities, there is an apparent equal susceptibility of regions with respiratory epithelium and olfactory epithelium. This lack of specificity supports a non-specific irritant mechanism. Likewise, also laryngeal epithelium shows irritant-related changes ranging from epithelial alterations, focal inflammatory infiltrations to epithelial squamous metaplasia especially at the ventral aspect of the larynx. In the lungs, bronchiolo-alveolar hypercellularity, minimal septal thickening, inflammatory infiltrates and increased alveolar macrophages with foamy appearance occurred. In addition, females of the dose group 0.25 mg/m3 showed minimal focal inflammatory infiltrations and epithelial alterations in the larynx. After 4 weeks of recovery, in the nasal cavities minimal or slight degeneration and/or atrophy of the olfactory epithelium was still apparent in all rats from the high dose group 3.96 mg/m3. Additionally, unusual nerve-like structures were obvious in the epithelium and neuronal degeneration of nerve bundles was detected in the lamina propria. In the lungs, minimal hypercellularity of the bronchiolo-alveolar region, an increased amount of macrophages and an increased incidence of minimal inflammatory infiltrates was still seen. Histopathological findings of the pharynx, larynx, and trachea recovered entirely. Overall, there was a tendency that female rats were mildly more susceptible than male ones. No respiratory tract lesions were still apparent in male and female rats at 0.25 and 1.02 mg/m3 after the 4-week recovery period.

In summary, this study demonstrates that the test substance causes respiratory tract irritation. The localization of irritation depends on the site of initial deposition and associated local response. Based on portal-of-entry-related endpoints NDI was tolerated without any local or systemic adverse effects up to 0.25 mg/m3. Conclusive evidence of respiratory irritation occurred at 1.02 and 3.96 mg/m3. With regard to histopathological changes, all changes observed were related to portal-of-entry; local irritant effects (nasal passages, larynx, airways and alveoli), i.e, changes that occurred at anatomical structures, to some extent rat-specific, favoring focal deposition of irritant particulates. The minimal laryngeal findings observed in female rats at 0.25 mg/m3 are assessed to be non-adverse. Based on the rationale given, taking all findings into account, 0.25 mg/m3 constitutes a no-observed-adverse-effect-level (NOAEL) based on effects related to respiratory tract irritation.