HDI oligomers, uretdione

Endpoint summary

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 guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2009)

GLP compliance:

yes (incl. QA statement)

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, The Netherlands
- Age at study initiation: 2 months old at first exposure day
- Weight at study initiation: At the study start the variation of individual weights did essentially not exceed ±10 per cent of the group means
- Housing: singly in conventional Makrolon® Type IIIh cages
- Diet and water: ad libitum
- Acclimation period: approximately 2 weeks

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:

air

Remarks:

unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Throughout the test groups the average MMAD was in the range of 1.3-1.5 μm (GSD ≈1.9).

Details on inhalation exposure:

- Exposure 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 is 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.
- Method of holding animals in test chamber: 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). The chambers used are commercially available (TSE, DE-61348 Bad Homburg, Germany) and the performance as well as their validation has been published (Pauluhn, J. Appl. Toxicology, 14, 55-62, 1994; Pauluhn and Thiel, J. Appl. Toxicol. 27, 160-167, 2007). Each segment of the aluminum inhalation chamber had 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 ‘two segment’ chamber were used in all groups.
- Method of conditioning 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. 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/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 on-line 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.
- System of generating particulates/aerosols: Under dynamic conditions the various concentrations of the test substance were atomized into the baffle (pre-separator) of the inhalation chamber. For atomization a binary nozzle and conditioned compressed air (15 L/min) was used. The representative dispersion pressure was approximately 600 kPa (constant liquid feed 10 μL/min through the nozzle maintained at 40°C to decrease the viscosity of the test article). The targeted concentrations were achieved by using additional air pull/push extraction/substitution dilution cascades. The neat test article was fed into the nozzle system using a digitally controlled pump (Harvard PHD 2000 infusion pump). In order to increase the efficiency of the generation of fine particles likely to evaporate and to prevent larger particles from entering the chamber a glass-pre-separator/baffle system was used (Tillery, Environ Health Perspect. 16, 25–40, 1976). The lower analytical concentrations compared with the nominal concentrations are attributed to the efficient removal of larger particles in the baffle/pre-separator system.
- Temperature, humidity, pressure in air chamber: Temperature and humidity are measured by the Data Acquisition and Control System using calibrated sensors (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 (Journal of Research of the National Bureau of Standards, Vol. 81 A, no. 1, Jan.-Febr. 1977) and Pauluhn (J. Appl. Toxicology, 14, 55-62, 1994) in a two-point calibration at 33% (MgCl2) and at 75% (NaCl) relative humidity. The calibration of the temperature sensors is also checked at two temperatures using reference thermometers. The mean chamber temperatures were 21.6, 22.0, 21.8 and 22.2°C for the control group and the 3 concentration groups, the mean chamber humidities 5.1, 5.2, 5.1 and 5.2%.
- Air change rate/ Air flow rate: 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 a chamber equilibrium is attained in less than one minute of exposure. 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. 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 (Bios DryCal Defender 510; http://www.smglink.com/bios/drycal_defender/drycaldefender.html) and were checked for correct performance at regular intervals.
- Method of particle size determination: 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 generally established procedures. The aerosol mass < 3 µm was 86.5, 90.2 and 86.7% for the 0.4, 2.0 and 10.0 mg/m³ target concentration group, respectively.
- Treatment of exhaust air: The exhaust air was purified via filter systems.

TEST ATMOSPHERE
- Brief description of analytical method used: The test-substance concentration was determined by gravimetric analysis (filter: glass-fiber filter, Sartorius, Göttingen, Germany; digital balance). The weight of filters (pre-conditioned) was determined shortly and after sampling. This method was used to define the actual total mass concentrations. Optimally, 2-3 samples per exposure day were collected from each exposure chamber.
To monitor the integrity and stability of the aerosol generation and exposure system was performed by using a Microdust Pro real-time aerosol photometer (Casella, USA).
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test-substance concentration was determined by gravimetric analysis (filter: glass-fiber filter, Sartorius, Göttingen, Germany; digital balance). Analytical and the real-time monitoring of the aerosol test atmosphere from the breathing zone indicated that the exposure conditions were temporally stable over the daily exposure period. Analysis of the aerosol particle-size distribution from the breathing zone samples demonstrated that the aerosol generated was of adequate respirability.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
0.4, 2, 10 mg/m³
Basis:
other: target conc.

Remarks:

Doses / Concentrations:
0.41, 2.2, 10.1 mg/m³
Basis:
analytical conc.

No. of animals per sex per dose:

5; additionally 5 animals/sex were used for recovery group (control and high-level exposure groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: As basis for dose selection served two Acute Inhalation Toxicity studies (see chapter Acute toxicity: inhalation; report AT03094; 2006 and report AT00075, 2002, both Bayer AG).

- Post-exposure recovery period in satellite groups:
5 further animals/sex were used for a post-exposure recovery period of 4 weeks for control and high dose group.
6 additional male rats per concentration were set up for examinations of bronchoalveolar lavage at the end of the 4 -weeks exposure period.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The appearance and behavior of each rat was examined carefully at least twice on exposure days (before and after exposure) and once a day during the exposure-free recovery period. If considered applicable due to unequivocal signs, in nose-only exposed rats observations were also made during exposure. 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 well 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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. The following reflexes were tested: visual placing response and for limb grip strength on wire mesh, abdominal muscle tone, corneal and pupillary reflexes, pinnal reflex, righting reflex, tail-pinch response, startle reflex with respect to behavioral changes stimulated by sounds (finger snapping) and touch (back).

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of all animals were measured before exposure, on a twice per week basis on Fridays and Mondays during the exposure period and once weekly (Mondays) during the postexposure period.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
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, pupil, 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.

HAEMATOLOGY: Yes
General clinical pathology was performed at the end of the exposure period on 5 animals/sex/group.The terminal blood samples were obtained by cardiac puncture of the deeply anesthetized, non-fasted rats (Narcoren®; intraperitoneal injection).
- Parameters examined: Hematrocrit, 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), Prothrombin time (PT, Quick value, “Hepato Quick”).

CLINICAL CHEMISTRY: Yes
General clinical pathology was performed at the end of the exposure period on 5 animals/sex/group.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 sampled after the urine collection period from the caudal vein of non-fasted rats.
Time schedule for collection of blood:
- Parameters examined: Aspartate aminotransferase optimized (ASAT), Alanine aminotransferase optimized (ALAT), Glutamate dehydrogenase (GLDH), γ-Glutamylaminotransferase (γ-GT), Lactate dehydrogenase (LDH), Alkaline phosphatase (APh), Albumin, Bilirubin (total), Blood glucose, Calcium,
Chloride, Cholesterol, Creatinine, Magnesium, Phosphate, Potassium, Sodium, Total protein, Triglycerides, Urea.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All surviving rats were sacrificed at the end of the exposure and postexposure observation period. All rats, irrespective of the day of death, were given a grosspathological 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.
The following organs were weighted at necropsy after exsanguination: Adrenal glands, Brain, Heart, Kidneys, Liver, Lung (incl. trachea), Ovaries, Spleen, Testes, Thymus. No organ weight data were collected from animals found dead. Paired organs were weighted together. Absolute weight, organ-to-body/brain weight ratios are reported.

HISTOPATHOLOGY: Yes
The entire respiratory tract was examined by light microscopy in all main and satellite group rats, whilst additional organs were examined in the control and high-level exposure groups only, unless treatment-dependent effects were observed.

Other examinations:

- Rectal temperatures: The rectal (colonic) temperatures were measured at several time points shortly after cessation of exposure (within ½ hour of cessation of exposure). Measurements were made in 5 rats/sex/main group following the first exposure day, at study midterm and towards the end of the exposure period.
- Bronchoalveolar Lavage: Samples of bronchoalveolar lavage fluid were collected from the lungs of rats (six male rats/group and time point) one day after the last challenge exposure. Within the acellular supernatant of BAL-fluid (BALF), several indicators of pulmonary damage were assessed: Lactate dehydrogenase (LDH), Total protein, N-Acetylglucosaminidase (ß-NAG), γ-Glutamyltransferase (γ-GT), Total number of lavaged cells, including the volume and diameter, Cytodifferentiation.

Statistics:

Descriptive analysis: All variables that are not dichotomous are described by 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 × 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.

Details on results:

CLINICAL SIGNS AND MORTALITY
All exposures were tolerated without test substance-induced mortality.
The number of animals showing signs and symptoms is depicted below:
(sex: concentration - number of animals with signs after exposure cessation/number of animals exposed (onset and duration of signs))
Males: 0 mg/m³ - 0/16, 0.41 mg/m³ - 0/11, 2.2 mg/m³ - 2/11 (23d-25d, 29d), 10.15 - 16/16 (2d-30d)
Females: 0 mg/m³ - 0/10, 0.41 mg/m³ - 0/5, 2.2 mg/m³ - 0/5, 10.15 mg/m³ - 10/10 (2d-45d)
One female rat (#81, 10.15 mg/m³) was necropsied in a moribund state after exposure on day 16. Some rats had nasal plugs (apparently polymerized test substance) which was mechanically removed from the nares. Neither gross necropsy nor histopathology revealed any specific cause of the state of morbidity of rat #81. This supports the notion that the death of rat#81 was causally related to nasal plugs which, therefore, is attributed not test article-induced.
The rats exposed up to 2.2 mg/m³ did not display substance-specific clinical signs. At the next higher concentration (10.15 mg/m³) the following clinical signs were recorded: bradypnea, labored breathing patterns, dyspnea, breathing sounds, stridor, runny nose (serous), nostrils: red encrustations, nose: nasal plugs removed, motility reduced, limp, gait high-legged, piloerection, hair-coat ungroomed, cyanosis, and emaciation.
The qualitative examination of reflexes did not reveal any differences between the groups.

BODY WEIGHT AND WEIGHT GAIN
The data in the respective representation show that there was no toxicologically consistent, i.e., concentration- or time-dependent effect on body weights up to the maximum concentration tested. 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.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmology performed did not reveal any conclusive evidence of test substance-induced changes in the dioptric media or in the fundus.

HAEMATOLOGY
There were no conclusive concentration-dependent changes with the exception of increased red blood cell (RBC) endpoints at 10 mg/m³ in male rats. Due to the absence of any RBC-related adverse characteristics this finding in male rats appears to be more reflective of a state of hypovolemia rather than erythrotoxicity. Therefore, the isolated statistical significances are considered to be of no pathodiagnostic significance.

CLINICAL CHEMISTRY
There were no concentration-dependent changes.

ORGAN WEIGHTS
Lung weights were increased at 10 mg/m³. No other significant changes in organ weights or the organ-to-body weight or organ-to-brain weight ratios considered to be of pathognostic significance occurred the remaining organs.

GROSS PATHOLOGY
Necropsy findings were unobtrusive up to 2 mg/m³. In rats exposed at 10 mg/m³ lung-associated lymph nodes appeared to be enlarged. Other respiratory tract-specific findings were not found.

HISTOPATHOLOGY: NON-NEOPLASTIC
Inflammatory findings and epithelial changes existed at larynx in all groups of rats exposed to the test substance with a concentration-dependent severity of lesions. In the lungs, minimal or slight thickening of the airway epithelium occurred at 10 mg/m³, associated with a minimal or slight hypercellularity of the bronchiolo-alveolar junction. This was complemented by alveolar histiocytosis (increased numbers of alveolar macrophages) at 2 and 10 mg/m³. In lung associated lymph nodes an increased cellularity of the paracortex (minimal or slight) was seen in rats exposed at 10 mg/m³. After the four week recovery period, increased inflammatory infiltrates and epithelial changes were still apparent in the larynx; however, with clear evidence of a diminution of intensity. The severity of lesions was more pronounced in female rats as compared to male rats. All other findings seen in the extrapulmonary organs/tissues evaluated were equally distributed across groups and/or are known as spontaneous findings from previous studies.

OTHER FINDINGS
- Rectal temperatures: Determinations did not reveal any significant differences between the groups.
- Bronchoalveolar Lavage: The average recovery of bronchoalveolar lavage fluid (BAL) was approximately 85% of the instilled volume and was similar
amongst the groups. The examination of Bronchoalveolar Lavage fluid show borderline statistically significant changes at 2.2 and definite changes at 10.15 mg/m³. These effects were characterized by increased neutrophilic granulocytes (PMNs), BAL-protein, LDH, and γ-GT. At 2.2 and 10.15 mg/m³ alveolar macrophages containing cellular debris and, possibly, polymerized reaction products with the test substance were apparent.

Dose descriptor:

NOAEL

Effect level:

0.41 mg/m³ air

Sex:

male/female

Basis for effect level:

other: Borderline but statistically significant changes in BAL fluid and alveolar histiocytosis in lung histopathology, both at next higher dose level (2.2 mg/m³)

Dose descriptor:

LOAEL

Effect level:

2.2 mg/m³ air

Sex:

male/female

Basis for effect level:

other: Adverse changes confined to portal-of-entry related local irritant effects (larynx, airways, and alveoli) at and above this dose level of 2.2 mg/m³

Critical effects observed:

not specified

The study revealed evidence of mild-to-moderate and transient laryngeal irritation at 2.2 and 10.1 mg/m³. The minimal epithelia laryngeal changes and squamous metaplasia occurring at 0.41 mg/m³ are not regarded to be adverse based on published evidence (Kaufmann et al., Exp. Toxicol. Pathol. 61: 591-603, 2009). Moreover, in this context, it is important to recall that in the rat the layout of the upper respiratory structures is tandem such that the air could travel in nearly linear fashion from the nose to the bifurcation of the trachea. The linear arrangement of the upper airways allows the larynx of rats to lie close to the posterior edge of the oral and nasal cavities. In the condition, the epiglottis lies against the soft palate. The apposition of the epiglottis to the soft palate in the resting condition isolates the oral cavity from the respiratory airways and makes the rat an obligatory nose breather (virtually no oropharynx). Thus, the direction of flow in rats is almost linear whereas in humans it is rectangular (DeSesso, Quality Assurance Good Practice, Regulation, and Law 2, 213-231, 1993). Thus, from an anatomical perspective, such laryngeal findings appear to be relatively rat specific.

Executive summary:

A subacute (4 weeks) inhalation toxicity study according to OECD TG 412 was conducted with 5 rats/sex for test and control groups.

Animals of the study were nose-only exposed to the aerosolised test substance at concentrations of 0 (vehicle control), 0.41, 2.2, and 10.1 mg/m³. The aerosol was of adequate respirability for the rats (MMAD 1.3-1.5 μm, GSD approx. 1.9).

In case of control and high dose group 5 further animals/sex were used for a post-exposure recovery period of 4 weeks. Furthermore, for examinations of bronchoalveolar lavage at the end of the 4 -weeks exposure period 6 additional male rats per concentration group were set.

As overall result, the study revealed evidence of mild-to-moderate and transient laryngeal irritation at 2.2 and 10.1 mg/m³. Minimal epithelia laryngeal changes and squamous metaplasia occurring at 0.41 mg/m³ were not regarded to be adverse based on published evidence (Kaufmann et al., Exp. Toxicol. Pathol., 61, 2009, 591 -603). Adverse changes in this study (on larynx, airways, alveoli) were confined to portal-of-entry related local irritant effects of the test substance aerosol. For 2.2 mg/m³ these effects were only apparent as borderline changes in bronchoalveolar lavage and lung histopathology (alveolar histiocytosis). Thus, 0.41 mg/m³ constituted 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

Study duration:

subacute

Species:

rat

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 guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2009)

GLP compliance:

yes (incl. QA statement)

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, The Netherlands
- Age at study initiation: 2 months old at first exposure day
- Weight at study initiation: At the study start the variation of individual weights did essentially not exceed ±10 per cent of the group means
- Housing: singly in conventional Makrolon® Type IIIh cages
- Diet and water: ad libitum
- Acclimation period: approximately 2 weeks

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:

air

Remarks:

unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Throughout the test groups the average MMAD was in the range of 1.3-1.5 μm (GSD ≈1.9).

Details on inhalation exposure:

- Exposure 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 is 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.
- Method of holding animals in test chamber: 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). The chambers used are commercially available (TSE, DE-61348 Bad Homburg, Germany) and the performance as well as their validation has been published (Pauluhn, J. Appl. Toxicology, 14, 55-62, 1994; Pauluhn and Thiel, J. Appl. Toxicol. 27, 160-167, 2007). Each segment of the aluminum inhalation chamber had 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 ‘two segment’ chamber were used in all groups.
- Method of conditioning 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. 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/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 on-line 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.
- System of generating particulates/aerosols: Under dynamic conditions the various concentrations of the test substance were atomized into the baffle (pre-separator) of the inhalation chamber. For atomization a binary nozzle and conditioned compressed air (15 L/min) was used. The representative dispersion pressure was approximately 600 kPa (constant liquid feed 10 μL/min through the nozzle maintained at 40°C to decrease the viscosity of the test article). The targeted concentrations were achieved by using additional air pull/push extraction/substitution dilution cascades. The neat test article was fed into the nozzle system using a digitally controlled pump (Harvard PHD 2000 infusion pump). In order to increase the efficiency of the generation of fine particles likely to evaporate and to prevent larger particles from entering the chamber a glass-pre-separator/baffle system was used (Tillery, Environ Health Perspect. 16, 25–40, 1976). The lower analytical concentrations compared with the nominal concentrations are attributed to the efficient removal of larger particles in the baffle/pre-separator system.
- Temperature, humidity, pressure in air chamber: Temperature and humidity are measured by the Data Acquisition and Control System using calibrated sensors (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 (Journal of Research of the National Bureau of Standards, Vol. 81 A, no. 1, Jan.-Febr. 1977) and Pauluhn (J. Appl. Toxicology, 14, 55-62, 1994) in a two-point calibration at 33% (MgCl2) and at 75% (NaCl) relative humidity. The calibration of the temperature sensors is also checked at two temperatures using reference thermometers. The mean chamber temperatures were 21.6, 22.0, 21.8 and 22.2°C for the control group and the 3 concentration groups, the mean chamber humidities 5.1, 5.2, 5.1 and 5.2%.
- Air change rate/ Air flow rate: 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 a chamber equilibrium is attained in less than one minute of exposure. 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. 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 (Bios DryCal Defender 510; http://www.smglink.com/bios/drycal_defender/drycaldefender.html) and were checked for correct performance at regular intervals.
- Method of particle size determination: 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 generally established procedures. The aerosol mass < 3 µm was 86.5, 90.2 and 86.7% for the 0.4, 2.0 and 10.0 mg/m³ target concentration group, respectively.
- Treatment of exhaust air: The exhaust air was purified via filter systems.

TEST ATMOSPHERE
- Brief description of analytical method used: The test-substance concentration was determined by gravimetric analysis (filter: glass-fiber filter, Sartorius, Göttingen, Germany; digital balance). The weight of filters (pre-conditioned) was determined shortly and after sampling. This method was used to define the actual total mass concentrations. Optimally, 2-3 samples per exposure day were collected from each exposure chamber.
To monitor the integrity and stability of the aerosol generation and exposure system was performed by using a Microdust Pro real-time aerosol photometer (Casella, USA).
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test-substance concentration was determined by gravimetric analysis (filter: glass-fiber filter, Sartorius, Göttingen, Germany; digital balance). Analytical and the real-time monitoring of the aerosol test atmosphere from the breathing zone indicated that the exposure conditions were temporally stable over the daily exposure period. Analysis of the aerosol particle-size distribution from the breathing zone samples demonstrated that the aerosol generated was of adequate respirability.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
0.4, 2, 10 mg/m³
Basis:
other: target conc.

Remarks:

Doses / Concentrations:
0.41, 2.2, 10.1 mg/m³
Basis:
analytical conc.

No. of animals per sex per dose:

5; additionally 5 animals/sex were used for recovery group (control and high-level exposure groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: As basis for dose selection served two Acute Inhalation Toxicity studies (see chapter Acute toxicity: inhalation; report AT03094; 2006 and report AT00075, 2002, both Bayer AG).

- Post-exposure recovery period in satellite groups:
5 further animals/sex were used for a post-exposure recovery period of 4 weeks for control and high dose group.
6 additional male rats per concentration were set up for examinations of bronchoalveolar lavage at the end of the 4 -weeks exposure period.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The appearance and behavior of each rat was examined carefully at least twice on exposure days (before and after exposure) and once a day during the exposure-free recovery period. If considered applicable due to unequivocal signs, in nose-only exposed rats observations were also made during exposure. 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 well 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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. The following reflexes were tested: visual placing response and for limb grip strength on wire mesh, abdominal muscle tone, corneal and pupillary reflexes, pinnal reflex, righting reflex, tail-pinch response, startle reflex with respect to behavioral changes stimulated by sounds (finger snapping) and touch (back).

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of all animals were measured before exposure, on a twice per week basis on Fridays and Mondays during the exposure period and once weekly (Mondays) during the postexposure period.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
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, pupil, 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.

HAEMATOLOGY: Yes
General clinical pathology was performed at the end of the exposure period on 5 animals/sex/group.The terminal blood samples were obtained by cardiac puncture of the deeply anesthetized, non-fasted rats (Narcoren®; intraperitoneal injection).
- Parameters examined: Hematrocrit, 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), Prothrombin time (PT, Quick value, “Hepato Quick”).

CLINICAL CHEMISTRY: Yes
General clinical pathology was performed at the end of the exposure period on 5 animals/sex/group.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 sampled after the urine collection period from the caudal vein of non-fasted rats.
Time schedule for collection of blood:
- Parameters examined: Aspartate aminotransferase optimized (ASAT), Alanine aminotransferase optimized (ALAT), Glutamate dehydrogenase (GLDH), γ-Glutamylaminotransferase (γ-GT), Lactate dehydrogenase (LDH), Alkaline phosphatase (APh), Albumin, Bilirubin (total), Blood glucose, Calcium,
Chloride, Cholesterol, Creatinine, Magnesium, Phosphate, Potassium, Sodium, Total protein, Triglycerides, Urea.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All surviving rats were sacrificed at the end of the exposure and postexposure observation period. All rats, irrespective of the day of death, were given a grosspathological 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.
The following organs were weighted at necropsy after exsanguination: Adrenal glands, Brain, Heart, Kidneys, Liver, Lung (incl. trachea), Ovaries, Spleen, Testes, Thymus. No organ weight data were collected from animals found dead. Paired organs were weighted together. Absolute weight, organ-to-body/brain weight ratios are reported.

HISTOPATHOLOGY: Yes
The entire respiratory tract was examined by light microscopy in all main and satellite group rats, whilst additional organs were examined in the control and high-level exposure groups only, unless treatment-dependent effects were observed.

Other examinations:

- Rectal temperatures: The rectal (colonic) temperatures were measured at several time points shortly after cessation of exposure (within ½ hour of cessation of exposure). Measurements were made in 5 rats/sex/main group following the first exposure day, at study midterm and towards the end of the exposure period.
- Bronchoalveolar Lavage: Samples of bronchoalveolar lavage fluid were collected from the lungs of rats (six male rats/group and time point) one day after the last challenge exposure. Within the acellular supernatant of BAL-fluid (BALF), several indicators of pulmonary damage were assessed: Lactate dehydrogenase (LDH), Total protein, N-Acetylglucosaminidase (ß-NAG), γ-Glutamyltransferase (γ-GT), Total number of lavaged cells, including the volume and diameter, Cytodifferentiation.

Statistics:

Descriptive analysis: All variables that are not dichotomous are described by 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 × 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.

Details on results:

CLINICAL SIGNS AND MORTALITY
All exposures were tolerated without test substance-induced mortality.
The number of animals showing signs and symptoms is depicted below:
(sex: concentration - number of animals with signs after exposure cessation/number of animals exposed (onset and duration of signs))
Males: 0 mg/m³ - 0/16, 0.41 mg/m³ - 0/11, 2.2 mg/m³ - 2/11 (23d-25d, 29d), 10.15 - 16/16 (2d-30d)
Females: 0 mg/m³ - 0/10, 0.41 mg/m³ - 0/5, 2.2 mg/m³ - 0/5, 10.15 mg/m³ - 10/10 (2d-45d)
One female rat (#81, 10.15 mg/m³) was necropsied in a moribund state after exposure on day 16. Some rats had nasal plugs (apparently polymerized test substance) which was mechanically removed from the nares. Neither gross necropsy nor histopathology revealed any specific cause of the state of morbidity of rat #81. This supports the notion that the death of rat#81 was causally related to nasal plugs which, therefore, is attributed not test article-induced.
The rats exposed up to 2.2 mg/m³ did not display substance-specific clinical signs. At the next higher concentration (10.15 mg/m³) the following clinical signs were recorded: bradypnea, labored breathing patterns, dyspnea, breathing sounds, stridor, runny nose (serous), nostrils: red encrustations, nose: nasal plugs removed, motility reduced, limp, gait high-legged, piloerection, hair-coat ungroomed, cyanosis, and emaciation.
The qualitative examination of reflexes did not reveal any differences between the groups.

BODY WEIGHT AND WEIGHT GAIN
The data in the respective representation show that there was no toxicologically consistent, i.e., concentration- or time-dependent effect on body weights up to the maximum concentration tested. 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.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmology performed did not reveal any conclusive evidence of test substance-induced changes in the dioptric media or in the fundus.

HAEMATOLOGY
There were no conclusive concentration-dependent changes with the exception of increased red blood cell (RBC) endpoints at 10 mg/m³ in male rats. Due to the absence of any RBC-related adverse characteristics this finding in male rats appears to be more reflective of a state of hypovolemia rather than erythrotoxicity. Therefore, the isolated statistical significances are considered to be of no pathodiagnostic significance.

CLINICAL CHEMISTRY
There were no concentration-dependent changes.

ORGAN WEIGHTS
Lung weights were increased at 10 mg/m³. No other significant changes in organ weights or the organ-to-body weight or organ-to-brain weight ratios considered to be of pathognostic significance occurred the remaining organs.

GROSS PATHOLOGY
Necropsy findings were unobtrusive up to 2 mg/m³. In rats exposed at 10 mg/m³ lung-associated lymph nodes appeared to be enlarged. Other respiratory tract-specific findings were not found.

HISTOPATHOLOGY: NON-NEOPLASTIC
Inflammatory findings and epithelial changes existed at larynx in all groups of rats exposed to the test substance with a concentration-dependent severity of lesions. In the lungs, minimal or slight thickening of the airway epithelium occurred at 10 mg/m³, associated with a minimal or slight hypercellularity of the bronchiolo-alveolar junction. This was complemented by alveolar histiocytosis (increased numbers of alveolar macrophages) at 2 and 10 mg/m³. In lung associated lymph nodes an increased cellularity of the paracortex (minimal or slight) was seen in rats exposed at 10 mg/m³. After the four week recovery period, increased inflammatory infiltrates and epithelial changes were still apparent in the larynx; however, with clear evidence of a diminution of intensity. The severity of lesions was more pronounced in female rats as compared to male rats. All other findings seen in the extrapulmonary organs/tissues evaluated were equally distributed across groups and/or are known as spontaneous findings from previous studies.

OTHER FINDINGS
- Rectal temperatures: Determinations did not reveal any significant differences between the groups.
- Bronchoalveolar Lavage: The average recovery of bronchoalveolar lavage fluid (BAL) was approximately 85% of the instilled volume and was similar
amongst the groups. The examination of Bronchoalveolar Lavage fluid show borderline statistically significant changes at 2.2 and definite changes at 10.15 mg/m³. These effects were characterized by increased neutrophilic granulocytes (PMNs), BAL-protein, LDH, and γ-GT. At 2.2 and 10.15 mg/m³ alveolar macrophages containing cellular debris and, possibly, polymerized reaction products with the test substance were apparent.

Dose descriptor:

NOAEL

Effect level:

0.41 mg/m³ air

Sex:

male/female

Basis for effect level:

other: Borderline but statistically significant changes in BAL fluid and alveolar histiocytosis in lung histopathology, both at next higher dose level (2.2 mg/m³)

Dose descriptor:

LOAEL

Effect level:

2.2 mg/m³ air

Sex:

male/female

Basis for effect level:

other: Adverse changes confined to portal-of-entry related local irritant effects (larynx, airways, and alveoli) at and above this dose level of 2.2 mg/m³

Critical effects observed:

not specified

The study revealed evidence of mild-to-moderate and transient laryngeal irritation at 2.2 and 10.1 mg/m³. The minimal epithelia laryngeal changes and squamous metaplasia occurring at 0.41 mg/m³ are not regarded to be adverse based on published evidence (Kaufmann et al., Exp. Toxicol. Pathol. 61: 591-603, 2009). Moreover, in this context, it is important to recall that in the rat the layout of the upper respiratory structures is tandem such that the air could travel in nearly linear fashion from the nose to the bifurcation of the trachea. The linear arrangement of the upper airways allows the larynx of rats to lie close to the posterior edge of the oral and nasal cavities. In the condition, the epiglottis lies against the soft palate. The apposition of the epiglottis to the soft palate in the resting condition isolates the oral cavity from the respiratory airways and makes the rat an obligatory nose breather (virtually no oropharynx). Thus, the direction of flow in rats is almost linear whereas in humans it is rectangular (DeSesso, Quality Assurance Good Practice, Regulation, and Law 2, 213-231, 1993). Thus, from an anatomical perspective, such laryngeal findings appear to be relatively rat specific.

Executive summary:

A subacute (4 weeks) inhalation toxicity study according to OECD TG 412 was conducted with 5 rats/sex for test and control groups.

Animals of the study were nose-only exposed to the aerosolised test substance at concentrations of 0 (vehicle control), 0.41, 2.2, and 10.1 mg/m³. The aerosol was of adequate respirability for the rats (MMAD 1.3-1.5 μm, GSD approx. 1.9).

In case of control and high dose group 5 further animals/sex were used for a post-exposure recovery period of 4 weeks. Furthermore, for examinations of bronchoalveolar lavage at the end of the 4 -weeks exposure period 6 additional male rats per concentration group were set.

As overall result, the study revealed evidence of mild-to-moderate and transient laryngeal irritation at 2.2 and 10.1 mg/m³. Minimal epithelia laryngeal changes and squamous metaplasia occurring at 0.41 mg/m³ were not regarded to be adverse based on published evidence (Kaufmann et al., Exp. Toxicol. Pathol., 61, 2009, 591 -603). Adverse changes in this study (on larynx, airways, alveoli) were confined to portal-of-entry related local irritant effects of the test substance aerosol. For 2.2 mg/m³ these effects were only apparent as borderline changes in bronchoalveolar lavage and lung histopathology (alveolar histiocytosis). Thus, 0.41 mg/m³ constituted 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:

LOAEC

2.2 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

NOAEC 0.41 mg/m³; at 2.2 (= LOAEC) only borderline but statistically significant changes in bronchoalveolar lavage and a finding in lung histopathology (alveolar histiocytosis) observed.

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

A subacute (4 weeks) inhalation toxicity study according to OECD TG 412 was conducted with 5 rats/sex for test and control groups. Animals of the study were nose-only exposed to the aerosolised test substance at concentrations of 0 (vehicle control), 0.41, 2.2, and 10.1 mg/m³. The aerosol was of adequate respirability for the rats (MMAD 1.3-1.5 μm, GSD approx. 1.9).

In case of control and high dose group 5 further rats/sex were used for a post-exposure recovery period of 4 weeks. Furthermore, for examinations of bronchoalveolar lavage at the end of the 4 -weeks exposure period 6 additional male rats per concentration were set.

As overall result, the study revealed evidence of mild-to-moderate and transient laryngeal irritation at 2.2 and 10.1 mg/m³. Adverse changes in this study (on larynx, airways, alveoli) were confined to portal-of-entry related local irritant effects of the test substance aerosol. For 2.2 mg/m³ these effects were only apparent as borderline changes in bronchoalveolar lavage and lung histopathology (alveolar histiocytosis). Thus, 0.41 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.

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

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

Justification for classification or non-classification

According to Regulation (EC) No 1272/2008, Annex I, no classification is warranted for repeated dose toxicity. Classification as STOT repeated is not justified, due to lack of cumulative toxicity. As it could be shown, no systemic effects were observed in the repeated dose study. The local effects observed did not give evidence for a major time-dependent change of the response threshold, as the changes associated with pulmonary irritation described in the repeated dose study match the single exposure (6 hours) pulmonary irritant potency study (report AT00075, Bayer AG, 2002; see chapter "Acute Toxicity: inhalation"). In this study 2.8 mg/m³ was considered to be the acute threshold of pulmonary irritation with changes in phospholipid homeostasis at higher exposure concentrations. The similarity of the acute irritant threshold concentration with the borderline pulmonary effect threshold concentration of 2.2 mg/m³ of the repeated dose inhalation toxicity study supports the conclusion that pulmonary effects are are caused by responses ensuing recurrent acute pulmonary irritation and are non-cumulative.