Registration Dossier

Administrative data

Description of key information

No repeated-dose toxicity tests are available for the oral or dermal route of exposure. Data waiver are claimed.

Furthermore, no subchronic inhalation toxicity study was performed with the registered substance. However, a grouping of substances and read-across approach according to Regulation (EC) No 1907/2006 (REACH), Annex XI 1.5 and following Scenario 4 of the ECHA Read-Across Assessment Framework, RAAF (2015) is accomplished for five blocked diisocyanate oligomers (also for the read-across target substance isophorone diisocyanate, oligomeristion product, blocked with caprolactam).

For repeated inhalation toxicity testing, the category is divided into two sub-groups – the liquid aerosol for blocked HDI-based oligomers and the powder aerosol for blocked IPDI-based oligomers. Thus, within the category the analogue approach is followed, i.e. ECHA RAAF Scenario 2.

Within this analogue approach, a GLP-conform and Guideline-compliant 90-day subchronic inhalation toxicity study was performed with the read-across source (isophorone diisocyanate, oligomeristion product, blocked with 2 -butanone oxime) for the registered substance on request of ECHA. In this subchronic inhalation toxicity study with 13 week exposure and 13 week recovery period the No-Observed-Adverse-Effect-Concentration (NOAEC) is 1.5 mg/m³ due to respiratory tract associated adverse findings seen in BAL, histopathology and weight increase of lung and LALN at 7.6 mg/m³. These effects represent portal-of-entry toxicity. Test substance related systemic toxicity was not observed and thus, the NOAEC for systemic toxicity is 7.6 mg/m³, the highest dose tested.

An updated outline of a grouping-strategy based on read-across of the available toxicological data for 5 blocked diisocyanate oligomers is attached to the endpoint summaries for 'Repeated dose toxicity' and 'Toxicity to reproduction' in IUCLID as a separate document.

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

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
(2009)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
GLP compliance:
yes (incl. certificate)
Remarks:
The diagnostic microbiological evaluation of blood, urine and renal tissue of rat number 21 was done under non-GLP condition. This deviation did not limit the assessment of the results obtained.
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (< 30°C) at darkness, headspace purge with N2 after opening of reservoir
- Stability under test conditions: Stability certified for the duration of study
- Solubility and stability of the test substance in the solvent/vehicle:
The integrity and stability of the aerosol generation and exposure system was measured by using a Microdust real-time aerosol photometer (Casella, Buffalo, UK) and at 0.3 mg/m³ the R&P Teom 1400a (amibent particulate monitor; Thermo Fisher Scientific Inc., USA). Samples were taken continuously from the vicinity of the breathing zone. Gravimetric analysis of filter samples and the real-time monitoring of the aerosol test atmosphere from the breathing zone area indicated that the exposure conditions were stable over the daily 6-h exposure period
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Healthy young adult SPF bred Wistar rats, strain Crl:(Wi)WU BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:(Wi)WU BR (SPF-bred)
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 2 months
- Weight at study initiation: Weight range at first exposure day: males 230-280 g. 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 with gnawing sticks
- Diet and water: ad libitum
- Acclimation period: at least 10-14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 40-60 %
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12 h/12 h
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
ca. 2.3 µm
Remarks on MMAD:
The mean Mass Median Aerodynamic Diameter’s (MMAD) measured by cascade impactor were in the range of 2.23 – 2.35 µm within the exposure groups (Geometric Standard Deviation: 1.86 – 2.30). MMADs measured by laser velocimetry ranged from 2.66 – 2.69 µm within the exposure groups (Geometric Standard Deviation: 1.72 – 1.73).
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Rats were exposed under dynamic directed-flow nose-only exposure conditions.
- Exposure apparatus: Details of the nose-only exposure system, including its validation, have been published previously (Pauluhn, Journal of Applied Toxicology 13, 1994, 55-62; Pauluhn and Thiel, J. Appl. Toxicol. 27, 2007, 160-167). 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).
- Method of holding animals in test chamber: Animals were exposed in polycarbonate 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.
- Source and rate of air: 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; SMG Interlink, USA) and TSI Mass Flow meter 4040 (TSI Incorporated, USA) and were checked for correct performance at regular intervals.
- 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 BEKO RA 55 compressed air dryer. Adequate control devices were employed to control supply pressure.
- Temperature, humidity: Temperature and humidity measurements are also performed by the computerized Data Acquisition and Control System using HC2-S3 sensors (Rotronic Messgeräte GmbH, Ettlingen, 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.
- Air flow rate: 5.85 L/min
- Air change rate: The test atmosphere generation conditions provide an adequate number of air exchanges per hour [45 L/min x 60 min/(11.4 L/chamber) = 237, continuous generation of test atmosphere]. Thus, under the test conditions used a chamber equilibrium is attained in less than one minute of exposure.
- Treatment of exhaust air: The exhaust air was purified via filter systems. These filters were disposed of by the laboratory.
The ratio between supply and exhaust air was selected so that 90 % of the supplied air was extracted via the exhaust air location and, if applicable, via sampling ports. Aerosol scrubbing devices were used for exhaust air clean-up. During sampling, the exhaust air was reduced in accordance with the sampling flow rate using a computerized Data Acquisition and Control System so that the total exhaust air flow rate was adjusted 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).
- Atmosphere generation: Test atmospheres were generated using a WRIGHT DUST FEEDER system (BGI Inc., Waltham, MA 02154, USA). For dry powder dispersion, conditioned compressed dry air (30 liters/min; generic dispersion pressure: 1.8 kPa) was used. The principle performance of the WRIGHT DUST FEEDER dust generating system can be described as follows: the test substance was metered in a reservoir and then was compressed to a pellet using approxi¬mately 1 metric ton by a carva laboratory press (F. S. Carver Inc., Wabash, IN 46992, USA). From this pellet defined amounts of test substance were scraped off and entrained into the main air flow.
The airborne powder was then conveyed into the inner cylinder of the inhalation chamber. Principles differences between chamber set-ups were related to the dilution cascades to attain the targeted concentrations.
- Method of particle size determination: The particle-size distribution was analyzed using a BERNER critical orifice cascade impactor. An adhesive stage coating (silicone spray) was used to minimize particle bounce due to the adhesive properties of the test article. Each impactor stage was covered with an aluminum foil. Collecting metrics were subjected to gravimetric analysis using a digital balance and pre-conditioning method as described in the previous section.

TEST ATMOSPHERE
- Brief description of analytical method used: The test-substance concentration was determined by gravimetric analysis (filter: glass-fiber filter, Sartorius, Göttingen, Germany; post-sampling conditioning period of 15 min at room temperature). This method was used to define the actual concentration.
- The integrity and stability of the aerosol generation and exposure system was measured by using a Microdust real-time aerosol photometer (Casella, Buffalo, UK) and at 0.3 mg/m³ the R&P Teom 1400a (amibent particulate monitor; Thermo Fisher Scientific Inc., USA). Samples were taken continuously from the vicinity of the breathing zone.
- Samples taken from breathing zone: yes
- Particle size distribution: The particle size distribution was analyzed using a TSI-Laser Velocimeter APS 3321. Calculations consider both a particle size distribution that encompasses aerodynamic diameters (Dae) of 0.5 to 7.4 µm and sample flows ranging from 8 to 80 ml/sec.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The mean Mass Median Aerodynamic Diameter’s (MMAD) measured by cascade impactor were in the range of 2.23 – 2.35 µm within the exposure groups (Geometric Standard Deviation: 1.86 – 2.30). MMADs measured by laser velocimetry ranged from 2.66 – 2.69 µm within the exposure groups (Geometric Standard Deviation: 1.72 – 1.73).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test-substance concentration was determined by gravimetric analysis. The concentrations of test atmosphere given are based on breathing zone concentrations from filter analyses which reflect the active substance.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day on 5 consecutive days/week for 13 weeks
Dose / conc.:
0.3 mg/m³ air (analytical)
Remarks:
target concentration: 0.3 mg/m³
Dose / conc.:
1.5 mg/m³ air (analytical)
Remarks:
target concentration: 1.5 mg/m³
Dose / conc.:
7.6 mg/m³ air (analytical)
Remarks:
target concentration: 7.5 mg/m³
No. of animals per sex per dose:
10 rats/sex/group plus 6 additional males/group for lung lavage plus 10 additional rats/sex of the control and high dose as recovery groups
Control animals:
other: rats exposed under otherwise identical test conditions to dry air served as concurrent control group
Details on study design:
Dose selection rationale:
Target concentrations for this repeated exposure study were based on the results of a 2 week inhalation toxicity study with study identification number T100111-4 (Exposure 9 x 6h/day, 5 days/week; recovery period of 2 weeks) using Vestanat B1358/100 as test item. See Pauluhn, 2013.
- Rationale for selecting satellite groups:
After two weeks of recovery in the 2 week inhalation study, the findings in lungs and LALN were still detectable. Additionally, accumulations of epitheloid cells occurred in BALT and LALN. Therefore satellite groups for lung lavage and recovery were included in this study.
- Post-exposure recovery period in satellite groups: 13 weeks
Positive control:
positive controls are not adequate for this study type
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, all animals
- Time schedule: Clinical signs were recorded twice daily, before and after exposure and once a day on exposure-free days and recovery period.

BODY WEIGHT: Yes, all animals
- Time schedule for examinations: During the exposure period, the body weights were determined twice per week to differentially record their gains over exposure-free weekends, and once per week during the postexposure period.

FOOD AND WATER CONSUMPTION: Yes.
- The individual food and water consumption of all animals (excluded lavage animals) were measured weekly (Mondays).

OPHTHALMOSCOPIC EXAMINATION: Yes.
- Prior the first exposure all animals (excluded lavage animals) and toward at the end of the dosing phase (on day 86) control and high dose animals were examined.

RECTAL TEMPERATURES: Yes, all animals
- Time schedule for examinations: Five animals per group 1-8 were subjected to body temperature measurements. The rectal temperatures were measured directly after cessation of exposure (approximately within ½hour after the end of exposure) using a digital thermometer with a rectal probe for rats during the dosing period on days rel. 0 and 85 as well as on day 85 of the recovery period.

FUNCTIONAL OBSERVATION BATTERY: Yes.
- The first five core rats of group 1-8 were used for functional observation battery investigation on day relative 0, 59 and 85. Each rat was firstly observed in the home cage and then individually examined. The following reflexes were evaluated: visual placing response and 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). Measurements of grip strength were measured qualitatively but defined as semi quantitative.

HAEMATOLOGY: Yes
- Blood samples (non-fasted) for hematology, coagulation and serum chemistry parameters were collected from all core animals (end of the exposure period) and recovery toxicology animals (end of the recovery period) during sacrifice by cardiac puncture.

CLINICAL CHEMISTRY: Yes
- Urines of core animals were collected on day 87 and 88 of the dosing phase as well as on day 87 and 88 at the recovery period, overnight (approximately 16 h). Therefore animals were housed in specific metabolism cages.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals
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.

ORGAN WEIGHTS: Yes, all animals
The following organs were weighed at necropsy: adrenal glands, brain, heart, kidneys, liver, lung, thymus,lung-associated lymph nodes, spleen, ovaries, uterus, testes, epididymis

HISTOPATHOLOGY: Yes
- Histopathology was performed on the following organs/tissues (core and recovery animals): nasal cavities (4 levels: L-I to L-IV) / Young, 1981), pharynx, larynx, trachea, lung, lung associated lymph nodes (hilus) and additionally in the control (control air) and high dose groups (core toxicology animals) the organs/tissues listed in the table Table 8. Tissues were fixed in 10% neutral-buffered formalin (NBF), except the left and two thirds of the right kidneys (fixed in Davidson’s fixative then transferred to 10% NBF) and followed routine processing in the histopathology laboratory. Lungs were instilled with 10% NBF and then transferred to 10% NBF solution. The tissue slides were examined by a veterinary pathologist. In addition, a formal cross-check was performed. The findings reported are those agreed-upon between the cross-checking and the study pathologist.

MICROBIOLOGICAL EVALUATION:
Animal number 21 (control group) was euthanized on 12-AUG-2016 due to blood in the urine in combination with clinical signs as e.g. piloerection, reduced body tone. To investigate the causality of the blood in the urine an additional microbiological diagnostic has been initiated. These microbiological investigation and evaluation took place at Bayer AG, Antiinfectiva, Bayer research Center Aprath, 42096 Wuppertal, Germany under non-GLP conditions.
Other examinations:
BRONCHOALVEOLAR LAVAGE: Yes, animals assigned (6 males/group)
Bronchoalveolar lavage fluid was sampled at the end of the exposure period using sodium pentobarbital. All rats, irrespective of the day of death, were given a gross-pathological examination. Consideration was given to perform 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 are recorded. Corresponding to the suitable SOP the cranium and the nasal cavities of some animals per group were opened.
In BAL-fluid (BALF) the following indicators of pulmonary damage were assessed: Total number of lavaged cells, including the volume and diameter, cytodifferentiation, lactate dehydrogenase (LDH9), total protein (PRQ9), gamma-glutamyltransferase (GGT9), phospholipids (PL9).

After complete exsanguination, the excised wet lungs were weighed and following ligation of the trachea, the lung lobes were then lavaged via a tracheal cannula with two volumes of 5-ml of physiological saline (nearly 37°C), which was left in the lungs for 30 sec, withdrawn, re-instilled for an additional 30 sec. This procedure was repeated once. From each rat the retrieved bronchoalveolar lavage fluid (BALF) from the lung lavage was then pooled and adjusted to 10-ml total volume. Prior to centrifuging the samples were kept on ice. Then the pooled lavage fluid was centrifuged at approximately 200 x g for approximately 10 min. at <10°C (Sigma 4-16K refrigerated centrifuge) and the resultant cell-free supernatant was analyzed for the endpoints/parameters listed below. The cell pellet was re-suspended in PBS-BSA (Dulbecco’s phosphate buffered saline with Ca2+ and Mg2+ containing 0.1% bovine serum albumin; Sigma, Deisenhofen, Germany) and then centrifuged (2 x 105 per cytospot) onto slides using a cytocentrifuge (Shandon Cytospin 4). Cell counts were determined in triplicates after 1:1000 dilution using a CASY cell counter + analyzer. Air-dried slides were fixed with a mixture of methanol:acetone, stained according to Pappenheim, and differentiated by light microscopy. Cytospots were differentiated by counting 300 cells/cytospot.
For the analysis of the BAL data the validated BalNew.exe and ANOVA.exe Software (Author Prof. Pauluhn, Bayer AG, Bayer Research Center Aprath, Wuppertal, Germany) has been used.
Statistics:
Analysis of variance (ANOVA BCTIC): This parametric method checks for normal distribution of data by comparing the median and mean. The groups are compared at a confidence level of (1-*) = 95% (P = 0.05). The test for the between-group homogeneity of the variance employed Box's test if more than 2 study groups were compared with each other. If the above F-test shows that the intra-group variability is greater than the inter-group variability, this is shown in the Appendix as "no statistical difference between the groups". If a difference is found then a pair-wise post-hoc comparison is conducted (1- and 2-sided) using the Games and Howell modification of the Tukey-Kramer significance post hoc test. This program (FORTRAN source code) was originally obtained from BCTIC and is used as means to evaluate off-line data. In most instances, data analyzed by ANOVABCTIC are also summarized in graphical form. In figures statistical significant differences are indicated by asterisks (* P < 0.05; ** P < 0.01).
BAL data: The individual results of the BAL-Investigation were manually entered in the program: BALnew.exe, Version 3.0 (Author Prof. J. Pauluhn, Bayer AG, Germany; Software was written in DIGITAL Fortran). The BALnew.exe software calculates group based arithmetic means and the standard deviations (SD) relating to 10 ml bronchoalveolar lavage volume. Means and SD were statistically evaluated using the ANOVABCTIC procedure (vide infra).
Description (incidence and severity):
No relevant concentration-dependent test item-related clinical symptoms were seen in the course of the study.
Red encrusted noses, reddish-brownish discolored fur around the eyes, the head or the neck/back area were partially seen in some control animals as well as in some animals exposed to the test item. These findings are well known in inhalation toxicity studies and are considered to be nose-only inhalation technique-related due to restraining in the inhalation tubes but not test item-induced. Isolated slight up to moderate piloerection was seen after exposure session in a few males of the control group as well in a few males exposed to the test item and is reflected to be not test item induced.
Description (incidence):
Three male rats (Animal number 21, 24 and 26) of the air control group and one male rat of the high exposure group (Animal number 60) were euthanized during the course of the study due to animal welfare reasons. All four euthanized animals showed blood in urine associated with reduced general condition e.g. reduced body tone, reduced motility and piloerection. Microbiological investigation of the urine and renal tissue of animal no. 21 demonstrated Proteus mirabilis (details are given in section 7.15). Since clinical signs as well as pathological findings in all four euthanized animals were similar, it is considered that the microbiological findings seen in rat number 21 are representative for all euthanized rats.
In summary, it is considered that the blood in the urine and the associated poor health condition of euthanized male animals are not test item-related since 3 control animals and 1 high concentration animal were affected. Furthermore the clinical and pathological findings are considered to be caused by an ascending urinary tract infection with Proteus mirabilis.
Description (incidence and severity):
Comparisons between the control and the exposure groups did not reveal any statistically concentration-dependent differences in body weights and cumulative body weight gain during the course of the study. Isolated significantly changed absolute body weights or cumulative body weight gain mean values are considered to be not test item associated due to lack of concentration-dependency.
Description (incidence and severity):
No relevant test item-related changes in food and water consumption were observed when compared to control animals.
Description (incidence and severity):
The ophthalmological examination did not reveal any test item-related findings.
Description (incidence and severity):
Reticulocytes were significantly increased in females at 7.6 mg/m³ at the end of the exposure period. At the end of the recovery period, reticulocytes were significantly decreased in males at 7.6 mg/m³. In females at the end of the exposure period, neutrophils were significantly increased at 7.6 mg/m³. At the end of the recovery period, leucocytes, lymphocytes, neutrophils, monocytes and atypical cells were significantly increased at 7.6 mg/m³ in comparison to the air control.
Furthermore isolated significantly changed parameters occurred and were reflected to be incidental.
Description (incidence and severity):
Isolated significantly changed parameters occurred and were reflected to be incidental.
Description (incidence and severity):
Quantitative urinalysis, microscopy of urine sediment and qualitative urinalysis were performed and did not reveal any concentration-dependent relevant differences in test item exposed animals when compared to air control rats.
Description (incidence and severity):
A battery of reflex measurements was made on day 3, 59 and 85. Differences between animals exposed to the test item compared to the control group did not occur.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the exposure period (main groups) test item-related increased lung-associated lymph nodes (LALN) were found in males and females exposed to 7.6 mg/m³. Furthermore absolute and relative (% of TBW and % of brain weights) lung weights were significantly increased in females at 7.6 mg/m³. Statistically significant higher absolute lung weights were observed in females at 1.5 mg/m³, whereas relative lung weight was unchanged; here it has to be taken into account that total body weights were also slightly higher when compared to controls. Consequently, relative lung weights (% of TBW and % of brain weight) at this dose level must be given preference. An Isolated significant increase in liver weight found in females exposed to 1.5 mg/m³ is considered to be not test item related due to lack of dose-dependency. Significantly raised relative spleen weights (% of TBW) were measured in female animals exposed to 7.6 mg/m³, whereas absolute spleen weights and relative spleen weights expressed as % of brain weights did not reach statistically significance.
At the end of the recovery period significantly increased absolute and relative (% of brain weight) adrenal gland, kidney, liver, LALN and lung weights were found in females exposed to 7.6 mg/m³, whereas additionally LALN weights were also significantly increased in males at 7.6 mg/m³. Relative spleen weights expressed as % of TBW were significantly increased in males in males at 7.6 mg/m³. Significantly changed adrenal gland, kidney and liver weights in comparison to the control group are interpreted to be not adverse in the absence of correlating histopathological and/or clinical pathological findings.
Significantly increased lung-associated lymph nodes and lung weights at 7.6 mg/m³ at the end of the exposure period as well as at the end of the recovery period are reflected to be test item-induced.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In males and females exposed to 7.6 mg/m³, lung-associated lymph nodes were increased in size (incidence males/females: 0/0-0/0-0/0-7/6). This finding was non-reversible after the post-exposure period (incidence males/females: 0/0-6/7).
A decrease in size and/or soft/flaccid consistency of testes were observed uni- or bilaterally throughout all exposure groups, including the control air group, that was as such not treatment related.
The three prematurely sacrificed male animals of the control air group with post-exposure observation period and the male animal at 7.6 mg/m³ assigned for bronchoalveolar lavage revealed findings at the urinary bladder consisting of increase in size, increased thickness of the wall and content changes. Furthermore, thymus was decreased in size in these animals. Since the three animals were affected with Proteus mirabilis no treatment relationship is seen.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of exposure:
In the nasal cavity of the exposed animals starting at 0,3 mg/m3 up to a moderate degree at 7.6 mg/m3, an increase in extent (whereas in level 2 and 3 nearly all animals were affected, more exposed animals showed the finding in level 1 when compared to control) and severity of eosinophilic globules predominantly at the transition between the respiratory and olfactory epithelium of the nasal septum, as well as the ventral conchae were observed. Eosinophilic globules were located partly apically, partly basally within the cells of the olfactory epithelium (most prominent in level 2 and 3; mean severity level 2 males: 1.0-1.5-1.6-2.8; females: 1.0-1.7-2.0-3.0; mean severity level 3 males: 1.4-1.7-1.6-2.7; females: 1.0-2.0-2.0-2.8). More severely affected animals had an increased incidence of epithelial degeneration and metaplasia.
Furthermore, an increase of goblet cell hypertrophy and hyperplasia in the respiratory epithelium was visible. These changes were associated with an increased infiltrate of eosinophils in the nasal respiratory epithelium.
The trachea revealed a minimally increased inflammatory cell infiltrate at 7.6 mg/m3 (incidence males: 5-4-4-7; incidence females: 5-5-5-4; mean severity of grading males: 1.2-1.0-1.5-1.3; mean severity of grading females. 1.0-1.0-1.0-1.3), as well as an increase of accumulated macrophages/giant cells in the area of the bifurcation starting at 1.5 mg/m3 (incidence males. 1-1-4-3; incidence females: 0-1-2-2).
In the lung, starting at 1.5 mg/m3 accumulation of alveolar macrophages/formation of giant cells of minimal to moderate degree were observed, focally at 1.5 mg/m3, multifocally throughout all lung lobes at 7.6 mg/m3 (incidence males: 0-0-3-10; incidence females: 0-0-1-10).
In animals exposed to 7.6 mg/m3, these accumulated macrophages/giant cells were associated with a chronic inflammation of slight degree affecting primarily the bronchiolo-alveolar junction and led to septal thickening and hypercellularity in this area (incidence males and females: 0-0-0-10).
In single animals, granuloma formation incorporating the macrophage accumulation/giant cells could be observed (incidence males and females: 0-0-0-2).
Furthermore, minimal accumulation of pigment-containing alveolar macrophages were observed (incidence males: 0-0-2-7; incidence females: 0-1-1-2).
The bronchus-associated lymphatic tissue (BALT) revealed an increase in incidence and severity of macrophage accumulation/giant cell/granuloma formation starting at 0.3 mg/m3 up to moderate degree at 7.6 mg/m3 (incidence males: 3-6-9-10; incidence females: 2-4-10-10; mean severity of grading males: 1.0-1.3-1.6-2.6; mean severity of grading females: 1.0-1.0-1.1-2.3). Single to multiple accumulations of macrophages/giant cells and isolated granulomas were observed as a continuum focally or multifocally within areas of BALT of control animals, especially in the left lung lobe or in the caudal aspect of the right lung lobe. In animals exposed to Vestanat B 1358/100, these accumulations/giant cells/granulomas increased in incidence and severity (larger accumulations/granulomas per area BALT; more areas of BALT affected, also in other lung samples) up to moderate degree at 7.6 mg/m3.
Furthermore, a minimal to slight lymphoid hyperplasia was observed starting at 1.5 mg/m3 (incidence males: 0-1-3-7; incidence females: 0-0-5-9).
Lung-associated lymph nodes (LALN) showed lymphoid hyperplasia starting in females at 0,3 mg/m3, in males at 1.5 mg/m3 (incidence males: 0-0-2-10; incidence females: 0-3-5-9), which is consistent with the reported increased size of lung-associated lymph nodes during necropsy observed in animals at 7.5 mg/m3. In addition, a predominantly moderate accumulation of macrophages with giant cell/granuloma formation was observed (incidence males: 0-0-2-10; incidence females: 0-0-0-10), which partly showed minimal to slight central degeneration/necrosis (incidence males: 0-0-0-3; incidence females: 0-0-0-7).
Furthermore, there was an increase in incidence and/or severity of histiocytes infiltrating the lymph nodes (incidence males: 6-6-6-10; incidence females: 4-4-8-10; mean severity of grading males: 1.2-1.2-1.5-2.1; mean severity of grading females: 1.0-1.3-1.5-2.7)

After the post-exposure observation period of 90 days:
Nasal cavity of all animals (including the control air group) revealed the same findings (eosinophilic globules, mean severity level 2 males: 2.0-3.0, females: 2.1-2.9; hypertrophy of goblet cells, mean severity level 2 males 1.8-2.0, females: 2.0-2.0
and hyperplasia of goblet cells, mean severity level 2 males:1.0-1.2, females: 1.0-1.0; infiltrate with eosinophils, mean severity level 2 males: 1.0-1.3, females 1.0-1.0). The findings in controls after the post-exposure observation period were thus observed with a similar severity compared with the exposed animals examined directly after the last day of exposure.
In animals with accumulation of eosinophilic globules of a higher degree incidences of focal degeneration and metaplasia increased.
In the lungs of all exposed animals, accumulation of alveolar macrophages/giant cells (incidence males and females: 0-10) and the slight chronic inflammation in the area of bonchiolo-alveolar junction (incidence males/females: 0-10) were still present. In addition, granuloma formation progressed in almost all animals (incidence males/females: 0-10/9), which showed central degeneration/necrosis in single animals (incidence males/females: 0-1/2).
Minimal to slight accumulation of pigment-containing alveolar macrophages increased in incidence in females and in severity in males compared to the findings directly after exposure (incidence males: 0-7; incidence females: 0-5).
The bronchus-associated lymphatic tissue (BALT) showed macrophage accumulation/giant cells/granuloma (incidence males: 5-10; incidence females: 4-10; mean severity of grading males: 1.4-2.4; mean severity of grading females: 1.0-2.2) and lymphoid hyperplasia (incidence males/females: 0-7/9).
The animals revealed a slight lymphoid hyperplasia within the lung-associated lymph nodes (incidence males and females: 0-10), which is consistent with the reported increased size of respective lymph nodes during necropsy. In addition, moderate accumulation of macrophages with giant cell/granuloma formation was observed (incidence males and females: 0-10), which partly showed minimal to slight central degeneration/necrosis (incidence males/females: 0-4/6).
Furthermore, there was an increase in incidence and/or severity of histiocytes infiltrating the lymph nodes (incidence males: 5-9; incidence females: 7-10; mean severity of grading males: 1.4-2.2; mean severity of grading females: 1.1-2.1).
After 13 weeks of exposure to Vestanat B 1358/100, spleen of some animals exposed to 7.6 mg/m3 revealed a minimally increased extramedullary hematopoiesis (incidence males: 0-1-1-3; incidence females: 0-0-0-2). There was no difference between control and exposed animals regarding this finding after the post-exposure observation period (incidence males: 2-3; incidence females: 3-3).
Three male animals of the control air group with post-exposure observation period and one male animal at 7.5 mg/m3 assigned for bronchoalveolar lavage were prematurely sacrificed for humane reasons. The observed macroscopic findings could be correlated to a marked to severe pyelonephritis and/or cystitis with microscopically detected bacteria. These animals also had a moderate to severe prostatitis. There were some more animals with less severe inflammatory processes within the urinary tract in other groups of exposure.
All other observations in these animals (especially liver: hepatocellular atrophy and vacuolation, Kupffer cell hypertrophy, spleen: atrophy of the white pulp, bone marrow: myeloid hyperplasia, thymus: involution/atrophy correlated to the macroscopically decreased thymus size etc.) were due to the bad condition of the animals.
A decrease in size and/or soft/flaccid consistency of testes were observed uni- or bilaterally throughout all exposure groups, including the control air group consistent with degeneration/atrophy of the testes up to a severe degree followed by aspermia or reduced sperm as well as increased cellular debris in the epididymides.
In female animals of the control air group as well as at 7.5 mg/m3, no cycle determination was possible due to ovarian atrophy and hypertrophy of interstitial cells. In consequence, the epithelium of the cervix and vagina showed no cycle-related changes and was affected by apoptosis/necrosis and sometimes slight pseudomucification.
As rare spontaneous finding, one male animal (animal 71 exposed to 7.6 mg/m3) revealed a malignant Schwannoma within the pancreas, which was observed grossly as a cystic, black-brown coloured nodule approximately 10 cm in diameter. This finding is considere to be incidential and not test item-related.
All other microscopic findings were incidental and spontaneous in nature and could not be attributed to the test item.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Rectal Temperature:
No significantly changed body temperatures were seen in animals exposed to the test item in comparison to control animals.

Bronchoalveolar Lavage:
adverse findings:
Total cell count (TCC), protein (PROT), lymphocytes (C-LYM), lactate dehydrogenase (LDH) and gamma-glutamyl transferase (GGT) were biologically relevant increased but did not reach statistically significance at 7.6 mg/m³.
LDH, an intracellular located enzyme, as well as GGT, a membrane bound enzyme, are established cytotoxic marker indicating cellular damage.
Consequently significantly increased polymorphonuclear cells and biologically relevant increase in protein, lymphocytes, LDH and GGT at 7.6 mg/m³ in the bronchoalveolar lavage are reflected as sings of inflammation also taking significantly increased LALN and lung weights into account (see section 7.9) and thus are considered to be adverse.

non adverse finding:
The average recovery of bronchoalveolar lavage fluid (BALF) was approximately 85-88 % of the instilled volume and was similar amongst all groups. An isolated significantly increase of foamy macrophages was found at 0.3 mg/m³. Adjusted alveolar macrophages (%) were significantly decreased at 0.3 and 7.6 mg/m³. Significantly increased alveolar macrophages with red blood cells were found at 1.5 mg/m³ only. These isolated findings are considered to be of no toxicological relevance.
At 7.6 mg/m³ the following parameter were significantly increased: mean cellular diameter (MCD), mean cellular volume (MCV) and polymorphonuclear cells (PMN and C-PMN). In this context it is remarkable that counted polymorphonuclear cells (C-PMN) at 7.6 mg/m³ were approx. 45-fold higher when compared to air control group. Percentage of alveolar macrophages (AM and AM-ADJ) were significantly decreased at 7.6 mg/m³ whereas absolute count of alveolar macrophages (C-AM) were biologically relevant increased at 7.6 mg/m³. Changes in MCD, MCV and C-AM at 7.6 mg/m³ are considered to be compensatory due to increased phagocytosis as normal part of the clearance mechanisms in the lung.
Total cell count (TCC), protein (PROT), lymphocytes (C-LYM), lactate dehydrogenase (LDH) and gamma-glutamyl transferase (GGT) were biologically relevant increased but did not reach statistically significance at 7.6 mg/m³.
LDH, an intracellular located enzyme, as well as GGT, a membrane bound enzyme, are established cytotoxic marker indicating cellular damage.
Consequently significantly increased polymorphonuclear cells and biologically relevant increase in protein, lymphocytes, LDH and GGT at 7.6 mg/m³ in the bronchoalveolar lavage are reflected as sings of inflammation also taking significantly increased LALN and lung weights into account (see section 7.9) and thus are considered to be adverse.

Microbiological Evaluation:
Male rat number 21 was investigated microbiologically. In the native urine microscopic slide, in all fields of view a lot of cells as well as a few bacteria were observed. Microscopically gram negative rod-shaped bacteria were found in urine and renal tissue. On the Columbia Agarplates pure cultures with swarming properties were seen. Aerob incubated plates showed dark discoloration. No colonies were observed on Agarplates treated with 100 µl blood whereas relevant number of colonies (pure culture) were found on plates incubated aerob as well as anaerob treated with urine and renal tissue. According to the evaluation of the API E20 test (Code 373600057) the bacterium isolated from the pure culture is up to 99.9% likelihood Proteus mirabilis. This result matches with the findings given above of gram negative rod-shaped bacteria and swarming colonies on Columbia Agarplates.
In summary bacteria have been detected in urine and renal tissue of rat number 21 but not in blood. Due to the characteristics, the bacterium is diagnosed as Proteus mirabilis. Proteus mirabilis is ubiquitous in the environment and is considered to be a normal part of human and animals gut flora (see section 12.21). Proteus mirabilis is not listed as recommended infectious agent to monitor for rats according to the FELASA (MÄHLER M. et al., 2014) and thus was not investigated prior to the start of the study. Consequently the microbiological diagnosis of Proteus mirabilis is considered to have no impact on the overall study validity. Nonetheless it is known that Proteus mirabilis may be involved in sporadic urinary tract infections.
Details on results:
Indicative for portal-of entry toxicity were significantly increased lung-associated lymph nodes (LALN) and lung weights, significantly increased polymorphonuclear cells as well as biologically relevant increased total cell count, protein, lymphocytes, LDH and GGT in the BAL at 7.6 mg/m³. These results correlated with gross pathological and histopathological findings as increased lung-associated lymph nodes after end of exposure and recovery period, chronic inflammatory reaction seen at the bronchiolo-alveolar junction of the lung as well as granuloma formation partly with central degeneration/necrosis of granuloma/giant cells also seen in the lung-associated lymph nodes at 7.6 mg/m³.
The daily inhalative exposure to Vestanat B 1358/100 to male and female rats over a period of at least 13 consecutive weeks revealed multiple histopathologically findings within the respiratory tract. Within the nasal cavity, an increase of eosinophilic globules, an increased hypertrophy and hyperplasia of goblet cells, an increased infiltrate with eosinophils as well an increased degeneration and metaplasia of epithelium were observed. At the end of the post-exposure observation period, also the animals of the control air group revealed these findings up to the same degree compared to animals exposed with 7.6 mg/m3. Thus, these findings are regarded as background findings, which occurred earlier and were slightly more prominent in animals exposed to the test item. A direct toxic effect on the nasal mucosa is not assumed.
At 7.6 mg/m3, granuloma formation was seen in the lungs at the end of the exposure period which partly progressed to granuloma with central degeneration and necrosis after the end of the recovery period. Furthermore, chronic inflammation of the bronchiolo-alveolar junction was induced at 7.6 mg/m3 that was still present after the post-exposure observation period. Accumulation of macrophages/giant cells/granuloma showed central degeneration and necrosis at 7.6 mg/m3, central degeneration and necrosis was still present after the recovery period. The moderate accumulation of macrophages/giant cells/granuloma partly with central degeneration/necrosis in the airways of the lung and in the associated lymphoid tissues (bronchus-associated lymphatic tissue, BALT and lung-associated lymph nodes, LALN) as well as the chronic inflammatory reaction seen at the bronchiolo-alveolar junction of the lung at 7.6 mg/m3 are considered to be adverse.
Extramedullary hematopoiesis in the spleen occurred in animals of the control group as well as in animals exposed to 7.6 mg/m3 test item after the recovery period. Incidences in males were 2-3 and in females 3-3 in control groups and at 7.6 mg/m3 respectively. Of note, the highest severity (moderate grade) was observed in a female control rat. At the end of the exposure period, minimal up to slight extramedullary hematopoiesis in the spleen was found in males (incidence 0-1-1-3) and females (incidence 0-0-0-2). Highest severity and indistinguishable incidence compared to test item exposed animals of extramedullary hematopoiesis was seen in control animals at the end of the recovery period. Thus, this finding is interpreted to be not test item-induced.
Relative body weight adjusted spleen weights were significantly increased in females at the end of the exposure period and significantly increased in males at the end of the recovery period at 7.6 mg/m³. Reticulocytes were significantly increased in females at the end of the exposure period and significantly decreased in males at the end of the recovery period at 7.6 mg/m³. Significantly decreased percentage of reticulocytes in the blood at 7.6 mg/m3 at the end of the recovery period in males do not correlate with significantly increased body weight-adjusted relative spleen weights at that concentration. Furthermore significantly decreased reticulocyte counts and significantly increased relative spleen weights were only seen in males and not in females. Additionally no test item induced histopathological findings were seen in the spleen at the end of the recovery period. Consequently these findings are reflected to be inconsistent and not of toxicological relevance.
At the end of the exposure period significantly increased reticulocytes and significantly increased body weight-adjusted relative spleen weights were found in females at 7.6 mg/m3. Of note, only body weight-adjusted spleen weights in female rats at 7.6 mg/m3 were significantly increased whereas absolute and brain weight-adjusted spleen weights did not reach statistically significance when compared to controls. Furthermore it has to be kept into account that only one gender was affected. Additionally, no further significantly changed red cell parameters were found at the end of the exposure period. Taking all those information into account it was considered that increased reticulocytes and increased body weight-adjusted relative spleen weights at the end of the exposure period are non-adverse.
Leucocytes and specifically neutrophils were increased at 7.6 mg/m³ in males and females at the end of the exposure period. At the end of the recovery period, leucocytes, lymphocytes, neutrophils, monocytes and atypical cells were increased at 7.6 mg/m³ in comparison to the air control. Systemically increased white cell parameters seen at clinical pathology mentioned above, are interpreted to be the consequence of cell recruitment due to inflammatory processes in the respiratory tract at 7.6 mg/m³. In this context the increased white blood cellsespacially neutrophils in the blood at 7.6 mg/m³ may be explained by raised recruitment due inflammatory processes in the lungs at 7.6 mg/m3.
Dose descriptor:
NOAEC
Effect level:
1.5 mg/m³ air
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: portal-of-enty toxicity in the respiratory tract associated tissues at 7.6 mg/m³; a NOEL could not be established
Dose descriptor:
NOAEC
Effect level:
7.6 mg/m³ air
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no systemic toxicity observed
Critical effects observed:
yes
Lowest effective dose / conc.:
7.6 mg/m³ air
System:
other: respiratory system
Organ:
other: portal-of-entry toxicity (local effects) in the respiratory tract; no systemic toxicity
Treatment related:
yes
Executive summary:

A repeated exposure 90-day (13 weeks) subchronic toxicity inhalation study with the solid aerosol of the test item was conducted in young adult male and female Wistar rats according to the procedures called for by OECD Test Guideline No. 413 (2009). 10 Wistar rats/sex were nose-only exposed for 6 hours/day on five consecutive days per week for 13 weeks to mean (± SD) actual concentrations of 0 [air control], 0.3 (±0.05), 1.5 (±0.20), and 7.6 (±0.80) mg/m³. The concentrations of the test item in the test atmosphere given are based on gravimetric analysis from breathing zone concentrations. Animals exposed to air under otherwise identical circumstances served as negative controls. Six additional male rats per concentration group were assigned to lung lavage at the end of the exposure period. Ten additional rats per sex of the control and the high concentration groups were allowed to recover during a 12-week postexposure period. The exposure took place in directed-flow nose-only inhalation chambers.

The characteristics of the test atmospheres were in compliance with the recommendations of the OECD Test Guideline No. 413. The mean Mass Median Aerodynamic Diameter’s (MMAD) were in the range of 2.23 – 2.35 µm within the exposure groups (GSD: 1.86 – 2.30).

Test item-related mortality did not occur.

There were no test-item induced adverse findings seen in clinical observations, rectal temperature, ophthalmology, absolute body weights, urinalysis, cumulative body weight gain, food and water consumption as well as in functional observation battery up to the highest dose of 7.6 mg/m³.

Absolute and relative lung weights were significantly increased in females at 7.6 mg/m³. Furthermore lung-associated lymph node (LALN) weights and size were increased at 7.6 mg/m³ at the end of the exposure period and at the end of the recovery period. Relative spleen weights were significantly increased in females at the end of the exposure period and significantly increased in males at the end of the recovery period at 7.6 mg/m³.

Reticulocytes were significantly increased in females at the end of the exposure period and significantly decreased in males at the end of the recovery period at 7.6 mg/m³. Changes in reticulocytes are considered to be not adverse in the absence of further significantly changed red blood cell parameters and also taking histopathology into account.

Leucocytes and specifically neutrophils were increased at 7.6 mg/m³ in males and females at the end of the exposure period. At the end of the recovery period, leucocytes, lymphocytes, neutrophils, monocytes and atypical cells were increased at 7.6 mg/m³ in comparison to the air control. Those findings may be interpreted to be the consequence of white cell recruitment due to relevant inflammatory processes in the lungs at 7.6 mg/m³.

Bronchoalveolar lavage analysis revealed significantly increased mean cellular diameter, mean cellular volume and polymorphonuclear cell counts at 7.6 mg/m³. Percentage of alveolar macrophages was significantly decreased at 7.6 mg/m³ whereas absolute count of alveolar macrophages was biologically relevant increased at 7.6 mg/m³. Changes in mean cellular diameter, mean cellular volume and alveolar macrophages at 7.6 mg/m³ are interpreted as compensatory effect due to increased phagocytosis, a physiological clearance mechanism in the lungs. Total cell count, protein, lymphocytes, lactate dehydrogenase (LDH) and gamma-Glutamyl transferase (GGT) were biologically relevant increased at 7.6 mg/m³ but did not reach statistical significance. LDH, a cytoplasmic enzyme, as well as GGT, a membrane bound enzyme, are established cytotoxicity marker indicating cellular damage. Consequently significantly increased polymorphonuclear cells and relevant increase in protein, lymphocytes, LDH and GGT in the BAL at 7.6 mg/m³ were reflected as signs of inflammation and thus considered to be adverse. Changes in bronchoalveolar lavage (BAL) parameters seen at 1.5 mg/m³ and below are considered to be not adverse.

Within the nasal cavity, an increase of eosinophilic globules, an increased hypertrophy and hyperplasia of goblet cells, an increased infiltrate with eosinophils as well an increased degeneration and metaplasia of epithelium were observed. At the end of the post-exposure observation period, also the animals of the control air group revealed these findings up to the same degree compared to animals exposed to 7.6 mg/m3. Thus, these findings are regarded as background findings, which occurred earlier and were slightly more prominent in animals exposed to Vestanat B 1358/100 compared to control. A direct toxic effect of the test item on the nasal mucosa is not assumed.

At exposure levels of 0.3 mg/m3 and 1.5 mg/m3 Vestanat B 1358/100 and in controls a minimal to slight accumulation of alveolar macrophages/giant cells in the lung as well as in the associated lymphoid tissues (BALT, LALN) was seen. In the tracheal

bifurcation, reversible minimal increases in inflammatory infiltrates with accumulation of partly foamy macrophages/giant cells were observed starting at 1.5 mg/m3. Bronchus-associated lymphatic tissue (BALT) was hyperplastic and also showed an increase in accumulation of macrophages/giant cells/granuloma, concentration-dependent starting at 0.3 mg/m3. A lymphoid hyperplasia was observed in the lung-associated lymph nodes starting at 0.3 mg/m3 at the end of the exposure period still detectable after the recovery period. Furthermore, there was an accumulation of macrophages/giant cells/granuloma in the lung associated lymph nodes starting at 1.5 mg/m3. In addition, accumulation of pigment-laden macrophages starting at 0.3 mg/m3 increased concentration-dependent. These abovementioned histopathological effects seen at 0.3 and 1.5 mg/m3 in the lungs and the lung associated lymphoid tissues (BALT, LALN) are considered to be part of physiological clearance mechanisms due to inhalation of solid particles and in the absence of any structural changes regarded as non-adverse.

At 7.6 mg/m3, granuloma formation was seen in the lungs at the end of the exposure period which partly progressed to granuloma with central degeneration and necrosis after the end of the recovery period. Furthermore, chronic inflammation of the bronchiolo-alveolar junction was induced at 7.6 mg/m3 that was still present after the post-exposure observation period. Accumulation of macrophages/giant cells/granuloma showed central degeneration and necrosis at 7.6 mg/m3, central degeneration and necrosis was still present after the recovery period. The moderate accumulation of macrophages/giant cells/granuloma partly with central degeneration/necrosis in the airways of the lung and in the associated lymphoid tissues (bronchus-associated lymphatic tissue, BALT and lung-associated lymph nodes, LALN) as well as the chronic inflammatory reaction seen at the bronchiolo-alveolar junction of the lung at 7.6 mg/m3 are considered to be adverse.

Observed extramedullary hematopoiesis in spleens of male and female rats are interpreted to be not test item induced as similar incidence and severity were also seen in control animals at the end of the recovery period.

In summary, no systemic toxicity was observed but there is evidence of portal-of-entry toxicity in rats after repeated inhalation (90 days) of the test item. Taking all findings into account, in this subacute inhalation toxicity study the No-Observed-Adverse-Effect-Level (NOAEL) is 1.5 mg/m³ due to respiratory tract associated adverse findings seen in BAL, histopathology and weight increase of lung and LALN at 7.6 mg/m³. A no-observed effect level (NOEL) could not be established.

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
A grouping of substances and read-across approach according to Regulation (EC) No 1907/2006 (REACH), Annex XI 1.5 and following the ECHA Read-Across Assessment Framework, RAAF (2015) is accomplished for the registration of five blocked diisocyanate oligomers (also for the registered substance) that are manufactured or imported in quantities of 10 (1/5), 100 (2/5) or 1000 (2/5) tons/anno or more. This is to fulfil the data requirements of REACH for the respective tonnage band in a scientifically based and efficient manner, taking also into account the aim to reduce vertebrate studies.

All five category members are oligomerisation products of diisocyanates (UVCBs), where the free isocyanate functionality is covalently reacted with blocking agents (decomposition at approximately 90 °C and above). The category members are either derived from 1,6-hexamethylene diisocyanate (HDI) oligomers or isophorone diisocyanate (IPDI) oligomers. Since the smallest constituent is the blocked trimer (n = 3) having a molecular weight of ≥ 700 g/mol, a very low water solubility and throughout high log Kow (> 4.4), absorption through biological membranes is limited. The chemical composition of the UVCBs is very similar and the free diisocyanate monomer content is throughout below 0.1%. The five blocked oligomeric diisocyanates thus show defined structural similarities and differences that allow the application of the ECHA RAAF Scenario 4.
In this category approach the physicochemical properties, environmental fate, ecotoxicity and most of the toxicological endpoints were experimentally determined for each of the five category members. The outcome of this testing proved the similarity of the category members not only in structure and composition but also in quality and quantity of reactivity/toxicity. The comparison of the environmental fate and pathways data (i.e. hydrolysis, biodegradation, bioaccumulation and adsorption on soil components) and ecotoxicological data (i.e. different tests for toxicity to aquatic organisms) reveals a very high similarity. The same is true for the toxicological endpoints acute toxicity, skin and eye irritation, skin sensitization and genotoxicity. None of the category members has to be classified according to GHS Regulation 1272/2008 for environmental fate, ecotoxicity, acute toxicity, skin and eye irritation, skin sensitization as well as genotoxicity.

Since inhalation exposure in occupational settings is potentially relevant for all category members acute and 14-day pilot repeated inhalation toxicity studies were conducted with all five UVCBs to allow a robust evaluation on mode of action and toxic potency and to further prove the group hypothesis.

The most noticeable difference in the performance of the inhalation studies was related to the physical properties of the substances. The blocked HDI-based oligomers had to be dissolved in ethyl acetate to generate a suitable test atmosphere because they became glue-like during testing. The blocked IPDI-based oligomers could be tested as powder aerosols. Thus, for inhalation toxicity testing the category is divided into two sub-groups – the liquid aerosol for blocked HDI-based oligomers and the powder aerosol for blocked IPDI-based oligomers. Thus, within the category the analogue read-across approach is followed, i.e. ECHA RAAF Scenario 2.
In consequence, two subchronic (90-days) inhalation studies, one for a blocked HDI oligomer and one for a blocked IPDI oligomer, were conducted. The remaining substances are covered by a read across to the category member with the closest structural analogy. In this case, the target substance (isophorone diisocyanate, oligomeristion product, blocked with hexahydro-2H-azepin-2 -one) is covered by read across to the source substance (isophorone diisocyanate, oligomeristion product, blocked with 2 -butanone oxime).

1. HYPOTHESIS FOR THE CATEGORY/ ANALOGUE APPROACH
For a detailed discussion of the hypothesis for grouping and read across of 5 blocked diisocyanate oligomers according to regulation No. 1907/2006 (REACH), Annex XI, 1.5 and following ECHA RAAF (2015) see the document attached below to this endpoint study record.
Note: The first outline of a grouping-strategy was prepared in May 2013 based on read-across of the available toxicological data for 4 blocked diisocyanate oligomers at that time. In the meantime, the group was enlarged to 5 blocked diisocyanate oligomers and 90-day inhalation toxicity studies for two of the category members have been performed on request of ECHA. The updated document refers to the REACH requirements according to Annex VIII - X (10 - > 1000 tonnes/a).

2. SOURCE AND TARGET CHEMICAL(S)
Please see document attached below to this endpoint study record.

3. CATEGORY / ANALOGUE APPROACH JUSTIFICATION
For a detailed discussion of the outcome of the testing strategy and for justification of the grouping and read across of 5 blocked diisocyanate oligomers according to regulation No. 1907/2006 (REACH), Annex XI, 1.5 and following ECHA RAAF (2015) see the document attached below to this endpoint study record.

4. DATA MATRIX
Please see document attached below to this endpoint study record.

Reason / purpose:
read-across source
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Healthy young adult SPF bred Wistar rats, strain Crl:(Wi)WU BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:(Wi)WU BR (SPF-bred)
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 2 months
- Weight at study initiation: Weight range at first exposure day: males 230-280 g. 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 with gnawing sticks
- Diet and water: ad libitum
- Acclimation period: at least 10-14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 40-60 %
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12 h/12 h
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
ca. 2.3 µm
Remarks on MMAD:
The mean Mass Median Aerodynamic Diameter’s (MMAD) measured by cascade impactor were in the range of 2.23 – 2.35 µm within the exposure groups (Geometric Standard Deviation: 1.86 – 2.30). MMADs measured by laser velocimetry ranged from 2.66 – 2.69 µm within the exposure groups (Geometric Standard Deviation: 1.72 – 1.73).
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Rats were exposed under dynamic directed-flow nose-only exposure conditions.
- Exposure apparatus: Details of the nose-only exposure system, including its validation, have been published previously (Pauluhn, Journal of Applied Toxicology 13, 1994, 55-62; Pauluhn and Thiel, J. Appl. Toxicol. 27, 2007, 160-167). 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).
- Method of holding animals in test chamber: Animals were exposed in polycarbonate 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.
- Source and rate of air: 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; SMG Interlink, USA) and TSI Mass Flow meter 4040 (TSI Incorporated, USA) and were checked for correct performance at regular intervals.
- 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 BEKO RA 55 compressed air dryer. Adequate control devices were employed to control supply pressure.
- Temperature, humidity: Temperature and humidity measurements are also performed by the computerized Data Acquisition and Control System using HC2-S3 sensors (Rotronic Messgeräte GmbH, Ettlingen, 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.
- Air flow rate: 5.85 L/min
- Air change rate: The test atmosphere generation conditions provide an adequate number of air exchanges per hour [45 L/min x 60 min/(11.4 L/chamber) = 237, continuous generation of test atmosphere]. Thus, under the test conditions used a chamber equilibrium is attained in less than one minute of exposure.
- Treatment of exhaust air: The exhaust air was purified via filter systems. These filters were disposed of by the laboratory.
The ratio between supply and exhaust air was selected so that 90 % of the supplied air was extracted via the exhaust air location and, if applicable, via sampling ports. Aerosol scrubbing devices were used for exhaust air clean-up. During sampling, the exhaust air was reduced in accordance with the sampling flow rate using a computerized Data Acquisition and Control System so that the total exhaust air flow rate was adjusted 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).
- Atmosphere generation: Test atmospheres were generated using a WRIGHT DUST FEEDER system (BGI Inc., Waltham, MA 02154, USA). For dry powder dispersion, conditioned compressed dry air (30 liters/min; generic dispersion pressure: 1.8 kPa) was used. The principle performance of the WRIGHT DUST FEEDER dust generating system can be described as follows: the test substance was metered in a reservoir and then was compressed to a pellet using approxi¬mately 1 metric ton by a carva laboratory press (F. S. Carver Inc., Wabash, IN 46992, USA). From this pellet defined amounts of test substance were scraped off and entrained into the main air flow.
The airborne powder was then conveyed into the inner cylinder of the inhalation chamber. Principles differences between chamber set-ups were related to the dilution cascades to attain the targeted concentrations.
- Method of particle size determination: The particle-size distribution was analyzed using a BERNER critical orifice cascade impactor. An adhesive stage coating (silicone spray) was used to minimize particle bounce due to the adhesive properties of the test article. Each impactor stage was covered with an aluminum foil. Collecting metrics were subjected to gravimetric analysis using a digital balance and pre-conditioning method as described in the previous section.

TEST ATMOSPHERE
- Brief description of analytical method used: The test-substance concentration was determined by gravimetric analysis (filter: glass-fiber filter, Sartorius, Göttingen, Germany; post-sampling conditioning period of 15 min at room temperature). This method was used to define the actual concentration.
- The integrity and stability of the aerosol generation and exposure system was measured by using a Microdust real-time aerosol photometer (Casella, Buffalo, UK) and at 0.3 mg/m³ the R&P Teom 1400a (amibent particulate monitor; Thermo Fisher Scientific Inc., USA). Samples were taken continuously from the vicinity of the breathing zone.
- Samples taken from breathing zone: yes
- Particle size distribution: The particle size distribution was analyzed using a TSI-Laser Velocimeter APS 3321. Calculations consider both a particle size distribution that encompasses aerodynamic diameters (Dae) of 0.5 to 7.4 µm and sample flows ranging from 8 to 80 ml/sec.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The mean Mass Median Aerodynamic Diameter’s (MMAD) measured by cascade impactor were in the range of 2.23 – 2.35 µm within the exposure groups (Geometric Standard Deviation: 1.86 – 2.30). MMADs measured by laser velocimetry ranged from 2.66 – 2.69 µm within the exposure groups (Geometric Standard Deviation: 1.72 – 1.73).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test-substance concentration was determined by gravimetric analysis. The concentrations of test atmosphere given are based on breathing zone concentrations from filter analyses which reflect the active substance.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day on 5 consecutive days/week for 13 weeks
Dose / conc.:
0.3 mg/m³ air (analytical)
Remarks:
target concentration: 0.3 mg/m³
Dose / conc.:
1.5 mg/m³ air (analytical)
Remarks:
target concentration: 1.5 mg/m³
Dose / conc.:
7.6 mg/m³ air (analytical)
Remarks:
target concentration: 7.5 mg/m³
No. of animals per sex per dose:
10 rats/sex/group plus 6 additional males/group for lung lavage plus 10 additional rats/sex of the control and high dose as recovery groups
Control animals:
other: rats exposed under otherwise identical test conditions to dry air served as concurrent control group
Details on study design:
Dose selection rationale:
Target concentrations for this repeated exposure study were based on the results of a 2 week inhalation toxicity study with study identification number T100111-4 (Exposure 9 x 6h/day, 5 days/week; recovery period of 2 weeks) using Vestanat B1358/100 as test item. See Pauluhn, 2013.
- Rationale for selecting satellite groups:
After two weeks of recovery in the 2 week inhalation study, the findings in lungs and LALN were still detectable. Additionally, accumulations of epitheloid cells occurred in BALT and LALN. Therefore satellite groups for lung lavage and recovery were included in this study.
- Post-exposure recovery period in satellite groups: 13 weeks
Positive control:
positive controls are not adequate for this study type
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, all animals
- Time schedule: Clinical signs were recorded twice daily, before and after exposure and once a day on exposure-free days and recovery period.

BODY WEIGHT: Yes, all animals
- Time schedule for examinations: During the exposure period, the body weights were determined twice per week to differentially record their gains over exposure-free weekends, and once per week during the postexposure period.

FOOD AND WATER CONSUMPTION: Yes.
- The individual food and water consumption of all animals (excluded lavage animals) were measured weekly (Mondays).

OPHTHALMOSCOPIC EXAMINATION: Yes.
- Prior the first exposure all animals (excluded lavage animals) and toward at the end of the dosing phase (on day 86) control and high dose animals were examined.

RECTAL TEMPERATURES: Yes, all animals
- Time schedule for examinations: Five animals per group 1-8 were subjected to body temperature measurements. The rectal temperatures were measured directly after cessation of exposure (approximately within ½hour after the end of exposure) using a digital thermometer with a rectal probe for rats during the dosing period on days rel. 0 and 85 as well as on day 85 of the recovery period.

FUNCTIONAL OBSERVATION BATTERY: Yes.
- The first five core rats of group 1-8 were used for functional observation battery investigation on day relative 0, 59 and 85. Each rat was firstly observed in the home cage and then individually examined. The following reflexes were evaluated: visual placing response and 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). Measurements of grip strength were measured qualitatively but defined as semi quantitative.

HAEMATOLOGY: Yes
- Blood samples (non-fasted) for hematology, coagulation and serum chemistry parameters were collected from all core animals (end of the exposure period) and recovery toxicology animals (end of the recovery period) during sacrifice by cardiac puncture.

CLINICAL CHEMISTRY: Yes
- Urines of core animals were collected on day 87 and 88 of the dosing phase as well as on day 87 and 88 at the recovery period, overnight (approximately 16 h). Therefore animals were housed in specific metabolism cages.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals
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.

ORGAN WEIGHTS: Yes, all animals
The following organs were weighed at necropsy: adrenal glands, brain, heart, kidneys, liver, lung, thymus,lung-associated lymph nodes, spleen, ovaries, uterus, testes, epididymis

HISTOPATHOLOGY: Yes
- Histopathology was performed on the following organs/tissues (core and recovery animals): nasal cavities (4 levels: L-I to L-IV) / Young, 1981), pharynx, larynx, trachea, lung, lung associated lymph nodes (hilus) and additionally in the control (control air) and high dose groups (core toxicology animals) the organs/tissues listed in the table Table 8. Tissues were fixed in 10% neutral-buffered formalin (NBF), except the left and two thirds of the right kidneys (fixed in Davidson’s fixative then transferred to 10% NBF) and followed routine processing in the histopathology laboratory. Lungs were instilled with 10% NBF and then transferred to 10% NBF solution. The tissue slides were examined by a veterinary pathologist. In addition, a formal cross-check was performed. The findings reported are those agreed-upon between the cross-checking and the study pathologist.

MICROBIOLOGICAL EVALUATION:
Animal number 21 (control group) was euthanized on 12-AUG-2016 due to blood in the urine in combination with clinical signs as e.g. piloerection, reduced body tone. To investigate the causality of the blood in the urine an additional microbiological diagnostic has been initiated. These microbiological investigation and evaluation took place at Bayer AG, Antiinfectiva, Bayer research Center Aprath, 42096 Wuppertal, Germany under non-GLP conditions.
Other examinations:
BRONCHOALVEOLAR LAVAGE: Yes, animals assigned (6 males/group)
Bronchoalveolar lavage fluid was sampled at the end of the exposure period using sodium pentobarbital. All rats, irrespective of the day of death, were given a gross-pathological examination. Consideration was given to perform 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 are recorded. Corresponding to the suitable SOP the cranium and the nasal cavities of some animals per group were opened.
In BAL-fluid (BALF) the following indicators of pulmonary damage were assessed: Total number of lavaged cells, including the volume and diameter, cytodifferentiation, lactate dehydrogenase (LDH9), total protein (PRQ9), gamma-glutamyltransferase (GGT9), phospholipids (PL9).

After complete exsanguination, the excised wet lungs were weighed and following ligation of the trachea, the lung lobes were then lavaged via a tracheal cannula with two volumes of 5-ml of physiological saline (nearly 37°C), which was left in the lungs for 30 sec, withdrawn, re-instilled for an additional 30 sec. This procedure was repeated once. From each rat the retrieved bronchoalveolar lavage fluid (BALF) from the lung lavage was then pooled and adjusted to 10-ml total volume. Prior to centrifuging the samples were kept on ice. Then the pooled lavage fluid was centrifuged at approximately 200 x g for approximately 10 min. at <10°C (Sigma 4-16K refrigerated centrifuge) and the resultant cell-free supernatant was analyzed for the endpoints/parameters listed below. The cell pellet was re-suspended in PBS-BSA (Dulbecco’s phosphate buffered saline with Ca2+ and Mg2+ containing 0.1% bovine serum albumin; Sigma, Deisenhofen, Germany) and then centrifuged (2 x 105 per cytospot) onto slides using a cytocentrifuge (Shandon Cytospin 4). Cell counts were determined in triplicates after 1:1000 dilution using a CASY cell counter + analyzer. Air-dried slides were fixed with a mixture of methanol:acetone, stained according to Pappenheim, and differentiated by light microscopy. Cytospots were differentiated by counting 300 cells/cytospot.
For the analysis of the BAL data the validated BalNew.exe and ANOVA.exe Software (Author Prof. Pauluhn, Bayer AG, Bayer Research Center Aprath, Wuppertal, Germany) has been used.
Statistics:
Analysis of variance (ANOVA BCTIC): This parametric method checks for normal distribution of data by comparing the median and mean. The groups are compared at a confidence level of (1-*) = 95% (P = 0.05). The test for the between-group homogeneity of the variance employed Box's test if more than 2 study groups were compared with each other. If the above F-test shows that the intra-group variability is greater than the inter-group variability, this is shown in the Appendix as "no statistical difference between the groups". If a difference is found then a pair-wise post-hoc comparison is conducted (1- and 2-sided) using the Games and Howell modification of the Tukey-Kramer significance post hoc test. This program (FORTRAN source code) was originally obtained from BCTIC and is used as means to evaluate off-line data. In most instances, data analyzed by ANOVABCTIC are also summarized in graphical form. In figures statistical significant differences are indicated by asterisks (* P < 0.05; ** P < 0.01).
BAL data: The individual results of the BAL-Investigation were manually entered in the program: BALnew.exe, Version 3.0 (Author Prof. J. Pauluhn, Bayer AG, Germany; Software was written in DIGITAL Fortran). The BALnew.exe software calculates group based arithmetic means and the standard deviations (SD) relating to 10 ml bronchoalveolar lavage volume. Means and SD were statistically evaluated using the ANOVABCTIC procedure (vide infra).
Description (incidence and severity):
No relevant concentration-dependent test item-related clinical symptoms were seen in the course of the study.
Red encrusted noses, reddish-brownish discolored fur around the eyes, the head or the neck/back area were partially seen in some control animals as well as in some animals exposed to the test item. These findings are well known in inhalation toxicity studies and are considered to be nose-only inhalation technique-related due to restraining in the inhalation tubes but not test item-induced. Isolated slight up to moderate piloerection was seen after exposure session in a few males of the control group as well in a few males exposed to the test item and is reflected to be not test item induced.
Description (incidence):
Three male rats (Animal number 21, 24 and 26) of the air control group and one male rat of the high exposure group (Animal number 60) were euthanized during the course of the study due to animal welfare reasons. All four euthanized animals showed blood in urine associated with reduced general condition e.g. reduced body tone, reduced motility and piloerection. Microbiological investigation of the urine and renal tissue of animal no. 21 demonstrated Proteus mirabilis (details are given in section 7.15). Since clinical signs as well as pathological findings in all four euthanized animals were similar, it is considered that the microbiological findings seen in rat number 21 are representative for all euthanized rats.
In summary, it is considered that the blood in the urine and the associated poor health condition of euthanized male animals are not test item-related since 3 control animals and 1 high concentration animal were affected. Furthermore the clinical and pathological findings are considered to be caused by an ascending urinary tract infection with Proteus mirabilis.
Description (incidence and severity):
Comparisons between the control and the exposure groups did not reveal any statistically concentration-dependent differences in body weights and cumulative body weight gain during the course of the study. Isolated significantly changed absolute body weights or cumulative body weight gain mean values are considered to be not test item associated due to lack of concentration-dependency.
Description (incidence and severity):
No relevant test item-related changes in food and water consumption were observed when compared to control animals.
Description (incidence and severity):
The ophthalmological examination did not reveal any test item-related findings.
Description (incidence and severity):
Reticulocytes were significantly increased in females at 7.6 mg/m³ at the end of the exposure period. At the end of the recovery period, reticulocytes were significantly decreased in males at 7.6 mg/m³. In females at the end of the exposure period, neutrophils were significantly increased at 7.6 mg/m³. At the end of the recovery period, leucocytes, lymphocytes, neutrophils, monocytes and atypical cells were significantly increased at 7.6 mg/m³ in comparison to the air control.
Furthermore isolated significantly changed parameters occurred and were reflected to be incidental.
Description (incidence and severity):
Isolated significantly changed parameters occurred and were reflected to be incidental.
Description (incidence and severity):
Quantitative urinalysis, microscopy of urine sediment and qualitative urinalysis were performed and did not reveal any concentration-dependent relevant differences in test item exposed animals when compared to air control rats.
Description (incidence and severity):
A battery of reflex measurements was made on day 3, 59 and 85. Differences between animals exposed to the test item compared to the control group did not occur.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the exposure period (main groups) test item-related increased lung-associated lymph nodes (LALN) were found in males and females exposed to 7.6 mg/m³. Furthermore absolute and relative (% of TBW and % of brain weights) lung weights were significantly increased in females at 7.6 mg/m³. Statistically significant higher absolute lung weights were observed in females at 1.5 mg/m³, whereas relative lung weight was unchanged; here it has to be taken into account that total body weights were also slightly higher when compared to controls. Consequently, relative lung weights (% of TBW and % of brain weight) at this dose level must be given preference. An Isolated significant increase in liver weight found in females exposed to 1.5 mg/m³ is considered to be not test item related due to lack of dose-dependency. Significantly raised relative spleen weights (% of TBW) were measured in female animals exposed to 7.6 mg/m³, whereas absolute spleen weights and relative spleen weights expressed as % of brain weights did not reach statistically significance.
At the end of the recovery period significantly increased absolute and relative (% of brain weight) adrenal gland, kidney, liver, LALN and lung weights were found in females exposed to 7.6 mg/m³, whereas additionally LALN weights were also significantly increased in males at 7.6 mg/m³. Relative spleen weights expressed as % of TBW were significantly increased in males in males at 7.6 mg/m³. Significantly changed adrenal gland, kidney and liver weights in comparison to the control group are interpreted to be not adverse in the absence of correlating histopathological and/or clinical pathological findings.
Significantly increased lung-associated lymph nodes and lung weights at 7.6 mg/m³ at the end of the exposure period as well as at the end of the recovery period are reflected to be test item-induced.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In males and females exposed to 7.6 mg/m³, lung-associated lymph nodes were increased in size (incidence males/females: 0/0-0/0-0/0-7/6). This finding was non-reversible after the post-exposure period (incidence males/females: 0/0-6/7).
A decrease in size and/or soft/flaccid consistency of testes were observed uni- or bilaterally throughout all exposure groups, including the control air group, that was as such not treatment related.
The three prematurely sacrificed male animals of the control air group with post-exposure observation period and the male animal at 7.6 mg/m³ assigned for bronchoalveolar lavage revealed findings at the urinary bladder consisting of increase in size, increased thickness of the wall and content changes. Furthermore, thymus was decreased in size in these animals. Since the three animals were affected with Proteus mirabilis no treatment relationship is seen.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of exposure:
In the nasal cavity of the exposed animals starting at 0,3 mg/m3 up to a moderate degree at 7.6 mg/m3, an increase in extent (whereas in level 2 and 3 nearly all animals were affected, more exposed animals showed the finding in level 1 when compared to control) and severity of eosinophilic globules predominantly at the transition between the respiratory and olfactory epithelium of the nasal septum, as well as the ventral conchae were observed. Eosinophilic globules were located partly apically, partly basally within the cells of the olfactory epithelium (most prominent in level 2 and 3; mean severity level 2 males: 1.0-1.5-1.6-2.8; females: 1.0-1.7-2.0-3.0; mean severity level 3 males: 1.4-1.7-1.6-2.7; females: 1.0-2.0-2.0-2.8). More severely affected animals had an increased incidence of epithelial degeneration and metaplasia.
Furthermore, an increase of goblet cell hypertrophy and hyperplasia in the respiratory epithelium was visible. These changes were associated with an increased infiltrate of eosinophils in the nasal respiratory epithelium.
The trachea revealed a minimally increased inflammatory cell infiltrate at 7.6 mg/m3 (incidence males: 5-4-4-7; incidence females: 5-5-5-4; mean severity of grading males: 1.2-1.0-1.5-1.3; mean severity of grading females. 1.0-1.0-1.0-1.3), as well as an increase of accumulated macrophages/giant cells in the area of the bifurcation starting at 1.5 mg/m3 (incidence males. 1-1-4-3; incidence females: 0-1-2-2).
In the lung, starting at 1.5 mg/m3 accumulation of alveolar macrophages/formation of giant cells of minimal to moderate degree were observed, focally at 1.5 mg/m3, multifocally throughout all lung lobes at 7.6 mg/m3 (incidence males: 0-0-3-10; incidence females: 0-0-1-10).
In animals exposed to 7.6 mg/m3, these accumulated macrophages/giant cells were associated with a chronic inflammation of slight degree affecting primarily the bronchiolo-alveolar junction and led to septal thickening and hypercellularity in this area (incidence males and females: 0-0-0-10).
In single animals, granuloma formation incorporating the macrophage accumulation/giant cells could be observed (incidence males and females: 0-0-0-2).
Furthermore, minimal accumulation of pigment-containing alveolar macrophages were observed (incidence males: 0-0-2-7; incidence females: 0-1-1-2).
The bronchus-associated lymphatic tissue (BALT) revealed an increase in incidence and severity of macrophage accumulation/giant cell/granuloma formation starting at 0.3 mg/m3 up to moderate degree at 7.6 mg/m3 (incidence males: 3-6-9-10; incidence females: 2-4-10-10; mean severity of grading males: 1.0-1.3-1.6-2.6; mean severity of grading females: 1.0-1.0-1.1-2.3). Single to multiple accumulations of macrophages/giant cells and isolated granulomas were observed as a continuum focally or multifocally within areas of BALT of control animals, especially in the left lung lobe or in the caudal aspect of the right lung lobe. In animals exposed to Vestanat B 1358/100, these accumulations/giant cells/granulomas increased in incidence and severity (larger accumulations/granulomas per area BALT; more areas of BALT affected, also in other lung samples) up to moderate degree at 7.6 mg/m3.
Furthermore, a minimal to slight lymphoid hyperplasia was observed starting at 1.5 mg/m3 (incidence males: 0-1-3-7; incidence females: 0-0-5-9).
Lung-associated lymph nodes (LALN) showed lymphoid hyperplasia starting in females at 0,3 mg/m3, in males at 1.5 mg/m3 (incidence males: 0-0-2-10; incidence females: 0-3-5-9), which is consistent with the reported increased size of lung-associated lymph nodes during necropsy observed in animals at 7.5 mg/m3. In addition, a predominantly moderate accumulation of macrophages with giant cell/granuloma formation was observed (incidence males: 0-0-2-10; incidence females: 0-0-0-10), which partly showed minimal to slight central degeneration/necrosis (incidence males: 0-0-0-3; incidence females: 0-0-0-7).
Furthermore, there was an increase in incidence and/or severity of histiocytes infiltrating the lymph nodes (incidence males: 6-6-6-10; incidence females: 4-4-8-10; mean severity of grading males: 1.2-1.2-1.5-2.1; mean severity of grading females: 1.0-1.3-1.5-2.7)

After the post-exposure observation period of 90 days:
Nasal cavity of all animals (including the control air group) revealed the same findings (eosinophilic globules, mean severity level 2 males: 2.0-3.0, females: 2.1-2.9; hypertrophy of goblet cells, mean severity level 2 males 1.8-2.0, females: 2.0-2.0
and hyperplasia of goblet cells, mean severity level 2 males:1.0-1.2, females: 1.0-1.0; infiltrate with eosinophils, mean severity level 2 males: 1.0-1.3, females 1.0-1.0). The findings in controls after the post-exposure observation period were thus observed with a similar severity compared with the exposed animals examined directly after the last day of exposure.
In animals with accumulation of eosinophilic globules of a higher degree incidences of focal degeneration and metaplasia increased.
In the lungs of all exposed animals, accumulation of alveolar macrophages/giant cells (incidence males and females: 0-10) and the slight chronic inflammation in the area of bonchiolo-alveolar junction (incidence males/females: 0-10) were still present. In addition, granuloma formation progressed in almost all animals (incidence males/females: 0-10/9), which showed central degeneration/necrosis in single animals (incidence males/females: 0-1/2).
Minimal to slight accumulation of pigment-containing alveolar macrophages increased in incidence in females and in severity in males compared to the findings directly after exposure (incidence males: 0-7; incidence females: 0-5).
The bronchus-associated lymphatic tissue (BALT) showed macrophage accumulation/giant cells/granuloma (incidence males: 5-10; incidence females: 4-10; mean severity of grading males: 1.4-2.4; mean severity of grading females: 1.0-2.2) and lymphoid hyperplasia (incidence males/females: 0-7/9).
The animals revealed a slight lymphoid hyperplasia within the lung-associated lymph nodes (incidence males and females: 0-10), which is consistent with the reported increased size of respective lymph nodes during necropsy. In addition, moderate accumulation of macrophages with giant cell/granuloma formation was observed (incidence males and females: 0-10), which partly showed minimal to slight central degeneration/necrosis (incidence males/females: 0-4/6).
Furthermore, there was an increase in incidence and/or severity of histiocytes infiltrating the lymph nodes (incidence males: 5-9; incidence females: 7-10; mean severity of grading males: 1.4-2.2; mean severity of grading females: 1.1-2.1).
After 13 weeks of exposure to Vestanat B 1358/100, spleen of some animals exposed to 7.6 mg/m3 revealed a minimally increased extramedullary hematopoiesis (incidence males: 0-1-1-3; incidence females: 0-0-0-2). There was no difference between control and exposed animals regarding this finding after the post-exposure observation period (incidence males: 2-3; incidence females: 3-3).
Three male animals of the control air group with post-exposure observation period and one male animal at 7.5 mg/m3 assigned for bronchoalveolar lavage were prematurely sacrificed for humane reasons. The observed macroscopic findings could be correlated to a marked to severe pyelonephritis and/or cystitis with microscopically detected bacteria. These animals also had a moderate to severe prostatitis. There were some more animals with less severe inflammatory processes within the urinary tract in other groups of exposure.
All other observations in these animals (especially liver: hepatocellular atrophy and vacuolation, Kupffer cell hypertrophy, spleen: atrophy of the white pulp, bone marrow: myeloid hyperplasia, thymus: involution/atrophy correlated to the macroscopically decreased thymus size etc.) were due to the bad condition of the animals.
A decrease in size and/or soft/flaccid consistency of testes were observed uni- or bilaterally throughout all exposure groups, including the control air group consistent with degeneration/atrophy of the testes up to a severe degree followed by aspermia or reduced sperm as well as increased cellular debris in the epididymides.
In female animals of the control air group as well as at 7.5 mg/m3, no cycle determination was possible due to ovarian atrophy and hypertrophy of interstitial cells. In consequence, the epithelium of the cervix and vagina showed no cycle-related changes and was affected by apoptosis/necrosis and sometimes slight pseudomucification.
As rare spontaneous finding, one male animal (animal 71 exposed to 7.6 mg/m3) revealed a malignant Schwannoma within the pancreas, which was observed grossly as a cystic, black-brown coloured nodule approximately 10 cm in diameter. This finding is considere to be incidential and not test item-related.
All other microscopic findings were incidental and spontaneous in nature and could not be attributed to the test item.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Rectal Temperature:
No significantly changed body temperatures were seen in animals exposed to the test item in comparison to control animals.

Bronchoalveolar Lavage:
adverse findings:
Total cell count (TCC), protein (PROT), lymphocytes (C-LYM), lactate dehydrogenase (LDH) and gamma-glutamyl transferase (GGT) were biologically relevant increased but did not reach statistically significance at 7.6 mg/m³.
LDH, an intracellular located enzyme, as well as GGT, a membrane bound enzyme, are established cytotoxic marker indicating cellular damage.
Consequently significantly increased polymorphonuclear cells and biologically relevant increase in protein, lymphocytes, LDH and GGT at 7.6 mg/m³ in the bronchoalveolar lavage are reflected as sings of inflammation also taking significantly increased LALN and lung weights into account (see section 7.9) and thus are considered to be adverse.

non adverse finding:
The average recovery of bronchoalveolar lavage fluid (BALF) was approximately 85-88 % of the instilled volume and was similar amongst all groups. An isolated significantly increase of foamy macrophages was found at 0.3 mg/m³. Adjusted alveolar macrophages (%) were significantly decreased at 0.3 and 7.6 mg/m³. Significantly increased alveolar macrophages with red blood cells were found at 1.5 mg/m³ only. These isolated findings are considered to be of no toxicological relevance.
At 7.6 mg/m³ the following parameter were significantly increased: mean cellular diameter (MCD), mean cellular volume (MCV) and polymorphonuclear cells (PMN and C-PMN). In this context it is remarkable that counted polymorphonuclear cells (C-PMN) at 7.6 mg/m³ were approx. 45-fold higher when compared to air control group. Percentage of alveolar macrophages (AM and AM-ADJ) were significantly decreased at 7.6 mg/m³ whereas absolute count of alveolar macrophages (C-AM) were biologically relevant increased at 7.6 mg/m³. Changes in MCD, MCV and C-AM at 7.6 mg/m³ are considered to be compensatory due to increased phagocytosis as normal part of the clearance mechanisms in the lung.
Total cell count (TCC), protein (PROT), lymphocytes (C-LYM), lactate dehydrogenase (LDH) and gamma-glutamyl transferase (GGT) were biologically relevant increased but did not reach statistically significance at 7.6 mg/m³.
LDH, an intracellular located enzyme, as well as GGT, a membrane bound enzyme, are established cytotoxic marker indicating cellular damage.
Consequently significantly increased polymorphonuclear cells and biologically relevant increase in protein, lymphocytes, LDH and GGT at 7.6 mg/m³ in the bronchoalveolar lavage are reflected as sings of inflammation also taking significantly increased LALN and lung weights into account (see section 7.9) and thus are considered to be adverse.

Microbiological Evaluation:
Male rat number 21 was investigated microbiologically. In the native urine microscopic slide, in all fields of view a lot of cells as well as a few bacteria were observed. Microscopically gram negative rod-shaped bacteria were found in urine and renal tissue. On the Columbia Agarplates pure cultures with swarming properties were seen. Aerob incubated plates showed dark discoloration. No colonies were observed on Agarplates treated with 100 µl blood whereas relevant number of colonies (pure culture) were found on plates incubated aerob as well as anaerob treated with urine and renal tissue. According to the evaluation of the API E20 test (Code 373600057) the bacterium isolated from the pure culture is up to 99.9% likelihood Proteus mirabilis. This result matches with the findings given above of gram negative rod-shaped bacteria and swarming colonies on Columbia Agarplates.
In summary bacteria have been detected in urine and renal tissue of rat number 21 but not in blood. Due to the characteristics, the bacterium is diagnosed as Proteus mirabilis. Proteus mirabilis is ubiquitous in the environment and is considered to be a normal part of human and animals gut flora (see section 12.21). Proteus mirabilis is not listed as recommended infectious agent to monitor for rats according to the FELASA (MÄHLER M. et al., 2014) and thus was not investigated prior to the start of the study. Consequently the microbiological diagnosis of Proteus mirabilis is considered to have no impact on the overall study validity. Nonetheless it is known that Proteus mirabilis may be involved in sporadic urinary tract infections.
Details on results:
Indicative for portal-of entry toxicity were significantly increased lung-associated lymph nodes (LALN) and lung weights, significantly increased polymorphonuclear cells as well as biologically relevant increased total cell count, protein, lymphocytes, LDH and GGT in the BAL at 7.6 mg/m³. These results correlated with gross pathological and histopathological findings as increased lung-associated lymph nodes after end of exposure and recovery period, chronic inflammatory reaction seen at the bronchiolo-alveolar junction of the lung as well as granuloma formation partly with central degeneration/necrosis of granuloma/giant cells also seen in the lung-associated lymph nodes at 7.6 mg/m³.
The daily inhalative exposure to Vestanat B 1358/100 to male and female rats over a period of at least 13 consecutive weeks revealed multiple histopathologically findings within the respiratory tract. Within the nasal cavity, an increase of eosinophilic globules, an increased hypertrophy and hyperplasia of goblet cells, an increased infiltrate with eosinophils as well an increased degeneration and metaplasia of epithelium were observed. At the end of the post-exposure observation period, also the animals of the control air group revealed these findings up to the same degree compared to animals exposed with 7.6 mg/m3. Thus, these findings are regarded as background findings, which occurred earlier and were slightly more prominent in animals exposed to the test item. A direct toxic effect on the nasal mucosa is not assumed.
At 7.6 mg/m3, granuloma formation was seen in the lungs at the end of the exposure period which partly progressed to granuloma with central degeneration and necrosis after the end of the recovery period. Furthermore, chronic inflammation of the bronchiolo-alveolar junction was induced at 7.6 mg/m3 that was still present after the post-exposure observation period. Accumulation of macrophages/giant cells/granuloma showed central degeneration and necrosis at 7.6 mg/m3, central degeneration and necrosis was still present after the recovery period. The moderate accumulation of macrophages/giant cells/granuloma partly with central degeneration/necrosis in the airways of the lung and in the associated lymphoid tissues (bronchus-associated lymphatic tissue, BALT and lung-associated lymph nodes, LALN) as well as the chronic inflammatory reaction seen at the bronchiolo-alveolar junction of the lung at 7.6 mg/m3 are considered to be adverse.
Extramedullary hematopoiesis in the spleen occurred in animals of the control group as well as in animals exposed to 7.6 mg/m3 test item after the recovery period. Incidences in males were 2-3 and in females 3-3 in control groups and at 7.6 mg/m3 respectively. Of note, the highest severity (moderate grade) was observed in a female control rat. At the end of the exposure period, minimal up to slight extramedullary hematopoiesis in the spleen was found in males (incidence 0-1-1-3) and females (incidence 0-0-0-2). Highest severity and indistinguishable incidence compared to test item exposed animals of extramedullary hematopoiesis was seen in control animals at the end of the recovery period. Thus, this finding is interpreted to be not test item-induced.
Relative body weight adjusted spleen weights were significantly increased in females at the end of the exposure period and significantly increased in males at the end of the recovery period at 7.6 mg/m³. Reticulocytes were significantly increased in females at the end of the exposure period and significantly decreased in males at the end of the recovery period at 7.6 mg/m³. Significantly decreased percentage of reticulocytes in the blood at 7.6 mg/m3 at the end of the recovery period in males do not correlate with significantly increased body weight-adjusted relative spleen weights at that concentration. Furthermore significantly decreased reticulocyte counts and significantly increased relative spleen weights were only seen in males and not in females. Additionally no test item induced histopathological findings were seen in the spleen at the end of the recovery period. Consequently these findings are reflected to be inconsistent and not of toxicological relevance.
At the end of the exposure period significantly increased reticulocytes and significantly increased body weight-adjusted relative spleen weights were found in females at 7.6 mg/m3. Of note, only body weight-adjusted spleen weights in female rats at 7.6 mg/m3 were significantly increased whereas absolute and brain weight-adjusted spleen weights did not reach statistically significance when compared to controls. Furthermore it has to be kept into account that only one gender was affected. Additionally, no further significantly changed red cell parameters were found at the end of the exposure period. Taking all those information into account it was considered that increased reticulocytes and increased body weight-adjusted relative spleen weights at the end of the exposure period are non-adverse.
Leucocytes and specifically neutrophils were increased at 7.6 mg/m³ in males and females at the end of the exposure period. At the end of the recovery period, leucocytes, lymphocytes, neutrophils, monocytes and atypical cells were increased at 7.6 mg/m³ in comparison to the air control. Systemically increased white cell parameters seen at clinical pathology mentioned above, are interpreted to be the consequence of cell recruitment due to inflammatory processes in the respiratory tract at 7.6 mg/m³. In this context the increased white blood cellsespacially neutrophils in the blood at 7.6 mg/m³ may be explained by raised recruitment due inflammatory processes in the lungs at 7.6 mg/m3.
Dose descriptor:
NOAEC
Effect level:
1.5 mg/m³ air
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: portal-of-enty toxicity in the respiratory tract associated tissues at 7.6 mg/m³; a NOEL could not be established
Dose descriptor:
NOAEC
Effect level:
7.6 mg/m³ air
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no systemic toxicity observed
Critical effects observed:
yes
Lowest effective dose / conc.:
7.6 mg/m³ air
System:
other: respiratory system
Organ:
other: portal-of-entry toxicity (local effects) in the respiratory tract; no systemic toxicity
Treatment related:
yes
Conclusions:
No subchronic inhalation toxicity study was performed with the registered substance. However, a grouping of substances and read-across approach according to Regulation (EC) No 1907/2006 (REACH), Annex XI 1.5 and following ECHA Read-Across Assessment Framework, RAAF (2015) is accomplished for five blocked diisocyanate oligomers (also for the read-across target substance isophorone diisocyanate, oligomeristion product, blocked with hexahydro-2H-azepin-2 -one).
For repeated inhalation toxicity testing the category is divided into two sub-groups – the liquid aerosol for blocked HDI-based oligomers and the powder aerosol for blocked IPDI-based oligomers. Thus, within the category the analogue read-across approach is followed, i.e. ECHA RAAF Scenario 2.

Within this analogue approach, a GLP-conform and Guideline-compliant 90-day subchronic inhalation toxicity study was performed with the read-across source for the registered substance (isophorone diisocyanate, oligomeristion product, blocked with 2 -butanone oxime) on request of ECHA. In this subchronic inhalation toxicity study with 13 week exposure and 13 week recovery period the No-Observed-Adverse-Effect-Concentration (NOAEC) is 1.5 mg/m³ due to respiratory tract associated adverse findings seen in BAL, histopathology and weight increase of lung and LALN at 7.6 mg/m³. These effects represent portal-of-entry toxicity. Test substance related systemic toxicity was not observed and thus, the NOAEC for systemic toxicity is 7.6 mg/m³, the highest dose tested.
Thus, according to EU Regulation 1272/2008 classification for specific target organ toxicity-repeated exposure Category 1 (STOT RE 1) is applicable for the source as well as the target substance.

An updated outline of a grouping-strategy based on read-across of the available toxicological data for 5 blocked diisocyanate oligomers is attached to the endpoint summaries for 'Repeated dose toxicity' and 'Toxicity to reproduction' in IUCLID as a separate document.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat
Quality of whole database:
All valid studies are valid without restriction (Klimisch score 1).

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
(2009)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
GLP compliance:
yes (incl. certificate)
Remarks:
The diagnostic microbiological evaluation of blood, urine and renal tissue of rat number 21 was done under non-GLP condition. This deviation did not limit the assessment of the results obtained.
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (< 30°C) at darkness, headspace purge with N2 after opening of reservoir
- Stability under test conditions: Stability certified for the duration of study
- Solubility and stability of the test substance in the solvent/vehicle:
The integrity and stability of the aerosol generation and exposure system was measured by using a Microdust real-time aerosol photometer (Casella, Buffalo, UK) and at 0.3 mg/m³ the R&P Teom 1400a (amibent particulate monitor; Thermo Fisher Scientific Inc., USA). Samples were taken continuously from the vicinity of the breathing zone. Gravimetric analysis of filter samples and the real-time monitoring of the aerosol test atmosphere from the breathing zone area indicated that the exposure conditions were stable over the daily 6-h exposure period
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Healthy young adult SPF bred Wistar rats, strain Crl:(Wi)WU BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:(Wi)WU BR (SPF-bred)
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 2 months
- Weight at study initiation: Weight range at first exposure day: males 230-280 g. 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 with gnawing sticks
- Diet and water: ad libitum
- Acclimation period: at least 10-14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 40-60 %
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12 h/12 h
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
ca. 2.3 µm
Remarks on MMAD:
The mean Mass Median Aerodynamic Diameter’s (MMAD) measured by cascade impactor were in the range of 2.23 – 2.35 µm within the exposure groups (Geometric Standard Deviation: 1.86 – 2.30). MMADs measured by laser velocimetry ranged from 2.66 – 2.69 µm within the exposure groups (Geometric Standard Deviation: 1.72 – 1.73).
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Rats were exposed under dynamic directed-flow nose-only exposure conditions.
- Exposure apparatus: Details of the nose-only exposure system, including its validation, have been published previously (Pauluhn, Journal of Applied Toxicology 13, 1994, 55-62; Pauluhn and Thiel, J. Appl. Toxicol. 27, 2007, 160-167). 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).
- Method of holding animals in test chamber: Animals were exposed in polycarbonate 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.
- Source and rate of air: 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; SMG Interlink, USA) and TSI Mass Flow meter 4040 (TSI Incorporated, USA) and were checked for correct performance at regular intervals.
- 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 BEKO RA 55 compressed air dryer. Adequate control devices were employed to control supply pressure.
- Temperature, humidity: Temperature and humidity measurements are also performed by the computerized Data Acquisition and Control System using HC2-S3 sensors (Rotronic Messgeräte GmbH, Ettlingen, 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.
- Air flow rate: 5.85 L/min
- Air change rate: The test atmosphere generation conditions provide an adequate number of air exchanges per hour [45 L/min x 60 min/(11.4 L/chamber) = 237, continuous generation of test atmosphere]. Thus, under the test conditions used a chamber equilibrium is attained in less than one minute of exposure.
- Treatment of exhaust air: The exhaust air was purified via filter systems. These filters were disposed of by the laboratory.
The ratio between supply and exhaust air was selected so that 90 % of the supplied air was extracted via the exhaust air location and, if applicable, via sampling ports. Aerosol scrubbing devices were used for exhaust air clean-up. During sampling, the exhaust air was reduced in accordance with the sampling flow rate using a computerized Data Acquisition and Control System so that the total exhaust air flow rate was adjusted 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).
- Atmosphere generation: Test atmospheres were generated using a WRIGHT DUST FEEDER system (BGI Inc., Waltham, MA 02154, USA). For dry powder dispersion, conditioned compressed dry air (30 liters/min; generic dispersion pressure: 1.8 kPa) was used. The principle performance of the WRIGHT DUST FEEDER dust generating system can be described as follows: the test substance was metered in a reservoir and then was compressed to a pellet using approxi¬mately 1 metric ton by a carva laboratory press (F. S. Carver Inc., Wabash, IN 46992, USA). From this pellet defined amounts of test substance were scraped off and entrained into the main air flow.
The airborne powder was then conveyed into the inner cylinder of the inhalation chamber. Principles differences between chamber set-ups were related to the dilution cascades to attain the targeted concentrations.
- Method of particle size determination: The particle-size distribution was analyzed using a BERNER critical orifice cascade impactor. An adhesive stage coating (silicone spray) was used to minimize particle bounce due to the adhesive properties of the test article. Each impactor stage was covered with an aluminum foil. Collecting metrics were subjected to gravimetric analysis using a digital balance and pre-conditioning method as described in the previous section.

TEST ATMOSPHERE
- Brief description of analytical method used: The test-substance concentration was determined by gravimetric analysis (filter: glass-fiber filter, Sartorius, Göttingen, Germany; post-sampling conditioning period of 15 min at room temperature). This method was used to define the actual concentration.
- The integrity and stability of the aerosol generation and exposure system was measured by using a Microdust real-time aerosol photometer (Casella, Buffalo, UK) and at 0.3 mg/m³ the R&P Teom 1400a (amibent particulate monitor; Thermo Fisher Scientific Inc., USA). Samples were taken continuously from the vicinity of the breathing zone.
- Samples taken from breathing zone: yes
- Particle size distribution: The particle size distribution was analyzed using a TSI-Laser Velocimeter APS 3321. Calculations consider both a particle size distribution that encompasses aerodynamic diameters (Dae) of 0.5 to 7.4 µm and sample flows ranging from 8 to 80 ml/sec.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The mean Mass Median Aerodynamic Diameter’s (MMAD) measured by cascade impactor were in the range of 2.23 – 2.35 µm within the exposure groups (Geometric Standard Deviation: 1.86 – 2.30). MMADs measured by laser velocimetry ranged from 2.66 – 2.69 µm within the exposure groups (Geometric Standard Deviation: 1.72 – 1.73).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test-substance concentration was determined by gravimetric analysis. The concentrations of test atmosphere given are based on breathing zone concentrations from filter analyses which reflect the active substance.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day on 5 consecutive days/week for 13 weeks
Dose / conc.:
0.3 mg/m³ air (analytical)
Remarks:
target concentration: 0.3 mg/m³
Dose / conc.:
1.5 mg/m³ air (analytical)
Remarks:
target concentration: 1.5 mg/m³
Dose / conc.:
7.6 mg/m³ air (analytical)
Remarks:
target concentration: 7.5 mg/m³
No. of animals per sex per dose:
10 rats/sex/group plus 6 additional males/group for lung lavage plus 10 additional rats/sex of the control and high dose as recovery groups
Control animals:
other: rats exposed under otherwise identical test conditions to dry air served as concurrent control group
Details on study design:
Dose selection rationale:
Target concentrations for this repeated exposure study were based on the results of a 2 week inhalation toxicity study with study identification number T100111-4 (Exposure 9 x 6h/day, 5 days/week; recovery period of 2 weeks) using Vestanat B1358/100 as test item. See Pauluhn, 2013.
- Rationale for selecting satellite groups:
After two weeks of recovery in the 2 week inhalation study, the findings in lungs and LALN were still detectable. Additionally, accumulations of epitheloid cells occurred in BALT and LALN. Therefore satellite groups for lung lavage and recovery were included in this study.
- Post-exposure recovery period in satellite groups: 13 weeks
Positive control:
positive controls are not adequate for this study type
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, all animals
- Time schedule: Clinical signs were recorded twice daily, before and after exposure and once a day on exposure-free days and recovery period.

BODY WEIGHT: Yes, all animals
- Time schedule for examinations: During the exposure period, the body weights were determined twice per week to differentially record their gains over exposure-free weekends, and once per week during the postexposure period.

FOOD AND WATER CONSUMPTION: Yes.
- The individual food and water consumption of all animals (excluded lavage animals) were measured weekly (Mondays).

OPHTHALMOSCOPIC EXAMINATION: Yes.
- Prior the first exposure all animals (excluded lavage animals) and toward at the end of the dosing phase (on day 86) control and high dose animals were examined.

RECTAL TEMPERATURES: Yes, all animals
- Time schedule for examinations: Five animals per group 1-8 were subjected to body temperature measurements. The rectal temperatures were measured directly after cessation of exposure (approximately within ½hour after the end of exposure) using a digital thermometer with a rectal probe for rats during the dosing period on days rel. 0 and 85 as well as on day 85 of the recovery period.

FUNCTIONAL OBSERVATION BATTERY: Yes.
- The first five core rats of group 1-8 were used for functional observation battery investigation on day relative 0, 59 and 85. Each rat was firstly observed in the home cage and then individually examined. The following reflexes were evaluated: visual placing response and 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). Measurements of grip strength were measured qualitatively but defined as semi quantitative.

HAEMATOLOGY: Yes
- Blood samples (non-fasted) for hematology, coagulation and serum chemistry parameters were collected from all core animals (end of the exposure period) and recovery toxicology animals (end of the recovery period) during sacrifice by cardiac puncture.

CLINICAL CHEMISTRY: Yes
- Urines of core animals were collected on day 87 and 88 of the dosing phase as well as on day 87 and 88 at the recovery period, overnight (approximately 16 h). Therefore animals were housed in specific metabolism cages.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals
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.

ORGAN WEIGHTS: Yes, all animals
The following organs were weighed at necropsy: adrenal glands, brain, heart, kidneys, liver, lung, thymus,lung-associated lymph nodes, spleen, ovaries, uterus, testes, epididymis

HISTOPATHOLOGY: Yes
- Histopathology was performed on the following organs/tissues (core and recovery animals): nasal cavities (4 levels: L-I to L-IV) / Young, 1981), pharynx, larynx, trachea, lung, lung associated lymph nodes (hilus) and additionally in the control (control air) and high dose groups (core toxicology animals) the organs/tissues listed in the table Table 8. Tissues were fixed in 10% neutral-buffered formalin (NBF), except the left and two thirds of the right kidneys (fixed in Davidson’s fixative then transferred to 10% NBF) and followed routine processing in the histopathology laboratory. Lungs were instilled with 10% NBF and then transferred to 10% NBF solution. The tissue slides were examined by a veterinary pathologist. In addition, a formal cross-check was performed. The findings reported are those agreed-upon between the cross-checking and the study pathologist.

MICROBIOLOGICAL EVALUATION:
Animal number 21 (control group) was euthanized on 12-AUG-2016 due to blood in the urine in combination with clinical signs as e.g. piloerection, reduced body tone. To investigate the causality of the blood in the urine an additional microbiological diagnostic has been initiated. These microbiological investigation and evaluation took place at Bayer AG, Antiinfectiva, Bayer research Center Aprath, 42096 Wuppertal, Germany under non-GLP conditions.
Other examinations:
BRONCHOALVEOLAR LAVAGE: Yes, animals assigned (6 males/group)
Bronchoalveolar lavage fluid was sampled at the end of the exposure period using sodium pentobarbital. All rats, irrespective of the day of death, were given a gross-pathological examination. Consideration was given to perform 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 are recorded. Corresponding to the suitable SOP the cranium and the nasal cavities of some animals per group were opened.
In BAL-fluid (BALF) the following indicators of pulmonary damage were assessed: Total number of lavaged cells, including the volume and diameter, cytodifferentiation, lactate dehydrogenase (LDH9), total protein (PRQ9), gamma-glutamyltransferase (GGT9), phospholipids (PL9).

After complete exsanguination, the excised wet lungs were weighed and following ligation of the trachea, the lung lobes were then lavaged via a tracheal cannula with two volumes of 5-ml of physiological saline (nearly 37°C), which was left in the lungs for 30 sec, withdrawn, re-instilled for an additional 30 sec. This procedure was repeated once. From each rat the retrieved bronchoalveolar lavage fluid (BALF) from the lung lavage was then pooled and adjusted to 10-ml total volume. Prior to centrifuging the samples were kept on ice. Then the pooled lavage fluid was centrifuged at approximately 200 x g for approximately 10 min. at <10°C (Sigma 4-16K refrigerated centrifuge) and the resultant cell-free supernatant was analyzed for the endpoints/parameters listed below. The cell pellet was re-suspended in PBS-BSA (Dulbecco’s phosphate buffered saline with Ca2+ and Mg2+ containing 0.1% bovine serum albumin; Sigma, Deisenhofen, Germany) and then centrifuged (2 x 105 per cytospot) onto slides using a cytocentrifuge (Shandon Cytospin 4). Cell counts were determined in triplicates after 1:1000 dilution using a CASY cell counter + analyzer. Air-dried slides were fixed with a mixture of methanol:acetone, stained according to Pappenheim, and differentiated by light microscopy. Cytospots were differentiated by counting 300 cells/cytospot.
For the analysis of the BAL data the validated BalNew.exe and ANOVA.exe Software (Author Prof. Pauluhn, Bayer AG, Bayer Research Center Aprath, Wuppertal, Germany) has been used.
Statistics:
Analysis of variance (ANOVA BCTIC): This parametric method checks for normal distribution of data by comparing the median and mean. The groups are compared at a confidence level of (1-*) = 95% (P = 0.05). The test for the between-group homogeneity of the variance employed Box's test if more than 2 study groups were compared with each other. If the above F-test shows that the intra-group variability is greater than the inter-group variability, this is shown in the Appendix as "no statistical difference between the groups". If a difference is found then a pair-wise post-hoc comparison is conducted (1- and 2-sided) using the Games and Howell modification of the Tukey-Kramer significance post hoc test. This program (FORTRAN source code) was originally obtained from BCTIC and is used as means to evaluate off-line data. In most instances, data analyzed by ANOVABCTIC are also summarized in graphical form. In figures statistical significant differences are indicated by asterisks (* P < 0.05; ** P < 0.01).
BAL data: The individual results of the BAL-Investigation were manually entered in the program: BALnew.exe, Version 3.0 (Author Prof. J. Pauluhn, Bayer AG, Germany; Software was written in DIGITAL Fortran). The BALnew.exe software calculates group based arithmetic means and the standard deviations (SD) relating to 10 ml bronchoalveolar lavage volume. Means and SD were statistically evaluated using the ANOVABCTIC procedure (vide infra).
Description (incidence and severity):
No relevant concentration-dependent test item-related clinical symptoms were seen in the course of the study.
Red encrusted noses, reddish-brownish discolored fur around the eyes, the head or the neck/back area were partially seen in some control animals as well as in some animals exposed to the test item. These findings are well known in inhalation toxicity studies and are considered to be nose-only inhalation technique-related due to restraining in the inhalation tubes but not test item-induced. Isolated slight up to moderate piloerection was seen after exposure session in a few males of the control group as well in a few males exposed to the test item and is reflected to be not test item induced.
Description (incidence):
Three male rats (Animal number 21, 24 and 26) of the air control group and one male rat of the high exposure group (Animal number 60) were euthanized during the course of the study due to animal welfare reasons. All four euthanized animals showed blood in urine associated with reduced general condition e.g. reduced body tone, reduced motility and piloerection. Microbiological investigation of the urine and renal tissue of animal no. 21 demonstrated Proteus mirabilis (details are given in section 7.15). Since clinical signs as well as pathological findings in all four euthanized animals were similar, it is considered that the microbiological findings seen in rat number 21 are representative for all euthanized rats.
In summary, it is considered that the blood in the urine and the associated poor health condition of euthanized male animals are not test item-related since 3 control animals and 1 high concentration animal were affected. Furthermore the clinical and pathological findings are considered to be caused by an ascending urinary tract infection with Proteus mirabilis.
Description (incidence and severity):
Comparisons between the control and the exposure groups did not reveal any statistically concentration-dependent differences in body weights and cumulative body weight gain during the course of the study. Isolated significantly changed absolute body weights or cumulative body weight gain mean values are considered to be not test item associated due to lack of concentration-dependency.
Description (incidence and severity):
No relevant test item-related changes in food and water consumption were observed when compared to control animals.
Description (incidence and severity):
The ophthalmological examination did not reveal any test item-related findings.
Description (incidence and severity):
Reticulocytes were significantly increased in females at 7.6 mg/m³ at the end of the exposure period. At the end of the recovery period, reticulocytes were significantly decreased in males at 7.6 mg/m³. In females at the end of the exposure period, neutrophils were significantly increased at 7.6 mg/m³. At the end of the recovery period, leucocytes, lymphocytes, neutrophils, monocytes and atypical cells were significantly increased at 7.6 mg/m³ in comparison to the air control.
Furthermore isolated significantly changed parameters occurred and were reflected to be incidental.
Description (incidence and severity):
Isolated significantly changed parameters occurred and were reflected to be incidental.
Description (incidence and severity):
Quantitative urinalysis, microscopy of urine sediment and qualitative urinalysis were performed and did not reveal any concentration-dependent relevant differences in test item exposed animals when compared to air control rats.
Description (incidence and severity):
A battery of reflex measurements was made on day 3, 59 and 85. Differences between animals exposed to the test item compared to the control group did not occur.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the exposure period (main groups) test item-related increased lung-associated lymph nodes (LALN) were found in males and females exposed to 7.6 mg/m³. Furthermore absolute and relative (% of TBW and % of brain weights) lung weights were significantly increased in females at 7.6 mg/m³. Statistically significant higher absolute lung weights were observed in females at 1.5 mg/m³, whereas relative lung weight was unchanged; here it has to be taken into account that total body weights were also slightly higher when compared to controls. Consequently, relative lung weights (% of TBW and % of brain weight) at this dose level must be given preference. An Isolated significant increase in liver weight found in females exposed to 1.5 mg/m³ is considered to be not test item related due to lack of dose-dependency. Significantly raised relative spleen weights (% of TBW) were measured in female animals exposed to 7.6 mg/m³, whereas absolute spleen weights and relative spleen weights expressed as % of brain weights did not reach statistically significance.
At the end of the recovery period significantly increased absolute and relative (% of brain weight) adrenal gland, kidney, liver, LALN and lung weights were found in females exposed to 7.6 mg/m³, whereas additionally LALN weights were also significantly increased in males at 7.6 mg/m³. Relative spleen weights expressed as % of TBW were significantly increased in males in males at 7.6 mg/m³. Significantly changed adrenal gland, kidney and liver weights in comparison to the control group are interpreted to be not adverse in the absence of correlating histopathological and/or clinical pathological findings.
Significantly increased lung-associated lymph nodes and lung weights at 7.6 mg/m³ at the end of the exposure period as well as at the end of the recovery period are reflected to be test item-induced.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In males and females exposed to 7.6 mg/m³, lung-associated lymph nodes were increased in size (incidence males/females: 0/0-0/0-0/0-7/6). This finding was non-reversible after the post-exposure period (incidence males/females: 0/0-6/7).
A decrease in size and/or soft/flaccid consistency of testes were observed uni- or bilaterally throughout all exposure groups, including the control air group, that was as such not treatment related.
The three prematurely sacrificed male animals of the control air group with post-exposure observation period and the male animal at 7.6 mg/m³ assigned for bronchoalveolar lavage revealed findings at the urinary bladder consisting of increase in size, increased thickness of the wall and content changes. Furthermore, thymus was decreased in size in these animals. Since the three animals were affected with Proteus mirabilis no treatment relationship is seen.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of exposure:
In the nasal cavity of the exposed animals starting at 0,3 mg/m3 up to a moderate degree at 7.6 mg/m3, an increase in extent (whereas in level 2 and 3 nearly all animals were affected, more exposed animals showed the finding in level 1 when compared to control) and severity of eosinophilic globules predominantly at the transition between the respiratory and olfactory epithelium of the nasal septum, as well as the ventral conchae were observed. Eosinophilic globules were located partly apically, partly basally within the cells of the olfactory epithelium (most prominent in level 2 and 3; mean severity level 2 males: 1.0-1.5-1.6-2.8; females: 1.0-1.7-2.0-3.0; mean severity level 3 males: 1.4-1.7-1.6-2.7; females: 1.0-2.0-2.0-2.8). More severely affected animals had an increased incidence of epithelial degeneration and metaplasia.
Furthermore, an increase of goblet cell hypertrophy and hyperplasia in the respiratory epithelium was visible. These changes were associated with an increased infiltrate of eosinophils in the nasal respiratory epithelium.
The trachea revealed a minimally increased inflammatory cell infiltrate at 7.6 mg/m3 (incidence males: 5-4-4-7; incidence females: 5-5-5-4; mean severity of grading males: 1.2-1.0-1.5-1.3; mean severity of grading females. 1.0-1.0-1.0-1.3), as well as an increase of accumulated macrophages/giant cells in the area of the bifurcation starting at 1.5 mg/m3 (incidence males. 1-1-4-3; incidence females: 0-1-2-2).
In the lung, starting at 1.5 mg/m3 accumulation of alveolar macrophages/formation of giant cells of minimal to moderate degree were observed, focally at 1.5 mg/m3, multifocally throughout all lung lobes at 7.6 mg/m3 (incidence males: 0-0-3-10; incidence females: 0-0-1-10).
In animals exposed to 7.6 mg/m3, these accumulated macrophages/giant cells were associated with a chronic inflammation of slight degree affecting primarily the bronchiolo-alveolar junction and led to septal thickening and hypercellularity in this area (incidence males and females: 0-0-0-10).
In single animals, granuloma formation incorporating the macrophage accumulation/giant cells could be observed (incidence males and females: 0-0-0-2).
Furthermore, minimal accumulation of pigment-containing alveolar macrophages were observed (incidence males: 0-0-2-7; incidence females: 0-1-1-2).
The bronchus-associated lymphatic tissue (BALT) revealed an increase in incidence and severity of macrophage accumulation/giant cell/granuloma formation starting at 0.3 mg/m3 up to moderate degree at 7.6 mg/m3 (incidence males: 3-6-9-10; incidence females: 2-4-10-10; mean severity of grading males: 1.0-1.3-1.6-2.6; mean severity of grading females: 1.0-1.0-1.1-2.3). Single to multiple accumulations of macrophages/giant cells and isolated granulomas were observed as a continuum focally or multifocally within areas of BALT of control animals, especially in the left lung lobe or in the caudal aspect of the right lung lobe. In animals exposed to Vestanat B 1358/100, these accumulations/giant cells/granulomas increased in incidence and severity (larger accumulations/granulomas per area BALT; more areas of BALT affected, also in other lung samples) up to moderate degree at 7.6 mg/m3.
Furthermore, a minimal to slight lymphoid hyperplasia was observed starting at 1.5 mg/m3 (incidence males: 0-1-3-7; incidence females: 0-0-5-9).
Lung-associated lymph nodes (LALN) showed lymphoid hyperplasia starting in females at 0,3 mg/m3, in males at 1.5 mg/m3 (incidence males: 0-0-2-10; incidence females: 0-3-5-9), which is consistent with the reported increased size of lung-associated lymph nodes during necropsy observed in animals at 7.5 mg/m3. In addition, a predominantly moderate accumulation of macrophages with giant cell/granuloma formation was observed (incidence males: 0-0-2-10; incidence females: 0-0-0-10), which partly showed minimal to slight central degeneration/necrosis (incidence males: 0-0-0-3; incidence females: 0-0-0-7).
Furthermore, there was an increase in incidence and/or severity of histiocytes infiltrating the lymph nodes (incidence males: 6-6-6-10; incidence females: 4-4-8-10; mean severity of grading males: 1.2-1.2-1.5-2.1; mean severity of grading females: 1.0-1.3-1.5-2.7)

After the post-exposure observation period of 90 days:
Nasal cavity of all animals (including the control air group) revealed the same findings (eosinophilic globules, mean severity level 2 males: 2.0-3.0, females: 2.1-2.9; hypertrophy of goblet cells, mean severity level 2 males 1.8-2.0, females: 2.0-2.0
and hyperplasia of goblet cells, mean severity level 2 males:1.0-1.2, females: 1.0-1.0; infiltrate with eosinophils, mean severity level 2 males: 1.0-1.3, females 1.0-1.0). The findings in controls after the post-exposure observation period were thus observed with a similar severity compared with the exposed animals examined directly after the last day of exposure.
In animals with accumulation of eosinophilic globules of a higher degree incidences of focal degeneration and metaplasia increased.
In the lungs of all exposed animals, accumulation of alveolar macrophages/giant cells (incidence males and females: 0-10) and the slight chronic inflammation in the area of bonchiolo-alveolar junction (incidence males/females: 0-10) were still present. In addition, granuloma formation progressed in almost all animals (incidence males/females: 0-10/9), which showed central degeneration/necrosis in single animals (incidence males/females: 0-1/2).
Minimal to slight accumulation of pigment-containing alveolar macrophages increased in incidence in females and in severity in males compared to the findings directly after exposure (incidence males: 0-7; incidence females: 0-5).
The bronchus-associated lymphatic tissue (BALT) showed macrophage accumulation/giant cells/granuloma (incidence males: 5-10; incidence females: 4-10; mean severity of grading males: 1.4-2.4; mean severity of grading females: 1.0-2.2) and lymphoid hyperplasia (incidence males/females: 0-7/9).
The animals revealed a slight lymphoid hyperplasia within the lung-associated lymph nodes (incidence males and females: 0-10), which is consistent with the reported increased size of respective lymph nodes during necropsy. In addition, moderate accumulation of macrophages with giant cell/granuloma formation was observed (incidence males and females: 0-10), which partly showed minimal to slight central degeneration/necrosis (incidence males/females: 0-4/6).
Furthermore, there was an increase in incidence and/or severity of histiocytes infiltrating the lymph nodes (incidence males: 5-9; incidence females: 7-10; mean severity of grading males: 1.4-2.2; mean severity of grading females: 1.1-2.1).
After 13 weeks of exposure to Vestanat B 1358/100, spleen of some animals exposed to 7.6 mg/m3 revealed a minimally increased extramedullary hematopoiesis (incidence males: 0-1-1-3; incidence females: 0-0-0-2). There was no difference between control and exposed animals regarding this finding after the post-exposure observation period (incidence males: 2-3; incidence females: 3-3).
Three male animals of the control air group with post-exposure observation period and one male animal at 7.5 mg/m3 assigned for bronchoalveolar lavage were prematurely sacrificed for humane reasons. The observed macroscopic findings could be correlated to a marked to severe pyelonephritis and/or cystitis with microscopically detected bacteria. These animals also had a moderate to severe prostatitis. There were some more animals with less severe inflammatory processes within the urinary tract in other groups of exposure.
All other observations in these animals (especially liver: hepatocellular atrophy and vacuolation, Kupffer cell hypertrophy, spleen: atrophy of the white pulp, bone marrow: myeloid hyperplasia, thymus: involution/atrophy correlated to the macroscopically decreased thymus size etc.) were due to the bad condition of the animals.
A decrease in size and/or soft/flaccid consistency of testes were observed uni- or bilaterally throughout all exposure groups, including the control air group consistent with degeneration/atrophy of the testes up to a severe degree followed by aspermia or reduced sperm as well as increased cellular debris in the epididymides.
In female animals of the control air group as well as at 7.5 mg/m3, no cycle determination was possible due to ovarian atrophy and hypertrophy of interstitial cells. In consequence, the epithelium of the cervix and vagina showed no cycle-related changes and was affected by apoptosis/necrosis and sometimes slight pseudomucification.
As rare spontaneous finding, one male animal (animal 71 exposed to 7.6 mg/m3) revealed a malignant Schwannoma within the pancreas, which was observed grossly as a cystic, black-brown coloured nodule approximately 10 cm in diameter. This finding is considere to be incidential and not test item-related.
All other microscopic findings were incidental and spontaneous in nature and could not be attributed to the test item.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Rectal Temperature:
No significantly changed body temperatures were seen in animals exposed to the test item in comparison to control animals.

Bronchoalveolar Lavage:
adverse findings:
Total cell count (TCC), protein (PROT), lymphocytes (C-LYM), lactate dehydrogenase (LDH) and gamma-glutamyl transferase (GGT) were biologically relevant increased but did not reach statistically significance at 7.6 mg/m³.
LDH, an intracellular located enzyme, as well as GGT, a membrane bound enzyme, are established cytotoxic marker indicating cellular damage.
Consequently significantly increased polymorphonuclear cells and biologically relevant increase in protein, lymphocytes, LDH and GGT at 7.6 mg/m³ in the bronchoalveolar lavage are reflected as sings of inflammation also taking significantly increased LALN and lung weights into account (see section 7.9) and thus are considered to be adverse.

non adverse finding:
The average recovery of bronchoalveolar lavage fluid (BALF) was approximately 85-88 % of the instilled volume and was similar amongst all groups. An isolated significantly increase of foamy macrophages was found at 0.3 mg/m³. Adjusted alveolar macrophages (%) were significantly decreased at 0.3 and 7.6 mg/m³. Significantly increased alveolar macrophages with red blood cells were found at 1.5 mg/m³ only. These isolated findings are considered to be of no toxicological relevance.
At 7.6 mg/m³ the following parameter were significantly increased: mean cellular diameter (MCD), mean cellular volume (MCV) and polymorphonuclear cells (PMN and C-PMN). In this context it is remarkable that counted polymorphonuclear cells (C-PMN) at 7.6 mg/m³ were approx. 45-fold higher when compared to air control group. Percentage of alveolar macrophages (AM and AM-ADJ) were significantly decreased at 7.6 mg/m³ whereas absolute count of alveolar macrophages (C-AM) were biologically relevant increased at 7.6 mg/m³. Changes in MCD, MCV and C-AM at 7.6 mg/m³ are considered to be compensatory due to increased phagocytosis as normal part of the clearance mechanisms in the lung.
Total cell count (TCC), protein (PROT), lymphocytes (C-LYM), lactate dehydrogenase (LDH) and gamma-glutamyl transferase (GGT) were biologically relevant increased but did not reach statistically significance at 7.6 mg/m³.
LDH, an intracellular located enzyme, as well as GGT, a membrane bound enzyme, are established cytotoxic marker indicating cellular damage.
Consequently significantly increased polymorphonuclear cells and biologically relevant increase in protein, lymphocytes, LDH and GGT at 7.6 mg/m³ in the bronchoalveolar lavage are reflected as sings of inflammation also taking significantly increased LALN and lung weights into account (see section 7.9) and thus are considered to be adverse.

Microbiological Evaluation:
Male rat number 21 was investigated microbiologically. In the native urine microscopic slide, in all fields of view a lot of cells as well as a few bacteria were observed. Microscopically gram negative rod-shaped bacteria were found in urine and renal tissue. On the Columbia Agarplates pure cultures with swarming properties were seen. Aerob incubated plates showed dark discoloration. No colonies were observed on Agarplates treated with 100 µl blood whereas relevant number of colonies (pure culture) were found on plates incubated aerob as well as anaerob treated with urine and renal tissue. According to the evaluation of the API E20 test (Code 373600057) the bacterium isolated from the pure culture is up to 99.9% likelihood Proteus mirabilis. This result matches with the findings given above of gram negative rod-shaped bacteria and swarming colonies on Columbia Agarplates.
In summary bacteria have been detected in urine and renal tissue of rat number 21 but not in blood. Due to the characteristics, the bacterium is diagnosed as Proteus mirabilis. Proteus mirabilis is ubiquitous in the environment and is considered to be a normal part of human and animals gut flora (see section 12.21). Proteus mirabilis is not listed as recommended infectious agent to monitor for rats according to the FELASA (MÄHLER M. et al., 2014) and thus was not investigated prior to the start of the study. Consequently the microbiological diagnosis of Proteus mirabilis is considered to have no impact on the overall study validity. Nonetheless it is known that Proteus mirabilis may be involved in sporadic urinary tract infections.
Details on results:
Indicative for portal-of entry toxicity were significantly increased lung-associated lymph nodes (LALN) and lung weights, significantly increased polymorphonuclear cells as well as biologically relevant increased total cell count, protein, lymphocytes, LDH and GGT in the BAL at 7.6 mg/m³. These results correlated with gross pathological and histopathological findings as increased lung-associated lymph nodes after end of exposure and recovery period, chronic inflammatory reaction seen at the bronchiolo-alveolar junction of the lung as well as granuloma formation partly with central degeneration/necrosis of granuloma/giant cells also seen in the lung-associated lymph nodes at 7.6 mg/m³.
The daily inhalative exposure to Vestanat B 1358/100 to male and female rats over a period of at least 13 consecutive weeks revealed multiple histopathologically findings within the respiratory tract. Within the nasal cavity, an increase of eosinophilic globules, an increased hypertrophy and hyperplasia of goblet cells, an increased infiltrate with eosinophils as well an increased degeneration and metaplasia of epithelium were observed. At the end of the post-exposure observation period, also the animals of the control air group revealed these findings up to the same degree compared to animals exposed with 7.6 mg/m3. Thus, these findings are regarded as background findings, which occurred earlier and were slightly more prominent in animals exposed to the test item. A direct toxic effect on the nasal mucosa is not assumed.
At 7.6 mg/m3, granuloma formation was seen in the lungs at the end of the exposure period which partly progressed to granuloma with central degeneration and necrosis after the end of the recovery period. Furthermore, chronic inflammation of the bronchiolo-alveolar junction was induced at 7.6 mg/m3 that was still present after the post-exposure observation period. Accumulation of macrophages/giant cells/granuloma showed central degeneration and necrosis at 7.6 mg/m3, central degeneration and necrosis was still present after the recovery period. The moderate accumulation of macrophages/giant cells/granuloma partly with central degeneration/necrosis in the airways of the lung and in the associated lymphoid tissues (bronchus-associated lymphatic tissue, BALT and lung-associated lymph nodes, LALN) as well as the chronic inflammatory reaction seen at the bronchiolo-alveolar junction of the lung at 7.6 mg/m3 are considered to be adverse.
Extramedullary hematopoiesis in the spleen occurred in animals of the control group as well as in animals exposed to 7.6 mg/m3 test item after the recovery period. Incidences in males were 2-3 and in females 3-3 in control groups and at 7.6 mg/m3 respectively. Of note, the highest severity (moderate grade) was observed in a female control rat. At the end of the exposure period, minimal up to slight extramedullary hematopoiesis in the spleen was found in males (incidence 0-1-1-3) and females (incidence 0-0-0-2). Highest severity and indistinguishable incidence compared to test item exposed animals of extramedullary hematopoiesis was seen in control animals at the end of the recovery period. Thus, this finding is interpreted to be not test item-induced.
Relative body weight adjusted spleen weights were significantly increased in females at the end of the exposure period and significantly increased in males at the end of the recovery period at 7.6 mg/m³. Reticulocytes were significantly increased in females at the end of the exposure period and significantly decreased in males at the end of the recovery period at 7.6 mg/m³. Significantly decreased percentage of reticulocytes in the blood at 7.6 mg/m3 at the end of the recovery period in males do not correlate with significantly increased body weight-adjusted relative spleen weights at that concentration. Furthermore significantly decreased reticulocyte counts and significantly increased relative spleen weights were only seen in males and not in females. Additionally no test item induced histopathological findings were seen in the spleen at the end of the recovery period. Consequently these findings are reflected to be inconsistent and not of toxicological relevance.
At the end of the exposure period significantly increased reticulocytes and significantly increased body weight-adjusted relative spleen weights were found in females at 7.6 mg/m3. Of note, only body weight-adjusted spleen weights in female rats at 7.6 mg/m3 were significantly increased whereas absolute and brain weight-adjusted spleen weights did not reach statistically significance when compared to controls. Furthermore it has to be kept into account that only one gender was affected. Additionally, no further significantly changed red cell parameters were found at the end of the exposure period. Taking all those information into account it was considered that increased reticulocytes and increased body weight-adjusted relative spleen weights at the end of the exposure period are non-adverse.
Leucocytes and specifically neutrophils were increased at 7.6 mg/m³ in males and females at the end of the exposure period. At the end of the recovery period, leucocytes, lymphocytes, neutrophils, monocytes and atypical cells were increased at 7.6 mg/m³ in comparison to the air control. Systemically increased white cell parameters seen at clinical pathology mentioned above, are interpreted to be the consequence of cell recruitment due to inflammatory processes in the respiratory tract at 7.6 mg/m³. In this context the increased white blood cellsespacially neutrophils in the blood at 7.6 mg/m³ may be explained by raised recruitment due inflammatory processes in the lungs at 7.6 mg/m3.
Dose descriptor:
NOAEC
Effect level:
1.5 mg/m³ air
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: portal-of-enty toxicity in the respiratory tract associated tissues at 7.6 mg/m³; a NOEL could not be established
Dose descriptor:
NOAEC
Effect level:
7.6 mg/m³ air
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no systemic toxicity observed
Critical effects observed:
yes
Lowest effective dose / conc.:
7.6 mg/m³ air
System:
other: respiratory system
Organ:
other: portal-of-entry toxicity (local effects) in the respiratory tract; no systemic toxicity
Treatment related:
yes
Executive summary:

A repeated exposure 90-day (13 weeks) subchronic toxicity inhalation study with the solid aerosol of the test item was conducted in young adult male and female Wistar rats according to the procedures called for by OECD Test Guideline No. 413 (2009). 10 Wistar rats/sex were nose-only exposed for 6 hours/day on five consecutive days per week for 13 weeks to mean (± SD) actual concentrations of 0 [air control], 0.3 (±0.05), 1.5 (±0.20), and 7.6 (±0.80) mg/m³. The concentrations of the test item in the test atmosphere given are based on gravimetric analysis from breathing zone concentrations. Animals exposed to air under otherwise identical circumstances served as negative controls. Six additional male rats per concentration group were assigned to lung lavage at the end of the exposure period. Ten additional rats per sex of the control and the high concentration groups were allowed to recover during a 12-week postexposure period. The exposure took place in directed-flow nose-only inhalation chambers.

The characteristics of the test atmospheres were in compliance with the recommendations of the OECD Test Guideline No. 413. The mean Mass Median Aerodynamic Diameter’s (MMAD) were in the range of 2.23 – 2.35 µm within the exposure groups (GSD: 1.86 – 2.30).

Test item-related mortality did not occur.

There were no test-item induced adverse findings seen in clinical observations, rectal temperature, ophthalmology, absolute body weights, urinalysis, cumulative body weight gain, food and water consumption as well as in functional observation battery up to the highest dose of 7.6 mg/m³.

Absolute and relative lung weights were significantly increased in females at 7.6 mg/m³. Furthermore lung-associated lymph node (LALN) weights and size were increased at 7.6 mg/m³ at the end of the exposure period and at the end of the recovery period. Relative spleen weights were significantly increased in females at the end of the exposure period and significantly increased in males at the end of the recovery period at 7.6 mg/m³.

Reticulocytes were significantly increased in females at the end of the exposure period and significantly decreased in males at the end of the recovery period at 7.6 mg/m³. Changes in reticulocytes are considered to be not adverse in the absence of further significantly changed red blood cell parameters and also taking histopathology into account.

Leucocytes and specifically neutrophils were increased at 7.6 mg/m³ in males and females at the end of the exposure period. At the end of the recovery period, leucocytes, lymphocytes, neutrophils, monocytes and atypical cells were increased at 7.6 mg/m³ in comparison to the air control. Those findings may be interpreted to be the consequence of white cell recruitment due to relevant inflammatory processes in the lungs at 7.6 mg/m³.

Bronchoalveolar lavage analysis revealed significantly increased mean cellular diameter, mean cellular volume and polymorphonuclear cell counts at 7.6 mg/m³. Percentage of alveolar macrophages was significantly decreased at 7.6 mg/m³ whereas absolute count of alveolar macrophages was biologically relevant increased at 7.6 mg/m³. Changes in mean cellular diameter, mean cellular volume and alveolar macrophages at 7.6 mg/m³ are interpreted as compensatory effect due to increased phagocytosis, a physiological clearance mechanism in the lungs. Total cell count, protein, lymphocytes, lactate dehydrogenase (LDH) and gamma-Glutamyl transferase (GGT) were biologically relevant increased at 7.6 mg/m³ but did not reach statistical significance. LDH, a cytoplasmic enzyme, as well as GGT, a membrane bound enzyme, are established cytotoxicity marker indicating cellular damage. Consequently significantly increased polymorphonuclear cells and relevant increase in protein, lymphocytes, LDH and GGT in the BAL at 7.6 mg/m³ were reflected as signs of inflammation and thus considered to be adverse. Changes in bronchoalveolar lavage (BAL) parameters seen at 1.5 mg/m³ and below are considered to be not adverse.

Within the nasal cavity, an increase of eosinophilic globules, an increased hypertrophy and hyperplasia of goblet cells, an increased infiltrate with eosinophils as well an increased degeneration and metaplasia of epithelium were observed. At the end of the post-exposure observation period, also the animals of the control air group revealed these findings up to the same degree compared to animals exposed to 7.6 mg/m3. Thus, these findings are regarded as background findings, which occurred earlier and were slightly more prominent in animals exposed to Vestanat B 1358/100 compared to control. A direct toxic effect of the test item on the nasal mucosa is not assumed.

At exposure levels of 0.3 mg/m3 and 1.5 mg/m3 Vestanat B 1358/100 and in controls a minimal to slight accumulation of alveolar macrophages/giant cells in the lung as well as in the associated lymphoid tissues (BALT, LALN) was seen. In the tracheal

bifurcation, reversible minimal increases in inflammatory infiltrates with accumulation of partly foamy macrophages/giant cells were observed starting at 1.5 mg/m3. Bronchus-associated lymphatic tissue (BALT) was hyperplastic and also showed an increase in accumulation of macrophages/giant cells/granuloma, concentration-dependent starting at 0.3 mg/m3. A lymphoid hyperplasia was observed in the lung-associated lymph nodes starting at 0.3 mg/m3 at the end of the exposure period still detectable after the recovery period. Furthermore, there was an accumulation of macrophages/giant cells/granuloma in the lung associated lymph nodes starting at 1.5 mg/m3. In addition, accumulation of pigment-laden macrophages starting at 0.3 mg/m3 increased concentration-dependent. These abovementioned histopathological effects seen at 0.3 and 1.5 mg/m3 in the lungs and the lung associated lymphoid tissues (BALT, LALN) are considered to be part of physiological clearance mechanisms due to inhalation of solid particles and in the absence of any structural changes regarded as non-adverse.

At 7.6 mg/m3, granuloma formation was seen in the lungs at the end of the exposure period which partly progressed to granuloma with central degeneration and necrosis after the end of the recovery period. Furthermore, chronic inflammation of the bronchiolo-alveolar junction was induced at 7.6 mg/m3 that was still present after the post-exposure observation period. Accumulation of macrophages/giant cells/granuloma showed central degeneration and necrosis at 7.6 mg/m3, central degeneration and necrosis was still present after the recovery period. The moderate accumulation of macrophages/giant cells/granuloma partly with central degeneration/necrosis in the airways of the lung and in the associated lymphoid tissues (bronchus-associated lymphatic tissue, BALT and lung-associated lymph nodes, LALN) as well as the chronic inflammatory reaction seen at the bronchiolo-alveolar junction of the lung at 7.6 mg/m3 are considered to be adverse.

Observed extramedullary hematopoiesis in spleens of male and female rats are interpreted to be not test item induced as similar incidence and severity were also seen in control animals at the end of the recovery period.

In summary, no systemic toxicity was observed but there is evidence of portal-of-entry toxicity in rats after repeated inhalation (90 days) of the test item. Taking all findings into account, in this subacute inhalation toxicity study the No-Observed-Adverse-Effect-Level (NOAEL) is 1.5 mg/m³ due to respiratory tract associated adverse findings seen in BAL, histopathology and weight increase of lung and LALN at 7.6 mg/m³. A no-observed effect level (NOEL) could not be established.

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
A grouping of substances and read-across approach according to Regulation (EC) No 1907/2006 (REACH), Annex XI 1.5 and following the ECHA Read-Across Assessment Framework, RAAF (2015) is accomplished for the registration of five blocked diisocyanate oligomers (also for the registered substance) that are manufactured or imported in quantities of 10 (1/5), 100 (2/5) or 1000 (2/5) tons/anno or more. This is to fulfil the data requirements of REACH for the respective tonnage band in a scientifically based and efficient manner, taking also into account the aim to reduce vertebrate studies.

All five category members are oligomerisation products of diisocyanates (UVCBs), where the free isocyanate functionality is covalently reacted with blocking agents (decomposition at approximately 90 °C and above). The category members are either derived from 1,6-hexamethylene diisocyanate (HDI) oligomers or isophorone diisocyanate (IPDI) oligomers. Since the smallest constituent is the blocked trimer (n = 3) having a molecular weight of ≥ 700 g/mol, a very low water solubility and throughout high log Kow (> 4.4), absorption through biological membranes is limited. The chemical composition of the UVCBs is very similar and the free diisocyanate monomer content is throughout below 0.1%. The five blocked oligomeric diisocyanates thus show defined structural similarities and differences that allow the application of the ECHA RAAF Scenario 4.
In this category approach the physicochemical properties, environmental fate, ecotoxicity and most of the toxicological endpoints were experimentally determined for each of the five category members. The outcome of this testing proved the similarity of the category members not only in structure and composition but also in quality and quantity of reactivity/toxicity. The comparison of the environmental fate and pathways data (i.e. hydrolysis, biodegradation, bioaccumulation and adsorption on soil components) and ecotoxicological data (i.e. different tests for toxicity to aquatic organisms) reveals a very high similarity. The same is true for the toxicological endpoints acute toxicity, skin and eye irritation, skin sensitization and genotoxicity. None of the category members has to be classified according to GHS Regulation 1272/2008 for environmental fate, ecotoxicity, acute toxicity, skin and eye irritation, skin sensitization as well as genotoxicity.

Since inhalation exposure in occupational settings is potentially relevant for all category members acute and 14-day pilot repeated inhalation toxicity studies were conducted with all five UVCBs to allow a robust evaluation on mode of action and toxic potency and to further prove the group hypothesis.

The most noticeable difference in the performance of the inhalation studies was related to the physical properties of the substances. The blocked HDI-based oligomers had to be dissolved in ethyl acetate to generate a suitable test atmosphere because they became glue-like during testing. The blocked IPDI-based oligomers could be tested as powder aerosols. Thus, for inhalation toxicity testing the category is divided into two sub-groups – the liquid aerosol for blocked HDI-based oligomers and the powder aerosol for blocked IPDI-based oligomers. Thus, within the category the analogue read-across approach is followed, i.e. ECHA RAAF Scenario 2.
In consequence, two subchronic (90-days) inhalation studies, one for a blocked HDI oligomer and one for a blocked IPDI oligomer, were conducted. The remaining substances are covered by a read across to the category member with the closest structural analogy. In this case, the target substance (isophorone diisocyanate, oligomeristion product, blocked with hexahydro-2H-azepin-2 -one) is covered by read across to the source substance (isophorone diisocyanate, oligomeristion product, blocked with 2 -butanone oxime).

1. HYPOTHESIS FOR THE CATEGORY/ ANALOGUE APPROACH
For a detailed discussion of the hypothesis for grouping and read across of 5 blocked diisocyanate oligomers according to regulation No. 1907/2006 (REACH), Annex XI, 1.5 and following ECHA RAAF (2015) see the document attached below to this endpoint study record.
Note: The first outline of a grouping-strategy was prepared in May 2013 based on read-across of the available toxicological data for 4 blocked diisocyanate oligomers at that time. In the meantime, the group was enlarged to 5 blocked diisocyanate oligomers and 90-day inhalation toxicity studies for two of the category members have been performed on request of ECHA. The updated document refers to the REACH requirements according to Annex VIII - X (10 - > 1000 tonnes/a).

2. SOURCE AND TARGET CHEMICAL(S)
Please see document attached below to this endpoint study record.

3. CATEGORY / ANALOGUE APPROACH JUSTIFICATION
For a detailed discussion of the outcome of the testing strategy and for justification of the grouping and read across of 5 blocked diisocyanate oligomers according to regulation No. 1907/2006 (REACH), Annex XI, 1.5 and following ECHA RAAF (2015) see the document attached below to this endpoint study record.

4. DATA MATRIX
Please see document attached below to this endpoint study record.

Reason / purpose:
read-across source
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Healthy young adult SPF bred Wistar rats, strain Crl:(Wi)WU BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:(Wi)WU BR (SPF-bred)
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 2 months
- Weight at study initiation: Weight range at first exposure day: males 230-280 g. 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 with gnawing sticks
- Diet and water: ad libitum
- Acclimation period: at least 10-14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 40-60 %
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12 h/12 h
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
ca. 2.3 µm
Remarks on MMAD:
The mean Mass Median Aerodynamic Diameter’s (MMAD) measured by cascade impactor were in the range of 2.23 – 2.35 µm within the exposure groups (Geometric Standard Deviation: 1.86 – 2.30). MMADs measured by laser velocimetry ranged from 2.66 – 2.69 µm within the exposure groups (Geometric Standard Deviation: 1.72 – 1.73).
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Rats were exposed under dynamic directed-flow nose-only exposure conditions.
- Exposure apparatus: Details of the nose-only exposure system, including its validation, have been published previously (Pauluhn, Journal of Applied Toxicology 13, 1994, 55-62; Pauluhn and Thiel, J. Appl. Toxicol. 27, 2007, 160-167). 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).
- Method of holding animals in test chamber: Animals were exposed in polycarbonate 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.
- Source and rate of air: 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; SMG Interlink, USA) and TSI Mass Flow meter 4040 (TSI Incorporated, USA) and were checked for correct performance at regular intervals.
- 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 BEKO RA 55 compressed air dryer. Adequate control devices were employed to control supply pressure.
- Temperature, humidity: Temperature and humidity measurements are also performed by the computerized Data Acquisition and Control System using HC2-S3 sensors (Rotronic Messgeräte GmbH, Ettlingen, 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.
- Air flow rate: 5.85 L/min
- Air change rate: The test atmosphere generation conditions provide an adequate number of air exchanges per hour [45 L/min x 60 min/(11.4 L/chamber) = 237, continuous generation of test atmosphere]. Thus, under the test conditions used a chamber equilibrium is attained in less than one minute of exposure.
- Treatment of exhaust air: The exhaust air was purified via filter systems. These filters were disposed of by the laboratory.
The ratio between supply and exhaust air was selected so that 90 % of the supplied air was extracted via the exhaust air location and, if applicable, via sampling ports. Aerosol scrubbing devices were used for exhaust air clean-up. During sampling, the exhaust air was reduced in accordance with the sampling flow rate using a computerized Data Acquisition and Control System so that the total exhaust air flow rate was adjusted 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).
- Atmosphere generation: Test atmospheres were generated using a WRIGHT DUST FEEDER system (BGI Inc., Waltham, MA 02154, USA). For dry powder dispersion, conditioned compressed dry air (30 liters/min; generic dispersion pressure: 1.8 kPa) was used. The principle performance of the WRIGHT DUST FEEDER dust generating system can be described as follows: the test substance was metered in a reservoir and then was compressed to a pellet using approxi¬mately 1 metric ton by a carva laboratory press (F. S. Carver Inc., Wabash, IN 46992, USA). From this pellet defined amounts of test substance were scraped off and entrained into the main air flow.
The airborne powder was then conveyed into the inner cylinder of the inhalation chamber. Principles differences between chamber set-ups were related to the dilution cascades to attain the targeted concentrations.
- Method of particle size determination: The particle-size distribution was analyzed using a BERNER critical orifice cascade impactor. An adhesive stage coating (silicone spray) was used to minimize particle bounce due to the adhesive properties of the test article. Each impactor stage was covered with an aluminum foil. Collecting metrics were subjected to gravimetric analysis using a digital balance and pre-conditioning method as described in the previous section.

TEST ATMOSPHERE
- Brief description of analytical method used: The test-substance concentration was determined by gravimetric analysis (filter: glass-fiber filter, Sartorius, Göttingen, Germany; post-sampling conditioning period of 15 min at room temperature). This method was used to define the actual concentration.
- The integrity and stability of the aerosol generation and exposure system was measured by using a Microdust real-time aerosol photometer (Casella, Buffalo, UK) and at 0.3 mg/m³ the R&P Teom 1400a (amibent particulate monitor; Thermo Fisher Scientific Inc., USA). Samples were taken continuously from the vicinity of the breathing zone.
- Samples taken from breathing zone: yes
- Particle size distribution: The particle size distribution was analyzed using a TSI-Laser Velocimeter APS 3321. Calculations consider both a particle size distribution that encompasses aerodynamic diameters (Dae) of 0.5 to 7.4 µm and sample flows ranging from 8 to 80 ml/sec.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The mean Mass Median Aerodynamic Diameter’s (MMAD) measured by cascade impactor were in the range of 2.23 – 2.35 µm within the exposure groups (Geometric Standard Deviation: 1.86 – 2.30). MMADs measured by laser velocimetry ranged from 2.66 – 2.69 µm within the exposure groups (Geometric Standard Deviation: 1.72 – 1.73).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test-substance concentration was determined by gravimetric analysis. The concentrations of test atmosphere given are based on breathing zone concentrations from filter analyses which reflect the active substance.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day on 5 consecutive days/week for 13 weeks
Dose / conc.:
0.3 mg/m³ air (analytical)
Remarks:
target concentration: 0.3 mg/m³
Dose / conc.:
1.5 mg/m³ air (analytical)
Remarks:
target concentration: 1.5 mg/m³
Dose / conc.:
7.6 mg/m³ air (analytical)
Remarks:
target concentration: 7.5 mg/m³
No. of animals per sex per dose:
10 rats/sex/group plus 6 additional males/group for lung lavage plus 10 additional rats/sex of the control and high dose as recovery groups
Control animals:
other: rats exposed under otherwise identical test conditions to dry air served as concurrent control group
Details on study design:
Dose selection rationale:
Target concentrations for this repeated exposure study were based on the results of a 2 week inhalation toxicity study with study identification number T100111-4 (Exposure 9 x 6h/day, 5 days/week; recovery period of 2 weeks) using Vestanat B1358/100 as test item. See Pauluhn, 2013.
- Rationale for selecting satellite groups:
After two weeks of recovery in the 2 week inhalation study, the findings in lungs and LALN were still detectable. Additionally, accumulations of epitheloid cells occurred in BALT and LALN. Therefore satellite groups for lung lavage and recovery were included in this study.
- Post-exposure recovery period in satellite groups: 13 weeks
Positive control:
positive controls are not adequate for this study type
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, all animals
- Time schedule: Clinical signs were recorded twice daily, before and after exposure and once a day on exposure-free days and recovery period.

BODY WEIGHT: Yes, all animals
- Time schedule for examinations: During the exposure period, the body weights were determined twice per week to differentially record their gains over exposure-free weekends, and once per week during the postexposure period.

FOOD AND WATER CONSUMPTION: Yes.
- The individual food and water consumption of all animals (excluded lavage animals) were measured weekly (Mondays).

OPHTHALMOSCOPIC EXAMINATION: Yes.
- Prior the first exposure all animals (excluded lavage animals) and toward at the end of the dosing phase (on day 86) control and high dose animals were examined.

RECTAL TEMPERATURES: Yes, all animals
- Time schedule for examinations: Five animals per group 1-8 were subjected to body temperature measurements. The rectal temperatures were measured directly after cessation of exposure (approximately within ½hour after the end of exposure) using a digital thermometer with a rectal probe for rats during the dosing period on days rel. 0 and 85 as well as on day 85 of the recovery period.

FUNCTIONAL OBSERVATION BATTERY: Yes.
- The first five core rats of group 1-8 were used for functional observation battery investigation on day relative 0, 59 and 85. Each rat was firstly observed in the home cage and then individually examined. The following reflexes were evaluated: visual placing response and 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). Measurements of grip strength were measured qualitatively but defined as semi quantitative.

HAEMATOLOGY: Yes
- Blood samples (non-fasted) for hematology, coagulation and serum chemistry parameters were collected from all core animals (end of the exposure period) and recovery toxicology animals (end of the recovery period) during sacrifice by cardiac puncture.

CLINICAL CHEMISTRY: Yes
- Urines of core animals were collected on day 87 and 88 of the dosing phase as well as on day 87 and 88 at the recovery period, overnight (approximately 16 h). Therefore animals were housed in specific metabolism cages.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals
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.

ORGAN WEIGHTS: Yes, all animals
The following organs were weighed at necropsy: adrenal glands, brain, heart, kidneys, liver, lung, thymus,lung-associated lymph nodes, spleen, ovaries, uterus, testes, epididymis

HISTOPATHOLOGY: Yes
- Histopathology was performed on the following organs/tissues (core and recovery animals): nasal cavities (4 levels: L-I to L-IV) / Young, 1981), pharynx, larynx, trachea, lung, lung associated lymph nodes (hilus) and additionally in the control (control air) and high dose groups (core toxicology animals) the organs/tissues listed in the table Table 8. Tissues were fixed in 10% neutral-buffered formalin (NBF), except the left and two thirds of the right kidneys (fixed in Davidson’s fixative then transferred to 10% NBF) and followed routine processing in the histopathology laboratory. Lungs were instilled with 10% NBF and then transferred to 10% NBF solution. The tissue slides were examined by a veterinary pathologist. In addition, a formal cross-check was performed. The findings reported are those agreed-upon between the cross-checking and the study pathologist.

MICROBIOLOGICAL EVALUATION:
Animal number 21 (control group) was euthanized on 12-AUG-2016 due to blood in the urine in combination with clinical signs as e.g. piloerection, reduced body tone. To investigate the causality of the blood in the urine an additional microbiological diagnostic has been initiated. These microbiological investigation and evaluation took place at Bayer AG, Antiinfectiva, Bayer research Center Aprath, 42096 Wuppertal, Germany under non-GLP conditions.
Other examinations:
BRONCHOALVEOLAR LAVAGE: Yes, animals assigned (6 males/group)
Bronchoalveolar lavage fluid was sampled at the end of the exposure period using sodium pentobarbital. All rats, irrespective of the day of death, were given a gross-pathological examination. Consideration was given to perform 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 are recorded. Corresponding to the suitable SOP the cranium and the nasal cavities of some animals per group were opened.
In BAL-fluid (BALF) the following indicators of pulmonary damage were assessed: Total number of lavaged cells, including the volume and diameter, cytodifferentiation, lactate dehydrogenase (LDH9), total protein (PRQ9), gamma-glutamyltransferase (GGT9), phospholipids (PL9).

After complete exsanguination, the excised wet lungs were weighed and following ligation of the trachea, the lung lobes were then lavaged via a tracheal cannula with two volumes of 5-ml of physiological saline (nearly 37°C), which was left in the lungs for 30 sec, withdrawn, re-instilled for an additional 30 sec. This procedure was repeated once. From each rat the retrieved bronchoalveolar lavage fluid (BALF) from the lung lavage was then pooled and adjusted to 10-ml total volume. Prior to centrifuging the samples were kept on ice. Then the pooled lavage fluid was centrifuged at approximately 200 x g for approximately 10 min. at <10°C (Sigma 4-16K refrigerated centrifuge) and the resultant cell-free supernatant was analyzed for the endpoints/parameters listed below. The cell pellet was re-suspended in PBS-BSA (Dulbecco’s phosphate buffered saline with Ca2+ and Mg2+ containing 0.1% bovine serum albumin; Sigma, Deisenhofen, Germany) and then centrifuged (2 x 105 per cytospot) onto slides using a cytocentrifuge (Shandon Cytospin 4). Cell counts were determined in triplicates after 1:1000 dilution using a CASY cell counter + analyzer. Air-dried slides were fixed with a mixture of methanol:acetone, stained according to Pappenheim, and differentiated by light microscopy. Cytospots were differentiated by counting 300 cells/cytospot.
For the analysis of the BAL data the validated BalNew.exe and ANOVA.exe Software (Author Prof. Pauluhn, Bayer AG, Bayer Research Center Aprath, Wuppertal, Germany) has been used.
Statistics:
Analysis of variance (ANOVA BCTIC): This parametric method checks for normal distribution of data by comparing the median and mean. The groups are compared at a confidence level of (1-*) = 95% (P = 0.05). The test for the between-group homogeneity of the variance employed Box's test if more than 2 study groups were compared with each other. If the above F-test shows that the intra-group variability is greater than the inter-group variability, this is shown in the Appendix as "no statistical difference between the groups". If a difference is found then a pair-wise post-hoc comparison is conducted (1- and 2-sided) using the Games and Howell modification of the Tukey-Kramer significance post hoc test. This program (FORTRAN source code) was originally obtained from BCTIC and is used as means to evaluate off-line data. In most instances, data analyzed by ANOVABCTIC are also summarized in graphical form. In figures statistical significant differences are indicated by asterisks (* P < 0.05; ** P < 0.01).
BAL data: The individual results of the BAL-Investigation were manually entered in the program: BALnew.exe, Version 3.0 (Author Prof. J. Pauluhn, Bayer AG, Germany; Software was written in DIGITAL Fortran). The BALnew.exe software calculates group based arithmetic means and the standard deviations (SD) relating to 10 ml bronchoalveolar lavage volume. Means and SD were statistically evaluated using the ANOVABCTIC procedure (vide infra).
Description (incidence and severity):
No relevant concentration-dependent test item-related clinical symptoms were seen in the course of the study.
Red encrusted noses, reddish-brownish discolored fur around the eyes, the head or the neck/back area were partially seen in some control animals as well as in some animals exposed to the test item. These findings are well known in inhalation toxicity studies and are considered to be nose-only inhalation technique-related due to restraining in the inhalation tubes but not test item-induced. Isolated slight up to moderate piloerection was seen after exposure session in a few males of the control group as well in a few males exposed to the test item and is reflected to be not test item induced.
Description (incidence):
Three male rats (Animal number 21, 24 and 26) of the air control group and one male rat of the high exposure group (Animal number 60) were euthanized during the course of the study due to animal welfare reasons. All four euthanized animals showed blood in urine associated with reduced general condition e.g. reduced body tone, reduced motility and piloerection. Microbiological investigation of the urine and renal tissue of animal no. 21 demonstrated Proteus mirabilis (details are given in section 7.15). Since clinical signs as well as pathological findings in all four euthanized animals were similar, it is considered that the microbiological findings seen in rat number 21 are representative for all euthanized rats.
In summary, it is considered that the blood in the urine and the associated poor health condition of euthanized male animals are not test item-related since 3 control animals and 1 high concentration animal were affected. Furthermore the clinical and pathological findings are considered to be caused by an ascending urinary tract infection with Proteus mirabilis.
Description (incidence and severity):
Comparisons between the control and the exposure groups did not reveal any statistically concentration-dependent differences in body weights and cumulative body weight gain during the course of the study. Isolated significantly changed absolute body weights or cumulative body weight gain mean values are considered to be not test item associated due to lack of concentration-dependency.
Description (incidence and severity):
No relevant test item-related changes in food and water consumption were observed when compared to control animals.
Description (incidence and severity):
The ophthalmological examination did not reveal any test item-related findings.
Description (incidence and severity):
Reticulocytes were significantly increased in females at 7.6 mg/m³ at the end of the exposure period. At the end of the recovery period, reticulocytes were significantly decreased in males at 7.6 mg/m³. In females at the end of the exposure period, neutrophils were significantly increased at 7.6 mg/m³. At the end of the recovery period, leucocytes, lymphocytes, neutrophils, monocytes and atypical cells were significantly increased at 7.6 mg/m³ in comparison to the air control.
Furthermore isolated significantly changed parameters occurred and were reflected to be incidental.
Description (incidence and severity):
Isolated significantly changed parameters occurred and were reflected to be incidental.
Description (incidence and severity):
Quantitative urinalysis, microscopy of urine sediment and qualitative urinalysis were performed and did not reveal any concentration-dependent relevant differences in test item exposed animals when compared to air control rats.
Description (incidence and severity):
A battery of reflex measurements was made on day 3, 59 and 85. Differences between animals exposed to the test item compared to the control group did not occur.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the exposure period (main groups) test item-related increased lung-associated lymph nodes (LALN) were found in males and females exposed to 7.6 mg/m³. Furthermore absolute and relative (% of TBW and % of brain weights) lung weights were significantly increased in females at 7.6 mg/m³. Statistically significant higher absolute lung weights were observed in females at 1.5 mg/m³, whereas relative lung weight was unchanged; here it has to be taken into account that total body weights were also slightly higher when compared to controls. Consequently, relative lung weights (% of TBW and % of brain weight) at this dose level must be given preference. An Isolated significant increase in liver weight found in females exposed to 1.5 mg/m³ is considered to be not test item related due to lack of dose-dependency. Significantly raised relative spleen weights (% of TBW) were measured in female animals exposed to 7.6 mg/m³, whereas absolute spleen weights and relative spleen weights expressed as % of brain weights did not reach statistically significance.
At the end of the recovery period significantly increased absolute and relative (% of brain weight) adrenal gland, kidney, liver, LALN and lung weights were found in females exposed to 7.6 mg/m³, whereas additionally LALN weights were also significantly increased in males at 7.6 mg/m³. Relative spleen weights expressed as % of TBW were significantly increased in males in males at 7.6 mg/m³. Significantly changed adrenal gland, kidney and liver weights in comparison to the control group are interpreted to be not adverse in the absence of correlating histopathological and/or clinical pathological findings.
Significantly increased lung-associated lymph nodes and lung weights at 7.6 mg/m³ at the end of the exposure period as well as at the end of the recovery period are reflected to be test item-induced.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In males and females exposed to 7.6 mg/m³, lung-associated lymph nodes were increased in size (incidence males/females: 0/0-0/0-0/0-7/6). This finding was non-reversible after the post-exposure period (incidence males/females: 0/0-6/7).
A decrease in size and/or soft/flaccid consistency of testes were observed uni- or bilaterally throughout all exposure groups, including the control air group, that was as such not treatment related.
The three prematurely sacrificed male animals of the control air group with post-exposure observation period and the male animal at 7.6 mg/m³ assigned for bronchoalveolar lavage revealed findings at the urinary bladder consisting of increase in size, increased thickness of the wall and content changes. Furthermore, thymus was decreased in size in these animals. Since the three animals were affected with Proteus mirabilis no treatment relationship is seen.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of exposure:
In the nasal cavity of the exposed animals starting at 0,3 mg/m3 up to a moderate degree at 7.6 mg/m3, an increase in extent (whereas in level 2 and 3 nearly all animals were affected, more exposed animals showed the finding in level 1 when compared to control) and severity of eosinophilic globules predominantly at the transition between the respiratory and olfactory epithelium of the nasal septum, as well as the ventral conchae were observed. Eosinophilic globules were located partly apically, partly basally within the cells of the olfactory epithelium (most prominent in level 2 and 3; mean severity level 2 males: 1.0-1.5-1.6-2.8; females: 1.0-1.7-2.0-3.0; mean severity level 3 males: 1.4-1.7-1.6-2.7; females: 1.0-2.0-2.0-2.8). More severely affected animals had an increased incidence of epithelial degeneration and metaplasia.
Furthermore, an increase of goblet cell hypertrophy and hyperplasia in the respiratory epithelium was visible. These changes were associated with an increased infiltrate of eosinophils in the nasal respiratory epithelium.
The trachea revealed a minimally increased inflammatory cell infiltrate at 7.6 mg/m3 (incidence males: 5-4-4-7; incidence females: 5-5-5-4; mean severity of grading males: 1.2-1.0-1.5-1.3; mean severity of grading females. 1.0-1.0-1.0-1.3), as well as an increase of accumulated macrophages/giant cells in the area of the bifurcation starting at 1.5 mg/m3 (incidence males. 1-1-4-3; incidence females: 0-1-2-2).
In the lung, starting at 1.5 mg/m3 accumulation of alveolar macrophages/formation of giant cells of minimal to moderate degree were observed, focally at 1.5 mg/m3, multifocally throughout all lung lobes at 7.6 mg/m3 (incidence males: 0-0-3-10; incidence females: 0-0-1-10).
In animals exposed to 7.6 mg/m3, these accumulated macrophages/giant cells were associated with a chronic inflammation of slight degree affecting primarily the bronchiolo-alveolar junction and led to septal thickening and hypercellularity in this area (incidence males and females: 0-0-0-10).
In single animals, granuloma formation incorporating the macrophage accumulation/giant cells could be observed (incidence males and females: 0-0-0-2).
Furthermore, minimal accumulation of pigment-containing alveolar macrophages were observed (incidence males: 0-0-2-7; incidence females: 0-1-1-2).
The bronchus-associated lymphatic tissue (BALT) revealed an increase in incidence and severity of macrophage accumulation/giant cell/granuloma formation starting at 0.3 mg/m3 up to moderate degree at 7.6 mg/m3 (incidence males: 3-6-9-10; incidence females: 2-4-10-10; mean severity of grading males: 1.0-1.3-1.6-2.6; mean severity of grading females: 1.0-1.0-1.1-2.3). Single to multiple accumulations of macrophages/giant cells and isolated granulomas were observed as a continuum focally or multifocally within areas of BALT of control animals, especially in the left lung lobe or in the caudal aspect of the right lung lobe. In animals exposed to Vestanat B 1358/100, these accumulations/giant cells/granulomas increased in incidence and severity (larger accumulations/granulomas per area BALT; more areas of BALT affected, also in other lung samples) up to moderate degree at 7.6 mg/m3.
Furthermore, a minimal to slight lymphoid hyperplasia was observed starting at 1.5 mg/m3 (incidence males: 0-1-3-7; incidence females: 0-0-5-9).
Lung-associated lymph nodes (LALN) showed lymphoid hyperplasia starting in females at 0,3 mg/m3, in males at 1.5 mg/m3 (incidence males: 0-0-2-10; incidence females: 0-3-5-9), which is consistent with the reported increased size of lung-associated lymph nodes during necropsy observed in animals at 7.5 mg/m3. In addition, a predominantly moderate accumulation of macrophages with giant cell/granuloma formation was observed (incidence males: 0-0-2-10; incidence females: 0-0-0-10), which partly showed minimal to slight central degeneration/necrosis (incidence males: 0-0-0-3; incidence females: 0-0-0-7).
Furthermore, there was an increase in incidence and/or severity of histiocytes infiltrating the lymph nodes (incidence males: 6-6-6-10; incidence females: 4-4-8-10; mean severity of grading males: 1.2-1.2-1.5-2.1; mean severity of grading females: 1.0-1.3-1.5-2.7)

After the post-exposure observation period of 90 days:
Nasal cavity of all animals (including the control air group) revealed the same findings (eosinophilic globules, mean severity level 2 males: 2.0-3.0, females: 2.1-2.9; hypertrophy of goblet cells, mean severity level 2 males 1.8-2.0, females: 2.0-2.0
and hyperplasia of goblet cells, mean severity level 2 males:1.0-1.2, females: 1.0-1.0; infiltrate with eosinophils, mean severity level 2 males: 1.0-1.3, females 1.0-1.0). The findings in controls after the post-exposure observation period were thus observed with a similar severity compared with the exposed animals examined directly after the last day of exposure.
In animals with accumulation of eosinophilic globules of a higher degree incidences of focal degeneration and metaplasia increased.
In the lungs of all exposed animals, accumulation of alveolar macrophages/giant cells (incidence males and females: 0-10) and the slight chronic inflammation in the area of bonchiolo-alveolar junction (incidence males/females: 0-10) were still present. In addition, granuloma formation progressed in almost all animals (incidence males/females: 0-10/9), which showed central degeneration/necrosis in single animals (incidence males/females: 0-1/2).
Minimal to slight accumulation of pigment-containing alveolar macrophages increased in incidence in females and in severity in males compared to the findings directly after exposure (incidence males: 0-7; incidence females: 0-5).
The bronchus-associated lymphatic tissue (BALT) showed macrophage accumulation/giant cells/granuloma (incidence males: 5-10; incidence females: 4-10; mean severity of grading males: 1.4-2.4; mean severity of grading females: 1.0-2.2) and lymphoid hyperplasia (incidence males/females: 0-7/9).
The animals revealed a slight lymphoid hyperplasia within the lung-associated lymph nodes (incidence males and females: 0-10), which is consistent with the reported increased size of respective lymph nodes during necropsy. In addition, moderate accumulation of macrophages with giant cell/granuloma formation was observed (incidence males and females: 0-10), which partly showed minimal to slight central degeneration/necrosis (incidence males/females: 0-4/6).
Furthermore, there was an increase in incidence and/or severity of histiocytes infiltrating the lymph nodes (incidence males: 5-9; incidence females: 7-10; mean severity of grading males: 1.4-2.2; mean severity of grading females: 1.1-2.1).
After 13 weeks of exposure to Vestanat B 1358/100, spleen of some animals exposed to 7.6 mg/m3 revealed a minimally increased extramedullary hematopoiesis (incidence males: 0-1-1-3; incidence females: 0-0-0-2). There was no difference between control and exposed animals regarding this finding after the post-exposure observation period (incidence males: 2-3; incidence females: 3-3).
Three male animals of the control air group with post-exposure observation period and one male animal at 7.5 mg/m3 assigned for bronchoalveolar lavage were prematurely sacrificed for humane reasons. The observed macroscopic findings could be correlated to a marked to severe pyelonephritis and/or cystitis with microscopically detected bacteria. These animals also had a moderate to severe prostatitis. There were some more animals with less severe inflammatory processes within the urinary tract in other groups of exposure.
All other observations in these animals (especially liver: hepatocellular atrophy and vacuolation, Kupffer cell hypertrophy, spleen: atrophy of the white pulp, bone marrow: myeloid hyperplasia, thymus: involution/atrophy correlated to the macroscopically decreased thymus size etc.) were due to the bad condition of the animals.
A decrease in size and/or soft/flaccid consistency of testes were observed uni- or bilaterally throughout all exposure groups, including the control air group consistent with degeneration/atrophy of the testes up to a severe degree followed by aspermia or reduced sperm as well as increased cellular debris in the epididymides.
In female animals of the control air group as well as at 7.5 mg/m3, no cycle determination was possible due to ovarian atrophy and hypertrophy of interstitial cells. In consequence, the epithelium of the cervix and vagina showed no cycle-related changes and was affected by apoptosis/necrosis and sometimes slight pseudomucification.
As rare spontaneous finding, one male animal (animal 71 exposed to 7.6 mg/m3) revealed a malignant Schwannoma within the pancreas, which was observed grossly as a cystic, black-brown coloured nodule approximately 10 cm in diameter. This finding is considere to be incidential and not test item-related.
All other microscopic findings were incidental and spontaneous in nature and could not be attributed to the test item.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Rectal Temperature:
No significantly changed body temperatures were seen in animals exposed to the test item in comparison to control animals.

Bronchoalveolar Lavage:
adverse findings:
Total cell count (TCC), protein (PROT), lymphocytes (C-LYM), lactate dehydrogenase (LDH) and gamma-glutamyl transferase (GGT) were biologically relevant increased but did not reach statistically significance at 7.6 mg/m³.
LDH, an intracellular located enzyme, as well as GGT, a membrane bound enzyme, are established cytotoxic marker indicating cellular damage.
Consequently significantly increased polymorphonuclear cells and biologically relevant increase in protein, lymphocytes, LDH and GGT at 7.6 mg/m³ in the bronchoalveolar lavage are reflected as sings of inflammation also taking significantly increased LALN and lung weights into account (see section 7.9) and thus are considered to be adverse.

non adverse finding:
The average recovery of bronchoalveolar lavage fluid (BALF) was approximately 85-88 % of the instilled volume and was similar amongst all groups. An isolated significantly increase of foamy macrophages was found at 0.3 mg/m³. Adjusted alveolar macrophages (%) were significantly decreased at 0.3 and 7.6 mg/m³. Significantly increased alveolar macrophages with red blood cells were found at 1.5 mg/m³ only. These isolated findings are considered to be of no toxicological relevance.
At 7.6 mg/m³ the following parameter were significantly increased: mean cellular diameter (MCD), mean cellular volume (MCV) and polymorphonuclear cells (PMN and C-PMN). In this context it is remarkable that counted polymorphonuclear cells (C-PMN) at 7.6 mg/m³ were approx. 45-fold higher when compared to air control group. Percentage of alveolar macrophages (AM and AM-ADJ) were significantly decreased at 7.6 mg/m³ whereas absolute count of alveolar macrophages (C-AM) were biologically relevant increased at 7.6 mg/m³. Changes in MCD, MCV and C-AM at 7.6 mg/m³ are considered to be compensatory due to increased phagocytosis as normal part of the clearance mechanisms in the lung.
Total cell count (TCC), protein (PROT), lymphocytes (C-LYM), lactate dehydrogenase (LDH) and gamma-glutamyl transferase (GGT) were biologically relevant increased but did not reach statistically significance at 7.6 mg/m³.
LDH, an intracellular located enzyme, as well as GGT, a membrane bound enzyme, are established cytotoxic marker indicating cellular damage.
Consequently significantly increased polymorphonuclear cells and biologically relevant increase in protein, lymphocytes, LDH and GGT at 7.6 mg/m³ in the bronchoalveolar lavage are reflected as sings of inflammation also taking significantly increased LALN and lung weights into account (see section 7.9) and thus are considered to be adverse.

Microbiological Evaluation:
Male rat number 21 was investigated microbiologically. In the native urine microscopic slide, in all fields of view a lot of cells as well as a few bacteria were observed. Microscopically gram negative rod-shaped bacteria were found in urine and renal tissue. On the Columbia Agarplates pure cultures with swarming properties were seen. Aerob incubated plates showed dark discoloration. No colonies were observed on Agarplates treated with 100 µl blood whereas relevant number of colonies (pure culture) were found on plates incubated aerob as well as anaerob treated with urine and renal tissue. According to the evaluation of the API E20 test (Code 373600057) the bacterium isolated from the pure culture is up to 99.9% likelihood Proteus mirabilis. This result matches with the findings given above of gram negative rod-shaped bacteria and swarming colonies on Columbia Agarplates.
In summary bacteria have been detected in urine and renal tissue of rat number 21 but not in blood. Due to the characteristics, the bacterium is diagnosed as Proteus mirabilis. Proteus mirabilis is ubiquitous in the environment and is considered to be a normal part of human and animals gut flora (see section 12.21). Proteus mirabilis is not listed as recommended infectious agent to monitor for rats according to the FELASA (MÄHLER M. et al., 2014) and thus was not investigated prior to the start of the study. Consequently the microbiological diagnosis of Proteus mirabilis is considered to have no impact on the overall study validity. Nonetheless it is known that Proteus mirabilis may be involved in sporadic urinary tract infections.
Details on results:
Indicative for portal-of entry toxicity were significantly increased lung-associated lymph nodes (LALN) and lung weights, significantly increased polymorphonuclear cells as well as biologically relevant increased total cell count, protein, lymphocytes, LDH and GGT in the BAL at 7.6 mg/m³. These results correlated with gross pathological and histopathological findings as increased lung-associated lymph nodes after end of exposure and recovery period, chronic inflammatory reaction seen at the bronchiolo-alveolar junction of the lung as well as granuloma formation partly with central degeneration/necrosis of granuloma/giant cells also seen in the lung-associated lymph nodes at 7.6 mg/m³.
The daily inhalative exposure to Vestanat B 1358/100 to male and female rats over a period of at least 13 consecutive weeks revealed multiple histopathologically findings within the respiratory tract. Within the nasal cavity, an increase of eosinophilic globules, an increased hypertrophy and hyperplasia of goblet cells, an increased infiltrate with eosinophils as well an increased degeneration and metaplasia of epithelium were observed. At the end of the post-exposure observation period, also the animals of the control air group revealed these findings up to the same degree compared to animals exposed with 7.6 mg/m3. Thus, these findings are regarded as background findings, which occurred earlier and were slightly more prominent in animals exposed to the test item. A direct toxic effect on the nasal mucosa is not assumed.
At 7.6 mg/m3, granuloma formation was seen in the lungs at the end of the exposure period which partly progressed to granuloma with central degeneration and necrosis after the end of the recovery period. Furthermore, chronic inflammation of the bronchiolo-alveolar junction was induced at 7.6 mg/m3 that was still present after the post-exposure observation period. Accumulation of macrophages/giant cells/granuloma showed central degeneration and necrosis at 7.6 mg/m3, central degeneration and necrosis was still present after the recovery period. The moderate accumulation of macrophages/giant cells/granuloma partly with central degeneration/necrosis in the airways of the lung and in the associated lymphoid tissues (bronchus-associated lymphatic tissue, BALT and lung-associated lymph nodes, LALN) as well as the chronic inflammatory reaction seen at the bronchiolo-alveolar junction of the lung at 7.6 mg/m3 are considered to be adverse.
Extramedullary hematopoiesis in the spleen occurred in animals of the control group as well as in animals exposed to 7.6 mg/m3 test item after the recovery period. Incidences in males were 2-3 and in females 3-3 in control groups and at 7.6 mg/m3 respectively. Of note, the highest severity (moderate grade) was observed in a female control rat. At the end of the exposure period, minimal up to slight extramedullary hematopoiesis in the spleen was found in males (incidence 0-1-1-3) and females (incidence 0-0-0-2). Highest severity and indistinguishable incidence compared to test item exposed animals of extramedullary hematopoiesis was seen in control animals at the end of the recovery period. Thus, this finding is interpreted to be not test item-induced.
Relative body weight adjusted spleen weights were significantly increased in females at the end of the exposure period and significantly increased in males at the end of the recovery period at 7.6 mg/m³. Reticulocytes were significantly increased in females at the end of the exposure period and significantly decreased in males at the end of the recovery period at 7.6 mg/m³. Significantly decreased percentage of reticulocytes in the blood at 7.6 mg/m3 at the end of the recovery period in males do not correlate with significantly increased body weight-adjusted relative spleen weights at that concentration. Furthermore significantly decreased reticulocyte counts and significantly increased relative spleen weights were only seen in males and not in females. Additionally no test item induced histopathological findings were seen in the spleen at the end of the recovery period. Consequently these findings are reflected to be inconsistent and not of toxicological relevance.
At the end of the exposure period significantly increased reticulocytes and significantly increased body weight-adjusted relative spleen weights were found in females at 7.6 mg/m3. Of note, only body weight-adjusted spleen weights in female rats at 7.6 mg/m3 were significantly increased whereas absolute and brain weight-adjusted spleen weights did not reach statistically significance when compared to controls. Furthermore it has to be kept into account that only one gender was affected. Additionally, no further significantly changed red cell parameters were found at the end of the exposure period. Taking all those information into account it was considered that increased reticulocytes and increased body weight-adjusted relative spleen weights at the end of the exposure period are non-adverse.
Leucocytes and specifically neutrophils were increased at 7.6 mg/m³ in males and females at the end of the exposure period. At the end of the recovery period, leucocytes, lymphocytes, neutrophils, monocytes and atypical cells were increased at 7.6 mg/m³ in comparison to the air control. Systemically increased white cell parameters seen at clinical pathology mentioned above, are interpreted to be the consequence of cell recruitment due to inflammatory processes in the respiratory tract at 7.6 mg/m³. In this context the increased white blood cellsespacially neutrophils in the blood at 7.6 mg/m³ may be explained by raised recruitment due inflammatory processes in the lungs at 7.6 mg/m3.
Dose descriptor:
NOAEC
Effect level:
1.5 mg/m³ air
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: portal-of-enty toxicity in the respiratory tract associated tissues at 7.6 mg/m³; a NOEL could not be established
Dose descriptor:
NOAEC
Effect level:
7.6 mg/m³ air
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no systemic toxicity observed
Critical effects observed:
yes
Lowest effective dose / conc.:
7.6 mg/m³ air
System:
other: respiratory system
Organ:
other: portal-of-entry toxicity (local effects) in the respiratory tract; no systemic toxicity
Treatment related:
yes
Conclusions:
No subchronic inhalation toxicity study was performed with the registered substance. However, a grouping of substances and read-across approach according to Regulation (EC) No 1907/2006 (REACH), Annex XI 1.5 and following ECHA Read-Across Assessment Framework, RAAF (2015) is accomplished for five blocked diisocyanate oligomers (also for the read-across target substance isophorone diisocyanate, oligomeristion product, blocked with hexahydro-2H-azepin-2 -one).
For repeated inhalation toxicity testing the category is divided into two sub-groups – the liquid aerosol for blocked HDI-based oligomers and the powder aerosol for blocked IPDI-based oligomers. Thus, within the category the analogue read-across approach is followed, i.e. ECHA RAAF Scenario 2.

Within this analogue approach, a GLP-conform and Guideline-compliant 90-day subchronic inhalation toxicity study was performed with the read-across source for the registered substance (isophorone diisocyanate, oligomeristion product, blocked with 2 -butanone oxime) on request of ECHA. In this subchronic inhalation toxicity study with 13 week exposure and 13 week recovery period the No-Observed-Adverse-Effect-Concentration (NOAEC) is 1.5 mg/m³ due to respiratory tract associated adverse findings seen in BAL, histopathology and weight increase of lung and LALN at 7.6 mg/m³. These effects represent portal-of-entry toxicity. Test substance related systemic toxicity was not observed and thus, the NOAEC for systemic toxicity is 7.6 mg/m³, the highest dose tested.
Thus, according to EU Regulation 1272/2008 classification for specific target organ toxicity-repeated exposure Category 1 (STOT RE 1) is applicable for the source as well as the target substance.

An updated outline of a grouping-strategy based on read-across of the available toxicological data for 5 blocked diisocyanate oligomers is attached to the endpoint summaries for 'Repeated dose toxicity' and 'Toxicity to reproduction' in IUCLID as a separate document.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
1.5 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
All relevant studies are valid without restriction (Klimisch score 1).

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

A GLP-conform and Guideline-compliant 90-day subchronic inhalation toxicity study in rats was performed with the read-across source (isophorone diisocyanate, oligomeristion product, blocked with 2 -butanone oxime) for the registered substance on request of ECHA. In this study portal-of-entry toxicity (local effects in the respiratory tract) was observed, but there was no indication of systemic toxicity and thus, of bioavailability. This result is in line with observations obtained in subacute and acute inhalation toxicity studies with the substance that also revealed no test substance related adverse systemic toxicity. Therefore, no hazard is anticipated with regard to systemic effects after long-term inhalation exposure.

Additional information

No results from repeated-dose toxicity tests are available for the oral and dermal route of exposure.

A sub-acute (14 day) repeated dose inhalation study (0, 42.6, 220, and 1029 mg/m3; 6 hours/day on five days/week over a 14 -day period; based on OECD TG 413, TNO, 2012) with Wistar rats is available for the test substance and indicates the respiratory tract to be the target organ. The effects seen in this study demonstrate a spectrum of effects in the high concentration group (1029 mg/m3, actual conc.) which decreased to only mild increases in neutrophils and one biochemical marker (GGT) in broncho-alveolar lavage fluid in the animals of the low concentration group (42.6 mg/m3, actual conc.). The increases in neutrophils and GGT in brancho-alveolar lavage fluid were considered adverse because these changes are known indicators of inflammation. However, because there are no histopathological abnormality at this level, the low concentration is considered a Minimal Observed Adverse Effect Level (MOAEL).

No subchronic inhalation toxicity study was performed with the test substance. However, a grouping of substances and read-across approach according to Regulation (EC) No 1907/2006 (REACH), Annex XI 1.5 and following ECHA Read-Across Assessment Framework, RAAF (2015) is accomplished for five blocked diisocyanate oligomers.

Within this read-across approach, a GLP-conform and Guideline-compliant 90-day subchronic inhalation toxicity study in rats was performed with the read-across source (isophorone diisocyanate, oligomeristion product, blocked with 2 -butanone oxime) for the registered substance on request of ECHA.

In this subchronic inhalation toxicity study, equivalent to OECD TG 413 and compliant with OECD GD 39, groups of 10 male/female Wistar rats were nose-only exposed for 6 hours/day, 5 days/week for 13 consecutive weeks to the test substance (powder aerosol) at actual concentrations of 0.3, 1.5, and 7.6 mg/m³. The dust was respirable to rats (average MMAD 2.23 -2.35 µm; GSD 1.86-2.3). The control group was exposed to dry air under otherwise identical test conditions. Six additional male rats per concentration group were assigned to lung lavage at the end of the exposure period. Ten additional rats per sex of the control and the high concentration groups were allowed to recover during a 12-week postexposure period. The exposure took place in directed-flow nose-only inhalation chambers.

All exposures were tolerated without test substance-induced mortality. Absolute and relative lung weights were significantly increased in females at 7.6 mg/m³. Furthermore, lung-associated lymph node (LALN) weights and size were increased at 7.6 mg/m³ at the end of the exposure period and at the end of the recovery period. Relative spleen weights were significantly increased in females at the end of the exposure period and significantly increased in males at the end of the recovery period at 7.6 mg/m³. Significantly increased polymorphonuclear cells and relevant increase in protein, lymphocytes, LDH and GGT in the BAL at 7.6 mg/m³ were reflected as signs of inflammation and thus considered to be adverse. At 7.6 mg/m3, granuloma formation was seen in the lungs at the end of the exposure period which partly progressed to granuloma with central degeneration and necrosis after the end of the recovery period. Furthermore, chronic inflammation of the bronchiolo-alveolar junction was induced at 7.6 mg/m3that was still present after the post-exposure observation period. Accumulation of macrophages/giant cells/granuloma showed central degeneration and necrosis at 7.6 mg/m3, central degeneration and necrosis was still present after the recovery period. The moderate accumulation of macrophages/giant cells/granuloma partly with central degeneration/necrosis in the airways of the lung and in the associated lymphoid tissues (bronchus-associated lymphatic tissue, BALT and lung-associated lymph nodes, LALN) as well as the chronic inflammatory reaction seen at the bronchiolo-alveolar junction of the lung at 7.6 mg/m3are considered to be adverse.

In summary, no systemic toxicity was observed in this study. However, there is consistent evidence of portal-of-entry toxicity in the respiratory tract at 7.6 mg/m³. Taking all findings into account, 1.5 mg/m³ constitutes the no-observed-adverse-effect-level (NOAEL) for respiratory tract toxicity. The NOAEL for systemic toxicity is the highest concentration tested of 7.6 mg/m³.

Update of grouping and read-across strategy of December 2018:

For a detailed discussion of the outcome of the testing strategy and for justification of the grouping and read across of 5 blocked diisocyanate oligomers according to regulation No. 1907/2006 (REACH), Annex XI, 1.5 and following ECHA RAAF (2015) see the document attached to the endpoint summaries for ‘Repeated Dose Toxicity’ and ‘Toxicity to reproduction’ in IUCLID.

Note: The first outline of a grouping-strategy was prepared in May 2013 based on read-across of the available toxicological data for 4 blocked diisocyanate oligomers at that time. In the meantime, the group was enlarged to 5 blocked diisocyanate oligomers and 90-day inhalation toxicity studies for two of the category members have been performed on request of ECHA. The updated document refers to the REACH requirements according to Annex VIII - X (10 - > 1000 tonnes/a).

Justification for classification or non-classification

In the 90-day inhalation toxicity study significant local toxicity in the respiratory tract was seen at doses below the Guidance value of 20 mg/m³. Thus, according to EU Regulation 1272/2008 classification for specific target organ toxicity-repeated exposure Category 1 (STOT RE 1; H372) is applicable for the registered substance.