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Description of key information

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

In a subchronic inhalation toxicity study with 13 week exposure and 13 week recovery period adverse findings were seen in histopathology of the respiratory tract at 0.3 mg/m³ and above. These effects represent portal-of-entry toxicity. A No-Observed-Adverse-Effect-Concentration (NOAEC) could not be established for portal-of-entry toxicity in the respiratory tract. Test substance related systemic toxicity was not observed and thus, the NOAEC for systemic toxicity is 7.4 mg/m³, the highest dose tested.

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)
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, dry, protected from light. After 1st opening storage under N2-atmosphere (headspace)
- Stability of the test substance: Stability certified for the duration of study
- Stability and homogeneity of the test substance in the solvent/vehicle: Analytically confirmed.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Healthy young adult SPF bred Wistar rats, strain Crl:(Wi)WU
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:(Wi)WU (SPF-bred)
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: approx. 10 -11 weeks
- Weight at study initiation: males approx. 280 g, females approx. 190
- Housing: in Makrolon® (polycarbonate) cages type III, two rats per cage
- Diet and water: ad libitum
- Acclimation period: approx. five weeks; 4 - 5 weeks prior to exposure animals were trained to become accustomed to nose-only tubes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 /12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
ca. 1.6 µm
Geometric standard deviation (GSD):
2.6
Remarks on MMAD:
The MMAD was 1.6, 1.3, and 1.6 μm, the corresponding GSD was 2.6, 2.6, and 1.7, in the low, mid and high dose group, respectively.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Rats were exposed by a flow-past nose-only inhalation exposure system.
- Exposure apparatus: Aerosols are supplied to each rat individually, and exhaled air is immediately exhausted. The airflow to each rat was approximately 1 L/min which was calculated to be laminar (rat minute volume: 0.2 L). Therefore measurement of the oxygen concentration was not necessary.
- Method of holding animals in test chamber: For exposure to the test item the rats were restrained in acrylic tubes with adjustable backstops. The exposure tubes were arranged around a cylinder capable to take up 16 tubes per platform. The rat nose was located at the front end of a tube being connected to a cylinder delivering the aerosol. Through the thin pipes, the aerosol was supplied to each rat nose individually and exhaled air was drawn off immediately by a cylinder surrounding the aerosol delivering cylinder. The position of exposure tubes of rats at the cylinder was changed daily according to a rotation plan to minimize exposure differences due to geometry. The exposure units (4 units) were located each under a separate hood to prevent contamination among different dose groups.
- Temperature, humidity: Air flow, temperature and relative humidity was measured continuously and recorded by 20-minute means. The limits were set at 22 °C + 2 °C for temperature. During the 6 hours of
exposure relative humidity was approx. between 15 % and 21 %, due to the fact that nebulization was conducted by dry pressurized air.
- Atmosphere generation: The aerosols were generated by nebulising Ethyl acetate (vehicle control) or test substance in in Ethyl acetate using two-substance nozzles (solution, air) with identical constructions for every dose group. The liquid was fed to the nebulizer nozzle using syringe pumps. Using aerosol photometers the actual aerosol concentrations were monitored. The ratio between photometer signal and concentration were determined throughout the study by comparing the signal to gravimetric concentrations. Photometer measurements were based on light scattering of the aerosol, if the size distribution of the aerosol measured was constant the signal obtained was proportional to aerosol concentration.
- Method of particle size determination: Prior to the 90-day exposure of rats, technical trials to adjust particle size distributions and exposure levels were conducted.

TEST ATMOSPHERE
- Brief description of analytical method used: The test-substance concentration was determined by gravimetric analysis.
- Samples taken from breathing zone: yes; samples for gravimetric analysis were collected at a port of the nose-only exposure unit, thus, under the same conditions the rats inhaled the aerosol.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The MMAD was determined at least once a week (and once before exposure start) by a cascade impactor (Marple impactor) and was in the range of 1.3 to 1.6 µm within the exposure groups (Geometric Standard Deviation: 1.7 – 2.6).
- A significant weight reduction in the high dose group observed during the first week of the study made necessary a short-term reduction of the solvent concentration in the breathing air both in the highest dose group and in the vehicle control group.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Glas fiber filter samples of the aerosols were taken once a day to control the aerosol concentrations and to correlate aerosol photometer signals to mass concentration. Gravimetric analysis was conducted using a digital balance of appropriate sensitivity. The filter samples were collected at a port of the nose-only exposure unit, thus, under the same conditions the rats inhaled the aerosol. The vapour pressure of ethyl acetate is 98 hPa at 20 °C. Therefore it can be assumed that almost all ethyl acetate evaporates directly after dispersion of the liquid. The residual aerosol was sampled on a filter and weighted gravimetrically. The evaluation of filter samples and hence aerosol concentrations therefore was conducted gravimetrical. A detailed drying protocol was developed and validated before study start and reported.
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.:
7.4 mg/m³ air (analytical)
Remarks:
target concentration: 7.5 mg/m³
No. of animals per sex per dose:
10 rats/sex and 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:
yes, concurrent vehicle
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 T100112-5 (Exposure 9 x 6h/day, 5 days/week; recovery period of 2 weeks) (Pauluhn, 2013)
- Rationale for selecting satellite groups: After two weeks of recovery in the 2 week inhalation study, findings in LALN were still detectable. Therefore satellite groups for lung lavage and recovery were included in this study.
- Post-exposure recovery period in satellite groups: 13 weeks

Furthermore, the study design was based on an ECHA decision (decision number: TPE-D-2114308303-64-01/F)
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: All animals were clinically observed in their cages at least once a day. Once a week, they were inspected outside their home cages and carefully examined for clinical symptoms, i.e. abnormalities concerning their general condition

BODY WEIGHT: Yes, all animals
- Time schedule for examinations: Individual body weight was recorded to the nearest 0.1 g twice a week in the first 4 weeks and once a week thereafter throughout the study for all animals.

FOOD AND WATER CONSUMPTION: Yes.
- Food and water consumption were recorded weekly during the study period. These data were collected per cage.

OPHTHALMOSCOPIC EXAMINATION: Yes.
- Ophthalmoscopy was performed prior to first exposure, during the last (13th) exposure week and towards end of the recovery period.

RECTAL TEMPERATURES: Yes, all animals
- Rectal temperature was recorded on day 1 (after first exposure), once during the last (13th) exposure week and once towards end of the recovery period.

FUNCTIONAL OBSERVATION BATTERY: Yes.
- A functional observational battery (FOB) based on Gad (J. Toxicol. Environ. Health 9, 691-704,1982) and Moser et al. (Toxicol. Appl. Pharmacol. 108, 267-283,1991) was utilized to assess the effects of the treatment during exposure weeks 1, 8, and 12. In addition to the determination of forelimb grip strength (Meyer et al., Neurobehav. Toxicol. 1, 233-236,1979), the FOB included the further endpoints.

HAEMATOLOGY: Yes
- The hematological investigations were done on day 1 (groups 1-4) and 3 months (groups 1, 4) after the end of exposure followed by a 16-hour fasting period (tap water ad libitum).
- The following parameter were determined: Hematocrit, Hemoglobin, Leukocytes, Erythrocytes, Mean corpuscular volume, Mean corpuscular hemoglobin concentration, Mean corpuscular hemoglobin, Thrombocyte count, Reticulocytes, Heinz Bodies, Leukocyte differential count.

CLINICAL CHEMISTRY: Yes
- The clinico-chemical investigations were done on day 1 (groups 1-4) and 3 months (groups 1, 4) after the end of exposure followed by a 16-hour fasting period (tap water ad libitum).
- The following parameter were determined: Aspartate aminotransferase, optimized (ASAT), Alanine aminotransferase, optimized (ALAT), Glutamate dehydrogenase (GLDH), γ-Glutamylaminotransferase (γ-GT), Lactate dehydrogenase (LDH), Alkaline phosphatase (APh), Albumin, Globulin, Bilirubin (total), Blood glucose, Calcium, Chloride, Cholesterol, Creatinine, Phosphate, Potassium, Sodium, Total protein, Triglycerides, Urea, Hemostasis, Prothrombin time (PT, Quick value, “Hepato Quick”).

URINALYSIS: Yes
- Urinalysis was done on day 1 (groups 1-4) and 3 months (groups 1, 4) after the end of exposure followed by a 16-hour fasting period (tap water ad libitum). 16 h urine was collected in plastic tubes using so-called metabolism cages.
- The following parameter were determined: Sediment composition (SQ), Urine osmolality (Q), pH (SQ), Volume (Q), Protein (SQ), Glucose (SQ), Blood (SQ), Bilirubin (SQ), Urobilinogen (SQ), Ketone bodies (SQ).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals
- All animals were subjected to a complete necropsy, which included careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
- Body weight was recorded prior to sacrifice in order to calculate organ-to-body weight ratios.

ORGAN WEIGHTS: Yes, all animals
- The following organs were preserved and wet weights were recorded: adrenals, brain, epididymides, heart, kidneys, liver, lung (incl. half of trachea, see above), ovaries, spleen, testes, uterus and thymus.
- The lung and the lower half of the trachea was weighed, and used for BAL or histopathology.

HISTOPATHOLOGY: Yes
- The respiratory tract was preserved as follows: Nasal passages (including nasal -associated lymphoid tissue-NALT), larynx, trachea, lungs, and LALN (mediastinal and tracheabronchial).
- All tissues listed in the following were collected and preserved for histopathology: Animal identification, Gross findings, Adrenal glands, Aorta, Bone and bone marrow section – sternum, Brain (cerebrum, cerebellum, pons/medulla), Epididymides, Esophagus, Extraorbital lacrimal glands, Eyes with optic nerve, Eyelids, Femur with knee joint, Heart, Head with (focus) bulbus olfactorius, Harderian glands, Intestine, large (cecum, colon, rectum), Intestine, small (duodenum, ileum, jejunum), Kidneys, including pelvis, Larynx (3 levels), Liver, Lungs plus trachea and main bronchi (all lobes), Lymph nodes (mandibular, mesenteric, popliteal, mediastinal, (LALN), Mammary gland (female), Muscle (biceps femoris), Nasal turbinates (4 levels), [Olfactory bulb], Ovaries, incl. oviducts, Pancreas, Parathyroid gland(s), Pharynx, Pituitary gland, Prostate gland, Sternum, Salivary glands, Sciatic nerve, Seminal vesicles with coagulation glands, Skin (flank, nose region and facial area), Spinal Cord (cervical, thoracic, lumbar), Spleen, Stomach, Teeth, Testes, Thymus, Thyroid gland, Tongue, Trachea, Urinary bladder, Uterus (plus cervix), Ureters, Vagina.
- Histopathology was performed on all tissue specimens shown below in the control and high-level exposure groups. The tissues of the respiratory tract were examined in all groups, including those of the recovery groups. Other groups (and/or tissues) were evaluated at the discretion of the pathologist only if warranted by specific changes.
For histopathology of the respiratory tract including bronchi and the lung-associated lymph nodes (LALN, mediastinal and tracheobronchial), trachea, larynx, pharynx and the nasal cavities (including NALT) tissue specimens were prepared according to Kittel (Exp Tox Path 55, 413-31, 2004) and OECD GD 125.

Other examinations:
BRONCHOALVEOLAR LAVAGE: Yes, animals assigned (6 males/group)
For bronchoalveolar lavage, performed after end of exposure (+1 day), the method of Henderson et al. (Exp. Lung Res. 13, 329-342, 1987) was used with minor modifications.
The following parameter were determined:
- Cytological parameters: total cell count (recruitment of lung leukocytes), differential cell count (inflammatory (PMNs) or immunological (lymphocytes)
reactions; a total of 400 leukocytes per rat are evaluated), cell viability count (check of living cell status)
- Biochemical parameters: lactic dehydrogenase (LDH = cytosolic marker enzyme; increased permeability of membranes, cell damage and lysis), β-glucuronidase (measure of phagocytic activity of macrophages; lysis of macrophages), total protein (marker of transsudation; damage of epithelial cells)
Statistics:
Differences between groups were considered statistically significant at p < 0.05. Data were analysed using analysis of variance. If the group means differ significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's test (Dunnett, 1955; 1964). The statistical evaluation of the histopathological findings were done with the two-tailed Fisher test by the PROVANTIS system. If necessary, further statistical procedures were applied upon agreement with the sponsor.
Description (incidence and severity):
No adverse compound-related clinical signs were observed in rats during the course of the study.
Description (incidence):
No mortality occurred during the course of the study.
Description (incidence and severity):
The body weight and absolute body weight gain was not statistically significantly influenced by the treatment with the substance.
Note: The significant weight reduction in the high dose group observed during the first week of the study made necessary a short-term reduction of the solvent concentration in the breathing air both in the highest dose group and in the vehicle-/ solvent-control group.
Occasional statistically significant differences found in body weight gain as compared to the control group are inconsistent and therefore considered to be incidental and of no toxicological relevance.
Description (incidence and severity):
The total food consumption was not statistically significantly influenced by the treatment with the substance.
Occasional statistically significant differences found in food consumption as compared to the control group are inconsistent and therefore considered to be incidental and of no toxicological relevance.
Description (incidence and severity):
The total water was not statistically significantly influenced by the treatment with the substance.
Occasional statistically significant differences found in fwater consumption as compared to the control group are inconsistent and therefore considered to be incidental and of no toxicological relevance.
Description (incidence and severity):
No adverse compound-related clinical signs were observed in rats during the course of the study.
Description (incidence and severity):
At day 92, occasional changes were observed in the hematology parameters. The mean erythrocyte hemoglobin concentration (MCHC) was marginally decreased in male rats in the mid dose group. Total leukocyte counts (WBC) and lymphocytes (LYMC) were slightly increased in females in the high dose group. The latter effect is most likely caused by the extraordinary low WBC counts observed in females in all dose groups.
At day 182, changes in hematology parameters were not observed.
Description (incidence and severity):
At day 92, slightly significantly increased urea values (UREA) were found in male rats in the low and high dose groups. Due to the lack of dose-dependency and because all values are within the normal range, this finding is considered accidental and not treatment-related.
At day 182, male rats in the high dose group showed significantly decreased creatinine values (CREA). This finding is considered due to biological variance and not treatment-related.
Single animals showed extraordinary high potassium levels (K). This phenomenon is most probably due to pre-analytical artefacts and of no toxicological relevance.
At both time points female rats showed strikingly high liver parameters (ALT, AST, GLDH). This is a problem frequently seen in this rat strain. Therefore, all values were in the normal range expected for this sex, age and strain and the elevated liver parameters are not treatment related.
Description (incidence and severity):
No treatment-related effects were obvious from the results of quantitative and semiquantitative urinalysis.
Description (incidence and severity):
Reflex Measurement (FOB): No influence on the parameters measured was observed.
Description (incidence and severity):
The absolute and relative organ weights were not statistically significantly influenced by the treatment with the test substance.
Description (incidence and severity):
No compound-related effects were observed during necropsy.
Description (incidence and severity):
Exposure-related findings were detected within the lung, at the tracheal bifurcation, in the larynx, in the nasal cavity, and in the lung-associated lymph nodes. No compound-related findings occurred in the remaining organs and tissues investigated.
Interpreted as adverse findings were the erosion/ulceration of the bronchiolar epithelium at the BALT and the epithelium at the terminal bronchioles within the lung, the increased mononuclear cell infiltration within the lung, tracheal bifurcation and nasal cavity as well as the occurrence of multinucleated giant cells in the lung and tracheal bifurcation. These lesions were seen in the high, mid and partially low dose exposed animals. The accumulation of macrophages in the BALT together with the occurrence of the multinucleated giant cells was interpreted as a developing granulomatous inflammation, in which macrophages fuse to multinucleated cells. After 90 days of exposure statistically significantly occurred the erosion/ulceration of the bronchiolar epithelium at the BALT within the lung in the low, mid and high dose male rats. In addition, the mononuclear cell infiltration was increased with statistical significance in the low (only males), mid and high dose male and female animals within the lung, in the mid and high dose male and high dose female rats at the tracheal bifurcation and in the high dose male and female rats within the nasal cavity.
As adverse findings after the additional 90 days of recovery following the 90 days inhalation only the mononuclear cell infiltration occurred. Though the occurrence appeared slightly elevated in the high dose rats compared to the control animals, this was not significant statistically.
The other findings in the respiratory tract such as the mucous cell hyperplasia/hypertrophy, the accumulation of macrophages, the lymphoid hyperplasia, the vacuolation of the bronchiolar epithelium at the BALT, the alveolar histiocytosis, and the bronchiolo-alveolar hyperplasia were interpreted as adaptive findings. The exposure of the control rats with the vehicle caused slight changes such as vacuolation of the bronchiolar epithelium at the BALT, alveolar histiocytosis, bronchiolo-alveolar hyperplasia, mononuclear cells infiltration and accumulation of macrophages in different parts of the respiratory tract.
Description (incidence and severity):
Rectal Temperature: No influence on this parameter was observed.
Occasional statistically significant differences found in rectal temperature as compared to the control group are inconsistent and therefore considered to be incidental and of no toxicological relevance.

Bronchoalveolar Lavage Analysis: Lymphocytes were dose-dependently increased reaching statistically significance at 1.5 and 7.4 mg/m³. Increase in percentage of lymphocytes was 0.6%, 4.8%, *7.2%, **9.7% at the vehicle control, 0.3, 1.5 and 7.4 mg test item/m³, respectively. In contrary, polymorphonuclear neutrophils (PMN) showed a statistically significant increase in the mid dose group only and a dose-dependency was missing. Thus, the effect in the mid dose group is considered as not relevant (incidental finding).
The cell viability test did not show statistically significant effects in the treatment groups as compared to vehicle controls.
For lactic dehydrogenase (LDH) and ß-glucuronidase no conclusive statistically significant increases were detected. The statistically significantly increase of LDH observed in the mid dose group was considered as incidental finding. For total protein the dose-dependent but statistically non-significant increases were considered as non-relevant.
Absolute and relative lung weights of rats used for BAL did not show statistically significant effects.
Dose descriptor:
LOAEC
Remarks:
local toxicity
Effect level:
0.3 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 starting at 0.3 mg/m³; a NOAEL could not be established
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
7.4 mg/m³ air
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no systemic toxicity observed up to the highest dose tested (7.4 mg/m³)
Critical effects observed:
yes
Lowest effective dose / conc.:
0.3 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

Based on the above results seen in the respiratory tract there was no NOAEC established.

Executive summary:

In a subchronic inhalation toxicity study, equivalent to OECD TG 413, 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 (liquid aerosol, vehicle ethyl acetate used for generation of atmosphere) at actual concentrations of 0.3, 1.5, and 7.4 mg/m³. The aerosol was respirable to rats (MMAD 1.6, 1.3, and 1.6 µm; GSD 2.6, 2.6, and 1.7). The control group was exposed to the vehicle alone under otherwise identical test conditions.

Six additional male rats per concentration group were assigned to lung lavage at the end of the exposure period. Further 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.

Overall, all exposures were tolerated without mortality and clinical signs. No compound-related effects were observed during necropsy.

BAL analysis showed a concentration-dependent increase in percentual lymphocyte levels reaching statistical significance at 1.5 and 7.4 mg/m³. In contrary, a clearly significant and dose-dependent inflammatory effect manifested by elevated PMN was not detected.

Exposure-related histopathology findings were detected within the lung, at the tracheal bifurcation, in the larynx, in the nasal cavity, and in the lung-associated lymph nodes. These lesions were seen in the high, mid and partially low dose exposed animals.

90 days of test item exposure resulted in a very slight but statistical significant

- erosion/ulceration of the bronchiolar epithelium at the BALT within the lung in the low, mid and high dose male rats;

- mononuclear cell infiltration in the low (only males), mid and high dose male and female animals within the lung, in the mid and high dose male and high dose female rats at the tracheal bifurcation and in the high dose male and female rats within the nasal cavity;

and in statistically non-significant

- erosion/ulceration of the bronchiolar epithelium at the BALT within the lung in the low, mid and high dose female rats;

- (multi)focal very slight multinucleated giant cells at the tracheal bifurcation and within the BALT in male and females of all dose groups. The multinucleated giant cells were interpreted to be part of a granulomatous inflammation in which accumulated macrophages fuse to multinucleated cells.

After the 90 -day recovery only the mononuclear cell infiltration occurred, but without statistical significance.

Overall, compound-related adverse findings were erosion/ulceration of the bronchiolar epithelium at the BALT and the epithelium at the terminal bronchioles within the lung, increased mononuclear cell infiltration within the lung, tracheal bifurcation and nasal cavity as well as occurrence of multinucleated giant cells in BALT and tracheal bifurcation.

In summary, no systemic toxicity was observed in rats after repeated (90 -day) inhalation exposure to the liquid aerosol of the test item. However, this study revealed consistent evidence of portal-of-entry toxicity in the respiratory tract at all concentrations. Taking all findings into account, a no-observed-adverse-effect-level (NOAEL) could not be established. The NOAEL for systemic toxicity is the highest concentration tested of 7.6 mg/m³.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline pilot study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
(2009)
Deviations:
yes
Remarks:
pilot study focussed on effects in respiratory tract
Qualifier:
according to
Guideline:
other: OECD Guidance Document No. 39 (2009)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: HsdCpb:WU (SPF-bred)
- Source: Harlan-Nederland , AD Horst, The Netherlands
- 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 (based on A. Spiegel and R. Gönnert, Zschr. Versuchstierkunde, 1, 38 (1961) and G. Meister, Zschr. Versuchstierkunde, 7, 144-153 (1965))
- Diet and water: ad libitum
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- 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:
other: ethyl acetate
Remarks on MMAD:
MMAD / GSD: The MMAD was in a range of 1.2-1.6 µm (GSD approx. 2.3).
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 aluminium 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 Plexiglas exposure restrainers. Restrainers were chosen that accommodated the animals' size. These restrainers were designed so that the rat's tail remained outside the restrainer, thus restrained-induced hyperthermia can be avoided.
- Source and rate of air: Compressed air was supplied by Boge compressors.
- Method of conditioning air: Compressed air was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer.
- Temperature, humidity: Temperature and humidity measurements are also performed by the computerized Data Acquisition and Control System using FTF11 sensors (ELKA ELEKTRONIK, Luedenscheid, Germany). The position of the probe was at the exposure location of rats.
- Air flow rate: At each exposure port a minimal air flow rate of 0.75 L/min was provided. The test atmosphere can by no means be diluted by bias-air-flows.
- Air change rate: The test atmosphere generation conditions provide an adequate number of air exchanges per hour [30 L/min x 60 min/(2 x 3.8 L/chamber) = 237, continuous generation of test atmosphere]. 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). The slight positive balance provides also adequate dead-space ventilation of the exposure restrainers.
- System of generating particulates/aerosols: Under dynamic conditions the test substance is atomized into the baffle (pre-separator) of the inhalation chamber. For atomization a binary Schlick-type nozzle and conditioned compressed air (15 L/min) was used. The representative dispersion pressure was in the range of 600 kPa. The nozzle was maintained at 10 °C by a water jacket connected to a digitally controlled water bath. The test article was injected into the nozzle system using a digitally controlled pump (Harvard PHD 2000 infusion pump). In the control group the vehicle ethyl acetate was used. The same aerosolisation principles were used in the vehicle and test article groups. The atomization temperature and concentration of the test article in the vehicle was optimized to attain stable and highly respirable aerosol concentrations at the expense of a high concentration of vehicle ethyl acetate. In order to increase the efficiency of the generation of fine particles likely to evaporate and to prevent larger particles from entering the chamber a glass-pre-separator/baffle system was used. Overall, this pre-separator system favours the evaporation of the test substance. Nonetheless, one has to admit that the evaporation kinetics is likely decreased due to the low temperature of aerosolisation.
- Method of particle size determination: The particle-size distribution was analysed using a BERNER-TYPE AERAS low-pressure critical orifice cascade impactor (Hauke, Gmunden, Austria).

TEST ATMOSPHERE
- Brief description of analytical method used: The test-substance concentration was determined by gravimetric analysis (filter: glass-'fiber filter, Sartorius, Gottingen, Germany; digital balance). This method was used to define the actual concentration. Prior to gravimetric analysis the filter was dried for 30 min @70 °C.
- To monitor the integrity and stability of the aerosol generation and exposure system either RAS-2 real-time aerosol photometer (MIE, Bedford, Massachusetts, USA) or Microdust Pro real-time aerosol photometer (Casella, USA) was used.
- Samples taken from breathing zone: yes
- Particle size distribution: The particle size distribution was analysed using a BERNER critical orifice cascade impactor. Aerosol mass < 3 µm: 86.7 % at 11.5 mg/m³, 78.9 % at 106.9 mg/m³.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The aerosol was generated so that it was respirable to rats, i.e. the MMAD was 1.23 µm (GSD 2.22) at 11.5 mg/m³ and 1.56 µm (GSD 2.29) at 106.9 mg/m³.

VEHICLE (if applicable)
Ethyl acetate, CAS No. 141-78-6
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:
6 hours/day
Frequency of treatment:
9x6 hours (5 days/1st week and 4 days/2nd week)
Remarks:
Doses / Concentrations:
0, 10, 100 mg/m³
Basis:
other: target concentrations
Remarks:
Doses / Concentrations:
0, 11.5, 106.9 mg/m³
Basis:
other: analytical concentrations
No. of animals per sex per dose:
10 males/group (6 animals + 4 animals for recovery)
Control animals:
yes, concurrent vehicle
Details on study design:
The test item was evaluated as aerosol for its portal-of-entry related toxicity following repeated (subacute) inhalation exposure in Wistar rats. Rats were exposed under dynamic directed-flow nose-only exposure conditions to targeted concentrations of 10 and 100 mg/m³ for approx. 2 consecutive weeks (6 hrs/day, 5-times/week on 9 x 6 hours/day). Rats exposed under otherwise identical test conditions (vehicle) served as concurrent control group. Six rats/group were sacrificed one day after the last exposure, the remaining four rats/group were allowed to recover during a 2-week postexposure period.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, all animals
- Time schedule: Clinical signs were recorded daily before and after exposure and once a day on exposure-free days.

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: No

OPHTHALMOSCOPIC EXAMINATION: No

RECTAL TEMPERATURES: Yes, all animals
- Time schedule for examinations: Rectal temperatures were determined at thrice during the course of study.

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No
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.
At each sacrifice, the weights of the lungs and lung-associated lymph nodes were determined.

HISTOPATHOLOGY: Yes, entire respiratory tract for all animals
The following tissues were collected: head, nasal cavities, larynx, pharynx, trachea, lungs (lavaged), LALN and gross findings. Tissues were fixed in 10 % neutral-buffered formalin (NBF). Additional collected: animal identification (transponder).
The entire respiratory tract was examined by light microscopy in all group rats at both the end of exposure and recovery periods.
Other examinations:
BRONCHOALVEOLAR LAVAGE: Yes, all animals
Bronchoalveolar lavage fluid was sampled at each sacrifice.
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 (LDH), N-acetylglucosaminidase (beta-NAG), total protein, gamma-glutamyltransferase (gamma-GT).

After complete exsanguination (severing the aorta abdominalis), the excised wet lungs were weighed and then lavaged via a tracheal cannula with two volumes of 5 mL of physiological saline (at 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 fluids (BALF’s) from the lung lavages were 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 1000 rpm for approximately 10 min. at <10 °C (Sigma 4-16K centrifuge) and the resultant cell-free supernatant was analysed for the various biochemical parameters. The cell pellet was re-suspended in PBS-BSA, and then centrifuged (2 x 10exp5 per cytospot) onto slides using a cytospin centrifuge (Cytospin 4, Thermo-Shandon, Electron Corporation). Air-dried slides were fixed with a mixture of methanol:acetone, stained according to Pappenheim, and differentiated by light microscopy (300 cells were counted/cytospot). Cell counts were determined in triplicates after 1:1000 dilution using a CASY cell counter + analyzer. While “relative neutrophil counts” were calculated as the percentage of the cells that are neutrophils, determined from 300 total cells counted from differential cytospots, the “absolute” counts were multiplied by yield of cells recovered from BAL (“total cell counts”).
Statistics:
Analysis of variance (ANOVA BCTIC): Selected endpoints were statistically evaluated using the ANOVA BCTIC procedure (vide infra). 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-alpha) = 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 analysed by ANOVA BCTIC are also summarized in graphical form. In figures statistical significant differences are indicated by asterisks (* P < 0.05; ** P < 0.01).
Rectal temperatures, bronchoalveolar lavage: Data were statistically evaluated using the ANOVA procedure. Prior to evaluation, parameters in BALF were corrected according to the recovered volume of the lavage fluid (dilution factor).
Details on results:
MORTALITY
All exposures were tolerated without test substance-induced mortality.

CLINICAL SIGNS
Specific findings were not observed or recorded.

RECTAL TEMPERATURES
Determinations did not reveal any significant differences between the groups. Additionally, the temperature measurements made on control animals demonstrate clearly that the animal restrainer had no apparent effect on the body temperature.

BODY WEIGHTS
Data did not show any toxicologically consistent, i.e., clearly concentration- and/or time-dependent effect on body weights.

ORGAN WEIGHTS
Lung weights were significantly increased at 11.5 and 106.9 mg/m³ with reversibility at the end of the 2-week recovery period. The significantly increased LALN weights especially occurred at 106.9 mg/m³.

GROSS PATHOLOGY
Necropsy findings were unremarkable at the low exposure level. In rats exposed at 106.9 mg/m³ the lungs had discoulorated areas and enlarged lung-associated lymph nodes.

HISTOPATHOLOGY
At the end of the exposure period most of the rats from the high concentration group showed enlarged lung-associated-lymph nodes (LALN). One rat had an increased cellularity of the paracortex. At the end of the exposure period, alterations of the airway epithelium (thickening, focal necrosis) of the lungs, including enlarged/foamy macrophages, hypercellularity of the bronchiolo-alveolar junction, were detected in rats exposed to the high concentration. After two weeks of recovery, some enlarged and/or foamy macrophages were still detectable in rats from the high concentration group. In LALN, accumulations of epitheloid cells occurred in all rats from the high concentration group. In 2/4 rats of the low concentration group, substance-laden macrophages were seen (focal, minimal) in LALN.

BRONCHOALVEOLAR LAVAGE
The average recovery of bronchoalveolar lavage (BAL) fluid was approximately 90 % of the instilled volume and was similar amongst all groups. The results did not show changes considered to be adverse at 11.5 mg/m³. Evidence of significantly increased total BAL-cells, macrophages and PMNs and foamy macrophages, concurrent with cytotoxicity, occurred at 106 mg/m³. Non-classifiable cells (macrophages) contained either red blood cells or their debris.
Dose descriptor:
other: borderline NOAEC
Effect level:
11.5 mg/m³ air
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: Significantly increased lung weights at 11.5 mg/m³ at the end of exposure period, but not after 2-weeks recovery; no other changes considered to be adverse at this concentration.
Dose descriptor:
LOAEC
Effect level:
107 mg/m³ air
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: At 107 mg/m³ increased total cells in BALF and effects on weight (increased) and histopathology of lung and lung-associated lymph nodes.
Critical effects observed:
not specified

This study revealed consistent evidence of adverse effects at 107 mg/m³. 11.5 mg/m³ did not constitute a no-observed-adverse-effect-level (NOAEL) due to the significantly increased lung weights.

Executive summary:

In a 2-week pilot inhalation study, equivalent to OECD TG 412 and compliant with OECD GD 39, groups of Wistar rats were nose-only exposed for 6 hours/day, 5 days/week for approx. 2 consecutive weeks to the aerosolized test substance at actual concentrations of 11.5 and 107 mg/m³. The aerosol was respirable to rats (average MMAD 1.2-1.6 mm; GSD approx. 2.3). For control a group of rats were exposed to the vehicle ethyl acetate under otherwise identical test conditions. In each group of 10 animals 6 rats were sacrificed one day after the last exposure, the remaining 4 rats were allowed to recover during a 2-week postexposure period.

All exposures were tolerated without test substance-induced mortality. Clinical signs, effects on rectal temperatures and on body weights were not observed in the course of the study. Analysis of bronchoalveolar lavage fluid (BAL) did not reveal changes considered to be adverse at 11.5 mg/m³. Evidence of significantly increased total BAL-cells, macrophages and PMNs and foamy macrophages, concurrent with cytotoxicity, occurred at 107 mg/m³. Likewise, at this exposure level the lung and lung-associated-lymph node (LALN) weights were significantly increased. Significantly increased lung weights were also apparent at 11.5 mg/m³ at the end of the exposure period but not at the end of the recovery period. Histopathology revealed enlarged LALN with increased cellularity of the paracortex and alterations of the airway epithelium (thickening, focal necrosis) in the lungs with enlarged/foamy macrophages and hypercellularity of the bronchiolo-alveolar junction at 107 mg/m³. After two weeks of recovery, some enlarged and/or foamy macrophages were still detectable at this exposure level.

In summary, this study revealed consistent evidence of adverse effects at 107 mg/m³. Taking all findings into account, 11.5 mg/m³ did not constitute a no-observed-adverse-effect-level (NOAEL) due to the significantly increased lung weights.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
7.4 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Guideline and GLP compliant study

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
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)
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, dry, protected from light. After 1st opening storage under N2-atmosphere (headspace)
- Stability of the test substance: Stability certified for the duration of study
- Stability and homogeneity of the test substance in the solvent/vehicle: Analytically confirmed.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Healthy young adult SPF bred Wistar rats, strain Crl:(Wi)WU
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:(Wi)WU (SPF-bred)
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: approx. 10 -11 weeks
- Weight at study initiation: males approx. 280 g, females approx. 190
- Housing: in Makrolon® (polycarbonate) cages type III, two rats per cage
- Diet and water: ad libitum
- Acclimation period: approx. five weeks; 4 - 5 weeks prior to exposure animals were trained to become accustomed to nose-only tubes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 /12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
ca. 1.6 µm
Geometric standard deviation (GSD):
2.6
Remarks on MMAD:
The MMAD was 1.6, 1.3, and 1.6 μm, the corresponding GSD was 2.6, 2.6, and 1.7, in the low, mid and high dose group, respectively.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Rats were exposed by a flow-past nose-only inhalation exposure system.
- Exposure apparatus: Aerosols are supplied to each rat individually, and exhaled air is immediately exhausted. The airflow to each rat was approximately 1 L/min which was calculated to be laminar (rat minute volume: 0.2 L). Therefore measurement of the oxygen concentration was not necessary.
- Method of holding animals in test chamber: For exposure to the test item the rats were restrained in acrylic tubes with adjustable backstops. The exposure tubes were arranged around a cylinder capable to take up 16 tubes per platform. The rat nose was located at the front end of a tube being connected to a cylinder delivering the aerosol. Through the thin pipes, the aerosol was supplied to each rat nose individually and exhaled air was drawn off immediately by a cylinder surrounding the aerosol delivering cylinder. The position of exposure tubes of rats at the cylinder was changed daily according to a rotation plan to minimize exposure differences due to geometry. The exposure units (4 units) were located each under a separate hood to prevent contamination among different dose groups.
- Temperature, humidity: Air flow, temperature and relative humidity was measured continuously and recorded by 20-minute means. The limits were set at 22 °C + 2 °C for temperature. During the 6 hours of
exposure relative humidity was approx. between 15 % and 21 %, due to the fact that nebulization was conducted by dry pressurized air.
- Atmosphere generation: The aerosols were generated by nebulising Ethyl acetate (vehicle control) or test substance in in Ethyl acetate using two-substance nozzles (solution, air) with identical constructions for every dose group. The liquid was fed to the nebulizer nozzle using syringe pumps. Using aerosol photometers the actual aerosol concentrations were monitored. The ratio between photometer signal and concentration were determined throughout the study by comparing the signal to gravimetric concentrations. Photometer measurements were based on light scattering of the aerosol, if the size distribution of the aerosol measured was constant the signal obtained was proportional to aerosol concentration.
- Method of particle size determination: Prior to the 90-day exposure of rats, technical trials to adjust particle size distributions and exposure levels were conducted.

TEST ATMOSPHERE
- Brief description of analytical method used: The test-substance concentration was determined by gravimetric analysis.
- Samples taken from breathing zone: yes; samples for gravimetric analysis were collected at a port of the nose-only exposure unit, thus, under the same conditions the rats inhaled the aerosol.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The MMAD was determined at least once a week (and once before exposure start) by a cascade impactor (Marple impactor) and was in the range of 1.3 to 1.6 µm within the exposure groups (Geometric Standard Deviation: 1.7 – 2.6).
- A significant weight reduction in the high dose group observed during the first week of the study made necessary a short-term reduction of the solvent concentration in the breathing air both in the highest dose group and in the vehicle control group.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Glas fiber filter samples of the aerosols were taken once a day to control the aerosol concentrations and to correlate aerosol photometer signals to mass concentration. Gravimetric analysis was conducted using a digital balance of appropriate sensitivity. The filter samples were collected at a port of the nose-only exposure unit, thus, under the same conditions the rats inhaled the aerosol. The vapour pressure of ethyl acetate is 98 hPa at 20 °C. Therefore it can be assumed that almost all ethyl acetate evaporates directly after dispersion of the liquid. The residual aerosol was sampled on a filter and weighted gravimetrically. The evaluation of filter samples and hence aerosol concentrations therefore was conducted gravimetrical. A detailed drying protocol was developed and validated before study start and reported.
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.:
7.4 mg/m³ air (analytical)
Remarks:
target concentration: 7.5 mg/m³
No. of animals per sex per dose:
10 rats/sex and 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:
yes, concurrent vehicle
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 T100112-5 (Exposure 9 x 6h/day, 5 days/week; recovery period of 2 weeks) (Pauluhn, 2013)
- Rationale for selecting satellite groups: After two weeks of recovery in the 2 week inhalation study, findings in LALN were still detectable. Therefore satellite groups for lung lavage and recovery were included in this study.
- Post-exposure recovery period in satellite groups: 13 weeks

Furthermore, the study design was based on an ECHA decision (decision number: TPE-D-2114308303-64-01/F)
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: All animals were clinically observed in their cages at least once a day. Once a week, they were inspected outside their home cages and carefully examined for clinical symptoms, i.e. abnormalities concerning their general condition

BODY WEIGHT: Yes, all animals
- Time schedule for examinations: Individual body weight was recorded to the nearest 0.1 g twice a week in the first 4 weeks and once a week thereafter throughout the study for all animals.

FOOD AND WATER CONSUMPTION: Yes.
- Food and water consumption were recorded weekly during the study period. These data were collected per cage.

OPHTHALMOSCOPIC EXAMINATION: Yes.
- Ophthalmoscopy was performed prior to first exposure, during the last (13th) exposure week and towards end of the recovery period.

RECTAL TEMPERATURES: Yes, all animals
- Rectal temperature was recorded on day 1 (after first exposure), once during the last (13th) exposure week and once towards end of the recovery period.

FUNCTIONAL OBSERVATION BATTERY: Yes.
- A functional observational battery (FOB) based on Gad (J. Toxicol. Environ. Health 9, 691-704,1982) and Moser et al. (Toxicol. Appl. Pharmacol. 108, 267-283,1991) was utilized to assess the effects of the treatment during exposure weeks 1, 8, and 12. In addition to the determination of forelimb grip strength (Meyer et al., Neurobehav. Toxicol. 1, 233-236,1979), the FOB included the further endpoints.

HAEMATOLOGY: Yes
- The hematological investigations were done on day 1 (groups 1-4) and 3 months (groups 1, 4) after the end of exposure followed by a 16-hour fasting period (tap water ad libitum).
- The following parameter were determined: Hematocrit, Hemoglobin, Leukocytes, Erythrocytes, Mean corpuscular volume, Mean corpuscular hemoglobin concentration, Mean corpuscular hemoglobin, Thrombocyte count, Reticulocytes, Heinz Bodies, Leukocyte differential count.

CLINICAL CHEMISTRY: Yes
- The clinico-chemical investigations were done on day 1 (groups 1-4) and 3 months (groups 1, 4) after the end of exposure followed by a 16-hour fasting period (tap water ad libitum).
- The following parameter were determined: Aspartate aminotransferase, optimized (ASAT), Alanine aminotransferase, optimized (ALAT), Glutamate dehydrogenase (GLDH), γ-Glutamylaminotransferase (γ-GT), Lactate dehydrogenase (LDH), Alkaline phosphatase (APh), Albumin, Globulin, Bilirubin (total), Blood glucose, Calcium, Chloride, Cholesterol, Creatinine, Phosphate, Potassium, Sodium, Total protein, Triglycerides, Urea, Hemostasis, Prothrombin time (PT, Quick value, “Hepato Quick”).

URINALYSIS: Yes
- Urinalysis was done on day 1 (groups 1-4) and 3 months (groups 1, 4) after the end of exposure followed by a 16-hour fasting period (tap water ad libitum). 16 h urine was collected in plastic tubes using so-called metabolism cages.
- The following parameter were determined: Sediment composition (SQ), Urine osmolality (Q), pH (SQ), Volume (Q), Protein (SQ), Glucose (SQ), Blood (SQ), Bilirubin (SQ), Urobilinogen (SQ), Ketone bodies (SQ).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals
- All animals were subjected to a complete necropsy, which included careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
- Body weight was recorded prior to sacrifice in order to calculate organ-to-body weight ratios.

ORGAN WEIGHTS: Yes, all animals
- The following organs were preserved and wet weights were recorded: adrenals, brain, epididymides, heart, kidneys, liver, lung (incl. half of trachea, see above), ovaries, spleen, testes, uterus and thymus.
- The lung and the lower half of the trachea was weighed, and used for BAL or histopathology.

HISTOPATHOLOGY: Yes
- The respiratory tract was preserved as follows: Nasal passages (including nasal -associated lymphoid tissue-NALT), larynx, trachea, lungs, and LALN (mediastinal and tracheabronchial).
- All tissues listed in the following were collected and preserved for histopathology: Animal identification, Gross findings, Adrenal glands, Aorta, Bone and bone marrow section – sternum, Brain (cerebrum, cerebellum, pons/medulla), Epididymides, Esophagus, Extraorbital lacrimal glands, Eyes with optic nerve, Eyelids, Femur with knee joint, Heart, Head with (focus) bulbus olfactorius, Harderian glands, Intestine, large (cecum, colon, rectum), Intestine, small (duodenum, ileum, jejunum), Kidneys, including pelvis, Larynx (3 levels), Liver, Lungs plus trachea and main bronchi (all lobes), Lymph nodes (mandibular, mesenteric, popliteal, mediastinal, (LALN), Mammary gland (female), Muscle (biceps femoris), Nasal turbinates (4 levels), [Olfactory bulb], Ovaries, incl. oviducts, Pancreas, Parathyroid gland(s), Pharynx, Pituitary gland, Prostate gland, Sternum, Salivary glands, Sciatic nerve, Seminal vesicles with coagulation glands, Skin (flank, nose region and facial area), Spinal Cord (cervical, thoracic, lumbar), Spleen, Stomach, Teeth, Testes, Thymus, Thyroid gland, Tongue, Trachea, Urinary bladder, Uterus (plus cervix), Ureters, Vagina.
- Histopathology was performed on all tissue specimens shown below in the control and high-level exposure groups. The tissues of the respiratory tract were examined in all groups, including those of the recovery groups. Other groups (and/or tissues) were evaluated at the discretion of the pathologist only if warranted by specific changes.
For histopathology of the respiratory tract including bronchi and the lung-associated lymph nodes (LALN, mediastinal and tracheobronchial), trachea, larynx, pharynx and the nasal cavities (including NALT) tissue specimens were prepared according to Kittel (Exp Tox Path 55, 413-31, 2004) and OECD GD 125.

Other examinations:
BRONCHOALVEOLAR LAVAGE: Yes, animals assigned (6 males/group)
For bronchoalveolar lavage, performed after end of exposure (+1 day), the method of Henderson et al. (Exp. Lung Res. 13, 329-342, 1987) was used with minor modifications.
The following parameter were determined:
- Cytological parameters: total cell count (recruitment of lung leukocytes), differential cell count (inflammatory (PMNs) or immunological (lymphocytes)
reactions; a total of 400 leukocytes per rat are evaluated), cell viability count (check of living cell status)
- Biochemical parameters: lactic dehydrogenase (LDH = cytosolic marker enzyme; increased permeability of membranes, cell damage and lysis), β-glucuronidase (measure of phagocytic activity of macrophages; lysis of macrophages), total protein (marker of transsudation; damage of epithelial cells)
Statistics:
Differences between groups were considered statistically significant at p < 0.05. Data were analysed using analysis of variance. If the group means differ significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's test (Dunnett, 1955; 1964). The statistical evaluation of the histopathological findings were done with the two-tailed Fisher test by the PROVANTIS system. If necessary, further statistical procedures were applied upon agreement with the sponsor.
Description (incidence and severity):
No adverse compound-related clinical signs were observed in rats during the course of the study.
Description (incidence):
No mortality occurred during the course of the study.
Description (incidence and severity):
The body weight and absolute body weight gain was not statistically significantly influenced by the treatment with the substance.
Note: The significant weight reduction in the high dose group observed during the first week of the study made necessary a short-term reduction of the solvent concentration in the breathing air both in the highest dose group and in the vehicle-/ solvent-control group.
Occasional statistically significant differences found in body weight gain as compared to the control group are inconsistent and therefore considered to be incidental and of no toxicological relevance.
Description (incidence and severity):
The total food consumption was not statistically significantly influenced by the treatment with the substance.
Occasional statistically significant differences found in food consumption as compared to the control group are inconsistent and therefore considered to be incidental and of no toxicological relevance.
Description (incidence and severity):
The total water was not statistically significantly influenced by the treatment with the substance.
Occasional statistically significant differences found in fwater consumption as compared to the control group are inconsistent and therefore considered to be incidental and of no toxicological relevance.
Description (incidence and severity):
No adverse compound-related clinical signs were observed in rats during the course of the study.
Description (incidence and severity):
At day 92, occasional changes were observed in the hematology parameters. The mean erythrocyte hemoglobin concentration (MCHC) was marginally decreased in male rats in the mid dose group. Total leukocyte counts (WBC) and lymphocytes (LYMC) were slightly increased in females in the high dose group. The latter effect is most likely caused by the extraordinary low WBC counts observed in females in all dose groups.
At day 182, changes in hematology parameters were not observed.
Description (incidence and severity):
At day 92, slightly significantly increased urea values (UREA) were found in male rats in the low and high dose groups. Due to the lack of dose-dependency and because all values are within the normal range, this finding is considered accidental and not treatment-related.
At day 182, male rats in the high dose group showed significantly decreased creatinine values (CREA). This finding is considered due to biological variance and not treatment-related.
Single animals showed extraordinary high potassium levels (K). This phenomenon is most probably due to pre-analytical artefacts and of no toxicological relevance.
At both time points female rats showed strikingly high liver parameters (ALT, AST, GLDH). This is a problem frequently seen in this rat strain. Therefore, all values were in the normal range expected for this sex, age and strain and the elevated liver parameters are not treatment related.
Description (incidence and severity):
No treatment-related effects were obvious from the results of quantitative and semiquantitative urinalysis.
Description (incidence and severity):
Reflex Measurement (FOB): No influence on the parameters measured was observed.
Description (incidence and severity):
The absolute and relative organ weights were not statistically significantly influenced by the treatment with the test substance.
Description (incidence and severity):
No compound-related effects were observed during necropsy.
Description (incidence and severity):
Exposure-related findings were detected within the lung, at the tracheal bifurcation, in the larynx, in the nasal cavity, and in the lung-associated lymph nodes. No compound-related findings occurred in the remaining organs and tissues investigated.
Interpreted as adverse findings were the erosion/ulceration of the bronchiolar epithelium at the BALT and the epithelium at the terminal bronchioles within the lung, the increased mononuclear cell infiltration within the lung, tracheal bifurcation and nasal cavity as well as the occurrence of multinucleated giant cells in the lung and tracheal bifurcation. These lesions were seen in the high, mid and partially low dose exposed animals. The accumulation of macrophages in the BALT together with the occurrence of the multinucleated giant cells was interpreted as a developing granulomatous inflammation, in which macrophages fuse to multinucleated cells. After 90 days of exposure statistically significantly occurred the erosion/ulceration of the bronchiolar epithelium at the BALT within the lung in the low, mid and high dose male rats. In addition, the mononuclear cell infiltration was increased with statistical significance in the low (only males), mid and high dose male and female animals within the lung, in the mid and high dose male and high dose female rats at the tracheal bifurcation and in the high dose male and female rats within the nasal cavity.
As adverse findings after the additional 90 days of recovery following the 90 days inhalation only the mononuclear cell infiltration occurred. Though the occurrence appeared slightly elevated in the high dose rats compared to the control animals, this was not significant statistically.
The other findings in the respiratory tract such as the mucous cell hyperplasia/hypertrophy, the accumulation of macrophages, the lymphoid hyperplasia, the vacuolation of the bronchiolar epithelium at the BALT, the alveolar histiocytosis, and the bronchiolo-alveolar hyperplasia were interpreted as adaptive findings. The exposure of the control rats with the vehicle caused slight changes such as vacuolation of the bronchiolar epithelium at the BALT, alveolar histiocytosis, bronchiolo-alveolar hyperplasia, mononuclear cells infiltration and accumulation of macrophages in different parts of the respiratory tract.
Description (incidence and severity):
Rectal Temperature: No influence on this parameter was observed.
Occasional statistically significant differences found in rectal temperature as compared to the control group are inconsistent and therefore considered to be incidental and of no toxicological relevance.

Bronchoalveolar Lavage Analysis: Lymphocytes were dose-dependently increased reaching statistically significance at 1.5 and 7.4 mg/m³. Increase in percentage of lymphocytes was 0.6%, 4.8%, *7.2%, **9.7% at the vehicle control, 0.3, 1.5 and 7.4 mg test item/m³, respectively. In contrary, polymorphonuclear neutrophils (PMN) showed a statistically significant increase in the mid dose group only and a dose-dependency was missing. Thus, the effect in the mid dose group is considered as not relevant (incidental finding).
The cell viability test did not show statistically significant effects in the treatment groups as compared to vehicle controls.
For lactic dehydrogenase (LDH) and ß-glucuronidase no conclusive statistically significant increases were detected. The statistically significantly increase of LDH observed in the mid dose group was considered as incidental finding. For total protein the dose-dependent but statistically non-significant increases were considered as non-relevant.
Absolute and relative lung weights of rats used for BAL did not show statistically significant effects.
Dose descriptor:
LOAEC
Remarks:
local toxicity
Effect level:
0.3 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 starting at 0.3 mg/m³; a NOAEL could not be established
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
7.4 mg/m³ air
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no systemic toxicity observed up to the highest dose tested (7.4 mg/m³)
Critical effects observed:
yes
Lowest effective dose / conc.:
0.3 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

Based on the above results seen in the respiratory tract there was no NOAEC established.

Executive summary:

In a subchronic inhalation toxicity study, equivalent to OECD TG 413, 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 (liquid aerosol, vehicle ethyl acetate used for generation of atmosphere) at actual concentrations of 0.3, 1.5, and 7.4 mg/m³. The aerosol was respirable to rats (MMAD 1.6, 1.3, and 1.6 µm; GSD 2.6, 2.6, and 1.7). The control group was exposed to the vehicle alone under otherwise identical test conditions.

Six additional male rats per concentration group were assigned to lung lavage at the end of the exposure period. Further 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.

Overall, all exposures were tolerated without mortality and clinical signs. No compound-related effects were observed during necropsy.

BAL analysis showed a concentration-dependent increase in percentual lymphocyte levels reaching statistical significance at 1.5 and 7.4 mg/m³. In contrary, a clearly significant and dose-dependent inflammatory effect manifested by elevated PMN was not detected.

Exposure-related histopathology findings were detected within the lung, at the tracheal bifurcation, in the larynx, in the nasal cavity, and in the lung-associated lymph nodes. These lesions were seen in the high, mid and partially low dose exposed animals.

90 days of test item exposure resulted in a very slight but statistical significant

- erosion/ulceration of the bronchiolar epithelium at the BALT within the lung in the low, mid and high dose male rats;

- mononuclear cell infiltration in the low (only males), mid and high dose male and female animals within the lung, in the mid and high dose male and high dose female rats at the tracheal bifurcation and in the high dose male and female rats within the nasal cavity;

and in statistically non-significant

- erosion/ulceration of the bronchiolar epithelium at the BALT within the lung in the low, mid and high dose female rats;

- (multi)focal very slight multinucleated giant cells at the tracheal bifurcation and within the BALT in male and females of all dose groups. The multinucleated giant cells were interpreted to be part of a granulomatous inflammation in which accumulated macrophages fuse to multinucleated cells.

After the 90 -day recovery only the mononuclear cell infiltration occurred, but without statistical significance.

Overall, compound-related adverse findings were erosion/ulceration of the bronchiolar epithelium at the BALT and the epithelium at the terminal bronchioles within the lung, increased mononuclear cell infiltration within the lung, tracheal bifurcation and nasal cavity as well as occurrence of multinucleated giant cells in BALT and tracheal bifurcation.

In summary, no systemic toxicity was observed in rats after repeated (90 -day) inhalation exposure to the liquid aerosol of the test item. However, this study revealed consistent evidence of portal-of-entry toxicity in the respiratory tract at all concentrations. Taking all findings into account, a no-observed-adverse-effect-level (NOAEL) could not be established. The NOAEL for systemic toxicity is the highest concentration tested of 7.6 mg/m³.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
0.3 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Guideline and GLP compliant study; a NOAEC for inhalation - local effects could not be established

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 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

In a subchronic inhalation toxicity study, equivalent to OECD TG 413, 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 (liquid aerosol, vehicle ethyl acetate used for generation of atmosphere) at actual concentrations of 0.3, 1.5, and 7.4 mg/m³. The aerosol was respirable to rats (MMAD 1.6, 1.3, and 1.6 µm; GSD 2.6, 2.6, and 1.7). The control group was exposed to the vehicle alone under otherwise identical test conditions.

Six additional male rats per concentration group were assigned to lung lavage at the end of the exposure period. Further 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.

Overal, all exposures were tolerated without mortality and clinical signs. No compound-related effects were observed during necropsy.

BAL analysis showed a concentration-dependent increase in percentual lymphocyte levels reaching statistical significance at 1.5 and 7.4 mg/m³. In contrary, a clearly significant and dose-dependent inflammatory effect manifested by elevated PMN was not detected.

Exposure-related histopathology findings were detected within the lung, at the tracheal bifurcation, in the larynx, in the nasal cavity, and in the lung-associated lymph nodes. These lesions were seen in the high, mid and partially low dose exposed animals.

90 days of test item exposure resulted in a very slight but statistical significant

- erosion/ulceration of the bronchiolar epithelium at the BALT within the lung in the low, mid and high dose male rats;

- mononuclear cell infiltration in the low (only males), mid and high dose male and female animals within the lung, in the mid and high dose male and high dose female rats at the tracheal bifurcation and in the high dose male and female rats within the nasal cavity;

and in statistically non-significant

- erosion/ulceration of the bronchiolar epithelium at the BALT within the lung in the low, mid and high dose female rats;

- (multi)focal very slight multinucleated giant cells at the tracheal bifurcation and within the BALT in male and females of all dose groups. The multinucleated giant cells were interpreted to be part of a granulomatous inflammation in which accumulated macrophages fuse to multinucleated cells.

After the 90 -day recovery only the mononuclear cell infiltration occurred, but without statistical significance.

Overall, compound-related adverse findings were erosion/ulceration of the bronchiolar epithelium at the BALT and the epithelium at the terminal bronchioles within the lung, increased mononuclear cell infiltration within the lung, tracheal bifurcation and nasal cavity as well as occurrence of multinucleated giant cells in BALT and tracheal bifurcation.

In summary, no systemic toxicity was observed in rats after repeated (90 -day) inhalation exposure to the liquid aerosol of the test item. However, this study revealed consistent evidence of portal-of-entry toxicity in the respiratory tract at all concentrations. Taking all findings into account, a no-observed-adverse-effect-level (NOAEL) could not be established. 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 summary 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 substance.