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Repeated dose toxicity: inhalation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
(2009)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Hexamethylene diisocyanate, oligomerisation product, blocked with 3,5-dimethyl-1H-pyrazole
EC Number:
605-318-9
Cas Number:
163206-31-3
Molecular formula:
Not applicable (UVCB substance)
IUPAC Name:
Hexamethylene diisocyanate, oligomerisation product, blocked with 3,5-dimethyl-1H-pyrazole
additive 1
Reference substance name:
Ethyl acetate
EC Number:
205-500-4
EC Name:
Ethyl acetate
Test material form:
liquid
Details on test material:
- Content: 70 % in ethyl acetate
- Appearance: colourless clear/light yellow liquid
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.

Test animals

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 or test system 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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
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)

Target system / organ toxicity

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.