Registration Dossier

Diss Factsheets

Toxicological information

Acute Toxicity: inhalation

Currently viewing:

Administrative data

acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 403 (Acute Inhalation Toxicity)
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method

Test material

Constituent 1
Reference substance name:
112-96-9 + 1943-84-6
112-96-9 + 1943-84-6
Constituent 2
Chemical structure
Reference substance name:
1-isocyanatooctadecane 1-isocyanatohexadecane
EC Number:
Molecular formula:
C17H33NO C18H35NO
1-isocyanatooctadecane 1-isocyanatohexadecane

Test animals

Details on test animals or test system and environmental conditions:
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Strain: Hsd Cpb:Wu (SPF)
- Age at study initiation: approx. 2 months
- Weight at study initiation: at the study start the variation of individual weights did not exceed ± 10 per cent of the mean for each sex
- Housing: singly in conventional Makrolon® Type II cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

- Temperature (°C): 22 +/- 2
- Humidity (%): approx. 50
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
other: conditioned dry air
Details on inhalation exposure:
- Mode of exposure: Animals were exposed to the aerosolized test substance in Plexiglas exposure tubes applying a directed-flow nose-only exposure principle.
- Vehicle: The neat test article was aerosolized undiluted.
- Aerosol generation: Under dynamic conditions the test substance was nebulized into a baffle (pre-separator) from which the substance was conveyed into the intake of the cylindrical inhalation chamber. The test substance was nebulized using a binary nozzle with conditioned compressed air (15 Llmin; dispersion pressure approximately 500 kPa). The solution was fed into the nozzle by either by a digitally controlled Hamilton Microlab or BrownMelsungen infusion pump.
- Inhalation chamber: The aluminium inhalation chamber has the following dimensions: inner diameter = 14 cm, outer diameter = 35 cm (two-chamber system), height = 25 cm (internal volume = about 3.8 L). Details of this modular chamber and its validation have been published previously (Pauluhn, Journal of Applied Toxicology 14: 55-62, 1994).
- Optimization of respirability: In order to increase the efficiency of the generation of respirable particles and to prevent larger particles from entering the chamber a preseparator/ baffle system was used.
- Conditioning the compressed air: Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer. Adequate control devices were employed to control supply pressure.
- Inhalation chamber steady-state concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour (> 200 x, continuous generation of test atmosphere). Under such test conditions steady state is attained within the first minute of exposure (t99% = 4.6 x chamber volume/flow rate). The ratio between the air supplied and exhausted was chosen so that approximately 80-90 % of the supplied air is removed via the exhaust system. The remainder provides adequate dead-space ventilation for the exposure tubes. At each exposure port a minimal air flow rate of 0.75 I/min was provided. The test atmosphere can by no means be diluted by bias-air-flows.
- Exhaust air treatment: The exhaust air was purified via cotton-wool/HEPA filters. These filters were disposed of by Bayer AG.
- Temperature and humidity measurements: Temperature and humidity control was performed using a computerized system (Hydra, Fluke-Philips). The test atmosphere temperature and humidity were measured at the exposure location using a FTF-sensor (Elka-Elektronik, Lüdenscheid). Data were recorded automatically at intervals of 5 min. The measured values were evaluated using spread-sheet software.

- Nominal concentration: The nominal concentration was calculated from the ratio of the total quantity of test substance discharged during the exposure period and the total throughput of air through the inhalation chamber. The lower analytical concentrations compared with the nominal concentrations are attributed to the efficient removal of larger particles in the baffle/preseparator system.
- Gravimetric evaluation: The test-substance concentration was determined by gravimetric analysis (filter: Glass-Fiber-Filter, Sartorius, Göttingen, Germany; digital balance). All analytical concentrations reported refer to mg of test substance/m3 air.
- Samples taken from breathing zone: yes
- Particle size distribution: The particle-size distribution was analyzed using a BERNER-TYPE AERAS low-pressure critical orifice cascade impactor (Hauke, Gmunden, Austria). The individual impactor stages had been covered by an aluminum foil and glass fiber filter which were subjected to
gravimetric analysis. An adhesive stage coating (silicone spray) was therefore not used to prevent particle bounce and re-entrainment. Gravimetric analyses were made using a digital balance. Impaction media were treated as filters.
- Respirability: The particle-size distribution achieved was adequate to reach all potential target structures of the respiratory tract.

- Particle size distribution: In the 385, 503, 631, 768 and 978 mg/m3 exposure groups 86, 83, 85, 89 and 85 % ,resp. of particles were < 3 µm.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): In the 385, 503, 631, 768 and 978 mg/m3 exposure groups MMAD was 1.5, 1.5, 1.5, 1.4 and 1.5 µm, resp. (GSD: 2.0, 2.1, 2.0, 1.9 and 2.0 resp.).

Analytical verification of test atmosphere concentrations:
Duration of exposure:
4 h
target concentrations: 400, 500, 650, 750, 1000 mg/m3
gravimetric concentrations: 385, 503, 631, 768, 978 mg/m3
No. of animals per sex per dose:
Control animals:
Details on study design:
- Duration of observation period following administration: 2 weeks
- Frequency of observations and weighing: clinical signs were examined several times on the day of exposure and at least once daily therafter; body weights were measured before exposure, on days 3 and 7, and weekly thereafter.
- Necropsy of survivors performed: yes
- Other examinations performed: rectaI temperatures were measured shortly after cessation of exposure (approx. within half an hour after the end of exposure); a battery of reflex measurements was made on the first post-exposure day.
-Necropsy flndings: If specific findings occur from the respiratory tract of surviving rats they are evaluated statistically using the pairwise Fisher test after the R x C chi-squared test.
-Body weights: Means and single standard deviations of body weights are calculated. Since in acute studies individual group means may differ prior to commencement of the first exposure, the body weight gain was statistically evaluated for each group. For these evaluations a one-way ANOVA (vide infra) is used.
-Calculation of the LC50: If calculation of a median lethal concentration (LC50) is possible, it is performed by computer (PC) according to the method of AP. Rosiello, I.M. Essigmann, and G.N. Wogan (1977) as modified by Pauluhn (1983). This method is based on the maximurn-likelihood method of C.I. Bliss (1938). If only 2 pairs of values with greater than 0% lethality and less than 100% are available then the first linear approximation is based on these values and a homogeneity test is not performed. In this case the interpolated concentration at 50% lethality is designated at approximate LC50. Additionally, the moving average interpolation according to Schaper et al. (1994) is used for calculation, if applicable.
-Analysis of variance (ANOVA): 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 as "no statistical difference between the groups". If a difference is found then a pairwise post-hoc comparison is conducted (1- and 2-sided) using the Games and Howell modification of the Tukey-Kramer significance test.

Results and discussion

Effect levels
Dose descriptor:
Effect level:
626 mg/m³ air
Based on:
test mat.
95% CL:
568 - 691
Exp. duration:
4 h
Remarks on result:
other: NOAEL: < 385 mg/m3 air
Exposure to concentrations up to 503 mg/m3 were tolerated without mortality whereas concentrations equal to or exceeding 631 mg/m3 resulted in mortality occurring within the first three post-exposure days. There was no evidence of any delayed onset of mortality.
Clinical signs:
other: In surviving rats clinical signs appeared to be largely related to respiratory effects and subsided almost entirely within the first post-exposure week. The following signs were observed: Dose groups 385-631 mg/m3: Piloerection, tachypnea, labored breat
Body weight:
Comparisons between control animals with those of the groups exposed to the test substance revealed transient, statistically significant effect on body weights at 385 mg/m3 and above.
Gross pathology:
Necropsy findings were unremarkable in surviving rats whilst in rats that succumbed evidence of lung edema existed.

- Animals sacrificed at the end of the observation period:
In rats exposed to the test compound an increased incidence of macroscopic findings was observed at 631 mg/m3 and above.

- Animals succumbing during the observation period:
Evidence of lung edema (lung less collapsed following opening of thoracic cavity; foamy content in trachea), consolidation, red discolorations and nasal discharge, nose with red encrustations.
Other findings:
- Rectal temperature:
Statistical comparisons between the control and the exposure groups revealed significant changes in body temperature in all exposure groups (for details see Table 1 below).

- Reflex measurements:
Rats of all dose groups exposed to the test substance experienced changes in reflexes: impaired grip strength, reduced tonus, reduced righting response as well as reduced startle reflex.

Any other information on results incl. tables

Table 1: Summary of acute inhalation toxicity (4 hrs, liquid aerosol) of Stearylisocyanate 65

 Sex Gravimetric concentration (mg/m3) Toxicological results  Onset and duration of signs   Rectal temperature (°C)

 Occurrence of mortality

 male 0 0 / 0 / 5 ---


  385 0 / 5 / 5 0d - 4d 33.2 ** ---
  503 0 / 5 / 5 0d - 6d 31.9 ** ---
  631 3 / 5 / 5 0d - 7d 31.5 ** 1d

768 3 / 5 / 5 0d - 7d 31.1 ** 1d, 3d 


978 5 / 5 / 5 0d - 1d

30.1 **

female  0 0 / 0 / 5 --- 38.5 ---
  385 0 / 5 / 5 0d - 6d 32.4 ** ---
  503 0 / 5 / 5 0d - 6d  31.1 ** ---


4 / 5 / 5  0d - 10d 30.4 **  1d, 2d, 3d
  768 5 / 5 / 5 0d - 1d 30.1 **   1d, 2d
  978 5 / 5 / 5 0d - 1d  28.5 **   1d 

Toxicological results:

number of dead animals / number of animals with signs after cessation of exposure / number of animals exposed

* = p < 0.05, ** = p < 0.01

Applicant's summary and conclusion

Executive summary:

A study on the acute inhalation toxicity of Stearylisocyanate 65 according to OECD TG 403 was conducted on male and female rats, which were nose-only exposed to liquid aerosol in concentrations of 385, 503, 631, 768, 978 mg/m3 (gravimetric concentrations). The liquid aerosol was generated so that it was respirable to rats.

Exposure to concentrations up to 503 mg/m3 were tolerated without mortality whereas concentrations equal to or exceeding 631 mg/m3 resulted in mortality occurring within the first three post-exposure days. In surviving rats the following clinical signs were observed: piloerection, bradypnea, tachypnea, labored and irregular breathing patterns, nostrils with red encrustations, cyanosis, motility reduced, limp, high-legged gait, ungroomed hair-coat, tremor, altered reflexes, hypothermia, and transiently decreased body weights. Clinical signs appeared to be largely related to respiratory effects and subsided almost entirely within the first post-exposure week. Necropsy findings were unremarkable in surviving rats whilst in rats that succumbed evidence of lung edema existed. With regard to the respirability of the aerosol generated internationally recognized recommendations such as of SOT (1992) were fulfilled i.e. the MMAD was approximately 1.5 µm (GSD ~2).

In summary, the aerosolized test substance (liquid aerosol) proved to have a moderate acute inhalation toxicity to rats (LC50: 626 mg/m3). Mortality appeared to be causally related to acute lung damage and ensuing lung edema.