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Acute Toxicity: inhalation

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Administrative data

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

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995
Reference Type:
publication
Title:
Acute inhalation studies with irritant aerosols: technical issues and relevance for risk characterization
Author:
Pauluhn J
Year:
2004
Bibliographic source:
Arch. Toxicol. 78: 243-251

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Desmodur 15
- Physical state: solid
- Purity: > 99.9 %
- Lot/batch No.: 5054 (BS 9507)
- Storage condition of test material: room temperature, darkness

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann, Borchen, Germany
- Age at study initiation: 2-3 months
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- 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
Vehicle:
other: conditioned air
Details on inhalation exposure:
EXPOSURE CONDITIONS:
- Mode of exposure: Animals were exposed to the aerosolized test substance in Plexiglas exposure tubes applying a directed-flow nose-only exposure principle (Moss and Asgharian, 1994). Tubes were chosen that accommodated the animals size. These tubes were designed so that the rat's tail remained outside the tube, thus restrained-induced hyperthermia can be avoided.
- Vehicle: The test substance was aerosolized as dust without a carrier or vehicle.

AEROSOL GENERATION AND EXPOSURE TECHNIQUE:
- Aerosol generation: Under dynamic conditions the test substance was fed into the intake of the cylindrical inhalation chamber.
- Description of apparatus: For powder dispersion, conditioned compressed air (28 liters of air/min; continuous operation) was used. The principle performance of the dust generating system can be described as follows: The test substance was entrained into a glass reservoir (approximately 0.5 kg). From this reservoir it was fed (by suction) into the orifice of a venturi tube. The airborne powder was then dispersed into the inner cylinder of the inhalation chamber. Reproducible and temporally stable dosing into the orifice of the venturi tube was achieved by an oscillating orifice. The orifice size was adjusted manually in order to obtain the targeted flow of powder. Stirring of the reservoir was performed using a minimum number of revolutions per unit of time. Thus the flowability of the test compound was maintained without inducing a vertical inhomogeneity of active ingredient particles.
- Inhalation chamber: The aluminum 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).
-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 (> 400 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 volumelflow rate; McFarland, 1976). The ratio between the air supplied and exhausted was chosen so that approximately 90% of the supplied air is removed as exhaust. The remainder provides adequate dead-space ventilation for the exposure tubes. At each exposure port a minimal air flow rate of 1.4 l/min was provided. This is equivalent to approximately 4 respiratory minute volumes of the rats. The test atmosphere can
by no means be diluted by bias-air-flows. The inhalation chamber was operated in a well ventilated chemical fume hood.
- Air flows: During the exposure period air flows were monitored continuously and, if necessary, readjusted to the conditions required. Air flows were measured with calibrated flowmeters and/or soap bubble meter (Gilibrator, Ströhlein Instruments, Kaarst) and were checked for correct performance at regular intervals.
- Treatment of exhaust air: The exhaust air was purified via cotton-wool/activated charcoal and HEPA filters. These filters were disposed of by Bayer AG.

ANALYSIS OF THE TEST ATMOSPHERE:
- General remark: A nominal concentration was not calculated since the construction and weight of the dust generator used did not allow for a precise measurement of the powder aerosolized.
- Gravimetric evaluation: The test-substanee concentration was determined by gravimetric analysis (filter: Cellulose-Acetate-Filter, Sartorius, Göttingen, Germany; balance: Mettler AE 100). Chamber samples were taken in the vicinity of the breathing zone. The number of sampIes taken was sufficient to characterize the test atmosphere and was adjusted so as to accommodate the sampling duration and/or the need to confirm specific concentration values. Optimally, samples were collected in hourly intervals. All analytical concentrations reported refer to mg of test substance/m3 air (gravimetrical method).

CHARACTERIZATION OF AERODYNAMIC PARTICLE-SIZE DISTRIBUTATION:
- General remark: The sampIes for the analysis of the particle-size distribution were also taken in the vicinity of the breathing zone. The particle-size distribution was analyzed using an BERNER-TYPE Aeras easeade impactor. The individual impactor stages were covered by aluminum foil and had been subjected to gravimetric analysis. An adhesive stage coating (silicone spray) was used to prevent particle bounce and re-entrainment, respectively. The aluminum foil used was evaluated gravimetrically using a Sartorius M3P balance.
- Evaluation of particle-size distributions: For the evaluation of the cascade impactor analyses the mass median aerodynamic diameter (MMAD) and the geometrie standard deviation (GSD) are determined from the probit-transformed cumulative particle mass frequency distribution (y-axis) and the logarithmic effective cut-off diameters (ECD's) (x-axis) of the individual impactor stages by linear regression. The GSD is calculated from the regression line: percentile 84/percentile 50. The relative mass with an aerodynamic diameter <= 3 µm ("respirable mass fraction") [Raabe, 1982; Snipes, 1989; SOT-Commentary, 1992] is calculated from the regression line. For probit transformation and linear regression FORTRAN algorithms published by Rosiello et al. (1977) are used. The MMAD was calculated using published formulas [Marple and Rubow, 1980; Pauluhn, 1994].

RESULTS OF PARTICLE-SIZE ANALYSES:
- Particle size distribution: In the 96, 189, 238, 314, 384 and 541 mg/m3 exposure groups 49, 46, 36, 31, 27 and 47 % ,resp. of particles were < 3 µm.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): In the 96, 189, 238, 314, 384 and 541 mg/m3 exposure groups MMAD was 3.1, 3.2, 4.0, 3.6, 3.8 and 3.1 µm, resp. (GSD: 1.6, 1.7, 2.1, 1.5, 1.5 and 1.6 resp.).
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
0, 96, 189, 238, 314, 384 and 541 mg/m³ (gravimetric)
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
ADMINISTRATION:
- Type of exposure: dynamic conditions, nose only
- Particle size: MMAD 3-4 µm, GSD approx. 1.6
- Observation period: 4 weeks

EXAMINATIONS:
- Body weights were recorded immediately prior to exposure, on days 3, 7 and  weekly thereafter.
- Clinical observations were made several times on the day of dosing and at least once a day during the 4 week observation period. Additionally examinations in a Funcional Observational Battery (FOB) were performed.
- Gross pathological examination was carried out on all animals.
Statistics:
-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 (HP 3000) 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. The interpolated concentration at 50% lethality in this case was designated at approximate LC50.
-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
Sex:
male/female
Dose descriptor:
LC50
Effect level:
270 mg/m³ air
95% CL:
>= 217 - <= 337
Exp. duration:
4 h
Remarks on result:
other: NOEL: < 96 mg/m³ air
Mortality:
Aerosol (dust) concentrations to 238 mg/m3 and above induced test substance related mortality within the first two post-exposure days.  Exposure to concentrations equal or less than 189 mg/m3 test compound  were tolerated without mortality (details see table 1).
Clinical signs:
All animals exposed to the test compound showed bradypnoea, laboured breathing pattern, nose/snout area with red encrustations, reduced motility, flaccid appearance, ungroomed hair-coat and piloerection starting at 96 mg/m3. In addition, rales, salivation, serous discharge from nose, cyanosis and apathy was seen at 189 mg/m3 and above (see also table 1 for onset and duration of signs).
Body weight:
Decreased body weights were observed in all groups exposed to the test compound (at 96 mg/m3 and above).
Gross pathology:
White foamy discharge from snout, red encrustation in the muzzle area, lungs with dark-red colourations and spongy (oedematous) appearance, foam in trachea, distended hydrothorax, lobulation of liver, and pale parenchymatous organs were observed in animals sacrificed during the observation period.

In rats sacrificed at the end of the observation period an increased incidence of macroscopical findings was observed on lungs. However, the findings appeared not to induced in a clear concentration-dependent manner.
Other findings:
All animals showed normal reflexes. At 96 mg/m3 and above a concentration-dependent decrease of body temperature was recorded

Any other information on results incl. tables

Table 1: Acute inhalation toxicity (aerosol) of NDI

 Sex Gravimetric concentration (mg/m3) Toxicological results  Onset and duration of signs  Onset of mortality
 male 0 0 / 0 / 5  --- --- 
  96 0 / 5 / 5 4h - 8d --- 
  189 0 / 5 / 5 4h - 7d  --- 
  238 3 / 5 / 5  4h - 7d 1d
  314 3 / 5 / 5 4h - 7d  1d - 2d
  384 4 / 5 / 5 4h - 11d  1d 
  541 4 / 5 / 5 4h - 11d  1d 
 female 0 / 0 / 5   --- --- 
  96 0 / 5 / 5  4h - 8d  --- 
  189 0 / 5 / 5 4h - 7d  --- 
  238  3 / 5 / 5 4h - 7d 1d - 2d 
  314  3 / 5 / 5 4h - 7d 1d - 2d
  384 4 / 5 / 5 4h - 6d 1d
  541 4 / 5 / 5 4h - 6d  1d

Toxicological results:

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

Applicant's summary and conclusion

Executive summary:

Aerosol (dust) concentrations to 238 mg/m3 and above induced test substance related mortality within the first two post-exposure days. Exposure to concentrations equal or less than 189 mg/m3 test compound were tolerated without mortality. Necropsy findings support the conclusion that a causal relationship between lethality and lung damage existed. Exposure to concentrations of 96 mg/m3 and higher were followed by concentration-dependent signs suggestive of irritation of the respiratory tract (e.g. bradypnoea, dyspnoea, laboured breathing pattern, rales, nose/snout area with red encrustations, serous discharge from nose, cyanosis, hypothermia) and non-specific signs such as reduced motility, body weight gain, emaciation, and flaccid muscle tone. The duration of signs (maximum duration up to day 11) was dependent on respiratory signs. According to OECD guideline 403 the LC50 (aerosol, 4 hrs) was 270 mg/m3 air for male and female rats.

The aerosolized test substance (dust) was of adequate respirability. Accordingly, mortality appeared to be related to damage of the respiratory tract in general and the lower tract in particular.