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Diss Factsheets

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
Justification for type of information:
Hypothesis: Acute inhalation toxicity is initiated by the reaction of the MDI substance with protective nucleophiles (primarily GSH) in the airway lining fluid (adduct formation) via bioaccessible NCO. Subsequent development of toxic effects is driven by the rate of depletion of GSH. The rate of nucleophile depletion by MDI-based substances is driven by the availability of the NCO-group, which itself is a function of (1) the NCO value of the substance and (2) the molecular weight of its constituents (driving bioaccessibility). The substances with the highest available NCO value and bioaccessibility (mMDI and three-ring oligomers) are the most toxic, while those with increasing amounts constituents less able to react with GHS demonstrate reduced toxicity. The mechanism for lethality is also consistent with respiratory irritation and all substances will be classified accordingly.
Justification: This is supported with high confidence by reliable acute inhalation data available for multiple MDI isomers and modified MDI substances across the entire category. The toxicity of the higher molecular weight MDI constituents decreases with increasing molecular weight as these molecules are less soluble and have a lower NCO value. For MDI substances that contain these constituents, higher exposure concentration is required to induce toxic effects, which is consistent with the observed results. Further testing on the substances with higher average molecular weight (and subsequent lower solubility and lower NCO value) will not provide additional information on potential toxicity compared to the mMDI and pMDI since these represent worst case and therefore upper boundary substances of the MDI category.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 403 (Acute Inhalation Toxicity)
according to guideline
EU Method B.2 (Acute Toxicity (Inhalation))
according to guideline
other: OECD Guidance Document 39 (Acute Inhalation Toxicity Testing)
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:

Test material

Constituent 1
Reference substance name:
PIR modified MDI
Cas Number:
PIR modified MDI

Test animals

Details on test animals or test system and environmental conditions:
- Source: Harlan-Nederland, AD Horst, Netherlands
- Strain: HsdRCCHan: (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 IIIH cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

- Temperature (°C): 22 +/- 3
- Humidity (%): 40-60
- 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 restrainers applying a directed-flow nose-only exposure principle.
- Aerosol generation: Under dynamic conditions the test substance is atomized into the baffle (pre-separator) of the inhalation chamber. For atomization a binary nozzle and conditioned compressed air (15 L/min) was used. The representative dispersion pressure was approximately 600 kPa. The neat test article was fed into the nozzle system using a digitally controlled pump (Harvard PHD 2000 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 fine particles likely to evaporate and to prevent larger particles from entering the chamber a glass-preseparator/ baffle system was used (Tillery et al., 1976).
- 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 equilibrium 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 chamber equilibrium is attained within the first minute of exposure. 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.
- Exhaust air treatment: The exhaust air was purified via cotton-wool/HEPA filters. These filters were disposed of by Bayer Pharma AG.
- Temperature and humidity measurements: Temperature and humidity measurements are also performed by the computerized Data Acquisition and Control System using HC-S3 sensors (Rotronic, The position of the probe was at the exposure location of rats. Temperature and humidity data are integrated for 30 seconds and displayed accordingly. The humidity sensors are calibrated using saturated salt solutions according to Greenspan (1977) and Pauluhn (1994) in a two-point calibration at 33% (MgCI2) and at 75% (NaCI) relative humidity. The calibration of the temperature sensors is also checked at two temperatures using reference thermometers.

- Nominal concentration - atomization: The nominal concentration was calculated from the ratio of the total quantity of test item consumed (nominally nebulized volume - mass of test article retained in the pre-separator) and the total throughput of air through the inhalation chamber. For calculation of the totally consumed content of test article a specific density of 1.24 g x cm-3 was used.
- Nominal concentration - nebulization: The nominal concentration was calculated from the ratio of the quantity of test item nebulized, i.e. the weight loss of nebulizer before and after each use, and the total throughput of air through the inhalation chamber.
- Total mass concentration: The test-substance concentration was determined by gravimetric analysis (filter: glass-fiber filter, Sartorius, Göttingen, Germany; digital balance). This method was used to define the actual concentration.
- Samples taken from breathing zone: yes
- Particle size distribution: The particle-size distribution was analyzed using a BERNER critical orifice cascade impactor. Each impactor stage was covered by an aluminum foil. Gravimetric analyses of filters used a digital balance under similar conditions as used for gravimetric analysis.
- Respirability: The particle-size distribution achieved was adequate to reach all potential target structures of the respiratory tract.

- Particle size distribution: In the 542, 1232 and 2147 mg/m3 exposure groups 70, 57 and 71 % ,resp. of particles were < 3 µm.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): In the 542, 1232 and 2147 mg/m3 exposure groups MMAD was 2.4, 2.7 and 2.2 µm, resp. (GSD: 1.6, 2.0 and 1.7 resp.).

Analytical verification of test atmosphere concentrations:
Duration of exposure:
4 h
target concentration: 500, 1200, 2000 mg/m3
gravimetric concentration: 542, 1232, 2147 mg/m3
No. of animals per sex per dose:
5 animals per sex (1200 and 2000 mg/m3, control groups)
3 animals per sex (500 mg/m3)
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 1, 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.
-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:
1 088 mg/m³ air
Based on:
test mat.
95% CL:
832 - 1 423
Exp. duration:
4 h
Remarks on result:
other: NOAEL: < 542 mg/m3
Mortality occurred at 1232 and 2147 mg/m3 in both sexes within 2 days post-exposure (for details see Table 1 below).
Clinical signs:
other: Controls (males and females): All rats tolerated the exposure without specific signs. Exposure groups (males): 542 mg/m3: bradypnea, dyspnea, labored breathing patterns, breathing sounds, motility reduced, atony, high-legged gait, piloerection, cyanosis,
Body weight:
Comparisons between the control and the exposure group 542 mg/m3 revealed transient changes in body weights of males and females while a more persistent reduction in body weights occurred in exposure group 1232 mg/m3 in both sexes.
Gross pathology:
Qualitative description of gross pathological key-findings:

- Animals succumbing during the observation period: The most salient findings are characterized by firm deposits within the nostrils, less collapsed lungs, acute lung edema and pleural effusions (hydrothorax).

- Animals sacrificed at the end of the observation period: The macroscopic findings of extrapulmonary organs were essentially indistinguishable amongst the groups. In surviving rats an increased incidence of focal pulmonary discolorations was noticed.
Other findings:
- Rectal temperature:
Statistical comparisons between the control and the exposure group revealed a concentration-dependent, statistically significant change in body temperature (for details see Table 1 below).

- Reflex measurements:
In comparison to the animals of the control groups, male and female rats of the exposure groups 542 and 1232 mg/m3 exhibited changes in reflexes (e.g. reduced tonus).

Any other information on results incl. tables

Table 1: Summary of acute inhalation toxicity (4 hrs, liquid aerosol) of MDI PIR

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

 Onset and duration of mortality

 male 0 0 / 0 / 5 ---


  542 0 / 3 / 3 0d - 14d 34.6  ---
  1232 4 / 5 / 5 0d - 11d 31.6 **  1d - 2d
  2147 5 / 2 / 5 0d 29.1 *   0d - 1d


0 0 / 0 / 5 ---


  542 0 / 3 / 3 0d - 5d 35.8 ** ---
  1232 2 / 5 / 5 0d - 14d 32.0 **  1d - 2d
  2147 5 / 2 / 5 0d  31.4 *  0d - 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

Interpretation of results:
Category 4 based on GHS criteria
Note: The aerosols were generated using sophisticated techniques in the laboratory, whereby extremely small particles are generated in order to meet international guidelines for testing. This size and concentration of aerosol is not generated in the workplace even under foreseeable worst-case conditions (Ehnes et al., 2019). The particle size distribution of aerosols formed during actual spraying applications has virtually no overlap with that of the highly respirable aerosol generated in inhalation studies (see EC (2005)).
Executive summary:

An acute inhalation study with MDI PIR according to OECD TG 403 was conducted on male and female rats, which were nose-only exposed to liquid aerosol in concentrations of 542, 1232 and 2147 mg/m3 (gravimetric concentrations). The aerosol was generated neat without any vehicle. The following clinical signs were observed: bradypnea, dyspnea, labored breathing patterns, breathing sounds, irregular breathing patterns, motility reduced, atony, highlegged gait, hair-coat ungroomed, piloerection, cyanosis, emaciation, nasal discharge (serous), nose reddened, nose with red encrustations, stridor, eyelids with red encrustations, tremor, nostrils: white deposits, hypothermia, decreased reflexes, and transient decrease in body weights. The lead pathodiagnostic effects were suggestive of portal of entry-related adverse effects (irregular and labored breathing patterns which lasted up to the end of the postexposure period in some rats). Mortality occurred at 1232 and 2147 mg/m3 up to day 2 post-exposure and was causally related to an acute pulmonary edema.

The respirability of the aerosol was adequate to achieve the objective of study, i.e. the average mass median aerodynamic diameter (MMAD) was 2.2-2.7 µm, the average geometric standard deviation (GSD) was 1.6-2.0.

In summary, the aerosolized test substance (liquid aerosol) proved to have a moderate acute inhalation toxicity in rats (LC50: 1088 mg/m3).