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Toxicological information

Acute Toxicity: inhalation

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Administrative data

acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2007 - June 2008 (study initiation date: September 20 2007, final report June 30 2008)
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

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

Test material

Constituent 1
Reference substance name:
Specific details on test material used for the study:
purity: 99.6%

Test animals

Details on test animals or test system and environmental conditions:
- Source: Harlan-Winkelmann GmbH, Borchen (Germany)
- Age at study initiation: ca. 2 month
- Weight at study initiation: males mean:189 (STD 8.8) to 201.4 (STD 4.2), females mean: 176.0 (STD 4.8) to 183.6 (4.3) g
- Housing: singly in conventional Makrolon Type IIIh cages
- Diet (e.g. ad libitum): standard fixed-formular diet (KLIBA 3883 PROVIMI KLIBA SA 4303 Kaiseraugust, Switzerland
- Water (e.g. ad libitum): tap-water
- Acclimation period: yes 5 days before exposure
- Method of randomisation in assigning animals to test and control groups: yes

- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 40-80%
- Air changes (per hr): ca. 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h/ 12 h artificial light

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Mass median aerodynamic diameter (MMAD):
>= 2.78 - <= 3.48 µm
Geometric standard deviation (GSD):
>= 2.23 - <= 2.72
Details on inhalation exposure:
- Exposure apparatus: modified BG 1-nozzle collison nebulizer (Type CN-25 MRE, BGl Inc. Waltham MA USA) was used
- Source and rate of air (airflow):
- Method of conditioning air: compressed air supplied by Boge compressor and condtioned by VIA compressed air dryer
- Method of particle size determination: BERNER TYPE AERAS cascade impactor
-Treatment of exhaust air: via cotton-wool/HEPA filters
- Temperature, humidity, pressure in air chamber: rel humidity: < 5.1 - <6.1 %, temperature: 22.6°C to 23.2 °C

Analytical verification of test atmosphere concentrations:
Duration of exposure:
4 h
target concentration: 250, 350, 550, 750 mg/m3
Nominal Conc.: 264, 461, 700, 1036 mg/m3
Gravimetric Conc.: 214, 355, 526, 730 mg/m3
No. of animals per sex per dose:
Five males and five females per dose
Control animals:
Details on study design:
- Duration of observation period following administration: 14 days: yes
- Frequency of observations and weighing:
- Necropsy of survivors performed: yes
- Clinical signs including body weight: yes
- Other examinations performed: clinical signs, body weight,organ weights, reflex masurments, rectal temperature, necropsy
analysis of variance (ANOVA), Box-test, Games and Howell modification of the Tukey-Kramer significance test

Results and discussion

Effect levelsopen allclose all
Key result
Dose descriptor:
Effect level:
387.46 mg/m³ air
Based on:
test mat.
95% CL:
>= 289.35 - <= 518.84
Key result
Dose descriptor:
Effect level:
645.57 mg/m³ air
Based on:
test mat.
95% CL:
>= 548 - <= 760.53
In males mortality occurred at 355 mg/m3 (gravimetric conc.) and higher. In females mortality was observed at the highest concentration tested (730 mg/m3) (gravimetric conc).
Clinical signs:
irregular respiration
Body weight:
Comparison between the control and the exposure groups revealed a consistent, concentration-dependent decrease in body weights.
Gross pathology:
Nose: with foamy discharge; brownish desposits; pleural cavity with yellowish clear fluid; lung: less collapsed, dark red, and marbled; trachea with white foamy content; liver, kidney and spleen with discolorations
Other findings:
Statistic comparasons between the control and the exposure groups revealed significant changes in body temperature in all exposure groups.

Applicant's summary and conclusion

Interpretation of results:
other: expert judgment with acute tox 4 H332 EU GHS 1272/2008 CLP classification
The aerolized test substance proved to have a high acute inhalation toxicity in rats. The signs observed demonstrated that the respirable aerosol of this test substance may cause marked respiratory tract irritation with mortality associated with lower respiratory tract irritation (alveolar edema).
Executive summary:

An acute inhalation study with the test substance according to OECD TG 403 was conducted on male and female rats, which were nose-only exposed to liquid aerosol in concentrations of 214, 355, 526 and 730 mg/m3 (gravimetric concentrations). The liquid aerosol was generated so that it was respirable to rats. Mortality occurred in a concentration-dependent manner at 355 mg/m3 and above in male rats while in the female rats mortality occurred at 730 mg/m3 only. The exposure caused irritant effects in the upper and lower respiratory tract which resolved in most rats within the first postexposure week. The following signs were observed: bradypnea, labored breathing patterns, irregular breathing patterns, dyspnea, piloerection, hair-coat ungroomed, motility reduced, flaccidity, nasal discharge (serous), stridor, breathing sounds, nose reddened, nose: red encrustations, muzzle: red encrustations, nostrils: red encrustations, eyelids: red encrustations, lacrimation, tremor, high-legged gait, cyanosis, emaciation, decreased body weights, altered reflexes, and hypothermia. Mortality is considered to be causally linked to acute lung edema and occurred in most cases within 1 day postexposure. Internationally recognised recommendations such as of SOT (1992) were fulfilled, in regard to the respirability of the aerosol generated, i.e. the MMAD was < 4 µm (MMAD 2.8-3.5 µm, GSD 2.2-2.7).

The aerosolized test substance (liquid aerosol) proved to have a high acute inhalation toxicity in rats with LC50 values (4 hrs) of 387 mg/m3 for males and 645 mg/m3 for females. The signs observed demonstrated that the respirable aerosol of 2,4'-MDI may cause marked respiratory tract irritation with mortality associated with lower respiratory tract irritation (alveolar edema). Such lower respiratory tract effects are dependent on a highly respirable aerosol not encountered in the workplace, which needs to be taken into account for classification.

Using the strict GHS LC50 cut-off for classification, the LC50 values obtained for the test substance would trigger a Category 2. However, classification for these substances according to GHS legal text allows for the application of scientific judgement. It must be considered that the LC50 cut-off of 500 mg/m3 (approximately 50 ppm for pMDI), is over 2,500-fold above the saturated vapor concentration for MDI.

Furthermore, 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)). In addition, the EU legislation for classification and labelling of chemicals, the 67/548/EEC Substances Directive in Article 1(d) makes it clear that the object of classification is to approximate the laws of the Member States in relation to substances dangerous to man or the environment. In Article 4 in points 1 and 2 it is clearly stated that substances shall be classified based on their intrinsic properties according to the categories of danger as detailed in Article 2(2) and that the general principles of classification shall be applied as in Annex VI. Intrinsic properties are those inherent in the substance. Due to a very low vapor pressure (<0.01 Pa) MDI substances are not inherently toxic by inhalation since the saturated vapor concentration would be orders of magnitude below toxic concentration. It is only with modification and input (in terms of heat, cooling and size screening) that MDI substances become toxic after inhalation. The European Chemical Industry Council have discussed and given guidance for these situations, and on the classification of respective aerosols. Classification of MDI as “Harmful” is consistent with this guidance.  

The acute inhalation data of pMDI and 4,4’-MDI data were considered by EU experts, and their conclusion that MDI be classified as “Harmful” and  reported in the 25th Adaptation to Technical Progress (ATP) to the Dangerous Substances Directive (67/548/EEC). This was endorsed in the 28th ATP and both MDI substances remain as “Harmful” in the 30th ATP (adopted by Member States on 16 February 2007 and published 15th September 2008). The original decision was upheld in the EU Risk Assessment of MDI (Directive 793/93/EEC, 3rd Priority List) published in 2005, noting that considering “the exposure assessment, it is reasonable to consider MDI as harmful only and to apply the risk management phrase ‘harmful by inhalation’. This classification was also endorsed by the Scientific Committee on Toxicity, Ecotoxicity and the Environment (CSTEE, now SCHER) in giving their opinion on the Risk Assessment (EC, 2008). With the enforcement of the CLP regulation (Regulation (EC) No 1272/2008) in 2009, the Dangerous Substance/Preparation Directive (DSD) was repealed and harmonized classifications were formally transferred to the CLP regulation. 2,4'-MDI is classified with Acute Tox. 4 H332 (Annex VI Regulation (EC) No 1272/2008 (CLP regulation).