Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 20 2007 (Study plan) to June 25 2008 (Final Report)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
yes
Remarks:
one control group and 4 exposure groups, an additional exposure group was included with modified generation principle to decrease particle size (group 3 target concentration 550 mg/m3)
GLP compliance:
yes
Test type:
traditional method
Limit test:
no

Test material

Constituent 1
Reference substance name:
2,2'-methylenediphenyldiisocyanate
IUPAC Name:
2,2'-methylenediphenyldiisocyanate
Constituent 2
Chemical structure
Reference substance name:
2,2'-methylenediphenyl diisocyanate
EC Number:
219-799-4
EC Name:
2,2'-methylenediphenyl diisocyanate
Cas Number:
2536-05-2
Molecular formula:
C15H10N2O2
IUPAC Name:
1,1'-methylenebis(2-isocyanatobenzene)
Constituent 3
Reference substance name:
2,2’ - MDI
IUPAC Name:
2,2’ - MDI
Constituent 4
Reference substance name:
MDI 22
IUPAC Name:
MDI 22
Details on test material:
Purity approx. 100%
MW: 250.3 g/mole
Specific details on test material used for the study:
Purity: 98.8%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann gmbH, Borchen (Germany)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: ca. 2 months
- Weight at study initiation: males: mean: 195- 205 g, females mean: 177- 185 g
- Housing: conventional Makrolon Type III h cages
- Diet (e.g. ad libitum): Standard fixed-formular diet (KLIBA 3883) PROVIMI KLIBA SA
- Water (e.g. ad libitum): tap water
- Acclimation period: 5 day before exposure
- Method of randomisation in assigning animals to test and control groups : yes

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

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 1.85 - <= 3.55 µm
Geometric standard deviation (GSD):
>= 1.87 - 2.29
Remark on MMAD/GSD:
An additional exposure group (group 3) was included with modified generation principle to decrease particle size. The finer aerosol at 546 mg/m3 had an MMAD/GSD 1.85 µm/1.87.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: modified BGI 1-nozzle collison nebulizer (Type CN-25 MRE, BGI Inc., Waltham MA USA)
- Method of holding animals in test chamber: plexiglas exposure tubes with direct-flow nose-only exposure
Inhalation Chamber: aluminum inhalation chamber internal volume= ca. 3.8 L
- Source and rate of air (airflow): compressed air, at eeach exposure port a minmal aor flow rate of 0.75 l/min
- Method of conditioning air: compressed air supplied by Boge compressor and conditioned automatically
- System of generating particulates/aerosols: modified BGI 1-nozzle collison nebulizer
- Method of particle size determination: BERNER-TYPE AERAS low-pressure critical orifice cascade impactor
- Treatment of exhaust air: exhausted air was purified via cotton-wool/HEPA filters
- Temperature, humidity: 22.9-23°C, <5.4-<9.6%

TEST ATMOSPHERE
- Brief description of analytical method and equipment used: nominal concentration calculated from ratio of quantity of test substance nebulized, gravimetric analysis with Glass-Fiber-Filter
- Samples taken from breathing zone: yes
Samples were collected after the equilibrium concentration had been attained in hourly intervals

VEHICLE
- Composition of vehicle (if applicable): no vehicle used, the test article was aerosolized neat as liquid aerosol
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
400, 550*,550, 750 mg/m3(target Conc.), 586.1, 750.0*, 794.4, 1183.3 mg/m3 (nominal Conc.), 431.3, 546.3*, 570.0, 825.0 mg/m3 (gravimetric Conc.)
*modified generation principle to decrease particle size
No. of animals per sex per dose:
five males and five females per dose
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 14 days
- Necropsy of survivors performed: yes
- Clinical signs including body weight : yes
- Other examinations performed: clinical signs, body weight,organ weights, histopathology, other: yes
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 (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 levelsopen allclose all
Key result
Sex:
female
Dose descriptor:
LC50
Effect level:
685.75 mg/m³ air
Based on:
test mat.
Exp. duration:
4 h
Key result
Sex:
male
Dose descriptor:
LC50
Effect level:
527.2 mg/m³ air
Based on:
test mat.
95% CL:
>= 445.2 - 624.3
Exp. duration:
4 h
Mortality:
Mortality occurred in male rats at 431 mg/m3 in a concentration-dependent manner while in female rats mortality occurred at 825 mg/m3 only and under conditions where the aerosol was in the range ≤ 3 µm. For either sex the more respirable aerosol (group 3) produced a distinctly higher mortality. Apart from group 3, where a modified aerosolization procedure was used to obtain finer particles, the particle size distribution was not essentially different between groups. The onset of mortality was typical for respirable aerosols penetrating the lower respiratory tract with subsequent acute lung edema.
Clinical signs:
irregular respiration
Remarks:
The exposure caused irritant effects in the upper and lower respiratory tract which resolved in most rats within the first post-exposure week.
Body weight:
Comparisons between the control and the exposure groups revealed a consistent, concentration-dependent decrease in body weights.
Gross pathology:
Animal sacrificed at the end of the observation period: The macroscopic findings were essentially indistinguishable amongst exposure and control groups.
Animal succumbing during the observation period: Nose: white foamy discharge; pleural cavity with yellowish clear fluid; lung: less collapsed, dark-red, and marbled; thrachea with white foamy content; kidney and spleen with discoloration
Other findings:
Rectal temperature: Statistical comparisons between the control and the exposure groups revealed significant changes in body temperature in all exposure groups.
Reflex measurments: a battery of reflex measurments was made on the first post-exposure day. In compaarision to the rats of the control group, rats of all exposure groups exhibited concentration-dependent changes in reflexes.

Any other information on results incl. tables

Table 1: Generation and characterization of chamber atmosphere (aerosolization of the liquid test article) -Mean values













































































 Group1Group 2Group 3Group 4Group 5
Target Conc (mg/m3)0400550*550750
Nominal Conc. (mg/m3)Control Air586.1750.0794.41183.3
Gravimetric Conc. (mg/m3)---431.3546.3570.0825.0
Recovery (%)---74727270
MMAD (µm)---2.911.853.552.73
GSD---2.221.872.291.94
Aerol Mass < 3 µm---51.878.141.956.6
Mass recovered (mg/m3)---460.4570.5597.4970.02

MMAD= Mass Median Aerodyynamic Diameter, GSD = Geometric Standard Deviation, *: Modified generation principle to decrease particle size


 


Table 2: Summary of acute inhalation toxicity (4 hrs, liquid aerosol) of 2,2'-MDI


 






























































































 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



---



38.1



 ---



 



431



1 / 5 / 5



0d - 6d



31.3 **



 1d



 



546



5 / 4 / 5



0d



29.2 **



   0d, 1d



 



570



3 / 5 / 5



0d - 4d



28.7 **



  1d



 



825



5 / 2 / 5



0d



26.9 **



   0d, 1d 



female 



0



0 / 0 / 5



--- 



38.2



   ---



 



431



0 / 5 / 5



0d - 6d



35.3 **



  ---



 



546



3 / 5 / 5



0d - 8d



30.7 **



 1d



 



570



0 / 5 / 5



0d - 4d 



33.4 **



  ---



 



825



5 / 4 / 5



0d



28.8 **



 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:
other: expert judgment with acute tox 4 H332 EU GHS 1272/2008 CLP classification
Conclusions:
In summary, the aerosolized test substance (liquid aerosol) proved to have a high acute inhalation toxicity in rats with a LC50 (4 hrs) of 527 mg/m3 for males and an approximate LC50 (4 hrs) of 686 mg/m3 for females. The signs observed demonstrated that the respirable aerosol of the test substance may cause marked respiratory tract irritation with mortality associated with lower respiratory tract irritation (alveolar edema).
Executive summary:

An acute inhalation toxicity study withthe 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 431, 546, 570 and 825 mg/m3 (gravimetric concentrations) (Pauluhn, 2008). The liquid aerosol was generated so that it was respirable to rats. Mortality occurred in male rats at 431 mg/m3 in a concentration-dependent manner while in female rats mortality occurred at 825 mg/m3 only and under conditions where the aerosol was in the range of ≤ 3 µm. For either sex the more respirable aerosol (546 mg/m3) produced a distinctly higher mortality. Apart from this group, where a modified aerosolization procedure was used to obtain finer particles, the particle size distribution was essentially identical between exposure groups. This group was not considered in the LC50-calculation. The onset of mortality was typical for respirable aerosols penetrating the lower respiratory tract with subsequent acute lung edema. 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, dyspnea, irregular breathing patterns, breathing sounds, motility reduced, flaccidity, tremor, high-legged gait, nose: reddened, nose: red encrustations, muzzle: red encrustations, piloerection, hair-coat ungroomed, nasal discharge (serous), stridor, nostrils: red encrustations, eyelids: red encrustations, periorbicular encrustations, cyanosis, lacrimation, bloated abdomen, decreased body weights, altered reflexes, and hypothermia. Mortality is considered to be causally linked to acute lung edema and occurred 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 2.7-3.6 µm, GSD 1.9-2.2). In the group exposed to the finer aerosol at 546mg/m3the MMAD/ GSD was 1.9µm / 1.9.


Using the strict GHS LC50 cut-off for classification, the LC50 values obtained for the test substance would trigger a Category 3. 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; 2,2'-MDI is classified with Acute Tox. 4 H332 (Annex VI Regulation (EC) No 1272/2008 (CLP regulation).