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Toxicological information

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: Study conducted in line with OECD Guideline 403. The study conditions were adjusted so as to fulfill both the Directive 92/69/EEC, OPPTS (1998), and Japan MAFF, Notification N° 12 Noussan-8147 (2000) Guidelines.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
yes
Remarks:
Humidity values lower than what is suggested in the test guidelines to prevent disintegration of the reactive diisocyanate moiety. This deviation had no apparent negative impact on the outcome of the study.
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-methylenediphenyl diisocyanate
EC Number:
202-966-0
EC Name:
4,4'-methylenediphenyl diisocyanate
Cas Number:
101-68-8
Molecular formula:
C15H10N2O2
IUPAC Name:
1-isocyanato-4-[(4-isocyanatophenyl)methyl]benzene
Constituent 2
Reference substance name:
Diphenylmethane-4,4'-diisocyanate (MDI-monomer)
IUPAC Name:
Diphenylmethane-4,4'-diisocyanate (MDI-monomer)
Test material form:
other: semi-solid of molten substance
Details on test material:
- Name of test material (as cited in study report): diphenylmethane, 4,4'-diisocyanate (4,4'-MDI) sourced from Bayer Material Science, Leverkusen,
Germany
- Molecular formula (if other than submission substance): C15H10N202
- Molecular weight (if other than submission substance): 250 g/mol
- Substance type: MDI-monomer
- Physical state: semi-solid of molten substance
- Analytical purity: 98.4% 4,4'-MDI; 1.6% 2,4'-MDI, < 0.01% 2-2'MDI and < 0.01% by-products (Reference is made to page 145 of the report for the
complete material balance)
- Lot/batch No.: P4DB001926 (Tox Id: 10064b)
- Expiration date of the lot/batch: 21-09-2007
- Stability under test conditions: stability certified for the duration of study
- Storage condition of test material: freezer (approximately 23°C)/ darkness; room temperature during the course of the study
- Handling: complete exclusion of air/humidity (head space of containers with MDI purged with nitrogen)

Test animals

Species:
rat
Strain:
other: Wistar rats, strain Hsd Cpb:WU (SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen (Germany)
- Age at study initiation: young adults - 2 months old
- Weight at study initiation: At the study start the variation of indvidual weigts did not exceed appr. 10% of the mean for each sex. Reference is made
to page 71 of the study report.
- Housing: During the acclimatization and study periods the animals were housed individually in conventional Makrolon® Type IIIH cages. Cages were
changed twice a week while unconsumed feed and water bottles were changed once per week. The legal requirements for housing experimental
animals (Directive 86/609 EEC) were followed. Bedding consisted of low-dust wood granulate from Lignocel BK 8-15 (Rettenmaier).
- Diet (e.g. ad libitum): standard fixed-formula diet (KLIBA 3883) provided ad libitum
- Water (e.g. ad libitum): drinking-quality municipality tap-water provided ad libitum in polycarbonate bottles containing appr. 300 ml
- Acclimation period: The animals were acclimatized to the animal room conditions for at least five days before use.
During this period rats were also acclimatized to the retaining tubes.
- Historical data on the physiology, diseases and spontaneous alterations of the rats are available at the test facility. The state of health of the strain is
regularly, randomly checked at the instance of the Laboratory Animal Services, Bayer Health Care AG, for the most important specific infectious
pathogens.
- Only health rats free of signs were used for this study. The animals were not vaccinated or treated with anti-infective agants either before their
arrival or during the acclimatization or study periods. The females were nulliparous and not pregnant. The rats were randomly assigned to the test
groups and identified by both individual color-marking and cage labels.
- The animal room was regularly cleaned and disinfected once a week. Contamination of the feed and contact with the test item were excluded.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C, continously monitored by means of a calibrated thermohygrograph
- Humidity (%): 40-80%, continously monitored by means of a calibrated thermohygrograph
- Air changes (per hr): approximatey 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h/12h; artificial light from 6.00 a.m. to 6.00 p.m. Central European time; light intensity 14 Watt/m2 floor area

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Remarks:
directed-flow
Vehicle:
other: Test article was aerosolized neat as liquid aerosol; no vehicle was thus used
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: aluminium inhalation chamber
- Exposure chamber volume: 3.8 L (internal volume)
- Method of holding animals in test chamber: Tubes accomodating the animals size were used.
- Source and rate of air: total air flow of 15 L/min through the inhalation chamber; compressed air supplied by Boge compressors
- Method of conditioning air: compressed air suppied by Boge compressors was conditioned (freed from water, dust, and oil) automatically by a VIA
compressed air dryer.
- System of generating particulates/aerosols: modified BGI 1-nozzle collision nebulizer (type CN-25 MRE, BGI Inc.; Waltham MA, USA, modified)
- nebulizer maintained at 60°C by means of a digitally controlled thermostat.
- dispersion pressure of approximately 45 kPa
- nebulization by primary flow of pure nitrogen through the collision nebulizer followed by dilution by conditioned, pressurized air.
- re-conditioning of athmosphere by means of a cooling chimney.
- additional dilution of air prior to entrainment in the inhalation chamber
- Method of particle size determination: analysed using a BERNER-TYPE AERAS low-pressure critical orifice cascade impactor.
- Treatment of exhaust air: purified via cotton-wool/HEPA filters
- Temperature, humidity in air chamber:
- temperature and humidity measured by compurterized system (Hydra, Fluke-Philips) at 5 min time intervals and measured at the
exposure locations. Temperature values in the range of what is suggested in the test guidelines. humidity values were lower than what
is suggested in the test guidelines to prevent degradation of the reactive diisocyanate moiety. No impact on the outcome of the study.


TEST ATMOSPHERE
- Brief description of analytical method used:
The nominal concentration was calculated from the ratio of the quantity of test substance nebulized (weight loss of nebulizer before and after each
use), and the total throughput of air in the inhalation chamber. The lower analytical concentrations compared with the nominal concentrations are
attributed to the condensation of MDI onto surfaces of the tubing system to the inhalation chamber.
Total mass concentration: determined by means of gravimetric analysis.
- Samples taken from breathing zone: yes - The number of samples taken was sufficient to characterize the test atmosphere and was adjusted to
accomodate the sampling duration and/or the need to confirm specific concentration values. Optimally samples were collected after the equilibrium
concentration had been attained in hourly intervals. Oxygen measurement sin the breathing zone of the rats were conducted.

VEHICLE
- No vehicle was used as the test article was aerosolized neat as liquid aerosol.

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: Reference is made to page 38 till page 57 of the study report.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): Reference is made to page 38 till page 57 of the study report.
- stability and integrity of the aerosol system and exposure system was measured by using a RAS-2 real-time aerosol photometer.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
ca. 4 h
Concentrations:
Rats were exposed to liquid aerosol in concentrations of 300.0, 354.2, 399.2, 500.0 and 553.8 mg/m3
No. of animals per sex per dose:
Five male & five female rats were simultaneously exposed to each concentration under nose-only conditions for 4 hours.
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 2 weeks post-exposure period
- Frequency of observations and weighing:
- body weights measured before exposure, on days 1, 3 and 7 and weekly thereafter. Individual weights were also recorder at death.
- appearance and behaviour of each rat were examined carefully several times on the day of exposure and at least once daily thereafter.
Weekend assessments were made once a day (morning). Assessments from restraining tubes were made only if unequivocal signs occurred
(e.g. spasms, abnormal movements, and severe respiratory signs).
- rectal temperatures measured shortly after cessation of exposure (within 30 min after the end of the exposure).
- Necropsy of survivors performed: yes
- Other examinations performed:
- Clinical signs: Cage-side observations included, but were not limited to, changes in the skin and fur, eyes, mucus menbranes, respiratory,
circulatory, autonomic and central nervous system, and somatomotor activity and behaviour pattern. Particular attention was directed to
observation of tremors, convulsions, salivation, diarrhea, lethargy, somnolence and prostration. The time of death is recoreded as precisely as
possible. Since these signs can only be assssed adequately from freely moving animals, no specific assessment was performed during exposure
while animals were restrained. In addition the following reflexes were tested: visual placing response and grip strenhth on wire mesh, abdominal
muscle tone, corneal and pupillary reflexes, pinnal reflex, righting reflex, tail-pinch response, startle reflex with respect to behaviour changes
stimulated by sounds (finger snapping) and touch (back)
-body weights, rectal temperatures were collected as well
Statistics:
Necropsy findings: pair-wise Fisher test after R x C chi-squared test
Body weights: one-way ANOCA for body weight gain
Physiological data: ANOVA
LC50: Rosiello et al. method (1977) as modified by Pauluhn (1983) or if applicable moving-average interpolation.

Results and discussion

Effect levelsopen allclose all
Sex:
male
Dose descriptor:
LC50
Effect level:
367.95 mg/m³ air
Based on:
other: aerosoled test material
95% CL:
> 295.71 - < 457.84
Exp. duration:
4 h
Sex:
male
Dose descriptor:
other: LC01
Effect level:
146.85 mg/m³ air
Based on:
other: aerosolized test material
Exp. duration:
4 h
Sex:
female
Dose descriptor:
LC50
Effect level:
558.98 mg/m³ air
Based on:
other: aerosoloized test material
Exp. duration:
4 h
Remarks on result:
other: Probit
Sex:
female
Dose descriptor:
other: LC01
Effect level:
146.93 mg/m³ air
Based on:
other: aerosolized test material
Exp. duration:
4 h
Sex:
male/female
Dose descriptor:
LC50
Effect level:
415.49 mg/m³ air
Based on:
other: aerosolized test material
95% CL:
> 369.85 - < 466.75
Exp. duration:
4 h
Remarks on result:
other: Moving Average Interpolation
Sex:
male/female
Dose descriptor:
LC50
Effect level:
431.18 mg/m³ air
Based on:
other: aerosolized test material
Exp. duration:
4 h
Remarks on result:
other: Probit
Sex:
male/female
Dose descriptor:
other: LC01
Effect level:
138.59 mg/m³ air
Based on:
other: aerosolized test material
Exp. duration:
4 h
Mortality:
Mortality occured in a concentration-dependent manner at 354.2 mg/m3 and above. Male rats appeared to be more susceptible than female rats.
Mortality is considered to be causally linked to acute lung edema and occurred in most cases within 1 day postexposure.
Clinical signs:
other: The exposure caused irritant effects in the upper and lower respiratory tract which resolved within the two postexposure weeks. The following signs were observed: bradypne, dyspnea, labored breathing patterns, breathing sounds, irregular breathing pattern
Body weight:
Comparisons between the control and exposure groups revealed a consistent concentration-dependent decrease in body weights.
Gross pathology:
Macroscopic findings were essentially indistinguishable amongst exposure and control groups for animals sacrificed at the end of the observation period. For animals succumbing during the observation period the following findings were noted: nose: white foamy discharge, yellowish and viscous mucous/deposits; pleural cavity with yellowish clear fluid, lung less collapsed, dark-red, and marbled; trachea with foamy content; liver, kidneys and spleen with discolorations.
Other findings:
Rectal Temperatures: statistical comparisons between control and exposure groups revealed significant changes in body temperature in all exposure groups.
Reflex measurements: In comparison to the rats of the control group, rats of all exposure groups exhibited concentrations-dependent changes in reflexes.

Any other information on results incl. tables

 N° group/sex  Target conc. (mg/m3)  Toxicological Result  Onset and duration of signs  Onset and duration of mortality  Rectal Temperature (°C)
 1/m  0  0/0/5  -  -  37.7
 2/m   300  0/5/5  0d -12d  -  32.0**
 3/m  350  3/5/5  0d-6d  0d  30.4*
 4/m  400  5/5/5  0d-2d  1d,2d  28.7**
 5/m  500  3/4/5  0d-8d  0d,1d  29.1**
 6/m  550  4/4/5  0d-5d   0d,1d  28.6**
 1/f  0  0/0/5  -  -  38.0
 2/f  300  0/5/5  0d-8d  -  33.0**
 3/f  350  2/5/5  0d-6d  1d,3d  32.4**
 4/f  400  2/5/5  0d-6d  1d  31.1*
 5/f  500  0/5/5  0d-10d  -  32.1**
 6/f  550  4/5/5  0d-5d  1d  29.9**

* p < 0.05; ** p< 0.01; 1st = number of dead animals, 2nd = number of animals with signs after cessation of exposure, 3rd = number of animals exposed

Applicant's summary and conclusion

Interpretation of results:
other: expert judgment with acute tox 4 H332 EU GHS 1272/2008 CLP classification
Conclusions:
The aerosolized test substance (liquid aerosol) proved to have a high acute inhalation toxicity in rats with an LC 50 (95% confidenc interval) of 368 (296-458) mg/m3 in the more susceptible sex. 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:

This study investigated the acute inhalation toxicity of diphenylmethane, 4,4'-diisocyanate (4,4'-MDI) on rats. The study was conducted in accordance with OECD Guideline 403. Test procedures were adapted so as to comply also with the EU Directive 92/69/EEC, OPPTS 870.1300 and Notification n° 12 Nousan-8147 guidelines. Five groups of rats were nose-only exposed to liquid aerosol in concentrations of 300.0, 354.2, 399.2, 500.0 and 553.8 mg/m3. The liquid aerosol was generated so it was respirable to rats. The results can be summarised as follows:


 


















LC50 inhalation (liquid aerosol, 4h)


 NO(A)EL
 

LC50 -males: 368 (296 -458) mg/m3*


 Males & females < 300 mg/m3 air*
 LC50- females: 559 mg/m3 

* all concentration data represent actual concentrations of the test substance in the rats' breathing zone


 


Mortality occured in a concentration-dependent manner at 354.2 mg/m3 and above. Male rats appeared to be more susceptible than female rats. The exposure caused irritant effects in the upper and lower respiratory tract which resolved within the two postexposure weeks. The following signs were observed: bradypnea, dyspnea, labored breathing patterns, breathing sounds, irregular breathing patterns, motility reduced, piloerection, hair-cut ungroomed, flaccidity, tremor, high-legged gait, nose reddened, nose: red encrustations, muzzle: red encrustations, nasal discharge (serous), stridor, nostril: red encrustations, eyelids with red encrustations, emaciation, cyanosis, prostration, 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 SOT (1992) were fulfilled, in regard to the respirability of the aerosol generated, i.e. the MMAD was < 4 µm (MMAD 3.0 -3.5 µm, GSD appr. 2.1). In one exposure group (399.2 mg/m3) the MMAD was 2.1 µm (GSD 2) which apperaed to result in slightly higher toxic potency of aerosol.


 


In summary, the respirable aerosol of test substance had a high acute inhalation toxicity to rats. The signs observed demonstrated 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 pMDI.


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; MDI is classified with Acute Tox. 4 H332 (Annex VI Regulation (EC) No 1272/2008 (CLP regulation).