Isocyanic acid, polymethylenepolyphenylene ester, 2-ethyl-1-hexanol-blocked

Acute Toxicity: inhalation

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: Fully reported guideline study to GLP.

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:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Test material

Test material information

Constituent 1

Reference substance name:

9016-87-9

Cas Number:

9016-87-9

IUPAC Name:

9016-87-9

Constituent 2

Reference substance name:

Isocyanic acid, polymethylenepolyphenylene ester

IUPAC Name:

Isocyanic acid, polymethylenepolyphenylene ester

Details on test material:

Test substance: DESMODUR 44 V 20 L
Chemical name: Polymeric Diphenylmethane-4,4'-diisocyanate (MDI-polymer)
Manufacturer: Bayer Material Science, Leverkusen, Germany
Purity: 35.1 % monomeric 4,4'-MDI
62.8 % higher oligomers of MDI
2.2% by-products
Batch no.: P4DB000489 (Tox Id: 10064~)
CAS#: 009016-87-9 (high purity monomeric 4,4'-MDI)
Stability: Stability certified for the duration of study.
Storage conditions: refrigerator (W 4 OC) 1 darkness; room temperature during the course of study.
Handling: Complete exclusion of airlhumidity (head space of containers with MDI purged with nitrogen)
Appearance: Brownish, viscous liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Healthy young adult SPF bred Wistar rats, strain Hsd Cpb:WU (SPF), from the experimental animal breeder Harlan-Winkelmann GmbH, Borchen (Germany), were used. Animals of this strain have been used at Bayer HealthCare AG in toxicological studies for years. Historical data on their physiology, diseases and spontaneous alterations are available. The state of health of the strain is randomly checked regularly at the instance of the Laboratory Animal Services, Bayer HealthCare AG, for the most important specific infectious pathogens.

Acclimatization: The animals were acclimatized to the animal room conditions for at least 5 days before use. During this period, rats were also acclimatized to the restraining tubes.

Age and weight: At the study start the variation of individual weights did not exceed ± 10 per cent of the mean for each sex (see Appendix). Animals of the weight class used are approximately 2 months old and hence fulfill the criterion for young adults (see Appendix).

Animal housing: During the acclimatization and study periods the animals were housed singly in conventional Makrolon® Type IllH cages (based on A. Spiegel and R. Gönnert, Zschr. Versuchstierkunde, 1, 38 (1961) and G. Meister, Zschr. Versuchstierkunde, 7, 144-1 53 (1 965)). 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 861609 EEC) were followed.

Bedding: Bedding consisted of low-dust wood granulate from Lignocel BK 8-15
(Rettenmaier).

Animal rooms: All animals were housed in a single room. The animal room environment was as follows:

Room temperature: 22 ± 2°C
Relative humidity: 40 – 80%
Dark/light cycle: 12h/12h; artificial light from 6.00 am to 6.00pm Central European Time
Light intensity: Approximately 14 watt/m2 floor area
Ventilation: Approximately 10 air changes per hour

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

Aerosol generation: Atmospheres of the test substance were generated under
dynamic conditions using a digitally controlled Havard PHD 2000 pump and a binary nozzle. For nebulization, conditioned (dry, oil free) compressed air (15 L/min, supply rate: see Table 1 in the Result section, dispersion pressure approximately 600 kPa) was used.

B

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

250, 300, 400, 440, 500, 550 mg/m3

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

Body weights were measured before exposure, on days 1, 3 and 7, and weekly thereafter. Individual weights are also recorded at death, if applicable. The period of observation was for 2 weeks.

Clinical Signs
The 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). Following exposure, observations are made and recorded systematically; individual records are maintained for each animal. Cage-side observations included, but were not limited to, changes in the skin and fur, eyes, mucus membranes, respiratory, circulatory, autonomic and central nervous system, and somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, alivation, diarrhoea, lethargy, somnolence and prostration. The time of death is recorded as precisely as possible, if applicable. Since these signs can only be assessed adequately from freely moving animals, no specific assessment was performed during exposure while animals were restrained.

Rectal Temperatures
The rectal temperatures were measured shortly after cessation of exposure
(approximately within %hour after the end of exposure) using a digital thermometer with a rectal probe for rats.

Necropsv
All surviving rats were sacrificed at the end of the observation period using sodium pentobarbital (Narcoreno) (approximately 300 mg/kg body weights, intraperitoneal injection). All rats, irrespective of the day of death, were given a gross-pathological examination. Consideration was given to performing a gross necropsy on animals as indicated by the nature of toxic effects, with particular reference to changes related to the respiratory tract. All gross pathological changes were recorded and evaluated.

Statistics:

Calculation of the LC50 is performed by computer according to the method of Rosiello et al. (1977) as modified by Pauluhn (1983). This method is based on the maximum likelihood method of 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 X2-homogeneity test is not performed. In this case the interpolated concentration at 50% lethality is designated the approximate LC50. Additionally, the moving average interpolation according to Schaper et al. (1994) is used for calculation, if applicable.

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

310 mg/m³ air

95% CL:

> 266 - < 361

Exp. duration:

4 h

Mortality:

mortality occurred at 237.3 mg/m3 in a concentration-dependent manner. Details of the method used to calculate the LCs0 are provided in the Appendix. The particle size distribution was not essentially different between groups.

LC50= 3 10.24 mg/m3
Confidence interval (95%)= 266.43 - 361.25 mg/m3
Slope= 2.76
LCol= 133.68 mg/m3

Clinical signs:

other: All exposed groups showed clinical signs not seen in controls (details below)

Body weight:

Comparisons between the control and the exposure groups revealed a consistent, concentration-dependent decrease in body weights.

Gross pathology:

A qualitative description, only of findings of toxicological importance and for toxicological evaluation, is given below.

Animals sacrificed at the end of the observation period: The macroscopic findings were essentially indistinguishable amongst exposure and control groups.

Animals succumbing during the observation period: Nose: white foamy discharge; yellowish and viscous mucous; pleural cavity with yellowish
clear fluid; lung: less collapsed, dark-red, and marbled; trachea with white foamy content; liver, kidneys and spleen with discolorations.
Discolouration of organs post mortem is not a direct compound related effect.

Other findings:

Reflex measurements
A battery of reflex measurements was made on the first post-exposure day. In comparison to the rats of the control group, rats of all exposure groups exhibited concentrations-dependent changes in reflexes

Rectal temperature
Results of the evaluation of the rectal temperature reveal significant changes in body temperature in all exposure groups compared to the controls

Any other information on results incl. tables

Generation and Characterization of Atmosphere

Technical information concerning generation of test atmospheres is provided in

Table 1.

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

Group	1	2	3	4	5	6	7
Target Conc. (mg/m^3)	0 (air)	250	300	400	440	500	550
Nominal Conc. (mg/m^3)	0	369.4	397.2	411.1	494.4	516.7	561.1
Gravimetric Conc. (mg/m^3)	-	237.3	299.3	391.7	435	488.8	540
Recovery (%) Temperature (mean, °C)	- 23.2	64 23.3	75 23.5	95 22.8	88 23.2	95 22.9	96 23.3
Rel. Humidity (mean, %)	10	6	6	8	6	9	6
MMAD (µm)	-	1.55	1.74	1.81	1.76	1.77	1.79
GSD	-	1.70	1.63	1.62	1.62	1.66	1.61
Aerosol Mass 3 µm (5)	-	89.3	86.8	85.4	86.9	85.1	86.1
Mass recovered (mg/m^3)	-	230.3	292.2	397.4	440.4	486.9	543.1

MMAD = Mass Median Aerodynamic Diameter, GSD = Geometric Standard Deviation; -- = not applicable.

Signs and Observations

The intensity and time-dependence of the observed signs are presented in the Appendix as incidence tables. A qualitative description, only for the signs of importance for the toxicological evaluation, is given below.

Group 1: All rats tolerated the exposure without specific signs.

Group 2/males: Bradypnea, labored breathing patterns, irregular breathing patterns, piloerection, hair-coat ungroomed, flaccidity, motility reduced, high-legged gait, nasal discharge (serous), nose red encrustations, stridor, nostrils: red encrustations, periorbicular encrustations, lacrimation.

Group 3/males: Bradypnea, labored breathing patterns, piloerection, hair-coat

ungroomed, flaccidity, motility reduced, high-legged gait, nose red encrustations,

stridor, nostrils: red encrustations, periorbicular encrustations, cyanosis, emaciation.

Group 4/males: Bradypnea, labored breathing patterns, irregular breathing patterns, piloerection, hair-coat ungroomed, tremor, flaccidity, motility reduced, high-legged gait, nasal discharge (serous), nose red encrustations, stridor, nostrils: red encrustations, cyanosis.

Group 5/males: Bradypnea, labored breathing patterns, irregular breathing patterns, piloerection, tremor, flaccidity, motility reduced, nasal discharge (serous), nose red encrustations, muzzle red encrustations, nostrils: red encrustations, cyanosis:

Group 6/males: Bradypnea, labored breathing patterns, irregular breathing patterns, breathing sounds, piloerection, hair-coat ungroomed, tremor, flaccidity, motility reduced, high-legged gait, nose red encrustations, stridor, nostrils: red encrustations, periorbicular encrustations, cyanosis.

Group 7/males: Bradypnea, labored breathing patterns, irregular breathing patterns, piloerection, tremor, flaccidity, motility reduced, nose red encrustations.

Group 2/females: Bradypnea, labored breathing patterns, irregular breathing

patterns, piloerection, hair-coat ungroomed, flaccidity, motility reduced, high-legged gait, nose reddened, nasal discharge (serous), nose red encrustations, muzzle red encrustations, stridor, nostrils: red encrustations, emaciation.

Group 3/females: Bradypnea, labored breathing patterns, irregular breathing

patterns, breathing sounds, piloerection, hair-coat ungroorned, flaccidity, motility

reduced, high-legged gait, nasal discharge (serous), nose red encrustations, muzzle red encrustations, stridor, nostrils: red encrustations, periorbicular encrustations, cyanosis, emaciation.

Group 4/females: Bradypnea, labored breathing patterns, irregular breathing

patterns, piloerection, hair-coat ungroorned, tremor, flaccidity, motility reduced, highlegged gait, nasal discharge (serous), nose red encrustations, stridor, nostrils: red encrustations, cyanosis.

Group 5/females: Bradypnea, labored breathing patterns, irregular breathing

patterns, dyspnea, breathing sounds, piloerection, hair-coat ungroomed, tremor,

flaccidity, motility reduced, high-legged gait, nasal discharge (serous), nose red

encrustations, stridor, nostrils: red encrustations, periorbicular encrustations,

cyanosis.

Group 6/lfemales: Bradypnea, labored breathing patterns, irregular breathing

patterns, piloerection, hair-coat ungroorned, tremor, flaccidity, motility reduced, highlegged gait, nasal discharge (serous), nose red encrustations, stridor, nostrils: red encrustations, cyanosis.

Group 7/females: Bradypnea, labored breathing patterns, piloerection, tremor,

flaccidity, motility reduced, nose red encrustations.

Toxicological Results

The results obtained during and after exposures of rats for 4 h to this test substance are summarized in Table 2.

Table 2:      Summary of acute inhalation toxicity - 4 hour exposure to the   aerosol of the liquid test article

N Group /sex	Target Concentration (mg/m^3)	Toxicological Result	Onset and Duration of Signs	Onset of Mortality	Rectal Temperature (°C)
1/m	0	0/0/5	-	-	37.7
2/m	250	2/5/5	0d – 7d	0d, 1d	29.5**
3/m	300	4/5/5	0d – 12d	0d, 1d	28.9**
4/m	400	4/5/5	0d – 7d	1d	29.6**
5/m	440	5/5/5	0d	0d, 1d	29.2**
6/m	500	4/5/5	0d – 7d	0d, 1d	28.5**
7/m	550	5/5/5	0d	1d	28.6**
1/f	0	0/0/5	-	-	38.0
2/f	250	0/5/5	0d – 8d	-	29.9*
3/f	300	1/5/5	0d – 8d	1d	29.8**
4/f	400	4/5/5	0d – 5d	1d	29.3**
5/f	440	3/5/5	0d – 8d	1d, 5d	28.9**
6/f	500	4/5/5	0d – 7d	1d	28.4**
7/f	550	5/5/5	0d	0d, 1d	28.0**

N = group assignment, m = males, f = females, * = p 0.05, ** = p 0.01

Values given in the 'Toxicological results' column are:

            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 of 310 (95% confidenc interval 266-361) mg/m3. 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:

A reliable (OECD TG 403) acute inhalation study is available for pMDI (Pauluhn, 2008b). Six groups of Wistar rats were nose-only exposed to liquid aerosol in concentration of 237, 299, 392, 435, 489 and 540 mg/m³ (gravimetric concentration). The liquid aerosol was generated so it was respirable to rats. Mortality occurred in a concentration-dependent manner at 237 mg/m³ and above, with a combined (males and females) LC50 of 310.2mg/m³. A particular sex difference in susceptibility was not apparent. The exposure caused irritant effects in the upper and lower respiratory tract which resolved within the first postexposure week. The following signs were observed: bradypnea, labored breathing patterns, irregular breathing patterns, dyspnea, breathing sounds, piloerection, hair-coat ungroomed, tremor, flaccidity, motility reduced, high legged gait, nose reddened, nasal discharge (serous), nose red encrustations, muzzle red encrustations, stridor, nostrils: red encrustations peri-orbicular encrustations, lacrimation, cyanosis, emaciation, decreased body weights, altered reflexes, and hypothermia. Mortality occurred in most cases within 1 d and was causally linked to acute lung edema.

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).