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

Endpoint summary

Description of key information

Acute Toxicity oral route: A reliable and valid guideline study is available for the oral route (OECD 423, Schuengel 2007). No toxic effects were indicated after gavage application of MDI MT in female Wistar rats. A dose of 2000 mg/kg body weight was tolerated by female rats without mortalities, clinical signs, effects on weight gain and gross pathological findings, the LD50 cut-off value was set ≥ 5000 mg/kg body weight. Based on the available study data MDI MT is regarded as relatively non-toxic for the endpoint acute oral toxicity and is not classified according EU GHS 1272/2008 CLP.

Acute Toxicity dermal route: There are no acute dermal toxicity studies available for MDI MT. An analogue read across with data from the source substances pMDI was performed. In a dermal acute toxicity study Wazeter et al., (1964a)  applied to the abraded skin of  albino rabbits liquid pMDI. Doses of 0, 2500, 3900, 6000 and 9400 mg/kg bw were applied (2 rabbits per sex and group). The animals were covered in rubberized cloth for 24 hours. At the end of exposure the test substance was removed  and animals were observed for 14 days post-exposure. No lethality was indicated up to the maximum dose tested, and the LD50 was greater than 9400 mg/kg bw. Based on the available study data from the source substance  the target substance MDI MT is regarded as relatively non-toxic for the endpoint acute dermal toxicity and is not classified according EU GHS 1272/2008 CLP.

Acute Toxicity inhalation route: There are no acute inhalation toxicity studies available for MDI MT. A read across with data from the source substances 4,4’-MDI and pMDI was performed with reliable acute inhalation toxicity studies. In a reliable OECD guideline study (TG 403) Pauluhn (2008a) exposed Wistar rats to respirable liquid aerosols of 4,4’-MDI. Five groups of Wistar rats were nose-only exposed to liquid aerosol concentration of 300.0, 354.2, 399.2, 500.0 and 553.8 mg/m³. Mortality occurred in a concentration-dependent manner at 354.2 mg/m³ and above, with LC50-males of  368 mg/m³ and an approximate LC50-females of  559 mg/m³. 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, dyspnoea, laboured breathing patterns, breathing sounds, irregular breathing patterns, motility reduced,  piloerection, hair-coat ungroomed, flaccidity, tremor, high legged gait, nose: reddened, muzzle with red encrustations, nasal discharge, stridor, nostrils: red encrustations, eyelids with red encrustations, emaciation, cyanosis, prostration, decreased body weights altered reflexes and hypothermia. Mortality occurred in most cases within 1 d and was causally linked to acute lung oedema.

In a reliable  guideline study( OECD TG 403) (Pauluhn, 2008b) six groups of Wistar rats were nose-only exposed to liquid aerosol of pMDI in concentration of 237, 299, 392, 435, 489 and 540 mg/m³. 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. 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, laboured breathing patterns, irregular breathing patterns, dyspnoea, 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 oedema.

In another acute inhalation study  Appelman and Jong (1982) evaluated the acute inhalation toxicity of  polymeric MDI (pMDI) in Wistar rats. Male and female rats were whole-body exposed for 4 hours to different  aerosol concentration of pMDI ( 384, 418, 523, 500 mg/m³). Mortality occurred in a concentration-dependent manner at 384 mg/m³, with a combined (males and females) LC50 of 490 mg/m³. Gross examination of animals killed immediately after the 4 hour exposure period revealed some hamorrhages or oedema in the lungs. In addition, the lungs of the highest dose group animals were greyish and somewhat wet. Similar changes were found in  animals killed at the end of the observation period. Mortality occurred in most cases within one to two days and was causally linked to acute lung oedema.

In summary, the study data of both source substances revealed that aerosolized test materials (liquid aerosol) proved to have a high acute inhalation toxicity  in rats. Using the strict GHS LC50 cut-off for classification, the LC50 values obtained for the source substances 4,4’-MDI and pMDI 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; the source substance 4,4’-MDI is officially classified with Acute Tox. 4 H332 (Annex VI Regulation (EC) No 1272/2008 (CLP regulation).

As the source substances 4,4’-MDI, pMDI and the target substance MDI MT contain sufficient monomeric MDI, the driver of toxicity, similarities in reactions leading to inflammation and irritation to the respiratory tract is predicted. In addition, as the higher molecular weight non-monomeric content of the UVCB substance MDI MT do not contains reactive centres and is consequently inert and thus do not contribute to the expected toxicity, it is reasonable to assume that using read across to the source substances 4,4’-MDI and pMDI is warranted and MDI MT should be classified with Acute Tox. 4 (H332) EU GHS 1272/2008 CLP.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 26 2006 to January 04 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
batch number of test material: LL6-077
- Purity: 100 % (given by sponsor)
Expire date: 2007-01-27

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Harlan-Winkelmann GmbH (Borchen, Germany)
- Age at study initiation: approx. 10-12 weeks
- Weight at study initiation: 176 -188 g
- Fasting period before study: 16-24 hours
- Housing: in groups
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 5
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

other: corn oil with the aid of 10 % acetone (dried with molecular sieve)

Details on oral exposure:

RATIONALE FOR THE SELECTION OF THE STARTING DOSE:
As described in the flow charts of Annex 2, OECD guideline 423, the starting dose level should be that which is most likely to produce mortality in
some of the dosed animals. Therefore, the limit dose 2000 mg/kg bw was chosen as starting dose.

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

6

Control animals:

no

Details on study design:

ADMINISTRATION
- Application volume: 10 ml/kg bw
- Post dose observation period: 14 days

EXAMINATIONS
- Clinical observations were made several times on the day of dosing and at least once a day during the 14 day observation period. Body
weights were recorded immediately prior to dosing and on days 7 and 14. Gross pathological examination was carried out on all animals.

Statistics:

none (limit test)

Key result

Sex:

female

Dose descriptor:

other: LD50 cut-off

Effect level:

>= 5 000 mg/kg bw

Based on:

test mat.

Mortality:

All 6 animals survived the treatment.

Clinical signs:

other: No clinical signs were observed.

Gross pathology:

No gross pathological findings were observed.

Other findings:

None

NOEL: 2000 mg/kg bw

Interpretation of results:

GHS criteria not met

Conclusions:

A dose of 2000 mg/kg bw was tolerated by female rats without mortalities, clinical signs, effects on weight gain and gross pathological findings.

Executive summary:

A single oral dose of 2000 mg/kg body weight was tolerated by female rats without mortalities, clinical signs, effects on weight gain and gross pathological findings. According to OECD guideline 423 the LD50 cut-off of Desmodur MT is >= 5000 mg/kg bw. for rats (Category 5 / unclassified of the Globally Harmonized Classification System).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

>= 5 000 mg/kg bw

Quality of whole database:

Reliable, according to OECD guideline 423

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Reference 1

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.

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

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

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.

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

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.

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).

 

Reference 2

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.

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

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

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.

Key result

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: aerosolized 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.

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

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).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

310 mg/m³ air

Physical form:

inhalation: aerosol

Quality of whole database:

Reliable, according to OECD guideline studies

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well documented study report which meets basic scientific principles

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

yes

Remarks:

Full details not in report. Numbers per dose = 2 and doses above limit dose used.

Principles of method if other than guideline:

Pre guideline study nevertheless fulfils the modern requirements.

GLP compliance:

no

Test type:

standard acute method

Limit test:

no

Species:

rabbit

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Mean body weight at study initiation: 2215-3180 g
- Housing: single
- Diet: ad libitum
- Water: ad libitum

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: 3 inch2 (19.35 cm2) in case of abraded skin
- Preparation of test site: The dorsal skin was prepared for treatment by closely clipping of the hair with an electric clipper in 2 rabbits (1 male and 1 female) per group. In addition, the skin of the rest of 2 rabbits per group was abraded by producing shallow incisions with a scalpel blade. The animals were confined in immobilising holders for 24 hours with their backs covered in rubberised cloth.


REMOVAL OF TEST SUBSTANCE
- Washing (if done): washed with lukewarm water
- Time after start of exposure: 24 hr

Duration of exposure:

24 hr

Doses:

2500, 3900, 6000 and 9400 mg/kg bw

No. of animals per sex per dose:

2

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: animals were observed daily for clinical signs and dermal irritation. Body weights were obtained for each animal at the end of the experiment.
- Necropsy of survivors performed: yes. All animals were sacrificed with air embolization.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 9 400 mg/kg bw

Based on:

test mat.

Mortality:

No mortalities were observed.

Clinical signs:

other: All the animals appeared normal throughout the 14 days observation period with the exception of dermal irritation.

Gross pathology:

No substance- related abnormalities were observed. Sub-endocardial haemorrhages, apparently dose related observed were of questionable significance due to sacrifice of animals by air embolisation.

Other findings:

Dermal irritation: The irritation noted was slight in all instances. The slight erytherma produced initially at all dosage levels was negative after seven
days. All animals at all dosage levels did exhibit a slight coriaceousness which in most instances continued throughout the 14-day observation period. Transient atonia was observed in a few animals at the three high dosage levels; however, this again was slight in nature. One animal at the highest dosage level exhibited slight edema during the first and second days of the study. There was no desquamation nor fissuring noted with this
compound.

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

In an acute dermal toxicity study with pMDI no lethality was indicated up to the maximum dose tested, and the LD50 was greater than 9400 mg/kg bw.

Executive summary:

In a dermal acute toxicity study Wazeter et al., (1964a)  applied to the abraded skin of  albino rabbits liquid pMDI. Doses of 0, 2500, 3900, 6000 and 9400 mg/kg bw were applied (2 rabbits per sex and group). The animals were covered in rubberized cloth for 24 hours. At the end of exposure the test substance was removed  and animals were observed for 14 days post-exposure. No lethality was indicated up to the maximum dose tested, and the LD50 was greater than 9400 mg/kg bw. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

>= 9 400 mg/kg bw

Quality of whole database:

reliable with restriction

Additional information

The findings from acute inhalation LC50 studies (OECD TG 403)  (Pauluhn 2008a, Pauluhn 2008b, Appelman and Jong 1982) revealed that aerosolized 4,4’-MDI or pMDI  proved to have a high acute inhalation toxicity  in rats under the test conditions used. The signs observed demonstrated that the respirable aerosol of this test substances may cause marked respiratory tract irritation with mortality associated with lower respiratory tract irritation leading to alveolar oedema.

Signs of respiratory tract irritation are confirmed by data of sub-lethal  acute inhalation toxicity studies. In a dose-range finding study Hotchkiss (2017) noted effects of respiratory tract irritation after single , acute 6 hours inhalation exposure to 4,4’-MDI. This study was designed to provide data on concentration – and time-dependent effects  for bronchoalveolar lavage (BAL) cells in the lung of Wistar rats. Groups of 12 male Wistar rats were exposed for six hours using a nose-only inhalation exposure system to time-weighted average concentrations of 0, 4, 12, and 27 mg 4,4'-MDI/m³. All animals survived the six-hour exposure to the test material. Endpoints indicative of inflammation, macrophage activation, apoptosis/necrosis, and oxidative stress were evaluated immediately following exposure or approximately 18 hours post-exposure (overnight). The single 6 hours exposure to respirable 4,4’-MDI to 4, 12 and 27 mg/m³ resulted in concentration- and time dependent increase in oxidative stress, inflammation, and markers of apoptosis. There was some evidence of inflammation, oxidative stress, and/or apoptosis at every dose, although the clearest effects were observed in the 12 and 27 mg/m³ exposures and especially by 18 hours post exposure. Randazzo (2017) performed a dose-range finding study  with 4,4’-MDI to  establish a maximum tolerated concentration for an in  vivo Comet assay. Male Wistar rats were exposed  once for 6 hours nose-only to  0, 3.2, 7.7, and 11.9 mg/m³. A bronchoalveolar lavage (BAL) was performed from all animals at the scheduled necropsies, and in the BAL fluid (BALF) marker for clinical chemistry, apoptosis and macrophage activation were evaluated and cytology was performed. All animals survived to the scheduled necropsies and no test substance related clinical observations or body weight effects were observed. Compared to control rats, dose dependent increases associated with macrophage activation (β- glucuronidase activity), inflammation (neutrophil infiltration and total protein)) at ≥3.2 mg MDI/m³, apoptosis (Annexin V activity; ≥ 7.7 mg MDI/m³), and necrosis (LDH; ≥ 11.9 mg MDI/m³) were noted in treated groups. From this range-finding study, inhalation exposure to MDI results in marked local acute toxicity at ≥10-12 mg/m³ for a 6 h exposure as identified by inflammation, apoptosis/necrosis and cytotoxicity supporting results from previous studies. The maximum tolerated concentration (MTC) was identified as 11.9 mg/ m³.

Pauluhn (2000) found after single exposure to highly respirable pMDI aerosols at high concentrations results in lung effects consistent with exposure to an irritant particulate. Wistar rats were exposed to respirable polymeric MDI (pMDI) aerosol of 0, 0.7, 2.4, 8, or 20 mg pMDI/m³ for 6 hours. The time-response relationship of MDI-induced acute lung injury was examined at 0 hours (directly after cessation of exposure), 3 hours, 1 day, 3 days, and 7 days after exposure. Bronchoalveolar lavage (BAL) fluid was analysed for markers indicative of injury of the bronchoalveolar region. Results suggested that respirable pMDI aerosol interacts directly with the air/blood barrier causing increased extravasation of plasma constituents because of increased permeability of capillary endothelial cells. A transient dysfunction of the pulmonary epithelial barrier occurred at a level as low as 0.7 mg/m³ and was interpreted as a dysfunction of pulmonary surfactant. In another study in rats with a similar design, single six-hours exposures to pMDI at 10, 30, or 100 mg/m³ resulted in signs of respiratory tract irritation (abnormal respiratory noise, breathing rate reduced and depth increased, mucous secretions from the nose) and a pattern of lung responses that was consistent with exposure to irritant aerosols (Kilgour et al., 2002). These effects were observed in all exposure groups. An exposure concentration related body weight loss and increase in lung weight were seen post-exposure, with complete recovery by day ten. Analysis of lung lavage fluid revealed irritation-related changes in the lung over the initial days following exposure. These consisted of a pattern of initial toxicity, rapid and heavy influx of inflammatory cells (alveolar macrophages) and soluble markers of inflammation and cell damage, increased lung surfactant, with a subsequent recovery and epithelial proliferative phase (e.g. bronchiolar and type II cell hyperplasia). Finally, by day 30 post-exposure, a return to the normal status quo of the lung was observed.

In summary, the findings from the source substances, indicating respiratory tract irritation, are in line with the hypothesized Mode of Action (MoA) with primary toxic effects on the  portal-of-entry, reflecting the high reactivity of the NCO functionalities. As the NCO groups reacts with nucleophiles at the deposition site (primarily glutathione (GSH), in the extracellular/aqueous interface in the lung the protective capacity is overwhelmed resulting in inflammation and cytotoxic effects.

As the source substances 4,4’-MDI, pMDI and the target substance MDI MT contain sufficient monomeric MDI, the driver of toxicity, similarities in reactions leading to inflammation and irritation to the respiratory tract is predicted. In addition, as the higher molecular weight non-monomeric content of the UVCB substance MDI MT do not contains reactive centres and is consequently inert and thus do not contribute to the expected toxicity, it is reasonable to assume that using read across to the source substances 4,4’-MDI and pMDI is warranted.

Justification for classification or non-classification

According to CLP Regulation (EC) No.1272/2008 the classification of 4,4'-MDI (CAS No.101-68-8) is considered for the classification of MDI MT (CAS No.147993-65-5): Acute Tox. 4 (H332) EU GHS 1272/2008 CLP.

MDI MT is regarded as relatively non-toxic for the endpoints acute oral and dermal toxicity and is not classified according EU GHS 1272/2008 CLP.