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Description of key information

The test substance is covered by the category approach of methylenediphenyl diisocyanates (MDI).

Hence, data of the category substances can be used to cover this endpoint. The read-across category justification document is attached in IUCLID section 13. It is important to note that the MDI category approach for read-across of environmental and human hazards between the MDI substances belonging to the MDI category is work in progress under REACH. Therefore the document should be considered a draft.

The MDI is harmful by inhalation according to EU (R20) and GHS (Cat. 4) classification. The MDI is non toxic after single oral and dermal exposure.

Key value for chemical safety assessment

Acute toxicity: via oral route

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
adverse effect observed

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

Acute oral toxicity

The animal studies demonstrated that MDI has low acute oral toxicity.

In the available acute oral toxicity key study (OECD Guideline 425) (Mallory, 2009), one female Sprague Dawley rat was dosed at 175 mg/kg. No mortality was observed at 175 mg/kg and dosing continued in another female at 550 mg/kg. Dosing continued in an additional female at 2000 mg/kg as per protocol guidelines and then in one female at 5000 mg/kg. Based on these results two additional females were dosed at 5000 mg/kg. A total of 6 females were dosed. Mortality checks were made once daily. Clinical observations were recorded prior to dosing, as well at 30 minutes, 4 hours post-dose, and daily thereafter through Day 15. Body weights were recorded on the day of dosing (Day 1), and on Days 8 and 15. All rats were euthanized by CO2 asphyxiation and necropsied on Day 15. For doses of 175, 550, 2000 or 5000 mg/kg, no mortality was observed. All animals appeared normal throughout the study at 175, 550, 2000 and 5000 mg/kg. No biologically significant effect was seen on body weights on Days 8 and 15. Terminal necropsy revealed no visible lesions in any of the animals at 175, 550, 2000 and 5000 mg/kg. Based on these LD50>5000 mg/kg was determined.

A supporting study describing the acute oral toxicity of MDI conducted as a limit test according to EU guideline (84/449/EEC) with GLP did not find any mortality up to the maximum dose tested (Bomhard, 1990). The acute LD50 was found to be > 2000 mg/kg bw. Another supporting study describing the acute oral toxicity of polymeric MDI (pMDI) conducted similar to OECD 401 guideline (Reliability 2) also did not find any mortality up to the maximum dose tested, hence the LD50 is > 10000 mg/kg bw (Wazeter, 1964). Other studies on MDI are consistent with this.


Acute dermal toxicity

Similar to acute oral toxicity data, the available animal studies demonstrated that the MDI has low acute dermal toxicity. The key study describing the acute dermal toxicity of pMDI in rabbits did not find lethality up to the maximum dose tested and the LD50 was > 9400 mg/kg bw (Wazeter et al., 1964). Other studies on pMDI or 4,4’-MDI are consistent with this.


Acute inhalation toxicity: Mortality studies

The key guideline acute inhalation study (OECD 403) was performed in male and female rats at five exposure concentrations of 4,4-MDI (Pauluhn, 2008). The animals were nose-only exposed for 4 hours to liquid aerosol in concentrations of 0, 300, 354, 399, 500 and 554 mg/m3 (analysed conc.), with a post exposure observation period of two weeks. Mass median aerodynamic diameter ranged between 2.1 and 3.5 µm across all groups. Male rats were apparently more susceptible than females. All MDI exposure groups exhibited clinical signs consistent with respiratory irritation, so the NO(A)EC was <300 mg/m3. Mortality occurred in a concentration related manner at 354 mg/m3 and above. The LC50 for male rats was calculated as 368 mg/m3 (95% confidence interval 296-458 mg/m3) and for females approximately 559 mg/m3.For both sexes combined, the LC50 was 431 mg/m3.


An acute inhalation study was performed in rats at only one concentration level of 2.24 mg/L/1h (Pauluhn 2003). This study was specifically designed to comply with NFPA 704, and also complied with the limit test of the OECD guideline 403 with deviations (only 1 hr exposure, concentration lower than limit test concentration) and is therefore reliable with restrictions. Exposure of 4,4’-MDI for 1 hr resulted in mortality shorty after exposure of one out of ten rats. Clinical signs were characterised by typical signs of respiratory tract irritation. Necropsy findings were unremarkable in surviving rats, whilst the rat that succumbed displayed signs of lung oedema which was considered to be the cause of death. The LC50 >2.24 mg/L/1h (analytical) in both males and females was determined.


Acute inhalation toxicity: Sub-lethal Mode of Action studies

In a study by Pauluhn (2000), Wistar rats were acutely (6 hours) exposed nose-only to pMDI (0, 0.7, 2.4, 8 or 20 mg/m3) and markers for lung injury were evaluated in bronchoalveolar lavage fluid (BALF) or cells (BALC) at various times up to 7-days post-exposure. The data provide evidence for an immediate (0 hours post-exposure) loss in barrier function and a change in surfactant homeostasis at all concentrations tested (≥0.7 mg/m3); these effects returned to control levels by 3 days post-exposure. In contrast, evidence of cytotoxicity (LDH) and oxidative stress (GSH) were seen at exposure concentrations ≥8 mg/m3.


The acute inhalation toxicity of the 4,4-MDI to male rats was investigated to characterize the mechanism of local effects in the lung following an acute inhalation exposure (Hotchkiss, et al. 2017). Groups of 12 Wistar rats were exposed to 4, 12 and 27 mg/m3 (analytical concentrations, determined by gravimetry) to an aerosol-generated form of the test substance via a single nose-only inhalation exposure for 6 hours. A concurrent control group received filtered air on a comparable regimen. Half of the rats in each exposure group were euthanized immediately after exposure with the remaining rats in each group euthanized approximately 18 hours later. At necropsy bronchoalveolar lavage was performed on all experimental animals. The lavage fluid supernatant was analysed for biomarkers of injury and inflammation (total protein, LDH, alkaline phosphatase, β-glucuronidase) and oxidative stress (GSH and GSSG levels). The lavage cells were analysed for total and differential cell counts, targeted gene expression (including signals for inflammation, macrophage activation, apoptosis, and oxidative stress), and markers of apoptosis (Annexin V and Caspase-3). All animals survived to the scheduled euthanasia and there were no test substance-related clinical observations or effects on body weight.

Results included:

·     Dose-dependent increases in total protein and β-glucuronidase was noted at the end of the exposure and 18 hours post-exposure was noted at ≥ 4 mg/m3 which was statistically significant in the high exposure group.

·     Small increases in LDH activity were detected at the end of exposure and 18 hours post-exposure (statistically significant in 4 and 27 mg/m3 exposure groups).

·     The ratio of GSH/GSSG was reduced (indicative of oxidative stress) compared to controls at the end of exposure at ≥4 mg/m3. GSH/GSSG ratio remained lower than control rats in 12 and 27 mg/m3 exposed rats at 18h but was slightly elevated in rats exposed to 4 mg/m3 due to a significant increase in GSH levels

·     Neutrophilic inflammation was noted at > 12 and 27 mg/m3 immediately and was still evident at 18hr post-exposure.

·     A concentration-dependent increase in % Annexin (+) / PI (-) cells in nucleated BAL cellswas observed in 4,4’-MDI exposed animals immediately after exposure and 18 hours postexposure


In summary, a single 6 hour exposure to respirable aerosols of 4,4’-MDI to 4, 12, and 27 mg/m3 resulted in concentration- and time dependent increases 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.


The acute inhalation toxicity of the test substance to male rats was investigated in a combined in vivo genotoxicity/acute inhalation toxicity study according to OECD TG 489 (genotoxicity assessed by Comet assay) under GLP conditions (Randazzo, 2017). Groups of 12 Wistar rats were exposed to an actual concentration of 2.5, 4.9 or 12 mg/m3(corresponding nominal concentrations: 2, 5, 11 mg/m3)of an aerosol-generated form of the test substance administered via a single nose-only inhalation exposure for 6 hours. A concurrent control group received filtered air on a comparable regimen. Bronchoalveolar lavage (BAL) was performed in all animals at the scheduled necropsies, and the BAL fluid (BALF) and cells (BALC) was assessed for biomarkers of cytotoxicity and inflammation.

The endpoints alkaline phosphatase (type II alveolar epithelial cell cytotoxicity), lactate dehydrogenase (tissue damage/cytotoxicity), Annexin V + flow cytometry (apoptosis and necrosis), and total protein (cytotoxicity, blood/air barrier dysfunction) were determined to assess the cytotoxicity. β-glucuronidase (indicator for macrophage activation: activated macrophages secrete various inflammatory mediators such as cytokines/chemokines) and cell differential (with particular focus on the % neutrophils, the influx of which is the hallmark of the typical acute inflammatory response in the rat lung) were determined to assess the inflammatory potential of the test substance. Six rats/group were sacrificed approximately 1 hour post-exposure (ca. 1 hour after termination of the 6 hour exposure) and the other six/group approximately 18 hours post-exposure. Samples of the BALC, liver, and glandular stomach were collected from all animals and processed for comet assay evaluation (see Comet assay in section 7.6.2 and Genetic Toxicity Endpoint Summary). All animals survived to the scheduled euthanasia and there were no test substance-related clinical observations or effects on body weight.

Results included:

·     Dose-dependent induction of β-glucuronidase was observed 1 h post-exposure at ≥ 4.9 mg/m3 and ≥ 2.5 mg/m3 at 18 h post-exposure, reaching statistical significance for the high dose-group.

·     Dose-dependent increases in LDH at ≥ 2.5 mg/m3 at 1h timepoint. At the 18h timepoint, LDH was only increased in BALF at 12 mg/m3.

·     Clear dose-dependent increases in total protein was observed ≥ 2.5 mg/m3 at both time points.

·     At both 1 and 18 h after cessation of exposure, the % neutrophils in BALF was increased in in the 4,4’-MDI-exposed groups at 12 mg/m3 indicating an acute inflammatory response.

·     An increased of late apoptosis and necrotic cells was observed 1 h post-exposure at≥ 2.5 mg/m3as identified by Annexin V expression which returned to baseline by 18 h. Similarly, the number of early apoptotic cells increased 1 h after exposure at ≥ 4.9 mg/m3. However, by 18 h, an increase was only observed at 12 mg/m3


In summary, local cellular toxicity, characterised by an increased concentration of total protein and the macrophage activation marker β-glucuronidase in BALF, an increase in apoptosis/necrosis, were observed in animals exposed to ≥ 2.5 mg 4,4’-MDI/m3. With the exception of β-glucuronidase and total protein, all other parameter returned to control levels by 18 h post-exposure at the low- and mid-dose groups. The influx of neutrophils (a hallmark of an acute inflammatory response) was induced at 12 mg 4,4’-MDI/m3 at both D0 and D1 confirming MDI induces a persistent inflammatory response at this dose level. No clinical signs were observed (e.g. laboured breathing, increase breathing rate). Based on the magnitude of the differences noted in the BALF endpoints support, 12 mg/m3 was considered to be the maximum tolerated concentration (MTC) for an in vivo comet assay.


Acute inhalation toxicity: discussion

Sub-lethal Mode of Action studies:

Taken together, data from non-lethal acute inhalation studies indicate that a disruption of surfactant homeostasis is an early event in the pulmonary response to the deposition of MDI and occur at relatively lower MDI concentrations (NOAEC 0.7 mg/m3). Aerosols of MDI deposited in the alveolar epithelial lining fluids (surfactant) react with macromolecular nucleophiles (e.g. GSH, proteins, peptides), and when exposure concentration and/or duration is sufficient, the nucleophilic capacity of this layer becomes overwhelmed and deterioration of cell membranes and cytotoxicity occurs (e.g. total protein, LDH, apoptosis). In addition to the disruption of the surfactant homeostasis, reaction products of the MDI with alveolar macromolecules are phagocytised by activated macrophages. When activated, these macrophages release pro-inflammatory cytokines which recruit neutrophils (with a possible subsequent oxidative burst and reactive oxidative species production). The available data demonstrate early indications for toxic lung effects such as inflammation / cytotoxicity (e.g. lactate dehydrogenase (LDH), Annexin V expression) and oxidative stress (e.g. BALF glutathione (GSH) levels) at concentrations ≥8mg/m3and with consistent moderate toxicity at ≥12mg/m3. This suggests that acute exposures to MDI concentrations ≥ 12mg/m3 results in significant portal of entry cellular toxicity.


It should be noted that in an analysis of acute and chronic inhalation studies with MDI, Pauluhn (2011) concurred with the report by Feron et al. (2001) that the pulmonary effects seen in chronic investigations are dosedependent but rely on both the concentration used and the time of exposure, i.e. C x T (day) rather than being concentration dependent. Using C x T (day) as a metric, Pauluhn (2011) showed that pulmonary effects thresholds from acute and short term inhalation studies are more predictive of effects in the chronic investigations than concentration per se without any evidence of any cumulative dose effects in the chronic studies.



For classification, there were two well conducted, guideline studies, with reliably generated aerosol atmospheres and analysed concentrations taken into account (Appelman and de Jong , 1982a&b, Pauluhn, 2008). It was preferred to use these studies with reliable valid data rather than taking a mean of data reported in databases which do not critically assess the experimental set-up and analysis of the test-atmosphere. The GHS guidance has cut off values for Category 1 of 0.5 mg/L (500 mg/m3) for vapours and 0.05 mg/L for aerosols. GHS guidance section note (d) states that where vapour and aerosol exist together the cut of value for Category 1 should be 100 ppmV. The result of Appelman and de Jong (1982a, b) is an LC50 of 490 mg/m3 (0.49 mg/L), which falls just under the cut off for GHS Category 1 for vapours and mixed vapours/aerosols, but would be Category 2 for aerosols. Similarly the Pauluhn (2003,2004) study result of LC50>2.24 mg/L and Pauluhn (2008) study result of 431 mg/m3 would also result in Category 2 (aerosol).


Classification of chemicals allows for the application of scientific judgement. It must be taken into account that the LC50 cut-off of 500 mg/m3 (approximately 50 ppm for pMDI), is over 2500-fold above the saturated vapour concentration for pMDI. At the saturated vapor concentration MDI has no effect on animals. Furthermore the aerosols were generated using sophisticated techniques in thelaboratory (often involving heating), were ofextremely small particle size only in order to meet international guidelines for testing of aerosols, and this sort and concentration of aerosol is not generated in the workplace (ISOPA, 2015). In spraying applications aerosols are formed where the particle size distribution has virtually no overlap with that of the highly respirable aerosol generated in inhalation studies (see EU Risk Assessment Report on methylene diphenyldiisocyanate, EINECS-No. 247-714-0, 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 on the basis of 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. MDI is not inherently toxic by inhalation, as evidenced by its lack of any effect at the saturated vapour concentration. It is only with modification and input (in terms of heat, cooling and size screening) that MDI becomes toxic after inhalation. The European Chemical Industry Council have discussed and given guidance for situations like that, and on the classification of respective aerosols (attached in 7.2.2 'Acute Toxicity Endpoint Summary Attached Documents'). Classification of MDI as “Harmful” is consistent with this guidance.


The Appelman and de Jong (1982a&b) data were considered by EU experts and their conclusion that MDI be classified as “Harmful” (Xn, R20) is 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 MDI remains 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. This classification is equvialant to H332 (Harmful if inhaled) under CLP Regulation.

This principle is considered to apply equally to the Appelman and de Jong (1982a&b) study and the Pauluhn (2008) results.


The classification as “Harmful”, is equivalent to GHS Category 4. For these reasons, the GHS proposal follows the EU Regulatory lead accepting that the animal data are inappropriate and classified pMDI as GHS acute toxicity category 4 (ISOPA 2007).



Assessment of the available acute toxicity data indicates that inhalation exposure to the aerosols of MDI results in toxicity confined predominantly to the respiratory tract. In terms of hazard characterization, MDI is harmful by inhalation according to EU (H332) and GHS (Cat. 4) classification. MDI is non-toxic after single oral and dermal exposure.

Justification for classification or non-classification

EU classification according to CLP: H332

GHS classification (GHS UN rev.2, 2007): Inhalation route (vapour): Acute Category 4.

Not toxic by the dermal or oral routes.