1-isocyanato-2(or 4)-(4-isocyanatobenzyl)benzene and their reaction products with [(methylethylene)bis(oxy)]dipropanol and butane-1,3-diol and propylene glycol

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

The test substance is covered by a category approach of methylenediphenyl diisocyanates (MDI) with existing data gaps filled according to ECHA guidance on Read Across (ECHA, 2017).  Hence, data of the category substances can be used to cover this endpoint. The read-across category justification document is attached in IUCILD Annex III. 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.  In this category Substances of the MDI category all share similar chemical features namely that they a) all contain high levels of mMDI, and b) contain have at least two aromatic NCO groups that are electronically separated from other aromatic rings by at least a methylene bridge. It is the NCO value (driven by the bioaccessible NCO groups on relatively soluble mMDI and low molecular weight species (e.g. three-ring oligomer) which is responsible for chemical and physiological reactivity and subsequent toxicological profile. The substances 4,4’-MDI, 4,4’-MDI/DPG/HMWP and pMDI are identified as the boundary substances within this MDI category. These three substances represent the extremes of key parameters (i.e. mMDI content and NCO value) within the MDI category that determine the hypothesized Mode of Action (MoA). Although NCO groups are present on the higher molecular weight constituents, they do not contribute to the toxicity profile because they are hindered due to their increased size and hydrophobicity.  

 

 

 

 Skin irritation:  

 

By dermal contact, the majority of the available NCO groups react with proteins and moisture on the skin, leading to the formation of an insoluble polymerized mass limiting absorption such that only a small fraction is available to penetrate into the viable skin layers. Residual toxicity, as demonstrated by mild irritation potential, is consistent with the hypothesized MoA that effects are driven by the rapid MDI-adduct formation with extracellular biological nucleophiles. No other toxicities that are inconsistent with this MoA are observed. Although skin irritation studies are not available for all category substances, the common consistency of effects in the data matrix can be assumed with high confidence as representatives from all subgroups show irritation in studies with sufficient quality. All tested substances caused signs of skin irritation including inflammation (erythema and oedema) and additionally in some cases hyperplasia (thickening (coriaceousness), scaling, flaking or fissuring). Although not all studies demonstrated full reversibility of these signs, their severity decreased towards the end of the studies, such that only mild symptoms remained by the end of the observation periods. Furthermore, no signs of irreversible skin damage (i.e. ulcers, bleeding, bloody scabs, skin blanching, alopecia, scars or other signs indicative of necrosis into the dermis) were reported in any of the available studies, justifying all substances of the MDI category being regarded as skin irritants of category 2 as

 

This is consistent with reports of reversible skin irritation in mine workers exposed to the chemical.

 

Therefore, the harmonized CLP classification as skin irritant category 2 (H315) for 4,4’-MDI is adopted for all category substances. Furthermore, as there is evidence of skin irritation in humans, skin protection is recommended. 

 

The data for all category substances is described below:

 

The ‘Monomeric MDI’ subgroup

 

In a guideline skin irritation study in rabbits with “generic MDI” (now assigned as MDI Mixed Isomers, test substance name according to study report Desmodur VP.PU1806) erythema and oedema were observed, which were most pronounced seven days after the application of the test substance (Märtins, 1991). Eschar formation was observed in 1 animal from 72 hours to seven days and in two animals on day seven but no longer on day fourteen, which indicates that this was reflective of tissue repair and not overt skin corrosion. Scaling was seen in two animals after seven days and in two additional animals on day fourteen (Märtins, 1991). Based on the results, the test substance was classified as a skin irritant according to CLP (Cat. 2, H315). Only mild irritation was observed in an older, well conducted non GLP-study with 4,4’-MDI applied under occlusive dressings (Schreiber, 1981a). Very slight (barely perceptible) to slight (clearly perceptible) oedema was observed during the first 72 hours after removal of the dressings. By day eight, only very slight oedema was visible in three of six animals. Erythema formation could not be evaluated due to substance-related discoloration of the skin. Transient skin staining is a known effect of mMDI and pMDI and does not indicate irritation but merely dermal exposure (Henschler, 1992; Petsonk et al., 2000). The skin discoloration observed in the study by Märtins is therefore not considered to represent persisting irritation. The reported oedema scores recorded until day eight do not correspond to those that would trigger classification as a skin irritant according to CLP. While the study did not include a reading fourteen days after application of the test substance, it is likely that the effects would have been fully reversible within that time based on the low severity of effects and the lack of tissue damage.

 

The ‘Oligomeric MDI’ subgroup

 

Although not a guideline irritation study, an acute dermal toxicity study performed in rabbits with pMDI points to it only having a slight skin irritation potential (Wazeter et al., 1964a). After 24 hours of exposure of the skin to undiluted pMDI under occlusive conditions, slight erythema was observed which subsided after seven days. One animal exhibited slight oedema during the first and second day of the study, but not at later timepoints. Desquamation or fissuring were not noted throughout the study. Although these findings cannot be compared with the CLP classification criteria for skin irritation, they indicate that pMDI is slightly irritating.

 

The ‘MDI and its condensation products’ subgroup

 

4,4’-MDI homopolymer was found to be irritating to the skin of rabbits in a GLP-compliant guideline study (Mallory, 2009h). Oedema (up to a moderate severity) and erythema (up to a moderate to severe severity) were observed. Although these effects decreased in severity towards the end of the 14-day observation period, they were not completely reversible within that time. On study days 13 and 14, sloughing was observed in one of three animals. The observed effects correspond to a CLP classification as skin irritant cat. 2 (H315).

 

The ‘MDI, its condensation products and the reaction products with glycols’ subgroup

 

4,4’-MDI/4,4’-MDI homopolymer/1,3-BD/TPG/PG exhibited moderate to severe erythema/flaking of the skin and moderate oedema in a GLP-compliant guideline study, with decreasing severity towards the end of the 14-day observation period (Mallory, 2010a).  A second study on this substance (originally wrongly identified as MDI Mixed Isomers/1,3-BD/TPG/PG, but subsequent corrected to 4,4’-MDI/4,4’-MDI homopolymer/1,3-BD/TPG/PG) exhibited moderate to severe erythema/flaking of the skin and moderate oedema in a GLP-compliant guideline study, with decreasing severity towards the end of the 14-day observation period (Mallory, 2010b). The observed effects in both correspond to a CLP classification as skin irritant cat. 2 (H315).

 

‘MDI and its reaction products with glycols’ products

 

In another GLP-compliant guideline study 4,4‘-MDI/DPG was found to produce delayed skin irritation, with severe erythema and severe oedema, including fissuring and flaking of the skin, at the application site occurring from day 7/8 (Vasquez, 2011). While not completely reversed by the end of the observation period, there was reduction in severity beginning at day 12.  Based on the type and severity of effects, the substance was classified as a skin irritant category 2 (H315) under CLP.

 

4,4'-MDI/TPG was tested in a skin irritation study in rabbits which is considered reliable with restrictions as it did not follow a guideline but meets generally accepted scientific standards, is sufficiently documented and considered acceptable for assessment (Birch, 1977). After a 24-hour exposure of rabbit skin to the undiluted substance under occlusive conditions, oedema and erythema with a maximal score of 2 (well defined erythema and slight oedema, respectively) were observed. Both effects were completely reversible within the 7-day observation period in that study. The substance was considered mildly irritating but based on the mean erythema and oedema scores and the fact that the effects were fully reversible, a CLP classification does not result from this study.

 

Reversibility

 

With all tested substances, signs of skin irritation were observed including inflammation (erythema and oedema) and additionally in some cases hyperplasia (thickening (coriaceousness), scaling, flaking or fissuring). In the study by Martins on “generic MDI” (now MDI Mixed Isomers) eschar (dry slough) formation was reported during the study, but this was reflective of tissue repair and not overt skin corrosion. Furthermore, no signs of irreversible skin damage (i.e. ulcers, bleeding, bloody scabs, skin blanching, alopecia, scars or other signs indicative of necrosis into the dermis) were reported in any of the other available studies justifying all substances of the MDI category being regarded as skin irritants of category 2. This is in line with the harmonized CLP classification of 4,4’-MDI as skin irritant category 2 (H315).

 

Human Information

The human data base on skin irritation is restricted to clinical case reports about occupational exposure to 4,4’-MDI ([Lyman and Berardinelli (1994), and cited in EC (2005)). These reports are available as a secondary source only, and only sparse detail is provided. The description in the EU RAR of the case reports of mine-workers dermally exposed to a 4,4’-MDI containing product does not allow for a clear assessment of the type of effect (“primary irritant inflammatory response, to local cytotoxicity, and/or sensitization”). The report describes chronic skin irritation after skin contact to rock glue containing monomeric 4,4’-MDI and continuing dermatitis after continuing exposure. Since no irreversible damage to the skin is reported, the findings are in line with the harmonized CLP classification as skin irritant category 2 (H315).

 

 

 

 Eye irritation:   

 

Reliable eye irritation data from animal studies are available for only representative substances of the MDI category: 4,4’-MDI, 2,4’-MDI, MDI Mixed Isomers, pMDI and one representative of the subgroup “MDI and its reaction products with glycols” (4,4’-MDI/TPG). With four of the five tested substances, signs of eye irritation, but not of corrosion, were observed. MDI Mixed Isomers did not cause any effects in an eye irritation study in rabbits. There is limited human information on mMDI (not further specified) which has been reported to cause eye irritation in exposed workers (Jennison et al., 1994).

 

On the basis that the aqueous matrix in the eye is similar to the environment within the lungs, it may be reasonable to predict that most MDI substances instilled into the eye will polymerize to form an insoluble polyurea that is subsequently incapable of causing ocular irritation. Any unpolymerized MDI constituents and bioaccessible NCO groups will be available to react with extracellular biological nucleophiles such as glutathione and protein resulting in ocular damage and an acute inflammatory response. This sequestering of unreacted MDI constituents via polyurea formation before they have opportunity to react with biological nucleophiles may explain the relatively slight responses observed in ocular irritation studies when compared with dermal studies, and the apparent lack of significant involvement of the cornea and iris.

 

Generally speaking, the speed of onset of irritating properties observed in the available studies is consistent with the hypothesized MoA and that local toxicity of the category substances is driven by the rapid reaction of the bioaccessible NCO group on mMDI with extracellular biological nucleophiles such as glutathione and protein, resulting in ocular irritation and an acute inflammatory response. Although eye irritation studies are not available for all substances of the MDI category, on the basis of a general consistency of effects in the data matrix and a common MoA, it can be assumed with high confidence that all MDI substance are eye irritants.

 

Therefore, the harmonized CLP classification as eye irritant category 2 (H319) for mMDI is used for read-across as worst case for all substances of the MDI category.

 

The data for all category substances is described below:

 

The ‘Monomeric MDI’ subgroup

 

4,4’-MDI was tested for eye irritation in a study comparable to OECD 405 (Schreiber, 1981b).  The test substance was not rinsed after instillation. Only minimal cornea opacity was observed, resulting in a mean opacity score of 0.05 over 6 animals at 24/48/72 h. No information on the reversibility of cornea opacity was reported. Conjunctivae redness and chemosis up to grade 2 were observed in all animals 1 hour after substance instillation, but the severity decreased within 72 hours (to grade 1 for both effects, only visible in 3/6 animals). Both effects were not fully reversible within 8 days of observation continued to subside so that on day 8, only one animal showed grade 1 redness and chemosis. No lesions of the iris were observed. The results of this study cannot be exactly compared to the CLP classification criteria as a reading on day 21 was not included. However, given the low initial severity and the observed decrease of the effects over 8 days, it is likely that the results would not trigger a CLP classification as irritating to the eyes.

 

In a guideline eye irritation study with the same batch of “generic MDI” (MDI Mixed Isomers, test substance name according to study report Desmodur VP.PU1806) as used for the skin irritation key study, no irritation effects were observed in the eyes of any of the three animals tested (eyes were rinsed after 24 hours of exposure) (Märtins, 1991).

 

2,2’-MDI showed only slight irritation in a guideline study in rabbits (Gmelin, 2010). One hour after instillation of the test substance, conjunctivae redness up to grade 2 was observed in all 3 animals tested, and grade 1 conjunctivae chemosis was observed in 1 animal. Chemosis had completely subsided by 24 hours after substance instillation, and redness decreased in severity such that all animals were free of signs of irritation by 72 hours. No effects on the cornea or iris were seen. Based on these findings, the substance is considered slightly irritating to the eyes, but the effects do not trigger a CLP classification as irritating to the eyes.

 

The ‘Oligomeric MDI’ subgroup

 

In another non-guideline study (Wazeter et al., 1964c) rabbits were exposed to pMDI (instillation into the conjunctival sac) for 8 days. Eye irritation was graded and recorded according to the method of Draize. Diffuse corneal opacity was noted, the iris remaining clearly visible. Circumcorneal injection of the iris was observed soon after application. Both effects were absent after 24 h. Moderate irritation of the conjunctivae was noted but was reversible within 7 days. Purulent ocular discharge was noted in four of the nine animals and in all animals on day 7, respectively. All findings were reversible by day 7 of the study. No indication of irreversible ocular damage (by means of fluorescein staining) was observed.

 

 

 

The ‘MDI and its oligomeric reaction products with glycols’ subgroup

 

4,4’-MDI/TPG was tested for eye irritation in a non-guideline study (Birch, 1977). One hour after instillation, slight erythema and copious discharge were noted. Twenty-four hours after instillation, areas of barely perceptible corneal dullness, slight to moderate erythema and copious discharge were observed. For the reading timepoints at 48-120 hours (test substance was removed after 24 hours of exposure), a gradual improvement of the effects was noted, until all parameters were scored zero after seven days. No further detail regarding the evaluated endpoints is given, but based on the slight effects and full reversibility after seven days, it was found that the results do not trigger a CLP classification as irritating to the eyes.

 

 

 

Human information

 

There is anecdotal evidence of eye irritation in humans, but that information is considered of low reliability. According to a NIOSH report (Jennison et al., 1994), workers exposed to MDI (substance identity not further specified) in the workplace during the production of MDI-based polyurethane foam reported eye irritation which they felt to be work-related (exposure also likely included other foam producing components). Individuals who reported high exposure to the polyurethane foaming process were more likely to report work-related nasal and eye irritation than were individuals in the other exposure categories (“intermediate”, “low”, or “never”). NIOSH concluded: “Current workers who work directly with the polyurethane foaming system were found to have a substantial prevalence of work-related nasal and ocular irritation, suggesting that there is ongoing exposure to an irritant.” Since no irreversible damage to the eyes is reported, the findings are in line with the results from animal studies (where signs of irritation but not of corrosion were observed for several category substances) and with the harmonized CLP classification as eye irritant category 2 (H319).

 

In the MAK documentation for 4,4’-MDI and pMDI (Henschler, 1992), two reports from the early years of pMDI application are cited that provide limited information on pMDI-induced eye irritation in humans. One describes exposure of twelve workers to an aerosol mixture used to apply rigid foam insulation to the insides of railway carriages and blown by the wind to the workers standing about 20-40 meter away (Longley, 1964). Within a few hours, all individuals showed symptoms such as pain behind the eyes, nasal discharge, retrosternal soreness, constriction of the chest, coughing and headaches. The symptoms regressed completely within a few days. Fitzpatrick et al. (1964) reported that “vapor concentrations of 56 ppb” during spraying of a pMDI/polyol mixture did not cause irritation (not specified if ocular or respiratory) of workers using respirators with particle filters. The MAK notes that since the saturated vapor concentration of MDI at 20°C is about 5 ppb, either the temperature at the sampling site was higher or the MDI was mostly present in the form of an aerosol; how much of this aerosol was retained in the mask filters is unclear.

 

Also cited in the MAK documentation is a report that direct pMDI contact with the eyes caused transient blepharo-conjunctivitis (Bertrand et al., 1984).

 

  

 

 Respiratory tract irritation:  

 

As described in the toxicokinetic chapter, the majority of inhaled MDI constituents polymerizes to inert polyurea and is removed via mucociliary clearance and excreted in the feces. The remaining NCO on low molecular weight MDI constituents is responsible for the observed irritating properties and are consistent with hypothesized MoA that local toxicity of the substances of the MDI category is driven by the rapid reaction of available NCO groups with extracellular biological nucleophiles (e.g. GSH, proteins, peptides). If the inhalation of the substances of the MDI category is within the protective glutathione (GSH) capacity in the epithelial lining fluid, there is little adverse effect. If, however, this GSH capacity is exceeded it will result in excess NCO being available to react with epithelial nucleophiles resulting acute and reversible irritation of the respiratory tract, as demonstrated by early changes in the BALF of inflammation indicators and, at higher concentrations, changes in respiratory function parameters. The data from non-lethal acute (and short-term) inhalation studies of substances of the ‘Monomeric MDI’, ‘Oligomeric MDI’ and ‘MDI and its reaction products with glycols’ subgroup, indicate an inflammatory reaction as a pulmonary response to the deposition of these MDI substances. Aerosols of MDI deposited in the alveolar epithelial lining fluids react with biological nucleophiles at the particle/fluid matrix, 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 nucleophile 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 expression) and oxidative stress (e.g. BALF glutathione (GSH) levels) at concentrations ≥8 mg/m3 and with consistent moderate toxicity at ≥12 mg/m3. This suggests that acute exposures to MDI concentrations ≥12 mg/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 substances, Pauluhn (2011a) concurred with the report by Feron et al. (2001) that the pulmonary effects seen in chronic investigations are dose‐dependent 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 (2011a) showed that pulmonary effect 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.

 

 Summarized MDI should be classified as a respiratory tract irritant (EU: H335, GHS: STOT SE Cat3).  

 

  Data demonstrating the mechanism for respiratory irritation in both single (acute) and short-term repeated dose studies for multiple category members is described below.  Longer term repeated dose studies is described in under “Repeat-dose: Inhalation” endpoint. 

 

 

 

Acute inhalation studies

Several acute inhalation studies are available on MDI substances. Three repeated exposure studies are also included as they inform on respiratory irritation. While most of the data is on a limited sample of category substances (4,4’-MDI, pMDI, and 4,4’-MDI/DPG/HMWP), the effects noted are highly consistent and support the proposed mechanism of respiratory irritation. These studies are outlined and  discussed in more details in the category justification document (IUCLID Annex III)

 

The ‘Monomeric MDI’ subgroup

 

In an acute inhalation study with mice, pulmonary irritation of 4,4’-MDI was assessed by recording respiratory rates (Weyel and Schaffer, 1985). Groups of four male Swiss-Webster mice were exposed for 240 minutes to 4,4’-MDI at 6.7, 10.2, 19.6, 25.8, 40.3, 58.5 mg/m³. The average respiratory rates were monitored during exposure, and all animals were killed 24 hours post exposure. Upon sacrifice, the lung weights were measured. RD50 (concentration required to reduce the respiratory rate by 50%) were determined. To confirm that 4,4’-MDI acted as a pulmonary and not solely as a sensory irritant, mice were also exposed via tracheal cannulation to MDI aerosol at a concentration of 23.6 mg/m3 with the aim of eliminating the influence of sensory irritation by bypassing the trigeminal nerve. The exposure concentration was that expected to be the RD50 in control mice. Except for the cannulation, all other experimental conditions were identical to those in the inhalation exposure groups. At the lowest concentrations (6.7 and 10.2 mg/m³), an increase in respiratory rate above the control was observed for almost three hours of the exposure, followed by a gradual decline in respiratory rate during the last hour. For the highest concentration (58.5 mg/m³) there was only a slight increase in respiratory rate, of short duration, followed by a rapid decline during the last three hours. For the intermediate concentrations, the respiratory rate was initially elevated above the control for approximately one hour and gradually declined for the last three hours of exposure. A plateau in response was reached during the last 30 minutes of exposure. Little or no recovery was observed during 20 minutes following each exposure. The RD50 was estimated to be 32 mg/m3. Dose-dependent increases in lung weight were found for all MDI concentrations, which the authors concluded were the result of pulmonary edema. In the tracheal cannulation test, a decrease in respiratory frequency was observed, which was interpreted by the authors as a clear pulmonary irritant response. Based on the results, the authors concluded that 4,4’-MDI acted primarily as a pulmonary irritant, evoking little or no sensory irritation.

 

Further, two studies which did not measure respiratory parameters but focused on the dose-response relationship of bronchoalveolar lavage (BAL) parameters in 4,4’-MDI-exposed rats are available:

 

A study by Hotchkiss et al. (2017) was designed to provide data on concentration- and time-dependent effects on BAL cells in the lung of Wistar rats after a single, acute 6-hour inhalation exposure to 4,4'-MDI aerosol and included evaluation of a) macrophage activation, b) oxidative stress, and c) cytotoxicity. Groups of twelve male Wistar rats were subjected to a single six hours aerosol exposure of 4,4’-MDI at 4, 12 or 27 mg/m3. 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 analyzed for biomarkers of injury and inflammation (total protein, LDH, alkaline phosphatase, β-glucuronidase) and oxidative stress (GSH and GSSG levels). The lavage cells were analyzed 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 were noted at the end of the exposure and 18 hours post-exposure at ≥4 mg/m3 (statistically significant in 27 mg/m3 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 18 hours 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 18 hours post-exposure.

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

In summary, a single six-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. Based on the increases in total protein in the BALF (which has been demonstrated to be a good marker for respiratory irritation of pMDI, see Pauluhn (2002b), it can be concluded that the LOAEC regarding respiratory irritation is 4 mg/m3 in that study.

 

This study was followed up with a second acute inhalation toxicity in a combination with an in vivo genotoxicity toxicity study according to OECD TG 489 (genotoxicity assessed by Comet assay) (Randazzo, 2017). Wistar rats were exposed 2.5, 4.9 or 12 mg/m3 (measured concentrations) of 4,4’-MDI administered via a single nose-only inhalation exposure for six hours. A concurrent control group received filtered air on a comparable regimen. Six rats/group were sacrificed approximately one hour post-exposure (ca. one hour after termination of the six-hours exposure) and the other six/group approximately 18 hours post-exposure. All animals survived to the scheduled euthanasia and there were no test substance-related clinical observations or effects on body weight. 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 + 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 percent 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.

 

The following results were found:

 

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

Dose-dependent increases in LDH at ≥2.5 mg/m3 at one-hour timepoint. At the 18 hour 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 hours after cessation of exposure, the percent 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 one-hour post-exposure at ≥2.5 mg/m3 as identified by Annexin V expression which returned to baseline by 18 hours. Similarly, the number of early apoptotic cells increased one hour after exposure at ≥4.9 mg/m3. However, by 18 hours, an increase was only observed at 12 mg/m3.

In summary, local cellular toxicity, characterized 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. Except for β-glucuronidase and total protein, all other parameter returned to control levels by 18 hour 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/m3 at both sacrifice days confirming MDI induces a persistent inflammatory response at this dose level. Based on the increases in total protein in the BALF (which has been demonstrated to be a good marker for respiratory irritation of pMDI, see Pauluhn (2002b), it can be concluded that the LOAEC regarding respiratory irritation is 2.5 mg/m3 in that study.

 

The ‘Oligomeric MDI’ subgroup

 

Pauluhn (2000a) examined the time course of the relationship between acute pulmonary irritation and acute pulmonary response of Wistar rats 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 analyzed 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. Such dysfunction is thought to correspond to a physiological response. These results show that single or repeat (sub-acute) exposure to highly respirable pMDI aerosols at high concentrations results in lung effects consistent with exposure to an irritant particulate but that recovery occurs upon cessation of exposure. As such, following a six-hours exposure to pMDI aerosol, the NOAEC is 0.7 mg/m³ air whereas the 2.4 mg/m³ air, caused borderline biochemical effects (= LOAEC). This study, among others, was used by Pauluhn (2002b) to estimate an acute irritant threshold concentration of 0.5 mg/m3 for pMDI.

 

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.

 

Repeated exposure studies

The ‘Oligomeric MDI’ subgroup

 

In a short-term inhalation toxicity study of polymeric MDI in rats, changes in respiratory parameters in a single-exposure scenario were correlated to the alteration of surfactant activity in a two-week repeated-dose scenario (Pauluhn et al., 1999b). For the single-exposure part of the study, rats were exposed to 0, 2.4, 6.7, 15.8 or 38.7 mg pMDI/m3 air for 150 minutes. Respiratory rate and tidal volume were examined before and during exposure using nose-only exposure restrainers modified to function as flow plethysmographs. Based on concentration-dependent changes in tidal volume, it was concluded that pMDI caused acute pulmonary irritation starting at 2.4 mg/m3 (minimal extent).

 

In the second part of the study, rats were exposed to 0, 1.1, 3.3 or 13.7 mg/m³ (original target concentration of 10 mg/m3 was raised to 16 mg/m3 during week two due to absence of marked effects) for 14 days (6 h/d, 5 d/week during first week 1, 7 d/week during second week). The rats of each group were allocated to three different examination groups: bromodeoxyuridine (BrdU) labeling, histopathology and organ weight determinations were performed in the first fraction of the animals per group, arterial blood-gas determinations and lung lavage in a second fraction, and lung function measurements, including two female rats/group for electron microscopy, in a third fraction. Rats assigned to lung lavage, blood gas measurements, histopathology, BrdU labeling, and organ weight determinations were sacrificed one day after the last exposure to pMDI. Rats assigned to ultrastructural examinations were sacrificed three days after the last exposure. Lung function measurements were made two to four days after the last exposure. All animals survived until the scheduled necropsies. At 3.3 mg/m3 and 13.7 mg/m3, clinical signs and changes in breathing patterns indicative of respiratory irritation (irregular and labored breathing, rales, dyspnea, tachypnea, bradypnea, at 13.7 mg/m3 additionally nasal discharge) were observed. These changes were partly transient at 3.3 mg/m3. Lung weights were statistically significantly increased in female rats at 13.7 mg/m3. Determination of arterial blood gases, lung function, and carbon monoxide diffusing capacity did not demonstrate specific effects. BAL revealed changes indicative of marked inflammatory response and/or cytotoxicity in rats exposed to 13.7 mg/m3 (statistically significantly increased activities of LDH, beta-NAG, and protein). In terms of non-inflammatory parameters, phospholipid concentrations and gamma-GT were already increased in rats exposed to 1.1 mg/m3 and above. Light and transmission electron microscopy revealed focal inflammatory lesions and an accumulation of refractile, yellowish-brownish material in alveolar macrophages with concomitant activation of type II pneumocytes at 3.3 and 13.7 mg/m3. A concentration-dependent increase of BrdU-labeled epithelial cells in the terminal bronchioles was observed in all exposure groups. In summary, the results show that rats experienced mild signs of respiratory tract irritation which appeared to exacerbate during the study. The LOAEC for respiratory irritation is considered 3.3 mg/m3 in this study. Furthermore, the findings obtained suggest that the interaction of pMDI with surfactants eventually leads to intracellular precipitates originating from precipitated surfactant or surfactant-pMDI complexes. The authors suggested that pMDI interacts directly with pulmonary surfactant lining fluids, the first line of pulmonary defense.

 

The ‘MDI and its oligomeric reaction products with glycols’ subgroup

 

In a five-day inhalation pilot study, used to select doses for a subsequent 28-day repeat-dose study, rats were exposed to 4, 10, and 20 mg 4,4’-MDI/DPG/HMWP/m3 (Ma-Hock, 2021).  Results showed effects consistent with respiratory irritation observed in previous studies with MDI substances including concentration-related changes in lavage fluid and morphological changes in lungs, as well some mild reactive changes in lung-draining lymph nodes, trachea and larynx. Moreover, significantly increased cell proliferation in large, medium and terminal bronchi in males and females and increased cell proliferation in alveoli in males (statistically significant) and females (not statistically significant) were observed. Concerning clinical pathology and other examined parameters, no systemic effect could be observed. Under the current study conditions, no observed adverse effect concentration (NOAEC) for local toxicity could not be determined with the LOAEC at 4 mg/m3. The NOAEC for systemic toxicity was 20 mg/m³.

 

 

 

Justification for selection of skin irritation / corrosion endpoint: 

 

OECD 404 Guideline study with GLP

 

Justification for selection of eye irritation endpoint: 

OECD 405 Guideline study with GLP

 

 Effects on skin irritation/corrosion: irritating

 

 

 Effects on respiratory irritation: irritating

Justification for classification or non-classification

The results of key studies (Märtins, 1991 and Schreiber, 1981) together with human occupational case reports (NIOSH 1994) support the official classification as skin irritant (EU: H315, GHS: Cat2).  

 

 The available animal data would not support classification of MDI as an eye irritant. But together with human occupational case reports ( NIOSH, 1994) in which symptoms of eye irritation were reported the legal classification as eye irritant (EU: H319; GHS: Cat2) should be applied.    

 

 The available data support the classification of MDI as a respiratory tract irritant (EU: H335, GHS: STOTsingleCat3).