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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.

ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint Specific Guidance (Version 3.0, August 2014; page 20 “Selection of the appropriate route of exposure for toxicity testing“) is indicating that “the overall objective of toxicity testing is to determine the potential hazard of the test substance to human beings”. It is stressed that “toxicity data obtained using the appropriate route of exposure are preferred” and that “route-to-route extrapolation should be considered on a case-by-case basis and may introduce additional uncertainties”. The evaluation of toxicokinetic information should be taken into account to address such uncertainties. For reactive substances like diisocyanates significant toxicokinetic differences can be demonstrated, e.g. with respect to primary reaction products at the portal of entry (oral, dermal, inhalation). Since the most relevant route of exposure is inhalation hazard characterization shall preferably be performed via inhalation.

 

Oral exposure:

Apart from reports on accidental ingestion in domestic animals no specific studies are available on the toxicokinetics of MDI following oral exposure in animals. However there is sufficient information from analogous isocyanates to describe the outcome with some certainty. 

The US NIEHS contracted studies on the aromatic diisocyanate, toluene diisocyanate (TDI) including metabolism and relevant reports available are Jeffcoat (1985, 1988) and one publication by Dieter et al., (1990). Radiolabelled 2,6-TDI isomer in corn oil was dosed by oral gavage to rats (60, 900 mg/kg bw in males) and excreta were collected.  In the high dose group polymerized solid mass was present in the stomach and the stomachs became greatly distended. Less than 1% of the radioactivity was recovered from the tissues 72 h after administration. The major excretory route was by the faeces, urine accounted for only 5% (900 mg/kg bw) or 12% (60 mg/kg bw) of the dose excreted. A significant shortcoming of this study is that dosing solutions were prepared in corn oil, which is known to result in partly degradation of TDI ahead of test substance application. In anin vitrostudy a TDI equivalent to a 60 mg/kg bw dose was added to rat stomach contents. Most TDI reacted in less than 10 minutes, although DMSO was used as solvent which is known to catalyse hydrolysis of TDI. A subsequent study with 2,4-TDI gave similar results for disposition. Overall, these studies show that TDI polymerized in the acid environment of the stomach to solid polyureas and therefore this unrealistic dose route leads to a specific polymerization reaction of TDI before absorption into the body. Timchalk et al. (1994) reported results similar to those of Jeffcoat, the majority of orally gavaged TDI was eliminated in the faeces, with less than 10% in urine. 

 

In analogy to TDI, for MDI it is expected that oral gavage dosing will result in a similar outcome, that is (1) reaction with stomach contents and (2) polymerization of MDI to solid polyureas.

(1) Reaction with stomach contents is very plausibly described in case reports of accidental ingestion of PMDI based glue in domestic animals. Extensive polymerization and CO2liberation resulting in an expansion of the gastric content is described in the stomach, without apparent acute chemical toxicity (Collins 2009). 

(2) Polyurea formation in organic and aqueous phases is best described by Yakabe et al (1994, see 5.1). In this generally accepted chemistry of hydrolysis of an isocyanate the initially produced carbamate decarboxylates to an amine which, as a reactive intermediate, then reacts very readily with the present isocyanate to produce a solid and inert polyurea. This urea formation acts as a pH buffer in the stomach, thus promoting transformation of MDI into polyurea, even under the acidic conditions.

 

At the resorptive tissues in the small intestine, these high molecular reaction products are likely to be of very low bioavailability, which is substantiated by the absence of systemic toxicity in acute oral bioassays with rats at the OECD limit dose (LC50>2 g/kg bw; Bomhard 1990).

It must be recognized, that oral exposure by direct intubation into the stomach of a fasted animal is an artificial exposure route, which will not be encountered by humans. Likewise swallowing of large doses cannot be anticipated as a forseeable exposure. The chemistry of reaction of MDI in biological milieu is such that in the event of a true exposure of small MDI doses to the mouth, reactions will commence at once with biological macromolecules in the buccal region and will continue along the digestive tract prior to reaching the stomach. Reaction products will be a variety of polyureas and macromolecular conjugates with mucus, proteins and cell components. This is corroborated by the results from the MDI inhalation study reported by Gledhill (2003a, b). Following an inhalation exposure of rats to radiolabelled MDI, 79% of the dose was excreted in faeces. The faecal excretion in these animals was considered entirely due to ingestion of radioactivity from grooming and ingestion of deposited material from the nasopharangeal region via the mucociliary escalator, i.e. not following systemic absorption. The faecal radioactivity was tentatively identified as mixed molecular weight polyureas derived from MDI. Diamine was not present. This shows that MDI intake to the digestive tract by grooming or by the mucocilicary escalator following inhalation results in formation of polyureas without detectable diamine formation. Thus, for MDI and diisocyanates in general the oral gavage dosing route is inappropriate for toxicological studies and risk assessment. 

 

Dermal exposure:

Two studies on the dermal absorption and distribution of radioactive labelled MDI in rats are available (Vock and Lutz, 1997; Leibold et al., 1999). While the EU Risk Assessment (JRC, 2005) suggested that neither of the two studies appear to be unreliable, the very low recoveries reported by Vock and Lutz and (1997) the reported opportunity for grooming of the unoccluded application site by animals housed together (evidenced by high radioactivity on the tongue) calls into question the validity of the excretion data, particularly for faeces. In the specifically designed study reported by Leibold et al (1999), published by Hoffmann et al (2010), animals were housed singly, the site of administration was protected with a silicone ring, a nylon mesh gauze and a porous bandage, to prevent contact by grooming and to minimise contact of the applied dose by the dressing. Nevertheless there was high recovery from the dressing which would also suggest removal and ingestion of radioactivity from the application site was easily possible in the study of Vock and Lutz (1997). 

 

Overall the results of Leibold et al (1999) must be considered reliable and indicate that dermal application of MDI results in low (less than 1%) penetration to the systemic circulation. This is compatible with the results of Bello et al (2006) who reported absorption of MDI into skin. Consequently, a dermal uptake of 1% (Leibold et al., 1999) is used to calculate the body burden in the dermal exposure assessment.

 

Inhalation exposure:

With respect to inhalation exposure, there are good and reliable data regarding distribution/excretion in experimental animals. In a two part full inhalation metabolism/toxicokinetics/distribution study performed by Gledhill (2003ab), approximately 5% of the dose was excreted in urine and 79% in faeces of rats. Bile duct cannulated animals excreted approximately 12% of the dose in urine, 14% in bile and 34% of the dose in faeces. In conclusion, most of the systemically available dose was excreted via bile, and a slightly lower amount via urine. No radioactivity was recovered in exhaled air.

Similar results were obtained in an older study by Centre d`Etudes (1977) were the faecal elimination of MDI and its metabolites was greater than the urinary elimination.

 

In both studies radioactivity was widely distributed with the respiratory and excretory organs containing the highest concentrations. The highest concentration of radioactivity was present in the respiratory nasal tissue (Gledhill 2003a).

 

Due to grooming and respiratory clearance a combined oral/inhaled exposure existed in the study of Gledhill (2003a). 25-32 % of the applied dose was systemically available. The data does not allow discrimination between absorption in the respiratory tract on the one hand, and the gastrointestinal tract on the other hand. However, taking into account the low urinary excretion in the study of Vock and Lutz (1997), together with the likelihood of oral intake due to grooming, and based on mechanistic in-vitro studies described below, the respiratory tract may be regarded as the main entry for systemically available MDI.

 

A detailed summary on urinary, plasma andin vitrometabolite studies is provided below. Taken together, all available studies provide convincing evidence that MDI-protein adduct and MDI-metabolite formation proceeds:

1) via formation of a labile isocyanate glutathione (GSH)-adduct,

2) then transfer to a more stable adduct with larger proteins, and

3) without formation of free MDA. MDA reported as a metabolite is actually formed by analytical workup procedures (strong acid or base hydrolysis) and is not an identified metabolite in urine or blood.

 

Other routes of exposure:

After intradermal administration of 14C-MDI to rats about 26% of the radioactivity was absorbed. Excretion was mainly via faeces (Leibold et al., 1999). Following intramuscular injection of 14C-MDI less than 25% of the applied dose was recovered in the excreta 120h after application. The amount of faecal elimination was larger if compared to urinary elimination, indicating that for absorbed MDI biliary transport is a significant route of excretion. The remaining radioactivity was found in the carcass.

Metabolites identified in vivo andin vitro:

 

Gledhill et al (2005) proposed a simple, single metabolic pathway for MDI, based on evidence from their guideline ADME radiolabel study. Glutathione is the likely carrier for MDI from the lung to the circulation. There, N-acetyl-4,4’-diaminophenylmethyl is the first identified metabolite, with further acetylation and hydroxylation thereafter. No MDA was detected and the metabolic pathway proposed does not require MDA as an intermediate. In urine and bile four main metabolites were identified (N-acetyl-4,4´-diaminophenylmethane, diacetyl-4,4´-diaminophenylmethane, N,N´-diacetyl-4,4´-diaminobenzhydrol and N,N´-diacetyl-4,4´-diaminobenzophenone) which shows the progressive acetylation and oxidation. None of these specified low molecular weight metabolites were found in faeces. The radiolabelled components of faeces and bile were tentatively identified as mixed molecular weight polyurea oligomers of MDI, derived from ingestion of MDI by grooming or via the mucociliary escalator of inhaled MDI from the respiratory tract. 

 

The role for glutathione as an intermediary in transport of diisocyanates is now supported by good evidence using various model compounds. Brown (1987) concluded that the most probable reactions of isocyanates with biological macromolecules are with the amine (mixed urea), the hydroxyl (carbamate) and the sulphydryl (thiolytic acid ester) and that latter is of a reversible nature. Mormann et al (2006) found that aqueous solutions of N-acetyl cysteine (an analogue for the sulphydryl group of glutathione) reacted with MDI to form a diacetyl cysteine-MDI conjugate. The majority of MDI formed insoluble polyureas and it was of note that no MDA was formed. Reisser et al (2002) synthesised the bis-glutathione-MDI conjugate and found it to be moderately stable in physiological conditions, and hydrolysis led to various products including polyureas, but not to the diamine MDA. 

As mentioned by Brown (1987), the thiocarbamate bond of isocyanate-sulphydryl is reversible, and various authors have found release and transfer of MDI moieties from thiocarbamate conjugates to other nucleophiles, notably protein. Mormann et al. (2008) reported transcarbamoylation of acetylcysteine adducts of MDI with model compounds. In aqueous buffer reaction rate was thiolysis > aminolysis > hydrolysis. No 4,4’-MDA could be detected and no free isocyanate groups could be detected in any of the reactions. Chipinda et al (2006) showed reaction of both MDI and MDI-cysteine methyl ester with albuminin vitro. Wisnewski et al (2013) reacted MDI with glutathionein vitro, and then by incubating the reaction products with human serum albumin, achieved transcarbamolyation to the protein. Specific lysines were the conjugation targets as well as the N-terminal amino group. Finally the conjugates were shown to be recognised by serum IgG from MDI exposed workers, demonstrating a non-enzymatic, thiol-mediated transcarbamolyating mechanism to protein. 

It should be noted that the very low vapour pressure and water solubility of MDI makes such studies technically challenging. There are similar and more extensive data available for the aromatic diisocyanate TDI. Of note is the study by Day et al (1997) who reported formation and transcarbamoylation reactions of glutathione-TDI adducts. Bis-adducts (rather than mono-) were the major product of incubations of glutathione with TDI. Incubation of the adduct with a peptide resulted in transfer of one of the glutathione cysteine bonds to the peptide to form the glutathione-TDI-peptide conjugate plus free glutathione. There was no formation of diamine. 

 

In vitroexperiments comparing the reactivity of the 2,4 and 2,2-MDI isomers with the 4,4-MDI show that the molecules generally react similarly with GSH (Wisnewski et al,. 2018). In these experiments, each isomer was reacted with 20 mM GSH at 0.1% w/v MDI, a 5:1 molar ratio of GSH to MDI, which is a slight excess of GSH’s reactive SH to MDI’s N=C=O groups (i.e., 2.5:1), for varying time periods ranging from 1 minute to 2 hours. Reaction products of different MDI isomers with GSH were assessed through LC-MS/MS and quantified based on UV light absorbance. The 2,2’- and 2,4’ MDI isomers were found to react rapidly with glutathione and demonstrate marked similarities with that previously described for 4,4’ MDI. Under physiologic conditions (aqueous phase, pH 7.4, 37oC), 2,2’ and 2,4’ MDI rapidly form primarily bis(GSH)-MDI reaction products with GSH, at initial rates that are indistinguishable from those observed with 4,4’ MDI and is dependent on the rate of dissolution of the MDI into the aqueous matrix. However, following prolonged reaction times with GSH (10 - 120 minutes), 2,2’ and 2,4’ MDI formed relatively greater amounts of Cyclized mono(GSH)-MDI conjugates (mono(GSH)-MDIcy). Observed kinetics suggest that these cyclized conjugates form via intra- or inter-molecular rearrangement of bis(GSH)-MDI (vs. direct reactivity of GSH with MDI). The presence of mono(GSH)-MDI*NH2, generally only at later time points, along with prior studies on 4,4’ MDI by Reisser et al., also supports potential dynamic formation of mono(GSH)-MDI secondary to bis(GSH)-MDI formation. Given that mono(GSH)-MDIcy may be stabilized by cyclization, and less likely to transcarbamylate self-molecules, we speculate the 2,2’ and 2,4’ isomers may be less likely to induce allergy or toxicity. 

 

Further since water in airway fluid may cause hydrolytic instability of MDI (and complete hydrolysis would result in its corresponding aromatic diamine, MDA) the potential hydrolysis of different MDI isomers under physiologic condition was evaluated via LC-MS and compared to spiked samples spiked with standards. MDA was below the limit of detection (0.03 μM or < 0.001% of the starting material) at all the time points tested (1 minute to 2 hours) following 2,2’, 2,4’, and 4,4’ MDI reactions with GSH in aqueous solution.

 

In conclusion the isocyanate moiety in thiocarbamates is readily transferred to sulfhydryl- and amino groups providing a mechanism for absorption of the reactive isocyanate in a masked form and subsequent transcarbamoylation with systemic proteins. All isomers of MDI react similarly with the primary nucleophilic molecules in the lung to initially form the same conjugates. In the case of the 2,4’ and 2,2’ isomers, these conjugates show a tendency to over time transform into more stable and potentially less toxicologically relevant variants, as compared to the 4,4’ isomer.

 

Application of biomonitoring to humans:

The methods to identify and quantify MDI-adducts to plasma proteins particularly albumin (Alb) and to haemoglobin (Hb) have now been applied to biological monitoring, particularly useful since the amount of adducts would be indicative of an integrated exposure. In addition these adducts are specific for MDI exposure. However the total amount of the analyte MDA, retrievable from Hb-adducts and urinary precursors, accounts for less than 0.5 % of the applied dose of MDI (Pauluhn et al, 2006), and the lack of linearity of biomarker to exposure dose makes uncertain the extrapolation from the yield of biomarkers in urine or blood towards inhalative MDI exposure. The use of protein adducts for biomonitoring appears to overcome some of these difficulties and benefits from specificity of the analyte. To date no diisocyanate specific urinary biomarker of MDI exposure has been identified. For blood, the MDI-specific methods developed are: Hb-conjugate derived hydantoin, Alb-lysine conjugates and peptide conjugates. On the basis of limits of detection, the Hb-hydantoin method is most sensitive compared to the Alb-lysine method which in turn is more sensitive than the signature peptide method. The Hb-hydantoin method covers a longer period of exposure than the Alb-lysine, due to the longer half life of the erythrocyte compared to serum albumin.

Biomonitoring for exposure to diisocyanates typically looks to assay derivatives (diamines) following hydrolysis of biological fluids and is routinely employed to measure occupational exposure to MDI and other diisocyanates. For diamine analysis, samples of urine are typically used, although blood samples can also be used. However, these markers are not specific for the diisocyanate exposure. Schütze et al (1995) was the first report of biomonitoring of MDI exposed workers for biomarkers in urine and in blood as Hb adducts. The report indicated that the urine biomarkers (after acid or base hydrolysis) reflected recent exposures whereas the Hb biomarkers did not necessarily correlate with the urine biomarkers, and were considered to reflect overall exposures over a longer term. The hydrolysis methods and conditions used release differing amounts of the diamine analyte (Sennbro et al 2003; Skarping et al 1994). Hydrolysis to release MDA as the analyte is not specific to MDI, for example MDA exposure will also give MDA as analyte after hydrolysis.  Hydrolysis analytes are at low concentrations and proportionally little of the dose is in urine or blood, and that there is no standardised method for measuring biomarkers in hydrolyzed urine. Investigation of diisocyanate specific biomarkers has focused on the conjugated molecules in blood. Typically, conjugates with Hb or albumin (Alb) have been assessed, and recently there has been progress in application of experiments in animals to biomonitoring of human exposures to MDI.

Gries and Leng (2013) have successfully developed a method to detect the haemoglobin adduct 5-isopropyl-3-[4-(4-aminobenzyl)phenyl]hydantoin (ABP-Val-Hyd) in human blood. They reported values up to 5.2 ng ABP-Val-Hyd/g globin (16 pmol/g) in blood samples of workers exposed to MDI. The analytical method focused on optimal sensitivity and selectivity, using gas chromatography–high resolution mass spectrometry (GC-MS). A detection limit of 0.02 ng ABP-Val-Hyd/g globin (0.062 pmol/g) was achieved, with a linear detection range up to 10 ngABP-Val-Hyd/g globin (31 pmol/g). The authors conclude the method described is optimized for screening studies of the human population.

Others have used the MDI-Alb conjugate to derive specific biomarkers. Sabbioni et al (2010) used post-shift blood and urine samples from potentially MDI-exposed workers. Alb was isolated and subject to a pronase digestion and the digest treated to isolate MDI-lysine and acetyl-MDI-lysine conjugates. Quantification was by LC-MS/MS. The MDI-Lysine conjugate was identified in about 63% of workers, the range being 0 – 900 fmol/mg Alb. The authors found the method to detect exposure in more individuals than did the hydrolysis of Hb method, but the urinary hydrolysis method detected exposure in all individuals including unexposed controls, which illustrates the need for a specific biomarker. Luna et al (2014) similarly used a trypsin digestion stage to derive signature peptide conjugates of MDI, but concluded that the peptide method was about 18 times less sensitive than the MDI-lysine method.    

The binding of MDI to Alb appears to be an important area of research for both the use of an MDI-specific biomarker of exposure, and for investigation of the ultimate antigen involved in the sensitisation process. Wisnewski et al (2010) identified 14 MDI conjugation sites (12 lysines and 2 asparagines) on human Alb and highlighted reaction specificity for the second lysine in di-lysine motifs which may be a common characteristic of ‘‘immune-sensitizing” chemicals. Several of the MDI conjugation sites are not conserved in albumin from other species, and this may suggest species differences in epitope specificity for albumin–isocyanate conjugates.

All methods require sophisticated laboratory facilities and capability, but can be used for practical biomonitoring for exposures specific to MDI. It may be that biomonitoring can be a two tier process, with urine analysis as a simple first step and protein adduct analysis for specific detailed investigations.