4,4'-methylenediphenyldiisocyanate and 2,4'-diisocyanatodiphenylmethane and their oligomerisation reaction products with 2,2'-oxydiethanol, propane-1,2-diol and butane-1,3-diol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro genotoxicity assays performed with MDI solubilized in DMSO should be interpreted with care, as in this solvent, MDI is chemically converted into MDA, a substance known to produce positive responses in in vitro genotoxicity assays.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Pauluhn and Gollapudi (2001) showed in a reliable rat micronucleus study that aerosolised, inhaled MDI at concentrations as high as 118 mg/m³ air failed to induce formation of micronuclei and cytogenetic damage in vivo.

Randazzo (2017) showed in a reliable in vivo Comet Assay that aersolized, inhlation MDI at the maximum tolerated concentration did not produce DNA strand breaks at the portal of entry (lung), stomach, or liver.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional 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.

Genetic toxicity in vitro

Aromatic diisocyanates are virtually insoluble in water and require a solvent to ensure homogeneous dispersion in in vitro genotoxicity assays. Dimethylsulphoxide (DMSO) has been used routinely as the solvent of choice for such assays and when used in an Ames test to assess diisocyanate mutagenicity, positive results were often obtained (only in TA98 and TA100 strains). However, further work demonstrated the selection of the solvent resulted in these positive results. Gahlmann et al. (1993) found there is a chemical conversion of MDI to MDA in the solvent which could explain a number of positive responses recorded in some genotoxicity assays in vitro.

 

A detailed evaluation of the stability of MDI in DMSO by Herbold et al. (1998b) and Seel et al. (1999) showed that there was a rapid breakdown of MDI in DMSO with less than 40% of the initial amount remaining after 15 minutes with almost complete breakdown within 2 hours. A HPLC examination of the breakdown products showed the presence of MDA which is known to produce positive responses in various in vitro genotoxicity assays including mutations in Salmonella typhimurium. To determine if the positive results seen in in vitro genotoxicity assays in which MDI was dissolved in DMSO were in fact a consequence of the chemical conversion of MDI to MDA, Herbold et al. (1998a) (key study with reliability 2) and Seel et al. (1999) undertook a series of mutagenic investigations using dry ethyleneglycol dimethylether (EGDE) as the organic solvent, as investigations indicated MDI was stable in this solvent with no formation of MDA.

 

The studies with Salmonella typhimurium showed quite clearly the absence of any mutagenic response when MDI was dissolved in EGDE. In contrast when MDI was dissolved in DMSO clear positive effects were seen in the presence of metabolic activation consistent with generation of MDA. Based on such evaluation it was concluded that the results of in vitro genotoxicity studies undertaken using solvents such as DMSO must be treated with caution as any positive response may very well be an artefact of the testing conditions caused by the breakdown of the isocyanate into the mutagenic amine.

 

Based on these observations the use of results from in vitro tests in aqueous cell systems are problematic because of physical chemical characteristics of the MDI and interaction with the test system components. Those in vitro studies not addressing problems of solvent selection and hydrolysis of the substance (which represent the majority of in vitro investigations) are considered to be invalid, and not useful for determining the genotoxic potential of MDI. Because of these technical problems conducting mammalian cell gene mutation assays in vitro is challenging and interpretation of data problematic, thus an assessment relies on data from in vivo studies.

 

Genetic toxicity in vivo: systemic

A number of in vivo genotoxicity studies have been undertaken using MDI. A mouse micronucleus study has been reported in summary form by JETOC (1982). In summary, MDI was dissolved in dry DMSO, mixed with corn oil and administered to mice by intra-peritoneal injection at doses of 32, 80 or 200 mg/kg. (Six male mice per treatment group). The mice were killed 24 hours following final treatment and incidence of polychromatic and normochromatic erythrocytes with micronuclei evaluated. There was no difference in incidence of micronuclei between animals treated with MDI and the untreated control group. It was concluded that MDI did not cause micronuclei, and therefore did not induce in vivo genotoxic effects.

 

Pauluhn and Gollapudi (2001) (key study with reliability 1) conducted a guideline, GLP, inhalation rat micronucleus study. In this study, Brown-Norway rats were exposed using two different technique: 1) using a Whole Body inhalation set-up, animals were exposed to aerosolised MDI at concentrations of 9.2 or 118 mg/m3 and 2) an additional group was exposed Nose Only to MDI aerosols at a concentration of 11.0 mg/m3. Micronuclei in polychromatic erythrocytes were counted in bone marrow smears prepared after the final exposure on post-exposure days 1, 2 and 7 and stained with acridine orange or Wright-Giemsa. The results of this study indicate that aerosolised, inhaled MDI at concentrations as high as 118 mg/m³ air (a concentration high enough to produce portal-of-entry specific toxic effects, including statistically significantly increased lung weights), failed to induce formation of micronuclei and cytogenetic damage in vivo. While effects on the ratio of polychromatic to normochromatic erythrocytes (PN ratio) or other indications of systemic toxicity are not observed, results from Gledhill (2001a) investigating the “Excretion and tissue distribution in the rat following inhalation exposure to [14C]-MDI at 2 mg/m³ for 6 hours”, indicate that it can be assumed there was indeed exposure of the bone marrow.

 

Lindberg et al. (2011) investigated the genotoxicity of inhaled MDI in male C57Bl/6J mice by examining micronucleated polychromatic erythrocytes (PCE) in bone marrow and peripheral blood. Mice were exposed head-only to MDI aerosol (mean concentrations 10.7, 20.9 and 23.3 mg/m³), 1 h/day for 5 consecutive days. Bone marrow and peripheral blood were collected 24 h after the last exposure. Haemoglobin adducts detected in the exposed mice resulted from direct binding to globin of MDI, and adducts originating from the diamine (MDA) were not observed. No significant increase in the frequency of micronucleated PCEs was detected in the bone marrow or peripheral blood of the mice exposed to MDI. The authors concluded that inhalation of MDI (1 h/day for 5 days), at levels that induced toxic effects (decreased respiratory frequency, decreased body weights and an influx of inflammatory cells into the lung were observed) and formation of MDI-specific adducts in haemoglobin, did not have detectable systemic genotoxic effects in mice, as investigated by the micronucleus assay.

 

In a study by Vock and Lutz (1997), female Wistar rats were treated with [14C] 4,4’-MDI in dried acetone on the back. Faecal excretion of radioactivity amounted to 20% of the administered radioactivity within 24 hours. Urinary excretion was below 1%. About 10% of the radioactivity was retained at the site of application. Epidermal nuclear protein exhibited very high specific radioactivity. 32P-postlabelling analysis did not reveal isocyanate-DNA adducts. The nuclear protein radioactivity in the liver, lung and kidney was much lower than in the epidermis. DNA radioactivity in the liver was at the limit of detection. Conversion to the units of the Covalent Binding Index, CBI = (µmol adduct/mol DNA nucleotide) per (mmol chemical administered/kg body weight) resulted in a value of <0.1. The presence of 2% MDA in the application solution could have contributed about 0.03 CBI-units to the measured values. In comparison with genotoxic carcinogens, the upper bound value is indicative of a very weak maximum possible systemic genotoxic potency of topically administered MDI.

 

Based on the available data it can be stated that even though exposure of the tissues is observed no genotoxic effects are observed.

 

Genetic toxicity in vivo: local

A non-guideline exploratory Comet Assay was employed to investigate the genotoxic potential of MDI in bronchoalveolar lavage (BAL) cells of male rats (Sutter, 2016). As there was no OECD guideline for the Comet Assay in force before Sept 2014, the study was performed according to published recommendations from international expert groups which were developed in accordance with OECD guidelines for other genotoxicity tests. In this exploratory study six male rats were nose-only exposed to the particulate aerosol of MDI at target concentrations of 10 – 180 mg/m3 for 3 or 6 hours. Air-exposed animals were used as negative control. As positive control, another group of male rats was treated by gavage with methylnitroso urea (MNU) at 50 mg/kg. In addition, paraffin was given by inhalation exposure as a non-genotoxic reference reported to activate inflammatory signalling in alveolar macrophages. Due to the physicochemical properties of MDI and paraffin in combination with the exposure setup used in this study it is considered that the test substances reached the lungs of the rats as solid particles. Six male rats of each treatment group were sacrificed within 0-3 days (most animals were sacrificed within 1 day post-exposure) after single exposure. After lavage of the lung, BAL cells were obtained. Cellular biological parameters were analysed to assess exposure and local cytotoxicity of the target cells, and to assess the biological relevance of Comet Assay results and included measurements for apoptosis, inflammation, necrosis and blood/air barrier dysfunction. After single exposure to MDI, all treated males showed compound-related symptoms (e.g. apathy, convulsions, piloerection, laboured breathing, eyelids narrowed, and weight loss) at all tested concentrations. More than 50% of animals treated with the positive control presented with piloerection and weight loss. In contrast, no symptoms were recorded for the negative control and paraffin groups. No animals died in any of these groups. Exposure of the target cells to MDI induced a significantly increase of at least one of the two assessed apoptosis parameters (Caspase and Annexin) at any dose / time point combination. Moreover, exposure to MDI increased lactate dehydrogenase activity and protein concentration in BAL fluid and decreased viability (indicated by Resazurin reduction, an indicator for metabolic activity) of BAL cells, pointing to cytotoxicity. Inflammation induced by MDI but not by MNU was evident from a concentration and time-dependent increase of polymorphonuclear cells (%) and foamy macrophages (%). Similarly, exposure to paraffin increased the number of Annexin V positive cells and caspase activity and alveolar macrophages with red blood cells and/or particular matters, pointing to induction of apoptosis and inflammation. Results are similar to previous studies demonstrating local apoptotic and inflammatory responses following acute inhalation exposures. The Comet Assay in BAL cells revealed statistically significant increases of mean tail intensity values between the air control groups and treatment groups exposed to 20 mg/m3 MDI (3 hr exposure) and above (3 hr and/or 6 hr exposure). Similarly, the non-genotoxic reference compound paraffin (25 mg/m3, 3hr, day 1) led to a statistically significant increase of tail intensity, which is a proof of concept demonstrating inflammation-induced genotoxicity, of a substance that is in itself non-genotoxic. In contrast, no statistically significant increase was found for BAL cells exposed to 10 mg/m3 MDI (6 hr exposure, day 1). The positive control MNU induced a pronounced increase in tail intensity, thus demonstrating the sensitivity of the test system to detect primary DNA damage. In conclusion, MDI induced DNA strand breaks in BAL cells of Wistar rats following a single inhalation exposure at concentrations of 20 mg/m3 (3 hr exposure, examined day 0) and above. Yet, these results do not point to primary, test substance-induced DNA damage as cytotoxicity, apoptosis and/or inflammation were induced in parallel and the non-genotoxic reference compound paraffin similarly induced apoptosis, inflammation and DNA strand breaks. The no observed adverse effect concentration (NOAEC) for MDI related to increase of tail intensity found in this exploratory Comet Assay is 10 mg/m3 over 6 hours; at that concentration acute direct respiratory toxicity (e.g. increase of protein and foamy macrophages) was still observed which is in line with what is known from earlier inhalation toxicity studies. Considering concentration and exposure duration role (Cxt) in observed toxicity, critical threshold for cytotoxicity induced DNA damage is around 60 mg/m3 x h (> 10 mg/m3 at 6h exposure and ≥ 20 mg/m3 at 3h exposure).

 

The genotoxic potential of the test substance to male rats was investigatedin a combined in vivo mammalian alkaline Comet Assay/acute inhalation toxicity study according to OECD TG 489 (genotoxicity assessed by Comet Assay) under GLP conditions (Randazzo, 2017). This study was performed to assess if 4,4’-MDI is a genotoxic substance at the site of contact. As the site of contact tissue, bronchoalveolar lavage cells were selected; these primarily consist of alveolar macrophages, which are the primary cells responsible for the removal of inhaled aerosols from the alveoli and are commonly selected in the assessment of pulmonary genotoxicity after inhalation or instillation. In addition, the liver was included since it is the site of primary metabolism; also, it was included to investigate systemic (as opposed to local) genotoxicity. Finally, the glandular stomach was included due to possible exposure after clearance of 4,4’-MDI via the mucociliary escalator. Groups of 12 Wistar rats were exposed to actual concentrations of2.5, 4.9 and 12mg/m3 (corresponding nominal concentrations: 2, 5, 11 mg/m3) to an aerosol-generated form of the test substance via a single nose-only inhalation exposure for 6 hours. The top concentration was defined as the maximum tolerated concentration (MTC) from a preliminary range-finding study and other supporting acute sub-lethal inhalation studies. The MTC was selected based on marked local acute toxicity as identified by biomarkers forinflammation, apoptosis/necrosis and cytotoxicity at concentrations ≥ 11.9 mg/m3. A concurrent control group received filtered air on a comparable regimen. A positive control group received ethyl methanesulfonate (EMS) via oral gavage (200 mg/kg/day) for 2 days. In preliminary studies at the performing laboratory, gavage administration of EMS resulted in a strong positive signal in all tissues examined as was determined appropriate for identifying direct acting genotoxicity. Bronchoalveolar lavage (BAL) wasperformed in all animalsat the scheduled necropsies, and the BAL fluid (BALF) was assessed for the following endpoints: clinical chemistry (alkaline phosphatase, lactate dehydrogenase, and total protein), cell differential (cytology), and measurement of Annexin V expression and β-glucuronidase activity.The alkaline phosphatase, lactate dehydrogenase, Annexin V expression + flow cytometry, and total protein were determined to assess the cytotoxicity of the test substance. The β-glucuronidase and cell differential (in particular % polymorphonuclear leukocytes) were determined to assess the inflammatory potential of the test substance.Six rats/groupwere sacrificed approximately1 hour post-exposure and the other six/group approximately 18 hours post-exposure, or 2 to 4 hours after the second dose for the positive control group.At the high concentration of 12 mg/m3, test substance-associated differences in BALF endpoints (neutrophil influx, total protein, β-glucuronidase, and Annexin V) were observed. Therefore, 12 mg/m3 was confirmed to be the MTC.The test substance gave a negative (non-DNA damaging) response in thisassay in the BAL cells, liver and stomach for both the 1 hour and 18 hour time points for males in % Tail DNA. It was therefore concluded that the test substance scored negative in the in vivo Comet Assay up to the maximum tolerated concentration.

 

Genetic toxicity human data

Some human exposure studies have reported possible alterations in DNA status (e.g. Szekely and Gundy, 2009; Pitarque, 1999; 2002; Marczynski et al., 1992; 1994a, 1994b; 2003), data from these studies have been critically reviewed by Greim in the MAK collection for Occupational Health and Safety (Greim, 2008). In this document, it was concluded that these studies suffer from substantial methodological problems or uncertainties. Therefore, the relevance of these results is considered questionable. In a valid comet assay conducted in accordance with present-day requirements, No increase in DNA strand breaks was observed in the lymphocytes of the exposed patients (Marczynski et al. 2005).

 

Overall conclusion

- Weight of scientific evidence supports the conclusion that MDI (or any reaction product) is not mutagenic or genotoxic either systemically or at the portal of entry.

- As MDI is unstable in solvents such as DMSO and degrades rapidly to MDA, results from the majority of in vitro genotoxicity test results are unsuitable for assessing the genotoxic potential of MDI. When an organic solvent was used that does not result in MDA formation, in vitro genotoxicity test were negative.

- In vivo micronucleus studies in mouse and rat using contemporary protocol design and conducted according to GLP are negative, demonstrating no systemic mutagenic potential.

- In vivo mammalian alkaline Comet Assay in lung (BAL cells), liver and glandular stomach according to OECD guideline 489 and GLP is negative. Results demonstrated that MDI (or any reaction products) is not genotoxic at the portal of entry (lung) for the most relevant route of exposure (inhalation). In addition no systemic genotoxicity of MDI or reaction products was observed in liver or glandular stomach of rats.

- MDI shows no capacity to form DNA adducts.

Justification for classification or non-classification

Not classified as mutagenic according to DSD or CLP.