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EC number: 701-124-4
CAS number: -
The test substance is part of a category approach of methylenediphenyl diisocyanates (MDI) with existing data gaps filled according to ECHA guidance on Read Across (ECHA, 2017). The read-across category justification document is attached in IUCLID section 13. 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.
MDI is not classified as a reproductive toxicant according to Regulation (EC) No 1272/2008 (CLP). For MDI, there is evidence that the strong respiratory irritation restricts exposure and ensures that there will be no exposure at levels at which there is a realistic possibility of reproductive toxicity.
Testing proposed to further support MDI category and proposed MoA for reproductive toxicity. Two OECD 443 Extended One-Generation Reproductive Toxicity Study is proposed to be conducted on 4,4'-MDI and 4,4'-MDI/DPG/HMWP (see test proposal for these substance for more details on design). As the boundary substances representing the material with the most and least bioaccessible NCO, respectively, within the MDI Substance category, results will confirm that MDI substances do not induce effects on fertility and reproduction.
OECD 422 Combined Repeated Dose and Reproduction and Development Screens will be conducted 9 MDI substance category members representing each sub-group and major polyol linkage (including the boundary substances). These studies will be used to support bridging of data from boundary substances. Additional definitive reproduction studies will be considered if results from the screening/bridging studies suggest potential unanticipated effects.
Additional supporting information is provided by the following:
(1) the absence of any type of systemic toxicity including toxicity to reproductive organs in subchronic and chronic inhalation studies in rats at concentrations resulting in irritation to the respiratory tract,
(2) Evidence for a lack of systemic availability of toxicologically active parent or metabolite, and
(3) Absence of effects on reproduction and fertility in a guideline two-generation reproductive toxicity study on an analogue aromatic diisocyanate TDI,
Analogous evidence from TDI
The toxicity of TDI on fertility was investigated in a two-generation inhalation study in rats (OECD 416). Rats were whole body exposed to vapors of an 80/20 mixture of 2,4´- and 2,6´-isomers of TDI at concentrations of 0, 0.2, 0.8, 2.9 mg TDI/m3 for 6 hours/day for 5 days/week during the pre-mating period and 7 days/week during mating period and subsequent in life period. No effect of exposure on any of the reproductive parameters (e.g. reproductive performance) or organ weights, sexual maturation, viability, or gross- or histopathology were observed in the F0 and F1 generation. The only signs of toxicity were transient irritations of the upper respiratory tract. The NOAEC for fertility was the highest tested concentration of 2.9 mg/m3 (Tyl et al., 1999b).
Lack of systemic toxicity
As confirmed by literature (Ulbrich and Palmer, 1996; Mangelsdorf et al., 2003; Dent, 2007; Janer et al., 2007; Sanbuissho et al., 2009) rodents’ histopathological examinations in repeated dose toxicity studies of reproductive tissues are of high value and high sensitivity for evaluation of reproductive toxicity in males and females. For example, the review of Mangelsdorf et al. (2003) outlines that of the endpoints investigated for detecting adverse effects of chemicals on male reproduction in animal species, the most sensitive proved to be histopathology of the testes. Only in some cases sperm motility was found to be more sensitive than histopathology. The above parameters showed a higher sensitivity than fertility parameters. Summarizing these reviews, histopathological changes in the reproductive organs in repeated dose toxicity studies are highly indicative of effects on fertility. In this respect repeated dose toxicity studies for MDI substances are considered in this weight of evidence, since they provide sensitive and sufficient information to evaluate toxicity on fertility if histological examination of the reproductive organs is available (Blackburn et al., 2015).
In a chronic inhalation study with respirable aerosols of 4,4’-MDI, 80 female rats per dose group were whole-body exposed to atmospheres of 0.23, 0.70 or 2.05 mg/m³ for 17 hours/day, 5 days /week for up to 24 months. Full pathological examination was done on 20 rats/dose of a 12 months exposure group and 20 rats/dose of a 24 months exposure group. Reproductive organs assessed included e.g. adrenals, ovaries, uterus, vagina and mammary gland. Again compound-related changes were found in the respiratory tract (LOAEC 0.23 mg/m³), but no treatment related findings on reproductive or any other systemic organ effects were reported (Hoymann et al. (1995); see repeated dose toxicity).
In a subchronic inhalation toxicity study with pMDI 30 males and 30 females each, were exposed to 0, 4.1, 8.4 and 12.3 mg respirable pMDI/m³ for 6 hours/day, 5 days/week for 13 weeks (followed by a four-week post-treatment). Reproductive organs and tissues were micro and macroscopically and gross pathologically assessed at the autopsy. A sensitive and detailed histopathological assessment was performed of 10 rats/sex of the control group and 20 rats/sex of the high-concentration group at the end of the exposure (week 14) and of 10 rats/sex of the control and high-concentration group at the end of the posttreatment period (in week 18). Histopathological assessment included e.g. adrenals, epididymides, mammary glands, seminal vesicles, testes and uterus. Neither gross examination at autopsy nor histopathological assessment revealed any treatment related systemic effects. The only treatment related effects were confined to the respiratory tract (increased relative lung weight ratios in the mid- and high concentration groups, histopathological and microscopic changes in all pMDI exposed groups) (Reuzel et al. (1994b), see repeated dose toxicity).
Data on macroscopy, gross pathology and histopathology in the reproductive organs of both sexes can furthermore be derived from a 24-month chronic inhalation toxicity and carcinogenicity study of respirable pMDI aerosol in rats. In this study 70 rats/sex/group (each group subdivided into one satellite group of 10 rats/sex and one main group of 60 rats/sex; exposure of the satellite group was limited to one year) had been exposed at concentrations of 0, 0.2, 1.0 and 6 mg/m³ for 6 hours/day, 5 days/week. In the satellite groups histopathological examinations were carried out on several organs and of all gross lesions of all rats of the two-year study (e.g. including epididymides, mammary glands, ovaries, prostate, seminal vesicles, testes and uterus). Compound-related changes were exclusively found in the respiratory tract (NOAEC 0.2 mg/m³) but no treatment related findings on reproductive or any other systemic organ effects were reported (Reuzel et al. (1994b); see repeated dose toxicity).
Collectively these supporting studies demonstrate that even with a chronic/lifetime exposure duration with group sizes equivalent to or greater than guideline reproductive studies, effects from pMDI and 4,4’-MDI aerosols are confined to the lungs. Effects on systemic organs including reproductive organs were not observed at exposure concentrations associated with marked respiratory tract toxicity.
Lack of systemic bioavailability
The common hypothesized MoA for acute and repeated toxicity of MDI substances through local reactivity of the NCO group is responsible for the predominance of effects on tissues at the portal of entry. Indeed, as aerosols of MDI substances enter the lung, most form insoluble and inert particles that are removed via mucociliary clearance with no absorption. The remaining NCO groups on low molecular weight bioaccessible MDI substances will react with biological nucleophiles (primarily GSH) in the protective lung fluid to become reactively solubilized and act as a shuttle for the conjugated MDI to move into circulation. Once in the more alkaline and GSH limiting environment of the cell and circulation, the labile GSH-MDI is transcarbamoylated to MDI-protein adducts or is metabolized via acetylation and hydroxylation and excreted in the urine. This lack of systemic exposure to the reactive NCO group is the basis of the lack of systemic toxicity and the claimed lack of concern for effects on the reproductive system.
Experimental in vitro reactivity studies (See Category Justificaiton Document Chapter 184.108.40.206) show that mMDI (which contain the most bioaccessible NCO groups) react similarly when they are alone (as mono-constituent substance) (Wisnewski et al., 2019a) or when part of a UVCB substances (Zhang et al., 2021). In vitro reactivity experiment by Zhang et al. (2021) demonstrated that the mMDI constituents in modified MDI substances followed a similar pattern of glutathione adduct formation seen previously (Wisnewski et al., 2019a). However, when pMDI was incubated with GSH, adducts with the higher molecular weight constituents (>3 ring oligomer) were not detected in subsequent GC/MS. Additional solubility experiments (Zhang et al., 2021) suggest that these constituents are not soluble and thus unable to react. In vivo inhalation experiments with pMDI by Pauluhn and Lewalter (2002) demonstrated that the increasing the molecular weight (e.g. 3-ring oligomers) reduces the likelihood of absorption of these molecules. Thus, next to mMDI even the most soluble constituent of the MDI category substances, the 3-ring oligomer showed a significant reduction in bioaccessibility. Further increasing the molecular weight (either by condensation, or glycol adduct formation) even further reduces this potential bioaccessibility of the molecule. Taken together, it is clear that the non-monomeric MDI constituents of modified MDI substances virtually eliminates the availability of these constituents to react with nucleophiles and elicit a toxicological effect.
Another aromatic diisocyanate substance, toluene diisocyanate (TDI) also contains reactive NCO groups and is used for analogous read-across. As with MDI substances, the highly reactive nature of the NCO group of TDI with the thiol of glutathione makes it the primary reaction partner (Wisnewski et al., 2011a) and acts as the carrier for systemic absorption in the form of conjugation adducts. Further, following absorption of adducts of both MDI and TDI substances, transcarbamoylation with proteins to form hemoglobin and albumin adducts will occur in the low GSH and alkaline pH environment of the cell and plasma (Wass and Belin, 1989; Day et al., 1997; Lange et al., 1999; Wisnewski et al., 2011a)
As described in detail in other sections (See AE C.6 in Chapter 3.2 of the Category Justification Document), differences in physico-chemical properties between TDI and MDI substances will result in differences in accessibility and deposition (TDI has higher vapor pressure and water solubility compared to 4,4’-MDI) as well as physical availability and deposition in the respiratory tract result in higher exposure, irritancy and toxicity of the respiratory tract of TDI versus that of 4,4’-MDI (Pauluhn, 2004). While this these differences are important for the quantification of local hazard potency, in for example acute and repeated dose studies (and secondary developmental toxicity), once the free NCO group is present in the alveolar fluid of the lung fluid the toxicokinetic behavior of each molecule is comparable. As fertility and direct toxicity on the fetus are systemic effects, TDI is an appropriate analogue for these effects. Thus, data on the reproductive hazard of TDI, or lack of it, can inform on the reproductive hazard of the substances of the MDI category.
A need for an oral hazard assessment for MDI is not indicated by its use or by toxicokinetic profile of exposure routes. Furthermore, oral toxicity data for MDI cannot be extrapolated for risk assessment of inhaled aerosols as the relevant route of exposure for human risk assessment. Therefore, there is no apparent benefit from any oral toxicity data for MDI. Toxicokinetic data for the inhalation route of exposure is sufficient, and the performance of an additional oral animal toxicity study would not create data that would influence the risk management measures and therefore would be in conflict with the principles of animal use and welfare. These differences are described below:
Route of exposure specific differences in MDI metabolism
4,4’-MDI (MDI) contains two highly reactive NCO-groups, which are responsible for the distinct portal of entry toxicity described by the available toxicological data. The NCO-group reacts readily with nucleophilic biomolecules, and depending on the chemical and physico-chemical composition of the interphase at the site of primary contact, distinct differences in primary reaction products can be described. Therefore significant differences in bioavailability and metabolic fate can be described for the oral, dermal and inhalation route of exposure (see end point summary on toxicokinetics\):
In conclusion the toxicokinetic behaviour of MDI needs to be considered with respect to the specific physico-chemical and chemical exposure conditions at the site of first contact. For MDI, significant differences in primary reaction products and by this in the subsequent bioavailability and metabolic fate imply a high level of uncertainty for route to route extrapolation of toxicological data. In accordance to REACh Annex I (0.3.), the chemical safety assessment of a substance shall be based on a “…known or reasonably foreseeable exposure…”, accidental exposures are not considered. Therefore, and in accordance to ECHA Guidance Chapter R.7a and the REACh Annexes on information requirements the potential hazard of MDI should be determined on the most relevant route of exposure for risk assessment which is inhalation.
Relevance of oral route of exposure for risk and hazard assessment:
ECHA Guidance Chapter R.14 (Occupational exposure estimation, R.14.2 Types and routes of exposure) indicates that, exposure through ingestion is “…generally not considered further in the assessment of workplace exposure“. For proof of concept, working processes for professional uses of MDI include handling and spray application of foams in concentrations up to max 30 % pbw. Other uses include handling and application of coatings, adhesives and paints in which MDI is not contained as such but as pre-reacted high viscous polymer with low monomer content. For spray applications in which aerosols are generated respiratory protection by full mask, and for all application’s effective dermal protection by full body protection and gloves is prescribed. These risk reduction measures effectively prevent from any oral exposure, e.g. via contaminated skin or clothing or inhalation cross-contamination. Cured PU applications, e.g. spray foam, do not contain residual unreacted MDI (see Topic 6 below). In conclusion, no significant uncertainties regarding a potential oral exposure can be anticipated in the holistic exposure assessment for professionals.
ECHA Guidance Chapter R.15 (Consumer exposure estimation, R.15.2.2 Reasonable worst-case situations) indicates that, “…the consumer exposure estimation should normally address the intended uses of the products that contain the substances under investigation. However, since consumers may not accurately follow instructions for use of products, an estimation of other reasonably foreseeable uses should be made. Consideration of deliberate abuse is not part of the exposure estimation process.” Consumer uses include one component rigid foam available in cans as well as coatings, adhesives & sealants, and paintings. These uses contain pre-reacted and polymerized MDI derivatives (prepolymers and higher oligomers originating form polymeric MDI (PMDI)) with low amounts of residual monomer. Reaction is readily with humidity in the air, resulting in entirely cured product, free of residual MDI.
Consumer uses of MDI are covered by national regulations, which e.g. restrictively prescribe selling in correspondingly equipped DIY stores, if the consumer agrees to information provided by the staff addressing the risk. In addition, the appropriate storage and handling is explicitly prescribed, e.g. by the recommendation to use gloves which are delivered with every can as part of an existing restriction under REACH Annex XVII as follow up of the 2005 Risk Assessment. Therefore, there is a very low possibility for oral exposure by the intended use, since e.g. skin contact should be prevented by the use of gloves, and by this dermal to oral cross-contamination is minimized. Since cured PU applications, e.g. spray foam, do not contain residual unreacted MDI (see Appendix 1 on Toxicological information, DNEL justification provided as a separate document) migration from articles through sucking, chewing or licking can likewise be excluded. In addition, no foreseeable misuse other than abuse has to reasonably be anticipated due to specific regulations of the consumer application.
Justification for selection of Effect on fertility via inhalation route:
In absence of an extended One- (or a Two Generation) Reproductive Toxicity Study for MDI a weight of evidence is presented based on three independent sources of information including
As all substances of the MDI category as well as the analogous substance TDI contain significant quantities of bioaccessible NCO groups required for the hypothesized MoA. Furthermore, it is likely that these bioaccessible NCO groups present in all substances of the MDI category have a similar reactivity profile with extracellular nucleophiles and are therefore not systemically available or toxic to reproduction. While modified MDI substances contain non-monomeric MDI constituents with NCO groups that may have slightly lower reactivity than NCO groups on unreacted mMDI, they are of higher molecular weight and therefore less soluble and less likely to be absorbed. No other effects are observed in any test on either the substances of the MDI category (specifically on reproductive tissues in repeated exposure studies) or in the analogues source substance TDI (full guideline study).
The key study performed on pMDI (which contains about 50 % mMDI) was conducted according to OECD Guideline 414 (Prenatal Developmental Toxicity Study) at concentrations of 0, 1, 4 and 12 mg/m3 (Gamer et al., 2000). At concentrations of 1 or 4 mg/m³, no signs of maternal toxicity and no substance-induced adverse effects on the gestational parameters or the fetuses were recorded. Maternal toxicity was substantiated by mortality, damage to the respiratory tract, reduced body weight development and reduced mean gravid uterus weights at 12 mg/m3. At this concentration clear signs of developmental (embryo-/feto-) toxicity in the form of reduced placental and fetal body weights and an increased occurrence of fetal skeletal (and overall) variations and retardation were recorded. However, no substance-induced teratogenic effects were observed up to and including the highest concentration (12 mg/m³). As the observed fetotoxic effects and adverse effects on the embryonic development are considered as minor signs of developmental toxicity and as these effects occur at the concentration inducing maternal toxicity, they are considered to be secondary to maternal toxicity, the NOAEC for maternal and fetal toxicity is 4 mg/m3. Consequently, pMDI is considered not to be a developmental toxicant.
In support of this conclusion, a study by Buschmann et al. (1996) performed according to OECD 414 using 4,4’-MDI at concentrations of 1, 3, and 9 mg/m3. The lung weights in the high-dose group were significantly increased compared to the sham-treated control animals. Treatment did not influence any other maternal and/or fetal parameters investigated (maternal weight gain, number of corpora lutea, implantation sites, pre- and post-implantation loss, fetal and placental weights, gross and visceral anomalies, degree of ossification), although a slight but significant increase in litters with fetuses displaying asymmetric sternebra(e) (within the limits of biological variability) was observed after treatment with the highest dose of 9 mg/m3. Conservatively, a no effect level of 3 mg/m3 for developmental and maternal toxicity was determined.
The findings of the studies were essentially the same with regard to the maternal and fetal NOAECs. Since the highest exposure concentration in the study on pMDI caused greater toxicity than in the study with 4,4’-MDI resulting in fetal effects (albeit secondary to maternal toxicity), this study is regarded as being of higher value and used as the key study for the assessment of developmental toxicity.
It is widely recognized that maternal conditions that cause decreased uterine-placental blood flow, decreased nutrient circulation, anemia, altered acid-base balance, hypoxia, obesity and others have been shown to contribute to fetal developmental toxicity. Some common developmental effects resulting from these maternal conditions include supernumerary ribs, delayed fetal growth, and skeletal variations, such as delayed ossification. For both 4,4’-MDI and pMDI, the only developmental effects noted are minor skeletal variations and slightly inhibited fetal growth and are only observed at concentrations that also induce significant reductions in maternal food consumption and body weight gain, and respiratory toxicity (e.g. labored breathing, increased lung weights). Reduced fetal weight and delayed ossification are common fetal manifestations induced by maternal toxicity (Banerjee and Durloo, 1973; Woo and Hoar, 1979; Tyl, 2012; Nitzsche, 2017) which suggests that the fetal effects noted in the 4,4’-MDI and pMDI studies are secondary.
While maternal toxicity should not automatically negate a fetal effect, there is also sufficient mechanistic data to support a lack of human relevance for fetotoxicity of MDI substances. OECD “Guidance Document on Inhalation Toxicity Studies” (no. 39) describes the rodent-specific physiological effects of respiratory irritants on inhalation developmental toxicity studies (OECD, 2018a). In short, rodents have a respiratory reflex that reduces respiration and body temperature and subsequently results in fetal hypoxia, hypercapnia, hypothermia and malnutrition. As the fetus is more sensitive to hypothermia and hypoxia, developmental defects and delays can develop. Isocyanates are known respiratory irritants and are included as an example of substances that induce the rodent-specific irritation reflex known to cause developmental effects. Thus, effects noted in these studies are not relevant to human exposure and MDI substances should not be classified as developmental toxicants.
Additional testing is also proposed to further support MDI category and proposed MoA for Developmental toxicity. An additional OECD 414 developmental toxicity study is proposed to be conducted on 4,4'-MDI/DPG/HMWP. As the boundary substance representing the material with the least bioaccessible NCO within the MDI Substance category, results will confirm that MDI substances do not induce effects on fetal development and toxicity.
OECD 422 Combined Repeated Dose and Reproduction and Development Screens will be conducted >= 8 MDI substance category members representing each sub-group and major polyol linkage (including the boundary substances). These studies will be used to support bridging of data from boundary substances. Additional definitive developmental toxicity studies will be considered if results from the screening/bridging studies suggest potential unanticipated effects.
Justification for selection of Effect on Developmental Toxicity via inhalation route:
Data gap filling is achieved using the category approach according to ECHA guidance on read-across (ECHA, 2017c). For the developmental toxicity endpoint, the fetal effects observed with 4,4’-MDI and pMDI are consistent and secondary to maternal toxicity as a consequence of respiratory irritation. Respiratory irritation, in turn, is consistent with the hypothesized MoA and direct electrophilic reactions of bioaccessible NCO groups. All substances of the MDI category share this common hypothesized MoA and since 4,4’-MDI and pMDI have the highest bioaccessible NCO content, they are considered the worst-case substance and can be used as the source for read-across to all substances of the MDI category with high confidence.
Since modified MDI substances are UVCB substances containing different non-monomeric MDI constituents, but all have in common a high content of unreacted mMDI responsible for presenting NCO reactivity, scenario 4 or 6 according to the RAAF (Different compounds have qualitatively similar properties) is considered as most appropriate. Selection between scenario 4 and 6 depends essentially upon the presence of variation in the properties i.e. magnitude of effect. As discussed in detail previous endpoints, it has been demonstrated that the bioaccessible NCO is primarily responsible for local toxicity and the non-monomeric MDI constituents (with the exception of oligomeric 3-ring constituents in pMDI) do not contribute to the respiratory irritation. On this basis, some variation in response between substances of the MDI category would be expected and use of scenario 4 (variations in the properties observed among source substances. Prediction based on a regular pattern or on a worst-case approach) could be justified over scenario 6. Accordingly, the available inhalation developmental toxicity data demonstrate consistent effects for the mechanism of toxicity the key (pMDI) and supporting (4,4’-MDI) source substances both in terms of qualitative and quantitative aspects for developmental toxicity. As discussed above in the fertility discussion, systemic exposure and toxicity is not observed and therefore direct developmental toxicity is not relevant. This points to the conclusion that like other effects determined by respiratory irritation and inflammation, while some variations in magnitude of response might be expected due the higher content of bioaccessible NCO in 4,4’-MDI and pMDI, inherent lack of sensitivity of the study designs are unable to detect this variation.
Not classified as a reproductive
toxicant according to Regulation (EC) No 1272/2008 (CLP).
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