1,1'-Methylenebis(4-isocyanatobenzene) and its oligomeric reaction products with [(methylethylene)bis(oxy)]dipropanol

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Justification for type of information:

Hypothesis: MDI substances lack genotoxic potential both at the site of contact and systemically and is consistent with the overall hypothesis that effects are driven by the toxicokinetic activities at the site of contact. Specifically for mutagenicity, this mechanism is reflected by a) the reactive NCO groups on the MDI substances is unavailable in the cell cytosol and MDI-adducts have not been found to be reactive with DNA either with cells at the site of contact (BALC) or systemically (bone marrow and blood cells), and b) absorption and metabolism of MDI substances occurs without detectable formation of the mutagenic diamine (MDA). As reactive NCO groups are a common feature of all substances of the MDI category, it is predicted that these have a similar reactivity profile with GSH and proteins and not available to react with DNA.

Justification: In vivo experimental evidence demonstrates that 4,4-MDI is not mutagenic systemically or at the point of contact. A clear understanding of the mechanism of this lack of toxicity with consistent supporting evidence from in vitro studies and other repeated dose studies (reporting no MDA formation), demonstrates that all category substances would also be considered not mutagenic and no further testing is warranted.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

75880-28-3

Cas Number:

75880-28-3

IUPAC Name:

75880-28-3

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In vivo genotoxicity data is unavailable on the target substance 4,4'-MDI/TPG. However, as described in the endpoint summary, genotoxicity for the entire category is addressed by a weight of evidence approach from both in vitro data on ALL MDI category substances and in vivo studies on the worst-case substance (4,4,’-MDI).
In vivo genotoxicity of 4,4’-MDI has been examined for both potential local and systemic effects in two key studies. In a guideline OECD 489 in vivo Comet assay via inhalation (Randazzo, 2017), the lung, stomach and the liver were examined for DNA damage up to the maximum tolerated concentration (as defined by local cytotoxicity, apoptosis and/or inflammation). Results from this study were clearly negative for DNA damage in all three tissues and was consistent with the hypothesis that local cellular toxicity in the lung is the critical mode of action of lung toxicity. When local cytotoxicity and inflammation is avoided, 4,4’-MDI does not induce DNA damage. This is also the case in the stomach (via secondary exposure from mucocilliary clearance) or in the liver (assessing potential systemic exposure).
Systemic genotoxicity of 4,4,’-MDI was also explored in a key OECD 474 guideline micronucleus study (Pauluhn et al., 2001). Rats were exposed both whole body and nose only to 4,4’-MDI for 1 hour per week for 3 weeks, with bone marrow examinations at one and two days post-exposure at concentrations up to 118 mg/m3. Although toxic effects at the portal of entry (e.g. respiratory distress, increased lung weights) were observed, there was no evidence of an MDI-induced increase in the frequency of MN-PCE at any of the time points selected. MN-PCEs were significantly increased in rats treated with the positive control when compared to both the negative control and MDI-exposure groups.
As with in vitro genotoxicity, read-across is based on the hypothesis MoA that all substances of the MDI category contain the reactive NCO group on the different constituents that is capable of reaction with biological nucleophiles. The chemical reactivity and toxicokinetic behavior of the NCO on the bioaccessible MDI substances is described in Toxicokinetics section and forms the basis of the hypothesized MoA for site of contact toxicity but is also the basis for the absence of mutagenicity. In this case, the hypothesized MoA recognizes that under physiological conditions (i.e. aqueous matrix of the lung) the MDI substance readily polymerizes at the MDI/aqueous interface forming insoluble polyureas and/or reacts with extracellular biological nucleophiles to form MDI-adducts rendering the free NCO completely unavailable to react with DNA. While hydrolysis to the amine is theoretically possible, reaction kinetics point to diamine formation being highly unlikely except under very specific experimental in vitro conditions (Herbold et al., 1998) which is supported by experimental evidence showing no amine formation in vivo following diisocyanate exposure (Gledhill, 2003b; Gledhill, 2003a). All MDI substances contain significant amount of the unreacted mMDI, which contain the most bioaccessible NCO groups, supporting this mechanism
The reaction mechanisms of isocyanates in the lung fluid also explains why the apparently ‘less reactive’ molecules (i.e. the 2,4’- and 2,2’-MDI isomers) are not more likely to be available intracellularly thus not more potent for DNA reactivity and genetic toxicity. As detailed in the Category Justification Document, toxicity of MDI substances is a function of adduct formation, and the extent of toxicity is determined by the dissolution kinetics (i.e. rate of dissolution of MDI-glutathione adducts) and not inherent reactivity. The faster the adducts are dissolved, the more rapidly glutathione is depleted and thus the greater likelihood of a toxic effect. However, as this relates to genetic toxicity, even the more ‘stable’ molecules must still react with nucleophiles (like GSH) prior to being systemically or intracellularly available. The rate of dissolution of the adducts into the aqueous lung fluid which dictates severity of site of contact toxicity is not relevant for genotoxicity (either local or systemic). This concept is demonstrated in experiments by Wisnewski et al. (2018) showing that the 2,2’- and 2,4’-MDI isomers were found to rapidly react with GSH and demonstrate marked similarities with that previously described for 4,4’-MDI. When fully dissolved, at physiological pH and temperature, 2,2’- 2,4’- and 4,4’-MDI-GSH reaction products are primarily bis(GSH)-MDI and form within minutes. The initial rate of bis(GSH)-MDI formation (assuming pseudo first order kinetics) with 2,2’-, 2,4’- and 4,4’-MDI were indistinguishable. Diamine hydrolysis products (MDA) were below the limit of detection (0.03 µM) for all MDI isomer reactions. These experiments show that regardless of the ‘reactivity’ of the molecule, they will equally form GSH adducts and hence the NCO will be unavailable for cellular transport and subsequent mutagenicity.
This observation is extended by a series of similar in vitro experiments with other modified MDI substances representing each of the sub-groups of the MDI category (Zhang et al., 2021) and including the boundary substances. In these experiments, 4,4’-MDI, pMDI, 4,4’-MDI/4,4’-MDI homopolymer, and 4,4’-MDI/DPG/HMWP were solubilized in solvent and added to the aqueous media containing radiolabeled GSH to qualitatively and quantitatively analyze in vitro adduct formation. A high amount of solvent was used with the intent to dissolve as much of the test substances as possible. Overall, results demonstrated that mMDI constituents (i.e. the most reactive soluble) in the MDI-based test materials react very rapidly (essentially instantaneously) to form adducts with glutathione (GSH). The formation of these GSH adducts with mMDI and their subsequent transcarbamoylation with amine functionalities proceed at similar rates for the four tested materials. The relative formation of various types of adducts (bis-, mono-, and cyclic) is consistent with prior experiments (Wisnewski, 2018), and depends on isomer of mMDI, but not on the presence of the higher molecular weight constituents such as oligomers, condensation adducts or glycol adducts in the test materials.
Conversely, no GSH adducts other than those with mMDI could be identified from radio-chromatographic separations of reaction mixtures containing these higher molecular weight constituents and radio-labeled GSH. The reason for the absence of GSH adducts with less soluble non-monomeric MDI constituents was due to their precipitation prior to addition of GSH to the test medium. Additional testing demonstrated that a GSH adduct of a three-ring MDI oligomer could be generated and detected under homogeneous (i.e. pre-solubilized in solvent) reaction conditions. Under heterogeneous reaction conditions (i.e. no solvent), the availability of such adducts may be further limited by solubility.
In summary, exposure to MDI substances can result in low levels of DNA adducts in vivo in some tissues at point of contact (e.g. skin, olfactory epithelium) but not others (e.g. respiratory epithelium or in lung tissue where Type II cell adenomas were observed in bioassays). Given the lack of genotoxicity in these point of contact tissues where MDI reactivity is expected to be highest (comet assay; Randazzo (2017)), it is not surprising there is no evidence of genotoxicity at more distant sites (i.e. negative micronucleus studies; Pauluhn et al. (2001)). This lack of genotoxicity is also seen in vitro when MDA formation is not favored and is demonstrated in multiple substances. These results and as well as toxicokinetic mechanisms support the use of the worst-case substance (4,4,’-MDI) as the source for read-across for all category substances.

Executive summary:

All substances of the MDI category contain the reactive NCO group on all constituents, and therefore a similar toxicokinetic response is indicated. In vitro GSH reactivity studies show similar qualitative and quantitative reaction profiles of the mMDI isomers when alone (i.e. in mono-constituent monomeric MDI) or as part of UVCB substances (pMDI, 4,4’-MDI homopolymer and 4,4’-MDI/DPG/HMWP). GSH adducts with non-monomeric MDI constituents were not identified and is attributed to precipitation of these constituents. These results and underlying mechanism clearly demonstrate that all substances of the MDI category are not expected to be mutagenic and is supported by Ames testing using several substances of the MDI category which all give negative responses. Taken together, the available data indicate that MDI substances lack genotoxic potential both at the site of contact and systemically and is consistent with the overall hypothesis that effects are driven by the toxicokinetic activities at the site of contact. Specifically for mutagenicity, this mechanism is reflected by a) the reactive NCO groups on the MDI substances is unavailable in the cell cytosol and MDI-adducts have not been found to be reactive with DNA either with cells at the site of contact (BALC) or systemically (bone marrow and blood cells), and b) absorption and metabolism of MDI substances occurs without detectable formation of the mutagenic diamine (MDA). As reactive NCO groups are a common feature of all substances of the MDI category, it is predicted that these have a similar reactivity profile with GSH and proteins and not available to react with DNA.  Therefore, all category substance are not classified for genotoxicity according to EU GHS 1272/2008 CLP.