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Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Justification for type of information:
Hypothesis: In the case of inhalation exposure, the MoA is expressed as a number of key events that ultimately could be proposed as an adverse outcome pathway (AOP) for inhalation toxicity by these substances:
Reactive NCO groups on MDI substances and the bioaccessible species react with, and deplete, protective electrophiles at the extracellular MDI/aqueous interface. As the nucleophilic scavenger capacity of the lung is overwhelmed this leads to compensation of extracellular GSH by exudation of intracellular GSH (Sies et al., 1980; Boehme et al., 1992; Reiners et al., 2000) and consequential reduction in intracellular GSH concentration (Scire et al., 2019). This in turn disturbs the redox balance inside the cell leading to elevated levels of reactive oxidative species (ROS; expressed by increased GSSG/GSH ratios). Redox imbalance subsequently induces cellular signaling mechanisms that trigger other responses such as extravasation of plasma proteins as a result of impaired tight junctions (Pauluhn et al., 1999b; Pauluhn, 2000a; Brooks, 2010; Siegrist et al., 2019) and/or neural reflexes by interaction with vagal receptors (Marek et al., 1992; Potthast et al., 1992; Marczynski et al., 1994b; Pauluhn, 2000a; Brooks, 2010). Whereas a reduction of intracellular GSH does not necessarily produce cytotoxicity (Pauluhn et al., 1999b; Pauluhn, 2000a), further depletion of intracellular GSH can lead to cytotoxic responses triggered by oxidative stress (Vock et al., 1998a; Rahman and MacNee, 1999; Mishra et al., 2009a; Mishra et al., 2009b; Mishra et al., 2009c; Azad et al., 2014; Robb et al., 2016; Hotchkiss et al., 2017; Ferreira et al., 2018; Melnikov et al., 2019; Siegrist et al., 2019), upregulated (Hur et al., 2009) or impaired enzymes (Rouzer et al., 1981; Jochheim and Baillie, 1994; Kassahun et al., 1994), and ultimately cell apoptosis or necrosis (Meister, 1988; Carpenter-Deyo et al., 1991; Vock et al., 1998b).
A reduction in extracellular GSH frequently leads to disrupted or denuded airways epithelium (Lange et al., 1999), which can exacerbate deleterious effects on airways and lungs. Low glutathione levels have been linked to abnormalities in the lung surfactant system and the increase in pro-inflammatory cytokines. Reduced functionality of pulmonary surfactant can be caused by reactive diisocyanates (Pauluhn, 2000a) and triggers signaling events similar to a reduction in GSH (increased permeability, oxidative stress) (Robb et al., 2016; Siegrist et al., 2019). Increased surface tension in the lung fluid is a potential cause for edema (Albert et al. (1979) [in dogs]). Accordingly, the physical chemical properties that drive GSH-adduct formation in the lung is good predictor for toxicity potential.


Justification: The available acute inhalation toxicity data demonstrate consistent and predictable results across the substances of the MDI category. As discussed above, in all cases, mortality is linked to local effects of the respiratory system (via NCO depletion of GSH) that includes severe irritation, pulmonary edema, and ultimately death occurring within one to two days following exposure consistent with the hypothesized MoA. The MDI substances with the highest NCO value, lowest average molecular weight, and highest solubility (i.e. mMDI and to a lesser extent the three-ring oligomers) are the most toxic. Thus, as expected, the toxicity of the higher molecular weight, less soluble substances demonstrate lower toxicity (e.g. monomer-depleted pMDI and monomerdepleted 4,4-MDI/DPG/HMWP). While there was some variability of LC50 values across the category, this is to be expected for complex test substances even when following standard regulatory testing guidelines (e.g. OECD, USEPA) due to inherent technical and biological variability.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008

Materials and methods

Test material

Constituent 1
Reference substance name:
1,1'-Methylenebis(4-isocyanatobenzene) and oligomeric reaction products of 1,1'-methylenebis(4-isocyanatobenzene) and oxydipropanol and oligomerization reaction products of oxydipropanol
EC Number:
701-041-3
Molecular formula:
C14 H10 N O [C21 H24 N2 O5]n N C O with n=0, 1, 2, 3
IUPAC Name:
1,1'-Methylenebis(4-isocyanatobenzene) and oligomeric reaction products of 1,1'-methylenebis(4-isocyanatobenzene) and oxydipropanol and oligomerization reaction products of oxydipropanol

Results and discussion

Effect levelsopen allclose all
Key result
Sex:
female
Dose descriptor:
LC50
Effect level:
559 mg/m³ air (analytical)
Exp. duration:
4 h
Remarks on result:
other: Weight of evidence
Key result
Sex:
male
Dose descriptor:
LC50
Effect level:
368 mg/m³ air (analytical)
Exp. duration:
4 h
Remarks on result:
other: Weight of evidence

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Remarks:
Members of the MDI category are officially classified with Acute Tox. 4 H332 (Annex VI Regulation (EC) No 1272/2008 (CLP regulation).
Conclusions:
No acute inhalation data exist for the target substance 44MDI/DPG. Accordingly, this endpoint is satisfied by a weight of evidence and read-across from valid acute inhalation toxicity studies on 8 category substances representing all sub-categories according to the hypothesized MoA. In these studies, the aerosolized test substance proved to have a high acute inhalation toxicity in rats. The signs observed demonstrated that the respirable aerosol of this test substance may cause marked respiratory tract irritation with mortality associated with lower respiratory tract irritation (alveolar edema). According to the hypothesis described in MDI Substance Category Justification Document (see Chapter 13), toxic severity for acute inhalation is a direct function of bioaccessible NCO with the most bioaccessible monomeric MDI driving toxicity with non-monomeric constituents attenuating the effects. The result is a trend of decreasing lethality as the amount of bioaccessible NCO declines. Reliable data on the substance with the most bioaccessible NCO is considered the worst-case and used as the basis for classification and hazard evaluation.
While testing is available on 8 MDI category members (including all sub-groups), acute toxicity testing (OECD 403) will be performed on an additional 4 MDI substances. This information will further support the category hypothesis as well as help to define substance selection and study design for repeat-dose bridging studies.
Executive summary:

Acute inhalation toxicity is a direct function of the toxicokinetic behavior of MDI substances at the extracellular/aqueous interface of the lung. As the NCO groups are highly reactive, nucleophiles (primarily GSH) at this extracellular interface play a key role in natural defense and detoxification mechanisms. The magnitude of the reaction of MDI substances with GSH is determined primarily by the NCO value and the rate of dissolution of the reacting MDI substance. In high dose animal studies, as the available NCO depletes the nucleophiles, the protective capacity is overwhelmed resulting in inflammation and cytotoxic effects ultimately leading to pulmonary edema and death within one to two days if concentration and duration of exposure is sufficient.  


The rate of nucleophile depletion by MDI-based substances is driven by the availability of the NCO-group, which itself is a function of (1) the NCO value of the substance and (2) the molecular weight of its constituents (driving its reactive dissolution). Monomeric MDI isomers have been shown to become available at a similar rate in toxicokinetic studies (Wisnewski, 2018; Wisnewski et al., 2019a) which is consistent with the generally comparable LC50 values for all of the isomers. Conversely, higher molecular weight constituents have both a reduced NCO value and exhibit reduced water solubility, making them less accessible to react with GSH. Therefore, the substances with the highest available NCO value and bioaccessibility (mMDI and three-ring oligomers) are the most toxic, while those with increasing amounts constituents less able to react with GHS demonstrate reduced toxicity.


Tests also show that toxicity is limited to portal-of-entry effects. The absence of systemic toxicity is due to the extracellular reactions, combined with transcarbamoylation to proteins constitute a detoxification mechanism. Acute toxicity is only observed when this protective mechanism becomes overwhelmed and is limited to the lung.


This mode of action is supported with high confidence by reliable acute inhalation data available for multiple MDI isomers and modified MDI substances across the entire category.


Using the strict GHS LC50 cut-off for classification, the LC50 values obtained for members of the MDI category would trigger in most cases a Category 2 or 3. However, classification for these substances according to GHS legal text allows for the application of scientific judgement. It must be considered that the LC50 cut-off of 500 mg/m3 (approximately 50 ppm for pMDI), is over 2,500-fold above the saturated vapor concentration for pMDI.


Furthermore, the aerosols were generated using sophisticated techniques in the laboratory, whereby extremely small particles are generated in order to meet international guidelines for testing. This size and concentration of aerosol is not generated in the workplace even under foreseeable worst-case conditions (Ehnes et al., 2019). The particle size distribution of aerosols formed during actual spraying applications has virtually no overlap with that of the highly respirable aerosol generated in inhalation studies (see EC (2005)).


In addition, the EU legislation for classification and labelling of chemicals, the 67/548/EEC Substances Directive in Article 1(d) makes it clear that the object of classification is to approximate the laws of the Member States in relation to substances dangerous to man or the environment. In Article 4 in points 1 and 2 it is clearly stated that substances shall be classified based on their intrinsic properties according to the categories of danger as detailed in Article 2(2) and that the general principles of classification shall be applied as in Annex VI. Intrinsic properties are those inherent in the substance. Due to a very low vapor pressure (<0.01 Pa) MDI substances are not inherently toxic by inhalation since the saturated vapor concentration would be orders of magnitude below toxic concentration. It is only with modification and input (in terms of heat, cooling and size screening) that MDI substances become toxic after inhalation. The European Chemical Industry Council have discussed and given guidance for these situations, and on the classification of respective aerosols. Classification of MDI as “Harmful” is consistent with this guidance.  


The acute inhalation data of pMDI and 4,4’-MDI data were considered by EU experts, and their conclusion that MDI be classified as “Harmful” and  reported in the 25th Adaptation to Technical Progress (ATP) to the Dangerous Substances Directive (67/548/EEC). This was endorsed in the 28th ATP and both MDI substances remain as “Harmful” in the 30th ATP (adopted by Member States on 16 February 2007 and published 15th September 2008). The original decision was upheld in the EU Risk Assessment of MDI (Directive 793/93/EEC, 3rd Priority List) published in 2005, noting that considering “the exposure assessment, it is reasonable to consider MDI as harmful only and to apply the risk management phrase ‘harmful by inhalation’. This classification was also endorsed by the Scientific Committee on Toxicity, Ecotoxicity and the Environment (CSTEE, now SCHER) in giving their opinion on the Risk Assessment (EC, 2008). With the enforcement of the CLP regulation (Regulation (EC) No 1272/2008) in 2009, the Dangerous Substance/Preparation Directive (DSD) was repealed and harmonized classifications were formally transferred to the CLP regulation; Members of the MDI category are officially classified with Acute Tox. 4 H332 (Annex VI Regulation (EC) No 1272/2008 (CLP regulation).


All substances of the MDI category share similar chemical features namely that they a) all contain a significant amount of mMDI, and b) contain at least two NCO functional groups per molecule which is bound to an aromatic ring and this ring is connected to a second aromatic ring by a methylene group. It is the NCO value (driven by the bioaccessible groups on monomeric MDI and low molecular weight constituents (e.g. three-ring oligomer) which is responsible for chemical and physiological reactivity and subsequent toxicological profile. 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 and a read across within the category is warranted (detailed information on the Mode of Action is available in Category Justification Document).

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