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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
chronic 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: Similarly to the mechanism of acute toxicity, as an MDI substance enters the lung, NCO groups react with biological nucleophiles at the MDI/lung fluid interface to form MDI-conjugates. Formation of these MDI-adducts depletes protective nucleophiles in the lung and results in pulmonary irritation and inflammatory cell influx. MDI only enters the systemic circulation in the form of MDI-GSH or protein adducts. Consequently, there is no systemic exposure to the toxic NCO functional group which is consistent with the lack of distal toxicity in any study conducted. As described above, due to its role in further reducing the solubility (and hence subsequent bioaccessibility), the non-monomeric MDI constituents of the substances do not add to toxicity profile.
Justification: The existing inhalation repeated dose data on the MDI boundary substances 4,4-MDI, pMDI and 4,4’-MDI/DPGHMWP, demonstrate that the NCO group drives local toxicity with no observations of toxicity distal to the portal of entry. The hazards of the modified MDI substances are comparable due to similar mMDI content and the lack of potential additional hazards from the non-monomeric MDI constituents.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990
Reference Type:
publication
Title:
Unnamed
Year:
1994
Reference Type:
publication
Title:
Chronic pulmonary effects of respirable methylene diphenyl diisocyanate (MDI) aerosol in rats: combination of findings from two bioassays
Author:
Feron VJ, Kittel B, Kuper CF, Ernst H, Rittinghausen S, Muhle H, Koch W, Gamer A, Mallett AK and Hoffmann HD
Year:
2001
Bibliographic source:
Arch Toxicol.75: 159-175.

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 levels

Dose descriptor:
NOAEC
Effect level:
0.2 mg/m³ air
Based on:
other: weight of evidence
Basis for effect level:
other:
Remarks on result:
other: No repeated dose toxicity studies exist on the target substance. This endpoint is satisfied by weight of evidence and read across from valid repeated dose toxicity studies, with the source substances 4'4_MDI and pMDI.
Remarks:
These studies of the source substances 4,4'-MDI and pMDI (Reuzel et al. 1994, Hoymann et al. 1995) were analyzed by Feron et al. (2001), normalization of inhalation parameter of both chronic inhalation studies revealed qualitiative and quantitative similarities with a NOAEC of 0.2 mg/m3 for the endpoint repeated dose toxicity

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 mg/m³ air
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes

Applicant's summary and conclusion

Conclusions:
No repeated dose toxicity studies exist on the target substance 44MDI/DPG. This endpoint is satisfied by weight of evidence and read across from valid repeated dose toxicity studies for the inhalation route. Reliable repeated dose inhalation data in animals is available for the boundary substances (4,4’-MDI, pMDI and 4,4’-MDI/DPG/HMWP). The chronic repeated dose toxicity study (Reuzel et al. 1994, reliability 2) performed with pMDI is a guideline study (OECD 451). In addition, valid sub-chronic and sub-acute repeated dose toxicity studies are available for pMDI (Reuzel et al. 1994, Kilgour et al. 2002, reliability 2). For the 4,4’-MDI a valid chronic inhalation toxicity study is available (Hoymann et al. 1995, reliability 2) and a limited documented sub-chronic inhalation toxicity study (Heinrich et al. 1991, reliability 4). For the boundary substance 4,4’-MDI/DPG/HMWP a sub-acute toxicity study is available (Ma-Hock, 2021, reliability 1). To support this weight of evidence and read across approach additional repeated dose toxicity testing is planned. It is considered to perform a sub-chronic inhalation toxicity study (OECD 413) with boundary substance 4,4’-MDI/DPG/HMWP. Additional, combined Repeated Dose Toxicity studies with Reproductive/ developmental Toxicity Screening tests (OECD 422) will be performed on 9 substances representing all sub-groups and key structural/chemical characteristics (see overview attached in Annex 27 in Chapter 13). These screening studies will confirm the proposed MoA on repeated dose toxicity or identify substances that may require additional testing.
The target substance share the common chemical characteristic of two NCO functional groups per molecule with each NCO group bound to an aromatic ring and this ring connected to a second aromatic ring by the methylene group with the source substances and all MDI category members . The target substance has a NCO functionality of 2 and a NCO value of 24.8 - 31.9%. The source substances 4,4’-MDI has a NCO content of up to 33.6 % and pMDI of up to 31.3 %. It is the monomeric NCO group which drives the reactivity and thus toxicity of MDI. Since all substances of the MDI category contain significant levels of mMDI, 4,4’-MDI represents a worst case with the highest levels of reactive free NCO groups. Based on the similarities in chemical characteristics and reactivity between the target substance and source substance 4,4’-MDI a read across is warranted, which can be seen as worst case approach. To support the weight of evidence read across to a 2nd source substance is done with the boundary substance pMDI, which has a 4,4’-MDI monomer content up to 41 %. Appropriateness of this conservative approach is supported by results of the boundary substance MDI/DPG/HMWP (Ma-Hock 2021), the substance with the least bioaccessible NCO groups, indicating a potential slight reduction in the most toxicologically relevant adverse effects in the respiratory tract. Since it has been demonstrated that NCO value (as attenuated by solubility) is responsible for toxicity and the higher molecular weight, low solubility components do not contribute to the observed toxicity (see endpoint toxicokinetic and Category Justification Document), it is reasonable to assume that using read across to the source substances 4,4’-MDI and pMDI is warranted.
The hypothesized MoA of MDI substances is point of contact reactivity of the NCO group with biological nucleophiles in the respiratory tract following inhalation. When exposure concentration and/ or duration exceeds the capacity of the alveolar epithelial lining fluids (surfactant) to detoxify NCO deterioration of cell membranes and cytotoxicity occurs. In addition to the disruption of the surfactant homeostasis, reaction products of the MDI with alveolar macromolecules are phagocytized by activated macrophages. When activated, these macrophages release pro-inflammatory cytokines which recruit neutrophils. On a relatively short (acute) timescale, these effects can be seen as increases in lung weight as well as BALF levels of intracellular enzymes (LDH, γ-GT), plasma protein (ACE) and inflammatory cells (see Category Justification Document). On a longer (chronic) timescale, when the acute effects become biologically significant, they can be seen as basal cell hyperplasia in the nasal cavity, pulmonary fibrosis, proliferation of the alveolar epithelium and bronchiolo-alveolar adenoma. In other words, chronic pathology following recurrent MDI inhalation is both dependent on the C x t and the degree of acute pulmonary irritation can be considered as an ‘acute-on-chronic’ effect (Pauluhn, 2011). This supports a simple, direct MoA at the site of contact in the lungs as described by the hypothesis.
This mode of action is clearly demonstrated in repeat dose studies in the two source substances (4,4’-MDI, pMDI) and supported by findings of the boundary substance. 4,4’-MDI/DPG/HMWP. As described by Feron et al. (2001) the effects between 4,4’-MDI and pMDI in chronic bioassays (by Reuzel et al. (1994a) and Hoymann et al. (1995) were remarkably similar even though the exposure regimens were significantly different (17 hours/day, 5 days /week for up to 24 months for 4,4’-MDI vs. 6 hours/day, 5 days/week for 24 months). The major pulmonary effects in the two studies were characterized by hyperplasia, interstitial fibrosis and a low incidence of bronchiolo-alveolar adenoma, the latter occurring in the high exposure groups of both studies (i.e. total inhalation exposures of 17,728 and 17,575 mg MDI h/m3) (table 2 overall remarks, attachments). Both studies also reported the presence of particle-laden macrophages predominantly in the alveoli close to the alveolar ducts which in some cases, particularly in high dose groups, were associated with areas of fibrosis.
Further, a comparison between similarly designed sub-acute studies on the source substances pMDI and 4,4’-MDI/DPG/HMWP also demonstrate this common mode of action Kilgour et al. (2002) and (Ma-Hock, 2021), respectively (table 3 overall remarks, attachments ). Both studies show dose-dependent increases in lavage fluid signals for inflammation and irritation, as well as morphological changes related to cell proliferation in the terminal bronchi and alveoli.
Since MDI toxicity is a function of NCO reactivity with biological nucleophiles (primarily GSH) the magnitude of the effect (severity) is determined by the rate of GSH depletion in the extracellular space which is a directly related to the concentration of available NCO groups. The main factors determining NCO availability in a substance is (1) NCO value and (2) its rate of reactive dissolution. A review of a potential quantitative trend between pMDI (Hoymann et al., 1995) and 4,4’-MDI (Reuzel et al., 1994 can be seen in the re-evaluation of the pulmonary effects from the two chronic inhalation toxicity studies as performed by Feron et al. (2001). A comparison of the relevant histopathology findings in females is presented in table 2 (see overall remarks, attachments). The apparent greater toxicity of pMDI over 4,4’-MDI, was abolished when exposure concentrations were normalized to exposure level and duration. The authors concluded that 0.19 mg/m3 (559 mgh/m3) could be considered the NOAEC for both studies for a 6 h/day, 5 days/week for 24 exposure. While a quantitative trend can theoretically be predicted from this hypothesized MoA, the fact that all category substances contain relatively high levels of low molecular weight constituents (e.g. mMDI) combined with the presence of a threshold to development of both acute and chronic effects makes quantitative differentiation of this trend may be difficult to demonstrate using standard testing tools. A similar semi-quantitative comparison can be conducted on comparably designed sub-acute studies for pMDI (Kilgour et al. 2002) and 4,4’-MDI/DPG/HMWP (Ma-Hock 2021) (table 3, overall remarks, attachments). Both studies were conducted at comparable concentrations, but when normalized to NCO value and concentration of bioaccessible two-ring, and 3-ring MDI oligomer, pMDI was 36-38% and 7-10% higher in these critical drivers of toxicity, respectively. The analysis of the Kilgour et al. (2001) and Ma-Hock (2021) studies reveals that the histological results are generally comparable between the high dose groups in all endpoints. However, at the mid and low-dose groups, pMDI induced minimal findings that were not observed at comparable exposure concentrations for 4,4'-MDI/DPG/HMWP (for more details, see Category Justification Document, IUCLID section 13). Taken together, this suggests that pMDI, having more bioaccessible NCO groups, is more toxic than 4,4’-MDI/DPG/HMWP. As discussed above, the repeated dose and chronic toxicity is a function of C x t and likely to be based on recurrent acute effects (acute on chronic inflammation) leading to hyperplasia, proliferation and eventually adenoma and interstitial fibrosis in the lung. Cell proliferation and histopathology in the 28-day studies of Kilgour and Ma-Hock can be considered the link between the acute effects (inflammation and irritation parameters) and the longer term effects of repeated exposures. On this basis it is justified that a worst-case approach is taken in which the data for the source substances 4,4’-MDI and pMDI can be used for read-across to all substances of the MDI category. Appropriateness of this conservative approach is confirmed by comparative analysis of test results of the source substances 4,4’-MDI and pMDI, demonstrating comparable results with the most bioaccessible NCO groups (and with a potential slight reduction in the most toxicologically relevant adverse effects in the boundary substance with the least bioaccessible NCO groups (4,4’-MDI/DPG/HMWP).
In summary, a weight of evidence with the source substances 4,4’-MDI and pMDI, with supporting data from the boundary substance 4,4’-MDI/DPG/HMWP, is used to read across to the target substance. As the source substances are classified with STOT RE 2 (H373) using the read across approach, a similar classification for the target substance is followed and the target substance is classified as STOT RE 2 (H373) EU GHS 1272/2008 CLP.
Executive summary:

The principal route of concern for human exposure is via inhalation. Reliable repeated dose inhalation toxicity data is available  for the source substances 4,4’-MDI and pMDI (Hoymann et al. 1995., Reuzel et al. 1994) and are read across to the target substance. Since all substances of the MDI category contain significant levels of monomeric MDI, 4,4’-MDI represents a worst case with the highest levels of reactive free NCO groups. For the repeated dose toxicity endpoint, the observation in the respiratory tract, especially the lung noted in both source substances is consistent with the hypothesized MoA. As a MDI substance enters the lung, NCO groups react with biological nucleophiles at the MDI/lung fluid interface to form MDI-conjugates. Formation of these MDI-adducts depletes protective nucleophiles in the lung and results in pulmonary irritation and inflammatory cell influx. As these acute effects become chronic this result in histopathological changes characterized by interstitial fibrosis, hyperplasia of the alveolar epithelium and a low incidence of bronchiolo-alveolar adenoma, the latter occurring in the high exposure groups of both chronic source substance studies (Hoymann et al. 1995., Reuzel et al. 1994). The available animal toxicity data shows that the content of bioaccessible NCO groups is the driving factor of lung toxicity after repeated inhalation with a negligible contribution from the higher molecular weight components and a lack of any systemic exposure or toxicity. This supports the conclusion that data generated for 4,4’-MDI and pMDI can be used as a worst-case source for read across to the target substance, which has in common with the source substances a high content of bioaccessible monomeric  MDI. Accordingly, the CLP classification (STOT RE 2, H373) for 4,4’-MDI and pMDI is adopted for all other substances in the MDI category, including the target substance 44MDI/DPG.


In summary, the available data on the endpoint repeated dose toxicity are considered adequate and sufficient for classification and labelling. To support the weight of evidence additional testing is planned. It is considered to perform a sub-chronic inhalation toxicity study (OECD 413) with boundary substance 4,4’-MDI/DPG/HMWP. Additional, combined Repeated Dose Toxicity studies with Reproductive/ developmental Toxicity Screening tests (OECD 422) will be performed on 9 substances representing all sub-groups and key structural/chemical characteristics (see Test Proposal Summary attached in Chapter 13).  These studies will confirm the proposed MoA on repeated dose toxicity or identify substances that may require additional testing.