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EC number: 700-674-2
CAS number: 147993-65-5
In order to better understand the toxicokinetic behaviour of MDI after
inhalation exposure, biological samples from the Gledhill (2001a)
experiments (urine, faeces, bile) were investigated in a separate study
on the metabolism of MDI. Urine, faeces and bile were collected for the
identification of metabolites at 6 (in urine and bile only), 12, 24, 36
and 48 h (and for intact animals at 24 hourly intervals until 7 days
after the end of exposure). Metabolites present in bile and excreta were
identified by LC/MS and/or by co-chromatography with reference standards
Identification/quantitation of metabolites:
There was no MDA detected in any of the samples analysed for this study.
With the exception of 1 minor metabolite, all low molecular weight
metabolites present in urine and bile were identified or characterised
Metabolite I: N,N'-diacetyl-4,4'-diaminobenzhydrol. This was the major
urinary metabolite in both intact and bile duct cannulated rats (1% and
6% of the dose respectively). It was also present in bile (1% of the
Metabolite II: N,N'diacetyl-4,4'-diaminophenylmethane This metabolite
was present in urine of intact and cannulated rats (0.5% and 4% of the
dose respectively) and was present as the major metabolite in bile (4%
of the dose).
Metabolite III: N-acetyl-4,4'diaminophenylmethane
Metabolite IV: N,N'-diacetyl-4,4'-diaminobenzophenone Metabolites III
and IV were minor urinary metabolites (< 0.5% of the dose) and were not
present in bile.
None of these specified low molecular weight metabolites were found in
The solvent extract of faeces and the major radioactive component in
bile (9% of the dose) was thought to consist of mixed molecular weight
polyurea oligomers derived from MDI (metabolite VI). The implication of
these results, made by the author, is that a proportion of the MDI dose
(10%) is converted to these metabolites via intermediary formation of an
amine group which is rapidly acetylated. Both formation and acetylation
are most likely to occur within specific cells or compartments. It is
not possible from the current data to elucidate whether this stage
- MDA, although none was detected
- bound-MDA, i.e. as a bound intermediate on an enzyme involved in the
formation of the metabolites,
- an amine group resulting from reversion of the expected
MDI-glutathione conjugates as proposed below:
Lung: MDI + 2GSH → GSH-MDI-SHG
Tissues: GSH-MDI-SHG → GSH-MD-NCO → GSH-MD-NH2 → GSH-MD-NHCOCH3
Reversal of the second glutathione link would lead to the formation of
Metabolite III, with subsequent metabolism but without free MDA having
been formed at any stage.
Table 1: Quantity of each metabolite present in % radioactivity
Quantity of each metabolite present in % radioactivity administered.
Samples from Gledhill study 2003.
10% of the radioactivity was excreted in urine in 0-24h, further
2% in 24-48h.
Bilary elimination accounted for approximately 14% of the dose in
0-48h after exposure and 34% was eliminated in faeces during the same
MDA and MDI metabolites in urine and heamoglobin following
Table 3: Amount of MDA and concentration of combined deacetylated
metabolite I and IV in urine.
metabolites I and IV (ng/ml)
in brackets the values expressed [C14] nmol equiv. MDI
Radioactivity in haemoglobin:
at the end of the exposure haemoglobin contained 25 µg equiv.
MDI/g mainly consisting of metabolites I and IV (as shown by GC-MS
analysis after acidic hydrolysis).
In order to better understand the toxicokinetic behavior of MDI after inhalation exposure, biological samples from the Gledhill (2001a) experiments (urine, feces, bile) were investigated in a separate study on the metabolism of MDI. Urine, feces and bile were collected for the identification of metabolites at 6 (in urine and bile only), 12, 24, 36 and 48 h (and for intact animals at 24 hourly intervals until 7 days after the end of exposure). Metabolites present in bile and excreta were identified by LC/MS and/or by co-chromatography with reference standards and quantified. The major urinary metabolite was N,N'-diacetyl-4,4'-diaminobenzhydrol (50% of urinary radioactivity). All other identified metabolites were intermediates on the same metabolic pathway (N-acetylation and CH2-hydroxylation). These metabolites were also identified in bile (7 -28% of biliary radioactivity) although the major components were identified as polyureas derived from MDI. The major radioactive components in feces were identified as polyureas derived from MDI. No free MDA was detected in any of the biomatrices investigated.
According to the author, the tissue distribution of radioactivity at
different time points after exposure, when considered with the excretion
data, imply that the systemic doses were mainly due to absorption of
radioactive material present in the gastrointestinal tract after
ingestion, with pulmonary absorption of radioactivity deposited in the
lungs accounting for only a minor, hardly quantifiable portion of the
Immediately at the end of the exposure period 32% of the received dose
was present in the gastrointestinal tract, which could be explained by
ingestion of radioactivity deposited on the head and the respiratory
tract during exposure. The residual carcass analysed immediately at the
end of the exposure period contained 37% of the received dose, with 36%
of this present on the skin and 1% present in the true residual carcass
(without skin). At 168 hours after dosing, the radioactivity in the
residual carcass had decreased to 5% of the received dose. This decrease
in radioactivity was accompanied by a concomitant increase in the
cumulative excretion of radioactivity in the faeces. It was found
reasonable by the author, to conclude therefore, that radioactivity
present on the skin was ingested during grooming. This conclusion was
supported by the excretion, from the bile duct cannuled rats, of 34% of
the received dose in faeces. If the exposure to radioactivity was
exclusively via the inhalation route, the radioactivity in faeces from
these animals would have been negligible.
Table 1: Distribution of radioactivity (%) in rats terminated
immediately after cessation of exposure.
skin from head
Table 2: Mean excretion of radioactivity in urine, faeces and bile
(expressed as % of received radioactivity +/-SD)
time after dosing [h]
urine (bile cannulated rats)
faeces (bile cannulated rats)
Table 3: Tissue and carcass residues of radioacticity. Values expressed
as % of received radioactivity (+/-SD) of n=4 rats. Only tissues with
>1% are listed.
nasal tissue (respiratory)
Following a 6 hour inhalation exposure to [14C]-MDI, radioactivity was
widely distributed. Initially, tissues associated with the respiratory
tract contained the highest concentration of radioactivity, but over the
168 hour time course after the exposure period, these declined markedly
to leave low residues in all tissues analysed.
Radioactivity deposited on the head and pelt during the exposure was
ingested during grooming resulting in a combined oral/inhaled dose, with
the ingestion derived dose predominating. Most of the systemically
available dose was excreted via bile (14%) with a slightly smaller (12%)
proportion excreted in urine.
The test substance was stable in the respective carriers.
Excretion, retention and tissue concentrations after dermal
application of 14C-MDI:
Table 1: Mean excretion and retention of radioactivity after a single dermal
administration of 14C-MDI (% of the radioactivity administered).
nominal dose [mg/cm2]
sacrifice time [h]
actual dose [mg/cm2]
skin (application site)
In all groups, the largest proportion of radioactivity was recovered
from the dressing and the skin of the application site. The amount of
radioactivity absorbed (including excreta, cage wash, tissues/organs and
carcass) increased in time, but remained below 1% at all dose levels.
Excretion occured via urine and feces. In similar amounts after 8 and
24h, but to a higher extend in feces after 120h. The radioactivity
absorbed was distributed in all organs and tissues. Due to the limited
dermal absorption, concentrations of radioactivity in organs and tissues
analyzed were considerably below 1 µg Eq/g (except for carcass). Levels
of tissue radioactivity were comparable at 8 and 24 h and declined until
120 h after application with highest levels generally being found in
carcass, thyroid, muscle, plasma and liver (not shown in table).
Excretion, retention and tissue concentrations after intradermal
application of 14C-MDI:
Table 2: Mean excretion and retention of radioactivity after a single intradermal
administration of 14C-MDI (% of the radioactivity administered).
The largest proportion of radioactivity was found at the application
site. The amount of radioactivity absorbed (including excreta, cage
wash, tissues/organs and carcass) during the 120 h observation period
amounted to 25.87 % of the dose applied. Excretion occurred mainly via
the feces and concentrations of radioactivity in organs and tissues were
rather low being below 1 µg Eq/g.
The absorption, distribution and excretion of radioactivity was studied in groups of four male Wistar rats following a single dermal and intradermal administration of 14C- MDI at nominal dose levels of 0.4 and 4.0 mg/m2 for dermal administration and 0.4 mg/animal for intradermal administration. Considering the animals weights, dose levels corresponded to about 14 mg/kg bw and 140 mg/kg bw (dermal administration) and 1.4 mg/kg bw (intradermal administration). In the experiment with dermal application, animals were exposed for 8 hours and scarified 8, 24, or 120 hours after treatment.
After dermal application of 14C-MDI, mean recoveries of radioactivity from all dose groups were in the range of 97.86 to 108.07% of the total radioactivity administered. Generally the largest proportion of radioactivity was found at the application site and dressing. The total amount of radioactivity absorbed (including excreta, cage wash, tissues/organs and carcass) increased with increasing sacrifice time. Dermal absorption was very low and quantitatively similar at both dose levels; maximally ca. 0.9% of the applied radioactivity was absorbed.
After intradermal administration of 14C-MDI, the mean recovery of radioactivity was 100.90% of the radioactivity applied. The largest proportion of radioactivity was found at the application site. The total amount of radioactivity absorbed (including excreta, cage wash, tissues/organs and carcass) amounted 26% of radioactivity applied.
Irrespective of the mode of administration of 14C-MDI, concentrations of radioactivity in tissues and organs generally were low 1 µEq/g at 120 hours after administration.
In summary, the results of this study comparing systemic availability of radioactivity after single dermal and intradermal administration of 14C-MDI clearly demonstrated very limited absorption after dermal administration but considerable absorption after intradermal administration.
Oral exposure: No information is available on the toxicokinetic of MDI following oral exposure in animals.
Dermal exposure: There are no study data available for the target substance MDI MT. A read across is performed to study data of the source substance 4,4’-MDI. Two in vivo studies on the dermal absorption and distribution of radioactive labelled MDI in rats are available. Both studies appear to be reliable, although they show contradictory results. From the study of Vock and Lutz (1997) it must be concluded that absorption through the skin is not negligible. Following un-occluded application of 14C-MDI 10% of the administered radioactivity was recovered in the epidermis, 20-30% in faeces and 1% in urine. In the GLP-study of Leibold et al. (1999) the largest proportion of radioactivity was recovered from the application site (32/61% for high dose/low dose) and the dressing (69/42%). Dermal absorption was rather limited following semi-occlusive application. Only 0.9% of the applied radioactivity was absorbed at the most. Absorbed radioactivity was excreted via urine and faeces, with the faecal route of excretion becoming more relevant during prolonged observation time. Although addressed in the study report, contamination from grooming probably explains high levels of absorption found by Vock and Lutz, whereas the high amount of competitive extraction of 14C-MDI to the dressing may contribute to low levels of absorption detected by Leibold et al. (1999).
An in vitro study (Clowes et al., 1999) confirmed the low levels of 14C-MDI absorption obtained by Leibold et al. (1999). No radioactivity was absorbed through human skin during a 54 hours continuous exposure, and only small amounts (maximally 0.23% of applied dose) were absorbed through rat and guinea pig skin. The majority of applied MDI equivalents were found to be associated with the skin.
As the source substance 4,4’-MDI and the target substance MDI MT contain sufficient monomeric MDI, the driver of toxicity, similarities in reactions with extracellular nucleophilic biomolecules at the site of contact are assumed. As the higher molecular weight non-monomeric content of the UVCB substance MDI MT do not contains reactive centres and is consequently inert and thus do not contribute to the observed toxicity, it is reasonable to assume that using read across to the source substances 4,4’-MDI is warranted.
Inhalation exposure: There are no inhalation study data available for the target substance MDI MT. A read across is performed to study data of the source substance 4,4’-MDI. In a two-part inhalation metabolism/toxicokinetics/ distribution study performed by (Gledhill, 2003a; Gledhill, 2003b), tissue distribution of radioactivity after nose-only inhalation exposure to radiolabelled 4,4’-MDI, when considered with excretion data, imply that tissue residues resulted from absorption of radioactive material primarily from the gastrointestinal (GI) tract after ingestion of an inhaled dose. Further, pulmonary absorption of radioactivity deposited in the lungs accounts for only a minor portion of the administered dose. Similar results were obtained in an older study by Centre d`Etudes (1977) where the faecal elimination of 4,4-MDI was greater than the urinary elimination. In both studies, radioactivity was widely distributed with the respiratory and excretory organs containing the highest concentrations. The highest concentration of radioactivity was present in the respiratory nasal tissue (Gledhill, 2003). Due to grooming and respiratory clearance a combined oral/inhaled exposure existed in the study of Gledhill (2003) with 25 to 32 % of the applied dose entered the systemic circulation. According to Gledhill (2003) no unreacted monomeric 4,4’-MDI were systemically available.
In order to better understand the toxicokinetic behaviour of MDI after inhalation exposure, biological samples from the Gledhill (2001) experiments (urine, faeces, bile) were investigated in a separate study on the metabolism of MDI. Urine, faeces and bile were collected for the identification of metabolites at 6 (in urine and bile only), 12, 24, 36 and 48 h (and for intact animals at 24 hourly intervals until 7 days after the end of exposure). Metabolites present in bile and excreta were identified by LC/MS and/or by co-chromatography with reference standards and quantified. The major urinary metabolite was N,N'-diacetyl-4,4'-diaminobenzhydrol (50% of urinary radioactivity). All other identified metabolites were intermediates on the same metabolic pathway (N-acetylation and CH2-hydroxylation). These metabolites were also identified in bile (7 -28% of biliary radioactivity). Although the major components in bile and faeces were identified as polyureas derived from MDI. No free MDA was detected in any of the biomatrices investigated.
Taken together, these data support that following exposure, most inhaled test material or reaction products (polymerized insoluble particles and other insoluble adducts) are removed via mucociliary transport and swallowed with a small portion absorbed via solubilized macromolecular adducts via lung into blood stream. Systemic toxicity has not been observed in any in vivo study with either 4,4’-MDI or pMDI which is attributed to the lack of systemic bioavailability of the reactive isocyanate functional group. As described by (Wisnewski et al. 2019a) the fraction of monomeric MDI absorbed by the lung is exclusively via MDI adducts, consisting primarily of soluble low molecular weight MDI-glutathione (GSH) adducts or MDI-protein adducts. These soluble low molecular weight MDI-adducts are enzymatically metabolized as described by Wisnewski et al. (2016, 2019b), Bruggeman et al. (1986), Hinchman et al. (1991) and Bartels et al. (2009), which is in line with the results of Gledhill (2003), with the almost exclusively detection of MDI-metabolites with no evidence of free MDI or MDA.
As the source substance 4,4’-MDI and the target substance MDI MT contain sufficient monomeric MDI, the driver of toxicity, similarities in reactions with extracellular nucleophilic biomolecules at the site of contact are assumed. As the higher molecular weight non-monomeric content of the UVCB substance MDI MT do not contains reactive centres and is consequently inert and thus do not contribute to the observed toxicity, it is reasonable to assume that using read across to the source substance 4,4’-MDI is warranted.
Other routes of exposure: After intradermal administration of 14C-MDI to rats about 26% of the radioactivity was absorbed. Excretion was mainly via faeces (Leibold et al., 1999). Following intramuscular injection of 14C-MDI less than 25% of the applied dose was recovered in the excreta 120h after application. The amount of faecal elimination was larger if compared to urinary elimination, indicating that for absorbed MDI biliary transport is a significant route of excretion. The remaining radioactivity was found in the carcass.
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