TDI Biuret

Endpoint summary

Additional information

Since no data on toxicokinetics, metabolism and distribution are available for the substance, such data have been 'read across' from TDI (justification of read-across see below).

Oral administration

Absorption:

Orally administered [14C]-2,4-TDI or [14C]-2,6-TDI is not very well absorbed. There was a consistent finding that when rats were gavaged with high doses of 2,4-TDI, much of the isocyanate polymerised in the stomach and was excreted in the faeces (Jeffcoat, 1988, 1985; Stolz et al., 1987). Hence proportional bioavailability increased with decreasing dose: e.g. 3.5% of the applied radioactivity was recovered in urine after gavage with 700 mg/kg, 6.3% after 70 mg/kg and 16% after 7 mg/kg 2,4-TDI (Jeffcoat, 1988). A similar proportional excretion pattern was described for the isomer mixture (23% of 6 mg/kg and 16% of 60 mg/kg, Stolz et al., 1987) or 2,6-TDI (12% of 59 mg/kg and 5% of 900 mg/kg bw dose, Jeffcoat, 1985).

 

These findings are consistent with the view that under the acidic conditions in the stomach TDI will hydrolyze to TDA which, in the presence of excess TDI, will react with it to form insoluble polyureas. Without the isocyanate as a reaction partner the TDA will be absorbed. The similarity of the urinary excretion kinetics and metabolite profiles for both orally administered TDI and TDA support this concept. Although the use of corn oil as the dosing vehicle and the stated degradation of the TDI in that vehicle to unidentified products rather compromises the data generated.

 

Excretion:

Urine is the predominant route of excretion of absorbed radioactivity and half of the [14C]-2,4-TDI derived total radioactivity which was recovered in urine was excreted in 7 hr (t1/2 = 7.5 hr, Timchalk et al.,1992). Excretion of radioactivity from urine was most rapid 0-6 hr, decreasing rapidly by 24 hr (Stolz et al., 1987). Results from i.m. applications suggest, that also bilary excretion plays a major role in the overall excretion of absorbed radioactivity (39% of applied radioactivity, Saclay, 1976).

 

Distribution:

In rats the largest part of a [14C]-2,4-TDI dose was recovered in the GI tract and excretory organs, including the stomach, caecum, large intestine and bladder (Jeffcoat, 1988). Following oral application of [14C]-2,6-TDI (Jeffcoat, 1985) or [14C]-2,4-TDI/2,6-TDI-isomer mixture (Stolz et al., 1987), [14C]-tissue concentrations were highest in blood, liver, kidney and stomach. Total recovery in tissue did not exceed 1% of the doses recovered at 4hr after dosing.

 

Metabolism:

Metabolite profiles from orally administered TDI, including the identification of free TDA, were not dissimilar to those for orally administered TDA.

 

Oral dosage with 2,4-TDI yielded qualitatively similar metabolic profiles in urine to those following i.v. dosing of 2,4-TDA (Timchalk et al.,1992). Six metabolic products from TDI metabolism co-chromatographed with those from 2,4-TDA. Less than 10% were 2,4-bis(acetylamino) toluene and 80% of the radioactivity was associated with 5 other peaks with identical chromatographic retention times as the metabolites of 2,4-TDA (Jeffcoat, 1988). Differences were described when comparing the metabolic profiles of 2,4-TDA with 2,6-TDA. More than half of the 2,6-TDA derived material in urine was 2,6-bis(acetylamino) toluene (Jeffcoat, 1985).

 

Following [14C]-2,4-TDI oral dosing, approximately 65% of the quantitated urinary metabolites existed as acid-labile conjugates. Monoacetyl, diacetyl and free TDA were detected (Timchalk et al.,1992). Analysis of plasma showed the majority of the radioactivity present to be in the high molecular weight fraction (> 10 kDa) and associated with a range of high molecular weight components. The majority of the radioactivity present in the low molecular weight fraction was tentatively identified as TDA.

 

Inhalation exposure

Absorption:

Acute inhalative administration of TDI as a vapor resulted in an almost complete absorbtion in rats (Timchalk et al., 1992 for 2,4-TDI; Stoltz et al., 1987 for the mixture). The major part of radioactivity was absorbed via the lungs while only a minor part was orally absorbed due to respiratory clearance. A proportional relationship between exposure and blood concentration was observed in rats (Kennedy, 1994) and guinea pigs (Kennedy, 1989).

 

Excretion:

The main proportion of the inhaled dose was excreted in the feces (>50%), resembling substance transported into the GI-tract via bilary excretion (Timchalk et al., 1992 for 2,4-TDI; Stoltz et al., 1987 for the mixture). The second greater proportion of excretion is via urine (20-24%) with a t1/2 of 20h, which is considerably higher than that for orally dosed TDI/TDA. No radioactivity was excreted via the expired air. In five days 86% of the dose was eliminated, and 8% was found in bile during the first 52 hours, with activity peaking between 6 and 9 hr. Faecal excretion (63.1% within 120 h) was greater than urinary excretion (23.4% within 120 h; Saclay, 1976).

 

Distribution:

Following a vapour exposure to mixed [14C]-TDI isomers (84% 2,4-, 16% 2,6-TDI) blood elimination of [14C] was biphasic and 90% of the radioactivity in plasma was associated with proteins. [14C] was distributed relatively uniformly throughout the body with a predominance for the stomach, small intestine, kidneys, lungs and thyroid (Saclay, 1976). In guinea pigs exposed to [14C]-2,4-TDI vapours, tissues showing highest levels of activity were trachea and lungs. Small amounts were found in kidney, liver, and heart (Kennedy et al., 1989). Similar findings were reported for the rat (Kennedy et al., 1994). Immediately after exposure of rats to [14C]-2,4-TDI the majority of radioactivity was detected in the carcass (74.5%), 48 h later the radioactivity in the carcass had declined to 10%, while 16.6% was found in the GI content. The total radioactivity in the carcass and tissues was approximately 34% 48 h after exposure (Timchalk et al.,1992; 1994). Slightly higher recoveries in the carcass (18% 96h after single application) were reported for the mixture (Stoltz et al., 1987).

 

Metabolism:

The urinary metabolite profiles between oral and inhalation exposure differed substantially, reflecting the different conditions on both application routes. In the lung (pH approx. 7), TDI-vapor conjugates with proteins, whereas in the stomach (pH below 2) protein binding is reduced and hydrolysis and formation of polyurea is facilitated.

 

Accordingly, even at a high inhalation exposure level of 2ppm [14C]-2,4-TDI for 4h, Timchalk et al. (1992; 1994) did not detect any free TDA in the urine. In rats orally exposed to 60 mg/kg bw [14C]-2,4-TDI small amounts of free TDA were detected (2.08 µg Eq/g urine). Furthermore, different ratios and absolute values of the mono- and di-acetylated derivates were determined in this study. Only very low total amounts of acetylated derivatives were detected following inhalation exposure (0.26 µg Eq/g urine) compared to the oral route (13.26 µg Eq/g urine). These acetylated derivatives are most likely not liberating free TDA. Even though, the low amount of acetylated derivatives detected following inhalation exposure guarantees that TDA would not be available in toxicologically relevant concentrations. Approximately 90% of the quantified urinary metabolites from inhaled 2,4-TDI existed as acid-labile conjugates, contrasting with only 65% for orally administered TDI (Timchalk et al., 1992).

 

When rats were exposed for 4 hours to [14C]-2,4-TDI vapours the majority of the label associated with the blood (74-87%) was recovered in the plasma. Plasma profiles showed that 97-100% of this radioactivity existed in the form of biomolecular conjugates. In contrast to oral dosing binding was predominantely associated with a single component of 70kDa, most likely representing albumin (Kennedy, 1994). The majority of the radioactivity present in the low molecular weight fraction was not identifiable as TDA but was spread across a number of unidentified components. The authors concluded that conjugation was the predominant reaction and that free TDA was not a primary in vivo reaction product following inhalation of 2,4-TDI vapour.

Further studies found polar and less polar metabolites following exposure to mixed TDI-isomers. Slightly less polar metabolites were recovered in urine, faeces and tissues with no apparent difference in distribution of polar and non-polar products due to dose (Stoltz et al., 1987). The most abundant derivative accounted for 25-30% of 14C in urine (Saclay, 1976).

 

Dermal administration

When applied in a mixture on to skin, both 2,4 - and 2,6 -TDI disappeared, with either about 16% or 3% respectively, remaining after 8 hours (Gamer 2007). Both Administration of 2,4 -TDI to the skin of rats for eight hours resulted in less than 1% of the applied dose reaching the systemic circulation. The absorption of 2,4-TDI was 0.27%, 0.50% and 0.90% after exposure periods of 0.5, 1 and 8 hours, respectively. Highest tissue concentrations of radioactivity were found in blood cells and plasma (OECD427; Fabian and Landsiedel, 2008).

 

Following occlusive dermal application of 0.2%, 1% and 5% TDI (80:20 2,4- : 2,6-isomers) to rats, the amount of hydrolysed urinary TDA correlates linearly with the amount of TDI applied, suggesting that the absorption is dose-dependent (Yeh et al., 2008). Excretion of hydrolysed TDA followed a first order kinetic and the apparent half lives were about 20 and 23 hours for the 2,4- and 2,6-TDI isomers respectively, increasing by an increase of dose. Although exposures were to 2,4- and 2,6-TDI isomers in 80:20 ratio, urinary hydrolysed amine isomer recoveries were essentially close to unity (1:1), which was attributed to the greater reactivity of the 2,4-TDI isomer forming polymers which were not absorbed.

 

The urinary elimination half life following dermal and inhalation administration is similar at about 20 hours, and markedly different from that following oral administration (3-5 hours), indicating a similarity in disposition and metabolism between inhalation and dermal exposure routes.

 

Other routes

Following a single intramuscular injection of [14C]-TDI (84% 2,4-, 16% 2,6-TDI) total urinary excretion after 360 h was 53%, faecal 39%, expired air was negligible, and the remaining activity in the carcass was 4% (Saclay, 1976).

 

In vitro metabolism:

According to an in vitro binding-study with blood proteins, 2,4-TDI binds preferentially to the N-terminal amino acids of globin (valine and lysine to a minor extend) and albumin (lysine and to a minor extent aspartic acid) (Mraz et al., 1997). N-terminal lysine adducts are the most abundant 2,4-TDI adducts. By this binding ureid adduct are being formed which can be converted to and determined as specific hydantoins.

 

In the presence of N-acetyl-L-cystein under aquaeous conditions, 2,4-TDI is predominantely forming (AcCys)-conjugates and insoluble urea with amino end groups. Free TDA/TDI is not detectable. The amount of conjugates being formed is increasíng with an excess of AcCys. With an excess of isocyanate insoluble urea is the predominant reaction product. Due to the hydrophobicity of TDI these reaction products are forming small droplets or solid perticles with an insoluble layer of urea at the surface and occluded TDI prevented from diffusion and therefore from further reaction (Morman, 2002).

 

Conclusions

When TDI was administered to rats by either the oral or inhalation routes, excretion of absorbed radioactivity was confined to urine and faeces. However, distinct exposure route-specific differences were apparent between urinary excretion kinetics and metabolite profiles, and plasma radioactivity profiles.

 

After oral administration of TDI physicochemical properties of the substance lead to the hydrolysis to TDA or formation of polyurea in the stomach. It is the TDA which is subsequently absorbed and metabolized. This does not happen by inhalation as supported by the data from several studies. While the chemical reactivity of TDI precludes the free isocyanate entering the systemic circulation from the lung, it has been postulated that TDI will conjugate or react with biological molecules in the lung which then enter the systemic circulation. Absorbtion as a glutathione conjugate may be a possible pathway.

 

In man there are several reports measuring either plasma or haemoglobin adducts of TDI, or urinary metabolites. For urinary biomarkers, methodology uses acid or base hydrolysis to release TDA which is subsequently quantified. Free TDA has not been detected in urine of humans exposed to atmospheric TDI. While a precise relationship between inhalation exposure and biomarker is not established, it is clear that urinary excretion reflects very recent exposures to TDI, while blood biomarkers may reflect exposures over the proceeding few weeks.

 

Justification of read-across from supporting substance (2,4-/2,6-TDI to the substance)

The 80:20 mixture of 2,4-/2,6-TDI (CAS No. 26471-62-5) is the monomeric component of the oligomeric substance. The examination of the material balance of Desmodur VP.PU 60WF14 (the substance) yielded amounts of 42 % 2,4-TDI, 13.7 % 2,6-TDI and ca. 44 % of the substance (Currenta, 2009). Thus, the substance contains ca. 56 % of a 80:20 mixture of 2,4-/2,6-TDI.

 

With regard to the toxicological comparability of the substance and 2,4-/2,6-TDI acute inhalation toxicity studies in rats revealed 4-hour LC50 values (aerosol) of 112 mg/m3 for the substance (based on sum of TDI isomers) and 107 mg/m3 for 2,4-/2,6-TDI (Folkerts, 2010). All qualitative cornerstones of TDI-induced respiratory tract injury were essentially identical. This included the typical delayed-onset mortality, likely as a result of a bronchiolitis obliterans. Of note is the over-proportional presence of TDI vapor relative to the the substance after inhalation exposure. This is consistent with the higher vapor pressure of TDI. In summary, the similarities of LC50s in the presence of the substance up to analytically verified breathing zone concentrations of 112 mg substance/m3 demonstrates that the inhalation toxicity of TDI per se isnot affected to any appreciable extent by the presence of the substance aerosol. This means, modulating factors due to physicochemical interactions (partitioning of the vapor phase with the liquid aerosol phase) were not apparent as this would have lead to a more immediate onset of mortality (immediate acute lung edema rather that delayed bronchiolitis obliterans). Overall, these data demonstrate that the acute inhalation toxicity of the substance is negligible relative to TDI and any dependence of acute hazards on specific use patterns (vapor vs. aerosol) cannot be envisaged(expert opinion of Prof. J. Pauluhn: Desmodur VP.PU 60 WF14 (TDI Biuret): Comparison of acute inhalation toxicities of TDI Biuret and TDI, dated Sep. 3, 2010; complete expert opinion attached in IUCLID chapter 7 “Endpoint summary: Toxicological information”).

In addition, the toxicity profiles of the substance and 2,4-/2,6-TDI also show a high degree of consistency regarding the endpoints acute oral toxicity, skin irritation, eye irritation, skin sensitization and genotoxicity in vitro.

Therefore, based on all available data the test results obtained for 2,4-/2,6-TDI can be transferred to the substance and based on such a read-across further testing of the substance is not required. This approach is in accordance with Annex XI, section 1.5 of the REACH Regulation (EC) No 1907/2006.

Discussion on bioaccumulation potential result:

For basic toxicokinetics endpoint summary refer to the 7.1 Toxicokinetics, metabolism and distribution endpoint summary.