2,4,6-triisopropyl-m-phenylene diisocyanate

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

Please refer to Read-across statement in section 13

Preliminary studies:

No further details on preliminary studies given (reference to endpoint study records of the cited publications Timchalk et al, 1994 and Kennedy et al, 1994).

Type:

distribution

Results:

Exposure of rats to radiolabelled TDI vapour results in the appearance of conjugated TDI in the plasma and the urine, but little or no free TDA can be detected. However, oral dosing leads to the appearance of free TDA in the plasma and the urine.

Type:

excretion

Results:

after oral exposure: major route is the faeces (80 % within 48 hours), second route is the urine (5-15 % within 48 hours)

Type:

excretion

Results:

after inhalation exposure: 48% within 48 hours in the faeces, 15 % within 48 hours via the urine

Type:

metabolism

Results:

The addition of TDI to an acidic aqueous medium, such as stomach contents, will give rise to some hydrolysis to TDA and subsequent urea oligomerisation.

Type:

metabolism

Results:

There are measurable quantities of free and acetylated TDA in the urine following oral TDI, such that a 60 mg/kg TDI oral dose is roughly equivalent to a 3 mg/kg TDA dose orally or intravenously.

Type:

metabolism

Results:

Following inhalation of 2 ppm TDI, the amount of acetylated TDA in urine was close to the limits of detection. Free TDA could not be detected in these samples.

Details on absorption:

No detailed information on absorption available (reference to endpoint study records of the cited publications Timchalk et al, 1994 and Kennedy et al, 1994).

Details on distribution in tissues:

No detailed information on distribution available (reference to endpoint study records of the cited publications Timchalk et al, 1994 and Kennedy et al, 1994).

Transfer type:

other: after oral exposure

Observation:

slight transfer

Remarks:

A relatively small portion is retained in the carcass (approximately 5% after 48 hours).

Transfer type:

other: after inhalation exposure

Observation:

distinct transfer

Remarks:

A rather large portion (of 34% recovered radioactivity) is found in the carcass 48 hours after termination of exposure, indicating a slow release of protein-bound material

Details on excretion:

Following oral administration, the major route of elimination is via the faeces. Within 48 hours 80% of the dose is excreted, presumably via the formation of oligoureas, which are not absorbed. The urine forms the second route of elimination (5-15% of dose within 48 hours), and a relatively small portion is retained in the carcass (approximately 5% after 48 hours).
Following inhalation of 2 ppm TDI, the excretion of radioactivity within 48 hours is different from that which follows oral dosing. Faeces contain approximately 48% (of recovered radioactivity) presumably arising from biliary excretion and from secondary oral uptake (escalation of polymeric material from the lung or from grooming). Urinary excretion accounts for approximately 15%, and a rather large portion (of 34% recovered radioactivity) is found in the carcass 48 hours after termination of exposure, indicating a slow release of protein-bound material, as suggested by in vitro data.

Metabolites identified:

yes

Details on metabolites:

There are measurable quantities of free and acetylated TDA in the urine following oral TDI, such that a 60 mg/kg TDI oral dose is roughly equivalent to a 3 mg/kg TDA dose orally or intravenously. Following inhalation of 2 ppm TDI, the amount of acetylated TDA in urine was close to the limits of detection. Free TDA could not be detected in these samples.

The addition of TDI to an acidic aqueous medium, such as stomach contents, will give rise to some hydrolysis to TDA and subsequent urea oligomerisation. The inability of TDI to form conjugates with biological molecules at low pH is consistent with the loss of strong nucleophilic substituents in these molecules at such pH levels. Following exceptionally high oral dosing with TDI, an insoluble residue is found in the stomach (Jeffcoat, 1988; Dieter et al., 1990). If animals are kept alive, then a major proportion of the administered and obviously polymerized radioactivity is excreted in the faeces, while a small amount is excreted via urine. At lower oral TDI doses polymerization is diminished and a relatively large percentage is absorbed and excreted via urine.

The in vitro data indicate also that TDI vapour preferentially forms conjugates with protein in an aqueous environment. The extent of conjugate formation is pH-dependent, with neutral pH favouring this reaction. In addition, the extent of conjugate formation is dependent on the composition of the aqueous environment.

Although it is known that after oral TDA administration metabolites other than TDA or acetylated TDA are excreted in urine (Waring and Pheasant, 1976), Timchalk et al. (1994) have shown that urinary excretion of both compounds can be used as indicator for the estimation of an internal TDA dose. It is therefore possible to use such information in the following risk estimation.

Conclusions:

This review on the toxicokinetic properties of Toluene diisocyanate was conducted by using several existing publications. Therefore no guideline has to be / was followed. The validity of the underlying experiments is described in the corresponding endpoint study records. Following oral dosing of TDI, dose-dependent percentages of it are converted to TDA by hydrolysis (carcinogenicity following TDI gavage studies). Following inhalation TDI is conjugated to protein preferentially before formation of oligoureas or hydrolysis.

Executive summary:

The substance of interest Toluene diisocyanate (TDI) was subject to a detailed review of the existing toxicological information by Doe and coworkers (1995). The absorption, distribution, and kinetics of TDI are qualitatively and quantitatively different following inhalation exposure when compared with oral dosing, with inhalation being the relevant route in humans. Following oral dosing of TDI, dose-dependent percentages of the compound are converted to TDA (a mutagen and rodent carcinogen) by hydrolysis (mainly at aqueous tissue surfaces), which is consistent with the carcinogenicity observed following TDI gavage studies. The lower pH levels in i.e. the stomach are leading to high protonation of biological NH2 groups and this facilitates hydrolysis of TDI to TDA and subsequent formation of polyureas. These observations are consistent with comparative toxicokinetic studies in rats, which demonstrate significant levels of TDA following oral dosing with TDI - due to the acidic environment in the stomach - but not after inhalation.

Upon inhalation exposure TDI is conjugated to protein preferentially before formation of oligoureas or hydrolysis to TDA takes place. After inhalation a rather large portion (of 34% recovered radioactivity) is found in the carcass 48 hours after termination of exposure, indicating a slow release of protein-bound material, as suggested by in vitro data. Overall these conclusions are consistent with the lack of carcinogenicity observed in inhalation two-year studies with TDI and the tumours observed in rodents after oral dosing of TDI in corn oil. Additionally there are human exposure data indicating that the metabolism and kinetics in humans might be similar to that in animals, therefore valid extrapolations can be made. The conventional hazard assessment and resulting risk evaluation comes to the conclusion that there is no risk for carcinogenicity associated with the inhalation of TDI. Considering additional data (gavage studies with TDI or TDA and mechanistic biochemical data), three approaches of risk characterization arrive at the conclusion that the highest possible risk associated with the inhalation of TDI at workplaces with TLV level exposure is about 5 x 10E-6. TDI inhalation at workplaces therefore will not present an unacceptable risk of carcinogenicity to man.

Reference 2

Endpoint:

basic toxicokinetics

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

Please refer to Read-across statement in section 13

Preliminary studies:

no data on preliminary studies given

Type:

metabolism

Results:

Free + acetylated TDA metabolites detected in urine (0-12 hr) after oral or inhalation exposure to 2,4-[14C]TDI were 15.37 & 0.26 ng eq 2,4-TDA, indicating that following oral TDI a larger amount of 2,4-TDA & acetylated metabolites (59-fold) were detected

Type:

excretion

Results:

81% of the oral 2,4-[14C]TDI dose was eliminated in the faeces, 8% of the dose was eliminated in the urine. The tissues/carcass accounted for approx. 4% of the dose & approx. 2/3 of this radioactivity (2.6 % of the dose) associated with the GIT-content.

Type:

excretion

Results:

Following inhalation exposure (excreta collected for 48 hr postexposure) faeces & urine accounted for 47 & 15% of the recovered radioactivity. The tissues/carcass accounted for 34%, & 1/2 of this ( 17% of the recovered RA) was associated with GIT-content.

Type:

excretion

Results:

Following oral administration and inhalation exposure to 2,4-[14C]TDI, peak urinary excretion of radioactivity occurred during the first 12-hr collection interval. However, the urinary 14C excretion was slower following inhalation exposure to 2,4-[14C]TDI

Type:

other:

Results:

Following oral exposure less than 1% of the recovered radioactivity was in the final cage wash.

Type:

other:

Results:

Following inhalation exposure approx. 4% of the radioactivity was recovered in the final cage wash & insufficient radioactivity was eliminated as 14CO2 or 14-volatile organics to quantify.

Type:

other:

Results:

data suggest that a larger fraction of the inhaled radioactivity was retained in the tissues/carcass for a longer period of time in comparison to orally administered 2,4-[14C]TDI.

Details on absorption:

no detailed information on absorption available.

Details on distribution in tissues:

The distribution of radioactivity recovered in the rat 48 hr after an oral 60 mg 2,4-[14C]TDI/kg body wt dose and 48 hr following a 2-ppm 4-hr inhalation exposure to 2,4-[14C]-TDI was just determined in the excreta.

Test no.:

#1

Transfer type:

other: transfer into tissue / carcass after oral administration of TDI

Observation:

slight transfer

Remarks:

The tissues/carcass accounted for approx. 4% of the dose & approx. 2/3 of this radioactivity (2.6 % of the dose) associated with the GIT-content.

Test no.:

#1

Transfer type:

other: transfer into tissue / carcass after inhalation administration of TDI

Observation:

distinct transfer

Remarks:

The tissues/carcass accounted for 34%, & 1/2 of this ( 17% of the recovered RA) was associated with GIT-content.

Details on excretion:

Following oral administration, approximately 94% of the administered radioactivity was recovered in the urine, faeces, tissues/carcass, and final cage wash. Insufficient radioactivity was eliminated as 14CO2 or 14C-volatile organics to quantify.
Following oral administration and inhalation exposure to 2,4-[14C]TDI, peak urinary excretion of radioactivity occurred during the first 12-hr collection interval. However, compared to the oral treatment group, the urinary 14C excretion was slower following inhalation exposure to 2,4-[l4C]TDI.

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: The half-live derived from the slope of the urinary 14C excretion time-course following 2,4-[14C]TDI oral administration was 7.5 h.

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: The half-live derived from the slope of the urinary 14C excretion time-course following 2,4-[14C]TDI inhalation exposure was 20 hr.

Metabolites identified:

yes

Details on metabolites:

Total amount of free + acetylated TDA metabolites detected in the 0- to 12-hr urine following oral or inhalation exposure to 2,4-[14C]TDI were 15.37 and 0.26 ng eq 2,4-TDA, respectively, indicating that following the oral 2,4-[14C]TDI dose a larger amount of 2,4-TDA and acetylated metabolites (59-fold) were detected in the urine compared to the inhalation exposure group.
Analysis of urine specimens for total acid-labile conjugates of 2,4-TDI/2,4-TDA detected 44.51 and 2.53 ng eq 2,4-TDA following the oral and inhalation exposure to 2,4-[14C]TDI, respectively. Comparison of the ratio between the free + acetylated TDA to the total acid-labile metabolites indicated that following an oral 60 mg/kg 2,4-[14C]TDI dose approximately 35% of detected metabolites existed as either free or acetylated 2,4-TDA with the remainder (65%) existing as other 2,4-TDI/2,4-TDA conjugates.
In contrast, analysis of the urine specimens from rats exposed to 2 ppm 2,4-[14C]TDI indicated that only 10% of the quantitated metabolites were identified as acetylated TDA while the remaining 90% were other conjugated forms of either 2,4-TDI or 2,4-TDA. No free 2,4-TDA was detected. Urine (0-12 hr) obtained from rats following an oral 60 mg/kg 2,4-[14C]TDI dose contained 48- and 25-fold less mono- and diacetyl-TDA and 88-fold less free 2,4-TDA than the urine obtained from rats administered an oral 60 mg/kg 2,4-[14C]TDA dose. The amount of free + acetylated TDA metabolites detected in the urine specimens following a 2-ppm inhalation exposure to 2,4-[14C]-TDI can be compared with the amounts detected following the oral 3 mg/kg 2,4-[14C]TDA dose. This comparison was reasonable since 2 ppm 2,4-TDI inhalation exposure and 3 mg/kg oral 2,4-TDA dose were nearly equivalent on a ng, eq basis (i.e., 899 vs 729 ng eq). Following 2,4-[14C]TDI inhalation exposure no free 2,4-TDA was detectable in the urine. In addition, the 2,4-[14C]TDI urine contained 38- and 101-fold less mono- and diacetyl-TDA than the urine obtained from rats administered an oral 2,4-[14C]-TDA dose. Comparison of the 2-ppm 2,4-[14C]TDI inhalation exposure and 3 mg/kg oral 2,4-[14C]TDA dose groups indicated that approximately 90 and 16% of the detected metabolites existed as acid-labile conjugates, respectively. Based on these data, the rats exposed via inhalation to 2,4-[14C]TDI had a much larger percentage of their TDI metabolites existing in a conjugated form, when compared to the 2,4-[14C]TDA treatment group.

The distribution of radioactivity recovered in the rat 48 hr after an oral 60 mg 2,4-[14C]TDI/kg body wt dose and 48 hr following a 2-ppm 4-hr inhalation exposure to 2,4-[14C]-TDI are presented in Table 1.

Following oral administration, approximately 94% of the administered radioactivity was recovered in the urine, faeces, tissues/carcass, and final cage wash. Insufficient radioactivity was eliminated as 14CO2 or 14C-volatile organics to quantify. Approximately 81% of the oral 2,4-[14C]TDI dose was eliminated in the faeces, which represented the primary elimination route, whereas 8% of the dose was eliminated in the urine. The tissues/carcass accounted for approximately 4% of the dose and approximately 2/3 of this radioactivity (2.6% of the dose) was associated with the gastrointestinal tract contents. Less than 1% of the recovered radioactivity was in the final cage wash.

Following inhalation exposure, excreta were collected through 48 hr postexposure and the faeces and urine accounted for 47 and 15% of the recovered radioactivity, respectively. The tissues/carcass accounted for 34% of the recovered radioactivity, and approximately 1/2 of this ( 17% of the recovered radioactivity) was associated with the gastrointestinal tract contents. Approximately 4% of the radioactivity was recovered in the final cage wash and insufficient radioactivity was eliminated as 14CO2 or 14C-volatile organics to quantify.

TABLE 1
Distribution of Radioactivity 48 hr after Male Fischer 344 Rats Were Given an Oral Dose of 60 mg 2,4-[^14C]TDI/kg body wt or Exposed to 2 ppm 2,4-[^14C]TDI Vapours for a 4-hr Period
	60mg/kg 2,4-TDI^a		2ppm 2,4-TDI^b
	Percentage of administered dose	Mgeq 2,4-TDI	Percentage of recovered radioactivity	Mgeq 2,4-TDI
Urine	8.38 ±0.41	1225±59	14.85±1.39	133±10
Faeces	80.67±5.30	11,795 ±718	47.29±11.98	424±102
Tissues/carcass	3.77±0.87 (2.56±0.84)	552±129 (375±124)	34.14±1 1.53 (16.63±9.18)	309±113 (150 ±86)
^14co2	NQ	NQ	NQ	NQ
^14C-volatile organics	NQ	NQ	NQ	NQ
Cage wash	0.99±0.52	145±77	3.73±1.95	33±17
Total	93.80±4.32	13,717±573	NA	899±35
Note: Values in( )represent^14C activity found in the gastrointestinal tract contents. NA, not applicable; NQ, not quantifiable.
^aValues represent mean ± SD for three animals.
^b Values represent mean ± SD for four animals.

The radioactivity recovered in the tissues/carcass at 2 and 48 hr following an oral 60 mg/kg dose, as well as immediately postexposure and 48 hr following a 2 ppm 4-hr inhalation exposure, are summarized in Table 2. Approximately 83 and 4% of the orally administered 60 mg/kg dose was recovered in the tissues/carcass at 2 and 48 hr postdosing, respectively. At 2 hr following oral administration the gastrointestinal tract and contents accounted for ~92% of the recovered radioactivity in the tissues/carcass with the remaining radioactivity evenly distributed among the remaining tissues. Whereas, at 48 hr postdosing the radioactivity levels in all tissues were fairly low with the gastrointestinal tract contents still accounting for the majority of what was recovered. Immediately following a 2-ppm 4-hr inhalation exposure, 90% of the recovered dose was in the tissues/ carcass with the remaining 10% associated with the gastrointestinal tract contents. At 48 hr postdosing, 18% of the recovered dose was associated with the tissues/carcass while 17% was in the gastrointestinal tract contents.

TABLE 2
Tissue Distribution of Radioactivity 2 and 48 hr after Male Fischer 344 Rats were Given an Oral Dose of 60 mg 2,4-[^14C]TDI/kg body wt Immediately and 48 hr Postexposure to 2 ppm 2,4-[^14C]TDI Vapors for a 4-hr Period
	Percentage of administered dose		Percentage of recovered dose
	60mg/kg 2,4-TDI		2ppm 2,4-TDI
	2hr postdosing ^a	48hr postdosing^b	Immediately postexposure^a	48hr postexposure^a
Blood	NA	0.05±0.02	NA	0.23 ±0.15
Carcass	5.50±3.62	0.77±0.20	71.54±2.99	10.02±2.69
Fat	<0.01	<0.01	0.02±0.00	<0.01
Kidney	0.08±0.02	0.02±0.00	0.69±0.08	0.25±0.04
Liver	0.50 ±0.15	0.11±0.00	1.68 ±0.13	0.37±0.01
Lung	0.99±0.52	<0.01	2.50±1.13	0.28 ±0.12
Skin	1.12±0.53	0.15 ±0.02	9.86 ±3.12	5.59±1.61
GI tract	10.10±3.09	0.10 ±0.05	3.75±1.56	0.76±0.37
GI contents	65.82±8.35	2.56±0.84	9.76±2.31	16.63±9.18
Total	83.18±7.19	3.77±0.87	---	34.14±11.53
Note: NA, not analysed.
^a Values represent Mean ± SD for four animals.
^bValues represent Mean ± SD for three animals.

Quantitative differences in the primary routes of elimination following 2,4-[14C]TDI (oral or inhalation) and 2,4-[14C]TDA (oral or iv) administration were observed. Whereas the urine was a minor excretion pathway following 2,4-[14C]TDI exposure, it represented the major excretion pathway following 2,4-[14C]TDA administration.

The urinary 14C-excretion time-course is expressed as ng eq (following either 2,4-[14C]TDI (oral and inhalation) or 2,4-[14C]TDA (oral and iv) administration). Following oral administration and inhalation exposure to 2,4-[14C]TDI, peak urinary excretion of radioactivity occurred during the first 12-hr collection interval. However, compared to the oral treatment group, the urinary 14C excretion was slower following inhalation exposure to 2,4-[14C]TDI. The half-lives derived from the slope of the urinary 14C excretion time-course following 2,4-[14C]TDI oral administration and inhalation exposure were 7.5 and 20 hr, respectively. The half-lives derived from the slope of the 14C urinary excretion time-course following an oral and iv administration of 2,4-[14C]TDA (3 and 60 mg/kg) were 8 and 4.6 hr, respectively. Comparison of the 14C urinary time-course following an oral 2,4-[14C]TDI (60 mg/kg) or 2,4-[14C]TDA (3 and 60 mg/kg) doses indicated that the 14C urinary excretion rates (t1\2 = 5-8 hr) were similar. In contrast, a 4-hr inhalation exposure to 2 ppm 2,4-[14C]TDI resulted in a much slower excretion of radioactivity in the urine {t1/2 = 20 hr).

TABLE 3
Concentration of Monoacetyl, Diacetyl, and Free 2,4-TDA Expressed as 2,4-TDA Equivalents in the 0- to 12-hr Pooled Urine Specimen Following Oral and Inhalation Exposure to 2,4-[^14C]TDI and Oral and iv Administration of 2,4 -[^14C]TDA in Male Fischer 344 Rats
	Mgeq 2,4-TDA^a
Treatment group	Monoacetyl	Diacetyl	Free
2,4-TDI
Oral(60mg/kg)	5.12 (4.96, 5.20)	8.17 (7.93, 8.41)	2.08 (2.00, 2.24)
Inhalation(2ppm)	0.16 (0.16, 0.16)	0.10(0.10, 0.10)	ND
2,4-TDA
Oral(60mg/kg)	247.90 (240.17, 255.74)	206.99 (194.47, 219.61)	183.48 (162.93. 204.04)
Oral(3mg/kg)	6.09 (5.87, 6.25)	10.13 (10.08, 10.18)	3.93 (3.77, 4.10)
iv(3mg/kg)	6.73 (6.43, 7.09)	10.63 (10.21. 11.05)	1.20(1.14, 1.26)
Note.ND, not detected. Detection limit, 100 ng eq 2,4-TDA/g urine.
^a Values are the means of two determinations; numbers in ( ) are the individual determinations.
TABLE4
Concentration of Free+Acetylated 2,4-TDA and Acid-Labile 2,4-TDI/2,4-TDA Conjugates Expressed as 2,4-TDA Equivalents in the 0-to 12-hr Pooled Urine Specimen Following Oral and Inhalation Exposure to 2,4-[^14C]TDI and Oral and iv Administration of 2,4 -[^14]TDA in Male Fischer 344 Rats
	Mgeq 2,4-TDA
Treatment group	Free+acetylated	2.4-TDI/2,4-TDA^a acid-labile conjugate	Free+acetylate ^b -X100%acid-labile
2,4-TDI
Oral(60mg/kg)	15.37	44.51 (42.59, 46.35)	35
Inhalation(2ppm)	0.26	2.53 (2.48,2.58)	10
2,4-TDA
Oral(60mg/kg)	638.37	1042.18(1025.49, 1058.97)	61
Oral(3mg/kg)	20.15	24.09 (23.39, 24.73)	84
iv(3mg/kg)	18.56	21.26(21.08,21.44)	87
Note: Acetylated, sum of the monacetyl- and diacetyl-2,4-TDA.
^aValues are the means of two determinations; numbers in ( ) are the individual determinations.
^bRatio of free + acetylated to acid-labile conjugates expressed as percentage.

Conclusions:

After inhalation exposure 34 % of the inhaled TDI are incorporated into the tissues /carcass (48 hr following exposure).
The study was conducted to reveal the toxicokinetic properties of TDI, applied orally or via inhalation to rats and the detection of TDI in the blood, urine faeces and cage-wash. The validity criteria of the test system are fulfilled, since the control groups showed the expected results. The study was conducted similar to OECD TG417 and its reliability is judged to be high (Klimisch 2). The results indicate, that the metabolic disposition and carcinogenic potential of 2,4-TDI are dependent upon the route of exposure. Oral administration enhances the hydrolysis of 2,4-TDI, forming 2,4-TDA which is readily absorbed, whereas inhalation exposure to 2,4-TDI primarily results in the formation of 2,4-TDI conjugates and only small amounts of acetylated 2,4-TDA are produced.

Executive summary:

The substance Toluene diisocyanate was investigated for its toxicokinetic properties by Timchalk and coworkers (1994) in Fischer 344 rats. The study was conducted similar to OECD TG417 with only minor deviations, why it was considered to be of high reliability (Klimisch 2). The rats received either 14C-radiolabelled TDI orally (60 mg/kg) or via inhalation (4 h, 2 ppm). The metabolism and excretion were determined also via HPLC and GC-MS.

The data suggest that orally administered 2,4-[14C]TDI is not very well absorbed. The rats eliminated approximately 8% of the radioactivity in the urine and cage wash while 4% was recovered in the tissues/carcass. Thus the minimum estimate for absorption was 12%, which assumed that the radioactivity recovered in the faeces (~ 81 %) represented un-absorbed material. A more realistic estimate for absorption was obtained by assuming that some of the radioactivity in the faeces was absorbed. In addition, orally administered TDI was reported to undergo a rapid hydrolysis under aqueous conditions to form TDA which reacted with available isocyanate groups (TDI) to form polyureas. Under appropriate conditions 2,4-TDI readily hydrolyses to form 2,4-TDA. The 2,4-TDA can react with free 2,4-TDI forming polyurea polymers, which appear to be poorly absorbed from the gastrointestinal tract. Absorbed 2,4-TDA can be excreted in the urine either unchanged or as acid-labile conjugates. Additionally, 2,4-TDA can be N-acetylated forming mono- and diacetylated TDA metabolites which are readily excreted in the urine. Based on the rapid reactivity it is doubtful that TDI was absorbed prior to its hydrolysis to TDA. Therefore, the 12-20% of the 2,4-[14C]TDI dose that was absorbed, most probably represented 2,4-[14C]TDA that had not reacted to form polyureas. Based on the above, it was assumed that the radioactivity that was absorbed and excreted in the urine following the 2,4-[14C]TDI oral dose was absorbed primarily as 2,4-[14C]TDA.

Rats which were exposed to 2,4-[14C]TDI vapours retained essentially all the radioactivity that they inhaled. Inhaled 2,4-[14C]TDI appeared to be retained by the rat, and the data suggest that a large percentage of the radioactivity was absorbed through the lungs into the blood. At 48 hr following a 2,4-[14C]TDI inhalation exposure, the urine and cage wash accounted for 19% of the recovered radioactivity, the tissues/carcass accounted for 34%, and 47% was recovered in the faeces. However, assuming 100% retention of inhaled TDI and minimal excretion over the 4-hr exposure, the quantitation of radioactivity detected in the tissues/carcass immediately postexposure would suggest that as much as 90% of the radioactivity was absorbed. The remaining radioactivity may have been rapidly cleared from the respiratory tract and subsequently swallowed. The pulmonary clearance of radioactivity from the lung to the gastrointestinal tract was supported by the fact that approximately 10% of the recovered inhalation dose was detected in the gastrointestinal tract contents of rats immediately postexposure. These data suggest that between 61 and 90% of the inhaled 2,4-[14C]TDI dose was absorbed and the remaining radioactivity was rapidly cleared from the respiratory tract, ingested, and then eliminated in the faeces. In conclusion, comparison of the total amount of radioactivity in the tissues/carcass of rats indicated that a larger fraction of the recovered radioactivity was in the tissues/carcass following the inhalation vs oral exposure to 2,4-TDI. These data suggest that following inhalation exposure, a large percentage of the 2,4-[14C]TDI was absorbed through the lungs and existed in a different form than what was absorbed following oral 2,4-TDI and/or 2,4-TDA doses. The urinary excretion of radioactivity by the kidneys was slower following inhalation exposure (t1\2 = 20 hr) when compared to the oral 2,4-[14C]TDI dose group (t1\2 = 7.5 hr), suggesting that inhaled 2,4-TDI was eliminated in the urine in a different form having a longer biological half-life than orally administered 2,4-TDI and/or 2,4-TDA. This suggests that in the rat, very little 2,4-TDA is formed following inhalation exposure to 2,4-[14C]TDI vapours. In addition 90% of the quantitated metabolites in the urine specimens following inhalation exposure to 2,4-[14C]TDI existed as acid-labile conjugates of TDI/TDA while only 10% existed as acetylated TDA. This indicated that following inhalation exposure, a larger percentage of the 2,4-[14C]TDI was excreted in the urine in a conjugated form and not as free or acetylated TDA.

Overall, these data suggest that the metabolic disposition and carcinogenic potential of 2,4-TDI are dependent upon the route of exposure. Oral administration enhances the hydrolysis of 2,4-TDI, forming 2,4-TDA which is readily absorbed, whereas inhalation exposure to 2,4-TDI primarily results in the formation of 2,4-TDI conjugates and only small amounts of acetylated 2,4-TDA are produced. These findings are consistent with the chronic bioassay data which indicated that 2,4-TDI was not carcinogenic following inhalation exposure, but did result in tumour formation following oral administration in corn oil. Considering that the primary route of occupational exposure to TDI is via the inhalation route, then the metabolic and bioassay data would suggest that the carcinogenic potential of TDI is low.

Reference 3

Endpoint:

basic toxicokinetics

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

Please refer to Read-across statement in section 13

Preliminary studies:

No information concerning any preliminary studies was given.

Type:

distribution

Results:

The highest levels (µgEq/g) were found in the airway tissues; however, over the concentrations tested, some form of the radioactive compound was distributed throughout the system.

Details on distribution in tissues:

The highest levels (µgEq/g) were, in fact, found in the airway tissues; however, over the concentrations tested, some form of the radioactive compound was distributed throughout the system.
Analysis of blood samples taken immediately upon termination of the 4 hr exposure showed that the 14C entered the bloodstream and that the level increased linearly as a function of exposure concentration.
In addition to understanding that the radioactivity, in some form, is taken into the bloodstream, it is perhaps more important to investigate the biochemical state of this radioactivity. One of the potential routes of uptake of TDI or its products into the bloodstream could be direct penetration from the respiratory tract surfaces into the blood. Based on the high degree of reactivity described above, this possibility appears unlikely. Another potential mechanism would involve the reaction of TDI within the aqueous environment of the airway which could result in hydrolysis and diamine formation. Thirdly, the isocyanate could react, in the airway, to form adducts with peptides, proteins, lipids and carbohydrates which then could be transferred into the bloodstream. To investigate these possibilities, biochemical analysis of the 14C-material in terminal blood was performed. The first level of characterization was to determine the distribution of 14C in the various blood components. For all exposure concentrations tested, the majority of radioactivity was plasma-associated. Further analysis of the plasma by molecular weight fractionation was also performed. The rationale for this separation was that if free TDI or TDA were the form of the labelled material in the bloodstream, the l4C would be associated with the tolyl group and would be of a low molecular weight. Alternatively, if biomolecular adducts were formed, the molecular weight of the 14C-adduct would be significantly higher. The results showed that greater than 95% of the plasma-associated radioactivity existed in a conjugated form with a molecular weight greater than 10 kDa. This data suggests that the in vivo reaction of TDI with biological macromolecules successfully competes with hydrolysis to the diamine. However, in the timeframe of the exposures, it is possible that hydrolysis to the diamine occurred. The diamine could have then been metabolised, either locally or systemically, to form a compound capable of in vivo reaction. Such an activation could also account for the high molecular weight components observed. Regardless of the pathway, the results support the conclusion that conjugation predominates under the experimental conditions tested.
Electrophoretic characterisation of the retentate fractions from plasma demonstrated a degree of specificity in the conjugation reactions. At all concentrations of TDI tested, the predominant radioactive component detected was a protein of 70 kDa molecular weight. Due to the relative abundance of serum albumin in plasma and the similarity in molecular weight, it was hypothesised that the 14C-labeled 70 kDa protein was TDI-modified serum albumin. An albumin affinity column was run and the profiles supported the hypothesis that some portion of the labelled material was the modified albumin. However, at the highest concentration, it appears a threshold of labeling is reached where upon another component is labelled which does not bind to the column. Alternatively, it is also possible that so many molecules of TDI had reacted with the albumin molecule that its binding affinity was altered.

It has been hypothesised that upon delivery to an acidic environment such as the conditions of the gastrointestinal tract, the hydrolysis of TDI to TDA would be favoured over conjugate formation. In this study, a significant fraction of the radiolabeled material was found associated with the tissues and materials of the gastrointestinal tract. Extraction and biochemical analysis of the stomach contents was performed to investigate the state of the 14C-material. Molecular weight fractionation studies showed that just as seen with the plasma, a conjugated form of the radioactive material predominated; however, a larger amount of low molecular weight material was observed. Based on the lower pH of the stomach, if reactive TDI reached this compartment, hydrolysis to the diamine would be predicted. This could occur by gasping and swallowing air. Alternatively, the material in the stomach could have first entered the airway and then could have been delivered through mucocilliary clearance, to the stomach. HPLC characterisation of the filtrate fraction of the stomach content extract demonstrated that the low molecular weight fraction had numerous radiolabeled components. A TDA co-migrating species was observed but this component was only one among a variety of other products. The prevalence of adducts suggests that the primary reactions occurred in the airway where the protonation state of the macromolecules would favour nucleophilic reactions. The low molecular weight of these products may be attributable to proteolysis once the conjugates entered the stomach environment. These results do not parallel the polymerisation and hydrolysis that was seen by gavage administration of TDI (Leffcoat 1985; Timchalk et al. 1994). This asserts that especially for reactive chemicals, the route of administration may severely influence the compound reactivity and subsequent fate.

Details on excretion:

The excretion was not determined.

Metabolites identified:

not measured

Details on metabolites:

No special analysis of the metabolites was conducted. However, the conjugation products in the blood have been described in more details.

Determination of reactive form and concentration of isocyanate in exposure atmosphere

A reversed phase HPLC analysis was conducted on exposure chamber atmospheric samples following derivatisation with PNBPA. Fractions were collected across the profile and counted. For each exposure, 95-99% of the injected radioactivity was recovered with 98-99% of the recovered radioactivity in the 2,4 TDI derivative peak. Retention times of unlabeled 2,4-TDI as well as 2,6-TDI, Mondur TD80 (80% 2,4-isomer/ 20% 2,6-isomer) (Miles) and toluenediamine (TDA) were analysed under identical conditions (Kennedy et al. 1989). This analysis showed that the radiolabeled material co-migrated with 2,4-TDI and did not contain other contaminating compounds such as the 2,6 isomer or the hydrolysis product, TDA. A concentration calibration curve was generated with unlabeled 2,4-TDI (Fluka) and the peak areas were used to determine the concentration of TDI in the radioactive samples. These analyses confirmed the exposure compound purity and concentration. The ability of the radioactive compound to react with the PNBPA demonstrated that the chemical form used for animal exposure was reactive isocyanate.

Quantitation of isocyanate exposure concentrations

A series of 14C-TDI exposures was conducted over a range of concentrations as given in Table 1. Three methods were used to monitor the exposure atmospheres: the Marcali assay, scintillation analysis of Marcali trapping solutions, and HPLC analysis of PNBPA-derivatized isocyanate, Table 1 summarises the compiled data available for each experiment. Multiple samples, were collected during the 4-h exposures at approximately 30-min intervals. The results of all determinations were averaged. This yielded exposure concentrations of 0.026, 0.143 and 0.821 ppm for the experimental series. Based on these concentrations, an estimated dose for each exposure group was calculated, assuming 100% retention of the reactive vapor using the following equation: concentration (mg/mL) x time x tidal volume x frequency of respiration. Values for tidal volume and frequency for rats were taken from Altman and Dittmer (1971). A summary of the results of these calculations is given in Table 2.

Table 1: Quantitation of isocyanate atmospheric concentration
Exposure#	Marcali(A550)			Marcali (cpm)			PNBPA (HPLC)			Average	SD
	n^a	ppm average	SD ^b	n	ppm average	SD	n	ppm average	SD	Cone, (ppm)
1	9	0.016	0.004	9	0.027	0.003	10	0.037	0.006	0.026	0.010
2	9	0.088	0.022	9	0.119	0.027	9	0.223	0.019	0.143	0.071
3	9	0.77	0.099	9	0.90	0.125	9	0.790	0.130	0.821	0.070
^an, number of samples taken during each exposure
^bSD, standard deviation

Table2: Calculation of estimated, inhaled dose
ppm	Concentration (mg/mL)	Time (min)	VT^a (mL)	f (breaths/min)	dose^b(µg)
0.026	1.9x10-7	240	1.5	100	6.7
0.143	1.0x10-6	240	1.5	100	37
0.821	5.9x10-6	240	1.5	100	210
^aVT, tidal volume
^bDose(mg),C(mg/mL) XT(min) XVT(mL) Xf(brcaths/min)

Distribution of 14C in tissues of TDI-exposed rats

The highest levels of radioactivity (µg Eq/g) were associated with the airway tissues. In addition, some form of the radioactivity was found associated with the other organs analysed. The specific activity (µgEq/g) of the 14C in all tissues increased with exposure concentration. The percentage of the calculated, estimated dose for each tissue is given in Table 3. The tissues are presented in three groupings: airway materials, gastrointestinal materials and blood and other systemic organs; each group showing decreasing levels of labeling as the material went through the system.

Table 3: Tissue distribution of radioactivity following^14C-TDI exposure
Organ system	Tissue	0.026 ppm		0.143 ppm		0.821 ppm
		µgEq/g  average ± SD	%of dose/  total tissue  ± SD	µgEq/g  average ± SD	%of dose/  total tissue  ± SD	µgEq/g average  ± SD	%of dose/ total tissue  ± SD
Airway	Trachea	1.045 ± 0.346	0.39± 0.16	14.25 ± 3.21	0.79  ± 0.18	73.02 ±40.08	0.63 ± 0.49
	Lung	0.124 ± 0.031	1.42 ± 0.36	0.431 ± 0.108	0.89 ± 0.22	2.687 ± 1.640	0.88  ± 0.49
Gastrointestinal	Esophagus	0.453 ± 0.273	0.70 ± 0.44	1.437 ± 0.308	0.39 ±0.13	3.867 ± 2.678	0.13  ± 0.08
	Stomach	0.162 ± 0.145	3.25 ± 3.71	5.663 ± 3.084	1.82 ±0.92	9.520 ± 2.808	2.07 ± 1.28
Systemic	Blood	0.050 ± 0.003	10.68 ± 1.03	0.190 ± 0.013	7.18 ± 0.37	0.577 ± 0.104	3.93 ± 0.72
	Heart	0.029  ± 0.009	0.28 ± 0.10	0.089 ± 0.016	0.10  ± 0.06	0.286 ± 0.089	0.07 ± 0.02
	Liver	0.024 ± 0.006	2.81 ±0.70	0.107 ± 0.005	2.00 ± 0.15	0.290 ± 0.041	0.90 ± 0.10
	Kidney	0.112 ± 0.031	2.39 ±0.63	0.379 ± 0.040	0.75 ±0.08	0.902 ± 0.151	0.55 ± 0.09
	Spleen	0.019 ± 0.002	0.11 ±0.01	0.076 ± 0.005	0.09 ± 0.01	0.207 ± 0.023	0.04 ± 0.01

Quantitation of 14C in the whole blood of TDI-exposed rats

Scintillation analysis of whole blood taken immediately following exposure showed that radioactivity reached the bloodstream. Using the calculated total dose, the percentage of the estimated value which was detected in the bloodstream decreased from 10.68 to 3.93 as exposure concentration increased (Table 3). Over the range of exposure concentrations tested, a direct relationship was found between the ppm*h and the µgEq tolyl group per mL immediate post-exposure terminal blood. The equation of the resultant line is y = 0.03+0.21x with an R value equal to 0.985.

Distribution of 14C in blood components of TDI-exposed rats

Analysis of radioactivity in whole blood clearly shows that for all exposures, some form of the labeled compound entered the bloodstream. Biochemical analyses of blood samples were performed to characterize the labeled constituents in the blood immediately following the 4-h exposures. Plasma and cell components were separated and subjected to scintillation analysis. Table 4 shows the results expressed as a percentage of total blood radioactivity. At all concentrations of TDI tested, the majority of radioactivity was found to be plasma-associated (74-87%); however, the amount of radioactivity in the cell pellet fraction was measurable (11-20%) and total µgEq increased with exposure concentration.

Table 4: Distribution of radioactivity in blood components
Exposure concentration (ppm)	Whole blood total µgEq	Plasma µgEq(%of total)	Cell pellet µgEq(%of total)
0.026	0.245	0.215 (87)	0.049 (20)
0.143	1.219	0.957 (79)	0.243 (20)
0.821	4.262	3.144 "(74)	0.485 (11)

Distribution of plasma radioactivity as a function of molecular weight

One of the primary questions regarding the fate of isocyanates in the blood following exposure is whether there are low molecular weight compounds (e.g., TDI, oligoureas or metabolites) and/or high molecular weight adducts. To address this question, plasma samples were subjected to molecular fractionation using Centricon-10 microcon-centrators which separate the high molecular weight (> 10 kDa) conjugates from low molecular weight (< 10 kDa) conjugates and metabolites. The distribution of radioactivity in the retentate (> 10 kDa) and filtrate (< 10 kDa) fractions was determined by scintillation analysis (Table 5). The results show that the predominant form (97-100%) of the radioactivity in the plasma immediately following a 4-h exposure is conjugated material greater than 10 kDa in molecular weight.

Table 5: Molecular sieve fractionation of plasma
Exposure cone. (ppm)	Analysis of fraction >10 kDa(retentatefraction)		Analysis of fraction <10 kDa (Filtrate fraction)
	Total ngEq average ± SD	%of original average ± SD	Total ngEq average ± SD	%of original average ± SD
0.026	18.4+ 3.7	100.9+14	0.91+0.59	5.1+1.3
0.143	58.8±7.3	97.0±13	2.41+0.84	3.9 + 3.9
0.821	138.0 ±31.3	100.3 + 20	5.29+1.41	3.8 + 1.1

Electrophoretic analysis of in vivo conjugates in plasma

Plasma retentate (>10 kDa) samples were subjected to SDS polyacrylamide gel electrophoresis to further characterize the nature of the in vivo, high molecular weight conjugates. The majority of radioactivity was associated with a 70-kDa protein band at all concentrations.

Affinity chromatography of plasma retentate fractions

To test if the 70-kDa labeled protein was serum albumin, retentate fractions were run through a Reactive Blue-4 agarose albumin affinity column. It is demonstrated that the absorbance profile is quite similar in samples from all three concentration levels tested. In contrast, the corresponding distributions of radioactive components varies with concentration. Relative to the amount of bound radioactivity associated with peak 2, the level of 14C in peak 1 increases with concentration. This is also supported by the representation of the data given in Table 6 which shows that as concentration increases, the percentage of radioactivity recovered in peak 1 also increases.

Table 6. Summary of blue agarose chromatography of plasmareténtatefractions
Exposure concentration (ppm)	Total ngEq	% of recovered ngEq
		Peak 1	Peak 2
0.026	26.8	20	68
0.143	69.9	21	60
0.821	129.2	48	18

Thin layer chromatography of plasma filtrate fractions

To examine the radioactive material in the low molecular weight filtrate fractions of plasma, thin layer chromatography was performed. In all samples, including control animal plasma filtrate, a component which co-migrated with TDA (Rf = 0.76) was detected. Notably, a unique TDI exposure related component (Rf = 0.65) was detected by this method. For all lanes, the plate was scraped and silica was subjected to scintillation analysis. The radioactivity was distributed throughout the lane and there was not a single band with a 14C level greater than twice background.

Extraction and fractionation of stomach contents

In addition to the blood and airway tissues, the other major system which showed an increased level of label was the gastrointestinal tract. To examine the nature of the radioactive material in the stomach contents, an aqueous extraction of the stomach content material from controls and the highest exposure concentration group was performed and analysed. The efficiency of the extraction procedure was determined by scintillation analysis of the extracts. The count distribution for each fraction, shows that the majority (77%) of the 14C was extractable in the saline wash and that 41% of the material was recovered as high molecular weight conjugates (> 10 kDa) and 28% was recovered in the filtrate (< 10 kDa) fraction. The percentage of material in the low molecular weight fraction is increased compared to the similar fraction in plasma.

Electrophoretic analysis of in vivo conjugates in stomach content reténtate fractions

Retentate ( > 10 kDa) fractions from stomach content extracts were subjected to SDS polyacrylamide gel electrophoresis to further characterise the nature of the in vivo, high molecular weight conjugates. The distribution of radioactive components in the gel was assayed by autoradiography. Two predominant bands of radioactivity were observed in the retentate fraction, one at the well (>200 kDa) and one co-migrating with the dyefront (< 15 kDa). In addition, a smear of other labeled products is observed throughout the lane. These high and low molecular weight bands were additionally observed in the pellet fraction as well.

High pressure liquid chromatography (HPLC) of stomach content filtrate fractions

Reverse phase HPLC analysis of the stomach content filtrate fractions was done to examine the distribution of low molecular weight radioactive components. A primary question addressed was whether the low molecular weight form of 14C was TDA, a conversion which may be favoured under the acidic conditions of the GI tract. Radioactivity was found not only associated with a TDA co-migrating component but also in numerous other products spread throughout the profile.

Conclusions:

The study was conducted to reveal the toxicokinetic properties of TDI, applied via inhalation to rats andsubsequently the investigation of its distibution. The validity criteria of the test system are fulfilled. The study was conducted similar to OECD TG417 and its reliability is judged to be high (Klimisch 2). The concentration of radioactivity in the bloodstream after exposure was linear with respect to dose. The majority (74-87%) of the label associated with the blood was recovered in the plasma, and of this, 97-100% of the 14C existed in the form of biomolecular conjugates. Thus, over the vapour exposure concentrations and time tested, it appears that conjugation is the predominant reaction and that free TDA is not a primary in vivo reaction product under the conditions tested.

Executive summary:

The substance Toluene diisocyanate was investigated for its toxicokinetic properties by Kennedy and coworkers (1994) in Sprague Dawley rats. The study was conducted similar to OECD TG417 with only minor deviations, why it was considered to be of high reliability (Klimisch 2). Rats were exposed to 14C-TDI vapors at concentrations ranging from 0.026 to 0.821 ppm for 4 h. The distribution was determined.

All tissues examined showed detectable quantities of radioactivity, with the airways, gastrointestinal system and blood having the highest levels which increased with exposure concentration. The concentration of radioactivity in the bloodstream after exposure was linear with respect to dose. The majority (74-87%) of the label associated with the blood was recovered in the plasma, and of this, 97-100% of the 14C existed in the form of biomolecular conjugates. Analysis of stomach contents shows that the majority of the label is also associated with high (>10 kDa) molecular weight species. While a larger percentage (28%) of the label is found in the low molecular weight fraction relative to blood, this low molecular weight labeled material represents at least eight different components. Thus, over the vapour exposure concentrations and time tested, it appears that conjugation is the predominant reaction and that free TDA is not a primary in vivo reaction product under the conditions tested.

Reference 4

Endpoint:

basic toxicokinetics

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well-documented publication which meets basic scientific principles

Justification for type of information:

Please refer to Read-across statement in section 13

Objective of study:

absorption

distribution

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

no individual metabolism cages, some deficiencies in reporting

Principles of method if other than guideline:

The uptake and distribution of radioactive toluene diisocyanate (TDI) is the focus of this paper. Biochemical analysis of the material derived from these exposures will further provide understanding of the mechanism of isocyanate toxicity. The overall goal of this research is to identify the in vivo target molecules labelled as a result of inhalation exposure to radioactive isocyanates and to determine whether a correlation can be made between the modification of these targets and the physiological responses which occur as a result of exposure.

GLP compliance:

not specified

Radiolabelling:

yes

Remarks:

2,4 [14C]TDI, with the 14 C incorporated in the benzene ring

Species:

guinea pig

Strain:

Hartley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazelton Research Products, Inc. (Denver, PA).
- Weight at study initiation: 300-400 g
- Individual metabolism cages: no, always 4 animals in one cage (during exposures)
- Diet (e.g. ad libitum): food provided ad libitum
- Water (e.g. ad libitum): water provided ad libitum
- Acclimation period: at least 7 days prior to exposure

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): on a 12 hr light/dark cycle

Route of administration:

inhalation: vapour

Vehicle:

other: air

Details on exposure:

Thirty-two guinea pigs were used in this study. Seven exposure levels were tested with four animals at each concentration, Four additional animals served as controls.

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMPER DESCRIPTION
- Exposure apparatus: A continuous airflow system was used for all seven radioactive TDI exposures. The system was designed according to the protocol of Alarie et al. (1987) with only minor alterations as previously presented (Ferguson et at.. 1988).
- Method of holding animals in test chamber: For each exposure, four cannulated animals were placed in separate, glass, whole-body plethysmographs which served as exposure chambers. These were attached to a central mixing chamber into which the desired exposure concentration was established. The radioactive TDI was shipped in sealed, glass mini-vials identical to those used for the MIC experiments.
- Source and rate of air: To generate the [14C]TDI vapour, house air was dried and delivered over the liquid in the vial once the internal glass septum was broken with a needle.
- Method of conditioning air:
- System of generating particulates/aerosols:
- Composition of vehicle (if applicable):
- Concentration of test material in vehicle (if applicable):
- Method of particle size determination:
- Treatment of exhaust air:

Exposure system.
Air flow was controlled by an appropriate flowmeter. Choice of flowmeters and rate settings, ranging from 2.75 mL/min to 5 litres/min, for the radioactive experiments was determined using identical mini-vials containing "cold" TDI and with animals in the exposure chambers. Due to the difference in vapor pressure between the two isocyanates, the icebath used to control vapor generation in the MIC exposures was eliminated for the TDI experiments. A 20-gauge needle also penetrated the top of the vial to deliver the vapor to the chamber. The vapor was drawn into the system by a vacuum pump. Exhaust rates varied between 7 and 30 litres/min depending on the isocyanate concentration desired.

Duration and frequency of treatment / exposure:

Most of the exposures were for 1 hr; however, two longer exposures were also performed to evaluate whether cumulative effects occur (4 and 5 hrs).

Remarks:

Doses / Concentrations:
0, 0.00005, 0.004, 0.016, 0.029, 0.084, 0.132 and 0.146 ppm

No. of animals per sex per dose / concentration:

4 males

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

No information on positive controls available.

Details on study design:

- Dose selection rationale: The range of concentrations was chosen to bracket the threshold limit value (TLV) for TDI.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, blood, other tissues (Trachea, lung, kidney, heart, spleen, and liver), bile
- Time and frequency of sampling: Analysis of clearance and postexposure effects was accomplished by euthanizing individual animals at times up to 3 days postexposure. One experiment extended this analysis to 2 weeks postexposure.
Analysis of 14C uptake into the bloodstream.
During the exposure 0.5 mL blood samples were collected at approximately 3, 6, 10, 15, and 20 min and every 10 min thereafter for the 1 hr exposures. Samples from the 4-hr exposure were taken following a similar schedule for the first hour and then every 30 min to the end of the exposure. For the 5 hr exposure, samples were taken at 15 min intervals for the first 2 hr and then every 30 min to the termination. All uptake samples were collected via the carotid cannulae. Alternate animals were sampled when blood flow from the cannula was adequate. The blood samples were immediately mixed in Vacutainer tubes containing 0.149 M buffered sodium citrate (Becton-Dickinson). An aliquot of 200 µL was removed from each sample and transferred to a glass scintillation vial for the determination of 14C content as follows. To each vial, 2.4 mL of NCS tissue solubilizer (Amersham) was added and the suspension was heated at 50°C for 20 min. An aliquot of 0.8 mL of a 20% benzoyl peroxide solution was added followed by an incubation at 50°C for an additional 30 min to decolourise the samples. After cooling to room temperature, organic scintillant (toluene, 2,5-diphenyloxazole, and 1,4,-bis2-(5-phenyloxazo-lyl)benzene) was added to bring the final volume to 20 mL. To reduce the level of background radioactivity due to cherrulurrunescence, the samples were stored in the dark for at least 24 hr before scintillation analysis. Total radioactivity was calculated on a counts per minute per milliliter of blood basis. The remaining portion of whole blood was spun at 478g for 15 min. Plasma was separated from cellular components and stored at -20°C. Cellular pellets were kept at 4°C to avoid freeze fracturing of the cell membranes.
Collection of terminal blood, body fluids, and tissues
After each exposure, animals were euthanised (2 mL sodium pentobarbital 50 mg/mL, ip). Typical times for euthanasia were 0, 6, 24, and 72 hr postexposure. Deviations from this schedule were demanded for exposures 4 and 6 because in both cases, one of the animals was visibly ill during exposure and was euthanised immediately. To assure relevant data for the 0 hr postexposure time point, an additional animal was euthanised and the 6 hr time point forfeited. Exposure 5 extended the schedule to 2 weeks postexposure. Terminal blood samples were collected via cardiac punctures, immediately mixed in a Vacutainer tube containing anticoagulant, and treated identically to the uptake samples previously described. A 2 hr post exposure blood sample was taken from arterial cannulae for exposures 5-7, and a 6 hr sample was taken for exposure 4. Additional cannulae and toebleed samples were collected following exposure 5. Bile was withdrawn from the gall bladder with a syringe and urine samples were similarly collected from the bladder. All fluids were stored at —60°C. To determine the level of HC in the bile and urine, 100 µL aliquots were counted in Scintiverse Bio-HP (Fisher Scientific, Pittsburgh, PA) and radioactivity was calculated on a counts per minute per milliliter basis following subtraction of background counts.
Trachea, lung, kidney, heart, spleen, and liver were dissected from each of the exposed animals as well as four control animals. For unpaired organs equal sections were taken: one was immediately frozen in liquid nitrogen and stored at -60°C for biochemical studies and the other portion was immersed in 10% buffered formalin for histological analysis. Paired organs were separated and processed similarly.
- Other: Tissue solubilization. A representative fragment was cut from each major organ and weighed. The fragment was transferred to a glass scintillation vial. NCS tissue solubilizer was added to a volume six times the total sample weight and the vials were then incubated at 50°C for 24 hr. Samples were cooled and neutralised to pH 7 with glacial acetic acid. Some samples required decolourization with benzoyl peroxide. Organic scintillant was added as described earlier and the samples were incubated in the dark for up to 4 weeks and then counted. Results were corrected for background and blood content using published percentages of blood volume for each organ (Wagner and Manning, 1976) and were then normalised on a counts per minute per gram basis.

Statistics:

No detailed information about statistical methods used was provided.

Preliminary studies:

No preliminary studies were described.

Type:

absorption

Results:

The uptake of radioactivity in arterial blood during exposure at all concentrations: the first blood sample showed a detectable level of radioactivity (except at 0.00005 ppm) which increased linearly throughout the exposure.

Type:

absorption

Results:

Following the linear uptake a postexposure increase in blood radioactivity was observed followed by a gradual decline over 72 hr.

Type:

distribution

Results:

As expected for inhalation exposures, the airway tissues show the highest level of radioactivity for all exposures. The 0.00005 ppm exposure did not show detectable levels in any of the tissues measured.

Type:

distribution

Results:

After correction for blood content, minimal levels of radioactivity were associated with other tissues, with the kidney demonstrating the most significant level.

Type:

distribution

Results:

Similar to the fluids, the tissues showed a measurable level of radioactivity was maintained in all tissues even after 2 weeks postexposure.

Type:

excretion

Results:

Highest levels of radioactivity was immediately postexposure. Maximum value for urine was always higher than the bile for equivalent samples on a counts per minute per millilitre basis.

Type:

excretion

Results:

A decline in radioactivity was evident by the 6 hr time point and returned toward a baseline level by 72 hr.

Details on absorption:

The uptake of radioactivity in arterial blood during exposure at all concentrations: the first blood sample showed a detectable level of radioactivity (except at 0.00005 ppm) which increased linearly throughout the exposure. Clearance of radioactivity from the bloodstream was analysed in terminal blood samples as well as postexposure arterial cannula samples or toebleeds. Following the linear uptake a postexposure increase in blood radioactivity was observed followed by a gradual decline over 72 hr. The level of radioactivity found at 72 hr postexposure did not show a significant decline over the subsequent 11 day period but instead, a level of approximately 1000 cpm/mL was maintained even after the second week. For all of the exposures above 0.029 ppm, a level of nearly 2000 cpm/mL (1938 cpm/mL, SD, 406) was observed at 72 hr, independent of the maximal amount of radioactivity seen in the bloodstream. Using the specific activity of the original compound, it is possible to calculate the molar quantity of the tolyl group represented by a cpm value irrespective of the form. For the average value of 1938 cpm/mL, the molar equivalent for the tolyl group is 8.3*10E-8 M.
As noted earlier, the uptake profiles and clearance of blood radioactivity were similar for all concentrations tested. For each exposure concentration, a direct relationship was found between the ppm*hr and the immediate postexposure terminal blood level of radioactivity. The slope of the line is 3.3*10E4 (r, 0.99). Using this value, the lowest ppm exposure (0.00005 ppm) blood level immediately postexposure was calculated and found to be 2 cpm which is below the detection limits and explains the absence of radioactivity at this exposure concentration.

Details on distribution in tissues:

As expected for inhalation exposures, the airway tissues show the highest level of radioactivity for all exposures. The 0.00005 ppm exposure did not show detectable levels in any of the tissues measured. After correction for blood content, minimal levels of radioactivity were associated with other tissues, with the kidney demonstrating the most significant level. Similar to the fluids, the tissues showed a measurable level of radioactivity was maintained in all tissues even after 2 weeks postexposure.

Test no.:

#1

Transfer type:

other: airway tissues

Observation:

distinct transfer

Details on excretion:

Aliquots of bile and urine were counted. The highest level of radioactivity for all exposures was immediately postexposure. The maximum value for the urine was always higher than the bile for equivalent samples on a counts per minute per milliliter basis. A decline in radioactivity was evident by the 6 hr time point and returned toward a baseline level by 72 hr.

Metabolites identified:

not measured

Details on metabolites:

No detailed investigations of the metabolites formed were conducted.

Isocyanate Exposure Concentrations

A series of 14C TDI exposures was conducted over a range of concentrations as given in Table 1. Three methods were used to monitor the exposure atmospheres: the Marcali assay, scintillation analysis of trapping solutions, and HPLC analysis of derivatised isocyanate. The Marcali determination involved the hydrolysis of isocyanate to toluenediamine and conversion to a colorimetric product. The HPLC method detected the derivative of reactive isocyanate with a nitroreagent (PNBPA). Marcali assay solutions were also subjected to liquid scintillation analysis. On the basis of the specific activity of the original compound, an additional measure of isocyanate concentration was thereby achieved. Table 1 summarises the compiled data available for each experiment. The data are comparable, independent of the assay method. In some cases a value was not available due to the detection limits (exposure 1) or trapping procedures. For the first series of experiments chamber atmospheres were bubbled directly into a solution of PNBPA and the derivatisation was not effective. Exposures 5 and 7 were monitored by the PNPBA glass fiber filter method. Average concentrations were assigned based on all values available. Multiple samples (n, Table 1) were collected during the exposures and showed only minor fluctuations in chamber atmospheres throughout the exposures (SD, Table 1).

TABLE 1
Summary of [^14C] TDI Exposure Concentrations
Isocyanate measurement
Exposure	Marcali^a assay Abs 550 nm (ppm)			Specific^b activity cpm (ppm)			PNBPA^c assay Abs 254nm (ppm)			Average concentration	Exposure duration	C*t
No.	x^d	SD^e	n^f	X	SD	n	X	SD	n	(ppm)	(hr)	(ppm*hr)
1	ND^g		l	0.00005	_	1	NA^h	_	0	0.00005	i	0.00005
2	0.0044	—	i	0.0038	0.0005	8	NA	—	0	0.004	1	0.004
4	0.0166	0.00185	10	0.0152	0.0009	10	NA	—	0	0.016	5	0.080
3	0.0299	0.0021	2	0.0270	—	1	NA	_	0	0.029	i	0.029
7	0.0895	0.036	15	0.0756	0.034	15	0.0877	0.062	5	0.084	4	0.336
5	0.1343	0.024	8	0.1047	0.024	7	0.1562	0.039	3	0.132	1	0.132
6	0.1667	0.026	6	0.1242	0.020	6	NA	—	0	0.146	1	0.146
^a Marcali assay (Marcali, 1957).
^b Quantitation of TDI based on the specific activity of the original compound.
^cPNBPA derivatization assay (Schroeder and Moore, 1985).
^d x the mean ppm value.
^e SD is the standard deviation
^fn is the number of samples collected during exposure.
^g ND, value is not detectable.
^h N A. value is not available due to inadequate trapping.

For all of the exposures above 0.029 ppm, a level of nearly 2000 cpm/mL (1938 cpm/mL, SD, 406) was observed at 72 hr, independent of the maximal amount of radioactivity seen in the bloodstream (Table 2).

TABLE 2
Levels of Radioactivity in Terminal Body Fluids as a Function of Exposure
Concentration and Time of Euthanasia
Exposure no.	^14C-TDI exposure (ppm-hr)^a	Animal no.	Time post-exposure (hr)	Terminal blood (cpm/mL)	Urine (cpm/mL)	Bile (cpm/mL)
1	0.00005	1	0	13	NA^b	NA
		2	6	0	NA	2
		3	24	0	425	39
		4	72	0	195	16
2	0.004	5	0	195	2,053	91
		6	6	567	886	40
		7	24	115	860	41
		8	72	104	377	27
3	0.029	9	0	741	4,355	465
		10	6	1,088	2,669	65
		11	24	580	1,331	6
		12	72	433	NA	NA
4	0.080 (0.016x5hr)	13	0	1,413	6,365	720
		14^c	0	811^c	NA	NA
		15	24	1,283	1,457	265
		16	72	1,389	2,001	176
5	0.132	17	0	5,086	86,835	2027
		18	72	2,202	5,330	376
		19	192	1,555	4,131	365
		20	360	1,055	1,065	27
6	0.146	21^c	0	2,738^c	NA	NA
		22	0	4,861	22,224	NA
		23	24	2,851	NA	186
		24	72	1,877	2,624	56
7	0.336 (0.084x4hr)	25	0	11,211	65,850	5766
		26	6	8,986	48,192	3042
		27	24	7,382	31,297	1960
		28^d	72	2,284	2,555	185
^a All exposures were for 1 hour except as noted.
^bNA, not available.
^cAnimal was visibly ill during exposure.
^dAnimal shown to be abnormal upon necropsy.

The uptake profiles and clearance of blood radioactivity were similar for all concentrations tested (Table 3).

TABLE 3
Linear Regression Analysis for ^14C Uptake in Carotid Arterial Blood during Exposure to [^14C]TDI
Exposure no.	Exposure concentration (ppmhr)	Animal no.	No. of uptake blood samples	Uptake in blood equation"	r value^a	Composite uptake in blood equation''	Composite r value^b
2	0.004	5	0	—	—	Y(X) = -8 + 1.9 X	0.96
		6	0	—	—
		7	5	Y(X)= -8 +2X	0.96
		8	1	—	—
3	0.029	9	2	Y(X)14 + 8X	1.00	Y(X)= -89 + 18X	0.87
		10	3	Y(X)-198 + 30X	0.98
		11	4	Y(X)-39+13X	0.99
		12	3	Y(X)-248 + 25X	0.99
4	0.080 (0.016 x 5 hr)	13	7	Y(X)31 + 2.3 X	0.99	Y(X)= 92 + 2.7X	0.86
		14	0	—
		15	7	Y(X)108 + 3.5X	0.97
		16	0	—	—
5	0.132	17	0	—	—	Y{X) =-407+ 98 X	0.82
		18	3	Y(X)-1146 + 75X	0.97
		19	3	Y(X)-478 + 138X	0.99
		20	3	Y(X)-884 + 149 X	0.99
6	0.146	21	1	—	—	Y(X) =-182 + 56X	0.99
		22	0	—	—
		23	2	Y(X)-364 + 65X	0.99
		24	5	Y(X)-i35 + 55X	1.00
7	0.336 (0.084 x 4 hr)	25	3	Y(X)-75 + 89X	0.99	Y(X)=179 + 76X	0.98
		26	3	Y(X)174 + 67X	0.99
		27	4	Y(X)176 + 76X	0.99
		28	4	—	—
^aLinear regression analysis for data from individual animals.
^bLinear regression analysis for all data at one exposure concentration.

The tissues showed a measurable level of radioactivity was maintained in all tissues even after 2 weeks postexposure (Table 4).

TABLE 4
Tissue Clearance of ^14C following Inhalation Exposure to^14C-TDI
Exposure no.	Animal no.	Postexposure time (hr)			CPM/G tissue
			Trachea	Lung	Kidney	Heart	Spleen	Liver
2	5	0	2,028	570	21	35	_	13
	8	72	349	81	40	—	19	—
3	9	0	10,391	1,232	198	—	102	—
	12	72	1,162	73	335	100	—
4	13	0	4,798	1,151	433	109	70	71
	16	72	2,849	1.109	440	242	96	-
5	17	0	103,598	9,058	2880	815	1238	564
	¡8	72	7,018	1,333	1080	499	70	240
	19	192	19,306	2,660	1078	433	419	151
	20	360	1,875	2,406	226	74	226	134
6	22	0	56,592	8,160	1354	—	697	301
	24	72	7,448	1,346	1301	220	182	176
7	25	0	211,300	30,435	2445	3246	3588	1390
	28	72	9,504	2,454	1377	607	222	384

Conclusions:

Interpretation of results (migrated information): low bioaccumulation potential based on study results It also demonstrates that the radioactivity clears from the bloodstream to a level corresponding to approximately a 100 nM concentration of tolyl group after 72 hr and persists at a nanomolar level even 2 weeks following the exposure.
An analysis of the toxicokinetic properties after inhalation of Toluene diisocyanate were conducted similar to OECD TG417 in guinea pigs. The different methods used (Marcali, PNBPA, and radioactivity values) yielded comparable exposure concentration results. This confirms that the exposure atmospheres contained reactive TDI at the desired levels, all animals received equivalent concentrations, and chamber atmospheres were maintained throughout the exposure. Therefore the reliability of the study is considered to be high (Klimisch 2). The results show that the rate of uptake into the blood is linear during exposure to concentrations ranging from 0.00005 to 0.146 ppm and that the uptake continues to increase slightly postexposure. It also demonstrates that the radioactivity clears from the bloodstream to a level corresponding to approximately a 100 nM concentration of tolyl group after 72 hr and persists at a nanomolar level even 2 weeks following the exposure.

Executive summary:

The substance Toluene diisocyanate was investigated for its toxicokinetic properties by Kennedy and coworkers (1989) in Hartley quinea pigs. The study was conducted similar to OECD TG417 with only minor deviations, why it was considered to be of high reliability (Klimisch 2). The guinea pigs received 14C-radiolabelled TDI via inhalation (1 h, in certain cases also 4 or 5 hours, 0 to 0.146 ppm). Exposures to 14C TDI were performed over a range of relatively low concentrations, including levels at and below the TLV for TDI which has been established at 0.005 ppm (Amer. Conf. Gov. Ind. Hyg., 1988). The absorption, distribution and excretion were determined via scintillation analysis and also via the Marcali- and the PNBPA method. The Marcali, PNBPA, and radioactivity values yielded comparable exposure concentration results. This confirms that the exposure atmospheres contained reactive TDI at the desired levels, all animals received equivalent concentrations, and chamber atmospheres were maintained throughout the exposure. Analysis of the uptake and distribution of radioactivity in the TDI-exposed fluids and tissues showed that some form of the labeled compound whether TDI, a conjugate, metabolite, or hydrolysis product, entered and penetrated throughout the entire system, even at the 0.004 ppm level. The urine and bile profiles demonstrate the rapid penetration of some form of the radioactivity through the system since at all concentrations the highest level of radioactivity in the bile and urine is found immediately following exposure. The post exposure increase of blood radioactivity is presumably due to the processing or desorption of the compound from the sites of entry (i.e., nasal passage, conducting airways, and alveoli) into the bloodstream for clearance. This study shows that the rate of uptake into the blood is linear during exposure to concentrations ranging from 0.00005 to 0.146 ppm and that the uptake continues to increase slightly postexposure. It also demonstrates that the radioactivity clears from the bloodstream to a level corresponding to approximately a 100 nM concentration of tolyl group after 72 hr and persists at a nanomolar level even 2 weeks following the exposure. After 2 weeks of recovery the animals still had measurable levels of blood radioactivity at a nearly constant amount (8.3 X 10E-8 M), regardless of initial dose, which suggests the saturation of a particular target which as a reacted form does not have a rapid turnover rate. The initial rate of 14C uptake is also a linear function of the concentration of TDI when expressed either as concentration (ppm) or as concentration multiplied by duration of exposure (ppm * hr). This is discussed in comparison with the toxic responses as a function of both ppm and ppm * hr.

Reference 5

Endpoint:

basic toxicokinetics

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

The model for Structural alerts: “Computational characterisation of chemicals and datasets in Terms of organic functional groups”; is aimed at the identification of organic functional groups in query chemicals. EUR 24871 EN -2011, Benigni R., Olga Tcheremenskaia O.,and A. Worth, Computational Characterisation of Chemicals and Datasets in Terms of Organic Functional Groups - a New Toxtree Rulebase.
SMARTCyp is a prediction model which identifies sites in a molecule that are labile for metabolism by Cytochromes P450 isoform 3A4. It is also a reactivity model which is applicable to all P450 isoforms (Toxtree User Manual, 2010)

Objective of study:

metabolism

Qualifier:

no guideline required

GLP compliance:

no

Radiolabelling:

other: not applicable

Species:

other: not applicable

Route of administration:

other: not applicable

Duration and frequency of treatment / exposure:

not applicable

Remarks:

Doses / Concentrations:
not applicable

No. of animals per sex per dose / concentration:

not applicable

Type:

other: Prediction of metabolism by modelling

Results:

2,4,6-triisopropyl-m-phenylene-diisocyanate is expected to be well metabolized by the Cytochrome P450 group of drugs metabolizing enzymes.

Results of identification of sites in the molecule which are labile for enzymes from group of drug metabolizing Cytochrome P450 family:

Q1.Rank1:Yes; Class:SMARTCyp primary sites of metabolism

Q2.Rank2:Yes; Class:SMARTCyp secondary sites of metabolism

Q3.Rank3:Yes; Class:SMARTCyp tertiary sites of metabolism

QRank>3.SMARTCyp:Yes;Class:SMARTCyp rank>3 sites of metabolism

Conclusions:

Interpretation of results (migrated information): other: well metabolized substance
2,4,6-triisopropyl-m-phenylene-diisocyanate is expected to be well metabolized by the Cytochrome P450 group of drugs metabolizing enzymes.

Executive summary:

The chemical structure of 2,4,6 -triisopropyl-m-phenylene diisocyanate was assessed by Toxtree (v.2.1.0) modelling tool for possible metabolism. SMART Cyp is a prediction model, included in the tool, which identifies sites in a molecule that are labile for the metabolism by Cytochromes P450.

2,4,6 -triisopropyl-m-phenylene diisocyanate is expected to be well metabolizedby the Cytochrome P450 group of drugs metabolizing enzymes.The molecule possesses equal or more than three sites of metabolism.The primary and secondary sitesof metabolism are the diisopropyl-groups, which are predicted to be subject to aliphatic hydroxylation. The tertiary sites of metabolism are the carbon-atoms of the aromatic ring, which are predicted to be subject to aromatic hydroxylations.

Reference 6

Endpoint:

basic toxicokinetics

Type of information:

other: Expert statement

Adequacy of study:

key study

Study period:

2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A read-across statement regarding the toxicological behaviour of the group of diisocyanates was performed, taking into account the chemical structure, the available physico-chemical-data and the available (eco-)toxicological data.

Qualifier:

no guideline required

GLP compliance:

no

Radiolabelling:

other: not applicable in this expert statement

Species:

other: not applicable

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

not applicable

Route of administration:

other: all routes of administration are discussed in the expert statement

Vehicle:

other: not applicable

Details on exposure:

all routes of administration are discussed in the expert statement

Details on study design:

not applicable

Details on dosing and sampling:

not applicable

Details on absorption:

In general, absorption of a chemical is possible, if the substance crosses biological membranes. The EU Technical Guidance Document on Risk Assessment (TGD, Part I, Appendix VI) gives a number of physico-chemical properties that normally determine oral, inhalation and dermal absorption (LINK to Guidance Document: http://ecb.jrc.ec.europa.eu/tgd/). This process requires a substance to be soluble both in lipid and in water and is also dependent on its molecular weight (substances with molecular weights below 500 are favourable for absorption). Firstly, 2,4,6-triisopropyl-m-phenylene-diisocyanate and TDI would be favourable for absorption, when only taking into account their molecular weights. However, as 2,4,6-triisopropyl-m-phenylene-diisocyanate is practically insoluble in water, it is apparent that its absorption is hindered. This is also seen in the value calculated for the LogPow that shows the substance to be better soluble in octanol than in water. Considering its low water solubility and the value for LogPow calculated to be above 4, the absorption into the body will not be favoured (LogPow between 0 and 4 are favourable for absorption). In general, the absorption of chemicals, which are surfactants or irritants may be enhanced, because of damage to cell membranes. This is the case for both substances of interest.

Absorption from the gastrointestinal tract

Regarding oral absorption, in the stomach, a substance will most likely be hydrolysed, as this is a favoured reaction in the acidic environment of the stomach. In accordance with the above mentioned principles it has been reported for TDI to be hydrolysed in the stomach to toluene diamine (TDA). The lower pH levels in i.e. the stomach are leading to high protonation of biological NH2 groups and this facilitates hydrolysis of TDI to TDA and subsequent formation of polyureas
In the small intestine absorption occurs mainly via passive diffusion or lipophilic compounds may form micelles and be taken into the lymphatic system. Additionally, metabolism may occur by gut microflora or by enzymes in the gastrointestinal mucosa. However, the absorption of highly lipophilic substances (Log P of 4 or above) may be limited by the inability of such substances to dissolve into gastrointestinal fluids and hence make contact with the mucosal surface. The absorption of such substances will be enhanced if they undergo micellular solubilisation by bile salts. Substances absorbed as micelles enter the circulation via the lymphatic system, bypassing the liver.
The toxicological data available for both substances show, that the substances resemble each other in the endpoints: acute toxicity oral (LD50 > 2000 mg/kg bw for both substances) and skin irritation (both irritating). As the results for these endpoints are identical, it can be presumed that these substances have in principle the same mode of action. The available toxicokinetic data for TDI suggest that orally administered 2,4-TDI is not very well absorbed (Timchalk et al., 1994). The minimum estimate for absorption was 12%, which assumed that the radioactivity recovered in the faeces (~ 81 %) represented un-absorbed material. A more realistic estimate for absorption was obtained by assuming that some of the radioactivity in the faeces was absorbed. However, the hydrolysed TDI can react as 2.4-TDA with free 2,4-TDI forming polyurea polymers, which appear to be poorly absorbed from the gastrointestinal tract. Based on the rapid reactivity it is doubtful that TDI was absorbed prior to its hydrolysis to TDA. Therefore, the 12-20% of the 2,4-TDI that was absorbed, most probably represented 2,4-TDA that had not reacted to form polyureas.
Based on the abovementioned data for the closest analogue, it can be presumed that the absorption of 2,4,6-triisopropyl-m-phenylene-diisocyanate via the oral route will be slower than that of TDI, which will result in a lower toxicity of the substance (also by the long-term exposure).

Absorption from the respiratory tract

Regarding absorption in the respiratory tract, any gas or vapour has to be sufficiently lipophilic to cross the alveolar and capillary membranes (moderate Log P values between 0-4 favourable for absorption). The rate of systemic uptake of very hydrophilic gases or vapours may be limited by the rate at which they partition out of the aqueous fluids (mucus) lining the respiratory tract and into the blood. Such substances may be transported out of the lungs with the mucus and swallowed or may pass across the respiratory epithelium via aqueous membrane pores. Lipophilic substances (Log P >0) would have the potential to be absorbed directly across the respiratory tract epithelium. Very hydrophilic substances might be absorbed through aqueous pores (for substances with molecular weights below around 200) or be retained in the mucus.
Even though 2,4,6-triisopropyl-m-phenylene-diisocyanate has a relatively low vapour pressure (0.19 Pa) and a high boiling point (calculated 325.14°C), which would indicate a low availability for inhalation, it is known, that isocyanates bear a high potential for respiratory sensitisation and irritation. As isocyanates are highly reactive, irritating compounds it is clear, that contact to the epithelium will produce irritation and therefore enhance absorption. The toxicokinetic data available for TDI show that no relevant hydrolysis occurs in the respiratory tract, as TDA as barely detectable in the urine, following inhalation of TDI (Timchalk et al., 1994). Additionally, essentially all the radioactivity inhaled via 2.4-TDI vapours was retained (Timchalk et al., 1994). The data suggest that a large percentage of the radioactivity was absorbed through the lungs into the blood. The data suggest that between 61 and 90% of the inhaled 2,4-[14C]TDI dose was absorbed and the remaining radioactivity was rapidly cleared from the respiratory tract, ingested, and then eliminated in the faeces.
Kennedy and co-workers found in all tissues examined detectable quantities of radioactivity, with the airways, gastrointestinal system and blood having the highest levels which increased with exposure concentration (Kennedy et al., 1989 and 1994). The concentration of radioactivity in the bloodstream after exposure was linear with respect to dose. The results showed that greater than 95% of the plasma-associated radioactivity existed in the form of biomolecular conjugates (reaction of TDI with biological macromolecules successfully competes with hydrolysis to the diamine). Thus, over the vapour exposure concentrations and time tested, it appears that conjugation (mainly with serum-albumin) is the predominant reaction and that free TDA is not a primary in vivo reaction product under the conditions tested.
Based on this data, it can be speculated that 2,4,6-triisopropyl-m-phenylene-diisocyanate might act in the same way as TDI. Presumably it is expected to be bonded to proteins of cells in the respiratory epithelium and/or be hydrolyzed to an amine derivative as well, triggering respiratory sensitization and irritation reactions.

Absorption following dermal exposure

In order to cross the skin, a compound must first penetrate into the stratum corneum and may subsequently reach the viable epidermis, the dermis and the vascular network. The stratum corneum provides its greatest barrier function against hydrophilic compounds, whereas the viable epidermis is most resistant to penetration by highly lipophilic compounds. Substances with a molecular weight below 100 are favourable for penetration of the skin and substances above 500 are normally not able to penetrate. The substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. Therefore if the water solubility is below 1 mg/l, dermal uptake is likely to be low. Additionally Log Pow values between 1 and 4 favour dermal absorption (values between 2 and 3 are optimal). Above 4, the rate of penetration may be limited by the rate of transfer between the stratum corneum and the epidermis, but uptake into the stratum corneum will be high. Above 6, the rate of transfer between the stratum corneum and the epidermis will be slow and will limit absorption across the skin. Uptake into the stratum corneum itself may be slow. Moreover vapours of substances with vapour pressures below 100 Pa are likely to be well absorbed and the amount absorbed dermally may be more than 10% of the amount that would be absorbed by inhalation. If the substance is a skin irritant or corrosive, damage to the skin surface may enhance penetration. During the whole absorption process into the skin, the compound may be subject to biotransformation.
In case of 2,4,6-triisopropyl-m-phenylene-diisocyanate and TDI, the molecular weight is above 100 and below 500, which would normally indicate low potential to penetrate the skin. Moreover, the logPow value of 2,4,6-triisopropyl-m-phenylene-diisocyanate is with 7.56 very high and this also indicates as stated above low absorption. Due to the vapour pressure of 0.19 Pa, 2,4,6-triisopropyl-m-phenylene-diisocyanate is once again represents rather an inhalation hazard than one by dermal route of exposure. One has to keep in mind, that absorption is influenced by the irritating potential of the two substances and might enhance penetration. It has been demonstrated that the absorption of 2,4- and 2,6-TDI through skin contact is possible, as toluene diamine was found in the urine (Yeh et al., 2008). A clear dose-dependent skin absorption for 2,4- and 2,6-TDI was demonstrated by the findings of AUC, Cmax and accumulative amounts (r ≥ 0.968) (Yeh et al., 2008).
Based on these factors, 2,4,6-triisopropyl-m-phenylene-diisocyanate is expected to be partially absorbed following dermal exposure into the stratum corneum. The transfer of the substance into the epidermis will be limited, due to its molecular weight and high lipophilicity. Hence, the systemic toxicity of 2,4,6-triisopropyl-m-phenylene-diisocyanate via the skin is assumed to be low and 10% adsorption via dermal route is proposed based on the high logPow and low water solubility.

Details on distribution in tissues:

In general, the following principle applies: the smaller the molecule, the wider the distribution. A lipophilic molecule (Log P >0) is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration particularly in fatty tissues. It’s not possible to foresee protein binding, which can limit the amount of a substance available for distribution. Furthermore, if a substance undergoes extensive first-pass metabolism, predictions made on the basis of the physico-chemical characteristics of the parent substance may not be applicable.
In case of 2,4,6-triisopropyl-m-phenylene-diisocyanate, no data is available for distribution patterns. However, there are data available for TDI. It has been demonstrated, that the absorbed amount of TDI is distributed after oral, dermal and inhalatory exposure (Timchalk et al., 1994, Kennedy et al., 1989, and 1994). The gastrointestinal tract and contents accounted for a high amount of the recovered radioactivity in the tissues/carcass with the remaining radioactivity evenly distributed among the remaining tissues (Timchalk et al., 1994).
The distribution of 2,4,6-triisopropyl-m-phenylene-diisocyanate is expected to be more extensive in fat tissues than in other tissues.

Details on excretion:

The major routes of excretion for substances from the systemic circulation are in the urine and/or the faeces (via bile and directly from the gastrointestinal mucosa). For volatile substances and metabolites exhaled air is an important route of excretion. Substances that are excreted favourable in the urine tend to be water-soluble and of low molecular weight (below 300 in the rat) and be ionized at the pH of urine. Most will have been filtered out of the blood by the kidneys, though a small amount may enter the urine directly by passive diffusion and there is the potential for reabsorption into the systemic circulation across the tubular epithelium. Substances that are excreted in the bile tend to be amphipathic (containing both polar and nonpolar regions), hydrophobic/strongly polar and have higher molecular weights and pass through the intestines before they are excreted in the faeces and as a result may undergo enterohepatic recycling which will prolong their biological half-life. This is particularly a problem for conjugated molecules that are hydrolysed by gastrointestinal bacteria to form smaller more lipid soluble molecules that can then be reabsorbed from the GI tract Those substances less likely to recirculate are substances having strong polarity and high molecular weight of their own accord. Other substances excreted in the faeces are those that have diffused out of the systemic circulation into the GIT directly, substances which have been removed from the gastrointestinal mucosa by efflux mechanisms and non-absorbed substances that have been ingested or inhaled and subsequently swallowed. Non-ionized and lipid soluble molecules may be excreted in the saliva, where they may be swallowed again, or in the sweat. Highly lipophilic substances that have penetrated the stratum corneum but not penetrated the viable epidermis may be sloughed off with skin cells.
For 2,4,6-triisopropyl-m-phenylene-diisocyanate no data is available concerning its elimination, but for TDI several studies have been undertaken. TDI is mainly eliminated via the faeces (80 %) after oral exposure; only 5 to 15 % are eliminated via the urine. However, this is in accordance with the above mentioned principles, as TDI reacts with hydrolysed TDA in the gastrointestinal tract to polyurea polymers, which have a high molecular weight and are subsequently not absorbed and therefore eliminated via the faeces. After inhalation 48 % is eliminated via the faeces and 15 % via the urine in 48 hours and no quantifiable elimination via exhalation occurred (Timchalk et al., 1994).
The urinary excretion by the kidneys was slower following inhalation exposure (t1\2 = 20 hr) when compared to the oral 2,4-TDI dose group (t1\2 = 7.5 hr), suggesting that inhaled 2,4-TDI was eliminated in the urine in a different form having a longer biological half-life than orally administered 2,4-TDI and/or 2,4-TDA (Timchalk et al., 1994).

Details on metabolites:

After oral exposure TDI, which is hydrolysed to TDA in the gastrointestinal tract and subsequently absorbed, it can be excreted in the urine either unchanged or as acid-labile conjugates. TDA, as a metabolite can be N-acetylated forming mono- and diacetylated TDA metabolites which are readily excreted in the urine (Timchalk, et al. 1994). After inhalatory exposure, very little 2,4-TDA is formed. In addition, 90% of the quantitated metabolites in the urine specimens following inhalation exposure to 2,4-TDI existed as acid-labile conjugates of TDI/TDA while only 10% existed as acetylated TDA. This indicated that following inhalation exposure, a larger percentage of the 2,4-TDI was excreted in the urine preferentially in a conjugated form (to proteins) and not as oligoureas or free or acetylated TDA (Timchalk et al., 1994).
Similar to TDI, 2,4,6-triisopropyl-m-phenylene-diisocyanate is expected to form conjugates with glutathione (and other peptides) because the carbon atoms in the isocyanate group represent an electrophile centre susceptible to nucleophile attack by such strong nucleophiles as lysine, cysteine and histidine (Smith and Hotchkiss, 2001). The isopropyl groups on aromatic ring can however affect the reactivity of electrophile carbon due probably to sterical hindrance. If 2,4,6-triisopropyl-m-phenylene-diisocyanate hydrolyses to its corresponding amine, the latter can be N-acetylated and then excreted in the urine. Aromatic and aliphatic hydroxylation can also occur, leading to a hydrophyle which is easily to be excreted.

Background:

There is little data available on physico-chemical properties of 2,4,6,-triisopropyl-m-phenylene diisocyanate. With the aid of the EPIWIN software some physical-chemical properties were calculated.

The substance is at room temperature a light yellowish liquid with a slight odour. The substance is insoluble in water (< 0.05 mg/L at 20°C) and has a logPow of 7.56. It has a low vapour pressure (0.19 Pa at 20°C). No exact value of melting point could be determined experimentally for 2,4,6-triisopropyl-m-phenylene-diisocyanate between -90°C and 50°C (Kintrup, 2012). Glass transition temperature (amorphous components) in the first heating run was determined to be -56°C. The calculated melting point was 82.9°C. The boiling point of 305.8°C was measured for 2,4,6-triisopropyl-m-phenylene-diisocyanate (Svobodova, 2012).

Hydrolysis as a function of pH has not been determined, but comparison to its structural analogue toluene diisocyanate revelaed a high likelihood for hydrolysis. The substance is not toxic when administered orally to ratsc (LD50 > 2000 mg/kg bw). However, it has been determined to be toxic after inhalation (LC50 < 110mg/m³). It is not an eye, but a skin irritant, and skin sensitising properties have been predicted. Additionally, the substance was shown to be not mutagenic in studies according to OECD471, 473 and 476.

Accumulation:

It is also important to consider the potential for a substance to accumulate or to be retained within the body. Lipophilic substances have the potential to accumulate within the body (mainly in the adipose tissue) if the dosing interval is shorter than 4 times the whole body half-life. Although there is no direct correlation between the lipophilicity of a substance and its biological half-life, substances with high log P values tend to have longer half-lives. On this basis, there is the potential for highly lipophilic substances (Log P >4) to accumulate in individuals that are frequently exposed. Highly lipophilic substances (Log P between 4 and 6) that come into contact with the skin can readily penetrate the lipid rich stratum corneum but are not well absorbed systemically. Although they may persist in the stratum corneum, they will eventually be cleared as the stratum corneum is sloughed off. A turnover time of 12 days has been quoted for skin epithelial cells

Following oral exposure little TDI is retained in the body (4 %) and following exposure via inhalation a larger amount of TDI (34%) is retained in the carcass, indicating a slow release of protein-bound material (Timchalk et al., 1994). However, to our knowledge the accumulation of TDI in the stratum corneum has not been investigated.

Metabolism:

Route specific toxicity may result from several phenomena, such as hydrolysis within the gastrointestinal or respiratory tracts, also metabolism by gastrointestinal flora or within the gastrointestinal tract epithelia (mainly in the small intestine), respiratory tract epithelia (sites include the nasal cavity, tracheo-bronchial mucosa (Clara cells) and alveoli (type 2 cells) and skin.

It has been shown, that after oral exposure TDI, which is hydrolysed to TDA in the gastrointestinal tract and subsequently absorbed, can be excreted in the urine either unchanged or as acid-labile conjugates. TDA, as a metabolite can be N-acetylated forming mono- and diacetylated TDA metabolites which are readily excreted in the urine (Timchalk, et al. 1994). After inhalatory exposure, very little 2,4-TDA is formed. In addition, 90% of the quantitated metabolites in the urine specimens following inhalation exposure to 2,4-TDI existed as acid-labile conjugates of TDI/TDA while only 10% existed as acetylated TDA. This indicated that following inhalation exposure, a larger percentage of the 2,4-TDI was excreted in the urine preferentially in a conjugated form (to proteins) and not as oligoureas or free or acetylated TDA (Timchalk et al., 1994).

There is no data on metabolism of 2,4,6-triisopropyl-m-phenylene-diisocyanate. Similar to TDI, the substance is expected to form conjugates with glutathione (and other peptides) because the carbon atoms in the isocyanate group represent an electrophile centre susceptible to nucleophile attack by such strong nucleophiles as lysine, cysteine and histidine (Smith and Hotchkiss, 2001). The isopropyl groups on aromatic ring can however affect the reactivity of electrophile carbon due probably to sterical hindrance. If 2,4,6-triisopropyl-m-phenylene-diisocyanate hydrolises to its corresponding amine, the latter can be N-acetylated and then excreted in the urine. Aromatic and aliphatic hydroxylation can also occur leading to a hydrophyle which is easily to be excreted.

Conclusions:

A read-across statement regarding the toxicological behaviour of several diisocyanates, taking into account the chemical structure, the available physico-chemical-data and the available (eco-)toxicological data is available.

Executive summary:

2,4,6-triisopropyl-m-phenylene-diisocyanate and TDI are aromatic diisocyanates with different alkyl rests attached to benzene ring (three isopropyl groups in 2,4,6-triisopropyl-m-phenylene-diisocyanate and one methyl group in TDI). As a result of the structural differences, such physic-chemical properties as melting point, LogPow and water solubility differ significantly from each other.

2,4,6-triisopropyl-m-phenylene-diisocyanate is expected to be absorbed to a lesser extent than TDI into the organism after oral exposure. Absorption via oral route is assumed to be low. Absorption after inhalation is considered to be rather fast. Dermal absorption, however will be limited by its molecular weight, its high lipophilicity and its high logPow. Due to high logPow, 2,4,6-triisopropyl-m-phenylene-diisocyanate is not expected to penetrate easily through the skin but tends to migrate towards fat tissues but certain reactivity of isocyanate groups with peptides and proteins might hinder accumulation. Inhalation and dermal routes can represent sensitising and irritating hazard for respiratory system. Nucleophilic substitution by SN2 mechanism with electron rich nucleophile amino acids of peptides and proteins is considered to be primary detoxifying mechanism of 2,4,6-triisopropyl-m-phenylene-diisocyanate. Excretion of 2,4,6-triisopropyl-m-phenylene-diisocyanate is expected via urine.

Reference 7

Endpoint:

dermal absorption in vivo

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

Please refer to Read-across statement in section 13

Signs and symptoms of toxicity:

not specified

Dermal irritation:

no effects

Remarks:

no skin irritation being found for any exposure or control group.

Absorption in different matrices:

not data on absorption in different matrices available

Total recovery:

- the recoveries were 95.7% (R.S.D 3.0%) and 97.0% (R.S.D 5.6%) for 2,6-TDA (100–500 ng/mL) and 2,4-TDA (20–100 ng/mL), respectively.
- Limit of detection (LOD): 1 ng/mL or TDAs

Conversion factor human vs. animal skin:

no data on conversion factors available

Urinary excretion

The peak urinary excretion of TDA (Cmax) occurred during the first 12 h collection interval among three doses of TDI. The Cmax of 2,4-TDA was found to be 0.062 ± 0.009, 0.238 ± 0.060 and 6.116 ± 0.429 μg/mL for low (0.2%), moderate (1%) and high (5%) TDI dose group, respectively.

Skin-absorbed 2,4-TDA was not completely eliminated by urinary excretion over 6 days in the high exposure group.

The elimination pattern of 2,6-TDA was similar to 2,4 -TDA. The Cmax was reached at 12 h after the end of exposure and found to be 0.056 ± 0.004, 0.268 ± 0.087 and 3.777 ± 0.384 μg/mL for low, moderate and high exposure groups, respectively.

The decrease trend slowed after 60 h for the moderate and high dose groups. However, the U-TDA concentration measurements were below the detection limit from 120 h, 72 h for the moderate and low exposure groups, respectively.

The accumulative amount profiles across 144 h for 2,4- and 2,6-TDA were similar. Excretory urinary TDA amount increased abruptly within 24 h since the end of exposure, the elimination amounts were becoming slow within 24– 60 h. The elimination amounts reached a plateau after 60 h.

Half-life

Apparent half-lives (t1/2) of excretory TDA were about 20.1 h (SD = 1.9) and 22.7 h (SD = 3.4) for 2,4- and 2,6- forms among three exposure groups with relatively narrow ranges.

An increasing t1/2 following by an increase of dose was found consistently for both 2,4- and 2,6-forms. The data indicating slower elimination and longer retention could occur at higher doses.

When the first-order kinetic linearity was tested, highly satisfactory coefficients of correlation (r = 0.930 ± 0.959 P < 0.05 for 2,4-TDA; r = 0.902 ±

0.953 P < 0.05 for 2,6- TDA) were obtained for U-TDA measurements since the exposure termination (time after tmax). These results suggested the elimination pattern of excretory TDA concentration profiles in 6-day consecutive urine samples were first-order kinetics. However, a non-linear saturation was found for high exposure at 60 h after tmax. The possible explanation for this observation could be: in lower doses, the TDA elimination process in the kidney could connect with the distribution process in highly perfused tissues with hardly any time lag. On the other hand, at a high dose, the TDA elimination process could not be immediately completed because of overwhelming residual TDA following the TDA distribution process in highly perfused tissues.

Comparison of urinary 2,4-TDA with urinary 2,6-TDA

The rat skins were originally exposed to a mixture of 2,4- and 2,6-TDI at a ratio of 80%:20% (m:m). The average ratios of 2,4-/2,6-TDA were found, however, to be 1.1, 0.9 and 1.6 in the low, moderate and high exposure groups for Cmax, respectively. For AUC results, the average ratios were 1.1, 0.8 and 1.2, respectively (Table 1). The overall ratios for both 2,4- and 2,6-form were close to unity, rather than 4:1, as expected from the exposure composition. The discrepancy between skin exposure application and urinary concentration might be attributed to the greater reactivity of 2,4-TDI, possibly related to higher self-polymerization to form polyurea polymers.

Table 1.  Kinetic parameters of urinary TDA, mean (SE)
	2,4-TDA			2,6-TDA			Ratio (CV%)
	0.2%	1%	5%	0.2%	1%	5%	0.2%	1%	5%
	(1)	(2)	(3)	(4)	(5)	(6)	[=(1)/(4)]	[=(2)/(5)]	[=(3)/(6)]
Tmax(h)	12	12	12	12	12	12	1	1	1
	(0)	(0)	(0)	(0)	(0)	(0)	(0)	(0)	(0)
Cmax(µg/ml)	0.062	0.238	6.116	0.056	0.268	3.777	1.1	0.9	1.6
	(0.009)	(0.060)	(0.429)	(0.004)	(0.087)	(0.384)	(8.5)	(8.0)	(29.1)
AUC(µg*h/ml)	2.186	8.395	158.599	2.046	10.558	133.994	1.1	0.8	1.2
	(0.376)	(0.919)	(5.517)	(0.263)	(0.538)	(20.350)	(8.2)	(5.9)	(18.5)
Accumulative amounts(µg)	2.682	12.940	83.843	2.622	14.978	69.810
	(0.631)	(4.224)	(29.542)	(0.779)	(2.628)	(11.541)
k (h^-1)^a	0.0376	0.0341	0.0325	0.0329	0.00339	0.0264
	(0.002)	(0.003)	(0.003)	(0.0020)	(0.0027)	(0.004)
t1/2(h)^a	18.4	20.4	21.5	21.1	20.5	26.6	0.9	1.0	0.8
	(0.8)	(1.5)	(2.2)	(1.3)	(1.6)	(3.7)	(3.0)	(0.8)	(13.8)

 ^aP > 0.05 by Kruskal-Wallis ANOVA test.

Dose-response Relationship between TDI exposed and AUC/Cmax/Accumulative Amounts

A linear increasing logarithm AUC trend for both forms of U-TDA with increasing TDI exposure was found (r = 0.968 for 2,4-TDA; r = 0.973 for 2,6-TDA) (Fig. 4a). A similar fashion for Cmax (r = 0.973 for 2,4-TDA; r = 0.984 for 2,6-TDA) and accumulative amounts (r = 0.998 for 2,4-TDA; r = 0.999 for 2,6-TDA) to AUC was also obtained. The above-mentioned findings suggested a clear dose-dependent fashion of skin absorption for 2,4- and 2,6-TDI.

Conclusions:

The study was conducted to reveal the toxicokinetic properties of TDI, applied dermally to the skin of rats and the detection of TDA in the urine after metabolisation of the test item to Toluene diamine (TDA). The validity criteria of the test system are fulfilled, since the control groups showed the expected results. The study was not conducted according to a certain guideline, but still its reliability is considered to be high (Klimisch 2). It has been demonstrated that the absorption of 2,4- and 2,6-TDI through skin contact is possible in this rat study.

Executive summary:

The toxicokinetics of the substance of interest Toluene diisocyanate (TDI) were investigated by Yeh et al. (2008) after dermal application in rats (dorsum, area approximately 3 * 5 cm). The exposure duration was 5 h, after which the substance was carefully washed of the skin, using a cleasing agent. It has been demonstrated that the absorption of 2,4- and 2,6-TDI through skin contact is possible in this rat study. A clear dose-dependent skin absorption for 2,4- and 2,6-TDI was demonstrated by the findings of AUC, Cmax and accumulative amounts (r ≥ 0.968). Excretory 2,4- and 2,6- TDA concentration profiles in 6-day consecutive urine samples were shown to fit in first-order kinetics, although higher order kinetics could not be excluded for high doses. The apparent half-lives for excretory urinary TDA were about 20 h at various skin exposures, similar to that from the inhalation exposure in the previous animal experiment. The overall yield ratios for 2,4- to 2,6-TDA in urine were found to be close to unity, apparently lower than the expectancy of 4:1, possibly due to the higher self-polymerization reactivity of 2,4- than 2,6-TDI.

It is concluded that skin absorption of TDI was confirmed in a rat model and a clear dose-dependent skin absorption relationship for 2,4- and 2,6-TDI was demonstrated. The findings in this study clearly demonstrate the skin absorption capability of topical TDI exposure based on the observation of the internal dose concentration profile of U-TDA across 6 days.

Description of key information

Short description of key information on bioaccumulation potential result:
1). Expert Statement, Chemservice S.A., 2011
2). Read across from TDI, basic toxikokinetics, Review Doe et al.,1995
3). Read across from TDI, basic toxicokinetics, Fischer 344 rats, oral administration or inhalation of vapour, Timchalk, 1994
4). Read across from TDI, basic toxicokinetics, Sprague Dawley rats, oral administration of TDI, Kennedy, 1994
5). Read across, basic toxicokinetics, Hartley guinea pigs, inhalation of vapours, Kennedy, 1989
6). Prediction using TOXTREE (v.2.1.0)

7). Read-across from MDI, basic toxicokinetic, Vock&Lutz

8.) read-across from MDI, basic toxicokinetik, Gledhill

9.) Read-across from MDI, basic toxicokinetik, Hoffmann

Key value for chemical safety assessment

Bioaccumulation potential:

low bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

10

Absorption rate - inhalation (%):

100

Additional information

Conclusion on toxicokinetical behaviour, metabolism, excretion and dermal penetration of TRIDI

TRIDI and TDI are both aromatic diisocyanates with different alkyl rests attached to benzene ring (three isopropyl groups in TRIDI and one methyl group in TDI). As a result of the structural differences, such physico-chemical properties as melting point, LogPow and water solubility differ significantly from each other. As no information on the toxicokinetic behaviour of TRIDI is available, the similar substance TDI was assessed as a possible read-across substance. Data revealed TDI to be an adequate read-across substance.

Taking into account the available information on the toxicokinetics of TDI and the detailed assessment of toxicokinetics of TRIDI, TRIDI is expected to be absorbed into the organism by all routes of exposure. However, absorption via the oral route is assumed to be low. Absorption after inhalation is considered to be rather fast. Dermal absorption, however will be limited by its molecular weight, its high lipophilicity and its high logPow. Due to high logPow, TRIDI is not expected to penetrate easily through the skin but tends to migrate towards fat tissues but certain reactivity of isocyanate groups with peptides and proteins might hinder accumulation. Inhalation and dermal routes can represent sensitising and irritating hazard for respiratory system. Nucleophilic substitution by SN2 mechanism with electron rich nucleophile amino acids of peptides and proteins is considered to be primary detoxifying mechanism of TRIDI. Excretion of TRIDI is expected via urine.

 

Discussion on bioaccumulation potential result:

There is no data on toxicokinetical behaviour of TRIDI. The data on its structural analogue TDI were taken into account to assess toxicokinetics of TRIDI. A detailed assessment of adsorption, distribution, metabolism pathways and excretion of target chemical was performed and is also attached in the section 13 "Assessment reports".

Toxicokinetics of TDI:

The substance Toluene diisocyanate (TDI) was subject of a detailed review of the existing toxicological information by Doe and coworkers (1995). The absorption, distribution, and kinetics of TDI are qualitatively and quantitatively different following inhalation exposure when compared with oral dosing, with inhalation being the relevant route in humans. Following oral dosing of TDI, dose-dependent percentages of the compound are converted to TDA (a mutagen and rodent carcinogen) by hydrolysis (mainly at aqueous tissue surfaces), which is consistent with the carcinogenicity observed following TDI gavage studies. The lower pH levels in i.e. the stomach are leading to high protonation of biological NH2 groups and this facilitates hydrolysis of TDI to TDA and subsequent formation of polyureas. These observations are consistent with comparative toxicokinetic studies in rats, which demonstrate significant levels of TDA following oral dosing with TDI - due to the acidic environment in the stomach - but not after inhalation.

Upon inhalation exposure TDI is conjugated to protein preferentially before formation of oligoureas or hydrolysis to TDA takes place. After inhalation a rather large portion (of 34% recovered radioactivity) is found in the carcass 48 hours after termination of exposure, indicating a slow release of protein-bound material, as suggested by in vitro data. Overall these conclusions are consistent with the lack of carcinogenicity observed in inhalation two-year studies with TDI and the tumours observed in rodents after oral dosing of TDI in corn oil. Additionally there are human exposure data indicating that the metabolism and kinetics in humans might be similar to that in animals, therefore valid extrapolations can be made. The conventional hazard assessment and resulting risk evaluation comes to the conclusion that there is no risk for carcinogenicity associated with the inhalation of TDI. Considering additional data (gavage studies with TDI or TDA and mechanistic biochemical data), three approaches of risk characterisation arrive at the conclusion that the highest possible risk associated with the inhalation of TDI at workplaces with TLV level exposure is about 5 x 10E-6. TDI inhalation at workplaces therefore will not present an unacceptable risk of carcinogenicity to man.

The substance Toluene diisocyanate was investigated for its toxicokinetic properties in Fischer 344 rats by Timchalk and coworkers (1994). The study was conducted similar to OECD TG417 with only minor deviations, why it was considered to be of high reliability (Klimisch 2). The rats received either 14C-radiolabelled TDI orally (60 mg/kg) or via inhalation (4 h, 2 ppm). The metabolism and excretion were determined also via HPLC and GC-MS.

The data suggest that orally administered 2,4-[14C]TDI is not very well absorbed. The rats eliminated approximately 8% of the radioactivity in the urine and cage wash while 4% were recovered in the tissues/carcass. Thus the minimum estimate for absorption was 12%, which assumed that the radioactivity recovered in the faeces (~ 81 %) represented un-absorbed material. A more realistic estimate for absorption was obtained by assuming that some of the radioactivity in the faeces was absorbed. In addition, orally administered TDI was reported to undergo rapid hydrolysis under aqueous conditions to form TDA which reacted with available isocyanate groups (TDI) to form polyureas. Under appropriate conditions 2,4-TDI readily hydrolyses to form 2,4-TDA. The 2,4-TDA can react with free 2,4-TDI forming polyurea polymers, which appear to be poorly absorbed from the gastrointestinal tract. Absorbed 2,4-TDA can be excreted in the urine either unchanged or as acid-labile conjugates. Additionally, 2,4-TDA can be N-acetylated forming mono- and diacetylated TDA metabolites which are readily excreted in the urine. Based on the rapid reactivity it is doubtful that TDI was absorbed prior to its hydrolysis to TDA. Therefore, the 12-20% of the 2,4-[14C]TDI dose that was absorbed, most probably represented 2,4-[14C]TDA that had not reacted to form polyureas. Based on the above, it was assumed that the radioactivity that was absorbed and excreted in the urine following the 2,4-[14C]TDI oral dose was absorbed primarily as 2,4-[14C]TDA.

Rats which were exposed to 2,4-[14C]TDI vapours retained essentially all the radioactivity that they inhaled. Inhaled 2,4-[14C]TDI appeared to be retained by the rat, and the data suggest that a large percentage of the radioactivity was absorbed through the lungs into the blood. At 48 hr following inhalation exposure, the urine and cage wash accounted for 19% of the recovered radioactivity, the tissues/carcass accounted for 34%, and 47% was recovered in the faeces. However, assuming 100% retention of inhaled TDI and minimal excretion over the 4-hr exposure, the quantitation of radioactivity detected in the tissues/carcass immediately postexposure would suggest that as much as 90% of the radioactivity was absorbed. The remaining radioactivity may have been rapidly cleared from the respiratory tract and subsequently swallowed. The pulmonary clearance of radioactivity from the lung to the gastrointestinal tract was supported by the fact that approximately 10% of the recovered inhalation dose was detected in the gastrointestinal tract contents of rats immediately postexposure. These data suggest that between 61 and 90% of the inhaled TDI dose was absorbed and the remaining radioactivity was rapidly cleared from the respiratory tract, ingested, and then eliminated in the faeces. In conclusion, comparison of the total amount of radioactivity in the tissues/carcass of rats indicated that a larger fraction of the recovered radioactivity was in the tissues/carcass following the inhalation vs oral exposure to 2,4-TDI. These data suggest that following inhalation exposure, a large percentage of the 2,4-[14C]TDI was absorbed through the lungs and existed in a different form than what was absorbed following oral 2,4-TDI and/or 2,4-TDA doses. The urinary excretion of radioactivity by the kidneys was slower following inhalation exposure (t1\2 = 20 hr) when compared to the oral 2,4-[14C]TDI dose group (t1\2 = 7.5 hr), suggesting that inhaled 2,4-TDI was eliminated in the urine in a different form having a longer biological half-life than orally administered 2,4-TDI and/or 2,4-TDA. This suggests that in the rat, very little 2,4-TDA is formed following inhalation exposure to 2,4-[14C]TDI vapours. In addition 90% of the quantitated metabolites in the urine specimens following inhalation exposure to 2,4-[14C]TDI existed as acid-labile conjugates of TDI/TDA while only 10% existed as acetylated TDA. This indicated that following inhalation exposure, a larger percentage of the 2,4-[14C]TDI was excreted in the urine in a conjugated form and not as free or acetylated TDA.

Overall, these data suggest that the metabolic disposition and carcinogenic potential of 2,4-TDI are dependent upon the route of exposure. Oral administration enhances the hydrolysis of 2,4-TDI, forming 2,4-TDA which is readily absorbed, whereas inhalation exposure to 2,4-TDI primarily results in the formation of 2,4-TDI conjugates and only small amounts of acetylated 2,4-TDA are produced. These findings are consistent with the chronic bioassay data which indicated that 2,4-TDI was not carcinogenic following inhalation exposure, but did result in tumour formation following oral administration in corn oil. Considering that the primary route of occupational exposure to TDI is via the inhalation route, then the data would suggest that the carcinogenic potential of TDI is low.

The substance Toluene diisocyanate was investigated for its toxicokinetic properties by Kennedy and coworkers (1994) in Sprague Dawley rats. The study was conducted similar to OECD TG417 with only minor deviations, and is considered to be of high reliability (Klimisch 2). Rats were exposed to 14C-TDI vapours at concentrations ranging from 0.026 to 0.821 ppm for 4 h. The distribution was determined. All tissues examined showed detectable quantities of radioactivity, with the airways, gastrointestinal system and blood having the highest levels, which increased with exposure concentration. The concentration of radioactivity in the bloodstream after exposure was linear with respect to dose. The majority (74-87%) of the label associated with the blood was recovered in the plasma, and of this, 97-100% of the 14C existed in the form of biomolecular conjugates. Analysis of stomach contents shows that the majority of the label is also associated with high (>10 kDa) molecular weight species. While a larger percentage (28%) of the label is found in the low molecular weight fraction relative to blood, this low molecular weight labelled material represents at least eight different components. Thus, over the vapour exposure concentrations and time tested, it appears that conjugation is the predominant reaction and that free TDA is not a primary in vivo reaction product under the conditions tested.

The substance Toluene diisocyanate was investigated for its toxicokinetic properties by Kennedy and coworkers (1989) in Hartley guinea pigs. The study was conducted similar to OECD TG417 with only minor deviations, why it was considered to be of high reliability (Klimisch 2). The guinea pigs received 14C-radiolabelled TDI via inhalation (1 h, in certain cases also 4 or 5 hours, 0 to 0.146 ppm). Exposures to 14C TDI were performed over a range of relatively low concentrations, including levels at and below the TLV for TDI which has been established at 0.005 ppm (Amer. Conf. Gov. Ind. Hyg., 1988). The absorption, distribution and excretion were determined via scintillation analysis and also via the Marcali- and the PNBPA method. The Marcali, PNBPA, and radioactivity values yielded comparable exposure concentration results. This confirms that the exposure atmospheres contained reactive TDI at the desired levels, all animals received equivalent concentrations, and chamber atmospheres were maintained throughout the exposure. Analysis of the uptake and distribution of radioactivity in the TDI-exposed fluids and tissues showed that some form of the labelled compound whether TDI, a conjugate, metabolite, or hydrolysis product, entered and penetrated throughout the entire system, even at the 0.004 ppm level. The urine and bile profiles demonstrated the rapid penetration of some form of the radioactivity through the system since at all concentrations the highest level of radioactivity in the bile and urine was found immediately following exposure. The post exposure increase of blood radioactivity is presumably due to the processing or desorption of the compound from the sites of entry (i.e., nasal passage, conducting airways, and alveoli) into the bloodstream for clearance. This study shows that the rate of uptake into the blood is linear during exposure to concentrations ranging from 0.00005 to 0.146 ppm and that the uptake continues to increase slightly postexposure. It also demonstrates that the radioactivity clears from the bloodstream to a level corresponding to approximately a 100 nM concentration of tolyl group after 72 hr and persists at a nanomolar level even 2 weeks following the exposure. After 2 weeks of recovery the animals still had measurable levels of blood radioactivity at a nearly constant amount (8.3 X 10E-8 M), regardless of initial dose, which suggests the saturation of a particular target which as a reacted form does not have a rapid turnover rate. The initial rate of 14C uptake is also a linear function of the concentration of TDI when expressed either as concentration (ppm) or as concentration multiplied by duration of exposure (ppm * hr). This is discussed in comparison with the toxic responses as a function of both ppm and ppm * hr.

Prediction for TRIDI using TOXTREE

The chemical structure of 2,4,6 -triisopropyl-m-phenylene diisocyanate was assessed by Toxtree (v.2.1.0) modelling tool for possible metabolism. SMART Cyp is a prediction model, included in the tool, which identifies sites in a molecule that are labile for the metabolism by Cytochromes P450.

2,4,6 -triisopropyl-m-phenylene diisocyanate is expected to be well metabolized by the Cytochrome P450 group of metabolizing enzymes.The molecule possesses equal or more than three sites of metabolism. The primary and secondary sites of metabolism are the diisopropyl-groups, which are predicted to be subject to aliphatic hydroxylation. The tertiary sites of metabolism are the carbon-atoms of the aromatic ring, which are predicted to be subject to aromatic hydroxylations.

 

Prediction of the toxicokinetic behaviour of TRIDI:

There is little data available on physico-chemical properties of 2,4,6,-triisopropyl-m-phenylene diisocyanate. With the aid of the EPIWIN software some physico-chemical properties were calculated.

The substance is at room temperature a light yellowish liquid with a slight odour. The substance is insoluble in water (< 0.05 mg/L at 20°C) and has a logPow of 7.56. It has a low vapour pressure (0.19 Pa at 20°C). No exact value of melting point could be determined experimentally for TRIDI between -90°C and 50°C (Kintrup, 2012). Glass transition temperature (amorphous components) in the first heating run was determined to be at -56°C. The calculated melting point was 82.9°C. The boiling point of 305.8°C was measured for TRIDI (Svobodova, 2012). Hydrolysis as a function of pH has not been determined, but comparison to its structural analogue toluene diisocyanate revealed a high likelihood for hydrolysis. The substance is not toxic when administered orally to rats (LD50 > 2000 mg/kg bw). However, it has been determined to be toxic after inhalation (LC50 < 110mg/m³). It is not an eye, but a skin irritant, and skin sensitising properties have been predicted. Additionally, the substance was shown to be not mutagenic in studies according to OECD 471, 473 and 476.

Absorption:

In general, absorption of a chemical is possible, if the substance crosses biological membranes. The EU Technical Guidance Document on Risk Assessment (TGD, Part I, Appendix VI) gives a number of physico-chemical properties that normally determine oral, inhalation and dermal absorption (LINK to Guidance Document:http://ecb.jrc.ec.europa.eu/tgd/). This process requires a substance to be soluble both in lipid and in water and is also dependent on its molecular weight (substances with molecular weights below 500 are favourable for absorption). Firstly, TRIDI and TDI would be favourable for absorption, when only taking into account their molecular weights. However, as TRIDI is practically insoluble in water, it is apparent that its absorption is hindered. This is also seen in the value calculated for the LogPow (7.56) that shows the substance to be better soluble in octanol than in water. Considering its low water solubility and the value for LogPow calculated to be above 4, the absorption into the body will not be favoured (LogPow between 0 and 4 are favourable for absorption). In general, the absorption of chemicals, which are surfactants or irritants may be enhanced, because of damage to cell membranes. This is the case for both substances of interest.

 

Absorption from the gastrointestinal tract

Regarding oral absorption, in the stomach, a substance will most likely be hydrolysed, as this is a favoured reaction in the acidic environment of the stomach. In accordance with the above mentioned principles it has been reported for TDI to be hydrolysed in the stomach to toluene diamine (TDA). The lower pH levels in i.e. the stomach are leading to high protonation of biological NH2 groups and this facilitates hydrolysis of TDI to TDA and subsequent formation of polyureas

In the small intestine absorption occurs mainly via passive diffusion or lipophilic compounds may form micelles and be taken into the lymphatic system. Additionally, metabolism may occur by gut microflora or by enzymes in the gastrointestinal mucosa. However, the absorption of highly lipophilic substances (Log Pow of 4 or above) may be limited by the inability of such substances to dissolve into gastrointestinal fluids and hence make contact with the mucosal surface. The absorption of such substances will be enhanced if they undergo micellular solubilisation by bile salts. Substances absorbed as micelles enter the circulation via the lymphatic system, bypassing the liver.

The toxicological data available for both substances show, that the substances resemble each other in the endpoints: acute toxicity oral (LD50 > 2000 mg/kg bw for both substances) and skin irritation (both irritating). As the results for these endpoints are identical, it can be presumed that these substances have in principle the same mode of action. The available toxicokinetic data for TDI suggest that orally administered 2,4-TDI is not very well absorbed (Timchalk et al., 1994). The minimum estimate for absorption was 12%, which assumed that the radio-labelled substance was recovered in the faeces (~ 81 %), represented un-absorbed material. A more realistic estimate for absorption was obtained by assuming that some of the radioactivity in the faeces was absorbed. However, the hydrolysed TDI can react as 2.4-TDA with free 2,4-TDI forming polyurea polymers, which appear to be poorly absorbed from the gastrointestinal tract. Based on the rapid reactivity it is doubtful that TDI was absorbed prior to its hydrolysis to TDA. Therefore, the 12-20% of the 2,4-TDI that was absorbed, most probably represented 2,4-TDA that had not reacted to form polyureas.

Based on the abovementioned data for the closest analogue, it can be presumed that the absorption of TRIDI via the oral route will be slower than that of TDI, which will result in a lower toxicity of the substance (also by the long-term exposure).

 

Absorption from the respiratory tract

Regarding absorption in the respiratory tract, any gas or vapour has to be sufficiently lipophilic to cross the alveolar and capillary membranes (moderate Log P values between 0-4 favourable for absorption). The rate of systemic uptake of very hydrophilic gases or vapours may be limited by the rate at which they partition out of the aqueous fluids (mucus) lining the respiratory tract and into the blood. Such substances may be transported out of the lungs with the mucus and swallowed or may pass across the respiratory epithelium via aqueous membrane pores. Lipophilic substances (Log P >0) would have the potential to be absorbed directly across the respiratory tract epithelium. Very hydrophilic substances might be absorbed through aqueous pores (for substances with molecular weights below around 200) or be retained in the mucus.

Even though TRIDI has a relatively low vapour pressure (0.19 Pa) and a high boiling point (calculated 305.8°C), which would indicate a low availability for inhalation, it is known, that isocyanates bear a high potential for respiratory sensitisation and irritation. As isocyanates are highly reactive, irritating compounds it is clear, that contact to the epithelium will produce irritation and therefore enhance absorption. The toxicokinetic data available for TDI show that no relevant hydrolysis occurs in the respiratory tract, as TDA was barely detectable in the urine, following inhalation of TDI (Timchalk et al., 1994). Additionally, essentially all the radioactivity inhaled via 2.4-TDI vapours was retained (Timchalk et al., 1994). The data suggest that a large percentage of the radioactivity was absorbed through the lungs into the blood. The data suggest that between 61 and 90% of the inhaled 2,4-[14C]TDI dose was absorbed and the remaining radioactivity was rapidly cleared from the respiratory tract, ingested, and then eliminated in the faeces.

Kennedy and co-workers found in all tissues examined detectable quantities of radioactivity, with the airways, gastrointestinal system and blood having the highest levels which increased with exposure concentration (Kennedy et al., 1989 and 1994). The concentration of radioactivity in the bloodstream after exposure was linear with respect to dose. The results showed that greater than 95% of the plasma-associated radioactivity existed in the form of biomolecular conjugates (reaction of TDI with biological macromolecules successfully competes with hydrolysis to the diamine). Thus, over the vapour exposure concentrations and time tested, it appears that conjugation (mainly with serum-albumin) is the predominant reaction and that free TDA is not a primary in vivo reaction product under the conditions tested.

Based on this data, it can be assumed that TRIDI might act in the same way as TDI. Presumably it is expected to be bonded to proteins of cells in the respiratory epithelium and/or be hydrolyzed to an amine derivative as well, triggering respiratory sensitization and irritation reactions.

 

Absorption following dermal exposure

In order to cross the skin, a compound must first penetrate into the stratum corneum and may subsequently reach the viable epidermis, the dermis and the vascular network. The stratum corneum provides its greatest barrier function against hydrophilic compounds, whereas the viable epidermis is most resistant to penetration by highly lipophilic compounds. Substances with a molecular weight below 100 are favourable for penetration of the skin and substances above 500 are normally not able to penetrate. The substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. Therefore if the water solubility is below 1 mg/l, dermal uptake is likely to be low. Additionally Log Pow values between 1 and 4 favour dermal absorption (values between 2 and 3 are optimal). Above 4, the rate of penetration may be limited by the rate of transfer between thestratum corneumand the epidermis, but uptake into the stratum corneum will be high. Above 6, the rate of transfer between the stratum corneum and the epidermis will be slow and will limit absorption across the skin. Uptake into the stratum corneum itself may be slow. Moreover vapours of substances with vapour pressures below 100 Pa are likely to be well absorbed and the amount absorbed dermally may be more than 10% of the amount that would be absorbed by inhalation. If the substance is a skin irritant or corrosive, damage to the skin surface may enhance penetration. During the whole absorption process into the skin, the compound may be subject to biotransformation.

In case of TRIDI and TDI, the molecular weight is above 100 and below 500, which would normally indicate low potential to penetrate the skin. Moreover, the logPow value of TRIDI is with 7.56 very high and this also indicates as stated above low absorption. Due to the vapour pressure of 0.19 Pa, TRIDI is once again represents rather an inhalation hazard than one by dermal route of exposure. One has to keep in mind, that absorption is influenced by the irritating potential of the two substances and might enhance penetration. It has been demonstrated that the absorption of 2,4- and 2,6-TDI through skin contact is possible, as toluene diamine was found in the urine (Yeh et al., 2008). A clear dose-dependent skin absorption for 2,4- and 2,6-TDI was demonstrated by the findings of AUC, Cmax and accumulative amounts (r ≥ 0.968) (Yeh et al., 2008).

Based on these factors, TRIDI is expected to be partially absorbed following dermal exposure into the stratum corneum. The transfer of the substance into the epidermis will be limited, due to its molecular weight and high lipophilicity. Hence, the systemic toxicity of TRIDI via the skin is assumed to be low and 10% adsorption via dermal route is proposed based on the high logPow and low water solubility.

Distribution

In general, the following principle applies: the smaller the molecule, the wider the distribution. A lipophilic molecule (Log Pow >0) is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration particularly in fatty tissues. It’s not possible to foresee protein binding, which can limit the amount of a substance available for distribution. Furthermore, if a substance undergoes extensive first-pass metabolism, predictions made on the basis of the physico-chemical characteristics of the parent substance may not be applicable.

In case of TRIDI, no data is available for distribution patterns. However, there are data available for TDI. It has been demonstrated, that the absorbed amount of TDI is distributed after oral, dermal and inhalatory exposure (Timchalk et al., 1994, Kennedy et al., 1989, and 1994). The gastrointestinal tract and contents accounted for a high amount of the recovered radioactivity in the tissues/carcass with the remaining radioactivity evenly distributed among the remaining tissues (Timchalk et al., 1994).

The distribution of TRIDI is expected to be more extensive in fat tissues than in other tissues.

Akkumulation

It is also important to consider the potential for a substance to accumulate or to be retained within the body. Lipophilic substances have the potential to accumulate within the body (mainly in the adipose tissue) if the dosing interval is shorter than 4 times the whole body half-life. Although there is no direct correlation between the lipophilicity of a substance and its biological half-life, substances with high log P values tend to have longer half-lives. On this basis, there is the potential for highly lipophilic substances (Log Pow >4) to accumulate in individuals that are frequently exposed. Highly lipophilic substances (Log Pow between 4 and 6) that come into contact with the skin can readily penetrate the lipid rich stratum corneum but are not well absorbed systemically. Although they may persist in the stratum corneum, they will eventually be cleared as the stratum corneum is sloughed off. A turnover time of 12 days has been quoted for skin epithelial cells

Following oral exposure little TDI is retained in the body (4 %) and following exposure via inhalation a larger amount of TDI (34%) is retained in the carcass, indicating a slow release of protein-bound material (Timchalk et al., 1994). However, to our knowledge the accumulation of TDI in the stratum corneum has not been investigated.

Metabolism

Route specific toxicity may result from several phenomena, such as hydrolysis within the gastrointestinal or respiratory tracts, also metabolism by gastrointestinal flora or within the gastrointestinal tract epithelia (mainly in the small intestine), respiratory tract epithelia (sites include the nasal cavity, tracheo-bronchial mucosa (Clara cells) and alveoli (type 2 cells) and skin.

It has been shown, that after oral exposure TDI, which is hydrolysed to TDA in the gastrointestinal tract and subsequently absorbed, can be excreted in the urine either unchanged or as acid-labile conjugates. TDA, as a metabolite can be N-acetylated forming mono- and diacetylated TDA metabolites which are readily excreted in the urine (Timchalk, et al. 1994). After inhalatory exposure, very little 2,4-TDA is formed. In addition, 90% of the quantitated metabolites in the urine specimens following inhalation exposure to 2,4-TDI existed as acid-labile conjugates of TDI/TDA while only 10% existed as acetylated TDA. This indicated that following inhalation exposure, a larger percentage of the 2,4-TDI was excreted in the urine preferentially in a conjugated form (to proteins) and not as oligoureas or free or acetylated TDA (Timchalk et al., 1994).

There is no data on metabolism of TRIDI. Similar to TDI, the substance is expected to form conjugates with glutathione (and other peptides) because a carbon atom in the isocyanate group represents an electrophile centre susceptible to nucleophile attack by such strong nucleophiles as liysine, cysteine and histidine (Smith and Hotchkiss, 2001). The isopropyl groups on aromatic ring can however affect the reactivity of electrophile carbon due probably to sterical hindrance. If TRIDI hydrolises to its corresponding amine, the latter can be N-acetylated and then excreted in the urine. Aromatic and aliphatic hydroxylation can also occur leading to a hydrophyle which is easily to be excreted.

Excretion

The major routes of excretion for substances from the systemic circulation are in the urine and/or the faeces (via bile and directly from the gastrointestinal mucosa). For volatile substances and metabolites exhaled air is an important route of excretion. Substances that are excreted favourable in the urine tend to be water-soluble and of low molecular weight (below 300 in the rat) and be ionized at the pH of urine. Most will have been filtered out of the blood by the kidneys, though a small amount may enter the urine directly by passive diffusion and there is the potential for reabsorption into the systemic circulation across the tubular epithelium. Substances that are excreted in the bile tend to be amphipathic (containing both polar and nonpolar regions), hydrophobic/strongly polar and have higher molecular weights and pass through the intestines before they are excreted in the faeces and as a result may undergo enterohepatic recycling which will prolong their biological half-life. This is particularly a problem for conjugated molecules that are hydrolysed by gastrointestinal bacteria to form smaller more lipid soluble molecules that can then be reabsorbed from the GI tract Those substances less likely to recirculate are substances having strong polarity and high molecular weight of their own accord. Other substances excreted in the faeces are those that have diffused out of the systemic circulation into the GIT directly, substances which have been removed from the gastrointestinal mucosa by efflux mechanisms and non-absorbed substances that have been ingested or inhaled and subsequently swallowed. Non-ionized and lipid soluble molecules may be excreted in the saliva, where they may be swallowed again, or in the sweat. Highly lipophilic substances that have penetrated the stratum corneum but not penetrated the viable epidermis may be sloughed off with skin cells.

For TRIDI no data is available concerning its elimination, but for TDI several studies have been undertaken. TDI is mainly eliminated via the faeces (80 %) after oral exposure; only 5 to 15 % are eliminated via the urine. However, this is in accordance with the above mentioned principles, as TDI reacts with hydrolysed TDA in the gastrointestinal tract to polyurea polymers, which have a high molecular weight and are subsequently not absorbed and therefore eliminated via the faeces. After inhalation 48 % is eliminated via the faeces and 15 % via the urine in 48 hours and no quantifiable elimination via exhalation occurred (Timchalk et al., 1994).

The urinary excretion by the kidneys was slower following inhalation exposure (t1\2 = 20 hr) when compared to the oral 2,4-TDI dose group (t1\2 = 7.5 hr), suggesting that inhaled 2,4-TDI was eliminated in the urine in a different form having a longer biological half-life than orally administered 2,4-TDI and/or 2,4-TDA (Timchalk et al., 1994).

 

Discussion on absorption rate:

There is no experimental data on dermal absorption of TRIDI. Therefore the available data on TDI , structurally similar analogue to TRIDI, was taken into account to assess dermal penetration of the target substance. A detailed assessment of possible dermal absorption is presented also in a separate file attached to the IUCLID file in section 13 "Assessment reports".

Toxicokinetic data on dermal absorption of TDI:

The toxicokinetics of the substance Toluene diisocyanate (TDI) were investigated in rats by Yeh et al. (2008) after dermal application in rats (dorsum, area approximately 3 * 5 cm). The exposure duration was 5 h, after which the substance was carefully washed of the skin, using a cleansing agent. It has been demonstrated that the absorption of 2,4- and 2,6-TDI through skin contact is possible. A clear dose-dependent skin absorption for 2,4- and 2,6-TDI was demonstrated by the findings of AUC, Cmax and accumulative amounts (r ≥ 0.968). Excretory 2,4- and 2,6- TDA concentration profiles in 6-day consecutive urine samples were shown to fit in first-order kinetics, although higher order kinetics could not be excluded for high doses. The apparent half-lives for excretory urinary TDA were about 20 h at various skin exposures, similar to that from the inhalation exposure in the previous animal experiment. The overall yield ratios for 2,4- to 2,6-TDA in urine were found to be close to unity, apparently lower than the expectancy of 4:1, possibly due to the higher self-polymerization reactivity of 2,4- than 2,6-TDI.

It is concluded that skin absorption of TDI was confirmed in a rat model and a clear dose-dependent skin absorption relationship for 2,4- and 2,6-TDI was demonstrated. The findings in this study clearly demonstrate the skin absorption capability of topical TDI exposure based on the observation of the internal dose concentration profile of urinary TDA across 6 days.

Toxicokinetic of MDI

The distribution and DNA adduct formation of radiolabeled methylenediphenyl-4,4'-diisocyanate (MDI) in the rat after topical treatment has been evaluated by Vock and Lutz, 1997.

29-30% of the dermally administered MDI dose was recovered in the faeces of female rats 48 h after treatment (~30 mg/kg bw). Unintentional oral exposure cannot be excluded in the study. Less than 1% of the applied radioactivity was detected in total in the lungs, liver, kidney and muscles of female rats upon topical application of radiolabelled 4,4’-MDI for 24 h (11-15 mg/kg bw) or 48 h (29-30 mg/kg bw). Detection of 29-30% of the radioactivity in the faeces during 48 h dermal exposure to 4,4’-MDI has been reported. The recovery in the urine was <1%. No measures were taken to prevent oral exposure (e.g. via grooming). No DNA adduct formation (epidermis) has been identified.

The absorption, distribution,  metabolism  and  excretion  of  an  inhalation  dose  of  [14C]  4,4'-methylenediphenyl diisocyanate in the male rat has been investigated by Gledhill et al, 2005.

Significant absorption of inhaled diisocyanates has been shown in the nasal and alveolar region of experimental animals. In rats, the absorption of inhaled MDI (2 mg/m³) has been estimated to be 32%. Exposure of male rats to 2 mg/m³ (0.20 ppm) of radiolabelled 4,4’-MDI (corresponding to 0.67 mg/m³ NCO) for 6 h resulted in distribution to several tissues, the concentrations being highest in the respiratory and gastrointestinal tract. The authors were not able to exclude the possibility of additional exposure by the oral route during the study. N-acetylated and N-acetylated hydroxylated MDI-metabolites were identified in the urine, faeces and bile of male rats after inhalation exposure to MDI . Free MDA was not detected. Mixed polyureas formed in spontaneous reactions of MDI were the primary products detected in the faeces.

The dermal uptake and excretion of 14C-methylene  diphenyl  diisocyanate  (MDI)  in  male  rats has been investigated by Hoffmann et al., 2010.Animals were housed singly, the site of administration was protected with a silicone ring, a nylon mesh gauze and a porous bandage, to prevent contact by grooming and to minimise contact of the applied dose by the dressing.

Investigations have been undertaken with MDI and TDI to assess dermal uptake and resulting systemic exposure. Absorption, distribution and excretion of MDI was studied in rats using a single dermal administration of 14C-MDI dissolved in acetone at nominal 165 mg/kg body weight and 15 mg/kg bw (4.0 and 0.4mg/cm²) and intradermal injection of 14C-MDI dissolved in corn oil at nominal 1.4 mg/kg bw. Dermal absorption of 14C-MDI (at both doses) was low; at or below 1% of the applied dose. Considerable amounts of the applied radioactivity were found at the application site which could not be washed off. By intradermal administration of 14C-MDI approximately 66% of applied radioactivity remained at the application site with approximately 26% recovered in excreta, cage wash, tissues and carcass. Overall it is concluded that, following dermal exposure to MDI, systemic exposures and resulting toxicity, other than the known sensitization, can be expected to be very low. The dermally absorbed amounts of MDI were estimated to be low (0.21-0.88%, 8-120 h after exposure) upon topical application of MDI (15 or 165 mg/kg bw) on rat skin. No radioactivity was detected in the tissues when radiolabelled 4,4’-MDI was used (8, 24 or 120 h after the exposure). No radioactivity was detected in the faeces. Very low levels of radioactivity were detected in the faeces and urine of male rats 8, 24 or 120 h after dermal exposure (8 h) to 4,4’-MDI.