Registration Dossier

Administrative data

Endpoint:
dermal absorption in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well-documented publication which meets basic scientific principles

Data source

Reference
Reference Type:
publication
Title:
Urinary excretion of toluene diisocyanates in rats following dermal exposure
Author:
Yeh, H.; Lin, W.; Shih, T.; Tsai, P.; Wang, S. and Chang, H.
Year:
2008
Bibliographic source:
J. Appl. Toxicol. 2008; 28: 189–195
Report Date:
2007

Materials and methods

Principles of method if other than guideline:
So far no studies in the literature have ever investigated the internal dose concentration profile for skin TDI absorption through an in vivo animal study. The aims of this study were to use a rat model to determine the skin absorption of TDI and to determine whether a dose-response pattern exists. Moreover, the kinetic parameters, including the apparent half-life and area under the curve (AUC), will also be investigated to facilitate an understanding of the uptake and elimination behaviors of TDI skin exposure.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,4-TDI: 2,6-TDI( = 80%:20%, m:m, purity of 95%) and 2,6-TDA (97%)
- Molecular formula (if other than submission substance): C9H6N2O2
- Molecular weight (if other than submission substance): 174,2 g/mol
- Smiles notation (if other than submission substance): Cc1ccc(cc1\N=C=O)\N=C=O
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: organic diisocyanate
- Physical state: liquid
- Analytical purity: 95 % or 97 % respectively
- Composition of test material, percentage of components: 2,4-TDI: 2,6-TDI = 80%:20%, m:m, purity of 95 %
- Isomers composition: 2,4-TDI: 2,6-TDI = 80%:20%, m:m

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: National Cheng Kung University, Laboratory Animal Center, Taiwan
- Age at study initiation: 10–12 weeks
- Weight at study initiation: 260–380 g
- Housing: each rat was kept in a separate cage to avoid any fighting.
- Individual metabolism cages: seperate cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): rats were housed in a temperature and humidity controlled room
- Humidity (%): rats were housed in a temperature and humidity controlled room
- Photoperiod (hrs dark / hrs light): 12 hours light 7 12 hours dark

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
olive oil
Duration of exposure:
5 hours
Doses:
0.2 %, 1 % and 5 % TDI in Olive oil
No. of animals per group:
Triplicates (n = 3) were performed for each group. The rats were killed on day 7.
Control animals:
yes
Remarks:
The rats in the control group were treated exactly as the exposure group except exposed to pure olive oil only.No detectable TDA was found in the control group urine at any time. Therefore, the control group was excluded from further data analysis.
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions: TDI was prepared in 1.5 ml olive oil for the topical application.

APPLICATION OF DOSE: dermal

VEHICLE
- Justification for use and choice of vehicle (if other than water): olive oil
- Purity: 0.2 %, 1 % or 5 %

TEST SITE
- Preparation of test site: the hair on the dorsum was clipped 24 h before the TDI was applied to avoid wounds to the skin.
- Area of exposure: chemicals were gently spread over a cotton pad approximately 3 × 5 cm then adhered to the test site.
- Type of cover / wrap if used: to assure no leakage on the tested skin, the experimental site was first covered with a sponge pad followed by an elastic bandage.

SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: yes: experimental site was first covered with a sponge pad followed by an elastic bandage. Throughout the experiment every part of the covered site on the rats was carefully examined to ensure that no loosened or broken pads were found

REMOVAL OF TEST SUBSTANCE
- Removal of protecting device: after 5 h
- Washing procedures and type of cleansing agent: at the end of exposure, the exposure site was washed three times to remove the residual TDI on skin using cleanser (D-TAM™, DOD Technologies, USA).
- Time after start of exposure: 5 h

SAMPLE COLLECTION
- Collection of urine and faeces: consecutive urine samples were collected for 6 days and U-TDA were analysed using GC/ECD. The collection frequency was from the end of exposure to day 6 every 12 h on the first 3 days and every 24 h on the last 3 days. The urine was collected in a 50 ml polypropylene conical tube and stored at -20 °C until analysis. The exact volume (ml) for each collected urine was measured.


SAMPLE PREPARATION
- Storage procedure: stored at -20 °C until analysis
- Preparation details: the accumulative U-TDA amount (in µg) for each collected urine sample was estimated using the multiplicative product of 2,4- and 2,6-TDA concentration in urine (µg ml-1) and its corresponding volume (ml). Before and at the end of the experiment, skin irritation was evaluated with no skin irritation being found for any exposure or control group. The urine samples were hydrolysed with 1.5 mL of 3 M H2SO4 for 16 h at 100°C, using a Firefox Dry Bath 6100 (Panttech, Taiwan) in 15 ml test tubes with Teflon screw caps. After hydrolysis, 5 ml of saturated NaOH solution and 2 ml of toluene were added to the samples. The mixtures were shaken for 2 min and then centrifuged for 10 min at 1500 rpm. One point five milliliters of the organic supernatant was transferred to a new test tube and 25 µl HFBA was added. The samples were immediately shaken vigorously for 2 min and allowed to stand for 10 min. Excess reagent was removed by extraction with 1 ml of 1 M phosphate buffer solution (pH 7.2). The toluene layer, containing the amide derivatives, was transferred into 1.5 ml auto sampler vials with Teflon seals for further GC analysis.

ANALYSIS
- Method type(s) for identification: all GC analyses were performed using an Agilent Model 6890 GC equipped with µ-electron capture detectors (µ-ECD, Agilent G2397A). A DB-5MS (J&W Scientific, Folsom, CA, USA) (60 m × 0.25 mm i.d.) with a film thickness of 0.25 µm fused silica capillary column was used to perform the separation. Ultra high-purity nitrogen (purity =99.99%) was used as the carrier and make-up gases. Their flow rates were set at 0.8 mL/min and 30 mL/min, respectively. The temperature program was initially set at 150 °C for 1 min, followed by an increase rate of 10 °C/min to 250 °C, at which the column temperature was held for 5 min. The injector and detector temperatures were 250 and 290 °C, respectively. The injection volume was 1 µL in the split mode with a split ratio of 30:1.
- Liquid scintillation counting results (cpm) converted to dpm as follows:
- Validation of analytical procedure: the retention times for 2,6- and 2,4-TDA derivatives were 10.796 min and 11.057 min in the GC chromatogram, respectively. The correlation coefficient (r) of 2,4- and 2,6-TDA calibration curves yielded at least 0.995 for both TDAs at high and low concentrations.
- Limits of detection and quantification: the limit of detection for TDAs was 1 ng/mL. 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.

OTHER:
- Because workers in industry could be exposed to liquid containing about 1% TDI during the manufacturing processes in industry, the rats in the experiment were dermally exposed at concentrations of 1%, 0.2% (lower 5-fold end), and 5% (upper 5-fold end), respectively for 5 h.

Details on in vitro test system (if applicable):
not applicable

Results and discussion

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.