Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliable without restriction; study conducted according to GLPs and OECD 428 guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
not specified
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
The structure of the test substance was confirmed using gas chromatography with mass spectrometric detection (GC/MS).

Prior to use on the study, the purity of the unlabeled test substance was determined by gas chromatography with flame ionization detection (GC/FID) and found to be 97.9 ± 0.2%.

The initial radiochemical purity of the 14C-labeled test substance was determined by gas chromatography with radiochemical detection (GC/RAD) and found to be >99.5%. The purity of the 14C-labeled test substance recovered from the donor chambers following the 29 hour skin absorption study at 32° C was determined by GC/RAD and GC/FID and found to be 100% and 97.7-100.2%, respectively. Based on these results, the test substance was considered to be stable during the test period.
Radiolabelling:
yes

Test animals

Species:
human
Sex:
not specified
Details on test animals and environmental conditions:
Skin was obtained from the abdominal region of human cadavers via an organ procurement agency (National Disease Research Interchange, Philadelphia, PA). In order to control for inter-individual variability in skin absorption, skin samples from three different human donors were used. The experiment utilized nine Franz-type diffusion cells containing three skin preparations from each of the three donors. Samples were collected using a dermatome; the specified thickness was approximately 200 to 500 micrometers.

During each phase of the experiment, the receptor chambers were filled with receptor solution (Dulbecco's phosphate buffered saline containing Penicillin 100 units/mL, Streptomycin 100 µg/mL, Amphotericin B as Fungizone ™ 0.25 µg/mL and Volpo-20™ 60 mg/mL). Duplicate background (0 hr) samples were taken from the receptor chamber from each cell.

Administration / exposure

Vehicle:
unchanged (no vehicle)
Duration of exposure:
29 hours
Doses:
300 µL (undiluted)
No. of animals per group:
Three skin preparations from each of three donors were used.
Control animals:
yes
Details on study design:
Study Design: The study consisted of a three phase experiment:

Phase 1 - Determination of the permeability to tritium;

Phase 2 - Determination of the permeability to the test substance;

Phase 3 - Determination of the permeability to tritium.

Procedure: In order to control for inter-individual variability in skin absorption, skin samples from three different human donors were used. The experiment utilized nine Franz-type diffusion cells containing three skin preparations from each of the three donors. Samples were collected using a dermatome; the specified thickness was approximately 200 to 500 micrometers.

During each phase of the experiment, the receptor chambers were filled with receptor solution. Duplicate background (0 hr) samples were taken from the receptor chamber from each cell.

Phase 1: 300 µL (specific activity 3.89 µCi/gm) of tritium was applied to the donor chambers and the chambers were incubated at 32 °C for 6 hours. Duplicate samples were removed from the receptor chambers at hourly intervals and analyzed for tritium by liquid scintillation spectrometry (LSS). The receptor chambers were refilled after each sampling. After collecting the last sample, the donor and receptor chambers were rinsed with phosphate buffered saline and refilled with antibiotic-antimycotic saline without Volpo-20™. The cells were allowed to stir overnight, after which they were emptied, rinsed, and filled with the appropriate solutions for Phase 2 of the experiment.

Phase 2: 300 µL of the neat test substance (specific activity 607.8 µCi/gm) was applied directly to the surface of the skin of the test substance cells and the chambers were incubated at 32 °C for 29 hours. Duplicate samples were removed from the receptor chambers at hourly intervals for 6 hours and then at 8, 11, 17, 25, 26, 27, 28, and 29 hours. The 14C-labeled test substance in the receptor solution was analyzed by LSS. The receptor chambers were refilled after each sampling. Control cells were conducted along with the cells containing the test substance in Phase 2.

Buffered isotonic saline was applied to the control cells donor chambers. Duplicate samples were removed from the receptor chambers at 0, 3, 6, and 29 hours and analyzed for tritium by LSS. The receptor chambers were refilled after each sampling.

At termination of Phase 2, all components of the test system, except skin specimens, were analyzed by LSS for determination of total 14C-labeled test substance recovery. Then the donor and receptor chambers were refilled with antibiotic-antimycotic saline without Volpo-20™. The chambers were allowed to stir overnight, after which they were emptied, rinsed, and filled with the appropriate solutions for Phase 3 of the experiment.

Phase 3: 300 µL of tritium was again applied to the donor chambers and the chambers were incubated at 32 °C for 6 hours. Duplicate samples were removed from the receptor chambers at hourly intervals and analyzed for tritium and the 14C-labeled test substance by LSS. The receptor chambers were refilled after each sampling.

Following the completion of Phase 3, the skin samples were removed, digested using a tissue solubilizer, and analyzed for the test substance. The samples collected following Phase 3 were analyzed by LSS for determination of total 14C-labeled test substance recovery.

The rate of increase in the concentration of the 14C-labeled test substance or tritium in the receptor chamber of each cell was used to calculate a permeability constant (cm/hr) and an absorption rate (µg/cm^2/hr) for the test substance and tritium.

Assessment of Membrane Integrity: The integrity of each skin sample was evaluated by measuring its permeability to tritium over a period of 6 hours during Phase 1 of the experiment. In addition, the ratio of the tritium permeability constants for Phase 3 and Phase 1 were used to calculate a damage ratio for each cell. This value was used to assess the effect of the test substance on the integrity of the skin sample.

Results and discussion

Total recovery:
The mean (± SD) total test substance recovery was 103.8% ± 6.3%.

Any other information on results incl. tables

Phase 1: The mean ( ± SD) absorption rate of tritium for Phase 1 was found to be 2.04 ± 0.95 mg/cm^2/hr for all of the cells.

Phase 2: The mean ( ± SD) absorption rate of di (2-ethylhexyl) terephthalate for Phase 2 was found to be 0.103 ± 0.053 µg/cm^2/hr for cells treated with the test substance.  The corresponding permeability constant was determined to be 8.39 x 10^-8 cm/hr.

The mean ( ± SD) total test substance recovery was 103.8% ± 6.3%.

Phase 3: The mean ( ± SD) absorption rate of tritium for Phase 3 was 2.10 ± 0.54 mg/cm^2/hr for the control cells.

The mean damage ratio for the control cells was 1.15 ± 0.18.  The mean damage ratio for the test cells exposed to di (2-ethylhexyl) terephthalate during Phase 2 was 1.14 ± 0.23.

Applicant's summary and conclusion

Conclusions:
The absorption rate of di (2-ethylhexyl) terephthalate through dermatomed human skin was found to be 0.103 ± 0.052 µg/cm^2/hr. According to the criteria set forth by Marzulli et al. (1969), di (2-ethylhexyl) terephthalate would be considered an "extremely slow" penetrant relative to other chemical species. The mean damage ratio for skin treated with di (2-ethylhexyl) terephthalate was determined to be 1.14 ± 0.23. This value is within the range of damage ratios for skin exposed to physiological saline (Dugard et al., 1984). Therefore, under the conditions of this study, di (2-ethylhexyl) terephthalate did not cause significant damage to the skin. Based on its low potential to penetrate the skin (0.103 µg/cm^2/hr), the potential for systemic exposure is limited.

References cited:
Marzulli et al., 1969. Techniques for studying skin penetration. Toxicol. Appl. Pharmacol.,Suppl. 3, 76-83.

Dugard et al., 1984. Absorption of some glycol ethers through human skin in vitro. Environ. Health Perspect. 57, 193-197.
Executive summary:

In a study designed to study the rate of percutaneous absorption of di (2-ethylhexyl) terephthalate through dermatomed sections of human skin it was determined that the absorption rate was 0.103 ± 0.052 µg/cm^2/hr. The integrity of the skin specimen was determined by measuring the skin’s permeability to tritiated water in two different study phases and the permeability was found to be 2.04 ± 0.95 mg/cm^2/hr. The damage ratio was calculated from the rates of tritiated water before and after di (2-ethylhexyl) terephthalate exposure and was similar to the negative control indicating that exposure to di (2-ethylhexyl) terephthalate for 29 hours does not significantly damage the skin. The total recovery of the test chemical was measured by determining the percentage of radiolabelled di (2-ethylhexyl) terephthalate (^14C-labelled) remaining in the test system components from each test cell with a mean recovery of 104 ± 6%. The data from the present study allows for an estimation of 1.06 µg/kg di (2-ethylhexyl) terephthalate uptake following a continuous 1 hour dermal exposure in an area of skin equivalent to both hands (approximately 720 cm^2, 70-kg person).