Sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2003-08-20 to 2003-10-04

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: non GLP but guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: COLIPA: Test Guidelines for In Vitro Assessment of Dermal Absorption and Percutaneous Penetration of Cosmetic Ingredients (1999).

Deviations:

no

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate

EC Number:

403-080-9

EC Name:

Sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate

Cas Number:

92484-48-5

Molecular formula:

C16 H17 N3 O4 S .Na

IUPAC Name:

sodium 3-(2H-1,2,3-benzotriazol-2-yl)-5-(butan-2-yl)-4-hydroxybenzene-1-sulfonate

Constituent 2

Details on test material:

- Name of test material: sodium 3(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonatesodium (FAT 75'309)
Analytical purity: formulations of 0.1 - 0.5 % test substance (nominal conc.) 0.04% - 0.214% (measured conc.)

Radiolabelling:

no

Test animals

Species:

human

Strain:

other: not applicable

Sex:

male/female

Details on test animals or test system and environmental conditions:

Percutaneous absorption was determined in vitro with human skin using the finite dose technique. Human cadaver trunk skin without obvious signs of skin disease, obtained within 24 hours of death, was used in this study. Following collection the skin is was sealed in a water-impermeable plastic bag, and stored at -20°C. Prior to the experiment, skin was removed from the bag, placed in about 37° C water for five minutes, and then cut into sections large enough to fit on 0.8 cm2 Franz Cells (Crown Glass Co., Somerville, NJ). The dermal chamber was filled with phosphate-buffered saline, pH 7.4 ± 0.1, and the epidermal chamber (chimney) left open to ambient laboratory conditions. All cells were mounted in a diffusion apparatus in which the dermal bathing solution was stirred magnetically at approximately 600 RPM and its skin surface temperature maintained at 33° ± 1 °C.
To assure the integrity of each skin section, its permeability to tritiated water was determined before application of the test products. Following a brief (0.5-1 hour) equilibrium period, 3H2O (NEN, Boston, MA, sp. Act. about 0.5 µCi/mL) was layered across the top of the skin by dropper so that the entire exposed surface was covered (approximately 100-150 µL). After 5 minutes the 3H2O aqueous layer was removed. At 30 minutes the receptor solution was collected and analyzed for radioactive content by liquid scintillation counting. Skin specimens in which absorption of 3H2O was less than 1.25 µL-equ were considered acceptable.

Administration / exposure

Type of coverage:

other: not applicable

Vehicle:

other: different formulation used (see below)

Duration of exposure:

30 min (formulation A), 48 h (formulation B,C)

Doses:

The test substance was measured in three different formluations (see below in any other information field).

No. of animals per group:

not applicable

Control animals:

yes

Details on in vitro test system (if applicable):

Dosing and Sample
Just prior to dosing with the test product, the chamber chimney was removed from the Franz Cell to allow full access to the epidermal surface of the skin. All formulations were then applied to the skin sections using a positive displacement pipette set to deliver a target dose of 0.8 mg/0.8 cm2 for formulations "A" and "B", and 1.6 mg/0.8 cm^ for formulation "C". Each formulation was applied to six replicate skin sections per donor. The dose was spread throughout the surface with the Teflon tip of the pipette. Five to ten minutes after application the chimney portion of the Franz Cell was replaced. Spare chambers were not dosed, but sampled, to evaluate for interfering substances during the analytical analysis.
At 30 minutes post dose, all skin sections dosed with formulation "A" were surface washed with de-ionized water. Skin sections dosed with formulations "B" and "C" were left with the applied dose remaining on the surface of the skin throughout the 48 hour study period. At pre-selected time intervals after test formulation application (2, 4, 8, 12, 24, 32, and 48) the receptor solution was removed in its entirety replaced with fresh solution (modified phosphate buffered saline), and an aliquot taken for analysis. Following the last receptor solution collection, the skin sections were surface washed twice with 0.5 mL volumes of deionized water. Following the 48 hour dose period, and surface washes, the skin was removed from each chamber, tape stripped 20 times to collect the stratum corneum (10 strips/vial), separated into epidermis and dermis, and allowed to extract for no less than 24 hours.

Analytical Methods
A Hewlett-Packard 1090 Series II system, with a diode-array ultraviolet detector was used throughout. The solvent system was an isocratic mixture consisting of 57% Acetonitrile, 32% Water, 5% Isopropanol, 10% 0.1 M potassium phosphate buffer (pH=8.0), and 0.2% Tetrabutylammoniumhydrogen sulphate. Solvent flow was maintained at 0.5 mL/min at room temperature through a reverse-phase, 3µ, Luna column (Phenomenex, 100 x 4.6 mm). Twenty microliter samples were injected. Eluting peaks were monitored at 320 nm (4 nm bandwidth) reference to 450 nm (80 nm bandwidth). Quantification was accomplished using an external standard curve (peak area) prepared from the Sponsor provided standard.

Pilot Study Details
A pilot study was conducted with Deo Formulation "A" to assess for measurable levels of drug in the receptor solution and sample collection time points. The pilot study was conducted in triplicate, from one cadaver skin donor (MA102202). The applied dose was 5 µI/0.8 cm2 and samples were collected at 2, 4, 8, 12, 24, 32, and 48 hours post dose.

Pivotal Study Details
This study was designed to assess the percutaneous absorption kinetics of FAT 75'309 from three Sponsor provided formulations. Three cadaver skin donors were prepared and mounted onto chambers. Each formulation was applied to six replicate sections from each donor where possible. Receptor solution samples were collected, in general, at 2, 4, 8, 12, 24, 32, and 48 hours after dosing. The receptor solution used throughout was 1:10 PBS. Differences between formulations were evaluated for statistical differences using the Student's t-Test.

Results and discussion

Signs and symptoms of toxicity:

not specified

Dermal irritation:

not specified

Total recovery:

Percutaneous absorption for the test substance was very low. The initial data calculations from the pivotal study demonstrated unusually low mass balance. To help address this issue, the content of each formulation were determined by HPLC by the Investigator. The results show that the measured content was less than 50% of labeled content. The mass balance results calculated in this study are based on the Investigator's analytical measured of the test formulation and not on the nominal values..

Percutaneous absorption

Percutaneous absorption 1

Dose:

0.1 %

Parameter:

percentage

Absorption:

11.735 %

Remarks on result:

other: 48 h

Remarks:

Formulation A

Percutaneous absorption 2

Dose:

0.5 %

Parameter:

percentage

Absorption:

1.887 %

Remarks on result:

other: 48 h

Remarks:

Formulation B

Percutaneous absorption 3

Dose:

0.5 %

Parameter:

percentage

Absorption:

0.483 %

Remarks on result:

other: 48 h

Remarks:

Formulation C

Any other information on results incl. tables

Total Absorption and Mass Balance Results Across Human Donors: Pivotal Study

Percutaneous Absorption of test substance through Human Cadaver Skin over 48 hours from a Single Application (n=3). Mean ± SE as Percent of Applied Dose and Total Mass (µg).

 

prarameter	Formulation A 0.1% Deo	Formulation B 0.5% Deo	Formulation C 0.5% Emulsion
Total Absorption (µg)	0.037 ± 0.035	0.029 ± 0.029	0.016 ± 0.016
Total Absorption (%)	11.735 ± 11.254	1.887 ± 1.887	0.483 ± 0.483
Tape Strip (%)	5.402 ± 1.405	5.119 ± 1.531	2.233 ± 0.671
Dermis (%)	0 ± 0	0 ± 0	0 ± 0
Epidermis (%)	0 ± 0	3.344 ± 1.770	2.126 ± 0.727
Surface(%)	124.664 ± 3.284	100.297 ± 3.213	80.663 ± 6.561
Total Recovery (%)	141.801 ± 14.816	110.648 ± 5.398	85.506 ± 5.402

Rate of Penetration Across Human Donors

Percutaneous Absorption of test substance (as measured in the receptor solution) through Human Cadaver Skin over 48 hours from a Single Application (n=3). Mean ± SE as µg/cm2/hr.

 

Sample #	Formulation A 0.1% Deo	Formulation B 0.5% Deo	Formulation C 0.5% Emulsion	Mid-Time (hr)*
1	0.001 ± 0.001	0.000 ± 0.000	0.000 ± 0.000	1
2	0.001 ± 0.001	0.000 ± 0.000	0.000 ± 0.000	3
3	0.001 ± 0.001	0.001 ± 0.001	0.000 ± 0.000	6
4	0.001 ± 0.001	0.001 ± 0.001	0.001 ± 0.001	10
5	0.002 ± 0.002	0.002 ± 0.002	0.001 ± 0.001	18
6	0.001 ± 0.001	0.001 ± 0.001	0.000 ± 0.000	28
7	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000	40

* Time = Mid-time of sample collection duration.

The data show that very little (< 100 ng) of the test substance (as measured in the receptor solution) penetrated the skin over the 48 hour test period. The lower limit of detection for this compound was approximately 50 ng/mL(neat standard), which is approximately 1.5 ng per receptor solution sample (the average recovery from concentrated receptor solution samples was found to be 100.8%).

The data also indicate that whether the test formulation is in contact with the skin for 30 minutes (Formulation "A") or 48 hours (Formulations "B" and "C"), essential the same amount of the test substance penetrates into and through the skin. There was no statistical difference (Student's t-Test) between the formulations in their ability to delivery the test substance into or through the skin (p>0.5). The surface wash content is very high, indicating that most of the vast majority of the applied dose remains on surface of the skin. Further, there is no evidence of dermal accumulation of test compound that may have penetrated the stratum corneum.

Overall, the mass balance from the three donors was found to be approximately 85% - 142% of the applied dose.

Applicant's summary and conclusion

Conclusions:

The data show that very little (< 100 ng) of sodium 3(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonatesodium penetrated the skin over the 48 hour test period.

Executive summary:

A percutaneous absorption pharmacokinetics study of sodium 3(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate was performed with three formulations according to COLIPA guidelines for in vitro assessment of dermal absorption and percutanous penetration of cosmetic ingredients (COLIPA, 1999). Absorption was measured in human cadaver skin, in vitro, using the finite dose technique and Franz Diffusion Cells. The products were tested on a minimum of six replicate sections, when possible, from three different cadaver skin donors for the percutaneous absorption of the test substance over a 48-hour dose period. At pre-selected times after dosing, the dermal receptor solution was removed in its entirety, replaced with fresh receptor solution, and an aliquot saved for subsequent analysis. The samples were analyzed for the test substance using high performance liquid chromatography (HPLC).

The data show that very little (< 100 ng) of the test substance penetrated the skin over the 48 hour test period. The lower limit of detection for this compound was approximately 50 ng/mL(neat standard), which is approximately 1.5 ng per receptor solution sample (the average recovery from concentrated receiver solution samples was found to be 100.8%).

The data also indicate that whether the test formulation is in contact with the skin for 30 minutes (Formulation "A") or 48 hours (Formulations "B" and "C"), essential the same amount of the test substance penetrates into and through the skin. There was no statistical difference (Student's t-Test) between the formulations in their ability to delivery the test substance into or through the skin (p>0.5). The surface wash content is very high, indicating that most of the vast majority of the applied dose remains on surface of the skin. Further, there is no evidence of dermal accumulation of test compound that may have penetrated the stratum corneum.

Overall, the mass balance from the three donors was found to be approximately 85% - 142% of the applied dose. Since no information was provided about the formulation matrix and stability of this compound, it is not known if it is unstable, or degrades in the presence of skin, in formulation, in the presence of UV light, or as a neat standard.