Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 3 September 2001 to 12 October 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

according to OECD Environmental Health and Safety Publications, Series on Testing and Assessment No. 28. Draft guidance document for the conduct of skin absorption studies

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

EC Number:

224-618-7

EC Name:

Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

Cas Number:

4430-18-6

Molecular formula:

C21H15NO6S.Na

IUPAC Name:

sodium 2-[(4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]-5-methylbenzenesulfonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by l'Oreal, Batch No. 437/3, formulation used : batch No. 473219
- Expiration date of the lot/batch: not specified
- Purity test date: 4 October 2001

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, in absence of light and humidity
- Stability under test conditions: stable when stored but no details in test conditions
- Solubility and stability of the test substance in the solvent/vehicle: the test item was soluble at 100% in receptor fluid.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Formulated test item was provided by l'Oreal

Radiolabelling:

no

Administration / exposure

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Remarks:

Formulation (components not described) containing 0.2% of test item

Duration of exposure:

24 hours

Doses:

- Nominal doses: 0.2% in formulation
- Actual doses: 0.11%
- Actual doses calculated as follows: nominal dose x purity = actual dose
- Dose volume: 20mg/cm2
- Rationale for dose selection: no justification

No. of animals per group:

9 cells were used

Control animals:

no

Details on study design:

APPLICATION OF DOSE:

VEHICLE
- Amount(s) applied (volume or weight with unit): 20 mg/cm2
- Concentration (if solution): 0.2% of test item in formulation
- Lot/batch no. (if required): 473219
- Purity: not specified

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: after 30 minutes of contact with distilled water, 2ml sodium lauryl sulfate aqueous solution, 3 cotton swabs to dry the skin
- Time after start of exposure: 30 minutes

SAMPLE PREPARATION
All skin biopsies were visually checked to ensure they were unaltered after clinical removal. After thawing, skin samples were dermatomed and cut into pieces of 2 cm X 2 cm ; their thickness was 380 ± 25 µm . Each skin sample was mounted in a diffusion cell, with an area of 2 cm2 and a receiver compartment of about 7 ml volume.
Skin samples were kept between two silicone membranes in order to maintain the cohesion with the cell compartments. The epidermal side of skin samples was exposed to the ambient conditions of the laboratory environment while the dermal side was in contact with the receptor fluid.

ANALYSIS
- Method type(s) for identification HPLC-MS-MS
- Validation of analytical procedure: validated by l'Oreal reported study
- Limits of detection and quantification: at 10 and 20 ng.mL in NaCI.

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: esthetic surgery, no more details
- Ethical approval if human skin: not specified
- Type of skin: human skin
- Preparative technique: All skin biopsies were visually checked to ensure they were unaltered after clinical removal. After thawing, skin samples were dermatomed and cut into pieces of 2 cm X 2 cm ; their thickness was 380 ± 25 µm. Each skin sample was mounted in a diffusion cell, with an area of 2 cm2 and a receiver compartment of about 7 ml volume.
Skin samples were kept between two silicone membranes in order to maintain the cohesion with the cell compartments. The epidermal side of skin samples was exposed to the ambient conditions of the laboratory environment while the dermal side was in contact with the receptor fluid.
- Thickness of skin (in mm): 0.380 ± 0.025 µm
- Membrane integrity check: Approximately one hour after the skin was mounted in the diffusion cell, and just before the application of the formulation, the integrity of each skin sample was checked by measuring Trans Epidermal Water Loss (TEWL).
- Storage conditions: -18 deg Celsius
- Justification of species, anatomical site and preparative technique: no justification

PRINCIPLES OF ASSAY
- Diffusion cell: diffusion cell, with an area of 2 cm2 and a receiver compartment of about 7 ml volume.
- Receptor fluid: The receptor fluid used was NaCI 0.9% solution. The receptor fluid was continuously stirred by a small Teflon-covered stirring bar. Skin surface temperature was 31.5 ± 0.3°C ; it was maintained by thermostating the receptor solution
- Solubility of test substance in receptor fluid: the test item was soluble at 100% in receptor fluid.
- Static system: yes
- Flow-through system: no
- Test temperature: 31.5±0.3 deg Celsius
- Humidity: no
- Occlusion: open
- Reference substance(s): not performed

Results and discussion

Absorption in different matrices:

Skin excess represented the majority of the recovery of ACID VIOLET 43 with 104.22 % of the applied dose. Visual observation showed no skin staining after the washing procedure 30 minutes after application. Very low amounts of ACID VIOLET 43 were found in the stratum corneum. i.e. below 0.33 ± 0.20 % of the applied dose, demonstrating the absence of storage effect in this compartment. The epidermis + dermis represented only 0.23 ± 0.28 % of the applied dose demonstrating the very low absorption of this compound. No test compound was found in the receptor fluid, which confirms of very low absorption of ACID VIOLET 43. The concentration of ACID VIOLET 43 in the receptor fluid was below the detection limit for the 8 diffusion cells. Total skin + receptor fluid (SC + epidermis + dermis + RF) represented 0.56 ± 0.46 % of the applied dose. The absorbed amount (epidermis + dermis + RF) corresponded to 0.23 ± 0.28 % of the applied dose. Physicochemichal properties of ACID VIOLET 43, an hydrophilic compound (Log Poct/H2o = -0.12) with a rather high molecular weight (MW = 432.41) may explain the low absorbed amount obtained.

Total recovery:

Total recovery: Skin excess represented the majority of the recovery of ACID VIOLET 43 with 104.22 % of the applied dose.
- Recovery of applied dose acceptable: 80%
- Results adjusted for incomplete recovery of the applied dose: No
- Limit of detection (LOD): - at 1, 2.5 and 5 ng/ml in methanol/ammonium acetate buffer (50/50, v/v).; - at 10 and 20 ng/ml in NaCI.
- Quantification of values below LOD or LOQ: no quantification, expressed as "Below detection limit"

Any other information on results incl. tables

Table 1 :Summaryof theresults

	Formulation 473219 (n = 8)
Skin excess µg/cm2 % of the applied dose	26.42 ± 2.50 104.22 ±3. 19
Stratum corneum (SC) µg/cm2 % of the applied dose	0.08 ± 0.05 0.33 ± 0.20
Epidermis + dermis µg/cm2 % of the applied dose	0.07 ± 0.06 0.28 ± 0.28
Receptor fluid (RF) ug/cm2 % of the applied dose	0.04±0.00 0.25±0.07
Total recovery % of the applied dose	105.12 ±3.06
Total skin + receptor fluid µg/cm2 % of the applied dose	0.20±0.11 0.86 ± 0.46
Absorbed amount (epidermis, dermis and receptor fluid) µg/cm2 % of the applied dose	0.11±0.11 0.53 ± 0.33

 

Applicant's summary and conclusion

Conclusions:

Under experimental conditions of this study, most of the test substance applied on the skin surface was removed with the washing procedure (about 104 % of the applied dose, in mean), and the total recovery rate was about 105 %. No test item Acid Violet 43 was measured in the receptor fluid. The mean absorbed amounts of test substance were estimated as follows: 0.11 ± 0.06 μg active dye/cm2 (0.53 ± 0.33 % of the applied dose). The no diffusion of the test item in receptor fluid showed a very low absoprtion of the test item.

Executive summary:

In this GLP compliant in vitro study,the test item Ext. D&C Violet 2 was assessed for its potential to permeate human skin samples.

Human skin samples were obtained from four female donors subjected to plastic surgery. Skin samples (380 ± 25 μm in thickness) were dermatomed and mounted in diffusion cells, using 0.9% NaCl in water as a receptor fluid. Skin integrity was checked before application of the formulation by measuring Trans Epidermal Water Loss. After exposure period, the remaining formulation on the skin surface was removed using a standardized washing procedure. Twenty four hours after application, the percutaneous absorption of Jarocol Violet 43 was estimated by measuring its concentration by LC/MS/MS in the following compartments: skin excess, stratum corneum (isolated by tape strippings), epidermis + dermis and receptor fluid. The no diffusion of the test item in receptor fluid showed a very low absoprtion of the test item.

Most of the test substance applied on the skin surface was removed with the washing procedure (about 104 % of the applied dose, in mean), and the total recovery rate was about 105 %. No test item Acid Violet 43 was measured in the receptor fluid. The mean absorbed amounts of test substance were estimated as follows (sum of amounts measured in epidermis, dermis and receptor fluid when assuming concentrations at the detection limit in the receptor fluid of 40 ng): 0.11 ± 0.06 μg active dye/cm2 (0.53 ± 0.33 % of the applied dose). The no diffusion of the test item in receptor fluid showed a very low absorption of the test item.