Registration Dossier

Toxicological information

Dermal absorption

Currently viewing:

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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
red powder
Batch # : 6718 Fass 10/20
Specific details on test material used for the study:
Hydroxyethyl-2-nitro-p-toluidine (B075) tested at a concentration of 1 % in a commercial hair dye formulation.
Radiolabelling:
no

Test animals

Species:
other: human
Sex:
male/female
Details on test animals and environmental conditions:
Full-thickness human skin samples (5 abdomen and 1 breast) were obtained from six patients aged 30 to 63 years old. The samples were obtained from patients attending the NHS Lothian, St Johns Hospital, Livingston, UK. All patients gave informed consent prior to undergoing surgery, for their excised skin to be used for scientific purposes. The skin was transferred to Charles River on ice, where it was cleaned of subcutaneous fat and connective tissue using a scalpel blade. The skin samples were washed in cold running water and dried using tissue paper. The skin samples were then cut into smaller pieces (where appropriate), wrapped in aluminium foil and placed into self sealing plastic bags. All skin samples were stored at ca -20°C until used in the study. The age and sex of the donor and site from which the skin was taken, were recorded centrally and in the study records.

Administration / exposure

Type of coverage:
open
Vehicle:
other:
Duration of exposure:
Receptor fluid was collected in 30 min fractions from 0 to 1 h post dose and hourly fractions
from 1 to 6 h post dose and then in 2 hourly fractions from 6 to 72 h post dose.
Doses:
Non-Oxidative Conditions test Preparation 1 (1%, w/w)
Oxidative Conditions Test Preparation 2 (1%, w/w)

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not specified
Total recovery:
Test Preparation 1 96.85%
Test Preparation 2: 98.32%
Percutaneous absorptionopen allclose all
Time point:
72 h
Dose:
Test Preparation 1 (1%, w/w)
Parameter:
percentage
Absorption:
3.08 %
Time point:
72 h
Dose:
Test Preparation 2 (1%, w/w)
Parameter:
percentage
Absorption:
2.14 %

Applicant's summary and conclusion

Conclusions:
In conclusion, following topical application of B075 in hair dye Test Preparations 1 and 2 (both 1%, w/w) to human skin in vitro, at 72 h post dose, the absorbed dose of B075 or the amount that had penetrated through the skin into the receptor fluid, was 3.08 μg/cm2 and 3.96 μg/cm2, respectively. The epidermis contained 0.01 μg/cm2 and 0.01 μg/cm2, respectively. The dermis contained 0.03 μg/cm2 and 0.08 μg/cm2, respectively.
Executive summary:

Hydroxyethyl-2-nitro-p-toluidine (B075, WR20883) is a hair dye molecule used in hair dye formulations. As part of the safety evaluation of B075, a study was required to assess the rate and extent of absorption of B075 following topical application of B075 to human skin, under in-use conditions. B075 was supplied ready-formulated in a typical hair dye formulation at 1% (w/w) for testing under non-oxidative conditions (Test Preparation 1). Additionally, for testing under oxidative conditions, a typical hair dye formulation containing 2% (w/w) B075 was mixed with peroxide developer (1:1, w/w, Test Preparation 2). Thereafter, the concentration of B075 in Test Preparation 2 applied to the skin was 1% (w/w). Split-thickness human skin membranes were mounted into flow-through diffusion cells. Receptor fluid, phosphate buffered saline containing sodium azide (ca 0.01%, w/v), was pumped underneath the skin at a flow rate of ca 1.5 mL/h. The skin surface temperature was maintained at ca 32°C throughout the experiment. A tritiated water barrier integrity test was performed and any human skin sample exhibiting absorption greater than 0.6% of the applied dose after 1 h was excluded from subsequent absorption measurements. Two test preparations containing B075 were applied, at an application rate of ca 20 mg test preparation/cm2, to human split-thickness skin membranes mounted into flow-through

diffusion cells in vitro. At 30 min post dose, the skin was washed with water, sodium dodecyl sulphate (SDS) solution (2% w/v) and water again. The skin was then dried with tissue paper swabs. The study was conducted according to the OECD principles of Good Laboratory Practice as set forth by the United Kingdom Department of Health and was performed following the SCCP, COLIPA and OECD Test Guideline No. 428 and the accompanying OECD Guidance Document No. 28. Absorption was assessed by collecting receptor fluid in 30 min fractions from 0 to 1 h post dose, then hourly fractions from 1 to 6 h post dose and then in 2-hourly fractions from 6 to 72 h post dose. At 72 h post dose, the skin surface was washed and dried in the same manner as described for the 30 min wash. The underside of the skin was rinsed with receptor fluid. The skin was then removed from the flow-through cells, dried and the skin divided into

exposed and unexposed skin (ie the area of skin under the cell flange). The stratum corneum from the exposed skin was removed by tape stripping. The exposed epidermis was separated from the dermis. All samples were analysed by LC-MS/MS. Following topical application of B075 in Test Preparation 1 (1%, w/w) to human skin in vitro, at 72 h post dose, the absorbed dose of B075 or the amount that had penetrated through the skin into the receptor fluid, was 1.55% (3.08 μg/cm2) of the applied dose. The epidermis contained <0.01% (0.01 μg/cm2) of the applied dose. The dermis contained 0.01% (0.03 μg/cm2) of the applied dose. For Test Preparation 1, the majority of the B075 was removed at 30 min post dose by washing the skin surface (94.83%). The mass balance was essentially complete (96.85%). These results are based on 9 samples of skin from 4 different donors Following topical application of B075 in Test Preparation 2 (1%, w/w) to human skin in vitro, at 72 h post dose, the absorbed dose of B075 or the amount that had penetrated through the skin into the receptor fluid, was 2.14% (3.96 μg/cm2) of the applied dose. The epidermis contained <0.01% (0.01 μg/cm2) of the applied dose. The dermis contained 0.04% (0.08 μg/cm2) of the applied dose. For Test Preparation 2, the majority of the B075 was removed at 30 min post dose by washing the skin surface (95.64%). The mass balance was essentially complete (98.32%). These results are based on 10 samples of skin from 4 different donors. In conclusion, following topical application of B075 in hair dye Test Preparations 1 and 2 (both 1%, w/w) to human skin in vitro, at 72 h post dose, the absorbed dose of B075 or the amount that had penetrated through the skin into the receptor fluid, was 3.08 μg/cm2 and 3.96 μg/cm2, respectively. The epidermis contained 0.01 μg/cm2 and 0.01 μg/cm2,

respectively. The dermis contained 0.03 μg/cm2 and 0.08 μg/cm2, respectively.