Skin absorption – OECD 428

The skin absorption test according to OECD guideline 428 is an internationally recognized in vitro method for assessing the amount of a substance that crosses the human skin barrier. This standardized test is essential for determining potential systemic exposure related to the use of cosmetic, plant protection, chemical, or biocidal products. The results obtained are used in regulatory dossiers (cosmetics, REACH, biocides), safety assessments, and ingredient safety studies.

OECD 428 Test Objectives:
The test allows us to:
• quantify the transdermal penetration of a substance applied to the skin
• measure its presence in the receiving fluid over time
• study the cutaneous distribution: surface, stratum corneum, epidermis, dermis, receiving fluid
• evaluate the absorption kinetics and the transdermal flow rate
• determine the fraction not absorbed or retained in the cutaneous layers
• document the parameters necessary for risk assessments (DIP, MoS, systemic exposure)

This is the most commonly used test to complete a regulatory dossier when a toxicological assessment requires experimental data on skin penetration.

Principle of the OECD 428 Method:
The method uses Franz or flow-through diffusion cells. An excised skin (preferably human, porcine as an alternative) separates:
• a donor chamber containing the formulation being tested
• a receiving chamber filled with a receiving fluid adapted to the solubility of the substance

The test generally includes:
• preparation and verification of skin integrity (TEWL, electrical resistance)
• application of the substance or formulation to be tested
• exposure for a defined duration (usually 24 h)
• collection of the receptor fluid at regular intervals
• extraction and quantification in the different skin layers
• analysis of any metabolites

The analysis is then carried out by LC-MS/MS, GC-MS, HPLC-UV or radioactivity depending on the nature of the substance.

Matrices analyzed:
OECD 428 applies to a wide variety of products intended to come into contact with human skin:
• cosmetic ingredients (active ingredients, preservatives, perfumes, UV filters, excipients)
• finished cosmetic formulations (creams, gels, lotions, serums, balms, oils)
• industrial chemicals likely to come into contact with skin
• biocidal or phytosanitary substances
• technical residues or contaminants in complex formulations
• raw materials and pure or diluted solutions

Industrial and regulatory applications
: OECD 428 testing is required or strongly recommended in the following contexts:
• cosmetic ingredient dossiers (Safety Report, Product Information File)
• REACH dossiers (registration, exposure assessment)
• product development (safety and efficacy)
• validation of natural or synthetic ingredients
• estimation of skin bioavailability
• occupational risk assessment
• evaluation of biocides coming into contact with human skin

It provides essential parameters such as absorbed dose, flow rate, total absorption and skin distribution, which are then used by toxicologists to calculate the safety margin.

Associated analytical techniques
: Depending on the substance, quantifications are performed by:
• LC-MS/MS
• GC-MS
• HPLC-UV
• scintillation (for radiolabeled substances)
• methods specific to the metabolites formed

Laboratories adapt the receiving fluid, the extraction method and the analytical sensitivity according to the physico-chemical properties of the molecule.

Recommended additional analyses:
For cosmetic or regulatory dossiers, the following tests are often combined:
• in vitro phototoxicity tests
• cytotoxicity or irritation tests

YesWeLab Service:
At YesWeLab , we collaborate with a rigorously selected network of laboratories specializing in OECD testing, in vitro toxicology, and regulatory analyses. Most are certified or accredited (ISO 17025, GLP, COFRAC).
We direct your request to the most suitable laboratory based on the substance to be tested, the matrix, the required analytical sensitivity, the applicable regulatory framework, and the desired turnaround time.
Our scientific team supports you in preparing your study, defining experimental parameters, interpreting results, and ensuring the security of your regulatory documentation. Since 2020, numerous industrial companies have entrusted us with their OECD testing through our digital platform.