How SPF is Measured (UVB Protection)

Based on FDA regulations, a product claiming SPF protection must be clinically tested on at least 10 (previously 20-25) human volunteer subjects. However, because such SPF measurement requires direct administration of UV radiation to the skin, there are efforts to replace the current in-vivo method with non-invasive methods that do not require human subjects. In recent years, several in-vitro tests have now been developed.

  • In-Vivo Methods: Typically, the SPF is measured on human volunteer subjects by applying 2 mg/cm2 of a sunscreen formula to an area of the mid-back, allowing the sunscreen to dry for 15 minutes, and administering a series of five increasing doses of UV radiation, simulating sunlight, to skin sites treated with the sunscreen. Another series of five increasing UV radiation doses is applied within a skin area without the sunscreen. After 16 to 24 hours, the irradiated skin sites are examined to determine the SPF. The SPF is the lowest dose of UV radiation that caused mild sunburn in the sunscreen-treated area divided by the lowest dose of UV radiation that caused mild sunburn in the area without sunscreen. The label SPF of a sunscreen formula is based on the average SPF for 10 volunteers
  • In-Vitro Methods: Current non-invasive methods for measurement of sunscreen SPF include in vitro measurements on artificial substrates that simulate the skin surface (e.g. polymethylmethacrylate or fused silica substrates) and computerized mathematical models based on the UV radiation absorbance spectra of active ingredients. However, there is no ISO Standard, regulatory agency protocol, or currently accepted method for in vitro measurements of SPF.

Free radicals are reactive oxygen species (ROS) reactive nitrogen species (RNS). ROS such as superoxide anions, hydrogen peroxide, hydroxyl radicals, singlet oxygen, and lipid peroxyl radicals are byproducts of aerobic life. Ultraviolet radiation (UVR) causes skin damage via two main mechanisms: directly, via absorption of energy by biomolecules and indirectly through increased production of ROS and reactive nitrogen species (RNS).he first line of defense against UVR is the presence of eumelanin, the dark pigment that acts as a scavenger of free radicals, shields genomic DNA and blocks the deeper penetration of UVR in the skin.

UVR is a potent generator of ROS in the skin and comprises three sub-categories based on the wavelength, UVA (315–400 nm), UVB (280–315 nm), and UVC (190-280 nm). UVA and UVB are the biologically relevant components of solar UVR; the extremely harmful UVC rays are blocked by the stratospheric ozone layer and therefore do not reach the Earth’s surface. UVB stimulates the production of superoxide anion radicals through the activation of NADPH oxidase and respiratory chain reactions. UVA produces singlet oxygen by an indirect mechanism that involves photosensitizing reactions with internal chromophores such as riboflavin and porphyrin in addition to superoxide anion radical through NADPH oxidase activation. The increased formation of ROS leads to the oxidation and damage of cellular molecules, resulting in altered function.

The carcinogenic effect of UVB is mainly due to its direct absorption by DNA and the generation of UV-signature mutations, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine pyrimidone dimers (6-4PP).

ROS are constantly generated in skin cells but are usually neutralized by a network of non-enzymatic (i.e., glutathione, ascorbic acid) and enzymatic antioxidants. Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase, and thioredoxin reductase (TRX) act in a coordinated manner to keep normal redox homeostasis.

In response to excessive presence of ROS a variety of transcription factors are activated including nuclear factor kappa B (NF-kB), activator protein 1 (AP-1) nuclear factor erythroid-derived 2-like 2 (Nrf2), and mitogen-activated protein kinase (MAPK) pathway. The transcription factor Nrf2 is a major transactivator of cytoprotective genes in response to oxidative stress and xenobiotic electrophiles. Nrf2 regulates the transcription of cytoprotective genes by binding to cis-acting elements, the antioxidant response elements (ARE) present in the enhancer regions of these genes. Activation of NF-kB and AP-1 contribute to the induction of matrix metalloproteinases (MMPs) by dermal fibroblasts that results in extracellular matrix (ECM) protein degradation and premature aging of the skin.

Oxidative stress plays a central role in both aging-associated and UV irradiation-initiated dermal extracellular matrix alterations. On irradiating the skin surface, UV light energy is readily absorbed by endogenous cellular chromophores such as NADH2/NADPH, tryptophan, riboflavin, or trans-urocanic acid (Hanson and Simon 1998). Chromophore excitation, which occurs in the presence of molecular oxygen, produces an array of oxidation products and radical oxygen species (ROS), including the highly reactive hydroxyl radical HO†