Some of these formulations are intended to treat and alleviate symptoms of irritation in partially compromised skin, while others, such as sunscreens or optically active powders, are intended to be applied to healthy intact skin and act merely on the very upper layer (no penetration needed) to protect from radiation or create an even-tone appearance, respectively. Some powders are incorporated to enhance feel.
When assessing potential route for penetration into the skin, the following can be outlined:
- Via the intercellular lipids;
- Via the coenocytes; and
- Via the follicular route.
Most compounds partitioning into the skin are known to intercalate via the intercellular lipid lamellae, however, penetration via keratinized corneocytes is most likely to occur too. Corneocytes are polyhedral, anucleate entities without cytoplasmic organelles, interlocked with each other and organized as vertical columns of 10-30 cells. They are embedded within a highly hydrophobic lipid matrix to form the stratum corneum. Filled with keratin, corneocytes exhibit limited capacity to absorb and retain water. Although some compounds such as ethanol and oleic acid were shown to penetrate corneocyte structure, a literature search reveals no clear evidence for penetration of solid particles into the corneocyte body.
Follicles are unevenly distributed throughout the body. The two extremes are probably the forehead, which is known to have the highest follicular content, and the soles of the feet, which contain no follicles. Studies are inconsistent with their reporting on the percentages of follicles distribution ranging from 0.1 to 1% or even 2%. Otbeg N. et al (2004) notes that the distribution as well as the size of the follicular opening differs between body sites. The follicle opening, according to this research, ranges between 70 and 170 micrometer.2
When you consider the follicle as a shunt route into the vasculated dermis, one may think that particles that are less than 70 microns in size can penetrate through the follicle into the blood. However, this is not accurate. The follicle is filled with sebum and the sebum flow is from the inside out. In addition, the follicle contains immune response mechanism protecting invasion of foreign substances and may respond in an inflammation reaction that will result in hyperkeratinization, will clog the pore and prevent further penetration. Such compounds, depending on the specifics of the reaction are called “comedogenic,” “acneogenic” or such that provoke “acne cosmetica.”
Some studies claim that depending on the size and other properties of nanoparticles and under specific conditions of penetration enhancement, they may generate a reservoir in the follicular shunt. For example, a study published in 2007 by Lademann J et al notes that nanoparticles with a 320nm diameter were found to be stored in the follicle for up to 10 days.3 It was theorized that the movement of the hairs may act as a pumping mechanism pushing the nanoparticles deep into the hair follicles. Other studies demonstrated that particulate substances penetrate preferentially into the hair follicles and that the penetration depth depends on the particle size or on the base formulation used (Patzelt A. et al 2009).4
To summarize this portion of the column, the potential routes for particle penetration, even at their nano-sized scale, are highly limited. If larger than a few nanometers, penetration via the intercellular lipids or through keratinized corneocytes may not be possible and while partition into the follicle is theoretically feasible, it is occurring under specific facilitating conditions or with the use of exceptional materials. Such discussion, of course, applies to healthy intact skin and may be very different when the skin is compromised for any reason.
The skin interaction of a compound in its molecular solubilized form differs profoundly from same chemistry when in its particulate solid form. In fact, solid particles, unless applied to a compromised skin or under unique conditions are not expected to penetrate the skin.
Similar assessment applies for large molecules such as polymers. Those are also anticipated to remain on the upper stratum corneum and not penetrate beyond it. The fact that the skin sheds one layer of corneocyets a day and will renew itself every month; adds yet a “dilution” safety factor to this calculation.
Considering the above analysis, when assessing safety of interaction of particles with skin, why do we need to test systemic endpoint such as cytotoxicity or genotoxicity? The scientific rationale for such can be explained by the following:
- Polymers and/or particles are not homogenous and may contain monomers and soluble impurities that potentially penetrate the skin. Since the cosmetic industry allows higher levels of impurities for its raw materials when compared to the pharmaceutical industry such long lasting cumulative exposure may be of concern since the impurities may penetrate beyond the stratum corneum.
- Compounds that are applied to skin do not have to physically penetrate it to impart a biological effect. We know for example that mere occlusion of the skin changes its biota population and leads to a cascade of biochemical events that can be detected in the living epidermis.
- Variety of cosmetic and personal care applications are aimed for compromised skin such as after shaving, after sun lotions, peels and after peels, and alike.
Adding to the challenge and complexity, because of the animal testing ban and the emergence of in vitro cell culture methodologies, testing of solid particles for variety endpoints is limited and needs to be designed with care. If a slurry of solids is introduced to cells in a culture such as keratinocytes or fibroblasts the cells will most likely die rapidly. Such phenomenon, while may be interpreted as a cytotoxicity reaction, is an artifact. This is because cells in a culture are much more vulnerable and sensitive when compared to an application to skin, and solid particles generate a toxic environment in the culture that cannot be paralleled to real life condition. If we test particles in such a manner, they will all be tested positive and will be wrongly classified as highly toxic. This would be a false positive result.
Following the above rationale that particles should be tested for their impurities and soluble components, a more relevant approach would be to suspend the powder in the culture media, vortex it, centrifuge it, remove the clear supernatant and introduce it to the culture in a variety of dilutions. This will cover the potential toxicity of present monomers and soluble impurities.
Another approach can be using the reconstructed live epidermis model such as Epiderm FT or fresh skin samples. Having a skin-like barrier, EpiDerm and similar models could be used to test for irritation resulting from particulate substances and sunscreen application. The EpiDerm Skin Irritation Protocol may be used for evaluating whether a particulate material has a potential to be a primary skin irritant. This protocol is validated by ECVAM (the European Centre for the Validation of Alternative Methods) and accepted by OECD (Organization for Economic Co-operation and Development) (TG 439) as a non-animal alternative to the rabbit Draize test. It is using 5% Sodium dodecyl sulfate (SDS) as a positive control and the endpoint of the study is cellular viability measured by MTT colorimetric assay. This assessment involves NAD(P)H-dependent cellular oxidoreductase enzymes that may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan, which has a purple color. According to this protocol, a compound that imparts mean tissue viability that is equal or less than 50% is considered an irritant while a compound that imparts more than 50% viability is considered a non-irritant.
It is also highly advisable to tailor and design the protocol in a manner that it covers potential safety concerns that are specific to the tested compound. Adding a tier of biomarkers tested at their protein level may be a wise approach if one wishes to gather an understanding on its potential effects upon chronic cumulative exposure.
In summary, particulate solid non-soluble compounds, unless extremely small, or applied to compromised skin, are not likely to penetrate healthy intact skin unless a means to compromise the barrier, such as permeation enhancers or others, are used.
Proximity to mucosal tissue (around the eyes or the mouth) should be addressed in a separate manner and considered if that is the intended use of the formulation. Study design in cell culture or skin model should be careful, so no false positive toxicity is generated due to sedimentation of the particles in the culture environment. Creative means and additional biological endpoints may be considered to assess potential known risk.
- Wertz P.W. and Hill J.R Molecular models of the intercellular lipid lamellae from epidermal stratum corneum. Biochimica et Biophysica Acta (BBA) - Biomembranes. Volume 1616, Issue 2, 13 October 2003, 121–126.
- Otberg N, Richter H, Schaefer H, Blume-Peytavi U, Sterry W, Lademann J. Variations of hair follicle size and distribution in different body sites. J Invest Dermatol. 2004 Jan;122(1):14-9.
- Lademann J1, Richter H, Teichmann A, Otberg N, Blume-Peytavi U, Luengo J, Weiss B, Schaefer UF, Lehr CM, Wepf R, Sterry W. Eur J Pharm Biopharm. 2007 May;66(2):159-64. Epub 2006.
- Alexa Patzelt Heike Richter Lars Dähne Peter Walden Karl-Heinz Wiesmüller, Ute Wank Wolfram Sterry and Jürgen Lademann Influence of the Vehicle on the Penetration of Particles into Hair Follicles Pharmaceutics 2011, 3, 307-314; doi:10.3390/pharmaceutics3020307.
- OECD http://www.oecd.org/chemicalsafety/testing/TG%20List%20EN%20Aug%202012.pdf
- Annex I EU Commission http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/uri=CELEX:32013D0674&qid=1395764232390&from=FR:PDF
Dr. Nava Dayan LLC
Nava Dayan Ph.D. is the owner of Dr. Nava Dayan L.L.C, a skin science and research consultancy and serving the pharmaceutical, cosmetic, and personal care industries. She has 25 years of experience in the skin care segment, and more than 150 publication credits.