Cosmetic product safety: Betting on the in vitro assays

Let the truth be told: we all love cosmetics! From the humble shampoo to the most extravagant eye shadow, cosmetics are a joy (and a necessity) in our daily lives and as we do love to pamper ourselves, we strive to provide the best for our bodies. Thankfully, scientists in the cosmetic industry also share this belief and try their best to discover novel ingredients, in order to improve an already existing product or introduce a completely new one to the market.

Of course, each and every ingredient (already existing or novel) that composes a cosmetic product undergoes exhaustive tests, which reveal the compounds’ potential, both in terms of stability and safety. As we all know, the safety of a product is the alpha and omega of every player in the cosmetic field, be that the manufacturer, the retailer or the consumer 1. No one wants their product to cause harm and, certainly, no one wants any harmful events to befall them.

Every cosmetic product and its ingredients are tested before being placed in the market.

But how much do we really know about these safety tests? Are they so important as to be governed by specific laws and regulations? Do companies need to invest their resources in order to constantly improve them? And finally, where do animals stand in this?

Two key points can help us find the answers to such questions: the EU Cosmetic Regulation and the in vitro methods.

Animal testing for cosmetic purposes was fully banned for the first time in Europe

with the introduction of the EU Cosmetic Legislation (1223/2009). Although whispers of abandoning animal tests were circulating from 1993, substantial steps were taken only after 2000, with the finality of the ban been placed in 2013 4. As a result, the cosmetic industry had to find alternative ways of testing its products and their ingredients, in order to comply with the new rules. This led to an increased focus on improving existing biochemical and cell-based assays, as well as inventing new methods, using the latest technological breakthroughs in computer science and skin engineering (like novel in silico models or 3D bio-printed human skin 5,6).

The case of in vitro assays is of particular interest as they constitute the main tool for evaluating the toxicity, and eventually the safety, of cosmetic ingredients. “In vitro” is translated from Latin as “in the glass”, meaning that these assays use biological components growing in a controlled environment in the laboratory 7.

Cells, in in vitro assays, are usually cultivated in well-plates, where the active substances to be tested are, later on, added.

The most common in vitro method is the cell culture system, be that the traditional two dimensional (2D) or the more advanced three dimensional (3D). Generally, cells, ideally isolated from human volunteers, are maintained and grown in a specific environment (nutrients, temperature, humidity). They may originate from various body sites (like the skin or the liver) and can quickly proliferate, forming a one-cell-type monolayer (2D cultures) or a complex, multicellular structure, on top of a supporting material (3D cultures) 7. While 2D cell cultures are a preferred tool for screening active ingredients in regards to their toxicity, 3D cultures offer a more complete image, in terms of structural complexity and biological interactions 8,9.

Picture A shows a skin cell culture (2D), while the next two pictures demonstrate a reconstructed (3D) skin equivalent (C) compared to normal human skin (B) 2,3

At this point, however, we should mention that not all procedures for safety evaluation of cosmetic ingredients are fully translated to non-animal testing. Even though in vitro methods have been established as the sole screening assays for a great number of endpoints, like dermal absorption and phototoxicity, in vivo follow-up tests are still considered a “safety net” by many, in aspects like carcinogenicity and genotoxicity 6,10.

We remain positive though, as more and more countries around the globe are making efforts to ban animal testing for cosmetics, while technological breakthroughs contribute to this goal, making it feasible in the years to come.



2 Pellevoisin, C. et al. SkinEthic™ RHE for in vitro evaluation of skin irritation of medical device extracts. Toxicology in Vitro 50, 418-425, doi: (2018).

3 Biggs, L. C. et al. Interferon regulatory factor 6 regulates keratinocyte migration. Journal of cell science 127, 2840, doi:10.1242/jcs.139246 (2014).

4 Comission, E. Ban on animal testing, <> (

5 Velasquillo, C., Galue, E., Rodriquez, L., Ibarra, C. & Ibarra-Ibarra, L. Skin 3D Bioprinting. Applications in Cosmetology. Journal of Cosmetics, Dermatological Sciences and Applications 03, 85-89, doi:10.4236/jcdsa.2013.31A012 (2013).

6 CosmeticsEurope. NON-ANIMAL APPROACHES TO SAFETY ASSESSMENT OF COSMETIC PRODUCTS, Cutting-Edge Science and Constant Innovation: The Keys to Success, <> (2017).

7 Srivastava, S. et al. in In Vitro Toxicology (eds Alok Dhawan & Seok Kwon) 21-43 (Academic Press, 2018).

8 Yan, W.-C. et al. 3D bioprinting of skin tissue: From pre-processing to final product evaluation. Advanced Drug Delivery Reviews 132, 270-295, doi: (2018).

9 Gebhardt, R. In vitro Screening of Plant Extracts and Phytopharmaceuticals:Novel Approaches for the Elucidation of Active Compounds and Their Mechanisms. Planta Med 66, 99-105, doi:10.1055/s-2000-11134 (2000).

10 Corvi, R. & Madia, F. In vitro genotoxicity testing-Can the performance be enhanced? Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 106, 600-608, doi:10.1016/j.fct.2016.08.024 (2017).

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