Human-based testing is the way forward for product validation

Introduction

In the personal care industry, developing products that target or affect the skin, scalp, vaginal, and oral microbiome requires robust scientific validation that meets both biological and regulatory standards. While in vitro (laboratory) testing provides valuable preliminary data, they cannot fully replicate the complex interactions occurring in the human body. Clinical testing in human subjects on the other hand captures the dynamic interactions between microbial communities and the host in their natural environment. Increasingly, multi-omic approaches, which combine genomic, transcriptomic, proteomic, and metabolomic data, are being used in these human studies to deliver a far more comprehensive understanding of how products influence both microbes and host biology simultaneously.

Complexity of the Human Skin

Human skin is a highly complex, multi-layered organ whose structure is essential for both barrier function and supporting a balanced microbiome. The epidermis, dermis, and hypodermis create distinct microenvironments with varying oxygen levels, nutrients, and physicochemical properties, while specialized structures, such as, hair follicles, sebaceous and sweat glands, and apocrine glands, provide unique habitats that shape microbial communities (Kolarsick et al., 2011). Host-microbe interactions are dynamic, with keratinocytes and immune cells continuously communicating with microbes to maintain equilibrium. This means that product effects cannot be separated from host responses, because microbial behavior depends on this three-dimensional architecture. Simplified in vitro systems often fail to predict real-world outcomes. 

To further complicate this interaction, skin biology and the microbiome influence each other at multiple molecular layers. Multi-omic analysis enables researchers to capture these layered responses by examining not only changes in microbial composition, but also shifts in gene expression, protein activity, and metabolite production in both the host and the microbiome. This depth of information is only achievable in vivo, where the full biological context, and environmental exposure remains intact.

Understanding in vitro testing 

In vitro testing involves experiments conducted in controlled laboratory environments. Common methods include,

• Co-culture systems: Culturing multiple microbial species together to study a product's impact on interspecies interactions and community dynamics (Rose et al., 2024). However this comes with several limitations including, the inability to include the complete microbial ecosystem, as it is difficult to control the ratios between bacterial strains (Shishkov et al., 2024). Another limitation is that standard liquid media do not accurately mimic the structured, biofilm-rich environment of the skin. Non-adherent bacteria can be lost during media changes, and competitive dynamics among strains can lead to overgrowth of certain species, reducing the physiological relevance of the model (Shishkov et al., 2024).

• 2D and Scaffold-Based 3D Skin Models: 2D skin models consist of keratinocytes grown as monolayers, sometimes co-cultured with fibroblasts, and are widely used for rapid, inexpensive screening of cytotoxicity, cellular uptake, and basic responses to ingredients, however, they lack the 3D architecture and barrier function of real skin. Scaffold-based 3D skin models, such as reconstructed human epidermis (RHE) and full-thickness human skin equivalents (FTHSE), use natural, synthetic, or decellularized scaffolds to mimic the extracellular matrix and support multilayered cell growth, differentiation, and fibroblast-keratinocyte interactions. These models better replicate the stratum corneum, barrier function, and cellular crosstalk, allowing testing of product effects on toxicity, irritation, penetration, and even disease-like conditions, nonetheless, they still lack appendages, full vascularization, and all native cell populations, limiting their ability to fully predict skin responses, particularly for microbiome interactions (Galvan, Pellicciari & Calderan, 2024).

While these methods offer controlled conditions for initial screening, and are continuously evolving, they cannot still replicate the complex microenvironment of human tissues that personal care products actually encounter during use.

Limitations of in vitro testing

While 3D skin models improve physiological relevance compared with 2D cultures, they still have important limitations for studying the human skin microbiome. Most models lack skin appendages such as hair follicles and sebaceous glands, making it difficult to culture anaerobic bacteria like Cutibacterium or maintain a complete microbial community. Simple synthetic membranes or 2D models cannot support symbiotic host-microbe interactions and are limited to very short-term experiments. Even simplified 3D models that recreate only the stratum corneum can maintain microbial populations for about a week but do not replicate the full tissue complexity needed for barrier function or product testing (Galvan, Pellicciari & Calderan, 2024). Additionally, environmental factors such as moisture, pH, sebum production, and exposure to cosmetic or hygiene products vary widely among individuals and are challenging to model in vitro. So while in vitro testing is essential for initial screening and mechanistic studies, its predictive value for real-world human outcomes is limited. 

What do the regulatory bodies say

According to the Cosmetic Supervision and Administration Regulation (CSAR) and the Standards for Cosmetic Efficacy Claim Evaluation, “claims of cosmetics need to be supported by scientific evidence”, and in vitro data alone may no longer be sufficient for substantiating personal care product claims (Ferreira et al., 2022). 

Similarly, the UK Advertising Standards Authority (ASA) acknowledges that while in vitro proof-of-concept studies are valuable in research, they should generally be supported by relevant human data, ideally reflecting the target population (CAP News, 2021). For “new” or “breakthrough” objective claims, the ASA and the Committee of Advertising Practice (CAP) require more stringent evidence, typically including at least one well-controlled experimental human study, often complemented by observational data (AdviceOnline, 2025).

Consistent with these standards, QVC also mandates that all product claims regarding safety and efficacy, whether made on-air or off-air, must be substantiated with adequate documentation, which may include third-party clinical testing where appropriate (Quality Assurance Overview, 2018).

Together these regulatory bodies highlight a clear expectation across markets, that claims must be grounded with credible, scientifically validated evidence. Through robust human studies and transparent documentation, brands are expected to demonstrate that their products perform safely and effectively as advertised.

Understanding Clinical testing

Clinical testing for personal care products involves evaluating formulations directly on human volunteers under conditions that simulate real-world use. Standard methodologies include,

• Randomized Controlled Trials (RCTs): Gold-standard studies where participants are randomly assigned to treatment or control groups to minimize bias (Braga et al., 2024).

• Skin Sampling Techniques: Methods such as skin swabs, tape strips, and punch biopsies are employed to collect microbial samples from various skin sites (Santiago-Rodriguez et al., 2023). 

• Microbiome Analysis: 16S rRNA gene sequencing, whole-genome shotgun metagenomics, and transcriptomic profiling to characterize microbial community changes (Santiago-Rodriguez et al., 2023).

• Biophysical Measurements: Non-invasive instrumentation to assess skin barrier function, hydration, sebum production, pH, and desquamation (Hwang et al., 2021).

• Perception Questionnaires: Standardized questionnaires capturing subjective experiences of relief, comfort, and product acceptability. They bridge the gap between biological data and user perception, ensuring that the observed microbial or biophysical data translate into meaningful benefits for consumers.

Clinical testing in humans captures the dynamic interactions between microbial communities and the host in their natural environment. By studying human participants, researchers can assess the full diversity of microbiomes that are absent in artificial culture systems. Moreover, clinical testing supports the development of personalized treatments by accounting for inter-individual variability in microbial composition, genetics, and lifestyle factors, which can significantly influence outcomes. Without clinical testing, laboratory findings, even those that appear promising in vitro, may fail to translate into real benefits for patients or consumers. 

In vitro models provide mechanistic understanding and cost-effective early screening, however, only well-designed clinical trials can deliver the comprehensive evidence necessary to substantiate product claims, ensure consumer safety, and demonstrate meaningful benefits reflective of the complex interactions in living humans. 

Moreover, human skin exhibits remarkable diversity across ethnicities, ages, genders, and body sites. Clinical testing captures this variability by enrolling participants representative of the target population, providing insights into how products perform across different skin types and conditions. This population-level understanding is essential for developing inclusive products that deliver consistent benefits to diverse consumer groups. 

Furthermore, clinical testing provides the foundation for consumer confidence and commercial success. Products supported by robust clinical evidence convey scientific credibility and a commitment to consumer well-being, setting them apart in an increasingly competitive marketplace. As consumers become more informed about skincare and microbiome science, there is a growing expectation for transparency and data-driven validation. Incorporating multi-omic analysis into clinical studies strengthens this further by demonstrating not only visible outcomes but also the underlying biological mechanisms, offering a deeper level of scientific clarity that resonates with both regulators and consumers. Therefore, it is essential that products are substantiated by rigorous scientific research and evaluated on real human skin to ensure both efficacy and trustworthiness.

About Sequential

Sequential helps simplify the entire process of generating strong scientific evidence for personal care products, by handling recruitment, sampling, analysis, and data reporting in one place, so that it runs faster and smoother. Our clinical testing approach shows how products perform in real-world conditions, across skin, scalp, vaginal, and oral sites, so brands can make confident decisions backed by high-quality microbial and molecular data (multi-omics). 

A proprietary non-invasive sampling method ensures consistent, rich biological material with minimal irritation for participants, supporting smoother studies and stronger datasets.

Each study is tailored to match the intended consumer population, supported by biophysical measurements and perception data to capture both measurable effects and user experience. This provides a complete dataset that demonstrates efficacy, and strengthens claim substantiation.

All data and results are presented in clear, detailed, easy-to-interpret reports, making it simple for anyone to understand the science, and translate the insights directly into formulation improvements, claims, and product strategy.

By handling all these processes under one roof, Sequential reduces complexity, shortens timelines, and provides brands with clear, actionable insights, making it easier to deliver products that are credible, effective, and trusted.

References 

AdviceOnline, 2025 June 23; Substantiation for health, beauty, and slimming claims. https://www.asa.org.uk/advice-online/substantiation-for-health-beauty-and-slimming-claims.html

Braga LH, Farrokhyar F, Dönmez Mİ, Nelson CP, Haid B, Herbst K, Garriboli M, Cascio S, Nieuwhof-Leppink A, Kaefer M, Bägli DJ, Kalfa N, Ching C, Fossum M, Harper L. Randomized controlled trials - The what, when, how and why. J Pediatr Urol. 2025 Apr;21(2):397-404. doi: 10.1016/j.jpurol.2024.11.021. Epub 2024 Dec 3. PMID: 39701869.

CAP News, 2021 Jan 28; Skin in the game - an update on microbiome claims for cosmetics. https://www.asa.org.uk/news/skin-in-the-game-an-update-on-microbiome-claims-for-cosmetics.html

Ferreira M, Matos A, Couras A, Marto J, Ribeiro H. Overview of Cosmetic Regulatory Frameworks around the World. Cosmetics. 2022; 9(4):72. https://doi.org/10.3390/cosmetics9040072

Galvan A, Pellicciari C, Calderan L. Recreating Human Skin In Vitro: Should the Microbiota Be Taken into Account? Int J Mol Sci. 2024 Jan 18;25(2):1165. doi: 10.3390/ijms25021165. PMID: 38256238; PMCID: PMC10816982.

Hwang BK, Lee S, Myoung J, Hwang SJ, Lim JM, Jeong ET, Park SG, Youn SH. Effect of the skincare product on facial skin microbial structure and biophysical parameters: A pilot study. Microbiologyopen. 2021 Oct;10(5):e1236. doi: 10.1002/mbo3.1236. PMID: 34713611; PMCID: PMC8494714.

Kolarsick, Paul A. J. BS; Kolarsick, Maria Ann MSN, ARHP-C; Goodwin, Carolyn APRN-BC, FNP. Anatomy and Physiology of the Skin. Journal of the Dermatology Nurses' Association 3(4):p 203-213, July 2011. | DOI: 10.1097/JDN.0b013e3182274a98

Quality Assurance Overview, 2018 July. https://www.qvcuk.com/content/dam/qvc_uk/pdf/vendor-relations/QVC%20UK%20Beauty%20Overview.pdf?srsltid=AfmBOor499tO1ighEImeZfonX-1M6ohWHkfnKQh_RC7k1buX5A91mI3-

Roso, A., Pecastaings, S., Cambos, S., Martin, R., Bauchet, L., Roubinet, B., ... & Garcia, C. (2024). An Innovative Model Based on Wild Type Bacteria Co-Culture to Identify Cosmetic Ingredients That Respect the Skin Microbiota. Journal of Cosmetic Science, 75(6).

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