Human-derived in vitro systems for cosmetics testing- the status and the road ahead

Subhadra Dravida, PhD and Lakshman Varanasi, PhD    November 4, 2020

The science behind product safety testing in the cosmetics industry is undergoing change. A model sports beauty products.

Introduction/ background

The United States Code (Title 21) defines cosmetics as “Articles intended to be rubbed, poured, sprinkled or sprayed on, introduced into, or otherwise applied to the human body, or any parts thereof, for cleansing beautifying, promoting attractiveness, or altering the appearance”; cosmetics are also “articles intended for use as a component of any such articles”.1Cosmetics from natural sources have existed since the origin of human civilisation. In the last few decades, the field of cosmetics has emerged as a major scientific discipline at the interface of a wide range of basic, translational and medical sciences, including but not limited to chemistry, physics, dermatology, microbiology, biochemistry, toxicology, and bioengineering. The trend, of late, is the development of functional cosmetics that go beyond merely improving ones appearance; these are intended to also benefit the targeted organ (i.e. have a visceral effect): skin, the hair, or the teeth. With this functional approach, product synthesis has become more complex with raw materials that are not only more innovative and effective but also raise the bar for safety and bioavailability. Their provenance is also proportionately varied: from natural sources and from chemical or biological synthesis, the latter in genetically modified microorganisms. New composites such as microparticles, liposomes and nanoparticles have found their way into the field. Materials science is key to these formulations, with the incorporation of raw materials such as titanium dioxide for sun protection, polymers for water resistance, zinc oxide for colours and pigments, and silicone blends for a variety of uses. The last includes products used to enhance sun protection factor values. Novel active ingredients, surfactants, and novel vehiclesare some of the components that set newer cosmetic formulations apart from traditional ones. Most cosmetic products contain water and are therefore conducive to microbial growth. Antimicrobial additives or preservatives to complex multiphasic formulations round off the list of new (age) ingredients. Cosmetic products can be broadly classified into the categories of skin care (including repair and anti-ageing products), makeup (colour cosmetics), hair care, and fragrances.

The problem with cosmetic safety assessment/ toxicity screening.

The introduction of new substances and ingredients in the cosmetics manufacture process has strained the capabilities of safety testing. Conventional methods of safety assessment do not measure up, and animal testing is frowned upon by a public that is increasing in favour of animal’s rights. There are problems with toxicological testing, an umbrella paradigm for cosmetics safety assessment, the primary one being the limited use of the animal model; how closely, or how well, does the animal model reflect the human system? The animal model, like any model, is an approximation of the real thing, and the continued use of the lab animal (mice, rats, rabbits, guinea pigs, and so on) has come under scrutiny. There are other problems with existing testing systems, including the study design, in this case, the dosage for the test, the use of multiple end-points (both of which lead to a higher number of false-positive results), and the degree to which chemicals in general cause health problems (a very small percentage of chemicals are known to cause disease in humans).2 The current discussion will restrict itself to the first of the problem of the stated problems as the others are beyond the scope of this manuscript. The use of human-derived in vitro systems for cosmetics testing can obviate regulatory concerns and the industry’s constraints. It can address the compelling need to balance human safety with animal rights.

Although regulation of cosmetic products differs between countries or regions such as India and the USA or EU, some homogeneity does exist.The regulations generally stipulate that all materials utilized in the production of cosmetics must be tested for their biological safety, viz skin irritation, sensitization, toxicity and carcinogenicity in preclinical settings. In vitro and in vivo tests screen for product safety and efficacy (for human application) before their release into a market. The US FDA does not have the legal authority to approve cosmetic products and ingredients but can regulate them.3 The international legal framework for safety assessment/ toxicological testing is coming together, and its acceptance and implementation is likely to be a challenge for governments. The reasons for eventual acceptance in various markets has been in parts economic (cheaper and at least as effective as the current standard), ethical (cosmetics without the taint of animal cruelty), and political (public sentiment has swung in favour of the animal).Cosmetic testing on animals to test the safety (toxicity, pyrogenicity,allergenic and other properties) of products for use by humans has been in practice for years, and it is only recently that it has fallen out of favour. Cosmetics labelled“animal-free testing” are generally preferred by the public. Indeed, the cruelty-free status accreditation, awarded by PETA, is a selling point for some brands. PETA’s views on this issue are not secret.4 Animal testing has received much negative publicity and is banned in the EU, the UK, Norway, Israel, India, South Korea, Turkey, Australia and New Zealand; the list is growing. These countries have passed legislation that bans or restricts the use of animals in testing, in hazard and risk assessment of cosmetic materials. The Humane Cosmetics Bill, introduced in the US Congress in 2019, and awaiting ratification, will substantially restrict the use of animal testing for cosmetics.5 India, in a landmark ruling in December2016, banned all animal testing in the cosmetics industry; it has further banned the import of all cosmetics tested on animals.6 The US FDA however continues to endorse animal testing, but it also encourages the humane use of experimental animals and the “Reduce, Refine, and Replace” (3Rs) principle in instances where animal testing cannot be done without. The Humane Cosmetics Bill in the United States would substantially reduce the use of animal testing for cosmetics (It is yet to become an act).5 The adoption of alternative testing methods by the largest markets forces change in the industry, as does its endorsement by industry leaders. A case in point is the call by cosmetics giant Unilever for the global ban of animal testing in the cosmetics industry; interesting because the effort came from within the industry.

A host of agencies and organisations now regulate the toxicological testing/ safety assessment of chemicals, including cosmetics. These include, but are not restricted to the following:
ICCR: The International Cooperation on Cosmetics Regulation.
It is an international group of national regulatory agencies that try to harmonise regulation of cosmetics to minimise trade barriers while ensuring customer protection uniformly in the member countries.
CAAT, Johns Hopkins University: Center for the Alternatives to Animal Testing.
In its words, it seeks to “promote humane science by the creation, development, validation, and use of alternatives in research, product safety testing, and education”.
IATA: Integrated approaches to testing and assessment.
These are approaches mandated by the ECVAM. These are modular approaches for chemical safety assessment that integrate and interpret data on a test material from a variety of sources.
REACH: Registration, evaluation, authorisation and restriction of chemicals (2007).
The regulation was passed in 2007 and is comprehensive and binding on the member states. The legislation is enforced by the ECHA, an EU agency. It is one of the most comprehensive pieces of legislation passed in this regard in an attempt to overhaul the architecture of toxicological screening and sorely needed.
ICCVAM: Interagency coordinating committee on the validation of alternative models. A permanent committee under the aegis of the NICEATM, NIEHS. The committee is tasked with reducing, replacing, and refining the use of animals in testing wherever it is feasible, and to ensure that new and revised test methods are validated to meet the requirements of the US federal agencies.
ECVAM: European centre for the validation of alternative models. It is an international reference centre for the development and scientific and regulatory acceptance of alternative testing methods for application in the biological sciences.
OECD: Organisation for economic cooperation and Development (with 37 member countries). An international agency which facilitates the harmonisation of new test requirements and protocols, “so that different countries and regions require and accept the same types of data for regulatory decisions. International acceptance in the form of an OECD guideline is an important milestone for a validated non-animal test method and can have a significant impact on reducing animal use in regulatory assessments”.
EPA: Environmental protection agency, originator of the ToxCast program
Toxicity Forecaster is, as the name suggests, a method for the automated prediction (or forecast) of a compound’s toxicity to humans. The method makes use of automated screening tools/ high throughput assays “to expose living cells, isolated proteins, or other biological molecules to chemicals” and to subsequently create predictive toxicity models for these chemicals using computational tools. A change in biological activity in the biological reagent indicates potential toxicity.

The new methods must be sufficiently well standardised and validated, and must be amenable to integration into existing processes in the industry. Mindsets, and established and entrenched practices die hard, and a new method must go beyond demonstrating its worth; It must be compelling enough to be accepted and implemented. The transition will be slow and will need to be helped along. Fortunately, the financial stakes are high enough, and the loss of revenue from the animal ban in a large market sufficiently dire, for cosmetics manufacturers to scramble for alternatives.

Cosmetics screening assays

Cosmetic candidates must run a gauntlet of tests, with a variety of end-points, before they makeit to the market; these comprise the following: Tests for acute systemic toxicity, carcinogenicity, dermal penetration, eco toxicity, endocrine disruption, genotoxicity, neurotoxicity, organ toxicity, phototoxicity, endocrine disruption, Reproductive and developmental toxicity, skin irritation/ corrosion, and skin sensitisation. Cosmetics, unlike drugs, do not require a pre-market approval from regulatory agencies before being introduced, but are required to conform to safety requirements. A cosmetics manufacturer can be prosecuted by law for a product that causes adverse reactions in consumers.

There are various other tests, and a company may perform some or all of these tests, as the cosmetic requires. The various non-animal alternatives to toxicological analysis and safety assessment for the chemical industry have been detailed in EU legislation, and the reader is directed to excellent resources freely available on the web on this subject.

Skin is the most common surface for application of a (chemical) cosmetic, and a brief description is in order here.The dermal penetration assay measures the rate and degree of absorption (movement) of the test material across skin into the bloodstream. The method/test has traditionally been performed on rats. The skin sensitization assay checks for allergenicity of the test material on guinea pigs. The test material is applied to a shaved patch of guinea pig skin to assess its sensitivity. The Draize test tests for the ability of the test material to cause corrosion to the skin or eye,and is expected to be gradually phased out by in vitro techniques.

The science behind product safety testing in the cosmetics industry is undergoing change. A model sports beauty products.

The brave new world of toxicological testing

Decades of advances in cell culture methodology, such as development of defined cell culture media, matrices, and growth factors have made cell and tissue culture have now become sufficiently viable as in vitro alternatives to animal testing. These are rapid, reliable, and consistent. Animal sourced tissues and derived cell lines were the immediate alternatives to live-animal models, but the test read-outs have limited predictive value (i.e. have a disconnect with human species) In other words, test-material safety proven in an animal does not guarantee safety in humans. Human biopsies followed, as in vitro test material. Artificial human skin developed by culturing skin cells harvested from human skin biopsies is one such alternative testing platform being practised. Sourcing (of biopsies), availability in required volumes, and the preparation and configuration of such cellular platforms in vitro for the established test are operational challenges in the industry.

It is known that many companies have not made the switch to animal-free models to test yet for many reasons, one of the key reasons being the time it takes for lab-grown tissues to be useable. Animals, on the other hand, have a defined lifecycle and lifespan, and breed prolifically. Their genetic backgrounds can be tailored and defined. Animals are entire mammalian organs, a few orders of organisation and complexity above the humble cell-line; importantly, the whole animal has the required biochemical and cellular milieu required for simulating the body’s response (such as an immune reaction) to a cosmetic/chemical. But ultimately, the disconnect still remains.

Enter the human stem cells, progenitor cells collected from human bone marrow, umbilical cord blood, that have special properties. A stem cell can give rise to clone of itself and a daughter cell, differentiate into and provide new cells for the body as it grows, replace specialized cells that are damaged or lost,andcan proliferate indefinitely in cultures. This gives them stem cells a special status. Their emergence has had consequences, andhas changed the perspective and fate of drug and cosmetics discovery, and research and developmental. Like primary cells (described below), cells differentiated from stem cells constitute a useful cellular surrogate reagent for testing cosmetics.

There are three types of stem cells: adult stem cells, embryonic (or totipotent) stem cells, and induced pluripotent stem cells while the respective sources are adult, embryo and adult de-differentiated cells. Among the types, adult stem cells with their proven credibility, features,and ease with sourcing are the preferred type. Biopsies should not and cannot be the source for human stem cell. An alternative source for clinical grade human stem cells is human biological discard, such as human umbilical cord blood/tissue. Adipose tissue, and tooth and bone marrow are some of the other sources. Hematopoetic stem cells and Mesenchymal stem cells are two different adult stem cell types that can be harvested from these biological discards and both their quality and quantity can be controlled to assure the process and product viability for given requirements. The proven clinical attributes of adult stem cells isan advantage in drug discovery and development.

Skin has been artificially cultured using skin from donors, and this is appropriate for some safety tests; assays based on artificial skin have already been validated. Stem cells have also been successfully grown into spheroids and organoids although their application to testing is yet to become routine. In instances where stem-cells are not suitable for toxicological assays, they can still be used for general screening purposes; the screen will eliminate harmful compounds, even if it cannot prove the safety of others.

One of the main problems with tissue culture is the culture of primary cells, and it is now overcome. Primary cells are cells that reflect the biology of cells in vivo, more than established cell lines. The latter are immortalised using either the Epstein Barr Virus, or irradiation. Primary cells have a limited lifespan, unlike established cell lines, but their advantages outweigh their deficiencies. Primary cells are truly representative of the (cell) donor, and reflect her attributes, such as age, medical history, race, and sex. These can be factored into the experimental design, and are especially important in this era of personalised medicine. Established cell lines are uninformative in this respect, and as a model, a degree further removed from the human than the primary cell line. Cell lines no longer have the characteristics of their tissue of origin, and serial passaging causes genotypic and phenotypic variation.

The way forward

A validated marker panel, be it transcriptomic or proteomic, for an outcome is desirable but this involves a lot of work. The oncology communityhasn’t had much success with validating putative biomarkers of diagnosis or prognosis of cancer, although markers of physically apparent symptoms may be easier to validate. Validation requires that the marker be a reliable predictor of a condition, or of the course of a disease and the chances of recovery. Few, if any, markers have made the cut, partly because of the lack of an appropriate method (for a long time); a methodological bottleneck. Validation of adverse reactions to chemicals may be conceptually easier to validate, because the endpoints are often easily observed gross symptoms, and because the symptoms manifest themselves in a shorter time span, provided candidate molecular markers of the reaction are known. As the biology of the allergic reaction, or of toxicity, becomes clearer with time, along with the role of the individual molecular actors, it will be possible to construct newer tests that are based on molecular endpoints not observable as gross symptoms; these could include for instance, the post-translational glycosylation of a protein (proteomic, observed by targeted mass spectrometry), or the upregulation of micro-RNA, or of an alternatively spliced mRNA (transcriptomic, measured by quantitative PCR). The readouts in this case will be proteomic and transcriptomic, obtained by targeted mass spectrometry (such as the DIA, SRM, or SWATH techniques) and by quantitative PCR respectively.

A multi-parametric analysis of several biomarkers can provide, such as those mentioned above, potentially, better prediction of chemical-induced adverseeffects on human health. (Health Risk Assessment, EFSA Journal, 2019) Transcriptomics and proteomics are often not in agreement and are now understood to be complementary sets of information about a biological system. The collation and integration of data from various sources is the basis for systems biology, a form of which is already being used with the EU’s REACH programme, and the US EPA’s ToxCast program.

A test material should beexamined for induced mutagenicity, carcinogenicity, genotoxicity, neurotoxicity, and haemotoxicity on human surrogate in vitro systems in preclinical exploratory settings, starting from a choice of raw material or ingredients to the finished product.A process that spans all aspects of the candidate’s testing is desirable. The genes and secreted proteins involved in toxicity pathwayscan best be utilized as predictive markers for the said toxicities. Transcriptomics and proteomics of human surrogate systems treated with test materials provide detailed information on the human genes and proteins involved in the response to the test– They constitute the next generation of preclinical cosmetics testing systems, that can dispense with the animal tests for regulatory approval.

A tiered approach to screening or safety assessment has been proposed, and is an integrated testing strategy (Hartung, 2009). It involves the evaluation of theoretical information about the compound/test material and possibly in silico analyses, followed by in vitro tests, and (as a last resort) in vivo analyses, with each stage predicated by the results of the previous test set, using a decision tree or flow diagram.The approach restricts animal testing until other avenues have been explored. Testing strategy will therefore eventually evolve into a systems biology- type of approach, as indeed the EPA’s ToxCast program partly is. Data and method sharing protocols will help speed up safety testing in all countries and regions, and ensure its uniformity across borders. A vision for safety testing into the 21st century has been outlined and continues to be shaped. The paradigm shift away from animals is a challenge, but the biomedical community is up to it.

1. Commissioner, O. of the. Federal Food, Drug, and Cosmetic Act (FD&C Act). FDA (2018).

2. Hartung, T. Toxicology for the twenty-first century. Nature460, 208–212 (2009).

3. Commissioner, O. of the. Federal Food, Drug, and Cosmetic Act (FD&C Act). FDA (2018).

4. Hartung, T. Toxicology for the twenty-first century. Nature460, 208–212 (2009).

5. Nutrition, C. for F. S. and A. FDA Authority Over Cosmetics: How Cosmetics Are Not FDA-Approved, but Are FDA-Regulated. FDA (2020).

6. Why Animal Rights? PETA (2016).

7. Ending Cosmetics Animal Testing. The Humane Society of the United States

8. Countries Around the World Work to Ban Cosmetics Testing on Animals. PETA (2015).

9. Unilever announces support for worldwide ban on animal-tested cosmetics. The Independent (2018).

10. DURA, A. IATA – Integrated Approaches to Testing and Assessment. EU Science Hub – European Commission (2018).

11. Registration, Evaluation, Authorisation and Restriction of Chemicals. Wikipedia (2020).

12. About ICCVAM.

13. European Centre for the Validation of Alternative Methods (ECVAM) – BfR.

14. Hazard Assessment | Toxicity Testing Overview.

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