Shampoo, perfume, lipstick, toothpaste – we all use at least some cosmetics in our everyday lives, and assume the manufacturers and regulatory agencies have our best health interests at heart. Here, we provide an overview on the use of laboratory animals in cosmetic safety testing, and discuss alternative non-animal-based test methods.
Testing of cosmetic products on laboratory animals
Historically, non-human species such as mice, rats, rabbits and guinea pigs were used to assess the safety of cosmetic products and ingredients, and several of the required or expected tests could result in these animals experiencing extreme discomfort and sometimes outright pain. The animal rights movement over the years have ensured that consumers and regulatory agencies were increasingly aware of the ethical issues of this type of applied research. Many countries now acknowledge that testing cosmetics on laboratory animals is unnecessary, given that (i) thousands of ingredients already have a history of safe use and (ii) non-animal testing methods are available that might provide the appropriate level of reassurance on any newer cosmetic chemical stars.
In the EU, animal testing of finished cosmetic products to ensure consumer safety was banned in 2004, and that of cosmetic ingredients was banned in 2009 (Regulation (EC) No. 1223/2009); in addition, a full ban on the marketing of products that have involved animal testing in their development came into force in the EU in 2013 [1, 2]. Since then, other countries, such as India, Israel, Norway, Iceland, Switzerland and Mexico, have passed similar laws. Several other countries and US states have also passed laws to end the sale of animal-tested cosmetics or ban/limit cosmetic animal testing . A number of countries (for example, China) still require animal testing data in order to bring some cosmetics to market , although the Chinese Government has recently removed the mandatory requirement for animal testing of imported cosmetics .
Despite the EU’s ban, under ECHA’s Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) regulation (Regulation (EC) No. 1907/2006), animal testing may still be required to ensure the safety of workers that manufacture the products or to protect the environment, if no alternative non-animal tests are available . Notably, just this year, in a landmark ruling that embodied divergence from the EU rules, the UK announced that it will no longer allow REACH-required animal tests intended to prove worker safety . Is this a Brexit benefit? The implication seems to be that either UK workers manufacturing cosmetics must now accept higher health risks than their wider European peers, or that at least some aspects of current REACH guidance on toxicity testing have no scientific merit. We may be entering some difficult philosophical waters.
Non-animal options for cosmetic safety testing
Non-animal approaches to cosmetic testing, often referred to as “new approach methodologies” (NAMs), can include in vitro, in silico, and grouping/read-across methods, often in combination . In addition, information obtained from studies in human volunteers can be useful.
In vitro methods typically involve the use of cultured human or animal cells or tissues, sometimes grown as 3D structures. A major breakthrough in non-animal cosmetics testing was the creation of a synthetic skin test named Corrositex, which enables testing of the skin corrosion potential of chemicals (OECD test guideline 435) .
The Scientific Committee on Consumer Safety (SCCS) has published a guide for the testing of cosmetic ingredients, which includes descriptions of validated NAMs for the evaluation of acute toxicity, corrosion and irritation, skin sensitisation, mutagenicity/genotoxicity, photo-induced toxicity . In addition, the EU Reference Laboratory for Alternatives to animal testing (EURL ECVAM) lists several validated alternative methods for toxicity testing. The table below provides an overview of several non-animal test methods that have been accepted by the OECD.
Table 1. In vitro OECD test guidelines (adapted from )
Human Health Endpoint
OECD Test Guideline
Skin corrosion/ irritation
430: In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER)
431: In Vitro Skin Corrosion: Human Skin Model Test
435: In Vitro Membrane Barrier Test Method for Skin Corrosion
439: In Vitro Skin Irritation - Reconstructed Human Epidermis Test Method
Eye damage/ irritation
437: Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants
438: Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants
460: Fluorescein Leakage Test Method for Identifying Ocular Corrosives and Severe Irritants
491: Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage
492: Reconstructed Human Cornea-like Epithelium (RHCE) Test Method for Eye Hazard Identification
494: Vitrigel-Eye Irritancy Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage
496: In vitro Macromolecular Test Method for Identifying Chemicals Inducing Serious Eye Damage and Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage
442C: In Chemico Skin Sensitisation : Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins
442D: In Vitro Skin Sensitisation : ARE-Nrf2 Luciferase Test Method
442E: In Vitro Skin Sensitisation : In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation
432: In Vitro 3T3 NRU Phototoxicity Test
471: Bacterial Reverse Mutation Test
473: In Vitro Mammalian Chromosomal Aberration Test
476: In vitro Mammalian Cell Gene Mutation Test
487: In Vitro Mammalian Cell Micronucleus Test
490: In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene
428: Skin Absorption: In Vitro Method
In silico testing uses computer-based methods to model the safety of compounds based on their physico-chemical properties and predicted chemical reactivity.
Quantitative structure–activity relationship modelling, or (Q)SAR, is gaining regulatory acceptance, although the use of this approach does require some expert knowledge. Among others, examples of free-access in silico systems are the OECD (Q)SAR ToolBox, the US EPA TEST tool and the Toxtree application.
Grouping and read-across
Grouping/read-across methods involve comparing the physico-chemical and structural characteristics of a new substances to those of similar existing substances for which safety/toxicity data are already available. In this way, the expected properties of the new substance can be predicted. Read-across approaches require expert knowledge and are typically carried out in addition to (Q)SAR, to increase the overall confidence in the predicted properties .
Human tissue samples can be donated after surgery or death for ex vivo testing of cosmetic ingredients/products. In addition, so-called micro-dosing of volunteers can be used to examine the effects of small amounts of substances on metabolic and physiological processes, as a way to predict their potential toxicity. Micro-dosing was considered by an ECVAM Expert Group as a potential replacement for most of the toxicokinetic and ADME data normally obtained from animal tests for chemicals used in cosmetics .
How can bibra help manufacturers developing a new cosmetic product?
Bibra scientists are skilled in planning and interpreting the results of in vitro studies and have a well-grounded appreciation of the current (Q)SAR models favoured by the regulators. We can help you to understand in vitro and (Q)SAR data in the context of literature identified in searches of databases, such as PubMed and bibra’s own TRACE database. A degree of scepticism is often of value in this area where over-excited claims of NAMs advocates are a understandable feature. Our team also has extensive experience of drafting read-across justification reports and advising on potential in silico or in vitro testing that could be used to support a read-across strategy.
Animal-based approaches to establish the safety of cosmetics for consumers are banned in the EU and UK, and are increasingly discouraged in several other countries. Alternative non-animal-based testing methods are available, several of which have been validated and/or approved by Expert Groups. The bibra team can help you to assess the existing toxicity and safety data for your cosmetic product or ingredient, and can suggest where additional in silico, in vitro or read-across approaches could be used.
 European Commission. Ban on animal testing. Accessed July 2023. https://single-market-economy.ec.europa.eu/sectors/cosmetics/ban-animal-testing_en
 EU Regulation (EC) No 1223/2009. https://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:342:0059:0209:en:PDF
 Human Society. Cosmetics animal testing FAQs. Accessed July 2023. https://www.humanesociety.org/resources/cosmetics-animal-testing-faq#required
 EURL ECVAM. EU Reference Laboratory for alternatives to animal testing. Frequently Asked Questions – General. Accessed July 2023. https://joint-research-centre.ec.europa.eu/eu-reference-laboratory-alternatives-animal-testing-eurl-ecvam/eurl-ecvam-faqs/frequently-asked-questions-general_en
 RSPCA. We welcome change in China's cosmetics testing regulations. May 2021. https://www.rspca.org.uk/-/news-rspca-welcomes-change-in-chinas-cosmetics-testing-regulations
 ECHA. European Chemicals Agency. Cosmetics Regulation and REACH – questions and answers on animal testing. Accessed July 2023. https://echa.europa.eu/documents/10162/0/cosmetics_reach_interface_animal_testing_en.pdf/a1c91da8-bad8-64c3-400a-82db0085406a
 SCCS (2023). Scientific Committee on Consumer Safety. Notes of guidance for the testing of cosmetic ingredients and their safety evaluation. 12th revision. 15 May 2023. https://health.ec.europa.eu/latest-updates/sccs-notes-guidance-testing-cosmetic-ingredients-and-their-safety-evaluation-12th-revision-2023-05-16_en
 OECD Test Guideline 435. In Vitro Membrane Barrier Test Method for Skin Corrosion. https://www.oecd-ilibrary.org/environment/test-no-435-in-vitro-membrane-barrier-test-method-for-skin-corrosion_9789264242791-en
 Pistollato F, Madia F, Corvi R, Munn S, Grignard E, Paini A, Worth A, Bal-Price A, Prieto P, Casati S, Berggren E, Bopp SK, Zuang V (2021). Current EU regulatory requirements for the assessment of chemicals and cosmetic products: challenges and opportunities for introducing new approach methodologies. Arch Toxicol. 95(6):1867-1897. doi: 10.1007/s00204-021-03034-y. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8166712/
 EURL ECVAM. EU Reference Laboratory for alternatives to animal testing. Computational Methods. Accessed July 2023. https://joint-research-centre.ec.europa.eu/eu-reference-laboratory-alternatives-animal-testing-eurl-ecvam/alternative-methods-toxicity-testing/computational-methods_en
 Langley G, Farnaud S (2010). Opinion: Microdosing: safer clinical trials and fewer animal tests. Bioanalysis. 2(3):393-5. doi: 10.4155/bio.09.168. https://www.future-science.com/doi/full/10.4155/bio.09.168?rfr_dat=cr_pub++0pubmed&url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org