Cosmetics & Personal Care

Alternatives to animal testing for cosmetics

By our Editorial Team - 13/04/2018

Advances in non-animal testing could provide new opportunities for the development of cosmetics ingredients, as novel testing approaches move to the fore.
A brief history
Animals have been used throughout the history of biomedical research. Early Greek physician-scientists, such as Aristotle and Erasistratus, reportedly performed experiments on living animals. In 1242, the Arab physician Ibn al-Nafis was famously the first to describe the pulmonary circulation of the blood in mammals.
Although considered by many to be barbaric by today’s standards, there can be little doubt that early scientists and their experiments in animals provided ground-breaking insights into the way in which our bodies work, and in understanding diseases and the path to developing effective therapies (in the 1880s, Louis Pasteur convincingly demonstrated the germ theory of medicine by giving anthrax to sheep).
Opposition to the use of animals in medical research arose in the United States during the 1860s, when Henry Bergh founded the American Society for the Prevention of Cruelty to Animals (ASPCA), with a similar movement emerging in Europe around the same time. Antivivisectionists of that time believed that the cruelty of vivisection was in conflict with the ‘spread of mercy’ – considered the great cause of civilization. However, their efforts had little effect because of the widespread support of an informed public for the careful and judicious use of animals.
Nevertheless, there has remained constant concern about both the methodology and the care of laboratory animals used in testing. Around the 1960s, this began to be particularly focussed on animal testing for cosmetics and cosmetics ingredients – considered a frivolous cause for the expenditure of animals’ lives. While tragic events such as the thalidomide disaster strengthened the cause of animal testing in pharmaceuticals, there was considered limited rationale for sacrificing animals in the name of superficial beauty.
It is perhaps remarkable that it was another 50 years or so before regulations caught up with this public opinion. However, regulators rightly needed to wait until appropriate alternatives had been found for testing.
The animal testing ban in the European Cosmetics Regulation (EC 1223/2009), which came into force in March 2013, provides the regulatory framework for the phasing out of animal testing for cosmetics purposes.1 Specifically, it establishes:
  • Testing ban – prohibition to test finished cosmetic products and cosmetic ingredients on animals
  • Marketing ban – prohibition to market finished cosmetic products and ingredients in the EU which were tested on animals.
The Swiss government officially followed suit in January 2017. Australia, India, New Zealand, South Korea and Taiwan have also banned – or are in the process of banning – animal testing for cosmetics.
However, while these regulatory bans finally demonstrate a ‘spread of mercy’ across the civilized world, the issues are compounded by the fact that other relevant regulations, such as REACH (EC 1907/2006) and the CLP (EC 1272/2008), as well as the European Scientific Committee on Consumer Safety (SCCS), still require animal testing in some circumstances, such as ensuring safe exposure levels for manufacturing workers. Nevertheless, the ban applies – irrespective of the availability of alternative non-animal tests.
This is a seemingly impossible block when trying to add new ingredients to the approved lists for cosmetics products, leaving companies with a difficult choice: test on animals, contravening the ban and risking a backlash from consumers, or follow a strict non-animal testing policy, with consequent limits on R&D and commercialization.
Alternatives to animal testing
Alternative methods to animal testing are one of the focuses of the International Cooperation on Cosmetics Regulation (ICCR). In fact, contrary to the diminishing belief that human safety can only be assured though animal tests, a number of in vitro tests can yield detailed, mechanistic, human-relevant data. Such tests have been extensively validated under strict criteria and scrutinized by international experts before acceptance as Test Guidelines. These strict criteria were not in place when many of the animal tests were developed and adopted for common use, which is why the animal tests often became the ‘gold standard’… frequently without being validated themselves.
Skin and eye irritation
Tests for skin and eye irritation were among the first non-animal methods to gain regulatory status (OECD Test Guidelines [TG] 439, 492 and 431), using tissue engineering technology to create reconstructed human skin and eye tissues as an in vivo representation of human physiology.
Test items (ingredients and formulations, as liquids or solids) are simply applied to the tissue surface. These tests, used for compliance across a broad range of chemical sectors, are among the most accepted regulatory in vitro methods.
Skin sensitization
A skin sensitizer is a chemical that has the intrinsic ability to lead to an allergic response in susceptible individuals following skin contact. Testing for substances that have a potential to be skin sensitizers is an important area of chemical regulation worldwide.
In 2016, REACH recognised the three non-animal methods for the adverse outcome pathway in skin sensitization: Proctor and Gamble’s Direct Peptide Reactivity Assay (DPRA) (TG442c); Givaudan’s ARE-Nrf2 Luciferase test (KeratinoSens) (TG442d); and Kao and Shiseido’s human Cell Line Activation Test (h-CLAT) (TG442e). Combining these tests leads gives a ‘2 out of 3’ result that classifies a product as a sensitizer or non-sensitizer.  Combining the findings from these in vitro tests can predict the sensitization potential of a compound. It enables regulatory compliance in most cases, while avoiding animal testing. The costs of performing these in vitro tests are comparable costs to those of the traditional animal-based Local Lymph Node Assay (OECD TG 429), and lower than those of the Guinea Pig Maximization Test (OECD TG 406).
Table 1 lists the non-animal methodologies currently accepted by REACH to assess skin sensitization. None of these methods are stand-alone methods, but need to be used in combination in a ‘2 out of 3’ process.
Table 1. Non-animal test methods for skin sensitization accepted by REACH
Accepted method
Molecular interaction with skin proteins
In silico assessment of metabolite or abiotic transformation product of the target chemical
Direct Peptide Reactivity Assay (DPRA) tests the ability of a chemical to bind covalently to a protein (TG442c)
Inflammatory response in keratinocytes
ARE-Nrf2 Luciferase test (KeratinoSens), composed of a cell line containing the luciferase gene which is up-regulated by contact sensitizers (TG442d)
Activation of dendritic cells
Human Cell Line Activation Test (h-CLAT) based on a monocytic leukaemia cell line (THP-1) that detects skin sensitizers via an increased expression of cell surface markers associated with the process of activation of monocytes and dendritic cells (TG442e)
Acute toxicity challenge
Testing acute toxicity is the only human health endpoint in Annex VII of the regulation that still has no validated in vitro test.
A conventional approach is the use of rat LD50-related tests – involving the administration of increasing doses of the substance in up to 100 rats, until a lethal dose to 50% of the animals (LD50) is identified. Aside from ethical concerns, the relevance of these tests to human safety has been questioned.2 A new approach is needed to assess human safety following acute exposure to chemicals.
In 2015, the European Commission reviewed the state of the science of non-animal alternatives for acute toxicity testing and explored ways to facilitate implementation of alternatives. Resources identified as necessary for meaningful progress in implementing alternatives included compiling and making available high-quality reference data, training on use and interpretation of in vitro and in silico approaches, and global harmonization of testing requirements.3
One approach could be a human-cell based screen currently under development,4 which in a feasibility study using 20 cosmetic ingredients demonstrated greater predictivity of toxicity than conventional rat LD50 methods. The screening test used human dermal fibroblasts in animal-product-free culture, measuring uptake of Neutral Red dye as an indicator of cell membrane integrity. The inhibitory concentration causing 50% reduction in cell viability (IC50) provided a sensitive, mechanistic in vitro alternative to LD50.  
According to the website of the company developing this test, XCellR8,5 the screening test is already used by its clients a part of a weight-of-evidence approach in REACH submissions, to avoid LD50-related animal tests. Now with support from the European Horizon 2020 programme, the company is planning to further develop the test and expand its applicability across a range of chemical sectors, as well as seeking regulatory approval for the screen to eventually be used as part of a non-animal testing strategy for human acute toxicity.
Significant advances in the development of alternative test methods for testing the safety and tolerability of cosmetics ingredients have been achieved in the past two decades. Validated and regulatory accepted non-animal methods are now available for many endpoints.
The current regulatory environment brings challenges in areas for which no validated non-animal alternatives currently exist – the gap in approved tests for acute has already been discussed, but other limitations include identification of sensitizers that require metabolic activation to exert sensitization, or testing of insoluble chemicals and metals. However, researchers are rising to these challenges. Higher ethical standards, embracing the latest scientific developments, may create opportunities for companies to demonstrate greater corporate responsibility while better adhering to regulatory guidelines and using available non-animal tests for their cosmetics products.
Further opportunities also exist along the supply chain. XCellR8 is just one company (already mentioned above) that is thriving on its investment in non-animal testing. Contract research company Envigo is also in the process of expanding R&D capabilities in non-animal technologies. In a 2017 press release, the company announced plans to develop and validate an integrated suite of in vitro tests for measuring cardiac risk potential, and to create up to 10 new in vitro tests per year for use in combination with in silico techniques across its contract research business, spanning the fields of genetic toxicology, electrophysiology, metabolism, endocrine disruption, skin sensitization, skin and eye irritation, genomics and gene expression.
Most industry leaders are using in vitro testing at some level, to demonstrate both safety and efficacy, but comprehensive strategies and expert guidance need to be made available to SMEs, enabling universal access to new technologies. The 21st century has been marked by remarkable progress in the movement away from animal testing, propelled by significant advances in non-animal testing alternatives. If we continue down this path, perhaps our children’s generation will be fortunate enough to look back in wonder at why we ever tested on animals at all.

2.    Prieto P et al. Publications Office of the European Union, 2014.
3.    Hamm J et al. Toxicol In Vitro 2047;41:245–259.
4.    CL Barker-Treasure et al. Altern Lab Anim 2015;43:199-203.