Cosmetics testing experts address Regulatory Summit

Toxicogenomics for genotoxicity testing and the potential of ex vivo organ culture testing were hot topics

The two-day Cosmetics Business Regulatory Summit (CBRS) returned to the Radisson Blu Royal Hotel in Brussels from 19-20 May 2015, gathering leading authorities in cosmetics regulation and testing to present topical issues faced worldwide by beauty and personal care companies when seeking to comply with legislation.

Over the next few days, will bring you highlights from this must-attend event, topic by topic; this edition will focus on cutting edge testing methods.

Mutagenicity/genotoxicity testing of active ingredients was the topic covered by Professor Vera Rogiers, Head of the Department of Toxicology, Dermato-Cosmetology and Pharmacognosy at Vrije Universiteit Brussel, who spoke on day one. She began by covering what the industry can expect in the context of the EU Cosmetics Regulation (1223/2009/EC) before delving into the results of a retrospective study of genotoxicity testing of Annex ingredients.

Rogiers helpfully opened by differentiating between mutagenicity, the induction of permanent transmissible changes in amount or structure of genetic material, and genotoxicity, processes which alter the structure, information content of segregation of DNA that are not necessarily associated with mutagenicity. Mutagenicity is part of genotoxicity, she explained, but both are important for the prediction of carcinogenic potential.

Historically, a two or three in vitro battery – comprising a mutation test in bacteria and/or a mutation test in mammalian cells for mutagenicity, or a chromosomal aberration test and/or micronucleus test for genotoxicity – was used to screen for undesirable genetic endpoints. In the case of a positive result, the substance was then tested in vivo in animals to see whether an ingredient posed a risk. “These in vitro tests were never intended to be a stand alone battery,” she said, “and the in vivo test overruled the in vitro test.” However, testing and marketing bans in regulation 1223/2009/EC means only in vitro tests are left to us as an industry.

She showed the results of a retrospective study by the EC's Scientific Committee on Consumer Safety (SCCS) of Annex II, III, IV, V and VI cosmetics substances. This looked at all submissions at European level from 2000 onwards – including those from before the marketing and testing bans with “quality” in vitro and in vivo mutagenicity and genotoxiticy data, and in vivo carcinogenicity data. Of a possible 249 ingredients, the 169 with the best quality data were chosen; for 44 ingredients, no in vivo follow up was required, but 125 required a further in vivo test. Of these, only 19 tested positive, meaning a significant 106 'misleading positive' ingredients would have been lost to the market under our current regulation, where an in vivo test cannot overrule.

A route towards improving performance, she said, would be to look at the specificity of an in vitro method, or the ability to detect a 'true negative'. Outlining possible improvements, Rogiers demonstrated that all the possible two test battery combinations (which still covered all endpoints) had a low specificity, similar to those of three test batteries, meaning the SCCS will revise its 9th revision guidance to recommend a two test in vitro battery later this year. Other conclusions for optimum accuracy were the recommended use of p53 functional human cells and a reduction in top concentration. 

Rogiers conceded that it's “not great news” for the industry “using a two test battery where you know you are going to get a lot of false positives”.

However she demonstrated how an new development – an in vitro toxicogenomics-based assay might provide a way forward. She drew delegates attention to the  CarcinoGENOMICS FP6 project studying responses to carcinogens and non-carcinogens of the RNA of HepaRG cells. The antibacterial agent triclosan – which tested positive in the in vitro chromosome aberration test, but negative in follow-up in vivo tests – was used as a case study alongside five well-known genotoxic carcinogens, five non-genotoxic carcinogens and five non-carcinogens. There are a whole set of pathways that are upregulated and down regulated by genotoxic carcinogens. “When you do a principal cluster analysis, you can see that for the well-known genotoxicants you get a certain pattern,” said Rogiers. “While you cannot make a distinction between non-genotoxic carcinogens and non-carcinogens, what you can see really clearly is whether your compound has a very typical genotoxic signature or not. And triclosan is clustered with the non-genotoxic compounds.”

Taking to the podium after Rogiers, Dr Ardeshir Bayat from the Manchester Institute of Biotechnology, spoke on a breaking area in the evaluation of today’s advanced cosmeceuticals: ex vivo organ culture testing. He described the method as a superior alternative to in vitro and in vivo animal testing in studying the effects of topicals on skin health and biology.

Traditional skin models – monolayer (2D) and an organotypic (3D) – lack a detailed human skin microenvironment, and cellular, molecular and biochemical interaction. They also exhibit different skin healing processes. Meanwhile, the EU animal testing ban nonwithstanding, Bayat asked: “What animal 100% represents human skin?”

“Why shouldn't we use human skin as an alternative?” he asked. He recommended organ cultures featuring 6mm epidermal biopsies (harvested from discarded skin from the cosmetic surgery industry and suspended in liquid culture media using transwell inserts) to ascertain both long term culture response to treatments and short term culture transdermal delivery.

He provided an example where the optimised whole tissue biopsy culture model could be used to quantify both the transdermal delivery of active components and the altered expression of key gene and protein markers in one study.

Having looked at biopsies from normal skin, Bayat conducted ex vivo studies on keloid scar tissue (a phenomenon that only occurs in humans) using a similar approach, whereby he and his team managed to maintain scar tissue outside of the body for up to six weeks to find mollecular targets for keloid scarring; using this ex vivo approach, for the first time mTOR was discovered to be a promising target and P529 (a dual PI3KAkt/mTOR inhibitor) a candidate agent for further exploration for the treatment of skin scarring.

“We built a whole map of how this drug might work by doing this organ culture test,” said Bayat.

Other experiments facilitated via ex vivo methods included one on wound healing organ culture, whereby a ‘donut’ within a punch was used to measure the rate of epithelialisation in biopsies with full thickness and half thickness wounds, compared with normal skin. This, said Bayat, could be useful for doctors wanting to reassure patients about speed of effect.

Another experiment on would healing involved Bayat using laser capture microarray (LCM) as part of in vivo experiments on human volunteers, analysing fibrotic outcomes of healing on 5mm punches, located on the inner arms to trial safe topicals against placebos. Optical coherence tomography (OCT) – which provides live data on how skin is healing – was shown to be a reliable method of assessing acute wound healing in human skin. Areas of quantification included surface area, volume, wound bed blood flow (measured using full field laser perfusion imaging), and collagen, melanin and haemoglobin (all measured via SIAscopy).

Bayat was excited about taking this to next level – studying the healing of different incisional models and types of wounds. “A puncture or biopsy heals with a considerable about of contraction, whereas an incision scar heals with less contraction. If a wound healing drug has no effect on contraction, there is no point on using it for a mole,” he gave as an example.

Keep reading for further insight into topics discussed at the Cosmetics Business Regulatory Summit 2015.