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Patch Testing vs. In Vitro Alternatives

Contact Author Barrie Drewitt-Barlow, Laura Marshall, and J. McPherson, Hill-Top Research
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The race to get newly formulated products through R&D and on the market shelves has long been the cause of many problems for formulators and marketing departments alike. Occasionaly, products going through months of extensive, costly and time-consuming internal research are only discovered not to meetin acceptability standards when tested by external R&D institutes.

We are all aware of the current European legislation on cosmetic testing using animals and the view of consumers demanding products that have not been tested on animals.

So, what is the way forward? How can you guarantee the safety of new products and how do you stay out of litigation? The possibilities do exist. Some scientists will argue that newly developed in vitro assays are the answer. With this argument in mind, we compare the 3T3 Neutral Red Uptake assay and a 3-application patch test to assess the dermal irritation potential of a selection of new and currently marketed personal-care products.

The 3T3 Neutral Red Uptake assay is a cytotoxicity assay used to assess eye and skin irritation. Fibroblasts are cultured in the presence and absence of the test articles. After 24 h, the reduction in uptake of the Neutral Red dye by the test articles is measured. Only viable cells will take up the dye.

In the 3-application patch test, occlusive patches are applied to the upper arm of healthy human volunteers to assess skin irritation potential of a variety of products. Three 24 h patches are applied to the same sites over a week and erythema levels graded visually by a trained observer.

In Vitro Test Validation

The quest to develop useful in vitro test methodologies to replace rabbit eye and skin irritation tests continues, but validation projects still depend on historical rabbit eye and skin
irritation data as the bench mark against which to measure the performance of the in vitro assays.

So far, only a limited amount of available data compares the results of in vitro methodologies with those of human volunteer trials. The aim of this project was to build a comprehensive inhouse database comparing various human trials with currently marketed in vitro assays.
We tested a selection of 10 personal care products in a 3T3 neutral red uptake (NRU) assay to fi nd the EC50 value: the Effective Concentration that causes a 50% reduction in cell viability.

We tested the same 10 products in a single-blind, within subject comparison, three-application patch test, done on a panel of 27 healthy human volunteers. We determined skin-irritation potential by assessing and scoring the volunteers’ reaction to the products. We compared products according to their irritancy potential in vivo and in vitro.

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