Publications

In vitro assays evaluating ocular irritation potential are routinely used by personal care companies. Two of these in vitro assays include the Chorioallantoic Membrane Vascular Assay (CAMVA) and the Bovine Corneal Opacity and Permeability Assay (BCOP). These assays do not require the use of live animals, provide reliable predictive data and are rapid to conduct. The BCOP uses excised bovine corneas to predict ocular irritation. The CAMVA uses the vascular network of fertilized chicken eggs as a conjunctival model to predict eye irritation. Both BCOP and CAMVA have been used for over fifteen years for product development, worker safety, and safety claims substantiation.

This poster describes procedures and considerations for demonstrating the inter-laboratory reliability of the BCOP and CAMVA. It is important to have a valid assay that can be implemented consistently at several different laboratories. For Kao Brands Company, a large BCOP and CAMVA database exists that covers multiple consumer product categories such as hair shampoos, skin cleansers, and hair styling sprays (containing ethanol). Therefore, a proper review of candidate laboratories is important for seamlessly generating consistent results that can be used for assessing potential ocular irritation of new products. First, a candidate laboratory should be audited for proper facility operation and personnel training. Second, the laboratory’s use of Good Laboratory Practices (GLPs) should be reviewed. Third, reference materials with known BCOP and CAMVA data (one irritant and two non-irritants for initial assessment) should be tested at each new laboratory for verification of proper assay performance.

The Bovine Corneal Opacity and Permeability Assay (BCOP) is an ex vivo assay, which may be used to assess the eye irritation potential of new chemicals and finished products. The BCOP assay has been accepted by several regulatory agencies for the identification of severe and corrosive ocular irritants, replacing the rabbit eye test. According to OECD Test Guideline 437 adopted in September 2009, two treatment protocols may be used; one for liquids and one for solids. Solids are tested as 20% (w/v) solutions or suspensions in deionized water. Freshly excised bovine corneas are mounted in special corneal holders and are treated with the 20% (w/v) test material dilutions for four hours at approximately 32°C. Changes in corneal opacity are measured using an opacitometer, and impairment of the corneal barrier function is determined by measuring fluorescein passage through the corneas. Histological evaluation of the treated corneas may be used to determine the degree and depth of injury at the tissue level. In this study, the reference standard solids recommended in the OECD TG 437 were tested in an inter-laboratory study. Overall, the results from the evaluation of solids were highly congruent between the two laboratories and to the historical data and for several substances histological evaluation improved the understanding of eye irritation effect. However, for chlorhexidine and dibenzoyl-L-tartaric acid there were inter-laboratory differences, which were further evaluated. For chlorhexidine, differences in results were attributed to different sources of the chemical. This study demonstrates the reproducibility of the BCOP assay when evaluating solid test substances. In parallel, the study compared the opacity scores from a newly developed opacitometer (BASF-OP2.0) to those of the standard device (OP-KIT). The comparison between the BASF-OP2.0 showed very little variability and overall corresponded very well with the OP-KIT values.

Topical antioxidants, which have the capacity to neutralize reactive oxygen species (ROS), have been shown to prevent skin damage and improve the appearance of sun-damaged skin. Accordingly, we have developed an in vitro method capable of evaluating the antioxidant performance of ingredients and final formulations which may be applied to the skin. For assessment of antioxidant potential, NHEKs (normal human epidermal keratinocytes) were subjected to UVA-irradiation to induce oxidative stress and then protection from oxidative stress was evaluated in cells incubated with antioxidants. Cells were seeded in 96-well plates and incubated with the fluorescent ROS-detecting probe, 5-(and-6)-chloromethyl-2?,7?-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA), for 40 minutes. Next, the cells were exposed to a serial dilution of antioxidants/antioxidant-containing formulations for 1 hour. ROS were generated by exposing the cultures to UVA light for 50 minutes (5 Joules/cm2), and then detected by fluorescence measurements of the cells. Cytotoxicity was assessed concurrently using the neutral red uptake (NRU) assay. Using this method, we evaluated several antioxidants and antioxidant-containing formulations for their ROS-reducing capabilities, including ascorbic acid, quercetin, resveratrol, kinetin, nicotinamide, and EGCG. Based on our results, several antioxidants and antioxidant-containing formulations noticeably reduced ROS levels compared to untreated controls. The greatest reduction was observed in cells treated with ascorbic acid, which has been qualified as the positive control for the assay. Other antioxidants which did not show this reduction were likely not water soluble and/or poorly bioavailable to cells. Overall, our results indicate that this method may provide a valuable in vitro tool for assessing antioxidant performance in a biologically relevant model.

Improvement of ocular irritancy prediction using a modified (shortened) 3-minute exposure in the BCOP assay was proposed for alcohols and ketones, which have been identified by ICCVAM as responsible for the most over-predictions in the BCOP. Eight alcohols and six ketones were tested using both the modified and the standard 10-minute exposure, and the data were compared with the GHS categories from the ICCVAM database. The evaluation of the 3-minute exposure data revealed that five of the 6 over-predicted alcohols showed an improved prediction, and of the 2 correctly predicted alcohols, one became an under-prediction and one remained the same. Two of the five over-predicted ketones showed an improved prediction, with the three other remaining the same. The one correctly predicted ketone remained the same. The results of the evaluation of the modified BCOP assay using the 3-minute exposures for alcohols and ketones suggest that improvements in the predictive capacity of the assay can be achieved by reducing the over-prediction of these small molecule, solvent-type chemicals, without an adverse impact upon the rate of under-prediction of similar chemicals. It is our recommendation that a) additional small molecule alcohols and ketones exhibiting solvent-like physical characteristics should be tested in the BCOP assay using the 3-minute (or shorter) and 10-minute exposures, and b) prior to any additional testing a more thorough evaluation of the supporting rabbit ocular irritation data be conducted to ascertain whether the correct standards are being used to calibrate the assay.

Chronic ulceration of the leg represents a major, underestimated problem of modern health care, involving physical and cosmetic impairment and social stigma along with high community costs for patients’ treatment. The increasing prevalence of chronic ulcers, currently reported to be as much as 0.3% in the general population, should stimulate identification of more efficacious therapeutic approaches to achieve complete healing. The strategies of regenerative medicine based on small molecules, biomimetic scaffolds, gene or cell therapy, and electromagnetic field manipulation represent some of the modern therapeutic alternatives for wound healing. Here we review in an integrated, interdisciplinary approach the modern cellular and molecular mechanistic concepts regarding the involvement of extremely low frequency electromagnetic fields (ELF-EMF) in the complex process of tissue repair, with particular focus on chronic wounds. The data analysis supports three main effects of electromagnetic fields on the wound healing pathways: 1) an anti-inflammatory effect, by modulation of cytokine profile that induces the transition of the healing process from a chronic pro-inflammatory to an anti-inflammatory state; 2) a neo-angiogenic effect, by increased endothelial cells proliferation and tubulization and production of fibroblast growth factor (FGF)-2; and 3) a re-epithelialization effect, by stimulation of collagen formation. We believe that utilization of ELF-EMF in larger clinical trials designed to optimize these functional parameters would facilitate a better understanding of ELF-EMF-induced healing mechanisms and lead to improved therapeutic outcomes for this disabling condition which is often totally resistant to treatment.

Mucosal surfaces, such as the vaginal epithelium, are natural barriers to infection that are constantly exposed to bacteria and viruses, and are therefore potential sites of entry for numerous pathogens. The vaginal epithelium can be damaged mechanically, e.g. by the incorrect use of objects such as tampons, and by chemicals that are irritating or corrosive. Consequently, this can lead to an increase in susceptibility to further damage or infection. Pharmaceutical, cosmetic and personal care products that are specifically formulated for application onto human external mucosae can occasionally induce undesirable local or systemic side-effects. Therefore, the compatibility of applied materials with this mucosal surface represents a key issue to be addressed by manufacturers. The most frequently used method for assessing vaginal mucosal irritation is the in vivo rabbit vaginal irritation test. However, the current emphasis in the field of toxicology is to use alternative in vitro methods that reduce, refine, and replace the use of animals, and which model and predict human, not animal, responses. Such an approach is of particular interest to the personal care and cosmetic industries in their effort to comply with European legislative measures, such as the 7th Amendment to the EU Cosmetics Directive that does not permit the marketing of cosmetic products if they, or their ingredients, have been tested for irritation responses in animals. The focus of this review is to provide an overview of the alternative and in vitro tests that are currently available for vaginal mucosal irritation assessment, and which are already used, or may become useful, to establish the safety of newly-designed products for human use.

The vaginal mucosa provides an effective barrier against numerous pathogens as one of the body’s host defense and immune surveillance components. However, some feminine-care and cosmetic products may induce irritation of the vaginal epithelium, consequently making the tissues more susceptible to infections. Therefore, it is important that the compatibility of newly developed cosmetic or personal care products with the human mucosal surface be assessed before the product is marketed. The most frequently used test to screen for vaginal mucosal irritation is the in vivo rabbit vaginal irritation model. However, the current emphasis and preference in toxicology is to use alternative, in vitro methods that Reduce, Refine, or Replace the use of animals in testing programs. To that end, a clear understanding of the current status, applicability, and limitations of available alternative tests for vaginal irritation assessment is critical when companies are building their safety testing strategies. We present an overview of the available alternative and in vitro techniques for vaginal irritation assessment, from simple cell cultures to more complex explants and reconstructed tissues. We further assess their advantages and disadvantages compared to whole animal test systems and their role in the safety assessment strategy used for a wide array of active ingredients or final formulations.

With the recent advancements and adoption of alternative methods to animal testing , and the global harmonization of the world commerce, the importance of providing education, throughout the world, on alternative methods with regulatory standing is becoming increasingly necessary. In particular, 1) the specifics of the in vitro test method protocols, 2) the reliability and relevance of the method for regulatory purposes, 3) the importance of ensuring good laboratory practices, and 4) the necessity for a laboratory to demonstrate proficiency, calls for education preferentially based on practical demonstrations and/or hands-on-training. Such training is essential to scientists performing in vitro tests, and is also key for regulators to make critical assessments of the in vitro data applied to their specific needs. Furthermore, such training could favor standardization of regulatory assessment and decisions on hazard properties of chemicals across the world. A critical step is the examination and interpretation of the resulting data for regulatory purposes. It is expected that when familiarized to the biological basis and relevance of alternative methods and the way in which the resulting data can be interpreted and utilized, the more acceptance will these methods gain.

In vitro assays evaluating ocular irritation potential are routinely used by personal care companies. Two of these in vitro assays include the Chorioallantoic Membrane Vascular Assay (CAMVA) and the Bovine Corneal Opacity and Permeability Assay (BCOP). These assays do not require the use of live animals, provide reliable predictive data and are rapid to conduct. The BCOP uses excised bovine corneas to predict ocular irritation. The CAMVA uses the vascular network of fertilized chicken eggs as a conjunctival model to predict eye irritation. Both BCOP and CAMVA have been used for over fifteen years for product development, worker safety, and safety claims substantiation.

This poster describes procedures and considerations for demonstrating the inter-laboratory reliability of the BCOP and CAMVA. It is important to have a valid assay that can be implemented consistently at several different laboratories. For Kao Brands Company, a large BCOP and CAMVA database exists that covers multiple consumer product categories such as hair shampoos, skin cleansers, and hair styling sprays (containing ethanol). Therefore, a proper review of candidate laboratories is important for seamlessly generating consistent results that can be used for assessing potential ocular irritation of new products. First, a candidate laboratory should be audited for proper facility operation and personnel training. Second, the laboratory’s use of Good Laboratory Practices (GLPs) should be reviewed. Third, reference materials with known BCOP and CAMVA data (one irritant and two non-irritants for initial assessment) should be tested at each new laboratory for verification of proper assay performance.

The purpose of this analysis was to evaluate the Reconstructed Human Epidermis (RhE) model as an in vitro method to predict skin corrosivity (OECD 431) for acid products with extreme pH (?2) when compared with in vivo data and the AISE Method (The Worst Case Table) of classification. Extreme pH can be a useful predictor of irritation but may lead to over classification in weakly buffered systems. Our objective was to determine whether the RhE method could accurately identify corrosive and non-corrosive acid products. When compared with the in vivo data, 4/7 products tested using the RhE method predicted the same skin classification. The skin classification of the remaining three formulas was over-predicted when compared with the in vivo data. There were no products for which the RhE under-predicted the skin classification when compared to the in vivo results. When compared with The AISE Method (which considers the results of the EU conventional method calculation and pH/acid reserve), 8/23 products tested using a RhE method predicted the same skin classification. The skin classification of the remaining fifteen formulas was over-predicted when compared with the AISE Method. There were no products in which the RhE under-predicted the skin classification when compared to the AISE method. Overall, the RhE did not reliably identify non-corrosive formulations when compared to either the in vivo data or the AISE Method. This presents significant challenges under hazard classification guidelines such as the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), which requires testing with a validated in vitro method to confirm a non-corrosive classification for an extreme pH product.