Use of the Bovine Udder Skin Model to Evaluate the Tolerability of Mesem Cosmetic Cream

Christa Raak, Friedrich Molsberger, Wolfgang Pittermann, Mathias Bertram, Sibylle Robens and Thomas Ostermann

Observational studies of Mesem cream (based on Mesembryanthemum crystallinum L. plant extract) found that it had positive effects on skin hydration and smoothing of the skin. However, some patients reported skin irritation effects. The current study evaluated the skin tolerability of Mesem cream, as compared to the carrier cream (without the active ingredient), by using the isolated perfused bovine udder skin model. The primary outcomes investigated were cytotoxicity (i.e. cell viability), assessed with the MTT assay, and irritancy and inflammation, assessed by measuring PGE2 tissue levels. A total reaction score was calculated by combining the results for each parameter. In the case of a single topical application, significant differences were found between the carrier cream and the Mesem cream. While the application of carrier cream resulted in low cytotoxicity (–8.4% change in viability, as compared to the untreated control), the Mesem cream was more cytotoxic (–18.7% change). In addition, one hour after application, PGE2 levels were higher in Mesem cream-treated skin, as compared to carrier cream-treated skin (16.6% versus 11.3%). Further experiments (tape-stripped skin and repeated application) also found significant differences between the two creams in the results obtained. Evaluation of the effectiveness, safety and tolerability of phyto-cosmetic products is important. Our results confirmed the findings of two previous human observational studies (the human patch test and open application study). Future experiments to understand the underlying principles of its effectiveness, safety and tolerability should include extracts of M. crystallinum L. juice, as well as the Mesem cream itself.
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Can Cytotoxic Effects Induced by Industrial Chemicals be Time-dependent?

Ewa Kuchowicz and Konrad Rydzynski

Short-term and delayed cytotoxic effects of selected water-soluble and waterinsoluble industrial chemicals (dimethyl sulphoxide, ethyl alcohol, methyl alcohol, ammonium nitrate, benzalkonium chloride, butoxyethanol and propylene glycol) were tested on confluent 3T3-L1 mouse fibroblasts by using the neutral red uptake (NRU) assay and the MTT assay. The NRU and MTT assays were performed after exposure to a chemical for 10 minutes and 3 hours and then again 7 days later. The results indicate that the system of testing used permits the assessment of both early and delayed cytotoxic effects of different classes of chemicals. Our experiments revealed that three out of the seven substances tested (butoxyethanol, dimethyl sulphoxide and propylene glycol) exhibited similar cytotoxic effects when assessed after exposure for 10 minutes and 7 days later. The results for ammonium nitrate, ethyl alcohol and methyl alcohol, tested after exposure for 10 minutes and 7 days later, did not significantly differ from each other. However, we noted that the cytotoxic effects observed 7 days after exposure were more pronounced than those found for the same concentration, after exposure for 3 hours. One chemical tested (benzalkonium chloride) had almost no cytotoxic effect after exposure for 10 minutes and 3 hours, but showed a strong cytotoxic effect 7 days after exposure. Our study indicated that the cytotoxic effect might be time-dependent in some chemicals, thus inducing a delayed effect in vitro. In our opinion, it is advisable to assess the cytotoxicity of a chemical after exposure for 3 hours (for immediate effects) and again 7 days later (for delayed effects).
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The In Vitro Acute Skin Irritation of Chemicals: Optimisation of the EPISKIN Prediction Model within the Framework of the ECVAM Validation Process

José Cotovio, Marie-Hélène Grandidier, Pascal Portes, Roland Roguet and Gilles Rubinstenn

In view of the increasing need to identify non-animal tests able to predict acute skin irritation of chemicals, the European Centre for the Validation of Alternative Methods (ECVAM) focused on the evaluation of appropriate in vitro models. In vitro tests should be capable of discriminating between irritant (I) chemicals (EU risk: R38) and non-irritant (NI) chemicals (EU risk: “no classification”). Since major in vivo skin irritation assays rely on visual scoring, it is still a challenge to correlate in vivo clinical signs with in vitro biochemical measurements. Being particularly suited to test raw materials or chemicals with a wide variety of physical properties, in vitro skin models resembling in vivo human skin were involved in prevalidation processes. Among many other factors, cytotoxicity is known to trigger irritation processes, and can therefore be a first common event for irritants. A refined protocol (protocol15min–18hours) for the EPISKIN model had been proposed for inclusion in the ECVAM formal validation study. A further improvement on this protocol, mainly based on a post-treatment incubation period of 42 hours (protocol15min–42hours), the optimised protocol, was applied to a set of 48 chemicals. The sensitivity, specificity and accuracy with the MTT assay-based prediction model (PM) were 85%, 78.6% and 81.3% respectively, with a low rate of false negatives (12%). The improved performance of this optimised protocol was confirmed by a higher robustness (homogeneity of individual responses) and a better discrimination between the I and NI classes. To improve the MTT viability-based PM, the release of a membrane damage marker, adenylate kinase (AK), and of cytokines IL-1α and IL-8 were also investigated. Combining these endpoints, a simple two-tiered strategy (TTS) was developed, with the MTT assay as the first, sort-out, stage. This resulted in a clear increase in sensitivity to 95%, and a fall in the false-positive rate (to 4.3%), thus demonstrating its usefulness as a “decision- making” tool. The optimised protocol proved, both by its higher performances and by its robustness, to be a good candidate for the validation process, as well as a potential alternative method for assessing acute skin irritation.
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An Evaluation of the In Vitro Cytotoxicities of 50 Chemicals by using an Electrical Current Exclusion Method versus the Neutral Red Uptake and MTT Assays

Toni Lindl, Birgit Lewandowski, Sonya Schreyögg and Andrea Stäudte

According to the 2001 National Institutes of Health guidance document on using in vitro data to estimate in vivo starting doses for acute toxicity, the performance of the electrical current exclusion method (ECE) was studied for its suitability as an in vitro cytotoxicity test. In a comparative study, two established in vitro assays based on the quantification of metabolic processes necessary for cell proliferation or organelle integrity (the MTT/WST-8 [WST-8] assay and the neutral red uptake [NRU] assay), and two cytoplasm membrane integrity assays (the trypan blue exclusion [TB] and ECE methods), were performed. IC50 values were evaluated for 50 chemicals ranging from low to high toxicity, 46 of which are listed in Halle’s Registry of Cytotoxicity (RC). A high correlation was found between the IC50 values obtained in this study and the IC50 data published in the RC. The assay sensitivity was highest for the ECE method, and decreased from the WST-8 assay to the NRU assay to the TB assay. The consistent results of the ECE method are based on technical standardisation, high counting rate, and the ability to combine cell viability and cell volume analysis for detection of the first signs of cell necrosis and damage of the cytoplasmic membrane caused by cytotoxic agents.
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The Skin Compatibility of Distilled Tall Oils: Evaluation With the Bovine Udder Skin In Vitro Model System

Wolfgang Pittermann, Fredrik Hopfgarten and Manfred Kietzmann

Distilled tall oil (DTO) is a natural product, often added as an emulsifying ingredient in cutting fluids used as lubricants and coolants in metal working. The in vitro model used to test the skin compatibility of these substances, was the isolated perfused ex vivo bovine udder skin (BUS) model. After three exposure periods (0.5, 1, and 5 hours), cytotoxic effects were determined by using the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and tissue levels of the pre-inflammatory mediator prostaglandin E2 (PGE2) in treated whole skin biopsies were assessed by using an enzyme immunoassay. The BUS standard study design, involving a single application, was previously developed to investigate the skin irritation potential of cosmetics and chemicals. In the current study, four different batches of undiluted DTO, and tall oil fatty acids as a reference compound, were applied both singly and repeatedly (three times), under open conditions which were in line with the potential usage conditions in the work place. Under the standardised single application conditions, no major differences in cytotoxic effects or PGE2 levels between the samples were apparent, so no indication of a skin irritation potential could be concluded. This result is in accordance with prior in vivo studies for acute dermal toxicity. Under repeated application conditions, signs of cytotoxicity were observed after the application of one of the DTO samples, which was known to be derived from different raw materials. Therefore, it was concluded that, generally, the presence of DTO at a concentration of up to 10% in cutting fluids, is not expected to result in any DTO-related deterioration of the skin.
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Validation Study of the In Vitro Skin Irritation Test with the LabCyte EPI-MODEL24

Hajime Kojima, Yoko Ando, Kenji Idehara, Masakazu Katoh, Tadashi Kosaka, Etsuyoshi Miyaoka, Shinsuke Shinoda, Tamie Suzuki, Yoshihiro Yamaguchi, Isao Yoshimura, Atsuko Yuasa, Yukihiko Watanabe11 and Takashi Omori

A validation study on an in vitro skin irritation assay was performed with the reconstructed human epidermis (RhE) LabCyte EPI-MODEL24, developed by Japan Tissue Engineering Co. Ltd (Gamagori, Japan). The protocol that was followed in the current study was an optimised version of the EpiSkin protocol (LabCyte assay). According to the United Nations Globally Harmonised System (UN GHS) of classification for assessing the skin irritation potential of a chemical, 12 irritants and 13 non-irritants were validated by a minimum of six laboratories from the Japanese Society for Alternatives to Animal Experiments (JSAAE) skin irritation assay validation study management team (VMT). The 25 chemicals were listed in the European Centre for the Validation of Alternative Methods (ECVAM) performance standards. The reconstructed tissues were exposed to the chemicals for 15 minutes and incubated for 42 hours in fresh culture medium. Subsequently, the level of interleukin-1 alpha (IL-1α) present in the conditioned medium was measured, and tissue viability was assessed by using the MTT assay. The results of the MTT assay
obtained with the LabCyte EPI-MODEL24 (LabCyte MTT assay) demonstrated high within-laboratory and between-laboratory reproducibility, as well as high accuracy for use as a stand-alone assay to distinguish skin irritants from non-irritants. In addition, the IL-1α release measurements in the LabCyte assay were clearly unnecessary for the success of this model in the classification of chemicals for skin irritation potential.
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