"Properties of a sericin-containing buffering shampoo', W. Engle, U. Hoppe, W. Pape, G. Sauermann, Reprint (vol. 2 Mar./Apr. 1987) from Medical Cosmetology 1,91-110 (1987). Copy of Application Serial No. 524,259, filed Aug. 18, 1983 "Cosmetics Containing Sericin', corresponds to Offen. 3,233,388. Yamazaki, 1974, vol. 80, p. 19390a, Chem. Abs. Prodenta, 1975, vol. 83, p. 33036h, Chem. Abs. Proserpio, 1984, vol. 101, p. 157457f, Chem. Abs. Hoppe, et al., 1984, vol. 100, p. 215305x, Chem. Abs. Weil, 1972, vol. 76, p. 87536v, Chem. Abs. Primary Examiner-Dale R. Ore Attorney, Agent, or Firm-Sprung Horn Kramer & Woods

The complex development process of cosmeceuticals is both a science and an art involving multiple stages and many different disciplines. A number of key factors must be evaluated and considered to insure the final product is both efficacious and aesthetically pleasing to the end user. From the initial consumer insights to the final product launch, many steps are taken to deliver a safe, effective product that delivers the desired product benefits. The final product launch is the culmination of years of scientific research, numerous product optimizations, extensive stability, safety, clinical and consumer testing, various quality tests, legal/regulatory evaluations, and marketing claims substantiation. Cosmeceuticals are a fast-paced and continuously evolving area of research driven by the competitive marketplace, changing trends and high consumer expectations. New technologies, more effective formulations, and great advances in skin research will offer a bright future for this growing segment.

We measured consumer exposure to formaldehyde (FA) from personal care products (PCP) containing FA-releasing preservatives. Six study subjects applied facial moisturiser, foundation, shower gel, shampoo, deodorant, hair conditioner, hair styling gel or body lotion at the 90th percentile amount of EU PCP consumer use. FA air concentrations were measured in the empty room, in the presence of study subjects prior to PCP use, and for one hour (breathing zone, area monitoring) after PCP use. The mean FA air concentration in the empty bathroom was 1.32 ± 0.67 μg/m3, in the presence of subjects it was 2.33 ± 0.86 μg/m3. Except for body lotion and hair conditioner (6.2 ± 0.1.9 or 4.5 ± 0.1.5 μg/m3, respectively), mean 1-h FA air concentrations after PCP use were similar to background. Peak FA air concentrations, ranging from baseline values (2.2 μg/m3; shower gel) to 11.5 μg/m3 (body lotion), occurred during 0–5 to 5–10 min after PCP use. Despite of exaggerated exposure conditions, FA air levels were a fraction of those considered to be safe (120 μg/m3), occurring in indoor air (22–124 μg/m3) or expired human breath (1.4–87 μg/m3). Overall, our data yielded evidence that inhalation of FA from the use of PCP containing FA-releasers poses no risk to human health.

Photoactivation and binding of photoactive chemicals to proteins is a known prerequisite for the formation of immunogenic photoantigens and the induction of photoallergy. The intensive use of products and the availability of new chemicals, along with an increasing exposure to sun light contribute to the risk of photosensitizing adverse reactions. Dendritic cells (DC) play a pivotal role in the induction of allergic contact dermatitis. Human peripheral blood monocyte derived dendritic cells (PBMDC) were thus perceived as an obvious choice for the development of a novel in vitro photosensitization assay using the modulation of cell surface protein expression in response to photosensitizing agents. In this new protocol, known chemicals with photosensitizing, allergenic or non-allergenic potential were pre-incubated with PBMDCs prior to UVA irradiation (1 J/cm(2)). Following a 48 h incubation, the expression of the cell surface molecules CD86, HLA-DR and CD83 was measured by flow cytometry. All tested photosensitizers induced a significant and dose-dependent increase of CD86 expression after irradiation compared to non-irradiated controls. Moreover, the phototoxicity of the chemicals could also be determined. In contrast, (i) CD86 expression was not affected by the chosen irradiation conditions, (ii) increased CD86 expression induced by allergens was independent of irradiation and (iii) no PBMDC activation was observed with the non-allergenic control. The assay proposed here for the evaluation of the photoallergenic potential of chemicals includes the assessment of their allergenic, phototoxic and toxic potential in a single and robust test system and is filling a gap in the in vitro photoallergenicity test battery.

Allergic contact dermatitis is a delayed T-cell mediated allergic response associated with relevant social and economic impacts. Animal experiments (e.g. the local lymph node assay) are still supplying most of the data used to assess the sensitization potential of new chemicals. However, the 7th amendment to the EU Cosmetic Directive will introduce a testing ban for cosmetic ingredients after 2013. In vitro alternative methods are thus being actively developed. Although promising results have been obtained with cell lines, their reduced functionality and inherent genomic instability led us to reinvestigate the use of peripheral blood monocyte-derived dendritic cells (PBMDCs) for the establishment of a reliable in vitro sensitization test. To solve the issues associated with the use of primary cells, the culture and exposure conditions (cytokine concentrations, incubation time, readout, pooled vs. single donors and cytotoxicity) were re-assessed and optimized. Here we propose a stable and reproducible protocol based on PBMDCs. This should allow a wider acceptance of PBMDCs as a reliable test system for the detection of human skin sensitizers and the inclusion of this protocol in an integrated testing strategy.

Based on the current weight of evidence of all available data, the risk for humans from the use of nano-structured titanium dioxide (TiO2) or zinc oxide (ZnO) currently used in cosmetic preparations or sunscreens is considered negligible. There is a large body of information that when viewed in its entirety is considered as sufficient to demonstrate that these nano-structured ultraviolet (UV) filters, irrespective of various treatments (coatings) or crystalline structure, can be regarded as safe for use at concentrations up to 25% in cosmetic products to protect the skin from harmful effects of solar UV radiation. "Nano" TiO2 and ZnO formulated in topically applied sunscreen products exist as aggregates of primary particles ranging from 30-150 nm in size. These aggregates are bonded such that the force of sunscreen product application onto the skin would have no impact on their structure or result in the release of primary particles. Multiple studies have shown that under exaggerated test conditions neither nano-structured TiO2 nor ZnO penetrates beyond the stratum corneum of skin. Further, the distribution and persistence of these nano-structured metal oxides is the same compared to larger pigment-grade (i.e., > 100 nm) particles, demonstrating equivalence in the recognition and elimination of such material from the body. Finally, the in vitro genotoxic and photogenotoxic profiles of these nano-structured metal oxides are of no consequence to human health. Whereas the most logical, straightforward conclusion based on data from internationally-recognized guideline studies and current 20+ year history of human use is that nano-structured TiO2 and ZnO are safe, there will continue to be questions as "nano" conjures images of technology gone awry. Despite this rather sober view, the public health benefits of sunscreens containing nano TiO2 and/or ZnO outweigh human safety concerns for these UV filters.

In spite of over 20 years of effort, no single in vitro assay has been developed and validated as a full regulatory replacement for the Draize Eye Irritation test. However, companies have been using in vitro methods to screen new formulations and in some cases as their primary assessment of eye irritation potential for many years. The present report shows the outcome of an Expert Meeting convened by the European Centre for the Validation of Alternative Methods in February 2005 to identify test strategies for eye irritation. In this workshop test developers/users were requested to nominate methods to be considered as a basis for the identification of such testing strategies. Assays were evaluated and categorized based on their proposed applicability domains (e.g., categories of irritation severity, modes of action, chemical class, physicochemical compatibility). The analyses were based on the data developed from current practice and published studies, the ability to predict depth of injury (within the applicable range of severity), modes of action that could be addressed and compatibility with different physiochemical forms. The difficulty in predicting the middle category of irritancy (e.g. R36, GHS Categories 2A and 2B) was recognized. The testing scheme proposes using a Bottom-Up (begin with using test methods that can accurately identify non-irritants) or Top-Down (begin with using test methods that can accurately identify severe irritants) progression of in vitro tests (based on expected irritancy). Irrespective of the starting point, the approach would identify non-irritants and severe irritants, leaving all others to the (mild/moderate) irritant GHS 2/R36 categories.

Dendritic cells (DC) are crucial in contact allergy induction. Here we report an assay based on monocyte-derived DCs applying CD86 surface expression changes and viability as indicators of lapten induced activation. Compared to LLNA data all tested substances were identified correctly. By using, DCs from single donors instead of DC-pools, the data were improved anticeably. This alternative method provides a basic application for assessing the allergic potential of chemicals.

In the complex aetiology of human actinomykoses, various Actinomyces species, especially A. israelii, play the pathogenetically leading role in the mixed flora so typical for these diseases. A number of other microaerophilic and anaerobic bacteria are also part of such mixed flora. Since these "concomitant" bacteria are themselves potentially pathogenic, it is important to have data on antibiotic sensitivity for the entire spectrum of microorganisms, to achieve effective and economic chemotherapy of human actinomykoses. Minimal inhibitory concentrations of antibiotics were determined for Actinomyces and the most important concomitant bacteria from actinomykotic processes: Actinobacillus actinomycetem-commitants, Bacteroides of the Melaninogenicus group, B. fragilis, B. theta-iotaomicron, Fusobacterium nucleatum, Fusobacterium spp. and Propionibacteria. The results indicate that aminopenicillines are the drug of first choice, if in the actinomykoses there are no Bacteroides strains resistant to beta-lactam antibiotics. These tests further indicate that clinically the combination of aminopenicillin and clindamycin, as well as aminopenicillin and metronidazol, are indicated. Minocycline and especially cefoxitin, a beta-lactamase resistant cephalosporin, inhibit with few exceptions all bacteria which are potentially involved in actinomykotic processes.

The European Partnership for Alternative App roaches to Animal Testing (EPAA) is a joint initiative of the European Commission and a number of companies and trade federations active in various industrial sectors. The Partnership was launched on 7 November 2005, at a major conference entitled Europe Goes Alternative, by Commissioners Ver heugen and Potočnik and industry representatives. The purpose of the Partnership is to promote the development of new ‘Three R’ methods (to refine, reduce, and replace the use of animals) as modern alternative approaches to safety testing. The Partnership’s work will focus on mapping existing research, developing new alternative approaches and strategies, and promoting communication, education, and the validation and acceptance of alternative approaches. In this article, EPAA Working Group 5 focuses on the validation process, identifying factors which might delay or hamper the validation of alternative methods.

The health of the ocular surface is intimately linked with quality of vision, and is also a determining factor in the success of surgical procedures. The ocular surface encompasses the outer structures of the eye (cornea, conjunctiva, and lids), the lacrimal gland and tear film and the innervation and immune structures that link them (Stern et al., 2004). Interactions within and between these components maintain the barrier to the environment and modulate responses to external stimuli (Cook et al., 2001; Stramer et al., 2003; Wilson et al., 2003; Hazlett, 2004; Holan et al., 2004; Wilson et al., 2004; Lema and Duran, 2005; Narayanan et al., 2005). The Colipa Strategy for the Development of in vitro Alternative Methods focuses on physiological processes – from organ level to gene activation – that can be used to monitor responses to toxicants and recovery from injury. Among the programme aims, central to the replacement R of the 3Rs and to cell based assays, is identifying quantitative endpoints predictive of the nature and severity of injury. The ultimate aim is to replace the Draize test – currently the gold standard of ocular toxicity testing – with a scientifically valid animal-free strategy. We needed to establish the minimal degree of complexity which is necessary and sufficient to represent the human cornea in cell culture models of toxicity. We have assessed the behaviour of undisturbed cultures and the response to a single toxicant application in constructs from monolayers to threedimensional cultures comprising stratified human corneal epithelia and quiescent human stromal cells, to probe stability and correlates to injury and recovery. Starting from the premise that depth (cell types) and area are the defining characteristics of an injury (Jester et al., 1998a; Jester et al., 1998b; Jester et al., 2000; Jester et al., 2001), we have explored endpoints related to metabolism and barrier function and sought correlations with patterns of cytokines, mediators that are known to be secreted by, and influence, the different cell types of the ocular surface. For the refinement R of the 3Rs and, more importantly, to anchor the in vitro tests with in vivo physiology, the add-on benefit of the latter endpoints is that signal molecules are readily detectable in (human) tears, and available through non-invasive tests.

The COLIPA eye irritation programme for development of in vitro eye irritation assays incorporates integrated research projects and collaborative activities with external partners. The research projects focus on understanding mechanisms of eye injury and identification of new in vitro endpoints more predictive of the in vivo human response to chemical injury resulting in new or improved in vitro methods that would proceed to formal validation. There are three projects: 1) investigation of whether kinetics/patterns of change in physiological function/signals of injury released from the cornea in vitro can predict a chemical's potential to damage the eye with a focus on recovery; 2) identification of endpoints related to magnitude of injury and repair in 3-dimensional human corneal constructs and 3) a genomics project using a pattern recognition approach to identify new endpoints for injury and repair for potential use in current/future in vitro assays. Equally important to achieve validated in vitro methods are other activities such as continued development/ optimisation of currently existing models and industry collaboration with academia, external scientific organisations and regulators. COLIPA is working with producers of Human Reconstructed Tissue (HRT) eye irritation models to further develop/optimise these models and with ECVAM through participation in its Eye Irritation Task Force and provision of an independent bio-statistician for post-hoc analysis of current in vitro methods.

Regulatory requirements: According to the current Notes for Guidance of the Scientific Committee on Cosmetic Products and Non-Food Products (SCCNFP), cosmetic ingredients and mixtures of ingredients absorbing UV light (in particular UV filter chemicals used, for example, to ensure the light stability of cosmetics or used in sun protection products) should be tested for acute phototoxic and photogenotoxic potential. Testing for photosensitisation (immunological photoallergy) potential is not specifically required, but it is nevertheless often performed. Acute phototoxicity: Due to a thorough multi-stage and multi-centre validation trial (1992-1998) the In Vitro 3T3 Neutral Red Uptake Phototoxicity Test (3T3-NRU-PT) had already gained acceptance by the SCCNFP in 1998, and it is recommended by the EMEA/CPMP as a basic preclinical test for acute phototoxicity. It was accepted as Method No. 41 in Annex V to Directive 67/548/EEC in the year 2000, and was accepted as the new Test Guideline 432 by the OECD in 2002. The 3T3-NRU-PT is regarded as a basic screen for identifying acute phototoxic potential. Two additional in vitro tests, formally evaluated in controlled blind trials, the RBC Phototoxicity Test (RBC-PT) and the Human 3-D Skin Model Phototoxicity Test (H3D-PT), are regarded as useful and important adjunct tests to overcome some limitations of the 3T3-NRU-PT, namely the fairly low UVB tolerance of the 3T3 fibroblasts and the inability to model the bioavailability of test materials topically applied to the skin. In addition, the RBC-PT permits an evaluation of the phototoxic mechanisms involved. In conclusion, the identification of acute phototoxic hazards is now regarded as being sufficiently covered by in vitro tests, so that animal testing for that endpoint can now be 100% replaced. Photogenotoxicity: In the area of photogenotoxicity, almost the whole battery of in vitro genetic toxicity tests have been (or are currently being) converted into test protocols of photogenotoxicity tests. Tests exclusively predictive for gene mutation, for example, the Photo-Ames (P-Ames) Test and the Photo-Thymidine Kinase Test (P-TKT), have become less important than tests for clastogenic effects (for example, the Photo-Chromosome Aberration Test [P-CAT] and the Photo-Micronucleus Test [P-MNT]). In addition, a number of promising indicator tests, such as the Photo-Comet Assay (P-Comet) have been developed. Although routinely used, to date none of the new photogenotoxicity tests have been formally validated. Therefore, the P-MNT and the P-Comet are currently being evaluated in a formal interlaboratory validation study. It is expected that these in vitro photogenotoxicity test methods may become available as validated and accepted methods within the next five years. Photoallergy (Photosensitisation): In the area of photoallergy (photosensitisation), as development of predictive in vitro tests for delayed contact sensitisation (allergenicity) potential without the involvement of light, due to a lack of ability to model the complex mechanisms underlying allergy, no promising in vitro methods to predict photo-sensitisation potential are currently in sight (see the section on skin sensitisation). One in vitro screening method, which models the covalent binding of a light activated chemical to human serum albumin, may become relevant. However, while the binding of an excited chemical to proteins is a prerequisite for photoallergy, this is not a sufficient predictor on its own. The only promising alternatives currently under development are in vivo refinements, like the Photo Local Lymph Node Assay (PLLNA). Once a reliable and predictive in vitro test battery and strategy for the assessment of dark sensitisation potential have been developed and accepted, their adaptation into similar photosensitisation testing will become possible.

This is the report of the forty-second of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM was established in 1992 by the Commission of the European Communities (EC) to carry into effect article 23 of Council Directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes (1). ECVAM is part of the Institute for Health & Consumer Protection (IHCP) of the Joint Research Centre of the European Commission, located ATLA 28, 777–814, 2000 777

In the present paper, we describe and analyse the performance and the results of the pollen tube growth (PTG) test applied to the COLIPA international validation study of in vitro alternatives to the Draize eye irritation test. The PTG test, based on photometric quantification of in vitro pollen tube mass production, was used by three independent laboratories to estimate the acute eye irritation potentials of 23 ingredients and 32 cosmetic formulations. Basing on historical Draize test data and on IC(50) values of previously tested cosmetic formulations, a mathematical formula was generated to predict rabbit eye irritation potentials from PTG test results. Statistical evaluation of the calculated modified maximal average scores (MMAS) revealed a high prediction capability of the PTG test in regard to the finished formulations but a relatively low one for alcohols, higher concentrated cationic surfactants, and acidic and alkaline materials. Furthermore, our results indicated that the PTG test was able to produce precise IC(50) values without any limitations from all of the 55 test substances with good intra- and interlaboratory reproducibility. From these findings we suggest that the PTG test is not a validated test at present but is considered to be a potent candidate for further validation processes. For this purpose an additional prediction model for ingredient classes as mentioned above must be generated.