Listed below are the latest publications added to IIVS’ online document library. To search our library, please visit our Resources page.
Background Determination of the potential for individual chemicals and product ingredients to induce allergic contact dermatitis (skin sensitization) is a key toxicological endpoint for the screening of novel ingredients used in consumer and industrial products. Although in vivo methods exist to evaluate the skin sensitization potential of chemicals, in vitro non-animal test methods have been developed using human cell lines to predict human skin sensitizers. In vitro human cell-based systems have been developed in response to international regulatory requirements prohibiting the use of animals in research, and to meet the needs of corporations proactively choosing to eliminate the use of animals in safety testing. The KeratinoSens assay was developed by Givaudan, and recently evaluated in an international multi-laboratory validation exercise. The KeratinoSens assay is a human immortalized keratinocyte cell-based reporter gene assay which is designed to identify chemicals likely to induce skin sensitization in humans. A feature of all chemical allergens is their intrinsic electrophilicity (or their potential to be transformed to electrophilic chemicals) and their reactivity with skin proteins to form haptens.
Materials and Methods Mechanistically, the intercellular Nrf-2-electrophile sensing pathway comprised of the repressor protein Keap1, the transcription factor Nrf2, and the antioxidant response element (ARE), is capable of detecting skin sensitizers. In the KeratinoSens assay, the induction of a luciferase gene, under the control of the antioxidant response element (derived from the human gene AKR1C2 gene) is determined by measuring the relative light output of treated cells. In parallel, viability of the treated cells is measured using the MTT assay.
Results and Discussion In the “ring trial” validation, 28 chemicals (19 sensitizers of varying potencies, and 9 nonsensitizers) were evaluated in 5 laboratories, and at least 3 experiments per chemical. The predictive capacity of the assay was found to be similar between labs and ranged from 85.7% to 96.4%. Subsequent application of the assay is targeted at further defining the applicability and predictivity of the assay by testing more neat chemicals, chemical mixtures, industrial solvents, and complex product matrices. Thus far, over 150 chemicals have been evaluated using the KeratinoSens assay and the results indicate a good predictive value (~79.5%). The results indicate that the KeratinoSens assay may be a relevant and reliable method for evaluating a broad range of materials. The presentation will highlight the assay performance and lessons learned from the validation program.
Background The Bovine Corneal Opacity and Permeability (BCOP) assay is an ex vivo test used to evaluate ocular irritation. According to the OECD Test Guideline (TG) 437, the BCOP assay can be used to identify chemicals which induce severe/corrosive eye irritation and those that do not require classification. However, BCOP has historically under-predicted certain anionic surfactants, when tested according to the standard liquid protocol. TG 437 specifies that liquid and solid surfactants may be tested as 10% aqueous dilutions for 10 minutes (although alternate dilutions and exposure times may be conducted with scientific rationale). The relevant guidance document (GD) No. 160 suggests that solid and concentrated liquid surfactants may be diluted to 10% for testing. However, GD No. 160 further directs that surfactant-based formulations are usually tested neat, but could be diluted with justification, imparting some confusion in identifying the most appropriate test methods. Additionally, as part of the EPA classification of ocular irritation, the BCOP assay may be used to assess anti-microbial products with cleaning claims. Such products may contain surfactants and are generally tested neat for classification purposes.
Methods Since neither the basis for selecting the appropriate surfactant test methods, nor the justification for modifications are clearly presented in TG 437 or GD No. 160, we present on the testing of a few common surfactant ingredients, including sodium lauryl sulfate (SLS), Triton X-100, and benzalkonium chloride, and surfactant based formulations in the BCOP assay using standard and modified dilutions and exposures to elucidate the impact of these variables on eye irritation prediction.
Results and Discussion As examples, in vitro scores of 20.7, 28.4, and 28.3 were obtained when testing SLS at concentrations of 50, 20, and 10% for 10 minutes, showing that irritation responses were not fully concentration-dependent. As a complement to the BCOP assay, histopathology was performed to assess the surfactant-induced corneal changes. Based upon these results, a framework for testing surfactant ingredients and surfactant-based formulations is proposed.
A TRPV1 expressing clone of the human SH-SY5Y neuroblastoma cell line (Figure 1) was obtained by stable transfection, using puromycin-containing selection medium. Prior to Ca2+ measurements the TRPV1-SH-SY5Y cells were cultured in 96-well plates to confluency. Acute increase in the intracellular free Ca2+ level was measured in a semi-HTS fluorescence reader (FlexStation II, Molecular Devices) using Fura-2/AM. The ratio of fluorescence at 340 (Ca2+-bound Fura-2)/380 (Fura-2) nm excitatory wavelengths was registered without interruption before and during the 2 min exposure to the test compounds. The mean value (% increase of basal Ca2+ level) from triplicate wells in the 96-well plate was monitored for each concentration from each experiment. The TRPV1 antagonist capsazepine was added simultaneously with each concentration of the chemicals in three sister wells to confirm TRPV1-mediated Ca2+ influx. The intracellular Ca2+ increase induced by the specific TRPV1-agonist capsaicin was set to 100% response for each experiment and the effect of the test products was calculated as percent of the capsaicin induced response. All test compounds were diluted in HKR-buffer and the addition to the cells was performed robotically during measurements by the FlexStation II reader.
Validation of In Vitro and Clinical Safety Assessment of Leave-On Body Lotions Using Post-Marketing Adverse Event Data
Behentrimonium chloride (BTC) is a straight-chain alkyltrimonium chloride compound commonly used as an antistatic, hair conditioning, emulsifier, or preservative agent in personal care products. Although the European Union restricted the use of alkyltrimonium chlorides and bromides as preservatives to ≤0.1%, these compounds have been safely used at ≤5% in hundreds of cosmetic products for other uses than as a preservative. In vitro, clinical, and controlled consumer usage tests in barrier-impaired individuals were conducted to determine if whole body, leave-on skin care products containing 1-5% BTC cause dermal irritation or any other skin reaction with use. BTC-containing formulations were predicted to be non-irritants by the EpiDerm®* skin irritation test and the bovine corneal opacity and permeability (BCOP)/chorioallantoic membrane vascular assay (CAMVA) ocular irritation test battery. No evidence of allergic contact dermatitis or cumulative dermal irritation was noted under the exaggerated conditions of confirmatory human occlusive patch tests. No clinically assessed or self-reported adverse reactions were noted in adults or children with atopic, eczematous, and/or xerotic skin during two-week and four-week monitored home usage studies. These results were validated by post-marketing data for five body lotions, which showed only 0.69 undesirable effects (skin irritation) reported per million shipped consumer units during 2006-2011. No serious undesirable effects were reported during in-market use of the products. Therefore, if formulated in appropriate conditions at 1-5%, BTC will not likely cause dermal irritation or delayed contact sensitization when used in a whole-body, leave-on product.
The Bovine Corneal opacity and Permeability (BCOP) assay can be used for predicting mild, moderate, and severe ocular irritation through quantitative assessment of the changes in opacity and permeability of the bovine cornea. In addition, histological evaluation of the corneas can be performed to assess the depth of damage. The BCOP assay with histology was used to determine the ocular irritation potential of prototype cleaning products with antimicrobial claims according to the guidance provided by the EPA-Office of Pesticide Program (OPP). Several prototype cleaners with similar formulation were evaluated along with a reference material. The results of the BCOP assay showed noticeable differences among the products. The in vitro score, determined by changes in opacity and permeability, of the corneas treated with products ranged from ~15 to 80. These scores indicate mild, moderate, and severe irritation according to the guideline provided in the EPAOPP document. In addition, the histological evaluation of the corneas showed differences in the depth of damage between moderate and severe category products, confirming the in vitro score.
The assay distinguished ocular irritation potential among similar prototypes demonstrating its effectiveness during product development. Additionally, the results demonstrate the utility of the BCOP assay with histology as a stand-alone assay for eye irritancy evaluation in the EPAOPP program.
Screening of Cosmetics Ingredients for Phototoxic Potential Using the In Vitro 3T3 Neutral Red Uptake Phototoxicity Test
Phototoxicity is an acute toxic response after exposure to a phototoxicant and either UV radiation or visible light (UV/VIS). Phototoxicity from substances applied topically typically occurs at the site of photo-irradiation. Phototoxicity is the result of direct cellular damage caused by a non-immunological inflammatory response. Clinically, phototoxicity resembles an exaggerated sunburn (erythema, increased skin temperature, pruritis and edema). Phototoxicity reactions have been reported for both synthetic substances and those which occur naturally (e.g., botanical extracts). Although symptoms generally subside quickly, the potential for substances used in topical products to cause phototoxicity is clearly of concern for manufacturers of cosmetics, personal care and other consumer products. Historically, the potential to cause phototoxicity from substances applied topically was evaluated by utilizing various animal models. However in 1997 the 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU PT) was validated by ECVAM’s Scientific Advisory Committee as an in vitro method for evaluating the phototoxic potential of chemicals shown to absorb in the UV/VIS range. To illustrate the utility of the 3T3 NRU PT as a useful screening tool in the safety evaluation of potential cosmetic ingredients, the results of the evaluation of 42 botanical extracts and 25 synthetic chemicals found to absorb in the UV/VIS range are reported. Most substances evaluated were found not to be phototoxic in vitro; however, 9 substances were identified as potentially/probably phototoxic in the 3T3 NRU PT and were eliminated from further consideration for use as cosmetic ingredients. Several substances found to be non-phototoxic in the 3T3 NRU PT were formulated with other ingredients in a prototype cosmetic formulation and subject to clinical testing. No manifestations of phototoxicity were observed in any of the test subjects in the prototype formulation containing any of the substances identified as non-phototoxic in vitro.
Surfactant Responses in the Bovine Corneal Opacity and Permeability Assay: Points to Consider for In Vitro Eye Irritation Testing
The Bovine Corneal Opacity and Permeability (BCOP) assay is an ex vivo test used to evaluate the ocular irritation of a broad range of chemicals. In the regulatory classification and labeling arena, BCOP can be used to identify severe and corrosive eye irritants according to the OECD Test Guideline (TG) 437. However, BCOP has historically under-predicted certain anionic surfactants, when tested according to the standard liquid protocol. TG 437 specifies that liquid surfactants may be tested as 10% aqueous dilutions for 10 minutes (although alternate dilutions and exposure times may be conducted with scientific rationale), and the relevant guidance document (GD) No. 160 suggests that solid and concentrated liquid surfactants may be diluted to 10% for testing. However, GD No. 160 further directs that surfactant-based formulations are usually tested neat, but could be diluted with justification, imparting some confusion in identifying the most appropriate test methods. Since neither the basis for selecting the appropriate surfactant test methods, nor the justification for modifications are clearly presented in TG 437 or GD No. 160, we present on the testing of sodium lauryl sulfate (SLS) in the BCOP assay, using standard and modified dilutions and exposures, to elucidate the impact of these variables on eye irritation prediction. For example, in vitro scores of 20.7, 28.4, and 28.3 were obtained when testing SLS at concentrations of 50, 20, and 10% for 10 minutes, showing that irritation responses were not fully concentrationdependent, but demonstrated optimally at intermediate doses. When tested using modified exposure times, SLS showed time-related responses, with improvements in irritation predictions at the 20 and 30 minute exposures. Histopathology was performed to assess the surfactant-induced corneal changes. Based upon these results, a framework for testing surfactants, and surfactant-based formulations is proposed.
The inter-laboratory reproducibility of the STE test for assessing eye irritation of cosmetic products
STE test is an in vitro eye irritation test using cell viability as an end point in SIRC cells following just a 5 minute treatment, and the good correspondence has been confirmed between the STE irritation categories (non irritant [NI] and irritant [I]) and GHS categories (NC and category 1 [Cat. 1]/category 2 [Cat. 2]). Generally, cytotoxicity tests using cultured cells have an advantage of being simple, a quick procedure, and a low evaluation cost. The STE test has the advantages not only easy-to-use but also evaluable the eye irritation potential of water insoluble substances by using mineral oil as test vehicle. The STE test is planned for peer review in 2013 and may be accepted as an OECD test guideline for classifying ocular irritation. In this study, the technical transferability and inter-laboratory reproducibility of the STE test were evaluated in 3 contract research laboratories as a naive laboratory.
Application of the KeratinoSens Assay for Prediction of Dermal Sensitization Hazard for Botanical Cosmetic Ingredients
An essential step in the safety review of cosmetic/personal care ingredients is hazard assessment for a series of endpoints, including dermal sensitization potential. In vitro methods have been developed to identify allergic (haptenic) potential for individual chemicals based on electrophilic interaction with marker peptides or cellular target systems. These assays generally use a specific molar ratio of the test chemical to the test system. Botanical extracts are used increasingly in formulas and, as mixtures, specific molar ratios cannot be determined for these assays. Often, the botanical extract portion is a relatively small portion of the complete ingredient. To assess these mixtures, the KeratinoSens assay was selected because it operates over a wide dose range and sets cytotoxicity limits on doses used to measure marker gene expression (Emter et al, 2010 ). In the KeratinoSens assay, the induction of a luciferase gene, under the control of the antioxidant response element (ARE) derived from the human gene AKR1C2 gene, is measured. In parallel, cytotoxicity is assessed by both Neutral Red Uptake (NRU) and MTT assays. Test concentrations ranged up to 1000 μg/mL (of complete ingredient) and a test concentration was considered positive if the relative viability was ≥ 70% and the fold induction of luciferase was 1.5x relative to the solvent controls. The goal of the study was to measure the activity of 3 known sensitizers (gluteraldehyde (GA) [strong], dimethyl maleate (DM) [moderate] and cinnamic aldehyde (CA) [moderate] spiked into four different botanical ingredients (each with a different excipient solvent systems). The “spiked” botanical ingredients were used as the test article as no sensitizing botanical ingredient was available. Activity of the spiked sample was measured relative to the EC1.5 of the neat sensitizer as a function of sensitizer concentration and extract composition. Three independent trials were performed on each test material. No appreciable cytotoxicity was observed with any of the samples. The recovery of the GA spike required at least a ~3 fold increase in concentration relative to the chemical alone and botanical ingredient #3 reduced the activity below detection. The DM and CA showed activity at about the same effective concentrations as the neat chemical although the DM showed reduced activity in botanical ingredient #3 as well. These data suggest that the KeratinoSens assay has the potential to identify electrophile allergens within a botanical ingredient matrix.
Choosing the Appropriate Solvent for Solid Materials Tested in the Bovine Corneal Opacity and Permeability (BCOP) In Vitro Assay
In compliance with OECD Test Guideline 437 for eye irritation (BCOP assay), non-surfactant solid materials are typically tested as 20% dilutions prepared in 0.9% sodium chloride solution, distilled water, or other solvent that has been demonstrated to have no adverse effects on the test system. However, the limited solubility of some chemicals adds technical challenges in finding a vehicle that would ensure the material’s availability to the excised corneas and that itself would not affect the test system. In this study, we evaluated five solvents frequently used in the BCOP assay: distilled water, mineral oil, corn oil, polyethylene glycol (PEG)-400, and methocel solution (0.5%). Based on the available classification systems, our preliminary data showed that water, methocel, mineral oil and corn oil were predicted as non-irritants, while PEG-400 was predicted as a mild irritant. To demonstrate the influence of the type of solvent on the outcome/prediction of the BCOP assay for solid materials, we tested a 20% suspension of benzoic acid (BA) prepared in these solvents. BA has a non-polar benzoic ring that would preferably dissolve in non-polar solvents and a polar acidic group with affinity for polar solvents, thus making it a good model for testing its effect on corneas when dissolved in various solvents. Previous animal tests reported moderate to severe eye irritation induced by BA. Our results demonstrated that when mixed in water, mineral oil, corn oil, or methocel, BA was predicted to be a corrosive/severe irritant, while it was predicted to be a moderate irritant when mixed in PEG-400. These results support the need for further investigation of the solvent’s influence in the BCOP assay to allow the correct prediction of the irritation potential of solid materials.