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Investigations on Reducing Ocular Irritation Associated with Harsh Ingredients by Altering Physicochemical Properties of the Formulation
With the upcoming implementation of the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) in various forms throughout the world and a global movement towards a reduction in animal testing, more emphasis is placed on utilizing the inherent hazards of chemicals for classification and labeling; however, the assessment of the toxicity of chemical mixtures, particularly ocular irritation, can be complex. The ability to formulate mixtures to be less irritating with minor modifications to the physical form would be very beneficial. The present study used the Bovine Cornea Opacity and Permeability (BCOP) assay, which is an OECD-approved in vitro method to assess ocular irritation, to investigate how physical properties (e.g., viscosity) affect ocular irritation. We found that the BCOP in vitro scores of irritating chemicals from several classes, including strong bases, were diminished by altering the viscosity of the aqueous medium or by dosing the solution as a foam. Our data show that a 1% NaOH solution in water produced an in vitro score of 165; however, when the medium was thickened with 1% Carbopol®, the in vitro score dropped to 67.6. A change of this nature is significant, and if this were an EPA-registered antimicrobial cleaner, for example, this reduction in the BCOP score would lower the hazard category. Product form and usage can clearly impact exposure, and the present results suggest that modifications to the physical properties of chemical mixtures can alter their ocular irritation potential; perhaps by affecting exposure to the eye. Although no formal comparisons were performed in animals, the BCOP assay is an OECD-validated method to assess ocular irritation, and studies have shown that the BCOP assay does not under-predict the results of traditional animal tests; thus, there are no obvious reasons to suggest that the present results would not correlate to animals or humans.
Testing Framework for Prediction of Ocular Irritation Using the Bovine Corneal Opacity and Permeability (BCOP) Assay and Chorioallantoic Membrane Vascular Assay (CAMVA)
In vitro ocular irritation assays, such as the Chorioallantoic Membrane Vascular Assay (CAMVA) and Bovine Corneal Opacity and Permeability (BCOP) test, are routinely used by personal care products companies because they are rapid and economical to conduct, do not require the use of live animals, and provide reliable predictive data. Previous research using an extensive CAMVA and BCOP database at Kao USA Inc. has shown that ocular irritation potential for new hair shampoos, ethanol-based hair stylers, skin cleansers, and skin lotions can be reliably predicted using a decision tree that systematically compares the ingredient composition, particularly ethanol and surfactant content, of the new formulation to previously tested formulations. Because the studies comprising this original database were conducted at a single contract laboratory, a follow-up study using a second contract laboratory was conducted to demonstrate inter-laboratory reliability of the CAMVA/BCOP data-derived decision tree for prediction of ocular irritation potential. Thirty-five personal care products were tested using the CAMVA and/or BCOP assays. The ethanol and surfactant content of each test material was evaluated, and the results of the assays were compared to the decision tree-based predictions of ocular irritation potential. Our data confirmed the ocular irritation predictions made using the decision tree model for 33 of 37 test samples (89% correlation rate) and verified the inter-laboratory reliability of the CAMVA and BCOP assays when conducted using appropriate controls. Our results also strengthened the ocular irritation decision tree model by confirming that deodorants are consistently predicted not to be ocular irritants based on composition.
In Vitro Safety Profile of Personal Care Products - Use of an In Vitro Testing Platform Based on a Reconstructed Vaginal Tissue Model
One of the common goals of industry is to confirm the safety of their products. Ethical concerns have led to the use of alternative testing methods in lieu of traditional testing methods. Several studies have shown good correlation between alternative test methods, traditional testing methods and human exposure. In the current study, the safety profile of three products with potential for vaginal exposure was assessed using the reconstructed human vaginal EpiVaginal™ model (MatTek Corporation, USA); the assay negative control (sterile, deionized water) and positive control (1% Triton® -X-100) were tested alongside. To increase the confidence in the test outcome, histopathology evaluation was conducted to assess the extent of cellular damage. Two liquid products were directly applied to the EpiVaginal™ tissues, while the wet wipe product was placed in direct contact with the tissue. Vaginal irritation expressed at ET50 values (3.32 and 12.71 hours) showed a higher irritation potential for the liquid formulations compared to the wipes (>24 hours). The lower irritation potential of the wipe product may be related to the availability of a rather limited amount of the liquid formulation in the wipes compared to the liquid formulations. Histology evaluations showed good correlation between the ET50 values and change in tissue structure. The results of this in vitro test methodology confirmed the safety profile of the products, should vaginal exposure occur during use. This two-endpoint testing platform (viability and histology) provided not only a correlative interpretation of the data, but also indication of the structural changes of the tissues exposed to the test article that are relevant to human exposure. Future plans include further exploring the capability of this in vitro testing platform for screening products before entering clinical trials.
Tiered Testing Strategy Using Validated In Vitro Assays for the Assessment of Skin and Eye Corrosion/Irritation of Pharmaceutical Intermediates
The safety of workers handling solid pharmaceutical intermediates was assessed using a tiered testing strategy based on regulatory validated in vitro assays. The Top-Down approach was initiated with the in vitro skin corrosion assay (OECD TG 431) followed by the in vitro skin irritation assay (OECD TG 439) using the reconstructed human epidermis model from MatTek Corporation. Of the ten pharmaceutical intermediates tested, nine were predicted to be non-corrosive to skin and were subsequently confirmed as non-irritants. The only intermediate predicted corrosive to skin was further tested using the Corrositex® assay (OECD TG 435) and was assigned a corrosive packing group II classification. Furthermore, three intermediates predicted non-corrosive/non-irritant to skin were tested as 20% dilutions in water in the in vitro Bovine Corneal Opacity and Permeability (BCOP) assay (OECD TG 437) and they were predicted as non-irritants to the eye. Our tiered skin and eye corrosion/irritation testing strategy proved to be a very useful platform for the assessment of the potential safety risk posed to workers during the manufacturing operations used for pharmaceutical intermediates.
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.
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.
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.
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.