Listed below are the latest publications added to IIVS’ online document library. To search our library, please visit our Resources page.
SUMMARY OF KEY THEMES
There are several plausible in vitro test systems, models, and assay endpoints currently available with the potential to be used for assessing adverse impacts of tobacco product exposure in respiratory tissues. These models and assay endpoints should be further evaluated for their use as hazard assessment tools in a regulatory arena.
Of particular interest are three-dimensional (3D) reconstructed, human, organotypic tissue models of the respiratory airways; these should reduce the need to extrapolate results across species, thus strengthening the relevancy of results.
Standardization of promising test methods should be a high priority.
Relevant reference standards should be developed and made easily available to the research community.
Access to information from non-invasive or minimally invasive clinical studies is needed to bridge the gap between in vitro and in vivo results.
Development of test methods for regulatory purposes is unlikely to be funded through the traditional grant process; more direct funding programs should be developed.
Close communication should be maintained between regulators and the research community to be most efficient in the development of useful in vitro methods to assess relative risk.
A Tiered In Vitro Irritation/Corrosion Testing Strategy for GHS Classification of Pharmaceutical Compunds
Irritation reactions are a frequently reported occupational health hazard. To reduce animal testing, BMS and IIVS have developed a testing strategy using three in vitro assays to assess the irritation/corrosive potential of pharmaceutical compounds (PC) for worker safety. The strategy allows for GHS classification by utilizing the Corrositex® assay for corrosivity (OECD TG 435), the Bovine Corneal Opacity and Permeability (BCOP) assay for ocular irritation (OECD TG 437), and the EpiDerm™ skin irritation test (SIT) for dermal irritation (OECD TG 439). Twenty-five solid PCs were evaluated in this tiered testing strategy. First the pH of each substance was determined. If the pH was ≥11 or ≤2, a Corrositex® assay was conducted. If the compound was negative in the Corrositex® assay or the pH was between 2 - 11, a BCOP assay was performed followed by a SIT assay. Based on their extreme pH, 4 compounds were tested in the Corrositex® assay, which resulted in corrosive predictions (packing group II or III) and thus no further testing was needed. Twenty-two compounds were evaluated in the BCOP assay (both neat and as a 20% dilution), with the higher response used for classification. The results were 5 Category 1 (score>55), and 8 non-irritants (score<3). There were 9 compounds with scores between 3 - 25, which were described as mild irritants on internal BMS hazard communications. Twenty-five compounds were evaluated using the SIT assay and were classified as non-irritants to skin. This is consistent with the BMS historical animal model results showing very low number of PCs as skin irritants. The comparison also confirmed 50% viability as an acceptable cut off for GHS dermal irritation classification. This tiered testing strategy, which replaces the use of animal studies, represents a rational platform that can be utilized for the prediction of ocular and dermal irritation/corrosive potential of PCs.
Using the Novel NociOcular Assay to Predict the Eye Sting Potential of Shampoos and Sunscreen Products
Although several in vitro eye irritation models exist, none have demonstrated the ability to predict eye stinging. The NociOcular assay, a novel neuronal in vitro model with high expression of capsaicin-responsive Transient Receptor Potential Vanilloid type 1 (TRPV1) channels, has been shown to distinguish stinging from non-stinging baby bath products. We sought to evaluate the eye stinging potential of additional surfactant-based products and sunscreen formulations. In the assay, SH-SY5Y neuroblastoma cells are cultured in 96-well plates and exposed to serially diluted test substance and TRPV1 channel activation is measured by acute increases in the intracellular free calcium. In separate wells, cells are treated with the TRPV1 antagonist capsazepine to confirm TRPV1-mediated calcium influx. The positive control, an adult shampoo that contains cocamide MEA, a known stinging ingredient, was the most active surfactant-based test substance evaluated in the assay. The negative control, a baby shampoo, was negative in the NociOcular assay and clinical tests. Four shampoo products demonstrated a range of responses between these controls and were classified as either stinging or non-stinging based on the percentage calcium influx as compared to capsaicin over the dose-response. During pilot studies with sunscreen formulations, several technical challenges arose including insolubility in assay buffers and pipetting the subsequent dilutions onto the cells. In order to achieve greater solubility, alternate solvents composed of detergents along with assay buffers were used. These alternate solvents allowed for increased solubility and dilutions were successfully administered onto the cells. Ten sunscreen formulations were evaluated and ranked according to TRPV-1 response and compared to available consumer experience reviews for eye stinging. Future research aims to assess the accuracy of the predictions for both the shampoos and sunscreen products through clinical data comparison.
The Transient Receptor Potential Vanilloid type 1 (TRPV1) receptor is one of the most well characterized pain-inducing receptors and has been recently identified as a valuable tool to predict eye stinging potential of surfactant based formulations. In this study we sought to predict eye stinging of non-surfactant based cosmetic formulations by studying TRPV1 activity using the NociOcular assay. In the NociOcular assay, TRPV1 expressing neuroblastoma cells are exposed to test substance and TRPV1 activity is measured by acute increases in intracellular calcium. Three of the formulations induced stinging in the human test and were also positive in the NociOcular assay. The other four formulations evaluated were classified as stinging in the human test, but a conclusive determination could not be made in the NociOcular assay as the formulations were not fully soluble in assay buffers. The formulations were also evaluated in the EpiOcularTM assay, an established in vitro model for eye irritation utilized by the cosmetics industry. The EpiocularTM assay results did not correlate with the human sting data. Our data support that the NociOcular assay may be a valuable in vitro tool to predict human eye stinging sensation for cosmetic formulations. Future efforts seeks to further expand the applicability of the assay to product types other than surfactant based formulations.
Investigation of Novel In Vitro Methods for Predicting the Dermal Sensitization Potential of Synthetic Process Intermediates
Skin Sensitization is a critical endpoint in the evaluation of synthetic process intermediates used in the manufacture of active pharmaceutical ingredients. Although the in vivo Local Lymph node assay (LLNA) has been traditionally used to qualitatively and quantitatively assess dermal sensitization, many novel alternative assays have reached an advanced stage of pre-validation and official testing guidelines are expected within the year. In this study we sought to evaluate two alternative assays, the KeratinoSensTM assay and the Direct Peptide Reactivity Assay (DPRA), for the prediction of dermal sensitization of synthetic process intermediates. The KeratinoSensTM assay is a cell-based reporter gene assay which identifies skin sensitizers by measuring the induction of luciferase under the control of the antioxidant response element (ARE) derived from the human AKR1C2 gene. Luciferase induction indicates activation of ARE-dependent genes which are involved in the dermal sensitization mechanism. The DPRA is an in chemico assay which identifies dermal sensitizers based on their reactivity with model peptides. Test substances are incubated with cysteine/lysine containing peptides and the depletion of each peptide is then used to determine the degree of reactivity and dermal sensitization potential. Ten process intermediates have been evaluated in KeratinoSensTM and DPRA and the results have been compared to existing LLNA data on these test substances. Compared to the LLNA data, nine of the ten test substances were identified correctly by the KeratinoSensTM assay; a positive response in either KeratinoSensTM assay or DPRA was considered to be potential sensitizer. Continued evaluation of these alternative assays for assessment of the dermal sensitization potential of synthetic process intermediates will be beneficial in establishing testing strategies to reduce animal testing while providing a useful tool for risk assessments.
Protocol Considerations for Testing Surfactants and Surfactant-based Formulations in the Bovine Corneal Opacity and Permeability Assay
The Bovine Corneal Opacity and Permeability (BCOP) assay is an ex vivo test for predicting ocular irritation. For regulatory classification, OECD Test Guideline (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. Guidance Document (GD) No. 160 also presents that solid and concentrated liquid surfactants may be diluted to 10% for testing. 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. Without question, surfactant solids should not be tested using the solid chemical protocol, since overexposure conditions are likely. In the absence of clear guidance from these regulatory documents, we present on the testing of a few common surfactant ingredients (sodium lauryl sulfate, Triton X-100, and benzalkonium chloride), and surfactant-based liquid and solid formulations in BCOP using standard and modified dilutions and exposures to evaluate the impact of these variables. Whereas the opacity values for the non-ionic and anionic surfactants were low, changes in the fluorescein permeability values correlated well to expected surfactant activities in all of the surfactant classes tested. Histopathology was performed to confirm corneal changes. We found that surfactants at very high concentrations may not exhibit dose-related effects, as irritation optima may occur at aqueous concentrations between 10 and 30%. Furthermore, since surfactants induce corneal erosion, we advocate that the fluorescein permeability endpoint in the BCOP assay should be evaluated individually from the In Vitro Irritation Score (IVIS) in a hazard assessment. Accordingly, a framework to guide the testing of surfactants and surfactant-based products is presented. Please contact us for more information on this work at firstname.lastname@example.org.
Toxic Insult to Rat Precision Cut Lung Slices Increases Tissue Cytokine Levels and Activation of Macrophages, and Causes Acute Damage, While Prolonged Insult May Lead to Increased Deposition of Collagen - a Marker of Fibrosis
The use of in vitro or ex vivo models is intended to provide meaningful data that will identify or predict the adverse effects of tissue exposure. Precision-cut lung slices (PCLS) are used as a model that retains the heterogenous population of cells in the native architecture of the organ. The retention of native cells allows the study of the initial, dynamic events (such as inflammation) that occur following a toxic insult prior to overt tissue damage. The purpose of the reported studies was to identify initial inflammatory signals, acute toxicities, as well as markers associated with chronic toxicities of PCLS exposed to a toxic insult as a way to qualify the model for identifying such endpoints. Rat PCLS were exposed to several chemotherapeutics known to cause acute and/or chronic pulmonary damage. Time points for respective endpoints were chosen based on known response times of when relevant endpoints may change. Cytokines and acute toxicity were evaluated during initial days of exposure while activation of macrophages and collagen deposition were evaluated through 4 weeks of culture in other studies. Exposure of PCLS for 24 hours resulted in increased cytokine levels and 72 hour exposure caused overt toxicity, as assessed using tissue protein content and histologically using H&E and ED-1 staining. Long term exposure of PCLS to two agents known to cause fibrosis (bleomycin and carmustine) resulted in elevated numbers of macrophages and also increased collagen deposition. PCLS generate inflammatory cytokine signals and, if levels persist after insult removal, these signals may predict subsequent tissue damage. The expression of adverse markers of chronic exposures (collagen deposition) in PCLS may signify risk of fibrosis. Cytokine responses, macrophage activation, and fibrosis are hallmarks of tobacco related exposures. PCLS may elucidate acute and chronic adverse pulmonary responses when exposed to tobacco products.
Evaluation of a Reconstructed Human Oral Buccal Tissue Model as a Testing Platform for Determining the Oral Irritation Potential of Tobacco Products
There is an increasing need by the tobacco industry to evaluate the irritation and inflammation potential of tobacco products to support product development goals, for sound product stewardship, and likely regulatory safety tests. The use of in vitro human cell and tissue-based test methods to replace in vivo animal models addresses the need for more human-relevant predictive tools, and is consistent with many corporate animal welfare policies. Although monolayer cell-based cytotoxicity and cytokine expression assays have been used, three-dimensional tissue constructs provide distinct advantages since tissue exposures and pharmacokinetics more closely resemble the in vivo events. In this study, we evaluated a reconstructed human oral buccal model for determining the oral irritation of oral tobacco products. A dilution series of tobacco extracts were applied topically onto the EpiOral™ reconstructed human oral buccal model (Cat no. ORL-200) (MatTek Corporation, Ashland, MA) for various exposure times (ranging from 2 to 16 hours) to estimate oral irritation potential based upon reduction in cell viability and the synthesis/release of the inflammatory mediators IL-1α and IL-8. We determined tobacco extract concentration-related increases in cytotoxicity for the highest tobacco extract concentrations. We also found that increases in IL-1α release (up to 19-fold) generally correlated with the cytotoxicity increases. Exposure time related increases in IL-8 release were generally observed in tissues treated with the three lower tobacco extract concentrations where relative viabilities were sufficiently high to allow for secondary cytokine production, but at the highest tobacco extract concentrations IL-8 release were below control levels where cytotoxic effects inhibited the cells’ ability to synthesize proteins. These results demonstrate the utility of reconstructed human epithelial models for evaluating the irritation potential of tobacco products. To expand upon this utility, we propose to apply these general methods for determining cytotoxicity and inflammatory cytokine profiles to evaluating inflammation responses in reconstructed human airway tissue models exposed to combustible tobacco product extracts, particulate matter, and whole smoke.
Preliminary Investigation on Reducing Ocular Irritation Potential of Harsh Ingredients By Increasing Formulation Viscosity
Formulations tested for ocular irritation using the Bovine Corneal Opacity and Permeability (BCOP) assay may be assigned a specific irritation label based on the resulting In Vitro Irritancy Score (IVIS) and specific regulatory guidelines (e.g. OECD, EPA, and CLP) that provide cutoff values for classifications. The ability to reduce ocular irritation by slightly adjusting the physical properties of a formulation is highly desirable. Laboratory investigations found that incrementally changing viscosity using increasing amounts of Carbopol® as a thickening agent reduced ocular irritation when mixed with a 1% NaOH solution in water. Following a 10-minute exposure in the BCOP assay, 1% NaOH was previously classified as a severe ocular irritant (IVIS=161.6). Increasing Carbopol® from 0.25% to 1.25% in a mixture with 1% NaOH decreased the In Vitro score to a range of values between 150.9 and 18.3 and decreased ocular irritation across a range of irritation classifications from severe to mild irritation (n= 3 corneas per treatment). Exposure to 1% Carbopol® alone exhibits minimal irritation (IVIS=1.3) and Carbopol® is consequently not considered to contribute to ocular irritation within the tested mixtures. Histopathology evaluation further supports that exposure to 1% Carbopol® results in damage similar to negative control treated corneas and that epithelial and stromal damage decreases as viscosity increases. Additionally, preliminary findings indicate that when a small amount of thickener is added to a complex formulation containing otherwise harsh ingredients, ocular irritation can be mitigated from a Category I label to a Category II label according to current EPA guidelines applicable to cleaning products making anti-microbial claims. Similarly, increasing the viscosity of a formulation containing more than 3% of a severe ingredient also resulted in a “Not Classified” label according to OECD criteria when it would have received a severe classification if left untested (according to CLP regulations). These results indicate that increasing viscosity may be an effective tool for reducing ocular irritation potential of a formulation. Viscosity, among other physical properties, may therefore be used to inform decision making during product development, ultimately affecting downstream users in such areas as marketing, labeling, packaging and distribution.
Assessing Increased Sensitivity and Variability Issues in an Established In Vitro Phototoxicity Testing Program
The 3T3 Neutral Red Uptake (NRU) Phototoxicity assay is an established in vitro assay used to evaluate the potential phototoxicity hazard of a test chemical. The assay methods and prediction model are described in The Organization for Economic Cooperation and Development (OECD) Test Guideline (TG) 432 “In Vitro 3T3 NRU phototoxicity test”1. IIVS’ routine assay performance is evaluated on the comparison of the positive control, chlorpromazine, and solvent control results to our historical database. Failure to consistently meet acceptance criteria calls for examination of assay performance. High assay sensitivity and low optical density values have contributed to the failure of recent assay results to meet acceptance criteria. Storage conditions, preparation, and manufacturer lot-to-lot consistency of assay reagents (DMSO, chlorpromazine, and neutral red) were evaluated. Variations in positive control or solvent control responses in different DMSO (lot-lot or catalog number). Preparation, storage condition, and filtration methods affected neutral red signal. UVA light source investigated for impacts on irradiance uniformity. Variations in UVA light intensity observed depending on plate placement under the light source. Several variables (reagents and light source) which likely impacted assay performance and may have contributed to increasing assay sensitivity were investigated.