United Nations Globally Harmonized System Research Articles

P21 Advancing dermatological care: a comprehensive evaluation of the steroid-free GL-1 formulation

Abstract Introduction and aims The prolonged use of topical steroids introduces notable dermatological risks, including the onset of permanent stretch marks, bruising, discoloration and vascular abnormalities. This study presents an innovative strategy by formulating GL-1, a steroid-free treatment amalgamating seaweed bioactives, sesame oil and naturally occurring salts and minerals. The objective of this study was to address the limitations of conventional topical steroids and introduce pioneering solutions to dermatological care. Methods To assess the safety and efficacy of GL-1 (product 1), a comprehensive battery of in vitro tests was employed. The skin corrosion test (OECD 431) established the noncorrosive nature of GL-1, validating its safety on reconstructed human epidermis. The skin irritation test (OECD 439) demonstrated the nonirritating attributes of GL-1, aligning with the United Nations Globally Harmonized System of Classification and Labelling guidelines. Additionally, the bovine corneal opacity and permeability test (OECD 437) affirmed the noninducing nature of GL-1 in relation to eye irritation or serious eye damage. Results Preliminary outcomes from dermal clinical trials indicate promising Results for GL-1. In vitro experiments on HaCaT and human dermal fibroblasts, adult (HDFa) cell lines highlighted the noncytotoxicity of GL-1, contrasting with the ‘Cortopin’, which exhibited cytotoxicity. GL-1 exhibited a proliferative effect in HDFa cells, signifying its potential for regenerative applications. Furthermore, both GL-1 and ‘Cortopin’ displayed antioxidant activity in HaCaT cells, underscoring the additional merits of our formulation. Conclusions Our steroid-free formulation, GL-1, demonstrates exceptional safety and efficacy through a meticulous series of in vitro assessments and initial clinical trials. These findings position GL-1 as a promising candidate for diverse dermal applications, offering a secure alternative to traditional topical steroids. The potential impact of GL-1 transcends its therapeutic efficacy, emphasizing its robust safety profile validated through multiple in vitro evaluations.

Development of an Eye Irritation Test Method Using an In-House Fabrication of a Reconstructed Human Cornea-like Epithelium Model for Eye Hazard Identification.

In a previous study, a novel human corneal-like epithelium model utilizing an immortalized human corneal epithelial cell line (iHCE-NY1) was developed as an alternative to animal models to identify chemicals not classified under the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (GHS) and was evaluated following the criteria of Test Guideline 492 of the Organization for Economic Co-operation and Development (OECD). In the present study, our aim was to establish an eye irritation test protocol using the iHCE-NY1 model to classify liquid chemicals under the GHS ocular hazard categories: no effect, no classification (No Cat.), Category 2 (Cat. 2) reversible effects, and Category 1 (Cat. 1) irreversible eye damage. The protocol involved exposing the iHCE-NY1 model to 31 liquid test chemicals for 5 min, followed by observation at post-incubation periods (PIPs) to assess recovery. Classification was based on cell viability, and histopathological findings on PIP days 7, 14, and 21. The outcomes were compared with an established database of classifications. All Cat. 1 liquid chemicals, 62.5% of No Cat., and 63.2% of Cat. 2 were correctly categorized. This study demonstrates that the iHCE-NY1 model can not only distinguish No Cat. test liquid chemicals but also differentiate between Cat. 2 and Cat. 1 liquid chemicals.

Retrospective evaluation of the eye irritation potential of agrochemical formulations

Multiple in vitro eye irritation methods have been developed and adopted as OECD health effects test guidelines. However, for predicting the ocular irritation/damage potential of agrochemical formulations there is an applicability domain knowledge gap for most of the methods. To overcome this gap, a retrospective evaluation of 192 agrochemical formulations with in vivo (OECD TG 405) and in vitro (OECD TG 437, 438, and/or 492) data was conducted to determine if the in vitro methods could accurately assign United Nations Globally Harmonized System for Classification and Labelling of Chemicals (GHS) eye irritation hazard classifications. In addition, for each formulation the eye irritation classification was derived from the classification of the contained hazardous ingredients and their respective concentration in the product using the GHS concentration threshold (CT) approach. The results herein suggest that the three in vitro methods and the GHS CT approach were highly predictive of formulations that would not require GHS classification for eye irritation. Given most agrochemical formulations fall into this category, methods that accurately identify non-classified agrochemical formulations could significantly reduce the use of animals for this endpoint.

How to resolve inconclusive predictions from defined approaches for skin sensitisation in OECD Guideline No. 497

In June 2021 the Organisation for Economic Co-operation and Development published Guideline No. 497 on Defined Approaches for Skin Sensitisation (DASS GL). There are two DAs published, known as the 2o3 and the ITS. The 2o3 uses two concordant results from either the DPRA, KeratinoSens™, or the h-CLAT assays to predict hazard (sensitiser/non-sensitiser). The ITS applies a score to results from the DPRA, the h-CLAT and an in silico model to predict United Nations Globally Harmonized System (GHS) sub-categories (1A/1B/Not Classified). The ITS can use Derek Nexus as the in silico model (known as ITSv1) or use OECD QSAR Toolbox (known as ITSv2).As limitations of the individual in chemico/in vitro assays and in silico predictions are carried through to the DAs, inconclusive predictions are possible for chemicals with results in the borderline range, and chemicals with out of domain results. However, these inconclusive predictions can be resolved by applying a weight of evidence approach.Herein, four case studies are presented, each ‘inconclusive’ for skin sensitisation potential according to both DAs. A weight of evidence approach was applied to each using a robust scientific approach to provide a conclusive prediction, where possible, based on several additional, non-animal lines of evidence.

Application of Defined Approaches for Skin Sensitization to Agrochemical Products

Skin sensitization testing is a regulatory requirement for safety evaluations of pesticides in multiple countries. Globally harmonized test guidelines that include in chemico and in vitro methods reduce animal use, but no single assay is recommended as a complete replacement for animal tests. Defined approaches (DAs) that integrate data from multiple non-animal methods are accepted; however, the methods that comprise them have been evaluated using monoconstituent substances rather than mixtures or formulations. To address this data gap, we tested 27 agrochemical formulations in the direct peptide reactivity assay (DPRA), the KeratinoSens™ assay, and the human cell line activation test (h-CLAT). These data were used as inputs to evaluate three DAs for hazard classification of skin sensitization potential and two DAs for potency categorization. When compared to historical animal results, balanced accuracy for the DAs for predicting in vivo skin sensitization hazard (i.e., sensitizer vs. nonsensitizer) ranged from 56 to 78%. The best performing DA was the “2 out of 3 (2o3)” DA, in which the hazard classification was based on two concordant results from the DPRA, KeratinoSens, or h-CLAT. The KE 3/1 sequential testing strategy (STS), which uses h-CLAT and DPRA results, and the integrated testing strategy (ITSv2), which uses h-CLAT, DPRA, and an in silico hazard prediction from OECD QSAR Toolbox, had balanced accuracies of 56–57% for hazard classification. Of the individual test methods, KeratinoSens had the best performance for predicting in vivo hazard outcomes. Its balanced accuracy of 81% was similar to that of the 2o3 DA (78%). For predicting potency categories defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), the correct classification rate of the STS was 52% and that of the ITSv2 was 43%. These results demonstrate that non-animal test methods have utility for evaluating the skin sensitization potential of agrochemical formulations as compared to animal reference data. While additional data generation is needed, testing strategies such as DAs anchored to human biology and mechanistic information provide a promising approach for agrochemical formulation testing.

Performance Statistics of Defined Approaches for Eye Hazard Identification of Non-Surfactant Liquids to Distinguish Between the Three United Nations Globally Harmonized System Categories

Introduction: Cosmetics Europe develops two defined approaches (DAs) for eye hazard identification, that is, addressing serious eye damage (Cat. 1), eye irritation (Cat. 2), and the absence thereof (No Cat.), for nonsurfactant liquid test substances. DAL-1 combines four physicochemical properties, the reconstructed human cornea-like epithelium (EpiOcular™ eye irritation test [EIT]: VRM1 and SkinEthic™ human corneal epithelium EIT: VRM2) test method (OECD test guideline [TG] 492), and bovine corneal opacity and permeability (BCOP) test method (OECD TG 437). DAL-2 combines the short time exposure test method (OECD TG 491) and the BCOP test method (OECD TG 437). In both DAs, the BCOP with the laser light-based opacitometer is used, however, only opacity is used to identify liquids that cause serious eye damage. Materials and Methods: The performance of the DAs to distinguish between the three United Nations Globally Harmonized System of Classification (UN GHS) categories was compared against the minimum performance values proposed by Cosmetics Europe to the Organisation for Economic Co-operation and Development expert Group on eye/skin irritation/corrosion and phototoxicity and agreed upon by the experts. Furthermore, the class-specific performance metrics are also provided for each DA. Results: The balanced accuracy of DAL-1 with validated reference method—EpiOcular EIT (VRM1), DAL-1 with validated reference method—SkinEthic HCE EIT (VRM2), and DAL-2 was 69.2%, 75.2%, and 74.3%, respectively. DAL-1 with VRM1 identified 76.5% of Cat. 1 (N = 17), 59.1% of Cat. 2 (N = 22), and 72.1% of No Cat. (N = 55) correctly. DAL-1 with VRM2 identified 76.5% of Cat. 1 (N = 17), 68.7% of Cat. 2 (N = 23), and 80.4% of No Cat. (N = 46) correctly. DAL-2 identified 81.2% of Cat. 1 (N = 17), 56.3% of Cat. 2 (N = 24), and 85.3% of No Cat. (N = 123) correctly. Conclusion: The percentage of correct predictions was greater than the proposed minimum performance values of 75% for Cat. 1, 50% for Cat. 2, and 70% for No Cat. Therefore, DAL-1 and DAL-2 have shown to successfully distinguish between the three UN GHS categories for eye hazard identification.

In silico prediction of the full United Nations Globally Harmonized System eye irritation categories of liquid chemicals by IATA-like bottom-up approach of random forest method

ABSTRACT As an alternative to in vivo Draize rabbit eye irritation test, this study aimed to construct an in silico model to predict the complete United Nations (UN) Globally Harmonized System (GHS) for classification and labeling of chemicals for eye irritation category [eye damage (Category 1), irritating to eye (Category 2) and nonirritating (No category)] of liquid chemicals with Integrated approaches to testing and assessment (IATA)-like two-stage random forest approach. Liquid chemicals (n = 219) with 34 physicochemical descriptors and quality in vivo data were collected with no missing values. Seven machine learning algorithms (Naive Bayes, Logistic Regression, First Large Margin, Neural Net, Random Forest (RF), Gradient Boosted Tree, and Support Vector Machine) were examined for the ternary categorization of eye irritation potential at a single run through 10-fold cross-validation. RF, which performed best, was further improved by applying the ‘Bottom-up approach’ concept of IATA, namely, separating No category first, and discriminating Category 1 from 2, thereafter. The best performing training dataset achieved an overall accuracy of 73% and the correct prediction for Category 1, 2, and No category was 80%, 50%, and 77%, respectively for the test dataset. This prediction model was further validated with an external dataset of 28 chemicals, for which an overall accuracy of 71% was achieved.

Performance of the GHS Mixtures Equation for Predicting Acute Oral Toxicity

Acute oral toxicity classifications are based on the estimated chemical dose causing lethality in 50 % of laboratory animals tested (LD50). Given the large number of pesticide registration applications that require acute toxicity data, an alternative to the in vivo test could greatly reduce animal testing. The United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS) Mixtures Equation estimates the acute toxicity of mixtures using the toxicities of mixture components. The goal of this study was to evaluate the concordance of LD50s predicted using the GHS Mixtures Equation and LD50s from the in vivo test results. Using the EPA classification system, concordance was 55 % for the full dataset (N = 671), 52 % for agrochemical formulations (N = 620), and 84 % for antimicrobial cleaning products (N = 51). Most discordant results were from substances LD50 > 2000 mg/kg (limit test) or 2000 < LD50 < 5000 mg/kg that were predicted as LD50 > 5000 mg/kg. A supplementary analysis combining all formulations with an LD50 > 500 mg/kg produced a concordance of 82 %. The lack of more toxic formulations in this dataset prevented a thorough evaluation of the GHS equation for such substances. Accordingly, our results suggest the GHS equation is helpful to predict the toxicity of mixtures, particularly those with lower toxicity.

Strategy Combining Nonanimal Methods for Ocular Toxicity Evaluation.

Historically, the ocular toxicity of manufactured consumer materials has been evaluated using the rabbit in vivo Draize rabbit eye test. The animal data obtained were used by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) to define the classification and labelling (C&L) for eye damage/irritation endpoint. However, the Draize test, a method which was never formally validated, has been widely criticized because of its technical limitations. In addition, ethical and economic issues and advances in scientific knowledge, and political and public pressures have made animal experimentation unsustainable. This scenario has consequently led to the development of nonanimal testing and protocols/approaches with considerable predictive value and relevance for humans. It is widely accepted that one single nonanimal method cannot cover all the criteria of damage/inflammation assessed by regulatory adopted in vivo animal testing. Thus, integrated testing strategies (ITS) have been proposed, including a tiered testing approach combining different nonanimal testing with different endpoints, which have been used for regulatory purposes, on a case-by-case basis and within integrated approaches to testing and assessment (IATA), to identify materials according to their ability to trigger eye damage. In particular, the top-down and bottom-up approaches have been recommended for the C&L of materials, which cause serious eye damage or eye irritation, respectively. This chapter describes detailed protocols for eye irritation testing based on cells (Short Time Exposure-STE, OECD No. 491/2017), a vascularized membrane (the Hen's Egg Test-Chorioallantoic Membrane-HET-CAM) and corneal tissue (Bovine Corneal Opacity and Permeability-BCOP, OECD No. 437/2017), which can be applied using top-down or bottom-up approaches. In addition, it suggests making a corneal histomorphometric evaluation as an additional parameter in the BCOP method to differentiate materials that cause serious eye tissue damage (UN GHS Cat. 1) from materials that have reversible eye irritation effects (UN GHS Cat. 2).

Overall performance of Bovine Corneal Opacity and Permeability (BCOP) Laser Light-Based Opacitometer (LLBO) test method with regard to solid and liquid chemicals testing

A prospective study of the Bovine Corneal Opacity and Permeability (BCOP) Laser Light-Based Opacitometer (LLBO) test method was conducted to evaluate its usefulness to identify chemicals as inducing serious eye damage (Cat. 1) or chemicals not requiring classification for eye irritation (No Cat.) applying United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).The aim was to demonstrate the reproducibility of the BCOP LLBO protocol for liquids and solids and define its predictive capacity. Briefly, 145 chemicals were simultaneously tested with BCOP LLBO and OP-KIT (OECD TG 437), one to two times in one laboratory. When used to identify Cat. 1, the BCOP LLBO has a false negative rate (FNR) of 24.1% (N = 56) compared to 34.8% (N = 56) for the BCOP OP-KIT, with a comparable false positive rate (FPR, N = 89) of 18.5% and 20.8%, respectively. When used to identify chemicals not requiring classification (No Cat.) the BCOP LLBO and BCOP OP-KIT had a FNR (N = 104) of 6.2% and 7.2% and a FPR (N = 41) of 45.1% and 42.7%, respectively.The OP-KIT and LLBO devices are interchangeable at no cost to data quality and reliability. The OP-KIT and LLBO devices are interchangeable at no cost to data quality and reliability. The performance of the LLBO is at least as good as the OP-KIT, both methods can be used to identify UN GHS Cat. 1 and UN GHS No Cat. chemicals.

Laser Light-Based Opacitometer ‘Peira LLBO 180’: A new and validated opacitometer for use in the Bovine Corneal Opacity and Permeability (BCOP) eye irritation test method

The “Peira LLBO 180” is a Laser Light-Based Opacitometer that can be used as an alternative for the standard OP-KIT device in the Bovine Corneal Opacity and Permeability (BCOP) test Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) 437 to identify chemicals inducing serious eye damage as defined by United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS), i.e. chemicals to be classified as UN GHS Category 1 and chemicals not requiring classification for eye irritation or serious eye damage under the UN GHS classification system (No Category).• The Peira LLBO 180 offers the advantage of analysing the complete corneal surface and is therefore able to detect more efficiently opaque spots located around the periphery of the excised corneas.• This new device will allow not only a more accurate definition of the eye irritating potential of compounds, but also a more precise ranking of moderate to mild and non-irritating compounds.• The value of Peira LLBO 180 is confirmed during in-house and multi-laboratory evaluation studies and is now included in the updated OECD TG 437, dated 26th of June 2020.The results demonstrate that the presented methodology is an improved new approach methodology (NAM) for ocular irritation testing of liquids and solids.

Determining the endocrine disruption potential of industrial chemicals using an integrative approach: Public databases, in vitro exposure, and modeling receptor interactions

Environmental and occupational exposure to industrial chemicals has been linked to toxic and carcinogenic effects in animal models and human studies. However, current toxicology testing does not thoroughly explore the endocrine disrupting effects of industrial chemicals, which may have low dose effects not predicted when determining the limit of toxicity. The objective of this study was to evaluate the endocrine disrupting potential of a broad range of chemicals used in the petrochemical sector. Therefore, 139 chemicals were classified for reproductive toxicity based on the United Nations Globally Harmonized System for hazard classification. These chemicals were evaluated in PubMed for reported endocrine disrupting activity, and their endocrine disrupting potential was estimated by identifying chemicals with active nuclear receptor endpoints publicly available databases. Evaluation of ToxCast data suggested that these chemicals preferentially alter the activity of the estrogen receptor (ER). Four chemicals were prioritized for in vitro testing using the ER-positive, immortalized human uterine Ishikawa cell line and a range of concentrations below the reported limit of toxicity in humans. We found that 2,6-di-tert-butyl-p-cresol (BHT) and diethanolamine (DEA) repressed the basal expression of estrogen-responsive genes PGR, NPPC, and GREB1 in Ishikawa cells, while tetrachloroethylene (PCE) and 2,2′-methyliminodiethanol (MDEA) induced the expression of these genes. Furthermore, low-dose combinations of PCE and MDEA produced additive effects. All four chemicals interfered with estradiol-mediated induction of PGR, NPPC, and GREB1. Molecular docking demonstrated that these chemicals could bind to the ligand binding site of ERα, suggesting the potential for direct stimulatory or inhibitory effects. We found that these chemicals altered rates of proliferation and regulated the expression of cell proliferation associated genes. These findings demonstrate previously unappreciated endocrine disrupting effects and underscore the importance of testing the endocrine disrupting potential of chemicals in the future to better understand their potential to impact public health.

Fundamental pharmacological expressions on ocular exposure to capsaicin, the principal constituent in pepper sprays

Eye irritation assessment is compulsory to anticipate health risks in military personnel exposed to riot control agents such as capsaicin, the principal constituent of oleoresin capsicum, or pepper sprays. The present work investigates certain fundamental yet unaddressed pharmacological manifestations on ocular exposure to capsaicin. Ocular pharmacology of capsaicin was studied using acute eye irritation (AEI), bovine corneal opacity and permeability (BCOP) assay, corneal fluorescein staining and indirect ophthalmoscopy studies, transcorneal permeation, Schirmer tear secretion test, nerve conduction velocity study and enzyme-linked immunosorbent assay (ELISA). Additionally, histopathology and scanning electron microscopy (SEM) of bovine corneas and rat optic nerves were done to further estimate capsaicin induced morphological variations. Our findings demonstrated that AEI, BCOP, corneal fluorescein staining and indirect ophthalmoscopy were useful in assessing capsaicin induced ocular irritation; AEI and BCOP also contributed towards indicating the eye irritation potential of capsaicin as per the United Nations Globally Harmonized System of Classification and Labelling of Chemicals categorization. Additional experimental observations include considerable transcorneal permeation of capsaicin, capsaicin induced reduction in tear secretions and nerve conduction velocity and increased expression of proinflammatory cytokines by ELISA. Histopathology and SEM were favourable techniques for the detection of capsaicin induced ocular physiological modifications.

Prioritization of reproductive toxicants in unconventional oil and gas operations using a multi-country regulatory data-driven hazard assessment

BackgroundRecent trends have witnessed the global growth of unconventional oil and gas (UOG) production. Epidemiologic studies have suggested associations between proximity to UOG operations with increased adverse birth outcomes and cancer, though specific potential etiologic agents have not yet been identified. To perform effective risk assessment of chemicals used in UOG production, the first step of hazard identification followed by prioritization specifically for reproductive toxicity, carcinogenicity and mutagenicity is crucial in an evidence-based risk assessment approach. To date, there is no single hazard classification list based on the United Nations Globally Harmonized System (GHS), with countries applying the GHS standards to generate their own chemical hazard classification lists. A current challenge for chemical prioritization, particularly for a multi-national industry, is inconsistent hazard classification which may result in misjudgment of the potential public health risks. We present a novel approach for hazard identification followed by prioritization of reproductive toxicants found in UOG operations using publicly available regulatory databases. MethodsGHS classification for reproductive toxicity of 157 UOG-related chemicals identified as potential reproductive or developmental toxicants in a previous publication was assessed using eleven governmental regulatory agency databases. If there was discordance in classifications across agencies, the most stringent classification was assigned. Chemicals in the category of known or presumed human reproductive toxicants were further evaluated for carcinogenicity and germ cell mutagenicity based on government classifications. A scoring system was utilized to assign numerical values for reproductive health, cancer and germ cell mutation hazard endpoints. Using a Cytoscape analysis, both qualitative and quantitative results were presented visually to readily identify high priority UOG chemicals with evidence of multiple adverse effects. ResultsWe observed substantial inconsistencies in classification among the 11 databases. By adopting the most stringent classification within and across countries, 43 chemicals were classified as known or presumed human reproductive toxicants (GHS Category 1), while 31 chemicals were classified as suspected human reproductive toxicants (GHS Category 2). The 43 reproductive toxicants were further subjected to analysis for carcinogenic and mutagenic properties. Calculated hazard scores and Cytoscape visualization yielded several high priority chemicals including potassium dichromate, cadmium, benzene and ethylene oxide. ConclusionsOur findings reveal diverging GHS classification outcomes for UOG chemicals across regulatory agencies. Adoption of the most stringent classification with application of hazard scores provides a useful approach to prioritize reproductive toxicants in UOG and other industries for exposure assessments and selection of safer alternatives.

Transformation/dissolution characterization of tungsten and tungsten compounds for aquatic hazard classification.

The transformation/dissolution protocol (T/DP) for metals and sparingly soluble metal compounds was applied to determine the transformation/dissolution (T/D) characteristics of yellow tungsten trioxide, WO3 ; blue tungsten oxide, WOx, x taken as 2.9; tungsten disulphide, WS2 ; tungsten metal, W; 3 samples of tungsten carbide, WC; sodium tungstate, Na2 WO4 · 2H2 O; ammonium paratungstate (APT), (NH4 )10 (H2 W12 O42 ) · 4H2 O; and ammonium metatungstate (AMT) (NH4 )6 (H2 W12 O40 ) · 3H2 O. The T/D data were used to derive aquatic hazard classification outcomes under the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) and European Union Classification, Labelling and Packaging of Substances and Mixtures (EU CLP) schemes by comparing the data with selected acute and chronic ecotoxicity reference values (ERVs) of 31 and 3.37 mg W/L, respectively. In addition to the concentration of total dissolved tungsten (W), the T/D solutions were analyzed for the concentration of the tungstate anion, because speciation can be an important factor in establishing the ecotoxicity of dissolved metals. Results show that the tungstate anion was the predominant W-bearing species in solution for all substances examined at pH 6 and 8.5. It was found that the 100 mg/L loadings of both the yellow WO3 and the blue WOx exceeded the 31 mg/L acute ERV, so they would classify as Acute 3-Chronic 3 under the UN GHS scheme but they would not classify under the EU CLP. An effect of pH on the reactivity of the W metal was observed with 3% and 16% W dissolution at pH 6 and 8.5, respectively. Tungsten metal would not classify under either the UN GHS or EU CLP schemes nor would the WS2 . The WCs were the least reactive in terms of the 1% or less dissolution of the contained W at pH 6. A critical surface area for WC was calculated. The sodium tungstate, APT and the AMT all yielded, at pH 8.5, total dissolved W concentrations that would result in UN GHS Acute 3-Chronic 3 classifications. Integr Environ Assess Manag 2018;14:498-508. © 2018 Her Majesty the Queen in Right of Canada. Integrated Environmental Assessment and Management © 2018 SETAC.

Study on feasibility of HaCaT epidermal model as an alternative to skin irritation in vitro

To construct the epidermal model with HaCaT cells and evaluate the feasibility of this model as an in vitro skin irritation test tool. The HaCaT model was reconstructed with HaCaT cells by adoption gas-liquid surface culture technique, and the EpiKutis ® model was reconstructed with human epidermal keratinocytes by the same techinique as control. Morphology changes of HaCaT and EpiKutis ® models were observed by HE staining. Barrier function assessment was performed with penetration test. According to Organization for Economic Cooperation and Development (OECD) Test Guideline 439, the surface of the HaCaT and the EpiKutis ® models were treated with 20 chemicals for 30 minutes, incubated for 42 hours, and determined tissue viability by MTT assay, to evaluate the irritation of chemicals. Then the results were compared with the irritation of chemicals with the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) system and validated reference method (VRM) for the classification of chemical, and evaluated the feasibility of this model as an in vitro skin irritation test tool. The results of HE staining showed that there was no complete stratified structure in the HaCaT model. The results of barrier function showed that the ET 50 was 0.99 hours. The results of skin irritation of chemicals showed that the sensitivity was 100% and 100%, the specificity was 50% and 70%, and the accuracy rate was 75% and 85% for HaCaT model and EpiKutis ® model respectively. The epidermal model of HaCaT cells does not possess the complete epidermal physiological structure, the barrier function as ET 50 of the HaCaT model is lower than EpiKutis ® model, the chemicals in vitro skin irritation test results do not meet the OECD criteria for the determination of stimulants, so the HaCaT model is not suitable as a replacement tool in vitro to determine the chemicals skin irritation.

Eye irritation potential: palm-based methyl ester sulphonates

Aim: To evaluate eye irritation potential of palm-based methyl ester sulphonates (MES) of different chain lengths; C12, C14, C16, C16:18.Methods: The Bovine Corneal Opacity and Permeability test method (BCOP), OECD Test Guideline 437, was used as an initial step to study the inducing effect of palm-based MES on irreversible eye damage. The second assessment involved the use of reconstructed human corneal-like epithelium test method, OECD Test Guideline 492 using SkinEthic™ Human Corneal Epithelium to study the potential effect of palm-based MES on eye irritancy. The palm-based MES were prepared in 10% solution (w/v) in deionized water and tested as a liquid and surfactant test substances whereby both test conducted according to the liquid/surfactant treatment protocol.Results: The preliminary BCOP results showed that palm-based MES; C12, C14, C16, C16:18 were not classified as severe eye irritants test substances with in vitro irritancy score between 3 and the threshold level of 55. The second evaluation using SkinEthic™ HCE model showed that palm-based MES; C12, C14, C16, C16:18 and three commercial samples were potentially irritants to the eyes with mean tissue viability ≤ 60% and classified as Category 2 according to United Nations Globally Harmonized System of Classification and Labelling of Chemicals. However, there are some limitations of the proposed ocular irritation classification of palm-based MES due to insolubility of long chain MES in 10% solution (w/v) in deionized water.Conclusion: Therefore, future studies to clarify the eye irritation potential of the palm-based MES will be needed, and could include; methods to improve the test substance solubility, use of test protocol for solids, and/or inclusion of a benchmark anionic surfactant, such as sodium dodecyl sulphate within the study design.

CON4EI: Development of serious eye damage and eye irritation testing strategies with respect to the requirements of the UN GHS/EU CLP hazard categories

The main objective of the CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project (2015–2016) was to develop tiered, non-animal testing strategies for serious eye damage and eye irritation assessment in relation to the most important drivers of classification. The serious eye damage and eye irritation potential of a set of 80 chemicals was identified based on existing in vivo Draize eye test data and testing was conducted using the following eight alternative test methods: BCOP (Bovine Corneal Opacity and Permeability)+histopathology, BCOP-LLBO (BCOP Laser Light-Based Opacitometer), ICE (Isolated Chicken Eye)+histopathology, STE (Short Term Exposure), EpiOcular™ EIT (EpiOcular Eye Irritation Test), EpiOcular™ ET-50 (EpiOcular™ Time-to-toxicity), SkinEthic™ HCE EIT (SkinEthic™ Human Corneal Epithelial Eye Irritation Test), and SMI (Slug Mucosal Irritation). Project management decided to not include the ICE data in this project since the execution showed relevant, and not predictable, deviations from Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) 438 and Guidance Document 160. At this stage, the outcome of these deviations has not been fully assessed. In addition to these alternative test methods, the computational models Toxtree and Case Ultra were taken into account. This project assessed the relevance of these test methods, their applicability domains and limitations in terms of ‘drivers of classification’, and their strengths and weaknesses. In this way, methods were identified that fit into a tiered-testing strategy for serious eye damage/eye irritation assessment to distinguish United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) Category 1 (Cat 1) chemicals from non-Cat 1 chemicals and address the gap namely distinguish between Category 2 (Cat 2) and Cat 1 chemicals.

Transformation/dissolution characteristics of a nickel matte and nickel concentrates for acute and chronic hazard classification.

For the purposes of aquatic hazard classification under the United Nations Globally Harmonized System of Classification (UNGHS), we have examined the transformation/dissolution (T/D) characteristics of a Ni matte and 4 Ni concentrates at pH 6 using the United Nations (UN) Transformation/Dissolution Protocol (T/DP) for metals and sparingly soluble metal compounds. Among the analytes Ni, Co, and Cu, Ni was released into the T/D solutions in the highest concentrations and was thus the main driver in establishing the hazard classification. We applied an extrapolation-scaling approach to obtain concentrations of total dissolved Ni at low loadings of 0.1 and 0.01 mg/L for derivation of chronic classification outcomes in the European Union (EU) classification, labeling, and packaging (CLP) scheme. The T/D data would classify the Ni matte as Acute 2-Chronic 2 under the Globally Harmonized System (GHS) scheme, and Chronic 1 under the EU CLP. Three of the 4 Ni concentrates would classify as GHS Acute 2-Chronic 2 and EU CLP Chronic 2, whereas the 4th would classify as GHS Acute 3-Chronic 3 and EU CLP Chronic 3. In applying the critical surface area (CSA) approach to the Ni concentrates, acute and chronic hazard classification outcomes were the same as those derived from direct application of the T/D data to the GHS and EU schemes. Such agreement provided confidence that the CSA approach could yield scientifically defensible acute and chronic hazard classification outcomes.

Toxicological evaluation of 6:2 fluorotelomer alcohol

6:2 fluorotelomer alcohol (6:2 FTOH; CF3[CF2]5[CH2]2OH, CAS# 647-42-7) was evaluated for acute, genetic, and subchronic toxicity using in vitro and in vivo methods. In rats, 6:2 FTOH was considered to be slightly toxic by the oral (LD50=1750mg/kg), and dermal (LD50>5000mg/kg) routes. In rabbits, 6:2 FTOH was not a primary skin or eye irritant, and it did not produce a dermal sensitization response in mice. In a 90-day subchronic study, 6:2 FTOH was administered to rats by oral gavage (0, 5, 25, 125, 250mg/kg/day). Mortality was observed at 125 and 250mg/kg/day; deaths occurred after approximately three weeks of dosing and continued sporadically. The NOAEL in the subchronic study was 5mg/kg/day based on hematology and liver effects. 6:2 FTOH was not mutagenic in the bacterial reverse mutation test or in the mouse lymphoma assay and was not clastogenic in a chromosome aberration assay in human lymphocytes. The hazard classification for human health endpoints of 6:2 FTOH according to the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (GHS) is Category 4 for acute oral toxicity based on an LD50 of 1750mg/kg. Other acute health endpoints including eye and skin irritation, skin sensitization, as well as genotoxicity, did not meet the criteria for hazard classification. Benchmark Dose Analysis was performed on the most sensitive endpoints from the 90-day oral gavage study and these levels were all above the study NOAEL of 5mg/kg/day. For risk assessment purposes, the recommended point of departure is the more conservative study NOAEL of 5mg/kg/day.