Bovine Corneal Opacity Research Articles

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.

An in vitro depth of injury prediction model for a histopathologic classification of EPA and GHS eye irritants

The purpose of this study was to develop Globally Harmonized System (GHS) and U.S. Environmental Protection Agency (EPA) prediction models for classifying irritant materials based on histopathologic in vitro depth of injury (DoI) measurements. Sixteen different materials were selected, representing all classes of toxicity, according to the GHS and EPA classification systems. Food-source rabbit eyes, similar to eyes used for the widely accepted Bovine Corneal Opacity and Permeability and Isolated Chicken Eye ocular irritation tests, were used. Tissues were exposed to test material for 1 min, and corneas were collected at 3- and 24-hours post-exposure. Tissues were then fixed and processed for live/dead biomarker fluorescent staining using phalloidin. DoI was then measured, and the percent DoI values for the epithelium and stroma were compared to the EPA and GHS classifications. Excluding surfactants, EPA nonclassified (category IV) materials showed no stromal and very slight epithelial damage (≤10%) to the cornea, whereas EPA corrosive (category I) materials showed significantly greater damage (P < 0.001), ranging from 39% to 100% of the stromal depth. Importantly, EPA reversible (categories II and III) materials showed significant damage to the epithelium (>10%, P < 0.005) but significantly less severe damage to the corneal stroma (P < 0.001), ranging from 1% to 38% of the stromal depth. GHS nonclassified (category NC) irritants caused damage to the epithelium but not to the stroma. All GHS class 2 materials showed damage to the stroma (1–11%), whereas GHS corrosives caused significantly greater damage to the stroma (38–100%; P < 0.001). Additionally, one corrosive material, which produced a stromal DoI of 99% at 24 h, produced no apparent damage at 3-hours post-exposure. Based on these findings, histopathologic EPA and GHS prediction models are proposed that appear to separate and identify reversible irritants from other irritant classes. Furthermore, GHS classification appears to require stromal damage, whereas NC materials may or may not damage the corneal epithelium.

Hydrocortisone loaded poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) nanoparticles for topical ophthalmic administration: Preparation, characterization and evaluation of ophthalmic toxicity.

Poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) nanoparticles (PHBV-NPs) to encapsulate hydrocortisone (HC) for topical ophthalmic administration were prepared and characterized. The technique used to prepare the nanoparticles (NPs) was emulsification/solvent evaporation. The obtained size was 237.3 ± 2.7 nm, suitable for topical ocular administration. The obtained results for the entrapment efficiency were between 1 and 2.5% and for the drug loading were around 0.5%. The release behaviour of HC from the PHBV-NPs was also analyzed, adjusting this to a Higuchi kinetic model. For the new drug delivery system developed the ocular toxicity profile was determined by viability studies carried out on bovine keratocytes, by a Hen's Egg Test - Chorioallantoic Membrane (HET-CAM) and by a Bovine Corneal Opacity and Permeability assay (BCOP). The obtained results concluded that the new system is no cytotoxic on bovine keratocytes and is neither irritating nor produces any alteration in the transparency and in the permeability of the cornea. Confocal studies were also performed and confirmed that PHBV-NPs are able to penetrate efficiently into the corneal tissue. This novel PHBV-based drug delivery system could be a good option for topical ophthalmic administration of drugs.

Corneal histomorphometric analysis: The depth of damage induced in the bovine cornea correlates with the severity of the ocular toxicity

The Bovine Corneal Opacity and Permeability (BCOP) assay is an alternative method used to ocular toxicity potential assessment of chemicals and mixtures. The standard BCOP test provides information about permeability and opacity, however, corneal histopathological analysis has been recommended as an additional parameter to better categorize eye irritants. Moreover, such analysis associated with depth of substance-induced corneal injury analysis may provide additional scientific measurement for the refinement of BCOP test. The aim of this study was to measure the depth of injury into the bovine cornea induced by eye irritants and associate it with the damage severity. For this purpose, BCOP assay was performed for 12 substances from different Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) categories and, additionally, corneal sections of 5 μm thickness were obtained and analyzed by fluorescence microscopy. The results showed that the fluorescein permeation depth was directly proportional to the substances irritation degree. Severe irritants promoted highest rates of permeation followed by moderate and mild irritants, while non-irritants showed similar permeation indexes to the negative control. The refinement of BCOP by the depth of injury analysis through epithelial permeation of fluorescein can be considered a useful quantitative parameter to better categorize eye irritants.

Development of a defined approach for eye irritation or serious eye damage for liquids, neat and in dilution, based on cosmetics Europe analysis of in vitro STE and BCOP test methods

The focus of Cosmetics Europe's programme on serious eye damage/eye irritation is on development of testing strategies and defined approaches for identification of ocular effects of chemicals in the context of OECD's Guidance Document on an Integrated Approach on Testing and Assessment (IATA) for Serious Eye Damage and Eye Irritation.Cosmetics Europe created a comprehensive database of chemicals for which in vitro data are available with corresponding historical in vivo Draize eye data. This database allowed further exploration of the initially proposed strategies from the CON4EI project and to identify opportunities for refinement. The current analysis focused on the development of a defined approach, applicable to liquid non-surfactant chemicals, neat and in dilution, that can distinguish between the three UN GHS categories (Cat. 1, Cat. 2, and No Cat.). Combining the modified-protocol Short Time Exposure (STE) test method (OECD TG 491 with extension to highly volatile substances) with the Bovine Corneal Opacity and Permeability Laser Light-Based Opacitometer (BCOP LLBO) test method in a Bottom-Up approach identified 81.2% Cat. 1, 56.3% Cat. 2, and 85.3% No. Cat correctly, with an NPV of 96.7% and a PPV of 68.6%. Therefore, the performance of the defined approach was better than the standalone test methods.

Assessing the dyeing efficiency and irritation potentials of plant hair dyes: A multi-analytical in vitro approach.

The interest toward dyeing hairs with plant colorants has grown in popularity considering its low-toxic nature. However, researches reporting plant hair dyes are limited and the potential adverse effects of irritation are unclear. This study is aimed to provide an avenue by which to more accurately assess the dyeing efficiency and irritation potentials of plant hair dyes. Four extracted plant colorants were incorporated in hydrogel hair dyes that were directly applied on unbleached gray human hairs. Their dyeing performances and the effect of an iron (Ⅱ) mordant were photometrically measured in CIELab coordinates and color strength. The eye and skin irritancy was assessed by combining various in vitro methods, including bovine corneal opacity and permeability (BCOP) assay in combination with histopathological analysis, Hen's egg test on chick chorioallantoic membrane (HET-CAM) and a test on reconstructed human epidermis models. The investigated hair dyes exhibited desirable dyeing efficiency on human hairs. Post-treatment with the iron (Ⅱ) mordant caused a significant increase in color strength with subtle changes to the hue of dyed color. In the irritation testing, the four hair dyes were categorized as slight-to-mild eye irritants but possessed no skin irritation potential, while the mordant was determined as a non-irritant. The results demonstrate the efficacy of a multi-analytical approach for in vitro assessment of various plant colorants for hair dyeing. The investigated plant extracts are suitable for producing viable colors on human hairs and may serve as a low-irritating alternative to the synthetic hair dyes.

Critical substances in bovine corneal opacity and permeability (BCOP) versus isolated chicken eye (ICE) assays

Critical substances in bovine corneal opacity and permeability (BCOP) versus isolated chicken eye (ICE) assays

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.

Evaluation of a tiered in vitro testing strategy for assessing the ocular and dermal irritation/corrosion potential of pharmaceutical compounds for worker safety

Introduction: Irritation reactions are a frequently reported occupational illness. The potential adverse effects of pharmaceutical compounds (PCs) on eye and skin can now be assessed using validated in vitro methods.Objectives: Our overall aim is to reduce animal testing by replacing the historically utilized in vivo test methods with validated in vitro test methods which accurately determine the ocular and dermal irritation/corrosion potential of PCs to inform worker safety within the pharmaceutical space. Bristol–Myers Squibb (BMS) and the Institute for In Vitro Sciences (IIVS) have therefore conceptualized and internally qualified a tiered in vitro testing strategy to inform occupational hazards regarding eye and skin irritation and corrosivity of PCs. For the small scale pre-qualification phase, we paired historical in vivo and newly generated in vitro data for 15 PCs to determine the predictive capacity of in vitro assays already validated for the eye and skin irritation/corrosion endpoints and accepted for certain regulatory submissions. During the post-qualification phase, a group of 24 PCs were subjected exclusively to the developed tiered testing strategy, which is based on three Organisation for Economic Co-operation and Development (OECD) in vitro methods.Materials and methods: The qualified in vitro testing strategy utilizes the Corrositex® assay for the corrosivity (OECD TG 435), the Bovine Corneal Opacity and Permeability (BCOP) assay for ocular irritation (OECD TG 437), and the EpiDerm™ tissue model-based Skin Irritation Test (SIT) for dermal irritation (OECD TG 439). In the first step, the pH of each PC was determined. For compounds with pH extremes ≥11 or ≤2, the Corrositex® assay was generally conducted first. For compound(s) that were incompatible with or were negative in the Corrositex® assay or had pH values between 2 and 11, the BCOP assay and SIT were performed first.Results: The results of the tiered testing strategy’s qualification phase demonstrated that the BCOP assay is sensitive enough to identify a wide range of eye irritation/corrosion potentials and its over-prediction rate was considered acceptable to inform occupational hazards and ensure the proper handling practices of PCs. The SIT correctly predicted the skin irritation potential of 14 out of the 15 PCs included in the qualification phase, only over-predicting one PC. In the post-qualification phase, four PCs out of four tested were predicted corrosive by the Corrositex® assay and thus no further testing was needed or conducted. The rest of the PCs were evaluated in the BCOP assay (both neat and as a 20% dilution), with the higher response being used for hazard classification. Four PCs were determined to be severe eye irritants, 1 a moderate irritant, 8 were mild irritants, and 8 were non-irritants. The same set of PCs was evaluated using the SIT and were classified as non-irritants to skin. These results are consistent with the BMS historical in vivo results showing a very low number of PCs as skin irritants.Conclusions: This tiered in vitro testing strategy, which replaces the use of animal studies, was found to be reasonably accurate in its predictive capacity when compared to historical in vivo results and represents a conservative and reliable platform that can be utilized for the prediction of ocular and dermal irritation/corrosion potential of PCs and for subsequent GHS classification and worker safety hazard communications.

Reprint of “CON4EI: Bovine Corneal Opacity and Permeability (BCOP) test for hazard identification and labelling of eye irritating chemicals”

Assessment of ocular irritation potential is an international regulatory requirement in the safety evaluation of industrial and consumer products. None in vitro ocular irritation assays are capable of fully categorizing chemicals as stand-alone. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI consortium assessed the reliability of eight in vitro test methods and computational models as well as established a tiered-testing strategy. One of the selected assays was Bovine Corneal Opacity and Permeability (BCOP). In this project, the same corneas were used for measurement of opacity using the OP-KIT, the Laser Light-Based Opacitometer (LLBO) and for histopathological analysis.The results show that the accuracy of the BCOP OP-KIT in identifying Cat 1 chemicals was 73.8% while the accuracy was 86.3% for No Cat chemicals. BCOP OP-KIT false negative results were often related to an in vivo classification driven by conjunctival effects only. For the BCOP LLBO, the accuracy in identifying Cat 1 chemicals was 74.4% versus 88.8% for No Cat chemicals. The BCOP LLBO seems very promising for the identification of No Cat liquids but less so for the identification of solids. Histopathology as an additional endpoint to the BCOP test method does not reduce the false negative rate substantially for in vivo Cat 1 chemicals.

Fatty Acid Based Microemulsions to Combat Ophthalmia Neonatorum Caused by Neisseria gonorrhoeae and Staphylococcus aureus.

The bacterial species Neisseria gonorrhoeae (N. gonorrhoeae) and Staphylococcus aureus (S. aureus) are amongst the main microorganisms that cause ophthalmia neonatorum. The current treatment involves the use of various antibiotics such as ciprofloxacin, cephalosporin, ceftriaxone and cefotaxime. However, this treatment strategy is becoming more ineffective due to the antibiotic resistance in N. gonorrhoeae. The current study explores the potential use of fatty acid based microemulsions (ME) to prevent N. gonorrhoeae and S. aureus infections in new-borns’ eyes without harmful side effects such as corneal or conjunctiva irritation. Pseudo-ternary phase diagrams were constructed to evaluate microemulsion regions and six different α-linolenic acid based microemulsions were prepared. The prepared formulations were characterized for α-linolenic acid content, size, transparency, zeta potential, Polarized light Microscopy, antimicrobial activity and ex vivo ocular toxicity. The mean droplet size of the ME formulations was in the range of 190.4 to 350.5 nm and polydispersity index (PDI) values were in the range of 0.102 to 0.561. All formulations were found stable upon storage for at least 8 weeks. In addition, self-diffusion coefficients determined by nuclear magnetic resonance (NMR) reflected that the diffusability of water increased at higher than 30% w/w water, while that of fatty acids and surfactants was in reverse. The antimicrobial efficacy of microemulsions was determined against N. gonorrhoeae and S. aureus. It was concluded that all microemulsions have strong antimicrobial effects against N. gonorrhoeae and S. aureus. Finally, bovine corneal opacity permeability (BCOP) and hen’s egg chorioallantoic (HET-CAM) tests results showed that all microemulsion formulations were not strong ocular irritants.

Methods for Testing Ocular Toxicity: Current Status

The developed ophthalmic formulations should be tested for ocular toxicity testing like draize eye test. Rapid growth in the field of ocular toxicity testing replaces the usage of animals and to adopt the modified protocols. The researchers have to develop properly validated and regulatory approved options to animal testing. Alternative ocular toxicity testing is based on corneal structure and mechanism through which the specific ocular formulation causes irritation to the eyes. Several alternative assay methods have been introduced in the field of ocular toxicity testing like in vitro organotypic model for testing ocular toxicity and several other methods like Bovine Corneal Opacity and Permeability Assay (BCOP Assay), isolated chicken eye test, isolated rabbit eye test and Hen's egg-test on the chorioallantoic membrane. The ultimate aim of alternate methods of ocular toxicity testing is to develop properly validated and regulatory approved best practices. Hence this review highlights the development in the field of alternative ocular toxicity testing methods.

CON4EI: Bovine Corneal Opacity and Permeability (BCOP) test for hazard identification and labelling of eye irritating chemicals

Assessment of ocular irritation potential is an international regulatory requirement in the safety evaluation of industrial and consumer products. None in vitro ocular irritation assays are capable of fully categorizing chemicals as stand-alone. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI consortium assessed the reliability of eight in vitro test methods and computational models as well as established a tiered-testing strategy. One of the selected assays was Bovine Corneal Opacity and Permeability (BCOP). In this project, the same corneas were used for measurement of opacity using the OP-KIT, the Laser Light-Based Opacitometer (LLBO) and for histopathological analysis. The results show that the accuracy of the BCOP OP-KIT in identifying Cat 1 chemicals was 73.8% while the accuracy was 86.3% for No Cat chemicals. BCOP OP-KIT false negative results were often related to an in vivo classification driven by conjunctival effects only. For the BCOP LLBO, the accuracy in identifying Cat 1 chemicals was 74.4% versus 88.8% for No Cat chemicals. The BCOP LLBO seems very promising for the identification of No Cat liquids but less so for the identification of solids. Histopathology as an additional endpoint to the BCOP test method does not reduce the false negative rate substantially for in vivo Cat 1 chemicals.

Selection of olive oil as solvent in Bovine Corneal Opacity and Permeability (BCOP) test

Selection of olive oil as solvent in Bovine Corneal Opacity and Permeability (BCOP) test

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.

Suggestion of the updated IVIS cut-off values for identifying non-ocular irritants in the bovine corneal opacity and permeability (BCOP) assay

The bovine corneal opacity and permeability (BCOP) assay is an alternative to the Draize test in rabbits. Although it can be used to evaluate ocular non-irritants and severe irritants, it is not used for the assessment of mild-irritating substances. In this assay, a chemical with an in vitro irritancy score (IVIS)≤3 is defined as a “non-irritant” while one with an IVIS>55 is defined as a “corrosive” or “severe irritant.” We attempted to evaluate mild eye irritants by assessing the recovery of corneal lesions histopathologically. Our results indicated that chemicals with IVIS≤6 may be defined as “non-irritants,” because the rapid recovery of lesions limited to the squamous layer was predicted by the histopathology. In cases where lesions extended to the wing and basal cell layers, when some intact basal cells remained, the lesions were also predicted to be reversible. Thus, chemicals which induce lesions in which basal cells remain intact can be considered to be mild irritants.

Prevention of Ophthalmia Neonatorum Caused by Neisseria gonorrhoeae Using a Fatty Acid-Based Formulation.

ABSTRACTOphthalmia neonatorum, also called neonatal conjunctivitis, acquired during delivery can occur in the first 28 days of life. Commonly caused by the bacterial pathogen Neisseria gonorrhoeae, infection can lead to corneal scarring, perforation of the eye, and blindness. One approach that can be taken to prevent the disease is the use of an ophthalmic prophylaxis, which kills the bacteria on the surface of the eye shortly after birth. Current prophylaxes are based on antibiotic ointments. However, N. gonorrhoeae is resistant to many antibiotics and alternative treatments must be developed before the condition becomes untreatable. This study focused on developing a fatty acid-based prophylaxis. For this, 37 fatty acids or fatty acid derivatives were screened in vitro for fast antigonococcal activity. Seven candidates were identified as bactericidal at 1 mM. These seven were subjected to irritation testing using three separate methods: the bovine corneal opacity and permeability (BCOP) test; the hen’s egg test—chorioallantoic membrane (HET-CAM); and the red blood cell (RBC) lysis assay. The candidates were also tested in artificial tear fluid to determine whether they were effective in this environment. Four of the candidates remained effective. Among these, two lead candidates, monocaprin and myristoleic acid, displayed the best potential as active compounds in the development of a fatty acid-based prophylaxis for prevention of ophthalmia neonatorum.

CON4EI: Bovine Corneal Opacity and Permeability (BCOP) test for hazard identification and labelling of eye irritating chemicals

Assessment of ocular irritation potential is an international regulatory requirement in the safety evaluation of industrial and consumer products. None in vitro ocular irritation assays are capable of fully categorizing chemicals as stand-alone. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI consortium assessed the reliability of eight in vitro test methods and computational models as well as established a tiered-testing strategy. One of the selected assays was Bovine Corneal Opacity and Permeability (BCOP). In this project, the same corneas were used for measurement of opacity using the OP-KIT, the Laser Light-Based Opacitometer (LLBO) and for histopathological analysis.The results show that the accuracy of the BCOP OP-KIT in identifying Cat 1 chemicals was 73.8% while the accuracy was 86.3% for No Cat chemicals. BCOP OP-KIT false negative results were often related to an in vivo classification driven by conjunctival effects only. For the BCOP LLBO, the accuracy in identifying Cat 1 chemicals was 74.4% versus 88.8% for No Cat chemicals. The BCOP LLBO seems very promising for the identification of No Cat liquids but less so for the identification of solids. Histopathology as an additional endpoint to the BCOP test method does not reduce the false negative rate substantially for in vivo Cat 1 chemicals.

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.

In vitro safety and efficacy evaluations of a complex botanical mixture of Eugenia dysenterica DC. (Myrtaceae): Prospects for developing a new dermocosmetic product

In the context of developing a new natural product-based cosmetic, the in vitro efficacy and safety evaluations of a complex botanical mixture based on Eugenia dysenterica leaf hydroalcoholic extract (EDE) (2.5–1000μg/mL) were carried out. Chromatographic analysis demonstrated the presence of the tannin (ellagic acid) and flavonoids (quercetin and gallic acid) which characterize the EDE as a polyphenol-rich mixture. Using HFF-1 fibroblasts, it was shown that EDE promoted cell regeneration after UVA exposure. It also led to the inhibition of the collagenase, elastase and tyrosinase enzymes, which are involved in skin-related disorders. In terms of toxicological evaluation, the EDE was classified as non-phototoxic through the 3T3 Neutral Red Uptake Phototoxicity Test (OECD N° 432, 2004) and non-eye irritant by Bovine Corneal Opacity and Permeability (OECD N° 437, 2013) assay, in conjunction with corneal histomorphometric analysis. Furthermore, the EDE has no skin sensitization potential as demonstrated by a two-out-of-three prediction model [protein-binding/haptenization (OECD N° 442C, 2015), keratinocyte and dendritic cell activations]. In addition, it was shown that the EDE seems to be non-genotoxic through the cytokinesis-block micronucleus assay (OECD N° 487, 2014) using HepG2 cells. When considered together, these findings support the use of EDE botanical mixture in cosmetic/pharmaceutical products.