Noémie Bonneau
email: noemie.bonneau@inserm.fr

Christophe Baudouin

Reaux-Le Goazigo

Françoise Brignole-Baudouin

Annabelle Reaux-Le Goazigo

AOP and IATA applied to ocular surface toxicity

Keywords: Draize Eye Test, OECD guidelines, Ocular Surface, In Silico, 3D Multicellular, Cornea-on-a-chip, Organoids

Introduction

Since the beginning of the 21 st century, modern toxicology has been focusing on the 3R principle, "Reduce, Refine, Replace", established in 1959 by Russell and Burch, stipulating that the use of laboratory animals should be only a last resort. Since 2013, in Europe, the cosmetic industry has been confronted with strict prohibition of evaluating its products on animals. Integrated approaches of testing and assessment (IATA), promoted by the OECD (Organisation for Economic Cooperation and Development) might enable validation of new compounds in this sector [START_REF] Canavez | Integrated approaches to testing and assessment as a tool for the hazard assessment and risk characterization of cosmetic preservatives[END_REF].

To date, validated alternative models have been available only for the evaluation of potential ocular surface irritation. Models to predict Toxicity-Induced Dry Eye (TIDE), anterior segment neuropathies or other ocular surface changes are still in the stage of basic science research. Furthermore, classification of ocular irritants is based on the United Nations Organization (UNO) system, i.e. the GHS "Globally Harmonized System of Classification and Labelling of Chemicals" [START_REF] Luechtefeld | Analysis of Draize eye irritation testing and its prediction by mining publicly available 2008-2014 REACH data[END_REF]). This international system distinguishes severe irritants (Category 1), moderate irritants (Category 2A), mild irritants (Category 2B) and non-irritants (No Category). However, unlike the Draize test, the in vivo reference model in rabbits, current alternative models for ocular irritation cannot distinguish Category 2A from 2B irritants. These irritants are usually differentiated based on the kinetics of the reversibility of damage. Of note, the lack of reproducibility of the in vivo test of reference, the Draize test, complicates the validation of alternative models by the ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods)(OECD Webinar 2019a).

In its first section, this review presents updates in the latest methodology for evaluation of ocular irritation, first presenting the five in vitro or ex vivo models validated by the OECD, in combination, to replace the Draize test (Guideline (GL) 405)(OECD, 2020a): Reconstructed human Cornea-like Epithelium (RhCE) viability tests (GL 492)(OECD, 2019a), Bovine Corneal Opacity and Permeability (BCOP) test (GL 437)(OECD, 2020b), Isolated Chicken Eye (ICE) test (GL 438)(OECD, 2018a), Fluorescein Leakage (GL 460) (OECD, 2017), and Short Time Exposure assay (STE, GL 491) (OECD, 2020c). The ocular irritation IATA indicates the combination of tests that should be considered depending on whether the product is suspected to be an irritant ('top-down' approach) or is thought to be in the non-irritant category ('bottom-up' approach). This review also presents models used by some cosmetic companies that either were or still are under evaluation by the OECD, such as the Isolated Rabbit Eye (IRE) test, Hen's Egg Test on the Chorio-Allantoic Membrane (HET-CAM).

Characteristics and protocol details of the models for ocular irritation are summarized in Table I.

Next, promising models mentioned in the OECD Guidance Document No. 263 (OECD, 2019b) are presented. These models, if optimized and validated, might represent a major asset in classifying new compounds into Categories 2A and 2B: PorCORA (Porcine Ocular Cornea Opacity/Reversibility Assay), EVEIT (Ex Vivo Eye Irritation Test), 3D Hemi-Cornea and SMI (Slug Mucosal Irritation assay).

Finally, in order to prevent complex toxicities as TIDE, glaucoma, cataract, some of which are rare topical side effects, new models presented in the literature could be validated and incorporated into new IATAs, taking into account real-life exposure, pharmacokinetics and knowledge already reported in the literature. Therefore, this final section provides an overview of in silico and in vitro models which could, in combination, enable complete evaluation of ocular surface toxicities within the framework of IATAs.

Alternative models to the Draize test according to OECD GL 405

Reconstructed human Cornea-like Epithelium (RhCE) Since the last update of GL 492 in 2019 (OECD, 2019a), four models of RhCE are now available to evaluate ocular surface irritation, two of which are considered Validated Reference Methods (VRM): EpiOcular™ (VRM1), SkinEthic™ HCE (VRM2), LabCyte CORNEA-MODEL24 and MCTT HCE TM . These RhCE models mimic human corneal epithelium morphologically, histologically, biochemically and physiologically and can be used first in a 'bottom-up' approach to identify non-irritant substances. Even though cellular damage can occur through several mechanisms, only cytotoxicity measurements are carried out. Indeed, cell viability is considered to be proportional to the severity of damage and representative of the global response of the ocular surface: mild irritants with low transcorneal penetration alter only the superficial corneal epithelium, whereas moderate or severe irritants can penetrate more deeply, reaching the corneal stroma and sometimes endothelium. This global response would be a correct representation of the damage that could occur in humans after toxic exposure, no matter the cellular mechanisms involved, ranging from slight conjunctival erythema or edema to severe changes such as corneal opacification.

While there may be differences between RhCE models (Table II), mainly concerning the cell types used and duration of epithelium culture, the testing method is similar: direct application of the tested compounds on the 3D epithelium and viability cytotoxicity assays reflecting the mitochondrial metabolic ability of viable cells. If corneal viability diminishes to below the fixed threshold (specific to each RhCE, see Table II), this will suggest classification of the compound as an ocular irritant. Above the threshold, the compound will not be classified and must be combined with another validated GL (437,438,460,491 or in last option, if the test compound is not a cosmetic, GL 405 i.e. Draize test). Nevertheless, the OECD Guidance Document No. 263 (OECD, 2019b) reports the ongoing OECD evaluation of the EpiOcular™ time-to-toxicity (ET50) assay, a test that could enable the differentiation of category 2A from 2B irritants [START_REF] Kandarova | CON4EI: CONsortium for in vitro Eye Irritation testing strategy -EpiOcular™ time-to-toxicity (EpiOcular ET-50) protocols for hazard identification and labelling of eye irritating chemicals[END_REF]. This new protocol is based on multiple time and concentration exposures. It could represent a major asset in the scope of IATA decision trees, since as of yet, no validated alternative model alone can distinguish between all the categories of irritants.

Another limitation is that GL 492 can only be used for solids, semi-solids, liquids and waxes, since gases and aerosols have not undergone validation procedures. Nonetheless, this aspect should be investigated, since many accidental ocular exposures are caused by volatile compounds (OECD 2019a).

Bovine Corneal Opacity and Permeability assay (BCOP)

Recommended as the first step of a 'top-down' strategy, the organotypic BCOP model described in GL 437 (OECD, 2020b) enables differentiation between severe irritants applied to isolated bovine cornea from slaughterhouses. It can also identify non-irritants in a 'bottomup' approach. The eyeballs are kept ex vivo for a brief period, during which physiological and biochemical functions remain unaltered. After excision, corneas are anchored on a corneal holder composed of two chambers, both filled with preservation medium. Briefly, the endothelial surface of the cornea is placed on the o-ring in the posterior chamber, while the epithelial surface is positioned in the anterior chamber.

Two application methods, adapted to the type of compound being tested, are described in the GL, but an important parameter is verification that the product covers the entire epithelial surface and that the washing step is sufficient to retrieve all of the compound. Irritancy potential is then measured through the In Vitro Irritancy Score (IVIS), which combines the diminution of light transmission capacity (corneal opacity, measured with an opacimeter) and the increase in fluorescein sodium passage (permeability, i.e. the amount of dye dropped in the anterior chamber and that crosses the corneal thickness). Of note, fluorescein sodium is an anionic compound, not retained by a healthy, negatively charged epithelium. A substance will be categorized as a severe irritant if the IVIS is greater than 55 and as a non-irritant if the IVIS is less than 3. However, if the IVIS is between 3 and 55, additional tests will be required to distinguish category 2 irritants. It is also possible to complement these results with a histologic analysis of the cornea, which procedure is described in the Guidance Document No.

(OECD, 2018b).

Of note, since the last guideline update in June 2020, a second opacimeter can be used (LLBO), requiring adaptation of the IVIS equation and decision criteria, but the performance is comparable to OP-KIT, the first opacimeter validated.

Isolated Chicken Eye (ICE)

Like the BCOP model, the ICE aims to discriminate Category 1 GHS substances in a 'topdown' strategy but can also be included in a 'bottom-up' approach to identify non-irritants.

The ICE is regulated by OECD GL 438, last updated in 2018(OECD, 2018a). This test uses enucleated eyes of chickens for human consumption. In this assay, corneas are not excised.

The whole eye is placed in a stainless-steel clamp with the cornea positioned vertically. The clamp is placed in a superfusion chamber to nourish the cornea. At the start of the test, the clamp is retrieved from the chamber and the cornea positioned horizontally in order to apply the tested compounds. A qualitative and quantitative evaluation of the cornea is conducted to establish potential opacities, epithelial morphological alterations (detected by fluorescein sodium retention) and edema. As for BCOP, corneal opacity and fluorescein retention are scored, and are associated to a morphological evaluation which is "subjective according to the interpretation of the investigator" (GL 438). The combination of these scores enables the GHS classification of test compounds. For instance, with three scores of I, the substance is considered a non-irritant.

Furthermore, since the last GL update, histological features after paraffin embedding can be analyzed notably for detergents and surfactant irritants (OECD, 2019b). Indeed, there should be a correlation between erosion, vacuole formation in the inferior area of the epithelium, presence of pycnotic nuclei and irreversibility of the damage. GL 438 proposes another table to score those parameters.

Fluorescein Leakage (FL)

The FL test follows GL 460 (OECD, 2017). The FL method is performed in vitro on a semipermeable membrane (insert) leading to a single-layer culture of renal tubular cells of Madin-Darby Canin (MDCK CB997). This is a well described cell line, known to form tight junctions and desmosomes. Its organization is similar to the non-proliferative apical corneal epithelium. Furthermore, permeabilization of corneal epithelium is known to be one of the first phenomena occurring in toxicity-induced ocular irritation.

Changes in tight junctions and desmosomes are proportional to the quantity of fluorescein sodium that diffuses into the basal chamber, evaluated though FL20%, that is to say the concentration of the tested compound that leads to an FL of 20% compared to negative controls (single layer of cells not exposed and insert without cells). The substance tested is categorized as a severe irritant on the GHS ocular irritation classification if the FL20% ≤ 100 mg/mL. Integrated into a 'top-down' strategy, this simple method enables distinguishing Category 1 chemicals without additional data. However, unlike the previous methods presented, this method can only be used with water-soluble compounds or mixtures. Indeed, solids in suspension will precipitate. In addition, it is not applicable to strong bases or acids, volatile compounds or cellular fixatives, because the toxic mechanisms for these types of compounds (such as protein coagulation or saponification) cannot be evaluated by FL. Finally, colored or viscous compounds should be tested with other methods, since their complete washout required before the fluorescent measurement is complicated. Of note, a compound with a strong affinity for the insert membrane can lead to the same problem. Therefore, this affinity must be tested, as described in the GL, before beginning the assay. While reversibility of the epithelial changes cannot yet be evaluated, this will be considered in the next update of the GL, with the possibility of using FL to separate Categories 2A and 2B of the GHS classification.

Short Time Exposure Assay (STE)

The STE, GL 491 (OECD, 2020c), can be considered in 'bottom-up' and 'top-down' strategies. STE enables the evaluation of all types of chemicals except volatile compounds with a vapor pressure above 6 kPa 1 and solid non-surfactants (not water-soluble after at least 5 min in NaCl). This in vitro model consists of a confluent monolayer culture of rabbit corneal fibroblasts (several cell lines are possible, such as CCL60 or SIRC). If cell viability (MTT assay, see Table 2) is less than 70% with both concentrations, the substance is placed in Category 1 without any additional assay. If cell viability is above 70% with at least one concentration, additional tests are required.

Other ocular irritation models evaluated by the OECD

Vitrigel-Eye Irritancy Test (EIT) method

While not mentioned in GL 405 as an alternative method, the OECD introduces this method in its Guidance Document n°263 (OECD, 2019b) and in the 2019 GL 494 (OECD, 2019c), establishing the protocol for the Vitrigel EIT method. This method can be used only in a 'bottom-up' approach to identify non-irritants. It evaluates the barrier function of a human corneal epithelium reconstructed on a Vitrigel matrix (ECh-T immortalized cell line ; collagen gel obtained by rehydration of a hydrogel that has undergone a vitrification process) [START_REF] Takezawa | Collagen vitrigel: a novel scaffold that can facilitate a three-dimensional culture for reconstructing organoids[END_REF][START_REF] Yamaguchi | Predictive performance of the Vitrigeleye irritancy test method using 118 chemicals[END_REF]) with a Transepithelial Electrical Resistance (TEER) measurement. Of note, this ohmmeter analysis is sensitive to the number of cell passages and to room temperature [START_REF] Srinivasan | TEER measurement techniques for in vitro barrier model systems[END_REF]. This measurement is characterized by three parameters: time lag, intensity and plateau level. A non-irritant product is identified by a time lag > 180 seconds, an intensity < 0.05% and a plateau level ≤ 5.0%. If one of these criteria differs, additional studies are required to classify the product. A limitation of this method is its small range of application, being limited to liquids or semiliquids with a pH > 5. However, unlike previous methods described, it can be used for volatile compounds and products that interfere with the detection of formazan in the MTT assay.

Ocular irritection®

Ocular Irritection® is an in vitro macromolecular test which is the subject of a GL drafted in 2019 (OECD, 2019d). It is suitable either in a 'bottom-up' or 'top-down' strategy for solids and liquids with a pH between 4 and 9. It is an acellular system composed of proteins, glycoproteins, carbohydrates, lipids and low molecular weight compounds. Ocular Irritection® test aims to mimic the organized and transparent structure of the cornea after rehydration [START_REF] Eskes | Validation study on the Ocular Irritection assay for eye irritation testing[END_REF]. This enables specific detection of protein coagulation or lipid saponification mechanisms. Nevertheless, since the system is devoid of cells, cytotoxicity cannot be evaluated. The matrix and the testing principle are presented in figure 1. Any change in the matrix organization leads to a modification of the turbidity and reflects the irritative capacity of the test compound. However, like many alternative models, this testing method alone is unable to distinguish mild irritants.

Cytosensor Microphysiometer (CM)

Because of the lack of commercial availability of the Cytosensor Microphysiometer technology, the preliminary GL version released in 2012 (OECD, 2012) for the evaluation of water-soluble compounds, solids, viscous substances or homogenous suspensions has seen its development discontinued in 2016 (European Commission 2020a). Nevertheless, it could be integrated into a 'bottom-up' or 'top-down' approach if similar instruments were to come to market. It consists of an adherent, confluent, single layer of mice fibroblasts (cell line L929) cultured on a polycarbonate insert. These cells are designed to represent conjunctival and corneal epithelia. The test endpoint is the Metabolic Rate Decrement of 50% (MRD50), that is to say the concentration that reduces the acidification rate by 50%. This measurement reveals irritation potential, since damaged cells will produce less acidic metabolites in the culture medium. On the one hand, if the MRD50 ≤ 2 mg/mL, the product is considered to be a severe irritant in a 'top-down' approach. On the other hand, if the MRD50 > 10 mg/mL, the test compound is classified as a non-irritant in a 'bottom-up' strategy. Of note, the GL mentions that this testing method could evaluate reversibility if optimized.

Neutral Red Release (NRR)

The NRR test evaluates cytotoxicity on a single layer fibroblast or keratinocyte culture loaded with neutral red 3 hours before the exposure to test compounds (OECD, 2019b). This vital dye incorporates itself into lysosomes of viable cells. Several protocols have been proposed in the literature [START_REF] Zuang | The neutral red release assay: a review[END_REF], such as the FRAME protocol based on mice embryonic fibroblasts (3T3-L1 cell line) or the Clonetics Corporation protocol using human keratinocytes. In both cases, the endpoint is the NRR50, that is to say the test compound concentration that releases, in the culture medium, 50% of the neutral red incorporated by lysosomes. The more toxic a substance is, the more cellular membranes, including lysosomal membranes, are altered, leading to leakage of intracellular compounds such as neutral red.

Validated by internal procedures in many industries, the ICCVAM is requesting supplementary data on inter-laboratory reproducibility before publishing a GL on the Neutral Red Release assay (OECD, 2019b). It is also being considered for use in combination with the EpiOcular time-to-toxicity assay on RhCE.

Red Blood Cell test (RBC)

The RBC test evaluates the ability of test compounds to disrupt red cell membranes (relation between hemolysis and oxyhemoglobin denaturation) and in this way, to classify products into GHS Categories 1 or non-classified (OECD, 2019b). RBC test can be conducted on red blood cells from various species (pig, sheep, rabbit) [START_REF] Lewis | A comparison of two cytotoxicity tests for predicting the ocular irritancy of surfactants[END_REF][START_REF] Mehling | Comparative studies on the ocular and dermal irritation potential of surfactants[END_REF]Pape et al. 1987;[START_REF] Pape | Standardization of an in vitro red blood cell test for evaluating the acute cytotoxic potential of tensides[END_REF]). The irritant potential score corresponds to the ratio between the leakage of red blood cell hemoglobin in the supernatant (H50 concentration inducing a red cell hemolysis of 50%) and oxyhemoglobin (denaturation index, DI). If H50/DI > 100, the substance is considered a non-irritant, between 10 and 100 the substance is categorized as a mild irritant (Category 2), between 1-10 as a moderate irritant, and if the H50/DI < 1, the compound is classified in Category 1 (severe irritant).

An application of this method on 12 shampoos and 7 conditioners was proposed by [START_REF] Alves | A reassessment of the in vitro RBC haemolysis assay with defibrinated sheep blood for the determination of the ocular irritation potential of cosmetic products: comparison with the in vivo Draize rabbit test[END_REF], attesting to a 91.6% sensitivity and 100% specificity of the method. However, in the Guidance Document n°263 (OECD, 2019b), the OECD underscores the necessity for more data on the types of compound that can be tested, in other words, the method's range of application. Indeed, while the literature reports other studies on surfactants, mixtures [START_REF] Mehling | Comparative studies on the ocular and dermal irritation potential of surfactants[END_REF]) and eyedrops [START_REF] Martins | Determination of Eye Irritation Potential of Low-Irritant Products: Comparison of in Vitro Results with the in Vivo Draize Rabbit Test[END_REF], the chemical and physical properties of test compounds must be further investigated.

Isolated Rabbit Eye (IRE)

Although the IRE test is similar to the ICE (compound exposure time, endpoints ; see Table 1), this organotypic model on the enucleated rabbit eye has not been validated by the ICCVAM since its 2010 evaluation, due to the lack of a standardized protocol, the lack of data on decision criteria, and the fact that rabbit eyes come from experimental animals and not from slaughterhouses as with BCOP or ICE [START_REF] Lee | Alternatives to In Vivo Draize Rabbit Eye and Skin Irritation Tests with a Focus on 3D Reconstructed Human Cornea-Like Epithelium and Epidermis Models[END_REF][START_REF] Prinsen | Justification of the enucleated eye test with eyes of slaughterhouse animals as an alternative to the Draize eye irritation test with rabbits[END_REF].

Nevertheless, the IRE is accepted in the European Union for distinguishing severe irritants (except alcohols, solids and surfactants, for which there are too many false negatives) (ICCVAM 2010).

Hen's Egg Test on Chorioallantoic Membrane (HET-CAM)

The HET-CAM is an alternative model developed by Luepke in 1985 and modified to classify irritant compounds. Indeed, the chorioallantoic membrane of the egg is considered to be a reasonable facsimile of the conjunctiva and its vasculature. Of note, from this model was derived another model, the Chorioallantoic Membrane Vascular Assay (CAMVA). The main nuance between the two (see figure 2) could enable differentiation of non-irritants from mild or moderate irritants.

The main advantages of using embryonated eggs are their accessibility, low cost and rapid growth. These eggs can be kept in an incubator for up to 13 days of maturation. After 14 days of growth, the development of the embryo is advanced, and the model is then considered an in vivo model [START_REF] Kue | Chick embryo chorioallantoic membrane (CAM): an alternative predictive model in acute toxicological studies for anticancer drugs[END_REF].

While the ICCVAM did not validate this testing method for distinguishing severe irritants (ICCVAM 2010), this method is still used by some industrials in their internal weight of evidence WoE, these methods being recognized in the European Union. The procedure for opening the eggs without breaking the vascular membrane is described in figure 2.

However, one should bear in mind that this testing method has been increasingly criticized, being considered an in vivo model even in the first days of embryonic development.

Models requiring optimization according to the OECD

The models introduced in the following section are models mentioned in the OECD Guidance Document n°263 as interesting models, if optimized, for evaluation of reversibility of ocular irritation/corrosion, which may thus be able to distinguish between all GHS categories, including category 2 compounds.

Porcine Ocular Cornea Opacity/Reversibility Assay (PorCORA)

PorCORA is an organotypic model similar to BCOP, since it is based on the maintenance ex vivo of porcine cornea obtained from slaughterhouses. Its added value lies in the air interface preservation allowing maintenance for 21 days (same as in the Draize reference test, the amount of time needed to evaluate reversibility of damage). Several steps are required to prepare the excised corneas [START_REF] Vij | Resolving Severe/Corrosive Irritant Ocular Classifications Using an Alternative Dual Ex Vivo Assay System[END_REF]. First, the corneas are placed in a 24-well plate, with the epithelium facing the bottom of the well. A mixture of agar/gelatin/medium is poured onto the corneas, which are then placed in Petri dishes after gelification. The test compounds are applied directly to the corneal surface (10 µL for liquids, 20 mg for solids) for 5 minutes.

The corneas are then washed with PBS [START_REF] Piehl | Porcine Corneal Ocular Reversibility Assay (PorCORA) predicts ocular damage and recovery for global regulatory agency hazard categories[END_REF]. Corneal alterations and their reversibility are then estimated and scored based on the area of staining with fluorescein sodium over the course of 1, 2, 3, 7, 10, 14 and 21 days after compound exposure (European Commission 2020b).

In this way, Piehl et al. demonstrated in 2011 that this method gave similar results to the Draize reference test (correlation coefficient of 0.98) with reproducible results for the five control test substances: phosphate buffered saline (PBS), absolute ethanol (EtOH), 3% sodium dodecyl sulfate (SDS), 1% benzalkonium chloride (BAK), and 10% sodium hydroxide (NaOH). Furthermore, in this study, PorCORA identified reversible and irreversible effects. By establishing a PorCORA score, it could be possible to distinguish GHS category 1 products (irreversible alterations before 21 days) from category 2 products (reversible damages before 21 days, with a score returning to 0). Nevertheless, additional data is needed. Indeed, in this study, Piehl et al. found that the method was too sensitive for surfactants. Similarly, in another study conducted on shampoos and hair dyes, PorCORA overestimated the irritant potential [START_REF] Donahue | Ocular irritation reversibility assessment for personal care products using a porcine corneal culture assay[END_REF]. Finally, a drawback of this model is the progressive opacification of the cornea due to the gel that prevents the endothelium from correctly regulating corneal stromal fluids [START_REF] Spöler | The Ex Vivo Eye Irritation Test as an alternative test method for serious eye damage/eye irritation[END_REF].

Ex Vivo Eye Irritation Test (EVEIT)

The EVEIT is an air-liquid interface culture system, enabling maintenance of excised rabbit corneas (from slaughterhouses) for 72 hours following compound application. Briefly, corneas with a scleral ring are removed and anchored in a chamber filled with a minimal volume of medium to maintain hydrostatic pressure. This ex vivo model reflects the biochemical activity of corneal epithelium and endothelium. Its advantage compared to the PorCORA system presented above is that the EVEIT does not lead to corneal opacification during culture [START_REF] Spöler | The Ex Vivo Eye Irritation Test as an alternative test method for serious eye damage/eye irritation[END_REF]. Decision criteria are evaluated four times over 72 hours enabling differentiation of non-irritants from category 2A irritants (OECD, 2019b;[START_REF] Spöler | The Ex Vivo Eye Irritation Test as an alternative test method for serious eye damage/eye irritation[END_REF]: macroscopic observation of corneal opacity, fluorescein sodium diffusion, corneal thickness and structural changes measured by optical coherence tomography. Each measurement results in a score, similar to those used in the ICE or Draize tests, which were described by [START_REF] Spöler | The Ex Vivo Eye Irritation Test as an alternative test method for serious eye damage/eye irritation[END_REF] If preservation time of the corneas ex vivo could be improved, this testing method could enable differentiation of all category 2 products. Of note, this method was used by [START_REF] Schrage | The Ex Vivo Eye Irritation Test (EVEIT) in evaluation of artificial tears: Purite-preserved versus unpreserved eye drops[END_REF] to evaluate the effect of artificial tears on corneal epithelial repair after mechanical damage. This study highlights the fact that the models presented in this section could serve equally well for toxicity studies as for pharmacological studies for the development of ophthalmic treatments.

3D Hemi-Cornea

The first in vitro system that may potentially discriminate GHS categories 1 and 2 alone, the 3D Hemi-Cornea combines, in an insert, a corneal human epithelium reconstituted from an immortalized cell line with human corneal immortalized keratinocytes which represent stromal cells [START_REF] Bartok | Development of an in vitro ocular test system for the prediction of all three GHS categories[END_REF][START_REF] Engelke | A human hemi-cornea model for eye irritation testing: quality control of production, reliability and predictive capacity[END_REF][START_REF] Zorn-Kruppa | Determining the Depth of Injury in Bioengineered Tissue Models of Cornea and Conjunctiva for the Prediction of All Three Ocular GHS Categories[END_REF]). The two cell types are separated by a collagen membrane allowing evaluation of the two cell lines independently after a 60 min-exposure of the chemical [START_REF] Zorn-Kruppa | Determining the Depth of Injury in Bioengineered Tissue Models of Cornea and Conjunctiva for the Prediction of All Three Ocular GHS Categories[END_REF]). This model is adapted for liquids as well as solids but is constraining since it has to be cultured during 7 days with a daily change of medium. The endpoint measured is metabolic activity and the cytotoxicity MTT test. The distinction between GHS categories non-irritant, 1 and 2 could be observed though the extension and/or localisation of corneal changes [START_REF] Tandon | Assessment of the eye irritation potential of chemicals: A comparison study between two test methods based on human 3D hemi-cornea models[END_REF].

Moderate irritants lead to a loss of viability of the corneal epithelium and can affect the stroma, whereas severe irritants lead to severe corneal epithelial and stromal alterations. As a result, this system properly classifies category 1 compounds and 80% of category 2 compounds, but only 50% of non-irritant substances, with an overestimation of their irritant potential. A hypothesis to explain these last, disappointing results is that the compounds in this category were frequently viscous and difficult to remove during the washing steps, leading to the deterioration of some epithelial layers [START_REF] Bartok | Development of an in vitro ocular test system for the prediction of all three GHS categories[END_REF]. Furthermore, in another study, the irritation potential of compounds with extreme pH were again overestimated, as in other in vitro tests, possibly because of the absence of the mucinous layer of the tear film, which has a buffer effect in vivo [START_REF] Zorn-Kruppa | Determining the Depth of Injury in Bioengineered Tissue Models of Cornea and Conjunctiva for the Prediction of All Three Ocular GHS Categories[END_REF].

Nonetheless, this test quoted in the Guidance Document n°263 of OECD seems to be an option for the evaluation of surfactants, alcohols, ketones, and volatile compounds, in other words, compounds that, in many other alternative models, lead to false positives results. This 3D hemi-cornea could at the same time allow the evaluation of compound diffusion, since the test substances need to cross an aqueous collagen membrane.

Slug Mucosal Irritation (SMI) assay

Described in the literature for the evaluation of reversible or irreversible ocular (Lenoir et al. 2011a) and nasal [START_REF] Lenoir | The Slug Mucosal Irritation (SMI) assay: a tool for the evaluation of nasal discomfort[END_REF] stinging, itching and/or burning (SIB), the SMI test measures the liberation of mucus proteins from Arion lusitanicus slugs. This method can screen for ocular discomfort generated by isolated ingredients or final products. As presented in the schematic protocol Figure 3, the slug's weight is compared before and after every contact period (CP) [START_REF] Lenoir | A New Application of the Slug Mucosal Irritation (SMI) Assay: Detecting Nasal Stinging, Itching and Burning (SIB)[END_REF](Lenoir et al. , 2011a[START_REF] Lenoir | The Slug Mucosal Irritation (SMI) assay: a tool for the evaluation of nasal discomfort[END_REF][START_REF] Cutuli | Potential Mucosal Irritation Discrimination of Surface Disinfectants Employed against SARS-CoV-2 by Limacus flavus Slug Mucosal Irritation Assay[END_REF].

Developed by Lenoir et al., this test was used to evaluate shampoos and artificial tears. The results were correlated with a clinical study (Spearman's Rank correlation of 0.986, p < 0.001) (Lenoir et al. 2011b). Similarly, [START_REF] Petit | Assessment of ocular discomfort caused by 5 shampoos using the Slug Mucosal Irritation test[END_REF] was able to reproduce this alternative model in 2017 to evaluate veterinary products. Recently, a new SMI alternative model, using a "Yellow slug", was reported to evaluate surface disinfectants used against SARS-CoV-2 [START_REF] Cutuli | Potential Mucosal Irritation Discrimination of Surface Disinfectants Employed against SARS-CoV-2 by Limacus flavus Slug Mucosal Irritation Assay[END_REF].

Since it can distinguish category 2 irritant products, optimization and validation of this test is mentioned to be of interest in the OECD Guidance Document n°263. However, depending on national regulations, this test might be considered animal experimentation (OECD, 2019b).

New innovative models for ocular surface toxicity evaluation

Mimicking ocular structures in vitro is challenging (lacrimal glands, conjunctiva, innervation, lens, …). New models are being developed in basic science research, notably using fluidic and three-dimensional approaches. These technologies of organ-on-a-chip originate from the area of pharmaceutical research and development [START_REF] Wilson | An overview of current techniques for ocular toxicity testing[END_REF]. In addition, this review will focus on in silico approaches, which are required to understand real-life exposure and thus aid in design of the in vitro strategy, reducing time and costs of development. Organoid models will be described in the final part of this section, even though these new cellular structures are mainly studied for the purpose of replacing deficient patient structures.

Table III proposes an overview of the selected models.

In silico models

In silico approaches, using computer and mathematical tools, aim to simulate in vivo biological processes, mimicking a multicellular organ crossed by biological flows and connected to other structures of the organism. Inspired by the "PB-PK", Physiologically Based Pharmacokinetics, approach (predicting absorption, distribution, metabolism and elimination), these in silico methods try to improve toxicological evaluation, taking into account local metabolism, barriers, …., and to estimate a toxic dose [START_REF] Knudsen | FutureTox II: in vitro data and in silico models for predictive toxicology[END_REF].

Once the organ is modelled, multiple scenarios can be tested by changing dose, time, method of exposure and other parameters that could influence the risk of toxicity (for instance, enzyme polymorphisms, pregnant women or pediatric differences in metabolism) [START_REF] Jones | Physiologically based pharmacokinetic modeling in drug discovery and development: a pharmaceutical industry perspective[END_REF].

For each product tested, an exhaustive knowledge of its physicochemical properties must come through computerized channels [START_REF] Brochot | Chapter13. Modelisation Toxico-Pharmacokinetic with Physiological Basis: Rle for risk and pharmacology evaluation[END_REF]. To this end, other in silico tools can contribute to the information in the literature: Qualitative and Quantitative Structure Activity Relationship (QSAR) models that can predict biological properties such as affinity, protein binding, based on chemical structure. These models are available as free access or commercial software (ECHA 2019). To encourage regulatory acceptance of these QSAR models, the OECD released Guidance Document n°69 and created a free access toolbox with some QSAR models (OECD 2020d). First developed for conception of possible pharmaceuticals, some QSAR models aim to predict ocular irritation and damage based on the compound's toxicodynamic properties: acidity, electrophilicity, chemical reactivity, surfactant effect (OECD, 2019b). For instance, [START_REF] Kulkarni | Prediction of eye irritation from organic chemicals using membrane-interaction QSAR analysis[END_REF] examined membrane interactions of compounds with the stratified lipophilic corneal epithelium to determine the irritant potential of substances already classified by the Draize test in vivo.

For local ocular toxicity, it is essential to mimic three main factors that influence ocular surface penetration and distribution: static barriers with different transport systems (claudins, zonula occludens), dynamic clearance (lacrimal fluids, Schlemm's canal drainage) and metabolic factors (enzymes, efflux pumps, receptors). In 2018, Pak et al. applied these principles to develop an in silico rabbit cornea model (epithelium, stroma, endothelium), the Quasi-3D CoBi (Computational Biology) model which includes passive transport (paracellular, transcellular) through the corneal epithelium (barrier to the passage of hydrophilic compounds), transport though the stroma (barrier to the passage of lipophilic compounds) and protein binding (such as glycosaminoglycans which can retain hydrophilic compounds). To do so, the research team created a precise geometric representation of the multilamellar corneal structure, applying complex mathematic equations to reflect the various flows. Nevertheless, this in silico model should be elaborated by adding all of the ocular structures (such as conjunctiva, tear film, neurons, retina) and should be based on human data to improve the predictions made through these models. The lack of human data on barriers, thickness and porosity of layers, local metabolism, physical constants, …, remains to this day a barrier to the development of in silico models and use as a high throughput tool.

Implementation of this work would be extremely time-consuming and would require a great deal of computing power but would improve extrapolations.

By enabling the identification of target structures, these models could guide the first steps of the AOP (adverse outcome pathway), which are currently the subject of toxicological development as supports for implantation of IATA, limiting unnecessary in vitro studies.

However, one should bear in mind that if an important metabolic pathway used by the compound tested is missed in the model, the predictions will not be accurate.

3D multicellular models

Numerous 3D models are described in the literature, improving the phenotype of the epithelium formed. Nevertheless, many of them neglect the tear film, which covers the epithelia of the ocular surface, as well as the innervation of the ocular surface, which are, however, two central structures in understanding and anticipating TIDE and anterior segment neuropathies. Thus, this section describes three models that could become assets in the development of IATAs for ocular surface toxicity: the first model presented would allow evaluation of tear film thickness and composition, while the following ones would permit analysis of the toxic impact on neurons interacting with corneal cells.

-Conjunctiva and lacrimal gland coculture

The literature is rich in alternative corneal models but delves less into conjunctival and lacrimal gland toxicity, although these structures which are essential for production of the tear film, a fundamental structure to be evaluated for the anticipation of TIDE. Nevertheless, in 2017, Lu et al. proposed a coculture between rabbit primary epithelial conjunctival cells and spheroids of rabbit primary lacrimal acinar cells. To our knowledge, this is the first in vitro 3D model capable of producing aqueous and mucinous layers of the tear film.

After testing several configurations, direct contact between the two cell types, as presented figure 4, was found to present the best configuration, with optimal epithelial morphology, permeability, phenotype and lacrimal fluid production, even though direct contact is not the most physiological configuration (no direct contact in humans between these types of cells).

To highlight the usefulness of their model, they demonstrated the protective effect of dexamethasone, a corticosteroid known to reduce inflammation of the ocular surface in TIDE, after exposure to pro-inflammatory IL-1β. This effect could not be seen on a simple monoculture of conjunctival cells. While this model does not allow the formation of a complete tear film with a lipid layer, it remains an interesting advance for the in vitro anticipation of TIDE. Further studies could be conducted by adding meibocytes in the culture, to obtain a complete tear film.

-3D model of nerve-stroma interactions

To date, only a few models consider corneal innervation in a toxic response. [START_REF] Sharif | Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea[END_REF] explored the corneal stroma-neuron interaction in depth by proposing a 3D coculture on an insert between HCF (human primary corneal fibroblasts) and SH-SY5Y neurons, a well-characterized human neuroblastoma cell line derived from bone marrow. This model is based on the de novo production of extracellular matrix by fibroblast cells and tries to mimic the in vivo nerve-stroma interaction in the cornea, improving the comprehension and anticipation of corneal cell damage as well as pathways of neuronal regeneration.

However, further studies are needed to characterize the neuronal phenotype of this model and therefore the ability of this model to mimic toxicity affecting the ocular surface. Indeed, SH-SY5Y neurons do not have the same phenotype as primary sensory neurons from trigeminal nerves, the main innervation of the ocular surface (Mélik-Parsadaniantz et al. 2018), since they can develop two distinct phenotypes (neuroblastic or epithelial-like). SH-SY5Y includes adherent cells but also floating viable cells whose biological significance is not yet understood. Also, neuroblastic SH-SY5Y cells express tyrosine hydroxylase and dopamine-βhydroxylase, two catecholaminergic markers, which are not characteristic of trigeminal neurons, which are primarily sensory neurons [START_REF] Kovalevich | Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology[END_REF].

Nevertheless, transfected SH-SY5Y could be considered to study certain ocular surface symptoms such as stinging or itching. This was the objective of the NociOcular test based on a 2D model of SH-SY5Y expressing the transient receptor potential cation channel subfamily V member TRPV1, known to be implicated in these ocular surface phenomena [START_REF] Dua | Neurotrophic keratopathy[END_REF]. Using this test, [START_REF] Forsby | Using novel in vitro NociOcular assay based on TRPV1 channel activation for prediction of eye sting potential of baby shampoos[END_REF] corneal tissue models are limited to one or two weeks of culture and do not include the nervous component. This ALI culture, integrating corneal interactions with neurons while conserving its integrity for 28 days, enables to evaluate toxic induced alterations of phenotype and viability. This model represents a progress in tissue engineering, promoting the importance of cell types interactions for better differentiation and maturation.

Cornea-On-a-Chip models

The focus of much attention in recent decades, organs-on-a-chip seek to miniaturize an organ, facilitate the assembly of cell types and recreate the dynamics of an organ [START_REF] Mandenius | Conceptual Design of Micro-Bioreactors and Organ-on-Chips for Studies of Cell Cultures[END_REF]).

These chips are mainly based on microfluidic technics, using biocompatible polymers such as polydimethylsiloxane (PDMS), a transparent, flexible and gas impermeable organomineral material. The advantage of these systems lies in the small amount of biological material needed, while improving the representation of dynamic in vivo parameters compared to a classic 2D cell culture. Nevertheless, protocols have not yet been standardized, scale-up remains unfeasible for routine experimentation, and the analytical challenge (because of the small quantity of cells) remains to be solved [START_REF] Sosa-Hernández | Organs-on-a-Chip Module: A Review from the Development and Applications Perspective[END_REF].

Because of the complexity of multicompartmental and multi-layered ocular structures, establishing an eye-on-a-chip is a hard task. If we focus on the anterior segment, some corneas-on-a-chip are described in the literature and attempt to include ocular surface flow (blinking of the eyelids, tear secretion, shear stress). Furthermore, microfluidics and compartmentalization on a chip are also being considered to improve the mimicry of ocular surface innervation, taking into account the fact that only nerve endings can be directly exposed to a topically applied toxicant.

-Cornea-on-a-chip, ocular flows and shear stress A current limitation of corneal barrier models is the lack of flow to mimic the shear stress caused on the epithelium by eyelid blinking, which is responsible for tear film movement, and as a result of drug or toxicant distribution and its effects on the ocular surface. Of note, this is also a limitation of the Draize test when attempting to most closely approximate human physiology, since rabbits blink less frequently than humans, resulting in a longer exposure time [START_REF] Maurice | The effect of the low blink rate in rabbits on topical drug penetration[END_REF].

In 2018, to study passage through the corneal barrier, [START_REF] Bennet | A microengineered human corneal epithelium-on-a-chip for eye drops mass transport evaluation[END_REF] proposed a corneaon-a-chip with a pulsatile flow to represent blinking or a continuous flow for tear secretion. A confluent epithelium of 5 to 7 layers with a stable phenotype and permeability was obtained on a PDMS chip with a fibronectin coated membrane (mimicking Bowman's layer) and immortalized human corneal epithelial cells. In this system, eyedrop pharmacokinetics and toxicity can be evaluated by applying either the continuous or pulsatile flow for 5 hours. After this experimentation time, 98% of the compounds were found to be eliminated; compared to a static model, it improves the evaluation of absorption, bioavailability and toxicity.

Nevertheless, additional studies are required to understand the impact of the two types of flow, since compound penetration appeared more significant with the pulsatile flow.

Similarly in 2020, Abdalkader and Kamei published a four chamber microfluidic model with uni-and bi-directional flow to study the impact of shear stress on corneal epithelium barrier phenotype. This PDMS system, composed of human corneal epithelial cells on a porous membrane, aims to simulate human cornea, with an apical side in contact with lacrimal fluid (bidirectional flow for eye blinking) and a proximal side with the aqueous humor (unidirectional flow mimicking drainage through Schlemm's canal). After having obtained a stratified (2-3 layers), permeable (evaluation by fluorescein diffusion), phenotyped (expression of tight junction proteins such as the zonula occludens proteins), they applied both flows for 24 hours and observed that shear stress did not alter cellular adhesion and improved the expression of cytokeratins, which are important proteins for flexibility, cellular elasticity and maintaining corneal barrier integrity. In addition, this model could take into account the compound real time of remanence in the tissue.

Nevertheless, these two models are limited in their representation of the cornea, since they lack formation of the stromal and endothelial layers, corneal elements that are notably essential for aqueous humor flow. This limitation is addressed by [START_REF] Bai | A Method for Developing Novel 3D Cornea-on-a-Chip Using Primary Murine Corneal Epithelial and Endothelial Cells[END_REF] with their cornea-on-a-chip, a PDMS compartmentalized chip using primary murine corneas; they simultaneously isolate both epithelial and endothelial corneal cells and plant them into two separate compartments with a collagen membrane to mimic Bowman's layer.

-Cornea and conjunctiva-on-a-chip Another approach to the 3D ocular model on-a-chip was designed in 2019 by Seo et al., combining human primary corneal epithelial cells and immortalized conjunctival cells (epithelial and glandular cells), cultured in an ALI system. The primary corneal cells are incorporated into a collagen matrix which mimics the stromal layer. A perfusion system mimics tear flow, while a biomimetic system recreates blinking of the eyelids. Their complementary data gives a better representation of this complex model. Seo et al. obtained a pluristratified epithelium with 7 to 8 layers like human cornea, expressing specific markers (ex. cytokeratins 3, 19) and producing a "tear film" of 6 µm comparable to the in vivo thickness. Like the previous models, they proved that shear stress induced cellular differentiation and limited pro-inflammatory cytokine production. To attest to the utility of their model, they demonstrated the anti-inflammatory action of lubricin, a protein-like mucin.

While this model does not include the vasculature or immune cells normally present in the conjunctiva nor the nerve endings of the ocular surface which participate in tear secretion, this chip represents a major improvement for pharmacological and toxicological compound evaluation, especially for a TIDE IATA.

-Corneal innervation compartmentalization Currently, most ocular surface models, like the flow systems just discussed, neglect toxic effects on ocular surface innervation, whereas during a toxic exposure, trigeminal nerve endings can be altered, with an indirect impact on neuronal cell bodies. Therefore, stimulating primary cell cultures of neurons directly does not mimic real life exposure, and, as a result, mechanisms of toxicity are impossible to analyze properly. In order to improve anatomical representation of the ocular surface innervation, [START_REF] Sarkar | Corneal neurotoxicity due to topical benzalkonium chloride[END_REF] used a Campenot device to evaluate morphological alterations (neurite fragmentation, axon breaks, lack of regeneration) of mice primary trigeminal neurons after exposition to BAK, preservative contained in many eyedrops. With this model, they highlighted a dose-dependent toxicity of BAK on neurites. Campenot devices were the first systems to allow neuronal compartimentalization but new microfluidic organ-on-a-chip devices could be considered.

Indeed, these microchips can be precisely designed to optimized axonal guidance of trigeminal neurons [START_REF] Courte | Reconstruction of directed neuronal networks in a microfluidic device with asymmetric microchannels[END_REF]). This innovative system also allows to analyze separately nerve ending and cell body responses. Finally, this model could be improved by adding corneal epithelial cells in the distal compartment to allow interaction between these cells and the nerve endings, coming even closer to corneal physiology. It could provide a better understanding of toxic mechanisms and facilitate establishment of TIDE AOPs and screening of new therapeutic agents (anti-inflammatory, axonal regeneration, neuroprotection). Nevertheless, a limitation of this model is the use of primary murine cells, which does not entirely respect the 3R rule to "Reduce, Replace, Refine," central in IATA development. Even if primary cells are a better representation of a peripheral neuronal phenotype, in the framework of alternative methods, induced Pluripotent Stem Cells should be considered, as in the organoid models described below.

Organoid models of the anterior segment of the eye

While the definition can vary between authors, organoids are 3D structures, derived from embryonic stems cells or induced Pluripotent Stem Cells (iPs), capable of self-organization on their framework (such as porous membrane and hydrogel) [START_REF] Duboule | Modèles QSAR[END_REF]. A Pubmed search with "eye organoid" as keywords reports mostly retinal organoids or organoids destined to be transplanted in humans to replace deficient structures. Few articles address anterior segment organoids for in vitro evaluation of pathologic or toxic pathways. However, some of the organoids described could be adapted for toxicological studies.

-Corneal organoids

In 2017, Foster et al. presented a corneal organoid derived from an IMR90.4 iPs cell line [START_REF] Foster | Cornea organoids from human induced pluripotent stem cells[END_REF] and published their precise methodology in 2020 [START_REF] Foster | A Guide to the Development of Human Cornea Organoids from Induced Pluripotent Stem Cells in Culture[END_REF].

Mature transparent organoids are obtained after 120 days of cellular sequential selection, forced aggregation and differentiation. Their lamellar structure is composed of epithelial, stromal and endothelial layers and expresses specific corneal markers (cytokeratins 3, 14, collagen of type I, V, VII). Even if any toxicological study has already been conducted, this model could be further optimized to evaluate the impact of toxic compounds on the interactions between the three main corneal layers (epithelium, stroma, and endothelium).

Nevertheless, cell differentiation sometimes appears incomplete, leading to the presence of some retinal cells within the corneal organoid. Other protocols presented to obtain corneal organoids for transplantation seem to result in pure corneal organoids, such as that of [START_REF] Susaimanickam | Generating minicorneal organoids from human induced pluripotent stem cells[END_REF], but additional studies are needed to evaluate the reproducibility of these models.

-Lens organoids

In 2018, Murphy et al. addressed the unsolved problem of obtaining pure lens cells from human embryonic pluripotent stem cells (CA-1 cell line). Their objective was to elaborate a simple, reproducible method to study lens pathologies and anticipate toxicity-induced cataracts. To this end, they put in place a complex, semi-automated selection protocol based on knowledge of embryonic development, with successive inhibition and activation of the FGF, TGF-β and Wnt pathways [START_REF] Yang | Efficient generation of lens progenitor cells and lentoid bodies from human embryonic stem cells in chemically defined conditions[END_REF]) and magnetic selection of ROR1+ expressing cells (orphan receptor expressed on epithelial lens cells). These organoids remain viable for 42 days, expressing, among others, α and β crystallins, present in vivo in lens fibers and necessary for focusing of light. In this study, they proved the ability of these microlenses to evaluate the toxic potential of a drug candidate, Vx-770, tested in 2016 for cystic fibrosis. This compound, which has induced toxic cataracts in rats, also altered the lens organoids' ability to focus light. To summarize, after reproducibility and intra-laboratory transferability is addressed, this innovative model could be used routinely for the evaluation of mechanisms of toxicity-induced cataract, which still remain poorly understood, as well as the efficacy of new treatments.

Conclusion

The 21 st century has seen an increase in the movement toward alternative methods to animal testing, especially since the complete ban of animal experimentation in cosmetics. Ocular toxicity studies are no exception, and studies still need to be conducted for new compounds.

Indeed, the alternative models to the Draize reference test present similar disadvantages, among which figure the absence of detection of conjunctival or iris damage, the absence of evaluation of systemic toxicity that can occur after ocular exposure and the possibility of false negatives or false positives. Furthermore, none of them alone is able to identify all of the GHS ocular irritant categories, and reversibility of damage is still difficult to evaluate, explaining the impetus of the OECD to optimize some other models. In recent decades, toxicology procedures have aimed to develop IATAs to circumvent these limitations of the alternative methods. Putting aside Draize reference test, known for its lack of reproducibility which complexifies the validation of alternative models by the ICCVAM (OECD Webinar 2019a), and constructing new models, from scratch, based on established AOPs, might be necessary to improve the robustness of the toxicology approaches and results for human use. Indeed, we need to break free from Draize eye irritation test and its poor quality of result to improve inter-laboratory validation of new models [START_REF] Spielmann | International Regulation of Toxicological Test Systems[END_REF]) that could enable the identification of a new category of compounds, very low irritants, which requires finer sensitivity methods. This validation step is essential to develop robust alternative approaches to animal testing in the ocular surface field, as it has been done for skin sensitization. Indeed, in June 2021, OECD released GL 497 on "Defined Approaches for Skin Sensitisation", describing the integrated testing strategy and combination of tests that can be used in toxicology studies in replacement of the reference test on rabbits, the Local Lymph Node Assay (OECD 2021).

In the field of ophthalmology, IATAs should extend the assessment of toxicity to pathologies other than irritation, especially Toxicity-Induced Dry Eye (TIDE), that can occur after chronic exposure to very low concentrations (Bonneau et al. in press). While much less frequent, a toxic compound can also lead to, cataract, glaucoma or ocular surface neuropathies after local exposure. These effects should be considered, taking into account real-life exposure to the compound, determined through literature searches and in silico models. As a result, new drugs, cosmetic compounds, or other chemicals, should be investigated for acute irritation and/or for chronic adverse events, depending on real-life use, requiring the development and validation of models and tests with short and/or repeated exposures.

Establishing integrated decision trees for these newly considered adverse events will require a precise understanding of toxic mechanisms, with the development of Adverse Outcome Pathways (AOP), a concept also promoted by the OECD with the establishment of new collaborative tools such as AOP wiki, Effectopedia and the e.AOP.Portal (OECD Webinar 2019b). The innovative models presented in the last section of this review could, after assessment of robustness and regulatory validation, be included in IATAs. They could be a key asset to understanding molecular mechanisms and establishing AOPs. Validation of new models will be a lengthy process, since they should be developed in such a way as to be as cost-effective and least constraining as possible (ethics and supply logistics).

Disclosures of Conflicts of Interests

CB is consultant for Aerie, Alcon, Allergan, Horus Pharma, Santen and Théa.

  completed an ocular tolerability study of 19 shampoos, resulting in only one false negative and two false positives compared to a clinical evaluation. NociOcular measures, by fluorescence, the intracellular calcium flux mediated by the activation of TRPV1 and correlated to ocular discomfort. A similar study was conducted by Narda et al. in 2019) on the ocular tolerance of sunscreens, confirming the need to evaluate disturbances in neuronal transmission and not just damage to the ocular surface epithelial cell in a comprehensive study. -Triculture of neuronal, epithelial and stromal cells Wang et al. 2017 proposed an air liquid interface (ALI) triculture between human primary corneal cells, human corneal stromal stem cells and Chicken Dorsal Root Ganglion (DRG) neurons, supported by silk proteins. The use of silk proteins aims to mimic the mechanical properties of the cornea, so as to favour neuronal development. Figure 5 explains the cellular organization of the model. Through this set up, Wang et al. obtained optimized axonal development as well as a better epithelium / stromal phenotype and viability. At the moment,

  CB and FBB has received research grants from Horus Pharma, Santen and Théa.NB has received funding from Horus Pharma and l'Agence Nationale de la Recherche et de la Technologie (ANRT) through a Convention Industrielle de Formation par la Recherche (CIFRE).

FiguresFigure 1 .

 1 FiguresFigure 1. Schematic presentation of the matrix created in the Red Blood Cell test and the principle of denaturation (modified from OECD Webinar 2019a)

Figure 2 .Figure 3 .Figure 4 .

 234 Figure 2. Procedure to prepare the embryonated egg in the HET-CAM testing method (personal images, not published) Figure 3. Schematic evaluation protocol for ocular discomfort in the slug irritation model (modified from Lenoir et al. 2011). CP: Contact Period; SIB: Stinging, Itching and Burning Figure 4. Schematic representation of the coculture established between conjunctival epithelial cells and lacrimal spheroids (modified from Lu et al. 2017).

Figure 5 .

 5 Figure 5. Schematic comparison of human corneal structure with 3D triculture model structure (modified from Wang et al. 2017).

Acknowledgment

The authors thank Dr Kevin CLARK, MD, for checking and editing the manuscript.


L Presmanes

A Chapelle

F Oudrhiri-Hassani

A Barnabé

Ph Tailhades

Synthesis and CO Gas-Sensing Properties of CuO and Spinel Ferrite Nanocomposite Thin Films

Keywords: Nanocomposites, Thin Films, Sputtering, CuO, Spinel Ferrite, Gas Sensors, Carbon Monoxide

A nanocomposite thin film composed of tenorite (CuO) and spinel ferrite (Cu x Fe 3-x O 4 has been prepared by radio-frequency sputtering method followed by a post-deposition thermal annealing. The nanostructure was examined by grazing X-ray diffraction, atomic force microscopy and scanning electron microscopy. CuO-Cu x Fe 3-x O 4 nanocomposite layer was deposited on the top of screenprinted gold electrodes onto SiO 2 (200 nm)/Si substrate. It was found that this material contains p-n nano-heterojunctions, was sensitive to 500 ppm carbon monoxide at the operational temperature of 400 C and exhibited a large response of 90%.

INTRODUCTION

Nanocomposites have received great attention due to their unique mechanical, optical, electrical, thermal or magnetic properties. [1][2][3] In this large family, oxide/oxide nanocomposites have been reported for many applications such as photocatalysis and photoelectric transition, 4 microelectronics, 5 solar cells, 6 and electroluminescence. 6 Nanocomposites based on semiconductor metal oxides are also of great interest for creation of selective gas sensors 7 8 and have been employed for the detection of carbon monoxide (CO). CO sensors are essential to the control of the emissions from combustion processes. In addition, the toxic nature of CO makes the detection of this gas for household and environmental applications necessary. The detection of CO is therefore an extremely important research area. Recently, composites made of p-type copper oxide and various n-type metal oxides such as CuO/SnO 2 and CuO/ZnO have been studied as gas sensors to detect H 2 S, H 2 and CO gas. [9][10][11] Several techniques such as ion implantation, 12 sputtering, [13][14][15] and electrodeposition 16 have been used for the preparation of nanocomposites films. In the form of thin films, these materials allow mass production of devices at low cost with high control and repeatability. Moreover, due to their high intrinsic surface-to-volume * Corresponding author; E-mail: presmane@chimie.ups-tlse.fr ratio, nanostructured thin films exhibit high capabilities for miniaturization. Among all the available processes, radio-frequency (RF) sputtering is particularly suitable for the preparation of nanocomposite films with a good homogeneity. In this context, it can be advantageous to prepare sensing layers containing p-n heterojunctions at the nanometric scales by RF sputtering process, and to use this process compatibility with the microelectronic technology, and with elaboration of micro-sensors.

We have already shown that RF sputtering can lead to the preparation of various nanocomposite thin films. [17][18][19] Additionally, we have recently highlighted the CO 2 sensing properties at low temperature (250 C) for nanocomposite thin layers made of p-type CuO and n-type Cu x Fe 3-x O 4 . 20 In this paper, we report the synthesis of a CuO/Cu x Fe 3-x O 4 thin film elaborated by RF sputtering followed by a post-deposition annealing-resulting in nanosized and well dispersed particles in the structure. We then present carbon monoxide sensing properties of this thin film at different temperatures.

EXPERIMENTAL DETAILS

Thin films were deposited with an Alcatel A450 apparatus using a home-made sintered ceramic target of pure CuFeO 2 with a relative density of 70%. The RF power was set at 4 W/cm 2 and the pressure inside the deposition chamber was lower than 5 × 10 -6 mbar prior to deposition. During the elaboration of the films, the argon pressure and the target-to-substrate distance were adjusted to 2 Pa and 8 cm, respectively. 300 nm thick films were deposited on glass substrate for structural and microstructural characterizations. The film thickness was fixed to a value of 50 nm on test device for the determination of CO sensing properties.

Structural phase analyses such as Grazing Incidence X-Ray Diffraction (GXRD) (grazing angle = 1 ) were carried out with a Siemens D5000 diffractometer using the copper K radiation. The morphology of the thin film was determined by Scanning Electron Microscopy using a Field Emission Gun (FEG-SEM) Jeol JSM 6400 and a Veeco Dimension 3000 Atomic Force Microscope (AFM) equipped with a super sharp TESP-SS Nanoworld© tip (nominal resonace frequency 320 kHz, nominal radius curvature 2 nm).

Our simplified test device consists of a silicon wafer substrate electrically insulated with a 1 m thick SiO 2 blanket-layer. Two gold interdigitated electrodes were screen-printed onto the surface of the silicon dioxide layer. The drying and densification of the gold paste was made by thermal annealing at 700 C. The sensing nanocomposite layer was then deposited on the top of the electrode by RF-sputtering with the electrical contacts at each side of the electrodes left uncovered.

The electrical response of the sensing layer was investigated by registering the variations in impedance with a RCL-meter (FLUKE PM6306) as a function of temperature and time. The test device was placed in a furnace operating under controlled atmosphere with the temperature range of 20-480 C. The total gas flow was fixed at 100 cm 3 /min. Two bottles equipped with mass flow regulators were used to set the CO concentrations at 500 ppm. Therefore, a low frequency was used to avoid the relaxation phenomenon and to acquire signals due to the spatialcharge phenomenon. An arbitrary value of 700 Hz was chosen.

The response R is defined by Eq. ( 1) as follows:

R % = 100 × Z CO -Z air /Z air (1)
where Z air and Z CO are the electrical impedances of the sensor measured in air and in CO respectively. Electrical responses were recorded at different temperatures, with alternating air and CO pulses.

RESULTS AND DISCUSSION

Synthesis and Characterization

The deposition of the film by RF sputtering constitutes the first step of the preparation process. In the Figure 1(a) the GXRD pattern of the as-deposited thin film shows a broad peak located at 2 = 35 5 deg, i.e., with d hkl ≈ 2 5 Å which could be attributed to CuFeO 2 (d 012 = 2 51 Å). However, delafossite phase cannot be unambiguously identified using GXRD, in the as-deposited film, due to the nanocrystallinity of the material. Apart from the preparation process, an additional thermal annealing under vacuum at 450 C was performed in order to increase the crystallite size of the phase and to clearly reveal the diffraction peak related to delafossite phase (Fig. 1(c)). By using high deposition pressure and large target to substrate distance, the oxidation state of copper which is +I in the delafossite target has been then preserved in the deposited layer. Moreover no metallic copper is detected contrary to others films deposited at low pressure and low target-to-substrate distance. 17 20 Indeed, with a high deposition pressure and large target to substrate distance during the sputtering process, the particles ejected from the target undergo many collisions during the transfer to the substrate, leading to lower energies and thus less oxygen ejected in the growing layer.

The second step in the preparation of CuO-Cu x Fe 3-x O 4 composite consists of heat treatment of the as-deposited film in air at 450 C. According to the phase diagram, 21 this treatment leads to the decomposition of nanocrystallized delafossite CuFeO 2 into tenorite (CuO) and cuprospinel (Cu x Fe 3-x O 4 with x close to 1) phases in line with the GXRD pattern (Fig. 1(b)).

This two steps process allowed us to obtain nanocomposites thin film containing p-type CuO 22 23 and n-type Cu x Fe 3-x O 4 24 25 semiconducting phases well dispersed. The average grain size for the thin film surface, determined from an AFM image was around 50-60 nm (Fig. 2(a)) and confirmed by a SEM-FEG image (Fig. 2(b)). By profile fitting in XRD pattern, we obtain a crystallite size of 50 nm for CuO particles by using Scherrer equation corrected by instrumental contribution. The nanometric p-n heterojunctions easily obtained from this process is of great interest for the field of sensing layers for semiconductor gas sensors.

CO Gas Sensing Properties

The CO gas sensing experiments of the simplified test device covered with the nanocomposite layer were performed at different temperatures in order to find out the optimum operating temperature. The response of our sensing layer to 500 ppm concentration of CO and in a temperature range of 200-450 C is illustrated in Figure 3. The operating temperature has an increased influence on the response to CO gas. A typical temperature dependant behavior showing a maximum response has been widely reported in the literature for a variety of semi-conducting materials. This phenomenon is generally explained by the thermally activated adsorption and desorption of oxygen and CO at the surface of the sensing layer. Other models take in account the width of a depletion layer between the semi-conducting grains which is temperature dependent. The temperature that gives the optimal response depends on the composition and the doping of the oxide used as sensitive layer. For a given material, the grain size and the grain boundaries have also a great influence on the optimal temperature. In our case, the evolution of the response of the layer shows that this maximum is located around 400 C.

Figure 4 shows the impedance transient of the nanocomposite layer recorded at the optimum temperature of 400 C with alternating air and air + CO (500 ppm) pulses. The positive response observed (i.e., Z CO > Z air is representative of the interaction of CO which is a reducing gas (electron donor) with a p-type semiconductor, by decreasing the number of holes. This suggests that the role of p-type CuO in the nanocomposite is predominant. We have seen in previous paper 20 that the interaction of CuO-Cu x Fe 3-x O 4 nanocomposite with an oxidizing gas such as CO 2 led to a negative response and then also behaved like in the presence of a p-type semiconductor. The best response was found to be approximately 90% at 400 C, which corresponds to a Z CO /Z air ratio of 1.9.

Response and recovery times, which are defined as the time to reach 90% of the final signal level, are important characteristics of a gas sensor. As depicted in Figure 4,

CONCLUSIONS

Based on the results and discussion, we can conclude that the nanocomposites CuO-Cu x Fe 3-x O 4 can be easily prepared by a two step process. A CuFeO 2 phase was first deposited and then annealed in air at 450 C to lead to the decomposition of the phase. In comparison with the elaboration of multi-layers composites artificially stacked, the interest of our method is that these nanocomposites have been obtained directly, after a simple thermal annealing, by using oxidation and decomposition phenomena expected from Fe-Cu-O phase diagram. Moreover, we can expect a good quality interface between the two n-type and p-type phases that are self-mixed at a nanometric scale. The particles sizes of the nanocomposites were obtained in the range of 50-60 nm. The CuO-Cu x Fe 3-x O 4 sensing layer showed a response of 90% to 500 ppm CO at 400 C, with a good reversibility.

Fig. 1 .

 1 Fig. 1. Grazing angle X ray diffraction patterns of the deposited film on glass substrate from a CuFeO 2 target, (a) As-deposited film. (b) After annealing in air at 450 C for 24 hours. (c) After annealing film under vacuum at 450 C for 15 hours. Thickness = 300 nm. P = 2 Pa, d = 8 cm.

Fig. 2 .

 2 Fig. 2. SEM-FEG (a) and AFM (b) images after annealing in air at 450 C for 12 hours. Thickness = 300 nm. P = 2 Pa, d = 8 cm.

Fig. 3 .

 3 Fig. 3. Response as a function of operating temperature of the CuO-Cu x Fe 3-x O 4 layer deposited on screen-printed gold electrodes under CO atmosphere. P = 2 Pa, d = 8 cm, f = 700 Hz, flow = 100 sccm, [CO] = 500 ppm, e = 50 nm.

Fig. 4 .

 4 Fig. 4. Impedance transient of CuO-Cu x Fe 3-x O 4 thin film deposited on screen printed gold electrodes. P = 2 Pa, d = 8 cm, T = 400 C, f = 700 Hz, flow = 100 sccm, [CO] = 500 ppm, e = 50 nm.

Acknowledgment:

The authors thank Xavier Hochart from Temex Ceramics Company for his help in preparation of gold coated Si/SiO 2 substrates.