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Collagen vitrigel membranes (CVMs) comprising high-density collagen fibrils equivalent to in vivo connective tissues have been widely used in cell culture applications. A human corneal epithelium (hCE) model was previously developed by the Takezawa group, by culturing HCE-T cells (derived from hCE cells) on a CVM scaffold in a chamber that provided an air–liquid interface culture system. This hCE model was used to establish a new test method, known as the Vitrigel-Eye Irritancy Test (Vitrigel-EIT) method, which can be used to estimate the ocular irritation potential of test chemicals by analysing relative changes in transepithelial electrical resistance (TEER) over time. The current study was conducted in order to assess the reliability and relevance of the Vitrigel-EIT method at three participating laboratories by determining the method’s within-laboratory reproducibility and between-laboratory reproducibility, as well as its capacity for distinguishing non-irritants from irritants in a bottom-up approach. The initial test sample size was found to be too low to evaluate the predictive capacity of the test method, and so it was evaluated with additional in-house data for a total of 93 test chemicals. The results showed 80–100% within-laboratory reproducibility and an excellent between-laboratory reproducibility that met the acceptance criteria of 80%. However, the method’s predictive capacity for distinguishing non-irritants (test chemicals not requiring classification and labelling for eye irritation or serious eye damage, i.e. United Nations Globally Harmonised System of Classification and Labelling of Chemicals (GHS) No Category) from irritants (GHS Categories 1 and 2) in a bottom-up approach was unacceptable because of false negative rates as high as 16.7%. After considerable review of the data with a view to using the method for regulatory purposes, it was determined that a more defined applicability domain, excluding test chemical solutions with a pH of 5 or less and solid test chemicals, improved the false negative rate to 4.2%. These results suggested that, within this carefully defined applicability domain, the Vitrigel-EIT method could be a useful alternative for distinguishing test chemicals that are ocular non-irritants from those that are irritants as part of a bottom-up approach.

Formic acid functions as a fragrance ingredient, preservative, and pH adjuster in cosmetic products, whereas sodium formate functions as a preservative. Because of its acidic properties, formic acid is a dermal and ocular irritant. However, when used as a pH adjuster in cosmetic formulations, formic acid will be neutralized to yield formate salts, for example, sodium formate, thus minimizing safety concerns. Formic acid and sodium formate have been used at concentrations up to 0.2% and 0.34%, respectively, with hair care products accounting for the highest use concentrations of both ingredients. The low use concentrations of these ingredients in leave-on products and uses in rinse-off products minimize concerns relating to skin/ocular irritation or respiratory irritation potential. The Cosmetic Ingredient Review Expert Panel concluded that formic acid and sodium formate are safe in the present practices of use and concentration in cosmetics, when formulated to be nonirritating.

To report a case of Fuchs' superficial marginal keratitis managed with circumferential marginal corneoscleral lamellar patch graft.Interventional case report.A 34-year-old man presented with several years' history of ill-defined symptoms of binocular ocular irritation associated with vision loss, mostly in the left eye. A superior marginal corneal thinning was found at biomicroscopy of the left eye, with 2 finely vascularized descemetoceles, and a gray epithelial demarcation line without lipid infiltrates. The right eye was clinically normal. Anterior segment optical coherence tomography demonstrated an important corneal thinning from the 7 o'clock to 3 o'clock positions, without scleral involvement. A circumferential marginal corneoscleral lamellar patch graft was done involving 3 mm of sclera and 3 mm of cornea.Fuchs' superficial marginal keratitis is a rare entity, mostly affecting young adults. It should be considered part of a spectrum of corneal thinning disorders, together with Terrien's marginal degeneration. Definitive treatment with a marginal corneoscleral lamellar patch graft with or without conjunctival autograft is suggested.

We believe the development and validation of in vitro alternatives to eliminate the need to use animals in ocular irritation testing must be based on a thorough understanding of the mechanisms of ocular irritation. We have recently undertaken the task of developing such an understanding for a panel of surfactants. The purpose of this study was to expand our current panel of surfactants for which the microscopic changes occurring over time have been characterized. Macroscopic and microscopic findings regarding the ocular irritation of 6 surfactants of relatively unknown irritancy were compared to those of 6 surfactants of known irritancy. The right eye of each rat was treated by placing 10 μl of a surfactant directly on the cornea. Untreated left eyes served as the controls. At 3 hr and on days 1, 3, and 35, eyes and eyelids were collected for microscopic examination. Collectively, the macroscopic and microscopic findings revealed 3 surfactants to be similar to the mildly irritating surfactants previously studied, and 3 surfactants to be similar to the moderately irritating surfactant previously studied. Information such as this will be important to develop mechanistically based in vitro alternatives to replace the use of animals for ocular irritation testing.

Current U.S. and Japanese regulatory guidelines mandate the use of at least six animals for dermal and ocular irritation testing, while EEC/OECD guidelines require only three animals. An analysis of data from this laboratory in general supports the use of as few as three animals for acute irritancy testing. Data from a series of 140 dermal and 118 ocular irritation tests were examined to determine injury rates associated with testing, and to generate six-, five-, four-, three-, and two-rabbit primary dermal irritation indices for dermal tests and to categorize ocular tests similarly. Results were then examined to determine whether fewer animals could have been used to obtain findings similar to those originally noted with six rabbits. These results suggest that the number of animals used in dermal and ocular irritation testing can be reduced, especially if a sequential approach involving a preliminary in vitro/SAR test is taken. In addition, low rates of severe injury were evident for dermal and ocular irritation tests, as well as low discomfort rates for ocular irritation tests using a topical anesthetic. Using a low level of dermal irritation as a screen for ocular testing could reduce injury and discomfort rates further.

An extensive computer search was conducted, and a comprehensive overview of the current status of alternatives to animal eye irritation tests was obtained. A search of Medline and Toxline databases (1988 to present) was supplemented with references from sources regarding in vitro eye irritation. Particular attention was paid to soap and detergent products and related ingredients. Eighty-five references are included in the review; the in vitro assays are categorized, and their predictive values for assessing acute ocular irritation are evaluated and compared with the Draize rabbit eye irritation assay and with each other. The present review shows that the increased activity of scientists from academia, industry, and regulatory agencies has resulted in substantial progress in developing alternative in vitro procedures and that a number of large, interlaboratory evaluations and international workshops have assisted in the selection process. However, none of these methodologies has obtained acceptance for regulatory classification purposes. Conclusions drawn from this review include that (a) no single in vitro assay is considered capable of replacing the Draize eye irritation test; (b) the chorioallantoic membrane vascular assay (CAMVA) or the hen egg test-chorio-allantoic membrane test (HET-CAM), the chicken or bovine enucleated eye test, the neutral red and plasminogen activation assays for cytotoxicity, and the silicon microphysiometer appear to have the greatest potential as screening tools for eye irritation; and (c) choosing a specific assay or series of assays will depend on the type of agent tested and the impact of false-negative or false-positive results. New assays will continue to be developed and should be included in future evaluations, when sufficient data are available.

The EYTEX™ system is an in vitro method for predicting in vivo ocular irritation via a target biomacromolecular approach. EYTEX™ uses changes of relevant macromolecules upon exposure to chemicals and formulations to predict in vivo irritancy and toxicity endpoints.The EYTEX™ system is based on alterations in the conformation and hydration of an ordered macromolecular matrix. An oligomeric protein with a molar mass of approximately 300,000g/mol is the major active component of the reagent. This oligomer, characterised by 12 subunits, associates itself into strands which are then incorporated into a gel network. This network also includes smaller proteins, peptides, amino acids, aminoglycans and mucopolysaccharides, which contribute to the overall response of the macromolecular matrix to a diverse array of test compounds, such as acids, bases, salts, solvents, surfactants, lubricants, preservatives, emulsions, dyes, amines, amides and many others. The method has been extensively evaluated using chemicals and formulations from diverse classes and with different ranges of toxicity, representing various mechanisms of ocular toxicity. Results using the in vitro test correlate broadly with the Draize in vivo eye irritancy test results. Quantification of the EYTEX™ method is based on measurement of changes in optical density of the matrix, which are sensitive to the conformation and hydration of protein, as well as the order in the matrix. These changes, which occur when the matrix is exposed to chemicals and formulations, can be used to predict in vivo ocular irritancy. Calibrators and controls provide assay standardisation with a 4–8% coefficient of variation. Multiple dose and time variables relating to irritancy potential can be assessed and compared with Draize in vivo results. The EYTEX™ system provides a comprehensive set of four protocols suitable for analysing chemicals and formulations with varying degrees and mechanisms of ocular irritancy.

The HET-CAM (hen's egg test-chorio-allantoic membrane), described by Luepke in 1985, permits the study of immediate effects following the administration of test substances to the chorioallantoic membrane of 10-day-incubated White Leghorn chicken eggs. The results of a study of 60 chemicals and 41 cosmetic formulations are presented here. Intralaboratory reproducibility was established with a double-blind study of 20 surfactants at two concentrations. The results show a high rank correlation between the scores given by both experimenters: the Spearman's rho is greater than 0.9 (p < 10-8). The 60 chemicals were studied at a concentration equivalent to 10% of the concentration tested in vivo. They were classified according to the three EEC categories of ocular irritancy, and when a correlation between the HET-CAM scores and historical Draize in vivo data was determined, the corresponding Spearman's rho value was 0.72 (p < 10-4). The 41 formulations (make-up removers, shower gels, shampoos, creams and body milks) were tested by two protocols: rinsed and non-rinsed. The correlation between the HET-CAM scores and the historical Draize in vivo maximum average scores was studied, and the rhos obtained were 0.77 and 0.76 (p < 10 -8), respectively. The advantages of the HET-CAM method lie in its sensitivity, rapidity and moderate cost. Both chemicals and cosmetics formulations can be tested, and specific families can be assessed using modified and more-appropriate protocols. However, to ensure that a good reproducibility and sensitivity is provided, the use of reference materials is strongly recommended. The satisfactory performances of the HET-CAM test in many previous evaluation studies (e.g. those of the BGA, CTFA, EEC and OPAL) show that it can be a useful assay as part of a battery of in vitro tests for the screening of new ingredients and formulations.

International guidelines for the evaluation of ocular irritation advise that all the available information, structural and physicochemical, should be taken into account in order to minimise the testing of substances under conditions that are likely to produce severe reactions in laboratory animals. In order to determine whether the pH and the acidic/alkaline reserve would be suitable parameters for the prediction of ocular irritation, we have measured, under standard conditions, the pH and the acidic/alkaline reserve of 166 chemicals (90 non-irritant, 22 irritant, and 54 severely irritant for the eye, according to the EEC classification and labelling system). The combination of these parameters permitted the correct identification of 74% of the severely irritant chemicals and 97% of the non-severely irritant (non-irritant or irritant) chemicals. The predictive value for a severely irritant potential was 93%, and 88% for non-severely irritant chemicals. This physicochemical approach, used as a first line screen for ocular irritation, is very simple and inexpensive.

The so-called Draize eye test, the offical method used worldwide for the evaluation of ocular irritancy, has been heavily criticised in recent years concerning its scientific credibility and its extensive use of animals. Numerous methods have been, or are being, developed with the aim of complementing and/or replacing the Draize test. Regardless of their nature, these new techniques will have to be validated to guarantee their scientific credibility. Used singly or in batteries, the alternative tests will have to evaluate all the components of ocular toxicity, and in particular the reversible or non-reversible nature of the observed effects.

Various cell culture models have been proposed as partial or total replacements for in vitro ocular irritation tests. The cytotoxic potential of substances can be evaluated according to various parameters for measuring cellular viability and metabolic capacity. Cell cultures have their limitations, but they also have advantages which justify the inclusion of some such models in a battery of in vitro tests that could replace the Draize test.

Four groups, each consisting of 10 male and 10 female Fischer-344 rats, were exposed 6 h/day, 5 days/week, for 9 days to diisobutyl ketone (DIBK) vapor at concentrations of 905, 300, 98, or 0 (con trol) ppm. An additional 10 rats/sex were assigned to the 905 and 0 ppm groups and allowed two weeks recovery prior to sacrifice. Rats exposed to 905 ppm had mild ocular irritation (lacrimation) and evidence of kidney toxicity, manifested as: 1) an increase in absolute and relative (as a percentage of body weight) kidney weights, 2) an increase in urine volume (and water intake) with a concomitant decrease in urine osmolality (males only), and 3) an increase in severity of hyalin droplet nephrosis in the proximal tubules (males only). Absolute and relative liver weights were also increased in both male and female rats of the 905 and 300 ppm groups. These effects either disappeared or lessened in severity fol lowing the 2-week recovery period. Male rats exposed to 300 ppm had similar renal alterations to the males of the 905 ppm group, although the alterations were fewer in number and smaller in mag nitude. Kidney weights and renal histology of the males of the 98 ppm group were similar to the control male rats, although an increase in urine volume with a decrease in urine osmolality was observed. The kidney findings in this study were not surprising because of the chemical relationship of DIBK with other aliphatic ketones (e.g., methyl isobutyl ketone, methyl isoamyl ketone) which, after repeated inhalation exposure, cause hyalin droplet nephropathy in male rats. The significance of this male rat nephro sis with regard to human exposure is unknown.

The biologic effects of various chlorhexidine acetate (ClAc) solutions containing isopropyl alcohol, Synperonic NP8, and carmoisin were evaluated (1) following intraperitoneal (IP) administration in rats for 5 consecutive days followed by a 5-day recovery period, (2) by measuring the primary irritation of the skin by a patch-test technique on intact and abraded skin of rabbits, and (3) following instillation into the eyes or rabbits followed by washing 50% of the treated eyes with lukewarm water in order to evaluate ocular irritation. Physiologic saline solution served as the control article throughout the studies. There was a high incidence of deaths (i.e., 28/32 and 11/16 for ClAc solutions with and without Synperonic NP8, respectively) among rats receiving IP treatment at a dosage of 40 ml/kg per day for 5 consecutive days. No toxicologically significant treatment-related changes were observed in rats treated IP with either surfactant or isopropyl alcohol with Synperonic NP8. Toxicity of ClAc solution appears to be due to a chemically induced peritonitis with no evidence of systemic toxicity. ClAc solution or the respective surfactants elicited either no reaction or a mild dermal irritation in the rabbit. Likewise, ClAc solution with or without Synperonic NP8 and the three respective surfactants did not cause any significant ocular irritation changes that are considered of toxicologic significance. It is concluded that (1) the multiple IP administration of ClAc solution elicited a toxic response in rats, and thus, caution must be exercised when the solution is used for the purposes of peritoneal lavage, and (2) ClAc solution is a mild dermal and ocular irritant in the rabbit.

A new technique is described for comparing the threshold concentrations that will produce olfactory, trigeminal, and ocular irritation when odorous irritants are exposed to the nose and eyes. It depends on the reduction of concentration of an odorous irritant in air, when the air is passed through an adsorbent. Of the four odorous irritants used in this investigation, three, viz. acetic anhydride, formaldehyde and triethylamine, first irritated the eye, then became perceptible to smell and finally irritated the nasal membranes as their concentration was gradually increased. The order of sensitivity of the three senses towards them was in each case: eye irritation > smell > nasal irritation. It was not found possible to separate the three sensations when ammonia was the odorous irritant, all three seeming to appear and to disappear together.