Changes In Corneal Transparency Research Articles

Evaluation of corneal backward light scattering in type 2 diabetes mellitus.

To compare the corneal backward light scattering values in type 2 diabetes mellitus (DM) patients with those of age and sex-matched healthy controls. The study included 30 patients (30 eyes) with type 2 DM and 30 control subjects (30 eyes). Duration of diabetes, most recent hemoglobin A1c levels, along with the status of diabetic retinopathy, and existing medical treatment of all subjects were recorded. All subjects underwent a complete ophthalmologic examination. In addition, backward light scattering (densitometry) was measured to assess changes in corneal transparency using tomography (Pentacam HR). The type 2 DM patients included 12 males and 18 females and control subjects included 16 males and 14 females. The age was 50.40±7.80y (range: 40-68y) of the diabetic group and 49.30±9.50y (rang: 40-73y) of control group. The diabetic group demonstrated significantly higher mean densitometry values of the anterior (6-10 mm) zone (P=0.047), the total anterior layer (P=0.036) and the total cornea (P=0.043) than control group. The corneal densitometry of the diabetic eyes demonstrated no significant correlation with hemoglobin A1c levels and DM duration. Diabetic group has higher densitometry in anterior corneal (6-10 mm) zone, total anterior cornea, and total cornea and with no correlation with hemoglobin A1c levels and DM duration.

Visual outcomes and corneal densitometry after allogenic and autologous lenticule intrastromal keratoplasty for the correction of moderate-to-high hyperopia.

This study aimed to evaluate the visual outcomes and corneal densitometry (CD) after allogenic lenticule intrastromal keratoplasty (AL-LIKE) and autologous lenticule intrastromal keratoplasty (AU-LIKE) for the correction of moderate-to-high hyperopia. Ten subjects (14 eyes) underwent AL-LIKE and eight (8 eyes) underwent AU-LIKE. Patients were examined preoperatively and 1 day, 1 month, and 6 months postoperatively. The visual outcomes and CD for both surgical methods were evaluated. No postoperative complications were observed with either method. The efficacy index was 0.85±0.18 and 0.90±0.33 in the AL-LIKE and AU-LIKE groups, respectively. The safety indices were 1.07±0.21 and 1.25±0.37 in the AL-LIKE and AU-LIKE groups, respectively. The CD values of the anterior, central, and posterior layers in the AL-LIKE group increased significantly at 1 day postoperatively (all P < 0.05). The CD values of the anterior and central layers remained significantly higher than the preoperative values at 6 months postoperatively (all P < 0.05). The CD values of the anterior layer in the AU-LIKE group increased significantly 1 day postoperatively (all P < 0.05) and decreased to preoperative values (all P > 0.05) 1 month postoperatively. Both AL-LIKE and AU-LIKE exhibit good efficacy and safety in correcting hyperopia. However, AU-LIKE may have a smaller affected area and faster recovery time than those associated with AU-LIKE related to changes in corneal transparency.

Corneal tissue changes following short-term soft contact lens wear of different materials.

To study the effect of different soft contact lens (CL) materials during short-term wear on corneal tissue. Twenty-two healthy participants wore both silicone hydrogel (MyDay, CooperVision) and hydrogel soft CLs (Biomedics 1day extra, CooperVision) for 8 h per lens. In each session, Scheimpflug images were captured before and immediately after CL removal. Images were analysed using the densitometry distribution analysis, a technique from which two parameters, α (corneal transparency) and β (corneal homogeneity), were estimated. In addition, the central corneal thickness changes after CL wear and the influence of the CL material on corneal transparency were evaluated. The β parameter (homogeneity) increased by 5% after wearing both CL materials (paired t-test, p< 0.001). However, the α parameter (transparency) only increased in half of the participants. No material was found to be more determinant in causing the corneal densitometry changes. Statistically significant but not clinically relevant changes in corneal thickness were observed. Biomarkers of corneal tissue integrity (α and β) were affected by short-term soft contact lens wear. The observed changes in corneal transparency and homogeneity were not clinically relevant but support the importance of participant-material biocompatibility.

Efficacy and Safety of Accelerated Corneal Cross-linking for Progressive Keratoconus: A 5-Year Follow-up Study.

To investigate the long-term efficacy and safety of continuous light-accelerated corneal cross-linking (CXL) in patients with progressive keratoconus. This retrospective study analyzed 29 eyes of 29 consecutive patients with progressive keratoconus treated with epithelium-off riboflavin-ultraviolet induced accelerated CXL (30 mW/cm2-7.2 joules/cm2). Uncorrected (UDVA) and corrected (CDVA) distance visual acuity, keratometry measurements at 3 and 6 mm (Kmax, Kmin, and Kmean), topographic astigmatism, central corneal thickness, anterior corneal aberrometric analysis (at 3 and 6 mm), endothelial cell density, and central foveal thickness were evaluated before and 24 and 60 months after surgery. UDVA significantly improved at 60 months postoperatively (P = .028). Kmax and Kmean at 3 mm significantly decreased at 24 (P = .009 and .006, respectively) and 60 (P = .017 and .034, respectively) months postoperatively, whereas Kmin significantly decreased only at 24 months postoperatively (P = .032). Kmax at 6 mm significantly decreased at 24 and 60 months postoperatively (P = .035 and .027, respectively). Cylinder error significantly decreased at 24 and 60 months postoperatively (P = .001 and .023, respectively). Wavefront aberration variables remained unchanged after surgery, except for spherical aberration at 6 mm that decreased at 24 and 60 months postoperatively (P = .038 and .031, respectively) and trefoil at 0° that significantly decreased at 24 months postoperatively (P < .005). Endothelial cell density and central foveal thickness showed no changes from baseline. Accelerated CXL was found to be effective in halting keratoconus progression and improving some of the visual, topographic, and wavefront aberration variables evaluated. High energy irradiance did not induce significant changes in corneal transparency, endothelial cell density, and central foveal thickness. [J Refract Surg. 2020;36(11):724-730.].

The changes in corneal transparency after orthokeratology with juvenile myopia

Objective: To evaluate the changes of corneal transparency undergoing overnight orthokeratology treatment. Methods: Prospective cohort study. Right eyes of juvenile myopic patients fitted with Ortho-K CL at Qingdao Eye Hospital were enrolled. Corneal optical density values were measured by the Pentacam Scheimpflug system before orthokeratology and at 1 day, 1 week, and 1, 3, 6, and 12 months after wearing. Four concentric radial zones centered on the apex of the cornea (≤2 mm, >2 mm and ≤6 mm, >6 mm and ≤10 mm, and>10 mm and ≤12 mm diameter) were applied. Anterior layer (anterior 120 μm), central layer, and posterior layer (posterior 60 μm) were defined according to corneal depth. The data analysis were used One-Way Repeated Measures ANOVA and LSD test. Results: Forty patients (40 eyes) with myopia (24 male and 16 female, age 10.76±1.74 years) were in enrolled. Corneal optical density value was 13.18±0.23 before orthokeratology, then showed a decreasing trend at the early stage, but there was no statistical difference (within 3months: t=-1.594, -1.472, 0.064, 1.971; P>0.05). The value increased significantly and reached the peak (15.31±0.23, t=7.613, P<0.01) at 6-month, and was still maintained in a high value at 1-year (15.15±0.24, t=7.227, P<0.01). Anterior, central, and posterior layer of corneal optical density values had a downward trend at early stage, and increased significantly in 6-and 12-month (anterior layer t=7.143, 6.177, central layer t=7.508, 6.563, posterior layer t=6.722, 8.533;P<0.01). Only the values decreased significantly in posterior layer at 1-week (baseline 10.21±0.14 vs. 1-week 9.91±0.14, t=-2.348, P=0.024). The corneal optical density value of ≤2 mm diameter began to increase significantly at 1-month (baseline 12.88±0.14 vs. 1-month 13.31±0.19, t=2.158, P=0.037),>2 mm and ≤6 mm diameter at 3-month (baseline 11.71±0.13 vs. 3-month 12.50±0.19, t=3.213, P=0.003), and>6 mm and ≤10 mm diameter at 6-month (baseline 11.40±0.30 vs. 6-month 13.70±0.33, t=7.635, P=0.000). The high values lasted for 1 year at this three areas. However, >10 mm and ≤12 mm diameter had no obvious change in optical density (F=1.668, P>0.05). In addition, the significant reduction of optical density values at early stage were mainly concentrated in the posterior layer of the cornea and the>6 mm and ≤10 mm diameter. Conclusions: Orthokeratology transiently increases corneal transparency of the posterior layer and the>6 mm and ≤10 mm diameter at early stage. However, long-term wearing can cause a decrease of transparency within a 10 mm diameter range. The corneal transparency changes first at the center of the optical zone, then gradually expanding to the periphery, and the anterior, central and posterior layers of cornea are all affected. We speculate orthokeratology affect the corneal oxygen supply and compress cornea mechanically, that leads to corneal stromal remodeling. (Chin J Ophthalmol, 2019, 55:435-441).

Changes in Corneal Transparency After Cross-linking for Progressive Keratoconus: Long-term Follow-up

To determine long-term changes in corneal transparency after riboflavin-ultraviolet A-induced corneal collagen cross-linking (CXL). Charts and anterior segment data of patients after CXL for progressive keratoconus were retrospectively reviewed. Patients were examined using the Scheimpflug-based Pentacam corneal densitometry module (Oculus Optikgeräte, Wetzlar, Germany) before CXL and at five postoperative follow-up visits: 1 to 3, 3 to 6, 6 to 12, 12 to 24, and 24 to 36 months. Forty-two eyes of 28 patients (mean age: 27.9 ± 8.6 years) were included. Total corneal light backscatter was higher 1 to 3 months after CXL than before CXL (P < .001). There were significant differences, especially in the anterior (P < .001) and central (P < .001) layer at total diameter and posterior layer (P = .014) and the three central annuli at total corneal thickness (0 to 2 mm: P < .001; 2 to 6 mm: P < .001; 6 to 10 mm: P = .002). Total corneal light backscatter at total corneal thickness and total diameter faded over time following CXL. The backscatter was significantly lower 24 to 36 months after CXL than before CXL (P < .001). Corneal densitometry peaks in the first months after CXL and returns to preoperative values approximately 1 year after CXL. Two years after CXL, corneal densitometry reaches values obtained for healthy, untreated corneas, thus achieving an improvement in corneal clarity over untreated keratoconic corneas.

Improvement of the Bovine Corneal Opacity and Permeability (BCOP) assay as an in vitro alternative to the Draize rabbit eye irritation test

Measurement of ocular irritancy is a necessary step in the safety evaluation of both industrial and consumer products. Assessment of the acute eye irritation potential is therefore part of the international regulatory requirements for testing of chemicals.The Bovine Corneal Opacity and Permeability (BCOP) assay is generally accepted as a valid in vitro alternative method to the Draize eye irritation test to detect corrosive and severe eye irritants (category 1), but has not proven sensitive enough to discriminate accurately moderate (category 2A/2B) to mild and non-irritating compounds. In the currently accepted BCOP assay, opacity is determined by the amount of light transmission through the cornea, and permeability is determined by the amount of sodium fluorescein dye that passes through all corneal cell layers. Both measurements are used to assign an In Vitro Irritancy Score (IVIS) for prediction of the in vivo ocular irritation potential of a test substance. Nowadays, opacity is measured by an OP-KIT opacitometer providing a center-weighted reading of light transmission by measuring changes in voltage when the transmission of white light passes through the cornea alters. As a consequence, this may underestimate opacity that develops as spots or heterogeneous opaque areas on the periphery of an isolated cornea.A prototype of a laser light-based opacitometer (PLLBO) allowing better measurement of opacities was developed by Van Goethem et al. (2010). This new device showed improved sensitivity to detect subtle changes in corneal transparency. Furthermore, the new opacitometer allowed the analysis of the complete corneal surface and was able to detect more efficiently opaque spots located along the sides of the excised corneas.A further improved prototype of the PLLBO was constructed in combination with a camera and a speckle noise reducer. Treatment conditions of the corneas in the cornea holders were optimized in order to mimic more the real in vivo situation. A set of test compounds with irritancy potencies especially in the mild and moderate range was tested. The improved LLBO showed some promising features which potentially could improve the usefulness of the BCOP test. Adaptation of cornea holders showed to be of limited value and only restricted to concentrations up to 15% which mimics more test conditions in industry.This 3-year research project was sponsored by the Stavros Niarchos Foundation (Greece).

The Reversibility of UV‐B Induced Alterations in Optical Properties of the Rabbit Cornea Depends on Dose of UV Irradiation

Solar UVB radiation evokes photokeratitis, accompanied by increased corneal hydration and changes in corneal transparency, resulting in increased light absorption. Corneal optical properties are disturbed and visual acuity decreased. The aim of this study was to investigate the reversibility of these UVB-induced changes. Rabbit corneas were irradiated with UVB doses of 0.5Jcm(-2) or 1.01Jcm(-2) during 4days. Some rabbits were sacrificed after the last irradiation and some 2months later. Corneas were investigated spectrophotometrically for light absorption, and corneal hydration was evaluated by central corneal thickness with an ultrasonic pachymeter. Corneal impression cytologies were examined immunohistochemically for proinflammatory cytokines and malondialdehyde. The increased corneal light absorption, hydration and the staining of immunohistochemical markers found in corneas after irradiation returned to normal values during 2months in corneas irradiated with the lower UVB dose. In contrast, in corneas irradiated with the higher UVB dose, a moderate but statistically significant increase in corneal light absorption, hydration and positive immunohistochemical stainings remained as residual changes. This was in contrast to normal corneas, where the staining of proinflammatory cytokines as well as malondialdehyde was negative. In conclusion, the reversibility of UVB-induced disturbances was dependent on UVB dose.

Corneal Transparency After Cross-linking for Keratoconus: 1-Year Follow-up

To evaluate the changes in corneal transparency and thickness measured objectively using Pentacam Scheimpflug (Oculus Optikgeräte GmbH) corneal tomography (densitometry) after corneal cross-linking (CXL). Twenty-two eyes from 15 patients with keratoconus underwent CXL. Corneal thickness and curvature as well as lens and corneal densitometry were measured at baseline and 1, 3, 6, and 12 months after CXL with Pentacam. Immediately after CXL, central corneal thickness significantly decreased (-40 μm) and corneal densitometry increased significantly. At 6-month follow-up, densitometry values were reduced and seemed to stabilize at a higher value than baseline. No statistically significant differences were noted (mean difference at 12 months: +30%). Changes in corneal densitometry were detected with the Pentacam even when clinical haze did not increase beyond grade 1 during follow-up. Corneal CXL is associated with a transient reduction in central corneal thickness, which recovered to baseline after 12 months. During the same time period, corneal densitometry experienced a transient increase that recovered to baseline values after 3 months. Changes in compactness of the corneal tissue after CXL may have an impact in corneal power not due to curvature changes, but to refractive index changes as a result of hydration changes. Pentacam is a useful objective method to monitor corneal transparency after CXL.

Development of a new opacitometer for the bovine corneal opacity and permeability (BCOP) assay

The bovine corneal opacity and permeability (BCOP) assay is an in vitro eye irritation test that relies on the measurement of induced opacity and permeability evaluated in isolated bovine corneas. Routinely, opacity is measured by an OP-KIT opacitometer which provides a centre-weighted reading of light transmission through the corneal tissue. However, this may underestimate opacity that develops as spots or heterogeneous opaque areas on the periphery of isolated cornea. Consequently, these findings question the reliability of the obtained data when opacity is manifested in a non-homogeneous manner. In addition, the BCOP test is only recommended to identify corrosive and severe eye irritants, and has not proven as sensitive in distinguishing among mild and moderate eye irritants. Within this context, a new opacitometer was developed which uses an adjustable laser beam in combination with a calibrated photometer. This work focused on a technical optimisation and describes the optical characteristics of the new device. The laser-based opacitometer allowed the analysis of the complete corneal surface, showed improved sensitivity to detect subtle changes in corneal transparency and was able to detect more efficiently opaque spots located along the sides of the excised corneas.

Comparison of cis- and trans-crotononitrile effects in the rat reveals specificity in the neurotoxic properties of nitrile isomers

The neurotoxic compound crotononitrile has two isomeric forms, cis and trans. We compared the effects of these two isomers isolated by distillation from the commercially available mixture. Adult male Long-Evans rats were administered vehicle control, cis-crotononitrile (80, 100, and 120 mg/kg/day), or trans-crotononitrile (250 mg/kg/day) for 3 days and the changes in corneal transparency and vestibular function were assessed. Surface preparations of the vestibular sensory epithelia and the organ of Corti were examined for hair cell loss by scanning electron microscopy. Concentrations in retina and brain regions of glial fibrillary acidic protein, a marker for reactive gliosis, were also determined in rats exposed to cis-crotononitrile. In a dose-dependent manner, cis-crotononitrile induced vestibular dysfunction, corneal opacity, and hair cell loss in both vestibular epithelia and organ of Corti, and gliosis in retina, olfactory bulb, superior colliculus, inferior colliculus, hypothalamus, hippocampus, and cingulate cortex, but not in cerebellum or striatum. This neurotoxic pattern is similar to that caused by 3,3′-iminodipropionitrile and allylnitrile. In contrast, trans-crotononitrile triggered rearing deficits but not vestibular dysfunction, hair cell loss, or corneal opacity. The isomeric specificity of crotononitrile isomers shows that the neurotoxic effects of nitriles depend on strict structural requirements, suggesting that they act through interaction with specific molecular targets.

Forward and backward light scatter measurements in the assessment of corneal oedema

As the cornea swells it loses its transparency, thereby increasing both forward and backward light scatter. The purpose of this study was to investigate the changes in corneal transparency by means of scatter assessment. Both forward and backward light scatter measurements were obtained from 18 normal corneas, before and after the induction of corneal oedema with sealed scleral lenses. Forward light scatter was measured at 3.5, 10 and 28 degrees, using a portable straylight meter as developed by van den Berg. Backward light scatter measurements were obtained by computerised linear scanning densitometry of Scheimpflug images using the Oxford Modular Cataract Image Analysis System. After the scleral lenses were worn, forward light scatter values increased significantly at all the three angles (3.5°: F1,17 = 65.69, P &lt; 0.001; 10° : F1,17 = 37.30, P &lt; 0.001; 28°: F1,17 = 26.66, P &lt; 0.001). Backward light scatter also increased significantly with the presence of corneal oedema (F1,17 = 3 4.80, P &lt; 0.001). A significant correlation was found between the differences on forward light scatter recorded at the different angles. However, no significant correlation was found between the increase in forward and backward scatter recorded by the two techniques. We conclude that both forward and backward light scatter can be good indicators of corneal transparency.

Corneal Haze after Excimer Laser Refractive Surgery: Objective Measurements and Functional Implications

Postoperative changes in corneal transparency is a major concern after PRK. Data were obtained from 69 human eyes treated with excimer laser photorefractive keratectomy in order to evaluate a relationship between objective measurements of corneal transparency and visual performance. A CCD-camera device was employed and by using polarizing filters the system could discriminate between reflected and scattered light. We observed two groups of postoperative behaviour in corneal transparency. Each group showed an increase in both scattered and reflected light signals with a maximum at around two months postoperatively. In the majority group (70%) this was followed by a subsequent decline of the scattered light signal, whereas the combined signal generated by reflected and scattered light showed a biphasic curve with a second peak at around 4 to 5 months postoperatively. In the minority patient group (30%) the timebase variations in pattern of both signals were indistinguishable throughout the period of observation. In all patients we observed a good correlation between the signal generated by scattered light alone and the reduction in the 5% contrast visual acuity performance, whereas correlation was poor when the combined signal of reflected and scattered light was considered. These disturbances in low contrast visual performance were only significant during the first three to four months postoperatively and thereafter most patients returned to their preoperative value. Eighteen percent of our patients discontinued topical steroids postoperatively. No differences in either corneal transparency or final refraction was observed. We consider that PRK is an effective form of refractive surgery and the marginal loss of corneal transparency should only be a problem during the first three or four months postoperatively.

Disturbances in the rabbit cornea after short-term and long-term wear of hydrogel contact lenses. Usefulness of histochemical methods.

The influence of soft contact lenses (SCL) with low (37%, L) and high (65%, H) water content on rabbit corneas was investigated. The lenses were worn continuously for 1, 2, 4, 7, 10, 14, 21 or 28 days. The changes in corneal transparency, hydration and enzyme activities were studied. A slight change in corneal transparency due to higher hydration caused by a decreased activity of Na+-K+-dependent adenosine triphosphatase (Na+-K+-ATPase) in the corneal endothelium is followed by a decrease in the activity of gamma-glutamyl transferase (GGT). Slight morphological disturbances appear within 4 days in animals wearing SCL (L). SCL (H) produce similar changes one week later. Subsequently, the corneal epithelium becomes thinner and changes in the size of corneal endothelial cells are obvious. Disturbances of enzyme activities in cells of all corneal layers are present. In the epithelium highly increased activities of acid glycosidases, acid phosphatase, and dipeptidyl peptidase I and II, in keratocytes decreased activities of alkaline phosphatase and GGT, and in the endothelium decreased activity of Na+-K+-ATPase and GGT were found. These changes are more severe after SCL (L). In this case, inflammatory cells displaying high activities of lysosomal hydrolases appear in the anterior part of the stroma during the 3rd and 4th weeks and local degradation of glycosaminoglycans and proteins takes place. In contrast, after SCL (H) a remarkable thinning of the corneas was observed during extended wear, accompanied by decreased stainability of stromal glycosaminoglycans and highly decreased enzyme activities in keratocytes. The histochemical methods proved very useful in the assessment of lesions caused by a continuous wear of SCL.

Amphotericin B-induced sodium transport and water flow across rabbit corneal epithelium

We have determined fluid translocation across the cellular layers lining the cornea by measuring changes in corneal transparency. The loss of 1.3 μ1/cm 2 fluid from the stroma causes an increase of +1% in transparency. Amphotericin B ( 2 · 10 -6 M ) when added to the tear side (=mucosal side) of the epithelium causes a rapid increase in potential difference of 12.3 ± 0.7 mV ( mean ± S.E., n=6 ) followed by a slower increase of 18.6 ± 1.5 mV . The electrical resistance is reduced from 3.2 ± 0.3 kΩ · cm 2 to 0.6 ± 0.1 kΩ · cm 2 . The resulting increase in calculated short circuit current is accompanied by a decrease in transparency at a rate of 3.6 ± 1.0% per h , corresponding to an uptake of fluid by the cornea of 4.7 μ l · cm -2 · h -1 . Replacement of the fluid bathing the endothelial side of the cornea, in order to prevent water movement from the aqueous compartment into the stroma, did not significantly alter this uptake of fluid. Thus the epithelial fluid transport which is reported to be normally slightly secretory, becomes absorptive in the presence of amphotericin B. Serosal hypertonicity (20 mM mannitol) increases the water influx into the cornea induced by amphotericin B. These results indicate that amphotericin B induces sodium-selective channels in the epithelium leading to an accumulation of NaCl and water in the stromal layer of the cornea. Ouabain reduces the potential and calculated short circuit current in epithelia pretreated with amphotericin B. Following addition of ouabain, the NaCl and water accumulated in the stroma leak away resulting in a transient increase in transparency. Finally, a model is proposed that includes a stromal compartment involved in fluid transport and that agrees with the results presented here.

298. Evaluation of cosmetic irritancy

Measurement of ocular irritancy is a necessary step in the safety evaluation of both industrial and consumer products. Assessment of the acute eye irritation potential is therefore part of the international regulatory requirements for testing of chemicals.The Bovine Corneal Opacity and Permeability (BCOP) assay is generally accepted as a valid in vitro alternative method to the Draize eye irritation test to detect corrosive and severe eye irritants (category 1), but has not proven sensitive enough to discriminate accurately moderate (category 2A/2B) to mild and non-irritating compounds. In the currently accepted BCOP assay, opacity is determined by the amount of light transmission through the cornea, and permeability is determined by the amount of sodium fluorescein dye that passes through all corneal cell layers. Both measurements are used to assign an In Vitro Irritancy Score (IVIS) for prediction of the in vivo ocular irritation potential of a test substance. Nowadays, opacity is measured by an OP-KIT opacitometer providing a center-weighted reading of light transmission by measuring changes in voltage when the transmission of white light passes through the cornea alters. As a consequence, this may underestimate opacity that develops as spots or heterogeneous opaque areas on the periphery of an isolated cornea.A prototype of a laser light-based opacitometer (PLLBO) allowing better measurement of opacities was developed by Van Goethem et al. (2010). This new device showed improved sensitivity to detect subtle changes in corneal transparency. Furthermore, the new opacitometer allowed the analysis of the complete corneal surface and was able to detect more efficiently opaque spots located along the sides of the excised corneas.A further improved prototype of the PLLBO was constructed in combination with a camera and a speckle noise reducer. Treatment conditions of the corneas in the cornea holders were optimized in order to mimic more the real in vivo situation. A set of test compounds with irritancy potencies especially in the mild and moderate range was tested. The improved LLBO showed some promising features which potentially could improve the usefulness of the BCOP test. Adaptation of cornea holders showed to be of limited value and only restricted to concentrations up to 15% which mimics more test conditions in industry.This 3-year research project was sponsored by the Stavros Niarchos Foundation (Greece).