Evaluation of surfactant cytotoxicity potential by primary cultures of ocular tissues: I. Characterization of rabbit corneal epithelial cells and initial injury and delayed toxicity studies.

This investigation was undertaken to develop cytotoxicity assay systems using primary cultures of rabbit corneal epithelial cells as an experimental model to evaluate oculotoxic agents and the ability of these in vitro assay systems to predict irritancy potential and delayed toxicity. We have characterized the epithelial nature of the cultures by identifying keratins with antikeratin antibodies (AE1/AE3) and by demonstrating metabolic enzymes important to the integrity of the cells: lactate dehydrogenase, glucose 6-phosphate dehydrogenase and aldolase. Eight surfactants were compared and ranked according to their cytotoxic potential. We evaluated cytotoxicity by measuring leakage of the cytosolic enzyme, lactate dehydrogenase, into the medium, by making morphological observations and by assessing lysosomal neutral red uptake and mitochondrial 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction. The cells were treated for 1 h with the surfactants and the possibility of delayed toxicity was evaluated 24 h after removal of the surfactant. The cytotoxicity of the different types of surfactants as shown by all the tests was cationic > anionic = amphoteric > non-ionic. Triton X-100, a non-ionic surfactant but a severe irritant, had a ranking similar to anionic surfactants. The in vitro rankings corresponded well to reported in vivo Draize rabbit eye test data. The 24-h test for lactate dehydrogenase leakage showed that mild and non-irritating surfactants did not demonstrate any subsequent damage after a 1-h exposure, but the extreme and severe surfactants continued to show further damage after the 1-h exposure. These in vitro findings were similar to reported in vivo results. The neutral red and MTT tests did not adequately predict the prolonged toxicity of the more irritating surfactants, as was demonstrated by the lactate dehydrogenase leakage test. We conclude that in vitro cytotoxicity assays using primary cultures of rabbit corneal epithelial cells may be used to rank the cytotoxic potential of surfactants, but only the lactate dehydrogenase leakage test was able to assess prolonged cell injury.     

An interlaboratory assessment of the Eytex system

Seventeen raw materials and chemical formulations were evaluated in the Eytex System to determine the ability of this assay in vitro to predict eye irritation potential in vivo. All the test samples, which represented a wide range of chemical types and eye irritancy potential in vivo, were provided by one of the participating laboratories. Historical data from tests in vivo were available for each of the test samples, so testing in vivo specifically for this study was not necessary. Samples were evaluated by both the membrane partition assay (MPA) and the rapid membrane assay (RMA). The sensitivity, specificity, predictivity and equivalence of the Eytex assay were determined by comparison with the rabbit eye irritation data, using each of the different Eytex Draize Equivalent (EDE) classification schemes. Regardless of the classification scheme used, the correlation between the scores in vivo and in vitro was poor. The Eytex System consistently overclassified materials of low irritancy in vivo and underclassified those test materials of moderate irritancy or above. On the basis of the results from the 17 materials tested in this study, the Eytex System appears unsuitable as a replacement in vitro for ocular irritancy testing of all types of chemical. However, Eytex may have a place as a pre-screening method used as part of a test battery.     

An in vitro screening test for the assessment of the irritant potential of parenteral formulations

A HeLa cell/neutral red cytotoxicity assay has been investigated as an in vitro screen for assessing the irritancy potential of parenteral formulations. Five commercially available intramuscular injectables, three irritants and two non-irritants, were examined for cytotoxic effects in this system. The ranking of the injectables tested, in order of decreasing cytotoxic effects, correlated with both previously reported qualitative in vitro data and their reported irritancy when administered to man. The results show that the HeLa cell/neutral red cytotoxicity assay may provide an alternative to animal studies for the assessment of the irritancy potential of parenteral formulations during the preliminary stages of their development.     

Cytotoxicity testing using neutral red and MTT assays on a three-dimensional human skin substrate

The use of a three-dimensional dermal culture system as a substrate in cytotoxicity assays is described. This substrate consists of several layers of dermal fibroblasts, derived from human foreskin, grown on pretreated nylon mesh. This physiological model of the human dermis has been used in conjunction with the neutral red assay and the MTT assay to assess the in vitro toxicity of a panel of 15 test agents from several different classes. NR₅₀ and MTT₅₀ endpoints (test agent concentrations yielding 50% viability) were obtained for compounds/formulations from the following groups: surfactants, alcohols, antimicrobial preservatives, metal chlorides and pesticides. In addition, the carboxylic ionophore, monensin, was tested in both assays. Limited comparisons of the in vitro neutral red and MTT results, using the three-dimensional culture system, with existing in vivo rabbit ocular irritancy data look promising. This three-dimensional model may afford several advantages over monolayer cultures.     

Toxicity monitored with a correlated set of cell-culture assays

A set of assays for toxicity has been developed in which cell cultures serve as an alternative to toxicity testing in vivo. One test is the assessment of the highest concentration of toxicant which produces minimal morphological alterations in cell cultures, followed by the determination of the amount of neutral red dye uptake by the cells. A second test is based on 50% inhibition of uptake of [3H]uridine after incubation of the cultures with the toxicant. There is good agreement between these assays in the rank correlation of a broad spectrum of compounds tested, as well as with the data from Draize rabbit eye irritancy tests in vivo.     

In vitro skin irritation testing with human skin cell cultures

Cultured human skin cells are a potentially useful model for skin irritancy testing. We have evaluated the effects of chemical irritants on human epidermal keratinocytes (NHEK) and on keratinocyte-dermal fibroblast (NHEK/DF) co-cultures. Cell viability in NHEK cultures, measured as incorporation of the vital dye neutral red (NR), was reduced in a dose-dependent manner in response to the chemical irritants tested. The half-maximal effective concentration (NR₅₀) values correlated with irritation scores in human patch tests with these materials. Certain materials were found to be incompatible with this test system. NHEK/DF cultures were treated with ten prototype surfactants, and were evaluated for cell viability (MTT incorporation), cytotoxicity (release of the enzymes lactate dehydrogenase and N-acetyl glucosaminidase), metabolism (glucose utilization), and inflammatory mediator (prostaglandin E₂) release. There was a close correlation of the dose-response characteristics for all the endpoints tested, and between the in vitro responses and human patch test scores for the surfactants tested. These results demonstrate the usefulness of human skin cell cultures and of cell viability, cytotoxicity, and inflammatory mediator release as endpoints, for the in vitro assessment of skin irritancy.     

Evaluation of tissue culture insert membrane compatibility in the fluorescein leakage assay

The Fluorescein Leakage (FL) test, a short-term in vitro assay measuring damage on exposure to eye irritants to a transepithelial barrier permeability system, has been assessed as an alternative to the Draize rabbit eye irritation assay in the EU/Home Office international validation trial. The assay is based on the degree of leakage of sodium fluorescein through a confluent layer of Madin-Darby canine kidney epithelial (MDCK) cells grown on a tissue culture insert following exposure to a potential irritant. Several different types of tissue culture inserts with different membrane growth surfaces, pore sizes and pore densities have been employed in the past. Problems with chemical binding and other incompatibilities with insert membranes can affect the sensitivity of MDCK cells. Cationic surfactants in particular appear to interfere with the passage of fluorescein, leading to underestimation of damage to MDCK cellular function. A blind comparison between MDCK cells cultured on Anopore^™ and the Millicell-HA inserts using the EU/Home Office set of surfactants indicate that the sensitivity of cells cultured on Anopore inserts appeared to correlate more closely with in vivo data. The ability to microscopically evaluate the status of MDCK cultures through Anopore further recommends this membrane as possibly the better choice for the FL assay.     

In vitro cytotoxicity tests on cultured human skin fibroblasts to predict skin irritation potential of surfactants.

Cultured human skin cells are a potentially useful model for skin irritancy testing. We have investigated the use of human skin fibroblasts for in vitro screening for skin toxicity. To assess the cytotoxic effects of surfactants, cell viability was measured by the NRU (neutral red uptake) assay and AB (Alamar blue) assay as in vitro methods. The skin irritation potential of surfactants by human skin patch test was assessed as in vivo methods. The close relationship was found between AB assay with human skin fibroblasts and human patch test (r=0.867). There was a relatively good agreement between the NRU and in vivo patch test (r=0.648). These results suggest that AB and NRU assay using cultured human fibroblast could be predictable methods for the irritancy of various surfactants in human.     

In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride

The aim of this study was to compare four in vitro cytotoxicity assays and determine their ability to detect early cytotoxic events. Two hepatoma cell lines, namely HTC and HepG2 cells, were exposed to cadmium chloride (0-300 microM) for 3, 5 and 8 h. Following exposure to the toxic metal cytotoxicity was determined with the lactate dehydrogenase leakage assay (LDH), a protein assay, the neutral red assay and the methyl tetrazolium (MTT) assay. In HTC cells no toxicity was observed for any incubation period when the LDH leakage, the MTT and the protein assay were employed whereas the neutral red assay revealed early cytotoxicity starting after incubation of HTC cells with CdCl(2) for 3 h. In the case of HepG2 cells the MTT assay reveals cytotoxicity due to CdCl(2) exposure after 3 h whereas no such effect is seen with the other three assays. Following 5 h exposure of HepG2 cells to CdCl(2), toxicity is observed with the MTT assay at lower concentrations compared to the ones required for detection of toxicity with the LDH leakage and the neutral red assay. In conclusion different sensitivity was observed for each assay with the neutral red and the MTT assay being the most sensitive in detecting cytotoxic events compared to the LDH leakage and the protein assay.     

Toxicity monitored with a correlated set of cell-culture assays

1. A set of assays for toxicity has been developed in which cell cultures serve as an alternative to toxicity testing in vivo.

2. One test is the assessment of the highest concentration of toxicant which produces minimal morphological alterations in cell cultures, followed by the determination of the amount of neutral red dye uptake by the cells.

3. A second test is based on 50% inhibition of uptake of [³H]uridine after incubation of the cultures with the toxicant.

4. There is good agreement between these assays in the rank correlation of a broad spectrum of compounds tested, as well as with the data from Draize rabbit eye irritancy tests in vivo.     

Evaluation of eye and skin irritation of arginine-derivative surfactants using different in vitro endpoints as alternatives to the in vivo assays

Arginine-derivative surfactants constitute a novel class of surfactants, which can be regarded as an alternative to conventional surfactants. Prior to human exposure, it is necessary to assess their irritation potential. The classical in vivo evaluation of the irritancy potential via the Draize test has been extensively criticized. In that regard, a great number of in vitro alternatives have been developed. Erythrocytes were chosen as the target cells for eye irritation assessment and hemolysis and hemoglobin denaturation were selected as appropriate endpoints. For skin irritancy assessment, the keratinocyte cell line NCTC 2544 was used and different in vitro endpoints were measured: two cytotoxicity assays (NRU and MTT) and the synthesis of the proinflammatory cytokine IL-1alpha. The eye and skin Draize tests were also performed for comparative purposes. The results point out that, according to in vivo and in vitro assays, the new arginine-derivative surfactants have lower eye and skin irritation potential than the synthetic surfactant SDS. Furthermore, in vitro methods were also able to detect differences in irritancy among the new surfactants not noticeable by the Draize tests, indicating that in vitro methods can be more sensitive than the in vivo test, offering the opportunity to detect subtle differences in irritancy.     

Comparative study of red blood cell method in rat and calves blood as alternatives of Draize eye irritation test.

INTRODUCTION: Red blood cell assay (RBC) is used to estimate potential irritation of tensioactive agents and detergents. Cell membrane lysis and cell protein denaturation are measured photometrically. This study was aimed to determine if rat blood cells can be used to predict eye potential irritation in the same way of calves blood cells in RBC assay. METHODS: We evaluated 20 cosmetic formulations using rat and calves blood according to INVITOX protocol No 37. Data of media hemolysis concentration, denaturation index and the ratio of both parameters were compared with in vivo data of eye irritancy. RESULTS: There was a significant difference (p<0.01) between H50 value when evaluated the standard SDS with red blood cell method in rat and calves blood. According to the exact probability of Fisher taking as approach the acceptance or rejection of the substance there are no significant differences between in vitro assay with calves blood and in vivo results. Not happening the same way for the RBC assay with rat blood where significant differences were obtained (p<0.01) among the classification of in vitro and in vivo test. DISCUSSION: The RBC assay using calves blood showed better results. Several test substances were false negatives with rat blood. This high false negative rate would be correctly identified by the animal test but it may also lead to increased animal consumption. For that RBC assay with calf blood cells is preferable to the employment of rat blood as screening method with a reduction and refinement strategy.     

Cytotoxicity screening of surfactant-based shampoos using a multiwell fluorescence scanner: Correlation with Draize eye scores

The irritancy potential of seven shampoos was evaluated by a rapid cytotoxicity assay in cultured human keratinocytes and rat hepatocytes. Loss of cell viability was estimated from increases in propidium iodide fluorescence measured using a multiwell fluorescence scanner. The concentration of shampoo causing a 50% loss of cell viability after 15 min of incubation (V₅₀) was determined by probit analysis. Log V₅₀ measured in human keratinocytes showed a strong negative correlation (r = −0.95; P <0.001) with Draize eye scores in rabbits. Log V₅₀ measured in rat hepatocytes did not show a statistically significant correlation with Draize eye scores. The results indicate that cytotoxicity screening of human keratinocytes using propidium iodide and a multiwell fluorescence scanner is highly predictive of Draize eye scores for surfactant-containing shampoos.     

Evaluation of eight in vitro assays for assessing the cytotoxicity of cigarette smoke condensate.

The accurate assessment of cytotoxicity is important for evaluating the potential of a test agent to induce pathologies that result from cell killing and to determine appropriate doses for other endpoints, such as genetic toxicology studies. The objective of this work was to determine the most sensitive assays for assessing cytotoxicity in Chinese hamster ovary (CHO) cells following short-term (1 h) and long-term (24 h) exposure to cigarette smoke condensate (CSC). Eight in vitro cytotoxicity assays with different endpoints were used to evaluate the cytotoxicity of Kentucky reference 1R4F (K1R4F) CSC in CHO cells. The assays used for this study were neutral red uptake, LDH release, kenacid blue binding, MTT formation, XTT formation, acid phosphatase activity, sulforhodamine B binding and resazurin binding. Four of the more widely used cytotoxicity assays (neutral red, MTT, kenacid blue and LDH) were also evaluated at 3-, 6-, 12- and 18-h time points. At the 1-h exposure time, LDH was the most sensitive with toxicity observed beginning at 100 microg/ml. None of the other assays demonstrated a concentration-dependent increase in toxicity after 1-h exposures even at the maximum concentration of 150 microg/ml of CSC. Following 24 h of exposure, neutral red and kenacid blue were the most sensitive. The results of our study indicate the assay that measured membrane integrity was the most sensitive for short exposure times, whereas the neutral red and kenacid blue assays that measured total cell number were more sensitive for longer exposure times.     

Comparison of cytotoxic effects of chemicals in four different cell types

Cytotoxic effects were compared using a colony-formation assay in three established cell lines (Balb 3T3, mouse whole embryo, ARLJ301-3, rat liver and FRSK, rat keratinocytes) and one primary cell culture (RC-1, rabbit cornea) with the Draize eye irritancy score in vivo. The cells were treated with 52 chemicals on the day after plating, then cultured for 7 or 8 days. The 50% inhibition dose (ID₅₀) for each chemical was calculated based on the colony number. With a few exceptions, the cytotoxicities of the chemicals were in the following order in all four cells: cationic detergents anionic detergents non-ionic detergents glycol or oil. These results were almost the same as the data in vivo. The correlation coefficients of the ID₅₀ to the Draize score of 20 in vivo were 0.57 (Balb 3T3), 0.61 (ARLJ301-3), 0.71 (FRSK) and 0.65 (RC-1). Balb 3T3 and ARLJ301-3 cells were slightly more sensitive to chemicals than FRSK and RC-1 cells. These results suggest that the colony-formation assay using established cell lines is an attractive method for the screening of chemicals in that large differences among cell types in their response to direct-acting chemicals, were not observed.     

Use of primary rabbit cornea cells to replace the Draize rabbit eye irritancy test

The Draize ocular irritancy test was compared with cytotoxicity determined from colony forming ability in cells freshly isolated from rabbit cornea (RC). The primary RC cells grew well and cloning efficiency ranged between 80 and 100% during early passages. We used 52 chemicals used in consumer products as test agents. There was a close correlation between the cytotoxicity in RC cells in vitro and the Draize score in vivo in response to the 52 chemicals. With a few exceptions, the cationic detergents appeared to be more toxic than the anionic or nonionic compounds tested. These data suggest that the cytotoxicity test in vitro using primary RC cells may be useful as a substitute for the Draize eye irritancy test.     

The SDA Alternatives Program Phase III: Comparison of in Vitro Data with Animal Eye Irritation Data on Solvents,Surfactants, Oxidizing Agents,and Prototype Cleaning Products

Abstract

Nine in vitro candidate tests for estimating eye irritation potential were evaluated as potential replacements for the Draize test. The tests examined were a cell protein assay, the chorioallantoic membrane vascularization assay, a cell protein assay, a fibroblast cytotoxicity assay, the Living Dermal Model and Living Skin Equivalent, two neutral red assays, an SIRC cytotoxicity assay, and a Tetrahymena thermophila motility assay. The results from these in vitro tests were compared to results from a modified Draize test with 22 test materials. The test materials were selected to represent various classes of cleaning products and ingredients. Ingredients were tested at concentrations representative of concentrations typically found in cleaning products. The correlation coefficients with all test materials considered ranged from 0.58 to 0.91. When only nonalkaline materials are considered, the correlation coefficients of all 10 tests were not significantly different from one another, ranging from 0.8 to 0.9. The assays least affected by the alkalinity of the test substances were the corneal epithelial plasminogen activator assay, the chorioallantoic membrane vascular assay, and the Tetrahymena motility assay. Further, six of the 10 tests were able to identify the five nonirritants in the study, although the relative irritation potentials of the irritants were not accurately predicted by any of the tests. Results from a low-volume eye irritation test (LVET) were also compared to results from a modified Draize test with the same 22 test materials. The LVET had a high correlation with the modified Draize test and will be useful for future comparison with other alternative eye irritation tests. Based on these data, a number of alternative tests developed to replace the Draize eye irritation test included in this phase of research are useful for screening the eye irritation potential of nonalkaline cleaning products, although some tests are better for identifying the eye irritation potential of test materials with alkaline or oxidation potential. Although the ability of the eye to recover from damage was not measured by any test, the tests show promise for the use of determining eye irritation potential.     

Formaldehyde cytotoxicity in three human cell types assessed in three different assays.

International standards for preclinical screening of the cytotoxicity of dental materials so far recommend the use of established cell lines. The aim of this study was to assess the relative susceptibility of human dental pulp fibroblasts (HPF), human buccal epithelial cells (HBE) and HeLa cervix cancer cells exposed to identical cytotoxic challenges. Formaldehyde, which may be released from dental materials such as dental composites, glassionomer cements, and endodontic sealers, was used as test chemical. Cytotoxicity data including dose-response relations and TC(50) values were assessed in three different assays: BrdU incorporation, neutral red uptake and MTT assays. HBE and HPF demonstrated statistically significant lower TC(50) values in both the neutral red and the BrdU assay in comparison to HeLa cells. In the MTT assay no statistically significant differences were observed between the cell types. In the two target-tissue cell types (HPF and HBE) the Neutral Red assay revealed lower TC(50) values in comparison to the BrdU assay. In HeLa cells no statistically significant differences were observed between the assays. In conclusion, the present study confirms that cytotoxicity data obtained by cell culture studies are influenced by both cell culture model and choice of assay. Under identical experimental conditions, human target tissue cells appeared to be more sensitive to formaldehyde toxicity than human HeLa cancer cells.     

Development of a human corneal epithelium model utilizing a collagen vitrigel membrane and the changes of its barrier function induced by exposing eye irritant chemicals

The brief TEER (trans-epithelial electrical resistance) assay after exposing chemicals to corneal epithelium in vivo is known as a suitable method for evaluating corneal irritancy and permeability quantitatively and continuously. A collagen vitrigel membrane we previously developed is a thin (about 20 μm thick) and transparent membrane composed of high density collagen fibrils equivalent to connective tissues in vivo, e.g. corneal Bowman’s membrane. To develop such a TEER assay system in vitro utilizing a human corneal epithelial model, HCE-T cells (a human corneal epithelial cell line) were cultured on the collagen vitrigel membrane substratum prepared in a Millicell chamber suitable for TEER measurement. Human corneal epithelium model possessing 5-6 cell layers sufficient for TEER assay was successfully reconstructed on the substratum in the Millicell chamber by culturing the cells in monolayer for 2 days and subsequently in air-liquid interface for 7 days. The exposure of chemicals to the model induced the time-dependent relative changes of TEER in response to the characteristic of each chemical within a few minutes. These results suggest that the TEER assay using the human corneal epithelial model is very useful for an ocular irritancy evaluation as an alternative to the Draize eye irritation test.