IRAG Working Group 4: Cell cytotoxicity assays

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Pergamon

Food and Chemical Toxicology 35 (1997) 79-126

IRAG WORKING GROUP 4

Cell Cytotoxicity Assays J. W. H A R B E L L * % S. W. KOONTZ:~, R. W. LEWIS§, D. L O V E L L ¶ a n d D. A C O S T A * II tMicrobiological Associates, Inc., Rockville, MD 20850, USA, ~:Johnson& Johnson Consumer Products, Inc., Skillman, NJ 08558, USA, §Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SKI0 4TJ, UK, ¶BIBRA International, Carshalton, Surrey SM5 4DS, UK and IlDivisionof Phalanacology and Toxicology, University of Texas at Austin, Austin, TX 78712, USA Abstract--Twenty-seven data sets from 12 cellular cytotoxicity assays, intended to predict ocular irritation, were submitted to the Interagency Regulatory Alternatives Group (IRAG) for review. These data consisted of paired in vivo (Draize) and in vitro responses to individual chemicals and formulations. In vivo data consisted of individual tissue scores so that the predictive value of the in vitro assay could be assessed for each tissue response normally measured in the standard Draize assay. Data were compiled and evaluated according to the IRAG Guidelines Document. The Pearson's linear correlation coefficient was used a:; the first step in assessing the relationship between the in vitro and in vivo responses. The majority of the data sets represented the study of surfactant-based materials. In many cases, there was good correlation between the in vitro scores and the in vivo tissue responses. Most pronounced were the particularly good correlations between the in vitro scores and conjunctival redness scores across most of the assays. Based on the data submitted, a number of the cell cytotoxicity assays show considerable promise as screens for ocular irritancy. None of the submitters recommended that their cell cytotoxicity assay be used as a sole replacement for in vivo assessment. For almost all of these assays, the materials being tested should be water-soluble/miscible.The toxicity of products with reserve acidity or alkalinity or with hig]~reactivity may be underestimated. A given user may prefer certain assays depending on the types of materials to be tested, the expected range of toxicities and the resources available. The cell cytotoxicity assays can serve as a valuable component of a tiered or battery testing program. As with any assay, a sufficient number of replicate values, concurrent positive and negative controls, and a strict adherence to assay acceptance criteria are essential to produce credible data. © 1997 Elsevier Science Ltd

Introduction Cell cytotoxicity assays were among the first in vitro bioassay methods used to predict toxicity of substances to various tissues. They have been used to examine organ-specific (e.g. liver) damage of chemicals, tissue inflammatory potential of medical devices and ocular irritancy of chemicals and formulations. A wide range of cell types and measures of cell viability have been used. This report reviews the data submitted to the lnteragency Regulatory Alternatives Group (IRAG) on the use of cell culture cytotoxicity assays to predict ocular irritation. The US Interagency Regulatory Alternatives Group (IRAG), composed of members from the Consumer Product Safety Commission, Environmental Protection Agency and Food and Drug Administration, proposed to examine the current state of non-whole animal methods for predicting ocular irritation. Quantitative in vitro data were to be compared directly with quantitative in vivo data generated on the same chemicals or formulations. To this end, data from a wide range of non-whole animal *Co-Chars.

test systems were solicited. By design, data submission was completely voluntary, and the data therefore represent only a portion of the total available. Because the goals of the workshop were to address the various needs of the broad scientific community (academic, corporate and regulatory), data were solicited in a form that would allow the greatest possible utility to each group. The submissions were divided by categories of in vitro assay types and reviewed by independent working groups drawn from academia and industry. Preliminary reports from these working groups were delivered to the Workshop on Eye Irritation Testing: Practical Applications of Non-whole Animal Alternatives, November 1993. After receipt of additional data and further review, the final reports were prepared for publication.

Methods The data for submissions followed the Guidelines for the Evaluation of Eye Irritation Alternative Tests: Criteria for Data Submissions. The details of this document are given in the report of Working Group 6. Data were reported from studies in which both in vivo and in vitro assays had been performed on the

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test materials. This arrangement allowed direct, quantitative determination of the correlation between the in vitro and the in vivo responses. To permit the greatest use of the data, individual tissue scores were solicited from the in vivo studies. Scores for the individual animals were requested, rather than just the average scores. This permitted the in vitro response to be compared to the response in each tissue and provided a measure of the variability of each tissue response among the (generally three to six) subjects tested. Content o f the submissions

The submissions were to be divided into the following seven parts, briefly stated: proposed context for the use of the in vitro assay; chemical characterization of the materials tested; the in vivo data; the in vitro data; plots prepared to show the relationship between the in vivo and in vitro data; statistical analysis of the plots; and results of the comparisons and conclusions. The proposed context for the use of the in vitro assay included information on the mechanistic basis of the assay, type of use (e.g. screen, preclinical screen, labelling or final product test), range of irritancy over which the assay might be applied, and the types of materials that were and were not amenable to test in the assay system. Chemical characterization of the materials tested or reported as a group included some indication of the chemical or product class (e.g. surfactant, petrochemical), the range of physical forms, water solubility and pH range (if appropriate). The in vivo data requested were very extensive and included a summary of the test method used. To address the format commonly used in the regulatory setting, animal responses were reported for each tissue parameter scored in the Draize assay (corneal opacity, corneal area, iris, conjunctival redness, conjunctival chemosis, discharge, days to clear). Tissue scores, used in the evaluation, were taken at >~24hr after exposure to the test material. In addition, most of the submitters included the maximum average score (at /> 24 hr). So that the variability of the in vivo response could be examined, the individual maximum scores for each tissue for each rabbit were requested. Extensive discussions of the in vitro method were also obtained. A detailed description of the test substrate (e.g. cells), culture and exposure conditions, physical form of the test article as tested, and endpoint(s) examined were provided. In discussing the endpoint(s), the submitters were asked to provide data on the type of response (e.g. neutral red uptake), the range of responses detectable [e.g. dynamic range of the endpoint such as range of optical density (absorbance) covered], and range of chemical toxicities which could be assessed by the test. Controls and acceptance criteria were requested, as well as any historical control data. If multiple trials

were used to determine the final numerical value, the number of trials and their means and standard deviations were to be included. The in vivo and in vitro responses were compared in a series of scatterplots (X-Y plots). Each tissue was plotted against the in vitro response. In some cases, the data for each rabbit were plotted but because this was not feasible for most of the submissions, means and standard deviations were used. The primary analysis performed on the plots was the Pearson's correlation for the best fit line through the data. The resulting r values provided some measure of the quality of the correlation. In the initial analysis, all points were included in the correlation analysis; this convention was adopted to provide a uniform method for all systems. The relative range of the values from certain in vitro assays required that a semi-log plot be used to graph the in vivo and in vitro data. Some submitters also included other methods. Not all submissions were amenable to this kind of correlation analysis. Conclusions were drawn from the data as appropriate. Most data sets submitted included most if not all of the requested data. Those lacking in vivo data were not reviewed by the committee and are not included in this report. The Results section details the specific submissions and is organized into sections according to the assay type. The committee reviewed the submissions to assess completeness, to provide some additional analysis of the data when appropriate and to integrate multiple submissions which used similar assay systems.

Importance of exposure and endpoint assessment times

Details of the specific methods for each assay are provided in the Results section. Many assays use the same cell types with different exposure and endpoint protocols. Consequently they may provide rather different information about the action of test materials on the cells. A number of critical factors define the method and therefore the data which it provides. First, how are the cells exposed to the test material? Second, what is the exposure time for the cells to the test article? Third, if the test material is removed from the cells before the endpoint is measured, what is that period (e.g. minutes, hours, days)? Finally, what is the endpoint measure (or measures) used to evaluate the effect of the test material on the cell population? Most but not all test materials were diluted in aqueous medium but the time of exposure varied from 5 min to 48 hr. Generally, those assays with long exposure times (i.e. />24 hr) assayed the effect of that exposure immediately at the end of the exposure period, for example the neutral red uptake assays. Some of the assays with shorter exposure times (e.g. SIRC clonal growth assay) provide a recovery and growth period before the assay is terminated. In that case, the exposure period is only 1 hr but the cells a r e

Working Group 4: Cell cytotoxicity assays allowed to grow for 7 days before the clones are fixed and counted. The neutral red release and K562 trypan blue exclusion assays measure toxicity immediately after the short exposures. These short-term exposures require that the toxicity be manifested much more rapidly if the endpoint is measured immediately after exposure. In contrast, the SIRC clonagenic assay with its 7-day post-exposure incubation period can detect delayed toxicity or recovery in the exposed cells. It should be remembered that the effective exposure time of a test material in vivo can be very short. In the absence of fixation of the test material to the ocular tissues, most of the test material will be flushed from the eye in <30min. If tearing is stimulated, the time', may be much less (MacDonald and Maurice, 1991). Therefore, the shorter exposure times (e.g. < 3 0 m i n ) may be expected to closely approximate those of the corneal and conjunctival epithelium experience in vivo. Assay endpoints

The endpoint measure is intended to show the degree of damage c~Lused by the test article. However, many endpoint measures are possible, and they do not necessarily provide comparable information. The range includes loss of membrane integrity, release of cytoplasmic enzymes, loss or decrease in metabolic processes (e.g. ATP production), cessation/reduction of D N A synthesis,; or inability to continue cell replication. Usually a control cell population is tested in parallel and the objective is to measure the difference between the control and treated populations. Among the most sensitive endpoints are the clonal growth (e.g. SIRC assay) (North-Root et al., 1983) and 3H-thymidine incorporation (DNA synthesis). Both measure the continued cell division (directly or indirectly), which is one of the first processes to cease in a cell exposed to a toxin or even poor culture conditions. Furthermore, the clonal growth assays generally begin with a very limited number of cells in each dish so that the effective dose per cell is quite high. This approach also allows the detection of delayed toxicity, such as might be seen with a D N A alkylator. In this case, the cells might be alive for several days after exposure but not be capable of continued replication. Recovery or reversibility of damage (e.g. sublytic membrane damage) can be seen, since the growth period allows cells which have temporarily ceasecL replication to begin and form visible clones. To see recovery, it is important to choose a cell type, like the SIRC cell, which does not readily release from the plastic when "injured". Some fibroblast lines (e.g. L929) tend to release from the plastic substrate w]aen damaged so that they are lost during the rinse steps. D N A synthesis measurements are usually taken at the end of the assay and thus provide a measure of this function at a point in time. They also assume that the populations are randomly

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cycling so that the treated and control populations can be compared. The endpoint of the Corneal Plasminogen Activator Assay (Chan, 1987) uses the constitutive synthesis and release of the enzyme to monitor continued cell function (e.g. protein synthesis and transport) after a short exposure. This endpoint integrates the functional health of the treated population during the 48 hr after treatment. Populations that do not die immediately may continue to produce the enzyme. A number of endpoints depend on the continued production of ATP by the cell to drive pH gradients or to alter a vital dye. Thus, they measure cell viability indirectly. Neutral red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride) is a vital dye which passively enters the cell. At physiological pH, it has little net charge and so passes through membranes. The lysosome maintains a pH much lower than the surrounding cytoplasm. This proton gradient across the lysosomal membrane is energy dependent. Neutral red passing into the lysosome becomes charged and thus does not freely pass out back into the cytoplasm. Loss of this pH gradient, through either mortality/morbidity of the cell or permeation of the membrane, will release retained neutral red or prevent its accumulation (Filman et al., 1975). The uptake of neutral red has been used both as a quantitative assay for viable cell number (Barstad et al., 1991) (e.g. Neutral Red Uptake Assay) and as a qualitative measure of cell viability in the agar overlay (agar diffusion) assays. The neutral red release assay depends on both the lysis of cells (high dose) and depolarization of the membranes (increased permeability and disruption of the proton gradient) (intermediate doses). Because the dye is not transformed, non-specific binding of the dye to the culture can give a false picture of viability. Precipitated test material is often the culprit. Binding to extracellular collagen (or other proteins) limits the use of this endpoint in organ cultures. Finally, materials which induce lysosome formation will give neutral red uptake readings which are not necessarily proportional to the cell number when compared to the controls. The tetrazolium dye MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide) and similar tetrazolium salts are converted from the oxidized form to the reduced form by the NADH÷-dependent reaction catalyzed by succinate dehydrogenase (Mosmann, 1983). In the oxidized form, MTT is yellow and somewhat water-soluble; on reduction, the colour turns blue-black and the dye precipitates. The oxidized form is provided to the cell population but only the reduced form is measured spectrophotometrically. This colour shift reduces interference from non-specific binding of the dye. Under stable conditions, the amount of MTT reduced per unit time is proportional to the cell number. It should be noted that this is also an indirect measure of cell viability and that test materials which increase mitochondrial

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activity also increase MTT reduction. Such increases are commonly seen when cells are treated with surfactants at doses which increase membrane permeability without lysing the cells. Increased membrane permeability increases the demands on the ion pumps, which in turn increase the need for ATP. This metabolic hormesis ("toxin"-induced increase in metabolic rate) normally does not interfere with the usefulness of the assay but may be recognized as an increase in MTT reduction in some portion of the test article dose-response curve. Fluorescein diacetate passes into the cell where esterases cleave the acetate groups. This cleavage is energy dependent and retards the release of the dye from the cell. It also enhances the fluorescence of the dye so that the cells appear "apple green". This stain is used both as a quantitative measure of viable cell number in a population and as a qualitative measure of viability in individual cells. The active, receptor-mediated transport of molecules into the cell can also be used to measure viable cell number and "normal function" in a population. Uptake of uridine is a receptor-mediated process that requires an intact membrane, ATP and flux in the intercellular uridine pool (e.g. RNA synthesis) (Shopsis, 1984). Uridine uptake is measured over a brief period so that the flux into the cells rather than incorporation into R N A is the endpoint. Under standard conditions, the uptake is expected to be proportional to the number of viable cells. As with the other endpoints discussed above, this assay endpoint would also be expected to measure morbidity (e.g. decrease in normal cellular functions) as well as mortality (cell death and lysis). As a practical matter, the two are not differentiated except perhaps by the shape of the dose-response curve. Membrane integrity endpoints measure cell viability as a function of the failure of a dye or enzyme to pass through the cell membrane. These endpoints include the trypan blue exclusion assay, in which cells with intact membranes do not stain with the dye whereas dead or damaged cells, with holes in their membranes, stain blue. Ethidium bromide staining of D N A relies on the same principle; this dye also will not pass through the intact cell membrane. Since these dyes are generally intended to differentiate between live and dead ceils, cells must be individually scored. Consequently the fraction of the total population being evaluated is small unless automated techniques are used (e.g. image analysis or fluorescent-activated cell sorting). Cell counts giving the percentage of live/dead cells are obtained. The measure of viability is not only indirect; it also requires that the cell undergo degeneration of its membrane before it will be counted as dead. Furthermore, enumeration of only dead cells has severe limitations. Fully degenerated cells will not be counted and thus toxicity would be underpredicted. Furthermore, cytostasis would go undetected unless the counting technique permits determination of the

total viable count in each treated and control population. For this reason, ethidium bromide is combined with a vital stain to count living cells (e.g. fluorescein diacetate). Cytoplasmic enzyme release can be another measure of membrane integrity (cell viability), especially in cells which do not divide (e.g. hepatocytes). The enzyme (i.e. lactate dehydrogenase) is released into the culture medium and detected quantitatively with a chromogenic substrate. The total available enzyme can be determined by lysing the population with 1% Triton X-100, which allows the amount of enzyme released to be expressed as a fraction of the total enzyme available. Caution should be exercised with lactate dehydrogenase, as the enzyme may be readily inactivated. Again, membrane leakage is required, so that these assays often are detecting cells in the last stages of cell death. Red blood cell lysis is an extreme example of a membrane damage endpoint. The release of haemoglobin is measured spectrophotometrically. Red blood cells are extremely fragile compared with nucleated cells, which have functioning cytoskeletons, so that the same exposure in one system is unlikely to give a similar response in both. The measurement of total protein in a population is a measure of relative cell number, but only a crude one. It relies on the relative growth of controls over the treated cultures to show differences in toxicity. However, depending on the cell type used and the number of cell replications in the controls, the dead cells may contribute significantly to the total protein. This endpoint measures viability very indirectly. The relative sensitivity of these endpoints is generally cell replication > metabolic > membrane integrity. For example, in a comparative study with human fibroblasts in three-dimensional culture, the doses giving 50% inhibition of D N A synthesis were as much as a log lower than those required to give 50% release of LDH (Harbeil et al., 1991). Thus, the endpoint chosen will provide specific information about the state of the cell population, but the information provided by each endpoint is not necessarily the same. Results

16 assay types were reviewed for a total of 27 data sets with close to 600 paired responses. The following assays were reviewed: SIRC Cell Cytotoxicity Assays (Dial Protocol and Biogir Protocol), Neutral Red Uptake Assay, Neutral Red Release Assay, Agar Diffusion Assay, Red Blood Cell Lysis Assay, K562 Trypan Blue Exclusion Assay, Fibroblast Cytotoxicity Assay, Corneal Plasminogen Activator Assay, Cell Protein Assay, and Dual Dye Assay. Submissions were received from Adelphi University; Ajinomoto Corporation; Allergan; Biogir, S.A.; Clonetics Corporation; Cosmetic, Toiletry and Fragrance Association (CTFA); Dial Corporation; Eli Lilly and Company; Instituto Superiore di Sanita;

Working Group 4: Cell cytotoxicity assays Johnson and Johnson; Kurabo Industries; L'Or6al; Mary Kay Cosmet:ics; Microbiological Associates, Inc.; The Procter & Gamble Company; RIKEN Cell Bank; RoC S.A.; Soap and Detergent Association (SDA); University of North Carolina; University of Washington; and Zeneca Ltd. In the text of this report, code numbers are used rather than corporate names. Assays were grouped whenever possible, but because of the number of different assays submitted, this report is composed of several smaller reviews. Most of the submitters proposed the use of their assay as screens to reduce animal testing, often to be used during product development. In many cases, the assay was part of a battery of non-in vivo assays (e.g. chemical class/ingredient review, chemistry and other in vitro tests). The majority of the data were provided for surfactants or surfactant-containing formulations. The vast rc~ajority of materials tested were water-soluble. This bias may have resulted from at least two factors: first, the large number of surfactant-containing materials under test in many industries, and second, the generally poor performance of the submerged cell culture systems with non-water-soluble materials. The C T F A Phase II study on creams and lotions tested many materials which were poorly water-soluble. The SIRC assay data (Dial Corporation) from that study were submitted to this workshop and provide a good indication of the performance of many of the cell cytotoxicity assays (e.g. neutral red) in that multi-assay study. Although plots comparing individual tissue scores with the in vitro scores were available for most submissions reviewed, it was not possible to present all of these plots in this review. Therefore, a shorthand summary, the Draize MAS (/> 24 hr), was chosen as a way of summarizing the general nature of the data. The plot of the MAS v. the in vitro score is included in the review for many of the data sets. This single plot is not intended to substitute for the complete analysis c,f the individual tissue plots. Inclusion of all points in a data set for the linear correlation analysis provided a very conservative measure of the in v,~vo/in vitro correlation. In several cases, the in vitro as:say was able to resolve differences in toxicity at the very mild end of the spectrum, which the in vivo assay could not. For example, the SIRC cell cytotoxicity as'say (Fig. 1) and the neutral red uptake assay (Fig. 5) were able to show differential toxicity among several surfactant-containing materials which had 24-hr Draize scores of very close to zero. The increased resolution of the in vitro assays would be considered a benefit when an assay was used to resolve very mild materials, but it did serve to reduce the linear correlation value for the assay data set. In several submissions, multiple independent trials of the in vitro assay were compared with a single in vivo trial which used multiple animals. For the

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in vitro assay, the mean and standard deviation from

the multiple trial values (generally three) were plotted. This gave a measure of the assay variability. Only a single in vivo trial was performed (although sometimes with a random block design) for each data set. To better represent the assay variability (rather than the animal-to-animal variability), the mean and standard error of the mean were plotted (J. Springer, personal communication).

SIRC Cell Cytotoxicity Assay (Dial Protocol) A. In vitro assay. S I R C Cell Cytotoxicity Assay Summary. Four data sets are included in this review. The materials were tested as part of either the CTFA or SDA studies. C T F A Phase III: Surfactant-Based Personal Care Prototype Formulations (all materials) C T F A Phase II: Oil-in-Water Emulsion Personal Care Prototype Formulations (some materials watersoluble; all materials tested as dispersions) C T F A Phase I: Hydro-alcoholic Personal Care Prototype Formulations (some materials water-soluble; all materials tested as dispersions) SDA Phase III: Generally surfactant-based materials (ingredients and formulations); some non-surfactant ingredients. Basis o f assay. The ability of individual cells to form colonies (clonal growth) in culture is a very sensitive measure of cell viability. In this assay, test article-induced damage to the cells is measured by the loss of clonal growth potential. This loss can reflect frank cell killing or sublethal damage which reduces the cell's replication rate. Protocol. The procedure is based on the work of North-Root et al. (1982) and Demetrulias and North-Root (1987) in which the toxicity of a given compound is evaluated by its actions on decreasing colony counts relative to untreated controls. Briefly, SIRC cell stock cultures were grown in Ham's F12 medium supplemented with 10% heat-treated (30 min, 56°C) foetal bovine serum; treated cultures were grown in the same serum-containing medium further supplemented with gentamicin at 50 #g/ml. Subconfluent stock cultures were briefly trypsinized to prepare a single cell suspension for seeding assay dishes. 400 cells were seeded into each dish 24 hr before dosing. Test material dispersions were prepared for dosing w/v with v/v dilutions thereafter in sterile Ham's FI2 without serum or antibiotics. Sample preparations were not subject to pH adjustment or sterilization. Sodium lauryl sulfatetreated cultures served as the positive control. Cells were exposed to the test or control materials for 1 hr, rinsed and subsequently cultured for 7 days. Colonies were fixed with methanol and stained with 0.1% crystal violet. The concentration of test material giving a 50% decrease in colony counts, relative to controls, was determined by probit analysis.

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Table 1. Summary of the correlation between the in vivo and in vitro values for the SIRC cytotoxicity assay in the CTFA Phase I study Laboratory: CCI00 Exposure: 1 hr In vivo/in vitro correlation (semi-log plot) Number of Material type Range of materials or class scores Tissue r= * CTFA Phase 1: MAS range Corneal opacity 0.03/0.03 10 Hydro-alcoholic personal care 0-50 Corneal area 0.03/0.02 prototype formulations SIRC LC50 range Iris 0.16/0.04 (/~g/ml) = 11-21,800 Conjunctival redness -0.06/-0.24 Chemosis - 0.05/- 0.04 Discharge --0.23/0.18 MAS - 0.09/- 0.01 Days to clear -0.08 *Correlation values for in vivo scores I hr post dosing/correlation values for in vivo scores 24 hr post dosing. P u r p o s e a n d p r o p o s e d use o f test. The assay is used to verify or identify irritation potential o f finished products. Each assay is conducted with a p p r o p r i a t e in-house controls representing the p r o d u c t class a n d the p r o d u c t f o r m u l a t i o n for which historical Draize in vivo data are available. S I R C LC50 a n d in vivo (usually M A S ) control test results are compared. Test material LC50 results are evaluated with respect to the product control LC50s. Should the p r o t o t y p e f o r m u l a t i o n prove to be more cytotoxic t h a n controls, the f o r m u l a t i o n will be labelled accordingly, or the ingredient or ingredients causing the increased cytotoxicity will be identified t h r o u g h additional in vitro testing a n d the formula modified to reduce the cytotoxicity. Use in risk a s s e s s m e n t . The submitters provided the following overview for the use o f in vitro data in p r o d u c t risk assessment. This a p p r o a c h is similar to t h a t proposed by several submitters (see, for example, Bruner et al., 1991). P r o d u c t safety, including ocular irritation, begins with c o m p u t e r literature searches a n d supplier data o n individual ingredients. In-house historical animal test data o n similar products are also reviewed. Promising ingredients or formulations m a y then be submitted to in vitro screening. In vitro tests include p r o d u c t controls to evaluate ocular irritation potential. P r o d u c t labels are carefully constructed based o n the test results. Once the p r o d u c t is in the marketplace, complaints o f any adverse effects are closely monitored.

B. D a t a on S I R C C e l l T o x i c i t y A s s a y

Tables 1-3 summarize the linear correlation between the in vivo tissue scores a n d the in vitro scores from the SIRC Cytotoxicity Assay submitted by l a b o r a t o r y CCI00. These studies were performed as part o f the C T F A Phase III, II a n d I studies. Figures 1-3 show the semi-log plots of the Draize M A S against the in vitro scores for these studies. Table 4 summarizes the linear correlation between the in vivo tissue scores a n d the in vitro scores from the S I R C Cytotoxicity Assay performed as part o f the S D A Phase III study a n d submitted by l a b o r a t o r y CC100. Table 5 shows the specific materials tested, their Draize M A S a n d in vitro scores.

C. In vivo database summary C T F A in vivo data sets: the study was a primary eye irritation study in rabbits as conducted by the C T F A E v a l u a t i o n o f Alternatives Program. T h e complete protocol has been published (Gettings et al., 1991). Six animals were treated with each test material. Anaesthetics were used. All in vivo protocols were essentially the same except for animal assignment. The r a n d o m block design was not used in Phase I. All in vivo studies were G L P studies. SDS in vivo data set: the s t a n d a r d Draize protocol was used with six animals per test material.

Table 2. Summary of the correlation between the in vivo and in vitro values for the SIRC cytotoxicity assay in the CTFA Phase II study Laboratory: CCI00 Exposure: 1 hr In vivo/in vitro correlation

(semi-log p l o t ) Number of materials 21

Material type or class CTFA Phase I1: Oil-in-water emulsion personal care prototype formulations

Range of scores MAS range 0-35 SIRC LC50 range 0tg/ml) = 632-24,100

Tissue Corneal opacity Corneal area Iris Conjunctival redness Chemosis Discharge MAS D a y s t o clear

r= * -0.49/-0.49 -0.50/-0.64 -0.46/-0.08 -0.51/-0.42 - 0.59/- 0.54 - 0 . 5 3 / - - 0.60 -0.59/-0.49 -0.64

*Correlation values for in vivo scores 1 hr post dosing/correlation values for in vivo scores 24 hr post dosing.

Working Group 4: Cell cytotoxicity assays

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Table 3. Summary of the correlation between the in vivo and in vitro values for the SIRC cytotoxicity assay in the CTFA Phase III study Laboratory: CC100 Exposure: I hr In

vivo/in

vitro

correlation

(semi-log plot) Number of Material type materials or class 25 CTFA Phase Ill: Surfactant-based personal care prototype formulations

Range of scores MAS range 0-40 SIRC LC50 range (/~g/ml) = 7-2153

O b s e r v a t i o n s were m a d e at 1, 24, 48 a n d 72 hr. As required, animals were m a i n t a i n e d up to 35 days. C o m p a r i s o n s were based o n scores o b t a i n e d at >~24hr after dosing. Toxicity range was well b a l a n c e d with approximately h a l f of the M A S scores between 1 a n d 20 (full range 1--41). Studies were c o n d u c t e d o n coded c o m p o u n d s ( b o t h in vivo a n d in vitro). In vivo studies were conducted u n d e r the principles of G L P s

D. Overall analysis of data P e a r s o n ' s correlations were conducted on each of the d a t a sets provided. Semi-log plots o f Draize score v. S I R C log LC50 in/~g/ml were conducted. The log o f the in vitro score was plotted against the in vivo scores. S I R C results were plotted as logs because of the b r o a d range of results. Semi-log plots provided better Pearson's correlation t h a n linear plots. T h e correlation is presented o n each plot (see s u m m a r y tables above).

Tissue Corneal opacity Corneal area Iris Conjunctival redness Chemosis Discharge MAS Days to clear

r= -0.70 -0.78 -0.72 - 0.78 - 0.75 - 0,72 -0.77 -0.75

C o n c u r r e n t positive control data were available for the SDS study a n d the C T F A Phase II a n d III studies. In the SDS study, the LC50 for SLS was 31.5 ___4.2 (n = 26) a n d in the C T F A Phase II study, it was 32.2 + 9.7 (n = 48). F o r the C T F A Phase III study, work was done in a different laboratory. The positive control, SLS, was tested in parallel with each trial o f each test material. The m e a n was 24.4 + 4.15 (n = 149). The acceptance criteria for the assay required that the SLS value be within two s t a n d a r d deviations o f the m e a n for the historical control.

E. Conclusions and recommendations The S I R C cell assay was designed to use a c o n t i n u o u s cell line derived from the r a b b i t cornea to predict the potential for a test material to cause ocular irritation. Studies using this assay r a n k e d surfactants a n d s h a m p o o formulations by the c o n c e n t r a t i o n of test material t h a t resulted in 50% cell death. In previously

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.

10oo

e

10000

.

.

.

.

.

.

.

.

IOG~GO

LC(r,o)(ugant)

Fig. 2. Semi-log plot of the Draize MAS (24 hr) values v. the SIRC cell cytotoxicity assay LCS0s from the CTFA Phase II study. The X-axis bars show one standard error of the mean rabbit scores; the Y-axis bars show one standard deviation of the mean for the multiple in vitro trials.

published studies, the rankings compared favourably with the in vivo irritation potential rankings resulting from modified Draize studies in rabbits ( N o r t h - R o o t et al., 1982 and 1985). These previous studies have provided CC100 with data to support the use of the S I R C cell toxicity test in the routine screening of its proprietary formulations. With these studies in mind, CC100 participated in the C T F A Evaluation of Alternatives Program in which three phases were conducted: Phase I (hydro-alcoholic products); Phase II (emul-

40

sion products); and Phase III (surfactants). The findings from the C T F A study of the S I R C cell toxicity test showed the importance of differentiating between water-soluble and non-water-soluble materials when submerged culture systems are used to predict ocular irritation potential. The truly lipophilic formulations tested (Phase II) showed poor correlations between the in vitro results and the in vivo Draize scores. In Phase I, the hydro-alcoholic formulations, which were described as dispersions and some of which were water-soluble, also showed

_0 ~OT 0 r =-0,7'/

3O

I

cO 5<

2O

tT 1

I 10-

1. •

1

10

100

1000

t~e

10000

100000

Fig. 3. Semi-log plot of the Draize MAS (24 hr) values v. the SIRC cell cytotoxicity assay LCS0s from the CTFA Phase I study. The X-axis bars show one standard error of the mean rabbit scores; the Y-axis bars show one standard deviation of the mean for the multiple in vitro trials.

Working Group 4: Cell cytotoxicity assays

87

Table 4. Summary of the correlation between the in vivo and in vitro values for the SIRC cytotoxicity assay in the soap and detergent association Phase I!I Study Laboratory: CCI00 Exposure: I hr In vivo/in vitro

correlation (semi-log plot) Number of materials 22 16 6

Material type or class Total Neat materials generally surfactants Formulations

Range of scores MAS range 1-41 SIRC LC50 range (/zg/ml) 25.6- > 500,000

Tissue

r= *

Corneal opacity Corneal area Iris Conjunctival redness Chemosis Discharge MAS Days to clear

-0.40/-0.60 NP'[" - 0.46/- 0.56 -0.80/-0.89 -0.55/-0.67 NP -0.63/-0.79 -0.30/-0.49

*The 22 test materials contained several materials with appreciable titratable alkalinity (/>0.1 meq HCI/g); one sample was 5.25% sodium hypochlorite. The analysis of the correlation data was performed on the complete set of 21 materials (ul9percorrelation number) and on only the neutral test materials (and without sodium hypochlorite) (lower number) (e.l;. MASr = -0.63 complete v. -0.79 restricted). The toxicity of the alkaline materials and hypochlorite was appreciably under-predicted in vitro. i'Not provided.

poor overall correlation with the in vivo irritation values. Similar results were reported with most of the s u b m e r g e d m o n o l a y , e r cell c u l t u r e s y s t e m s u s e d to test

the lipophilic formulations in the Phase II trials. The Phase III trials consisted of surfactant formulations, and all materials were water-soluble. This last group gave the best correlations between the in vitro data and the in vivo irritation scores. Correlations ranged from r = 0.70 to r = 0.78, with redness and area scores giving the better correlations. The SDA data set showed the difficulties inherent in testing materials with significant reserve alkalinity or high reactivity (ihypochlorite) but supported the use of the assay for more neutral surfactant-based materials. Because the SIRC method has been published (with satisfactory peer review of the method and its results with shampoos and surfactants), these

additional CTFA results provide further evidence for selective use of the test in evaluating certain formulations. It is clear that water-insoluble products cannot be adequately evaluated for potential ocular irritation and toxicity by the SIRC cell toxicity assay; too many problems are associated with access of the test material to the biological model system. However, if water solubility of the test formulations was good, the SIRC cell toxicity assay provided good correlations to the different in vivo irritation tests. In conclusion, the SIRC cell toxicity assay can serve as a g o o d in v i t r o screening m e t h o d for assessing the

potential

formulations.

ocular

irritation

However,

because

of

water-soluble

of

the

product

types are

needed.

The

Working

Identity of sample Ethanol, 15% Polyethylene glycol, 400 mw, neat Isopropa nol, 15% Butoxyethanoi, 5% Glycerol, neat Alcohol ethoxylate, 10% Dodecyla mine oxide, 1% Dimethylditallowammonium chloride, 3% Alkylethoxysulfate, 10% Triethylammonium lauryl sulfate, 10% Sodium lauryl sulfate, 10% Ammoni am lauryl sulfate, 10% Alkylglyceryl sulfate, 10% Sodium perborate Sodium alkyl sulfate, 10% Sodium hypochlorite, 5.25% Liquid cleaner Powdered cleaner Liquid hand soap Powdered cleaner Powdered bleach with protease Pine oil q;leaner

Group

r e c o m m e n d s t h a t in view o f the c o n s i d e r a b l e p r o m i s e

Table 5. SIRC cytotoxicity assay from the Soap and Detergent Association Phase Ill study: Laboratory: CC100 Physical form

limited

n u m b e r o f c o m p a n i e s u s i n g the a s s a y o n a r o u t i n e basis, m o r e d a t a o n the suitability o f the test for o t h e r

Alkalinity (meq HCI/g)

Maximum averageDraize score

assay score (,ug/ml)

In vitro

Liquid Liquid Liquid Liquid Liquid Liquid Liquid

0.0 0.0 0.0 0.0 0.0 0.0 0.0

1.0 2.0 2.3 2.7 13.8 14.7 14.8

>500,000 148,300 462,600 196,100 233,000 25.6 5465

Liquid Liquid Liquid Liquid Liquid Liquid Solid Liquid Liquid Liquid Solid Liquid Solid Solid Liquid

0.0 0.004 0.0 0.0 0.0 0.0 5.36 0.0 0.0 0.044 0.118 0.0 0.816 4.7 0.0

18.8 18.8 25.7 27.0 32.7 34.3 34.3 36.2 41.0 15.8 17.3 24.5 34.3 34.3 41.3

755 200 259.6 123.6 151.0 268.5 4922 182.1 1632 434.9 29.6 149.6 256.3 2275 178.2

88

J.W. Harbell et al.

it shows, the SIRC Cytotoxicity Assay warrants additional research. NEUTRAL RED ASSAYS

12 data sets using the quantitative neutral red endpoint were submitted. To facilitate the review, the data sets were divided into three sets, depending on the exposure time of the assay: 48 hr, 24 hr and ~<30 min. The neutral red assay has been one of the most extensively used assays. These submissions may comprise only a fraction of the data available but are probably representative of how the assay has been used in the cosmetic, personal care and household products industries for evaluating raw materials and formulations. Neutral Red Assay (48-hr exposure)

A. In vitro assay. Neutral Red Assay using a 48-hr exposure time Summary. Eight data sets are included in this review. Most of the materials tested were surfactants or surfactant-based products. The standard Draize assay was used for seven data sets and the low-volume eye test was used for one data set. Full tissue scores were available for five of the eight studies. Six of the studies used the normal human epidermal keratinocyte (NHEK) as the target cell and followed the standard protocol proposed by the Clonetics Corporation. The other two studies followed a similar protocol but used either normal rabbit corneal epithelial (NRCE) cells (in serum-free medium) or SIRC cells (in serum-containing medium). Basis of assay. The assay is designed around short-term monolayer cultures, often using normal human cells as the target and cytotoxicity as the endpoint. The procedure is based on the work of Guess, Borenfreund and their respective collaborators (see also Babich and Borenfreund, 1990). Neutral red, the marker of cell viability, has been shown to be selectively retained by the lysosomes of living cells because of the differential pH between the inside of the lysosome and the surrounding cytoplasm. The amount of neutral red taken up by the population of keratinocytes is directly proportional to the number of viable cells in the culture (Barstad et al., 1991). The test material-induced cytotoxicity (and cytostasis) is measured over a wide range of concentrations, and the concentration yielding a 50% reduction in neutral red uptake is used as the measure for comparison between test materials. The test is based on the observation that some materials which are damaging to the eye appear to be cytotoxic to a number of cell types (e.g. corneal epithelium, corneal endothelium, conjunctival epithelium, conjunctival endothelium). This toxicity may be manifest in actual cell killing (loss of

epithelium) and/or less severe (and obvious) toxicity and release of inflammatory mediators which these cells are believed to produce. Surface-active agents would be expected to be cytolytic by disrupting cell membranes. N H E K cells are selected for this assay because of their similarity to the corneal and conjunctival epithelial cells. Protocol. The Keratinocyte Neutral Red Bioassay, used in most of the studies presented here, has been adapted from the protocol of Borenfreund and Puerner (1984). The general protocol is summarized as follows: secondary cultures of proliferating normal human epidermal keratinocytes (NHEK) were harvested (when 50-80% confluent) by trypsinization. The single cell suspensions were centrifuged and then resuspended in Keratinocyte Growth Medium (KGM). The cells were plated at 2500 cells/well (total volume of 250 #l) into each well of a 96-well tissue culture plate. The plates were incubated for 3 days at 37°C (5% CO2/humidified atmosphere). Depleted K G M was removed from the wells by careful aspiration; then 250 #1 fresh K G M was added to control "untreated" wells and 250#1 of various concentrations of test agents diluted in K G M were added to test wells. Replicate wells (generally four) were used for each dilution and for the untreated (or solvent) control. The plates were incubated for 48 hr at 37°C. The spent medium/test agent solutions in all wells except the blanks were removed and replaced with K G M supplemented with neutral red dye (final concentration of 50 #g/ml in KGM); 250 #1 portions of K G M (without dye) were added to the blanks. The plates were then incubated for 3 hr at 37°C. Each well then received 250 #1 of wash/fix solution (an aqueous 1% formaldehyde-1% calcium chloride solution) for 2 min (room temperature). The wash/fix solution was decanted and each well received 100 #1 of a solvent solution (1% glacial acetic acid-50% ethanol) for 20 min at room temperature. Absorbances were measured at 540 or 550 nm in a microplate reader (after the appropriate blank correction) and "percentage of untreated control" values were calculated for each dilution of test agent. The "percentage of untreated control" values v. the test agent concentrations were plotted (semi-log plot) and the concentration of test material resulting in a 50% inhibition of neutral red uptake (NRUs0) was determined by extrapolation. This is the "standard" protocol developed by Clonetics Corporation for N H E K cells. For testing with SIRC or NRCE cells, similar procedures were followed except that the medium was changed to reflect the cell type. Serum was used in the medium for the SIRC cells; all of the other assays were performed without serum. Proposed use. The uses of the assay depend on the product line and the company. The majority of submissions came from trade associations and raw material suppliers that are not manufacturing products for direct commercial use. The manufacturer of consumer and household products who

Working G r o u p 4: Cell cytotoxicity assays

89

Table 6. Summary of the correlation between the in vivo and in vitro values for the neutral red uptake assay in the SDS Phase Ill study Laboratory: (CCI01) Exposure: 48 hr

In vivo/in vitro correlation (semi-log plot) Number of materials

Material type or class

22

Total

16

Neat materials generally surfactants Formulations

6

Range of scores

Tissue

MAS range =1-41 NR50 range (/~g/ml) 7-> 150,000

r= *

Corneal opacity

-0.31/-0.61

Corneal area Iris Conjunctival redness Chemosis Discharge MAS Days to clear

NPt -0.41/-0.56 -0.76/-0.91 - 0.46/- 0.65 NP -0.59/-0.82 -0.20/-0.46

*The 22 test materials contained several materials with appreciable titratable alkalinity (/> 0.1 meq HC1/g); one sample was 5.25% sodium hypoehlorite. The analysis of the correlation data was performed on the complete set of 21 materials (upper correlation number) and on only the neutral test materials (and without sodium hypochlorite) (lower number) (e.g. MAS r = -0.59 complete v. -0.82 restricted). The toxicity of the alkaline materials and hypochlorite was appreciably underpredicted in vitro. tNot provided.

s u b m i t t e d d a t a u s e s t h o s e d a t a as p a r t o f a tiered t e s t i n g p r o g r a m . F o r s o m e m a n u f a c t u r e r s o f pers o n a l - c a r e p r o d u c t s , t h e a s s a y h a s b e e n u s e d to select f o r m u l a t i o n s for d e v e l o p m e n t a n d to q u a l i f y final products for use by/on a human panelist (but p r o b a b l y n o t direct i n s t i l l a t i o n i n t o t h e eye). T h e m a j o r i t y o f u s e r s su~ggest t h a t this a s s a y be u s e d in d e v e l o p m e n t ( s c r e e n i n g ) a n d as a n a d j u n c t a s s a y w i t h o t h e r tests. I n s o m e cases, t h e s e d a t a h a v e b e e n u s e d in final s a f e t y assess~naent. Range o f responses covered. See below.

Range o f test materials amenable f o r assessment in the assay. B a s e d o n t h e s e s u b m i s s i o n s a n d o t h e r d a t a a v a i l a b l e in t h e l i t e r a t u r e (see C T F A P h a s e II trials), t h e s u b m e r g e d cell m o n o l a y e r - b a s e d c y t o t o x i c i t y assays are appropriate for water-soluble or -miscible materials. Water-insoluble materials, particularly

t h o s e less d e n s e t h a n w a t e r , h a v e b e e n s h o w n to p e r f o r m p o o r l y in t h e s e a s s a y s . I n a d d i t i o n , m a t e r i a l s w i t h a p p r e c i a b l y h i g h o r l o w p H v a l u e s a r e likely to be less t o x i c in t h e c u l t u r e s y s t e m s b e c a u s e o f t h e buffering capacity of the culture medium. Use in risk assessment. See t h e d i s c u s s i o n u n d e r S I R C Cell C y t o t o x i c i t y .

B. Summary data tables Soap and Detergent Association, Phase I I I (CC101). T a b l e 6 s u m m a r i z e s t h e l i n e a r c o r r e l a t i o n b e t w e e n t h e in vivo t i s s u e s c o r e s a n d t h e in vitro s c o r e s f r o m t h e 4 8 - h r n e u t r a l red u p t a k e a s s a y p e r f o r m e d as p a r t o f t h e S o a p a n d D e t e r g e n t A s s o c i a t i o n P h a s e III study and submitted by laboratory CC101. Table 7 s h o w s t h e specific m a t e r i a l s tested, t h e i r D r a i z e M A S a n d in vitro scores.

Table 7. Neutral red uptake assay (48 hr) from the Soap and Detergent Association Phase llI study: Laboratory: CCI01 Maximum In vitro Physical Alkalinity average Draize assay score Identity oJ" sample form (meq HCI/g) score (pg/ml) Ethanol, 15% Polyethylene glycol, 400 mw, neat lsopropanol, 15% Butoxyethanol, 5% Glycerol, neat Alcohol ethoxylate, 10% Dodecylaraine oxide, 1% Dimethyld itallowammonium chloride, 3% AIkylethoxysulfate, 10% Triethylammonium lauryl sulfate, 10% Sodium lauryl sulfate, 10% Ammonium lauryl sulfate, 10% Alkylglyceryl sulfate, 10% Sodium pc,'rborate Sodium alkyl sulfate, 10% Sodium h,.zpochlorite, 5.25% Liquid cleaner Powdered cleaner Liquid haod soap Powdered cleaner Powdered bleach with protease Pine oil cleaner

Liquid Liquid Liquid Liquid Liquid Liquid Liquid

0.0 0.0 0.0 0.0 0.0 0.0 0.0

1.0 2.0 2.3 2.7 13.8 14.7 14.8

> 150,000 67,590 > 150,000 94,850 120,300 29 39

Liquid Liquid Liquid Liquid Liquid Liquid Solid Liquid Liquid Liquid Solid Liquid Solid Solid Liquid

0.0 0.004 0.0 0.0 0.0 0.0 5.36 0.0 0.0 0.044 0.118 0.0 0.816 4.7 0.0

18.8 18.8 25.7 27.0 32.7 34.3 34.3 36.2 41.0 15.8 17.3 24.5 34.3 34.3 41.3

61 27 25 12 22 9 2915 7 1066 63 9 21 39 3029 38

J. W. Harbell et al.

90

Table 8. Summary of the correlation between the in vivo and in vitro values for the neutral red uptake assay in the SDS Phase ii! study Laboratory: CC102 Exposure: 48 hr

In vivo/in vitro correlation (semi-log plot) Number of materials

Material type or class

22

Total

16

Neat materials generally surfactants Formulations

6

Range of scores

Tissue

MAS range 1-41 NR50 range (pg/ml) 4.4-450,000

r= *

Corneal opacity

-0.38/-0.67

Corneal area Iris Conjunctival redness Chemosis Discharge MAS Days to clear

NP't - 0.44/- 0.60 -0.79/-0.92 -0.51/-0.69 NP - 0.63/-0.87 -0.28/-0.55

*The 22 test materials contained several materials with appreciable titratable alkalinity (>t 0.1 meq HCI/g); one sample was 5.25% sodium hypochlorite. The analysis of the correlation data was performed on the complete set of 21 materials (upper correlation number) and only on the neutral test materials (and without sodium hypochlorite) (lower number) (e.g. MAS r = -0.63 complete v. -0.87 restricted). The toxicity of the alkaline materials and hypochlorite was appreciably under-predicted in vitro. tNot provided.

In vivo d a t a set: t h e s t a n d a r d D r a i z e p r o t o c o l w a s u s e d w i t h six a n i m a l s p e r test m a t e r i a l . O b s e r v a t i o n s were m a d e at 1, 24, 48 a n d 72 hr. A s r e q u i r e d , a n i m a l s were m a i n t a i n e d u p to 35 d a y s . C o m p a r i s o n s were b a s e d o n s c o r e s o b t a i n e d at >/24 h r a f t e r d o s i n g . T o x i c i t y r a n g e w a s well b a l a n c e d , w i t h approximately half of the MAS scores between 1 and 20 (full r a n g e ! - 4 1 ) . S t u d i e s were c o n d u c t e d o n c o d e d c o m p o u n d s ( b o t h in vivo a n d in vitro). In vivo s t u d i e s were c o n d u c t e d u n d e r t h e p r i n c i p l e s o f G L P . In vitro d a t a set: t h e in vitro r e s p o n s e s t e n d e d to be p o l a r i z e d . T h e v e r y m i l d m a t e r i a l s ( M A S 1-13) yielded N R U s 0 v a l u e s o f ~> 6 7 , 5 9 0 / ~ g / m l ; m a t e r i a l s t h a t were n o t m u c h m o r e t o x i c ( M A S 15-20) s h o w e d v a l u e s o f a b o u t 30 p g / m l . T h e best c o r r e l a t i o n w a s o b t a i n e d w i t h c o n j u n c t i v a l r e d n e s s ; in t h a t case, t h e in vitro v a l u e s were well d i s t r i b u t e d relative to t h e

in vivo r e s p o n s e s . A l k a l i n e a n d h y p o c h l o r i t e m a t e r i a l s were u n d e r - p r e d i c t e d . N o d i s c u s s i o n o f c o n c u r r e n t p o s i t i v e c o n t r o l s o r a c c e p t a n c e criteria were p r o vided. Soap and Detergent Association, Phase III (CC102). T a b l e 8 s u m m a r i z e s t h e l i n e a r c o r r e l a t i o n b e t w e e n t h e in vivo t i s s u e s c o r e s a n d t h e in vitro s c o r e s f r o m t h e 4 8 - h r n e u t r a l red u p t a k e a s s a y p e r f o r m e d as p a r t o f t h e S o a p a n d D e t e r g e n t A s s o c i a t i o n P h a s e III study and submitted by laboratory CC102. Table 9 s h o w s t h e specific m a t e r i a l s tested, t h e i r D r a i z e M A S a n d in vitro scores. In vivo d a t a set: t h e s t a n d a r d D r a i z e p r o t o c o l w a s u s e d w i t h six a n i m a l s p e r test m a t e r i a l . O b s e r v a t i o n s were m a d e at 1, 24, 48 a n d 72 hr. A s r e q u i r e d , a n i m a l s were m a i n t a i n e d u p to 35 d a y s . C o m p a r i s o n s were b a s e d o n s c o r e s o b t a i n e d a t >124 h r a f t e r

Table 9. Neutral red uptake assay (48 hr) from the Soap and Detergent Association Phase Ill study: Laboratory: CCI02

Identity of sample Ethanol, 15% Polyethylene glycol, 400 mw, neat Isopropanol, 15% Butoxyethanol, 5% Glycerol, neat Alcohol ethoxylate, 10% Dodecylamine oxide, 1% Dimethylditallowammonium chloride, 3% Alkylethoxysulfate, 10% Triethylammonium lauryl sulfate, 10% Sodium lauryl sulfate, 10% Ammonium lauryl sulfate, 10% Alkylglyceryl sulfate, 10% Sodium perborate Sodium alkyl sulfate, 10% Sodium hypochlorite, 5.25% Liquid cleaner Powdered cleaner Liquid hand soap Powdered cleaner Powdered bleach with protease Pine oil cleaner

Physical form

Alkalinity (meq HCI/g)

Maximum average Draize score

In vitro assay score (,ug/ml) 450,000 6200 190,000 155,000 66,000 67 45

Liquid Liquid Liquid Liquid Liquid Liquid Liquid

0.0 0.0 0.0 0.0 0.0 0.0 0.0

1.0 2.0 2.3 2.7 13.8 14.7 14.8

Liquid Liquid Liquid Liquid Liquid Liquid Solid Liquid Liquid Liquid Solid Liquid Solid Solid Liquid

0.0 0.004 0.0 0.0 0.0 0.0 5.36 0.0 0.0 0.044 0.118 0.0 0.816 4.7 0.0

18.8 18.8 25.7 27.0 32.7 34.3 34.3 36.2 41.0 15.8 17.3 24.5 34.3 34.3 41.3

62 11.5 20 17 21 7.4 1700 5.2 2200 68 4.4 13 34 1750 4.9

Working Group 4: Cell cytotoxicity assays

91

Table 10. Summary of the correlation between the in vivo and in vitro values for the neutral red uptake study conducted on surfactant compounds L.'Lboratory: CC103 Exposure: 48 hr In vivo/in vitro correlation

Number of materials 9

(semi-log plot) Range of scores Tissue MAS range 3--22 Corneal opacity NRU50 range Corneal area (,ug/ml) = 4-306 Iris Conjunctival redness Chemosis Discharge MAS Days to clear

Material type or class Pure surfactants

•= * -0.79 -0.65 -0.82 - 0.94 - 0.93 - 0.82 -0.92 NP*

*Not provided.

dosing. Toxicity range was well balanced, with approximately half c f the M A S scores between 1 a n d 20 (full range 1-41). Studies were conducted on coded c o m p o u n d s ( b o t h in vivo a n d in vitro). In vivo studies were c o n d u c t e d u n d e r principles o f G L P . In vitro d a t a set: tile in vitro responses tended to be polarized. T h e very mild materials ( M A S 1-13) yielded NRUs0 values of /> 67,590 #g/ml; materials t h a t were not m u c h more toxic ( M A S 15-20) showed values o f a b o u t 60 tlg/ml. The best correlation was o b t a i n e d with conju~actival redness; in t h a t case, the in vitro values were well distributed relative to the in vivo responses. Alkaline a n d hypochlorite materials were underpredicted. N o discussion of c o n c u r r e n t positive controls or acceptance criteria were provided. A c o m p a r i s o n between the two laboratories performing the neutral red assay on these materials (CCI01 a n d CC102) showed a n i n t e r l a b o r a t o r y correlation of 0.99 (see also Triglia et al., 1989).

Surfactants (CCI03). Table 10 summarizes the linear correlation between the in vivo tissue scores a n d the in vitro scores from the 48-hr neutral red u p t a k e assay submitted by l a b o r a t o r y C C 103. This study was performed o n a series of individual surfactants. Figure 4 shows the semi-log plot o f the Draize M A S against the in vitro scores for this study. In vivo d a t a set: The Draize assay was performed o n 5 % solutions o f neat surfactants with four animals per test group. Two materials showed low M A S scores (3); the remainder were grouped between 10 a n d 22. Specific surfactants tested were given by name, m a k i n g this a very useful d a t a set for future investigations. Use o f sample coding or G L P s was not discussed. In vitro data set: the data set showed a range of neutral red 50% values between 300 a n d 3/~g/ml with the majority between 20 a n d 3. These values reflect the distribution o f the in vivo toxicities. Values are the means of at least two trials. C o n c u r r e n t positive

30-

1

W

TI

T

20-

@ .L

i

r=-O.~

T

10-

0

°

-

I

.

.

.

.

.

.

.

i

.

.

.

.

10

.

.

.

i

100

.

.

.

.

.

.

.

.

1000

NRU(~l)(ug/ml)

Fig. 4. Semi-log plot of the Draize MAS (24 hr) values v. the neutral red uptake assay (48 hr) NRU50s for a series of :;urfactants. The X-axis bars show one standard error of the mean rabbit scores; the Y-axis bars show one standard deviation of the mean for the multiple in vitro trials.

92

J.W. Harbell et al. Table I I. Summary of the correlation betweenthe in vivoand in vitro values for the neutral red uptake assays conducted in the CTFA Phase Ill study Laboratory: CC104 Exposure: 48 hr In vivo/in vitro correlation (semi-log plot) Number of Materialtype Range of materials or class scores Tissue r= 25 Surfactants-based MAS range Corneal opacity -0.59 formulations = 0-40 NRU50 range Cornealarea -0.74 (pg/ml) = 7-800 Iris -0.68 Conjunctival redness -0.72 Chemosis -0.67 Discharge - 0.70 MAS -0.73 Days to clear -0.77

controls (sodium lauryl sulfate) and negative controls were performed with each assay. Acceptance criteria for a given trial were based on the positive control NRUs0 value. An assay was acceptable if the NRUs0 value was within two standard deviations of the historical mean (4.3 _ 0.87), so that the value would fall between 2.63 and 5.97 #g/ml. Samples were tested under code. The spirit of G L P was used for all in vitro testing. C T F A Phase III (CC104). Table 11 summarizes the linear correlation between the in vivo tissue scores and the in vitro scores from the 48-hr neutral red uptake assay submitted by laboratory CC 104. This study was performed as part of the C T F A Phase III study on surfactant-containing formulations. Figure 5 shows the semi-log plot of the Draize M A S v. the in vitro scores for this study. In vivo data set: the standard Draize assay was used with six rabbits per test material. Anaesthetic was used. Testing was performed under code and with full GLPs. The in vivo M A S scores were distributed

over the whole range (0-10, six materials; 11-20, seven materials; 21-30, four materials; 31--40, eight materials). All the materials were surfactantbased formulations (!1 were shampoos). The standard deviations for the six animals were quite large--in the mid-range of toxicity (average CV approx. 40%). This CV value is consistent with other published data and is not a negative reflection on the laboratory performing these tests. The variability in the in vivo responses should be considered in comparing the in vivo results with the in vitro data. As previously indicated, the standard error of the in vivo scores, rather than the standard deviation, has been used in plots of the data to better approximate the error expected in multiple independent trials. However, since the standard of practice in toxicology has been to use only one in vivo trial, the standard deviation remains an important indicator of variability in the in vivo standard against which the in vitro assays are judged.

40

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Working Group 4: Cell cytotoxicity assays

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Table 12. Summaryof the correlationbetweenthe in vivo and in vitro valuesfor the neutral red uptake assays conducted on a range of formulations Laboratory: CC105 Exposure: 48 hr In vivo/in vitro correlation (semi-log plot) Number of Material type Range of materials or class scores Tissue r= 45 10 classes of generally MAS range Cornealopacity -0.42 water-soluble products; 0-44 (LVET) Corneal area -0.40 most contained some Iris -0.57 surfactant Conjunctival redness -0.52 Chemosis - 0.47 Discharge - 0.37 MAS - 0.54 Days to clear -0.29

In vitro data set: ,in vitro data were generated by using the standard neutral red protocol. Three independent NRUs0 determinations were made. The mean NRU~o values ranged from 9 to 761 #g/ml. The average CV was 25% for the 25 materials tested. Concurrent positive controls (sodium lauryl sulfate) and negative controls were performed with each assay. Acceptance criteria for a given trial were based on the positive control NRUs0 value. An assay was acceptable if the NRUs0 value was within two standard deviations of the historical mean (4.3 _ 0.87; n = 141 trials over 2 yr). Therefore the value would fall between 2.63 and 5.97#g/ml. Samples were tested under code. The spirit of G L P was used for all in vitro testing. Surfactant formuhztions (CC105). Table 12 summarizes the linear correlation between the in vivo tissue scores and the in vitro scores from the 48-hr neutral red uptake assay submitted by laboratory CC105. This series of studies was performed on a variety of surfactant-.containing formulations. Figure

6 shows the semi-log plot of the Draize M A S v. the in vitro scores for this study. In vivo data set: this was a large data set containing 10 classes of formulations (personal care and household) and some individual surfactants. The largest classes were hard-surface cleaners (7) and shampoos (10). The low-volume eye test (LVET) was used. This assay differs from the standard Draize assay in that only 10-/~1 or 10-mg portions are used and they are applied directly to the cornea. Scoring is performed in the normal manner. All tissue scores were provided, and assays were performed under full GLPs. The physical forms tested were both solids (e.g. granular laundry powder) and liquids. M A S scores were balanced over the range of toxicities. In vitro data set: the standard neutral red protocol was used to generate in vitro data. Three independent NRUs0 determinations were made. The mean NRUs0 values ranged from 0.34 to 2000/~g/ml and were balanced over the range of toxicities. Concurrent positive controls (sodium lauryl sulfate) and negative

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94

J.W. Harbell et al. Table 13. Summary of the correlation between the in vivo and in vitro values for the neutral red uptake assays conducted with three different cell types on surfactants Laboratory: CC106 In vivo/in vitro correlation

Number of materials 17

Material type or class Named surfactants

Range of scores

Log:log plot

In vh,o

In vivo dose giving MAS

dose response

of 20 v. NR50: Neutral red assay with N H E K cells Neutral red assay with normal rabbit corneal epithelial cells Neutral red assay with SIRC cells

controls were performed with each assay. Acceptance criteria for a given trial were based on the positive control NRUs0 value. An assay was acceptable if the NRUs0 value was within two standard deviations of the historical mean (4.3 +0.87; n = 141 trials). Therefore the value would fall between 2.63 and 5.97 pg/ml. Samples were tested under code, and full GLP was used for all in vitro testing. Surfactants (CCI06). Table 13 summarizes the linear correlation between the in vivo tissue scores and the in vitro scores from the neutral red uptake assays submitted by laboratory CC106. These studies were performed on individual surfactant materials with three different cell types used as target cells in the assay. In vivo data set: an in vivo dose-response study was performed with at least four doses of each surfactant. Three to six rabbits were used for each dose (100 #1 or 100 mg per rabbit). Eyes were scored at 1, 3, 6, 24, 96 and 168 hr after dosing. The arithmetic mean Draize score, calculated from individual scores of three to six rabbits at six different exposure times, was plotted v. each chemical concentration tested to obtain a dose-response plot. The Draize score equal to 20 units from a possible total of 110 was selected as a standard measure (DS20) for calculating comparative potency. Values for the DS20 ranged from 1.5 to 50% (w/w) for the 17 materials. Mild to severe scores ranged from 14 to 1.5%. The data set was balanced with similar numbers of non-toxic, mild, moderate and severe materials. Although only DS20 scores were given, the in vivo data are valuable because the specific surfactant names were provided and because this type of experiment would not be possible today. Some of the dose-response curves and additional in vivo data have been published (Watanabe et al., 1989). In vitro data sets: all three studies used the same basic protocol except that the medium was changed to match the target cells. The NHEK and NRCE cells were grown and tested in serum-free medium; the SIRC cells were grown and tested in Ham's medium containing 10% serum. Three trials were performed for each assay. SLS was used as the positive control, although specific acceptance criteria or normal ranges were not addressed. NRUs0 values for the SIRC cells

0.72 0.65 0.69

(in serum-containing medium) were generally higher than those for the other two cell types. NHEK cell values ranged from 0.5 to 393/~g/ml, NRCE cell values ranged from 0.8 to 583 #g/ml and SIRC values ranged from 8.7 to 650/zg/ml. The submitters suggest that serum-free conditions were more sensitive and therefore preferable for this assay (Torishima et al., 1990). They indicate that the assay was capable of discriminating between non-irritants and mild irritants and could be used as a screening test. Data suggest, however, that some irritants were underpredicted; thus differentiation between non-irritating and moderate/severe might be more appropriate. Water-insoluble materials are not appropriate for these assays.

Overall analysis In vivo data

Complete tissue data were presented for 56 materials (neat chemicals and formulations) tested by the standard Draize assay. An additional 17 materials were tested by the standard Draize protocol, but in a dose-response fashion. Complete tissue data were presented for 45 materials (mostly formulations) tested with the LVET protocol. The range of irritancy for the standard protocol studies was 0-40; for the LVET the range was 0-44. Although not all of the assays were conducted under full GLPs, all met acceptance criteria for animal number and scoring. Almost all of the studies showed in vivo responses well distributed over the range of toxicities (balanced). Multiple independent in vivo trials were not available for analysis of in vivo variability. Variability among the animals in a single trial was marked. CVs ranged, conservatively, around 40% for MAS and individual tissue scores. In vitro data

78 data sets were presented against the standard Draize tissue scores, 45 against the LVET, and 51 against the DS20 scores. In general, the range and magnitude of the in vitro scores were proportional to those of the in vivo scores. Some polarization was observed between the extremely mild materials and the remaining materials. Some of the NRU~0 values for the non-toxic materials were a lot greater than

Working Group 4: Cell cytotoxicity assays those of the mild metteriais in a given data set. This spread may have detracted from the numeric correlation coetficients but does not diminish the usefulness of the data. In most cases, in vitro scores were based on replicate determinations (often three). All methods have been published in peer-reviewed journals, and laboratories using the N H E K cells appear to have used essentially the same protocol. Three of the studies were performed in the same laboratory. Interlabc,ratory comparisons between the two laboratories testing the SDA Phase III materials showed a correlatio~ of r = 0.99. Intralaboratory or interassay variability (CV) over an extended period of using SLS as a positive control (2yr) was approximately 20%. Interassay variability (CV) for three assays performed on each of 25 materials (CTFA Phase III) averaged about 25%. The weaker correlations between the NRUs0 and tissue scores from the CC105 data set, relative to the other sets, may reflect several factors independent of the execution of the in vivo or in vitro assay. First, the data set combines assays on l0 classes of materials. Although these classes of materials are generally water-miscible, some of them may not be amenable to testing with this assay system. Data from the C T F A Phase I study on hydroalcoholic preparations illustrate this point. Long-term exposure to the alcohol preparations over-predicted the toxicity of these materials compared with their actual in vivo scores (see also the SIRC assay report). Other exposure times (e.g. short-term) or different in vitro methods may be required for certain of these classes. Second, some materJLals tested as solids in vivo were tested as solutions in vitro. For poorly soluble surfactant products, such as laundry detergents, the solid form is known to be much less toxic than the solution. These data suggest the importance of considering in vivo exposure (e.g. as a function of solubility) in selecting in vitro assays and evaluating the resulting data. Analysis

Most submitters stipulated that test materials must be water-soluble to be tested in this type of assay. Neutral red assay data from the CTFA Phase II study (not submitted) strongly support this conclusion (see also the SIRC assay report). Materials need not necessarily be completely in solution to be tested, but specific data on suspension preparations, distinct from solutions, were not presented for review. Extremes of pH will be muted by the buffering capacity of the medium, and therefore the toxicity of those materials may be under-predicted. In particular, those materials with high acid or alkaline reserve may cause problems in this type of assay. Uniquely (or highly) reactive species, such as hypochlorite, may be under-predicted because they react with culture medium. In general, the best correlations were obtained with conjunctival redness and MAS. Corneal and iris

95

scores showed generally lower correlation in most of the data sets. Days to clear also showed limited correlation with the NRUs0.

Conclusions In general, the data support the use of this assay as a screening and adjunct assay over the range of toxicities found in personal care/household products. It performed very well in separating non-irritating materials from those at the higher end of the in vivo scale. Within the limitations discussed below, the assay shows the potential to identify moderate and severe irritants. Use of the assay should be limited to water-soluble materials for which extremes of pH and reactivity are not potential sources of toxicity. Data support use of this assay for surfactant materials (both neat and formulations) but attention should be paid to evaluating each product class. For example, fabric softeners may not perform well, yet shampoos do. Physical form should be considered in testing. Toxicity of the solution will not necessarily predict the toxicity of the solid.

Neutral Red Assay (24-hr exposure) A. In vitro assay: Neutral Red Assay using a 24-hr exposure time Summary. Data sets for two in vitro assays were submitted by CC107. These sets used essentially the same in vivo data set. Full tissue scores were provided. The first assay, the SIRC neutral red uptake (NRU) cytotoxicity test (Roguet et al., 1992), used the SIRC rabbit corneal cell line; the second assay, the V79 N R U cytotoxicity test, used the V79 Chinese hamster lung fibroblast line (see general discussion, Marinowich et al., 1990; Shadduck et al., 1987). Basis o f assay. Both assays are based on short-term cultures of continuous cell lines with cytotoxicity as the assay endpoint. The neutral red procedure is derived from the work of Guess, Borenfreund and their respective collaborators (see the 48-hr neutral red assay report for more details). The use of the SIRC cell line as a target for ocular irritation studies is based on the work of North-Root et al. (1982). The V79 cells, often used in genetic toxicology studies, are not derived from the eye but do provide targets for generalized cytotoxicity. The test mechanism is based on the observation that some materials that are damaging to the eye appear to be cytotoxic to a number of cell types (e.g. corneal epithelium, corneal endothelium, conjunctival epithelium, conjunctival endothelium). This toxicity may be manifest in actual cell killing (loss of epithelium) and/or less severe (and obvious) toxicity and release of inflammatory mediators which these cells are believed to produce. Surface active agents would be expected to be cytotoxic in vivo by disrupting cell membranes. The relative potency of test materials in vitro would be determined by the dose giving a 50% reduction in viable cells.

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Protocols. S I R C neutral red assay: S I R C cells were obtained from the American Type Culture Collection. They were grown at 37°C in Earle's balanced salt solution ( D M E M ) containing 10% heat-inactivated FCS (30min; 56°C), l mM L-glutamine, streptomycin at 100 ttg/ml, amphotericin at 0.25/~g/ ml and penicillin at 1 0 0 I U / m l in a humidified atmosphere containing 5% CO2 in air. For treatment, cells were seeded at 2 x 104 cells per cm 2 in 96-well plates and incubated for 18hr to allow cell attachment. The medium was removed, and 100 #1 of seven dilutions of surfactant or formulation were added in culture medium. Each experiment was performed in triplicate. The controls, containing culture medium only, were included in each experiment. After 24 hr of treatment, the neutral red uptake assay was performed on each well. SDS was used as the positive control. The optical densities (absorbances) of the treated wells were compared with those of the untreated controls and the relative survival was determined. The NRUs0 values were taken from semi-log plots of the relative survival v. the log of the test material concentration (/~g/ml). V79 neutral red assay. V79 Chinese hamster lung fibroblasts were obtained from the European Collection of Animal Cell Culture. They were grown in minimal essential medium ( M E M ) containing 10% heat-inactivated FCS, 1 mM L-glutamine, streptomycin at 100 pg/ml and penicillin at 100 IU/ml in a humidified atmosphere containing 5% CO2 in air at 37°C. A robot ( B I O M E K 1000 Beckman) was used for seeding, preparation of test compound dilutions, distribution of reactives, measurement and calculations. Cells were seeded in 96-well plates at a density of 3 × 103 cells per well. Test materials were added in culture medium 24 hr later. After 24 hr of treatment, the neutral red uptake assay was performed on each well. Tween 20 was used as the positive control. The IC50 values were taken from log-transformed probit plots of the relative survival v. the test material concentration (#g/ml). At least three trials were performed to produce the mean IC50.

Proposed use. The submitters propose that the S I R C N R U cytotoxicity test be used "for screening purpose, inclusion in a battery of tests to eliminate the need of animals in the screening of ocular safety." They propose a similar use for the V79 N R U cytotoxicity test: " F o r a screening purpose and inclusion in a battery of tests to eliminate the need for animals in the screening of ocular safety of surfactants and surfactant-based formulations." In both cases, these assays are used in a battery of ocular irritation assessment methodologies. Range o f responses covered. The in vivo irritancy scores ranged from 0 to 50 (MAS). The in vivo data sets for the two assays differed by only a very few materials of the 38 (or 37) tested. Both in vitro assays can address a wide range of toxicities because they can test over a wide range of dilutions. Range o f materials amenable to use in the assays. Tests were limited to water-soluble materials only. Although the point was not addressed in the data sets submitted, additional limitations on extremes of reserve acid or alkaline potential may also be important (see the 48-hr neutral red assay). Use in risk assessment. Although the point was not specifically addressed, the assay is used as part of a battery of tests to determine product safety. See also the discussion for the S I R C cell cytotoxicity assay, above.

B. In vivo assay Adult New Zealand white albino rabbits were used. Generally six but no fewer than three were treated. Each rabbit received 0.1 ml of liquid test material placed into the everted lower lid of the right eye. Lids were held together for 10see. Animals were restrained for only 1 hr after dosing. Readings were performed at 1, 24, 48, 72 and 96 hr and at 7 days after dosing according to the scoring procedures of the Journal Officiel de la R~publique Franfaise (9 February 1985). Sodium fiuorescein was instilled to detect or confirm corneal lesions and area of involvement. Surfactants were tested at 10% concen-

Table 14. Summary of the correlation between the in vivo and in vitro values for the neutral red uptake assay using SIRC ceils on formulations Laboratory: CC107 Exposure: 24 hr In vivo/in vitro correlation (semi-log) (Pearson's) Number of Materialtype Range of materials or class scores Tissue r=* 38 Total MAS = 5-50 Corneal o p a c i t y -0.85/-0.84 20 Surfactants NR50 range Corneal area -0.82/-0.79 11 Lotions (/~g/ml) = 120-25,400 Iris - 0.84/- 0.79 7 Shampoos Conjunctival redness -0.83/-0.77 Chemosis - 0.84/-0.79 Discharge - 0.79/- 0.76 MAS NPt Days to clear NP/-0.75 *Pearson's Linear Correlation Coeflicient/Spearman'sRank Correlation Coefficient. tNot provided.

Working Group 4: Cell cytotoxicity assays

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plot of the corneal scores v. the in vitro scores for this study. Figure 8 shows the semi-log plot of the conjunctival redness scores v. the in vitro scores for this study. The M A S values were not included with this submission. In vivo data set: data from all three classes of materials were combined. The tissue responses were balanced over the milder ranges of toxicity (e.g. corneal scores generally ~<2, iris scores ~< 1 and conjunctival scores < 3). Mean data were plotted, but individual data were also provided. Because all of the

C. Summary data tables Assay with SIRC Cells (CC107). Table 14 summarizes the lirLear correlation between the in vivo tissue scores and the in vitro scores from the 24-hr neutral red uptake assay using S I R C cells submitted by laboratory CC107. This study was performed on three types of materials: surfactant formulations, lotions and shampoos. Figure 7 shows the semi-log

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J.W. Harbell et al. Table 15. Summary of the correlation between the in vivo and in vitro values for the neutral red uptake assay using V79 cells on formulations Laboratory: CC107 Exposure: 24 hr In vivo/in vitro correlation (semi-log) (Pearson's) Number of Material type Range of materials or class scores Tissue r=* 37 Total MAS range Corneal o p a c i t y -0.75/-0.81 19 Surfactants = 0-50 11 Lotions 1C50 range Corneal area -0.72/-0.77 7 Shampoos (#g/ml) = 19-8827 Iris -0.74/- 0.79 Conjunctival redness -0.73/-0.76 Chemosis -0.85/- 0.78 Discharge - 0.79/- 0.74 MAS NPt Days to clear NP/-0.76 *Pearson's Linear Correlation Coefficient/Spearman's Rank Correlation Coefficient. tNot provided.

test materials were liquids, physical form was not a n issue (see the 48-hr neutral red assay report). In vitro data set: in vitro scores were the m e a n o f at least three determinations. NRUs0 values were balanced over the range o f toxicities. The m o d a l CV for this set was between 20 a n d 30%, which is consistent with similar in vitro assays. The test material-dependent response for a given well ranges from 0 to 100% of controls (0 to 0.946 + 0.18 O D (absorbance) units). Historical positive control (SDS) values for the assay (n = 25) were NRUs0 = 85 + 13/~g/ml (mean + SD). Acceptance criteria are a positive control within one s t a n d a r d deviation of the historical m e a n (85 ___ 13 pg/ml) ( + 15%) and a negative control O D (absorbance) within 20% o f the historical m e a n (0.946 + 0.18 O D (absorbance) units). All tests were performed in the spirit of GLPs.

Subrnitter's conclusions. " D u e to the choice of the biological material (corneal cells) a n d e n d p o i n t m e a s u r e m e n t for cellular d a m a g e (neutral red uptake), the S I R C N R U test could be related to some of the mechanisms involved in ocular irritancy induced by surfactant a n d surfactant-containing f o r m u l a t i o n s . " Correlations with the in vivo data are very good, particularly for corneal opacity. The assay is highly reproducible a n d available to a large n u m b e r of laboratories. A l t h o u g h the assay is limited to water-soluble materials, b o t h formulations a n d ingredients m a y be tested. Assay with V79 cells (CC107). Table 15 summarizes the linear correlation between the in vivo tissue scores a n d the in vitro scores from the 24-hr neutral red uptake assay using V79 cells submitted by l a b o r a t o r y CC107. This study was performed o n three types o f materials: surfactant formulations, lotions a n d shampoos. Figure 9 shows the semi-log

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Working Group 4: Cell cytotoxicity assays

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Fig. 10. Semi-log plot of the conjunctival redness scores (24 hr) v. the neutral red uptake assay (24 hr) NR50s using V79 cells for three classes of cosmetic/personal care formulations. plot of the corneal scores v. the in vitro scores for this study. Figure 10 shows the semi-log plot of the conjunctival redness ~,;cores v. the in vitro scores for this study. Standard cleviations were not provided. The MAS values were not included with this submission. In vivo data set: see above. In vitro data seL: in vitro scores were the mean of at least three determinations except when no toxicity was observed at the upper dose of 5000/zg/ml. IC50 values were balanced over the range of toxicities, although the scores were shifted down relative to the SIRC-based assay. The IC50s are obtained from log probit plots. Not all standard deviations were provided but the CVs appear to be < 20%. Historical positive control values (Tween 20) were 183.8 _+ 41.8 with n = 17. Acceptance criteria were based on both positive and negative control values. The acceptable range was based on the 95% confidence interval (_+ 2SD). For Tween 20 the IC50 should be between 120 and 260/tg/ml, and the negative control optical density (absorbance) values should be between 0.2 and 0 . 4 0 D units. The optical density from the V79 is much lower th~Ln that for the SIRC cells. Assays were performed in the spirit of the GLPs. Submitter's conclusions. Correlation values were good for all tissues. Of particular interest was the good separation for corneal opacity and chemosis scores. The assay possesses many of the same positive attributes as the SIRC-based system: universal endpoint, well-documented use limits and high reproducibility. Tile assay is suggested for use as part of a battery of in vitro methods for testing water-soluble surfactant-based products.

D. Overall analysis of the data These studies have been conducted on 38 liquid, water-soluble surfactants or surfactant preparations.

The /n vivo toxicities are reasonably balanced over the range of responses from 0 to 50 (MAS) on a scale of 0 to 110. This range would be consistent with personal care and cosmetic products. The in vitro data sets were produced from multiple trials and show a high level of interassay reproducibility. Historical control and acceptance criteria further document the reproducibility of these assays. The Pearson's correlations for the SIRC NRU cytotoxicity assay were particularly impressive. The V79 cells were more sensitive than the SIRC cells, as reflected by the 50% toxicity values. This difference may be the result of the difference in the number of cells plated per well (2 x 104/well SIRC v. 3 × 103/ well V79) rather than anything to do with the origin of the cells. Parallel studies with equal cell numbers might be of value in determining the basis for the difference.

E. Conclusions and recommendations The data presented support the proposed use of the SIRC NRU and V79 NRU cytotoxicity tests as screens in a battery of tests. However, certain cautions must be emphasized. The test materials must be water-soluble and should be surfactant based. Furthermore, the present data set does not contain solids (e.g. granular surfactant products), which may be more toxic as solutions than as solids. As has been demonstrated with the 48-hr neutral red assay, the toxicity of such products may be markedly over-predicted by a solution in a cell culture assay. Finally, the toxicity of products with high or low pH (specifically high acid or alkaline reserve) or high reactivity may be markedly under-predicted by these assays. These concerns are appropriate for all of the submerged cell culturebased assay systems.

100

J.W. Harbell et al.

Neutral Red Release Assay (5-30 min Exposure) A. In vitro assay: Neutral Red Release Assay using a 5--30 min exposure time Summary. Two data sets from two slight variations of this in vitro assay were submitted. Although one study examined only very mild materials (surfactants intended for eye care use), the other study was conducted over a wider range of toxicities (CTFA Phase I!I surfactant formulations). Full tissue scores were provided. Both assays used the normal human epidermal keratinocyte (NHEK) as the target cell. Basis of assay. Both assays are based on short-term cultures of normal human cells with cytotoxicity (loss of lysosomal integrity) as the assay endpoint. The neutral red procedure is derived from the work of Guess, Borenfreund and their respective collaborators (see the 48-hr neutral red assay report for more details). The uptake procedure was modified for short-term exposures by Reader et al. (1990). The test material-induced cytotoxicity is measured over a wide range of concentrations, and the concentration yielding a 50% reduction in retained neutral red (within the population of cells) is used as the measure for comparison between test materials. The exposure period for the assays reported in these two studies differs from that originally used by Reader et al. In their work, the exposure time was 1 min, whereas in these protocols, a 5 or 30min exposure time was used. The 5 min time was selected for technical reasons so that slight differences in exposure time (e.g. time between wells) would not represent a large percentage of the total exposure time. The 30 min exposure time was required for the very mild surfactants being studied. These short exposure times are intended to focus on the test material's ability to interact with the membrane of the cells and resulting liberation of the dye; therefore this is an assay for immediate toxicity. The dose giving a 50% reduction in dye retention is considerably higher than the dose of the same material which gives a 50% reduction in neutral red uptake. The test is based on the observation that some materials that are damaging to the eye appear to be cytotoxic to a number of cell types (e.g. corneal epithelium, corneal endothelium, conjunctival epithelium, conjunctival endothelium). This toxicity may be manifest in actual cell killing (loss of epithelium) and/or less severe (and obvious) membrane damage and/or release of inflammatory mediators which these cells are believed to produce. Surface-active agents would be expected to be cytolytic at high concentrations by disrupting cell membranes. NHEK cells are selected for this assay because of their similarity to the corneal and conjunctival epithelial cells. Protocol. Secondary cultures of proliferating normal human epidermal keratinocytes (NHEK) were harvested (when 50-80% confluent) by trypsinization. The single cell suspensions were

resuspended in Keratinocyte Growth Medium (KGM) (Clonetics Corporation). The cells were plated at 2500 cells/well (total volume of 250/~1) into each well of a 96-well tissue culture plate. The plate was incubated for 4--5 days at 37°C (5% CO2/humidified atmosphere) until the cells reached approximately 80% confluence. Depleted KGM was decanted from the wells. Cells were preloaded with neutral red dye (NR) by adding 250 ~1 of a 50/~g/ml solution of neutral red dye in KGM to each well (except for two 'blank' wells, which received KGM only) and incubating the cells for 3 hr at 37°C. The K G M / N R was decanted from the plates and the cells were exposed to various dilutions of KGM/test agent at room temperature for 5 or 30 min. The KGM/test agent solutions were decanted from the wells by inversion. Cells were washed twice with wash/fix solution (an aqueous 1% formaldehyde-1% calcium chloride solution) for 1 min. The wash/fix solution was decanted and each well received 100#1 of a solvent solution (1% glacial acetic acid 50% ethanol) for >-20 min at room temperature. Absorbances (optical densities) were measured at 540 or 550 nm in a microplate reader (after the appropriate blank correction) and "percentage of untreated control" values were calculated for each dilution of test agent. The "percentage of untreated control" values v. the test agent concentrations were plotted (semi-log plot) and the concentration of test material resulting in a neutral red release which was 50% of controls (NRRs0) was determined by extrapolation. Proposed use. The uses of the assay depend on the product line and the company. For the 30 min exposure assay, CC108 proposes that the assay be used in the developmental process to eliminate irritating compounds and formulations before proceeding to in vivo tests on the final product formulations. Because eye care solutions are necessarily very mild, the robustness of the in vitro methods is desirable to eliminate even "mild" ocular responses. The second data set (CC104) was obtained from the CTFA Phase III trials of surfactant-containing personal care materials. For personal care products, the assay has been used to select formulations for development. Range of responses covered. The data set from CC108 contains five nonionic surfactants and one cationic surfactant/preservative. All but the last material was very mild. Scoring of the in vivo responses was based on a modification of the Draize system. The CTFA data set covered a range of 0-40 (MAS) and used standard Draize scoring. Range of materials amenable to use in the assay. All of the data submitted were taken from water-soluble materials. In addition to these data, experience also suggests that the assay should be used only on materials which are water-soluble or at least watermiscible. As with the other neutral red assays, additional restrictions on testing materials with extremes of reserve acidity or alkalinity should be

Working Group 4: Cell cytotoxicity assays

101

Table 16. Summary of the correlation between the in vivo and in vitro values for the neutral red release assay on very mild materials of interest to the ophthalmic industry Laboratory: CC108 Exposure: 30 rain In vivo/in vitro correlation (semi-log plot)

Number of materials 6

Material type or class Surfactants

Range of scores

NRR50 range = 0.002-10 mg/ml

Tissue Rose Bengal staining of cornea Corneal area Ocular discomfort Conjunctival redness Chemosis Discharge MAS Days to clear

r= -0.62 -

0.23 0.49 0.70 0.77 NA* NA

*NA = not applicable.

noted, since buffering by the culture m e d i u m will reduce the toxicity. Use in risk assessment. Use in risk assessment was not addressed by either of the submitters. B. In viva assays

Please see below.

C. Summary data tables M i l d surfactant products, 30-min Exposure (CCI08). Table 16 summarizes the linear correlation between the in vivo tissue scores a n d the in vitro scores from the neutral red release assay submitted by l a b o r a t o r y CC108. This study was performed o n extremely mild surfactant solutions which would be o f interest to the o p h t h a l m i c industry. In vivo d a t a set: d a t a were provided o n six surfactants: P o l o x a m e r 407, Propylene Glycol, Polysorbate 80, P E G 300, P E G 24 H y d r o g e n a t e d L a n o l i n a n d Benz~dkonium Chloride (C 14 isomer). A modified Draize scoring system was used that was a d a p t e d to the relatively " m i l d " effects o f eye care products. 1-day acute ocular responses were measured in rabbits. G r o u p s o f four rabbits were treated with 1 d r o p (50/tl) eight times at 1 hr intervals for 1 day with the different test solutions in each eye. G r o s s observations for ocular discomfort a n d irritation were performed at the time o f instillation. All rabbits were examined with a slit-lamp following

the last instillation a n d on days 2 a n d 3. A l t h o u g h multiple c o n c e n t r a t i o n s were tested, only the results from the lowest c o n c e n t r a t i o n that produced a n in vivo response were given (for example, b e n z a l k o n i u m chloride). Since three of the surfactants did not produce any in vivo response, the in vivo scores were highly skewed. In vitro d a t a set: the in vitro data were generated from a 30 min exposure to the doses o f the test materials. F o u r c o n c e n t r a t i o n s o f the test articles were tested in quadruplicate. Two replicate experiments were performed, a n d the d a t a presented were the average of the NRRs0 values from these two trials. Tests were conducted u n d e r the spirit o f G L P s . T h e n u m b e r o f replicate assays, if any, was not given. SDS was used as the positive control, a l t h o u g h no historical ranges or acceptance criteria were provided. The a u t h o r did indicate t h a t the m a n u f a c turer's suggested NRRs0 range was met for this control. The NRRs0 scores covered a far greater range t h a n did the in vivo responses; they ranged from 10 to 0.002 mg/ml. Thus, the in vitro test resolved differences a m o n g the test materials that m a y have detracted from the numeric correlation b u t m a y be quite useful in p r o d u c t development. C T F A Phase I I I (CCI04). Table 17 summarizes the linear correlation between the in vivo tissue scores a n d the in vitro scores from the neutral red release assay submitted by l a b o r a t o r y CC104. This study was

Tab le 17. Summary of the correlation between the in vivo and in vitro values for the neutral red release assays conducted in the CTFA Phase III study Laboratory: CC104 Exposure: 5 rain In vivo/in vitro correlation (semi-log plot) Number of Material type Range of materials or class scores Tissue r= 25 Surfactant-based MAS = 0-40 Corneal opacity -0.72 formulations NRR50 range Corneal area -0.75 =0.55-1000 mg/ml Iris -0.71 Conjunctival redness -0.86 Chemosis - 0.75 Discharge - 0.70 MAS -0.77 Days to clear -0.69

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Fig. 11. Semi-logplot of the Draize MAS (24 hr) values v. the neutral red release assay (5 min) NRR50s from the CTFA Phase III study. The X-axis bars show one standard error of the mean rabbit scores; the Y-axis bars show one standard deviation of the mean for the multiple in vitro trials. performed as part of the CTFA Phase III studies. Figure 11 shows the semi-log plot of the Draize MAS against the in vitro scores for this study. In vivo data set: in vivo data were generated by the standard Draize assay using six rabbits per test material. Anaesthetic was used. Testing was performed under code and with full GLP. In vivo MAS scores were distributed over the whole range (0-10, six materials; 11-20, seven materials; 21-30, four materials; 31-40, eight materials). All the materials were surfactant-based formulations (11 were shampoos). The standard deviations for the six animals were quite large--in the midrange of toxicity (average CV approx. 40%). This CV value is consistent with other published data and is not a negative reflection on the laboratory performing these tests. The variability in the in vivo responses should be considered in comparing in vivo and in vitro data. In vitro data set: the standard neutral red release protocol was used to generate in vitro data. Three independent NRRs0 determinations were made. The mean NRRs0 values ranged from 0.55 to 1000 mg/ml. The least toxic materials ranged from 1000 to 40 mg/ml, mild/moderate materials gave scores from 10 to 2 mg/ml and the most toxic materials had scores of ~< 1 mg/ml. This wide range at the very mild end of the scale tended to reduce the apparent linear correlation. The average CV was 17% for the 25 materials tested. Concurrent positive (Triton X-100) and negative controls were performed with each assay. Acceptance criteria for a given trial were based on the positive control NRRs0 value; an assay was acceptable if the NRRs0 value was within two standard deviations of the historical mean. This mean + 1 SD was 0.204 ___0.038 mg/ml

(CV = 18.6%; n = 23 trials) over 2 yr. Samples were tested under code, and the spirit of GLP was used for all in vitro testing.

D. Overall analysis of the data In vivo/in vitro correlations for the five very mild surfactants were highly skewed by the absence of an in vivo response to three of the materials. The in vitro responses did show appreciable differences between the materials, which may be very useful in product development. There was a 4-log spread among NRR~0 scores in the absence of reported in vivo toxicity. Data were provided according to the guidelines. The second study covered a wider range of toxicities with a better balanced data set. Given the variability of the in vivo responses, the in vivo/in vitro correlations were good, especially for conjunctival redness. More important, there was very good separation between the non-toxic, mild/moderately toxic and most toxic materials for redness, MAS and days to clear. In this case, the linear correlation is a conservative estimate of the utility of the test. Although the data submitted focused on surfactants, other studies (e.g. CTFA Phase II) have suggested that the assay should be applied with caution to non-water-soluble materials. In addition, the short-term exposure may not be appropriate for testing non-surfactant materials (see Wallace et al., 1992).

E. Conclusions and recommendations The neutral red release assay "was developed to assess the immediate toxic action of surfactants. The very short exposure time (approx. 5 min) corresponds to the retention time an irritant may remain in the eye before being flushed away by tears. On the basis of

Working Group 4: Cell cytotoxicity assays the data from the CTFA Phase III study, the assay looks promising as a screen for testing surfactantbased materials over the range of toxicities normally found in person~tl care products. For the CTFA Phase III compounds, the neutral red release assay showed better correlation and separation than did the neutral red uptake, assay performed with the same cell type (see CCi04 48-hr neutral red exposure). The assay may also be useful in screening surfactant materials in the very mild range, but the present data are very limited. Currently, the number of studies comparing NRRse values with in vivo responses is also very limited. Further work would seem to be warranted. The assay should be used with caution for non-surfactant formulations. In addition, the neutral red release assay would be expected to have similar test article limitations to those of the other neutral red assay: water-soluble/miscible materials with limited reserve acidity and alkalinity.

AFar Diffusion Assay A. In vitro assay: Agar Diffusion Method Summary. Three data sets are included in this review. The first ,;et was submitted from laboratory CC107 and used the same in vivo data presented for the 24-hr neutral red assay using SIRC and V79 cells. The second submission (CC120) used a slightly different method and tested cosmetic creams. The third submission used a protocol developed for testing pharmaceutical intermediates. Basis of assay. The test material and target cells are separated by a layer of agar (or agarose). Leachable material passes through the agar to interact with the target cells. Since the cell monolayer is largely developed before 'Lhe agar is added and the exposure times are relatively short, this is an assay for immediate cell killing (e.g. membrane lysis) rather than for the cessation of cell growth. Cell viability can be scored by direct observation or more precisely by vital dye uptake; neutral red is commonly used for this purpose. The agar overlay (diffusion) assay is a USP-approved test for cytotoxicity from medical device materials. Protocol. The method is based on a technique first described by Guess et al. (1965) for the toxicity screening of plastics. The model uses a diffusion matrix (agar or agarose) for delivery of the test material to a monolayer of cultured cells. Protocol (CC107)--V79 cells were cultured in Eagle's modified minimal essential medium (EMEM) supplemented with 10% foetal calf serum and antibiotics at 37°C in a humidified atmosphere containing 5% OD2. After trypsinization, V79 cells were suspended (500,000 cells per ml) in culture medium containing 1% agarose, and 4 ml portions of this medium were poured into 60 mm dishes and coloured with neutral red. A 6 mm non-cytotoxic filter paper was placed in the centre of the agarose

103

surface and 10/A or 10 mg of test sample was added. Dishes were capped and incubated at 37°C for 18 hr. At the end of the incubation period, cultures were examined microscopically to determine whether the cells were viable. When cytotoxicity was observed, the diameter of the zone of lysis was measured in two directions. Each product was tested in three to five independent experiments. The range of mean diameters of lysis was 0-5 cm. A negative control was used with each experiment (10#1 of medium) and assays showing cytotoxicity in this control were discarded (acceptance criterion for the assay). Protocol (CC120)---L-929 cells were grown in EMEM with 10% foetal calf serum at 37°C in a humidified atmosphere of 5% CO2. 24 hours before the assay was started, a 4-ml portion of medium containing 4 x 10Scells/ml was seeded into each 60 mm petri dish. To begin the assay, the growth medium was removed and replaced with EMEM (complete) containing 1% agarose and 0.2% neutral red. This medium was placed onto the cells at 42°C and allowed to solidify for 10 min. A 9-mm filter paper saturated with the test material was placed on the agar and the culture was incubated for 24 hr. Three cultures were used for each test material. Latex and the filter alone served as the positive and negative controls, respectively; specific acceptance criteria were not provided. At the end of the incubation period, the cell cultures were examined microscopically for changes in cell morphology and for signs of cell discolouration around the disc. Next, the agarose overlay gel was removed and the cell culture was examined macroscopically. Any zone of toxicity was traced onto paper and the tracing was weighed to determine the score. Protocol (Reboulet et al., 1994) (CC109)--SIRC cells were maintained in Dulbecco's modified Eagle's medium/Nutrient Mixture F-12 with 10% foetal calf serum at 37°C in a humidified atmosphere with 5% CO2. Test cultures were obtained by seeding 100 mm dishes with 1.4 × 107 cells in 16 ml. After incubation for 24 hr, the growth medium was removed and the cells were overlayered with 16ml of a solution containing 1% Bacto agar, 25% Earle's balanced salts and 0.01% neutral red in complete growth medium (exposure temperature approx. 41°C). Cultures were examined after 16 hr to verify viability and uniform dye uptake. Solid and liquid test articles were applied to the agar at a dose of 25 mg or 25 #1 per plate. Test materials were applied in cloning rings (solids) or on paper discs (liquids). The cultures were incubated for an additional 24 hr and the general viability was determined microscopically. The size of the lytic zone around the test article was determined in two dimensions. Three replicate assays were performed for this study. No discussion of controls was provided. This third protocol is intended only to predict corneal opacity and identify severe irritants. Purpose, basis and proposed use of test. The three submitters propose a range of uses for this assay

104

J.W. Harbell et al. Table 18. Summary of the correlation between the in vivo and in vitro values for the agar diffusion assay (Method l) on formulations Laboratory: CCI07 Exposure: 18 hr In vivo/in vitro correlation (linear/linear plot) Number of Materialtype Range of materials or class scores Tissue r= * 20 Surfactants MAS range Corneal opacity 0.83/0.72 12 Lotions = 0-60 9 Gels AG18 score range Corneal area 0.73/0.68 7 Shampoos = 0.5~1.8 Iris 0.78/0.71 Conjunctival redness 0.80/0.67 Chemosis 0.66/0.62 Discharge 0.63/0.64 MAS NPt Days to clear 0.72 *Pearson's Linear Correlation Coeflicient/Spearman'sRank Correlation Coeflficient. tNot provided.

system. The test serves the pharmaceutical company (CC109) as an initial biological screen to reduce the chance of exposing animals to severe corneal irritants during in vivo ocular testing. For the cosmetic users, solid, liquid, water-soluble and water-insoluble formulations, representing the broad range of physical and chemical properties routinely encountered in live phase testing, have been evaluated with this model. For example, multiple formulations (such as gels, creams and lotions) may be screened preclinically with this method; in addition, interactions of chemical ingredients may be studied. Use in risk assessment. Submitters CCI07 and CC120 suggest that the method be used as a preclinical screen of cosmetic ingredients and products, especially hydrosoluble ingredients present in lipophilic finished products such as oils and mascaras and for other cosmetics such as lotions, shampoos, gels and creams. At CC109, the agar diffusion method is currently

used routinely as the first biological model in the corneal irritation testing tier of hazard evaluation of chemical intermediates. B. Data on agar diffusion method Cosmetic and personal care formulations (CC107).

Table 18 summarizes the linear correlation (Pearson's) and rank correlation (Spearman's) between the in vivo tissue scores and the in vitro scores from the agar diffusion assay (method 1) submitted by laboratory CC107. This study was performed on three types of materials: surfactant formulations, lotions and shampoos. For this study, Fig. 12 shows the semi-log plot of the corneal scores v. the in vitro scores, and Fig. 13 shows the semi-log plot of the conjunctival redness scores v. the in vitro scores. The M A S values were not included with this submission. In vivo data set: data from all three classes of materials were combined. The tissue responses were balanced over the milder ranges of toxicity (e.g.

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Fig. 13. Plot of the conjunctival redness scores (24 hr) v. the agar diffusionassay (18 hr) (Method 1) AGI8 scores using V79 cells for three classes of cosmetic/personal care formulations. corneal scores generally ~<2, iris scores ~<1 and conjunctival scores < 3). Mean data were plotted but individual data were also provided. All of the test materials were liquids. For the correlation of the in vitro results v. the days to clear, the in vivo data were converted into four reversibility classes (0-1 day, Class 1; 1-3 days, Class 2; 3-7 days, Class 3; and > 7 days, Class 4). In vitro data: in vitro scores were the mean of at least three determinations. In vitro values were balanced over tile range of toxicities. Acceptance criterion is that the negative control is not cytotoxic. No positive control acceptance criteria or historical data were presented. All tests were performed in the spirit of GLPs. Cosmetic formulations (CC120). Table 19 summarizes the linear correlation between the in vivo tissue scores and the in vitro scores from the agar diffusion assay (method 2) submitted by laboratory CC129. This study was performed on a range of cosmetic formulations. Figure 14 shows the

plot of the Draize MAS v. the in vitro scores for this study. In vivo data set: adult New Zealand white albino rabbits were used; generally six but no fewer than three were treated. Each rabbit received 0.1 ml of liquid test material placed into the everted lower lid of the right eye. Lids were held together for l0 sec. Readings were performed at l, 24, 48, 72 and 96 hr and at 7 days after dosing according to the scoring procedures of the Journal Officiel de la R~publique Franf~aise (9 February 1985). As would be expected with the types of products tested, the vast majority of the responses were in the milder range (e.g. MAS of ~<10) with only three materials giving scores of >i 30. This tended to polarize the data. In vitro data: In vitro scores were based on the average of three scores. Scores ranged from 0 to 594. Because the in vivo data were somewhat polarized, the correlation coefficients may have been higher than the predictive value of the data would have warranted. Agar scores of <200 tended to reflect milder

Table 19. Summary of the correlation between the in vivo and in vitro values for the agar diffusion assay (Method 2) on formulations Laboratory: CC120 Exposure: 24 hr In vivo/in vitro correlation (linear/linear plot)

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materials, but very low scores (e.g. 0) were not predictive of non-toxicity. Higher scores ( > 200) were seen with relatively mild products and some of the more aggressive materials as well. The acceptance criteria for the negative control required that the non-toxic filter alone have a score of zero and that the score for the positive control (latex) be 711-977. Pharmaceutical intermediates (CC109). Table 20 summarizes the data from the agar diffusion assay submitted by laboratory CC109. This study was performed on pharmaceutical intermediates. Figure 15 shows the plot of the data for 68 materials tested. The r value reflects the Pearson's linear correlation analysis. The dashed lines show the cutoff points suggested by the submitter for a Cooper's analysis. In vivo data set: adult New Zealand white albino rabbits were used; generally three were treated, except for potentially severe irritants in which case only one rabbit was used. Each rabbit received 0. I ml of liquid test material placed into the everted lower lid of the right eye. Lids were held together briefly. Readings were performed at 1, 24, 48, 72 and 96 hr and at 7 days after dosing according to the.scoring procedures of the EPA and O E C D Guidelines. Tests were performed under GLP. All appropriate tissue scores

were taken but only the corneal scores are reported here. In vitro data set: the submitter focused the in vitro assay only on prediction of severe irritation to the corneal tissue. To this end, Cooper's analysis was used to evaluate the data. Such an approach was actively discouraged by the I R A G Guidelines document because the cutoff values are often chosen without reference to some specific standard and because eye irritation data are continuous (e.g. cover a range of responses) rather than discontinuous (e.g. presence or absence of a tumour). However, the submitter did provide the data set and rationale by which the in vitro assay cutoff was developed. This value was then applied to the data set of 68 materials. The data were also subjected to linear correlation analysis by the reviewer. A "training set" of 24 test articles was used to set the in vitro cutoff point (Critical Lytic Zone). The test materials in this initial study included sodium periodate, sodium meta-periodate, citric acid, Triton X-100, propylparaben, 1-butanol, ethoxyethanol monoethyl ester, 2-butanone, talc and 14 chemical intermediates isolated from synthetic processes. Evaluation of the in vitro result is based on estimation of the Critical Lytic

Table 20. Summary of the correlation between the in t,it,o and in t~itro valuesfor the agar diffusion assay (Method 3) on formulations Laboratory: CC109 Exposure: In t,it~o/in z,itro correlation Number of Materialtype Range of materials or class scores Tissue r= 68 Pharmaceutical Cornealopacity Corneal 0.46 intermediates 0-4 Lytic zone score Iris NP* range = 0-54.5 Conjunctivae NP *Not provided. See text and Fig. 15 for discussion.

Working Group 4: Cell cytotoxicity assays

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Zone Size (CLS). The CLS was determined by using a 4-step algorithm: (!) One of the 24 lytic zone values at the 25 mg or 25 pl dose was selected as the interim lytic zone size (Int CLS) to which all 24 lytic zone values were compared. Test articles with lytic zone sizes less than the Int CLS were classified as non-severe, and those with values greater than or equal to the Int CLS were classified as severe. (2) The overall predictivity (relative to the in vivo responses) was calculated. (3) The process was repeated until all 24 lytic zone sizes had been tried as the Int CLS. (4) The Int CLS associated with the highest predictivity was selected as the CLS. On the basis of the training set of 24 test materials, a CLS of >~42mm was determined. A corneal opacity score of 3 or 4 was considered to be a severe in vivo ocular response. This type of analysis is used when discontinuous data are being evaluated or when limited predictive correlation is observed between the two assays over the full range of continuous responses in the assays. The corneal opacity score was used for making comparisons to the in vitro test because of its importance in ocular damage by severe irritants. The submitter reported generally weak correlation between the CLS value and conjunctival irritation responses (data not provided). The r value of 0.46 for the correlation between the CLS and corneal scores would suggest limited correlation over the full range of irritation. The Cooper's plot showed that 11 of the 15 severe irritants were correctly predicted but that four false negatives and six false positives were observed. The pH of the test material may contribute significantly to the observed toxicity in vivo whereas the buffering capacity of the culture medium may

mitigate the effect & vitro. The submitter divided the test materials between two groups, those with pH values > 4 and < 10.5 and those with pH values of ~<4 or /> 10.5. Of the 11 severely irritating materials in the first group, nine were correctly placed at >/42; of the four severely irritating materials in the second group, two were correctly placed. Only 15 of the 68 materials tested were in the severe category. This ratio is probably representative of pharmaceutical intermediates but does make this a strongly unbalanced data set for a Cooper's analysis. Historical controls for two materials were provided. For solids, sodium periodate showed a mean lytic zone of 55 with a standard deviation of 2.8 for 19 triplicate assays. For liquids, 5% Triton X-100 showed a mean of 45 with a standard deviation of 3.7 for 21 triplicate pairs. No acceptance criteria for the assay were provided. C. Conclusions and recommendations Previous studies have demonstrated the value of the agar diffusion method in assessing the cytotoxicity of a variety of compounds, including plastics (Guess et al., 1965). This technique has been adopted as a test for cytotoxic potential by the United States Pharmacopeia. In this case, the in vitro response is used to predict irritation from implantation into the muscle of a medical device. This application for the in vitro assays (agar diffusion and extract cytotoxicity) has gained wide industry and regulatory acceptance because of their speed and high sensitivity. The basic technique has also been used to evaluate cosmetics (Milstein and Hume, 1991; WaUen et al., 1987). Acosta and co-workers have shown that cultured corneal epithelial cells (primary cultures)

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gave different responses than cultured conjunctival epithelial cells (primary cultures) to several surfactants under similar experimental conditions (Grant et al., 1992; Yao and Acosta, 1992). The CC107 study showed good correlations between the in vitro method and many of the in vivo ocular responses, including conjunctival and iridal scores. The data are consistent with those presented for the other two assay systems tested against these in vivo data. In contrast, the results from the CC120 study were less promising. Although this difference may reflect the different technique used, it is more likely to result from the type of materials tested. Like other cell culture-based assays reviewed by this Working Group, the agar diffusion assay is much more likely to address water-soluble test materials (or extracts) than those which are not water-soluble.

Cell Protein Assay

measurement of the protein content of cultured cells after a 48-hr exposure to the test material. (See the 48-hr neutral red assay report for the full protocol, CC101.) In this assay, however, total cellular protein is measured, rather than neutral red uptake. PRs0 represents the concentration of test material (/ag/ml) which reduces protein content of N H E K cells by 50%. Purpose, basis and proposed use of test. Changes in protein content of cultured cells exposed to potential ocular irritants are thought to represent eye irritation potential. Use in risk assessment. The cell protein assay has been proposed as a replacement for animal testing, but it has not been used as such. It is reported that the cell protein assay can distinguish severe from mild irritants. However, because of the delivery medium and/or other parameters, the assay was not useful for solutions with highly alkaline materials or for water-insoluble materials.

A . In vitro assay: cell protein assay Basis o f assay. The test is based on the observation

that some materials that are damaging to the eye appear to be cytotoxic to a number of cell types (e.g. corneal epithelium, corneal endothelium, conjunctival epithelium, conjunctival endothelium). This toxicity may be manifest in actual cell killing (loss of epithelium) and/or less severe (and obvious) toxicity and release of inflammatory mediators which these cells are believed to produce. Surface-active agents would be expected to be cytolytic by disrupting cell membranes. Cell death (with resulting cell loss) in the population is measured by the total cell protein, relative to the control cultures. Depending on the incubation time with the test material, the cessation of cell replication may also contribute to the overall difference in protein content. Protocol. The procedure is based on the method of Shopsis and Eng (1985) which presupposes that specific cellular functions may be determined by the

B. Data on cell protein assay

Table 21 summarizes the linear correlation between the in vivo tissue scores and the in vitro scores from the cell protein assay performed as part of the Soap and Detergent Association Phase lII study and submitted by laboratory CC101. Table 22 shows the specific materials tested, their Draize MAS and in vitro scores. C. Overall analysis o f data In vivo data set: the standard Draize protocol was used with six animals per test material. Observations were made at 1, 24, 48 and 72 hr. As required, animals were maintained up to 35 days. Comparisons were based on scores obtained at />24hr after dosing. The toxicity range was well balanced, with approximately half of the MAS scores between 1 and 20 (full range 1-41). Studies were conducted on coded compounds (both in vivo and in vitro). In vivo studies

Table 21. Summary of the correlation between the in vivo and in vitro values for the cell protein assay in the SDS Phase Ill study Laboratory: CCIOI Exposure: 48 hr In vivo/in vitro correlation

(semi-log plot) Number of materials 22 16 6

Material type or class Total Neat materials generally surfactants Formulated products

Range of scores MAS range = 0~1 PC50 score range (/~g/ml) = 1 6 - > 150,000

Tissue

r= *

Corneal opacity

-0.34/-0.61

Corneal area Iris Conjunctival redness Chemosis Discharge MAS D a y s to clear

NPt -0.41/-0.75 -0.77/-0.91 -0.48/-0.67 NP -0.58/-0.81 -0.23/-0.48

*The 22 test materials contained several materials with appreciable titratable alkalinity (>~0.1 meq HCI/g); one sample w a s 5.25% sodium hypochlorite. The analysis of the correlation d a t a w a s performed on the complete set of 21 materials (upper correlation number) and on only the neutral test materials (and without sodium hypochlorite) (lower number) (e.g. MAS r = - 0 . 5 8 complete v. - 0 . 8 1 restricted). The toxicity of the alkaline materials and hypochlorite w a s appreciably underpredicted in vitro. t N o t provided.

Working Group 4: Cell cytotoxicity assays

109

Table 22. Cell protein assay from the Soap and Detergent Association Phase III study: Laboratory: CCI01 Maximum In vitro Physical Alkalinity averageDraize assay score IdentiLy of sample form (meq HCI/g) score (,ug/ml) Ethanol, 15% Liquid 0.0 1.0 > 150,000 Polyethylene glycol, 400 mw, neat Liquid 0.0 2.0 51,130 Isopropanol, 15% Liquid 0.0 2.3 > 150,000 Butoxyethanol, 5% Liquid 0.0 2.7 57,840 Glycerol, neat Liquid 0.0 13.8 > 135,800 Alcohol ethoxylate, 10% Liquid 0.0 14.7 23 Dodecylamine oxide, 1% Liquid 0.0 14.8 89 Dimelhylditallowammonium Liquid 0.0 18.8 l01 chloride, 3% Alkyh;thoxysulfate, 10% Liquid 0.004 18.8 32 Triethylammonium lauryl sulfate, 10% Liquid 0.0 25.7 57 Sodium lauryl sulfate, 10% Liquid 0.0 27.0 19 Ammonium lauryl sulfate, 10% Liquid 0.0 32.7 24 Alkyll;lycerylsulfate, 10% Liquid 0.0 34.3 17 Sodium perborate Solid 5.36 34.3 2389 Sodium alkyl sulfate, 10% Liquid 0.0 36.2 16 Sodium hypochlorite, 5.25% Liquid 0.0 41.0 2432 Liquid cleaner Liquid 0.044 15.8 97 Powd,:red cleaner Solid 0.118 17.3 13 Liquid hand soap Liquid 0.0 24.5 27 Powd,.~red cleaner Solid 0.816 34.3 89 Powd,.~red bleach with protease Solid 4.7 34.3 1850 Pine nil cleaner Liquid 0.0 41.3 35

were conducted under the principles of GLPs. Test materials included representative surfactants, bleach ingredients and formulated products. When appropriate and possible, the cleaning product ingredients were tested at concentrations likely to be found in cleaning products. The test materials and test concentrations were selected to obtain a range of eye irritation responses. In vitro data: the in vitro responses tended to be polarized, with the very mild materials ( M A S 1-13) yielding PR~0 values of >~ 50,000/~g/ml and the more toxic materials ( M A S > 15) showing values of about 100/,g/ml. The best correlation was obtained with conjunctival redness, and in that case, the in vitro values were more balanced relative to the in vivo responses. Alkaline and hypochlorite materials were under-predicted. No discussion of concurrent positive controls or acceptance criteria were provided. D. Conclusions and recommendations As indicated above, the cell protein assay is not very predictive for alkaline or water-insoluble materials. O f the several in vivo tests conducted on the test materials, conjunctival redness showed the best correlation to the cell protein assay. Depending on the cell type and test articles used in this assay, dead cells may either remain attached to the plastic (NHF, K cells) or be lost into the medium (rodent fibroblasts). Test materials which kill and fix the cells will allow more cellular protein to remain on the dish than these which disrupt the membrane and release cytosolic proteins. Total protein is not a measure of current viability. Thus, dose-response curves tend to be very different for different types of cells and different classes of test articles. Because very few laboratories or companies have conducted comprehensive studies on the cell protein

assay, much more research is necessary before its value as an in vitro irritation screen can be determined.

Fibroblast Cytotoxicity Assay A. In vitro assay: Fibroblast Cytotoxicity Assay Basis of assay. The test is based on the observation that some materials that are damaging to the eye appear to be cytotoxic to a number of cell types (e.g. corneal epithelium, corneal endothelium, conjunctival epithelium, conjunctival endothelium). This toxicity may be manifest in actual cell killing (loss of epithelium) and/or less severe (and less obvious) toxicity and release of inflammatory mediators which these cells are believed to produce. Surface-active agents would be expected to be cytolytic by disrupting cell membranes. Protocol. The method of Kruse and Patterson (1973) was used. Swiss albino mouse embryo fibroblasts (3T3 cells) were used as the target tissue and exposed to doses of the test material for 1 hr. Values were reported as the dose (#g/ml) which is lethal to 50% of the cells (EC50). Viability was determined by vital dye uptake in the treated populations relative to the untreated controls. Purpose and proposed use of test. The fibroblast cytotoxicity test has been proposed as a screen to be used before in vivo testing. Use in risk assessment. See above. B. Data on Fibroblast Cytotoxicity Assay Table 23 summarizes the linear correlation between the in vivo tissue scores and the in vitro scores from the fibroblast cytotoxicity assay performed as part of the Soap and Detergent Association Phase III study and submitted by laboratory CC114. Table 24 shows

110

J. W. Harbell et al. Table 23. Summary of the correlation between the in t,n,o and in t,itro values for the fibroblast cytotoxicity assay in the SDS Phase 111 study Laboratory: CC114 Exposure: 1 hr In vit,o/in vitro correlation (semi-log plot) Number of materials 22 16 6

Material type or class Total Neat materials, generally surfactants Formulated products

Range of scores

Tissue

MAS range =0~-1 EC50 score range (,ug/ml) = 5-> 5500

r= *

Corneal opacity

-0.40/-0.57

Corneal area Iris Conjunctival redness Chemosis Discharge MAS Days to clear

NPt -0.38/-0.59 -0.69/-0.83 - 0 . 5 3 / - 0.65 NP - 0.52/- 0.80 -0.46/-0.50

*The 22 test materials contained several materials with appreciable titratable alkalinity (/>0.1 meq HCI/g); one sample was 5.25% sodium hypochlorite. The analysis of the correlation data was performed on the complete set of 21 materials (upper correlation number) and on only the neutral test materials (and without sodium hypochlorite) (lower number) (e.g. MAS r = -0.52 complete v. -0.80 restricted). The toxicity of the alkaline materials and hypochlorite was appreciably underpredicted in vitro. tNot provided.

t h e specific m a t e r i a l s tested, t h e i r D r a i z e M A S a n d in vitro scores. C . Overall analysis o f data In vivo d a t a set: t h e s t a n d a r d D r a i z e p r o t o c o l w a s u s e d w i t h six a n i m a l s p e r test m a t e r i a l . O b s e r v a t i o n s were m a d e at 1, 24, 48 a n d 72 hr. A s r e q u i r e d , a n i m a l s were m a i n t a i n e d u p to 35 d a y s . C o m p a r i s o n s were b a s e d o n s c o r e s o b t a i n e d at ~ > 2 4 h r a f t e r d o s i n g . T o x i c i t y r a n g e w a s well b a l a n c e d , w i t h approximately half of the MAS scores between 1 and 20 (full r a n g e 1-41). S t u d i e s were c o n d u c t e d o n c o d e d c o m p o u n d s ( b o t h in vivo a n d in vitro), a n d in vivo s t u d i e s were c o n d u c t e d u n d e r t h e p r i n c i p l e s o f G L P . Test materials included representative surfactants, bleach ingredients and formulated products. When

a p p r o p r i a t e a n d possible, t h e c l e a n i n g p r o d u c t i n g r e d i e n t s were t e s t e d at c o n c e n t r a t i o n s likely to be f o u n d in c l e a n i n g p r o d u c t s . T h e test m a t e r i a l s a n d test c o n c e n t r a t i o n s were selected to o b t a i n a r a n g e o f eye i r r i t a t i o n r e s p o n s e s . In vitro d a t a : t h e in vitro r e s p o n s e s t e n d e d to be s o m e w h a t p o l a r i z e d , w i t h E C 5 0 v a l u e s o f >~ 1250 # g / m l o n o n e h a n d a n d < 2 0 0 / ~ g / m l o n t h e other. T h u s , t h e ability to r e s o l v e toxicities b e t w e e n m i l d a n d severe w a s limited, e v e n t h o u g h t h e r v a l u e s were s i m i l a r to t h o s e f r o m t h e o t h e r a s s a y s u s i n g t h e s e s a m e m a t e r i a l s . A s w i t h t h e o t h e r a s s a y s in this series, toxicities o f a l k a l i n e a n d h y p o c h l o r i t e m a terials were u n d e r p r e d i c t e d . No discussion of c o n c u r r e n t positive c o n t r o l s o r a c c e p t a n c e criteria was provided.

Table 24. Fibroblast cytotoxicity assay from the Soap and Detergent Association Phase Ill study Laboratory: CC114

Identity of sample Ethanol, 15% Polyethylene glycol, 400 mw, neat lsopropanol, 15% Butoxyethanol, 5% Glycerol, neat Alcohol ethoxylate, 10% Dodecylamine oxide, 1% Dimethylditallowammonium chloride, 3% Alkylethoxysulfate, 10% Triethylammonium lauryl sulfate, 10% Sodium laury| sulfate, 10% Ammonium lauryl sulfate, 10% Alkylglyceryl sulfate, 10% Sodium perborate Sodium alkyl sulfate, 10% Sodium hypochlorite, 5.25% Liquid cleaner Powdered cleaner Liquid hand soap Powdered cleaner Powdered bleach with protease Pine oil cleaner

Physical form

Alkalinity (meq HCl/g)

Maximum average Draize score

In t,itro assay score (pg/ml)

Liquid Liquid Liquid Liquid Liquid Liquid Liquid Liquid

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

1.0 2.0 2.3 2.7 13.8 14.7 14.8 18.8

> 1250 > 5500 > 1250 > 1250 > 1250 88 > 1250 81

Liquid Liquid Liquid Liquid Liquid Solid Liquid Liquid Liquid Solid Liquid Solid Solid Liquid

0.004 0.0 0.0 0.0 0.0 5.36 0.0 0.0 0.044 0. I 18 0.0 0.816 4.7 0.0

18.8 25.7 27.0 32.7 34.3 34.3 36.2 41.0 15.8 17.3 24.5 34.3 34.3 41.3

153 162 176 101 130 5 67 > 1300 317 42 162 367 530 108

Working Group 4: Cell cytotoxicity assays

111

Table 25. Summaryof the correlationbetweenthe in vivo and in vitro valuesfor the cornealplasminogenactivator assay in the SDS Phase 111study Laborat,ary: CC117 Exposure: 5 min In vivo/in vitro correlation

(semi-log plot) Number of materials 22 16 6

Material type or class Total Neat ingredients generally surfactants Formulated products

Range of scores MAS range =(~41 In vitro score range = 0.99-973

Tissue

r= *

Corneal opacity

-0.53/-0.67

Corneal area Iris Conjunctival redness Chemosis Discharge MAS Days to clear

NPt - 0 . 5 9 / - 0.61 -0.84/-0.87 - 0 . 6 6 / - 0.70 NP -0.77/-0.89 -0.45/-0.54

*The 22 t,.~st materials contained several materials with appreciable titratable alkalinity (>/0.1 meq HCI/g); one sample was 5.25% sodium hypochlorite. The analysis of the correlation data was performed on the complete set of 21 materials (upper correlation number) and on only the neutral test materials (and without sodium hypochlorite) (lower number) (e.g. MAS r = - 0 . 7 7 complete v. - 0 . 8 9 restricted). The toxicity of the alkaline materials and hypochlorite was appreciably underpredicted in ritro. t N o t provided.

D. Conclusions a~d recommendations

The relatively limited resolution of this assay on this set of test mal:erials may warrant further analysis. Even with the understanding that such assays are not appropriate for certain classes of test materials, the data from this assay appeared more polarized and less predictive than similar assays using the same test set (e.g. neutral red uptake, SIRC, or corneal plasminogen activator). Perhaps the exposure time or scoring method might be examined and revised. Most of the assays in the SDA submission were best able to predict conjunctival redness. The fibroblast cytotoxicity assay also did best with this endpoint, although other assays gave more definite resolution over the full range of toxicities. The 3T3 cells are used routinely by several laboratories for cytotoxicity assays. Most protocols call for a 24 hr test article exposure period. An assay using the 3T3 cell system with a 24 hr exposure and neutral red uptake endpoint was included in the European Comnmnity/British Home Office study. Data from that study should be available in late 1996.

Corneal Plasminogen Activator Assay A. In vitro assaT: Corneal Plasminogen Activator Assay (CEPA) Basis of assay Corneal epithelial cells in culture secrete plasminogen activator into the medium. The presence of the enzyme is measured with a colourimetric substrate to determine the concentration of the enzyme. Decreases in the enzyme may come from inhibition of protein synthesis or cell loss. Protocol. The procedure is based on the work of Chan (1985 and 1987). The release of corneal plasminogen activator from rabbit corneal epithelial cells in a primary culture may be measured and used to determine eye irritation potential after a 5 min exposure to a test material and a 48 hr recovery

period. Treated cultures are compared with control cultures. The results are reported as the inverse of the concentration of test material which reduces plasminogen activator release by 50% relative to controls. This value (expressed as percent of neat material or a saturated solution) is the CEPA score and has no units. References suggest that three trials are generally performed for each test material. Pearson's correlation coefficients are supplied below for each of the endpoints determined. Purpose and proposed use of test. Release of plasminogen activator from cultured cells exposed to ocular irritants is thought to represent eye irritation potential. Use in risk assessment. The submitter reports that CEPA has been proposed as a replacement for animal testing; however, it has not been used as such. It is reported that CEPA can distinguish severe from mild irritants. B. Data on Corneal Plasminogen Activator Assay

Table 25 summarizes the linear correlation between the in vivo tissue scores and the in vitro scores from the corneal plasminogen activator assay performed as part of the Soap and Detergent Association Phase III study and submitted by laboratory CC117. Table 26 shows the specific materials tested, their Draize MAS and in vitro scores. C. Overall analysis o f data In vivo data set: the standard Draize protocol was used with six animals per test material. Observations were made at 1, 24, 48 and 72 hr. As required, animals were maintained up to 35 days. Comparisons were based on scores obtained at >/24 hr after dosing. The toxicity range was well balanced, with approximately half of the MAS scores between 1 and 20 (full range 1 4 1). Studies were conducted on coded compounds (both in vivo and in vitro). In vivo studies were conducted under the principles of GLP. Test

J.W. Harbell et al.

112

materials included representative surfactants, bleach ingredients a n d formulated products. W h e n appropriate a n d possible, the cleaning p r o d u c t ingredients were tested at concentrations likely to be f o u n d in cleaning products. The test materials a n d test concentrations were selected to o b t a i n a range of eye irritation responses. In vitro data: the in vitro responses tended to be less polarized t h a n some o f the other in vitro data sets in this series. The best correlations were obtained with conjunctival redness a n d MAS. Alkaline a n d hypochlorite materials were underpredicted. N o discussion o f concurrent positive controls or acceptance criteria was provided.

D. Conclusions and recommendations The C E P A test is not as predictive for alkaline or water-insoluble materials. Conjunctival redness a n d M A S showed the best correlation to the C E P A test. Like the SIRC cytotoxicity assay in this series, the C E P A examines the cell p o p u l a t i o n after it has h a d a n o p p o r t u n i t y to recover from the exposure to the test article. Because very few laboratories or companies have conducted comprehensive studies on the C E P A test, m u c h more research is necessary before its value as a n in vitro irritation screen for diverse chemicals/ p r o d u c t groups can be determined.

SIRC Cell Cytotoxicity Assay (Biogir S.A. Protocol)

A. In vitro assay: S I R C Cell Cytotoxicity Assay Basis o f assay. The assay is based on short-term cultures o f the S I R C rabbit corneal cell line with cytotoxicity as the assay endpoint. The test m e c h a n i s m is based o n the observation that some

materials that are d a m a g i n g to the eye a p p e a r to be cytotoxic to a n u m b e r of cell types. Protocol. S I R C cells are g r o w n in M E M with 10% foetal bovine serum a n d antibiotics at 37°C in a humidified a t m o s p h e r e of 5 % CO2. F o r the assay, cells are seeded at 2-3 × 104 cells per well (200 pl medium) in a Transwell cell culture c h a m b e r with 0.4 p m pores. The inserts are then placed into wells of a corresponding c h a m b e r plate containing 600/~1 of medium. The cells are incubated for 48 hr to allow m o n o l a y e r formation. After incubation, the inserts are transferred to a new cluster plate. The wells of this new plate contain the test materials. F o r cosmetic ingredients, the wells contain 100 pl of the test material overlayered with 400 #1 of mineral oil; control wells contain mineral oil alone. The Transwell inserts are placed o n t o the mineral oil in such a way that there is a b o u t a 1-mm space between the test material a n d the filter b o t t o m o f the Transwell. F o r surfactant-containing formulations, the test solutions are prepared in culture m e d i u m a n d placed directly into the well w i t h o u t mineral oil. The c o n c e n t r a t i o n o f the surfactant test materials is determined by the c o n c e n t r a t i o n used to test in vivo. F o r these studies, those materials tested at 30% or at 10% in vivo were tested at 0.1% or 0.033% in vitro, respectively. Each material is tested at only one concentration. Wells containing only culture medium serve as controls. Nine Transwells are prepared for each test material. In m a n y of the surfactant studies, 12 wells were used for each time point. Three Transwells are assessed for viability at each of three time points (30, 60 a n d 240 min of exposure). M T T reduction is used as the basis for determining viability unless the test material interferes with this

Table 26. Corneal plasminogenactivator assay (CEPA) from the Soap and Detergent Association Phase Ill study: Laboratory: CC117 Maximum In vitro Physical Alkalinity averageDraize assay score Identity of sample form (meq HCI/g) score (no units) Ethanol, 15% Liquid 0.0 1.0 973 Polyethylene glycol, 400 mw, neat Liquid 0.0 2.0 405 Isopropanol, 15% Liquid 0.0 2.3 650 Butoxyethanol, 5% Liquid 0.0 2.7 290 Glycerol, neat Liquid 0.0 13.8 249 Alcohol ethoxylate, 10% Liquid 0.0 14.7 3.17 Dodecylamine oxide, 1% Liquid 0.0 14.8 20.9 Dimethylditallowammonium Liquid 0.0 18.8 64.8 chloride, 3% Alkylethoxysulfate, 10% Liquid 0.004 18.8 3.55 Triethylammonium lauryl sulfate, 10% Liquid 0.0 25.7 2.19 Sodium lauryl sulfate, 10% Liquid 0.0 27.0 0.99 Ammonium lauryl sulfate, 10% Liquid 0.0 32.7 1.24 Alkylglyceryl sulfate, 10% Liquid 0.0 34.3 8.8 Sodium perborate Solid 5.36 34.3 6.27 Sodium alkyl sulfate, 10% Liquid 0.0 36.2 8.21 Sodium hypochlorite, 5.25% Liquid 0.0 41.0 1.12 Liquid cleaner Liquid 0.044 15.8 4.87 Powdered cleaner Solid 0.118 17.3 1.3 Liquid hand soap Liquid 0.0 24.5 1.53 Powdered cleaner Solid 0.816 34.3 3.74 Powdered bleach with protease Solid 4.7 34.3 6.98 Pine oil cleaner Liquid 0.0 41.3 1.35

Working Group 4: Cell cytotoxicity assays

113

']?able27. Summaryof the correlationbetweenthe in vivo and in vitro valuesfor the SIRC cell cytotoxicityassay on surfactants Laboratory: CC119

Exposure: 0.5-4hr ]n vitro~in ritro correlation (linear/linear plot)

Number of

Material type

Range of

materials

or class

scores

35

Surfactantbased products

MAS range = 1-49 In vitro score range = 0-50

endpoint. In that case, the neutral red uptake assay is used. Dye reduction is compared in the treated and control cultures ~tt each time point. The Mean Cytotoxicity Inde~ (MCI) is determined from the formula: MCI =

Tissue

r=

Corneal opacity

0.44

Corneal area Iris Conjunctival redness Chemosis Discharge MAS (I hr) MAS (24 hr)

0.49 0.22 0.12 0.11 0.55 0.24 0.52

Figure 16 shows the plot of the Draize MAS v. the in vitro scores for this study.

Table 28 summarizes the linear correlation between the in vivo tissue scores and the in vitro scores from the SIRC cell cytotoxicity assay submitted by laboratory CC119 for cosmetic products. It should be noted that most of the cosmetic formulations tested were creams, lotions and powders, which are traditionally not compatible with submerged cell culture-based systems. Figure 17 shows the plot of the Draize MAS v. the in vitro scores for this study.

C39 + 0.5"C60 4- 0.125"C240 3

where C30, C60 and C240 are the percent cytotoxicity (with 100% being complete kill) recorded after 30, 60 and 240 min, respectively. This method was published by Boue-Grabot e,' al. in 1992. Purpose and proposed use o f test. The submitters did not propose a specific application for this assay. Use in risk assessment. Not addressed.

C. Overall analysis o f data In vivo data set" adult New Zealand white albino rabbits were used; three were treated with each test material. Each rabbit received 0.1 ml of liquid test material placed into the everted lower lid of the right eye. Lids were held together for l0 sec. Animals were restrained for only 1 hr after dosing. Readings were performed at l, 24, 48, 72 and 96 hr and at 7 days after dosing according to the scoring procedures of

B. Summary data tables Table 27 summarizes the linear correlation between the in vivo tissue scores and the in vitro scores from the SIRC cell cytotoxicity assay submitted by laboratory CCl19 for surfactant-based products. 60.

r -

50.

O

40. • i

o

20.

oa

O

OO O

30-

10.

0.52

,0

•

o

O o

o

O

O

# O •

O

O

O

Oo

o

O

In Vitro$¢m¢o Fig. 16. Plot of the Draize MAS (24 hr) values v. the SIRC cell cytotoxicity assay (Biogir method) in vitro scores for a series of surfactant-based formulations.

114

J.W. Harbell et al. Table 28. Summary of the correlation between the in vivo and in vitro values for the SIRC cytotoxicity assay on cosmetics Laboratory: CC119 Exposure: 0.5-4 hr In vivo/in vitro correlation (linear/linear plot)

Number of materials 55

Material type or class Cosmetics

Range of scores MAS range = 1-45 In vitro score range = 0-50

the Journal Officiel de la Rkpublique Fran¢aise (9 February 1985). Sodium fluorescein was instilled to detect or confirm corneal lesions and area of involvement. Cosmetic products were administered as supplied; surfactant-based formulations were tested at 30% or 10% concentration. In vivo responses for the surfactant-containing formulations ranged from 3 to almost 50 (MAS at 24 hr). The majority of the values were < 25 but the distribution of scores was fairly uniform over the range up to about 40. Individual tissue scores tended to show much more clustering. The majority of the opacity scores were 2, and all but one of the products gave a conjunctival redness score of 1 (irrespective of the other tissue scores). In vivo responses for the cosmetic ingredients covered a range of 1-44 (MAS at 24 hr) with the greatest concentration of scores < 10. This is not surprising for cosmetic formulations, but did lead to some polarization of the data between the very mild formulations and those few in the more aggressive

Tissue

r=

Corneal opacity

0.63

Corneal area Iris Conjunctival redness Chemosis Discharge MAS (1 hr) MAS (24 hr)

0.71 0.67 0.23 0.72 0.69 0.19 0.79

category. Individual tissue scores showed some polarization except for conjunctival redness, for which most scores were 1. The corneal scores, in particular, showed polarization between a large group at 0 and a second group at 2. In vitro data sets: in vitro scores for the surfactant-containing products ranged from 0 to 50. Correlation with individual tissue scores was generally weak (particularly for iris and conjunctival effects). In vitro scores for the cosmetic formulations ranged from 0 to 52 with the majority about 10 or less. The correlation was particularly good between the conjunctival chemosis and discharge scores and the in vitro scores for which the data were better distributed. The higher correlation values for opacity and iris reflect polarization of the in vivo data. The comparison between the MAS (at 24 hr) and the in vitro score was generally good for resolving between the less irritating ( < 25 MAS) and the more irritating. The apparent resolution between very mild and less mild formulations was weak. However, it should be

NI

40

r =0.79

O O O O 0

i

20, •

O

O O O

0

10~ oo

,~ITf

, 0, *

In VIIIra__~,e_. Fig. 17. Plot of the Draize MAS (24 hr) values v. the SIRC cell cytotoxicity assay (Biogir method) in vitro scores for a series of cosmetic formulations.

Working Group 4: Cell cytotoxicity assays

115

Table 29. Summaryof the correlationbetweenthe in vivo and in vitro valuesfor the dual dye cytotoxicityassay in the CTFA Phase Iil study laboratory: CC112 F.xposure: 4 hr In vivo/in vitro correlation (semi-logplot) Number of Materialtype Range of materials or class scores Tissue r= 25 Surfactant-based MAS range Corneal opacity -0.48 formulations = 0-42 DDA score range Cornealarea -0.82 = 3-55 Corneal score -0.82 Iris -0.73 Conjunctivalredness -0.63 Chemosis - 0.70 Discharge - 0.72 Conjunctivalscore -0.72 MAS -0.79 Days to clear NP* *Not provided.

remembered that tile in vivo values in this range are highly variable and therefore provide poor prediction themselves. Conclusions regarding correlation in this range should be made with caution. D. Conclusions and recommendations

This submission represents 90 materials tested, The surfactant protocol tested with 35 formulations did not produce data with high correlation to any of the individual tissue scores or the MAS scores. On the other hand, the cosmetic formulation protocol tested on 55 formulations does show considerable promise. This data set was taken from formulations which are traditionally not compatible with submerged cell culture systems (i.e,. creams, lotions, powders, etc.). Therefore, the good correlation between the in vitro scores and certain in vivo parameters suggests that this assay may be able to address a unique range of test articles and should undergo further evaluation in several laboratories.

Dual Dye Cytotoxieity Assay A . In vitro assay: Dual Dye Cytotoxicity Test Basis o f assay. This assay is classified as a live--dead assay and evaluates the viability of cells on the basis of the presence of cytoplasmic esterases (live) and the ipenetration of a damaged cell membrane by a substance which would be excluded by a normal, intact cell membrane. Fluorescein diacetate is the dye used to evaluate the viability of cells in this assay. It readily diffuses into the cells, where the presence of esterase in the cytoplasm cleaves it, resulting in the creation of a compound which fluoresces green under UV light and will only diffuse out of intact cells at a slow rate. Ethidium bromide will fluoresce red in UV and does not readily penetrate the membrane of intact cells. This compound has a high affinity for nucleic acids and the nucleus of a dead cell (permeable membrane) will thus appear red. Protocol. The test protocol was adapted from the method previously described by Scaife (1985). L929

mouse fibroblast-like cells (ATCC-CCLI) are grown as monolayer~s in Eagle's basal medium (BME) with 5% foetal bovine serum and antibiotics. Cells are trypsinized to obtain cell suspensions at l0 6 cells/ml for the testing. Serial dilutions of the test samples and standard control are prepared by diluting the samples with culture medium. Sodium dodecyl sulfate (SDS 10% in water) is used as the control to which all other values are normalized. Aliquots of 5 ml of cell suspension are prepared, and 50/d portions of test sample (dilution) are added. The tubes are incubated at 37°C for 4 hr on a rocking platform. At the end of the incubation period, 5 ml of saline containing fluorescein diacetate at 5/~g/ml and ethidium bromide at 300 pg/ml is added to each tube. The percentage of cells staining with each dye is determined by using a fluorescent microscope and image analyser. Viable cells stain green and dead cells stain red. Probit analysis is used to determine the dose giving 50% toxicity (EC50). All data are normalized to the SDS EC50, and the normalized data are expressed as Relative Units (RU). Purpose and proposed use o f test. CC112 participated in the third phase of the CTFA study on evaluation of in vitro alternatives to the Draize ocular irritation test. This phase evaluated a panel of 25 surfactant products. The two in vitro procedures (dual dye and transepithelial permeability assays) used in this study have been validated at this company using data generated in-house by a modified rabbit ocular irritation test, as described previously, and may not be relevant to most of those laboratories doing ocular screening on strong irritants (Kemp et al., 1983). These methods may serve as potential screening methods for detecting ocular irritation of proprietary formulations. Use in risk assessment. The submitter did not indicate the present use of the method for ocular irritation testing of consumer products in the United States. It has been used in the U K by this company for evaluating surfactant products.

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B. Data f r o m the Dual Dye Cytotoxicity Assay

point. The scores for the corneal area were well distributed over the entire range of the scoring system. When the corneal opacity and corneal area scores were used to compute the corneal scores, the values showed a fairly uniform distribution at the lower end of the range, 20 and below. Iris scores were well distributed at 1.0 and below (maximum range limit of 2.0). Conjunctival redness, chemosis and discharge were fairly evenly distributed, generally in the moderate or less regions of the ranges. The conjunctival scores were fairly well distributed over the range below 15, where the maximum score possible is 20. The MADS were evenly distributed below 40, with the maximum possible score being 110. It is clear that the range of samples tested in the study were generally mild irritants or less. This would be expected, since they were all consumer products consisting of cosmeticr toiletries and household cleansers. This limits the ability of the in vitro models used in this study to make distinctions concerning the degree of irritation greater than that of the samples used in the test panel. The in vitro data were transformed to common logarithms for the statistical analyses. All data were evaluated by using BMDP statistics to prepare scatterplots and calculate the regression coefficients. In the cases of censored data, for which an actual value could not be calculated because of extreme mildness or the limitation of the assay procedure, that data point was not plotted. Two such cases occurred for each of the in vitro models. Positive controls are performed with each assay. However, no indication of acceptance criteria or historical control data were provided. Normalization of the test material responses against the SDS EC50 would tend to reduce assay-to-assay variability. The

Table 29 summarizes the linear correlation between the in vivo tissue scores and the in vitro scores from the dual dye assay submitted by laboratory CC112. This study was performed as part of the CTFA Phase 111 study. Figure 18 shows the semi-log plot of the Draize MAS v. the in vitro scores for this study. C. In vivo database summary

The study was a primary eye irritation study in rabbits as conducted by the CTFA Evaluation of Alternatives Program. The complete protocol has been published (Gettings et al., 1991). The standard Draize assay was used with six rabbits per test material. Anaesthetic was used. Testing was performed under code, using full GLPs. In vivo MAS scores were distributed over the whole range (0-10, six materials; 11-20, seven materials; 21-30, four materials; 31-40, eight materials). All the materials were surfactant-based formulations (11 were shampoos). The standard deviations for the six animals were quite large--in the mid-range of toxicity (average CV approx. 40%). This CV value is consistent with other published data and is not a negative reflection on the laboratory performing these tests. This variability in the in vivo responses should be considered in comparing the in vivo results with the in vitro data. D. Overall analysis o f data

The data selected from the in vivo scores was first used to plot histograms by using the BMDP software programs (BMDP Statistical Software Inc., Los Angeles, CA). Each tissue was graphed separately, and the combined scores were used as well. The data for opacity showed most of the data clustered at one 60

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E. Conclusions and recommendations These data showed rather consistent good correlations between the in vivo tissue responses and the in vitro scores. The corneal opacity scores were an exception. In this case, the in vivo scores tended to fall mostly between zero and one (see also SIRC, CCI00, and neutral red assays, CC104, using the same in vivo data set) so that the distribution of in vivo scores was limited. The submi'Lters point out, however, that the correlation value may not be strictly indicative of a truly useful correlztion. In some cases, particularly the iris and conjunctival scores, the wide standard deviations observed for the in vivo scores and narrow range of in vitro scores would make it difficult to use these plots to predict the irritation potential of unknown materials. The corneal scores (opacity x area) and MAS scores gave very useful plots, particularly for the identification of the more strongly irritating materials in the data set. Because this company's proprietary products generally produce mild irritation, it has attempted to develop in vitro and in vivo methods which can assess these products for potential ocular irritation. One conclusion that may be drawn from this study is that the in vivo data vary much more than the in vitro results. However, decisions on the marketability of a formulation are usually based on the in vivo results. Furthermore, in making comparisons between in vivo and in vitro data, Jlt is often better to use combined or total eye scores (cornea, conjunctiva, or maximum average daily scores) to assess the relevance and reliability of the it; vitro methods. Although these data are encouraging, the dual dye assay has generally been used by the submitters in testing their proprietary (and usually mild) formulations. Additional users will need to assess its potential and value for evaluating other test materials before a definitive statement can be made about its utility as a general in vitro screen for ocular irritation.

K562 Tryl[mn Blue Exclusion Assay A. In vitro assay: K562 Trypan Blue Exclusion Assay Basis o f assay. The assay is based on short-term

cultures of transformed human cells with cytotoxicity (membrane leakag,~) as the assay endpoint. Cells with intact membranes :;hould exclude the dye trypan blue and thus will remain unstained. Cells with damaged membranes (although some disagreement exists as to how much damage is required) will allow the dye to pass and stain cytoplasmic components. For a given cell, the response is all or none. The submitters point out that most test batteries involve a cytotoxicity assay, the justification being that necrosis or cell death (associated with changes in corneal permeability) is an important factor in the determination of ocular irritancy in vivo. Since the mechanism of

117

action of all surfactants on the cell surface is related to membrane disruption, simple cytotoxicity models could be expected to predict in vivo irritancy for this class of materials. Protocol. The target cell line was originally derived from a chronic myelogenous leukaemia patient (Lozzio and Lozzio, 1975) and is non-adherent and fast growing. Cells were seeded at 105cells/ml in complete RPMI 1640 containing 10% heat-inactivated newborn calf serum and 2 mM L-glutamine, and 48-well plates were loaded with complete medium containing dilutions of the test surfactants (100/~!/ well). The prepared plates were preincubated for 30 min in a humidified atmosphere containing 5% CO2 at 37°C. The test was started by the addition of 100/zl of cell suspension (107cells/ml) into the surfactant or control wells. Wells were incubated for 15 min (duplicate samples). After the incubation period, 800 pl of Hanks' balanced salt solution was added to each well and mixed. Trypan blue dye (0.5%) was added to all wells immediately before cell counts were performed on a haemocytometer. At least 100 cells per well were counted and the percentage of viable (non-staining) cells was determined. Average percentage viabilities were calculated and expressed relative to control values. This method has been published (Lewis et al., 1993). Protocol use. This assay is used as part of an in vitro test battery (Lewis et al., 1994) which is routinely used as a pre-screen to ensure humane in vivo testing. Range o f responses. The Maximum Mean Total Draize Scores (MMTS) of the 14 surfactants ranged from 0 to 98.5. Although the majority of the scores were at the milder end of the scale, the data set was reasonably well balanced. In vitro scores (TC 50) ranged from 0.35 to > 10 mM. Range o f materials amenable to use in the assay. All of the materials tested for the data submitted were surfactants. Based on other in vitro cell cytotoxicity assays, it might be expected that only water-soluble compounds, without extremes of pH or reactivity, would be appropriate for this assay. Use in risk assessment. Use in risk assessment is not addressed by the submitters.

B. Summary data table Table 30 summarizes the linear correlation between the in vivo tissue scores and the in vitro scores from the K562 trypan blue exclusion assay submitted by laboratory CC115. This study was performed on surfactants as part of a company study. Figure 19 shows the semi-log plot of the Draize MAS against the in vitro scores for this study. C. Overall analysis o f data In vivo data set: the in vivo data were taken from the database of studies of these surfactants performed for or by the submitter. Individual tissue scores were provided, but no details of the experimental protocol

118

J.W. Harbell et al. Table 30. Summary of the correlation between the in vivo and in vitro values for the K562 trypan blue exclusion assay on surfactants Laboratory: CC115 Exposure: 15 min In z:ivo/in vitro correlation (linear/linear plot) Number of Material type Range of materials or class scores Tissue r 14 Surfactants MM'I'S Corneal opacity -0.59 =0-98.5 TC50 range Corneal area -0.71 =0.35-> l0 mM Iris -0.51 Conjunctival redness -0.72 Chemosis - 0.66 Discharge - 0.67 MAS -0.52 Days to clear -0.39 =

D. Conclusions and recommendations

were given. The n u m b e r o f animals varied between one a n d six per test material but the n u m b e r was not related to toxicity. Thus, the in vivo data set is weak. In vitro data set: in vitro scores tended to fall at log intervals between 0.35 a n d > 10 mM. The n u m b e r o f replicate assays c o n t r i b u t i n g to the reported scores was not provided. Historical control ranges or acceptance criteria were not provided. The in vivo scores were c o m p a r e d with the in vitro scores t h r o u g h linear plots. Limited i n f o r m a t i o n a b o u t the conduct o f these experiments m a k e s analysis difficult. W h e n the data were c o m p a r e d with individual tissue scores, some o f the d a t a were polarized, which m a y have produced correlation coefficients that exceeded the actual predictive value o f the data. Scoring of this assay m a y be s o m e w h a t subjective a n d the a m o u n t of trypan blue t a k e n up by the cells is dependent on b o t h time o f exposure a n d the nature o f the m e m b r a n e damage.

A t present, this assay does not a p p e a r to resolve differences a m o n g surfactants over the range o f toxicities tested in this study. A l t h o u g h additional work may address some o f these problems, other cell cytotoxicity assays reviewed by this committee m a y be more a p p r o p r i a t e to this task. Red Blood Cell Lysis Assay A . In vitro assays: Red Blood Cell Lysisa Assay Basis o f assay. The assay is based on short-term exposure to r a b b i t red b l o o d cells with cytotoxicity (haemoglobin release) as the assay endpoint. The submitters point out t h a t most test batteries involve a cytotoxicity assay, the justification being t h a t necrosis or cell d e a t h (associated with changes in corneal permeability) is a n i m p o r t a n t factor in the determination o f ocular irritancy in vivo. Since the m e c h a n i s m of action o f all surfactants o n the cell surface is related to m e m b r a n e disruption, simple

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119

Table 31. Summary of the correlation between the in vivo and in vitro values for the red blood cell lysis assay on surfaetants Laboratory: CC115 Exposure: 60 rain In vivo/in vitro correlation (linear/linear plot) Number of Materialtype Range of materials or class scores Tissue r= 14 Surfactants MMTS Corneal opacity -0.76 = 0-98.5 TC50 range Corneal area -0.93 =0.05-> 10 mM Iris -0.75 Conjunctival redness -0.82 Chemosis - 0.79 Discharge - 0.84 MAS -0.81 Days to clear -0.67

cytotoxicity models could be expected to predict in vivo irritancy for this class of materials (see O k a m o t o et al., 1990). Protocol. The procedure was a modification of that described by Harington et al. (1971). Packed red blood cells recovered from whole rabbit blood were suspended at a co~acentration of 2% in phosphate buffered saline. Test surfactants (final concentrations from 10 -2 to l0 -6 M in assay buffer) were incubated with the red blood cells at 20°C for 60 min with gentle shaking. The degree of haemolysis was quantified by spectrophotometric measurement of the absorbance (optical density) at 541 nm to determine haemoglobin release. Results were expressed relative to a sample totally lysed in deionized water. Fragility controls were performed. Endpoint data were expressed as the concentration giving 50% haemoglobin release relative to the total lysis control. Protocol use. This assay is used as part of an in vitro test battery which is routinely used as a pre-screen to ensure humane in vivo testing.

Range o f responses. The Maximum Mean Total Draize Scores (MMTS) of the 14 surfactants ranged from 0 to 98.5. Although the majority of the scores were at the milder end of the scale, the data set was reasonably well balanced. In vitro scores (TC 50) ranged from 0.05 to > 10 mM. Range o f materials amenable to use in the assay. All of the materials tested for the data submitted were surfactants. Based on other in vitro cell cytotoxicity assays, it might be expected that only water-soluble compounds, without extremes of pH, would be appropriate for this assay. Use in risk assessment. Use in risk assessment was not addressed by the submitters.

B. Summary data table Table 31 summarizes the linear correlation between the in vivo tissue scores and the in vitro scores from the red blood cell iysis assay submitted by laboratory CC115. This study was performed on surfactants as part of a company study. Figure 20 shows the plot of

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the Draize MAS against the /n vitro scores for this study. C. Overall analysis In vivo data set: the in vivo data were taken from the database of studies of these surfactants performed for or by the submitter. Individual tissue scores were provided, but no details of the experimental protocol were given. The number of animals varied between one and six per test material but the number was not related to toxicity. Thus, the in vivo data set is weak. In vitro data set: the in vitro data were extremely polarized; six values were 0.05 mM and six values were > 10 raM. Only two scores were intermediate (40 and 5raM). All of the scores of ~>40mM came from surfactants with MMTS of ~<10.2; the scores of ~<5mM came from surfactants with MMTS of 17.2 98.5. The number of replicate assays contributing to the reported scores was not provided. Historical control ranges or acceptance criteria were not provided. Linear plots were used to compare the in vivo scores with the in vitro scores. The limited information on the conduct of these experiments makes analysis somewhat difficult. The extreme polarization of the in vitro data probably leads to over-prediction of the real correlation between the in vivo and in vitro data over the whole range of the assay. However, the clear demarcation between the very mild (or very severe) surfactants and the remaining test materials suggests that the assay may be appropriate for differentiating surfactants (and potentially surfactant formulations) in this very mild subgroup.

D. Conclusions and recommendations

Although the assay shows promise in differentiating between very mild and non-mild surfactants (and formulations), considerable additional data would be required. In particular, more examples of anionic, amphoteric and cationic surfactants should be tested. Also, a battery of surfactant formulations should be used to address the use of this assay for formulations. Discussion 12 cell cytotoxicity assays (27 data sets) have been reviewed. A wide range of target cell types, exposure times and assay endpoints have been used. The majority of the data sets were derived from the study of surfactant-based materials. In many cases, there was good correlation between the in vitro scores and the in vivo tissue responses. Most pronounced were the particularly good correlations between the in vitro scores and conjunctival redness scores across most of the assays when water-soluble materials were tested. The S1RC cell cytotoxicity assay (Dial protocol), neutral red uptake assays, neutral red release, corneal plasminogen activator assay, dual dye assay and RBC assay were among those that showed good correlations with conjunctival redness for water-soluble test materials. These assays also showed very

credible correlations with the MAS scores in the same studies. In contrast, the SIRC cytotoxicity assay (Biogir protocol) showed generally poor correlations when surfactant materials were tested. However, this assay showed good correlations to most tissue scores, except conjunctivai redness, when non-water-soluble cosmetic formulations were tested (see Tables 27 and 28). This assay's special method of test material presentation to the cells may have circumvented the usual problems seen in testing non-water-soluble materials with monolayer cell systems. The poor correlation with conjunctivai redness scores may have resulted from the unique distribution of in vivo scores; in almost all cases, a conjunctival redness score of l was obtained, irrespective of the other tissue scores for the test material. The mechanism by which a class of materials exerts its damage in the eye will have a significant impact on the type of in vitro assay which will be able to detect that type of damage. The mechanism by which surfactants damage (and thus kill) cells in vitro is thought to involve membrane disruption with resulting influx of ions and water. This disruption is dose-dependent, with the cell responding to sublethal challenge by increased active ion transport. The metabolic response in a cell population to such a sublethal surfactant dose has been well documented (see Rhoads et al., 1994; K. Miller et al., 1992, unpublished). With increasing concentrations, the cell's ability to maintain membrane integrity and ion balance is overwhelmed. The mechanistic basis for the correlation between cytotoxicity and conjunctival redness may relate to the common denominator of membrane perturbation. The surfactant's ability to perturb the cell membrane may provide the key to its ability to irritate the cells of the conjunctivae (and cornea). It is perhaps significant that conjunctival redness is often one of the first signs of irritation observed with most of the chemicals and products reported in this review. The relative ability to perturb cell membranes, as measured by the doses of test materials leading to cell death, may be important in predicting in vivo irritation. The generally lower linear correlation between in vitro scores and corneal opacity does not imply that cell membrane damage, with resulting lysis, is not a significant mechanism by which chemicals induce corneal damage (and resulting opacity). Surfactants and alkalies act in this fashion (Burns and Paterson, 1989; Green et al., 1978). Rather, the lower linear correlation may suggest that the scoring of the in vivo response (0-4) does not represent uniform increments of actual pathology. That is, the amount of cellular and tissue damage required to progress from a score of 0 (no opacity) to l (scattered or diffuse opacity, details of the iris clearly visible) to 2 (easily discernible translucent areas, details of the iris slightly obscured) to 3 (nacreous areas, no details of iris visible) to 4

Working Group 4: Cell cytotoxicity assays (complete corneal opacity) may not be uniform. If this is the case, a non-linear relationship between an in vitro score (e.g. the concentration of test material giving 50% toxicity) and corneal damage (as measured by opacity at 24 hr) might be expected. Although conjunctival redness may not represent the major area of concern in ocular safety, it is of considerable interest in situations in which even minor irritation would be unacceptable. For those products which are instilled in or applied around the eye, the ability of an in vitro assay to predict conjunctival redness would be of significant use. As a practical matter, historical data on a particular class of formulations may suggest that a prospective new or modified formulation is not going to be a severe eye irritant. However, the degree of absolute mildness may be the information required of an assay system. Several of the assay systems were able to resolve appreciable differences in toxicity among formulations which showed essentially no toxicity in vivo at 24 hr. The SIRC cell cytotoxicity (Dial protocol), neutral red uptake and neutral red release assays provided good examples of this resolution. At the other extreme, the ability to identify severe irritants would be a very useful feature for an assay. Such an assay could be used as part of a screening system to identify and eliminate such chemicals or formulations from further development (in cases in which such toxicity was unacceptable) or from in vivo testing for labelling purposes. Very few severe irritants were included among the data submitted. However, the ability of several assays to distinguish between mild and moderate irritants, particularly in the surfactant category, suggests that they would be able to identify severe irritants as well. Use of the assays to identify severe irritants should be limited to those types of test articles which are compatible with the systems (see below). How does exposure time affect the data produced by an assay? Acute in vivo exposures, particularly to humans, may range from 1 min to approximately 30 min, depending on natural tearing or intervention by rinsing. Chronic exposures, such as might occur from materials applied to the face or eyes, are also a concern, and the exposure times may be longer and recurrent. The in vitro studies reviewed in this report used test agent expcsure times ranging from 5 min to 48 hr. The very short exposure time followed directly by the endpoint measurement (i.e. neutral red release and K562 trypan blue exclusion assays) is intended to measure immediate toxicities such as membrane damage from surt~ctants. The SIRC cytotoxicity assay (Biogir protocol), with its 30, 60 and 240 min time points, also assesses more immediate damage. The SIRC cytotoxicity assay (Dial protocol) and CEPA limit exposure to 60 min but allow several days for recovery or delayed toxicity to be manifested. The dual dye assay uses a 4 hr exposure whereas most of the neutral red assays use 24 or 48 hr exposures. The neutral red assays measure both

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immediate and delayed (or cytostatic) effects, since one or more rounds of cell replication are possible during exposure. In addition, the long-term exposure means that the effective dose to the cells is higher, since equilibrium between the medium and cells can be reached. Certainly doses giving 50% toxicity among the assays were quite different. For example, a 10% SDS solution gave 50% toxicity at 176/~g/ml (fibroblast cytotoxicity), 124/~g/ml (SIRC-Dial) and 12-17 pg/ml (48 hr neutral red uptake). There is probably no universally optimum exposure time for in vitro assays (Bagley et al., 1992; Shopsis et al., 1987). The shorter exposure times may better approximate the actual in vivo exposure time, particularly for acute exposures to surfactants. For that reason, a short exposure may be appropriate for measuring irritation potential from certain classes of surfactants or surfactant products. For these classes of material, the correlations between the in vitro and in vivo data have been quite promising, especially for predicting conjunctival irritation. For the CTFA Phase III data set (surfactant materials), the neutral red release assay did provide a much better separation of the materials than did the 48 hr neutral red uptake assay performed in the same cells and medium. However, a short exposure may not prove sensitive enough for non-surfactants or formulations intended for use around the face or eyes. Cell replication in the presence of the test material provides a very sensitive measure of toxicity and may be essential for detecting certain types of delayed toxicity (such as those which interfere with cell replication or differentiation). Again, a good correlation was observed between the neutral red uptake assays (particularly the 24 hr exposures) and in vivo irritation. The longer exposure may be the more prudent approach for testing mixtures where both surfactant and non-surfactant materials are of concern. This report is not intended to stand alone. Although the actual submissions are too voluminous to include, review of the data plots is essential to understand the value of the assays. IRAG is working with submitters to make these individual tissue responses available. Wherever possible, sample graphs of the data (usually MAS v. in vitro scores) have been provided in the report, but these "samples" are not intended to substitute for the full set of tissue responses. These plots can be used to assess the overall spread and usefulness of the data. The r values also provide a "first approximation" of the relationship between the in vivo and in vitro responses for each tissue endpoint. The regression coefficient provides one estimate, but by no means the only one, of the strength of the correlation between X and Y. This measure of strength comes from an examination of the variance. The total variance is the sum of the regression variance and the error variance. The strength of the correlation is determined by the portion of the total

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where N is the number of values forming the regression. Thus, a specific r value determined for a large data set would yield a larger t value than the same r value determined for a small data set. It would be inappropriate to judge an assay (or submission) strictly or solely on the basis of the linear correlation between the in vivo and in vitro responses. The linear correlation was selected by the Guidelines Committee because it could be applied as a conservative and uniform measure across all data sets. However, several additional factors which can be discerned from the plots must be considered. Are the data balanced over the range of responses (both in vivo and in vitro)? Is the response linear over the

whole range of responses? How much variation is present in the in vivo and in vitro values? Was the same physical form tested in vivo and in vitro? Balanced data sets provide a much more rigorous test of an assay. Polarized data sets may sometimes appear to show a better correlation than is warranted. Many of the data sets show that the in vitro responses are not linear over the full range of the assay (compared with the in vivo responses). This non-linearity is seen especially at the low-toxicity end of the spectrum and suggests that the relative toxicity may be resolved to a far greater extent by the in vitro assays than the in vivo assay. Although this non-linearity does not necessarily detract from the usefulness of an assay or particular data set, it does decrease the correlation coefficient. How good can the real (statistically valid) correlation coefficient be, given the variability within the two assays being compared? The ideal of unity is approached only if there is very little variance in the data sets (B. Bruce, personal communication, 1993). The reality is that there is significant variance in the animal responses which often exceeds that found in the in vitro assay. Thus, the best real correlation coefficient may be far less than 1. The values of 0.7 to 0.8 may be very good when variances of 40% and 20% are seen in the in vivo and in vitro data sets, respectively. As a practical matter in setting our expectations for correlations between the in vitro and the in vivo data, we might look to the correlation between in vivo data generated in two species tested in parallel. Such a study was performed under the auspices of the Soap and Detergent Association (Green et al., 1978). In this study 11 materials (or dilutions) were tested in albino rabbits and rhesus monkeys. Standard Draize

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scoring, slit-lamp evaluation and histology were performed. Figures 21-23 show the comparison of the maximum average tissue responses in the two species. Figure 21 shows the complete corneal scores (opacity x area x 5~ which gave a linear correlation o f r = 0.42. Figure 22 shows the complete iris scores (iris × 5) which gave a correlation of r = 0.45. Figure 23 shows the complete conjunctival scores (2 x sum o f chemosis + redness + discharge) which gave an r value of 0.20. In general, the monkey was much less sensitive than the rabbit. If these data were evaluated in terms of the rabbit as the "regulatory standard," the monkey test might be judged to have limited

predictive potential. Data such as these should be considered in setting the requirements for new (in vivo and in vitro) assays. Finally, the physical form of the test material can have a major impact on its toxicity. Almost all of these assays require that test materials be placed in culture medium. If the material is insoluble in aqueous medium, contact with the submerged cells may be irregular at best and probably not dosedependent. The agar diffusion methods do not require the test material to be placed in medium but do require the "toxicologically active" substance to cross through an aqueous medium to reach the target

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cells. The encouraging results with cosmetic formulations from the SIRC cell cytotoxicity assay (Biogir protocol) suggest that this alternative approach may allow hydrophobic materials to reach the cell monolayer. This technique may have wider application, and other laboratories may wish to investigate this possibility. Buffering effects of the medium in vitro can alter extremes of pH. If the pH (or reserve acidity or alkalinity) accounts for the test material's toxicity, these in vitro methods will generally underestimate the toxicity. For some products (e.g. granular laundry powder), the solid form is far less toxic than a solution of the same product. Such materials, tested as solids in vivo, will appear far more toxic when tested as solutions in vitro. Of particular concern to this Working Group was the failure by many of the submitters to address concurrent controls performed with their assays. The Working Group must emphasize that the positive and negative controls are an essential part of all in vitro assays. In fact, concurrent positive and negative controls would be an essential part of all assays (in vitro and in vivo) were it not for concerns about additional animal use. In many cases, negative controls (medium alone) were used to determine 100% viability, but a concurrent positive control was lacking. The role of the concurrent positive control is to ensure that the assay is performing in a reproducible manner during each trial and to control for variations in cell number, incubation conditions, reagents and technique. It should be at least one of the acceptance criteria for a given trial. Generally, this is accomplished by taking the historical mean value and setting limits based on the standard deviation of that mean, the approach used by a number of the submitters (e.g. CCI00, CC103, CC104, CCI05, CCl12 and CC107). Acceptance criteria for a given assay trial are essential to both the generation of credible data and use of those data in the regulatory setting. The positive control responses are also extremely important in evaluating the reproducibility of an assay system, both within-laboratory and between-laboratories. Determination of the interlaboratory variability for an assay has a major impact on the use of data by the regulatory community, where interpretation of data from many sources is necessary. Finally, performing in vitro assays in the absence of concurrent positive controls reflects a lack of scientific rigour. Although such controls have been eliminated from in vivo ocular irritation studies for animal welfare concerns, no such difficulty exists with in vitro methods. Those laboratories performing in vitro assays without concurrent positive controls severely limit the credibility and utility of their data. Efforts to choose a "best" assay are confounded by many factors. Rather, it may be more productive to identify assays which perform less well for a given set of materials. The Committee was fortunate to have a

number of submissions with common in vivo data sets, thereby facilitating such selections. Six assays using the SDA Phase III data set, three assays using the CTFA Phase III data set, three assays using the corporate (CC107) data set and several other sets with multiple assays (CC106 and CC115) have been reported. Some of these same in vivo data sets have been used for in vitro assays covered by other Working Groups. Therefore, as a committee, we have not attempted to "rank" the assays but have tried to provide the analysis of the cell cytotoxicity assays which will aid the reader in making these choices. One should be extremely careful to consider more than comparison of simple correlation coefficients presented in this analysis. Many more factors can influence one's overall assessment (e.g. variability, resolution, assay mechanics and range of compatible test materials). Based on the data submitted, a number of the cell cytotoxicity assays show considerable promise as screens for ocular irritancy. None of the submitters recommended that their cell cytotoxicity assay be used as a sole replacement for in vivo assessment. Parallel testing of unknowns with "benchmark materials" (materials of similar composition which have "known" toxicities) in an in vitro assay will greatly increase its predictive value. For almost all of these assays, the materials being tested should be water-soluble/miscible. The toxicity of products with reserve acidity or alkalinity or high reactivity may be under-estimated. Certain assays may be preferred by a given user depending on the types of materials, range of toxicities and resources available. The cell cytotoxicity assays may serve as a valuable component of a tiered or battery testing program (Lewis et al., 1994). As with any assay, a sufficient number of replicate values, concurrent positive and negative controls, and a strict adherence to assay acceptance criteria are essential to produce credible data. REFERENCES

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