Stagewise, adaptive dose allocation for quantal response dose-response studies.

A principal design objective of many dose-response studies is to estimate extreme percentiles of a dose-response distribution, e.g., the ED95 dose for a particular drug therapy, as precisely as feasible using the smallest number of experimental subjects possible. Such a design requirement necessitates that allocation of subjects to drug doses be carried out in a stagewise fashion to maximize the information obtained from each subsequent experimental observation in light of what has previously been determined. This paper describes and illustrates specialized methods and associated computer programs to evaluate, on a stagewise basis, the anticipated relative sensitivities of alternative experimental plans in the case of dichotomous responses. Following each stage of experimentation, the current estimates of the dose-response distribution parameters, as well as the uncertainties in these estimates, are updated and are used to assign subjects to experimental dose levels for the next stage of testing. Competing dose allocations are compared with respect to anticipated improvement in estimation precision. The adoption of such a stagewise dose allocation strategy is illustrated by example.

Stagewise, group sequential experimental designs for quantal responses. one-sample and two-sample comparisons.

The use of stagewise, group sequential experimental designs with dichotomous responses in toxicity or drug screening programs is discussed. Such designs represent a compromise between the standard, fixed sample size designs and fully sequential designs. Stagewise group sequential designs place specified numbers of animals on test at each stage, up to a maximum number of stages. The greatest increases in sample size efficiency occur with small numbers of stages, particularly when going from one stage to two. Two-stage designs can result in a 15 to 20 percent reduction in average sample size. Five-stage designs can result in a 30 to 40 percent reduction in average sample size, with no appreciable decrease in Type 1 error or power. Examples of the efficiencies that arose in actual screening programs are given. This paper demonstrates that the routine use of stagewise, group sequential designs in standardized screening protocols can result in substantial savings in animal use with virtually no sacrifice of statistical sensitivity.

Absolute bioavailability of lead acetate and mining waste lead in rats.

The primary purpose of this study was to generate data that could be used to determine the absolute bioavailability of lead using data from a previous study in which soil containing lead from mining waste was mixed with feed. Young male and female Sprague-Dawley rats (7-8 weeks of age, five/sex/group) were given either soluble lead acetate mixed in a purified diet (AIN-76) at three different dose levels (1, 25, and 250 ppm Pb for 30 consecutive days) or intravenously at doses of 0.02, 0.20, and 2.0 mg Pb/kg BW for 29 days. A control group (purified diet only) was also included. The intravenous groups were used to provide maximal absorption (lead presumed to be 100% bioavailable) and accumulation data for lead in blood, bone, and liver. The lead acetate groups were used to evaluate the comparability of the present study with a previous study that compared bioavailable lead from ingested soil and lead acetate. Group mean whole blood, bone and liver lead concentration values increased with increasing dose levels for all treatment groups. A linear relationship was observed between blood lead concentration and dose following intravenous administration of lead and this provided empirical support for using blood lead concentrations at supposed steady state (approximately 30 days) to compute the bioavailability of lead administered by different routes and from different sources. The absolute bioavailability values of mining waste lead in soil were low based on the results for all tissue types. Absolute bioavailability values for lead acetate in dosed feed for blood, bone, and liver were approximately 6-, 19-, and 20-fold greater, respectively, than mining waste lead. Based on the current design and test system used, the absolute bioavailability of mining waste lead in soil administered in feed was approximately 3% based on blood data and less than 1% based on bone and liver data. These data are consistent with the low solubility of the constituent lead mineral phases in Butte soils.

Evaluation of compounds as barriers to dermal penetration of organophosphates using acetylcholinesterase inhibition.

An efficient, objective method for evaluating the efficacy of barrier compounds in preventing dermal penetration of organophosphates (OP) in rabbits was developed using time-dependent reduction in erythrocyte (RBC) acetylcholinesterase (AChE) activity as an endpoint. Anesthetized rabbits, with or without a dermal application of a mixture of high- and low-molecular-weight polyethylene glycols (mean molecular weight of 540 daltons; PEG 540), were exposed to different percutaneous doses of 3 highly toxic OP compounds. Dose-response curves were generated for RBC AChE inhibition as a function of percutaneous dose for each OP test material over time. From data generated, a single dose of each OP was selected to challenge PEG-540-protected and unprotected animals to validate the method as a means of differentiating effective from ineffective barriers to skin penetration. Data for a complete evaluation of a PEG 540 test barrier application were obtained within 4 h and anesthesia was maintained for the entire period.

Comparing and evaluating alternative (in vitro) tests on their ability to predict the Draize maximum average score.

The Cosmetic, Toiletry, and Fragrance Association (CTFA) Evaluation of Alternatives Program comprised a multi-phased study of the relationship between Draize eye irritation test data and comparable data from a selection of promising alternative (in vitro) tests. The CTFA Program was designed to determine the effectiveness and limitations of several in vitro tests over a range of different cosmetic and personal-care product types. Test materials constituted experimental formulations representative of three distinct product types. Each material was tested in vivo (according to a modified Draize eye irritation test protocol) and in vitro (according to one of up to forty different protocols). A statistical ranking and selection procedure ("concordance analysis") was used to identify those in vitro tests where the relationships between in vitro and in vivo score was sufficiently well defined to warrant further statistical analysis. In vitro test performance was then evaluated by regression modelling of these relationships. Maximum average Draize score (MAS) was utilized as the primary quantitative measure of eye irritation potential in vivo. The goodness-of-fit of the observed data to the regression model and comparison of the magnitude of upper and lower prediction-bounds on the range of probable MAS values associated with the regression model fit (prediction intervals) provide a means by which the performance of each in vitro test may be measured relative to Draize test outcome. The narrower the prediction interval (i.e. the more precise the fit), the more predictive of in vivo score (MAS) is the in vitro test result. The prediction interval thus represents uncertainty associated with Draize test prediction. Such uncertainty depends heavily on the degree of irritancy. In Phases I and II, the widths of the prediction intervals were narrowest in the region corresponding to low irritation potential; increasing widths were observed as irritation potential increased. In Phase III, relatively narrow prediction interval widths were observed at both the low and high end of the observed range of irritation potential; wider intervals were observed in the middle of the observed range. In general, the selected endpoints in each phase had similar average prediction interval widths and thereby differed only slightly in their ability to predict MAS to a given level of precision; any differences between endpoints tended to occur at the low and/or high ends of the observed range of irritation potential. The primary contributor to total variability associated with prediction of MAS is the deviation between the Draize score as observed in the laboratory and what is predicted by the model for a given formulation. Consistently, this component is responsible for 70% to 95% of the total variability. The other components (i.e. variability among replicate MAS and in vitro scores) could be reduced simply by increasing the number of replicate tests performed on each test formulation. However, this would have relatively little impact on the overall precision of prediction.

Statistical planning and analysis considerations in the evaluation of in vitro alternatives to whole animal use for eye irritation testing.

Reports from the IRAG Working Groups assessing the current status of in vitro alternatives to whole animal eye irritation tests reflect some common approaches. Although each Working Group studied a particular class of assay, typically all Groups evaluated the in vitro alternatives on the basis of correlation with an in vivo test and the statistical significance of that correlation. However, the data furnished to them by the testing organizations had been obtained with little or no standardization of procedures for testing and evaluation of results. This paper presents issues of design and execution of such test programs that are of statistical concern, including objectives of the evaluation process; limitations of correlation; sources of variation and distinction between actual replication and repeated measurements; evaluation of the predictive ability of in vitro tests; association criteria; and other approaches to such evaluation programs. The distinction between statistical significance and toxicological significance is pointed out. Suggestions are presented for standardization of test protocols, test materials and evaluation programs for both in vitro and in vivo tests. A central repository for such standardization, under the aegis of a government agency, professional standards organization, university or research institute, would be a valuable asset. An addendum is furnished to illustrate assertions about a number of factors influencing correlations and their statistical significance.

Comparison of low-volume, Draize and in vitro eye irritation test data. I. Hydroalcoholic formulations.

The first phase in a series of investigations of the relationship between low-volume eye test (LVET) data, Draize eye irritation test data, and comparable data from 25 in vitro assay protocols is presented. These investigations utilize Draize eye test and in vitro assay data generated previously as part of the Cosmetic, Toiletry and Fragrance Association (CTFA) Evaluation of Alternatives Program. LVET data were generated de novo using the same 10 representative hydroalcoholic personal-care formulations. The linear correlation between maximum average score (MAS) as determined by the Draize test and the LVET (LVET-MAS) was 0.93. Comparison of in vitro assay performance with that of the LVET was determined by statistical analysis of the relationship between LVET-MAS and in vitro endpoint. As in the CTFA program, regression modelling is the primary means of enabling such a comparison. The objective is to predict LVET-MAS for a given test material (and to place upper and lower prediction interval bounds in the range in which the LVET-MAS is anticipated to fall with high probability) conditional on observing an in vitro assay score for that material. The degree of confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves. Four assays [EYTEX MPA (membrane partition assay), HET-CAM (hen's egg test-chorioallantoic membrane HET-CAM) I, neutral red release and HET-CAM II] were shown to have the greatest agreement with the LVET. These assays were also among those with low discordance rates relative to the Draize test. Prediction of LVET-MAS values from experimentally determined in vitro scores was more accurate for hydroalcoholic formulations with lower rather than higher irritancy potential.

A comparison of low volume, draize and in vitro eye irritation test data. II. Oil/water emulsions.

The second phase in a series of investigations of the relationship between low volume eye test (LVET) data, Draize eye irritation test data, and comparable data from in vitro eye irritation test protocols is presented. These investigations utilize Draize eye test and in vitro endpoint data generated previously as part of the CTFA Evaluation of Alternatives Program. LVET data were generated de novo using the same 18 representative oil/water based personal-care formulations. In general, these formulations were minimally to mildly irritating; only three were classified as moderate eye irritants. The linear correlation between maximum average score as determined by the Draize test (MAS) and the LVET (LVET-MAS) was 0.85; LVET-MAS values were typically about half the corresponding MAS values. Comparison of in vitro assay performance with that of the LVET was determined by statistical analysis of the relationship between LVET-MAS and each in vitro endpoint. Regression modelling was the primary means of enabling such a comparison, the objective being to predict LVET-MAS for a given test material (and to place upper and lower 95% prediction bounds on the range in which the LVET-MAS is anticipated to fall with high probability) based on observation of an in vitro score for that material. The degree of confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves. Sixteen endpoints were shown to have the greatest agreement with the LVET (all but two were selected for modelling when compared with the Draize procedure). While the lower maximum average scores values (compared with the Draize test) in the LVET led to lower variability in LVET-MAS compared to MAS, the upper and lower bounds on predicted LVET-MAS values conditional on observed in vitro scores were still wide. Because there was overlap in the range of scores determined by the prediction bounds for many formulations, each of the selected endpoints was frequently unable to distinguish between test formulations in terms of statistically different predicted LVET-MAS values. In summary, none of the in vitro endpoints evaluated were able to reliably predict values of LVET-MAS among the set of oil/water emulsions considered here.

A comparison of low volume, Draize and in vitro eye irritation test data. III. Surfactant-based formulations.

The third phase in a series of investigations of the relationship between low volume eye test (LVET) data, Draize eye irritation test data, and comparable data from in vitro assay protocols is presented. These investigations utilize Draize eye test and in vitro endpoint data generated previously as part of the CTFA Evaluation of Alternatives Program. LVET data were generated de novo using the same 25 representative surfactant-based personal-care formulations. In general, these formulations were minimally to moderately irritating. The linear correlation between maximum average score as determined by the Draize test (MAS) and the LVET (LVET-MAS) was 0.87; LVET-MAS values were typically about 30% lower then corresponding MAS values. Comparison of in vitro assay performance with that of the LVET was determined by statistical analysis of the relationship between LVET-MAS and in vitro endpoint. Regression modelling was the primary means of enabling such a comparison, the objective being to predict LVET-MAS for a given test material (and to place upper and lower prediction bounds on the range in which the LVET-MAS is anticipated to fall with high probability) based on observation of an in vitro score for that material. The degree of 95% confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves. Twenty in vitro endpoints were shown to have the greatest agreement with the LVET (these endpoints included those with low discordance rates relative to the Draize test) and were therefore selected for regression modelling. Although prediction interval widths tended to be narrower when predicting LVET-MAS compared with predicting MAS, the confidence with which the selected in vitro endpoints predicted both LVET-MAS and MAS for surfactant-based formulations was greatest when values were close to the lower or upper limits of the observed irritation range (i.e. 95% prediction interval widths were most narrow in these areas). Overall precision of LVET-MAS prediction for surfactant-based formulations was similar to that previously reported for hydroalcoholic formulations and considerably better than was reported for oil/water emulsions.

IRAG working group 5. Other assays. Interagency Regulatory Alternatives Group.

As part of the Interagency Regulatory Alternatives Group (IRAG) program to evaluate the state of the art in the development of alternative (non-whole animal) eye irritation tests, academic and industrial organizations were invited to submit in vitro eye irritation data generated in their laboratories to one of several working groups for review. The assays reviewed in this report (from Working Group 5. "Other Assays") were the EYTEX assay, tissue equivalent assay, a cytotoxicity assay using three-dimensional human fibroblast constructs, the Microtox assay, and other miscellaneous assays. Each submission consisted of raw data for chemicals and products tested, a description of the methodology, and an analysis (generally by regression analysis and Pearson's correlation coefficient) for the performance of the in vitro test relative to its ability to predict individual ocular tissue scores or total ocular score. In vivo data were generated according to the scoring methods proposed by Draize. Working Group 5 evaluated the submissions and commented on the utility of the assays. The variability of the in vivo data made conclusions difficult in many situations. Most of these assays were deemed useful (within limited chemical classes) for screening purposes or for use in conjunction with other toxicological information.

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase II) oil/water emulsions.

The Cosmetic, Toiletry and Fragrance Association (CTFA) Evaluation of Alternatives Program is an evaluation of the relationship between Draize ocular safety test data and comparable data from a selection of in vitro tests. In Phase II, 18 representative oil/water-based personal-care formulations were subjected to the Draize primary eye safety test and 30 in vitro assay protocols (14 different types of in vitro endpoints were evaluated; the remainder were protocol variations). Correlation of in vitro with in vivo data was evaluated using analysis of sensitivity/specificity and statistical analysis of the relationship between maximum average Draize score (MAS) and in vitro endpoint. Regression modelling is the primary approach adopted in the CTFA Program for evaluating in vitro assay performance. The objective of regression analysis is to predict MAS for a given test material (and to place upper and lower prediction interval bounds on the range in which the MAS is anticipated to fall with high probability) conditional on observing an in vitro assay score for that material. The degree of confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves: the narrower the prediction interval, the more predictive of the Draize score is the in vitro test result. 16 assays were shown to have the greatest agreement with the Draize procedure and were therefore selected for regression analysis. Based on the magnitude of the 95% prediction bounds of each of the 16 selected assays over the range of test data, it may be inferred that prediction of MAS values from experimentally determined in vitro scores is more accurate for oil/water-based formulations with lower rather than higher irritancy potential. The assays selected for modelling in Phase II generally exhibited weaker relationships with MAS than those selected in Phase I (evaluated using hydroalcoholic formulations), even though several assays were common to both Phases.

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase III) surfactant-based formulations.

The CTFA Evaluation of Alternatives Program is an evaluation of the relationship between data from the Draize primary eye irritation test and comparable data from a selection of promising in vitro eye irritation tests. In Phase III, data from the Draize test and 41 in vitro endpoints on 25 representative surfactant-based personal care formulations were compared. As in Phase I and Phase II, regression modelling of the relationship between maximum average Draize score (MAS) and in vitro endpoint was the primary approach adopted for evaluating in vitro assay performance. The degree of confidence in prediction of MAS for a given in vitro endpoint is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curve. Prediction intervals reflect not only the error attributed to the model but also the material-specific components of variation in both the Draize and the in vitro assays. Among the in vitro assays selected for regression modeling in Phase III, the relationship between MAS and in vitro score was relatively well defined. The prediction bounds on MAS were most narrow for materials at the lower or upper end of the effective irritation range (MAS = 0-45), where variability in MAS was smallest. This, the confidence with which the MAS of surfactant-based formulations is predicted is greatest when MAS approaches zero or when MAS approaches 45 (no comment is made on prediction of MAS > 45 since extrapolation beyond the range of observed data is not possible). No single in vitro endpoint was found to exhibit relative superiority with regard to prediction of MAS. Variability associated with Draize test outcome (e.g. in MAS values) must be considered in any future comparisons of in vivo and in vitro test results if the purpose is to predict in vivo response using in vitro data.

Development, evaluation and use of a pharmacokinetic model for hexachlorobenzene.

This study discusses the background, biological basis, development characteristics, application and evaluation of six physiologically based multicompartment models that describe the absorption, distribution and elimination of hexachlorobenzene (HCB) in growing rats and growing humans. The models for rats and for humans have similar structures but differ in specific physiological parameters. The goal of the modelling effort was to obtain toxicological information about HCB based on its pharmacokinetics. Comparisons were made between estimated tissue concentrations based on the rat models and observed tissue distributions based on pharmacokinetic animal studies with HCB, using both chronic and single-dose studies. The estimates from the female model agreed reasonably well with experimental results, and estimated a long half-life of approximately 180 days in all tissues; it did not, however, duplicate the biphasic tissue efflux of HCB that has been reported in the literature. The male model estimated a half-life of approximately 55 days in all tissues, shorter than that observed experimentally. The estimated yield of metabolites agreed well with values reported in the literature. A pregnancy and offspring model predicted minimal transfer of HCB to the fetal compartment during gestation and extensive mobilization of HCB to the offspring during lactation. This agrees with results reported in the literature. Correlations were obtained between experimentally observed liver toxicity and estimated yield of metabolites; between experimentally observed effects on the central nervous system and estimated HCB concentrations in the brain; and between experimentally observed offspring mortality and estimated extent of lactational HCB transfer. This paper also discusses the effects on tissue concentrations and half-lives of trapping HCB in the intestines by sequestering a large portion of it there. Various characteristics of the human models are discussed. The female model has a biphasic elimination pattern, the second elimination phase having a half-life of greater than 200 days. The pregnancy and offspring model projects extensive transfer of HCB via the milk to the nursing offspring, which correlates with the greater mortality of nursing infants whose mothers were exposed to HCB in Turkey.

Relative bioavailability of lead from mining waste soil in rats.

The purposes of this study were to determine the extent of absorption of lead (Pb) in mining waste soil from Butte, Montana, and to investigate the effect of mining waste soil dose (g soil/day) on tissue lead concentrations. Young, 7- to 8-week-old male and female Sprague-Dawley rats (5/sex/group) were given mining waste soil that contained 810 or 3908 ppm lead mixed in a purified diet (AIN-76) at four different dose levels (0.2, 0.5, 2, and 5% dietary soil) for 30 consecutive days. Standard groups included untreated controls and dosed feed soluble lead acetate groups (1, 10, 25, 100, and 250 micrograms Pb/g feed). The test soil dose levels bracketed a pica child's soil exposure level and the lead acetate concentrations bracketed the test soil dose levels of lead. Liver, blood, and femur were analyzed for total lead concentration using graphite furnace atomic absorption spectroscopy. Clinical signs, body weight, food consumption, and liver weights for test soil and standard groups were similar to control. Tissue lead concentrations from test soil animals were significantly lower than the tissue concentrations for the lead acetate group. Relative percentage bioavailability values, based on lead acetate as the standard, were independent of the two different test soils, dose levels, and sex and were only slightly dependent on the tissue (blood > bone, liver). Mean relative percentage bioavailability values of lead in the Butte mining waste soil were 20% based on the blood data, 9% based on the bone data, and 8% based on the liver data. The results of this study will provide the information needed to determine the significance of lead exposure from Butte soils in assessing human health risks as part of the Superfund Remedial Investigation/Feasibility Study process.