Robert D. Bruce

Dose-response studies with chemical irritants in the albino rabbit eye as a basis for selecting optimum testing conditions for predicting hazard to the human eye

Abstract Twenty-one chemicals, solutions, and mixtures with different degrees of recognized irritant potential to the human eye were tested in albino rabbit eyes at dose volumes of 0.003, 0.01, 0.03, and 0.1 ml. Materials were administered directly to the central corneal surface. Irritation that developed was followed up to 21 days and graded by the Draize scale. Maximum irritation scores and median number of days for eyes to return to normal were compared with available data on human experience as a basis for selecting the dose volume that would best predict human response. Using irritation categories of negligible = clearing within 24 hr, moderate = clearing within 7 days, substantial = clearing within 21 days, and severe or corrosive = persisting beyond 21 days, a dose volume of 0.01 ml most often gave results that were consistent with information on effects of human exposures. This dose of hexane, triethanolamine, 0.1% benzalkonium chloride, 0.5% sodium hydroxide, 5% hydrochloric acid, and 20% sodium chloride caused negligible irritations. Granular sodium chloride, 10% sulfuric acid, 5% sodium hypochlorite, isopropyl alcohol, cocount soap powder and 10% solution, a powdered laundry soap, two phosphate laundry detergents, sodium carbonate-sodium sulfate mixtures, 3% acetic acid, 1% silver nitrate, and 10 and 40% sodium lauryl sulfate gave moderate irritation. Sodium lauryl sulfate, 29%, in another experiment was substantially irritating, and 10% acetic acid, 10% sodium hydroxide, formaldehyde, and calcium hydroxide were severely irritating or corrosive. Corneal application of 0.01 ml of material is proposed as a more realistic test of eye hazard than is the Draize test.

Spontaneous Corneal Dystrophy and Generalized Basement Membrane Changes in Fischer-344 Rats

Groups of young, sexually mature Fischer-344 rats (n = 25/sex) obtained from 3 commercial breeders were examined ophthalmologically and histopathologically to determine the prevalence and severity of corneal basement membrane lesions (corneal dystrophy) and basement membrane changes in select nonocular tissues. Results disclosed a high incidence of corneal basement membrane dystrophy in rats of both sexes from all breeders; however, severity levels were significantly increased in rats obtained from one breeder when compared to others. Furthermore, rats that displayed the most advanced corneal lesions also exhibited more severe basement membrane changes in other organs, especially renal tubules and vascular internal laminae. These findings suggest that both ocular and nonocular dystrophic changes may have been linked through common physiologic (or genetic) mechanisms. Animals that displayed basement membrane lesions were not considered to represent compromised biologic test systems.

Reducing the number of rabbits in the low-volume eye test

Although the Draize eye irritation test has provided important and useful information for eye safety assessments, considerable effort has been directed toward refining the assay procedure, reducing the number of animals used, and replacing this assay with alternative methods. The low-volume eye test (LVET) is a refinement of the Draize eye irritation test that uses 1/10 the volume of test substance placed directly on the cornea. The level and duration of eye irritation in the LVET are less than those in the Draize procedure, which means that it is a less stressful test. Furthermore, LVETs are more predictive of human response. Statistical studies have been conducted to determine the effects of reducing the number of animals used in the Draize test. These results suggested that a three-animal test would provide essentially the same information as the six-animal test. A similar analysis has not been performed on results from the LVET. Accordingly, the present study was undertaken to evaluate previously existing LVET data to determine if the number of animals used in a LVET can be decreased as has been shown for the Draize test. The results of the analysis are consistent with the findings of earlier evaluations of classical Draize data. Three-animal subsets from 119 six-animal LVETs provided the correct classification greater than 92% of the time for three different classification schemes. Furthermore, the discrepancies between the three-animal subsets and the six-animal maximum average score tended to be smaller than those observed for the Draize test.(ABSTRACT TRUNCATED AT 250 WORDS)

A report on the workshop on biological and statistical implications of the ED01 study and related data bases

Summary The following is a summary of the consensus reached on various issues discussed at the Deer Creek State Park Workshop. The key conclusions agreed to by the biologists, statisticians and pathologists during the Workshop were: o 1) The determination of time-to-tumor is important in carcinogenicity studies. The ED01 study demonstrates that the observed risk is more adequately expressed in a time and dose continuum rather than simply as a function of dose. 2) There is a need for time-to-tumor adjustments in the estimation of tumor rates, particularly in the presence of different mortality patterns due to intercurrent disease. This requires a determination of whether or not the animal died from the tumor of interest, since statistical methodology for fatal tumors differs from that for nonfatal tumors. Although there is disagreement as to whether the cause of an animals death can be determined, it may be possible to establish whether a particular tumor caused death. 3) Two types of dose responses were observed in the ED01 study in the two target organs for the test compound 2-AAF. The bladder tumors exhibited a no-observed effect level against a low background rate, while the liver tumors showed much less curvature in the dose-response relation. Older animals showed a high spontaneous rate of hepatic-cell tumors. 4) Even with a study as large as the ED01 study, statistical uncertainty makes it impossible to establish the true shape of the dose-response curve at low tumor rates. Neither can such studies prove or disprove the existence of thresholds. 5) Although a definitive model for low-dose extrapolation is not currently known, upper limits on risk at low dose can be estimated by the use of linear extrapolation. However, this procedure may be unduly conservative when the actual dose-response curve exhibits thresholdlike behavior. 6) Development of better models for low-dose extrapolation can be aided by the incorporation of more biological information, such as pharmacokinetics, metabolism and comparitive physiology. 7) The ED01 study is not the only large-scale, whole animal study of carcinogenesis. Other studies of possible interest for further investigation include radiation and skin-painting studies at Oak Ridge National Laboratories, formaldehyde studies by the Chemical Industries Institute of Toxicology, and the nitrosamine study conducted by the British Industrial Biological Research Association.

Morphometric Assessment of Thymic Size Variation in Laboratory Rabbits

Sections of thymus from New Zealand white rabbits used as controls in 28-day and 91-day percutaneous toxicity studies conducted at different laboratories were morphometrically assessed. Measurements of total thymic area, medullary area, and cortical area were greater for 28-day vs 91-day studies conducted at a given laboratory, but varied from one laboratory to another. Relative thymic measurements (percent medulla, percent cortex, and medullarcortex ratio) were similar for studies conducted at each laboratory and from one laboratory to another regardless of study duration. A decrease in thymic size occurred between approximately 16 and 25 weeks of age (i.e., 28-day studies vs 91-day studies) due to proportionally equivalent decreases in both the cortical and medullary areas. The consistency of the relative measurements can be used to assist in distinguishing changes in thymic size due to aging from changes in size due to stress or toxicity which would be expected to differentially affect the cortical and medullary areas. Appreciation of the normal variation in thymic size is needed when evaluating results of toxicity testing in rabbits. Data are provided as to the degree of normal variation of rabbit thymic size expected within and across percutaneous toxicity studies, with considerations for interpreting such data.

Hepatocellular Vacuolization in Rabbits: Effects of Feed Restriction, Orchiectomy and Ovariectomy

Previous studies have reported that striking variations in hepatocellular vacuolization occurs in rabbits and the magnitude of vacuolization correlated independently with weight and sex. The current study evaluated the effects of feed restriction and gonadectomy on this hepatocellular vacuolization. For 28 days, rabbits were fed either ad libitum (ad lib group), 100% of the National Research Councils recommended feed intake required for growth (100% group), or 50% of the NRC recommended feed intake required for growth (50% group). Feed consumption, weight gain, final body weight, absolute liver weight, and relative liver weight were not significantly different between the ad lib and 100% groups. Values for these parameters for both groups were significantly greater than for the 50% group. Rabbits in the 50% group had significantly less hepatocellular vacuolization than rabbits in the 100% group. Hepatocellular vacuolization in the 100% group did not differ from rabbits fed ad libitum. Hepatocellular vacuolization in the ad lib group was greater than in the 50% group but this difference was not significant. Ovariectomy and orchiectomy did not significantly alter hepatocellular vacuolization in either female or male rabbits, respectively, that were fed ad libitum for 28 days. However, intact females had significantly greater hepatocellular vacuolization than either intact or orchiectomized males. Conversely, hepatocellular vacuolization in ovariectomized females was not significantly different from that in intact and orchiectomized males. There were no significant differences in feed consumption, weight gain, final body weight, and absolute and relative liver weights among these intact and gonadectomized groups. Results of these studies indicate feed consumption can affect the degree of hepatocellular vacuolization in rabbits. In addition, intact female rabbits have greater hepatocellular vacuolization than males. This difference is reduced by ovariectomy.