Robert Leo Kanerva

Comparison of short-term renal effects due to oral administration of decalin or d-limonene in young adult male Fischer-344 rats

Groups of young adult male Fischer-344 rats given the vehicle (corn oil) or either decalin or d-limonene at dose levels of 75, 150 or 300 mg/kg body weight by a single daily gavage on 5 days/wk were killed on study days 6 or 27, approximately 24 hr after the fifth or 20th dose, to determine whether the specific time- and dose-related triad of renal alterations characterizing decalin-associated nephrotoxicity in the adult male rat also occurs in response to d-limonene. Dose-related hyaline droplet formation associated with renal accumulation of a specific protein alpha 2u-globulin) is considered the primary response in the morphogenesis of decalin-induced nephrotoxicity in the male rat and was present to a maximal degree in all decalin- and d-limonene-treated groups by day 6. Alterations considered to be sequelae of the hyaline droplet response, including granular casts in the outer zone of the medulla and multiple cortical changes collectively classified as chronic nephrosis, were present in the kidneys of both decalin- and d-limonene-treated rats killed on day 27. These findings demonstrate a uniformity of primary and secondary renal responses to the two chemicals, strongly suggesting that the morphogenesis of d-limonene-associated nephrotoxicity in the adult male rat is consistent with that of decalin. The response of the male rat kidney to decalin treatment has been shown to be uniquely different, by virtue of anatomical, physiological and biochemical peculiarities involving the proximal convoluted tubule, from that in female rats and higher mammalian species.

Assessment of the subchronic oral toxicity of d-limonene in dogs☆

Several hydrocarbons, including d-limonene, have been shown to produce a male-rat-specific nephrotoxicity that is manifested acutely as exacerbation of hyaline droplet formation. In a study to assess the presence or absence of this response in a non-rodent species, the dog was selected as a relevant model because of an earlier report suggesting that d-limonene may be nephrotoxic in this species. Five male and five female adult beagle dogs per treatment group were gavaged twice daily over a 6-month period with tap-water (control) or d-limonene at 0.12 or 1.2 ml/kg body weight/day (100 or 1000 mg/kg body weight/day). The highest daily dose was determined in a pilot study to be close to the maximum tolerated dose for emesis (ED50 1.6 ml/kg body weight). The test compound was administered in divided doses to minimize the incidence of emesis. Feed consumption and body weight were unaffected by treatment. Linear regression analyses indicated a positive dose-related trend for absolute and relative female kidney weight and relative male kidney weight. There were no histopathological changes in the kidneys, evaluated by both haematoxylin and eosin and Mallory-Heidenhain staining, that could be associated with the organ-weight changes. Furthermore, there was no evidence of hyaline droplet accumulation nor of any other sign of hydrocarbon-induced nephropathy typical of those seen in male rats treated with d-limonene. Thus, dogs are refractory to the hyaline droplet nephropathy observed in male rats, thereby providing additional evidence that the male rat kidney is uniquely sensitive to hydrocarbons like d-limonene, and that this specific male rat nephropathic response may be inappropriate for interspecies extrapolation and human risk assessment.

Intraduodenal delivery of intrinsically and extrinsically labelled CaCO3 in the rat: effect of solubilization on calcium bioavailability*

Abstract— The dissolution of CaCO3 before intraduodenal administration was found to be an important factor determining calcium (Ca) bioavailability. Extrinsically and intrinsically labelled 47CaCO3 preparations were sequentially dissolved by serial additions of HCl. Aliquots of these preparations were collected before (no HCl added) and during the solubilization process and administered intraduodenally to rats. Whole body 47Ca retention 72 h post‐dose was used as a measure of Ca bioavailability. Although dissolution of CaCO3 significantly increased Ca bioavailability (p < 0·001), Ca from both intrinsically and extrinsically labelled CaCO3 was absorbed and retained to some extent without prior acid dissolution. Due to a disproportionately high concentration of 47Ca on the particle surface, extrinsically labelled 47CaCO3 overestimated bioavailability when unsolubilized or partially solubilized CaCO3 preparations were used (P < 0·05). These data indicate that dissolution is a determining factor for Ca bioavailability from CaCO3. Incomplete dissolution will significantly limit but not completely prevent Ca bioavailability. The disintegration and dissolution characteristics of commercial CaCO3 preparations, which vary widely, may produce important differences in Ca absorption.

Effects of Variation of Necropsy Time and Fasting on Liver Weights and Liver Components in Rats

In rats, percent liver weight loss is greater than percent body weight loss within the 8 a.m.-4 p.m. period of the working day. The liver weight loss is principally the result of decreased water content, either carbohydrate (glycogen) bound water in rats with access to feed, or protein bound water in rats fasted overnight. During this period, percent kidney weight loss is approximately equal to percent body weight loss. To optimize the sensitivity of kidney and liver weight evaluation, it is recommended that rats be fasted overnight and that relative liver and kidney weights be expressed based on body weights taken immediately prior to necropsy. Since these procedures will not entirely eliminate necropsy time-related organ weight differences, the animal necropsy sequence must be randomized to distribute the remaining differences across all treatment groups.

Comparison of Fresh and Fixed Organ Weights of Rats

Male and female rats (20 of each sex) were killed, and a broad sample of organs were excised, weighed, and immersed in 10% neutral buffered formalin. Following 72 hours fixation the organs were reweighed. Comparison of fresh and fixed organ weights revealed statistically significant organ weight changes due to fixation. Although the fixation-induced organ weight changes varied in both direction and magnitude among organs and between sexes, the changes were consistent throughout samplings of each specific organ. The results of this study therefore suggest that fixed organ weights may be a valid alternative to fresh organ weights. A significantly larger data base must be generated, however, to determine the influence of fixation prior to weighing in the presence of the various pathologic tissue alterations observed in safety evaluation studies.

The Effect of Uniform Exsanguination on Absolute and Relative Organ Weights, and Organ Weight Variation

The effect of uniform exsanguination on absolute and relative organ weights was assessed. The data demonstrate that the simple, rapid, and uniform procedure described for exsanguination of rats prior to necropsy significantly (P < .05) reduces both absolute and relative liver and kidney weights with respect to weights from animals that are not exsanguinated. In addition, liver weight standard deviation relative to controls is significantly (P < .05) reduced, increasing sensitivity of the measurement. Exsanguination also decreases agonal change related variation in macroscopic tissue appearance, enhancing the gross examination.

Optimization and Standardization of Male Gonad Weight Determinations in Rats

In order to develop an optimal method for measuring male gonad weight, one that is both statistically dependable (i.e. not subject to wide fluctuations within similarly treated groups) and appropriate with respect to end uses of data, testis, epididymis, and epididymal fat pad weights were obtained from normal adult male rats and subjected to statistical analyses. The results underscore the necessity for omitting the epididymal fat pad from “gonad” weight measurements since there appeared to be no relationship between its weight and the weight of either the testis or epididymis. Furthermore, the inclusion of epididymal fat resulted in large variation in total unit weight. An interdependent relationship was shown for testis and epididymis weights, indicating it may be appropriate to report a combined value for these entities, though this decision should be based on the intent of the study. A significant saving in time was effected by obtaining the weight of the testis alone. In studies which require male gonad weight assessment, therefore, the testis, excluding epididymis and epididymal fat, should be weighed to maximize the combined factors of time/cost effectiveness and scientific validity.