Evaluation of organ weights for rodent and non-rodent toxicity studies: a review of regulatory guidelines and a survey of current practices

The Society of Toxicologic Pathology convened a working group to evaluate current practices regarding organ weights in toxicology studies. A survey was distributed to pharmaceutical, veterinary, chemical, food/nutritional and consumer product companies in Europe, North America, and Japan. Responses were compiled to identify organs routinely weighed for various study types in rodent and non-rodent species, compare methods of organ weighing, provide perspectives on the value of organ weights and identify the scientist(s) responsible for organ weight data interpretation. Data were evaluated as a whole as well as by industry type and geographic location. Regulatory guidance documents describing organ weighing practices are generally available, however, they differ somewhat dependent on industry type and regulatory agency. While questionnaire respondents unanimously stated that organ weights were a good screening tool to identify treatment-related effects, opinions varied as to which organ weights are most valuable. The liver, kidneys, and testes were commonly weighed and most often considered useful by most respondents. Other organs that break were commonly weighed included brain, adrenal glands, ovaries, thyroid glands, uterus, heart, and spleen. Lungs, lymph nodes, and other sex organs were weighed infrequently in routine studies, but were often weighed in specialized studies such as inhalation, immunotoxicity, and reproduction studies. Organ-to-body weight ratios were commonly calculated and were considered more useful when body weights were affected. Organ to brain weight ratios were calculated by most North American companies, but rarely according to respondents representing veterinary product or European companies. Statistical analyses were generally performed by most respondents. Pathologists performed interpretation of organ weight data for the majority of the industries.     

Relationships between organ weight and body/brain weight in the rat: what is the best analytical endpoint?

Analysis of organ weight in toxicology studies is an important endpoint for identification of potentially harmful effects of chemicals. Differences in organ weight between treatment groups are often accompanied by differences in body weight between these groups, making interpretation of organ weight differences more difficult. Using data from control rats that were part of 26 toxicity studies conducted under similar conditions, we have evaluated the relationship between organ weight and body/brain weight to determine which endpoint (organ weight, organ-to-body weight ratio, or organ-to-brain weight ratio) is likely to accurately detect target organ toxicity. This evaluation has shown that analysis of organ-to-body weight ratios is predictive for evaluating liver and thyroid gland weights, and organ-to-brain weight ratios is predictive for evaluating ovary and adrenal gland weights. Brain, heart, kidney, pituitary gland, and testes weights are not modeled well by any of the choices, and alternative analysis methods such as analysis of covariance should be utilized.     

The effect of exsanguination on organ weight of rats

In three experiments, rats were anesthetized and killed by exsanguination or were killed by an overdose of anesthetic and not exsanguinated. The second group of rats in each experiment mimicked rats found dead on toxicology studies. Liver and kidney weights of rats killed by exsanguination were statistically significantly lower than those of rats not exsanguinated both in terms of gross organ weight and of percentage of body weight. The results of these experiments indicate that organ weight data of rats killed by exsanguination should be segregated from those of rats found dead. In fact, because of uncontrollable variables, organs of rats found dead should not be weighed.     

Toxic effects of citrinin on the male reproductive system in mice

This study investigated the effects of citrinin (CTN) on male mouse reproductive organs. Adult male mice were exposed to intraperitoneal injection of CTN at 0–6.25 mg/kg body weight daily for 7 days, and then mated with sexually mature untreated female mice. Reproductive organ relative weights, semen quality, serum testosterone concentrations and fertility of treated mice were assessed. CTN significantly increased relative weights of the testes, epididymis, seminal vesicle and preputial gland, increased the number of abnormal spermatozoa and decreased the number of live spermatozoa. A significantly lower pregnancy rate was observed when females were mated with CTN-exposed males. The histological results indicated that distance of testicular seminiferus tubule increased. The sperm count and serum testosterone concentrations were significantly reduced in a dose-dependent manner in mice treated with CTN. The results suggest that CTN has adverse effects on the reproductive system of adult male mice.     

Degeneration of the seminiferous epithelium following epididymal obstruction in prepubertal rats

The development of the testes was studied in rats following prepubertal obstruction of the epididymis. Male rats received bilateral ligation of the corpus epididymidis or a sham operation at 10 days of age, and temporal changes in testicular morphology and weights of reproductive organs were determined at intervals spanning sexual maturation. Development of the testes was normal through 35 days of age. The initial histological changes in the testes of ligated animals, observed at 56 days, included an increased diameter of the seminiferous tubule lumen, depletion of spermatids, and the presence of multinucleate spermatids. Subsequently, germ cells were greatly depleted in the testes of 91‐ and 128‐day‐old rats with ligated epididymides. After puberty, testicular weight and volume declined relative to corresponding sham‐operated animals. On the other hand, the weights of the epididymides in ligated animals prior to puberty significantly exceeded those of sham‐operated rats but weighed significantly less than those of rats in the sham group after sexual maturation. Testicular alterations occurred after increases in the weights of the epididymides. Testicular changes may have contributed to rather than resulted from an autoimmune response to spermatozoa because testicular alterations preceded increases in antisperm autoantibodies. Anat Rec 254:76–86, 1999. © 1999 Wiley‐Liss, Inc.     

Administration of an antagonist of neurokinin receptors 1, 2, and 3 results in reproductive tract changes in beagle dogs, but not rats

SCH 206272, an antagonist of neurokinin receptors 1, 2, and 3, was administered orally by gavage for 1 month to 8- to 10-month-old dogs at doses of 0, 15, 30, or 60 mg/kg, and to 6-week-old rats at doses of 0, 30, 100, or 300 mg/kg. The most important changes occurred in the reproductive tract of the dogs at all doses. Absolute and relative group mean organ weights for the testes, prostate gland, epididymides, ovaries, and uterus were 33-86% lower than concurrent controls in groups receiving SCH 206272. Organ weight changes were not dose-related. Microscopic changes that correlated with the organ weight changes occurred in all groups receiving SCH 206272. For males, they included minimal to severe atrophy of the testes, epididymides, and prostate gland. In addition, the epididymides exhibited severe oligospermia or aspermia, minimal epithelial apoptosis and mild epithelial vacuolation. In female dogs, the ovaries and uteri appeared immature. Microscopic changes were similar in incidence and severity in dogs receiving 30 or 60 mg/kg, but were slightly less in dogs receiving 15 mg/kg. In contrast, similar findings were not observed in the reproductive tract of male or female rats, despite overlapping systemic, hypothalamic, and pituitary gland concentrations of SCH 206272.     

Application of Animal Toxicology Data to Clinical Study of Experimental Drugs

The pharmaceutical industry performs safety studies in animals to underwrite administration of new chemical entities to man. Early clinical evaluation of drugs in volunteers is performed, generally, using highdose acute studies in rodents and 1-month repeat dose studies in one rodent species and one non-rodent species as a basis for risk assessment. Results from the 1-month toxicology studies enable a ceiling for initial clinical doses to be set and give some indication of the major target organs for toxicity. Extrapolation of these results to man helps determine the preliminary safety endpoints, including clinical chemistry parameters, for use in early clinical studies in man.Originally presented at ECCP 93.     

The minipig in toxicology

The use of pigs (Sus scrofa) in biomedical research is well established in particular in surgical and physiological research. For years both pigs and minipigs have been used in pharmacology and toxicology to answer specific questions when the more conventional species have been found unsuitable. The development of minipigs has resulted in strains of more manageable size than the domestic pig. Because of their well-accepted physiological and other similarities to humans, minipigs are becoming increasingly attractive toxicological and pharmacological models. There are several strains of minipigs (Göttingen, Yucatan, Sinclair, Hanford and other). This presentation is based on experience primarily with the Göttingen minipigs. In toxicology in Europe minipigs have become very popular for pharmaceutical studies in place of dogs and primates. Minipigs have been shown to be sensitive to a wide variety of drugs and chemicals. It has become obvious that minipigs can be used for all routes of administration, and in many cases are preferable to dogs or primates for metabolic or pharmacological reasons. There are advantages over the traditional non-rodent species in relation to specific responses to particular drug classes. Their use in general toxicology testing employing the continuous intravenous infusion, dermal or inhalation route has been described in detail in the literature. Background data on toxicological endpoints (ophthalmology, clinical pathology, ECG, organ weight, histopathology and reproduction parameters) have been well-established allowing studies to be interpreted. In the context of this conference, histopathology and toxicopathology data of spontaneous or drug-induced origin are available in the scientific literature. Now there is good supply of high-quality minipigs of known disease status. There are advantages over the traditional non-rodent species in relation to the ethical difficulties of use of animals in biomedical research. Consequently, there are scientific, economic and sociological reasons that make minipigs good toxicological and pharmacological models. The principal disadvantage is that toxicity testing in minipigs may require more test compound than the traditional species.     

Environmental influence on development,behavior, and endocrine morphology of gerbils

Gerbils reared under standard laboratory conditions grow and develop more rapidly, achieve sexual maturity when younger, and are less reactive to stimulation than gerbils reared in environments providing access to a tunnel-like shelter. Gerbils reared in open cages have lighter adrenal glands, heavier pituitary glands, and heavier reproductive organs than gerbils raised with access to shelter. Comparison of gerbils reared in cages providing access either to opaque or transparent shelter with animals maintained in open cages either on a 12-hr light-dark cycle or in constant darkness revealed that different aspects of environments providing shelter affected different characteristics. Opportunity to move from an open area to a sheltered one increased behavioral reactivity. Reduced exposure to illumination affected developmental rates, reproductive organ and pituitary gland weights. Both reduced exposure to illumination and access to a shelter affected adrenal gland and body weights. These data suggest that expression of the phenotype typical of domesticated gerbils requires deprivation of a variety of stimuli normally experienced by burrow-dwellers during ontogeny.     

The impact of maternal protein malnutrition on pre-weaning skeletal and visceral organ growth in neonatal offspring of Rattus norvegicus

Most studies of malnutrition focus on adult size, or limited durations of malnutrition. Little is known about the impact of life-long maternal malnutrition on young, pre-weaning offspring, in part because working with such infants is difficult. We created a maternal generation of malnourished dams by feeding female Sprague-Dawley rats, from weaning, either a control diet high in protein (CT) or an isocaloric low protein diet (LPT). The offspring of matings between these dams and control fathers were weighed daily and radiographed three times before sacrifice at 22d, when several visceral organs and muscles were dissected out and weighed. We compared lengths of craniofacial and limb bones, and organ and muscle weights, between the two diet treatments. Allometric cancellation was used to assess integration of growth among organs and muscles. The offspring of LPT dams had body, organ and muscle weights smaller than the offspring of CT dams. When scaled to body mass, some organs of the LPT offspring were relatively larger. Although the CT offspring skeletons were significantly larger than the LPT skeletons, considerable variation existed in the patterns of growth between the two treatments. The CT offspring had a higher level of correlation among muscles, and most organs, than did the offspring of LPT dams. The organs that did maintain a correlation in growth, or linkage, were pairs of organs more likely to be protected (heart-lung or eye-brain) from the insult of protein malnutrition. The ability to protect some organs may be the result of their tighter developmental program, one that is more resistant to differences in available nutrition.     

Variability in body and organ weights in the newborn dog and cat compared with that in the adult

Body and organ weights of 24 newborn dogs and 35 newborn cats were studied. All of the measurements, except weights of hypophysis, spinal cord and testes are larger in the dogs. As percentages of body weight, the organs are more equally divided, with seven organs relatively heavier in the dogs and eight in the cats. All except two of the measurements of the newborn dogs are more variable than in the newborn cats. All of the organs are significantly correlated with body weight in the dogs and all except one, in the cats. All 15 of the organs are significantly correlated with body length in the dogs and 13, in the cats. The intercorrelations of the organ weights are somewhat higher in the dogs. The coefficients of variation of the newborn are compared with similar coefficients in adult dogs and cats. Body weight, body length and the kidneys are more variable in the adult dogs and the other organs, so far as data are available, are more variable in the newborn dogs of both sexes. Seven organs are more variable in adult male cats and three in females. The newborn dog is more variable in body and organ weights than the newborn cat, but weights of body and organs are better correlated in the newborn dog than in the newborn cat.     

Retinal electrophysiology for toxicology studies: Applications and limits of ERG in animals and ex vivo recordings

Assessing retinal drug toxicity is becoming increasingly important as different molecules are now developed for the treatment of neurodegenerative diseases and vascular disorders. In pharmacology and toxicology, the electroretinogram (ERG) and the multielectrode array (MEA) recording techniques can be used to quantify the possible side effects of retino-active xenobiotics. Toxicity testing requires the use of rodent as well as non-rodent models for extrapolation to the human model when determining risk and safety. Animal species differ in their retinal anatomo-physiology: most rodents used in toxicology studies are essentially nocturnal species, whereas the non-rodent laboratory species normally used (e.g. dogs, pigs and monkeys) are diurnal. The ratio between the photoreceptor populations which varies from species to species, should be considered when designing the experiment protocol and the interpretation. The described ERG procedures are designed to comply with all applicable good laboratory practice standards. Use of these procedures should yield an acceptable level of intra- and inter-subject variability for compiling a historical database, and for detecting possible retinal toxicity in animal studies. They could therefore be used as specific and standardized tools for screening of potential retinotoxic molecules in drug discovery and development in order to compare methods and results with those obtained in human electrophysiological assessments. Recording of ganglion cell light responses on ex vivo retina with the MEA technique can further demonstrate how retino-active xenobiotics affect retinal visual information processing by eliminating potential obstacles related to bioavailability and blood barrier permeability.     

Growth patterns and hormonal profile of male rats with protein-calorie malnutrition

A condition of protein-calorie malnutrition was precipitated in young Sprague-Dawley male rats at 20 days of age using an 8% low protein diet (LPD). At five-day intervals for up to 50 days of age, the rats were studied to determine the effect of an LPD on the reproductive axis of the endocrine system. Daily monitoring of the body weight, as well as the consumption of food, kilocalories, and protein was conducted. The same parameters were followed over the identical time period in a group of animals desigated as controls which were fed a standard laboratory diet (SLD) containing 27% protein. The controls showed a linear growth rate over the 30-day experimental period. In comparison, the malnourished rats grew more slowly so that by 50 days of age, their mean body weight was 68.9 +/- 3.1 g as compared to 248.1 +/- 6.1 g for the controls. The daily food, kilocalorie, and protein intake by the experimental animals were also appreciably less. The pituitary gland, ventral prostate gland, testes and liver were smaller in the animals fed the LPD. This was observed as early as five days after initiating the dietary regimes and remained a consistent observation until the end of the experiment. In general, the absolute weights of these organs in the 50 day-old malnourished rats were similar to those found in 25 to 26-day-old animals fed the SLD. The relative weights of the pituitary gland and liver remained similar between the two animal groups. The testes and ventral prostate gland, however, were relatively smaller in the malnourished animals at nearly every time interval studied. On light microscopic examination of the testes, it was found that normal maturation of the germ cells failed to occur in all but one of the experimental animals, whereas maturation proceeded normally in the rats fed the SLD. Serum luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL), and testosterone were lower in the malnourished animals at all ages studied. These hormones not exhibit the fluctuations that were seen in the controls and are typical in rats that are becoming sexually mature. The effect of protein deficiency on the concentration of the pituitary gonadotrophins was more varied. FSH concentrations were consistently lower, PRL was moderately affected, and LH remained essentially unchanged. Hypothalamic LH-releasing hormone was measured and found to be significantly less in the rats fed the LPD at most of the time intervals examined. These results indicate that the hypothalamo-hypophyseal-gonadal axis is impaired when the consumption of proteins and calorie is decreased. The possible involvement of extrahypothlamic centers in the control of hormone secretion in the protein-deficient rat is discussed.     

Reproductive toxic effect of bisphenol A dimethacrylate in mice

The current study evaluated the effect of bisphenol A dimethacrylate (Bis-DMA) on mouse fertility. Adult male and female mice were exposed to intragastric Bis-DMA (0, 5, 25, and 100 microg/kg) daily for 28 days and then mated with sexually mature untreated mice and after mating fertility was assessed. Females mated by males that had been exposed to Bis-DMA had significant reductions in pregnancy rates and significant increases in the total number of resorptions out of the total number of implantations. Bis-DMA exposed males had significant reductions in body weights and relative testes weights and significant increases in seminal vesicle and preputial gland weights. Testicular and epididymal sperm counts as well as the efficiency of sperm production were also significantly reduced in these groups. Female mice exposed to Bis-DMA showed significant reductions in pregnancy rates, number of implantation sites, number of viable fetuses, and total number of resorptions out of the total number of implantations. Significant reductions in the body weights were observed at all doses, and significant increases were found in the relative weights of the ovaries and the uterus. The results suggest that Bis-DMA has adverse effects on the fertility and reproductive systems of male and female mice.     

The role of histopathology in the identification of immunotoxicity

Recently finalized regulatory guidance documents concerned with the identification of immunotoxicity (CPMP: Note for Guidance on Repeated Dose Toxicity; FDA: Guidance for Industry, Immunotoxicology Evaluation of Investigational New Drugs; ICH S8) state that immunotoxicity testing should be performed on all new investigational drugs or medicinal products. In addition, all documents clearly identify gross and microscopic examination of lymphoid tissues as necessary and pivotal first steps in the assessment of new xenobiotics for immunotoxic potential. However, as is true for the evaluation of other organs systems, there are numerous approaches to the histopathologic examination of lymphoid tissues. To assist in a more uniform and consistent histopathologic assessment of the immune system, the Society of Toxicologic Pathology (STP), has recently prepared "best practice" recommendations concerning the collection, interpretation and reporting of organ weights, gross and microscopic observations, and other pathology data relevant to the immune system. The STP recommendations are intended to provide a scientifically sound and well-considered guidance document for routine pathology evaluation of the immune system. This presentation will consider the implications of this "best practice" document and place these recommendations in the context of normal animal tissue variability.     

The ability to predict weight gain,individual organ weight,and corresponding food intake in the rat by the four-parameter model for physiological responses

The applicability of the four-parameter model for physiological responses to the prediction of food intake and corresponding weight gain and individual organ weight gain was studied further in 40-day postpartum male rats. Seven groups of animals were maintained on diets in which protein content ranged from 0 to 23.54% casein. Food intake and weight gain were recorded every other day for each animal for 21 days. At the termination of the experiment the following organs were removed and weighed: liver, heart, lungs, spleen, kidneys, adrenals, and testes. When these weight values are fitted by use of the four-parameter model, food intake and total animal and organ weight gains can be predicted in relation to the amount of protein in the diet. It was found that liver, heart, lungs, spleen, and whole animal had similar K(0.5) values. However, it was also shown that there is variation in response of organs when relating organ weight as a percentage of body weight. For example, heart, lungs, and testes show an increased ratio on low protein diet while liver, kidneys, and adrenals maintain a fairly constant ratio and the spleen shows a decreased ratio. Additionally, it was noted that the animals on low protein diet consumed more food per gram body weight but did so at a slower rate. Possible future applications of the four-parameter model for physiological reponses are discussed.     

A vision of cell death: insights into immune privilege

Summary: Immune privilege is a term applied to several organs that have a unique relationship with the immune response. These sites prohibit the spread of inflammation since even minor episodes can threaten organ integrity and function. The most prominent examples of these are the eye, brain and reproductive organs where immune responses either do not proceed, or proceed in a manner different from other areas. Once thought to be a passive process relying on physical barriers, immune privilege can now be viewed as an active process that utilizes multiple mechanisms to maintain organ function. Recently there has been a renewed interest in immune privilege when it was shown that two privileged sites (the eye and testes) constitutively express FasL, which functions by killing lymphoid cells that invade these areas. Here we will examine the role of FasL in immune privilege and discuss how this molecule interacts with other elements of the inflammatory response to maintain organ integrity in the face of potentially damaging immune reactions.     

Relaxin is a key mediator of prostate growth and male reproductive tract development

Male mice deficient in relaxin showed retarded growth and marked deficiencies in the reproductive tract within 1 month of age. At 3 months of age, male reproductive organ weight (including the testis, epididymis, prostate, and seminal vesicle) from relaxin null (RLX-/-) mice were significantly (p < 0.05) smaller than those of wild-type (RLX+/+) male mice. Histologic examination of RLX-/- mouse tissues demonstrated decreased sperm maturation (testis), increased collagen, and decreased epithelial proliferation in the prostate compared with tissues obtained from RLX+/+ animals. The degree of sperm maturation in the testes of sexually mature RLX-/- mice (3 months) resembled that of immature (1 month) RLX+/+ mice and correlated with a decrease in fertility in RLX-/- mice. The marked differences in the extracellular matrix of the testis and prostate in RLX-/- males also correlated with an increase in the rate of cell apoptosis. Relaxin and LGR7 (relaxin receptor) mRNA expression was demonstrated in the prostate gland and testis of the normal mouse. Data from this study demonstrate that relaxin is an important factor in the development and function of the male reproductive tract in mice and has an essential role in the growth of the prostate and maintenance of male fertility. Relaxin may mediate its effects on growth and development by serving as an antiapoptotic factor.     

Factors related to parathyroid weight in normal persons

In a study of the parathyroid glands from 100 subjects who consecutively underwent autopsy, we found that median individual gland weight was 25.7 mg (95% weighed between 8.2 and 75.0 mg) and median individual gland parenchymal weight was 17.2 mg (95% weighed between 5.3 and 49.3 mg). Values were significantly skewed toward higher weights. Glandular weights were lower in patients with chronic illnesses, lower in women than in men, and lower in whites than in blacks. We found an inverse correlation between parenchymal weight and serum calcium concentration. Our results suggested that both total and parenchymal weights have a wider range of normal than is generally appreciated, and that a variety of factors probably affect parathyroid gland weight. A reevaluation of the weight of the parathyroid gland in normal persons is needed.