Barry L. Yano

Society of Toxicologic Pathology position paper: organ weight recommendations for toxicology studies.

The evaluation of organ weights in toxicology studies is an integral component in the assessment of pharmaceuticals, chemicals, and medical devices. The Society of Toxicologic Pathology (STP) has created recommendations for weighing organs in GLP general toxicology studies lasting from 7 days to 1 year. The STP recommends that liver, heart, kidneys, brain, testes, and adrenal glands be weighed in all multidose general toxicology studies. Thyroid gland and pituitary gland weights are recommended for all species except mice. Spleen and thymus should be weighed in rodent studies and may be weighed in non-rodent studies. Weighing of reproductive organs is most valuable in sexually mature animals. Variability in age, sexual maturity, and stage of cycle in non-rodents and reproductive senescence in female rodents may complicate or limit interpretation of reproductive organ weights. The STP recommends that testes of all species be weighed in multidose general toxicology studies. Epididymides and prostate should be weighed in rat studies and may be weighed on a case-by-case basis in non-rodent and mouse studies. Weighing of other organs including female reproductive organs should be considered on a case-by-case basis. Organ weights are not recommended for any carcinogenicity studies including the alternative mouse bioassays. Regardless of the study type or organs evaluated, organ weight changes must be evaluated within the context of the compound class, mechanism of action, and the entire data set for that study.

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 thatbreak 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.

Comparison of the teratogenic potential of inhaled ethylene glycol monomethyl ether in rats, mice, and rabbits.

Studies to assess the effects of inhaled ethylene glycol monomethyl ether (EGME) on embryonal and fetal development were conducted on groups of Fischer 344 rats, CF-1 mice, and New Zealand White rabbits. Rabbits and rats were exposed to vapor concentrations of 0, 3, 10, or 50 ppm for 6 hr/day on Days 6 through 18, or Days 6 through 15 of gestation, respectively; mice were exposed to 0, 10, or 50 ppm on Days 6 through 15 of gestation. Exposure of pregnant rabbits to 50 ppm produced significant increases in the incidence of malformations, minor variations, and resorptions, as well as a decrease in fetal body weight. Rats and mice exposed to 50 ppm showed no evidence of a teratogenic effect, although indications of slight fetotoxicity were observed in both species. Transient decreases in maternal body weight gain among rats, mice, and rabbits exposed to 50 ppm were the only consistent signs of maternal effects. No significant treatment-related effects on fetal development were observed in any of the species tested at 10 ppm of EGME or below.

Recommended Tissue List for Histopathologic Examination in Repeat-Dose Toxicity and Carcinogenicity Studies: A Proposal of the Society of Toxicologic Pathology (STP)

The Executive Committee of the Society of Toxicologic Pathology (STP) appointed an ad hoc task force to devise and recommend a standard list of tissues to be evaluated histopathologically in repeat-dose toxicity and carcinogenicity studies that are used to support the registration of new pharmaceutical products. The recommended tissue list is intended to be a minimum core list that can be used for all types of repeat-dose toxicity and carcinogenicity studies, regardless of route of administration, species or strain of mammalian laboratory animal, duration, or class of drug to be tested. The resulting recommendations of the task force, presented here, were subsequently reviewed by the STP membership and endorsed by the STP Executive Committee.

Abnormal auditory brainstem responses and cochlear pathology in rats induced by an exaggerated styrene exposure regimen.

Groups of 12 male 42-day-old rats were exposed to 0 or 800 ppm styrene vapors for 14 hr/day, 5 days/ week for 3 weeks. Tone-pip auditory brainstem responses (ABRs) at 4, 8, 16, and 30 kHz were obtained after the last exposure. ABRs were minimally affected at 4 kHz and moderately to severely affected at 8, 16, and 30 kHz as indicated by waveforms which had a decreased amplitude and increased latency as compared to the controls. Missing outer hair cell(s) were evident in the basal and lower middle turns of the organ of Corti. Outer hair cell loss was least in the first row and greatest in the second and third rows. Occasional inner hair cells were also missing in regions of severe outer hair cell loss. The distribution of hair cell loss within the cochlea was consistent with the pattern of ABR alterations. These data document mid-frequency auditory dysfunction in styrene-exposed young adult rats with significant damage to the organ of Corti following an exaggerated styrene exposure regimen.

Chronic Dietary Toxicity/Oncogenicity Studies on 2,4-Dichlorophenoxyacetic Acid in Rodents

Forms of 2,4-dichlorophenoxyacetic acid (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants. Doses in the 2-year chronic/oncogenicity rat study were 0, 5, 75, and 150 mg/kg/day. The chronic toxicity paralleled subchronic findings, and a NOEL of 5 mg/kg/day was established. A slight increase in astrocytomas observed (in males only) at 45 mg/kg/day in a previously conducted chronic rat study was not confirmed in the present study at the high dose of 150 mg/kg/ day. Doses in the 2-year mouse oncogenicity studies were 0, 5, 150, and 300 mg/kg/day for females and 0, 5, 62.5, and 125 mg/ kg/day for males. No oncogenic effect was noted in the study. In summary, the findings of these studies indicate low chronic toxicity of 2,4-D and the lack of oncogenic response to 2,4-D following chronic dietary exposure of 2,4-D in the rat and mouse.

Propylene glycol monomethyl ether: A 13-week inhalation toxicity study in rats and rabbits

Fischer 344 rats (10/sex/exposure concentration) and New Zealand White rabbits (7/sex/exposure concentration) were exposed to 0, 300, 1000, or 3000 ppm (0, 1.09, 3.62, or 10.9 mg/L) of propylene glycol monomethyl ether (PGME) for 6 hr/da, 5 da/wk, for 13 weeks. Minimal effects were observed in animals exposed to 3000 ppm. Indications of a transient central nervous system depression were observed in rats and rabbits exposed to 3000 ppm. There were also small increases (6 to 8%) in mean relative liver weights of 3000 ppm exposed male and female rats relative to controls. Minimal histologic effects were observed in the livers of 3000 ppm exposed female rats. These were suggestive of hepatocellular hypertrophy but were without evidence of degenerative changes. There was an increase in the urinary pH of male rats exposed to 3000 ppm PGME for 4 weeks, but this was not evident after 12 weeks of exposure. There was no indication of histopathological effects in the kidneys of either species, and there were no hematological effects. No treatment-related effects were found in either rats or rabbits exposed to 300 or 1000 ppm.

The chronic toxicity and oncogenicity of inhaled technical-grade 1,3-dichloropropene in rats and mice

Male and female Fischer 344 rats and B6C3F1 mice were exposed by inhalation to target concentrations of 0, 5, 20, or 60 ppm (0, 22.7, 90.8, or 272 mg/m3) technical-grade 1,3-dichloropropene (DCPT) 6 hr/day, 5 days/week, for up to 2 years. Ancillary groups of rats and mice were exposed for 6- and 12-month periods. Significant treatment-related nonneoplastic changes following exposure for 2 years were morphological alterations in the nasal tissues of rats exposed to 60 ppm and mice exposed to 20 or 60 ppm DCPT. In addition, mice exposed to 20 or 60 ppm had hyperplasia of the transitional epithelium lining the urinary bladder. Survival of male and female rats and mice exposed to DCPT was similar to that of the corresponding controls. No statistically increased tumor incidence was observed in treated rats. The only neoplastic response observed in mice was an increased incidence of benign lung tumors (bronchioloalveolar adenomas) in male mice exposed to 60 ppm DCPT (22/50 versus 9/50 in controls).

An F1-extended one-generation reproductive toxicity study in Crl:CD(SD) rats with 2,4-dichlorophenoxyacetic acid.

2,4-Dichlorophenoxyacetic acid (2,4-D) was assessed for systemic toxicity, reproductive toxicity, developmental neurotoxicity (DNT), developmental immunotoxicity (DIT), and endocrine toxicity. CD rats (27/sex/dose) were exposed to 0, 100, 300, 600 (female), or 800 (male) ppm 2,4-D in diet. Nonlinear toxicokinetic behavior was shown at high doses; the renal clearance saturation threshold for 2,4-D was exceeded markedly in females and slightly exceeded in males. Exposure was 4 weeks premating, 7 weeks postmating for P1 males and through lactation for P1 females. F1 offspring were examined for survival and development, and at weaning, pups were divided in cohorts, by sex and dose, and by systemic toxicity (10), DNT (10), DIT (20), and reproductive toxicity (≥ 23). Remaining weanlings were evaluated for systemic toxicity and neuropathology (10–12). Body weight decreased during lactation in high-dose P1 females and in F1 pups. Kidney was the primary target organ, with slight degeneration of proximal convoluted tubules observed in high-dose P1 males and in high-dose F1 males and females. A slight intergenerational difference in kidney toxicity was attributed to increased intake of 2,4-D in F1 offspring. Decreased weanling testes weights and delayed preputial separation in F1 males were attributed to decreased body weights. Endocrine-related effects were limited to slight thyroid hormone changes and adaptive histopathology in high-dose GD 17 dams seen only at a nonlinear toxicokinetic dose. 2,4-D did not cause reproductive toxicity, DNT, or DIT. The “No Observed Adverse Effect Level” for systemic toxicity was 300 ppm in both males (16.6mg/kg/day) and females (20.6mg/kg/day), which is approximately 6700- to 93 000-fold higher than that reported for 2,4-D exposures in human biomonitoring studies.

Neurotoxicologic Examination of Rats Exposed to 1,1,2,2-Tetrachloroethylene (Perchloroethylene) Vapor for 13 Weeks

Large evoked potential and EEG changes occurred in a pilot study in Fischer 344 rats during exposure to 800 ppm of 1,1,2,2-tetrachloroethylene [perchloroethylene (Perc)], a cleaning solvent with anesthetic properties. In the main study, rats were evaluated for persistent nervous system effects the week following exposure to 0, 50, 200, or 800 ppm Perc for 6 h/day, 5 days/week, for 13 weeks. The only effect related to treatment was in the flash evoked potential (FEP-V), recorded from the visual cortex. The longer latency potentials (N3) of the FEP-V had a greater amplitude in the 800 ppm Perc group. The FEP-Vs were of normal shape and latency. Although mild neurotoxicity could not be ruled out completely, amplitude changes in N3 can occur for a variety of psychophysiological reasons other than neurotoxicity. Consequently, as a stand-alone finding, the toxicologic significance of the larger FEP in the 800 ppm exposure group was unknown. Other data did not support a diagnosis of neurotoxicity. No treatment-related alterations were noted in expanded clinical observations, in the FEP recorded from the cerebellum (as opposed to visual cortex FEP-V), or in auditory, somatosensory, or caudal nerve evoked potentials. No treatment-related lesions were noted during histopathologic examination of eyes, optic nerves, optic tract, or multiple sections of brain, spinal cord, peripheral nerves, or limb muscles. The no-observed-effect-level (NOEL) was 200 ppm, based on increased amplitude of the longer latency potentials of the FEP at 800 ppm.