The in vitro pharmacology and non-clinical cardiovascular safety studies of a novel 5-HT4 receptor agonist, DSP-6952.

The pharmacological activity of DSP-6952, a novel compound was investigated, compared to that of clinically efficacious gastrointestinal (GI) prokinetic 5-hydroxytryptamine4 (5-HT4) receptor agonists. DSP-6952 had a strong affinity of Ki = 51.9 nM for 5-HT4(b) receptor, and produced contraction in the isolated guinea pig colon with EC50 of 271.6 nM and low intrinsic activity of 57%, similar to tegaserod and mosapride. In the development of the 5-HT4 receptor agonists, cardiovascular risk was deliberately evaluated, because some related prokinetics were reported to cause with cardiovascular adverse events, such as ventricular arrhythmias or ischemia. DSP-6952 showed minimal effects up to 100 µM in human ether-a-go-go-related gene (hERG) channels or guinea pig cardiomyocytes. In telemetered conscious monkeys, DSP-6952 did not affect blood pressure or any electrocardiogram (ECG) up to 180 mg/kg, p.o.; however, DSP-6952 transiently increased heart rate, as well as in anesthetized dogs. The positive chronotropic effects of DSP-6952 were completely antagonized by a 5-HT4 receptor antagonist, and another 5-HT4 receptor agonist, TD-5108 also increased heart rate. These effects are considered a class effect seen in clinically developing and marketed 5-HT4 receptor agonists, and have not been regarded as a critical issue in clinical use. DSP-6952 did not induce contraction in the rabbit coronary artery up to 100 µM, which differed from tegaserod or sumatriptan. These results show that DSP-6952 does not have cardiac ischemic risk via coronary vasoconstriction. In conclusion, DSP-6952 is a promising GI prokinetic compound with partial 5-HT4 receptor agonistic activity as well as a favorable cardiovascular safety profile.

CSAHi study-2: Validation of multi-electrode array systems (MEA60/2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes: Assessment of reference compounds and comparison with non-clinical studies and clinical information.

With the aim of reconsidering ICH S7B and E14 guidelines, a new in vitro assay system has been subjected to worldwide validation to establish a better prediction platform for potential drug-induced QT prolongation and the consequent TdP in clinical practice. In Japan, CSAHi HEART team has been working on hiPS-CMs in the MEA (hiPS-CMs/MEA) under a standardized protocol and found no inter-facility or lot-to-lot variability for proarrhythmic risk assessment of 7 reference compounds. In this study, we evaluated the responses of hiPS-CMs/MEA to another 31 reference compounds associated with cardiac toxicities, and gene expression to further clarify the electrophysiological characteristics over the course of culture period. The hiPS-CMs/MEA assay accurately predicted reference compounds potential for arrhythmogenesis, and yielded results that showed better correlation with target concentrations of QTc prolongation or TdP in clinical setting than other current in vitro and in vivo assays. Gene expression analyses revealed consistent profiles in all samples within and among the testing facilities. This report would provide CiPA with informative guidance on the use of the hiPS-CMs/MEA assay, and promote the establishment of a new paradigm, beyond conventional in vitro and in vivo assays for cardiac safety assessment of new drugs.

Comprehensive in vitro cardiac safety assessment using human stem cell technology: Overview of CSAHi HEART initiative.

Recent increasing evidence suggests that the currently-used platforms in vitro IKr and APD, and/or in vivo QT assays are not fully predictive for TdP, and do not address potential arrhythmia (VT and/or VF) induced by diverse mechanisms of action. In addition, other cardiac safety liabilities such as functional dysfunction of excitation-contraction coupling (contractility) and structural damage (morphological damage to cardiomyocytes) are also major causes of drug attrition, but current in vitro assays do not cover all these liabilities. We organized the Consortium for Safety Assessment using Human iPS cells (CSAHi; http://csahi.org/en/), based on the Japan Pharmaceutical Manufacturers Association (JPMA), to verify the application of human iPS/ES cell-derived cardiomyocytes in drug safety evaluation. The main goal of the CSAHi HEART team has been to propose comprehensive screening strategies to predict a diverse range of cardiotoxicities by using recently introduced platforms (multi-electrode array (MEA), patch clamp, cellular impedance, motion field imaging [MFI], and Ca transient systems) while identifying the strengths and weaknesses of each. Our study shows that hiPS-CMs used in these platforms have pharmacological responses more relevant to humans in comparison with existent hERG, APD or Langendorff (MAPD/contraction) assays, and not only MEA but also other methods such as impedance, MFI, and Ca transient systems would offer paradigm changes of platforms for predicting drug-induced QT risk and/or arrhythmia or contractile dysfunctions. Furthermore, we propose a potential multi-parametric platform in which field potential (MEA)-Ca transient-contraction (MFI) could be evaluated simultaneously as an ideal novel platform for predicting a diversity of cardiac toxicities, namely whole effects on the excitation-contraction cascade.

CSAHi study: Validation of multi-electrode array systems (MEA60/2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes -assessment of inter-facility and cells lot-to-lot-variability.

In vitro screening of hERG channels are recommended under ICH S7B guidelines to predict drug-induced QT prolongation and Torsade de Pointes (TdP), whereas proarrhythmia is known to be evoked by blockage of other ion channels involved in cardiac contraction and compensation mechanisms. A consortium for drug safety assessment using human iPS cells-derived cardiomyocytes (hiPS-CMs), CSAHi, has been organized to establish a novel in vitro test system that would enable better prediction of drug-induced proarrhythmia and QT prolongation. Here we report the inter-facility and cells lot-to-lot variability evaluated with FPDc (corrected field potential duration), FPDc10 (10% FPDc change concentration), beat rate and incidence of arrhythmia-like waveform or arrest on hiPS-CMs in a multi-electrode array system. Arrhythmia-like waveforms were evident for all test compounds, other than chromanol 293B, that evoked FPDc prolongation in this system and are reported to induce TdP in clinical practice. There was no apparent cells lot-to-lot variability, while inter-facility variabilities were limited within ranges from 3.9- to 20-folds for FPDc10 and about 10-folds for the minimum concentration inducing arrhythmia-like waveform or arrests. In conclusion, the new assay model reported here would enable accurate prediction of a drug potential for proarrhythmia.

Availability of human induced pluripotent stem cell-derived cardiomyocytes in assessment of drug potential for QT prolongation.

Field potential duration (FPD) in human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), which can express QT interval in an electrocardiogram, is reported to be a useful tool to predict K(+) channel and Ca(2+) channel blocker effects on QT interval. However, there is no report showing that this technique can be used to predict multichannel blocker potential for QT prolongation. The aim of this study is to show that FPD from MEA (Multielectrode array) of hiPS-CMs can detect QT prolongation induced by multichannel blockers. hiPS-CMs were seeded onto MEA and FPD was measured for 2min every 10min for 30min after drug exposure for the vehicle and each drug concentration. IKr and IKs blockers concentration-dependently prolonged corrected FPD (FPDc), whereas Ca(2+) channel blockers concentration-dependently shortened FPDc. Also, the multichannel blockers Amiodarone, Paroxetine, Terfenadine and Citalopram prolonged FPDc in a concentration dependent manner. Finally, the IKr blockers, Terfenadine and Citalopram, which are reported to cause Torsade de Pointes (TdP) in clinical practice, produced early afterdepolarization (EAD). hiPS-CMs using MEA system and FPDc can predict the effects of drug candidates on QT interval. This study also shows that this assay can help detect EAD for drugs with TdP potential.

Translational research into species differences of endocrine toxicity via steroidogenesis inhibition by SMP-028--for human safety in clinical study.

SMP-028 is a drug candidate developed for the treatment of asthma. In a 13-week repeated dose toxicity study of SMP-028 in rats and monkeys, differences of endocrine toxicological events between rats and monkeys were observed. In rats, these toxicological events mainly consisted of pathological changes in the adrenal, testis, ovary, and the other endocrine-related organs. On the other hand, in monkeys, no toxicological events were observed. The goal of this study is to try to understand the reason why only rats, but not monkeys, showed toxicological events following treatment with SMP-028 and to eventually predict the possible toxicological effect of this compound on human endocrine organs. Our results show that SMP-028 inhibits neutral cholesterol esterase more strongly than other steroidogenic enzymes in rats. Although SMP-028 also inhibits monkeys and human neutral cholesterol esterase, this inhibition is much weaker than that of rat neutral cholesterol esterase. These results indicate (1) that the difference in endocrine toxicological events between rats and monkeys is mainly due to inhibition of steroidogenesis by SMP-028 in rats, not in monkeys, and (2) that SMP-028 may not affect steroidogenesis in humans and therefore might cause no endocrine toxicological events in clinical studies.

Effect of SMP-028 on steroidogenesis in rats; mechanism of toxicological events on endocrine organs of rats.

SMP-028 is a new compound for treatment of asthma. Oral administration of SMP-028 to rats was associated with toxicological events in endocrine organs. These events mainly consisted of pathological changes in the adrenal gland, testis, prostate, seminal vesicle, ovaries, and uterus. In this study, we set to clarify whether SMP-028 inhibits steroidogenesis in primary culture cells obtained from rat endocrine organs in vitro. Adrenal cells, testicular cells, and ovarian cells were treated with SMP-028 and the production of steroid hormones, i.e., progesterone, aldosterone, corticosterone, total testosterone, and estradiol from these cells was measured by radioimmunoassay. We found that the production of progesterone from these cells treated with SMP-028 at 1 µM decreased to 16-67% that of the control. These findings indicate that SMP-028 inhibits steroidogenesis in rat endocrine organs in vitro. Considering that free maximum concentration in rats treated with SMP-028 are higher than the IC50 values for the inhibition of steroidogenesis in vitro, it is therefore believed that the toxicological events seen in rats following treatment with SMP-028 are due to inhibition of steroidogenesis in vivo.

The effects of clobazam treatment in rats on the expression of genes and proteins encoding glucronosyltransferase 1A/2B (UGT1A/2B) and multidrug resistance-associated protein-2 (MRP2), and development of thyroid follicular cell hypertrophy.

Clobazam (CLB) is known to increase hepatobiliary thyroxine (T4) clearance in Sprague-Dawley (SD) rats, which results in hypothyroidism followed by thyroid follicular cell hypertrophy. However, the mechanism of the acceleration of T4-clearance has not been fully investigated. In the present study, we tried to clarify the roles of hepatic UDP-glucronosyltransferase (UGT) isoenzymes (UGT1A and UGT2B) and efflux transporter (multidrug resistance-associated protein-2; MRP2) in the CLB-induced acceleration of T4-clearance using two mutant rat strains, UGT1A-deficient mutant (Gunn) and MRP2-deficient mutant (EHBR) rats, especially focusing on thyroid morphology, levels of circulating hormones (T4 and triiodothyronine (T3)) and thyroid-stimulating hormone (TSH), and mRNA or protein expressions of UGTs (Ugt1a1, Ugt1a6, and Ugt2b1/2) and MRP2 (Mrp). CLB induced thyroid morphological changes with increases in TSH in SD and Gunn rats, but not in EHBR rats. T4 was slightly decreased in SD and Gunn rats, and T3 was decreased in Gunn rats, whereas these hormones were maintained in EHBR rats. Hepatic Ugt1a1, Ugt1a6, Ugt2b1/2, and Mrp2 mRNAs were upregulated in SD rats. In Gunn rats, UGT1A mRNAs (Ugt1a1/6) and protein levels were quite low, but UGT2B mRNAs (Ugt2b1/2) and protein were prominently upregulated. In SD and Gunn rats, MRP2 mRNA and protein were upregulated to the same degree. These results suggest that MRP2 is an important contributor in development of the thyroid cellular hypertrophy in CLB-treated rats, and that UGT1A and UGT2B work in concert with MRP2 in the presence of MRP2 function to enable the effective elimination of thyroid hormones.

Evaluation of ovarian toxicity of mono-(2-ethylhexyl) phthalate (MEHP) using cultured rat ovarian follicles.

Mono-(2-ethylhexyl) phthalate (MEHP) is the most toxic metabolite of di-(2-ethylhexyl) phthalate (DEHP). It has been reported that DEHP causes abnormal reproductive development in women, and suppresses estradiol synthesis and ovulation in female rats with diminished size of preovulatory follicles. The present study was conducted to evaluate the ovarian toxicity of MEHP using cultured rat ovarian follicles. Secondary follicles were isolated from the ovaries of 14-day-old female rats and cultured for 48 hr with MEHP (0, 10, 30, and 100 µg/ml). At 0, 24, and 48 hr of MEHP treatment, follicular diameters were measured. After the culture, viability and apoptosis of follicles were assessed, and progesterone, androstenedione, testosterone, and estradiol levels in culture media were measured. At 100 µg/ml, suppression of follicular development was observed, which is associated with decreased viability of follicles and apoptosis of granulosa cells. At this concentration, progesterone level increased markedly, whereas androstenedione, testosterone, and estradiol levels decreased. At 10 and 30 µg/ml, follicular development was not suppressed, no apoptotic change was observed, and the levels of all measured steroid hormones tended to increase. The combined levels of all steroid hormones increased at all concentrations of MEHP, and the increase implies that MEHP activates the synthetic pathway from cholesterol to estradiol including de novo synthesis of cholesterol. However, the progesterone/androstenedione ratio increased extremely at 100 µg/ml, and the increase implies that MEHP inhibits the conversion of progesterone to androstenedione. In conclusion, MEHP induces ovarian toxicity via suppression of follicular development and abnormal steroid hormone synthesis in cultured rat ovarian follicles.

Evaluation of ovarian toxicity of sodium valproate (VPA) using cultured rat ovarian follicles.

Sodium valproate (VPA) is a major antiepileptic drug that is widely used for the treatment of epilepsy as well as other neuropsychiatric diseases. The present study was conducted to evaluate the ovarian toxicity of VPA using cultured rat ovarian follicles. Secondary follicles were isolated from the ovaries of 14-day-old female rats and cultured for 48 hr with VPA (0, 0.2, 1.0, and 5.0 mM). At 0, 24, and 48 hr of VPA treatment, follicular diameters were measured. After the culture, viability of follicles and expression of aromatase in the follicles were assessed, and progesterone, androstenedione, testosterone, and estradiol levels in culture media were measured. At all concentrations of VPA, follicular development was suppressed, and androstenedione, testosterone, estradiol, and combined levels of all steroid hormones tended to decrease in association with suppression of aromatase expression in granulosa cells. Additionally, the suppression of follicular development was associated with decreased viability of follicles and an increased progesterone level at 5.0 mM of VPA. The decrease in the combined levels of all steroid hormones implies that VPA suppresses the synthetic pathway from cholesterol to estradiol including de novo synthesis of cholesterol. In conclusion, VPA induces ovarian toxicity via suppression of development and abnormal steroid hormone synthesis in cultured rat ovarian follicles.

GC-MS-based metabolomics reveals mechanism of action for hydrazine induced hepatotoxicity in rats.

Gas chromatography-mass spectrometry (GC-MS) has great advantages for analyzing organic/amino acids, which are often targets in efficacy and/or toxicity studies. Although GC-MS has been used for the detection of many metabolic disorders, applications of GC-MS-based metabolomics in pharmacology/toxicology are relatively underdeveloped. We intended to investigate applicability of a GC-MS-based metabolomics approach for toxicological evaluation, and tried to elucidate the mechanism of hydrazine-induced hepatotoxicity. Rats were administered hydrazine chloride orally (120 and 240 mg kg⁻¹), and urine, plasma and liver samples were collected at 24 or 48 h post-dosing. Conventional clinical chemistry and liver histopathology were performed, urine and plasma were analyzed by GC-MS, and metabolic profiles were assessed using chemometric techniques. Principal component analysis score plots showed clear separation of the groups, indicating dose-dependent toxicity and recovery. The mechanism of toxicity was investigated based on semi-quantification data of identified metabolites. Amino acid precursors of glutathione (cystein, glutamate and glycine) and a product of glutathione metabolism (5-oxoproline) were elevated dose-dependently, accompanied with elevation of ascorbate levels. In addition, intermediates of the TCA cycle were decreased, whereas participants of the urea cycle and other amino acids were increased. These alterations were associated with histopathological changes such as fatty degeneration and glycogen accumulation. Application of GC-MS-based metabolomics revealed that oxidative stress and GSH consumption play important roles in the etiology of hydrazine-induced hepatotoxicity, demonstrating that this approach is a useful tool in pharmacology and toxicology for screening, elucidating mode of action and biomarker discovery.

The antipsychotics haloperidol and chlorpromazine increase bone metabolism and induce osteopenia in female rats.

The main objective of this study was to evaluate the effects of the antipsychotic drugs haloperidol (HAL) and chlorpromazine (CPZ) on bone mineral density (BMD) in female rats and to examine the relationship between the effects on bone and reproductive organs or hormone concentrations. Female rats were orally administered HAL (2 or 10 mg/kg) or CPZ (25 or 50 mg/kg) once daily (7 days/week) for 6 months resulting in a significant increase in prolactin. Hyperprolactinemia resulted in enlarged corpora lutea in the ovary, because prolactin has a luteotropic activity. Thus, atrophy in the uterus, epithelial mucification in the vagina and continuous diestrus stages were observed. These events in the reproductive organs induced a decrease in estradiol, elevation of biochemical markers of bone metabolism, significant reductions of BMD in trabecular bone of the femur and decreased trabecular bone in the femur. The bone loss is associated with an increase in bone resorption due to decreased estradiol derived from the luteotropic activity of prolactin. The mechanism of dopamine blockers to induce bone loss in female rats is considered to be rodent specific because the luteotropic effects of prolactin are confined primarily to rodents. Also, it appears that chronic hyperprolactinemia and maintained corpora lutea leading to bone loss are commonly inducible in female rats receiving long-term treatment with antipsychotic drugs possessing dopamine D2 receptor antagonist activity.

Influences of biofluid sample collection and handling procedures on GC-MS based metabolomic studies.

Sample collection procedures of pharmacology and toxicology studies might have a great impact on interpretation of metabolomic study results. Characterization of range variation among sample collection methods is necessary to prevent misinterpretation, as is use of optimal methods in animal experiments to minimize biological/technical variation. Here, we investigated the influence of urine and plasma sample collection and handling procedures on GC-MS based metabolomic studies as follows: for urine, pooling period and tube conditions during collection; for plasma, sampling sites, anesthesia and anticoagulants. Metabolic profiles of urine varied dramatically depending on urine pooling period and tube conditions, underscoring the importance of determining appropriate sampling periods in consideration of diurnal effects and targets of effect/toxicity, and suggesting it would be preferable to keep tubes in metabolic cages under iced conditions for urine sampling. Metabolic profiles of plasma differed depending on blood sampling sites. Anesthesia was not effective in reducing individual variation, although the anesthesia was beneficial in reducing discomfort in rats. In GC-MS based metabolomic studies, we recommend that EDTA be used as anticoagulant in plasma sample preparation, because peaks derived from heparin might overlap with endogenous metabolites, which may induce inter-sample variation. The present study demonstrated that biofluid sample collection and handling procedures provide great impact on metabolic profiles, at the very least for minimizing biological/technical variation, sampling period for urine collection should not be set as a short period, and the use of EDTA is recommended as anticoagulant in preparing plasma for analysis by GC-MS.

Comparison of potential risks of lactic acidosis induction by biguanides in rats.

Lactic acidosis has been considered to be a side effect of some biguanides, after phenformin was withdrawn from the market because of its association with lactic acidosis. The potential of lactic acidosis induced by biguanides at human therapeutic exposure levels, however, has not been examined. Then, we compared the risk of lactic acid at doses providing exposure levels comparable to human therapeutic doses. Metformin and phenformin were orally administered to rats for up to 28 days, and plasma drug concentrations and blood lactic acid levels were examined. Metformin did not elevate lactic acid levels at the dose corresponding to higher systemic drug exposure than human therapeutic level, even for repeated doses. In contrast, phenformin elevated lactic acid levels at the dose corresponding to lower exposure than human therapeutic level, and sustained high levels were observed up to 24h post-dose; furthermore, these changes were enhanced by repeated doses. Direct comparison at each rat equivalent dose clearly indicated that lactic acid levels of phenformin were higher than those of metformin. These non-clinical findings suggest that metformin dose not increase lactic acid levels like phenformin does, and therefore may not increase the risk for lactic acidosis at human therapeutic exposure level.

Evaluation for reliability and feasibility of the draft protocol for the enhanced rat 28-day subacute study (OECD Guideline 407) using androgen antagonist flutamide.

As part of an international validation project to establish a test protocol for the 'Enhanced OECD Test Guideline no. 407', a 28-day repeated dose study of flutamide was performed (1) to examine which of the current and/or additional parameters can detect endocrine effects of test chemicals most reliably and sensitively, (2) to investigate whether it is actually feasible to routinely include all additional parameters into the testing routine, and (3) to assess intra-laboratory variability by performing two identical studies (experiments A and B) in parallel using groups of five animals each per dose and sex. Groups of five male and five female CD(SD)IGS rats were treated by oral gavage with 0, 1, 10 and 100 mg flutamide/kg body weight for at least 28 days. The dose level considered to be around the MTD (100 mg/kg) exerted the expected antiandrogenic effects on androgen related tissues: significant decrease of the weights of androgen dependent organs and the sperm count and increase in histopathological lesions. At the middle dose (10 mg/kg), significant decrease of prostate weight (ventral and dorso-lateral parts combined) was observed and it was suggested that weight measurement of androgen dependent organs provides the most reliable and sensitive endpoint with this protocol. As for the feasibility, because of many items in this protocol, selection should be based on the sensitivity. From our data, addition of weight measurement of androgen dependent organs to the items of the existing OECD 407 guideline might allow accurate screening for endocrine disruptors. At the dose level considered to be around the MTD, the findings achieving statistical significance in one experiment with five animals/dose/sex could be reproduced in the second experiment, and evaluation with the small groups was consistent with findings using the combined groups of 10 animals/dose/sex. The results demonstrate that the protocol can reliably detect antiandrogenic effects of flutamide.

Enhanced rat Hershberger assay appears reliable for detection of not only (anti-)androgenic chemicals but also thyroid hormone modulators.

Development of an internationally recognized standard for the Hershberger assay as a screening tool to detect potential (anti-)androgenic chemicals is in progress. In the present preliminary study, we evaluated the reliability of the enhanced Hershberger assay to detect thyroid hormone modulating activity, while concentrating attention on possible confounding influence on evaluation of (anti-)androgenic activity. Castrated or testosterone propionate (TP; 0.2 or 0.25 mg/kg/day)-injected castrated male Crj:CD(SD) IGS rats (seven weeks of age) were dosed for 10 days by oral gavage with vehicle (corn oil) or the following chemicals: propylthiouracil (PTU; 2.5 mg/kg/day), a potent inhibitor of thyroid hormone synthesis, phenobarbital (PB; 125 mg/kg/day) and 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE; 100 mg/kg/day), two hepatic enzyme inducers that enhance the clearance of thyroid hormones. PTU markedly increased thyroid weights, and decreased serum T3 and T4, and increased serum TSH, also causing marked microscopic alteration of the thyroid gland. In comparison, PB and p,p'-DDE only significantly affect serum T4 and revealed some histopathological findings. The alterations appeared to be more robust in the presence of TP. Furthermore, data for p,p'-DDE demonstrated its anti-androgenic effects, whereas PTU and PB had little or no effects on the weights of androgen-related accessory glands/tissues: the ventral prostate, dorso-lateral prostate, seminal vesicles with coagulating glands, glans penis, Cowper's glands, and levator ani plus bulbocavernosus (LABC) muscles. Weight of the LABC muscles was decreased by PB treatment in TP-treated castrated rats. These findings in the present study suggests that the enhanced Hershberger assay, with evaluation of thyroid histopathology and weights, and hormone levels, appears to be reliable for screening for not only (anti-)androgenic chemicals but also thyroid hormone modulators. In order to evaluate whether the sensitivity and specificity of such a thyroid assay is great enough for routine screening purposes, future experiments including dose-response studies using lower dose levels have to be performed.

Lack of (anti-) androgenic or estrogenic effects of three pyrethroids (esfenvalerate, fenvalerate, and permethrin) in the Hershberger and uterotrophic assays.

Synthetic pyrethroids are among the most common pesticides and insecticides currently in use worldwide. Recently, chemicals classified as synthetic pyrethroids are suspected as being endocrine disrupting chemicals. However, no study has been conducted to assess their potential hormonal activities using in vivo test specifically focused on endocrine disruption. In the present study, we evaluated the interaction of three pyrethroids (esfenvalerate, fenvalerate, and permethrin) with androgen receptor (AR)- and estrogen receptor (ER)-mediated mechanisms using in vivo short-term assays. While internationally standardized protocols for the Hershberger and uterotrophic assays have not yet been fully developed, both are widely used and are being considered by OECD as short-term screening assays for hormonal activity. A 5-day Hershberger assay using castrated male rats measures agonistic and androgenic ability of the test chemicals to AR of several accessory glands/tissues (the ventral prostate, dorsolateral prostate, seminal vesicles with coagulating glands, and levator ani plus bulbocavernosus muscles). Esfenvalerate (5, 10, or 20 mg/kg/day), fenvalerate (20, 40, or 80 mg/kg/day), or permethrin (25, 50, or 75 mg/kg/day) was administered by oral gavage for 5 days to castrated male Crj:CD(SD)IGS rats (1 week after the castration, 11 weeks of age) with or without coadministration of 0.25 mg/kg/day testosterone propionate (subcutaneous injection on the dorsal surface). The highest dose levels tested for each chemical were considered the maximum level that could be used without causing excessive systemic toxicity. None of esfenvalerate, fenvalerate, and permethrin showed any androgenic or antiandrogenic effects. Reference control of p,p'-DDE and methyltestosterone (100 mg/kg/day) provided significant effects in this assay protocol. Potential effects of these pyrethroids mediated through the ER were evaluated by means of 3-day uterotrophic assay using ovariectomized Crj:CD(SD)IGS rats (2 weeks after the ovariectomy, 8 weeks of age). No increase in weight of uterus (wet or blotted) was observed following oral exposure to esfenvalerate (5, 10, or 20 mg/kg/day), fenvalerate (20, 40, or 80 mg/kg/day), or permethrin (37.5, 75, or 150 mg/kg/day), respectively. Again, the highest dose levels tested for each chemical were considered the maximum level that could be used without causing excessive systemic toxicity. Reference controls consisting of ethynyl estradiol (0.03 mg/kg/day) and methoxychlor (125 mg/kg/day) both showed a significant effect in this assay protocol. It is concluded that, based on the results of these two reliable in vivo assays, none of esfenvalerate, fenvalerate, or permethrin exhibit any potential to cause adverse (anti-) androgenic or estrogenic effects at dose levels below that of those causing excessive systemic toxicity.