The effects of oral treatment with transfluthrin on the urothelium of rats and its metabolite, tetrafluorobenzoic acid on urothelial cells in vitro.

Transfluthrin, a pyrethroid insecticide, induced urinary bladder tumors in rats but not in mice in 2-year bioassays. We investigated the urothelial effects of transfluthrin in vivo in rats and the effects of its major metabolite tetrafluorobenzoic acid (TFBA) in vitro on rat (MYP3) and human (1T1) urothelial cell lines. Rats were fed diet containing 0, 2000 or 5000 (with and without 1.25% NH(4)Cl) ppm transfluthrin for 4 weeks or 0 or 2000 ppm transfluthrin for 13 weeks. After 4 weeks, there was no evidence of hyperplasia or increased proliferation in any treatment group. After 13 weeks treatment with 2000 ppm, cytotoxicity and necrosis of the rat urothelial superficial layer were detected by scanning electron microscopy. The urinary concentration of TFBA in rats fed 2000 ppm transfluthrin was 2.94±0.67 mM. The LC(50) of TFBA was 2.25 mM for MYP3 cells and 2.43 mM for 1T1 cells. These studies support cytotoxicity and regenerative proliferation as the mechanism for induction of bladder tumors with high oral doses of transfluthrin due to metabolism of transfluthrin to the weakly cytotoxic TFBA which is excreted at high concentrations in the urine of rats administered high doses of transfluthrin (≥2000 ppm) for an extended period.

A 1-year toxicity study in dogs is no longer a scientifically justifiable core data requirement for the safety assessment of pesticides.

A review of publications on pesticides assessing the need for 1-year toxicity studies in dogs was performed. Four key peer-reviewed papers with different approaches investigated the value of a 1-year dog study in addition to a 3-month study. Despite different databases and approaches, each concluded with the recommendation to limit the testing of pesticides in dogs to a duration of 3 months. The combined weight of evidence presented in this review reinforces these independent conclusions. Therefore, the routine inclusion of a 1-year dog study as a mandated regulatory requirement for the safety assessment of pesticides is no longer justifiable and a globally harmonized approach should be taken to match the latest legislation of the European Union and the US EPA.

Respiratory control, respiratory sensations and cycling endurance after respiratory muscle endurance training.

Respiratory muscle endurance training (RMET) was shown to increase endurance performance in healthy subjects. Reduced adverse respiratory sensations might contribute to this improvement. In the present study, we aimed to assess the relationship between changes in respiratory sensations and changes in ventilation and endurance performance after RMET. Fourteen healthy subjects completed either forty 15-min RMETs (n = 8) or did no training (control, n = 6). Respiratory endurance increased significantly after RMET while breathlessness and respiratory exertion were significantly reduced. Cycling endurance did not change while average ventilation was increased and perception of respiratory exertion was reduced. We conclude that (1) RMET reduces adverse respiratory sensations during isolated and exercise-induced hyperpnea even in the face of increased respiratory drive, and (2) the reduction in adverse respiratory sensations after RMET does not per se cause an increase in endurance performance. Whether the reduced perception of adverse respiratory sensations during exercise after RMET might be the cause of the increased respiratory drive remains to be clarified.

The acquisition and application of absorption, distribution, metabolism, and excretion (ADME) data in agricultural chemical safety assessments.

A proposal has been developed by the Agricultural Chemical Safety Assessment (ACSA) Technical Committee of the ILSI Health and Environmental Sciences Institute (HESI) for an improved approach to assessing the safety of crop protection chemicals. The goal is to ensure that studies are scientifically appropriate and necessary without being redundant, and that tests emphasize toxicological endpoints and exposure durations that are relevant for risk assessment. Incorporation of pharmacokinetic studies describing absorption, distribution, metabolism, and excretion is an essential tool for improving the design and interpretation of toxicity studies and their application for safety assessment. A tiered approach is described in which basic pharmacokinetic studies, similar to those for pharmaceuticals, are conducted for regulatory submission. Subsequent tiers provide additional information in an iterative manner, depending on pharmacokinetic properties, toxicity study results, and the intended uses of the compound.

Tissue-specific effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the activity of 5'-deiodinases I and II in rats.

Thyroid hormones play a complex role in the toxicity of polychlorinated dibenzo-p-dioxins and furans and related compounds. We investigated the toxicological significance of 5'-deiodinases I and II (5'-DI and 5'-DII) in the altered thyroid hormone status of TCDD-treated rats. Time courses and dose responses were determined for serum thyroxine (T4) and triiodothyronine (T3) levels, for 5'-DI activity in thyroid gland, liver and kidney, and for 5'-DII activity in brown adipose tissue (BAT). TCDD-treatment resulted in prompt and dose-dependent decrease in circulating T4 followed by a decrease in liver 5'-DI activity 1-2 days later and an apparent increase in BAT 5'-DII activity. Changes in liver 5'-DI and BAT 5'-DII activity were secondary to decreased T4 levels. Thyroid and kidney 5'-DI activities as well as circulating T3 levels were not affected. The results suggest that altered 5'-DI or 5'-DII activities do not significantly influence the circulating levels of T4 or T3 in TCDD-treated rats.

Effects of the new herbicide fentrazamide on the glucose utilization in neurons and erythrocytes in vitro.

Treatment of rats with fentrazamide for 2 years at 3000 ppm (males) and 4000 ppm (females) led to an increased incidence and degree of axonal degeneration in sciatic nerve as well as to effects on red blood cells. The mechanism underlying these effects was investigated in vitro using various cell cultures (permanent rodent cell lines from the nervous system, liver, kidney, skeletal and heart muscle and fibroblasts, primary cortical neurons and erythrocytes from the rat). Added to cultured rat cortical neurons for 1 week, fentrazamide considerably decreased glucose consumption, ATP levels and mitochondrial membrane potential and lowered the GSH level, however, it had little impact on viability and neurofilaments and did not induce oxidative stress (ROS) over the first 2 h. After recovery for 1 week, in addition some destruction of neurofilaments had occurred probably secondary to the disturbance of energy production. These effects were prevented by pyruvate. Further studies indicated that fentrazamide primarily inhibited glucose utilization, most likely by interfering with glycolysis. Similar effects were found in erythrocytes treated with fentrazamide over a period of 7 days. Primarily, the glucose consumption was reduced after 1-day treatment, followed by a marked reduction of the energy supply at days 3 and 7. Comparable to the neurons, the GSH level was significantly reduced. A marked hemolysis of the red blood cells was then observed after prolonged treatment. The extensive energy demand and exclusive dependency on glucose utilization of neurons and erythrocytes may explain the specific vulnerability of motor neurons and erythrocytes. When comparing the concentrations necessary for inducing effects in vitro on neuronal cells and erythrocytes to the very low plasma concentrations of fentrazamide in treated rats it is suggested that only a small impact of fentrazamide on the energy status at high doses will occur in vivo. Therefore, aging of the rat as another factor compromising mitochondrial energy production in motor neurons must be considered as additional contribution for the induction of axonal degeneration. It is concluded that this effect of fentrazamide in rats poses no specific risk under the exposure conditions relevant to humans.

Effects of the dithiocarbamate fungicide propineb in primary neuronal cell cultures and skeletal muscle cells of the rat.

After repeated-dose toxicity studies with the fungicide propineb, reversible effects on muscle functions were found. Therefore, mechanistic investigations should contribute to clarification of its mode of action in relation to disulfiram and diethyldithiocarbamate neurotoxicity or direct effects on muscle cells. In principle, besides the dithiocarbamate effects, two different mechanisms have been discussed for this fungicide. One mechanism is the degradation to carbon disulfide (CS(2)) and propylenthiourea (PTU) and the other are direct effects of zinc. Primary neuronal cell cultures of the rat are a well established model to identify neurotoxic compounds like n-hexane or acrylamide. In this cell culture model, endpoints such as viability, energy supply, glucose consumption and cytoskeleton elements were determined. Additionally, skeletal muscle cells were used for comparison. Propineb and its metabolite PTU were investigated in comparison to CS(2), disulfiram and diethyldithiocarbamate. The toxicity of zinc was tested using zinc chloride (ZnCl(2)). It was clearly shown that propineb exerted strong effects on the cytoskeleton of neuronal and non-neuronal cell cultures (astrocytes, muscle cells). This was similar to ZnCl(2,) but not to CS(2). With CS(2) and disulfiram effects on the energy supply were more prominent. In conclusion, the toxicity of propineb is not comparable to disulfiram, diethyldithiocarbamate or CS(2) neurotoxicity. In regard to these findings, a direct reversible effect of propineb on skeletal muscle cells seems to be more likely.

Sensitive detection of the endocrine effects of the estrogen analogue ethinylestradiol using a modified enhanced subacute rat study protocol (OECD Test Guideline no. 407).

Groups of five male and female Wistar rats were treated by gavage with 0, 0.01, 0.05 or 0.2 mg/kg body weight of the known synthetic estrogen ethinylestradiol for 28-32 days according to a modified enhanced OECD Test Guideline no. 407 in order to investigate which of the current and/or additional parameters would detect effects on the endocrine system reliably and sensitively and to provide data on intra-laboratory variability. Two identical studies (A and B) were run concurrently. The modified enhanced protocol requests the additional determination of triiodothyronine, thyroxine and thyroid-stimulating hormone (TSH), of the stage of the estrous cycle to ensure necropsy of all females in diestrus, of the number and morphology of cauda epididymal spermatozoa, and of additional organ weights (ovaries, uterus, thyroid, and male accessory reproductive organs), and histopathology of additional organs (pituitary, epididymides, coagulation glands, pancreas, and vagina). There were no treatment-related mortalities, clinical signs or changes in behavioral parameters. In male rats, 0.2 mg/kg was the maximum tolerated dose (MTD) resulting in reduced body weight gain. The only treatment-related alteration in hematological parameter was prolonged blood clotting time in high-dose females of both studies. Changes in clinical chemistry observed in study A were elevated alkaline phosphatase activity (high-dose females) and triglyceride levels (mid- and high-dose females and high-dose males). Changes in thyroid hormones and TSH of treated animals showed high variability with no clear dose-dependency, and could not be clearly related to estrogenic activity. In accordance to a suppression of the hypothalamic-pituitary-gonadal axis, decreased relative organ weights of the male accessory reproductive organs were obtained in both studies at the high dose. Corresponding histological changes were degeneration of the testicular germinal epithelium and atrophy of Leydig cells and of all accessory sex glands. Atrophy of the coagulating gland (study A) and seminal vesicles (study B) was also seen at 0.05 mg/kg. A marked increase in relative adrenal weight in male rats, accompanied by decreased vacuolization of zona fasciculata cells observed in both studies at the high dose seems to reflect an activation of the hypothalamic-pituitary-adrenal axis. The male mammary gland was sensitively affected. Increased numbers of small basophilic over large acidophilic cells indicated an estrogen-mediated feminisation and were detected at the low (study A) or mid dose (study B). Co-mitogenic properties of estrogens in rat liver were reflected by increased relative liver weights in females at the mid and high dose of study A and also at the high dose in study B. No treatment-related changes in endocrine organ weights were observed in treated females. Histological changes in the ovaries were increased numbers of apoptotic corpora lutea (from mid dose, study B) and of early stage follicles at the high dose in both studies. Classical direct estrogenic effects on the uterus, i.e. an increased height of luminal and glandular epithelium and increased granulocytic infiltration of the endometrium, were observed even at the low dose in both studies. Uterine findings occurring with a greater variability were dilation, squamous metaplasia of glands and thickened walls. Although females were necropsied in diestrus, as diagnosed by vaginal cytology, typical signs of estrogenic action in the vagina such as keratinization (indicative of estrus in normally cycling rats), mucification (indicative of proestrus), or thickened epithelia were observed in both studies even at the lowest dose. This unexpected discrepancy between vaginal cytology and vaginal and uterine morphology of treated females was considered to be treatment-related as it was not observed in the controls. Studies on liver enzymes that were performed outside the scope of the enhanced protocol showed that ethinylestradiol at 0.2 mg/kg decreased the activity of the sex-specific testosterone-dependent liver enzyme CYP2C11 in male rats. A simulation of doubling group size (to ten animals) by combining both studies did not increase the sensitivity of detection of endocrine-mediated effects above the level already obtained by histopathological examination of groups containing five animals. Only some of the enhancements to the current OECD Test Guideline no. 407 evaluated in this study (additional organs weights and additional histopathological investigations) were helpful in detecting the endocrine-mediated effects of ethinylestradiol, while other enhancements did not contribute towards this aim. Spermatology was completely insensitive at the MTD and measurement of thyroid hormones and TSH did not contribute to increased sensitivity. Vaginal cytology appeared to be an unreliable procedure for estrous cycle staging in estrogen-treated animals. Ongoing investigations, according to the modified version of the enhanced OECD Test Guideline no. 407 protocol, into the interference of ten compounds with the endocrine system by different mechanisms will result in the identification of the most appropriate enhancements.