The U.S. federal framework for research on endocrine disruptors and an analysis of research programs supported during fiscal year 1996.

The potential health and ecological effects of endocrine disrupting chemicals has become a high visibility environmental issue. The 1990s have witnessed a growing concern, both on the part of the scientific community and the public, that environmental chemicals may be causing widespread effects in humans and in a variety of fish and wildlife species. This growing concern led the Committee on the Environment and Natural Resources (CENR) of the National Science and Technology Council to identify the endocrine disruptor issue as a major research initiative in early 1995 and subsequently establish an ad hoc Working Group on Endocrine Disruptors. The objectives of the working group are to 1) develop a planning framework for federal research related to human and ecological health effects of endocrine disrupting chemicals; 2) conduct an inventory of ongoing federal research programs; and 3) identify research gaps and develop a coordinated interagency plan to address priority research needs. This communication summarizes the activities of the federal government in defining a common framework for planning an endocrine disruptor research program and in assessing the status of the current effort. After developing the research framework and compiling an inventory of active research projects supported by the federal government in fiscal year 1996, the CENR working group evaluated the current federal effort by comparing the ongoing activities with the research needs identified in the framework. The analysis showed that the federal government supports considerable research on human health effects, ecological effects, and exposure assessment, with a predominance of activity occurring under human health effects. The analysis also indicates that studies on reproductive development and carcinogenesis are more prevalent than studies on neurotoxicity and immunotoxicity, that mammals (mostly laboratory animals) are the main species under study, and that chlorinated dibenzodioxins and polychlorinated biphenyls are the most commonly studied chemical classes. Comparison of the inventory with the research needs should allow identification of underrepresented research areas in need of attention.

Research to strengthen the scientific basis for health risk assessment: a survey of the context and rationale for mechanistically based methods and models.

Assessment of health risks is an integral part of regulatory decision-making that occurs at the interface between science (e.g. facts) and policy (e.g. values). Because existing scientific knowledge and understanding are often inadequate to answer the most critical risk-related questions, regulatory agencies have developed sets of formalized 'science policies' to extrapolate from existing data to real-life events and situations. These science policies, as, for example, the use of default assumptions or exposure scenarios, can introduce significant uncertainties into the final risk estimate. We survey the rationale for research to reduce extrapolation-related uncertainties, focusing specifically on the need to develop mechanistically based methods and models, including test methods to identify and characterize health effects, integrated human exposure models, physiologically based pharmacokinetic (PBPK) models and biologically based dose-response (BBDR) models.

Age-dependent differences in the susceptibility of rats to deltamethrin.

Separate groups of weanling and adult rats were exposed to both behaviorally active and lethal doses of deltamethrin to examine age-dependent toxicity of a pyrethroid over a wide dose range. The acoustic startle response (ASR) was selected for comparison at low doses since it is a sensitive, quantifiable biological indicator of pyrethroid effects in rats. Acute mortality was included for comparison at the upper limit of the dose-response. Deltamethrin was administered by gavage as a single dose in corn oil for all tests. Effects on the ASR were comparable in 21- and 72-day-old rats, with a 4-mg/kg dose decreasing ASR amplitude by approximately 50% (ED50) at both ages. By comparison LD50 values in 11-, 21- and 72-day old male rats were 5.1, 11, and 81 mg/kg, respectively. Thus, 11- and 21-day-old male rats were 16 and 7 times, respectively, more sensitive than adults to acute lethality. The concentration of deltamethrin was measured in whole-brain tissue from weanling and adult males treated with ED50 and LD50 doses. The brain concentration of deltamethrin at the ED50 dose of 4 mg/kg was higher in weanling rats than adults. This suggests a possible functional difference, with weanling rats being less susceptible than adults to a low dose. By comparison, there was an equivalent concentration of deltamethrin in brain tissue following an LD50 dose of 12 mg/kg in weanling rats and 80 mg/kg in adults. These results support age-related differences in pharmacokinetics as the basis for the markedly greater sensitivity of young rats to a lethal dose of deltamethrin.

Interlaboratory comparison of motor activity experiments: implications for neurotoxicological assessments.

Motor activity is an important functional measure used in neurotoxicology. The effects of chemicals on motor activity, however, may depend on variables such as type of measurement apparatus, physical and environmental testing conditions, and many other experimental protocol and organismic variables. Due to the increasing use of motor activity in neurotoxicology, a major question concerns the potential for differences in experimental findings due to variations in sensitivity and reliability between different laboratories and devices used to measure motor activity. This study examined historical data from a number of laboratories that employed different devices and experimental protocols to measure motor activity. Four aspects of the motor activity data were compared: 1) within-laboratory control variability across time; 2) within-laboratory replicability of control data; 3) between-laboratory variability in the effects of chemicals; and 4) between-laboratory comparison of the control rates of habituation. The analyses indicated that there was a relatively restricted range of within-laboratory variability and reliability in control values, and that these ranges were comparable across laboratories. Similar profiles of habituation were also seen across the different laboratories. Moreover, in virtually every case, all laboratories were capable of detecting qualitatively similar changes in motor activity following acute exposure to a variety of chemicals. These data indicate a high degree of comparability in the data generated by the different devices and experimental protocols.

Serotonergic modulation of the acoustic startle response in rats during preweaning development.

The involvement of serotonin (5-HT) in modulating the acoustic startle response (ASR) is well established in adult rats, but 5-HT involvement during the preweaning period, when 5-HT neurons undergo extensive development, has not previously been described. Three 5-HT receptor subtypes are reported to modulate the ASR in adult rats: 5-HT1A and 5-HT2 receptor agonists facilitate the ASR, whereas 5-HT1B agonists decrease the response. In the present study, the effects of 5-HT agonists and generalized 5-HT depletion on the ASR were studied in preweanling animals, using independent groups of Long-Evans rats tested on postnatal day (PND) 13, 17 and 21. 8-Hydroxy-2-(di-n-propylamino) tetralin (8OHDPAT, 62-1000 micrograms/kg), a 5-HT1A receptor agonist, and 5-methoxy-N,N-dimethyl tryptamine (MeODMT, 2-4 mg/kg), a nonselective 5-HT agonist, had no effect on PND 13 and then increased the ASR on PND 17 and 21. The 5-HT2 receptor antagonists cyproheptadine (5 mg/kg) and ketanserin (5 mg/kg) blocked the effect of MeODMT at both ages, providing some evidence that MeODMT increased the ASR through 5-HT2 receptors. 1-(m-Chlorophenyl) piperazine (mCPP, 1-5 mg/kg), a 5-HT1B agonist, had no effect on ASR amplitude on PND 13 or 17 and then produced a dose-related decrease in the response on PND 21. Generalized depletion of 5-HT by 80-90% in whole-brain and spinal cord, using p-chlorophenylalanine (PCPA, 300 mg/kg 24 hr prior to testing), did not alter ASR amplitude at any age.(ABSTRACT TRUNCATED AT 250 WORDS)

Prenatal or postnatal exposure to bis(tri-n-butyltin)oxide in the rat: postnatal evaluation of teratology and behavior.

The results of a series of screening tests to determine the potential teratogenicity and neurotoxicity of developmental exposure to TBTO in rats are presented in this paper. For prenatal exposure, pregnant Long Evans rats were intubated with 0-16 mg/kg/day bis(tri-n-butyltin)oxide TBTO from Days 6 to 20 of gestation (GD 6-20). For postnatal exposure, rat pups were intubated with 0-60 mg/kg TBTO on Postnatal Day 5 (PND 5). Following prenatal exposure, dams were allowed to litter and pups were evaluated using a postnatal teratology screen. Postnatal evaluation for both exposures included motor activity (PND 13-64), the acoustic startle response (PND 22-78), growth, and brain weight. The maximally tolerated dose (MTD) in pregnant rats was 5 mg/kg/day, which is one-third the MTD in nonpregnant rats. There were decreased numbers of live births, and decreased growth and viability at dosages greater than or equal to 10 mg/kg/day. Cleft palate was found in 3% of the 12 mg/kg/day group. There was mortality following postnatal exposure to 60 mg/kg and all prenatal dosages greater than or equal to 10 mg/kg/day. Preweaning body weight was significantly decreased for all postnatal dosages, and all prenatal dosages greater than 2.5 mg/kg/day. Body weight reductions persisted to the postweaning period only in the high dose groups (10 mg/kg/day and 60 mg/kg). Behavioral evaluation demonstrated transient alterations in motor activity development (prenatal exposure only) and the acoustic startle response (postnatal exposure only). Persistent behavioral effects were observed only at dosages that produced overt maternal toxicity and/or postnatal mortality. The demonstration of the teratogenic and neurotoxic potential of TBTO in rats is confounded by associated maternal toxicity and/or pup mortality.

Ontogeny of the acoustic startle response and sensitization to background noise in the rat.

The purpose of this study was to characterize the ontogeny of the acoustic startle response (ASR) and response sensitization to background noise in preweanling rats. Animals were tested daily from 11 to 21 days of age using one of four sets of background white noise levels [45-80 dB(A)]. With constant low-level (45 dB, SPL) background noise, response latency decreased steadily with age, whereas both response incidence and amplitude increased nonmonotonically with age. Two approaches were used to examine the ontogeny of sensitization to background noise: The first compared the ASR of animals tested at 75 dB background noise with ones tested at 45 dB; the second compared the ASR of animals tested at three background levels (30 dB range) within the test session. Sensitization was not evident before 15-16 days of age. By comparing these results with the results from naive animals, it was found that daily test experience does not alter ASR amplitude, latency, incidence, or the development of sensitization.

Alkyltin inhibition of ATPase activities in tissue homogenates and subcellular fractions from adult and neonatal rats.

Inhibition of ATPase activities by triethyltin (TET), diethyltin (DET), monoethyltin (MET), and trimethyltin (TMT) was studied in homogenates of brain and liver from adult and neonatal rats. In the adult, sensitivities were as follows: mitochondrial ATPase of liver much greater than Na+, K+-ATPase of brain approximately equal to mitochondrial ATPase of brain greater than nonspecific ATPase of brain and liver. MET did not produce significant inhibition. ATPase activities in brain and liver homogenates from TET-treated adult rats did not differ from controls. Mitochondrial ATPase in brain homogenates from 5-day-old rats was two orders of magnitude more sensitive to TET than brain homogenates from adult rats (IC50 of 2.5 microM in the 5-day-old neonate vs 260 microM in the adult). By contrast, isolated mitochondria and synaptosomal fractions from adult and neonatal brains were equally sensitive to TET (IC50 = 1-3 microM). At 10 days of age, following the onset of myelination, the IC50 for TET inhibition of brain mitochondrial ATPase increased to 71 microM. Myelin added directly to isolated mitochondria also reduced TET-induced inhibition. It is concluded that in vivo brain tin concentrations in 5-day-old rats following a neurotoxic dose of TET are sufficient to inhibit brain mitochondrial ATPase, whereas in adults, tin concentrations are insufficient for inhibition. In the adult rat, TET binding to myelin appears to prevent inhibition of brain mitochondrial ATPase, and the target of toxic action may be myelin. In the neonateal rat, TET may inhibit oxidative phosphorylation in unmyelinated brain tissue, leading to neuronal cell death.

The effects of type I and II pyrethroids on motor activity and the acoustic startle response in the rat.

Recent data have demonstrated that the in vivo effects of low dosages of two pyrethroids, cismethrin and deltamethrin, can be differentiated. Two behavioral tests, locomotor activity and the acoustic startle response (ASR), were utilized to separate the behavioral actions of Type I and II pyrethroids using permethrin, RU11679, cypermethrin, RU26607, fenvalerate, cyfluthrin, flucythrinate, fluvalinate and p,p'-DDT. Dosage-effect functions for all compounds were determined for both figure-eight-maze activity and the ASR in the rat. All compounds were administered po in 1 ml/kg corn oil 1.5-3 hr prior to testing. All compounds produced dosage-dependent decreases in locomotor activity. The Type I compounds, permethrin and RU11679, along with p,p'-DDT, increased amplitude and had no effect on latency to onset of the ASR. In contrast, the Type II pyrethroids, cypermethrin, cyfluthrin, and flucythrinate, decreased amplitude and increased the latency to onset of the ASR. Fenvalerate increased the amplitude, had no effect on latency, but unlike the other compounds tested, increased ASR sensitization. Fluvalinate had no effect on any measure of the ASR. These data provide further evidence of the differences between the in vivo effects of low dosages of Type I and II pyrethroids, and extend the findings of our previous work to other representatives of the two classes of pyrethroids.

Hyperactivity induced by triadimefon, a triazole fungicide.

Triadimefon is an agriculturally important triazole fungicide. The present experiments were conducted to characterize the effects of triadimefon on a measure of motor activity. Dosage-effect, time-effect, and the effect of repeated dosing (7 days) were determined following triadimefon exposure. Male Long Evans hooded rats, approximately 70 days old, received triadimefon po in 2.0 ml/kg corn oil. Motor activity testing was conducted for 1 hr in figure-eight mazes. For the dosage-effect determination, triadimefon (50-400 mg/kg) was administered 1 hr prior to testing. In the time-course study, triadimefon (200 mg/kg) was administered either 0.5, 1, 2, 4, 8, or 24 hr prior to testing. In the repeated dosing experiment animals received triadimefon (100 mg/kg) daily for 7 days and were tested 24 hr after the last exposure. Triadimefon produced significant hyperactivity following dosages of 100 and 200 mg/kg. This hyperactivity was rapid in both onset (0.5 hr) and recovery (8.0 hr). Repeated dosing with 100 mg/kg/day revealed no cumulative effects nor tolerance. These results indicate that triadimefon produces a transient hyperactivity at dosages 17 to 33% of the reported LD50.

Pyrethroid insecticides and the gamma-aminobutyric acidA receptor complex: motor activity and the acoustic startle response in the rat.

Two behavioral tests, motor activity and the acoustic startle response (ASR), were used to test for dose-addition of cismethrin, a Type I, or deltamethrin, a Type II pyrethroid, with compounds active at the gamma-aminobutyric acid (GABAA) receptor complex (picrotoxin, muscimol and chlordiazepoxide). Additivity was assessed using a simplified version of isobolographic analysis using chlorpromazine and haloperidol as positive controls for dose-additivity. Dosage-effect functions for all compounds were determined for both motor activity and the ASR. The effects of various combinations of chlorpromazine (0.5-4.0 mg/kg) and haloperidol (0.05-0.2 mg/kg) on motor activity indicate dose-addition. To test for dose-addition of pyrethroids and GABAergic compounds, cismethrin (3-18 mg/kg) or deltamethrin (2-6 mg/kg) were administered 90 min before testing, either alone, or before treatment with picrotoxin (0.25-2.0 mg/kg), muscimol (0.6-2.5 mg/kg) or chlordiazepoxide (2.5-10 mg/kg) administered 20 to 30 min before testing. All compounds produced dosage-dependent decreases in motor activity. Muscimol and picrotoxin decreased ASR amplitude, increased ASR latency and reduced ASR sensitization to increasing background noise levels. Chlordiazepoxide had no effect on any measure of the ASR. Results from the interaction studies indicate dose-addition of the effects of picrotoxin and deltamethrin on motor activity and the ASR. Additivity of dose was not seen with any other combination. These data suggest that the in vivo effects of the Type II pyrethroid deltamethrin may be due in part to interaction with the picrotoxinin binding site of the GABAA receptor-ionophore complex. In addition, these results are consistent with reported differential effects of the two classes of pyrethroids on the GABAA receptor complex.

Pyrethroid insecticides and radioligand displacement from the GABA receptor chloride ionophore complex.

Radioligand binding displacement studies were conducted to determine the effects of Type I and II pyrethroids on [3H]flunitrazepam (FLU), [3H]muscimol (MUS), and [35S]t-butylbicyclophosphorothionate (TBPS) binding. Competition experiments with [3H]FLU and [3H]MUS indicate a lack of competition for binding by the pyrethroids. Type I pyrethroids failed to compete for the binding of [35S]TBPS at concentrations as high as 50 microM. Type II pyrethroids inhibited [35S]TBPS binding to rat brain synaptosomes with Ki values ranging from 5-10 microM. The data presented here suggest that the interaction of Type II pyrethroids with the gamma-aminobutyric acid (GABA) receptor-ionophore complex is restricted to a site near the TBPS/picrotoxinin binding site.

Neurotoxicology in regulation and risk assessment.

It is well recognized that a wide variety of chemicals exert toxic effects on the structure and function of the nervous system. Regulatory agencies, charged with the protection of human health from toxicant exposures, should therefore consider neurotoxicological evaluations in the risk assessment process. Over the last decade, several expert panels have recommended testing schemes for evaluating neurobehavioral toxicity. In general, these panels have recommended behavioral measures performed in tandem with neuropathological evaluations. In keeping with these recommendations, the Environmental Protection Agency has developed seven neurotoxicity test guidelines for use in evaluating new and existing chemicals. Further refinement and extension of these test guidelines will flow from a better understanding of the cellular/molecular events which underly neurotoxicant-induced functional alterations.

Collaborative Behavioral Teratology Study: protocol design and testing procedures.

This paper presents background information on the methods used in the Collaborative Behavioral Teratology Study (CBTS), the rationale behind the experimental design, and the design and specific procedures used in the CBTS. Each of the following methods is discussed: negative geotaxis, olfactory discrimination, auditory startle habituation, one-hour activity in the figure-8 maze, visual discrimination learning, 23-hour activity in the figure-8 maze, and amphetamine-stimulated activity. The CBTS was designed to determine the intra- and interlaboratory reliability of these test methods and the detection sensitivity of each method, as well as to determine the importance of several major variables (early test experience, gender, litter). The important design features which permitted these evaluations are discussed. Each laboratory conducted two independent experiments: one using d-amphetamine sulfate as the test agent and one using methylmercuric chloride. Other than the use of different agents and dosing regimens in the two studies, all other characteristics of experimental design were identical. Each study was conducted in four replicates with 4 litters/each of 4 treatment groups/replicate. The replicate design was an important feature which permitted reliability of the tests to be addressed under conditions in which several other sources of variation in responding could be identified and accounted for in the model. Other methods by which optimal testing conditions were implemented in the participating laboratories included the "blind" testing of all subjects in specific orders which were counterbalanced for treatment group, time of day, and the apparatus in which the animals were placed.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of locomotor activity of rat pups in figure-eight mazes.

In a series of four experiments, social and experiential factors that influence the development of motor activity in rat pups were examined. Motor activity was monitored from postnatal Days 13 to 21 as photocell interruptions in figure-eight mazes and comparisons were made between pups maintained in a nest box containing a dam and siblings and allowed access to the maze for 23 hr/day, pups tested daily for 1 hr/day vs pups tested only on postnatal Days 15, 18, or 21, pups tested daily for either 5 min, 30 min, or 1 hr/day, and pups tested daily for 30 min/day either singly in a maze, paired with a littermate, or paired with an anesthetized pup of the same age. A monotonic increase in activity was seen for nest-box testing, minimal developmental change was seen for pups tested on only a single day or for pups tested with an anesthetized pup, whereas all other groups showed an inverted U-shaped profile of activity which was influenced by the duration of testing and/or the presence of a littermate. These data emphasize the relevance of environmental factors as determinants of preweaning behavior.

Development of locomotor activity of rat pups exposed to heavy metals.

Cadmium (Cd), triethyltin (TET), and trimethyltin (TMT) are heavy metals which are neurotoxic to developing animals. In the present experiment, preweaning assessment of locomotor activity was used to detect and differentiate between the developmental toxicity of these metals. On postnatal day (PND) 5, rat pups received a single injection of either Cd, TET, or TMT. A within-litter design was used for dosing; 1 male and 1 female pup from each litter (N = 10 litters/compound) received either the vehicle, low, medium, or high dosage of the compound. Preweaning motor activity was assessed in 30-min sessions in figure-eight mazes from PND 13 to 21. Motor activity of control animals progressively increased in the initial days of testing, and then both within-session and between-session habituation developed. A single exposure to Cd, TET, and TMT produced hyperactivity by the end of the preweaning period but these metals differed in the day of peak activity, the onset of hyperactivity, and the development of habituation.

Tin distribution in adult rat tissues after exposure to trimethyltin and triethyltin.

The time course of distribution of tin in the adult rat was determined in brain, liver, kidneys, heart, and blood following single ip administrations of trimethyltin hydroxide (TMT) and triethyltin bromide (TET). Adult Long-Evans rats were killed 1, 4, 12, and 24 hr, and at 5, 10, or 22 days following injection of TMT and TET (N = 6/time), and tissues were analyzed for total tin by atomic absorbance spectroscopy. TET exposure resulted in higher tin concentrations in brain, liver, and kidney tissues, while the two trialkyltins resulted in approximately equal tin concentrations in the heart and blood. Rates of elimination of tin (expressed as elimination rate constants, Kel) were greater in all tissues following TET exposure than following TMT exposure. The concentration of tin in the brain 12 hr after TMT exposure was 4.4, 8.5, and 12.7 ng tin/mg protein for dosages of 3.0, 6.0, and 9.0 mg/kg, respectively. Tin was evenly distributed across the cerebellum, medulla-pons, hypothalamus, hippocampus, and striatum following TMT exposure. These results describe major differences in the disposition and rates of elimination of tin from body tissues after TMT and TET exposure, and demonstrate that the regional disposition of tin is not related to the region-specific pathology reported following TMT exposure.

Effects of two pyrethroid insecticides on motor activity and the acoustic startle response in the rat.

To better characterize the behavioral toxicity of pyrethroid insecticides, comparisons were made of the effects of cismethrin and deltamethrin exposure on motor activity and the acoustic startle response in male Long-Evans rats. Acute dose-effect, acute time course, and 30-day repeated-exposure determinations of 1-hr motor activity were made using figure-eight mazes. The acoustic startle response was measured to a 13-kHz, 120-dB(A), 40-msec tone at each of three background white noise levels (50, 65, and 80 dB). Deltamethrin (0, 2, 6, or 8 mg/kg) or cismethrin (0, 6, 12, 18, or 24 mg/kg) were administered po in 0.2 ml/kg corn oil. Cismethrin and deltamethrin produced similar dosage-dependent decreases in motor activity. The time course of onset and recovery for this decreased activity was rapid (1 to 4 hr) No cumulative effects on motor activity of a 30-day exposure to 2 mg/kg/day deltamethrin or 6 mg/kg/day cismethrin were found. The effects of cismethrin and deltamethrin on the acoustic startle response were dissimilar: deltamethrin produced a dosage-dependent decrease in amplitude and an increase in latency, and cismethrin produced an increase in amplitude and no change in latency. The differential effects of cismethrin and deltamethrin on the acoustic startle response may be related to the contrasting effects previously shown with neurophysiological and/or neurochemical techniques.