Análisis de un protocolo basado en la norma USEPA 1.213 para la evaluación regulatoria de la eficacia de las cebaderas en el control seguro de roedores pequeños / Analysis of a protocol based on the USEPA 1.213 standard for the regulatory evaluation of the efficacy of the bait station system in the safe control of small rodents

Varios rodenticidas modernos son formulados con compuestos anticoagulantes superwarfarínicos. Debido a su alta toxicidad en humanos, los cebos suelen ser formulados con otros ingredientes destinados a generar respuestas de rechazo en el caso de ingestión accidental o intento de suicidio; aun así, las unidades hospitalarias reportan anualmente numerosos casos de intoxicaciones con rodenticidas. Se han desarrollado cebaderas con el propósito de brindar al usuario alta eficacia y seguridad extendida. Dado que en muchos países existe experiencia limitada para la evaluación regulatoria de cebos combinados con cebaderas, en este trabajo se presenta un protocolo adaptado a partir de la guía USEPA- 1.213/1990, el cual se propone para verificar la aptitud de las cebaderas para controlar roedores en ambientes hogareños, ocupacionales y otros espacios urbanos. Se incluye una discusión de aspectos técnicos que se espera que sean de ayuda para los asesores de las agencias regulatorias intervinientes al interpretar los resultados en términos de eficacia y seguridad. El sistema cebadera-cebo examinado mostró alta letalidad en ratas adultas jóvenes. La inspección, carga y recarga de producto fresco se realizó en forma simple, rápida y segura; sólo excepcionalmente se observó dispersión del cebo fuera de la cebadera. Sin embargo, debe tenerse en cuenta que el sistema que solicita un registro ante la autoridad regulatoria debe ser adecuado a la especie que se busca controlar, considerando las diferencias de peso corporal entre ratas y ratones, entre machos y hembras, y entre animales jóvenes y adultos, lo cual determina la facilidad de ingreso al interior del dispositivo (cebadera) donde se coloca el cebo contenido en su embalaje unitario original. (AU)

Several modern rodenticides are formulated with anticoagulant superwarfarin compounds. Due to their high toxicity in humans, these baits are usually formulated with other ingredients intended to generate rejection responses in the case of accidental ingestion or suicide attempt. However, hospital units report numerous cases of poisoning with rodenticides every year. Box-like bait stations have been developed to provide the user with high efficacy and extended safety. Given that in many countries there is limited experience for the regulatory consideration of bait stations, this work presents a protocol adapted from the USEPA-1.213/1990 guideline, proposed to verify the aptitude of these devices to control wild rodents in home, work, and other urban spaces. The work includes a discussion of technical issues that are expected to be helpful to advisors of the regulatory agencies involved in interpreting the results in terms of efficacy and safety. The bait-station system examined showed high efficacy in young adult rats. The inspection, loading and reloading of fresh product were carried out in a simple, fast and safe way; bait dispersal outside the station was only rarely observed. However, it must be taken into account that the system that require a registration by the regulatory authority must be appropriate to the species sought to be controlled. This is important due to differences in body weight between rats and mice, between males and females, and between young and aged animals, which determines the ease of entry into the station where the bait contained in its original unitary packaging is placed. (AU)

Turbidity matters: differential effect of a 2,4-D formulation on the structure of microbial communities from clear and turbid freshwater systems.

We evaluated the effect of AsiMax 50®, a commercial formulation of 2,4-D (2,4-dichlorophenoxyacetic acid), on the structure of both micro + nano phytoplankton (>2 µm; species composition and abundance) and cytometric populations (photosynthetic picoplankton (PPP, 0.2-2 µm), which included prokaryotic phycocyanin-rich picocyanobacteria (PC-Pcy), phycoerythrin-rich picocyanobacteria (PE-Pcy) and eukaryotic phototrophs (PEuk); and bacterioplankton (HB), heterotrophic bacteria), using a microcosms-based approach and a single 7-day exposure. Assays were performed on two different microbial assemblages sampled from freshwater bodies of two contrasting turbidity status: clear (chlorophyll a = 7.6 µgL-1, turbidity = 1 NTU) and organic turbid systems (chlorophyll a = 25.0 µgL-1, turbidity = 9 NTU). For each system, the herbicide was applied to 500 mL-Erlenmeyer flasks, at seven concentration levels of the active ingredient (a.i.): 0 (control = no addition), 0.02, 0.2, 2, 20, 200 and 2,000 mg a.i.L-1. The impact of AsiMax 50® seemed to be greater in the turbid system. In this system, total abundance of living (live) micro + nano phytoplankton showed a significant increase at lower concentrations and data were fitted to a humped-shaped curve. For both clear and organic turbid systems, micro + nano phytoplankton decreased in species richness and abundance at higher herbicide concentrations. These results suggest that 2,4-D may mimic hormonal function. Some species, such as Ochromonas sp. and Chlamydomonas sp., showed different responses to herbicide exposure between water systems. In the turbid system, the increase in abundance of the PPP fraction observed at 7-d exposure was probably due to either an increase in PE-Pcy (thus suggesting the existence of auxin pathways) or a reduction in competitive pressure by micro + nano plankton. Our results provide some evidence of the importance of using community-scale approaches in ecotoxicological studies to predict changes in freshwater ecosystems exposed to a 2,4-D-based formulation. However, caution must be taken when extrapolating these effects to real scenarios, as assays were based on a laboratory microcosm experiment.

Critical consideration of the multiplicity of experimental and organismic determinants of pyrethroid neurotoxicity: a proof of concept.

Pyrethroids (PYR) are pesticides with high insecticidal activity that may disrupt neuronal excitability in target and nontarget species. The accumulated evidence consistently showed that this neurophysiologic action is followed by alterations in motor, sensorimotor, neuromuscular, and thermoregulatory responses. Nevertheless, there are some equivocal results regarding the potency of PYR in lab animals. The estimation of potency is an important step in pesticide chemical risk assessment. In order to identify the variables influencing neurobehavioral findings across PYR studies, evidence on experimental and organismic determinants of acute PYR-induced neurotoxicity was reviewed in rodents. A comprehensive analysis of these studies was conducted focusing on test material and dosing conditions, testing conditions, animal models, and other determinants such as testing room temperature. Variations in the severity of the neurotoxicity, under lab-controlled conditions, was explained based upon factors including influence of animal species and age, test material features such as chemical structure and stereochemistry, and dosing conditions such as vehicle, route of exposure, and dose volume. If not controlled, the interplay of these factors may lead to large variance in potency estimation. This review examined the scope of acute toxicological data required to determine the safety of pesticide products, and factors and covariates that need to be controlled in order to ensure that predictivity and precaution are balanced in a risk assessment process within a reasonable time-frame, using acute PYR-induced neurotoxicity in rodents as an exemplar.

Neurobehavioral toxicology of pyrethroid insecticides in adult animals: a critical review.

Pyrethroids are pesticides with high selectivity for insects. In order to identify strengths and gaps in the database for pyrethroid neurobehavioral toxicology, we have critically analyzed the data from peer-reviewed literature. This review includes dose-response data that have been recently generated demonstrating consistent findings for low-dose, acute, oral exposure to pyrethroids in small rodents. All pyrethroids tested (i.e., about twenty compounds), regardless of structure, produce a decrease in motor activity in a variety of test protocols. The range of relative potencies varies more than two orders of magnitude, and thresholds for motor activity were found well below doses that produce overt signs of poisoning. Six compounds (allethrin, permethrin, cis-permethrin, deltamethrin, cypermethrin, and fenvalerate) impair schedule-controlled operant responding, seven compounds (pyrethrum, bifenthrin, S-bioallethrin, permethrin, beta-cyfluthrin, cypermethrin, and deltamethrin) decrease grip strength, and two compounds (deltamethrin and alpha-cypermethrin) produce incoordination using the rotarod. In addition, while compounds lacking an alpha-cyano group (e.g., cismethrin, permethrin, bifenthrin) induce an increase in acoustic-evoked startle response amplitude, cyano compounds (e.g., deltamethrin, cypermethrin, cyfluthrin) produce the opposite outcome. Other endpoints (e.g., tremor intensity, sensory response) have been only occasionally explored. A synthesis of the neurobehavioral evidence relating to the action of pyrethroids indicates that some differences in the experimental findings across compounds are also present in the low-effective dose range. For risk assessment purposes, a strategy that takes into account data from an array of neurobehavioral endpoints is needed to capture the heterogeneity of pyrethroid-induced adverse effects and accurately inform policy decisions.

Influence of dosing volume on the neurotoxicity of bifenthrin.

Pyrethroids are pesticides with high insecticidal activity and relatively low potency in mammals. The influence of dosing volume on the neurobehavioral syndrome following oral acute exposure to the Type-I pyrethroid insecticide bifenthrin in corn oil was evaluated in adult male Long Evans rats. We tested bifenthrin effects at 1 and 5 ml/kg, two commonly used dose volumes in toxicological studies. Two testing times (4 and 7 h) were used in motor activity and functional observational battery (FOB) assessments. Four to eight doses were examined at either dosing condition (up to 20 or 26 mg/kg, at 1 and 5 ml/kg, respectively). Acute oral bifenthrin exposure produced toxic signs typical of Type I pyrethroids, with dose-related increases in fine tremor, decreased motor activity and grip strength, and increased pawing, head shaking, click response, and body temperature. Bifenthrin effects on motor activity and pyrethroid-specific clinical signs were approximately 2-fold more potent at 1 ml/kg than 5 ml/kg. This difference was clearly evident at 4 h and slightly attenuated at 7 h post-dosing. Benchmark dose (BMD) modeling estimated similar 2-fold potency differences in motor activity and pyrethroid-specific FOB data. These findings demonstrate that dose volume, in studies using corn oil as the vehicle influences bifenthrin potency. Further, these data suggest that inconsistent estimates of pyrethroid potency between laboratories are at least partially due to differences in dosing volume.

Relative potencies for acute effects of pyrethroids on motor function in rats.

The prevalence of pyrethroids in insecticide formulations has increased in the last decade. A common mode-of-action has been proposed for pyrethroids based on in vitro studies, which includes alterations in sodium channel dynamics in nervous system tissues, consequent disturbance of membrane polarization, and abnormal discharge in targeted neurons. The objective of this work was to characterize individual dose-response curves for in vivo motor function and calculate relative potencies for eleven commonly used pyrethroids. Acute oral dose-response functions were determined in adult male Long Evans rats for five Type I (bifenthrin, S-bioallethrin, permethrin, resmethrin, tefluthrin), five Type II (beta-cyfluthrin, lambda-cyhalothrin, cypermethrin, deltamethrin, esfenvalerate) and one mixed Type I/II (fenpropathrin) pyrethroids (n = 8-18 per dose; 6-11 dose levels per chemical, vehicle = corn oil, at 1 ml/kg). Motor function was measured using figure-8 mazes. Animals were tested for 1 h during the period of peak effects. All pyrethroids, regardless of structural class, produced dose-dependent decreases in motor activity. Relative potencies were calculated based on the computed ED30s. Deltamethrin, with an ED30 of 2.51 mg/kg, was chosen as the index chemical. Relative potency ratios ranged from 0.009 (resmethrin) to 2.092 (esfenvalerate). Additional work with environmentally-based mixtures is needed to test the hypothesis of dose-additivity of pyrethroids.

Exogenous NGF alters a critical motor period in rat striatum.

In previous studies we found that there is a critical period during rat postnatal development when motor training starting at age 30 days (P30) but not before or after this age, induces a bilateral lifetime drop in Bmax of the muscarinic radioligand [3H]QNB in striatum. We examined the possibility that striatal NGF level would be a determining factor for the normal occurrence of this synaptic plasticity. With this aim, rats underwent training at P30-37 with or without simultaneous NGF perfusion into the left striatum. At P70, we found the expected bilateral enduring fall of striatal [3H]QNB sites in trained controls. While the non-cannulated side of NGF-treated trained rats showed a similar drop in [3H]QNB binding, the perfused striata from these animals were not affected by training. Thus, the findings add new evidence in favour of a major role of NGF in this critical period of activity-dependent permanent adjustment in the striatal muscarinic system.

Decreased GAP-43/B-50 phosphorylation in striatal synaptic plasma membranes after circling motor behavior during development.

We evaluated the in vitro phosphorylation of the presynaptic substrate of protein kinase C (PKC), GAP-43/B-50 and the PKC activity in the striatum of rats submitted to a circling training (CT) test during postnatal development. Motor activity at 30 days of age, but not at other ages, produced a unilateral reduction (-29.5%; p<0.001) in the level of GAP-43/B-50 endogenous phosphorylation in the contralateral striatum with respect to the ipsilateral side, while non-trained control animals did not show asymmetric differences. Compared to controls, the contralateral striatum of trained animals also showed a significant reduction (-29.3%; p<0. 001) in the incorporation of 32P-phosphate into GAP-43. This decreased in vitro GAP-43 phosphorylation was seen at 30 min, but not immediately after circling motor behavior. This contralateral change in GAP-43 phosphorylation correlated with the running speed developed by the animals [(r=0.9443, p=0.0046, n=6, relative to control group) and (r=0.8813, p=0.0203, n=6, with respect to the ipsilateral side of the exercised animals)]. On the contrary, GAP-43/B-50 immunoblots did not show changes in the amount of this phosphoprotein among the different experimental groups. Back phosphorylation assays, performed in the presence of bovine purified PKC, increased the level of GAP-43/B-50 phosphorylation in the striatum contralateral to the sense of turning [(+22%; p<0.05, with respect to ipsilateral side of the same trained group) and (+21%; p<0.05, relative to control group)]. Taken together, these results demonstrate that the activity developed in the CT test induces a reduction in the phosphorylation state of GAP-43/B-50 in the specific site for PKC. We conclude that general markers of activity-dependent neuronal plasticity are also altered in the same period that long-lasting changes in striatal neuroreceptors are triggered by circling motor behavior.

Nerve growth factor preserves a critical motor period in rat striatum.

We previously found the occurrence of a critical motor period during rat postnatal development where circling training starting the 7-day schedule at 30 days-but not before or after-induces a lifetime drop in the binding to cholinergic muscarinic receptors (mAChRs) in striatum. Here, we studied whether nerve growth factor (NGF) participates in this restricted period of muscarinic sensitivity. For this purpose, we administered mouse salival gland 2.5S NGF (1.4 or 0.4 microg/day, infused by means of ALZA minipumps) by intrastriatal unilateral route between days 25 and 39, and then trained rats starting at 40 days. Under these conditions, NGF induced a long-term reduction in the striatal [3H] quinuclidilbenzylate (QNB) binding sites despite the fact that motor training was carried out beyond the natural critical period. Thus, at day 70, measurement of specific QNB binding in infused striata of trained rats showed decreases of 42% (p < .0004) and 33% (p < .02) after administration of the higher and lower NGF doses, respectively, with respect to trained rats treated with cytochrome C, for control. Noncannulated striata of the NGF-treated rats also showed a decrease in QNB binding sites (44%; p < .0001) only at the higher infusion rate. This effect was not found in the respective control groups. Our observations show that NGF modulates the critical period in which activity-dependent mAChR setting takes place during rat striatal maturation.

Co-alteration of dopamine D2 receptor and muscarinic acetylcholine receptor binding in rat striatum after circling training.

CIRCLING training (CT) decreases muscarinic acetyl-choline receptor (mAchR) binding in rat striatum. As cholinergic and dopaminergic systems interact strongly we evaluated the expression of D2-subtype dopamine receptor (DA D2) and mAchR together after CT. Animals trained from 30 to 37 days of age and sacrificed 2 months later showed an enduring drop in Bmax of 40% in DA D2 and 34% in mAchR. Plotting the percentage of binding drop of both receptors for each animal showed that the reduction of one system correlates with the other (r2 = 0.71, p < 0.01; n = 8). Neither mAchR nor DA D2 were affected when training started at 20 or 60 days. We conclude that the presence of a period where CT exerts long term alterations during development involves both cholinergic and dopaminergic systems.