Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny - Part IV: the ECETOC and CLE Proposal for a Thyroid Function-Related Neurodevelopmental Toxicity Testing and Assessment Scheme (Thyroid-NDT-TAS).

Following the European Commission Endocrine Disruptor Criteria, substances shall be considered as having endocrine disrupting properties if they (a) elicit adverse effects, (b) have endocrine activity, and (c) the two are linked by an endocrine mode-of-action (MoA) unless the MoA is not relevant for humans. A comprehensive, structured approach to assess whether substances meet the Endocrine Disruptor Criteria for the thyroid modality (EDC-T) is currently unavailable. Here, the European Centre for Ecotoxicology and Toxicology of Chemicals Thyroxine Task Force and CropLife Europe propose a Thyroid Function-Related Neurodevelopmental Toxicity Testing and Assessment Scheme (Thyroid-NDT-TAS). In Tier 0, before entering the Thyroid-NDT-TAS, all available in vivo, in vitro and in silico data are submitted to weight-of-evidence (WoE) evaluations to determine whether the substance of interest poses a concern for thyroid disruption. If so, Tier 1 of the Thyroid-NDT-TAS includes an initial MoA and human relevance assessment (structured by the key events of possibly relevant adverse outcome pathways) and the generation of supportive in vitro/in silico data, if relevant. Only if Tier 1 is inconclusive, Tier 2 involves higher-tier testing to generate further thyroid- and/or neurodevelopment-related data. Tier 3 includes the final MoA and human relevance assessment and an overarching WoE evaluation to draw a conclusion on whether, or not, the substance meets the EDC-T. The Thyroid-NDT-TAS is based on the state-of-the-science, and it has been developed to minimise animal testing. To make human safety assessments more accurate, it is recommended to apply the Thyroid-NDT-TAS during future regulatory assessments.

Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny-part III: how is substance-mediated thyroid hormone imbalance in pregnant/lactating rats or their progeny related to neurodevelopmental effects?

This review investigated which patterns of thyroid- and brain-related effects are seen in rats upon gestational/lactational exposure to 14 substances causing thyroid hormone imbalance by four different modes-of-action (inhibition of thyroid peroxidase, sodium-iodide symporter and deiodinase activities, enhancement of thyroid hormone clearance) or to dietary iodine deficiency. Brain-related parameters included motor activity, cognitive function, acoustic startle response, hearing function, periventricular heterotopia, electrophysiology and brain gene expression. Specific modes-of-action were not related to specific patterns of brain-related effects. Based upon the rat data reviewed, maternal serum thyroid hormone levels do not show a causal relationship with statistically significant neurodevelopmental effects. Offspring serum thyroxine together with offspring serum triiodothyronine and thyroid stimulating hormone appear relevant to predict the likelihood for neurodevelopmental effects. Based upon the collated database, thresholds of ≥60%/≥50% offspring serum thyroxine reduction and ≥20% and statistically significant offspring serum triiodothyronine reduction indicate an increased likelihood for statistically significant neurodevelopmental effects; accuracies: 83% and 67% when excluding electrophysiology (and gene expression). Measurements of brain thyroid hormone levels are likely relevant, too. The extent of substance-mediated thyroid hormone imbalance appears more important than substance mode-of-action to predict neurodevelopmental impairment in rats. Pertinent research needs were identified, e.g. to determine whether the phenomenological offspring thyroid hormone thresholds are relevant for regulatory toxicity testing. The insight from this review shall be used to suggest a tiered testing strategy to determine whether gestational/lactational substance exposure may elicit thyroid hormone imbalance and potentially also neurodevelopmental effects.

Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny - part II: how can key events of relevant adverse outcome pathways be addressed in toxicological assessments?

The current understanding of thyroid-related adverse outcome pathways (AOPs) with adverse neurodevelopmental outcomes in mammals has been reviewed. This served to establish if standard rodent toxicity test methods and in vitro assays allow identifying thyroid-related modes-of-action potentially leading to adverse neurodevelopmental outcomes, and the human relevance of effects - in line with the European Commission's Endocrine Disruptor Criteria. The underlying hypothesis is that an understanding of the key events of relevant AOPs provides insight into differences in incidence, magnitude, or species sensitivity of adverse outcomes. The rodent studies include measurements of serum thyroid hormones, thyroid gland pathology and neurodevelopmental assessments, but do not directly inform on specific modes-of-action. Opportunities to address additional non-routine parameters reflecting critical events of AOPs in toxicological assessments are presented. These parameters appear relevant to support the identification of specific thyroid-related modes-of-action, provided that prevailing technical limitations are overcome. Current understanding of quantitative key event relationships is often weak, but would be needed to determine if the triggering of a molecular initiating event will ultimately result in an adverse outcome. Also, significant species differences in all processes related to thyroid hormone signalling are evident, but the biological implications thereof (including human relevance) are often unknown. In conclusion, careful consideration of the measurement (e.g. timing, method) and interpretation of additional non-routine parameters is warranted. These findings will be used in a subsequent paper to propose a testing strategy to identify if a substance may elicit maternal thyroid hormone imbalance and potentially also neurodevelopmental effects in the progeny.

Recommendations for harmonization of data collection and analysis of developmental neurotoxicity endpoints in regulatory guideline studies: Proceedings of workshops presented at Society of Toxicology and joint Teratology Society and Neurobehavioral Teratology Society meetings.

The potential for developmental neurotoxicity (DNT) of environmental chemicals may be evaluated using specific test guidelines from the US Environmental Protection Agency or the Organisation for Economic Cooperation and Development (OECD). These guidelines generate neurobehavioral, neuropathological, and morphometric data that are evaluated by regulatory agencies globally. Data from these DNT guideline studies, or the more recent OECD extended one-generation reproductive toxicity guideline, play a pivotal role in children's health risk assessment in different world areas. Data from the same study may be interpreted differently by regulatory authorities in different countries resulting in inconsistent evaluations that may lead to inconsistencies in risk assessment decisions internationally, resulting in regional differences in public health protection or in commercial trade barriers. These issues of data interpretation and reporting are also relevant to juvenile and pre-postnatal studies conducted more routinely for pharmaceuticals and veterinary medicines. There is a need for development of recommendations geared toward the operational needs of the regulatory scientific reviewers who apply these studies in risk assessments, as well as the scientists who generate DNT data sets. The workshops summarized here draw upon the experience of the authors representing government, industry, contract research organizations, and academia to discuss the scientific issues that have emerged from diverse regulatory evaluations. Although various regulatory bodies have different risk management decisions and labeling requirements that are difficult to harmonize, the workshops provided an opportunity to work toward more harmonized scientific approaches for evaluating DNT data within the context of different regulatory frameworks. Five speakers and their coauthors with neurotoxicology, neuropathology, and regulatory toxicology expertise discussed issues of variability, data reporting and analysis, and expectations in DNT data that are encountered by regulatory authorities. In addition, principles for harmonized evaluation of data were suggested using guideline DNT data as case studies.

A critical review of neonicotinoid insecticides for developmental neurotoxicity.

A comprehensive review of published and previously unpublished studies was performed to evaluate the neonicotinoid insecticides for evidence of developmental neurotoxicity (DNT). These insecticides have favorable safety profiles, due to their preferential affinity for nicotinic receptor (nAChR) subtypes in insects, poor penetration of the mammalian blood-brain barrier, and low application rates. Nevertheless, examination of this issue is warranted, due to their insecticidal mode of action and potential exposure with agricultural and residential uses. This review identified in vitro, in vivo, and epidemiology studies in the literature and studies performed in rats in accordance with GLP standards and EPA guidelines with imidacloprid, acetamiprid, thiacloprid, clothianidin, thiamethoxam, and dinotefuran, which are all the neonicotinoids currently registered in major markets. For the guideline-based studies, treatment was administered via the diet or gavage to primiparous female rats at three dose levels, plus a vehicle control (≥20/dose level), from gestation day 0 or 6 to lactation day 21. F1 males and females were evaluated using measures of motor activity, acoustic startle response, cognition, brain morphometry, and neuropathology. The principal effects in F1 animals were associated with decreased body weight (delayed sexual maturation, decreased brain weight, and morphometric measurements) and acute toxicity (decreased activity during exposure) at high doses, without neuropathology or impaired cognition. No common effects were identified among the neonicotinoids that were consistent with DNT or the neurodevelopmental effects associated with nicotine. Findings at high doses were associated with evidence of systemic toxicity, which indicates that these insecticides do not selectively affect the developing nervous system.

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.

Developmental neurotoxicity study of dietary bisphenol A in Sprague-Dawley rats.

This study was conducted to determine the potential of bisphenol A (BPA) to induce functional and/or morphological effects to the nervous system of F(1) offspring from dietary exposure during gestation and lactation according to the Organization for Economic Cooperation and Development and U.S. Environmental Protection Agency guidelines for the study of developmental neurotoxicity. BPA was offered to female Sprague-Dawley Crl:CD (SD) rats (24 per dose group) and their litters at dietary concentrations of 0 (control), 0.15, 1.5, 75, 750, and 2250 ppm daily from gestation day 0 through lactation day 21. F(1) offspring were evaluated using the following tests: detailed clinical observations (postnatal days [PNDs] 4, 11, 21, 35, 45, and 60), auditory startle (PNDs 20 and 60), motor activity (PNDs 13, 17, 21, and 61), learning and memory using the Biel water maze (PNDs 22 and 62), and brain and nervous system neuropathology and brain morphometry (PNDs 21 and 72). For F(1) offspring, there were no treatment-related neurobehavioral effects, nor was there evidence of neuropathology or effects on brain morphometry. Based on maternal and offspring body weight reductions, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was 75 ppm (5.85 and 13.1 mg/kg/day during gestation and lactation, respectively), with no treatment-related effects at lower doses or nonmonotonic dose responses observed for any parameter. There was no evidence that BPA is a developmental neurotoxicant in rats, and the NOAEL for developmental neurotoxicity was 2250 ppm, the highest dose tested (164 and 410 mg/kg/day during gestation and lactation, respectively).

Comparative functional observational battery study of twelve commercial pyrethroid insecticides in male rats following acute oral exposure.

Twelve commercial pyrethroid insecticides (technical-grade active ingredients) were evaluated individually for acute neurobehavioral manifestations of toxicity under conditions suited to assist with determining whether they act by a common mechanism of toxicity. The pyrethroids that were tested reflect a diversity of structures, including six with an alpha-cyano phenoxybenzyl moiety (beta-cyfluthrin, lambda-cyhalothrin, cypermethrin, deltamethrin, esfenvalerate and fenpropathrin) and six without this moiety (bifenthrin, S-bioallethrin, permethrin, pyrethrins, resmethrin and tefluthrin). These chemicals also present a variety of behavioral effects, including ones that are historically classified as causing a T (tremor), CS (choreoathetosis with salivation) or intermediate syndrome of intoxication, and others that have not previously been classified. Each pyrethroid that was tested consisted of the complement of isomers that occur in commercial products--a key factor for relevance for environmental and human exposure and for comparisons, since the biological activity of the individual isomers can vary tremendously. Young-adult male Sprague-Dawley rats (10 per dose group) were administered a single dose of pyrethroid by oral gavage, in corn oil, at a volume of 5 ml/kg. Each was tested at a range of two or three dose levels, including a minimally toxic dose, to establish the more sensitive manifestations of toxicity, and a more toxic dose, to establish a more complete spectrum of neurobehavioral manifestations. Animals were evaluated using a functional observational battery (FOB) that was designed to characterize and distinguish effects classically associated with T or CS syndromes of intoxication. The FOB was performed when manifestations of toxicity were most apparent at the time of peak effect (2, 4, or 8 h post-dosing) by observers who were blinded to dose group assignment, thus avoiding possible bias. The results from this study indicate that some pyrethroids clearly exhibit the historic classification symptoms of the T and CS syndromes while others do so less obviously. Use of the statistical technique of Principal Component Analysis (PCA) further helped interpret the study findings, as described in the accompanying paper (Breckenridge et al., 2009). These results establish manifestations of neurotoxicity in vivo that can be used as weight of evidence to determine whether pyrethroid insecticides act through a common mechanism of toxicity in mammals. Based on a review of the FOB data, analyzed by PCA, and other published data, two common mechanism groups are proposed. Group 1 would include pyrethrins, bifenthrin, resmethrin, permethrin, S-bioallethrin and tefluthrin. Group 2 would include cypermethrin, deltamethrin, esfenvalerate, beta-cyfluthrin and lambda-cyhalothrin. Fenpropathrin exhibited features of both groups.

Evidence for a separate mechanism of toxicity for the Type I and the Type II pyrethroid insecticides.

Neurotoxicity and mechanistic data were collected for six alpha-cyano pyrethroids (beta-cyfluthrin, cypermethrin, deltamethrin, esfenvalerate, fenpropathrin and lambda-cyhalothrin) and up to six non-cyano containing pyrethroids (bifenthrin, S-bioallethrin [or allethrin], permethrin, pyrethrins, resmethrin [or its cis-isomer, cismethrin] and tefluthrin under standard conditions. Factor analysis and multivariate dissimilarity analysis were employed to evaluate four independent data sets comprised of (1) fifty-six behavioral and physiological parameters from an acute neurotoxicity functional observatory battery (FOB), (2) eight electrophysiological parameters from voltage clamp experiments conducted on the Na(v)1.8 sodium channel expressed in Xenopus oocytes, (3) indices of efficacy, potency and binding calculated for calcium ion influx across neuronal membranes, membrane depolarization and glutamate released from rat brain synaptosomes and (4) changes in chloride channel open state probability using a patch voltage clamp technique for membranes isolated from mouse neuroblastoma cells. The pyrethroids segregated into Type I (T--syndrome-tremors) and Type II (CS syndrome--choreoathetosis with salivation) groups based on FOB data. Of the alpha-cyano pyrethroids, deltamethrin, lambda-cyhalothrin, cyfluthrin and cypermethrin arrayed themselves strongly in a dose-dependent manner along two factors that characterize the CS syndrome. Esfenvalerate and fenpropathrin displayed weaker response profiles compared to the non-cyano pyrethroids. Visual clustering on multidimensional scaling (MDS) maps based upon sodium ion channel and calcium influx and glutamate release dissimilarities gave similar groupings. The non-cyano containing pyrethroids were arrayed in a dose-dependent manner along two different factors that characterize the T-syndrome. Bifenthrin was an outlier when MDS maps of the non-cyano pyrethroids were based on sodium ion channel characteristics and permethrin was an outlier when the MDS maps were based on calcium influx/glutamate release potency. Four of six alpha-cyano pyrethroids (lambda-cyfluthrin, cypermethrin, deltamethrin and fenpropathrin) reduced open chloride channel probability. The R-isomers of lambda-l-cyhalothrin reduced open channel probability whereas the S-isomers, antagonized the action of the R-isomers. None of the non-cyano pyrethroids reduced open channel probability, except bioallethrin, which gave a weak response. Overall, based upon neurotoxicity data and the effect of pyrethroids on sodium, calcium and chloride ion channels, it is proposed that bioallethrin, cismethrin, tefluthrin, bifenthrin and permethrin belong to one common mechanism group and deltamethrin, lambda-cyhalothrin, cyfluthrin and cypermethrin belong to a second. Fenpropathrin and esfenvalerate occupy an intermediate position between these two groups.

A retrospective performance assessment of the developmental neurotoxicity study in support of OECD test guideline 426.

OBJECTIVE: We conducted a review of the history and performance of developmental neurotoxicity (DNT) testing in support of the finalization and implementation of Organisation of Economic Co-operation and Development (OECD) DNT test guideline 426 (TG 426). INFORMATION SOURCES AND ANALYSIS: In this review we summarize extensive scientific efforts that form the foundation for this testing paradigm, including basic neurotoxicology research, interlaboratory collaborative studies, expert workshops, and validation studies, and we address the relevance, applicability, and use of the DNT study in risk assessment. CONCLUSIONS: The OECD DNT guideline represents the best available science for assessing the potential for DNT in human health risk assessment, and data generated with this protocol are relevant and reliable for the assessment of these end points. The test methods used have been subjected to an extensive history of international validation, peer review, and evaluation, which is contained in the public record. The reproducibility, reliability, and sensitivity of these methods have been demonstrated, using a wide variety of test substances, in accordance with OECD guidance on the validation and international acceptance of new or updated test methods for hazard characterization. Multiple independent, expert scientific peer reviews affirm these conclusions.

Identification and interpretation of developmental neurotoxicity effects: a report from the ILSI Research Foundation/Risk Science Institute expert working group on neurodevelopmental endpoints.

The reliable detection, measurement, and interpretation of treatment-related developmental neurotoxicity (DNT) effects depend on appropriate study design and execution, using scientifically established methodologies, with appropriate controls to minimize confounding factors. Appropriate statistical approaches should be optimized for the specific endpoints in advance, analyzing effects across time and functional domains as far as possible. If available, biomarkers of exposure are useful to assess the bioavailability of toxicants to the dam and offspring in utero and after birth. Finally, "weight of evidence" principles are used to aid assessment of the biological significance of differences from concurrent controls. These effects should be interpreted in light of available information from historical controls, positive controls, maternal and offspring systemic toxicity, and other relevant toxicological data. This review provides a framework for the integration of all these types of information in the interpretation of DNT studies.

A tiered approach to life stages testing for agricultural chemical safety assessment.

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. The ACSA Life Stages Task Force proposes a tiered approach to toxicity testing that assesses a compound's potential to cause adverse effects on reproduction, and that assesses the nature and severity of effects during development and adolescence, with consideration of the sensitivity of the elderly. While incorporating many features from current guideline studies, the proposed approach includes a novel rat reproduction and developmental study with enhanced endpoints and a rabbit development study. All available data, including toxicokinetics, ADME data, and systemic toxicity information, are considered in the design and interpretation of studies. Compared to existing testing strategies, the proposed approach uses fewer animals, provides information on the young animal, and includes an estimation of human exposure potential for making decisions about the extent of testing required.

Developmental toxicology evaluations--issues with including neurotoxicology and immunotoxicology assessments in reproductive toxicology studies.

Developmental and reproductive toxicology (DART) has routinely been a part of safety assessment. Attention is now focused on the effects of chemicals on the developing nervous and immune systems. This focus on developmental neurotoxicology (DNT) and developmental immunotoxicology (DIT) is based on the premise that children differ from adults in some aspects of their biology and, thus, may also differ in their responses to chemicals. This session's objective was to discuss issues common to DNT and DIT as they relate to DART protocols, including high dose selection and maternal toxicity, adequacy of pup exposure during lactation, use of a different dosing paradigm for DART versus DNT or DIT studies, and whether DIT and DNT endpoints can be incorporated into a single DART study for hazard identification purposes. Consensus was achieved on all topics except the adequacy for risk assessment purposes of the use of a limited number of endpoints for DIT and DNT, with the DNT endpoints being the primary focus of disagreement. Panelists indicated that a combination study design for hazard identification was feasible, though flexibility to meet the scientific needs of the project was emphasized. The adequacy of existing triggers for additional developmental studies was also questioned. Panelists iterated the importance of understanding pup exposure during the various life stages and the use of toxicokinetic data in designing these studies. The group agreed to consider the HESI ACSA Life Stages Task Force recommendations as a next step to address some of the issues and challenges raised during this session.

Behavioral test methods workshop.

A one and a half day workshop on behavioral testing was conducted in order to discuss experimental procedures and practices that may help enhance the utility of behavioral data as a reliable index of neurotoxicity and in the safety evaluation of chemical substances. The workshop was open to participation by all sectors of the neuroscience community including academia, government, testing laboratories, and industry. The level of confidence with which changes in behavior can reliably signal adverse effects on the nervous system depends, in part, on the scientific quality of the data generated. With an emphasis on education and problem solving, the workshop focused on the practical aspects and scientific rationale underlying valid and high quality testing. In behavioral testing, there are numerous experimental factors that may impact on the quality of data. These include such elements as experimental design, selection of test methods, the care and precision in the conduct of behavioral testing, procedures to minimize bias and potential confounds, appropriateness of statistical analyses, and data interpretation. In plenary session investigators experienced in behavioral testing discussed the significance of these various experimental factors to data quality, outlined problematic issues, and presented a synopsis of approaches for addressing each of the factors as outlined in a draft of a primer developed by the Interagency Committee on Neurotoxicology (ICON). During the remainder of the workshop, open discussions in small breakout groups were used to address the problematic issues identified by the plenary speakers and explore alternative approaches for dealing with them. Finally, all workshop participants were reconvened in plenary session for summation of breakout group discussions and final recommendations. Information from the workshop was used to form the basis of this manuscript and will be used to help finalize a behavioral test methods primer being drafted by the ICON. The overall conclusions from the workshop were that consensus can be reached on the fundamentals of behavioral assessment, and that aspects of behavioral assessment including experimental design, test method selection, training, validation, control of confounds, data variability, data analysis, and data interpretation need to be carefully considered in the planning and conduct of behavioral safety assessments.

Neurobehavioral assessment: a survey of use and value in safety assessment studies.

This report describes the results of a survey designed to evaluate the contribution of F1 neurobehavioral testing to hazard identification and characterization in safety assessment studies. (To review the details of the distributed survey, please see the supplementary data for this article on the journal's Web site.) The survey provided information about studies completed in industrial laboratories in the United States, Europe, and Japan since 1990 on 174 compounds. The types of compounds included were pharmaceutical (81%), agricultural (7%), industrial (1%), or were undefined (10%). Information collected included the intended use of the test agent, general study design and methodology, the types and characteristics of F1 behavioral evaluations, and the frequency with which agents affected neurobehavioral parameters in comparison to other F0 and F1 generation parameters. F1 general toxicology parameters such as mortality, pre- and postweaning body weight, and food intake were assessed in most studies and were affected more frequently than other parameters by the test agents. F1 behavioral parameters were assessed less consistently across studies, and were less frequently affected by the agents tested. Although affected by agents less often than general toxicology parameters, F1 behavioral parameters along with other parameters defined the no-observed-effect level (NOEL) in 17/113 (15%) of studies and solely defined the NOEL in 3/113 (2.6%) of studies. Thus, F1 behavioral parameters sometimes improved on the standard toxicological measures of hazard identification. While not detecting agent effects as readily as some measures, the F1 behavioral parameters provide information about agent effects on specialized functions of developing offspring not provided by other standard measures of toxicity. The survey results emphasize the need for further research into the methods of behavioral assessment as well as the mechanisms underlying the neurobehavioral alterations.

Mechanisms of pyrethroid neurotoxicity: implications for cumulative risk assessment.

The Food Quality Protection Act (FQPA) of 1996 requires the United States Environmental Protection Agency to consider the cumulative effects of exposure to pesticides having a 'common mechanism of toxicity.' This paper reviews the information available on the acute neurotoxicity and mechanisms of toxic action of pyrethroid insecticides in mammals from the perspective of the 'common mechanism' statute of the FQPA. The principal effects of pyrethroids as a class are various signs of excitatory neurotoxicity. Historically, pyrethroids were grouped into two subclasses (Types I and II) based on chemical structure and the production of either the T (tremor) or CS (choreoathetosis with salivation) intoxication syndrome following intravenous or intracerebral administration to rodents. Although this classification system is widely employed, it has several shortcomings for the identification of common toxic effects. In particular, it does not reflect the diversity of intoxication signs found following oral administration of various pyrethroids. Pyrethroids act in vitro on a variety of putative biochemical and physiological target sites, four of which merit consideration as sites of toxic action. Voltage-sensitive sodium channels, the sites of insecticidal action, are also important target sites in mammals. Unlike insects, mammals have multiple sodium channel isoforms that vary in their biophysical and pharmacological properties, including their differential sensitivity to pyrethroids. Pyrethroids also act on some isoforms of voltage-sensitive calcium and chloride channels, and these effects may contribute to the toxicity of some compounds. Effects on peripheral-type benzodiazepine receptors are unlikely to be a principal cause of pyrethroid intoxication but may contribute to or enhance convulsions caused by actions at other target sites. In contrast, other putative target sites that have been identified in vitro do not appear to play a major role in pyrethroid intoxication. The diverse toxic actions and pharmacological effects of pyrethroids suggest that simple additivity models based on combined actions at a single target are not appropriate to assess the risks of cumulative exposure to multiple pyrethroids.