Feasibility of the extended one-generation reproductive toxicity study (OECD 443).

The extended one-generation reproduction toxicity study (OECD 443, adopted 28-July-2011) produces more information with fewer animals than the two-generation study (OECD 416), by including F1 neurotoxicity and immunotoxicity assessments, and omitting an F2 generation if there are no relevant F1 findings. This saves >1000 animals per compound. Feasibility studies based on draft OECD443 were conducted in industrial GLP laboratories in Europe and USA, using vinclozolin, methimazole and lead acetate. A fourth study was conducted with 2,4-dichlorophenoxyacetic acid (2,4-D) in response to a regulatory request for reproduction and developmental neurotoxicity data. The studies effectively profiled vinclozolin as an anti-androgenic developmental toxicant, methimazole as a developmental anti-thyroid agent, and lead acetate as a systemic and developmental toxicant. The 2,4-D study demonstrated the value of toxicokinetic data in dose setting and data interpretation. These results illustrate the variety of reproductive and developmental endpoints which can be captured in this complex but manageable study design. Time constraints for triggering further (F2) testing are summarized.

Methods to identify and characterize developmental neurotoxicity for human health risk assessment. I: behavioral effects.

Alterations in nervous system function after exposure to a developmental neurotoxicant may be identified and characterized using neurobehavioral methods. A number of methods can evaluate alterations in sensory, motor, and cognitive functions in laboratory animals exposed to toxicants during nervous system development. Fundamental issues underlying proper use and interpretation of these methods include a) consideration of the scientific goal in experimental design, b) selection of an appropriate animal model, c) expertise of the investigator, d) adequate statistical analysis, and e) proper data interpretation. Strengths and weaknesses of the assessment methods include sensitivity, selectivity, practicality, and variability. Research could improve current behavioral methods by providing a better understanding of the relationship between alterations in motor function and changes in the underlying structure of these systems. Research is also needed to develop simple and sensitive assays for use in screening assessments of sensory and cognitive function. Assessment methods are being developed to examine other nervous system functions, including social behavior, autonomic processes, and biologic rhythms. Social behaviors are modified by many classes of developmental neurotoxicants and hormonally active compounds that may act either through neuroendocrine mechanisms or by directly influencing brain morphology or neurochemistry. Autonomic and thermoregulatory functions have been the province of physiologists and neurobiologists rather than toxicologists, but this may change as developmental neurotoxicology progresses and toxicologists apply techniques developed by other disciplines to examine changes in function after toxicant exposure.

A consideration of age-dependent differences in susceptibility to organophosphorus and pyrethroid insecticides.

Evidence that neonates are more sensitive than adults to organophosphorus (OP) and pyrethroid insecticides is largely based on studies that compare toxicity at acute lethal doses. Under such circumstances, the greater susceptibility of the neonate appears to be due to limited metabolic capacity rather than an inherent difference in the sensitivity of target sites. For purposes of risk assessment with food-use pesticides, the more relevant issue is whether the neonate is more sensitive than the adult to lower levels of exposure, approximating levels used to establish acceptable residue limits (tolerances) on various food products. If infants and children are not more sensitive to environmentally-relevant levels of exposure, then the existing tolerances for dietary exposure will provide adequate protection. If, on the other hand, they may be more sensitive, then additional studies with young animals or an additional uncertainty factor may be needed for added protection. This paper examines two sets of studies that address this issue. The first involves multi-generation reproduction studies with rats that were treated with OP insecticides (coumaphos, fenamiphos, tribufos, trichlorfon, or oxydemeton-methyl) through the diet and examined for effects, including cholinesterase (ChE) inhibition. The second involves rats that were treated by gavage with an acute dose of a pyrethroid (cismethrin, permethrin, deltamethrin or cypermethrin) to establish relative sensitivity to either a lethal dose or to a low, behaviorally-active dose. The results with the OP insecticides support ChE inhibition as the most sensitive measure of exposure and the critical effect (i.e., the lowest NOEL) for each study was based on ChE inhibition in the adult. The magnitude of ChE inhibition in pups (measured on postnatal day (PND) 4 and 21) was consistently less than for adults at a given dietary level. For the representative Type I pyrethroids, there was no evidence that pups are more sensitive than adults at any dose level. For both Type II pyrethroids, young rats were considerably more sensitive than adults to a lethal dose but not to lower doses. Levels of deltamethrin in whole-brain tissue support kinetics as the basis for the greater sensitivity of young rats to a lethal dose, with the immature systems involved with detoxification being overwhelmed at such high dose levels. These findings indicate that young animals are not more sensitive than adults to lower doses of OP or pyrethroid insecticides. This outcome supports the conclusion that infants and children are protected by existing tolerances, without the need for an additional uncertainty factor.

A combined reproduction, neonatal development, and neurotoxicity study with 1,6-hexamethylene diisocyanate (HDI) in the rat.

1,6-Hexamethylene diisocyanate (HDI), a chemical widely used in commercial polyurethane products, was evaluated in a combined reproductive/developmental/neurotoxicity study. Sprague-Dawley rats (n = 120; 15 per sex/dose group) were administered via whole-body inhalation exposure either 0, 0.005, 0.05, or 0.3 ppm HDI for 6 h/day during a 14-day premating phase, up to a 14-day mating phase, and a 21-day gestation phase. The dams and their litters were maintained for a 4-day lactation phase during which exposure to HDI was discontinued. Neurobehavioral testing (automated measures of activity and a functional observational battery) was conducted before exposure, after the premating phase, and before termination. Body weight and clinical observations were recorded throughout the study. Terminal examinations included a gross necropsy, hematology, and clinical chemistry. Tissues retained for microscopic examination included the reproductive organs, neural tissues, nasal turbinates (multiple sections), trachea, larynx, and lung. The animals were also evaluated for effects on mating, fertility, gestation length, litter size, pup sex ratio, and pup viability. In the 0.300 ppm dose group a statistically significant decrease in body weight was observed in the females on day 4 of the study. Also observed at this dose level, in both males and females, were microscopic alterations in the nasal cavity, primarily epithelial hyperplasia, squamous metaplasia, chronic-active inflammation, and more seriously, degeneration of the olfactory epithelium. Similar microscopic effects were also observed, albeit to a lesser extent, in the males and females of the 0.05 ppm dose level. No histopathologic effects were observed in the 0.005 ppm dose level. No effects on any reproductive or neurotoxicologic parameters, hematology, clinical chemistry, or any effects on pup growth and development were observed at any exposure level.

Carcinogenicity testing in the CD-1 mouse of a prospective insect repellent (KBR 3023) using the dermal route of exposure.

The carcinogenic potential of 1-(1-methyl-propoxycarbonyl)-2-(2-hydroxyethyl)-piperidine (KBR 3023), a prospective new insect repellent intended for human use, was studied in mice using the dermal route of application. Relying upon the toxicology profile that emerged in the subchronic rat bioassay that was conducted using dermally applied dosages of 0, 80, 200, 500, and 1000 mg KBR 3023/kg body weight per day, it was determined, in concert with the EPA, that dermally applied dosages of 0, 50, 100, or 200 mg KBR 3023/kg body weight per day would be used in the conduct of all definitive forms of subchronic, chronic, and lifetime descriptive testing performed with the chemical. Using this testing approach, the specific results of this 18-month study are as follows. All in-life parameters, which included body weight, food consumption, clinical observations, survival, and hematology were unaffected by exposure to KBR 3023. Similarly, postmortem analyses, which included organ weights and gross pathology, and histopathology were also unchanged following exposure to KBR 3023. No evidence of a compound-induced neoplasia was suggested in this bioassay.

Chronic toxicity and carcinogenicity testing in the Sprague-Dawley rat of a prospective insect repellant (KBR 3023) using the dermal route of exposure.

The chronic toxicology and carcinogenic potential of 1-(1-methyl-propoxycarbonyl)-2-(2-hydroxyethyl)-piperidine (KBR 3023), a prospective new insect repellent intended for human use, was studied in rats using the dermal route of application. Relying upon the toxicology profile that emerged in the subchronic rat bioassay that was conducted using dermally applied dosages of 0, 80, 200, 500 and 1000 mg KBR 3023/kg body wt/day, it was determined, in concert with the Environmental Protection Agency (EPA), that dermally applied dosages of 0, 50, 100 or 200 mg KBR 3023/kg body wt/day would be used in the conduction of all definitive forms of subchronic, chronic, and lifetime descriptive testing performed with the chemical. Using this testing approach, the specific results of this 2-year study are as follows. All in-life parameters, which included body weight, food consumption, clinical observations, survival, ophthalmology, clinical chemistry, hematology, and urinalysis, were unaffected by exposure to KBR 3023. Similarly, postmortem analyses, which included organ weights and gross pathology, were also unchanged following exposure to KBR 3023. Histopathology at the dose site/skin was characterized by a pattern of acanthosis and/or hyperkeratosis across all doses in 1- and 2-year rats. Beyond the dosing site, cystic degeneration of the liver was described in 2-year 200-mg KBR 3023/kg body wt/day males. No other compound-related non-dosing site lesion was identified at any dose tested. No evidence of a compound-induced neoplasia was suggested in this bioassay.

Common mechanism of toxicity: a case study of organophosphorus pesticides.

The Food Quality Protection Act of 1996 (FQPA) requires the EPA to consider "available information concerning the cumulative effects of such residues and other substances that have a common mechanism of toxicity ... in establishing, modifying, leaving in effect, or revoking a tolerance for a pesticide chemical residue." This directive raises a number of scientific questions to be answered before the FQPA can be implemented. Among these questions is: What constitutes a common mechanism of toxicity? The ILSI Risk Science Institute (RSI) convened a group of experts to examine this and other scientific questions using the organophosphorus (OP) pesticides as the case study. OP pesticides share some characteristics attributed to compounds that act by a common mechanism, but produce a variety of clinical signs of toxicity not identical for all OP pesticides. The Working Group generated a testable hypothesis, anticholinesterase OP pesticides act by a common mechanism of toxicity, and generated alternative hypotheses that, if true, would cause rejection of the initial hypothesis and provide criteria for subgrouping OP compounds. Some of the alternative hypotheses were rejected outright and the rest were not supported by adequate data. The Working Group concluded that OP pesticides act by a common mechanism of toxicity if they inhibit acetylcholinesterase by phosphorylation and elicit any spectrum of cholinergic effects. An approach similar to that developed for OP pesticides could be used to determine if other classes or groups of pesticides that share structural and toxicological characteristics act by a common mechanism of toxicity or by distinct mechanisms.

Subchronic neurotoxicity screening studies with six organophosphate insecticides: an assessment of behavior and morphology relative to cholinesterase inhibition.

Sulprofos, disulfoton, azinphos-methyl, methamidophos, trichlorfon, and tebupirimphos were screened for neurotoxic potential, in accordance with U.S. EPA (FIFRA) requirements. Each organophosphate was administered through the diet for 13 weeks to separate groups of Fischer 344 rats at four dose levels, including a vehicle control. For each study, 12 rats/sex/dietary level were tested using a functional observational battery (FOB), automated measures of activity (figure-8 maze), and detailed clinical observations, with half of the animals perfused at term for microscopic examination of neural and muscle tissues. Separate groups of satellite animals (6/sex/dietary level) were used to measure the effect of each treatment on plasma, erythrocyte (RBC), and brain cholinesterase (ChE) activity. The results show that measures of ChE activity were consistently the most sensitive indices of exposure and assisted in the interpretation of findings. All treatment-related neurobehavioral findings were ascribed to cholinergic toxicity, occurring only at dietary levels that produced more than 20% inhibition of plasma, RBC, and brain ChE activity. Neurobehavioral tests provided no evidence of additional cumulative toxicity after 8 weeks of treatment. The FOB and motor activity findings did not alter the conclusions and generally did not reduce the neurobehavioral no-observed-effect level (NOEL) for any of the six compounds, relative to the results from detailed clinical observations as conducted in these studies. The one exception occurred with tebupirimphos, where the NOEL for motor activity was one dose level lower than the NOEL for the FOB and clinical observations. These results support the value of detailed clinical observations to screen for the neurotoxic potential of organophosphates and a general standard of more than 20% inhibition of brain ChE activity for cholinergic neurotoxicity.

Method for screening drug and chemical effects in laboratory rats using computerized quantitative electroencephalography.

A minimally-invasive method of quantitative electroencephalography (qEEG) that requires no anesthetics and parallels techniques of naturalistic stimulation was developed and validated for regulatory testing of drugs and chemicals in rats. Male and female Fischer 344 rats were utilized in a randomized-block design to measure qEEG target parameters associated with a range of cholinesterase inhibition. For this study, physostigmine was administered ip at doses of 0.05, 0.2 or 1.0 mg/kg, resulting in average cholinesterase inhibition in plasma (28, 38 and 70%), erythrocytes (19, 24 and 36%), and brain (2, 10 and 31%) which correlated well with increased total power and amplitude changes. Additional treatment-related effects consisted of decreased relative alpha and beta, increased relative delta, and a left-shift in the spectral-edge frequency. In a second study, male and female Sprague-Dawley rats were utilized in a treatment-by-subjects design to determine qEEG target parameter changes due to the M2 autoreceptor agonist oxotremorine. Repeated incremental doses (0.05, 0.1, 0.2 mg/kg; ip) of oxotremorine resulted in increased beta contribution, a right-shift in the spectral-edge frequency and decreased alpha contribution. These qEEG results with physostigmine and oxotremorine correlate well with receptor-specific and general muscarinic effects, making it a reliable contribution to analysis of agonist and antagonist effects of cholinergic compounds.

Method for screening drug and chemical effects in laboratory rats using computerized quantitative electroencephalography.

A minimally-invasive method of quantitative electroencephalography (qEEG) that requires no anesthetics and parallels techniques of naturalistic stimulation was developed and validated for regulatory testing of drugs and chemicals in rats. Male and female Fischer 344 rats were utilized in a randomized-block design to measure qEEG target parameters associated with a range of cholinesterase inhibition. For this study, physostigmine was administered ip at doses of 0.05, 0.2 or 1.0 mg/kg, resulting in average cholinesterase inhibition in plasma (28, 38 and 70%), erythrocytes (19, 24 and 36%), and brain (2, 10 and 31%) which correlated well with increased total power and amplitude changes. Additional treatment-related effects consisted of decreased relative alpha and beta, increased relative delta, and a left-shift in the spectral-edge frequency. In a second study, male and female Sprague-Dawley rats were utilized in a treatment-by-subjects design to determine qEEG target parameter changes due to the M2 autoreceptor agonist oxotremorine. Repeated incremental doses (0.05, 0.1, 0.2 mg/kg; ip) of oxotremorine resulted in increased beta contribution, a right-shift in the spectral-edge frequency and decreased alpha contribution. These qEEG results with physostigmine and oxotremorine correlate well with receptor-specific and general muscarinic effects, making it a reliable contribution to analysis of agonist and antagonist effects of cholinergic compounds.

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.

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.