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Human complement receptor 1 (HuCR1) is a pivotal regulator of complement activity, acting on all three complement pathways as a membrane-bound receptor of C3b/C4b, C3/C5 convertase decay accelerator, and cofactor for factor I-mediated cleavage of C3b and C4b. In this study, we sought to identify a minimal soluble fragment of HuCR1, which retains the complement regulatory activity of the wildtype protein. To this end, we generated recombinant, soluble, and truncated versions of HuCR1 and compared their ability to inhibit complement activation in vitro using multiple assays. A soluble form of HuCR1, truncated at amino acid 1392 and designated CSL040, was found to be a more potent inhibitor than all other truncation variants tested. CSL040 retained its affinity to both C3b and C4b as well as its cleavage and decay acceleration activity and was found to be stable under a range of buffer conditions. Pharmacokinetic studies in mice demonstrated that the level of sialylation is a major determinant of CSL040 clearance in vivo. CSL040 also showed an improved pharmacokinetic profile compared with the full extracellular domain of HuCR1. The in vivo effects of CSL040 on acute complement-mediated kidney damage were tested in an attenuated passive antiglomerular basement membrane antibody-induced glomerulonephritis model. In this model, CSL040 at 20 and 60 mg/kg significantly attenuated kidney damage at 24 h, with significant reductions in cellular infiltrates and urine albumin, consistent with protection from kidney damage. CSL040 thus represents a potential therapeutic candidate for the treatment of complement-mediated disorders., Competing Interests: Conflict of interest S. W., H. C., A. B. M., T. R., and M. P. H. are listed as inventors on International Patent Publication number WO2019/218009. All authors with the exception of A. G. N., G. A. P., G. M.- R., M. M., and M. S. are CSL shareholders., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The ability to engineer monoclonal antibodies (mAbs) with high specificity made mAbs the fastest growing segment in the drug market. mAbs represent 8 of the top 20 selling drugs with combined sales of more than 57 billion US$ per year. The ability to purify large numbers of mAbs with sufficient yields for initial screening campaigns has direct impact on the timelines of a project. Automated liquid handling (ALH)-based mAb purification platforms have been used to facilitate the production of large numbers of mAbs. However, the ongoing pressure to de-risk potential lead molecules at an early development stage by including bio-physical characterization of mAbs has further increased the demand to produce sufficient quantities from limited sample volumes. A bottleneck so far has been the limited dynamic binding capacity of these systems, which is partly due to the binding properties of commonly used Protein A affinity matrices. The present publication suggests that by using a Protein A matrix optimized for continuous chromatography applications the yields of ALH-based but also standard lab-scale mAb purifications can be significantly increased without the need to change established protocols., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development. Miniaturization and automation of chromatography using micro columns (RoboColumns(®) from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO(®) from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH. The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A-desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The Flavivirus NS5 protein possesses both (guanine-N7)-methyltransferase and nucleoside-2'-O methyltransferase activities required for sequential methylation of the cap structure present at the 5' end of the Flavivirus RNA genome. Seventeen mutations were introduced into the dengue virus type 2 NS5 methyltransferase domain, targeting amino acids either predicted to be directly involved in S-adenosyl-l-methionine binding or important for NS5 conformation and/or charged interactions. The effects of the mutations on (i) (guanine-N7)-methyltransferase and nucleoside-2'-O methyltransferase activities using biochemical assays based on a bacterially expressed NS5 methyltransferase domain and (ii) viral replication using a dengue virus type 2 infectious cDNA clone were examined. Clustered mutations targeting the S-adenosyl-l-methionine binding pocket or an active site residue abolished both methyltransferase activities and viral replication, demonstrating that both methyltransferase activities utilize a single S-adenosyl-l-methionine binding pocket. Substitutions to single amino acids binding S-adenosyl-l-methionine decreased both methyltransferase activities by varying amounts. However, viruses that replicated at wild type levels could be recovered with mutations that reduced both activities by >75%, suggesting that only a threshold level of methyltransferase activity was required for virus replication in vivo. Mutation of residues outside of regions directly involved in S-adenosyl-l-methionine binding or catalysis also affected methyltransferase activity and virus replication. The recovery of viruses containing compensatory second site mutations in the NS5 and NS3 proteins identified regions of the methyltransferase domain important for overall stability of the protein or likely to play a role in virus replication distinct from that of cap methylation.

Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration. In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. However, the mechanism of MACPF function is unknown. We determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens, to 2.0 angstrom resolution. The MACPF domain reveals structural similarity with poreforming cholesterol-dependent cytolysins (CDCs) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterial and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.

Dengue virus nonstructural protein 5 (NS5) is a large multifunctional protein with a central role in viral replication. We previously identified two nuclear localization sequences (NLSs) within the central region of dengue virus type-2 (DENV-2) NS5 ('aNLS' and 'bNLS') that are recognized by the importin alpha/beta and importin beta1 nuclear transporters, respectively. Here, we demonstrate the importance of the kinetics of NS5 nuclear localization to virus production for the first time and show that the aNLS is responsible. Site-specific mutations in the bipartite-type aNLS or bNLS region were introduced into a reporter plasmid encoding green fluorescent protein fused to the N-terminus of DENV-2 NS5, as well as into DENV-2 genomic length complementary DNA. Mutation of basic residues in the highly conserved region of the bNLS did not affect nuclear import of NS5. In contrast, mutations in either basic cluster of the aNLS decreased NS5 nuclear accumulation and reduced virus production, with the greatest reduction observed for mutation of the second cluster (K(387)K(388)K(389)); mutagenesis of both clusters abolished NS5 nuclear import and DENV-2 virus production completely. The latter appeared to relate to the impaired ability of virus lacking nuclear-localizing NS5, as compared with wild-type virus expressing nuclear-localizing NS5, to reduce interleukin-8 production as part of the antiviral response. The results overall indicate that NS5 nuclear localization through the aNLS is integral to viral infection, with significant implications for other flaviviruses of medical importance, such as yellow fever and West Nile viruses.

Viruses of the family Flaviviridae are important human and animal pathogens. Among them, the Flaviviruses dengue (DENV) and West Nile (WNV) cause regular outbreaks with fatal outcomes. The RNA-dependent RNA polymerase (RdRp) activity of the non-structural protein 5 (NS5) is a key activity for viral RNA replication. In this study, crystal structures of enzymatically active and inactive WNV RdRp domains were determined at 3.0- and 2.35-A resolution, respectively. The determined structures were shown to be mostly similar to the RdRps of the Flaviviridae members hepatitis C and bovine viral diarrhea virus, although with unique elements characteristic for the WNV RdRp. Using a reverse genetic system, residues involved in putative interactions between the RNA-cap methyltransferase (MTase) and the RdRp domain of Flavivirus NS5 were identified. This allowed us to propose a model for the structure of the full-length WNV NS5 by in silico docking of the WNV MTase domain (modeled from our previously determined structure of the DENV MTase domain) onto the RdRp domain. The Flavivirus RdRp domain structure determined here should facilitate both the design of anti-Flavivirus drugs and structure-function studies of the Flavivirus replication complex in which the multifunctional NS5 protein plays a central role.

Serpins fold into a native metastable state and utilize a complex conformational change to inhibit target proteases. An undesirable result of this conformational flexibility is that most inhibitory serpins are heat sensitive, forming inactive polymers at elevated temperatures. However, the prokaryote serpin, thermopin, from Thermobifida fusca is able to function in a heated environment. We have determined the 1.8 A x-ray crystal structure of thermopin in the native, inhibitory conformation. A structural comparison with the previously determined 1.5 A structure of cleaved thermopin provides detailed insight into the complex mechanism of conformational change in serpins. Flexibility in the shutter region and electrostatic interactions at the top of the A beta-sheet (the breach) involving the C-terminal tail, a unique structural feature of thermopin, are postulated to be important for controlling inhibitory activity and triggering conformational change, respectively, in the native state. Here we have discussed the structural basis of how this serpin reconciles the thermodynamic instability necessary for function with the stability required to withstand elevated temperatures.

A newer locomotor activity system for rodents is described. The system consists of a black, ventilated test chamber, internally lighted with a ceiling mounted video camera. The camera's image is transmitted to a contrast-sensitive tracker which maps the point of highest contrast and relays the digitalized coordinates to a PC. Dedicated software stores the information and simultaneously displays a map of the tracked subject. To illustrate the system's utility, results from an experiment are presented using an established behavioral teratogen, phenytoin. Pregnant Sprague-Dawley CD rats were exposed to 0 or 200 mg/kg of phenytoin by gavage on embryonic days 7-18 and the offspring tested in the videotracker activity monitoring device. Phenytoin is known to induce hyperactivity and circling behavior in the offspring. The system revealed that phenytoin-exposed offspring that exhibit neurological impairment were hyperactive compared to controls and the effect was predominantly seen in females. These animals exhibited more section transitions and more central and peripheral activity. When peripheral activity was subdivided into that occurring along corners versus sides, it was found that corner activity represented only a small component of the group differences whereas the side component represented most of the effect. Moreover, phenytoin offspring exhibiting the circling defect were found to display a qualitatively different pattern than noncirclers or controls. Videotracking represents a different approach to the analysis of locomotor activity patterns in experimental animals compared to older methods. Two advantages of this method are higher spatial resolution and not having to pre-specify data capture intervals. Other features are detailed regional movement information and qualitative mapping of ambulatory patterns.

A Work Group was formed to evaluate the criteria considered important in determining when to require developmental neurotoxicity testing in animal studies (i.e., triggers for testing). The primary objective of the Work Group was to determine whether there is sufficient scientific evidence to support the triggers identified by the Environmental Protection Agency and determine whether there is sufficient evidence to use structure activity relationships (SAR) to trigger automatic testing of certain classes of chemicals. A weight of evidence (WOE) approach was recommended by the Work Group in order to assist in determining which agents should undergo developmental neurotoxicity testing and to what level of testing. Evaluation of biological effects, length and duration of exposure, and quality and quantity of data available on an agent should be used in the WOE approach. Agents that are teratogenic to the central nervous system (CNS) were considered of highest priority for developmental neurotoxicity testing, especially if there is the potential for a high degree of exposure. Neuropathic and neuroactive compounds, chemicals with hormone-like activity, and developmental toxicants (with effects other than structural abnormalities of the CNS) were also considered likely candidates for such testing. Although reluctant to recommend testing based solely on SAR or chemical class, the Work Group recognized the importance of considering SAR along with other toxicity data, pharmacokinetic data and potential human exposure in making final requirements or recommendations for further testing.(ABSTRACT TRUNCATED AT 250 WORDS)

Two experiments were conducted to evaluate FD and C Red Dye #3 for its developmental toxicity and psychotoxicity. Adult Sprague-Dawley rats were fed diets containing the dye for 2 weeks and were then bred. The diets were continued for the females throughout gestation and lactation and were provided continuously to their offspring thereafter. The treatment groups for Experiment 1 were Red Dye #3 as 0.0, 0.25, 0.5, or 1.0% of the diet (w/w), and a positive control group treated with the toxin hydroxyurea on days 2-10 of life (50 mg/kg/day, s.c.); Experiment 2 was a replication of Experiment 1 with the same dose groups, but without the positive control group. Parental animals were evaluated for weight and food consumption, and females for reproductive success. The offspring were assessed on a series of tests using the Cincinnati Psychoteratogenicity Screening Test Battery, plus weight, food consumption, physical landmarks of development, and brain weight. Red-3 produced no reductions in parental or offspring weight or food consumption. Red-3 significantly increased preweaning offspring mortality in the first experiment, but not in the second. Behaviorally, Red-3 produced no dose-dependent effects that replicated across the two experiments. It was concluded that no evidence was obtained that dietary exposure to FD and C Red Dye #3 (erythrosine) is psychotoxic to developing rats.

Regulatory guidelines have produced a need to develop behavioral screening techniques to accompany teratology and reproduction testing. In the repeated use of a provisional test battery, we have found that conclusions about the behavioral teratogenic potential of test compounds are most likely to be revealed using tests having intermediate levels of variability. The results obtained from examining animals exposed developmentally to brominated vegetable oil (BVO as 2.0, 1.0, 0.5, or 0.25 percent of the diet) and comparisons of the tests' coefficients of variation, offer an empirical example of this concept. Preweaning tests of locomotion and reflex development demonstrated numerous instances of developmental delay in the BVO-treated subjects, but postweaning tests revealed few abnormalities. Behavioral testing revealed functional deficits from BVO administration at doses lower than those which have adverse effects on reproduction. Examination of the tests' variability by using coefficients of variation as a comparative index, disclosed that the postweaning test variability was almost twice that of the preweaning tests. Thus, the lack of effects of BVO on most of the postweaning tests should not be conclusively interpreted as indicative of recovery of function, because this pattern is equally likely to have resulted from the lower sensitivity of these tests. An acceptable standard for future behavioral teratology screening requires a close examination of test variability, as it appears to be an important element in the sensitivity and, hence, the interpretation of such procedures.

Potassium iodide (KI) was fed to male and female rats before and during breeding, to females only during gestation and lactation, and to their offspring after weaning (day 21 after birth) through to day 90, at levels of 0, 0.025, 0.05 or 0.1% (w/w) of the diet. Dams in a fifth group (positive controls) were given 4 mg/kg ip of the anti-mitotic/cytotoxic drug 5-azacytidine on day 17 of gestation. All offspring were reared by their natural dams and were evaluated blind with respect to treatment in a battery of standardized behavioural tests between 3 and 90 days of age. KI produced no significant reductions in parental body weight or food consumption, though it significantly reduced litter size and increased offspring mortality at the highest dose, and decreased weight gain at the two highest doses throughout the first 90 days after birth. Functionally, KI delayed auditory startle at the two highest doses, delayed olfactory orientation to the home-cage scent at the middle dose and decreased female running-wheel activity at all dose levels. In rats killed on day 90 after birth KI reduced brain and body weight at a dose of 0.1% of the diet, and reduced body but not brain weight at a dose of 0.05% of the diet. No significant effect was found on absolute or relative thyroid weight at 90 days of age. Several additional behavioural effects were observed in the low-dose KI group, but because these effects were not dose-dependent, they were not regarded as reliable. 5-Azacytidine produced evidence of substantially greater developmental toxicity than KI. It was concluded that KI produced evidence of developmental toxicity consistent with a picture of impaired thyroid function. The inclusion of tests of functional development added useful evidence to the overall picture of KI developmental toxicity.

Adult Sprague-Dawley rats were fed diets containing 0, 0.25, 0.5, 1.0, or 2.0% of the food additive brominated vegetable (soybean) oil (BVO) for 2 weeks prior to mating. After conception, the diets were continued throughout gestation and lactation for the females. The same diets were also provided to the dams' offspring throughout their development (up to 90-120 days of age). BVO at 2.0% of the diet completely blocked reproduction. BVO at 1.0% of the diet severely impaired conception, reduced maternal body weight, and produced slightly reduced litter sizes but no evidence of malformations. At this dose postnatal mortality was high, and survivors showed impaired growth and severe behavioral impairments on a battery of standardized tests of functional development. After weaning, adequate data could not be obtained because of the high mortality rate in this group. BVO at 0.5% of the diet produced less reproductive interference and much less offspring mortality or impairment of growth, but produced behavioral impairments almost as severe as seen in the BVO 1.0% group. In addition, this group exhibited severely reduced postweaning activity, delayed vaginal patency development, and reduced day-90 weight. BVO at 0.25% of the diet produced reproductive deficits similar to the BVO 0.5% group, but less severe effects on growth and behavioral development. This group showed no significant increase in offspring mortality. The data demonstrate clear evidence of dose-related physical and behavioral developmental toxicity.

Behavioral effects have been found to result from the prenatal administration of substances known to be teratogenic to the CNS. These effects occur at dose levels lower than those producing gross malformations and when the agent is administered at times other than that optimal for CNS relevant technique for detecting adverse consequences of prenatal exposure to drugs and chemicals. Behavioral testing, however, also appears to have attributes that dictate a thoughtful approach to its role as a method for assessing risk, and additional research is needed to obtain usable techniques. The need for such research is intensified by the present inability to identify potential behavioral teratogens by means other than laboratory investigation.

Butylated hydroxyanisole (BHA) was fed to rats throughout development (from prior to conception through 90 days of postnatal age) in doses of 0, 0.125, 0.25 or 0.5 percent (w/w) of the diet. A fifth group was also prepared as a positive control by administering 50 mg/kg/day of the antimitotic agent hydroxyurea on days 2-10 of postnatal age. Offspring from all groups were reared by their natural dams and were evaluated blind with respect to treatment assignment in a battery of standardized behavioral tests between 3 and 90 days of age. BHA at 0.5% of the diet impaired offspring growth during the last week of preweaning development and increased preweaning mortality (13.5%). No changes in maternal weight, reproductive performance or mortality were observed. No reductions in offspring growth after weaning or changes in day 90 brain weights were found. BHA at 0.25 and 0.125% of the diet had no effect on growth, reproduction or mortality; although a marginal increase was seen in the 0.25% BHA offspring mortality up to 30 days of age (8.3%, p = 0.06). BHA at 0.5 and 0.25% of the diet delayed startle development and showed a marginal trend towards increased diurnal running wheel activity; no other behavioral effects were found. Comparison of the present results to a similar study using BHT clearly indicates that BHA at equivalent dietary doses is considerably less toxic than BHT. The present results also suggest that BHA is not a potent behavioral toxin, although it is developmentally toxic using non-behavioral measures.

Experimental progress in the development of an accurate and useful model of phenylketonuria (PKU) during the last 15 years is reviewed in detail. From this review it is clear that the recent emergence of models using the combined administration of phenylalanine (phe) and p-chlorophenylalanine (PCPA) constitutes a major success that lays the groundwork for future research into the pathogenesis and treatment of PKU. Biochemical evidence on the pathophysiology of PKU is also briefly reviewed in the context of the behavioral and biochemical adequacy of the models used. It appears that in the past biochemical investigations into PKU have been impaired by use of inadequate models, a situation that should now change if the best of the phe-PCPA models are more widely adopted. New trends in PKU research involve the role of large neutral amino acids other than phe as potential aids in the treatment of PKU and the appearance of a new model based on the use of alpha-methylphenylalanine (AMPhe) combined with phe. It appears that PKU research may be on the brink of a new and productive era as investigations into these promising areas unfold and as new emerge through the full utilization of existing models.

In an effort to determine the relationship between time of administration and consequent behavioral effects on progeny, a uniform subteratogenic dose of vitamin A (80,000 I.U./KG) was administered to gravid Sprague-Dawley rats during one of five periods of gestation (days 5-7, 8-10, 11-13, 14-16 and 17-19). Offspring were examined for changes in rate of weight gain, locomotor activity and maze learning ability (T-maze with return to nest as reward and multiple T water maze escape). Vitamin A 8-10 animals were hyperactive, vitamin A 11-13 animals acquired T-maze slower than controls and both vitamin A 8-10 and 11-13 acquired water maze slower than controls. Vitamin A 11-13 animals were significnatly lighter than controls and all other vitamin A groups.

Sodium nitrite (NaNO2) was fed to male and female rats before and during breeding, to females only during gestation and lactation, and to their offspring after weaning (day 21 after birth) through day 90, at levels of 0, 0.0125, 0.025 or 0.05% (w/w) of the diet. Dams in a fifth group (positive controls) were given 4 mg/kg ip of the anti-mitotic/embryotoxic drug 5-azacytidine on day 16 of gestation. All offspring were reared by their natural dams and were evaluated blind with respect to treatment in a battery of standardized behavioural tests between 3 and 90 days of age. NaNO2 produced no significant reductions in parental body weight or food consumption, though it significantly increased offspring mortality and decreased weight gain at the two highest doses during the preweaning period. Functionally, NaNO2 delayed swimming development and decreased open-field activity. The open-field effect was not linearly dose dependent. In rats killed on day 90 after birth, NaNO2 produced no effects on brain or body weights. 5-Azacytidine produced evidence of substantially greater developmental toxicity than did NaNO2. NaNO2 produced a moderate degree of developmental toxicity, but no evidence was found to suggest that the central nervous system was the target organ for the toxic effects. The inclusion of tests of functional development added useful confirmatory evidence to the overall picture of NaNO2 toxicity.

Behavioral measures used in the Collaborative Behavioral Teratology Study (CBTS) were negative geotaxis (PNDs 7-10), olfactory discrimination (PNDs 9-11), auditory startle habituation (PNDs 18-19 and 57-58), 1-hr activity (PNDs 21, 60, 100 and 120), 23-hr activity (PND 100), activity following a pharmacological challenge (PND 120), and an operant, discrete trial visual discrimination task. Maternal and offspring body weights and the appearance of certain physical landmarks of development were also monitored. The design of the CBTS allowed evaluation of the reproducibility and detection sensitivity of these behavioral test methods, as well as the impact of early testing experience on later behavioral assessment, offspring sex differences in response levels and variability, and the contribution of litter-to-litter and animal-to-animal variation to behavioral measures in a standardized test protocol. The results obtained in this test system are discussed in relation to each of these factors and to the degree of overt toxicity obtained using prenatal treatment with 0, 0.5 or 2.0 mg/kg d-amphetamine sulfate, SC, on gestation days 12-15 (Study 1) or methylmercuric chloride, 0, 2.0 or 6.0 mg/kg by gavage, on gestation days 6-9 (Study 2).

New requirements by several regulatory agencies for testing the psychotoxic potential of new drugs, chemicals, and environmental contaminants raise unique problems. In order to assess intra- and interlaboratory reliability of behavioral tests a model animal maze learning procedure was designed and run in 3 cooperating laboratories. Uniform procedures were written and identical mazes were constructed. Normal control animals of identical age and sex, but of different strains, were used by the participants. A positive control group of neurologically impaired rats was run by one laboratory. Significant differences in test results among the laboratories were found. Data obtained from the positive control animals (mean errors=28.3) indicated a learning impairment statistically significant compared to the negative control data (mean errors=12.7) from any of the participating laboratories. Based on the results of this study, a reasonable standard of interlaboratory reliability in behavioral testing appears an attainable goal.

Adult Sprague-Dawley rats were fed diets containing FD and C red dye No. 40 for 2 weeks and were then bred. The diets were continued for the females throughout gestation and lactation and were provided continuously to their offspring thereafter. The treatment groups were: FD and C red dye No. 40 as 0.0, 2.5, 5.0 or 10.0% of the diet, and a positive control group treated with the toxin hydroxyurea on days 2-10 of life with 50 mg/kg/day given s.c. as a positive control group. Parental animals were evaluated for weight and food consumption, and females for reproductive success. The offspring were assessed on a series of tests using the Cincinnati Psychoteratogenicity Screening Test Battery. Additional measures were weight, food consumption, physical landmarks of development, and brain weight. Red-40 significantly reduced reproductive success, parental and offspring weight, brain weight, survival, and female vaginal patency development. Behaviorally, R40 produced substantially decreased running wheel activity, and slightly increased postweaning open-field rearing activity. Overall, R40 produced evidence of both physical and behavioral toxicity in developing rats at doses of up to 10% of the diet.

The permanence of developmental abnormalities from prenatal methylmercuric chloride (MMC) was examined in the offspring of Sprague-Dawley rats exposed on Days 6-15 of gestation to 0.00, 0.25, 1.25, 2.50, or 5.0 mg/kg MMC. The effects of MMC on reproduction and early physical development were examined together with tests of negative geotaxis, righting, pivoting, swimming, locomotor activity in an open field, and startle responses to either tactile or acoustic stimuli. No live offspring were produced by females treated with 5.0 mg/kg MMC. Offspring from the 2.50 mg/kg dose group displayed impaired performance on almost all pre-weaning measures and continuing abnormalities on longitudinal tests of locomotor activity in an open field and startle response performance.

Coffee and caffeine solutions were administered as the sole source of fluid to male and female Sprague-Dawley rats (F0) beginning 60 days before breeding and continuing until the litters (F1) from these animals were weaned. Treatments were administered as 100% brewed coffee (COF-100), and a 25% dilution of coffee (COF-25), together with solutions of caffeine in water that paralleled the caffeine content of the coffee groups, 0.056% caffeine (CAF-100) and 0.014% (CAF-25). Controls received measured amounts of plain water (CNL) and another group received vitamin A (40,000 IU/kg) on Days 7-20 of gestation (positive control treatment). During pregnancy all groups receiving COF and CAF consumed significantly more fluid than CNLs. Offspring from the COF-100 and CAF-100 dams were significantly lower in weight than CNLs. No abnormalities of reproductive performance were observed. Of 10 preweaning tests, COF-100 and CAF-100 litters displayed delayed incisor eruption, delayed swimming development, and altered activity. On 7 postweaning measures, these groups showed decreased running wheel activity and increased open-field ambulation and/or defecation. The CAF-25 group, by contrast, showed an increase in running wheel activity. Vitamin A (Vit-A) offspring showed multiple effects, including delayed incisor eruption, increased pre- and postweaning open-field activity, and reduced running wheel activity. COF and CAF produced effects on tests for psychoteratogenesis that appear consistent with the morphological consequences (delayed development) known to be associated with pre- and neonatal administration of caffeine, alone or in coffee, at high doses. The data indicate that most of the behavioral effects observed from caffeine exposure were consistent with the expected effects of concurrent administration of this agent, while the postweaning exposure effects suggest a longer-term change in activity. No effects of caffeine were found, however, on measures of learning, memory, or motoric functioning.

Three psychotropic drugs were administered to pregnant rats and were then evaluated for their behavioral and reproductive effects in the offspring. Control rats received either saline or vitamin A. Prochlorperazine had the most disruptive effects on reproduction and growth, but had the least effect on behavior. Propoxyphene had no apparent effects on reproduction or growth, but produced a variety of behavioral changes. Fenfluramine was intermediate in its effects on reproduction and growth and had behavioral effects that were revealed in tests of preweaning development. The data suggest that systematic tests of behavior add important information to evaluations of reproductive toxicity that cannot, at present, be obtained by other means.

Pregnant Sprague-Dawley rats received 25 mg/kg of prochlorperazine, 20 mg/kg of fenfluramine, 75 mg/kg of propoxyphene or 200 mg/kg of diazepam daily between the 7th and 20th days of gestation. Vehicle control groups and a positive control group (vitamin A 40,000 IU/kg/day) were similarly prepared. Observations of reproductive performance were made and the offspring examined in a battery of neurobehavioral tests. Fenfluramine and prochlorperazine produced abnormalities in both the reproductive measures and neurobehavioral testing. Propoxyphene produced developmental delays and other signs of "pure" behavioral teratogenesis in that these effects were not anticipated in any of the observations of reproductive performance. Diazepam appeared to have the mildest effect on all the measurements taken. The test methods used in this study appear to be a reasonable initial approach to the development of neurobehavioral screening procedures which are comprehensive, sensitive, and usable.

Phenylketonuria (PKU) was induced in rats by the combined feeding of 3 per cent excess phenylalanine and 0.12 per cent of p-chlorophenylalanine, an inhibitor of phenylalanine and tryptophan hydroxylases. Increased concentrations of phenylalanine and increased ratio of phenylalanine to tyrosine were demonstrated in blood from pregnant rats fed the experimental PKU diet from day 10 to 20 of pregnancy, in fetal blood and amniotic fluid of fetal animals from mothers fed the PKU diet, and in blood of rats fed the PKU diet for 28-30 days beginning at 20-21 days of age. Both phenylpyruvic acid and orthohydroxyphenylacetic acid were excreted by rats fed the PKU diet, but neither were detected in urine in animals fed either excess phenylalanine or excess inhibitor alone. Reduced serotonin concentrations were found in brains of rats fed p-chlorophenylalanine, either alone or in combination with excess phenylalanine in the PKU diet. These biochemical changes in rats with induced PKU and the behavioral changes described earlier are similar to those of the human condition. The animal model should prove useful in searching for the mechanism of the disease.

Newborn rats injected on Days 1-8 of life with L-phenylalanine (2 g/kg) and p-chlorophenylalanine (80 mg/kg) displayed biochemical symptoms analogous to human phenylketonuria (PKU) and maze learning impairments. The behavioral effects were less evident in rats treated on Days 9-16 or 7-24. None of the symptoms observed were alleviated by simultaneous administration of melatonin (10 mg/kg/day).

Behavioral Teratology as it exists today is viewed as having developed from the combination of theory and test methods from Developmental Psychology with the principles of morphological examination and an orientation toward public health issues from Teratology. In the recent past, the search for useful screening methods for safety evaluation has used two basic strategies: (1) having tests chosen by the consensus of experts in the field or through surveys of tests actually used in Behavioral Teratology; and (2) exploring the utility of test procedures in small, informal interlaboratory reliability studies. Neither of these approaches succeeded in identifying appropriate screening methods; rather, these attempts have made clear the need for research efforts specifically directed at test selection and interlaboratory reliability.

Rats easily discriminate between two types of subcortical brain shock which differ in reinforcing properties. When both stimuli are either neutral or positively reinforcing subjects have difficulty in responding differentially to the two types of electrical stimulation of the brain. Possible implications for a theory concerning a generalized or diffuse reinforcement system are discussed.