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.     

QT PRODACT: usability of miniature pigs in safety pharmacology studies: assessment for drug-induced QT interval prolongation

To investigate whether miniature pigs are useful for evaluating the potential of drugs for drug-induced prolongation of the QT interval, we performed an in vivo QT assay using conscious and unrestricted miniature pigs. Compared with the vehicle average baseline values, haloperidol at 3 and 10 mg/kg, p.o. prolonged the QTcF interval (Fridericia's formula) by 8%-16%. The plasma concentration of haloperidol at which QT interval was prolonged (Cmax=42.9 ng/mL) was almost equal to that in humans. dl-Propranolol at 3, 10, and 30 mg/kg, p.o. caused no alterations in QT interval. dl-Propranolol at 3, 10, and 30 mg/kg, at which plasma concentrations were lower than in humans treated with dl-propranolol at the therapeutic dose level, shortened QTcF interval by 7%-12%. dl-Sotalol at 10 mg/kg, p.o. prolonged QTcF interval by 7%. From the above results, we considered that the miniature pig can be used for prediction of drug-induced prolongation of QT interval in humans, and thus, it is one of the useful animal species for assessing electrocardiograms in safety pharmacology studies.     

Immunotoxicology: role in the safety assessment of drugs.

The immunotoxic effects of drugs are divided into immunosuppression, immunostimulation, hypersensitivity and autoimmunity. The major adverse consequences of immunosuppression are infectious complications and virus-induced malignancies. Flu-like reactions, more frequent autoimmune diseases and hypersensitivity reactions to unrelated allergens, and inhibition of drug-metabolising enzymes are the adverse effects related to immunostimulation. Hypersensitivity reactions are the most frequent immunotoxic effects of drugs. They include immune-mediated ('allergic') and non immune-mediated ('pseudoallergic') reactions. Drug-induced autoimmune reactions, either systemic or organ-specific, are seemingly rare. A review of drug-induced immunotoxic effects demonstrates that immunotoxicity is a significant cause of morbidity and even mortality. As immunotoxicologists have long focused on immunosuppression, the nonclinical immunotoxicity safety assessment of unexpected immunosuppression is based on a number of relatively well standardised and validated animal models and assays. However, there is no general consensus regarding the minimal requirement for this assessment. Many different assays can be used to extend the assessment case by case. Few animal models and assays have been validated for use in the nonclinical safety assessment of unexpected immunostimulation. The situation is worse regarding the prediction of hypersensitivity and autoimmune reactions. Our limited understanding of the molecular and cellular mechanisms of immunotoxicity accounts, at least partly, for this situation. Recent guidelines for the immunotoxicity safety assessment of drugs, even though conflicting on several points, will serve as an impetus not only to refine current animal models and assays, but also to search for better alternatives. The new data generated will have to be interpreted and extended to animal species other than just rodents. Likewise, animal results will have to be compared with findings in humans. The search for immunological endpoints that can be used in several animal species and in humans will therefore become essential. Specific endpoints and clinical criteria that can be included in clinical trials to further investigate the potential for immunotoxicity of new drugs will have to be defined. Because immunotoxicity plays a key role in drug-induced adverse effects, the role of immunotoxicology in drug safety assessment is indisputable and the systematic nonclinical as well as clinical immunotoxicity assessment of every new drug is deemed essential.     

Natural feed additive of Macleaya cordata: Safety assessment in rats a 90-day feeding experiment

Macleaya cordata (Willd.) (Papaveraceae) is used as an active component in the natural feed additive Sangrovit^®. Sangrovit^® contains mixture of the intact aerial parts and the fraction of quaternary benzo[c]phenanthridine alkaloids from M. cordata (FQBA). In a 90-day pilot toxicity trial, Sangrovit^® and the FQBA were tested for safety. Male Wistar rats were fed for 90 days with 100, 7000 or 14000 mg of Sangrovit^® or 600 mg of FQBA in 1 kg of feed. Body and organ weights, clinical chemistry and hematology markers, oxidative stress parameters, morphological structure of tongue, liver, ileum, kidney and heart samples, and total cytochrome P450 in liver were monitored. The results showed no statistically significant alterations in any parameter between control and treated animals, except for the group treated with 14000 ppm Sangrovit^® that resulted in elevation of reduced glutathione level and superoxide dismutase activity in liver.     

SNPs and chips: genomic data in safety evaluation and risk assessment.

The biotechnology revolution is impacting all of the biologicalsciences, and toxicology is no exception. Perusal of the abstractsof the Society’s recent annual meetings confirms thatour field is already actively using the powerful new tools ofmolecular biology to identify and understand the mechanismsof toxic responses. Thus, the Workshop on Toxicogenomics andRisk Assessment sponsored by the Society and discussed in thisissue by Cunningham et al. was a timely undertaking. This was not the first workshop to focus on the applicationof genomic approaches to risk assessment, but it was uniquein a number of respects. First, it brought together thought     

Effects of intracerebroventricular injection of naloxone on operant feeding and drinking in pigs

Operant feeding and drinking to satiation were studied in prepubertal pigs deprived of food or water for 18 hours and then given intracerebroventricular (ICV) injections of a solution of naloxone hydrochloride. In feeding tests there was no difference in the amount of food consumed, or in the rate at which reinforcements were obtained, between pigs given ICV injections of 0.4 or 0.8 mg naloxone and those receiving a control injection of saline. However, in drinking tests, injection of both 0.2 and 0.4 mg naloxone significantly (p less than 0.01) reduced the quantity of water drunk and slowed the rate at which reinforcements were obtained. No significant effects on operant water intake were seen after intravenous injection of 0.4 mg naloxone.     

Effect of intracerebroventricular administration of the GABAB-receptor agonist baclofen on operant feeding in satiated pigs.

1. The present study investigated the effects of intracerebroventricular (i.c.v.) administration of the GABAB-receptor agonist baclofen on food and water intake in satiated pigs previously trained to make operant responses for food and water, which were available ad libitum. 2. Baclofen (25-100 nmol) i.c.v. produced a dose-related increase in food intake. Baclofen (50 nmol) increased feeding during the first 15 min after administration (P less than 0.01), while the 100 nmol dose increased feeding during the first 30 min (P less than 0.01). None of these doses of baclofen had any affect on the daily (24 h) food intake. 3. The effect of baclofen (50 nmol) on feeding was prevented by pretreating the animals with the GABAB antagonist phaclofen (500 nmol, i.c.v.). 4. Baclofen (25-100 nmol) i.c.v. had no significant effects on water intake. 5. Intravenous administration of baclofen (100 nmol) had no effect on food intake, thus eliminating the possibility that i.c.v. baclofen might have stimulated feeding by a peripheral mode of action. 6. These results show that baclofen increases food intake in satiated pigs, and that this effect is mediated by the drug acting at central GABAB-receptors.     

Effects of feeding regimen on ethanol intake by guinea pigs

During daily two-hr sessions, guinea pigs licked a drinking tube filled with either 0 (tap water), 2, 4 or 8% (v/v) ethanol solution under three feeding regimens. Consumption of each solution was highest when sufficient food to maintain subjects at 90% of free-feeding weight was provided during sessions, lower when the same food ration was provided after sessions, and lowest when ad lib access to food was provided within and between sessions. However, this decrease in consumption across feeding regimens was inversely related to ethanol concentration. Under all feeding regimens, volume of solution consumed decreased with increasing ethanol concentration while milligrams ethanol consumed increased with ethanol concentration. These results are similar in some respects to previous findings with rats and monkeys, suggesting that further studies of oral ethanol self-administration by guinea pigs may be merited.     

Food safety: risk assessment methodology and decision-making criteria

As our scientific technology grows, risk assessment methods become more complex and, therefore, open to greater scientific debate. Risk assessment has always been a part of the regulatory notification and approval process for foods. However, the methodologies applied to risk assessment and decision-making have become diverse, dependent on a number of features, including the areas of the world in which one operates, the need to use cumulative risk assessment for pesticides and other ingredients or alternative risk assessment considerations for evaluating nontraditional or bioengineered foods. Diverse institutional structures within a single federal regulatory authority may tend to lead to diversity in risk outcomes that creates policy decisions that complicate and confuse the risk management process. On top of this challenge, decisions become more complicated by the need to examine beneficial factors of foods rather than the adverse effects of foods and food additives. Foods are a complex mixture of ingredients. Regulatory groups recognize the need to use new approaches for evaluating the safety and risks associated with foods and food additives, and to do so in a timely manner. The United States Food and Drug Administration (US FDA) in its need to ensure standards of "reasonable certainty of no harm" continues to explore alternative means to be responsive to petitioners as well as continue to examine scientifically validated means, e.g., quantitative structure-activity relationship (QSAR), and computer-assisted programs, within the approval process to assist in the evaluation of risks. Another means to improve the risk management process would include the cumulative risk assessment of pesticides that will, no doubt, be the beginning of more intensive efforts to understand cumulative exposures and the inherent risks from multiple pathways of exposure. The passage of the Food Quality Protection Act (FQPA) resulted in developing additional risk assessment methodologies and approaches to assess the potential for multiple exposures and risks. Addressing the international criteria used in decision-making related to foods safety assessment has resulted in acceptable intake values for food ingredients for carcinogens and noncarcinogens that, in general, tend to be more stringent in the United States compared to Europe. Clearly, the need for harmonization of risk assessment criteria and the impact of the decision process on regulatory approvals and safety assessment is a future need for the continued assurances of food safety. The topics presented in this paper are based on a symposium held in November 2002 at the annual meeting of the American College of Toxicology.     

加米霉素注射液对靶动物猪的安全性试验研究

加米霉素(gamithromycin)是一种新型动物专用大环内酯类半合成抗生素,目前国内暂无加米霉素在靶动物猪的相关用药研究。为了研究加米霉素注射液按照临床推荐剂量(6mg/kg·B·W)用于靶动物猪的安全性,试验选用24头健康二元断奶仔猪(70日龄左右),随机分为4组,每组6头,设1组为对照组,其余为1倍剂量组(6mg/kg·B·W),3倍剂量组(18mg/kg·B·W)和5倍剂量组(30mg/kg·B·W)。猪颈部肌肉注射给药,连续3d,每天1次,持续至最后1次给药后第15天。结果显示：与未给药前相比,加米霉素各剂量组和对照组猪精神状态、呼吸特征、采食、饮水和粪尿排泄均正常,未表现异常反应。与对照组相比,加米霉素注射液各剂量组猪给药前(第0天)、最后一次给药后的第8天、第15天的增重和增重率无显著差异;且给药前(第0天)、最后1次给药后的第1天、第8天、第15天血常规和血液生化指标差异均不显著;宰杀后经肉眼观察,加米霉素注射液各剂量组猪的心、肝、脾、肺、肾、胰腺、胃、十二指肠、空肠、回肠、直肠、淋巴结、肾上腺和脑组织表面未出现显著的病理变化。HE染色,与对照组相比,5倍剂量组猪的上述组织均未发生显著病理变化。表明加米霉素注射液在临床使用中按“肌肉注射,每千克体重,猪6mg,每天1次,连续给药3d”的用法和用量使用,安全、无明显不良反应。     

Final report on the safety assessment of HC Yellow No. 5

HC Yellow No. 5 is a direct hair dye. Hair dyes containing HC Yellow No. 5, as "coal tar" hair dye products, are exempt from the principal adulteration provision and from the color additive provision of the Federal Food, Drug, and Cosmetic Act of 1938 when the label bears a caution statement and "patch test" instructions for determining whether the product causes skin irritation. Preliminary testing on or by individuals should be done using an open patch test that is evaluated at 48 h after application of the test material. Users, therefore, would be able to determine their individual reactions to hair dye products containing HC Yellow No. 5. Absorption of HC Yellow No. 5 is minimal through skin (< 0.2%). The oral LD(50) for rats is 555.56 mg/kg. No significant toxic effects were observed after chronic oral exposure of HD Yellow No. 5 to dogs. Mild dermal irritation, but no dermal sensitization or ocular irritation was observed in laboratory animals. Results of fertility and reproductive performance, teratology, and developmental studies were negative. HC Yellow No. 5 was found to be nonmutagenic and noncytotoxic in standard laboratory assays. A current review of the hair dye epidemiology literature identified that use of direct hair dyes, although not the focus in all investigations, appears to have little evidence of an association with cancer or other adverse events. Based on the available safety test data on HC Yellow No. 5, the Panel determined that this ingredient likely would not have carcinogenic potential as used in hair dyes. The Cosmetic Ingredient Review (CIR) Expert Panel concluded that HC Yellow No. 5 is safe as a hair dye ingredient in the practices of use and concentration as described in this safety assessment.     

Final report on the safety assessment of HC Red No. 7

HC Red No. 7 functions as a semipermanent (direct) hair colorant in one cosmetic product at 1%. Analytical studies found the relative purity of HC Red No. 7 to be > 98.5%. Impurities may include 2-nitro-benzene-1,4-diamine; 3-(4-amino-3-nitro-phenyl)-oxazolin-2-one; 2-chloroethyl 4-amino-3-nitrophenylcarbamate; residual solvents ethanol, DMF, or isopropyl acetate; chloride ions; and heavy metals. Around 0.10% of the applied HC Red No. 7 was absorbed in human dermatomed skin samples. In an acute oral toxicity study in rats, the maximum nonlethal dose was 300 mg/kg. The no observed effect level (NOEL) in a subchronic oral toxicity study in rats was 50 mg/kg day(- 1). HC Red No. 7 was not a dermal or ocular irritant in rabbits, but lymphoproliferative responses in mice indicated that HC Red No. 7 should be considered a moderate sensitizer. The NOEL for maternal toxicity was 50 mg/kg/day and the no observed adverse effect level (NOAEL) for embryonic development was 200 mg/kg/day in a prenatal toxicity study of HC Red No. 7 using rats. HC Red No. 7 was nonmutagenic at the hprt locus but mutagenic at the TK locus in mouse lymphoma cells, was mutagenic in several Salmonella typhimurium strains, was not active in an unscheduled DNA synthesis assay, and was unclear in a micronucleus assay in human lymphocyte cultures. No carcinogenicity studies were available, nor were any clinical tests reported. Available hair dye epidemiology studies are insufficient to conclude a causal relationship between hair dye use and cancer or other diseases, but more relevant is that direct hair dyes, although not the focus in all investigations, appear to have little evidence of an association with adverse events as reported in epidemiology studies. As reviewed by the Cosmetic Ingredient Review (CIR) Expert Panel, HC Red No. 7 appears to be a moderate sensitizer in animals. No human sensitivity data concerning this ingredient have been reported. However, hair dyes containing HC Red No. 7, as coal tar hair dye products, are exempt from the principal adulteration provision and from the color additive provisions in sections 601 and 706 of the Federal Food, Drug, and Cosmetic Act, when the label bears a caution statement and patch test instructions for determining whether the product causes contact dermatitis. The Expert Panel expects that following this procedure will identify prospective individuals who would have an irritation/sensitization reaction and allow them to avoid significant exposures. The CIR Expert Panel also noted that mutagenicity studies available for HC Red No. 7 gave both positive and negative results. Based on the available data, it was concluded that, at most, this ingredient is a weak mutagen. Due to its low dermal absorption potential and its use as a semipermanent hair dye, the CIR Expert Panel believes there is low risk of genotoxicity and that HC Red No. 7 is safe as a hair dye ingredient in the practices of use and concentrations as described in this safety assessment.     

Cardiovascular parameters in anaesthetized guinea pigs: a safety pharmacology screening model

INTRODUCTION: Assessment of cardiovascular functions in vivo is part of the core battery of guideline ICH S7A and is thereby required by regulatory authorities. The haemodynamic effects of repeated intravenous administrations of reference compounds were analyzed in order to validate the guinea pig model for safety pharmacology studies under GLP conditions. METHODS: Male guinea pigs (n=54, weighing 565-762 g) were anaesthetized using 1.5 g/kg, i.p., urethane. Systolic arterial blood pressure (SAP), diastolic arterial blood pressure (DAP), heart rate (HR), left ventricular pressure (LVP), cardiac contractility (dp/dt(max)), and ECG (RR, QT, and QTc intervals) were recorded continuously. Animals received vehicle i.v. followed by cumulative doses of reference compounds. RESULTS: Vehicle did not produce any relevant changes, either in cardiovascular or ECG parameters. Isoproterenol caused a rapid and significant increase in HR, LVP, and dp/dt(max), in contrast to a dose-dependent decrease in SAP and DAP. Epinephrine led to a potent increase in all cardiovascular parameters. Nifedipine produced a slight decrease in HR and LVP, and a potent decrease in blood pressure and dp/dt(max). Verapamil caused a dose-dependent decrease in all cardiovascular parameters. Ouabain resulted in a significant increase in SAP, DAP, LVP, and dp/dt(max); ECG showed an atrioventricular block and arrhythmia. Terfenadine, cisapride, and sotalol prolonged QT and QTc intervals, whereas vehicle and the other tested compounds did not produce any prolongation of the QTc interval. DISCUSSION: Our results on HR, blood pressure, and ECG obtained after i.v. administration of reference compounds show the usefulness of the guinea pig in assessing cardiovascular safety. The anaesthetized guinea pig allows the measurement of cardiac contractility and the use of doses higher than in conscious animals. Using this animal model, several cardiovascular parameters can be recorded simultaneously at a modest cost in terms of test compound and the number of animals required.     

Application of emerging technologies in toxicology and safety assessment: regulatory perspectives

Emerging technologies applied in the regulatory field encompass a group of technologies that are used in addition to or in replacement of the standard toxicology studies conducted to support an Investigational New Drug Application (IND) or New Drug Application (NDA). The standard package includes general toxicology studies of various duration, safety pharmacology studies, genetic toxicology studies, and reproductive toxicology studies. New and emerging technologies applied to the regulation of new drugs include the use of novel biomarkers, transfected cells and transgenic animals, and the "omics" technologies (toxicogenomics, proteomics, and metabonomics). These technologies are at various stages of regulatory development and acceptance. For example, the use of transgenic animals have gained acceptance by regulatory authorities to replace a 2-year carcinogenicity assay. Alternatively, the "omics" technologies are not sufficiently advanced to achieve regulatory acceptance as replacements, although these assays have a role early in drug development and they may prove useful as supplements to standard studies. Data from these assays have been used to address specific mechanistic questions in combination with standard toxicology assays.     

Preclinical safety assessment: in vitro -- in vivo testing

In vitro--and in vivo preclinical safety tests on drug candidates needed before first dose in man and before marketing authorisation are as follows: The acute and repeated dose toxicity studies, the reproductive toxicity studies, the genotoxicity studies, the carcinogenicity studies and finally the safety pharmacology studies. The Safety Assessment of the results with respect to predictability for humans is discussed, as well as new tests under validation. Suggestions for changes in the future of Non-Clinical Safety tests are mentioned.     

Investigation of sanguinarine and chelerythrine effects on CYP1A1 expression and activity in human hepatoma cells.

Quaternary benzo[c]phenanthridine alkaloids (QBA) sanguinarine and chelerythrine exhibit a wide spectrum of biological activities whence they are used in dental care products. Recent studies indicated that cytochrome P450 CYP1A attenuates sanguinarine toxicity both in vivo [Williams, M.K., Dalvi, S., Dalvi, R.R., 2000. Influence of 3-methylcholanthrene pretreatment on sanguinarine toxicity in mice. Vet. Hum. Toxicol. 42, 196-198] and in vitro [Vrba, J., Kosina, P., Ulrichová, J., Modriansky, M., 2004. Involvement of cytochrome P450 1A in sanguinarine detoxication. Toxicol. Lett. 151, 375-387]. However, CYP1A converts sanguinarine to the products that form DNA adducts [Stiborová, M., Simánek, V., Frei, E., Hobza, P., Ulrichová, J., 2002. DNA adduct formation from quaternary benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine as revealed by the 32P-postlabeling technique. Chem. Biol. Interact. 140, 231-242]. In our work we examined the effects of sanguinarine and chelerythrine on CYP1A1 expression and catalytic activity in human hepatoma cells-HepG2. Sanguinarine and chelerythrine did not affect basal and dioxin-inducible expression of CYP1A1 mRNA and protein in HepG2 cells. The enzymatic activity of CYP1A1 was assessed by the fluorescent measurement of 7-ethyxoresorufin-O-deethylase (EROD) activity. We observed a slight decrease of dioxin-induced EROD activity in HepG2 cells by sanguinarine and chelerythrine. This decrease was attributed to the inhibition of CYP1A1 catalytic activity, as revealed by enzyme kinetic studies on recombinant CYP1A1 protein. The IC50 values for the inhibition of CYP1A1 by sanguinarine and chelerythrine were 2.1 and 1.9muM, respectively. In conclusion, albeit the CYP1A modulates QBA cytotoxicity and genotoxicity, the QBA themselves do not affect CYP1A1 expression. The data indicate that studied alkaloids do not have specific cellular target and their biological effects are rather pleiotropic.