Assessment of three approaches for regulatory decision making on pesticides with endocrine disrupting properties.

Recent EU legislation has introduced endocrine disrupting properties as a hazard-based "cut-off" criterion for the approval of active substances as pesticides and biocides. Currently, no specific science-based approach for the assessment of substances with endocrine disrupting properties has been agreed upon, although this new legislation provides interim criteria based on classification and labelling. Different proposals for decision making on potential endocrine disrupting properties in human health risk assessment have been developed by the German Federal Institute for Risk Assessment (BfR) and other regulatory bodies. All these frameworks, although differing with regard to hazard characterisation, include a toxicological assessment of adversity of the effects, the evaluation of underlying modes/mechanisms of action in animals and considerations concerning the relevance of effects to humans. Three options for regulatory decision making were tested upon 39 pesticides for their applicability and to analyze their potential impact on the regulatory status of active substances that are currently approved for use in Europe: Option 1, based purely on hazard identification (adversity, mode of action, and the plausibility that both are related); Option 2, based on hazard identification and additional elements of hazard characterisation (severity and potency); Option 3, based on the interim criteria laid down in the recent EU pesticides legislation. Additionally, the data analysed in this study were used to address the questions, which parts of the endocrine system were affected, which studies were the most sensitive and whether no observed adverse effect levels were observed for substance with ED properties. The results of this exercise represent preliminary categorisations and must not be used as a basis for definitive regulatory decisions. They demonstrate that a combination of criteria for hazard identification with additional criteria of hazard characterisation allows prioritising and differentiating between substances with regard to their regulatory concern. It is proposed to integrate these elements into a decision matrix to be used within a weight of evidence approach for the toxicological categorisation of relevant endocrine disruptors and to consider all parts of the endocrine system for regulatory decision making on endocrine disruption.

Assessment strategies and decision criteria for pesticides with endocrine disrupting properties relevant to humans.

There is growing concern that environmental substances with a potential to modulate the hormonal system may have harmful effects on human health. Consequently, a new EU regulation names endocrine disrupting properties as one of the cut-off criteria for the approval of plant protection products, although it currently fails to provide specific science-based measures for the assessment of substances with such properties. Since specific measures are to be presented by the European Commission in 2013 the development of assessment and decision criteria is a key challenge concerning the implementation of this new EU regulation. Proposals of such decision criteria for substances with potential endocrine disrupting properties in human health risk assessment were developed by the German Federal Institute for Risk Assessment (BfR) and discussed at an expert workshop in November 2009. Under consideration of the requirements laid down within the new plant protection product legislation and the scientific discussions during the workshop, a conceptual framework on evaluation of substances for endocrine disrupting properties in a regulatory context is presented in this paper. Central aspects of the framework include assessment of adversity of effects, establishment of a mode/mechanism of action in animals, considerations concerning the relevance of effects to humans and two options for a regulatory decision.

Combined retrospective analysis of 498 rat multi-generation reproductive toxicity studies: on the impact of parameters related to F1 mating and F2 offspring.

The multi-generation reproductive toxicity study (OECD TG 416 and USEPA 870.3800) has been extensively used internationally to assess the adverse effects of substances on reproduction. Recently the necessity of producing a second generation to assess the potential for human health risks has been questioned. The present standardized retrospective analysis of the impact of the second generation on overall study outcome combines earlier analyses and includes 498 rat multi-generation studies representing 438 different tested substances. Detailed assessment of study reports revealed no critical differences in sensitivities between the generations on the basis of a consideration of all endpoints evaluated. This analysis indicates that the second generation mating and offspring will very rarely provide critical information. These findings are consistent with the conclusions of previous retrospective analyses conducted by RIVM, USEPA and PMRA and support adoption of the proposed OECD extended one-generation reproductive toxicity study protocol in regulatory risk assessment testing strategies.

The cult of culling.

It is difficult to understand why culling (reduction of litter size) has become such a widely used procedure in reproductive toxicity studies since there appear to have been no prior investigations to ascertain that it would improve the efficiency of studies with respect to detecting adverse effects. Perhaps the only provable advantage of culling is with respect to economics and convenience. Post hoc rationalizations for culling lack conviction because many of the claims made for culling are erroneous, inconsistent, vague, and contradictory. Mostly, they are based on part truths derived from minimal studies, conducted for totally different purposes. That experimental animals have to be killed sooner or later is unquestioned, but for ethical and scientific reasons, it is imperative that the maximum amount of information is obtained from them. Currently, the most common practice is to cull litters to four per sex (total eight) on Day 4 postpartum. This is totally divorced from natural values for most rat strains and involves elimination, usually without adequate examination, of between 30 and 45% of offspring. Without culling most of these would survive, unless there was a treatment effect. Intuitively, it would seem that removal of such a proportion of offspring would severely limit the possibility of detecting the postnatal equivalent of fetal malformations. Culling totally nullifies litter size as an indicator of toxicity. Indirectly, it also nullifies the value of mean pup weight as an indicator of toxicity because it greatly increases the variation in mean pup weight. This is quite contrary to the claim that culling reduces variance. Further, the increased growth of offspring in culled litters can have long-term consequences of a shorter overall and reproductive life span.

Risk assessment for neurobehavioral toxicity: SGOMSEC joint report.

Behavioral end points for neurotoxicity risk assessment have been developed and examined over the past three decades. They are now ready to move from simple qualitative guidelines, such as exemplified by reference doses, to more quantitative models, such as benchmark doses, based on dose-response information. Risk assessors, confronted by a wider array of methodologies and data than in the past, should be offered guidance in interpretation because now they have to deal with unaccustomed questions and problems. These include reversibility, susceptible populations, multiple end points, and the details of dose-response and dose-effect distributions.

Neurobehavioral aspects of developmental toxicity testing.

Tests for detection of neurobehavioral changes in the offspring have been a regulatory requirement in developmental toxicity testing of drugs for almost 20 years. Keeping their purpose of hazard identification and risk assessment for humans in mind, investigators and agency reviewers have become deeply ingrained with some stereotyped behaviors with respect to such relevant issues as choice of animal species and data evaluation. Other problematic areas of study design and conduct, selection of litter representatives for testing, what methods to combine in a testing battery, and statistical treatment of results and their interpretation, will need more research and discussion in the future.

Peri- and postnatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin: effects on physiological development, reflexes, locomotor activity and learning behaviour in Wistar rats.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the development of rat offspring were studied after administration of a loading dose of 300 or 1000 ng TCDD/kg body wt on day 19 of pregnancy, followed by weekly maintenance doses of 120 or 400 ng TCDD/kg body wt. The dose regimens led to a fluctuation of average TCDD concentrations in the liver of the offspring of 4.9-14.9 ng/g (TCDD1000/400 group) or 1.4-6.3 ng/g (TCDD300/120 group) during the course of the experiment. In both TCDD-exposed groups the body weight of the offspring was significantly lower on postnatal day 7 (PND 7); in the high dose group from PND 7 to PND 31. Some landmarks of postnatal development were retarded in the exposed groups; in particular, the vaginal opening was delayed for several days in both TCDD-exposed groups. The TCDD-exposed animals revealed a reduced ability to remain on a rotating rod. During reflex testing, the rate of successfully responding animals was higher in the exposed groups. No statistically significant differences in the locomotor activity between controls and TCDD-exposed offspring were detectable under our experimental conditions. In a discrimination learning test no effects on the learning ability were found. However, TCDD-exposed offspring showed an increase in unanswered trials during critical phases of the task. They also exhibited increased locomotor activity in a novel environment; prior to an amphetamine challenge dose of 1 mg/kg body weight. Amphetamine-induced activity was decreased in a dose-dependent manner.

Requirements of preclinical examinations.

The eye is an important and sensitive target organ not only for ophthalmic drugs but also generally for adverse drug effects and has to be taken into consideration accordingly when performing toxicological studies. Depending on the drug in question protocols for oculotoxicity studies have to be determined on a case by case basis. The test battery used to detect possible eye damage should be an intelligent combination of pharmacokinetic/metabolic studies, clinical examinations, pathomorphologic examinations, and biochemical tests. For special studies on oculotoxicity only pigmented animals should be used. The legal basis for the necessity to conduct oculotoxicity studies and ophthalmologic examinations in toxicity studies is given by national law (AMG) and guidelines and by EEC-Guidelines. Studies have to be performed according to the principles of Good Laboratory Praxis.

A new biscoumarin glucoside ester from Ruta chalepensis cell cultures.

The investigation of a plant cell culture of RUTA CHALEPENSIS L. yielded three compounds: isorutarin, rutacridone, and rutarensin, a so far unknown ester of daphnoretin glucoside ("daphnorin"), as well as 3-hydroxy-3-methylglutaric acid.

Detection limits of different approaches in behavioral teratology, and correlation of effects with neurochemical parameters.

Five laboratories collaborated in the evaluation of detection limits of different testing concepts in behavioral teratology. In one laboratory, rat dams were treated by gavage with five doses of methylmercury (0.0, 0.25, 0.05, 0.5, and 5.0 mg/kg/day). The treatment period was restricted to days 6 to 9 of gestation. The usual reproduction parameters were assessed in the dams. The offspring (88-99 per group) were subjected to a routine developmental and behavioral testing battery. After completion of these tests, random samples of the animals were further investigated in four other laboratories using the following techniques: auditory startle habituation, visual discrimination and figure-8 activity monitor; wheel-shaped activity monitor and spatial alternation operant conditioning; two-compartment locomotor activity, passive avoidance and male ultrasonic vocalization during sexual behavior; assays of the weight of different brain areas, their glial fibrillary acidic (GFA) protein and S-100 protein concentration. The following dose-dependent effects were noted in ascending dose sensitivity order: delayed vaginal opening; increased and more variable passiveness in spatial alternation; impaired swimming behavior, increased GFA protein concentration in the cerebellar vermis; increased auditory startle amplitude, decreased intertrial interval pokes in the visual discrimination test, increased percentage of visits in passive area of figure-8 activity monitor, increased path iteration frequencies and decreased local activity in the wheel-shaped activity monitor, decreased locomotor activity in the two-compartment monitor, increased cerebellar vermis weight, and decreased S-100 protein in the hippocampus. Therefore, this study showed comparable sensitivities for the behavioral testing battery, for some automated multiparametric test systems and for the neurochemical assays.

Specific toxicologic aspects of the quinolones.

Possible targets of quinolone toxicity include the juvenile joint, the kidney, the central nervous system (CNS), the eye, and the cardiovascular system. In immature animals all quinolones studied cause arthropathies of the major diarthrodial joints. Arthropathies have also developed in adult dogs after 12 months of pefloxacin treatment. At high doses the quinolones exert effects on renal function that are related to a foreign-body reaction caused by crystals; nephropathologic changes seem not to occur without crystalluria. In humans quinolones can have various CNS effects. The subcellular "substrate" for these effects is unknown. Further understanding of severe CNS reactions (confusion, hallucination, anxiety, agitation, nightmares, convulsive seizures, and depression) is needed. Pefloxacin causes cataracts in dogs after treatment for 8-12 months. Low-dose quinolones (administered as an intravenous bolus) cause pronounced but transient systolic hypotension in dogs and cats; cardiovascular effects may be mediated by histamine release. Quinolones inhibit the bacterial enzyme DNA gyrase. To exclude the possibility of damage to mammalian DNA, mutagenicity studies have been performed. Since all but two tests (which may give false-positive results) have been negative, quinolones appear to be nonmutagenic. Photosensitivity has occurred in humans given quinolones. Drug interactions can be clinically important.

Modulation of soluble auxin-binding proteins in soybean cell suspensions.

Rapidly growing cell suspensions of soybean were analyzed for the presence of cytoplasmic high-affinity binding sites for auxin. Cytosol preparations were studied in lag, log and early stationary phase of the growth cycle. Two binding sites were detected, which show some similarities with binding sites previously reported from etiolated pea epicotyls. While the number of both sites declined in the cytoplasm during the growth cycle, the number of one of the two sites increased at the onset of rapid cell divisions. In parallel, both sites exhibited an increase in binding affinity during the growth cycle. The data will be discussed in relation to other reports on soluble auxin binding as well as to possible physiological functions.

Testing strategies in behavioral teratology: IV. Review and general conclusions.

Three laboratories collaborated to evaluate and compare different test concepts to be used for routine testing in behavioral teratology. Rat dams were treated orally with methylmercury starting two weeks prior to pairing until weaning of their offspring. In the first laboratory, the usual reproduction parameters were assessed in the dams, and all offspring (96-103 per group) were subjected to a routine developmental and behavioral test battery. After termination of the tests a random selection of these animals was further tested by multiparametric automated techniques in the other two laboratories. In one of these laboratories 12-13 males and females per group (one of each per litter) were tested in a visual discrimination reversal schedule, using nose-poking as operand. In the other laboratory the free behavioral of eight randomly selected males per dose group was studied in a wheel-shaped activity monitor with respect to locomotion magnitude and structure. In the same laboratory eight other males per group were trained in a discrete trial spatial alternation schedule, using lever press as operand. Both the developmental and behavioral testing battery as well as the automated techniques showed some significant effects in the offspring even at the low dose, where no reproduction effects had been noted. However, whereas the testing battery results were of unspecific nature, results of the automated techniques gave precise and specific information. In order to obtain optimal information, it is proposed to combine both approaches for routine testing. This combination could be achieved by using a balanced testing battery in young pups followed by an appropriate operant conditioning schedule in selected young adults.

Testing strategies in behavioral teratology: II. Discrimination learning.

Male and female Wistar rats exposed to methylmercury chloride prenatally via drinking water (1.5 and 5.0 mg/l) were tested in a microcomputer-directed learning task (visual discrimination reversal) at the age of two months. Differences were observed between control and high dose group for several parameters, the most obvious being an increase in passiveness and in response latency, as well as a decrease in intertrial interval response rates in the methylmercury group. No effects were seen in the low dose group. Performances of male and female animals were quite similar. However, females showed longer response latencies and passiveness scores were somewhat higher than in males.

Antiinflammatory Compounds from Plagiorhegma dubium Cell Culture1.

Successive fractionation of a crude methanolic extract of cultured PLAGIORHEGMA DUBIUM cells, followed by assay of the biological activity using cobra venom factor induced paw oedema, led to the detection and isolation of three compounds with antiinflammatory activity. The substances were identified as the protoberberine alkaloid jatrorrhizine and the lignaneglucosides dehydrodiconiferyl-alcohol-4-beta- D-glucoside and its isomer dehydrodiconiferyl-alcohol-gamma-beta- D-glucoside, the latter a compound not previously described in the literature.