Satellite rats are redundant in embryo-fetal development studies.

Routinely in many laboratories, satellite rats are added to embryo-fetal development (EFD) studies for pharmaceuticals to assess toxicokinetic (TK) properties, because it is assumed that collection of multiple blood samples with relatively large volumes might affect the study outcome. With recent refinement of blood sampling techniques, this belief requires reevaluation. The current work showed successful implementation of jugular vein blood sampling in an EFD rat study without satellite animals, thereby reducing the number of rats in standard EFD studies for pharmaceuticals by 20%. Although not evaluated in this study, microsampling has shown to be very successful and eliminates the need of satellite animals. However, currently not all laboratories have implemented this method and regularly the bioanalytical method is already developed with a limit of quantification that is insufficiently sensitive. Therefore in those cases, a quick win to omit satellite animals can be established by using jugular vein blood sampling.

Effects of Capillary Microsampling on Toxicological Endpoints in Juvenile Rats.

Blood sampling during juvenile rat toxicology studies is required to determine the toxicokinetic (TK) profile of compounds. Juvenile rats are too small to undergo repeated blood sampling using conventional methods, which collect 200-300 µl blood at each time point. Recently, capillary microsampling (CMS) gained interest because sample sizes are almost 10 times smaller enabling multi-sample collection from 1 rat. Here, we evaluated the use of CMS in juvenile rats in support of reduced animal usage. Juvenile rats at postnatal day (PND) 4, 10, and 17 underwent CMS via the submandibular, tail, and jugular veins. The CMS methods for pups at different ages were evaluated based on sample quality and technical practicality as well as on acute and chronic changes of toxicological parameters. The best location for CMS was the submandibular vein for PND 4 and 10 pups and the tail vein for PND 17 pups. No effects were found on clinical signs, body and organ weights and biochemistry parameters when 2 × 32 µl of blood was withdrawn from PND 4 pups or when 3 × 32 µl was taken from PND 10 and 17 pups within 24 h. Significant changes in several hematology parameters were observed 24 h after CMS due to a decrease of red blood cells and renewed production. These values had recovered to normal 7 days after CMS. CMS is feasible in juvenile rats for TK assessment. Utilizing this method could decrease the number of additional animals by 75%.

Update of OECD DART guidelines with endocrine disruptor relevant endpoints: Practical considerations.

In 1998, the OECD initiated a high-priority project aimed at revising existing test guidelines and developing new test guidelines for screening of potential endocrine disruptors. In 2011, OECD 443 was adopted, and in 2015 OECD 421 and OECD 422 were updated with endocrine disruptor relevant endpoints. A feasibility study for the enhancement of OECD 414 with endocrine disruptor relevant endpoints is currently ongoing. The addition of these endpoints is considered crucial for gaining more information on endocrine disruptor potency of tested chemicals, however it should be noted that these additions have a major impact on the study designs and give rise to several practical challenges. The aim of this review is to discuss important aspects of these challenging study designs and to share our knowledge on their implementation in our laboratory. Together, this review can be used as guidance for other laboratories, study monitors and registration officers.

Visualizing Compound Distribution during Zebrafish Embryo Development: The Effects of Lipophilicity and DMSO.

The predictability of the zebrafish embryo model is highly influenced by internal exposure of the embryo/larva. As compound uptake is likely to be influenced by factors such as lipophilicity, solvent use, and chorion presence, this article focuses on investigating their effects on compound distribution within the zebrafish embryo. To visualize compound uptake and distribution, zebrafish embryos were exposed for 96 hr, starting at 4 hr postfertilization, to water-soluble dyes: Schiff's reagent (logP -4.63), Giemsa stain (logP -0.77), Van Gierson stain (logP 1.64), Cresyl fast violet (logP 3.5), Eosine Y (logP 4.8), Sudan III (logP 7.5), and Oil red O (logP 9.81), with and without 1% dimethyl-sulfoxide (DMSO). Three additional compounds were used to analytically determine the uptake and distribution: Acyclovir (logP -1.56), Zidovudine (logP 0.05), and Metoprolol Tartrate Salt (logP 1.8). Examinations were performed every 24 hr. Both methods (visualization and specific analysis) showed that exposure to higher logP values results in higher compound uptake. Specific analysis showed that for lipophilic compounds >90% of compound is taken up by the embryo. For hydrophilic compounds, >90% of compound within the complete egg could not be associated to embryo or chorion and is probably distributed into the perivitelline space. Overall, internal exposure analyses on at least two occasions (i.e., before and after hatching) is crucial for interpretation of zebrafish embryotoxicity data, especially for compounds with extreme logP values. DMSO did not affect exposure when examined with the visualization method, however, this method might be not sensitive enough to draw hard conclusions.

From cutting edge to guideline: A first step in harmonization of the zebrafish embryotoxicity test (ZET) by describing the most optimal test conditions and morphology scoring system.

In the last couple of years, the interest in the zebrafish embryotoxicity test (ZET) for use in developmental toxicity assessment has been growing exponentially. This is also evident from the recent proposal for updating the ICHS5 guideline. The methodology of the ZET used by the different groups varies greatly. To further evaluate its successfulness and to take the ZET to the next level, harmonization of procedures is crucial. In the present study, based on literature and empirical data, the most optimal study design regarding temperature, test chamber, exposure period, presence of chorion, solvent use, exposure method, choice of concentrations, and teratogenic classification is proposed. Furthermore, our morphology scoring system is reported in detail as protocol to further enhance study design harmonization.

The underestimated value of OECD 421 and 422 repro screening studies: putting it in the right perspective.

To assess the efficacy of reproduction/developmental screening studies (OECD 421 and 422), a retrospective evaluation of 134 studies was performed. The major findings were: (1) for up to half of the studies with developmental and reproductive toxicity, these effects would have been missed in other types of studies, which underscores that reproduction/developmental screening studies should not be waived by default based on negative 28-day and/or prenatal developmental data, (2) the required number of animals as stated in the guidelines, is appropriate for detecting developmental and reproductive toxicity, and (3) adding measurements like anogenital distance, internal sex determination and nipple retention, plus extending the postnatal period would add predictive value. Overall, the current reproduction/developmental screening studies are effective in providing unique data, especially considering the limited number of animals used. Some simple additions would enrich its value in risk assessment even further.

Subchronic and reproductive/developmental (screening level) toxicity of complexation products of iron trichloride and sodium tartrate (FemTA).

A complexation/reaction product, termed FemTA, of sodium tartrate [D(-)- and L(+)-tartaric acid and mesotartaric acid], sodium hydroxide, and iron trichloride may have use as an anticaking agent in salt preparations. FemTA is composed of about 4% sodium tartrate, approximately 10% mesotartaric acid, approximately 7% chloride, approximately 4% iron, approximately 7% sodium, approximately 0.3% sodium oxalate, and approximately 65% water. FemTA was tested in a 90-d oral toxicity study, which included a screening level reproductive/developmental toxicity phase, in Harlan Wistar rats. FemTA was administered by oral gavage at 500, 1000, and 2000 mg/kg body weight/d prior to and during mating, or about 20, 40, or 80 mg of iron/kg body weight/d, such that males received 90/91 d of treatment and females 104 to 109 d. Treatment was associated with inflammatory lesions of the lower GI tract at the mid- and high-dose levels, increased liver and kidney weights, increased serum bile acids and blood urea nitrogen, decreased chloride, and changes to hematological parameters consistent with inflammation. The effects were considered the result of iron overload. There were no effects on reproductive/developmental toxicity parameters. The no-observed-adverse-effect level (NOAEL), based on gastrointestinal tract effects was 500 mg/kg body weight/d. The NOAEL for reproductive/developmental toxicity was 2000 mg/kg body weight/d, the highest dose tested.

Differential performance of Wistar Han and Sprague Dawley rats in behavioral tests: differences in baseline behavior and reactivity to positive control agents.

Developmental neurotoxicity (DNT) testing assesses potentially adverse effects on the developing nervous system. The present DNT study was conducted to generate historical data with the Wistar Han (WH) and Sprague Dawley (SD) rat strains, commonly used in Europe and the US, respectively. Potential differences between these strains in DNT endpoints have not been extensively investigated. Motor activity, startle response, learning and memory testing, and neurological (quantitative and qualitative) examinations were conducted using three groups of control, prenatally exposed (to Methylazoxymethanol [MAM] on gestation Day 15) and acutely treated (with IDPN, MK-801 or Chlorpromazine) animals for each strain. The positive controls produced clear effects in most endpoints investigated, with limited functional differences in baseline behavior and positive control sensitivity. However, SD rats were considerably more susceptible to MAM-induced learning and memory impairments and neurological damage. These data highlight differential sensitivity between the strains, which may require risk assessment consideration for developmental neurotoxicants.

The role of annoyance in the relation between transportation noise and children's health and cognition.

On the basis of this study it cannot be ruled out that the appraisal of the noise affects the association between air and road traffic noise exposure and children's health and cognition. However, the conclusion is limited due to the relatively small group of annoyed children, which may have influenced our group comparisons. Furthermore, the observed relation between annoyance and perceived health is possibly biased due to the fact that both were measured within the same questionnaire. These are the main conclusions of a cross-sectional multi-center study carried out among 2,844 schoolchildren (age 9-11 years) attending 89 primary schools around three European airports. The aim was to investigate how annoyance affects the relation between air and road traffic noise exposure and children's health and cognition. Different, sometimes competing, working mechanisms of how noise affects children's health are suggested. Some effects are supposed to be precipitated through (chronic) stress, while others may arise directly. There is still no theory that can adequately account for the circumstances in which noise will affect cognitive performance.

Neurobehavioral effects of transportation noise in primary schoolchildren: a cross-sectional study.

BACKGROUND: Due to shortcomings in the design, no source-specific exposure-effect relations are as yet available describing the effects of noise on children's cognitive performance. This paper reports on a study investigating the effects of aircraft and road traffic noise exposure on the cognitive performance of primary schoolchildren in both the home and the school setting. METHODS: Participants were 553 children (age 9-11 years) attending 24 primary schools around Schiphol Amsterdam Airport. Cognitive performance was measured by the Neurobehavioral Evaluation System (NES), and a set of paper-and-pencil tests. Multilevel regression analyses were applied to estimate the association between noise exposure and cognitive performance, accounting for demographic and school related confounders. RESULTS: Effects of school noise exposure were observed in the more difficult parts of the Switching Attention Test (SAT): children attending schools with higher road or aircraft noise levels made significantly more errors. The correlational pattern and factor structure of the data indicate that the coherence between the neurobehavioral tests and paper-and-pencil tests is high. CONCLUSIONS: Based on this study and previous scientific literature it can be concluded that performance on simple tasks is less susceptible to the effects of noise than performance on more complex tasks.

To mate or not to mate: a retrospective analysis of two-generation studies for evaluation of criteria to trigger additional mating in the extended one-generation design.

An extended one-generation study is proposed to replace the standard two-generation study, and would eliminate breeding F1 animals unless triggered by effects on parental reproduction or pup development. Nine two-generation studies in rats were reviewed to determine whether reproductive or developmental toxicity was more pronounced in the F2 generation. Three studies lacked reproductive effects, one had reproductive toxicity only in parental animals, three had mostly equivalent effects between generations, and two showed greater toxicity in the second generation. In each study with relevant effects, criteria proposed by Cooper et al. [Cooper RL, Lamb IV JC, Barlow SM, Bently K, Brady AM, Doerrer NG, et al. A tiered approach to life stages testing for agricultural chemical safety assessment. Crit Rev Toxicol 2006;36:69-98.] were applied to determine whether additional breeding would be triggered in the extended one-generation design. Additional mating was triggered for all but one study with more pronounced F1 reproductive toxicity. Thus, while the extended one-generation design may be a useful substitution for the two-generation study, more stringent criteria must first be developed to determine whether additional mating is actually needed.

Effects of perinatal exposure to PCBs on neuropsychological functions in the Rotterdam cohort at 9 years of age.

PCBs are known for their neurotoxic properties, especially on the developing brain. To increase insight into the neurotoxic effects of PCB exposure, the authors studied the effects of perinatal exposure to environmental levels of these compounds on different neuropsychological domains. In 9-year-old children of the Rotterdam PCB--dioxin cohort, higher prenatal PCB levels were associated with longer response times (RTs), more variation in RTs, and lower scores on the Tower of London (TOL; Shallice, 1982). A longer breast-feeding duration was associated with lower TOL scores and with better spatial organizational skills. There was some evidence of negative effects of lactational exposure to PCBs on scores on the TOL.