Perinatal ethinyl oestradiol alters mammary gland development in male and female Wistar rats.

Increased attention is being paid to human mammary gland development because of concerns for environmental influences on puberty onset and breast cancer development. Studies in rodents have showed a variety of changes in the mammary glands after perinatal exposure to endocrine disrupting chemicals, indicating progressed development of mammary glands when exposed to oestrogens early in life. However, laboratories use different parameters to evaluate the development of mammary glands, making studies difficult to compare. Moreover, studies of whole mounts in Wistar rats are lacking. In the present study, Wistar rats were exposed to 0, 5, 15 or 50 µg/kg of ethinyl oestradiol per day during gestation and lactation. A wide range of morphological parameters were evaluated in whole mounts of mammary glands from male and female offspring PD21-22. This study showed that in both male and female pre-pubertal Wistar rats, mammary gland development was accelerated after perinatal oestrogen exposure with increase in size, density and number of terminal end buds (TEBs). In female rats, the most sensitive parameters were the distance to the fifth gland, the relative growth towards the lymph node and the overall density. The sensitive endpoints in male rats were TEB numbers, both in the whole gland and in the zone C, the overall- and the highest density. The overall density was sensitive in both male and female rats and was considered a good representative of both branching and budding of the gland. The number of TEBs in zone C was representative of the number of TEBs in the whole gland. Further studies in older Wistar rats and with weak oestrogenic compounds could be performed to validate mammary gland examination as an endpoint in reproductive toxicity studies and to examine how early life environmental exposures may alter mammary gland development, disrupt lactation and alter susceptibility to breast cancer.

Mixtures of endocrine disrupting contaminants modelled on human high end exposures: an exploratory study in rats.

By diminishing the action of androgens during gestation, certain chemicals can induce irreversible demasculinization and malformations of sex organs in the male rat after gestational exposure. Studies with mixtures of such anti-androgens have shown that substantial combined effects occur even though each individual chemical is present at low, ineffective doses, but the effects of mixtures modelled based on human intakes have not previously been investigated. To address this issue for the first time, we selected 13 chemicals for a developmental mixture toxicity study in rats where data about in vivo endocrine disrupting effects and information about human exposures was available, including phthalates, pesticides, UV-filters, bisphenol A, parabens and the drug paracetamol. The mixture ratio was chosen to reflect high end human intakes. To make decisions about the dose levels for studies in the rat, we employed the point of departure index (PODI) approach, which sums up ratios between estimated exposure levels and no-observed-adverse-effect-level (NOAEL) values of individual substances. For high end human exposures to the 13 selected chemicals, we calculated a PODI of 0.016. As only a PODI exceeding 1 is expected to lead to effects in the rat, a total dose more than 62 times higher than human exposures should lead to responses. Considering the high uncertainty of this estimate, experience on lowest-observed-adverse-effect-level (LOAEL)/NOAEL ratios and statistical power of rat studies, we expected that combined doses 150 times higher than high end human intake estimates should give no, or only borderline effects, whereas doses 450 times higher should produce significant responses. Experiments indeed showed clear developmental toxicity of the 450-fold dose in terms of increased nipple retention (NR) and reduced ventral prostate weight. The 150-fold dose group exhibited significantly increased NR. These observations suggest that highly exposed population groups, especially women of reproductive age, may not be protected sufficiently against the combined effects of chemicals that affect the hormonal milieu required for normal male sexual differentiation.

Dietary Inflammatory Index and Cross-Sectional Associations with Inflammation, Muscle Mass and Function in Healthy Old Adults.

IMPORTANCE: Inflammaging is considered a driver of age-related loss of muscle mass and function (sarcopenia). As nutrition might play a role in this process, the Dietary Inflammatory Index® (DII) has been developed to quantify the inflammatory potential of an individual diet. OBJECTIVES: We aimed to examine associations between the DII, inflammation, oxidative stress and sarcopenia-related parameters in healthy old compared to young adults. DESIGN, SETTING, AND PARTICIPANTS: This cross-sectional study included data of 79 community-dwelling, healthy old adults (65-85 years) and 59 young adults (18-35 years) who participated in a randomized controlled trial from April to December 2019. MEASUREMENTS: The DII was computed with dietary data collected from 24-h recall interviews. Associations between the DII, inflammatory and oxidative stress markers as well as bioimpedance-derived body composition, handgrip strength and gait speed were determined with multiple linear regression analyses adjusted for age, sex, physical activity and insulin resistance. RESULTS: Regression analyses revealed significant relationships between a higher interleukin (IL) 6 and IL-6:IL-10-ratio and higher percentage fat mass (%FM), waist-to-height-ratio (WHtR) as well as lower percentage skeletal muscle mass (%SMM) and gait speed exclusively in old adults. Subsequent analyses showed that IL-6 was associated with a pro-inflammatory diet as indicated by a higher DII, again exclusively in old adults (beta coefficient (ß)= 0.027, standard error (SE) 0.013, p=0.037). While the DII was not related with handgrip strength or oxidative stress in neither old nor young adults, linear models confirmed that a higher DII was inversely associated with gait speed in old participants (ß= -0.022, SE 0.006, p<0.001). Finally, a pro-inflammatory diet was significantly associated with higher %FM, WHtR and lower %SMM in both age groups. CONCLUSION AND RELEVANCE: A pro-inflammatory diet reflected by the DII is associated with higher systemic inflammation, slower gait speed as well as lower muscle mass in old adults. Intervention studies are needed to examine whether anti-inflammatory dietary approaches can help to improve muscle mass and function and thus minimize the risk for sarcopenia in the long-term.

Combined exposure to endocrine disrupting pesticides impairs parturition, causes pup mortality and affects sexual differentiation in rats.

Risk assessment is currently based on the no observed adverse effect levels (NOAELs) for single compounds. Humans are exposed to a mixture of chemicals and recent studies in our laboratory have shown that combined exposure to endocrine disrupters can cause adverse effects on male sexual development, even though the doses of the single compounds are below their individual NOAELs for anti-androgenic effects. Consequently, we have initiated a large project where the purpose is to study mixture effects of endocrine disrupting pesticides at low doses. In the initial range-finding mixture studies, rats were gavaged during gestation and lactation with five doses of a mixture of the fungicides procymidone, mancozeb, epoxyconazole, tebuconazole and prochloraz. The mixture ratio was chosen according to the doses of each individual pesticide that produced no observable effects on pregnancy length and pup survival in our laboratory and the dose levels used ranged from 25 to 100% of this mixture. All dose levels caused increased gestation length and dose levels above 25% caused impaired parturition leading to markedly decreased number of live born offspring and high pup perinatal mortality. The sexual differentiation of the pups was affected at 25% and higher as anogenital distance was affected in both male and female offspring at birth and the male offspring exhibited malformations of the genital tubercle, increased nipple retention, and decreased prostate and epididymis weights at pup day 13. The results show that doses of endocrine disrupting pesticides, which appear to induce no effects on gestation length, parturition and pup mortality when judged on their own, induced marked adverse effects on these endpoints in concert with other pesticides. In addition, the sexual differentiation of the offspring was affected. This as well as the predictability of the combination effects based on dose-additivity modelling will be studied further in a large dose-response study.

Endocrine-disrupting properties in vivo of widely used azole fungicides.

The endocrine-disrupting potential of four commonly used azole fungicides, propiconazole, tebuconazole, epoxiconazole and ketoconazole, were tested in two short-term in vivo studies. Initially, the antiandrogenic effects of propiconazole and tebuconazole (50, 100 and 150 mg/kg body weight/day each) were examined in the Hershberger assay. In the second study, pregnant Wistar rats were dosed with propiconazole, tebuconazole, epoxiconazole or ketoconazole (50 mg/kg/day each) from gestational day (GD) 7 to GD 21. Caesarian sections were performed on dams at GD 21. Tebuconazole and propiconazole demonstrated no antiandrogenic effects at doses between 50 and 150 mg/kg body weight/day in the Hershberger assay. In the in utero exposure toxicity study, ketoconazole, a pharmaceutical to treat human fungal infections, decreased anogenital distance and reduced testicular testosterone levels, demonstrating a demasculinizing effect on male fetuses. Tebuconazole, epoxiconazole and ketoconazole induced a high-frequency of post-implantation loss, and both ketoconazole and epoxiconazole caused a marked increase in late and very late resorptions. Overall the results show that many of the commonly used azole fungicides act as endocrine disruptors in vivo, although the profile of action in vivo varies. As ketoconazole is known to implicate numerous endocrine-disrupting effects in humans, the concern for the effects of the other tested azole fungicides in humans is growing.

Combined exposure to anti-androgens causes markedly increased frequencies of hypospadias in the rat.

The incidence of hypospadias is increasing in young boys, but it remains unclear whether human exposure to endocrine disrupting chemicals plays a role. Risk assessment is based on estimation of no-observed-adverse-effect levels for single compounds, although humans are exposed to combinations of several anti-androgenic chemicals. In a mixture (MIX) study with three androgen receptor antagonists, vinclozolin, flutamide and procymidone, rats were gavaged during gestation and lactation with several doses of a MIX of the three chemicals or the chemicals alone. External malformations of the male reproductive organs were assessed on PND 47 using a score from 0 to 3 (normal to marked) for hypospadias. Markedly increased frequencies were observed after exposure to a MIX of the three chemicals compared to administration of the three chemicals alone. Anogenital distance at PND 1, nipple retention at PND 13, and dysgenesis score at PND 16 were highly correlated with the occurrence of hypospadias, and MIX effects were seen at doses where each of the individual chemicals caused no observable effects. Therefore, the results indicate that doses of anti-androgens, which appear to induce no hypospadias when judged on their own, may induce a very high frequency of hypospadias when they interact in concert with other anti-androgens.

EDC IMPACT: Reduced sperm counts in rats exposed to human relevant mixtures of endocrine disrupters.

Human semen quality is declining in many parts of the world, but the causes are ill defined. In rodents, impaired sperm production can be seen with early life exposure to certain endocrine-disrupting chemicals, but the effects of combined exposures are not properly investigated. In this study, we examined the effects of early exposure to the painkiller paracetamol and mixtures of human relevant endocrine-disrupting chemicals in rats. One mixture contained four estrogenic compounds; another contained eight anti-androgenic environmental chemicals and a third mixture contained estrogens, anti-androgens and paracetamol. All exposures were administered by oral gavage to time-mated Wistar dams rats (n = 16-20) throughout gestation and lactation. In the postnatal period, testicular histology was affected by the total mixture, and at the end of weaning, male testis weights were significantly increased by paracetamol and the high doses of the total and the anti-androgenic mixture, compared to controls. In all dose groups, epididymal sperm counts were reduced several months after end of exposure, i.e. at 10 months of age. Interestingly, the same pattern of effects was seen for paracetamol as for mixtures with diverse modes of action. Reduced sperm count was seen at a dose level reflecting human therapeutic exposure to paracetamol. Environmental chemical mixtures affected sperm count at the lowest mixture dose indicating an insufficient margin of safety for the most exposed humans. This causes concern for exposure of pregnant women to paracetamol as well as environmental endocrine disrupters.

Low-dose effects of bisphenol A on mammary gland development in rats.

Bisphenol A (BPA) is widely used in food contact materials, toys, and other products. Several studies have indicated that effects observed at doses near human exposure levels may not be observed at higher doses. Many studies have shown effects on mammary glands at low doses of BPA, however, because of small number of animals or few doses investigated these data have not been used by EFSA as point of departure for the newly assessed tolerable daily intake (TDI). We performed a study with perinatal exposure to BPA (0, 0.025, 0.25, 5, and 50 mg/kg bw/day) in rats (n = 22 mated/group). One of the aims was to perform a study robust enough to contribute to the risk assessment of BPA and to elucidate possible biphasic dose-response relationships. We investigated mammary gland effects in the offspring at 22, 100, and 400 days of age. Male offspring showed increased mammary outgrowth on pup day (PD) 22 at 0.025 mg/kg BPA, indicating an increased mammary development at this low dose only. Increased prevalence of intraductal hyperplasia was observed in BPA females exposed to 0.25 mg/kg at PD 400, but not at PD 100, and not at higher or lower doses. The present findings support data from the published literature showing that perinatal exposure to BPA can induce increased mammary growth and proliferative lesions in rodents. Our results indicate that low-dose exposure to BPA can affect mammary gland development in male and female rats, although higher doses show a different pattern of effects. The observed intraductal hyperplasia in female rats could be associated with an increased risk for developing hyperplastic lesions, which are parallels to early signs of breast neoplasia in women. Collectively, current knowledge on effects of BPA on mammary gland at low doses indicates that highly exposed humans may not be sufficiently protected.

Multiple Endocrine Disrupting Effects in Rats Perinatally Exposed to Butylparaben.

Parabens comprise a group of preservatives commonly added to cosmetics, lotions, and other consumer products. Butylparaben has estrogenic and antiandrogenic properties and is known to reduce sperm counts in rats following perinatal exposure. Whether butylparaben exposure can affect other endocrine sensitive endpoints, however, remains largely unknown. In this study, time-mated Wistar rats (n = 18) were orally exposed to 0, 10, 100, or 500 mg/kg bw/d of butylparaben from gestation day 7 to pup day 22. Several endocrine-sensitive endpoints were adversely affected. In the 2 highest dose groups, the anogenital distance of newborn male and female offspring was significantly reduced, and in prepubertal females, ovary weights were reduced and mammary gland outgrowth was increased. In male offspring, sperm count was significantly reduced at all doses from 10 mg/kg bw/d. Testicular CYP19a1 (aromatase) expression was reduced in prepubertal, but not adult animals exposed to butylparaben. In adult testes, Nr5a1 expression was reduced at all doses, indicating persistent disruption of steroidogenesis. Prostate histology was altered at prepuberty and adult prostate weights were reduced in the high dose group. Thus, butylparaben exerted endocrine disrupting effects on both male and female offspring. The observed adverse developmental effect on sperm count at the lowest dose is highly relevant to risk assessment, as this is the lowest observed adverse effect level in a study on perinatal exposure to butylparaben.

Low-dose effect of developmental bisphenol A exposure on sperm count and behaviour in rats.

Bisphenol A is widely used in food contact materials and other products and is detected in human urine and blood. Bisphenol A may affect reproductive and neurological development; however, opinion of the European Food Safety Authority (EFSA) on bisphenol A (EFSA J, 13, 2015 and 3978) concluded that none of the available studies were robust enough to provide a point of departure for setting a tolerable daily intake for bisphenol A. In the present study, pregnant Wistar rats (n = 17-21) were gavaged from gestation day 7 to pup day 22 with bisphenol A doses of 0, 25 µg, 250 µg, 5 mg or 50 mg/kg bw/day. In the offspring, growth, sexual maturation, weights and histopathology of reproductive organs, oestrus cyclicity and sperm counts were assessed. Neurobehavioural development was investigated using a behavioural testing battery including tests for motor activity, sweet preference, anxiety and spatial learning. Decreased sperm count was found at the lowest bisphenol A dose, that is 25 µg/kg/day, but not at the higher doses. Reproductive organ weight and histology were not affected and no behavioural effects were seen in male offspring. In the female offspring, exposure to 25 µg/kg bw/day bisphenol A dose resulted in increased body weight late in life and altered spatial learning in a Morris water maze, indicating masculinization of the brain. Decreased intake of sweetened water was seen in females from the highest bisphenol A dose group, also a possible sign of masculinization. The other investigated endpoints were not significantly affected. In conclusion, the present study using a robust experimental study design, has shown that developmental exposure to 25 µg/kg bw/day bisphenol A can cause adverse effects on fertility (decreased sperm count), neurodevelopment (masculinization of spatial learning in females) and lead to increased female body weight late in life. These results suggest that the new EFSA temporary tolerable daily intake of 4 µg/kg bw/day is not sufficiently protective with regard to endocrine disrupting effects of bisphenol A in humans.

Low-dose developmental exposure to bisphenol A alters the femoral bone geometry in wistar rats.

BACKGROUND: Bisphenol A (BPA) is a chemical produced in large volumes for use in manufacturing of consumer products and industrial applications, and an endocrine disruptor known to affect several hormonal systems. Bone produces hormones and is additionally a sensitive hormone target tissue, and is thus potentially sensitive to low doses of endocrine disruptors such as BPA, especially during development. METHODS: 110 pregnant Wistar rats were gavaged with 0; 25 µg; 250 µg; 5000 µg or 50,000 µg BPA/kg bodyweight (bw)/day from gestational day 7 until weaning at postnatal day 22. The three-month-old offspring were sacrificed and right femurs collected for length measurements, geometrical measurements by peripheral quantitative computed tomography (pQCT), as well as for analyses of biomechanical properties using the three-point-bending method. RESULTS: The femur was elongated in female offspring of dams exposed to 25 or 5000 µg BPA/kg bw/day (1.8% and 2.1%, respectively), and increased cortical thickness (4.7%) was observed in male offspring of dams exposed to 25 µg BPA/kg bw/day, compared to controls (p < 0.005). The biomechanical properties of the bone were not significantly altered. CONCLUSIONS: In utero and lactational exposure to the lowest BPA dose used in this study altered femoral geometry in both male and female offspring. This was observed at 25 µg BPA/kg bw/day, a dose lower than the Human Equivalent Dose (HED) applied by EFSA to set a temporary TDI (609 µg BPA/kg bw/day), and far lower than the No-Observed-Adverse-Effect-Level (NOAEL) (5000 µg BPA/kg bw/day) on which the US FDA TDI is based.

Burden of disease and costs of exposure to endocrine disrupting chemicals in the European Union: an updated analysis.

A previous report documented that endocrine disrupting chemicals contribute substantially to certain forms of disease and disability. In the present analysis, our main objective was to update a range of health and economic costs that can be reasonably attributed to endocrine disrupting chemical exposures in the European Union, leveraging new burden and disease cost estimates of female reproductive conditions from accompanying report. Expert panels evaluated the epidemiologic evidence, using adapted criteria from the WHO Grading of Recommendations Assessment, Development and Evaluation Working Group, and evaluated laboratory and animal evidence of endocrine disruption using definitions recently promulgated by the Danish Environmental Protection Agency. The Delphi method was used to make decisions on the strength of the data. Expert panels consensus was achieved for probable (>20%) endocrine disrupting chemical causation for IQ loss and associated intellectual disability; autism; attention deficit hyperactivity disorder; endometriosis; fibroids; childhood obesity; adult obesity; adult diabetes; cryptorchidism; male infertility, and mortality associated with reduced testosterone. Accounting for probability of causation, and using the midpoint of each range for probability of causation, Monte Carlo simulations produced a median annual cost of €163 billion (1.28% of EU Gross Domestic Product) across 1000 simulations. We conclude that endocrine disrupting chemical exposures in the EU are likely to contribute substantially to disease and dysfunction across the life course with costs in the hundreds of billions of Euros per year. These estimates represent only those endocrine disrupting chemicals with the highest probability of causation; a broader analysis would have produced greater estimates of burden of disease and costs.

The calcium antagonist nifedipine inhibits the uptake of acetylated LDL into endothelial cells.

The uptake of LDL and modified LDL into macrophages via specific receptors is one of the crucial steps during atherogenesis. Recently, similar receptors for acetylated and oxidized LDL were characterized in endothelial cells. It is known that dihydropyridine calcium antagonists may attenuate the formation of atherosclerotic plaques presumably by an increased nitric oxide (NO) release from endothelial cells. Therefore, we investigated whether the uptake of acetylated LDL into endothelial cells may be altered by the calcium antagonist nifedipine with special emphasis on the NO metabolism. Treatment of porcine endothelial cell cultures with nifedipine induced a significant concentration-dependent inhibition of the uptake of acetylated LDL. This effect could be completely prevented by coincubation with L-nitro-N-arginine, a competitive NO synthase inhibitor. Treatment with the NO donor SNAP resulted in a similar significantly reduced uptake of acetylated LDL. To test whether this effect is due to an NO-mediated cGMP mechanism, we incubated cells with 8-bromo-cGMP and coincubated cells with nifedipine and the soluble guanylyl cyclase inhibitor ODQ. 8-bromo-cGMP partly mimicked the nifedipine effect and ODQ partly reversed the nifedipine effect but not to a significant extent. Therefore, we conclude that the calcium antagonist nifedipine inhibits the uptake of acetylated LDL into endothelial cells via an NO- but presumably not by a cGMP-mediated process, which may possibly contribute to the antiatherogenic action of this drug.

An approach to risk assessment.

A strategy for delineating risk factors from use of neurotoxic chemicals was applied to the Danish working environment. An analysis using this strategy disclosed the need for internationally adopted criteria for neurotoxicity, and consequently a working group was established by the Nordic Council of Ministers with the task to propose criteria for neurotoxicity. Functional effects on the nervous system, such as reduction in memory and learning ability, decrease in attention, and alteration of behavior due to toxic chemicals in the environment is now being acknowledged as an important public health problem. This change in concern from obvious effects of high dose exposure to the more subtle effects of environmental exposure to neurotoxicants was incorporated in the criteria. The new criteria for evaluating neurotoxicity data has subsequently been used on 79 common industrial chemicals. The results indicate that numerous persons are exposed in the working as well as in the general environment to several chemicals, for which almost no data on the effect on subtle neurophysiological functions are available. Development of an approach to risk assessment dealing with this problem is a major challenge in the nineties. Different approaches to risk assessment are discussed, the quality of the databases available for hazard assessment are evaluated, and the needs for further research are identified.

Long-lasting neurobehavioral effects of prenatal exposure to xylene in rats.

The persistence of neurobehavioral effects in female rats (Mol:WIST) exposed to 500 ppm technical xylene (dimethylbenzene, CAS-no 1330-20-7) for 6 hours per day on days 7-20 of prenatal development was studied. The dose level was selected so as not to induce maternal toxicity or decreased viability of offspring. Investigations of learning and memory abilities were performed using a Morris water maze. This task requires rats to spatially navigate, using distal extramaze cues to locate a small platform under the surface of the water in a large pool. At the age of 16 weeks, the exposed offspring showed impairments when the platform was relocated in the pool. Impaired performances after platform relocation were also observed in exposed offspring at 28 and 55 weeks of age, although the difference was not statistically significant at 55 weeks. These data could indicate that the effect was partly reversible, although over a long time period. However, another explanation could be that the animals became more practised at solving the problem (finding the platform) as continued testing occurred and therefore were able to compensate for the neurotoxic effect of the prenatal xylene exposure. Further studies are planned to investigate whether neurobehavioral effects resulting from prenatal xylene exposure can interact with neurophysiological aging processes.

Toxicity of the styrene metabolite, phenylglyoxylic acid, in rats after three months' oral dosing.

Male Wistar rats were dosed with 0, 1250, 3750 or 5000 mg/l of phenylglyoxylic acid (PGA) (CAS no. 611-73-4) in the drinking water ad libitum for 3 months. During the entire treatment period, there were no gross signs of toxicity related to PGA. No changes in neurobehavior were found after using a functional observational battery or radial arm maze. An increased relative kidney weight was seen in the highest dose-group (Controls: 0.504 +/- 0.031 g/100 g b.wt.; 5000 mg PGA/l: 0.579 +/- 0.033 g/100 g b.wt.; p<0.01). No other organ weights were affected. Histopathology revealed no change in kidney structure. No changes in clinical biochemistry. In the highest dose-group three animals out of ten showed reduction in peripheral nerve myelin sheath thickness. No such changes were seen in the control group. The study revealed no changes in auditory brain stem response but minor changes in electroretinography. The noradrenaline (NA) concentration decreased in pons and thalamus whereas it increased in medulla oblongata and whole brain. The dopamine (DA) concentration increased in cerebellum, hippocampus, pons, and whole brain. The most marked DA increase was seen in hippocampus (Controls: 0.56 +/- 0.10 nmol/g tissue; 5000 mg/l: 1.04 +/- 0.11 nmol/g tissue; p<0.001). The 5-hydroxytryptamine (5-HT) concentration decreased in cerebellum, cerebral cortex, hippocampus, and medulla oblongata, whereas it increased in thalamus. The yield of synaptosomal protein, synaptosomal NA, DA, and 5-HT concentrations, and DA uptake rate were not affected. When dosed males were mated with naive females, there were no differences between groups in the pregnancy rate, number of corpora luteae, implantations, live or dead fetuses, resorptions, preimplantation loss, or postimplantation loss. It is concluded that a part of the effects on kidney, peripheral nerves, and vision, which have previously been reported after exposure to styrene, might be induced by the styrene metabolite, PGA. If PGA has ototoxic effects in rats, the dosing in the present study is not sufficient to induce the necessary ototoxic concentration in blood. Alternatively, the ototoxicity of styrene, like toluene, may be caused the parent compound itself and not by a metabolite like PGA.

Current status of developmental neurotoxicity: regulatory view.

The need for developmental neurotoxicity testing has been recognized for decades and guidelines are available, as the USEPA guideline and the OECD draft TG 426. Regulatory testing of industrial chemicals for developmental neurotoxicity is required to some extent, especially for pesticides in the US. Until recently, however, developmental neurotoxicity testing of industrial chemicals has not been a clear regulatory requirement in EU, probably due to the lack of an accepted OECD TG. The revised EU Technical Guidance Document for Risk Assessment (EU-TGD) has now included the OECD draft TG 426 in the testing strategy for new and existing substances, and biocides. Hopefully, this will lead to an improved database for risk assessment of potential developmental neurotoxicants. However, the regulatory authorities and toxicologists will also be faced with the challenge that decisions have to be made concerning e.g. when testing should be requested, how testing should be performed, as well as evaluation of the results and the regulatory consequences. In this paper, these three issues will be discussed based on the recommendations given in the EU-TGD.

Effects of prenatal exposure to N-methylpyrrolidone on postnatal development and behavior in rats.

Pregnant rats (Mol:WIST) were exposed to 150 ppm N-methylpyrrolidone for 6 hours per day on gestation days 7-20. The dose level was selected so as not to induce maternal toxicity or decrease viability of offspring. In the preweaning period, the exposed offspring had a lower body weight and their physical development was delayed. Neurobehavioral evaluation of the male pups revealed no effects on basal functions of the central nervous system. The animals appeared normal and motor function (rotarod), activity level (open field), and performance in learning tasks with a low grade of complexity were similar in the two groups. However, in more difficult tasks such as the reversal procedure in Morris water maze and operant delayed spatial alternation (Skinner boxes), performance was impaired in exposed offspring.

Effects of prenatal exposure to xylene on postnatal development and behavior in rats.

The effects of prenatal exposure to the organic solvent xylene (dimethylbenzene, CAS-no 1330-20-7) on postnatal development and behavior in rats were studied. Pregnant rats (Mol:WIST) were exposed to 500 ppm technical xylene 6 h per day on gestation days 7-20. The dose level was selected so as not to induce maternal toxicity or decrease the viability of offspring. In the exposed offspring, a delay in the ontogeny of the air righting reflex, a lower absolute brain weight, and impaired performance in behavioral tests for neuromotor abilities (Rotarod) and for learning and memory (Morris water maze) were found. Generally, the effects were most marked in the female offspring.

Effects of prenatal exposure to toluene on postnatal development and behavior in rats.

Development and neurobehavioral effects of prenatal exposure to toluene (CAS 108-88-3) were studied after exposing pregnant rats (Mol:WIST) to 1800 ppm of the solvent for 6 h daily on days 7-20 of gestation. Body weights of exposed offspring were lower until day 10 after parturition. Neurobehavioral evaluation of the pups revealed no effects on motor function (rotarod), activity level (open field), acoustic startle, and prepulse inhibition. Measurements of hearing function using auditory brain stem response revealed small effects in male-exposed offspring. Performance in a Morris water maze during initial learning gave some indications of impaired cognitive functions, which was confirmed during further testing, especially in reversal and new learning. Effects on cognitive functions seemed most marked in female offspring.