Greet Schoeters

A roadmap for the development of alternative (non-animal) methods for systemic toxicity testing - t4 report

Systemic toxicity testing forms the cornerstone for the safety evaluation of substances. Pressures to move from traditional animal models to novel technologies arise from various concerns, including: the need to evaluate large numbers of previously untested chemicals and new products (such as nanoparticles or cell therapies), the limited predictivity of traditional tests for human health effects, duration and costs of current approaches, and animal welfare considerations. The latter holds especially true in the context of the scheduled 2013 marketing ban on cosmetic ingredients tested for systemic toxicity. Based on a major analysis of the status of alternative methods (Adler et al., 2011) and its independent review (Hartung et al., 2011), the present report proposes a roadmap for how to overcome the acknowledged scientific gaps for the full replacement of systemic toxicity testing using animals. Five whitepapers were commissioned addressing toxicokinetics, skin sensitization, repeated-dose toxicity, carcinogenicity, and reproductive toxicity testing. An expert workshop of 35 participants from Europe and the US discussed and refined these whitepapers, which were subsequently compiled to form the present report. By prioritizing the many options to move the field forward, the expert group hopes to advance regulatory science.

Birth weight and prenatal exposure to polychlorinated biphenyls (PCBs) and dichlorodiphenyldichloroethylene (DDE): A meta-analysis within 12 European birth cohorts

Objectives: Exposure to high concentrations of persistent organochlorines may cause fetal toxicity, but the evidence at low exposure levels is limited. Large studies with substantial exposure contrasts and appropriate exposure assessment are warranted. Within the framework of the EU (European Union) ENRIECO (ENvironmental Health RIsks in European Birth Cohorts) and EU OBELIX (OBesogenic Endocrine disrupting chemicals: LInking prenatal eXposure to the development of obesity later in life) projects, we examined the hypothesis that the combination of polychlorinated biphenyls (PCBs) and dichlorodiphenyldichloroethylene (DDE) adversely affects birth weight. Methods: We used maternal and cord blood and breast milk samples of 7,990 women enrolled in 15 study populations from 12 European birth cohorts from 1990 through 2008. Using identical variable definitions, we performed for each cohort linear regression of birth weight on estimates of cord serum concentration of PCB-153 and p,p´-DDE adjusted for gestational age and a priori selected covariates. We obtained summary estimates by meta-analysis and performed analyses of interactions. Results: The median concentration of cord serum PCB-153 was 140 ng/L (range of cohort medians 20–484 ng/L) and that of p,p´-DDE was 528 ng/L (range of cohort medians 50–1,208 ng/L). Birth weight decreased with increasing cord serum concentration of PCB-153 after adjustment for potential confounders in 12 of 15 study populations. The meta-analysis including all cohorts indicated a birth weight decline of 150 g [95% confidence interval (CI): –250, –50 g] per 1-µg/L increase in PCB-153, an exposure contrast that is close to the range of exposures across the cohorts. A 1-µg/L increase in p,p´-DDE was associated with a 7-g decrease in birth weight (95% CI: –18, 4 g). Conclusions: The findings suggest that low-level exposure to PCB (or correlated exposures) impairs fetal growth, but that exposure to p,p´-DDE does not. The study adds to mounting evidence that low-level exposure to PCBs is inversely associated with fetal growth.

Renal function, cytogenetic measurements, and sexual development in adolescents in relation to environmental pollutants: a feasibility study of biomarkers

BACKGROUND Human exposure to chemicals is normally monitored by measurement of environmental pollutants in external media. We investigated whether biomarkers in adolescents can show exposure to, and health effects of, common environmental pollutants. METHODS We recruited 200 17-year-old adolescents (120 girls) from a rural control area and from two suburbs polluted by a lead smelter and two waste incinerators. We measured biomarkers of exposure and of effect in blood and urine samples, and obtained questionnaire data. School doctors measured testicular volume and staged sexual maturation. FINDINGS Internal exposure was mostly within current standards. Concentrations of lead and cadmium in blood, PCBs (polychlorinated biphenyls) and dioxin-like compounds in serum samples, and metabolites of VOCs (volatile organic compounds) in urine were higher in one or both suburbs than in the control area. Children who lived near the waste incinerators matured sexually at an older age than others, and testicular volume was smaller in boys from the suburbs than in controls. Biomarkers of glomerular or tubular renal dysfunction in individuals were positively correlated with blood lead. Biomarkers of DNA damage were positively correlated with urinary metabolites of PAHs (polycyclic aromatic hydrocarbons) and VOCs. Interpretation Biomarkers can be used to detect environmental exposure to pollutants and measure their biological effects before overt disease develops. Our findings suggest that current environmental standards are insufficient to avoid measurable biological effects.

The Faroes statement: Human Health effects of developmental exposure to chemicals in our environment

The periods of embryonic, foetal and infant developmentare remarkably susceptible to environmental hazards. Toxicexposures to chemical pollutants during these windows ofincreased susceptibility can cause disease and disability ininfants, children and across the entire span of human life.Among the effects of toxic exposures recognized in the pasthave been spontaneous abortion, congenital malformations,lowered birthweight and other adverse effects. These outcomesmay be readily apparent. However, even subtle changes causedby chemical exposures during early development may leadto important functional deficits and increased risks ofdisease later in life. The timing of exposure during early lifehas therefore become a crucial factor to be considered intoxicological assessments.During 20–24 May 2007, researchers in the fields of environmentalhealth, environmental chemistry, developmentalbiology, toxicology, epidemiology, nutrition and paediatricsgathered at the International Conference on Fetal Programmingand Developmental Toxicity, in Torshavn, FaroeIslands. The conference goal was to highlight new insightsinto the effects of prenatal and early postnatal exposure tochemical agents, and their sustained effects on the individualthroughout the lifespan. The conference brought togetherresearchers to focus on human data and the translationof laboratory results to elucidate the environmental risks tohuman health.

Intrauterine Exposure to Environmental Pollutants and Body Mass Index during the First 3 Years of Life

Objective We investigated the association between body mass index (BMI) standard deviation score (SDS) and prenatal exposure to hexachlorobenzene, dichlorodiphenyldichloroethylene (DDE), dioxin-like compounds, and polychlorinated biphenyls (PCBs). Methods In this prospective birth cohort study, we assessed a random sample of mother–infant pairs (n = 138) living in Flanders, Belgium, with follow-up until the children were 3 years of age. We measured body mass index as standard deviation scores (BMI SDS) of children 1–3 years of age as well as pollutants measured in cord blood. Results DDE correlated with BMI SDS, with effect modification by maternal smoking and the child’s age. At 1 year, children of smoking mothers had higher BMI SDS than did children of nonsmoking mothers. At 3 years, this difference was reduced because of the faster rate of decline in BMI SDS in the former group. This relationship held except for children with high levels of DDE. DDE had a small effect on BMI SDS at 3 years of age in children of nonsmoking mothers (difference in BMI SDS for DDE concentrations between the 90th and 10th percentiles = 0.13). On the other hand, smoking enhanced the relation between DDE and BMI SDS at 3 years (difference in BMI SDS for DDE concentrations between the 90th and 10th percentiles = 0.76). Increasing concentrations of PCBs were associated with higher BMI SDS values at all ages (parameter estimate = 0.003 ± 0.001; p = 0.03). Conclusion In this study we demonstrated that intrauterine exposure to DDE and PCBs is associated with BMI during early childhood. Future studies are warranted to confirm our findings and to assess possible mechanisms by which these pollutants could alter energy metabolism.

Endocrine disruptors and abnormalities of pubertal development.

Onset and development of puberty is regulated by the neuroendocrine system. Population-based studies worldwide have observed secular trends towards earlier puberty development. These changes are apparently caused by environmental factors such as improved socio-economic status, improved health care and nutrition. However, they may also partly result from endocrine-disrupting chemicals in the environment. Epidemiological studies have investigated the relationship between pubertal development and exposure to endocrine-disrupting chemicals (polychlorinated biphenyls, polybrominated biphenyls, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, phthalate esters, furans and the pesticide endosulfan). Associations with both perinatal and postnatal exposure have been reported. Studies in experimental animals support some of these findings and point to differential endocrine regulatory mechanisms linked to pubertal development acting in the perinatal and the pre-pubertal period. Pubertal development is naturally associated with growth and body composition. There is increasing evidence for a link between prenatal development and pubertal onset. In girls born small for gestational age (SGA), pubertal onset and age at menarche often are advanced, especially if there has been an extensive catch-up growth during the first months of life. In utero growth retardation may have multiple causes including exposure to xenobiotic substances as was suggested for some endocrine-disrupting chemicals. An abnormal perinatal environment of children born SGA may alter the endocrine status and the sensitivity of the receptors for endocrine and metabolic signalling that may have effects on maturation of brain and gonads. However, the causal pathways and the molecular mechanisms that may link the pubertal growth pattern of children born SGA, pubertal development and endocrine-disrupting chemicals need further study.

Prevalidation of a model for predicting acute neutropenia by colony forming unit granulocyte/macrophage (CFU-GM) assay

This report describes an international prevalidation study conducted to optimise the Standard Operating Procedure (SOP) for detecting myelosuppressive agents by CFU-GM assay and to study a model for predicting (by means of this in vitro hematopoietic assay) the acute xenobiotic exposure levels that cause maximum tolerated decreases in absolute neutrophil counts (ANC). In the first phase of the study (Protocol Refinement), two SOPs were assessed, by using two cell culture media (Test A, containing GM-CSF; and Test B, containing G-CSF, GM-CSF, IL-3, IL-6 and SCF), and the two tests were applied to cells from both human (bone marrow and umbilical cord blood) and mouse (bone marrow) CFU-GM. In the second phase (Protocol Transfer), the SOPs were transferred to four laboratories to verify the linearity of the assay response and its interlaboratory reproducibility. After a further phase (Protocol Performance), dedicated to a training set of six anticancer drugs (adriamycin, flavopindol, morpholino-doxorubicin, pyrazoloacridine, taxol and topotecan), a model for predicting neutropenia was verified. Results showed that the assay is linear under SOP conditions, and that the in vitro endpoints used by the clinical prediction model of neutropenia are highly reproducible within and between laboratories. Valid tests represented 95% of all tests attempted. The 90% inhibitory concentration values (IC(90)) from Test A and Test B accurately predicted the human maximum tolerated dose (MTD) for five of six and for four of six myelosuppressive anticancer drugs, respectively, that were selected as prototype xenobiotics. As expected, both tests failed to accurately predict the human MTD of a drug that is a likely protoxicant. It is concluded that Test A offers significant cost advantages compared to Test B, without any loss of performance or predictive accuracy. On the basis of these results, we proposed a formal Phase II validation study using the Test A SOP for 16-18 additional xenobiotics that represent the spectrum of haematotoxic potential.

Cadmium and children: exposure and health effects.

Cadmium exposure and accumulation in the body start at young age. Exposure routes in children are mainly via food, environmental tobacco smoke and house dust. Excretion from the body is limited. Cadmium accumulation in the kidney is responsible for effects such as nephrotoxicity and osteoporosis which are observed at adult age. Cadmium exposure through inhalation is also associated with lung cancer in adulthood. Although transfer to the neonate through the placenta and through breast milk is limited, teratogenic and developmental effects were observed in experimental animals. The database on human studies involving children is limited, yet effects on motoric and perceptual behaviour in children have been associated with elevated in utero cadmium exposure. In school age children urinary cadmium levels were associated with immune suppressive effects. More studies are needed to confirm these results. Experimental data in vitro and in animals refer to effects of cadmium on the hypothalamus‐pituitary axis at different levels. This may lead to disorders of the endocrine and/or immune system. Cadmium exposure at early age should be limited as much as possible to prevent direct effects on children and to prevent accumulation of cadmium which may have serious health effects only becoming manifest at older age.

Health risk assessment of dioxin emissions from municipal waste incinerators: the Neerlandquarter (Wilrijk, Belgium)

Two municipal waste incinerators in the vicinity of a residential area close to the city of Antwerp caused concern to local habitants. Risk assessment was performed combining chemical, toxicological measurements and model calculations. As the first step in risk assessment an inventory was made of historic emissions from both incinerators with emphasis on dioxins. The operational atmospheric transport and deposition model for priority substances (OPS) was used to calculate the deposition of dioxins in the vicinity of incinerators. The observed soil contamination pattern did not correspond to the calculated deposition pattern, indicating that other sources may contribute at least partly to the local PCDD/PCDF contamination of the area. Dioxin exposure of people in the Neerlandquarter as a function of the food consumption behavior was calculated using a mathematical model (VLIER-HUMAAN) combined with transfer factors. According to the results of these calculations, just residing in the impact area does not result in a meaningful risk. Only if locally produced food was consumed (milk, meat and vegetables), exposure in the Neerlandquarter was enhanced compared to the average dioxin exposure estimated for the Flemish population. Exposure in 1997 was below the exposure in 1980. As a consequence of different eating habits and lower bodyweight, children are subjected to significantly higher exposure than adults. Adverse health outcomes from dioxin exposure in the past cannot be excluded. There was no evidence for enhanced exposure to genotoxicants based on a comparison of chromosomal damage to blood cells of children from the study area to those from a control group.

Comparative sensitivity of 20 bioassays for soil quality

Increasing evidence suggests that the use of a single bioassay will never provide a full picture of the quality of the environment. Only a test battery, composed of bioassays of different animal and plant species from different trophic levels will reduce uncertainty, allowing an accurate assessment of the quality of the environment. In the present study, a test battery composed of 20 bioassays of varying biological endpoints has been compared. Apart from lethality and reproductive failure in earthworms, springtails, nematoda, algae and vascular plants, these endpoints also included bioavailibility of metals (bacteria), heat-shock induction (nematodes, algae), DNA damage (bacteria, earthworm, vascular plants), beta-galactosidase (Daphnia) and esterase activity (algae) and a range of immunological parameters (earthworm). Four chemicals (cadmium, phenol, pentachlorophenol and trifluralin)--each representing a different toxic mode of action--were applied in a dilution series (from 1 mg/kg up to 1000 mg/kg) onto OECD standard soil. The tests have been performed both on these artificially contaminated soil samples and on aqueous extracts subsequently obtained from these soils. The results show that the immunological parameters and the loss of weight in the earthworms were among the most sensitive solid-phase assays. Esterase inhibition and heat-shock induction in algae were shown to be extremely sensitive when applied to soil extracts. As previously shown at the species level, no single biological endpoint was shown to be the most sensitive for all four modes of toxic action.