Inflammation and (secondary) genotoxicity of Ni and NiO nanoparticles

Abstract

Nanoparticle-induced genotoxicity can arise through different mechanisms, and generally, primary and secondary genotoxicity can be distinguished where the secondary is driven by an inflammatory response. It is, however, yet unclear how a secondary genotoxicity can be detected using in vitro methods. The aim of this study was to investigate inflammation and genotoxicity caused by agglomerated nickel (Ni) and nickel oxide (NiO) nanoparticles and, furthermore, to explore the possibility to test secondary (inflammation-driven) genotoxicity in vitro. As a benchmark particle to compare with, we used crystalline silica (quartz). A proteome profiler antibody array was used to screen for changes in release of 105 different cytokines and the results showed an increased secretion of various cytokines including vascular endothelial growth factor (VEGF) following exposure of macrophages (differentiated THP-1 cells). Both Ni and NiO caused DNA damage (comet assay) following exposure of human bronchial epithelial cells (HBEC) and interestingly conditioned media (CM) from exposed macrophages also resulted in DNA damage (2- and 3-fold increase for Ni and NiO, respectively). Similar results were also found when using a co-culture system of macrophages and epithelial cells. In conclusion, this study shows that it is possible to detect a secondary genotoxicity in lung epithelial cells by using in vitro methods based on conditioned media or co-cultures. Further investigation is needed in order to find out what factors that are causing this secondary genotoxicity and whether such effects are caused by numerous nanoparticles.     

Derivation of inhalation toxicity reference values for propylene oxide using mode of action analysis: Example of a threshold carcinogen

Propylene oxide (PO) is an important industrial chemical used primarily in the synthesis of other compounds. Inhalation carcinogenesis studies in rodents, with no-observed-adverse-effect levels (NOAELs) of 100 and 200?ppm, have revealed that chronic, high exposure to PO can induce tumors at the site of contact. Despite these characteristics, there is no evidence that typical environmental or occupational exposures to PO constitute a health risk for humans. The nongenotoxic effects of PO (glutathione depletion and cell proliferation) that augment its DNA-reactive and non-DNA-reactive genotoxicity are expected to be similar in humans and rodents. Available evidence on mode-of-action suggests that cancer induction by PO at the site of contact in rodents is characterized by a practical threshold. Human toxicity reference values for potential carcinogenic effects of PO were derived based on nasal tumors identified in rodent studies and specified uncertainty factors. The 95% lower confidence limit on the dose producing a 10% increase in additional tumor risk (LED10) was calculated using the rat and mouse data sets. The human reference values derived from the rat and mouse LED10 values were 0.7 and 0.5?ppm PO, respectively. A similar noncancer reference value, 0.4?ppm, was derived on the basis of non-neoplastic nasal effects in rats.     

Mechanisms of cobalt(II) uptake into V79 Chinese hamster cells

V79 Chinese hamster cells were used as a model for the characterization of the Co(II) uptake into mammalian cells as well as the mechanisms involved. Co(II) was taken up in a dose and time dependent manner. The uptake was exponential without saturation in the tested concentration range up to 400 M. CoCl₂. Furthermore, there was a high intracellular cobalt accumulation at elevated extra-cellular Co(II) doses (up to 16 fold at 200 M). The time course of Co(II) uptake showed a maximum after about 8–12 h with no further change after the longest tested incubation time (24 h). The uptake of Co(II) into V79 cells seems to be mediated by multiple mechanisms: active, energy consuming transport like ion pumps and endocytosis, since the Co(II) uptake was significantly reduced by ouabain (an inhibitor of the Na⁺/K⁺ATPase), N-ethyl-maleinimide (an inhibitor of the Ca²⁺/Mg²⁺ATPase and the Na⁺/K⁺ATPase), chlorpromazine (a calmodulin antagonist and inhibitor of the Ca²⁺/Mg²⁺ ATPase) as well as by the endocytosis inhibitor chloroquine. Furthermore, the two agents iodoacetate and potassium cyanide, which produce ATP depletion, resulted in a diminution of the intracellular cobalt concentration. An uptake through anion channels could be excluded, since 4,4-diisothiocyanostilbene-2,2-disulphonic acid was not inhibitory     

Multigenerational exposure to cobalt (CoCl2) and WCCo nanoparticles in Enchytraeus crypticus

Abstract

Cobalt and cobalt nanoparticles have many applications, for example, in the hard metal industry and in tires. The assessment of long term effects is crucial, as these materials are persistent. For many organism groups, multigenerational (MG) exposure is a highly relevant scenario for persistent materials. In this study, the biological effect of CoCl₂ (salt) and Tungsten Carbide Cobalt nanoparticles (WCCo NPs) exposure was assessed in an MG test (4 generations in spiked + 2 generations in clean soil) using the OECD/ISO standard soil test species Enchytraeus crypticus. To ensure transgenerational survival, sublethal concentrations were used to assess the MG impact. MG exposure did not increase toxicity (survival, reproduction). There was an increase in reproduction at low concentrations of Co. Materials were characterized in the exposure media and the organisms in terms of Co content. Uptake of Co-occurred from exposure to both CoCl₂ and WCCo, although without toxicity for WCCo. Cobalt from CoCl₂ exposure seemed to be stored, whereas for WCCo it was eliminated.     

Assessment of thyroid endocrine system impairment and oxidative stress mediated by cobalt ferrite (CoFe2O4) nanoparticles in zebrafish larvae

Fascinating super paramagnetic uniqueness of iron oxide particles at nano‐scale level make them extremely useful in the state of the art therapies, equipments, and techniques. Cobalt ferrite (CoFe₂O₄) magnetic nanoparticles (MNPs) are extensively used in nano‐based medicine and electronics, results in extensive discharge and accumulation into the environment. However, very limited information is available for their endocrine disrupting potential in aquatic organisms. In this study, the thyroid endocrine disrupting ability of CoFe₂O₄ NPs in Zebrafish larvae for 168‐h post fertilization (hpf) was evaluated. The results showed the elevated amounts of T4 and T3 hormones by malformation of hypothalamus pituitary axis in zebrafish larvae. These elevated levels of whole body THs leads to delayed hatching, head and eye malformation, arrested development, and alterations in metabolism. The influence of THs disruption on ROS production and change in activities of catalase (CAT), mu‐glutathione s‐transferase (mu‐GST), and acid phosphatase (AP) were also studied. The production of significantly higher amounts of in vivo generation of ROS leads to membrane damage and oxidative stress. Presences of NPs and NPs agglomerates/aggregates were also the contributing factors in mechanical damaging the membranes and physiological structure of thyroid axis. The increased activities of CAT, mu‐GST, and AP confirmed the increased oxidative stress, possible DNA, and metabolic alterations, respectively. The excessive production of in vivo ROS leads to severe apoptosis in head, eye, and heart region confirming that malformation leads to malfunctioning of hypothalamus pituitary axis. ROS‐induced oxidative DNA damage by formation of 8‐OHdG DNA adducts elaborates the genotoxicity potential of CoFe₂O₄ NPs. This study will help us to better understand the risk and assessment of endocrine disrupting potential of nanoparticles. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 2068–2080, 2016.     

Long-term exposures to low doses of cobalt nanoparticles induce cell transformation enhanced by oxidative damage

Abstract

A weak aspect of the in vitro studies devoted to get information on the toxic, genotoxic and carcinogenic properties of nanomaterials is that they are usually conducted under acute-exposure and high-dose conditions. This makes difficult to extrapolate the results to human beings. To overcome this point, we have evaluated the cell transforming ability of cobalt nanoparticles (CoNPs) after long-term exposures (12 weeks) to sub-toxic doses (0.05 and 0.1?µg/mL). To get further information on whether CoNPs-induced oxidative DNA damage is relevant for CoNPs carcinogenesis, the cell lines selected for the study were the wild-type mouse embryonic fibroblast (MEF Ogg1^+/+) and its isogenic Ogg1 knockout partner (MEF Ogg1^?/?), unable to properly eliminate the 8-OH-dG lesions from DNA. Our initial short-term exposure experiments demonstrate that low doses of CoNPs are able to induce reactive oxygen species (ROS) and that MEF Ogg1^?/? cells are more sensitive to CoNPs-induced acute toxicity and oxidative DNA damage. On the other hand, long-term exposures of MEF cells to sub-toxic doses of CoNPs were able to induce cell transformation, as indicated by the observed morphological cell changes, significant increases in the secretion of metalloproteinases (MMPs) and anchorage-independent cell growth ability, all cancer-like phenotypic hallmarks. Interestingly, such changes were significantly dependent on the cell line used, the Ogg1^?/? cells being particularly sensitive. Altogether, the data presented here confirms the potential carcinogenic risk of CoNPs and points out the relevance of ROS and Ogg1 genetic background on CoNPs-associated effects.     

A comparison of the effects of silver nanoparticles and silver nitrate on a suite of soil dwelling organisms in two field soils

Nanomaterials are increasingly used in a wide range of products, leading to growing concern of their environmental fate. In order to understand the fate and effects of silver nanoparticles in the soil environment, a suite of toxicity tests including: plant growth with Elymus lanceolatus (northern wheatgrass) and Trifolium pratense (red clover); collembolan survival and reproduction (Folsomia candida); and earthworm avoidance, survival and reproduction (Eisenia andrei) was conducted. The effect of silver nanoparticles (AgNP) was compared with the effect of ionic silver (as AgNO₃) in two agricultural field soils (a sandy loam and a silt loam). Lethal (LC50) or sub lethal (IC50) effect levels are presented for all endpoints and demonstrate that in most cases AgNO₃ (i.e. ionic silver) was found to be more toxic than the AgNP across test species. The difference in effects observed between the two forms of silver varied based on test species, endpoint and soil type. In tests that were conducted across different soil types, organisms in the sandier soil had a greater response to the Ag (ionic and nano) than those in soil with a high silt content. Earthworms (avoidance behavior and reproduction) were the most sensitive to both AgNP and AgNO₃, while plant emergence was the least sensitive endpoint to both forms of Ag. The use of a test battery approach using natural field soils demonstrates the need to better quantify the dissolution and transformation products of nanomaterials in order to understand the fate and effects of these materials in the soil environment.     

An investigation of cobalt toxicity on blood parameters and evaluation of deferasirox and desferrioxamine chelators in removing cobalt from biological system

Abstract

This experiment was undertaken to study the toxic effects of cobalt chloride in rats. Sixty-five rats weighing 207?±?7?g were used. Cobalt chloride was administrated orally in low and high doses for 90?d. The aim of study was to evaluate the effects of cobalt on blood (RBC, WBC, Hb, PLT and HCT) and to investigate the abilities of deferasirox (DFX) and desferrioxamine (DFO) as chelators in removing cobalt from the blood. Cobalt and iron concentrations were determined by flame atomic absorption spectrometer (FAAS). Results show that both of chelators are able to remove cobalt but DFX was more effective, whereas the combined therapy is more efficient.     

Genetic toxicity assessment: employing the best science for human safety evaluation Part VII: Why not start with a single test: a transformational alternative to genotoxicity hazard and risk assessment.

A transformational alternative for genotoxicity hazard and risk assessment is proposed to the current standard regulatory test battery. In principle, the proposed approach consists of a single in vitro test system with high genomic sequence homology to humans that addresses the relevant principal genetic lesions assessed in the current test battery. The single test system also possesses higher throughput attributes to permit the screening of large numbers of compounds and allow for an initial differentiation of genotoxic mechanisms (i.e., direct vs. indirect mechanisms) by how the hazard end point is measured. To differentiate compounds showing positive results, toxicogenomic analysis can be conducted to evaluate genotoxic mechanisms and further support risk assessment. Lastly, the results from the single test system can be followed up with a complementary in vivo assessment to establish mechanistic relevance at potential target tissues. Here, we propose the in vitro (yeast) DNA deletion (DEL) recombination assay as a single test alternative to the current genotoxicity test battery with a mechanistic follow up toxicogenomic analysis of genotoxic stress response as one approach that requires broader evaluation and validation. In this assay, intrachromosomal recombination events between a repeated DNA sequence lead to DNA deletions, which have been shown to be inducible by a variety of carcinogens including those both negative and positive in the standard Salmonella Ames assay. It is hoped that the general framework outlined along with this specific example will provoke broader interest to propose other potential test systems.     

A comparison of fate and toxicity of selenite,biogenically, and chemically synthesized selenium nanoparticles to zebrafish (Danio rerio) embryogenesis

Microbial reduction of selenium (Se) oxyanions to elemental Se is a promising technology for bioremediation and treatment of Se wastewaters. But a fraction of biogenic nano-Selenium (nano-Se^b) formed in bioreactors remains suspended in the treated waters, thus entering the aquatic environment. The present study investigated the toxicity of nano-Se^b formed by anaerobic granular sludge biofilms on zebrafish embryos in comparison with selenite and chemogenic nano-Se (nano-Se^c). The nano-Se^b formed by granular sludge biofilms showed a LC₅₀ value of 1.77?mg/L, which was 3.2-fold less toxic to zebrafish embryos than selenite (LC_50?=_?0.55?mg/L) and 10-fold less toxic than bovine serum albumin stabilized nano-Se^c (LC_50?=_?0.16?mg/L). Smaller (nano-Se^cs; particle diameter range: 25–80?nm) and larger (nano-Se^cl; particle diameter range: 50–250?nm) sized chemically synthesized nano-Se^c particles showed comparable toxicity on zebrafish embryos. The lower toxicity of nano-Se^b in comparison with nano-Se^c was analyzed in terms of the stabilizing organic layer. The results confirmed that the organic layer extracted from the nano-Se^b consisted of components of the extracellular polymeric substances (EPS) matrix, which govern the physiochemical stability and surface properties like ζ-potential of nano-Se^b. Based on the data, it is contented that the presence of humic acid like substances of EPS on the surface of nano-Se^b plays a major role in lowering the bioavailability (uptake) and toxicity of nano-Se^b by decreasing the interactions between nanoparticles and embryos.     

In vitro assessment of cobalt oxide particle toxicity: Identifying and circumventing interference

The continuing development of nanotechnology necessitates the reliable assessment of potential adverse health consequences associated with human exposures. The physicochemical properties of nanomaterials can be responsible for unexpected interactions with components of classical toxicity assays, which may generate erroneous interpretations.In this paper, we describe how particle interference can be observed in in vitro toxicity tests (CellTiter Blue, CyQUANT, WST-1 and CellTiter-Glo assay) and in cell biology tests using flow cytometry (cell cycle analysis). We used cobalt oxide (Co₃O₄) particles as an example, but these assays can be performed, in principle, regardless of the nanoparticle considered. We have shown that cobalt particles interfere with most of these tests. We adapted the protocol of the CellTiter-Glo assay to circumvent this interference and demonstrated that, using this protocol, the toxicity level is consistent with results obtained using the clonogenic assay, which is considered to be the reference test.Before assessing particle toxicity using in vitro toxicity tests, interference testing should be performed to avoid false interpretations. Furthermore, in some cases of interference, protocol adaptation can be considered to allow the reliable use of these quick and convenient in vitro tests.     

Cellular uptake and toxicity effects of silver nanoparticles in mammalian kidney cells

The rapid progress and early commercial acceptance of silver‐based nanomaterials is owed to their biocidal activity. Besides embracing the antimicrobial potential of silver nanoparticles (AgNPs), it is imperative to give special attention to the potential adverse health effects of nanoparticles owing to prolonged exposure. Here, we report a detailed study on the in vitro interactions of citrate‐coated AgNPs with porcine kidney (Pk15) cells. As uncertainty remains whether biological/cellular responses to AgNPs are solely as a result of the release of silver ions or whether the AgNPs themselves have toxic effects, we investigated the effects of Ag⁺ on Pk15 cells for comparison. Next, we investigated the cellular uptake of both AgNPs and Ag⁺ in Pk15 cells at various concentrations applied. The detected Ag contents in cells exposed to 50 mg l^?1 AgNPs and 50 mg l^?1 Ag⁺ were 209 and 25 µg of Ag per 10⁶ cells, respectively. Transmission electron microscopy (TEM) images indicated that the Pk15 cells internalized AgNPs by endocytosis. Both forms of silver, nano and ionic, decreased the number of viable Pk15 cells after 24 h in a dose‐dependent manner. In spite of a significant uptake into the cells, AgNPs had only insignificant toxicity at concentrations lower than 25 mg l^?1, whereas Ag⁺ exhibited a significant decrease in cell viability at one‐fifth of this concentration. The Comet assay suggested that a rather high concentration of AgNP (above 25 mg l^?1) is able to induce genotoxicity in Pk15 cells. Further studies must seek deeper understanding of AgNP behavior in biological media and their interactions with cellular membranes. Copyright © 2014 John Wiley & Sons, Ltd.     

Toxicological evaluation of metal oxide nanoparticles and mixed exposures at low doses using zebra fish and THP1 cell line

Metal and metal oxide nanoparticles are being used in different industries now‐a‐days leading to their unavoidable exposure to humans and animals. In the present study, toxicological testing was done using nanoparticles of copper oxide, cerium oxide and their mixture (1:1 ratio) on zebra fish embryos and THP‐1 cell line. Zebrafish embryos were exposed to 0.01 μg/ml to 50 μg/ml concentrations of dispersed nanoparticles using a 96 well plate and their effects were studied at different hours post fertilization (hpf) i.e. 0 hpf, 24 hpf, 48 hpf, 72 hpf and 96 hpf respectively. Results showed that copper oxide nanoparticles has drastic effects on the morphology and physiology of zebra fish whereas cerium oxide nanoparticles and mixture of these nanoparticles did not show much of the effects. Comparable results were obtained from in vitro study using human monocyte cell line (THP‐1). It is concluded that these nanoparticles may cause toxicological effects to humans and environment.     

Identification of the appropriate dose metric for pulmonary inflammation of silver nanoparticles in an inhalation toxicity study

A number of studies have shown that induction of pulmonary toxicity by nanoparticles of the same chemical composition depends on particle size, which is likely in part due to differences in lung deposition. Particle size mostly determines whether nanoparticles reach the alveoli, and where they might induce toxicity. For the risk assessment of nanomaterials, there is need for a suitable dose metric that accounts for differences in effects between different sized nanoparticles of the same chemical composition. The aim of the present study is to determine the most suitable dose metric to describe the effects of silver nanoparticles after short-term inhalation. Rats were exposed to different concentrations (ranging from 41 to 1105?µg silver/m³ air) of 18, 34, 60 and 160?nm silver particles for four consecutive days and sacrificed at 24?h and 7 days after exposure. We observed a concentration-dependent increase in pulmonary toxicity parameters like cell counts and pro-inflammatory cytokines in the bronchoalveolar lavage fluid. All results were analysed using the measured exposure concentrations in air, the measured internal dose in the lung and the estimated alveolar dose. In addition, we analysed the results based on mass, particle number and particle surface area. Our study indicates that using the particle surface area as a dose metric in the alveoli, the dose–response effects of the different silver particle sizes overlap for most pulmonary toxicity parameters. We conclude that the alveolar dose expressed as particle surface area is the most suitable dose metric to describe the toxicity of silver nanoparticles after inhalation.     

Cobalt chloride exposure dose‐dependently induced hepatotoxicity through enhancement of cyclooxygenase‐2 (COX‐2)/B‐cell associated protein X (BAX) signaling and genotoxicity in Wistar rats

Cobalt chloride (CoCl₂) is one of the many environmental contaminants, used in numerous industrial sectors. It is a pollutant with deadly toxicological consequences both in developing and developed countries. We investigated toxicological impact of CoCl₂ on hepatic antioxidant status, apoptosis, and genotoxicity. Forty Wistar rats were divided into four groups, 10 rats per group: Group 1 served as control and received clean tap water orally; Group 2 received CoCl₂ solution (150 mg/L); Group 3 received CoCl₂ solution (300 mg/L); and Group 4 received CoCl₂ (600 mg/L) in drinking water for 7 days, respectively. Exposure of rats to CoCl₂ led to a significant decline in hepatic antioxidant enzymes together with significant increase in markers of oxidative stress. Immunohistochemistry revealed dose‐dependent increase in cyclooxygenase‐2 and BAX expressions together with increased frequency of Micronucleated Polychromatic Erythrocytes. Combining all, CoCl₂ administration led to hepatic damage through induction of oxidative stress, inflammation, and apoptosis.     

Expression and Regulation of Xenobiotic-Metabolizing Cytochrome P450 (CYP) Enzymes in Human Lung

Pathogenesis of lung diseases, such as lung cancer and chronic obstructive pulmonary disease, is tightly linked to exposure to environmental chemicals, most notably tobacco smoke. Many of the compounds associated with these diseases require an enzymatic activation to exert their deleterious effects on pulmonary cells. These activation reactions are mostly catalyzed by cytochrome P450 (CYP) enzymes. Interindividual differences in the in situ activation and inactivation of chemical toxicants may contribute to the risk of developing lung diseases associated with these compounds. This review summarizes in detail the expression of individual CYP forms in human pulmonary tissue and gives a view on the significance of the pulmonary expression of CYP enzymes. The localization of individual CYP enzymes in various cell types of human lung and the emerging field of regulation of human pulmonary CYP enzymes are discussed. At least CYP1A1 (in smokers), CYP1B1, CYP2B6, CYP2E1, CYP2J2, and CYP3A5 proteins are expressed in human lung, and also other CYP forms are likely to be expressed. Xenobiotic-metabolizing CYP enzymes are mostly expressed in bronchial and bronchiolar epithelium, Clara cells, type II pneumocytes, and alveolar macrophages in human lung, although individual CYP forms have different patterns of localization in pulmonary tissues. Problems in animal to human lung toxicity extrapolation and several specific aspects requiring more detailed assessment are identified.     

Differential genotoxicity of chemical properties and particle size of rare metal and metal oxide nanoparticles

Nanoparticles of rare metal compounds are used in various products. However, their carcinogenicity and genotoxicity have not been sufficiently evaluated. The tumor-initiating and -promoting potentials of four rare metals, indium oxide (In2O3), dysprosium oxide (Dy2O3), tungsten oxide (WO3) and molybdenum (Mo), with a well-defined particle diameter were evaluated. The mutagenicity of these rare metals was investigated by Ames test using five bacteria strains, and transformability of these rare metals was investigated by cell-transformation assay using v-Ha-ras-transfected BALB/c 3T3 cells (Bhas 42 cells). Nano-sized Dy2O3 showed strong mutagenesis in all five bacteria strains tested with and without metabolic activation, while micro-sized particles showed weak mutagenesis in two bacterial strains. Dy2O3 induced transformation colonies of Bhas 42 cell dose-dependently, although there was no difference in the number of transformed foci between nano-sized and micro-sized particles. Nano-sized In2O3 and WO3 showed positive mutagenic response in TA1537 and TA98, respectively, whereas the micro-sized metal oxide particles showed no mutagenesis in the test bacterial strains. Both nano-sized and micro-sized In2O3 showed similar levels of transformability. However, nano-sized and micro-sized WO3 did not show any transformability. Both nano-sized and micro-sized Mo particles showed neither mutagenesis nor transformability. These results suggest that mutagenicity of rare metals depends on their particle size, although transformability depends on their chemical components but not on their particle size.     

Formaldehyde exposure and leukemia: Critical review and reevaluation of the results from a study that is the focus for evidence of biological plausibility

Abstract

A recent study (Zhang et al., 2010) has provided results attributed to aneuploidy in circulating stem cells that has been characterized as providing potential support for proposed mechanisms for formaldehyde to impact bone marrow. A critical review of the study, as well as a reanalysis of the underlying data, was performed and the results of this reanalysis suggested factors other than formaldehyde exposure may have contributed to the effects reported. In addition, although the authors stated in their paper that “all scorable metaphase spreads on each slide were analyzed, and a minimum of 150 cells per subject was scored,” this protocol was not followed. In fact, the protocol to evaluate the presence of monosomy 7 or trisomy 8 was followed for three or less samples in exposed workers and six or less samples in non-exposed workers. In addition, the assays used (CFU-GM) do not actually measure the proposed events in primitive cells involved in the development of acute myeloid leukemia. Evaluation of these data indicates that the aneuploidy measured could not have arisen in vivo, but rather arose during in vitro culture. The results of our critical review and reanalysis of the data, in combination with recent toxicological and mechanistic studies, do not support a mechanism for a causal association between formaldehyde exposure and myeloid or lymphoid malignancies.     

Developmental toxicity of polychlorinated biphenyls (PCBs): a systematic review of experimental data

Experimental reproductive and developmental toxicity studies with polychlorinated biphenyls (PCBs) are reviewed in brief to determine their relevance for current environmental exposure of humans during the prenatal and postnatal developmental periods. Additional material is published in electronic form only, which contains graphic overviews on individual PCBs and various mixtures that are linked with the relevant citations. In this comprehensive article we focus on interactions of PCBs with biological substrates that could mediate adverse effects observed in experimental animals and in children, and the shortcomings of many of the animal studies available. A main point of criticism involves the relative lack of animal data on several of those persistent congeners, either as individual compounds or as environmentally relevant mixtures, which are currently used as a measure of human exposure. Experimental studies in animals are frequently conducted with commercial PCB mixtures, a test design that does not reflect the exposure situation in humans. Important improvements of animal experiments could be achieved by more complete reporting of litter data (pre- and post-natal losses, toxic signs in the dam and the offspring, birth weights and postnatal growth data), the inclusion of endpoints that have been found previously to be affected by PCBs, and measurements of internal exposure data.An erratum to this article can be found at