No Adverse Effect Level Research Articles

An assessment of the reliability of 52 enhanced preliminary embryofetal development studies to detect developmental toxicity.

Preliminary embryofetal development (pEFD) data from two species are currently recommended before inclusion of women of child-bearing potential (WOCBP) in clinical trials in Europe or Japan, but not before trials in the United States. The ICH S5(R3) guideline advises an "enhanced" study design for this purpose. The reliability of pEFD studies was assessed by comparing the outcome of 52 enhanced pEFD studies (25 rat, 23 rabbit, and 4 mouse) with the results of the definitive nonclinical EFD assessment. Four pEFD studies revealed severe developmental hazard without the need for a main EFD study. Only one pEFD study failed to detect drug-related teratogenicity or pregnancy failure subsequently detected in the main study. There were, however, some false positive and some equivocal pEFD study results. Of the 48 pEFD studies for which a main EFD study was performed, 16 (33%) failed to accurately predict (within two-fold) the no adverse effect level (NOAEL) for developmental toxicity subsequently defined in the main EFD study. Skeletal examination of fetuses in the pEFD study was necessary was to detect drug-induced malformations. One quarter (23%) of EFD investigations revealed malformations and/or pregnancy failure at one dose level or more. pEFD studies are effective for the detection of serious or irreversible effects on embryofetal development, provided that full fetal examinations are completed. They are not, however, sufficiently powered to reliably define the NOAEL for developmental toxicity. The regulatory impact of pEFD studies remains obscure since maximum pregnancy prevention precautions are required in clinical trials in all regions until the results of the main EFD studies are available.

Safety and toxicologic evaluation of Edible Pongamia Oil: A novel food ingredient

Edible Pongamia Oil (EPO) was evaluated in an acute oral toxicity study, GLP 14-Day and 90-Day repeated dose isocaloric dietary toxicity studies in rats, and in vitro Bacterial Reverse Mutation, and in vivo Mammalian Bone Marrow Chromosome Aberration genotoxicity studies for potential use as a food ingredient. In a non-GLP acute study, an LD50 > 5000 mg/kg was determined. Subacute 14-day repeated dose dietary administration of 0, 5, 10 and 15% oil revealed no adverse changes in clinical pathology, liver histology, body weight or weight gain, food consumption or food efficiency. In a 90-day dietary study fed 0, 2.5, 5.0, 7.5 and 10.0%, no mortalities, clinical or ophthalmologic signs, body weight, body weight gain, food consumption, food efficiency or Functional Observational Battery/Motor Activity changes occurred with EPO consumption, nor were there any adverse changes in hematology, clinical chemistry, coagulation, urinalysis, or thyroid hormone values. There were no adverse macroscopic, estrus cycle, histopathologic or spermatogenesis findings, or absolute or relative organ weight changes related to administration of EPO. The No-Adverse-Effect-Level (NOAEL) was 10% in the diet, the highest dose tested, equivalent to 5163 (male) and 6469 (female) mg/kg/day in rats. No mutagenic or clastogenic genotoxic potential was reported.

Safety Evaluation of Goat Milk Added with the Prebiotic Inulin Fermented with the Potentially Probiotic Native Culture Limosilactobacillus mucosae CNPC007 in Co-culture with Streptococcus thermophilus QGE: Analysis of Acute and Repeated Dose Oral Toxicity.

Despite functional goat milk products having emerged due to their importance for human nutrition and health, few studies have assessed the safety of consumption of goat dairy products containing potentially probiotic autochthonous lactic acid bacteria supplemented with prebiotic carbohydrates. Aiming this field, this study evaluated the safety of goat's milk fermented with Streptococcus thermophilus QGE, the autochthonous Limosilactobacillus mucosae CNPC007 culture, and the prebiotic inulin, through single- and repeated-dose oral toxicity tests (SDT and RDT, respectively) in animals. Ten female Swiss Webster mice were used for SDT evaluation - 2 groups, SDTc (20mL/kg of filtered water) and SDTt (20mL/kg of fermented milk) - and 40 Wistar rats for RDT - RDT3, RDT6, and RDT12 (treated with fermented milk at doses of 3mL/kg, 6mL/kg, and 12mL/kg, respectively) and also RDTc (12mL/kg of filtered water). For SDT, no signs of mortality or toxicity were observed, and the animals maintained the expected weight gain and feed intake. The RDT trials did not show mortality or signs of toxicity, as well as no change in body weight and organs, in the hematological and biochemical parameters, and also in relation to morphology and histology. Since the fermented milk did not cause any toxic effect in the conditions evaluated, it can be said that its no-adverse effect level (NOAEL) was considered to be higher than 20mL/kg/day. Thus, the fermented milk with L. mucosae CNPC007 and inulin was considered to be of low toxicity, safe for use in rodents, and allowed for use in further studies.

In Vivo Evaluation of the Oral Toxicity of the Chlorobutanol.

Chlorobutanol (CB) is used as a preservative in cosmetics and has antibacterial activity. This study investigated the single- and repeated-dose 28-day oral toxicity of a CB solvent in Sprague Dawley (SD) rats. For the single-dose oral toxicity study, a dose of 62.5, 125, or 250 mg per kg of body weight (mg/kg b.w.) of CB was given once orally via gavage. For the repeated-dose 28-day toxicity study, the high dose was set as 100 mg/kg b.w./day, and the middle, middle-low, and low doses were set to 50, 25, and 12.5 mg/kg b.w./day, respectively. Body weight was not significantly changed in the repeated-dose toxicity study. Relative liver and kidney weights were significantly increased in both sexes of the 100 mg/kg b.w./day treatment group. However, there were histopathological changes in liver and kidney for females and males, respectively. These data suggested that the approximate lethal dose (ALD) of CB was over 250 mg/kg b.w./day in the single-dose study, and the no adverse effect level (NOAEL) for CB was over 50 and 12.5 mg/kg b.w./day for female and male rats in the repeated-dose toxicity study.

IND-Enabling Studies of a Switchable Chimeric Antigen Receptor-T Cell (CLBR001+SWI019) to Support First in Human Clinical Study

Despite the tremendous clinical benefits, adverse events associated with CAR-T cell therapy remain a challenge. Most frequent adverse events are cytokine release and immune effector cell-associated neurotoxicity syndromes due to the inability to modulate the level of activity of current CAR-T cell products after administration to patients. Additional challenges include on target, off tumor toxicities and antigen loss mediated relapse of disease.To address these challenges, we have developed a “switchable” CAR-T (sCAR-T) where the activity of the sCAR-T cells is controlled by an antibody-based switch. The switch targets the tumor antigen, and the sCAR recognizes a unique peptide engrafted on the switch. The switch creates a bridge between the sCAR-T cell and the tumor cell, activating the sCAR-T cells and inducing tumor cell killing. Combined, the switch and sCAR-T cells afford complete elimination of tumors in xenograft and syngeneic models, but individually, each is designed to be inactive. A short half-life of the switch allows for a rapid modulation of sCART-cell activity through the switch dosing. Moreover, by swapping different switches, sCAR-T cells can be modularly redirected against other tumor targets. Further, we have shown the cyclical on/off stimulation of the sCAR-T cells affords improved memory and persistence of the sCAR-T cells. Here, we report IND-enabling studies for an optimized CD19-targeted switch (SWI019) and sCAR-T cell (CLBR001) to support a first in human (FIH) clinical study of the combination.The preclinical development of a platform which includes a sCAR-T cell that lacks any endogenous antigen target, in combination with an antibody-based switch molecule that lacks intrinsic activity in the absence of the sCAR-T cell, necessitated development of novel approaches. Fidelity of such a system is essential to control, thus, to confirm CLBR001 cells did not activate in the presence of normal tissues, in vitro activity studies were performed by co-culturing CLBR001 cells, in the presence or absence of SWI019, and a panel of 14 primary cells. This panel represented a survey of vital tissues throughout the body. CLBR001 did not demonstrate activity in any of the 14 cell types tested, supporting a high fidelity of CLBR001 recognition for SWI019.Because SWI019 lacks activity in the absence of CLBR001 cells, traditional toxicology studies to identify the no adverse effect level (NOAEL) in support of the first in human starting dose were not applicable. In such cases, a minimal anticipated biological effect level (MABEL) is commonly used to support starting human dose based on the predicted Cmax; however, the femtomolar-level in vitro activity of SWI019 in combination with CLBR001 resulted in starting doses that were modeled to be far outside of the range of potential clinical activity. Therefore, an in vivo-based approach to determine MABEL was developed. CLBR001 cells were administered in NSG mice bearing CD19+ Nalm-6 cell tumors and a single dose titration of SWI019 was performed. Comparison of the SWI019 efficacious dose (ED 50) values for anti-tumor activity, peripheral cytokines, and CLBR001 cells in peripheral blood demonstrated that reduction in Nalm-6 tumor burden was the most sensitive marker of activity. Extravasation of CLBR001 cells from peripheral blood and all three cytokines (IFN-γ, IL-2, and TNF-α) exhibited weaker ED 50 values. Therefore, antitumor activity (Nalm-6 tumor burden reduction) was chosen as the parameter for which to determine in vivo MABEL. Allometric scaling, using mouse and NHP SWI019 PK data, was used to model a SWI019 recommended dose in humans corresponding to the ED 20 of the in vivo MABEL study. Compared to the dose modelled using the in vitro MABEL approach, the in vivo MABEL approach afforded a first in human starting point which was ~13000-fold higher. We expect this approach provides an excellent starting point for the first in human study which balances safety and the potential for patient benefit.In summary, the results presented in this abstract led to the initiation of clinical trial NCT04450069, the combination of CLBR001+SWI019 for the treatment of relapse/ refractory B cell malignancies. DisclosuresStone: Abbvie: Current Employment, Current equity holder in publicly-traded company. Trikha: Abbvie: Current Employment, Current equity holder in publicly-traded company. Young: Abbvie: Research Funding; Qihan Bio: Membership on an entity's Board of Directors or advisory committees; Shoreline Bio: Membership on an entity's Board of Directors or advisory committees.

Combined Exposure to Diazinon and Nicotine Exerts a Synergistic Adverse Effect In Vitro and Disrupts Brain Development and Behaviors In Vivo.

A real-life environment during pregnancy involves multiple and simultaneous exposures to toxic chemicals. Perinatal exposures to toxic chemicals have been reported to exert an inhibitory effect on mouse neural development and behaviors. However, the effect of combined exposures of organophosphate and nicotine has not been previously reported. In this study, we investigated whether a combined exposure of diazinon and nicotine can have a synergistic effect. The effects of the combined chemical exposure on cell viability and neuronal differentiation were examined using mouse Sox1-GFP cells. Additionally, mice were maternally administered 0.18 mg/kg diazinon, a no adverse effect level (NOAEL) dose, combined with 0.4, 1, and 2 mg/kg nicotine. Mice offspring underwent behavior tests to assess locomotor, depressive, cognitive, and social behaviors. Morphological change in the brain was investigated with immunolocalization. We revealed that the combined exposure to diazinon and nicotine can have a synergistic adverse effect in vitro. In addition, the chemical-treated mouse offspring showed abnormalities in motor learning, compulsive-like behaviors, spatial learning, and social interaction patterns. Moreover, 0.18 mg/kg diazinon and 2 mg/kg nicotine co-exposure resulted in an increase in tyrosine hydroxylase (TH)-positive dopaminergic neurons. Thus, the findings suggest that perinatal co-exposure to nicotine and diazinon can result in abnormal neurodevelopment and behavior, even at low-level administration.

Toxicological Evaluation of Kenaf Seed Supercritical Carbon Dioxide-Oil in Male Sprague Dawley Rats

Kenaf seed supercritical carbon dioxide oil (KSO-SFE) is known for its numerous health benefits such as high antioxidant activity, lower plasma cholesterol level, chemopreventive to cervical and colon cancer. As there has been no report of toxicological evaluation, present study was conducted to assess the toxicity effects of KSO-SFE at 500, 1000, 1500 mg/kg body weight/day on male Sprague Dawley rats for 90 days. Parameters measured were body and organs weight, hematology, serum biochemistry, and histopathology of liver and kidneys. Induction with KSO-SFE showed lower creatinine level at all doses when compared with control group. Whereas lower plasma cholesterol level has been observed at 1000 and 1500 mg/kg bw/day, respectively. These slight changes were considered no toxicological significance. No mortality or treatment-related adverse effects in any of the parameters measured throughout the administration period even at the highest dose (1500 mg/kg bw). Based on these results, the no-adverse-effect level (NOAEL) for KSO-SFE under the condition of this study corresponds to the highest dose (1500 mg/kg bw/day).

Calculating safety margins using total plasma concentration versus unbound plasma concentration - does it make a difference?

Safety margin, a key aspect of any non-clinical toxicity studies, is calculated by dividing the systemic exposure (AUC) at NOAEL (No Adverse Effect Level) in toxicity studies by the clinical exposure. The validity of using total plasma concentration (Cp) to calculate AUC is often discussed, as it is the unbound plasma concentration (Cup) that elicits the pharmacological and toxicological effects. Data regarding plasma protein binding across species was collected for 114 MSD small molecule compounds which had been discontinued from development either due to non-clinical toxicity or due to clinical Adverse Effects. A >3-fold difference in unbound fraction in plasma (fup) was selected as a meaningful difference in plasma protein binding between non-clinical species and humans. In rats, dogs and non-human primates, approximately 3–5% of the compounds had a >3-fold difference in plasma protein binding than humans. Following assessment of toxicity profile of these compounds, it was concluded that calculation of safety margins after incorporating fup would have still led to the discontinuation of these compounds. Therefore, although fup can still be used for calculation of safety margin on a case by case basis, the routine use of fup for calculation of safety margins is not warranted.

Toxicologic evaluation of repetitive 4-week intravenous injections of midkine antisense oligonucleotide nanoliposomes in rats

Midkine antisense oligonucleotide (MK-ASODN) nanoliposomes have previously been shown to have inhibitory activity against hepatocellular carcinoma growth. Herein we report the 4-week sub-chronic toxicity of MK-ASODN nanoliposomes in SD rats. The adverse effects included loss of body weight gain and food consumption, peri-rhinal bleeding, piloerection, peri-anal filth, and kidney, liver, spleen, thymus, lung, and injection site lesions at high doses. Macroscopic changes were observed in the kidneys of the high-dose group, accompanied by a variation in urine protein and white blood cells, blood urea nitrogen, and serum creatinine. The increased spleen and liver coefficient, and the variation in circulating white blood cells, lymphocytes, and eosinophils in the high-dose group demonstrated that inflammation was caused by MK-ASODN nanoliposomes and was consistent with the macroscopic changes in the spleen and liver. The main necropsy findings of the animals that died were macroscopic changes in the lung. No severe toxic effects or mortalities occurred in the low- and medium-dose groups. However, a No Adverse Effect Level (NOAEL) was not identified since there were changes in organs deemed to be adverse at all dose levels. Thus, the maximum tolerated dose of MK-ASODN nanoliposomes for rats was considered to be 6 mg/kg/day.

Risk assessment for migration of styrene oligomers into food from polystyrene food containers

Regulation EU 10/2011 requires a risk assessment of Non Intentionally Added Substances (NIAS) migrating into food for food contact plastics within the EU. Styrene oligomers are important potential components of NIAS in polystyrene used for food packaging and so far only dimers and trimers have been identified. They are not genotoxic in vitro, and there is good evidence that they are not endocrine disruptors. Hazard characterization to establish “safe” exposure levels is based on 1. The No Adverse Effect Level (NOAEL) of 1 mg/kg bw/d in an oral rat study during pregnancy and lactation and 2. The concept of Threshold of Toxicological Concern (TTC). Likely human exposure is derived from 1. the concentrations of dimers and trimers in food simulants or 2. in food and 3. the probabilistic FACET exposure estimation based on dimer and trimer concentrations in polystyrene and their potential for migration. The Margin of Safety as the relation of potential consumer exposure and the “safe” exposure level was always above 1 (apart from migration with 95% ethanol which is no longer recommended as an official food simulant for overall migration into fatty food) demonstrating that dimers and trimers in PS food packaging present a low risk for consumers.

In vitro and in vivo safety evaluation of Nephure™

Nephure™ is a proprietary oxalate decarboxylase (OxDC) enzyme being developed as a food ingredient. In this study, the safety of Nephure™ was evaluated in a bacterial mutagenicity assay and in a sub-chronic (13-week) oral toxicity study in rats. Nephure™ did not show any mutagenic properties in the mutagenicity assay. In the 13-week sub-chronic oral toxicity study in which 10 Sprague Dawley rats per sex were administered 0, 118, 235 and 475 mg/kg bw/day (8260, 16450 and 33,250 Units/kg bw/day, respectively) of Nephure™ by gavage, male and female rats did not show any test article-related clinical observations or effects on body weight, body weight gain, food consumption, food efficiency, ophthalmology, functional observational battery parameters or motor activity. Furthermore, there were no changes in coagulation, clinical chemistry, urinalysis or hematology parameters, macroscopic/microscopic findings or organ weights that could be attributed to the test article. Based on these results, Nephure™ was not mutagenic and the no-adverse-effect level (NOAEL) in the 13-week study was determined to be 475 mg/kg bw/day (33,250 Units/kg bw/day). Evaluation of the estimated consumption of Nephure™, generation of the metabolite formate, and the current safety studies resulted in a conclusion of a tolerable upper limit of 3450 Units of OxDC activity/day (57.5 Units activity/kg bw/day), when Nephure™ is added to food to decrease dietary oxalate.

Acute and repeated dose (28 days) toxicity studies in rats and dogs of recombinant batroxobin, a snake venom thrombin-like enzyme expressed from Pichia pastoris

Recombinant batroxobin is a thrombin-like enzyme of Bothrops atrox moojeni venom. To evaluate its toxicological effect, it was highly expressed in Pichia pastorisand successfully purified to homogeneity from culture broth supernatant following Good Manufacturing Practice (GMP). The maximum tolerated dose of the recombinant batroxobin was examined in Sprague-Dawley (SD) rat and Beagle dogs following Good Laboratory Practice (GLP) regulations. The approximate lethal dose of recombinant batroxobin was 10 National Institute of Health (NIH) u/kg in male and female rats. Slight test substance-related effects were clearly in male and female dogs at more than 10 NIH u/kg. The maximum tolerated dose (MTD) was considered to be greater than 30 NIH u/kg in dogs. To investigate the repeated dose toxicity of batroxobin, the test item was intravenously administered to groups of SD rat and Beagle dog every day for 4 weeks. We observed that all animals survived the duration of the study without any effects on their mortality. There were no effects in both rats and dogs regarding their clinical signs, body weight, food consumption, ophthalmological examination, urinalysis, hematology, clinical chemistry, organ weightand gross post mortem examinations. The no adverse effect level (NOAEL) of recombinant batroxobin for both males and females is considered to be greater than 2.5 NIH u/kgin rats and 1 NIH u/kg in dogs, respectively. No toxic effects were noted in target organs. In conclusion, these results show a favorable preclinical profile and may contribute clinical development of recombinant batroxobin.

Paracelsus Revisited: The Dose Concept in a Complex World.

At the time that Paracelsus coined his famous dictum, 'What is there that is not poison? All things are poison and nothing is without poison. Solely the dose determines that a thing is not a poison', embryonic toxicology was a fairly focused discipline that mainly dealt with occupational poisonings and side effects of pharmaceuticals, such as mercury. While Paracelsus paved the way for the modern threshold concept and the no-adverse effect level, modern-day toxicology is now tussling with highly complex issues, such as developmental exposures, genetic predisposition and other sources of hypersusceptibility, multiple causes of underestimated toxicity, and the continuous presence of uncertainty, even in regard to otherwise well-studied mercury compounds. Further, the wealth of industrial chemicals now challenges the 'untested-chemical assumption', that the lack of documentation means that toxic potentials can be ignored. Unfortunately, in its ambition to provide solid evidence, toxicology has been pushed into almost endless replications, as evidenced by the thousands of toxicology publications every year that focus on toxic metals, including mercury, while less well-known hazards are ignored. From a public health viewpoint, toxicology needs to provide better guidance on decision-making under ever-present uncertainty. In this role, we need to learn from the stalwart Paracelsus the insistence on relying on facts rather than authority alone to protect against chemical hazards.

Acute and subchronic oral toxicity studies in rats with nanoscale and pigment grade titanium dioxide particles

Data generated using standardized testing protocols for toxicity studies generally provide reproducible and reliable results for establishing safe levels and formulating risk assessments. The findings of three OECD guideline-type oral toxicity studies of different duration in rats are summarized in this publication; each study evaluated different titanium dioxide (TiO2) particles of varying sizes and surface coatings. Moreover, each study finding demonstrated an absence of any TiO2 -related hazards. To briefly summarize the findings: 1) In a subchronic 90-day study (OECD TG 408), groups of young adult male and female rats were dosed with rutile-type, surface-coated pigment-grade TiO2 test particles (d50 = 145 nm − 21% nanoparticles by particle number criteria) by oral gavage for 90 days. The no-adverse-effect level (NOAEL) for both male and female rats in this study was 1000 mg/kg bw/day, the highest dose tested. The NOAEL was determined based on a lack of TiO2 particle-related adverse effects on any in-life, clinical pathology, or anatomic/microscopic pathology parameters; 2) In a 28-day repeated-dose oral toxicity study (OECD TG 407), groups of young adult male rats were administered daily doses of two rutile-type, uncoated, pigment-grade TiO2 test particles (d50 = 173 nm by number) by daily oral gavage at a dose of 24,000 mg/kg bw/day. There were no adverse effects measured during or following the end of the exposure period; and the NOAEL was determined to be 24,000 mg/kg bw/day; 3) In an acute oral toxicity study (OECD TG 425), female rats were administered a single oral exposure of surface-treated rutile/anatase nanoscale TiO2 particles (d50 = 73 nm by number) with doses up to 5000 mg/kg and evaluated over a 14-day post-exposure period. Under the conditions of this study, the oral LD50 for the test substance was >5000 mg/kg bw. In summary, the results from these three toxicity studies – each with different TiO2 particulate-types, demonstrated an absence of adverse toxicological effects. Apart from reporting the findings of these three studies, this publication also focuses on additional critical issues associated with particle and nanotoxicology studies. First, describing the detailed methodology requirements and rigor upon which the standardized OECD 408 guideline subchronic oral toxicity studies are conducted. Moreover, an attempt is made to reconcile the complex issue of particle size distribution as it relates to measurements of nanoscale and pigment-grade TiO2 particles. Clearly this has been a confusing issue and often misrepresented in the media and the scientific literature. It is clear that the particle-size distribution for pigment-grade TiO2, contains a small (“tail”) component of nanoscale particles (i.e., 21% by particle number and <1% by weight in the test material used in the 90-day study). However, this robust particle characterization finding should not be confused with mislabeling the test materials as exclusively in the nanoscale range. Moreover, based upon the findings presented herein, there appears to be no significant oral toxicity impact contributed by the nanoscale component of the TiO2 Test Material sample in the 90-day study. Finally, it seems reasonable to conclude that the study findings should be considered for read-across purposes to food-grade TiO2 particles (e.g., E171), as the physicochemical characteristics are quite similar.

Optimization and Performance Assessment of the Chorion-Off [Dechorinated] Zebrafish Developmental Toxicity Assay.

The Dechorinated Zebrafish Embryo Developmental toxicity assay was originally developed from a training set of 31 compounds and reported to be 87% concordant with in vivo teratogenicity data (Brannen, K. C., Panzica-Kelly, J. M., Danberry, T. L., and Augustine-Rauch, K. A. (2010). Development of a zebrafish embryo teratogenicity assay and quantitative prediction model. Birth Defects Res. 89, 66-77.). The assay includes scoring larva treated in a concentration range for malformations of specific morphological structures/organ systems. The model includes identifying a no-adverse-effect-level (NOAEL) and the concentration resulting in 25% lethality (LC25) at 5 days postfertilization. An LC25/NOAEL ratio ≥10 classifies a compound positive for teratogenic potential. A consortium effort evaluated a modified version of this assay which involved enzymatic chorion treatment instead of manual dissection and used experimental replicates for final classification. The modified assay achieved an 85% overall predictivity (Gustafson, A. L., Stedman, D. B., Ball, J., Hillegass, J. M., Flood, A., Zhang, C. X., Panzica-Kelly, J., Cao, J., Coburn, A., Enright, B. P., et al. (2012). Inter-laboratory assessment of a harmonized zebrafish developmental toxicology assay - progress report on phase I. Reprod. Toxicol. 33, 155-164.). The objective of this study was to perform a thorough performance evaluation of the dechorinated assay by repeating the original training set and testing additional compounds in experimental replicates. When the initial training set was repeated with inclusion of experimental replicates, the overall predictivity was 83%. Model performance was tested with an additional 34 compounds and achieved overall predictivity of 74%. When the training and test sets were combined (63 compounds) the assay's final sensitivity was 83% and the specificity was 71%. Total predictivity was 78% with relatively balanced predictivity for nonteratogens (77%) and teratogens (78%). The chorion-off assay achieved superior sensitivity (83%) compared with sensitivity (63-74%) reported by consortium efforts testing a similar assay with chorion-intact embryos (Ball, J. S., Stedman, D. B., Hillegass, J. M., Zhang, C. X., Panzica-Kelly, J., Coburn, A., Enright, B. P., Tornesi, B., Amouzadeh, H. R., Hetheridge, M., et al. (2014). Fishing for teratogens: a consortium effort for a harmonized zebrafish developmental toxicology assay. Toxicol. Sci. 139, 210-219.). Additional protocol modifications were made to increase assay throughput.

Benchmark Dose Calculation for Ordered Categorical Responses

The use of benchmark dose (BMD) calculations for dichotomous or continuous responses is well established in the risk assessment of cancer and noncancer endpoints. In some cases, responses to exposure are categorized in terms of ordinal severity effects such as none, mild, adverse, and severe. Such responses can be assessed using categorical regression (CATREG) analysis. However, while CATREG has been employed to compare the benchmark approach and the no-adverse-effect-level (NOAEL) approach in determining a reference dose, the utility of CATREG for risk assessment remains unclear. This study proposes a CATREG model to extend the BMD approach to ordered categorical responses by modeling severity levels as censored interval limits of a standard normal distribution. The BMD is calculated as a weighted average of the BMDs obtained at dichotomous cutoffs for each adverse severity level above the critical effect, with the weights being proportional to the reciprocal of the expected loss at the cutoff under the normal probability model. This approach provides a link between the current BMD procedures for dichotomous and continuous data. We estimate the CATREG parameters using a Markov chain Monte Carlo simulation procedure. The proposed method is demonstrated using examples of aldicarb and urethane, each with several categories of severity levels. Simulation studies comparing the BMD and BMDL (lower confidence bound on the BMD) using the proposed method to the correspondent estimates using the existing methods for dichotomous and continuous data are quite compatible; the difference is mainly dependent on the choice of cutoffs for the severity levels.

Domestic Duck Eggs: An Important Pathway of Human Exposure to PBDEs around E-Waste and Scrap Metal Processing Areas in Eastern China

Although consumption of local foods is recognized as an important pathway of human exposure to PBDEs in areas of China involved in rudimentary recycling of electronic waste (e-waste), dietary intake studies to date have not considered the contribution from consumption of duck eggs, despite being a common dietary component. Fresh duck eggs (n = 11) were collected from each of five farms located within 500 m of e-waste recycling workshops in the Wenling and Luqiao districts of Taizhou City, Eastern China, in November 2011, along with eggs from a control site located 90 km to the northeast. Average ΣPBDE yolk concentrations in eggs from the Taizhou farms ranged from 52.7 to 1778 ng/g lipid weight (8 ng/g lipid weight at the control site), at the high end of values previously reported for PBDEs in chicken eggs from the same locations and with BDE-209 predominant in over 60% of samples. Estimated typical adult daily ΣPBDE intakes due to consumption of duck eggs were in the range of 159-5124 ng/person per day. For the pentabrominated BDE-99 congener, estimated intakes from duck eggs alone were substantially above the no adverse effect level (NAEL) for impaired human spermatogenesis proposed by Netherlands researchers.

Metabolic disruption in male mice due to fetal exposure to low but not high doses of bisphenol A (BPA): Evidence for effects on body weight, food intake, adipocytes, leptin, adiponectin, insulin and glucose regulation

Exposure to bisphenol A (BPA) is implicated in many aspects of metabolic disease in humans and experimental animals. We fed pregnant CD-1 mice BPA at doses ranging from 5 to 50,000μg/kg/day, spanning 10-fold below the reference dose to 10-fold above the currently predicted no adverse effect level (NOAEL). At BPA doses below the NOAEL that resulted in average unconjugated BPA between 2 and 200pg/ml in fetal serum (AUC0–24h), we observed significant effects in adult male offspring: an age-related change in food intake, an increase in body weight and liver weight, abdominal adipocyte mass, number and volume, and in serum leptin and insulin, but a decrease in serum adiponectin and in glucose tolerance. For most of these outcomes non-monotonic dose–response relationships were observed; the highest BPA dose did not produce a significant effect for any outcome. A 0.1-μg/kg/day dose of DES resulted in some but not all low-dose BPA outcomes.

Introduction of risk size in the determination of uncertainty factor UFL in risk assessment

The methodology for using uncertainty factors in health risk assessment has been developed for several decades. A default value is usually applied for the uncertainty factor UFL, which is used to extrapolate from LOAEL (lowest observed adverse effect level) to NAEL (no adverse effect level). Here, we have developed a new method that establishes a linear relationship between UFL and the additional risk level at LOAEL based on the dose–response information, which represents a very important factor that should be carefully considered. This linear formula makes it possible to select UFL properly in the additional risk range from 5.3% to 16.2%. Also the results remind us that the default value 10 may not be conservative enough when the additional risk level at LOAEL exceeds 16.2%. Furthermore, this novel method not only provides a flexible UFL instead of the traditional default value, but also can ensure a conservative estimation of the UFL with fewer errors, and avoid the benchmark response selection involved in the benchmark dose method. These advantages can improve the estimation of the extrapolation starting point in the risk assessment.

Polybrominated diphenyl ethers exposure in breast milk in Shanghai, China: Levels, influencing factors and potential health risk for infants

The use of polybrominated diphenyl ethers (PBDEs) is increasing annually in China. Although several studies have reported significant contamination by these compounds in biological samples in China, little is known about the factors that may influence the levels of exposure and the potential health risk for infants. We investigated PBDE exposure levels in breast milk, its influencing factors and the potential health risk for infants in Shanghai, China by enrolling 48 healthy women between September 2006 and April 2007. PBDE exposure was assessed by analyses of seven PBDE congeners in breast milk. The estimated daily intake of PBDEs by infants via breastfeeding was determined for evaluating the potential health risk for infants. The median (range) of total PBDE content in breast milk was 8.81 (1.92–41.55)ng/g lipid. 34 (71%) of the 48 samples tested positive for all seven congeners. Maternal education level and household monthly salary were positively associated with PBDE concentration in breast milk (r=0.337, p=0.019 and r=0.436, p=0.004, respectively). The median of estimated daily intake of BDE-99 (6.67ng/kg/day) by infants via breastfeeding was below the human no adverse effect level (NAEL) set for BDE-99 for neurodevelopmental toxicity (18.8ng/kg/day). However, 3 (6%) of the samples exceeded the human NAEL. Taking the high detection rate of PBDEs in breast milk together with 6% of their EDI higher than NAEL in Shanghai, more studies on the effects of maternal PBDE exposure on fetal growth and child neurodevelopment are warranted.