Hexamoll® DINCH: Lack of in vivo evidence for obesogenic properties.

Hexamoll® DINCH is an important alternative to phthalate plasticizers. Although regulatory reviews have not identified any potential hazards even in sensitive populations, an in vitro study by Campioli et al. (2015) suggested Hexamoll® DINCH might alter fat storage in adipocytes resulting in obesity. To evaluate this hypothesis, data from studies with Hexamoll® DINCH were reviewed for evidence of deposition in fat, changes in body weight, or changes in serum chemistry reflecting altered metabolic status. Body weights of F1 and F2 pups in a two-generation study did not differ from controls even at 1000 mg Hexamoll® DINCH/kg body weight. Mean relative liver weights from the 1000 and 300 mg/kg bw groups were increased, but without histopathologic changes. Triglyceride and cholesterol levels in serum were not affected. In addition, subchronic and chronic studies in rats did not give evidence of an obesogenic effect. Radioactivity from 20 or 1000 mg/kg bw 14C-labelled Hexamoll® DINCH dosed orally remained 2-3 times longer in adipose tissue than in well-perfused tissues; however, levels were 20-500% below other tissues at 1 and 8 h post dosing. Radioactivity concentrations in organs and tissues excluding the GI tract declined rapidly and continuously, and decreased in parallel to the concentration in plasma during the following 20 h. Both, initial and terminal half-lives of radioactivity concentration do not indicate a potential for accumulation. Furthermore, a metabolomic comparison of Hexamoll® DINCH with DEHP and other phthalates shows complete separation of the metabolomic profile of these two chemical classes, meaning that their effects on the body and the body's reaction to the substance are different. Hence, comprehensive in vivo data do not show any evidence of Hexamoll® DINCH altering fat metabolism or having obesogenic properties.

Expert consensus on an in vitro approach to assess pulmonary fibrogenic potential of aerosolized nanomaterials.

The increasing use of multi-walled carbon nanotubes (MWCNTs) in consumer products and their potential to induce adverse lung effects following inhalation has lead to much interest in better understanding the hazard associated with these nanomaterials (NMs). While the current regulatory requirement for substances of concern, such as MWCNTs, in many jurisdictions is a 90-day rodent inhalation test, the monetary, ethical, and scientific concerns associated with this test led an international expert group to convene in Washington, DC, USA, to discuss alternative approaches to evaluate the inhalation toxicity of MWCNTs. Pulmonary fibrosis was identified as a key adverse outcome linked to MWCNT exposure, and recommendations were made on the design of an in vitro assay that is predictive of the fibrotic potential of MWCNTs. While fibrosis takes weeks or months to develop in vivo, an in vitro test system may more rapidly predict fibrogenic potential by monitoring pro-fibrotic mediators (e.g., cytokines and growth factors). Therefore, the workshop discussions focused on the necessary specifications related to the development and evaluation of such an in vitro system. Recommendations were made for designing a system using lung-relevant cells co-cultured at the air-liquid interface to assess the pro-fibrogenic potential of aerosolized MWCNTs, while considering human-relevant dosimetry and NM life cycle transformations. The workshop discussions provided the fundamental design components of an air-liquid interface in vitro test system that will be subsequently expanded to the development of an alternative testing strategy to predict pulmonary toxicity and to generate data that will enable effective risk assessment of NMs.

Toxicity of Hexamoll(®) DINCH(®) following intravenous administration.

Alternative plasticizers to di(2-ethylhexyl) phthalate (DEHP) for blood bags have been sought for many years. Cyclohexane-1,2-dicarboxylic acid, diisononylester (Hexamoll(®) DINCH(®)) is an alternative that has been evaluated in preliminary studies for compatibility and efficacy to preserve whole blood. While Hexamoll(®) DINCH(®) has an extensive database for mammalian toxicity via oral administration, data were needed to evaluate toxicity from intravenous (IV) administration to support the use of the plasticizer Hexamoll(®) DINCH(®) in blood bags. A series of studies was performed by slow IV injection or IV infusion of Hexamoll(®) DINCH(®), a highly viscous, hydrophobic substance, suspended in Intralipid(®) 20% (20% intravenous fat emulsion). Rats were injected once, followed by 14 days of recovery; injected daily for 5 days followed by 5 days of recovery, or infused for 29 days (4h/day) followed by 14 days of recovery. Dose levels were 0, 62, 125, and 250-300mg/kg body weight/day. These dose levels represent the limits of suspension and far exceed any anticipated exposures from migration out of plasticized blood bags. Animals were observed for signs of toxicity; body weight and feed consumption were measured; blood collected for clinical chemistry and hematology; and tissues collected and processed for histopathology. Special emphasis was placed on evaluating endpoints and tissues that are commonly associated with plasticizer exposure in rodents. Urine was collected during the 4-week study to quantify urinary metabolites of Hexamoll(®) DINCH(®). The results of the studies indicate that no substance-related toxicity occurred: no effects on behavior, no effects on organ weight, no effect on serum chemistry including thyroid hormones; and no effect on major organs, especially no testicular toxicity and no indication for peroxisome proliferation in the liver. The only effects seen were petechia and granulomas related to dissipation of suspended Hexamoll(®) DINCH(®) in the aqueous environment of the blood. However, the results of metabolite analyses demonstrate that Hexamoll(®) DINCH(®) was bioavailable. Therefore, based on the lack of Hexamoll(®) DINCH(®)-related systemic toxicity with the exception of the physical limitations, the no-observed-adverse-effect level for parenterally administered Hexamoll(®) DINCH(®) is considered to be 300mg/kg bw/day.

Potential release scenarios for carbon nanotubes used in composites.

The expected widespread use of carbon nanotube (CNT)-composites in consumer products calls for an assessment of the possible release and exposure to workers, consumers and the environment. Release of CNTs may occur at all steps in the life cycle of products, but to date only limited information is available about release of CNTs from actual products and articles. As a starting point for exposure assessment, exploring sources and pathways of release helps to identify relevant applications and situations where the environment and especially humans may encounter releases of CNTs. It is the aim of this review to identify various potential release scenarios for CNTs used in polymers and identify the greatest likelihood of release at the various stages throughout the life-cycle of the product. The available information on release of CNTs from products and articles is reviewed in a first part. In a second part nine relevant release scenarios are described in detail: injection molding, manufacturing, sports equipment, electronics, windmill blades, fuel system components, tires, textiles, incineration, and landfills. Release from products can potentially occur by two pathways; (a) where free CNTs are released directly, or more frequently (b) where the initial release is a particle with CNTs embedded in the matrix, potentially followed by the subsequent release of CNTs from the matrix. The potential for release during manufacturing exists for all scenarios, however, this is also the situation when exposure can be best controlled. For most of the other life cycle stages and their corresponding release scenarios, potential release of CNTs can be considered to be low, but it cannot be excluded totally. Direct release to the environment is also considered to be very low for most scenarios except for the use of CNTs in tires where significant abrasion during use and release into the environment would occur. Also the possible future use of CNTs in textiles could result in consumer exposure. A possibility for significant release also exists during recycling operations when the polymers containing CNTs are handled together with other polymers and mainly occupational users would be exposed. It can be concluded that in general, significant release of CNTs from products and articles is unlikely except in manufacturing and subsequent processing, tires, recycling, and potentially in textiles. However except for high energy machining processes, most likely the resulting exposure for these scenarios will be low and to a non-pristine form of CNTs. Actual exposure studies, which quantify the amount of material released should be conducted to provide further evidence for this conclusion.

Exploratory in vitro study of red blood cell storage containers formulated with an alternative plasticizer.

BACKGROUND: The plasticizer di-2-ethylhexyl phthalate (DEHP) is a common component in medical plastics. There is motivation to replace this component; however, DEHP is necessary to prevent excessive hemolysis in stored red blood cells (RBCs). Our objective is to evaluate a candidate replacement plasticizer (Hexamoll, di-isononyl cyclohexane-1,2-dicarboxylic acid [DINCH], BASF Corp.) compared to DEHP in an in vitro feasibility study. We hypothesize that the candidate will provide at least equivalent protection against hemolysis for RBCs stored for 42 days and periodic mixing of RBCs will add additional protection against hemolysis. STUDY DESIGN AND METHODS: Whole blood was collected into citrate-phosphate-dextrose; combined into pools of 2 ABO identical whole blood units; and divided, leukoreduced, centrifuged, and separated into plasma and RBCs. Additive solution was added, and the RBCs were stored for 42 days at 1 to 6°C. In three parts of this study, split pools were paired as DINCH-polyvinyl chloride (PVC) with weekly mixing versus DINCH-PVC with no mixing, DINCH-PVC mixed versus DEHP-PVC no mix, and DINCH-PVC versus DEHP-PVC with neither mixed. A standard panel of in vitro RBC characteristics was determined on Days 0 and 42. RESULTS: Mixing DINCH-PVC weekly improved Day 42 hemolysis (0.36 ± 0.07% vs.0.56 ± 0.15%, p = 0.002), and mixed DINCH-PVC bags were noninferior to unmixed DEHP-PVC bags (p ≤ 0.05). DINCH-PVC bags stored without weekly mixing were inferior to unmixed DEHP-PVC bags for hemolysis on Day 42, although no individual bag exceeded 0.8% hemolysis. CONCLUSION: Periodic mixing of RBCs stored in DINCH-PVC provides additional protection against hemolysis. Unmixed DINCH-PVC bags were inferior to DEHP-PVC bags for prevention of hemolysis, but remain a candidate for replacement DEHP in RBC storage bags.

Developing a registry of workers involved in nanotechnology: BASF experiences.

OBJECTIVE: To assist BASF in the establishment of a registry of workers involved in nanotechnology. METHODS: The initial step was a complete inventory of nanomaterials and sites of use. Guidance was developed to clarify which particulate nanomaterials were to be included in the survey. Site management was then contacted by the medical department to obtain a list of workers. RESULTS: The time line for collecting data ranged from several months to a year, depending on the information needed, and presented challenges based on the lack of global definition and labeling of nanomaterials. Less than 50 nanomaterials are used as raw materials in less than 10% of the sites globally. In North America, less than 5% of sites and 5% workers use nanomaterials. CONCLUSIONS: Further work is required to integrate the inventory, registry, and exposure assessments.

Assessing the human carcinogenic potential of tetrahydrofuran: I. Mode of action and human relevance analysis of the male rat kidney tumor.

In 1998, the National Toxicology Program concluded that inhalation exposure to tetrahydrofuran resulted in increased incidences of renal adenomas and carcinomas (combined) in male F344 rats and of hepatocellular adenomas and carcinomas (combined) in female B6C3F1 mice. In the present paper, the bioassay results and additional information are evaluated using the IPCS/ILSI Mode of Action/Human Relevance Framework to determine if the data are sufficient to describe the possible mode(s) of action (MOA) underlying the reported results for the rat renal tumor and to determine if any of these modes of action could be operative in humans. Preliminary analysis of the rat renal tumor data and related information suggested that a MOA could be described, but questions remained concerning the role that chronic progressive nephropathy (CPN) may play in the development of the lesions. In 2009, a Pathology Working Group concluded that the rat renal lesions resulted primarily from regenerative processes associated with advanced CPN. The renal tumor finding is considered not relevant to humans and should not be considered in any further risk assessment efforts on this chemical. A companion paper describes a similar analysis of the female mouse liver tumor finding.

Modeling Human Exposure to Phthalate Esters: A Comparison of Indirect and Biomonitoring Estimation Methods.

Humans are potentially exposed to phthalate esters (PEs) through ingestion, inhalation, and dermal contact. Studies quantifying exposure to PEs include "biomarker studies" and "indirect studies." Biomarker studies use measurements of PE metabolites in urine to back-calculate exposure to the parent diester, while indirect studies use the concentration of the PE in each medium of exposure and the rate of intake of that medium to quantify intake of the PE. In this review, exposure estimates from biomarker and indirect studies are compiled and compared for seven PEs to determine if there are regional differences and if there is a preferred approach. The indirect and biomarker methods generally agree with each other within an order of magnitude and discrepancies are explained by difficulties in accounting for use of consumer products, uncertainty concerning absorption, regional differences, and temporal changes. No single method is preferred for estimating intake of all PEs; it is suggested that biomarker estimates be used for low molecular weight PEs for which it is difficult to quantify all sources of exposure and either indirect or biomarker methods be used for higher molecular weight PEs. The indirect methods are useful in identifying sources of exposure while the biomarker methods quantify exposure.

Histopathologic changes in the kidneys of male F344 rats from a 2-year inhalation carcinogenicity study of tetrahydrofuran: a pathology working group review and re-evaluation.

Risk evaluation and hazard classification for tetrahydrofuran (THF) is based partly on the incidences of renal tumors in male F344/N rats reported in a 2-year carcinogenicity study by the National Toxicology Program (NTP). A Pathology Working Group (PWG) was commissioned to conduct an independent review of the kidney slides from this bioassay (along with two subchronic studies) to assess renal changes in light of recent scientific work on pathogenesis of pre-neoplastic and neoplastic lesions in rat kidney. PWG pathologists confirmed the NTP assessment that adenomas were non-statistically increased in animals exposed to the highest level of THF. However, when pre-neoplastic and neoplastic lesions were combined, there was no difference between control and THF-exposed groups. Also, the majority of these proliferative lesions were in rats with severe chronic progressive nephropathy (CPN). Accordingly, the PWG concluded that renal lesions in the control and THF-exposed groups resulted primarily from regenerative processes associated with advanced CPN. Based on an alpha(2u)-globulin/hyaline droplet response observed in a 4-week study with THF, the PWG could not exclude the possibility of both advanced CPN and low-grade alpha2u-g nephropathy contributing to the renal proliferative lesions developing chronically in high-dose males. Neither condition has a pathologic counterpart in humans.

Nanomaterial characterization: considerations and needs for hazard assessment and safety evaluation.

Nanotechnology is a rapidly emerging field of great interest and promise. As new materials are developed and commercialized, hazard information also needs to be generated to reassure regulators, workers, and consumers that these materials can be used safely. The biological properties of nanomaterials are closely tied to the physical characteristics, including size, shape, dissolution rate, agglomeration state, and surface chemistry, to name a few. Furthermore, these properties can be altered by the medium used to suspend or disperse these water-insoluble particles. However, the current toxicology literature lacks much of the characterization information that allows toxicologists and regulators to develop "rules of thumb" that could be used to assess potential hazards. To effectively develop these rules, toxicologists need to know the characteristics of the particle that interacts with the biological system. This void leaves the scientific community with no options other than to evaluate all materials for all potential hazards. Lack of characterization could also lead to different laboratories reporting discordant results on seemingly the same test material because of subtle differences in the particle or differences in the dispersion medium used that resulted in altered properties and toxicity of the particle. For these reasons, good characterization using a minimal characterization data set should accompany and be required of all scientific publications on nanomaterials.

Effect of di(2-ethylhexyl) phthalate (DEHP) on genital organs from juvenile common marmosets: I. Morphological and biochemical investigation in 65-week toxicity study.

Recent studies demonstrated that preadolescent male rats are more sensitive to testicular damage from exposure to DEHP than adults. Male and female marmosets were treated daily with 0, 100, 500, or 2500 mg/kg DEHP by oral gavage for 65 wk from weaning (3 mo of age) to sexual maturity (18 mo). No treatment-related changes were observed in male organ weights, and no microscopic changes were found in male gonads or secondary sex organs. Sperm head counts, zinc levels, glutathione levels, and testicular enzyme activities were comparable between groups. Electron microscopic examination revealed no treatment-related abnormalities in Leydig, Sertoli, or spermatogenic cells. Histochemical examination of the testis after 3beta-hydroxysteroid dehydrogenase (3beta-HSD) staining did not reveal any alterations in steroid synthesis in the Leydig cells. Thus, although marmoset monkeys were treated with 2500 mg/kg DEHP, throughout the pre- and periadolescent period, no histological changes were noted in the testes. For females, increased ovarian and uterine weights and elevated blood estradiol level were observed in higher dosage groups, 500 and 2500 mg/kg. These increased weights were associated with the presence of large corpus luteum, a common finding in older female marmosets. Although an effect on the female ovary cannot be completely ruled out, no abnormal histological changes were observed in the ovaries or uteri in comparison to controls. No increases in hepatic peroxisomal enzyme activities were noted in treated groups; isolated hepatic enzyme activities (P-450 contents, testosterone 6beta-hydroxylase, and lauric acid omega-1omega-hydroxylase activities) were increased in males and/or females of either the mid- or high-dose groups, but no consistent dose-related trend was observed.

Proposed mode of action for in utero effects of some phthalate esters on the developing male reproductive tract.

Phthalate esters containing a straight-chain backbone of 4-6 carbons have demonstrated testicular toxicity and infertility in adult and pre-adolescent rats, mice, hamsters, and ferrets. In recent years, these same phthalates have been shown to interfere with the normal development of the male reproductive tract in rodents and rabbits. The review presented here summarizes studies that provide evidence of a mode of action for these effects. The data indicate that C4-C6 phthalate esters inhibit processes in the Leydig cell, such as the synthesis of testosterone (T) and production of insulin-like factor 3 (insl3), both of which are required for normal development of male genitalia. A proposed secondary effect of reduced androgen production is on Sertoli cells, resulting in failure to proliferate and interference with cell-cell communication (gap-junction intracellular communication) leading to the development of large multinucleate gonocytes. The possibility that phthalates act directly on the Sertoli cells to interfere with intracellular communication is not excluded. The strength, consistency, and plausibility of the proposed mode of action and alternate modes of action are discussed.

Revised assessment of cancer risk to dichloromethane II. Application of probabilistic methods to cancer risk determinations.

An updated PBPK model of methylene chloride (DCM, dichloromethane) carcinogenicity in mice was recently published using Bayesian statistical methods (Marino et al., 2006). In this work, this model was applied to humans, as recommended by Sweeney et al.(2004). Physiological parameters for input into the MCMC analysis were selected from multiple sources reflecting, in each case, the source that was considered to represent the most current scientific evidence for each parameter. Metabolic data for individual subjects from five human studies were combined into a single data set and population values derived using MCSim. These population values were used for calibration of the human model. The PBPK model using the calibrated metabolic parameters was used to perform a cancer risk assessment for DCM, using the same tumor incidence and exposure concentration data relied upon in the current IRIS entry. Unit risks, i.e., the risk of cancer from exposure to 1 microg/m3 over a lifetime, for DCM were estimated using the calibrated human model. The results indicate skewed distributions for liver and lung tumor risks, alone or in combination, with a mean unit risk (per microg/m3) of 1.05 x 10(-9), considering both liver and lung tumors. Adding the distribution of genetic polymorphisms for metabolism to the ultimate carcinogen, the unit risks range from 0 (which is expected given that approximately 20% of the US population is estimated to be nonconjugators) up to a unit risk of 2.70 x 10(-9) at the 95th percentile. The median, or 50th percentile, is 9.33 x 10(-10), which is approximately a factor of 500 lower than the current EPA unit risk of 4.7 x 10(-7) using a previous PBPK model. These values represent the best estimates to date for DCM cancer risk because all available human data sets were used, and a probabilistic methodology was followed.

Revised assessment of cancer risk to dichloromethane: part I Bayesian PBPK and dose-response modeling in mice.

The current USEPA cancer risk assessment for dichloromethane (DCM) is based on deterministic physiologically based pharmacokinetic (PBPK) modeling involving comparative metabolism of DCM by the GST pathway in the lung and liver of humans and mice. Recent advances in PBPK modeling include probabilistic methods and, in particular, Bayesian inference to quantitatively address variability and uncertainty separately. Although Bayesian analysis of human PBPK models has been published, no such efforts have been reported specifically addressing the mouse, apart from results included in the OSHA final rule on DCM. Certain aspects of the OSHA model, however, are not consistent with current approaches or with the USEPA's current DCM cancer risk assessment. Therefore, Bayesian analysis of the mouse PBPK model and dose-response modeling was undertaken to support development of an improved cancer risk assessment for DCM. A hierarchical population model was developed and prior parameter distributions were selected to reflect parameter values that were considered the most appropriate and best available. Bayesian modeling was conducted using MCSim, a publicly available software program for Markov Chain Monte Carlo analysis. Mean posterior values from the calibrated model were used to develop internal dose metrics, i.e., mg DCM metabolized by the GST pathway/L tissue/day in the lung and liver using exposure concentrations and results from the NTP mouse bioassay, consistent with the approach used by the USEPA for its current DCM cancer risk assessment. Internal dose metrics were 3- to 4-fold higher than those that support the current USEPA IRIS assessment. A decrease of similar magnitude was also noted in dose-response modeling results. These results show that the Bayesian PBPK model in the mouse provides an improved basis for a cancer risk assessment of DCM.

Dose-dependent transitions in mechanisms of toxicity.

Scientists and decision makers from all sectors agree that risk assessments should be based on the best available science. Several years ago, the Health and Environmental Sciences Institute (HESI), a global branch of the International Life Sciences Institute (ILSI), identified the need for better scientific understanding of dose-dependent transitions in mechanisms of toxicity as one avenue by which the best and latest science can be integrated into the decision making process. In July 2001, the HESI Project Committee on Dose-Dependent Transitions in Mechanisms of Toxicity established a group of academic, government, and industry scientists to engage in active technical discourse on the issue of dose-dependent transitions in mechanisms of toxicity. Over the next 18 months, case studies were examined. These case studies included acetaminophen, butadiene, ethylene glycol, formaldehyde, manganese, methylene chloride, the peroxisome proliferator-activated receptor, progesterone/hydroxyflutamide, propylene oxide, vinyl acetate, vinyl chloride, vinylidene chloride, and zinc (Slikker, W., Jr., Andersen, M.E., Bogdanffy, M.S., Bus, J.S., Cohen, S.D., Conolly, R.B., David, R.M., Doerrer, N.G., Dorman, D.C., Gaylor, D.W., Hattis, D., Rogers, J.M., Setzer, R.W., Swenberg, J.A., Wallace, K., 2004. Dose-dependent transitions in mechanisms of toxicity: case studies. Toxicol. Appl. Pharmacol. 201(3), 226-294 (this issue)). The HESI Project Committee sponsored two technical workshops in 2003. The first of these workshops took place on February 12-13, 2003, and was co-sponsored by the Agency for Toxic Substances and Disease Registry, the American Chemistry Council, the National Institute of Environmental Health Sciences, the Society of Toxicology, and the U.S. Environmental Protection Agency. Additional support was provided by Health Canada. Invited experts from government, academia, and industry provided scientific perspectives and recommendations at the workshop. The purpose of the workshop was to examine approaches to dose-response analysis, learn from the case study examples, and gather feedback from invited participants on the impact of dose-dependent transitions on the risk assessment process. The second forum consisted of a workshop in March 2003 at the Society of Toxicology Annual Meeting in Salt Lake City, UT. This paper addresses the issues discussed at both workshops, and presents the consensus conclusions drawn by expert participants.

Dose-dependent transitions in mechanisms of toxicity: case studies.

Experience with dose response and mechanisms of toxicity has shown that multiple mechanisms may exist for a single agent along the continuum of the full dose-response curve. It is highly likely that critical, limiting steps in any given mechanistic pathway may become overwhelmed with increasing exposures, signaling the emergence of new modalities of toxic tissue injury at these higher doses. Therefore, dose-dependent transitions in principal mechanisms of toxicity may occur, and could have significant impact on the interpretation of reference data sets for risk assessment. To illustrate the existence of dose-dependent transitions in mechanisms of toxicity, a group of academic, government, and industry scientists, formed under the leadership of the ILSI Health and Environmental Sciences Institute (HESI), developed a series of case studies. These case studies included acetaminophen, butadiene, ethylene glycol, formaldehyde, manganese, methylene chloride, peroxisome proliferator-activated receptor (PPAR), progesterone/hydroxyflutamide, propylene oxide, vinyl acetate, vinyl chloride, vinylidene chloride, and zinc. The case studies formed the basis for technical discourse at two scientific workshops in 2003.

Phthalate treatment does not influence levels of IgE or Th2 cytokines in B6C3F1 mice.

Bronchial asthma is mediated, in part, by the immunoregulatory cytokines interleukins 4 and 13 (IL-4 and IL-13). These cytokines stimulate IgE synthesis that in turn is associated with airway hyper-responsiveness. Compounds that stimulate IgE synthesis and elicit bronchial reactivity are generally considered to be respiratory sensitizers. Recently, it has been hypothesized that exposure to phthalates may contribute to childhood asthma. To address this question, di-(2-ethylhexyl) phthalate (DEHP) was tested using a protocol adapted from work by Dearman that involves topical application (and challenge) of test substances to mice followed by measurements of total serum IgE. In addition, auricular lymph nodes were harvested for measurement of IL-4 and IL-13 proteins and their corresponding messenger RNAs. Because skin absorption of high molecular weight phthalates is limited, liver weight increase, a measure of peroxisomal proliferation, was monitored to assure that internal dosing had been achieved. ELISA and RNAse protection assays demonstrated that DEHP treatment did not significantly affect IgE, IL-4, or IL-13 levels. Similarly, IL-4 and IL-13 mRNA levels were not elevated. In contrast, all of these were significantly elevated by trimellitic anhydride (TMA), a respiratory sensitizer used as the positive control in this assay. Liver weights were significantly elevated by DEHP, providing evidence of sufficient percutaneous absorption to induce physiological responses. To extend these observations, three other commercial phthalate ester plasticizers, di-isononyl phthalate (DINP), di-isohexyl phthalate (DIHP), and butyl benzyl phthalate (BBP), were assessed using the same protocol. As above, ELISA and RNAse protection assays showed that IgE, IL-4, and IL-13 proteins, and IL-4 and IL-13 mRNAs in the phthalate-treated animals were all at levels similar to that of control values. The positive control, TMA, produced large, statistically significant increases in all parameters, demonstrating responsiveness of the assay. Another control, dinitrochlorobenzene (DNCB), a contact sensitizer, also responded as expected, producing smaller but statistically significant increases in IgE and in mRNA for IL-4 and IL-13 but not in the levels of these cytokines. In summary, treatment with DEHP, DINP, DIHP, and BBP did not result in significant elevations in total serum IgE, IL-4, or IL-13. As such it is unlikely that these substances would produce antibody-mediated respiratory allergy.

Activation of mouse and human peroxisome proliferator-activated receptors (PPARs) by phthalate monoesters.

Administration of phthalates is known to cause toxicity and liver cancer in rodents through the activation of peroxisome proliferator-activated receptors (PPARs), and the monoesters appear to be the active metabolites that function as ligands of PPARs. There is evidence that PPARs exhibit significant species differences in response to ligand activation. In this study, the activation of mouse and human PPARalpha, PPARbeta, and PPARgamma by a broad class of phthalate monoesters was investigated using a trans-activation assay, functional analysis of PPARalpha target gene expression, and a PPARgamma-mediated differentiation assay. These studies demonstrated a range in the ability of various phthalate monoesters to activate PPARalpha, with the mouse PPARalpha generally being activated at lower concentrations and exhibiting a greater response than human PPARalpha. Similarly, a range in the trans-activation of mouse PPARbeta by phthalate monoesters was also observed, but this effect was not found with human PPARbeta. A number of phthalate monoesters activated both mouse and human PPARgamma, with similar sensitivity being exhibited by both receptors. These studies show that the potency and efficacy of phthalate monoesters for the activation of PPARalpha and PPARgamma increase with increasing side-chain length. These studies also show that mouse PPARalpha and PPARbeta are generally activated at lower concentrations of phthalate monoesters than human PPARalpha and PPARbeta, and that both mouse and human PPARgamma exhibit similar sensitivity to phthalate monoesters. Lastly, there is a good relationship between the relative ability of phthalate monoesters to trans-activate PPARalpha and PPARgamma, and the relative induction of PPARalpha target gene mRNA and PPARgamma-mediated adipocyte differentiation, respectively.

NTP center for the evaluation of risks to human reproduction reports on phthalates: addressing the data gaps.

Between 1998 and 2000 an Expert Panel convened by the National Toxicology Program's Center for the Evaluation of Risks to Human Reproduction (NTP-CERHR) reviewed information related to the developmental and reproductive toxicity of seven phthalate esters; DBP, BBP, DnHP, DEHP, DnOP, DINP, and DIDP. Information on exposures was also considered. The objectives were to determine whether any of these phthalates posed potential human reproductive risks, and if so, to define the circumstances. The Expert Panel also identified some areas of uncertainty. These assessments, ultimately published in 2002, concluded that reproductive risks were minimal to negligible in most cases although some specific uses were considered potentially more problematic. Since the evaluations were completed, numerous studies dealing with both hazard characterization and underlying mechanism have been carried out. Additionally, exposures of the general population have been much better characterized through the use of urinary measurements developed by the Centers for Disease Control (CDC). This additional information makes several important points. First, calculations based on the urinary metabolite measurements indicate that exposures within the general population are at levels similar to or lower than the estimates used by the NTP-CERHR. The demonstration that exposures were not underestimated by the CERHR has removed a substantial portion of the uncertainty. Second, new hazard characterization studies on several phthalates have established NOAELs similar to or higher than those used by the Expert Panel. Thus, these data demonstrate that, to the extent that the rodent data are useful for human health risk assessment, the no effect levels and dose-response relationships are now more precisely defined. In some cases, the no effect levels may be substantially higher than those estimated by the Expert Panel. Third, studies of underlying mechanism and/or hazard characterization studies in other species suggest that primates may be less sensitive than rodents to the reproductive effects of certain phthalates. Finally, the two specific situations that the CERHR identified as potentially problematic, the exposure of young children to DINP through the use of toys or to DEHP from medical devices, have been assessed by the responsible regulatory authorities. The Consumer Product Safety Commission concluded that exposure to DINP from toys was well below effect levels in animals, and, therefore, there was no risk. The Food and Drug Administration estimates of exposures from medical devices indicated that for some limited, intensive medical procedures, DEHP exposures could be similar to or greater than the NOAELs selected by the NTP-CERHR. However, the FDA also acknowledged that more recent information indicates that the NOAELs identified in rodent studies may be substantially higher than values previously proposed by the NTP-CERHR. In summary, much of the uncertainty identified by the CERHR has now been addressed, and the overall conclusions that levels of concern are minimal to negligible in most situations are much better established. The overall objective of this report is to summarize this new research and comment on its relevance to the NTP-CERHR assessments.