National Toxicology Program Report Research Articles

Dose-response analyses of the 27 human developmental neurotoxicity studies ranked highest quality by National Toxicology Program (NTP) systematic review

BACKGROUND AND AIM: The National Toxicology Program (NTP) has been conducting a systematic review of the evidence for fluoride’s neurotoxicity. Drafts of the NTP report conclude fluoride is a presumed hazard for developmental neurotoxicity, but have not included rigorous dose-response analyses to estimate a safe dose. This is of great public health importance because millions of people are exposed to fluoride added to public water supplies or table salt; or naturally elevated fluoride in drinking water. METHODS: We conducted several dose-response analyses using data extracted by NTP from 27 human studies NTP ranked high quality. The number of studies finding adverse effects were counted at three exposure levels: 0.7, 0.7–1.5, 1.5mg/L. Meta-analyses were performed with the 14 studies having individual-level exposure data, in two subgroups by mean exposure: ≤1.5mg/L or 1.5mg/L. The same 14 studies were then assessed using meta-regression of IQ loss at mean exposure against mean exposure. RESULTS:Of the 27 higher quality studies, 25 found adverse neurocognitive effects with 11 at exposures below 0.7mg/L; 4 at 0.7–1.5mg/L; and 10 at 1.5mg/L. None found significant beneficial effects. In the meta-analysis, all 14 studies found loss of IQ, including the 7 in subgroup ≤1.5mg/L which had a pooled effect size of -4.0 (95%CI -6.03, -2.05) IQ points per 1mg/L fluoride. The meta-regression fit a linear dose-response with pooled effect size of -2.94 (95%CI -4.03, -1.84) IQ points per 1mg/L. CONCLUSIONS:Dose-response analyses of 27 studies ranked high quality by NTP found a remarkable consistency of adverse effects down to very low doses. The magnitude of predicted IQ loss is of considerable concern. Pregnant women and infants should limit exposure to fluoride. KEYWORDS: fluoride, developmental neurotoxicity, dose-response, IQ, National Toxicology Program, meta-analysis

Health Effects of Naphthalene Exposure: A Systematic Evidence Map and Analysis of Potential Considerations for Dose-Response Evaluation.

Background:Naphthalene is a polycyclic aromatic hydrocarbon that has been associated with health effects, including cancer. As the state of the science on naphthalene toxicity continues to evolve, updated toxicity reference value(s) may be required to support human health risk assessment.Objectives:We present a systematic evidence map of studies that could be used to derive toxicity reference value(s) for naphthalene.Methods:Human and animal health effect studies and physiologically based pharmacokinetic (PBPK) models were identified from a literature search based on populations, exposures, comparators, and outcomes (PECO) criteria. Human and animal studies meeting PECO criteria were refined to a smaller subset considered most informative for deriving chronic reference value(s), which are preferred for assessing risk to the general public. This subset was evaluated for risk of bias and sensitivity, and the suitability of each study for dose–response analysis was qualitatively assessed. Lowest observed adverse effect levels (LOAELs) were extracted and summarized. Other potentially relevant studies (e.g., mechanistic and toxicokinetic studies) were tracked as supplemental information but not evaluated further. Existing reference values for naphthalene are also summarized.Results:We identified 26 epidemiology studies and 16 animal studies that were considered most informative for further analysis. Eleven PBPK models were identified. The available epidemiology studies generally had significant risk of bias and/or sensitivity concerns and were mostly found to have low suitability for dose–response analysis due to the nature of the exposure measurements. The animal studies had fewer risk of bias and sensitivity concerns and were mostly found to be suitable for dose–response analysis.Conclusion:Although both epidemiological and animal studies of naphthalene provide weight of evidence for hazard identification, the available animal studies appear more suitable for reference value derivation. PBPK models and mechanistic and toxicokinetic data can be applied to extrapolate these animal data to humans, considering mode of action and interspecies metabolic differences. https://doi.org/10.1289/EHP7381

Quantifying association between liver tumor incidence and early-stage liver weight increase - An NTP data analysis.

Two-year toxicology and carcinogenesis rodent studies conducted at the National Toxicology Program (NTP) are used to identify potential adverse health effects in humans due to chemical exposure, including cancer. Liver tumor, the most frequently diagnosed tumor type of chemically induced neoplastic effects documented in NTP's carcinogenicity studies, is usually difficult to be detected at early stage due to the inconspicuous symptoms. However, the abnormal growth of liver cells can lead to liver weight increase, so it is hypothesized that liver tumor incidence is associated with early stage liver weight increase. In this study, the association between liver weight increase and liver tumor incidence are quantified by (1) calculating the correlation coefficient of and (2) building quantitative linear relationship between benchmark dose estimates derived from these two types of data collected from NTP studies. Together with 151 chemical/species/sex combinations of liver tumor data showing positive evidence collected from 76 NTP long-term studies, short-term liver weight data reported in the same NTP report were extracted to be paired with the liver tumor data for the analyses. Results show that the estimated correlation coefficients (as high as 0.78) along with the adequately fitted linear models suggest that the association between relative liver weight increase and aggregated liver tumor incidence are relatively strong. Additional analyses focused on some more specific situations (e.g., specific tumor type or specific strain/sex combination) further confirmed the strong association. Given the design of this study, the interpretation of the findings is not that liver weight increase can be used to predict liver tumor incidence, instead, evident increase in liver weight might be used as a reason to prioritize the test article for a two-year toxicology and carcinogenesis study.

Free Radical Metabolism of Methyleugenol and Related Compounds

Methyleugenol, the methyl ether of eugenol, both of which are flavorant constituents of spices, has been listed by the National Toxicology Program’s Report on Carcinogens as reasonably anticipated to be a human carcinogen. This finding is based on the observation of increased incidence of malignant tumors at multiple tissue sites in experimental animals of different species. By contrast, eugenol is not listed. In this study, we show that both methyleugenol and eugenol readily undergo peroxidative metabolism in vitro to form free radicals with large hyperfine interactions of the methylene allylic hydrogen atoms. These large hyperfine splittings indicate large electron densities adjacent to those hydrogen atoms. Methyleugenol undergoes autoxidation such that the commercial product contains 10–30 mg/L hydroperoxide and is capable of activating peroxidases without the presence of added hydrogen peroxide. Additionally, the hydroperoxide is not a good substrate for catalase, which demonstrates that these antioxidant defenses will not be effective in protecting against methyleugenol exposure.

Developmental programming: Impact of prenatal exposure to bisphenol-A and methoxychlor on steroid feedbacks in sheep

Bisphenol-A (BPA), a polymer used in plastics manufacturing, and methoxychlor (MXC), a pesticide, are endocrine disrupting compounds with estrogenic and anti-androgenic properties. Prenatal BPA or MXC treatment induces reproductive defects in sheep with BPA causing prepubertal luteinizing hormone (LH) hypersecretion and dampening of periovulatory LH surges and MXC lengthening follicular phase and delaying the LH surge. In this study, we addressed the underlying neuroendocrine defects by testing the following hypotheses: 1) prenatal BPA, but not MXC reduces sensitivity to estradiol and progesterone negative feedback, 2) prenatal BPA, but not MXC increases pituitary responsiveness to gonadotropin releasing hormone (GnRH), and 3) prenatal BPA dampens LH surge response to estradiol positive feedback challenge while prenatal MXC delays the timing of the LH surge. Pregnant sheep were treated with either 1) 5mg/kg/day BPA (produces approximately twice the level found in human circulation, n=8), 2) 5mg/kg/day MXC (the lowest observed effect level stated in the EPA National Toxicology Program's Report; n=6), or 3) vehicle (cotton seed oil: C: n=6) from days 30 to 90 of gestation. Female offspring of these ewes were ovariectomized at 21months of age and tested for progesterone negative, estradiol negative, estradiol positive feedback sensitivities and pituitary responsiveness to GnRH. Results revealed that sensitivity to all 3 feedbacks as well as pituitary responsiveness to GnRH were not altered by either of the prenatal treatments. These findings suggest that the postpubertal reproductive defects seen in these animals may have stemmed from ovarian defects and the steroidal signals emanating from them.

The Weight of Evidence Does Not Support the Listing of Styrene as “Reasonably Anticipated to be a Human Carcinogen” in NTP's Twelfth Report on Carcinogens

ABSTRACT Styrene was listed as “reasonably anticipated to be a human carcinogen” in the twelfth edition of the National Toxicology Program's Report on Carcinogens based on what we contend are erroneous findings of limited evidence of carcinogenicity in humans, sufficient evidence of carcinogenicity in experimental animals, and supporting mechanistic data. The epidemiology studies show no consistent increased incidence of, or mortality from, any type of cancer. In animal studies, increased incidence rates of mostly benign tumors have been observed only in certain strains of one species (mice) and at one tissue site (lung). The lack of concordance of tumor incidence and tumor type among animals (even within the same species) and humans indicates that there has been no particular cancer consistently observed among all available studies. The only plausible mechanism for styrene-induced carcinogenesis—a non-genotoxic mode of action that is specific to the mouse lung—is not relevant to humans. As a whole, the evidence does not support the characterization of styrene as “reasonably anticipated to be a human carcinogen,” and styrene should not be listed in the Report on Carcinogens.

Toxicologic Assessment of a Commercial Decolorized Whole Leaf Aloe Vera Juice, Lily of the Desert Filtered Whole Leaf Juice with Aloesorb

Aloe vera, a common ingredient in cosmetics, is increasingly being consumed as a beverage supplement. Although consumer interest in aloe likely stems from its association with several health benefits, a concern has also been raised by a National Toxicology Program Report that a nondecolorized whole leaf aloe vera extract taken internally by rats was associated with intestinal mucosal hyperplasia and ultimately malignancy. We tested a decolorized whole leaf (DCWL) aloe vera, treated with activated charcoal to remove the latex portion of the plant, for genotoxicity in bacteria, acute/subacute toxicity in B6C3F1 mice, and subchronic toxicity in F344 rats. We found this DCWL aloe vera juice to be nongenotoxic in histidine reversion and DNA repair assays. Following acute administration, mice exhibited no adverse signs at 3- or 14-day evaluation periods. When fed to male and female F344 rats over 13 weeks, DCWL aloe led to no toxicity as assessed by behavior, stools, weight gain, feed consumption, organ weights, and hematologic or clinical chemistry profiles. These rats had intestinal mucosal morphologies—examined grossly and microscopically—that were similar to controls. Our studies show that oral administration of this DCWL aloe juice has a different toxicology profile than that of the untreated aloe juice at exposures up to 13 weeks.

Lack of human tissue-specific correlations for rodent pancreatic and colorectal carcinogens

To better understand the relationships between chemical exposures and human cancer causation, incidence data for human cancer types were identified and pancreatic and colorectal cancers were studied in-depth to assess whether data supporting the causation of pancreatic or colorectal tumors by chemicals in rodents is predictive of causation by the same chemicals of the same tumors in humans. A search of the Carcinogenic Potency Database, the National Toxicology Program (NTP) technical report database, and the published literature identified 38 and 39 chemicals reported to cause pancreatic and colorectal tumors, respectively, in mice or rats. For each of these chemicals, searches were conducted of the International Agency for Research on Cancer monographs, the NTP Report on Carcinogens, and the published literature for evidence of induction of the same tumors in humans. Based on this evaluation, no conclusive evidence was identified to suggest that chemicals reported to cause pancreatic or colorectal tumors in rodents also cause these tumors in humans. These findings suggest that pancreatic tumor data from mouse and rat bioassays are of limited utility with regard to predicting similar tumor induction in humans. For colorectal cancer, a lack of correlation was noted for the vast majority of chemicals.

Carcinogenic Food Contaminants

A large number of scientific studies and reviews have addressed the potential for dietary components to influence the risk of developing cancer. One topic of particular interest has been the impact of food contaminants. Two complementary programs, among others, have reviewed and synthesized information on the carcinogenic potential of food contaminants and judged the degree of evidence linking different food contaminants to the risk of cancer in humans. These programs, the International Agency for Research on Cancer's IARC Monographs on the Evaluation of Carcinogenic Risks to Humans and the US National Toxicology Program's Report of Carcinogens have reviewed hundreds of chemicals, mixtures, and natural products and then graded the cancer risk posed to humans. Contaminants with the highest level of evidence include aflatoxin, alcoholic beverages, 2,3,7,8-tetracholordibenzo-p-dioxin. Agents with a moderate level of evidence include acetaldehyde, polycyclic aromatic hydrocarbons, some nitrosamines, and yerba mate. Agents with a low level of evidence include bracken fern, fumonsin B1, ochratoxin, and others. This review presents a summary of the evidence for the carcinogenicity of these and other agents and the ranks provided by two important assessment programs.

Chemical Exposures: Will DEA Findings Wash?

A common soap and shampoo ingredient restricted in Europe for its suspected link to cancer is raising new concerns as study results suggest it can thwart brain development in mice. Researchers at the University of North Carolina at Chapel Hill reported in the August 2006 FASEB Journal that diethanolamine (DEA) irreversibly damaged the memory capacity of animals exposed before birth. Author Steven Zeisel believes DEA could induce fetal neural abnormalities in humans, too. “It’s hard to estimate human exposure, but we believe the mice had exposures about ten times higher,” he says, assuming that people bathe and shampoo daily with DEA-containing products, and use DEA-containing sunscreen. “There’s no reason to believe we wouldn’t see similar effects in humans.” The authors note, however, that dermal absorption of DEA is less efficient in humans than in rats. Further, most DEA used in personal care products is conjugated with fatty acids, which may not have the same effects as just DEA. Procter & Gamble principal scientist Tim Long calls Zeisel’s estimates of human exposure “grossly inaccurate,” saying that exposures from consumer product uses are actually thousands of times lower. John Bailey, executive vice president for science at the Cosmetic, Toiletry, and Fragrance Association, points out that “when you look at the exposure [of] humans, [you must take] into account the ability of the skin to protect against exposure and the fact that shampoos are rinsed off.” DEA and its condensates are used as foaming agents in many personal products. According to the public education group known as the Cancer Prevention Coalition, DEA by itself is not harmful, but it can combine with other ingredients in cosmetics to form N-nitrosodiethanolamine, which the National Toxicology Program (NTP) has deemed reasonably anticipated to be a human carcinogen. According to the International Agency for Research on Cancer, levels of N-nitrosodiethanolamine in personal care products have declined substantially since the 1980s. DEA first drew attention a decade ago, when animal studies suggested it might be carcinogenic. Both the International Agency for Research on Cancer and the NTP have considered listing it as a carcinogen, but ultimately decided there was too little evidence of human carcinogenicity. The more cautious European Union opted to limit the concentration of DEA allowed in personal care products sold there to 1%. In his article, Zeisel cited a 2002 NTP report stating that products sold in the United States may contain up to 25% DEA, although Bailey claims U.S. products contain 1% or less. Zeisel and his colleagues exposed fetal mice by painting DEA dissolved in ethanol on a shaven patch of their mothers’ skin for 11 days. DEA inhibited cell development and increased cell apoptosis in the hippocampus of the fetal mice. Zeisel says such abnormalities would permanently impair the mice’s memory. Zeisel acknowledges that even if effects are similar in humans, most babies exposed to DEA before birth would probably escape ill effects, though others may be more vulnerable to harm. He bases this belief on his 10-year studies of the nutrient choline. The body uses choline to produce acetylcholine. Zeisel and his colleagues found that adequate choline is crucial for fetal brain development. They also discovered that individuals’ choline needs vary. In a study published in the July 2006 FASEB Journal, the investigators found that some human subjects placed on a choline-deficient diet quickly suffered liver and muscle dysfunction, while others did not. They traced the effects to a genetic polymorphism that raises people’s choline requirements. Zeisel says most men and about half of women carry this inherited trait. Because the DEA molecule is similar to choline, Zeisel speculated it could perturb choline metabolism and cause the same effects as choline deficiency. “We’re not saying mothers shouldn’t shampoo their hair or use sunscreen during pregnancy,” he says. “Just look at the label. Plenty of products don’t use DEA.”

David Rall and the National Toxicology Program

Concerning the 25 year history and milestones of the National Toxicology Program (NTP), McGovern (2004) failed to acknowledge the huge conceptual and leadership contributions provided by David P. Rall (1926–1999) in the creation, development, and continuing achievements of the NTP (Huff 2000). Of course, Rall was the individual most responsible for conceiving, nurturing, and establishing the National Institute of Environmental Health Sciences (NIEHS) as a world-recognized leader in environmental health sciences. The NTP, a natural and independent partner of the NIEHS, was the innovative idea of Rall, who with a few other like-minded collegial giants in the fields of public and occupational health, recognized the need to better coordinate the disparate and often redundant toxicology and health hazard identification activities in the Department of Health and Human Services.

A review of evidence from short-term studies leading to the prediction that diazoaminobenzene (1,3-diphenyltriazine) is a carcinogen

The National Toxicology Program (NTP) is responsible for providing comprehensive toxicology evaluations of substances, while at the same time incorporating approaches to reduce, refine or replace laboratory animals in routine toxicity/carcinogenicity studies. Consistent with this, a series of metabolism studies in rodents and human liver slices, electron spin resonance spectroscopy (ESR) studies, short-term dermal toxicity studies in rodents, and acute bone marrow micronucleus studies in mice were performed on diazoaminobenzene (DAAB, also known as 1,3-diphenyltriazine). These studies demonstrated that DAAB is metabolized and shares similar genotoxic and toxicological properties to the known human carcinogen, benzene, and the known rodent carcinogen, aniline. These data were used to evaluate the potential carcinogenicity of DAAB without doing a 2-year rodent bioassay. Based on this analysis, DAAB was predicted to be carcinogenic if evaluated in a 2-year rodent bioassay. These data were evaluated to support listing DAAB in the NTP Report on Carcinogens as a substance 'reasonably anticipated to be a human carcinogen'. The purpose of this article is to review the data developed for predicting the carcinogenicity of DAAB.

Summary of the National Toxicology Program's report of the endocrine disruptors low-dose peer review.

At the request of the U.S. Environmental Protection Agency (U.S. EPA), the National Toxicology Program organized an independent and open peer review to evaluate the scientific evidence on low-dose effects and nonmonotonic dose-response relationships for endocrine-disrupting chemicals in mammalian species. For this peer review, "low-dose effects" referred to biologic changes that occur in the range of human exposures or at doses lower than those typically used in the standard testing paradigm of the U.S. EPA for evaluating reproductive and developmental toxicity. The demonstration that an effect is adverse was not required because in many cases the long-term health consequences of altered endocrine function during development have not been fully characterized. A unique aspect of this peer review was the willing submission of individual animal data by principal investigators of primary research groups active in this field and the independent statistical reanalyses of selected parameters prior to the peer review meeting by a subpanel of statisticians. The expert peer-review panel (the panel) also considered mechanistic data that might influence the plausibility of low-dose effects and identified study design issues or other biologic factors that might account for differences in reported outcomes among studies. The panel found that low-dose effects, as defined for this review, have been demonstrated in laboratory animals exposed to certain endocrine-active agents. In some cases where low-dose effects have been reported, the findings have not been replicated. The shape of the dose-response curves for reported effects varied with the end point and dosing regimen and were low-dose linear, threshold-appearing, or nonmonotonic. The findings of the panel indicate that the current testing paradigm used for assessments of reproductive and developmental toxicity should be revisited to see whether changes are needed regarding dose selection, animal-model selection, age when animals are evaluated, and the end points being measured following exposure to endocrine-active agents.

Comparative carcinogenicity of 1,3-butadiene, isoprene, and chloroprene in rats and mice

1,3-Butadiene, isoprene (2-methyl-1,3-butadiene), and chloroprene (2-chloro-1,3-butadiene) are high-production-volume chemicals used mainly in the manufacture of synthetic rubber. Inhalation studies have demonstrated multiple organ tumorigenic effects with each of these chemicals in mice and rats. Sites of tumor induction by these epoxide-forming chemicals were compared to each other and to ethylene oxide, a chemical classified by the National Toxicology Program (NTP) and by the International Agency for Research on Cancer (IARC) as carcinogenic to humans. For this group of chemicals, there are substantial species differences in sites of neoplasia; neoplasia of the mammary gland is the only common tumorigenic effect in rats and mice. Within each species, there are several common sites of tumor induction; these include the hematopoietic system, circulatory system, lung, liver, forestomach, Harderian gland, and mammary gland in mice, and the mammary gland and possibly the brain, thyroid, testis, and kidney in rats. For studies in which individual animal data were available, mortality-adjusted tumor rates were calculated, and estimates were made of the shape of the exposure–response curves and ED10 values (i.e. exposure concentrations associated with an excess risk of 10% at each tumor site). Most tumorigenic effects reported here were consistent with linear or supralinear models. For chloroprene and butadiene, the most potent response was for the induction of lung neoplasms in female mice, with ED10 values of 0.3 ppm. Based on animal cancer data, isoprene and chloroprene are listed in the NTP's Report on Carcinogens (RoC) as reasonably anticipated to be a human carcinogen. Butadiene is listed in the RoC as known to be a human carcinogen ‘based on sufficient evidence of carcinogenicity from studies in humans, including epidemiological and mechanistic information’, with support from experimental studies in laboratory animals. Epidemiology data for isoprene and chloroprene are not considered adequate to evaluate the potential carcinogenicity of these agents in humans.