Evaluating the evidence for non-monotonic dose-response relationships: A systematic literature review and (re-)analysis of in vivo toxicity data in the area of food safety.

This study aims to evaluate the evidence for the existence of non-monotonic dose-responses (NMDRs) of substances in the area of food safety. This review was performed following the systematic review methodology with the aim to identify in vivo studies published between January 2002 and February 2015 containing evidence for potential NMDRs. Inclusion and reliability criteria were defined and used to select relevant and reliable studies. A set of six checkpoints was developed to establish the likelihood that the data retrieved contained evidence for NMDR. In this review, 49 in vivo studies were identified as relevant and reliable, of which 42 were used for dose-response analysis. These studies contained 179 in vivo dose-response datasets with at least five dose groups (and a control group) as fewer doses cannot provide evidence for NMDR. These datasets were extracted and analyzed using the PROAST software package. The resulting dose-response relationships were evaluated for possible evidence of NMDRs by applying the six checkpoints. In total, 10 out of the 179 in vivo datasets fulfilled all six checkpoints. While these datasets could be considered as providing evidence for NMDR, replicated studies would still be needed to check if the results can be reproduced to rule out that the non-monotonicity was caused by incidental anomalies in that specific study. This approach, combining a systematic review with a set of checkpoints, is new and appears useful for future evaluations of the dose response datasets regarding evidence of non-monotonicity.

Overview on legislation and scientific approaches for risk assessment of combined exposure to multiple chemicals: the potential EuroMix contribution.

This article reviews the current legislative requirements for risk assessment of combined exposure to multiple chemicals via multiple exposure routes, focusing on human health and particularly on food-related chemicals. The aim is to identify regulatory needs and current approaches for this type of risk assessment as well as challenges of the implementation of appropriate and harmonized guidance at international level. It provides an overview of the current legal requirements in the European Union (EU), the United States and Canada. Substantial differences were identified in the legal requirements for risk assessment of combined exposure to multiple chemicals and its implementation between EU and non-EU countries and across several regulatory sectors. Frameworks currently proposed and in use for assessing risks from combined exposure to multiple chemicals via multiple routes and different durations of exposure are summarized. In order to avoid significant discrepancies between regulatory sectors or countries, the approach for assessing risks of combined exposure should be based on similar principles for all types of chemicals. OECD and EFSA identified the development of harmonized methodologies for combined exposure to multiple chemicals as a key priority area. The Horizon 2020 project "EuroMix" aims to contribute to the further development of internationally harmonized approaches for such risk assessments by the development of an integrated test strategy using in vitro and in silico tests verified for chemical mixtures based on more appropriate data on potential combined effects. These approaches and testing strategies should be integrated in a scientifically based weight of evidence approach to account for complexity and uncertainty, to improve risk assessment.

Dioxin contamination in food. Bayreuth, Germany, from September 28 to October 1, 2000.

Dioxin and PCB monitoring programs for food and feeding stuff in most countries of the world, including many European Countries are currently inadequate. Better control of food production lines and food processing procedures is needed to minimize entry of dioxin to the food chain and will help to avoid dioxin contamination accidents. This would also improve the ability to trace back a possible contamination to its source. European guidelines for monitoring programs should be established to ensure comparable and meaningful results. These guidelines should define the minimum requirements for the design of monitoring programs, analytical methods, and quality assurance. Though data from Northern Europe shows that the general population exposure to dioxin and PCB has decreased during the last ten years these compounds continue to be a risk of accidental contamination of the food chain. The most prominent recent example is the Belgian dioxin contamination of feeding stuff in 1999. The Belgian dioxin contamination was not detected due to dioxin monitoring programs but by their direct biological effects seen in animals. Four other cases of dioxin contamination have been detected in Europe since 1997 due to local monitoring programs. One of them (citrus pulp pellets 1998) was in a much larger scale than the Belgian dioxin contamination. The general population's exposure to dioxins and PCBs is still in the same range (1-4 pg WHO-TEQ/kg body weight and day) as the recently revised WHO tolerable daily intake (TDI). There is concern that short-term high level exposure to dioxins, furans, and PCB may cause biological effects on the human fetal development and further research is required. Further actions to control sources building on considerable advances already made in many countries may need to be supplemented by measures to prevent direct contamination of feeding stuff or food to reduce general population exposure further.

Hepatic vitamin a depletion is a sensitive marker of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in four rodent species.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-treated animals show altered retinoid homeostasis and exhibit signs of toxicity similar to those of vitamin A-deficient animals. In this study we established dose-response curves for sublethal oral doses of TCDD and hepatic vitamin A gain in four rodent species. This was done to evaluate any potential correlation between decreased hepatic vitamin A gain and other TCDD-induced effects, particularly depressed body weight gain and hepatic CYP1A induction. Young Hartley guinea pigs, Sprague-Dawley rats, C57BL/6 mice, and Golden Syrian hamsters were given single oral doses of TCDD at up to 2.5, 100, 1000, and 1000 microg/kg bw, respectively, and killed 28 days after treatment. Hepatic vitamin A gain was decreased 25% compared to controls at estimated doses of 0.1, 0.9, 1.1 and 3.6 microg/kg bw in guinea pigs, hamsters, rats, and mice, respectively. CYP1A induction and hepatic vitamin A gain were affected at similar dose levels and showed similar, but inverse dose-response curves in each of the four species, consistent with the hypothesis that altered vitamin A homeostasis is Ah-receptor mediated. In addition, there was an apparent correlation between the dose-response curves for decreased hepatic vitamin A gain and decreased body weight gain in all species. Taken together with the known importance of vitamin A in body weight regulation, this result was consistent with a contributing role for altered retinoid homeostasis in the wasting syndrome induced by TCDD.

Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife.

An expert meeting was organized by the World Health Organization (WHO) and held in Stockholm on 15-18 June 1997. The objective of this meeting was to derive consensus toxic equivalency factors (TEFs) for polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and dioxinlike polychlorinated biphenyls (PCBs) for both human, fish, and wildlife risk assessment. Based on existing literature data, TEFs were (re)evaluated and either revised (mammals) or established (fish and birds). A few mammalian WHO-TEFs were revised, including 1,2,3,7,8-pentachlorinated DD, octachlorinated DD, octachlorinated DF, and PCB 77. These mammalian TEFs are also considered applicable for humans and wild mammalian species. Furthermore, it was concluded that there was insufficient in vivo evidence to continue the use of TEFs for some di-ortho PCBs, as suggested earlier by Ahlborg et al. [Chemosphere 28:1049-1067 (1994)]. In addition, TEFs for fish and birds were determined. The WHO working group attempted to harmonize TEFs across different taxa to the extent possible. However, total synchronization of TEFs was not feasible, as there were orders of a magnitude difference in TEFs between taxa for some compounds. In this respect, the absent or very low response of fish to mono-ortho PCBs is most noticeable compared to mammals and birds. Uncertainties that could compromise the TEF concept were also reviewed, including nonadditive interactions, differences in shape of the dose-response curve, and species responsiveness. In spite of these uncertainties, it was concluded that the TEF concept is still the most plausible and feasible approach for risk assessment of halogenated aromatic hydrocarbons with dioxinlike properties.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the lymphatic absorption of a single oral dose of [3H]retinol and on the intestinal retinol esterification in the rat.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters the turnover of vitamin A in the body and inhibits the normal hepatic accumulation of dietary vitamin A. Vitamin A is absorbed in the small intestine, where it is incorporated into chylomicrons as retinyl esters for release into the lymph and further distributed via blood to the liver for storage. The aim of the present study was to investigate if the decreased hepatic vitamin A levels in TCDD-exposed rats could be due to impaired intestinal absorption of vitamin A via lymph. Male Sprague-Dawley rats were given a single oral dose of TCDD (10 microg/kg). Five days after administration, the main intestinal lymph duct of the rats was cannulated. After a 24-h recovery from surgery, the rats were each given a single dose of [3H]retinol in corn oil via gavage and the lymph was collected for 24 h. The cumulative radiolabel recovered in the intestinal lymph was significantly lower in TCDD-treated than in control rats during the first 6 h of absorption. However, no significant differences in radiolabel recovered in lymph were seen when looking at the entire 24-h collection period. In the intestinal mucosa, retinol esterification catalyzed by the enzyme lecithin:retinol acyl transferase (LRAT) or acyl coenzyme A (CoA):retinol transferase (ARAT) was not statistically different between the groups. However, mucosal retinyl palmitate levels were significantly increased in TCDD-treated rats. In conclusion, a small and transient reduction was found of the uptake of vitamin A into the lymph of TCDD-treated rats. It is obvious that this finding cannot explain the TCDD-induced decrease in hepatic vitamin A levels in the rat. Rather, a combination of inhibited retinol esterification in hepatic stellate cells, increased release of endogenous vitamin A, and increased hepatic catabolism of retinoids could explain the effect of TCDD on liver retinoid levels.

Pesticides and cancer.

Epidemiologic evidence on the relationship between chemical pesticides and cancer is reviewed. In animal studies, many pesticides are carcinogenic, (e.g., organochlorines, creosote, and sulfallate) while others (notably, the organochlorines DDT, chlordane, and lindane) are tumor promoters. Some contaminants in commercial pesticide formulations also may pose a carcinogenic risk. In humans, arsenic compounds and insecticides used occupationally have been classified as carcinogens by the International Agency for Research on Cancer. Human data, however, are limited by the small number of studies that evaluate individual pesticides. Epidemiologic studies, although sometimes contradictory, have linked phenoxy acid herbicides or contaminants in them with soft tissue sarcoma (STS) and malignant lymphoma; organochlorine insecticides are linked with STS, non-Hodgkin's lymphoma (NHL), leukemia, and, less consistently, with cancers of the lung and breast; organophosphorous compounds are linked with NHL and leukemia; and triazine herbicides with ovarian cancer. Few, if any, of these associations can be considered established and causal. Hence, further epidemiologic studies are needed with detailed exposure assessment for individual pesticides, taking into consideration work practices, use of protective equipment, and other measures to reduce risk.

Effect of 2,3,7,8-tetrachlorodibenzo-P-dioxin (TCDD) on the hepatic stellate cell population in the rat.

TCDD inhibits the normal accumulation of vitamin A in the hepatic stellate cells, which constitute the main storage site for vitamin A. In this study we investigated if the reduced capacity of stellate cells to store vitamin A could be due to cell transformation or cytotoxicity. Livers from rats exposed to TCDD were immunohistochemically stained for markers of normal and transformed stellate cells. The results show that the TCDD-induced inhibition of hepatic vitamin A accumulation is neither due to a reduction in the number of stellate cells nor to transformation of the cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin affects retinol esterification in rat hepatic stellate cells and kidney.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a highly toxic environmental contaminant, interferes with retinoid homeostasis. To elucidate the underlying mechanism, the activities of lecithin : retinol and acyl-CoA : retinol acyltransferase (LRAT and ARAT) were determined in liver, kidney, and hepatic parenchymal and nonparenchymal cell fractions from rats 7 days after a single oral dose of 10 µg TCDD/kg body weight (b.w). Severely depressed LRAT activity in hepatic stellate cells, and greatly increased LRAT activity in kidneys, as well as decreased ARAT activity in stellate cells, were seen in TCDD-treated rats. Although the relevance of decreased ARAT activity under physiological conditions is not clear, the changed LRAT activities most likely contributes significantly to the TCDD-induced effects on tissue retinyl ester levels. It is intriguing that TCDD affects LRAT activity in hepatic stellate cells and kidney in opposite directions. The results suggest that effects of TCDD on retinyl ester tissue levels could be due to a specific interaction with retinoid metabolism.

Organochlorine compounds and estrogen-related cancers in women.

The organochlorines, a diverse group of some 15,000 compounds, have been implicated increasingly as being harmful to humans. Some congeners of DDT and PCB elicit very weak estrogenic responses in animals, while the dioxin TCDD and related compounds have antiestrogenic properties. This review summarizes the evidence regarding whether certain organochlorine compounds, usually as persistent food-chain contaminants, increase the risk of breast and endometrial cancers through their estrogenic potential. In humans, neither ecologic data nor occupational studies provide clear support for an association between organochlorine exposure and the occurrence of these cancers. In our summary analysis of occupational exposure, the rate ratio of breast cancer for exposed cf unexposed women was 0.84 (95 percent confidence interval [CI] = 0.50-1.33) for PCBs and 1.08 (CI = 0.68-1.58) for TCDD. Similarly, effect estimates close to unity were found in summary analysis of breast cancer case-control studies regarding levels of DDE and PCB in adipose tissue or serum. In two recent nested case-control studies using stored specimens, the odds ratio per standard deviation increase in serum p,p'-DDE was 1.27 (CI = 0.95-1.69). Although estrogenic effects of certain organochlorine compounds should be easier to detect on the endometrium, we know of no analytic epidemiologic studies of endometrial cancer published to data. We conclude that available data do not indicate that organochlorines will affect the risk of these two cancers in any but the most unusual situation.

Organochlorine compounds in relation to breast cancer, endometrial cancer, and endometriosis: an assessment of the biological and epidemiological evidence.

There is an increasing public and scientific concern that certain chlorinated compounds, recognized as environmental pollutants, may cause estrogen-related neoplastic disease in humans. The main hypothesis has been that certain organochlorines, through their estrogenic actions, might cause breast cancer. From experimental studies, both in vitro and in vivo, there is evidence that certain organochlorine compounds may cause estrogenic effects, whereas others may cause antiestrogenic effects. In limited studies, some of these compounds in high doses have also been shown to increase and reduce the frequency of estrogen-related tumors in animals. The epidemiological findings regarding the association between organochlorines and breast cancer are inconclusive. However, the largest and best designed study has been interpreted as negative with respect to DDT and polychlorinated biphenyls (PCB) in relation to breast cancer. Associations between organochlorine exposure and endometrial cancer or endometriosis have even more limited empirical basis. The hypothesis that human exposure to environmental levels or organochlorines would favor an estrogenic overactivity leading to an increase in estrogen-dependent formation of mammary or endometrial tumors is not supported by the existing in vitro, animal and epidemiological evidence. It can, however, not be conclusively rejected on the basis of available data.

Swedish dioxin survey: evaluation of the H-4-II E bioassay for screening environmental samples for dioxin-like enzyme induction.

The H-4-II E enzyme induction bioassay was used for testing both pure reference substances and extracts of wildlife samples. Polychlorinated naphthalenes were found to be as active as enzyme inducers as certain coplanar polychlorinated biphenyls (PCBs). Also a mixture of polybrominated diphenyl ethers (Bromkal 70-5DE) was shown to induce enzyme activity. In extracts of herring, containing polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), bioassay and chemically derived TCDD-equivalents (TEQs) were nearly identical. When extracts containing other types of dioxin-like compounds as well were tested, the bioassay TEQs for most of them agreed well with chemical TEQs calculated for PCDDs, PCDFs and non-ortho PCBs. However, for ringed seal and whitefish, TEQs obtained from the bioassay were higher than those from the chemical analysis. Our results indicate that this bioassay is an excellent complement to chemical residue analysis and a useful tool in understanding the complex interactions of halogenated hydrocarbons. For risk assessment, such results should, however, be used most carefully as they are measured in vitro.

The distribution of [14C]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and its effect on the vitamin A content in parenchymal and stellate cells of rat liver.

Isolated liver cells from male Sprague-Dawley rats given a single dose of [14C]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 micrograms, 0.9 microCi/kg body wt in corn oil, p.o.) or vehicle only were separated into parenchymal and nonparenchymal cell fractions 4 h, and 1, 4, 7, 25, 50, and 147 d after treatment. Vitamin A content and TCDD-derived radioactivity were estimated in the parenchymal cells and in the stellate cells, which were identified and quantified in these fractions. Similar levels of vitamin A (0.3 +/- 0.4 nmol per million cells or 0.5 +/- 0.7 mumol per liver; values are mean +/- SD for 56 rats) were found in parenchymal cells from both control and TCDD-treated rats. However, while the vitamin A content of stellate cells increased from 14 to 46 nmol per million cells (i.e., from 1.7 to 7.7 mumol per liver) in control rats over the course of the study, stellate cells from TCDD-exposed rats showed no increase in vitamin A level until at least 25 d after exposure and remained at a level about 30% below the controls thereafter. TCDD-derived radioactivity resided mainly in the parenchymal cell compartment, although stellate cells contained more radioactivity per cell. Most of the radioactivity in parenchymal cells was eliminated with a half-life of 13 d, whereas the remainder persisted with an elimination half-life of 70 d. The elimination half-life in stellate cells was estimated to be 52 d. Thus, TCDD inhibited storage of vitamin A in stellate cells until 60-90% of the TCDD-derived radioactivity had been eliminated from the liver.