Wout Slob

Endocrine effects of tetrabromobisphenol-A (TBBPA) in Wistar rats as tested in a one-generation reproduction study and a subacute toxicity study.

Endocrine effects of the brominated flame retardant tetrabromobisphenol-A (TBBPA) were studied in a one-generation reproduction assay in Wistar rats via repeated dietary exposure, applying eight dose groups at 0-3-10-30-100-300-1,000-3,000 mg/kg body weight/day (mkd). This design enables dose-response analysis and calculation of benchmark doses (BMDL). This reproduction study was preceded by a 28-day repeat dose subacute toxicity study, at 0-30-100-300 mkd. Major effects in the reproduction study included decreased circulating thyroxine (T4) with BMDLs of 31 (m) and 16 (f) mkd, and increased weight of testis and male pituitary (BMDLs of 0.5 and 0.6 mkd). The hypothyroxinemia correlated to a cluster of developmental parameters including delayed sexual development in females, decreased pup mortality, and effects on brainstem auditory evoked potentials [Lilienthal, H., Verwer, C.M., Van der Ven, L.T.M., Piersma, A.H., Vos, J.G., 2008. Neurobehavioral effects of tetrabromobisphenol A (TBBPA) in rats after pre- and postnatal exposure. Toxicology]. A second cluster of parameters in F1 animals was correlated to increased testis weight, and included female gonad weight, endometrium height, CYP19/aromatase activity in the ovary, and plasma testosterone levels in males. These two correlation clusters suggest a dual action of TBBPA. The only effects in the subacute study were decreased circulating T4 and increased T3 levels in males (BMDLs 48 and 124mkd), and non-significant trends for these parameters in females, suggesting that the other effects in the reproduction study were induced during development. Combined with data of human exposure to environmental TBBPA, the margin of exposure for highly exposed populations can be calculated at 2.6, and current use of TBBPA may therefore be a matter of concern for human health.

Assessment factors for human health risk assessment: A discussion paper

The general goal of this discussion paper is to contribute toward the further harmonization of human health risk assessment. It first discusses the development of a formal, harmonized set of assessment factors. The status quo with regard to assessment factors is reviewed, that is, the type of factors to be identified, the range of values assigned, as well as the presence or absence of a scientific basis for these values. Options are presented for a set of default values and probabilistic distributions for assessment factors based on the state of the art. Methods of combining default values or probabilistic distributions of assessment factors are also described. Second, the effect parameter, the no-observed-adverse-effect level (NOAEL), is discussed. This NOAEL as selected from the toxicological database may be a poor substitute for the unknown, true no-adverse-effect level (NAEL). New developments are presented with respect to the estimation of the NAEL. The already widely discussed Benchmark Dose concept can be extended to obtain an uncertainty distribution of the Critical Effect Dose (CED). This CED distribution can be combined with estimated uncertainty distributions for assessment factors. In this way the full distribution of the Human Limit Value will be derived and not only a point estimate, whereas information on dose-response relations is taken into account. Finally, a strategy is proposed for implementation of the new developments into human health risk assessments.

Endocrine effects of hexabromocyclododecane (HBCD) in a one-generation reproduction study in Wistar rats.

The brominated flame retardant (BFR) hexabromocyclododecane was tested in a one-generation reproduction assay in Wistar rats, enhanced for endocrine parameters. A solution of the compound in corn oil was mixed in the feed, targeting at dietary exposure of 0-0.1-0.3-1-3-10-30-100 mg/kg body weight/day (mkd) in parental rats during 10 (males) or 2 (females) weeks premating, during gestation and lactation, and in their F1 offspring from weaning until final necropsy. Effects were assessed in F1 animals. Livers of these animals showed increased HBCD concentrations, in a dose-dependent way. The trabecular bone mineral density of the tibia was dose-dependently decreased in females (BenchMark Dose Lower confidence bound, BMDL=0.056 mkd). The IgG response after immunization with sheep red blood cells (SRBC) was increased in males (BMDL=0.46 mkd). Further sensitive effects were decreased weight of the testis (BMDL=1.5 mkd), increased fraction of neutrophilic granulocytes (BMDL=7.7 mkd), decreased concentration of apolar retinoids in female livers (BMDL=1.3 mkd), and decreased plasma alkaline phosphatase in females (BMDL=8.6 mkd). CYP19/aromatase activity in the ovary was correlated to the concentration of gamma-HBCD in the liver. A developmental origin of these effects is considered, and this is also true for sensitive effects observed in neurobehavioural testing in littermates from the same experiment, i.e. in the brainstem auditory evoked potentials and in a catalepsy test [Lilienthal, H., Van der Ven, L.T.M., Piersma, A.H., Vos, J.G. Neurobehavioral effects of the brominated flame retardant hexabromocyclododecane (HBCD) in rats after pre- and postnatal exposure, in press]. The low BMDLs of these effects may raise concern for human health, particularly when based on body burdens of HBCD, which leads to critical margins of exposure particularly for the occupational setting.

A quantitative method for assessing the sensitizing potency of low molecular weight chemicals using a local lymph node assay: Employment of a regression method that includes determination of the uncertainty margins

Risk assessment of sensitizing chemicals requires, besides hazard identification, the assessment of potency. To examine the sensitizing capacity of low molecular weight chemicals, a murine local lymph node assay (LLNA) was used. The sensitizing capacity of known allergens was quantified by dose-response modeling. At a stimulatory index (SI) of 3, the corresponding estimated concentration was calculated (EC(3)), together with a confidence interval to take account of the quality of the particular data set. We tested ten allergens (ethyl-p-aminobenzoate (benzocaine), diethylamine (DEA), 2,4-dinitrochlorobenzene (DNCB), 2-mercaptobenzothiazole (MBT), 4-ethoxymethylene 2-phenyl oxazol-5-one (oxazolone), phthalic anhydride (PA), toluene diisocyanate (TDI), trimellitic anhydride (TMA), tetramethylthiuramdisulfide (TMTD) and zincdimethyldithiocarbamate (ZDMC)). Oxazolone showed the strongest sensitizing potency followed in this order by DNCB, TDI, TMA, PA, TMTD, ZDMC, MBT, benzocaine and DEA. The approach performed in this study is a way to accurately assess the potency of sensitizing chemicals and thus a possibility for classification.

Developmental toxicity of butyl benzyl phthalate in the rat using a multiple dose study design

The developmental toxicity of butyl benzyl phthalate (BBP) was investigated in the rat using ten dose groups between 270 and 2100 mg/kg/day. Exposure was by daily gavage from gestation day 5 through 16 or gestation day 5 through 20. Dose-response data were analyzed using the benchmark approach by fitting dose-response models to the various endpoints. BBP induced increased liver and kidney weights in dams, accompanied by liver enzyme increases in maternal serum. Extramedullary hematopoiesis, which was already substantial in control pregnant animals, was increased after BBP treatment. Fetotoxicity included increased resorptions, reduced fetal weights, increased incidence of skeletal anomalies, and reduced fetal testis weights in the presence of an increased incidence of retarded testicular descent. As embryotoxicity was found at lower dosages compared to observed maternal toxicity, BBP appeared to be a specifically embryotoxic compound. The extended exposure protocol (gestation day 5 through 20) appeared more sensitive for measuring fetotoxic effects. We recommend the use of more doses in toxicity tests, together with the benchmark approach as an appropriate and more accurate method for analyzing dose-response data compared to the NOAEL approach.

A 28-day oral dose toxicity study enhanced to detect endocrine effects of a purified technical pentabromodiphenyl ether (pentaBDE) mixture in Wistar rats

A 28-day subacute oral toxicity study was performed in Wistar rats with a purified preparation of the commercial pentabromodiphenyl ether (pentaBDE), DE-71. The applied OECD407 protocol was enhanced for endocrine and immune parameters, and to enable benchmark dose analysis. A vehicle control group and 7 dose groups were included, which received 0.27, 0.82, 2.47, 7.4, 22.2, 66.7 or 200 mg pentaBDE/kg bw/d (mkd). The liver appeared to be a key target organ, showing a marked increase of weight and centrilobular hepatocellular hypertrophy, probably due to the observed induction of P450 enzymes, notably CYP1A and CYP2B. A marked decrease of circulating total thyroxine (TT4) and an increase of plasma cholesterol were probably secondary to the liver effects. Furthermore, dose-dependently decreased weight of epididymis, seminal vesicles, and prostate, as well as sperm head deformities in males, and induction of CYP17 activity in adrenals in females were observed, all possibly related to anti-androgenic activity. Finally, we observed a substantial increase of large unstained cells in the blood and a decrease of apolar retinoids in the liver. All these effects had benchmark doses at the lower confidence bound (BMDL) in the low- or mid-dose range, but particular sensitive, potentially adverse effects were TT4 decrease (BMDLs 1.1 in males and 1.8 mkd in females), and decrease of hepatic apolar retinoids (BMDLs 0.5 mkd in males and 2.3 mkd in females). These results contribute to refinement of the hazard identification of pentaBDE and improved risk assessment of human exposure to this industrial chemical and environmental pollutant.

Shape and steepness of toxicological dose–response relationships of continuous endpoints

Abstract A re-analysis of a large number of historical dose–response data for continuous endpoints indicates that an exponential or a Hill model with four parameters adequately describes toxicological dose–responses. No exceptions were found for the datasets considered, which related to a wide variety of endpoints and to both in vivo and in vitro studies of various types. For a given endpoint/study type dose–response shapes were found to be homogenous among chemicals in the in vitro studies considered, while a mild among-chemical variation in the steepness parameter seemed to be present in the in vivo studies. Our findings have various practical consequences. For continuous endpoints, model selection in the BMD approach is not a crucial issue. The often applied approach of using constraints on the model parameters to prevent “infinite” slopes at dose zero in fitting a model is not in line with our findings, and appears to be unjustified. Instead, more realistic ranges of parameter values could be derived from re-analyses of large numbers of historical dose–response datasets in the same endpoint and study type, which could be used as parameter constraints in future individual datasets. This approach will be particularly useful for weak datasets (e.g. few doses, much scatter). In addition, this approach may open the way to use fewer animals in future studies. In the discussion, we argue that distinctions between linear, sub/supralinear or thresholded dose–response shapes, based on visual inspection of plots, are not biologically meaningful nor useful for risk assessment.

Risk Assessment of Deoxynivalenol in Food: Concentration Limits, Exposure and Effects

The mycotoxin, deoxynivalenol (DON), is produced world-wide by the Fusarium genus in different cereal crops. We derived a provisional TDI of 1.1 microg/kg body weight (bw) and proposed a concentration limit of 129 microg DON/kg wheat based on this TDI and a high wheat consumption of children. In the period September 1998-January 2000, the average DON concentration in wheat was 446 microg/kg (n = 219) in The Netherlands. During this period, the dietary intake of DON exceeded the provisional TDI, especially in children. Eighty percent of the one-year-olds showed a DON intake above the provisional TDI and 20% of these children exceeded twice the provisional TDI. Our probabilistic effect assessment shows that at these exposure levels, health effects may occur. Suppressive effects on body weights and relative liver weight were estimated at 2.2 and 2.7%. However, the large confidence intervals around these estimates indicated that the magnitudes of these effects are uncertain.

Deriving a Data-Based Interspecies Assessment Factor Using the NOAEL and the Benchmark Dose Approach

In deriving human health-based exposure limits from animal data, differences in sensitivity to a compound between animals and humans must be taken into account. These interspecies differences can be caused by differences in toxicokinetics and or toxicodynamics. Apart from that, species differ in body size, and this is usually accounted for by scaling doses to body weight (i.e., expressed as mg/kg body weight1.0/day).Adefault assessmentfactor (AF) of 10 is commonly applied to this dose metric to account for potential toxicokinetic and toxicodynamic differences. However, both proportional body weight (BW)scalingand the defaultAFas often applied are not directly based on empirical findings. Attempts have been made to derive data-based assessment factors and allometric scaling powers using various toxicological values such as no-observedadverse-effect-levels (NOAELs). In thisstudy both the NOAEL approach and the benchmark dose (BMD) approach are applied to deriveNOAEL ratios and BMD ratios from mouse and rat studies and, based on that information, toestimate an allometric scaling power and an interspecies AF. To account for interspecies differences in body size, our results confirm earlier findings that allometric body weight scaling with a power of around 0.7 is appropriate. The factor needed to rescale the dose in terms of mg/kgBWto the allometric dose scale ranges from around 1.7 (for dogs) to 10(for mice), similar to other findings. The additional factor required for taking into account interspecies toxicokinetic and toxicodynamic differences, when based on the 95th percentile of the relevant ratio distribution, would be 3.1 for a lower Confidence limit of theBMD (BMDL), and 8.3 for a NOAEL (to be applied to the allometrically scaled dose). These results indicate that the generally used defaultAFof 10 may not cover potential interspecies differences, in particular when applied to results from smaller test species. Therefore, using the default AF of 10 could lead to human exposure limits that are insufficiently protective. Further, our results show that a data-based AF that would be needed for interspecies extrapolation is smaller when the point of departure is aBMDLrather than a NOAEL. In the context of a probabilistic hazard characterization, our results indicate that the (geometric) SD of the interspecies AF distribution should be around 2.0 when the BMDL (or BMD uncertainty distribution) is used, and around 3.4 when the NOAEL is used as a point of departure for further risk assessment.

A 28-day oral dose toxicity study in Wistar rats enhanced to detect endocrine effects of decabromodiphenyl ether (decaBDE)

Decabromodiphenyl ether (decaBDE) is a widely used brominated flame retardant, considered to be of low toxicity. However, previous toxicity studies applied exposure methods with low bioavailability of this compound, and the actual hazard of decaBDE for humans, which are environmentally exposed to decaBDE, may thus be underestimated in current risk assessments. The present 28 days oral toxicity study in Wistar rats was designed to facilitate detection of endocrine and immune modulating effects of decaBDE using an exposure protocol with improved bioavailability. A technical preparation of high purity decaBDE was thus tested by daily exposure through gavage with an emulsion of soy phospholipon/lutrol as a carrier. Most sensitive effect in males were increased weight of seminal vesicle/coagulation gland with BMDL of 0.2mg/kg bw/day and increased expression of hepatic CYP1A and CYP2B (BMDLs 0.5-0.7 mg/kg bw/day). In females the most sensitive effect was decreased activity of P450c17 (CYP17), which is a key enzyme in the androgen synthesis pathway, in adrenals (BMDL 0.18 mg/kg bw/day). These results suggest that decaBDE may represent an as yet unreported hazard for reproductive health.