Experiments were conducted to assess the in vivo potency of binary mixtures of estrogenic chemicals using plasma vitellogenin (VTG) concentrations in juvenile rainbow trout (Oncorhynchus mykiss) as ...the endpoint. The estrogenic potencies of estradiol-17β (E2), 4-tert-nonylphenol (NP), and methoxychlor (MXC) were determined following 14 day exposures to the individual chemicals and binary mixtures of these chemicals. E2, NP, and MXC all induced concentration dependent increases in plasma VTG, with lowest observed effect concentrations of 4.7 and 7.9 ng L-1 for E2, 6.1 and 6.4 μg L-1 for NP, and 4.4 and 6.5 μg L-1 for MXC. Concentration−response curves for fixed ratio binary mixtures of E2 and NP (1:1000), E2 and MXC (1:1000), and NP and MXC (1:1) were compared to those obtained for the individual chemicals, using the model of concentration addition. Mixtures of E2 and NP were additive at the concentrations tested, but mixtures of E2 and MXC were less than additive. This suggests that while NP probably acts via the same mechanism as E2 in inducing VTG synthesis, MXC may be acting via a different mechanism(s), possibly as a result of its conversion to HPTE which is an estrogen receptor α agonist and an estrogen receptor β antagonist. It was not possible to determine whether mixtures of MXC and NP were additive using VTG induction, because the toxicity of MXC restricted the effect range for which the expected response curve for the binary mixture could be calculated. The data presented illustrate that the model of concentration addition can accurately predict effects on VTG induction, where we know that both chemicals act via the same mechanism in mediating a vitellogenic response.
Pain is a major symptom in many medical conditions, and often interferes significantly with a person’s quality of life. Although a priority topic in medical research for many years, there are still ...few analgesic drugs approved for clinical use. One reason is the lack of appropriate animal models that faithfully represent relevant hallmarks associated with human pain. Here we propose zebrafish (Danio rerio) as a novel short-term behavioral model of nociception, and analyse its sensitivity and robustness. Firstly, we injected two different doses of acetic acid as the noxious stimulus. We studied individual locomotor responses of fish to a threshold level of nociception using two recording systems: a video tracking system and an electric biosensor (the MOBS system). We showed that an injection dose of 10% acetic acid resulted in a change in behavior that could be used to study nociception. Secondly, we validated our behavioral model by investigating the effect of the analgesic morphine. In time-course studies, first we looked at the dose-response relationship of morphine and then tested whether the effect of morphine could be modulated by naloxone, an opioid antagonist. Our results suggest that a change in behavioral responses of zebrafish to acetic acid is a reasonable model to test analgesics. The response scales with stimulus intensity, is attenuated by morphine, and the analgesic effect of morphine is blocked with naloxone. The change in behavior of zebrafish associated with the noxious stimulus can be monitored with an electric biosensor that measures changes in water impedance.
To support a mixture risk assessment with a focus on male reproductive health, we conducted a systematic review of associations between bisphenol A (BPA) exposures and declines in semen quality, ...based on animal and epidemiological studies. Contrary to a widely held view that there is “conflicting” evidence of such associations, our review and confidence rating approach reveals that animal studies provide convincing evidence of declines of semen quality after gestational BPA exposures. Many of the reported negative findings can be attributed to deficiencies in study sensitivity, insufficient control of background contamination and probable confounding through hormonal interference due to the use of soy-containing diets. We did not evaluate animal studies of adult BPA exposures. Divergent findings in “medium to high” and “medium” confidence epidemiological studies can be explained in terms of differences in exposure conditions. We attempted the estimation of a BPA reference dose based on animal studies. Due to variations in the no-observed adverse effect levels (NOAELs) in high confidence studies, possible reference doses ranged from 0.0001 to 0.0099 μg/kg/d. In choosing 0.003 μg/kg/d we struck a balance between caution suggested by studies at the lower end of the doses and the weight of evidence from studies with higher NOAELs. This weighting was motivated by the intended use of the value in a mixture risk assessment which meant arriving at a reasonable estimate of BPA exposures likely without effects on semen quality. We realise that our approach does not conform with the standards necessary for deriving tolerable daily intakes (TDIs) for single chemical exposures, which is not our interest here. BPA exposures currently experienced by European populations and beyond are in excess of 0.003 μg/kg/d and even fall in the range where some epidemiological studies observed effects on semen quality as a result of BPA exposures in adulthood.
•Mixture risk assessments of declining semen quality require a reference dose for bisphenol A.•To derive a bisphenol A reference dose for poor semen quality we used a systematic review with confidence rating approach.•We found convincing evidence of bisphenol A and poor semen quality in animal studies after gestational exposures.•Divergent findings in epidemiological studies can be explained by different exposure conditions.•0.003 μg/kg/day is derived from animal studies as a reference dose for poor semen quality for mixture risk assessments of male reproductive health.
The downward revision of the bisphenol A (BPA) Health-based Guidance Value (HBGV) by the European Food Safety Authority (EFSA) has led to disagreements with other regulatory agencies, among them the ...German Federal Institute for Risk Assessment (BfR). The BfR has recently published an alternative Tolerable Daily Intake (TDI), 1000-times higher than the EFSA HBGV of 0.2 ng/kg/d. While the EFSA value is defined in relation to immunotoxicity, the BfR alternative TDI is based on declines in sperm counts resulting from exposures in adulthood. Earlier, we had used semen quality deteriorations to estimate a BPA Reference Dose (RfD) of 3 ng/kg/d for use in mixture risk assessments of male reproductive health. We derived this estimate from animal studies of gestational BPA exposures which both EFSA and BfR viewed as irrelevant for human hazard characterisations. Here, we identify factors that drive these diverging views. We find that the fragmented, endpoint-oriented study evaluation system used by EFSA and BfR, with its emphasis on data that can support dose-response analyses, has obscured the overall BPA effect pattern relevant to male reproductive effects. This has led to a disregard for the effects of gestational BPA exposures. We also identify problems with the study evaluation schemes used by EFSA and BfR which leads to the omission of entire streams of evidence from consideration. The main driver of the diverging views of EFSA and BfR is the refusal by BfR to accept immunotoxic effects as the basis for establishing an HBGV. We find that switching from immunotoxicity to declines in semen quality as the basis for deriving a BPA TDI by deterministic or probabilistic approaches produces values in the range of 2.4-6.6 ng/kg/d, closer to the present EFSA HBGV of 0.2 ng/kg/d than the BfR TDI of 200 ng/kg/d. The proposed alternative BfR value is the result of value judgements which erred on the side of disregarding evidence that could have supported a lower TDI. The choices made in terms of selecting key studies and methods for dose-response analyses produced a TDI that comes close to doses shown to produce effects on semen quality in animal studies and in human studies of adult BPA exposures.
House dust contains many organic contaminants that can compete with the thyroid hormone (TH) thyroxine (
) for binding to transthyretin (TTR). How these contaminants work together at levels found in ...humans and how displacement from TTR
relates to
-TTR binding is unknown.
Our aims were to determine the TTR-binding potency for contaminant mixtures as found in house dust, maternal serum, and infant serum; to study whether the TTR-binding potency of the mixtures follows the principle of concentration addition; and to extrapolate the
TTR-binding potency to
inhibition levels of
-TTR binding in maternal and infant serum.
Twenty-five contaminants were tested for their
capacity to compete for TTR-binding with a fluorescent FITC-
probe. Three mixtures were reconstituted proportionally to median concentrations for these chemicals in house dust, maternal serum, or infant serum from Nordic countries. Measured concentration-response curves were compared with concentration-response curves predicted by concentration addition. For each reconstituted serum mixture, its inhibitor-TTR dissociation constant (
) was used to estimate inhibition levels of
-TTR binding in human blood.
The TTR-binding potency of the mixtures was well predicted by concentration addition. The
inhibition in FITC-
binding observed for the mixtures reflecting median concentrations in maternal and infant serum was extrapolated to 1.3% inhibition of
-TTR binding in maternal and 1.5% in infant blood. For nontested mixtures reflecting high-end serum concentrations, these estimates were 6.2% and 4.9%, respectively.
The relatively low estimated inhibition levels at median exposure levels may explain why no relationship between exposure to TTR-binding compounds and circulating
levels in humans has been reported, so far. We hypothesize, however, that 1.3% inhibition of
-TTR binding may ultimately be decisive for reaching a status of maternal hypothyroidism or hypothyroxinemia associated with impaired neurodevelopment in children. https://doi.org/10.1289/EHP5911.
A key question in the risk assessment of exposures to multiple chemicals is whether mixture effects may occur when chemicals are combined at low doses which individually do not induce observable ...effects. However, a systematic evaluation of experimental studies addressing this issue is missing.
With this contribution, we wish to bridge this gap by providing a systematic assessment of published studies against well-defined quality criteria.
On reviewing the low-dose mixture literature, we found good evidence demonstrating significant mixture effects with combinations of chemicals well below their individual no observable adverse effect levels (NOAELs), both with mixtures composed of similarly and dissimilarly acting agents.
The widely held view that mixtures of dissimilarly acting chemicals are "safe" at levels below NOAELs is not supported by empirical evidence. We show that this view is also based on the erroneous assumption that NOAELs can be equated with zero-effect levels. Thus, on the basis of published evidence, it is difficult to rule out the possibility of mixture effects from low-dose multiple exposures.
The risks associated with human exposures to chemicals capable of antagonising the effects of endogenous androgens have attracted considerable recent interest. Exposure is typically to large numbers ...of chemicals with androgen receptor (AR) antagonist activity, yet there is limited evidence of the combined effects of multi-component mixtures of these chemicals. A few
in vitro studies with mixtures of up to six AR antagonists suggest that the concept of concentration addition (CA) provides good approximations of experimentally observed mixture effects, but studies with larger numbers of anti-androgens, and with more varied structural features, are missing. Here we show that the mixture effects of up to 17 AR antagonists, comprising compounds as diverse as UV-filter substances, parabens, perfluorinated compounds, bisphenol-A, benzo(α)pyrene, synthetic musks, antioxidants and polybrominated biphenyls, can be predicted well on the basis of the anti-androgenicity of the single components using the concept of CA. We tested these mixtures in an
in vitro AR-dependent luciferase reporter gene assay, based on MDA-kb2 cells. The effects of further mixtures, composed of four and six anti-androgens, could be predicted accurately by CA. However, there was a shortfall from expected additivity with a ten-component mixture at two different mixture ratios, but attempts to attribute these deviations to differential expression of hormone-metabolising CYP isoforms did not produce conclusive results. CA provides good approximations of
in vitro mixture effects of anti-androgens with varying structural features.
► Humans are exposed to a large number of androgen receptor antagonists. ► There is limited evidence of the combined effects of anti-androgenic chemicals. ► We modelled the predictability of combined effects of up to 17 anti-androgens. ► We tested the mixture predictions in an
in vitro reporter gene assay. ► Dose addition provides good approximations of mixture effects of anti-androgens.
The way in which mixture risk assessment (MRA) should be included in chemical risk assessment is a current topic of debate. We used data from 67 recent pesticide evaluations to build a case study ...using Hazard Index calculations to form risk estimates in a tiered MRA approach in line with a Framework proposed by WHO/IPCS. The case study is used to illustrate the approach and to add detail to the existing Framework, and includes many more chemicals than previous case studies.
A low-tier MRA identified risk as being greater than acceptable, but refining risk estimates in higher tiers was not possible due to data requirements not being readily met. Our analysis identifies data requirements, which typically expand dramatically in higher tiers, as being the likely cause for an MRA to fail in many realistic cases. This forms a major obstacle to routine implementation of MRA and shows the need for systematic generation and collection of toxicological data. In low tiers, hazard quotient inspection identifies chemicals that contribute most to the HI value and thus require attention if further refinement is needed. Implementing MRA requires consensus on issues such as scope setting, criteria for performing refinement, and decision criteria for actions.
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•Case study using JMPR data from 2006 to 2010 for 67 pesticides to illustrate options for mixture risk assessment (MRA).•Using Hazard Index (HI) approach allows data gaps and critical values/drivers to be identified.•Higher tiers in MRA have substantial data requirements, which can grow as a multiple of the number of endpoints considered.•Selection of realistic approaches can simplify MRA and allow resources to be concentrated where they are most needed.
Many xenobiotics have been identified as in vitro androgen receptor (AR) antagonists, but information about their ability to produce combined effects at low concentrations is missing. Such data can ...reveal whether joint effects at the receptor are induced at low levels and may support the prioritisation of in vivo evaluations and provide orientations for the grouping of anti-androgens in cumulative risk assessment. Combinations of 30 AR antagonists from a wide range of sources and exposure routes (pesticides, antioxidants, parabens, UV-filters, synthetic musks, bisphenol-A, benzo(a)pyrene, perfluorooctane sulfonate and pentabromodiphenyl ether) were tested using a reporter gene assay (MDA-kb2). Chemicals were combined at three mixture ratios, equivalent to single components' effect concentrations that inhibit the action of dihydrotesterone by 1%, 10% or 20%. Concentration addition (CA) and independent action were used to calculate additivity expectations. We observed complete suppression of dihydrotestosterone effects when chemicals were combined at individual concentrations eliciting 1%, 10% or 20% AR antagonistic effect. Due to the large number of mixture components, the combined AR antagonistic effects occurred at very low concentrations of individual mixture components. CA slightly underestimated the combined effects at all mixture ratios. In conclusion, large numbers of AR antagonists from a wide variety of sources and exposure routes have the ability of acting together at the receptor to produce joint effects at very low concentrations. Significant mixture effects are observed when chemicals are combined at concentrations that individually do not induce observable AR antagonistic effects. Cumulative risk assessment for AR antagonists should apply grouping criteria based on effects where data are available, rather than on criteria of chemical similarity.
•Mixtures of AR antagonists at low individual concentrations cause complete inhibition.•Concentration addition was an appropriate prediction model for observed effects.•Risk assessment for AR antagonists should use grouping criteria based on effects.
Several countries have experienced rises in cryptorchidisms, hypospadias and testicular germ cell cancer. The reasons for these trends are largely unknown, but Skakkebaek has proposed that these ...disorders form a testicular dysgenesis syndrome and can be traced to androgen insufficiency in foetal life. This suggests that antiandrogenic chemicals might contribute to risks, but few chemicals have been linked to these diseases in epidemiological studies. In animal studies with p,p'-dichlorodiphenyldichloroethylene, effects typical of disruptions of male sexual differentiation became apparent when the foetal levels of this androgen receptor (AR) antagonist approached values associated with responses in in vitro assays. This prompted us to analyse whether the 22 chemicals with AR antagonistic properties would produce mixture effects in an in vitro AR antagonism assay when combined at concentrations found in human serum. Other antiandrogenic modalities could not be considered. Two scenarios were investigated, one representative of average serum levels reported in European countries, the other in line with levels towards the high exposures. In both situations, the in vitro potency of the 22 selected AR antagonists was too low to produce combined AR antagonistic effects at the concentrations found in human serum, although the high exposure scenario came quite close to measurable effects. Nevertheless, our analysis exposes an explanation gap which can only be bridged by conjuring up as yet undiscovered high potency AR antagonists or, alternatively, high exposures to unknown agents of average potency.
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