Review of 2,4-dichlorophenoxyacetic acid (2,4-D) biomonitoring and epidemiology

A qualitative review of the epidemiological literature on the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and health after 2001 is presented. In order to compare the exposure of the general population, bystanders and occupational groups, their urinary levels were also reviewed. In the general population, 2,4-D exposure is at or near the level of detection (LOD). Among individuals with indirect exposure, i.e. bystanders, the urinary 2,4-D levels were also very low except in individuals with opportunity for direct contact with the herbicide. Occupational exposure, where exposure was highest, was positively correlated with behaviors related to the mixing, loading and applying process and use of personal protection. Information from biomonitoring studies increases our understanding of the validity of the exposure estimates used in epidemiology studies. The 2,4-D epidemiology literature after 2001 is broad and includes studies of cancer, reproductive toxicity, genotoxicity, and neurotoxicity. In general, a few publications have reported statistically significant associations. However, most lack precision and the results are not replicated in other independent studies. In the context of biomonitoring, the epidemiology data give no convincing or consistent evidence for any chronic adverse effect of 2,4-D in humans.     

Review of 2,4-dichlorophenoxyacetic acid (2,4-D) biomonitoring and epidemiology

A qualitative review of the epidemiological literature on the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and health after 2001 is presented. In order to compare the exposure of the general population, bystanders and occupational groups, their urinary levels were also reviewed. In the general population, 2,4-D exposure is at or near the level of detection (LOD). Among individuals with indirect exposure, i.e. bystanders, the urinary 2,4-D levels were also very low except in individuals with opportunity for direct contact with the herbicide. Occupational exposure, where exposure was highest, was positively correlated with behaviors related to the mixing, loading and applying process and use of personal protection. Information from biomonitoring studies increases our understanding of the validity of the exposure estimates used in epidemiology studies. The 2,4-D epidemiology literature after 2001 is broad and includes studies of cancer, reproductive toxicity, genotoxicity, and neurotoxicity. In general, a few publications have reported statistically significant associations. However, most lack precision and the results are not replicated in other independent studies. In the context of biomonitoring, the epidemiology data give no convincing or consistent evidence for any chronic adverse effect of 2,4-D in humans.     

Human milk biomonitoring data: interpretation and risk assessment issues

Biomonitoring data can, under certain conditions, be used to describe potential risks to human health (for example, blood lead levels used to determine children's neurodevelopmental risk). At present, there are very few chemical exposures at low levels for which sufficient data exist to state with confidence the link between levels of environmental chemicals in a person's body and his or her risk of adverse health effects. Human milk biomonitoring presents additional complications. Human milk can be used to obtain information on both the levels of environmental chemicals in the mother and her infant's exposure to an environmental chemical. However, in terms of the health of the mother, there are little to no extant data that can be used to link levels of most environmental chemicals in human milk to a particular health outcome in the mother. This is because, traditionally, risks are estimated based on dose, rather than on levels of environmental chemicals in the body, and the relationship between dose and human tissue levels is complex. On the other hand, for the infant, some information on dose is available because the infant is exposed to environmental chemicals in milk as a "dose" from which risk estimates can be derived. However, the traditional risk assessment approach is not designed to consider the benefits to the infant associated with breastfeeding and is complicated by the relatively short-term exposures to the infant from breastfeeding. A further complexity derives from the addition of in utero exposures, which complicates interpretation of epidemiological research on health outcomes of breastfeeding infants. Thus, the concept of "risk assessment" as it applies to human milk biomonitoring is not straightforward, and methodologies for undertaking this type of assessment have not yet been fully developed. This article describes the deliberations of the panel convened for the Technical Workshop on Human Milk Surveillance and Biomonitoring for Environmental Chemicals in the United States, held at the Hershey Medical Center, Pennsylvania State College of Medicine, on several issues related to risk assessment and human milk biomonitoring. Discussion of these topics and the thoughts and conclusions of the panel are described in this article.     

Sensory irritation: risk assessment approaches

Irritation of eyes and upper airways--sensory irritation--is commonly used as a parameter for setting occupational exposure limits and is a common complaint in occupants of non-industrial buildings. Sensory irritation occurs from stimulation of receptors on trigeminal nerves. In general, chemically reactive compounds are more potent than non-reactive congeners. Animal studies allow prediction of sensory irritation effects in humans; the concentration-effect relationships are often steep. In humans, thresholds and suprathreshold effects can be obtained from short-term ( approximately seconds) exposures and from longer exposures ( approximately hours). Sensory irritation may develop over time and odour cues may influence reported sensory irritation symptoms; generally, the slope of the irritant effect is steeper than the slope of odour cues. A best available no-observed-adverse-effect level (NOAEL) should be based on a combined estimate from the three types of study. The NOAEL/5 is considered sufficient to protect individuals not especially sensitive. The present knowledge suggests that especially sensitive individuals may be protected by an additional uncertainty factor (UF) of 2, suggesting a combined UF of 10. In published studies, the combined UF is up to 300, highlighting the need of evidence-based UFs. Combined effects of sensory irritants can be considered additive as a first approximation.     

Comparison of the cytotoxicity and DNA-damaging properties of 2,4-D and U 46 D Fluid (dimethylammonium salt of 2,4-D)

U 46 D Fluid (the dimethylammonium salt of 2,4-dichlorophenoxyacetic acid in a commercial formulation) was more toxic to human fibroblasts than 2,4-dichlorophenoxyacetic acid (2,4-D). Moreover, U 46 D Fluid induced single-strand breaks at apurinic/apyrimidinic (AP) sites of heat-acid treated PM2 DNA while 2,4-D did not. The¹H-NMR spectrum of the dimethylammonium (DMA) salt of 2,4-D indicated the formation of a complex via a six-membered aggregate of the DMA ion and the acid group of 2,4-D. The role of complex formation of the DMA salt of 2,4-D as a cause of single-strand breaking activity and increased cytotoxicity is discussed.     

Benchmark dose approaches in chemical health risk assessment in relation to number and distress of laboratory animals

Use of benchmark dose (BMD) approaches is expected to increase substantially, with growing awareness among researchers and inclusion in regulatory testing guidance documents such as REACH. The BMD approach has clear advantages over the No-Observed-Adverse-Effect-Level (NOAEL) approach in defining toxicological thresholds, risk levels, and points of departure as the basis for setting guidance and limit values. Several aspects of the BMD may increase the use of laboratory animals; the optimal number of dose groups for BMD calculation is between five and ten, rather than the current standard of four; also, experiments with more animals will result in narrow confidence intervals. However, this paper presents several counterarguments suggesting that design of experiments suited for BMD analyses might be used to decrease the distress and use of laboratory animals. If experiments are performed with unequal group size, with fewer animals in the high response dose groups and more animals close to toxicological threshold, the aggregated distress might be reduced. In addition, there is a need to evaluate how the total number of animals affects the quality of BMD (e.g. in terms of confidence intervals). Development of strategies for optimal design of experiments requires tools which evaluate experimental designs from an ethical perspective; a concept of distress-adjusted number of animals is suggested.     

Benchmark dose approaches in chemical health risk assessment in relation to number and distress of laboratory animals

Use of benchmark dose (BMD) approaches is expected to increase substantially, with growing awareness among researchers and inclusion in regulatory testing guidance documents such as REACH. The BMD approach has clear advantages over the No-Observed-Adverse-Effect-Level (NOAEL) approach in defining toxicological thresholds, risk levels, and points of departure as the basis for setting guidance and limit values. Several aspects of the BMD may increase the use of laboratory animals; the optimal number of dose groups for BMD calculation is between five and ten, rather than the current standard of four; also, experiments with more animals will result in narrow confidence intervals. However, this paper presents several counterarguments suggesting that design of experiments suited for BMD analyses might be used to decrease the distress and use of laboratory animals. If experiments are performed with unequal group size, with fewer animals in the high response dose groups and more animals close to toxicological threshold, the aggregated distress might be reduced. In addition, there is a need to evaluate how the total number of animals affects the quality of BMD (e.g. in terms of confidence intervals). Development of strategies for optimal design of experiments requires tools which evaluate experimental designs from an ethical perspective; a concept of distress-adjusted number of animals is suggested.     

Exposure assessment and risk of gastrointestinal illness among surfers

Surfing is a unique recreational activity with the possibility of elevated risk for contracting gastrointestinal (GI) illness through ingestion of contaminated water. No prior studies have assessed exposure from ingestion among surfing populations. This study estimated the magnitude and frequency of incidental water ingestion using a Web-based survey and integrated exposure distributions with enterococci distributions to predict the probability of GI illness at six Oregon beaches. The mean exposure magnitude and frequency were 170 ml of water ingested per day and 77 days spent surfing per year, respectively. The mean number of enterococci ingested ranged from approximately 11 to 86 colony-forming units (CFU) per day. Exposure-response analyses were conducted using an ingested dose model and two epidemiological models. Risk was characterized using joint probability curves (JPC). At the most contaminated beach, the annualized ingested dose model estimated a mean 9% probability of a 50% probability of GI illness, similar to the results of the first epidemiological model (mean 6% probability of a 50% probability of GI illness). The second epidemiological model predicted a 23% probability of exceeding an exposure equivalent to the U.S. Environmental Protection Agency (EPA) maximum acceptable GI illness rate (19 cases/1000 swimmers). While the annual risk of GI illness for Oregon surfers is not high, data showed that surfers ingest more water compared to swimmers and divers and need to be considered in regulatory and public health efforts, especially in more contaminated waters. Our approach to characterize risk among surfers is novel and informative to officials responsible for advisory programs. It also highlights the need for further research on microbial dose-response relationships to meet the needs of quantitative microbial risk assessments (QMRA).     

Comparison of cancer risk estimates based on a variety of risk assessment methodologies

The EPA guidelines recommend a benchmark dose as a point of departure (PoD) for low-dose cancer risk assessment. Generally the PoD is the lower 95% confidence limit on the dose estimated to produce an extra lifetime cancer risk of 10% (LTD(10)). Due to the relatively narrow range of doses in two-year bioassays and the limited range of statistically significant tumor incidence rates, the estimate of the LTD(10) is constrained to a relatively narrow range of values. Because of this constraint, simple, quick estimates of the LTD(10) can be readily obtained for hundreds of rodent carcinogens from the Carcinogenic Potency Database (CPDB) of Gold et al. Three estimation procedures for LTD(10) are described, using increasing information from the CPDB: (A) based on only the maximum tolerated dose (the highest dose tested); (B) based on the TD(50); and (C) based on the TD(50) and its lower 99% confidence limit. As expected, results indicate overall similarity of the LTD(10) estimates and the value of using additional information. For Method (C) the estimator based on the [[(TD(50))(0.36) x (LoConf)(0.64)]/6.6] is generally similar to the estimator based on the one-hit model or multistage model LTD(10). This simple estimate of the LTD(10) is applicable for both linear and curved dose responses with high or low background tumor rates, and whether the confidence limits on the TD(50) are wide or tight. The EPA guidelines provide for a margin of exposure approach if data are sufficient to support a nonlinear dose-response. The reference dose for cancer for a nonlinear dose-response curve based on a 10,000-fold uncertainty (safety) factor from the LTD(10), i.e., the LTD(10)/10,000, is mathematically equivalent to the value for a linear extrapolation from the LTD(10) to the dose corresponding to a cancer risk of <10(-5) (LTD(10)/10,000). The cancer risk at <10(-5) obtained by using the q(1)(*) from the multistage model, is similar to LTD(10)/10,000. For a nonlinear case, an uncertainty factor of less than 10,000 is likely to be used, which would result in a higher (less stringent) acceptable exposure level.     

Comparison of two approaches to amitriptyline dose individualisation.

Individualisation of an amitriptyline dose regimen offers substantial advantages over non-individualised treatment. In our study, we have compared both clinical effects, adverse effects and plasma steady-state concentrations of amitriptyline in 15 patients with major depressive disorder divided in three groups; (i) patients in group A were taking non-individualised doses of amitriptyline; (ii) patients in group B were taking doses of amitriptyline individualised by modified Bayesian method; and (3) patients in group C were taking doses of amitriptyline individualised by the multiple point method. The treatment course was 8 weeks long, in the setting of a psychiatric clinic. The patients in group A were taking significantly higher doses throughout the treatment course; the initial doses for the patients in group B were higher than doses for the patients in group C, but after corrections based on measured steady-state plasma concentrations they became similar. While Hamilton score descended uniformly in all three groups, both adverse effects and steady-state plasma concentrations of amitriptyline were higher in non-individualised group during the whole treatment course. The results of our study suggest that the multiple points method is the most precise, but tedious and not practical. The modified Bayesian method with correction based on first measured plasma steady-state concentration of amitriptyline offers similar therapeutic outcome and adverse effects score combined with low cost and being easy-to-use.     

On the assessment of dose proportionality: a comparison of two slope approaches

The problem for assessment of dose proportionality (or linearity) is studied. Various methods for assessment of dose proportionality (or linearity) such as ANOVA type F-test have been proposed. Cheng et al. (2006) proposed an alternative approach based on the slopes of adjacent dose levels under a crossover design. They showed that when dose proportionality (or linearity) cannot be established, their proposed slope approach is useful for evaluation of the degree of departure from dose proportionality (or linearity). In this article, we propose the use of slopes between the dose level and the initial dose (baseline), which we refer to as the baseline slope approach. The two slope approaches are compared under a parallel group design by means of an ANOVA type F-test and other tests. Simulation studies show that the proposed method has a satisfactory small sample performance.     

Skin irritation and sensitization: mechanisms and new approaches for risk assessment

Allergic contact dermatitis (ACD) is a common skin disease with a significant social and economic impact. In contrast to irritation, skin sensitization is a response of the adaptive immune system, in which there is a delayed T-cell-mediated allergic response to chemically modified skin proteins. The chemicals that can covalently modify the skin proteins and trigger an allergic reaction are referred to as haptens or sensitizers. Attempts have been made in many countries to reduce the problems of ACD by the implementation of legislations related to skin-sensitizing chemicals, as well as by the early detection and risk assessment of substances with sensitizing properties. For many years, the simple identification of sensitizing chemicals was performed in guinea pig tests. A murine test, the local lymph node assay (LLNA), has been validated as a replacement for the guinea pig tests. Despite the recent introduction of in vitro methods for the identification of sensitizing chemicals, the LLNA results (when coupled with good exposure data) can be used as the starting point for a quantitative risk assessment. The quantitative risk assessment is aimed to identify the safe use thresholds for any potential skin sensitizer.     

Regulatory cancer risk assessment based on a quick estimate of a benchmark dose derived from the maximum tolerated dose

The proposed U.S. Environmental Protection Agency carcinogen risk assessment guidelines employ a benchmark dose as a point of departure (POD) for low-dose risk assessment. If information on the carcinogenic mode of action for a chemical supports a nonlinear dose-response curve below the POD, a margin-of-exposure ratio between the POD and anticipated human exposure would be considered. The POD would be divided by uncertainty (safety) factors to arrive at a reference dose that is likely to produce no, or at most negligible, cancer risk for humans. If nonlinearity below the POD is not supported by sufficient evidence, then linear extrapolation from the incidence at the POD to zero would be used for low-dose cancer risk estimation. The carcinogen guidelines suggest that the lower 95% confidence limit on the dose estimated to produce an excess of tumors in 10% of the animals (LTD10) be used for the POD. Due to the relatively narrow range of doses in 2-year rodent bioassays and the limited range of statistically significant tumor incidence rates, the estimate of the LTD10 obtained from 2-year bioassays is constrained to a relatively narrow range of values. Because of this constraint, a simple, quick, and relatively precise determination of the LTD10 can be obtained by the maximum tolerated dose (MTD) divided by 7. All that is needed is a 90-day study to establish the MTD. It is shown that the LTD10 determined by this relatively easy procedure is generally within a factor of 10 of the LTD10 that would be estimated using tumor incidence rates from 2-year bioassays. Estimates of cancer potency from replicated 2-year bioassays, and hence estimates of cancer risk, have been show to vary by a factor of 4 around a median value. Thus, there may be little gain in precision of cancer risk estimates derived from a 2-year bioassay, compared to the estimate based on the MTD from a 90-day study. If the anticipated human exposure were estimated to be small relative to the MTD/7 = LTD10, there may be little value in conducting a chronic 2-year study in rodents because the estimate of cancer risk would be low regardless of the results of a 2-year bioassay. Linear extrapolation to a risk of less than 1 in 100,000 and use of an uncertainty factor, e.g., of 10,000, would give the same regulatory "safe dose." Linear extrapolation to a virtually safe dose associated with a cancer risk estimate of less than one in a million would be 10 times lower than the reference dose based on the LTD10/10,000.     

Noninvasive biomonitoring approaches to determine dosimetry and risk following acute chemical exposure: analysis of lead or organophosphate insecticide in saliva

There is a need to develop approaches for assessing risk associated with acute exposures to a broad range of metals and chemical agents and to rapidly determine the potential implications to human health. Noninvasive biomonitoring approaches are being developed using reliable portable analytical systems to quantitate dosimetry utilizing readily obtainable body fluids, such as saliva. Saliva has been used to evaluate a broad range of biomarkers, drugs, and environmental contaminants, including heavy metals and pesticides. To advance the application of noninvasive biomonitoring a microfluidic/electrochemical device has also been developed for the analysis of lead (Pb), using square-wave anodic stripping voltametry. The system demonstrates a linear response over a broad concentration range (1-2000 ppb) and is capable of quantitating saliva Pb in rats orally administered acute doses of Pb acetate. Appropriate pharmacokinetic analyses have been used to quantitate systemic dosimetry based on determination of saliva Pb concentrations. In addition, saliva has recently been used to quantitate dosimetry following exposure to the organophosphate insecticide chlorpyrifos in a rodent model system by measuring the major metabolite, trichloropyridinol, and saliva cholinesterase inhibition following acute exposures. These results suggest that technology developed for noninvasive biomonitoring can provide a sensitive and portable analytical tool capable of assessing exposure and risk in real-time. By coupling these noninvasive technologies with pharmacokinetic modeling it is feasible to rapidly quantitate acute exposure to a broad range of chemical agents. In summary, it is envisioned that once fully developed, these monitoring and modeling approaches will be useful for evaluating acute exposure and health risk.     

Toxicokinetics as a key to the integrated toxicity risk assessment based primarily on non-animal approaches

Toxicokinetics (TK) is the endpoint that informs about the penetration into and fate within the body of a toxic substance, including the possible emergence of metabolites. Traditionally, the data needed to understand those phenomena have been obtained in vivo. Currently, with a drive towards non-animal testing approaches, TK has been identified as a key element to integrate the results from in silico, in vitro and already available in vivo studies. TK is needed to estimate the range of target organ doses that can be expected from realistic human external exposure scenarios. This information is crucial for determining the dose/concentration range that should be used for in vitro testing. Vice versa, TK is necessary to convert the in vitro results, generated at tissue/cell or sub-cellular level, into dose response or potency information relating to the entire target organism, i.e. the human body (in vitro–in vivo extrapolation, IVIVE). Physiologically based toxicokinetic modelling (PBTK) is currently regarded as the most adequate approach to simulate human TK and extrapolate between in vitro and in vivo contexts. The fact that PBTK models are mechanism-based which allows them to be ‘generic’ to a certain extent (various extrapolations possible) has been critical for their success so far. The need for high-quality in vitro and in silico data on absorption, distribution, metabolism as well as excretion (ADME) as input for PBTK models to predict human dose–response curves is currently a bottleneck for integrative risk assessment.     

Application of PBPK model for 2,4-D to estimates of risk in backpack applicators

A PBPK model for 2,4-D was developed that involves flow-limited pH trapping modified to consider tissue binding, binding to plasma, and high-dose inhibition of urinary excretion. The PBPK model provides reasonable estimates of the kinetics of 2,4-D in rats as well as in humans, providing a common metric for expressing risk. The risk characterization for 2,4-D based on the PBPK model is consistent with that based on standard risk assessment methods, except that the apparent variability in the risk characterization is reduced. The model demonstrates that non-linear pharmacokinetics and inhibition of urinary excretion would not be expected in occupational exposures. This case study suggests that preliminary PBPK models could be developed for numerous pesticides based on commonly available data. If properly validated with well-designed worker exposure studies, such models may be useful in more complete assessments of risks to workers as well as members of the general public.     

Review of 2,4-dichlorophenoxyacetic acid (2,4-D) epidemiology and toxicology

The scientific evidence in humans and animals relevant to cancer risks, neurologic disease, reproductive risks, and immunotoxicity of 2,4-D was reviewed. Despite several thorough in vitro and in vivo animal studies, no experimental evidence exists supporting the theory that 2,4-D or any of its salts and esters damages DNA under physiologic conditions. Studies in rodents demonstrate a lack of oncogenic or carcinogenic effects following a lifetime dietary administration of 2,4-D. Epidemiologic studies provide scant evidence that exposure to 2,4-D is associated with soft tissue sarcoma, non-Hodgkin's lymphoma, Hodgkin's disease, or any other cancer. Overall, the available evidence from epidemiologic studies is not adequate to conclude that any form of cancer is causally associated with 2,4-D exposure. There is no human evidence of adverse reproductive outcomes related to 2,4-D. The available data from animal studies of acute, subchronic, and chronic exposure to 2,4-D, its salts, and esters show an unequivocal lack of systemic toxicity at doses that do not exceed renal clearance mechanisms. There is no evidence that 2,4-D in any of its forms activates or transforms the immune system in animals at any dose. At high doses, 2,4-D damages the liver and kidney and irritates mucous membranes. Although myotonia and alterations in gait and behavioral indices are observed after overwhelming doses of 2,4-D, alterations in the neurologic system of experimental animals are not observed with the administration of doses in the microgram/kg/day range. It is unlikely that 2,4-D has any neurotoxic potential at doses below those required to induce systemic toxicity.