Time-Cumulative Toxicity of Neonicotinoids: Experimental Evidence and Implications for Environmental Risk Assessments.

Our mechanistic understanding of the toxicity of chemicals that target biochemical and/or physiological pathways, such as pesticides and medical drugs is that they do so by binding to specific molecules. The nature of the latter molecules (e.g., enzymes, receptors, DNA, proteins, etc.) and the strength of the binding to such chemicals elicit a toxic effect in organisms, which magnitude depends on the doses exposed in a given timeframe. While dose and time of exposure are critical factors determining the toxicity of pesticides, different types of chemicals behave differently. Experimental evidence demonstrates that the toxicity of neonicotinoids increases with exposure time as much as with the dose, and therefore it has been described as time-cumulative toxicity. Examples for aquatic and terrestrial organisms are shown here. This pattern of toxicity, also found among carcinogenic compounds and other toxicants, has been ignored in ecotoxicology and risk assessments for a long time. The implications of the time-cumulative toxicity of neonicotinoids on non-target organisms of aquatic and terrestrial environments are far reaching. Firstly, neonicotinoids are incompatible with integrated pest management (IPM) approaches and secondly regulatory assessments for this class of compounds cannot be based solely on exposure doses but need also to take into consideration the time factor.

Letter to the editor "The resilience of the beehive".

A recent quantitative weight of evidence (QWoE) assessment of higher tier studies on the toxicity and risks of neonicotinoids in honeybees by Solomon and Stephenson reported a colony-level no-observed-adverse effect concentration (NOAEC) of 25 µg/kg (ppb) for imidacloprid and clothianidin. The toxicity of these insecticides to honeybees is however known to be reinforced with chronic exposure, and extrapolation of time-to lethal-effect toxicity plots compiled from published studies indicate that an imidacloprid level of 0.25 ppb, i.e. one-hundredth of the reported colony NOAEC, would kill a large proportion of bees nearing the end of their life. This huge discrepancy points to the impressive resilience of beehives in counteracting lethal effects of neonicotinoids, as long as the colony remains otherwise healthy with a productive queen that is able to maintain the colony population. The explicit connection between innate immunity loss and the neonicotinoids leading to infestation with a wide variety of pathogens appears to be the decisive factor that ultimately bring down stressed colonies.

Dose-response analysis indicating time-dependent neurotoxicity caused by organic and inorganic mercury-Implications for toxic effects in the developing brain.

A latency period preceding neurotoxicity is a common characteristic in the dose-response relationship induced by organic mercury. Latency periods have typically been observed with genotoxicants in carcinogenesis, with cancer being manifested a long time after the initiating event. These observations indicate that even a very small dose may cause extensive adverse effects later in life, so the toxicity of the genotoxic compound is dose and time-dependent. In children, methylmercury exposure during pregnancy (in utero) has been associated with delays in reaching developmental milestones (e.g., age at first walking) and decreases in intelligence, increasing in severity with increasing exposure. Ethylmercury exposure from thimerosal in some vaccines has been associated, in some studies, with autism and other neurological disorders in children. In this paper, we have examined whether dose-response data from in vitro and in vivo organic mercury toxicity studies fit the Druckrey-Küpfmüller equation c·t(n)=constant (c=exposure concentration, t=latency period), first established for genotoxic carcinogens, and whether or not irreversible effects are enhanced by time of exposure (n≥1), or else toxic effects are dose-dependent while time has only minor influence on the adverse outcome (n<1). The mode of action underlying time-dependent toxicity is irreversible binding to critical receptors causing adverse and cumulative effects. The results indicate that the Druckrey-Küpfmüller equation describes well the dose-response characteristics of organic mercury induced neurotoxic effects. This amounts to a paradigm shift in chemical risk assessment of mercurial compounds and highlights that it is vital to perform toxicity testing geared to investigate time-dependent effects.

Delayed and time-cumulative toxicity of imidacloprid in bees, ants and termites.

Imidacloprid, one of the most commonly used insecticides, is highly toxic to bees and other beneficial insects. The regulatory challenge to determine safe levels of residual pesticides can benefit from information about the time-dependent toxicity of this chemical. Using published toxicity data for imidacloprid for several insect species, we construct time-to-lethal-effect toxicity plots and fit temporal power-law scaling curves to the data. The level of toxic exposure that results in 50% mortality after time t is found to scale as t(1.7) for ants, from t(1.6) to t(5) for honeybees, and from t(1.46) to t(2.9) for termites. We present a simple toxicological model that can explain t(2) scaling. Extrapolating the toxicity scaling for honeybees to the lifespan of winter bees suggests that imidacloprid in honey at 0.25 µg/kg would be lethal to a large proportion of bees nearing the end of their life.

Anti-androgenicity can only be evaluated using a weight of evidence approach.

Preputial separation (PPS) is a commonly used external marker for the onset of male puberty in experimental animal studies. While treatment-related delays in PPS may be indicative of specific anti-androgenic activity, impaired general growth also alters the onset of puberty. To differentiate between specific and non-specific effects on the age at PPS--and thereby evaluate the validity of the endpoint PPS-two-generation toxicity studies of 23 substances were evaluated. The 23 substances were assessed regarding anti-androgenicity using all available data and external assessments in a weight-of-evidence evaluation (WoE). Correlation of individual pup body weight with age at PPS revealed that delays in pubertal development coincided with reduced pup body weight. After comparison with the WoE assessment, we concluded that inclusion of body weight analysis into the PPS evaluation of each study was able to correctly identify three compounds which specifically induced delayed PPS and 16 which only showed unspecific changes. A further two compounds which might be categorized as anti-androgens based on delayed PPS, were correctly regrouped using our refined methodology. Based on this analysis and in comparison to the WoE evaluation, it was found, that caution should be exercised when using the endpoint PPS in hazard assessment.

The molecular basis of simple relationships between exposure concentration and toxic effects with time.

Understanding the toxicity of chemicals to organisms requires considering the molecular mechanisms involved as well as the relationships between exposure concentration and toxic effects with time. Our current knowledge about such relationships is mainly explained from a toxicodynamic and toxicokinetic perspective. This paper re-introduces an old approach that takes into account the biochemical mode of action and their resulting biological effects over time of exposure. Empirical evidence demonstrates that the Druckrey-Küpfmüller toxicity model, which was validated for chemical carcinogens in the early 1960s, is also applicable to a wide range of toxic compounds in ecotoxicology. According to this model, the character of a poison is primarily determined by the reversibility of critical receptor binding. Chemicals showing irreversible or slowly reversible binding to specific receptors will produce cumulative effects with time of exposure, and whenever the effects are also irreversible (e.g. death) they are reinforced over time; these chemicals have time-cumulative toxicity. Compounds having non-specific receptor binding, or involving slowly reversible binding to some receptors that do not contribute to toxicity, may also be time-dependent; however, their effects depend primarily on the exposure concentration, with time playing a minor role. Consequently, the mechanism of toxic action has important implications for risk assessment. Traditional risk approaches cannot predict the impacts of toxicants with time-cumulative toxicity in the environment. New assessment procedures are needed to evaluate the risk that the latter chemicals pose on humans and the environment. An example is shown to explain how the risk of time-dependent toxicants is underestimated when using current risk assessment protocols.

The significance of the Druckrey-Küpfmüller equation for risk assessment--the toxicity of neonicotinoid insecticides to arthropods is reinforced by exposure time.

The essence of the Druckrey-Küpfmüller equation dtn = constant (where d = daily dose and t = exposure time-to-effect, with n > 1) for chemical carcinogens is that the total dose required to produce the same effect decreases with decreasing exposure levels, even though the exposure times required to produce the same effect increase with decreasing exposure levels. Druckrey and Küpfmüller inferred that if both receptor binding and the effect are irreversible, exposure time would reinforce the effect. The Druckrey-Küpfmüller equation explains why toxicity may occur after prolonged exposure to very low toxicant levels. Recently, similar dose-response characteristics have been established for the toxicity of the neonicotinoid insecticides imidacloprid and thiacloprid to arthropods. This observation is highly relevant for environmental risk assessment. Traditional approaches that consider toxic effects at fixed exposure times are unable to allow extrapolation from measured endpoints to effects that may occur at other times of exposure. Time-to-effect approaches that provide information on the doses and exposure times needed to produce toxic effects on tested organisms are required for prediction of toxic effects for any combination of concentration and time in the environment.

The stability of historical control data for common neoplasms in laboratory rats and the implications for carcinogenic risk assessment.

Time-related changes in the incidences of spontaneous neoplasms in skin (fibroma and keratoacanthoma), thyroid (C-cell and follicular cell adenomas/carcinomas), uterus (stromal polyp), testes (Leydig cell tumor) and hemolymphoreticular system (mesenteric lymph node hemangioma and malignant granular lymphocytic leukemia) were assessed statistically in Wistar, Sprague-Dawley and F344 rats employed by the BASF, Germany and major European contract research organizations over the last 20 years. Negative trends (5 out of 80 cases) were observed for skin fibromas in F344 males, for follicular cell adenomas in Han Wistar females and in Sprague-Dawley males and females, and for follicular cell carcinomas in Sprague-Dawley males. Positive trends (8 out of 80 cases) were observed for skin keratoacanthomas in Han Wistar males, for C-cell adenomas in BASF Wistar males and females, for stromal polyps in Han Wistar and Sprague-Dawley females, and for mesenteric lymph node hemangiomas in Han Wistar and Sprague-Dawley males and in BASF Wistar females. In 67 out of 80 cases there were no statistically significant trends. Tumor drift was not common but occurred far more often in outbred rat strains (Wistar and Sprague-Dawley) than in the inbred rat strain (F344). This observation suggests that tumor predisposition is genetically determined, that tumor drift is primarily caused by genetic drift and that non-genotoxic carcinogens operate by facilitating the expression of tumor predisposition in target cells.

The stability of historical control data for common neoplasms in laboratory rats: adrenal gland (medulla), mammary gland, liver, endocrine pancreas, and pituitary gland.

Time-related changes in the incidences of spontaneous neoplasms in adrenals (medulla), mamma, liver, pituitary, and (endocrine) pancreas were assessed statistically in Wistar, Sprague-Dawley, and F344 rats employed by BASF, Germany and major European contract research organizations over the last 20 years. Negative trends (7 of 80 cases) were observed for pituitary pars distalis adenomas in Sprague-Dawley males and females, for pancreas islet cell adenomas in BASF Wistar males and females, for benign adrenal pheochromocytomas in Sprague-Dawley males, for malignant pheochromocytomas in F344 males, and for mammary gland fibroadenomas in BASF Wistar females. Positive trends (13 of 80 cases) were observed for benign pheochromocytomas, mammary gland adenocarcinomas, and pancreas islet cell carcinomas in HanWistar females, for malignant pheochromocytomas and islet cell carcinomas in BASF Wistar males, for benign pheochromocytomas and islet cell adenomas in F344 males, for mammary gland fibroadenomas in Sprague-Dawley females, and for benign hepatocellular tumors in HanWistar males and females, and in BASF Wistar and Sprague-Dawley females. In 60 of 80 cases there were no statistically significant trends. These results indicate that the majority of tumor types showed no time trends and that, in each rat strain, certain tumor types are susceptible to slight positive or negative time trends. Accordingly, the validity and use of historical control data should be based on an organ- and strain-specific statistical analysis of tumor incidence over time.

A Wistar rat strain prone to spontaneous liver tumor development: implications for carcinogenic risk assessment.

The European Union legislation considers nongenotoxic substances that only cause liver tumors in certain sensitive strains of mice as raising no concern for man. The EU legislation, however, also clearly stipulates that cases where the only available tumor data are the occurrence of neoplasms at sites and in strains where they are well known to occur spontaneously with a high incidence are relevant arguments which exclude a concern for man. We have analyzed the spontaneous liver tumor incidence in Wistar rats and in B6C3F(1) and C57Bl mice used in carcinogenicity trials at the BASF facility in Ludwigshafen, Germany, over the past 15 years and compared the spontaneous liver tumor incidence in BASF Wistar rats with those observed in rat strains employed in major European contract research organizations (CROs). The results of these analyses indicate that the incidence of spontaneous liver tumors in the BASF Wistar rat strain is very high, similar to that seen in the B6C3F(1) mouse and much higher than that seen in the C57Bl mouse and other commonly used strains of rat. The analyses also revealed signs of increasing variability and liver tumor drift in several rat strains. Moreover, the incidence of spontaneous preneoplastic liver cell foci was far higher in the BASF Wistar rat strain than reported for other rat strains in the literature. The analyses provide relevant arguments which exclude a concern for man for nongenotoxic chemicals that only tested positive for liver tumors in this sensitive rat strain.