Alternative toxicological methods for establishing residue definitions applied for dietary risk assessment of pesticides in the European Union.

Consumers are constantly exposed to trace levels of residues present in food commodities, arising from the use of pesticides. For this reason, assessing the risk caused by pesticide residues present in food requires not only identification and toxicological properties assessment of the active substance, but also of its metabolites, isomers, and degradates. This requires the use of many laboratory animals. On the other hand, currently there is an emphasis on minimizing the use of animals in toxicological research. This review article presents current activities of the European Food Safety Authority (EFSA) and the European Commission's Joint Research Centre (JRC) aiming to replace at least a part of toxicological tests on substances of unknown toxicity with the alternative methods. Quantitative Structure-Activity Relationship (QSAR) and Threshold of Toxicological Concern (TTC) can be used for this purpose in procedure of establishing residue definitions applied for dietary risk assessment.

Effect of chlorpyrifos on the profile of subpopulations immunocompetent cells B, T and NK in in vivo model.

BACKGROUND: Current studies have indicated many environmental factors, such as pesticides, that cause immune system disorders through inducing changes in humoral and cellular responses thereby increasing the risk of contracting infectious diseases and cancer. The literature suggests that low exposures to certain organophosphorus pesticides stimulate the immune system, whilst high exposures result in decreased function. Precise mechanisms for the fall in immunocompetence are often unclear, however it can be predicted that the intimate interaction between the nervous and immune systems can potentially lead to toxicity. OBJECTIVES: To determine the effects of organophosphorus pesticide, chlorpyrifos that is often used in Poland, on selected immunological responses, such as immune-competent cell proportions formed experimentally in-vivo by cells of Wistar rats during subchronic exposures after 45 and 90 days. MATERIALS AND METHODS: The test was carried out on ten male and ten female Wistar rats in each of three test groups, who received 3 chlorpyrifos doses for 90 days intragastrically, according to OECD guidelines (No. 401). Two control groups were given olive oil. After completion, the animals were deeply anaesthetised by a mixture of ketamine (Vetaketam) and xylazine (Vetaxym). Immuno-competent cells were profiled by a commercial monoclonal antibody method. In order to measure the dynamics of any changes, the aforementioned immunological responses were investigated after 45 days using the same procedures for obtaining the relevant biological test material. RESULTS: Test animals exposed to chlorpyrifos had altered number of white bood cells which were either increased or decreased relative to controls after 45 and 90 days for all exposure levels used. CONCLUSIONS: The study demonstrated changes in white-blood cell (lymphocyte) response profiles, reflecting an immunomodulation although such changes were equivocal, where both suppression and stimulation were observed. KEY WORDS: immunomodulation, immune system, lymphocyte, organophosphorus pesticides, chlorpyrifos.

Computer modelling as a tool for the exposure assessment of operators using faulty agricultural pesticide spraying equipment.

BACKGROUND: Faulty but still operating agricultural pesticide sprayers may pose an unacceptable health risk for operators. The computerized models designed to calculate exposure and risk for pesticide sprayers used as an aid in the evaluation and further authorisation of plant protection products may be applied also to assess a health risk for operators when faulty sprayers are used. OBJECTIVE: To evaluate the impact of different exposure scenarios on the health risk for the operators using faulty agricultural spraying equipment by means of computer modelling. MATERIAL AND METHODS: The exposure modelling was performed for 15 pesticides (5 insecticides, 7 fungicides and 3 herbicides). The critical parameter, i.e. toxicological end-point, on which the risk assessment was based was the no observable adverse effect level (NOAEL). This enabled risk to be estimated under various exposure conditions such as pesticide concentration in the plant protection product and type of the sprayed crop as well as the number of treatments. Computer modelling was based on the UK POEM model including determination of the acceptable operator exposure level (AOEL). Thus the degree of operator exposure could be defined during pesticide treatment whether or not personal protection equipment had been employed by individuals. Data used for computer modelling was obtained from simulated, pesticide substitute treatments using variously damaged knapsack sprayers. These substitute preparations consisted of markers that allowed computer simulations to be made, analogous to real-life exposure situations, in a dose dependent fashion. Exposures were estimated according to operator dosimetry exposure under 'field' conditions for low level, medium and high target field crops. RESULTS: The exposure modelling in the high target field crops demonstrated exceedance of the AOEL in all simulated treatment cases (100%) using damaged sprayers irrespective of the type of damage or if individual protective measures had been adopted or not. For low level and medium field crops exceedances ranged between 40 - 80% cases. CONCLUSIONS: The computer modelling may be considered as an practical tool for the hazard assessment when the faulty agricultural sprayers are used. It also may be applied for programming the quality checks and maintenance systems of this equipment.

Influence of chlorpyrifos on the profile of subpopulations of immunoactive cells and their phagocytic activity in an experimental in vivo model.

Many environmental factors, including pesticides, cause immunological system disorders by inducing changes in humoral and cellular response. They may stimulate or trigger immunological autoaggression, hypersensitivity and allergy, as well as lead to immunosuppression, thus increasing the incidence of infectious diseases and cancers. Such activity is also attributed to organophosphorus compounds used in agriculture as insecticides, and commonly in households as biocides. The aim of the study was to define possible mechanisms of the immunotoxic activity of the chlorpyrifos (an organophosphorus compound) on experimental animals following their exposure to the compound via the oral route. The present study attempts to define the influence of chlorpyrifos on the profile of subpopulations of immunoactive cells: B, T, CD4+, CD8+, and NK, and on their phagocytic activity in an experimental in vivo model. For this purpose, the Wistar rats, were exposed orally to increasing doses of chlorpyrifos: 0.1 LD(50), 0.15 LD(50), 0.2 LD(50), 0.3 LD(50) and 0.4 LD(50) for 28 days. In the study animals, we failed to demonstrate a statistically significant decrease in the phagocytic activity of the granulocyte.

[Toxicological evaluation of crop protection chemicals in the EU registration process]. / Ocena toksykologiczna srodków ochrony roslin w procesie rejestracyjnym UE.

Authorisation of crop protection chemicals prior to placing into the market is being regulated with standardized regulations in the European Community territory. The Member States are obliged to introduce all provisions constituting the base for the evaluation of protection chemicals and determining their safety for man and environment. The rules governing toxicological evaluation of plant protection products and their active substances have been discussed and the practical relevance of the harmonized provisions for the safety assessment of pesticides in the EU were also presented. Introducing the assessment of risk resulting from treatments with chemical crop protection chemicals in the registration process widens the safety margin for users of plant protection products as well as fixes new safety standards at agrochemical works.

Di-butyl phthalate-induced hypomethylation of the c-myc gene in rat liver.

Peroxisome proliferators (PPs)-induced DNA hypomethylation has been proposed as a mechanism of their toxicity, including carcinogenic action. The effect of di-butyl phthalate (DBP), a known peroxisome proliferators, on the methylation level of the c-myc promoter region in rat liver was studied. Changes in the methylation status of the c-myc gene were correlated with changes in DNA synthesis, DNA methyltransferase (DNMTs) activity and liver weight. Male Wistar rats received DBP in one, three or fourteen daily oral doses of 1800 mg/kg body weight (b.w.) x day(-1) (this dose is close to the dose that increases the numbers of peroxisomes in male Wistar rats). We have demonstrated that DBP decreased the methylation of the c-myc gene. Cytosine hypomethylation in the analyzed CpG sites of the c-myc gene promoter occurred during the whole period of study, although after 14 doses of DBP the difference from control was only on the borderline of significance (p = 0.066). An increase in DNA synthesis was only observed after 24 hours of treatment with DBP, and it preceded liver growth. We hypothesize that DBP-induced demethylation of the c-myc gene was an active mechanism, not associated with DNMTs activity and DNA replication.

[Phenobarbital-induced hypermethylation of the p53 promoter region in the liver of Wistar rats]. / Indukowana fenobarbitalem hipermetylacja rejonu promotorowego genu p53 w watrobie szczurów szczepu Wistar.

Non-genotoxic carcinogens (NGCs)-induced changes of DNA methylation has been proposed as a mechanism of their toxicity, including carcinogenic action. The effect of phenobarbital (PB), a rodent liver carcinogen on the methylation level of the p53 promoter region in rat liver was studied. Changes in the methylation status of the p53 gene were correlated with changes in DNA synthesis, DNA methyltransferase (DNMTs) activity and liver weight. Male Wistar rats received PB in one, three or fourteen daily oral doses of 92.8 mg/kg b.w. x day(-1). We have demonstrated that PB increased the methylation of the p53 gene. Cytosine hypermethylation in the analyzed CpG sites of the p53 gene promoter occurred during the whole period of study. However, an increase in DNA synthesis was only observed after 1 and 3 days of treatment with PB and it preceded liver growth. Treatment of rats with PB for 1 and 3 days also produced an increase in nuclear DNMTs activity. After prolonged administration (14 days), no changes in DNMTs activity nor DNA synthesis were observed. It is proposed that PB-induced de novo methylation of the p53 gene was not associated with DNMTs activity.

Hepatocellular peroxisome proliferation and DNA synthesis in Wistar rats treated with herbicide fluazifop.

The aim of this study was to determine the effect of herbicide fluazifop, on the early occurring changes in rat liver regarded as hepatic markers of peroxisome proliferators (PPs). Fluazifop was administered orally to male Wistar rats at increasing doses from 5.6 to 891 mg/kg body weight per day for 1, 2, 4, 7 and 14 consecutive days and peroxisome proliferation, induction of some peroxisome-associated enzymes and mitogenesis (S-phase, M-phase and percentage of binucleated hepatocytes) were studied. Short-term treatment of rats with fluazifop resulted in hepatomegaly due to time dependent proliferation of smooth endoplasmic reticulum (SER) and peroxisomes. The increase in the number of peroxisomes in the hepatocytes was supported by an increase in peroxisomal palmitoyl-CoA oxidation and catalase activity. In contrast to other PPs fluazifop induced low rate of rcplicative DNA synthesis and did not affect mitoses (M-phase). DNA synthesis was accompanied by the appearance of binucleated hepatocytes. Thus, we can conclude that fluazifop produces in male Wistar rats hepatomegaly due to cellular hypertrophy. The threshold dose for palmitoyl-CoA oxidation and DNA synthesis was 112 and 223 mg/kg body weight per day, respectively. The value for hepatomegaly and catalase activity was 56 mg/kg body weight per day. The results presented in this paper demonstrated that fluazifop can be classified as a weak rodent PPs.

[Effect of diclofop on the activity of some drug-metabolizing enzymes in the liver of male Wistar rats]. / Wplyw diklofopu na wybrane enzymy metabolizujace substancje obce w watrobie szczurów rasy wistar.

The study was designed to determine whether diclofop, introduced to environment as herbicide, would exert properties of chemical inducers of rat liver monooxygenase system related to CYP1A and CYP2B isozymes. For this purpose, the effect of diclofop on 7-etoxyresorufin O-dealkylase and p-nitroanisole O-demethylase activities specific for CYP1A as well as on CYP2B mediated 7-pentoxyresorufin O-dealkylase activity was studied in male Wistar rats. This biochemical method permits to determine whether tested compound belongs to one of two main types of chemical inducers. Diclofop was dosed by gavage for 4 days at 0; 5.6, 11.2 and 56 mg/kg b.w. per day. Treatment of rats with diclofop resulted in significant increase in relative liver weight (RLW), to 19% and 31% above the control, respectively. Diclofop administered at the dose of 11.2 and 56 mg/kg b.w. per day induced a 3-fold (p < 0.001) and a 5-fold (p < 0.001) increase in the metabolism of 7-pentoxyresorufin (CYP2B-mediated reaction), respectively. No effect level for CYP2B induction was 5.6 mg/kg b.w per day. However, diclofop induced only slight increase in 7-etoxyresorufin O-dealkylase and did not show any effect on p-nitroanisole O-demethylase activity (CYP1A-mediated reactions). The results revealed that diclofop not show the ability to induce CYP1A and moreover it was weak inducer of CYP2B isozymes in the liver of male Wistar rats. Diclofop was also examined for its ability to affect the activity of phenol UDP-glucuronosyltransferase in rat liver. The results suggest that diclofop suppressed phenol form of UDP-glucuronosyltransferase activity. The isozyme activity decreased by 50% and 35% at the dose of 11.2 and 56 mg/kg b.w. per day, respectively. It should be noted that this form of enzyme inactivates during II phase of biotransformation most chemical carcinogens and other xenobiotics.