In Vivo Investigation of Dietary Exposure to 5

In Vivo Investigation of Dietary Exposure to 5 Pesticides

Summary and conclusions

We are daily exposed to a number of pesticides through the food we eat. In fruit pesticide residues were found in approximately 30% of Danish produced fruit and in approximately 70% of the fruit produced outside Denmark in the test samples examined by the Danish Veterinary and Food Administration. In fruit and vegetables produced outside Denmark residues of pesticides, that are forbidden to use in Denmark, are also found.

The possible adverse health effects of pesticides have traditionally been evaluated on the basis of the individual properties of single substances. There are few animal studies on combination of pesticides and other compounds, and therefore only few data available for risk assessment. Those available show that the combined effect of two or more chemical substances at low concentration is either not detectable or at most additive. There is still no internationally agreed method for evaluation of combination of compounds. The Danish Veterinary and Food Administration uses as a first cut-of an approach of summing all pesticide residues and compare the intake based on the acceptable daily intake (ADI) for the most toxic pesticide involved for the risk assessment. If the summarized ADI for all the pesticides exceeds the most toxic compounds ADI, a Hazard-Index is calculated. A Hazard-Index is calculated as the sum of the theoretical intake of the individual pesticides in the food item in relation to the ADI of the individual pesticides.

The purpose of the studies in this report was to provide further data on possible combined effects of pesticides that may occur as residues in food.

Five pesticides (alphacypermethrin, bromopropylate, carbendazim , chlorpyrifos and mancozeb) were chosen. All five pesticides are often found as pesticide residues in test samples of food examined by the Danish Veterinary and Food Administration. The rats were fed the pesticides in various concentrations through the diet for 28 days. In the initial study the 5 pesticides were all given in increasing doses. Due to the results of the initial study a dose-response study of chlorpyrifos was done and at last a combined exposure study was performed where the rats were fed increasing doses of chlorpyrifos and a constant dose of the other four pesticides (alphacypermethrin, bromopropylate, carbendzim and mancozeb).

The rats in the high dose group of the initial study were intoxicated with signs of disturbances of the central nervous system (CNS) with incoordinated movements and changed behaviour, this group was terminated. In the mid and low dose groups an inhibition of 12 and 8% in brain acetylcholinesterase in female rats was found and a decrease in the number of white blood cells in male rats was found. The mid dose group had increased liver weight and appeared more sensitive to sounds and were more active at autopsy. These result were surprising and there was a need for further investigation of the results.

Two of the pesticides, alphacypermethrin and chlorpyrifos, have known effects on CNS. Chlorpyrifos by inhibiting the enzyme acetylcholinesterase. Alphacypermethrin by changing permeability of sodium in nerve membranes causing general stimulation of neurotransmitters. Since an inhibition of brain acetylcholinesterase was found in female rats in the intial study in the low and mid dose group, we focused on chlorpyrifos and a possible combination effect. A dose-response study of chlorpyrifos was performed. There was no effect in the animals and no inhibition of acetylcholinesterase in the brain. Another combination study was performed where chlorpyrifos was given in increasing doses and the four other pesticides kept at the same level. No effect was found on CNS or on brain acetylcholinesterase. The results from the initial study could thus not be confirmed. However, other effects were seen with weight changes of liver, thymus and thyroid gland, changes in haematology and in histology of the liver and thyroid gland.

The conclusion of the 3 animal studies was that effects are seen at lower levels when rats are exposed to the five pesticides in a combination than when the rats are exposed to the individual pesticides. Such a combined effect shows that risk assessment of health effects of residues of pesticides is complicated.

The unexpected effect on CNS and on inhibition of brain acetylcholinesterase seen in the initial study could not be repeated in the second combination study. However, the level of mancozeb in the diet in the last combination study was only 18-26% of the intended, which means that the effect on CNS in the initial study still might be a true effect.

Combination effects were seen in the last combination study, as there was weight increase in the relative liver weight, the thyroid gland weight and a decrease in the weight of the thymus in the combination groups. There were changes in the histology of the liver and the thyroid gland and changes in both white and red blood cells in the combination groups.

In the initial study a possible increase in liver metabolism of chlorpyrifos to the active metabolite chlorpyrifos-oxon was considered. However, increased liver metabolism of chlorpyrifos to chlorpyrifos-oxon could not be confirmed, since chlorpyrifos-oxon was not found in plasma. However, there were some problems with the method used to measure chlorpyrifos-oxon. The effects seen in the initial study might be through other mechanism e.g. change of toxico-kinetics (absorption, distribution, metabolism and elimination).

From a regulatory point of view the CNS symptoms seen in the intial study still give reason to concern, and there is a need to explain these findings. Which of the pesticides in the combination gave rise to the CNS symptoms -alphacypermethrin, chlorpyrifos or a combination?

There is furthermore a need to perform some toxicokinetic studies of the single compounds and the compounds in combination. Toxicokinetic studies might elucidate possible modes of action, since there might be changes in absorption, distribution, metabolism and elimination of a single compound in the combinations.