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In Vivo Investigation of Dietary Exposure to 5 Pesticides

2 Background – initial study

2.1 Objectives

In 1998/1999 a study was initiated with the objective of studying a possible combination effect of simultaneous exposure to five pesticides alphacypermethrin, bromopropylate, carbendazim, chlorpyrifos and mancozeb in rats. These pesticides were chosen as representative of pesticides occurring together in food produced in or imported into Denmark (for detailed results, see Appendix A)

2.2 Material and methods

The initial study was performed as a 28 days oral toxicity study. A combination of the 5 pesticides was administered to the rats in their diet for 28 days in four dose groups with 6 males and 6 females in each dose group. The dose levels were based on literature data and were set to 1/5 times NOAEL (low-dose), NOAEL (mid-dose), and 5 times NOAEL (high dose) of each pesticide, a control group was also included. A number of organs were examined by macroscopic and microscopic pathology, biochemical blood analyses were performed, and acetylcholinesterase activity in plasma and brain was measured.

2.3 Results

After one day of exposure via the test diet, the rats in the highest dose group showed severe toxic effects in the form of changed behaviour and ataxia indicating central nervous system disorder. Four rats were euthanized, while the remaining rats were put on control diet for two days, during which all toxic signs disappeared. The high dose group was terminated at this point. At sacrifice after 28 days’ dosing, the rats in the mid-dose group appeared more active and sensitive to sound stimuli. There were no treatment-related differences in weight gain. The most marked biochemical effect was statistically significant acetylcholinesterase inhibition of the brain of 8% in low- and 12% in the mid-dose groups in female rats. Plasma acetylcholinesterase was decreased in females and most pronounced in the mid-dose group. A decreased WBC was found in males in the low- and mid-dose group. Differential count revealed a higher number of neutrophiles in the mid-dose group. In females this tendency was also found, but statistical significance was not reached. The only effect on organ weight was an increased absolute and relative liver weight in both sexes in the mid-dose group.

2.4 Discussion and conclusions

The high level of toxicity in the initial study was unexpected. Since a specific effect in the form of acetylcholinesterase inhibition was identified in low- and mid-dose groups, interest was focused on chlorpyrifos, the only acetylcholinesterase inhibitor among the five pesticides in the combination. The result could be explained as a true combination effect. However, since the dose of chlorpyrifos had been set on the basis of literature data, the possibility that chlorpyrifos had been administered at effective levels above those intended could not be excluded / the NOAEL had to be reaffirmed under the conditions in our laboratory
A possible way, in which other compounds can enhance the toxic effect of chlorpyrifos, is by increasing the bio activation of chlorpyrifos to the active metabolite chlorpyrifos-oxon. This can occur e.g. by the other compounds causing an increase in the metabolic activity of the liver, thereby causing an increased production of the oxon. Another explanation could be that one or more of the other compounds interfere with the metabolism of the oxon resulting in a decreased rate of removal of the oxon.

The observed symptoms were signs of an acute toxic effect indicating changed CNS function. The two pesticides in the mixture that have an effect on CNS are chlorpyrifos and alphacypermethrin. However, as acetylcholinesterase inhibition was the critical effect in the study, further studies focused on a possible enhanced effect of chlorpyrifos caused by one or more of the other four pesticides.

It was therefore decided to conduct a series of two experiments.

A dose-response study for chlorpyrifos to clarify whether the literature-based dose used in the initial study was indeed a NOAEL.

Secondly, another combination study to try and confirm the previous findings, and to show a dose-response for inhibition of brain acetylcholinesterase by increasing the dose of chlorpyrifos while keeping a constant dose of the other four pesticides (alphacypermethrin, bromopropylate, carbendazim and mancozeb). Furthermore the study should provide data for explanation of the mode of interaction with focus on liver metabolism.

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