Letter

Gerald P. Schoenig, Ph.D.

Toxicology/Regulatory Services, Inc. 2365 Hunters Way Charlottesville, VA 22911 E-mail: trs{at}toxregserv.com

To the Editor:

The article entitled "Locomotor and Sensorimotor Performance Deficit in Rats following Exposure to Pyridostigmine Bromide, DEET, and permethrin, Alone and in Combination," by Abou-Donia et al.(2001) Toxicological Sciences 60, 305–314, describes experiments that were intended to address potential sensorimotor and cholinergic effects of the three chemicals given alone or in combination.

The authors claim that "these results suggest that exposure to physiologically relevant doses of [pyridostigmine bromide] PB, DEET and permethrin, alone or in combination, leads to neurobehavioral deficits and region-specific alterations in AchE and acetylcholine receptors." They also claim that "These results further suggest that exposure with these chemicals, alone or in combination, may have played a role in the development of long-term health consequences associated with the [Persian Gulf War] veterans."

Because of major deficiencies in study design coupled with inappropriate and/or incomplete statistical treatment of the data, the reliability of the purported findings in this set of experiments is questionable at best. The major deficiencies in study design are that the study consisted of one control group and seven treatment groups, each consisting of only five rats. Therefore, the data for five control rats were compared to data for seven different treatment groups. In addition, the solvent (70% ethanol) that was used to apply the two dermally administered materials during the entire experimental period, was administered to the control animals only during the last 15 days of the 45-day experiment. Deficiencies in the statistical treatment of the data include the fact that interactions were not analyzed, the degrees of freedom appear to be incorrect, inadequate adjustments have been made for multiple comparisons, and none of the combination treatment groups was directly compared to the control group.

There also are numerous confusing and/or inconsistent findings in all of the data sets, some of which are not discussed and for some of which the authors’ explanations and/or conclusions are not supported by the data or are overly speculative. For example, with regard to sensorimotor effects, the authors claim that "each drug given alone had a significant behavioral effect, which tended to become more evident over time" and that "Most significant deficits were observed in animals given PB or a combination of PB with other chemicals." The data that were provided to support these statements (Figures 1 and 2) present a different and confusing picture. First of all, effects on beam-walking score (BW Score), beam-walk time (BW Time), incline plane (IP) and grip response (Grip) are shown and discussed for the PB group at the 30-day point in the experiment. However, PB administration was not initiated until day 31 of the 45-day experiment! If effects can be produced in the PB group before drug administration was started, how reliable can the data be for other findings in this set of experiments? Second, in no case are the effects observed on these behavioral tests significantly different with any of the combination treatments than they are with PB alone. Third, while a significant group x time interaction effect is claimed for BW Score, IP and Grip, it is unlikely that a proper statistical treatment of the data would support such a claim given the small differences between the data presented in Figure 1 (30 days of treatment) and Figure 2 (45 days of treatment), coupled with the small group sizes and the large standard errors. The data from these sensorimotor experiments at best support some questionable effects produced by PB. The nonsensical statement that "The animals were examined blindfolded for BW score, BW time, IP and Grip," which appears in the captions for both Figures 1 and 2, also does not do much to instill confidence in the data or in the level of peer review to which this manuscript was subjected.

Another example of inconsistent and confusing data coupled with inappropriate text interpretations is provided by the data set developed to examine effects on plasma and brain cholinesterase. First of all, the expected inhibitory effect on plasma cholinesterase by the known peripheral anticholinesterase agent, PB, was not observed. Since this evaluation represents the positive control for this set of experiments, the reliability of all other analyses for cholinesterase inhibition is highly questionable. This obvious deficiency is not addressed by the authors. Second, inconsistencies in the findings defy any logical explanation. For example, according to the data in Figure 3, DEET and permethrin, two chemicals that are structurally unrelated to other known anticholinesterase agents, when given alone each cause a significant (p < 0.001) increase in ChE activity in one or more regions of the brain. However, when these two chemicals are combined, the significant increases in ChE activity no longer are observed in any region of the brain. Instead a significant (p < 0.001) decrease in ChE activity is observed in the brainstem. The authors’ interpretation of these findings is that they "suggest that the brainstem may be the most susceptible to combined exposure." The totally inconsistent nature of the findings and the fact that there is no known mechanism to account for them, are never discussed. The authors go on to state that this inhibition by DEET and permethrin in the brainstem may be mediated by PB. However, PB was not administered in the set of experiments under discussion. Further, when PB was administered along with DEET and permethrin, less of an inhibition was observed in the brainstem than when only DEET and permethrin were administered together. Therefore, the statement "Treatment with three chemicals together could lead to added neurotoxic effects" is not supported by the data. Further, there is no case where brain or plasma ChE was significantly lower with any combination of PB, DEET and/or permethrin below that observed with PB alone. Therefore, as was the case with the purported behavioral effects, the data at best support some questionable effects produced by PB.

The same type of inconsistent findings and questionable claims of treatment-related effects are present in the data and text for the other two parameters evaluated in this study, i.e., brain choline acetyl transference activity and m2 muscarinic and nicotinic acetylcholine receptor activity. Specific examples are not provided due to space limitations, but the bottom line conclusion is that none of the data for the combination treatments are significantly different from the PB data alone. Therefore, there is no support for the authors’ claim that "The results suggest that exposure to these chemicals, alone or in combination, causes significant sensorimotor deficit and differential regulation of AchE and m2 muscarinic and nicotinic acetylcholine receptors in the CNS."

With regard to the authors’ claim that "The results of these experiments suggest that exposure to these chemicals, alone or in combination, may have played a role in the long-term health consequences associated with the [Persian Gulf War] veterans," the data do not support this conclusion since the purported effects are for concurrent drug treatment and the experimental design does not address the issue of enduring or residual effects.

One final point is that a key publication that represents the benchmark for neurotoxicity testing with DEET (Schoenig, et al., 1993Go) is not discussed or referenced in this article. In the studies that were the subject of this publication, groups of 10–20 male and 10–20 female rats were included in each treatment group and multiple trials were performed on each animal for each endpoint. For the behavioral test that was most closely related to an endpoint evaluated in the Abou-Donia study (grip strength), no effect was observed at acute oral doses up to 500 mg/kg/day or at dietary dose levels up to 250 mg/kg/day after 8 months of exposure. This is in contrast to the reported treatment related effects after 30 and 45 days of dermal exposure to DEET at a dose level of 40 mg/kg/day.

In conclusion, based on both study design and execution, the study that is the subject of this article is fatally flawed. In addition, the presentation, the statistical evaluation and the interpretation of the data have numerous deficiencies. Besides the poor quality of the science, one also has to wonder how a manuscript with these obvious deficiencies gained access to publication in the world’s most prestigious toxicology journal. If this is not a rare exception to the rule, the peer-review process for Toxicological Sciences needs to be reevaluated.

REFERENCE

Schoenig, G. P., Hartnagel, R. E., Jr., Shardein, J. L., and Vorhees, C. V. (1993). Neurotoxicity evaluation of N, N-diethyl-m-toluamide (DEET) in rats. Fundam. Appl. Toxicol. 21, 355–365.[ISI][Medline]





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