Arena Tests with Piperonal , A New Louse

Piperonal, once used co kill lice in Australian hospitals, was acclaimed as an effective pediculicide (Corlecce, 1925) by the standards ofthe day. It is unusual in also exhibiting a repellent actionagainstlice,apropertyonlyrecentlyrealisedAnew,easy to use, low-fragrance, pump action spray, which incorporates 1% piperonal, was tested in die laboratory using clothing lice in an arena cesc and was found co exhibit consistently high repellency after halfan hour, dropping only slighdy after 24 hours. A well known multi-purpose insect repellent, diechylcoluamide (DEET), was then rested against piperonal. A 2% solurion of piperonal was found to be almost twice as effective as a 50% solution ofDHETIn arena tescs using lice with the rip segments of both antennae removed, no behavioural differences or statistically significant differences from a random distribution could be found between uncreaced and Rappell-created areas. This strongly suggests that sense organ(s) on the tip of the antenna are necessary for detection of the repellent. Although there can be no strict correlation between results in the laboratory and potential efficacy in the field, materials such as insecticides found to be effective in the laboratory have been found co be equally effective in the field. Furthermore, what with the threat of resistance ro head lice insecticides, ineffectual treatmentand the lack ofcontact tracing, a repellent would be ofobvious use in the control ofhead lice. INTRODUCTION The best method ofprotecting people from any blood-sucking insect is co destroy the insect. Often this is impossible or impractical. However, a high degree of protection can be obtained by the proper use ofan effective insect repellent. None ofour modern repellents are ideal. They may have someodour, feel oily,sofcen paincsorplastics, need to beapplied in high doses or ac high concentrations and may be effective for only a few hours. Even the powerful multi-purpose insect repellenc.diechyltoluamide (DEET) is known tocause harmful side effects above a concentration of 50% (Lancet, 1988) although high strength preparations are still in general use. Therefore, it is noc surprising that any new insect repellent would meet with scepticism and caution from health professionals. Head liceconstitureaninfectiousdisease(pedicuiosiscapitis) well within our powers ro conquer. However, nearly3 million units ofhead lice and scabies treatments are supplied annually in the UK, much otic being used prophylaccicaliy or inappropriately (Burgess, 1992) and infection levels srill remain unacceptable. As yet, the public are still often poorly informed as to the mostproficient treatments. Insecricidal shampoos, ineffectual as treatments, are frequently employed by the public in the hope of preventing lice (Maunder, 1995). Thus an efficient, pleasant, easy co use, long-lascing head lice repellent .would lower the level of abuse of pesticides and may even reduce infection races or, when correctly used after creacmenc with insecticide, limit re-infeccions while contact tracing is being carried out. Rappell (Charwell Pharmaceuricals), a new head lice repellent available from pharmacists, is applied to the hair daily as a fine mist spray. The active ingredient is 1% piperonal, a natural material produced by planes, presumably as a repellent againstharmful insects. This relarively non-roxic, non-volatile compound has long been used extensively in cosmetics and in vanillaand cherry flavourings and has an excellentsafety record (Merck Index, 1989). Dechier (1947) described a repellent as ’a chemical thac causes insects to make oriencaied movements away from its source’ but the repellent narure of piperonal has not, until recently, been described. However, ic has now been reported chac, in the laboratory, ;i high degree ofrepellency ofclothing lice (fediwius hunianns hitiimnns) co creaced hair and filter paper discs (arena cesc) has been observed (Burgess, 1993). The present tests shed further iighc on che behaviour of lice to piperonal. The theory thac piperonal provokes a negacive behavioural response mediated by ancennat receptors, causing lice toavoid treatedareas(Rappe!l Information Leaflec),canalso be examined by using the arena cesc. MATERIALS AND METHOD Head lice {Pedicfilf/s h/iineinnj capitis} cannot live on other parrs ofche body or oft che body. However, human clothing lice (Pedicnins hinii^uns humaniis} can be grown in large numbers in laboratory colonies and provide the best available model. Arena rescs, first developed by Wigglesworch (1941), are based on lice discribucing randomly in an inert environment. This random distribution does not occur when an atcraccant or repellent is present. The tesc is quick, simple and does not require elaborate analysis. For each test paper, the control or test substance was dissolved as necessary in isopropanol, and 50pl ofthe solution was applied as evenly as possible to one halfofa filcerpaperdisc (5.5cm in diameter). Two cest papers were prepared for each solution. Thepapers were then dried ina flowofmoving airfor 30 minutes. The effecc of this drying was to concentrate the active material so that the essencial difference between the papers lay in the absolute quantities ofactive material which were evenly distributed upon chem. Rappell itself contains 30% alcohol, which likewise evaporates away after applicationEach paper was then placed in the centre ofa 10cm glass Petri dish. In the case ofthe 24 hour cesc, the test papers were left ac room temperature for a further 231 hours in open Petri dishes. 5-7 hours after engorgemenc, 10 young adult female lice (which are thought to be important in initiating infection), were placed in the centre of the disc and the Pecri dish was covered. The dishes were then placed in an incubator at 30 + 2C and approximately 50% humidity. After 5 minutes, which allowed time for the lice to deaggregate and distribute randomly, the lice on the created side were counted. The dishes were then re-examined after each of a further 4 incubarion periods of2 minutesAny lice found off thefilterpaperwereexcluded from thetotalsamplenumberbut were placed back on the filter paper co be counted at the next inspection. 5 repeats of this tesc were completed on che same day, where possible. Tesc scores were summed, as were the tocal number oflice sampled,and theconcrot wascheckedforarandomdiscriburion by usingaChi-square cesi with Yaces’correction. The 5 repeats were summed co produce a percentage repellency value using the following formula:% Repeilency f^. treacecLcgntrQt),-: C^ltrgaregLcestj made without the active ingredient, was also employed. Anothertescwasdevised t-ofmdourwhetherrepellentshave any enecc on the choice of egg laying site. This used 9cm diameterfilter papercircles. each torn, noccuc, inroasquareand bisected by a pencil line into 2 triangles. The rough edges normally form an attractive site for the laying ofeggs. Onehalf ofeach paper was impregnated with 200pl ofeither placebo or Rappell and the papers were dried for halfan hour. A batch of 20 young adult females and 20 young adult males were incubated on the papers over a 24 hour period. The eggs laid were then counted. These tests were repeated over 5 days and egg counts summed for each type ofarea. RESULTS A repellency value, for clothing lice, co created filter discs was obtained for a 10% solution ofDEBT and for 10% piperonal. A high repellency of 95.8% was obtained for piperonal but only 42.9% of lice rested, were repelled by DEBT. On the next day, a 2% solution ofpiperonal was found to be almost twice as effective (81.4% repellency) as a 50% solution cf DEET againsc [ice (41.0% repellency) (Table 1). When Rappell created papers were left exposed for half TABLE 1 IN VITRO ARENA TESTS TO DETERMINE REPELLENCY OF CLOTHING LICE TO DEET AND PIPERONAL Test Solutions Sample % Repellency S Treated Side 90% Isopropanol (control! 234 464 10% OEET in isopropanal 131 455 42.9% 10% Piperonal in isopropanol 10 473 95.8% Control (untreated) 219 476 50% DEET in isopropanol 123 453 41.0% 2% Piperonal in isopropanol 41 478 81.4% *!f=0.04,P>0.05 "Xs= 3.04. P>0.05 (Neither’of’the controls were sign/ficant/ydifferentfromarandom distribution asP=0.05atX’=3.84) (S concrol sample) (S test sample) x 100 Sjreaced control S control sample This formula, adapted from Schreck (1977), was originally used co evaluate new compounds, as repellents, for crawling archropods such as ricks, fleas and mices. If chere were no repellent effecc whatsoever, the lice would be expected to be found in approximately equal numbers on each side of the boundary. The percentage repellency describes the degree of variation from this equality. The fifth ancennal segments were removed from another group ofadulc female lice, under a dissection microscope using a very fine blade. Tescs were then repeated as before except that the lice were given approximately 20 hours to recover, so thai chey were used 24 hours after cheir lasr feed. The lice were chen watched in order to determine whether the repellenc response wasabolished. Aconcrolsample, consistingofplacebo Rappell, TABLE 2 ARENA TEST TO’ DETERMINE REPELLENCY OP CLOTHING LICE TOTREATED FILTER DISCS, LEFT IM LOW LIGHT FOR 24 HDURS Test Solutions Sample % Repellency Treated Side Control (Placebo) Rappell (24 hour) Rappell (1 hour) 2 237