Report of the WHO Informal Consultation on the evaluation and testing of insecticides WHO, Geneva We Ola) Lae)¥ CTDWHOPESICI96.1 Distr: LIMITED ENGLISH ONLY REPORT OF THE WHO INFORMAL CONSULTATION ON THE “EVALUATION AND TESTING OF INSECTICIDES” WHO/HQ, Geneva, 7 to 11 October, 1996 WHO PESTICIDE EVALUATION SCHEME (WHOPES) WORLD HEALTH ORGANIZATION DIVISION OF CONTROL OF TROPICAL DISEASES (CTD) This document is not issued to the general public, and all rights are reserved by the World Health Organization (WHO). The document may not be reviewed, abstracted, quoted, reproduced or translated, in part of in whole, without the prior written permission of WHO. No part of this dacument may be stored in a retrieval systam or transmitted in any form or By any means - electro mechanical or other ~ without the prior written permission of wi. ‘The views expressed in documents by named authors are solely the responssility of those authors. Ce document n'est pas destné & dire distribué au grand public et tous les droits y afférens sont réservés par Organisation mondiale de la Santé (OMS). Il ne peut Gtre commenté, résumé, cit, reproduit ou sans une autorisation préalable adult, partillement ou en total ferite de 'OMS. Aucune partie ne recherche dacurentaire ou ditfusée sous quelque forme ou par quelque moyen que ce soit «électronique, mécanique, ou autre - sans une autorisation préalabe érite de OMS, Les opinions exprimées dans les documents par des auteurs cités ‘nommément n’angagent que lesdits auteursCTDIWHOPES/IC/96.1 The consultation was generously supported by the members of the Insecticide Resistance Action Committee (IRAC) of GIFAPCTDWHOPESIIC/96.1 page 3 Contents Page 41. Introduction 4 1.1, Introduction to the meeting 4 1.2. Introduction to WHO Pesticide Evaluation Scheme 5 (WHOPES) 2. The present status of vector-borne diseases in the world 9 3. The present status of vector-control 1 4. The status of vector resistance to insecticides 13 5. The future requirements for pesticides for vector control 14 5.1. Synthetic active ingredients with new modes of action 15 5.2. Insect growth regulators 15 5.3. Biopesticides 15 5.4, Genetically engineered biopesticides 16 5.5. The need for new formulations of insecticides and repellents 16 5.6. New strategies for deployment of insecticides 7 5.7. Repellents 18 5.8. Attractants 18 5.9. Appropriate insecticides and repellents for integration into 19 primary health care 6. Specifications and quality control of pesticides 19 7. Relations with industry and international organizations 21 7.1, Industries 22 7.2. FAO 23 7.3. AOAC and CIPAC 23 7.4, UNEP 23 7.5, OECD 23 7.6. Collaborating Centres 24 7.7. Global Collaboration for Development of Pesticides 24 for Public Health (GCDPP) 8. Future directions for WHOPES 25 9. Recommendations 27 Annex |. Protocols for the laboratory and field evaluation of insecticides 29 and repellents Annex II. List of participants 65 Annex Ill. Useful references 68CTDWHOPESIICI9t page 4 41. INTRODUCTION 1.1, Introduction to the meeting The consultation was opened on behalf of the Director-General of the WHO by the Assistant Director General, Dr. R. H. Henderson. He recalled that the last consultation on this subject was held in November 1987' and the present consultation was convened with the following objectives: * to review and update the methods and criteria for the evaluation and testing of pesticides; * to provide information to the pesticide industry about the requirements for pesticides in public health programmes; ¢ to draw the attention of the industry to the problems faced in vector-borne disease control, especially those due to the spread of resistance to insecticides; * to stimulate the development of alternative chemicals and formulations for public health use; and + to strengthen the relationship between WHO, industry and the WHO Collaborating Centres. Dr. Henderson emphasized that the vector-borne diseases and those with intermediate hosts are among the major causes of illness and death in many tropical and subtropical countries. Such diseases, which include malaria, filariasis, schistosomiasis, dengue, trypanosomiasis and _ leishmaniasis, represent a significant impediment to social and economic development. Selective vector control is an integral part of many vector-bone disease control activities, Its implementation envisages targeted site-specific use of available vector contro! methods, taking into consideration the technical and operational feasibility, resources and infrastructures. The available vector control methods are mainly based on environmental management, biological control and the use of chemicals. With rare exceptions, environmental management and biological control have limited applicability on their own, and chemical control is still considered as the most important element in the integrated control of vector-borne diseases. However, due to the resistance of important vectors to some pesticides and the need for the replacement of those whose use has been abandoned for safety, environmental * Meeting of Directors of WHO Collaborating Centres on the Evaluation and Testing of New Pesticides, Geneva, 9-13 November 1987 (WHO/VBC/88.957).(CTDWHOPESIICI96.1 page S or other considerations, the need for new chemical agents with different modes of action, as well as new formulations to be used in various ecological and epidemiological conditions and different application methods is clearly recognized. The WHO Pesticide Evaluation Scheme (WHOPES) is the only international programme which promotes the development and evaluation of new pesticide products and formulations for use in public health. It functions through the participation of representatives of governments, the pesticide industry, WHO Collaborating Centres and universities, associate laboratories as well as other WHO Programmes, notably the Programme for the Promotion of Chemical Safety (PCS) WHOPES should remain a technical resource for the validation, consolidation and dissemination of experiences in the use of pesticides as well as an important body to assist member states in the use of pesticides for public health. Necessary actions should be taken to ensure that the specifications for pesticides, will become widely available and used by all Member States for the purchase and quality control of pesticides. There is, therefore, a strong motivation to further strengthen the programme and to extend its activities to cover a greater variety of public health pests and pesticides. Several activities have already been initiated to strengthen the scheme including moves to harmonize the many activities related to pesticide development and use. 1.2. INTRODUCTION TO WHO PESTICIDE EVALUATION SCHEME (WHOPES) A programme for the evaluation of pesticides for use in public health was initiated by WHO in 1960. After more than a decade, during which over 1000 pesticides with potential public health use were tested, the number of compounds submitted to the programme began to diminish as interest in products such as herbicides and fungicides increased. Stricter regulation of pesticide toxicity was also a factor. The reduction in the number of pesticides for public health purposes resulted in a sharp drop in the productivity of some WHO Collaborating Centres and WHO field units responsible for the medium and/or large-scale field evaluation of the products. This also coincided with the global attrition of medical entomologists as well as general lack of interest of certain Member States in the evaluation and use of insecticides for public health use. These Collaborating Centres and field units were therefore gradually abandoned. Nevertheless, programmes for the control of vector-bome diseases continuedCTDWHOPESIIC/96.1 Page 6 to demand new products, particularly because the problem of vector resistance to pesticides was worsening. The meeting of directors of WHO Collaborating Centres held in March 1982 in Geneva dealt with this problem; the meeting decided to set up a new evaluation programme. This was given the name of WHO Pesticide Evaluation ‘Scheme (WHOPES). Unlike the previous programme, which comprised seven stages of evaluation, WHOPES has only four phases. The new programme also planned for a new method of collaboration between WHO, Member States and industry, for the field evaluation of pesticides. WHOPES was reviewed in November 1987 by a further meeting of directors of Collaborating Centres in order to make the changes that were found to be essential after the new programme had been in operation for five years: ‘The activities which are conducted under each phase of WHOPES are summarized as follows (see table 1): Phase |: Evaluation of the new technical products or their formulations is performed on laboratory bred arthropods. This phase also comprises a study of cross-resistance with the various classes of pesticides currently available, together with toxicological studies and evaluation. Phase Il-The evaluation is performed in the field, on a small-scale (e.g. individual houses), under well-controlled conditions. The trials are performed on natural vector populations with the aid of formulated products. Where appropriate, the action of the products on the non- target fauna is verified. Phase II is also the first opportunity for making observations on any harmful effects of the product upon the operators. Phase Ill: This is a joint undertaking with the participation of WHO, industry and one or more institutions located in endemic countries. The purpose of this phase is to evaluate a product on a large-scale (e.g. several whole villages) on a specified disease vector. The institution supplies qualified staff for implementation, while the industry supplies the insecticide and the funds needed for the trial. WHO bears the technical responsibility for the operation and is involved in the field through independent consultants. All three parties participate in drafting the trial protocol which needs to be adapted to each situation, The final report is drafted by the institution, which submits it to WHO for evaluation. The report is then submitted for review to the manufacturer and distributed in the form of a WHO document. The study itself comprises entomological, safety and, where appropriate, epidemiological evaluation.CTDWHOPESIC/96.1 page 7 Phase IV: When a pesticide has met the criteria of the first three phases, interim specifications are drawn up in close collaboration with the manufacturer for the technical product and the formulations which have been evaluated. The specifications include a description of the pesticide concemed and the formulations suitable for use in public health, together with sections concerning their physical and chemical characteristics. If necessary, the maximum contents of impurities are also included in the specifications. Methods for measuring the characteristics of the products are described. These methods should have been approved either by AOAC (Association of Official Analytical Chemists) or by CIPAC (Collaborative International Pesticide Analytical Council). The specifications also include indications on the safe use of the products and recommendations on the labeling of packaging. The interim specifications are distributed in a special series of WHO documents which can be used for the purchase and quality control of pesticides. Every five or six years, they are reviewed by the WHO Expert Committee on Vector Biology and Control, which may recommend their publication in the manual on Specifications for Pesticides Used in Public Health. The sixth edition of the manual was published in English in 1985, in Spanish in 1986 and in French in 1988. It should be noted that due to the high cost of trials, WHOPES intends to review the results of trials already carried out (especially laboratory trials) and submitted by the manufacturer, and, if appropriate, accept them as a part of the WHOPES requirements. ‘The WHOPES Working Group, a scientific committee, will assist WHOPES in the planning, monitoring and evaluation of pesticides in the scheme. The committee will review the dossiers submitted by the manufacturers and will make recommendations to WHOPES on the complementary trials needed for the full evaluation of the product.CTDWHOPESIICI96.1 page 8 Table 1. WHOPES, four phase evaluation and testing programme Phases | Type of studies | Agencies in Activities charge 1 | Laboratory Collaborating Efficacy Centres - Persistence - Resistance assessment - Toxicity on human and nontarget organisms i | Small-scale field | Collaborating | - Efficacy under different trials Centres ecological conditions - Rate and method of application - Persistence - Effect on non-target organisms (when appropriate) - Safety observations il | Medium- and/or | Member States, | - Efficacy large-scale field | manufacturer, | - Entomological studies trials Collaborating (impact on vectorial Centres capacity and behaviour) - Persistence - Effect on non-target organisms (where appropriate) - Epidemiological studies (where appropriate) - Physical characteristics of formulation - Safety observations (operators and inhabitants) - Acceptance of treatment by residents - Ease of application and equipment performance - Cost-effectiveness W__ | Laboratory Manufacturer | - Drawing up specifications and (physical and chemical Collaborating properties of ai. and Centres formulations; analytical methods; safety recommendations, etc.)CTDWHOPESIIC/S6.1 page 9 2. THE PRESENT STATUS OF VECTOR-BORNE DISEASES IN THE WORLD The vector, snail and rodent-borne diseases impose heavy public health burdens on the citizens of many countries especially in the tropical areas of the world. This section reviews the most important of these diseases. It has been estimated that more than half of the world's population is at risk from tropical diseases and that at least 500 million people suffer from at least one of these infections every year. Not only are the vector-borne diseases the cause of a high level of morbidity and much mortality but they are also responsible for great economic loss and social disruption to the disease endemic countries due to the costs of medical treatment and from lost work and school time resulting from illness. Malaria is the most important of the vector-borne diseases both in terms of its geographical distribution, its incidence and the extent of the morbidity and mortality it causes. About 100 countries are affected by malaria and the WHO has estimated that there may be between 300 and 500 million clinical cases each year with between 1.4 and 2.6 million deaths world wide. More than 90% of these deaths occur in Africa, where malaria is one of the most important causes of morbidity and mortality among infants and young children. It is also apparent that there is a recrudescence of malaria in many geographical areas. The problem of managing malaria has become more difficult due to the development of vector resistance to a number of the insecticides used for its control as well as the spread of resistance in Plasmodium falciparum to antimalarial drugs. Renewed emphasis must be given to vector control especially in areas of multidrug resistance. Chagas disease or American trypanosomiasis, caused by Trypanosoma cruzi, is endemic only in Central and South America. At least 21 countries are affected. About 35 million people are exposed to infection and 16 to 18 million are thought to be already infected and 2 to 3 million people may have already developed chronic disease with its attendant complications and likelinood of early death. In the absence of a vaccine or an effective chemotherapeutic agent, control depends on the control of the bug vector and much progress has been made in the “southern cone" countries of South America in a large scale vector control campaign based on the application of residual insecticides. Lymphatic filariasis, caused mainly by Wuchereria bancrofti, infects some 80 million people of which about 30 million suffer from chronic disease. In Africa, control of the rural anopheline vectors of this parasite is difficult to achieve and in south Asia, East Africa and foci in South America, urbanization has caused great increases in the population densities of the urban mosquito vector, Culex quinquefasciatus. Recent field trials with the drug ivermectin have provided very encouraging results in the treatment of the infection.CTDWHOPESIIC/96.1 page 10 Onchocerciasis transmitted by blackflies has been the cause of hundreds of thousands of cases of blindness-'river blindness"-in Africa and Central and South America, The large scale WHO Onchocerciasis Control Programme based on application of larvicides to rivers to control Simulium larvae followed by the distribution of ivermectin has largely suppressed transmission of the infection in countries of west Africa where the programme has been carried out. In other countries in Africa and in the Americas, a large scale programme based on ivermectin distribution has been launched. Schistosomiasis of which snails are the intermediate host, is very wide spread throughout much of Africa, the Middle East and parts of Asia and the ‘Americas. It is the second most prevalent tropical disease after malaria. It is estimated that there are 200 million persons infected with schistosomiasis worldwide. It is responsible for severe morbidity and economic losses. While an effective drug is available for the treatment of infection by the parasites, water resource development projects have been spreading the infection to new areas. African trypanosomiasis or sleeping sickness is transmitted by tsetse flies. While there has been successful contro! of the vector fly by the use of traps and insecticide treated targets in some countries, nevertheless there were estimated to be some 25 to 50,000 cases of sleeping sickness every year and about 25,000 deaths. Major epidemics are occurring in some countries where control efforts have not been sustained. Leishmaniasis is endemic in many tropical and subtropical regions of the world. Cutaneous leishmaniasis is the most common clinical form and there are 1 to 1.5 million new cases per year which are the cause of great suffering. Visceral leishmaniasis is the cause of a severe systemic infection which is usually fatal if untreated. In the Indian sub-continent and certain areas of the Eastern Mediterranean and East African regions there has been serious recrudescence of visceral leishmaniasis; this increase has been a consequence of the return of the sandfly vector following cessation of indoor spraying by the malaria eradication programme. Visceral leishmaniasis has been the cause of epidemics of hundreds of thousands of cases and possibly some 100,000 deaths a year. Arboviral infections are responsible for millions of cases of disease every year, most of these are due to dengue with its severe and often fatal syndromes of dengue haemorrhagic fever and dengue shock syndrome, (DHF/DSS) which is a problem in more than 100 countries. This infection is responsible for about 500,000 hospitalizations every year with an average mortality rate of 5% causing tens of thousands of deaths. As there is neither a chemotherapeutic agent nor an effective vaccine available, control of the transmission of dengue virus must depend on the control of the vector mosquitoes.CTDWHOPESIIC/96.1 page 11 Yellow fever virus is widespread in Africa and South America. The natural reservoirs are monkeys and the disease may be transmitted by mosquito vectors to man when they enter the forest or jungle. Despite the availability of an efficient vaccine, there has been a serious recrudescence of the disease, especially in Africa including its spread to areas where it had been uncommon, The urban vector of yellow fever is Aedes aegypti and there is a constant threat of urban outbreaks of the disease as have recently occurred in Africa. In the Wester Pacific and Southeast Asia Japanese encephalitis has been reported in 14 countries. The causative virus of this severe disease is transmitted by mosquitoes breeding in rice fields. Increased irrigation projects have resulted in increases in vector mosquito densities and increased rearing of the amplifying host, the pig, has been the cause of increased outbreaks of the disease. In southem Africa and the middle east, Rift Valley Fever transmitted by Culex mosquitoes has been the cause of great epidemics among animal herds along with a substantial number of serious human cases including many deaths. There are several species of vector mosquitoes which breed in both manmade and natural larval habitats. The infection is of great economic and public health importance when outbreaks occur. Tick-borne diseases, e.g. Lyme disease and relapsing fever, are emerging as serious problems in widespread geographical areas of the world Among the rodent-borne diseases, plague persists in all foci in which it has been previously known; new strains of haemorrhagic fever with renal syndrome are emerging and rapidly spreading. Scrub typhus and murine typhus, both of which have rodent reservoirs persist in most of the foci in which they have previously been reported and, in some areas, their incidence is increasing, House dust mites are @ major cause of asthma and are responsible for increasing morbidity in the developed world. Their control is difficult and requires much further study. There is also evidence that control of house flies reduces the incidence of diarheal diseases. These diseases are estimated to cause about 3 million deaths per year. 3. THE PRESENT STATUS OF VECTOR-CONTROL For a substantial number of these diseases, the group agreed that the use of pesticides for vector control will continue to play the major role in the programmes for disease control. These pesticides will include synthetic andCTDWHOPESIIC/96.1 page 12 biologically derived compounds. There have, however, been important changes in the concepts of vector control; it is now emphasized that the methods in use must be environmentally acceptable and that they should be sustainable. There has been a trend towards the use of pesticides which are less persistent in the environment and of low mammalian toxicity. As a result of the increased emphasis on environmental acceptability and sustainability of the programme, there have also been important changes in their management strategies. The changes in management strategies involve more selective and effective application of pesticides. Examples of more selective applications are the use of insecticide-impregnated bednets for mosquito control and traps and targets for tsetse control. Most vector control programmes will continue to depend on large scale pesticide applications; these include the use of indoor residual sprays, and insecticide impregnated bednets for malaria and leishmaniasis vector control, the use of larvicides for the control of Culex mosquito pests and vectors of filariasis and adulticide space sprays and larvicides for the control of the mosquito vectors of dengue. Indoor residual sprays remain the main method for the control of the triatomine vectors of Chagas disease. Louse and flea control are based almost entirely on pesticides. Wherever possible biological and environmental control methods are integrated into vector control programmes but pesticides remain the major control tool. Where the ‘community is to be involved in vector control, whatever the agents and methods applied, it is necessary to ensure that they have adequate training and sustained support of their efforts. The use of pesticides has been impeded by the growing concern with their possible effects on the environment, their toxicity to non-target organisms, their increased costs and the growth and spread of resistance. This, in part, has been the cause of a substantial decline in the number of candidate insecticides submitted to WHOPES by the chemical industry. ‘As noted above, much attention has been given to the development of alternatives to pesticides in vector control. Environmental, biological and genetic methods have been considered. The successful use of many environmental methods depends on the presence of a developed infrastructure including underground sewage, piped water supply, adequate housing and regular refuse collections, all of which depend on adequate capital investment. There have been some outstanding success with the use of environmental management and other alternatives particularly against vectors of malaria in several regions. Such methods have also proved successful with the use of traps for the control of tsetse vectors of African trypanosomiasis. Biological control implies the use of predators, parasites or pathogens. While many field trials have been carried out against vectors with each of these(CTDIWHOPESIIC/96.4 page 13 agents, only fish predators of mosquito larvae have been extensively applied in some areas with success. Predators and parasitoids of housefly larvae and pupae have been effectively used in limited areas. The toxins produced by Bacillus species and used in mosquito and black fly control should be regarded as pesticides though of biological rather than chemical origin. With the exception of the sterile insect release technique for tsetse fly control, there has been no other example of successful vector control using genetic methods. Genetic control of vectors using presently available methods is expensive. Its use may be justified where eradication of a vector from a defined area is feasible. It must be emphasized that with greater knowledge of the biology and behaviour of the target insects, insecticide use has become more selective and less environmentally polluting. The continued development of new pesticides and pesticide groups, such as the insect growth regulators to replace those to which resistance has developed, will require the continued improvement and support of the insecticide industry, greater support from the WHO, continued research on the behaviour of the vectors and field evaluation. 4, THE STATUS OF VECTOR RESISTANCE TO INSECTICIDES Pesticide resistance has appeared in almost every major group of insect and tick vectors of diseases as well as among rodent reservoirs. By 1991 resistance to insecticides in public health use was reported in more than 150 species of vectors and nuisance pests, to all major classes of insecticides, and the number has been steadily increasing. Furthermore, cross-resistance has occurred between older insecticides such as DDT and pyrethroids in different species of mosquitoes and among the different pyrethroids themselves. As an example Anopheles gambiae s.s. in several West African countries has been reported as resistant to permethrin with delayed knockdown (decreased susceptibility) to deltamethrin and lambdacyhalothrin. In Turkey, An. sacharovi is now resistant to DDT, propoxur, bendiocarb, permethrin, deltamethrin, lambdacyhalothrin and cypermethrin. Such multiple resistance also exists in populations of An. albimanus in Central and South America. In Indonesia Aedes aegypti is reported as highly resistant to propoxur, bendiocarb, and pyrethroids. High levels of multiresistance involving OP, carbamate and pyrethroid insecticides has been reported in Cx. quinquefasciatus populations in many countries. It is considered that there is considerable under-reporting of insecticide resistance and there are other instances of multiresistant species of mosquitoes and other insects of public health importance in various geographical areas,CTDIWHOPESIIC/96.1 page 14 The presence of insecticide resistance has impeded many vector control programmes and the necessity to develop altemative, environmentally acceptable and cost effective compounds is the main purpose of WHOPES and remains an even greater necessity as insecticide resistance spreads Techniques exist, and in some cases the relevant reference strains of test insects exist, both of which are necessary to enable cross-resistance and multiple resistance phenomena to be recognized when screening existing and new compounds through WHOPES. 5. THE FUTURE REQUIREMENTS FOR PESTICIDES FOR VECTOR CONTROL There have been major successes in solving some vector problems. However, many problems remain and these are likely to be augmented by the following factors:- «Emergence of physiological or behavioural resistance to one or more of the existing categories of pesticide in increasing numbers of vector species and areas of the world; * Increasing sensitivity to environmental issues causing demand for pesticides with reduced hazard to humans and other non-target organisms; Rapid unplanned urbanization in many tropical areas leading to increased problems of urban vectors and pests; * Construction of dams, water resource development and colonization of jungle areas for agriculture, logging and mining, leading to increased problems of the mosquito or snail vectors/intermediate hosts of malaria, schistosomiasis and Japanese encephalitis; + The possibility that global warming will lead to spread of vector problems to higher altitudes in the tropics and return of some of these problems to temperate areas from which they were eradicated in earlier decades. For all of these reasons, in the next decade or so, one can expect increased demand for more appropriate pesticides. To make new active ingredients more useful, formulations and strategies for their use in integrated management will be necessary. WHOPES should play a major role in the evaluation of such innovations, develop liaison between industry, researchers and Member States and serve as a clearing house for pesticides testing and evaluation.CTDWHOPESIIC/96.1 page 15 5.1, Synthetic active ingredients with new modes of action The four well known categories of insecticides are organochiorines, organophosphates, carbamates and pyrethroids. Examples exist among vector populations of resistance to each of these categories. However, recently, synthetic insecticides of new categories, such as phenyl pyrazoles and others, have been developed and their testing against vectors has begun, including addressing the vital question whether existing resistance mechanisms confer cross resistance to the innovations. WHOPES should do everything possible to encourage the industry to bring forward compounds, developed with the agricultural market in mind, for testing against vectors. 5.2, Insect Growth Regulators Many chemicals exhibiting insect growth inhibiting properties have been developed or are being developed at the present time. Most of the compounds discovered thus far are grouped as juvenile hormone mimics or chitin synthesis inhibitors. These compounds in general have a high margin of safety to humans and little or no toxicity to most non target organisms. They are relatively specific in their action against insects, showing a high degree of activity against insects of public health importance. They are primarily active against the immature stages of mosquitoes, flies and other insects, but some IGRs induce sterility and other reproductive anomalies in the adult stage. IGRs have a great potential for the control of mosquitoes especially container breeding species, as well as species breeding in pools, waste water accumulations, wells, impoundments and other developmental sites. Some IGRs have also been found to be highly efficacious against fleas, house flies and cockroaches. It is desirable to evaluate currently available, as well as newly discovered, IGRs against a variety of vectors and pestiferous insects. Because of their relatively low toxicity to fish and other wildlife, IGRs can be successfully used in integrated control of mosquitoes in conjunction with fish or other predators. 5.3. Biopesticides The term biopesticides is now finding acceptance among vector and pest control technologists. Biopesticides include microbial control agents, phytochemicals with pesticidal activity, toxins produced by algae and other organisms. In recent years great strides have been made in isolating, identifying and developing spore-forming bacterial agents for vector control Among these, Bacillus thuringiensis H-14 and B. sphaericus have been commercialized and used for a number of years in the control of disease vectors and pest mosquitoes and blackflies. Currently, emphasis is placed onCTDWHOPES/ICI96.1 page 16 extracting and evaluating extracts of plants for insecticidal activity. Plants may be considered as factories synthesizing many chemicals, a number of which serve to provide defense against phytophagous insects. Naturally, these types of chemicals have potent insecticidal activity and provide models for the design and synthesis of new chemicals with novel modes of action Some plant extracts have been found to show considerable activity against mosquitoes and flies. Exploitation of locally available plants with insecticidal properties will avail us new modes of action and new tools for vector and pest control in developing countries. 5.4. Genetically engineered biopesticides Genetically improved bacterial agents could provide us with new control agents for the future in the form of biopesticides which may solve some of the problems of resistance. In addition, products which can be more cheaply produced, have higher efficacy and/or expanded range of bioactivity will make them more likely to be usable in low income tropical countries. Following the identification of new, natural, entomopathogenic bacterial strains of B. thuringiensis H-14 and Clostridium bifermentans serovar malaysia, characterization of novel mosquitocidal toxins is now in progress, supported by WHO. Such strains may be a great improvement on the widely used B. thuringiensis H-14 and B. sphaericus. They may also allow toxin diversification by the construction of recombinant strains combining toxin genes from different origins which may have synergistic effects. This will require cloning of characterized toxin genes and their introduction into new hosts. Such a strategy may be particularly useful with B. sphaericus to combine its good qualities with higher toxicity and a wider range of toxicity to insects. Such transgenic bacteria would be expected to have a reduced likelihood of insect resistance because of their possession of a mixture of several unrelated toxins, each requiring evolution of a separate resistance gene if an insect is to survive exposure to the transgenic bacteria. Attempts have also been made to introduce toxin genes into species of bacteria which could colonize larval habitats so that these become permanently farvicidal WHOPES should have a role in evaluating the efficacy and safety of new strains produced and. in considering the legal questions surrounding the irrevocable release of transgenic organisms. 5.5. The need for new formulations of insecticides and repellents Much can be done to improve the effectiveness, persistence and/or safety of insecticides by improvement of the formulations used in vector control.(CTDIWHOPESIICI96.1 page 17 ‘An example is the recent development of a slow-release formulation designed to last longer when sprayed onto walls for triatomine control. Micro- encapsulated formulations of several insecticides and repellents to improve persistence have been developed. It would seem possible to extend this technology to other compounds. ‘The safety of space sprays has been increased by development of water based formulations; the same is true of replacement of emulsifiable concentrates by suspension concentrates, and other improved formulations for making the mixtures with which bednets are treated. Larviciding, especially with bacterial toxins or insect growth regulators, may be made more persistent and affordable by developing formulations which release the agent slowly and at the appropriate depth to be likely to be ‘encountered by the target larvae. Mosquito coils, vaporizing mats and liquid vaporizers may be considered as other types of insecticide formulations. There is a developing market in these devices and the many different brands need to be monitored by national authorities using approved protocols. In the area of packaging and distribution, advances have been made in limited areas, To reduce contact of spraymen with toxic pesticides the pesticides are made available in pre-weighed sachets. 5.6. New strategies for deployment of insecticides Over the last decade, work on insecticide impregnated bednets and curtains has grown from small scale tests, to trials and operations with a total of more than 10 million treated nets, The concept is to place a small quantity of a quick acting insecticide of low mammalian toxicity directly in the path of the host seeking mosquito. This chemical barrier improves on the physical barrier provided by the net or curtain. Also, in some cases, the mass killing due to widespread use of impregnated material lowers the local vectorial capacity and provides protection to non-users of the material. Considerable reductions in all- cause child mortality have recently been recorded in WHO sponsored African trials, and operational use in China has lowered hypo-endemic levels of malaria. Barriers to the extension of this method include the difficulty of many communities in low income countries to pay for it and the threat of resistance developing to the pyrethroids, the only insecticides currently used for impregnation of nets and curtains. WHOPES should encourage the development of cheaper and longer lasting pyrethroid formulations and assist in field trials of them for the control of malaria transmission. The threat of resistance could be met by the use of compounds other than pyrethroids whichCTDIWHOPES/IC/96.1 page 18 show no cross resistance or which even possess a negative cross resistance in anopheline species which have developed resistance to existing pyrethroids, The deployment of pyrethroids or insect growth regulators which sterilize females on odour- or visually-baited tsetse traps and screens operates on essentially the same principles as impregnated bednets. 5.7. Repellents Repellents are the most common means of personal protection against mosquitoes and they provide the only method available against the biting of exophagic mosquitoes and sandflies. So far a limited number of synthetic or plant-based compounds have been shown to provide satisfactory protection against mosquitoes. By far the most commonly used active ingredient has been di-ethyl-3-methyl benzamide (deet). However, questions have been raised about its safety and effectiveness against some species of Anopheles and it attacks paint, varnish and some hard plastics. Derivatives of certain plants, including lemon eucalyptus, neem and citronella, have shown some potential and conventional or non-ester pyrethroids such as permethrin or etofenprox applied to the skin or clothing have been shown to provide adequate protection against mosquito bites. Major chemical companies are actively involved in development of synthetic substitutes for deet and/or formulations to prolong efficacy or increase consumer acceptability. All of these developments need to be critically tested in the laboratory and the field. 5.8. Attractants The field of attractants of vectors and human pests is not adequately studied. It is conceivable that insect attractants either from the insects themselves or of host or habitat origin could offer some new avenues for the management and control of vectors and pests. Currently, several kinds of feeding attractants (synthetic and natural) as well as a sex pheromone attractant are employed for the control of synanthropic flies. An attractant of host origin in now employed in traps and in conjunction with pesticides for the control of savanna species of tsetse flies. An oviposition pheromone of Culex mosquitoes has been chemically identified and the use of this pheromone offers some possibility for managing mosquito vectors of filariasis. An even more potent blend of oviposition attractants of these mosquitoes has been chemically identified and shown to induce oviposition by gravid females under field conditions. It is envisaged that larvicide treated pots of water baited with Culex oviposition attractants could usefully be integrated with treatment of major breeding sites in pit latrines and cess pits with polystyrene beads. The baitedCTD WHOPES/ICI96.1 page 19 pots may attract the egg laying of females deterred from ovipositing in the treated pits and avoid the risk of them being diverted to inaccessible but productive sites. It is anticipated that in the future, insect attractants might become more widely available and be incorporated in the integrated control of disease vectors and human pests 5.9. Appropriate insecticides and repellents for integration into primary health care Insecticides possessing low toxicity to non-target organisms may be safely used for application by villagers who may have no training in safe handling of more toxic compounds. If indigenous plants and their metabolites can be shown to be efficacious against vectors, their safe local use is to be encouraged as it should be affordable even by the most deprived communities. Though devices such as vaporizing mats generally require electricity, similar effects can be achieved by appropriate use of volatile pyrethroids in conjunction with widely used oil lamps Use of plant based repellents by children and pregnant women, who are vulnerable to malaria, may be an appropriate means for their protection against the bite of anopheline vectors. Although these ideas are simple and appealing they need to be critically tested before giving any overt encouragement to their use. 6. SPECIFICATIONS AND QUALITY CONTROL OF PESTICIDES WHO has developed specifications for chemical pesticides used in public health since 1953. These have been regularly published after periodic meetings of the WHO Expert Committee on Vector Biology and Control, called to consider and approve the draft specifications drawn up in consultation with Collaborating Centres and indusiry (see Table 2). The specifications have proven to be of great importance to national governments in ensuring the procurement of quality pesticides for use in disease vector and reservoir control programmes. It would be most useful if harmonization could be achieved, whenever possible (due to differences in purpose and requirements), between specifications issued by WHO and that of Food and Agriculture Organization of the United Nations (FAO)CTDWHOPESICI96.1 page 20 Specifications for compounds which have successfully passed WHOPES should be drawn up and published as soon as possible. The information on such specifications and on newly available pesticides should then be brought to. the attention of vector control programmes in disease endemic countries, so that it can be effectively used in purchase and quality control of the products. Table 2. Pesticide products which have passed the WHO Pesticide Evaluation ‘Scheme (WHOPES) and for which specifications are available DDT (technical, WDP, EC, dustable powder) Lindane (technical, WDP, EC, dustable powder) Methoxychlor (technical, EC) Pyrethrum Diazinon (technical, WOP, EC) Malathion (technical, WDP, EC, dustable powder) Bendiocarb (technical, WDP, dustable powder) Deltamethrin (technical, WOP, EC, dustable powder, ULV liquid) Larvicidal oil Diflubenzuron (preconcentrate, WDP) Dimethoate (technical, EC) Endosulfan (technical, EC) Permethrin (technical, EC) Phoxim (technical, EC) Pirimiphos-methyl (technical, WDP, EC) Trichlofon (technical, water soluble powder) Fenthion (technical, WOP, EC) Dichlorvos (technical, EC) Fenitrothion (technical, WOP, EC) Propoxur (technical, WOP) ‘Temephos (technical, EC, sand granules) Chloropyrifos (technical, EC) lodofenphos (technical, WOP) Niclosamide (technical, WDP, EC) DEET (technical) * Interim specifications are available for lambdacyhalothrin (technical, WDP, EC) and Brodifacoum (Technical, concentrates, baits).CTD WHOPESIIC/96.1 page 21 Biopesticides are already in use in agriculture as well as vector control programmes, though there are no written specifications to assist in their acquisition Existing biopesticides and those being considered for use in public health will require standardization and specification for use in quality control. A variety of biochemical and molecular analytical techniques are available for evaluation, but standardization of these methods would be important in establishing specifications, Very large quantities of household insecticidal products are used throughout the world of which a large proportion is used for the control of arthropods of public health importance. These include aerosols, coils, electric fumigation mats, liquid vaporizers, baits, and traps. Industry sources have reported that consumers spent approximately US $ 6.1 billion on such products in 1994. The use of insecticides for pest control in the household can be considered a form of community participation in vector and pest control. At present only a few countries have established specifications and laboratory and bioefficacy test protocols, for this group. Because of their importance and widespread use, it is considered that the establishment of international guidelines including chemical and physical specifications and methods for determining their biological efficacy is important. Because of the distinct nature of this group, it is considered desirable that a meeting be called to assist WHOPES in drawing up specifications and evaluation methods Large-scale use of rodenticides is made in both agriculture and public health. A number of new rodenticides have been developed and marketed over the last two decades but there are practically no specifications for this group. To remedy this situation and establish specifications, guidelines are needed. As both WHO and FAO are concerned with this subject, the organizations could work together to establish guidelines in cooperation with GIFAP. The quality control of pesticides is intended to ensure that the product used is effective and safe for both humans and the environment. As public health pesticides are frequently applied in close contact with the spray team and the population to be protected, considerations of human and environmental safety are of particular importance (requiring specifications which might be different from that of agricultural: pesticides). Problems may frequently occur with products that are no longer under patent and are produced by a large number of manufacturers some of whom have: inadequate experience in manufacturing/formulations as well as with products that have been stored for long periods of time, often under extreme climatic conditions. WHOPES shall continue to assist Member States in such quality control. 7. RELATIONS WITH INDUSTRY AND INTERNATIONAL ORGANIZATIONS Research in the field of pesticide development is best advanced by assisting, coordinating and making use of the activities of collaborating agencies, institutions and industry. Therefore, WHOPES should furtherCTDWHOPES/IC/96.1 page 22 strengthen its relationship with the named partners in order to carry out its responsibilities WHOPES should be a focal point for collection, consolidation and dissemination of information on pesticides and their uses for vector control WHOPES should actively collect information from member states, through WHO regional offices; the mechanisms for dissemination should include: * publication of test results. © preparation or updating of manuals on vector control and specifications of pesticides. * preparation of computerized data bases on pesticides, which may be made available through internet. * contribution to the publication of data sheets and, eventually, the compilation of monographs on specific pesticides Alll these documents should be made widely available to member states through the WHO Regional Offices. Exchange of information should not be limited to the list of agencies directly participating in WHOPES, but should also be made available to the international donor community, e.g. UNICEF, UNDP, UNHCR, World Bank, and bilateral agencies, e.g. USAID, ODA, JICA, SIDA, CIDA, DANIDA, and internationally operating NGOs which are collaborating in health programmes. 7.1. Industries There are three types of industry which should closely collaborate with WHOPES: pesticide manufacturers, pesticide application equipment manufacturers, and consultant firms; each of these groups has its own international associations, e.g. GIFAP, and working groups, e.g. IRAC, which have relations with WHOPES. WHOPES should strengthen working relations at all these levels. There is a need to widen and strengthen WHO/Industry collaboration over the whole range of vector contro! activities, with particular reference to the development and testing of new products, drawing up of specifications, exchange and dissemination of information, resistance monitoring, development of resistance management strategies and promoting selective vector control programmes. The exchange of information should aim at an open discussion for joint planning of product development. WHOPES should take a pro-active role in its relationship with industry not only accepting the testing and evaluation of products submitted by industry but, following the identification of needs, activelyCTDWHOPESIIC/96.1 page 23 requesting the submission of products which may fulfil those requirements or promoting the development of suitable new formulations or products. WHOPES should co-ordinate collection of information on needs and actual or projected consumption of pesticides used in public health programmes, which should be consolidated by WHO regions and disease control programmes for analysis. Subsequently, as required, special investigations of total pesticide consumption, including users other than public health programmes (mainly agriculture, nuisance control and household pesticides) should be undertaken by WHOPES in conjunction with industry and market research companies. 7.2. FAO WHO and FAO have a long history of collaboration derived from their mutual interest in pesticide testing and evaluation. WHOPES should strengthen its collaboration with FAO in the following areas: sharing of experiences with common active ingredients, harmonization of specifications, pesticide application technology, and resistance management strategies. WHOPES has fully adopted the FAO Code of Conduct for Pesticide Distribution and Use. It would be most useful if harmonization could be achieved whenever possible between specifications issued by WHO and FAO. 7.3. AOAC and CIPAC WHOPES should continue and further strengthen its collaboration with AOAC (Association of Official Analytical Chemists) and CIPAC (Collaborative International Pesticide Analytical Council) in developing standard methods of analysis and physical tests for inclusion in the specifications. 7.4. UNEP WHOPES should assist and collaborate with UNEP in disseminating relevant information to promote the use of effective vector control methods by disease control programmes which are safe for humans and the environment. 7.5. OECD WHOPES should provide relevant information to the collaborative programmes of OECD (Organization for Economic Cooperation and Development), GIFAP, IPCS and other agencies including national authorities,CTDAWHOPESIICI96.1 page 24 particularly those aimed at the improvement of the efficiency of pesticide re- registration and the promotion of intemational harmonization in pesticide registration requirements. 7.6. Collaborating Centres WHOPES should review the existing network of collaborating centres in order to identify necessary actions to adapt the network to the new requirements of the Scheme in its different phases. Terms of reference for phase III should be appropriate to the centre's competence in specific diseases and the needs of the Scheme, The review should identify necessary WHOPES inputs in order to maintain the activities of the centres, both in funding and in basic technical support. Collaborating centres should not only participate in the testing and evaluation of new pesticides, through the phases of the Scheme, but should also play an essential role in training (e.g. research training) at all levels, problem identification and need assessment, and * technical support to WHOPES, e.g. in providing technical consultation to national registration trials, if required WHOPES has an important contribution to make to CTD training activities and the preparation of training materials, not only by the resources of its collaborating centres, but also by its documentation, data bases and technical competence; it should, therefore, become an active member of the MANTEAU (Managing Tropical Diseases through Education and Understanding) programme. 7.7. Global Collaboration for Development of Pesticides for Public Health iCDPP” The Division of Control of Tropical Diseases has recently taken a new initiative to achieve these goals and further strengthen WHOPES activities, by initiating a new partnership in the field of pesticide development for public health, named WHOPES GLOBAL COLLABORATION FOR DEVELOPMENT OF PESTICIDES FOR PUBLIC HEALTH (GCDPP), participants of which will include collaborating agencies (e.g. UN and bilateral agencies), foundations and institutions (WHO Collaborating Centres), organizations such as GIFAP (International Group of National Associations of Agrochemical Manufacturers) as well as pesticide and pesticide application equipment manufacturers. In addition to the aforesaid participants, ‘distinguished scientists and officers inCTDIWHOPESIICI96.1 page 25 charge of vector control activities in the WHO Regional Offices may also be invited to attend the meetings of GCDPP, as and when the need arises. The GCDPP will provide a forum for the exchange of technical information and ideas, and will offer opportunities for participants to make suggestions with respect to programme strategies and priorities, progress achieved, the proposed WHOPES biennial plan and collaboration with other organizations and institutions. WHO/CTD will establish and administer a trust fund in which contributions from the participants and other financial support designated to support the activities of the GCDPP will be deposited. This trust fund, entitled the "Global Collaboration for Development of Pesticides for Public Health (°GCDPP Trust Fund") will be administered in accordance with WHO's financial rules, regulations and practices. The available funds will be used for the convening of meetings, production and dissemination of related documentation, the general exchange and dissemination of information on the public health needs for pesticides and pesticide application equipment and otherwise for activities which promote the development of alternative, safer and more cost- effective pesticides and application methodologies, as agreed upon by the GCDPP. 8. FUTURE DIRECTIONS FOR WHOPES ‘As has been noted earlier, the control of the vector-borne tropical diseases will continue to rely on the use of pesticides for the foreseeable future. There are, however, problems constraining the use of many currently available pesticides; these include the development of insecticide resistance among virtually every major group of vectors, heightened concem with the possible effect of pesticides on the environment and concern with pesticide toxicity to non-target organisms. To meet the continuing and, indeed, increasing need for new, selective and safe compounds, it is essential that WHOPES encourage the development of altemative pesticides by the pesticide industry. WHOPES priority must be given to pesticides which may be effective in the control of important groups of arthropod vectors or rodent reservoirs of diseases in which high levels of resistance has developed. This necessitates the use of alternative compounds to which no cross resistance exists and this will be taken into account in the areas selected for field trials. Special effort will also be made in the development of pesticides which are cheaper, more acceptable to people and suitable for use by the community in their own protection. ‘Such insecticides should be of low mammalian toxicity, easily formulated for use and highly effective against the target vectors or pests.CTDWHOPESIICI96.1 page 26 Many of the newer pesticides including both chemical and biopesticides being submitted to WHOPES would belong to new groups whose modes of action frequently differ from those of older compounds, WHOPES will need to regularly adapt its testing methods to ensure that laboratory and field trials of the candidate compounds accurately measure their efficacy. This will necessitate close consultation with both industry and the collaborative centres responsible for the testing and implementation of efficacy studies. The insecticide industry, globally, is faced by many regulations related to averting environmental or toxic hazards; WHOPES should take these regulations into account, where possible. A number of insecticides important to public health vector control programmes are being withdrawn from registration by industry, How this problem can be dealt with should be the subject of discussions with national authorities and industry. Increasing attention must be given to the evaluation of pesticides intended for the control of vectors which were not previously given priority by WHOPES but whose control is of concern to Member States. This will include the arthropod vectors of infections such as arboviruses in addition to dengue and the tick-borne diseases. Furthermore, the recrudescence of body louse populations will require testing methods for pesticides intended for the control of body lice to be developed Existing test methods of compounds intended for the control of mechanical vectors of diseases such as cockroaches and houseflies or mite agents of allergies will also have to be developed or reviewed. The control of the nuisance caused by insects such as bedbugs, and many species of mosquitoes which are not necessarily vectors of disease represents an already considerable and increasing expenditure in both the developed and developing world for the purchase of household insecticides. With the participation of Collaborating Centres, methods for the testing of compounds used in preparations for the control of pest mosquitoes, fleas, bedbugs, head lice and ticks may have to be developed by WHOPES to reflect the needs of governments, industries, research groups and regulatory bodies. This may necessitate the calling of a special consultation.9, RECOMMENDATIONS 1, WHOPES should encourage and coordinate efforts to promote the evaluation and development of safe, cost-effective and environmentally acceptable pesticides for vector and human pest control 2. WHOPES should continue to support the development of new pesticides including the insect growth regulators and biologically produced toxins to cope with the development of insecticide resistance. 3. Continued encouragement should be given, by WHO/CTD, to the development and application of management strategies which lead to the more selective use of pesticides. 4. WHOPES should inform industry and research institutions of the current and future needs for pesticides and their formulations for use in vector and pest control programmes. 5, WHOPES, in collaboration with industry and research institutions, should develop workable schemes for the testing and evaluation of pesticides possessing novel modes of action, with special emphasis on microbial control agents and natural products. 6. WHOPES in collaboration with research institutions and industry, should promote the development of specific and tailor-made formulations of effective pesticides for use in specific and area-wide vector control programmes. 7. There is a need for finding and developing new insect repellents for personal protection against biting arthropods. Industry should be encouraged to promote the development of new insect repellents. 8. The scope of the activity of WHOPES should be broadened to include important groups of human pests such as synanthropic flies, cockroaches, house dust mites, rodents and others. 9. WHOPES should be an important focal point for collection, consolidation and dissemination of information on pesticides and their uses for vector control. WHOPES should actively collect information from Member States, through WHO regional offices. 10. WHOPES should take a proactive role in its relationship with industry, not ‘only promoting the testing and evaluation of products submitted by industry but, following the identification of needs, actively request the submission of products which may fulfil those requirements or promote the development of suitable new formulations.CTDWHOPESICI96.1 page 28 11.WHOPES Collaborating Centres should seek to standardize the establishment and maintenance of characterized resistant and susceptible strains of given vector species for evaluating cross resistance to new candidate pesticides. 12. Informal consultations should be organized in collaboration with FAO to establish uniform specifications for rodenticides and biopesticides. 13. WHOPES should convene consultative meetings to develop protocols for bio-efficacy assessment of household pesticides as well as establishing Quidelines for specification of such products. 14. It would be most useful if harmonization could be achieved whenever possible between specifications issued by WHO for pesticides used in public health and FAO for plant protection products; this will be the task of the WHO Expert Committee on Vector Biology and Control 15. Consultations between WHO and FAO should continue for the purpose of reviewing pesticide application technology and equipment. 16. WHOICTD should promote consultations between WHO and FAO, with active involvement of WHOPES, for developing resistance management strategies. 17. WHOPES should continue collaboration with the Member States in quality control assurance on products entering the market or after long-term storage. This quality control must be undertaken by designated WHO Collaborating Centres or by the local recognized laboratories under WHO supervision, and certification of analysis from the laboratories mentioned above should be included in the requirement. WHOPES should continue to encourage national authorities and international agencies and bilateral organizations to use WHO specifications for quality control. 18. WHO should improve the quality of the Regional Collaborative Centres and national laboratories by supporting training and supervision through reference Collaborating Centres.CTDWHOPESIICI96.1 page 29 ANNEX | PROTOCOLS FOR LABORATORY AND FIELD EVALUATION OF INSECTICIDES AND REPELLENTS CONTENTS Page 4. PHASE | (LABORATORY STUDIES) 32 4.4 Mosquito adults 32 4.1.1 Topical applications 32 1.1.2. Tarsal contact with deposits on filter paper 33 1.1.3. Residual action of deposits on plywood, plaster, mud, 33 thatch and synthetic substrates 1.1.4 Tarsal contact with deposits on bednet fabrics 34 1.1.5 Irritability test 34 1.1.6 Space spray 34 1.4.7. Repellents 35 4.1.8 Household insecticides 36 1.2. Mosquito larvae 37 1.2.1. Conventional insecticides 37 1.2.2 Insect Growth Regulators (IGRs) 37 1.2.3. Assay of bacterial larvicides 38 1.2.3.1. Standardisation of assays 38 1.2.3.2. Preparation of a stock standard suspension 38 1.2.3.3 Exposure of larvae to standard suspensions 39 1.2.3.4 Preparations of suspensions of unknown activity 39 1.2.3.5 Titration 39 4.2.3.6 Standardization of larvae 40 1.3 Blackflies 40 1.3.1 Bacterial larvicides 40 1.3.2. Chemical larvicides 42 1.4 Tsetse flies 42 1.4.1 Topical application of insecticides 42 1.4.2. Topical application of IGRs 42 1.4.3. Residual activity of insecticides or IGRs on target fabrics 43CTDWHOPES/IC/96.1 page 30 1.4.4 Residual activity of pour-on formulations of insecticide on livestock 1.4.5. Residue analysis on target fabrics 1.5 Triatomine bugs 1.5.1. Topical application 1.5.2. Treated mud bricks 1.6 Fleas 1.6.1. Contact with filter paper 1.6.2 Dusts 1.6.3. Ovicidal effect 1.6.4. Treatment of flea hosts 1.7 Resistance assessment 1.7.1 Cross resistance 1.7.2 Standard reference strains 1.7.3 Methods of characterising resistance mechanisms 1.7.4 Insecticide resistance tests in Triatomines 1.8 Toxicological evaluation 1.8.1 Toxicological requirements within WHOPES 1.8.2, Eco-toxicological evaluation 2. PHASE Il (SMALL SCALE FIELD TRIALS) 2.1. Introduction 2.2 Mosquito adults 2.2.1. Residual spraying of houses 2.2.2 Impregnated bednets 2.2.3. Space spraying 2.2.4 Repellents and personal protection devices 2.4 Sandflies 25 Fleas 43 43 44 44 45 45 45 46 47 47 47 48 43 49 49 50 50 51 52 52 53 542.6. Safety observations CTDWHOPES/ICI96.1 page 31 3. PHASE Ill (TRIALS IN WHOLE VILLAGES OR LARGER AREAS) 55 3.1 Introduction 3.2 Malaria vectors 3.2.1 Entomological evaluation 3.2.2 Impact on malaria 3.2.3. Operational acceptability 3.3. Culicine vectors of urban filariasis and arboviruses 3.4 Sandflies 3.5. Blackflies 3.5.1. Efficacy assessment 3.5.2 Operational trials 3.6 Tsetse flies 3.7. Triatomine bugs 3.8 Safety evaluation in phase 3 trials, 3.8.1. Design of protocols 3.8.2. Safety criteria for acceptance 3.8.3 Criteria for acceptance of environmental risk under field conditions 61 62 63 63 64 64CTDWHOPESIICI96.1 page 32 1. PHASE | (LABORATORY STUDIES) It is recognized that laboratory testing procedures (phase |) should reflect as closely as possible the procedures to be used in later phases of testing, e.g. with respect to materials and surfaces to be treated with pesticides, and in the monitoring of persistence of compounds. Phase | is conducted in the laboratory. It involves the cooperation of Collaborating Centres which receive samples from industry either as technical products or as formulations. The activity of the material is evaluated against adults or larvae of the main insect vectors. Phase | also aims at determining the possible field of application of the new products, e.g. residual indoor spraying, bednet impregnation, larviciding, space spraying etc. Mammalian toxicity and resistance assessments are an integral part of Phase |. It is noteworthy that standard rearing and testing conditions are “key” factors as far as the reliability of insecticide testing is concerned. 1.1 Mosquito adults New insecticides should be tested against the mosquito species of greatest importance: Anopheles gambiae s.s, Aedes aegypti, and Culex quinquefasciatus. If possible, tests should also be carried out with vector species which are important in specific areas of the world (An. dirus, An. minimus, An. stephensi, An. culicifacies, An. sacharovi, An. albimanus, etc ). 1.1.1 Topical applications The objective of topical application is to determine the intrinsic toxicity of insecticides with an accurately measured quantity of insecticide deposited on each mosquito This is not necessarily the case with the filter paper test where the amount of chemical picked up by tarsal contact varies significantly depending on whether an insecticide is irritant such as most pyrethroids, or non-irritating, such as most organophosphates. ‘A micro-droplet of 0.1 il acetone solution is individually dropped onto the pronotum of each test mosquito (larger volumes may result in significant mortality due to the toxicity of the solvent) . Micro-pipettes are available for delivering the required small volume, the most appropriate applicators being those which allow treatment of mosquitos without moving them. For this reason, micro-syringes are unsuitable. A tube fitted with a micro-capillary, comparable to the WHO kits for testing houseflies, can be made with a finer capillary delivering 0.1 instead of 0.36 ul. Alternatively appropriate automatic applicators are now commercially available.CTD WHOPES/ICI96.1 Mosquitos are briefly anaesthetized with COz and maintained on an ice tray during the test. A total of 30 unfed 2 to 5 day old females are used per concentration, with at least 5 concentrations tested. Three replicates with insects from different rearing batches are tested and a composite probit mortality/iog dose regression is computed or plotted on appropriate graph paper. Individual mosquitoes from the control are weighed after the test and the LDso and LDss are calculated in ng/mg of fresh mosquito. 1.1.2. Tarsal contact with deposits on filter paper Tests are conducted with a graduated series of dosages applied to filter paper (Whatman no. 1 or equivalent) 12 x 15 cm in 2 ml acetone solution with silicone oil (Dow Corning 556 cosmetic grade 3.6 mg/cm’). The papers are air dried for 24 hours and then inserted into WHO tubes for testing susceptibility of adult mosquitoes. Groups of 20 to 25 non-blood-fed female mosquitoes, 2 to 5 days old, are placed in each tube, exposed to the treated paper for 1 hour and returned to holding tubes for the determination of the 24 hour dosage/mortality relationship. The dosage/mortality curve is replicated on three separately reared batches to allow for inter-batch variability. From the results the probit mortality/log dose regression, and hence the LDso is computed or plotted on appropriate graph paper. Criterion: LDso<0.16mg/cm’ at 24 h. Mortality may also be recorded after longer periods of time with slow acting insecticides 1.1.3 Residual action of deposits on plywood, plaster, mud, thatch and synthetic substrates Samples are sprayed singly, e.g. in a Potter Tower, with the chosen formulation of a candidate compound. The suspension or solution concentration in de-ionized water is adjusted and applied at a rate of 30 mlm? which corresponds to the ratio of liquid to surface area usually employed in spraying in the field, The sprayed surfaces are stored on open shelves in a room kept at 25°C, 50-55% relative humidity and constant darkness. Air can diffuse freely around the samples but there is no forced ventilation and the door is kept closed At intervals after treatment, 2 to 5 day old non-blood-fed females are exposed to the treated surface for 30 min (air temperature: 25°C, R.H.: 50- 55%). Batches of 15 females are introduced into standard WHO test cones and allowed to alight and rest on the vertical treated surfaces. After 30 mins, 3CTDIWHOPESIIC/96.1 page 34 the mosquitoes are removed and transferred for observation and mortality count after 24 hours. Criterion: At least 70% mortality, 24 hours after exposure of 30 mins, 8 weeks after treatment. 1.1.4 Tarsal contact with deposits on bednet fabrics impregnated bednet fabrics (preferably, multiilament, polyester fibres, 80-100 denier) are tested by exposing 5 non-blood-fed, 2-5 day old, female mosquitoes for 3 minutes under standard WHO cones, after which they are held for 24 hours with access to sugar solution. At least 20 replicates should be tested. Hence, 100 mosquitos are used and monitored for mortality 24 hours later. Controls and a range of doses of the test compound should be used to enable estimation of LDso values. These are compared to a benchmark standard such as permethrin (25/75 cisitrans). The dosage chosen to go forward for phase 2 testing should give at least 80% mortality after exposure to a freshly treated net. The knockdown time is also a good indication for fast acting insecticides and can be determined by exposing batches of 11 female mosquitos (minimum five replicates) and observing the time at which the sixth (median) mosquito is knocked down for the first time. (Mosquitos may temporarily recover and fall down again, but confusion is avoided by removing each mosquito when it is first knocked down). The mean KT is calculated. A KTs0 up to 10 minutes indicates a good knockdown performance. 1.1.5. Iritability test WHO standard plastic cones are used with filter paper impregnated at the concentration giving 100% mortality with susceptible strains exposed for 1 hour in WHO test tubes for adult mosquitoes. Individual 2 to 5 day old non- blood-fed females are gently introduced into the cone The criterion used is the time for the first observed take off after a 1.5 min pre-observation time. During the test, constant conditions of light, air temperature (25°C) and 70 to 80% relative humidity have to be maintained. A control with silicone oil only is used. As far as possible, permethrin should be tested in parallel as a reference for the test product. 4.1.8 Space spray In these tests 2 to 5 day old mosquitoes are exposed to various concentrations of the insecticides in a wind tunnel. This apparatus consists essentially of a cylindrical tube 15 cm in diameter through which a column of air moves at 6.5 km/h, The mosquitos are confined in a cylindrical screen cagepage 35 which is placed in the centre of the tube. 0.25 mi of the insecticide in an acetone solution is atomized at 105 g/cm? (10.2 kPa) into the mouth of the tube, and the mosquitoes are then transferred to untreated 500-ml cardboard cartons. The covers of the cartons are replaced with plastic screens through which the mosquitoes may feed on a 20% sugar-water solution on an absorbent cotton pad. Alll tests are run in duplicate, using 25 adult females in each cage. The duration of exposure is 0.35 s. Criterion: LCso<0.05% at 24 h. 1.1.7 Repellents Deet (diethyl 3 methylbenzamide) is the active ingredient of most commercially available repellents and should be the standard against which the effectiveness of alternative repellents is judged. Use of laboratory animals or artificial membranes may inadequately simulate the situation in which repellents for use on human skin are intended to perform (e.g. laboratory animals do not sweat) Therefore, it is preferable to conduct tests on human volunteers. They should be selected from those who show mild or no allergic reaction to mosquito bites. It is conventional to use Aedes aegypti mosquitoes for the tests, but people generally show milder reactions to Anopheles bites. Tests are generally conducted with mosquitoes held in large laboratory cages into which the forearm(s) of the volunteer is introduced with the hand protected by a glove to make it unattractive to mosquitoes. The whole forearm may be exposed, or only an area of 25 sq cm of skin, the remainder being covered with a rubber sleeve. Alternatively, the repelient may be applied to a cotton stocking on which repellents are much more persistent than on skin. The stocking is drawn over another stocking which has been drawn ‘over the arm to prevent skin contact with a repellent compound which may not have been thoroughly evaluated toxicologically Different laboratories which carry out extensive comparative testing of repelients give different emphasis to either (i) % protection in relation to dose, (ii) protection time after treatment, with tests of only a single large dose or (iii) both of these parameters. The protocols used to measure these are as follows:~ (i) treatment of one arm with a measured quantity of test material dissolved in isopropanol and the other with solvent only. Only a 25 sq cm of skin on each arm is exposed and both arms are introduced simultaneously into the cage. The number of bites is counted in 5 mins during which mosquitoes which are not repelled feed to repletion. The % protection is calculated by comparing the counts on the two arms (ii) treatment of one arm with 1 ml of a 25% solution of the test compound in ethanol. The arm is exposed for 3 mins in every 30 mins and the first time after treatmentCTOAVHOPESIICI96.1 Page 36 noted at which a bite occurs followed by a “confirmatory” bite in the same or the following exposure period. (ii) treatment of 280 sq cm of stocking with 1.gm of test material dissolved in enough acetone to saturate the stocking. Exposure of an arm covered by the stocking to 1500 hungry mosquitoes for 1 min at daily or longer intervals until at least 5 bites are obtained. (iv) sequential exposure of an arm with zero, and then progressively higher, doses of repellent for 30 secs to cages each containing approximately 50 hungry An. gambiae (or 45 secs with An. stephensi). The number biting at the end of the short exposure is quickly counted (preferably with the help of an assistant) and the mosquitoes are then shaken off before they can imbibe any blood. Hence the same mosquitoes can be used for testing each dose and their continued hunger can be checked by exposing the other untreated arm. Probit analysis is used to calculate the EDs. After reaching a dose which gives 100% repellency, the arm is. re-exposed hourly until repellency declines to 50% compared with contemporary counts on the untreated arm. (iv) treatment of the feet and lower legs and counting of bites in a 10 min period, after release of 25 hungry mosquitoes into a small mosquito proof room. This more closely simulates a field test, but with the advantage that the number of mosquitoes is controlled and not subject to the vagaries of natural populations. 1.1.8 Household insecticides Aerosols, coils, mats and vaporizers may be tested in chambers made of non- absorbent material (e.g. glass) which can be thoroughly cleaned to remove residues of earlier treatments. The chambers may range in size form 70 to 180 cm cube (the latter size in known as a Peet Grady chamber). The air in the chamber is circulated with a fan and may be vented to the outside by an extractor fan to simulate a room with a slight through draught rather than still air. Rapid knockdown is desired by users of household insecticides and emphasis is therefore given to observed times of knockdown of a sample of introduced mosquitoes, from which the KTs0 (time for 50% knockdown) is calculated by probit analysis. After 20 min exposure all mosquitoes are collected up and held with access to sugar for 24 hrs before counting final mortality. Emphasis is given to standard rearing conditions of the mosquitoes (which are tested when 2-5 days old) and to replications of tests 3-4 times. With coils in a small test chamber, half a gram of coil may be burned to completion before introducing the mosquitoes. With a larger chamber the coil may be burned continuously throughout the test.For vaporizing mats, test may be carried out after 1,2,4,6,8 and 10 hours of heating the mat in a ventilated chamber on its electric heater, to test for decline in emission of insecticidal vapour. The mat and heater are brought into the test chamber after the above mentioned periods of heating. With a small chamber the mat is heated in the chamber for 3 mins and mosquitoes are then introduced into the chamber for 20 mins. With a large chamber the mat is heated throughout a 60 min exposure of mosquitoes. Electrically heated liquid vaporizers are tested as for mats but the periods of heating outside the chamber between tests are 5,10,20 and 25 days. For testing aerosols, mosquitoes are released into the chamber before emission of the insecticide. The amount emitted is measured by weighing the container before and after emission. 4.2. Mosquito larvae 1.2.1 Conventional insecticides Screening of candidate compounds is performed by exposing larvae in de-ionized or distilled water treated with a series of at least 5 concentrations of the insecticide in alcohol. For certain compounds, an organic: solvent other than alcohol is appropriate . One mi of a standard wiv concentrate in an organic solvent is added to 99 ml of water. Twenty late 3rd- or young 4th-instar larvae are used per cup with 4 cups per concentration (water temperature 25+1° C). Tests should be replicated 3 times, each time from separately reared batches of larvae. Mortality is observed after 24 hours and the probit mortality/iog dose regression is computed or plotted on appropriate graph paper. Criterion: LCso<0.1 mg/l at 24 h. 1.2.2 Insect Growth Regulators (IGRs) The test procedures for insect growth regulators and natural products should utilize the same procedures as those for quick-acting compounds except that assessment procedures for mortality and activity determination are different, With most IGRs, mortality is delayed beyond the instar treated. For accurate determination of activity of IGRs and natural products, it is necessary to assess mortality every two or three days up to emergence of the adults or death of the last larva or pupa. Due to the long duration of the test, the larvae have to be provided with food at intervals during the test period. From the overall results of the test, activity of the compound is determined from the magnitude of the inhibition of emergence of the adult mosquitoes.CTDMWHOPESIC/96.1 Page 38 1.2.3. Assay of bacterial larvicides 1.2.3.1. Standardisation of assays Various methods are being developed to assay bacterial larvicides, including biochemical or molecular approaches. However, at present the bioassay method, based on direct observation of the larvicidal potency of bacterial preparations, remains the most reliable procedure for their standardization. The procedure is the same for B.tH-14 and B. sphaericus and is applicable to’ laboratory preparations, to primary powders or formulated preparations. At present there are two reference powders, recognized internationally, that allow one to determine the toxicity of bacterial preparations towards mosquito larvae and to compare their efficacy between laboratories. B.tH-14 products are titrated against a reference IPS82 (strain 1884) lyophilized powder with young fourth instar Aedes aegypti larvae (strain Bora- Bora). A titre of IPS82 has been assigned 15000 ITU (International Toxic Units)mg powder on this insect. B. sphaericus products are titrated against SPH88 (strain 2362) standard lyophilized powder with young fourth instar Culex pipiens pipiens larvae (strain Montpellier). The SPH88 titre is 1700 ITU/mg on this insect. The larvicidal activity of both standard powders is checked monthly and the mean of their titres is recorded in the WHO Collaborating Centre (Institut Pasteur, Paris). All bacterial preparations based on B.tH-14 or B. sphaericus can be titrated against their respective standard powders. The titre of the product is determined according to the formula:- Titre of product “X" = titre standard (ITU/ma) x LCso(ma/!) standard LCs0 (mg/l) of product *X" 1.2.3.2. Preparation of a stock standard suspension Fifty mg of the standard powder are weighed and placed in a 20 ml penicillin flask, to which is added 10 ml de-ionized water and 15 glass beads (6 mm diameter), The contents are thoroughly homogenized on a vibrating pulverisor (e.g. Dangoumau type) for 10 minutes at 700 strokes/minute. This homogenate (5.000 mg/l) can be stored at 4°C, sealed and used for several months. From this homogenate, a stock solution is made in a tube (22mm diameter) by adding 0.1 ml of the homogenate to 9.9 mi de-ionized water, followed by mixing on a Vortex agitator at maximum speed for a few seconds,(CTDWHOPES/ICI96.1 page 39 1.23.3. Exposure of larvae to standard suspensions A series of plastic cups is filled with 150 mi de-ionized water. To each cup, 25 early 4th instar larvae of Aedes aegypti or Culex pipiens (depending on the bacterial suspension to be tested: Aedes for B.tH-14 and Culex larvae for B. sphaericus) are added with a pasteur pipette and the volume of water added with the larvae is re-aspirated and discarded to avoid a change of the volume in the cup. With micropipettes, 120 pil, 90 il, 60 pl, 30 pl, 24 yl, and 15 yl of stock solution (prepared as described in the previous section) are added to the cups in order to obtain final concentrations of 0.04, 0.03, 0.02, 0.01, 0.008 and 0,005 mg/l, respectively, of the standard. Two to four replicates are used for each concentration and for the control, which contains 150 ml de-ionized water only. 1.2.3.4. Preparation of suspensions of unknown activity For bioassay of preparations of unknown activity, an initial homogenate is made in the same manner as described for the standard powder. When titrating a liquid formulation, 100 mg are weighed instead of 50 mg (homogenate will then correspond to 10000 mg/l). Cups and larvae are prepared as described above and comparable dilutions prepared as for the standard. The range of concentrations (at least six) should exceed that of the standard in order to obtain a reliable regression line. Time can be saved by making a preliminary range-finding bioassay, with widely spaced concentrations of the unknown preparation. The data obtained are used to fix the subsequent concentrations for an exact bioassay. 1.2.3.5. Titration No food is added for Aedes larvae. For the Culex bioassay, a finely powdered yeast extract is previously added to the water at a rate of 10 mg/l. All tests should be conducted at 23-27°C. Each bioassay series should preferably involve 6 replicates of 50 larvae for the standard and 100 larvae for the control, For preparations of unknown activity, at least 6 replicates of 50 larvae should be used, The aim is to define a range of concentrations which give a range of mortalities between 10 and 90%. Mortality is determined at 24 and 48 hr and is based mainly on the counting of live larvae. Because of the very rapid killing action of B.tH-14, there is usually no difference between the 24 and 48 h mortality. But the 48 hr reading confirms the 24 hr reading and also checks for the possible influence of factors other than Bacillus components. For B. sphaericus preparations, due to theirCTDWHOPES/IC/96.1 page 40 slower toxic action, mortality is recorded at 48 hr. If pupation occurs, the pupae should be removed and their numbers excluded from the data base. When control mortality exceeds 5%, the mortalities of treated groups should be corrected by Abbott's formula. Tests with control mortality greater than 10 % should be discarded. Probit mortality/log concentration regression lines should be computed or drawn on appropriate graph paper. Based on the LCs: of standard and unknown preparations, the titre of the latter is determined. For increased accuracy, bioassays should be repeated on at least three different days simultaneously with the standard, and the standard deviation of the means calculated. 1.2.3.6 Standardization of larvae It is very important to use a homogeneous population of early fourth instar larvae reared by the following standardised methods:-. For Aedes aegypti, standardisation of larval rearing is relatively easy. Eggs are laid on a filter paper (and can be kept several months in a sealed plastic bag) and, when required, are immersed to hatch in dechlorinated water. 24 hr after hatching, first-instar larvae are transferred to a container (25 x 25 cm) filled with 2 litres of dechlorinated water in order to obtain 500 to 700 larvae per container. Two cat biscuits containing meat are provided on the first day. After five days at 25°C adequately homogeneous young fourth instar larvae (4 to 5 mm in length) should be obtained. For Culex pipiens, it is more difficult to achieve homogeneity. Initially an adequate number of rafts must be laid on the same day. First instar larvae are fragile and should not be manipulated. Production of second instar larvae occurs 3-4 days after oviposition at 25°C. Young second instar larvae are placed in a container (35 x 35 cm) in 3 litres of water in order to produce a larval density of around 800-1000 young fourth instar larvae. Food (cat biscuits containing meat and yeast extract powder) is provided daily as the previously provided food is eaten. The correct stage for testing should be obtained within 7 days (sometimes 8 or 9 days) from egg hatching 1.3 Blackflies 1.3.1 Bacterial larvicides Larvae are collected in a river by cutting off pieces of their larval support or of artificial substrates (palm fronds tied in the river) which they have colonized. This material is transported to the bioassay laboratory in openCTDAWHOPESIIC/96.1 page 41 plastic bags lying on the top of ice in a cooler. In the laboratory, larvae are used immediately or maintained until needed in a closed circuit trough system River water is used for the test after adjustment to a turbidity of 10 NTU (Hach Ratio, using Turbidimeter model 18900) by adding filtered or distilled water if the river water is too turbid, or filtrate from river water if it is not sufficiently turbid, The adjusted water is kept in a plastic drum where it is aerated by an aquarium pump and agitated by a magnetic stirrer. Forty round bottomed flasks (250 mi capacity) are filled with 200 ml of this adjusted water, and then 30 mature larvae are placed in each flask. The bioassay system employs an orbital shaker (Digital gyrorotary shaker, Mode! G-10, New Brunswick Scientific Co, Edison, N.J., U.S.A.) which generates a swirling motion in the flasks. The flasks are individually fixed in clamps on the top plate of the shaker and a board is then placed above them, which holds the syringes used for treatment. The orbital motion is gradually increased up to a speed of 150 rpm over a 30 min period. The orbital movement is then stopped, and larvae not yet attached drop to the bottom of the flask, where they readily attach. The orbital motion is then slightly increased up to 250 rpm and the larvae are left to adapt for 90 min. The water temperature for adaptation and treatment is 26° C. Solutions of B. thuringiensis H-14 are prepared by diluting 1 mi of each sample in 1 litre of distilled water. Serial dilutions are made with distilled water. For each flask an aliquot of 2 ml of B.tH-14 solution is prepared in a plastic syringe fitted with a large-bore, 5 om long needle. Fifteen minutes before treatment, the syringes are placed in the holes of the top board. At the time of ‘treatment, the plungers of the syringes are depressed simultaneously in rows using a wooden board, and injection is completed in less than 15 seconds. The larvae are then left exposed to B.t. H-14 for 10 min. The shaker is stopped, and the flasks are placed for 4 hours.in an insulated cabinet where the temperature is kept at 22°C and air is circulated with a fan. After 4 hours, pupae are discarded and the remaining larvae are classified as dead, moribund or alive, and preserved separately in vials containing ethanol, A graph is plotted or the regression is computed of probit mortality {including moribund tarvae) on log of the dose expressed in mg B.tH-14/lisec. In the analysis, zero and 100% mortalities are subject to Berkson's correction. ‘The probit analysis provides an estimate of the LCs: for each sample of B.tH- 14 tested The final estimate of the LCso of a given sample of B.t.H-14 is calculated as the average of 2 valid tests performed on different days. The validity of a test depends on the following criteria: there must be: (i) at least 3 flasks treated at each dose, (ii) at least five doses resulting in a mortality above control and less than 100%, (iii) mortality in control flasks less than 4%, (iv) slope of theCTDAWHOPESIICI96.1 page 42 regression line equal to or greater than 2.4, and (v) heterogeneity chi-square about the regression line not significant. 1.3.2. Chemical larvicides The methodology is similar to that described for bacterial larvicides using an orbital shaker but water current in beakers is provided by a rotating multi- agitator. The water quality is adjusted as described above. During the adaptation time, the rotational speed of the beakers is 250 rpm. During the 10 min exposure, it is raised to 400 rpm and it is then returned to 250 rpm for the observation time. Just after the 10 min exposure, the water with insecticide is removed from the beakers which are briefly rinsed with distilled water and filled again with adjusted water. The observation time for delayed mortality is 5 hours, then pupae are discarded and larvae are removed from the beakers for counting (as with the orbital shaker method). The interpretation of results is as described above. 1.4 Tsetse flies 4.4.1. Topical application of insecticides Applications of solutions in 0.2 - 1.0 yl acetone are made, e.g. from a Hamilton micro-syringe mounted in a standard press-button applicator. Controls are treated with acetone only. Unfed, adult flies, 24 hours after emergence are used without anesthesia. Each fly is held by the legs and the volume of solution is applied to the dorsal surface of the thorax. Three batches of 10 flies of each sex are treated with an appropriate range of doses. Treated flies are placed in-unwaxed- paper, or_ clear polystyrene, cups with filter paper perches and gauze tops. They are held at 25° C and 70% R.H. for mortality assessment at 24 hours post-treatment. (Results are only accepted if control mortality is less than 10%). The median lethal dose is determined for each sex separately after computing, or plotting on appropriate graph paper, the regression of probit mortality on log dosage. Criterion: LDso for the test compound should be compared to deltamethrin as standard, but should also be based on safety and cost effectiveness rather than dose alone. 1.4.2, Topical application of IGRs Methodology is the same as for insecticides but assessment of effect is different. Test insects are either recently mated females, or 6-7 day old malesCTDIWHOPES/IC/96.1 page 43 which will be mated to untreated females after exposure. Test and untreated control female insects are held in standard colony cages and fed daily for at least 45 days. Offspring are harvested and held to monitor survival. Reduction in offspring viability of test insects is observed, with reference to control offspring viability, for each reproductive cycle following direct exposure to the test compound, or after mating with treated males 1.4.3. Residual activity of insecticides or IGRs on target fabrics Flies are exposed by tarsal contact to a piece of treated fabric on a wooden peg, mounted on a board, over which a glass tube containing a test fly is inverted. The tube is only slightly larger than the peg and this forces the fly into contact with the treated surface. Flies are exposed to insecticides for 5 seconds and to IGRs for up to 5 minutes. Effects are monitored in the same way as described for topical applications. For persistence tests, treated fabrics are weathered out of doors under conditions as close as possible to the conditions to be faced in the field, and re- tested at intervals for up to one year. Candidate compounds are compared to deltamethrin as the benchmark standard. 1.4.4. Residual activity of pour-on formulations of insecticides on livestock Bioassays are recommended where cages of test flies are held in continuous contact with treated animals at intervals following treatment of the animal. Knockdown times are monitored and the median knockdown time is used to compare different compounds. Mortality effects are monitored by exposure of test insects for pre-determined times which have not yet been standardized. 1.4.5 Residue analysis on target fabrics Standard High Pressure Liquid Chromatography techniques have been developed to analyze residues of insecticides and IGRs on target fabrics. However, fabrics differ in their abilities to retain compounds and in the ability of the retained compounds to affect test insects following tarsal contact. Hence, the persistence of a compound as indicated by residue analysis may not reflect its efficacy as measured in a bioassay and hence not reflect useful persistence under field conditions.CTDWHOPESIICI96.41 page 44 1.5, Triatomine bugs 1.5.1 Topical application Triatoma infestans, laboratory reared from a susceptible strain, are the bugs generally used for testing. For other species it is necessary to determine the optimum laboratory rearing conditions and standardise the nymphs to be used, Different genera (and possibly species) are likely to differ in susceptibility to each compound tested. Laboratory conditions should be maintained at 25- 30°C, 50-70% R.H. and 12:12 hours photoperiod. Blood feeding intervals should not exceed 15 days. Fifth instar nymphs are used. They are selected soon after ecdysis, blood-fed 7-8 days later and used 7-8 days after feeding with a mean weight of 140 + 20 mg. ‘A range of doses of the active ingredient in an acetone solution is applied (1 il) to the dorsal part of the abdomen of the 5th instar nymph, using a micro-applicator. At least 10 nymphs are used per dose and at least two replicates are carried out. At first, 3-4 doses, with a dilution factor 10 fold between each, are used, Subsequently a narrower range is tested, with the aim of selecting at least 3 doses that yield between 10% and 90% mortality, 72 hours after treatment. During this time insects are kept at 25-30°C and 50-70% R.H. in clean glass containers fitted with filter paper resting places. In making the mortality counts a triatomine is normally considered dead when it is knocked down and unable to move when touched. However, when testing pyrethroids, some may be knocked down but recover and the insects should be observed for at least a week to check for such recovery. The LDsp and LDao are derived by computing the regression of probit mortality on log dose or plotting the data on appropriate graph paper. Criterion: LDso less than 2 jg a.i./mg of insect weight. 1.5.2. Treated mud bricks Water dilutions of insecticide formulations are sprayed on to unbaked, dry mud bricks (surface pH: 6-8). Usually 1.5 ml of diluted formulation is applied to each brick (11x11 cm), using a sprayer fitted with a teejet 8002 or similar nozzle, at a flow rate of 760 ml/min at a pressure of 3.5 kg/cm and 45 cm distance from the target surface. The following day the Sth instar nymphs in two replicates are placed in contact with the treated surface under WHO cones for wall bioassays. They are kept in this situation for 21 days. without feeding and then mortality is observed. To determine residual effect, this procedure can be repeated at 3 month intervals until the insecticide activity falls below 70% mortality. The treated surfaces are aged indoors at 25-30°C, 50-70% RH, under indirect sunlight.(CTDAWHOPESIIC/96.1 page 45 Alternatively, filter paper (Whatman No.1) can be treated with formulated products using 16 jl of the final aqueous dilution for each cm? of surface (e.g. 1 mi for a petri-dish of 9 cm diameter). After 24 hours, ten Sth instar nymphs are exposed for 1 hour. After a holding time of 72 hours in a clean container, the mortality is observed. Treated surfaces are aged as described above. However, it is preferable to test candidate compounds on the type of surface likely to be found in the field. Criterion to proceed to field studies: at least 95% mortality at a dose less than 2.5 g a.i./m? (or less than 0.5 g a.i/ m? for pyrethroids) after 21 days of continuous exposure to a recently treated surface. 1.6 Fleas 1.6.1 Contact with filter papers Strips of Whatman No. 1 or 2 filter paper, 15 mm by 50 mm (one end tapered) are impregnated with 0.13 ml of an acetone solution in silicone oil containing 0.031% of the insecticide under test. This impregnation rate produces a deposit of 5.4 yg of toxicant per om*. After drying for one hour, each treated paper (tapered end down) is inserted in the bottom of a glass test-tube (18mm x 150 mm). Ten unfed female fleas, <24 hrs after emergence, are placed in each test-tube, which is then capped with organdie cloth. There should be five replicates of each treatment plus untreated controls. Exposure is at 25°C and 80% R.H. in darkness. After the 24 hr exposure period, the test fleas are removed and the mortality is counted. The treated papers are aged in darkness in the tubes and tested at 1, 2, and 4 weeks, and every 4 weeks thereafter. Criterion: At least 90% mortality at each test for 4 weeks after impregnation of the papers. 1.6.2 Dusts Promising compounds are tested in dust formulations over soil in the laboratory. Thirty grams of a standard pulverised soil of high organic content (PH 6-8) is placed in the top half of a 100 mm petri dish and leveled. The surface of the soil is dusted through a piece of nylon stocking (15 denier, 51 gauge) at not more than of 20 g/m’, Dusts containing 0.01%, 0.05%, 0.25%, 1.0% and 5.0% of the insecticide are used. The exposure chamber consists of a plastic dish, 95 mm in diameter and 70 mm high, with a circular opening 25 mm in diameter cut in the bottom. The chamber is inverted and inserted into the treated soil so that the soil becomes the floor of the exposure chamber and the opening is at the top. Fifty adult fleas, <24 hr after emergence, areCTDWHOPESIIC/96.1 page 46 introduced through the opening in the top of the chamber with a funnel and exposed to the treated soil for a period of 24 hr at 25°C and 80% RH. in darkness. Additional petri dishes are dusted on day 0, and aged at 25°C and 80% R.H. in darkness, in preparation for tests which are carried out after 1, 2, and 4 weeks, and every 4 weeks thereafter. Criterion: At least 90% mortality after 24 hrs exposure at a dose of 20 gim’ or less. Persistence should be for at least 4 weeks. 1.6.3 Ovicidal effect Filter papers treated with a range of concentrations of the compound dissolved in acetone solution in silicone oil are dried for 24 hours. Five replicates of ten eggs (<24 h old) are exposed to the treated filter papers at 25°C and 80% R.H. for 24 hours. Filter papers treated only with the solvent constitute the controls. Criterion: At least 80% inhibition of hatching after 24 hours. 1.6.4 Treatment of flea hosts ‘When treatments on the host are made for flea control, the compounds can be applied to the fur of the host or administered orally. The assessment of the effect of the compounds is from comparisons, before and after treatment, of flea infested host animals. The evaluation of adulticides is based on counting the flea egg production of individually caged host animals and/or combing the host for fleas. For IGRs this should be combined with an evaluation of the hatchability of the flea eggs or the prevention of the fleas from developing into adults (larval food has to be provided during the tests) 1.7. Resistance assessment When a compound is submitted for evaluation in WHOPES, it is important to assess whether there is cross-resistance with known types of resistance in those vector species (e.g. Culex quinquefasciatus) where resistance is diverse and widespread, For others, e.g. tsetse flies, resistance is so far of no practical significance. 1.7.1 Cross-resistance As it is practical to screen only a small number of resistant insect populations, and because resistance in diverse insects is generally based on aCTDWHOPESIICI96.4 Page 47 limited number of mechanisms, the screening of a candidate compound can be carried out by observing the response to each of these mechanisms. Therefore, initially new candidate insecticides should be tested against a small number of distinct multi-resistant strains. The compounds should also be tested simultaneously against a susceptible strain. If cross-resistance is detected the exact mechanisms of resistance is determined by testing the insecticide against strains each possessing a single resistance mechanism. 1.7.2 Standard reference strains For some insect species, susceptible strains are already maintained in one or more laboratories. For other species, susceptible strains should be collected in the field if truly susceptible populations still exist. If not, one or more susceptible strains may be artificially selected using bioassays and assays for individual resistance mechanisms and selection between families derived from individual mated females. Resistant strains to be used in the WHOPES should be identified using well- established assay techniques. The strains should preferably be homozygous for one or more known resistance mechanisms. If homozygosity cannot be achieved, periodic selection is usually necessary to prevent natural selection favouring the susceptible, at the expense of the resistant, type. Established reference strains should be monitored regularly by bioassays and biochemical and/or molecular assays so that any such changes in resistance and underlying mechanisms can be assessed and rectified by selection. 1.7.3. Methods for characterising resistance mechanisms In order to characterise the resistant reference strains, biochemical and/or molecular assay methods should be used in conjunction with conventional bioassays. Standard published methods are available for all of these assays. 1.7.4 Insecticide resistance tests in Triatomines The base line of susceptibility is derived from Sth instar nymphs from a laboratory colony or collected in an area that has not been treated for at least 5 years. The impregnated filter paper technique is used, with different concentrations of technical product diluted in olive oil (for organophosphates and carbamates) or silicone oil (for pyrethroids). The amount of oil should be 3.7 mg/cm* , e.g. 1 mg of active ingredient is mixed with 0.25 ml of oil and 1 ml acetone which is enough to impregnate a filter paper of 9 cm diameter (64 cm’),