148 J Anat. Soc. India 50(2) 148-152 (2001) Toxic Effects Of Diethyltoluamide And Dimethylphthalate Creams As Mosquito Repellents On Rabbit s Skin Al-Sagaff, Sammar, Shahid, Rehana & Nayeem, Fouzia. Department of Anatomy, Girls Medical College, King Abdul Aziz University, Jeddah-21551, SAUDI ARABIA Abstract. In this study an attempt has been made to examine and analyse the toxic effects of Diethyltoluamide and Dimethylphthalate creams as mosquito repellents. Rabbits skin on the back of the body and also on back of the ears was investigated by application of the creams. Twelve rabbits were used in this experiment and these were divided into 3 groups. Four rabbits were included in each group. These were labelled as A, B, C, D, and E in each group was for skin of the back of the ear. Thus these were placed as below : Group I A, B, C, D + E Group II A, B, C, D, + E Group III A, B, C, D, + E Rabbits labelled A in each group were used as control and applied with water in all the three groups. Group I B, C, D + E were applied with Dimethylphthalate cream for 2 months. Group II B, C, D, + E were applied with Diethyltoluamide cream for 2 months. Group III B, C, D + E were applied with a combination of 50% each of the above creams. Rabbits labelled B in each group were sacrificed at the end of 2 months while those labelled C + D were left without application of any cream for 2 more months. The animals labelled C were sacrificed at the end of 4 th month to see the still remaining irritating effects of the creams on the skin after an interval of 2 months. At the end of 4 th month the animals labelled D in each group were reapplied with the same creams as in group I, Group II and Group III. At the end of 6 th month the last 4 animals were sacrificed and their skin was compared with the control. The results showed that with application of creams skin reaction showed infoldings of epidermis with less hairs & thickened dermis with more vascularity. The effects of irritation were present even after two months of cream free interval. Reapplication of the creams after an interval of two months showed severe reaction, in combination of the two creams in 50% in group III while Diethyl toluamide in Group II was less in reaction than with. Dimethylphthlate cream in Group I. It was concluded that these creams as Mosquito repellents had toxic effects on rabbit s skin so also on human beings. Hence these should not be used as repellents even in low concentrations. Key words : Rabbit s skin, Diethyl toluamide and Dimethylphthalate, mosquito repellents. Introduction : Malaria is common in countries like Pakistan, India, South Saudi Arabia and parts of Africa. Usually it assumes an epidemic form which retards the growth of children and leaves them vulnerable to other infections. People exposed to these parasites face serious problems. Hence they use mosquito repellents to protect themselves from mosquito bite but in exchange get more serious problems of skin irritation, erythema, desquamation and bullae formation etc. (Lamberg & Mulennen, 1969). People are not acquainted with the side effects of the mosquito repellents. Hence this study was specially done to make the people aware of the use of these repellents and their toxic effects. Ackerman (1978), worked on inflammatory skin disease and found that keratinization takes 31 days till they are fully cornified. Cook, (1974) Ham s Haemotoxylin & Eosin reagents : A. Haematoxylin 5 G Ethyl Alcohol 50 ml Potassium Alum 100 G Distilled water 950 ml. Mercuric oxide 2.5 G. Glacial acetic acid 4 ml. B. 1% HCl in 70% alcohol.
Al-Sagaff, S. et al C. Eosin 1 G Distilled water 100 ml. Thymol 1 small crystal The above reagents were used to stain the slides in the present study. Dipalma, (1971) found that Dimethylphthalate and Diethyltoluamide are of low toxicity when ingested by mouth. Both these drugs are irritant to the eyes and mucous membranes. They possess bitter taste hence usually children dislike them. Ghosh, (1977) found that Dimethylphthalate is very effective as insect repellent so also on mosquitoes. When applied to skin as 35% cream or lotion, it is effective for 3 5 hours or more due to its low volatility and much longer action can be obtained by using impregnated clothings. Gilbert, (1966) concluded that the most effective repellents were Diethyltoluamide, Dimethylphthalate, Ethylexanediol, Dimethylcarbonate and Butopyronoxyl. Hocking, (1963) states that oil of citronella and camphor were used as repellents before world war II but were discarded as they gave very little protection from mosquito attack. Junqueero and Carneiro, (1971) observed closely that human skin on fingers and palms show definite patterns that are never exactly the same in any two individuals not even in twins. They were unique and these patterns such as whorls, or arches are dermatoglyphics used for medico legal purpose and are of anthropological interest. Khan et al (1974) reported that Diethyltoluamide provides the longest protection time as a repellent. On application to the skin some of it penetrates the skin and is lost, a part of it is absorbed on the surface and is available as contact repellent and the rest evaporates in the form of vapours and thus repels the mosquitoes. It was Lamberg, and Mulrenner in (1969) who used the standard 48 hours patch testing procedures on human beings with undiluted Diethyltoluamide and found that there was dermatitis with erosions and blisters were produced in 49% of cases. The biopsy of these areas showed multiloculated vesicles and large bullae within and below the epidermis, containing scattered polymorphunuclear leucocytes and eosinophils. The dermis was edematous with a perivascular lymphocytic and eosinophilic infiltrate along the dilated vessels. The antecubital fossae are more 149 susceptible to reactions because of their greater likelihood to be occluded during sleep and to become macerated. The insect repellent issued to the military for personal use was 75% Diethyltoluamide. Luna (1968) used the following reagents for staining the slides. A. 5% acid fuchsin solution, Acid fuchsin 5 G., Distilled water 100 ml. B. Aniline blue orange G. Solution, Aniline blue, water soluble 0.5 G., Orange G. 2.0 G. This method was tried for the present study but the results were not satisfactory hence only H & E stain was used. Smith, (1966) proved that the length of time a repellent remained effective depended on the rate at which it was lost by rubbing, evaporation or absorption. There is no evidence that repellents become less effective by dilution with sweat, carbon dioxide or water, which are attractive to insects. Wong and Yew, (1978), studied that Dimethylphthalate and Diethyltoluamide are effective insect repellents but are irritant to the mucous membranes and eyes. However, these have been regarded as the least dangerous mosquito repellents and are safe for use on human skin. Material & Methods : This study was done in the histology laboratory of the Anatomy department, in the Girls Medical College, Faculty of Medicine, King Abdul Aziz University, Jeddah. Twelve (12) rabbits were used in this experiment. These were divided into three groups I, II, III. Each group included 4 rabbits. These were labelled as Group I A, B, C, D and E was labelled for the skin of back of ear of each group. Similarly group II A, B, C, D & E (for ear) and Group III, A, B, C, D and E for ear. Skin of back of all animals was shaved with razor for an area of 2 square. The skin of the back of both the ears was also shaved. The rabbits labelled A in all the 3 groups were used as control for both the skin of the back of the body and also back of the ear. These were applied with 0.5 ml. of water daily with a soft brush for 2 months. Rabbits labelled B, C, D & E
150 in group. I. were applied with Dimethylphthalate cream on back of skin of body and back of the ear for 2 months daily on the shaved area with a soft brush. In group II all the rabbits labelled B, C, D & E were applied with Diethyltoluamide cream daily with a soft brush on the shaved area of back of body and ear for 2 months. In Group III all the rabbits labelled B, C, D + E were applied with a mixture of 50% of each of Diethyltoluamide and Dimethylphthalate creams daily for 2 months on back of body skin & on back of ear. group B rabbits of each group were sacrificed at the end of 2 nd month and the skins of the back and ear were taken and after staining these were compared with the control labelled A of all the groups. The rabbits labelled C in all groups were left without application of any drug for 2 months and were sacrificed at the end of 4 th month to see the presence of the effects of the drugs even without application and were again compared with the control. The rabbits labelled D were reapplied with the creams in Group I with Dimethylphthalate; in Group II with Diethyltoluamide and in Group III with combination of 50% mixture of both creams daily for 2 months on the shaved area of back of the skin of the body and ear. At the end of 6 th month all the rabbits labelled D were sacrificed and were compared with the control for both the skin of the back of body and the back of ear. During the experimental period of 6 months all the rabbits had free access to tap water and laboratory food. After each experiment, histological examination of the skin of the back of the body and the skin of back of ear was compared with the control. The tissue was fixed in neutral buffered formalin solution and latter processed for section cutting. Five micron sections were cut. These were stained with methods of Cook, (1974) & Luna (1968). The results were better with Cook s method and these were photographed. Results : Fig. 1 Group I A : Control Application of water daily for two months on the skin of back of the body and on back of ear. Normal skin with very few infoldings of epidermis, diffuse hair follicles, few blood vessels in the dermis were seen in both the skin of the back of the body and in the skin of the ear. Fig. 2 Group I B : Application of Dimethylphthalate cream daily for 2 months on both the skin of the back and back of ear of the rabbit. It showed many infoldings of the epidermis, less hair follicles, thickened dermis with more blood vessels. Tissue spaces were filled with fluid and bundles of collagen fibres were seen in both the sites. Fig. 3 Group I C : No application of any cream for 2 months. After that interval the skin still showed signs of irritation like many infoldings of the epidermis, few blood vessels in the dermis and few tissue spaces filled with tissue fluid, in both the skin of the back of the body as well as in the skin of back of ear. Fig. 4 Group I D : Reapplication of Dimethyl phthalate cream after an interval of two months. The skin on the back of the body showed severe reaction. Epidermis showed breaks, infoldings and keratin was irregularly distributed. Dermis was thickened, edema was more and fibroblasts with collagen fibre bundles were seen. Fig. 5 Group I E : Skin of the back of ear showed dermatitis, increased intercellular spaces as there was more accumulation of tissue fluid. Fig. 6 Group II B : Diethyl toluamide cream application for 2 months on both sites. The skin showed thin and infolded epidermis. Dermis showed edema, bundles of collagen fibres, dilated blood vessels surrounded by inflammatory cells and tissue spaces filled with fluid. Fig. 7 Group II C : Interval of 2 months No Cream. Skin of rabbit still showed signs of irritation. Infolding of thin epidermis, dermis was edematous with intercellular spaces filled with tissue fluid, signs of irrtability were still present, on both sites. Fig. 8 Group II D : Reapplication of the Diethyltoluamide cream after 2 months interval on the back of the body. Rabbit s skin showed severe reaction, keratin thin and dark coloured. Dermis was thickened, with bundles of collagen fibres, blood vessels dilated and surrounded by inflammatory cells. Tissue spaces filled with lymph. Subcutaneous adipose tissue was filled with fat.
Al-Sagaff, S. et al Fig. 9 Group II E : Skin of ear back (after 2 months interval). Reapplication of Diethyl toluamide cream. Epidermis was thin and broken at intervals, the dermis was thickened with more of collagen and elastic fibres, tissue spaces filled with fluid, blood vessels dilated and surrounded by inflammatory cells. It was seen that the reactions in group II with Diethyltoluamide cream were more severe than that in group I. with dimethylphthalate cream. Fig. 10 Group III B : Combined creams 50% each of Dimethylphthalate and Diethyltoluamide applied on both skin of back of the body as well as on back of ear. Similar effects were seen in both the places. Epidermis showed breaks in the keratin. Dermis was thick with more of bundles of collagen fibres, blood vessels were dilated and surrounded by inflammatory cells. Tissue spaces were seen to be filled with fluid. Fig. 11 Group III C : No cream was applied both on the back of the body and the skin on back of the ear for two months. Still signs of irritation were present. Epidermis was thickened, infolded and keratinized. Dermis edematous, blood vessels dilated and surrounded by inflammatory cells. Tissue spaces filled with tissue fluid; more of fibroblasts and elastic fibres seen. Fig. 12 Group III D : Reapplication of combined 50% each of the creams after an interval of 2 months. Epidermis thick, infolded and keratinized. Dermis edematous. Blood vessels dilated and surrounded by inflammatory cells. Tissue spaces filled with tissue fluid. These severe reactionary changes were seen in the skin of the back of the body. Fig. 13 Group III E : Skin of the back of the ear seen after reapplication of combined 50% each of the creams after an interval of 2 months showed severe reaction. Signs of dermatitis such as thin epidermis, infolded thick dermis more intercellular spaces filled with fluid and more of elastic and collagen fibres. It was seen that most severe reaction was seen with combination of the 50% each of the two creams, than with each of the creams alone. 151 Discussion : In Group I, A, B, C, D, & E of Dimethyl phthalate cream application for two months on rabbits skin both on the back of the body and on the back of ear showed signs of irritation. There were many infoldings of epidermis, less hairs, dermis was thick with profuse vascularity. After an interval of two months without application of the cream signs of irritation were still present. Reapplication for two months after that interval gave severe effect. Diethyl toluamide cream application for two months gave comparatively more severe effects in Group II, A, B, C, D, & E, than those seen in group-i. These effects were still present although no cream was applied for a duration of two months and more severe effects were seen by reapplication of the same cream. In group III A, B, C, D, & E labelled rabbits, combination of 50% each of the above creams were applied for two months. The effects were comparatively more severe than those seen with the application of single cream. After an interval of two months there were still signs of irritation. Reapplication of the creams after a duration of two months gave maximum reaction. Conclusion : It is concluded that combination of the creams is very harmful. Each of the single creams are also dangerous. Hence Diethyltoluamide cream or Dimethylphthalate creams should not be used even in small quantities as mosquito repellents. References : 1. Ackerman, A.B.: Histologic Diagnosis of inflammatory skin Diseases. : pp. 33 49 (1978) 2. Cook, H.C. : Manual of Histological Demonstration Techniques. Butter worth and Co., Ltd., London, pp. 1 7 (1974). 3. Dipalma, J.R. : Drill s phamacology in medicine. 4th Edition, McGraw Hill, Co. New York. (1971) 4. Ghosh, R. : Pharmacology, Materia Medica and Therapeutics, 7 th Edition. pp. 765 766, (1977). 5. Gilbert, I.H. (1966) : Journal of American Medical Association 196, (3), pp 253-255. 6. Hocking, B. (1963) : The use of attractants and repellents in vector Control. Bulletin of World Health Organisation 29: pp. 121 126.
152 7. Jungueiro, L.C. & Carneiro, J: Basic Histology 3 rd Edition, pp. 378 391, (1971). 8. Khan, A.A., Maibach, H.I., and Skidmore, D.L. (1974) : Effect of Mosquito and Repellent related factors on protection Time. Journal of Economic Entomology. Vol. 68, No. 1 : pp. 43 45. 9. Lamberg, S.I. & Mukrennen, J.A. (1969) : Bullous Reaction to Diethyltoluamide. Archives of Dermatology Vol. 100: pp 582-86. 10. Luna, L.G.H.T. (ASCP) : Manual of Histological staining methods 3rd Edn, pp 77-78, 82-83 (1969) 11. Smith, C.N. : Personal Protection from blood sucking arthropods. Journal of American Medical Association 196(3) : 236-9. 12. Wong, M.H. & Yew, D.T. (1978) : Dermotoxicity of the mosquito repellent related to rabbit ears. Acta Anatomica 100: pp 129-131. This Article Can be Downloaded / Printed Free from http:\\jasi.net
Opp. 150(A) Fig. 1 Group I A : Normal skin of the back of the body : Few infoldings of epidermis, diffuse hair follicles, few blood vessels in the dermis. Fig. 2 Group I B : Dimethylphthalate cream on back of the ear : Epidermis showing many infoldings, less hair follicles, dermis thick with more blood vessels and collagen bundles and tissue spaces filled with fluid. Fig. 3 Group I C : No Applicaton of cream for 2 months on back of the ear : Signs of irritation still present. Many infoldings of epidermis. Few tissue spaces in the dermis are filled with tissue fluid.
Opp. 150(B) Fig. 4 Group I D : Reapplication of Dimethylphthalate cream for 2 months. Severe reaction seen in the skin of back of the body. Epidermis was broken & infolded and keratin was irregularly distributed. Dermis was thick with more of edema, tissue fluid and bundles of collagen fibres. Fig. 5 Group I E : Reapplication of DImethylphthalate cream on ear : Signs of dermatitis like more of intercellular spaces filled with tissue fluid. Fig. 6 Group II B : Diethylatoluamide cream application for 2 months on the skin of back of the body. Epidermis was thin, dermis was edematous, bundles of collagen fibres, dilated blood vessels were surrounded by inflammatory cells, tissue space filled with fluid.
Opp. 150(C) Fig. 7 Group II C : No cream was applied for 2 months. The skin on the back of the body showed that epidermis was thin and infolded. Dermis was edematous with intercellular spaces filled with lymph. Fig. 8 Group II D : Reapplicaton of cream on the back of the body showed severe reaction. Keratin of epidermis was thin dark. Dermis was thick with more collagen fibres, blood vessels dilated and surrounded by inflammtory cells, tissue spaces filled with lymph. Subcutaneous adipose cells filled with fat. Fig. 9 Group II E : Skin of back of ear after interval of 2 months when diethyltoluamide cream was reapplied showed severe reaction. Epidermis was thin and broken at intervals Dermis was thick with more bundles of collagen fibres, elastic fibres, blood vessels dilated and surrounded by inflammatory cells. Tissue spaces were filled with tissue fluid.
Opp. 150(D) Fig. 10 Group III B : Combined 50% each of the two creams for 2 months. Skin of back of the body showing breaks in epidermis + in keratin. Dermis thick, more of bundles of collagen fibres, blood vessels dilated and surrounded by inflammatory cells & tissue spaces filled with fluid. Fig. 11 Group III C : No cream in interval of 2 months. Skin of back of the body : Signs of irritation still present. Epidermis is having infoldings. Dermis thickened with more of fibroblasts, elastic fibres, dilated blood vessels and inflammatory cells around them. Fig. 12 Group III D : Reapplication of combined 50% of creams. Epidermis is thickened, keratinized and infolded. Dermis is edematous, blood vessels dilated, surrounded by inflammatory cells. Tissue spaces filled with tissue fluid seen in the skin of the back of the body. Fig. 13 Group III E : Skin of back of ear reapplication of 50% each of the creams after 2 months of interval. Epidermis has thin layer of keratin and is infolded. Dermis had edema, with numerous elastic and collagen fibres and tissue spaces filled with tissue fluid.