L.Bui


 * Final Project**

Linh Bui Professor: Dr. Jean-Claude Bradley CHEM 367

=Final Project: DEET, and Essential oils, Popularly Used Insect Repellent Agents Today=

Abstract
Arthropod and insect bites have been a thread of infectious and inflammatory diseases. This led to the starting of researches on insect repellents to prevent insect-transmitted illnesses. There are synthetic chemical and plant-based insect repellent products available on the market today. DEET which is a synthetic insect repellent, essential oils, which is a plant-based insect repellent, will be discussed in this paper. The introduction, ways of DEET synthesis and effect assessments and toxicity of insect repellent products containing DEET will be discussed. In addition, the background of plant-based insect repellents, and essential oils as an example will also be found in this paper.

Introduction
Arthropod bites are a main reason for patient morbidity. These arthropod bites can cause infectious or inflammatory wounds. Popularly serious diseases caused by arthropods, insects or arachnoid are malaria, Lyme disease, dengue fever, and West Nile fever. It was necessary for researches on insect repellents to protect people and prevent the spread of arthropod transmitted diseases throughout the world [1,2,3].

Back in ancient time, tars, types of smoke, plant oils, camel urine were used as insect repellents. Before World War II, there were four commonly used insect repellents: oil of citronella (discovered in1901), dialkyl phthalates (discovered in 1929), indalone (available in 1937), and Rutgers 612 (available in 1939). In 1946, DEET (N,N-diethyl-m-toluamide) was developed by The U.S Department of Agriculture for the military use in insect infested area. In 1956, DEET was registered for use by population [3,4,5]. Besides synthetic chemical insect repellents such as DEET, permethrin, picaridin [3,4,6], there are natural insect repellent products on the market also such as plant-based insect repellents like citronella oil, neem oil, pyrethrum, lavender, para-menthane-3,8-diol (oil of lemon eucalyptus) [3,7,8].

Insect repellents are available in many various forms such as pump sprays, lotions, creams, aerosols, powder, oils, grease roll-on sticks and cloth-impregnating laundry emulsions. Without regard to the forms, the period of protection of one repellent depends on the active ingredients, the body type of the users, the environment, and the insect species [3,4,18].

Insect repellent agents can be applied directly on skin or clothing. They form a vapor barrier layer which has an unpleasant smell or bad taste to keep insects away from coming into contact with the skin. Insects turn away only when they get close (about less than 4 cm) to the area where the repellent agent is applied. For the most efficiency, insect repellents must be reapplied every couple hours because the abrasion by clothing, sweating, or washing with water reduces the duration of protection of insect repellent products. The efficiency of the repellents is related to the boiling of the active ingredient chemicals. The desired boiling range is from 230*C to 260*C at atmospheric pressure [4]. Chemicals have low boiling point tend to vaporize quickly so they decompose rapidly. Vice versa, chemicals have high boiling point do not vaporize as much as needed. Active ingredient chemicals in repellent products have lower or higher boiling point than the desired range will provide an insufficient repellent environment [3,4].

An ideal insect repellent product should include these following criteria [4]: + Have long-durable efficiency and against as many types of arthropods and insects as possible. + Give nonirritating and pleasant feel to the skin after application. + Resist removing by wiping, washing, or sweating after topical application. + Have no smell or have refreshing smell. + Be harmless to clothes such as non-discolor, non-bleaching, weakening the fiber but be able to resist repeated washing. + Be inactive to commonly used plastics. + Use stable, economically available active ingredient chemicals for widely produce and use.

There are many insect repellents used currently. However, DEET (N,N-diethyl-m-toluamide or N, N-diethyl-3-methylbenzamide), which is a synthetic insect repellent, essential oils, which is a plant-based insect repellent, are popularly and efficiently used today, will be discussed in this research paper.

Introduction of DEET
Most researches on insect repellents have been done by the U.S Armed Forces and Department of Agriculture because soldier who served during World War II, the Korean War, the Vietnam Conflict, and Operation Desert Storm had many diseased caused by arthropod bites. The first clinical trials started in 1942. In 1946, DEET was developed by The U.S Department of Agriculture for the military only. DEET became available on the market in 1956. DEET has been known as the most popular (used by approximately 30% U.S population) and effective insect repellent against to a wide range of arthropods and insects. Therefore, it is the active ingredient in most insect repellent products which are currently available on the market since it first marketed [1,3,4,9].

DEET is safe for cotton, wool, and nylon fabric, but it is not safe for spandex, rayon, acetate, pigmented leather, plastic and vinyl [3,4].

Synthesis of DEET
There are three common ways to synthesize DEET in the laboratory.

• The first method is to use m-toluic acid (1) and thionyl chloride SOCl2 (2) as starting reactants and followed by aqueous condensation with an amine to yield N,N-diethyl-m¬-toluamide. The acid is first converted to the corresponding acid chloride using thionyl chloride. The product of this reaction is m-toluoyl chloride (5). Then this product is allowed to react with diethyl amine (6) to form N,N-diethyl-m¬-toluamide (DEET) (8). In this method, using thionyl chloride should be done cautiously because the byproducts are hydrogen chloride (HCl) and sulfur dioxide (SO2) which are toxic and corrosive gasses [11]. (Figure 1)



• The second method is to use m-toluic acid (1) and excess oxalyl chloride (9) in hexane (10) and DMF (N,N-dimethyl formamide) (11) acting as catalysts. This reaction gives m-toluoyl chloride (5). The byproducts of this reaction are carbon monoxide (CO), carbon dioxide (CO2) and hydrogen chloride (HCl) (gases). The m-toluoyl chloride (5) (acyl chloride) then is converted into DEET (8) by a non-aqueous addition of diethyl amine (6) [11]. (Figure 2)



Both two ways accomplish the same goal producing the same intermediates and products. The first method (using thionyl chloride (2)) is a nucleophilic substitution by chloride ions to remove acyl chlorosulfite intermediates which decompose to sulfur dioxide and hydrogen chloride. Pyridine (3) is used as a catalyst to speed up the reaction. The second method (using oxalyl chloride (9)) is an exchange reaction which converts a carboxylic acid to acid chloride and vice versa. Oxalyl chloride (9) degrades to gaseous products: hydrogen chloride (HCl), carbon dioxide (CO2), and carbon monoxide (CO). DMF (11) is used as a catalyst to accelerate the reaction. Oxalyl chloride (9) is a milder and selective reagent, compared to thionyl chloride (2) [11]. The first method is commonly used in organic chemistry laboratory for teaching purpose. The second method is rarely used because DMF is a hazardous agent causing cancer in human and birth effects [20].

• The third method is to convert a nitrile to an amide. In this method, DEET (8) is synthesized from m-methyl benzonitrile (16) and N,N-dimethylaminomagnesiumbromide (15). First of all, N,N-dimethylaminomagnesiumbromide (15) is produced by the reaction of diethylamine (12) and ethylmagnesiumbromide (13) in diethyl ether (4) and tetrahydrofuran (THF) (14). Then N,N-dimethylaminomagnesiumbromide (15) is added to m-methyl benzonitrile (16) in tetrahydrofuran (THF) (14) and the mixture of dichloromethane (17) and aqueous hydrochloric acid (18). The collected organic layer is washed with brine solution and dried with drying agent sodium sulfate. Evaporation of the organic layer yields DEET [19]. (Figure 3)



Effect assessments and toxicity of insect repellent containing DEET
Studies on the side effects causing by the use of insect repellent products containing DEET showed that side effects happen on young children more frequently than adults because they had oral contact with the repellent agents. It happened on girls (56%) more than boys (44%) [9]. Parental carelessness is one of the reasons causing adverse effects of DEET to occur on their children. Parents often did not read instructions on the label, left repellent agent on their children’s skin during the night, or use repellent products directly on their children’s faces. Cases of older children, adolescents, and adults happened mostly because they had sprayed the repellent products into their eyes or drawn into their lungs by breathing. Results occurring on adults are often more severe than on children [1,3,4,9,10].

According to reports on adverse effects of DEET, the common problems caused by the use of DEET are inflammation of skin, allergic responses, disorders on nervous system, and problems with heart or blood vessels (e.g. abnormally low blood pressure), paroxysm. Besides that, there are also reports on DEET ingestion. Consumption of DEET can cause seizure, hypotonic reactions, syncope, coma respiratory distress or even death, which depends on how much the amount of concentrated DEET was ingested [1,3,4,9,10]. The cases resulting in the death involved in ingestion of large amount (50 mL) of repellent products containing 11% to 95% of concentration of DEET [4,10].

It is concluded that the risk of serious side effects from the use of repellent agents containing DEET is insignificant. DEET is not a selective toxin damaging or destroying nerve tissues. The increase in DEET concentration does not change the severity of reported symptoms [4,10].

In order to reduce the chance of occurring of adverse effects of using DEET, the minimum efficient concentration of DEET for a particular situation should be used. For repellent products containing 10% to 30% of DEET, supply a decent protection. For products containing 40% to 50% of DEET, the amount of two to four tablespoons should be enough for covering arms, legs, and face of an average adult [4]. Studies of DEET toxicity were done on animals such as rat, dog [1].

• Acute toxicity: a report about oral LD50 in the rats was 3664 mg/kg body weight (BW). Another report about acute dermic toxicity in dogs with different doses of 356, 1426, 1782, and 7128 mg/kg BW. Dogs receiving doses of 1782 mg/kg BW had no signs of toxicity. The reported LD50 in rabbits was 3180 md/kg BW. The application of 75% and 100% DEET on skin caused no sign of reactions in treated animals [1].

• Subchronic toxicity: the application of DEET on skin with doses of 0, 100, 300, 1000 mg/kg BW/day for 13 weeks did not show any sign of toxicity. Therefore, the lowest-observed-effect-level (LOEL) was set at 1000 mg/kg BW/day and the no-observed effect-level (NOEL) at 300 mg/kg BW/day. Rats which were treated with dermal application of DEET in ethanol at 4, 40, 400 mg/kg BW daily did not show any toxicity signs. However, continuous use of DEET at 40, and 400 mg/kg BW for 60 days decreased the permeableness of blood-brain barrier in some brain areas. A case on the study of the microscopic structure of abnormal and diseased tissues showed subchronic dermic exposure to DEET at 40 mg/kg BW/day which caused considerable neuronic cell death and cytoskeletal abnormalcies in surviving nerve cells. More severe signs of toxicity on neuronic system owing to DEET appeared at high doses only [1].

• Chronic toxicity: application of DEET on skin in treated animals caused acute and subchronic toxicity only. Therefore, the oral route of administration was studied in 2-year feeding in rats, mice, and dogs to valuate the chronic toxicity of DEET. In female rats, there was a decrease in body weights and food ingestion along with a slight rise in cholesterol serum with high dose at 400 mg/kg BW/day. The NOEL was set to be 100 mg/kg BW/day. In both male and female mice, there was a slight drop in body weight and food ingestion at the highest dose of 1000 mg/kg BW/day. The NOEL of the study on mouse was determined to be 500 mg/kg BW/day. In dogs, there was a drop in body weight and food ingestion along with a rise relative frequency of vomiting and excessive flow of salvia, and alterations in some clinical pathobiology factors at 400 mg/kg BW/day. The NOEL was 100 mg/kg BW/day [1].

The NOEL for acute and subchronic toxicity was 200 and 300 mg/kg BW/day respectively. The NOEL for chronic toxicity was 100 mg/kg BW/day [1].

Human health risk assessments were estimated by combining toxicity and exposure using the Margin of Exposure method (MOE). The value of an MOE for each subgroup in a population was the ratio of the appropriate toxic endpoint (for instance NOEL) and the everyday exposure. MOE less than 100 usually regard to be over a regulative level of concern. [1].

• Acute toxicity: for everyday exposure ranging of 2 to 59 mg/kg BW/day, possible acute MOE for DEET ranges from 85 to 2127. To reach the MOE of 100, the concentration range of DEET for children and adults should be from 33.8% to more than 100%. In other words, children under 12 years old have an MOE below 100 if using 40% concentration of DEET products [1].

• Subchronic toxicity: the subchronic MOE range is from 25 to 638. At 25% concentration of DEET, children under 17 years old have MOE below 100. At 40% concentration of DEET, every population subgroup has MOE below 100. The concentration of DEET should range from 10.1% to 31.9% in order for the MOE of all subgroups to reach 100 [1].

• Chronic toxicity: the chronic MOE ranges from 42 to 1064. At 25% concentration of DEET, under-12-year-old children and at 40% concentration of DEET, under-17-year-old children have MOE of below 100. For MOE value of 100, the DEET concentration must be in the range of 16.9% to 34.2% for under-17-year-old children, and 45.3% to 53.2% for male and female adults [1].

History of plant-based insect repellents
Besides synthetic chemical insect repellents, there are plant-based insect repellents ((botanicals) currently available on the market. Without any doubt, synthetic insecticides have been effectively used in agriculture among crop pesticides. However, they also cause unexpected issues related to human and environment such as at 25% acute and chronic diseases to users, farmers or consumers, devastation of wildlife living environment, impurity of underground-water, interruption of pollination, possible threats to human health and environmental concerns, and the process of resistance of insecticide in the pestis population [6]. Therefore, health hazards and environmental concerns caused by the use of chemical synthetic insect repellents result in the development of plant-based insect repellents, which are safe to environment and human health [6,7,12,15].

Plant-based insect repellents were actually used popularly in China, Egypt, Greece, and India two thousand years ago. The use of botanical insect repellents in Europe and North America appeared 150 years ago. This means botanical insecticides were used before the discoveries of synthetic chemical insect repellents in the mid-1930s to 1950s [6]. Botanical insect repellents are considered to be efficient and safer to human health and environment [7].Therefore, during recent years, there has been an increase in the use of plant-based insect repellents [12,15].

There are some main kinds of plant-based insect repellents used popularly today. They are pyrethrum, rotenone, neem (Azadirachtin), and essential oils. Beside those four kinds above, there are also other botanical products with restricted use because of their danger to humans: ryania, nicotine, strychnine, and sabadilla [6,14].

Here are some examples of toxicity of plant-base insect repellents to mammals. Based on the report, pure pyrethrums whose value of oral acute LD50 in rats is from 350 to 500 mg/kg are mild toxic to mammals, but the formulated pyrethrum whose LD50 value of 1500 mg/kg is less toxic. In contrast to pyrethrums, neem (azadichatin) is not toxic to mammals, fish and pollinators. Its rat oral acute LD50 value is above 5000mg/kg. In purity, rotenone is toxic to mammals. The value of rat oral LD50 is 132mg/kg. Sabadilla in really toxic to mammals. The rat oral LD50 value is approximately 13mg/kg. Because of its high toxicity, commercial products usually contain no more than 1% active ingredient. The LD50 value of pure nicotine is 50mg/kg in rats. So, it is extremely toxic to mammals as well [6].

Introduction of plant essential oils
Essential oils of many plants have been found to have repellent properties against different hematophagous arthropods, and insect. Essential oils are the main classification that started to grow with researched in 1980s among botanical insect repellents. Essential oils are also used in other industries such as perfume, cosmetics, detergents, household cleaning products, pharmacology, food and beverage productions, agriculture, fine chemistry, and more recent for aromatherapy and as herbal medicines. Essential oils are used widely over the world especially in Europe, Japan, and North America because of their safety today [6,12,14,16].

Since plant essential oils are used in various industries, they have different activities such as anticancer, antiphlogistic to remove injury or infection, penetration improvement to enhance drug delivery, insect repellent, antiviral, and antioxidant activity [16].

Plant essential oils, also known as volatile oils, are extracted from natural aromatic botanical sources [6,13-17,21]. Essential oils can be obtained from hydro distillation, steam distillation, dry distillation, or mechanical cold pressing of plant. The traditional preparation way is based on the Clevenger steam distillation apparatus developed in 1928. This method is now used commonly for industrial production. The disadvantages of this method are that large containers required because of the low yield (generally <1%) from biomass and high value of production because of high temperature need for distillation process. However, the advantage of the steam distillation method is that it separates the relatively clean secondary products of metabolism from plants, consisting of mainly low-molecular-weight volatile phytochemicals, especially unsaturated hydrocarbons found in essential oils and oleoresins of plants (terpene) and compounds derived from phenol (phenolic compounds), while it excludes most primary metabolite and high-molecular-weight secondary compounds [12].

Essential oils exist in different parts of plant such as flowers (bergamot orange, Citrus bergamia), leaves (lemon grass, Citronella, eucalyptus), wood (sandalwood, Santalum), roots (vetiver grass, Chrysopogon zizanioides), rootstalks (ginger, Zingiber officinale; turmeric, Curcuma longa), fruits (anise, Pimpinella anisum), and seeds (nutmeg, Myristica fragrans) [12].

Synthetic chemical pesticides are not only more expensive than botanical ones, but also cause problems relating to human health and environment issues. In developing countries, farmers might not afford synthetic chemical insecticides. They use tradition plants and plant derivatives as crop protectants instead [12,13]. Botanical essential oils are known as environmentally friendly insect repellent products. Generally, essential oils are safe to mammals. The acute oral LD50 value of pure plant essential oils ranges from 800 to 3000 mg/kg. The value of LD50 for essential oils prepared according to a specific formula is approximately 5000 mg/kg [13]. Beside safe essential oils, some essential oils can cause cancer or diseases. For instances, essential oil from Salvia sclarea and Melaleuca quinquenervia raise estroden secretions which leads to higher chance of estrogen-dependent cancers; flavins, cyanin, porphyrins, and hydrocarbures can cause skin redness or even cancer; essential oils from citrus bergamia can  cause skin cancer, etc [21].

Conclusion
There is a variety of insect repellent products available on the market today. Every type has its own efficiency, toxicity, safety and risk concerns. Therefore, choosing a right repellent product for different purposes of usage, ages and following safety guidelines are very important to maximize the effectiveness and minimize the toxicity of an insect repellent.