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The combined efficacy of neem (Azardirachta indica) seed oil and orange
(Citrus sinensis) peel oil cream as a mosquito repellent

Article · July 2022

DOI: 10.30574/gscarr.2022.12.1.0184

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The combined efficacy of neem (Azardirachta indica) seed oil and orange (Citrus
sinensis) peel oil cream as a mosquito repellent
Godfred Yaw Boanyah 1, 2, * and Ruth Carre Brenyah 2
1International Centre of Insect Physiology and Ecology, P.O. Box 30772, Nairobi 00100, Kenya.
2Department of Clinical Microbiology, College of Health Sciences, Kwame Nkrumah University of Science and Technology,
Kumasi, Ghana.

GSC Advanced Research and Reviews, 2022, 12(01), 057–067

Publication history: Received on 09 June 2022; revised on 16 July 2022; accepted on 18 July 2022

Article DOI: https://doi.org/10.30574/gscarr.2022.12.1.0184

Abstract
Essential oils obtained from certain plants have repellent potency against insects such as mosquitoes. Most repellents
on the market have the synthetic formulation of N, N-diethyl-3-methylbenzamide (DEET) as the main active ingredient.
This study is an attempt to contribute to the search for an effective plant-extracted essential oil, Neem and Orange as
alternative to DEET.

The oil from Citrus sinensis (sweet orange) peels was extracted by steam distillation and that for Azadirachta indica
(Neem) seed by Soxhlet extraction respectively. Creams were formulated at four concentrations of 5%, 15%, 25% and
30% v/w respectively for Neem, Orange peel and combined Neem-Orange oils, after which laboratory reared
mosquitoes were used to assess the efficacy of each of the concentrations. A DEET-based cream of 13% concentration
was used as the standard.

Wild mosquito larvae were brought to the laboratory from which the adults female after mating were given a blood
meal. Second generation mosquitoes were used for the arm in cage test (AIC).

The combined Neem and Orange-based cream at 30% was as effective as the DEET-based repellent which recorded the
highest repellent time, followed by Neem cream and the Orange peel based cream recorded the least repellent time even
at 30% concentration. The statistically significant difference at 30% between DEET and Orange peel, Neem, and
combined Neem-Orange creams were 0.030, 0 .499 and 0.195, respectively.

This study suggests that the combined Neem and Orange Peel Oil can be used to produce repellent creams due to their
synergistic potency and good smell.

Keywords: Natural mosquito repellent; Essential oil; Anopheles; Aedes

1. Introduction
Mosquitoes are important public health concern especially in developing countries as a result of being vectors to many
pathogenic organisms that infect humans[1, 2]. They serve as vectors for the transmission of dengue fever, malaria,
filariasis, yellow fever just to mention few[3, 4].

According to the 2021 World Malaria Report, there was an estimated figure of 241 million cases of malaria worldwide
in the year 2020 with Africa constituting 95% of the world burden. Additionally, the World Health Organization, Africa

Corresponding author: Godfred Yaw Boanyah
International Centre of Insect Physiology and Ecology, P.O. Box 30772, Nairobi 00100, Kenya.
Copyright © 2022 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0.
 GSC Advanced Research and Reviews, 2022, 12(01), 057–067

Region carried 96% burden of the 627 000 total global malaria deaths estimated in 2020 [5]. Malaria is also responsible
for a significant maternal deaths in Ghana and Africa as a result of contracting malaria during pregnancy[6]. From the
findings of Ameme et al, (2014), Malaria accounts for 40% of outpatients cases in Ghana from their cross sectional study.
Transmission cycle of human-vector contact can be broken by the use of repellent [8]. Additionally, the application of
proper mosquito repellent on clothing and bed nets have yielded massive positive results especially in Africa, conferring
protection to the individual using them [9, 10]. Moreover, topical repellent is one of the most effective ways of
controlling outdoor biting of mosquitoes[11].

Furthermore, N, N-diethyl-3-methylbenzamide (DEET) based repellents are the standard and most common synthetic
products in our markets today. Even though very effective, the repercussions of their continuous use cannot be
overlooked. The fear of the side effects of DEET by people is gradually giving market to the natural repellents [12]. The
findings of Colucci and Müller[2018] showed that DEET showed at least 6 hours of protection against Aedes aegypti,
Anopheles stephensi and Culex quinquefasciatus in a field studies in Switzerland at 15% [14–16]. Additionally, increasing
insecticide resistance has posed a major obstacle to mosquito control programmes and this calls for the development
of new repellents[12, 17].

The use of botanicals and derivatives as insect repellent particularly against mosquitoes is a feasible option to achieve
protection against the mosquitoes, and thus, reducing the risk of acquiring mosquito borne diseases[16, 18, 19]. Among
the various natural organic chemicals, products from the neem tree, Azadirachta indica, like neem seed oil have long
been established as a potential mosquito repellent[20]. Azadirachtin, a complex tetra-terpenoid limonoid compound in
neem seeds, is the major component responsible for the repellent action in insects [21]. Moreover, repellents from
essential oils are environmentally friendly, biodegradable and not harmful to non-target organism as compared to the
synthetic DEET[22].

Citrus sinensis on the other hand considered in this study has several usefulness. Juice from the fruits is used in the
production of different beverages in large quantities. Flavour from the oil of peels are used to season foods and it has
repellent effect as well due to the presence of limonene [23–25]. Peels of Citrus fruits (Citrus sinensis) has also been
proven to possess repellent properties [26]. Exploration of different botanicals against mosquitoes should be continued,
thereby reducing the contact of human and mosquitoes as well as the risk of transmission of diseases to humans [27,
28].

This present manuscript elaborates the evaluation of the combined efficacy of neem seed oil and orange peel oil as
mosquito repellent against Aedes aegypti and Anopheles gambiae using DEET as the standard.

2. Material and methods

2.1. Study design

This is an experimental study conducted in the insectary of the Department of Theoretical and Applied Biology at
Kwame Nkrumah University of Science and Technology, KNUST Kumasi, Ghana within the period of September 2019 to
February 2020.

2.2. Sample collection, extraction, and phytochemical screening

The neem (Azadirachta indica) seed oil and sweet orange (Citrus sinensis) peel oil were purchased from Agape Moringa
Processing in Tamale Northern Region of Ghana. The seed and the peels were collected from Tamale as well. Coloured
rind of Citrus sinensis of two oranges were put into Clevenger apparatus and distilled water of 100 cm 3 was added. The
flask containing the peels was heated just below 100oC and distillation took place steadily. Distillate of 50 cm 3 was
collected in a measuring cylinder and the oil layer is removed with pipette completing the steam distillation method
[29].This was done till 200ml of oil was obtained.

Azadirachta indica from Tamale were dried and milled into powder. Oil was extracted from the powder (458.65 g) using
Petroleum ether (2.2 L) with the Soxhlet apparatus for 4 days. The extract was concentrated using the rotary evaporator
at temperature 45oC and a percentage yield of 43.6% v/w obtained. The oil was then stored in an amber bottle at a cool
dry place until ready for use.

𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑛𝑒𝑒𝑚 𝑠𝑒𝑒𝑑 𝑜𝑖𝑙 𝑥 100%

Yield of extract obtained =
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑝𝑙𝑎𝑛𝑡 𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙 𝑡𝑎𝑘𝑒𝑛

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 GSC Advanced Research and Reviews, 2022, 12(01), 057–067

The extracted Citrus sinensis and Azadirachta indica oils were phytochemically screened according to the method used
by [30] and [31] to confirm limonene and azardirachthin respectively which were the active ingredient responsible for
the repellent effect before formulating it into a cream.

2.3. Processing of active ingredient into cream

Twenty (20g) of Aqueous Cream was weighed and a calculated measured volume of the active ingredient (neem seed
oil, orange peel oil or both) was added to the cream base. The volume of the oil or active ingredient added was
determined by the standard formula for preparing aqueous cream which is the quantity to be prepared divided by
quantity in the master formula. The mixture was stirred, slightly heated and stirred again with a spatula. The mixture
was then homogenized to form the cream which was allowed to cool. Varying concentrations of 5%, 15%, 25% and
30% v/w of the active ingredient (neem seed oil or orange peel oi) was added to the emulsifying ointment. The
homogenized cream was allowed to cool or solidify and then packaged into containers. The above concentrations were
chosen because Citrus oil contains a compound called bergapten which usually above 30% concentration causes
phototoxic skin irritation [32].

2.4. Repellent test by arm in cage test (AIC)

Repellent test was done using the World Health Organisation Pesticide Evaluation Scheme (WHOPES) 2013 guidelines
with slight modification [33] thus, larva of Anopheles gambiae and Aedes aegypti were brought to the laboratory and the
second generation of 5 days old were used for the repellent testing.

The twelve (12) volunteers were put into four (4) groups of three (3). Each group of three volunteers was assigned to
one concentration of the repellents, thus 5%, 15%, 25% and 30% respectively for orange cream and thereafter Neem
only and combined creams.

A confined netted cage on all sides made with wooden bar 20 cm by 20 cm by 20cm. A piece of hollow cloth, big enough
to allow for an arm or a glass screen to enter was fixed at one side of the nets. The netted cage was filled with 30
laboratory-reared mated and sorted female mosquitoes of Anopheles gambiae and Aedes aegypti of 5 days old.

Bare hand without repellent was first introduced into the cage as a negative control followed by aqueous cream base
which was used to process the essential oils into repellent creams to see if it has any repellent effect and time recorded.
The cream of each concentration of a particular extract was smeared on the arm and the hand was put into the repellent
cage with mosquitoes starved for 12 hours. The hand was observed and the time it took for mosquito to land was
recorded. This was done by putting the hand in the box every 5 minutes (min) interval after the first 5 min till 30 min.
If no mosquito landed after 30 min, the interval time was increased to 15 minutes. Moreover, if no mosquito landed till
6 hours, the six hours was recorded as the complete protection time of the repellent. Also, the conditions of the
participants were observed during and after the test for any adverse reactions.

2.5. Statistical analysis

The results were entered into Microsoft Excel and analysed using SPSS. One-Way Analysis of Variance (ANOVA) and
independent student t-test were used to compare the Complete Protection Time of the various creams. All tests were
performed at a significant level of 0.05.

3. Results

3.1. Repellent results for Aedes aegypti mosquitoes

Figure 1 compares the complete protection time (CPT) between neem seed oil cream and orange peel oil cream. At 5%
concentration, the CPT for neem seed oil cream was 0.35 ± 0.04 minutes while orange peel oil cream recorded CPT of
0.34 ± 0.05 minutes. The trend did not change when concentrations were increased to 15% and 25%. The CPTs for neem
seed and orange peel oil creams were 1.01 ± 0.19 and 0.83 ± 0.07 minutes respectively at 15% concentration and 3.60
± 0.25 and 1.87 ± 0.23 minutes respectively at 30% concentration. However, at 25% concentration, the CPT of neem
seed oil cream (1.53 ± 0.13 minutes) was higher than that orange peel oil cream (1.48 ± 0.07 minutes). Generally, an
independent t-test showed that there was no statistically significant difference (p > 0.05) between the CPT of neem seed
and orange peel oil creams at 5%, 15% and 25% concentrations. However, there was a statistically significant difference
(p = 0.001) between the CPT of the two products at 30% concentration.

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 GSC Advanced Research and Reviews, 2022, 12(01), 057–067

Figure 1 Comparison of Repellent Time of Neem and Orange Creams (Aedes aegypti)

Figure 2 Comparison of natural creams at 15% against DEET (Aedes aegypti)

Figure 3 Comparison of natural creams at 30% against DEET (Aedes aegypti)

The 13% DEET is standard product on the market and the study compared the CPTs of the prepared products to the
standard. The 13% DEET (5.06 ± 0.10 minutes) had a higher CPT compared to the neem seed (1.01 ± 0.19 minutes),
orange peel (0.83 ± 0.07 minutes) and combined neem seed and orange peel (1.69 ± 0.20 minutes) oil creams at 15%
concentration (Fig 2). There was a statistically significant difference between 15% neem seed vs. 13% DEET (p < 0.001);
15% Neem seed vs. 15% combined neem seed and orange peel (p = 0.002); 15% orange peel vs. 15% combined neem
seed and orange peel (p = 0.001); 15% orange peel vs. 13% DEET (p < 0.001); and 15% combined neem seed and orange
peel vs. 13% DEET (p < 0.001).

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When the 13% DEET was compared to the CPTs of the prepared products at 30% concentration, the 13% DEET (5.06 ±
0.10 minutes) still had a higher CPT compared to the neem seed (3.60 ± 0.25 minutes), orange peel (1.87 ± 0.23 minutes)
and combined neem seed and orange peel (4.17 ± 0.07 minutes) oil creams at 30% concentration (Fig 3). Generally,
there was a statistically significant difference (p < 0.001) between 13% DEET and all the products at 30% concentration.

3.2. Repellent results for Anopheles gambiae mosquitoes

For Anopheles mosquitoes, at 5% concentration, there was no difference between the mean CPTs of neem seed (0.33 ±
0.08 minutes) and orange peel (0.33 ± 0.12 minutes) oil creams. However, at concentrations of 15%, 25% and 30%, the
mean CPTs of neem seed (3.43 ± 1.15 minutes, 10.07 ± 0.39 minutes, and 25.38 ± 0.48 minutes respectively) was higher
than that of orange peel oil cream (1.83 ± 0.66 minutes, 2.91 ± 1.59 minutes, and 10.01 ± 5.76 minutes respectively) as
shown in Fig 4. Statistically, significant difference existed between the two products only at 25% concentration (p =
0.001) and 30% concentration (p = 0.010).

Figure 4 Comparison of neem and orange creams (Anopheles gambiae)

The 13% DEET (20.90 ± 0.50 minutes) had a higher CPT compared to the neem seed (3.43 ± 1.15 minutes), orange peel
(1.83 ± 0.66 minutes), and the combined neem seed and orange peel (3.75 ± 0.03 minutes) oil creams at 15%
concentration (Fig 5). Statistically, significant difference existed between 15% neem seed vs. 13% DEET (p < 0.001);
15% orange peel vs. 15% combined neem seed and orange peel (p = 0.042); 15% orange peel vs. 13% DEET (p < 0.001);
and 15% combined neem seed and orange peel vs. 13% DEET (p < 0.001).

Figure 5 Comparison of natural creams at 15% against DEET (Anopheles gambiae)

When concentrations of the products were increased to 30%, the CPTs of 30% neem seed (25.38 ± 0.48 minutes) and
30% combined neem seed and orange peel (27.72 ± 4.75 minutes) were higher than the CPT of 13% DEET (20.90 ± 0.50

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minutes) as shown in Fig 6. Statistically significant difference existed only between 30% neem seed vs. 30% orange peel
(p = 0.004), 30% orange peel vs. 30% combined neem seed and orange peel (p = 0.002), and 30% orange peel and 13%
DEET (p = 0.030).

Figure 6 Comparison of natural creams at 30% against DEET (Anopheles gambiae)

3.3. Negative Controls

The time recorded for the bare hand and the aqueous cream base used to prepare the repellent creams were the same.
Thus, no repellent property in aqueous cream (S1 Table).

Table 1 Comparison of the complete protection time between Anopheles and Aedes mosquitoes

Complete Protection Time (in minutes)

Product p – value
Anopheles Aedes
5% concentration
Neem seed 0.33 ± 0.08 0.35 ± 0.04 0.778
Orange peel 0.33 ± 0.12 0.34 ± 0.05 0.897
Neem seed & orange peel 1.27 ± 0.07 0.42 ± 0.08 < 0.001*
15% concentration
Neem seed 3.43 ± 1.15 1.01 ± 0.19 0.023*
Orange peel 1.83 ± 0.66 0.83 ± 0.07 0.118
Neem seed & orange peel 3.75 ± 0.03 1.69 ± 0.20 < 0.001*
25% concentration
Neem seed 10.70 ± 0.39 1.48 ± 0.07 < 0.001*
Orange peel 2.91 ± 1.59 1.53 ± 0.13 0.274
Neem seed & orange peel 11.02 ± 0.07 1.88 ± 0.08 0.001*
30% concentration
Neem seed 25.38 ± 0.48 3.60 ± 0.25 < 0.001*
Orange peel 10.01 ± 5.76 1.87 ± 0.23 0.071
Neem seed & orange peel 27.72 ± 4.75 4.17 ± 0.07 0.013*
Control
13% DEET 20.90 ± 0.50 5.06 ± 0.10 < 0.001*
Numbers with asterisk (*) indicate the mean difference is significant at the 0.05 level.

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4. Discussion
Previous study [34] indicated that 2% neem oil mixed in coconut oil was sufficient to provide 12h protection from the
bites of anopheline mosquitoes. In another study, the combination of the neem oil with kerosene oil showed protection
against mosquitoes [35]. In 2004, a commercial product called Bite Blocker which was made up of glycerine, lecithin,
oils of coconut, geranium and 2% soyabean showed 7.2 hours protection against Aedes albopictus[36].

Moreover, a laboratory study of Citrus sinensis oil at 0.02 mg/cm against Anoheles dirus provided 40% protection. A
recent study from Ethiopia has shown about 20% neem oil combination provide 3-hour protection against Anopheles
arabiensis in a field study [8]. Application of neem oil in water, (5 g/l; 0.3% Azadiractin in 1 litre containing the
emulsifier Sorbitan, tri-9-octadecenoate, poly(oxy-1,2-ethanediyl) was effective in reducing the abundance and
oviposition of the mosquito Aedes albopictus [37]. However, a systemic review in 2018 demonstrated that Ligusticum
sinense extract was able to give 11 hours of protection against Anopheles mosquitoes[38].

From Figs 1 and 4, an increase in concentration corresponded with an increase in complete protection time (CPT) thus,
5%, 15%, 25% to 30% in that order. This trend was not different from an earlier research conducted on neem seed oil
as mosquito repellent and repellent action of orange peels oil on mosquitoes [26, 35]. Generally, there was a statistically
significant difference (p < 0.001) between the various concentrations of Neem seed and Citrus oil creams. However, a
post hoc test showed no significant difference (p = 0.079) between orange peel oil creams at 25% and 30%
concentrations for Aedes aegypti and 5% vs. 30% concentrations (p = 0.018), and 15% vs. 30% concentrations (p =
0.042) for Anopheles gambiae.

Furthermore, the high volatility of citrus could be responsible for almost an equal protection time between Neem and
Orange oil Creams at lower concentrations but drastically reduced at higher concentrations, especially at 30% [39].
Interestingly, the combined neem seed and orange peel oil cream did better than the individual creams (Figs 2, 3, 5, 6
and Table 1). The results indicated synergy between orange and neem oils because the formulation was 50:50 v/v. For
instance, the CPT of Anopheles gambiae at 30% for the combined cream gave (27.72 ± 4.75), this constituted 15% neem
seed oil and 15% orange peel oil which would have given (3.43 ± 1.15) and (1.83 ± 0.66) CPT individually (Table 1). The
synergistic repellent effect of combined essential oils observed confirmed recent findings by Noosidum et al, [2014]
and Feroz, [2020].

Repellent test involving skin application of Citrus peel oil at 20% and other essential oil produced irritancy in previous
studies [26, 40]. On the contrary, there was no record of any adverse reaction by all the participants during and after
this study probably due to incorporation of the oil into cream.

Also, DEET had excellent protection against Aedes aegypti compared to the natural creams at all concentrations.
However, Anopheles gambiae at 30% of neem only, and combined neem-orange creams (Figs 3 and 6) compared with
DEET gave a CPT mean difference of 4.48 and 6.82 respectively without significant difference between them. This is an
important indication that Combined Neem and Orange peel oil cream can be used as an alternate effective mosquito
repellent in Malaria endemic regions.

Finally, it is evident from the results that the CPTs of Aedes aegypti were much lower than that of Anopheles gambiae,
similar to research conducted in 2018 to evaluate CPTs of both Arm In-Cage Test (AIC) and field for two standard
mosquito repellents in Switzerland. For the AIC, the CPTs for Aedes aegypti and Anopheles gambiae were 0.5 and 2 hours
respectively for DEET [13]. However, the results here for DEET (Table 1) are much lower than that recorded in their
work probably due to lower concentration used or environmental changes. This could also be due to continuous
reduction in repellence of DEET to Aedes aegypti upon previous exposure conferring genetic resistance [42]. As well as
an indication of resistance of mosquitoes to repellents especially the standard DEET on the market.

5. Conclusion
The observation from this study has shown clearly that DEET-based repellent is more effective against Aedes aegypti
than the plant products. However, Combined Neem Seed and Orange Peel Oil Cream at 30% is as effective as DEET and
therefore can be used as an effective mosquito repellent in Malaria endemic regions. There was generally reduced
complete protection time compared to previous studies for the standard, DEET and the plant repellents.

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Compliance with ethical standards

Acknowledgments
I say a big thank you to Mr. Akuamoah Yaw and Patrick Obuam for their assistance for the field work and Esther Naanbir
(Agape Moringa Processing) for the extraction of the essential oils. And, the Department of Theoretical and Applied
Biology, KNUST for giving us the insectary for this research.

Disclosure of conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.

Statement of ethical approval

Ethical clearance with reference CHRPE/AP/417/19 was obtained from the Committee on Human Research Publication
and Ethics Kwame Nkrumah University of Science and Technology and Komfo Anokye Teaching Hospital on the 3 rd of
July 2019. Also, participant information and consent forms were given to volunteers with the explanation of the purpose
of the research. The twelve volunteers who consented were enrolled into the research for the repellent test.

Supporting information
Table S1 Raw results for anopheles gambiae

Anopheles gambiae mosquito

Concentration Neg. Ctrl Replicates (in sec.) CPT (in sec.) CPT (in min.)
barehand
1 2 3 Average Avg. – Control
(sec.)
Complete Protection Time (CPT) for Neem Seed Oil Cream
5% 10 35 30 25 30 20 0.33
15% 5 145 205 283 211 206 3.43
25% 4 640 672 626 646 642 10.70
30% 6 1503 1524 1560 1529 1523 25.38
Complete Protection Time for Orange Peel Oil Cream
5% 9 22 29 36 29 20 0.33
15% 5 89 95 160 115 110 1.83
25% 3 145 102 285 177 174 2.91
30% 5 647 241 928 605 600 10.01
Complete Protection Time for Orange Peel Oil and Neem Seed Oil Cream
5% 6 86 82 78 82 76 1.27
15% 5 232 228 230 230 225 3.75
25% 3 668 664 660 664 661 11.02
30% 6 1830 1340 1838 1669 1663 27.72
Complete Protection Time for Aqueous Cream (Negative control) and Positive Control (DEET)
13% DEET 6 1290 1260 1230 1260 1254 20.90
Aqueous Cream 5 5 6 6 6 1 0.01
Note: *There is no mean difference between the Negative controls of bare hands (without repellent) and Aqueous Cream Base used to prepare the
repellent.

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Table S2 Raw results for Aedes aegypti mosquitoes

Aedes aegypti mosquito

Concentration Replicates (in seconds) CPT (in min.)
1 2 3 Average
Complete Protection Time (CPT) for Neem Seed Oil Cream
5% 19 20 24 21 0.35
15% 60 49 72 60 1.01
25% 89 93 85 89 1.48
30% 231 201 216 216 3.60
Complete Protection Time for Orange Peel Oil Cream
5% 20 18 24 21 0.34
15% 50 46 54 50 0.83
25% 92 84 100 92 1.53
30% 126 98 112 112 1.87
Complete Protection Time for Orange Peel Oil and Neem Seed Oil Cream
5% 25 20 30 25 0.42
15% 100 114 90 101.3 1.69
25% 118 108 112 112.7 1.88
30% 250 254 246 250.0 4.17
Complete Protection Time for Aqueous Cream Base (Negative Control) and Positive Control (DEET)
13% DEET 300 310 300 303 5.06
Aqueous Cream 3 1 2 2.0 0.03

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