## 2 Materials and Methods
### Insect Repellent Test  
The efficacy of the action of NLC EO as an insect repellent was estimated based on the protocol of the World Health Organization (WHO) with modifications [24]. The study was conducted on 25 adult volunteers living in the study area to avoid any possible exposure to unusual contamination. The volunteers were men and women aged 25-60 years. Exclusion criteria were a history of allergy, any medication, and known sensitivity to mosquito bites. The volunteers were asked to sign an informed consent document after they were informed about the test protocol and the potential risks that might arise during the study. Finally, all volunteers were advised to avoid the application of perfume as well as smoking twelve hours before and during the study. The volunteers were randomly divided into two groups. NLC EO was applied to the 15 volunteers of group A, while the equivalent amount of water was applied to the 10 volunteers of group B (control) to determine the number of mosquitoes that would approach the skin in the absence of the tested sample.  
NLC EO was applied to the entire area of the forearm (600 cm\({}^{2}\) skin area) of each volunteer in an amount equal to 2-3 applications of the product (2-3 presings of the pump), and the time of product application was noted. Volunteers were placed outdoors in random positions in the selected area, at least 20 m apart. The study was carried out in a single (1 day) test during the hours of 21:00-22:00 in July. The temperature and relative humidity during the test were 20-22 \({}^{\circ}\)C and 70-75%, respectively, while the mosquito genera found in the areas were _Aedes_ spp., _Anopheles_ spp., and _Culex_ spp. [2]. The locations of the test were Arnea Halkidiki (5 volunteers in each group), and Patras, Greece (10 volunteers in group A and 5 in group B). The complete protection time (CPT) was calculated as the number of minutes between the time of repellent application (or exposure) and the first mosquito landing. The number of applications that had the same CPT was defined as the frequency of full protection time.  
The resulting data were analyzed using the Kaplan-Meier survivor function, and the median CPT value and corresponding confidence interval were reported. The Kaplan-Meier function is a non-parametric statistic that can be used to calculate the mean complete protection time (CPT) of a repellent as well as its corresponding confidence interval. To achieve this, the \"survival function\" of the CPT must be determined, which is based on the total protection time recorded during the test. The data needed for this purpose are the CPT in minutes (t) for each application (i) in a control consisting of n applications, the number of applications (r) that have the same CPT (j) (where r < n), and the number of applications that do not have the same CPT.  
To generate the Kaplan-Meier equation table, the minimum protection times tj are placed in ascending order. Thus, for each time tj, there are nj applications with a CPT greater than tj. The number of repellent failures recorded in the interval between tj and tj+1 is defined as dj. The Kaplan-Meier (St) statistical analysis defines the probability of sample survival-efficacy (or conversely the failure time) during this interval as nj-dj/nj. The standard error of the equation at time t is defined as s.e [S(t)]. The median complete protection time (median CPT) t(50) is defined as the time above which 50% of CPTs are recorded. The estimated mean CPT is the smallest CPT for which S(t) \(\leq\) 0.5. The 95% confidence interval of this estimate is given by t(50) \(\pm\) 1.96 s.e.{t(50)}.