DEAS 1120 -1: 2022  
ICS 65.100  
 
 
© EAC 2022                                                                                                                              First Edition 2022   
 DRAFT EAST AFRICAN STANDARD  
Mosquito repellent  — Performance test guideline s — Part 1: Skin applied  
 
EAST AFRICAN COMMUNITY  DEAS 1120 -1: 2022  
ii © EAC  2022  – All rights reserved  
 Copyright notice  
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Violators may be prosecuted . DEAS 1120 -1: 2022  
© EAC 2022  – All rights reserved  iii 
 Contents  Page 
Foreword  ................................ ................................ ................................ ................................ ............................  iv 
1 Scope  ................................ ................................ ................................ ................................ ......................  1 
2 Normati ve references  ................................ ................................ ................................ ............................  1 
3 Terms and definitions  ................................ ................................ ................................ ...........................  1 
5 Laboratory studies  ................................ ................................ ................................ ................................  2 
5.1 General  ................................ ................................ ................................ ................................ ...................  2 
4.2 Estimation of effective dose (technical material)  ................................ ................................ ...............  3 
4.3 Estimation of complete protection time (technical mat erial and formulated product)  ..................  4 
5 Field trials  ................................ ................................ ................................ ................................ ...............  5 
5.1 General  ................................ ................................ ................................ ................................ ...................  5 
5.2 Efficacy and persistence of technical material  ................................ ................................ ..................  5 
5.3 Efficacy and persistence of formulated products  ................................ ................................ ..............  8 
Annex  A (Informative)  Guidelines for development of the informed consent form  ................................ ... 9 
A.1 For: (name the group of individuals for whom this consent is written)  ................................ ..........  9 
A.2 Part I: Information sheet  ................................ ................................ ................................ .......................  9 
A.2.1  Introduction  ................................ ................................ ................................ ................................ ............  9 
A.2.2  Propose of the research  ................................ ................................ ................................ .......................  9 
A.2.3  Type of the research  ................................ ................................ ................................ .............................  9 
A.2.4  Participant selection  ................................ ................................ ................................ .............................  9 
A.2.5  Voluntary participation  ................................ ................................ ................................ .........................  9 
A.2.6  Information on the repellent (name of the repellent)  ................................ ................................ .........  9 
A.2.7  Participant protection against malaria or other vector -borne diseases  ................................ ........  10 
A.2.8  Description of the process, procedures and protocol  ................................ ................................ .... 10 
A.2.9  Duration  ................................ ................................ ................................ ................................ ................  10 
A.2.10  Side -effects  ................................ ................................ ................................ ................................ ..........  10 
A.2.11  Risks  ................................ ................................ ................................ ................................ .....................  10 
A.2.12  Discomforts  ................................ ................................ ................................ ................................ ..........  10 
A.2.13  Benefits  ................................ ................................ ................................ ................................ ................  10 
A.2.14  Incentives  ................................ ................................ ................................ ................................ .............  10 
A.2.15  Confidentiality  ................................ ................................ ................................ ................................ ...... 10 
A.2.16  Sharing the results  ................................ ................................ ................................ ..............................  10 
A.2.17  Right to refuse or withdraw  ................................ ................................ ................................ ................  11 
A.2.18  Whom to contact  ................................ ................................ ................................ ................................ . 11 
A.3 Part II: Certificate of consent  ................................ ................................ ................................ .............  11 
Annex  B (informative)  Measurement of the surface area of the skin of limbs  ................................ ..........  12 
Annex C (Informative)  Estimation of median and confidence interval of complete protection time 
using the Kaplan -Meier survivor function  ................................ ................................ ........................  13 
Annex D (normat ive)  Examples of calculation  ................................ ................................ .............................  15 
D.1 Example A  ................................ ................................ ................................ ................................ ............  15 
D.2 Example B  ................................ ................................ ................................ ................................ ............  15 
D.3 Example C  ................................ ................................ ................................ ................................ ............  16 
Bibliography  ................................ ................................ ................................ ................................ ......................  17 
 DEAS 1120 -1: 2022  
iv © EAC  2022  – All rights reserved  
 Foreword  
Development of the East African Standards has been necessitated by the need for harmonizing requirements 
governing  quality of products and services in the East African Community. It is envisaged that through 
harmonized standardization, trade barriers that are encountered when goods and services are exchanged 
within the Community will be removed.  
The Community has established an East African Standards Committee  (EASC)  mandated to develop and 
issue East African Standards  (EAS) . The Committee is composed of representatives of the National 
Standards Bodies in Partner States, together with the representatives from the public and private sector 
organizations  in the community .  
East African Standards are developed through  Technical Committees that are representative of key 
stakeholders including government, academia, consumer groups, private sector and other interested partie s. 
Draft East African Standards are circulated to stakeholders through the National Standards Bodies in the 
Partner States. The comments received are discussed and incorporated before finalization of standards, in 
accordance with the Principles and procedu res for develop ment of East African Standards.  
East African Standards are subject to review, to keep pace with technological advances. Users of the East 
African Standards are therefore expected to ensure that they always have the latest versions of the sta ndards 
they are implementing .  
The committee responsible for this document is Technical Committee EASC/TC 078, Healthcare and medical 
devices.  
Attention is drawn to the possibility that some of the elements of this document may be subject of patent 
rights.  EAC shall not be held responsible for identifying any or all such patent rights.  
DEAS 1120 consists of the following parts, under the general title Mosquito repellent — Performance tests 
guideline : 
Part 1 : Skin applied  
Part 2: Spatial  DEAS 1120 -1: 2022  
© EAC 2022  – All rights reserved  v 
 Introduction  
The purpose of these guidelines is to provide specific and standardized procedures and criteria for efficacy 
testing and evaluation of mosquito repellents for human skin. Their aim is to harmonize the  testing procedures 
carried out in different laboratories and institutions in order to generate comparable data for registering and 
labelling such products by the national regulatory authorities. However, the requirements for registration of 
pesticides, in cluding repellents, are determined by the national regulatory authorities.  
The guidelines provide guidance and procedures on laboratory studies, field trials and evaluation of technical 
material used in mosquito repellent products and on the methods used t o determine their application rate(s) 
and effectiveness. Guidance is also provided on the single -dose evaluation of formulated repellent products. 
With some modification, the guidelines can be used to determine the repellency of candidate compounds for 
other flying insects that blood -feed on humans.  
Detailed treatment and analysis of repellent safety and toxicity data are beyond the scope of these guidelines, 
and it is assumed that preliminary human safety assessments have been undertaken before the materia l(s) 
are applied to human skin. Any side -effects and/or undesirable characteristics experienced in association with 
the application and use of repellents in laboratory studies and field trials should be recorded and reported. The 
protocol must include prov ision for medical care and the reporting of adverse events.  
Products submitted for laboratory studies and/or field trials should be accompanied by the Material Safety 
Data Sheet, the labelling recommendation and the manufacturer’s certification that the pr oduct is within the 
company's manufacturing specifications for that product. Independent physical and chemical assessment may 
be required before initiating the efficacy studies.  
Biological tests are subject to the variation that accompanies living organism s. Studies should therefore be 
conducted under the close supervision of personnel familiar with biological testing of repellents and with 
sound scientific and experimental procedures; the principles of good laboratory practice or other suitable 
quality sch emes such as the International Organization for Standardization should be applied.  
 
 DRAFT EAST AFRICAN STANDARD  DEAS 1120 -1:2022  
 
© EAC 2022  – All rights reserved  1 
 Mosquito repellent  — Performance test guideline s — Part 1: Skin applied  
1 Scope   
This D raft East African  standard provides guidelines for the design and execution of stud ies to evaluate the 
performance of mosquito repellents formulated and prepared for application directly to human skin.  
These guidelines apply to products in any formulation intended to be applied directly to human skin .  
2 Normative references   
There are n o normative references in this document.  
3 Terms and definitions   
For the purposes of this standard, the following terms and definitions apply.  
ISO and IEC maintain terminological databases for use in standardization at the following addresses:  
— ISO Onl ine browsing platform: available at http://www.iso.org/obp  
3.1 
landing  
act of flying on to or approaching and settling on to a human s kin, without probing or biting  
3.2 
probe  
act of penetrating human skin by mosquito mouthp arts without ingestion of blood  
3.3 
bite 
act of penetrating human skin by mosquito mouthparts with ingestion of blood, typically associated with 
abdominal swelling and colour change  
3.4 
crossing  
act of passage by mosquitoes from an area of untreated skin to an area of treated skin. A cr ossing may be 
quantified either or both by the distance the mosquitoes moves onto treated skin or by how long the 
mosquit oes remains on the treated skin  
3.5 
unconfirmed event  
landing, probe, bite, or crossing not followed by another similar event within 3 0 min  
3.8 
confirming event  
one landing, probe, bite, or crossing followed by another similar event within 30 minutes. The first event is 
confirmed by the second; the secon d event in the confirming event  DEAS 1120 -1: 2022  
2 © EAC  2022  – All rights reserved  
 3.9 
human subject  
living individual about whom an inv estigator conducting research obtains either data through intervention or 
interaction with the individual or identifiable private information  
3.10 
questing  
behaviour of mos quitoes actively seeking a host  
3.11 
repellent  
product that deters the host -seeking behaviour of mosquito, from approaching or settling on treated human 
skin 
3.12 
Complete Protection Time (CPT)  
time from application of repellent until efficacy failure as it is defined in each study – for example, the time 
from application until the first efficacy failure event confirmed within 30 min utes by a second similar event  
3.13 
dose determination  
testing procedure used to estimate a “typical human dose” of a topical repellent  
4 Laboratory studies  
4.1 General  
4.1.1 The objective of laboratory studies  is to estimate the effective dose of a repellent and the complete 
protection time provided by a repellent after application on the skin.  
4.1.2 The specific aims of these tests are to:  
a) estimate dose –response lines and effective doses (EDs) of a repellent  corresponding to 50% (ED 50) 
and 99.9 % (ED 99.9) protection from mosquito landing and/or probing (NOTE 1); and  
b) estimate the complete protection time (CPT) of a repellent.  
NOTE 1  Landing and/or probing behavior signifies the end -point of the repellent eff icacy test. However, landing is not 
always associated with probing, and separate recordings of each behavior may be needed. A repellent may provide 
efficacy by a reduction in biting activity but not in landing. Alternatively, it may provide efficacy in ter ms of landing, but 
those mosquitoes that land may all bite. Both scenarios may be important in determining the efficacy of a repellent.  
4.1.3 DEET (N,N -diethyl -3-methylbenzamide) is the active ingredient of most commercially -available 
repellents and is re commended as the positive control (usually 20 % in ethanol) against which the 
effectiveness of alternative mosquito repellents is judged.  
4.1.4 Testing of repellents on human subjects is the method of choice as it utilizes the repellent end -user in 
the te sting process and yields results that are relevant to the actual conditions of use. Use of laboratory 
animals or artificial membranes may inadequately simulate the situation in which repellents for use on human 
skin are intended to perform. Tests are carri ed out on adult human volunteers who may be selected from 
among candidates exhibiting mild or no sensitivity to mosquito bites. Equal numbers of male and female test 
volunteers are preferred.  
4.1.5 In preparation for the laboratory studies, the test area of the volunteers’ skin should be washed with 
unscented soap and rinsed with water, then rinsed with a solution of 70% ethanol or isopropyl alcohol in water 
and dried with a towel. Given the possibility that various factors may alter a person's attractiven ess to 
mosquitoes, and that this may in turn affect the outcome of repellency assays, test volunteers should avoid 
the use of fragrance and repellent products for 12 hours before and during testing. Volunteers should                    DEAS  1120 -1: 2022  
© EAC 2022  – All rights reserved  3 
 preferably not be tobacco users, or at least to have refrained from tobacco use for 12 hours prior to and during 
testing.  
4.1.6 Standardized mosquito rearing and laboratory testing conditions are essential to ensure the reliability 
and reproducibility of data. Mosquitoes should be reared, main tained and tested (in a sep arate space or room) 
at 27 ± 2 °C temperature, ≥ 80 ± 10 % relative humidity, and a 12:12 (light:dark) photoperiod. Temperate 
mosquito species may require modifications to rearing conditions. Stock populations of adult mosquitoes  
should have access to sugar solution but not have been blood -fed. Observations of repellency should be 
made using female mosquitoes starved for the preceding 12 h and, where practical, during times in the diel 
period that correspond with biting activity b y that species.  
4.1.7  Mosquito repellency tests should be conducted with three or more of the more anthropophilic 
Anopheles (preferably, An. gambiaes .l.) species. The test species, strain and age should be reported. 
Mosquitoes should be contained during t esting using a cage (suggested metal frame for ease of 
decontamination, size: 35 –40 cm per side) with a solid bottom and top, screen or netting on the back, a clear 
acrylic sheet (for viewing) on the right and left sides, and a fabric sleeve for access on  the front. Female 
mosquitoes should be collected from a stock population cage in which both sexes have been maintained to 
allow mating to occur. They should be host -seeking, of uniform age, preferably 5  – 7 days post -emergence 
(use different ages of mosqu itoes when it is more suitable for a particular species and justify such use in the 
study report). Active host -seeking females should be selected to ensure a good response from the test 
mosquitoes using an aspirator or an appropriate airflow apparatus.  
4.2 Estimation of effe ctive dose (technical material)  
4.2.1  Serial dilutions of repellent are made with ethanol or another suitable diluent and tested to identify an 
effective dose range. Dosages giving responses between 10 % and 90 % are used for this analy sis, preferably 
2 - 3dosages that give <50% repellent response and 2 – 3 dosages that give >50%.  
4.2.2  Each volunteer uses incremental doses on the test forearm so that at least five successive 
applications of increasing dose are used by each volunteer. A  single test comprises continuous use of the 
same mosquitoes by the same volunteer and is completed in one day. Replicate tests repeat this process 
using different batches of mosquitoes over several days. It is recommended that a minimum of three 
replicate s be conducted per volunteer, with the number of volunteers sufficient to allow for statistical analysis.  
4.2.3  One mL of ethanol or the same diluent used in the preparation of the test repellent is applied evenly 
using a pipette to ≈600 cm2 of the forearm skin between the wrist and elbow (Annex B) and allowed to dry 
(approximately 1 min for ethanol). Before inserti on of the arm into the cage containing 50 –100 female 
mosquitoes, the hands are protected by gloves made of material through which the mosquitoes cannot bite. 
The first step is to insert the forearm applied with diluent into the cage and to count the number  of mosquitoes 
that land on and/or commence to probe the skin during a 30 -second period. During testing, the volunteer 
should avoid movement of the arm. For the test to proceed, the biting rate must be ≥10 landings and/or 
probings in the 30 -second period. The control forearm is carefully withdrawn and this arm is then treated with 
the lowest dose of repellent in 1 mL diluent and allowed to dry. The treated arm is place d in the cage for 
another 30 -second period and observed for mosquito landings and/or probings. This procedure is repeated for 
each additional incremental repellent dose. Successive tests should be carried out one after the other without 
delay and the repel lent dose at each test calculated as the sum of the doses applied to arrive at the 
cumulative dose for each test (Table 1) . DEAS 1120 -1: 2022  
4 © EAC  2022  – All rights reserved  
 Table 1 — Example of successive doses applied to arrive at a cumulative dose for a sample 
experiment  
Application sequence  Repellen t solution 
concentration to be applied in 1 
mL (mg/mL)  Cumulative amount of repellent 
(mg/600 cm2area)  
Left-arm control  Pre-treated with alcohol onlya  - 
Left-arm dose 1  1 1 
Left-arm dose 1  1 2 
Left-arm dose 1  2 4 
Left-arm dose 1  4 8 
Left-arm dose 1  8 16 
Right -arm control  Pre-treated with alcohol onlya - 
a Alcohol or the same diluent as that used in the preparation of the repellent solution  
 
4.2.4  If, at any time, the landing and/or probing rate is too high to accurately count the number o f 
mosquitoes landing and/or commencing to probe the skin, the mean landing and/or probing rate for the test 
should be calculated from a series of three readings, each five seconds long, and the sum multiplied by two to 
estimate the landings and/or probings  that would occur in a 30 -second period. Testing should not proceed 
when the mosquito landing and/or probing rate on the exposed forearm is <10 females in 30 seconds.  
4.2.5  This procedure should be used consistently throughout the experiment. The trained volunteer will 
record the number of landings and/or probings. At the conclusion of the dose – response experiment, 1 mL 
alcohol/diluent is applied on the other forearm and allowed to dry. This forearm is inserted in the cage for 
30seconds to verify that th e number of landings and/or probings is approximately ≥10 per 30 seconds, as was 
observed at the beginning of the experiment. If the rate is <10 females in 30 sec, the results of this experiment 
should be discarded.  
4.2.6  Protection (p) is expressed as a proportion of the number of mosquitoes landings and/or  probings on 
the treated arm (T) in relation to the number of landings and/or probings on the control arm (C) of the same 
individual:  
𝑝 = 1 − (𝑇/𝐶) = (𝐶 − 𝑇)/𝐶 
where,  
C is the average of the landings/probings on the two untreated arms (the diluent -applied test arm 
before repellent treatment and the other arm at the end of the experiment).  
4.2.7 Data are analysed using probit -plane regression analysis from which the ED 50 and ED 99.9 and their 
confidence limits can be estimated.  
4.3 Estimation of com plete protection time (technical material and formulated product)  
4.3.1  The complete protection time, or CPT, of a repellent can be determined in one of two ways. 
Preferably, the ED 99.9 dose should be estimated using the procedures outlined in section 5.2;  1 mL of the 
repellent is then tested at the ED 99.9 level against 1 mL of the standard 20% ethanolic DEET. Alternatively, 1 
mL of the 20 % ethanolic DEET solution can be compared with the same amount (weight/weight) of the 
candidate repellent on the other arm. In both cases, treatments are applied to  ≈600 cm2area (Annex B) of the 
forearm skin between the wrist and elbow.  
4.3.2  Two mosquito cages (size: 35 cm – 40 cm per side) each containing 200 –250 non -blood -fed females 
are normally used. One cage is designated for testing the candidate repellent an d the other for the positive                    DEAS  1120 -1: 2022  
© EAC 2022  – All rights reserved  5 
 control (ethanolic DEET). During testing, the hands are protected by gloves made of material through which 
the mosquitoes cannot bite while the volunteer avoids movement of the arm.  
4.3.3  Initially, the readiness of mosquitoes  to land and/or probe must be assessed by inserting an untreated 
(alcohol - or diluents treated) arm into a cage for 30 sec or until 10 landings/probings are counted. The 
procedure is repeated with the other arm in the second cage. If this level of landing and/or probing is not 
achieved in either cage, the experiment should be discarded.  
4.3.4  Before testing commences, 1 mL of the candidate repellent prepared in alcohol/diluent solution is 
applied to one arm and 1 mL of the DEET standard solution is applied  to the other arm. After 30 minutes, the 
repellent -treated arm is inserted into the appropriate cage and exposed for 3 minutes to determine landing  
and/or probing activity. Next, the DEET -applied arm is exposed to determine landing and/or probing activity.  
This procedure is repeated at 30 or 60 -minute intervals and should be used consistently throughout the 
experiment. The occurrence of one landing and/or probing in a 3 -minute test interval concludes the test for 
that repellent dose. Complete protection tim e is calculated as the number of minutes elapsed between the 
time of repellent application and the first mosquito landing and/or probing. Most repellent studies of technical 
material are completed in 8 hours or less.  
4.3.5  The number of volunteers include d in the test should be sufficient to allow for statistical analysis. The 
median CPT and confidence interval can be estimated from the Kaplan –Meier Survival Function (Annex C).  
5 Field trials  
5.1 General  
5.1.1  The objective of field trials is to extend th e results of laboratory testing to estimate the optimum 
application dose, persistence and efficacy of a repellent material, in terms of repellency and protection time, 
against one or more mosquito vectors and/or pest species in different ecological and/or geographical settings. 
A minimum of two field tests are recommended, one each in different ecological and or geographical settings 
suitable for the target mosquito species where the human exposure occurs.  
5.1.2  Assessment is made by human volunteers colle cting mosquitoes landing and/or probing on one or 
more bare limbs (knee to ankle; elbow to wrist), depending upon the biting behavior of the mosquito species. 
The volunteers should be:  
a) from the same settings where the test is conducted, such that they are  not exposed to unusual risk of 
infection;  
b) if appropriate and applicable, protected by chemoprophylaxis and/or vaccination; and/or  
c) where possible, at sites where there is no disease transmission but high abundance of target mosquito 
species.  
5.1.3  The d etermination of proportional end -points of repellent efficacy and persistence is recommended. 
These allow concurrent estimation of EDs, effective period of time for a range of doses, repellent half -life and 
the CPT.  
5.2 Efficacy and persistence of technic al material  
5.2.1  Collections are performed by volunteers skilled in the use of aspirators (blow -type or mechanical) or 
collecting tubes to catch all mosquitoes landing on an exposed limb before the mosquitoes commence to 
probe. Aspirated mosquitoes are tr ansferred into holding cups that are labelled and changed every half -hour 
or, if collected in tubes, transferred to labelled holding cups that are changed every half -hour. Inclusion of 
equal numbers of male and female volunteers in the test is recommended.   DEAS 1120 -1: 2022  
6 © EAC  2022  – All rights reserved  
 5.2.2  Human landing catches are performed during the period of biting activity of the target mosquito 
species and indoors and/or outdoors depending upon mosquito behaviour. Where targeted species have short 
duration of main biting activity, tests with re pellent material should begin such that the expected endpoints as 
described above occur within the biting period of such species.  
5.2.3  Volunteers should avoid using fragrance products 12 hours prior to and during the test. Volunteers 
should preferably no t be tobacco users or at least have refrained from tobacco use for 12 hours prior to and 
during testing.  
5.2.4  The surface area of the skin of the limbs of each volunteer is measured (Annex B) and the volume of  
repellent material needed to treat the area at a specific dose is determined. To do this, the technical material 
is diluted in ethanol (or any other suitable diluent specified by the manufacturer) in sufficient quantities to 
remain effective for the duration of the study. The appropriate dose of tes t repellent should be applied  
uniformly to a limb of the volunteer using a pipette and allowed to dry before the start of the first collection 
period. A blind test, in which volunteers are not aware of the nature of the treatment, is preferred.  
5.2.5  A negative control volunteer should be used in the test, to which the corresponding volume of alcohol 
or a suitable diluent is applied to the skin in the same manner as the repellent treatment. A positive control 
(20% ethanolic DEET) may also be used.  
5.2.6  A preliminary assessment of the suitability of collection sites by performing human landing catches 
prior to the test is recommended to minimize variation in the number of mosquitoes landing and/or probing 
among sites. This will result in the selection of s ites that yield the most homogeneous densities of mosquitoes 
and with suitable biting rates.  
5.2.7  Within each collection site, volunteers should be placed singly and separated from each other by >20 
meters. A range of four doses is recommended to be test ed for each repellent and may be approximated from 
laboratory studies (see clause 4.2). The doses are selected to produce a comprehensive range of protection 
rates at the end of the exposure period. Normally, the effective dose that provided 99.9% protecti on in 
laboratory studies is used as a guide to establish the dosages for field trials.  
5.2.8  A completely randomized design is used. The number of test volunteers and the number of collection 
sites are equal to the number of doses to be tested plus the un treated control. If a positive control (20% DEET 
in ethanol) is used, an extra collection site and volunteer will be required. A single (1 -day) test comprises 
rotation of each volunteer at random among all collection sites at 1 -hour intervals (Table2). Eac h treatment 
combination of dose, negative and/or positive control and volunteer should be tested for a suitable period on 
two or more dates. Replicate tests should each be similarly randomized. The mosquito collection at each 
collection site for the repell ent treated volunteers and the negative and/or positive controls should be chosen 
to be suitable for the target species. For example, under high biting pressure, this could be one minute each 
beginning at minute 15, 30, and 45 into the observation period, with the first measurement in the initial hour of 
observation period (0 –1 h) starting at minute 15 (i.e. minute15 –16) after repellent application.  
5.2.9  Mosquitoes landing and/or probing on the skin should be collected with an aspirator for accurate 
count ing and species identification. Species composition should be reported. Tests should not be conducted 
in windy conditions, but records should be made throughout each test of wind speed, temperature, relative 
humidity and precipitation amount(s) for possibl e later consideration and/or analysis.  
5.2.10  At the conclusion of the experiment, the number of mosquitoes collected within each observation 
period (i.e. at 15  min – 16 min, 30 min – 31 min and 45 min – 46 min), at each dose, is averaged for each 
replica te.  
5.2.11  Protection (efficacy) afforded by the repellent (p) at each test period and for each dose is calculated 
as:  
𝑝 = 1 − (𝑇 /𝐶) = (𝐶 − 𝑇) / 𝐶 
where,  
T is the number of mosquitoes collected from the treatment volunteer; and                     DEAS  1120 -1: 2022  
© EAC 2022  – All rights reserved  7 
 C is the number o f mosquitoes collected from the negative (or positive) control volunteer.  
5.2.12  The persistence of the repellent efficacy is assessed by the median effective dose (ED 50) and the 
repellent half -life. These end -points are estimated by fitting the data to a  probit –plane regression model, which 
relates the protection afforded by the repellent to the test period and the natural logarithm (ln) of the dose of 
repellent applied:  
ln[𝑝/(1 − 𝑝)] = 𝑎 + 𝑏1(𝐷0) + 𝑏2𝑡1  
where,  
p is the protection afforded by the repellent,  
D0 is the dose calculated as the natural logarithm of the dose applied (ln [dose]),  
T is the time post -treatment in hours, and  
a, b 1 and b 2 are coefficients est imated using the probit -plane regression model.  
5.2.13  The ED 50 corresponds to the application dose providing 50% protection (p = 0.5) at the time of 
repellent application (t = 0). Taking the value of 0.5 for p and 0 for t in equation iii (above), and sol ving for D0, 
provides an estimate of the ED 50 (see Example A, Annex D).  
5.2.14  Other percentiles can be estimated by setting t = 0 and calculating p for a specific application dose. 
For example, to estimate the ED 90 using the coefficients provided in Exam ple A (above), put t = 0 and p = 0.90 
in equation iii and calculate (see Example B, Annex D).  
5.2.15 The effective time (ET 50) and other percentiles, such as the ET99 (see Example C, Annex D), for a specified application dose (D 0) can also be estimated fr om equation mentioned in 5.2.12. The repellent half -life can be estimated using the following e quation:  
ln[1/2]×(𝑏1/𝑏2) 
5.2.16  The complete protection time for a given dose is estimated from the time elapsed up to the first 
mosquito landing and/or probing in each replicate. The median CPT and its confidence interval can be 
estimated using the Kaplan –Meier survivor function procedure (see Annex C).  
Table 2 – Example of the allocation of four doses (D) of the candidate repellent, a negative control 
(CN) and a positive control (CP) to six volunteers (V) among six collection sites in a completely 
randomized design  
Begin ning of test 
period (hours)  Observation period 
(hours)a  Collection sites  
1  2  3  4  5  6  
0  0-1  V5CN  V4D4  V6D2  V1D3  V2CP  V3D1  
1  1-2  V4D4  V6D3  V5D1  V2D2  V1CN  V3CP  
2  2-3  V2D1  V4CN  V1D4  V3D2  V6CP  V5D3  
3  3-4  V1D2  V5CN  V2D3   V6D1  V4D4  V3CP  
4  4-5  V2D3  V5CN  V1D1  V6D2  V3D4  V4CP  
5  5-6  V4CP  V3D2  V5D4  V2D3  V1CN  V6D1  
6  6-7  V4D1  V3D3  V5D2  V2CN  V6CP  V1D4  
7  7-8  V1CN  V2D1  V4D2  V6D3  V5D4  C3CP  
8  8-9  V5D4  V4D1  V3D2  V1D3  V6CN  V2CP  
9  9-10  V3D2  V6CN  V5CP  V1D3  V2D4  V4D1  
a in this example, within each observation period, three measurements (at 15, 30 and 45 minutes) are taken at each collection si te  
 DEAS 1120 -1: 2022  
8 © EAC  2022  – All rights reserved  
 5.3 Efficacy and persistence of formulated products  
5.3.1  The single -dose evaluation o f formulated repellent products should be performed under field 
conditions at a minimum of two ecologically and geographically distinct locations. A preliminary assessment of 
collection sites by performing human landing catches prior to the test is recomme nded to minimize variation in 
the number of mosquitoes collected among such sites. This will include a selection of sites that yield the most 
homogeneous densities of mosquitoes and with suitable biting rates. The appropriate number of volunteers  
trained i n mosquito aspiration will be required for the study, including individuals serving as positive controls 
and individuals serving as untreated test subjects (for ongoing monitoring of mosquito biting pressure). A 
minimum number of volunteers necessary to de monstrate statistical significance should only be included in 
the testing for ethical reasons.  
5.3.2  In a single (1 -day) test, at location 1, the study investigator should apply the formulated product to an 
exposed limb of each volunteer at the rate of 1 mL of formulated product (or 1 mL of 20% DEET  in ethanol for 
the positive control) on ≈600 cm2 area. The limb is allowed to dry and the time recorded of application to each 
volunteer. Volunteers are individually assigned at random to collection sites arran ged ≥20 meters apart. The 
exposure time to mosquito populations at each collection site for the repellent treated volunteers and the 
positive control should be chosen to be suitable for the target species. For example, under high biting 
pressure, this coul d be 1 -minute each at 15, 30 and 45 minutes into the observation period, with the first 
measurement in the initial observation period (0 – 1 hours) starting at minute 15 (i.e. minute 15 – 16) after 
repellent application. A suitable number of additional vol unteers (untreated) should monitor biting pressure 
throughout the study. All mosquitoes landing on the exposed limb of each volunteer are collected by aspiration 
and transferred to a holding container for later counting and species identification. A single  (1-day) test 
comprises rotation of each test volunteer, the positive control and an untreated control at random among all 
collection sites at 1 -hour intervals.  
5.3.3  Human landing catches should be performed during the period of biting activity of the ta rget mosquito 
species. This may require treatment with repellent well in advance to ensure the expected end -point occurs 
within the biting period of the target species. The study should be otherwise conducted until the time to first 
landing and/or probing by a mosquito on a volunteer. Complete protection time is calculated as the number of 
minutes elapsed between the time of repellent application and the first mosquito landing and/or probing. The 
resulting data can be analyzed using the Kaplan –Meier survivo r function (Annex C) and the median CPT and 
corresponding standard errors reported.  
5.3.4  Repellency  (p), expressed as percent in field trials for each hour of the test can be determined as:  
=
  
where,  
T is the average number of mosquitoes collected from  the treatment volunteer(s) in a given hour of a 
test, and  
C is the average number of mosquitoes collected from the untreated or positive control volunteer in the 
same hour of the test.  
5.3.5  The calculation is repeated for each hour to result in a profi le of hourly change in % repellency over  
the 12 -hour test period.                     DEAS  1120 -1: 2022  
© EAC 2022  – All rights reserved  9 
 Annex  A 
(Informative)  
 
Guidelines for development of the informed consent form  
A.1 For: (name the group of individuals for whom this consent is written)  
Name of principal investigator: ………… …………………………………………………………………  
Name of organization: ……………………………………………………………………………………..  
Name of sponsor: …………………………………………………………………………………………...  
Name of proposal: ………………………………………………………………………………………….  
A.2 Part I: Information sheet  
This sheet is a suggestion or example that can be modified according to the national rules and guidelines.  
A.2.1  Introduction  
State briefly who you are and explain to the participants that you are inviting them to take part in research that 
you are doing.  
A.2.2  Propose of the researc h 
State briefly in lay terms why you are doing the research.  
A.2.3  Type of the research  
State briefly the type of intervention that will be undertaken.  
A.2.4  Participant selection  
State why this participant has been chosen for this research (adult males  and females will preferably be 
recruited among the inhabitants of the study site, after having announced in the district, through oral 
advertisements, that the project is looking for volunteers. The selection will ensure that equal opportunities are 
provi ded to everybody. Report how many volunteers are expected to participate in the study. Criteria for 
inclusion and exclusion of volunteers needs to be reported.  
A.2.5  Voluntary participation  
Indicate clearly that volunteers can choose to participate or not . State that they will still receive all the services 
they usually do whether they choose to participate or not.  
A.2.6  Information on the repellent (name of the repellent)  
Explain to the participant why you are testing a repellent product. Provide as much  information as is 
appropriate and understandable about the repellent product, such as its manufacturer or location of 
manufacture, and the reason for its development. Explain the known experience with this repellent product. 
Explain comprehensively, if an y, all the known side -effects or toxicity of this repellent product.  DEAS 1120 -1: 2022  
10 © EAC  2022  – All rights reserved  
 A.2.7  Participant protection against malaria or other vector -borne diseases  
Explain to each participant the safeguards that will be provided (e.g. chemoprophylaxis, where relevant) to 
protect them from malaria or other vector -borne diseases and, if necessary, their treatment.  
A.2.8  Description of the process, procedures and protocol  
Describe or explain to the participant the exact procedures that will be followed on a step -by-step basis a nd 
the tests that will be done.  
A.2.9  Duration  
Include a statement about the time commitments of the research for the participant, including the duration of 
the research and volunteer follow -up.  
A.2.10  Side -effects  
Potential participants should be told i f there are any known or anticipated side -effects and what will happen in 
the event of a side effect or an unexpected event. State who will provide medical care should a volunteer be 
injured in the study, and who will provide funds for treatment .  
A.2.11  Risks 
Explain and describe any possible or anticipated risks. Describe the level of care that will be available in the 
event that harm does occur, who will provide it and who will pay for it.  
A.2.12  Discomforts  
Explain and describe the type and source of a ny anticipated discomforts that are in addition to the side -effects 
and risks discussed above.  
A.2.13  Benefits  
Mention only those activities that will be actual benefits (as an additional protection from mosquito bites) and 
not those to which they are ent itled regardless of participation.  
A.2.14  Incentives  
State clearly what you will provide the participants with as a result of their participation. WHO does not 
encourage incentives. However, it recommends that reimbursements for expenses incurred as a res ult of 
participation in the research be provided.  
A.2.15 Confidentiality  
Explain how the research team will maintain the confidentiality of data, especially with respect to the 
information about the participant, which would otherwise be known only to the Principal Investigator/physician 
but would now be available to the entire research team. State any other groups that will have access to 
individually identifiable information collected in this study.  
A.2.16  Sharing the results  
Where relevant, your plan fo r sharing the findings with the participants should be provided.                     DEAS  1120 -1: 2022  
© EAC 2022  – All rights reserved  11 
 A.2.17  Right to refuse or withdraw  
This is a reconfirmation that participation is voluntary and includes the right to withdraw.  
A.2.18  Whom to contact  
Provide the name and contact informatio n of someone who is involved, informed and accessible (a local 
person who can actually be contacted). State also that the proposal has been approved, and how.  
This proposal has been reviewed and approved by [name of the local ethical committee], whose tas k is to 
make sure that research participants are protected from harm. If you wish to find out about more the Local 
Ethical Committee, please contact [name, address and telephone number] .  
A.3 Part II: Certificate of consent  
This section can be written in t he first person. It should include a few brief statements about the research and 
be followed by a statement similar to the one in bold below. If the participant is illiterate but gives oral consent, 
a witness must sign. A researcher or the person checking the informed consent must sign each consent form.  
I have read the foregoing information, or it has been read to me. I have had the opportunity to ask questions 
about it, and any questions that I have asked have been answered to my satisfaction. I consent voluntarily to 
participate as a participant in this research and understand that I have the right to withdraw from the research 
at any time without in any way affecting my medical care.  
Print name of participant: …………………………………………………………………………………………  
Signat ure of participant: …………………………………………………………………………………………  
Date: …………/………………/………………..  
If illiterate  
A literate witness must sign (if possible, this person should be selected by the participant and should have no 
connection to the research team).  
I have wit nessed the accurate reading of the consent form to the potential participant, and the individual has 
had the opportunity to ask questions. I confirm that the individual has given consent freely.  
Print name of participant: …………………………………………………………………………………… and  
Thumb print of participant  
Signature of witness: ………………………………………………………………………………………………  
Date: …………/………………/………………..  
I have accurately read or witnessed the accurate reading of the consent form to the potential participant, and 
the individual has had t he opportunity to ask questions. I confirm that the individual has given consent freely.  
Print name of researcher: …………………………………………………………………………………………  
Signature of researcher: …………………………………………………………………………………………  
Date: …………/………………/………………..  
A copy of this I nformed Consent Form has been provided to participant………………………………….. 
(initiated by the researcher/assistant ). DEAS 1120 -1: 2022  
12 © EAC  2022  – All rights reserved  
 Annex  B 
(informative)  
 
Measurement of the surface area of the skin of limbs  
B.1 The surface area  of skin used on a limb for repellent testing can be approximated on the basis of the 
surface area of a cylinder. Three dimensions are required for this purpose: the length of the treatment area, 
and the circumference of the limb at the proximal and distal  boundaries of the treatment area.  
B.2 The repellent should be applied evenly over the total surface area around the arm from wrist to elbow. 
The treatment area can be approximated by measuring the circumference (cm) of the wrist, the circumference 
(cm) a t the elbow -cubital fossa when the arm is extended, and the distance (cm) from the elbow to the wrist.  
B.3 The surface area (in cm2) of skin is then calculated by:  
Area = ½ (C w + C e) Dwe  
Where,  
Cw is the circumference of the wrist in cm,  
Ce s the elbo w-cubital fossa circumference in cm, and  
Dwe  is the distance in cm between c e and c w.  
B.4 Example  
A repellent study is conducted using the arm. For the volunteer, the researcher measures the circumference 
of the wrist as 18 cm and an elbow region circum ference of 27 cm, where the treatment area ends. The 
distance from wrist to elbow -cubital fossa is measured as 26 cm. Using the equation above:  
a) Area = ½ (18 cm + 27 cm) x 26 cm  
b) Area = ½ (45 cm) x 26 cm  
c) Area = 22.5 cm x 26 cm  
d) Area = 585 cm2                    DEAS  1120 -1: 2022  
© EAC 2022  – All rights reserved  13 
 Annex C  
(Informative)  
 
Estimation of median and confidence interval of complete protection 
time using the Kaplan -Meier survivor function  
C.1 The Kaplan –Meier function is a non -parametric statistic that can be used to calculate the median 
complete protection time (CPT) of a repellent and its corresponding confidence interval. To do this, the CPT 
"survivor function", which is based on the complete protection times recorded during testing, must be 
determined. The data required for this purpose are:  
a) The CPT in minutes (t) for each replication (i) of a test with n replications (i.e. t i [i = 1, 2, … n]).  
b) The number of replications ® of the test sharing the same CPT (j), where r ≤ n (i.e., tj [j = 1, 2, … r]).  
c) The number of replications of the test not sharing the same CPTs (i.e. [n - r].  
C.2 To construct the table of survivor function estimates, the tj times are arranged in ascending order. For 
each tj, there will be nj observations with a CPT greater than tj. The number of repellent failures (CPTs) 
recorded between the tim e interval tj and tj+1 is denoted by dj. The Kaplan –Meier statistic (S(t)) estimates the 
probability of survival (or, inversely, the time to repellent failure) through this interval as (nj – dj)/ nj,. The 
standard error of the survivor function estimate at  time t is designated as s.e.{S(t)}.  
C.3 The median complete protection time t(50)  is that beyond which 50% of the CPTs are recorded (i.e. 
S{t(50)} =0.5). The estimated median CPT is the smallest observed CPT for which S(t) ≤0.5. The 95% 
confidence interval of this estimate is given by: t(50) ± 1.96 s.e.{t(50)}.  
Example  
In a hypotheti cal 10 -hour test of the efficacy of a repellent applied on human skin, CPTs were recorded in 48 
replications (Table C1). In 11 of the replications, no landings and/or probing were received prior to the end of 
the study period (10 hours) and the CPT for the se 11 replications is unknown.  
Table C1 — Summary of the frequency of the complete protection time (CPT) of a candidate repellent 
applied on human skin.  
CPT 
(min)  120  150  210  390  420  450  480  510  600  
Frequen
cy (dj)  1  3  3  10  7  4  4  2  3  
 
C.4 Using the data from this test, estimates of (nj – dj)/nj and S(t) and their corresponding standard error 
are constructed, as presented in Table C2.  DEAS 1120 -1: 2022  
14 © EAC  2022  – All rights reserved  
 Table C2 —— Estimates of (nj – dj)/nj and their corresponding standards errora 
Time interval 
(tj) in mi n.  nj  dj  (nj – dj)/nj  S(t)  Standards error 
S(t)  
0  48  0  1.0000  1.0000  0.0000  
120  47  1  0.9787  0.9787  0.0210  
150  44  3  0.9318  0.9120  0.0420  
210  41  3  0.9268  0.8453  0.0538  
390  31  10  0.6774  0.5726  0.0798  
420  24  7  0.708 3  0.4056  0.0776  
450  20  4  0.8000  0.3245  0.0719  
480  16  4  0.7500  0.2434  0.0643  
510  14  2  0.8571  0.2086  0.0597  
600  11  3  0.7273  0.1517  0.0516  
a Data processing software for calculation of these variables is available on the Intern et. Search key word: Kaplan Meier survival function online 
calculator. 
 
Median complete protection time is the smallest CPT for which S(t) ≤0.5.  
In this case, at tj = 390, S(t) = 0.5726, and at tj = 420, S(t) = 0.4056.  
Our best estimate of the median CP T in this example is t(50) = 420 min.  
C.5 To calculate the 95% confidence interval we set S{upper(50)} = 390 and S{lower(50)} = 420. The 
probability density function at t(50) is estimated as:  
f {t(50)} = {[ 𝑆(390)− 𝑆(420)] ÷ (420−390 )} = ( 0.5726−0.4056 ) ÷30 = 0.005567   
The standard error of the survivor function at t(50) is 0.0776 and the standard error of the median CPT is 
estimated by:  
s.e. {t(50)} = (1 ÷ 0.005567) 𝑥 0.0776 =13.94   
The 95% confidence interval for the estimated median CPT for the repel lent in this example has boundaries of 
420 ± 1.96 13.94 minutes. On this basis, in 95 out of 100 observations, we would expect the median CPT to 
lie between 393 to 447 minutes.                     DEAS  1120 -1: 2022  
© EAC 2022  – All rights reserved  15 
 Annex D  
(normative)  
 
Examples of calculation  
D.1 Example A  
The following coeffi cients were estimated using the probit –plane regression model based on protection 
against Anophelesgambiae s.l. by DEET ( 4):  
a = 8.160  
b1 = 2.209  
b2 = - 0.532  
The ED 50 is calculated as:  
1) ln [p/(1 – p)] = a + b 1(D0) + b 2t1  
2) ln [0.5/(1 – 0.5)] = 8.160 + 2.209(D 0) + - 0.532 (0)  
3) (ln (1) = 8.160 + 2.209 (D 0) + 0  
4) ln (1) = 0 = 8.160 +2.209 (D0)  
5) 8.160/2.209 = D 0  
6) 3.694 = D 0  
7) ln (ED 50) = D 0  
8) ED 50 = exp (D 0)  
9) ED 50 = 2.7183 (-3.694)  
10) ED 50 = 0.025 mg/cm2  
D.2 Example B  
The ED 90 is calculated as:  
i. ln [p/(1 – p)] = a + b 1(D0) + b 2t1  
ii. ln [0.9/(1 – 0.9)] = 8.160 + 2.209(D 0) + - 0.532 (0)  
iii. ln (9.0) = 8.160 + 2.209 (D 0) + 0  
iv. – 5.963 = 8.160 +2.209 (D 0)  
v. – 5.963/2.209 = D 0 
vi. – 2.699 = D 0  DEAS 1120 -1: 2022  
16 © EAC  2022  – All rights reserved  
 vii. ln (ED 90) = D 0  
viii. ED 90 = exp (D 0)  
ix. ED 90 = 2.7183 (-2.699)  
x. ED 90 = 0.067 mg/cm 2  
D.3 Example C  
A volunteer applies a dose of 0.50 mg/cm 2to the arm (i.e. D0 = ln [0.50] mg/cm 2). Determine the ET 99 (using 
the same coefficients as provided in Example A):  
a) ln [p/(1 – p)] = a + b 1(D0) + b 2t  
b) ln [0.99/(1 – 0.99)] =a + b 1(D0) + b 2t  
c) ln (99) - 8.160 – (2.209 x – 0.693) = ( - 0.532) t  
d) 4.595 - 8.160 – (-1.531) = - 0.532 t  
e) – 2.034 = - 0.532 t  
f) t = ( - 2.034/ - 0.532) = 3.6 hours after application  
                    DEAS  1120 -1: 2022  
© EAC 2022  – All rights reserved  17 
 Bibliography  
[1] RS 394 -1: 2018, Mosquito repellents — Performance tests guidelines — Part 1: Skin applie d 
repellents  
[2] US 2373 -1:2022, Mosquito repellents - Performance tests guidelines - Part 1: Skin applied repellents  
 WDEAS/TC078 -1: 2022    
 
© EAC 2022  – All rights reserved