DRAFT UGANDA STANDARD  
  DUS 2373 -1 
First  Edition  
2021 -mm-dd 
 
 
 Reference number  
DUS 2373 -1:2021 
© UNBS 2021  
  
Mosquito repellents — Performance tests guidelines — Part 1: 
Skin applied repellents  
 DUS 2373 -1:2021  
ii © UNBS 2021  – All rights reserved  
 Compliance with this standard does not, of itself confer immunity from legal obligations  
A Uganda Standard does not purport to include all necessary provisions of a con tract. Users are 
responsible for its correct application  
© UNBS 2021.  
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilised in any 
form or by any means, electronic or mechanical, including photocopying a nd microfilm, without prior written 
permission from UNBS.  
Requests for permission to reproduce this document should be addressed to  
The Executive Director  
Uganda National Bureau of Standards  
P.O. Box 6329  
Kampala  
Uganda  
Tel: +256 41 4 333 2 50/1/2/3  
Fax: +256 414 286 123  
E-mail: info@unbs.go.ug  
Web: www.unbs.go.ug  DUS 2373 -1:2021  
© UNBS 2021  – All rights reserved  iii 
 Contents  Page 
Foreword  ................................ ................................ ................................ ................................ .............................  v 
1 Scope.  ................................ ................................ ................................ ................................ .....................  1 
2 Normative references.  ................................ ................................ ................................ ...........................  1 
3 Terms and definitions.  ................................ ................................ ................................ ..........................  1 
4  Laboratory studies  ................................ ................................ ................................ ................................  2 
4.1  General  ................................ ................................ ................................ ................................ ...................  2 
4.2  Estimation of effective dose (technical material)  ................................ ................................ ..............  3 
4.3  Estimation of complete protection time (technical material 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 f ormulated products  ................................ ................................ ..............  7 
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 ................................ ........  13 
B.3 The surface area (in cm2) of skin is then calculated by:  ................................ ................................ . 13 
B.4 Example:  ................................ ................................ ................................ ................................ ...............  13 
Annex C  (informative)   Estimation of median and confidence interval of complete protection 
time using the Kaplan -Meier survivor function  ................................ ................................ ................  14 
Anne x D  (informative)   Examples of calculation  ................................ ................................ .........................  16 
D.1 Example A  ................................ ................................ ................................ ................................ ............  16 
D.1.1  The ED50 is calculated as:  ................................ ................................ ................................ .................  16 
D.2 Example B  ................................ ................................ ................................ ................................ ............  16 
D.2.1  The ED90 is calculated as:  ................................ ................................ ................................ .................  16 
D.3 Example C  ................................ ................................ ................................ ................................ ............  17 
Bibliography  ................................ ................................ ................................ ................................ ......................  18 
  DUS 2373 -1:2021  
iv © UNBS 2021  – All rights reserved  
  . DUS 2373 -1:2021  
© UNBS 2021  – All rights reserved  v 
 Foreword  
Uganda National Bureau of Standards (UNBS) is a parastatal under the Ministry of Trade , Industry and 
Cooperatives established under Cap 327 , of the Laws of Uganda , as amended .  UNBS is mandated to co -
ordinate the elaboration of standards and is  
(a) a member of International Organisation for Standardisation (ISO) and  
(b) a contact point for the WHO/FAO Codex Alimentarius Commission on Food Standards, and  
(c) the National Enqui ry Point on TBT  Agreement  of the World Trade Organisation (WTO).  
The work of preparing Uganda Standards is carried out through Technical Committees. A Technical 
Committee is established to deliberate on standards in a given field or area and consists of key stakeholders 
including government, academia, consumer groups, private sector and other interested parties.  
Draft Uganda Standards adopted by the Technical Committee are widely circulated to stakeholders and the 
general public for comments. The committee  reviews the comments before recommending the draft standards 
for approval and declaration as Uganda Standards by the National Standards Council.  
The committee responsible for this document is Technical Committee UNBS /TC 301- Chemis try. 
  
DUS 2373  consists  of the following parts, under the general title Introductory element — Main element :  
 Part 1: Skin applied repellents.  
  Part 2: Spatial repellents.  
  DUS 2373 -1:2021  
vi © UNBS 2021  – All rights reserved  
  Introduction  
The purpose of these guidelines is to provide specific and standardized procedures and c riteria 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 su ch products by the national regulatory authorities. However, the requirements for registration of 
pesticides, including 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 to determine their application rate(s) 
and effectiveness. Guidance is also provided on the single -dose evaluation of formulated repellent product s. 
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 material(s) 
are applied to human skin. Any side -effects and/or undesirable characteristics experienced in association with 
the application and use of r epellents in laboratory studies and field trials should be recorded and reported. The 
protocol must include provision 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 product 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 organisms. 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 schemes such as the International Organization for Standardization should be applied.  
 DRAFT UGANDA STANDARD  DUS 2373 -1:2021  
 
© UNBS 2021  – All rights reserved  1 
 Mosquito repellents — Performance tests guidelines — Part 1 : 
Skin applied repellents  
1 Scope . 
This Draft Uganda Standard  provides guidelines  for the design and execution of studies 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 no normative ref erences 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 Online browsing platform: available at http://www.iso.org/obp   
3.1  
landing  
The act of flying on to or approaching and settling on to a human skin , without probing or biting.   
3.2  
probe  
act of penetrating human skin by mosquito  mouthparts 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 crossing may be 
quantified either or both by the distance the mosquitoes moves onto tr eated skin or by how long the 
mosquitoes remains on the treated skin  
3.5  
unconfirmed event  
landing, probe, bite, or crossing not followed by another similar event within 30 min   
2 © UNBS 2021  – All rights reserved  
 3.6  
confirming event  
one landing, probe, bite, or crossing followed by an other similar event within 30 minutes. The first event is 
confirmed by the second; the second event in the confirming event  
3.7  
human subject  
living individual about whom an investigator conducting research obtains either data through intervention or 
interaction with the individual or identifiable private information  
3.8  
questing  
behaviour  of mosquitoes actively seeking a host  
3.9  
repellent  
product that deters the host -seeking behaviour of mosquito, from approaching or settling on treated human 
skin 
3.10  
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 minutes by a second similar  event.  
3.11  
dose determination  
testing procedure used to estimate a “typical consumer 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 c omplete 
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) protect ion 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 efficacy test. However, landing is not 
always associate d 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 terms of landing, bu t 
those mosquitoes that land may al l 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 recommended 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 testing 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 carried out on adult human volunteers who may be select ed from 
among candidates exhibiting mild or no sensitivity to mosquito bites. Equal numbers of male and female test 
volunteers are preferred.  DUS  2373 -1:2021  
© UNBS 2021  – All rights  reserved  3 
 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 attractiveness to 
mosquitoes, and that this may in turn affe ct 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 
preferably not be tobacco users, or at least to have refrained from tobacco use for 12 h ours 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, maintained and tested (in a separate space or room) 
at 27 ± 2 0C 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 ha ve 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 by that species.  
4.1.7   Mosquito repellency test s 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 testing 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 collect ed 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 mosquitoes when it is more suitable for a particular s pecies 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 effective dose (technical materi al)  
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 analysis, preferably 
2 - 3dosages that g ive <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 
replicates 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 cm2of the forearm skin between the wrist and elbow (Annex B) and allowed to dry 
(approximately 1 min for ethanol). Before insertion 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 th e 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 placed 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 repellent dose at each test  calculated as the sum of the doses applied to arrive at the 
cumulative dose for each test (Table 1)  
Table 1 – Example of successive doses applied to arrive at a cumulative dose for a sample experiment  
Application sequence  Repellent solution concentration to 
be applied in 1 mL (mg/mL)  Cumulative amount of repellent 
(mg/600 Cm2area)  
Left-arm control  Pre-treated with alcohola onlya -  
4 © UNBS 2021  – All rights reserved  
 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 of 
mosquitoes landing and/or comme ncing 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 the number of landings and/or prob ings 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 mos quitoes 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). 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 complete 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 – 40 cm per side) each containing 200 –250 non -blood -fed females are 
normally used. One cage is designated for testing the candidate repellent and the other for the positive 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 repeat ed 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 DUS  2373 -1:2021  
© UNBS 2021  – All rights  reserved  5 
 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 time 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 c ompleted in 8 hours or less.  
4.3.5  The number of volunteers included 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 the 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 collecting 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 determination of proportional end -points of repellent efficacy and persistence is recommended. 
These allow concurrent estimation of EDs, effective period of time for a r ange of doses, repellent half -life and 
the CPT.  
5.2  Efficacy and persistence of technical 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 land ing on an exposed limb before the mosquitoes commence to  
probe. Aspirated mosquitoes are transferred 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.  
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 repellent 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 us ing fragrance products 12 h ours prior to and during the test. Volunteers 
should preferably not be tobacco users or at least have refrained from tobacco use for 12 h ours 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 suffic ient quantities to 
remain effective for the duration of the study. The appropriate dose of test repellent should be applied  
6 © UNBS 2021  – All rights reserved  
 uniformly to a limb of the volunteer using a pipette and allowed to dry before the start of the first collection 
period. A blind tes t, 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 sites that yield the most homogeneous densities of mosquitoes 
and with suitable biting rates.  
5.2.7   Within each collection site, volunteers should be placed si ngly and separated from each other by >20 
meters. A range of four doses is recommended to be tested 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% protection 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 volun teers and the number of collection 
sites are equal to the number of doses to be tested plus the untreated 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). Each 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 s hould each be similarly randomized. The mosquito collection at each 
collection site for the repellent treated volunteers and the negative and/or positive controls should be chosen 
to be suitable for the target species. For example, under high biting pressu re, 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 
counting and species identification. Species composition should be reported. Tests should not be conducted 
in windy conditions, but records should be made through out each test of wind speed, temperature, relative 
humidity and precipitation amount(s) for possible 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. a t 15 – 16 min, 30 – 31 min and 45 – 46 min), at each dose, is averaged for each replicate.  
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   
C  is the number of 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 app lied:  
ln[𝑝/(1 − 𝑝)] = 𝑎 + 𝑏1(𝐷0) + 𝑏2𝑡1  
  
where,  DUS  2373 -1:2021  
© UNBS 2021  – All rights  reserved  7 
  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 estimated 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 solving 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 coeffic ients provided in Example 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 5.2.1 2. The repellent half -
life can be estimated using the following equation:  
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 survivo r 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  
Beginning of 
test period 
(hours)  Observation 
period 
(hours)2 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  
2 In this example, within each observation period , three measurements (at 15, 30 and 45 minutes) are taken at each collection site  
 
5.3 Efficacy and persistence of formulated products  
5.3.1   The single -dose evaluation of formulated repellent products should be performed under field 
conditions at a mini mum of two ecologically and geographically distinct locations. A preliminary assessment of 
collection sites by performing human landing catches prior to the test is recommended to minimize variation in 
the number of mosquitoes collected among such sites. T his will include a selection of sites that yield the most 
homogeneous densities of mosquitoes and with suitable biting rates. The appropriate number of volunteers  
8 © UNBS 2021  – All rights reserved  
 trained in 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 demonstrate statistical significance should only be included in 
the testing for ethica l 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% DEETin ethanol for 
the positive control) o n ≈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 arranged ≥20 meters apart. The 
exposure time to mosquito populations at each collection s ite 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 could be 1 -minute each at 15, 30 and 45 minutes into the observation period, with the fi rst 
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 volunteers (untreated) should monitor biting pressure 
throughout the study. All mosquit oes 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 target 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 o f 
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 survivor function (Annex C) and the median CPT and 
corresponding standard errors reported.  
5.3.4   Percentage repellency (%p) in field trials can be determined for each hour of the test as:  
%p = [(C – T)/C] x 100  
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 ave rage 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 profile of hourly change in % repellency over 
the 12 -hour test period . 
 DUS  2373 -1:2021  
© UNBS 2021  – All rights  reserved  9 
 Anne x 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 guidelin es. 
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 research  
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 s ite, after having announced in the district, through oral 
advertisements, that the project is looking for volunteers. The selection will ensure that equal opportunities are 
provided to everybody. Report how many volunteers are expected to participate in th e 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 t hey 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, s uch as its manufacturer or location of 
manufacture, and the reason for its development. Explain the known experience with this repellent product. 
Explain comprehensively, if any, all the known side -effects or toxicity of this repellent product.   
10 © UNBS 2021  – All rights reserved  
 A.2.7 Parti cipant 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 t reatment.  
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 and 
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 if 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 c are 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 any anticipated discomforts that are in addition to the side -effects 
and risks discuss ed 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 entitled regardless of participation.  
A.2.14 Incentives  
State clearly what you will provi de the participants with as a result of their participation. WHO does not 
encourage incentives. However, it recommends that reimbursements for expenses incurred as a result of 
participation in the research be provided.  
A.2.15 Confidentiality  
Explain how th e 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 for sharing the findings with the participants should be provided.  DUS  2373 -1:2021  
© UNBS 2021  – All rights  reserved  11 
 A.2.17 Right to refuse o r 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 information 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 task 
is to make sure that research participants are protected from harm. If you wish to find o ut 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 the 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 r ight 
to withdraw from the research at any time without in any way affecting my medical care.  
Print name of participant: …………………………………………………………………………………………  
Signature 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 witnessed the accurate reading of the consent form to the potential participant, and the 
individual ha s 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 the opportunity to ask questions. I confirm that the individual 
has given consent freely.  
Print name of r esearcher: …………………………………………………………………………………………  
Signature of researcher: …………………………………………………………………………………………  
Date: …………/………………/………………..   
12 © UNBS 2021  – All rights reserved  
 A copy of this Informed Consent Form has been provided to participant………………………………….. 
(initiated by the researcher/assistant ). DUS  2373 -1:2021  
© UNBS 2021  – All rights  reserved  13 
 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 pu rpose: 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) at 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 calcula ted by:  
Area = ½ (C w + C e) Dwe 
Where,  
Cw  is the circumference of the wrist in cm,  
Ce  is the elbow -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 volu nteer, the researcher measures the circumference 
of the wrist as 18 cm and an elbow region circumference 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:  
Area = ½ (18 cm  + 27 cm) x 26 cm  
Area = ½ (45 cm) x 26 cm  
Area = 22.5 cm x 26 cm  
Area = 585 cm2  
14 © UNBS 2021  – All rights reserved  
 Annex C  
 (informative ) 
 
 Estimation of median and confidence interval of complete protection 
time using the Kaplan -Meier survivor function  
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 t esting, must be determined. The 
data required for this purpose are:  
The CPT in minutes (t) for each replication (i) of a test with n replications (i.e. t i [i = 1, 2, … n]).  
The number of replications ® of the test sharing the same CPT (j), where r ≤ n (i.e ., tj [j = 1, 2, … r]).  
The number of replications of the test not sharing the same CPTs (i.e. [n - r]. 
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 C PT greater than tj. The number of repellent failures (CPTs) recorded 
between the time 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)}.  
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 hypothetical 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 these 11 replications is unknown.  
Table C1 – Summary of the frequency of the complete protection ti me (CPT) of a candidate repellent 
applied on human skin.  
CPT (min)  120  150  210  390  420  450  480  510  600  
Frequency (dj)  1  3  3  10  7  4  4  2  3  
 
Using the data from this test, estimates of (nj – dj)/nj and S(t) and their corresponding standa rd error are 
constructed, as presented in Table C2.  
Table C2 – Estimates of (nj – dj)/nj  and their corresponding standards error  (4) 
Time interval 
(tj) in min.  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  DUS  2373 -1:2021  
© UNBS 2021  – All rights  reserved  15 
 390  31  10  0.6774  0.5726  0.0798  
420  24  7  0.7083  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  
(4) Data processing software for calculation of these variables is available on the Internet. Search key word: Kaplan Meier s urvival function online calculator . 
 
Median complete protecti on 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 CPT in this example is t(50) = 420 min.  
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 m edian 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 repellent 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.   
16 © UNBS 2021  – All rights reserved  
 Annex D  
(informative )  
 
Examples of calculation  
D.1 Example A  
The following coefficients 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  
D.1.1  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/cm 2  
D.2 Example B  
D.2.1  The ED 90 is calculated as:  
(1) ln [p/(1 – p)] = a + b 1(D0) + b 2t1  
(2) ln [0.9/(1 – 0.9)] = 8.160 + 2.209(D 0) + - 0.532 (0)  
(3) ln (9.0) = 8.160 + 2.209 (D 0) + 0  
(4) – 5.963 = 8.160 +2.209 (D 0)  
(5) – 5.963/2.209 = D 0  DUS  2373 -1:2021  
© UNBS 2021  – All rights  reserved  17 
 (6) – 2.699 = D 0  
(7) ln (ED 90) = D 0  
(8) ED 90 = exp (D 0)  
(9) ED 90 = 2.7183 (-2.699)  
(10) 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):  
(1) ln [p/(1 – p)] = a + b 1(D0) + b 2t  
(2) ln [0.99/(1 – 0.99)] =a + b 1(D0) + b 2t  
(3) ln (99) - 8.160 – (2.209 x – 0.693) = ( - 0.532) t  
(4) 4.595 - 8.160 – (-1.531) = - 0.532 t  
(5) – 2.034 = - 0.532 t  
(6) t = ( - 2.034/ - 0.532) = 3.6 hours after application  
  
18 © UNBS 2021  – All rights reserved  
 Bibliography  
[1] RS 394 -1: 2018, Mosquito repellents — Performance tests guidelines — Part 1: Skin applied 
repellents  
  DUS 2373 -1:2021  
© UNBS 2021  – All rights reserved  19 
 Certification marking  
Products that conform to Uganda standards may be marked with Uganda National Bureau of Standards 
(UNBS) Certification Mark shown in the figure below.  
The use of the UNBS Certification Mark is governed  by the Standards Act, and the Regulations made 
thereunder. This mark can be used only by those licensed under the certification mark scheme operated by 
the Uganda National Bureau of Standards and in conjunction with the relevant Uganda Standard. The 
prese nce of this mark on a product or in relation to a product is an assurance that the goods comply with the 
requirements of that standard under a system of supervision, control and testing in accordance with the 
certification mark scheme of the Uganda Nationa l Bureau of Standards. UNBS marked products are 
continually checked by UNBS for conformity to that standard.  
Further particulars of the terms and conditions of licensing may be obtained from the Director, Uganda 
National Bureau of Standards.  
 
 DUS 2373 -1:2021    
 
ICS 65.100.10  
Price based on nn pages  
© UNBS 2021 – All rights reserved