AFRICAN 
STANDARD  DARS  
1474  
First Edition 2018  
 
 Reference No. ARS 1474: 2018 (E) 
ICS 71.100.40  
 
 
© ARSO 2018  
  
 
 
 
 
 
 
 
 
 
Antibacterial bathing bars — Specification  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 DARS 1474: 2018  
ii © ARSO 2018 — All rights reserved   
Table of contents  
 
1 Scope  ................................ ................................ ................................ ................................ .............  1 
2 Normative reference s ................................ ................................ ................................ .....................  1 
3 Terms and definitions  ................................ ................................ ................................ .....................  1 
4 Requirements  ................................ ................................ ................................ ................................ . 1 
4.1 General requi rements  ................................ ................................ ................................ ............  1 
4.2 Specific requirements  ................................ ................................ ................................ ............  2 
5 Sampling  ................................ ................................ ................................ ................................ .........  3 
6 Packaging a nd labelling  ................................ ................................ ................................ .................  3 
6.1 Packaging  ................................ ................................ ................................ ..............................  3 
6.2 Labelling ................................ ................................ ................................ ................................ . 3 
Annex A (normativ e)  Test for lather volume  ................................ ................................ ..........................  4 
Annex B (normative)  Evaluation of the mushing properties of a bathing bar  ................................ ........  6 
Annex C (normative)  D etermination of grittiness in antibacterial bathing bar  ................................ ........  8 
Annex D (normative)  Determination of TCC and TCN in antibacterial bathing bar by HPLC  ................  9 
Annex E (normative)  Determination of chloroaniline  ................................ ................................ ...........  13 
Annex F (normative)  Determination of active detergent content  ................................ .........................  16 
Annex G (informative)  Permitted structuring and processing aids  ................................ ......................  21 
Annex H (informative)  Permitted antibacterial agents  ................................ ................................ .........  22 
Annex J (normative)  Determination of microbial inhibition of cosmetic soap bars and liquid hand and 
body washes  ................................ ................................ ................................ ................................ .........  23 
Bibliography  ................................ ................................ ................................ ................................ ..........  27 
 DARS 1474: 2018  
© ARSO 2018 — All rights reserved  iii  
Foreword  
 
The Af rican Organization for Standardization (ARS O) is an African intergovernmental organization 
established by the United Nations Economic Commission for Africa (UNECA) and the Organization of 
African Unity (AU)  in 1977 . One of the fundamental mandates of ARSO is to develop and harmoniz e 
African Standards (ARS) for the purpose of enhancing Africa’s internal trading capacity, increase 
Africa’s product and service competitiveness globally and uplift the welfare of African communities. 
The work of preparing African  Standards is normally carried out through ARSO  technical committees. 
Each Member State  interested in a subject for which a technical committee has been established has 
the right to be represented on that committee. International organizations, Regional Economic 
Communities (RECs), governmental and non -governmental  organizations , in liaison with ARSO , also 
take part in the work.  
 
ARSO  Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.  
 
The main task of technical co mmittees is to prepare ARSO  Standards. Draft ARSO  Standards 
adopted by the technical committees are circulated to the member bodies for voting. Publication as an 
ARSO  Standard requires approval by at least 75 % of the member bodies casting a vote.  
 
Attenti on is drawn to the possibility that some of the elements of this document may be the subject of 
patent rights. ARSO  shall not be held responsible for identifying any or all such patent rights.  
 
This African Standard was prepared by the ARSO Technical Harmo nization Committee Number 5 on 
Chemistry and Chemical Engineering (ARSO/THC 05).  
 
 
 
 
 
© African Organisation for Standardisation  2018 — All rights reserved* 
 
ARSO Central Secretariat  
International House 3rd Floor  
P. O. Box 57363 — 00200 City Square  
NAIROBI , KENYA  
 
Tel. +254 -20-2224561, +254 -20-3311641, +254 -20-3311608  
 
E-mail: arso@arso -oran.org  
Web: www.arso -oran.org  
                                                 
* 2018 ARSO — All rights of exploitation reserved worldwide for African Member States’ NSBs. 
 DARS 1474: 2018  
iv © ARSO 2018 — All rights reserved   
 
Copyright notice  
 
This AR SO document is copyright -protected by ARSO. While the reproduction of this document 
by participants in the ARSO standards development process is permitted without prior 
permission from ARSO, neither this document nor any extract from it may be reproduced, stored 
or transmitted in any form for any other purpose without prior written permission from ARSO.  
 
Requests for permission to reproduce this document for the purpose of selling it should be 
addressed as shown below or to ARSO’s member body in the country  of the requester:  
 
 
© African Organisation for Standardisation 2018 — All rights reserved  
 
ARSO Central Secretariat  
International House 3rd Floor  
P.O. Box 57363 — 00200 City Square  
NAIROBI, KENYA  
 
Tel: +254 -20-2224561, +254 -20-3311641, +254 -20-3311608  
 
 
E-mail: arso@arso -oran.org  
Web: www.arso -oran.org  
 
Reproduction for sales purposes may be subject to royalty payments or a licensing agreement. 
Violators may be p rosecuted.  
 
 
 DARS 1474: 2018  
© ARSO 2018 — All rights reserved  v  
Introduction  
 
Performance of soaps, for a long time, has been primarily based on Total Fatty Matter (TFM).  
 
Whereas the above is a fact, technological trends have shown that performance of soap can be 
enhanced by accepta ble and safe surface active agents where TFM levels have been reduced.  
 
In this standard the TFM levels have been reduced from that of antibacterial toilet soap with 
introduction of surface active agents whilst serving the same purpose. However this does not 
necessarily imply substitution of antibacterial toilet soap but rather an alternative and affordable 
antibacterial soap product.  
 
This standard therefore sets minimum requirements for performance and safety characteristics of 
antibacterial bathing bar . 
 
 DARS 1474: 2018  
© ARSO 2018 — All rights reserved  1  
Antibacterial bathing bars — Specification  
 
 
1 Scope  
 
This African Standard specifies the requirements and methods of sampling and test for solid 
antibacterial bathing bars  used for personal care .  
 
This Standard applies to antibacterial bathing bar s supplied in the form of bars and produced from 
vegetable or animal oils or fats, fatty acids, or from a blend of all or part of these materials, with or 
without the addition of rosins or non -soapy /synthetic surfactants . 
 
2 Normative references  
 
The follo wing referenced documents are indispensable for the application of this document. For dated 
references, only the edition cited applies. For undated references, the latest edition of the referenced 
document (including any amendments) applies.  
 
CD-ARS 1462 :2018, Powder detergents — Biodegradability  
 
CD-ARS 1468 :2018, Anti-bacterial liquid toilet soap — Specification  
 
ISO 456, Surface active agents — Analysis of soaps — Determination of free caustic alkali  
 
ISO 685, Analysis of soap — Determination of alkali content and total fatty matter content  
 
ISO 4315, Surface active agents -- Determination of alkalinity -- Titrimetric method  
 
Regulation (E C) No 1223/2009 of the European Parliament and of the Council of 30 November 2009 
on Cosmetic Products  
 
3 Terms and definitions  
 
For the purpose of this standard the following definitions apply.  
 
antibacterial bathing bar  
is a product in the form of a bar  containing an antibacterial agent and soap of fatty acids and synthetic 
surface active agents listed in 5.4.1 as ac tive ingredients and which could be used for bathing 
purposes in soft and hard water  
 
4 Requirements  
 
4.1 General requirements  
 
4.1.1  Antibacterial bathing bars shall be in the form of bars .  
 
4.1.2  The colour of the bar shall generally be uniform, except for multi -coloured bars.  
 
4.1.3  The product shall not be  harmful to skin.  
 
4.1.4  The bar shall not have an unpleasant odour.  
 
4.1.5  The antibacterial bathing bars shall be firm.  
 
4.1.6  They may contain suitable quantities of colouring matter, perfume, op acifiers and optical 
brightening agents.  
 AFRICAN STANDARD  DARS 1474: 2018  
2 © ARSO 2018 — All rights reserved  4.1.7  Antibacterial bathing bars shall not contain any ingredients in amounts that are harmful to the 
human body and environment  
 
4.1.8  Synthetic surface active agents, may be used and when used shall not be more  than 4% by 
mass when tested in accordance to Annex F  
 
4.1.9  The antibacterial bathing bar shall contain permitted antibacterial agent. The label shall 
clearly state the antibacterial agent used and its level.  
 
4.1.10  The antibacterial bathing bar shall pass the antibacterial activity test when determined by the 
method given in  Annex J.  
 
4.2 Specific requirements  
 
4.2.1  Ingredients  
 
The antibacterial bathing bar shall have one or more of the following surfactants:  
 
4.2.1.1  There is no restriction on the use of soap of fatty acids, fatty acid ester sulphonates, fatty 
alkalonamide, fatty alcohol ethoxylates, sarcosinates, taurides, fatty isothionates, alpha olefin 
sulphonates, alcohol sulphates and amphoterics such as betaines and fatty alcohol ethoxy sulph ate, 
linear alkyl benzene sulphonates (LAS) and alkyl poly glycosides (APG) and fatty alcohol 
sulphosuccinate and fatty alkanol amido sulphosuccinate.  
 
4.2.1.2  In addition to the surfactants and perfume, the antibacterial bathing bar may contain other 
Ingredients such as electrolytes, bar structuring and processing aids, colouring matter, permitted 
antioxidants, preservatives, permissible germicides, super fatting agents, humectants  and plant 
extracts.  
 
4.2.1.3  Rosins, as % of total fatty matter, shall no t exceed 2 %  m/m.  
 
4.2.1.4  The synthetic surface active agents shall pass the biodegradability test as given in  CD-ARS 
1462:2017  
 
4.2.2  All ingredients shall be declared on the label following descending order in terms of quantity.  
 
4.2.3 The bathing bar  shall not contain any material s prohibited by regulations such as Regulation 
(EC) No 1223 . 
 
4.2.4  Performance and safety requirements  
 
Antibacterial bathing bars shall also comply with the requirements given in Table 1 when tested 
against the methods pres cribed therein.  
 
Table 1 — Performance and safety requirements  
 
No. Characteristic  Requirement  Test method  
(ii) Lather, ml, min.  200 Annex A  
(iii) Mush (loss in mass due to mushing on a wet 
surface), g/30 cm2, max.)  10 Annex B  
(iv) Freedom from grittine ss Pass test  Annex C  
(v) Total alkalinity (as NaOH), % by mass, max.  1.0 ISO 685/ ISO 4315  
(vi) Free caustic alkali (as NaOH), % by mass, max.  0.1 ISO 456  
(vii) Triclosan (TCN) and Trichlorocarbanilide (TCC) a) ≤1% by mass  Annex D  
(viii) Chloroaniline content  a) ≤10 ppm  Annex E  DARS 1474: 2018  
© ARSO 2018 — All rights reserved  3 NOTE  Trichlorocarbanilide (TCC) is not heat stable and decomposes into chloroanilines on prolonged heating above 
60°C. If TCC is used in soap, the manufacturer should take care that such  soap is not subjected to temperature above 
60°C during the entire manufacturing process or during storage  
a) Delete. On the label you specify what actives you have used. Alternative active agents to be researched and to replace 
the two.  
 
5 Sampling  
 
Sampling shall be done in accordance to with CD-ARS 1468:2017, Annex D.  
 
6 Packaging and labelling  
 
6.1 Packaging  
 
Each bar shall be wrapped  and packed in suitable boxes, packages or cartons to avoid contamination 
or damage during transportation.  
 
6.2 Label ling 
 
Each antibacterial bathing bar shall be marked legibly and indelibly with the following particulars:  
 
a) the words “Antibacterial Bathing Bar”;  
 
b) manufacturer’s name and physical address;  
 
NOTE  The name, physical address of the distributor/suppl ier and trade mark may be added as required.  
 
c) nominal weight of each bar at the time of packaging;  
 
d) number of bars  contained in the package;  
 
e) batch number or code number; (Check bathing bar)  
 
f) all ingredients;  
 
g) date of manufacture and expiry date;  
 
h) antibacterial agents used and their levels;  
 
i) country of origin.  
 DARS 1474: 2018  
4 © ARSO 2018 — All rights reserved   
Annex A  
(normative)  
 
Test for lather volume  
 
 
 
A.1 General  
 
Strict attention shall be paid to all details of the procedure in order to ensure concordant results. 
Particul ar care should be taken to invert the cylinder exactly as described.  
 
A.2 Outline of the method  
 
A suspension of the material in standard hard water is taken in a graduated cylinder and given 12 
inversions under prescribed conditions. The volume of the f oam formed is observed after keeping the 
cylinder for 5 minutes.  
 
A.3 Reagents  
 
A.3.1  Calcium chloride CaCl 2.2H 2O, AR  
 
A.3.2  Magnesium sulphate MgSO 4.7H 2O, AR  
 
A.3.3  Distilled water  
 
A.4 Apparatus  
 
A.4.1  Graduated cylinder — Glass stoppered with gradu ation from 0 to 250 mL, with 2 mL 
divisions. Overall height about 35 cm and the height of the graduated portion about 20 cm.  
 
A.4.2  100-mL glass beaker  
 
A.4.3  Thermometer of range 0 – 110 C  
 
A.5 Preparation of standard hard water  
 
Dissolve 0.220 g of calcium chloride dihydrate and 0.246 g of magnesium sulphate heptahydrate in 
distilled water. Dilute to 5 L with distilled water.  
 
NOTE  This standard hard water has a hardness of approximately 50 ppm calculated as calcium carbonate.  
 
A.6 Sample preparati on  
 
Cut away the outer edges of bathing bar using a knife  
 
Using a stand up type of grater, grate up to 10 g – 15 g of the bathing bar into small chips.  
 
A.7 Procedure  
 
Weigh 1 g of the grated chips antibacterial bathing bar accurately in a 100 -mL glass  beaker. Add 10 
mL of the standard hard water. Cover the beaker with a watch glass and allow to stand for 30 min. 
The operation is carried out to disperse the antibacterial bathing bar.  
 
Stir the contents of the beaker with a glass rod and transfer the sl urry to a 250 -mL graduated cylinder 
ensuring that not more than 2 mL foam is produced. Repeat the transfer of the residue left in the 
beaker with further portions of 20 mL of standard hard water ensuring that all the matter in the beaker 
is transferred to the cylinder.  
 DARS 1474: 2018  
© ARSO 2018 — All rights reserved  5 Adjust the contents in the cylinder to 100 mL by adding sufficient standard hard water. Bring the 
contents of the cylinder to 30  C. Stir the contents of the cylinder with a glass rod or thermometer to 
ensure a uniform suspension.  
 
As soon  as the temperature of the contents of the cylinder reach 30  C, stopper the cylinder and give 
it 12 complete inversions, each inversion comprising movements in a vertical plane, upside down and 
vice versa. After the 12 inversions, let the cylinder stand f or 5 min. Take the following readings as 
shown in Figure A.1:  
 
a) foam plus water (V1 mL).  
 
b) water only (V2mL).  
 
 
 
Figure A.1 — Measurement of foam  
 
A.8 Calculation  
 
Lather volume = V1 - V2  
where  
 
V1 = Volume, in mL of foam + water;  
 
V2 = Volume, in mL of water only.  
 
 DARS 1474: 2018  
6 © ARSO 2018 — All rights reserved   
Annex B 
(normative)  
 
Evaluation of the mushing properties of a bathing bar  
 
 
 
 
B.1 Principle  
 
A test piece of defined size is cut from the sample bar to remove harder outer layers. The test piece is 
preconditioned by giving 18 x 18 0 degree twists under running water at 25 °C or in a bowl of water at 
25 °C. The bar is left for six hours on a piece of fabric that has been wetted and drained of excess 
water. During the six hours the soap/ cloth are covered to prevent drying. At the end  of the test period 
mush is removed from the test piece face in contact with the cloth. Weight loss from the test piece is 
expressed as mush per 30 cm2 of original surface area in contact with the cloth.  
 
B.2 Equipment  
 
B.2.1  For sample preparation  
 
B.2.1.1 Coarse kitchen cheese grater  
 
B.2.1.2  Sharp thin blade knife or carpenters plane  
 
B.2.1.3  Callipers  or ruler, to ensure the sample dimensions  
 
B.2.1.4  Other equipment/ materials for the test  
 
Plastic or non -corrodible trays which are suitable size d for the test piece. Plastic soap dishes 7 cm x 
11 cm x 2 cm are quite suitable.  
 
Cotton cloth pieces cut and folded to fit as a triple layer inside the trays. Normal, flat weave, cotton 
sheeting as used for bed sheets will be quite suitable.  
 
B.3 Bar p reparation  
 
B.3.1  Three (3) individual bars of a type should be tested. A test piece is cut from each bar. The test 
piece should if possible have a working face (to be applied to the fabric) of 6 cm ±1 cm x 4 cm ±1 cm.  
 
All bars in a set shall be cut to have the same face size. If the smallest of the range of bars to be 
tested at a given time is too small to allow a working face within these limits, then all bars should be 
cut to the maximum size possible from the smallest bar.  
 
The longest axis of the t est piece (6 ±1) cm should be from a direction parallel to the longest axis of 
the original bar sample.  
 
The working face should be a fresh surface from the interior of the bar sample. The face opposite the 
working face should be identified by making a sm all hole with a sharp object. This enables the 
working face to be identified after the preconditioning step.  
 
B.3.2  To cut the bar it is convenient to first trim it to the approximate size using a coarse kitchen 
cheese grater and then to make the final ad justments to a smooth surface with a sharp thin -bladed 
knife or carpenters plane. If a plane is used, it is better to move the bar over the plane blade.  
 
B.4 Test procedure  
 
For each test piece  
 DARS 1474: 2018  
© ARSO 2018 — All rights reserved  7 B.4.1  The tray plus triple thickness of cloth is filled with  demineralised water. The tray is then held 
vertically to drain the water from the cloth. The vertical position is maintained until water ceases to run 
from the dish in a continuous stream i.e. starts to drip.  
 
B.4.2  The area of the working face of the te st piece is measured (A).  
 
B.4.3  The working face of the bar is placed onto the damp fabric and then the tray plus soap are 
covered e.g. with a sealed plastic bag, to prevent water loss.  
 
B.4.4  The covered test piece and holder are maintained at 25 C for 6 h.  
 
B.4.5  The mushed soap test piece is removed from the tray and is weighed (W 1).  
 
B.4.6  Mush is removed from the working face of the soap test piece by scraping with the edge of a 
blunt sided spatula or plastic ruler.  
 
B.4.7  The test piece is rewe ighed (W 2) and the amount of mush removed is calculated as in D.5. 
The mush is expressed as grams per 30  cm2 of original test piece surface area.  
 
NOTE  The procedure for weighing the bar and removing the mush will take some minutes. During that time the r emaining 
soaps will continue to form mush. While this time is not critical for a set of three test pieces from a given product, if mor e than 
one product is under test it is advised to stagger the start of the test for the second product. This will give ade quate time to 
complete work on the first set before the 6 -hour storage time of the subsequent set is completed.  
 
B.5 Calculation  
 
Weight of mush (grams) W = W 1 – W2  
 
Surface area of bar (cm2) A = (width x breadth)  
 
Mush = 
2cm 30 per 30
Ag W  
 
B.6 Criteria for conformity  
 
The test is done with three (3) separate samples of each product type, and the mean value from three 
samples is quoted (X). The range of values (R) is quoted as the difference between the highest and 
lowest values obtained for a gi ven product type.  
 
The sample lot of products shall be declared as conforming to the requirements for this standard if X + 
0.6R is less than the maximum value given in Table 1.  
 DARS 1474: 2018  
8 © ARSO 2018 — All rights reserved   
Annex C 
(normative)  
 
Determination of grittiness in antibacterial bathing b ar 
 
 
 
C.1 Procedure  
 
Either  
Hold the antibacterial bathing bar under a smooth stream of running water at a temperature of 30 °C 
and gently rub the two sides of the bar on the palm of one hand for one minute each side.  
 
or  
 
Immerse the soap in a bowl co ntaining 5 L of water at 30 °C and gently rub two opposite bar faces 
with the palm of one hand for 30 s (15 s per bar face). Remove the bar from the water and continue to 
gently rub the two opposite bar faces for a further 30 s (15 s per face).  
 
Allow the  used bar to dry in the open for 4 hours and examine the surface.  
 
A set of 3 samples will be tested for each product.  
 
NOTE 1  Hands will become hydrated and insensitive with prolonged immersion in water. Testers should wait 15 min between 
testing every 3 sets of products (9 grit tests).  
 
NOTE 2  If using a bowl rather than running water use fresh water after testing every set of 3 samples.  
 
C.2 Criteria for conformity  
 
The performance criteria are:  
 
During manipulation under running water the washing bar will not have a visibly rough surface and will 
feel smooth to the touch. No gritty particles will be observed on the surface of the dried bar 4 h after 
the washing test.  
 
 DARS 1474: 2018  
© ARSO 2018 — All rights reserved  9  
Annex D 
(normative)  
 
Determination of TCC and TCN in antibacterial bathing bar  by HPLC  
 
 
 
D.1 Principle  
 
TCC and TCN are antibacterial agents, which are separated from other components in soap by 
normal phase or reverse phase liquid chromatography, detected spectrophotometrically and 
quantified by comparison with standard TCC and TC N. The method can estimate as low as 1 ppm of 
the above compounds:  
 
Procedures for both normal and reverse HPLC has been described and provide the option to use 
either method whichever is available to the users. Both methods are comparable.  
 
D.2 Normal p hase HPLC  
 
D.2.1  Reagents  
 
D.2.1.1  Iso-octane , HPLC grade.  
 
D.2.1.2  Iso-propanol (2 -propanol) , HPLC grade  
 
D.2.1.3  Hexane , HPLC grade.  
 
D.2.1.4  Standard TCC , 99 % pure  
 
D.2.1.5  Standard TCN , 99 % pure  
 
D.2.2  Apparatus  
 
D.2.2.1  High Performance Liquid Chrom atograph consisting of a pump, a sample injector of fixed 
volume with UV detector having variable wavelengths and a recorder.  
 
D.2.2.2  Standard volumetric flasks  
 
D.2.2.3  Pipettes  
 
D.2.2.4  Magnetic stirrer  
 
D.2.2.5  Millipore filter apparatus with 0.5 μ filter  
 
D.2.2.6  Column  
 
D.2.2.6.1 Silica column , stainless steel 25 cm x 0.46 cm packed with Normal phase -silica 5 
(Lichrosorb Si -60)  
 
D.2.2.6.2 Cyano column , stainless steel 25 cm x 0.40 cm packed with (Lichrospher 100) cyano 5 . 
 
NOTE  Either , of the above columns can be used depending on the availability.  
 
D.2.2.7  Mobile phase  
 
D.2.2.7.1 For silica column — Transfer 20 ml of iso -propanol into a 500 ml volumetric flash and make 
upto mark with iso -octane and mix well. Assemble millipore filter  apparatus and filter the solvent 
system prior to use.  
 DARS 1474: 2018  
10 © ARSO 2018 — All rights reserved  D.2.2.7.2 For cyano column — Transfer 50 ml of HPLC grade iso -propanol (2 -propanol) into a 500 ml 
volumetric flask, fill upto the mark with hexane and mix well assemble milllipore filter apparatus and  
filter the solvent system prior to use.  
 
D.2.2.7.1 For silica column — Transfer 20 ml of iso -propanol into a 500 -ml volumetric flask and 
make up to mark with iso -octane and mix well. Assemble millipore filter apparatus and filter the 
solvent system prior  to use.  
 
D.2.2.7.2 For cyano column — Transfer 50 ml of HPLC grade iso -propanol (2 -propanol) into a 500 - 
mL volumetric flask, fill up to the mark with hexane and mix well. Assemble millipore filter apparatus 
and filter the solvent system prior to use.  
 
D.2.2.8  HPLC conditions  
 
Detector wavelength flow rate:  280 nm  
 
Flow rate:  0.5 ml/min  
 
Injection volume:  20 l  
 
Retention time  
 
Silica column  
TCN - 7.5 min  
TCC - 19.2 min  
 
Cyano column  
TCN - 4.0 min  
TCC - 7.5 min  
 
D.2.3  Procedure  
 
D.2.3.1  Standa rd preparation (see note under D.3.4)  
 
Weigh accurately 25 mg of triclosan (TCN) and 25 mg of TCC into a 100 -ml volumetric flask and 
make up to volume with the mobile phase and mix well. Pipette 1.0 ml of this solution in a 50 ml 
volumetric flask and dilut e with mobile phase. Final concentration of TCC and TCN is 250 g/50 ml 
(5.0 ppm).  
 
D.2.3.2  Sample preparation  
 
Weigh accurately 1 g of homogenized sample into a 100 -ml standard flask, and dilute to the mark with 
mobile phase. Pipette 10 ml of the superna tant liquid to a 50 -ml volumetric flask, dilute with mobile 
phase, to the mark, and filter through 0.45 μm filter.  
 
D.2.3.3  Chromatography  
 
Equilibrate the column, maintained at a temperature of 30 °C, with the mobile phase with a flow rate 
of 0.5 ml /min for iso -octane - iso-propanol mobile phase and 1.0 ml/min for Hexane - iso-propanol 
mobile phase for 3 0 min. Set the wavelength at 280 nm. Inject 20 l of standard solution and then 
sample solutions.  
 
Measure area of the peaks of respective retention time for standard and sample.  
 
D.2.4  Calculation  
 
100sample of ion Concentrat  TCN standard of AreaTCN standard of ion Concentrat  TCN for sample of Area  massby  % TCN, 
 
 DARS 1474: 2018  
© ARSO 2018 — All rights reserved  11 
100sample of ion Concentrat  TCC standard of AreaTCC standard of ion Concentrat  TCC for sample of Area  massby  % TCC,  
 
D.3 Reverse phase  
 
D.3.1  Reagents  
 
D.3.1.1  Methanol — HPLC grade.  
 
D.3.1.2  Sodium Dihydrogen Phosphate  
Monohydrate — Chemical grade.  
 
D.3.1.3  Standard TCC  
 
D.3.1.4  Standard TCN (TCS)  
 
D.3 Reverse phase  
 
D.3.1  Reagents  
 
D.3.1.1  Methanol , HPLC grade.  
 
D.3.1.2 Sodium Dihydrogen Phosphate Monohydrate , Chemical grade.  
 
D.3.1.3  Standard TCC  
 
D.3.1.4  Standard TCN (TCS)  
 
D.3.2  Apparatus  
 
D.3.2.1  Column  
 
Octyldimethylsilyl (C -DB)  
 
Supercosil LC -8-DB - 15 cm x 4.6 mm. 5  
 
D.3.2.2  Mobile phase  
 
MeOH/0.01 M Phosph ate buffer 62:38 v/v  
 
0.01 M Phosphate buffer: Dissolve 1.38 g sodium dihydrogen phosphate monohydrate in 1 000 ml of 
distilled water. Prepare to pH 3.0 by 10 % phosphate solutions.  
 
D.3.3  Procedure  
 
D.3.3.1  Standard preparation (see Note under D.3.4)  
 
D.3.3.1.1 Weigh accurately about 90 mg of TCN. Dissolve in methanol and make up to 1 000 ml 
volumetric flask with methanol.  
 
D.3.3.1.2 Weigh about 110 mg of TCC, dissolve well with methanol, and make up the volume to 1 000 
ml.  
 
D.3.3.1.3 Accurately pipet te 10 ml of the solution prepared in (D.3.3.1.1) into the (D.3.3.1.2) 
volumetric flask containing TCC. And make up to the volume with methanol. Then accurately pipette 5 
ml of the solution into a 50 -ml volumetric flask. Make up to the volume with methanol.  Filter this 
standard solution through 0.45 m filter.  
 
D.3.3.2  Sample preparation  
 DARS 1474: 2018  
12 © ARSO 2018 — All rights reserved  Weigh accurately about 1.0 g of product, dissolve in methanol and make up to 100 ml in a volumetric 
flask with methanol. Filter this sample solution through 0.45 m filter.   
 
D.3.3.3  HPLC conditions  
 
Detector wavelength  280 nm  
Column temperature  35 ºC  
Flow rate  1.0 ml/min  
Injection volume  10 l  
 
Prepare the standard solution and the sample solution at the same time. Inject the standard solution 
three times and calculate the average of each ingredients peak count. Inject 10 g the sample 
solution and determine each ingredients percentage by the calculation shown.  
 
D.3.4  Calculations  
 
100massby  % TCN,
t sr s
MAFA M
 
 
100massby  % TCC,
t sr s
MAFA M
 
 
where  
Ar is the peak area of the test s ample,  
 
As is the averaged peak area of the standard,  
 
F is the purity of standard (percent).  
 
Ms is the mass, in grams, of the standard, and  
 
Mt is the mass, in grams, of the test sample,  
 
NOTE  Both TCC and TCN are photosensitive, hence standards sho uld be freshly prepared.  
 DARS 1474: 2018  
© ARSO 2018 — All rights reserved  13  
Annex E 
(normative)  
 
Determination of chloroaniline  
 
 
E.1 Principle  
 
The chloroanilines are extracted from soap with dimethyl sulfoxide and diazotized with nitrous acid. 
The reaction products are then coupled with N -1-(naphthyl)  ethylenediamine hydrochloride to produce 
coloured compounds which are estimated spectrophotometrically.  
 
E.2 Safety precautions  
 
Dimethyl sulfoxide (DMSO) is readily absorbed into the skin. Inhalation or skin penetration shall be 
avoided.  
 
DMSO should n ever be pipette using mouth. Always use pipette bulb. The standard chloroanilines 
and N -1-(naphthyl) – ethylenediamine hydrochloride shall not be allowed to come into contact with the 
skin. If they should, then wash the contaminated parts thoroughly with s oap and water.  
 
A supply of diluted sodium hypochlorite should be at hand at all times to deal with accidental spillages 
of chloraniline solution. Spillage on laboratory surface should be treated immediately with the sodium 
hypochlorite solution, followed  by water.  
 
E.3 Reagents  
 
E.3.1  Dimethyl Sulphoxide(DMSO) , AR grade.  
 
E.3.2  Hydrochloric Acid – Concentrated (specific gravity — 1.18).  
 
E.3.3  Sodium Nitrite – 0.4 percent w/v analytical grade, freshly prepared (aqueous).  
 
E.3.4  Ammonium Sulphamate , 2 %  w/v solution freshly prepared, (aqueous).  
 
E.3.5  N-1-(naphthyl) Ethylene , 0.1 % w/v solution diamine hydrochloride freshly prepared 
(aqueous).  
 
E.3.6  n-Butanol , AR grade  
 
E.3.7  Sand, acid purified 40 — 100 micron mesh.  
 
E.3.8  Solvent mixture  
 
DMSO  5 volumes  
 
n-Butanol  2 volumes  
 
distilled water  2 volumes  
 
hydrochloric acid  1 volume  
 
Mix n -butanol, water and HCL, cool the mixture and add DMSO.  
 
E.3.9  4-Chroroaniline and 3, 4 -Dichloroaniline, AR grade.  
 
E.4 Apparatus  
 
E.4.1  Spectrophotometer, suita ble for use at 554 nm  
 
E.4.2  Cuvettes — Glass (matched pair) 10 mm  
 DARS 1474: 2018  
14 © ARSO 2018 — All rights reserved  E.4.3  Water bath — Thermostatically controlled at 25 °C  
 
E.4.4  Stop watch  
 
E.4.5  Standard laboratory glassware  
 
E.4.6  Filter Paper, Whatman No. 541  
 
E.5 Procedure  
 
E.5.1  Preparation o f Calibration Curve  
 
E.5.1.1  Dissolve 0.349 8 g of 3, 4 -dichloroaniline and 0.2753 g of 4 -chloroaniline in solvent mixture 
(see C.2.8) in a 250 ml amber volumetric flask.  
 
Dilute to mark with solvent mixture. 1 ml = 2.5 mg mixed chloroanilines (stock solu tion).  
 
E.5.1.2  Dilute this stock solution with solvent mixture as given below:  
 
a) Take 5 ml of stock solution and dilute it to 250 ml with solvent mixture  
 
1 ml = 50 μg mixed chloroanilines.  
 
b) Take 5 ml of the above solution [see E.5.1.2(a)] and further dilute to 250 ml with solvent 
mixture.  
 
1 ml = 1 μg mixed chloroanilines.  
 
Use this solution for preparation of calibration curve.  
 
Transfer using a bur ette 0, 1 ml, 2 ml, 5 ml, 10 ml, 20 ml, 40 ml into 50 ml amber volumetric flasks.  
 
E.5.1.3  From a burette, add sufficient solvent mixture to make total volume to 40 -mL in each flask. 
The flasks are incubated in a water bath at 25 °C for 20 min: After exac tly 20 min, add 2 -mL of 
reagent (see E.3.3) into each flask and return them to the water bath for exactly 10 min (measure with 
a stop watch).  
 
Then add 2 -mL of reagent (see C.3.4) into each flask and return them to the water bath for exactly 10 
min. Swirl  the flask occasionally.  
 
Then add 2 -ml of reagent (see E.3.5) into each flask and remove them from the water bath. Dilute to 
volume with distilled water, mix and allow to stand for 30 min. Measure absorbance at 554 nm against 
the blank solution as prepar ed in E.5.1.4.  
 
E.5.1.4  In preparing the blank solution, take 40 ml of solvent mixture in a 50 ml amber volumetric 
flask. Incubate the flask in a water bath at 25 °C for 20 min. After exactly 20 min, add 2 ml of reagent 
(see E.3.3) into the flask and retu rn it to the water bath for exactly 10 min. Then add 2 ml of reagent 
(see E.3.4) into the flask and return it to the water bath for exactly 10 min (swirl the flask 
occasionally). Then add 2 ml of reagent (see E.3.5) into the flask and remove it from the wa ter bath. 
Dilute to volume with distilled water, mix and allow to stand for 30 min. Use this blank solution for 
preparation of calibration curve only.  
 
E.5.1.5  Prepare a graph by plotting weight ( g) of chloroanilines contained in each 50 ml flask against  
absorbance. The linear calibration will pass through the origin/or determine the average absorbance 
(AA) of 1 g of mixed chloroanilines by dividing sum of absorbances of all different aliquots of the 
standard by sum of g of chloroanilines in all differe nt aliquots of standard.  
 
E.6 Determination of chloroanilines  
 
E.6.1  Weigh to the nearest mg 3.0 - 15 g of finely grated soap add 10.0 g - 15.0 g of acid purified 
sand. Transfer quantitatively the sample and the sand into a mortar and grind the mixture th oroughly 
with a pestle to give a homogenous mass. Transfer the mass to a previously weighed 250 ml flat DARS 1474: 2018  
© ARSO 2018 — All rights reserved  15 bottom flask quantitatively and reweigh. Add DMSO (100 ml), stopper firmly and attach the flask to an 
automatic shaker. Shake for 1 h. Filter the DMSO e xtract through Whatman No. 541 into a 250 ml 
amber volumetric flask. Wash the flask and filter paper with small aliquots of DMSO. Allow the filtrate 
to drain completely, dilute to volume with DMSO and mix. Transfer 20 ml DMSO extract into a 50 ml 
amber vol umetric flask. Add 20 ml of solvent mixture. The flask is incubated in a water bath at 25 °C 
for 20 min. After exactly 20 min, add 2 ml of reagent (see E.3.3) into the flask and return it to the 
water bath for exactly 10 min (measure with a stop watch). Th en add 2 ml of reagent (see E.3.4) into 
the flask and return it to the water bath for exactly 10 min (swirl the flask occasionally). Then add 2 ml 
of reagent (see E.3.5) into the flask and remove it from the water bath. Dilute to volume with distilled 
wate r, mix and allow to stand for 30 min. Read the absorbance at 554 nm against blank (prepared as 
below).  
 
E.6.2  Prepare the blank solution by mixing 20 ml of DMSO extract of sample and 20 ml of solvent 
mixture in a 50 ml amber volumetric flask. Incubate the  flask in a water bath at 25 °C for 20 min.  
 
After exactly 20 min, add 2 ml of distilled water into the flask and return it to the water bath for exactly 
10 min. Then add 2 ml of reagent (see E.3.4) into the flask and return it to the water bath for exact ly 
10 min (swirl the flask occasionally). Then add 2 ml of reagent (see E.3.5) into the flask and remove it 
from the water bath. Dilute to volume with distilled water, mix and allow to stand for 30 min. Use this 
solution as a blank for reading sample only.   
 
E.6.3  Deduce the amount of chloroanilines ( g) from the calibration graph curve.  
 
NOTE  The determination should be completed in one day.  
 
E.7 Calculations  
 
Determine the amount of mixed chloroanilines in the aliquot of test solution from the calibratio n graph.  
 
Chloroaniline content (in ppm) = 
MMMMM
23 1
20) (250  
 
where  
M is the mass, in grams, of soap  
 
M1 is the mass, in grams, of sand  
 
M2 is the mass, in grams, of soap and sand transferred to the flask  
 
M3 is the mass, in micrograms, (μg) of m ixed chloroanilines found from calibration graph/or it can 
be calculated as given below:  
 
(AA) ine chloroanil mixed g1 of absorbance Averagesample the of Mass
3M
 
 
where  
 
g in ines chloroanil standard of ion concentrat of Sumstandards the of OD the of Sum
3M
 
 
Weight of soap actually used, in g = 
) (12
MMMM
  
 DARS 1474: 2018  
16 © ARSO 2018 — All rights reserved   
Annex F 
(normative)  
 
Determination of active  detergent content  
 
 
 
F.1 Outline of the method  
 
When equivalent amounts of cationic and anionic detergents are present in a two -phase mixture of 
water and chloroform, methylene blue will colour the two phases to the same degree. Sodium alkyl 
benzene sulph onate and sodium lauryl sulphate or any other detergent can be titrated with a standard 
solution of cetyl trimethyl ammonium bromide.  
 
F.2 Reagents  
 
Weigh 1.5 ± 0.001 g of cetyl trimethyl ammonium bromide into a 250 mL beaker. Add 100 mL of 
distilled wate r and stir until dissolved. Transfer quantitatively to a 1 litre volumetric flask and make to 
volume. Mix thoroughly and standardize against solution B. (See F.2.1).  
 
F.2.1  Anionic solution (Solution B)  
 
Weigh accurately such amount of standard alkyl sul phate of known combined SO 3 or active content 
so as to give exactly 0.320 g of combined SO 3 into a 250 mL beaker. Dissolve in 100 mL to 200 mL of 
warm water. Transfer quantitatively to 1 -litre volumetric flask and make to volume with water at room 
temperat ure. Mix thoroughly. This is the primary standard against which solution A, is standardized. 
Solution B is 0.004 N.  
 
F.2.2  Methylene blue indicator  
 
Dissolve 0.1 g of methylene blue in 100 mL of water. Transfer 30 mL of this solution to a 1 -L flask. 
Add 500 mL of water, 6.8 mL of concentrated sulphuric acid, 50 g of sodium dihydrogen phosphate 
monohydrate (NaH 2PO 4H2O) and shake until solution is complete. Dilute to the mark.  
 
F.2.3  Chloroform  
 
Analytical reagent grade  
 
F.3 Procedure  
 
F.3.1  Weigh accur ately a sample of sufficient size to give approximately 0.320 g of combined SO3 
into a 250 mL beaker. Sample size is crucial (see Note). Use 700 mL to 800 mL of warm water to 
transfer quantitatively to a 1 -L volumetric flask. Warm on steam bath and shake g ently until the 
sample is dissolved and solution is clear. Cool, dilute to the mark and mix thoroughly.  
 
NOTE  The titration value V should be as near as to 10 mL as possible, say between 8 mL and 12 mL but never outside 5 
mL and 15 mL.  
 
F.3.2  Pipette 10. 0 mL of the sample solution into a 100 mL glass stoppered cylinder (25 x 300 mm). 
Add 25.0 ± 0.5 mL of methylene blue solution and 10 ± 0.5 ml of chloroform (see Note). Titrate with 
solution A to the correct end point, shaking the cylinder carefully after such addition to avoid emulsion 
and maintaining temperature within prescribed limits of 20 C - 30 C by immersion in water bath, if 
necessary. As the end point is approached, the rate of transfer of colour increases and solution A 
shall be added dropwise with vigorous shaking after each addition. If the approximate titration is 
known, before shaking since this avoids emulsion formation.  
 
Application of vacuum to the titration cylinder may help to break some emulsions, if formed. The end 
point is reached wh en both layers have same colour intensity. The end point is very sharp and 0.05 
mL will cause a distinct change in colour distribution at or near the equivalence point.  
 DARS 1474: 2018  
© ARSO 2018 — All rights reserved  17 NOTE  The titration value V should be as near to 10 mL as possible, say between 8 mL a nd 12 mL but never outside 5 mL 
and 15 mL.  
 
F.3.3  Pipette 10.0 mL of the sample solution into a 100 -mL glass stoppered cylinder (25 x 300 mm). 
Add 25.0 mL ± 0.5 mL of chloroform (see Note). Titrate with solution A to the correct end point, 
shaking the cyl inder carefully after such addition to avoid emulsion and maintaining temperature 
within prescribed limits of 20  C to 30  C by immersion in water bath if necessary. As the end point 
solution A shall be added dropwise with vigorous shaking after each addit ion. If the approximate 
titration is known, 80 % of the required titrating solution should be added before shaking since this 
avoids emulsion formation.  
 
Application of vacuum to the titration cylinder may help to break some emulsions, if formed. The end 
point is reached when both layers have same colour intensity. The end point is very sharp and 0.05 
mL will cause a distinct change in colour distribution at or near equivalence point.  
 
NOTE  The volume of methylene blue solution and chloroform may be chang ed if found advantageous provided the same 
volumes are used in standardizing solutions A and B.  
 
F.3.4  Calculation  
 
F.3.4.1  The percent combined SO 3 shall be expressed as follows:  
 
% combined SO 3 = 
MNV 0.8  
where: 
V volume, in millilitres,  of solution A used in the titration;  
 
N normality of solution A; and  
 
M mass, in grams, of the sample in the aliquot.  
 
F.3.4.2  The percent active detergent content shall be expressed as follows:  
Percent active detergent content = percent combined SO 3  Mol. weight of active detergent.  
 
NOTE  The molecular weight of active detergent should be supplied by the manufacturer on request.  
 
F.4 Alternative method for determination of active detergent content  
 
(To be used only if the first method (Clause H.1)  fails to work on the product).  
 
F.4.1  Field of application  
 
This method is applicable to the analysis of alkylbenzene sulphonates, alkyl sulphonate, sulphates 
and hydroxy -sulphates, alkylphenol and fatty alcohol ethoxysulphates and dialkyl sulphosuccinat es 
and to the determination of active materials containing one hydrophilic group per molecule.  
 
F.4.2  Principle  
 
Determination of anionic -active matter in a medium consisting of an aqueous and chloroform phase, 
by volumetric titration with a standard cati onic-active solution (benzethonium chloride), in the 
presence of an indicator which consists of a mixture of a cationic dye (dimidium bromide) and an 
anionic dye (acid blue 1).  
 
F.4.3  Reagents  
 
F.4.3.1  The water used shall be of distilled quality.  
 
F.4.3. 2 Chloroform, (sp. gravity = 1.48 g/m, distilling between 59.5  ºC and 61.5  ºC).  
 
F.4.3.3  Sulphuric acid, 2.5 M solution.  
 DARS 1474: 2018  
18 © ARSO 2018 — All rights reserved  F.4.3.4  Sulphuric acid, 0.5 M solution.  
 
F.4.3.5  Sodium hydroxide, 1.0 M standard volumetric solution.  
 
F.4.3.6  Sodium lauryl sulp hate (sodium dodecyl sulphate) (CH 3(CH 2) (11 OSO 3Na), 0.004 M standard 
volumetric solution.  
 
Check the purity of the sodium lauryl sulphate and simultaneously prepare the standard solution.  
 
F.4.3.6.1 Determination of purity of sodium lauryl sulphate — Weigh to the nearest 1 mg, 5 g ± 0.2 g 
of the product into a 250 -mL round bottom flask with ground glass neck. Add exactly 25 mL of the 
sulphuric acid solution (H.4.3.4) and reflux into a water condenser.  
 
During the first 5 min - 10 min, the solution will  thicken and tend to foam strongly; control this by 
removing the source of heat and swirling the contents of the flask.  
 
In order to avoid excessive foaming, instead of refluxing the solution may be left on a boiling water 
bath for 60 min.  
 
After a furth er 10 min the solution clarifies and foaming ceases. Reflux for further 90 min. Remove the 
source of heat, cool the flask and carefully rinse the condenser with 30 mL of ethanol followed by 
water.  
 
Add a few drops of the phenolphthalein solution (F.4.3.8)  and titrate the solution with the sodium 
hydroxide solution (F.4.3.5).  
 
Carry out a blank test by titrating 25 mL of the sulphuric acid solution (F.4.3.4) with the sodium 
hydroxide solution (F.4.3.5).  
 
The purity of the sodium lauryl sulphate, expressed  as a percentage,  
 
10 0 1 ) (84.28
MMVV
 
 
 
where,  
V0 volume, in millilitres, of sodium hydroxide solution used for the blank test;  
 
V1 volume, in millilitres, of sodium hydroxide solution used for the sample;  
 
M1 mass, in grams, of the sodium lauryl sulphate to be checked; and  
 
M0 exact molarity of the sodium hydroxide solution.  
 
F.4.3.6.2 Weigh 0.004 M sodium lauryl sulphate standard volumetric solutions. Weigh, to the nearest 
1 mg between 1.14 g and 1.16 g of sodium lauryl sulphate and dissolve in  200 mL of water. Transfer 
to a ground glass stoppered 1 -L one -mark volumetric flask and dilute to the mark with water.  
Calculate the molarity, M1, of the solution by means of the solution by means of the formula:  
 
100 288.4(%) purity 2
1mM
 
where,  
m2 mass in grams of sodium lauryl sulphate.  
 
F4.3.7  Benzethonium chloride 0.004 M standard volumetric solution  
 
Weigh, to the nearest 1 mg, between 1.75 g and 1.85 g benzethonium chloride and dissolve in water. 
Transfer to a ground glass -stoppered 1 -L one -mark v olumetric flask and dilute to the mark with water.  
 DARS 1474: 2018  
© ARSO 2018 — All rights reserved  19 NOTE  In order to prepare a 0.004 M solution, dry the benzethonium chloride at 105EC, weigh 1.792 g, to the nearest 1 mg, 
dissolve in water and dilute to 1 L.  
 
F.4.3.8 Phenolphthalein, ethanolic solution  containing 10 g/L. Dissolve 1 g of phenolphthalein in 100 
mL of 95 % (v/v) ethanol.  
 
F.4.3.9 Mixed indicator  
 
F.4.3.9.1 Stock solution  
 
Weigh to the nearest 1 mg 0.5 g ± 0.005 g dimidium bromide into a 50 -mL beaker, and 0.025 g ± 
0.005 g of acid blue 1  into a second 50 -mL beaker.  
 
Add between 20 mL and 30 mL of hot 10 %. (v/v) ethanol to each beaker. Stir until dissolved and 
transfer the solutions to a 250 -mL one mark volumetric flask. Rinse the beakers into the volumetric 
flask with ethanol and dilute  to the mark with 10 % (v/v) ethanol.  
 
F.4.3.9.2 Mixed acid indicator solution  
 
Take 20 mL of the stock solution prepared above, put it in a 500 -mL one -mark volumetric flask. Add 
200 mL of water, and 20 mL of 2.5 M sulphuric acid (F.4.3.3) mix and dilute  to the mark with water. 
Store away from direct sunlight.  
 
F.4.4  Apparatus  
 
Ordinary Laboratory apparatus, and  
 
a) bottles, 200 -mL, glass stoppered, or measuring cylinders, glass stoppered.  
 
b) burettes, 25 -mL and 50 -mL.  
 
c) one -mark volumetric flask,  1-L capacity glass stoppered.  
 
d) one -mark pipette, 25 -mL.  
 
F.4.5  Procedure  
 
F.4.5.1  Standardization of benzethonium chloride solution  
 
By means of the pipette transfer 25 mL of the 0.004 M sodium lauryl sulphate solution to a bottle or 
measuring cyli nder, add 10 mL of water, 15 mL of the chloroform and 10 mL of the mixed indicator 
solution.  
 
Titrate with the 0.004 M benzethonium chloride solution. Stopper the bottle or measuring cylinder after 
each addition and shake well. The lower layer will be col oured pink. Continue the titration with 
repeated vigorous shaking. As the end point approaches, the emulsions formed during shaking tend 
to break easily continue the  titration drop by drop. Shaking after each addition of titrant, until the end point is 
reached. This is at the moment when the pink colour is completely discharged from the chloroform layer, which 
becomes a faint greyish blue.  
 
The molarity, M, of the benzethonium chloride solution is given by the formula:  
 
2125
VMM
 
where,  
M1 molarity of the sodium lauryl sulphate solution; and  
 
V2 Volume, in millilitres, of benzethonium chloride added.  
 
 DARS 1474: 2018  
20 © ARSO 2018 — All rights reserved  F.4.5.2  Determination  
 
Weigh to the nearest 1 mg a sample of 30 g; dissolve the test portion in water. Add a few drops of the 
phenolphthale in solution and neutralize to a faint pink colour with the sodium hydroxide solution or 
sulphuric acid solution as required.  
 
Transfer to a 1 -L one -mark volumetric flask and dilute to the mark with water. Mix thoroughly and, by 
means of the pipette transf er 25 mL of this solution to a bottle or measuring cylinder, , and add 10 mL 
of water, and 15 mL of chloroform. Titrate with the benzethonium chloride solution as described in 
F.4.5.1.  
 
F.4.6  Expression of results  
 
The content as a percentage by mass, of anionic -active matter  
 
MVMM MV
300 3
4100025100 1000

 
 
The amount of active matter, expressed in milliequivalents per gram,  
 
01 3 40
MMV
 
 
where,  
 
M0 mass, in grams, of the test portion;  
 
M relative molar mass of anionic -active matter;  
 
M1 molarit y of the benzethonium chloride solution;  
 
V3 volume, in millilitres, of benzethonium chloride solution used for the titration of a 25 -
mL aliquot of anionic -active matter solution.  
 
 DARS 1474: 2018  
© ARSO 2018 — All rights reserved  21  
Annex G  
(informative)  
 
Permitted structuring and processing aids  
 
 
The following is the list of structuring and processing aids used generally in bathing bars.  
 
1) Starch and derivatives  
 
2) Cellulose and derivatives  
 
3) Propylene glycol  
 
4) Sorbitol  
 
5) Glycerol  
 
6) Dextrin  
 
7) Kaolin  
 
8) Talc  
 
9) Bentonite  
 
10) Calcite  
 
11) Sodium lactate  
 
12) Soda ash  
 
13) Vegetable/animal oil fatty acids and salts  
 
14) Phosphates  
 
15) Sodium chloride  
 
16) Sodium sulphate  
 
17) Dolomite  
 
18) Fatty alcohol  
 
19) Rosin and rosin salts  
 
20) Fatty acid ethanolamide  
 
21) Dieth ylene glycol monostearate  
 
22) Paraffin  
 
23) Polyoxyethylene glycol  
 
24) Glycerol monostearates  
 
25) Silicates  
 
26) Sodium citrate  
 
27) Chelating agents  
 
28) Any other internationally accepted builder  DARS 1474: 2018  
22 © ARSO 2018 — All rights reserved   
Annex H  
(informative)  
 
Permitted antibacteria l agents  
 
 
 
The following is the list of antibacterial agents used generally in antibacterial bathing bars.  
 
a) Triclosan (TCN)  
 
b) Trichlorocarbanilide (TCC)  
 
c) Zinc oxide  
 
d) Hexa chlorophene  
 
e) Chloro xylenols  
 
f) Plant extracts  
 
g) Any other i nternationally accepted antibacterial agent.  
 DARS 1474: 2018  
© ARSO 2018 — All rights reserved  23  
Annex J 
(normative)  
 
Determination of microbial inhibition of cosmetic soap bars and liquid hand 
and body washes  
 
 
 
J.1 Scope  
This annex  specifies a method for testing and comparing the microbial inhibition properties of 
cosmetic soap bars and liquid hand and body washes.  
 
J.2 Application  
 
This method applies to the verification of efficacy claimed by antibacterial soaps in the inhibition of 
microorganisms.  
 
J.3 Principle  
 
When soap bars and liquid hand and b ody washes that have antimicrobial properties are inoculated 
into medium No.1 (see J.4.3.1.1), specific active ingredients diffuse into the surrounding agar thereby 
creating a zone of inhibition around the product, which is measured as an indication of the  microbial 
inhibition properties of the product.  
 
NOTE  The organisms referenced in this standard are the indicator organisms used when testing for hygiene purposes.  
 
J.4 Test method  
 
J.4.1 General  
 
Sampling and testing shall be carried out by personnel fa miliar with microbiological procedures.  
 
J.4.2 Accuracy  
 
Except where otherwise specified, all the following tolerances:  
temperatures  ± 2 °C;  
masses  ± 1.0 %;  
volumes  ± 1.0 %; and  
pH values  ± 0.1 pH unit.  
 
J.4.3 Culture medium, reagents, reference  cultures and controls  
 
J.4.3.1  Culture medium  
 
NOTE 1  The culture medium listed in J.4.3.1.1 is commercially available in dehydrated form and is made up in accordance 
with the manufacturer’s instructions.  
 
NOTE 2  Glass distilled water or deionised wate r should be used  
 
J.4.3.1.1 Medium No. 1  
 
Ingredients  
agar 15.0 g  
beef extract  15.0 g  
glucose  1.0 g  
pancreatic digest of casein  4.0 g  
peptone  6.0 g  
Yeast extract  3.0 g  
Water 1 000 mL  
 DARS 1474: 2018  
24 © ARSO 2018 — All rights reserved  J.4.3.1.2  Preparation  
 
J.4.3.1.2.1 Dissolve the ingredients in  approximately 900 mL of water and mix.  
 
J.4.3.1.2.2 Make up to 1 L.  
 
J.4.3.1.2.3 Adjust the pH so that after sterilization the pH value is 6.6 ± 0.05.  
 
J.4.3.1.2.4 Dispense 20 mL ± 0.2 mL volumes into suitable containers and sterilize in an autoclave f or 
15 min ± 0.5 min at 121 °C.  
 
J.4.3.2  Horse serum  
 
Sterile inactivated horse serum that is free from preservatives.  
 
J.4.3.3  Reference cultures  
 
J.4.3.3.1 Test organisms  
 
Use the following test organisms:  
Staphylococcus aureus  Sta 10 ATCC 6538;  
Escherichia coli  Esc 20 ATCC 8739; and  
Pseudomonas aeruginosa  Pse 16 ATCC 15442.  
 
NOTE Other reference organisms may also be used in addition to the ones specified in J.4.3.3.1.  
 
J.4.3.3.2  Preparation of test organism suspensions  
 
From a newly opened fre eze-dried culture or recently received agar culture, subculture the test 
organisms into bottles of 10 mL nutrient medium ( J.4.3.3.2.1)  
 
J.4.3.3.2.1 Nutrient medium  
 
J.4.3.3.2.1.1 Ingredients  
 
peptone  5.0 g  
sodium chloride  5.0 g  
yeast extract  2.0 g  
beef extract  1.0 g  
water  1 000 mL  
 
J.4.3.3.2.1.2 Preparation  
 
Dissolve the ingredients in the water and adjust the pH value to 7.1. Dispense in 10 mL volumes into 
suitable bottles and sterilize by autoclaving at 121 ±2 °C for 15 minutes.  
 
J.4.3.3.3  Incuba te the bottles at 37 °C for 24 h. Subculture onto nutrient agar slopes (see J.4.3.3.3.1). 
Incubate the slopes at 37 °C for 24 h.  
 
J.4.3.3.3.1 Nutrient agar  
 
J.4.3.3.3.1.1 Ingredients  
Agar 15.0 g  
peptone  5.0 g  
sodium chloride  5.0 g  
yeast extract  2.0 g   
beef extract  1.0 g  
water  1 000 mL  
 
 
 DARS 1474: 2018  
© ARSO 2018 — All rights reserved  25 J.4.3.3.3.1.2 Preparation  
 
Dissolve the ingredients in the water and adjust the pH value to 7.1.  Dispense 10 mL and 15 mL 
volumes into suitable bottles and sterilize by autoclaving at 121 ±2 °C for 15 minutes. Allow  only the 
10 mL volumes to solidify in a sloped position.  
 
J.4.3.3.4  From each of these slope cultures, prepare four subcultures (stock cultures) of each test 
organism onto 10 mL nutrient agar slopes (see J.4.3.3.3.1). Incubate the stock cultures at 37 °C  for 
24 h and then store in a refrigerator maintained at 4 °C, except for Pseudomonas aeruginosa which is 
stored at ambient temperature.  
 
J.4.3.3.5  Use the stock cultures to prepare further subcultures for the test, but do not make more than 
six serial su bcultures from each stock culture. After the sixth serial subculture, resort to n new freeze -
dried culture.  
 
J.4.3.3.6  Preparation of cultures for test suspensions  
 
J.4.3.3.6.1 For each of the test organisms, inoculate a nutrient agar slope (see J.4.3.3. 3.1) from a 
stock culture (see J.4.3.3.4) and incubate at 37 °C for 24 h.  
 
J.4.3.3.6.2 For the test, use a 24h culture that has been subcultured for two successive days. After six 
subcultures, restart the process using a fresh stock culture (see J.4.3.3.4 ).  
 
NOTE  The physiological condition of the test organisms is important and might influence inter -laboratory and intra -
laboratory variations in test results.  
 
J.4.3.3.6.3 After incubation, wash the bacterial growth from the slope using 10 mL sterile wate r and, if 
necessary, scrape the agar surface. Carefully decant the suspension into a sterile Erlenmeyer flask 
and shake vigorously to suspend all growth in the water. Standardize the suspension, by using a 
spectrophotometer in conjunction with a standard c urve, a haemocytometer, Petroff -Hausser counting 
chamber or any other suitable means, so that it contains 105 cfu/mL ± 104 cfu/mL. Use the suspension 
within 3 h of preparation.  
 
J.4.3.4  Control  
 
J.4.3.4.1  Positive control  
 
Natural honey with no additives,  e.g. colourants  
 
J.4.3.4.2  Negative control  
 
Sterile deionised water.  
 
J.4.4 Procedure  
 
J.4.4.1  Melt the contents of a bottle of the medium No. 1 (see J.4.3.1.1) and cool to 45 ± 2 °C. Add 1 
mL of the sterile inactivated horse serum (see J.4.3.2) and 1  mL of S. aureus test organism 
suspension prepared in accordance with J.4.3.3.2. Mix well and avoid the formation of air bubbles, 
then pour the mixture into a Petri dish of 90 mm diameter and allow to solidify.  
 
J.4.4.2  Using as sterile cutter that produc es cylindrical wells (holes) of 8 mm diameter, make five 
evenly spaced straight cylindrical wells in the solidified medium. Remove and discard the plugs of 
medium. Seal the bottom of each well using one or two drops of the molten medium in J.4.3.1.1 and 
allow to solidify.  
 
J.4.4.3  Introduce the test sample into the wells in such a way that each well is completely filled with 
the sample, taking care not to form bubbles. When the test sample is a soap bar, grind a sufficient 
amount to a powder, using a steri le pestle and mortar, and introduce the powder into the wells. 
Ensure that the surface of the agar remains free from the sample.  
 
J.4.4.4  Incubate the petri dish at 37 ± 1 °C for 18 h to 24 h.  DARS 1474: 2018  
26 © ARSO 2018 — All rights reserved   
J.4.4.5  At the end of this period (see J.4.4.4), remove the Petri dish from the incubator. Measure the 
diameter of the zone of inhibition diagonally across the well to the nearest millimeter  
 
J.4.4.6  Take the average of the five diameters and record this to the nearest millimeters .  
 
J.4.4.7  Repeat the procedure d escribed in J.4.4.1 to J.4.4.6 (inclusive) using the E. coli and P. 
aeruginosa test suspensions successively.  
 
J.4.4.8  Repeat the procedure described in J.4.4.1 to J.4.4.6 (inclusive) for the positive control and the 
negative control  
 
J.5 Interpretation of results  
 
J.5.1 For the product to pass the test, the average diameter of the zone of inhibition for each of the 
test organisms shall be at least 10 mm.  
 
J.5.2 If the zones are not clearly defined for one of the reference organism (e.g . hazy, incomplet e 
zone, or distorted shape), the test shall be repeated for the specified reference organism.  
 
 DARS 1474: 2018  
© ARSO 2018 — All rights reserved  27  
Bibliography  
 
 
Working Group to identify and acknowledge useful literature used in the preparation of this standard.  
 
EAS 794:2013, Kenya Standard — Determina tion of the microbial inhibition of cosmetic soap bars 
and liquid hand and body washes — Test method  
 
EAS 878:2016, Antibacterial bathing bars — Specification  
 
 DARS 1474: 2018 
© ARSO 2018 — All rights reserved