FINAL DRAFT UGANDA 
STANDARD  
  FDUS EAS 336 
First  Edition  
2013-mm-dd 
 
 Reference number  
FDUS EAS 336: 2013 
© UNBS 201 3 
  
Chemical depilatories  — Specification  
 FDUS EAS 336: 2013 
ii                © UNBS  2013 - 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 contract. Users are 
responsible f or its correct application  
© UNBS 20 13 
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 and microfilm, without prior wri tten 
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 41 505 995  
Fax: 256 41 286 123  
E-mail: unbs@infocom.co.ug  
Web: www.unbs.go.ug  
 FDUS EAS 336: 2013 
© UNBS  2013 - All rights reserved                 iii National foreword  
Uganda National Bureau of Standards (UNBS) is a parastatal under the Ministry of Tourism, Trade and 
Industry established under Cap 327, of the Laws of Uganda. UNBS is mandate d 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 Enquiry Point on TBT/SPS Agreemen ts 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 representatives of 
consum ers, traders, academicians, manufacturers, government and other stakeholders.  
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 rec ommending the draft standards 
for approval and declaration as Uganda Standards by the National Standards Council.  
This Final Draft Uganda Standard, FDUS EAS 336: 2013, Chemical depilatories  — Specification , is identical 
with and has been r eproduced from an East African  Standard, EAS 336: 2013, Chemical depilatories  — 
Specification , and is being proposed for adoption as a Uganda Standard.  
Wherever the words, “ East African  Standard" appear, they should be replaced by "Uganda Standard."   EAS 336: 201 3 
ICS 71.100.40  
© EAC 2013                                                                                                                         Second Edition 201 3  
 
EAST AFRICAN STANDARD  
 
Chemical depilatories — Specification  
EAST AFRICAN COMMUNITY  EAS 336: 201 3 
ii © EAC 201 3 – All rights reserved  
 Copyright notice  
This EAC document is copyright -protected by EAC. While the reproduction of this document by 
participants in the EAC standards development process is permi tted without prior permission from EAC, 
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 EAC.  
Requests for permission to reproduce this document f or the purpose of selling it should be addressed 
as shown below or to EAC’s member body in the country of the requester:  
© East African Community 2013 — All rights reserved  
East African Community   
P.O.Box 1096  
Arusha  
Tanzania  
Tel: 255 27 2504253/8  
Fax: 255  27 2504481/2504255  
E-mail: eac@eachq.org  
       Web: www.eac -quality.net  
Reproduction for sales purposes may be subject to royalty payments or a licensing agreement.  
Violators may be persecuted  EAS 336: 2013  
© EAC 2013  – All rights reserved  iii 
 Contents  Page 
Foreword  ................................ ................................ ................................ ................................ .............................  v 
1 Scope  ................................ ................................ ................................ ................................ ......................  1 
2 Description  ................................ ................................ ................................ ................................ .............  1 
3 Normative references  ................................ ................................ ................................ ............................  1 
4 Requirements  ................................ ................................ ................................ ................................ .........  2 
4.1 General requirements  ................................ ................................ ................................ ...........................  2 
4.2 Specific quality requirements  ................................ ................................ ................................ ..............  2 
5 Packaging and labelling ................................ ................................ ................................ ........................  3 
5.1 Packaging  ................................ ................................ ................................ ................................ ...............  3 
5.2 Labelling  ................................ ................................ ................................ ................................ .................  3 
6 Sampling  ................................ ................................ ................................ ................................ ................  3 
Annex  A (normative)  Determinatio n of pH neat  ................................ ................................ .............................  4 
A.1 Apparatus  ................................ ................................ ................................ ................................ ...............  4 
A.2 Reagents  ................................ ................................ ................................ ................................ ................  4 
A.3 Procedure  ................................ ................................ ................................ ................................ ...............  4 
Annex B (normative)  Determination of calcium thioglycollate  ................................ ................................ ..... 5 
B.1 Reagents  ................................ ................................ ................................ ................................ ................  5 
B.2 Procedure  ................................ ................................ ................................ ................................ ...............  5 
B.3 Calculation  ................................ ................................ ................................ ................................ .............  5 
Annex C (normative)  Determination of thermal stability  ................................ ................................ ...............  6 
C.1 Apparatus  ................................ ................................ ................................ ................................ ...............  6 
C.2 Procedure  ................................ ................................ ................................ ................................ ...............  6 
C.3 Results  ................................ ................................ ................................ ................................ ....................  6 
Annex D (normative)  Determination of free alkali content  ................................ ................................ ............  7 
D.1 Outline of the method  ................................ ................................ ................................ ...........................  7 
D.2 Reagents  ................................ ................................ ................................ ................................ ................  7 
D.3 Procedure  ................................ ................................ ................................ ................................ ...............  7 
D.4 Calculation  ................................ ................................ ................................ ................................ .............  7 
Annex E (normative)  Determinatio n of total viable count  ................................ ................................ .............  9 
E.1 Outline of the method  ................................ ................................ ................................ ...........................  9 
E.2 Apparatus  ................................ ................................ ................................ ................................ ...............  9 
E.3 Medial buffer  ................................ ................................ ................................ ................................ ..........  9 
E.4 Sterilization of apparatus  ................................ ................................ ................................ ...................  10 
E.5 Procedure  ................................ ................................ ................................ ................................ .............  10 
E.6 Expression of results  ................................ ................................ ................................ ..........................  10 
Annex F (normative)  Determination of Pseodomonas aeruginosa, Staphylococcus  aureus  and 
Candida albicans  in cosmetic products  ................................ ................................ ............................  11 
F.1 Introduction  ................................ ................................ ................................ ................................ ..........  11 
F.2 Principle of the method ................................ ................................ ................................ .......................  11 
F.3 Apparatus  ................................ ................................ ................................ ................................ .............  11 
F.4 Reagents  ................................ ................................ ................................ ................................ ..............  12 
F.5 Micro -organisms  ................................ ................................ ................................ ................................ .. 13 
F.6 Product quality check  ................................ ................................ ................................ .........................  13 
F.7 Product preparation  ................................ ................................ ................................ ............................  14 
F.8 Bacterial inoculum preparation  ................................ ................................ ................................ .........  14 
F.9 Fungal i noculum preparation  ................................ ................................ ................................ .............  14 EAS 336: 201 3 
iv © EAC 201 3 – All rights reserved  
 F.10   Inoculum pools  ................................ ................................ ................................ ................................ .. 14 
F.11   Inoculation  ................................ ................................ ................................ ................................ ..........  15 
F.12   Sampling intervals  ................................ ................................ ................................ .............................  15 
F.13   Aerobic Plate Count (APC)  ................................ ................................ ................................ ...............  15 
F.14   Neutralization check  ................................ ................................ ................................ ..........................  15 
F.15   Data analysis  ................................ ................................ ................................ ................................ ...... 16 
Annex G (normative)  Test for lead using atomic absorption spectrophotometer (AAS)  ........................  17 
G.1 Scope  ................................ ................................ ................................ ................................ ...................  17 
G.2 Reagents  ................................ ................................ ................................ ................................ ..............  17 
G.3 Standard solutions  ................................ ................................ ................................ .............................  17 
G.4 Sample preparation  ................................ ................................ ................................ ............................  17 
G.5 Instrument conditions  ................................ ................................ ................................ ........................  18 
G.6 Analysis  ................................ ................................ ................................ ................................ ...............  19 
G.7 Calculations  ................................ ................................ ................................ ................................ .........  19 
G.8 Interferences  ................................ ................................ ................................ ................................ ....... 19 
Annex H (normative)  Test for arsenic using atomic absorption spectrophotom eter (AAS)  ...................  20 
H.1 Scope  ................................ ................................ ................................ ................................ ...................  20 
H.2 Reagents  ................................ ................................ ................................ ................................ ..............  20 
H.3 Instrument condit ions ................................ ................................ ................................ ........................  20 
H.4 Sample preparation  ................................ ................................ ................................ ............................  21 
H.5 Analysis  ................................ ................................ ................................ ................................ ...............  21 
H.6 Calculatio n ................................ ................................ ................................ ................................ ...........  21 
Annex I (normative)  Test for mercury using the atomic absorption spectrophotometer (AAS)  ............  22 
I.1 Scope  ................................ ................................ ................................ ................................ ...................  22 
I.2 Reagents  ................................ ................................ ................................ ................................ ..............  22 
I.3 Instrument conditions  ................................ ................................ ................................ ........................  22 
I.4 Sample preparation  ................................ ................................ ................................ ............................  23 
I.5 Analysis  ................................ ................................ ................................ ................................ ...............  23 
I.6 Calculations  ................................ ................................ ................................ ................................ .........  23 
 EAS 336: 2013  
© EAC 2013  – All rights reserved  v 
 Foreword  
Development of the East African Standards has been necessita ted by the need for harmonizing requirements 
governing quality of products and services in the East African Community. It is envisaged that through 
harmonized standardization, trade barriers that are encountered when goods and services are exchanged 
within  the Community will be removed.  
In order to achieve this objective, the Community established an East African Standards Committee mandated 
to develop and issue East African Standards.   
The Committee is composed of representatives of the National Standards  Bodies in Partner States, together 
with the representatives from the private sectors and consumer organizations. Draft East African Standards 
are circulated to stakeholders through the National Standards Bodies in the Partner States. The comments 
received  are discussed and incorporated before finalization of standards, in accordance with the procedures 
of the Community.  
East African Standards are subject to review, to keep pace with technological advances. Users of the East 
African Standards are therefore expected to ensure that they always have the latest versions of the standards 
they are implementing .  
EAS 336 was prepared by Technical Committee EAS/TC 071, Cosmetics and cosmetic products.  
This second edition cancels and replaces the first edition (EAS 3 36:2004), which has been technically revised.  EAS 336: 201 3 
vi © EAC 201 3 – All rights reserved  
 Introduction  
Depilatories are preparations applied to the body to remove unwanted hair. Such preparations may involve 
epilation or chemical depilation. Epilation is a process in which hair is embedded in an ad herent material 
which can then be pulled away from the skin bringing the hair with it. Epilatory preparations are essentially 
based on rosin and beeswax. Chemical depilatories on the other hand cause chemical degradation of the hair, 
and may have organo -mercapto compounds (the most common being thioglycollic acid and its salts), metal 
sulphides or stannites (tin) -based composition.  
Since most individuals are allergic to chemical depilatories, the instructions regarding "patch test"  have been 
included in th e standard. Other requirements include pH, thioglycollic acid content, thermal stability, 
microbiological examination, and limits for lead, mercury and arsenic.  
 EAST AFRICAN STANDARD  EAS 336:2013  
 
© EAC 2013  – All rights reserved  1 
 Chemical depilatories — Specification  
1 Scope  
This East African Standard specifies the requi rements and methods of  sampling  and test for chemical 
depilatories  of alkaline -thioglycollic acid composition .   
This East African S tandard  does not cover depilatories of epilatory type and those having  metallic sulphides or 
stannite composition .  
2 Descri ption  
Depilatories are preparations applied to the body to remove unwanted hair. They may be in the form of a 
cream, gel or lotion.  
3 Normative references  
The following 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.  
AOAC  official method 998.10, Efficacy of preservation of non -eye area water -miscible cosmetic and toiletry  
formulations   
EAS 346 , Labelling of cosmetics — General requirements  
EAS 377 -1, Cosmetics and cosmetic products — Part 1: List of substances prohibited in cosmetic products  
EAS 377 -2, Cosmetics and cosmetic products — Part 2: List of substances which cosm etic products must not 
contain except subject to the restrictions laid down  
EAS 377 -3, Cosmetics and cosmetic products   Part 3: List of colorants allowed in cosmetic products  
EAS 377 -4, Cosmetics and cosmetic products   Part 4: List of preservatives allow ed in cosmetic products  
EAS 377 -5, Cosmetics and cosmetic products  Part 5: Use of UV filters in cosmetic products  
ISO 6887 -1, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension 
and decimal dilutions for micro biological examination — Part 1: General rules for preparation of the initial 
suspensions and decimal dilutions  
ISO 11930, Cosmetics  — Microbiology — Evaluation of the anti -microbial protection of a cosmetic product  
ISO 24153, Random sampling and randomiza tion procedures  EAS 336: 201 3 
2 © EAC 201 3 – All rights reserved  
 4 Requirements  
4.1 General requirements  
4.1.1  The chemical depilatory shall com ply with the requirements specified in all parts of EAS 377 .  
4.1.2  In addition, the product shall:   
a) convert human hair completely, in accordance with the instru ctions of use,  to a soft plastic mass 
which may be easily removed from the skin by wiping  or rinsing ; 
b) be non toxic systemically and non irritant to skin even on long contact ; 
c) be easily applied ; and   
d) be stain -free to clothing .  
4.2 Specific quality requirem ents 
4.2.1  The depilatories shall comply  with the requirements given in Table 1 when tested in accordance with 
the methods prescribed therein.  
Table 1 — Chemical r equirements for chemical depilatories  
S/No  Characteristic  Requirement  Method of test  
(i) pH neat* 11.0- 12.7 Annex A  
(ii) Calcium thioglycol late, calculated as thioglycollic acid, 
% by mass  2.5- 5.0 Annex B  
(iii) Thermal stability  To pass the test  Annex C  
(iv) Free alkali  To pass test  Annex D  
*pH neat not diluted ( refer to Annex A)  
 
4.2.2  Microbiological limits shall comply with the limits specified in Table 2.  
Table 2   Microbiological limits  
S/N Micro -organisms  Limits, max.  
Cfu/g  Method of test  
i.  Total viable count  100 in 0.5 g  Annex B 
ii.  Pseudomonas aeruginosa  
Not detected in 0.5 g  Annex C 
iii.  Staphylococcus aureus  
iv.  Candida albicans  
 
4.2.3 The limits for the  heavy metal  contaminants shall comply with the limits specified in Table 3.  EAS 336: 2013  
© EAC 2013  – All rights reserved  3 
  
Table 3 — Limits  for heavy metal contaminants  
S/N Contaminant  Limits  Method of test  
i.  Lead (as Pb) ppm max 20 Annex G  
ii.  Arsenic (as As 2O3), ppm 
max 2 Annex H  
iii.  Mercury, ppm, max.  2 Annex I  
NOTE  The total amount of heavy metals as lead, mercury and arsenic, in combination, in the 
finished product should not exceed 20 ppm.  
 
5 Packaging and labelling  
5.1 Packaging 
The product shall be pack aged in suitable well -sealed containers that shall protect the contents a nd shall not 
cause any contamination or react with the product.  
5.2 Labelling  
5.2.1  The labelling shall be in English, Kiswahili or French or in combi nation as agreed between the 
manufacturer and supplier . 
5.2.2  The labelling shall comply with the requirements of EAS 346 . In addition , each package shall be 
legibly and indelibly marked with the following :  
a) contact time permitted for the product to comple tely remove hair from the body ; and 
b) instructions for use.  
The following instructions shall appear on the container or on the leaflet which shall be supplied with 
the pack:  
i. never use the product beyond the time  as specified on the package  by the manufactu rer. 
ii. never use the material on inflamed or broken skin or near the eyes . Should this occur, wash  with 
boric acid solution (approximately 3 %) and rinse with water If symptoms persist, seek 
medical advice.  
iii. if used for the first time, carry out the following  patch test.  
Use a little hair remover over  5 cm2 of skin on the inner elbow. If 24 h  later the skin is normal, the 
material may be safely used.  
6 Sampling  
Random samples of the product shall be drawn for test in accordance with ISO 24153 from the market,  factory 
or anywhere else.  EAS 336: 201 3 
4 © EAC 201 3 – All rights reserved  
 Annex  A 
(normative)  
 
Determination of pH  neat 
A.1 Apparatus  
A.1.1  pH meter , equipped  with a glass electrode  
A.1.2 Beaker , of 100 mL capacity   
A.2 Reagents  
A.2.1 pH 7.0 buffer solution  
A.2.2 pH 4.0 and pH 9.0 buffer solutions  
A.2.3 Deionised water  
A.3 Procedure  
A.3.1 Dip the pH meter into about 50 mL of pH 7.0 buffer solution. Ensure that the reading is 7.0.  
A.3.2 Rinse the meter with deionised water, and dip it into about 50 mL of pH 4.0 buffer solution.  Ensure 
that the reading  is 4.0. Repeat using pH 9.0 buffer solution.  
A.3.3  Determine the pH directly in case of lotion or cream using pH meter with glass electrode.  EAS 336: 2013  
© EAC 2013  – All rights reserved  5 
 Annex B  
(normative)  
 
Determination of calcium thioglycollate  
B.1 Reagents  
B.1.1  Concentrated hydrochloric acid  
B.1.2 Standard iodine solution , 0.1 N  
B.1.3  Starch indicator solution , 0.5 % (m/v), freshly prepared  
B.2 Procedure  
Accurately weigh about 5  g of the sample in a 250 m L conical flask. Add about 75 m L of water and 15 m L of 
concentrated hydrochloric acid and h eat on a water bath for 10 min. Cool  to room temperature and then titrate 
with iodine solution using starch solution as the indicator.  
B.3 Calculation  
Calculate on the basis that each m illilitre of 0.1 N iodine solution is equivalent to 0.00921 g of thiogl ycollic acid  
as follows  
% of thioglycollic acid, m/m = 
MV 100x 0.00921x  
where 
M is the mass , in grams,  of sample taken for test ; and  
V is the volume, in millilitres,  of 0.1  N iodine solution used  EAS 336: 201 3 
6 © EAC 201 3 – All rights reserved  
 Annex C  
(normative)  
 
Determination of thermal stabili ty 
C.1 Apparatus  
Thermostatically controlled oven , capable of maintaining a temperature of 37  C ± 10 C 
C.2 Procedure  
Place a fresh unopened sample of the product in its original container into a thermostatically controlled oven 
at 37  C ± 1 C for 48 h, making sure that the sample is securely sealed. If the product is packed in an opaque 
container ( for example,  a tube), remove 50 g of the sample and place into an effectively sealed tube or vial, 
and test as above.  
C.3 Results  
The product shall be taken t o have passed the test if, on removal from the oven, the following indications of 
instability are not observed:  
a) change of colour ; 
b) change of smell or odour ; 
c) phase separation ;  
d) formation of granules or crystal growth ; and 
e) shrinkage due to evaporation of wat er. 
An unopened sample, stored at room temperature and pressure, shall be used as a control sample.  EAS 336: 2013  
© EAC 2013  – All rights reserved  7 
 Annex D  
(normative)  
 
Determination of free alkali content  
D.1 Outline of the method  
This method consists of dissolving the sample in alcohol, and titrating against standard acid.  
D.2 Reagents  
D.2.1 Phenolphthalein indicator solution , dissolve 1  g of phenolphthalein in 100 mL of 95 % (v/v) rectified 
spirit.  
D.2.2 Ethyl alcohol, freshly boiled, and neutral to phenolphthalein, 95 % (v/v) 
D.2.3 Standard hydrochlo ric acid, 0.1 N  
D.3 Procedure  
Dissolve 2 g of the product  in 100 mL of ethyl alcohol by warming, if necessary. Cool and add a few drops of 
phenolphthalein indicator. Titrate with standard hydrochloric acid.  
D.4 Calculation  
The free alkali shall be calculat ed as follows:  
Free alkali, % by mass =
.C
MVN  
where  
C is a Constant  
C = 2.4 for LiOH  
C = 4 for NaOH, KOH and LiOH  
C = 5.6 for KOH  
C = 3.7 for Ca(OH) 2 
C = 3.5 for NH 4OH 
V is the volume, in millilitres, of standard hydrochloric acid;  
N is the  normality of standard hydrochloric acid; and  
M is the mass, in grams, of the sample.  EAS 336: 201 3 
8 © EAC 201 3 – All rights reserved  
 Where mixtures of sodium, lithium and potassium hydroxide are present in the product, the free alkali content 
shall not exceed 2.5 % by mass when calculated as sodium hyd roxide.  EAS 336: 2013  
© EAC 2013  – All rights reserved  9 
 Annex E 
(normative)  
 
Determination of total viable count  
E.1 Outline of the method  
The test consists of plating a known dilution of the sample or any digest agar medium (soya bean casein is 
recommended) suitable for the total count of bacteria and fungi after incubating them for a specified period to 
permit the development of visual colonies.  
IMPORTANT  Take precaution in ascertaining that only fresh samples, from carefully sealed containers that 
had not been opened before, are used for this test.  This is very necessary for getting accurate results.  
E.2 Apparatus  
E.2.1 Tubes , of resistant glass, provided with closely fitting metal caps  
E.2.2 Autoclaves , of sufficient size. They shall keep uniform temperature within the chamber up to and 
including th e sterilizing temperature of 122  C. They shall be equipped with an accurate thermometer, located 
so as to register the minimum temperature within the sterilizing chamber, a pressure gauge and properly 
adjusted safety valves.  
E.2.3 Petri dishes , of 100 mm diameter and 15 mm depth. The bottom of the dishes shall be free from 
bubbles and scratches and shall be flat so that the medium is of uniform thickness throughout the plate.  
E.2.4 Colony counter , an approved counting aid, such as Quebec colony counter. I f such a counter is not 
available, counting may be done with a lens giving a magnification of 1.5 diameter. In order to ensure 
uniformity of conditions during counting, illumination equivalent to that provided by Quebec colony counter 
shall be employed.  
E.2.5 Balance  
E.2.6 pH meter  
E.2.7 Water bath  
E.3 Medial buffer  
E.3.1 Soya bean casein digest agar medium  
Dissolve 15 g of pancreatic digest of casein 5 g of papaic digest of soya bean meal, and 5 g of sodium 
chloride in 100 m L of distilled water contained  in a 2 -litre beaker by heating in a water bath. Add 15 g of 
powdered agar and continue boiling until the agar is completely digested. Adjust the pH to 7.5 with sodium 
hydroxide solution. Distribute in 20 -mL quantities, close the tubes with metal caps and autoclave at 123 C for 
20 min. After autoclaving, store the tubes in a cool place and use them within three weeks.  EAS 336: 201 3 
10 © EAC 201 3 – All rights reserved  
 E.3.2 Stock solution pH 7.2 phosphate buffer  
Dissolve 34 g of monobasic potassium phosphate in about 100  mL of water contained in a 500 -mL volumetric 
flask. Adjust the pH 7.2  ± 0.1 by the addition of 4 % sodium hydroxide solution. Add water to volume and mix. 
Sterilize at 122 C for 20 min. Store under refrigeration.  
E.3.3 Dilute phosphate buffer solution pH  7.2  
Dilute 1 m L of stock solution  with distilled water in the ratio of 1:800. Fill 50  mL in each of the conical flask of 
100 m L capacity. Plug the flasks with cotton and sterilize at 122 C for 20 min.  
E.4 Sterilization of apparatus  
E.4.1 Tubes  
These shall be sterilized in the autoclave a t 122  C temperature and 1.05  kg/cm2 pressure for 20 min or in a 
hot oven at 160 C for one hour.  
E.4.2 Petri dishes   
These shall be packed in drums and autoclaved at 122 C temperature and 1.05  kg/cm2 pressure for 20 min or 
individually wrapped in kraft paper and sterilized  in a hot air oven at 160 C for one hour.  
E.4.3 Pipettes  
These shall be placed in pipette cones (copper, stainless steel or aluminium) after plugging the breeder end 
with cotton and sterilized in the autoclave at 122 C temperature and 1.05  kg/cm2 pressure for 20 min, or at 
160 C for one hour in the air oven.  
E.5 Procedure  
E.5.1 Melt a sufficient number of soya bean casein digest agar medium tube in a hot water bath and 
transfer while hot in to a constant temperature water bath main tained at 48  C ± 20 C.  
E.5.2 Weigh and transfer aseptically 1 g of the sample to a conical flask containing sterile 50 m L or any 
suitable dilution factors of dilute phosphate buffer at pH 7.2. Shake well. Pipette out in 1 m L portions into three 
sterile petri dishes. Pour melted and cooled (at 45 C) soya bean casein digest agar medium over it, and 
rotate to mix thoroughly. Incubate the plate at 32 C for 72 h in an inverted position.  
E.6 Expression of  results  
Get the average number of colonies on soya b ean casein digest agar medium plates and determine the 
number of micro -organisms per gram of the sample. If no colony is recovered from any of the plates micro -
organisms can be stated as being less than 50 per gram. For calculations, refer to ISO 6887-1. EAS 336: 2013  
© EAC 2013  – All rights reserved  11 
 Annex F  
(normative)  
 
Determination of Pseodomonas aeruginosa, Staphylococcus  aureus  and Candida 
albicans  in cosmetic products  
F.1 Introduction  
A general aseptic and safety procedures should be followed.  Table F.1 gives the results of the interlaboratory 
study supporting the acceptance of the method.  
Table F.1 — Inter -laboratory study results for determination of preservation of non -eye area water -
miscible cosmetic and toiletry formulations  
 
Incidence of false - negatives among total positive samplesa) Inciden ce of false -positives among total 
negative samplesb) 
Product name  Number  Percentage  Sensitivity 
rate Number  Percentage  Sensitivity 
rate 
Shampoo  2/49 4 96 0/53 0 100 
Conditioner  3/48 6 94 0/54 0 100 
Water -in-oil emulsion  0/52 0 100 1/50 2 98 
Oil-in-water emulsion  0/51 0 100 0/51 0 100 
All combined  5/200  2 98 1/208  0.5 99.5 
a) False -negative analysis indicates a sample is adequately preserved.  
b) False -positive analysis indicates a sample is not adequately preserved.  
 
F.2 Principle of the method  
Bacte ria yeast and mould grown on laboratory media, harvested, calibrated, and inoculated into test products.  
Using serial dilutions and plate counts; the numbers of organisms surviving in the test products are 
determined over time.  Products meeting the speci fied criteria are considered adequately preserved for 
manufacture and consumer use.  Products not meeting criteria are considered inadequately preserved.  
F.3 Apparatus  
F.3.1 Jars, 2-4 oz wide -mouth, straight -side flint glass ointment jars with linerless me tal, polypropylene or 
teflon lined scre w caps  
F.3.2 Disposable borosilicate glass culture tubes , 16 mm x 125 mm, with caps  
F.3.3 Disposable borosilicate glass culture tubes, 20 mm x 150 mm, with screw caps  
F.3.4 Petri plates Z , 100 mm x 15 mm  
F.3.5 Sterile  2.2 mL pipettes  EAS 336: 201 3 
12 © EAC 201 3 – All rights reserved  
 F.3.6 Steri le swabs  
F.3.7 Glass beads  
F.3.8 Sterile gauze  
F.3.9 10 l -20 l inoculating loops  
F.3.10   Vortex mixer  
F.4 Reagents  
F.4.1 Preparation  
For convenience, dehydrated media of any brand equivalent in function may be used. Test each  lot of medium 
for sterility and growth -promotion using suitable organisms.  
F.4.2 List of reagents  
F.4.2.1 Letheen agar, contains 5.0 g pancreatic digest of casein 1.0 g dextrose, 3.0 g beef extract, 1.0 g 
lecithin, 7.0 g polysorbate 80 g, and 15.0 g agar per L.  Prepare according to manufacturer’s directions.  
Dispense into suitable containers and sterilize by autoclaving at 121 C for 15 min. Final pH should be 7.0  
0.2 at 25 C . Place in 45 C water bath until agar is 45 C  2 C.  Use for pour plates . 
F.4.2.2 D/E Neutralizing broth (Dey/Engley), contains 5.0 g pancreatic digest of casein, 2.5 g yeast 
extract, 10 g dextrose, 1.0 g sodium thiogycollate, 6.0g Na 2 S2O3 . 5H2O, 2.5 g NaHSO 3, 7.0 g lecithin, 5.0 g 
polysobate 80 g, and 0.02 g bromcresol purp le per L.  
Prepare according to manufacturer’s directions. Dispense 9  mL or 9.9 mL aliquot into tubes and sterilize by 
autoclaving at 121 C for 15 min.  Final pH should be 7.6  0.2 at 25 C.  Use for aerobic plate count, L, 
dilutions.  
F.4.2.3 Nutrient aga r, contains 5.0 g pancreatic digest of gelatin 3.0 g beef extract, and 15.0 g agar per 
L. Prepare according to manufacturer’s directions. Dispense into tubes and sterilize by autoclaving at 121 C 
for 15 min. Final pH should be 6.8  0.2 at 25 C. Cool in inclined position to form a slant. Use for bacterial 
culture maintenance and inoculum preparation.  
F.4.2.4 Y/M agar (yeast/malt extract),  contains 3.0 yeast extract, 3.0 g malt extract , 5.0 g peptone.10.0 
g dextrose  and 20.0 g agar per L. Prepare according  to manufacturer’s directions.  Dispense into tubes and 
sterilize by autoclaving at 121 C for 15 min.  Final pH should be 6.2  0.2 at 25 C. Cool in slanted position.  
Use for yeast culture maintenance and inoculum preparation.  
F.4.2.5 Potato dextrose ag ar (PDA), contains 200 g potato infusion, 20.0 g dextrose, and 15.0 g agar 
per L. Prepare according to manufacturer’s directions. Dispense into tubes and sterile by autoclaving at 121 
C for 15 min. Final pH should be 5.6  0.2 at 25 C.  Cool in slanted p osition. Use for mould culture 
maintenance and inoculum preparation.  
F.4.2.6 0.85 % Saline, dissolve 8.50 g NaCI in water and dilute to 1 L.  Dispense into flasks or bottles 
and sterilize by autoclaving at 121 C for 15 min.  
F.4.2.7 0.85 % Saline with 0.05  % Polysorbate 80,  dissolve 8.5 g NaCI in water, mix in 0.50 g 
polysorbate 80 g, and dilute to 1  L.  Dispense into suitable containers and sterilize by autoclaving at 121 C 
for 15 min.  
F.4.2.8 Barium sulphate standard No. 2   EAS 336: 2013  
© EAC 2013  – All rights reserved  13 
 F.4.2.8.1 Prepare a 1.0 % BaCI 2 solution by dissolving 1.0 g BaCI 2. 2H 2O in 100 mL water. Let this be 
referred to as solution 1.  
F.4.2.8.2 Prepare a 1.0 % H 2SO 4  solution by mixing 1.0 mL H 2SO 4 in 100 mL water. Let this be referred to 
as solution 2.  
F.4.2.8.3 Mix 0.2 mL of solution (1)  with9.8 mL solution (2), in scre w-capped test tube. Cap tightly  and store 
in the dark at room temperature.  
F.4.2.9 Barium sulphate standard No. 7, use solutions from F.4.2.8. Mix 0.7 mL of solution F.4.2.8.1, 
with 9.3 mL of solution F.4.2.8.2, in a screw -capped test tube. Cap tightly and store in the dark at room 
temperature.  
F.5 Micro -organisms  
F.5.1 Staphylococcus aureus   — ATCC 6538  
F.5.2 Staphycloccuss epidermidis  — ATCC12228  
F.5.3 Klebsiella pneumoniae   — ATCC10031  
F.5.4 Escherichia coli    — ATCC 8739  
F.5.5 Enterobacter gergoviae   — ATCC 33028  
F.5.6 Pseudomonas aeruginosa  — ATCC 9027  
F.5.7 Burkholderia cepacia   — ATCC25416  
F.5.8 Acinetobacter baumannii  — ATCC 19606  
F.5.9 Candida albicans    — ATCC10231  
F.5.10 Aspergillus niger    — ATCC 16404  
NOTE  Enviro nmental micro organisms (s) likely to be contaminants of concern during product manufacture or use be 
included as a separate inoculum. Predominant environmental microbes isolated during manufacturing, equipment 
cleaning, and sanitizing, or from related dei onized water systems are used as supplemental test inocula).  
F.6 Product quality check  
F.6.1 Weigh 1.0 g product into a screw -capped culture tube containing 9.0 mL sterile neutralizing broth to 
make a 1:10 dilution.  If necessary to disperse product, add t en to twenty 3 -mm diameter glass beads to tube. 
Mix on Vortex mixer until homogeneous.  
F.6.2 Pipette 1.0 mL of the 1:10 dilution into each of four sterile petri plates. Pour 15 mL - 20 mL sterile 
molten Letheen agar (45 C  2 C) into each plate. Mix by ro tating plates to disperse the dilution thoroughly. 
Let solidify.  
F.6.3 Invert and incubate 2 plates at 35 C  2 C for 48 h and two plates at 25 C  2 C for 5 days.  
F.6.4 Count the number of colonies on all plates, add, and multiply by 2.5 to determine the number of 
colony forming units per gram (cfu./g) in the product.  
F.6.5 Save plates to be used for the neutralization validation by refrigerating.  EAS 336: 201 3 
14 © EAC 201 3 – All rights reserved  
 F.7 Product preparation  
F.7.1 Measure 20 mL sterile saline into four sterile jars, F.3.1. Cap tightly and store at room temperature.  
F.7.2 Weigh 20 g produc t into each of four sterile jars, F .3.1. Cap tightly and store at room temperature.  
F.8 Bacterial inocul um preparation  
F.8.1 Streak each bacteria culture, F.5.1 – F.5.10, onto a nutrient agar  slant , F.4.2 .3. Incubate for 48 h at 35  
C  2  C. Wash each slant with 5.0 mL sterile saline, loosening the culture from the agar surface. Transfer 
the suspension into a sterile tube. Repeat the wash with second 5.0 mL aliquot of saline. Combine washes 
and mix on Vort ex mixer to disperse evenly.  
F.8.2 Adjust each wash with sterile saline to yield a suspension of ca 108 cfu/mL using Mc Farland BaSO 4 
standard No, 2,  F.4.2.8, direct microscopic count, turbidimetry, absorbance, or other method correlated to  an 
aerobic plat e count (APC), F.13 . Perform an APC, F.13, on each suspension to confirm standardization.  
F.9 Fungal inoculum preparation  
F.9.1 Streak  C. albicans,  F.5.9, on 3 slants of Y/M agar, F.4.2 .4. Incubate at 25 C  2 C for 48 h. Wash 
each slant sequentially wit h 5.0 mL aliquot of sterile saline.  Repeat with a second 5.0 mL aliquot of sterile 
saline. Combine washes to produce 10 mL suspension. Mix on Vortex mixer to disperse evenly.  
F.9.2 Adjust the wash with sterile saline to yield a suspension of ca 107 cfu/ml  using a Mc Farland BaSO 4 
standard No. 7, F.4.2 .8, direct microscopic count, turbidimetry, absorbance, or other method that has been 
correlated to an APC, F.13. Perform an APC, F.13, on the suspension to confirm standardization.  
F.9.3 Streak  A. Niger , F.5.10, on 5 slants of potato dextrose agar F.3.5. Incubate at 25 C  2 C for 10 
days. Dislodge mould spores by adding 5.0 mL sterile saline containing 0.05 % polysorbate 80 to each tube 
and vigorously rubbing the surface of the agar slant with a sterile swa b.  Repeat with a second 5.0 mL aliquot 
in each tube.  Combine the 10 washes to produce 50 mL suspension. Filter into a sterile container through 3  - 
5 layers of sterile gauze supported in funnel.  Perform an APC, F.13, using appropriate dilutions. Adjust mould 
suspension to ca 107 per ml using sterile saline.  Use immediately or refrigerate at 2 C - 5 C for up to 1 
month. Verify mould viability by an APC, F.13, before each use.  
F.10  Inoculum pools  
F.10.1 Pool equal parts of the S. aureus  and S . epiderm idis  suspensions, F.8.2 in a sterile container to 
make incoculum pool 1: Gram -postive cocci.  
F.10.2 Pool equal parts of the K pneumoniae  , E . coli, and E . gergoviae  suspensios, F.8.2, in  a sterile  
container to make inoculum pool  2: Gram - negative ferm entors.  
F.10.3 Pool equal parts of the P. aeruginosa , B .cepacia  and A. baumanii suspensions, F.8.2, in a sterile 
container to make Inoculum Pool 3: Gram -negative nonfermentors.  
F.10.4 Pool equal parts of C. Albicans, F.9.2, and A. Niger, F.9.3, suspension s in a sterile container to make 
innoculum pool 4: Fungi.  
F.10.5 Use organism pools immediately or refrigerate them at 2 C - 5 C for more than 72 h.  EAS 336: 2013  
© EAC 2013  – All rights reserved  15 
 F.11  Inoculation  
F.11.1 Inoculate each of the four 20.0 mL aliquots of sterile saline, F.7.1, with 0.2 m L of its respective 
inoculum pool, F.10.1 - F.10.4. Mix thoroughly.  Use these suspensions to determine inoculums counts (see 
Ka). 
F.11.2 Inoculate each of the four 20 g products suspensions, with 2.0 mL of its respective inoculums , F.10.1 
– F.10.4 . Mix th oroughly by shaking, Vortex mixing or stirring, so that each suspension contains 106 bacteria 
or 10 5 fungi per gram, evenly distribute throughout the product. Tightly close inoculated containers and store 
at ambient temperature (20 C - 25 C). 
F.12  Samp ling intervals  
F.12.1 Sample each inoculated saline suspension, F.11.1, for APC, F.13, within 1 h after inoculation to obtain 
inoculum count.  
F.12.2 Test each inoculated product, F.11.2 for APC, F.13, at 7, 14 and 28 days after inoculation to obtain 
produc t interval count.  
F.13  Aerobic Plate Count (APC)  
F.13.1 Mix suspension thoroughly . Weigh 1.0 g product into screw -capped culture tube containing 9.0 mL 
sterile neutralizing broth for a 1:10 dilution. If necessary to disperse product, add 10 - 20 sterile 3  mm 
diameter glass beads to the tube. Mix on Vortex mixer until homogeneous.  
F.13.2 Aseptically pipette 0.1 mL of the 1: 10 dilution into 9.9 mL tube of neutralizing broth to obtain a 1:1000 
dilution. Vortex mix. Pipette 0.1 mL of the 1:1000 dilution into 9.9 mL neutralizing broth to obtain a 1: 100 000 
dilution.  
The number of dilutions may be decreased if previous counts microbial populations show reduction.  
F.13.3 Using a 2.2 mL pipette, aseptically pipette 1.0  mL and 0.1 mL aliquots from the 1:10 dilutio n into 
duplicate petri dishes for the 1:10 and 1:100 plates. If necessary, transfer duplicate 1.0  mL and 0.1 mL 
aliquots from the 1: 1000 dilution for plates 1:1000 and 1:10 000, and from the 1:100 000 dilution for plates 
1:100 000 and 1:1000 000. Pour 15  mL - 20 mL sterile Letheen agar F.3.1, (45 C  2 C into each plate. Mix 
by rotating the plates to disperse the suspension thoroughly, and let solidify.  
F.13.4 Invert bacterial plates and incubate at 35 C  2 C.  Examine bacterial plates after 48 h - 72 h.  Count 
in suitable range (30 - 300 colonies). If no countable plates fall in that range, count the plate(s) nearest that 
range showing distinct colonies. Average duplicate plates counts and express results as cfu/g of product.  
F.13.5 Invert and incubat e fungal plates at 25 C  2 C.  Read fungal plates at 2 - 3 days and record results. 
Count plates in a suitable range (30 - 300 colonies). If no countable plates fall in that range, count the plate(s) 
nearest that range showing distinct colonies. Re -incubate plates for another 2 - 3 days. Read and record 
additional colonies. Add to previous results to obtain total counts. Average duplicate plate counts and record 
as cfu/g of products.   
F.14  Neutralization check  
Make a 1:10 000 dilution in steri le saline  of pools 1, 2 and 3, F .10.1 - F.10.3, and  a 1:1000 dilution of pool 4, 
F.10.4. Streak each dilution for isolation with a 10 l loop on the plates s aved from F .6.5.  If plates are not 
usable due to either desiccation or surface growth, repeat F .6, and stre ak freshly pr epared plates.  Incubate 
as in F. 13.4 - F.13.5 EAS 336: 201 3 
16 © EAC 201 3 – All rights reserved  
 F.15  Data analysis  
F.15.1 Product quality check , F.6.4, should  be found to contain, <100 cfu/g to proceed with the challenge 
test. 
F.15.2 Inoculums counts , F.12.1, should be between 1 to 9.9 x 106 cfu /g product for bacteria and 1 to 9.9 x 
105 cfu/g product for fungi, or the test should be repeated with different dilutions.  
F.15.3 Neutralization check , F.14, should  show significant growth of all pools to confirm adequate 
neutralization.  A neutral izing broth other than D/E broth can be used.  If neutralization does not occur, the test 
is invalid.  Refer to references 4 -6 in AOAC  official method 998.10  for assistance.  
F.15.4 Calculate the percentage reduction:  
count Inoculum100 count interval product count Inoculum% Reduction,
 
 
F.15.5 The tes t product is considered adequately preserved if  
a) bacteria show at least 99.9 % (3 log) reduction within one week following challenge and remain at or 
below that level thereafter, and  
b) fungi show at least a 90 % (1 log) reduction within one week following c hallenge, and remain at or below 
that level thereafter. This criteria applies to freshly prepared formula tions.  EAS 336: 2013  
© EAC 2013  – All rights reserved  17 
 Annex G 
(normative)  
 
Test for lead using atomic absorption spectrophotometer (AAS)  
G.1 Scope  
This method describes the determination of lead in various cosmetic products. No interference occurs  from 
the high concentrations of bismuth which do interfere in the dithzone calorimetric procedure.  
G.2 Reagents  
G.2.1 Lead nitrate , Pb(NO 3)2 
G.2.2 Dimethylacetamide  DMA  
G.2.3 Nitric acid  HNO 3, 3N. Prepare by diluting 195  mL of concentrated HNO 3 (15.4  N) to one litre with 
deionized water.  
G.2.4 Ethanol  C2H5OH  
G.2.5 Bismuth oxychloride  BiOCl 
G.2.6 Hydrochloric acid  HCl, 6 N. Prepare by diluting 516 m L of concentrated HC l (11.6  N) to  one litre with 
deionized water.  
G.2.7 Hydrochloric acid  HCl, 2 N. Prepare by diluting 172 m L of concentrated HC l (11.6  N) to one litre with 
deionized water.  
G.2.8 Hydrochloric acid  HCl, 0.5 N. Prepare by diluting 25 m L of 2 N HC l to 100 m L with deionized water.  
G.3 Standard soluti ons 
G.3.1 Lead standard solution , 100 μg/mL. Dissolve 0.1598  g of Pb(NO 3)2 in 10 mL of dilute HNO 3 and 
dilute to 1000 m L with deionized water . 
G.3.2 Lead standard solution , 1000  μg/mL in DMA . Dissolve 0.1598  g of Pb(NO 3)2 in DMA. Dilute to  100 
mL.  
G.4 Sample preparation  
G.4.1 Aerosols  
For hai r, deodorant spray or similar aerosols, weigh accurately about 5  g of sample and dissolve in  50 mL of 
ethanol. For shaving cream spray or similar aerosols weigh accurately about 1 g of sample and dissolve in 50 
mL of ethanol.  EAS 336: 201 3 
18 © EAC 201 3 – All rights reserved  
 G.4.2 BiOCl or cosmetics conta ining B IOCl 
Dissolve 1 g of sample (5  g if the lead level is expected to be less than 10  μg/g) in 15 m L of 6 N HCI and  dilute 
to 100 m L with 0.5  N HCI. If the sample is coated with an organ ic material, it is necessary to ignite the sample 
to 500  C to ash before analysis.  
G.5 Instrument conditions  
G.5.1 Standard atomic absorption conditions fo r lead  
G.5.1.1  Recommended flame air -acetylene , oxidizing (lean, blue)  
G.5.1.2  Data obtained with a standard nebulizer and flow spoiler. Operation with a High sensitivity 
nebulizer or impact bead will typically p rovide a 2 -3x sensitivity improvement.  
G.5.1.3 Characteristic concentration with a N 2O-C2H2 flame at 283.3  nm: 2.7  mg/L.  
Table G.1 — HCl data EDL sensitivity values  for lead  
Wavelength , 
nm Slit  
nm Relative noise  Characteristic 
concentration , 
mg/L  Characteristic 
concentration 
check ,  
mg/L  Linear range , 
mg/L  
283.3  0.7 0.43 0.45 20.0 20.0 
217.0  0.7 1.0 0.19 9.0 20.0 
205.3  0.7 1.4 5.4 250.0  - 
202.2  0.7 1.8 7.1 350.0  - 
261.4  0.7 0.35 11.0 500.0  - 
368.3  0.7 0.40 27.0 1200.0  - 
364.0  0.7 0.33 67.0 3000.0  - 
 
G.5.2 Standard flame emission condition s for lead  
Stock standard solution , lead, 1000  mg/L . Dissolve 1.598  g of lead nitrate, Pb( NO3)2 in 1 %(v/v)  HNO 3 and 
dilute to 1 litre with 1 % (v/v) HNO 3 
Table G.2 — Flame  emission conditions for lead  
Wavelength (nm)  Slit (nm)  Flame  
405.8  0.2 Nitrous oxi de- acetylene  
 
G.5.3 Light sources  
Both Electrodeless Discharge Lamps (EDLS) and Hollow Cathode Lamps are available for lead. EDLs  provide 
greater light output and longer life than Hollow Cathode Lamps. For lead, both EDLs and  Hollow Cathode 
Lamps provide  approximately the same sensiti vity and detection limit. With multi -element lamps containing 
copper, the Cu 216.5  nm resonance line may interfer e with lead determinations at the lead 217.0 nm line. The 
lead 283.3  nm line should be used instead.  EAS 336: 2013  
© EAC 2013  – All rights reserved  19 
 G.5.4 Interferences  
Large excesses of other elements ( for example , 10 000 mg/L Fe) May interfere with the lead signal .  
G.6 Analysis  
Determine the concentration of lead in the sample solutions of aerosols and similar products using the  
standard conditions for lead a nd standards prepared in ethanol. The standard solutions shall be the following 
concentrations: 0  ppm, 10 ppm, 20 ppm, 30 ppm, 40 ppm and 50 ppm. Determine the concentration  of lead in 
the BiOCI solutions using the st andard conditions for lead and the meth od of additions.  
G.7 Calculations  
The total amount of lead shall be calculated as follows:  
µg/g Pb =
sampleofgPbμg/mL x 50 
G.8 Interferences  
No interference was found from 100 0 μg/mL of Na, Mg, K, or Ca, or from 500  μg/mL of Mn, Co or Ni. A 
background  absorption interference was noted with 100 0 μg/mL of Al, Fe or Bi, which can be eliminated by 
correcting the lead absorption at 283 nm by any absorption observed at 280 nm, or by using the Deuterium 
Background Corrector.  
 EAS 336: 201 3 
20 © EAC 201 3 – All rights reserved  
 Annex H 
(normative)  
 
Test for ars enic using atomic absorption spectrophotometer (AAS)  
H.1 Scope  
This method describes the determination of arsenic in various cosmetic products.  
H.2 Reagents  
The reagents used should be of analytical reagent grade. Water shall be distilled or di -ionised.  
H.3 Instrument conditions  
H.3.1 Standard atomic absorption conditions for arsenic  
H.3.1.1  Recommended flame; air -acetylene, reducing (rich, slightly yellow)  
H.3.1.2  Data obtained with a standard nebulizer and flow spoiler. Operation with a High Sensitivity  
nebulizer or impact bead will typically provide a 2 -3X sensitivity improvement.  
H.3.1.3  Characteristic concentration with a N 2O-C2H2 flame at 193.7 M: 1.4mg/L  
H.3.1.4  Table contains EDL data.  HCL sensitivity values are more than 25 % poorer.  
Table H.1 — HCl data EDL sensitivity values  for arsenic  
Wavelength ,  
nm Slit,  
nm Relative noise  Characteristic 
concentration ,  
mg/L  Characteristic  
concentration 
check ,  
mg/L  Linear range , 
mg/L  
193.7  0.7 1.0 1.0 45.0 100.0  
189.0  0.7 1.8 0.78 40.0 180.0  
197.2  0.7 0.95 2.0 90.0 250.0  
 
H.3.2 Stock standard solution  
Arsenic , 1000  mg/L . Dissolve 1.320 g of Arsenious oxide A s2O3, in 25  mL of 20  % (w/v) KOH solution. 
Neutralize with 20  % (v/v) H 2SO 4 to a phenolphthalein endpoint. D ilute to one litre with 1  % (v/v) H2SO 4. The 
standard solutions shall be of the following concentrations: 0  ppm, 2 ppm, 4 ppm, 6 ppm, 8 ppm and 10 ppm.  
H.3.3 Flames  
The air -acetylene flame absorbs or scatters more than 60  % of the light source radiation at the  193.7  nm 
arsenic line. Flame absorp tion is reduced with the use of the nitrous oxide -acetylene flame, although EAS 336: 2013  
© EAC 2013  – All rights reserved  21 
 sensitivity is also reduced. Use of background correction is r ecommended, as it will correct for flame 
absorption and thus improve the signal to noise ratio.  
H.3.4 Light  sources  
Both HCL and EDL sources are available for arsenic. EDLS, which are more intense, provide better 
performance and longer life.  
H.3.5 Interferences  
Sample with high total salt content (greater than 1  %) can produce non -specific absorption at the  193.7  mn 
arsenic line, even when the metal is absent. It is there fore advisable to se t background correction.  
H.4 Sample preparation  
As in G.4.  
H.5 Analysis  
As in G.6.  
H.6 Calculation  
As in G.7 EAS 336: 201 3 
22 © EAC 201 3 – All rights reserved  
 Annex I 
(normative)  
 
Test for mercury using the atomic absorption spectro photometer (AAS)  
I.1 Scope  
This method describes the determination of mercury in various cosmetic products.  
I.2 Reagents  
The reagents used should be analytical reagent grade.  Water shall be distilled or de -ionised.  
I.3 Instrument conditions  
I.3.1 Standar d atomic absorption conditions for mercury  
I.3.1.1  Recommended flame: air -acetylene, oxidizing (lean, blue)  
I.3.1.2  Data obtained with a standard nebulizer and flow spoiler. Operation with a High Sensitivity 
nebulizer or impact bead will typically provide a 2-3X sensitivity improvement.  
I.3.1.3  Characteristic Concentration with a N 2O-C2H2 flame at 253.7 nm: 12 mg/L  
I.3.1.4  Table contains EDL data.  HCL sensitivity values more than 25 % poorer  
Table I.1 — HCl data EDL sensitivity values  for mercury  
Waveleng th,  
nm Slit,  
nm Relative noise  Characteristic 
concentration ,  
mg/L  Characteristic 
concentration 
check ,  
mg/L  Linear range , 
mg/L  
253.7  0.7 1.0 4.2 200.0  300.0  
 
I.3.2 Standard flame emission conditions for mercury  
Table I.2 — Flame  emission conditions fo r mercury  
Wavelength (nm)  Slit (nm)  Flame  
253.7  0.2 Nitrous oxide - acetylene  
I.3.3 Stock standard solution  
Mercury , 1000  mg/L — Dissolve 1.080 g of mercury (II) oxide, HgO, in a minimum volume of (1 + 1) HCI. 
Dilute to one litre with deionized water. The  standard solutions shall be of the following concentrations: 0  ppm, 
2 ppm, 4 ppm, 6 ppm, 8 ppm and 10 ppm.  EAS 336: 2013  
© EAC 2013  – All rights reserved  23 
 I.3.4 Light sources  
Both Electrodeless Discharge Lamps (EDLS) and Hollow Cathode Lamps are available for Mercury. However, 
the light output of mercu ry Hollow Cathode Lamp is significantly poorer than with EDLs, and the sensitivity and 
detection limit achieved also are much poorer. In addition, the life of Hollow Cathode Lamps is much shorter.  
I.3.5 Interferences  
Large co ncentrations of cobalt will absorb at the mercury 253.7  nm resonance line. A 1000  mg/L cobalt 
solution  produces approximately 10 % absorption. Ascorbic acid, stannous chloride, or other reducing agents 
may reduce the mercury present to Hg  (I) or elemental mercury. These give higher sensitivities than Hg  (II), 
and their presence can generate erroneously high results.  
I.4 Sample preparation  
As in G.4 
I.5 Analysis  
As in G.6 
I.6 Calculations  
As in G.7 
 
. EAS 336: 2013    
 
© EAC 2013 – All rights reserved