FINAL DRAFT UGANDA 
STANDARD  
  FDUS EAS 339 
First  Edition  
2013-mm-dd 
 
 Reference number  
FDUS EAS 339: 2013 
© UNBS 201 3 
  
Hair creams, lotions and gels —  Specification  
 FDUS EAS 339: 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 for its correct application  
© UNBS 2013 
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 written 
permission from UNBS. 
Requests for permission to reproduce this document should be addressed to 
The Executive Director 
Uganda National Bureau of Standards 
P.O. Box 6329 
Kampala 
Uganda 
Tel: 256 41 505 995 
Fax: 256 41 286 123 
E-mail: unbs@infocom.co.ug  
Web: www.unbs.go.ug  
 FDUS EAS 339: 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 3 27, of the Laws of Uganda. UNBS is mandated to co-ordinate the elaboration 
of standards and is  
(a) a member of International Organisation for Standardisation (ISO) and  
(b) a contact point for the WHO/FAO Codex Alimentarius Commission on Food Standards, and  
(c) the National Enquiry Point on TBT/SPS Agreements 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 
consumers, 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 recommending the draft standards 
for approval and declaration as Uganda Standards by the National Standards Council. 
This Final Draft Uganda Standard, FDUS EAS 339: 2013, Hair creams, lotions and gels  — Specification , is 
identical with  and has been r eproduced from an East African  Standard, EAS 339: 2013, Hair creams, lotions 
and gels  — 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 339: 2013 
ICS 71.100.70  
© EAC 2013                                                                                                                         Second Edition 2013  
 
EAST AFRICAN STANDARD 
 
Hair creams, lotions and gels — Specification  
EAST AFRICAN COMMUNITY  EAS 339: 2013 
ii © EAC 2013 – 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 permitted 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 for 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 339: 2013 
© EAC 2013 – All rights reserved  iii 
 Contents Page 
Foreword ............................................................................................................................................................  iv 
1 Scope  ................................ ................................ ................................ ................................ ...................... 1 
2 Normative references  ................................ ................................ ............................................................  1 
3 Description  ................................................................ ................................ ................................ ............. 1 
4 Requirements  ................................................................ ................................ ................................ ......... 2 
4.1 General requirements  ................................ ................................ ...........................................................  2 
4.2 Specific requirements  ................................ ................................ ...........................................................  2 
5 Packaging and labelling ................................ ................................ ................................ ........................ 3 
5.1 Packaging  ................................ ................................ ................................ ................................ ............... 3 
5.2 Labelling  ................................ ................................ ................................ ................................ ................. 3 
6 Sampling  ................................ ................................ ................................ ................................ ................ 3 
Annex A (normative)  Determination of pH ...................................................................................................... 4  
A.1 Apparatus  ................................ ................................ ................................ ................................ ............... 4 
A.2 Reagents  ................................ ................................ ................................ ................................ ................ 4 
A.3 Procedure  ................................ ................................ ................................ ................................ ............... 4 
Annex B (normative)  Determination of thermal stability ............................................................................... 5  
B.1 Apparatus  ................................ ................................ ................................ ................................ ............... 5 
B.2 Procedure  ................................ ................................ ................................ ................................ ............... 5 
B.3 Results  ................................ ................................ ................................ ................................ .................... 5 
Annex C (normative)  Test for rancidity ........................................................................................................... 6  
C.1 Reagent  ................................ ................................ ................................ ................................ .................. 6 
C.2 Procedure  ................................ ................................ ................................ ................................ ............... 6 
Annex D (normative)  Determination of total viable count ............................................................................. 7  
Annex E (normative)  Determination of Pseudomonas aeruginosa, Staphylococcus  aureus  and 
Candida albicans  in cosmetic products  ................................ ..............................................................  9 
Annex F (normative)  Test for lead using atomic absorption spectrophotometer (AAS) .........................  15 
Annex G (normative)  Test for arsenic using atomic absorption spectrophotometer (AAS) ................... 18 
Annex H (normative)  Test for mercury using the atomic absorption spectrophotometer (AAS) ........... 20 
Bibliography ...................................................................................................................................................... 22 
 EAS 339: 2013 
iv © EAC 2013 – All rights reserved  
 Foreword 
Development of the East African Standards has been necessitated by the need for harmonizing requirements 
governing quality of products and services in the East African Community. It is envisaged that through 
harmonized standardization, trade barriers that are encountered when goods and services are exchanged 
within the Community will be removed.  
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 339 was prepared by Technical Committee EAS/TC 071, Cosmetics and cosmetic products.  
This second edition cancels and replaces the first edition (EAS 339:2004), which has been technically revised   
 EAST AFRICAN STANDARD  EAS 339:201 3 
 
© EAC 2013 – All rights reserved  1 
 Hair creams, lotions and gels — Specification 
1 Scope 
This East African Standard specifies the requirements and methods of sampling and test for hair creams, 
lotions and gels based on vegetable oil or mineral oil, or any combination of the above, with fatty acids or fatty 
acid emulsions.  
It also applies to hair conditioners and setting lotions.   
This East African standard does not cover hair sprays, hair sheens or hair oils including hair creams, lotions 
and gels for which therapeutic claims are made.  
2 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. 
EAS 334 , List by category of cosmetic products  
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 cosmetic 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 allowed in cosmetic products  
EAS 377- 5, Cosmetics and cosmetic products  Part 5: Use of UV filters in cosmetic products 
ISO 11930, Cosmetics — Microbiology — Evaluation of the anti-microbial protection of a cosmetic product  
ISO 24153, Random sampling and randomization procedures  
3 Description  
Hair creams, lotions and gels are products used to enhance glossy appearance on the hair; stimulate the 
scalp and used as an anti-dandruff; and condition the hair. EAS 339: 2013 
2 © EAC 2013 – All rights reserved  
 4 Requirements 
4.1 General requirements 
4.1.1 The hair cream and lotion shall be in the form of an emulsion and of a uniform colour showing no 
signs of separation nor visible impurities. 
4.1.2 Substances used, including dyes, colours, pigments, perfumes or preservatives if added, shall be of 
pharmaceutical or cosmetic grade and shall comply with all parts of EAS 377.  
4.1.3 The product shall not be harmful to the scalp or hair when used as intended by the manufacturer. 
4.2 Specific requirements 
4.2.1 The hair cream, lotion or gel shall comply with the chemical requirements as specified in Table 1 
when tested according to the specified methods of test. 
Table 1 — Chemical requirements for hair creams, lotions and gels 
SI No  Characteristic  Requirements  Test method  
i.  pH: 
 Hair cream gel or lotion  
 Hair conditioners*  
5 - 8 
3 - 6  
Annex A  
ii.  Thermal stability  To pass test  Annex B  
iii.  Rancidity  To pass test  Annex C  
*Hair conditioners shall inclu de lotions as well as cream treatment conditioners (rinse off).  
 
4.2.2  Microbiological limits shall comply with the limits specified in Table 2. 
Table 2   Microbiological limits  
Micro -organisms  Limits, max.  
Cfu/g  Method of test  
Total viable count  100 in  0.5 g  Annex D  
Pseudomonas aeruginosa  
Not detect ed in 0.5 g  Annex E  
Staphylococcus aureus  
Candida albicans  
 
4.2.3  The products shall comply with the limits for heavy metal contaminants in accordance with Table 3. EAS 339: 2013 
© EAC 2013 – All rights reserved  3 
 Table 3 — Limits for heavy metal contaminants 
S/N Characteristic  Limit  Method of test  
i.  Lead, ppm, max  20 Annex F 
ii.  Arsenic, ppm, max  2 Annex G 
iii.  Mercury, ppm, max  2 Annex H 
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 and 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 combination as agreed between the 
manufacturer and supplier. 
5.2.2  The labelling shall comply with the requirements of EAS 346.  
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 339: 2013 
4 © EAC 2013 – All rights reserved  
 Annex A 
(normative) 
 
Determination of pH 
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 For gels and oil- in-water emulsion creams or lotions, weigh 5 g ± 0.01 g of the sample in a 100-mL 
beaker. Add 45 mL of water and disperse the sample in it. Determine the pH of the suspension at 25 C using 
the pH meter. 
A.3.4 For water- in-oil emulsion creams and lotions, weigh 10 g of the cream to the nearest 0.1 g. Add 90 mL 
of rectified spirit previously adjusted to pH 6.5 to 7.0. Warm, if necessary, to 45 C and stir thoroughly for 15 
min. Filter the alcohol layer through a filter paper and measure the pH of the filtrate at 25 C using pH using 
meter. 
Determine the form of cream by placing some of it on spot tile and sprinkling with a mixed indicator consisting 
of an intimate mixture of oil soluble dye of one colour; for example, oil orange, and a water-soluble dye of a 
different colour for example, methylene blue. After a few minutes, the predominant colour indicates whether 
the continuous phase is oil or water. In case of doubt, matter is confirmed by checking whether the product is 
capable of conducting electricity: if so the cram is deemed to be water continuous. EAS 339: 2013 
© EAC 2013 – All rights reserved  5 
 Annex B  
(normative) 
 
Determination of thermal stability 
B.1 Apparatus 
Thermostatically controlled oven , capable of maintaining a temperature of 37 C ± 1 C 
B.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 (e.g. a tube), remove 50 g of the sample and place into an effectively sealed tube or vial, 
and test as above. 
B.3 Results 
The products shall be taken to 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 water. EAS 339: 2013 
6 © EAC 2013 – All rights reserved  
 Annex C 
(normative) 
 
Test for rancidity 
C.1 Reagent 
C.1.1  Concentrated hydrochloric acid 
C.1.2  Phloroglucinol solution 
Dissolve 0.1g of phloroglucinol in 100 mL of diethyl ether. 
C.2 Procedure 
C.2.1  Shake 10 mL of the material, melted if necessary, with 10 mL of concentrated hydrochloric acid and 
10 mL of phloroglucinol solution. Shake for 1 min. 
C.2.2  The sample shall be taken to have passed the test if no pink colour develops. EAS 339: 2013 
© EAC 2013 – All rights reserved  7 
 Annex D 
(normative) 
 
Determination of total viable count 
D.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. 
D.2 Apparatus 
D.2.1 Tubes, of resistant glass, provided with closely fitting metal caps 
D.2.2 Autoclaves , of sufficient size. They shall keep uniform temperature within the chamber up to and 
including the 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. 
D.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.  
D.2.4 Colony counter , an approved counting aid, such as Quebec colony counter. If 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.  
D.2.5 Balance 
D.2.6 pH meter 
D.2.7 Water bath 
D.3 Medial buffer 
D.3.1 Soya bean casein digest agar mediu m  
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 mL 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 339: 2013 
8 © EAC 2013 – All rights reserved  
 D.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. 
D.3.3 Dilute phosphate buffer solution pH 7.2  
Dilute 1 mL of stock solution with distilled water in the ratio of 1:800. Fill 50 mL in each of the conical flask of 
100 mL capacity. Plug the flasks with cotton and sterilize at 122 C for 20 min. 
D.4 Sterilization of apparatus 
D.4.1 Tubes 
These shall be sterilized in the autoclave at 122 C temperature and 1.05 kg/cm2 pressure for 20 min or in a 
hot oven at 160 C for one hour.  
D.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.  
D.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. 
D.5 Procedure 
D.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 maintained at 48 C ± 20 C.  
D.5.2 Weigh and transfer aseptically 1 g of the sample to a conical flask containing sterile 50 mL or any 
suitable dilution factors of dilute phosphate buffer at pH 7.2. Shake well. Pipette out in 1 mL 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.  
D.6 Expression of results 
Get the average number of colonies on soya bean 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 EAS 217- 1. EAS 339: 2013 
© EAC 2013 – All rights reserved  9 
 Annex E 
(normative) 
 
Determination of Pseu domonas aeruginosa, Staphylococcus  aureus  and 
Candida albicans  in cosmetic products  
E.1 Introduction 
A general aseptic and safety procedures should be followed. Table E.1 gives the results of the interlaboratory 
study supporting the acceptance of the method. 
Table E .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) Incidence 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 in dicates a sample is adequately preserved.  
b) False -positive analysis indicates a sample is not adequately preserved.  
 
E.2 Principle of the method  
Bacteria 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 specified criteria are considered adequately preserved for 
manufacture and consumer use.  Products not meeting criteria are considered inadequately preserved. 
E.3 Apparatus  
E.3.1  Jars, 2-4 oz wide-mouth, straight-side flint glass ointment jars with linerless metal, polypropylene or 
teflon lined screw caps 
E.3.2  Disposable borosilicate glass culture tubes , 16 mm x 125 mm, with caps 
E.3.3  Disposable borosilicate glass culture tubes, 20 mm x 150 mm, with screw caps 
E.3.4  Petri plates Z , 100 mm x 15 mm 
E.3.5  Sterile 2.2 mL pipettes EAS 339: 2013 
10 © EAC 2013 – All rights reserved  
 E.3.6 Sterile swabs 
E.3.7 Glass beads 
E.3.8 Sterile gauze 
E.3.9 10 l -20 l inoculating loops 
E.3.10  Vortex mixer 
E.4 Reagents  
E.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. 
E.4.2 List of reagents 
E.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. 
E.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 purple 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. 
E.4.2.3 Nutrient agar,  contains 5.0 g pancreatic digest of gelatin 3.0 g beef extract, and 15.0 g agar per 
L. Prepare accord ing 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. 
E.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. 
E.4.2.5  Potato dextrose agar (PDA), contains 200 g potato infusion, 20.0 g dextrose, and 15.0 g agar 
per L. Prepare according to m anufacturer’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 position. Use for mould culture 
maintenance and inoculum preparation. 
E.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. 
E.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. 
E.4.2.8  Barium sulphate standard No. 2   EAS 339: 2013 
© EAC 2013 – All rights reserved  11 
 E.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. 
E.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. 
E.4.2.8.3 Mix 0.2 mL of solution (1) with9.8 mL solution (2), in screw-capped test tube. Cap tightly and store 
in the dark at room temperature. 
E.4.2.9  Barium sulphate standard No. 7, use solutions from E .4.2.8.  Mix 0.7 mL of solution E.4.2.8.1, 
with 9.3 mL of solution E.4.2.8.2, in a screw-capped test tube. Cap tightly and store in the dark at room 
temperature. 
E.5 Micro-organisms  
E.5.1  Staphylococcus aureus  — ATCC 6538 
E.5.2 Staphycloccuss epidermidis  — ATCC12228 
E.5.3 Klebsiella pneumoniae   — ATCC10031 
E.5.4 Escherichia coli    — ATCC 8739 
E.5.5 Enterobacter gergoviae  — ATCC 33028 
E.5.6 Pseudomonas aeruginosa  — ATCC 9027 
E.5.7 Burkholderia cepacia   — ATCC25416 
E.5.8 Acinetobacter baumannii  — ATCC 19606 
E.5.9 Candida albicans    — ATCC10231 
E.5.10 Aspergillus niger    — ATCC 16404 
NOTE Environmental 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 deionized water systems are used as supplemental test inocula). 
E.6 Product quality check  
E.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 ten to twenty 3-mm diameter glass beads to tube. 
Mix on Vortex mixer until homogeneous. 
E.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 rotating plates to disperse the dilution thoroughly. 
Let solidify. 
E.6.3 Invert and incubate 2 plates at 35 C  2 C for 48 h and two plates at 25 C  2 C for five days. 
E.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. 
E.6.5 Save plates to be used for the neutralization validation by refrigerating. EAS 339: 2013 
12 © EAC 2013 – All rights reserved  
 E.7 Product preparation  
E.7.1  Measure 20 mL sterile saline into four sterile jars, E.3.1. Cap tightly and store at room temperature. 
E.7.2 Weigh 20 g product into each of four sterile jars, E.3.1. Cap tightly and store at room temperature. 
E.8 Bacterial inoculum preparation  
E.8.1  Streak each bacteria culture, E.5.1 – E.5.10, onto a nutrient agar slant, E.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 Vortex mixer to disperse evenly. 
E.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,  E.4.2.8, direct microscopic count, turbidimetry, absorbance, or other method correlated to an 
aerobic plate count (APC), E.13. Perform an APC, E.13, on each suspension to confirm standardization. 
E.9 Fungal inoculum preparation  
E.9.1  Streak  C. albicans,  E.5.9, on 3 slants of Y/M agar, E.4.2.4. Incubate at 25 C  2 C for 48 h. Wash 
each slant sequentially with 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. 
E.9.2 Adjust the wash with sterile saline to yield a suspension of ca 107 cfu/ml using a Mc Farland Ba SO 4 
standard No. 7, E.4.2.8, direct microscopic count, turbidimetry, absorbance, or other method that has been 
correlated to an APC, E.13. Perform an APC, E.13, on the suspension to confirm standardization. 
E.9.3 Streak  A. Niger , E.5.10, on 5 slants of potato dextrose agar E.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 swab.  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, E.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, E.13, before each use. 
E.10  Inoculum pools  
E.10.1  Pool equal parts of the S. aureus  and S . epidermidis   suspensions, E.8.2 in a sterile container to 
make incoculum pool 1: Gram-postive cocci. 
E.10.2  Pool equal parts of the K pneumoniae  , E . coli, and E . gergoviae  suspensios, E.8.2, in a sterile  
container to make inoculum pool  2: Gram- negative fermentors. 
E.10.3 Pool equal parts of the P. aeruginosa , B .cepacia  and A. baumanii suspensions, E.8.2, in a sterile 
container to make Inoculum Pool 3: Gram-negative nonfermentors. 
E.10.4 Pool equal parts of C. Albicans, E.9.2, and A. Niger, E.9.3, suspensions in a sterile container to make 
innoculum pool 4: Fungi. 
E.10.5 Use organism pools immediately or refrigerate them at 2 C - 5 C for more than 72 h. EAS 339: 2013 
© EAC 2013 – All rights reserved  13 
 E.11  Inoculation 
E.11.1 Inoculate each of the four 20.0 mL aliquots of sterile saline, E.7.1, with 0.2 mL of its respective 
inoculum pool, E.10.1 - E.10.4. Mix thoroughly.  Use these suspensions to determine inoculums counts (see 
Ka). 
E.11.2 Inoculate each of the four 20 g products suspensions, with 2.0 mL of its respective inoculums, E.10.1 
– E.10.4. Mix thoroughly 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). 
E.12  Sampling intervals  
E.12.1  Sample each inoculated saline suspension, E.11.1, for APC, E.13, within 1 h after inoculation to 
obtain inoculum count. 
E.12.2 Test each inoculated product, E.11.2 for APC, E.13, at 7, 14 and 28 days after inoculation to obtain 
product interval count. 
E.13  Aerobic Plate Count (APC) 
E.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. 
E.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. 
E.13.3 Using a 2.2 mL pipette, aseptically pipette 1.0 mL and 0.1 mL aliquots from the 1:10 dilution 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 m L - 20 mL sterile Letheen agar E.3.1, (45 C  2 C into each plate. Mix 
by rotating the plates to disperse the suspension thoroughly, and let solidify. 
E.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. 
E.13.5 Invert and incubate 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.   
E.14  Neutralization check 
Make a 1:10 000 dilution in sterile saline of pools 1, 2 and 3, E.10.1 - E.10.3, and a 1:1000 dilution of pool 4, 
E.10.4. Streak each dilution for isolation with a 10 l loop on the plates saved from E.6.5.  If plates are not 
usable due to either desiccation or surface growth, repeat E.6, and streak freshly prepared plates.  Incubate 
as in E.13.4 - E.13.5 EAS 339: 2013 
14 © EAC 2013 – All rights reserved  
 E.15  Data analysis 
E.15.1  Product quality check , E.6.4, should be found to contain, <100 cfu/g to proceed with the challenge 
test. 
E.15.2 Inoculums counts , E.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. 
E.15.3 Neutralization check , E.14,  should show significant growth of all pools to confirm adequate 
neutralization.  A neutralizing 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. 
E.15.4 Calculate the percentage reduction: 
count Inoculum100 count interval product count Inoculum% Reduction,
 
 
E.15.5 The test 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 challenge, and remain at or below 
that level thereafter. This criteria applies to freshly prepared for mulations.  EAS 339: 2013 
© EAC 2013 – All rights reserved  15 
 Annex F 
(normative) 
 
Test for lead using atomic absorption spectrophotometer (AAS) 
F.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. 
F.2 Reagents 
F.2.1 Lead nitrate,  Pb(NO 3)2 
F.2.2 Dimethylacetamide  DMA  
F.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. 
F.2.4 Ethanol  C 2H5OH  
F.2.5 Bismuth oxychloride  BiOCl 
F.2.6 Hydrochloric acid  HCl, 6 N. Prepare by diluting 516 mL of concentrated HCl (11.6 N) to one litre with 
deionized water. 
F.2.7 Hydrochloric acid  HCl, 2 N. Prepare by diluting 172 mL of concentrated HCl (11.6 N) to one litre with 
deionized water. 
F.2.8 Hydrochloric acid  HCl, 0.5 N. Prepare by diluting 25 mL of 2 N HCl to 100 mL with deionized water. 
F.3 Standard solutions 
F.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 mL with deionized water . 
F.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.  
F.4 Sample preparation 
F.4.1 Aerosols 
For hair, 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 339: 2013 
16 © EAC 2013 – All rights reserved  
 F.4.2 BiOCl or cosmetics containing BIOCl 
Dissolve 1 g of sample (5 g if the lead level is expected to be less than 10 μg/g) in 15 mL of 6 N HCI and dilute 
to 100 mL with 0.5 N HCI. If the sample is coated with an organic material, it is necessary to ignite the sample 
to 500 C to ash before analysis. 
F.5 Instrument conditions 
F.5.1 Standard atomic absorption conditions for lead 
F.5.1.1  Recommended flame air-acetylene, oxidizing (lean, blue)  
F.5.1.2  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.  
F.5.1.3  Characteristic concentration with a N 2O-C 2H2 flame at 283.3 nm: 2.7 mg/L.  
Table F .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  - 
 
F.5.2 Standard flame emission conditions for lead 
Stock standard solution, lead, 1000 mg/L. Dissolve 1.598 g of lead nitrate, Pb(NO 3)2 in 1 %(v/v) HNO 3 and 
dilute to 1 litre with 1 % (v/v) HNO 3 
Table F .2 — Flame emission conditions for lead 
Wavelength (nm)  Slit (nm)  Flame  
405.8  0.2 Nitrous oxi de- acetylene  
 
F.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 sensitivity and detection limit. With multi-element lamps containing 
copper, the Cu 216.5 nm resonance line may interfere with lead determinations at the lead 217.0 nm line. The 
lead 283.3 nm line should be used instead.  EAS 339: 2013 
© EAC 2013 – All rights reserved  17 
 F.5.4 Interferences 
Large excesses of other elements (for example, 10 000 mg/L Fe) May interfere with the lead signal.  
F.6 Analysis 
Determine the concentration of lead in the sample solutions of aerosols and similar products using the 
standard conditions for lead and 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 standard conditions for lead and the method of additions. 
F.7 Calculations 
The total amount of lead shall be calculated as follows: 
µg/g Pb =
sampleofgPbμg/mL x 50 
F.8 Interferences 
No interference was found from 1000 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 1000 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 339: 2013 
18 © EAC 2013 – All rights reserved  
 Annex G 
(normative) 
 
Test for arsenic using atomic absorption spectrophotometer (AAS) 
G.1 Scope 
This method describes the determination of arsenic in various cosmetic products.  
G.2 Reagents 
The reagents used should be of analytical reagent grade. Water shall be distilled or di-ionised.  
G.3 Instrument conditions 
G.3.1 Standard atomic absorption conditions for arsenic 
G.3.1.1  Recommended flame; air-acetylene, reducing (rich, slightly yellow) 
G.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. 
G.3.1.3  Characteristic concentration with a N 2O-C 2H2 flame at 193.7 M: 1.4mg/L 
G.3.1.4  Table contains EDL data.  HCL sensitivity values are more than 25 % poorer. 
Table G .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  
 
G.3.2 Stock standard solution 
Arsenic , 1000 mg/L. Dissolve 1.320 g of Arsenious oxide As 2O3, in 25 mL of 20 % (w/v) KOH solution. 
Neutralize with 20 % (v/v) H 2SO 4 to a phenolphthalein endpoint. Dilute to one litre with 1 % (v/v) H 2SO 4. The 
standard solutions shall be of the following concentrations: 0 ppm, 2 ppm, 4 ppm, 6 ppm, 8 ppm and 10 ppm.  
G.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 absorption is reduced with the use of the nitrous oxide-acetylene flame, although EAS 339: 2013 
© EAC 2013 – All rights reserved  19 
 sensitivity is also reduced. Use of background correction is recommended, as it will correct for flame 
absorption and thus improve the signal to noise ratio.  
G.3.4 Light sources 
Both HCL and EDL sources are available for arsenic. EDLS, which are more intense, provide better 
performance and longer life. 
G.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 therefore advisable to set background correction. 
G.4 Sample preparation 
As in F.4.  
G.5 Analysis 
As in F.6.  
G.6 Calculation 
As in F .7 EAS 339: 2013 
20 © EAC 2013 – All rights reserved  
 Annex H 
(normative) 
 
Test for mercury using the atomic absorption spectrophotometer (AAS) 
H.1 Scope 
This method describes the determination of mercury in various cosmetic products.  
H.2 Reagents 
The reagents used should be analytical reagent grade.  Water shall be distilled or de-ionised. 
H.3 Instrument conditions 
H.3.1 Standard atomic absorption conditions for mercury 
H.3.1.1  Recommended flame: air-acetylene, oxidizing (lean, blue) 
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-C 2H2 flame at 253.7 nm: 12 mg/L 
H.3.1.4  Table contains EDL data.  HCL sensitivity values more than 25 % poorer  
Table H .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  
 
H.3.2 Standard flame emission conditions for mercury 
Table H .2 — Flame emission conditions for mercury 
Wavelength (nm)  Slit (nm)  Flame  
253.7  0.2 Nitrous oxide - acetylene  
H.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 339: 2013 
© EAC 2013 – All rights reserved  21 
 H.3.4 Light sources 
Both Electrodeless Discharge Lamps (EDLS) and Hollow Cathode Lamps are available for Mercury. However, 
the light output of mercury 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. 
H.3.5 Interferences 
Large concentrations 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.  
H.4 Sample preparation 
As in G.4.  
H.5 Analysis 
As in G.6.  
H.6 Calculations 
As in G.7. 
 
 
. 
 
 
 
 
 
. EAS 339: 2013 
22 © EAC 2013 – All rights reserved  
 Bibliography 
[1] IS: 7670-1975 Specification for hair creams. 
[2] 76/768/EEC The European Economic Community Cosmetics Directive. 
 EAS 339: 2013    
 
© EAC 2013 – All rights reserved