fdus eas 786 final draft uganda standard first edition …€¦ · this final draft uganda...

FINAL DRAFT UGANDA

STANDARD

FDUS EAS 786

First Edition 2013-mm-dd

Reference number FDUS EAS 786: 2013

© UNBS 2013

Skin care creams, lotions and gels — Specification

FDUS EAS 786: 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: [email protected] Web: www.unbs.go.ug

mailto:[email protected]

FDUS EAS 786: 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 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 786: 2013, Skin care creams, lotions and gels — Specification, is identical with and has been reproduced from an East African Standard, EAS 786: 2013, Skin care 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 786: 2013

ICS 71.100.70

© EAS 2013 First Edition 2013

EAST AFRICAN STANDARD


EAST AFRICAN COMMUNITY

EAS 786: 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: [email protected]

Web: www.eac-quality.net

Reproduction for sales purposes may be subject to royalty payments or a licensing agreement. Violators may be persecuted

EAS 786: 2013

© EAC 2013 – All rights reserved iii

Contents Page

Foreword ............................................................................................................................................................. v

1 Scope ...................................................................................................................................................... 1

2 Normative references ............................................................................................................................ 1

3 Terms and definitions ........................................................................................................................... 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 thermal stability ............................................................................... 4 A.1 Apparatus ............................................................................................................................................... 4 A.2 Procedure ............................................................................................................................................... 4 A.3 Results .................................................................................................................................................... 4

Annex B (normative) Determination of pH ...................................................................................................... 5 B.1 Apparatus ............................................................................................................................................... 5 B.2 Reagents ................................................................................................................................................ 5 B.3 Procedure ............................................................................................................................................... 5

Annex C (normative) Determination of total fatty substance content ......................................................... 6 C.1 Outline of the method ........................................................................................................................... 6 C.2 Reagents ................................................................................................................................................ 6 C.3 Procedure ............................................................................................................................................... 6 C.4 Calculation ............................................................................................................................................. 6

Annex D (normative) Determination of total viable count ............................................................................. 7 D.1 Outline of the method ........................................................................................................................... 7 D.2 Apparatus ............................................................................................................................................... 7 D.3 Medial buffer .......................................................................................................................................... 7 D.4 Sterilization of apparatus ..................................................................................................................... 8 D.5 Procedure ............................................................................................................................................... 8 D.6 Expression of results ............................................................................................................................ 8

Annex E (normative) Determination of Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans in cosmetic products .............................................................................................. 9

E.1 Introduction ............................................................................................................................................ 9 E.2 Principle of the method......................................................................................................................... 9 E.3 Apparatus ............................................................................................................................................... 9 E.4 Reagents .............................................................................................................................................. 10 E.5 Micro-organisms .................................................................................................................................. 11 E.6 Product quality check ......................................................................................................................... 11 E.7 Product preparation ............................................................................................................................ 11 E.8 Bacterial inoculum preparation ......................................................................................................... 12 E.9 Fungal inoculum preparation ............................................................................................................. 12 E.10 Inoculum pools ................................................................................................................................... 12 E.11 Inoculation .......................................................................................................................................... 12 E.12 Sampling intervals .............................................................................................................................. 13 E.13 Aerobic Plate Count (APC) ................................................................................................................ 13 E.14 Neutralization check........................................................................................................................... 13 E.15 Data analysis ....................................................................................................................................... 13

EAS 786: 2013

iv © EAC 2013 – All rights reserved

Annex F (normative) Test for lead using Atomic Absorption Spectrophotometer (AAS) ....................... 15 F.1 Principle of the method ...................................................................................................................... 15 F.2 Reagents .............................................................................................................................................. 15 F.3 Procedure ............................................................................................................................................ 15 F.4 Calculation ........................................................................................................................................... 15

Annex G (normative) Test for arsenic using Atomic Absorption Spectrophotometer (AAS) ................. 16 G.1 Scope ................................................................................................................................................... 16 G.2 Reagents .............................................................................................................................................. 16 G.3 Instrument conditions ........................................................................................................................ 16 G.4 Sample preparation ............................................................................................................................ 17 G.5 Analysis ............................................................................................................................................... 17 G.6 Calculation ........................................................................................................................................... 17

Annex H (normative) Test for mercury using the Atomic Absorption Spectrophotometer (AAS) ......... 18 H.1 Scope ................................................................................................................................................... 18 H.2 Reagents .............................................................................................................................................. 18 H.3 Instrument conditions ........................................................................................................................ 18 H.4 Sample preparation ............................................................................................................................ 19 H.5 Analysis ............................................................................................................................................... 19 H.6 Calculations ......................................................................................................................................... 19

Bibliography ..................................................................................................................................................... 20

EAS 786: 2013

© EAC 2013 – All rights reserved v

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 786 was prepared by Technical Committee EAS/TC 071, Cosmetics and cosmetic products.

EAS 786: 2013

vi © EAC 2013 – All rights reserved

Introduction

Skin creams, lotions and gels are useful not only as skin beautifiers, but also guard the skin against adverse climatic conditions. The performance of any skin cream, lotion or gel depends on the type of skin on which it is applied, and the nature of ingredients added to effect the intended end use. This Uganda Standard covers the following preparations, vanishing creams and lotions, foundation creams and lotions, cold creams and lotions, night creams, moisturizers, cleansers, hands creams and lotions, body creams and lotions, sun-protection preparations, toners, emollients, purifiers, nourishers and any other special treatment skin preparations e.g. AHA.

EAST AFRICAN STANDARD EAS 786:2013

© EAC 2013 – All rights reserved 1


1 Scope

This East African Standard specifies requirements and methods of sampling and test for creams, lotions and gels for skin care.

This East African Standard does not apply to skin care products, for which therapeutic claims are made and also does not apply to non-emulsified lotions and gels.

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 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 6887-1, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension and decimal dilutions for microbiological 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 randomization procedures

3 Terms and definitions

For the purposes of this Final East African Standard, the following term and definition shall apply

special purpose product include cleansers, toners, purifiers, facial washes, nourishers, Alpha Hydroxy Acids (AHA) skin lightening preparations based on vitamins and acids, anti-wrinkle/aging preparations, facial scrubs, facial masks, facial wash, non-emulsified lotions and gels and such other products

EAS 786: 2013

2 © EAC 2013 – All rights reserved

4 Requirements

4.1 General requirements

4.1.1 All ingredients used including dyes, pigments and colours shall comply with all the parts of EAS 377.

4.1.2 The preparation shall be clear or of uniform colour.

4.1.3 The cream, lotion or gel shall be free from visible impurities.

4.2 Specific requirements

4.2.1 All creams, lotions and gels for skin care shall comply with the chemical requirements given in Table 1 when tested according to the methods described therein.

Table 1 — Chemical requirements for creams, lotions and gels for skin care

SL No. Characteristic Requirement Method of test

i Thermal stability To pass test Annex A

ii

pH range

Hand and body lotion, creams and gels (including baby products)

Special purpose products*

4.5 - 8.5

3.5 - 8.5

Annex B

iii Total fatty substance content, % by mass, min 5 Annex C

* These include cleansers, toners, purifiers, facial washes, nourishers, AHA’s skin lightening preparations based on

vitamins and acids, anti-wrinkle/aging preparations, facial scrubs, facial masks, facial wash, non-emulsified lotions and

gels and such products

4.2.2 All creams, lotions and gels for skin care shall comply with the microbiological limits given in Table 2 when tested in accordance with the methods indicated therein.

Table 2 Microbiological limits

Micro-organisms Limits, max.

cfu/g

Method of test

Total viable count 100 in 0.5 g1) Annex D

100 in 0.1 g2)

Pseudomonas aeruginosa3)

Not detectable

Annex E

Staphylococcus aureus3)

Candida albicans3)

1) For products specifically intended for children under 3 years, eye area and mucous membranes

2) For other products

3) For Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans, the limits shall not be

detectable in 0.5 g for products specifically intended for children under 3 years, eye area and mucous

membranes and in 0.1 g for other products.

EAS 786: 2013


4.2.3 The products shall comply with the limits for heavy metal contaminants in accordance with Table 3.

Table 3 Limits for heavy metal contaminants

S/N Characteristic Limitst 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 packaged 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.

5.2.3 Special purpose products shall in addition bear the following warning on the label:

a) These products shall not be used on children below the age of 12 years;

b) This product may cause irritation. If irritation persists discontinue use; and

c) Precaution/warning shall be ‘'do patch test before use’’.

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 786: 2013


Annex A (normative)

Determination of thermal stability

A.1 Apparatus

Thermostatically controlled oven, capable of maintaining a temperature of 37 °C ± 1 °C

A.2 Procedure

Place a fresh, unopened sample of the cream or lotion 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 glass tube or vial and test as above.

A.3 Results

The product shall be taken to have passed the test if, on removal from the oven, the following indications of instability are not observed:

(i) change of colour;

(ii) change of smell or odour;

(iii) phase separation;

(iv) formation of granules or crystal growth; and

(v) shrinkage due to evaporation of water.

EAS 786: 2013


Annex B (normative)

Determination of pH

B.1 Apparatus

B.1.1 pH meter, equipped with a glass electrode

B.1.2 Beaker, of 100 mL capacity

B.2 Reagents

B.2.1 pH 7.0 buffer solution

B.2.2 pH 4.0 and pH 9.0 buffer solutions

B.2.3 Deionised water

B.3 Procedure

B.3.1 Dip the pH meter into about 50 mL of pH 7.0 buffer solution. Ensure that the reading is 7.0.

B.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.

B.3.3 For oil-in-water emulsion creams, weigh accurately 5 g ± 0.01 g of the cream in a 100-mL beaker. Add 45 mL of water and disperse the cream in it. Determine the pH of the suspension at 25 °C using the pH meter.

B.3.4 For water-in-oil emulsion creams, 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 alcoholic layer through a filter paper and measure the pH of the filtrate at 25 °C using the pH meter.

The type of cream is determined by placing some of it on a spot tile and sprinkling with a mixed indicator consisting of an intimate mixture of an oil soluble dye of one colour, e.g. 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 this matter is confirmed by checking whether the product is capable of conducting electricity and, if so, then the cream is deemed to be water-continuous.

EAS 786: 2013


Annex C (normative)

Determination of total fatty substance content

C.1 Outline of the method

The emulsion is broken with dilute mineral acid and the fatty matter is extracted with petroleum ether. It is weighed after removal of the solvent.

C.2 Reagents

C.2.1 Dilute hydrochloric acid 1:1 (v/v)

C.2.2 Petroleum, B.P. 40 °C to 60 °C

C.2.3 Methyl orange indicator solution — Dissolve 0.1 g of methyl orange in 100 mL of water.

C.2.4 Sodium sulphate, desiccated

C.3 Procedure

Weigh accurately about 2 g of the material into a conical flask; add 25 mL of dilute hydrochloric acid, fit a reflux condenser into the flask and boil the contents until the solution is perfectly clear. Pour the contents of the flask into a 300 mL separation funnel and allow it to cool to 20 °C. Rinse the conical flask with 50 mL of petroleum ether in portions of 10 mL. Pour the ether rinsings into the separation funnel shake the separation funnel well and leave until the layers separate. Separate out the aqueous phase and shake it out with 50 mL portions of ether twice. Combine all the ether extracts and wash them with water until free of acid (when tested with methyl orange indicator solution). Filter the ether extracts through a filter paper containing sodium sulphate into a conical flask which has been previously dried at a temperature of 60 °C ± 2 °C and then weighed. Wash the sodium sulphate on the filter with ether and combine the washings with the filtrate. Distil off the ether and dry the material remaining in the flask at a temperature of 60 °C ± 2 °C to constant mass.

C.4 Calculation

The total fatty substance shall be calculated as follows:

Total fatty substance, percent by mass =2

1100

M

M,

where,

M1 is the mass, in grams, of the residue, and

M2 is the mass, in grams, of the material taken for the test.

EAS 786: 2013


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 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 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.

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.

EAS 786: 2013


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 1 h.

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 1 h.

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 1 h 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 ISO 6887-1.

EAS 786: 2013


Annex E (normative)

Determination of Pseudomonas 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 samples

b)

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.

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

E.3.6 Sterile swabs

EAS 786: 2013


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 Na2 S2O3 . 5H2O, 2.5 g NaHSO3, 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 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.

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 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 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

E.4.2.8.1 Prepare a 1.0 % BaCI2 solution by dissolving 1.0 g BaCI2. 2H2O in 100 mL water. Let this be referred to as solution 1.

EAS 786: 2013


E.4.2.8.2 Prepare a 1.0 % H2SO4 solution by mixing 1.0 mL H2SO4 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 5 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.

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.

EAS 786: 2013


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 BaSO4

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 SO4

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.

E10.3 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.

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).

EAS 786: 2013


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 10

6 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 mL - 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 F.6, and streak freshly prepared plates. Incubate as in E.13.4 - E.13.5

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.

EAS 786: 2013


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:

countInoculum

100countintervalproductcountInoculum%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 formulations.

EAS 786: 2013


Annex F (normative)

Test for lead using Atomic Absorption Spectrophotometer (AAS)

F.1 Principle of the method

The sample is passed through wet digestion using pressure decomposition. The amount of lead is then determined using AAS.

F.2 Reagents The reagents used shall be of analytical reagent grade. Water shall be distilled or de-ionised.

F.3 Procedure

F.3.1 Weigh accurately 0.4 g of sample and put this into a 50 mL decomposition pressure tube. Add 7.0 mL of concentrated nitric acid. Add 2.0 mL of water. Close the pressure vessel, and apply between 15 Nm- 20 Nm of pressure. Digest for about 2 h. Transfer to a 20 mL volumetric flask and make to the mark.

F.3.2 Prepare the standard solutions for lead. Aspirate into the flame each of the standard solutions in ascending order of concentration. Take the absorbance reading for each concentration using the AAS. Plot a calibration graph of the concentration of lead in the standard solutions against the corresponding values of absorbances.

F.3.3 Aspirate into the flame the sample solution. Take the absorbance reading from the AAS and give it a value X. From the graph, use the value of absorbance X to read the corresponding value of concentration. Let this value of concentration be C.

F.4 Calculation

The amount of lead shall be calculated as follows:

Amount of lead = M

DC

where

C is the concentration of the sample solution;

M is the mass of the sample in grams; and

D is the Dilution factor.

EAS 786: 2013


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 N2O-C2H2 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 As2O3, in 25 mL of 20 % (w/v) KOH solution. Neutralize with 20 % (v/v) H2SO4 to a phenolphthalein endpoint. Dilute to one litre with 1 % (v/v) H2SO4. 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 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.

EAS 786: 2013


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 786: 2013


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 N2O-C2H2 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

Wavelength,

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 786: 2013


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 F.4.

H.5 Analysis

As in F.6.

H.6 Calculations

As in F.7

EAS 786: 2013


Bibliography

[1] IS 6608: 1972, Specification for skin creams;

[2] IS 4011: 1982, Methods for dermatological tests for cosmetics

[3] KS 03-580: 1998, Specification for creams, lotions and gels for skin care

[4] Official Journal of the European Communities N. L26217

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