Microbiology in Cosmetics – Challenges in

Cosmetic Manufacturing

DONALD J. ENGLISH
SENIOR MANAGER
R&D MICROBIOLOGY
AVON PRODUCTS, INC.

1
 Drug vs. Cosmetic
 Drug – is a finished product dosage form that contains a drug substance that is intended to furnish
pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment or
prevention of disease or to affect the structure or any function of the human body.
 Cosmetic – is a product intended to be applied to the human body for cleansing, beautifying, and
promoting attractiveness, or altering the appearance without affecting the body’s structure or
function.
 Some products produced by a cosmetic company is an OTC Drug product such as:
 Acne
 Antifungal
 Antimicrobial
 Antiperspirant
 Dandruff
 External Analgesic
 Sunscreen
 Skin Protectant

2
Preservative Challenge Testing of Formulations

3
 What is a Preservative?

A chemical agent that will either kill or inhibit the growth

of microorganisms

4
 Purpose for Using Preservatives in Formulations
 Prevent the Development of Adverse Risks:
 Finished Product:
 Malodors
 Viscosity Changes
 Discoloration
 Presence of Visible Microbial Growth

 Consumer:
 Prevent Infections:
 Eyes – an infection could lead to blindness.
 Development of skin infections if the consumer has open sores or cuts.
 Death for those consumers that are either immunocomprised or has a pre-existing condition.

5
 Types of Preservatives
 Traditional Preservatives:
 Parabens (e.g. Methylparaben, Ethylparaben)
 Formaldehyde Donors (Imidazolidinyl urea, DMDM Hydantoin)
 Isothiazolinones (e.g. MCT/MI)
 Phenolic Types (e.g. Phenoxyethanol, Benzyl Alcohol)
Acidic (e.g. Sorbic Acid, Benzoic Acid)
Halogenated Compounds (e.g. Chlorphenesin)
Alcohols (e.g. Ethyl Alcohol)
Quaternia (e.g. Benzalkonium chloride)

 Non-Traditional or Alternative Preservatives:

 Has antimicrobial activity by itself.
 Can be an ingredient marketed for some other function in combination with a traditional preservative.
 Can boost the antimicrobial activity of a traditional preservative by which:
 A lower concentration of a traditional preservative can be used in a product formulation.
 Allows for a switch to a less powerful or potent preservative that does not have a marketing issue to
guarantee preservative adequacy of a formulation.

6
Non-Traditional or Alternative Preservatives
 Marketed for Some Other Function in a Formulation:
 Antioxidant
 Emollient
 Fragrance Additive
 Humectant
 Moisturizer
 Skin and hair Conditioner
 Viscosity Regulator
 Regulatory Aspects:
 Not listed on an approved preservative list such as:
 Cosmetic Ingredient Review (CIR)
 Japan Ministry of Health and Welfare Positive Preservative List
 EU No. 1223/2009 Annex V – List of Preservatives Allowed in Cosmetic Products

7
Regulatory Status of Traditional Preservatives - Europe
 Europe – EU No 1223/2009, Annex V - List of Preservatives Allowed in
Cosmetic Products
 Maximum Authorized Concentration.

 Limitation and Requirements.

 Rinse-off products only.
 Prohibited in aerosols.

 Conditions of Use and Warnings which must be printed on the product label.
 Not to be used for children under 3 years of age.
 Contains XYZ.

8
Regulatory Status of Traditional Preservatives - Japan
 Ministry of Health and Welfare Positive Preservative List – 2 Parts
 Preservatives that can be used in all cosmetic products.
 Preservatives that are restricted depending upon the type of cosmetic product that
they are going to be used in:
 Rinse-off products not applied to a mucous membrane.
 Leave-on products not applied to a mucous membrane.
 Cosmetics applied to a mucous membrane.

9
 Regulatory Status of Traditional Preservatives - USA
No Positive Preservative List.
 Food and Drug Administration has the ability to restrict or prohibit the use of a preservative due to
safety reasons.
 Prohibited or Restricted Preservatives:
 Hexachlorophene (21 CFR 250.250)
 Has neurotoxic effects and ability to penetrate human skin
 May be used only when an alternative preservative has not shown to be effective.
 Level cannot exceed 0.1% and may not be applied to a mucous membrane
 Mercury Compounds (21 CFR 700.13)
 Readily absorbed through the skin on topical application and is able to accumulate in the skin.
 Limited use to the eye-area only at a concentration of 65 ppm provided no other effective and safe preservative can be
used.
 Bithionol (21 CFR 700.11) – causes photo-contact sensitization.
 Halogenated Salicylanilides (21 CFR 700.15) – causes photo-contact sensitization.

10
 Cosmetic Ingredient Review (CIR) – Traditional Preservatives
 Unsafe Cosmetic Ingredient (Preservative)
- Chloroacetamide – unsafe due to its potential as a human sensitizer.

 Insufficient Data to Support Use

- Benzylparaben - Glyoxyl
- Benezoxiquine - p-cresol
- Captan - mixed cresols
- Chlorophene
- Dichlorophene
- Dimethyl lauramine

11
 Formulation Factors Affecting the Antimicrobial
Activity of Preservatives
 Water Activity

 pH

 Solubility of Preservatives

 Compatibility with Other Raw Ingredients

12
 Microbial Metabolism and Growth
 Need a source of available water and nutrients.

 Byhaving a reduction in the amount of available water in a formulation,

microorganisms will be affected by having a longer generation time or
reduce metabolic activity.

13
 Water Activity
 Water Activity is defined as the ratio of vapor pressure of a substance to that of pure
water at a specified temperature.

 Water Activity can be expressed mathematically as a function of Raoult’s Law:

Aw = P/Po = n1/ (n1 + n2)
where P = vapor pressure of a solution , Po = vapor pressure of pure water, n1 is the
number of moles of solvent, and n2 is the number of moles of solute.
 Relationship between Aw and Equilibrium Relative Humidity (ERH) can be expressed as
follows:
Aw = P/Po and ERH (%) = Aw x 100
14
 General Water Activity Values Required for Microbial Growth
Water Activity Value Types of Microorganisms Capable of Proliferation
0.96 to 0.99 Gram-positive and Gram-negative bacteria (e.g. Ps. species), mold and yeasts

0.90 to 0.95 Several Gram-negative and most Gram-positive bacteria (e.g. Enterobacter
aerogenes, Escherichia coli, Bacillus species), mold and yeasts

0.80 to 0.89 Gram-positive bacteria (e.g. S. aureus), mold and yeasts

0.70 to 0.79 Halophilic bacteria, mold and yeasts

0.65 to 0.69 Osmotolerant yeasts

Below 0.6 None

15
Typical Water Activity Value Examples for Cosmetic Product Formulations
Type of Cosmetic Product Aw Value*
Foundation 0.68
Hand Cream 0.96
Loose Body Powder 0.76
Lipstick 0.68
Mascara 0.96
Powdered Eyeshadow 0.76
Shampoo 0.99
Shampoo Conditioner 0.97
Toothpaste 0.86
Wet/Dry Eyeshadow 0.57
* = Aw Value is formulation dependent.

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 Formulation Water Activity and Preservative
Spectrum of Antimicrobial Activity

Water Activity Level Antimicrobial Spectrum of a Preservative for Inclusion

Below 0.6 Inclusion of a preservative system may not be necessary.

0.70 – 0.79 Preservative system needs to be active against yeast and mold.

0.80 - 0.89 Preservative system needs to be active against Gram-positive bacteria, yeast
and mold.

0.90 – 0.99 Preservative system needs to have a broad spectrum of antimicrobial activity
(e.g. Gram-negative and Gram-positive bacteria, yeast and mold)

17
 Examples of Product Formulations that are Least and Highly
Susceptible to Microbial Contamination
 Least Susceptible
 Lipsticks

 Nail Enamels

 Powders
 Highly Susceptible
 Creams

 Lotions

 Shampoos

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 Examples of Raw Ingredients Affecting Water
Activity Values of Product Formulations
 Lowers Water Activity Values by Absorbing Water
 Amino acids
 Butylene glycol
 Dextrin’s
 Ethanol
 Glycerol
 Propylene glycol
 Sodium chloride
 Xanthan gum
 Causes an Increase in the Osmotic Pressure
 High Sugar Concentrations (e.g. glucose, sucrose, sorbitol)

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 Formulation Factors - pH and Microbial Growth
 Bacteria:
Optimum pH for growth is between 5.5 and 8.5.

 Fungi (Yeasts and Mold):

Optimum pH for growth is between 4.0 and 6.0.

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 pH Microbiological Affects
 For product formulations with a pH less than 4.0 or greater than 10.0, microorganisms
are not able to proliferate or survive in a formulation due to:
 Metabolic injury to microbial cells
 Cellular stress by which microorganism expend a greater amount of energy to maintain intracellular
pH. After energy has been used up, microbial cells will die.
 The function of many microbial cellular enzymes is dependent on the maintenance of proper
intracellular pH.

 Examples of extreme pH product formulations without preservatives

 Acidic product formulations (e.g. pH of 3.0 to 5.0)
 Salicyclic acid containing product formulations, Hair conditioners, Aluminum chlorohydrate antiperspirants.
 Alkaline product formulations (e.g. pH of 10.0-14.0).
 Hair relaxers, Depilatory creams

21
 pH Effects on the Chemical Stability of
Traditional Preservatives
 Benzyl alcohol - will degrade at low pH values.
 Bronopol - will decompose at alkaline pH values.
 Chlorobutanol - unstable at neutral or alkaline pH values.
 4, 4 – Dimethyl-1,3- oxazolidine - is unstable at acidic pH values.
 Iodopropylnyl butylcarbamate (IPBC) - will decompose at pH values above 9.0.
 Methyldibromo glutaronitrile (MDGN) - is only stable at pH values below 8.5.
 Parabens - will undergo chemical hydrolysis at alkaline pH values.
 Quaternium-15 - is unstable at pH values below 4.0 and greater than 10.0.

22
 pH Range for Optimum Antimicrobial
Activity of Traditional Preservatives
 Organic Acid Preservatives
 Benzoic acid – only has antimicrobial activity at pH’s below 5.0.
 Dehydroacetic acid – only has antimicrobial activity at pH’s up to 7.0.
 Sorbic acid – only has antimicrobial activity at pH’s below 5.0.

 Parabens – antimicrobial activity becomes inactive by dissociating into the salt form at
pH’s above 8.0.
 Quaternary Ammonium Preservatives – have antimicrobial activity at neutral to alkaline
pH.
 Benzalkonium chloride
 Benzethonium chloride

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pH Range for Optimum Antimicrobial Activity of
Non-Traditional or Alternative Preservatives
 Anisic Acid: < 5.5
 Glyceryl Caprate: 4.0 - 7.5
 Glyceryl Caprylate: 4.0 - 7.5
 Sodium levulinate and phenylpropanol: <5.5
 Caprylyl Glycol: Has a broad pH range.
 Phenoxyethanol and Caprylyl Glycol: Optimum pH range is 4.0 to 7.0
 1,2-Decanediol (Decylene Glycol): Optimum pH range is 3.0 to 8.0

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 Formulation Factors - Water and Microbial Growth
 Water is necessary for microbial growth to occur.
 Microorganisms will only proliferate in the water phase of a product
formulation.
 In emulsions, microorganisms will grow in the aqueous phase, but will also collect
at the interface between the oil and water phases of the formulation.

To prevent microorganisms from growing, a traditional or a non-

traditional/alternative preservative has to be present in the aqueous
phase of a product formulation.

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 Water Solubility of Preservatives
 Water Soluble Preservatives
Traditional Non-traditional/Alternative
Diazolidinyl urea Phenoxyethanol, Caprylyl glycol and Hexylene glycol Bld.
DMDM hydantoin Phenethyl alcohol, Caprylyl glycol, and Trideceth-8
Methylchloroisothiazolinone/Methylisothiazolinone Bld.

 Limited Water Soluble Preservatives

Traditional Non-traditional/Alternative
Chlorphenesin – up to 1% Caprylyl glycol – up to 0.5%
Dichlorobenzyl alcohol – up to 0.1% Phenoxyethanol and Caprylyl glycol – up to 1.2%
Methylparaben – up to 0.25% Ethylhexylglycerin – up to 0.2%

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 2 Ways for Incorporating a Limited Water Soluble
Preservative into an Aqueous Product Formulation
 Use a water miscible solvent to dissolve a water insoluble or limited
water soluble preservative.
 Ethanol
 Glycerin
 Glycerol
 Propylene glycol

 Use heat to warm up the water phase of a product formulation to

dissolve a water insoluble preservative.
 Parabens – are more soluble in water at 80oC than at 25.0oC.

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 Partition Coefficients of Preservatives
 Some preservatives will exhibit both oil and water solubility.
 Examples: Benzoic acid and parabens
 Oil and water soluble preservatives will migrate or naturally partition themselves between
the water and oil phases of a formulation (e.g. oil-in-water and water-in-oil emulsions).
 By migrating into the oil phase of a formulation, there might not be a sufficient concentration of the
preservative in the water phase where microorganisms are located to protect the formulation.
 Solutions:
 Use a higher concentration of the preservative in water phase.
 Limit the solubility of the preservative by adding 10% or more of glycerin, ethanol, butylene glycol,
hexylene or by adding greater than 5% of propylene glycol to the water phase of the formulation.

 This phase distribution of the preservative is called partition coefficient.

 If a preservative has a high concentration in the oil phase of an emulsion, the partition coefficient of that
preservative will be large.

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 Compatibility with Other Raw Ingredients
 Some raw ingredients can be:
 Microbial Nutrients
 Preservative Inactivators
 Preservative Absorbers
 Preservative Potentiators

29
 Examples of Raw Ingredients that Can Serve as a
Microbial Nutrient in a Product Formulation
 Botanical Extracts (e.g. aloe vera)
 Carbohydrates (sugars or cellulose)
 Proteins
 Amino Acids
 Emulsifiers (e.g. anionic, cationic, nonionic, and amphoteric surfactants)
 Lipids (e.g. waxes, fatty acids or fatty alcohols)
 Gums
 Vitamins

30
Examples of Raw Ingredients that Can Inactivate the Antimicrobial
Activity of Preservatives in a Product Formulation
 Polysorbate (Tween)
 Lecithin
 Cellulose derivatives
 2-hydroxypropyl-ß-cyclodextrin
 Gelatin
 Proteins or Protein hydrolysates
 Raw ingredients that have sulfhydryl groups
 Avobenzone
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 Examples of Raw Ingredients that Can Absorb
Preservatives in a Product Formulation
 Bentonite
 Calamine
 Carbonates
 Diatomaceous earth
 Kaolin
 Silicon dioxide
 Zinc oxide
 Some color pigments
 Talc
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 Examples of Raw Ingredients that are Preservative Potentiators
 A raw ingredient that is able to enhance or increase the antimicrobial
activity of a preservative:
 Examples:
 Propylene Glycol
 Nonionic surfactants
 EDTA
 Antioxidants
 Caprylyl glycol
 Ethanol
 Ethylhexylglycerin
 Pentylene Glycol
 Caprylic/Capric glycerides
 Essential Oils Fragrances or Fragrance Components

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Manufacturing Conditions Can Have an Affect on Preservatives

 Raw ingredient order of addition.

 pH of the formulation at the time of preservative addition.

 Temperature during processing.

34
Raw Ingredient Order of Addition During Manufacturing
 Water-Soluble preservatives should always be added to the
water phase or to the emulsified portion of a formulation during
the cool down stage.
 Limited water soluble preservatives should first be dissolved in a water
miscible solvent for addition to the water phase of a product formulation.
 May affect the partitioning of an oil and water-soluble
preservative if added to the incorrect phase of an emulsion.

35
 pH Manufacturing Affects on Preservatives

 Antimicrobial Activity
 Organic acid preservatives (benzoic acid, sorbic acid, and dehydroacetic acid)
are only antimicrobially active at acidic pH values.
 If Sodium benzoate, Potassium sorbate of Sodium dehydroacetate is used a
preservative, the pH of the product formulation needs to be adjusted by using Citric acid
to cause the disassociation of the inactive salt into the acid.

 Physical Stability
 Parabens and IPBC are not physically stable at alkaline pH conditions.

36
 Manufacturing Temperature Affects on Preservatives
 Manufacturing temperature tolerance for all preservatives should be known.
 Purpose :
 To prevent the accidental decomposition of a preservative in a formulation during processing:
 Examples:
 Diazolidinyl urea – will start to break down at 60oC.
 Imidazolidinyl urea – will decompose if held at a emulsification temperature for longer than 4 hours.
 IPBC – will degrade above 45oC.
 Parabens – will undergo hydrolysis with prolonged heating especially if the aqueous phase of the formulation is at an
alkaline pH.

 Prevent evaporation of an alcohol component of a preservative in a formulation during processing:

 Examples:
 Phenoxyethanol
 Ethanol
 Phenoxyethanol and Caprylyl Glycol
 Phenethyl Alcohol and Caprylyl Glycol

37
 Examples of Packaging Affects on Preservatives
 Chemical Composition of a Product Package.
 Leaching of PVC plasticizers can inactivate phenolic preservatives.
 Polyurethane is able to reduce the antimicrobial activity of phenolic and quaternary ammonium
preservatives.
 Low to medium density polyethylene is able to absorb parabens from a formulation.
 Incompatibility: Benzyl alcohol is known to interact with polyethylene and polystyrene.

 Light Exposure.
 Certain types of preservatives are more susceptible to decomposition if they are exposed to light. (e.g.
Bronopol, sorbic acid).
 If a formulation contains a light sensitive preservative, the use of opaque packaging is recommended.

38
 Risk Factor Analysis for in Evaluating Packaging Affects on
the Preservative Adequacy of a Product Formulation

 Are Refills Used for a Product?

 What is the Size of the Packaging?
 What is the Predicted Use Up Rate?
 What is the Mode of Product Dispensing?
 Open Jar
 Spray Bottle
 Pump
 Type of Container Closure (e.g. Slit Cap, Flip cap, Screw cap)
 Mode of Product Application?
 Fingers of the Hand
 Brushes
 Pads
 Puffs
 Sponges
 Is the Package Pressurized?

39
 Preservative Challenge Test

 Definition: A microbial challenge test that determines the

antimicrobial effectiveness of a preservative in a
product formulation or an unpreserved product
formulation against selected test microorganisms.

40
 Preservative Challenge Test Method
GENERAL ASPECTS

Standardized Inoculated Removal of Neutralization of Recovery of

Microbial Product 1.0 gram Preservative Surviving
Suspension Aliquots at Antimicrobial Microorganisms
Specified Activity
Intervals

Calculation of
Percent or Log10
Reduction

41
 Different Categories of Microbial
Challenge Test Methods
 Pharmacopoeia Challenge Test Methods
 USP Antimicrobial Effectiveness Test
 BP/EP Test for Efficacy of Antimicrobial Protection
 JP Preservatives - Effectiveness Test

 Consensus Challenge Test Methods

 CTFA (PCPC) – 5 types of challenge test methods
 ASTM E640-78 – Standard Test Method for Preservatives in Water Containing Cosmetics

 Standard Challenge Test Methods

 AOAC 998.10 – Preservative Challenge Efficacy Test of Non-Eye Area Water Miscible Products
 ISO 11930 – Cosmetics - Microbiology - Efficacy Test and Evaluation of Preservation of a Cosmetic Product

 In-House Challenge Test Methods

 Other Challenge Methods - D-Value (Linear Regression), Direct Contact Membrane Filtration, PET
Method for Powdered Eye Shadows
42
 CTFA Microbial Challenge Test Methods
 CTFA M-3 Determination of Preservative Adequacy of Water Miscible Cosmetics
 CTFA M-4 Method for Preservative Testing of Eye Area Cosmetics
 CTFA M-5 Methods for Preservation Testing of Nonwoven Substrate Personal Care
Products
 CTFA M-6 Method for Preservation Testing of Atypical Personal Care Products
 A challenge test method for oils, water-in-oil emulsions, water-in-silicone, Semi-solid (<20% water content), loose
powders, and pressed powder product formulations.

 CTFA M-7 A Rapid Method for Preservative Testing of Water-Miscible Personal Care
Products
 A rapid challenge test method for screening different preservative systems for a water miscible product formulation.

43
 In-House Microbial Challenge Test Method

 A microbial challenge test method that has been developed and used by a
company to determine the preservative adequacy of a product
formulation.

 Usually based upon a compendial challenge test method (e.g. USP, BP/EP).

44
 Main Differences Between the Various Types of
Preservative Challenge Test Methods
 Types of Challenge Test Microorganisms

 Inoculum Preparation (e.g. Broth vs. Agar)

 Inoculum Levels

 Mix Culture verses Pure Culture Inoculums

 Sampling Time-Points After Inoculation

 Preservative Effectiveness Acceptance Criteria

45
CTFA Survey Results for Microorganism Source Used in
Preservative Challenge Testing

100 95
90

% Respondents
80 68 68
70
60
50
40
30
20
10
0
USP Test Other ATCC In-House
Strains Strains Strains
Type of Microbial Strains

46
Challenge Testing Parameters-Topical Product Formulations
Challenge Test Method Inoculum Level in Product Testing Intervals for Performing
(CFU/gram) Plate Counts (Days)

Bacteria Yeast/Mold
USP 1.0x105-6 1.0x105-6 14, 28
BP/EP 1.0x105-6 1.0x105-6 2, 7, 14, 28

JP 1.0x105-6 1.0x105-6 14, 28

PCPC 1.0x106 1.0x105 7,14,21, 28

ASTM 1.0x106 1.0x105 7, 14, 28

ISO 1.0x105-6 1.0x104-5 7, 14, 28

AOAC 1.0-9.9x106 1.0x105-6 7, 14, 28

47
 Various Challenge Testing Acceptance Criteria for Topical
Product Formulations
Challenge Test Challenge Acceptance Criteria (Log10 Reduction)
Method

Day 2 Day 7 Day 14 Day 21 Day 28

Bacteria Y/M Bacteria Y/M Bacteria Y/M Bacteria Y/M Bacteria Y/M
USP NT NT NC NC 2 NI NT NT NI NI
BP/EP A: 2 NC A: 3 NC NC 2 NT NT A: NI NI NT = Not Tested

B: 3 B: NI B: NI NI = No Increase
NC = No Criteria
JP NT NT NT NT 2 NI NT NT NI NI
PCPC NT NT 3 1 NI NI NI NI NI NI
ASTM NT NT 3 NC NI NI NT NT NI 1
ISO NT NT 3 1 NI NI NT NT NI NI
AOAC NT NT 3 1 NI NI NI NI NI NI

48
 In-House Preservative Challenge Acceptance Criteria

 Water Miscible/Dispersible Products

 Generally more stringent in terms of log reductions at certain time-periods than
compendial and guideline recommendations.
 For example, a 5-log reduction for bacteria and a 2-3 log reduction for mold at 7-days after
inoculation.

 Anhydrous Atypical Products

 Risk assessment needs to be perform to justify the use of alternative preservative
challenge criteria in place of criteria for aqueous based products.
 For example, stasis in the microbial counts for those challenge microorganisms that do not need
water to survive.

49
CTFA Survey Results in the Types of Preservative Challenge
Test Methods Being Used by the Cosmetic Industry

80 71
% Respondents

60 47
40 29
20 13 11 8
0
0
In- USP M-3 M-4 EP AOAC ASTM
House
Challenge Test Methods

50
 Microbial Content Testing
 Raw Ingredients
 Packaging Applicators
 Finished Product Formulations

51
Microbial Methods for Evaluating Product Quality

 Aerobic Plate Count

 Enrichment Test

52
 Aerobic Plate Count Method

GENERAL ASPECTS

1:10 Dilution Dispense

Plate Count
Neutralizing &
Sample Diluent Incubate

Count Number of
Microorganisms/gram or ml

53
 Enrichment Test Method

GENERAL ASPECTS
1.0 or 10.0 Incubate Streak onto
Microbial Selective/
grams
Enrichment Differential
Sample Broth Microbial
with Growth
Neutralizers Agars

Gram-stain and Identify

Recovered Microbial Isolates

54
 Microbial Content Test Methods
 In-House Microbial Content Test Methods
 USP/EP/JP Harmonized Chapters <61> and <62>
 FDA Bacteriological Analytical Manual – Chapter 23- Microbiological Methods for
Cosmetics
 PCPC (CTFA) Microbial Content (M-1) and Examination for S. aureus, E. coli, and Ps.
aeruginosa (M-2)
 ISO Microbial Content Standard Test Methods for Cosmetics (7)
 Rapid Microbial Content Test Methods such as ATP Bioluminescence and Flow Cytometry

55
ISO Cosmetic Microbial Content Standard Test Methods
 21148 - Cosmetics - Microbiology - General Instruction for Microbiological Examination
 21149 - Cosmetics - Microbiology - Examination and Detection of Aerobic Mesophilic
Bacteria
 18415 - Cosmetics - Microbiology - Detection of Specific and Nonspecific
Microorganisms
 18416 - Cosmetics - Microbiology - Detection of Candida albicans
 21150 - Cosmetics - Microbiology - Detection of Escherichia coli
 22717 - Cosmetics - Microbiology - Detection of Pseudomonas aeruginosa
 22718 - Cosmetics - Microbiology - Detection of Staphylococcus aureus

56
Microbial Content Testing of Cosmetic
Raw Ingredients

57
 Microbial Classifications of Raw Ingredients
 Hostile Raw Ingredients

 Inert Raw Ingredients

 Chemically Preserved Raw Ingredients

 Raw Ingredients Supporting Microbial Growth

58
 Hostile and Inert Raw Ingredients
 Hostile Raw Ingredients
 Inherently self-preserved/microbiocidal
 Have extreme pH’s (<4 or > 10.0).
 Contain greater than 20% alcohol concentration.
 Contain greater than a 25% Propylene glycol concentration.
 Have low Water Activity levels (e.g. <0.60).
 Examples: Perfume Oils, Essential Oils, Salts, Preservatives.

 Inert Raw Ingredients

 Not microbiocidal
 Generally have a low Water Activity level.
 Cannot support the growth or proliferation of microorganisms, but still can be contaminated with
viable microorganisms.
 Examples: Dyes, Waxes, Glycerin, Powders.

59
 Chemically Preserved Raw Ingredients and Raw
Ingredients Supporting Microbial Growth

 Chemically Preserved Raw Ingredients

Preservatives have been added to the raw ingredient to protect against the
introduction of microbial contamination and microbial proliferation during use in
manufacturing.
Water-based or have an aqueous vehicle.
Examples: Surfactants, Natural Extracts, Botanical Solutions.

 Raw Ingredients Supporting Microbial Growth

 May act as a microbial growth medium and do not contain preservatives.
 May serve as a microbial nutrient.
 Examples: Botanical Ingredients, Starches, Sugars, Cellulose, Proteins, Amino
Acids, Vitamins, Natural Gums , and Aqueous Dye Solutions.

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 Microbial Risk Assessment of Raw Ingredients
 Hostile Raw Ingredients
 Risk of causing microbial contamination in a finished product is low or non-existent.
 Do not require microbial content testing provided as long as scientific judgment is documented and the
processing controls for a hostile raw ingredient is well documented.
 Inert Raw Ingredients
 Have a moderate risk in being contaminated and causing finished product to be contaminated with
microorganisms.
 Microbial testing may be performed to either develop a historical database to stop testing in the future or
conducted microbial content testing is for monitoring purposes.
 Chemically Preserved Raw Ingredients and Raw Ingredients Supporting Microbial Growth
 Are a high risk in being found to be contaminated and causing finished products to be contaminated with
microorganisms.
 Routine microbial content testing should always be performed.

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 Microbial Content Stability of Raw Ingredients
 For those raw ingredients that are determined to be susceptible to microbial
proliferation:
 Effects of storage conditions on microbial content should be examined.
 e.g. Ambient or refrigerated conditions.

 Should be retested at certain intervals to determine whether the microbial bioburden present in
the ingredient at the time of receipt or from the introduction during usage had increased over
time.

 For those raw ingredients that are highly susceptible to microbial contamination, they should
always be retested prior to use in manufacturing to confirm acceptability.

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 Two Ways for Establishing Microbial Limits for Raw Ingredients

 Use Compendial Information Chapters or Trade Association Guidelines

 USP Section <1111> - Acceptance Criteria Nonsterile Substances for Pharmaceutical
Use
 Aerobic Plate Count: 1000 CFU/gram or ml
 Yeast/Mold Count: 100 CFU/gram or ml
 Objectionable Microorganism Risk Assessment

 CTFA Guideline – Raw Materials Microbial Content

 All synthetic and natural raw materials – < 100 CFU/gram
 No raw material should have a microbial content recognized as harmful to the user or product.

 Conduct a Microbial Risk Assessment for Establishing Microbial Limits

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 Risk Assessment Model for Establishing
Microbial Limits for a Raw Ingredient
 Need to ask the following questions:
 What is the proposed use level of a raw ingredient (RI) in a finished product (FP)?

 What is the microbial test specification of the FP?

 What is the suppliers actual microbial test history for a RI?

 Calculations to determine whether a suppliers RI microbial specification is acceptable:

 Microbial Count:
Supplier RI Test Specification x % Use Level in FP = Possible Contributing Number of CFU from RI
in the microbial bioburden of a FP.
 Objectionable Microorganisms:
Absence in 10 g of FP x % Use Level in FP = Absence/gram

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 Example of a Risk Assessment Calculation
 RI Use concentration in FP: 1%
 FP microbial test specification:
 Aerobic Plate Count - <100 CFU/gram and absence of objectionable microorganisms per 10 grams
 Suppliers RI microbial test history:
 RI specification of 1000 CFU/gram and absence of objectionable microorganisms in 1 gram.
 98% of the lots had a microbial count of <10 CFU/gram and the absence of objectionable organisms per 1 gram.
 Calculation to determine RI acceptability:
 Microbial Count:
 Supplier RI Test Specification x % Use Level in FP = Possible Contributing Number of CFU from RI in the FP microbial
bioburden
 1000 CFU/gram x 1% = 10 CFU/gram is a possible contributing microbial count from used RI in a finished product.
 Objectionable Microorganisms
 Absence in 10 g of FP x % Use Level in FP = Absence/gram
 10 g x 1% = 0.1 gram. Raw ingredient specification requires absence of objectionable microorganisms in 1 gram.
 Risk Assessment: Based upon the suppliers RI specification, establish a 10-fold safety margin in the microbial
test specification of the finished product. APC of <100 CFU/gram and absence of objectionable in 10 grams
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 Packaging Applicators
 Product Applicators (e.g. sponges, pads, brushes)
 Composition: Natural vs. synthetic.
 Natural applicators are usually treated by using an antimicrobial treatment such as gamma irradiation,
ethylene oxide or electron beam to reduce the microbial bioburden levels.
 Natural applicators are known to be contaminated with microorganisms (e.g. pony hair brushes).

 Incorporate antimicrobial agents in the composition of synthetic material (e.g. Microban, Zinc zeolite).

 How are Applicators Used?

 Stored outside the product formulation after use by the consumer.
 Powder/Sponge applicators.
 Doe foot applicators of click pens.

 Stored within the product formulation (e.g. mascara brushes).

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Microbial Content Testing of
Cosmetic Formulations

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 Non-Susceptible Cosmetic Formulations to Microbial
Contamination During Manufacturing
 Anhydrous Formulations with Processing Temperatures ≥ 68oC.
 Lipsticks
 Wax-based Eye and Lip Pencils
 Soap Bars
Formulations with Low Water Content (<1%).
 Bath Salts/Powder Bubble Baths
 Aqueous Formulations with a pH ≥ 10.0.
 Depilatory or Hair Removal Creams/Gels
 Hair Colorants/Dyes
 Formulations Containing Hostile Raw Ingredients
 Alcohol (e.g. with >20% concentration) – Colognes, Hair Sprays, Deodorant Sprays
 Antiperspirants with greater than 25% concentration of Aluminum chlorohydrate
 Nail Enamels with a solvent base (e.g. ethyl acetate and butyl acetate)

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Reasons for Establishing Cosmetic Microbial Content Limits for Products
that are Susceptible to Contamination During Manufacturing
 No product should have a microbial content that can be considered:
 Harmful to the user such as:
 Excessive numbers of microorganisms.
 Specific types of microorganisms:
 e.g. Pseudomonas aeruginosa in an eye formulation.
 e.g. Staphylococcus aureus in a topical formulation.
 Can compromise product esthetics due to microbial growth such as:
 Malodors.
 Phase Separation.
 Color Changes.
 e.g. Growth of Ps. aeruginosa in a product will produces a green pigment.
 e.g. Growth of Serratia marcescens in a product will produce a red pigment.
 Presence of Visible Microbial Colonies (e.g. Mold Colonies).

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 Cosmetic Product Formulations Susceptible to
Contamination During Manufacturing
 Product Formulations Containing >1% water.
 Creams and Lotions
 Shampoos and Conditioners
 Bubble Baths
 Aqueous Eyeshadows
 Makeup Formulations
 Anhydrous Liquid Formulations
 Body Oils
 Lip Glosses with a processing Temperature <68oC
 Anhydrous Powder Formulations
 Eyeshadows
 Blushes
 Powder Foundations

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 Guidelines for Cosmetic Microbial Limit Specifications
 FDA’s BAM Chapter 23 – Microbiological Methods for Cosmetics
 Eye-area Products - < 500 CFU/gram
 Non-eye-area Products - <1000 CFU/gram
 Absence of known pathogens and opportunistic pathogens
 CTFA (PCPC) Microbiological Guidelines – Establishing Microbial Quality of
Cosmetic Products
 Baby and Eye Area Products - < 100 CFU/gram or ml
 All Other Products - <1000 CFU/gram or ml
 No product should have a microbial content recognized as either harmful to the user
or able to compromise product integrity.

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 ISO Standard for Cosmetic Microbial Limit Specifications
 ISO 17516 – Cosmetic – Microbiology – Microbiological Limits
 Enumeration Limits
 Children under 3 years of age, eye area or mucous membranes - <100 CFU/g or ml
 Other Products - <1000 CFU/g or ml
 Enrichment Requirements:
 Escherichia coli – Absence in 1 g or ml
 Ps. aeruginosa – Absence in 1 g or ml
 S. aureus – Absence in 1 g or ml
 C. albicans – Absence in 1 g or ml

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USP/EP/JP Harmonized Informational Chapter <1111>
 <1111> Microbiological Examination of Nonsterile Products:
Acceptance Criteria for Pharmaceutical Preparations and Substances for
Pharmaceutical Use
 Cutaneous Use - Aerobic Plate Count: 100 CFU/gram
- Yeast/Mold Count: 10 CFU/gram
- Absence of S. aureus (1 g or ml)
- Absence of Ps. aeruginosa (1 g or ml)
 Aqueous Preparations for Oral Use - Aerobic Plate Count: 100 CFU/gram
- Yeast/Mold Count: 10 CFU/gram
- Absence of E. coli (1 g or ml)

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 In-House Cosmetic Microbial Test Specifications
 Are generally based upon the finished product microbial test specifications
of a guideline, standard or compendia.
 Tend to be more stringent than the finished product specifications of a
guideline, standard or compendia:
 For example:
 Total Microbial Count: <100 CFU/gram
 Absence of Gram-negative bacteria
 Absence of Staphylococcus aureus
 Absence of Candida albicans

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 Presumptive Identification Methods of
Recovered Microbial Isolates
 Characteristic microbial growth on selective/differential agars
 Gram stain results (e.g. positive or negative)
 Morphology (e.g. cocci, bacilli, yeast)
 Diagnostic tests:
 Coagulase
 Catalase
 Slide Agglutination
 O/F Tests

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Phenotypic Microbial Identification Methods
 Biochemical Identification Kits
 Vitek
 Remel
 API
 BBL
 Biolog
 MALDI-TOF Mass Spectrometry
 MIDI Sherlock (A GC FAME Method)

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Genotypic Microbial Identification Methods
 Genotypic
 DNA Base Ratio
 Restriction fragment analysis
 DNA probes
 Phylogenetic
 DNA-DNA Hybridization
 16s and 23s rRNA sequencing
 Strain Specific PCR

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PCPC Survey Results for Identification Methods
Being Used by the Cosmetic Industry
Identification Methods
4.20%

8.30%

29.20%
58.30%

Biochemical Identification Kits Genotypic (Riboprinter, 16S rRNA Sequencing)

MALDI-TOF Mass Spectrometry MIDI Sherlock

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 Microorganisms of Concern to the Cosmetic Industry
 Primary Microorganisms of Concern
Pseudomonas aeruginosa Burkholderia cepacia complex
Staphylococcus aureus Escherichia coli
Candida albicans
 Secondary Microorganisms of Concern
Any Gram-negative bacilli
Enterococcus species
Mold species

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Recommended Microbial Strains for Conducting Validation
of Cosmetic Microbial Test Methods
 Plate Count - Preservative Challenge and Microbial Content Test Methods
 Pseudomonas aeruginosa ATCC 9027
 Escherichia coli ATCC 8739
 Staphylococcus aureus ATCC 6538
 Burkholderia cepacia ATCC 25416
 Candida albicans ATCC 10231
 Aspergillus brasiliensis ATCC 16404
 Enrichment
 Pseudomonas aeruginosa ATCC 9027
 Escherichia coli ATCC 8739
 Staphylococcus aureus ATCC 6538
 Burkholderia cepacia ATCC 25416

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Validation of Microbial Plate Count Test Method
GENERAL ASPECTS
Add to
0.1 ml separate
(<1000 CFU) 2 x 1.0 aliquots 100 x 15 mm Count # of
Petri Dishes microbial colonies
Prepare a 104 1g + 8.9 ml and add 18 to per Petri dish,
(<100 CFU) Incubate
CFU/ml Neut. Diluent 20-ml/Petri average counts,
Suspension Dish of convert average
melted counts to Log10
0.1 ml 2 x 1.0 aliquots Microbial values, and
(<1000 CFU) Growth Agar, compare
mix, and
9.9 ml of
solidify.
Neut. Diluent

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Example of Microbial Count Validation Test Data for a Cosmetic Cream

Test Organism Microbial Counts Microbial Counts Log10 Value of Log10 Value of Log Difference
with Sample without Sample Counts with Sample Counts with Sample
Counts Ave. Counts Ave.
Ps. aeruginosa 122, 172 147 209, 190 199.5 2.17 2.3 (+) 0.13
ATCC 9027
S. aureus 72, 90 81 92, 91 91.5 1.91 1.96 (+ ) 0.05
ATCC 6538
E. coli 78, 90 84 81, 93 87 1.92 1.94 (+) 0.02
ATCC 8739
B. cepacia 25, 35 30 28, 40 34 1.48 1.53 (+) 0.05
ATCC 25416
C. albicans 8, 5 6.5 15, 9 12 0.81 1.08 (+) 0.27
ATCC 10231
A. brasiliensis 54, 68 61 44, 47 45.5 1.78 1.65 (-) 0.13
ATCC 16404

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 Validation of an Enrichment Test Method
GENERAL ASPECTS

0.1-ml of Microbial Streak onto After If microbial

test Enrichment Selective/ incubation, growth is
organism Broth Differential examine for present,
at a conc. Agars and presence or confirm
of <100 TSA for absence of identification of
CFU isolation microbial recovered
growth organism

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Conclusions

84
Thank You

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