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

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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) Acidic (e.g. Sorbic Acid, Benzoic Acid)

Formaldehyde Donors (Imidazolidinyl urea, DMDM Hydantoin) Halogenated Compounds (e.g. Chlorphenesin)

Isothiazolinones (e.g. MCT/MI) Alcohols (e.g. Ethyl Alcohol)

Phenolic Types (e.g. Phenoxyethanol, Benzyl 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.

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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

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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.

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.

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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.

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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

12

Formulation Factors Affecting the Antimicrobial Activity of Preservatives

Water Activity

pH

Solubility of Preservatives

Compatibility with Other Raw Ingredients

13

Microbial Metabolism and Growth

Need a source of available water and nutrients.

By having 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.

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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

15

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

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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)

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

Least Susceptible Lipsticks

Nail Enamels

Powders

Highly Susceptible Creams

Lotions

Shampoos

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.

pH Microbiological AffectsFor 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

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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.

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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.

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

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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

31

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.

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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.

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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.

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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

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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.

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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?

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.

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Preservative Challenge Test Method

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GENERAL ASPECTS

Standardized Microbial

Suspension

Inoculated Product

Removal of 1.0 gram

Aliquots at Specified Intervals

Neutralization of Preservative

Antimicrobial Activity

Recovery of Surviving

Microorganisms

Calculation of Percent or Log10

Reduction

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

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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.

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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).

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Main Differences Between the Various Types ofPreservative 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

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CTFA Survey Results for Microorganism Source Used in Preservative Challenge Testing

95

68 68

0102030405060708090

100

USP Test

Strains

Other ATCC

Strains

In-House

Strains

Type of Microbial Strains

% R

esp

on

de

nts

Challenge Testing Parameters-Topical Product Formulations

Challenge Test Method Inoculum Level in Product (CFU/gram)

Testing Intervals for Performing 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

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Various Challenge Testing Acceptance Criteria for Topical Product Formulations

Challenge Test Method

Challenge Acceptance Criteria (Log10 Reduction)

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

B: 3 B: NI B: NI

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

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NT = Not Tested

NI = No Increase

NC = No Criteria

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In-House Preservative Challenge Acceptance Criteria

Water Miscible/Dispersible ProductsGenerally 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 ProductsRisk 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.

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

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47

29

13 11 80

0

20

40

60

80

In-

House

USP M-3 M-4 EP AOAC ASTM

Challenge Test Methods

% R

espo

nden

ts

Microbial Content Testing

Raw Ingredients

Packaging Applicators

Finished Product Formulations

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Microbial Methods for Evaluating Product Quality

Aerobic Plate Count

Enrichment Test

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Aerobic Plate Count Method

GENERAL ASPECTS

Plate Count Neutralizing

Diluent

1:10 Dilution Dispense

&

Incubate

Count Number of Microorganisms/gram or ml

Sample

Enrichment Test Method

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GENERAL ASPECTS

1.0 or 10.0 grams

Microbial Enrichment

Broth with

Neutralizers

Streak onto Selective/

Differential Microbial Growth Agars

Incubate

Gram-stain and Identify Recovered Microbial Isolates

Sample

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

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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

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Microbial Content Testing of Cosmetic Raw Ingredients

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Microbial Classifications of Raw Ingredients

Hostile Raw Ingredients

Inert Raw Ingredients

Chemically Preserved Raw Ingredients

Raw Ingredients Supporting Microbial Growth

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Hostile and Inert Raw IngredientsHostile 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.

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Chemically Preserved Raw Ingredients and Raw Ingredients Supporting Microbial Growth

Chemically Preserved Raw IngredientsPreservatives 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.

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).

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).

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

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58.30%29.20%

8.30%

4.20%

Identification Methods

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

MALDI-TOF Mass Spectrometry MIDI Sherlock

Microorganisms of Concern to the Cosmetic Industry

Primary Microorganisms of ConcernPseudomonas aeruginosa Burkholderia cepacia complex

Staphylococcus aureus Escherichia coliCandida albicans

Secondary Microorganisms of Concern

Any Gram-negative bacilli

Enterococcus species

Mold species

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Recommended Microbial Strains for Conducting Validationof 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

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GENERAL ASPECTS

Prepare a 104

CFU/ml Suspension

1g + 8.9 ml Neut. Diluent

9.9 ml of

Neut. Diluent

0.1 ml

(<1000 CFU)

0.1 ml (<1000 CFU)

2 x 1.0 aliquots

2 x 1.0 aliquots

Incubate

Count # of microbial colonies

per Petri dish, average counts, convert average counts to Log10

values, and compare

Add to separate

100 x 15 mm Petri Dishes

and add 18 to 20-ml/Petri

Dish of melted

Microbial Growth Agar,

mix, and solidify.

(<100 CFU)

Example of Microbial Count Validation Test Data for a Cosmetic Cream

Test Organism Microbial Counts with Sample

Microbial Counts without Sample

Log10 Value of Counts with Sample

Log10 Value of Counts with Sample

Log Difference

Counts Ave. Counts Ave.

Ps. aeruginosa ATCC 9027

122, 172 147 209, 190 199.5 2.17 2.3 (+) 0.13

S. aureus ATCC 6538

72, 90 81 92, 91 91.5 1.91 1.96 (+ ) 0.05

E. coli ATCC 8739

78, 90 84 81, 93 87 1.92 1.94 (+) 0.02

B. cepacia ATCC 25416

25, 35 30 28, 40 34 1.48 1.53 (+) 0.05

C. albicans ATCC 10231

8, 5 6.5 15, 9 12 0.81 1.08 (+) 0.27

A. brasiliensis ATCC 16404

54, 68 61 44, 47 45.5 1.78 1.65 (-) 0.13

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Validation of an Enrichment Test Method

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GENERAL ASPECTS

0.1-ml of test

organism at a conc. of <100

CFU

Microbial Enrichment

Broth

Streak onto Selective/

Differential Agars and

TSA for isolation

After incubation, examine for presence or absence of microbial growth

If microbial growth is present, confirm

identification of recovered organism

Conclusions

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Thank You

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