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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) 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.
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
8
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.
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
11
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.
14
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
16
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.
17
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)
18
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)
19
20
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
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
23
24
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
25
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%
26
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.
27
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.
28
Compatibility with Other Raw Ingredients Some raw ingredients can be:
Microbial Nutrients
Preservative Inactivators
Preservative Absorbers
Preservative Potentiators
29
30
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
32
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
33
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
34
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.
35
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.
36
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.
37
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
38
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.
39
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.
40
Preservative Challenge Test Method
41
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
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
45
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
46
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
47
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
48
NT = Not Tested
NI = No Increase
NC = No Criteria
49
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.
50
CTFA Survey Results in the Types of Preservative Challenge Test Methods Being Used by the Cosmetic Industry
71
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
51
Microbial Methods for Evaluating Product Quality
Aerobic Plate Count
Enrichment Test
52
53
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
54
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
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
58
Microbial Classifications of Raw Ingredients
Hostile Raw Ingredients
Inert Raw Ingredients
Chemically Preserved Raw Ingredients
Raw Ingredients Supporting Microbial Growth
59
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.
60
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.
61
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.
62
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
63
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
64
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
65
66
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
67
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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