English Don Pres
English Don Pres
English Don Pres
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?
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)
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.
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.
11
Formulation Factors Affecting the Antimicrobial
Activity of Preservatives
Water Activity
pH
Solubility of Preservatives
12
Microbial Metabolism and Growth
Need a source of available water and nutrients.
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.
0.90 to 0.95 Several Gram-negative and most Gram-positive bacteria (e.g. Enterobacter
aerogenes, Escherichia coli, Bacillus species), mold and yeasts
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.
16
Formulation Water Activity and Preservative
Spectrum of Antimicrobial Activity
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
18
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
Formulation Factors - pH and Microbial Growth
Bacteria:
Optimum pH for growth is between 5.5 and 8.5.
20
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.
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
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
24
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.
25
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.
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
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.
28
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
31
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
32
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
33
Manufacturing Conditions Can Have an Affect on Preservatives
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.
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
39
Preservative Challenge Test
40
Preservative Challenge Test Method
GENERAL ASPECTS
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
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 Levels
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
47
Various Challenge Testing Acceptance Criteria for Topical
Product Formulations
Challenge Test Challenge Acceptance Criteria (Log10 Reduction)
Method
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
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
Enrichment Test
52
Aerobic Plate Count Method
GENERAL ASPECTS
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
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
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.
59
Chemically Preserved Raw Ingredients and Raw
Ingredients Supporting Microbial Growth
60
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.
61
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.
62
Two Ways for Establishing Microbial Limits for Raw Ingredients
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
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).
66
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)
68
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).
69
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
70
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.
71
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
72
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)
73
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
74
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
75
Phenotypic Microbial Identification Methods
Biochemical Identification Kits
Vitek
Remel
API
BBL
Biolog
MALDI-TOF Mass Spectrometry
MIDI Sherlock (A GC FAME Method)
76
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
77
PCPC Survey Results for Identification Methods
Being Used by the Cosmetic Industry
Identification Methods
4.20%
8.30%
29.20%
58.30%
78
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
79
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
80
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
81
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
82
Validation of an Enrichment Test Method
GENERAL ASPECTS
83
Conclusions
84
Thank You
85