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RIGHT S O L U T I O N S | RIGHT PARTNER
Microbiology for Oral and Topical Products - The basics
Scott Colbourne
Business Manager NSW – ALS Food & Pharmaceutical
Contents
• TGO 77 - Introduction
• Tests Performed
– Common Tests
– Preservative Efficacy
– Endotoxin
– Bioburden
• Method Turn Around Times (TAT)
• The Confirmation Process
• Method Validation
• Causes of counts being high initially, then low when retested
• Topical Products
2Microbiology for Oral and Topical
Products - The Basics
TGO 77 - General
• Therapeutic Goods Order No. 77 – Microbiological
Standards for Medicines (TGO 77)
• This Order specifies the minimum microbiological
requirements with which a medicine must comply
throughout its shelf life in Australia.
• On and from 1 January 2010, each medicine to which this
Order applies must comply with this Order.
3Microbiology for Oral and Topical
Products - The Basics
TGO 77 - General
• Acceptance criteria is the same as used in the BP, Ph.Eur. and
the USP:
• A medicine is considered to be non-compliant with a microbial
count acceptance criterion if the CFU (colony forming unit)
count exceeds two (2) times the stated limit.
• When the Order establishes a limit of 102
CFU, the maximum
acceptable count is 200 (which allows for microbial testing
variability)
4Microbiology for Oral and Topical
Products - The Basics
TGO 77
• Section 7: Sterility and Bacterial Endotoxin testing
– A medicine must comply when these tests are required by
an individual or general monograph
– Where a sponsor labels or packages a medicine in a way
that states or implies that the medicine is sterile
5Microbiology for Oral and Topical
Products - The Basics
TGO 77
• Section 8: Efficacy of antimicrobial preservation of
multidose medicines
– Applies to multidose sterile medicines (e.g. eye drops)
– Applies to multidose non-sterile medicines (e.g. oral liquids)
6Microbiology for Oral and Topical
Products - The Basics
TGO 77
• Subsection 9(1):
– Applies to prescription, OTC and many complementary
medicines
– Acceptance Criteria
• BP Appendix XVI (Ph. Eur. 5.1.4): Limits defined by route
of administration
• USP chapter <1111>: Almost identical to the BP criteria
– Testing consists of some/all of the following:
7Microbiology for Oral and Topical
Products - The Basics
Total Aerobic Microbial Count
Total CombinedYeasts/MouldsCount
Escherichia coli Staphylococcus aureus
Pseudomonas aeruginosa
Candida albicans
Bile-tolerant Gram-negative bacteria
TGO 77
• Subsection 9(2):
– Applies to a complementary medicine oral dosage form containing raw
material of natural origin
* A herbal medicinal product consisting solely of one or more herbal substances
(whole, reduced or powdered) to which boiling water is added before use
8Microbiology for Oral and Topical
Products - The Basics
Microbiological Quality Units Schedule 1:Containing
Schedule 2:Solely herbal substances *
Total aerobic microbial count CFU per g or mL ≤ 104 ≤ 107
Total yeast and mould count CFU per g or mL ≤ 102 ≤ 105
Bile-tolerant Gram negative bacteria
CFU per g or mL ≤ 102 ≤ 102
Salmonella in 10g or 10mL Absent Absent
Escherichia coli in 1g or 1mL Absent Absent
Staphylococcus aureus in 1g or 1mL Absent Not applicable
Common Tests – what and why?
Microbiology for Oral and Topical
Products - The Basics9
• Total Aerobic Microbial Count (TAMC)
What?
A generic count of micro-organisms present. The results are
non-specific
Why?
Gives a general indication of the level of micro-organisms
present in the sample tested
Can be referred to as…
Total Microbial Count (TMC), Standard Plate Count (SPC), TVC,
TVAC
Common Tests – what and why?
• Yeast and Mould
What?
Generic test for the levels of these present. Non-specific.
Why?
• They cause biodegradation of natural materials
• Some yeasts are useful in fermentation (e.g. Bread and Beer)
• However some (e.g. Candida albicans) are opportunistic
pathogens and can cause infections
Microbiology for Oral and Topical
Products - The Basics10
Common Tests – what and why?
• Bile Tolerant gram negative bacteria
What?
• The name comes from their gram staining method of bacterial
differentiation. Basically they do not retain crystal violet.
• Large family of bacteria including Enterobacteriaceae, Salmonella and
E.Coli
Why?
• In humans, disease is produced by both invasive action and production of
toxin.
• Species not normally associated with disease are often opportunistic
pathogens.
• Enterobacteriaceae have been responsible for up to half of hospital
infections annually in the United States
Microbiology for Oral and Topical
Products - The Basics11
Common Tests – what and why?
• Salmonella
What?
Pathogen found in cold/warm blooded animals and the
environment
Why?
• It causes food poisoning, such as gastrointestinal issues
• To ensure there is no presence of pathogens
Microbiology for Oral and Topical
Products - The Basics12
Common Tests – what and why?
• E. coli
What?
A coliform almost exclusively of faecal origin
Why?
• E. coli presence is an effective confirmation of faecal
contamination.
• Most strains of E. coli are harmless, but some can cause
serious illness in humans.
Microbiology for Oral and Topical
Products - The Basics13
Common Tests – what and why?
• Staphylococcus Aureus
What?
There are over 30 Staphylococcus types of bacteria, but
Staphylococcus aureus causes most staph infections
Why?
• Can cause skin infections (most common), Pneumonia, food
poisoning, toxic shock syndrome and blood poisoning
(bacteremia)
• A staph skin infection is more likely if you have a cut or scratch
Microbiology for Oral and Topical
Products - The Basics14
Common Tests – what and why?
• Pseudomonas aeruginosa
What?
It is found in soil, water and most man-made environments
Why?
• An opportunistic human pathogen of immunocompromised
individuals
• Because it thrives on moist surfaces, this bacterium is also
found on and in medical equipment, including catheters,
causing cross-infections in hospitals and clinics
Microbiology for Oral and Topical
Products - The Basics15
Common Tests – what and why?
• Candida Albicans
What?
• A fungus that is a constituent of the normal gut flora that live in the
human mouth and gastrointestinal tract.
• Present in 80% of the human population without causing harmful
effects
Why?
• Causes opportunistic oral and genital infections in humans
• Has emerged as an important cause of morbidity and mortality in
immunocompromised patients (e.g., AIDS, and cancer chemotherapy)
• Routinely required for Vaginal use medicines
Microbiology for Oral and Topical
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Preservative Efficacy Testing (PET)
Microbiology for Oral and Topical
Products - The Basics17
• What is preservative efficacy?
• The test consists of challenging the preparation by inoculating with suitable
micro-organisms, at a prescribed temperature, withdrawing samples at specified
intervals and counting the organisms present.
• The preservative properties of the preparation are adequate if there is a
significant fall (or no increase)
• Acceptance criteria vary for different types of preparations
Preservative Efficacy Testing (PET)
Microbiology for Oral and Topical
Products - The Basics18
• What is preservative efficacy?
• The test consists of challenging the preparation by inoculating with suitable
micro-organisms, at a prescribed temperature, withdrawing samples at specified
intervals and counting the organisms present.
• The preservative properties of the preparation are adequate if there is a
significant fall (or no increase)
• Acceptance criteria vary for different types of preparations
• What samples are tested ?
• Samples with preservative systems e.g. liquid products, creams and lotions
Preservative Efficacy Testing (PET)
Microbiology for Oral and Topical
Products - The Basics19
• What is preservative efficacy?
• The test consists of challenging the preparation by inoculating with suitable
micro-organisms, at a prescribed temperature, withdrawing samples at specified
intervals and counting the organisms present.
• The preservative properties of the preparation are adequate if there is a
significant fall (or no increase)
• Acceptance criteria vary for different types of preparations
• What samples are tested ?
• Samples with preservative systems e.g. liquid products, creams and lotions
• How long does it take?
• Samples are tested at intervals such as 0, 1, 2, 7, 14 and 28 days.
• Therefore the completed full PET will be 5-7 days after the final time point.
Preservative Efficacy Testing (PET)
Microbiology for Oral and Topical
Products - The Basics20
• What is preservative efficacy?
• The test consists of challenging the preparation by inoculating with suitable
micro-organisms, at a prescribed temperature, withdrawing samples at specified
intervals and counting the organisms present.
• The preservative properties of the preparation are adequate if there is a
significant fall (or no increase)
• Acceptance criteria vary for different types of preparations
• What samples are tested ?
• Samples with preservative systems e.g. liquid products, creams and lotions
• How long does it take?
• Samples are tested at intervals such as 0, 1, 2, 7, 14 and 28 days.
• Therefore the completed full PET will be 5-7 days after the final time point.
• How often is it performed?
• Generally not required to release batches of product.
• When finalising the formulation and to check the preservative system.
Endotoxin (LAL)
Microbiology for Oral and Topical
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• What is endotoxin testing?
• The outer membrane of a Gram-negative bacterium is composed of
lipopolysaccharide (an endotoxin).
• The qualitative determination of bacterial endotoxin concentration is
performed by the Limulus Amebocyte Lysate (LAL) gel clot test
Endotoxin (LAL)
Microbiology for Oral and Topical
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• What is endotoxin testing?
• The outer membrane of a Gram-negative bacterium is composed of
lipopolysaccharide (an endotoxin).
• The qualitative determination of bacterial endotoxin concentration is
performed by the Limulus Amebocyte Lysate (LAL) gel clot test
• What samples are tested ?
• The primary application for LAL is the testing of parenteral pharmaceuticals
and medical devices that contact blood or cerebrospinal fluid
Endotoxin (LAL)
Microbiology for Oral and Topical
Products - The Basics23
• What is endotoxin testing?
• The outer membrane of a Gram-negative bacterium is composed of
lipopolysaccharide (an endotoxin).
• The qualitative determination of bacterial endotoxin concentration is
performed by the Limulus Amebocyte Lysate (LAL) gel clot test
• What samples are tested ?
• The primary application for LAL is the testing of parenteral pharmaceuticals
and medical devices that contact blood or cerebrospinal fluid
• Why is it performed?
• When the endotoxin is at a significant level it produces a pyrogenic effect
• A pyrogen is an agent which elevates the body temperature of an animal or
human
Bioburden
Microbiology for Oral and Topical
Products - The Basics24
• What is Bioburden testing?
• This testing does not constitute sterility testing, but is a method used
to determine the microbial bioburden of the sample.
• Bioburden is normally defined as the number of bacteria living on a
surface that has not been sterilised
Bioburden
Microbiology for Oral and Topical
Products - The Basics25
• What is Bioburden testing?
• This testing does not constitute sterility testing, but is a method used
to determine the microbial bioburden of the sample.
• Bioburden is normally defined as the number of bacteria living on a
surface that has not been sterilised
• What samples are tested?
• Medical Devices
Bioburden
Microbiology for Oral and Topical
Products - The Basics26
• What is Bioburden testing?
• This testing does not constitute sterility testing, but is a method used
to determine the microbial bioburden of the sample.
• Bioburden is normally defined as the number of bacteria living on a
surface that has not been sterilised
• What samples are tested?
• Medical Devices
• How long does it take?
• The test requires 5 days of incubation
Bioburden
Microbiology for Oral and Topical
Products - The Basics27
• What is Bioburden testing?
• This testing does not constitute sterility testing, but is a method used
to determine the microbial bioburden of the sample.
• Bioburden is normally defined as the number of bacteria living on a
surface that has not been sterilised
• What samples are tested?
• Medical Devices
• How long does it take?
• The test requires 5 days of incubation
• Why is it performed?
• The aim of bioburden testing is to measure the total number of viable
micro-organisms on a medical device prior to its final sterilisation
before implantation or use.
Method Turn Around Times
Microbiology for Oral and Topical
Products - The Basics28
Method
Turnaround Time (Days)
If CleanIf Confirmation is
Required
Total aerobic microbial count 5 n/a
Yeast and Mould 5-7 n/a
Bile tolerant Gram negative bacteria 2-3 n/a
Salmonella 3-4 5-6
E. coli 3-6 4-8
Staphylococcus Aureus 2-4 4-6
Extras not listed in TGO 77 section 9(2)
Pseudomonas aeruginosa 2-4 4-6
Candida albicans 6-7 10+
Confirmation Process
Microbiology for Oral and Topical
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Hi John,
Please note that the below sample is suspect for E.Coli:
Lot: 12345 sample A, DOM 15/05/2015
The sample is under confirmation and the final result will
be updated once complete
Thank you and best regards
Jenny Bloggs
Microbiologist
• Have you ever received an email like this?
Confirmation Process
Microbiology for Oral and Topical
Products - The Basics30
Hi John,
Please note that the below sample is suspect for E.Coli:
Lot: 12345 sample A, DOM 15/05/2015
The sample is under confirmation and the final result will
be updated once complete
Thank you and best regards
Jenny Bloggs
Microbiologist
• Have you ever received an email like this?
For a number of microbiological tests the initial test is generic and any positive or SUSPECTresult requires more testing. A suspect may be:• The micro-organism being analysed• Other micro-organisms present that interfere• Matrix interference
Synonyms: Presumptive
Confirmation Process
Microbiology for Oral and Topical
Products - The Basics31
Hi John,
Please note that the below sample is suspect for E.Coli:
Lot: 12345 sample A, DOM 15/05/2015
The sample is under confirmation and the final result will be updated once complete
Thank you and best regards
Jenny Bloggs
Microbiologist
• Have you ever received an email like this?
Confirmation Process
Microbiology for Oral and Topical
Products - The Basics32
Hi John,
Please note that the below sample is suspect for E.Coli:
Lot: 12345 sample A, DOM 15/05/2015
The sample is under confirmation and the final result will be updated once complete
Thank you and best regards
Jenny Bloggs
Microbiologist
• Have you ever received an email like this?
What is confirmation?• Carrying out further steps of a test for identifying a particular type of bacteria.• Therefore CONFIRMING its presence.• When the steps have been completed we say that the test has been “Confirmed”.• After confirmation, the result will be finalised.
Synonyms: Follow up
Confirmation Process
• What tests can require confirmation?
Microbiology for Oral and Topical
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Method
Turnaround Time (Days)
If CleanIf Confirmation is
Required
Total aerobic microbial count 5 n/a
Yeast and Mould 5 n/a
Bile tolerant Gram negative bacteria 3 n/a
Salmonella 4 2-3
E. coli 5 7
Staphylococcus Aureus 3 6
Extras not listed in TGO 77 section 9(2)
Pseudomonas aeruginosa 3 5
Candida albicans 6-7 10+
Confirmation Process
• What tests can require confirmation?
Microbiology for Oral and Topical
Products - The Basics34
Method
Turnaround Time (Days)
If CleanIf Confirmation is
Required
Total aerobic microbial count 5 n/a
Yeast and Mould 5 n/a
Bile tolerant Gram negative bacteria 3 n/a
Salmonella 4 2-3
E. coli 5 7
Staphylococcus Aureus 3 6
Extras not listed in TGO 77 section 9(2)
Pseudomonas aeruginosa 3 5
Candida albicans 6-7 10+
Method Validation
• What is microbiological method validation?
• Verifying that the method used including media and sample
dilution are suitable to recover the micro organism
Microbiology for Oral and Topical
Products - The Basics35
Method Validation
• What is microbiological method validation?
• Verifying that the method used including media and sample
dilution are suitable to recover the micro organism
• How is it done?
• By inoculating the sample with the bacteria, incubating and
recovering >70% of the micro organism
Microbiology for Oral and Topical
Products - The Basics36
Method Validation
• What is microbiological method validation?
• Verifying that the method used including media and sample
dilution are suitable to recover the micro organism
• How is it done?
• By inoculating the sample with the bacteria, incubating and
recovering >70% of the micro organism
• How long does it take?
• The validation is the same duration as the test
Microbiology for Oral and Topical
Products - The Basics37
Method Validation
• What is microbiological method validation?
• Verifying that the method used including media and sample
dilution are suitable to recover the micro organism
• How is it done?
• By inoculating the sample with the bacteria, incubating and
recovering >70% of the micro organism
• How long does it take?
• The validation is the same duration as the test
• How often is it performed?
• Once per product every 5 years
Microbiology for Oral and Topical
Products - The Basics38
Method Validation
• Why do they fail?
• Due to antimicrobial efficacy of the product
• Presence of preservatives
• <70% recovery of the validation test
Microbiology for Oral and Topical
Products - The Basics39
Method Validation
• Why do they fail?
• Due to antimicrobial efficacy of the product
• Presence of preservatives
• <70% recovery of the validation test
• How can I avoid validation delays on new products?
• By sending in samples for validation in advance, e.g.
process validation or pilot scale batches
Microbiology for Oral and Topical
Products - The Basics40
High to Low Counts
• How can a result be high in one test and then low when
retested?
• This can be caused by a number of factors
– The sample may not be completely homogenous
– Lab error
– The sample has anti-bacterial properties
– Preservative taking effect
Microbiology for Oral and Topical
Products - The Basics41
High to Low Counts: Sample Not Homogeneous
• The laboratory generally uses 10g of the sample in the required media, e.g. 90 mL
Microbiology for Oral and Topical
Products - The Basics42
High to Low Counts: Sample Not Homogeneous
• The laboratory generally uses 10g of the sample in the required media, e.g. 90 mL
• Of that 90 mL, 0.1ml to 1ml is removed for each test
Microbiology for Oral and Topical
Products - The Basics43
High to Low Counts: Sample Not Homogeneous
• The laboratory generally uses 10g of the sample in the required media, e.g. 90 mL
• Of that 90 mL, 0.1ml to 1ml is removed for each test
• A reported result of 500 cfu/mL, may only be 5 colonies on an agar plate. This is a
low number and with repeat testing small variations will affect the result by 100’s of
cfu/mL
Microbiology for Oral and Topical
Products - The Basics44
High to Low Counts: Sample Not Homogeneous
• The laboratory generally uses 10g of the sample in the required media, e.g. 90 mL
• Of that 90 mL, 0.1ml to 1ml is removed for each test
• A reported result of 500 cfu/mL, may only be 5 colonies on an agar plate. This is a
low number and with repeat testing small variations will affect the result by 100’s of
cfu/mL
• In general, acceptable variation would be within half a log, e.g.
Microbiology for Oral and Topical
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Result Possible Variation
5,000 (or 5 x 103) ± 500
3,000,000 (or 3 x 106) ± 500,000
High to Low Counts: Lab Error
• This is always a possibility and our quality procedures and controls
minimise this risk. However it can never be 100% removed.
Microbiology for Oral and Topical
Products - The Basics46
High to Low Counts: Lab Error
• This is always a possibility and our quality procedures and controls
minimise this risk. However it can never be 100% removed.
• If we believe lab error is a possibility we will try to look for a pattern,
e.g.
• Do all the similar samples have a high count?
• Does the sample have a history of high counts?
• Do unrelated samples tested concurrently have high counts?
Microbiology for Oral and Topical
Products - The Basics47
High to Low Counts: Lab Error
• This is always a possibility and our quality procedures and controls
minimise this risk. However it can never be 100% removed.
• If we believe lab error is a possibility we will try to look for a pattern,
e.g.
• Do all the similar samples have a high count?
• Does the sample have a history of high counts?
• Do unrelated samples tested concurrently have high counts?
• We also look at other factors, e.g.
• Experience of the technician
• Environmental monitoring results
• Media testing
Microbiology for Oral and Topical
Products - The Basics48
High to Low Counts: Anti-Bacterial Properties
• The sample may contain substances that are anti-bacterial
(e.g. preservative systems)
• Samples are stored for up to 5 days before a retest can
occur and substances in the sample itself may lower the
bacterial count
• Therefore possibly high levels upon initial testing have been
killed off or had their numbers lowered before the retest.
Microbiology for Oral and Topical
Products - The Basics49
Topical Products
• What should I test?
• Based on Cutaneous acceptance criteria in the Pharmacopoeias
(BP, USP, Ph. Eur)
Microbiology for Oral and Topical
Products - The Basics51
Microbiological Quality Units Pass Criteria
Total aerobic microbial count CFU per g or mL ≤ 102
Total yeast and mould count CFU per g or mL ≤ 101
Staphylococcus aureus in 1g or 1mL Absent
Pseudomonas aeruginosa in 10g or 10mL Absent
Topical Products
• What should I test?
• Based on Cutaneous acceptance criteria in the Pharmacopoeias
(BP, USP, Ph. Eur)
Microbiology for Oral and Topical
Products - The Basics52
Microbiological Quality Units Pass Criteria
Total aerobic microbial count CFU per g or mL ≤ 102
Total yeast and mould count CFU per g or mL ≤ 101
Staphylococcus aureus in 1g or 1mL Absent
Pseudomonas aeruginosa in 10g or 10mL Absent
• Preservative Efficacy Testing (PET) – for liquid, multi-use products,
using Pharmacopoeial methodologies
Topical Products
• What should I test?
• Based on Cutaneous acceptance criteria in the Pharmacopoeias
(BP, USP, Ph. Eur)
Microbiology for Oral and Topical
Products - The Basics53
Microbiological Quality Units Pass Criteria
Total aerobic microbial count CFU per g or mL ≤ 102
Total yeast and mould count CFU per g or mL ≤ 101
Staphylococcus aureus in 1g or 1mL Absent
Pseudomonas aeruginosa in 10g or 10mL Absent
• Preservative Efficacy Testing (PET) – for liquid, multi-use products,
using Pharmacopoeial methodologies
• Method Validation?
• This is critical to ensure the method used is suitable to recover the
micro organisms. Per product (formulation) every 5 years
Further Reading
British Pharmacopoeia (2015): Appendix XVI D. Microbiological Quality of Non-sterile
Pharmaceutical Preparations and Substances for Pharmaceutical Use
United States Pharmacopoeia – National Formulary (38): <1111> MICROBIOLOGICAL
EXAMINATION OF NONSTERILE PRODUCTS: ACCEPTANCE CRITERIA FOR PHARMACEUTICAL
PREPARATIONS AND SUBSTANCES FOR PHARMACEUTICAL USE
Therapeutic Goods Order No. 77 - Microbiological Standards for Medicines (22/09/2008)
Microbiology for Oral and Topical
Products - The Basics54
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