Disinfectant efficacy testing

Wednesday 22nd March 2023

 Introductions

Annette Russell
Sterile & Non-Sterile Annette.russell@rssl.com
RSSL Microbiology Lead
+44(0)118 918 4075
www.rssl.com

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 RSSL – Who are we?

Reading based
40 mins from London with
excellent airport and rail links

Best CRO 2022 & MHRA and FDA Over 30 years of Committed CRO,
Employer of the approved experience and Flexible, Agile,
Year 2022 ISO 17025 expertise, from a Quality across the
accredited team of 250 whole
scientists in 12 organisation.
specialisms

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 Annex 1 regulations support
Contamination Control Quality Risk Management New Technologies

Environmental Extractables and Cleaning and

monitoring and leachables of SUP and disinfectant
consultancy packaging validation

Raw material testing

Microbial
including bioburden
identification
and endotoxins

Vial and stopper

Particulate control Sterility assurance testing, container
integrity

Training and consultancy

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Today’s speaker – Dr Tim Sandle

• 25 years experience in the field of pharmaceutical microbiology

• Member of several editorial boards
• Extensive publication record
• Works in the pharmaceutical industry
• Tutor at University of Manchester and UCL

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 Introduction

● How to use disinfectants effectively

● Points for success

● Approaching disinfectant validation: Differences between European and U.S.

standards
● Practical approach to disinfectant validation: what is best for pharma?

● Legal requirements for disinfectants: Europe and U.S.

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 Objectives of cleaning and disinfection
Cleaning Disinfection

• Cleaning is the process of • Keeping cleanrooms and

removing residues and soil (dirt, equipment:
dust, protein, skin cells and so 1. With microbial numbers below
on) from surfaces to the extent cleanroom classification levels
that they are visually clean. 2. With no problematic trends of
• So a disinfectant will work organisms that are difficult to
effectively and interact with the kill
microbial cell • With ‘1’ and ‘2’, this is for an
appropriate period of time.

Incorporated as part of a wider contamination control strategy

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 What are disinfectants? #1
Disinfectants are:
• A diverse group of chemicals
• Part of a wider group called biocides
• Some are bacteriostatic, others are bactericidal
• Factors influenced by the active ingredient
Non-Oxidizing Disinfectants
• Specific mode of action against microorganisms
• Generally have a lower spectrum of activity
• Include: alcohols, aldehydes, amphoterics, acid anionics, biguanides, phenolics, quaternary Ammonium Compounds
(QACs).
Oxidizing Disinfectants
• Non-specific modes of action against microorganisms.
• A wider spectrum of activity.
• Most are sporicidal.
• Pose greater risks to human health.
• Includes: halogens, oxidizing agents, such as peracetic acid and hydrogen peroxide.

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 Regulations
The main regulatory documents relating to the use of disinfectants in pharmaceutical
manufacturing are:
‒ U.S. Code of Federal Regulations:
• 21 CFR 211.56b and 21 CFR 211.56c (which refer to sanitation)
• CFR 211.67 (which refer to equipment and maintenance)
• CFR 211.182 (which describes the need for a cleaning programme)
• CFR 211.113b (control of microbiological contamination)
‒ FDA Aseptic Processing Guide, revised 2004.
‒ USP (General Chapter <1072> Disinfectants and Antiseptics).
‒ PIC/S Recommendation on the Validation of Aseptic Processes
‒ Annex 1 to the EU Guide to Good Manufacturing Practice, 2022.

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 Rotation
– Typically two disinfectants are rotated.
– The theoretical argument for rotating two disinfectants is to reduce the possibility of
resistant strains of micro-organisms developing.
• Concern for antibiotics, but little evidence for biocides used on surfaces
– A requirement of some regulatory bodies
• EU GMP Guide states that “where disinfectants are used, more than one type should
be employed” (Annex 1).
• FDA Guidance on Aseptic Processing suggests a disinfectant programme should
include a sporicide and trends should be monitored
• USP (<1072>) is less exacting and poses some questions about the scientific need for
rotation: “It is prudent to augment the daily use of a bactericidal disinfectant with
weekly (or monthly) use of a sporicidal agent.”

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 EU GMP Annex 1 (2022)

• The references to disinfection have been expanded.

• The need to rotate between two different
disinfectants remains.
‒ One of these should be a sporicidal agent is a
change.
‒ Reference made to disinfectant qualification.
• This needs to be carried out within each facility
because of the different types of surfaces.
• Need to assess the bioburden of non-sterile
disinfectants.

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 EU GMP Annex 1 (2022)

• With cleaning, the text indicates that cleaning needs to

be undertaken prior to each use of disinfectant.
‒ In fact cleaning before every disinfection is not
necessary, especially in higher grade areas where
there is very little soiling.
‒ With disinfection, the confusing reference to
‘resistant strains’ remains. Here the phrase
''development of resistant strains “is often
misinterpreted as development of acquired
resistance (a theory which has largely been
discredited).

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 Factors affecting efficiency #1

• The intrinsic biocidal activity.

• The concentration of the disinfectant.
• Contact time.
• Nature of the surface disinfected.
‒ Physico-chemical conditions

• Hardness of water used to dilute the disinfectant.

• The level of organic materials present on the surface.
• Type and the number of microorganisms present.

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 Factors affecting efficiency #2
What makes a disinfectant work well or badly?
Number of microorganisms:
• More effective against a low number of microorganisms than a higher number.
Types of microorganisms:
• Some microorganisms are more resistant than others.

Most Resistant Microorganism

Prions
Bacterial spores
Protozoal oocysts
Mycobacteria
Fungal spores
Gram-negative rods
Least Resistant
Gram-positive bacteria

Adapted from: McDonnel “Antisepsis, Disinfection and Sterilization”, ASM Press, 1987
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 Factors affecting efficiency #3

Location of microorganisms
• How likely are they to be fixed Suspended
to surfaces?

Reduction
Contact time

Log.
• Time taken for a disinfectant to
kill the microorganism. Surface attached
• Must be left in contact with the (monolayer, not biofilm)
surface.
• Typically - 1 to 5 minutes. Concentration

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 Factors affecting efficiency #4
Temperature
‒ Most disinfectants work poorly at low temperatures e.g. cleanrooms
‒ To meet European disinfectant standards, disinfectants are qualified at ±20oC
‒ Is temperature an issue?
Different surfaces
‒ Surfaces differ, studies show variability of different disinfectants on different surfaces.
‒ Surface conditions
‒ Surfaces must be clean
• Sporicides especially have particularly poor penetrative ability
Method of application
‒ Spray and wiping out performs spraying every time
Time
‒ As surfaces age, some studies suggest that spores become harder to kill.

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 Factors affecting efficiency #5
Environment and impact on the contact time
‒ Under what conditions has the contact time been assessed?
• Standard environment
• Turbulent flow cleanroom (e.g. Grade B)
• Unidirectional airflow (e.g. Grade A)
‒ Does an increased drying time matter? How many reapplications to keep a surface
‘wet’?
Stability
‒ How stable is the disinfectant?
‒ Is it stable ‘ready-to-use’ or does it require preparation and use within a short time
period?
‒ What is the shelf-life?
‒ Has the end of use been assessed with disinfectant efficacy studies?

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 Disinfectant Efficacy Testing
• All disinfectants used in Good Manufacturing Practice (GMP)
cleanrooms must be validated in order to demonstrate its
efficacy.
• Different global standards
• Standard approach for disinfectant validation consists of:
‒ A basic suspension test.
‒ Two-part simulated-use test.
‒ A field trial.

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 Different standards, similar approaches

AOAC / ASTM For bactericidal, European Committee

international analyses sporicidal, and for Normalization
fungicidal activity (CEN) tests
• Include carrier tests • Tests are: • Phase 1: A basic
(E2111 & E2197) • Mainly qualitive suspension test.
• In use-dilution tests • Different criteria • Two-part test:
(955.14, 955.15, for different • Phase 2/1:
964.02, 966.04) organisms. Quantitative
suspension test
• Phase 2/2: Surface
test
• Phase 3: A field trial.

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 Disinfectant Efficacy Testing
Basic Suspension Test
• Sometimes called the ‘kill time method’
• Designed to measure the efficacy of a disinfectant
against selected microorganisms after a
predetermined contact time
• The selection of a suitable sterile neutralizer is
required
• After challenging a disinfectant solution with a
microbial population the mixture is plated after the
required contact time, and the surviving
microorganisms enumerated

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 Disinfectant Efficacy Testing
Quantitative suspension test
‒ The purpose of the quantitative suspension test is to
evaluate the activity of a disinfectant against a range of
microorganisms under conditions which more closely
simulate practical use.
‒ The test consists of inoculating a prepared sample of
the disinfectant under test in simulated ‘clean’ and
‘dirty’ conditions using a challenge suspension of
bacteria or fungi.

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 Disinfectant Efficacy Testing
Surface test
‒ Sometimes called ‘carrier test’ or ‘coupon
test’
‒ Representative surface samples are
inoculated with a selection of microbial
challenge organisms.
‒ A disinfectant is applied to the inoculated
surfaces and exposed for a predetermined
contact time.
‒ Any surviving organisms are recovered
using a disinfectant-neutralising broth and
test method (surface rinse, contact plate, or
swab)

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 Field trials #1

To ensure the effectiveness of a cleaning and disinfection programme

microbiological environmental monitoring of surfaces is performed.
‒ Primary methods:
• Cotton swabs
• Contact plates.
• Plating agars used should contain appropriate neutralising
agents.

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 Field trials #2
Field Trails
‒ To establish how often to clean and disinfect
‒ Establish how often to rotate
‒ Establish if residues need to be removed
• Some disinfectants, such as chlorine dioxide, attack stainless steel.
– Cause discoloration or rusting

– Residues should be wiped with (sterile) WFI or 70% IPA.

‒ Often called ‘phase 3 of disinfectant validation’

Assess areas before, after and in-between cleaning and disinfection
‒ Lots of EM samples
‒ Lots of trend monitoring
‒ Microbial identification to assess resistant strains

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 What do you need to do?

Test Do you need to do this? Comment

Basic suspension test No Accept manufactures data
Quantitative suspension test Only when selecting between A potentially useful screening test
candidate disinfectants
Surface tests Yes Need to decide on several variables
Field trials Yes The ultimate proof
Decisions are required

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 Surface tests
Need to decide:
‒ Test variables:
• Different surfaces
• Clean or dirty conditions
• Optimal contact times
• Types of microorganisms (reflective
of the natural microflora of the
facility)
• Level of microbial kill

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 Which surfaces?
• Determine the effectiveness of inactivation of the
desiccated microorganism by disinfectants on appropriate
surfaces.
• Impractical to use all surfaces
• Select surfaces that are more common and representative
• Generally this is 4 to 6 surfaces, such as:
‒ Stainless steel
‒ Acrylic
‒ Glass
‒ Vinyl
‒ Plastic

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 Contact times and cleanliness
Contact times

• Contact times need to be short to:

• Achieve rapid kill
• Avoid or minimise the need for reapplication
• Cope with the rapid air change rates found in cleanrooms

Bacteria and fungi (vegetative)

• Target 5 minutes

Spores

• Target 10 to 15 minutes

Clean or dirty conditions

• Clean conditions should be representative of most pharmaceutical facilities

• If dirty conditions are present, then cleaning prior to disinfection is important.

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 Microorganism selection for validation
Typically this will be:
‒ A Gram-positive coccus e.g. Staphylococcus or
Micrococcus species
‒ A Gram-negative rods e.g. a Pseudomonas type organism
‒ A Gram-positive non-spore forming rod e.g. a
Coryneform
‒ A yeast-like fugus e.g. Candida
‒ A filamentous fungus e.g. Cladosporium
For sporicides
‒ A Gram-positive sporing rod in the spore state e.g.
Bacillus species
‒ A filamentous fungus in the spore state.

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 Level of microbial kill
• Recommended: 3 log reduction in the viable microbial count should be demonstrated for
bacteria and fungi (vegetative state)
‒ Cleanroom surface limits:
• A Grade D cleanroom is only permitted 50 CFU.
• Grade C / ISO class 8 (in operation) = 25 CFU
• Grade B / ISO class 7 (in operation) = 5 CFU
• Grade A / ISO class 5 (in operation) = 1 CFU
‒ Spores are less common than vegetative organisms. A 2-log reduction is a suitable target
value.
‒ Log reductions have different meanings depending on the starting point.
• 3 log reduction from 107 cells to 104 viable cells constitutes a relatively large reduction in
the microbial cell count.
• 3 log reduction from 104 cells to 101 cells constitutes a much smaller reduction in the cell
count.
• An analysis of the cell count reduction is important.

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 Techniques as part of validation
• With disinfection:
‒ Wiping more effective against microorganisms than spraying –
physical removal
‒ Spraying alone is least effective – especially not effective against
fungi and bacteria spores.
‒ Spray/wipe or wipe/spray combination more effective than spray or
wipe alone – order is not important
• Best assessed in field trials
‒ Can also assess cleaning efficacy.

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 Validation summary
Review materials used in construction of the facility

• Select appropriate surfaces

Review environmental monitoring data

• What types of microorganisms are found?

• What numbers of microorganisms are typical?

Assess the suitability of the disinfectant against the microorganisms

• Is the spectrum of activity suitable?

Perform efficacy tests on surfaces, using appropriate microorganisms and realistic contact times

Review cleaning and disinfection procedures, and methods of application, and rotation patterns

• Conduct a field trial

• Train personnel

On-going – review environmental monitoring trends

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 Validation file
Document
‒ Anti-microbial activity on different surfaces – including in
house isolates
‒ Contact time
‒ In use concentration
‒ Shelf life, unopened and in-use
‒ Compatibility with cleanroom materials and equipment

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 Perform identity checks on receipt
General condition of packages/containers
• Clean and undamaged

Cleanroom compatible packaging as appropriate

Sterility statement if appropriate

• Aseptically manufactured/terminally sterilized

Water quality statement

Lot certification
• Manufacturer’s testing frequency
• What defines a batch
• Test results
• At which point are samples taken for testing
Batch release procedures in place
• Stock location
• Stock status labelling

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 Good Quality Control

Entry into Method of Methods and Operator

Shelf life Disposal
cleanroom areas preparation Records of use training

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 On-going monitoring
• Ultimate proof of disinfectant control
‒ Good environmental and material
control systems
‒ Periodic environmental monitoring and
trending of isolates
• Care with culture media selection
and neutralisers
‒ Periodic re-assessment of the
disinfectant
• If resistant microorganisms are found
in the environment, should they be
challenged as part of a disinfectant
efficacy test?

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 Contact plates
‒ Only good for flat surfaces, finger dabs and garments
‒ Carry a risk of leaving media traces on the surfaces sampled and
encouraging microbial growth – disinfection after use required
‒ If surfaces were disinfected, media containing “neutralizers” must be
used.
‒ Consistency can be introduced by applicators:
• Standardise pressure
• Standardise time

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 Resistant strains
• Need to profile EM data for unexpected organisms and be concerned about:
• Shifts in endospore forming
bacteria Most Resistant Microorganism
• Fungi
Prions
• Gram-negative rods
• Patterns may signal issues with Bacterial spores
disinfectant efficacy or application Protozoal oocysts
• Reduced susceptibility or the
resistance of bacteria may be Mycobacteria
occurring. Fungal spores
Gram-negative rods
Least Resistant Gram-positive bacteria

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 Data review
Include appropriate information with tables and graphs. This helps to identify patterns and possible reasons for
a given trend.
Such information includes:
‒ Locations;
‒ Dates;
‒ Times;
‒ Identification results;
‒ Changes to room design;
‒ Operation of new equipment;
‒ Shift or personnel changes;
‒ Seasons;
‒ HVAC problems (e.g. an increase in temperature).

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 Effective contamination control

Design controlled Control people

Clean Disinfect
environments well and materials
• Good detergents • Suitable • Air • Gowning
disinfectant • Cleanability • Changing rooms
• Spray and wipe • Multi-pack
• Never simply sterile items
spray
• Check
saturated
wipes actually
work

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 Q&A session

Dr Tim Sandle

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How we can support you with key areas of Annex 1
Webinars - next in the Annex 1 series is ‘Designing a Cleaning Validation Strategy’ (19th April). Register now via
our website

Analytical services to support the manufacture of your sterile medicinal product

Disinfectant efficacy and validation Sterility testing Extractable and leachables
Environmental monitoring Bacterial Endotoxins Microbial identification (MALDI-ToF)
Cleaning validation Raw materials testing Water testing (inc WFI)

Training courses available both on our open schedule and tailored, delivered at your site
12-13 April Manufacturing sterile products
25 Apr, 7 Sept Water systems and microbiological control
27 July Cleaning validation
31 July-2 Aug Pharmaceutical microbiology
2 Nov Environmental monitoring

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 Thank you for your time

Annette Russell
Sterile & Non-Sterile Annette.russell@rssl.com
RSSL Microbiology Lead
+44(0)118 918 4075
www.rssl.com

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