The ABCs of disinfectant validation

Despite ambiguities inherent in the regulatory inspection process, a few absolute expectations
should be met
By Elaine Kopis Sartain, Steris Corporation
I recently attended a meeting with several members of the pharmaceutical, biotechnology and
medical device industries. During our discussions of cleanroom decontamination, it quickly
became apparent that the subject of disinfectant validation was a considerable source of both
regulatory pressure and internal frustration. One of the key concerns was that regulatory
authorities have provided very little guidance as to how they expect a disinfectant validation to
look. Some also stated that what may be acceptable to one investigator might not be acceptable
to another.
While I agree that there may be some variability inherent in the regulatory inspection process
and consequently, some variability in what is considered an acceptable validation study, there are
a few absolute expectations that anyone involved in disinfectant validation should meet.
A: Acquire thorough knowledge of your process before
designing your validation protocol
One cannot validate a process without first understanding it. Knowledge in the following areas is
essential for developing a disinfectant validation protocol:
Antimicrobial products (i.e., disinfectants, sporicides, and decontamination agents )
Application procedures
Environmental conditions (e.g., airflow, temperature, humidity)
Microorganisms: Include environmental isolates
When discussing microorganisms, regulatory agencies have been clear and consistent: isolates
from the processing environment must be included in disinfectant studies. Because these are the
microorganisms most likely to present a contamination risk to your product, it is critical
tosubstantiate that your microbial control program is capable of controlling them. It is no
accident that firms receive FDA-483 observations for failure to identify microorganisms:
“Organisms are not identified…when counts exceed the firm’s established specification
of…organisms recovered from gowning validations and routine monitoring are not identified.”
The fact that a microorganism was isolated may be an indication that your microbial control
program is not capable of handling that particular organism. The only way to rule it out as the
potential cause of the excursion (and thus prevent future excursions) is to identify the organism
and to confirm that your disinfectant validation has substantiated performance against it or a very
similar strain. Although all isolates need not be included in the disinfectant validation, there
should be a valid rationale for the organisms that are included in the study.
What is sufficient microbial reduction?
Another question that must be addressed during protocol development is: What level of
microbial reduction is sufficient to demonstrate satisfactory antimicrobial efficacy? The answer
to this will not be found in the new Aseptic Processing Guideline or in any other regulatory
document. However, the USP informational chapter 1072, Disinfectants and Antiseptics, In-
process Revision, states the following: “In practice, sufficient organisms need to be inoculated
on a 2-inch x 2-inch square of the surface being decontaminated, i.e., a coupon, to demonstrate at
least a 2-(for bacterial spores) to 3-(for vegetative bacteria) log reduction…”

It is important to consider that the microbial load on cleanroom surfaces, especially those that are
kept dry, is extremely low to begin with; therefore, a 2- to 3-log reduction should be sufficient.
Additionally, during testing the organisms are dried onto the surface and then recovered using
additional steps, all of which may lead to reductions in recoverable and viable organisms. This
makes it very difficult to prove higher log reductions. Suspension tests may allow for better
recoveries and overall higher log reductions, but they do not provide substantive proof of
performance under application conditions.
Antimicrobial products
There are fairly significant differences in antimicrobial capabilities among different types of
antimicrobial agents. More than one type is required to achieve the proper balance of effective
microbial control, minimal substrate damage, and personnel comfort and safety.
Disinfectants typically contain a surfactant (synthetic detergent) and an antimicrobial agent (e.g.,
phenol). The surfactant allows the disinfectant to handle a fairly substantial soil load, making it
an ideal product for heavy traffic areas such as floors. These agents are also designed for
frequent use and are relatively safe for both personnel and surfaces.
Sporicides are typically formulated with highly reactive chemicals (e.g., sodium hypochlorite
and peroxygen compounds) and may cause damage to surfaces, even stainless steel, if used too
frequently. They are a “necessary evil” for cleanrooms, since routine disinfectants are not
capable of controlling bacterial endospores or resistant molds (i.e., A. niger). However, they
should be used judiciously to avoid damage to substrates and to reduce the potential for irritation
to the personnel involved in cleaning operations.
Isopropyl alcohol (IPA) is capable of good broad-spectrum efficacy, but the manner in which it
is routinely used (i.e., minimal contact time) and its flammability effectively limit its broad
applicability as a primary mic robial control product. It is more appropriately classified as a
decontamination or residue control agent.
All three product types are required to properly maintain a cleanroom environment and control
microorganisms. Therefore all should be included in the disinfectant validation study, although
they may not necessarily be tested against all of the same challenge organisms.
Application procedures
To some degree, antimicrobial products are only as effective as the manner in which they are
applied. Cleanroom housekeeping guidelines generally emphasize the importance of “clean to
dirty” application techniques.
The purpose here is to ensure that a previously clean surface, such
as a wall, is not inadvertently contaminated with residue from another surface (i.e., the floor) via
the cleaning device and procedure. This technique is generally included in cleaning SOPs, but
there are other application-related issues, just as important to the success of the microbial control
program, that are not always well understood, documented, or executed. These include
maintaining sufficient wet contact time and balancing the surface area-to-antimicrobial solution
Cleaning SOPs often indicate that a surface should be allowed to remain wet for a defined period
(for example, ten minutes) after disinfectant application. This timing is often derived from
product label instructions or from a validation study. However, seldom have I seen an operator
actually time the application to determine if the “ten minutes” is being achieved. It is also rare to
see a validation protocol in which a predefined wet contact time was qualified during in situ
evaluations. What I have seen, however, is FDA-483 observations that make a point of this
oversight: “The qualification of the various disinfectants used in sanitizing surfaces in the aseptic
processing area (sterile core)…failed to assess the disinfectants in the manner that they are used,
including …disinfectant exposure time.”

The surface area-to-antimicrobial agent ratio is somewhat related to contact time in that if
sufficient solution is applied to a surface, and environmental conditions (temperature, humidity,
airflow) are accounted for, then sufficient wetting will be achieved to attain the desired contact
time and thus, the expected microbial control.
Surface wetting effectiveness may also be related to the application device (e.g., wiper, mop, or
sprayer) and to the technique. Therefore, application procedures and methods should be included
in the disinfectant validation work, in order to avoid a citation: “…Disinfectant agents used to
sanitize surfaces in the aseptic process areas (APA) have not been adequately qualified to assure
that they provide the intended microbial decontamination when used in the manner as specified
in standard operating procedures … The qualification study immersed the test surface in the
disinfectant …instead of wiping the surface as specified in cleaning SOPs.”

The actual cleanroom surfaces being cleaned also affect the antimicrobial efficacy of agents. At
one time, surface studies were conducted using either stainless steel or glass almost exclusively.
In fact, most industrial protocols (e.g., AOAC, EN) involve the use of suspension testing or
stainless-steel panels. Over the last few years I have reviewed the results of disinfectant
validation studies that document the differences in performance between suspension and surface
testing methods and show that surface condition and type have an impact on cleanability (i.e.,
particulate removal efficiency). Consider the difference in surface texture between a cast iron
skillet and a Teflon -coated pan; or more to the point, between a heavily-textured, epoxy-coated
floor and a smooth, seamless, polymeric floor.
The regulatory authorities have made the same observations. They have made it clear that
surface evaluations should be included in the disinfectant validation and that in vitro studies
should include surface materials that represent the types of surfaces being cleaned: “The
qualification of the various disinfectants used in sanitizing surfaces in the aseptic processing area
…failed to assess the disinfectants in the manner that they are used, including types of surfaces

Environmental conditions
Another consideration is whether or not the actual environmental conditions of the cleanroom
can be captured during an in vitro study. Airflow, humidity and temperature are all important
parameters that have an impact on wet contact time and consequently on disinfectant
performance. Temperature is an especially important consideration where cold-room applications
are involved. An attempt should be made to replicate these conditions during in vitro testing. For
example, a laminar flow hood can be used for drying inoculated coupons.
B: Build a consensus on the validation protocol and
prevalidation steps based on a solid scientific rationale and
the best available guidance tools.
The steps leading to development of a protocol, and ultimately the protocol itself, should at least
address the following questions:
Do SOPs need to be revised?
Which antimicrobial products are going to be included in the validation study?
Which microorganisms will be included in testing and what criteria will be used for selection of
these microorganisms?
Which substrates will be included in surface studies and what criteria will be used to select the
What application techniques will be utilized for the surface studies?
Will suspension testing be conducted?
How will the in situ evaluation be conducted?
What recovery techniques will be used?
Will testing be done internally or at a contract-testing facility?
How will the testing facility be selected and qualified?
How will failing data be addressed?
How often and under what circumstances will revalidation be conducted?
The results from in vitro studies give an indication of how the disinfectants will perform under
actual use conditions, but no matter how sound the in vitro protocol is, FDA-483 observations
have shown that there is no substitute for evaluation under actual use conditions: “The
Disinfection Qualification Testing Report was incomplete in that the study failed to simulate
actual use (i.e., contact on production surfaces).”
Hence most facilities will agree to conduct an
in situ qualification as a part of the protocol.
The in situ study can be conducted under worst-case conditions, such as during a PM shut-down.
This type of qualification generally involves sampling several locations, including a
preponderance of worst-case locations, prior to and after disinfectant application. Data is
subsequently reviewed to determine that the disinfectant effectively reduced the bioburden in
those areas.
C: Correlate data gathered during the in vitro and in situ
studies with results obtained during routine EM monitoring
This approach is also supported by the USP 1072 In-process Revision: “To demonstrate the
efficacy of a disinfectant within the pharmaceutical manufacturing environment, it may be
deemed necessary to conduct the following tests: … a statistical comparison of the frequency of
isolation and numbers of microorganisms isolated prior to and after the implementation of a new

There is an “X factor” involved in disinfectant performance. This factor may be defined as the
manner in which the disinfectant performs under use conditions when considering the following
The condition of working cleanroom surfaces
The room environment (e.g., temperature, humidity), including seasonal fluctuations
The condition of application devices (e.g., integrity, bioload)
The compliance of cleanroom personnel (i.e., disinfectant preparation, application, cleanroom-
appropriate behavior)
The cleaning frequency
The use of other, potentially interfering, substances in the room
The X factor may have tremendous impact on the success of a microbial control program. The
only way to evaluate the impact of this X factor is by reviewing EM data and correlating the
results to those obtained during qualification studies. Careful evaluation of this data should
provide an indication of the origin of cleanroom microbial control problems and may help in the
design of a roadmap toward greater regulatory compliance.
ABCs can yield results
Disinfectant validation is a precise and demanding function that must account for many potential
variables in order to secure a validatable aseptic manufacturing environment. These ABCs
address key issues raised in FDA-483 observations, and provide basic guidelines that can help to
establish an effective validation protocol.
Elaine Kopis Sartain is the director of technical service for STERIS Corporation. She can be
reached at
1. GMP Trends, Issue No. 622, December 15, 2002.
2. USP Chapter 1072, Disinfectants and Antiseptics, In-Process Revision, Vol. 30 No. 6, Nov.-
Dec. 2004.
3. IEST-RP-CC018.3 Cleanroom Housekeeping: Operating and Monitoring Procedures,
December 2002.
4. GMP Trends, Issue No. 666, October 15, 2004.
5. GMP Trends, Issue No. 631, May1, 2003.
6. See reference 4.
7. GMP Trends, Issue No. 665, October 1, 2004.
8. See reference 2.