I hope you're doing well and enjoying the start of spring. I also hope that you'll find the following
article about the USP <797> environmental monitoring by Dr. Joseph Manfrida both interesting
With best wishes,
USP <797> Environmental Monitoring Program Design and Application
By Joseph P. Manfrida, Ph.D., EMLab P&K Analyst
The proper design and execution of an environmental sampling plan is a central component of
A good environmental sampling program will not only allow a pharmaceutical compounding
laboratory to know whether or not it is within the recommended action levels of USP <797>,
but will also provide valuable information for determining sources of potential contamination
and counteracting them.
Testing Requirements and Methodology
Successful implementation of a USP <797> environmental sampling program starts with an
understanding of the different tests required by USP <797>. The most basic division
between the required tests is nonviable particle sampling and viable particle sampling.
Nonviable airborne particle testing seeks to measure the density of airborne particles based
strictly on their size (0.5 µm in diameter or larger) without regard for the nature of
the particles themselves. Viable particle sampling only measures those particles that are
living organisms (typically bacterial and fungal spores). USP <797> breaks viable
particle testing into several different categories, each of which is designed to test a
separate aspect of pharmaceutical compounding for potential contamination. These tests are
viable airborne particle testing, viable surface particle testing, gloved fingertip sampling
and media-fill testing (also called aseptic manipulation testing).
Nonviable airborne particle sampling must be performed by a qualified operator using an
electronic particle counter.
Testing needs to occur once every six months at a minimum. Additional testing is required any
time that the primary engineering controls (laminar flow hoods, isolators, etc.) are moved or
altered, or in the event of major changes to the facilities surrounding the primary engineering
controls. At a minimum, each separate ISO class 5, 7 or 8 area needs to be tested (1). A thorough
testing plan will also investigate areas and items that could be potential sources of nonviable
particulates. Potential sources of nonviable particles include, but are not limited to, potential
leaks in a clean room's containment (possibly at windows, doors and pass-through cabinets),
mechanical and electrical equipment (refrigerators, computer printers, etc.) and areas with high
personnel traffic during compounding operations. A map of the laboratory being sampled can be
very useful for pinpointing critical areas for sampling. If a map or blueprint of the compounding
laboratory cannot be obtained from the laboratory manager, a hand-sketched map can prove to be
useful. All data collected must be thoroughly documented. Documentation should include the
specific locations sampled, the time of day sampling took place, copies of the calibration
certificate for the particle counter used to collect the data, and training certificates for the
individual(s) who performed the sampling.
Airborne bacteria and fungi can pose a significant threat of contamination during the
manufacture of compounded sterile preparations. Viable airborne particle testing is performed
to monitor this threat and to ascertain that physical and procedural controls in place are
keeping the airborne microbial load of a facility's air to an acceptable level. The frequency
of testing for airborne viable particulates is identical to that for airborne nonviable
particulates (2). It is recommended that sampling take place during ongoing compounding
operations. If it is not possible to take viable air samples while compounding is taking place,
then sampling should take place immediately after compounding has ended for the day (3).
Volumetric sampling devices must be used and 400 to 1000 liters of air must be tested for each
sample (4). Gravimetric sampling may be used to supplement volumetric sampling, but gravimetric
sampling is not sufficient for meeting the requirements of USP <797>. Air samples taken
for the purpose of measuring bacterial load utilize
TSA (Tryptic Soy Agar)
or soybean casein digest as growth media for collected organisms, while air samples taken to
examine airborne fungal load are collected using MEA (Maltose Extract Agar)
or Sabouraud Dextrose Agar. It is important to note that testing for fungi is only required for
compounding categorized as high risk (5).
Viable surface sampling is required by USP <797> in order to assess the success of
the laboratory's cleaning program in keeping surfaces free of microbial contamination. While USP
<797> mandates periodic surface sampling, it does not specify a definitive period or time
frame for sampling. A feasible option is for surface sampling to take place at the same time as
air sampling. Sampling surfaces in parallel with air sampling allows for a more thorough
investigation of any detected contamination and maximizes convenience for personnel performing
the sampling. At least one surface sample must be taken from each ISO 5, 7 and 8 area after
compounding has concluded (6). Additional samples can be taken from any surface that personnel,
materials or produced compounds are exposed to regularly. Work surfaces, storage surfaces, door
handles and equipment are all good targets for surface sampling. Surface samples may be collected
using TSA contact plates with added lecithin and Polysorbate 80 (TWEEN 80) or swabs. Contact
plates are generally the preferred method for surface testing because of their ease of use, but
sample collection with swabs is perfectly acceptable under USP <797>.
are also the only means of sampling from curved and oddly shaped surfaces, such as door handles
or sink faucets.
Gloved fingertip sampling is performed to evaluate the efficacy of compounding personnel's
hand washing and garbing techniques. Workers wash their hands and don all of their protective
equipment while being observed by their supervisor, in order to make certain that their technique
is in accordance with the laboratory's standard operating procedures. Once they are completely
garbed, the worker presses four fingers and a thumb to a TSA plate (one for each hand) with
lecithin and Polysorbate 80 (7). It should be noted that only TSA plates with lecithin and
Polysorbate 80 are utilized for this test. There are no requirements for a separate gloved
fingertip test specifically to check for fungal contamination. Gloved fingertip tests must be
performed prior to any new personnel beginning work at a compounding facility, annually for
established personnel performing low and medium risk compounding, and once every six months for
personnel performing high risk compounding.
Media-fill testing (also called aseptic manipulation testing) is performed to measure
the ability of procedures, personnel and equipment to successfully produce a sterile end product.
This test is a simulation of the actual compounding taking place in a facility. During media-fill
testing, personnel perform all of the steps for a given compounding procedure using the same
equipment and facilities as they would when compounding, except the typical medications and
diluents normally used in the procedure, are replaced with soybean casein digest media. It is
important for the laboratory manager or head pharmacist of a facility to participate extensively
in the development and execution of this particular test. This is because extensive knowledge
of the laboratory's compounding procedures is necessary to properly set up a media-fill test
that closely resembles the compounding taking place in their lab or facility. A media-fill test
is considered successful if no growth is seen in the final containers prepared by the test after
incubation. Media-fill tests must be performed by all personnel prior to being allowed to begin
compounding at a given facility, annually for personnel working in low and medium risk
compounding facilities, and once every six months for personnel engaging in high risk compounding (8).
While performing viable or nonviable sampling it is important to be aware of quality control.
Copies of calibration certificates should be provided to the laboratory manager for all equipment
used in sampling. Likewise, a copy of the sampling personnel's training qualifications should
also be made available. All media used for viable sampling should have an accompanying certificate
of analysis that also should be provided to the laboratory manager. It is generally considered
good practice to utilize positive and negative controls for viable testing, however USP <797>
does not require these controls. Positive controls require that a viable sample plate be exposed
to a known quantity of microorganisms and incubated alongside samples taken in a compounding
facility. In practice, it is very difficult for personnel working in the field to obtain and
maintain stock cultures of organisms with known concentrations. In the field, personnel seeking
to make a positive control, sometimes sample from an area that is not ISO rated. Sampling air
outside of an ISO classified area is likely to detect microorganisms of an unknown concentration.
This type of "positive control" will demonstrate the media's ability to support
microbial growth if microorganisms are in the area sampled, but without any specific data
available for microbial concentrations in a specific area, it is impossible to be certain how
well the media performs. As a result, this type of sample represents a compromise between the
precision of a true positive control and the ambiguity of having no positive control at all.
Negative controls are much easier for field personnel to obtain. A negative control is provided
by sealing an unopened media plate taken from the same batch as the sample plates, then sending
it to a laboratory for incubation and analysis without ever exposing the plate to an air sample.
For media plates that are received sterile, the negative controls should show no growth after
Data interpretation begins by comparing the results of nonviable and viable air particle testing
and viable surface particle testing to the recommended action levels in USP <797>. These
recommended action levels have been reproduced in Table 1 (9). If the number of particles
detected by nonviable air particle testing, viable air particle testing or viable surface
particle testing, exceeds the levels given in table 1, then it is necessary to begin a full root
cause investigation of the source of contamination, followed by whatever steps are necessary to
bring the particulate levels detected to within acceptable limits.
|Table 1: Recommended Action Levels. (9)
||≥ 0.5 µm Nonviable
For example, if a viable airborne particle sample from an ISO 7 clean room yielded a total of
11 cfu/m3, it would be higher than the recommended action level of greater than 10
cfu/m3 and a full investigation into the source of contamination would be necessary.
Alternatively, if the same sample had only 9 cfu/m3, the recommended action level
would not have been exceeded and a full investigation would not need to be initiated. It is also
necessary to implement an investigation and remediation in the event that there are particulates
identified above historically detected levels for a given facility.
There is also a special requirement for viable air particle samples. If any cfu's are
detected on a viable airborne particle test plate from an ISO 5, 7 or 8 area, then USP <797>
requires that the colonies growing on that plate be identified to at least the genus level,
even if the number of colonies is below the recommended action level (10). The reasoning
behind this requirement for genus identification is that there are some organisms that cannot
be tolerated in a clinical environment at any concentration.
For example, Methicillin-resistant Staphylococcus aureas (MRSA) is a very dangerous organism
in hospitals, and has been discussed extensively in the popular media. If even one cfu of MRSA is
detected in the air of a pharmaceutical compounding laboratory, then extensive investigation and
remediation would be warranted. Full speciation of viable colonies is highly recommended in order
to avoid expending extensive resources for remediation of non-dangerous organisms in the same
genus as highly dangerous ones.
To return to our previous example, Staphylococcus epidermidis would not warrant the same
response as Staphylococcus aureas. However, a genus identification for these two organisms
would yield the same result, Staphyloccoccus. A laboratory manager would not be able to
distinguish which of the two organisms were present in the laboratory and would have to respond
as if the more dangerous organism was present. Species level identification allows for a more
precise response by laboratory personnel to the actual threat posed by any given organism.
Interpretation of data from gloved fingertip and media-fill tests is less complex than
interpretation of air and surface plates. For gloved fingertip plates, a new worker must have
three successful tests with 0 cfu's, prior to beginning work in a compounding laboratory or
facility. For experienced workers, the recommended action level is >3 cfu's per test total,
whereby the counts from both the left and right hand plates are added together. Media-fill
tests are either positive or negative for growth. Any positive media-fill tests require a
root cause investigation and the implementation of remediation to fix the problem (11).
Investigation and Remediation
Investigation of viable particle concentrations above action levels, or away from historical
baseline levels of contamination, must take into account both physical and personnel factors.
Physical factors include items such as making certain that all equipment is working properly.
The maintenance records of all equipment should be reviewed to be certain that everything in
the lab has been properly maintained. Regularly scheduled cleaning of the lab itself is also
critical. Cleaning logs should be reviewed and cleaning equipment inspected to be certain that
it is still capable of cleaning properly. Cleaning equipment should be non-shedding and
dedicated for use strictly in the controlled environment. Cleaning agents need to be rotated
on a regular basis. Not all cleaning agents are equally effective against all microorganisms.
For example, 70% ethanol is a cleaning agent that is highly effective against bacteria, but
has very little effect on bacterial endospores, some fungal spores and certain viruses (12, 13).
Rotating through a series of different cleaning agents over time increases the likelihood
that all organisms in a laboratory are eventually exposed to a susceptible substance. Cleaning
agents must also be stored and used according to their labels. Changes in the weather or in
the building containing the compounding facility also need to be investigated. A rapid climate
change can cause previously quiescent organisms to begin growing. Changes to the physical
structure of the building could affect the ability of the compounding laboratory to prevent
environmental incursions. Personnel factors are the ability of compounding personnel to
successfully implement the laboratory's standard operating procedures to prevent contamination.
Direct observation of personnel during ongoing compounding operations should be conducted to
verify proper implementation of all laboratory standard operating procedures. The procedures
themselves should be reviewed to make certain they are adequate to the task of preventing
At all stages of USP <797> implementation it is very important to remember to document
every action taken and every test result. The historical record is the only means that a
laboratory manager has for recognizing trends and spotting potential problems before they become
severe. Ideally, observing how the contamination levels change over time in a laboratory will
allow potential contamination issues to be isolated and solved early, before they have a chance
to cause injury or illness in pharmacy patients or laboratory personnel. In the event that a
patient illness leads to an investigation by legal authorities, these records are the only
proof that a laboratory has been following the regulations established by USP <797>.
Without thorough documentation that the laboratory has been meeting the requirements of
USP <797>, they will likely be subject to legal consequences.
Design and execution of a USP <797> environmental sampling plan is a task that requires
attention to detail, extensive knowledge of the tests required and precision in reporting data.
At EMLab P&K we are dedicated to providing the technical expertise and laboratory
capabilities necessary to meet the challenge of implementing a rigorous USP <797> program.
We are prepared to provide the commitment to quality that you have come to expect from EMLab P&K.
Contact us for USP <797> services.
1. The United States Pharmacopeial Convention. <797> Pharmaceutical Compounding -
Sterile Preparations. Revision Bulletin. 2008, p. 1-61.
2. Ibid., Revision Bulletin, p. 25.
3. The United States Pharmacopeial Convention. <1116> Microbiological Evaluation of
Clean Rooms and Other Controlled Environments. National Formulary. 2000, p. 2099-2106.
4. Ibid., Revision Bulletin, p. 25.
5. Ibid., Revision Bulletin, p. 25.
6. Ibid., Revision Bulletin, p. 33.
7. Ibid., Revision Bulletin, p. 31-32.
8. Ibid., Revision Bulletin, p. 30.
9. Ibid., Revision Bulletin, p. 2, 26 and 34.
10. Ibid., Revision Bulletin, p. 26.
11. Ibid., Revision Bulletin, p. 31-32.
12. Allen, L.V., Jr. and Okeke, C.C. 2007. Basics of Compounding: Considerations for
Implementing United States Pharmacopeia Chapter <797> Pharmaceutical Compounding -
Sterile Preparations, Part 4: Considerations in Selection and Use of Disinfectants and
Antiseptics. International Journal of Pharmaceutical Compounding. 11(6): 492-496.
13. Utama, I.M.S., Wills, R.B.H., Ben-yehoshua, S. and Kuek, C. 2002. In Vitro Efficacy
of Plant Volatiles for Inhibiting the Growth of Fruit and Vegetable Decay Microorganisms.
Journal of Agricultural Food Chemistry. 50(22): 6371-6377.