By Lionel Karcher, Head of Microbiology Laboratory, BU Confarma

Container
Closure
Integrity
Tests (CCIT)
for Single-Use
Systems

Container Closure Integrity Tests have traditionally been performed

using dye testing or microbial challenge testing. The article entitled
“Development of a Dye Ingress Method to Assess Container-Closure
Integrity: Correlation to Microbial Ingress”, published in PDA Journal1
is one of the key reference texts on the subject. There are currently
many more sensitive physical methods that in particular allow
high-throughput in-process integrity testing; that said, dye tests
using methylene blue and microbial challenges remain relevant and
offer various advantages.
In 2016, the “Package integrity evaluation – sterile products” Chapter2 of the USP was
updated, significantly calling into question the use of traditional methods like dye testing
or microbial challenge testing. In this Chapter, the microbial challenge is referred to as
“Microbial Challenge, Immersion Exposure” and the dye test is included among “Tracer
Liquid” methods. These two methods are classified as “probabilistic methods”. Probabilistic
methods are characterized by the random number of events allowing the transfer of liquid
into the samples. By contrast, the USP defines “deterministic methods” as physicochemical
techniques that yield quantifiable and reproducible results with clearly defined and
predictable detection limits.

Principle and key points

The microbial challenge test and the dye test both share a similar basic principle: they
consist of detecting the transfer of liquid into the container. The transfer of liquid is
revealed by coloration or microbial growth.

• MICROBIAL CHALLENGE
The samples are first filled with a medium that allows microbial growth. A standard
Trypticase soya (TS) growth medium is generally used. A product containing a sufficient
amount of nutrients can be directly used as a growth medium. In this case, the
product’s fertility compared to the test germ must be assessed before performing the
assays. Using the product as the growth medium has the advantage of not producing a
specific batch and/or allows the test to be performed in the context of an investigation.

Using the product as the growth medium has the advantage

of not producing a specific batch and/or allows the test to be
performed in the context of an investigation.

The samples are then immersed in a microorganism solution. The most commonly used
microorganisms are Brevundimonas diminuta, Serratia marcesans and Escherichia coli. In
order to challenge a sample with a specific strain, other microorganisms, such as
in-house strains may be used. It should be noted that certain microorganisms require
special growth parameters that must be taken into account when choosing the test
strain. This, for instance, is the case with the strictly aerobic Brevundimonas diminuta,
for which the growth medium must contain oxygen.

At the end of the immersion cycle, the samples are cleaned and then incubated for a
sufficient amount of time to allow the growth of microorganisms. There is no standard in
terms of incubation time; seven or 14 days are generally used by default, but if microbial
growth can be detected in a shorter time, then this shorter interval may be chosen.

After this incubation period, the samples are directly observed to detect any cloudiness.
Generally speaking, if the test conditions were correctly chosen, there will be no doubt
if there is any growth. That said, an analysis of sample contents may also be performed
to detect the presence of any microorganisms. This analysis must be performed if the
container or the growth medium does not allow cloudiness to be assessed or if there is
any doubt concerning microbial growth.
• DYE TEST
The samples are first filled with product. An empty sample can also be tested, but
generally speaking, transfer between two liquids is preferred. The samples are then
immersed in a dye solution. Various dyes can be used, but methylene blue has been
used traditionally. At the end of the immersion cycle, the samples are cleaned and read.
The samples can be read visually or using a spectrophotometer.

Positive samples
Alongside the tests, positive controls must be performed using samples in which “holes”
have been made. By using samples rather than standard controls, the test can be actually
simulated. Compared to certain deterministic methods, using positive controls allows the
detection limit on the actual sample to be re-verified during every test session. Glue

It should be noted that detection limits vary by container type. The larger the container Transfer
(several liters, for instance), the higher the detection limit. The detection limit is correlated
to the liquid’s capacity to penetrate the container. For example, for a 20 liter flexible pouch,
the detection limit will generally be greater than 100µm whereas for a syringe or a few
milliliter bottle, the detection limit will decrease to 2-5µm given sufficient pressure
variations. By comparison, according to the USP, a deterministic method used for rapid
in-process testing detects defects of 25-100µm. Meanwhile, these methods can be used to Figure 1:
Figure 1: Transfer
Transferalong
alongthe
a microcapillary
microcapillary
detect defects of under 1µm when used out-of-process.

In order to create positive controls, microtubes or microcapillaries can be used to simulate

microholes in the samples. Creating positive controls is a delicate task. During the
preparation and during the insertion of the capillaries in the samples, it is essential not to
plug or break the capillaries.

Another key point is the attachment of the capillaries. If a capillary is not sealed or correctly
sealed to the sample, the liquid will transfer next to it rather than inside of it (see figure 1)
even if the capillary is introduced through a rubber stopper. The stopper does not retract
perfectly around the capillary and leaves a path for the liquid. There is also a correlation
between capillary length and the probability of transfer through it, which makes it
necessary to perform a validation before starting routine tests.

If a capillary is not sealed or correctly sealed to the sample,

the liquid will transfer next to it rather than inside

Drawbacks
The two methods have certain known drawbacks:
• They are “destructive” methods, meaning that the samples cannot be reused in either case,
• As a direct result of the aforementioned aspect, the generated waste needs to be managed,
• While the detection limits are relatively high, they can approach those of deterministic
methods if significant pressure variations are applied,
• The analysis time is long, particularly for microbial tests, ranging between one and 14 days,
• For microbial tests, a medium enabling the growth of the chosen germ must be used.
Advantages
Despite the relatively significant drawbacks of these methods, they still offer some advantages.

1 PRESSURE VARIATIONS
One of the main advantages is the possibility to expose the samples to different
pressure variations.

At present, various companies challenge their products using specific conditions that
most closely mimic the transportation conditions of their products. Some samples are
thus immersed in a solution and undergo a series of dips in pressure to simulate
multiple flights.

That said, the pressure variation must be large enough to set air bubbles free that
prevent the transfer of the liquid through the microtubes. The difference in pressure is
therefore directly correlated to the test’s detection limit.

As mentioned above, using significant pressure variations allows us to approach the

detection limits offered by deterministic technologies, but the structure of the samples
must be taken into account. For samples that have a mobile component, such as the
rubber stoppers of syringes, an excessive variation in pressure significantly shifts the
stopper and presents the risk of a false positive result. By using pressure that is equal to
the pressure variations that the product is subject to during air transport, the shifting of
mobile components can be mimicked without generating false positive results. This
latter point allows the verification of the risk of microbial contamination when the
product’s mobile components shift as a result of pressure variations. We should keep in
mind that pressure variations mimicking air transport are relatively minor, therefore the
detection limits of these tests are relatively high.

2 LARGE VOLUMES
For many sample integrity testing methods, sample size can quickly become a limiting
factor. For dye testing and microbial challenge testing, the size limit for conducting a
test is relatively high. To conduct the tests, special large size containers are
manufactured (see photo 1). Then a large volume of immersion solution must be
prepared to make sure that all sample surfaces are challenged. Photo 1: Containers used for CCIT

The containers are then introduced into a chamber large enough to perform cycles of
pressure variations (see photo 2). When studying large or very large volume containers,
the studies are relatively long because every sample needs to be tested individually. In
addition, for microbial tests, the incubation chamber must be sufficiently large. Lastly,
the destruction of samples is also more difficult.

Photo 2: Pressure variation chamber

3 EASE OF SET-UP
Dye tests and microbial challenges also have the advantage of being relatively simple
to set up. For microbial challenges, if the reading is performed visually, only a device for
implementing pressure variations is needed in addition to standard microbial
laboratory equipment.

What is needed:
• A sterilizable and/or sterile container to immerse and challenge all of the sample surfaces,
• Samples containing a medium enabling the growth of the germ,
• A device for implementing changes in pressure,
• Incubators or incubation chambers,
• Growth media and microorganisms to prepare the immersion solutions,
• Capillaries or microtubes if these are chosen to make the positive controls.

To perform dye tests, standard chemical laboratory equipment is enough in addition to

the equipment for implementing pressure variations.
• A container for immersing the samples,
• Samples,
• A device for implementing changes in pressure,
• A reading booth or a spectrophotometer depending on the type of reading,
• Capillaries or microtubes if these are chosen to make the positive controls.

In these two cases, a single device may be used to conduct the tests. In function of the
size of the device, containers of various volumes can be assessed.

Other applications
There are other applications for microbial tests and for the dye test that is not described in
the USP. These include two often used examples:

• In the case of the dye test, there is a test method that detects seal leaks in porous
packaging (ASTM F 19293). When performing this test, there are two possibilities: either
the packaging is filled with dye or conversely, the packaging is dipped into solution.
• For microbial challenges, another option is to use bacteria in aerosol form to challenge
the integrity of the sample by simulating an atmosphere saturated with microorganisms.
Conclusion
In recent years, traditional dye tests using methylene blue and microbial
challenge tests have been seeing competition from highly effective
“new” technologies. These technologies, referred to as deterministic
methods, guarantee known detection limits. But despite the known
drawbacks of traditional methods, they nevertheless remain useful
thanks to their relative simplicity, rapid set-up and low cost. Moreover,
these methods offer a solution for the integrity testing of large-volume
products. Even more importantly, they can be used to test the impact of
any shifting of a product’s mobile components (such as the stopper) on
the risk of microbial contamination.

References
1. USP 39 (1207.1) Package integrity testing in the product life cycle - test method selection and validation.
2. PDA, Development of a Dye Ingress Method to Assess Container-Closure Integrity: Correlation to Microbial Ingress
3. ASTM, F 1929 Standard Test Method for Detecting Seal Leaks in Porous Medical Packaging by Dye Penetration

Originally published in French in La Vague in October 2017

Confarma, a Solvias Group company, provides biological analysis

Confarma France SAS

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info@confarma.fr | www.confarma.fr