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á1207ñ PACKAGE INTEGRITY EVALUATION—STERILE PRODUCTS

1. INTRODUCTION

This chapter provides guidance on the integrity assurance of nonporous packages intended for sterile pharmaceutical
products. Background instruction is provided on the topics of leaks, leakage rate, and package sealing/closure mechanisms.
Explanation is given as to how packages that conform to specified leakage limits help to ensure the contained product meets
and maintains sterility and relevant physicochemical specifications. The integration of package integrity assurance as a key
component of the entire product life cycle is stressed. Guidance in the selection, validation, and use of leak test methodologies
as well as package seal quality tests is included. Detailed recommendations are presented in three subchapters listed below:
• Package Integrity Testing in the Product Life Cycle—Test Method Selection and Validation á1207.1ñ
• Package Integrity Leak Test Technologies á1207.2ñ
• Package Seal Quality Test Technologies á1207.3ñ
At the end of this chapter, the Glossary section defines terms as they are used in the context of this set of four general
information chapters.
The term product–package refers to the container–closure system plus the product contents. The container–closure system
consists of the primary packaging components, those components that are or may be in intimate contact with the product, as
well as secondary packaging components vital to ensuring correct package assembly, for example, the aluminum cap used to
seal a stoppered vial package.
Suitable container–closure systems adequately store and protect the contained pharmaceutical product. Thus, sterile

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product–package integrity is the ability of a sterile product container–closure system to keep product contents in, while keeping
detrimental environmental contaminants out. Specifically, leaks of concern for sterile product–packages include the following
three categories described in Table 1. In other words, the leaks of concern for a given product–package are a function of the
degree of package protection demanded by the product to ensure that all relevant product physicochemical and microbiological
quality attributes are met through product expiry and use.

Leaks of Concern
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Table 1. Product Quality Risks Posed by Leaks of Concern
Product Quality Risks Posed by Leaks
Capable of allowing entry of microorganisms Failure of product sterility quality attribute
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Capable of allowing escape of the product dosage form or allowing entry of
external liquid or solid matter Failure of relevant product physicochemical quality attributes
Capable of allowing change in gas headspace content. For example, loss of
headspace inert gases (e.g., nitrogen), loss of headspace vacuum, and/or entry Failure of relevant product physicochemical quality attributes and/or
of gases (e.g., oxygen, water vapor, air). hindrance of product access by the end-user
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Package integrity is synonymous with container–closure integrity; these terms are used interchangeably throughout this
chapter. In the past, to say a sterile product–package has container–closure integrity commonly meant that the package either
had passed or was capable of passing a microbiological challenge test. This guidance chapter defines the concept of container–
closure integrity more broadly, encompassing the absence of all package leaks that risk product quality. By this definition, a
package is considered to have integrity if it allows no leakage greater than the product–package maximum allowable leakage
limit. In other words, the largest and smallest leaks of concern are absent.
Leakage differs from permeation. Leakage is the unintentional entry or escape of matter (solids, liquids, or gases) through a
breach in a package wall or through a gap between package components. Leakage can also refer to the leaking matter itself.
Leakage flow rate is a function of the absolute and/or partial pressure gradient of leaking matter that exists across the package
barrier. In the context of this chapter, permeation is the passage of fluid (e.g., gas) into, through, and out of a nonporous
package wall. Permeation, not leakage, occurs when only a small fraction of molecules is able to move through a barrier by
way of any one hole. A nonporous package is able to permit permeation, but not the volumetric flow of air. Package permeation
test methodologies fall outside of the chapter scope and are not described.
Package integrity tests are leak test methods. A leak test is a method that detects the presence of (and in some cases, the
magnitude or location of) a package breach or gap.
Package seal quality tests are techniques used to characterize and monitor the quality and consistency of a package seal or
closure system parameter, which can influence the package’s ability to maintain integrity. An example is the peel force test
widely used to test seal strength. Seal quality tests are not leak tests but can play a valuable role by monitoring a characteristic
of the seal itself, the package materials, the package components, and/or the processes required to create the seal or closure
mechanism.
Integrity test methods vary not only in their application and detection limit, but also in terms of detection range, precision,
and specificity, for example. No one test is appropriate for all packages or for all leak testing applications. Selection criteria for
package integrity test methods, as well as method comparison aids, are presented to guide the user in the selection process.
Leak tests, even many commonly recognized industry standard testing approaches, require optimization and validation for
each product–package application. A science- and risk-based approach may allow some tests to be leveraged for broader
application under certain circumstances. For example, small differences in product formulation or package design and materials
may permit the use of one test for multiple product–packages. Package integrity verification plays an important role throughout
the product life cycle, starting with product development and continuing through marketed product stability studies.

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2. SCOPE

The package integrity leak tests and seal quality tests outlined in this chapter apply to the testing of both large and small
volume container–closure systems for sterile pharmaceutical dosage forms. Such package systems include, but are not limited
to, the following examples.
• Vials or bottles closed with elastomeric closures or screw-thread caps
• Form-fill-seal plastic or glass ampules
• Syringes or cartridges
• Flexible bags or pouches
• Packages for some drug/device combination products (e.g., packages cased inside autoinjectors)
Outside the chapter scope are packaging systems and processing equipment used in the preparation, storage, and
manufacture of sterile pharmaceutical products. Examples include containers for active pharmaceutical ingredients and
containers for product intermediate or final bulk product.
Outside the chapter scope are packages used for sterile diagnostic products or medical devices, and some packages used for
sterile drug/device combination products.
Outside the chapter scope are products having a primary package composed of porous barrier package materials, i.e.,
materials designed with pores or openings to allow volumetric air flow while preventing airborne microbial contamination.

3. LEAKS AND LEAKAGE RATE

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Leaks are commonly conceptualized as holes of a defined diameter, or channels of distinctive diameter and length, although
leaks that occur naturally are generally complex, multicavity tortuous paths and are rarely uniform in size or shape. Even
artificially created leaks such as laser-drilled defects (also called holes) used for leak-test method development and validation
are irregular in size, shape, and depth. When stating the size of a leak, it is important to define the measurement approach. In
some cases leaks are measured dimensionally, but quite often, leak size is determined based on gaseous leakage rate. For
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example, a package wall laser-drilled defect having a nominal diameter of 5 ± 2 µm may have been size-certified by matching
the airflow rate through the drilled defect to that of a 5 ± 2 µm hole present in a thin metal plate reference standard when
pressurized with dry air at specified differential pressure and temperature conditions.
Gaseous leakage rate is a measure of the rate of gas flow (in mass or volume units) that passes through a leak path under
specific conditions of temperature and the concentration or pressure differential across the barrier wall. Therefore, gaseous
leakage rate has dimensions of pressure multiplied by volume, divided by time. The international standard SI nomenclature is
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pascal cubic meter per second (Pa · m3 · s−1). These leakage measurement units refer to the quantity of leaking gas (Pa · m3) per
unit of time. When a leakage rate is described and no test conditions are noted, standard conditions of one standard atmosphere
differential pressure with dry air at 25° are assumed. For a more complete discussion of gaseous leakage rates and units of
measure refer to reference 1.
Unintentional leaks in packages are often detected or sized using gas as a tracer element. Given a situation in which a tracer
gas partial pressure difference exists across the package barrier wall and no absolute pressure difference exists, gaseous leakage
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is predominantly diffusional in nature as the tracer gas moves from a region of higher concentration to a region of lower
concentration. For example, the headspace in a vial package low in oxygen concentration and at ambient pressure at time of
closure will exhibit a rise in headspace oxygen concentration over time as a function of diffusion rate (relative to package leak
size) plus permeation rate (relative to permeability through the package). Such a change can be monitored instrumentally, as
is discussed later in the chapter.
Given the situation in which an absolute pressure difference exists across the package barrier wall, gas leakage through
package gaps is more rapid (flux being primarily convective) as gas moves from the higher pressure region to one of lower
pressure. For most package materials, the permeation rate of the gas of concern through the package wall is insignificant in
comparison. In this example, the rate of leakage into the package is determined by monitoring the change in headspace absolute
pressure as a function of time.
For many instrumental leak test methods described in this chapter, gas flow into or out of package leaks is induced by
exerting a pressure gradient across the package. In this way, a wide range of leak types and sizes can be identified.
Liquid leakage rate is a measure of the volume of liquid that moves through a leak path as a function of time under specified
conditions of temperature and absolute differential pressure across the barrier wall. Liquid movement through a leak path occurs
only when leak size/shape, package materials of construction, the absence of leak obstruction, tracer liquid composition, and
test parameters all work together. All sterile product–packages within the chapter scope are intended to prevent liquid leakage
and block microbial entry, thus it is the absence of liquid leakage flow or the absence of liquid in the leak path, rather than the
rate at which liquid leakage occurs that is typically verified in relevant package leak tests. Liquid leakage rate measurements are
only useful in measuring larger size leaks.

4. CLOSURE TYPE AND MECHANICS

An understanding of closure mechanics makes it possible to better characterize, monitor, and test packages for integrity.
This section discusses various closure systems and how each type functions to ensure package integrity.

4.1 Physically Mated Closures

Closure can be achieved by the close physical mating of two surfaces that often are dissimilar in material composition.
Examples include the interference fit of a plunger inserted in a syringe barrel, the compression fit of an elastomeric closure

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capped onto a vial finish surface, and the application force of a screw-cap torqued onto a bottle. Physically mated surfaces are
not bonded together; therefore, a tiny gap exists even between well-closed components. Nevertheless, when properly designed
and assembled, closure systems fit together such that liquid leakage (and microbial ingress) is prevented and gas migration is
limited. Regarding screw-thread closures such as those used for ophthalmic-product dropper bottles, the barrier to leakage is
afforded by inner cap surfaces pressing against the package opening, in some cases aided by a secondary gasket or plug. The
winding path afforded by the threads of a screw-cap does not provide an optimal barrier to gas or liquid leakage, or to microbial
ingress in the event of liquid presence in the cap threads.

4.2 Physicochemically Bonded Closures

Seals are formed when two similar or dissimilar surfaces are physicochemically bonded together. One example is the
formation and sealing of contiguous containers from a single material (e.g., glass or plastic ampules). In other form, fill, and
seal systems, sheets of material are mated by means of a heat or ultrasonic welding process. Two dissimilar materials may be
joined using an intermediate bonding material. Fully fused seals inherently block liquid leakage and microbial ingress; however,
gaseous leakage and permeation may occur.

4.3 Multiple-Dose Package Closures

Multiple-dose package closures are designed to allow product access while limiting microbial ingress and product leakage
between doses. For example, elastomeric closures for parenteral products requiring needle-puncture access are designed to
offer reseal protection against microbial ingress and product leakage. Some multiple-dose ophthalmic dosage form package
closures are designed with filters, plugs, or other mechanical means that allow the product to be dispensed while restricting

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microbial ingress and product leakage. For example, ophthalmic product–package closure systems have been designed to
automatically pinch shut between intermittent dosing to limit microbial entry and product loss.

5. PRODUCT–PACKAGE QUALITY REQUIREMENTS AND THE MAXIMUM ALLOWABLE

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LEAKAGE LIMIT

As noted in Introduction, package integrity is necessary to maintain product critical quality attributes within physicochemical
label-claim specifications and to ensure product sterility until time of use. Detrimental contaminants include microorganisms
and any substances that threaten patient safety or product quality. Product leakage can cause a product to fail content or
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potency specification limits. For certain products to maintain product physicochemical stability, the package needs to maintain a
headspace of nonreactive gases and/or low water vapor content, sealed under atmospheric or reduced-pressure conditions.
Headspace vacuum conditions may also be necessary to facilitate product ease of use, e.g., product reconstitution using a
diluent injected into the container.
Most package types display very low but definite gaseous leakage flow through the gap that exists even between well-fitted
closures. Therefore, it is not practical to require that packages be absolutely leak-free. Rather, it is the significance of leakage in
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relation to product quality that needs to be considered. In other words, the package should not permit leakage beyond the
product’s maximum allowable leakage limit. Such leaks of concern should be absent.
Identifying the maximum allowable leakage limit for a product–package is a science- and risk-based decision. The smaller
the leak path, the less likely the product can escape and the less likely microorganisms or other contaminants can enter.
Eventually, leak paths may be so small that only headspace gas exchange is realistically possible. Package construction and
assembly, package contents, and the range of environments a given product–package may be exposed to during its life cycle
are to be considered when specifying the maximum allowable leakage limit.
There are two major product–package quality requirement categories used when specifying the maximum allowable leakage
limit and one subcategory that applies only to multiple-dose packages. These are discussed below.

5.1 Sterility and Product Formulation Content must be Preserved; Gas Headspace Content
Preservation is not Required
This category includes product–packages for which the maximum allowable leakage limit correlates to the prevention of
product formulation escape, or product contamination by external liquid or solid matter or by microorganisms, while gas
headspace preservation (i.e., ambient pressure air) is of no concern. The smallest leak paths that allow only limited gas exchange
are irrelevant as they pose no real risk to product quality.
Considerable published research exists exploring rigid package leaks and their relationship to risk of aqueous solution leakage
and/or microbial ingress. A key study series found orifice leaks of approximately 0.1 µm in nominal diameter (using
micropipettes) placed in rigid packages demonstrate a small risk of aqueous liquid passage, while orifice leaks as small as
approximately 0.3 µm in nominal diameter first demonstrate some risk of microbial ingress by liquid challenge (2,3). For a
summary of similar studies comparing risks of microbial ingress and liquid leakage to leak size and type, the reader may refer
to reference 4.
Given this body of evidence, a maximum allowable leakage limit of less than 6 × 10−6 mbar · L/s (measured by helium mass
spectrometry in the vacuum mode) can be adopted for products in this category packaged in rigid container–closure systems.
This leakage rate equates to the presence of an orifice of nominal diameter of between 0.1 and 0.3 µm. At this leakage rate,
the probability of microbial ingress was determined to be <0.10 (2). Selecting this conservative maximum allowable leakage
limit will ensure a low risk of microbial ingress and liquid leakage and can eliminate the need to perform additional microbial
ingress or liquid challenge studies as a function of leak size.

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For other container systems such as those made using flexible materials or those with complex, lengthy closure/seal interfaces,
or those meant to contain a product of markedly greater leakage potential, the risk of microbial ingress or liquid passage through
leak paths is not as widely publicized or perhaps understood. In such cases, where the relationship between defect size/type to
the risks of microbial ingress and/or liquid passage is less prescriptive, a study exploring these relationships could be useful. Test
results can be used to establish a meaningful maximum allowable leakage limit for the given product–package system, which
can be employed for package integrity verification by other validated leak test methods of choice. Once established, this
maximum allowable leakage limit can be applied to similar product–packages with appropriate justification.
Finally, before classifying a product–package in this category, one may consider the potential impact of product life cycle
processing, storage, distribution and use scenarios on package integrity. For example, elastomeric closures have been found to
shrink and lose their viscoelastic properties during ultra-cold storage (≤−80°) to such an extent that gas influx into stoppered
vial packages may occur. During warming, package closure is restored, trapping gases and notably raising internal package
pressure. In such cases, gas headspace preservation may be a product quality concern, even if the product does not require
specific headspace content or pressure for optimum stability.

5.2 Sterility, Product Formulation Content, and Gas Headspace Content must be Preserved
The second category includes product–packages for which the package should prevent product formulation escape and
product contamination by external liquid or solid matter or by microorganisms, but in addition, the package must preserve the
gas headspace content or absolute pressure. Preservation of headspace content and/or pressure is needed to maintain product
stability within physicochemical specification limits, and/or to aid end-user product access. Thus, the maximum allowable
leakage limit for such products is likely more stringent than that described in the first category. Although outside the chapter
scope, the influence of gas permeation through the package itself may also need to be considered when establishing the

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maximum allowable leakage limit as permeation plus leakage can impact package headspace content.
The maximum allowable leakage limit for products in this category may be expressed in terms of the maximum allowable
package headspace content or pressure change as a function of time. This limit, established for each product–package system,
may be applied to other similar product–packages systems with appropriate justification.
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5.3 Sterility must be Preserved; Product Access is Required
This subcategory represents a quality requirement applicable only to multiple-dose product–packages included in either of
the above two categories. Once the product–package has reached the end-user and the closure has been activated or otherwise
compromised to allow dosage access and delivery, the maximum allowable leakage limit at this life cycle phase (called the
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in-use maximum allowable leakage limit) is defined in terms of microbial ingress and product loss prevention between and
during dosage access. For example, elastomeric closures of multiple-dose vials or cartridges containing antimicrobial-preserved
parenteral products are designed to afford reseal protection against microbial ingress and product formulation leakage as
product is accessed via needle puncture as well as between doses.
To establish the in-use maximum allowable leakage limit for multiple-dose package closure systems, a study will likely be
required to explore the relationship between product access attempts, product loss risk and/or microbial ingress risk, versus
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leakage measurement by an alternative adequately sensitive leak test method of choice. These data can be used to establish a
meaningful in-use maximum allowable leakage limit expressed in units of measure reflective of the preferred leak test method
and that will assure that product loss and microbial ingress risk during use is minimal. The in-use maximum allowable leakage
limit established for a given product–package may be applied to other similar product–packages systems with appropriate
justification.

6. INHERENT PACKAGE INTEGRITY AND THE PACKAGE INTEGRITY PROFILE

Inherent package integrity is the leakage characteristic of a well-assembled container–closure system using no-defect package
components. Inherent package integrity is first determined during product–package development and qualification and is a
measure of the leak tightness of a container–closure system, given anticipated variables of material composition, dimension,
processing, and assembly. Inherent package integrity may also be determined as a function of anticipated final product storage,
distribution, and use. Acceptable inherent package integrity for a container–closure system conforms to the specific product–
package maximum allowable leakage limit. Inherent package integrity is expressed in terms that allow a meaningful comparison
to the maximum allowable leakage limit.
Confirmation that the inherent package integrity conforms to the maximum allowable leakage limit is the first step in
product–package integrity verification. Verification of package integrity continues throughout the product life cycle. The
package integrity profile is an ongoing database of product life cycle package leak and seal quality test results. This profile
provides information regarding package integrity given operative variations in package component design and material;
package assembly and processing; and product storage, distribution, and stability. These concepts are more fully explored in
Package Integrity Testing in the Product Life Cycle—Test Method Selection and Validation á1207.1ñ, Test Instrument Qualification,
Method Development, and Method Validation.
In summary, a product–package system having integrity is one in which the inherent package integrity conforms to the
required product–package maximum allowable leakage limit. Further, the package integrity profile database operates as a risk
management tool to ensure that finished product container–closure systems are intact, able to block microbial ingress, restrict
loss of product contents including critical headspace gases, and prevent entry of detrimental gases or other substances, thus
ensuring that the product meets all relevant physicochemical and microbiological label-claim specifications through expiry and
final end-use.

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REFERENCES

1. Jackson CN, Sherlock CN, Moore PO, editors. In: Nondestructive testing handbook. 3rd ed. Vol. 1. Leak testing.
Columbus, OH: The American Society for Nondestructive Testing; 1997.
2. Kirsch LE, Nguyen L, Moeckly CS, Gerth R. Pharmaceutical container/closure integrity. II: The relationship between
microbial ingress and helium leak rates in rubber-stoppered glass vials. PDA J Pharm Sci Technol. 1997;51(5):195–202.
3. Kirsch LE. Pharmaceutical container/closure integrity. VI: A report on the utility of the liquid tracer methods for evaluating
the microbial barrier properties of pharmaceutical packaging. PDA J Pharm Sci Technol. 2000;54(4):305–314.
4. Guazzo DM, Singer DC, Stevens-Riley M, Walker D, Kirsch LE, Buus P, et al. Proposed revisions to general chapter sterile
product packaging—integrity evaluation á1207ñ. Stimuli to the revision process. Pharmacopeial Forum. 2014;40(5).

GLOSSARY
For definitions of container, materials of construction, packaging component, packaging system, primary packaging
component, and secondary packaging component, see Packaging and Storage Requirements á659ñ. In the context of this chapter
and its subchapters, the following definitions relevant to packaging and package integrity methods, seal and closure
mechanisms apply. For definitions of specific leak test and seal quality test methods, refer to á1207.2ñ and á1207.3ñ, respectively.
Accuracy: The accuracy of a leak test method is a measure of the ability of the method to correctly differentiate packages
that leak above the claimed detection limit from those that leak below this limit (do not leak); a measure of false positive and
negative occurrence. Alternatively, for those methods that deliver an outcome that is a direct quantitative measure of gas leakage
rate (or of gas content or pressure), accuracy is a measure of the method’s ability to produce an outcome comparable to a true
standard, such as a nationally recognized gas standard.

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Container–closure integrity: Container–closure integrity is the ability of a package to prevent product loss, to block
microorganism ingress, and to limit entry of detrimental gases or other substances, thus ensuring that the product meets all
necessary safety and quality standards. Synonymous with Package integrity.
Container–closure integrity test: A container–closure integrity test is any package leak test (either physicochemical or
microbiological) that detects the presence of a package breach or gap. Some tests may also be able to identify the leak
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magnitude and/or location. The term container–closure integrity test is synonymous with package leak test or package
integrity test.
Container–closure system: See Packaging and Storage Requirements á659ñ, General Definitions, Packaging System.
Detection limit: The leak test detection limit is a measure of test method sensitivity. The detection limit is the smallest
leakage rate (or leak size) that a leak test method can reliably detect. Also called limit of detection.
Deterministic leak test method: A deterministic leak test method is one in which the leakage event being detected or
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measured is based on phenomena that follow a predictable chain of events. In addition, the measure of leak detection is based
on physicochemical technologies that are readily controlled and monitored, yielding objective quantitative data.
In-use maximum allowable leakage limit: See Maximum allowable leaking limit.
Inherent package integrity: Inherent package integrity is the leakage rate (or leak size) of a well-assembled container–
closure system using no-defect package components. Inherent package integrity is a measure of the leak tightness of a
container–closure system, given anticipated variables of material composition, dimension, processing, assembly; package
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storage, distribution and use.

Leak: A leak is a breach in a package wall or a gap between package components that is capable of permitting the passage
of gas, liquid, or solid matter. Leak is synonymous with leak path.
Leakage: Leakage is the unintentional escape or entry of matter (solid, liquid, or gas) through a breach in the package wall
or through a gap between package components. Leakage can also refer to the substance that enters or escapes from a
compromised package. For example, “Leakage from the cracked container stained the package label.”
Leakage rate: Gaseous leakage rate is a measure of the rate of gas flow (in mass or volume units) that passes through a
leak path under specific conditions of temperature and absolute pressure or concentration differential. Leakage rate has
dimensions of pressure multiplied by volume, divided by time. For example, the international standard SI nomenclature is pascal
cubic meter per second (Pa · m3 · s−1). Other common units of measure include standard cubic centimeters per second (std ·
cm3 · s−1 or sccs) and mbar liters per second (mbar · L · s−1). Liquid leakage rate is a measure of the volume of liquid that moves
through a leak path as a function of time under specified conditions of temperature and absolute differential pressure across
the barrier wall. Liquid leakage rate measurement, possible for larger leak tests, requires that leak size, materials of construction;
absence of leak obstruction; tracer liquid composition; and test parameters work together to ensure liquid movement through
the leak path.
Linearity: Leak test method linearity is the ability of the method to elicit test results that are mathematically proportional
to leak path size or leakage rate.
Master: A master is a package prototype, model, or facsimile made to simulate an actual package in shape and design.
Masters may be made of solid plastic or metal, or they may be simply a designated container–closure unit. Masters are designed
to simulate a no-leaking package, and are often used in leak test system suitability tests to verify instrument performance.
Maximum allowable leakage limit: The maximum allowable leakage limit is the greatest leakage rate (or leak size)
tolerable for a given product–package that poses no risk to product safety and no or inconsequential impact on product quality.
The maximum allowable leakage limit for a sterile pharmaceutical dosage form package will ensure the content’s sterility,
preserve product contents, and prevent entry by detrimental gases or other substances, thus ensuring that the product meets
relevant physicochemical and microbiological specifications through expiry and use. For multiple-dose product–packages, the
in-use maximum allowable leakage limit is defined as the degree of protection demanded of the closure to limit microbial ingress
and product formulation leakage between and during dosage access.
Microbiological challenge test: A microbiological challenge test is a package leak test whereby package integrity is
evaluated by exposing containers filled with growth-supportive media to microorganisms suspended in submersion media (a

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liquid-borne challenge test). Leakage is evidenced by the subsequent growth of the challenge microorganisms in the package
contents. Synonymous with microbial challenge test.
Negative control: A negative control is a package with no known leak. Negative controls used for leak test method
development and validation studies represent packages optimally assembled using normally processed components. Negative
controls should duplicate the container–closure system of the product under integrity investigation. For some methods, it may
be necessary for negative controls to simulate test product headspace and formulation content as well.
Nonporous: Nonporous packaging does not have pores or openings to allow volumetric air flow. In other words, NMT a
small fraction of molecules is able to pass through any one hole. Nonporous materials may be impermeable, semi-permeable,
or permeable to the passage of fluid through the package barrier wall.
Nominal diameter: In the context of this chapter, nominal diameter is a means of expressing the size of a package leak.
Package leaks are typically irregular tortuous paths or matrices that cross a package wall or barrier. Such leaks are difficult to
size in accurate or meaningful terms. The nominal diameter of a leak path is defined as the width of an orifice of relatively short
depth, through which gas (e.g., dry air) at equivalent airflow rate may pass when subject to one atmosphere differential pressure
at controlled temperature conditions.
Package integrity: See Container–closure integrity.
Package integrity test: See Container–closure integrity test.
Package integrity profile: The package integrity profile is a database of product life cycle package leak and seal quality
test results that denotes product–package integrity given operative variations in package component design and material;
package assembly and processing; and product storage, distribution, and stability.
Package leak test: See Container–closure integrity test.
Package seal quality: Package seal quality relates to the consistency of a package seal’s performance within required
specification limits. Examples of package seal quality attributes include heat seal bond strength and capped vial package residual
seal force.

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Package seal quality test: A package seal quality test is used to characterize and monitor the quality of a product–
package seal or closure system to ensure that package assembly is consistently kept within established limits. Package seal quality
tests are not and cannot substitute for leak tests. However, they can provide some assurance of the package’s ability to maintain
integrity by monitoring a characteristic of the seal itself, the package materials, the package components, and/or the processes
required to create the seal or closure mechanism. Examples include the seal peel force test and the capped vial package residual
seal force test.
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Permeation: Permeation is the passage of fluid into, through, and out of a nonporous package wall. Permeation, not
leakage, occurs when the package barrier has no holes large enough for more than a small fraction of molecules to pass through
any one hole.
Physicochemical package integrity test: A physicochemical package integrity test is a leak test that detects the
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presence of a package leak, or detects/measures package leakage rate, via physical or chemical means. All leak test methods
that do not use microorganisms for leak detection are physicochemical leak test methods.
Porous: A porous package or package material has pores or openings to allow volumetric air flow while still blocking airborne
microbial passage. Examples include coated paper or spun-bonded polyolefin barrier materials that permit package penetration
by ethylene oxide gas or steam.
Positive control: A positive control is a package with a known, intentional defect. Positive controls used for leak test
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method development and validation studies should duplicate study negative controls in terms of materials of construction,
package assembly, and component processing. Positive controls are used during leak test method development and validation.
Some methods require the use of positive controls during routine testing as well.
Precision: Leak test method precision is a measure of the method’s ability to yield reliable, repeatable data. Precision
includes repeatability (repeat testing of a single homogeneous test sample population set), ruggedness (within laboratory tests
performed by multiple operators on multiple days, using multiple instruments; also known as intermediate precision), and
reproducibility (tests among multiple laboratories).
Probabilistic leak test method: A probabilistic leak test method is the converse of a deterministic leak test method,
being stochastic in nature. Probabilistic tests rely on a series of sequential and/or simultaneous events, each associated with
random outcomes described by probability distributions. Thus, the findings are associated with uncertainties that necessitate
large sample sizes and rigorous test-condition controls to obtain meaningful results. Typically, sample size and test condition
rigor are inversely related to leak size.
Product: The pharmaceutical product includes the pharmaceutical formulation as well as the packaged headspace, which
may consist of ambient air or nonreactive gases with specified water-vapor content under full or sub-atmospheric pressure
conditions.
Product–package: The product–package includes the primary package with critical secondary components (the
container–closure system) plus the packaged product (the contents).
Qualitative measure of analysis: A qualitative measure of analysis for leak testing is a measurement approach based
on a subjective evaluation of some quality, attribute, or characteristic of the test sample. Visual inspection is an example of a
qualitative measure of analysis.
Quantitation limit: The quantitation limit is a leak test method characteristic defining the lowest leakage rate or leak size
that can be differentiated with accuracy and precision under the stated experimental conditions.
Quantitative measure of analysis: A quantitative measure of analysis for leak testing is a measurement approach based
on objective, numeric data that either directly or indirectly correlates with leak presence, leak location, or leakage rate. Examples
include the mass-of-gas-per-time reading generated by the helium mass spectrometry tracer-gas leak test, or the pressure
reading as a function of test time measurement produced by the vacuum-decay method.
Range: The range of a leak test method is that interval between the smallest to largest leak size (or leakage rate) that can
be detected by a given leak test method with a suitable level of accuracy and precision.

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Robustness: Robustness of a leak test method is the method’s ability to accurately identify leaking versus nonleaking
packages despite small but deliberate variations in procedural parameters, providing an indication of the method’s suitability
during normal usage.
Specificity: The specificity of a leak test method is the ability of the method to accurately differentiate leaking and
nonleaking packages, despite interfering factors that may cause false detection.
System suitability: System suitability is a manner of ensuring that the leak test method including all factors, which may
be subject to variability, that may impact test results (such as instrumentation, analysts, test sample preparation steps, and the
test environment) are adequately controlled and maintained in such a fashion that the method is rugged and robust.
System suitability test: A system suitability test is a test to verify that the leak test method and all key factors that may
impact test results are correctly controlled and set prior to method performance.
Tortuous path: As applied to leaks, a tortuous path is a convoluted, complex leakage pathway. Most naturally occurring
leaks, such as cracks and tears, are tortuous in nature, rather than pristine holes. As applied to sealing mechanisms, a sealing
material that has tortuous barrier qualities can block microbial entry. [NOTE—The winding path afforded by the threads of a
screw-cap (e.g., an ophthalmic dropper bottle closure) does not provide an optimal barrier to gas or liquid leakage, nor does
it provide an optimal barrier to microbial ingress in the event of a liquid presence in the cap threads.]
Type defect: A type defect is a positive-control package that represents realistic package flaws. Type-defect positive controls
may be included in leak test method feasibility and development studies before method validation. An example of a type defect
is a heat seal wrinkle or a loose cap. Type defects are inherently irregular in size and shape and are often described qualitatively
instead of being described in terms of leak size or leakage rate.

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á1207.1ñ PACKAGE INTEGRITY TESTING IN THE PRODUCT LIFE CYCLE

—TEST METHOD SELECTION AND VALIDATION
1. INTRODUCTION
2. PACKAGE INTEGRITY TESTING IN THE PRODUCT LIFE CYCLE
2.1 Development and Validation
2.2 Product Manufacturing
2.3 Commercial Product Stability
3. TEST METHOD SELECTION CRITERIA
3.1 Package Contents
3.2 Package Design, Materials of Construction, and Mechanics
3.3 Closure Type and Mechanics
3.4 Maximum Allowable Leakage Limit
3.5 Deterministic or Probabilistic Methods
3.6 Physicochemical or Microbiological Methods
3.7 Method Outcome
3.8 Quantitative or Qualitative Methods of Analysis
3.9 Leak Test Detection Limit

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3.10 Leak Test Method Range
3.11 Nondestructive or Destructive Methods
3.12 Off-Line or On-Line Methods
4. TEST INSTRUMENT QUALIFICATION, METHOD DEVELOPMENT, AND METHOD VALIDATION
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4.1 Instrumentation and Equipment Qualification
4.2 Method Development and Validation
4.3 System Suitability
4.4 Microbial Ingress Risk Comparison
4.5 Negative and Positive Controls
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1. INTRODUCTION

Package Integrity Evaluation—Sterile Products á1207ñ provides guidance in the integrity assurance of product packages. This
chapter describes package integrity verification during three product life cycle phases: 1) package development, and package
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processing and assembly validation; 2) product manufacturing; and 3) commercial product shelf-life stability assessments.
Further, this chapter provides information on how to select, develop, and validate leak test methods.

2. PACKAGE INTEGRITY TESTING IN THE PRODUCT LIFE CYCLE

Appropriate packaging for a sterile product can be determined on the basis of a knowledge-gaining effort conducted during
the product’s life cycle.

2.1 Development and Validation

2.1.1 PACKAGE DEVELOPMENT
Package development begins with the preparation of a product–package profile (e.g., user requirements specification), which
considers the product end use, stability requirements, and method of manufacture, as well as the anticipated storage, shipment,
and distribution environments. This profile also defines the proper product–package quality requirements and the package’s
maximum allowable leakage limit (see Package Integrity Evaluation Sterile Products á1207ñ, Product–Package Quality Requirements
and the Maximum Allowable Leakage Limit). With this prospectively developed body of information, one can select each package
component’s materials of construction, choose suitable component sources, and establish critical physical attributes and
component dimensional tolerances. Each component material, with its critical dimensional tolerances, directly affects the
integrity of the final packaged product. Assurance of package integrity originates from the use of appropriate materials, accurate
and optimum closing properties, matching dimensional fit, appropriate multi-component stack heights and tolerances, and
consistent control of processes used to assemble the closed package.
The manner in which the package is processed, formed, or assembled is an important consideration in package integrity
assurance. A preliminary assessment of package integrity at the end of the development phase under conditions representative
of the marketed product manufacturing system is prudent.
These conditions include processes such as sealing operations and component sterilization. Where possible, processes are
performed according to established and approved user requirement specifications. Establishing appropriate physical
characteristic specifications for container materials, considering lot-to-lot variations, can ensure that the most extreme

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processing conditions anticipated (e.g., multiple sterilization cycles) do not physically damage materials in a manner that would
adversely affect package integrity.
Finally, the robustness of the manufactured product–package system may be evaluated during the development phase by
exposing a representative number of product samples to specified storage, shipment, distribution, and final product-use
environments. These efforts may include studies that evaluate package integrity at the extremes of the finished product–package
profile, not simply at optimal conditions. Given the complexity of some sterile packages, it may be useful for package integrity
development studies to incorporate multi-point analyses of test packages manufactured within specified process parameters.
Utilizing a common container–closure for which the producer has significant experience and process knowledge can
supplement the overall development process and may help reduce the efforts needed. A battery of integrity and seal quality
test methods may be employed during product–package development, starting with techniques able to measure the product’s
inherent package integrity. Inherent package integrity of a viable package system conforms to the product’s maximum allowable
leakage limit.
Test package quantities for inherent package integrity verification may vary on the basis of: 1) the complexity of the product–
package, 2) the specifics of the user specification requirements, and 3) the prior experience of the producer. Test quantity
choice is also influenced by the confidence that can be placed on the package integrity test results as well as the level of integrity
assurance desired. In some cases inherent package integrity verification may be more readily and economically determined by
using empty or placebo-filled container–closure systems, thereby enabling larger sample quantities to be tested by the most
sensitive and quantitative leak test methods without the risk of product formulation interference with the test method.
The outputs of the packaging development phase include the final user requirement specifications, which form the basis of
production purchasing specifications for package components. Also during the development phase, the final equipment user
requirement specifications are developed for package material cleaning, sterilization, and forming; sealing or assembly
equipment; and allied materials supply and component feed systems. These user requirement specifications provide purchase
specifications for the acquisition of equipment or for the vetting of potential contract manufacturers.

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2.1.2 PACKAGE PROCESSING AND ASSEMBLY VALIDATION
Final confirmation of acceptable inherent package integrity is generally part of a larger process validation activity for the
overall production process. The scope depends upon the product type and whether the organization has previous experience
with the container–closure system.
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All processes germane to the sterilization and formation of a package having integrity are to be evaluated against the user
requirement specifications established in the package development phase, including likely process extremes. For example,
inherent package integrity verification may consider extremes of package assembly variables such as line speed, heat-sealing
temperature, screw-cap application torque, and vial-capping forces, as well as resterilization processes, labeling, and secondary
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and tertiary packaging processes. Validation test requirements and scope should fit the statistical requirements and capabilities
of each process, taking into account both package and package-line complexity, as well as prior experience with similar product–
packages.
Testing done during technical transfer from the product development site to the manufacturing sites will assist in determining
whether the user requirement specification targets and the control ranges established in development require any modification
when packages are made on a full manufacturing scale.
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Integrity test methods for package processing and assembly validation studies are meant to verify the packages’ continued
conformance to the product’s maximum allowable leakage limit. For some product–packages, the most fitting integrity test
methods for this life cycle phase may have a detection limit greater than the maximum allowable leakage limit. Methods able
to reject largely leaking packages such as those caused by defective or out-of-specification components, package damage, and/
or package misassembly are appropriate. Seal quality tests suitable for use include those able to monitor package processing
and/or assembly consistency.
Successful validation will result in a package that meets its user requirement specifications. The primary objective in package
development and subsequent validation is to arrive at a quality product–package prepared using processes that reliably and
consistently run within specified operating parameters as defined in the user requirement specifications, yielding critical package
defects at a satisfactorily low rate. When performed, in-process and end-product package integrity testing should complement,
not replace, thorough package development efforts.

2.2 Product Manufacturing

To ensure the quality of the manufactured product–package, it is critical to specify components of sufficient quality and to
select vendors carefully. The factors to consider include:
• Acceptable results of the initial vendor or supplier evaluation
• Appropriate vendor acceptance quality limits and statistical sampling plan(s), or relevant certification
• Incoming component quality verification, including statistical assessment of quality against purchase specifications
• Appropriate procedure(s) for establishing corrective and preventive action when a vendor falls short of quality expectations
Package integrity re-evaluation is considered when changes occur in the product, package design, package materials, or
manufacturing/processing conditions. The extent of change control efforts are evaluated on a case-by-case basis, while the level
of testing required to support the change is determined based on impact assessment.
The product’s package integrity profile is compiled over the course of commercial manufacture. This database of ongoing
leak and seal quality test results serves to flag potentially harmful package integrity excursions that may be linked to operative
variations in package component design/material and package assembly/processing. Not all packaged-product damage can
be readily detected post-assembly; therefore, processing procedure controls and monitors may be relied upon to safeguard
against such problems. The proper combination of package leak tests, complementary package seal quality tests, and visual
inspection is largely based on package failure results observed in earlier production validation and package development studies.

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Testing production lot samples can provide a measure of package integrity confirmation, while entire testing by
nondestructive means is able to yield an ongoing assessment of integrity assurance. In some cases regulatory requirement
dictates the level of testing performed. For other product–packages, justification for the level of testing is based on statistical
process control results generated during the validation phase, and later, on the basis of routine manufacturing product-quality
trending analyses.
For example, glass or plastic ampules closed by heat fusion are customarily subjected to 100% nondestructive leak testing.
Products sealed under vacuum require appropriate package assembly validation supplemented by testing over time to ensure
that the vacuum is retained. Similarly, integrity assurance of packages that require a specific, non-reactive, inert gas headspace
is based on appropriate package assembly validation along with testing for rise in reactive gas or water vapor content over time.

2.3 Commercial Product Stability

Container–closure integrity tests have been recommended as alternatives to sterility testing as part of commercial product
stability programs. The goal is to ensure package integrity as a function of long-term product storage (1). [NOTE—Package
integrity tests do not replace product release sterility tests.] Testing product for sterility is a poor measure of product–package
integrity and also will not ensure product–package integrity over the shelf life of the product when performed as part of the
stability program. In addition, a package may be in no danger of microbial ingress and yet be unable to maintain the gas
headspace content necessary for product quality. Validated package integrity test methods using technologies such as those
described in this chapter are more sensitive and reliable than product lot sterility testing for detecting a breach in package
integrity that could lead to sterility loss or failure in relevant product physicochemical quality attributes.
Ideally, package integrity tests selected to support marketed-product stability studies are able to verify the absence of the
smallest leaks of concern for a given product–package system. In other words, the product’s maximum allowable leakage limit

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falls within the chosen test method leak detection range. However, the test methodology most appropriate for a particular
product–package system may be unable to detect the very smallest leaks of concern. In some cases the package contents
interfere with the ability of leak test methodologies to detect the smallest leaks. For example, proteinaceous ingredients or even
salts may clog leak pathways, inhibiting leak detection by gas flow methods such as vacuum decay or mass extraction. It is
prudent to understand the product’s potential to interfere with the selected leak test method, both initially after package
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assembly and over time.
In situations such as those described above, the package integrity test chosen to support stability studies should have a
detection limit as close as feasible to the product’s maximum allowable leakage limit. An understanding of what the method
is capable of evaluating and how this is applicable to microbial integrity assurance is warranted.
For products that demand package headspace content preservation, it is appropriate that the integrity test for stability studies
verify the continued presence of specific headspace gases or subatmospheric pressure over time. Satisfactory results verify the
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absence of leaks that could jeopardize product sterility as well as relevant physicochemical quality attributes. The required
duration for monitoring container vacuum or headspace gas content maintenance that equates to microbial barrier assurance
and liquid product loss prevention can be predicted on the basis of gas flow kinetics, should a leak pathway exist that is large
enough to allow microbial entry and product formulation loss.
Stability test samples intended for package integrity evaluation are kept at labeled storage conditions for the marketed
product. Test sample quantities chosen for each testing time point are to be relevant and sufficiently representative of the
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purpose of stability testing, which is to indicate whether integrity is affected by the stability conditions. Sample quantity selection
takes into account all prior development and validation testing.
As noted in reference (1), if an integrity test is nondestructive to the product or package, samples that pass package integrity
testing may be further used in the stability testing for that specific test period or interval. However, samples should not be tested
for package integrity at one time point (e.g., 12 months), then stored for further stability testing at a later time point (e.g., 24
months). While not specifically noted in reference (1), it is logical to assume that test samples earmarked for integrity verification
over the course of the stability study could be checked for integrity before placement on stability if a nondestructive leak test
method is used. This would be akin to the common practice of visually inspecting test samples prior to placing them on stability.
In this way, subsequent integrity failures can be attributed to stability storage, rather than to other causes.

Change to read:
3. TEST METHOD SELECTION CRITERIA

No single package leak test or package seal quality test method is applicable to all product–package systems. Test method
selection is made on a product–package on a case-by-case basis. Often more than one test method is employed during a given
product’s life cycle. Package test method selection as a function of product life-cycle phase, along with important integrity
considerations, are discussed in Package Integrity Testing in the Product Life Cycle. A broad discussion of additional selection
criteria for leak test methods follows. Specific leak test method examples are provided for informational purposes but are not
intended as recommendations. The attributes and general capabilities of tests can be found in Package Integrity Leak Test
Technologies á1207.2ñ and are helpful in the method selection process.

3.1 Package Contents

When selecting a leak test method, the first determining factor is the nature of the package contents. Whether the package
contains a liquid or solid formulation, and whether it contains a headspace of inert gas, air, vacuum, or even no headspace at
all, will influence leak test method choice. For example, if testing liquid-filled packages by vacuum decay or mass extraction,
test vacuum conditions may trigger some formulations to solidify inside leak paths, thereby blocking gas leakage flow and

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making the test ineffective. Alternatively, electrical conductivity and capacitance tests can be used, but only if the liquid product
is more electrically conductive than the package materials.

3.2 Package Design, Materials of Construction, and Mechanics

Packages vary widely in their design, their materials of construction, and the mechanisms whereby package closure is affected.
Each of these variables influences the leak test method choice, as the following examples illustrate.

3.2.1 RIGID OR FLEXIBLE PACKAGES

Rigid (nonporous) packages can tolerate the pressure or vacuum-challenge conditions required in several leak tests, including
tracer liquid ingress, vacuum decay, mass extraction, and some tracer gas tests. In contrast, some flexible packages can tolerate
differential-pressure test conditions only if special tooling is used to restrict package expansion and prevent subsequent seal
damage. Package restriction is necessary to ensure consistent differential pressure conditions across the package barrier.

3.2.2 MOVEABLE OR FIXED COMPONENTS

Moveable package components, such as syringe stoppers (plungers or pistons), may require restraint to prevent their
dislocation during the differential pressure test conditions required for a majority of leak test procedures (e.g., tracer liquid test,
pressure or vacuum decay test, mass extraction test, bubble emission test, and microbial challenge by immersion test).

3.2.3 POLYMERIC MATERIALS

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When exposed to test vacuum conditions, package materials such as plastics and some elastomers may outgas volatiles that
raise vacuum-decay leak test results as well as mass extraction results, falsely implicating package leakage. Plastic packages that
are highly permeable to tracer gases may not be compatible with helium tracer gas leak detection, as helium permeating
through the package could be mistaken for package leakage or may mask small leaks. Special fixtures to limit tracer gas
permeation effects and isolate tracer gas exposure to the seal area under test have been used to mitigate such difficulties.
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3.2.4 METALLIC MATERIALS
Packages made of foil laminate materials may prove incompatible with electrical conductivity and capacitance leak detection,
which works best with relatively nonconductive package materials. However, aluminum caps used to secure vials closed with
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elastomeric closures pose no hindrance to electrical conductivity and capacitance tests, even for finding leaks located between
the closure and the vial finish.

3.2.5 TRANSPARENT OR OPAQUE MATERIALS

Packages made of transparent or translucent materials allow for visual inspection and electromagnetic wave passage.
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Therefore, transparent or translucent materials can be tested by laser-based gas headspace analysis techniques, as well as
tracer-liquid ingress or microbial ingress. Opaque packages are incompatible with testing approaches that require visual
inspection of the package contents.

3.3 Closure Type and Mechanics

Refer to Package Integrity Evaluation—Sterile Products á1207ñ, Closure Type and Mechanics for a discussion of the closure type
categories represented by the various product–package systems. The design of the closure system and its leakage restriction
function (i.e., the maximum allowable leakage), plus the types of defects anticipated, influence the integrity test method choice.

3.4 Maximum Allowable Leakage Limit

The maximum allowable leakage limit as a function of the product–package quality requirement is discussed in Package
Integrity Evaluation—Sterile Products á1207ñ, Product–Package Quality Requirements and the Maximum Allowable Leakage Limit.
The following integrity test method selection considerations are offered in light of that discussion.

3.4.1 STERILITY AND PRODUCT FORMULATION CONTENT MUST BE PRESERVED; GAS HEADSPACE PRESERVATION IS NOT
REQUIRED
Integrity tests for this product quality category include those able to verify that the maximum allowable leakage limit that
prevents liquid and microbial ingress is not exceeded. Tracer gas tests performed using the vacuum mode and laser-based gas
headspace analysis test methods are two examples. Both have been shown to be sensitive enough to quantitatively analyze
leakage through the smallest leak paths found to pose the smallest chance of liquid leakage or microbial ingress in rigid
packaging. Such tests have also proven useful for defining relationships among package design, component fit, package
assembly parameters, and leakage rate, even in the absence of package defects.
Leak testing of product-filled packages during later product-life-cycle phases often requires other tests. Leak test methods
available for this phase of the product life cycle are able to be validated to reliably detect defects a few micrometers and larger.
Examples of such physicochemical leak tests include vacuum or pressure-decay tests, mass extraction methods, electrical
conductivity and capacitance tests, and liquid tracer tests.

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3.4.2 STERILITY, PRODUCT FORMULATION CONTENT, AND GAS HEADSPACE CONTENT MUST BE PRESERVED
For this product quality category, leak test options include those that directly check for package headspace pressure and/or
content, such as laser-based gas headspace analysis techniques. The detection limit for such methods is a function of the
method’s ability to accurately measure package headspace content or absolute pressure at the product acceptance limit, given
the package headspace volume and the time lapse after package assembly. Such methods have broad application throughout
the product life cycle.

3.4.3 STERILITY MUST BE PRESERVED; PRODUCT ACCESS IS REQUIRED

The product quality subcategory relates to the additional integrity requirement for those products that are contained in
multiple-dose packages once the product is accessed by the end user. As discussed in Package Integrity Evaluation—Sterile
Products á1207ñ, Product–Package Quality Requirements and the Maximum Allowable Leakage Limit, Sterility Must Be Preserved;
Product Access is Required, physicochemical as well as microbiological test methods designed to characterize and verify the
unique barrier properties specific for the given container–closure system and its end-use application may be required (2).
Elastomeric closures of multiple-dose parenteral products are meant to reseal, limiting microbial ingress and product leakage
during and between product access via needle puncture. The test in ▲Elastomeric Closure Functionality in Pharmaceutical
Packaging/Delivery Systems á382ñ, Functionality Tests, Self-Sealing Capacity▲ (CN 1-Dec-2020) is a blue dye liquid tracer test intended
to screen closures for their ability to prevent gross liquid leakage post puncture. Caution is advised when solely relying on this
test to prove a given closure’s ability to reseal. Other test method(s) may need to be designed and implemented that more
fully characterize the closure’s leak prevention capabilities for the given product–package system and product end-use
application.

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3.5 Deterministic or Probabilistic Methods
A “deterministic leak test method” is one in which the leakage event is based on phenomena that follow a predictable chain
of events, and leakage is measured using physicochemical technologies that are readily controlled and monitored, yielding
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objective quantitative data. Most deterministic leak test methods rely on the predictable movement of gas that inevitably occurs
through an open leak path, given specific differential pressure and/or partial pressure test conditions (e.g., tracer gas, laser-based
gas headspace analysis, pressure decay, vacuum decay, and mass extraction). The electrical conductivity and capacitance test
is a deterministic leak test approach that relies on the more predictable presence of liquid near a leak path, rather than the less
predictable movement of liquid through a leak. Deterministic methods are characterized as being capable of reproducibly
detecting leaks at clearly defined and predictable detection limits. Because the majority of deterministic leak test methods
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described in this chapter require no special test sample preparation, sample preparation error is eliminated.
A “probabilistic leak test method” is stochastic in nature in that it relies on a series of sequential and/or simultaneous events
each associated with uncertainties, yielding random outcomes described by probability distributions. Thus, the findings are
associated with uncertainties that necessitate larger sample sizes and rigorous test condition controls to obtain meaningful
results. Typically, sample size and test condition rigor are inversely related to leak size. Therefore probabilistic leak test methods
are more challenging to design, develop, validate, and implement, especially when used to find leaks near the upper and lower
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limits of the test method’s detection range. Probabilistic methods include microbial challenge tests, as well as some
physicochemical tests. These methods include bubble emission tests, tracer liquid tests (employing either qualitative or
quantitative measurement methods), and tracer gas tests by the sniffer probe approach.
A deterministic leak test method having the ability to detect leaks at the product’s maximum allowable leakage limit is
preferred when establishing the inherent integrity of a container–closure system. Deterministic methods may also be chosen if
test sample quantities are limited, when checking for rarely occurring leaks of concern and/or when the potential risk for failing
to find leaks of a given size or type is too great. Probabilistic methods are best chosen when the method outcome requirements
demand a specific probabilistic testing approach. Probabilistic tests are further discussed in Method Outcome and Quantitative
or Qualitative Methods of Analysis below.

3.6 Physicochemical or Microbiological Methods

“Physicochemical leak test methods” are those that use physical and/or chemical analysis techniques to test for package
integrity. Physicochemical methods include both deterministic and probabilistic leak test methods. “Microbiological leak test
methods” are probabilistic methods of analysis that use viable microorganisms to evaluate test sample integrity. This chapter
includes the microbial challenge test by immersion exposure.
The following section includes additional information on the test method outcome criterion that is related to the
microbiological challenge test method.

3.7 Method Outcome

The outcome(s) sought from leak test results will often drive the test method choice. These desired outcomes may include:
• Leak path presence of detection
• Leak path location determination
• Leakage rate measurement
• Liquid egress/ingress potential
• Microbial ingress potential

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All leak test methods are meant to identify the leak path presence. Often, leak tests are able to provide additional outcome
information. Methods that also provide evidence of leak path location include electrical conductivity and capacitance tests,
bubble emission tests, tracer gas sniffer probe tests, and some tracer liquid tests. Methods that provide a measure of
whole-package leakage rate include laser-based gas headspace analysis, mass extraction, pressure and vacuum decay methods,
and tracer gas tests by the vacuum mode.
Microbial challenge tests provide information on the degree of protection afforded by the product–package against microbial
ingress that occurs via active growth or motility through leak pathways and/or by liquid carrier passive transport through leak
pathways. Microbiological challenge tests help to clarify the risks to product sterility posed by specific package materials,
package designs, or potential package barrier breaches. Sterility risks linked to particular environmental exposure or product
use conditions may also demand a microbiological challenge methodology. In summary, all leak test methods at minimum
detect leaks; some methods may provide more information, but no single method alone can yield all four outcomes listed
above.

3.8 Quantitative or Qualitative Methods of Analysis

The measure of analysis can play a part in leak test method selection. In other words, does the method yield quantitative
data that allow for objective analysis, or are the data strictly qualitative and require more subjective interpretation? Leak test
methods that use a “quantitative measure of analysis” include electrical conductivity and capacitance tests, laser-based gas
headspace analysis, mass extraction, pressure and vacuum decay, tracer gas tests (especially when testing via the vacuum
mode), and tracer liquid tests that use quantitative analysis (e.g., spectrophotometric analysis).
Conversely, a “qualitative measure of analysis” is based on subjective observation of a specific quality, attribute, or
characteristic of the test sample, e.g., a visual check for turbidity when evaluating microbial challenge test samples. Bubble

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emission tests that report visible evidence of continuous bubbling, and tracer liquid tests that rely on visible evidence of dye
migration are other examples of subjective and qualitative analysis. Because qualitative measurement results are subject to
interpretation, they may be prone to human error. When method considerations permit, leak test methods that yield
quantitative measurements are preferred.
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3.9 Leak Test Detection Limit
The detection limit of a leak test is the smallest leakage rate or leak size that the method can reliably detect, given the product–
package of interest. A large variety of measurement units are used to describe leakage rates and leak sizes when specifying the
detection limits (and detection ranges) of leak test methods. This often leads to confusion when comparing the performance
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claims of various instrument manufacturers or examining test results generated by multiple methods.
To address this, Table 1 presents the relationship between orifice size (assuming a perfect hole of negligible length) and the
rate at which dry air would pass through such a hole when exposed to 1 atmosphere (atm) differential pressure at a specified
temperature.
These leakage rates and leak sizes are theoretical approximations and are not definitive.
Table 1 services two purposes. First, it is meant to help the reader better grasp the relationship between theoretical hole
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diameter and the gas leakage rate. Second, it provides a common measurement scale that can be referred to later in this chapter
to more simply state leak detection limits for the various technologies described. For example, test technology X found in
published studies to detect leaks as small as about 8 µm would be referred to in á1207.2ñ as having an approximate leak detection
limit of row 4 in Table 1.
Leak detection limit should not be the only or perhaps even the primary basis for choosing a test method. Often, the best
method for a given application is dictated by other factors. For example, a tracer gas leak test method having an extremely
small leakage rate detection limit may be the proper choice for establishing the inherent package integrity of a stoppered glass
vial as a function of capping machine parameters during package development. Yet this method would be an inappropriate
choice for rapid on-line testing in routine manufacturing. Instead, an electrical conductivity and capacitance test with a larger
leak detection limit, able to test product-filled packages at on-line speeds, may be the best option. Refer to Detection Limit for a
discussion on how to determine the limit of detection.

Table 1. Gaseous Leakage Rate versus Orifice Leak Sizea

Row Air Leakage Rateb (std · cm3/s) Orifice Leak Sizec (µm)
1 <1.4 × 10−6 <0.1

2 1.4 × 10 to 1.4 × 10
−6 −4
0.1 to 1.0

3 >1.4 × 10−4 to 3.6 × 10−3 >1.0 to 5.0

4 >3.6 × 10−3 to 1.4 × 10−2 >5.0 to 10.0

5 >1.4 × 10−2 to 0.36 >10.0 to 50.0

6 >0.36 >50.0

a This table is not intended for ranking test methods but is offered as an aid for expressing test method leak detection capabilities in this chapter.
b Dry air leakage rate measured at 1 atm differential pressure across an orifice leak (i.e., leak inlet pressure of 1 atm versus outlet pressure of approximately 1 Torr)
at 25°. The theoretical correlations of orifice sizes to air leakage rates were provided by Lenox Laser, Glen Arm, MD. Leakage rates are approximation ranges.
c Nominal diameter orifice sizes assume a leak path of negligible length. Orifice sizes are approximation ranges.

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3.10 Leak Test Method Range

Leak test method range is the interval between the smallest and largest leak size (or leakage rate) that can be detected by a
given leak test method with a suitable level of accuracy and precision. All leak test methods have an optimum detection range.
Therefore, it is possible that additional tests may be needed to catch those leaks of concern that fall outside the chosen leak
test method’s detection range. For example, a test method able to find the smallest leaks may miss gross leakage such as a
missing package component. Vision systems that check for major package defects such as a missing package component or a
package crack may be required. Finally, before using a test method to check for large leaks, the impact of gross defects on leak
test instrumentation should be considered; some instruments may malfunction or become damaged upon exposure to a leaking
product or damaged packages. Refer to Range for additional detection range information.

3.11 Nondestructive or Destructive Methods

The need to preserve the test product–packages may influence the decision to select a nondestructive leak test method,
rather than a destructive one. Destructive test methods damage the test sample and/or expose it to potential contaminants;
hence, the product is not recoverable. Only nondestructive test methods are appropriate for leak testing product–package units
earmarked for commercial or clinical study distribution.
Examples of destructive leak test methods include tracer liquid ingress tests, bubble emission tests, and microbiological
challenge tests. Tracer gas and pressure decay leak tests are considered destructive if they require that the package barrier be
compromised in order to introduce tracer or pressurizing gas into the assembled package. Examples of nondestructive leak test
methods include mass extraction and vacuum decay leak tests, as well as noninvasive gas headspace analysis tests. Electrical
conductivity and capacitance leak tests are deemed nondestructive if it can be shown that electrical current exposure causes

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no harm to the product; in rare instances, exposure has triggered headspace ozone formation, causing product oxidation (3).

3.12 Off-Line or On-Line Methods

Off-line leak testing is generally performed on a stratified random sampling of the product lot, away from the manufacturing
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line. Off-line package evaluation allows for the use of any validated nondestructive or destructive leak test and seal quality test
option compatible with the product–package. Off-line testing can accommodate slower test cycle times; for methods in which
test time is a performance factor, a slower off-line test is often more sensitive than its on-line counterpart. Off-line tests typically
are less costly to perform as they utilize bench-top or smaller scale equipment without the sophisticated product-handling
machinery required to support higher speed on-line processes.
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On-line leak testing is commonly integrated into a continuous fill and seal product–package manufacturing process. A
prerequisite for an on-line leak test method for entire lot testing is that it be nondestructive to the package and its contents.
On-line testing can potentially provide greater assurance that all packages have integrity and can yield instant feedback in the
event of package misassembly or breakage, enabling real-time line corrections. For some test systems, incorporating large-scale
leak detection equipment into a complex high-speed product–package filling and assembly line can be prohibitive. Higher line
speeds leading to shorter leak detection test cycle times can limit test method detection capability. In addition, the impact of
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instrument downtime on the production run as a result of leaking packages or possible equipment malfunction is an important
consideration. A separate leak testing line may be set up outside the sterile manufacturing suite to allow for full lot testing
without the complications of leak test integration with package filling/sealing operations. A few examples of on-line leak test
technologies include electrical conductivity and capacitance, vacuum decay leak, and noninvasive laser-based gas headspace
analysis tests.

4. TEST INSTRUMENT QUALIFICATION, METHOD DEVELOPMENT, AND METHOD VALIDATION

Leak test methods are validated in order to demonstrate method effectiveness. Method validation is preceded by instrument/
equipment qualification, followed by test method development. The following discussion specific to leak test methods is
intended to supplement the guidance for analytical instrument qualification presented in Analytical Instrument Qualification
á1058ñ, plus the guidance for method validation provided in Validation of Compendial Procedures á1225ñ.

4.1 Instrumentation and Equipment Qualification

The qualification of instruments or equipment to be used for leak testing includes: 1) evaluation of instrument/equipment
functionality, and 2) determination of test system detection capabilities using appropriate calibration tools or reference
standards to simulate with-leak test conditions.

4.2 Method Development and Validation

After successful instrument/equipment qualification, leak test method parameters are developed and optimized to ensure a
leak test method is able to meet all relevant leak detection performance criteria specific for the test product–package system.
The following properties of a valid test method are defined as they relate to package integrity tests.

4.2.1 ACCURACY
“Accuracy” is a measure of the method’s ability to correctly differentiate packages that leak above the claimed detection
limit from those that leak below this limit (i.e., do not leak). Accuracy provides a measure of false positive and false negative

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occurrence. Alternatively, for those methods that deliver an outcome that is a direct quantitative measure of gas leakage rate
(or of gas concentration or gas pressure), accuracy is a measure of the method’s ability to produce an outcome comparable
to a true standard. For example, helium mass spectrometry provides a direct measure of helium leakage rate. Accuracy is the
closeness of the instrument reading to the certified leakage rate of a nationally recognized traceable standard.

4.2.2 PRECISION
“Precision” is the ability of the method to yield reliable, repeatable data. Precision includes repeatability (e.g., repeat testing
of a homogeneous test sample population), ruggedness (within laboratory tests performed, for example, by multiple operators
on multiple days, using multiple instruments; also known as intermediate precision), and reproducibility (among laboratories
tests). The level of precision to which a leak test method is validated is often a function of resource availability (e.g., one
instrument versus multiple instruments) and intended test method application (e.g., use of the method at one test site only
versus across multiple test sites).

4.2.3 SPECIFICITY
“Specificity” is the ability of the method to accurately differentiate between leaking and nonleaking packages, despite
interfering factors that may cause false detection. For example, when employing tracer gas leak detection using helium mass
spectrometry (vacuum mode), excessive helium permeation through the package wall may mask small package leaks or may
be falsely interpreted as leakage in no-defect packages.

4.2.4 DETECTION LIMIT

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Refer to Leak Test Detection Limit for an introduction to this topic. The detection limit of a leak test is specific for a given
testing approach when performed using a specific instrument make/model in evaluating a given product–package system.
Utilizing the principles described in á1225ñ, detection limit is demonstrated by challenging packages with and without known
defects by the leak test method for multiple test days by multiple operators. The intended application of the method will dictate
the level of precision required (i.e., whether to incorporate multiple operators/instruments/laboratories, etc.).
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A common challenge with small leak detection is the potential for interfering factors to be misconstrued as leakage presence.
For example, vacuum decay leak tests measure the rise in pressure inside an evacuated chamber containing the test package.
Package leakage causes chamber pressure to rise, but package material volatiles, test system moisture, and package expansion
may also do the same. Gas permeating through a package wall detected by a tracer gas test may be mistaken for leakage.
Bubbles emitted during a bubble test may actually be the result of package surface outgassing, volatilization of dissolved gases
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in the immersion fluid, or the release of trapped air between package components.
The absence of a leak detection signal may also be misinterpreted as the absence of leaks. For example, tracer liquid tests
may fail to reliably detect small leaks due to any one of a number of factors including air locks, product, or debris in the leak
path; liquid surface tension; leak path geometry; or insufficient differential pressure test conditions. The same is true for microbial
ingress tests that are further subject to the inherent variability of living microorganisms.
In short, false negative results that miss leaks and false positive results that incorrectly suggest leak presence are possible with
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any leak test method. Therefore, test method detection limit is determined by comparing readings of intentionally defective
packages to nondefective ones. Leak detection limit determination test units consist of a randomly ordered population mix of
negative and positive control units (refer to Negative and Positive Controls). Nondestructive test methods can employ the same
set of units for multiple test exposures, while destructive methods will require a new set for each test series. Control subset unit
quantities are chosen based on several factors: 1) the deterministic or probabilistic nature of the outcome, 2) the inherent
package-to-package variability that may influence test results, and 3) the statistical confidence level required by the test
acceptance criterion. The positive control subset includes units with defects sized to the anticipated detection limit, in addition
to units with leaks bracketing this size limit. If the detection range is to be established, controls having large defects are included.
Positive controls representing a wide defect size range are especially important for probabilistic methods in order to clearly
understand leak detection likelihood as a function of leak size.
Because of the many product–packages and leak-testing options available, the resultant leak detection limit is more
meaningfully stated when the negative and positive control subsets used, the test precision level, and the test results are
summarized. The following is an example of expressing a test method’s limit of detection:
“The detection limit for method X was determined to be 5 ± 2 µm. Validation studies found defects of this nominal size were
detected 95% of the time; all larger defects were detected 100% of the time. Studies included three replicate test series
performed on multiple days by multiple operators in a single laboratory using one instrument. Detection limit was determined
using product-filled packages. Test units in each series included a negative control subset of 300 units (each without defect)
and a positive control subset of 90 units (each having a laser-drilled defect ranging in nominal size from 1.5 ± 0.6 µm to 15 ± 3
µm). Each defect was independently size-certified by comparing the dry air leakage rate at 1 atm differential pressure (leak inlet
pressure of 1 atm versus outlet pressure of approximately 1 Torr) at 25° to that of standard orifice leaks.”
[NOTE—This is one example of how test method detection limit could be expressed and is not to be considered compulsory
or restrictive either in content or level of detail.]
Leak test technologies exist that are able to detect leaks even smaller than can be artificially created in a positive control test
set. Two examples follow. In both cases, a limited number of positive controls can serve to verify that the instrument set-up is
able to detect leaks of specific type and in specific package locations but not to determine leak size detection limit capability.
• Tracer gas tests by helium mass spectrometry performed in the vacuum mode can detect leaks as small as about 10−11
mbar · L/s, which is about 5 logarithmic leakage units smaller than leakage through a hole 0.2 µm in size. For such methods,
leakage rate detection ability can only be verified by using nationally recognized gas leakage rate standards to introduce
leaks into the test system. Because gas permeation can be mistaken for leaks, it is important to experimentally establish

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the relationship between tracer gas leakage rate and permeation rate as a function of test cycle time for a given package
system.
• Laser-based gas headspace analysis is another approach that may be able to identify the presence of leaks smaller than
can be artificially created. For such methods, the limit of detection can be mathematically predicted on the basis of gas
flow kinetics and is a function of the time lapse between analyses, and the smallest gas content or pressure change that
can be reliably detected by the instrument for the given package system.

4.2.5 QUANTITATION LIMIT

“Quantitation limit” is that lowest leakage rate or leak size that can be determined with acceptable accuracy and precision
under the stated experimental conditions. Laser-based gas headspace analysis is a method that may be evaluated for
quantitation limit. For most leak test methods, detection limit is more meaningful.

4.2.6 LINEARITY
“Linearity” is the ability of the method to elicit test results that are mathematically proportional to leak path size or leakage
rate. Deterministic leak test methods that exhibit linearity include laser-based gas headspace analysis and tracer gas analysis
(vacuum mode). Other methods such as vacuum decay, pressure decay, and mass extraction also produce results that correlate
to leak size or leakage rate. However, test findings are generally intended to identify leak presence and perhaps to understand
relative leak size; they are not typically relied upon for leak size or leakage rate quantitation. Electrical conductivity and
capacitance tests and all referenced probabilistic methods are not validated for linearity.

4.2.7 RANGE

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Leak detection range is defined in Leak Test Method Range. Leak test method range is explored in test method development
to better understand leak test method detection limitations. For methods being relied upon to detect leaks within a specified
size range, detection at the upper range limit may be confirmed in validation. Range is evaluated by using sets of negative
controls and appropriately sized larger-defect positive controls. The large-defect positive control subset may include defects of
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various types likely to occur for the given product-package system (refer to Type Defects).

4.2.8 ROBUSTNESS
“Robustness” is the method’s ability to accurately identify leaking versus nonleaking packages despite small but deliberate
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variations in procedural parameters, providing an indication of the method’s suitability during normal usage. One way to
evaluate robustness is to perform the test using test parameters bracketing optimal or normal test specifications. Parameters to
be varied are those having the greatest impact on test results; variation should reflect instrument performance accuracy. For
example, the robustness of a vacuum decay leak test with a test cycle time of 30 s (accurate to within 0.5 s) might be
demonstrated during method development by verifying method performance at set test cycle times of 29.5 and 30.5 s.
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4.3 System Suitability

One outcome of test method development and validation is the establishment of a system suitability test(s), also called a
performance verification test(s). “System suitability” is a manner of ensuring that the leak test method, including all factors that
may be subject to variability that may impact test results (e.g., instrumentation, analysts, test sample preparation, and the test
environment), is adequately controlled and maintained in such fashion that the method is rugged and robust. System suitability
is important for all leak test methods. Extra care may be required for probabilistic methods more prone to variability.
For example, a highly instrumental method such as a mass extraction test may be checked for system suitability by leak
testing a master package unit both with and without the added challenge of external air introduced to the test system via a
calibrated leak adjusted to the method’s limit of detection. If the method requires preliminary test package preparation (e.g., a
drying step), then it would be appropriate to demonstrate that an intact package prepared in the prescribed manner elicits the
anticipated mass flow rate response. System suitability can be performed at the beginning and end of each testing sequence
for added method assurance.
System suitability for a test more probabilistic in nature takes into account multiple test method variables and may require
greater numbers of challenge samples (i.e., positive and negative controls) for adequate method assurance. For example, tracer
gas tests via a sniffer probe may require a routine demonstration that the operator is able to successfully differentiate packages
without defect from those with leaks (ranging in size from smallest to largest, located at various package positions) in a blinded
challenge study. Alternatively, the operator could be challenged by randomly introducing defective samples (unknown to the
inspector) among the test sample population during routine test procedures.

4.4 Microbial Ingress Risk Comparison

Appropriate leak test method validation practices mirror compendial analytical test validation guidelines. In the past,
validation of physicochemical leak test methods used for pharmaceutical sterile product packages also routinely included a
direct or indirect comparison of physicochemical leak test results to microbiological challenge data. While an understanding of
the relationship between physicochemical leak test method capabilities and microbial ingress risk may be important, an
experimental comparison may not be needed or useful in all cases. [NOTE—The reader may refer to Package Integrity Evaluation
—Sterile Products á1207ñ, Product–Package Quality Requirements and the Maximum Allowable Leakage Limit for a discussion of
maximum allowable leakage limit and inherent package integrity.]

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The following situations illustrate when a comparison study of the microbial ingress or liquid leakage risk to physicochemical
leak test method capability relationship is likely not needed. [NOTE—This is not intended to be a complete or exhaustive listing
of all situations for which comparison studies would not be required or useful but is provided for illustration only.]
• If the validated physicochemical leak test method has a proven detection limit at or below the product-package maximum
allowable leakage limit.
• If the validated physicochemical leak test method is not being used to verify the absence of all leaks of concern. Instead,
the method is being used to find leaks notably larger than the maximum allowable leakage limit. For example, a rapid
on-line test shown to reliably detect leaks of 25–150 µm in nominal diameter is being used to reject damaged or
misassembled product-filled packages within the method’s leak detection range.
The following situations illustrate when an experimental indirect or direct comparison of the microbial ingress risk (or liquid
leakage risk) to physicochemical leak test method capability relationship may be useful. [NOTE—This is not intended to be a
complete or exhaustive listing of all situations for which comparison studies could prove beneficial but is provided for illustration
only. These examples are not compulsory but are provided for instruction only.]
• If the validated physicochemical leak test method is being relied upon to measure or confirm the inherent package integrity
of a product–package system, but the method’s limit of detection is notably greater than the maximum allowable leakage
limit. For example, a package has a maximum allowable leakage limit of less than 6 × 10−6 mbar · L/s as measured by
helium mass spectrometry (equivalent to holes less than approximately 0.2 ± 0.1 µm in nominal diameter). This limit was
chosen based on published literature references. But the leak test method of choice for testing product-filled packages
placed on stability is able to detect leaks equivalent to holes 3 µm in nominal diameter and larger. A study correlating
microbial ingress or liquid leakage risk to defect type/size can provide a measure of the likelihood of microbial ingress
(and/or liquid leakage) at the leak test method detection limit, and thus can provide an understanding of ability of the
stability leak test to identify leaks of concern.

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• If the validated physicochemical leak test method is being used to measure or verify the inherent package integrity of a
product–package system, but the maximum allowable leakage limit is either lacking or not well defined. For example, a
unique package is being used for which the maximum allowable leakage limit that will ensure absence of product loss or
microbial ingress has not been determined. A study comparing the risk of microbial ingress or liquid leakage to leak type/
size, and in turn to the likelihood of detection by the physicochemical leak test method may be useful.
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When performing microbial ingress risk assessment studies it is important to keep in mind the probabilistic nature of microbial
ingress. To achieve the most meaningful data, large population sets of negative and positive controls should be used. Test
protocols should be thorough and well designed, taking into consideration the multiple factors and variables that can influence
results. As suggested above, liquid leakage risk assessment studies may substitute for microbial ingress risk studies assuming the
risk of liquid leakage is equivalent to or greater than that of microbial ingress, with appropriate justification.
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4.5 Negative and Positive Controls
“Negative controls” are packages with no known leak, and “positive controls” are packages with intentional or known leaks.
Negative and positive controls are designed and assembled for use in method development and validation with consideration
given to container–closure design, materials of construction, characteristics of anticipated package leaks, and impact of product
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contents on test results. Negative and positive controls should represent packages assembled in a typical manner as the product
being tested using normally processed components, the exception being the intentionally created leak in each unit of the
positive control subset. Some leak test methods may necessitate positive controls that simulate test product headspace and
formulation contents as well. System suitability checks for some test methods employ negative and positive controls (refer to
System Suitability).
Test blanks should not be confused with negative controls. For example, liquid tracer leak detection by spectrophotometric
analysis may require a blank solution without the liquid tracer element to confirm instrument baseline performance.
A “master” is a type of negative control test unit. It is a package prototype, model, or facsimile made to simulate the test
package in shape and design. Masters may be made of solid material such as plastic or metal, or they may be simply a designated
container–closure unit. Masters are no-leaking mock packages often used in system suitability verification tests for leak tests to
verify instrument performance, such as for vacuum decay or mass extraction testing.

4.5.1 DEFECT CREATION METHODS

The positive control set typically represents a range of package defect sizes and types. Numerous approaches have been used
to create package defects. When creating positive controls, a fundamental awareness of leakage dynamics as a function of
different defect types and materials of construction is important.
Placing a so-called “hole” or break in the package wall is one positive control creation approach. In this case the defect
materials of construction are identical to the package itself, thus potential test method interference due to product exposure
to the package material can be readily identified. An example of such interference is the clogging of small leak paths by product
formulation observed during vacuum decay, mass extraction, or tracer gas (vacuum mode) tests.
Laser drilling is often used to create package defects commonly referred to as holes. However, these so-called holes are not
pristine orifices but are non-cylindrical and asymmetric and may consist of a tortuous matrix of micro-cracks. While laser-drilled
defects are not dimensionally ideal, they offer the advantages of closely simulating actual package defects (e.g., cracks) and do
not require the introduction of foreign materials, such as wires, tubes, or epoxies, that may influence leakage dynamics.
Laser-drilled defects are often sized for nominal diameter by comparing dry air leakage rate through the defect at specified
conditions of pressure and temperature to flow rates through standard orifice leaks in thin metal plates. Currently, laser-drilled
defects in rigid glass or plastic components can be made as small as about 2–3 µm in nominal diameter and to about 5–10 µm
in nominal diameter in flexible thicker wall package materials. Smaller diameter defects tend to clog from handling,
environmental debris, or flexible package wall deflection.

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Glass micropipettes can be used to simulate single-orifice defects as small as about 0.1 µm in diameter. The tip diameter can
be nominally sized using air flow measurements. When creating positive controls, micropipettes are inserted through a break
in the package wall, then an appropriate sealant is applied to the insertion site. Challenges to micropipette use include ensuring a
complete seal between the micropipette perimeter and the package wall and avoiding micropipette tip damage.
In addition, air trapped in the pipette tube can interfere with leak test methods that depend on fluid flow through the leak
path, such as tracer liquid tests and microbial challenge ingress tests.
Microtubes (also called microcapillaries) inserted through the package wall and fixed in place with sealant are another means
of creating positive control defects. Microtubes can be made of a variety of materials, can be cut to any length and can be as
narrow as 2 µm in cross-sectional diameter. Microtubes are often employed as a substitute for a smaller-bore, shorter-length
leak path when performing leak tests that rely on gas flow measurements. However, caution is advised before choosing lengthy,
larger-bore microtubes to simulate an orifice leak of smaller diameter for leak tests that rely on the passage of liquids or
microorganisms. Fluid dynamic theory correlating fluid flow through capillary tubes to pass through smaller bore holes is based
on the unimpeded passage of ideal liquids through capillaries at equilibrium pressure conditions. Liquid product formulation
and aqueous media flow into and through a microtube is complicated by numerous factors including liquid surface tension,
liquid viscosity, surface contact angle, airlocks, particulate blockage, and tube-wall and tube-end finishes. Microbial ingress
through microtube defects relies more on the presence of liquid in the tube than on the physical barrier to passage or
grow-through afforded by the tube diameter (4). Microtubes are a logical choice when creating defects representing channel
defects. In this case microtube length should mimic as closely as possible the actual package barrier thickness (package wall or
seal width). Microtube use challenges include effecting smooth, perpendicular cuts of microtube ends and adequately sealing
microtubes into the test sample wall.
Other commonly used defect creation methods include inserting a needle through the package wall; placing a wire,
microfilament, or film between sealing surfaces; and adhering a holed, thin metal plate onto package surfaces. It is important
to note that defects made by using an object foreign to the package (e.g., needle, film, wire plate) may display gas, liquid, or

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microbial leakage dynamics markedly different from that of actual defects (5). Positive controls made by such means are easy
and inexpensive approaches for creating larger size defects useful for test method feasibility studies and for exploring a test
method’s detection range upper limit.

4.5.2 TYPE DEFECTS

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“Type defects” are positive controls representing realistic package flaws. Type defects are important to include in method
validation studies to explore a leak test method’s practical application in detecting realistic package failures and flaws. A few
types of defect examples are listed below:
• Heat sealed bag: 1) weak seal, 2) wrinkled seal, 3) seal gap, 4) seal channel, 5) product entrapment in seal
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• Stoppered vial package: 1) vial finish channel defect, 2) loosely capped stopper, 3) product trapped between stopper
and vial
• Prefilled syringe: 1) needle shield punctured by staked needle, 2) defective plunger
• Ophthalmic dropper bottle: 1) loose cap, 2) missing or poorly inserted dropper tip, 3) defective tip or cap
Assigning precise sizes to type defects is generally not meaningful or feasible because of their inherent irregularity and
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complexity. Type defects are defined in more qualitative, descriptive terms such as those listed above. Because no leak test
method can find all possible defects in a given container–closure system, information collected from type defect tests can be
used to identify alternative approaches to detecting or limiting the occurrence of critical imperfections not readily found by the
chosen leak test method.

REFERENCES

1. Department of Health and Human Services. Container and closure system integrity testing in lieu of sterility testing as a
component of the stability protocol for sterile products. Guidance for industry. Rockville, MD: U.S. Food and Drug
Administration, Center for Biologics Evaluation and Research (CBER), Center for Drug Evaluation and Research (CDER),
Center for Devices and Radiological Health (CDRH), Center for Veterinary Medicine (CVM); 2008.
2. Bagel S, Wiedemann B. Extension of in-use stability of preservative-free nasalia. Eur J Pharm Biopharm. 2004;57(2):353–
358.
3. Bigwarfe P Jr, Domin-Turza A, Hwang E, Leidner S, McGinley C, Olson K, Nachtigall M. Presentation. Annual Meeting of
the American Association of Pharmaceutical Scientists 2008. Elucidation of an unusual mechanism of drug product
degradation caused by a leak detection instrument. Lake Forest, IL: Hospira; 2008.
4. Keller S, Marcy J, Blakistone B, Hackney C, Carter H, Lacy G. Application of fluid and statistical modeling to establish the
leak size critical to package sterility. J Appl Pack Res. 2006;1:11–21.
5. Morrical BD, Goverde M, Grausse J, Gerwig T, Vorgrimler L, Morgen R, Büttiker J-P. Leak testing in parenteral packaging:
establishment of direct correlation between helium leak rate measurements and microbial ingress for two different leak
types. PDA J Pharm Sci Technol. 2007;61(4):226–236.

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á1207.3ñ PACKAGE SEAL QUALITY TEST TECHNOLOGIES

1. INTRODUCTION
2. CLOSURE APPLICATION AND REMOVAL TORQUE
3. PACKAGE BURST
4. PACKAGE SEAL STRENGTH
5. RESIDUAL SEAL FORCE
6. AIRBORNE ULTRASOUND

1. INTRODUCTION

The purpose of this chapter is to briefly summarize test methods useful for characterizing and monitoring package seal quality
and to guide the reader in their selection and use. These methods are not leak tests but provide additional data regarding
package seal characteristics that may affect package integrity and leakage.
“Package seal quality tests” are checks used to characterize and monitor the quality and consistency of a parameter related
to the package seal, providing some assurance of the package’s ability to maintain integrity. Seal quality tests ensure that seal
attributes, package materials, package components, and/or the assembly process are consistently kept within established limits,
thus further supporting package integrity. Seal quality tests differ from leak tests in that they provide no information relative to
actual package integrity; thus, a package that meets the requirements of a seal quality test may still be defective and leak. For

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example, a flexible pouch package that passes a seal strength test may leak through a puncture in the pouch face. A bottle that
meets closure application and removal torque tests may have a scratch on the bottle finish surface that allows product leakage.
In contrast, a pouch or bottle that is poorly assembled could pass leak tests at the time of product manufacture, yet develop
leaks later, before reaching the end user.
Therefore, seal quality tests and leak tests work together to ensure package integrity. The package seal quality tests described
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in this chapter were selected for inclusion on the basis of data in peer-reviewed scientific publications and/or data regarding
recognized standard tests (e.g., precision and bias study results). Standard test methods (e.g., ASTM) are referenced where
applicable (the reader is advised to utilize the most recent versions). In some cases, the scope of referenced standard test
methods does not include the package types of the scope in Package Integrity Evaluation—Sterile Products á1207ñ. All methods
and literature references are cited to provide benchmark information useful for the application and use of pharmaceutical
package seal quality test methods.
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Unlike package leak tests, seal quality tests are qualified for use rather than being fully validated. Qualification includes a
demonstration of instrument performance and in some cases proof of appropriate instrument set-up specific for the package
to be tested.
Finally, this chapter is not intended to provide an exhaustive listing of all seal quality technologies that could be used. Nor
is the use of a methodology cited meant to be compulsory. Other qualified tests that are not included in this chapter may be
used, as appropriate.
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2. CLOSURE APPLICATION AND REMOVAL TORQUE

The closure application torque test measures the force exerted during the application of a screw-thread cap onto a threaded
container. Conversely, the closure removal torque test measures the force required to initiate screw-cap removal. The container–
closure systems of some ophthalmic solution products are examples of sterile product packaging closed with screw-thread caps.
Cap application torque is kept within an optimum range to prevent leakage from loose caps and to preclude component
distortion and compromised seals from over-torqued caps. Cap removal torque is less than application torque due to stress
relaxation and closure “back off” that may occur as a function of time and other environmental variables. A properly designed
and applied cap will retain sufficient sealing force until the package is opened at the time of use. Caps that back off excessively
during shipping, storage, or distribution increase the risk of product leakage. For multiple-dose packages, cap design and
application forces should be such that the end-user population may be able to open and reclose the package in a manner that
properly preserves the pharmaceutical product.
Reproducibility of application/removal torque test results is improved when tests are performed using automatic
instrumentation that applies and removes caps at uniform speeds, with fixtures to lock the bottle and cap into proper test
position. Application and removal torque tests are described in several standard test methods that have been written to support
various continuous-thread and child-resistant cap designs, including the ASTM methods referenced (1–7).

3. PACKAGE BURST

The package burst test is performed by inserting a pressure source into a test package and applying pressure until the package
seal(s) burst open. Packages having seals that could be compromised when exposed to a net positive pressure force inside the
package may be tested by this method. Such package types include flexible bags and pouches formed by heat-sealing processes.
The package burst strength test result (reported in pressure units) provides an indication of relative seal strength and evaluates
the most likely location and mode of package failure when the package is exposed to a pressure differential. However, note
that this test method cannot provide a measure of package seal uniformity or overall package integrity.
Package burst tests are described in ASTM F2054 (8) and ASTM F1140 (9) (Test method A). Method ASTM F2054 uses a
restraining plate to limit package expansion during inflation. A restraining plate ensures that the stress is uniformly applied to

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all seal areas, allowing for identification of the weakest point along the seal. Additional factors can influence the test results,
including package inflation rate, tooling dimensions (including gap height), and pressure-sensing mechanisms.
Method ASTM F1140 uses no restraining plate; therefore, this approach provides a snapshot of package performance when
challenged with differential pressures in a typical sterilization cycle or distribution environment, for example. However, without
restraining plates, stress applied to the package is highest at the middle of the package where the package inflates to the greatest
diameter; therefore, the weakest area of the seal may not be identified by this approach. Package inflation rate, as well as
pressure-sensing mechanisms and their detection limits, can influence test results.

4. PACKAGE SEAL STRENGTH

The package seal strength test, commonly known as the peel test, measures the force required to peel apart two bonded
surfaces. This test is applicable for testing seals between two bonded, flexible surfaces (e.g., a pouch or bag) or between a
flexible material and a rigid material (e.g., a lidded tray). Force results provide a measure of seal strength between the bonded
surfaces. Maximum seal force, as well as average force to open the seal, may be determined with this method. The results are
also useful for monitoring the consistency of package assembly.
The test is performed using a universal stress-strain instrument set to extension force mode, with special tooling for correctly
positioning and holding the test sample. The seal strength test is described in ASTM F88 (10). Test results are affected by the
peel test fixture design, the peel angle, the pull direction, pull speed, and properties of the test sample itself.

5. RESIDUAL SEAL FORCE

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The residual seal force (RSF) test provides an indirect measure of the compressive force exerted by an elastomeric closure
onto a parenteral vial finish after package assembly (capping). RSF tests can be performed on glass or plastic vial packages of
all dimensions. A consistent and sufficiently significant RSF value provides a useful indicator of capping process consistency. RSF
is linearly related to closure compression: more tightly capped vials yield higher RSF values.
The RSF test is performed using a universal stress-strain instrument, set to compression force mode, and a metal tool (called a
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cap anvil) designed to fit on top of the sealed vial package. A constant rate of compressive strain is exerted onto the anvil, which
rests on top of the vial package, yielding a typical plot of stress as a function of time (or compressive distance). The point in the
stress-response curve immediately before the terminal slope change corresponds to the capped closure’s RSF, reported in either
newtons or pound-force units. RSF tests are nondestructive in terms of package integrity. Test results are influenced by cap anvil
tooling design, test compression rate, closure viscoelastic properties, closure dimensions, and the inclusion of an aluminum seal
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plastic top (more reproducible results are possible if plastic tops are removed before testing). Research articles describing the
development and application of this method are referenced (11–14).

6. AIRBORNE ULTRASOUND
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The airborne ultrasound test checks seal quality by passing an ultrasound signal through the sealed area of a package or
item. The signal strength transmitted through the package seal under test is compared to that previously established for so-called
good package seals. Poorly sealed areas will not transmit as much ultrasonic energy as properly sealed areas.
The types of package seals that can be tested using airborne ultrasound include flexible pouch seals and rigid tray seals made
of metallic or plastic laminates. This technology can be used to check for the presence and location of package seal defects such
as an incomplete seal, a channel, a wrinkle, or extraneous material inclusion, as well as for package seal bond weakness. The
method may be used off-line to perform an x–y coordinate scan of a seal area, or it may be used to linearly scan seals during
on-line manufacturing processes. Airborne ultrasound is a noncontact testing technology that requires no package preparation
and no coupling media, such as a liquid or gel, to propagate sound; thus, it is nondestructive to the package under test.
The airborne ultrasound method is described in ASTM F3004 (15). The ASTM method test system is composed of a transducer
that provides an ultrasonic signal, a means of holding/transporting the item under test within an air gap between the two
transducers, and the detection transducer, which captures the intensity of the signal that passed through the air gap and the
item under test. The capability of airborne ultrasound to detect specific package seal quality problems will vary on the basis of
package material of construction, seal structure (e.g., smooth versus textured surface), scanning speed, and scanning signal
strength.

REFERENCES

1. ASTM D2063/D2063M. Standard test methods for measurement of torque retention for packages with continuous thread
closures using non-automated (manual) torque testing equipment. West Conshohocken, PA: ASTM International; 2012.
2. ASTM D3198. Standard test method for application and removal torque of threaded or lug-style closures. West
Conshohocken, PA: ASTM International; 2007.
3. ASTM D3469. Standard test methods for measurement of vertical downward forces to disengage type IIA lug-style
child-resistant closures. West Conshohocken, PA: ASTM International; 2007.
4. ASTM D3470. Standard test method for measurement of removal lug strippage of type IIA child-resistant closures. West
Conshohocken, PA: ASTM International; 2007.
5. ASTM D3472. Standard test method for reverse-ratchet torque of type IA child-resistant closures. West Conshohocken,
PA: ASTM International; 2007.

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6. ASTM D3810. Standard test method for minimum application torque of type IA child-resistant closures. West
Conshohocken, PA: ASTM International; 2007.
7. ASTM D3968. Standard test method for monitoring of rotational torque of type IIIA child-resistant closures. West
Conshohocken, PA: ASTM International; 2007.
8. ASTM F2054/F2054M. Standard test method for burst testing of flexible package seals using internal air pressurization
within restraining plates. West Conshohocken, PA: ASTM International; 2014.
9. ASTM F1140/F1140M. Standard test methods for internal pressurization failure resistance of unrestrained packages. West
Conshohocken, PA: ASTM International; 2013.
10. ASTM F88/F88M. Standard test method for seal strength of flexible barrier materials. West Conshohocken, PA: ASTM
International; 2009.
11. Ludwig JD, Nolan PD, Davis CW. Automated method for determining Instron residual seal force of glass vial/rubber
stopper closure systems. PDA J Pharm Sci Technol. 1993;47(5):211–253.
12. Ludwig JD, Davis CW. Automated method for determining Instron residual seal force of glass vial/rubber closure systems.
Part II. 13 mm vials. PDA J Pharm Sci Technol. 1995;49(5):253–256.
13. Morton DK, Lordi NG. Residual seal force measurement of parenteral vials. I. Methodology. J Parenteral Sci Technol.
1988;42(1):23–29.
14. Morton DK, Lordi NG. Residual seal force measurement of parenteral vials. II. Elastomer evaluation. J Parenteral Sci Technol.
1988;42(2):57–61.
15. ASTM F3004. Standard test method for evaluation of seal quality and integrity using airborne ultrasound. West
Conshohocken, PA: ASTM International; 2013.

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á1207.2ñ PACKAGE INTEGRITY LEAK TEST TECHNOLOGIES

1. INTRODUCTION
2. DETERMINISTIC LEAK TEST TECHNOLOGIES
2.1 Electrical Conductivity and Capacitance (High-Voltage Leak Detection)
2.2 Laser-Based Gas Headspace Analysis
2.3 Mass Extraction
2.4 Pressure Decay
2.5 Tracer Gas Detection, Vacuum Mode
2.6 Vacuum Decay
3. PROBABILISTIC LEAK TEST TECHNOLOGIES
3.1 Bubble Emission
3.2 Microbial Challenge, Immersion Exposure
3.3 Tracer Gas Detection, Sniffer Mode
3.4 Tracer Liquid

1. INTRODUCTION

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The purpose of this chapter is to provide information guiding the selection and proper use of leak test technologies (also
called methodologies, approaches, or methods). The leak test technologies described in this chapter were selected on the basis
of relevant research study data published in peer-reviewed journals and/or precision and bias study data generated in support
of recognized test method standards. When referencing standard test methods (e.g., ASTM), the reader is advised to refer to
the most recent versions. In some cases, the scope of referenced standard test methods does not include the package types of
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the scope in Package Integrity Evaluation—Sterile Products á1207ñ. In all cases, methods and literature studies are cited to provide
benchmark information useful for pharmaceutical package leak test method development and validation.
The technologies described in this chapter are not prescriptive methods but represent testing concepts that may be applied
when leak testing sterile product–packages. Test technologies vary in terms of their potential detection limits, reliability, and
applications; therefore, none are universally appropriate for leak testing all product–packages. This chapter provides information
to allow a thorough comparison of testing approaches so that the most appropriate technology for a given situation can be
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identified.
After a methodology has been selected for use, the test equipment operation and performance is qualified. Test method
parameters are optimized during method development and confirmed during validation. Thus, a final leak test method is specific
to a particular container–closure or product–package system.
The leak test methods included are divided into two categories: deterministic and probabilistic. Deterministic leak test
methods (Table 1) are preferred over probabilistic methods when other key method selection criteria permit. Probabilistic leak
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test methods (Table 2) are best used when the product–package system proves incompatible with deterministic methods, or
when method outcome requirements demand a particular probabilistic testing approach.
In this chapter’s Table 1 and Table 2, the “leak size detection limit” provided for each methodology refers to leakage rates/
leak sizes listed by row in Package Integrity Testing in the Product Life Cycle—Test Method Selection and Validation á1207.1ñ, Table
1. The reported leak detection limits were chosen on the basis of literature sources, as well as commonly accepted experience.
This information is intended to aid in the selection of the test technology but should not be used as a definitive statement of
test method performance for any specific leak test method applied to any given product–package system. Instead, leak detection
limit and range should be established during leak test method development and validation for the respective product–package
or container–closure system. For instance, an approach cited as capable of detecting row 6 leaks may be validated by the user
to detect leaks as small as those in row 3. Conversely, a method described as capable of detecting leaks in row 3 may be
determined by the user to detect leaks only as small as row 5.
This battery of testing technologies and the information provided are intended to aid, not limit, the selection, development,
validation, and use of leak test methods. Unlisted methodologies shown by the user to meet the qualification and validation
requirements for a satisfactory leak test may be used. In addition, listed technologies may demonstrate expanded testing
capabilities beyond those currently identified.

Table 1. Deterministic Leak Test Technologiesa

Deterministic Package Package Effect of Test Time
Leak Test Content Require- Leak Detection Lim- Measurement Outcome and Method Order of
Technologies Requirements ments itb Data Analysis on Package Magnitude
Quantitative measure of electrical cur- Nondestruc-
Liquid (with no com- rent passing through the test sample: tive, al-
bustion risk) must be Row 3 provides an indirect determination of though im-
more electrically con- Varies with product– leak presence and leak location as pact of test
Electrical conductivity ductive than pack- Less electrical- package, instrument, shown by a drop in test sample elec- exposure on
and capacitance age. ly conduc- test sample fixtures, trical resistivity, with a resultant in- product sta-
(high-voltage leak Product must be tive than liq- and method parame- crease in voltage reading above a pre- bility is rec-
detection) present at leak site. uid product. ters. determined pass/fail limit. ommended Seconds

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Table 1. Deterministic Leak Test Technologiesa (continued)

Deterministic Package Package Effect of Test Time
Leak Test Content Require- Leak Detection Lim- Measurement Outcome and Method Order of
Technologies Requirements ments itb Data Analysis on Package Magnitude
Quantitative measure of gas head-
space content of the test sample by
laser-based gas analysis, for a product
requiring a headspace low in oxygen,
carbon dioxide, or water vapor con-
Gas volume, path centration; and/or low in absolute
length, and content Row 1 pressure.
must be compatible Allows trans- Varies as a function of Whole test sample leakage rate is de-
Laser-based gas head- with instrument’s de- mission of time span between termined by compiling readings as a Nondestruc-
space analysis tection capability. near-IR light. analyses. function of time. tive Seconds

Quantitative measure of mass flow rate

resulting from test sample headspace
escape or liquid product volatilization
within an evacuated test chamber
housing the test sample.
Gas or liquid must be Quantitative pressure readings early
present at leak site. in the test cycle indicate larger leak
Presence of liquid at Row 3 presence.
leak site requires test Rigid, or flexi- Varies with product– Whole test sample leakage rate is de-
pressures below va- ble with package, instrument, termined by comparing the test sam-
por pressure. package re- test fixtures/cham- ple mass flow results to results using
Product must not straint mech- ber, and method pa- leak rate standards and positive con- Nondestruc- Seconds to

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Mass extraction clog leak path. anism. rameters. trols. tive minutes

Nondestruc-
Compatible tive, unless
with pres- Quantitative measure of pressure drop the means Minutes to
sure detec- within a pressurized test sample. Pres- used to ac- days, de-
Gas must be present tion mode. sure drop readings are a measure of cess test pending on
at leak site.
Product (especially ible with
liquids or semi-solids) package re-
must not cover po-
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Rigid, or flex- Row 3
Varies with product–
gas escape through leak paths.
Whole test sample leakage rate is de-
package, instrument, termined by comparing pressure de-
straint mech- and method parame- cay results to results using leak rate
sample inte-
rior compro-
mises test
package vol-
ume and re-
quired leak
sample barri- limit of de-
Pressure decay tential leak sites. anism. ters. standards and positive controls. er. tection
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Able to toler-
ate high-vac-
uum test
conditions Quantitative measure by spectroscopic Nondestruc-
Rigid, or flex- analysis of tracer gas leak rate emitted tive, unless
ible with from a tracer-flooded test sample tracer gas in-
Tracer gas must be package re- positioned in an evacuated test cham- troduction
added to package. straint mech- Row 1 ber. into the
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Tracer gas must have anism Varies with instru- Whole test sample leakage rate is cal- package
access to package Limited trac- ment culated by normalizing the measured compromis-
Tracer gas detection, surfaces being tested er gas per- capability and test tracer leak rate by tracer concentra- es test sam- Seconds to
vacuum mode for leaks. meability sample fixtures. tion in the test sample. ple barrier. minutes

Quantitative measure of pressure rise

(vacuum decay) within an evacuated
test chamber housing the test sample;
vacuum decay readings are a measure
Gas or liquid must be of headspace escape from the test
present at leak site. sample, or liquid product volatiliza-
Presence of liquid at Row 3 tion.
leak site requires test Rigid, or flexi- Varies with product– Whole test sample leakage rate is de-
pressures below va- ble with package, instrument, termined by comparing vacuum de-
por pressure. Product package re- test sample chamber, cay results for the test sample to re-
must not clog straint mech- and method parame- sults of tests performed using leak rate Nondestruc- Seconds to
Vacuum decay leak path. anism ters. standards and positive controls. tive minutes

a All methods apply to nonporous, rigid and flexible packages as per the scope of á1207ñ.
b The leak detection limit cited for each technology refers to Package Integrity Evaluation—Sterile Products á1207ñ, Table 1 and is provided for information only. This
information is intended to assist in early methodology selection. The validated leak detection limit for a product–package test method may deviate from these
values.

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Table 2. Probabilistic Leak Test Technologiesa

Probabilistic Package Package Effect of Test Time
Leak Test Content Require- Leak Detection Lim- Measurement Outcome and Method Order of
Technologies Requirements ments itb Data Analysis on Package Magnitude
Qualitative measure by visual inspec-
tion of bubble emission caused by es-
cape of test sample headspace while
Gas must be present Row 4 sample is submerged and exposed to
at leak site. Varies with product– differential pressure conditions. Alter-
Product (especially Rigid, or flexi- package, test sample natively, sample surfaces may be ex-
liquids or semi-solids) ble with fixtures and position- posed to surfactant.
must not cover pack- package re- ing, method parame- Continuous bubble emission indi-
age surfaces to be straint mech- ters, and analyst cates leak presence, location, and rel-
Bubble emission leak tested. anism. technique and skill. ative size. Destructive Minutes

Qualitative measure by visual inspec-

tion of microorganism growth inside
test samples filled with growth-sup-
Able to toler- portive media or product, post im-
ate pressure mersion in heavily contaminated chal-
and immer- Row 4 lenge media while exposed to differ-
sion chal- Varies with contain- ential pressure conditions, followed
Growth-supportive lenge. er–closure, test sam- by incubation to encourage microbial
media or product. Rigid, or flex- ple fixtures and posi- growth.
Presence of liquid at ible with tioning, challenge Growth in the test sample indicates
the leak site required package re- condition severity, the presence of test sample leak site(s)

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Microbial challenge, for method reliabili- straint mech- and inherent biologi- capable of allowing passive or active
immersion exposure ty. anism. cal variability. entry of microbes. Destructive Weeks

Row 2
Varies with test sam- Nondestruc-
ple, method parame-
ci tive, unless
ters, test sample fix- Quantitative measure by spectroscopic tracer gas in-
Tracer gas must be Leak site ac- tures, and analyst analysis of tracer gas near the outer troduction to
added to package. cessible to technique and skill. surfaces of the tracer-flooded test the package
Tracer gas must have probe. Smaller leak detec- sample, sampled using a sniffer interior com-
access to package Limited trac- tion may be possible probe. promises test
Tracer gas detection, surfaces to be tested er gas per- under optimum test Tracer presence above a pass/fail limit sample barri- Seconds to
sniffer mode for leaks. meability. conditions. indicates leak presence and location. er. minutes
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Measure of tracer in test sample previ-
ously submerged in tracer-charged
liquid while exposed to differential
Row 4 pressure conditions. Alternatively,
Varies with contain- tracer-charged test samples may be
Rigid, or flexi- er–closure, test sam- submerged in tracer-free collection
ble with ple fixtures and posi- fluid.
O

package re- tioning, challenge Tracer migration measurement may

straint mech- condition severity, be quantitative (by chemical analysis;
anism. and tracer liquid con- preferred approach for small leak de-
Able to toler- tent. tection) or qualitative (by visual in-
ate liquid im- Smaller leak detec- spection).
Contents must be mersion. tion may be possible Tracer presence indicates leak site(s)
compatible with liq- Compatible under optimal test capable of allowing tracer passage.
uid tracer. with liquid conditions employ- Tracer magnitude may indicate rela-
Product must not tracer detec- ing chemical analysis tive leak size (assuming a single-leak Minutes to
Tracer liquid clog leak path. tion mode. tracer detection. pathway). Destructive hours

a All methods apply to nonporous, rigid and flexible packages as per the scope of á1207ñ.
b The leak detection limit cited for each technology refers to Package Integrity Evaluation—Sterile Products á1207ñ, Table 1 and is provided for information only. This
information is intended to assist in early methodology selection. The validated leak determination limit for a product–package specific test method may deviate
from these values.

2. DETERMINISTIC LEAK TEST TECHNOLOGIES

2.1 Electrical Conductivity and Capacitance (High-Voltage Leak Detection)

2.1.1 DESCRIPTION
The electrical conductivity and capacitance leak test (high-voltage leak detection, or HVLD) is an approach for detecting the
presence, and potentially the location, of a leak(s) in the wall of a nonporous, rigid or flexible package containing liquid or
semi-liquid product. Test analysis is based on quantitative electrical conductance measurements (1–5). HVLD leak tests are
generally nondestructive to the package and to the product, although an evaluation of HVLD exposure impact on product
physicochemical stability is advised.
The test is performed by first positioning the test sample (containing liquid product) onto an electrically grounded instrument
test fixture. Alternatively, the test sample may be placed onto a transport system that will carry the test sample through an
electrically grounded testing zone. Upon test start, an electrode uniquely designed for the product–package type under test
exposes all or part of the test sample to a high-frequency, high-voltage, low-amperage current. The presence of a leak path in
the proximity of an electrically conductive, liquid-formulation product results in a drop in the electrical resistance of the test

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sample, as shown by a spike in current passing through the test sample above a predetermined pass/fail limit established using
negative controls.

2.1.2 APPLICATION
Rigid or flexible packages of nonporous components containing liquid or semi-liquid product may be tested:
• Package components must be relatively electrically nonconductive.
• Product must be electrically conductive, relative to the package.
• Product must not be flammable (i.e., not a combustion risk).
• Product must be near or at the leak inspection location at the time of the leak test.
• Solidified, electrically conductive product that blocks leak paths may be detected.
• Metal caps used to seal stoppered vial or cartridge packages conduct current, improving the likelihood of finding leaks
under the cap.
HVLD tests are rapid, requiring no more than several seconds for a full scan of the test sample, thus making them appropriate
for off-line testing, or as an on-line, 100% product inspection test method. This technology is useful for any product life cycle
phase.

2.1.3 TEST EQUIPMENT

HVLD instrumentation comes equipped with tooling and/or a test sample transport system for proper test sample and probe/
ground positioning, an internal high-voltage transformer, electrode voltage and ground potentiometer adjustment capabilities,
and a test result output display. The design and materials of construction used for the electrode probe and electrical ground

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are product–package specific.

2.1.4 TEST PARAMETERS

The following are test parameters for the electrical conductivity and capacitance leak test, also known as HVLD:

sensitivity
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• Conductivity of test sample product relative to test sample package: a greater difference will improve leak detection

• Test voltage set point: voltage is set high enough to ensure leak detection, but not so high that current will arc, falsely
rejecting the test sample
• Test sensitivity set point (potentiometer or gain set point): sensitivity should be maximized to ensure leak detection without
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triggering a false reject result
• Package content proximity to potential leak paths: leak detection sensitivity is directly related to the proximity of product
to the leak path
• Electrode probe position relative to potential leak paths: probe proximity to the leak is directly related to the test
method sensitivity
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• Speed at which the electrode passes over the test sample surface: although the test is very rapid, test speeds too rapid
may cause leaks to be missed
• Moisture presence on the package: test sample surface condensation can potentially trigger a false reject reading

2.2 Laser-Based Gas Headspace Analysis

2.2.1 DESCRIPTION
Gas headspace analysis via laser-based techniques provides a quantitative, nondestructive measure of oxygen content, water
vapor content, and low internal pressure in the headspace of a nonporous, rigid or nonrigid package (6–8). Some instruments
are capable of measuring headspace carbon dioxide concentration as well.
The test is performed by first placing the test sample in a fixture designed for precise test sample positioning. Upon test start,
frequency-modulated spectroscopy is used to cause a near-infrared (IR) diode laser light to pass through the gas headspace
region of the sealed test sample. Light is absorbed as a function of gas concentration and pressure. The absorption information
is processed using phase-sensitive detection techniques; a mixer demodulates the signal. The output voltage, which is
proportional to the absorption line shape, is digitally converted and further analyzed by a microprocessor, yielding test sample
signal results. Final test sample readings are automatically generated based on a comparison of test sample signals to a
calibration curve. This curve is pre-established by using control packages flooded with traceable gas reference standards. Gas
headspace analysis, as a function of time, provides a quantitative measure of the total leakage rate of the test sample. Leakage
rates are judged acceptable or unacceptable on the basis of predetermined limits, calculated to ensure proper gas headspace
content maintenance over the product life cycle.

2.2.2 APPLICATION
Rigid or flexible packages made of nonporous components (transparent or semi-transparent material, either amber or
colorless) that allow transmission of near-IR diode laser light may be tested. Test samples require a minimum headspace volume
and headspace path length. The requirements vary on the basis of the gas moiety to be tested and may be specific to the
instrument as well as to the construction and design of the package materials.
Test samples that may be analyzed fall into these categories:
• Products that require low-oxygen or low-carbon-dioxide headspace content

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• Products that require low water vapor content (e.g., lyophilized or powdered products)
• Products that require low internal package pressure (e.g., lyophilized products)
Package integrity, or absence of leakage, is confirmed by replicating tests on a given test sample as a function of time. Longer
time periods between tests are needed to detect smaller leaks. Mathematical models appropriate to leak flow dynamics may
be used to predict the time required for detecting leaks of various sizes or rates.
Headspace analysis at a single time point provides the headspace content result, which may or may not be indicative of
package integrity.
• A test result not meeting specification could be due to package leakage, or could result from improper package filling or
assembly processes that caused the package headspace to be out of specification.
• A test result that meets specification may confirm package integrity if enough time has elapsed since product–package
preparation for measurable leakage to have occurred, assuming that the initial preparation of the test sample met
manufacturing standards.
Methods of laser-based gas analysis may be used during any phase of the product life cycle. Tests are rapid and are appropriate
for off-line testing using lab-scale equipment (typical measurement time, 2 s) or as an on-line, 100% product inspection method
(typical measurement time, 0.2 s).

2.2.3 TEST EQUIPMENT

Test instrumentation for laser-based gas headspace analysis is capable of accurate and reproducible near-IR diode laser light
emission, light detection, and signal analysis. Tooling specific to product–package test samples is used to properly position test
samples, ensuring reproducible laser-light transmission and detection. Standards with components identical to the packages
under test, in terms of both the materials of construction and the dimensions (at the point where light is to be transmitted),

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are required. These standards also need to contain headspace content that is representative of the gas mixture under test (i.e.,
oxygen, carbon dioxide, water vapor, pressure).

2.2.4 TEST PARAMETERS

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The following are test parameters for laser-based gas headspace analysis:
• Test sample position with respect to laser beam transmission and detection points: imprecise test sample handling,
and/or dimensional irregularity of the package, can increase measurement standard deviation
• Test sample headspace volume: the volume must meet minimum requirements for test instrumentation
• Test sample headspace absolute pressure: headspace absolute pressure will influence the detection limit and range for all
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test types
• Test sample speed: increase in testing speed will increase standard deviation of measurement
• Test sample temperature: temperature can influence moisture and pressure test results (e.g., lower temperatures are
associated with lower internal pressure of the test sample) (9)
• Test sample outer surface moisture: presence of moisture may hamper test performance
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• Time allotted between the replicate tests: performing replicate tests over a period of time allows calculation of continuous
package leakage

2.3 Mass Extraction

2.3.1 DESCRIPTION
The mass extraction test is a nondestructive, quantitative measurement approach for detecting leakage in nonporous, rigid
or flexible packages (10). Leakage of package headspace gases and/or leakage below the product fill level may be detected,
given appropriately designed equipment and test parameters.
The test is performed by first placing the test sample inside a test chamber that is pneumatically connected to a mass
extraction leak test system equipped with a vacuum generator package. The test chamber is uniquely designed to contain the
test package, which is fitted with appropriate tooling to limit movement or expansion of moveable or flexible components,
respectively.
Upon test start, the chamber is quickly evacuated for a predetermined time to reach a predetermined vacuum level. A series
of such evacuation cycles may be performed, each intended to identify smaller leakage rates. After each cycle, the test system
is isolated from the vacuum source and measurements of absolute pressure, pressure decay rate, and/or gas mass flow rate are
captured. Readings greater than predetermined limits that were established using negative controls are indicative of container
leakage, triggering test cycle abort.
For those test samples passing all previous larger leak vacuum cycles, a final vacuum is drawn. The test system is then isolated
from the vacuum source. With all flow from the test chamber directed through the mass flow sensor, the mass flow rate is
measured. Mass flow above a predetermined limit established using negative controls is indicative of container leakage.

2.3.2 APPLICATION
Nonporous, rigid or flexible packages may be tested. Packages containing gas, liquid, and/or solid materials can be tested:
• Flexible packages or packages with nonfixed components require tooling to restrict package expansion or movement,
respectively, when exposed to test vacuum conditions. Tooling minimizes the seal stress of flexible packages and maintains
consistent package volume and differential pressure conditions across the leak path.
• Gas headspace must be at atmospheric pressure or at a pressure notably greater than test vacuum conditions.

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• Package surfaces below the product-fill level may be leak tested for those solid-formulation products that do not block
leak-site gas flow and for those liquid products that volatilize at test vacuum but do not solidify and so block leak paths.
• Packages ranging in volume from a few milliliters to several liters may be tested.
Tests require anywhere from several seconds to a few minutes to perform. Longer test times are necessary for testing
larger-volume packages. Lengthening test cycles also allows for detection of smaller leaks.
Mass extraction leak tests are useful in any phase of the product life cycle. Tests may be performed in a laboratory setting or
off-line in the production environment. Longer laboratory or off-line test cycle times are generally capable of detecting smaller
leaks. Higher speed on-line tests are restricted to larger leak detection.

2.3.3 TEST EQUIPMENT

Mass extraction test instrumentation consists of a system of conduits and valves that pneumatically connect a test chamber
with a test system pressure sensor, micro-flow mass sensor, and vacuum generator package, including an external vacuum
source. The instrument includes appropriate timers, electronic controls, and monitors. A fixed-size orifice is included for periodic
system performance verification. The test chamber is uniquely designed to contain the test package, which is fitted with
appropriate tooling to limit movement or expansion of moveable or flexible components, respectively.

2.3.4 TEST PARAMETERS

The following are test parameters for mass extraction:
• Pressures
○ Test system pressure reading after initial gross leak check. At evacuation stage, pressure is a function of test system

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volume, time allotted for evacuation, and the vacuum source pressure level.
○ Test system pressure reading after the secondary evacuation stage(s): pressure above a predetermined limit is due
to test package leakage. The pressure level above baseline is a function of leak size, available headspace volume, and/
or volatile liquid in the test sample.
○ The final absolute pressure of the test cycle must be lower than the headspace pressure of the test package for

located below the liquid-product fill level.

• Mass flow
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detection of gas headspace leaks, and/or lower than the volatilization pressure of liquid product formulation for leaks

○ Mass flow rate reading after secondary evacuation stage(s).

○ The mass extracted from the test sample is monitored for larger leak detection after the first secondary evacuation
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stage; mass extracted from the test sample is monitored for the smallest leak detection after the last evacuation stage.
○ Background flow level (i.e., the baseline flow or noise level) is the flow rate for packages without leaks. Baseline flow
is a function of test package and system outgassing, test system volume, and the time allowed for evacuation.
○ The mass flow rate at steady-state conditions, when extracted from the test chamber, is equal to test sample leakage
into the test chamber, assuming that outgassing from the sample and external leakage from the test system are
O

insignificant. Leakage is identified once the mass flow rate notably exceeds the rate of negative controls.
• Times
○ Time allotted for system evacuation for gross leak detection: enough time is allotted to draw off most of the test
chamber gases, without exhausting the headspace gases of the test package, or without drawing off the liquid
contents from grossly leaking packages.
○ Time allotted for large-leak check through the mass flow sensor: a brief time is required for detection of large- and
medium-sized defects.
○ Time allotted for system evacuation for small leak detection: enough time should be allowed to establish the desired
vacuum equilibrium of the test chamber. Insufficient time will not adequately draw off gases sorbed onto package
surfaces or entrapped between components.
○ Time allotted for mass flow to stabilize: after the secondary evacuation stage(s), monitor the flow as the flow rate
approaches steady state. Enough time is allotted so that flow from the smallest allowed defect is statistically greater
than baseline (no-leak) flow.
○ Time allotted for the final leak test by mass flow: enough time should be allotted so that the mass flow rate exceeds
baseline readings for negative controls.

2.4 Pressure Decay

2.4.1 DESCRIPTION
The pressure decay test is a quantitative measurement approach for detecting leakage in nonporous, rigid or flexible
packages. The test is destructive if the introduction of pressurized gas creates a break in the package wall or seal. The test is
nondestructive if the introduction of gas into the test sample does not compromise the package barrier. Pressure decay testing
is intended for integrity testing of the gas headspace region of the test sample.
To perform the test, a dry air or inert gas pressure source is attached to the test sample that is fitted with an internal pressure
monitoring device. The test sample is pressurized to a predetermined pressure, after which the pressure source is isolated from
the test sample. The decay in pressure is monitored for a predetermined time. Pressure decay that exceeds a predetermined
limit established using negative controls indicates container leakage.

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The referenced ASTM F2095 method (11) is intended for testing flexible packages (pouches and foil-sealed trays). Seals or
surfaces being tested cannot be in contact with product such as water, oils, or other liquids. ASTM method A describes testing
packages without use of a restraint mechanism. The method requires that the package reach a stable volume configuration
(i.e., it stops stretching) to take a measurement. ASTM method B requires that the test sample is kept between restraining plates
during the test to limit the volume of the pressurized package.

2.4.2 APPLICATION
Nonporous, rigid or flexible packages may be tested:
• Package surfaces that can be tested are those unobstructed by product (e.g., oils, water, or other liquids); small leaks below
the liquid-fill level would not be detected by this method.
• Flexible packages or packages with nonfixed components require tooling to restrict package expansion or movement,
respectively, when exposed to test pressure conditions. Tooling minimizes the seal stress of the flexible package and
maintains consistent package volume and differential pressure conditions across the leak path.
• Pressure decay can be used for testing packages anywhere from a few milliliters in volume to large, bulk-storage vessels.
Tests require anywhere from a few seconds to a several hours to perform. Longer test times are necessary for testing
larger-volume containers. Lengthening test cycles also allows for detection of smaller leaks.
Pressure decay tests are useful in any phase of the product life cycle.
Tests may be performed in a laboratory setting or off-line in the production environment. Laboratory or off-line test
equipment that allows for longer test times is generally capable of detecting smaller leaks. Higher-speed, on-line pressure decay
equipment may be used to check for defects in open packages before package filling and closure.

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2.4.3 TEST EQUIPMENT
Pressure decay test instrumentation includes conduits to connect the test sample with test system pressure transducers
(absolute, differential, or a combination of both) and a pressure source (12). Instrumentation includes appropriate timers,
electronic controls, and monitors. Greatest test method sensitivity and reproducibility are achieved when the instrument is kept
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in a temperature-controlled environment; test samples (especially larger-volume samples) are kept at a controlled, constant
temperature during test; and dry pressurizing gas at constant temperature is used. It is optional to use tooling uniquely designed
to limit movement or expansion of moveable or flexible components, respectively, thereby keeping test sample volume constant
and limiting seal stress.

2.4.4 TEST PARAMETERS

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The following are test parameters for pressure decay:
• Test sample internal pressure after pressurization:
○ The initial pressure reached after sample pressurization is a function of the test system volume, time allotted for
pressurization, pressure source capacity, and temperature of the test sample headspace.
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○ Higher pressure creates the potential for more rapid and sensitive leak testing. However, the selection of maximum
test pressure should take into consideration personnel safety risks and potential damage to equipment and package.
• Pressure decay baseline: the baseline pressure decay (i.e., noise level) is the pressure drop that occurs for packages without
leaks.
○ Baseline pressure drop is a function of test package volume, temperature conditions, and the length of time allowed
for pressure to rise.
○ Baseline pressure drop requiring longer time periods is affected by gas sorption onto test package surfaces, gas
moisture content (dry gas should be used), and gas temperature. Techniques to limit baseline pressure drop include
the use of dry gases, and keeping the test container and the pressurized gas at a constant temperature.
• Pressure decay due to test package leakage: the extent of pressure decay above baseline is a function of leak size, available
headspace volume in the test sample, the initial pressure inside the test sample, temperature control, and the time allotted
for pressure to rise.
• Times
○ Time allotted for test sample pressurization: enough time is allotted to establish the desired pressure inside the test
sample.
○ Time allotted after pressurization for pressure decay: enough time should be allotted so that the pressure decay from
the smallest leaks can be detected (i.e., baseline decay is exceeded).
○ A time lag may be incorporated before monitoring for pressure decay to allow for gas equilibrium within the container
and test system.
• Temperatures
○ Temperature of the pressurized gas can significantly affect test method sensitivity and reliability, especially when
testing larger-volume containers.
○ An increase in gas temperature causes a rise in pressure, and conversely, a rise in gas pressure triggers an increase in
gas temperature. Therefore, upon initial test package pressurization, the gas temperature will spike, causing a further
spike in pressure. The subsequent drops in temperature and pressure during system equilibrium may be mistaken for
leakage.

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○ Effects of temperature variation may be minimized by: 1) allowing the temperature in the pressurized system to come
to equilibrium before starting the pressure decay test, 2) minimizing temperature variation outside the test system,
3) minimizing test duration, and/or 4) applying a mathematical correction to the pressure readings.

2.5 Tracer Gas Detection, Vacuum Mode

2.5.1 DESCRIPTION
The leak detection method for tracer gas detects leakage from nonporous, rigid or flexible packages. The method can be
destructive or nondestructive, depending on the test approach used. The test requires the presence of tracer gas inside the test
sample package. Helium is the most commonly used tracer gas, and hydrogen is also used. The leakage rate of tracer gas is
quantitatively measured using a spectrometric analytical instrument specific for the tracer gas. Instruments are designed to
check for tracer gas leaking out of a test package, either by means of a test chamber that can be evacuated to draw gas out of
test sample leaks (the vacuum mode) or by use of a vacuum wand (the sniffer mode) for scanning the outer surfaces of the test
package (see Tracer Gas Detection, Sniffer Mode). The vacuum-mode tracer gas test is used more commonly than the sniffer
mode for integrity testing sterile pharmaceutical product–packages. The vacuum mode is both quantitative and deterministic
and is used to capture and quantify leakage from an entire test package, or it can be used to test for leakage along a test package
surface or seal, given proper sample fixtures. Both vacuum and sniffer testing modes using helium as the tracer gas are described
in ASTM F2391 (13).
To perform the vacuum mode test, test samples that have been fully or partially flooded with tracer gas are placed inside an
evacuation chamber that is pneumatically connected to the tracer gas analysis instrument. Alternatively, the test sample may
be fitted in such a manner that only the surface or seal of interest is exposed to the instrument, allowing for targeted leak

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detection at one specific seal or surface. In some cases, test samples that cannot withstand the high-vacuum test conditions
may be tested with the use of tooling to restrict package expansion or movement.
At test start, the instrument’s vacuum pump evacuates the test chamber or fixture, drawing leaking tracer gas through the
analyzer. The absolute leak rate of the test sample is calculated by normalizing test results by the partial pressure of the tracer
gas within the test sample at the time of test. For accurate results, tracer gas concentration within the sample must be uniform
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and consistent at the time of test; also, there should be minimal tracer gas permeation out of the test sample that can mask
test sample leakage. Calibration tracer gas reference standards can be used for understanding the relationship between true
leak rates and measured leak rates under actual test conditions.
The vacuum-mode tracer gas leak test is a nondestructive test, unless tracer gas introduction into the test sample requires
package wall compromise (e.g., piercing), or if the presence of tracer gas is detrimental to the package contents.
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2.5.2 APPLICATION
Rigid or flexible packages made of nonporous components:
• Flexible packages or packages with nonfixed components may require tooling to restrict package expansion or movement,
respectively.
O

• Tracer gas permeation through the package material must not be so great that the leakage rate of concern is masked.
• A wide range of package sizes may be tested.
Leak paths must be clear of liquid or solid materials that could potentially block tracer gas flow.
Caution is advised when testing liquid-filled packages, because vapors or liquid drawn into the test system can seriously
damage instrumentation.
Detection capabilities range from large leaks to the smallest leaks.
• Method capability is related to the size of the unobstructed leak path.
• Large leaks in the smallest packages may be missed because of the rapid loss of tracer gas (e.g., during the evacuation
phase of the vacuum mode test).
• Significant tracer gas permeation through the package itself can interfere with the test by swamping leakage rate.
The test is nondestructive if the tracer gas is introduced into the package at the time of package assembly or closure, but
the inclusion of tracer gas may prevent introduction of these packages into commercial or clinical markets. The test is destructive
if the introduction of tracer gas compromises assembled package integrity (e.g., package puncture). Following test sample
preparation, the actual leak test generally takes less than 1 min.
Tracer gas leak test methods may find application in any product life cycle phase. They are generally used in a laboratory
environment. Tracer gas methods can also be used in production as an off-line testing approach; they can be used on-line if
tracer gas is introduced into the test samples before final package closure.

2.5.3 TEST EQUIPMENT

Analytical instrumentation specific for tracer gas detection (e.g., mass spectrometer for helium detection) is required, typically
equipped with an internal leak rate standard for instrument calibration at time of use. Additional equipment needed includes
external calibration reference standards of tracer gas, to be used for comparisons of true leak rate versus measured leak rate
under actual test conditions; a tracer gas source with a means for introducing tracer gas into the test sample; a test chamber
or fixture to pneumatically connect the test sample with the instrument; tooling to restrict package expansion or movement,
as appropriate; a means for accessing and analyzing the test sample headspace for tracer gas partial pressure after execution
of the vacuum-mode leak test; and ventilation to remove tracer gas from the test area, or a tracer gas recapture system.

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2.5.4 TEST PARAMETERS

The following are test parameters for tracer gas detection, vacuum mode:
• Tracer gas partial pressure within the test sample at the time of test:
○ When added by soaking the intact package, the concentration of the tracer gas is dependent on the time allotted,
the positive pressure applied, and the tracer gas leak rate, plus the rate of permeation into the package
○ When added before package closure, tracer gas concentration is dependent on the gas flooding rate and time, the
tooling and/or enclosure used to concentrate gas inside the package, and the efficiency of package closure for
preventing escape of tracer gas
○ When added after package assembly, tracer gas concentration is dependent on the tooling used to pierce, flush, and
vent the package; the gas flooding rate and time; and the sealant material applied to reseal the puncture site
• Differential pressure applied to the test sample during the vacuum mode will drive tracer gas out of the test sample,
increasing method sensitivity
• Time allotted to allow for leakage to reach steady state should not exceed tracer gas permeation lag time

2.6 Vacuum Decay

2.6.1 DESCRIPTION
The vacuum decay test is a nondestructive, quantitative measurement approach for detecting leakage in nonporous, rigid
or flexible packages. Leakage in the package headspace gas region and/or below the product-fill level may be detected given

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appropriately designed test parameters and if product properties allow (as detailed below).
To perform the test, the test sample is placed in a closely fitting evacuation test chamber pneumatically connected to the
leak test system, which is equipped with an external vacuum source. The test chamber is uniquely designed to contain the test
package. Test samples with moveable or flexible components require appropriate tooling to limit the movement or expansion
of such components, respectively. ci
Upon test start, the test chamber plus test system dead space are evacuated for a predetermined period of time. The targeted
vacuum level chosen for the test is predetermined on the basis of the test sample type, size, and content. The vacuum source
is then isolated from the test system. After a short time has elapsed to allow for system equilibration, the rise in dead space
pressure (i.e., vacuum decay) is monitored for a predetermined length of time using absolute and/or differential pressure
transducers. A pressure increase that exceeds a predetermined pass/fail limit established using negative controls indicates
container leakage. ASTM F2338 may be referenced (14).
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2.6.2 APPLICATION
Nonporous, rigid or flexible packages may be tested. Packages containing gas, liquid, and/or solid materials can be tested:
• Flexible packages or packages with nonfixed components require tooling to restrict package expansion or movement,
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respectively, when exposed to test vacuum conditions. Tooling minimizes flexible package seal stress and maintains
consistent package volume and differential pressure conditions across the leak path.
• Product–package gas headspace must be at atmospheric pressure or at a pressure notably greater than test vacuum
conditions.
• Package surfaces below the product-fill level may be tested for leaks for those solid dosage formulation products that do
not block leak-site gas flow or for those liquid dosage form products that volatilize at test vacuum without solidifying and
blocking leak paths.
• Packages ranging in volume from a few milliliters to several liters may be tested.
Vacuum-decay leak tests are useful in all phases of the product life cycle. Tests require anywhere from a few seconds to a few
minutes to perform. Longer test times are necessary for testing larger-volume packages or for detection of the smallest leaks.
Longer test times are more appropriately performed in a laboratory setting or off-line in the production environment.
Higher-speed on-line equipment is generally used for detecting larger leaks.

2.6.3 TEST EQUIPMENT

Vacuum-decay leak test instrumentation consists of a system of conduits and valves that pneumatically connect a test
chamber with the test system pressure sensors and an external vacuum source. The instrument includes appropriate timers,
electronic controls, and monitors. An external gas flow meter allows for periodic system performance verification. The test
chamber is uniquely designed to closely contain the test package and may be fitted with tooling to limit movement or expansion
of moveable or flexible package components, as appropriate.

2.6.4 TEST PARAMETERS

The following are test parameters for vacuum decay:
• Pressures
○ Test chamber pressure after evacuation: the initial pressure reached during test chamber evacuation is a function of
test system volume, time allotted for evacuation, and vacuum pump capacity. The absolute pressure of the test system
must be lower than the headspace pressure of the test package for detection of gas headspace leaks, and/or lower
than the volatilization pressure of the liquid-product formulation for leaks located below the liquid-product fill level.

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○ Pressure rise baseline: the baseline pressure rise (i.e., noise level) is the pressure increase that occurs for packages
without leaks. Baseline pressure rise is a function of test package and system outgassing, test system volume, and the
time allowed for pressure rise.
○ Pressure rise due to test package leakage: the extent of pressure rise above baseline is a function of leak size, test
chamber vacuum level at test start, available headspace volume or volatile liquid in the test sample, and the time
allotted for pressure rise.
• Times
○ Time allotted for system evacuation: enough time should be allowed for establishing the desired vacuum pressure
level of the test chamber, plus draw off gases sorbed onto package surfaces or entrapped between components.
Excessive time will evacuate headspace from largely leaking packages, risking that there will be insufficient headspace
gas for leak detection. Times should not be so great that leaking package headspace gases are exhausted or the liquid
product floods and contaminates the test system.
○ Time allotted after evacuation for pressure rise (vacuum decay): enough time should be allotted so that the pressure
rise from the smallest leaks to be detected exceeds baseline. A time lag may be incorporated after evacuation and
before vacuum decay monitoring to allow for gas equilibrium within the container and test system.

3. PROBABILISTIC LEAK TEST TECHNOLOGIES

3.1 Bubble Emission

3.1.1 DESCRIPTION

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The bubble emission leak test is a destructive, qualitative measurement approach for detecting and locating leaks in
nonporous, rigid or flexible packages containing headspace gas.
The test is performed in one of two ways. The first is an internal pressurization method referenced in ASTM F2096 (15) in
which a positive pressure air source with pressure monitor is inserted into the test sample. The test sample is then submerged
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in water, and air pressure is applied to a predetermined level, for a predetermined time period. The second approach is
referenced in ASTM D3078 (16). The intact test sample is submerged in water or other suitable submersion fluid contained in a
vacuum chamber. Vacuum is established to a predetermined level, for a predetermined time period.
With both approaches, leakage can be observed as a continuous stream of bubbles emitted from the leak site. Bubble
diameter and emission rate may provide some indication of relative leak size. An alternative to test sample submersion is coating
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the test sample with surfactant, in which case any leakage is seen as foaming or bubbling at the leak site. The surface tension
of the submersion fluid or surfactant allows for smaller bubble formation, potentially improving test sensitivity. Use of
submersion fluid with low gas solubility may also improve test sensitivity.
The bubble emission test is categorized as a probabilistic leak test method. Although this method relies on the predictable
flow of gas through leak paths, escaping gas can become entrapped within or between package components; false-leak
outgassing events may occur; gas emitted from small leaks may solubilize in the immersion fluid before bubble formation; and
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test sample set up may be inadequate to ensure sufficient differential pressure conditions and appropriate bubble visibility. The
use of negative and positive controls along with test samples provides evidence of test method limit of detection.

3.1.2 APPLICATION
Nonporous, rigid or flexible packages with gas headspace may be tested by bubble leak methods:
• Packages must be able to tolerate wetting or submersion
• Flexible packages or packages with nonfixed components generally require tooling to restrict package expansion or
movement, respectively, when exposed to vacuum conditions. Tooling minimizes the seal stress of the flexible package
and maintains consistent package volume and differential pressure conditions across the leak path. However, tooling may
block leak paths or hinder bubble emission visibility
• Only leak sites that are present in the gas headspace region of the package can be detected
• This test is generally used for testing smaller-volume packages that are less than a few liters in size
Bubble tests are applicable in any product life cycle phase. Bubble tests require several minutes or longer for test sample
analysis and subsequent cleaning and/or drying. Bubble tests are most commonly used in laboratory settings as part of a research
investigation to verify leak presence and location. They can also be used as an off-line production leak test. Bubble tests are also
used for integrity testing of aerosol–package products in a research or production setting. In this application, the test may be
considered nondestructive to the product–package test sample.

3.1.3 TEST EQUIPMENT

Bubble tests require a pressure or vacuum source (as appropriate) equipped with pressure monitors and controls; tooling to
restrict expansion of flexible packages or movement of nonfixed components; submersion fluid or surfactant to be applied to
the package surface; submersion vessel equipped for external vacuum or internal pressure test mode; and visual inspection aids
such as lighting, magnification, and/or background, as needed.

3.1.4 TEST PARAMETERS

The following are test parameters for bubble emission:

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• Differential pressure: greater differential pressure evokes more rapid bubble emission. Differential pressure should not be
so great that package seals are compromised or that gas escapes through large leaks so rapidly that it may be confused
with package surface outgassing
• Time allotted during differential pressure application: longer test times allow for smaller leak detection
• Times allotted for inspection (pacing) and for inspection breaks to lessen operator fatigue
• Package positioning during inspection
• Package mode of restraint for moveable or flexible components
• Submersion fluid (or surfactant) surface tension: lower surface tension improves method sensitivity
• Inspection environment parameters: lighting intensity and angle, degree of magnification, background color

3.2 Microbial Challenge, Immersion Exposure

3.2.1 DESCRIPTION
The immersion exposure microbial challenge is a destructive, qualitative measurement approach for confirming leaks in
nonporous, rigid or flexible packages.
The test is performed by first filling test samples with sterile, growth-supporting media, followed by incubation and visual
inspection of samples to ensure sample sterility before microbial challenge. Samples are then immersed in a concentrated
bacterial suspension for a predetermined time. Samples can be exposed during immersion to a predetermined vacuum for a
predetermined time, followed by release of vacuum while the packages remain immersed at ambient pressure for a
predetermined time. Samples are then incubated under growth-promoting conditions, followed by examination of package

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contents for evidence of microbial growth by visual inspection or other appropriate analytical means. Alternative approaches
can include exposure of immersed test samples to positive pressure conditions, or to multiple cycles of vacuum and/or pressure
conditions. Test sample leakage is evidenced by visible growth of the challenge microorganism(s) inside test samples. Immersion
microbial challenge tests rely on the presence of a liquid carrier in the leak path that sweeps microorganisms into the package
or provides a means whereby microorganisms can actively migrate and/or grow into the test sample. The use of negative and
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positive controls along with test samples provides evidence of test method limit of detection.
The microbial challenge by immersion test is categorized as a probabilistic leak test because of the multiple events that must
occur sequentially and/or simultaneously for leak detection to take place. All such events are difficult to predict or control,
especially for detection of smaller leaks. For example, the microorganisms must be physically present at the leak site. The
necessary presence of liquid in the leak path, and/or flowing through the leak path, is influenced by the package materials of
construction, leak path tortuosity and topography, media surface tension, and leak path blockage by product, extraneous debris,
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or air locks. Microorganisms must not be hindered from entering the package by getting trapped in a tortuous leak path, and
enough microorganisms must enter the package to allow for sufficient growth that can be detected visually after test sample
incubation.

3.2.2 APPLICATION
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Microbial challenge tests by immersion are most useful when an appropriate and validated physicochemical leak test method
does not exist, or when the test outcome demands direct evidence of the prevention of microbial entry.
Nonporous packages of rigid or flexible components may be tested by immersion microbial challenge methods:
• Packages must be able to tolerate submersion.
• Flexible packages or packages with nonfixed components may require tooling to restrict package expansion or movement,
respectively. Tooling minimizes stress on flexible package seals and maintains consistent differential pressure conditions
across the package seal.
Immersion microbial challenge tests are performed in a laboratory environment, not as an on-line test of the finished product.
A test requires several days to prepare; the test samples are pre-incubated before microbial challenge to ensure initial package
content sterility. The challenge itself, plus post-challenge sample decontamination, may take several hours. Final incubation
followed by sample inspection may take 1–2 weeks. The immersion microbial challenge test is primarily used in product–package
development and validation studies.

3.2.3 TEST EQUIPMENT

The test chamber for conducting the immersion microbial challenge test is designed and equipped to maintain suspension
uniformity and appropriate temperature while exerting the required differential pressure condition. Fixtures for restraining and/
or positioning packages during immersion are also required. Small, motile microorganisms are preferred for the challenge;
examples include Brevundimonas diminuta and Serratia marcescens. The immersion challenge media should support challenge
microorganism growth to the desired concentration. Soybean-casein digest medium is one commonly used medium.
The media filled into test samples may match the immersion challenge media formulation. Alternatively, product that has
been shown to support microbial growth may be used. Media should allow sufficient growth of the particular challenge
organism so that package contamination can be detected in the positive controls. Verification of growth promotion in the
immersion media and the test sample media should be performed each time the immersion challenge test is conducted (see
Sterility Tests á71ñ, Growth Promotion Test of Aerobes, Anaerobes, and Fungi). Use of an incubation chamber is required for test
samples, both before and after exposure. A means for determining microbial growth inside test packages is required.

3.2.4 TEST PARAMETERS

The following are test parameters for microbial challenge, immersion exposure:

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• Microorganism concentration: in the immersion challenge media, microorganism concentration should meet appropriate
predetermined levels throughout the challenge test (a minimum concentration of 105 CFU/mL is commonly used).
• Media fill volume: the fill volume of media in test samples should be sufficient to ensure a liquid path at each potential
leak site. Exposure of package seals and leak sites to the media may also be accomplished by test sample positioning during
the immersion challenge. It is also necessary to have sufficient volume and correct composition of the package headspace
to encourage growth of the selected challenge microorganism. Note that when the composition of the package headspace
does not include oxygen, other test conditions (e.g., anaerobic) may be applicable.
• Vacuum/pressure conditions: exposing immersed test samples to differential pressure conditions is an important method
parameter that serves multiple purposes:
○ Differential pressure helps eliminate trapped air and ensures the presence of liquid media between package
components and at leak sites.
○ Differential pressure simulates the pressure changes incurred during air or land freight transport of the product.
Absolute pressure conditions that correspond to various altitudes anticipated during land and/or air freight transport
are provided in ASTM D6653/D6653M (17). Differential pressure conditions of the test may be modified on the basis
of knowledge of the product–package shipping environment.
○ Differential pressure exposure can simulate conditions experienced by the product during some sterilization
treatments.
• Test times: longer exposure times improve the likelihood of microbial ingress into defective test samples and positive
controls. However, a possible decrease in the ability of media to support growth over time must be considered.
○ Time allotted for immersion exposure during differential pressure exposure (vacuum and/or pressure)
○ Time allotted for immersion exposure at ambient pressure conditions

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• Temperature during challenge: temperatures sufficient to support microbial growth are recommended. Temperature
cycling can also be used as a tactic to eliminate airlocks and promote the presence of liquid media at package seal sites.
• Pre- and post-challenge test incubation temperature and times: the temperature selected should allow sufficient microbial
growth. Incubation times should be sufficient to ensure visualization of growth; these times are determined on the basis
of positive controls and samples from growth-promotion studies.
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• Parameters for detecting post-incubation microbial growth (e.g., lighting and background color for visual inspection,
handling procedure, and pacing).

3.3 Tracer Gas Detection, Sniffer Mode

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3.3.1 DESCRIPTION
The following information is specific to the tracer gas detection performed in the sniffer mode. (For additional information,
see Tracer Gas Detection, Vacuum Mode.)
Tracer gas detection using a sniffer attachment is used to detect leak presence and location in nonporous, rigid or flexible
packages. This is a nondestructive leak test, unless tracer gas introduction into the test sample requires package wall compromise
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(e.g., piercing) or if tracer gas presence is detrimental to package contents. The sniffer mode test using helium as the tracer gas
is described in ASTM F2391, Procedure A (13). Briefly, test samples are flooded completely or partially with the tracer gas via
one of several options. These options may include piercing a closed test sample to introduce pressurized tracer gas (sealant is
applied to close the puncture site); flooding the test sample before package closure; or “soaking” a closed test sample by
pressurizing with tracer gas (most applicable to larger leak detection). Test samples are checked for leakage by scanning the
outer package surfaces using a vacuum wand that is pneumatically connected to the tracer gas analytical test instrument (e.g., a
mass spectrometer for helium detection). Calibration reference standards of tracer gas can be used for understanding the
relationship between true leak rates and measured leak rates under actual test conditions. The use of negative and positive
controls along with the test samples provides evidence of test method limit of detection.
The sniffer mode of tracer gas leak testing is a probabilistic leak test method. This is because the presence of concentrated
tracer gas near the test sample surface is not a well-defined or predictable event, and the sniffer scanning procedure is prone
to variability related to human technique. The sniffer mode is generally chosen when the leak location is to be identified.

3.3.2 APPLICATION
Rigid or flexible packages made of nonporous components may be tested:
• Flexible packages or packages with nonfixed components may require tooling or manual manipulation to force tracer gas
through leak paths.
• Tracer gas permeation through the package material must not be so great that the leakage rate of concern is masked.
• A wide range of package sizes may be tested, ranging from small packages to large multi-liter vessels.
Leak paths must be clear of liquid or solid materials that could potentially block tracer gas flow. The leak size detection
capability is related to an unobstructed leak path.
The sniffer probe must not be allowed to draw liquid or hazardous vapors into the test system, as this would risk serious
instrument damage.
Tracer gas leak test methods require time for the introduction of tracer gas into the test package, and up to several minutes
to scan the package. Large leaks in the smallest packages may be missed because of the rapid loss of tracer gas.
Methods of tracer gas leak tests in the sniffer mode are generally used in the laboratory environment for locating package
leaks. Tracer-gas sniffer mode tests are useful in any product life cycle phase.

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3.3.3 TEST EQUIPMENT

Test equipment for tracer gas leak detection is described in Tracer Gas Detection, Vacuum Mode. However, a sniffer probe
with an enclosure and/or tooling for concentrating tracer gas passing through smaller leaks is used, rather than the test chamber
or test fixtures described in Tracer Gas Detection, Vacuum Mode.

3.3.4 TEST PARAMETERS

The following are test parameters for tracer gas detection, sniffer mode:
• Differential pressure applied to the test sample will drive tracer gas out of the test sample, increasing the method’s
sensitivity. For flexible packages, differential pressure can be exerted by package compression.
• Tracer gas partial pressure within the package at the time of the test is discussed, with instructions, in the Tracer Gas
Detection, Vacuum Mode.
• Aspects of the sniffer mode vacuum wand, such as sweeping speed and distance from the package surface, as well as the
tooling or enclosure used for concentrating leaking gas.

3.4 Tracer Liquid

3.4.1 DESCRIPTION
The tracer liquid test method is a destructive approach for detecting and potentially locating leaks in nonporous, rigid or
flexible packages. Tracer liquid tests provide an indication of leak presence and may provide a measure of relative leak size.

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Tracer liquid tests using liquid submersion work by the diffusive flow of the tracer element through a liquid-filled leak path and/
or the effusive flow of tracer solution through the leak path.
The liquid submersion test uses one of two basic approaches. In the first approach, test samples are submerged in a
tracer-element solution formulation contained in an evacuation chamber. Examples of tracer elements include dyes,
radionuclides, or metallic ions. In the second approach, test samples containing tracer formulation are submerged in tracer-free
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liquid contained in an evacuation chamber. For both approaches, test samples may be fitted with tooling to ensure proper
positioning and to restrict flexible or moveable components. The submerged test samples are subjected to vacuum at a
predetermined pressure level for a predetermined time. After vacuum release, test samples remain submerged for a
predetermined time. Additional test options include the use of positive pressure exposure or multiple cycles of differential
pressure conditions to encourage effusive flow of the tracer element through the leak path.
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In the first approach, after the challenge is complete, test sample outer surfaces are cleaned, and the contents are checked
for evidence of tracer ingress (18). In the second approach, after the challenge is complete, the immersion fluid is checked for
evidence of tracer liquid egress out of the test sample. In both cases, measurement of tracer liquid migration may be performed
in a quantitative manner by using chemical analysis techniques (preferred for small leak detection). Alternatively, the presence
of leakage may be determined qualitatively by visual inspection if the tracer element can be discerned visually. The use of
negative and positive controls along with test samples provides evidence of test method limit of detection.
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Liquid tracer tests are categorized as probabilistic methods. Successful liquid tracer detection relies on a combination of tracer
solution wicking, tracer solution effusion, and tracer element diffusion through a liquid-filled leak path and are events that are
difficult to predict or control, especially for detection of smaller leaks. These events are influenced by numerous factors, including
the package materials of construction, leak path tortuosity and topography, tracer liquid surface tension, and leak path blockage
by product, extraneous debris, and air locks.

3.4.2 APPLICATION
Rigid or flexible packages of nonporous components may be tested using tracer liquid submersion methods:
• Packages must be able to tolerate wetting or submersion.
• Flexible packages or packages with nonfixed components may require tooling to restrict package expansion or movement,
respectively.
○ Testing by submerging a test sample in tracer liquid is used when the test sample allows for visual examination of
the tracer (e.g., dye) ingress, or when the tracer element is to be contained within the test sample after testing.
○ Testing by submerging tracer-filled test samples in tracer-free liquid may be used when the sample interior cannot
be visually examined or when the tracer element is best captured for analysis outside the test sample.
Tests may require up to 1 h or longer for test sample exposure, cleaning, and inspection or analysis. Tracer liquid tests are
primarily used for laboratory testing or off-line product sample testing. Tracer liquid submersion tests can be used in any product
life cycle phase.

3.4.3 TEST EQUIPMENT

A test vessel equipped for challenge conditions of vacuum and/or positive pressure is required and is pneumatically connected
to a pressure and/or vacuum source, as appropriate, and equipped with pressure monitors and controls (19). The tracer liquid
formulation should be physicochemically compatible with the test sample components and the tracer-free solution to be filled
into the test samples or used as the immersion bath, to ensure optimal tracer solution functionality. Formulation considerations
include tracer element type and concentration; surfactant use, type, and concentration; and solvent system. Examples of tracer
liquid incompatibilities apparent upon contact with the test product include tracer dye fading, tracer element precipitation,
and tracer element sorption onto package components. Tooling is often needed to restrict flexible package expansion or

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nonfixed component movement upon differential pressure exposure. Tooling minimizes stress on flexible package seals and
ensures consistent differential pressure conditions across the leak path.
Tracer detection requires the use of either analytical detection instrumentation [e.g., UV-Vis spectrophotometry (20), phase
induction spectrophotometry, or other] or visual inspection aids. Analytical detection offers the advantage of minimizing the
error that is inherent in visual discernment of low dye concentrations. Optimal visual inspection requires the use of controlled
inspection conditions, such as background color, lighting, pacing, fatigue breaks, and negative controls for comparison.

3.4.4 TEST PARAMETERS

The following are test parameters for tracer liquid:
• Differential pressure conditions used (vacuum and/or pressure): greater pressure differentials encourages tracer liquid
passage.
• Submersion times during and after differential pressure application: longer times allow for greater tracer liquid passage
through leak paths.
• Holding time between tracer liquid challenge and final inspection: some tracer liquids visibly fade or are sorbed onto
package surfaces over time.
• Tracer liquid surface tension: lower surface tension allows for smaller leak detection.
• Tracer detection parameters
○ Analytical detection
■ Test sample content extraction procedure
■ Method-specific performance parameters

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○ Visual inspection
■ Inspection environment parameters: lighting intensity and wavelength, background color, viewing angle, and
test sample visibility
■ Time allowed for inspection (pacing) and breaks to lessen operator fatigue
■ Comparison with negative controls
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