Stability Testing For The Shelf Life Determination of Supplements EBOOK (ISBN 978 1 912787 17 3)

Stability Testing for the
Shelf Life Determination of Supplements

The International Alliance of Dietary/Food Supplement Associations (IADSA) brings together
over 50 associations of dietary/food supplement manufacturers and distributors from across
the world. IADSA’s central goal is to ensure a greater exchange of information about the
science and regulation of dietary supplements and ingredients across government, industry
and the scientific community.
The publication Stability Testing for the Shelf Life Determination of Supplements has been
developed by an international group of experts within the supplement industry. These
experts represented countries from five continents around the globe.
IADSA acknowledges the leading role of the Council for Responsible Nutrition UK (CRN
UK) in the development of these guidelines, and particularly Peter Berry Ottaway, Technical
Director to CRN UK, as lead author of the publication.
Table of
Contents
STABILITY TESTING FOR THE SHELF LIFE DETERMINATION OF SUPPLEMENTS
1. Introduction 8
2. Stability by design 10
3. Mechanisms of product deterioration 12

3.1 Moisture/water vapour content and transfer 13
3.2 Temperature 13
3.3 Oxygen 14
3.4 Oxidising/Reducing agents 14
3.5 Light 15
3.6. Chemical hydrolysis 15
3.7 pH 15
3.8 Chemical interactions 16
4. Physical stability 18
4.1 Tablets 19
4.2 Capsules 19
5. Product verification 23
6. Test parameters for stability testing 25

6.1 Selection of test parameters 25
6.2 Analysis of parameters 26
7. Shelf life studies 28

7.1 General criteria 29
7.2 Analytical requirements 29
7.3 Product samples 30
8. Types of stability studies to support shelf life determination 32

8.1 Stress testing 33
8.2 Accelerated testing 34
8.3 Long term testing 34
8.4 In-use testing 35
8.5 Transit (excursion) testing 35
9. Shelf life evaluation 36

9.1 Analysis of appropriate stability data 37
9.2 Extrapolation of stability data 38
10. Stability study protocols 40

10.1 Protocol development 41
11. Bracketing and matrixing 44

11.1 Bracketing 45
11.2 Matrixing 45
5
TABLE OF CONTENTS
11.3 Points to consider 46
12. Use of published sources on stability 48

12.1 Before formulation of product 49
12.2 Before shelf life testing of product 49
Annex I Glossary of terms 50
Annex II Chemical interactions of vitamins that may

affect the stability of a food supplement 54
Annex III Comparison of vitamin stability 58
Annex IV Microbiology of supplements 60
Annex V Predictive analysis of stability data 66
Annex VI Bracketing and matrixing 72
Annex VII Useful resources 78
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7
1. Introduction
‘Stability Testing for Shelf Life Determination of Supplements’ is part of a series of

Technical Guidances for the supplement industry, which have been produced by the
IADSA Technical Group over the past few years.
This document should be read in conjunction with the IADSA ‘Global Guide to Good
Manufacturing Practice for Supplements’ and ‘Shelf–life Recommendations for
Supplements’.
‘Stability Testing for Shelf Life Determination of Supplements’ outlines the principles for
setting up scientifically based stability studies, which are specifically designed to assess
the stability and potential shelf life of supplements. It discusses the various important
aspects that need to be taken into consideration when designing a stability study.
As the product category of supplements covers a wide variety of product forms and
compositions, a stability study has to be developed around the specific form, composition
and proposed packaging for the product. This document gives guidance on the resolution
of issues that may arise in relation to the stability of a particular product.
‘Stability Testing for Shelf Life Determination of Supplements’ is intended as guidance and
in no way should be taken as prescriptive.
9
2. Stability by
Design
The principles of ‘Quality by Design’ should be applied to supplement products. During

the development of a product the proposed formulation should be critically reviewed to
assess the impact on its stability by:
– the combination of the proposed components in terms of moisture levels, the known
interactions between certain ingredients and the possible effects on stability if the
ingredients are used where critical parameters (for example, moisture) are at the
extremes of their specification ranges.
The formulation should also be examined for potential chemical interactions between
components in the same product matrix.
– the handling of the product throughout the manufacturing and packaging operations,
including the periods when the product can be exposed to ambient climatic conditions,
such as can occur between the manufacturing and packaging stages.
– the barrier properties of the proposed packaging in terms of material porosity to air
(oxygen) and moisture, seal integrity, head space of containers, barrier properties in
terms of ultra-violet light transmission, and the potential for migration of substances
(for example, label glue solvents).
– the intended storage, distribution including transportation, and anticipated retail

storage conditions. If the product is likely to be exported from the country of origin, the
environmental conditions in the receiving countries should be taken into consideration.
– the amount of additional active ingredient (overage) required to ensure that the labelled
amount is still present at the end of shelf life.
The failure to consider all of the above at an early stage of the product development
can potentially lead to a product recall/withdrawal from the marketplace or a significant
reduction in the shelf life. Both of these situations can have serious commercial
consequences.
(See also Chapter 12)
11
3. Mechanisms
of Product
Deterioration
During each stage of product development and before undertaking any form of stability
testing, it is essential that all the known mechanisms of product deterioration are fully
understood and, where possible, counteracted.
There are a number of chemical and physical effects that, singly and in combination, can
affect the shelf life of a supplement
A supplement formula may be comprised of a mixture of a large number of diverse

chemical substances (active ingredients, technological additives, flavours, colours, carriers
and bulking agents), all of which can potentially affect the shelf life of the product.
It is important that the main mechanisms of deterioration are fully understood in the
context of the product under development. Some of the important aspects are described
below and an outline of the affect of factors on the stability of different vitamins is given in
Annex III.
3.1 Moisture/water vapour content and transfer

Many of the processes of deterioration in supplements can be associated with moisture
and its gain, and sometimes loss, in a product.
Uncontrolled variations in moisture content can cause physical and chemical changes in
the product, alter the organoleptic characteristics such as flavour, colour and texture and
promote microbiological growth.
To ensure maximum stability of a product, moisture must be controlled at every stage

of the product life, from the selection of ingredients through to consumption by the
consumer.
Moisture and water vapour exchange can be either between the product and its
environment or by movement within a product. In the latter case, the moisture content and
its origins in the final product becomes a critical factor. For example, a high moisture level
in a cereal-starch based carrier could affect the stability of other ingredients.
For supplements marketed in a dry form such as tablets, hard-gel capsules and powders,
the moisture content of the ingredients should be at the lowest practical level, consistent
with the manufacturing process for the product. In some cases it may be necessary
to select ingredient batches/lots with the lowest moisture content, or even to consider
additional drying of the ingredient.
3.2 Temperature
Temperature changes can, in many ways, have a very profound influence on the nature
and rate of deterioration caused by other physical or chemical mechanisms.
13
3. MECHANISMS OF PRODUCT DETERIORATION
As a generalisation, an increase in the temperature increases the rate of a chemical

reaction. This effect forms the basis of most of the stability studies, as the higher the
temperature, the shorter the potential shelf life.
A further consideration is that fluctuations in storage temperature can affect product

packed in moderate to high ambient humidity conditions. These fluctuations may
precipitate condensation in the container, providing localised conditions for chemical
reactions or microbiological growth. This is a particular risk where there is a large head-
space in the container. Temperature cycling may also impact the pack seal integrity of a
package, due to thermal expansion and contraction of the packaging materials.
3.3 Oxygen
In terms of supplement stability, oxygen is one of the more critical factors, as many
common active ingredients found in supplements, such as vitamins and fish oils, are
susceptible to oxidation. For example, 11.2mg of ascorbic acid is destroyed by 1.0mg
of oxygen. To put this into perspective, if a liquid supplement is made with un-deaerated
water (that is, water saturated with air), the oxygen present in the product is sufficient to
oxidise up to 75mg ascorbic acid (vitamin C) per litre.
Where ingredients that are susceptible to oxidation have to be included in the product,
suitable precautions may have to be taken to ensure product stability. Examples of such
precautions include minimising the head-space of a container, using packaging with a
good oxygen barrier or packaging with an inert gas, such as nitrogen.
A specific problem with fats and oils, and particularly some nutritional oils, is rancidity.
Oxidative rancidity develops either via metal ion catalysis or by enzyme-initiated oxidative
degradation. The latter involves lipoxygenases, which are widely distributed in plant and
animal sources.
Whether or not the autoxidation is initiated by an enzyme or a metal ion such as Cu2+, the
intermediate is a hydroperoxide which, when cleaved to form smaller molecules such as
volatile aldehydes and ketones, contributes to the typical rancid off-flavours.
3.4 Oxidising/Reducing Agents

The presence of strong oxidising or reducing agents in a multi-component formula can
have a deleterious effect on the stability of a number of active ingredients, particularly
vitamins. For example, vitamins A, D and C are very sensitive to oxidants, whilst vitamin
B12 is sensitive to reducing agents. The Fe2+ ion, such as in iron (II) sulphate, is a
common reducing agent, as are a number of organic acids. It is essential that proposed
formulations are checked at an early stage of development to remove these potential
sources of instability.
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3.5 Light
A critical factor in the stability of a supplement is light, particularly the ultra-violet
component. Light can play a number of roles in the deterioration of a product and can
affect the organoleptic properties and label claims.
Exposure to light can reduce the activity of a number of vitamins, including riboflavin and
vitamins A (as retinol or retinyl palmitate), D and K.
A number of colours and other nutrients such as the chlorophylls, turmeric (curcumin) and
betanin also have poor light stability. Adverse changes to these can have an effect both on
the product appearance and its label claims.
Studies have shown significant differences in the vitamin contents of supplement tablets
stored in clear glass bottles compared to almost identical amber glass bottles, with the
product in the clear glass containers losing vitamin activity at a faster rate than the amber
glass.
Photo-oxidation of lipids can also occur when a product is exposed to a light source whilst
on display in a retail outlet. Certain spectra of fluorescent lighting have been shown to
initiate photo deterioration. In addition, photo-oxidation of proteins, although less prevalent
than the photo-degradations outlined above, has been demonstrated in certain product
matrixes.
3.6 Chemical Hydrolysis

Depending upon the combination of components, chemical hydrolysis can occur in
liquid supplements. For example, under certain pH and temperature conditions, the
high-intensity sweetener aspartame will hydrolyse, resulting in a gradual reduction in
sweetness.
3.7 pH
The pH of a liquid supplement or the ‘apparent’ pH of paste-filled soft-gel capsules can
significantly affect the shelf life of a number of active components. For example, about
half the vitamins are susceptible to low pH conditions, whilst the other half are affected
by high pH. Thus, in the case of a multivitamin liquid supplement, the pH may need to
be adjusted to around 7.0 in order to maximise shelf life. The pH of a liquid supplement
can also have an effect on the microbiological stability and on the stability of a number of
other ingredients and additives such as colours. There is also the problem that a number
of the preservatives used in liquid supplements can only work effectively within specific pH
ranges.
15
3. MECHANISMS OF PRODUCT DETERIORATION
3.8 Chemical Interactions

Chemical interactions between common ingredients is a large and diverse area that can
seriously affect the stability and physiological activity of a supplement. Those that have
been identified show that a number of unexpected chemical reactions can occur between
ingredients during the shelf life. Some common examples are given in Annex II.
As can be seen from these examples, there are a number of different chemical reactions
that can take place in a product during storage. Searches should be undertaken in the
scientific literature on the ingredients proposed for inclusion in the product, to ensure that
all known and established interactions are taken into consideration during the formulation
of the product. For example, ascorbic acid (vitamin C) is reactive both as an acid and as a
reducing agent and is implicated in a diverse range of interactions in supplements.
Due to the focus of attention over decades, many interactions have been identified for the
13 recognised vitamins. There is far less information available in the scientific literature on
the potential for interactions with many of the more recent popular active ingredients found
in supplements. However, from a review of the chemistry of some of these substances,
it is possible that similar reactions may be found. It is therefore very important that the
possibility of such interactions is considered and the molecular structure and the known
chemistry of the substance assessed, in the context of the product form and matrix.
In situations where there is a need to combine components which are known, or

suspected, to be interactive, it is advisable to use a coating for one or more of the
ingredients, or to select alternative forms of the ingredient.
For example, if ascorbic acid and certain iron salts are present in a supplement, there is
a high risk of an interaction. This can present as black spots on the surface of a tablet
or as a black deposit on the inside of the shell of a soft-gel capsule. A common solution
is to coat the iron source powder (a number of forms are commercially available) or,
alternatively, to use a less reactive iron source.
The need for a detailed assessment of the chemistry of a proposed formulation cannot be
over emphasised.
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17
4. Physical Stability
An aspect of stability that must also be considered during the early development of a
product is that of the physical characteristics of a product.
4.1 Tablets
The hardness and friability of a tablet can change significantly during storage and in many
cases these changes are related to the composition of the formulation, particularly the
use of carriers and additives which are inappropriate for the particular combination of
ingredients. The choice of the inert carrier/tablet base can affect the long-term physical
characteristics of a tablet, with some combinations of inorganic salts (such as phosphates)
contributing to increasing hardness with time. In extreme cases, this can reach a point
where the tablet fails to disintegrate.
In other cases, high moisture levels in ingredients and in the head-space of packaging can
lead to a softening and increased friability of the tablet. Interaction of ingredients with film
coatings can also cause extension of disintegration times and/or discolouration.
For modified release tablets, the dissolution profile may change over time and it is
important to include an appropriate test in a stability study for this type of product.
4.2 Capsules
A physical/chemical problem that is often under-appreciated is cross-linking of the gelatin
in both hard and soft gel capsules. Cross-linked gelatin can very considerably increase
the dissolution time of a capsule and, in more extreme cases, prevent the capsule from
dissolving in the human gut.
Cross-linking arises from the polymerisation of the gelatin. This reaction can be facilitated
by high temperature, high humidity, ultra-violet radiation and a range of substances that
can be present either in the capsule shell or, more commonly, in the capsule fill.
Gelatin cross-linking can be catalysed by a number of chemical substances either found

in the capsule ingredients or present as impurities of the components. Common chemical
groups are the aldehydes and ketones, which can be found in a number of botanicals,
marine oils and marine oil concentrates used in supplements.
It has been demonstrated that many compounds frequently used in supplement capsules
such as polyethylene glycols, polysorbates and esters of unsaturated fatty acids can
undergo autoxidation to form aldehydes of higher molecular weight. These can react with
the gelatin in the capsule shell, particularly in soft-gel capsules and result in a product with
poor dissolution/disintegration.
A number of commonly used food colours have been shown to interact with gelatin via
hydrophobic and hydrogen bonding.
19
4. PHYSICAL STABILITY
In addition, leaking capsules caused by poor manufacturing practices (for example, over-
drying or inappropriate formulation) can be an issue for the physical stability of soft gel
capsules.
It is important that all new supplement formulations using gelatin capsules are carefully
evaluated for the potential of components to induce cross-linking in the capsule shell or
other physical defects. Additionally, the specification and purity criteria for the ingredients
should be evaluated for possible components or contaminants that could cause reactions.
It is also important that dissolution/disintegration tests are carried out on capsules during
both accelerated and real-time stability studies and also on retained samples from
production batches.
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21
5. Product
Verification
Stability testing can be costly, both in a monetary sense and time. It is important that
consideration is given to all the many factors that can affect stability and shelf life before
the stability studies are commenced.
A stability study should be the final confirmation that the product in a particular packaging
will remain stable throughout the length of its intended shelf life. This requires that all
possible sources of instability in terms of the formulation, processing and packaging have
been evaluated and necessary action taken before any stability tests are undertaken.
This may require that smaller, selective studies are carried out before the formulation is
finally agreed, to check for potential sources of instability, such as ingredient interactions.
Similarly, small scale tests may be needed to ensure that the selected packing is suitable
for the formulation in terms of oxygen, moisture and light barriers.
23
6. Test Parameters
for Stability Testing
When designing a stability test, it is essential that all the parameters tested are both
appropriate for the product form (e.g. tablet, capsule, liquid) and are also a qualitative or
quantitative measure of product stability. All parameters selected should be able to be
used to detect changes to the physical, chemical and microbiological characteristics of the
product during the storage period.
6.1 Selection of test parameters

The selection of the parameters will be dependent on the type of study being conducted
and it may be that not all of the parameters are necessary for a particular supplement. Also,
not all parameters need to be tested at all time points in a study. For example, for many
tablet and capsule formulations, the risk of microbiological growth is very low and tests may
only need to be carried out at the beginning and end of the study. By contrast, certain liquid
supplements may require microbiological tests at every time point. See Annex IV for further
detail on the microbiology of supplements.
As a guide, the following lists are an indication of parameters that could be considered for
each supplement form.
a) Tablets
• Appearance, colour, odour, taste and/or texture
• Chemical fingerprints
• Assay [i.e. label claim ingredient(s)]
• Ingredient degradation products
• Hardness/friability
• Disintegration or dissolution rate
• Moisture content/loss on drying
• Fat oxidation (where applicable)
• Microbial content (e.g. Total Bacterial Count, Total Yeast and Mould count etc.)
b) Capsules (hard and soft shell)

• Appearance, colour, odour, taste and/or texture
• Dissolution rate
• Capsule integrity such as brittleness or leakage
• Moisture content/loss on drying
25
6. TEST PARAMETERS FOR STABILITY TESTING
c) Liquids and semi-solids (gels)

• Appearance, colour, odour, taste and/or mouth feel
• pH
• Viscosity and/or texture
• Moisture content and/or water activity
• Preservative content
• Microbiological challenge test / Preservative efficacy test (where applicable)
• Emulsion stability (where applicable)
• Precipitation
d) Powders
• Appearance, colour, odour and taste
• Moisture content and/or water activity
• Hygroscopisity
• Particle size distribution
• Powder flow/adhesion properties
6.2 Analysis of parameters

For products where there are quantitative label claims for one or more active ingredients,
tests for the active components should be carried out at all time points and the values
plotted on a time-line to ensure any changes are identified at the earliest point.
For a product with a number of claimed active ingredients, such as a multivitamin, it is

advisable to assay for all the active components unless there is previous knowledge that
there are one or more which are stable beyond the anticipated shelf life in the particular
matrix. Minerals and trace elements provide an example where there may be prior
knowledge of good stability properties. In such cases, consideration could be given to
testing for content at the start and end of the study only.
26
27
7. Shelf Life Studies
The primary requirement of a shelf life study for supplements is to confirm that the product
can sustain a commercially realistic shelf life. In other words, it should confirm that all
label claims can still be met at the end of the shelf life and that there are no obvious or
unacceptable organoleptic changes during the declared shelf life of the product.
Whilst it is a part of good manufacturing practice (GMP) that it can be demonstrated that
a product should meet qualitative and quantitative specifications throughout its shelf life,
some countries may have specific requirements. In those countries where supplements
are required to be registered there is often a requirement for ‘ongoing stability’, whilst
others may specify that stability data is generated on more than one batch of the product.
Other economic areas and countries may require that the date of the product’s durability
(expiry dating) is declared on the label. It is therefore important that the design of the shelf
life study should accommodate the requirements of the countries in which the product is
intended to be marketed.
7.1 General criteria

Shelf life studies for the purpose of setting an expiration date for a product should be
carried out in the proposed retail packaging. Whenever possible, the processing and
packaging of the test samples should simulate those proposed for the commercial
production of the product.
As has been already stated, all shelf life studies are expensive, so it is important that
sufficient thought is given to the design of the study. Studies for supplements containing a
number of ingredients subject to quantitative claims may have to be customised to meet
the specific needs of the product matrix and content.
The properties and parameters selected for testing during a shelf life study should include
those characteristics that may be subject to change during storage and which could affect
the label claims, product quality, safety and/or efficacy.
When possible, provision should be made to test and monitor all those ingredients subject
to a quantitative label claim at all time points, unless there is good documented evidence
that they are unlikely to be significantly affected by the storage conditions. For example,
most of the chemical forms of the mineral and trace element sources are relatively stable,
whereas in many circumstances the vitamins can be regarded as inherently unstable.
7.2 Analytical requirements

After the selection of the test parameters, it is essential to ensure that the analytical
methods are appropriate and are validated for the particular product matrix.
It is important that all analytical procedures are critically reviewed in the context of the
product composition, to ensure that none of the components has the potential to interfere
29
7. SHELF LIFE STUDIES
with the results. The analysis of some active ingredients in certain matrices can be difficult;
for example, the levels of vitamin D in multivitamin tablets and capsules. Where practical,
assays should be carried out on a number of samples of the product to be used before
the stability study commences. This helps to identify the range of analytical variation that
could be expected and this information may be important when assessing the results of
the study. Every effort should be made to minimise analytical variation. For example, where
possible, the same technician and instrumentation should be used for the analysis of the
samples from all the time points of the study.
It cannot be overemphasised that care should be taken to standardise the analytical

procedures as much as possible, as it has been shown that variations in technique,
extraction procedures, and even in analytical reagents can invalidate the results of a study.
7.3 Product samples

Before proceeding with the selection of samples for a stability study, the product should
first be verified to confirm the batches meet the expected specification, as outlined in
Section 5 of the IADSA Good Manufacturing Practice Guide for Supplements (see Annex
VII). Ideally, the analyses should be on samples from three consecutive batches.
The product samples selected for a stability study should be taken from batch(es)/lot(s)
which are considered to be truly representative of that to be sold commercially. Batches
should not be selectively chosen on the basis of specific parameters. Samples should also
be checked to ensure homogenous distribution of the ingredients, particularly those that
are the subject of label claims.
The packaging used for the samples in the study should be identical to that intended to
be used for the commercial product in terms of internal volume, barrier and migration
properties and seal integrity.
Where products are intended to be sold in more than one pack size, both the smallest
and largest of the packs should be tested. This is necessary as the larger pack sizes could
have a greater relative head space or packaging surface area, factors which can affect
stability.
With shelf life testing, and particularly accelerated testing of multiactive products (see
Section 8.2), it is advisable to budget for more, rather than less, testing. It is also advisable
to place additional packs of the product in storage (as reserve samples) so that one extra
pack can be removed at each time point to be stored at low temperature (circa 1ºC) to
allow for re-testing in the event of future stability anomalies.
It should be borne in mind that shelf life studies can be one of the most expensive aspects
of product development, both in terms of financial cost and time. It is therefore important
that provisions such as the addition of reserve samples, as described above, are given
due consideration.
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31
8. Types of
Stability Studies to
Support Shelf Life
Determination
There are a number of types of stability studies and the supplement manufacturer should
select that which is most appropriate to the required results.
The studies can fall into the following categories:
– Stress Testing, to determine an ingredient’s or combination of ingredients’ susceptibility

to degradation.
– Accelerated Testing, where the storage conditions are enhanced to accelerate the rate
of any changes, and to reduce the study time.
– Long-Term Testing, where the storage time is at, or over, the intended shelf life. This is
also known as ‘real time’ testing.
– In-Use Study, which is used to simulate the stability of the product after opening and
during use by the consumer.
– Transit (Excursion) Testing, which is used to simulate the transportation and storage
conditions during product distribution.
The main testing regimes are explained in the following sections.
8.1 Stress testing

Stress testing is used to determine a material’s susceptibility to degradation caused
by elevated temperature, humidity, light, acidic or basic conditions, and/or oxidising or
reducing substances.
One common procedure is to store the material in open containers under a variety of
temperature and humidity regimes, for example with temperature increasing in 10°C
increments above 40°C and humidity increasing above 75% relative humidity (RH). This
type of test is called an “open dish” study and serves as a worst-case scenario because
the open container provides little or no protection from the environment.
Another common procedure is to store portions of the material in solution or suspension

across a wide range of pH values, or combined with a variety of known oxidising or
reducing substances to evaluate the effects of acids, bases, oxidants and reductants
on the material.
Stress testing is normally conducted over short periods of time ranging from days to a few
weeks. The stress test is normally conducted on ingredients, rather than supplements,
and is helpful in deciding how best to formulate, package, and label supplements
containing the tested ingredient.
Stress testing normally does not yield results which can be extrapolated to establish an
appropriate shelf life for the commercial product, except in cases where the product
33
8. TYPES OF STABILITY STUDIES TO SUPPORT SHELF LIFE DETERMINATION
formulation, packaging, and storage instructions are chosen to ensure any sources of
degradation identified during the stress test are strictly avoided.
8.2 Accelerated testing

Accelerated testing utilises the fact that, in general, the rate of a chemical reaction doubles
with approximately every 10°C rise in temperature. Therefore, accelerated studies involve
storage of the product at temperatures considerably above the expected ambient storage
in the market place. Thus, if the ambient average temperature is 30°C, the accelerated
storage should be at temperatures of 40°C and above.
The accelerated studies should be carried out at two or more elevated temperature
points, particularly where extended shelf life is required. A study where there is only one
temperature point at 10°C above the ambient may in some cases only give confidence of
2 x accelerated storage time. That is, if the accelerated storage time for certain products is
6 months, the confidence for the shelf life is only 12 months. As a consequence, if a study
has to be limited to only one temperature point, then the temperature selected may need
to be 15°C above recommended storage conditions.
Accelerated testing can also be used to evaluate the effect of brief excursions outside the
desired storage conditions, for example during transportation, when the product may be
subject to greater extremes of temperature.
The advantage of accelerated testing is that it provides preliminary shelf life data in a
relatively short time frame; however, data from accelerated testing should be confirmed
through long-term shelf life testing, since the assumptions involved are not always reliable.
Accelerated testing may also be unreliable where the harsh accelerated conditions cause
physical changes in the product, such as melting, softening, cracking, or phase separation
or for temperature sensitive active ingredients.
8.3 Long term testing

Also known as “real time” testing, long term testing is performed on ingredients or
supplements stored under the same environmental conditions as commercial batches and
for lengths of time similar to those recommended for the shelf life of commercial batches.
The environmental conditions should be representative of regional conditions in which the

product is to be sold. For example, product destined for a tropical region will have higher
temperature and humidity requirements than those distributed in more temperate climates.
Conditions such as temperature should be maintained and controlled for the length of the
study. The use of stability chambers or dedicated and controlled rooms should be used,
where practical.
34
Long term testing may include retesting of retained samples and retesting of ingredient or
supplement batches remaining in stock. However, such tests may be of limited value if the
storage conditions are uncontrolled or unknown.
Long term/real time testing provides more reliable shelf life information than accelerated
testing.
8.4 In-use testing

In-use studies are used to evaluate the stability of a material in a multi-dose container once
the container has been initially opened. The repeated opening and closing of the container
increases the material’s exposure to oxygen, moisture and microorganisms that may cause
important changes during the period of time the container is used. This is due to the air
changes that can take place each time the container is opened to remove a dose.
It is recommended that at least two batches are studied, with one chosen towards the
end of its shelf life. If the material is sold in different strengths or container sizes, the in-use
study examines the configuration in which significant changes are most likely to occur, such
as the largest of the containers in a range of sizes.
The study is designed to simulate the actual conditions of use of the product, including
the normal environmental conditions of storage and use, the ongoing reduction in fill level
during the course of use, and any dilution or reconstitution which occurs prior to use. The
appropriate physical, chemical, microbiological or other specifications are examined at the
beginning and end of use as well as at appropriate intermediate time points.
Where the product is known or is expected to be susceptible to microbiological growth, it

may be advisable to also conduct microbiological challenge testing using organisms which
could be potential contaminants.
8.5 Transit (excursion) testing

Transit testing, or excursion testing, investigates the possible effects on the product of
environmental challenges introduced during distribution and transportation. This may be of
particular interest where a product is likely to be transported across or between continents.
A transit test study aims to simulate the extremes of conditions, such as temperature and
humidity, that are expected to be encountered by the product during distribution. The
ambient temperatures and humidities of the countries and regions to which, and through
which, the product is transported should be taken into consideration.
Transit testing uses the normal stability testing procedures, but with the cycling of the
environmental conditions to simulate the distribution conditions as closely as possible.
35
9. Shelf Life
Evaluation
The shelf life of a product is determined by the stability of the most unstable ingredient in
the context of the formulation selected, the packaging used and the anticipated storage
conditions.
One of the most important elements of a stability study is the scientific evaluation of the
data generated by the study, using applicable statistical techniques.
9.1 Analysis of appropriate stability data

The design of the stability protocol should be such as to provide the data from the study in
a form and quantity that permits a realistic statistical analysis.
This requires that a systematic approach is adopted in the presentation and evaluation
of the information derived from the study. The totality of the information should be
considered and this should include, where appropriate, results from physical, chemical
and microbiological testing, including tests related to particular attributes of the dosage
form (for example, disintegration/dissolution rates for oral dosage forms).
The data generated from the studies and, where appropriate, any supporting data, should
be evaluated to determine the critical attributes which are likely to influence the quality of
the product during the anticipated shelf life. Each attribute should be assessed separately
and then collectively, and an overall assessment of all the findings should be made before
a shelf life/expiry date is predicted.
The shelf life proposed should not exceed that predicted for any single attribute.
Before the evaluation is commenced, it is necessary that the physical chemistry and
thermokinetics of the major ingredients, particularly those with label claims, should be
understood. For example, the degradation of most of the vitamins follows ‘first order’ or
‘zero order’ kinetics. This knowledge allows predictive statistical models to be used, such
as those based on the Arrhenius Equation (see Annex V).
The understanding of the kinetics is particularly important when interpreting the data
from short-term stability studies, such as accelerated studies, where the shelf life has to
be based on an extrapolation of trends identified over relatively short periods (i.e. 3 to 6
months). A failure to use appropriate statistical models can lead to invalid extrapolations.
The precision of a statistical model has been found to be related to the number of storage
temperatures that can be used for the short-term study and the number of samples
that can be taken from each temperature at each time point. Ideally, at least three
temperatures at 5ºC or 10ºC increments should be used for a 24 week study on a multi-
active product with a commercial requirement of a 2 – 3 year’s shelf life.
Such a study, when properly conducted, can provide sufficient data to allow analysis using
the Arrhenius equation to enable shelf life predictions to be made for the individual actives
37
9. SHELF LIFE EVALUATION
and to enable estimates to be made of the ‘overage’ requirements for the less stable
components. It can also help to identify potential stability problems.
Although it can be demonstrated that the technique may have some limitations,
experience has shown that, if all experimental controls are maintained, useful predictions
of a product’s stability can be obtained.
9.2 Extrapolation of stability data

Extrapolation is the practice of using known data to predict or infer future data, particularly
in terms of trends.
The principle of extrapolation forms the basis of the accelerated study, in that the data
obtained from a 3 or 6 month test is used to predict the behaviour of the product over two
or three years.
Whether the extrapolation of stability data is appropriate for the product depends upon the
extent of the information available about the change pattern in the product, the goodness
of fit of any mathematical model and the existence of any relevant supporting data (for
example, from studies on similar products or from the scientific literature).
An extrapolation assumes that any change patterns identified in the short-term studies
will continue to apply until the end of the period of the anticipated shelf life, provided that
the external conditions (temperature, moisture, oxygen, light etc.) all remain within an
acceptable range.
A shelf life proposed on the basis of extrapolation should always be verified by additional
long-term stability data as soon as such data becomes available.
38
39
10. Stability Study
Protocols
The objective of a long-term (real time) stability study is to simulate the conditions to which
the product, in normal circumstances, would be subjected between manufacture and retail
sale, assuming package integrity throughout that period. The data obtained from the study
allows the manufacturer to set or confirm the shelf life and expiry date of the product.
Protocols for long-term stability studies should be set up so that the study is continued to
at least the anticipated shelf life of the product and preferably to a point past that time.
The objective of an accelerated stability study is to subject the product to stress in

the form of high temperature and high humidity (moisture), in order to induce potential
reactions and interactions over a short time period.
With accelerated studies a number of assumptions have to be made from the limited
available data.
When determining the storage conditions to be used for the study, they need to reflect the
temperature and humidity conditions in all intended markets.
For example, for the Northern European market, the accepted ambient temperature
for the purposes of the study is 25ºC and an ambient humidity of 60%RH. For markets
in tropical climates, and particularly those where a high proportion of retail outlets do
not have air-conditioned storage, the temperature and humidity base-lines have to be
increased to represent the higher ambient conditions.
As a general rule, the higher the ambient temperature to which the product is exposed,
the shorter the potential shelf life.
Nevertheless, it cannot be assumed that there is a linear relationship between the

temperature and a product’s shelf life. This is particularly so in the case of multivitamin
products, where some vitamins have differing degradation patterns.
10.1 Protocol development

There are a number of internationally recognised protocols for the conducting of stability
studies. However, many have their origins in the drug industry and have different objectives
and requirements than those for supplements. The selected protocol, including customised
protocols, should be in the form of a written procedure that describes in detail the shelf life
study programme used to assess the stability characteristics of supplements. The results of
the stability testing should be used in determining appropriate storage conditions and shelf
life. The protocol should be defined prior to study commencement (e.g. in the design of the
shelf life study or in applicable standard operating procedures etc.). This protocol should
include the following:
41
10. STABILITY STUDY PROTOCOLS
a. Name, strength and package quantity of each product included in the study
b. Type, size and composition of each container and closure system
c. Total number of containers required of each pack size to complete the study (including
reserve samples in case of re-test)
d. Procedures for confirming integrity of packaging seal closures
e. Recognition that the supplement should be tested in the same type of container-closure
system as that in which the supplement is/will be marketed
f. Each formulation included
g. Each batch code included
h. Sample size and test intervals (e.g. frequency of sample testing) for each attribute
i. Storage conditions of samples retained for testing
j. Test parameters
k. Acceptance criteria / critical values
l. Use of reliable, meaningful, specific and preferably validated test methods
m. Method of data evaluation, including any statistical analysis used to establish product
shelf life
Although the storage facilities for conducting stability trials can be of various types and
sizes, from cabinets to rooms, the facility should be capable of controlling the storage
conditions (temperature and humidity) within the ranges specified in the protocol for the
study. The storage facilities should be validated for uniformity of conditions throughout the
room/cabinet.
Provision should be made to monitor temperature, humidity and, where applicable, light
levels throughout the storage period. These records should be retained and documented.
Procedures should also be developed for dealing with unexpected episodes, such as
equipment or power failure. This should include a maximum permissible time for a failure,
which should also be related to the period when the monitored conditions are significantly
outside the acceptable ranges. All such episodes should be described in the study report
and their potential effect on the outcome of the study assessed.
The protocol should additionally include some requirements and controls related to the
handling, intermediate storage and assay of the test samples after removal from storage.
42
43
11. Bracketing and
Matrixing
Bracketing, and its companion procedure matrixing, are internationally recognised

procedures for a reduced sampling plan for shelf life studies.
Unlike a full stability study design, in which samples for every combination of all the design
factors are tested at all time points, a reduced design is one where samples for every
factor combination are not all tested at all time points. A reduced stability design can be
a valid alternative to a full testing regime when multiple design factors are involved. The
reduced design selected should have the ability to provide data to adequately predict the
product shelf life.
11.1 Bracketing
Bracketing is where the stability testing protocol is designed in such a way that only
samples at the extremes of certain design factors, such as potency or container size, are
tested at all time points. This design assumes that the stability of any of the intermediate
levels is represented by the stability of the extremes subjected to testing.
Bracketing may be used where a study encompasses different potencies of identical or

closely related formulations where the same additives/carriers are used. It is also useful
where different packs of the same composition and closure system are required to be
tested in different sizes for different product fills.
Bracketing, whilst a useful tool, particularly in helping to reduce the cost of a study, should
be applied with caution. Before it is applied to a study, its effect on the confidence of the
shelf life estimations should be assessed.
In the case where the stability of the extremes is shown to differ, the intermediates should
be considered no more stable than the least stable extreme. In order to accommodate
such eventualities, the study should be set up as a full study design and the reduced
testing pattern applied to the selected extreme samples and time points. In the case
where anomalies are found, the testing of the intermediates can be carried out.
An example of where bracketing could be applied effectively for supplements would be for
a vitamin C product being sold at three different potencies (250mg, 500mg and 1000mg),
in three pack sizes, where both the formula and packaging composition are essentially the
same.
11.2 Matrixing
Matrixing is where a shelf life study is designed in such a way that a selected sub-set
of the total number of possible samples would be tested for relevant parameters at
a specified time point. At a subsequent time point, another sub-set of samples of all
combinations of factors would be tested.
45
11. BRACKETING AND MATRIXING
Such a design is based on the assumption that the stability of each sub-set of samples
tested represents the stability of all samples at a given time point.
Matrixing can be applied to different strength products with closely related or identical
formulations, or may include batches made by using the same process and equipment, or
container sizes/fill quantities with the same container closure system.
Where matrixing is appropriate to the study, it can lead to a one-third to one-half reduction
in testing. A ‘one-third’ reduction would initially remove the need to test one in every three
samples, with the consequent savings.
When considering whether matrixing is appropriate for the study, a number of factors
should be considered, such as:
• the expected stability of the product, based on previous data;

• knowledge of data variability;
• availability of supporting data; and
• stability differences in the product, within a parameter or among parameters.
As a general comment, a matrixing design may be applicable if the supporting data

indicate predictable product stability and/or when the supporting data exhibit only small
variability.
If the matrixing design is considered to be applicable, the degree of reduction of testing

that can be made from the full design depends on the number of factor combinations
being evaluated. The more parameters that are associated with the product and the more
levels in each parameter, the larger the degree of reduction that may be considered.
Whatever the degree of reduction of testing selected, the designs should have the ability
to adequately predict the product shelf life.
Due to the reduced amount of data collected, a matrixing design based on parameters
other than time points may, in general, have less precision in shelf life estimation than
a corresponding full design. There is a risk that it may lead to a shorter shelf life being
established. The statistics in a matrixing design should also have sufficient power to detect
certain effects such as interactions.
Whilst matrixing can have the advantage of reducing the test burden in a study, it should
only be introduced after a full consideration of its positive and negative aspects.
11.3 Points to consider

Both bracketing and matrixing may result in unanticipated ‘out-of trend’ results, which
can be caused by one or more poorly closed or badly sealed containers. This problem is
often difficult to eliminate, as testing the integrity of each container submitted for a stability
trial can be difficult, given that most common forms of integrity testing of packaging are
destructive (for example, vacuum or water ingress methods).
46
Close attention to product total pack weights, especially for liquids, and individual tablet /
capsule weights is often critical. If unreasonable weight gain or loss can be demonstrated,
there may be good justification for challenging the integrity of a particular pack and
treating the results as an outlier.
It is important that the rationale for selecting the bracketing or matrixing procedure is
clearly documented.
Both bracketing and matrixing are described in more detail in Annex VI.
47
12. Use of
Published Sources
on Stability
It is strongly recommended that a literature search be carried out before undertaking the
formulation of a new supplement product to ensure stability by design (see Chapter 2).
It can also be helpful to undertake a literature search before preparing a protocol for a
stability study.
Many stability problems commonly found in supplements can be avoided, if a

comprehensive review of the scientific literature is undertaken prior to formulation and
shelf life testing.
12.1 Before formulation of product

The scientific literature contains important information and data on the stability of vitamins,
both individually and in combination with other vitamins, and also on the stability of the
vitamins in different supplement matrices. This information can be useful in determining
potential overages (see Chapter 2).
When formulating a new product, it is important that potential interactions between the
ingredients are researched, as these can have a very significant effect on the stability and
shelf life of a product.
Where there is a choice of different forms of an ingredient, such as glucosamine sulphate

and glucosamine hydrochloride, it is prudent to investigate the relative stability of these
forms. There are a number of publicly available scientific papers that discuss the relative
stability of glucosamine salts.
12.2 Before shelf life testing of product

There are two key aspects that may benefit from a literature search when developing a
shelf life study protocol:
1. Not all methods of analysis for the active components in supplements apply without
modification to all product matrices. The use of an inappropriate analytical method may
invalidate the results of a study. For example, modification to the extraction procedures
for vitamins may be necessary for some tablet matrices.
2. The statistical methods to be used for evaluating the results of the shelf life study should
be appropriate for the study being undertaken.
49
ANNEX I
Glossary of Terms
50
Autoxidation [of an Spontaneous oxidation of an ingredient at ambient temperatures

ingredient] in the presence of oxygen.
Batch See ‘Lot’.
Disintegration The time taken for a supplement dosage form to break up

into particles of a specified size (or smaller) in a solution under
carefully controlled specified conditions. Normally applied to
tablets.
Dissolution A process by which a solid substance such as a supplement

capsule dissolves in the solvent to yield a solution. The
dissolution time is the time taken to achieve dissolution under
specified controlled conditions.
Finished product A supplement which has undergone all the stages of

manufacture.
Homogeneity Uniform in structure or composition throughout. When applied to

mixing, all parts of a multi-component mix should have the same
composition.
Hygroscopisity The ability of a substance to attract and hold water molecules

from the surrounding environment by absorption or adsorption.
Lot A quantity of any supplement produced during a given cycle of

manufacture and from a specific formulation order, that is uniform
in character and quality (the essence of a manufacturing lot is its
homogeneity).
Manufacture The complete cycle of production and quality control of a

supplement from the acquisition of all materials through all
stages of subsequent processing, packaging and storage to the
distribution or release of the finished product.
Manufacturer The person or business that is involved in the manufacture of a

finished product.
Microbiological Standard microbiological method designed to evaluate the

challenge test effectiveness of preservatives in supplements and foods
Mycology The branch of biology dealing with fungi. This includes moulds
found as spoilage organisms in supplements and foods.
51
ANNEX I GLOSSARY OF TERMS
Organoleptic Relating to the sensory qualities of a substance such as taste,

characteristics / odour, texture and colour.
properties
Overage The quantity of a substance above the amount claimed on the

label that is added to the supplement during manufacture to
cover losses that may occur from degradation during processing
and storage of the product.
Packaging All operations, including filling, sealing and labelling, that a bulk
product has to undergo in order to become a finished product.
Packaging material Any material, including printed material, employed in the

packaging of a supplement, such as containers, closures, bags,
packing, label materials (labels, inserts, etc.), seals, binding
materials, adhesives and tapes.
Potency In the context of supplements, the measurable activity of a

substance with a nutritional or physiological effect in a defined
weight/dose of a product.
Preservative efficacy See Microbiological challenge test.

test
Reducing agent An element or compound that loses (or donates) an electron

to another chemical species in a redox chemical reaction.
Also called a reducer or reductant.
Shelf life The period during which a finished product retains its specific
properties when properly stored.
Stability Ability of a substance to remain unchanged over time under

stated or reasonably expected conditions of storage and use.
Thermokinetics The study of the rates and types of reactions during the thermal
(of an ingredient) decomposition of a substance.
Water activity (aw) In the context of supplements, water activity is a measure of the
propensity for microbiological growth and chemical reactions.
The higher the aw, the more unstable the product may be.
52
53
ANNEX II
Chemical
interactions of
vitamins that may
affect the stability of
a food supplement
54
Below are some well-established examples of interactions of vitamins with other vitamins,
minerals or preservatives that may affect the stability of food supplements. The list is not
exhaustive and other interactions may emerge with greater knowledge over time.
For interest value, a few interactions are also included that have been noted in
experimental studies, but which have not been established as occurring in final products.
i) Vitamin – Vitamin Interactions

− A
scorbic acid and Folate
Cleavage of folic acid molecules can occur in liquid supplements where ascorbic acid
is also present.
This is due to the reducing effect of ascorbic acid. This effect is most rapid in the pH
range 3.0 – 3.3 and slowest at pH 6.5 – 6.7.
− A
scorbic acid and Vitamin B12
Vitamin B12 (as cyanocobalamin) has been shown to be unstable in the presence of
ascorbic acid in aqueous solution. The instability has been shown to be pH dependent.
In relative terms, cyanocobalamin has been found to be more stable in the presence of
ascorbic acid than hydroxycobalamin.
− T
hiamin and Folic acid
The stability of folic acid can be significantly affected in the presence of thiamin,
particularly in solutions in the pH range 5.9 – 7.0.
It has also been found that the decomposition of thiamin in solutions can affect the rate
of the breakdown of folic acid, which is accelerated in the presence of the degradation
products of thiamin, particularly hydrogen sulphide.
− T
hiamin and Vitamin B12
The decomposition of thiamin can increase the rate of breakdown of cyanocobalamin
due to substances formed during thiamin cleavage.
− R
iboflavin and Thiamin
Riboflavin can exert an oxidative action on thiamin leading to the formation and
precipitation of thiochrome.
This reaction appears to be specific to solutions containing the B-vitamins and is not
seen in solutions containing riboflavin, thiamin and ascorbic acid.
− R
iboflavin and Folic acid
The stability of folic acid can be affected by the combined actions of riboflavin and
light, which can produce an oxidative reaction resulting in the cleavage of the folic acid.
This effect occurs most rapidly at pH 6.5 and can be reduced, but not eliminated, by
deaeration.
55
ANNEX II CHEMICAL INTERACTIONS OF VITAMINS THAT MAY AFFECT THE STABILITY OF A FOOD SUPPLEMENT
− R
iboflavin and Ascorbic acid
The oxidation of ascorbic acid exposed to light can be catalysed by the presence of
riboflavin, which acts as a light-energy receptor.
− R
iboflavin and Niacinamide
Although not so applicable to stability, it has been observed that the presence of
niacinamide at concentrations above 1% in aqueous solution can increase the
solubility of riboflavin.
This is possibly due to a complex formation between the two vitamins.
− N
iacinamide and Ascorbic Acid
A niacinamide-ascorbic acid complex, containing one molecule each of niacinamide
and ascorbic acid, forms readily in solution by what appears to be a charge-transfer
reaction.
Pre-forming of this complex may prevent difficulties with thickening and hardening of
the mixtures employed in soft gelatin capsules.
− N
iacinamide and Folic Acid
Although not so applicable to stability, it has been observed that niacinamide acts as a
solubiliser of folic acid.
A 10% solution of niacinamide can maintain a concentration of 5 mg/ml of folic acid at
pH as low as 5.6, whereas the normal solubility of folic acid at pH 6.0 is 2 mg/ml.
ii) Vitamin – Mineral Salt Interactions

− A
scorbic acid and metal ions
Traces of a number of metal ions can catalyse the degradation of ascorbic acid.
Studies have shown that the order of effectiveness of the main metallic ions is Cu+2
> Fe+2 > Zn+2 and that the presence of copper ions as low as 0.85ppm (mg/kg) was
sufficient to catalyse oxidation of ascorbic acid.
− A
scorbic acid and Iron salts
The presence of ascorbic acid and iron salts in tablet and capsule formulations can
result in black spots appearing in the product.
This can be avoided by coating the iron source.
− R
etinol and Trace minerals
The oxidation of retinol and its esters is catalysed by the presence of trace minerals in
a formulation.
− T
hiamin and Copper ions
The stability of thiamin can be adversely affected by the presence of copper ions.
This appears to occur mainly when the copper is capable of forming complex anions
with other constituents of the matrix.
56
iii) Vitamin – Preservative Interactions

− T
hiamin and Sulphites
Thiamin is very sensitive to sulphites and bisulphites, both of which cleave the
molecule.
The presence of sulphites in glucose syrup, glucose powder and fruit juices or
concentrates used as flavours, can be sufficient for this reaction to occur.
− A
scorbic acid and Benzoates
The presence of both ascorbic acid and benzoates in liquid products can result in the
production of benzene.
iv) Other vitamin interactions indicated by experimental studies

− N
iacinamide, Riboflavin-5’-phosphate sodium and Ascorbic acid
Niacinamide added to a solution of riboflavin-5’-phosphate sodium and ascorbic acid
significantly increased the loss of riboflavin-5’-phosphate during photolysis, whereas
added tryptophan stabilized both vitamins.
− P
yridoxal-5-phosphate and Thiamin, Thiamin diphosphate, Riboflavin-5’-
phosphate, Cobamamide, Pyridoxal or Pyridoxine
The stability of pyridoxal-5-phosphate is poor at pH 6 in aqueous solution.
An increase in the degradation rate can be caused by thiamin, thiamin diphosphate,
riboflavin-5’-phosphate, cobamamide, pyridoxal or pyridoxine.
− E
rgocalciferol and Ascorbic acid, Folic acid, Thiamin hydrochloride or
Pyridoxine hydrochloride.
Ergocalciferol in powder preparations may be readily isomerised by ascorbic acid, folic
acid, thiamin hydrochloride or pyridoxine hydrochloride.
57
ANNEX III
Comparison of
Stability of Vitamins
58
Below are some examples of how different factors may affect the stability of the 13 recognised vitamins and beta-carotene. In some
cases, one factor can influence the effect of another. The degree of influence of the different factors will depend upon the product matrix.
Temperature Light Atmospheric Humidity pH <7 pH >7 Oxidising Reducing Metallic Ionising Additional
(UV) oxygen agents agents ions radiation information
Vitamin A uu uuu uu u uu u uuu u u u Susceptible to

isomerisation
B-Carotene u u u
Vitamin D uu uuu u u uu uu uuu u Susceptible to
isomerisation
Vitamin E uu uu u u uu uu u u Vitamin esters

very stable
Vitamin K u uu u u u uuu uu u u Susceptible to

isomerisation
Vitamin C uu uu u uu uuu uuu uuu u Cu and Unstable with

Fe dissolved oxygen
in solutions
Thiamin uuu uu uu u uuu u u u Cleaved by
(Vitamin B1) sulphites
Riboflavin u uuu u u uuu u uu

(Vitamin B2)
Niacin u u u u u u uu Normally very

stable
Vitamin B6 u uu u uu uu u u
Folic acid u uu u uu uu uuu uuu
Vitamin B12 u uu uu uuu uuu u uuu
Biotin u u u uu uu u u uu u? Inactivated by
avidin
Pantothenic uu u uu uuu uuu u u Free pantothenic

acid acid very unstable
59
Key: u Slightly sensitive uu Sensitive uuu Highly sensitive
ANNEX IV
Microbiology of
Supplements
60
Supplements come in a variety of physical forms such as tablets, hard and soft-gel
capsules, powders and various liquids. Each form has its own specific microbiological
concerns with regard to product stability. As a consequence, the microbiological
evaluation of a supplement for stability should be designed specifically for its physical form
and the relevant risks.
As a generalisation, microbiological contamination is mainly a problem in liquid and

moist products. Dry supplements such as tablets, capsules and most powders can be
considered to have a relatively low microbiological risk, provided the essential principles of
GMP are followed, including:
• The selection of ingredients with low microbiological counts;

• The sanitising of all production and handling equipment.
If the raw materials conform to realistic microbiological specifications, the main problems
that are likely to occur are:
• The use of unsanitised equipment;
• The handling of the product or ingredients by untrained personnel, for example:
o The handling of product without gloves;
o Personnel coughing or sneezing over exposed product.
IV.1 Microorganisms of Concern in Supplements

The microbiology of food can be classified into four main groups:
1. Spoilage organisms
2. Pathogenic or potentially pathogenic organisms
3. Organisms producing toxic metabolites
4. Organisms deliberately added for technological or nutritional purposes
IV.1.1 Spoilage organisms

The main concern with most foods and supplements is the presence of spoilage
organisms. These can contaminate liquid supplements and moist products such as fruit /
cereal bars.
The most common spoilage organisms in supplements are the yeasts and moulds, many
of which have airborne spores and are ubiquitous in an environment where the air is not
filtered. Yeasts can cause fermentation in the nutrient-rich liquid supplements.
61
ANNEX IV MICROBIOLOGY OF SUPPLEMENTS
A number of species of bacteria can also cause spoilage and, in general, whilst not
pathogenic, these bacteria can cause serious organoleptic problems in terms of adverse
changes to colour, odour and taste.
Spoilage organisms are the most important group to be monitored during stability studies.
IV.1.2 Pathogenic or potentially pathogenic organisms

Pathogenic and potentially pathogenic organisms are normally only found in supplements
if the product or one or more of its ingredients has been contaminated with the organism.
Contamination usually arises from animal sources such as rodents, flies and unhygienic
human practices. Routes of contamination from humans include:
• Unwashed/unsanitised hands;
• Coughing, spitting or sneezing in areas where the product is exposed.
The presence of pathogenic organisms in all forms of supplements is very rare. In the
small number of cases where they have been detected, the cause is invariably traced to a
breakdown of GMP systems.
From the point of view of stability, if human pathogens are not detected in the initial test
samples, it is unlikely that their presence will be found at the later stages of a stability
study.
IV.1.3 Organisms producing toxic metabolites

A number of microorganisms can produce toxic metabolites, particularly if certain
conditions are met. Probably the most well known bacterium in this class is Clostridium
botulinum.
Mycotoxins, which are produced by moulds/fungi on plant material, have recently

increased in importance, mainly due to growing knowledge of their carcinogenic potential.
Mycotoxins can be found to occur on many of the botanicals used in supplements,

particularly if Good Agricultural or Collection Practices (GACP) are not observed.
If all relevant botanical sources have been checked for mycotoxins before production,
there should be no further need to test for them during a stability study.
IV.1.4 Organisms deliberately added for technological or nutritional purposes

For centuries, microorganisms have been employed in food and drink production, such as
in the use of yeasts for baking and in the fermentation of alcoholic drinks. More important
to the supplement industry is the use of microorganisms for health purposes, mainly as
probiotics.
62
Stability studies on probiotic supplements need to be carefully controlled. It is strongly

recommended that real-time studies are carried out, as it is almost impossible to
extrapolate bacterial survival rates from accelerated studies.
IV.1.5 Microorganisms and stability studies

In terms of stability studies, it can be seen from the above that spoilage organisms are
normally of far greater concern than pathogens.
If the principles of GMP are followed, the microbiological status of all susceptible
ingredients should be checked before the product is manufactured.
If all the ingredients have been shown to be free of pathogens, there is a very low risk of
contamination during the processing and handling of most products.
IV.2 Chemical Preservation

With regard to spoilage organisms in liquid and moist products, it is likely that chemical
preservation will be necessary. This is particularly the case for products where there are
multiple servings in the container.
Before a liquid/moist formulation is confirmed, trials should be carried out to evaluate the
efficacy of permitted preservatives and their required levels.
Each group of preservatives (e.g. sorbates and benzoates), play a different role in
preservation, with some being more effective against yeasts and moulds and others
against bacteria.
For liquids where there is a high nutrient content, particularly carbohydrates, a blend of
chemical preservatives may be required.
IV.2.1 Microbiological Challenge Testing

There are standard microbiological methods designed to evaluate the effectiveness of
preservatives in supplements and foods, a technique generally known as Microbiological
Challenge Testing (MCT).
A challenge test can be defined as a ‘microbiological evaluation of a product’s ability to kill

or prevent the growth of microorganisms over a relatively long period of time’.
MCT involves four stages:
1. An appropriate experimental design relevant to the product and the required shelf life.
2. An inoculation procedure with the test microoganisms.
3. The test procedure.
4. The interpretation of results.
63
ANNEX IV MICROBIOLOGY OF SUPPLEMENTS
MCT should be carried out during the development stages of microbiologically susceptible
products.
Guidelines for the conducting of MCTs are available from a number of authoritative
organisations (see Annex VII).
IV.3 Microbiological Examination of Supplements

The microbiological examination of supplements should, in general, be carried out to the
standards and protocols for food.
IV.3.1 Testing for spoilage organisms for stability purposes

• Microbiological testing during shelf life studies should have a focus on spoilage
organisms.
• In the case of liquid and moist products, the mycology (particularly yeasts and moulds)
is a concern that requires monitoring.
• In terms of spoilage bacteria, Total Viable Counts should be enumerated.
IV.3.2 T esting for pathogenic and potentially pathogenic organisms for stability
purposes
• The presence of pathogenic and potentially pathogenic organisms should be
investigated at the start and the end of the study, but should not normally be required
at each time point in the study.
• If pathogens are not detected in the first examination, it is unlikely that they will be
detected in subsequent examinations, unless the product is not homogenous or has
been subsequently contaminated.
IV.3.3 Testing of different supplement forms
IV.3.3.1 Liquid and powder forms

• Standard methods for the microbiological analysis of foods can be used.
IV.3.3.2 Tablet forms

• Tablets can introduce problems in the analysis depending upon their composition,
as some formulae can contain a higher proportion of ingredients that are not water
soluble.
• In such cases, the tablets need to be ground under sterile conditions into a fine
powder, which needs to be kept suspended in the diluent when pippetting.
IV.3.3.3 Capsule forms

• Capsules, both soft gel and hard gel can be treated either as a single unit or as two
distinct components, the shell and the fill.
64
• If any microbiological contamination of a capsule is identified, it is often useful to test

the components separately to discover the source.
• When this is necessary, soft gel capsule shells should be cut aseptically with a
sterile scalpel and the contents emptied out. Both shell and the fill should be tested
separately.
• A similar procedure applies for hard gel capsules.
• An advantage of this approach is that it can more easily identify contamination of the
shell surface from post-production handling.
65
ANNEX V
Predictive Analysis
of Stability Data
66
One of the major problems affecting those working on product stability is making effective
use of the data generated.
Over the years, a number of statistical and kinetic models have been developed, and the
most commonly used is based on the Arrhenius Equation.
Whilst this technique has some limitations, it has been shown that, if all the experimental
controls are maintained, useful time predictions of product stability can be made.
In terms of physical chemistry, the degradation of most of the vitamins and a number of
other organic active ingredients commonly found in supplements follow ‘first order’ or
‘zero order’ kinetics. As a consequence, a classical Arrhenius model can be developed,
which allows predictions to be made on both shelf lives and overages for a product. This
has to be based on the following assumptions:
• That the model holds for all the reactions being studied;
• The same reaction mechanism occurs throughout the temperature range of the study;
• The energy of activation is in a defined range; and
• That the effects of moisture at ambient temperature are equivalent to maintaining the
same relative humidity at the higher temperatures.
V.1 Arrhenius Equation

A brief mathematical explanation of the Arrhenius Equation is given in Figure 1. The
activation energy (EA) is the energy barrier that the reactant substances must surmount
in order to react. Thus, the activation energy can be seen as an energetic threshold for a
reaction.
Figure 1: Mathematical explanation of the Arrhenius Equation
Rates of reaction (i.e. rates of change of concentration) are typically modelled using:
dc = – kc n
dt
where d
= differential c = concentration t = time n = order of reaction
k = rate constant
To employ this equation to find shelf life and suitable overages, it is necessary to
estimate the rate constant k for the reaction in question for any given room temperature.
67
ANNEX V PREDICTIVE ANALYSIS OF STABILITY DATA
The Arrhenius Equation uses the model:
EA
k = Ae –
RT
where R
= universal gas constant T = absolute temperature in kelvin
EA = energy of activation (the minimum energy needed for the reaction to occur,
expressed in joules per mole)
A = frequency factor / pre-exponential factor
e = mathematical quantity
If the Arrhenius Equation is used in the logarithmic form, it is possible to apply

experimentally established rate constants at known temperatures, and to extrapolate the
assumed linear relationship between the logarithm of the rate constant (ln k), versus the
inverse temperature, 1 to estimate the values of EA and A for the reaction in question:
T
EA 1
ln k = ln A – x
R T
Once the values of EA and A for the reaction in question have been found, it becomes
easy to estimate k for any given room temperature, and hence the rate of reaction, from
which shelf life may be calculated.
The approximation of the rate of a reaction doubling for a 10 degree rise in temperature
generally only applies to reactions with activation energies of about 50 kJ mol-1, relatively
close to ambient room temperature.
V.1.1 Application of Arrhenius Equation in supplement stability testing
The application of the Arrhenius Equation in supplement stability testing is relatively

straightforward. In the commonly used isothermal method, the product to be investigated
is stored under two or more controlled high temperatures, with all other parameters /
conditions remaining fixed.
68
The higher thermal exposure from the controlled high temperatures accelerates the
degradation of the product, and therefore allows the rate constants at each temperature
to be determined in a shorter time period.
Although it is possible to calculate the A and EA in the Arrhenius Equation by just using
the rate constants at two temperatures, it is more useful and reliable to determine at least
three rate constants at three different temperatures and determining the A and EA for
each.
Having determined A and EA, the Arrhenius Equation can be used to project the rate
constant at any temperature.
V.1.1.1 Theoretical example of predicted shelf life determination
Table 1 shows the effect of activation energy and storage temperature on the predicted
shelf life of product found to be stable for six months at 40˚C. For this illustration, three
activation energies were selected: 50, 83.144 and 100 kJ/mol/K.
The middle activation energy (83.144 kJ/mol/K) was selected because this is a ‘typical’
value recommended by the United States Pharmacopeia for the purpose of illustration.
Coincidentally, this is also the value needed to make EA/R = 10,000k-1.
Table 1. Effect of activation energy and storage temperature on the predicted shelf life of
product found to be stable for six months at 40°C*
EA = 50 kJ/mol/K EA = 83.144 kJ/ EA = 100 kJ/mol/K

mol/K
25 °C 15 months 2.5 years 3.4 years
30 °C 11 months 1.4 years 21 months
40 °C 6 months
50 °C 14 weeks 9 weeks 8 weeks
60 °C 8 weeks 4 weeks 18 days
70 °C 34 days 11 days 6 days
80 °C 21 days 5 days +
90 °C 13 days + +
69
ANNEX V PREDICTIVE ANALYSIS OF STABILITY DATA
+ For practical reasons having to do with the rate at which thermal equilibration with the
test environment can be achieved, exposure to high temperatures for less than several
days is not recommended.
* Porter W. R. Journal of Validation Technology [Summer 2011]
In Table 1, the values at 25˚C are the predicted shelf life at an ambient temperature for
each of the activation energies selected.
The progressive reduction in shelf life can be seen as the storage temperature is increased
in 10˚C increments from 30˚C to 90˚C.
The above principles can also be applied to a product containing more than one active
ingredient, such as a multivitamin product.
V.1.1.2 Real-life example of predicted and actual vitamin losses over six months
Table 2 shows an actual example of the losses predicted from an Arrhenius model for four
vitamins in the same product matrix. These predictions are compared to the actual losses
assayed in the same product after storage.
Table 2. Comparison of predicted and actual percentage losses in a multivitamin tablet

after six months in plastic containers at 298K and 75%rh
Vitamin Predicted Loss (%) Actual Loss (%)

Vitamin A 43.0 44.0
Vitamin C 24.0 23.0
Vitamin B12 9.2 7.7
Folic acid 12.0 10.5
(Berry Ottaway, 2008)
V.1.2 Computer modelling
There are a number of computer programmes now available which are based on the
Arrhenius Equation and which help to simplify the procedures for the prediction of shelf
lives and estimation of overages.
Discussion may be required with the chosen software company to ensure that any
selected programme is suitable for the specific stability requirements of predictive analysis.
70
71
ANNEX VI
Bracketing
and Matrixing in
Stability Studies
72
As outlined in Chapter 11, the techniques of bracketing and matrixing have been
developed to allow the use of a reduced sampling plan for shelf life studies without
jeopardising the scientific value of the study.
VI.1 Bracketing
Bracketing is a procedure whereby the stability study is designed in such a way that only
the extremes of the selected parameters are tested at all the time points.
It is an appropriate procedure for the stability assessment of different potencies of an

active ingredient in a supplement matrix, or where the same product is to be marketed in
different container sizes or fills (for example, 30, 60, 120 tablets).
Bracketing is not appropriate if the samples to be tested do not represent the extremes.
VI.1.1 Use of bracketing for similar formulations
Bracketing may potentially be used in studies of supplements with closely related

formulations, such as those where the differences relate only to minor components such
as colours and flavours. If the formulation differences are significant, for example, different
ingredients or additives are used or the levels of the proportions of the components are
greatly different, then the use of bracketing is inappropriate.
Figure 1 shows a simplistic example of bracketing for a study on a vitamin C tablet with
the following particulars:
• To be marketed in three potencies: 100mg, 250mg and 500mg;
• All three potencies to be in a matrix of the same ingredients and additives;
• All three potencies intended to be marketed in three pack sizes of 30, 60 and 120
tablets.
In this example, only the extremes of each parameter are to be tested, thus reducing the
testing requirements from a potential nine samples to just four samples.
73
ANNEX VI BRACKETING AND MATRIXING IN STABILITY STUDIES
Figure 1: Simplistic example of bracketing for a study on a vitamin C tablet
Container fill VITAMIN C CONTENT (mg)

(tablets) 100mg 250mg 500mg
30 T T
60
120 T T
VI.1.2 Use of bracketing for assessing packaging and closure systems
Bracketing is a useful tool for evaluating the stability of a product in different containers
and closure systems, particularly where the container size and fill can vary.
In order to assess the effect on stability of different containers or packaging systems,

extremes of the following characteristics should be selected:
• Container surface area to volume ratio;

• Container head-space to volume ratio;
• Headspace per unit (for example, mL/tablet or mL/softgel);
• Container composition and wall thickness;
• Water vapour permeation rate;
• Oxygen permeation rate;
• Closure geometry and integrity.
VI.2 Matrixing
Matrixing is defined in Chapter 11 as ‘the design of a shelf life study in such a way that
a selected sub-set of the total number of possible samples would be tested for relevant
parameters at a specific time point’. At a subsequent time point, another sub-set of
samples of all combinations of factors would be tested.
Matrixing assumes that the stability of each sub-set of samples tested adequately
represents the stability of all samples at a given time point.
VI.2.1 Application and design of matrixing
For the effective use of matrixing, the different potencies of the product should:
74
• Have identical or closely related formulations;

• Be from batches manufactured using the same process and equipment; and
• Be packaged in the same container size with the same fill and closure system.
The design of a matrix should be such that each combination of parameters is tested to
the same extent over the duration of the study.
Initial and end point values must be obtained for all samples for all parameters being
tested.
VI.2.1.1 Other factors to consider
When considering a reduction in sampling through matrixing, consideration should be

given to a number of factors, including:
• The expected stability of the product;

• Prior knowledge of data variability;
• Potential stability differences in the product; and
• The availability of supporting data.
VI.2.1.2 Accelerated studies
In addition to consideration of the above factors, in order to enable a valid analysis of

the stability data, at least three time points are necessary for accelerated studies. The
matrix design should allow an adequate ability to detect stability differences both within
parameters and among parameters.
VI.2.2 When to consider matrixing
Matrixing may potentially be considered if the supporting data, as outlined above, indicate
a probable product stability and little variability.
Matrixing should not be considered if the supporting data indicate the possibility of large
variabilities.
In addition, where matrixing is applied, the amount of reduced testing from the full design
is dependent on the number of parameters and parameter combinations being evaluated.
Matrixing is more applicable to long-term (real time) stability studies, and can result in a
reduction of one third to one half in testing requirements.
75
ANNEX VI BRACKETING AND MATRIXING IN STABILITY STUDIES
Figure 2 provides an example of a matrix plan where only three or four of the six time
points from the second to the penultimate point are tested. In every case all samples must
be tested at the first and last time points.
Figure 2: Example of a matrix design on time points for a vitamin C product with two
potencies
Time Point (months) 0 3 6 9 12 18 24 36
P P1 Batch 1 T T T T T T
O 100mg
T Batch 2 T T T T T T
E Batch 3 T T T T T
N
C P2 Batch 1 T T T T T
I 250mg
E Batch 2 T T T T T T
S Batch 3 T T T T T
VI.3 Caution
Whilst both bracketing and matrixing can provide the benefit of cost savings by the
reduction of the number of analytical tests carried out, they can also have limitations.
For example, an errant result or an indication of poor stability at one or more conditions
or time points can call into question the validity of the reduced study. In such cases, if
reliance has been placed on the reduced testing and only those conditions and time
points indicated by the bracketing or matrixing plan have been used, the whole study may
have to be re-run, leading to significant delays and costs.
One precaution against such an eventuality is to work to a protocol whereby all conditions
and time points are covered by stored samples, but where only those identified by a
bracketing or matrixing plan are analysed in the first instance. The intermediate samples
which are not tested should be removed from the controlled temperature/humidity storage
at the relevant time point and stored at 0°C until the completion of the study.
76
77
ANNEX VII
Useful resources
78
General
Food and beverage stability and shelf life. Ed. Kilcast D.

Woodhead Publishing, UK. 2011. ISBN 1 84569 701 4, ISBN-13: 978 1 84569 701 3
Global Guide to Good Manufacturing Practice for Supplements. The International Alliance
of Dietary / Food Supplement Associations (IADSA). 2011
http://www.iadsa.org/
Shelf Life Dating of Botanical Supplement Ingredients and Products. Ed. Eisner S.
The American Herbal Products Association, USA. 2011.
http://www.ahpa.org
Shelf-life Recommendations for Supplements: Guidelines for Manufacturers.

The International Alliance of Dietary / Food Supplement Associations (IADSA). 2014
http://www.iadsa.org/
Stability testing guideline for dietary supplements.

NSF International, USA. 2011.
http://www.nsf.org
The Shelf Life of Foods and Beverages. Proceedings of the Fourth International Flavour
Conference, Rhodes, Greece, 23–26 July 1985. Ed. Charalambous G,
Developments in Food Science 12. Elsevier Science, 1986. ISBN 0-444-42611-6
The Stability and Shelf-life of Food. Eds. Kilcast D and Subramaniam P.

Woodhead Publishing Series in Food Science, Technology and Nutrition, No 48.
Woodhead Publishing. 2000. ISBN 978 1 85573 500 2
Understanding and Measuring the Shelf-life of Food. Ed Steele R.

CRN Press, USA. 2004. ISBN 0-8493-2556-0
Chapter 3: Mechanisms of Product Deterioration
Chemical deterioration and physical instability of food and beverages. Eds. Skibsted L,
Risbo J and Andersen M.
Woodhead Publishing, UK. 2010. ISBN 1 84569 495 3, ISBN-13: 978 1 84569 495 1
79
Food Shelf Life Stability: Chemical, Biochemical, and Microbiological Changes. Eds. Eskin
M, Robinson DS.
CRC Press, USA. 2000. ISBN 9780849389764
Oxidation in foods and beverages and antioxidant applications Volume 1: Understanding

mechanisms of oxidation and antioxidant activity. Eds. Decker EA, Elias RJ and
McClements DJ.
Woodhead Publishing, UK. 2010. ISBN: 978 1 84569 648 1
Oxidation in foods and beverages and antioxidant applications Volume 2: Management in

different industry sectors. Eds. Decker EA, Elias RJ and McClements DJ.
Woodhead Publishing, UK. 2010. ISBN: 978 1 84569 983 3
‘The stability of vitamins in fortified foods and supplements’. Berry Ottaway P.

In Food Fortification and Supplementation Ed. Peter Berry Ottaway.
CRC Press Ltd., USA. 2008. ISBN 978 1 42007 201 3
Also in Russian (ISBN 978-5-93913-188-9) and Chinese (ISBN 978-7-5019-7888-5)
Chapter 6: Test Parameters for Stability Testing
Sensory Evaluation of Food: Principles and Practices, 2nd Edition. Harry T Lawless and
Hildegarde Heymann.
Springer-Verlag, USA. 2010. ISBN 978-1-4419-6487-8
Sensory Evaluation Techniques, Fourth Edition. Morten C Meilgaard, B Thomas Carr, Gail
Vance Civille.
CRC Press, USA. 2006. ISBN 978-0-8493-3839-7
Chapter 9 and Annex V: Arrhenius Equation
Labuza T P and Riboh D (1982). ‘Theory and Application of Arrenhius kinetics to the
prediction of nutrient losses in foods’. Food Technol 36 (10) 66-74.
Porter W R (2011), ‘Stability by Design’ J. of Validation Techn. Summer 2011.
Stability System. Arrenhius Equation: Effect of Temperature on Reaction. 2006, ScienTek

Documents on Stability Theory, ScienTek Software, www.StabilitySystem.com (accessed
October 2015)
80
Chapter 11 and Annex VI: Bracketing and Matrixing
International Conference On Harmonisation Of Technical Requirements For Registration

Of Pharmaceuticals For Human Use (ICH) Harmonised Tripartite Guideline: Bracketing
And Matrixing Designs For Stability Testing Of New Drug Substances And Products
(Q1D). 2002
http://www.ich.org/
81
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Stability Testing for the

Shelf Life Determination of Supplements

3. Mechanisms of product deterioration 12

6. Test parameters for stability testing 25

7. Shelf life studies 28

8. Types of stability studies to support shelf life determination 32

9. Shelf life evaluation 36

10. Stability study protocols 40

11. Bracketing and matrixing 44

11.3 Points to consider 46

12. Use of published sources on stability 48

Annex I Glossary of terms 50

Annex II Chemical interactions of vitamins that may

Annex III Comparison of vitamin stability 58

Annex IV Microbiology of supplements 60

Annex V Predictive analysis of stability data 66

Annex VI Bracketing and matrixing 72

Annex VII Useful resources 78

‘Stability Testing for Shelf Life Determination of Supplements’ is part of a series of

The principles of ‘Quality by Design’ should be applied to supplement products. During

– the intended storage, distribution including transportation, and anticipated retail

(See also Chapter 12)

A supplement formula may be comprised of a mixture of a large number of diverse

3.1 Moisture/water vapour content and transfer

To ensure maximum stability of a product, moisture must be controlled at every stage

As a generalisation, an increase in the temperature increases the rate of a chemical

A further consideration is that fluctuations in storage temperature can affect product

3.4 Oxidising/Reducing Agents

3.6 Chemical Hydrolysis

3.8 Chemical Interactions

In situations where there is a need to combine components which are known, or

Gelatin cross-linking can be catalysed by a number of chemical substances either found

6.1 Selection of test parameters

b) Capsules (hard and soft shell)

c) Liquids and semi-solids (gels)

6.2 Analysis of parameters

For a product with a number of claimed active ingredients, such as a multivitamin, it is

7.1 General criteria

7.2 Analytical requirements

It cannot be overemphasised that care should be taken to standardise the analytical

7.3 Product samples

The studies can fall into the following categories:

– Stress Testing, to determine an ingredient’s or combination of ingredients’ susceptibility

The main testing regimes are explained in the following sections.

8.1 Stress testing

Another common procedure is to store portions of the material in solution or suspension

8.2 Accelerated testing

8.3 Long term testing

The environmental conditions should be representative of regional conditions in which the

8.4 In-use testing

Where the product is known or is expected to be susceptible to microbiological growth, it

8.5 Transit (excursion) testing

9.1 Analysis of appropriate stability data

9.2 Extrapolation of stability data

The objective of an accelerated stability study is to subject the product to stress in

Nevertheless, it cannot be assumed that there is a linear relationship between the

10.1 Protocol development

b. Type, size and composition of each container and closure system

d. Procedures for confirming integrity of packaging seal closures

f. Each formulation included

g. Each batch code included

i. Storage conditions of samples retained for testing

k. Acceptance criteria / critical values

– the intended storage, distribution including transportation, and anticipated retail

– Stress Testing, to determine an ingredient’s or combination of ingredients’ susceptibility

b. Type, size and composition of each container and closure system

d. Procedures for confirming integrity of packaging seal closures

l. Use of reliable, meaningful, specific and preferably validated test methods

• If any microbiological contamination of a capsule is identified, it is often useful to test