PRESERVATIVE CHALLENGE TESTING

OVERVIEW

PRESERVATIVE CHALLENGE SCREEN

Preservative challenge testing is a
easy way to learn about the efficacy
preservative system. Setting up a
preservative challenge test with
Test Labs is easy. Simply request a
quote.

fast and
or your
Cosmetic
price-

Preservatives are ingredients used

to protect
a product from deterioration and
help it
perform as intended for the lifetime
of the
product.The activity of a
preservative depends on the combination of all the product ingredients and the packaging
used. Therefore, preservative efficacy of a product cannot be predicted based on its individual
ingredients alone. It must be established through microbial challenge testing of a complete
formulation. The Preservative Challenge Test Screen is a pooled preservative challenge test
for cosmetics that is recommended as an early indicator for preservative efficacy of cosmetics
and personal care products. This is a test in which we purposely introduce specific
microorganisms into the product, then watch the sample over a specified amount of time to
see whether your preservative system is sufficient to eliminate those microorganisms.
The Preservative Challenge Screen utilizes 5 microorganisms, 3 bacteria and 2 fungi,
specified in the USP <51> test method to challenge the products. However, this test is
typically faster, more cost effective, and more challenging, making it a good indicator of
efficacy for your preservation system. A preservative challenge screen for cosmetics consists
of the 3 bacteria being pooled together and the 2 fungi being pooled together to be added to
the product. One container of the product is challenged with the pooled bacteria and another
with the pooled fungi. After the specified contact time, typically 7 days, the samples are
analyzed to determine the level of bacteria and fungi remaining in the samples. If there are no
microorganisms detected after the contact time, you can be fairly certain that your
preservative system is working sufficiently. If the microorganisms are detected in the sample,
don't fret, there is still hope as long as there are less remaining than we started with.
However, if there are more or the same level of microorganisms remaining as when we
started, then you should begin looking into ways to improve your preservation system.

WHY USE PRESERVATIVES?

Consumer products are
expected by the FDA to
withstand microbial attack
within the intended period of
use of the product. Without
preservatives, cosmetics and
personal care products are
likley to become contaminated
with various microorganisms,
leading to product spoilage and
possibly irritation or infections
caused by harmful
microorganisms.
From the time a cosmetic or personal care product is opened to the time it is discarded, it is
subject to constant microbial contamination from the environment and the consumers' hands
and body. For example, touching an applicator to your face which has low levels of
microorganisms on it and then placing the applicator back in the product is a common source
of potential contamination. Temperatures and humidity levels in home environments where
cosmetic and personal care products are typically located, such as in warm, moist bathrooms
are perfect for enhancing microbial growth in a product unless it is well preserved. Now, that
same microorganism that has just been inoculated in the product by the applicator has been
allowed to grow in the product in the bathroom. Therefore, the next use will be a selfinoculation with higher levels of the microorganism, which can cause various unpleasant
problems for consumers, including skin and eye area infections. Preservatives are used to
help prevent these product contamination problems.
USP <51> - PRESERVATIVE CHALLENGE TEST FOR COSMETICS
Starting a USP 51 test with Cosmetic Test Labs is simple - just contact the lab or request a
price-quote.
The USP <51> - Preservative
Test is an antimicrobial
effectiveness test from chapter 51
United States Pharmacopeia. Much
Preservative Challenge Screen, it
the activity of preservatives used in

Challenge
of the
like the
evaluates
cosmetics

and personal care products. Preservatives are anitmicrobial ingredients that are added to
product formulations to help maintain the safety of the product by inhibiting the growth and
reducing the amount of microbial contaminants. The USP <51> challenge test for cosmetics
utilizes 5 microorganisms, 3 bacteria and 2 fungi, for challenge testing. Each of the
microorganisms are known strains of pathogenic microorganisms. The first time a product is
challenge tested for antimicrobial effectiveness under the USP <51> method, a neutralization
and recovery validation is required to ensure that the microorganisms are able to be recovered
from the product if they are present.

SUMMARY of USP <51> CHALLENGE TEST PROCEDURE

The product is evaluated using the USP <61> - Microbial Enumerations Test for the
presence of pathogens after manufacturing.

The product is separated out into 5 containers, each being challenged with one of the
5 method-specified microorganisms (S. aureus, E. coli, P. aeruginosa, C. albicans,
and A. brasiliensis) at fairly high concentrations.

The initial concentration of each microorganism is determined by standard dilution

and plating techniques.

A separate aliquot of the product is diluted along with a control substance, to be used
in the neutralization and recovery validation

The product is held at room temperature for a period of no less than 28 days.

The product is evaluated at specific intervals within the 28 day period that depend on
the category of the product specified by the method. Click here for USP <51> product
categories.

Test sample colonies are counted at each specified interval to determine the amount of
microorganisms remaining.

The log reduction of each microorganism at each interval is reported.

The effectiveness of the preservative system is determined based on the USP <51>
passing criteria.

OPTIONAL Customization: The product is re-inoculated with even more

microorganisms and allowed a longer incubation period (specified by study sponsor).
WHEN TO CONDUCT A USP<51> CHALLENGE
TEST

It is important to determine if the preservative chosen for a product is compatible with the
formulation soon after manufacture. It may also be helpful to challenge test again within 2-3
months of manufacture or as appropriate to the intended shelf-life as part of your stability
testing regimen. It is also necessary to re-evaluate preservative effectiveness in your product
whenever a formulation, or manufacturing process has been changed or when product
packaging changes
occur.

PRESERVATIVE EFFECTIVENESS TEST (BASED ON USP 51)

Let's say you are a cosmetic or personal care product maker and you have formulated your
product, then run tests for microbial counts and chemical contaminants and they look good.
Congratulations! Now it's time to figure out how long the microbial counts will keep in
check.
Most cosmetic and personal care products are made from a few to several dozen ingredients.
Most of those ingredients are biodegradable. To a microbiologist, "biodegradable" means
"germ food." In fact, one could comfortably define a biodegradable ingredient as anything
easily degraded by microorganisms.
Therefore, a cosmetic or personal care product can be free from germs right after
manufacturing, but can support their growth over the course of time. Sometimes this growth
can be quite substantial and dangerous to users. Microorganisms are remarkable. In a
cosmetic without an adequate preservative just one cell can multiply to several billion over
the course of just a couple of days. This is especially likely in aqueous (water-based) products
with neutral pH.
To make sure this doesn't happen, makers of personal care and cosmetic products conduct
preservative effectiveness tests, sometimes called "challenge tests." Preservative challenge
tests for cosmetics are easy to understand. The product is simply inoculated with a variety of
microorganisms then evaluated at intervals, much later, to see if the germs have grown or
died.
The typical preservative challenge test for cosmetics is modeled after a standard preservative
challenge test method, USP <51> Preservative Challenge Test. For this test method, a product
is inoculated with 5 germs consisting of 3 bacteria and 2 fungi. For bacteria, Staphylococcus
aureus, Escherichia coli, and Pseudomonas aeruginosa are used. For fungi, a yeast
called Candida albicans and a mold called Aspergillus niger are put to work.

For USP 51, the product is first evaluated to make sure that inoculated microorganisms can be
accurately recovered from the product. Then it is inoculated separately with each of the 5
strains and initial microbial concentrations are determined. After that, it is held for 14 days at
room temperature then evaluated again for the presence of contaminant microorganisms.
Finally, it is held for another 14 days (making 28 total) and then evaluated again. Of course,
the desired result is that the microbial population is killed off completely by the formulation
over the study period. In the event that some germs survive, the method provides guidance
with regard to whether or not that is significant.
Preservative challenge studies run for a long period in the laboratory and require a great deal
of scientific expertise and labor. Thus, they are relatively expensive, running about $300$1500 per sample, depending on the laboratory.

Some companies find the USP 51 challenge test method to be a low-level product challenge,
and prefer to further challenge test cosmetics by customizing the method to give them extra
confidence that the products won't support microbial growth on the store shelf or in the hands
of consumers. The stringency or challenge level of a preservative challenge test can be
increased using a number of approaches. The most common approach is to supplement the
inoculum with additional microorganisms isolated from manufacturing environments.
Secondarily, the number of inoculated microorganisms can be increased to greater than 10^6
per milliliter. Thirdly, the product can be diluted prior to testing. Finally, the product can be
repeatedly inoculated.
By challenge testing cosmetics with a qualified laboratory, the makers of a product gain a
critical bit of information about their product: They know whether or not the incorporated
preservative will fight off microorganisms introduced into the product during manufacturing
or introduced during use by consumers.
If a cosmetic product fails preservative challenge testing, a knowledgeable laboratory can
help determine the cause and suggest formulation changes that will result in a more
microbiologically stable product.
PCPC (FORMERLY CTFA)
METHODS FOR COSMETICS

TEST

Cosmetics and personal care

are perfect environments for
microorganisms to grow unless
measures are taken during

products
proper

formulation and manufacture to preserve them. Cosmetics and personal care products are
intended for use in direct contact with the body and this makes it imperative for these
products to be free of harmful contamination and prepared with effective preservative
systems. Microbial content tests are designed to determine the contamination levels in the
products and preservative challenge testing is designed to verify the ability of cosmetics to
avoid microbial growth upon contamination that may be introduced during manufacturing or
through normal consumer use.
The Personal Care Products Council (formerly Cosmetics, Toiletries, and Fragrance
Association - CTFA) has set forth guidelines specific testing of cosmetic and personal care
product manufacturers to aid in determining the microbial content and the effectiveness of the
preservative system of many different types of cosmetic products.
Cosmetic Test Labs offers microbial examination and challenge testing following these CTFA
guidelines:

CTFA M-1 - Determination of the Microbial Content of Cosmetic Products

CTFA M-2 - Examination for Staphylococcus aureus, Escherichia coli and

Pseudomonas aeruginosa

CTFA M-3 - A Method for Preservation Testing of Water-Miscible Personal Care

Products

CTFA M-4 - Method for Preservation Testing of Eye Area Cosmetics

CTFA M-5 - Methods for Preservation Testing of Nonwoven Substrate Personal Care
Products

CTFA M-6 - A Method for Preservation Testing of Atypical Personal

Care Products

TOWELETTE PRESERVATIVE CHALLENGE TEST FOR COSMETICS

Many cosmetics and personal care products are now available in the form of presaturated towelettes or wipes. Like the majority of personal care products having an
aqueous base, pre-saturated towelettes are susceptible to microbial contamination and
require a preservation system to protect them from degradation. Cosmetics and
personal care products made in the form of towelettes or wipes can harbor harmful
microorganisms just like any other consumer product that is not properly preserved.
Their preservation system must also be verified as appropriate through empirical
challenge testing. The efficacy of such preservatives could be evaluated on the liquid
formulation, however, this alone cannot be considered as good representation for the
formulation when added to the towelettes or wipes.

Cosmetic Test Labs has modified methods for the USP <51> challenge test method,
and the Preservative Challenge Screen, along with the CTFA M-5 method to include

cosmetics and personal care products in the form of pre-saturated towelettes or wipe
products. These modified methods allow the efficacy of preservatives used in
towelette and wipe products to be evaluated while being kept in their original form.

CUSTOM PRESERVATIVE CHALLENGE TESTS FOR COSMETICS

All cosmetic and personal care

products are expected by the FDA to
withstand microbial attack within the
use period of the product. However,
not all cosmetic products are made the
same or fall into the same categories.
Therefore, standard preservative
challenge test methods may not be
suitable for all types of cosmetic
products. For example, a standard USP
<51> Challenge Test is defined for
products with an aqueous base. Obvious problems can occur in products with a high
water content, but seemingly harmless products made with talc or powder can become
contaminated with harmful microorganisms as well.

Often time, products during the manufacturing process become contaminated with
microorganisms that are not included in standard preservative challenge tests for
cosmetics. In this case, it may be beneficial to challenge the products with those
specific microorganisms to ensure the preservative system is able to inhibit the growth
of them as well as those typically used in challenge testing. In addition to the
microorganisms used in the standard preservative test methods, Cosmetic Test Labs
has access to an extensive list of problematic microorganisms, including some that
have previously been isolated from contaminated consumer products.

NON-ANIMAL PRODUCT SAFETY TESTING

Once a company has formulated a cosmetic product, tested it to ensure it is free from
microbial and chemical contamination after production, and run a preservative challenge test,
and assessed stability, then it is time to conduct product safety testing.
Product safety testing is another way of saying toxicity testing. It is a very important class of
testing because of the way that cosmetic and personal care products are used. They have

direct and repeated exposure to the body. If they are irritating to the skin or eyes or cause
unwanted immune reactions, consumers will know right away.
Historically, a great number of cosmetic product safety tests were done on animals. Cosmetic
Test Labs likes to think that fewer animal tests are run now than in the past, but that data is
hard to come by. Nevertheless, non-animal toxicity tests are available for cosmetics that are
reliable and accurate. Cosmetic Test Labs does not endorse animal testing for safety purposes,
so the rest of this section focuses on in vitro (non-animal) methods.
Product safety testing is typically the most expensive phase of cosmetic product
development. Therefore, it may be advisable to conduct it last, just before marketing the
product.
There are three key product safety tests personal care product manufacturers should consider
for every finished product. By running all three, on each formulation class, companies can
rest assured their products are safe for use.
The first component of cosmetic product safety testing is dermal irritancy. It is used to
evaluate the potential of a product to cause skin irritation when used by the consumer. Some
cosmetic products can cause skin irritation by passing through the outer layer of the skin and
entering the underlying layers, harming those cells. This, in turn, causes an immediate
physiological response characterized by redness, and itching or pain in the affected area. It is
done by placing the product to be tested on a laboratory-grown bed of cells, designed to
mimic human skin. After a period of time, the cells are evaluated for negative effects
indicative of dermal irritancy.
The second component of cosmetic product safety testing is ocular irritancy. It uses a 3dimensional model of eye tissue to determine the potential for eye irritation from a consumer
product or cosmetic. This test is fundamentally similar to skin irritancy, but done on a labgrown tissue model that more closely resembles the cornea.
The last part of product safety testing for cosmetics is dermal sensitization testing. Dermal
sensitization is crucial for products intended to go on the skin - especially products that
contain potentially sensitizing agents such as certain essential oils. Dermal sensitization takes
place when the body's immune system identifies a component of a cosmetic product as being
harmful. When that happens, the immune system is recruited to fight the "invader," resulting
in rash and swelling. This phenomenon is also known as allergic contact dermatitis. Nonanimal tests are available to investigate the likelihood that a given product will be a skin
sensitizer.
Traditional (animal) product safety tests are very expensive and are burdened by a number of
serious drawbacks, in addition to the pain and suffering they cause animals. In vitro
(laboratory-based) methods offer an excellent alternative to animal testing and are proving to
be equally useful.

NATURAL VS. SYNTHETIC PRESERVATIVES IN COSMETICS

Customers often ask Cosmetic Test Labs questions about the choice between natural and
synthetic preservatives. Choosing whether to use natural or synthetic preservatives for
cosmetics is an important decision that has many implications. It will affect product stability,
odor , appearance, texture and most importantly, safety.
To help customers choose the best preservative for their products, Cosmetic Test Labs has
assembled the following list of benefits and drawbacks of natural and synthetic preservatives.
Of course, generalizations must be made in order to fit all the different types of natural and
synthetic chemicals into categories. Remember that no matter which preservative a cosmetics
maker chooses, they are responsible for confirming safety and stability.

Benefits of Synthetic Preservatives

Broad-spectrum of activity against bacteria and fungi.

Many options are available.

Produced by established chemical manufacturing firms that understand a great deal

about the chemicals.

Cosistent from batch to batch.

Relatively low cost

Safety/toxicity profiles well understood.

Compatibility with other cosmetic and personal care product ingredients is well
understood.

Low concentrations required to effectively preserve products.

Generally do not interfere with fragrance, lather, color, or other aspects of a given
formulation.

Companies that produce synthetic preservatives often have in-house microbiology

testing laboratories and can offer fee-based support to cosmetics formulators.

Drawbacks of Synthetic Preservatives

Many have developed negative reputations in popular media related to cosmetics.

Often petroleum based.

Some cosumers find them irritating to the skin.

Interfere with "natural" and "organic" product labeling.

May require a narrow pH range to be effective.

Benefits of Natural Preservatives

Allow "natural" and sometimes "organic" product labeling.

Consumers generally prefer natural preservatives over synthetic preservatives.

Often function over a broad pH range.

Many confer natural fragrances to products.

Drawbacks to Natural Preservatives

Fewer options available.

Often do not inhibit microbial growth as well as synthetic counterparts.

Often lack broad-spectrum activity. For example, a natural preservative may inhibit
the growth of Staphylococcus aureus or "Staph" in a formulation, but may have no
mold-inhibiting effect.

May require relatively high concentrations in formulations to be effective.

Often have undesirable impact to other aspects of formulations, such as color,

fragrance, or lather.

May result in skin sensitization (immunological reaction to the natural preservative

that increases over time.

Consistency and potency may vary considerably from batch to batch.

In summary, synthetic preservatives have been around for decades and have successfully
preserved thousands of cosmetics. Lately, the market has been driven toward natural
preservatives by evidence of toxicity of certain traditional synthetic preservatives.
Unfortunately, selection of natural preservatives is limited, they can vary in terms of potency
from manufacturer to manufacturer and batch to batch, and sometimes affect other aspects of
the formulation in an undesirable way.
Regardless of whether your company chooses to use a natural cosmetic preservative or
synthetic cosmetic preservative, Cosmetic Test Labs can verify preservative effectiveness,
ensuring a given formulation remains safe and stable over the course of normal usage.