DETERMINATION OF PRESERVATION EFFICACY IN

WATER-MISCIBLE PERSONAL CARE PRODUCTS

INTRODUCTION

Many personal care products provide conditions that are conducive to microbial
growth such as water, nutrients, and favorable pH. Preservation efficacy testing
(PET) is performed to assure that each personal care product that is susceptible to
microbial growth is not affected by the introduction of microorganisms during normal
or reasonably anticipated use by the consumer. However, some personal care
products, due to characteristics that create an environment hostile to microbial
growth and/or survival, are considered low microbiological risk1 (see also Section
15 - Microbiological Risk Factor Assessment of Atypical Cosmetic Products and
Reference #1). These products are not routinely subject to preservative efficacy
testing.

The design of preservation tests and subsequent interpretation of results is a

complex process. The technical personnel responsible for preservation testing
should therefore, be professionally educated and experienced in conducting
challenge test procedures and evaluating the data generated (see Sections 4 and 9
– Microbiology Staff Training and Microbial Validation and Documentation).

It is important to remember that microorganisms are ubiquitous and capable of

adaptation. No method can guarantee adequate microbiological control under all
conditions. In addition, the importance of adhering to good manufacturing practices
in the production of personal care products cannot be overstated. Preservatives
should not be used as a substitute for good manufacturing practices2.

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Personal care products constitute a wide and expanding variety of formulations and
package configurations that differ significantly from each other in their composition,
intended use, and physical characteristics. This guideline reflects current
considerations and practices used within the personal care industry in conducting
preservation efficacy testing of water-miscible products. Other standardized
methods as well as those developed by the user may also be employed to assess
preservation efficacy of water-miscible personal care products3,4 .For guidance on
testing atypical products, see Section 15 - Microbiological Risk Factor Assessment
of Atypical Cosmetic Products.

SCOPE

The purpose of this guideline is to provide guidance to those developing and

performing PET of water-miscible personal care products such as are described in
Method M-3, A Method for Preservation Testing of Water-Miscible Personal Care
Products (Section 19) and Method M-7, A Rapid Method for Preservation Testing
of Water-Miscible Personal Care Products (Section 22).

GENERAL CONSIDERATIONS

The preservation efficacy test (PET) is an important tool in evaluating the

robustness of a formulation to withstand microbial contamination during consumer
use. Various factors can have an effect on the performance of a preservative
system in a formulation and on the interpretation of results generated from these
studies1,5-7.

The following factors may be considered when performing a preservation

efficacy test and interpreting results:

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 The nature and compatibility of the raw materials in the formulation 6.
 Information on preservation of similar formulations.
 Manufacturing conditions, e.g. temperature of processing and filling 6.
 Types of packaging used to contain and deliver the product 1,5, 7.
 Information on the product’s intended use, including area of application,
frequency of use, shelf-life, intended users, etc.

PRODUCT TESTING

Microbial Content Test

It is recommended that the bioburden of the test sample(s) be determined by a

microbial content test, such as Method M-1 - Determination of the Microbial Content
of Cosmetic Products (Section 17), prior to performing the preservation efficacy
test. The purpose is to verify that the level and type of microorganisms in the test
sample will not interfere with the recovery of the test organisms or interfere with the
interpretation of the challenge test data. Furthermore, an initially high microbial
bioburden in the test sample before inoculation with microorganisms could
compromise the preservation system.

Neutralization

Antimicrobial or preservative neutralizers are added to microbial plate count diluents

and recovery agars to inhibit or inactivate the antimicrobial properties or activity of
the formulas being tested8. Carryover of antimicrobial activity from the product
formulation into the microbial plate count diluent and recovery growth agar may
partially or completely inhibit the growth of surviving challenge test microorganisms9.
Antimicrobial or preservative neutralization may normally be accomplished by use of
chemical neutralizing agents, physical dilution, or a combination of both. Prior to

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the start of the neutralization step of the test, verification of the lack of neutralizer
toxicity to each of the challenge test microorganisms must be confirmed 10. Once
this has been confirmed, this step does not need to be repeated during routine
neutralization studies of formulations using the same microbial count diluent or
recovery agar. If a new preservative neutralizing agent is added to the microbial
count diluent or recovery agar, verification of the lack of neutralizer toxicity to each
of the challenge test microorganisms must be re-confirmed.

Examples of chemicals and agents that may be used to neutralize the antimicrobial
activity of preservatives are listed in Table 13-111. Verification of the antimicrobial or
preservative neutralization is generally performed by inoculating the product dilution
and a microbial count diluent without product (control) with a low level of challenge
microorganisms to yield a final count of approximately 30 to 300 Colony-Forming
Units (CFU)/milliliter (ml) for each challenge test microorganism in a product test
dilution and diluent control. Enumeration of the microorganisms from these dilutions
is performed. Neutralization of the antimicrobial or preservative activity is verified if
microbial recoveries of both test and control dilutions are within 0.5 log of each
other. If one or more challenge test microorganisms cannot be recovered, the use
of a higher dilution and/or the investigation of additional chemical neutralizers may
be considered.

TEST PROCEDURE

Organisms

The organisms listed in Table 19-1 of Method M-3 (Section 19) and Table 22-1of
Method M-7 (Section 22) are representative types of the microbial species that a
formulation may encounter during manufacture and use: Gram-positive cocci, Gram-
negative fermentative bacilli, Gram-negative non-fermentative bacilli, yeast and

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mold. It is recommended that at least one microorganism from each group be
included in the challenge test. Examples of other organism types, relevant to the
formulation, may include microbial isolates that have been recovered from raw
materials, formulations returned from consumers or other sources (e.g. product in or
after use studies). Either pure or mixed microbial culture suspensions may be used
to conduct challenge testing of formulations. Decisions to use pure or mixed
cultures may be influenced by the factors discussed below.

Inocula consisting of only pure microbial cultures will yield specific data on each
test microorganism employed in the challenge study. Mixed culture inocula may
serve to simulate real world conditions during use. When conducting mixed culture
challenge studies, it is recommended that closely related types of microorganisms
such as Gram-positive bacteria (e.g. cocci), Gram-negative fermentative bacilli,
Gram-negative non-fermentative bacilli, and yeasts and molds be pooled into
separate distinct groups. Antagonism between different types of organisms may
occur due to differences in growth factors and nutritional requirements12. A rapidly
growing organism may impede the detection of a more slowly growing organism.
For example, Escherichia coli has a shorter generation time and may obscure
detection or growth of microorganisms such as Pseudomonas aeruginosa that have
a longer generation time. Competition for growth factors or production of inhibitory
byproducts and other factors may result in antagonism between different types of
microorganisms12, 13.

Inoculation and Enumeration Procedures

A typical challenge test consists of the following steps: inoculation of the test
formulation, followed by enumeration of the inoculated formulation at various time
points, and interpretation of data to determine adequacy of preservation. Details of
these procedures may be found in the challenge test methods of M-3 (Section 19)

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and M-7 (Section 22). It is recommended that the volume of inoculum added to
the sample of the formulation does not exceed 1% by volume. Larger volumes of
inocula (e.g. >1.0%) may result in undesirable dilution of the test formulation.

A preservative challenge test usually employs a single inoculation of each

microorganism or pool of microorganisms. A rechallenge consisting of a second
inoculation may be considered if more information is desired, e.g. to determine if a
formulation is marginally preserved.

OTHER CONSIDERATIONS

Modified Formulations

When changes are made to the composition of a formulation that has already been
challenge tested, a rapid screening test such as described in Method M-7 (Section
22) may be used to determine whether the change has an adverse effect on the
preservation adequacy of the formulation. The decision to perform additional
challenge testing for these types of formulations is dependent upon, but not limited
to the type of finished package used, pH changes to the formulation, the addition of
new or deletion of raw ingredients from the previously tested formulation, and the
challenge test data of the original tested formulation.

Scale-Up/Pilot Batches

Changes in processing conditions during scale-up from laboratory to production size

batches may alter the performance of the preservation system. Processing
conditions (e.g. order of raw ingredient addition, pH of a batch during processing,
and temperature of a batch during processing) may alter the antimicrobial activity or
the physical stability of the preservative system in a formulation. Therefore,

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preservation tests may be performed on scale-up batches to confirm the
effectiveness of the preservation system.

Product Stability

During product development, the stability of the preservation system in a formulation

should be considered. Challenge tests may be performed on either bulk material or
product from a filled container that has been aged by using accelerated aging
conditions such as holding at specific temperature and/or humidity conditions or
real time aging at ambient conditions. Accelerated aging may cause a formulation
to undergo chemical and physical changes more rapidly than would otherwise occur
during real time aging. A decrease in preservative effectiveness over a period of
time may occur due to a variety of factors. These factors include preservative
degradation, partitioning, interaction with other formula ingredients, and chemical
reaction with or absorption into the packaging material. The PET may be used to
assess the degree of preservative effectiveness after accelerated or real time aging
of a formulation has occurred. The preservative challenge test criteria for
accelerated or real time aged product may or may not differ from the challenge test
criteria for fresh product.

RECOMMENDATIONS

Since many personal care products are used on a regular basis, an effective
preservation system should ensure the reduction of bacteria to a low and steadily
decreasing level and fungi should remain static or decrease over time, even after
repeated use by the consumer. The following challenge test criteria are the minimal
recommendations for evaluating preservation system performance in water- based
product formulations:

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 Vegetative Bacteria
There should be greater than or equal to 3 log (>99.9%) reduction of
vegetative bacteria by aerobic plate count or quantitative spread plate methods
within 7 days following each challenge and no increase to the end of the test
period.

 Yeast and Molds

There should be greater than or equal to 1 log (>90%) reduction of yeasts and
molds by aerobic plate count or quantitative spread plate methods within 7 days
following each challenge and no increase for the duration of the test period.

 Spore-Forming Bacteria
If spore-forming bacteria are included in the test, there should be bacteriostatic
activity against these microorganisms throughout the entire test period.

The above minimal challenge test criteria are suggested to aid manufacturers in
evaluating the adequacy of preservation in personal care products. If a product
does not meet these criteria, it is the responsibility of the manufacturer to select the
appropriate challenge test criteria that will ensure product integrity. For example,
single use packaging or use of pressurized delivery systems may be factors that
could be considered in selecting appropriate criteria. More stringent challenge test
criteria may be considered where deemed appropriate.

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Table 13-1 – Examples of Antimicrobial Preservatives and Recommended
Neutralizing Agents

Antimicrobial Preservatives Recommended Neutralizing Agents:

Alcohol Dilution, Nonionic Surfactants (e.g. Polysorbates)

Bronopol (2-Bromo-2-Nitropropane-1,3,-Diol) Sulfhydryl Compounds (e.g. Cysteine,

Thioglycollate, Thiosulfate, and Metabisulfite)

Chlorhexidine Nonionic Surfactants (e.g.Polysorbates) and

Lecithin, Anionic Surfactants

Formaldehyde donors Dilution, Protein, Gelatin, Sodium bisulfite,

Histamine, Histidine, Nonionic Surfactants (e.g.
Polysorbates), Lecithin

Glutaraldehyde Dilution, Sodium bisulfite, Sodium sulfite, Sodium

thioglycollate, Glycine

Isothiazolinones Dilution, Amines, Sulfites, Sodium bisulfite,

Sodium thioglycollate, Mercaptans

Organic Acid Preservatives (e.g. benzoic and Nonionic Surfactants (e.g. Polysorbates),
sorbic acids) increasing pH

Mineral Acids (e.g. sulfuric and hydrochloric Increasing pH, peptones

acids)
Parabens Lecithin, Nonionic Surfactants
(e.g.Polysorbates),
Phenolic Compounds (e.g. Phenylphenol, Nonionic Surfactants (e.g.Polysorbates) and
chloroxylenol, cresols, chlorocresols) Lecithin

Quaternary Ammonium Compounds Lecithin, Nonionic Surfactants (e.g.Polysorbates),

Protein, Anionic Surfactants

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REFERENCES:

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assessment and identification of microbiologically low-risk products.
www.iso.org.
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Personal Care Products Council, Washington, DC.
3. United States Pharmacopeia. 2009. <51> Antimicrobial effectiveness testing.
USP 36-NF31, United States Pharmacopeia and the National Formulary.
Rockville, MD, pp.67-69.
4. ISO. 2012. ISO 11930, Cosmetics – Microbiology – Evaluation of the
antimicrobial protection of a cosmetic product. www.iso.org .
5. McCarty, T. J. 1984. Formulated factors affecting the activity of preservatives.
In: Kabara, JJ, Orth, DS, ed. Cosmetic and Drug Preservation, Principles and
Practice. Marcel Dekker Inc., New York, pp.359-402.
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Cosmetic and Drug Microbiology. Informa Healthcare, New York, pp.57-108.
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contamination of cosmetics during consumer use. Appl. Environ. Microbiol.
56(5): 1476-1479.
8. Singer, S. 1987. The use of preservative neutralizers in diluents and plating
media. Cosmetics and Toiletries. 102(12): 55-60.
Sutton, S.W. 1996 Neutralizer evaluations as control experiments for
antimicrobial efficacy Test. In: J.M. Ascenzi ed. Handbook of Disinfectants and
Antiseptics. Marcel Dekker, Inc., New York. pp 43-61.
9. American Society for Testing Materials. 2008. ASTM E1054-08 – Standard
test methods for evaluation of inactivators of antimicrobial agents.
http://www.astm.org..
11. Anon. 2003. Disinfectants and Antiseptics. Pharmacopeial Forum Vol. 29 (3)
May-June, 726-735, 2003.
12. Fredrickson, A.G. and Stephanopoulos, G. Microbial Competition. Science:
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 213(4511): 972-979, 1981.
13. Neilands, JB. Microbial Iron Compounds. Ann. Rev. Biochem. 50: 715-731,
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