The Mechanism of Action and Efficacy

Evaluation Methods for Preservatives

Hongyu Xue, Guiqi Yin, Evelyn G. Su

Nanjing Zhongshi Chemical Co., Ltd.

Abstract:
This paper discussed and reviewed the mechanism of action and factors
influencing the preservative system in cosmetics. Three major evaluation
methods (USP method, CTFA method and Linear-regression analysis) of
Microbial challenge test have been compared to test preservatives’ efficacy. It
has been pointed out that the microbial challenge test is an important means to
evaluate the efficacy of preservatives.

Keywords:
Cosmetic preservation; Preservative System Evaluation; Microbial Challenge
Test.

Introduction

In summary, preservation has drawn increased attention in cosmetic

manufacturing and distribution process nowadays. Preservatives are playing
more and more important a role in cosmetic formulation. It is essential for a
cosmetic product that its preservative system keeps the product safe and
stable. With rapid growth of cosmetic industry, more and more anti-microbial
agents are widely used.

Microbial challenge test, which is established by international practice, is a

most widely used method to evaluate the efficacy of preservatives. The test is
performed for a period of time to simulate clinical scenarios in the process of
manufacturing and handling. In this test, samples are inoculated with test
microorganisms and then inspected visually and by plate count to determine if
the preservative system is effective. The above method is the classical means
to evaluate preservative efficacy because it closely mimicks the practical

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situation.

1. The Important Action of Preservatives on Cosmetics

1.1 The Influence of Microorganism
There is no doubt that all kinds of microorganisms are widely spread around us.
Although we have killed successfully an amount of microorganism in
cosmetics products through sterilization in the manufacturing procedure, there
are some still alive more or less. After choosing suitable preservative system
and packaging, cosmetic products can be preserved well. But maybe they
would be re-contaminated during consumption. Some ingredients in cosmetics,
such as esters, hydrocarbon and soluble polymers tend to provide not only
abundant nutrition with carbon and nitrogen sources that are necessary for
microbes to grow but also chemical energy with its adduction. The
microorganisms in cosmetics would grow, reproduce, and even cause the
products to be spoiled once they live under appropriate conditions. The
following cosmetic deteriorations are caused by microorganisms, opacities,
deposition and color change by decomposing the substance; changing the
product’s pH value through the catastate and the appearance through the
gases created. The microorganisms may even secrete toxins, which can
cause harm and skin anaphylaxis.

The microorganisms that exist in used or unused cosmetics normally include

bacteria (either gram positive or gram negative), mold, and yeast. Some
microbes are harmless, but some, such as Staphylococcus aureus,
Pseudomonas aeruginosa, are greatly harmful to human beings.

At present, there are still not universal criteria for microbiological evaluation of
drugs and cosmetics. In general, there are strict standards for baby care and
eye care products. In China, according to cosmetics health standard
requirement, the colonies of bacteria in cosmetics should not be more than
1000 cfu/mL (g), the colonies in baby care or mucosa products should not be
more than 500 cfu/mL (g), the colonies of mold and yeast should not be more
than 10 cfu/mL (g), harmful microbes such as Escherichia coli, Pseudomonas
aeruginosa and staphylococcus aureus and so on should not be detected.

1.2 The Mechanism of Action of Preservative

A preservative actually is a protective agent to keep cosmetics unspoiled
through killing or inhibiting microorganisms in cosmetic products. It doesn’t
show strong instant killing effect until the preservative contacted the microbes’
cells directly at enough concentration.

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Some preservatives can destroy the cleavage of cells to inhibit the microbes
grow by acting on the target nucleus of microbes, such as cell membranes, cell
wall, enzymes of cell. Actually, it affected the activity of enzymes or the
structure of heredity particles in bioplasm. For example, the acting target
nucleus of phenoxy ethanol or alcohol was cell membrane, Bronopol
(2-Bromo-2-nitropropane-1, 3-diol) acted on the SH enzymes, the preservative
with formaldehyde donator, such as EverguardTM series, acted on the COOH
and NH2 enzymes in the cells, and that, the phenols and aldehydes could
cause the protein denaturation. In short, the preservatives interfere cells
growth through inhibiting synthesis of enzymes, proteins and nucleic acids.

1.3 Factors of Influencing Activity of Preservatives

The preservatives’ efficacy depends on many factors. The preservative may be
highly effective in a special system but it may be of no efficacy in others. Many
factors can cause the preservative inactivation in a formula. The following are
several major factors that affect preservatives’ efficacy.

1.3.1 Influence of pH
The pH of the system can affect the preservative’s activity through influencing
the dissociation of organic acid. For example, Bronopol
(2-Bromo-2-nitropropane-1, 3-diol) is very stable when its pH is 4; it also could
keep active for a year when its pH is 6; but its activity only lasts several months
when its pH is 7.

1.3.2 Influence of Particles and Gels

Some particles like Kaolin or Magnesium Aluminum silicate in cosmetics could
reduce the preservative activity because of its adsorption of preservatives.
However, the particles could sometimes strengthen the bacteriostatic effects
through adsorbing the microbes as well.

The preservatives’ performance may be affected by combination with

water-soluble high molecular weight polymer, which can bring down the
concentration of free preservatives in the formulation.

1.3.3 Influence of Nonionic Surfactant

All kinds of surfactants in cosmetics, especially the nonionic surfactants, can
interfere the activity of preservatives by solubilization and complexation. The
oil soluble nonionic surfactant (HLB value is 3-6) has more de-active effect to
preservatives than the water soluble one with higher HLB value. The
preservative’s activity decreases with its free concentration reduction. So, the
use level of preservative should be increased in the nonionic surfactants
system in order to keep the same preservation power.

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1.3.4 Influence of Preservative Decomposition
Some factors may reduce the preservation efficacy through the preservatives’
decomposition. For example, light or heat cause preservative decomposition;
chemical reaction may lead to preservation inactivation or radiation sterilization
may reduce preservatives’ activity.

1.3.5 Other Influencing Factors

In addition, there are many other factors, such as preservative distribution in
oil/water phase, packaging, fragrance, chelating agents, may all interfere with
preservative activity.

1.4 Ideal Preservatives

No single preservative can provide a broad-spectrum anti-microbial activity at
very low use levels. But, it is important for us to assume “ideal” preservative
criteria so that we can develop a broad –spectrum preservative system
successfully. We think an excellent preservative should at least have the
following characteristics:
1) It should have broad-spectrum anti-microbial activity against both bacteria
and fungi.
2) It should have excellent anti-microbial activity at low levels in cosmetic
products.
3) It should be effective over a wide pH range.
4) It should be safe, non-toxic and non-irritating.
5) It should be inert, non-reactive with other ingredients in formula or
container materials.
6) It should have suitable distribution coefficient of oil-water phase in order to
get effective concentration in water phase.
7) It should be compatible with essentially all cosmetic materials and not to
affect the color and fragrance in the finished products.
8) It should be cost effective and easy to obtain.

It is the trend that several preservatives are obtained to form an effective,

broad-spectrum mixture system. An effective, broad-spectrum antisepsis
performance also depends on the final formula. How to make a preservative
system resist microorganisms successfully? The first thing is to avoid being
contaminated in manufacturing process; on the other hand, we should avoid
contamination in packaging and use procedure.

2. Preservative Efficacy Test Methods:

There are several different preservative efficacy test methods of evaluating the
preservative performance in different countries, including USP method, BP

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 method and EP method. In 1995, BP method was revised in accordance with
EP method. The CTFA method is the internal method for Cosmetic, toiletries
and fragrance Association in USA. Besides standard USP method and CTFA
method, there are some rapid evaluation techniques, such as linear regression
method, presumptive challenge test and accelerated preservation test
methods. The test process of above three methods are almost the same, but
there were some differences in the time needed for analysis and performance
criteria. These differences could lead to different results and whether a sample
passes the challenge test.

So, the three evaluation methods of USP, CTFA and Linear Regression
methods are discussed below, and selection of microorganisms, inoculums
level, measuring intervals and acceptance criteria are compared. Table 1 gives
the details for these three methods.

Table1 The Comparison of Three Evaluation Methods

Methods Linear
USP Method CTFA Method Regression
Items Method
Staphylococcus Gram-positive and Staphylococcus
aureus, gram-negative aureus,
Escherichia coli, bacteria, mold, Escherichia coli,
Pseudomonas yeast and aerobic sporeforming
aeruginosa, spore-forming bacillus (separated
Strains
Candida albicans bacillus some strain), Aspergillus
and Aspergillus strains separated niger and
niger from Aspergillus flavus
anti-preservative
samples
1.0×106 cfu/ml (g)
the level of yeast
Inoculums level 5 6
1.0×10 -1.0×10 and mold in ＞1.0×106
Unit: cfu/ml (g)
eye-cosmetic is
4
1.0×10 cfu/ml (g)
0, 7, 14, 21 and 28 0, 7, 14, 21 and 28 0, 2, 4, 24, 28 and
days after original days after original 168 hours after
Measuring
inoculation inoculated. Take original inoculated.
Process
sample additional Or up to the last
of Sampling
after 28 days if it APC is less than
was re-challenged. 10 cfu/ml (g).

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 A 99.9% reduction A more than The D value of
of bacteria by the 99.9% reduction of pathogenic
14th day. The bacteria within 7 bacteria is less
concentration of days. The than 4 hours; the D
viable yeasts and concentration of value of
molds remain at or yeast and mold non-pathogenic
below the original reduced and bacteria is less
Performance inoculated within continued than 28 hours.
Criteria 14 days. The reduction for the
concentration of duration of the
test organism test. Bacteriostatic
remains at or activity against
under required spores through the
levels of the entire test.
28-day test period.

3. Discussion of Preservative Efficacy Test Methods

3.1 How to choose microorganism in challenge test
From the above, we can find that the same microorganisms in the challenge
test can be adopted in the three evaluation methods of USP, CTFA and Linear
Regression: staphylococcus aureus, Pseudomonas aeruginosa and
Aspergillus niger. The test procedure shows the organisms changes during the
products manufacture and consumption. Although these recommended strains
are representative, the seudomonas cepacia is recommended mightily to get
the broad-spectrum preservative system. Seudomonas cepacia is a
non-fluorescent monad, such as Pseudomonas aeruginosa, which is more
sensitive to preservatives than fluorescent monad. Furthermore, the
organisms separated from environment or housing microbes recommended by
CTFA shall be considered as well, because these microbes are more likely to
be present in the products. It is the most ideal if the challenge test can be
performed with both the standard trains and the separated organisms partly if
the condition permits.

3.2 Rationality of Re-challenge Test

The aim of re-challenge test is to reassess contamination during product use
period. It shows the number of challenge test that the preservative system
could hold before the system breads down by repeated inoculation with special
microorganisms. It is un-necessary to go on re-challenge test for linear

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regression method, because the microorganisms’ death rate of tested formula
that contained enough preservatives really has nothing to do with the amount
of microorganisms during the test time.

3.3 Acceptance Standard

Linear regression method determined (expressed by ‘D’ value) the
decimal-reduction time of microorganism in challenge test, namely, the time
that a 90% microorganism reduction required. This method is used by
traditional microorganism measurement –APC. The evaluation speed by the
linear regression method is quicker than CTFA and USP method.

The results of USP and CTFA methods indicated ‘pass’ or ‘fail’. The linear
regression method result is showed by the D value that could be determined
from a survivor curve by the construction method and least-squares method.
The curve is prepared by plotting the number of viable organisms recovered at
various times.

We could compare the test standard of microorganism death rate of USP and
CTFA method with linear regression method through translating the D value.
The D value for bacteria in USP method is 112; the D values for bacteria and
fungi in CTFA are 56 and 168, respectively. They all are far greater than the D
value 4 and 28 in linear regression method. It is clearly that the standard of
linear regression method is stricter than USP and CTFA methods. It has been
shown that no re-contamination of adaptation microbes have been found in
products that passed the acceptance standard of linear regression method.
The preservative system, if it passed the test of linear regression method, has
less possibility of failure than if it only passes the test of USP or CTFA methods.
It is illustrated that the linear regression method provides a more reliable
means of performing preservative efficacy tests.

3.4 Experimental Time

Linear regression method is a rapid preservative evaluation method. The test
of pathogens such as staphylococcus aureus and Pseudomonas aeruginosa
could be compared within 3 days and the test of non-pathogens could be
completed in two weeks. The test period of USP and CTFA method are both 28
days, it even needs 8 weeks if it is continued with re-challenge test. But the
evaluation time is greatly shortened for linear regression method because it is
unnecessary to go on for re-challenge test.

There is still not a fully standardized method by now to evaluate the

preservative systems in cosmetics and personal care products. In fact, many

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large-scale companies have built internal criteria for evaluation according to
the USP method or CTFA method. Under the same condition, it is suggested
that Linear Regression method could be alternatives to evaluate the efficacy of
preservative systems because it is easy, quick and reliable evaluation method.

Reference:
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[M], 1984
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Toiletries, No. 3, 1991
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Development and Evaluation, [J] Cosmetic & Toiletries, No. 4, 1989
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Toiletries, No. 3, 1981
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