Preservative-Free and Self-Preserving Cosmetics

Preservative-Free
and Self-Preserving
Cosmetics
and Drugs
COSMETIC SCIENCE AND TECHNOLOGY
Series Editor
~RIC JUNGERMANN
Jungennann Associates, Inc.

Phoenix, Arizona
1. Cosmetic and Drug Preservation: Principles and Practice, edited by

Jon J. Kabara
2. The Cosmetic Industry: Scientific and Regulatory Foundations, edi-
ted by Norman F. Estrin
3. Cosmetic Product Testing: A Modern Psychophysical Approach,
Howard R. Moskowitz
4. Cosmetic Analysis: Selective Methods and Techniques, edited by P.
Bore
5. Cosmetic Safety: A Primer for Cosmetic Scientists, edited by James
H. Whittam
6. Oral Hygiene Products and Practice, Morton Pader
7. Antiperspirants and Deodorants, edited by Karl Laden and Carl B.
Felger
8. Clinical Safety and Efficacy Testing of Cosmetics, edited by William
C. Waggoner
9. Methods for Cutaneous Investigation, edited by Robert L. Rietschel
and Thomas S. Spencer
10. Sunscreens: Development, Evaluation, and Regulatory Aspects, edi-
ted by Nicholas J. Lowe and Nadim A. Shaath
11. Glycerine: A Key Cosmetic Ingredient, edited by Eric Jungermann
and Norman 0. V. Sonntag
12. Handbook of Cosmetic Microbiology, DonaldS. Orth
13. Rheological Properties of Cosmetics and Toiletries, edited by Dennis
Laba
14. Consumer Testing and Evaluation of Personal Care Products,
Howard R. Moskowitz
15. Sunscreens: Development, Evaluation, and Regulatory Aspects.
Second Edition, Revised and Expanded, edited by Nicholas J. Lowe,
Nadim A. Shaath, and Madhu A. Pathak
16. Preservative-Free and Self-Preserving Cosmetics and Drugs: Principles
and Practice, edited by Jon J. Kabara and DonaldS. Orth
ADDITIONAL VOLUMES IN PREPARATION
Hair and Hair Care, edited by Dale H. Johnson

Preservative-Free
and Self-Preserving
Cosmetics
and Drugs
Principles and Practice
edited by
Jon J. Kabara
Technology Exchange, Inc.
Galena, Illinois, and
Michigan State University
East Lansing, Michigan
Donald S. Orth
Neutrogena Corporation
Los Angeles, California
~ Taylor & Francis

V Taylor&Francis Group
Boca Raton London New York
CRC is an imprint of the Taylor & Francis Group,

an informa business
Library of Congress Cataloging-in-Publication Data
Preservative-free and self-preserving cosmetics and drugs:

principles and practice I edited by Jon J. Kabara, DonaldS. Orth.
p. em.- (Cosmetic science and technology series; v. 16)
Includes index.
ISBN 0-8247-9366-8 (hardcover: alk. paper)
1. Cosmetics. 2. Chemical preservatives. I. Kabara, Jon J.
IT. Orth, Donald S. ill. Series.
TP983.P8917 1996
668'.55-dc21
96-47675
CIP
The publisher offers discounts on this book when ordered in bulk quantities. For more
information, write to Special Sales/Professional Marketing at the address below.
This book is printed on acid-free paper.
Copyright© 1997 by Marcel Dekker, Inc. All Rights Reserved.
Neither this book nor any part may be reproduced or transmitted in any form or by any
means, electronic or mechanical, including photocopying, microfilming, and recording, or
by any information storage and retrieval system, without permission in writing from the
publisher.
Marcel Dekker, Inc.
270 Madison Avenue, New York, New York 10016
Current printing (last digit):
10 9 8
About the Series
The Cosmetic Science and Technology series was conceived to permit discussion
of a broad range of current knowledge and theories of cosmetic science and
technology. The series is made up of books written by one or more authors or edited
volumes with a number of contributors. Authorities from industry, academia, and
the government are participating in writing these books.
The aim of this series is to cover the many facets of cosmetic science and
technology. Topics are drawn from a wide spectrum of disciplines ranging from
chemistry, physics, biochemistry, and analytical and consumer evaluations to
safety, efficacy, toxicity, and regulatory questions. Organic, inorganic, physical,
and polymer chemistry, emulsion technology, microbiology, dermatology, and
toxicology all play a role in cosmetic science.
There is little commonality in the scientific methods, processes, or formula-
tions required for the wide variety of cosmetics and toiletries manufactured.
Products range from hair care, oral care, and skin care preparations to lipsticks, nail
polishes and extenders, deodorants, body powders and aerosols to over-the counter
products, such as antiperspirants, dandruff treatments, antimicrobial soaps, and
acne and sunscreen products.
Cosmetics and toiletries represent a highly diversified field with many
subsections of science and "art." Indeed, even in these days of high technology,
"art" and intuition continue to play an important part in the development and
evaluation of formulations and the selection of raw materials. There is a move
Iii
iv About the Series
toward more sophisticated scientific methodologies in the fields of preservative

efficacy testing, claim substantiation, safety testing, product evaluation, and chemi-
cal analyses.
Emphasis in the Cosmetic Science and Technology series i~ placed on
reporting the current status of cosmetic technology and science in addition to
historical reviews. The series includes books on safety, efficacy testing, oral
hygiene, scientific and regulatory foundations, cosmetic product testing, analytical
methods and techniques, antiperspirants and deodorants, glycerine, rheological
properties, cosmetic microbiology, sunscreens, methods of cutaneous investiga-
tion, and consumer testing and evaluation of personal care products. Several of the
books have found a wider audience and have been translated into Japanese.
Contributions range from highly sophisticated and scientific treatises to primers,
practical applications, and pragmatic presentations. Authors are encouraged to
present their own concepts as well as established theories. Contributors have been
asked not to shy away from fields that are still in a state of transition, nor to hesitate
to present detailed discussions of their own work. Altogether, we intend to develop
in this series a collection of critical surveys and ideas covering diverse phases of
the cosmetic industry.
Preservative-Free and Self-Preserving Cosmetics and Drugs is the sixteenth
book in the series. It investigates the concept of self-preserving formulations and
promotes the idea of either eliminating or greatly reducing the use of chemical
preservatives. In so doing, this volume introduces a new paradigm in the formula-
tion approach to cosmetic and drug products. The book is divided into eleven
chapters ranging from basic discussions of the principles of self-preserving prod-
ucts, survival strategies of microorganisms, and "water activity" to a review of
different classes of chemicals that can provide antimicrobial activity. Included are
certain surfactants, fatty acids and their esters, phospholipids, "aroma" chemicals,
chelating agents, and antioxidants. This is the first book published that brings
together the principles of preservation and different classes of formulation compo-
nents.
I want to thank the editors, Dr. Jon J. Kabara and Dr. Donald S. Orth, for
collaborating in the writing and editing of this book. It is the second time that both
have contributed to the Cosmetic Science and Technology series. Dr. Kabaraedited
Cosmetic and Drug Preservation, and Dr. Orth wrote the Handbook of Cosmetic
Microbiology. Special recognition is also due to Sandra Beberman and the editorial
staff at Marcel Dekker, Inc. In addition, I would like to thank my wife, Eva, without
whose constant support and editorial help I would never have undertaken this
project.
Eric Jungennann, Ph.D.

Series Editor
Preface
The term preservative{ree means "without preservative chemicals." Usually,

multiple-use aqueous consumer products in their current containers cannot be made
preservative-free merely by removing preservatives from the formula. It is possible
to have preservative-free aqueous products only if they are sterilized and packaged
and stored appropriately.
The term self-preserving is more appropriate than preservative-free for most
aqueous products containing a chemical system that kills microorganisms and/or
prevents their growth. The concept of "preservative-free" is controversial; how-
ever, the emphasis of this book is to focus on the principles of preservation that
may be used to make products that are microbiologically safe at the time of
manufacture and during use by the consumer. No other publication presently
available addresses this subject to the depth presented in this book.
Preservative-free and self-preserving products may be developed by under-
standing and applying the principles of preservation as they relate to each product.
Some of the principles discussed are water activity, pH, special lipids, surfactants,
antioxidants (phenols), aroma chemicals, chelators, and packaging. Products that
are adequately preserved and microbiologically safe may be developed by
selection of chemicals to create conditions that have static or cidal effects on
microorganisms.
Although we could continue to use standard preservative systems for domes-
tic products, globalization requires that we think beyond traditional formula
v
vi Preface
development. Self-preserving products offer many advantages for formulation

because they reduce and/or eliminate chemical preservatives that are a source of
skin irritation and contact sensitization; they meet the demands of a growing
segment of consumers who want natural products; they may encourage the use of
contamination-resistant packaging; they allow the use of global formulas without
the regulatory issues surrounding the use of chemical preservatives, and they may
reduce formula costs by avoiding addition of preservatives.
Bacteria, yeasts, and molds cope with stressful conditions by using a wide
range of survival strategies designed to prevent or repair injury so that they can
survive and grow. Several mechanisms are discussed to illustrate how microorgan-
isms deal with unfavorable conditions that are used to limit their growth in products.
Consumer-friendly, environment-friendly products made without preservatives are
possible, but this may require modification of current formulations and packaging.
Opportunities exist for innovative workers who want to look beyond current
technologies and apply the principles of preservation in developing novel, preserv-
ative-free and self-preserving cosmetics and drugs. We hope this book will be a
platform for future discoveries.
We wish to acknowledge the efforts of our contributing authors and the
cooperation of the staff at Marcel Dekker, Inc. We are pleased to recognize Betty
Kabara and Barbara Jo Orth for their patience, understanding, encouragement, and
continued support because this greatly helped our writing and editing.
Suggestions and comments about the book are welcomed.
Jon J. Kabara, Ph.D.

DonaldS. Orth, Ph.D.
Contents
About the Series (Eric Jungermann) iii

Preface v
Contributors ix
1. Principles for Product Preservation

Jon J. Kabara and DonaldS. Orth
2. The Effect of Acid pH on Microorganisms and Survival Strategies

that Permit Growth in Products 15
DonaldS. Orth
3. Water Activity and Self-Preserving Formulas 45

Davin C. Enigl and Kent M. Sorrells
4. The Roles of Surfactants in Self-Preserving Cosmetic Formulas 75

Oreste Cozzoli
5. Fatty Acids and Esters as Multifunctional Components 119

Jon J. Kabara
6. Biomimetic Phospholipids: Components for Self-Preservation 139

Dennis L. Fost and John I. Yablonski
vii
viii Contents
7. Use of Antioxidants in Self-Preserving Cosmetic and

Drug Formulations 159
A. Larry Branen and P. Michael Davidson
8. Aroma Chemicals as Preservatives 181

Jon J. Kabara
9. Chelating Agents as Preservative Potentiators 209

Jon J. Kabara
I 0. The Role of Packaging in Product Preservation 227

Daniel K. Brannan
II. Preservative-Free and Self-Preserving Cosmetic and Drug

Products: The Future 243
Jon J. Kabara and DonaldS. Orth
Index 263
Contributors
A. Larry Bran en, Ph.D. Department of Food Science and Toxicology, University
of Idaho, Moscow, Idaho
Daniel K. Brannan, Ph.D. Department of Biology, Abilene Christian University,

Abilene, Texas
Oreste Cozzoli, Dr. Stazione Sperimentale perle Industrie degli Oli e dei Grassi
(SSOG), Milan, Italy
P. Michael Davidson, Ph.D. Department of Food Science and Toxicology, Uni-

versity of Idaho, Moscow, Idaho
Davin C. Enigl, M.S., M.E.A.S. Hazard Analysis Critical Control Points,

HACCP Validations-sm. Sacramento, California
Dennis L. Fost, Ph.D. Marketing and Commercial Development, Mona Indus-

tries, Inc., Paterson, New Jersey
Jon J. Kabara, Ph.D. Technology Exchange, Inc., Galena, Illinois, and Professor
Emeritus, Michigan State University, East Lansing, Michigan
lx
X Contributors
Donald S. Orth, Ph.D. Research and Development Department, Neutrogena

Corporation, Los Angeles, California
Kent M. Sorrells, M.B.A., Ph.D. Quality Assurance, National Steak and Poultry,
Owasso, Oklahoma
John I. Yablonski, M.S. Bio-Control Consultants, Inc., Westfield, New Jersey

1
Principles for Product Preservation
Jon J. Kabara
Technology Exchange, Inc.
Galena, Illinois
Michigan State University
East Lansing, Michigan
Donald S. Orth
Neutrogena Corporation
Los Angeles, California
Preservatives are used in aqueous cosmetics and drugs to prevent microbial

contamination. While this has solved the problem of contamination in most
instances, it has created other problems. No preservative is free of allergenic
potential, and many consumers want "natural" products that do not contain pre-
servatives. Consequently, views on cosmetic preservation and "the greening of
cosmetics" are on a collision course.
In recent years, there has been increasing interest in developing and market-
ing natural cosmetics, including those that are "preservative-free" (i.e., products
that do not contain ingredients commonly recognized as antimicrobial preserv-
atives), to meet the consumer demand for natural products. A glance at product
labeling in local retail outlets reveal scores of cosmetics and over-the-counter
(OTC) drugs with labeling that indicates these products are "natural," "fragrance-
free," "hypoallergenic," "noncomedogenic," "PABA-free," "chemical free," etc.
"Preservative-free" has been added to the labeling of a few products. What
is the scientific basis for the creation of this new class of products? Is there a need
1
2 Kabara and Orth
for them?" Are they safe? Are they satisfactorily preserved? Do they have suitable
"cosmetic elegance"?
The term preservative-free means without preservative chemicals. It is
possible to have preservative-free aqueous products if they are sterilized and
packaged/stored appropriately. The term self-preserving is more appropriate than
preservative-free for most aqueous cosmetic and OTC drug products in multiple-
use containers because formulas without any form of preservation will spoil if they
are not packaged or stored in such a manner that microbial contamination and
growth are prevented. Most aqueous products have a "preservative system" created
by the physicochemical composition of the formula (with or without the addition
of chemical preservatives) that kills microorganisms and/or prevents their growth.
Kabara (1) discussed formulation of self-preserving products using a systems
approach to cosmetic preservation.
Although cosmetics and OTC drugs are not necessarily intended to be sterile,
satisfactorily preserved aqueous formulas in multiple-use containers have a pre-
servative system that makes them self-sterilizing (2). A self-preserving formula is
one that either prevents microbial growth (i.e., is bacteriostatic) or kills microor-
ganisms (i.e., is bactericidal/microbicidal). A self-preserving formula may or may
not contain antimicrobial preservatives.
Relatively anhydrous products (e.g., talcum powder, cornstarch, mineral oil,
and stick deodorants) do not require preservatives because they do not have
sufficient water content to support microbial growth. However, manufacturers may
incorporate preservatives into these products to prevent microbial growth that may
occur due to inadvertent addition of water by consumers during use (or abuse).
Aqueous products, on the other hand, require preservatives unless their
composition, packaging, or storage conditions prevent microbial contamination
and growth. The physicochemical make-up of the product, including low water
activity (aw), high or low pH, and multifunctional materials that have antimicrobial
properties (i.e., alcohols, surfactants, quaternary ammonium compounds), deter-
mines whether a formula is "self-preserving." Aqueous products in multiple-use
containers generally require the addition of preservatives if they do not prevent
microbial growth.
The type of packaging and consumer use habits determine the preservative
requirements of a formula. The required D-value (RDV) concept was introduced
to relate the preservative system of the formula with product packaging and
consumer use/abuse (2,3). Although an unpreserved formula in a multiple-use
container would not be acceptable because it may become contaminated during
use, the formula would be acceptable if it were manufactured, sterilized, and
supplied in a unit-dose container. The same formula in a multiple-use container
may be suitable for restricted use if it were handled as we now handle perishable
foods, such as milk, which is refrigerated when not being used. Alternately,
unpreserved products could be frozen and thawed immediately before use.
Principles for Product Preservation 3
Currently available antimicrobial preservatives may cause some adverse

reactions including skin irritation, eye irritation, and contact sensitization (2). The
North American Contact Dermatitis Group identified preservatives as a group of
ingredients most commonly associated with skin irritation due to the use of
cosmetics (4,5). Recognizing that preservatives may be a source of irritation
provides a driving force for attempting to find milder, less irritating preservative
systems.
Although it would be desirable to eliminate preservatives that cause adverse
reactions (e.g., skin and eye irritation, contact sensitization) in some individuals,
this is not possible with most existing formulations in their current fonn/packaging.
Preservatives are used in these products because self-preserving products may be
unattainable without accepting specific formula and/or packaging constraints. This
chapter will introduce topics covered in detail in later chapters. The material
presented includes 1) a discussion of the principles of preservation providing
background information on the control of microorganisms, 2) a discussion of the
preservative system concept, which shows how the principles of preservation are
used to design self-preserving products, 3) an overview of survival strategies of
microorganisms to illustrate mechanisms used by microorganisms to survive in
hostile environments, and 4) some advantages and disadvantages of self-preserving
products.
1.1 PRINCIPLES OF PRESERVATION

Bacteria, yeasts, and molds are ubiquitous in nature. Cosmetic raw materials and
finished products provide a wide spectrum of inorganic and organic compounds,
which are available for microbial growth when sufficient water is present. Micro-
bial growth will occur in aqueous systems held at ambient temperatures found in
manufacturing plants, in trade channels, and in consumers' homes if control
measures are not instituted (2). The principles of preservation are used globally to
control microbial growth in foods, drugs, and cosmetics. These principles are:
1. Asepsis (keeping microorganisms out of the process stream)

adherence to current good manufacturing practices (GMPs)
microbiological validation of processes
microbiological validation of cleaning and sanitization programs
2. Removal of microorganisms
trimming and washing
filtration and clarification
sedimentation and centrifugation
3. Retarding growth or killing microorganisms
high temperatures (heat sterilization, pasteurization)
low temperatures (refrigeration, freezing)
4 Kabara and Orth
drying conditions (low aw)

high/low pH
removal of substrates
anaerobic atmosphere (COz, Nz gas packing)
preservatives/biocides
irradiation
mechanical disruption (grinding, high pressure)
The quality of finished products and the microbiological problems that occur
during manufacturing may be influenced by the kinds and numbers of microorgan-
isms present. Quality defects including altered color, flavor, odor, viscosity, and
texture become evident when sufficient microbial growth has occurred. Preventing
unnecessary microbial contamination of the process stream is an important consid-
eration in making products in accordance with GMPs. Frequently, the extent of
microbial contamination of in-process materials and finished products depends on
the microbia/load of raw materials and conditions that permit growth of microor-
ganisms (2). This is particularly true for "natural" raw materials.
The principles of preservation are applied to manufacturing and storage of
products to minimize the microbial load and to retard growth or kill microorgan-
isms that may gain access to the process stream and finished products. Microor-
ganisms have physical and chemical requirements for growth including suitable
temperature, pH, water (osmotic pressure or aw), nutrients, minerals, oxygen [or
the proper oxidation/reduction (0/R) potential], organic growth factors, and free-
dom from harmful radiation and chemicals (preservatives, antibiotics). It is evident
that microbial growth may be prevented by interfering with these requirements.
The principles of preservation utilized most successfully in self-preserving
products are low or high pH, low aw, multifunctional materials that have antimi-
crobial activity, and combinations of these. Generally, microorganisms of interest
in raw materials or in cosmetic products grow best around neutrality (pH 7.0). The
growth rate of microorganisms generally decreases as the pH departs from neutral-
ity (or from the optimum pH for growth for each different organism). Although
many yeasts and molds are able to tolerate acid pH conditions (i.e., pH< 4.0), many
microorganisms are metabolically injured or "stressed" by extreme pH condi-
tions-where the pH is <4 or >10 (6). It should be noted that the excess acidity or
alkalinity in low- or high-pH products may make these products harsh or in·itating
to the skin.
1.2 INJURY OF MICROORGANISMS

When conditions are suitable, a population of microorganisms utilizes available
substrates and grows. When conditions are unfavorable, the population stops
growing. Individual cells within the population may die or become metabolically
injured or "stressed" as a result of exposure to adverse physical and chemical

conditions. Cellular injury may be caused by altered membrane permeability,
altered enzyme function, degradation of ribosomes, and increased sensitivity to
selective agents (6-8).
Sublethally injured microorganisms may be more susceptible than nonin-
jured microorganisms to secondary stresses, such as elevated temperatures, low aw,
and the presence of inhibitory chemicals; consequently, they are unable to grow
under certain environmental conditions. The significance of this is that stressed
cells may escape detection during routine microbiological testing, when in fact they
may be capable of recovery and growth after an extended lag period or after
removal of the adverse conditions. Injury can be reversible, and injured microor-
ganisms may revive and grow (6).
Injured microorganisms can be detected in several ways. One of the most
convenient means is to compare the aerobic plate count (APC) on a noninhibitory
growth medium with the APC on a selective medium (6,9). After a period of
exposure to stress, some cells in the population lose their characteristic tolerance
to the selective agent-with concomitant decrease in the observed APC on the
selective medium. Nevertheless, the APC on nonselective media remains the same,
unless cell death has occurred.
A hypothetical example of the effect of injury on the APC during the lag
phase of growth is illustrated in Figure 1.1. Here, a suspension of 105 bacteria/ml
has been injured by exposure to low pH. All viable cells retain their ability to grow
on the nonselective plating medium (open circles), but only a portion of the cells
(2.5 x 102 bacterialml) is able to form colonies on the selective medium (closed
circles). The difference in APCs on the selective and nonselective media reflects
the extent of injury of cells in the population. In this case, more than 99% of the
cells have been injured.
After removal of the source of stress (i.e., by neutralizing the acid pH in this
example), the injured cells must recover before they can begin dividing. This
recovery period requires resuscitation and/or repair and causes an extended lag
phase-a period in which the population appears to be dormant because it is not
growing (6). Cellular repair may take a period of hours in a favorable environment
and longer when conditions are not optimum. After recovery, the cells are able to
grow on the selective medium and the APCs become the same on both the selective
and nonselective media. After all cells have undergone the repair process, the
population goes into a log growth phase (Fig. 1.1 ).
Microorganisms in self-preserving cosmetic and OTC drug products may be
exposed to several different kinds of physical and chemical stresses including low
pH, low aw, chelating agents, surface active agents, free fatty acids, and nutrient
depletion (starvation). Stressed microorganisms that are viable but nonculturable
represent a hazard because they may recover and grow in the product at some later
time (6).
6 Kabara and Orth
7r-------------------------------------------------~
-0
,_
Q)
..04
E
::J
z
_ga
C)
Recovery Period
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (Units)
FIGURE 1.1 Hypothetical example of the effect of sublethal injury on the ability of
bacteria to grow. This figure represents the first portion of the idealized bacterial
growth curve. Here, a portion of the population was injured, as determined by plating
on a nonselective medium (open circles) and on a selective medium (closed circles).
The APCs on the nonselective medium remain at the same level until injured
members of the population recover. The APCs on the selective medium are lower
than those on the nonselective medium until the recovery period is complete. This
recovery period results in an extended lag phase. The population goes into the log
phase of growth at the completion of the lag phase.
1.3 PRESERVATION OF PRODUCTS WITH LOW

OR HIGH pH
The pH of a product may be lowered using mineral acids or organic acids. Freese
et a!. (10) reported that lipophilic acids (e.g., lauric acid, myristic acid) are
antimicrobial because they inhibit membrane transport of oxidizable substrates.
Kabara (11) examined the structure-function relationships of anionic surfactants
as antimicrobial agents and rep01ted the minimum inhibitory concentration (MIC)
of free fatty acids for gram-positive bacteria. He found the most active chain length
for saturated fatty acids to be C 12 (lauric acid), the most active monounsaturated
fatty acid C 16 (palmitoleic acid), and the most active polyunsaturated fatty acid
cl8:2 (linoleic acid) when using pneumonococci, group A streptococci, and P-
hemolytic streptococci (non-group A) as test organisms. Kabara reported that free
fatty acids do not affect gram-negative bacteria except for short-chain fatty acids
with fewer than eight carbon atoms. This work suggests that free fatty acids will
contribute to the antimicrobial action of a formula, especially at pH values acid to
the pKa of carboxylic acids (i.e., pH < 4.5).
1.4 PRESERVATION OF PRODUCTS WITH LOW WATER

ACTIVITY
Water is a requirement for growth of all known living organisms, from bacteria to
humans. As would be expected, different microorganisms have different minimum
water requirements. The water requirement of microorganisms may be expressed
in terms of available water, or aw. The aw is the vapor pressure of the solution
(solutes in water) divided by the vapor pressure of the solvent (water). The aw of
pure water would be 1.00, and a 1.0 M solution of an ideal solute would have an
aw of0.9832. If a solute such as glucose is dissolved in water, it will lower the water
vapor pressure. One mole of an ideal solute dissolved in one kilogram of water will
depress the vapor water by 1.77%, and the solution will have an aqueous vapor
pressure that is 98.23% of the vapor pressure of pure water (without solutes) at the
same temperature. The atmosphere above this solution will have a relative humidity
(R.H.) of 98.23%, and the solution will have an aw of 0.9823. At equilibrium, the
aw is a numerical expression equal to the R.H./100% (2,12).
Several generalizations may be made about the moisture requirements of
microorganisms as follows:
Each microorganism has a characteristic optimal aw· and range of aw for
growth for a given set of environmental conditions.
Factors affecting the aw requirements of microorganisms include the kind of
solutes present, the nutritional value of the substrates available, tempera-
ture, Oz supply, pH, and the presence of inhibitors.
An unfavorable aw will decrease the growth rate and the maximum yield of
cells.
The lag time before initiation of growth or germination of spores is increased
as the aw becomes more unfavorable.
Bacteria generally have higher aw requirements than yeasts, and yeasts
generally have higher aw requirements than molds.
Different classes of microorganisms have different tolerance to low aw, and
this determines whether a particular bacterium, yeast, or mold is able to grow in a
specific product. The reported lower limits of aw for several microorganisms of
interest to the cosmetic industry are presented in Table 1.1. Although the reported
aw requirements for specific microorganisms depend on the experimental condi-
tions and the physicochemical conditions of the environment, studies indicate that
the aw requirements for Pseudomonas species are quite high (minimum aw = 0.97).
Addition of solute to decrease the aw to <0.96 may be a convenient means of
8 Kabara and Orth
TABLE 1.1 Lowest Reported aw Values

Permitting Growth of Microorganisms
Microorganism Minimum aw value
Many bacteria 0.91

Pseudomonas spp. 0.97
Escherichia coli 0.95
Bacillus subtifis 0.945
Staphylococcus aureus 0.86
Halophilic bacteria 0.75
Many yeasts 0.88
Osmophilic yeasts 0.60
Many molds 0.80
Xerophilic fungi 0.65
Source: Adapted from Ref. 2.
controlling the growth of pseudomonads in many aqueous systems (including

cosmetic products). Escherichia coli and Enterobacter aerogenes have minimum
aw requirements around 0.95. This suggests that the growth of most coliforms and
pseudomonads may be prevented by decreasing the aw to <0.94. Review of the data
in Table 1.1 reveals that the moisture requirements of Staphylococcus aureus are
much lower (aw = 0.86) than are those of the above gram-negative organisms. As
noted above, the moisture requirements for any given microorganism depend on
other factors, including nutrient availability, the type of solute used to decrease the
aw, the pH, and other physicochemical factors.
Certain raw materials decrease the aw of a formulation. Humectants such as
glycerol, propylene glycol, and sorbitol and other water-soluble materials decrease
the aw and increase the stress imposed on microorganisms. Inorganic salts, acids,
and alkalis used to adjust the viscosity and pH decrease the aw. Curiously, some
raw materials contain a considerable amount of salt. For example, protein hydro-
lysates may contain> 15% salt so that use of 1-5% protein hydrolysate will add up
to 0.75% NaCI. Hydrocolloids (xanthan gum, guar gum, etc.) used for increasing
viscosity and alcohols (SD alcohol 40, isopropyl alcohol) used for enhancing the
penetration of specific ingredients, decreasing viscosity, or solubilizing ingredients
may also decrease the aw. Greater than 15% alcohol is effective in inhibiting
bacteria, yeasts, and molds, especially when the pH is acidic (pH < 4.5). Higher
concentrations of alcohol destabilize cell membranes, denature proteins, and kill
microorganisms. Lower concentrations of alcohol contribute to the preservative
system of the product.
The above discussion reveals that the aw of a product has broad implications
because it determines whether microbial growth is possible. Although determina-
tion of moisture content is useful for understanding the composition of the product,
the moisture content may tell us very little about the chemical or microbial
dynamics of the system-unless the moisture available for microbial growth is
determined. For example, a 25% NaCl solution has an aw of about 0.75, whereas
a 25% sucrose solution has an aw of about 0.98. The differences in aw are due to
differences in molar concentrations obtained with 25% N aCl and sucrose. Although
both solutions have the same moisture content, their ability to support microbial
growth is different. Many bacteria, yeasts, and molds are able to grow in the
presence of 25% sucrose, but only the halophilic bacteria, osmophilic yeasts, and
xerophilic fungi are able to grow in the presence of 25% NaCl (2).
Low aw by itself may not be sufficient to kill microorganisms. For example,
an aw of 0. 90 will not permit E. coli to grow, but viable cells may persist for weeks
or months in a dormant state. The survival of the E. coli population depends on
other stresses imposed by the formula (acidic pH,lack of nutrients, membrane-des-
tabilizing surfactants or chelating agents, etc.) and storage conditions including
temperature. Microorganisms are preserved by lyophilization (freeze-drying), and
it is reasonable to think that products with very low aw values, such as powders,
may allow microorganisms to survive for extended periods. Thus, cells may enter
a dormant state because removal of virtually all water causes metabolism to cease.
1.5 PRESERVATION OF PRODUCTS WITH

CHELATING AGENTS
Removal of substrates and essential nutrients helps prevent the growth of micro-
organisms. Chelating agents sequester divalent and trivalent metal ions and fre-
quently are used to bind metal ions that are prooxidants and cause deterioration in
fragrance, color, and appearance of cosmetic formulations. Sequestration of metal
ions interferes with microbial growth because divalent metal ions are required as
enzyme cofactors and are required to maintain the integrity of the gram-negative
bacterial cell envelope. Chelating agents commonly used in cosmetic products
include tetrasodium EDTA, trisodium EDTA, tetrasodium etidronate, and citric
acid. Several reports indicate that chelating agents function as preservative system
potentiators and/or synergists (2,13-15). It is not possible to control microbial
growth using levels of chelating agents normally found in cosmetic products (i.e.,
~1 %) because some microorganisms accumulate reserve materials, including
calcium, which may be complex with poly ~-hydroxybutyric acid (PHB) or
dipicolonic acid (6,13).
1.6 PRESERVATION OF PRODUCTS WITH

SURFACTANTS
Many components in cosmetic products have the potential to interfere with micro-
bial growth if they are present in high enough concentrations or when their action
is combined with that of other formula ingredients. Anionic surfactants such as
10 Kabara and Orth
ammonium lauryl sulfate (ALS) and sodium lauryl ether sulfate are commonly used
in shampoos, bath gels, and cleansers because they help solubilize lipids (e.g.,
sebum on the scalp and hair, greasy residues). When present in low concentrations,
various surfactants may be used as substrates for energy and growth, which is why
aqueous anionic surfactants require preservation. At higher concentrations, anionic
surfactants destabilize cell membranes and the lipopolysaccharide (LPS) cell
envelope of gram-negative bacteria. Different species of bacteria differ in their
tolerance to anionic surfactants. Gram-positive bacteria such as the staphylococci
generally cannot grow in 10% surfactant, some species of spore-forming bacilli
and E. coli have moderate tolerance to surfactants, and some strains of Pseudo-
monas aeruginosa are able to grow in 28% ALS. Low concentrations of surfactant
(1-5%) may add to the stress imposed on microorganisms when other factors in
their environment are unfavorable. The antimicrobial effects of surfactants, low
pH, low aw, chelating agents, etc. are additive or synergistic in some cases.
Phosphate esters, glyceryl stearate, and glyceryl dilaurate generally have no
significant antimicrobial activity at levels used for emulsifying creams and lotions.
Kabara reported that esterification of a fatty acid to a anhydric alcohol yields an
ester with no antimicrobial activity, whereas esterification to a polyhydric alcohol
produces an antimicrobial compound (16). Glyceryl monolaurate has antibacterial
activity against gram-positive bacteria and fungi at use levels ofO.l-2.0% (1,14).
Antibacterial activity against gram-negative bacteria is achieved by combining
glyceryl monolaurate with a chelating agent such as disodium EDTA.
Quaternary ammonium compounds (quats) are used for hair conditioning,
skin feel, emulsification, and their antimicrobial activity. Different quats possess
different antimicrobial activities. Benzalkonium chloride (BAC) is used as an
antimicrobial agent and is found in mouthwashes and contact lens cleaning solu-
tions. This quat is effective against most microorganisms at a concentration of
0.1 %; however, gram-negative bacteria--especially the pseudomonads-are more
resistant than are the gram-positive bacteria (17). Linoleamidopropyl PG-di-
monium chloride phosphate is a primary cationic emulsifier that is reported to leave
a smooth after-feel on the skin, reduce tackiness, and have an MIC of s;300 ppm
for gram-positive bacteria, C. albicans, and molds. Cocamidopropyl PG-dimonium
chloride phosphate is reported to contribute to foaming and detergency of a
formula, to condition the skin, and to have MICs of s;300 ppm for gram-positive
bacteria, s;I250 ppm for gram-negative bacteria, and s;sooo ppm for yeasts and
molds (18). Ethyl N-cocoyl-L-arginate PCA salt is a cationic surfactant that is
reported to have a MIC of s;soo ppm for bacteria and yeast (19).
1.7 PRESERVATION WITH MULTIFUNCTIONAL

MATERIALS
Each ingredient in a formula may have more than one effect on the physical and/or
chemical properties of the formula. As noted by Kabara (1 ), multifunctional
ingredients may be used to help preserve cosmetic and drug products because they
affect the ability of microorganisms to grow. Although antioxidants are used to
stabilize fragrances and colors, the phenolic antioxidants are reported to potentiate
the antimicrobial action of other formula components (2, 14,20,21 ). Fragrances and
essential oils may contribute to the preservative action of the formula because they
contain alcohols, phenols, esters, organic acids, aldehydes, and terpenes, which
may have antimicrobial activity. Short-chain alcohols (e.g., ethyl alcohol and
isopropyl alcohol) are used to solubilize ingredients, to give fragrances a "lift," and
to contribute to the antimicrobial action of the formula.
1.8 PRESERVATIVE SYSTEM

The preservative system of a product (2,14) includes both compounds with known
antimicrobial activity and formula components that may contribute directly or
indirectly to antimicrobial activity. The physicochemical make-up of a formula,
which includes factors such as product pH, aw, nutrient availability, surfactant
concentration, sequestering agents, alcohols, interfering materials, etc., determines
whether a formula is self-preserving and/or whether specific preservatives are
needed for adequate preservation.
Some familiar products use low pH as part of their preservative system. The
low pH and low aw obtained using 25% aluminum chlorohydrate in a roll-on
antiperspirant helps create conditions unfavorable to microbial growth-especially
gram-negative organisms like P. aeruginosa and E. coli. The acid pH of cationic
hair conditioners (around pH 4) and cationic conditioning agents (quats) contribute
to the antimicrobial action of these products.
Liquid soaps with an alkaline pH (pH 9.5-10.5) present an unfavorable
environment for the growth of some microorganisms due to the chaotrophic (i.e.,
membrane destabilizing) effects of ionized fatty acids and the free alkalinity due
to NaOH. Liquid soaps may contain substantial quantities of dissolved solutes (e.g.,
glycerol, salts), which decrease the aw. Chelating agents and phenolic antioxidants,
such as butylated hydroxyanisole (BHA), become part of the product preservative
system.
1.9 SURVIVAL STRATEGIES OF MICROORGANISMS

Microorganisms respond to the physical and chemical conditions in their environ-
ment. The global responses of microorganisms are regulated and coordinated with
all metabolic responses to provide bacteria, yeasts, and molds with efficient means
of dissimilating substrates for use as sources of energy or as nutrients for growth.
The processes by which microorganisms respond to conditions in the environment
are termed survival strategies (6). The survival strategies discussed in Chapter 2
include acid tolerance, osmoregulation, and the production of siderophpres.
12 Kabara and Orth
1.10 ADVANTAGES AND DISADVANTAGES OF

PRESERVATIVE-FREE AND SELF-
PRESERVING PRODUCTS
One of the primary objectives in creating preservative-free and self-preserving
products is to minimize the irritation potential of a formula. Whether a reduction
in irritation could be observed by consumers depends on the severity of the
undesirable reactions caused by the preservatives in a specific formula. A second
benefit of preservative-free and self-preserving products is in marketing, because
this class of products would fill a real and/or perceived need on the part of some
consumers.
It must be recognized that use of low pH and low aw imposes physicochemi-
cal constraints on self-preserving formulas, and many products in their current form
may not be preserved adequately without the use of preservatives. Decreasing the
pH and increasing inorganic salts to nonionic emulsions that use acrylic acid
co-/homopolymers, magnesium aluminum silicate, or gums as thickening agents
may cause substantial decreases in viscosity or alter the solubility of other formula
components. Modification of the product with alternative thickening agents may
affect skin feel and/or the kinetics of percutaneous absorption. Decreasing the aw
by addition of nonionic materials (e.g., sugars, glycerol, propylene glycol, butylene
glycol, sorbitol) may create problems on product application (e.g., sticky, tacky,
dragy skin feel).
As noted above, self-preserving products may be microbiostatic-not micro-
bicidal. This means that contaminants would be prevented from growing, but that
they may remain in the product for extended periods. The acceptance criteria for
preservative efficacy testing of self-preserving products would not necessmily be
the same as for conventionally preserved products. Testing would need to establish
that:
Both routine test organisms and contaminants reasonably expected to be

introduced during manufacturing and/or use by the consumer are unable
to grow.
Microorganisms do not change the physicochemical composition of the
product (raise the pH by use of the arginine deiminase system, decrease
the aw as a result of uptake or utilization of salts and/or water-soluble
solutes).
Mixed flora do not have associative growth effects so that one organism
could alter the system, thereby allowing another to grow.
Microorganisms do not grow following abuse studies that subject the product
to conditions reasonably expected from normal use and abuse, including
dilution with water.
Packaging prevents contamination during consumer use/abuse.
Ptlnclples for Product Preservation 13
The safety of self-preserving products will have to be evaluated on an individ-

ual product basis. Although contamination with nonobjectionable microorgan-
isms may not present a consumer health hazard (providing they were not capable
of growing or rendering the product susceptible to contamination and growth by
other microorganisms), opportunistic pathogens including P. aeruginosa or C.
albicans may present an unacceptable risk to consumers. The hazard would
depend on the kinds and numbers of contaminating microorganisms, on the
site/frequency of use, and on an individual's susceptibility to the microorganism
and its metabolic by-products (enzymes, toxins, etc.). For example, low levels of
contamination by these microorganisms may present minimal risks for products
applied to normal skin; however, the risks increase when products are applied to
damaged skin (e.g., skin with cuts and abrasions, chapped skin with cracks and
fissures) or to sensitive areas (e.g., around the eyes, on baby skin). In some
instances, creative new approaches to product packaging may be needed to pro-
te~t the formula from microbial contamination. As in determining RDVs, the
type of packaging determines the preservation requirements of a product. Careful
studies are required to assess the safety of aqueous products in multiple-use
containers.
1.11 SUMMARY
Preservative-free and self-preserving products are an emerging class of cosmetics
intended to provide the same performance benefits as existing formulations and
reduced irritation potential. Many contemporary haircare, skincare, and eyecare
formulas may not simply be converted to preservative-free products by elimination
of preservative chemicals or to self-preserving products by formula changes that
produce low pH and/or low aw because these products may have unacceptable
"cosmetic elegance." Preservative-free and self-preserving products may be devel-
oped by understanding and applying the principles of preservation as they relate to
each product. This may require modification of current formulations, more protec-
tive packaging, and testing to ensure that microbial survival strategies will not
allow bacteria, yeasts, or molds to grow. The development of these new products
will require creativity and cooperation among formulators, microbiologists, engi-
neers, and package designers.
Technology has developed to the point where we understand microbial
physiology and are able to use the principles of preservation to create products that
are adequately preserved. Consumer-friendly, environment-friendly products
made without preservatives are possible. The trend to use of multipurpose formula
components to create self-preserving products is the wave of the future. The
following chapters provide insights into the creation of preservative-free and
self-preserving products.
14 Kabara and Orth
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4 3 r d e d . 1 9 8 1 , p p . 2 5 0 2 6 9 .
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8 . L . S t r y e r . B i o c h e m i s t r y , 3 r d e d .
W . H . F r e e m a n a n d C o . , N e w Y o r k , 1 9 8 8
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9 . M . A . L a m p e , M . L . W i l l i a m s , a n d P .
M . E l i a s . H u m a n e p i d e r m a l l i p i d s : c
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1 0 . P . M . E l i a s . S t r u c t u r e a n d f u n c t
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1 1 . G . K . M e n o n , S . G r a y s o n , a n d P . M .
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1 2 . P . W . W e r t z , D . C . S w a r t z e n d r u b e
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1 3 . P . W . W e r t z , W . A b r a h a m , J . J a n d m
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1 4 . G . B r o o k s a n d B . I d s o n . S k i n l i p i
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Preservative-Free

Jungennann Associates, Inc.

1. Cosmetic and Drug Preservation: Principles and Practice, edited by

ADDITIONAL VOLUMES IN PREPARATION

Hair and Hair Care, edited by Dale H. Johnson

~ Taylor & Francis

CRC is an imprint of the Taylor & Francis Group,

Preservative-free and self-preserving cosmetics and drugs:

toward more sophisticated scientific methodologies in the fields of preservative

Eric Jungennann, Ph.D.

The term preservative{ree means "without preservative chemicals." Usually,

development. Self-preserving products offer many advantages for formulation

Jon J. Kabara, Ph.D.

About the Series (Eric Jungermann) iii

1. Principles for Product Preservation

2. The Effect of Acid pH on Microorganisms and Survival Strategies

3. Water Activity and Self-Preserving Formulas 45

4. The Roles of Surfactants in Self-Preserving Cosmetic Formulas 75

5. Fatty Acids and Esters as Multifunctional Components 119

6. Biomimetic Phospholipids: Components for Self-Preservation 139

7. Use of Antioxidants in Self-Preserving Cosmetic and

8. Aroma Chemicals as Preservatives 181

9. Chelating Agents as Preservative Potentiators 209

I 0. The Role of Packaging in Product Preservation 227

II. Preservative-Free and Self-Preserving Cosmetic and Drug

Daniel K. Brannan, Ph.D. Department of Biology, Abilene Christian University,

P. Michael Davidson, Ph.D. Department of Food Science and Toxicology, Uni-

Davin C. Enigl, M.S., M.E.A.S. Hazard Analysis Critical Control Points,

Dennis L. Fost, Ph.D. Marketing and Commercial Development, Mona Indus-

Donald S. Orth, Ph.D. Research and Development Department, Neutrogena

John I. Yablonski, M.S. Bio-Control Consultants, Inc., Westfield, New Jersey

Preservatives are used in aqueous cosmetics and drugs to prevent microbial

Currently available antimicrobial preservatives may cause some adverse

1.1 PRINCIPLES OF PRESERVATION

1. Asepsis (keeping microorganisms out of the process stream)

drying conditions (low aw)

1.2 INJURY OF MICROORGANISMS

injured or "stressed" as a result of exposure to adverse physical and chemical

1.3 PRESERVATION OF PRODUCTS WITH LOW

1.4 PRESERVATION OF PRODUCTS WITH LOW WATER

TABLE 1.1 Lowest Reported aw Values

Many bacteria 0.91

controlling the growth of pseudomonads in many aqueous systems (including

1.5 PRESERVATION OF PRODUCTS WITH

1.6 PRESERVATION OF PRODUCTS WITH

1.7 PRESERVATION WITH MULTIFUNCTIONAL

1.8 PRESERVATIVE SYSTEM

1.9 SURVIVAL STRATEGIES OF MICROORGANISMS

1.10 ADVANTAGES AND DISADVANTAGES OF

Both routine test organisms and contaminants reasonably expected to be

The safety of self-preserving products will have to be evaluated on an individ-

1 1. Principles for Product Preservation

1. D. S. Orth. In: Handbook ofCosmetic Microbiology. Marcel

2. D. S. Orth. The required D-value: evaluating product

3. J. J. Kabara. Food-grade chemicals in a systems approach

4. D. S. Orth and S. R. Milstein. Rational development of

5. D. S. Orth. In: HandbookofCosmetic Microbiology. Marcel

6. D. S. Orth. In: Handbook of Cosmetic Microbiology.

7. P. Gacesa and N.J. Russell. The structure and properties

8. T. B. May, D. Shinabarger, R. Maharaj, J. Kato, L. Chu,