IFSCC MONOGRAPH

Number 5

An Introduction to Cosmetic
Microbiology

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IF_SCC MONOGRAPH
Number 5

An Introduction to Cosmetic
Microbiology

Donald S. Orth

Published on behalf of the

International Federation of Societies
of Cosmetic Chemists
by

~ MICELLE PRESS

Weymouth, Dorset, England

Copyright © International Federation of the Societies of Cosmetic
Chemists 1999

All rights reserved. No part of this publication may be reproduced,

stored in a retrieval system or transmitted in any form or by any means,
electronic, mechanical, photocopying, recording or otherwise, without
permission in writing from the International Federation of the Societies
of Cosmetic Chemists

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ISBN 1-870228-18-9

Published by
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on behalf of the
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Printed and bound in Great Britain by Black Bear Press, Cambridge

 IFSCC Benefactors
The current list of benefactors, to whom we offer our profound thanks
for their continuing support ofthe IFSCC, are shown below:

Amerchol Corporation[ USA] International Specialty Products [USA]

Arval SA [Switzerland] Iwasa Cosfa Co Ltd [Japan]
Aston Chemicals Ltd [UK] Karlshamns AB [Sweden]
BASF AG [Germany] Kemira Pigments OY [Finland]
S. Black (Import & Export) Ltd [UK] Lipo Chemicals, Inc. [USA]
Bronson & Jacobs Pty Ltd [Australia] Main Camp Tea Tree Oil Group
Centre de Recherches Biocosm6tiques [Australia]
SA [Switzerland] Matsumoto Trading Co Ltd [Japan]
Cosmetics & Toiletries [USA] Metrolab Industries, Inc. [Philippines]
Croda, Inc [USA] Mitzuho Industrial Co Ltd [Japan]
Dow Corning Europe [Belgium] Mona Industries [USA]
Dragoco Gerberding & Co GmbH Nikko Chemicals Co Ltd [Japan]
[Germany] Nipa Laboratories Ltd [UK]
DROM Fragrances International Pentapharm AG [Switzerland]
[Germany] Pentapharm Japan Corp. [Japan]
Firmenich SA [Switzerland] Plantapharm GmbH [Austria]
Gattefoss6 Etablissements [France] Provital SA [Spain]
Th. Goldschmidt AG [Germany] Quest International [UK]
Haarmann + Reimer GmbH [Germany] Rh6ne-Poulenc [France]
Henkel KGaA [Germany] Rohm & Haas Ltd [UK]
Hoechst AG [Germany] Takasago International Corp. [Japan]
Induchem Ltd [Switzerland] Union Chemical Corp. [Japan]
Witco Surfactants GmbH [Germany]

Should members of other Member Societies be interested in inviting their own com-
panies or those of their colleagues to become IFSCC benefactors, the annual donation
is a modest Sw.Frs. 500 per annum. Benefactors receive a lapel pin showing that they
are an IFSCC benefactor. Names of benefactors are recorded in future IFSCC publi-
cations such as Congress/Conference programmes, newsletters, etc.
Letters of invitation for benefactors and invoices for payment of the annual donation
are obtainable from the Luton secretariat.

The following companies also make a substantial contribution to the

IFSCC in supporting members of the Praesidium, and our grateful
thanks therefore also go to

Amerchol Corporation [USA] Lemmel SA [Spain]

Arval SA [Switzerlandl Les Colorants Wackherr [France]
Beiersdorf AG [Germany] Lipo Chemicals, Inc. [USA]
Ego Pharmaceuticals Pty [Australia] Pentapharm Ltd [Switzerland]
ISPE srl [Italy] Quest International [UK]
Kemira Agro OY [Finland] Shiseido Co Ltd [Japan]
Laboratoires Adesil SA [Argentina] Unil-It Spa [Italy]
 General Preface to the Series

There are many excellent, authoritative tex&books covering the different

areas which comprise Cosmetic Science. However, certain topics cut
across these various disciplines and are best studied individually. From
the study of such a topic, a better appreciation can be achieved of the
practical use of that topic in the cosmetic field. This series of IFSCC
monographs is a collection of such intersecting themes.
It is hoped that the knowledge gained from identifying activities
common to a number of areas will be transferable when a chemist
moves from project to project. This series of monographs will cover a
wide range of themes compiled by experts in their fields, providing both
the novice and the experienced individual with valuable reference books
on the major topics of Cosmetic Science.

Monographs already published in this series are

IFSCC Monograph No . 1 : Principles of Product Evaluation -
Objective Sensory Methods
IFSCC Monograph No . 2 : The Fundamentals of Stability Testing
IFSCC Monograph No. 3 : An Introduction to Rheology
IFSCC Monograph No . 4 : Introduction to Cosmetic Emulsions
and Emulsification
IFSCC Monograph No . 6 : Antiperspirants and Deodorants :
Principles of Underarm Technology

and those in preparation include:

IFSCC Monograph No . 7 : Micro-emulsions in Cosmetics

In a further series, the IFSCC has also published

Cosmetic Raw Material Analysis and Quality, Vol. 1. Hydrocarbons,
Glycerides, Waxes and Other Esters, edited by Hilda Butler
 Foreword

Within the field of cosmetic science, cosmetic microbiology is a dy-

namic discipline which is growing to meet the needs of scientists in the
cosmetic and pharmaceutical industries. Cosmetic microbiology was, in
its first fifty years, primarily concerned with the microbiological as-
pects of plant sanitation, determining aerobic plate counts of finished
products, and preservative efficacy testing.
Today, we are seeing changes in cosmetic products and claims. Com-
panies are introducing products that are more therapeutically posi-
tioned, that claim increased performance in treating specific conditions,
and that meet changed consumer needs.
These developments are causing a shift of focus in cosmetic micro-
biology from formula development and manufacturing plant sanitation
concerns to clinical studies on the effects of active ingredients in drug
products and microorganisms on the skin. This monograph discusses
issues relevant to cosmetic and drug microbiology today.

The IFSCC is grateful to Dr. Donald S. Orth for preparing this

monograph on cosmetic microbiology.
 Contents

Page
IFSCC Benefactors iii
General Preface to the Series iv
Foreword v

1 Introduction to Cosmetic Microbiology 1

2 Microbial Contamination of Cosmetic Products 3
2.1 The occurrence of microorganisms in raw materials 3
2.2 Microbial risk assessment of raw materials 11
2.3 Microbial contamination of cosmetic products 11
3 Plant Sanitization, GMPs and House Organisms 15
3.1 Plant sanitation 15
3.2 Process validation 18
3.3 House organisms 20
3.4 Preservative efficacy testing 20
3.5 Finished product testing 20
3.6 Cleaning and sanitization procedures 21
4 Preservatives and Preservative Systems in Cosmetic Products 24
4.1 Preservatives and preservative systems 25
4.2 The preservative system concept 25
4.3 Mild preservative systems 26
5 Preservative Efficacy Test Methods, Acceptance Criteria and
Rapid Methods 29
5.1 Preservative efficacy test methods 29
5.2 Types of organisms used in preservative efficacy testing 31
5.3 Rechallenge testing 32
5.4 Acceptance criteria 32
5.5 Rapid screening method 33
5.6 Preservation of products during use 33

Vi
6 FD&C Act and Regulations Pertaining to Cosmetics and OTC
Drugs 36
6.1 Definitions 36
6.2 Adulterated drugs and cosmetics 37
6.3 Inspections by regulatory agencies 37
6.4 Current good manufacturing practices 37
6.5 Good laboratory practices 38
6.6 Monographs for OTC drugs 38
7 Topical/Oral Products that Prevent, Correct or Conceal Conditions
Caused by Microorganisms 41
7.1 Antiperspirant deodorants and deodorants 41
7.2 Products that control dandruff, seborrheic dermatitis and
psoriasis 42
7.3 Products that treat acne 43
7.4 Fungal infections of the skin 44
7.5 Oral-care products 44
8 Effects of Microorganisms on Skin Physiology and the Skin
Immune System 47
8.1 Inflammatory mediators and immunomodulators o f
R aeruginosa 47
8.2 Inflammatory mediators and immunomodulators of
R acnes 49
9 Harmonization of Test Methods and Globalization of Preservative
Systems 52
9.1 Microbiological test methods 52
9.2 Preservative efficacy test acceptance criteria 53
9.3 Microbial limits on finished products 54
9.4 Globalization of preservative systems 55
10 Self-Preserving Cosmetic and Drug Products 58
10.1 Self-preserving products 58
10.2 Packaging 59
10.3 Summary 60

Vii
 Introduction to Cosmetic Microbiology 1

1 Introduction to Cosmetic Microbiology

Historically, cosmetic microbiology,was primarily concerned with mi-
crobiological aspects of plant sanitation and preservative efficacy test-
ing [ 11. Today, we are seeing changes in cosmetic products and claims.
Companies are introducing products that are more therapeutically posi-
tioned, with claims for increased performance in treating specific condi-
tions such as reducing the appearance of aging [i.e., use of alpha-hy-
droxy acids (AHAs) for decreasing fine lines and wrinkles] and reduc-
ing the numbers of microorganisms on the skin with antibacterial hand-
washes. Products are being introduced to meet the (perceived) need of
consumers who want products that are "cruelty-free" (e.g., non-animal
tested), "preservative-free" (e.g., without chemical preservatives), and
natural [2].
This shift of focus in marketing is causing changes in cosmetic mi-
crobiology. Scientists in government, industry and academia have been
working on microbiological issues pertaining to product development,
product preservation, plant sanitation, process validation, governmental
regulations, test methods, and toxicity. Research in many areas is be-
coming more medically orientated as the cosmetic industry introduces
more therapeutic products. This is expanding the role of cosmetic mi-
crobiology from formula development and concerns in the manufactur-
ing plant to clinical studies involving active ingredients in drug prod-
ucts, determining the effects of microorganisms and their metabolic
products on human skin, and the use of in vitro testing to predict effects
of raw materials and products on the body (e.g., tests for skin and eye
irritation).
The increasing pace of product development has placed heavy de-
mands on microbiologists to meet product introduction timetables. This
has created a need for faster, more reliable test methods in all areas -
from preservative efficacy testing in product development to release
testing of finished products. Corporate growth on an international scale
has also created new concerns - the need for harmonization of test
methods in different countries and the "globalization" of formulas (and
preservative systems). It is becoming apparent that there are opportuni-
ties for use of standardized test methods and more rigorous acceptance
criteria than currently specified in some official compendia. This mono-
graph discusses microbiological issues that are relevant to the world-
wide cosmetic and drug industries.
2 IFSCC Monograph No. 5

1.1 References
1 . Curry, J . C . Cosmetic microbiology - 50 years of change . Household Pers.
Prod Ind 8,44-52 (1983).
2 . Kabara, J . J . and Orth , D . S . (eds .). Preservative-Free and Self-Preserving Cos -
metic and Drug Products . Marcel Dekker, NewYork ( 1997).
 Introduction to Cosmetic Microbiology 3

2 Microbial Contamination of Cosmetic Products

Microbial contamination of cosmetic products has been - and contin-
ues to be - a matter of great importance to the industry. It is recog-
nized that aqueous cosmetic and drug products are subject to microbial
contamination and spoilage. Satisfactorily preserved products that have
been made with proper microbiological controls are self-sterilizing.
Products must be protected from environmental contamination during
manufacturing, distribution and use by consumers [ 1].

2.1 The occurrence of microorganisms in raw materials

Bacteria, yeasts, and molds are ubiquitous in nature. Bacteria typically
are unicellular rods or spheres, 0·2-1 kim in diameter/length, and grow
by binary fission. Yeasts are unicellular spherical/elliptical microorgan-
isms, around 5 Mim in diameter, and grow asexually by forming spores
or "buds". Molds are multicellular microorganisms that generally grow
on the surface of substrates and form filamentous colonies (mycelia and
fruiting structures that contain spores). People often see colorful green,
yellow and black mold colonies on foods that have been stored under
conditions that allowed growth. Specific microorganisms can grow on
or in aqueous raw materials and change physical and chemical charac-
teristics (color, odor, pH, viscosity, etc.). Microorganisms can also
cause specific infections and disease. The types of microorganisms that
become the microbial flora of a raw material depend on the presence of
microorganisms, on their ability to utilize the materials available for
energy and growth, and on the suitability of conditions for growth in
that raw material. Raw materials used by the cosmetic and drug indus-
tries provide a wide range of inorganic and organic compounds in aque-
ous and non-aqueous systems [2].
The microbial problems encountered during manufacturing and in
finished products often may be traced to the microbial quality of raw
materials used [3-6]. The bioburden of raw materials may be reduced
by processing conditions that kill microorganisms (i.e., heat treatment,
spray-drying, acid neutralization), but many factors in manufacturing
determine whether microbial contamination of raw materials will carry
through to the finished product. The 6th Amendment to the Cosmetics
Directive requires manufacturers to determine physicochemical and mi-
crobiological specifications of raw materials based on the type and use
4 IFSCC Monograph No. 5

of the raw material concerned and the product type in which it will be
used [7,8].
The principles of preservation in Table 1 are used to control micro-
bial growth in raw materials as well as in foods, drugs and cosmetics. If
proper control measures are not used, microbial growth will occur in
aqueous raw materials held at ambient temperatures for several hours
(or more). The microbial risks of several raw materials are discussed
below.

Table 1
Principles of preservation

1. Asepsis, or keeping microorganisms out of the process stream

2. Removal of microorganisms
a. Trimming
b. Washing
c. Sedimentation and centrifugation
d. Clarification or filtration

3. Retarding growth or killing microorganisms by physical or chemical means

a. High temperatures
b. Low temperatures
c. Drying conditions (low water activity)
d. High or low pH
e. Removal of substrates (e.g., cleaning equipment to remove product
residues)
f. Anaerobic atmosphere (e.g., (02,142)
g. Preservatives, disinfectants, sanitizers
h. Irradiation
i. Mechanical destruction by grinding or high pressure

Source: Adapted from reference [2]

2.1.1 Water
Water is the major ingredient in many cosmetic and drug products.
Water is required for microbial growth, it is susceptible to microbial
contamination, and it has been the source of finished product contami-
nation [3]. Most manufacturers recognize that municipal water supplies
frequently are not suitable for use in manufacturing because of the dis-
solved minerals. Although potable water supplies have been chlorinated
so that the coliform level is <1 colony forming unit (CFU)/100 ml,
manufacturers should be aware of the potential microbial load of incom-
ing chlorinated municipal water supplies [2]. It is known that microor-
ganisms in biofilms are more resistant to sanitizing chemicals, and
 Introduction to Cosmetic Microbiology 5

LeChevallier, Cawthon, and Lee [9] reported that microbial adherence

to solid surfaces increased their resistance to chlorination. Reasoner et
al. [10] reported on the presence of pigmented bacteria in municipal
water supplies and determined that their presence does not demonstrate
that a problem exists. However, they observed that if the disinfectant
concentration is too low to inhibit the growth of these organisms, chlo-
rine disinfection may select for pigmented bacteria which are more
chlorine-tolerant than non-pigmented bacteria. [Note: some species of
bacteria are able to form red or yellow carotenoid pigments or brown
melanoidin pigments in response to adverse environmental conditions
(sunlight, chlorine, disinfectants)]. The work of Reasoner and co-work-
ers emphasized the need to control bacterial regrowth in water distribu-
tion systems by use of available chlorine residuals >0·2 ppm and by
elimination of stagnation in dead-end portions (i.e., "dead legs") of the
water system.
Manufacturers treat municipal water by passing it through anion and
cation exchange resins to remove dissolved salts that may interfere with
the stability, clarity, and fragrance of finished 'products. This removes
the chlorination that prevents many microorganisms from growing in
the municipal water system. Thus, distilled water and demineralized
water supplies will allow microorganisms to grow unless preventative
measures are instituted. In 1971, Favero et al. (11) reported that
Pseudomonas aeruginosa is capable of rapid growth in distilled water,
with populations growing from 102 to 107 organisms/ml in 48 hours.
They also reported that high densities of this organism (106 to 107
organisms/ml) remained viable for up to 42 days and that cells grown in
distilled water were more resistant to stress (i.e., inactivation by various
disinfectants) than cells cultured on trypticase soy agar. The formation
of stress (heat shock) proteins in response to nutrient depletion is one
explanation for this increased resistance (1,12).
Carson et at. [13] examined the growth and survival patterns of
Burkholderia (Pseudomonas) cepacia and found that this organism was
able to grow rapidly after being inoculated into distilled water. Favero,
Carson and co-workers [ll, 13] regarded distilled water in their hospital
as a source of contamination. In addition, they found that several other
Gram-negative bacteria, including species of Achromobacter and Flavo-
bacterium, were capable of growing in distilled water. [Note: bacteria
are classified based on their reaction to a staining procedure known as
the Gram stain. Gram -positive bacteria typically have a more dense cell
wall and retain the crystal violet (blue/purple) stain; whereas Gram-
6 IFSCC Monograph No. 5

negative bacteria are decolorized and pick up the safranin (red) stain.]
In 1975, Malcolm and Woodroffe [14] reported that the most frequently
reported contaminants of cosmetic products included Pseudomonas,
Klebsiella, Achromobacter. and Alcaligenes . They observed that these
genera are commonly found in water and that water was the likely
source of organisms found in contaminated cosmetic products. More
recently, Blachman and Elowitz-Jeffes [15] reported that pseudomonads
were the most common bacterial contaminant recovered from aqueous
products. These reports and others indicate that contaminated water
may cause product contamination.

2.1.2 Acids, alkalis, and salts

Generally, microorganisms of interest in raw materials or in cosmetic
products grow best around neutrality (pH 7·0). Although yeasts and
molds are able to tolerate acid pH conditions (i.e., pH <4), many micro-
organisms are metabolically injured or "stressed" by adverse pH condi-
tions, such as when the pH is <4 or >10 [2]. The extreme pH values in
acids and alkalis prevent microbial survival.
Dry inorganic salts (i.e., marine salts, sea salts, Dead Sea salts, etc.)
may contain low levels of microorganisms that were present in the start-
ing waters. Bacteria, yeasts and molds must have sufficient water to
grow; consequently dry inorganic salts, such as NaCl which is used for
viscosity control in shampoos, present little microbial risk. Aqueous
salt solutions may support microbial growth where there is sufficient
water [i.e., where the water activity (aw) is high enough]. The aw is an
expression of the relative humidity in the headspace above a product,
and it is different from the total moisture in a product. Enigl and Sor-
rells [16] reported the minimum aw permitting growth of selected bacte-
ria and fungi , with salient aw values being 0 · 97 for Pseudomonas fluo-
rescens, 0·95 for Escherichia coli, 0·86 for Staphylococcus aureus, 0·90
for bread yeast (Saccharomyces cerevisiae) and 0·11 for Aspergillus
niger (a common mold).

2.1.3 Oils, waxes, fatty acids, alcohols, and esters

Anhydrous lipids do not support microbial growth. The levels of micro-
organisms contained in oils, waxes and paraffins are, in most cases,
negligible. Proper storage of lipids under anhydrous conditions-in-
cluding freedom from condensation in storage tanks - prevents micro-
bial growth.
 Introduction to Cosmetic Microbiology 7

As raw materials, fatty acids, alcohols and esters do not pose micro-
biological problems because they do not support growth unless water is
present. Freese, Sheu, and Galliers [17] reported that lipophilic acids,
such as laurie acid and myristic acid, are antimicrobial because they
inhibit membrane transport of oxidizable substrate. Kabara [18] exam-
ined the structure-function relationship of anionic surfactants as antimi-
crobial agents. Based on minimum inhibitory concentration (MIC) data
for Gram-positive bacteria, he reported that the most active chain length
for saturated fatty acids is laurie (C 12), the most active monounsatu-
rated fatty acid is palmitoleic ((16:1), and the most active polyunsatu-
rated fatty acid is linoleic (C 18:2). Moderate concentrations of free fatty
acids generally do not affect Gram-negative bacteria, except for short-
chain fatty acids with fewer than eight carbon atoms. Kabara also indi-
cated that yeasts are inhibited by fatty acids with chain lengths of 10-12
carbons [18,19].
Alcohols are used in cosmetic and drug products for solubilizing
other ingredients, increasing the percutaneous absorption of materials in
the formula, giving fragrances a "lift", creating a cooling sensation on
the skin due to evaporative cooling, viscosity control, and as compo-
nents of product preservative systems [2]. The OTC Review Panel for
Topical Antiseptics determined that ethyl alcohol (48 - 95% by vol.)
and isopropyl alcohol (50 - 91·3% by vol.) are effective first-aid anti-
septics [20]. Opened alcoholic beverages, such as wines which are
around pH 3 ·5, generally require at least 18% ethanol for satisfactory
preservation. Lower concentrations of ethyl alcohol, other short-chain
alcohols (<(6), and glycols may contribute to the antimicrobial action
of preservative systems.
Esters generally are used in cosmetic formulations to improve the
performance of emulsions by giving them "cushion" or rub-in charac-
teristics and providing the desired "skin-feel" after the product is ap-
plied. Esters that are condensation products of fatty acids and fatty
alcohols are anhydrous and present no risk of microbial contamination
unless they are stored improperly, which results in the introduction of
water. Some esters are reported to have antimicrobial activity (21).

2.1.4 Surfactants and emulsifiers

Surfactants are compounds that reduce the interfacial tension at water/
oil interfaces. Compounds displaying surface activity have hydrophilic
(i.e., polar) and hydrophobic (i.e., hydrocarbon or non-polar) groups.
Surfactants are used as wetting agents, detergents, and emulsifiers. Al-
8 IFSCC Monograph No. 5

though dry (i.e., flaked or powdered) surfactants do not support micro-

bial growth due to the lack of water, many surfactants are supplied to
the industry as 28 - 35% aqueous solutions. Gram-positive cocci, such
as S. aureus, are inactivated readily by anionic surfactants such as so-
dium lauryl sulfate or ammonium lauryl sulfate. Gram-negative bacteria
including E coli, Enterobacter aerogenes, and pseudomonads are not
necessarily killed in anionic surfactants; consequently, preservatives
must be used to prevent growth of these organisms and concomitant
utilization of the surfactant as the carbon and energy source for growth.
Cationic surfactants, such as quaternary ammonium compounds, are
used extensively in the cosmetic and pharmaceutical industries for hair
conditioning, skin-feel, and emulsification. Many quaternary ammo-
nium compounds (i.e., "quats") have antimicrobial activity, and are the
most effective wide-spectrum germicides, especially at alkaline pH and
with low organic loads [22]1. Benzalkonium chloride (BAC) is used
widely as an antimicrobial agent because it is effective against most
organisms at a level of 0·1%. Gram-negative bacteria - and especially
pseudomonads - are more resistant to BAC than are Gram-positive
bacteria [2]. In some cases, the presence of a cationic ingredient does
not allow the use of other preservatives. BAC (0·1 - 0·13%) and ben-
zethonium chloride (0·1 - 0·2%) are permitted active first-aid antisep-
ties in the United States [20].
Formulating with surfactants may create problems for product pres-
ervation. Surfactants form micelles which can entrap hydrophobic mole-
cules including many lipophilic preservatives. This reduces the effec-
tive concentration of preservatives in the aqueous phase of the product,
thereby reducing the potency of the preservative system [23].

2.1.5 Tale and days

Tale is used to help dry moist parts of the body and to provide a smooth
skin-feel. Moisture uptake by tale reduces the water available for micro-
bial growth and retards the development of body odor. Clays, such as
kaolin, bentonite, and magnesium-aluminum silicate, are used to give
products different viscosities and skin-feel characteristics. Tale and
clays are mined from the earth; consequently, one would not expect
them to be free from microorganisms. They are processed without the
addition of water, and little, if any, microbial contamination occurs.
Sakamoto et al. [24] reported that tale and clays interfere with the pre-
servative potency of parabens, presumably due to adsorption. Raw ma-
terials including jojoba oil, starch, clays, and botanicals may contain
 Introduction to Cosmetic Microbiology 9

Bacillus spores . Each formula needs to be checked to determine whether

raw materials have contributed to the microbial load of the product.

2.1.6 Protein hydrolysates, starches, botanicals, gums, and resins

Naturally occurring raw materials including plant proteins, starches, bo-
tanicals, and gums are subject to contamination during growth, harvest-
ing, and processing. Similarly, animal-derived raw materials including
hydrolyzed proteins, hyaluronic acid, and collagen may be exposed to
air, water and soil during processing. European manufacturers are now
required to process animal proteins using special processes (e.g., re-
moval of brains and spinal cords) to prevent transmission of prion dis-
eases (bovine spongiform encephalopathy, "mad cow disease") in ani-
mal proteins. The nature and type of contaminants occurring in natural
products, the availability of water, and the level of sanitation and atten-
tion to time/temperature relationships during processing determine the
extent of microbial contamination [2,25].

2.1.7 Humectants
Humectants are hygroscopic materials that help to control the moisture
exchange between the product and the air and the product and skin
(after product application). Humectants help to prevent emulsions from
drying out, they affect the skin-feel during rub-down by delaying the
phase inversion of oil-in-water (0/w) emulsions, and they help to mois-
turize skin. The most commonly used humectants in the cosmetic and
pharmaceutical industries are glycerol, propylene glycol, sorbitol, buty-
lene glycol, and methyl propanediol. Hyaluronic acid use is increasing
and it is used where its high water-holding capacity is considered to be
a benefit. Aqueous hyaluronic acid requires preservation [2]. As sup-
plied, 95% or 99% glycerol, 70% sorbitol, and neat propylene glycol or
butylene glycol have such low aw values that they do not support micro-
bial growth. The use of high concentrations of humectants may reduce
or eliminate the need for chemical preservatives in a formula. For exam-
ple, 40% glycerin in a hand cream may reduce the aw to 0·83 so that
growth of problem microorganisms is virtually eliminated. Such prod-
ucts are self-preserving [26,27].

2.1.8 Colors and pigments

In 1973, Bonorden [4] reported that colors and pigments used in cos-
metic products contained <10 to >104 CFU/g Gram-negative bacteria of
 10 IFSCC Monograph No. 5

various species. Today, it is believed that manufacturers of certified

colors are more aware of the need to control microbial growth than
when Bonorden collected data for his report. The USA requires use of
certified colors and lakes; the European Union (EU) permits only colors
found in Annex IV with specified Colour Index numbers [7]. All ingre-
dients used must comply with the guidelines established for safety in
European Community (EC) countries [28].

2.1.9 Preservatives, antioxidants, and chelating agents

Preservatives, antioxidants and chelating agents are used in cosmetic
products to stabilize physical and chemical attributes of a product. Pre-
servatives are intended to be biocidal, and, as supplied, present no risks
of microbial contamination. Antioxidants and reducing agents help to
poise the oxidation/reduction potential in a system, terminate free-radi-
cal reactions, and quench singlet oxygen, thereby preventing oxidative
deterioration that may lead to the generation of rancidity and non-enzy-
matic browning reaction products. Butylated hydroxyanisole (BHA) and
butylated hydroxytoluene (BHT) are two of the most commonly used
phenolic antioxidants in cosmetic products. BHA and BHT generally
are supplied as dry powders which do not support microbial growth.
Chelating agents such as tetrasodium EDTA and citric acid are used to
sequester divalent metal ions (i.e., Ca2+ , Fe2+ ), may be pro-oxidants
and initiate free-radical reactions in aqueous systems. Tetrasodium
EDTA does not permit microbial growth and must not contain NaCN.
Citric acid 50% or powdered citric acid has a pH that is too low for
microbial growth. Chelating agents and antioxidants contribute to the
potency of a preservative system (see Chap. 4).

2.1.10 Fragrances and essential oils

The aromatic chemicals extracted from botanicals (i.e., rose petals, gar-
denia blossoms, jasmine flowers, etc.) are used neat or are combined to
create essential oils. Alternatively, alcohol extracts may be prepared for
compounding fragrances. Fragrances and essential oils may have an-
timicrobial activity because they contain mixtures of chemicals with
varying antimicrobial properties, such as alcohols, phenols, esters, or-
ganic acids, aldehydes, and terpenes [2,26].
 Introduction to Cosmetic Microbiology 11

2.2 Microbiological risk assessment of raw materials

Because water is required for growth, anhydrous products present a
negligible risk of microbial contamination-provided they did not have
aqueous stages in their manufacture that allowed contamination. On the
other hand, raw materials containing water present a risk. The degree of
risk depends on how well a product is able to support the growth of
microorganisms. Conducting a microbiological risk assessment involves
professional judgement and microbiological examination of raw materi-
als to determine the aerobic plate counts (APCs).
The EU Cosmetics Directive notes that microbiological criteria or
specifications are appropriate for some raw materials [8]. Loprieno [28]
reviewed the guidelines for safety evaluation of cosmetic ingredients in
the EC countries. Orth [2,25] used the degree of risk associated with
each raw material to implement a program of sampling and analysis of
raw materials. This risk classification is presented in Table 2.

Table 2
Microbiological risk classification for raw materials

Classification Relative degree of risk Sampling frequency and

analysis
0 No risk No test needed
1 Slight risk Test once (for the record)
2 Low risk Test once/year
3 Moderate risk Test each lot
4 High risk Test daily

Source: Adapted from reference [25]

2.3 Microbial contamination of cosmetic products

The Cosmetics Directive requires microbiological specifications for all
finished products [8]. The relative hazards created by contaminated cos-
metics may be related to the severity of the infection or disease they
cause. Bacteria, yeasts and molds are regarded as pathogens if they
produce virulence factors that enable them to produce recognized infec-
tions and disease . Microorganisms are considered to be opportunistic
pathogens if they generally are unable to cause infection or disease in
healthy individuals, yet they are able to produce adverse reactions in the
elderly, in infants, in sick persons who have compromised immune sys-
12 IFSCC Monograph No. 5

tems, and in persons on immunosuppressive drugs. Microorganisms are

considered to be non-pathogens if they generally are unable to produce
infection or disease. The classification of objectionable microorganisms
in different types of products is shown in Table 3 [2].

Table 3
Classification of objectionable microorganisms by product type

¤ Sterile Drugs Any organism or pyrogen in a sterile product is ~

objectionable.
¤ Eye Products P. aeruginosa is always objectionable. Other
Pseudomonas spp., S. aureus, Serratia
marcescens and S. liquifaciens are usually
objectionable.
¤ Non - Sterile Oral Products Any enteric pathogen ( i . e ., Salmonella spp .) and
E. co/i are always objectionable. Other enteric
organisms , such as Enterobacter spp„ Citrobacter
spp., Pseudomonas spp., proteolytic C/ostridium
spp ., enterotoxigenic S. aureus, pathogenic yeasts
(i.e, Candida a/bicans), and mycotoxin-producing
fungi are usually objectionable.
¤ Non-sterile topical P. aeruginosa, Klebsiella spp, S. aureus, S. marc-
products escens and clostridia are usually objectionable.
¤ Genito- urinary tract E. coli, Proteus spp., S. marcescens, P. aeruginosa,
products and Fl mu/tivorans are always objectionable.
Klebsiella spp., Acinetobacter anitratus, and A.
calcoaceticus are usually objectionable .

Source: Adapted from reference [2]

The occurrence of contaminated cosmetic products was brought into

prominence by the landmark report of Kallings, Ernerfeldt and Silver-
stolpe [29], who published findings of their work on the microbiologi-
cal content of non-sterile pharmaceuticals and selected toiletries. Proper
product preservation requires an examination of the preservative system
of the formula, and of the effect of packaging and consumer use/abuse
on product preservation (see Chap. 5).

2.4 References
1. Orth, D.S. The linear regression method: a quantitative method for determin-
ing preservative requirements and evaluation of cosmetics and OTC Drugs.
Presentation at the SCC Annual Scientific Seminar in Boston, on May 9,1996.
2 . Orth, D . S . Handbook of Cosmetic Microbiology. Marcel Dekker, New York
(1993), pp. 15-54.
 Introduction to Cosmetic Microbiology 13

3 . Goldman , C . L . Microorganisms isolated from cosmetics . Drug Cosmet. Ind.

117 (1), 4041 (1975)
4. Bonorden, R. The nature and extent o f bacteria found in some raw materials
used in the cosmetic industry. CTFA Cos,net. J. 5 (3), 23-28 (1973).
5. Davis, J.G. Fundamentals of microbiology in relation to cleansing in the cos-
metics industry . J. Soc. Cosmet. Chem. 23, 45 -71 ( 1972 ).
6 . Croshaw, B . Preservatives for cosmetics and toiletries . 1 Soc. Cosmet. Chem .
28,3-16 (1973).
7, Official Journal of the European Communities No. L 151/32, the 6th Amend-
ment to the Cosmetics Directive. (Directive 76/768/EEC). June 14,1993.
8 . Steinberg , D . C . A Guide to European Cosmetic Regulations . Independent
Cosmetic Manufacturers & Distributors and American Beauty Association,
(1998), pp. 48-50.
9, LeChevallier, M.W., Cawthon, C.D., and Lee, R.G. Inactivation of biofilm
bacteria. Appl. Environ. Microbiol. 54, 2492 -2499 ( 1988).
10, Reasoner, D.J., Blannon, J.C., Geldreich, E.E., and Barnick, J. Nonphotosyn-
thetic pigmented bacteria in a potable water-treatment and distribution system.
Appl. Environ. Microbiol. 55, 912 -921 ( 1989).
11 . Favero, M . S ., Carson, L . A ., Bond, W. W., and Petersen, N . J . Pseudomonas
aeruginosa: growth in distilled water from hospitals . Science 173 , 836 -838
(1971).
12 . Orth, D . S . Handbook ofCosmetic Microbiology. Marcel Dekker, New York
(1993), pp. 151-219.
13. Carson, L.A., Favero, M.S., Bond, W.W., and Petersen, N.J. Morphological,
biochemical , and growth characteristics of Pseudomonas aeruginosa from dis -
tilled water. Appl. Microbiol. 25, 476 -483 ( 1973 ).
14. Malcolm, S.A. and Woodroffe, R.C.S. The relationship between water-
borne bacteria and shampoo spoilage . J. Soc. Cosmet. Chem. 26, 277-288
(1975).
15. Blachman, U. and Elowitz-Jeffes, L.S. Microbiology of cosmetics - regula-
tory and quality-assurance aspects. Cosmet. Technol. 4 (1), 24-26,28-30,32,
34,36,37,54 (1982).
16. Enigl, D.C. and Sorrells, K.M. Water activity and self-preserving formulas.
In Preservative-Free and Self-Preserving Cosmetics and Drugs. Principles and
Practice 0.3. Kabara and D . S . Orth , eds .), Marcel Dekker, New York ( 1997 ),
pp. 45-73.
17. Freese, E., Sheu, C.W., and Galliers, E. Functions of lipophilic acids as an-
timicrobial food additives . Nature 241 , 321 -325 ( 1973 ).
18 . Kabara, J . J . Medium-chain fatty acids and esters . In Antimicrobials in
Foods CA.L. Branen and P. M . Davidson , eds .), Marcel Dekker, New York
(1983), pp. 109-140.
14 IFSCC Monograph No. 5

19. Kabara, J.J. Structure-function relationships of surfactants as antimicrobial

agents. J. Soc. Cosmet. Chem. 29, 733-741 (1978).
20. Food and Drug Administration. Topical Antimicrobial Drug Products for
Over-the-Counter Human UsE,_Tentative Final Monograph for First Aid Anti-
septic Drug Products; Proposed lillie. Federal Register 56 (140), 33644-33680
(1991).
21. Kabara, J.J. Lauricidin. The nonionic emulsifier with antimicrobial proper-
ties . In Cosmetic and Drug Preservation. Principles and Practice 0.1 . Kabara,
ed.), Marcel Dekker, New York (1984), pp. 305-322.
22. Scott, E.M. and Gorman, S.P. Chemical disinfectants, antiseptics and pre-
servatives . In Pharmaceutical Microbiology (W.B. Hugo and A . D . Russel,
eds.), 4th ed., Blackwell Scientific Publications, London (1988), pp. 226-252.
23 . Orth , D . S . Inactivation of preservatives by surfactants . In Surfactants in
Cosmetics, 2nd ed., (M.M. Rieger and L.D. Rhein, eds.), Marcel Dekker, New
York (1997), pp. 583-603.
24. Sakamoto, T., Yanagi, M., Fukushima, S., and Mitsui, T. Effects of some
cosmetic pigments on the bacterial activities of preservatives . 1 Soc. Cosmet.
Chem. 38,83-98 (1987).
25. Orth, D.S. Microbiological considerations in cosmetic formula development
and evaluation. I. Microbiological quality of a product. Cosmet. Tbiletr 104 (4),
49-57,59-64 (1989).
26 . Kabara, J . J ., and Orth , D . S . ( eds .). Preservative-Free and Self-Preserving
Cosmetics and Drugs. Principles and Practice. Marcel Dekker, New York
(1997).
27. Orth, D.S. and Kabara, J.J. Preservative-free and self-preserving cosmetics
and drugs . Application of hurdle technology. Cosmet. Toiletr. 113 (4), 51 , 52,
54,56-58 (1998).
28. Loprieno, N. Guidelines for safety evaluation of cosmetics ingredients in ~
the EC countries . Fd. Chem . Toxic. 9, 809 -815 ( 1992).
29. Kallings, L.O., Ernerfeldt, F., and Silverstolpe, I. Microbiological contami-
nation of medical preparations. Report to the Royal Swedish Medical Board, Fi-
nal Report, Feb. 1965. Stockholm. Cited in C.L. Goldman. Microorganisms
isolated from cosmetics. Drug Cosmet. Ind 117 (1), 40-41 (1975).
 Introduction to Cosmetic Microbiology 15

3 Plant Sanitation, GMPs and House Organisms

Plant sanitation historically has been a significant concern for microbi-
ologists. In the United States, drug products are required to adhere to
manufacturing procedures that follow current good manufacturing prac-
tices (cGMPs), and guidelines for cosmetic cGMPs were enacted in
1997 [1,2]. Cosmetics manufactured in the EU are regulated under the
6th Amendment to the Cosmetics Directive, which was passed on June
14, 1993 [3]. This Directive requires that manufacturers comply with
cGMPs laid down by Community law or by the law of the Member
State concerned. Failure to comply with cGMPs in controlling the mi-
crobial load of raw materials and/or inadequate plant sanitation may
allow microorganisms to adapt and become house organisms [4]1.

3.1 Plant sanitation

Microorganisms may gain access to the manufacturing plant in air and
water or via animal vectors (birds, rodents), insects, and humans. Bacte-
ria, yeasts and molds become a problem when they are allowed to grow
in aqueous systems, including water, aqueous raw materials, and prod-
uct residues in tanks, filling lines, pumps, etc. Each manufacturing plant
has a unique set of physical and chemical conditions that can provide
suitable moisture, substrates, and temperature for microbial growth.
Quality assurance procedures must be established to prevent micro-
organisms that are present in the plant environment from growing to
unacceptable levels. It is necessary to recognize conditions that may
encourage growth, to determine whether this constitutes a microbiologi-
cal risk to the manufacturing environment, and to institute control
measures in those areas in which growth is unacceptable. This is accom-
plished by: (1) control of raw materials [discussed in Chap. 2], (2)
microbiological validation of processes, (3) implementation of an effec-
tive cleaning and sanitization program, and (4) training personnel.
Training of personnel and prevention of cross-contamination are per-
haps the most important aspects of implementing a successful cleaning
and sanitization program.
Sanitary manufacturing requirements differ according to the product.
The monograph on The Hygienic Manufacture and Preservation of Toi-
letries and Cosmetics, prepared by the Council of the Society of Cos-
metic Chemists of Great Britain [3], contains a great deal of useful
information on controlling manufacturing procedures. The Cosmetic,
16 IFSCC Monograph No. 5

Toiletry, and Fragrance Association (CTFA) guideline on the microbio-

logical evaluation of the plant environment notes that environmental
influences can be grouped into four basic categories: buildings, equip-
ment, personnel, and sanitization/housekeeping [5].
The ease of cleaning and sanitization of equipment should be a pri-
mary consideration in the selection of equipment. Product contact sur-
faces should be of a clean design and non-porous (i.e., stainless steel) to
facilitate cleaning and sanitization. Components of fillers, pumps, and
line connectors should be designed so that they may be readily disas-
sembled for inspection, cleaning and sanitization. The manufacturing
environment should be maintained in a clean, orderly manner.
Sterilization by heat (i.e., live steam) is the most effective means of
ensuring the destruction of all contaminating microorganisms. Sum-
cient contact time must be allowed for the most massive pieces of metal
in the pumps, lines, and fillers to be effectively sterilized. Use of steam
flowing continuously for a minimum of 30 minutes generally is suffi-
cient; however, the most massive and/or difficult to heat pieces of this
equipment must reach a minimum of 80 °C for the entire 30 minutes.
Intermittent periods (i.e., two 15-min. periods) may not produce the
same killing action as a single 30-minute treatment.
Small amounts of product residues that remain in regions of equip-
ment that are difficult to clean (i.e., beneath the 0-rings on piston fill-
ers) are a potential source of microbial contamination. These residues
may provide nutrients for surviving microorganisms, and growth in di-
lute product residues may enable these microorganisms to adapt to the
product preservative system and contaminate subsequent batches of
product passing through the filler [4,6,7]. Such adapted microorgan-
isms may become house organisms (see below).
Chemical sterilization is possible when it follows thorough washing
to remove soil and product residues. Although chlorine is somewhat
corrosive to metal surfaces, it is an excellent sterilizing agent [8]. Cold
solutions of hypochlorite with 200-250 ppm available chlorine will
sterilize clean metal and glass in 5 minutes. Cold solutions of formalde-
hyde (0·5% formalin; 0·2% formaldehyde) will sterilize clean surfaces
in 10 minutes. A 0·5% benzalkonium chloride solution (pH not stated)
at 60 °C will sterilize clean surfaces in less than 10 minutes [8]. Davis
[9] summarized the advantages and disadvantages of various sterilizing
agents and recommended certain types, as shown in Tables 4 and 5.
 Table 4
Sterilizing agents: advantages and disadvantages
Characteristic Steam Hot water Chlorine-releasing Quaternary ammonium Iodophores
(90 °C) compounds compounds
Cost Varies Varies Low High Intermediate
Convenience Dependson layout Recirculatory Very convenient Very convenient Very convenient

Introduction to Cosmetic Microbiology

system
necessary
Penetration Good if adequate Good Clean plant Clean plant essential Has detergent
supply essential action
Heating effect Tanks and other Less than None According to temperature None
equipment may steam
require hours to
cool. Undesirable
stresses may be
set up
Suitability Very suitable for Very suitable All purposes All purposes All purposes
enclosed systems for pipelines
and small articles
Persistence Not persistent Not persistent Not persistent Persistent Not persistent
Corrosion None None Very corrosive None Not corrosive if
unless maintained thoroughly rinsed
at PH 9 or above away
Odor None None Marked None None below 50 °C
Rinsing Unnecessary Unnecessary Good rinsing Good rinsing essential Good rinsing
essential essential

Note: AU chemical sterilants can be corrosive if improperly used, e.g., at too high a concentration, at too high a temperature, for too
long, and/or not adequately rinsed away.

17
Source: Adapted from reference [9]
18 IFSCC Monograph No. 5

Table 5
Recommendations on types of sterilizing agents

Sterilant Circumstances where Circumstances where

indicated inadvisable
Hypochlorites, Where drastic action is Where odor
chlorocyanuric required, where low cost is inadvisable, where
acids, and so- important, where all types of corrosion likely
dium phosphate microorganisms are likely to
hypochlorite be encountered, where
alkaline detergent is required
Quaternary Where odor, taste, and tox- Where low cost is
ammonium com- icity are to be avoided, important, where
pounds where safety in use is Gram-negative
important, where Gram- organisms are a likely
positive organisms are chief danger, where rinsing
danger, where alkaline is difficult (e.g. rough
detergent is desired surfaces)
lodophores Where calcium scale is a With galvanized iron
problem
Orthophenyl- For equipment not coming
phenol into contact with food
Nitric acid Where calcium scale is a Where equipment is
problem not all stainless, where
strong acid is
dangerous, where fat-
protein films are a
special problem
Chlorhexidine As "antiseptic" Where cost is import-
ant
Amphoterics Where neutral conditions are Where cost is import-
required ant
Source: Adapted from reference [9]

3.2 Process validation

Process validation is a deliberate and systematic approach to demon-
strate that each step in manufacturing and packaging operations is per-
formed in accordance with guidelines that have been demonstrated to
keep production of the product in control. Mead [10]1 explained that a
validated process is one that has been proved to do what it is purported
to do. A process may be out of control if (1) it has not been validated,
(2) a validated process has not been revalidated after significant
changes have been made, or (3) the established controls are not fol-
 Introduction to Cosmetic Microbiology 19

towed exactly. Equipment performance, installation and operation re-

quire validation.

3.2.1 Validation of deionized water systems

Microbial contamination of deionized water has been correlated with
contamination of finished products (see Chap. 2). This provides an ex-
cellent reason for validating the deionized water used in manufacturing.
Water may be sterilized by UV radiation, passage through sterilizing
membranes (i.e., ultrafilters or 0·2 micron membranes), ozonation, use
of H2O2, or heating. Storing deionized water at >60 °C and recirculation
through piping that has no "dead legs" virtually guarantees that water
will not become contaminated with microorganisms that may be a prob-
lem in cosmetic products.

3.2.2 Validation of cleaning and sanitizing procedures

Failure to adhere to validated cleaning and sanitizing procedures and
inadequate preservative systems are the most common reasons why
products become contaminated during manufacturing, because they may
allow the development of adapted organisms [4]. Cleaned and sanitized
equipment should not be allowed to stand for over 6 hours before being
put into service unless it is dried completely.

3.2.3 Hazard Analysis Critical Control Point (HACCP) concept

A useful means of maintaining microbiological control over manufac-
turing operations is by use of the Hazard Analysis Critical Control
Point (HACCP) concept, as reported in 1974 by Bauman [11]. The
HACCP concept is a preventative system of controlling microbiological
hazards because it estimates the risk associated with each step in the
manufacturing process and considers the raw materials and the process-
ing steps that provide suitable conditions for growth. Conditions to
avoid are time/temperature relationships in which adequate moisture
and nutrients may be available for use by contaminating microorgan-
isms. It is advisable to use the "bracketing" technique - analysis of
samples collected before and after a particular processing step (i.e.,
before and after a holding tank, line pump, filler, etc.) - to demonstrate
whether that step may add to the microbiological load of the product
stream.
20 IFSCC Monograph No. 5

3.3 House organisms

Often, viable microorganisms in products are metabolically injured as a
result of adverse physical or chemical conditions (i.e., brief exposure to
high processing temperatures, cleaning and sanitization agents, or
sublethal concentrations of preservatives/biocides). Injured microorgan-
isms typically exhibit an extended lag phase of growth and have re-
duced tolerance to secondary stresses, so culture media and growth tem-
peratures must provide suitable conditions for repair/recovery. If viable
microorganisms are undetected during routine quality control checks,
the surviving bacteria may recover and grow to cause product contami-
nation that is detected later. This situation is known as the Phoenix
phenomenon [4,12,13]. The Phoenix phenomenon is an artifact caused
by the failure of the recovery system and culture conditions to allow
growth of injured microorganisms that are present.
Occasional or frequent recovery of microorganisms from products
suggests the presence of microorganisms that carry-over from raw mate-
rials or microorganisms that are adapted to the plant environment. Some
manufacturers may isolate the same organism(s) for months or years.
These isolates are house organisms [4]. Inadequate preservative systems
and inadequate test methods for routine microbial examination of fin-
ished products may lead to the development of house organisms in the
manufacturing plant [14-17]1.

3.4 Preservative efficacy testing

The goal of preservative efficacy testing is to determine the type(s) and
minimum effective concentration of antimicrobial preservatives re-
quired for adequate preservation of cosmetic and pharmaceutical prod-
ucts. Products are judged to be satisfactorily preserved if testing demon-
strates that they meet appropriate acceptance criteria and have
packaging that prevents contamination and/or dilution of the product
( 12 , 17 ). (See Chap . 5 .)

3.5 Finished product testing

Finished products must be tested to demonstrate that they meet micro-
bial limit guidelines - that they are free from unacceptable levels of
contamination. Aqueous cosmetic and pharmaceutical products should
have microbial limits of<1 CFU/g or <10 CFU/g (i.e., none found). Not
all conditions affecting outgrowth of injured organisms are known, so
 Introduction to Cosmetic Microbiology 21

the presence of any living microorganisms in aqueous products is unac-

ceptable unless it is known that microorganisms cannot grow in the
product. Conditions that prevent microbial growth include lack of water
[low water activity (aw)], extreme pH, unsuitable oxidation/reduction
potential, storage temperatures (i.e., freezing), or the presence of inhibi-
tory materials [18]. The presence of low levels of non-objectionable
microorganisms in anhydrous products or products that are self-preserv-
ing is acceptable.
Test methods used for routine (release) testing of finished products
must be capable of detecting microorganisms of interest (i.e., those that
may cause product defects and/or spoilage and those that may be a
public health risk). The recovery procedures, including incubation con-
ditions and the recovery system (i.e., diluent and culture media) must
allow the recovery of stressed microorganisms. Test methods must be
validated with each product to show that preservative carry-over does
' not affect the growth of microorganisms present [14]. This may be done
I by inoculation of product dilutions with test organisms and showing
that the number recovered is not significantly different from the number
recovered on control media which contain no product (or preservatives).
Furthermore, suboptimal incubation temperatures (i.e., 30 °C for bacte-
ria and 25-28 °C for yeasts and molds) should be used to ensure that
temperature stress does not cause an extended lag phase and/or prevent
the growth of injured microorganisms. Use of incorrect incubation con-
ditions or culture media may result in the Phoenix phenomenon [19]1.

3.6 Cleaning and sanitization procedures

Warwick [20] noted that adequate preservative levels, stable preserv-
atives and cGMPs are necessary for preventing microbial contamina-
tion. Orth et al. [4] reported that adequate preservative systems, adher-
ence to cGMPs (with special emphasis for proper implementation of
cleaning and sanitization programs), and suitable test methods/micro-
bial limits are necessary to prevent the development of house organ-
isms. Key features of good cleaning and sanitization programs are that
they work (i.e., that they have been validated) and that they are carried
~
out properly - all the time. Proper implementation of the cleaning and
sanitization program is of paramount importance for controlling micro-
organisms in the plant environment. Controlling the microbiological
aspects of cGMPs is essential, and data showing the absence of micro-
organisms in the process stream or finished products may be a better
22 IFSCC Monograph No. 5

indicator of compliance with cGMPs than a plant that has a shiny, clean
appearance.

3.7 References
1. Human and Veterinary Drugs. Current good manufacturing practice in manu- ~
facture , processing, packing , or holding . Federal Register 43 0 90), 45014 -
45087 (1978).
2 . Cosmetic good manufacturing practice guidelines . Federal Register Oct . 30 ,
1997
3. Cosmetics Directive 76/768/EEC, 6th Amendment. June 14,1993.
4. Orth, D.S., Dumatol, C., and Zia, S. House organisms - Dealing with the
bug in the plant. Cosmet. Toiletr. 111 (61 59-66,68-70 (1996)
5. Quality Assurance Subcommittee of the CTFA Microbiological Committee.
Microbiological Evaluation of the Plant Environment Guideline. The Cosmetic,
Toiletry and Fragrance Association, Inc. (1985), pp. 1-6.
6 . Orth, D . S . Handbook of Cosmetic Microbiology . Marcel Dekker, New York
(1993),pp. 151-219.
7. Orth, D.S. Evaluation of preservatives in cosmetic products. In J.J. Kabara
( ed .). Cosmetic and Drug Preservation. Principles and Practice. Marcel Dek-
ker, New York (1984), pp 403-421.
8. Van Abbe, N.J., Dixon, H., Hughes, 0. and Woodroffe, R.C.S. The hygienic
manufacture and preservation=of toiletries and cosmetics . J. Soc. Cos,net. Chem.
21,719-800 (1970)
9. Davis, J.G. Fundamentals of microbiology in relation to cleansing in the cos- ~
metics industry. J. Soc. Cosmet. Chem . 23 , 45 -71 ( 1972 ).
10 . Mead , W. J . Process validation in cosmetic manufacturing . Drug Cosmet.
Ind. 126 (2), 46-48 (1980).
11. Bauman, H.E. The HACCP concept and microbiological hazard categories.
Food Technot. 32 (9), 31,32,34,74 (1974).
12. Orth, D.S. Microbiological considerations in cosmetic formula development
and evaluation. I. Microbiological quality of a product. Cosmet. Tbiletr. 104 (4),
49-57,59-64 (1989).
13 . Orth , D . S . Handbook of Cosmetic Microbiology . Marcel Dekker, New York
(1993),pp. 119-149.
14 . Microbiological tests , antimicrobial preservatives - effectiveness . United
States Pharmacopoeia XXIII . United States Pharmacopoeia Convention . Rock-
ford, MD, 9 1681 (1995). 1
15. Preservation Subcommittee of the CTFA Microbiological Committee. A
guideline for the determination of adequacy of preservation of cosmetics and
toiletry formulations . TGA Cosmet. J. 2: 20 - 23 ( 1970).
 Introduction to Cosmetic Microbiology 23

16. Orth, D.S. Linear regression method for rapid determination of cosmetic pre-
servative efficacy. J. Soc. Cosmet. Chem. 30, 321-332 (1979).
17 . Orth, D . S . Standardizing preservative efficacy test data . Cosmet. Toiletn
106 (3), 45-48,51 (1991).
18 . Orth, D . S . Handbook of Cosmetic Microbiology. Marcel Dekker, New York
(1993),pp. 491-519.
19 . Orth, D . S . Putting the Phoenix phenomenon into perspective . Cosmet.
Tbiletr 114(4): 61-66 (1999).
20. Warwick, E.F. Preventing microbial contamination in manufacturing. Cos-
met. Toiletr. 108 ( 10), 77, 78 , 80-82 ( 1993 ).
24 IFSCC Monograph No. 5

4 Preservatives and Preservative Systems in

Cosmetic Products
The objective of product preservation is to ensure that cosmetic and
pharmaceutical products are microbiologically safe and stable. Preserv-
atives are defined in the 6th Amendment to the Cosmetics Directive as
substances added to products for the primary purpose of inhibiting mi-
croorganisms from growing [1]. The preservative requirements of each
product are unique and depend on the physicochemical composition of
the product. If there were an "all-purpose" preservative combination
that was effective for all products, there would be no reason for this
monograph! Cowen and Steiger [2] noted that the preservative system
must be tailored to a specific product. Sterile drugs in multiple-dose
containers must have a preservative system that is capable of self-steril-
izing these products if contamination occurs. Similarly, non-sterile
aqueous products in multiple-use containers need preservative systems
that are capable of reducing the microbial bioburden to an acceptable
level in a reasonable time [3].
Anhydrous products, such as talcum powder, mineral oil, and stick
deodorants, do not require preservatives because they do not have suffi-
cient water to support microbial growth. Nevertheless, some manufac-
turers add preservatives to anhydrous products to prevent microbial
spoilage that may occur due to inadvertent addition of water by the
consumer during use (or abuse). Aqueous products generally require
preservatives unless they are hostile to microbial growth due to low
water activity (aw), low or high pH, or antimicrobial components such
as alcohols, surfactants, and quaternary ammonium compounds. Al-
though cosmetic products in multiple-use containers are not necessarily
intended to be sterile, an adequate preservative system renders the prod-
uct self-sterilizing for vegetative cells and bacteriostatic or bactericidal
for spores. [Vegetative cells are those that are metabolizing and/or
growing (dividing). Spores are a dormant state of some species of bac-
teria and fungi-much like the seeds of plants. Bacterial spores gener-
ally are much more resistant than seeds to adverse conditions (heat,
drying, ionizing radiation).] This enables a product to "cleanse" itself of
and prevent outgrowth of microorganisms if contamination occurs dur-
ing manufacturing, in trade channels, or during consumer use [4].
 Introduction to Cosmetic Microbiology 25

4.1 Preservatives and preservative systems

Steinberg [5]1 listed preservatives allowed in EU countries along with
CTFA names, maximum concentrations allowed, and limitations of use.
Methylparaben and propylparaben have been used more than twice as
often as any other preservative products registered with the U.S. Food
& Drug Administration for the past 15 years [4,6]. Preservatives at-
lowed in the USA, the EC and Japan are presented in the Table 6.

Table 6
Selected preservatives allowed in the USA, the EC and Japan

Maximum
Preservative allowable
level %
Benzyl alcohol 1.0
Chlorhexidine 0.3
Chlorphenesin 0.3
Methylparaben, Propylparaben, Ethyl- 0·8
paraben, Butylparaben, Isopropylpara-
ben, Isobutylparaben
Phenoxyethanol 1.0
Sorbic acid 0.6

4.2 The preservative system concept

The preservative action of a formula is often considered to be due
solely to the preservatives used; however, the preservative system of a
product involves both specific preservative chemicals and the physico-
chemical constitution of the product [7]. Preservative chemicals do not
act independently of the product, so factors such as pH, aw, nutrient
availability, surfactant concentration, sequestering agents, etc. will in-
fluence the preservative action of any given formula [3]1.
Formula components that may contribute to the preservative system
of a product are shown in Table 7. Antibiotics and preservatives are
bactericidal for susceptible microorganisms. Although most microor-
ganisms capable of causing problems in cosmetic products and raw
materials are able to grow when the pH is around neutrality (i.e., pH 5
to 8), extreme pH conditions created by acids and alkalis may contrib-
ute. to the preservative system of a product by stressing bacteria, yeasts
1
26 IFSCC Monograph No. 5

and molds. The type of acid or alkali and the concentration present
determine the extent to which they contribute to the preservative system
of the product. Some emulsifiers have antimicrobial action. Free fatty
acids (i.e., laurie and myristic acid) in soap-based emulsions or liquid
soap formulas are antimicrobial and are reported to inhibit membrane
transport of oxidizable substrates into cells (8). Nonionic surfactants,
such as Polysorbate 80, may enhance preservative efficacy when at con-
centrations below the critical micelle concentration (CMC). Above the
CMC, Polysorbate 80 and a number of other surfactants may inhibit
various classes of preservatives [9].

Table 7
Formula components that may contribute to product preservative systems

¤ Preservatives, biocides, antibiotics

¤ Acids, alkalies
¤ Alcohols (e.g., ethyl, isopropyl, benzyl)
¤ Cationic surfactants (e.g., benzalkonium chloride, cetyl pyridinium chloride)
¤ Anionic surfactants (e.g., soap, sodium lauryl sulfate)
¤ Esters (e.g., glyceryl monolaurate, sucrose hexadecanoate)
¤ Humectants and glycols (e g., glycerin, propylene glycol, butylene glycol,
sorbitol)
¤ Aqueous solutes (e.g., sugars, dextrins, salts)
¤ Phenolic antioxidants (e.g., t-butyl hydroxyanisole [BHA] and t-butyl
hydroxytoluene [BHT])
¤ Botanicals (e.g., tea tree oil, tannins)
¤ Chelating agents (e.g., citric acid, tetrasodium EDTA, sodium etidronate)
¤ Colors
¤ Fragrances and flavors

4.3 Mild preservative systems

By their very nature, preservatives are biocides - compounds that in-
terfere with microbial cell envelope/membrane integrity, metabolism,
enzyme function, protein conformation, macromolecular synthesis, etc.
Although selective action against microorganisms is desirable so that
the product is effective, many preservatives can be somewhat toxic to
humans. A desirable goal of formulation chemists is to have a mild but
effective preservative system. The preservatives listed in Table 6 are
safe and effective at the concentrations allowed.
 Introduction to Cosmetic Microbiology 27

Products that are adequately preserved and microbiologically safe

may be developed by a selection of chemicals to create conditions that
have bacteriostatic and/or bactericidal effects on microorganisms.
Aqueous systems with >20% alcohol, or which contain chemicals that
maintain low pH (i.e., pH <3) or low aw (i.e., aw <0·6) will not permit
growth of microorganisms that have been a problem to the cosmetic and
drug industries. Combinations of ingredients may be used to interfere
with the ability of microorganisms to grow in products [10,11]. Orth et
al. [12] reported that methylparaben and acrylic acid homopolymer/co-
polymers have synergistic anti-Pseudomonas activity in vitro. Similar
action was observed with EDTA and methylparaben by these workers.

4.4 References
1. Cosmetics Directive 76/768/EEC, 6th Amendment. June 14,1993.
2. Cowen, R.A. and Steiger, B. Why a preservative system must be tailored to a
specific product. Cosmet. Toiletr 92 (31 15-16,18-20 (1977).
3 . Orth , D . S . Principles of preservation . In Guide to Microbiological Control in
Pharmaceuticals CSR Denyer and R . Baird, eds .), Ellis-Horwood, London
(1990), pp. 242-250.
4 . Orth, D . S . Handbook of Cosmetic Microbiology. Marcel Dekker, New York
(1993), pp. 75-102.
5 . Steinberg , D . C . A Gl*de to European Cosmetic Regulations. Independent
Cosmetic Manufacturers & Distributors and American Beauty Assoc. (1998),
pp 34-40.
6. Steinberg, D.C. Frequency of use of preservatives in the United States, 1996.
Presentation at the Annual Scientific Seminar, May 1997.
7. Orth, D.S., Lutes, C.M., Milstein, S.R. and Allinger, J.J. Determination of
shampoo preservative stability and apparent activation energies by the linear re-
gression method of preservative efficacy testing . J. Soc. Cosmet. Chem . 38 ,
307-319 (1987).
8. Freese, E., Sheu C.W., and Galliers, E. Function of lipophilic acids as antimi-
crobial food additives . Nature 241 , 321 -325 ( 1973 ).
9 . Orth , D . S . Inactivation of preservatives by surfactants . In Surfactants in Cos-
,netics, 2nd ed. (M.M. Rieger and L.D. Rhein, eds.), Marcel Dekker, New York
(1997), pp. 583-603.
10 . Kabara, J . J . and Orth , D . S . ( eds .). Preservative-Free and Self- Preserving
Cosmetics and Drugs. Principles and Practice. Marcel Dekker, New York
(1997),
28 IF'SCC Monograph No. 5

11. Orth, D.S. and Kabara, J.J. Preservative-free and self-preserving cosmetics
and drugs . Application of Hurdle Technology. Cosmet. Toiletr. 113 (4), 51 , 52 ,
54,56-58 (1998).
12. Orth, D.S., Lutes Anderson, C.M., Smith, D.K. and Milstein, S.R. Syner-
gism o f preservative system components: Use of the survival curve slope
method to demonstrate anti-Pseudomonas synergy of methylparaben and acrylic
acid homopolymer/copolymers in vitro. J. Soc. Cosmet. Chem. 40, 347-365
(1989).
 Introduction to Cosmetic Microbiology 29

5 Preservative Efficacy Test Methods,

Acceptance Criteria and Rapid Methods
Preservative efficacy testing, or "challenge testing", is performed on
aqueous cosmetic and pharmaceutical products to determine the mini-
mum effective concentration of antimicrobial preservatives required for
adequate preservation. Products are satisfactorily preserved if they meet
appropriate acceptance criteria. Preservative efficacy testing may also
be performed to determine antimicrobial synergism of formula ingredi-
ents because use of ingredients with synergistic action against mieroor-
ganisms may result in product cost savings and mildness that cannot be
obtained by other means. This chapter reviews the basic methods for
preservative efficacy testing, how they are performed, and how the test
results should be interpreted.

5.1 Preservative efficacy test methods

Compendial methods of preservative efficacy testing used in different
countries include the United States Pharmacopoeia (USP), British Phar-
macopoeia (BP), and European Pharmacopoeia (EP) methods [1-3]. In
1995, the BP was updated to bring it in line with the EP for harmoniza-
tion of test methods in European countries. There are trade association
methods, such as the Cosmetic, Toiletry, & Fragrance Association
(CTFA) method [4-] and rapid procedures such as the linear regression
method [5], the accelerated preservative test [6], and the presumptive
challenge test [7].
All of these methods have a number of similarities, including test
organisms used, recovery systems, and the method of performing aero-
bic plate counts (APCs). Preservative efficacy tests typically are per-
formed by adding 0· 1 ml of a saline suspension of organisms to 50 g of
a product sample to give an initial count of about 106 CFU/ml. The
samples are mixed by shaking or stirring, and aliquots are withdrawn at
various times (typically, initially 4 or 6 hours, 24 hours, 7 days, 14
days, 21 days and 28 days) and colony counts are made to determine the
remaining CFU/ml of each test organism at each time point. The num-
ber of organisms remaining (CFU/ml at a particular time) is used to
3 determine whether the product preservative system meets acceptance
criteria, which are expressed in the number of log reductions at a par-
ticular time (e.g., reduction from 106 to 103 CFU/ml at 7 days) or the
decimal reduction time (D-value). The D-value is the time required for ~
30 IFSCC Monograph No. 5

killing 90% of the population o f test organisms (a 1 -log reduction) and

is determined by calculating the negative reciprocal of the slope of the
survival curve. The survival curve for each test organism is constructed
by plotting the log number of viable microorganisms recovered from the
inoculated sample as a function of the time at which that sample was
taken. This is illustrated in Figure 1.

D-Value
6

5
Log No. S. aureus/m

0
0 5 10 15 20 25
Hours

Figure 1 Survival curve for S. aureus showing a decrease in the population from 106 1
to less than 10 organisms per milliliter after 24 hr. The D-value in this example is 4 9
hr and is shown to be the time required for a 1 -log reduction in the aerobic plate I
count.
 Introduction to Cosmetic Microbiology 31

The main differences in these methods are the growth temperatures

used, procedures used for preparing inocula, times at which aerobic
plate counts (APCs) are determined, use of rechallenge testing, accep-
tance criteria, and the ability to use statistical controls to ensure that the
methods are in control [8-10]. In addition, these differences may cause
variations in test results and affect whether a product passes or fails the
challenge test. Different acceptance criteria may make it difficult for a
laboratory (or a country) which requires more stringent criteria to ac-
cept products that have been tested and found to be acceptable by a
laboratory (or a country) that relies on more lenient criteria.

5.2 Types of organisms used in preservative efficacy testing

In general, compendial (USB EP) and CTFA methods, and the linear
regression method use the same "core" test organisms, which include: S.
aureus ATCC 6538 , A aeruginosa ATCC 9027 , E. coli ATCC 8739,
Candida albicans ATCC 10231 , and Aspergillus niger ATCC 1 6404 . It
is believed that use of these microorganisms will provide a diverse
range of morphological and physiological types of test organisms rea-
sonably expected to be encountered in the manufacturing environment
and during use by consumers. Some laboratories use additional microor-
ganisms including Burkholderia (Pseudomonas) cepacia (a non-fuores-
cent pseudomonad), and Bacillus cereus (ATCC 11778) or B. subtilis
(ATCC 6633) (Gram-positive spore-forming bacteria). Some products
may require testing with anaerobes, and enteric pathogens such as Sal-
monella spp. may be used for preservative efficacy testing of oral
preparations.
The use of environmental isolates (e.g., microorganisms recovered
from manufacturing plant) or house organisms is not necessary for rou-
tine testing because it is unlikely that any laboratory will ever find all
environmental isolates that may occur when formulas are inadequately
preserved, when current good manufacturing practices (cGMPs) are not
satisfactory, or when test methods are inadequate. It is believed that
products that meet appropriate acceptance criteria will kill house organ-
isms that are grown on laboratory media. Isolates may be adapted by
growth in dilute product residues, and resistant organisms may be ob-
tained by exposure to stressful conditions [11]. Adapted microorgan-
isms can grow in many adequately preserved products and do not need
to be used for routine testing [12].
 32 IFSCC Monograph No. 5

5.3 Rechallenge testing

The use of rechallenge testing has been discussed in several publica-
tions [4,5,13]. The rationale for the use of repeated inoculations is that
it represents the repeated contamination of a product that may occur
during use. Repeated inoculation with specific test organisms shows the
number of challenges a product can withstand before the preservative
system fails for that organism. An alternative to rechallenge testing is to
increase the inoculum. Testing 10 times with 106 organisms/ml is the
same as testing once with 107 organisms/ml [10,14,15].

5.4 Acceptance criteria

Unlike the USB EP and CTFA methods which determine the percentage
of the original population present after 2,3,7 and/or 14 days, the linear
regression method determines the D-values. The slowest rates of killing
allowed for bacteria used in preservative efficacy testing (i.e., the larg-
est D-values) are D-values of 5112, 556, 524, 54, and 528 hours for the
USP, CTFA, BP, pathogens (linear regression method), and non-patho-

E 4

m 3
-A
-1
2

~0 1 2 3 4 5 6 ~ 8 9 1011 12131 15
Days

A usp * CTFA ~ LRM NONPATHOGENS ~ LRM PATHOGENS

Figure 2 Slowest rates of killing a population of 106 organisms/ml based on D-values

Source: From reference [13]
 Introduction to Cosmetic Microbiology 33

gens (linear regression method) testing methods, respectively, as shown

in Figure 2 [13,16]. Larger D-values indicate a slower rate of killing
than smaller D-values. Reference to Figure 2 reveals that the USP and
CTFA methods allow <2-log and 3-log reductions at 7 days to pass,
respectively; whereas the linear regression method requires 6-log reduc-
tions at 7 days. Orth, Delgadillo and Dumatol [17] reported that the
maximum permissible D-values for unadapted Gram-negative bacteria
were around 30 hours. This means that Gram-negative bacteria rou-
tinely used in preservative efficacy testing persisted or grew if they
were not killed with initial D-values of 530 hours.
The EP method notes that in justified cases, where the "A" criteria
cannot be attained (i.e., for reasons of an increased risk of adverse
reactions), the "B" criteria must be satisfied. The "B" criteria state that
there must be a 3-log (99·9%) reduction in bacteria and a 1-log (90%)
reduction in fungi by 14 days and no increase in bacteria or fungi at 28
days. These criteria are too lenient and should not be used for products
in multiple-use containers unless the containers prevent microbial con-
tamination of the product.

5.5 Rapid screening method

Orth and Enigl [18] reported use of a rapid screening method for esti-
mation of D-values. Although this method uses conventional microbio-
logical procedures (i.e., plating on agar media and counting colonies
after 48 hours of growth), it is faster than CTFA, USP, and EP methods
because tests for pathogenic bacteria such as S. aureus and R aerugi-
nosa may be completed within three days and tests for non-pathogenic
bacteria (such as E. coli), yeasts, and molds may be completed within
two weeks (typically, 5-7 days are allowed for fungal growth after the
7-day test period).

5.6 Preservation of products during use

Adequacy of preservation must be maintained during use of the product.
It is recommended that manufacturers consider home-use tests of the
product - in its final packaging - to determine whether the product
remains uncontaminated during use. Maintenance of preservative effi-
cacy (as indicated by testing samples of product returned after a period
of use) and an APC of <10 CFU/g (for aqueous products) indicates that
the product preservative system is satisfactory in the packaging used
and in the manner used.
34 IFSCC Monograph No. 5

Orth, Barlow and Gregory [19] reported on the use of the required
D-value (RDV) for evaluating the effect of formula, packaging, and
consumer use/abuse on product preservation. Three variables that deter-
mine whether a product will become contaminated are the preservative
system of the formula, the packaging factor, and the consumer
use/abuse factor. Use of the RDV enables one to consider factors that
have the greatest impact on product contamination during use.

5.7 References
1 . Microbiological tests , antimicrobial preservatives-effectiveness . United
States Pharmacopoeia XXIII . United States Pharmacopoeial Convention , Rock-
ford, MD (1995), p. 1681.
2 . Medicines Commission . E fficacy of antimicrobial preservation . British Phar-
macopoeia 1993, Vol. II. Her Majesty's Stationery Office, London (1995), pp.
A191-192; Addendum App. XVI C (1995).
3. European Pharmacopoeia Commission. Efficacy of antimicrobial preserva-
tion . European Pharmacopoeia, 3rd ed; Council of Europe , Strasbourg ( 1996 ),
pp. 286-287.
4. Preservation Subcommittee of the CTFA Microbiological Committee. A
guideline for the determination of adequacy of preservation of cosmetics and
toiletry formulations . TGA Cosmet. J. 2, 20 -23 ( 1970).
5. Orth, D.S. Linear regression method for rapid determination of cosmetic pre-
servative efficacy. J. Soc. Cosmet. Chem. 30, 321-332 (1979).
6. Scibienski, E.J., O'Niel, J.J. and Mead, C.A. An accelerated preservation
test. Presentation at Annual Scientific Meeting of the Society of Cosmetic
Chemists, Dec. 11,1981.
7. Chan, M. and Bruce, H.N. A rapid screening test for ranking preservative effi-
cacy. Drug Cos,net. Ind. 129, 34-37, 80-81 (1981).
8 . Orth , D . S . Evaluation of preservatives in cosmetic products . In Cosmetic and
Drug Preservation. Principles and Practice O.J . Kabara, ed .), Marcel Dekker,
New York (1984), pp. 401-421.
9. Orth, D.S, Lutes, C.M. and Smith, D.K. Effect of culture conditions and
method of inoculum preparation on the kinetics of bacterial death during pre-
servative efficacy testing . 1 Soc . Cosmet. Chem. 40, 193 -204 ( 1989 ).
10. Orth, D.S. and Brueggen, L.R. Preservative efficacy testing of cosmetic
products. Rechallenge testing and reliability of the linear regression method.
Cosmet. Toilet,·. 97 (5), 61 -65 ( 1982 ).
1 I. Orth, D.S., Dumatol, C. and Zia, S. House organisms - Dealing with the
bug in the plant. Cosmet. Toiletr. 111 (6), 59-66,68-70 ( 1996)
 Introduction to Cosmetic Microbiology 35

12 . Orth, D . S . Principles of preservative efficacy testing . Cosmet. Toiletr. 96

(3), 43,44,48-52 (1981).
13 . Orth, D . S . Handbook of Cosmetic Microbiology. Marcel Dekker, New York
(1993),pp. 491-519.
14. Barnes, M. and Denton, G.W. Capacity tests for the evaluation of preserv-
atives in formulation. Soap, perf. Cosmet. 42, 729-733 ( 1969).
15 . Bean, H . S . Preservatives for Pharmaceuticals . 1 Soc. Cosmet. Chem . 13,
703-720 (1972).
16 . Orth, D . S . Standardizing preservative efficacy test data. Cosmet. Toiletr.
106 (3), 45-48,51 (1991)
17. Orth, D.S, Delgadillo, K.S. and Dumatol, C. Maximum allowable D-values
for Gram-negative bacteria. Determining killing rates required in aqueous prod-
ucts. Cosmet. Tbiletr 113 (9), 53-56,58,59 (1998).
18. Orth, D.S. and Enigl, D.C. Preservative efficacy testing by a rapid screening
method of estimating D-values . J Soc. Cosmet. Chem. 44, 329 -336 ( 1993 ).
19. Orth, D.S., Barlow, R.F. and Gregory, L.A. The required D-value:Evaluat-
ing product preservation in relation to packaging and consumer use/abuse. Cos-
met. Toiletr. 101 ( 12), 39-43 ( 1992).
36 IFSCC Monograph No. 5

6 FD&C Act and Regulations Pertaining to

Cosmetics and OTC Drugs
The cosmetic and drug industries in the United States, the EU and
Southeast Asia are controlled by several laws. Laws regulating cosmetic
and drug quality, safety and labeling include the Federal Food, Drug &
Cosmetic Act (FD&C Act) in the United States, the 6th Amendment to
the Cosmetics Directive in the EU, and the Pharmaceutical Affairs Law
in Japan [1-3].

6.1 Definitions
The FD&C Act defines drugs as "articles...intended to affect the struc-
ture or any function of the body". This law defines cosmetics as "arti-
cles intended to be rubbed, poured, sprinkled or sprayed on, introduced
into, or otherwise applied to the human body or any part thereof for
cleansing, beautifying, promoting attractiveness, or altering the appear-
ance, and articles intended for use as a component of any such articles"
[1].
Article 1 of the Cosmetics Directive describes a cosmetic product as
"any substance or preparation intended to be placed in contact with the
various external parts of the human body (epidermis, hair system, nails,
lips and external genital organs) or with the teeth and the mucous mem- ~
branes of the oral cavity with a view primarily or exclusively to clean-
ing them, perfuming them, changing their appearance and/or correcting
body odours and/or protecting or keeping them in good condition".
Such a product must not cause damage to human health when applied
under normal or reasonably foreseeable conditions of use, and take into
account the product's presentation, labelling and instructions for use
[2].
The Japanese Pharmaceutical Affairs Law defines drugs as articles
recognized in the official Japanese Pharmacopoeia and articles (other
than quasi-drugs) which are intended for use in the diagnosis, cure or
prevention of disease in man or animals, and which are not equipment
or instruments. Quasi-drugs are articles that have specific purposes and
exert mild actions on the human body. Cosmetics are articles "intended
to be used by means of rubbing, sprinkling or by similar application to
the human body for cleaning, beautifying, promoting attractiveness, al-
tering the appearance of the human body, and for keeping the skin and
hair healthy, provided that the action...is mild" [3]. In 1996, the Japa-
 Introduction to Cosmetic Microbiology 37

nese Ministry of Health and Welfare consolidated product types into the
following preparations: cleaning, hair care, treatment, make-up, fra-
grant, suntan/sunscreen, nail makeup, eyeliner, lip, oral and bath prepa-
rations [3].

6.2 Adulterated drugs and cosmetics

Drugs and cosmetics in the United States shall be deemed to be adulter-
ated if they have been prepared, packed, or held under insanitary condi-
tions whereby they may have become contaminated with filth , or
whereby they may have been rendered injurious to health. Microbial
contamination of the process stream with objectionable microorganisms
constitutes this type of adulteration. This probably is more difficult for
the U.S. Food & Drug Administration (FDA) to establish; however, it
may be done through establishment inspections that reveal insanitary
conditions (i.e., processing stream samples that are contaminated), even
though finished product samples collected and analyzed may reveal no
viable microorganisms. Frequently, a terminal heating step in many
processes (i.e., "pasteurization" or holding a batch of cream or lotion at
making temperatures [70-80 °C], filter-sterilizing of parenterals, etc.)
may kill or remove contaminating microorganisms.

6.3 Inspections by regulatory agencies

Cosmetic and drug manufacturers must be prepared to deal with inspec-
tions by regulatory agencies. From a microbiological point of view, the
most significant observations are those that demonstrate microbial con-
tamination of the proce5s stream and contamination of finished products
with the same microorganisms.
If the regulatory agent collects samples during the inspection, the
microbiologist should collect an identical set to test in the same manner.
The microbiologist may be able to use the results of these tests in
responses to the regulatory agency and/or as part of the company's
defense in court.

6.4 Current good manufacturing practices

In the United States, the current good manufacturing practices (cGMPs)
regulation published in 1978 [4] sets forth the minimum cGMPs for
production of drugs and over-the-counter drugs (OTC drugs). Failure to
comply with cGMPs shall render a drug (or OTC drug) adulterated, and
38 IFSCC Monograph No. 5

the person who is responsible for failure to comply shall be subject to

regulatory action. In 1997, the U.S. FDA published cosmetic GMP
guidelines [5]. These guidelines provide a checklist for manufacturers
to use to help ensure that they are complying with regulatory require-
ments.
In a recent review, Steinberg [6] noted that the Council of Europe
published Guidelines for Good Manufacturing Practice of Cosmetic
Products (GMPC). The objectives of the GMPC are to eliminate and
prevent quality defects and to provide a model for EU Member States.
Following the GMPC will ensure compliance with EU regulations on
manufacturing cosmetics.
Products must not be released for shipment before tests on repre-
sentative samples from each lot (typically, samples from the beginning,
middle, and end of each batch, or from each shift during which a batch
is being packed into finished product containers) are performed to dem-
onstrate that the product meets finished-product microbial specifica-
tions, which should be <10 CFU/g for aqueous products. Preservative
efficacy tests, physical tests, and chemical analyses must be done at
designated time intervals during stability studies (i.e., initial time and at
3, 6, 9,12, 18, 24 and 36 months for compliance with the International
Council on Harmonization stability testing guidelines) to demonstrate
the stability and efficacy of the preservative system for the expected
shelf-life of the product [7]. The Hazard Analysis Critical Control Point
(HACCP) concept [8] is an excellent way to maintain microbiological
control over manufacturing operations (see Chap . 3 ).
6.5 Good laboratory practices
In the United States, the FDA established regulations on good labora-
tory practices (Gifs) to ensure the quality and integrity of the safety
data submitted to the Agency in support of the approval of regulated
products [9]. Although these regulations generally are thought to cover
non- clinical studies involved with animals , they apply to both in vitro
and in vivo experiments using animal , plant, or microorganism test sys -
tems.

6.6 Monographs for OTC drugs

The 1962 amendments to the FD&C Act created the requirements that a
drug had to be both safe and effective. In 1978, the FDA issued a
proposal to establish a monograph for OTC topical antimicrobial prod-
 Introduction to Cosmetic Microbiology 39

ucts for repeated daily human use [10]. Since that time, several final
monographs for topical OTC drugs have been published in the Federal
Register Gee Chap . 7). Although many formulations are considered to
be OTC drugs in the United States, they are regulated and sold as
cosmetics in the EU. Steinberg [11]1 summarized substances allowed in
the EU, maximum levels allowed, product uses, limitations, and condi-
tions of use/warnings required.
The quasi-drug regulations in Japan stipulate the purpose of use,
primary product form and scope of indications/effects for the following
types of quasi-drugs: mouth refreshers, body deodorants, talcum pow-
ders, hair growers, depilatories, and hair dyes [3]. These products exert
mild effects on the body to improve appearance and promote attractive-
ness by combating body odor, preventing perspiration, removing hair
and altering hair color.
The Scientific Committee on Cosmetology of the Commission of the
European Communities was established in 1978 to assist the Commis-
sion in the application of the 76/768 Cosmetics Directive. The Commit-
tee includes specialists in several scientific fields, including microbiol-
ogy, Loprieno [12] published guidelines for the safety evaluation of
cosmetics ingredients in the EC countries.

6.7 References
1. Federal Food, Drug, and Cosmetic Act as Amended. U.S. Code, Title 21:301-
392.
2. Cosmetics Directive 76/768/EEC, 6th Amendment. June 14,1993.
3. The Comprehensive Licensing Standards of Cosmetics by Category, 1994
(CLS 1994). Yakuji Nippo, Tokyo, Japan 1994.
4. Human and veterinary drugs. Current good manufacturing practice in manu-
facture , processing , packing , or holding . Federal Register 43 090), 45014-
45087 (1978).
5. U.S. Food & Drug Administration/Center for Food Safety and Applied Nutri-
, tion. Cosmetic good manufacturing practice guidelines, Oct. 30,1997, pp. 1-5.
6 . Steinberg, D . C . Regulatory Review : GMPC and GMP. Cosmet. Toiletr. 113
(8),41-44 (1998).
1 7, The European Agency for the Evaluation of Medicinal Products, Human
Medicines Evaluation Unit. Stability testing of new drug substances and prod-
ucts. ICH Harmonized Tripartite Guideline, Dec. 1993.
8, Bauman, H.E. The HACCP concept and microbiological hazard categories.
~ Food Technol. 32 (9), 31,32,34,74 (1974).
40 IFSCC Monograph No. 5

9. Food and Drug Administration. Good laboratory practice regulations. Fed-

eral Register 51 ( 172), 33768 -33782 ( 1987 ).
10. Over-the-counter drugs generally recognized as save, effective and not mis-
branded . OTC Topical Antimicrobial Products . Federal Register 43 (4), 1210-
1248 (1991).
11 . Steinberg, D. C . A Guide to European Cosmetic Regulations. Independent
Cosmetic Manufacturers & Distributors and American Beauty Assoc. (1998),
pp. 18-26.
12. Loprieno, N. Guidelines for safety evaluation of cosmetics ingredients in
the EC countries . Fd. Chem . Toxic. 9, 809-815 ( 1992).
 Introduction to Cosmetic Microbiology 41

7 Topical/Oral Products that Prevent, Correct

or Conceal Conditions Caused by
Microorganisms
The distinction between a cosmetic and a drug product may depend
solely on how it is marketed. Curiously, two products may have identi-
cal compositions, yet one may be designated a drug because therapeutic
(i.e,, drug) claims are made, and the other may be a cosmetic because of
its "cosmetic" positioning.
Many cosmetics and OTC drugs are intended to prevent, correct, or
conceal conditions caused by microorganisms and/or their metabolic
products. Powders reduce the water activity (aw) when applied to the
body; alcohols have an antimicrobial action; and active ingredients in
deodorant soaps, antiperspirants (APs), and antidandruff products help
to prevent the growth of microorganisms that produce odors or un-
sightly conditions which OTC drugs and cosmetics prevent and/or cor-
rect. Blemishes caused by microorganisms (e.g., acne lesions) or other
causes are concealed with products that contain opaque pigments in-
cluding iron oxides and titanium dioxide. The objective of this chapter
is to present some cosmetics and OTC-drug products that prevent, cor-
rect, or conceal conditions caused or exacerbated by microorganisms.

7.1 Antiperspirant deodorants and deodorants

Antiperspirant deodorants or antiperspirants (APs) are OTC-drug prod-
ucts that contain active ingredients that retard perspiration, microbial
growth and the production of odor in the axillary region. It is believed
that the first APs probably appeared around 1900 as simple aqueous
solutions of aluminum chloride. "Everdry" was introduced in 1902 and
is given credit for being the first AP product on the market [1].
Underarm odor is caused by the growth of bacteria in the axillae.
These bacteria are predominantly coagulase negative staphylococci and
aerobic diphtheroids. APs provide deodorant protection by preventing
microorganisms from growing in the axillae [2]. Although apocrine se-
cretion is sterile when it is formed, it quickly becomes contaminated
with bacteria in the axillae. The characteristic axillary odor is due to
volatile, short-chain free fatty acids (including acetic, propionic, bu-
tyric, valerie, and isovaleric acid) and steroids [3-5]1.
42 IFSCC Monograph No. 5

APs help prevent sweating. This decreases the availability of mois-

ture and decreases the aw, which helps prevent microbial growth and
odor production. APs in the United States are OTC drugs because they
affect a function of the body - perspiration. The active ingredients in
most formulations sold in the United States today are aluminum salts,
including aluminum chlorohydrate, aluminum zirconium tetrachlorohy-
drex gly (glycinate), and aluminum zirconium trichlorohydrex gly [6].
Aluminium [British spelling] zirconium chloride hydroxide complexes
and zinc 4-hydroxybenzenesulphonate are allowed in APs in EC coun-
tries [7]. The Ministry of Health and Welfare (MHW) in Japan allows
ingredients that help control body odor and perspiration in quasi-drugs
[8].
Although APs are deodorants, deodorants are not APs. Deodorancy
may be obtained (1) by use of fragrances which mask or "hide" the
offensive odor of the axillary vault, such as the "deo-colognes," which
are natural (perfume) oils that have some antimicrobial/deodorant activ-
ity [9]; or (2) by use of antimicrobial compounds, such as alcohols,
triclosan and zinc 4-hydroxybenzenesulphonate [2,7]. These products
remain cosmetics unless antimicrobial claims are made.

7.2 Products that control dandruff, seborrheic dermatitis

and psoriasis
Dandruff is a condition of the scalp characterized by excessive clini-
cally non-inflammatory scaling. Dandruff scales are white or gray and
are shed from small round patches that may extend to cover the entire
scalp [ 10, 11]. Dandruff scales appear different from the greasy scales
of seborrheic dermatiti5 or from the silvery scales of psoriasis. The
turnover rate of skin cells is much higher in seborrheic dermatitis than
in dandruff. In addition, itching and inflammation are more common
and shedding of skin cells is more widespread in seborrheic dermatitis
than in dandruff [11]. Psoriasis is a chronic inflammatory skin disorder
of the skin and/or scalp, and it is characterized by pink or dull red areas
covered with silvery scales.
Current methods of treating dandruff involve the use of shampoos
which contain antimicrobial ingredients that inhibit Pityrosporum ovale
(Malassezia ovalis), a yeast that is a member of the resident scalp mi-
croflora [2 , 12]. Excessive shedding of scales disappears when P ovale
is eradicated by use of antidandruff shampoos or antibiotics [2,13]1.
 Introduction to Cosmetic Microbiology 43

P ovale is considered to be the etiological agent in dandruff and

seborrheic dermatitis [2,14,15]; however, it has been difficult to deter-
mine the specific role of this yeast in seborrheic dermatitis because it is
a member of the resident human skin flora. Sebum appears to be another
major contributing factor in seborrheic dermatitis because it provides
substrates for lipid peroxidation mediated by P. ovale lipoxygenase [ 2 ].
lhe OTC drug monograph for dandruff, seborrheic dermatitis, and pso-
riasis [12] lists active ingredients for the control or treatment of dan-
druff, seborrheic dermatitis, and psoriasis. The Ministry of Health and
Welfare (MHW) in Japan allows the use of quasi-drugs for dandruff and
itching of the scalp [8].

7.3 Products that treat acne

Acne vulgaris ( i . e ., acne) is a pathological condition of the pilose-
baceous unit which is composed of a hair follicle, the sebaceous glands
that are attached to it, the products of the follicle and sebaceous glands
(i.e., hair and sebum), and microorganisms. Sebaceous follicles occur
primarily on the face, neck, upper chest, shoulders and back. Acne usu-
ally occurs during adolescence; however, it is common in adults and is
the most common disorder seen in dermatological offices [16].
Holland et al. [2,17] indicated that acne is a multifactorial disease
because microbial, hormonal, sebum production, keratinization, and
skin immune system factors influence its occurrence and effects. The
primary lesions in acne are microcomedones that are formed as a result
of hyperkeratinization (i.e., an increased production of keratinized
cells), and retention hyperkeratosis (i.e., plugging of the follicle with
clumps of shed keratinocytes and sebum). Retention keratosis is be-
lieved to help in establishing anaerobic conditions and abundant sup-
plies of substrates and moisture that favor the development of Propioni-
bacterium acnes . Acne lesions are thought to develop from
microcomedones to give closed comedones - non-inflamed whiteheads
that progress to various types of inflamed lesions (i.e., papules, pus-
tules, and nodules) and open comedones - non-inflamed blackheads.
Comedogenicity is a term that refers to abnormal differentiation of
the epithelium of a follicular canal that results in the formation of mi-
crocomedones. The rabbit ear assay and human testing have been used
for predicting comedogenicity [2,18,19]. Several cosmetic and OTC-
drug products are designed for preventing, treating or concealing acne,
or are positioned for treating acne and acne-prone skin. In the United
44 IFSCC Monograph No. 5

States, the OTC-drug monograph for topical acne drug products lists
active ingredients allowed for topical treatment of acne (20).
Although benzoyl peroxide (BPO) at 2·5-10% was removed from
the list of Category I ingredients for safety reasons, OTC-drug products
with BPO remain the most popular acne treatments in the United States
and in the EU [2,7]1. In the EU, BPO is not permitted in cosmetics - all
products with this active ingredient are drugs. Complexion bars, medi-
cated soaps, antibacterial soaps, and anti-acne soaps are used as "ad-
junct therapy" along with acne medications for cleansing skin (i.e., acne
washes with salicylic acid) and removing bacteria to help care for acne-
prone skin. The active ingredients in the majority of medicated/antibac-
terial soaps and complexion bars are triclocarban and triclosan.

7.4 Fungal infections of the skin

Superficial mycoses are caused by fungi that invade only keratinized
tissue, such as skin and nails, when sufficient moisture and nutrients are
available. Tight-fitting clothing and shoes that prevent perspiration
from evaporating may increase the aw to the point needed for growth,
while sebum and the stratum corneum provide substrates for growth.
The toe web of the feet and the groin area are particularly susceptible to
fungal growth, and this results in objectionable foot odor and/or the
development of fungal infections known as "athlete's foot". Dermato-
phyte infections in the groin area are known as "jock itch" [2].
Products used for foot-care include foot bath salts, salicylic acid ~
corn treatment products, fungicidal dusting powders, antiperspirant foot
sprays, deodorant foot powder, tale/cornstarch powders that are used to
keep feet and inguinal regions dry, and antifungal foot creams, lotions
and powders [2,7,8].

7.5 Oral-care products

The oral cavity provides ample moisture, nutrients and environmental
conditions well suited to the development of a number of different types
of microorganisms. Growth of microorganisms in the mouth is one of
the primary causes of bad breath. Bacteria adhere to the enamel surfaces
of teeth and cause the build-up of dental plague. The production of
organic acids by these microorganisms leads to the development of den- i
tal caries. Microorganisms adhering to epithelial cells and growing in
the gingival crevice cause gingivitis [2,21,22].
 Introduction to Cosmetic Microbiology 45

Oral-care products inclzide cosmetic or OTC-drug products intended

for preventing, treating or concealing bad breath, dental plague, dental
caries and the build-up of tartar, and periodontal disease. Dentifrices are
oral-care products that are intended to be used for removal of food
particles on and around teeth, for removal of superficial plague and
stains, for polishing teeth, and for refreshing breath. Fluoride-contain-
ing dentifrices may be therapeutic and may help to reduce dental caries
by hardening external tooth surfaces [2,7,8]. In 1998, the Colgate-Pal-
molive Company introduced Total®, a toothpaste with 0·24% stannous
fluoride to fight tooth decay and 0·3% triclosan to help control gingivi-
tis.

7.6 References
1 , Jass , H . E . The history of antiperspirant product development , Cosmet.
Toiletr. 95 CD, 25-31 (1980).
2 . Orth, D . S . Handbook of Cosmetic Microbiology . Marcel Dekker, New York
(1993) pp. 221-323.
3 . Shelley, W. B ., Hurley, H . J . and Nichols , A . C . Arch. Derm. Syphilol. 68, 430
(1953). Cited in H.E. Jass. The history of antiperspirant product development.
Cosmet. Toiletr 95 (7), 25-31 (1980).
4. Eigen, E. Letter to the Editor. J. Soc. Cosmet. Chem. 41, 147-149 (1990).
5. Froebe, C., Simone, A., Charig, A. and Eigen, E. Axillary malodor produc-
tion: A new mechanism. J. Soc. Cosmet. Chem. 41, 173-185 (1990).
6. Food and Drug Administration. Antiperspirant drug products for over-the-
counter human use ; tentative final monograph (proposed rule). Federal Register
47 (162), 36492-36505 (1982).
7 . Steinberg D.C. A Guide to European Cosmetic Regulations. Independent
Cosmetic Manufacturers & Distributors and American Beauty Assoc. (1998),
pp. 14-26.
8. The Comprehensive Licensing Standards of Cosmetics by Category, 1994
(CLS 1994). Yakuji Nippo, Tokyo, Japan. 1994.
9 . Scrase, J . The deo cologne : New way to sell fragrance . Drug Cosmet. Ind.
130 (3), 46,47,78 (1982).
10. Kligman, A.M., McGinley, K.J. and Leyden, J.J. The nature of dandruff. J.
Soc. Cosmet. Chem. 27,11 1-139 (1976)
11 . Hale, E . Brushing off dandruffand other flaky afflictions , FDA Consumer
22 (4), 28-31 (1988).
46 IFSCC Monograph No. 5

12. Food and Drug Administration. Dandruff, seborrheic dermatitis, and psoria-
sis drug products for over- the-counter human use ; final monograph . Federal
Register 56 ( 233 ), 63544 -63569 ( 1991 ).
13. Saint-Leger, D., Kligman, A.M. and Stoudemayer, T.J. The role of the resi-
dent microflora in the pathogenesis of dandruff. J. Soc. Cosmet. Chem. 40,109-
117 (1989).
14 . Shuster, S . Dandruff, seborrhoeic dermatitis , and Pityrosporum ovale. Cos-
met. 7biletr. 103 (3), 87,90,91 (1988). 1
15 . Bergbrant , I . and Faergemann , J. The role of Pityrosporum ovale in sebor-
rheic dermatitis . Seminars Dermatol. 9 (4), 262 -268 ( 1990).
16 . Shalita, A . Acne vulgaris : Its pathogenesis and forms . Tomorrow 's Pharm.
9,4-6 (1987).
17. Holland, K.T., Ingham, E. and Cunliffe, W.J. A review. The microbiology of
acne . 1 Appl. Bacteriol. 51 , 195 -215 0 981 ).
18. Special Report: American Academy of Dermatology Invitational Sympo-
sium on Comedogenicity. J. Am. Acad. Dermatol. 20
(2), 272-277 ( 1989).
19. Mills, Jr., O.H. and Kligman, A.M. A human model for assessing comedo-
genie substances . Arch . Dermatol. 118, 903 -905 ( 1982 ).
20. Food and Drug Administration. Topical acne drug products for over-the-
counter human use ; final monograph . Federal Register 56 1 159), 41008 -41020
(1991).
21, Anon. Oral health care drug products for over-the-counter human use; estab-
lishment of a monograph . Federal Register 41 (101), 22760 -22930 ( 1982 ).
22. Swartz, M.N., Gibbons, R. and Socransky, S. Indigenous bacteria; oral mi-
crobiology. In Microbiology (B.D. Davis , R . Delbecco , H . N . Eisen and H . S . Gis -
berg, eds.), 4th ed. J.B. Lippincott Co., Philadelphia (1990), pp. 727-736.
 Introduction to Cosmetic Microbiology 47

8 Effects of Microorganisms on Skin Physiology

and the Skin Immune System
adapt-
Microorganisms have survival strategies that enable them to be
and use a wide range of
able so they can withstand adverse conditions
are the morph ologica l fea-
substrates as nutrients. Survival strategies
mi-
tures that protect and the physiological characteristics that enable
ns in their environ ment.
croorganisms to respond to changing conditio
be-
Survival strategies become virulence factors in the disease process
cause they enhance the invasive ness and/or toxigen icity of microor gan-
isms.
Human skin is an extremely effective barrier against infection, and
no examples have been reported on bacteria that can penetrate intact
lack of ~
skin unaided [1]. Protective mechanisms of the skin include the
moisture [low water activity (aw)], the mild acid pH (acid mantle condi-
continu ous exfolia tion of corneo cytes, the skin's
tion of pH 5-5·5),
microfl ora which compet e with potentia l invader s for nutrien ts
resident
and colonization sites, lysozyme (in pores, hair follicles, and sweat
[1].
glands) that degrades bacterial cell walls, and antimicrobial lipids
This chapter gives an overview of two bacteria to illustrat e mecha-
nisms that microorganisms use to affect skin physiology and the skin
immune system ( SIS ). Pseudomonas aeruginosa was selected because it
is perhaps the most "notorious" microorganism in the cosmetic industry.
This Gram-negative bacterium is very adaptable, it can use a wide range
of nutrients for growth, and it is a virulent opportunistic pathogen that
other
has caused severe eye infections and corneal ulcers [2,3]. The
bacterium to be considered is Propionib acterium acnes . This bacterium
rphic
was selected because it is an anaerobic, Gram-positive, pleomo
bacillus that is a member of the skin's resident microflora.

8.1 Inflammatory mediators and immunomodulators of P.

aeruginosa
R aeruginosa is somewhat unique among the Gram - negative bacteria
because it secretes a number of proteins and other substances into its
environment [4 , 5 ]. Several virulence factors of P. aeruginosa listed in |
8. Viru-
the Handbook Of Cosmetic Microbiology [6] appear in Table
or
, lence factors may interact with the SIS and contribute to toxicity
I establishment and spread of microbial infection.

It
 48 IFSCC Monograph No. 5

Table 8
Virulence factors of P. aeruginosa

1. Morphological features
Capsule (alginate glycocalyx)
Cell envelope (outer membrane)
Porins
Pili (adhesins)

11. Physiological capabilities

Enzymes (exotoxin A, exotoxin S, proteases, hemolysin [phospho-
lipase C], alkaline phosphatase)
Toxins (exotoxin A, exotoxin S, hemolysins [phospholipase C and glyco-
lipid], endotoxin (LPS, lipid A)
Phenazine pigments (pyocyanin)
Siderophores (pyoverdin, pyochelin)

111. Inflammatory mediators and immunomodulators

Endotoxin (LPS)
Exotoxin A
Proteases
Hemolysins (phospholipase C and glycolipid)
Alginate
Pili
Phenazine pigment

Source: Adapted from reference [6]

Exotoxin A (ETA) is a protein toxin and is the most toxic substance

produced by P. aeruginosa [7]. ETA is cytotoxic to a wide range of
mammalian cells, because affected cells are unable to modulate protein
(enzyme) synthesis in response to changes in their environment. ETA
suppresses the immune response which may result in a delay in forma-
tion of antibodies to P aeruginosa and the extracellular products it
produces [ 8 ]. ETA thus provides P aeruginosa with a mechanism to
become a virulent pathogen because it prolongs the exposure of the host
cells to the destructive action of other virulence factors produced by
this organism.
Phenazine pigment (pyocyanin) is reported to inhibit cytochrome
electron transport, which further impairs the ability of infected cells to
resist the spread of infection. Siderophores such as pyoverdin and pyo-
chelin are iron-binding molecules. Siderophores are produced when iron
is limited in the environment and serve to sequester iron and make it
available to P. aeruginosa [6]1.
 Introduction to Cosmetic Microbiology 49

The Gram-negative bacterial cell envelope contains lipopolysaccha-

ride (LPS). The LPS of P. aeruginosa plays an important role in patho-
genicity and has many biological activities in common with LPS from
other Gram-negative bacteria. As with the LPS from the enterobacte-
riaceae , the LPS from Pseudomonas exhibits a wide range of pathologi -
cal effects [6,9,10].
Growing pseudomonads frequently are surrounded by a glycocalyx
(extracellular polysaccharide). Fluorescent pseudomonads, such as P,
aeruginosa and R fluorescens, produce a polymer composed of alginic
acid (mannuronic acid and guluronic acid). The alginate appears to pro-
tect invading pseudomonads [6,11,12]1. The cell envelope (outer mem-
brane) and porins (channels through the envelope) help protect cells
from the toxic effects of antibiotics, while allowing selective permeabil-
ity through the outer membrane. Pili are tiny, thread-like appendages of
bacteria. They enable bacteria to attach to surfaces, including host tis-
sues [6].

8.2 Inflammatory mediators and immunomodulators of P.

acnes
The propionibacteria are reported to be the only anaerobic bacteria that
have been demonstrated to be resident members of the cutaneous mi-
croflora in humans [ 13 , 14]. P. acnes, R granulosum and R avidum are
associated with acne vulgaris. Several virulence factors of these organ-
isms are shown in Table 9 [6]. Some of the interactions of the propioni-
bacteria with the SIS and human physiology will be presented below
(see Table 9).
P. acnes appears to be involved in the inflammatory stages of acne .
Allaker et al. [ 15] reported the production of inflammatory materials by
the cutaneous propionibacteria including short-chain fatty acids
(propionic and acetic acids) that could cause direct cytotoxic and in-
flammatory effects as well vasoactive amines (histamine and try-
pamine). Other workers have reported the involvement of these organ-
isms in inflammation due to the production of chemoattractants and
immunostimulatory properties of the cell wall of P. acnes [16-18].
Acne is a multifactorial disease because the microbial effects are
influenced by host physiological, immunological and hormonal condi-
tions [6]. We now recognize that the pathogenesis of acne involves
overproduction of sebum, abnormal desquamation of the sebaceous fol-
 50 IFSCC Monograph No. 5

licle epithelium, and growth of P. acnes which triggers inflammation [6 ,

18].

Table 9
Virulence factors ofP. acnes

1. Morphological features
Cell wall (teichoic acid)

11. Physiological capabilities

Enzymes (hyaluronate Iyase, lipase, neuraminidase, phosphatase, protease,
hemolysin [lecithinase, phospholipase], DNase and RNase
Anaerobic growth

111. Inflammatory mediators and immunomodulators

Metabolic products: histamine, tryptamine, short-chain fatty acids (acetic,
propionic), phospholipase C
Cell wall (teichoic acid)
Complement activation
Chemotactic factors
Stimulation of reticuloendothelial system
Immune complexes

Source: Adapted from reference [6]

8.3 References
1 . Salyers , A . A . and Whitt, D . D . Bacterial pathogenesis. A molecular ap-
proach. ASM Press, Washington, D.C. (1994), pp. 3-15. L
2. Wilson, L.A., Kuehne, W., Hall, S.W. and Ahearn, D.G. Microbial contamina-
tion in ocular cosmetics . Am. J. Ophthamol. 71, 1298 - 1302 ( 1971 ).
3. Wilson, L.A. and Ahearn, D.G. Pseudo,nonas-induced corneal ulcers associ-
ated with contaminated eye mascaras. Am. J. Ophtha,not. 84, 112-119 (1 977).
4 . Iglewski , B . Probing Pseudomonas aeruginosa, an opportunistic pathogen .
ASM News 55, 303 -307 ( 1989 ).
5. Doring, G., Maier, M., Muller, E., Bibi, Z., Tummler, B. and Kharazmi, A.
Virulence factors of Pseudomonas aeruginosa. Antibiot. Chemother. 39, 136-
148 (1987).
6 . Orth , D . S . Handbook Of Cosmetic Microbiology. Marcel Dekker, New York
(1993),pp. 417-489.
7. Wick, M.J., Frank, D.W., Storey, D.G. and Iglewski, B.H. Structure, function
' and regulation of Pselidomonas aeruginosa exotoxin A. Annu. Rev, Microbiol.
44,335-363 (1990).
 Introduction to Cosmetic Microbiology 51

8. Holt, P.S. and Misfeldt, M.L. Alteration of murine immune response by

Pseudomonas aeruginosa exotoxin A . Infect. Immun . 45, 227-233 ( 1 984).
9. Henderson, B., Poole, S. and Wilson, M. Bacterial modulins: a novel class of
virulence factors which cause host tissue pathology by inducing cytokine syn-
thesis. Microbiol. Rev. 60, 316-341 (1996).
10. Jakway, J.P. and DeFranco, A.L.. Pertussin toxin inhibition of B Cell and
macrophage responses to bacterial lipopolysaccharide . Science 234 , 743 -746
(1986).
11 , Pier, G. B ., Small, G .J . and Warren, H .B . Protection against mucoid Pseudo-
monas aeruginosa in rodent models of endobronchial infections . Science 249 ,
537-540 (1990).
12. Deretic, V., Dikshit, R., Konyecsni, W.M., Chakrabarty, A.M. and Misra,
T. K . The algR gene, which regulates mucoidy in Pseudomonas aeruginosa, be-
longs to a class of environmentally responsive genes . J. Bacteriol. 171 , 1278 -
1283 (1989).
13. McGinley, K.J., Webster, G.F. and Leyden, J.J. Regional variations of cuta-
neous propionibacteria . Appl. Environ. Microbi01 . 35 , 62 -66 ( 1978 )
14. McGinley, K.J., Webster, G.F., Ruggieri, M.R. and Leyden, J.J. Regional
variations in density of cutaneous propionibacteria: Correlation of Propionibac-
terium acnes populations with sebaceous secretions . 1 Clin. Microbiol. 12, 672-
675 (1980).
15. Allaker, R.P., Greenman, J. and Osborne, R.H. The production of inflamma-
tory compounds by Propionibacterium acnes and other skin organisms . Br. J.
Dermatol. 117, 175 - 183 ( 1987 ).
16. Leeming, J.P., Holland, K.T. and Cunliffe, W.J. The microbial colonization
of inflamed acne vulgaris lesions . Br J. Dermatol. 118 , 203 -208 ( 1988 ).
17 . Webster, G . F. Inflammation in acne vulgaris. J. Am. Acad. Dermatol . 33 ,
247-253 (1995).
18 . Leyden, J .J . New understandings of the pathogenesis of acne . J. Am . Acad.
Dermatol. 32 (5), S 15-S25 (1995).
52 IFSCC Monograph No. 5

9 Harmonization of Test Methods and

Globalization of Preservative Systems
The FD&C Act considers a cosmetic to be adulterated if it contains any
poisonous or deleterious substance which may render it injurious to
users under the conditions of use prescribed in the labeling, if it con-
tains any filthy, putrid or decomposed substance (e.g., material contami-
nated with microorganisms), and if it has been manufactured under con-
ditions whereby it may have become contaminated [ 1 ]. The Cosmetic
Handbook addresses adequacy of preservation by stating that cosmetics
do not need to be sterile. However, it requires that cosmetics must not
be contaminated with pathogenic microorganisms and that the density
of non-pathogenic microorganisms should be low from the time of
manufacture until they are used by consumers [2]. Article 2 of the 6th
Amendment to the Cosmetics Directive addresses product quality by
stating that "a cosmetic...must not cause damage to human health when
applied under normal or reasonably foreseeable conditions of use" [3].
This means that the product must be free from unacceptable contamina-
tion. Article 8 of the 6th Amendment stipulates microbiological criteria
and test methods [4]1.
Historically, countries have approached regulation of cosmetics and
drugs with little regard to the regulatory requirements of other coun-
tries. In recent years, countries have been involved with reaching con-
sensus or "harmonization" (i.e., standardization) of test methods used
by different European Union (EU) countries so that they do not become
an issue for registration and marketing of products throughout Europe.
This chapter considers the harmonization of preservative efficacy tests,
microbial limits for finished products, and globalization of preservative
systems.

9.1 Microbiological test methods

To meet regulatory requirements in the United States and the EU, cos-
metic microbiology laboratories generally perform two types of testing.
Preservative efficacy testing is done to determine whether a formula is
adequately preserved, and aerobic plate counts (APCs) are performed to
determine the microbial load of raw materials and finished products.
In reviewing the USP, BP, CTFA, and linear regression methods,
Orth [5] pointed out differences that may affect the kinetics of death of
test organisms during preservative efficacy testing, and hence the accep-
 Introduction to Cosmetic MicrobioloKY 53

tance criteria based on these methods. The type of growth media used
and the way inocula are prepared may affect the results of preservative
efficacy testing. The growth media allowed with the different methods
include surface growth on agar media and broth cultures which may or
may not be harvested. Orth, Lutes and Smith [6] demonstrated that
D-values obtained using saline inocula prepared from cultures of
Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus spp.
grown on tryptic soy agar (TSA) were smaller (i.e., faster death) than
broth inocula of these organisms following growth in tryptic soy broth
without glucose (TSB). Also, addition of 0·2% sterile TSB (0.1 ml in a
50 g test sample) to test samples at the time of inoculation with saline
suspensions of test organisms gave larger D-values (i.e., slower death)
than were obtained with control samples that received only the saline
inocula, as shown in Table 10 [6,7]. These data indicate that it is neces-
sary to standardize the method of inoculum preparation to achieve har-
monization of test methods.
The test organisms recommended in the USP, EP, CTFA, and linear
regression methods are basically the same and represent a broad range
of morphological and physiological types of microorganisms reasonably
expected to be encountered during manufacturing and/or use by the
consumer (see Chap . 5 ). All laboratories should use the same cultures ,
grow them on the same agar media for no more than five subcultures,
use the same growth temperatures, and prepare saline suspensions of
inocula to help standardize the way in which preservative efficacy tests
are performed. This will aid significantly in the harmonization of test
methods.

9.2 Preservative efficacy test acceptance criteria

The USP, EP, CTFA and linear regression methods have different
acceptance criteria [8-11]. The acceptance criteria of the USP, EP and
CTFA methods are given in log reductions which may be converted to
D-values, which are 5112,524, and 556 hours, respectively. The maxi-
mum D-values allowed by the linear regression method for pathogens
and non-pathogens are 54 hours and 528 hours [5,7,11]. The D-value
is the standard way of reporting the rate of microbial death and gener-
ally is used in the food industry, in medicine, in sterilization science,
and research on antibiotics/biocides. Harmonization requires stand-
ardization of acceptance criteria.
 54 IFSCC Monograph No. 5

Table 10
Effect of growth medium and suspending liquid on D-values obtained using
S. aureus and E. coli in preservative efficacy testing of a cream and several
test lotions

Test Growth Cream Lotion C Lotion D Lotion E Lotion F

organisms medium/sus-
pending D- value X D-value X D-value X D-value X D- value X
liquid
S. aureus TSALT/saline 15 20 14 5 .4 9.2
15* 2lt 14* 5.8* 7.9t
15 22 14 6·2 6.5
S. aureus TSB 60 94 49 10 20
63* 86t 52* 10* 2Ot
65 78 55 10 19
E. coli TSALT/saline 22 55 5, 1 11 45
2lt 58t 5,3 12 44*
20 60 5,4 12 42
E. coli TSB 47 86 9,0 12 60
44t 84t 7,6 11 57*
40 80 6·2 10 53

Table values are D-values in hours.

* Mean D-values obtained in TSALT/saline and TSB were significantly different (p = 0.01)
t Mean D-values obtained in TSALT/saline and TSB were significantly different (p = 0.05)
* Mean D-values obtained in TSALT/saline and TSB were significantly different (p = 0.10)
Source: Adapted from reference [6]

The preservative systems: the face cream contained sodium borate; lotions C and D
contained phenoxyethanol, methylparaben and propylparaben; lotions E and F con-
tained methylparaben, propylparaben and quaternium-15.

9.3 Microbial limits on finished products

Finished products are tested to demonstrate that they meet microbial
limit guidelines - that they are free from unacceptable levels of con-
tamination so that they will not be injurious to consumers. The CTFA
microbiological limit guideline for cosmetics recommends 500 or 1000
microorganisms per g or ml (depending on product type) and no harm-
ful microorganisms [10].
These guidelines may be suitable for anhydrous products, in which
the unavailability of water prevents microbial growth. However, manu-
 Introduction to Cosmetic Microbiology 55

facturers should recognize that these are finished product guidelines,

which means that the microbial load must never increase after products
are released to the trade. The only way to ensure this is to have micro-
bial limits of APC <10 g (i.e., "none found") for finished aqueous prod-
ucts in multiple-use containers. An exception to this is if the products
are "self-preserving", which means that the physicochemical conditions
of the product (i.e., low aw, low or high pH, high alcohol concentration,
etc.) preclude microbial growth [12,13].

9.4 Globalization of preservative systems

The International Cosmetic Ingredient Dictionary and Handbook [ 14]
lists many preservatives, Annex VI of the European Cosmetics Direc-
tive lists permitted preservatives and acceptable use levels, and the
Ministry of Health and Welfare (MHW) has published a list of ingredi-
ents and concentrations allowed for use in Japan. The MHW considered
information on formaldehyde donors for several years before permitting
their use in products which contain <500 ppm formaldehyde, and only
in wash-off products with appropriate warning labeling. (No manufac-
turer has agreed to market products with these preservatives and warn-
ings, so, in effect, they are not permitted.) As manufacturers move prod-
ucts to global markets, they are learning that it is to their advantage to
use preservative systems containing chemicals permitted in all markets,
Use of the principles of preservation to create self-preserving prod-
ucts that may contain 50·5% tetrasodium EDTA, fragrance chemicals,
mild surfactants, antioxidants, formulations with low pH or low aw, and
protective packaging helps eliminate the need for chemical preserva-
tives [13]. Where chemical preservatives are needed to give these sys-
tems more effective preservation, addition of 50·8% paraben esters and
51·0% phenoxyethanol generally is sufficient. Manufacturers should
consider the use of preservative-free or self-preserving formulas to ex-
pedite product globalization Gee Chap . 10).
In November 1991, the First Pan-European Conference on the Sig-
nificance and Validation of Test Methods for the Efficacy of Preserv-
atives in Cosmetics, Toiletries and Pharmaceuticals was held in London
[15]. The objectives of this symposium were to consider the currently
available challenge tests and to discuss the prospects for international
harmonization of these methods. The enactment of regulations that are
accepted by EU countries indicates that significant progress has been
made in Europe. The United States is in the process of adopting many
56 IFSCC Monograph No. 5

guidelines set forth by the International Council on Harmonization

(ICH); however, global harmonization still appears to be several years
away.

9.5 References
1. Federal Food, Drug, and Cosmetic Act as Amended. U.S. Code, Title 21:301-
392.
2 . Food and Drug Administration . Cosmetic Handbook. FDA/IHS ( 1992 ).
3. Cosmetics Directive 76/768/EEC, 6th Amendment. June 14,1993.
4 . Steinberg , D . C . A Guide to European Cosmetic Regulations. Independent
Cosmetic Manufacturers & Distributors and American Beauty Assoc. (1998), p.
45.
5 . Orth, D . S . Standardizing preservative efficacy test data . Cosmet. Toiletr. 106
(3), 45-58,51 (1991).
6. Orth, D.S., Lutes, C.M. and Smith, D.K. Effect of culture conditions and
method of inoculum preparation on the kinetics of bacterial death during pre-
servative efficacy testing . 1 Soc. Cosmet. Chem. 40, 193 -204 ( 1989).
7 . Orth, D.S. Handbook of Cosmetic Microbiology. Marcel Dekker, New York
(1993), pp. 521-582.
8 . Microbiological tests , antimicrobial preservatives - effectiveness . United
States Pharmacopoeia XXIII . United States Pharmacopoeia Convention , Rock-
ford, MD (1995), p. 1681.
9. European Pharmacopoeia Commission. Efficacy of antimicrobial preserva-
tion . European Pharmacopoeia, 3rd ed., Council of Europe, Strasbourg ( 1996),
pp. 286-287.
10. Preservation Subcommittee of the CTFA Microbiological Committee. A
guideline for the determination of adequacy of preservation o f cosmetics and
toiletry formulations . TGA Cosmet. J. 2, 20 -23 ( 1970).
11. Orth, D.S. Linear regression method for rapid determination of cosmetic pre-
servative efficacy. J. Soc . Cosmet. Chem. 30, 321 -332 ( 1979).
12 . Orth , D . S . Handbook of Cosmetic Microbiology. Marcel Dekker, New York
(1993),pp. 103-118.
13. Kabara, J.J. and Orth, D.S. (eds.). Preservative-Free and SelAPreserving
Cosmetics and Drugs. Principles and Practice. Marcel Dekker, New York
(1997).
14 . Wenninger, J . A . and McEwen, G . N . International Cosmetic Ingredient Dic-
tionary and Handbook, 1th ed. The Cosmetic , Toiletry, and Fragrance Associa-
tion, Washington, DC (1997).
15. The First Pan-European Conference on the Significance and Validation of
Test Methods for the Efficacy of Preservatives in Cosmetics, Toiletries and
 Introduction to Cosmetic Microbiology 57

Pharmaceuticals, held November 20 and 21, 1991 at the Commonwealth Insti-

tute, London.
58 IFSCC Monograph No. 5

10 Self-Preserving Cosmetic and Drug Products

In recent years, we have witnessed the emergence of products that are
responding to the (perceived) consumer need for natural cosmetics. La-
beling on these products indicates that they contain "natural ingredi-
ents" and they may be fragrance-free or "preservative-free". The devel-
opment of these new products is causing a change in the way we think
about product preservation.
Preservative-free means without preservative chemicals . In the EU ,
this term means without chemicals listed as preservatives in Annex 6 of
the Cosmetics Directive. In most cases, 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 if they are sterilized and pack-
aged/stored appropriately. The term self-preserving is more appropriate
than "preservative-free" for most aqueous cosmetic and pharmaceutical
products in multiple-use containers because these products must have a
preservative system that kills microorganisms and/or prevents their
growth. This chapter introduces the reader to these emerging categories
of cosmetics and drugs.

10.1 Self-preserving products

Self-preserving products may be formulated by applying the principles
of preservation or hurdle technology Il, 21. These principles are used to
control microbial growth in foods, drugs and cosmetics and are:
(a) asepsis - keeping microorganisms out of the process stream
and keeping microorganisms out of finished products using pro-
tective packaging
(b) removal of organisms - trimming, sorting, washing, filtra-
tion, sedimentation, centrifugation, etc.
(c) retarding growth or killing microorganisms using hostile en-
vironmental conditions such as high temperatures (heat steriliza-
tion or pasteurization), fragrances, surfactants, chelators, low
temperatures (refrigeration or freezing), low aw, high or low pH,
removal of substrates required for microbial growth, biocides
(preservatives, disinfectants), irradiation, and mechanical disrup-
tion.
 Introduction to Cosmetic Microbiology 59

In reviewing these principles, it is apparent that they are used to

control unacceptable microbial growth during manufacturing and stor-
age as well as in finished products. These principles work against the
physical and chemical requirements for microbial growth, which are
suitable temperature and pH, availability of water, adequate nutrients
and minerals, suitable oxidation/reduction potential, and freedom from
harmful chemicals (including preservatives).
The physicochemical composition of self-preserving products pre-
vents microbial growth and/or survival. Generally, microorganisms of
interest in raw materials or in cosmetic products grow best around neu-
trality (i.e., pH 7), and their growth rate generally decreases as the pH
departs from neutrality (or from the optimum pH for growth for each
different organism). The low pH (typically, pH 3-4) and other ingredi-
ents in alpha-hydroxy products, antiperspirants and conditioners pre-
vent the growth of most microorganisms that could cause problems in
cosmetic products.
True liquid soaps, which are alkaline products made from the salts
of fatty acids, typically have a pH in the range of pH 8-9·5. These
liquid soaps present a hostile environment for the survival and growth
of many microorganisms due to the chaotrophic (i.e., membrane desta-
bilizing) effects of the free alkalinity (see Chap. 2). In addition, liquid
soaps may contain substantial quantities of dissolved solutes including
glycerin, inorganic salts and salts of free fatty acids which decrease the
aw· Soaps and shower products typically contain chelating agents, such
as tetrasodium EDTA, and antioxidants, such as butylated hydroxytolu-
ene (BHT), which contribute to the antimicrobial action of the formula.
Self-preserving cosmetics and drugs must meet the same preserv-
ative efficacy test criteria and microbial limits as do products with con-
ventional preservatives. The exception to this may be aw-based systems
that may be microbiostatic rather than microbiocidal, as long as it is
known that the aw is too low to prevent growth of microorganisms that
present a health hazard or could cause quality defects [1,2].

10.2 Packaging
, The packaging is especially important for self-preserving aqueous for-
mulas because proper packaging prevents environmental factors from
altering the formula [3]. Packages with sterilizing membranes fitted into
dispensing caps virtually prevent the possibility of microbial contami-
nation of aqueous products that are used repeatedly [3-6].
60 IFSCC Monograph No. 5

10.3 Summary
Self-preserving products reduce and/or eliminate chemical preservatives
that are a source of skin irritation and contact sensitization; they reduce
costs; they meet the demands of consumers who want natural products
(i.e., products without chemical preservatives); they may encourage the
use of contamination-resistant packaging; and they may allow develop-
ment of global formulas without the regulatory issues relating to the use
of chemical preservatives.

10.4 References
1. Kabara, J.J and Orth, D.S. (eds.). Preservative-Free and Seifpreserving Cos-
metics and Drugs. Principles and Practice. Marcel Dekker, New York (1997').
2, Orth, D.S. and Kabara, J.J. Preservative-free and self-preserving cosmetics
and drugs . Application of hurdle technology. Cosmet. Toiletr. 113 (4), 51 , 52 ,
54,56-58 (1998).
3 . Brannan , D . K . The role of packaging in product preservation . In Preservative-
Free and Self-Preserving Cosmetics and Drugs. Principles and Practice 0.1
Kabara and D.S. Orth, eds.). Marcel Dekker, New York (1997) pp. 227-242.
4. Ranalletta, J.V., Williams, Jr., RE., and Kanner, R.W. Liquid dispenser nozzle
assembly. U.S. patent 5,025,957, assigned to Ryder International Corp. (June
25,1991).
5. Ryder, RE., Kanner, R.W. and Rabenau, R. Solution delivery nozzle and sys-
tem with antimicrobial features. U.S. patent 5 154 325, assigned to Ryder Inter-
national Crop. (Oct. 13, 1992).
6. Kanner, R.W. and Nevelli, G.M. Liquid dispenser nozzle assembly. U.S. pat-
ent 5 431 310, assigned to Ryder International Corp. (July 11, 1995).