International Journal of Cosmetic Science, 2005, 27, 223–236
Method for evaluation of the efficacy of antimicrobial
preservatives in cosmetic wet wipes
A. Crémieux*, S. Cupferman  and C. Lensà
*Micraam, Faculté de Pharmacie, 13385 Marseille Cedex, France,  L’Oréal Recherche, 94550 Chevilly-Larue, France
and àKeybio, ZI Les Paluds, BP 1427, 13785 Aubagne Cedex, France
Received 19 September 2004, Accepted 13 March 2005
Keywords: wipes, challenge test, preservatives, dry inoculum
Synopsis
Many cosmetic formulations are now available in
the form of wet wipes packaged in sealed sachets
or packets. Like the majority of cosmetic products
having an aqueous phase, wipes are susceptible to
microbial contamination and require the addition
of preservatives. The efficacy of such preservatives
can be evaluated using a standard challenge test
performed on the wetting liquid but this test can-
not be regarded as representative for this new type
of formulation. The method presented here evalu-
ates the efficacy of preservatives used in wet wipes
kept in their original packaging. Dried inoculums
were prepared by membrane filtration followed by
drying in an incubator. The method is applicable
to bacteria (Pseudomonas aeruginosa, Escherichia
coli, Staphylococcus aureus and Enterococcus faecalis),
Bacillus cereus spores and fungi (Candida albicans
and Aspergillus niger). These inoculated carriers
were inserted between two wipes in the original
package, which was then re-sealed immediately.
The test requires one dry inoculum per packet and
one packet for each control or test. After incuba-
tion at 22.5
C for 1, 2, 7, 14 or 28 days and, for
the control, immediately after insertion of the
membrane (time 0), microorganism counts were
performed on the germ-carrier membranes as well
as on adjacent wipes, after transfer into a suitable
neutralizing agent. The membranes were shaken
in the presence of glass beads and microorganisms
Correspondence: S. Cupferman, L’Oréal Recherche, 94550
Chevilly-Larue, France. E-mail: scupferman@rd.loreal.com
ª 2005 Society of Cosmetic Scientists and the Société Française de Cosmétologie
were dissociated from the wipes by means of a Sto-
macher. The supernatants recovered after being
left to stand for 20 min are counted by pour plate
method or membrane filtration. The feasibility of
the method was demonstrated for each of the
seven above-mentioned strains. The repeatability
and reproducibility of the results obtained is sim-
ilar to that obtained for preservative efficacy tests
in the Pharmacopoeias. The lethal rate of microor-
ganisms during the preparation of dry inoculums
ranges from 50 to 90% depending on the strain
and the test (generally, a spontaneous reduction of
about 1 log up to a maximum of 2 log). The
recovery rate for microorganisms from dry inocu-
lums (on membranes) at time 0 (control ¼ T0) is
around 90%, regardless of the strain or the test.
The number of microorganisms recovered from the
wipes (W0) is between 2 and 10% of the number
recovered from membranes (T0) and may be con-
sidered negligible. Application of this method to
different types of wipes demonstrates that the effic-
acy of preservatives, expressed as the logarithmic
reduction in the number of microorganisms at
each time point, depends on the type of wipe and
on the strain tested. The results obtained are con-
siderably different from those found with the
standard challenge tests applied to wetting liquids
for wipes. The differences found confirm the need
for a specific method applicable to wipes.
Résumé
De nombreuses formulations cosmétiques sont
maintenant disponibles sous forme de lingettes imp-
régnées emballées dans des sachets ou pochettes
223
Efficacy of preservatives in wet wipes
étanches. Comme la majorité des produits cosmé-
tiques renfermant une phase aqueuse, les lingettes
sont sensibles à la contamination microbienne et
nécessitent, de ce fait, la présence de conservateurs.
L’efficacité de ces conservateurs peut être évaluée
par un challenge test classique portant sur le liquide
d’imprégnation, mais cet essai ne peut être consid-
éré comme représentatif de ces nouvelles présenta-
tions. La méthode décrite ici évalue l’efficacité des
conservateurs au sein de lingettes imprégnées
maintenues dans leur emballage d’origine. Des ino-
culums desséchés sont préparés par filtration sur
membrane et séchage à l’étuve. La mèthode est
applicable aux bactéries (Pseudomonas aeruginosa,
Escherichia coli, Staphylococcus aureus et Enterococcus
faecalis) aux spores de Bacillus cereus et aux fongi
(Candida albicans et Aspergillus niger). Ces porte ger-
mes sont introduits entre deux lingettes dans leur
emballage d’origine qui est immédiatement refermé.
L’essai nécessite au minimum un inoculum sec par
emballage et un emballage pour chaque essai ou
témoin. Après 1, 2, 7, 14, ou 28 jours d’incubation
à 22.5
C, et, pour le témoin, immédiatement après
insertion de la membrane (temps 0), les dénombre-
ments de microorganismes sont réalisés sur les
membranes porte germes et sur les lingettes adja-
centes après transfert dans un neutralisant validé.
Les membranes y sont agitées en présence de billes
de verre et les lingettes sont dissociées à l’aide d’un
Stomacher. Les surnageants récupérés après
20 min de repos sont dénombrés par inclusion en
boite de Pétri ou par filtration sur membrane. La fai-
sabilité de la méthode a été démontrée pour chac-
une des sept souches citées ci-dessus. La répétabilité
et la reproductibilité des résultats obtenus sont
semblables à celles que l’on observe dans les essais
d’efficacité des conservateurs décrits dans les Phar-
macopées. Le taux de mortalité des microorganis-
mes pendant la préparation des inoculums secs
varie de 50% à 90% (réduction spontanée en géné-
ral voisine de 1 log, au maximum 2 log) selon la
souche et l’essai. Le taux de recouvrement des
microorganismes constituant les inoculums secs
(sur membranes) au temps 0 (témoins ¼ T0) est vo-
isin de 90% quelque soit la souche et l’essai. Le
nombre de microorganismes récupérés des lingettes
(W0) est compris entre 2% et 10% de celui trouvé
sur les membranes (T0) et il peut être considéré
comme négligeable. L’application de cette méthode
à différents types de lingettes montre que l’effica-
cité des conservateurs, exprimée par la réduction
logarithmique du nombre de microorganismes en
224 ª 2005 International Journal of Cosmetic Science, 27, 223–236
A. Crémieux et al.
fonction du temps, dépend du type de lingette et
de la souche testée. Les résultats obtenus sont
sensiblement différents de ceux provenant des chal-
lenge-tests classiques appliqués aux liquides d’imp-
régnation des lingettes et les différences observées
montrent l’intérêt d’une méthode spécifique applic-
able aux lingettes.
Introduction
Cosmetic wipes, which originally appeared on the
market some 20 years ago as baby hygiene
products, are now widely used in a variety of
applications, such as for face and eye cleansing
and make-up removal, treatment of oily skin, deo-
dorants, aftershaves, sun protection and self-tan-
ning lotions.
They consist of two main components: an absor-
bent delivery system and a cosmetic formulation
to saturate the delivery system. The absorbent
delivery system is most commonly fibre-based but
other materials, especially foams, can also be used.
The fibrous delivery system is generally non-
woven and consists of a mixture of viscose and
polyethylene-tetraphthalate (PET) fibres. Mixtures
containing cotton or cellulose fibres also exist.
Wet cosmetic formulations in non-woven sys-
tems can be aqueous, hydro-alcoholic, an emul-
sion or oil. There is no limitation with regard to
the composition of products with the exception of
viscosity, a key parameter. For acceptable satura-
tion to be obtained, in other words uniform distri-
bution throughout the delivery system, the
cosmetic composition must be a low-viscosity
liquid. In fact, practically all industrial processes
now consist of spraying the cosmetic product onto
the non-woven system or soaking it in a bath.
The most commonly applied rate of saturation is
300–400%, which is 3–4 g of liquid per gram of
delivery system. With these saturation rates, a
liquid sedimentation phenomenon is often observed
in the case of multi-dose products. While wipes are
sometimes presented in single sachets, most wipes
on the market are sold in packs containing 20–25
wipes, and up to 80 wipes for baby products. They
are either folded or stacked in the packet or criss-
crossed so that the packet is always ready to be used
after the first wipe is removed. The packaging
material is usually plastic (polyethylene/polyester)
or a plastic and aluminium complex.
As for the majority of cosmetic products, multi-
dose wipes saturated with a cosmetic formulation
Efficacy of preservatives in wet wipes
including an aqueous phase are susceptible to
microbial contamination. Preservatives are gener-
ally needed to maintain constant microbiological
quality during manufacture and storage and until
the wipes are used. Suitable molecules for antimi-
crobial protection of cosmetic products are listed in
Appendix 6 of the European Cosmetics Directive,
which also specifies the maximum level of use [1].
The efficacy of preservatives depends on a num-
ber of factors. Efficacy cannot be estimated solely
on the basis of data in the literature (ex. M.I.C.)
but must be checked using standard methods such
as the challenge test [2–5]. The challenge test is
based on the principle of artificial contamination
of the product to be tested by different microbial
strains. The level of contamination is then monit-
ored at several time intervals. During the first step
of the test, the microbial inoculum in liquid form
is carefully mixed with the sample. The protocol
was originally designed to test cosmetic or phar-
maceutical products in liquid, fluid or semi-solid
form but is difficult to apply directly to wet wipes.
However, it is important to evaluate the efficacy of
preservation of these products in their final form
rather than simply testing the wetting liquid. It is
likely that microbial growth, and especially mould
growth, is not the same in a liquid and at the sur-
face of a saturated delivery system.
Various studies published in the literature reveal
the advantages of techniques which allow micro-
bial growth to be monitored over the course of
time [6–8] or methods which focus in particular
on studying materials such as paper or textiles
[3, 5, 9–11]. However, they do not cover adapta-
tion of the challenge test to the evaluation of anti-
microbial protection in wipes and other saturated
cosmetic delivery systems. This was the purpose of
the work reported in this paper.
The main source of contamination of cosmetic
wipes is the non-woven delivery system, through
the presence of bacterial and fungal spores [12].
These organisms are represented in the present
method by Bacillus cereus spores and Aspergillus
niger spores. Moreover, as the manufacture of cos-
metic products is not, with the exception of a few
cases, performed under sterile conditions, acciden-
tal contamination by species from the production
environment, especially from water, is possible.
Finally, as these products come into contact with
users’ skin, they should have a degree of antimi-
crobial protection against skin pathogens. Microor-
ganisms representing those which the wipes may
ª 2005 International Journal of Cosmetic Science, 27, 223–236
A. Crémieux et al.
encounter during manufacturing process and use
were therefore chosen for this test: Escherichia coli,
Pseudomonas aeruginosa, Enterococcus faecalis,
Staphylococcus aureus, B. cereus (spores), Candida
albicans and A. niger (spores).
The principle of the method was based on the
use of inoculums dried on a membrane which acts
as a carrier. Wipes were inoculated by the carrier
and the development of contamination was monit-
ored both on the carrier and on the wipes in con-
tact with it to take into account all surviving
microorganisms, regardless of the rate of transfer
from the carrier to the wipes.
Preliminary tests were performed to study the
survival of the most fragile microorganisms (bac-
teria) on the carrier inserted into wipes impregna-
ted with diluent and to define, under real
conditions, the parameters to be retained for the
evaluation of antimicrobial activity at various crit-
ical times. The results obtained made it possible to
identify a trial protocol to validate the method and
then simplify the protocol so that it could be easily
used in routine tests.
Materials and methods
Microbial strains
The strains used are collection strains stored at
)74
C in the laboratory under the conditions laid
out in standard EN 12353 [13]. Inoculums were
prepared from working cultures where the number
of transfers from the frozen batch was strictly lim-
ited to 4. The reference strains were: P. aeruginosa
(ATCC 19429), E. coli (ATCC 8739), S. aureus
(ATCC 6538), E. faecalis (ATCC 33186), B. cereus
(ATCC 33019), C. albicans (ATCC 10231) and
A. niger (ATCC 6275).
Wipes
Several types of non-woven wipes packaged in a
flexible sachet and saturated with oil in water emul-
sions containing preservatives were used in the
study. They differed in terms of size, wetting liquid
and packaging. They were either commercially
available products or products being developed.
Type I wipes
Packs of 25 make-up removal wipes containing
phenoxyethanol and parabens. The sedimentation
profile for the wetting liquid in type I wipes was
225
Efficacy of preservatives in wet wipes
9.5
9
8.5
8 ile, France). Diluents for microbial suspensions
7.5
7 salt solution with polysorbate 80 (0.05%; v/v) for
6.5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Figure 1 Sedimentation profile of type I wipes. Packs
(three) of wipes were stored flat for 48 h. The wipes were
then weighed separately, starting from the top of the
pack (wipe 1) to the bottom (wipe 25). The graph gives
the mean weight values for three wipe packs.
determined by weighing three packs and is repor-
ted in Fig. 1.
Type II wipes
Type II-1: packs of 25 aftershave wipes containing
chlorhexidine digluconate and parabens.
Type II-2: packs of 72 baby wipes containing
phenoxyethanol, parabens and potassium sorbate.
Type II-3: packs of 80 baby wipes containing
imidazolidinyl urea and parabens.
Type II-4: packs of 25 make-up removal wipes
containing quaternium 15 and parabens.
Each wetting liquid corresponding to type II wipes
underwent a challenge test to evaluate the efficacy
of preservation at day 7, 14 and 28 (see Table VII).
Also included in the test were non-woven wipes
– 25 per flexible sachet – impregnated with a ster-
ile diluent (tryptone–salt solution) [control wipes].
Inoculated carriers
Sterile cellulose ester membranes, diameter 47 mm,
porosity 0.45 lm (GN-6 metricel S-Pack; Pall Life
Science, New York, NY, USA). Membranes were
inoculated by filtration of about 10 mL of diluent
containing 1 mL of standard inoculum protected by
milk then dried in Petri dishes placed in a ventilated
incubator for 15–20 min (see Procedure). The carri-
ers are used for contamination of the wipes and for
easy recovery and counting of surviving microor-
ganisms.
Media and reagents
Trypcase soya agar (TSA) and Sabouraud dextrose
agar (with and without Chloramphenicol), malt
226
A. Crémieux et al.
extract agar and manganese sulphate agar were
prepared by the laboratory using dehydrated bases
(Difco, Becton Dickinson, Le Pont de Claix, France)
or supplied ready-to-use (Biomérieux, Marcy l’Eto-
were: tryptone–salt solution (tryptone, 0.1%; NaCl,
0.9%; w/v) for bacteria and yeasts and tryptone–
moulds.
The desiccation protector was a powdered skim-
med milk solution in sterile water (10% w/v). The
diluent-neutralizer was Eugon LT 100 medium
prepared by the laboratory using dehydrated base
(A.E.S., Combourg, France).
Apparatus
The method requires the use of a Stomacher (Bag-
Mixer, Interscience, Saint Nom la Bretêche,
France) for recovery of microorganisms from the
wipes, a heat-sealer (Wuhrlin-Soplamed, AMIS,
Trilport, France), a borosilicate glass filtration
apparatus (Millipore, Bedford, MA, USA), 250-mL
wide-neck Erlenmeyer flasks and glass beads
(Ø 3 mm).
Procedure
Preparation of inoculated carriers
Inoculums were prepared from two successive sub-
cultures on TSA at 24-h intervals for bacteria. For
C. albicans the inoculum was prepared from a sin-
gle 24-h subculture on Sabouraud dextrose agar
and for A. niger from a single 7–9-day subculture
on Malt extract agar. Bacillus cereus spores were
obtained from a single 8–10-day subculture on
manganese sulphate agar. Following collection
and heat treatment for 10 min at 80
C, the spores
were centrifuged and washed and then resuspend-
ed in physiological saline and stored at 4
C until
use. All strains were incubated at 36 ± 1
C.
For bacteria and the yeast, the culture collected
from the surface of the agar medium using glass
beads was diluted in a tryptone–salt solution and
the optical density adjusted using a spectropho-
tometer (544 nm) so as to obtain 1–3 · 108
cfu mL)1, for bacteria and 1–3 · 107 cfu mL)1,
for the yeast.
The A. niger spore suspension diluted in tryptone
salt solution with polysorbate 80 was filtered on
sintered glass and adjusted to 1–3 · 107 cfu mL)1
ª 2005 International Journal of Cosmetic Science, 27, 223–236
Efficacy of preservatives in wet wipes
using a counting device (e.g. Malassez) (cf. EN
1275 [14]).
Adjusted suspensions were protected from desic-
cation by the addition of a protector (rehydrated
powdered milk) at a rate of 0.5 mL of milk per
9.5 mL of adjusted suspension [15]. The adjusted
and protected suspensions were filtered (1 mL dilu-
ted in about 10 mL of diluent) on membranes
placed in Petri dishes and left half-open for
15–20 min in a ventilated incubator at 36
C until
completely dry.
Counting of the adjusted suspension (N) and the
dry inoculum (I) was carried out in order to deter-
mine mortality caused by desiccation.
Inoculation of wipes and incubation
Inoculated membranes were inserted between the
wipes at a rate of one or more membrane per pack
and maintained in the pack until counting (at 1,
2, 7, 14 or 28 days). Wipes were kept in their ori-
ginal packaging which was then resealed (heat-
sealed).
Each test required the preparation of a number
of contaminated wipe packs equivalent to the
number of exposure times, with an additional pack
acting as a control. Contact between the inocula-
ted carriers and wipes was maintained for 1, 2,
7, 14 and/or 28 days at a temperature of
22.5 ± 2.5
C depending on the purpose of each
particular test.
Controls
The control (T0) was performed according to the
same procedure except that the contaminated
membrane was removed immediately (time
£2 min) after its insertion into the wipe pack.
Recovery and counting of inoculated
microorganisms
Recovery and counting of microorganisms on carrier
membranes
Membranes removed with tweezers were placed
in an Erlenmeyer flask containing 100 mL of
Eugon LT 100 broth and glass beads (5 g). After
stirring for 3 min, 1 mL of broth was removed
and diluted to 1:10 (1:100 for the control at
t ¼ 0) in Eugon LT 100. For counting, 1 mL of
the 1:10 dilution and 1 and 0.1 mL of the
1:100 dilution were carried out (in duplicate) by
ª 2005 International Journal of Cosmetic Science, 27, 223–236
A. Crémieux et al.
inclusion in TSA agar for bacteria and Sabou-
raud dextrose agar for fungi.
Recovery and counting of microorganisms on wipes
Two wipes on either side of the previously extrac-
ted membrane (four wipes in total) were trans-
ferred to a Stomacher bag containing 100 mL of
Eugon LT 100 broth. After stomaching for 10 min
before being left to stand for 20 min at room tem-
perature, the supernatant was recovered and the
microorganisms counted in agar medium as above
and by filtering the remaining liquid (
90 mL) by
membrane filtration (porosity 0.45 lm).
Test parameters
The various experimental values obtained over the
course of these trials were:
N: control count for the calibrated inoculum
(»108 cfu mL)1 for bacteria, »107 cfu mL)1 for
fungi).
I: control count for the dry inoculum (microbial
load introduced into the wipe by the carrier).
T0: initial contamination control (number of
recoverable microorganisms at time 0 from the
carrier membrane inserted into the wipes).
W0: number of microorganisms recovered from
four wipes in immediate contact with the carrier
at time 0.
M1, M2, M3, M7, M14, M28: number of recover-
able microorganisms from carrier membranes after
1, 2, 3, 7, 14 and 28 days contact (in cfu/car-
rier), W1, W2, W3, W7, W14, W28: number
of recovered microorganisms from the four wipes
(in immediate contact with the carrier) after 1, 2,
3, 7, 14 and 28 days (in cfu/four wipes).
Preliminary tests
These tests assessed the feasibility of the method
and evaluated the conditions for artificial contam-
ination of wipes by dry inoculums.
Microbiological content of wet wipes and control
wipes
Four wipes were removed from the middle of the
pack and placed in 100 mL of neutralizing diluent
and stomached for 10 min. After being left to
stand for 20 min, the supernatant was recovered
and total aerobic microorganisms (bacteria, yeasts,
moulds) were counted.
227
Efficacy of preservatives in wet wipes
Lethal rate of Gram-negative bacteria during
preparation of dry inoculums and in inoculated
carriers inserted into control wipes
The test was limited to two strains of Gram-negat-
ive bacteria known to be sensitive to desiccation.
It was performed on control wipes saturated in a
sterile diluent. The test was repeated three times.
Counting of surviving bacteria was performed at
day 1, 2, 3, 7 and 14 on both the carrier mem-
branes and the wipes.
Effect on test results of the level of insertion of
carrier in packs of wipes
Taking into account the sedimentation of the wet-
ting liquid in packs of wipes (see Fig. 1), varying
degrees of preservative efficacy can be observed. In
type I wipes, three carriers were inserted between
the 4th and 5th, 14th and 15th, and 24th and
25th wipes. The test was performed with two bac-
terial strains (E. coli and S. aureus) and for short
exposure times (24 and 48 h).
Test parameters involved in the evaluation of
preservative efficacy
The test was conducted on the seven microbial
strains selected using type I wipes inoculated by
insertion of the carrier in the centre of the pack of
wipes.
Monitoring the level of microorganisms in the
contaminated wipes over time
The number of viable cells was counted at day 0,
2, 7, 14 and 28. The number of microorganisms
on the carrier membranes and on adjacent wipes
was monitored and the levels of reduction with
reference to the number of recoverable microor-
ganisms at time 0 were calculated.
Repeatability and reproducibility
To assess the repeatability of the method, a first
series of two independent tests was carried out on
the same day using the same calibrated microor-
ganisms suspension. Reproducibility was estimated
from a third independent test carried out 1 week
later using a different calibrated suspension. In
this phase of development of the method, all tests
were performed by the same analyst.
228
A. Crémieux et al.
‘Routine’ test for evaluation of preservative
efficacy in wet wipes
After consideration of the results from preliminary
testing, parameters for routine testing were
chosen:
• Seven microorganisms.
• Insertion of the carrier in the centre of the pack
of wipes.
• Evaluation time points of 0, 7, 14 and 28 days.
Evaluation of microbial content on the carrier
only (not on adjacent wipes). This routine test was
performed on type II multi-dose wipes.
Results
Preliminary tests
Microbial content of wet wipes
None of the wipes tested (control wipes and satur-
ated wipes) showed the presence of microorgan-
isms, which suggests that the number of
microorganisms recoverable from four wipes under
the conditions of this test was <10.
Lethal rate of Gram-negative bacteria during prepar-
ation of dry inoculums and on inoculated carriers
inserted into control wipes
The results for P. aeruginosa and E. coli, reported
in Table I, show a high rate of mortality of the
inoculum (N) on the carrier membrane (I).
Although predictable, this loss is not negligible as
it represents about 1–1.8 log per carrier. This
result reveals the need to perform time 0 controls
for each strain and for each type of wipe.
On the other hand, counts performed after 24 h
and up to the 14th day reveal the growth of inocu-
lated microorganisms both on the inoculated carri-
ers and adjacent wipes (>3 · 105 on membranes
and >3 · 106 on wipes). As the survival of microor-
ganisms considered to be fragile is proven under
these test conditions, any reduction in the number
of microorganisms in routine testing of cosmetic
wipes can be attributed to antimicrobial activity.
It should also be noted that because of the
abundant growth observed, the exact level of dry
inoculum recovery could not be established.
Effect on test results of the level of insertion of carrier
in packs of wipes
The results are reported in Table II. In this test,
the lethal rate in the dry inoculum is 60% for
ª 2005 International Journal of Cosmetic Science, 27, 223–236
Efficacy of preservatives in wet wipes A. Crémieux et al.

S. aureus and only 45% for E. coli. The recovery

106
106
106
106
106
106

N, control count for the standard inoculum; I, control count for the dry inoculum; M1, M2, M3, M7, M14, number of recoverable survivors on membranes after 1, 2, 3, 7 and 14 days of contact (in cfu/
rates on the germ carrier at time 0 are 100% for

·
·
·
·
·
·
W14

>3
>3
>3
>3
>3
>3
Table I Variation in the number of Gram-negative bacteria during preparation of dry inoculums and in inoculated carriers inserted into control wipes (without preservative)
S. aureus and 44–61% for E. coli.
After 24 and 48 h, no difference in the number

105
105
105
105
105
105
of survivors, and therefore in the efficacy of preser-

·
·
·
·
·
·
vation, was observed for the inoculated carriers
M14

>3
>3
>3
>3
>3
>3
inserted at the bottom, centre and top of packs of
wipes (see Table II) whereas the sedimentation

106
106
106
106
106
106
profile in the wetting liquid in type I wipes (see
·
·
·
·
·
·
W7

>3
Fig. 1) is significant. Under the conditions of this
>3
>3
>3
>3
>3
test and for this type of wipe, the level of insertion
of the carrier has no effect on the results.
105
105
105
105
105
105
·
·
·
·
·
·
M7

>3
>3
>3
>3
>3
>3
Test parameters affecting the evaluation of
preservative efficacy
106
106
106
106
106
106
·
·
·
·
·
·

For each of the seven strains selected and for the

W3

>3
>3
>3
>3
>3
>3

same batch of type I wipes, we attempted to estab-

membrane); W1, W2, W3, W7, W14, number of recovered microorganisms on wipes after 1, 2, 3, 7 and 14 days (in cfu/four wipes).

lish the various parameters considered to be

105
105
105
105
105
105

important in the course of preliminary tests and to

·
·
·
·
·
·

evaluate the efficacy of the preservative system

M3

>3
>3
>3
>3
>3
>3

used for these wipes.

Results concerning the lethal rate for the micro-
106
106
106
106
106
106

organisms in the course of dry inoculum prepar-

·
·
·
·
·
·
W2

>3
>3
>3
>3
>3
>3

ation are grouped together in Table III. The

number of microorganisms recovered from the car-
105
105
105
105
105
105

riers inserted into wipes at time 0 and after 2, 7,

·
·
·
·
·
·

14 and 28 days are reported in Table IV.

M2

>3
>3
>3
>3
>3
>3

The efficacy of the preservative system at different

incubation times, given as log reduction (R ¼ log
106
106
106
106
106
106

T0 ) log M2,7,14,28 or R¢ ¼ log W0 ) log W2,7,14,28)

·
·
·
·
·
·

for carriers and wipes is reported in Table V. The

W1

>3
>3
>3
>3
>3
>3

following points are clear from these results.

105
105
105
105
105
105

Survival of microorganisms in the course of dry

·
·
·
·
·
·
M1

>3
>3
>3
>3
>3
>3

inoculum preparation
The data in Table III confirm the stability of
104
105
104
105
105
105

Gram-positive bacteria and fungal inoculums

(maximum lethal rate of about 50%) and the vul-
·
·
·
·
·
·
4.0
1.1
4.0
1.5
1.7
1.5

nerability of P. aeruginosa for which lethality is

I

90% (i.e. 1 log). Although the lethal rate found

106
106
106
106
106
106

for E. coli in our tests was similar to that for

·
·
·
·
·
·

Gram-positive bacteria, previous results [15]

2.3
2.3
2.3
2.6
2.6
2.6
N

showed higher susceptibility of this species during

drying. Consequently, systematic controls of the
P. aeruginosa ATCC 9027

rate of survival for each strain in each test should

continue to be performed [unpublished data].
E. coli ATCC 8739

Recovery of viable microorganisms from carrier mem-

Parameters

branes and from wipes in contact with the membranes

In order to evaluate the reduction in the number
of recoverable microorganisms from the carriers

ª 2005 International Journal of Cosmetic Science, 27, 223–236 229

Efficacy of preservatives in wet wipes A. Crémieux et al.

Table II Effect of level of insertion of germ carrier in wipe packs (type I)

T0 M1 M2

Parameters N I Top(1) Middle(2) Bottom(3) Top Middle Bottom Top Middle Bottom

S. aureus 1.6 · 107 6.4 · 106 7.1 · 106 6.0 · 106 6.7 · 106 3.7 · 105 4.1 · 105 3.5 · 105 4.2 · 104 4.1 · 105 7.3 · 104
ATCC 6538
E. coli 2.0 · 107 1.1 · 107 4.4 · 106 6.2 · 106 4.3 · 106 1.4 · 105 1.3 · 105 1.5 · 105 1.3 · 104 6.1 · 103 3.8 · 103
ATCC 8739

N, control count for the standard inoculum; I, control count for the dry inoculum; T0, control for initial recoverable contamination on the
carrier membrane; M1, M2, number of recoverable survivors on carrier membranes after 1 and 2 days of contact (in cfu/membrane).
(1)
Membrane inserted between fourth and the fifth wipe.
(2)
Membrane inserted between 14th and the 15th wipe.
(3)
Membrane inserted between 24th and the 25th wipe.

Table III Number of microorganisms deposited on be considered the rate of initial transfer from the
inoculated carriers and number of survivors in dry carrier to the wipes with which it comes in con-
inoculums tact. This rate is the same for all tested strains.
However, the behaviour of the strains on the carri-
Strains N I ers and on the wipes for the duration of the test
differs (for example, for S. aureus, the ratio W2/
P. aeruginosa ATCC 19429 2.2 · 107 2.6 · 106–2.4 · 106(1) M2 ¼ 10)2; for A. niger, W2/M2 ¼ 1). Neverthe-
2.8 · 107 3.2 · 106(2) less, the preservative activity is demonstrated on
E. coli ATCC 8739 2.3 · 107 1.3 · 107–1.2 · 107
both the carriers and the wipes.
2.3 · 107 1.4 · 107
S. aureus ATCC 6538 2.9 · 107 2.1 · 107–2.1 · 107
In all cases, until the 28th day, when surviving
2.7 · 107 1.9 · 107 microorganisms were detected, they were consis-
E. faecalis ATCC 33186 2.2 · 107 1.2 · 107–1.4 · 107 tently detected on the carrier membranes.
2.1 · 107 1.7 · 107
B. cereus ATCC 33019 7.3 · 106 5.6 · 106–6.0 · 106
3.2 · 107 2.9 · 107 Efficacy of the preservative system in type I wipes
C. albicans ATCC 10231 1.8 · 107 1.0 · 107–1.2 · 107
2.0 · 107 1.5 · 107 The experimental results are reported in Table IV
A. niger ATCC 6275 1.6 · 107 1.2 · 107–1.4 · 107 and the rates of reduction in Table V.
1.3 · 107 9.5 · 106 Reproducibility of the results is within the range
expected for standard challenge test [unpublished
N, control count for standard inoculum; I, control count for dry data] with the exception of B. cereus. For the initial
inoculum.
(1)
two tests, the preservative system exhibited stasis
Results obtained in two tests performed on the same day with
the same calibrated suspensions.
against B. cereus spores. This was not the case in
(2)
Results obtained in a third test performed on a different day the third separate test where it appears that growth
with different calibrated suspensions. on the wipes occurred. This phenomenon could not
be explained.
resulting from scraping during wipe contact (and The efficacy of the preservatives was noted from
not as a result of the effect of preservatives), we day 2 for most strains. With C. albicans and
compared dry inoculum (I) counts with counts for A. niger, differences in efficacy were noted at day 2
the membrane that was in contact with the wipe but disappeared by day 7. For a given test, and
and then recovered immediately (T0 membrane). therefore for a given inoculum, the variation in
The differences noted between I values and T0 val- the number of microorganisms at different times is
ues are minor and appear to be attributable to the consistent and interpretation of results concerning
normal variability of the counts (see Table IV). efficacy of the preservative system is justified. For
The results of counts carried out on wipes (W0) better quantitative evaluation of the results,
are also uniform and represent about 10% of the these discrepancies, which were significant for the
population of the dry inoculum (T0), which may shorter times but subsequently disappeared, could

230 ª 2005 International Journal of Cosmetic Science, 27, 223–236

 Table IV Number of microorganisms counted on membranes (T0 and M) and on wipes (W) at time 0 and after 2, 7, 14 and 28 days

I T0 W0 M2 W2 M7 W7 M14 W14 M28 W28

P. aeruginosa ATCC 19429 2.6 · 106(1) 7.0 · 106 1.3 · 105 1 1 0 1 0 0 0 0

2.4 · 106(1) 8.8 · 106 1.2 · 105 0 1 0 1 0 0 0 0
Efficacy of preservatives in wet wipes

3.2 · 106(2) 5.1 · 106 5.0 · 104 0 0 0 0 0 0 0 0

E. coli ATCC 8739 1.3 · 107(1) 6.0 · 106 2.4 · 105 0 0 0 0 0 0 0 0
1.2 · 107(1) 6.4 · 106 2.4 · 105 0 0 0 0 0 0 0 0
1.4 · 107(2) 1.2 · 107 4.4 · 105 0 0 0 0 0 0 0 0
S. aureus ATCC 6538 2.1 · 107(1) 2.1 · 107 5.4 · 105 2.2 · 104 92 1 3 0 0 0 0
2.1 · 107(1) 2.1 · 107 7.0 · 105 1.1 · 104 57 1 6 0 0 0 0
1.9 · 107(2) 1.8 · 107 1.7 · 105 7.4 · 104 1.4 · 102 0 0 0 0 0 0
E. faecalis ATCC 33186 1.2 · 107(1) 1.4 · 107 1.3 · 105 0 2 1 0 0 0 0 0
1.4 · 107(1) 1.3 · 107 1.9 · 105 4 3 1 0 0 0 0 0
1.7 · 107(2) 1.6 · 107 9.2 · 104 1 0 0 0 0 0 0 0

ª 2005 International Journal of Cosmetic Science, 27, 223–236

B. cereus ATCC 33019 5.6 · 106(1) 3.7 · 106 1.0 · 105 9.2 · 104 1.4 · 104 9.5 · 104 3.8 · 103 4.5 · 104 1.1 · 103 3.4 · 104 0
6.0 · 106(1) 2.8 · 106 5.5 · 104 1.0 · 105 8.2 · 103 1.4 · 105 6.1 · 103 1.2 · 105 1.3 · 103 6.2 · 104 0
2.9 · 107(2) 3.1 · 107 3.0 · 106 >3.0 · 107 >3.0 · 107 >3.0 · 107 >3.0 · 107 >3.0 · 107 >3.0 · 107 >3.0 · 107 >3.0 · 107
C. albicans ATCC 10231 1.0 · 107(1) 1.2 · 107 4.2 · 106 2.0 · 104 2.2 · 104 0 0 0 0 0 0
1.2 · 107(1) 1.7 · 107 4.4 · 106 2.6 · 104 2.2 · 105 0 0 0 0 0 0
1.5 · 107(2) 1.4 · 107 5.1 · 106 1.8 · 106 2.2 · 105 17 0 0 0 0 0
A. niger ATCC 6275 1.2 · 107(1) 12 · 107 2.9 · 106 2.2 · 106 2.8 · 106 42 47 1 0 0 0
1.4 · 107(1) 1.2 · 107 2.3 · 106 >3.0 · 106 >3.0 · 106 8.0 · 103 5.3 · 103 1 0 0 0
9.5 · 106(2) 8.6 · 106 3.0 · 106 2.5 · 106 2.1 · 106 5.1 · 104 1.1 · 104 0 0 0 0

I, control count for dry inoculum; T0, W0, number of recoverable microorganisms at time 0 on membranes and wipes; M2, M7, M14, M28, number of recovered survivors on membranes after 2, 7, 14 and
28 days of contact (in cfu/membrane); W2, W7, W14, W28, number of recovered survivors on wipes after 2, 7, 14 and 28 days (in cfu/four wipes).
(1)
Results obtained in two tests performed on the same day with the same calibrated suspensions.
(2)
Results obtained in a third test performed on a different day with different calibrated suspensions.

231
A. Crémieux et al.
Efficacy of preservatives in wet wipes A. Crémieux et al.

Table V Reduction rates obtained at the level of carrier membranes and type I wipes

Reduction rates in membranes Reduction rates in wipes

R2 R7 R14 R28 R ¢2 R ¢7 R ¢14 R ¢28

P. aeruginosa ATCC 19429 >6.00 >6.00 >6.00 >6.00 >5.00 >5.00 >5.00 >5.00
>6.00 >6.00 >6.00 >6.00 >5.00 >5.00 >5.00 >5.00
>6.00 >6.00 >6.00 >6.00 >4.70 >4.70 >4.70 >4.70
E. coli ATCC 8739 >6.00 >6.00 >6.00 >6.00 >5.00 >5.00 >5.00 >5.00
>6.00 >6.00 >6.00 >6.00 >5.00 >5.00 >5.00 >5.00
>6.00 >6.00 >6.00 >6.00 >5.00 >5.00 >5.00 >5.00
S. aureus ATCC 6538 2.98 >7.00 >7.00 >7.00 3.77 >5.00 >5.00 >5.00
3.28 >7.00 >7.00 >7.00 4.09 >5.00 >5.00 >5.00
2.39 >7.00 >7.00 >7.00 3.08 >5.00 >5.00 >5.00
E. faecalis ATCC 33186 >7.00 >7.00 >7.00 >7.00 >5.00 >5.00 >5.00 >5.00
>7.00 >7.00 >7.00 >7.00 >5.00 >5.00 >5.00 >5.00
>7.00 >7.00 >7.00 >7.00 >4.96 >4.96 >4.96 >4.96
B. cereus ATCC 33019 1.61 1.59 1.92 2.04 0.85 1.42 1.96 >5.00
1.45 1.30 1.37 1.66 0.83 0.95 1.63 >4.74
Increase Increase Increase Increase Increase Increase Increase Increase
C. albicans ATCC 10231 2.78 >7.00 >7.00 >7.00 2.28 >6.00 >6.00 >6.00
2.82 >7.00 >7.00 >7.00 1.30 >6.00 >6.00 >6.00
0.89 5.92 >7.00 >7.00 1.37 >6.00 >6.00 >6.00
A. niger ATCC 6275 0.74 5.46 >7.00 >7.00 0.01 4.79 >6.00 >6.00
Increase 3.18 >7.00 >7.00 Increase 2.64 >6.00 >6.00
0.53 2.22 >7.00 >7.00 0.16 2.44 >6.00 >6.00

R2, R7, R14, R28: R (¼log T0 ) log M) after 2, 7, 14 and 28 days; R ¢2, R ¢7, R ¢14, R ¢28: R ¢ (¼log W0 ) log W) after 2, 7, 14 and 28 days.

have been minimized by using several carriers for
each time and taking into account the average of
two results in calculating the reduction. It was
deemed preferable to evaluate the method on a
wide range of strains rather than multiply the
number of trials for the same strain, in order to
better define the field of application and ensure it
is actually useful.
Given that the method is to be applied to var-
ious types of wipe, the test parameters were modi-
fied to simplify the test:
• Minimum observation time of 7 days, in compli-
ance with evaluation criteria generally used for
preservatives,
• Counts on carriers only, according to the results
reported in Table IV.
However, controls N, I and T0 were retained in
order to verify the validity of the dry inoculums
prepared for each test.
Routine test for evaluation of the efficacy of
preservatives in wet wipes
Application of the ‘routine test’ to four types of
wipes demonstrates its feasibility and its usefulness
in evaluating preservative efficacy. The results are
reported in Table VI.
Among the seven microbial strains tested, only
B. cereus (in the form of spores) showed the ability
to survive after 28 days of incubation in all wipes,
although the survival rate was considerably
reduced in type II-3 and II-4 wipes, suggesting
spore germination and an effect of the preserva-
tives on vegetative cells. With fungi, on the other
hand, type II-1 and, especially, type II-2 wipes
demonstrated faster activity than type II-3 and
II-4 wipes, with the effect being more pronounced
with C. albicans than with A. niger. The efficacy of
preservatives against the four bacterial strains was
more pronounced for type II-2 wipes than for type
II-1, II-3 and II-4 wipes, which allowed the survi-
val of E. faecalis for 7 days. With type II-1 wipes,
four bacterial species were detectable at day 7 and
it was not until day 14 that their numbers fell
below the method’s threshold of detection. The
control values I and T0 are similar to those
observed previously, confirming the significant
lethal rate of P. aeruginosa during the desiccation
phase and the good levels of microorganism recov-
ery in carrier membranes. These results need to be

232 ª 2005 International Journal of Cosmetic Science, 27, 223–236

 Efficacy of preservatives in wet wipes A. Crémieux et al.

compared with those from wetting liquids

I, control count for the dry inoculum; T0, number of microorganisms recoverable on carrier membranes at time 0; M7, M14, M28, number of recoverable survivors on carrier membranes after 7, 14 and 28 days of contact (in cfu/membrane).
2.7 · 106 6.8 · 106 4.6 · 105 3.0 · 106 2.8 · 106 4.5 · 105 8.2 · 105 5.5 · 105 9.6 · 106 9.7 · 105 4.6 · 105 4.2 · 105 2.8 · 104 2.8 · 105 2.6 · 106 9.4 · 104 9.4 · 104 5.6 · 102

2.4 · 106 2.2 · 106 6.6 · 105 2.6 · 105 9.2 · 102

1.9 · 106 3.7 · 106 1.0 · 106 5.1 · 105 6.4 · 103 2.2 · 106 5.2 · 106 9.5 · 105 6.8 · 105 9.8 · 104
(Table VII, Plate 1). Generally speaking, it was

M28

0
0

0
found that wetting liquids show greater antimicro-
bial activity than the corresponding saturated

M14
delivery systems. This difference in behaviour is

0
0

2.6 · 105 3.4 · 105 2.7 · 105 2

particularly marked with fungi: the type II-1 wet-
M7 ting liquid eliminates A. niger in 7 days while type

4.8 · 105 1.5 · 105 0

8.8 · 105 8.0 · 104 0

3.7 · 105 4.5 · 105 0
II-1 wipes need 28 days for this to occur. In the
case of type II-3, A. niger disappears within
T0

14 days but survives until day 28 on the mem-

Wipes II-4

brane inserted into the corresponding wipes.

I

Discussion
M28

0
0

0 The set of results presented above make clear that

the efficacy of preservation must be demonstrated
M14

0
0

1.8 · 106 1.4 · 105 4.7 · 102 0

1.9 · 106 1.8 · 106 2.8 · 104 0

for the impregnated wipes, not the wetting liquids

alone. Challenge tests performed on the wetting
M7

liquids showed, in two out of four cases, much

1.2 · 106 6.8 · 104 0

1.2 · 106 1.8 · 104 0

1.6 · 106 6.2 · 104 0

higher antimicrobial activity than was observed

with the corresponding wet wipes, and the rate of
T0
Wipes II-3

kill was slower, especially in the case of fungi. This

is particularly unacceptable as development of
I

moulds inside packs are a major microbiological

Table VI Changes in the number of microorganisms on membranes inserted into type II wet wipes

risk in this type of product.

M28

The chosen method uses ‘dry’ inoculums depos-

0

0
0

ited on a carrier membrane which is inserted

M14

between the wipes and then removed at a specific

0

0
0

time in order to count surviving organisms. The

use of a carrier membrane improves the standard-
M7

2.2 · 105 6.3 · 105 0

6.4 · 105 9.0 · 105 0

7.5 · 105 5.3 · 105 0

3.7 · 106 2.6 · 106 0

1.8 · 106 2.3 · 106 0

1.2 · 106 1.3 · 106 0

ization of the inoculum step and makes it easier to

recover survivors. Although it is possible to
T0

recover the microorganisms from the wipes, this

Wipes II-2

requires using a special apparatus (Stomacher)

and necessitates adapting the volume of diluent
I

used according to the absorbing power of the

wipes.
M28

0
0

1.8 · 106 4.6 · 102 0

This method makes it possible to measure the

rate of survival of inoculated microorganisms and
M14

to detect differences in preservative efficacy as a

5.6 · 105 0

0
0

1.7 · 103 0

function of the microbial strain and type of wipe

tested. The method involves the systematic use of
M7

3.1 · 106 88
5.6 · 106 4

controls to monitor microorganism survival and

2.1 · 107

5.4 · 106

3.2 · 106

3.7 · 106

3.2 · 106

recovery rates. Control values act as a reference to

T0

establish the reduction due to the preservatives.

Wipes II-1

1.7 · 107

2.0 · 106
6.4 · 106

6.2 · 106

6.4 · 106

2.2 · 106

6.2 · 106

The method was applied to seven microorgan-

ism strains in three series of tests. In spite of
I

anomalies with bacterial spores, repeatability and

E. coli ATCC 8739

B. cereus ATCC
33019 (spores)

reproducibility were satisfactory and similar to

ATCC 19429

ATCC 33186

ATCC 10231
P. aeruginosa

ATCC 6538

ATCC 6275

that obtained in conventional challenge tests

C. albicans
E. faecalis
S. aureus

A. niger

[Schnittger, Fisher & Gruner, unpublished results]

or Pharmacopoeia preservative tests.

ª 2005 International Journal of Cosmetic Science, 27, 223–236 233

 Efficacy of preservatives in wet wipes A. Crémieux et al.

Although previous experiments [15] have led to

8.8 · 105
the standardization of inoculums, several protocols

<200

<200
<200
<200

<200

<200
M28
had to be evaluated in order to develop the pro-

1.2 · 106
posed methodology and determine the test param-

<200

<200
<200
<200

<200

2.6 · 106 4.0 · 102 <200

eters, regardless of the type of wipe. These
M14

parameters are:

1.8 · 106
• Initial level of wipe contamination.
2.2 · 106 <200

<200
<200
<200

3.7 · 106 <200

• Control of microorganism survival in the dry
M7

inoculum.
Wipes II-4

106
106
106
106
• Recovery rate of microorganisms from mem-
·
·
·
·

T0, number of surviving microorganisms recoverable at time 0 (in CFU mL)1); M7, M14, M28, number of survivors after 7, 14 and 28 days/contact (in CFU mL)1).
branes at time zero.
1.8
1.1
2.2
3.4
T0

• Transfer rate of microorganisms from the carrier

1.0 · 106

membrane in contact with the wipes.

<200

<200
<200
<200

1.6 · 106 6.4 · 103 2.8 · 103 <200

<200
M28

• Preservative activity on both wipes and carriers.

Insofar as survivors remain, they are always
1.8 · 106

detected on the membrane. It was decided that for

<200

<200
<200
<200

1.8 · 106 2.8 · 103 <200

M14

the routine test only this membrane would be tes-

2.0 · 103
1.4 · 106

ted, which considerably simplifies the analysis to

be performed.
1.8 · 106 <200

<200
<200
M7

All other parameters, such as the duration of

Wipes II-3

106
106
106
106

tests, contact times, choice of strains, insertion

·
·
·
·

level of carrier and number of test repetitions or

3.9
1.6
2.6
1.5
T0

replications, can be adapted as a function of the

1.4 · 106

required objectives, according to the nature and/or

<200

<200
<200
<200

<200

<200

expected usage of wipes. In all cases, the test must

M28

be adapted to deal with the principal risks for

1.2 · 106

users and for the manufacturer in the event of

<200

<200
<200
<200

<200

<200
M14

shortcomings in the preservation process.

Table VII Results of challenge tests performed on saturation liquids in type II wipes

All types of wipe used in this study were tes-

1.1 · 106

ted for initial bioburden and found to be clean.

2.2 · 106 <200

<200
<200
<200

2.1 · 106 <200

2.4 · 106 <200

M7

In other cases resident microorganisms could be

Wipes II-2

present in sufficient numbers to disrupt the test.

106
106
106
106

Therefore, practicing the preliminary test is justi-

·
·
·
·
3.2
1.8
2.8
1.4
T0

fied to avoid performing a long and costly test

4.6 · 105

which would show a lack of preservative

efficacy.
<200

<200
<200
<200

<200

<200
M

Many new types of wipes are being developed in

6.8 · 106

the cosmetic and pharmaceutical industries, and

<200

<200
<200
<200

<200

<200

for domestic and industrial use. Other adaptations

M14

of the protocol may be considered to assess not

2.7 · 106

only the efficacy of preservatives but also the

2.7 · 106 <200

<200
<200
<200

2.7 · 106 <200

1.8 · 106 <200

microbial efficacy of antiseptics and disinfectants.

M7

The nature and titre of inoculums and the mode

Wipes II-1

106
106
106
106

of action (static or lethal effects), as well as the

·
·
·
·
1.9
2.2
2.0
5.0

required reduction criteria, should be defined in

T0

order to justify and validate protocols.

E. faecalis ATCC 33186
S. aureus ATCC 6538

A. niger ATCC 6275

E. coli ATCC 8739

B. cereus ATCC
33019 (spores)

Conclusion
ATCC 19429

ATCC 10231
P. aeruginosa

C. albicans

The method proposed for evaluation of the efficacy

of preservative systems in wet wipes makes use of
dried inoculums on a carrier membrane. It util-

234 ª 2005 International Journal of Cosmetic Science, 27, 223–236

Efficacy of preservatives in wet wipes A. Crémieux et al.

Type II-1 wet wipes Type II-1wetting liquid

Number of CFU/membrane
1.0E + 07 1.0E + 07

–1
Number of CFU ml
1.0E + 06 1.0E + 06

1.0E + 05 P.aruginosa
1.0E + 05 P.aruginosa
E.coli E.coli
S.aureus
1.0E + 04 S.aureus
E.faecalis
1.0E + 04 E.faecalis
B.cereus B.cereus
C.albicans
1.0E + 03 C.albicans 1.0E + 03 A.niger
A.niger

1.0E + 02 1.0E + 02

1.0E + 01 1.0E + 01
0 7 14 21 28 0 7 14 21 28
Time (days) Time (days)

Type II-2 wet wipes Type II-2 wetting liquid

Number of CFU/membrane

1.0E + 07 1.0E + 07

–1
1.0E + 06

Number of CFU ml
1.0E + 06

1.0E + 05 P.aruginosa 1.0E + 05 P.aruginosa

E.coli E.coli
S.aureus S.aureus

1.0E + 04 E.faecalis 1.0E + 04 E.faecalis

B.cereus
B.cereus C.albicans
C.albicans A.niger
1.0E + 03 A.niger 1.0E + 03

1.0E + 02 1.0E + 02

1.0E + 01 1.0E + 01
0 7 14 21 28 0 7 14 21 28
Time (days) Time (days)

Type II-3 wet wipes Type II-3 wetting liquid

Number of CFU/membrane

1.0E + 07 1.0E + 07
–1

1.0E + 06
Number of CFU ml

1.0E + 06

1.0E + 05 1.0E + 05 P.aruginosa

E.coli
P.aruginosa S.aureus

1.0E + 04 E.coli
1.0E + 04 E.faecalis
B.cereus
S.aureus
C.albicans
E.faecalis A.niger
1.0E + 03 B.cereus
1.0E + 03
C.albicans
A.niger
1.0E + 02 1.0E + 02

1.0E + 01 1.0E + 01
0 7 14 21 28 0 7 14 21 28
Time (days) Time (days)

Plate 1 Comparison of results obtained for wet wipes by the specific challenge test with results obtained for the corres-
ponding wetting liquids using standard challenge test.

izes bacteria (P. aeruginosa, E. coli, S. aureus and
E. faecalis), bacterial spores (B. cereus) and fungi
(C. albicans and A. niger). The feasibility of the
method for various types of wipe has been demon-
strated. The results obtained demonstrated differ-
ent levels of preservative efficacy which vary as a
function of strain, incubation time and type of
wipe. The repeatability and reproducibility of the
results are similar to those for challenge tests des-
cribed in the Pharmacopoeias.
Results of this study showed that the lethal rate is
slower on impregnated wipes than in corresponding
wetting liquids tested by the standard challenge test.
Consequently, using a challenge test adapted to wet
wipes appears to be essential within the context of
developing this type of product.
Moreover, our results prove that the method
may be applicable to all types of microorganism
and therefore to any microorganism of interest in
the cosmetic industry as long as the production of

ª 2005 International Journal of Cosmetic Science, 27, 223–236 235

Efficacy of preservatives in wet wipes
a dry inoculum is controlled. This methodology
may therefore also be applied, with the appropriate
adaptation of various parameters, to the evalua-
tion of antimicrobial activity in wipes or other sim-
ilar materials.
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ª 2005 International Journal of Cosmetic Science, 27, 223–236