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

were dissociated from the wipes by means of a Sto-

Synopsis
macher. The supernatants recovered after being
Many cosmetic formulations are now available in left to stand for 20 min are counted by pour plate
the form of wet wipes packaged in sealed sachets method or membrane filtration. The feasibility of
or packets. Like the majority of cosmetic products the method was demonstrated for each of the
having an aqueous phase, wipes are susceptible to seven above-mentioned strains. The repeatability
microbial contamination and require the addition and reproducibility of the results obtained is sim-
of preservatives. The efficacy of such preservatives ilar to that obtained for preservative efficacy tests
can be evaluated using a standard challenge test in the Pharmacopoeias. The lethal rate of microor-
performed on the wetting liquid but this test can- ganisms during the preparation of dry inoculums
not be regarded as representative for this new type ranges from 50 to 90% depending on the strain
of formulation. The method presented here evalu- and the test (generally, a spontaneous reduction of
ates the efficacy of preservatives used in wet wipes about 1 log up to a maximum of 2 log). The
kept in their original packaging. Dried inoculums recovery rate for microorganisms from dry inocu-
were prepared by membrane filtration followed by lums (on membranes) at time 0 (control ¼ T0) is
drying in an incubator. The method is applicable around 90%, regardless of the strain or the test.
to bacteria (Pseudomonas aeruginosa, Escherichia The number of microorganisms recovered from the
coli, Staphylococcus aureus and Enterococcus faecalis), wipes (W0) is between 2 and 10% of the number
Bacillus cereus spores and fungi (Candida albicans recovered from membranes (T0) and may be con-
and Aspergillus niger). These inoculated carriers sidered negligible. Application of this method to
were inserted between two wipes in the original different types of wipes demonstrates that the effic-
package, which was then re-sealed immediately. acy of preservatives, expressed as the logarithmic
The test requires one dry inoculum per packet and reduction in the number of microorganisms at
one packet for each control or test. After incuba- each time point, depends on the type of wipe and
tion at 22.5
C for 1, 2, 7, 14 or 28 days and, for on the strain tested. The results obtained are con-
the control, immediately after insertion of the siderably different from those found with the
membrane (time 0), microorganism counts were standard challenge tests applied to wetting liquids
performed on the germ-carrier membranes as well for wipes. The differences found confirm the need
as on adjacent wipes, after transfer into a suitable for a specific method applicable to wipes.
neutralizing agent. The membranes were shaken
in the presence of glass beads and microorganisms
Résumé
De nombreuses formulations cosmétiques sont
Correspondence: S. Cupferman, L’Oréal Recherche, 94550 maintenant disponibles sous forme de lingettes imp-
Chevilly-Larue, France. E-mail: [email protected] régnées emballées dans des sachets ou pochettes

ª 2005 Society of Cosmetic Scientists and the Société Française de Cosmétologie 223
Efficacy of preservatives in wet wipes A. Crémieux et al.

étanches. Comme la majorité des produits cosmé- fonction du temps, dépend du type de lingette et
tiques renfermant une phase aqueuse, les lingettes de la souche testée. Les résultats obtenus sont
sont sensibles à la contamination microbienne et sensiblement différents de ceux provenant des chal-
nécessitent, de ce fait, la présence de conservateurs. lenge-tests classiques appliqués aux liquides d’imp-
L’efficacité de ces conservateurs peut être évaluée régnation des lingettes et les différences observées
par un challenge test classique portant sur le liquide montrent l’intérêt d’une méthode spécifique applic-
d’imprégnation, mais cet essai ne peut être consid- able aux lingettes.
éré comme représentatif de ces nouvelles présenta-
tions. La méthode décrite ici évalue l’efficacité des
Introduction
conservateurs au sein de lingettes imprégnées
maintenues dans leur emballage d’origine. Des ino- Cosmetic wipes, which originally appeared on the
culums desséchés sont préparés par filtration sur market some 20 years ago as baby hygiene
membrane et séchage à l’étuve. La mèthode est products, are now widely used in a variety of
applicable aux bactéries (Pseudomonas aeruginosa, applications, such as for face and eye cleansing
Escherichia coli, Staphylococcus aureus et Enterococcus and make-up removal, treatment of oily skin, deo-
faecalis) aux spores de Bacillus cereus et aux fongi dorants, aftershaves, sun protection and self-tan-
(Candida albicans et Aspergillus niger). Ces porte ger- ning lotions.
mes sont introduits entre deux lingettes dans leur They consist of two main components: an absor-
emballage d’origine qui est immédiatement refermé. bent delivery system and a cosmetic formulation
L’essai nécessite au minimum un inoculum sec par to saturate the delivery system. The absorbent
emballage et un emballage pour chaque essai ou delivery system is most commonly fibre-based but
témoin. Après 1, 2, 7, 14, ou 28 jours d’incubation other materials, especially foams, can also be used.
à 22.5
C, et, pour le témoin, immédiatement après The fibrous delivery system is generally non-
insertion de la membrane (temps 0), les dénombre- woven and consists of a mixture of viscose and
ments de microorganismes sont réalisés sur les polyethylene-tetraphthalate (PET) fibres. Mixtures
membranes porte germes et sur les lingettes adja- containing cotton or cellulose fibres also exist.
centes après transfert dans un neutralisant validé. Wet cosmetic formulations in non-woven sys-
Les membranes y sont agitées en présence de billes tems can be aqueous, hydro-alcoholic, an emul-
de verre et les lingettes sont dissociées à l’aide d’un sion or oil. There is no limitation with regard to
Stomacher. Les surnageants récupérés après the composition of products with the exception of
20 min de repos sont dénombrés par inclusion en viscosity, a key parameter. For acceptable satura-
boite de Pétri ou par filtration sur membrane. La fai- tion to be obtained, in other words uniform distri-
sabilité de la méthode a été démontrée pour chac- bution throughout the delivery system, the
une des sept souches citées ci-dessus. La répétabilité cosmetic composition must be a low-viscosity
et la reproductibilité des résultats obtenus sont liquid. In fact, practically all industrial processes
semblables à celles que l’on observe dans les essais now consist of spraying the cosmetic product onto
d’efficacité des conservateurs décrits dans les Phar- the non-woven system or soaking it in a bath.
macopées. Le taux de mortalité des microorganis- The most commonly applied rate of saturation is
mes pendant la préparation des inoculums secs 300–400%, which is 3–4 g of liquid per gram of
varie de 50% à 90% (réduction spontanée en géné- delivery system. With these saturation rates, a
ral voisine de 1 log, au maximum 2 log) selon la liquid sedimentation phenomenon is often observed
souche et l’essai. Le taux de recouvrement des in the case of multi-dose products. While wipes are
microorganismes constituant les inoculums secs sometimes presented in single sachets, most wipes
(sur membranes) au temps 0 (témoins ¼ T0) est vo- on the market are sold in packs containing 20–25
isin de 90% quelque soit la souche et l’essai. Le wipes, and up to 80 wipes for baby products. They
nombre de microorganismes récupérés des lingettes are either folded or stacked in the packet or criss-
(W0) est compris entre 2% et 10% de celui trouvé crossed so that the packet is always ready to be used
sur les membranes (T0) et il peut être considéré after the first wipe is removed. The packaging
comme négligeable. L’application de cette méthode material is usually plastic (polyethylene/polyester)
à différents types de lingettes montre que l’effica- or a plastic and aluminium complex.
cité des conservateurs, exprimée par la réduction As for the majority of cosmetic products, multi-
logarithmique du nombre de microorganismes en dose wipes saturated with a cosmetic formulation

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

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

including an aqueous phase are susceptible to encounter during manufacturing process and use
microbial contamination. Preservatives are gener- were therefore chosen for this test: Escherichia coli,
ally needed to maintain constant microbiological Pseudomonas aeruginosa, Enterococcus faecalis,
quality during manufacture and storage and until Staphylococcus aureus, B. cereus (spores), Candida
the wipes are used. Suitable molecules for antimi- albicans and A. niger (spores).
crobial protection of cosmetic products are listed in The principle of the method was based on the
Appendix 6 of the European Cosmetics Directive, use of inoculums dried on a membrane which acts
which also specifies the maximum level of use [1]. as a carrier. Wipes were inoculated by the carrier
The efficacy of preservatives depends on a num- and the development of contamination was monit-
ber of factors. Efficacy cannot be estimated solely ored both on the carrier and on the wipes in con-
on the basis of data in the literature (ex. M.I.C.) tact with it to take into account all surviving
but must be checked using standard methods such microorganisms, regardless of the rate of transfer
as the challenge test [2–5]. The challenge test is from the carrier to the wipes.
based on the principle of artificial contamination Preliminary tests were performed to study the
of the product to be tested by different microbial survival of the most fragile microorganisms (bac-
strains. The level of contamination is then monit- teria) on the carrier inserted into wipes impregna-
ored at several time intervals. During the first step ted with diluent and to define, under real
of the test, the microbial inoculum in liquid form conditions, the parameters to be retained for the
is carefully mixed with the sample. The protocol evaluation of antimicrobial activity at various crit-
was originally designed to test cosmetic or phar- ical times. The results obtained made it possible to
maceutical products in liquid, fluid or semi-solid identify a trial protocol to validate the method and
form but is difficult to apply directly to wet wipes. then simplify the protocol so that it could be easily
However, it is important to evaluate the efficacy of used in routine tests.
preservation of these products in their final form
rather than simply testing the wetting liquid. It is
Materials and methods
likely that microbial growth, and especially mould
growth, is not the same in a liquid and at the sur-
Microbial strains
face of a saturated delivery system.
Various studies published in the literature reveal The strains used are collection strains stored at
the advantages of techniques which allow micro- )74
C in the laboratory under the conditions laid
bial growth to be monitored over the course of out in standard EN 12353 [13]. Inoculums were
time [6–8] or methods which focus in particular prepared from working cultures where the number
on studying materials such as paper or textiles of transfers from the frozen batch was strictly lim-
[3, 5, 9–11]. However, they do not cover adapta- ited to 4. The reference strains were: P. aeruginosa
tion of the challenge test to the evaluation of anti- (ATCC 19429), E. coli (ATCC 8739), S. aureus
microbial protection in wipes and other saturated (ATCC 6538), E. faecalis (ATCC 33186), B. cereus
cosmetic delivery systems. This was the purpose of (ATCC 33019), C. albicans (ATCC 10231) and
the work reported in this paper. A. niger (ATCC 6275).
The main source of contamination of cosmetic
wipes is the non-woven delivery system, through
Wipes
the presence of bacterial and fungal spores [12].
These organisms are represented in the present Several types of non-woven wipes packaged in a
method by Bacillus cereus spores and Aspergillus flexible sachet and saturated with oil in water emul-
niger spores. Moreover, as the manufacture of cos- sions containing preservatives were used in the
metic products is not, with the exception of a few study. They differed in terms of size, wetting liquid
cases, performed under sterile conditions, acciden- and packaging. They were either commercially
tal contamination by species from the production available products or products being developed.
environment, especially from water, is possible.
Finally, as these products come into contact with Type I wipes
users’ skin, they should have a degree of antimi- Packs of 25 make-up removal wipes containing
crobial protection against skin pathogens. Microor- phenoxyethanol and parabens. The sedimentation
ganisms representing those which the wipes may profile for the wetting liquid in type I wipes was

ª 2005 International Journal of Cosmetic Science, 27, 223–236 225

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

9.5 extract agar and manganese sulphate agar were

9
prepared by the laboratory using dehydrated bases
(Difco, Becton Dickinson, Le Pont de Claix, France)
8.5
or supplied ready-to-use (Biomérieux, Marcy l’Eto-
8 ile, France). Diluents for microbial suspensions
were: tryptone–salt solution (tryptone, 0.1%; NaCl,
7.5
0.9%; w/v) for bacteria and yeasts and tryptone–
7 salt solution with polysorbate 80 (0.05%; v/v) for
moulds.
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 The desiccation protector was a powdered skim-
med milk solution in sterile water (10% w/v). The
Figure 1 Sedimentation profile of type I wipes. Packs
diluent-neutralizer was Eugon LT 100 medium
(three) of wipes were stored flat for 48 h. The wipes were
then weighed separately, starting from the top of the
prepared by the laboratory using dehydrated base
pack (wipe 1) to the bottom (wipe 25). The graph gives (A.E.S., Combourg, France).
the mean weight values for three wipe packs.
Apparatus
determined by weighing three packs and is repor-
ted in Fig. 1. The method requires the use of a Stomacher (Bag-
Mixer, Interscience, Saint Nom la Bretêche,
Type II wipes France) for recovery of microorganisms from the
Type II-1: packs of 25 aftershave wipes containing wipes, a heat-sealer (Wuhrlin-Soplamed, AMIS,
chlorhexidine digluconate and parabens. Trilport, France), a borosilicate glass filtration
Type II-2: packs of 72 baby wipes containing apparatus (Millipore, Bedford, MA, USA), 250-mL
phenoxyethanol, parabens and potassium sorbate. wide-neck Erlenmeyer flasks and glass beads
Type II-3: packs of 80 baby wipes containing (Ø 3 mm).
imidazolidinyl urea and parabens.
Type II-4: packs of 25 make-up removal wipes
Procedure
containing quaternium 15 and parabens.
Each wetting liquid corresponding to type II wipes
Preparation of inoculated carriers
underwent a challenge test to evaluate the efficacy
of preservation at day 7, 14 and 28 (see Table VII). Inoculums were prepared from two successive sub-
Also included in the test were non-woven wipes cultures on TSA at 24-h intervals for bacteria. For
– 25 per flexible sachet – impregnated with a ster- C. albicans the inoculum was prepared from a sin-
ile diluent (tryptone–salt solution) [control wipes]. 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
Inoculated carriers
obtained from a single 8–10-day subculture on
Sterile cellulose ester membranes, diameter 47 mm, manganese sulphate agar. Following collection
porosity 0.45 lm (GN-6 metricel S-Pack; Pall Life and heat treatment for 10 min at 80
C, the spores
Science, New York, NY, USA). Membranes were were centrifuged and washed and then resuspend-
inoculated by filtration of about 10 mL of diluent ed in physiological saline and stored at 4
C until
containing 1 mL of standard inoculum protected by use. All strains were incubated at 36 ± 1
C.
milk then dried in Petri dishes placed in a ventilated For bacteria and the yeast, the culture collected
incubator for 15–20 min (see Procedure). The carri- from the surface of the agar medium using glass
ers are used for contamination of the wipes and for beads was diluted in a tryptone–salt solution and
easy recovery and counting of surviving microor- the optical density adjusted using a spectropho-
ganisms. 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.
Media and reagents
The A. niger spore suspension diluted in tryptone
Trypcase soya agar (TSA) and Sabouraud dextrose salt solution with polysorbate 80 was filtered on
agar (with and without Chloramphenicol), malt sintered glass and adjusted to 1–3 · 107 cfu mL)1

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

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

using a counting device (e.g. Malassez) (cf. EN inclusion in TSA agar for bacteria and Sabou-
1275 [14]). raud dextrose agar for fungi.
Adjusted suspensions were protected from desic-
cation by the addition of a protector (rehydrated Recovery and counting of microorganisms on wipes
powdered milk) at a rate of 0.5 mL of milk per Two wipes on either side of the previously extrac-
9.5 mL of adjusted suspension [15]. The adjusted ted membrane (four wipes in total) were trans-
and protected suspensions were filtered (1 mL dilu- ferred to a Stomacher bag containing 100 mL of
ted in about 10 mL of diluent) on membranes Eugon LT 100 broth. After stomaching for 10 min
placed in Petri dishes and left half-open for before being left to stand for 20 min at room tem-
15–20 min in a ventilated incubator at 36
C until perature, the supernatant was recovered and the
completely dry. microorganisms counted in agar medium as above
Counting of the adjusted suspension (N) and the and by filtering the remaining liquid (
90 mL) by
dry inoculum (I) was carried out in order to deter- membrane filtration (porosity 0.45 lm).
mine mortality caused by desiccation.
Test parameters
Inoculation of wipes and incubation
The various experimental values obtained over the
Inoculated membranes were inserted between the course of these trials were:
wipes at a rate of one or more membrane per pack N: control count for the calibrated inoculum
and maintained in the pack until counting (at 1, (»108 cfu mL)1 for bacteria, »107 cfu mL)1 for
2, 7, 14 or 28 days). Wipes were kept in their ori- fungi).
ginal packaging which was then resealed (heat- I: control count for the dry inoculum (microbial
sealed). load introduced into the wipe by the carrier).
Each test required the preparation of a number T0: initial contamination control (number of
of contaminated wipe packs equivalent to the recoverable microorganisms at time 0 from the
number of exposure times, with an additional pack carrier membrane inserted into the wipes).
acting as a control. Contact between the inocula- W0: number of microorganisms recovered from
ted carriers and wipes was maintained for 1, 2, four wipes in immediate contact with the carrier
7, 14 and/or 28 days at a temperature of at time 0.
22.5 ± 2.5
C depending on the purpose of each M1, M2, M3, M7, M14, M28: number of recover-
particular test. 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
Controls
of recovered microorganisms from the four wipes
The control (T0) was performed according to the (in immediate contact with the carrier) after 1, 2,
same procedure except that the contaminated 3, 7, 14 and 28 days (in cfu/four wipes).
membrane was removed immediately (time
£2 min) after its insertion into the wipe pack.
Preliminary tests
These tests assessed the feasibility of the method
Recovery and counting of inoculated
and evaluated the conditions for artificial contam-
microorganisms
ination of wipes by dry inoculums.
Recovery and counting of microorganisms on carrier
membranes
Microbiological content of wet wipes and control
Membranes removed with tweezers were placed
wipes
in an Erlenmeyer flask containing 100 mL of
Eugon LT 100 broth and glass beads (5 g). After Four wipes were removed from the middle of the
stirring for 3 min, 1 mL of broth was removed pack and placed in 100 mL of neutralizing diluent
and diluted to 1:10 (1:100 for the control at and stomached for 10 min. After being left to
t ¼ 0) in Eugon LT 100. For counting, 1 mL of stand for 20 min, the supernatant was recovered
the 1:10 dilution and 1 and 0.1 mL of the and total aerobic microorganisms (bacteria, yeasts,
1:100 dilution were carried out (in duplicate) by moulds) were counted.

ª 2005 International Journal of Cosmetic Science, 27, 223–236 227

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

Lethal rate of Gram-negative bacteria during ‘Routine’ test for evaluation of preservative
preparation of dry inoculums and in inoculated efficacy in wet wipes
carriers inserted into control wipes
After consideration of the results from preliminary
The test was limited to two strains of Gram-negat- testing, parameters for routine testing were
ive bacteria known to be sensitive to desiccation. chosen:
It was performed on control wipes saturated in a • Seven microorganisms.
sterile diluent. The test was repeated three times. • Insertion of the carrier in the centre of the pack
Counting of surviving bacteria was performed at of wipes.
day 1, 2, 3, 7 and 14 on both the carrier mem- • Evaluation time points of 0, 7, 14 and 28 days.
branes and the wipes. Evaluation of microbial content on the carrier
only (not on adjacent wipes). This routine test was
performed on type II multi-dose wipes.
Effect on test results of the level of insertion of
carrier in packs of wipes
Results
Taking into account the sedimentation of the wet-
ting liquid in packs of wipes (see Fig. 1), varying
Preliminary tests
degrees of preservative efficacy can be observed. In
type I wipes, three carriers were inserted between Microbial content of wet wipes
the 4th and 5th, 14th and 15th, and 24th and None of the wipes tested (control wipes and satur-
25th wipes. The test was performed with two bac- ated wipes) showed the presence of microorgan-
terial strains (E. coli and S. aureus) and for short isms, which suggests that the number of
exposure times (24 and 48 h). microorganisms recoverable from four wipes under
the conditions of this test was <10.
Test parameters involved in the evaluation of
Lethal rate of Gram-negative bacteria during prepar-
preservative efficacy
ation of dry inoculums and on inoculated carriers
The test was conducted on the seven microbial inserted into control wipes
strains selected using type I wipes inoculated by The results for P. aeruginosa and E. coli, reported
insertion of the carrier in the centre of the pack of in Table I, show a high rate of mortality of the
wipes. 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
Monitoring the level of microorganisms in the
result reveals the need to perform time 0 controls
contaminated wipes over time
for each strain and for each type of wipe.
The number of viable cells was counted at day 0, On the other hand, counts performed after 24 h
2, 7, 14 and 28. The number of microorganisms and up to the 14th day reveal the growth of inocu-
on the carrier membranes and on adjacent wipes lated microorganisms both on the inoculated carri-
was monitored and the levels of reduction with ers and adjacent wipes (>3 · 105 on membranes
reference to the number of recoverable microor- and >3 · 106 on wipes). As the survival of microor-
ganisms at time 0 were calculated. ganisms considered to be fragile is proven under
these test conditions, any reduction in the number
of microorganisms in routine testing of cosmetic
Repeatability and reproducibility
wipes can be attributed to antimicrobial activity.
To assess the repeatability of the method, a first It should also be noted that because of the
series of two independent tests was carried out on abundant growth observed, the exact level of dry
the same day using the same calibrated microor- inoculum recovery could not be established.
ganisms suspension. Reproducibility was estimated
from a third independent test carried out 1 week Effect on test results of the level of insertion of carrier
later using a different calibrated suspension. In in packs of wipes
this phase of development of the method, all tests The results are reported in Table II. In this test,
were performed by the same analyst. the lethal rate in the dry inoculum is 60% for

228 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 in evaluating preservative efficacy. The results are
each time and taking into account the average of reported in Table VI.
two results in calculating the reduction. It was Among the seven microbial strains tested, only
deemed preferable to evaluate the method on a B. cereus (in the form of spores) showed the ability
wide range of strains rather than multiply the to survive after 28 days of incubation in all wipes,
number of trials for the same strain, in order to although the survival rate was considerably
better define the field of application and ensure it reduced in type II-3 and II-4 wipes, suggesting
is actually useful. spore germination and an effect of the preserva-
Given that the method is to be applied to var- tives on vegetative cells. With fungi, on the other
ious types of wipe, the test parameters were modi- hand, type II-1 and, especially, type II-2 wipes
fied to simplify the test: demonstrated faster activity than type II-3 and
• Minimum observation time of 7 days, in compli- II-4 wipes, with the effect being more pronounced
ance with evaluation criteria generally used for with C. albicans than with A. niger. The efficacy of
preservatives, preservatives against the four bacterial strains was
• Counts on carriers only, according to the results more pronounced for type II-2 wipes than for type
reported in Table IV. II-1, II-3 and II-4 wipes, which allowed the survi-
However, controls N, I and T0 were retained in val of E. faecalis for 7 days. With type II-1 wipes,
order to verify the validity of the dry inoculums four bacterial species were detectable at day 7 and
prepared for each test. 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
Routine test for evaluation of the efficacy of
observed previously, confirming the significant
preservatives in wet wipes
lethal rate of P. aeruginosa during the desiccation
Application of the ‘routine test’ to four types of phase and the good levels of microorganism recov-
wipes demonstrates its feasibility and its usefulness 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 Results of this study showed that the lethal rate is
E. faecalis), bacterial spores (B. cereus) and fungi slower on impregnated wipes than in corresponding
(C. albicans and A. niger). The feasibility of the wetting liquids tested by the standard challenge test.
method for various types of wipe has been demon- Consequently, using a challenge test adapted to wet
strated. The results obtained demonstrated differ- wipes appears to be essential within the context of
ent levels of preservative efficacy which vary as a developing this type of product.
function of strain, incubation time and type of Moreover, our results prove that the method
wipe. The repeatability and reproducibility of the may be applicable to all types of microorganism
results are similar to those for challenge tests des- and therefore to any microorganism of interest in
cribed in the Pharmacopoeias. 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. Crémieux et al.

a dry inoculum is controlled. This methodology 8. Lrasfjk, D. Performance standards for antimicrobial
may therefore also be applied, with the appropriate disk susceptibility tests. Approved Standard, 8th edn,
adaptation of various parameters, to the evalua- Vol. 23, no. 1, NCCLS M2-A8 (2003).
tion of antimicrobial activity in wipes or other sim- 9. Microbial contamination of products not required to
comply with the test for sterility (total viable aerobic
ilar materials.
count). In: European Pharmacopoeia, 4th edn.
2.6.12 (2001).
References 10. Standard test method for preservatives in water-con-
taining cosmetics. ASTM Standard test method E
1. European Cosmetics Directive 76/768/CEE. Available 640-78 (1998).
at: http://europa.eu.int/eur-lex/lex/hexUriServ/LexUri 11. Antibacterial finishes on textile materials, American
Serv.do?uri=CELEX:31976L0768:EN:HTML Association of Textile chemists and Colorists. AATCC
2. Efficacy of antimicrobial preservation. In: European Test Method 100 (1989).
Pharmacopoeia, 4th edn (2001). 12. Itoe, R.D. and Wright, J. Avoiding contamination of
3. Antimicrobial preservatives effectiveness. In: US wet wipes. Personal care (2003).
Pharmacopoeia, 26th edn, 51 (2003). 13. EN 12353 – Chemical disinfectants and antiseptics –
4. Torres-Anjel, M.J., Gouzalez, G.H., Giraldo, M.E. et al. preservation of microbial strains used for the deter-
Enterotoxigenecity of Bacillus cereus. Rev. Latinoam. mination of bactericidal and fungicidal activity. Euro-
Microbiol. 21:5–10 (1979). pean Standard (1999).
5. Methods of test for determination of the resistance of 14. EN 1275 – Chemical disinfectants and antiseptics –
textiles to microbiological deterioration. British Basic fungicidal activity – Test method and require-
Standard BS 6085 (1981). ments (phase 1 test) (1997).
6. Standard test methods for mildew (fungus) resistance 15. Eyraud, A. Preparation of dry inoculums aimed at
of paper and paperboard. ASTM Standard test testing antiseptic activity Obtention d’inoculums dés-
method D 2020-92 (1999). séchés pour évaluer l’activité de certains antisep-
7. Connolly, P. et al. The use of impedance for preser- tiques. DES de Sciences Biologiques. Université de
vative efficacy testing of pharmaceutical and cos- Provence, Marseille (1980).
metic products. J. Appl. Bacteriol. 76:68–74 (1994).

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