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W. Siegert:
ISO 11930 –
A Comparison to
other Methods to Evaluate the
Efficacy of Antimicrobial
Preservation
7-2012
English Edition
International Journal for Applied Science
• Personal Care • Detergents • Specialties
44 SOF W-Journal | 138 | 7-2012
COSMETICS
PRESERVATION
•Ph. Eur. 7 - 5.1.3 »Efficacy of Antimi-
crobial Preservation«(3)
•USP 35 »Antimicrobial Effectiveness
Testing« (4)
•ASEAN ACM MAL 08 »Preservative Ef-
ficacy Test for Cosmetic Product« (5)
•CTFA M-3 »A Method for Preservation
Testing of Water-Miscible Personal
Care Products« (6)
•CTFA M-4 »Method for Preservation
Testing of Eye Area Cosmetics« (6).
W. Siegert*
ISO 11930 – A Comparison to other Methods
to Evaluate the Efficacy of Antimicrobial
Preservation
■Legal Regulations
Only in the SCCS notes of guidance, the
demands for challenge testing are speci-
fied. In paragraph 4-4 »Guidelines on Mi-
crobiological of the Finished Cosmetic
Product« in the 7th revision (December
2010) (7) of the SCCS publication »Notes
of Guidance for the Testing of Cosmetic
Ingredients and their Safety Evaluation«
is an obligation for carrying out a preser-
vation test with all cosmetic products
which could be contaminated under
normal storage and usage conditions or
if an infection risk for the consumer ex-
ists.
The use of Pseudomonas aeruginosa,
Staphylococcus aureus and Candida al-
bicans from official collection strains
from any state within the EU is obliga-
tory. In addition, the use of specific germs,
which are known to lead to spoilage of
cosmetic products, is recommended.
■ISO 11930
Compared to other methods ISO 11930 is
an overall standard to evaluate the anti-
microbial stabilisation of a cosmetic prod-
uct.
With the decision diagram in Annex
A also formulations intrinsically hostile
to microbial growth, specific manufac-
turing conditions, packaging and/or con-
ditions
for use and the reference to ISO
29261 (Guidelines for the risk assess-
ment and identification of microbiologi-
cally low-risk products) (7) are covered.
Nevertheless, the preservative efficacy
test described in ISO 11930 is primarily
designed for water-soluble or water-mis-
cible
products.
This publication compares the following
test methods:
•ISO 11930 »Evaluation of the antimi-
crobial protection of a cosmetic prod-
uct« (1)
•SCCS »Notes of Guidance for the Test-
ing of Cosmetic Ingredients and their
Safety Evaluation« (7)
•schülke KoKo test »Determination of
the Preserving Effect of Chemical
Preservatives in Cosmetic Formula-
tions«
The first edition of ISO 11930 (1) was published in April 2012 under the
title »Cosmetics – Microbiology – Evaluation of the antimicrobial pro-
tection of a cosmetic product«. This test can be used to evaluate the
preservation of a cosmetic formulation.
While the regulation on cosmetic products (EC 1223/2009) (2) requires
that the cosmetic product safety report demonstrates the results of preser-
vation challenge test to prove the microbiological stability, it does not
specify the test procedure for the challenge test. In addition to ISO 11930
tests from the EU (3) and US pharmacopoeia (4), the ASEAN preservative
efficacy test (5), the test from the CTFA Microbiology Guidelines (6) as well
as several in-house test protocols have been established for many years.
This paper includes a comparison of the different methods and compara-
tive test results for ISO 11930, Ph. Eur. 7 and the schülke KoKo Test.
Introduction
Other test methods not included in this
study might be of interest for special ap-
plications:
•CTFA M-5 Methods for Preservation
Testing of Nonwoven Substrate Per-
sonal Care Products (6)
•CTFA M-6 A Method for Preservation
Testing of Atypical Personal Care
Products (6)
•CTFA M-7 A Rapid Method for Preser-
vation Testing of Water-Miscible Per-
sonal Care Products (6)
•
schülke FeuTuKo test »Method to
Determine the Preserving Effect of
Chem
ical Preservatives in Wet Tissues«.
Test microorganisms
The microorganisms used for the differ-
ent tests are listed in Table 1 to 3. The test
methods from the CTFA Micro
biology
Guidelines differentiate between
testing
of eye area cosmetics and other water
miscible cosmetic products. Eye cosmet-
ics are recommended to be tested with
more species. Nevertheless, the CTFA
methods have the biggest variability, on-
ly the use of different types of mi
croor-
ganisms is specified (Table 2 and 3).
The ISO 11930, the Ph. Eur 7, the USP 35
and the ASEAN tests use only pathogenic
microorganisms, they only partly fulfil the
recommendation of the SCCS to use ad-
ditional specific germs which are known
to lead to spoilage of cosmetic products.
The CTFA includes in their selection lists
typical product spoiling microorganisms,
SOF W-Journal | 138 | 7-2012 45
COSMETICS
PRESERVATION
Table 1 Microorganisms used for ISO 11930, SCCS recommendation Ph. Eur. 7, USP 35, ASEAN
Table 2 Microorganisms recommended for CTFA / M-3 A Method for Preservation
Testing of Water-Miscible Personal Care Products
they can be tested instead ore in addi-
tion to pathogenic species.
The schülke KoKo test includes these mi-
croorganisms, selected using decades of
experience from schülke’s support ser-
vices to cosmetic producers through the
scope of MQM (Microbiological Quality-
Management). Only the schülke KoKo
test completely fulfil recommendations
of the SCCS Notes of Guidance.
■Cultivation of
Test Microorganisms
The cultivation of the test germs is com-
parable for all methods including the
schülke KoKo test. Fig. 1 shows the pro-
cedure of single-strain cultivation on
agar plates.
■Production of Inoculum
For the production of an inoculum, mi-
croorganisms are washed off from the
nutrient media. For the production of an
inoculum, microorganisms are washed
off from the nutrient media plates and
adjusted to the required starting germ
count by dilution. The inoculum is then
directly used to inoculate the test sam-
ples according to ISO 11930, Ph. Eur. 7
and ASEAN. The CTFA tests accepts the
use of either as well pure or mixed cul-
ture suspensions, the schülke KoKo test
uses mixed culture suspensions (i.e. sin-
gle cultivated microorganisms brought
together into a mixed suspension). To
guarantee a constant composition of the
mixed inoculum the mixed suspension is
stored for maximum of three days in a
refrigerator. A new inoculation suspen-
sion is prepared for each inoculation cy-
cle.
■Inoculation of Samples
The microorganism counts used and the
quantity of the inoculum are demon-
strated in Table 4.
All tests have similar germ loads in the
inoculated sample. The total germ count
of the inoculum for the schülke KoKo
test was slightly higher to ensure safe
preservation to compensate for less clean
production facilities. As there is now the
legal demand to produce cosmetic prod-
ucts according to ISO 22716 (Guidelines
on Good Manufacturing Practices) (9),
the germ count has recently been adapt-
ed bringing it into line with the other test
models.
■Criteria of Acceptance
ISO 11930 (Table 5)
If the formulation meets criteria A, the
microbiological risk is considered to be
tolerable (the cosmetic product is pro-
tected against microbial proliferation
that may present a potential risk for the
user) and the cosmetic product is deemed
to meet the requirements of this Inter-
national Standard without additional ra-
tionale (1).
If the formulation meets criteria B, the
microbiological risk analysis shall demon-
strate
the existence of control factors
not related to the formulation; for ex-
ample, a protective package such as a
pump provides a higher level of protec-
tion than a jar (see Annex D, ISO 11930).
46 SOF W-Journal | 138 | 7-2012
COSMETICS
PRESERVATION
Table 3 Microorganisms used for CTFA / M-4 Method for Preservation Testing of
Eye Area Cosmetics
Fig. 1 Cultivation of test germs for the schülke KoKo Test
This would be considered a protective
device for risk reduction (1).
schülke KoKo test (Table 6)
If a sample passes criteria A, this means
that even after the sixth inoculation no
microbial growth can be observed, the
product can be considered to be well
preserved. From many years of experi-
ence in the use of this test method, if a
product passes criteria A we can state the
microbiological stability of 30 months,
which is recommended for cosmetic
products.
If the formulation meets criteria B, the
microbiological risk analysis shall demon-
strate the existence of control factors not
related to the formulation; for example,
a protective package such as a pump pro-
vides a higher level of protection than a
48 SOF W-Journal | 138 | 7-2012
COSMETICS
PRESERVATION
Table 4 Microorganism counts and the quantity of inoculum used for challenge testing
Table 5 ISO 11930 / Criteria of acceptance
4
4
5
5
3
3
4
4
jar and/or following strong demands on
good manufacturing practice.
Ph. Eur. 7 (Table 7)
The A criteria express the recommended
efficacy to be achieved. In justified cas-
es where the A criteria cannot be at-
tained, for example, for reasons of an in-
creased risk of adverse reactions, the B
criteria must be satisfied.
USP 35 (Table 8)
ASEAN (Table 9)
CTFA / M-3 (Table 10)
CTFA / M-4 (Table 11)
■Preservative Stability
Preservative stability is an important
factor to ensure the microbial quality.
The SCCS requires (7):
•The efficacy of the preservation of a
cosmetic product under development
has to be assessed experimentally in
order to ensure microbial stability and
preservation during storage and use.
To ensure a safe preservation over the
shelf life of a cosmetic product a single
inoculation of a fresh prepared sample is
insufficient. Degradation or inactivation
of actives may occur, e.g. methylchloro-
isothiazolinone or bronopol at alkaline
pH.
ISO 11930
There is no hint to preservative stability
in ISO 11930.
schülke KoKo test
The six fold inoculation includes auto-
matically a preservative stability check,
the sixth inoculation is performed on at
least six weeks old sample. Practical ex-
perience has shown that this time scale
is sufficient to ensure microbial quality.
Ph. Eur. 7
The Ph. Eur. stipulates to investigate the
antimicrobial activity with stored sam-
ples:
SOF W-Journal | 138 | 7-2012 49
COSMETICS
PRESERVATION
Table 6 schülke KoKo test / Criteria of acceptance
Table 7 Ph. Eur 7 / Criteria of acceptance
Table 9 ASEAN / Criteria of acceptance
Table 10 CTFA / M-3 / Criteria of acceptance
Table 11 CTFA / M-4 Eye area cosmetics / Criteria of acceptance
Table 8 USP 35 / Criteria of acceptance
•
The antimicrobial activity of the prepa-
ration
in its final container is investi-
gated over the period of validity to
ensure that such activity has not been
impaired by storage.
USP 35
The USP 35 does not address the issue of
stability; there is only the statement:
•The tests and criteria for effectiveness
apply to a product in the original, un-
opened container in which it was dis-
tributed by the manufacturer.
ASEAN
There is no hint to preservative stability
in the ASEAN test.
CTFA / M-3
Under »Other Considerations« the stor-
age stability is included:
•It is important that challenge tests al-
so be conducted on samples aged in
the final container in order to deter-
mine the stability of the preservative
system.
CTFA / M-4
For eye area cosmetic it is clearly stated
in the guidelines:
•Product should be evaluated for
preservative stability in commercial
packaging by testing after storage
that simulates warehouse, shipping
and shelf-life conditions.
■Experimental Data
Using a body lotion containing Q10 from
an actual market request, we ran the
preservative efficacy tests according to
ISO 11930 in comparison to Ph. Eur. 7 and
the schülke KoKo test. Formulations con-
taining coenzyme Q-10 (ubichinone) are
known to be more susceptible to mould
growth. Table 12 shows the preservative
systems that have been selected for the
challenge test.
For the ISO 11930 and Ph. Eur. test re-
duction in microbial counts are expressed
in log units (Rx=lgN0– lgNx). Negative
figures indicate an increase of the mi-
crobial count in the test sample.
For the KoKo test, the preserving effect
is evaluated semi-quantitatively by the
growth of different streaks. The micro-
bial growth is classified in bacteria, yeasts
and moulds according to the following
legend:
B = Bacteria
M = Moulds
Sp = Sporeforming bacteria
Y = Yeasts
- = free of growth
+ = slight growth
++ = moderate growth
+++= heavy growth
T
able 14 to 19 display the results achieved
with the different preservative systems.
■Summary Experimental Data
Table 20 summarises the assessment of
the antimicrobial protection with re-
gards to the different test methods.
The criteria of acceptance for ISO 11930
appear to be the easiest to fulfil. It is
questionable if the demands of protec-
tion for a cosmetic product should not
be stricter than for pharmaceutical
products. The test for efficacy of antimi-
crobial preservation according to EU
Pharmacopoeia and the criteria of ac-
ceptance are designed for the produc-
50 SOF W-Journal | 138 | 7-2012
COSMETICS
PRESERVATION
Table 13 Test results unpreserved lotion
Preservative System
Unpreserved lotion -
Lotion preservative 1
1.0 % combination phenoxyethanol / ethylhexylglycerin
1)
Lotion preservative 2
1.1 % combination phenoxyethanol / ethylhexylglycerin
1)
Lotion preservative 3
1.0 % combination phenoxyethanol / ethylhexylglycerin
1)
0.1 % ethylhexylglycerin (preservative booster)
2)
Lotion preservative 4
1.0 % combination phenoxyethanol / ethylhexylglycerin
1)
0.06 % combination ethylhexylglycerin / methyliso-
thiazolinone
3)
Lotion preservative 5
1.0 % combination phenoxyethanol / benzyl alcohol /
potassium sorbate
4)
(pH adjusted to 5.3)
Lotion preservative 6 1 % combination phenoxyethanol / benzoic acid /
dehydroacetic acid / ethylhexylglycerin / polyamino-
propyl biguanide
5)
(pH adjusted to 5.1)
1) euxyl® PE 9010
2) sensiva® SC 50
3) euxyl® K 220
4) euxyl® K 700
5) euxyl® K 702
Table 12
Selected preservative systems used in the challenge test
tion of drugs under the strong require-
ments of the pharmaceutical GMP (Good
Manufacturing Practice). Additionally the
useable lifespan after opening of cos-
metic products are generally longer than
for pharmaceutical products.
The schülke KoKo test is specifically ver-
ified for the assessment of cosmetic
products. From more than 25 years of ex-
perience (11, 12) with this test method,
we can state a 30 month microbiologi-
cal stability for a product passing crite-
ria A, which is recommended for cos-
metic products.
The lotion with the preservative system
1 passed only ISO 11930 and Ph. Eur. cri-
teria B. Nevertheless, even 28 days after
t
he inoculation with Aspergillus brasilien-
sis it was still detectable. Within 7 days
the decrease of moulds is insufficient,
the KoKo failed.
The lotion with the preservative system
2 and 3 passed ISO 11930 and Ph. Eur.;
KoKo showed only slight growth in the
last cycles, it fulfils criteria B.
Precision and reproducibility of
preservative efficacy tests
To assess the differences of the results in
the various test methods, it is important
to realise the variation in microbiologi-
cal test results. The GÖCH has published
the
results of a round robin test for Ph.
Eur. -
5.1.3 »Efficacy of Antimicrobial
Preservation« (10). The microbial counts
differed widely between the laborato-
ries. Fig. 2 shows an overview of the re-
sults including expanded uncertainty.
For the other methods this data is not
available, probably however it can be as-
sumed that it will be in the same range.
Borderline results with low reduction in
microbial counts might fail in a replicate.
Uncertain contaminations during pro-
duction combined with an insufficient
speed of killing may lead to detection of
contamination in the market place. The
worst case is the inclusion in the RAPEX
notifications (13).
■Conclusion
The ISO 11930 as new standard to eval-
uate the antimicrobial protection of a
cosmetic product includes reference
method to evaluate the preservation, as
well as a decision diagram to evaluate
the microbiological risk. The recommend-
ed
preservation efficacy test is less strict
than the Ph. Eur. and the KoKo test (see
Experimental data). The microbial strains
SOF W-Journal | 138 | 7-2012 51
COSMETICS
PRESERVATION
Table 14 Test results lotion preservative 1
Table 15 Test results lotion preservative 2
Table 16 Test results lotion preservative 3
Table 17 Test results lotion preservative 4
did not include typical product spoiling
microorganisms alongside the pathogen-
ic
germs as required by the SCCS (7). The
stability of a preservative is not taken in-
to account by ISO 11930.
Formulations passing KoKo or Ph. Eur.
criteria A can be also evaluated as safe
according to ISO 11930 criteria A.
It is likely that products tested according
to CTFA M-3 or M-4 will be safe as the
requirement for a germ count reduction
within seven days is more rigorous than
that of ISO 11930 and comparable to the
KoKo test.
The ASEAN test is comparable to ISO
11930.
The USP 35 can be considered as a weak
standard; the demands are below those
for ISO 11930. It is advisable to perform
a test according to one of the methods
designed for cosmetics.
References
(1) ISO 11930, Cosmetics - Microbiology - Eval-
uation of the antimicrobial protection of a
cosmetic product
(2) Regulation (EC) No 1223/2009 of the Euro-
pean parliament and the council of Europe of
30 November 2009 on cosmetic products
http://eur-lex.europa.eu/LexUriServ/LexUri
Serv.do?uri=OJ:L:2009:342:0059:0209:EN:
PDF
(3) European Pharmacopeia 7th edition, 5.1.3. Ef-
ficacy of antimicrobial preservation
(4) United States Pharmacopoeia (USP 35); 51
Antimicrobial effectiveness testing
(5)
Association of Southeast Asian Nations, Preser-
vative efficacy test for cosmetic product,
Document N° ACM MAL 08
(6) Personal Care Products Council (PCPC - for-
merly the Cosmetic, Toiletry and Fragrance
Association); CTFA Microbiology Guidelines
2007
(7) Scientific Committee on Consumer Safety
(SCCS), The SCCS's Notes of Guidance for the
Testing of Cosmetic Ingredients and their
Safety Evaluation, 7th Revision
(8) ISO 29621, Cosmetics - Microbiology - Guide-
lines for the risk assessment and identifica-
tion of microbiologically low-risk products
52 SOF W-Journal | 138 | 7-2012
COSMETICS
PRESERVATION
Table 18 Test results lotion preservative 5
Table 19 Test results lotion preservative 6
Table 20 Summary experimental data
Fig. 2 GÖCH results of round robin test including expanded uncertainty
(9) ISO 22716; Cosmetics - Good Manufacturing
Practices (GMP) – Guidelines on Good Manu-
facturing Practices
(10)
GÖCH Arbeitskreis »Mikrobiologie«, Zwischen-
bericht Ringversuch Konservierungsbelas-
tungstest (KBT) kosmetische Mittel http://
www.goech.at/files/2011_GOECH_Arbeits
kreis_1_Veroeffentlichung_110118.pdf
(11) Siegert W., Evaluation of the Microbiological
Safety of Finished Cosmetic Products, Euro
Cosmetics 3-2010
(12) Brill H., Prüfverfahren zur Bestimmung der
mikrobiologischen Wirksamkeit con chemis-
chen Konservierungsmitteln für Kosmetika,
Jahrbuch für den Praktiker 1987
(13) European Commission, RAPEX – Latest notifi-
cations, http://ec.europa.eu/consumers/dyna/
rapex/rapex_archives_en.cfm
* Author’s address:
Wolfgang Siegert
Schülke &Mayr GmbH
Robert-Koch-Straße 2
22851 Norderstedt
Germany
Email: wolfgang.siegert@schuelke.com
■
SOF W-Journal | 138 | 7-2012 53
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