W. Siegert:

ISO 11930 –A Comparison to

other Methods to Evaluate theEfficacy of Antimicrobial

Preservation

7-2012

English EditionInternational Journal for Applied Science

• Personal Care • Detergents • Specialties

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COSMETICSPRESERVATION

• Ph. Eur. 7 - 5.1.3 »Efficacy of Antimi-crobial Preservation«(3)

• USP 35 »Antimicrobial EffectivenessTesting« (4)

• ASEAN ACM MAL 08 »Preservative Ef-ficacy Test for Cosmetic Product« (5)

• CTFA M-3 »A Method for PreservationTesting of Water-Miscible PersonalCare Products« (6)

• CTFA M-4 »Method for PreservationTesting of Eye Area Cosmetics« (6).

W. Siegert*

ISO 11930 – A Comparison to other Methodsto Evaluate the Efficacy of AntimicrobialPreservation

■ Legal Regulations

Only in the SCCS notes of guidance, thedemands for challenge testing are speci-fied. In paragraph 4-4 »Guidelines on Mi-crobiological of the Finished CosmeticProduct« in the 7th revision (December2010) (7) of the SCCS publication »Notesof Guidance for the Testing of CosmeticIngredients and their Safety Evaluation«is an obligation for carrying out a preser-vation test with all cosmetic productswhich could be contaminated undernormal storage and usage conditions orif an infection risk for the consumer ex-ists.The use of Pseudomonas aeruginosa,Staphylococcus aureus and Candida al-bicans from official collection strainsfrom any state within the EU is obliga-tory. In addition, the use of specific germs,which are known to lead to spoilage ofcosmetic products, is recommended.

■ ISO 11930

Compared to other methods ISO 11930 isan overall standard to evaluate the anti-microbial stabilisation of a cosmetic prod-uct. With the decision diagram in AnnexA also formulations intrinsically hostileto microbial growth, specific manufac-turing conditions, packaging and/or con-ditions for use and the reference to ISO29261 (Guidelines for the risk assess-ment and identification of microbiologi-cally low-risk products) (7) are covered.Nevertheless, the preservative efficacytest described in ISO 11930 is primarilydesigned for water-soluble or water-mis-cible products.

This publication compares the followingtest 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 theirSafety Evaluation« (7)

• schülke KoKo test »Determination ofthe Preserving Effect of ChemicalPreservatives in Cosmetic Formula-tions«

The first edition of ISO 11930 (1) was published in April 2012 under thetitle »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) requiresthat the cosmetic product safety report demonstrates the results of preser-vation challenge test to prove the microbiological stability, it does notspecify the test procedure for the challenge test. In addition to ISO 11930tests from the EU (3) and US pharmacopoeia (4), the ASEAN preservativeefficacy test (5), the test from the CTFA Microbiology Guidelines (6) as wellas 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 thisstudy might be of interest for special ap-plications:

• CTFA M-5 Methods for PreservationTesting of Nonwoven Substrate Per-sonal Care Products (6)

• CTFA M-6 A Method for PreservationTesting of Atypical Personal CareProducts (6)

• CTFA M-7 A Rapid Method for Preser-vation Testing of Water-Miscible Per-sonal Care Products (6)

• schülke FeuTuKo test »Method toDetermine the Preserving Effect ofChemical Preservatives in Wet Tissues«.

Test microorganisms The microorganisms used for the differ-ent tests are listed in Table 1 to 3. The testmethods from the CTFA MicrobiologyGuidelines differentiate between testingof eye area cosmetics and other watermiscible cosmetic products. Eye cosmet-ics are recommended to be tested with

more species. Nevertheless, the CTFAmethods have the biggest variability, on-ly the use of different types of microor-ganisms is specified (Table 2 and 3).The ISO 11930, the Ph. Eur 7, the USP 35and the ASEAN tests use only pathogenic

microorganisms, they only partly fulfil therecommendation of the SCCS to use ad-ditional specific germs which are knownto lead to spoilage of cosmetic products.The CTFA includes in their selection liststypical product spoiling microorganisms,

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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 PreservationTesting 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 ofexperience from schülke’s support ser-vices to cosmetic producers through thescope of MQM (Microbiological Quality-Management). Only the schülke KoKotest completely fulfil recommendationsof the SCCS Notes of Guidance.

■ Cultivation of Test Microorganisms

The cultivation of the test germs is com-parable for all methods including theschülke KoKo test. Fig. 1 shows the pro-cedure of single-strain cultivation onagar plates.

■ Production of Inoculum

For the production of an inoculum, mi-croorganisms are washed off from thenutrient media. For the production of aninoculum, microorganisms are washedoff from the nutrient media plates andadjusted to the required starting germcount by dilution. The inoculum is thendirectly used to inoculate the test sam-ples according to ISO 11930, Ph. Eur. 7and ASEAN. The CTFA tests accepts theuse of either as well pure or mixed cul-ture suspensions, the schülke KoKo testuses mixed culture suspensions (i.e. sin-gle cultivated microorganisms broughttogether into a mixed suspension). Toguarantee a constant composition of themixed inoculum the mixed suspension isstored for maximum of three days in arefrigerator. A new inoculation suspen-sion is prepared for each inoculation cy-cle.

■ Inoculation of Samples

The microorganism counts used and thequantity of the inoculum are demon-strated in Table 4. All tests have similar germ loads in theinoculated sample. The total germ countof the inoculum for the schülke KoKotest was slightly higher to ensure safepreservation to compensate for less clean

production facilities. As there is now thelegal demand to produce cosmetic prod-ucts according to ISO 22716 (Guidelineson Good Manufacturing Practices) (9),the germ count has recently been adapt-ed bringing it into line with the other testmodels.

■ Criteria of Acceptance

ISO 11930 (Table 5)If the formulation meets criteria A, themicrobiological risk is considered to be

tolerable (the cosmetic product is pro-tected against microbial proliferationthat may present a potential risk for theuser) and the cosmetic product is deemedto meet the requirements of this Inter-national Standard without additional ra-tionale (1).If the formulation meets criteria B, themicrobiological risk analysis shall demon-strate the existence of control factorsnot related to the formulation; for ex-ample, a protective package such as apump provides a higher level of protec-tion than a jar (see Annex D, ISO 11930).

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Table 3 Microorganisms used for CTFA / M-4 Method for Preservation Testing ofEye Area Cosmetics

Fig. 1 Cultivation of test germs for the schülke KoKo Test

This would be considered a protectivedevice for risk reduction (1).

schülke KoKo test (Table 6)If a sample passes criteria A, this meansthat even after the sixth inoculation nomicrobial growth can be observed, theproduct can be considered to be wellpreserved. From many years of experi-ence in the use of this test method, if aproduct passes criteria A we can state themicrobiological stability of 30 months,which is recommended for cosmeticproducts.

If the formulation meets criteria B, themicrobiological 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

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Table 4 Microorganism counts and the quantity of inoculum used for challenge testing

Table 5 ISO 11930 / Criteria of acceptance

jar and/or following strong demands ongood manufacturing practice.

Ph. Eur. 7 (Table 7)The A criteria express the recommendedefficacy 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 Bcriteria 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 importantfactor to ensure the microbial quality.The SCCS requires (7):

• The efficacy of the preservation of acosmetic product under developmenthas to be assessed experimentally inorder to ensure microbial stability andpreservation during storage and use.

To ensure a safe preservation over theshelf life of a cosmetic product a singleinoculation of a fresh prepared sample isinsufficient. Degradation or inactivationof actives may occur, e.g. methylchloro-isothiazolinone or bronopol at alkalinepH.

ISO 11930There is no hint to preservative stabilityin ISO 11930.

schülke KoKo testThe six fold inoculation includes auto-matically a preservative stability check,the sixth inoculation is performed on atleast six weeks old sample. Practical ex-perience has shown that this time scaleis sufficient to ensure microbial quality.

Ph. Eur. 7The Ph. Eur. stipulates to investigate theantimicrobial activity with stored sam-ples:

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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 toensure that such activity has not beenimpaired by storage.

USP 35The USP 35 does not address the issue ofstability; there is only the statement:

• The tests and criteria for effectivenessapply to a product in the original, un-opened container in which it was dis-tributed by the manufacturer.

ASEANThere is no hint to preservative stabilityin the ASEAN test.

CTFA / M-3Under »Other Considerations« the stor-age stability is included:

• It is important that challenge tests al-so be conducted on samples aged inthe final container in order to deter-mine the stability of the preservativesystem.

CTFA / M-4For eye area cosmetic it is clearly statedin the guidelines:

• Product should be evaluated forpreservative stability in commercialpackaging by testing after storagethat simulates warehouse, shippingand shelf-life conditions.

■ Experimental Data

Using a body lotion containing Q10 froman actual market request, we ran thepreservative efficacy tests according toISO 11930 in comparison to Ph. Eur. 7 andthe schülke KoKo test. Formulations con-taining coenzyme Q-10 (ubichinone) areknown to be more susceptible to mouldgrowth. Table 12 shows the preservativesystems that have been selected for thechallenge test.For the ISO 11930 and Ph. Eur. test re-duction in microbial counts are expressedin 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 effectis evaluated semi-quantitatively by thegrowth of different streaks. The micro-bial growth is classified in bacteria, yeastsand moulds according to the followinglegend:

B = Bacteria M = MouldsSp = Sporeforming bacteriaY = Yeasts

- = free of growth+ = slight growth++ = moderate growth+++= heavy growth

Table 14 to 19 display the results achievedwith the different preservative systems.

■ Summary Experimental Data

Table 20 summarises the assessment ofthe antimicrobial protection with re-gards to the different test methods.The criteria of acceptance for ISO 11930appear to be the easiest to fulfil. It isquestionable if the demands of protec-tion for a cosmetic product should notbe stricter than for pharmaceuticalproducts. The test for efficacy of antimi-crobial preservation according to EUPharmacopoeia and the criteria of ac-ceptance are designed for the produc-

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Table 13 Test results unpreserved lotion

Preservative System

Unpreserved lotion -

Lotion preservative 1 1.0 % combination phenoxyethanol / ethylhexylglycerin1)

Lotion preservative 2 1.1 % combination phenoxyethanol / ethylhexylglycerin1)

Lotion preservative 3 1.0 % combination phenoxyethanol / ethylhexylglycerin1)

0.1 % ethylhexylglycerin (preservative booster)2)

Lotion preservative 4 1.0 % combination phenoxyethanol / ethylhexylglycerin1)

0.06 % combination ethylhexylglycerin / methyliso-thiazolinone3)

Lotion preservative 5 1.0 % combination phenoxyethanol / benzyl alcohol / potassium sorbate4) (pH adjusted to 5.3)

Lotion preservative 6 1 % combination phenoxyethanol / benzoic acid / dehydroacetic acid / ethylhexylglycerin / polyamino-propyl biguanide5) (pH adjusted to 5.1)

1) euxyl® PE 90102) sensiva® SC 503) euxyl® K 2204) euxyl® K 7005) 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 (GoodManufacturing Practice). Additionally the

useable lifespan after opening of cos-metic products are generally longer thanfor pharmaceutical products.

The schülke KoKo test is specifically ver-ified for the assessment of cosmeticproducts. 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 system1 passed only ISO 11930 and Ph. Eur. cri-teria B. Nevertheless, even 28 days afterthe inoculation with Aspergillus brasilien-sis it was still detectable. Within 7 daysthe decrease of moulds is insufficient,the KoKo failed.The lotion with the preservative system2 and 3 passed ISO 11930 and Ph. Eur.;KoKo showed only slight growth in thelast cycles, it fulfils criteria B.

Precision and reproducibility ofpreservative efficacy testsTo assess the differences of the results inthe various test methods, it is importantto realise the variation in microbiologi-cal test results. The GÖCH has publishedthe results of a round robin test for Ph.Eur. - 5.1.3 »Efficacy of AntimicrobialPreservation« (10). The microbial countsdiffered widely between the laborato-ries. Fig. 2 shows an overview of the re-sults including expanded uncertainty.For the other methods this data is notavailable, probably however it can be as-sumed that it will be in the same range.Borderline results with low reduction inmicrobial counts might fail in a replicate.Uncertain contaminations during pro-duction combined with an insufficientspeed of killing may lead to detection ofcontamination in the market place. Theworst case is the inclusion in the RAPEXnotifications (13).

■ Conclusion

The ISO 11930 as new standard to eval-uate the antimicrobial protection of acosmetic product includes referencemethod to evaluate the preservation, aswell as a decision diagram to evaluatethe microbiological risk. The recommend-ed preservation efficacy test is less strictthan the Ph. Eur. and the KoKo test (seeExperimental data). The microbial strains

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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 spoilingmicroorganisms alongside the pathogen-ic germs as required by the SCCS (7). Thestability 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 safeaccording to ISO 11930 criteria A. It is likely that products tested accordingto CTFA M-3 or M-4 will be safe as therequirement for a germ count reductionwithin seven days is more rigorous thanthat of ISO 11930 and comparable to theKoKo test. The ASEAN test is comparable to ISO11930.The USP 35 can be considered as a weakstandard; the demands are below thosefor ISO 11930. It is advisable to performa test according to one of the methodsdesigned for cosmetics.

References

(1) ISO 11930, Cosmetics - Microbiology - Eval-uation of the antimicrobial protection of acosmetic product

(2) Regulation (EC) No 1223/2009 of the Euro-pean parliament and the council of Europe of30 November 2009 on cosmetic productshttp://eur-lex.europa.eu/LexUriServ/LexUriServ.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); 51Antimicrobial 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 FragranceAssociation); CTFA Microbiology Guidelines2007

(7) Scientific Committee on Consumer Safety(SCCS), The SCCS's Notes of Guidance for theTesting of Cosmetic Ingredients and theirSafety Evaluation, 7th Revision

(8) ISO 29621, Cosmetics - Microbiology - Guide-lines for the risk assessment and identifica-tion of microbiologically low-risk products

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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 ManufacturingPractices (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_Arbeitskreis_1_Veroeffentlichung_110118.pdf

(11) Siegert W., Evaluation of the MicrobiologicalSafety of Finished Cosmetic Products, EuroCosmetics 3-2010

(12) Brill H., Prüfverfahren zur Bestimmung dermikrobiologischen 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 GmbHRobert-Koch-Straße 2

22851 NorderstedtGermany

Email: wolfgang.siegert@schuelke.com

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