163-2010 EURO COSMETICS

1. Introduction

Cosmetic products should be safe under normal or reasonably fore-seeable conditions of use. The regulation on cosmetic products (EC1223/2009) requires for the cosmetic product safety report the resultsof preservation challenge test to prove the microbiological safety.The regulation itself didn’t specify the test procedure for the challengetest. Beside the test from the EU and US pharmacopoeia in-house testprotocols are established since many years. The applicability of theschülke KoKo test for the evaluation of the microbiological safety offinished cosmetic products is assessed in this paper.

2. Legal Regulations

In paragraph 6.4 “Guidelines on Microbiological of the Finished Cos-metic Product“ in the 6th revision (December 2006) of the SCCP pub-lication “Notes of Guidance for Testing of Cosmetic Ingredients andTheir Safety Evaluation” there is an obligation for carrying out apreservation test with all cosmetic products which could be contam-inated under normal storage and usage conditions or if an infectionrisk for the consumer exists.

The use of Pseudomonas aeruginosa, Staphylococcus aureus andCandida albicans from official collection strains from any state withinthe EU is obligatory. In addition, the use of specific germs which areknown to lead to spoilage of cosmetic products is recommended.

Evaluation of the Microbiological Safety of Finished Cosmetic ProductsBy Wolfgang Siegert*

* Technical Support Manager International · Schülke & Mayr GmbH · Special Additives International · Telephone +49 40 52100 528 · mailto:wolfgang.siegert@schuelke.com ·www.schuelke.com

3. Test micro-organisms

SCCP Recommendation

Pseudomonas aeruginosaof an official reference stock in the EU

Staphylococcus aureusof an official reference stock in the EU

Candida albicansof an official reference stock in the EU

specific germs which areknown to lead to spoilageof cosmetic products

schülke KoKo Test

Enterobacter gergoviaeATCC 33028

Escherichia coliATCC 11229

Klebsiella pneumoniaeATCC 4352

Pseudomonas aeruginosaATCC 9027

Pseudomonas fluorescensATCC 17397

Pseudomonas putidaATCC 12633

Kocuria rhizophilaATCC 9341

Staphylococcus aureusATCC 6538

Candida albicansATCC 10231

Aspergillus nigerATCC 16404

Penicillium funiculosumATCC 36839

EU Pharmacopoeia

Pseudomonas aeruginosaATCC 9027; NCIMB 8626; CIP 82.118.

Staphylococcus aureusATCC 6538; NCTC 10788;NCIMB 9518; CIP 4.83.

Candida albicansATCC 10231; NCPF 3179; IP 48.72.

Aspergillus nigerATCC 16404; IMI 149007; IP 1431.83.

US Pharmacopoeia

Pseudomonas aeruginosa ATCC 9027

Escherichia coliATCC 11229

Staphylococcus aureusATCC 6538

Candida albicansATCC 10231

Aspergillus niger ATCC 16404

183-2010 EURO COSMETICS

In difference to the pharmacopoeia tests which uses only thepathogen microorganisms, the schülke KoKo test includes productspoiling microorganisms. The choice of the product spoiling microor-ganisms used is based on decades of experience from schülke’s sup-port services to cosmetic producers through the scope of MQM(Microbiological Quality-Management).

4. Cultivation of Test Micro-Organisms

The cultivation of the test germs that is carried out by the methodsschülke KoKo Test, US Pharmacopoeia and EU Pharmacopoeia arecomparable as single-strain cultivation on agar plates (Figure 1).

5. Production of Inoculum

For the production of an inoculum, microorganisms are washed offfrom the nutrient media plates and adjusted to the required startinggerm count by dilution. The inoculum is then directly used to inocu-late the test samples according to US Pharmacopoeia and EU Phar-macopoeia. For the schülke KoKo Test, the single cultivated microor-ganisms brought together into a mixed suspension. To guarantee a con-stant composition of the mixed inoculum the mixed suspension isstored for maximum of three days in a refrigerator. A new inoculationsuspension is prepared for each inoculation cycle.

6. Inoculation of Samples

The microorganism counts used and the quantity of the inoculumare demonstrated in the following table:

Figure 1: Cultivation of test germs for the schülke KoKo Test

Used germ count

0.4 % each inoculation(2.4 % at 6 inoculations)

4x105 – 4x106 germs/geach inoculation

2.4x106 – 2.4x107 germs/gat 6 inoculations

Quantity of the inoculum

schülke KoKo Test EU Pharmacopoeia US Pharmacopoeia

≤ 1 % 0.5 – 1.0 %

105 – 106 germs/ml 105 – 106 germs/ml

The total germ count of the inoculum for the schülke KoKo Test isslightly higher in order to guarantee a comparable high contaminationload for the single germs.

7. Criteria of acceptance

In the schülke KoKo test a sample can be called well preserved, if itpasses a period of six weeks under the above described laboratoryconditions without showing microbial growth on the test batches.That means even after the sixth inoculation no microbial growth canbe observed.

In the EU Pharmacopoeia for topical preparations the followingacceptance criteria are given:

Criteria Log Reduction

2 d 7 d 14 d 28 d

BacteriaA 2 3 - No increase

B - - 3 No increase

FungiA - - 2 No increase

B - - 1 No increase

The A criteria express the recommended efficacy to be achieved. Injustified cases where the A criteria cannot be attained, for example, forreasons of an increased risk of adverse reactions, the B criteria mustbe satisfied.

In the US Pharmacopoeia for topical preparations the followingacceptance criteria are given:

14 d 28 d

Bacteria 2 Log reduction No increase

Fungi No increase No increase

The comparisons of the criteria of acceptance are shown in the fol-lowing graphs:

8. Conclusion

The test for efficacy of antimicrobial preservation according to USPharmacopoeia resp. to EU Pharmacopoeia and the criteria of accept-ance are valid for the production of drugs under the strong require-ments of the pharmaceutical GMP (Good Manufacturing Practice). Atransfer to the assessment of cosmetic products is only limitedly pos-sible. The additional use of spoiling microorganisms required by theSSCP is not covered by these tests. Additional tests are necessary. Bythe use single microorganisms as inoculum, a symbiotic growth ofmicroorganisms, like in nature, is disabled.

The schülke KoKo test is specifically verified for the assessment ofcosmetic products. The germ spectrum includes typical product spoil-ing microorganisms besides the pathogen germs as required by theSCCP. By inoculation of a mixed germ spectrum, a symbiotic growthaccording to the natural conditions is possible. To future equivalentdetection of bacteria as fungi, the tests are carried out on tryptone-soy-agar as well as on sabouraud-dextrose-agar. A wrong assessment of theefficacy of the preservative by inhibition of the growth of onemicroorganism by the growth of other germs is impossible as growthof microorganisms’ means that the sample is not sufficiently pre-served.

The actual situation during production, filling and the use of a cos-metic product will be additionally simulated by testing with multipleinoculations. The schülke KoKo Test is a reliable test method for theassessment of the efficacy of antimicrobial preservation of cosmeticproducts. ❘❘❙❙