A guidance document on

microbiological control of cosmetic

products




Ann Detmer
Claus Jrgensen
Dorthe Nyln

DHI

















Envi r onment al Pr oj ect No. 1336 2010
Mi l j pr oj ekt





























T he D ani sh Envi ronmental Protecti on A gency wi ll, when opportuni ty
offers, publi sh reportsand contri buti onsrelati ng to envi ronmental
research and development projectsfi nanced vi a the D ani sh EPA .

Please note that publi cati on does not si gni fy that the contents of the
reportsnecessari ly reflect the vi ews of the D ani sh EPA .

T he reportsare, however, publi shed because the D ani sh EPA fi ndsthat
the studi es represent a valuable contri buti on to the debate on
envi ronmental poli cy i n D enmark.



3
Index





PR EFA C E 5
SA M M EN FA T N I N G O G K O N K L U SI O N ER 7
SU M M A R Y A N D C O N C L U SI O N S 9
1 I N T R O D U C T I O N 11
1.1 BA C K G R O U N D 12
1.2 L EG I SL A T I O N 12
1.3 D EFI N I T I O N S 13
1.4 H U M A N SA FET Y 13
1.5 D O SSI ER 13
1.6 G O O D M A N U FA C T U R I N G PR A C T I C E 13
1.7 D U R A BI L I T Y L A BEL I N G 14
2 M I C R O BI O L O G I C A L C O N T R O L 15
2.1 M I C R O BI O L O G I C A L C O N T R O L O F FI N A L PR O D U C T 15
2.2 M I C R O BI A L C O N T A M I N A T I O N 16
2.2.1 During manufacturing 16
2.2.2 After opening 17
2.3 PR ESER V A T I O N 17
3 L A BO R A T O R I ES 19
3.1 Q U A L I T Y M A N A G EM EN T 19
3.1.1 Quality management standards 19
3.1.2 Approach to quality management in microbiology laboratories 20
3.1.3 Requirements to quality management 23
4 A N A L Y T I C A L M ET H O D S 29
4.1 ST A N D A R D S U N D ER D EV EL O PM EN T 29
4.2 N EU T R A L I Z A T I O N A N D PR EPA R A T I O N O F WA T ER -I M M I SC I BL E
SA M PL ES. 29
4.3 EX A M I N A T I O N O F M I C R O BI A L Q U A L I T Y O F PR O D U C T S 30
4.3.1 ISO 21149 Cosmetics Microbiology Enumeration and
detection of aerobic mesophilic bacteria 30
4.3.2 ISO 18415 Cosmetics Microbiology Detection of specified
and non-specified micro-organisms. 30
4.3.3 ISO- methods for thedetection of specific microorganisms:
E. coli (ISO 21150), Pseudomonas aeruginosa (ISO 22717),
Staphylococcus aureus (ISO22718) and Candida albicans
(ISO 18416). 30
4.3.4 ISO/FDIS 16212 Cosmetics Microbiology Enumeration
of yeast and mould. 31
4.4 EFFI C A C Y O F PR ESER V A T I O N - C H A L L EN G E T EST 31
4.4.1 Proposed procedurefor challengetesting-non validated 31
5 R EFER EN C ES 33

4


A ppendi x 1 Standardi sati on mandate assi gned to C EN concerni ng good
manufacturi ng practi ce for cosmeti cs products.

A ppendi x 2 C hallenge test of water mi sci ble cosmeti c products.








5
Preface
T he purpose of thi s gui deli ne i s to help those concerned wi th the producti on
or i mport of cosmeti c products to mai ntai n a good mi crobi ologi cal quali ty all
through the li fe of the product. T he gui deli ne was prepared for the D ani sh
Envi ronmental Protecti on A gency ( D EPA ) i n 2007-2009 by D H I Water,
Envi ronment and H ealth ( D H I ) .

T he contri butors at D H I were:

A nn D etmer
C laus Jrgensen
D orte N yln

A steeri ng commi ttee from D EPA followed the proj ect:

D orri t Skals
A nette A lbj erg Ej ersted
Betti na rsnes A ndersen

6

7
Sammenfatning og konklusioner
En gui de ti l mi krobi ologi sk kontrol af kosmeti kprodukter blev udarbej det vi a
et proj ekt, som blev fi nansi eret af M i lj styrelsens V i rksomhedsordni ng.
Formlet med gui den er at i ntroducere passende testmetoder og at ge
kendskabet ti l de generelle krav i nden for mi krobi ologi ske testlaboratori er.
G ui den anbefaler, at man anvender de nyli gt offentli ggj orte I SO standarder,
der er speci elt beregnet ti l mi krobi ologi sk kontrol af kosmeti ske produkter, fr,
i lbet af, og ti l slut i processen. D a der endnu i kke foreli gger en I SO standard
for provokati ons testni ng, prsenteres en provokati onstest baseret p ti dli gere
beskrevne metoder. D en beskrevne provokati onstest er i kke blevet efterprvet,
og derfor har man i kke kendskab ti l, hvordan den fungerer i de forskelli ge
laboratori er. T i l trods herfor fandt vi det vi gti gt at medtage en
provokati onstest, og den skal ses som et forslag ti l, hvordan en sdan test kan
desi gnes..


8


9
Summary and conclusions

A gui dance document on mi crobi ologi cal control of cosmeti c products was
created wi thi n a proj ect from V i rksomhedsordni ngen of the D EPA . T he
i ntenti on of the gui dance document i s to i ntroduce adequate methods of
testi ng and knowledge of the general demands on mi crobi ologi cal testi ng
laboratori es. T he gui dance document recommends the use of the newly
publi shed I SO standards especi ally produced for mi crobi ologi cal control of
cosmeti c products, before, duri ng and at end of use. A s no I SO standard for
challenge testi ng i s yet avai lable a challenge test based on earli er descri bed
methods i s presented i n thi s gui dance document as a suggesti on to how a
challenge test can be constructed. U nfortunately the proposed challenge test
has not been vali dated or tested i n laboratori es and therefore we do not
have the knowledge of how i t performs i n di fferent laboratori es.






10


11
1 Introduction
T he purpose of thi s gui deli ne i s to help those concerned wi th the producti on
or i mport of cosmeti c products to mai ntai n a good mi crobi ologi cal quali ty all
through the li fe of the product. C osmeti cs refer to products i ntended to be
placed i n contact wi th the vari ous external parts of the human body
( epi dermi s, hai r system, nai ls, li ps and external geni tal organs) or wi th the
teeth and the mucous membranes of the oral cavi ty wi th a vi ew exclusi vely or
mai nly to cleani ng them, perfumi ng them, changi ng thei r appearance and/or
correcti ng body odours and/or protecti ng them or keepi ng them i n good
condi ti on. For defi ni ti on of cosmeti c product i n D ani sh see
K osmeti kbekendtgrelsen, K ap.1 3 ( 1) and i n Engli sh see A rti cle 1 i n
C ounci l D i recti ve 76/768/EEC ( 2) . C ontami nati on of cosmeti cs duri ng the
producti on process can cause adverse effects when used by sensi ti ve
i ndi vi duals. A cosmeti c product placed on the market must not cause damage
to human health. T hi s gui deli ne wi ll descri be and recommend vali dated
methods for measuri ng mi crobi ologi cal contami nati on of the cosmeti c
product before, duri ng and at end of use. T hi s gui deli ne wi ll also recommend
how laboratori es used for self-control of cosmeti cs products can be equi pped.

T hi s gui deli ne i s prepared by:
A nn D etmer
C laus Jrgensen
D orte N yln
D H I , C entre for Envi ronment and T oxi cology

T he proj ect i s fi nanced by V i rksomhedsordni ngen by T he D ani sh A gency
of Envi ronmental Protecti on, 2007.

C o-ordi nators of T he D ani sh EPA was:
D orri t Skals
A nette A lbj erg Ej ersted
Betti na rsnes A ndersen


12
1.1 Backgr ound
D ue to the wi de range of formulati ons, manufacturi ng procedures and
condi ti ons of consumer use, the control of mi crobi ologi cal growth i n
cosmeti cs i s complex. L egi slati on i n relati on to mi crobi ologi cal growth i n
cosmeti cs i s not detai led, and concerned bodi es are worki ng on developi ng
more detai led standards. O ne stakeholder i s PEM SA C ( Platform of European
M arket Survei llance A uthori ti es i n C osmeti cs) . PEM SA C i s cooperati on
between European authori ti es wi thi n cosmeti cs. PEM SA C has assi gned a
standardi sati on mandate to the European Standards O rgani sati ons ( C EN )
concerni ng G ood M anufacturi ng Practi ces for cosmeti cs products, see
appendi x 1. G M P i s supposed to ensure that products, that are not
necessari ly steri le contai n no harmful organi sms and that the beni gn
populati on i s of low and stable order and/or decli nes over the product li feti me.
A t the same ti me I nternati onal O rgani zati on for Standardi zati on, techni cal
commi ttee for cosmeti cs, I SO /T C 217 i s worki ng on a seri es of standards for
the detecti on and i denti fi cati on of mi croorgani sms i n cosmeti c products.
D enmark has no representati ve i n thi s techni cal commi ttee. T he exi stence of
these new standards wi ll help create safe cosmeti c products.

1.2 Legi sl at i on
A cosmeti c product i s regulated i n C ounci l D i recti ve 76/768/EEC of 27 July
1976 on the approxi mati on of the laws of the M ember States relati ng to
cosmeti c products ( C osmeti cs D i recti ve) . M ore than 55 amendments and
adaptati ons have changed the C osmeti c D i recti ve through the years. T he
C osmeti cs D i recti ve i ntroduces a legal responsi bi li ty for compani es assuri ng
that products reachi ng the market place are not only mi crobi ologi cally safe
but wi ll also conti nue to be safe throughout the products li fe. I n close co-
operati on wi th M ember States the C ommi ssi on has i ssued a number of
gui deli nes to provi de a coherent i nterpretati on of vari ous provi si ons of the
cosmeti cs-D i recti ve i n the i nterest of M ember States authori ti es and
stakeholders, such as i ndustry. T he C osmeti cs D i recti ve i s transposed i nto the
i ndi vi dual nati onal legal frameworks ( law) and i n D enmark i t i s i mplemented
i n the Statutory O rder of the mi ni stry of the Envi ronment no. 422 of 4. M ay
2006 ( Bekendtgrelse nr. 422 af 4. maj 2006 om kosmeti ske produkter ( 1) .
T he C osmeti cs and M edi cal D evi ces uni t of the European C ommi ssi on/
D i rectorate G eneral Enterpri se and I ndustry i s i n charge of admi ni steri ng the
C osmeti cs D i recti ve and supervi ses a correct i mplementati on.

T he cosmeti c D i recti ve consi sts of a body text and ei ght annexes:
I ndi cati ve li st of cosmeti c product types
O ffi ci ally recogni zed symbols ( two annexes)

T he "negati ve li sts"
Substances prohi bi ted i n cosmeti cs ( over 1200)
I ngredi ents wi th li mi tati on when used ( over 150)

T he "posi ti ve li sts"
C olorants wi th li mi tati ons
Preservati ves wi th li mi tati ons
U V -fi lters wi th li mi tati ons

13
1.3 Def i ni t i ons
A cosmeti c product i s defi ned as any substance or preparati on i ntended to be
placed i n contact wi th the vari ous external parts of the human body
( epi dermi s, hai r system, nai ls, li ps and external geni tal organs) or wi th the
teeth and the mucous membranes of the oral cavi ty wi th a vi ew exclusi vely or
mai nly to cleani ng them, perfumi ng them, changi ng thei r appearance and/or
correcti ng body odours and/or protecti ng them or keepi ng them i n good
condi ti on.

1.4 Human Saf et y
T he safety of a cosmeti c product i n the EU i s the full responsi bi li ty of the
manufacturer, the fi rst i mporter i nto the EU market or the marketer. A
cosmeti c product put on the market must not cause damage to human health
when appli ed under normal or reasonably foreseeable condi ti ons of use,
accordi ng to A rti cle 2 i n the C osmeti cs D i recti ve and i mplemented i n 10 i n
the D ani sh cosmeti cs regulati on.

1.5 Dossi er
T he manufacturer or hi s agent or the person to whom a cosmeti c product i s
manufactured or the person responsi ble for placi ng an i mported cosmeti c
product on the C ommuni ty market shall for control purposes keep a dossi er
readi ly accessi ble for i nspecti on by the competent authori ti es of the M ember
State i ndi cated by the address speci fi ed on the label. T he dossi er i s not
di rectly avai lable i n each M ember State but only through the competent
authori ty i n the M ember State, whi ch the manufacturer or hi s agent speci fi ed
on the label. T he i nformati on requi red to produce a dossi er i s descri bed i n
33 i n the D ani sh cosmeti cs regulati on ( 1) and i n arti cle 7a i n the C osmeti cs
D i recti ve 76/768/EEC ( 2) . Each dossi er must contai n a safety assessment of
the product and i nformati on on mi crobi ologi cal speci fi cati ons of the raw
materi als used for producti on and i n the product. R ecords should be
mai ntai ned for all aspects of mi crobi ologi cal testi ng duri ng development and
manufacture of the cosmeti c product. A gui deli ne on Safety assessment of
cosmeti c products and how to comply a dossi er i s avai lable i n G ui deli ne on
safety assessment of cosmeti c products:
http://www.mst.dk/U dgi velser/Publi cati ons/2001/03/87-7944-336-2.htm from
the D ani sh EPA and i n a D ani sh V ersi on i n Envi ronmental G ui deli ne N o 9
2000. http://www.mst.dk/U dgi velser/Publi kati oner/2001/03/87-7944-335-
4.htm Both gui deli nes can be found vi a the homepage of the D ani sh EPA .

1.6 Good Manuf act ur i ng Pr act i ce
Producers of cosmeti c products are legally obli ged to comply wi th the
pri nci ples and gui deli nes of G M P. T he requi rements were formulated i n
D i recti ve 93/35/EEC , the 6th amendment to the C osmeti cs D i recti ve. T he
I SO standard D S/EN I SO 22716:2007, C osmeti cs - G ood M anufacturi ng
Practi ces ( G M P) - G ui deli nes on G ood M anufacturi ng Practi ces, gi ves
gui deli nes for the producti on, control, storage and shi pment of cosmeti c
products. T hese gui deli nes cover the quali ty aspects of the product, but as a
whole they do not cover safety aspects for the personnel engaged i n the plant,
nor do they cover aspects of protecti on of the envi ronment. T he gui deli nes i n

14
I SO 22716:2007 are not appli cable to research and development acti vi ti es and
di stri buti on of fi ni shed products. T he standard can be acqui red vi a D ansk
Standards homepage, D S/EN I SO 22716:2007. C O L I PA ( 3) and the C ounci l
of Europe ( 4) have also produced G M P gui deli nes.
G M P should ensure that products, whi lst not necessari ly steri le, contai n no
harmful organi sms and that the mi crobi ologi cal populati on i s of a low and
stable order and/or decli nes over the product li feti me. G M P i ncludes speci fi c
cleani ng procedures to keep all apparatus and materi als appropri ately clean.
Procedures also i nclude mi crobi ologi cal control of raw materi als, bulk and
fi ni shed products, packagi ng materi al, personnel, equi pment and preparati on
and storage rooms.

1.7 Dur abi l i t y l abel i ng
From M arch 2005 i t was legally demanded ( D i recti ve 2003/15/EC , amendi ng
D i recti ve 76/768/EC ) to label durabi li ty on cosmeti c products. T hi s D i recti ve
i s i mplemented as 21 i n the D ani sh cosmeti cs regulati on.

I ndi cati on of the exact date of durabi li ty i s not mandatory for cosmeti c
products wi th a mi ni mum durabi li ty of more than 30 months. I nstead, such
products must be labelled wi th a symbol i ndi cati ng the peri od of ti me i n
month/year after openi ng ( PaO ) , for whi ch the product can be used wi thout
any harm to the consumer. Such a symbol i s the open j ar found i n annex ei ght
of the D ani sh cosmeti cs Statutory:



Products wi th durabi li ty less than 30 months must be labelled wi th durabi li ty
peri od and a fi xed date: best used before the end of i nsert date .

T he manufacturer must have i nformati on supporti ng the mi crobi ologi cal
stabi li ty of the product. T he manufacturer must demonstrate that no
unacceptable alterati ons of the product occur wi thi n the i ndi cated durabi li ty
peri od. Each product's PaO must be assessed wi th relevant methods. N o
offi ci al methodology i s avai lable but examples of sources of i nformati on for
assessi ng a product s PaO may i nclude:
- mi crobi ologi cal challenge tests
- stabi li ty data
- analyti cal data ( e.g. preservati ve analysi s)
- type of packagi ng
- experi ence wi th si mi lar formulati ons and products
- consumer habi ts and practi ces.

15
2 Microbiological control
T he di fferent needs for mi crobi ologi cal exami nati ons of cosmeti c products are
establi shed from the mi crobi ologi cal ri sk analysi s, whi ch are carri ed out i n
order to determi ne the type of cosmeti c product ( low mi crobi ologi cal ri sk etc)
you have. T he mi crobi ologi cal ri sk analyses i nclude consi derati on of the type
of user, si te of appli cati on, potenti al alterati on of cosmeti c products as well as
the pathogeni ci ty of mi croorgani sms.

Speci fi ed mi croorgani sms are aerobi c mesophi li c bacteri a or yeast undesi rable
i n a cosmeti c product and recogni sed as a ski n pathogen speci es that may be
harmful for human health or as an i ndi cati on of hygi eni c fai lure i n the
manufacturi ng process. M i croorgani sms consi dered as speci fi ed
mi croorgani sms are Staphylococcus aureus, Pseudomonas aeruginosa, C andida
albicansand Escherichia coli.

2.1 Mi cr obi ol ogi cal cont r ol of f i nal pr oduct
N o li mi ts for mi crobi al contami nati on of cosmeti cs are enclosed i n the
C osmeti cs D i recti ve or i n the nati onal D ani sh i mplementati on of thi s
D i recti ve. R ecommendati ons on li mi ts of mi crobi al contami nati on i n cosmeti c
products can be found i n the notes of gui dance for the testi ng of cosmeti c
i ngredi ents and thei r safety evaluati on prepared by EU s Sci enti fi c C ommi ttee
of C onsumer Products ( SC C P) ( 5) . T he D ani sh Envi ronmental Protecti on
A gency recommends the use of these levels.

I n SC C P s notes of gui dance, two separate categori es of cosmeti cs products
are defi ned, as vari ous ski n areas can be di fferently sensi ti ve.


C ategory 1


Products speci fi cally i ntended for
chi ldren under 3 years, eye areas and
mucous membranes, leave-on
products.


C ategory 2


O ther products, ri nse-off products.


T he quanti tati ve speci fi cati ons are G enerally acceptable levels:



C ategory 1


T otal vi able count for aerobi c
mesophi li c mi croorgani sms ( bacteri a,
yeast and moulds) not more than 10
2
cfu/g or ml i n 0, 5g or 0.5 ml of the
product




16

C ategory 2


T otal vi able count for aerobi c
mesophi li c mi croorgani sms not more
than 10
3
cfu/g or ml i n 0, 1g or 0.1 ml
of the product



I t i s not acceptable that the followi ng potenti ally pathogeni c mi croorgani sms
are present i n cosmeti c products:

Staphylococcus aureus
Pseudomonas aeruginosa
Candida albicans

T he occurrence of i ndi cator bacteri a i s not menti oned i n SC C P s notes of
gui dance ( 5) . But i t i s generally acknowledged that nei ther the occurrence of
E. coli nor other members of Enterobacteriaceaeare acceptable i n cosmeti c
products.

Q uali tati ve li mi ts:



C ategory 1


Staphylococcus aureus, Pseudomonas
aeruginosa, Candida albicans or E.coli
must not be detectable i n 0.5g or 0.5
ml of the product.


C ategory 2


Staphylococcus aureus, Pseudomonas
aeruginosa, Candida albicansor E.coli
must not be detectable i n 0.1g or
0.1ml of the product


2.2 Mi cr obi al cont ami nat i on
M i crobi ologi cal durabi li ty depends on product composi ti on, content of
preservati ves, manufacturi ng hygi ene, packagi ng, transport and storage. T he
abi li ty of mi croorgani sms to grow and reproduce i n cosmeti c products i s well
known. Water i s essenti al for mi crobi al growth and water-based products
often have a li mi ted durabi li ty, as they are sensi ti ve to mi crobi al growth. M ore
are cosmeti cs i deal nutri ent medi a for mi croorgani sms.

2.2.1 During manufacturing
C ontami nati on duri ng producti on and fi lli ng i n cosmeti c products may occur.

R aw materi als can contri bute to a si gni fi cant level of mi crobi al contami nati on
to the fi ni shed product. T esti ng of raw materi als before use, especi ally those
of natural ori gi n i s i mportant. T he speci fi cati ons of the raw materi als must
i nclude mi crobi ologi cal puri ty. Water i s a raw materi al, and the most common
i ngredi ent. Water must be tested conti nuously for mi crobi al growth. I t mi ght
be necessary to steri li se dei oni sed water to obtai n a suffi ci ent puri ty.


17
M any other condi ti ons of producti on may i nfluence the contami nati on duri ng
manufacturi ng, such as contami nated areas, i nsuffi ci ent manufacturi ng
hygi ene, personnel hygi ene and i nsuffi ci ent preservati on. Effecti ve cleani ng i s
very i mportant.

2.2.2 After opening
From the moment of openi ng the cosmeti c product i s subj ect to constant and
vari able mi crobi al contami nati on from the domesti c envi ronment and the
consumer's hands and body ( the ski n) . Si nce mi croorgani sms are ever present
i n the home, especi ally i n warm, moi st areas, such as bathrooms and ki tchens,
cosmeti cs are exposed to contami nati on wi th both spoi lage and potenti ally
hazardous mi cro-organi sms duri ng use.

Puri ty after openi ng depends on the preservati ve abi li ty of the product,
sui tabi li ty of the packagi ng, storage and appli cati on.

T he followi ng scenari os can contri bute to contami nati on of a cosmeti c
product, fi ngers di pped i n product, spi llage of water i nto product, shampoo
used by several di fferent people

2.3 Pr eser vat i on
T he functi on of preservati on i s for consumer protecti on and preventi on of
spoi lage duri ng normal and reasonable product use. T he preservati ves i nhi bi t
the growth of contami nati ng mi croorgani sms duri ng manufacturi ng, storage
and use by consumers after openi ng.

T he preservati ve effi cacy of a formulati on cannot be predi cted i n every detai l
and must be confi rmed by mi crobi al challenge testi ng ( see secti on 4.4) si nce
the acti vi ty of the preservati ve i s dependent on the effect of i ndi vi dual
i ngredi ents and the packagi ng i n whi ch i t i s stored. Preservati ves must be used
at the lowest concentrati on that ensures thei r effi cacy and thi s must be
determi ned duri ng the product development process.

T he effi cacy of anti mi crobi al preservati on i n cosmeti cs can be tested wi th the
C hallenge test.

T he use of preservati ves i n cosmeti cs cannot replace good manufacturi ng
practi ce.


18

19
3 Laboratories
I n D enmark, there i s no speci fi c regulati on on quali ty requi rements or
accredi tati on of mi crobi ologi cal laboratori es used i n self-control of cosmeti c
products. T hi s gi ves the producer of cosmeti c products freedom but also
responsi bi li ty. When conducti ng mi crobi ologi cal exami nati ons of cosmeti c
products i t i s necessary to pay attenti on to personal hygi ene and to use
appropri ate worki ng techni ques to ensure that only those mi croorgani sms that
are present i n the samples are enumerated. T o help producers wi th thei r own
laboratori es for self-control of cosmeti cs products establi sh a reasonable
laboratory quali ty level, the followi ng chapter on quali ty management i s
i ncluded. M atters related to the worki ng envi ronment are not covered. Please
refer to the D ani sh Worki ng Envi ronment A uthori ty G ui deli ne C .0.18.

3.1 Qual i t y Management
3.1.1 Quality management standards
Q uali ty management i s defi ned i n I SO 9000 ( 6) as coordi nated acti vi ti es to
di rect and control an organi sati on wi th regard to quali ty. Q uali ty management
generally i ncludes establi shment of a quali ty poli cy wi th quali ty obj ecti ves,
and quali ty planni ng, quali ty control, quali ty assurance and quali ty
i mprovement. For laboratori es, I SO 17025 ( 7) speci fi es the general
requi rements for the competences of testi ng and cali brati on laboratori es, and
i ts i mplementati on wi ll ensure compli ance wi th I SO 9000 as well. I SO 17025
descri bes both management requi rements and techni cal requi rements.

I f your company has i mplemented a quali ty management system i n
accordance wi th I SO 9000, all management requi rements and some of the
techni cal requi rements i n I SO 17025 are already i mplemented and wi ll be
easy to extend to the laboratory. I n thi s case i t i s recommended to fully
i mplement I SO 17025.

I f your company has i mplemented a G ood M anufacturi ng Practi ce accordi ng
to D S/EN I SO 22716, a number of both management and techni cal
requi rements si mi lar to those of I SO 17025 are i mplemented and wi ll be easy
to extent to the laboratory. I n thi s case i t i s recommended to follow the I SO
17025 as close as reasonable for the speci fi c laboratory.

Below, we descri be the recommended mi ni mum requi rements for the control
laboratory.


20
3.1.2 Approach to quality management in microbiology laboratories
A common approach for i mplementati on of a practi cal Q A /Q C system i s the
5 D s .

Decidewhere i t i s relevant to perform quali ty management
Describewho does what, how and when
Do what i s deci ded and descri bed
Document what has actually been done
Deemwhether procedures and practi ces gi ve the desi red results and
make i mprovement, i f necessary

I n the followi ng paragraphs each of the D s are descri bed and suggesti ons are
gi ven on how to do the D s.

3.1.2.1 Decide
I n order to deci de where i t i s relevant to perform quali ty management i t i s
suggested fi rst to draw a flowchart of the analyti cal flow from sampli ng to
reporti ng the results. A n example of a flowchart i s seen i n Fi gure 1.



Fi gure 1: Example of a flowchart descri bi ng the steps of a mi crobi al analysi s.

T he flow chart should i denti fy all the cri ti cal steps of an analysi s. T he i dea i s
now to carefully go through each step and deci de whi ch of steps needs a
wri tten operati onal procedure. I n some cases more than one step may be
needed. I t i s i mportant to do thi s very carefully because mi ssi ng procedures
may hamper the analyti cal quali ty. O n the other hand, the number of
procedures should be li mi ted to a mi ni mum; because the more procedures to
be followed the hi gher the chance i s that one or more of them are not
followed.

Sampli ng
R egi strati on
Storage
Pretreatment
A nalysi s
Producti on of medi a
I noculati on
I ncubati on
C ounti ng/i nterpretati on
C alculati on
D ata management
R eport
Q uali ty control
of report
C li ent A rchi ve

21
D eci di ng where to perform quali ty management and i n what detai l i s a
deli cate balance. For i nstance the temperature i s a cri ti cal factor i n a mi crobi al
analysi s, and a number of questi ons ari se on how to manage the temperature.
For i nstance: should the i ncubator temperature be moni tored weekly, dai ly or
recorded conti nuously? I s i t O K to moni tor the i ncubator temperature i n one
poi nt, or should the spati al vari ati on also be known? Should the thermometer
be cali brated at each C at all the temperatures, where the thermometer i s
used, or i s i t O K to i ntra- or extrapolate from two cali brati on poi nts? T he art
of i mplementi ng good quali ty management i s to fi nd a sati sfactory level of
Q A /Q C wi thout overdoi ng i t. A s a rule of thumb, procedures or devi ces that
di rectly i nfluence the analyti cal results should be gi ven the hi ghest pri ori ty.

3.1.2.2 Describe
When the steps requi ri ng an operati onal procedure have been deci ded, the
procedures should be wri tten as short and clear as possi ble wi thout mi ssi ng
any poi nts. T he procedures may be very si mple consi sti ng of a reference to a
standard, for i nstance the analyti cal methods or sampli ng procedures, a
reference to a mai ntenance manual, or i t may be procedures enti rely wri tten
by the laboratory, for i nstance procedures for control of i ncubators or
thermometers. T he procedures may have annexes such as templates for
sample regi strati on. A number of the procedures may be shared wi th other
laboratori es i n the company, such as procedures for regi strati on of chemi cals,
and cali brati on of pi pettes and balances. I t i s suggested that the procedures
follow a common format, whi ch i ncludes at least the followi ng:

1. A uni que ti tle
2. T he purpose of the procedure
3. T he process
4. R esponsi bi li ti es
5. N ame of the author and the approvi ng person
6. D ate of approval, date of expi ry and edi ti on.

3.1.2.3 Do
T hi s part i s qui te si mple: Y ou j ust have to do what was deci ded and
descri bed. H owever, thi s i s also the hard part. T he experi ence shows that
procedures are often forgotten and left alone i n the bi nder or i n the drawer. A
few thi ngs can be done to reduce the li keli hood of forgotten and unfollowed
procedures:

T he procedures should be revi ewed by the staff doi ng the work to
assure that the procedures are practi cal and i n accordance wi th the
way the work i s actually carri ed out i n the lab ( assumi ng the work i s
done i n a proper way) .
T he procedures should be readi ly avai lable to all relevant staff.
A year plan for mai ntenance and cali brati on should be made and
followed, and made readi ly avai lable ( e.g. posted on the wall) .
Educati on of and di scussi on among the staff members.


22
3.1.2.4 Document
D ocument what has actually been done. T hi s requi rement i s i ncluded for at
least four reasons:

I t provi des a tool for i denti fyi ng errors and thereby preventi ng the
same errors to take place i n the future work.
I t enables your company to perform i nternal audi t to veri fy that the
acti ons to be taken were actually taken.
I t enables audi t to be done by an i ndependent thi rd party, i f necessary.
I n case of complai nts, or i f unusual results have been obtai ned, the
laboratory can control and prove that the quali ty of the analysi s i s
suffi ci ent and the results are reli able.

T he requi rement for documentati on coversall operati ons that may i nfluence
the quali ty of the analysi s. I n a good Q A /Q C system, all relevant ( and only the
relevant) operati ons are descri bed i n the procedures, and must be
documented. T he best way i s to provi de templates where the work carri ed out
can be recorded. Examples of documentati on are si gned templates for
sampli ng, control and cali brati on of volumetri c equi pment, substrate control,
employees educati on, quali ty control of reports etc.

A short rule of thumb i s: i t must be possi ble for an audi tor by a si gned
document to veri fy that a certai n operati on has been performed when and by
whom.

3.1.2.5 Deem
Even the best Q A /Q C system can be i mproved and fi ne-tuned. T herefore i t i s
necessary to evaluate the system peri odi cally. T hi s i s done through several
methods.

O ne of the methods i s audi t, i nternal ( and external) , for whi ch requi rements
usually are lai d down i n the quali ty management /G M P system. D uri ng the
audi ts, i nconsi stenci es between procedures and the actual work are i denti fi ed.
I t must be deci ded i n each case, i f the procedure or the practi se should be
changed.

A nother method i s i nternal quali ty control, whi ch i s a program carri ed out by
the laboratory to show that the vari abi li ty i s under control, usi ng tools such as
standards, repli cate samples and parti ci pati on i n profi ci ency tests. I f the
vari abi li ty i s deemed to be too hi gh, acti ons must be taken to i mprove the
procedures.

A thi rd mechani sm, and maybe the most i mportant, i s the dai ly di scussi ons
among the staff and wi th colleagues from other laboratori es. I t i s i mportant to
encourage open di scussi ons between all lab employees, and to be wi lli ng to
make changes accordi ngly.


23
3.1.3 Requirements to quality management
T hi s secti on descri bes a recommended mi ni mum of requi rements to the
mi crobi al control laboratory. T he recommendati ons are based on I SO 17025,
EA - 4/10 - Accreditation in Microbiological Laboratories (8) and D S/EN I SO
22716. H owever, full i mplementati on of I SO 17025 i s preferable to obtai n
consi stently reli able results.

3.1.3.1 Document control
T he laboratory must establi sh and mai ntai n procedures to control all
documents related to the quali ty of the analyses. T hese procedures should
follow the requi rements of I SO 17025 or I SO 22716.

3.1.3.2 Personnel
T he laboratory management must ensure the competences of all personnel
i nvolved i n planni ng, performi ng, i nterpreti ng and reporti ng of tests and/or
cali brati ons. T esti ng and cali brati on must be performed or supervi sed by an
experi enced person wi th a degree i n mi crobi ology or equi valent, or wi th
extensi ve relevant experi ence. T he staff must have relevant practi cal worki ng
experi ence and have recei ved adequate trai ni ng i n basi c techni ques such as
plate pouri ng, counti ng of coloni es, asepti c techni ques etc.

Where a method or a techni que i s not regular i n use, veri fi cati on of personnel
performance before testi ng i s undertaken may be necessary. C ri ti cal i nterval
between performances of tests should be establi shed.

T he laboratory must mai ntai n j ob descri pti ons and documentati on of staff
quali fi cati ons.

3.1.3.3 Environment
T he laboratory must ensure that the envi ronmental condi ti ons do not
i nvali date the results or adversely affect the requi red quali ty of any
measurement.

T he laboratory must moni tor, control and record the envi ronmental
condi ti ons as requi red by the relevant methods or procedures or when they
i nfluence the quali ty of the result. D ue attenti on must be pai d, for example, to
bi ologi cal steri li ty and temperature.

T he laboratory should be arranged so as to mi ni mi se the ri sks of cross
contami nati on. T hi s can be achi eved for example by constructi ng the
laboratory accordi ng to the no way back pri nci ple, where all samples and
cultures only travel i n one di recti on through the laboratory. For i nstance,
cultures or i ncubated plates should never enter medi a and sample preparati on
rooms. A lternati vely, acti vi ti es can be separated by ti me and space and
appropri ate precauti ons can be taken to ensure test and sample i ntegri ty, such
as use of sealed contai ners and hygi eni c practi ses.

I t i s good practi ce to have separate locati ons or clearly desi gnated areas for:
sample recei pt and storage
sample preparati on and challenge test preparati ons i ncludi ng steri le
room or steri le cupboards. Powdery products should be handled
separately.
i ncubati on and sample exami nati on

24
medi a and equi pment preparati on i ncludi ng steri li sati on
decontami nati on

T he area for washi ng after decontami nati on may be shared wi th other
laboratori es provi ded that transfer of substances that could adversely affect
mi crobi al growth i s prevented.

Space should be suffi ci ent to allow work areas to be kept clean and ti dy.

R ooms should be appropri ately venti lated and at a sui table temperature.

R educti on of contami nati on may be achi eved by havi ng:
smooth surfaces on walls, cei li ngs, floors and benches. T i les are not
recommended as bench coveri ng materi al
concave j oi nts between the floor, walls and cei li ng;
mi ni mal openi ng of wi ndows and doors whi le tests are bei ng carri ed
out
sun shades placed on the outsi de;
flui d conveyi ng pi pes not passi ng above work surfaces unless placed i n
hermeti cally sealed casi ngs
a dust-fi ltered ai r i nlet for the venti lati on system;
separate hand-washi ng arrangements, preferably non-manually
controlled;
cupboards up to the cei li ng;
no rough and bare wood;
wooden surfaces of fi xtures and fi tti ngs adequately sealed;
stored i tems and equi pment arranged to faci li tate easy cleani ng;
no furni ture, documents or other i tems other than those stri ctly
necessary for testi ng acti vi ti es.

T here must be a cleani ng programme for laboratory fi xtures, equi pment and
surfaces.

L aboratory coats must be worn i n the laboratory and must be removed before
leavi ng the area.

A ccess to the mi crobi ology laboratory should be restri cted to authori sed
personnel.

See also I SO 21148 G eneral i nstructi ons for mi crobi al exami nati on.

3.1.3.4 Test and calibration methods and method validation
T he laboratory must use appropri ate methods and procedures for all tests
and/or cali brati ons. T hese i nclude sampli ng, handli ng, transport, storage and
preparati on of i tems to be tested or cali brated, and where appropri ate, an
esti mati on of the measurement uncertai nty as well as stati sti cal techni ques for
analysi s of test and/or cali brati on data.

I f laboratory-developed methods or non-standard methods are used, they
must be vali dated i n house. Standard methods used on matri ces not speci fi ed
i n the standard must be vali dated as well.

V ali dati on of a mi crobi al method requi res a substanti al amount of work. T he
standards for vali dati on ( D S/EN I SO 16140 or D S/EN V I SO /T R 13843)

25
should be followed. See also EA - 4/10. T o avoi d the vali dati on procedure i t i s
strongly recommended to use the I SO methods descri bed i n secti on 4.
H owever, even when a complete vali dated method i s used, the laboratory sti ll
needs to veri fy on a regular basi s that performance can be met, e.g. by use of
spi ked samples or reference materi als.

T he laboratory must have and must apply procedures for esti mati ng the
uncertai nty of measurements. R i gorous, metrologi cally and stati sti cally vali d
calculati on of uncertai nty of mi crobi al analyses cannot be performed. I t i s
appropri ate to base the esti mate of uncertai nty on repeatabi li ty and
reproduci bi li ty data, combi ned wi th bi as esti mati on from parti ci pati on i n
profi ci ency testi ng or use of standard materi als when possi ble. T he i ndi vi dual
components of uncertai nty must be demonstrated to be under control and
thei r contri buti on to the vari abi li ty of the evaluated results. Some components
such as pi petti ng, wei ghi ng and di luti on effects can be readi ly measured and
evaluated. O ther components such as sample stabi li ty and sample preparati on
cannot be evaluated i n a stati sti cal manner but thei r i mportance should be
consi dered. Se also secti on 3.1.3.6

C alculati ons and data transfers must be subj ect to appropri ate checks i n a
systemati c manner.

3.1.3.5 Equipment
T he laboratory must be furni shed wi th all i tems of sampli ng, measurements
and test equi pment requi red for the correct performance of the tests.

T he laboratory must operate a documented programme for the mai ntenance,
cali brati on, and performance veri fi cati on of i ts equi pment.

M ai ntenance of essenti al equi pment must be carri ed out at speci fi ed i ntervals
and detai led records must be kept.

T he laboratory must establi sh a programme for the cali brati on and
performance veri fi cati on of equi pment whi ch di rectly i nfluences the test
result. Examples of cali brati on and performance checks are gi ven i n EA -4/10.
Before taken i nto servi ce, equi pment must be checked to establi sh that i t
meets the requi rements.

T he temperature i s an i mportant parameter and the laboratory must have
temperature measuri ng devi ces of an appropri ate quali ty. C ali brati on of the
devi ces must be traceable to nati onal or i nternati onal standards for
temperature.

T he stabi li ty of temperature and the uni formi ty of the temperature
di stri buti on i n i ncubators, water baths and ovens must be establi shed i ni ti ally
and documented wi th respect to typi cal uses. T he i ni ti al vali dati on must be
checked and recorded after each si gni fi cant repai r or modi fi cati on, and
operati ng temperatures must be moni tored and recorded.

A utoclaves must be capable of meeti ng the ti me and temperatures speci fi ed i n
the methods used. Pressure cookers only equi pped wi th a pressure gauge are
not acceptable. I ni ti al vali dati on must i nclude operati ng cycles and load
confi gurati ons used i n practi ce. T emperature sensors should be posi ti oned
i nsi de contai ners fi lled wi th li qui d.


26
M oni tori ng of autoclavi ng should be carri ed out usi ng a thermocouple and a
recorder to produce a chart or pri ntout, or by the use of chemi cal or bi ologi cal
i ndi cators. A utoclave tape and i ndi cator stri ps should only be used to show
that the load has been processed.

Balances and wei ghts must be cali brated traceably at regular i ntervals.

L aboratori es must carry out i ni ti al veri fi cati on of volumetri c equi pment and
make regular checks to ensure that the equi pment i s performi ng wi th the
requi red speci fi cati on. V eri fi cati on should not be necessary for glassware
whi ch has been certi fi ed to a speci fi c tolerance. Equi pment should be checked
for the accuracy of the deli vered volume agai nst the set volume ( for several
di fferent setti ngs i n the case of vari able volume i nstruments) and the preci si on
of the repeat deli veri es should be measured. Si ngle use di sposable volumetri c
equi pment should be obtai ned from I SO 9000 certi fi ed manufacturers. A n
i ni ti al vali dati on of the sui tabi li ty of the equi pment must be carri ed out.

C onducti vi ty meters, oxygen meters, pH meters and other si mi lar i nstruments
should be veri fi ed regularly or before each use. T he buffers used for
veri fi cati ons purposes should be stored i n appropri ate condi ti ons and should
be marked wi th an expi ry date, and thei r use documented.

3.1.3.6 Reagents and culturemedia
L aboratori es must have procedures for selecti ng and purchasi ng of the
servi ces and suppli es i t uses that affect the quali ty of the tests. T o ensure that
the quali ty of the reagents and medi a used i s appropri ate for the test
concerned, i t i s recommended to obtai n reagents and medi a from I SO 9001
certi fi ed manufacturers. I n thi s case an i ni ti al vali dati on of the sui tabi li ty of
the equi pment must be carri ed out. I n case i n-house prepared medi a or ready-
to-use medi a from non-certi fi ed manufacturers are used, each batch should be
vali dated accordi ng to I SO 11133.

L aboratori es must ensure that all reagents ( i ncludi ng stock soluti ons) , medi a,
di luents, and other suspendi ng flui ds are adequately labelled to i ndi cate, as
appropri ate, i denti ty, concentrati on, storage condi ti ons, preparati on date,
vali dated expi ry date and /or recommended storage peri ods. T he person
responsi ble for preparati on should be i denti fi able from records.

T he laboratory must keep a record of all purchased reagents, medi a etc.

3.1.3.7 Internal quality control
I nternal quali ty control consi sts of all the procedures undertaken by a
laboratory for the conti nuous evaluati on of i ts work. T he mai n obj ecti ve i s to
ensure the consi stency of results day-to-day and thei r conformi ty wi th defi ned
cri teri a.

A programme of peri odi c checks i s necessary to demonstrate that vari abi li ty
( i .e. between analysts and between equi pment and materi als etc.) i s under
control. A ll tests i ncluded i n the control of the products need to be covered.
T he programme may i nvolve:
the use of spi ked samples
the use of reference materi als ( i ncludi ng profi ci ency testi ng scheme
materi als)
repli cate testi ng


27
I t i s recommended to follow opti on 1 descri bed i n I SO /T S 19036:2006 ( 9) .

A laboratory may use a test at rare. I t i s recogni sed that i n such cases an
ongoi ng i nternal quali ty control programme may be i nappropri ate and that a
scheme for demonstrati ng sati sfactory performance whi ch i s carri ed out i n
parallel wi th the testi ng, may be more sui table.

3.1.3.8 External quality assessment (proficiency testing)
I f avai lable laboratori es should regularly parti ci pate i n profi ci ency testi ng
whi ch are relevant to thei r acti vi ti es, bi as should be assessed and the vali di ty of
the whole quali ty system should be checked.

3.1.3.9 Internal audit
T he company must peri odi cally, and i n accordance wi th a predetermi ned
schedule and procedure conduct i nternal audi ts of the acti vi ti es of the control
laboratory to veri fy that i ts operati ons conti nue to comply wi th the
requi rements of the laboratory quali ty management system. T he i nternal audi t
programme must address all elements of the laboratory management system.
I nternal audi t i s a requi rement of both I SO 9001 and I SO 22716 and can be
extended to cover the laboratory as well. T he audi t must be carri ed out by
speci ally desi gnated personnel havi ng both quali ty management competence
and techni cal competence. I f the company does not have i ndependent
techni cal competence, external techni cal advi sors can be i ncluded i n the audi t.

I nternal audi t follow-up must confi rm the sati sfactory completi on of the audi t
or sati sfactory i mplementati on of correcti ve acti ons.

T he area of acti vi ty audi ted, the audi t fi ndi ngs and correcti ve acti ons that
ari se from them must be recorded.

28

29
4 Analytical Methods
T hrough i ts techni cal commi ttee for C osmeti cs I SO /T C 217, the i nternati onal
organi zati on for standardi zati on ( I SO ) has presented a number of new
i nternati onal standards for mi crobi ologi cal exami nati on of cosmeti c products.
T hese standards are detai led and cover the needs of a large part of avai lable
cosmeti c products. I t i s strongly recommended to i ncorporate the use of I SO
standards i n mi crobi ologi cal testi ng of cosmeti c products. I n the followi ng
some of these standards are presented.

I n genera, the effi cacy of anti mi crobi al preservati on i n cosmeti cs can be tested
by the C hallenge test. T he test shows the abi li ty of the cosmeti c product to
reduce the count of mi cro-organi sms after a contami nati on. C hallenge testi ng
i s mandatory for all cosmeti c products that under normal condi ti ons of
storage and use may deteri orate or form a ri sk to the consumer. A s nei ther a
legal nor a uni versal challenge test method i s avai lable; i t i s up to the
manufacturer to deci de on the detai ls of the test to be used.

4.1 St andar ds under devel opment
T he techni cal commi ttee for C osmeti cs I SO /T C 217 i s developi ng two new
gui deli nes. T he laboratori es should be updated on the status of the standards.

I SO /C D 29621 i s a gui deli ne for the ri sk assessment and i denti fi cati on of
mi crobi ologi cally low-ri sk products. I n the commi ttee draft stage the gui deli ne
has been approved for regi strati on as a draft i nternati onal standard.

I SO /N P 11930 i s general i nformati on on evaluati on of the anti mi crobi al
protecti on and has been approved as a new proj ect.

4.2 Neut r al i zat i on and pr epar at i on of wat er -i mmi sci bl e sampl es.
M i crobi al exami nati on of cosmeti cs has at least two i nherent problems,
namely the toxi c properti es of the conservati on systems and water-
i mmi sci bi li ty of some products. T hese problems are very i mportant to deal
wi th i n order to achi eve a correct result.

C osmeti c products are usually conserved to mai ntai n a hygi eni cally good
quali ty duri ng storage and to prevent growth of mi croorgani sms duri ng use of
the product. T he conservati on system i s li kely to i nhi bi t growth on agar plates
or i n enri chment broths and thereby li kely to gi ve ri se to false negati ve results.
T herefore the conservati on system must be i nacti vated or neutrali zed before
analysi s.

T he i ni ti al steps of mi crobi al exami nati ons i nvolve preparati on of an i ni ti al
suspensi on of the mi croorgani sms i n the sample. T hi s suspensi on i s
subsequently di luted to achi eve sub-samples wi th appropri ate concentrati ons
of mi croorgani sms. I f the cosmeti c product to be tested i s water-i mmi sci ble,

30
the di luents should contai n a sui table amount solubi li si ng agents such as
Polysorbat 80.

Samples wi th anti mi crobi al properti es must be neutrali zed before analysi s.
T hi s i s done by addi ng neutrali zers to the di luents. R elevant neutrali zers are
suggested i n the speci fi c standards ( see secti on 4.3) and i n A ST M E 1054
( 10) . I n all cases and whatever methodology, the neutrali zati on of the
anti mi crobi al properti es of the product must be checked and vali dated. T he
vali dati on procedures are descri bed i n each speci fi c standard. T he pri nci ple of
the vali dati on procedure i s to add a known amount of the relevant test
strai n( s) to the i ni ti al sample suspensi on and compare the number of mi cro-
organi sms wi th a control wi thout the sample. I n case of quali tati ve or
presence/absence tests, growth and characteri sti cs of the coloni es are
exami ned.

4.3 Exami nat i on of mi cr obi al qual i t y of pr oduct s
C osmeti c products must be subj ected to mi crobi ologi cal control as descri bed
i n C hapter 2. A number of I SO standards have been developed to gi ve
gui deli nes for the manufacturers. I t i s recommended that all laboratori es use
the I SO standards descri bed i n thi s secti on.

4.3.1 ISO 21149 Cosmetics Microbiology Enumeration and detection of
aerobic mesophilic bacteria
T he standard contai ns gui deli nes for enumerati on and detecti on of mesophi li c
aerobi c bacteri a i n cosmeti cs by counti ng coloni es on agar medi um after
aerobi c i ncubati on or by checki ng absence of bacteri al growth after
enri chment.

4.3.2 ISO 18415Cosmetics Microbiology Detection of specified and non-
specified micro-organisms.
T he standard contai ns gui deli nes for the detecti on and i denti fi cati on of
speci fi ed mi croorgani sms i n cosmeti c products as well as for the detecti on and
i denti fi cati on of other ki nds of aerobi c mesophi li c non-speci fi ed
mi croorgani sms i n cosmeti c products. T he standard contai ns gui deli nes for
the detecti on of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus,
or Candida albicans. T he detecti on i s carri ed out by enri chment i n a non-
selecti ve broth followed by i solati on and i denti fi cati on. T he i denti fi cati on
consi sts of gram stai ni ng, catalase- and oxi dase test followed by the use of an
i denti fi cati on test ki t.

4.3.3 ISO- methods for the detection of specific microorganisms: E. coli (ISO
21150), Pseudomonas aeruginosa (ISO 22717), Staphylococcus aureus
(ISO22718) and Candida albicans (ISO 18416).
I n all four standards, the fi rst step i s enri chment i n a non-selecti ve broth to
i ncrease the number of mi croorgani sms wi thout the ri sk of i nhi bi ti on by the
selecti ve i ngredi ents present i n the growth medi a. T he second step i s i solati on
on selecti ve medi a followed by i denti fi cati on tests.


31
4.3.4 ISO/FDIS 16212 Cosmetics Microbiology Enumeration of yeast and
mould.
T hi s standard i s currently avai lable as a draft. T he method i nvolves
enumerati on of coloni es on Sabouraud dextrose chlorampheni col agar
medi um. Enumerati on may be carri ed out as a pour plate, surface spread or
membrane fi ltrati on method.

4.4 Ef f i cacy of pr eser vat i on Pr oposal f or a Chal l enge t est -not
val i dat ed
A challenge test i s a procedure i n whi ch a product i s challenged by exposure
to speci fi ed types of bacteri a and fungi . T he product i s then i ncubated at a
gi ven temperature, samples are taken at speci fi ed i ntervals and the number of
mi croorgani sms i s determi ned. N ormally the product i s challenged to the
mi croorgani sms Staphylococcus aureus, Pseudomonas aeruginosa and Candida
albicans, but i n-house mi croorgani sms, found as contami nati ons i n the
products, may be used for addi ti onal speci fi c purposes of challenge testi ng.
T he anti mi crobi al properti es of the product are acceptable, i f a si gni fi cant
decrease or no i ncrease i n vi able count of mi cro-organi sms i s seen, when the
product i s tested under consi derati on to storage and use.

T he challenge test should be performed both duri ng development of the
preservati ve system and as an evaluati on of the protecti on effi cacy i n i ntact,
i n-use or i n endi ng cosmeti c products.

T here are no approved I SO standards avai lable for challenge tests. U nti l the
new I SO standard i s avai lable, the procedure below i n secti on 4.4.1 i s
recommended. T he procedure has not been vali dated why i t i s suggested to
perform an i n-house vali dati on before use. Si mi lar challenge tests are
descri bed i n the European Pharmacopoei a ( 11) , the U S Pharmacopoei a ( 12)
and i n A ST M ( the A meri can Soci ety for T esti ng and M ateri als) ( 13) . T he
methods are si mi lar but di ffer i n the detai led procedures, test organi sms,
cri teri a for passi ng the test and requi rements for vali dati on. I nexperi enced
laboratori es should send the samples to an accredi ted laboratory. A nother
alternati ve i s to contact the producer/deli verer of the preservati ve; they can
usually provi de laboratory capaci ty.

4.4.1 Proposed procedure for challenge testing
T he product i s challenged wi th cell/spore suspensi ons ( 10
8
cells/spores pr. ml)
at a concentrati on of 10
5
10
6
cells/spores pr. ml. T he challenged product i s
i ncubated at 22 C 1 C i n the dark. Samples for determi nati on of plate
counts are taken after 0, 7, 14, 21 and 28 days.

C hallenge the product wi th

Staphylococcus aureus
Pseudomonas aeruginosa
Candida albicans
Aspergillus niger

O ther relevant organi sms, such as commonly observed contami nati ng
organi sms, should also be used as test organi sms.

32

U se L etheen agar whi ch contai ns the neutrali sers polysorbat 80 and leci thi n,
or C asei n soya bean di gest agar for bacteri a, and Saboraud-glucose agar
wi thout anti bi oti cs for fungi .

T he i ni ti al plate counts are determi ned i mmedi ately after addi ti on of the test
organi sms. T he concentrati on of bacteri a should reach a log 3 reducti on after
14 days, and there should be no i ncrease i n the concentrati on after day 14.
T he concentrati on of fungi should reach a log 2 reducti on after 14 days and
there should be no i ncrease i n concentrati on after day 14.

T he effi cacy of the neutrali sers shall be vali dated accordi ng to the speci fi c
I SO standards ( se secti on 4.3) or accordi ng to A ST M E 1054.









33
5 References
1.BEK nr 422 af 04/05/2006 ( retsi nfo.dk)

2. C ounci l D i recti ve 76/768/EEC of 27 July 1976 on the approxi mati on of the
laws of the M ember States relati ng to cosmeti c products.

3. C oli pa gui deli nes; cosmeti c good manufacturi ng practi ces, 1994 ( pdf can be
downloaded free of charge from coli pa.com) .

4. V an der M aren L . G ui deli nes for good manufacturi ng practi ce of cosmeti c
products ( G M PC ) . C ounci l of Europe, H ealth protecti on of the consumer,
I SBN : 98-871-2849-9 ( 1995) .

5. T he SC C P s notes of gui dance for the testi ng of cosmeti c i ngredi ents and
thei r safety evaluati on 6th revi si on D ecember 2006 li nk.

6. Standards I SO 9000:2005 or I SO 9001:2000 can be obtai ned from D ansk
Standards net shop.

7. Standard I SO 17025:2005 can be obtai ned from D ansk Standards net shop.

8. EA -4/10 ( rev.2) A ccredi tati on for L aboratori es Performi ng M i crobi ologi cal
T esti ng can be downloaded from http://www.european-accredi tati on.org

9. I SO /T S 19036:2006 M i crobi ology of food and ani mal feedi ng stuffs --
G ui deli nes for the esti mati on of measurement uncertai nty for quanti tati ve
determi nati ons

10. E 1054 02 T est M ethods for Evaluati on of I nacti vators of A nti mi crobi al
A gents astm.org. N ew versi on A ST M E1054 - 08 Standard T est M ethods for
Evaluati on of I nacti vators of A nti mi crobi al A gents

11. European Pharmacopoei a C ommi ssi on Effi cacy of anti mi crobi al
preservati on. Strasbourg, France: European Pharmacopoei a C ommi ssi on;
European Pharmacopoei a EP 5.1.3, 1997

12. U .S. Pharmacopei a. U SP X X I

13. E 640 78( 1998) T est M ethod for Preservati ves i n Water-C ontai ni ng
C osmeti cs astm.org N ew versi on A ST M E640 - 06 Standard T est M ethod
for Preservati ves i n Water-C ontai ni ng C osmeti cs.


34

Responsible person: Barabra Mentre: barbara.mentre@cec.eu.int



EUROPEAN COMMISSION
ENTERPRISE AND INDUSTRY DIRECTORATE-GENERAL

Chemicals and construction
Chemicals

Brussels, 28
th
November 2005
M/375


STANDARDISATION MANDATE ASSIGNED TO CEN CONCERNING
GOOD MANUFACTURING PRACTICE FOR COSMETICS PRODUCTS
1. MOTIVATION
This standardisation mandate relates to Council directive 76/768/EEC of 27 July 1976 on the
approximation of the laws of the Member States relating to cosmetic products (hereinafter the
Cosmetics Directive). The directive based on article 95 of the Treaty aims to insure free
circulation of cosmetic products into the Community market. To that end it determines at
Community level the regulations which must be observed as regards the composition,
labelling and packaging of cosmetic products
According to article 7a (1) of the Cosmetics Directive the manufacturer or his agent or the
person to whose order a cosmetic product is manufactured or the person responsible for
placing an imported cosmetic product on the Community market shall for control purposes
keep [inter alia] readily accessible to the competent authorities [] the method of
manufacture complying with the good manufacturing practice [].

However, no good manufacturing practice in the cosmetic sector is currently defined at
Community level. In order to avoid unnecessary legislation and in view of better regulation
and simplifying Community legislation , creation of a standard in this area would be the best
approach. Indeed the standard would allow to relate to a common reference in this technical
field without creating burdensome and avoidable legislation.
2. DESCRIPTION OF THE MANDATED WORK
The Commission invites the ESO to establish a European standard giving guidance for the
production, control, storage and shipment of cosmetic products.
For the purpose of this mandate, Cosmetic products shall mean any substance or
preparation intended to be placed in contact with the various external parts of the human
body (epidermis, hair system, nails, lips and external genital organs) or with the teeth and the
mucous membranes of the oral cavity with a view exclusively or mainly to cleaning them,
perfuming them, changing their appearance and/or correcting body odours and/or protecting
them or keeping them in good condition. (article 1 of the Cosmetics Directive).

In order to facilitate a wide acceptance of the standard, the ESO will take into account, as
much as possible, the work undertaken by the international standards organisations on the
same subject, and, in particular, the standard(s) or other standardisation deliverables under
preparation or published as a result of ISO/TC 217 Cosmetics, particularly the draft under
preparation under reference ISO/CD 22716 Cosmetics - Good manufacturing practice
(GMP). The ESO will avoid any unnecessary duplication of work with the international
standards organisations, particularly by using the provisions for parallel approval procedures
provided for in the existing co-operation agreements (Vienna Agreement)
3. BODIES TO BE ASSOCIATED
As appropriate, the ESO will ensure that the representative organisations of consumers
interests (ANEC), environmental protection (ECOS), workers (ETUI-REHS), small and
medium-size enterprises (NORMAPME) and every relevant industrial organisation, in
particular COLIPA
1
, take part in the elaboration of the standard.
4. EXECUTION OF THE MANDATED WORK
The ESO will deliver a draft European standard and submit it to a public enquiry by
2006-02-28.
The ESO will publish a final European standard by 2007-08-31. By that date the standard will
be available in English, French and German, and the correct title of the standard will be
available in the other Community languages.
At the latest six months after the publication of the European standard by the ESO, it will be
implemented as a national standard by all national standards institutes in all Member States
and every conflicting national standard will be withdrawn.
The acceptance of this mandate by one of the ESO will trigger the standstill period referred to
in Article 7 of Directive 98/3/EC of 22 June 1998.


1
The European Cosmetic Toiletry and Perfumery Association.

37
Appendix 2









Challenge test of water miscible
cosmetic products
Claus Jrgensen, DHI
Ann Detmer, DHI

38

39



Table of contents




1.1 PU R PO SE 40
1.2 PR I N C I PL E 40
1.3 SC O PE 40
1.4 R EFER EN C ES 40
1.5 EQ U I PM EN T 41
1.6 T EST O R G A N I SM S 41
1.7 C H EM I C A L S A N D SU BST R A T ES 41
1.8 PR O C ED U R E 41
1.8.1 Preparation of inocula 41
1.8.2 Preparation of challengetest samples 42
1.8.3 Sampling and incubation 42
1.8.4 Analysis of samples 42
1.9 V A L I D I T Y C R I T ER I A 43
1.9.1 Validity of analyses 43
1.9.2 Validity of test organisms and neutralising agent 43
1.10 A C C EPT A N C E C R I T ER I A 43










40
1.1 Pur pose
T he purpose of thi s procedure i s to determi ne the effecti veness of
anti mi crobi al preservati ves used to stop proli ferati on or to prevent mi crobi al
contami nati on i n cosmeti c products.

T he procedure i s parti cularly useful duri ng development of new products.

1.2 Pr i nci pl e
T he cosmeti c product i s challenged by addi ng 105-106 C FU /ml or g of a
si ngle strai n of test mi croorgani sm vi a cell suspensi ons of approxi mately 108
cells/spores pr. ml and i s i ncubated at 20-25 C protected from li ght. A fter 0,
7, 14, 21 and 28 days of i ncubati on, samples are taken to determi ne the
number of mi croorgani sms by plate count. T he product wi ll pass the test, i f
the analyses are vali d and results are i n compli ance wi th the acceptance
cri teri a.

1.3 Scope
T hi s procedure can be used for water mi sci ble products.

1.4 Ref er ences
European Pharmacopoei a ( 6.0) . M ethod 5.1.3 Effi cacy of anti mi crobi al
preservati on. 01/2008:50103.

A ST M E 640 78 Standard test method for preservati ves i n water contai ni ng
cosmeti cs. A meri can Soci ety for T esti ng and M ateri als, 1991 ( reapproved
1998) . A nnual Book of A ST M Standards. A ST M , Phi ladelphi a, Pa.

I SO 16212:2008 C osmeti cs M i crobi ology Enumerati on of yeast and mold.
Fi rst edi ti on 2008-10-07.

I SO 22149:2006 C osmeti cs M i crobi ology Enumerati on and detecti on of
aerobi c mesophi li c bacteri a. Fi rst edi ti on 2006-03-01.

I SO 22717:2007 C osmeti cs M i crobi ology D etecti on of C andi da albi can.
Fi rst edi ti on 2007-07-15.

I SO 22717:2006 C osmeti cs M i crobi ology D etecti on of Pseudomonas
aerugi nosa. Fi rst edi ti on 2006-02-01.

I SO 22718:2006 C osmeti cs M i crobi ology D etecti on of Staphylococcus
aureus. Fi rst edi ti on 2006-02-01.

41
1.5 Equi pment
Balance ( 0.01g)

Equi pment for homogeni sati on ( e.g. stomacher)

pH meter ( 0.1 pH uni ts)

O ne autoclave to clean equi pment and medi a, and another autoclave to treat
contami nated waste

I ncubators: 20-25 C and 30-35 C .

O ther standard equi pment for mi crobi ologi cal laboratori es necessary to
perform steri le work, sample handli ng and work wi th cultures.

1.6 Test or gani sms
Pseudomonas aeruginosa C C U G 22801 ( A T C C 9027; N C I M B 8626; C I P
82.118)
Staphylococcus aureusC C U G 10778 ( A T C C 6538; N C T C 10788; N C I M B
9518; C I P 4.83)
Candida albicansC C U G 19915 ( A T C C 10231; N C PF 3179; I P 48.72)
Aspergillus niger C C U G 18919 ( A T C C 16404; I M I 149007; I P 1431.83)

T he organi sms can be obtai ned from type culture collecti ons.

1.7 Chemi cal s and subst r at es
For P. aeruginosa, S. aureusand C. albicansa tryptone ( 1, 0 g/l) sodi um
chlori de ( 8, 5 g/l) di luent i s used.

For A. niger a tryptone ( 1, 0 g/l) sodi um chlori de ( 8, 5 g/l) soluti on wi th
Polysorbat 80 ( 0.5 g/l) i s used.

A gar for culti vati on and quanti fi cati on of P. aeruginosa, S. aureus and C.
albicans: L etheen agar

A gar for culti vati on and quanti fi cati on of A. niger: Saboroud-glucose
( Saboroud-dextrose) agar wi thout anti mi crobi als.

L etheen agar contai ns polysorbat 80 and leci thi n, whi ch i nacti vates many
anti mi crobi al preservati ves.

1.8 Pr ocedur e
1.8.1 Preparation of inocula
Prepare stock and worki ng cultures of the test organi sms as descri bed by the
suppli er.


42
Before the test, inoculate the surface of the agar with recently grown stock or
working cultures of each of the specified microorganisms. Incubate the agar plates
with P. aeruginosa and S. aureus at 30 C to 35 C in 18 to 24 hours, agar plates
with C. albicans at 20 C to 25 C in 48 hours, and agar plates with A. niger at 20
C to 25 C in 1 week or until good sporulation is obtained. Examine the agar
plates for contamination before use.

To produce challenge suspensions, harvest the bacterial and fungal cultures by
transferring colonies from the agar plates to diluents to a concentration of
approximately 10
8
P. aeruginos or S. aureus pr. ml or approximately 10
7
C.
albicans or A. niger pr. ml. Determine the number of colony forming units (CFU)
pr. ml immediately after resuspension by plate count on the specified agar.

In order to make quick assessments of the concentration of organisms in the
challenge suspensions, it is advised that the laboratory compares different
concentrations of the specific test organisms (determined by plate count) in diluent
against rapid detection methods, i.e. absorbance at 620 nm (typical absorbance of
10
8
bact/ml is 0.15 to 0.46 at 1 cm), turbidometry or visual comparison to
McFarland standards.

1.8.2 Preparation of challenge test samples
Transfer 10 times 100 g or 10 times 100 ml of the product to be tested into sterile
double Stomacher bags (one bag inside the other). Of these, two are used for each
of the four test organisms and two are used as uninoculated controls.

Inoculate the test samples by adding no more than 1 ml of challenge suspension pr.
100 ml (or g) test product. Homogenise the test samples in the Stomacher for 30
seconds.

1.8.3 Sampling and incubation
Take a zero sample for each microorganism immediately after homogenisation (see
8.4) and incubate as described in 8.1. Incubate the Stomacher bags with the
challenged test samples at 20 25 C. It is important to seal the bags to avoid
evaporation. Leave approximately 10 % headspace in the Stomacher bags.

Take further samples after 7, 14, 21 and 28 days. Homogenise before each
sampling.

1.8.4 Analysis of samples
Transfer 10 g or 10 ml to 90 ml diluents (or 1 g/1ml to 9 ml). The density of the
product must be known if the transfer is based on weight. Homogenise the dilutions
and spread 100 l of dilutions in duplicate on the surface of the agar. Use the 10
-1
,
10
-2
, 10
-3
, 10
-4
and 10
-5
dilutions for the bacterial challenges and 10
-1
, 10
-2
, 10
-3
, 10
-4

for mold and yeast challenges at time zero. For subsequent sampling select the
dilutions to be analysed on the basis of the results of the initial analyses. It is
suggested to use the 10
-1
, 10
-2
and 10
-3
on day 7 for P. aeruginos and S. aureus and
10
-1
and 10
-2
for C. albicans and A. niger. Incubate as described in 1.8.1, count the
number of colonies and calculate the plate count according to ISO 21149:2006, and
plot the log
10
transformed results versus time.


43
1.9 Val i di t y cr i t er i a
1.9.1 Validity of analyses
If the organisms grow readily on the plates, and if there is a 1:10 relation between
the dilutions used for calculation of each analysis, the result of the analysis is valid.
The relation between dilutions can be tested by a
2
test with 1 degree of freedom:



where C1 and C2 are the number of colonies counted in dilutions V1 and V2. If
2

3.84 then the dilution is significantly different from 1:10 at the 5% level. If a low
dilution (more sample) has a relatively low count compared to a higher dilution
(less sample) it may be caused by inhibition by the antimicrobial preservative in
the tested product, and the test should be repeated with an alternative neutralizer.
See for instance ISO 22717 annex B for information on alternative neutralizers.

1.9.2 Validity of test organisms and neutralising agent
If no growth is observed on the agar plates it may be caused either by poor viability
of the test organisms or infectivity of the neutralising agent.

If no growth is observed, test organisms are spread in duplicate on the 10
-1
and 10
-2

plates and on clean control plates. Poor viability of the test organisms is shown, if
no growth is observed on the control plates. In this case the entire test should be
repeated with new and viable test organisms.

If no growth is observed on the 10
-1
or 10
-2
plates, and growth is observed on the
control plates, then the neutralizing agent has been ineffective. The lab should try
to wash away/neutralize the antimicrobial preservative with a sterile diluent to
which an alternative neutralizer has been added. Again, the test organisms are
spread on the plates. If no growth is observed on the washed plates and growth is
observed on control plates treated in a similar way, then the antimicrobial
preservative is accepted. If growth is observed, the test is repeated with an
alternative neutralizer.

1.10 Accept ance cr i t er i a
The antimicrobial preservation system is accepted if:

the performed analyses were valid according to section 1.9.1, and

bacteria are reduced by 3 log
10
units after 14 days, and no increase in plate
counts after day 14.
mold and yeast are reduced by 2 log
10
units after 14 days, and no increase
in plate counts after day 14.
or if

no growth is observed after washing with alternative neutralizers according to
section 1.9.2.