Professional Documents
Culture Documents
Introduction
Cosmetic was defined in Cosmetic Law (24.03.2005 / No. 5324)
published by The Ministry of Health of Turkey as Cosmetics are all the
preparations that were prepared to be used for epidermis, nails, hair,
lips, genital organs and teeth and mouth mucosa and their only aim is to
clean, give odors, change the morphological appearance and/or to regulate the body odors and keep them in good positions 1.
According to The Federal Food and Drug Cosmetic Act criteria, cosmetic means the articles intended to be rubbed, poured, sprinkled, or
sprayed on, introduced into, or otherwise applied to the human body or
any part thereof for cleansing, beautifying, promoting attractiveness, or
altering the appearance, and articles intended for use as a component of
any such articles; except that such term shall not include soap 2.
Contamination of microorganisms in cosmetics may cause spoilage
of the product and when pathogenic, they represent a serious health risk
for consumers 3.
Most of the cosmetics are not sterile and they are made of non-sterile raw material 4,5,6. Although cosmetics do not have to be sterile, limit values have been reported according to the type of the cosmetics 5.
Mannitol Salt Agar (Merck), Selenit-F (Merck) and XLD Agar (Merck) were
used to grow microorganisms during microbial contamination tests. Ethanol (Merck) was diluted to 70% (v/v) and used for the disinfection of the
sample packages and Tween-80 (Merck) was used to disperse insoluble
samples.
Microorganisms
Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC
27853, Candida albicans ATCC 10231 and Escherichia coli ATCC 25922
were used in the study for the Microbial Challenge Test.
Streptococcus
spp.
Bacillus spp.
E.coli
Candida spp.
Total fungi
(CFU/g)
Total aerobic
bacteria
(CFU/g)
+*
EL3
10.25103
LP1
LP2
ES1
20.1103
* Coagulase positive
S. epidermidis
EL1 EL2
S. aureus
Sample
M.o.
10.18103
ES2
20.11103
ES3
10.17103
FD1
TABLE I
40.06103
40.17103
FD2
10.21104
2.50.46105
5.51.5105
71.18104
71.4104
EL2
EL3
EL4
EL5
EL6
EL7
EL8
EL9
EL10
EL20
EL19
EL18
EL17
EL16
EL15
EL14
EL13
EL12
EL11
1.60.1105
80.35104
82.73104
10.16106
50.04104
CFU/ml
41104
1.20.13105
FD11
2.4 0.08105
1.20.16105
30.24104
CFU/ml
FD10
FD9
FD8
FD7
FD6
FD5
FD4
FD3
FD2
FD1
1.40.11105
EL1
CFU/ml
ES9
ES8
ES7
ES6
ES5
ES4
ES3
ES2
ES1
FD13
FD12
40.17104
40.12104
20.2104
40.5104
10.05104
60.47104
1.40.03105
CFU/ml
ES20
ES19
ES18
ES17
ES16
ES15
ES14
ES13
ES12
ES11
ES10
CFU/ml
40.2104
20.05104
1.40.2105
1.60.1105
1.20.08105
1.20.05105
20.24104
81.3104
20.23104
20.28104
20.2104
Number of Candida albicans cells at time zero (CFU/ml) after Microbial Challenge Test.
TABLE II
6
HACETTEPE UNIVERSITY JOURNAL OF THE FACULTY OF PHARMACY
80.2105
1.20.1106
3.80.2105
tntc
20.2106
1.50.1106
1.20.1106
1.50.11106
80.21105
EL2
EL3
EL4
EL5
EL6
EL7
EL8
EL9
EL10
EL20
EL19
EL18
EL17
EL16
EL15
EL14
EL13
EL12
EL11
tntc
tntc
tntc
6.50.31105
tntc
1.20.11106
tntc
tntc
tntc
tntc
CFU/ml
tntc
tntc
FD11
tntc
tntc
tntc
tntc
tntc
tntc
tntc
tntc
tntc
CFU/ml
FD10
FD9
FD8
FD7
FD6
FD5
FD4
FD3
FD2
FD1
tntc
EL1
CFU/ml
ES9
ES8
ES7
ES6
ES5
ES4
ES3
ES2
ES1
FD13
FD12
tntc
tntc
tntc
tntc
81106
1.50.1106
tntc
tntc
tntc
tntc
tntc
CFU/ml
ES20
ES19
ES18
ES17
ES16
ES15
ES14
ES13
ES12
ES11
ES10
Number of Candida albicans ATCC 10231 cells after 3 days (CFU/ml) after Microbial Challenge Test.
TABLE III
tntc
tntc
tntc
tntc
tntc
tntc
tntc
tntc
tntc
tntc
tntc
CFU/ml
10.35104
1.70.12105
20.27104
30.32104
EL2
EL3
EL4
EL5
EL6
EL7
EL8
EL9
EL10
EL20
EL19
EL18
EL17
EL16
EL15
EL14
EL13
EL12
EL11
1.40.15106
2.60.07106
9.51.15105
10.23104
20.25104
CFU/ml
10.06106
3.60.62105
FD11
40.9105
3.60.16106
10.17104
10.09104
2.2 0.3105
10.15104
CFU/ml
FD10
FD9
FD8
FD7
FD6
FD5
FD4
FD3
FD2
FD1
71.33104
EL1
CFU/ml
ES9
ES8
ES7
ES6
ES5
ES4
ES3
ES2
ES1
FD13
FD12
10.04105
1.60.1105
2.40.26105
1.60.14105
10.15104
1.60.02106
2.40.33105
CFU/ml
ES20
ES19
ES18
ES17
ES16
ES15
ES14
ES13
ES12
ES11
ES10
6.20.22105
7 1.73105
1.20.18106
5.40.26106
5.20.7106
6 0.53104
1.80.17105
1.40.05105
1.40.09105
80.84104
CFU/ml
Number of Pseudomonas aeruginosa cells at time zero (CFU/ml) after Microbial Challenge Test.
TABLE IV
8
HACETTEPE UNIVERSITY JOURNAL OF THE FACULTY OF PHARMACY
number of cells were 4 106, 2.8 106, 3.2 106 and 1 107 CFU/ml in
EL2, EL3, EL6 and EL9 numbered samples respectively. Although number of cells reduced in these samples, the reduction was not enough. After 14, 21 and 28 days in all the samples, number of C.albicans cells were
too numerous to count. As a result preservative activity was determined
only in 19 samples at time zero for C.albicans.
At time zero, in 17 samples, Pseudomonas aeruginosa ATCC 27853
growth was not observed so 99.9% reduction was determined in 17 samples at time zero. However, after 3, 7, 14, 21 and 28 days number of Pseudomonas aeruginosa ATCC 27853 cells were too numerous to count in all
the samples. As a result preservative activity was determined only in 29
samples at time zero for Pseudomonas aeruginosa ATCC 27853.
For E.coli ATCC 25922, only in 5 samples no growth was determined.
In other samples numbers of cells were between 1 104 - 2.1 106 CFU/
ml at time zero. After 3 days in all the samples, number of E. coli ATCC
25922 cells were too numerous to count. After 7, 14, 21 and 28 days
in all the samples number of E. coli ATCC 25922 cells were too numerous to count. As a result, preservative activity was determined only in 8
samples at time zero.
40.21104
40.7104
40.8104
60.87104
70.12104
40.46105
20.26104
4 0.61104
41.26104
EL2
EL3
EL4
EL5
EL6
EL7
EL8
EL9
EL10
EL20
EL19
EL18
EL17
EL16
EL15
EL14
EL13
EL12
EL11
71.8105
1.20.04106
7.60.85105
30.17104
50.16105
20.43104
10.05104
80.43104
30.5104
40.8104
CFU/ml
1.20.09106
10.23106
FD11
9.20.52105
1.10.28106
31.32104
30.95104
30.43104
6 0.75104
CFU/ml
FD10
FD9
FD8
FD7
FD6
FD5
FD4
FD3
FD2
FD1
10.13104
EL1
CFU/ml
ES9
ES8
ES7
ES6
ES5
ES4
ES3
ES2
ES1
FD13
FD12
2.44.58105
1.75.1106
40.2104
30.43105
2.40.26105
70.2104
10.15104
20.26104
1.10.26106
1.60.17105
CFU/ml
ES20
ES19
ES18
ES17
ES16
ES15
ES14
ES13
ES12
ES11
ES10
Number of E. coli ATCC 25922 cells at time zero (CFU/ml) Microbial Challenge Test.
TABLE V
8.64.2105
70.43105
1.20.26106
2.10.29106
10.06106
2.20.53105
10.12105
3.40.35105
2.60.36105
2.80.26105
CFU/ml
10
HACETTEPE UNIVERSITY JOURNAL OF THE FACULTY OF PHARMACY
50.87104
40.91104
20.26104
30.28104
40.24105
40.27104
40.33104
20.43104
EL2
EL3
EL4
EL5
EL6
EL7
EL8
EL9
EL10
EL20
EL19
EL18
EL17
EL16
EL15
EL14
EL13
EL12
EL11
20.12106
30.12106
4.30.26106
30.7104
90.19105
10.31104
40.2104
40.34104
50.21104
40.56 104
CFU/ml
2.70.67106
2.60.19106
FD11
2.10.36106
FD10
FD9
FD8
3.40.53106
10.21104
FD6
FD7
30.1104
50.26104
51.05104
60.7104
10.02105
CFU/ml
FD5
FD4
FD3
FD2
FD1
70.2104
EL1
CFU/ml
1.80.36105
1.10.05106
ES9
4.40.28105
1.60.17105
1.30.38105
70.13104
90.43104
20.07104
5.10.36106
3.50.5106
ES8
ES7
ES6
ES5
ES4
ES3
ES2
ES1
FD13
FD12
CFU/ml
ES20
ES19
ES18
ES17
ES16
ES15
ES14
ES13
ES12
ES11
ES10
3.90.56106
30.46106
20.09106
1.90.47106
5.50.49106
3.60.11106
1.50.1105
50.69105
10.36105
CFU/ml
Number of Staphylococcus aureus ATCC 29213 cells at time zero (CFU/ml) Microbial Challenge Test.
TABLE VI
MICROBIOLOGICAL INVESTIGATION OF USED COSMETIC SAMPLES
11
12
13
observed in the in-use product. Also in the study, the preservative systems of the two tested products were studied and they have showed long
lasting antimicrobial activity 3.
Ravita et al. have studied with post-consumer use cosmetic products
in case of microbial contamination. In this study, densities of culturable
aerobic microorganisms of used cosmetic products containing global
(GPC) and non-global (NPC) was compared. Among the 96 samples, 28
samples did not yield culturable microorganisms. There was no significant difference between GPC and NPC samples 6. Preservative activity
was not detected by a microbial challenge test in the previous study.
Hugbo et al. have studied with ten brands of commercially available
cosmetic creams and lotions. After microbial investigations they have
determined that all the products were contaminated to varying degrees.
They came to the conclusion that the samples that they were tested did
not generally meet the standards for microbial limits. In this study the
investigators made a microbial challenge test for preservative activity but
in the challenge test they have only used S. aureus as a bacteria and
Aspergillus and Penicillum as molds. After the challenge test they have
concluded that the preservatives did not possibly possess adequate preservative capacity 14.
Zhang et al. has reported a study on hygienic microbe pollution for
imported cosmetics during 2003-2006 and determined that 0.27% of the
4764 cosmetic samples have exceeded the maximum limits of Hygienic
Standard for Cosmetic in China. As a result of their study they have
reported that the quality of the imported cosmetics is satisfactory except
sea-mud products whose microbes proportion exceeds the limitation severely 15.
In our study, among 73 samples, in 10 samples microbial contamination was observed. In 5 samples total aerobic bacteria numbers were
off the limits. Salmonella spp. and Pseudomonas aeruginosa were not
observed but Candida spp., Staphylococcus aureus and E.coli that are not
allowed to be founding cosmetics were determined.
After the challenge test the preservative activity of all the products
was shown to be ineffective because the microbial growth was not limited with a reduction of 99.9%. At time zero, in 29 samples, C. albicans
growth was not observed so the growth was limited with a reduction
14
Summary
In this study, our aim was to study the cosmetic samples that were
used before and that have not an expiry date report, in case of microbial
contamination and preservative activity. A total of 73 samples including 20 lipsticks, 20 eye shadows, 13 foundations and 20 eyelashes that
were used before were studied. Microbial contamination was studied
according to the guidelines of U.S. Food and Drug Administration (FDA)
method: Microbiological Methods for Cosmetics. Among the samples,
in 3 eyelashes, 3 eye shadows, 2 lipsticks and 2 foundations of 10 samples microbial contamination were observed. In 5 samples total aerobic
bacteria numbers were off the limits. Salmonella spp. and Pseudomonas aeruginosa were not observed but Candida spp., Staphylococcus
aureus and E.coli that are not allowed to be found in cosmetics were
determined.
15
zet
Kullanlm Kozmetik rneklerinin Mikrobiyolojik Ynden
Aratrlmas
Bu almada, daha nce kullanlan ve son kullanma tarihleri
belirtilmemi olan kozmetiklerin mikrobiyal kontaminasyon ve prezervatif madde aktivitesi ynnden aratrlmasn amaladk. almada
daha nce kullanlm 20 adet ruj, 20 adet gz far, 13 adet fondten,
20 adet rimel olmak zere toplam 73 adet rnek kullanld. Mikrobiyal
kontaminasyon, Gda ve la Dairesi (FDA)nin: Kozmetikler iin Mikrobiyal Yntemler metodu ile allmtr. 3 rimel, 3 gz far, 2 ruj
ve 2 fondten rnei olmak zere toplam 10 rnekte mikrobiyal kontaminasyon tespit edildi. rneklerin 5inde total aerobik bakteri says
izin verilen snrlar dnda bulundu. Salmonella spp. ve Pseudomonas
aeruginosa gzlenmemekle birlikte, kozmetiklerde bulunmamas gereken Candida spp., Staphylococcus aureus ve E.coli baz rneklerde
saptand.
Prezervatif madde aktivitesi, Challenge Test ile saptand. 0, 3, 7, 14,
21 ve 28 gn srelerde, hcre canll koloni sayma yntemiyle saptand
ve 7 gn boyunca her mikroorganizmann saysnda %99.9lk azalma
olmas halinde test sonucu olumlu olarak deerlendirildi. Deney sonucunda hibir rnde mikroorganizma saysnda %99.9luk azalma
grlmediinden hibir rnde prezervatif aktivitenin yeterli olmad
sonucuna ulalmtr.
Anahtar kelimeler: Mikrobiyal kontaminasyon, ruj, gz far, fondten,
rimel, prezervatif aktivitesi, Challenge test
16
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