Title
Revision n°
Date
Document No
EVALUATION OF THE
ANTIMICROBIAL PROTECTION
OF A COSMETIC PRODUCT
1
28/11/13
ACM 008
Page 1/6
1.0
SCOPE AND FIELD OF APPLICATION
To evaluate the overall antimicrobial protection of a cosmetic product.
2.0
PRINCIPLE
Each of the microorganism tests is evaluated by placing the formulation in
contact with a calibrated inoculum, and then measuring the changes in the
microorganism count at set time intervals for a set period and a set
temperature.
3.0
MATERIALS
3.1
Biohazard cabinet
3.2
Autoclave
3.3
Hot Air Oven
3.4
Incubator: 32.5 ± 2.5C and 22.5 ± 2.5C
3.5
Vortex mixer
3.6
Glass beads
3.7
Pipettes
3.8
Petri dishes
3.9
Haemocytometer & Phase-contrast Microscope (if available)
3.10
Spectrophotometer / Colorimeter (if available)
3.11
Colony counter
3.12
Media bottles
3.13
Sterile universal bottles / Test tubes
3.14
Inoculating loops / spreader
3.15
Bunsen burner (or Bacticinerator)
3.16
Waterbath
3.17
Top pan balance
3.18
pH meter
4.0
MEDIA AND REAGENTS
For convenience, dehydrated media of any brand equivalent in function may
be used. Media should be tested for sterility and growth promotion using
suitable organisms.
4.1
Nutrient Agar (or other suitable equivalent media).
4.2
Letheen Agar (or other suitable equivalent media e.g. TSA with 1%
Tween 80, “TSA t”.
4.3
Mycophil Agar, pH 4.7 (or other suitable equivalent media e.g. “PDA
a” / “SDA a”, SDA with 1% Tween 80 “SDA t”).
4.4
Letheen Broth (or peptone saline with 1% Tween 80).
4.5
Chloride buffer (or similar buffer).
4.6
Diluent 1 – Sterile solution containing 0.9% sodium chloride and 0.1%
peptone.
Note : "a" means with antibiotic
Title
Revision n°
Date
Document No
EVALUATION OF THE
ANTIMICROBIAL PROTECTION
OF A COSMETIC PRODUCT
1
28/11/13
ACM 008
Page 2/6
5.0
TEST ORGANISMS
5.1
Pseudomonas aeruginosa ATCC 9027, CIP 82.118, or equivalent
5.2
Staphylococcus aureus ATCC 6538 (NCIMB 9518, CIP 4.83, NCTC
10788)
5.3
Candida albicans ATCC 10231 (NCPF 3179, IP 48.72)
5.4
Enterobacter aerogenes ATCC 13048
5.5
Aspergillus brasiliensis (previously A.niger) ATCC 16404, IP 1431,
IMI 149007 or equivalent
6.0
PREPARATION AND ENUMERATION OF INOCULA
6.1
Bacteria and yeast
6.1.1
Prepare a subculture from the stock culture by streaking slant
tubes or plates (TSA for bacteria, SDA for Candida albicans)
in order to obtain a confluent culture. Incubate at (32.5 ±
2.5)°C for 18 – 24 hours.
6.1.2
Wash each slant of bacteria and yeast culture with 10 mL of
diluent 1, loosening the culture from the agar surface with the
help of sterile glass beads. Mix by using mechanical mixer to
disperse evenly and transfer the suspension into sterile
universal bottle
6.1.3
Adjust the number of cells in the suspension to 1 X 10
7
cfu/mL to 1 X 10
8
cfu/mL (bacteria) or 1 X 10
6
cfu/mL to 1 X
10
7
cfu/mL (yeast) using diluent 1 either by using McFarland
BaSO
4
standard No.2, direct microscopic count, turbidimetry,
absorbance or other method correlated to an aerobic plate
count
6.1.4
At the time of the test, check the initial capacity of the
suspension, N. Make successive tenfold dilutions of the
calibrated suspension in the diluent 1. Perform the
enumeration by duplicating 1 ml of the suitable dilutions into
TSA for bacteria and into SDA for Candida albicans. Incubate
the dishes at (32.5 ± 2.5)°C for 24-48 hours
6.2
Aspergillus brasiliensis spore suspension
6.2.1
Streak Aspergillus brasiliensis culture onto SDAa slant and
incubate at (22.5 ± 2.5)°C for 7 – 11 days or until full
sporulation is achieved.
6.2.3
Wash each slant of fungi culture with 10 mL of diluent 2,
loosening the spores from the culture surface with the help of
sterile glass beads. mix by using mechanical mixer to
disperse evenly and transfer the suspension into sterile
universal bottle
6.2.4
Adjust the number of spores in the suspension to a value about 1 X
10
6
spores/mL to 1 X 10
7
spores/mL using diluent 2 and any
appropriate means.
Title
Revision n°
Date
Document No
EVALUATION OF THE
ANTIMICROBIAL PROTECTION
OF A COSMETIC PRODUCT
1
28/11/13
ACM 008
Page 3/6
6.2.5
At the time of the test, check the initial capacity of the
suspension, N. Make successive tenfold dilutions of the
calibrated suspension in the diluent 2. Perform the
enumeration by duplicating 1 mL of the suitable dilutions into
SDA plates. Incubate the dishes at (22.5 ± 2.5)°C for 3-5
days.
7.0
DEMONSTRATION OF THE NEUTRALIZER EFFICACY
7.1
In the test, the neutralization of the possible antimicrobial activity of
the tested sample shall be checked and demonstrated.
7.2
The suitability and effectiveness of the neutralizing agent with respect
to the test strains used and to the tested formulation shall be
demonstrated.
7.3
A calibrated suspension of microorganism (about 103 cfu/mL) is
inoculated in the neutralizer in the presence (test) and in the absence
(control) of the formulation.
7.4
The neutralizer efficacy is demonstrated if the counts performed on
the inoculum, Nv, and on the control, Nvn (mixture of the neutralizer
and diluent) are equivalent and if the count in the test, Nvf (mixture of
the neutralizer and the formulation) is at least 50% of Nvn.
7.5
If the results do not comply with the requirements, it is necessary to:-
i. modify the neutralizer, or
ii. make a further dilution of the sample , or
iii. carry out a membrane filtration, if possible
7.6
If the results still do not comply with the requirements, it is unlikely
that the formulation can be contaminated by the strain concerned
8.0
DETERMINATION OF THE PRESERVATION EFFICACY OF THE
FORMULATION
8.1
Procedure
Run the test separately for each strain.
8.2
Aliquoting of Test Product
For each strain, dispense 20 gram or 20 ml of the test formulation into
a sterile container.
8.3
Inoculation of the test microorganisms
8.3.1
Add to each container 0.2 mL of calibrated inoculum to obtain
1 X 10
5
cfu/mL and 1 X 10
6
cfu/mL or gram for bacteria, and
between 1 X 10
4
cfu/mL and 1 X 10
5
cfu/mL or gram for
Candida albicans and Aspergillus brasiliensis in the
formulation (final concentration).
8.3.2
Mix thoroughly to ensure a homogeneous distribution of the
inoculum.
8.3.3
The initial concentration of microorganisms present in the
inoculated product, N
0
, is calculated using the results of the
enumeration of the calibrated inoculum, N.
Title
Revision n°
Date
Document No
EVALUATION OF THE
ANTIMICROBIAL PROTECTION
OF A COSMETIC PRODUCT
1
28/11/13
ACM 008
Page 4/6
8.4
Incubation of the Inoculated Formulation
Store the containers holding the inoculated formulation at ( 22.5 ±
2.5)°C.
8.5
Sampling and Enumeration
8.5.1
Remove 1 gram or 1 mL of sample at each specified
sampling interval, 7 days (T7), 14 days (T14) and 28 days
(T28) according to the test strain into peptone saline
containing 1% Tween 80. Mix until homogeneous.
8.5.2
Leave in contact for 15 - 45 minutes at room temperature.
Proceed with 10-fold serial dilutions using peptone saline
containing 1% Tween 80.
8.5.3
Determine the number of viable microorganisms in duplicate
by surface spread technique on TSA for bacteria , and pour
plate technique using SDA for yeast and fungi.
8.5.4
Incubate at ( 32.5 ± 2.5)°C for 48-72 hours for the bacteria
and C.albicans and at (22.5± 2.5)°C for 3-5 days for
A.brasiliensis.
8.5.5
Count the number of surviving microorganisms per gram or
ml of product.
9.0
CALCULATIONS
9.1
Determination of the initial numbers of microorganisms , N and N
0
9.1.1
Calculate N, the number of microorganisms present in the
calibrated suspensions in colony-forming units per millilitre,
using Equation :
N = Ć / ( V x d)
Where,
Ć is the mean number of colonies counted in duplicate over
the plates.
V is the volume of inoculum applied to each dish, in millilitres.
d is the dilution factor of the counted dilution.
N shall be between 1 X 10
7
cfu/mL and 1 X 10
8
cfu/mL for
bacteria, and between 1 X 10
6
cfu/mL and 1 X 10
7
cfu/mL for
C.albicans and A.brasiliensis
9.1.2
Calculate N
0
, the number of microorganisms inoculated in the
formulation at time t
0
using Equation :
N
0
= N/100
N
0
shall be between 1 X 10
5
cfu/mL and 1 X 10
6
cfu/mL or
gram for the bacteria, and between 1 X 10
4
cfu/mL and 1 X
10
5
cfu/mL or gram for C.albicans and A.brasiliensis
Title
Revision n°
Date
Document No
EVALUATION OF THE
ANTIMICROBIAL PROTECTION
OF A COSMETIC PRODUCT
1
28/11/13
ACM 008
Page 5/6
9.2
Enumeration of the microorganisms at each sampling time, Nx
Calculate Nx the number of surviving microorganisms in the
contaminated formulation, in colony-forming units per millilitre or
grams, at each sampling time, tx, (T7, T14 or T28) using Equation :
Nx = C / (V x d)
Where,
C is the mean number of colonies counted in duplicate over the
plates.
V is the volume of inoculum applied to each dish, in millilitres.
d is the dilution factor corresponding to the retained and counted
dilution.
9.3
Reduction in Microbial Counts
Calculate the reduction values, Rx, expressed in log units, obtained at
each sampling time using Equation :
Rx = lgN0 - lgNx
Where
N
0
is the number of microorganisms inoculated at time t
0
Nx is the number of surviving microorganisms at each sampling time,
tx
There may be no reduction and there may be an increase in the
microorganism count.
10.0
INTERPRETATION OF TEST RESULTS
10.1
The obtained log reduction values, Rx are compared to the minimum
values required for evaluation criterion A or B (Table 1)
Table 1
Log Reduction Values (R
x
= lgN
0
– lgN
x
) required
a
Microorg
anisms
Bacteria
C.albicans
A.brasiliensi
s
Sampling
Time
T7
T14
T28
T7
T14
T28
T14
T28
Criteria A
≥3
≥3
and
NI
b
≥3
and
NI
≥1
≥1
and
NI
≥1
and
NI
≥0
c
≥1
Criteria B
Not
performed
≥3
≥3
and
NI
Not
performed
≥1
≥1
and
NI
≥0
≥0
and
NI
a
In this test, an acceptable range of deviation of 0.5 log is accepted
b
NI: no increase in the count from the previous contact time
c
R
x
= 0 when lgN
0
– lgN
x
(no increase from the initial count )
Title
Revision n°
Date
Document No
EVALUATION OF THE
ANTIMICROBIAL PROTECTION
OF A COSMETIC PRODUCT
1
28/11/13
ACM 008
Page 6/6
10.2
The criterion representing the protection capacities are : -
10.2.1
Criterion A, whereby the formulation is protected against
microbial proliferation that may present a potential risk for
the user and no additional factors are considered.
10.2.2
Criterion B, whereby the level of protection is acceptable if
the risk analysis demonstrates the existence of control
factors not related to the formulation indicating that the
microbiological risk is tolerable for the cosmetic product.
10.2.3
The criteria are expressed either by a minimum log
reduction value or by “NI” when the requirement is that there
be no increase in the microbial population.
11.0
REFERENCES
11.1
The American Society for Testing and Materials, Designation : E 640-
78 (Reapproved 1998), Standard Test Method for Preservatives in
Water-Containing Cosmetics,pp.141-142.
11.2
AOAC Official Methods of Analysis (2000), Chapter 15, Efficacy of
Preservation of Non-Eye Area Water Miscible Cosmetic and Toiletry
Formulations, pp 3-5.
11.3
British Pharmacopoeia 2012, Volume 4, Appendix XVI C, Efficacy of
Antimicrobial Preservation.
11.4
ISO 11930, Cosmetics-Microbiology-Evaluation of the antimicrobial
protection of a cosmetic product, First Edition 2012-04-01
12.0
HISTORY
12.1
Issued by the microbiological analysis group at the harmonization
workshop in Kuala-Lumpur, September 13th to 17th 2004
12.2
Approved by the harmonization workshop delegates workshop in
Kuala-Lumpur, September 13th to 17th, 2004,
12.3
Modified after the Kuala-Lumpur training, Dec 6th to 10th 2004
12.4
Modified and approved after the Brunei workshop, Aug 30th to 31st,
2005
12.5
Modified and approved after the final review in Singapore, Nov 30th to
Dec 2nd, 2005
12.6
Modified and approved after the Regional Cosmetic Workshop in
Malaysia, July 10th to 12th 2006
12.7
Modified and approved after the final review in Malaysia on November
28th, 2013
