Mıcrobıology ın Personal Care - kmoetkinlik.org · Mıcrobıology ın Personal Care: ... Klebsiella Micrococcus Proteus Pseudomonas Sarcinia Serratia Staphylococcus Streptococcus

Mıcrobıology ın Personal Care:

Bıologıcal Evaluatıon Test Methods

ICPC 2016 – Symposium & Exhibition

3rd of November 2016

THOR SPECIALTIES SRL

TÜRKİYE ISTANBUL ŞUBESİ

Koşuyolu Mah. Cenap Şahabettin Sok. No:17

34718 Kadıköy / Istanbul

Tel +90 216 658 62 85 – dahili (ext): 118

Info.istanbul@thor – www.thor.com

Juri Masuri

Technical Service of Cosmetic Microbiological Analysis

[email protected]

Technical Service

mailto:Info.istanbul@thor

Summary

- MICROBIOLOGY IN COSMETIC PRODUCTS

- Introduction

- Types of Microorganisms

- Cosmetic Stability

- Preservative Systems and New Trend

- MICROBIOLOGICAL COSMETIC EVALUATION

- List of Test Methods

- Sterility Test

- Total Viable Count - TVC

- Research of Specific Microorganism

- Challenge Test Single Inoculum

- Challenge Test Multi Inoculum

- Single Inoculum VS Multi inoculum

- RESUME & CONCLUSION

MICROBIOLOGY IN COSMETIC PRODUCTS

Introduction

Cosmetic Product (short definition):

substances or Mixtures applied on skin surfaces and

mucoses to give appearance change, protection and/or

keeping them in good condition

Cosmetic Stability: The product must be stable with

appropriate:

- Appearance

- Purity (including controlled microbial contaminations)

- Efficacy (active ingredients)

For a period not less than Expiry date and PAO declaration


Introduction

Physical stability: cosmetic raw materials must

remain inside the cosmetic products without change

of cosmetic properties and without producing toxic

compounds

Biological stability: No external biological source

(ex: Microrganism) must influence the properties of

the cosmetic products


Introduction

Cosmetic Product Instability

Customers Negative Feed Back

Return to Production

ECONOMY LOSS!!


Types of Microorganisms

PROBLEM OF BIOLOGICAL STABILITY 99% MICRORGANISMS!!

- Which Kind of Microorganisms can Growth in Cosmetic Products?

- What is the contamination source for Microorganisms Founded in

Cosmetic Products?



Micro-organisms

Bacteria Fungi Algae*

Yeasts Moulds Lichens*

Wet-State Wet-State Wet-State Dry-Film* Dry-Film*

Dry-Film*

Powders

* Not relevant in Personal Care applications

Which Kind of Microorganisms can Grow in Cosmetic Products?

• Single-celled, variously shaped

• Very small (0.5 -2.5 m : 10-12 grams)

• Favourable growth factors:

• Water

• Nutrients (C,H,N sources)

• Temperature (kind dependent)

• pH (Neutral to Alkaline)

• Oxygen (kind dependent)

• Quick reproduction

• Partition every 20 min. possible

• Multicellular organisms

• fibre growth (hyphae)

• Formation of a mesh work

(mycelium)

• Size 10-20 µm up to ...m


• Water

• Nutrients (C, H, N-sources)


• pH (Acidic to Neutral)

• Oxygen

• Augmentation by formation of spores

• Single-celled, often typical oval form

• Small (4-10 m)


• Water

• Nutrients (C,H,N sources)


• pH (Acidic)

• Oxygen (for respiration),

fermentation

• Produce a lot of different metabolites

Bacteria Yeasts Moulds



Absidia

Alternaria

Aspergillus

Citromyces

Cladosporium

Dematium

Fusarium

Geotrichim

Helminthosporium

Hormodendrum

Mucor

Paecylomyces

Penicillium

Phoma

Pullularia

Rhizophus

Stemphylium

Thamnidium

Trichothecium

Acinetobacter

Alcaligenes

Bacillus

Diphteroids

Enterobacter

Enterococcus

Escherichia

Klebsiella

Micrococcus

Proteus

Pseudomonas

Sarcinia

Serratia

Staphylococcus

Streptococcus

Candida

Monilia

Torula

Zygosaccharomyces

Bacteria Yeasts Moulds



Microorganism Species founded during the History of Cosmesis Most Common Microorganism Species founded in Cosmesis



What is contamination source for the Microorganisms

Founded in Cosmetic Products?

WATER NO GOOD QUALITY OF PRODUCTION WATER

AIR WINDY AND NOT CLEAN CONDITIONS OF THE AIR INSIDE THE

FACTORY

DIRTY CONDITIONS PRESENCE OF DIRTY POWDERS/LIQUIDS IN THE PLANT

PLANT PRESENCE OF HIGH AMOUNT OF MICROORGANISMS INSIDE THE

PLANT AND/OR PARTICULAR ADAPTED SPECIES

PROPABLE PRESENCE OF BIOFILM

RAW MATERIAL PRESENCE OF NOT CONTROLLED AMOUNT OF MICROORGANISMS

INSIDE THE RAW MATERIALS

OPERATOR UNCORRECTED USE OR NOT USE OF CLEAN DISPOSABLE

PACKAGING USE OF DIRTY PACKAGING


Cosmetic Stability Economy Loss

& Time Loss

No Good Aspect

& Bad Smell

Reduced Expiry

date & PAO

Refused Product

Withdrawal from

the market Legal Problems

Microbial Contamination

Not Healthy

Product


Cosmetic Stability

How is Possible Avoid the Growth of Microrganisms??

Preservative System is the most common used solution

Microbicide and Microbiostatic effect


Preservative System – Preservative List

Parabens

Isothiazolinone

Amine Oxide Organic Acid

Phenoxyethanol

Benzyl Alcohol

Mercurials Not-Listed Preservatives

Formaldehyde

Formaldehyde Donors

Alogenated Compounds

Biguanide

Quaternary ammonium Compound

Pyrithione derivates

Etc…

?

15


Preservative System

Cosmetic Type

Products

• Hair Care

• Skin Care

• Sun Care

• Cosmetic Detergents

• Conditioner

• Shampoos

• Wet Wipes

• O/W or W/O

• Etc..

Preservative

System

• A

• B

• C

• D

• E

• F

• G

• Etc…

Effective Against

Microbial Growth

YES

NOT

How is possible evaluate the Cosmetic Biological Stability??

? Stable? Cost? Toxic? Effective?

16

MICROBIOLOGICAL

COSMETIC

EVALUATION

17

MICROBIOLOGICAL COSMETIC EVALUATION

List of Test Methods

WHICH IS THE CORRECT METHOD TO FOLLOW ?

All these methods are only guideline.

Every company can develop and validate a

method to define the preservative

adequacy of a product

18



Cosmetic Microbiology Test Methods:

Know if the Microorganism are Growing in the product

Sterility Test

Know the Amount of Microorganisms

TVC – Total Viable Count

Know if the Microorganisms are Pathogenic

Research of Specific Microorganism

Test the Preservative System Speed

Challenge Test Single-Inoculum

Test the Preservative System Robustness

Challenge Test Multi-Inoculum

19



STERILITY TEST ( example of approch )

Cosmetic

Products

Detection Methods

(ex: Streak Plate Method)

Evaluation for Presence or Absence

of Microbial Growth

20



STERILITY TEST ( evaluation )

Absence of Microrganisms: No microorganisms are growing

in this cosmetic product

Presence of Microorganisms: Some microrganisms are

growing in this cosmetic product. The amount of microrganism

can indicate if is possible recover or not the cosmetic product

In which guideline is possible to find these methods?

• Internal Methods

Properties?

• Short, Fast, Simple

21



TVC – Total Viable Count ( example of approch )

Cosmetic

Products

Neutralization

and diluition

Technics

Amount

evaluation the of

microrganisms

Detection Methods

(ex: Plate Count Method)

22



TVC - Total Viable Count ( evaluation )

Absence of Microorganisms: No microorganisms are

founded in this cosmetic product

Number of Microorganisms: a defined amount of

microorganisms are founded in this cosmetic product. The

level of contamination should stay under a specific limit

In which guideline is possible to find this method?


• Pharmacopoeia ( Europe 8.0 – American USP 39–NF 34 – etc.. )

• ISO Guide Line 21149 ( Bacteria ) and 16212 ( Yeasts and Moulds )

• SCCS (Scientific Committee on Consumer Safety) 9th Revision SCCS/1564/15 ( for Europe )

• Etc..

23



TVC - Total Viable Count ( methods comparations )

Guide

Line

Neutralizing

solution Growth Medium Detection Method

Enumeration Limit

(cfu/gr)

Evaluation Limit

(cfu/gr)

EP LPT;

Universal

TSABacteria;

SDAYeast/Mould

Plate Count; Membrane

Filtration

MPN

Minimum: 101 Type dependant

ISO Eugon LT;

Universal

TSABacteria;

SDAYeast;

PDAMould

Plate Count; Minimum: 101 <102 for children

<103 others

USP Biocide

Dependant

TSABacteria;

SDAYeast/Mould


Filtration

MPN

Minimum: 101 Type dependant

Properties?

• Correct amount evaluation, common use, repeatable

24



Research of Specific Microorganism ( example of approch )

Cosmetic

Products Neutralization and

enrichment method

Isolation on Selective agar medium

Multiple Biochemical and Physical

identification methods

Identification of Pathogenic Microorganism

25



Selection of Specific Microorganism ( evaluation ) -1

Absence of Microorganisms: No microorganisms are

founded in this cosmetic product

Presence of Microorganisms: One or more microorganisms

are founded in this cosmetic product. But:

Not Pathogenic: No pathogenic microorganisms are

founded. We need to know if the amount of

microrganisms are on the limit

Pathogenic: Presence of pathogenic microorganisms

in this cosmetic products ( S.aureus, P.aeruginosa,

C.albicans, E.coli, etc.. )

26



Selection of Specific Microorganism ( evaluation ) -2



• Pharmacopoeia ( Europe 8.0 – American USP 39–NF 34 – etc.. )

• ISO Guide Line 18415, 18416, 22717, 22718 and 21150


• Etc..

Properties?

• Good microbial identification, Avoid future healthy problem

27



Selection of Specific Microorganism ( methods comparations )

Guide

Line

Neutralizing

solution

Selective

Medium Used

Microorganisms

detected Detection method Evaluation Limit

EP LPT;

Universal

MSA; CET;

MCA; VRB;

EEB; SDAC

E.coli;

P.aeruginosa;

S.aureus;

C.albicans;

GRAM- Bile Tolerants

Biochemical and

physical tests Absence in 10gr

ISO Eugon LT;

Universal

MSA; CET;

MCA; SDAC

E.coli;

P.aeruginosa;

S.aureus;

C.albicans;

Biochemical and


USP Universal MSA; CET;

MCA; SDAC

E.coli;

P.aeruginosa;

S.aureus;

C.albicans;

Biochemical and


28



CHALLENGE TEST – Single Inoculum ( example of approch )

Standardized

Microbial

Suspension

Cosmetic

Products Inoculated

Sample

Time

0

Time

1

Time

2

Time

n

At different points the inoculated samples are

evaluated with appropriate methods

(ex: Neutralization - Diluition method +

plate count meyhod )

With all the obtained results is possible evaluate

the microbial decrease speed after the inoculum

29



Challenge Test – Single Inoculum ( evaluation ) - 1

Reduction of Microorganism: all the viability data are

comparated to have the microbial Log10 reduction value in

differents time points. This value need to stay under specific

limits:

Pass the test: the preservative system is sufficient to

have a fast microbial decrease and can be considered

«Efficient»

Not Pass the test: the preservative system is not

sufficient to have a fast microbial decrease and can be

consideredd «Not Efficient»

30



Challenge Test – Single Inoculum ( evaluation ) - 2



• Pharmacopoeia (Europe 8.0 – American USP 39–NF 34 – etc.. )

• ISO Guide Line 11930


• Etc..

Properties?

• Preservative system Efficacy evaluation, common use

31



Challenge Test – Single Inoculum ( methods comparations )

Guide Line Neutralizing

solution

Growth Medium

and Neutralizer Detection Method Strian for Inoculum

EP LPT;

Universal

TSABacteria;

SDAYeast/Mould


Filtration;

MPN

E.coli; P.aeruginosa; S.aureus;

C.albicans; A.brasiliensis;

Z.rouxii

ISO Eugon LT;

Universal

TSABacteria;

SDAYeast;

PDAMould

Plate Count; E.coli; P.aeruginosa; S.aureus;


USP Biocide

Dependant

TSABacteria;

SDAYeast/Mould


Filtration

MPN

E.coli; P.aeruginosa; S.aureus;


32



Challenge Test – Single Inoculum ( Evaluation comparations )

Guide Line Bacteria- Mould and Yeast

EP

≥2 by 2 days

And ≥3 by 7 days

No increase thereafter

≥2 by 14 days

and no increase thereafter

ISO ≥3 by 7 days

And No increase thereafter

Yeat ≥1 by 7 days

and no increase thereafter

Mould ≥1 by 28 days

and No increase in count throughout test

USP ≥2 by 14 days

and no increase thereafter No increase in count throughout test

33



CHALLENGE TEST – Multi Inoculum ( example of approch )

Standardized Microbial Suspension

Cosmetic

Products

Inoculated

Sample

n Time

1°

2° 3°

4°

n Time n Time n Time

Detection Methods

(ex: Streak Plate

Method)

Evaluations the

sample after every

inoculum

34



Challenge Test – Multi Inoculum ( evaluation ) - 1

Evaluation of Microorganism Amount: after every inoculum

an analysis of the sample gives results about the microbial

amount inside:

Pass the test: the microbial amount is low after every

inoculum. The preservative system can be considered

«Efficient and Robust»

Not Pass the test partially: the microbial amount is

low after the first and the second inoculum but high in

the following. The preservative system can be

considered «Efficient but not Robust»

Not Pass the test completely: the microbial amount is

not low after every inoculum. The preservative system

can be considered «Not Efficient and not Robust»

35



Challenge Test – Multi Inoculum ( evaluation ) - 2

Properties?

• Preservative system Efficacy evaluation,

• Preservative system Robustness evaluation

• New trend of evaluation

• High stress conditions

In which guideline is possible to find these methods?


36

• Uses individual inoculum

• Single challenge

• Monitor the survival of the

inoculum by performing a TVC

at 0, 2, 7, 14 and 28 days

• Validate the used Neutralizer

in the test

• How QUICK the preservative

system works?

• Uses Individual or

mix inocula

• Multi challenge

• Monitor the survival

microorganisms after 6 days

from every inoculum

• Neutralizer dependent from

the used protocol

• How STRONG the preservative

system is?

≠ Challenge Test Multi Inoculum


Single Inoculum VS Multi Inoculum

Challenge Test Single Inoculum

37


Single Inoculum VS Multi Inoculum

Taking in consideration: - The continuous use of cosmetic products in a multitude of different an

high stressconditions (like high humidity, temperature, sunlight, etc) of cosmetic products

- The new trend of preservation (preservative-free, mildness, green) and the continuous product avoided for the collateral effect (like Parabens and Methylisothiazolinone)

- The continuous interest to use methods more predictive of the classical used, to have the maximum performance and avoid future problem of contamination from different possible sources.

We can conclude that the Challenge Test Multi Inoculum give results more predictive than the Challenge Test Single Inoculum

38

RESUME

&

CONCLUSIONS

39


Resume & Conclusions

- This presentation is only the resume for evaluation of cosmetic

products Biological Stability and for all the cosmetic products is

important take in consideration both the Physical and the

Biological Stability

- The physical stability is «chained» with biological stability when we

have presence of microorganisms

- All the source of contamination must be reduced to avoid loss of

preservative system and to avoid future problem (like economy

loss or Healthy damage)

40


Resume & Conclusions

- To choose a test it’s important know the parameter that the

producer want to know (like number of microorganisms or efficacy

of preservative system)

- Every company/producer can use an existing method or develop

and validate a new one to determine the preservative adequacy of

the product

- The Challenge Test Multi Inoculum give results more predictive

than the Single Inoculum taking in consideration the use and high

stress conditions

41

Any question?

Thanks for the attention

Documents

Mıcrobıology ın Personal Care - kmoetkinlik.org · Mıcrobıology ın Personal Care: ... Klebsiella Micrococcus Proteus Pseudomonas Sarcinia Serratia Staphylococcus Streptococcus