Post on 27-Jun-2018
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An Overview of Preservatives and their Modes of ActionKevin RodenThor Specialties
A substance that maintains (preserves) the original performance/characteristics/integrity of the product by controlling the growth of contaminating microbes
• Chemical agents added to products to prevent the growth of microorganisms
• protect the product from microorganisms added to the product during use
• to stop organisms growing, not to treat contaminated material or to make up for poor production methods
• Also physical means, water activity, pH, packaging will not be discussed
What is a Preservative?
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Preservation - Disinfection
time
Cellcount
without preservation insufficient preservation
Microbiostasis
sufficient preservationdisinfection
Australian Regulations
Preservatives for use in cosmetics in Australia are regulated by several means:
NICNAS
AICS
Cosmetic guidelines
ACCC
Cosmetic regulations
TGA
SUSMP
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EU Cosmetics Regulation Annex V
The preamble to Cosmetics Directive Annex VI stated: “1. Preservatives are substances which may be added to
cosmetic products for the primary purpose of inhibiting the development of micro-organisms in such products.
3. Other substances used in the formulation of cosmetic products may also have anti-microbial properties and thus help in the preservation of the products, as, for instance, many essential oils and some alcohols. These substances are not included in this Annex.”
Cosmetic Ingredients which may assist Preservative Efficacy
Alcohols Potentiators
Glycols, sugars Control water availability
Cationic surfactants Affect integrity of microbial cell membrane
Chelating agents Interfere with Gm negative bacterial outer membrane by binding Mg2+ & Ca2+
Natural (essential oils) Exhibit selective anti-microbial activity
Acids/alkalis Reduce spectrum of microbial activity through pH control
Fatty acid esters Exhibit selective anti-microbial activity
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EU Cosmetics Regulation Annex V
The preamble to Cosmetic Directive Annex VI stated: “1. Preservatives are substances which may be added to
cosmetic products for the primary purpose of inhibiting the development of micro-organisms in such products.
3. Other substances used in the formulation of cosmetic products may also have anti-microbial properties and thus help in the preservation of the products, as, for instance, many essential oils and some alcohols. These substances are not included in this Annex.”
The EU regulations Annex V definition is: “Preservatives mean substances which are
exclusively or mainly intended to inhibit the development of micro-organisms in the cosmetic product”
Commonly used preservatives
Benzoic acid Propionic acid Salicylic acid Dehydroacetic acid sorbic acid
formaldehyde
DMDM Hydantoin
Diazolidinylurea
Sodium hydroxymethylglycinate
Imidazolidinylurea
MIT/CIT Methyl isothiazolinone
bronopol chlorphenesin IPBC
parabens phenoxyethanol Benzyl alcohol
Polyaminopropylbiguanide
hexamidine
Undecylenicacid
hexetidine Bromo-nitro-dioxane
Formic acid Dichlorobenzylalcohol
trichlocarban chlorocresol triclosan chloroxylenol methanamine
OPP Zinc pyrithione Sodium sulphite
Quaternium-15 climbazole
Benzalkoniumchloride
Ethyl lauroylarginate HCl
Piroctone olamine
Bromo chlorophene Isopropylcresol
phenylmercuric chlorophene chloracetamide Benzylhemiformal Phenoxyisopropanol
Cetrimonium chloride
Dimethyl oxazoladine
chlorobutanol Ethylbicyclo oxazolidine Benzethoniumchloride
Chlorhexadine thiomerosal Silver chloride Dibromohexamidine glutaraldehyde
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Regulation: EUSubstance Maximum concentration (%)
Potassium sorbate 0.6% (acid)
Parabens Methyl, ethyl, propyl, butyl 0.4% individual, 0.8% total (as acid)
Dehydroacetic acid 0.6% (acid)
Phenoxyethanol 1.0%
Benzyl alcohol 1.0%
CIT/MIT 1:3 0.0015%
Chlorphenesin 0.3%
MIT 0.01%
Substance Rinse off Leave on Other
Sodium benzoate 2.5% (acid) 0.5% (acid) 1.7% oral (acid)
IPBC 0.02% 0.01% 0.0075% deodorant
Not to be used for lip or oral,not for children under 3 years except rinse offNot for products applied to large part of the body
Biocides Mode of Action
Biocide Mode of Action
Formaldehyde Bronopol Isothiazolinone
Oxidation of thiol groups and inhibition of sulphydryl groups in cytoplasmic membrane and intracellular enzymes
Imidazolidinyl urea Mercurials Hexamine Derivatives Parabens
Transmembrane pH gradient effecting Proton transfer across the cell wall and cytoplasmic membrane
Formaldehyde Hexamine Derivatives
Amino groups in the cytoplasm Including proteins, DNA, RNA
Quaternary Ammonium Compounds (QAC’s) Alcohols
Potassium, Ribosome leakage through cell wall
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Effects of Preservatives
Disruption of the transmembrane proton motive force leading to uncoupling of oxidative phosphorylation and inhibit active transport across membrane
Inhibition of respiration or catabolic/anabolic reactions
Disrupt replication
Lose membrane integrity
Lysis
Coagulation of intracellular components
Effects Metabolic imbalance after sublethal injury may lead
to free radical production and self destruction
Differentiates highly metabolising exponential growth cells from those in stationary phase.
Time
Log 1
0 C
ell C
ount
Lag phase
Log phase
Stationary phase Decline phase
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Preservative Actives
Commonly used preservatives
Christmas pictures
1996 2005 2007 2010
Methylparaben methylparaben methylparaben methylparaben
propylparaben propylparaben propylparaben propylparaben
Imidazolidinyl urea butylparaben phenoxyethanol phenoxyethanol
butylparaben ethylparaben butylparaben butylparaben
ethylparaben phenoxyethanol ethylparaben ethylparaben
phenoxyethanol Imidazolidinyl urea isobutylparaben Isobutylparaben
DMDMH DMDMH isobutylparaben MI
MI/MCI MI/CMI MI MI/CMI
Quaternium 15 Diazolidinyl urea MI/CMI DMDMH
USA Canada
2007 2010 2007 2010
Caprylol glycol 591 1712 887 4466
Pentylene glycol 200 705 718 1882
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• Chemical Type: • Phenolics, Esters of parahydroxybenzoic acid (para acid).
• Spectrum of Activity:• Fungi & Gram positive bacteria• They are inactivated by strong hydrogen bonders such as ethoxylated compounds, cellulose gums, lecithin.• Incompatible with some nonionics• World wide approval for use• Effective across pH 4 – 8
•Since April 2014
Paraben estersmethyl, ethyl, propyl, butyl, isobutyl
Paraben Maximum concentrations (as acid)
Methyl, Ethyl, Propyl, Butyl 0.4% for one ester 0.8% for esters blend
Isobutyl, isopropyl, phenyl Risk can not be evaluated due to lack of data
C
O
OCH3HO
•Increase in chain length gives increase in activity and decrease in water solubility•Methyl paraben
• Ethyl paraben
•Propyl paraben
• Butyl paraben
Paraben estersmethyl, ethyl, propyl, butyl
C
O
OCH3HO
Paraben Water (w/w 25OC)
Water (w/w 80OC)
Ethanol(w/w)
MIC Ps. aeruginosa
Methyl 0.25 2.0 52 2000
Ethyl 0.17 0.86 70 1000
Propyl 0.04 0.3 95 600
Butyl 0.02 0.15 210 500
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Parabens• Bad press regarding breast cancer and hormone mimic has resulted in reduced usage.
•Japanese Study suggests that under influence of UV light Methyl paraben increases skin aging
• no claims proven
•Chemical Type: •Phenolic like
•Spectrum of Activity:•active against Gram positive bacteria, but is weak against mould. It acts more as a bacteriostat
•Characteristic: • Volatile and may be inactivated by nonionics• should be used with an antioxidant and <pH 7• Listed in EU as a fragrance allergen
•Typical Dose (%) : 1.0 - 3.0•Maximum dose: 1%
•Used in combination with other actives at relatively high concentrations
Benzyl Alcohol
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Phenethyl alcohol
Found in nature in a variety of essential oils
Colourless liquid with warm rose and honey aroma
Better activity than benzyl alcohol
Non volatile
Not a listed preservative
Used in combination with other actives at relatively high concentrations
Usually mixed with caprylyl glycol or phenoxyethanol
“Although phenethyl alcohol is known for its antimicrobial properties, it’s not on EU, Japanese or the U.S. list of recognized cosmetic preservatives and so theoretically this system can be considered a ‘preservative-free’preservative,” added Ms. Kasprzyk
•Chemical Type: • Phenolic like• Spectrum of Activity:• Some activity against bacteria, yeast and moulds, especially Gram –ve bacteria (Pseudomonas sp.)
•Characteristic: •Good solvent for parabens and some other preservatives. • World wide approval for use• Wide pH range 3 – 10• May be inactivated by nonionics.
• Typical Dose (%) : 0.1 -1.0• Maximum dose: 1%
• Used in combination with other actives at relatively high concentrations
Phenoxyethanol
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MIC Values (ppm)
Mixture St.aureus Ps. aeruginosa C. albicans
phenoxyethanol 9000 8000 4000
phenethyl alcohol 10000 5000 100000
70% phenoxyethanol 30% phenethyl alcohol
2500 1250 >5000
69% phenoxyethanol 30% phenethyl alcohol1% lactic acid
2500 635 2500
Lactic acid 781 781 >100,000
Alcohols enhance the permeability of micro-organisms and causes disruption of the cell membrane. The destabilization of the membrane permeability barrier allows the entry of materials that are normally unable to penetrate the cell. It also allows the loss of important cell components that lead to the inhibition of DNA synthesis.
• Formaldehyde/formalin
• Formaldehyde releasers
• Formaldehyde is listed in Annex V with a maximum use concentration of 0.2%, but is limited to 0.1% in oral products and prohibited from use in aerosols. Products with >0.5% formaldehyde must be labelled “Contains formaldehyde”.
HO CH2 O
CH2 CH2
O CH2 OH
O CH2
Formaldehyde Preservatives
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Formaldehyde Releasers
Advantages :• Fast acting
• Broad spectrum against bacteria
• Sporicidal
• Can give “headspace” protection
• Activity unaffected by pH
• Low cost
Disadvantages :• Odour
• Perceived toxicity; carcinogen, sensitiser, irritant
• Poor efficacy against moulds and yeasts
Head Space Protection
HCHO
HCHO
Full Jar Used Jar
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Formaldehyde Donors
HCHO Donor Activity spectrum
Typical
Use conc (%)
Stability Max
level
HCHO biocide/ product
Imidazolidinyl urea
Gram +ve
Gram –ve
No fungi
0.1 – 0.5 600C
pH 4 - 9
0.6% a) 31
b) 0.186
Diazolidinyl urea
Gram +ve
Gram -ve
Some Fungi
0.1 – 0.3 600C
pH 4 - 9
0.5% a) 43
b) 0.215
DMDM Hydantoin
Gram +ve
Gram –ve Weak Fungi
0.15 – 0.4 800C
pH 3 -10
0.6% a) 33
b) 0.198
Sodium hydroxymethyl glycinate
Gram +ve
Gram –ve
Some fungi
Weak yeast
0.05 – 0.5 500C
pH 4 -12
0.5% a) 24
b) 0.118
a) % total formaldehyde bound in biocide – based on 100% active
b) % free formaldehyde released into cosmetic at maximum addition level of active
ECHA Chemicals for substitution
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Concentration (%) of organic acid in undissociated state
Organic acid pH5 pH6 pH7 Water solubility
Salicylic acid 0.94 0.094 0.0094 0.2
Benzoic acid(sodium benzoate)
13 1,5 0,15 0.3
Sorbic acid(Potassium sorbate)
37 6 0.6 0.2
Propionic acid 42 6.7 0.71 miscible
Dehydroacetic acid 65 15.8 1.9 <0.1
Organic acid pka
Salicylic acid 3.0
Formic acid 3.8
Benzoic acid 4.2
Sorbic acid 4.8
Propionic acid 4.9
Dehydroacetic acid 5.4
Propyl paraben 8.1
Methyl paraben 8.5
Boric acid 9.1
The effect of pH on organic acids
The activity is pH dependent, efficacy in a very narrow pH range around their pKa values. pH Range 3.7 – 5
Undissociated state enables the uncharged acid to pass through the negatively charged cell
Typical use rates are 0.05 – 0.2% Recommend to add organic acid in water for emulsion system.
Microbes exist in water phase. Preservatives also must be in water phase. If added in oil phase, undissociated form may stay in oil phase.
Organic Acids
28
Cell
Na+
R-COO - Na+
R-COO - Na+
R-COO - Na+
R-COO - Na+
R-COO - Na+
R-COO - Na+
H+
R-COOH
Na+
R-COO - Na+
ATP level
R-COO-H
R-COO-H
R-COO-H
R-COO-H
• The cell will actively try to restore its optimal pH by exchanging H + ions by Na + ions. To do this it will use its energy source the ATP
• Use in combination with active transport inhibitors for greater effect
Organic Acids – mode of action
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MIC values
Extract St. aureus Ps. aeruginosa C. albicans
Lactic acid 781 781 100,000Citric acid 781 781 100,000Anisic acid 3125 3125 3125Levulinic acid/sodium levulinate
>10000 >10000 >10000
sample 10% 5% 2.5% 1.25% 0.68% 0.34% 0.17% 0.09% 0.05% pKA
anisic 4.66 4.72 grow 4.47
Lactic 1.76 2.03 2.31 2.67 2.93 3.35 3.79 4.38 grow 3.86
Citric 1.59 1.91 2.21 2.49 2.87 3.30 3.97 4.31 grow 3.08
Levulinic 4.5
Lactic Citric Anisic Levulinic
Case Study
Baby moisturising cream pH 5.0; preserved with 1% Microcare SB
Potassium sorbate plus sodium benzoate
Pass Preservative Efficacy Test, production contaminated At pH 5 sorbic acid (pKa 4.8) only 37% activity
benzoic acid (pKa 4.2) only 13% activity Factory hygiene very important to stop development of
tolerant organisms Action Lower pH to 4.7, increased Microcare SB to 1.2%
Sorbic acid from 37% to 80% active Benzoic acid from 13% to 35% active
Small increase in preservative and small drop in pH achieve 300% increase in the preservation strength
No contamination in production
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Halogenated Compounds
Methylchloroisothiazolinone (MCI)
Methylisothiazolinone (MI)
Bronopol
Methyl dibromo gluteronitrile
Iodopropynyl Butylcarbamate (IPBC)
Chlorophenesin
• Chemical Type: • Halogenated Compounds
• Spectrum of Activity: • Excellent broad spectrum activity against all bacteria and fungi
• Characteristics MCIT:• Water soluble • Poor stability above pH7 in presence of amines such as coco-DEA and inactivated by zinc pyrithione• Skin sensitiser, restricted to15ppm in rinse off and leave-on products• Compatible with all types of surfactants.
• SCCS opinion released: safe at 15ppm for rinse off, not to be used in combination with MIT
methylchloroisothiazolinone/methylisothiazolinone
(MCI/MI) mix 3:1
SN-CH3
Cl
O
SN-CH3
O
+
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• Chemical Type: • Heterocyclic N, S Compounds
• Spectrum of Activity: •broad spectrum activity against bacteria, poor fungicide
• Characteristics: •Miscible with water, propylene glycol and lower alcohols •Lower sensitisation than CIT/MIT (~30X) •May be used for leave-on applications•Compatible with all types of surfactants.•Effective alternative to formaldehyde donors•Excellent inherent stability over wide pH and temperature range •
• Typical Dose (%) : 0.01• Maximum allowed concentration 100ppm
Methylisothiazolinone (MI)
SN
O
CH3
Tricarboxylic acid cycle
Pyruvate
Acetyl-coenzyme A
Pyruvate dehydrogenase
Citrate
Cis-aconitate
Isocitrate
Ketogluterate
Succinyl-coenzyme ASuccinate
Fumerate
Malate
Oxalacetate
Citric synthaseAconitase
Aconitase
Isocitratedehydrogenase
Succinyl kinase
KetogluteratedehydrogenaseSuccinate
dehydrogenase
Fumerase
Malatedehydrogenase
CO2
CO2
CO2
Aconitase
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Enzyme Active Site
Effect of isothiazolinones
Pyruvate
Acetyl-coenzyme A
Citrate
Cis-aconitate
Isocitrate
Ketogluterate
Succinyl-coenzyme ASuccinate
Fumerate
Malate
Oxalacetate
KetogluteratedehydrogenaseSuccinate
dehydrogenase
Malatedehydrogenase
Pyruvate dehydrogenaseTricarboxylicacid cycle
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Electron transport chain
Produces large amounts of ATP
Transports protons across cell membrane
Interior alkaline and negatively charged
Oxidative phosphorylation
AH2AH2
AA
Carrier I OXCarrier I OX
Carrier I REDCarrier I RED
Carrier II REDCarrier II RED
Carrier II OXCarrier II OX
ADPADP
ATPATP
Carrier III REDCarrier III RED
Carrier III OXCarrier III OX
ADPADP
ATPATP
H2OH2O
1/2 O21/2 O2
TerminaloxidaseTerminaloxidase
CIT associationoccurs in minutes
0
5
10
15
20
25
30
35
0.5 5 10 15 20 25 30
P. aerug. 1
P. aerug. 2
P. florec.
Minutes
ug CIT per mg protein
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• Chemical Type: •Halogenated Compounds
• Spectrum of Activity:• Fungal only
• Characteristics:• hydrolysis in strong alkaline conditions• Heat stable to 400C
•Typical Dose (%) : 0.002 – 0.01%• Maximum allowed concentration;
•rinse off products 0.02%•leave on products 0.01%•deodorants/antiperspirants 0.0075%
• Not allowed in oral or lip products, children cosmetics or body lotions
IodoPropinylButylCarbamate (IPBC)
Chlorphenesin
Listed preservative
Fungicide
Coming into favour with loss of parabens and IPBC
Used in combination with other preservativeslike phenoxyethanol
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Efficacy – Comparative MICs
Sources: Thor data, trade & technical literature
MIC (ppm)
Preservative Pseudomonas aeruginosa
Staphylococcus aureus
Aspergillusbrasiliensis
Candidaalbicans
MCI/MI 4 3 4 5
MI 40 30 750 100
Bronopol 50 50 3200 400
Formaldehyde 60 20 300 1000
DMDMH 290 290 1455 -
Diazodinyl urea 1000 800 3000 >6000
IPBC 625 156 10 39
Chlorphenesin 2500 2500 600 1250
Phenoxyethanol 3200 6400 3200 3200
Methyl paraben 2000 1500 1000 1000
Caprylyl glycol 6300 12500 1630 3100
Decylene glycol 400 300 200 300
Combinations of Actives
Why?
Broader Activity
Ease of Incorporation
Increased stability
Dilution of Actives
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Combination Preservatives
MCIT/MIT & Benzyl Alcohol
Diazolidinyl urea or Imidazolidinyl urea & methyl and propyl paraben
Methyl, ethyl and propyl parabens & phenoxyethanol
Methyl, ethyl, propyl, butyl and isobutyl parabens & phenoxyethanol
MCIT/MIT & Formaldehyde
MCIT/MIT & Bronopol
MIT & lots
Preservative mixtures
“Preservative” Phenoxyethanol & organic acids
Phenoxyethanol & glycols
Phenoxyethanol & phenols
Phenoxyethanol & everything (extracts)
Glycols & organic acids
Sodium benzoate & potassioum sorbate
Benzyl alcohol & dehydroacetic acid
“Preservative” free Glycols
Glycols & phenols
Glycols & caprylates
Glycols & everything (extracts)
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What’s wrong with preservatives
Active Concern
Parabens Cancer, hormone mimic
Formaldehyde and donors Cancer, sensitiser
Bronopol Nitrosamine, formaldehyde
MI Sensitiser
MI/MCI Sensitiser
IPBC Sensitiser, Iodine
Triclosan Everything bad
Synthetic preservatives Toxic
Marketing
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Natural
Product Content
Citramed Grapefruit peel extract
Citrazole Citrus extract
Plantservative Lonicera caprifolium & japonica extract
Natacide Vegetable extract, vanilla and almond odour
Biosecure A520D Hydro-glycerin Citrus extract, no grapefruit
Leucidal liquid Leuconostic fermentation of Radish root
Phytocide Elderberry Elderberry fruit extract
Grape fruit seed extract Grapefruit pulp
Chemical structure of 1,2-alkanediols
O H
O H
O H
O H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
C2: 1,2 ethanediol
C8: 1,2 octanediol(Caplyryl glycol)
C4: 1,2 butanediol
C5: 1,2 pentanediol
C6: 1,2 hexanediol
C10: 1,2 decanediol
C3: 1,2 propanediol
OH
OH
1,3 butane diol
OH
OH
OH 1,2,3 propane triol(glycerin)
OHOH
OHOH
1,5 pentane diol
2,4 pentane diol
O
OH OHDi propylene
glycol
OO
HOH
n
PEG
1,2-alkanediols Isomers etc
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Membrane inhibition Model of 1,2-alkanediol
1,2-diol
phospholipids
Lipid bilayer
1,5-pentanediol
Substances outside of cell
Substances inside of cell(K+, ribosomes)
Outer
Inner
Efficacy and Skin irritation potentialThor recommendation
-
5,000
10,000
15,000
20,000
25,000
30,000
35,000
40,000
PTG C5 HXD C6 CLG C8 DCG C10
ppm
S. aureus
E.coli
Ps. aeruginosa
C. albicans
A. niger
3.5% needed
2.5% needed
0.25% needed0.06% needed
2.0% MAX
1.5% MAX
0.5% MAX0.3% MAX
Blue box: highest MIC ≈ minimum required concentration
Green box: Thor recommendation of maximum dosage from risk assessment (NOAEL, MoS)
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Migration Model of 1,2-alkanediol
OH
OH
1,2- alkanediol
Oil phase
Aqueous phase
OH
OH
Alkane diols more soluble in polar oil phase, > solubility in caprylic/capric triglyceride than in mineral oil
Reduce polarity of oil phase or add solvent to water phase
Increase emulsifier concentration or use mixture of ionic and nonionic
Other actives
Caprylic acid
Glyceryl capralate
Caprylhydroxamic acid
Sorbitan caprylate
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Preservative Selection
critical that the preservative selected for a product is matched to the physical and chemical requirements of the product and will provide protection against the full spectrum of microorganisms likely to be encountered.
The preservative must be used correctly Addition point
Concentration
pH, temperature, surfactants, water/oil phases
The toxicity, or the perceived toxicity of the active must also be compatible with the intended use and market.
Basing the selection of preservative actives on these criteria and ensuring good manufacturing practices should enable production of Personal Care Products with no fear of contamination or adverse effects
Antidandruff shampoo
1% zinc pyrithione
Sodium benzoate
Benzyl alcohol
Methylchloroisothiazolinone
Methylisothiazolinone
pH 5.87
Sodium benzoate• inactive but high solubility• Convert to benzoic acid above pKa• Useful at pH <4.7, pKa 4.2• Mainly antifungal activity , low antibacterial activity
1% Zinc pyrithione • 10,000ppm• MIC for: St.a 4ppm
Ps.a 512ppm• ZPT solubility in water 8ppm• Mainly antifungal activity, low antibacterial activity
methylchloroisothiazolinone• degraded by free pyrithione• will give initial preservation
methylisothiazolinone • Present at 4ppm• Need higher concentration
Suggested Preservative: Methyl Isothiazolinone
Now: reduce the pH and use benzoic acid/sorbic acid
0
2
4
6
8
10
12
14
16
0 2 days 4 days 6 days 8 days 10 days
no ZPT
1% ZPT
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Get the Balance Wrong
Batch Numbers:
Sold:
Defect:
Hazard:
What to do:
Contact Details:
www.recalls.gov.au
Defect:
Hazard: