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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: