An Overview of Preservatives and Their Modes of Action Kevin Roden Thor Specialties

10/13/2014

An Overview of Preservatives and their Modes of Action Kevin Roden Thor 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

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Preservation - Disinfection

without preservation insufficient preservation

Cell Microbiostasis count

sufficient preservation disinfection

time

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 formaldehyd DMDM Diazolidinyl Sodium Imidazolidinyl e Hydantoin urea hydroxymethyl urea glycinate MIT/CIT Methyl bronopol chlorphenesin IPBC isothiazolinone parabens phenoxyethanol Benzyl Polyaminopropyl hexamidine alcohol biguanide Undecylenic hexetidine Bromo-nitro- Formic acid Dichlorobenzyl acid dioxane alcohol trichlocarban chlorocresol triclosan chloroxylenol methanamine OPP Zinc pyrithione Sodium Quaternium-15 climbazole sulphite Benzalkonium Ethyl lauroyl Piroctone Bromo chlorophene Isopropylcresol chloride arginate HCl olamine phenylmercuric chlorophene chloracetamide Benzylhemiformal Phenoxy isopropanol Cetrimonium Dimethyl chlorobutanol Ethylbicyclo oxazolidine Benzethonium chloride oxazoladine chloride Chlorhexadine thiomerosal Silver chloride Dibromohexamidine glutaraldehyde

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Regulation: EU Substance 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 off Not for products applied to large part of the body

Biocides Mode of Action

Biocide Mode of Action Formaldehyde Oxidation of thiol groups and inhibition Bronopol of sulphydryl groups in cytoplasmic Isothiazolinone membrane and intracellular enzymes Imidazolidinyl urea Transmembrane pH gradient effecting Mercurials Proton transfer across the cell wall Hexamine Derivatives and cytoplasmic membrane Parabens Formaldehyde Amino groups in the cytoplasm Hexamine Derivatives Including proteins, DNA, RNA Quaternary Ammonium Potassium, Ribosome leakage Compounds (QAC’s) through cell wall Alcohols

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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.

Stationary phase Decline phase

Log phase Cell Count 10 Log

Lag phase

Time

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Preservative Actives

Commonly used preservatives

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 phenoxyethanolChristmas picturesImidazolidinyl 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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Paraben esters methyl, ethyl, propyl, butyl, isobutyl • Chemical Type: • Phenolics, Esters of parahydroxybenzoic acid (para acid). • Spectrum of Activity: O • Fungi & Gram positive bacteria HO C OCH3 • 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 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

Paraben esters methyl, ethyl, propyl, butyl •Increase in chain length gives increase in activity and decrease in water solubility O •Methyl paraben HO C OCH3

• Ethyl paraben

•Propyl paraben

• Butyl paraben

Paraben Water Water Ethanol MIC (w/w 25OC) (w/w 80OC) (w/w) 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

Benzyl Alcohol •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

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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

Phenoxyethanol •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

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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 2500 1250 >5000 30% phenethyl alcohol 69% phenoxyethanol 2500 635 2500 30% phenethyl alcohol 1% lactic acid 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 Preservatives

• Formaldehyde/formalin

O CH2 • Formaldehyde releasers

H2C CH2 CH HO CH2 O O 2 OH • 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”.

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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

Full Jar Used Jar

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Formaldehyde Donors

HCHO Donor Activity Typical Stability Max HCHO spectrum Use conc level biocide/ (%) product Imidazolidinyl Gram +ve 0.1 – 0.5 600C 0.6% a) 31 Gram –ve urea pH 4 - 9 b) 0.186 No fungi Diazolidinyl Gram +ve 0.1 – 0.3 600C 0.5% a) 43 Gram -ve urea pH 4 - 9 b) 0.215 Some Fungi DMDM Gram +ve 0.15 – 0.4 800C 0.6% a) 33 Hydantoin Gram –ve Weak Fungi pH 3 -10 b) 0.198 Sodium Gram +ve 0.05 – 0.5 500C 0.5% a) 24 Gram –ve hydroxymethyl pH 4 -12 b) 0.118 glycinate Some fungi Weak yeast

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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The effect of pH on organic acids Organic Acids

Concentration (%) of organic acid in undissociated state Organic acid pka Salicylic acid 3.0 Organic acid pH5 pH6 pH7 Water solubility Formic acid 3.8 Salicylic acid 0.94 0.094 0.0094 0.2 Benzoic acid 4.2 Benzoic acid Sorbic acid 13 1,5 0,15 0.3 4.8 (sodium benzoate) Propionic acid 4.9 Sorbic acid 37 6 0.6 0.2 Dehydroacetic (Potassium sorbate) acid 5.4 Propionic acid 42 6.7 0.71 miscible Propyl paraben 8.1 Dehydroacetic acid 65 15.8 1.9 <0.1 Methyl paraben 8.5 Boric acid 9.1  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 – mode of action

Cell

R-COO - Na+ R-COO - Na+

R-COO-H

R-COO - Na+ R-COO - Na+ ATP level H+ R-COO-H R-COOH

R-COO - Na+ Na+ + Na R-COO - Na+

R-COO-H

R-COO - Na+ • 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 28 transport inhibitors for greater effect

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MIC values

Extract St. aureus Ps. aeruginosa C. albicans Lactic acid 781 781 100,000 Citric acid 781 781 100,000 Anisic acid 3125 3125 3125 Levulinic acid/sodium >10000 >10000 >10000 levulinate

Lactic Citric Anisic Levulinic

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

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

methylchloroisothiazolinone/methylisothiazolinone (MCI/MI) mix 3:1 • Chemical Type: O O • Halogenated Compounds N-CH N-CH 3 + 3 • Spectrum of Activity: Cl S S • 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

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Methylisothiazolinone (MI)

• Chemical Type: O • Heterocyclic N, S Compounds N • Spectrum of Activity: S CH3 •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

Tricarboxylic acid cycle

Pyruvate

CO2 Pyruvate dehydrogenase Acetyl-coenzyme A Citric synthase Citrate Aconitase Oxalacetate Malate dehydrogenase Cis-aconitate Aconitase Malate Isocitrate Isocitrate Fumerase dehydrogenase

CO2 Fumerate  Ketogluterate  Ketogluterate Succinate dehydrogenase dehydrogenase Succinate Succinyl-coenzyme A Succinyl kinase CO2

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Enzyme Active Site

Effect of isothiazolinones

Tricarboxylic Pyruvate Pyruvate dehydrogenase acid cycle Acetyl-coenzyme A Citrate Malate Oxalacetate dehydrogenase Cis-aconitate

Malate Isocitrate

Fumerate  Ketogluterate  Ketogluterate Succinate dehydrogenase dehydrogenase Succinate Succinyl-coenzyme A

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Electron transport chain Oxidative phosphorylation ADP ADP AH H O 2 Carrier I OX Carrier II RED Carrier III OX 2 Terminal oxidase A Carrier II Carrier III 1/2 O Carrier I RED OX RED 2 ATP ATP

 Produces large amounts of ATP  Transports protons across cell membrane  Interior alkaline and negatively charged

CIT association occurs in minutes 35 30 25 20 P. aerug. 1 ug CIT per mg 15 P. aerug. 2 protein 10 P. florec. 5 0 0.5 5 10 15 20 25 30 Minutes

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IodoPropinylButylCarbamate (IPBC)

• 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

Chlorphenesin

 Listed preservative  Fungicide  Coming into favour with loss of parabens and IPBC  Used in combination with other preservatives like phenoxyethanol

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Efficacy – Comparative MICs

MIC (ppm) Preservative Pseudomonas Staphylococcus Aspergillus Candida aeruginosa aureus brasiliensis albicans 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 Sources: Thor data, trade & technical literature

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

1,2-alkanediols Isomers etc

OH C2: 1,2 ethanediol OH

OH C3: 1,2 propanediol HO OH 1,2,3 propane triol OH OH (glycerin)

OH OH C4: 1,2 butanediol 1,3 butane diol OH OH

OH C5: 1,2 pentanediol HO OH 1,5 pentane diol OH 2,4 pentane diol OH OH OH C6: 1,2 hexanediol OH OH OH Di propylene OH C8: 1,2 octanediol O glycol (Caplyryl glycol) OH

OH C10: 1,2 decanediol O H PEG OH HO O n

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Membrane inhibition Model of 1,2-alkanediol

Substances outside of cell

1,5-pentanediol 1,2-diol Outer

Lipid bilayer

Inner phospholipids Substances inside of cell (K+, ribosomes)

Efficacy and Skin irritation potential Thor recommendation

ppm 40,000 3.5% needed S. aureus E.coli 35,000 Ps. aeruginosa C. albicans 30,000 A. niger 2.5% needed 25,000

2.0% MAX 20,000 1.5% MAX 15,000

10,000 0.5% MAX 0.3% MAX 5,000 0.25% needed 0.06% needed

- PTG C5 HXD C6 CLG C8 DCG C10 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

1,2- alkanediol

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 OH OH

Oil phase

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

16  1% zinc pyrithione 14 1% Zinc pyrithione 12  Sodium benzoate • 10,000ppm no ZPT 10  Benzyl alcohol • MIC for: St.a 4ppm1% ZPT 8 Ps.a 512ppm  Methylchloroisothiazolinone 6 • ZPT solubility in water 8ppm  Methylisothiazolinone 4 2 • Mainly antifungal activity, low  pH 5.87 0 antibacterial activity 0 2 days 4 days 6 days 8 days 10 days Sodium benzoate methylchloroisothiazolinone • inactive but high solubility • degraded by free pyrithione • Convert to benzoic acid above pKa • will give initial preservation • Useful at pH <4.7, pKa 4.2 • Mainly antifungal activity , low methylisothiazolinone antibacterial activity • Present at 4ppm • Need higher concentration Suggested Preservative: Methyl Isothiazolinone Now: reduce the pH and use benzoic acid/sorbic acid

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Get the Balance Wrong

Defect: Batch Numbers: Hazard: Sold:

Defect:

Hazard:

What to do:

Contact Details: