NZ620616B2 - Disinfecting formulations and uses thereof - Google Patents

Abstract

The present disclosure provides a disinfecting formulation useful, for example, for cleaning and disinfecting human or animal body parts, and in particular for disinfecting human hands. The disinfecting formulation comprises alcohol including ethanol; an essential oil comprising cineole, in particular eucalyptus oil; an emollient including glycerin; and other ingredients comprising piroctone olamine, acrylic acid based polymer and 2-amino-2-methyl-1-propanol. The disclosure also provides methods of disinfection of human and animal body parts and methods for preparing the formulation. ar eucalyptus oil; an emollient including glycerin; and other ingredients comprising piroctone olamine, acrylic acid based polymer and 2-amino-2-methyl-1-propanol. The disclosure also provides methods of disinfection of human and animal body parts and methods for preparing the formulation.

Description

DISINFECTING FORMULATIONS AND USES THEREOF ‘ FIELD OF THE INVENTION The present invention provides a disinfecting formulation useful, for example, for cleaning and disinfecting human or animal body parts, and in particular for ecting human hands. The invention also provides methods of disinfection of human and animal body parts and methods for preparing the ation.

BACKGROUND OF THE INVENTION There is an increasing need to develop improved methods and products for ng and disinfecting human and animal body parts, and in particular human hands. In addition to the normal need for individuals to maintain clean and ected hands in order to minimise the potential for er of pathogens, the members of a large number of professions are required to clean their hands during the course of their normal work. For example, those involved in the provision of human health care, in the preparation of food and beverage, the handling of animals, in child and geriatric care and in ng and waste management will all need to ensure their hands are rly cleaned to aVoid the transmission of pathogens that may cause disease either to themselves or to others. The conventional approach for hand cleaning is to use soap and water and in many workplaces where hygiene is paramount, liquid soaps or alcohol based hand rubs (gels or foams that include denatured l that has been made unpalatable for human consumption) are adopted, many of which may include antimicrobial active agents such as isopropanol, chlorhexidine, triclosan, quaternary um compounds, iodinated compounds and the like or parabens, glycols and synthetic fragrances. The problem with many of these agents is that they are harsh on the skin, and in the case of the antimicrobials many microbes have mutated to develop ance to them. It is therefore desirable to develop fast acting hand cleaning formulations that exhibit high efficiency of killing or inactivating pathogens while being relatively gentle on the skin, to thereby allow for regular use (by not only trained professionals but also the general ) without development of skin irritation, ation, dryness, cracking, redness or an allergic response (particularly in the case of pre- and post-operative patients). Owing to some of the additives used in conventional hand cleaning formulations, it is necessary to also use a separate barrier cream and/or skin ioner to t and/or rehydrate the skin.

Indeed the use of a separate barrier cream and/or skin ioner is recommended by the World Health Organisation (WHO) in its “WHO Guidelines on Hand Hygiene in Health Care” published in 2009 (the sures of which are included herein in their entirety by way of reference) for subject susceptible to skin tion. It would be preferable if the additional use of such barrier creams and skin conditioners was not required. Ideally, hand cleaning formulations should neither dehydrate the skin nor cause skin irritation, inflammation, dryness, cracking, redness or an allergic response.

There are also a range of settings in which it is desirable to minimise the need for the use of water in ction with cleansing. In terms of daily hand ing this is a particular issue at the moment in parts of the world where climatic and rainfall conditions are changing and where water is becoming increasingly . In parts of Australia, for example, availability of water is of increasing importance due to limits being placed at least in some areas on household water consumption. It is also desirable to have access to means of effectively cleansing for military applications or in the case of activities such as outdoor labour, camping, bushwalking and the like, where limited amounts of water may be available and where any available water will be for consumption rather than for washing.

It is in this context that the present inventors have conceived a disinfecting formulation that may be used in the absence of additional water. The inventors have adopted substantially l products that exhibit surprising efficacy against a broad range of pathogenic microorganisms, and which can be used repeatedly on human and animal skin generally t significant irritation, inflammation, dryness, cracking, redness or allergic reSponse. There is also no evidence of the development of microbial ance against the formulations of the invention. Primary ingredients of the formulations according to the invention include alcohol, one or more essential oils comprising cineole and a moisturiser, which is preferably a plant d oil. Further, gh it is well understood that alcohols such as ethyl alcohol exhibit antimicrobial activity (due to their ability to denature protein) it is also understood that the effects of the alcohol do not persist on the skin due to ation without residue. The inventors have determined, however, that other components of the inventive formulation do leave a residue, which is not sant or irritant for users, but which results in tence of antimicrobial activity on the skin surface. Thus the formulations of the invention are useful as a professional hygienic handrub, for example for-health professionals, as a al handrub and for preoperative skin preparation for patients about to undergo surgery. It has also been observed that the formulations of the present ion improve symptoms of skin irritation and dryness that may have been caused by the application of other cleansing formulations in some subjects. _ German Patent Application No. 202007002978 discloses a gel composition comprising specified amounts of alcohol, thickener, at least one active agent selected from ves, healing promoters and/or anti—inflammatory agents, as well as water.

For effective disinfection activity this formulation appears to require the presence of biguanide compounds, phenol compounds, iodine compounds or the like, which are not required for disinfection in the present ion. In preferred embodiments of the present invention such compounds are excluded from the ation ing to the present invention, such that disinfecting activity is contributed to by essential oils and alcohol.

International Patent Publication No. discloses a cleaning solution comprising l, and essential oil with specified content of cineole. While the cleansing and disinfecting properties of alcohol such as ethanol and essential oils comprising cineole were tood, it was not expected that such agents could be combined into an alcohol based hand rub (ABHR) formulation that has moisturising properties. Before the work of the present inventors it was generally understood that it was not possible to ively moisturise using such formulations without either compromising upon anti—microbial effectiveness or requiring the need for the inclusion of irritant anti-microbial agents such as quaternary ammonium compounds, chlorhexidine or chlorhexidine gluconate, chloroxylenol, benzalkonium chloride, flurosalan, hexachlorophene, phenol, tribromosalan, triclocarban, triclosan and isopropanol. Indeed WHO notes in its Guideline referred to above that hand rub formulations should contain 1% to 2% of moisturiser and that adding more than 3% of moisturiser can compromise antimicrobial activity.

Y OF THE ION In one aspect of the invention there is provided a disinfecting formulation sing: (a) alcohol; (b) one or more essential oils comprising cineole; and (c) an emollient.

In r aspect the present invention relates to a ecting formulation for topical application to human or animal skin comprising: (a) l; (b) eucalyptus oil; (c) glycerine; (d) one olamine; and (e) water.

In another aspect the present invention relates to’ a disinfecting formulation for topical application to human or animal skin comprising: (a) ethanol; (b) eucalyptus oil; (c) glycerine; (d) terpinenol; (e) piroctone olamine; and (f) water. 2012/000841 In another aspect the present invention relates to a ecting ation for tOpical application to human or animal skin comprising: (a) ethanol; (b) ptus oil; (c) glycerine; (d) piroctone e; and (e) water.

In another aspect the present invention relates to a disinfecting formulation for topical application to human or animal skin comprising: (a) ethanol; (b) eucalyptus oil; (c) glycerine; (d) acrylic acid based polymer; and (e) water.

In another aspect the present invention relates to a disinfecting formulation for tOpical application to human or animal skin comprising: (a) ethanol; (b) eucalyptus oil; (0) glycerine; (d) acrylic acid based polymer; (e) 2-aminomethyl—Il—propanol; and (f) water.

In a preferred embodiment the formulations referred to above are applied to human skin. ably the formulation comprises one or more C] to C10 alcohol, preferably one or more of methanol or ethanol. It is particularly preferred that the alcohol comprises ethanol of analytical grade (AR).

Preferably the one or more essential oils is selected from eucalyptus, tea tree, bayleaf, int and rosemary oils, ably eucalyptus oil and most preferably eucalyptus oil of BF. (British Pharmacopoeia) grade. In an embodiment the formulation may optionally comprise tea tree oil or tea tree oil extract such as terpinenol.

In an embodiment the emollient is or comprises one or more of lanolin, mineral, vegetable and synthetic oils and ants. For example, humectants may include glycerine, propylene glycol, glyceryl triacetate, sorbitol, xylitol, melitol and polydextrose and vegetable oils may comprise coconut oil, jojoba oil, shea butter, mango butter and palm oil. ably the emollient is glycerine (glycerol) and preferably it is plant derived glycerine.

In another embodiment where the formulation is a hand gel where the formulation comprises one or more gelling or thickening agents.

In one embodiment the formulation comprises, by volume, from about 60% to about 80% ethanol, from about 5% to about 15% ptus oil and from about 2% to about % glycerine.

In another embodiment of the invention there is ed a disinfecting ation comprising, by volume: (a) from about 60% to about 80% ethanol; (b) from about 5% to about 15% eucalyptus oil; (0) from about 2% to about 10% glycerine; (d) from about 0.01% to about 0.1% piroctone olamine; and (e) water.

In another embodiment of the invention there is ed a disinfecting formulation comprising, by volume: (a) from about 60% to about 80% ethanol; (b) from about 5% to about 15% eucalyptus oil; (c) from about 2% to about 10% glycerine; (d) from about 0.1% to about 1% terpinenol; (e) from about 0.01% to about 0.1% piroctone olamine; and (t) water.

In another embodiment the formulation comprises, by volume: (a) about 73% of 95% l; (b) about 10% eucalyptus oil; (0) about 5% glycerine; (d) about 0.5% terpinenol; (e) about 0.05% piroctone e; and (1) water.

In another embodiment of the invention there is provided a disinfecting formulation comprising, by volume: (a) from about 60% to about 80% ethanol; (b) from about 5% to about 15% eucalyptus oil; (0) from about 2% to about 10% glycerine; (d) from about 1% to about 2% acrylic acid based r; and (e) water.

In another embodiment of the invention there is provided a ecting formulation comprising, by volume: (a) from about 60% to about 80% ethanol; (b) from about 5% to about 15% eucalyptus oil; (0) from about 2% to about 10% glycerine; (d) from about 1% to about 2% acrylic acid based polymer; and (e) from about 0.8% to about 2% 2-aminomethyl—l -propanol; and (t) water.

The invention also includes a method of disinfecting a human or animal body part comprising applying to the body part a formulation as defined above.

In one embodiment there is ed a method of disinfecting a human or animal body part comprising applying to the body part a formulation comprising, by volume: (a) from about 60% to about 80% ethanol; (b) from about 5% to about 15% eucalyptus oil; (0) from about 2% to about 10% glycerine; (d) from about 0.1% to about 1% terpinenol; (e) from about 0.01% to about 0.5% piroctone e; and (f) water.

In another embodiment the formulation used in the method ses, by volume: (a) about 73% of 95% ethanol; (b) about 10% eucalyptus oil; (c) about 5% glycerine; (d) about 0.5% terpinenol; (e) about 0.05% piroctone olamine; and (f) water.

In another embodiment there is provided a method of disinfecting a human or animal body part comprising applying to the body part a ation comprising, by volume: (a) from about 60% to about 80% ethanol; (b) from about 5% to about 15% ptus oil; (c) from about 2% to about 10% glycerine; (d) from about 0.01% to about 0.1% piroctone olamine; and (e) water.

In one embodiment the method is for disinfecting human hands and in another embodiment the formulation is for pre-operative treatment disinfection of human skin.

In another embodiment there is provided a method of disinfecting a human or animal body part comprising applying to the body part a formulation comprising, by volume: (a) from about 60% to about 80% ethanol; (b) from about 5% to about 15% eucalyptus oil; (c) from about 2% to about 10% glycerine; (d) from about 1% to about 2% c acid based polymer; and (e) water.

In another ment there is provided a method of disinfecting a human or animal body part comprising applying to the body part a formulation comprising, by volume: (a) from about 60% to about 80% ethanol; (b) from about 5% to about 15% eucalyptus oil; (c) from about 2% to about 10% glycerine; (d) from about 1% to about 2% c acid based polymer; and (e) from about 0.8% to about 2% 2—aminomethy1propanol; and (0 water.

DETAILED DESCRIPTION OT THE INVENTION The nce to any prior art in this specification is not, and should not, be taken as an acknowledgment or any form of suggestion that the prior art forms part of the common general knowledge in Australia.

Throughout this specification and the claims which , unless the context requires otherwise, the word ”comprise", and variations such as "comprises" and ”comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Throughout this cation and the claims which follow, unless the context requires ise, the phrase "consisting essentially of“, and variations such as "consists essentially of‘ will be understood to indicate that the recited element(s) is/are essential ie. necessary elements of the invention. The phrase allows for the presence of other non—recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

In a preferred embodiment, the disinfecting formulation comprises alcohol such as a C1 to C10 alcohol, preferably selected from one or more of methanol or ethanol. The alcohol should be non-toxic to s and especially humans on the basis of dermal contact, and inhalation exposure since with normal use the formulation will come into contact with skin and its vapour will be inhaled. It is preferred that the alcohol is not denatured and it is particularly red that the alcohol is ethanol of analytical grade (AR). Preferably the formulation includes alcohol .in an amount by volume of from about 30% to about 85% (% v/v), preferably from about 60% to about 80%, more preferably from about 70% to about 75% and most preferably about 72% or about 73%. In a preferred ment the formulation does not e isopropanol.

The ation comprises one or more ial oils and/or fractions thereof comprising cineole, which are lly obtained from distillation of fresh, dried or partially dried plants or plant derived materials. The essential oil may be obtained from components such as leaves, branches, shoots, stems, bark, seeds, fruit, roots, nuts or the like from one or more plants. Essential oil fractions may be obtained from distillation, purification, refining or the like of essential oils or components thereof.

The ial oils and/0r fractions are ably selected from the group eucalyptus, tea tree, bayleaf, Spearmint and rosemary oils, although other plant species may also give rise to ial oils containing cineole compounds, preferably neole, and preferably in an amount of 20% to 100% of the oil. In a still further preferred embodiment, eucalyptus oil of BF. grade (where it complies with British Pharmacopoeia requirements) is used and preferably a visibly clear grade is used so as to produce a clear formulation, which is generally more aesthetically appealing to consumers. Preferably the amount by weight of cineole in the essential oil, preferably eucalyptus oil, is at least about 60% (% v/v), preferably from about 75% to about -11_ 85%. In a preferred embodiment, the amount by volume of the essential oil, preferably eucalyptus oil, within the formulation is from about 5% to about 30%, preferably from about 10% to 25%, more preferably from about 10% to about 15% and most preferably about 10% or about 11%.

The formulatiOns may also include tea tree oil or an t of tea tree oil, such as enol, Which is cially available and is understood to be the primary active agent of tea tree oil. The presence of tea tree oil or an t thereof in the formulation has been demonstrated by the inventors to assist with preservation of the formulation, to make the formulation unpalatable for human consumption (which is necessary in some jurisdictions for regulatory purposes if a non—denatured l is used, as is preferred) and contributes to antimicrobial activity, particularly due to leaving a residue on the Skin (which is not unpleasant or irritant) that improves persistence of the antimicrobial activity. The structure of terpinen-4~ol is as s: Terpinen—4-ol chemical structure If present, the tea tree oil, extract thereof or terpinenol can be present in an amount of from about 0.1% to about 1% v/v, ably from about 0.2% to about 0.8% and preferably about 0.5% v/v.

The formulations of the invention also include water. Preferably the water will be UV light treated or purified to remove pathogens such as by mi‘crofiltration and/or distillation. The formulation includes water in an amount by volume of from about 5% to about 20% (% v/v), ably from about 5% to about 15%, preferably from about % to about 15%, such as about 10%, about 11%, about 12%, about 13%, about 14% or about 15%.

A preferred further ingredient in some embodiments of the invention‘which are ated to produce a hand rub (ABHR), is one olamine roxy —6-(2,4,4—trimethylpentyl)-2(1H)—pyridone in combination with 2-aminoethanol (1:1); also known as Octopirox®) which is commercially available from Clariant and is extensively used in hair care formulations as an anti—dandruff agent. It is also an anti-oxidant that has been shown to t antimicrobial activity against a range of pathogens, including Gram-positive and Gram-negative bacterial and fungi. It is derived form organic salts using green chemistry. Without wishing to be bound by theory, it appears that in the present invention the presence of one olamine not only assists in the preservation of the formulation by acting as a pH buffer but also contributes to sustained anti—microbial action. In relation to the former advantage, it has been found that previous formulations encountered marked decreases in pH over their shelf life which in turn negatively impacts on antimicrobial performance. It has now been shown that the addition of piroctone olamine stabilises the pH of formulations which in turn enhances the antimicrobial effects of the formulation over an increased shelf life time. The piroctone olamine will generally be t in the formulation in ‘an amount of from about 0.01% to about 0.5% by volume, preferably from about 0.02% to about 0.1% or about 0.05% v/v, and ably about 0.02%, 0.03%, 0.04% or about 0.05% v/v. It is also postulated that the combination of, for instance, eucalyptus oil and piroctone e may provide for a persistant or sustained anti-microbial effect on the surface of the skin. In this way it is postulated that this increased anti-microbial residence time or prolonged anti—microbial effect may be created through a synergistic interrelationship with, for instance, eucalyptus] oil and piroctone olamine. As a further advantage it has been found that the one olamine decreases the usually noticeable smell of the eucalyptus oil which makes the overall aroma of the formulation more pleasant.

The ation may also include one or more emollient agents which serve to soften the skin, usually by improving skin hydration (i.e. moisturising the skin). Examples of suitable emollients are lanolin, mineral, Vegetable and synthetic oils and humectants. Humectants are hygroscopic agents that have the ability to form hydrogen bonds and attract water to thereby have a moisturising effect. For . example, humectants may comprise glycerine, propylene glycol, glyceryl triacetate, sorbitol, xylitol, melitol and polydextrose. es of vegetable oils include coconut oil, jojoba oil, Shea , mango butter and palm oil. The emollients utilised should all be xic in dermal use and substantially non-irritant and non-allergenic.

Preferably the emollient will be present in an amount by volume of from about 2% to about 10%, preferably from about 3% to about 8% and more preferably from about 4% to about 6%, such as about 5% v/v. In a particularly preferred embodiment the emollient agent is glycerine, preferably vegetable glycerine which is readily cially available, and which may be t in an amount by volume as mentioned above.

In another embodiment the invention provides a disinfecting formulation consisting of, by volume: (a) from about 60% to about 80% ethanol; (b) from about 5% to about 15% eucalyptus oil; (0) from about 2% to about 10% glycerine; (d) from about 0.01% to about 0.1% one olamine; and (e) water.

In another embodiment the invention provides a disinfecting formulation consisting essentially of, by volume: (a) from about 60% to about 80% ethanol; (b) from about 5% to about 15% ptus oil; (c) from about 2% to about 10% glycerine; (d) from about 0.01% to about 0.1% piroctone olamine; and (e) water.

The above embodiments which exclude thickeners (gelling agents) may be beneficially used as handrubs, for instance, in a surgical setting where a sustained antimicrobial effect is required.

In on to the above two embodiments this includes a method of disinfecting a human or animal body part rably human) comprising applying to the body part a formulation as defined above. It will be appreciated that in the context of the above embodiment "consisting essentially of“ will exclude the use of additional emollients (other than glycerine), further disinfectant agents, thickeners (gelling agents), but may include small amounts of pH adjusting agents such as, for instance, lactic acid, (i.e. for instance in an amount of from 0.02% - 0.2% v/v).

In other embodiments of the invention additional ingredients not otherwise ed may be included within the formulation such as for e ial oils that do not include cineole to any significant extent (e. g. clove oil, sWeet orange oil), other agents active against microorganisms, aromatic scents, isers, preservatives and the like that are ely used in dermal ations, which should all be non-toxic in dermal use and substantially non—irritant and non—allergenic. Preferably such agents are naturally derived.

In another embodiment where the formulations are intended to be used as handgels they may additionally comprise one or more gelling agents in an amount suitable to modify the formulations into a gelled state. Preferably the gel is in a form that allows it to be readily sed, for example from a tube, tub or pump pack, and can be easily spread upon the surface to be disinfected. By reference to a gel it is intended to convey that there is formation of a colloid that is to some extent immobilised and exhibits solid or olid characteristics. Gelling agents that serve to thicken or impart a level of structural form to the formulation such as vegetable, animal, mineral, WO 06917 petroleum or synthetic waxes, vegetable gums, es, pectins, gelatine, chitin, chitosan, collagen, silica, cornstarch, glycols and carbomer (polyacrylic acid) can be used. For example, vegetable gums include locust bean gum, guar gum, xanthan gum, alginates, ‘ agar, carageenan, beta-glucan, gellan‘ gum, gum arabic, gum tragacanth, karaya gum, locust bean gum, mastic gum, um gum, spruce gum, ghatti gum and glucomannan and vegetable, animal, mineral, petroleum or synthetic waxes include x, shellac. wax, ceti, lanolin, bayberry wax, candelilla wax, ba wax, castor wax, esparto wax, jojoba oil, ouricury wax, rice bran wax, soy wax, ceresin waxes, montan wax, ozocerite, paraffin wax, microcrystalline wax, polyethylene waxes, chemically modified waxes, Fischer-Tropsch waxes, substituted amide waxes and polymerised a—olefins. To be useful within the formulations of the present invention the gelling agents should be non-toxic in dermal use and substantially non—irritant and non-allergenic. The gelling agents may be t in amounts by volume of from about 0.1% to about 5%, preferably from about 0.5% to about 3% and more preferably from about 1% to about 2%, for instance, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, or 1.9%.

In an embodiment the gelling agents are petroleum waxes, examples of which include paraffin wax and microcrystalline wax. A preferred petroleum wax is the proprietary formulation known as "Anhydro Wax", which is commercially available from Intelisol Pty Ltd of 1 / 57 Malvern Street, Bayswater, Victoria, 3153, Australia. If "Anhydro Wax" is adopted in the formulation it is preferably used in an amount by volume of from about 0.5% to about 2%, preferably from about 1% to about 2% or preferably about 1.5%. Another preferred gelling agent is polymeric ic acid, which may be partially lised with ammonia, such as the AristotlexTM t available from Clariant, Frankfurt, Germany.

In a preferred embodiment theformulation comprises a gelling or ning agent which is an acrylic acid based polymer such as Carbopol® or Pemulen®. Carbopol® and Pemulen® are high molecular weight homo- and copolymers of acrylic acid ~16— crosslinked with a kenyl polyether. Preferably the acrylic. acid based polymer is present in conjunction with a neutralising agent which may be an organic base.

Preferably the base is 2-aminomethyl—1-propanol. More preferred the base is 95% 2—amino—2-methylpropanol. Preferably the base is added in an amount of about 0.8% - about 2% v/v of the formulation. Preferably the gelling or thickening agent is Carbopol, and preferably in an amount of about 1% to about 2% v/v, such as about 1.1%. The present formulation presented as a gel has the added advantage of being able to dry quicker than conventional gels which is important in clinical settings. The drying time has been noted to be around 25 — 40 seconds, and on average 30 seconds.

Conventional gels can take much longer, for instance, up to 1 minute.

In a preferred embodiment the gelling or thickening agent is added to achieve ity of from 5,000 — 8,000 cps,land preferably about 6,000 — 7,000, and more preferably about 6,000cps.

In a preferred embodiment the pH of any of the formulations referred to herein is from about 4.5 to 8.5, such as from about 6-8.5.

In a further embodiment the formulation is about 8, such as within the range 7.8-8.5.

The formulation is a disinfectant in that it has icrobial activity and hence will kill, slow growth and/or ar division of microorganisms and other pathogens such as bacteria (Gram positive and Gram negative), viruses, fungi, protozoa, mites, algae, nematodes and the like ctively referred to as ”flora"). By application of the solution to the skin preferably at least 90%, more preferably at least 95%, particularly preferably at least 98% or 99% and most preferably‘at least 99.5% or 99.9% of flora on the skin will be killed or otherwise inactivated. The superior disinfecting nature of the preferred formulation of the ion (comprising aqueous ethanol, eucalyptus oil, glycerine, and one olamine) is attributed to the prolonged presence of ethanol on the skin and persistence of a non-irritant residue of ptus oil, and piroctone olamine. It is r postulated that upon ation of the formulation, the eucalyptus oil and glycerine components act to delay evaporation of the ethanol component, thereby allowing the ethanol component to kill any flora present on the skin and prevent regrowth for an extended period of time. In contrast, conventional disinfecting ations contain ous chemicals to achieve such persistent anti— microbial activity.

In an embodiment the formulation provides a Log reduction of at least 4.0, about 10- seconds after application.

In an ment the formulation provides a Log reduction of at least 4.0, about 15- seconds after application.

In an embodiment the formulation provides a Log reduction of at least 4.0, about 30- 60 seconds after application.

In an embodiment the formulation provides a Log reduction of at least 4.0 for at least about 30 minutes.

In an ment the formulation provides a Log ion of at least 4.0 for at least about 1 hour.

In an embodiment the formulation provides a Log reduction of at least 4.0 for at least about 2 hours.

In a further embodiment the formulation provides a Log reduction of at least 4.0 for at least about 3 hours.

In a further embodiment the formulation provides a Log reduction of at least 4.0 for at least about 4 hours. —18— The formulation will be logically ible with skin. In addition to disinfecting, the formulation may assist to clean skin (by removing dead skin cells, grease, grime and the like) and/or manage minor wounds and skin ers (e. g., cuts, scratches, abrasions, eczema, dermatitis, tinea, fungal skin disorders, acne, herpes sores). The formulation can also symptomatically assist with management of minor wounds, abrasions, infections, insect bites, stings, sun burn and minor skin burns. It can be used as a treatment or preventative against tinea/ athletes foot.

The ation sooths itchy sore red skin, conditions noted in people that have eczema. The formulation kills polyps/herpes, and when applied does not dry and crack the skin. The formulation demonstrates high anti toxic ance due to its natural chemistry. For instance, the formulation may be used to treat insect bites in which the bite contains toxins. Applying the formulation quickly may counter attack the toxins/infection and heal the skin quickly.

The formulation can, for e, be dispensed from a pump container, spray container canister, tub or tube, or alternatively a single dose of the formulation may be ' provided in a sealed foil or laminated plastic (polypropylene) sachet. In a preferred embodiment when the formulation is a hand rub it has a viscosity which allows it to be readily sprayable, such as, for instance 7 to 100 cps. It can be provided on or with swabs for skin cleansing or in sealed packaging with sponges for surgical theatre'use.

In use an amount sufficient to coat the hands or skin to be ed will be applied to the skin. The formulation is a "leave on" application and dries naturally. However, excess formulation may be removed by wiping with paper towel or a dry cloth (which should be sterile in the case of medical/surgical ations).

The disinfecting formulation of the invention can, for example, be ed by adding aqueous ethanol to a stainless steel mixing container and then adding the preservative (if present) with mixing to aid dissolution. Eucalyptus oil and ent are then separately added with thorough mixing, with subsequent addition of tea tree oil or an extract of tea tree oil, if present. The mixture is then made up to the desired volume with purified water.

The present invention will now be bed r with reference to the following non-limiting examples: EXAMPLES Example 1 — Preparation of Disinfecting Formulation A formulation (hand rub) was prepared containing the ingredients listed in Table 1 below in the percentage amounts by volume as indicated.

Table l 73% AR. grade aqueous ethanol (95% v/v) % BR grade eucalyptus oil 5% plant derived ine 0.5% terpinenol 0.05% piroctone olamine distilled water up to volume The formulation was prepared by adding 43.8kg of the ethanol solution to a stainless steel mixing container and then adding 0.03 kg of piroctone olamine with mixing to aid ution. Eucalyptus oil (6 kg) and glycerine (3 kg) were each then separately added with thorough mixing, with subsequent addition of 0.3 kg of the terpinen-4—ol.

The e was then made up to 60 L with purified water.

Example 2 A formulation (hand rub) was prepared containing the ingredients listed in Table 1a below in the tage amounts by volume as indicated.

Table 1a Theoretical Actual Test RAW INGREDIENT 1.000 1.000 4.000 COMPONENT (% V/V) 0(/oW/V) ("/oW/V) Purified Water 12. 03 1.000 12.03 48.13 Glycerol BP 5.00 1.262 6.31 25.24 Octopirox 0.05 1.000 0.05 0.20 Total Phase 1 Materials 5.05 6.36 25.44 l, Undenatured 72.92 0.809 58.95 235.81 96% BP Eucalyptis Oil BP 10.00 0.917 9.17 36.66 Total Phase 2 Materials 82. 92 68.12 2 72.4 7 (Target pH: 8.1) Total I 00. 000 86. 511 Grams 346. 046 Grams Units (%) per 100.0 ML per 400. 0 ML 2012/000841 INSTRUCTION: NOTE: all als to be at room temperature 1). Add Phase 1 materials in the main mixing vessel in the stated order. Mix moderately until all materials have been dissolved 2). Slowly add phase 2 into main vessel. Mix each component into the batch with an air mixer until homogeneous. 3). Mix for 15 s. Adjust with Lactic Acid to target pH value of 8.1. Mix for an additional 10 minutes.

RELEASE SPECEFIC’ATION Retease Specifications for the Formulated Gel ?reduct Gas Chromatogzaphi; Ethane: Etha no? Confer-i Senscf‘y’ Pctezfliemelry TC-LA Tapcasl Liam: 2‘ TBA Topical Liauid Teal; 713A Tape-'31 Liauéd Test 16¢: Topéeal Liquid Test Example 3 A formulation (hand gel) was prepared containing the ingredients listed in Table lb below in the percentage amounts by volume as indicated.

Table 1b Theoretical Actual Test RAW INGREDIENT 1.000 1.000 (%W/V) COMPONENT (% V/V) (%W/V) Purified Water 11 13 1. 000 11.13 22.26 Glycerol BP 4.00 1.262 5.05 10.10 Ethanol, Undenatured 72.92 0.809 58.95 117.91 96% BP Carbopol Ultrez 20 1.05 1.000 1.05 2.10 Total Phase 1 Materials 77. 97 65. 05 130.10 AMP PC-2000 0.90 0.942 0.85 1.70 Eucalyptus Oil BP (A) 10.0 0.917 9.17 18.33 Total Phase 2 Materials 10. 90 10.02 20. 03 (TopH.°6.5-7.5) Total 100.000 86.197 Grams 1 72.395 Grams Units (%) per 100. 0 ML per ML INSTRUCTION: NOTE: all materials to be at room ature 1). Add Phase 1 als in the main mixing vessel in the stated order. Mix moderately until all carbopol have been dissolved 2). Add Phase 2 into main vessel and mix for 30 minutes.

RELEASE SPEClT-ECATEON Release cations forthe ated Gel Product Test Parameter Test Method Specification Test Site identification of Gas Chromatography Retention tE‘me of the ALS ".'€elbr:ylarne Ethanol saznole matches the retention time of the standard Ethanol Content Bias Chromatography ALE: ’-:€elbou:°'ne Appeazanee El Ceiour ‘ Hath slight Baxter Laboiatoriee white Inge Odour Eiawtzer Laboratories: Baxter Laboratories Baxter Laboratozies- Santeria? Count EML ltlteéboume 7823" Tomcat Uguéd ”zest EML Sza‘reibout‘ne 7 3A aé timid Est EML aléeiboume 781%. Topical Litgutd test None in lg EML lutegboume Example 4 — Test against surgical handrub standard Objective To determine whether the disinfecting formulation produced according to the ol of Example 1 meets the European standard for a surgical handrub (EN 12791 of 2005), which requires a significant log reduction in hand microflora both immediately and over a 3 hour sustained , when compared against a standard formulation (60% propan-l—ol).

Testprotocol The protocol is based upon European standard EN 127912005 designed for testing “Chemical disinfectants and antiseptics — Surgical hand disinfectant”.

The clean and healthy hands (free of cuts and abrasions and with short, clean fingernails) of 5 volunteers were lly washed for 1 min with soap and water and dried with paper towel. To establish base line flora levels the fingertips and thumb .24~ were rubbed for 1 minute on the base of a petri dish containing 10 ml of Tryptone Soy Broth (TSB). A separate petri dish was used for each hand. Dilutions of 10'2 and 10‘3 were ed from the TSB that were assayed by the pour-plate method using Tryptone Soy Agar (TSA) plates. The time between sampling and g did not exceed 30 mins.

Immediately after sampling for the baseline and without recontamination of the hands the subjects performed the handrub test. The subjects were split into two groups which were alternately d with the standard formulation or the test ation initially and then the other formulation, separately by a week to allow onisation of native flora.

The handrub test involved the hands of each volunteer being exposed to treatment with tWO 4.6 ml aliquots of both the test and standard formulations. The treatment involved an initial application of the formulation (test or standard) for 30 s and then immediate further treatment for a further 30 seconds for a total exposure in each case of 60 seconds.

The finger tip and thumb of the left hand were then rubbed for 1 min on the base of a petri dish containing 10ml of PDE neutraliser to provide the immediate effect results.

The right hand was covered by a sterile glove for 3 hours, followed by the same testing protocol for the left hand, to provide the sustained effect results.

The testplates were diluted 100 and 10'1 and were d to the pour-plate assay using TSA. All plates were incubated for 18 to 24 hours at 36°C i2°C.

Loglo values for immediate effect and sustained effect were then determined and a Log Reduction Factor for both test formulation and standard formulation were calculated based on the e difference between the log”) pre-value and the logo post—value in each subject.

WO 06917 _25- The standard formulation reduced hand microflora by 1.61 Logs immediately and 1.89 Logs after 3 hours, whereas the test formulation d hand microflora by 1.61 Logs ately and 2.02 Logs after 3 hours. The test formulation was therefore more effective than the standard ation and meets the requirements of the EN 12791 standard.

Example 5 — Test against hygienic handwash standard Objective To determine whether the disinfecting formulation ed according to the protocol of Example 1 meets the European standard for a hygienic handwash (EN 1500 of 1997), which requires a significant log reduction in hand contaminant ia for artificially contaminated hands, when compared t a standard formulation (60% propanol).

Test protocol The protocol is based upon European standard EN 1500:1997 designed for testing “Chemical disinfectants and antiseptics — Hygienic handwash”.

The subjects (15 volunteers with clean and healthy hands that were free of cuts and abrasions and having short, clean fingernails) were split into two approximately equal groups which were alternately treated with the standard formulation or the test formulation initially and then the other formulation, tely by a one day.

The hands of the subjects were initially washed for 1 min with soap and water and dried with paper‘towel. The contamination fluid (Escherichia coli (NCTC 10538) bacterial suspension of between 2 x 108 and 2 x 109 cfu/mL) was placed into a tray . 30 and each subject immersed his/her hands up to the mid-metacarpals for 5 seconds.

Excess fluid was allowed to drain back into the container. Hands were air dried for 3 WO 06917 minutes, holding them in a ntal position. Each t was encouraged to rotate their hands to avoid the formation of droplets. Each subject was treated from the same batch of contamination fluid.

After drying, fingertips and thumb were rubbed for 1 minute on the base of a Petri— dish, which contained 10mL of TSB. A separate Petri-dish for each hand (Left and Right) was used. This was the Pre-value. Dilutions of 10'4 and 10'5 were prepared using sterile Peptone Water (PEP). Each dilution was spread onto pre-poured, dried TSA plates. The time between sampling and plating did not exceed 30 minutes.

Immediately after sampling for the pre-values and without re-contaminating the hands, the group performed the ‘handwash’ procedure, initially with either test formulation or standard ation, with the standard formulation being applied to Group 1 volunteers on Day 1 and Group 2 volunteers on Day 2 and the test formulation being applied to Group 1 on Day 2 and Group 2 on Day 1.

For the standard formulation, 3mL of 60% propan-2—ol was dispensed onto the subject’s hands and rubbed over for 30 seconds using the standard procedure as instructed. This was done twice to give total of 6mL of 60% propan—2-ol over a 60 second rub. This procedure was completed with a 5 second rinse of fingers under running tap water.

For the test formulation, 3mL of test t was dispensed onto the subject’s hands and rubbed over for 30 seconds using the standard procedure as instructed. This was done twice to give total of 6mL test product over 60 second rub. The test product procedure was completed with a 5 seconds rinse of fingers under g tap water.

The alue was obtained by rubbing the fingertips and thumb on the base of a Petri-dish containing 10mL of either T6 or PDE Broth as inactivating agent (dilution of 100). Dilutions of 100, 10'1 and 10'2 were spread on pre-poured and dried TSA plates. All plates were incubated at 36°C i 2°C for 24 — 48 hours. _27_ Results and ations After the incubation period, all the plates were checked and colony forming units on both standard and test sample were counted. Dilutions with counts that are within 15 — 300 cfu were used in the computation. Plates showing less than 15 cfu or no growth were also counted.

Data were collected from 15 volunteers, but only 14 were taken into account as the results of plate count from one subject were evidently improbable and hence excluded.

With 14 subjects, the test remains in compliance with the minimum requirement of 12.

No other deviation to the experimental protocol was ed.

To meet the requirements of EN 1500 all results from at least 12 subjects shall be available and the overall mean of the log ues for the standard and test product shall be at least 5.00. In this study, the mean was 7.13 and 7.01 respectively. r, in each procedure there must not be more than three subjects with log reduction factor lower than 3.00. In this study, the lowest log reduction recorded was 3.94.

It is also necessary for the mean log reduction factor ed not to be significantly smaller than that of the standard (propan-2—ol). In this study, the mean log reduction for the test formulation is higher than that of the standard formulation, with a mean difference of 0.54.

The log reduction of contaminant ia of the test formulation by 5.7 may be regarded as comparable, albeit close, having surpassed that of the standard formulation (5.16 logs).

The requirements of EN 1500 have been ed by the test formulation of Example Example 5a Objective/Summary WO 06917 This study was designed to evaluate the efficacy of Example 2 by the European Standard — EN 1500 : 1997. In this in-vz‘vo test, the product in the recommended volume, time and frequency has achieved an average of 5.70 log reduction of the test organism, Escherichia coli (NCTC 10538), as opposed to the average log reduction of .16 from the application reference al 60% (v/v) propan-2—ol. From statistical analysis of data obtained, Example 2 can be considered as effective having shown a significantly higher log reduction than that of the reference on propan 2-ol, as well as demonstrated compliance to all other parameters as specified in the guidelines.

PRODUCT UNDER EVALUATION Antimicrobial Handrub, in 250mL containers, Batch No. BE079, was received on 08/04/2011. Testing commenced 13th April, 2011.

Application Details: 0 Volume' 3.0 mL 0 Contact Time: 30 seconds 0 Frequency of application: twice ' EERS Fifteen individuals (volunteers) were recruited whose 'hand—skin’ were healthy, without cuts or abrasions and with short and clean fingernails.

All volunteers are asked to sign a consent form and specify their age range. All volunteers should be at least 18 years of age or older.

No ction was applied in terms of gender distribution/selection.

MATERIALS Culture Strain: The test e used in this study was obtained from the University of New South Wales Culture Collection — Escherichia coli (NCTC 10538) Reagents and Suppliers: o Tryptone Soy Broth (TSB) was supplied by Oxoid Pty. Ltd. 0 Tryptone Soy Agar (TSA) was supplied by Oxoid Pty. Ltd. 0 10.1% Peptone Water (PEP), supplied by Oxoid Pty. Ltd. 0 T6 Broth, prepared within ams Laboratories. 0 PDE Broth, prepared within ams tories. 0 Propan-2—ol, supplied by BDH. o Tween 80, supplied by um Distributor. 0 Soft Soap, 200g/L per EN 1500 : 1997, prepared within ams Laboratories Equipment and Apparatus o Incubator 36°C i 2°C 0 Thermometer o Timer Jadco, supplied by Biolab. o Pipettors and sterile pipette tips were supplied by Biolab. 7.3.5. Sterile Spreaders were supplied by Biolab. 0 Sterile bottles 0 Petri Plates (90mm), e 0 Stainless tray deep enough to immerse hands to mid- metacarpals.

METHODS Neutralization Study A neutralizer validated according to prEN 12054 were incorporated in the sampling fluids and diluents for the assessment of post—values in both test and reference procedure. PDE Broth and T6 were found to be a le neutralizer for test products and reference respectively.

Inoculum Preparation The test organism was freshly revived from frozen t beads by sub culturing in two ney bottles containing 5mL of TSB. They were incubated at 37°C.

These cultures are then inoculated into two bottles containing 1L TSB. They were incubated at 37°C for 18 — 24 hours The cultures were pooled into a sterile bottle to prepare the bacterial suspension. The suspension prepared in this way should be between 2 x 108 and 2 x 109 cfu/mL.

Test Procedures For testing this product, a cross—over design was used. The subjects were ly divided into two groups of approximately the same size. The test was first performed with Group 1 applying the reference b (60% Propan-Z-Ol) and Group 2 applying the test sample and following the ‘handrub operation as demonstrated in the guidelines. The test was then repeated on the next day with the two groups switching over the test sample and reference solution.

Establishing Pre—values Each subject/volunteer washed hand to remove normal dirt and natural flora using the soft soap for l min. The hands were then dried thoroughly on paper towels.

The contamination fluid (test suspension) was placed into the tray and each subject immersed his/her hands'up to the mid—metacarpals for 5 seconds. Excess fluid was allowed to drain back into the container.

Hands were air dried for 3 minutes, g them in a horizontal position. Each subject was encouraged to rotate hands to avoid the formation ofdroplets.

Each subject was treated out of the same batch of contamination fluid.

After drying, fmgertips and thumb were rubbed for 1 minute on the base of a Petri-dish, which contained 10mL of TSB. A separate Petri-dish for each hand (Left and Right) was used. This was the Pre-value.

Dilutions of 10"4 and 10'5 were ed using e Peptone Water (PEP). Each on was spreaded onto pre-poured, dried TSA plates. The time between sampling and plating did not exceed 30 minutes.

Immediately after sampling for the pre—values and t re-contaminating the hands, the group performed the ‘handwash' procedure in accordance with 8.5 and 8.6. -3]- Reference Handwash Procedure These steps were followed by Group 1 recruits on Day 1 and Group 2 recruits on Day 3mL of 60% propanol was dispensed onto the subject's hands and rubbed over for 30 seconds using the standard procedure as instructed. This was done twice to give total of 6mL of 60% propan—*2~ol over a 60 second rub.

The reference procedure was completed with a 5 second rinse of fingers under g tap water.

Test Product Procedure These steps were followed by Group 1 on Day 2 and Group 2 on Day 1. 3mL of test product was dispensed onto the subject's hands and rubbed over for 30 s using the rd procedure as instructed. This was done twice to give total of 6mL test product over 60 second rub.

The test product procedure was completed with a 5 seconds rinse of fingers under running tap water.

Post-values - The Post-value was obtained by rubbing the fingertips and thumb on the base of a Petri dish containing. lOmL of either T6 or PDE Broth as inactivating agent (dilution of 10°). Dilutions of 10°, 10'1 and 10'2 were Spreaded on pre—poured and dried TSA plates.

All plates were incubated at 36°C :t 2°C for 24 — 48 hours.

RESULTS AND ATIONS After the incubation period, all the plates were checked and colony forming units on both reference and test sample were counted. Dilutions with counts that are within — 300 cfu were used in the computation. _32_ Plates showing less than 15 cfu or no growth were also counted.

Data were ted from 15 volunteers, but only 14 were taken into account as the results of plate count from one subject were evidently improbable and hence excluded. With 14 ts, the test remains in compliance with the minimum requirement of 12. No other ion to the experimental protocol was required.

Raw data and statistical evaluations are given in Table 9.1 —— Table 9.6 (pages 7 - l2).

ANCE CRITERIA In ance with the criteria specified in EN 1500: All results from at least 12 subjects shall be available. In this study, 14 set ofdata are available.

The over all mean of the log prevalues for the reference and test product shall be at least 5.00. In this study, the mean was 7.13 and 7. 01 respectively.

In each procedure, R and P, not more than three subjects with log ion factor IOWer than 3.00. In this study, the lowest log reduction recorded was 3.94.

For any product, the mean log reduction factor obtained shall not be significantly smaller than that of the reference propan—2-ol. In this study, the mean log reductionfor P is higher than that ofR, with a mean diflerence of 0. 5 4.

If the mean log reduction factor of a test product is smaller than that of the reference propan —2—ol, the difference shall be tested for statistical significance. In this study, the mean log reduction factor of the test product is greater than that of the nce; nofurther statistical analysis is deemed necessary.

DISCUSSION The log reduction of contaminant bacteria by 5.7 may be regarded as comparable, albiet close, having surpassed the reference preparation (5.16 logs) and according to the acceptance criteria, this is within the acceptable level of confidence Experimental Results — nce (Propan—2—13l) Solution ation: 60% -Z-ol Application: rub-in 3mL' for 30 seconds, repeat once (Total 6mL for te) Test Organism: Escherichia coli (NCTC 10538) Suspension: 1.0 x 109 cfu/mL Number of cfu per Plate from Dilution l e Prevalues Postvalues -= TNTC/TNTCTNTC/TNTC 96 113 19/22 0/0 0/0 92 86 54/69 8/8 0/1 2-—90 TNTC/TNTC 1/5 0/0 2-L NTC 18502 72 86/62 1/11 0/0 -LR TNTC/TNTC 170 3/10 0/0 0/0 TNTC/TNTC 155 21/22 1/2 0/0 L TNTC/"NTC 108/94 5/16 R TNTC/TNTC 42/41 9/3 0/3 L TNTC/TNTC 177/129 11/19 R TNTC/TNTC 92 96 —_2/4 6 L TNTC/TNTC 144 123 0/0 1/0 0/0 - R TNTC/TNTC 117 112 5/4 5/3 0/1 7 L TNTC/TNTC 93 76 0/0 0/0 0/0 R TNTC/TNTC 126 119 0/0 0/0 0/0 8 L T.\TC/T.\TC 245 256 TNTC/TNTC 20/27 —l_2‘/1 R TNTC/TNTC_ TNTC/TNTC 50/41 3/9 9 L xrc 113 105 58/46 0/0 0/0 TNTC/TNTC 104 97' 134/120 2/0 0/0 -R—167 159 TNTC/TNTC 26/22 7/3 -T\TC/T.\TC 133 135 TNTC/TNTC 141/142 11 "I: TNTC/TNTC 415 325 TNTC/TNTC TNTC/TNTC 16/ TNTC/TNTC 308 260 TNTC/TNTC TNTC/TNTC 56/ 12 L TNTC/TNTC 197 179 R TNTC/TNTC 206 214 5/5 0/5 0/0 F_’_—18/15 on R TNTC/TNTC 115 TNTC/TNTC 9/16 1/0 _—_- 117 111 TNTC/TNTC 62/76 — —16/ 183 182 246/275 26/24 1/2 212 174 232/182 19/23 3/5 _ 34 _ Underline counts were used in the computation Experimental s — Test (Handrub) Solution Preparation: Test Sample Neat Application: rub-in 3mL for 30 seconds, repeat once Test Organism: Escherichia coli (NCTC 10538) Suspension: 1.0 x 109 cfu/mL Number of cfu per Plate from Dilution 10" 81113?“ Hand Prevalues Postvalucs '4 10'5 10'0 104—7— 10'2 L TNTC/T\TC 76 100 6/3 0/0 0/0 R TNTC/TNTC 69 77 0/5 0/0 0/0 “TNTC/TNTCTNTC/TNTC 52 58 4/3 1/1 0/0 12 5 2/3 1/1 0/0 L TNTC/Tthc 84 107 15/9 1/3 0/0 -TNTC/TNTC 46 35 4/1 0/0 0/0 4 L TNTC/TNTC 89 100 TNTC 108/94—l_/16 R NTC 100 92 42/41 9/3 3/0 L TNTC/TNTC 135 118 TNTC 54/66 6/5 |——:R TNTC/TNTC 141 130 TNTC/TNTC 160/148 27/28 6 _ —~——1———4 - 3/8 0/0 0/0 55/42 3/0 0/0 L A 10/12 0/0 0/0 R TNTC/TNTC 0/0 0/0 8 L TNTC/TNTC TNTC/TNTC 12/15 0/0 R TNTC/TNTC TNTC/TNTC 20/22 0/1 r l 9 L TNTC / TNTC 84 ‘i129 50/44 0 / 0 0 / 0 R TNTC/TNTC 52 148 58/ 78 1/1‘ 0 / 0 TNTC / TNTC 60 37 TNTC / TNTC 79/90 8/ 14 TNTC / TNTC 28 36 59/ 83 L TNTC/TNTC 517 496 TNTC/TNTC 405 419 1/0 12 -TZ\'LTC/TNTC 159 147 0/1 __.____.._I TNTC / TNTC Underline counts were used in the computation List of Log Values (Mean of Left & Right Hand) - Reference Wash Prevalues Postvalues lSubject No LogX Ave. Lo_g_X L_o_gY _]_Ave.Lo_gY L 10450000 7.02 21 1.32 1 —1— 6.98 |—————1———— 1.55 R. 8900000 6.95 61 1.79 J— L 8850000 6.95 30 1.48 2 6.91 ‘T———J 1.67 R 7600000 6.88 74 1.87 L 16100000 7.21 7 0.85 3 1— 7.18 —|— 1.09 R 14500000 7.16 22 1.34 L 7550000 6.88 1530 3.18 4 6.93 [—————— 2.99 R 9400000 6.97 625 2.80 L 13350000 7.13 5 0.70 7.09 r———‘———‘ 0.70 R 11450000 4 7.06 5 0.70 L 8450000 6.93 0 1.00 6 7.01 g—L———{ 1.00 l— R 12250000 7.09 0 1.00 L 00 7.40 235 2.37 7 7.37 2.51 R 21550000__ 7.33 455 2.66 _| L 10900000 7.04 52 1.72 8 7.02 1 J 1.91 R 00 7.00 }__ 127 2.10 L 16300000 7.21 240 2.38 9 7.17 2.77 R 13400000_J 7.13 1415 3.15 L 37000000 7.57 2050 1— 3.31 7] n21000000 12 an 1.3 11—- Ia_- N = Number of Subjects List of Log Values (Mean of Left & Right Hand) — SunnyWipcs (Test Sample) Sub'ectNo Prevalues L0 1 E_- -I_-2 3 II- 4 E- . .

I 00 SUBSTITUTE SHEET (RULE 26) R 7250000 210 2.32 . _i L 10650000 47 1.67 7.01 1.75 10000000 7.00 68 A 1.83 4850000 6.69 845‘ 2.93 6.60 2.39 3200000 6.51 ‘ ‘ 71 _j 1.85 L 50650000 7.70 3 0.48 —1— 7.66 1.11 41200000 7.61 55 1.74 L 15300000 7.18 0.00 _' 11 7.28 035 _ T R 24150000 7.38 5 0.70 1_ 1— L 5650000 6.75 11 1.04 12 6.86 0.87 9350000 6.97 5 t: 0.70 L 16700000 7.22 54 1.73 13 7.15 1.73 R 00 7.08 53 1.72 L--22750000 7.36 3 0.48 14 7.25 j —] m 1— _l_ R 14650000 7.17 9 0.95 ~—* —1 Average 7.01 1.31 SD j_ 0.32 0.79 L 14.00 _1 14 ___, N = Number of Subjects Log Reduction Derived from Reference and Test Sample Reference (R) T Test Product ( P ) -Z-ol 60% (v/v) J Sunnywipe Handwash Log Y Log Z Log X Log Y Log Z 1.55 5.43 6.90 0.59 6.32 ‘_{ ' —l 167 624 633 054 580 __J __J 109 609 679 078 602 299 394 742 300 442 .__l _38_ 0.70 6.39 7.18 1.23 5.95 —-1.00 0.01 7.00 0.00 m0.00 —- 4.40 6.60 2.39 4.21 0.00 7.00 1.11 000“ 11 7.30 6.65 7.28 0.35 6.93 12‘ 6.96 4.81 6.86 0.87 5.99 2.60 4.44 7.15 1.73 5.42 2.36 4.91 7.26 0.72 6.55 Mcan 7.13 1.97 5.16 7.01 1.31 5.70 SD 0.18 0.89 0.86 0.32 0.79 0.86 Example 6 — Bacterial ection test Objective To determine r the formulation of Example 1 demonstrated bactericidal activity required to meet the Australian Therapeutic Goods Administration (TGA) disinfection test, option D against six significant infective bacteria. The test followed the protocol of method TM122—04.

Conditions Test organisms: 1. Staphylococcus aureus NCTC 4163 2. Escherichia coli NCTC 8196 3. Pseudomonas nosa NCTC 6749 4. Proteus vulgaris NCTC 4635 . Methicillin Resistant Staphylococcus aureus (MRSA) 6. Vancomycin Resistant Enterobacteria (VRE) lnoculum Level: Approx. 1.7 to 3.6 x106 Colony Forming Units (CFU) / mL Test Concentration: Neat Contact Times: 30 seconds Test Temperature: Ambient Results No growth of any of the bacterial organisms tested for was identified after exposure to the test formulation, which the control inoculations behaved as ed. The test formulation successfully demonstrated bactericidal activity according to the TGA disinfection test, option D rd. e 7 — Anti—microbial Activity Challenge Test (TMl 10) Objective To ine whether the formulation of Example 1 demonstrated anti—microbial activity against Methicillin-resistant Staphylococcus aureus and Vancomycin—resistant Enterococcusfaecalis.

Conditions Test organisms: 1. Methicillin-resistant Staphylococcus aureus (MRSA) 2. Vancomycin-resistant Enterococcusfaecalis (VRE) Test Concentration: Neat Contact Times: 30 s and 2 minutes Test Temperature: Ambient Results Table 2: Surviving MRSA after exposure to test formulation Sample Details Surviving sms (CFU/mL ) and Log“) Reduction Log Reduction CFU/mL Log Reduction (10 I l SunnyWipes <10 Handmb (<1) Inoculum 5,3 x 105 (5.72 Logs) CFU = Colony Forming Unit Table 3: Surviving VRE after exposure to test formulation Surviving organisms (CFU/mL ) and Logm Reduction CFU/mL Log Reduction CFU/mL (10-10) (10.10) ipes <10 <10 Gel >4.61 >4.61 (<1) ‘ <1) 4.1x 105 ‘ (5.61 Logs) CFU = Colony Forming Unit Conclusions The test formulation successfully demonstrated icrobial activity against Methicillin-resistant Staphylococcus aureus and Vancomycin~resistant EnterocOccus faecalz‘s by more than 4 log reduction (kill >99.99%) after 30 seconds and 2 minutes of contact time, when tested neat in conditions described above.

Example 7a — crobial Activity Challenge Test (TM 110—04) Objective To determine whether the formula of Example 2 demonstrates anti-microbial activity against pseudomonas aeruginosa, inter alia.

CONDITIONS: Test Organisms: pseudomonas aeruginosa ATCC 15442 Neutraliser: T6 1:100 (TSB with Lecithin and Tween 80) Test Concentration: Neat Contact Time: 15 and 30 seconds Test Temperature: t RESULTS: ing Organisms after 15 and 30 s exposure to Antimicrobial Hand Rub Pseudomonas aeruginosa Contact Time CFU/mL Log ion (10g10) seconds <100 >570 (2.00) seconds <100 >570 (2.00) Inoculum count .0x 107 (7.70) _42_ COMMENTS: The sample Example 2 has demonstrated bactericidal activity against Pseudomonas aeruginosa by showing greater than 5 Log reduction which s to 99.999% kill at and 30 seconds t time when tested under the conditions described above.

EXAMINATION: TGA (Australian Therapeutic Goods Administration) Disinfectant Test, Option D DILUTION: Neat METHOD: TM122-04 NEUTRALISER: T6 RESULTS: Test Date Organisms Subculture Inoculum Challenge Controls N0. (CFU/mL) Result Staphylococcus 5 1.1 x106 C1— aureus Escherichia coli C2- Pseudomonas C3 +++ aeruginosa +++ Proteus is 16/05/2012 Methicillin C4+++ Resistant Staphylococcus aureus Vancomycin 1.6x106 Resistant Enterococcus faecalis lococcus 1.9x106 C1— aureus Escherichia coli 2.3x 106 C2- Pseudomonas 2.1 x106 C3+++ aeruginosa s vulgaris 2.1x106 17/05/2012 Methicillin 6 C4+++ Resistant lococcus aureus Vancomycin 6 1.6x 106 Resistant Enterococcus faecalis Staphylococcus 7 1.7x 106 C1— aureus Escherichia coli 7 1.7x106 C2- Pseudomonas 7 2.1x106 c3+++ aeruginosa +++ s vulgaris 7 2.4x 106 18/05/2012 Methicillin 7 2.3 x106 C4+++ Resistant +++ Staphylococcus aureus Vancomycin 7 6 Resistant Enlerococcus faecalls Where "—" Denotes No Growth "+" s Growth "Cl " Denotes media sterility check. Must be negative for a valid result "C2" Denotes sample sterility check. Must be negative for a valid result "C3" Denotes organism viability check. Must be positive for a valid result "C4" Denotes neutralizer toXicity check. Must be positive for a valid result FINAL RESULTS: The sample Example 2 passed the TGA Disinfectant Test, Option D when tested under the conditions described above. -44_ Notes: 1. Strains of Organisms used were as follows Organism AMS Culture No. NCTC N0.

Staphylococcus aureus AMS 028 NCTC 4163 Escherichia coli AMS 007 NCTC 8196 Pse'udomonas aeruginosa AMS 018 NCTC 6749 Proteus vulgaris AMS 016 NCTC 4635 Methicillin Resistant AMS 077 NA Staphylococcus aureus Vancomycin ant AMS 084 NA Enterococcusfaecalis 2. Results of controls confirm to expected outcome Example 8 — Evaluation of Trans-epidermal Water Loss Objective The effect of the formulation of Example 1 on skin ion was evaluated using a TEWA Meter and compared with untreated skin on the same test subjects at 30 min, 2 days and 6 days.

Standardsfor Inclusion in a Study 1. Individuals n the ages of 18 and 70. 2. Individuals not taking medication or under the care of a physician for a period of one month prior to commencement and throughout the entire test period.

Individuals who have completed a inary l history questionnaire.

Individuals who have read, understood and signed an informed consent document.

Individuals who understand the ctions for use and are willing to cooperate with the program as stated.

Individuals free of any dermatological or systemic disorder that would interfere with the s, at the discretion of the Investigator. 7. Individuals able to cooperate with the Investigator and the research staff and willing to complete the full course of the study.

Standardsfor Exclusionfrom a Study 1. . Individuals who are under doctors' care. 2. duals who are tly taking medication which in the opinion of the Investigator would mask or interfere with the results. 3. Individuals with any history of sensitivity to ics in general and moisturisers in particular. 4. Individuals with any form of skin cancer, melanoma, lupus, psoriasis, a, porphyria cutanea tarda, connective tissue disease, or any e that would interfere with the test results.

Individuals diagnosed with chronic skin allergies.

Female volunteers who indicate that they are pregnant or nursing an infant. 9°.\‘.O\.V‘ Individuals with excessive hair on the test sites.

Individuals with known hypersensitivity to cosmetic products.

Informed Consent A signed informed consent was ed from each panelist prior to initiating the study describing reasons for the study, possible adverse effects, associated risks and potential benefits of the treatment and their limits of liability.

Methodology One distinct method was employed for the tion procedure. Biophysical ements were made pre-application (t = 0) and following a single application of the test material after 30 min. onal readings were also taken at 2 days and 6 days. Before each set of measurements, subjects were required to equilibrate in a closed environment with constant temperature (20°C+/—2°C) and humidity (45% to 55% RH). Measurements are expressed as g/hmz.

Moisture Measurement 2012/000841 -46— Model TM 210 TEWA Meter — Courage + Khazala References Tewameter TM 210 Information and ing Instructions (manual). pidermal Water Loss, Bioengineering of the Skin: Methods and Instrumentation, CRC Press 1995.

Dermatest SOP DESOP — 030 Procedure for Determining Transepidermal Water Loss (TEWL).

Study Design 11 healthy subjects between the ages of 33 to 58 years were inducted into this study.

In order to precondition the test sites and keep all topical treatments constant for all - test subjects they were required to abstain from using deodorant soaps, moisturising soaps or ic risers on the test area for a period of one week prior to study commencement and during the course of the study. At the completion of the one week ‘washout’ period, subjects were required to return to the test facility at the time specified by the technician for the study commencement. On the day of the study, test al was delivered to the test sites by applying to the back of the hands liberally.

Subjects were blinded as to the nature of the material being applied. Biophysical measurements with a TEWA Meter were taken at t = 0 (pre—application). Subjects were required to return to the lab at each subsequent designated period for repeat biophysical measurements.

Results Table 4 — Torans-epidermal water loss % diff Conclusions After repeat applications over a period of 6 days, the formulation was shown to have a positive impact as trans-epidermal water loss was significantly reduced. There was no incidence of skin irritation under the test conditions.

Example 9 — Skin irritation testing [.0 Objective Consumer products or raw materials designed for consistent reapplication to areas of the skin may, under proper conditions, prove to be contact sensitizers or nts in certain individuals. It is the intention of a Repeat Insult Patch Test (RIPT) to provide a basis for evaluation of this irritation/sensitization potential if such . 2.0 Reference The method is modified to test 50 panelists and not the 200 cited in the reference sal of the Safety of Chemicals in Food, Drugs and Cosmetics, hed by the Association of Food and Drug Officials of The United States The method also employs nine inductive patchings and not the ten cited in the reference under semi— occlusive patch conditions. 3.0 Test Material: Formulation produced ing to Example 1. 4.0 Panel Selection." 4.1 Standardsfor Inclusion in a Study Individuals who are not currently under a doctor’s care.

Individuals free of any dermatological or systemic disorder which would interfere with the results, at the discretion of the Investigator.

Individuals free of any acute or chronic disease that might interfere with or increase the risk of study participation.

Individuals who will complete a preliminary medical history form and are in general good health.

Individuals who will read, understand and sign an informed t nt relating to the specific type of study they are subscribing.

Individuals able to cooperate with the Investigator and research staff, willing to have test materials d according to the protocol, and complete the full course of the study. 4.2 Standardsfor Exclusionfrom a Study Individuals under 18 years of age.

Individuals who are under ’s-care.

Individuals who are currently taking any medication (topical or systemic) that may mask or interfere with the test results.

Subjects with a history of any acute or c disease that might interfere with or increase the risk of study participation.

Individuals diagnosed with c skin allergies.

Female volunteers who indicate that they are pregnant or nursing. 4.3 Recruitment Panel selection is accomplished by advertisement in local periodicals, ity bulletin , phone solicitation, electronic media or any combination thereof.

WO 06917 4. 4 Informed Consent and Medical y Forms An informed t was obtained from each volunteer prior to initiating the study describing reasons for the study, possible adverse effects, associated risks and potential benefits of the ent and their limits of liability. Panelists signed and dated the informed consent document to indicate their authorization to proceed and acknowledge their understanding of the contents. Each subject was assigned a permanent identification number and completed an ive medical history form. 5.0 Population Demographics Number of subjects enrolled ................................................................ 54 Number of subjects completing study ................................................. 53 Age Range ...................................................................................... 21-67 Sex........— ............................. Male ......................................................... 14 Female ' ...... ......................................... 40 Race ................................... Caucasian ................................................. l 9 Hispanic ..................................................... 1 Asian .......................................................... 5 African American .................................... 29 6.0 Equipment - Patch Description: Parke-Davis Hypoallergenic Readi Bandages (20 x 20 mm Webril affixed to the centre of a 40 x 40 mm adhesive bandage) or the equivalent, trimmed at right angles on opposite sides to the opening of the paper g of patch, allowing air flow. - 1 ml tric syringe without a needle. 7.0 Procedure — Subjects are requested to bathe or wash as usual before arrival at the facility. — 0.2 ml of the test material was dispensed onto a semi-occlusive, hypoallergenic patch.

WO 06917 - The patch was then affixed directly to the skin of the infrascapular regions of the back, to the right or left of the midline and the subject was sed with ctions not to wet or expose the test area to direct sunlight.

— After 24 hours the patch was removed by the panelist at home.

— This procedure was repeated until a series of nine consecutive 24 hour exposures have been made for every Monday, Wednesday and Friday for thee consecutive weeks.

- In the event of an adverse reaction, the area of erythema and edema is ed. The edema is estimated by the evaluation of the skin with t to the contour of the cted normal skin. Reactions are scored just before applications two through nine and the next test date following application nine. s are notified immediately in the case of adverse reaction and ' determination is made as to treatment program if necessary.

- Subjects were then given a 10-14 day rest period after which a challenge or retest dose was applied once to a previously unexposed test site. The retest dose is equivalent to any one of the original nine exposures. Reactions are scored 24 and 48 hours after application.

— Comparison ‘was made between the nine sensitizing doses and the retest dose.

- At the end of the study, the consulting Dermatologist revised this data and confirmed the stated conclusions. 8. 0 Results No adverse reactions of any kind were noted during the course of this study.

. Conclusions The test material when tested under semi—occlusive conditions as described herein, can be considered as a non-primary irritant and a non-primary sensitizer to the skin according to the reference.

Example 10 — Surface persistence test Objective To determine whether terpinen—4—ol from the formulation of Example 1 will t on a surface for a period of time after application Method The test surface was a smooth melamine laboratory bench top. The e was prepared by cleaning with 100% EtOH and allowing to dry. The test formulation (one pump from a plastic hand pump (+/— 2g)) was evenly spread over the estimated surface ' area of a human hand (400 cm2) using a rubber finger cot. Duplicate sample areas were prepared. Samples were taken after 3 hours and 6 hours with a cotton swab by wiping the whole area with 20 swipes of the swab. The swab samples were placed in test tubes containing 5mL (10% methanol in acetone) and analyzed h gas chromatography (GC) using the validated method MTCS - 220.

Results Analyte tested for - Terpinen—4-ol 3 Hours - Area A Present 3 Hours — Area B Present 6 Hours — Area A None Detected 6 Hours - Area B None Detected Standard Check Terpinenol (0.45 mg/mL) 98.0% These results demonstrate tence of terpinen—4—ol on a surface at least 3 hours following application.

Example 11 — Skin hydration test The effect of the ation of Example 1 on skin hydration was evaluated using a Corneometer, compared with untreated skin on the same test subjects at 30 min, 2 days and 6 days. _ 52 - Standardsfor Inclusion in a Study 1. Individuals between the ages of 25 and 65. 2. Individuals not taking medication or under the care of a physician for a period of one month prior to commencement and hout the entire test period. 3. Individuals who have completed a preliminary medical history questionnaire. 4. Individuals who have read, understood and signed an informed consent document.

. Individuals who understand the instructions for use and are g to cooperate with the m as stated. 6. duals free of any dermatological or systemic disorder that would interfere with the s, at the discretion of the Investigator. 7. Individuals able to cooperate with the Investigator and the research staff and willing to complete the full course of the study.

Standardsfor Exclusion.from a Study 1. Individuals who are under doctors' care. 2. Individuals who are currently taking medication which in the opinion of the Investigator would mask or interfere with the results. 3. Individuals with any history of sensitivity to cosmetics in general and moisturisers in ular. 4. Individuals with any form of skin cancer, melanoma, lupus, psoriasis, rosacea, porphyria cutanea tarda, connective tissue disease, or any disease that would interfere with the test results.

Individuals diagnosed with chronic skin allergies.

°‘.V' Female eers who indicate that they are pregnant or nursing an .

Individuals with excessive hair on the test sites.

Individuals with known hypersensitivity to cosmetic products.

Informed Consent A signed ed t was obtained from each panelist prior to initiating the study. describing reasons for the study, possible adverse effects, associated risks and potential benefits of the treatment and their limits of liability. ology One distinct method was employed for the evaluation procedure. Biophysical measurements were made pre-application (t = O) and following a single application of the test material after 30 min. onal gs were also taken at 2 days and 6 days. Before each set of measurements, subjects were required to equilibrate in a closed environment with constant ature (20°C+/—2°C) and humidity (45% to 55% RH).

Moisture Measurement - Corneometer Model CM 825 PC Corneometer — Courage + Khazala Study Design healthy subjects between the ages of 33 to 58 years were inducted into this study. In order to precondition the test sites and keep all topical treatments constant for all test subjects they were required to abstain from using deodorant soaps, moisturising soaps or cosmetic moisturisers on the test area for a period of one week prior to study commencement and during the course of the study. At the completion of the one week ‘washout’ period, subjects were required to return to the test facility at the time specified by the technician for the study commencement.

On the day of the study, test material was delivered to the test sites by applying to the back of the hands liberally. Subjects were blinded as to the nature of the al being applied. Biophysical measurements with a Corneometer were taken at t = 0 (pre— ation). Subjects were required to return to the lab at each subsequent designated period for repeat biophysical measurements. _ 54 _ Results Table 5 — Skin hydration t = 0 t = 30min t = 2 days t = 6idays 26.4 33.7 43.0 46.2 % diff I 27.7% 63.0% i 74.8% Conclusions After repeat applications over a period of 6 days, the formulation was shown to provide ive moisturisation under the conditions of the test. There was no nce of skin irritation under the test conditions.

Example 12 EXAMINATION: Anti-microbial Activity Challenge Test (Time—Kill Study) METHOD: TMF 1 10-04 CONDITIONS: Test organisms: Pseudomonas aeruginosa ATCC 15442 Neutraliser: T6 1:100 (TSB with Lecithin and Tween 80) Test Concentration: Neat Contact Time: 15 and 30 seconds Test Temperature: t RESULTS: Table 1: Surviving Organisms after 15 and 30 seconds exposure to Antimicrobial Hand Gel Pseudomonas aefuginosa Contact Time CFU/mL Log ion (logm) seconds <100 >570 (2.00) seconds <100 >370 (2.00) Inoculum count 5.0x 107 (7.70) COMMENTS: The sample e 3 (Hand Gel) has demonstrated bactericidal activity against Pseudomonas aeruginosa by showing greater than 5 Log reduction which equates to 99.999% kill at 15 and 30 seconds contact time when tested under the conditions described above.

EXAMINATION: TGA (Australian Therapeutic Goods Administration) Disinfectant Test, Option D ON: Neat METHOD: TM122-04 NEUTRALISER: T6 RESULTS: Test Date Organisms Subculture um nge Controls No. (CFU/mL) Result Staphylococcus 5 1.1 X 106 _____ C 1 - aureus Escherichia coli 5 2‘3 X 106 ..... C2- Pseudomonas 5 2.1 x 106 ————— C3 +++ aeruginosa +++ Proteus vulgaris 5 2.0 X 106 _____ 2012 Methicinin 5 2.9 x 106 ————— C4+++ Resistant ' Staphylococcus aureus Vancomycin 5 1_6 x 106 _____ Resistant Enterococcus —56— faecalis Staphylococcus C1- aureus ichia coli C2- Pseudomonas c3+++ aeruginosa +++ Proteus vulgaris 2012 Methicillin C4+++ Resistant Staphylococcus aureus Vancomycin 1.6x106 Resistant Enterococcus faecalis lococcus 1.7x106 C1— aureus Escherichia coli 1.7x106 C2- Pseudomorzas 2.1x106 c3+++ aeruginosa +++ Proteus vulgaris 2.4x 106 18/05/2012 Meth icillin 2.3 x106 C4+++ Resistant lococcus aureus Vancomycin 2.0 x 106 Resistant Enterococcus faecalis REF N0: 1206546 —C0ntd.

Where "-" Denotes No Growth "+" Denotes Growth "C1" Denotes media sterility check. Must be negative for a valid result "C2" Denotes sample sterility check. Must be negative for a valid result WO 06917 -57_ "C3" Denotes organism viability checlc. Must be positive for a valid result "C4" Denotes neutralizer toxicity check. Must be positive for a valid result FINAL RESULTS: The sample Example 3 (Hand Gel) passed the TGA Disinfectant Test, Option D when tested under the conditions bed above.

Notes: 1. Strains of Organisms used were as follows Organism AMS Culture No. NCTC No.

Staphylococcus aureus AMS 028 NCTC 4163 Escherichia coli AMS 007 NCTC 8196 Pseudomonas aeruginosa AMS 018 NCTC 6749 s vulgar/Ts AMS 016 NCTC 4635 Methicillin Resistant AMS 077 NA Staphylococcus aureus Vancomycin Resistant AMS 084 NA Enterococcusfaecalis 2. Results of controls confirm to expected outcome Example 13 EXAMINATION: Anti-microbial Activity Challenge Test (Time—Kill Study) METHOD: TMF 110-04 CONDITIONS: Test organisms: Pseudomonas aeruginosa ATCC 15442 Neutraliser: T6 1:100 (TSB with Lecithin and Tween 80) Test Concentration: Neat t Time: 15 and 30 seconds Test Temperature: Ambient RESULTS: Table 1: Surviving Organisms after 15 and 30 seconds exposure to Antimicrobial Hand Gel Pseudomonas nosa Contact Time CFU/mL Log Reduction (10g10) seconds <100 >5 70 (2.00) seconds <100 >5 70 (2.00) Inoculum count 5.0x 107 (7.70) COMMENTS: The sample Example 2 (Hand Rub) has demonstrated bactericidal activity against » Pseudomonas aeruginosa by showing r than 5 Log reduction which equates to 99.999% kill at 15 and 30 s contact time when tested under the conditions described above.

EXAMINATION: TGA (Australian Therapeutic Goods Administration) Disinfectant Test, Option D DILUTION: Neat METHOD: TM122-04 NEUTMLISER: T6 RESULTS: Test Date Organisms Subculture um Challenge Controls N0. (CFU/mL) Result Staphylococcus 5 1.1 x 106 _____ C1- aureus ichia coh‘ 5 2,3 x 106 _____ C2— Pseudomonas 5 2.1 x 106 ----- C3 +++ aeruginosa -59_ Proteus vulgaris +++ 16/05/2012 +++ Methicillin 2.9x 106 C4+++ Resistant lococcus aureus Vancomycin Resistant Enterococcus faecalis Staphylococcus C1— aureus Escherichia coli .C2- Pseudomortas C3+++ aerugiriosa Proteus vulgaris 17/05/2012 Meth ic i 11in C4+++ Resistant Staphylococcus aureus Vancomycin 1.6x106 Resistant Enterococcus faecalis Staphylococcus 1.7x106 C1- aureus Escherichia coli 6 C2- Pseudomonas 2.1x106 C3+++ nosa Proteus vulgaris 2.4 x106 18/05/2012 Meth icillin 2.3 x106 C4+++ Resistant Staphylococcus aureus Vancomycin 7 2_() X 106 _____ Resistant Enterococcus faecalis REF NO: 1206548~Contd.

Where "-" s No Growth "+" Denotes Growth "C1" Denotes media sterility check. Must be negative for a valid result "C2" Denotes sample sterility check. Must be negative for a valid result "C3" Denotes organism viability check. Must be positive for a valid result "C4" Denotes neutralizer toxicity check. Must be ve for a valid result FINAL RESULTS: The sample Example 2 (Hand Rub) passed the TGA Disinfectant Test, Option D when tested under the conditions described above.

Notes: 1. Strains of Organisms used were as follows Organism AMS Culture No. NCTC N0.

Staphylococcus aureus AMS 028 NCTC 4163 Escherichia coli AMS 007 NCTC 8196 Pseudomonas aerugiflw'a AMS 018 NCTC 6749 Proteus is AMS 016 I\CTC 4635 Methicillin Resistant AMS 077 RA Staphylococcus aureus Vancomycin Resistant AMS 084 NA Enterococcusfaecalis 2. Results of controls confirm to expected outcome It will be appreciated that the present invention has been described by way of example only and that modifications and additions may be made thereto without ing from the scope of the invention as defined in the ed claims.

Claims (4)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A disinfecting formulation sing: (a) ethanol; 5 (b) eucalyptus oil; (c) ine; (d) piroctone olamine; and (e) water.

2. The disinfecting formulation according to claim 1 comprising, by volume: (a) from about 60% to about 80% ethanol; (b) from about 5% to about 15% eucalyptus oil; (c) from about 2% to about 10% glycerine; 15 (d) from about 0.01% to about 0.1% piroctone olamine; and (e) water.

3. The disinfecting formulation according to claim 1 or claim 2 sing, by volume: 20 (a) from about 60% to about 80% ethanol; (b) from about 5% to about 15% eucalyptus oil; (c) from about 2% to about 10% glycerine; (d) from about 0.1% to about 1% terpinenol; (e) from about 0.01% to about 0.1% piroctone olamine; and 25 (f) water.

4. The disinfecting ation according to claim 1 or claim 2 comprising, by volume: (a) about 73% of 95% ethanol; 30 (b) about 10% eucalyptus oil; (c) about 5% glycerine; H:\fmt\Interwoven\NRPortbl\DCC\FMT\9902160_1.docx-