AU2021202506A1

AU2021202506A1 - Disinfectant formulation

Info

Publication number: AU2021202506A1
Application number: AU2021202506A
Authority: AU
Inventors: Sydney Hawthorne
Original assignee: Clinotec Pty Ltd
Current assignee: Clinotec Pty Ltd
Priority date: 2020-04-24
Filing date: 2021-04-23
Publication date: 2021-11-11

Abstract

An antimicrobial formulation suitable for use as a disinfectant on hard surfaces id disclosed. The formulation comprises at least one antimicrobial agent that is a noncyclic aliphatic 5 hydrocarbon having from 7 to 20 carbon atoms in a liquid carrier. The noncyclic aliphatic hydrocarbon has at least one substituent selected from the group consisting of a primary amine, a secondary amine, a tertiary amine, an amine salt, or a halogen.

Description

DISINFECTANT FORMULATION FIELD

The present disclosure relates to a disinfectant formulation having particular application for in the disinfecting and sanitising surface or for use as a hand sanitiser.

BACKGROUND

The reference to background art in this specification is not intended to, and should not be taken as, an acknowledgment, statement, suggestion or admission that the referenced prior art forms part of the common general knowledge in Australia or in any other country.

Surface and hand hygiene have become not only important but critical in control of disease .0 associated with antimicrobial resistant pathogens and viral infections such as the present COVID - 19 pandemic.

The world Health Organisation (WHO) recommends the following products for surface disinfection; Ethanol 70-90%, Chlorine-based products (e.g., hypochlorite) at 0.1% (1000 ppm) for general environmental disinfection or 0.5% (5000 ppm) for blood and body fluids .5 large spills and Hydrogen peroxide >0.5%.

The TGA advises that disinfectants containing > 70% alcohol, quaternary ammonium compounds (such as benzalkonium chloride) or diluted household bleach (including products containing sodium hypochlorite) are suitable for surface use.

The World Health Organization (WHO) recommends alcohol-based hand rubs as the gold standard for hand hygiene in health care. The two WHO recommended alcohol based hand rub formulations are as follows;

Formulation 1

Ethanol 96%v/v 833.3 ml

H 2 02 3%,14.5 ml

Glycerol 98%,14.5ml

Make up to 1000ml with distilled water

Formulation 2

Isopropyl alcohol 75.5ml

H 2 02 3%, 41.7 ml

Glycerol 98%,14.5 ml

Make up to 1000 ml with distilled water.

The TGA has followed this advice from WHO such that hand sanitizers that comply with the WHO recommendations do not need to be TGA approved.

Alcohol quickly and effectively denatures the lipid viral coating and evaporation of the alcohol essentially dehydrates and denatures the viral proteins.

However, it is desirable to provide an effective sanitizer with alcohol levels below 60% and .0 the remainder of the liquid carrier comprising water. The reduction in alcohol may reduce costs and also provide a choice or option in areas where there may be a shortage of alcohol.

There are other reasons where it may be desirable to provide a disinfectant formulation with less than 75% alcohol such as in areas near heat and flame and workers with skin allergies. There are also social concerns with allowing young adults access to alcohol based .5 formulations. For example, some schools or other institutions do not permit the use of alcohol based hand sanitizers.

It is therefore desirable to be able to provide an alternative to sanitizers and disinfectants that rely on alcohol as the active agent.

DEFINITION

In the specification the term "comprising" shall be understood to have a broad meaning similar to the term "including" and 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. This definition also applies to variations on the term "comprising" such as "comprise" and "comprises".

SUMMARY

In one aspect there is disclosed an antimicrobial formulation comprising at least one antimicrobial agent that is a noncyclic aliphatic hydrocarbon having from 7 to 20 carbon atoms in a liquid carrier, wherein the noncyclic aliphatic hydrocarbon has at least one substituent selected from the group consisting of a primary amine, a secondary amine, a tertiary amine, an amine salt, or a halogen.

The liquid carrier is suitably water and a solubilising agent for the aliphatic hydrocarbon. A suitable non-toxic agent is a lower alcohol such as ethanol or isopropyl alcohol below 75%

The presence of the at least one noncyclic aliphatic hydrocarbon in the antimicrobial formulation may in some aspects, provide the formulation with antimicrobial properties against microbes including viruses, bacteria, fungi and prions.

Surprisingly, the antimicrobial activity of the formulation does not require the presence of 75% alcohol, thereby allowing for a more cost effective formulation or to obtain an .0 antimicrobial formulation where there is a limited supply of alcohol.

This is shown in the present test Examples discussed below in which the formulation solvent comprised 20% w/w isopropyl alcohol.

These examples also show a very surprising and unexpected efficacy against COVID - 19 proteins in that the protein may be deactivated in 60 seconds or less.

.5 Preferably, the amount of alcohol in the formulation is 75% or less.

The liquid solvent preferably comprises water and no more than 60% alcohol. Preferably, the amount of alcohol in the solvent is the minimum amount required to provide a stable solution of the water insoluble hydrocarbons. It will be appreciated that the amounts will vary according to the amounts and the different solubilities of different hydrocarbons. However, o0 modifying a mixed solvent concentrations to obtain a stable solution is well within the ordinary skill of a formulation chemist.

It will also be appreciated that the formulation may be provided in a concentrated form and diluted with water prior to use. This may reduce transport costs.

In some embodiments, the formulation is in a concentrated form.

Suitably, the antimicrobial agent is present in a concentrate a concentration from about 10% to about 80% by weight of the formulation. Preferably, the antimicrobial agent is present in a concentration from about 20% to about 50% by weight of the formulation.

The aliphatic hydrocarbon may be branched or unbranched, preferably non branched.

The aliphatic hydrocarbon may be saturated or unsaturated, preferably unsaturated or mono-saturated.

In one aspect, the aliphatic hydrocarbon is unsaturated or monosaturated and comprises a single primary amine substituent in the 1 position.

Exemplary unsaturated amines include aminoheptane, aminooctane, aminononane, aminodecane, aminoundecane, aminoundecane, aminododecane, aminotridecane, aminotetradecane, aminopentadecane, aminohexadecane, aminoheptadecane, aminooctodecane, aminononadecane and aminoeicosane.

If the aliphatic hydrocarbon is mono-unsaturated, the alkene moiety is suitably towards the .0 opposite end of the carbon chain to the substituent.

In another aspect, the substituent may be a tertiary amine such asdimethylaminoheptane, dimethylaminooctane, dimethylaminononane, dimethylaminodecane, dimethylaminoundecane, dimethylaminoundecane, dimethylaminododecane, dimethylaminotridecane, dimethylaminotetradecane, dimethylaminopentadecane, .5 aminohexadecane, aminoheptadecane, aminooctodecane, aminononadecane and aminoeicosane.

In another aspect, the amine substituent may be present at the 2 or 3 position.

The substituent may also be a quaternary ammonium ion.

The substituent may also be a halogen. Preferably the halogen is chlorine.

As for the amine substituent, it is also preferred that the halogen is substituted at the 1, 2 or 3 positions.

The halogen may also be substituted at the 1 and terminal position for example, 1,7 dicloroheptane, 1,8-diclorooctane, 1,9-dichlorononane, 1,10-dichloroodecane, 1,11 dichloroundecane, 1,12-dichloroundecane, 1,13-dichlorodecane, 1,14-dichlorotridecane, 1,15-dichlorotetradecane, 1,16-dichloropentadecane, 1,17-dichlorohexadecane, 1,18 dichloroheptadecane, 1,19 dichlorooctodecane, 1, 20-dichlorononadecane and 1,21 dichlorooeicosane.

In an embodiment, the formulation comprises one or more substituted aliphatic hydrocarbons. There has been a report in the literature, almost 50 years ago, (Kabara et al, Relationship of Chemical Structure and Antimicrobial Activity of Alkyl Amides and Amines,

Antimicrobial Agents and Chemotherapy, 1972, p492-498) that aliphatic amines have antimicrobial action that is more effective against Gram-positive bacteria than Gram-negative bacteria. The test data compared the MIC (Minimum Inhibitory Concentration) of the compounds. MIC measures the minim concentration of that inhibits growth after overnight incubation and is the basic initial tests to determine any antimicrobial activity. It will be appreciated that MIC does not provide any information as to whether a test microorganism is actually being killed and if so within what period of time.

However, the Applicant is unaware of any reports as to antimicrobial activity of halogen substituted aliphatic hydrocarbons.

.0 For sanitisation and disinfectant purpose, and in particular disinfection or sanitation of hard surfaces, there are other factors that require consideration. Obviously the formulation must be non-toxic, non-irritant and non-allergenic. Touching or rubbing the treated hard surfaces dislodges disinfecting chemicals to hands (surface-to-hand transfer); subsequent touching the face further transfers these compounds to the mouth (hand-to-mouth transfer). Some .5 agents such as hypochlorite' or iodophors may cause corrosion and/or staining. It is also important that the active agents can kill the microorganisms within a short period of time. Still further, the active agent must be effective when applied to a range of hard surfaces.

Another factor to be considered is volatility. It is desirable for hand sanitisers to be volatile to avoid having to dry the hands. The active agents must be able to be effective within very .0 short time periods due to the short handwashing time. The requirement for volatility for hard surface cleaning may be less important. It is considered desirable for an active agent to remain on the surface to provide ongoing antimicrobial action.

The present inventor has surprisingly and unexpectedly discovered that the formulations as disclosed herein may provide broad spectrum activity against Gram positive bacteria, Gram negative bacteria, viruses and biofilm.

Biofilm formation is a mechanism, in which bacterial resistance towards a sanitizer can occur. Certain bacteria secrete a polysaccharide which is a constituent of their membrane. These secretions are very sticky and attach themselves firmly to metal surface. The resulting film so formed containing trapped bacteria is referred to as a biofilm. Bacteria which are responsible for biofilm formation may in themselves not be harmful or pathogenic. However, the gelatinous matrix which they excrete is capable of attracting to itself and embedding pathogenic bacteria, such as Lysteria monocytogenes. Although the pathogens themselves do not contribute towards the integrity of the film, they nevertheless are capable of contaminating products which come into contact with the surface.

Biofilms are often very difficult to remove, since their matrix is very resistant to chemical attack by detergents. They often require higher than normal concentrations of alkaline detergents and strong oxidizing levels of sodium hypochlorite in order to remove them. Several applications may be required before the biofilm can be totally removed.

As discussed below in the experimental section, the it has been surprisingly and unexpectedly observed that the disclosed formulation in may also have activity against biofilms.

.0 In another aspect the formulation comprises a first antimicrobial agent that is at least one of a C7 to C21 aliphatic hydrocarbon substituted with at least one amine and a second antimicrobial agent and that is at least one of a C7 to C21 aliphatic hydrocarbon substituted with at least one halogen.

Preferably the or each of the first and second antimicrobial agents have a carbon chain .5 length of from 9 to 15.

Preferably at least one of the amine hydrocarbons are tertiary amines.

Preferably the or each amine hydrocarbon is at the C1 position.

Preferably the halogen is chlorine.

Preferably, the halogenated aliphatic hydrocarbon is substituted at the C1 position and the end Carbon.

The relative amounts of the first and second antimicrobial agents in the formulation suitably corresponds to the relevant tests as required under the relevant Government authority such as the Therapeutic Goods Administration (TGA) in Australia https://www.tqa.qov.au/publication/tga-instructions-disinfectant-testinc, as carried out using the dilution according to the recommended instructions.

Suitable amounts of first and second antimicrobial agents are from 0.001w/w% to 10.00w/w%, or about 0.01% to about 5% or about 0.25% to 1.00% or about 0.30% to 0.70%.

Whilst not being bound by theory, it is believed that the combination of the halogenated hydrocarbon and amine hydrocarbons are complimentary so as to provide the observed broad spectrum antimicrobial effect. Further the volatility of aliphatic amines and aliphatic are very different. For example, aminodecane has a vapour pressure of 0.1mm Hg at 25°C and 1,10-dichlorodecane has a vapour pressure of 0.00515mm Hg at 25°C. Thus, dichlorodecane will remain on a hard surface for longer than the alkylamine that may provide extended antimicrobial action.

The Example formulation in the detailed description below compriseddimethylaminodecane and 1,2 dichlrodecane, that each have a carbon chain length of 10. It is therefore reasonable to extrapolate that C7 to C20 hydrocarbons, or mixtures thereof may also have antimicrobial effect. Preferred carbon chain lengths are from C7 to C16, C7 to C15, C7 to C14, C7 to C13, C7 to C12, C7 to C11.

.0 The formulations of the present disclosure may additionally include adjunct ingredients conventionally found in formulations in their art-established fashion and at their art established levels. For example, the formulations may comprise additional compatible active materials such as antioxidants, anti-parasitic agents, antipruritics, antifungals, antiseptic actives, biological actives, astringents and combinations thereof. Other suitable additives .5 that may be included in the compositions of the present disclosure include colorants, deodorants, fragrances, perfumes, emollients, emulsifiers, anti-foaming agents, lubricants, solubilizing agents, suspending agents, wetting agents, preservatives, pH adjusting ingredients, chelators, propellants, dyes and/or pigments, embittering agents, and combinations thereof.

.0 Preferably the formulation includes an emollient such as glycerine.

Preferably, the formulation includes a fragrance that is a natural product or derived from a natural product. Preferred fragrances are essential, oils and preferably essential oils of Australian native plants.

Preferably, the formulation is safe for humans, animals and the environment. Suitably, formulations of a dangerous nature are avoided. For example, formulations such as flammables, combustibles, explosives, agents of acute toxicity, agents with a low LD50 when tested intraperitoneally, dermally, cutaneously or topically are avoided.

The formulation can be used for domestic, industrial and medical applications, such as food service, food processing, cosmetic, pharmaceutical and health care industries, especially such as in hospitals. Suitably, the formulation can be applied to a wide variety of surfaces, for example, articles adapted for medical use, including scalpels, needles, scissors and other devices used in invasive surgical, therapeutic or diagnostic procedures; implantable medical devices, including artificial blood vessels, catheters and other devices for the removal or delivery of fluids to patients, artificial hearts, artificial kidneys, orthopedic pins, plates and implants; catheters and other tubes, urinary devices, shunts; prostheses, vascular catheter ports, wound drain tubes, hydrocephalus shunts, pacemakers and implantabledefibrillators, and the like. Other examples will be readily apparent to practitioners in these arts.

In other embodiments, the formulation can be applied to hard surfaces, such as floors, counter tops, furniture, fixtures in areas used for medical, veterinary and cosmetic procedures or for preparing medical, veterinary and cosmetic apparatus etc., such as are found in, for example, the health care industry.

Further, the formulation may be suitable for oral, rectal, topical, buccal (e.g., sub- lingual), .0 vaginal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), topical (i.e., both skin and mucosal surfaces, including airway surfaces) and transdermal administration or application to humans and animals.

For example, the formulation can be applied topically to prevent or treat skin infections, burns, diaper rash, other irritations and various skin care products. Alternatively, the .5 formulation can be used internally such as in the mouth, such as for mouthwashes, toothpastes, and various other disinfecting solutions that can be employed in dental industries.

The antimicrobial formulation of the invention may be effective to reduce concentrations of bacteria and other microbes on surfaces more effectively in comparison to conventional formulations known in the art.

In another aspect, the invention provides a method of sanitizing, disinfecting or sterilizing a surface, comprising applying an effective amount of the antimicrobial formulation of the first aspect.

For the purposes of this invention, an "antimicrobial formulation" can refer to compositions that are capable of killing, inactivating, neutralising, disintegrating or inhibiting the growth of microbes and still be within the scope of the present disclosure.

As generally used herein, the term "antimicrobialagent" refers to a compound capable of killing, inactivating, neutralising, disintegrating or inhibiting the growth of microbes.

As generally used herein, "contacting" or "contact" refers to the application or spread of the formulations of the present invention onto any surface.

As generally used herein, a "surface" may be continuous or discontinuous, solid or porous, soft or hard, synthetic or natural, fibrous or non-fibrous, metallic or non-metallic, or have any other form, shape or character in or on which antimicrobial action is desired.

The term "effective amount' describes a quantity of an antimicrobial formulation sufficient to achieve a desired effect on a microbe being contacted with that formulation. In some embodiments, an "effective amount'is sufficient to kill the microbe. In other embodiments, an "effective amount' is an amount sufficient to achieve a desired biological effect, for example an amount that is effective to inhibit the growth of or kill a microbe.

As generally used herein, the term "microbe" should be taken to mean any bacterial, virus, .0 fungi or prion. For example, a bacterial or fungal isolate, a viral particle, or a monomicrobial or polymicrobial biofilm.

As generally used herein, "kil/'or "killing" is demonstrated by death of the microbe, including by cell death, for example, by inhibition of cell wall synthesis or other vital protein synthesis of the microbes or the synthesis of their nucleic acids. Methods of demonstrating or .5 determining microbial death are well known in the art. Further, it is to be understood that such killing need not be absolute with respect to a particular population of microbes in order to be beneficial from a sanitation, disinfection or sterilization standpoint.

As generally used herein, "denature" or "denaturing"is demonstrated by destruction of a virus, a viral particle or a prion, including by structural deformation or function inhibition or .0 inactivation, for example, by chemical binding, induction, blockage. Methods of demonstrating or determining protein denaturing are well known in the art. Further, it is to be understood that such denaturing need not be absolute with respect to a particular quantity or concentration of protein molecules in order to be beneficial from a sanitation, disinfection or sterilization standpoint.

As generally used herein, "inhibiting" or "inhibit'is demonstrated by reduction or suppression of microbial growth. In particular embodiments, inhibiting microbial growth and/or killing microbes is when the number of microbes is reduced.

In another aspect there is provided a method of killing and/or inhibiting the growth of a microbe, the method including the step of contacting the microbe with an effective amount of the disclosed antimicrobial formulation.

In another aspect, the disclosure provides a method of sanitizing, disinfecting or sterilizing a surface, comprising applying an effective amount of the antimicrobial formulation of the first aspect.

The surface is suitable a hard surface such as such as floors, counter tops, furniture, fixtures in areas used for medical, veterinary and cosmetic procedures or for preparing medical, veterinary and cosmetic apparatus

The antimicrobial formulation can be applied directly or can be initially diluted from its concentrated form prior to application.

DETAILED DESCRIPTION

.0 FORMULATION PREPARATION

The following formulations were prepared by mixing (as v/v%) as shown in Table 1;

F1 F2 F3 F4 F5

Isopropanol 20 20 20 20 20

1,10- 0.08 0.25 0.50 1.00 0.50 Dichlorodecane Dimethyl 0.01 0.25 0.50 1.00 0.35 aminodecane Corymbia 0.38 0.38 0.38 0.38 0.38 citriodra Leaf extract Glycerol 0.1 0.1 0.1 0.1 0.1

Water to 100%

EXAMPLE 1 TESTING USING CULTURE GROWTH AND COLONY COUNTING Laboratory methods, equipment, microbial isolates, media, solutions, procedures, protocols and accreditations were used in compliance with instructions published by the Australian Therapeutic Goods Administration for the elaboration and testing of disinfectants (TGA instructions for disinfectant testing - Version 2.0, October 2019 https://www.tqa.qov.au/publication/tqa-instructions-disinfectant-testin). The antimicrobial formulations as described above tested at various dilutions, whereby the concentration of each component ranged from 0.001% to 10% v/v. For disinfectant challengeeach bacterial inoculum comprising the pathogens Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli, Staphylococcus aureus, Candida albicans or the probiotic microorganisms Bacillus spp., Lactococcus lactis, Lactobacillus spp., Pisolithus spp. were aseptically mixed to various concentrations of the antimicrobial formulation of the invention, and a sample was collected at specified post-exposure time-points for growth into recovery medium. After a second time interval the mixture was challenged again by a second culturing step, and samples were considered to pass or fail the test according to the extent of growth shown .0 in the two cultures sampled. In this test, commercial yeast preparations and the probiotic fungus Pisolithus spp. were used as controls, colony counting was used to determine inoculum density, both among controls and cultures that were exposed to the invention.

Briefly, yeast inoculumwas prepared using hard water as per protocol orientation, aiming to .5 demonstrate yeast growth (0.304g anhydrous calcium chloride and 0.065g anhydrous magnesium chloride were dissolved in glass-distilled water to one litre). Wright and Mundy dextrose medium and the AOAC Synthetic Broth were used for growth of the test microorganisms (pathogens and probiotic bacteria and fungi). Nutrient broth used as a recovery medium was prepared from a commercial product, whereby the following .0 composition was used: Beef Extract Powder 1Og, Peptone 1Og, Sodium Chloride 5g were added to 970ml of deionised water and were dissolved by stirring and heating. To each litre of nutrient broth prepared, 30g polysorbate 80 was added. pH of the culture media used for all growth cultures was set between 6.9 and 7.1, and autoclavation was conducted at 121 ±1°C for 15 minutes to ensure complete media sterilisation before start.

Ensuing to that, cultures were grown overnight at 370± 1C in Wright and Mundy dextrose medium or AOAC, followed by their inoculation onto nutrient agar. Colonies were then sub cultured into 10-15ml of Wright and Mundy dextrose or AOAC medium, and incubated at 370± 1C for 24± 2 hours. Liquid cultures were then sub-cultured into fresh medium, and incubatefor further 24± 2 hours at 370± 1C. After five successive sub-culturing procedures the cultures were exposed to different concentrations of the invention, aiming to conduct both dose-response and time-response kinetic assessments.

Briefly, three millilitres of diluted disinfectant were added to capped glass containers. Timing was then started, and 1ml of each culture was immediately added and mixed by swirling. At the 10, 20, 30 and 60-second time-point, as well as 2, 5, 8, 10 and 18 minutes, 20 microlitres of the mixture were inoculated into five tubes containing recovery or nutrient broth. The contents of all tubes were incubated at 370± 1C for 48± 2 hours for the observation of turbidity, and for use in colony counting. The test was repeated twice for each microbe across 2 subsequent days, using a fresh disinfectant dilution and a freshly prepared bacterialsuspension.

All test cultures were centrifuged until cells were compacted, supernatant was removed, and the test organisms were resuspended in the original volume (10ml or 15ml using sterile hard water) for further spreading on nutrient agar plates. All disinfectant exposure and efficacy testing was conducted at 21° 1C. The functional concentration of active .0 constituents, in ready-to-use disinfectant versions, was confirmed ranging from 0.25 to 5%. Product shelf-lifestability was confirmed across all active constituent concentrations i.e. formulas 2, 3, 4 and 5.

EXAMPLE 2 .5 TESTING USING MOLECULAR METHODS In addition to growth kinetics, microbial reduction due to sanitation, disinfection and sterilisation upon immediate exposure to the antimicrobial formulation of the invention hereinwas confirmed by PCR. Briefly, the culture samples collected for plate counting were also used for DNA extraction (including controls), whereby either 16S or ITS sequences .0 were amplified by PCR before and after exposure, following standard DNA extraction and amplifications protocols determined by the manufacturers of each respective kits and enzymes.

EXAMPLE 3 .5 TESTING USING A BIOFILM INHIBITION APPROACH Biofilm load reduction by functional concentrations of the present invention was also determined, using the microbial growth protocol from Example 1 in which laboratory flasks containing control and the antimicrobial formulation of the invention-exposed cultures were grown for 48h aiming to observe the formation of a biofilm ring. Culture flasks were emptied and filled up with either crystal violet or other dyes to promote biofilm staining for 2min, whichwas followed by immediate pouring of the solution and air-drying of the stained biofilms whichwere then resuspended in 1ml of 100% ethanol, and used for immediate quantification by spectrophotometry. A 10-fold dilution of each biofilm area was used for quantification. Similarly, empty flasks or those containing an unstained mixture (invention plus microbial cultures) were emptied after 30 and 60 seconds as well as after 2 and 5 minutes post- exposure, and were further filled with sterile nutrient broth medium for incubation at 37 0 C for 48h, aiming to assess the antimicrobial formulations ability to respectively prevent microbial growth due to residue formation, as well as biofilm establishment or microbial re-growth fromleftover cells/biofilms.

EXAMPLE4 TESTING USING THE DENATURING OF VIRAL PROTEINS AND ENZYMES Loss or alteration of protein function due to denaturing was determined by Dot Blot, wherebyCOVID-19 recombinant proteins corresponding to viral antigens with a known role in eukaryotic cell infection, as well as specific polyclonal antibodies were used. Briefly, COVID-19 recombinant proteins were mixed with functional concentrations of the invention .0 asspecified in Example 1, for time intervals ranging from 30, 45 and 60 seconds to 2, 5 and 10 minutes at 21C. Treated and untreated proteins were used for spot-binding onto Western Blotmembrane, blocking was conducted using 1% casein or powder milk, and a solution with eachrespective antibody was deposited until complete air-drying. Membrane washing was conducted three times using sterile 1X PBS-T, and anti-IgG conjugated with .5 peroxidase was added and incubated for 30 minutes as a liquid solution. The conjugate solution was poured,and the membrane was washed as previously described. Hydrogen peroxide was then addedand used to confirm COVID-19 protein denaturing.

Similarly, for demonstration of loss of enzymatic activity Taq DNA polymerase was exposed .0 to the invention following the aforementioned time-interval and concentrations. Untreated vs treated enzymes were then used to amplify a control DNA fragment using a standard PCR protocol established by the manufacturer, confirming enzymatic inactivation.

The results are summarized below. .5 It is noted that both active compounds had antiviral activity.

Table 2: Testing COVID-19 proteins.

Concentration Quantified component (pg) (A 28o-determ.) Read1 Read 2 COVID-19 Spike S1 protein (1pg PEF/UQ-determined) 1 0.927 0.08% 1,10-Dichlorodecane 0 0.025 0.01% N,N Dimethyl-1-decanamine 0.01 0.01 Chemical combo 0.01 0.01

Post-mixing (15-sec treatment) Outcome COVID-19 protein + 0.08% 1,10-Dichlorodecane 0 0

COVID-19 protein + 0.01% N,N Dimethyl-1-decanamine 0 0 COVID-19 protein + Chemical combo 0.01 0.01

ALTERNATIVE FORMULATION

A further formulation was also prepared as follows (w/w%)

Water 50.788

Isopropyl Alcohol 47.705

.0 Lemon eucalyptus Oil 0.363

1,10 Dichlordecane 0.551

Dimethyl aminodecane 0.301 .5 Glycerine 0.139

Lactic Acid 0.153

Total 100

This alternative formulation has a higher amount of isopropyl alcohol and added lactic acid as a pH modifier to reduce the pH to 8.5 to 9.5. This alternative formulation was simply modified to have a clear appearance as compared to the original formulation that had a slightly yellow colour. No effect on antimicrobial activity of the second formula was observed.

CONCLUSION As a result of all the testing exemplified, bacterial and fungal isolates were found to be killed, mono- and polymicrobial biofilms were found to be both inhibited (pre-establishment) and destroyed (post-establishment), microbial cultures and surfaces were found to be sterilised, viral proteins were found to be promptly denatured, and enzymes were found to be inactivated under various concentrations and combinations of the compounds used in the invention described herein.

It will be appreciated that various changes and modifications may be made to the invention as described and claimed herein without departing from the spirt and scope thereof.

Claims

1. An antimicrobial formulation comprising at least one antimicrobial agent that is a noncyclic aliphatic hydrocarbon having from 7 to 20 carbon atoms in a liquid carrier, wherein the noncyclic aliphatic hydrocarbon has at least one substituent selected from the group consisting of a primary amine, a secondary amine, a tertiary amine, an amine salt, or a halogen.

2. The formulation of claim 1, the liquid carrier comprises water and an alcohol selected from ethanol and/or isoprene. .0

3. The formulation of claim 2, wherein the formulation comprises 75% v/v or less alcohol.

4. The formulation of claim 2, wherein the formulation comprises 15% tv/v to 55% v/v alcohol. .5

5. The formulation of any one of claims 1 to 4, wherein the at least one antimicrobial agent is present in a concentration from about 0.01% to about 10% by weight of the formulation.

.0

6. The formulation of any one of claims 1 to 5, wherein at least one aliphatic hydrocarbon is non branched.

7. The formulation of any one of claims 1 to 6, wherein the at least one aliphatic hydrocarbon is unsaturated or mono-saturated.

8. The formulation of any one of claims 1 to 7, wherein the at least one aliphatic hydrocarbon is a primary amine.

9. The formulation of any one of claims 1 to 7, wherein the at least one substituent is a halogen.

10. The formulation of claim 9, wherein the halogen is chlorine.

11. The formulation of claim 7, wherein the at least one hydrocarbon is selected from the group consisting of 6,7-dicloroheptane, 1,8-diclorooctane, 1,9-dichlorononane, 1,10 dichloroodecane, 1,11 dichloroundecane, 1,12-dichloroundecane, 1,13 dichlorodecane, 1,14-dichlorotridecane, 1,15-dichlorotetradecane, 1,16 dichloropentadecane, 1,17-dichlorohexadecane, 1,18-dichloroheptadecane, 1,19 dichlorooctodecane, 1, 20-dichlorononadecane and 1,21-dichlorooeicosane.

12. The formulation of any one of claims 1 to 11 comprising two or more aliphatic hydrocarbons.

13. The formulation of claim 12, wherein the formulation comprises a first antimicrobial agent that is at least one of a C7 to C20 aliphatic hydrocarbon substituted with at least one amine and a second antimicrobial agent and that is at least one of a C7 to C20 .0 aliphatic hydrocarbon substituted with at least one halogen.

14. The formulation of claims 12, wherein the or each of the first and second antimicrobial agents have a carbon chain length of from 9 to 15.

.5 15. The formulation of claim 13 or claim 14 wherein the at least one amine hydrocarbon is a tertiary amine.

16. The formulation of any one of claims 13 to 15, wherein the at least one second antimicrobial agent is selected from the group consisting of 6,7-dicloroheptane, 1,8 ldiclorooctane, 1,9-dichlorononane, 1,10-dichloroodecane, 1,11 dichloroundecane, 1,12-dichloroundecane, 1,13-dichlorodecane, 1,14-dichlorotridecane, 1,15 dichlorotetradecane, 1,16-dichloropentadecane, 1,17-dichlorohexadecane, 1,18 dichloroheptadecane, 1,19 dichlorooctodecane, 1, 20-dichlorononadecane and 1,21 dichlorooeicosane. '5

17. The formulation of any one of claims 13 to 16, wherein the amounts of first and second antimicrobial agents are from 0.001w/w% to 10.00w/w%, about 0.01% to about 5% or about 0.25% to 1.00% or about 0.30% to 0.70%.

18. The formulation of any one of claims 13 to 17 when the at least one first antimicrobial agent is dimethylaminodecane and the at least one second antimicrobial agent is 1,2 dichlorodecane.

19. A method of sanitizing or disinfecting a surface, comprising applying an effective amount of the I formulation of any one of claims 1 to 19 to a surface.

20. A method of killing and/or inhibiting the growth of a microbe, the method including the step of contacting the microbe with an effective amount of the formulation to any one of claims 1 to 19.