WO2010035008A2

WO2010035008A2 - Composition

Info

Publication number: WO2010035008A2
Application number: PCT/GB2009/002304
Authority: WO
Inventors: Peter Bacon
Original assignee: Deonissi Limited
Priority date: 2008-09-26
Filing date: 2009-09-24
Publication date: 2010-04-01
Also published as: GB2463928A; WO2010035008A3; GB0817656D0

Abstract

An aqueous biocidal composition comprises: i. from 0.5 to 5% by weight of a chelating component comprising one or more chelating agents or salts thereof; ii. from 0.4 to 4% by weight of a surfactant component comprising one or more surfactants selected from non-ionic surfactants, amphoteric surfactants and mixtures thereof; and iii. from 0.6 to 5% by weight of a quaternary ammonium component comprising one or more trialkyl benzylammonium salts having at least one C₆-C₂₀ alkyl substituent, wherein the surfactant component and quaternary ammonium component are different from each other.

Description

COMPOSITION

The present invention relates to an aqueous biocidal composition. In particular, the invention relates to an aqueous biocidal composition thai is suitable for killing, inhibiting or preventing the growth of spores of Clostridium difficile.

Antiseptics and disinfectants are used extensively in hospitals and other health care settings for a variety of topical and hard-surface applications. In particular, they are an essential part of infection control practices and aid in the prevention of nosocomial infections¹'². Mounting concerns over the potential for microbial contamination and infection risks in the food and general consumer markets have also led to increased use of antiseptics and disinfectants by the general public. A wide variety of active chemical agents (or "biocides") are found in these products, many of which have been used for hundreds of years for antisepsis, disinfection, and preservation³.

A bacterium causing increasing concern is Clostridium difficile. C. difficile is a commensal bacterium of the human intestine in a minority of the population⁴. For C. difficile to establish itself and proliferate in the colonic mucosa, the normal flora of the colon must be disrupted (antibiotic use is the major cause) and C. difficile must be ingested⁵'⁶. Once established, C. difficile is associated with gastrointestinal infections ranging in severity from asymptomatic colonization to severe diarrhoea, life-threatening pseudomembranous colitis, fulminant colitis, toxic megacolon and intestinal perforation⁵. Their spores are resistant to all antibiotic, commonly used disinfectants such as alcohol, heating and drying⁴'⁷'⁸. C. difficile spores can survive up to 5 months in the environment⁶.

It has been observed that several disinfectants commonly used in hospitals may fail to kill the bacteria C. difficile, and may actually promote spore formation.

Hence, there is a need for new biocidal compositions for use in hospital environments having a broad spectrum efficacy. In particular, there is a need for new biocidal compositions that are capable of killing the spores of C. difficile.

US 7,192,601 relates to a germicidal composition which is stated to have enhanced microbiocidal activities for killing microbiological spores, microbiological outgrowth from spores, and vegetative microorganisms. The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

According to a first aspect of the invention, there is provided an aqueous biocidal composition comprising: i. from 0.5 to 5% by weight of a chelating component comprising one or more chelating agents or salts thereof; ii. from 0.4 to 4% by weight of a surfactant component comprising one or more surfactants selected from non-ionic surfactants, amphoteric surfactants and mixtures thereof; and iii. from 0.6 to 5% by weight of a quaternary ammonium component comprising one or more trialkyl benzylammonium salts having at least one C₆-C₂O alky! substituent, wherein the surfactant component and quaternary ammonium component are different from each other.

In a second aspect, there is provided a container comprising an aqueous biocidal composition according to the first aspect of the invention.

In a third aspect of the invention, there is provided a wet wipe impregnated or coated with an aqueous biocidal composition according to the first aspect of the invention.

In a fourth aspect of the invention, there is provided a process for the preparation of an aqueous biocidal composition according to the first aspect of the invention, comprising forming a mixture comprising the chelating component, the surfactant component and the quaternary ammonium component.

In a fifth aspect of the invention, there is provided the use of an aqueous biocidal composition according to the first aspect of the invention as an in vitro anti-microbial agent.

Figures 1 and 2 are photographs of slides of Cleaning Test 1, Figures 3 to 6 are photographs of tiles of Cleaning Test 2, Figure 7 is a photograph of slides of Cleaning Test 3, and Figures 8 and 9 are photographs of slides of Cleaning Test 4. The present invention is based in part on the surprising finding that a quaternary ammonium- based composition shows efficacy against the spores of C. difficile. The compositions of the invention may have one or more advantages compared to known anti-microbial agents that demonstrate efficacy against the spores of C. difficile, the most commonly used being hypochlorite. For example, the composition of the invention is virtually odourless, is less damaging to surfaces, is safer to personnel and/or provides residual protection on hard surfaces for up to 5 days after treatment, preferably up to 10 days, such as up to 15, 20 or 25 days, most preferably up to 28 days after treatment.

The term "biocidal" as used herein refers to an anti-microbial agent suitable for killing, inhibiting (at least in part, e.g. ,10%, 20%, 50%, or 100% growth inhibition) or preventing the growth of micro-organisms such as bacteria, viruses, fungi and/or protozoa.

The biocidal composition of the invention is an aqueous biocidal composition. The term "aqueous" as used herein describes a water-based solution comprising at least 5% by weight water, for example at least 10%, preferably at least 25%, such as at least 50% or at least 75%, most preferably at least 95% such as at least 99%, by weight of water.

The aqueous biocidal composition of the present invention comprises a chelating component comprising one or more chelating agents or salts thereof. Preferably, the chelating component comprises or consists of one or more chelating agents selected from the group consisting of nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), N-(hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA), methylglycinediacetic acid (MGDA), hydroxyethyiiminodiacetic acid (HEIDA), glutaric acid-N,N-diacetic acid (GLDA), iminodisuccinic acid (IDS) or salts thereof. Suitable salts include, for example, sodium, potassium, ammonium or amine salts. Sodium salts are particularly preferred. A particularly preferred chelating agent for use in the aqueous biocidal composition is trisodium nitrilotriacetic acid (Na₃NTA). Thus, the chelating component preferably comprises or consists of Na₃NTA. In a particularly preferred embodiment, Na₃NTA is the sole chelating agent in the composition of the invention.

The chelating component constitutes from 0.5 to 5% by weight of the composition, preferably from 1 to 4.5% by weight, such as from 1.5 to 4% by weight, more preferably from 2 to 3.5% by weight or from 2 to 2.5 % by weight (e.g., 2.27% by weight) of the composition. The aqueous biocidai composition comprises a surfactant component comprising one or more surfactants selected from non-ionic surfactants (e.g. alkoxyiated alcohols, alkylphenols, amides, amines, esters, fatty acids or glycerides; alkanolamides, amine oxides, esters and/or alkyl polyglucosides), amphoteric surfactants (e.g. betaines, glycinates, propionates, acetates and/or imidazolines) and mixtures thereof.

Preferably, the surfactant component comprises or consists of one or more alcohol ethoxylates having an alkyl group with a pendant chain of the structure -(OCH₂CH₂)_mOH, wherein m is from 3 to 20, more preferably from 4 to 15, most preferably from 5 to 10 (such as 7). The alkyl group is preferably a C₄- C ₂o alkyl, more preferably a C₈-Ci₈ alkyl or a Ci₀-C₁₆ alkyl, most preferably a C₁₃-Ci₅ alkyl, which may be linear or branched, cyclic or acyclic. For example, the alcohol ethoxylates may be derived from a primary, secondary or tertiary alcohol. Preferably, the alkyl group is an unsubstituted alkyl, more preferably a straight chained, unsubstituted alkyl. In a particularly preferred embodiment, the surfactant component comprises or consists of one or more alcohol ethoxylates having a C₁₃-C₁₅ alkyl group with a pendant chain of the structure -(OCH₂CH₂)_rτiOH, wherein m is from 5 to 10.

The surfactant component constitutes from 0.4 to 4% by weight of the composition, preferably from 0.5 to 3.5% by weight, more preferably from 0.7 to 3% by weight, such as from 0.9 to 2.5% by weight, most preferably from 1 to 2% by weight or from 1.25 to 1.75% by weight (e.g., 1.45% by weight) of the composition.

The aqueous biocidai composition comprises a quaternary ammonium component comprising one or more trialkyl benzylammonium salts having at least one C₆-C₂₀ alkyl substituent. The alkyl substituent may be linear or branched, cyclic or acyclic. Preferably, the alkyl group is an unsubstituted alkyl, more preferably a straight chained, unsubstituted alkyl. In a preferred embodiment, the quaternary ammonium component comprises or consists of one or more trialkyl benzylammonium salts having at least one C₁₂-C₁₆ alkyl substituent. Suitable salts include, for example, chloride, bromide, iodide and methosulphate. Chloride salts are particularly preferred.

In a preferred embodiment, the trialkyl benzylammonium salts for use in the aqueous biocidai composition of the invention include the chloride salts of alkyldimethylbenzylammonium having a C₁₂-Ci₆ alkyl substituent and dialkylmethylbenzylammonium having at least one C₁₂-Ci₆ alkyl substituent. Most preferably, the quaternary ammonium component comprises or consists of one or more chloride salts of alkyldimethylbenzylammonium having a Ci₂-Ci₆ alkyl substituent.

The quaternary ammonium component constitutes from 0.6 to 5% by weight of the composition, preferably from 1 to 4.5% by weight, such as from 1.5 to 4% by weight, more preferably from 2 to 3.5% by weight or from 2.5 to 3% by weight (e.g., 2.83% by weight) of the composition.

In a preferred embodiment, the aqueous biocidal composition comprises:

(i) from 1 to 4.5% by weight of a chelating component comprising one or more chelating agents or salts thereof;

(ii) from 0.5 to 3.5% by weight of a surfactant component comprising one or more surfactants selected from non-ionic surfactants, amphoteric surfactants and mixtures thereof; and

(iii) from 1 to 4.5% by weight of a quaternary ammonium component comprising one or more trialkyl benzylammonium salts having at least one C₆-C₂O alkyl substituent, wherein the surfactant component and quaternary ammonium component are different from each other.

In another preferred embodiment, the aqueous biocidal composition comprises:

(i) from 2 to 3.5% by weight of a chelating component comprising one or more chelating agents or salts thereof;

(ii) from 1 to 2% by weight of a surfactant component comprising one or more surfactants selected from non-ionic surfactants, amphoteric surfactants and mixtures thereof; and

(iii) from 2 to 3.5% by weight of a quaternary ammonium component comprising one or more trialkyl benzylammonium salts having at least one C₆-C₂₀ alkyl substituent, wherein the surfactant component and quaternary ammonium component are different from each other.

In another preferred embodiment, the aqueous biocidal composition comprises:

(i) from 1 to 4.5% by weight of a chelating component, wherein the chelating component comprises or consists of Na₃NTA;

(ii) from 0.5 to 3.5% by weight of a surfactant component, wherein the surfactant component comprises or consists of one or more alcohol ethoxylates having a Ci₃-C₁₅ alkyl group with a pendant chain of the structure -(OCH₂CH₂)_mOH, wherein m is from 4 to

15; and (iii) from 1 to 4.5 % by weight of a quaternary ammonium component, wherein the quaternary ammonium component comprises or consists of one or more chloride salts of alkyldimethylbenzylammonium having a Ci₂-Ci₆ alky! substituent, wherein the surfactant component and quaternary ammonium component are different from each other.

In a particularly preferred embodiment, the aqueous biocidal composition comprises:

(i) from 2 to 3.5% by weight of a chelating component, wherein the chelating component comprises or consists of Na₃NTA;

(ii) from 1 to 2% by weight of a surfactant component, wherein the surfactant component comprises or consists of one or more alcohol ethoxylates having a Ci₃-Ci₅ alkyl group with a pendant chain of the structure -(OCH₂CH₂)_mOH, wherein m is from 5 to 10; and

(iii) from 2 to 3.5% by weight of a quaternary ammonium component, wherein the quaternary ammonium component comprises or consists of one or more chloride salts of alkyldimethylbenzylammonium having a C1₂-C16 alkyl substituent, wherein the surfactant component and quaternary ammonium component are different from each other.

In another particularly preferred embodiment, the aqueous biocidal composition comprises:

(i) from 2 to 2.5% by weight of a chelating component, wherein the chelating component comprises or consists of Na₃NTA;

(ii) from 1.25 to 1.75% by weight of a surfactant component, wherein the surfactant component comprises or consists of one or more alcohol ethoxylates having a Ci₃-C₁₅ alkyl group with a pendant chain of the structure -(OCH₂CH₂)_I11OH₁ wherein m is from 5 to 10; and

(iii) from 2.5 to 3.0% by weight of a quaternary ammonium component, wherein the quaternary ammonium component comprises or consists of one or more chloride salts of alkyldimethylbenzylammonium having a Ci₂-Ci₆ alkyl substituent, wherein the surfactant component and quaternary ammonium component are different from each other.

Preferably, the aqueous biocidal composition comprises one or more additional components, such as rheology modifiers, fragrances and colouring agents (e.g., acid dyes such as Cl acid blue 80). These optional components may be present in the composition of the invention in an amount of less than 1 % by weight, such as less than 0.5% or less than 0.05% or less than 0.005% by weight.

In a preferred embodiment, the aqueous biocidal composition further comprises components which may enhance the anti-microbial activity of the composition. These further components may include, for example, solvents (such as alcohols and/or glycol ethers) and/or alkalis (such as hydroxides, carbonates, phosphates, silicates and/or amines).

The aqueous biocidal composition of the invention may be in any suitable form, such as, for example, a liquid or foam.

According to the second aspect of the invention, there is provided a container comprising an aqueous biocidal composition according to the first aspect of the invention. The container preferably comprises a spray nozzle, which facilitates the formation of spray to distribute the aqueous biocidal composition over an area. For example, the composition of the invention may be packaged and sold in the form of a hand operated spray (e.g., a trigger spray) or a pressurised aerosol spray.

The third aspect of the invention provides a wet wipe. Thus, in one embodiment, the composition of the invention may be packaged and sold in the form of a wet wipe i.e., a cloth or fabric which is impregnated or coated with the aqueous biocidal composition of the invention.

According to the fourth aspect of the invention, there is provided a process for preparing the biocidal composition according to the first aspect of the invention, comprising forming a mixture comprising the chelating component, the surfactant component and the quaternary ammonium component.

Thus, compositions of the invention will typically be prepared by dissolving the constituent components in water to the desired concentration. In one embodiment, the constituent components are provided in the form of a concentrate comprising: i. from 5 to 20% by weight of a chelating component comprising one or more chelating agents or salts thereof; ii. from 2 to 15% by weight of a surfactant component comprising one or more surfactants selected from non-ionic surfactants, amphoteric surfactants and mixtures thereof; and iii. from 5 to 20% by weight of a quaternary ammonium component comprising one or more trialkyl benzylammonium salts having at least one C₆-C₂₀ alkyl substituent, wherein the surfactant component and quaternary ammonium component are different from each other.

The preferred features of the chelating component, the surfactant component and the quaternary ammonium component are as defined in respect of the first aspect of the invention.

It will be appreciated that the concentrate may be provided in any suitable form, such as a solid (e.g., as a powder or granulate) or as a liquid.

The concentrate may be prepared using methods well know in the art. For example, a powder or granular concentrate may be prepared by blending the constituent components to produce a homogeneous mixture. This may be achieved, for example, simply by mechanically stirring the mixture for a suitable amount of time. Alternatively, a liquid concentrate may be produced by dissolving the constituent components in water to the desired concentration.

The aqueous biocidal composition of the invention may be used as a anti-microbial agent, particularly in the biomedical and/or healthcare fields. For example, the composition may be used in the form of a single stage disinfecting solution or wash to kill or prevent the growth of microorganisms on a surface or substrate, such as in a clinical environment {e.g. surgical theatre) or a domestic environment (e.g. a kitchen work surface). In all cases, the composition may be applied to the surface to be disinfected.

Thus, the invention also contemplates the use of the aqueous biocidal composition according to the first aspect of the invention as an in vitro anti-microbial agent. For example, the composition may be used for killing, inhibiting or preventing the growth of bacteria, viruses, fungi and/or protozoa. The bacteria may be Gram-positive or Gram- negative bacteria. Thus, the bacteria may be, for example, selected from the group consisting of Mycobacteria spp. (e.g., Mycobacterium tuberculosis, Mycobacterium terrae, Mycobacterium avium or Mycobacterium Bovis), Staphylococcus spp. (e.g., methicillin-resistant Staphylococcus aureus), Clostridium spp. (e.g., Clostridium difficile) and Enterococcus spp. (e.g., vancomycin-resistant Enterococcus). In one embodiment, the bacteria are spore forming. For example, the composition of the invention may be used for killing, inhibiting or preventing the growth of spores of Clostridium difficile.

In an alternative embodiment, the composition of the invention may be used for killing, inhibiting or preventing the growth of viruses. The viruses may be selected, for example, from the group consisting of noroviruε, influenza (e.g., human or avian influenza), human immunodeficiency virus, Hepatitis B virus and Hepatitis C virus.

In another embodiment, the composition of the invention may be used for killing, inhibiting or preventing the growth of fungi. The fungi may be selected, for example, from Penicillium spp., Candida spp. (e.g., Candida albicans) and Aspergillus spp. (e.g., Aspergillus niger).

The composition of the invention can be applied to a surface or object to be disinfected using any suitable technique known in the art. For example, the composition may be sprayed onto a surface (in the form of a liquid or foam) or transferred onto a surface using, for example, an absorbent paper, cloth, sponge, mop or using the wet wipe described herein. Alternatively, objects to be disinfected may be immersed in the composition of the invention, the composition being optionally agitated. In yet another method of application, the composition may be diffused into the atmosphere of a closed area, for example, by means of diffusion/fogging equipment. Alternatively, the composition may be released into the atmosphere by using an aerosol can that is able to discharge its contents into the surrounding atmosphere in a single rapid discharge.

The following non-limiting examples illustrate the invention but do not limit its scope in any way. In the examples and throughout the specification, all percentages, parts and ratios are by weight unless indicated otherwise. It will be appreciated that the various percentage amounts of the different components that are present in the products of the invention, including any optional components, will add up to 100%.

Examples

Example 1 Comparative study of the sporicidai activity of a biocidal composition of the invention, Peracetic acid and Sodium hypochlorite (bleach) against Clostridium difficile

Samples tested

The three products tested were a biocidal composition of the invention (Composition A), a proprietary peracetic acid based formulation, and a sodium hypochlorite based product.

The exemplary biocidal composition of the invention (Composition A) had the following composition:

Preparation of inoculums

The test strain used was Clostridium difficile, NCTC 11209. An overnight culture of NCTC 11209 was grown up in fastidious anaerobe broth at 37°C and the final count ascertained by the surface drop (Miles and Misra) technique, incubated in an anaerobic jar.

Test for residual activity

A 1 ml volume of the Composition A was pipetted and spread over a defined area of 5 x 5 cm (25 cm²) and allowed to dry in a laminar flow cabinet. On the tile there were eight 5 x 5 cm² squares, each receiving 1 ml of Composition A. The squares were labelled neat 0 hrs, neat 4 hrs, neat day 7, neat day 28, -2 dilution 0 hrs, -2 dilution 4 hrs, -2 dilution day 7 and -2 dilution day 28. The same was carried out with the peracetic acid based formulation at 5%v/v dilution and the sodium hypochlorite at 10%v/v dilution. The peracetic acid product was tested at 5%v/v dilution. The product manufacturers recommend the product to be used at 3-5%, therefore the product was tested at the highest concentration. The sodium hypochlorite sample was a nominal 10% available chlorine product. This was tested at 10%v/v, which means that the final concentration of the test solution was 10000 ppm of available chlorine.

A volume of 0.1 ml of a neat Clostridium difficile suspension containing a known number of colony forming units was spread over the neat 0 hrs square on the Composition A, peracetic acid and sodium hypochlorite tile. 0.1 ml of a -2 dilution from the neat Clostridium difficile was spread on the - 2 dilution 0 hr square on the Composition A, peracetic acid and sodium hypochlorite tile. For each chemical, the tile was swabbed 5 minutes after inoculation to determine the level of surviving organisms. Each swab was then placed in 10 ml of TLTR neutralising solution (containing Tween 80, lecithin, sodium thiosulphate, ringers and maximum recovery diluent), pulverised vigorously, then plated onto blood agar plates using 0.5 ml, 0.1 ml and 10μl volumes.

The TLTR neutralising solution is the standard neutralising solution used in the HPA- WEMS laboratory for challenge and residual activity testing.

Once the inoculum had been absorbed, the plates were incubated in an anaerobic jar at 37⁰C for 18- 24 hours, and the number of colonies counted and recorded.

Four hours after the initial inoculation of the tiles, 0.1 ml of the same neat and -2 Clostridium difficile overnight suspension was spread over a new defined 5 x 5 cm (25 cm²) area, neat 4 hrs for the neat Clostridium difficile and -2 dilution 4 hrs for the -2 Clostridium difficile dilution, on each tile, and the same method as above was followed. One week later, and again at day 28, each tile was reinoculated in the same way using a fresh neat and -2 dilution Clostridium difficile suspension and the procedure previously described repeated.

A control was carried out by pipetting 0.1 ml of a neat Clostridium difficile suspension onto a defined 5 x 5 cm (25 cm²) area on a tile with no chemicals added. After 5 minutes, the tile was swabbed, placed in 10 ml of TLTR neutralising solution, pulverised vigorously, then plated onto blood agar plates and incubated in an anaerobic jar with the test plates. Results

Table 1

Recovery of Clostridium difficile from coated tiles

1 log reduction is equivalent to a 10 fold drop in numbers Table 2

Colony counts on plates

TNTC = Too numerous to count Discussion

For the zero hour time period for the three tiles inoculated with the Clostridium difficile organism, the sodium hypochlorite covered tiie produced a 3.1 log reduction in counts. The peracetic acid covered tile returned a 2.6 log reduction in counts and the tiie covered in Composition A returned a 6.8 log reduction in counts. The actual counts for each plate can be seen in Table 2 above.

After the tiles had been coated with the three chemicals for 4 hours, the microbial counts {Clostridium difficile) returned for the Composition A tile gave a 7.1 log reduction, but it had fallen for the sodium hypochlorite (a 1.4 log reduction in actual counts) and for the peracetic acid (a 1.7 log reduction in actual counts). When the tiles were re-inoculated with Clostridium difficile one week later, the Composition A tile log reduction was 5.4 log. This log reduction is lower than at the other 3 time points due to the initial Clostridium difficile inoculum being only 10⁵ colony forming units (cfu) compared to 10⁷ cfu at 0 hours, 4 hours and 28 days. The sodium hypochlorite tile had fallen to a 0.5 log reduction in counts, while the peracetic acid tile had a 0.6 log reduction in counts.

After 28 days, the log reduction, for Clostridium difficile, for the Composition A tile was 7.1 log in counts. The sodium hypochlorite overall log reduction had fallen to 0.6 log in counts while the peracetic acid overall log reduction was also 0.6 log in counts.

The control result shows that there is a 0.4 log recovery reduction using the above method when no disinfectant is added to the tile

On the tile dosed with sodium hypochlorite, it was also noticed that on the 4 hour, day 7 and day 28 testing times, the bleach had dried and left behind a grainy white crusty residue.

Conclusion

Results on day 0 after the first challenge indicate that Composition A coated onto a tile is most effective in reducing very high levels (10⁷ cfu) of Clostridium difficile within 5 minutes. The sodium hypochlorite based formulation is the next most effective, with the peracetic acid based formulation the least effective. Following re-inoculation of Clostridium difficile at various time periods, residual activity could still be demonstrated 28 days after the Composition A coating was applied to the tiles. The log reduction is slightiy lower after day 7 but this is due to a lower initial concentration of Clostridium difficile compared to the other time points. In comparison, with sodium hypochlorite the log reduction has fallen from 3.1 log to 0.6 log, and with the peracetic acid based formulation from 2.6 log to 0.6 log over this time.

Comparative Example 2

Assessment of the activity against Clostridium Difficile

Sample tested

Composition B having the following composition:

Appearance of sample

Clear blue solution

Storage conditions

Room temperature, in a dry place out of direct sunlight.

Test organisms

Clostridium difficile NCTC 11209

Test method and validation

No European Phase 2/ Step 1 test has as yet been described to establish sporicidal activity of chemical disinfectants against Clostridium difficile in the medical area. The only Phase 2/ Step 1 test published to date is EN 13704 which is designated for food, industrial, domestic and industrial areas. This test looks for a > 3 log₁₀ reduction in 60 minutes under clean conditions only. The test product 'Composition B' was tested against C. difficile spores following the test conditions described in other Phase 2/ Step 1 tests for the medical area.

Sample test concentration

5% solution in standard hard water

Contact times

1 , 5, 10, 15, and 30 minutes.

An additional contact time of 60 minutes was included to meet the mandatory test criteria.

Test temperature

2O⁰C

Interfering substance

Clean conditions - 0.03 % bovine albumin (final concentration).

Dirty conditions - 0.3 % bovine albumin (final concentration) plus 3% washed sheep erythrocytes.

Inhibition method

Dilution/neutralization

Neutralizer

Tween 80 (3Og/ L), Sodium Lauryl Sulphate (4g/ L), Lecithin (3g/ L). Tests were performed to establish the suitability of this neutralizer in inhibiting the activity of the disinfectant without being toxic to the test organisms (method described in EN 14348).

Summary of the test methods

The disinfectant was prepared in accordance with EN 13704, in sterile hard water, immediately prior to testing.

A suspension of Clostridium difficile was prepared, containing at least 10⁷ viable spores/ml. The EN 13704 test method involves mixing 1 ml of the test bacteria with 1 ml of soil (0.3% albumin or 3% albumin plus 3% sheep erythrocytes) and then adding 8 ml of test disinfectant. After the required contact time, 1 ml is removed to 9 ml of recovery broth (8ml neutralizer and 1 ml diluent). Surviving test bacteria were detected by plating onto blood agar and incubated anaerobically for 3 -5 days.

Requirement

The test requirements for EN 13704 (Phase2 Step 1 sporicidal test) is for a 3 log™ reduction in 60 minutes.

Results

Table 3

Sporicidal activity of Composition B under clean and dirty conditions

All tests were carried out in duplicate. Conclusion

Composition B does not demonstrate sporicidal activity at 20⁰C under clean (0.03% albumin) and dirty (0.3% albumin/ 0.3% sheep erythrocytes) conditions against Clostridium difficile spores.

Published EN tests for sporicidal activity have a requirement for a 3 log₁₀ reduction in 60 minutes. This was not achieved after a contact time of 60 minutes under either clean or dirty conditions; therefore Composition B failed the test criteria.

Example 3

Assessment of the activity of a biocidal composition of the invention against Clostridium Difficile

Sample tested

Biocidal composition of the invention (Composition A) having the following composition:

Comparative tests were carried out using a proprietary chlorine releasing agent at lOOOppm.

Appearance of product

Clear blue solution

Storage conditions

Room temperature, in a dry place out of direct sunlight.

Test organisms

Clostridium difficile NCTC 11209 Test method and validation

No European Phase 2/ Step 1 test has as yet been described to establish sporicidal activity of chemical disinfectants against Clostridium difficile in the medical area. The only Phase 2/Step 1 test published to date is EN 13704 which is designated for food, domestic and industrial areas. This test looks for a > 3 log₁₀ reduction in 60 minutes under clean conditions only. The test product "Composition A" was tested against C. difficile spores using the test conditions ie temperature, organic load, described in other Phase 2/ Step 1 tests for the medical area.

Contact times

5, 10, 20 and 30 minutes. An additional contact time of 60 minutes was included to meet the mandatory test criteria.

Test temperature

2O⁰C

interfering substance

Clean conditions - 0.03 % bovine albumin (final concentration).

Inhibition method

Dilution/neutralization

Neutalizer

Composition A: Tween 80 (3Og/ L), Sodium Lauryl Sulphate (4g/ L), Lecithin (3g/ L).

Chlorine releasing agent: Tween 80 30g/1 , lecithin 3g/1 , sodium thiosulphate 7.5g/1 , Histidine 1g/l, Saponin 30g/l, TSB 30g/l.

Tests were performed to establish the suitability of this neutralizer in inhibiting the activity of the disinfectant without being toxic to the test organisms (method described in EN 14348). Summary of the test method

A suspension of Clostridium difficile was prepared, containing at least 10⁷ viable spores/ml.

The EN 13704 test method involves mixing 1 m! of the test bacteria with 1 ml of soil (0.3% albumin or 3% albumin plus 3% sheep erythrocytes) and then adding 8 ml of test disinfectant. After the required contact time, 1 ml is removed to 9 ml of recovery broth (8ml neutralizer and 1 ml diluent). Surviving test bacteria were detected by plating onto blood agar and incubated anaerobically for 3 -5 days.

Requirement

Results

Table 4

Sporicidal activity of Composition A under clean and dirty conditions

All tests were carried out in duplicate. I able 5

Sporicidal activity of a chlorine releasing agent dOOOppm) under clean and dirty conditions

All tests were carried out in duplicate.

Conclusion

Tests carried out with Composition A demonstrated activity against spores of Clostridium difficile at 20⁰C under clean (0.03% albumin) and dirty (0.3% albumin/ 0.3% sheep erythrocytes) conditions.

Published EN tests for sporicidal activity i.e. EN 13704 have a requirement for a 3 logio reduction in 60 minutes. This was achieved after a contact time of 20 minutes under clean or dirty conditions; therefore Composition A fulfils the test criteria.

Example 4

Soil removal efficiency

Samples tested

A biocidal composition of the invention (Composition A) was compared with IOOOppm Chlorine and 10,000ppm Chlorine from liquid bleach (NaCIO) and bleach tablets (NaDCC). Composition A (also referred herein as Anovium) had the following composition:

Cleaning Test 1

Test Soil: Whole Milk

Test Surface Preparation: A Stilbene Derivative Optical Brightener was added to the soil (Whole milk) before it was applied to the test surfaces. The test surfaces used in this test were 38mm x 76mm glass slides. The soil was applied to 6 slides by pipette. 1 ml of soil was drawn across each test surface. The slides were then stood up and 1 ml of soil was allowed to run over the test surface to cover. The slides were then allowed to dry at room temperature.

Products under test:

Test Procedure: The test was carried out a: ambient temperature. 50ml of each test product were placed in 100m! beakers. Each siide was placed in the corresponding beaker to soak. After 3 seconds, the slides were removed from the beakers and rinsed under a running water tap at a constant flow. Each slide in turn was then dipped back into the test solution and the treated area was wiped over 3 times with a 25mm pure bristle brush and the slides rinsed again. The slides were then left to dry.

Obtaining Results: The processed slides were photographed in a dark room under UV light to detect any soil residue left behind.

Photographic Results:

It can be seen from Figure 1 that the slides cleaned with Chlorine at both IOOOppm and 10,000ppm still show a film of soil across the cleaned area. The slide cleaned with Composition A (Anovium) shows virtually no soil residue left on the cleaned area. The soil can be clearly seen on the control which was not subjected to cleaning.

Again it can be seen from Figure 2 that the slides cleaned with Chlorine show a film of soil residue, however, it can also be seen that the 10,000ppm Chlorine from NaCIO has left less residue than the IOOOppm and than the NaDCC Chlorine test slides. Composition A (Anovium) shows virtually no residue of soil left on the cleaned area. The soil can be clearly seen on the control slide which was not subjected to cleaning.

Cleaning Test 2

Test Soil: Blood#1

Test Surface Preparation: The test surfaces used in this test were 150mm x 150mm white glazed ceramic tiles. 2ml of soil was applied to an area of 50mm x 150mm via pipette and drawn across the surface of the test area. Each tile was then stood vertically to allow excess liquid to drain off. The tiles were then allowed to dry at room temperature. Products under test:

Test Procedure: The test was carried out at ambient temperature. Each test solution was sprayed onto a portion of the soiled tile using a mist sprayer (Internal reference: 125MLTK). The tiles were then left to dry.

Obtaining Results: The processed slides were photographed to enable assessment of any visual soil residue left behind.

Photographic Results:

It can be seen in comparative Figures 3 to 6 that Composition A (Anovium) gave a better clean than the other test solution. In each Chlorine source, the 10,000ppm solution gave a slightly inferior clean to the IOOOppm solution.

Cleaning Test 3

Test Soil: Blood#2

Test Surface Preparation: The test surfaces used in this test were 150mm x 150mm white glazed ceramic tiles. 1 ml of soil was applied to an area of 50mm x 150mm via pipette and drawn across the surface of the test area. Each tile was then stood vertically to allow excess liquid to drain off. The tiles were then allowed to dry at room temperature. Once dried, the tiles were stored at 4O⁰C for 15 hours. Before testing commenced the tiles were removed from 4O⁰C conditions and allowed to cool at room temperature for 30 minutes.

Products under test:

Test Procedure: The test was carried out at ambient temperature. For each test solution, 4ml was allowed to run over half of the soiled area under gravity with the tiles standing vertically. Each test surface was then rinsed under a running tap of standard speed. The slides were then allowed to dry at room temperature.

Photographic Results:

The results demonstrated by Figure 7 clearly show that Chlorine at 10,000ppm from NaDCC showed the least cleaning power. Composition A (Anovium) and 10,000ppm Chlorine from NaCIO showed to clean the best leaving behind the least quantity of soil.

Cleaning Test 4

Test Soil: Whole Egg

Test Surface Preparation: A Stilbene Derivative Optical Brightener was added to the soil (Whole Egg) before it was applied to the test surfaces. The test surfaces used in this test were 38mm x 76mm glass slides. The soil was applied to 6 slides by pipette. O.βg of soil was drawn across each test surface and the slides were allowed to dry at room temperature.

Products under test:

Test Procedure: The test was carried out at ambient temperature. 50ml of each test product were placed in 100ml beakers. Each slide was placed in the corresponding beaker to soak. After 30 seconds, the slides were removed from the beakers and rinsed under a running water tap at a constant flow. Each slide in turn was then dipped back into the test solution and the treated area was wiped over 3 times with a 25mm pure bristle brush and the slides rinsed again. The slides were then left to dry. The cleaning cycle was then repeated soaking each slide for 30 seconds, agitating with a brush and rinsing. The slides were then left to dry again at ambient temperature.

Photographic Results:

It can be seen from Figure 8 that the slides cleaned with Chlorine at IOOOppm and Composition A (Anovium) have significantly less soil remaining on the cleaned area than the slide cleaned with 10,000ppm Chlorine. The soil can be clearly seen on the control which was not subjected to cleaning.

It can be seen again from Figure 9 that the 10,000ppm Chlorine solutions had inferior clean to the IOOOppm Chlorine solution. Composition A (Anovium) showed the best clean leaving the smallest quantity of soil behind. The soil can be clearly seen on the control which was not subjected to cleaning.

Example 5

Feline Calicivirus (Human Norovirus Surrogate)

EN 14476:2005 Chemical disinfectants and antiseptics - Virucidal quantitative suspension test for chemical disinfectants and antiseptics used in human medicine - Test method and requirements (phase 2/step 1 )

Sample tested

A biocidal composition of the invention (Composition A) having the following composition:

Storage conditions Room temperature and darkness

Product diluent Sterile Hard Water

Product test concentrations 80% V/V Test Method and its validation

Method

1 part interfering substance + 1 part virus suspension + 8 parts biocide were mixed and incubated at the indicated contact temperature for the indicated contact times. Assays were validated by a cytotoxicity control, virotoxcity control and a formaldehyde internal standard.

Dilution-neutralization Neutraiizer Dulbecco's modified Eagles medium + 5% v/v foetal bovine serum at 4⁰C.

Experimental Conditions

Product diluent used Sterile Hard Water Product test concentration 80% V/V Contact times 10 minutes + 10s

5 minutes + 10s

Test temperature 2O⁰C + 1⁰C Interfering substances 0.6 g/l foetal bovine serum + 0.03%

V/V BSA.

Temperature of incubation 37⁰C + 1⁰C + 5% CO₂ Identification of virus FELINE CALICIVIRUS/CRFK CELLS

Results

Conclusion

According to EN 14476: 2005, Composition A possesses virucidal activity at 80.0% V/V against FELINE CALiCIVIRUS at 2O⁰C following 5 minutes contact under clean conditions.

Example 6

Activity against EN 14348 under 'clean' and 'dirty' conditions.

Sample tested

A biocidal composition (Composition C) having the following composition:

Product stored at room temperature in the dark.

Experimental conditions:

Product test concentrations 10% v/v Product diluent used during test Sterile hard water 300mg/kg CaCO₃ Contact time 5 min Test Temperature 20⁰C + 0.5⁰C Interfering substance As table Neutralising solution 3% Tween 80, 3% Saponin, 0.1% Histidine, 0.1 % Cysteine

Temperature of incubation 3O⁰C + VC Identification of bacteria! strains used Mycobacterium terrae ATCC 15755

Test Results

10% v/v

Count at: 0.3g/l 3 .0g/l serum / 6 .0g/l serum / 9 .0g/l serum /

Reduction bovine 3 .Oml/lerythrocytes 6 .Oml/lerythrocytes 9 .Oml/lerythrocytes in numbers serum

5 min 0 0 0 0 exposure

15 min 0 0 0 0 exposure

Vc = Viable Count.

N = Number of cfu/m! of the bacterial test suspension.

Nv = Number of cfu in bacterial suspension.

R = Reduction in viability.

Na = Number of cfu/ml in the test mixture Conclusion

According to EN 14348 Composition C when diluted 1 :10 in sterile hard water possesses satisfactory mycobactericidal activity in 5 minutes at 2O⁰C under extreme dirty conditions (9.0g/l bovine albumin / 9.0ml/l sheep erythrocytes) for the reference organism detailed.

REFERENCES

1. Larson, E. L. 1996. Antiseptics, p. 19-1-19-7, G1-G17. In R. N. Olmstad (ed.), APIC infection control & applied epidemiology: principles & practices. Mosby-Year Book, Inc., St. Louis, Mo.

2. Rutala, W. A. 1995. APIC guidelines for selection and use of disinfectants. Am. J. Infect. Control 23:313-342.

3. Block, S. S. 1991. Historical review, p. 3-17. In S. S. Block (ed.), Disinfection, terilization, and preservation, 4th ed. Lea & Febiger, Philadelphia, Pa.

4. Kuijper EJ et al. Emergence of Clostridium cf/fficZ/e-associated disease in Canada, the United States of America and Europe. Second Concept March 3^rd, 2006. Report from European CDC www.ecdc.eu.int/documents/pdf/CI dif v2.pdf

5. Sunshine RH et al. Clostridium difficile-associated disease: New challenges from an established pathogen. Cleveland Journal of Medicine (2006); 73(2): 187-97.

6. Tonna I et al. Pathogenesis and treatment of Clostridium difficile infection. Postgraduate Medical Journal (2005); 81 : 367-9.

7. McFarland LV. Alternative treatments for Clostridium difficile disease: what really works? Journal of Medical Microbiology (2005); 54: 101-11.

8. Fordtran JS. Colitis due to Clostridium difficile toxins: underdiagnosed, highly virulent, and nosocomial. Baylor University Medical Center Proceedings (2006); 19(1): 3-12.

Claims

1. An aqueous biocidal composition comprising: i. from 0.5 to 5% by weight of a chelating component comprising one or more chelating agents or salts thereof; ii. from 0.4 to 4% by weight of a surfactant component comprising one or more surfactants selected from non-ionic surfactants, amphoteric surfactants and mixtures thereof; and iii. from 0.6 to 5% by weight of a quaternary ammonium component comprising one or more trialkyl benzylammonium salts having at least one

C₆-C₂o alkyl substituent, wherein the surfactant component and quaternary ammonium component are different from each other.

2. The composition according to Claim 1 , wherein the chelating component comprises or consists of one or more chelating agents selected from the group consisting of nitrilotriacetic acid, ethylenediaminetetraacetic acid, N-(hydroxyethyl)- ethylenediaminetriacetic acid, methylglycinediacetic acid, hydroxyethyliminodiacetic acid, glutaric acid-N,N-diacetic acid, iminodisuccinic acid or salts thereof.

3. The composition according to Claim 1 or Claim 2, wherein the chelating component comprises or consists of trisodium nitrilotriacetic acid.

4. The composition according to any of the preceding claims, wherein the surfactant component comprises or consists of one or more surfactants selected from the group consisting of alkoxylated alcohols, alkylphenols, amides, amines, esters, fatty acids or glycerides; alkanolamides, amine oxides, esters, alkyl polyglucosides, betaines, glycinates, propionates, acetates and imidazolines.

5. The composition according to any of the preceding claims, wherein the surfactant component comprises or consists of one or more alcohol ethoxylates having an alkyl group with a pendant chain of the structure -(OCH₂CH₂)_mOH, wherein m is from 4 to 15.

6. The composition according to Claim 5, wherein the alkyl group is a C₄-C₂O alkyl.

7. The composition according to any of the preceding claims, wherein the quaternary ammonium component comprises or consists of one or more trialkyt benzylammonium salts having at least one Ci₂-C₁₆ alky! substituent.

8. The composition according to Claim 7, wherein the quaternary ammonium component comprises or consists of one or more chloride salts of alkyldimethylbenzyiammonium having a C₁₂-C₁₆ alkyl substituent.

9. The composition according to any of the preceding claims, wherein the chelating component constitutes from 1 to 4.5% by weight of the composition, preferably from 2 to 3.5% by weight, most preferably from 2 to 2.5% by weight of the composition.

10. The composition according to any of the preceding claims, wherein the surfactant component constitutes from 0.7 to 3% by weight of the composition, preferably from 1 to 2% by weight, most preferably from 1.25 to 1.75% by weight of the composition.

11. The composition according to any one of the preceding claims, wherein the quaternary ammonium component constitutes from 1.5 to 4% by weight of the composition, preferably from 2 to 3.5% by weight, most preferably from 2.5 to 3% by weight of the composition.

12. A container comprising an aqueous biocidal composition according to any of Claims 1 to 1 1.

13. The container according to Claim 12, wherein the container comprises a spray nozzle.

14. A wet wipe impregnated or coated with an aqueous biocidal composition according to any of Claims 1 to 11.

15. A process for the preparation of an aqueous biocidal composition according to any of Claims 1 to 11 comprising forming a mixture comprising the chelating component, the surfactant component and the quaternary ammonium component.

16. The process according to Claim 15, wherein the chelating component, the surfactant component and the quaternary ammonium component are provided in the form of a concentrate comprising: i. from 5 to 20% by weight of a chelating component comprising one or more chelating agents or salts thereof; ii. from 2 to 15% by weight of a surfactant component comprising one or more surfactants selected from non-ionic surfactants, amphoteric surfactants and mixtures thereof; and iii. from 5 to 20% by weight of a quaternary ammonium component comprising one or more trialkyl benzylammonium salts having at least one

17. Use of an aqueous biocidal composition according to any of Claims 1 to 11 as an in vitro anti-microbial agent.

18. The use according to Claim 17, for killing, inhibiting or preventing the growth of bacteria, viruses, fungi and/or protozoa.

19. The use according to Claim 18, wherein the bacteria are selected from the group consisting of Mycobacteria spp., Staphylococcus spp., Clostridium spp. and Enterococcus spp.

20. The use according to Claim 19, wherein the bacteria are selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium terrae, Mycobacterium avium, Mycobacterium Bovis, methicillin-resistant Staphylococcus aureus, Clostridium difficile and vancomycin-resistant Enterococcus.

21. The use according to any of Claims 18 to 20, wherein the bacteria are spore forming.

22. The use according to Claim 17 or Claim 18, for killing, inhibiting or preventing the growth of spores of Clostridium difficile.

23. The use according to Claim 18, wherein the virus is selected from the group consisting of norovirus, influenza, human immunodeficiency virus, Hepatitis B virus and Hepatitis C.