WO2008122773A2

WO2008122773A2 - Biocidal composition

Info

Publication number: WO2008122773A2
Application number: PCT/GB2008/001179
Authority: WO
Inventors: Frances Anderson
Original assignee: Alexander Ross Holdings Limited
Priority date: 2007-04-05
Filing date: 2008-04-04
Publication date: 2008-10-16
Also published as: WO2008122773A3; GB0706739D0

Abstract

An composition which comprises a chelating agent such, as nitrilotriacetic acid or its derivatives, at least one biocide such as a quaternary ammonium compounds and a metasilicate. The composition can be use as a disinfectant for work or hard surfaces and/or body parts.

Description

Biocidal composition

The invention relates to a biocidal composition which is advantageously effective against infections and in particular nosocomial infections.

Background of invention

It has been estimated that as many as one in ten patients become infected by nosocomial (or hospital acquired) infections. Such infections are generally defined as those which occur as a result of hospital or hospital- type treatment but are secondary to the patient's original condition. Such infections are considered to be nosocomial if they appear either 48 hours or later from admittance to hospital or within 30 days of discharge therefrom. Nosocomial infections are becoming both more prevalent and extensive as their resistance to a number of antibiotics increases. Additional reasons for the increase in incidence of such nosocomial infections is due to the high concentration and close proximity of patients; the fact that such patients have suppressed or depressed immune systems; the increased use of outpatient treatment means that inpatients are sicker on average; and the fact that medical staff move from one patient to another thereby providing a means for spreading pathogens.

Thorough hand washing by all medical personnel before and after each patient contact is one of the most effective ways to combat transfer of such infections. Further, washing hands as promptly and thoroughly as possible between patient contacts and after contact with blood, body fluids, secretions, excretions, and equipment or articles contaminated by them is an important component of control and isolation procedures. In addition, surface cleaning is also essential and has to be carried out regularly and thoroughly with an appropriate anti-infectious agent. More careful and considered use of anti-microbial agents, such as antibiotics, is also considered vital to try to prevent further resistance to the small number of active antibiotics still available. Alternatively, investigations into new antibiotics are required. In the meantime, in order to bridge the gap, in case resistance to all known antibiotics becomes a reality and before new ones have been sufficiently developed, investigations have been ongoing to provide biocidal compositions which would be effective against such nosocomial infections.

A great variety of biocidal formulations are known. US 3,507,796 describes an antibacterial composition which comprises a chelating agent, a zwitterionic detergent and an alkaline buffering salt. The alkaline buffering can be chosen in a wide group of compounds such as carbonates, bicarbonates, orthophosphates, tripolyphosphate, metaphosphates, and silicates. Sodium carbonate and sodium trypolyphosphate are the buffering agents preferred.

The general use of silicates as buffering or pH controlling agents is generally known and is mentioned for example in US 2006/0128585, US4597887, US 6,184,195 B1 or US 2005/0055787. However sodium metasilicate pentahydrate is seldomly used for biocidal composition actually in the market.

However, these existing biocidal compositions have still been shown to be insufficient in the fight against such infections as is demonstrated by the current Methicillin-resistant Staphylococcus aureus (MRSA) epidemic in UK hospitals which shows that there is still a need for more efficient compositions effective against, in particular, nosocomial infections. Such currently available biocidal compositions also have a number of disadvantages such as their high level of toxicity (due mainly to the fact that they are alcohol based and generally include a high level of surfactant), their low biodegradability and their limited specificity. Such limited specificity results in their efficiency often being limited to a particular type of microbe (i.e. bacteria, viruses, spores, fungi) or even to a particular genus or species.

Object of the Invention

Accordingly there is a need for a biocidal composition which would obviate or mitigate at least one if not more of the above-mentioned disadvantages and provide a biocidal composition which is effective when used against a wide range of microbes of different types, including bacteria, virus, fungi and spores and/or different species thereof.

Summary of the Invention

According to the present invention there is provided a biocidal composition comprising a chelating agent, at least one bactericidal biocide, a surfactant and metasilicate.

The chelating agent is preferably chosen from nitrilotriacetic acid (NTA), and its derivatives, including but not limited to, its salts, its esters, phosphonate or phosphonic acids thereof. The calcium, copper, iron and sodium salts of NTA are preferred, amongst which the trisodic form of NTA, or trisodium nitrilotriacetate, is particularly preferred.

Alternative chelating compounds which are similar or equivalent in structure or characteristics thereto are also encompassed by the invention. Amongst these compounds are ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), diethylene triamine pentaacetic acid (DTPA), their salts, esters and their phosphonates or phosphonic acids (NTMP, EDTMP and DTPMP). NTMP, EDTMP and DTPMP are structural analogues to the known aminopolycarboxylates NTA, EDTA and DTPA and are effective chelating agents. The major disadvantage of such structural analogues is their relatively low biodegradability. EGTA has a very high affinity of calcium ions and DTPA can chelate heavy metal ions very efficiently as well, making both these chemical compounds possible alternatives to NTA, though NTA is preferred.

Whilst the concentrations (% by weight) of both chelating agent and the biocide can be altered as desired it is preferred according to one embodiment that their respective concentrations be at least about 5%.

Alternatively the concentration (% by weight) of the chelating agent can advantageously range from 3 to 10 %, and more preferably 4 to 8%.

The biocide should have anti-bacterial properties and can be chosen amongst a wide variety of compounds. However the bactericide is optimally selected from a group of quaternary ammonium compounds and/or alkyl dimethyl betaines and a mixture thereof. Of course, more than one biocidal agent may be suitable for inclusion in the composition. Also, if more than one biocide is included in the composition, it is preferred that they have a different mode of action such as, for example, different efficacies for killing different forms of living organisms.

Advantageously the concentration (% by weight) of the biocide ranges from 5-35%. However the optimal concentration depends upon the potency of the particular biocide chosen. For example the concentration of benzalkonium chloride in a ready-to-use composition advantageously ranges from 5-25%, more preferably from 5-10%.

Sodium metasilicate, Na₂SiO₃ (or Disodium trioxosilicate) and in particular its pentahydrate form Na₂SiO₃ ^#5H₂O are particularly preferred.

The concentration (wt %) of the metasilicate may range from 2-15%. For example the concentration of metasilicate in a ready-to-use composition advantageously ranges from 3 to 15%, more preferably from 4 to 11% and even more preferably from 5 to 8 wt%. Advantageously the concentration of metasilicate ranges from 3 to 6 wt%.

Additionally a surfactant may also be included in the composition but preferably at a low concentration, for example less than 15 wt%.

According to a particularly preferred aspect of the invention the composition is an aqueous (or water based) composition. In such a case the chelating agent and/or the biocide are advantageously water-soluble.

According to another aspect of the invention the composition does not include any substantial amount of alcohol and, in particular, does not include alcohol(s) as a solvent.

Surfactants are preferably chosen by their ability to be soluble in water and for their compatibility with the other components of the composition. They can be selected from the group consisting of anionic, cationic, non-ionic and ampholytic surfactants. Non-ionic surfactants are preferred as they can show a weak bactericidal effect, especially on Gram positive bacteria. Common surfactants used are: sodium dodecylsulfate (SDS), sodium deoxycholate (DOC), lauroylsarcosine sodium salt, lauryldimethylamine oxide (LSAO), cetyltrimethylammonium bromide (CTAB), sulfosuccinate sodium salt, Craymul 7050 (an acrylic polymer dispersion), Licomet F3 (a fluorinated anionic surfactant containing butyl glycol), liquid sodium lauryl ether sulphate (SLES), Synperonic 9110 ( a fatty alcohol ethoxylate) , Surfactant UN90/90 (C₉-Cn alcohol ethoxylate with 9 MEO), Amphoteric 400 (an amphoteric surfactant), Synperonic LF/RA 280 (an alcohol alkoxylate surfactant), Synperonic LF/RA 30 (an alcohol alkoxylate surfactant), Synperonic 9110:85% (a fatty alcohol ethoxylate 85%) and

Surfactant CDE/G (coconut diethanalamide). Synperonic 9110, Surfactant UN90/90, Synperonic LF/RA 280 and Synperonic LF/RA 30 are preferred. Advantageously the chelating agent on the one hand and metasilicate on the other hand are present in the composition in a ratio of 1 :1 (% by weight).

According to a preferred embodiment of the invention the composition is an aqueous biocidal composition which comprises a water-soluble chelating agent in the form of NTA, a water-soluble bactericide in the form of benzalkonium chloride together with sodium metasilicate, preferably pentahydrate sodium metasilicate.

According to a preferred embodiment of the invention the composition further comprises a surfactant in the form of Synperonic UN 90/90, C₉-Cn alcohol ethoxylate with 9MEO.

The invention also extends to the use of the composition of the invention as a biocide or a disinfectant, particularly for use as a liquid disinfectant for body parts (e.g. hand wash) or as a surface cleaner. The composition of the invention is particularly useful in hospitals or other communal buildings where hygiene is important such as care homes and schools.

According to the invention it is also disclosed as a method of disinfecting and/or cleaning which comprises the step of applying the composition of the invention onto a surface and/or body part to be disinfected.

Detailed Description of the Invention

Whilst the concentrations of each component of the formulation of the invention can be modified as desired, the following concentrations (% by weight) are preferred: Biocide: 3%, 5%, 7%, 10%, 11 % 15%, 23%, 25%, 27%, 35%. Chelating Agent: 3%, 4%, 5%, 6%, 7%, 10% Metasilicate: 3%, 4%, 5%, 6%, 7%, 10%. Advantageously the chelating agent is chosen from nitrilotriacetic acid (NTA), C₆H₉NO₆, its salts or esters thereof. NTA is a chemical compound which is generally used as a chelating agent and which forms coordination compounds with metal ions (chelates) such as Ca²⁺, Cu²⁺Or Fe³⁺. NTA is easily biodegradable and is almost completely removed during wastewater treatment. Most preferred is the NTA in the form of its salts, of which the trisodic form of NTA orTrisodium Nitrilotriacetate is most preferred. Other variants of NTA such as nitrilotriacetic acid sodium salt, nitrilotriacetic acid monosodium salt, nitrilotriacetic acid disodium salt, nitrilotriacetic acid disodium salt monohydrate, nitrilotriacetic acid trisodium salt monohydrate could be used as suitable alternatives to NTA.

According to the present invention it was found that such a composition combining a chelating compound, such as NTA (or one of its derivatives such as salts, esters, phosphates etc), at least one biocide and a metasilicate is surprisingly effective and has a wide biocidal properties spectrum that extends to bacteria, viruses, spores and fungi.

Advantageously the composition of the invention is a high level or "Ievel2" disinfectant according to European standard.

The biocide itself can be chosen form a range of compounds.

According to the Biocidal Products Directive 98/8/EC (BPD), the classification of biocides can be broken down into 23 product types (i.e. application categories), with several comprising different subgroups. Whilst the present invention is not limited to any particular type of biocide, it is particularly preferred that the composition of the invention to belong to disinfectants and general biocidal products of the classification and more particularly to Product-type one of this main group, that is human hygiene biocidal products.

However, it is envisaged that other product-types are encompassed by the invention including the following sub-groups: ^* Product-type 2: Private area and public health area disinfectants and other biocidal products

^* Product-type 3: Veterinary hygiene biocidal products

^* Product-type 4: Food and feed area disinfectants * Product-type 5: Drinking water disinfectants

Of course the present invention is not limited to the use of only one biocide but can in fact include one of more together with the metasilicate and chelating agent in the composition.

A preferred biocide(s) is to be found amongst the bactericides and comprises certain types of quaternary ammonium compounds (quats) which are well known for their biocidal activity. For example, Benzalkonium chloride (alkyl dimethyl benzyl ammonium chloride) is a mixture of alkylbenzyl dimethylammonium chlorides of various alkyl chain lengths. It is commonly used as an antiseptic. The greatest bactericidal activity is associated with the Ci₂-Ci₄ alkyl derivatives. It has been considered one of the safest synthetic biocides known, and has a long history of efficacious use. Benzalkonium chloride is readily soluble in water, alcohol, and acetone. However, formulation requires great care as benzalkonium can be inactivated by certain organic compounds, including soap, and must not be mixed with anionic surfactants.

Standard concentrates are manufactured as 50% and 80% w/w solutions, and sold under trade names such as BC50, BC80, BAC50, BAC80, etc. The 50% solution is purely aqueous, while more concentrated solutions require incorporation of rheology modifiers (alcohols, polyethylene glycols, etc.) to prevent increases in viscosity or gel formation under low temperature conditions. Because of ease of handling and manufacture, BAC50 formulations are generally preferred in the composition of the invention. Benzalkonium chloride solutions are rapidly acting anti-infectious agents with a moderately long duration of action. They are in fact active against bacteria and also some viruses, fungi, and spores. However, on the whole, bacterial spores have been shown to be resistant to such compounds. Solutions are bacteriostatic or bactericidal according to their concentration. Gram-positive bacteria are generally more susceptible than gram-negative. Activity is not greatly affected by pH, but increases substantially at higher temperatures and prolonged exposure times.

Metasilicates are used as degreasing agents and as buffering agents.

Also they are known as having some anti-viral properties. According to a preferred embodiment of the invention, water soluble sodium metasilicates Na₂SiO₃ , and especially sodium metasilicate pentahydrate, are preferably used in the composition.

In a further embodiment a surfactant may be included in the aqueous composition but preferably at a low concentration such as less than 10%. Preferably the ratio of chelating agent to biocide in the composition remains unchanged. Use of such a surfactant can increase the efficacy of the aqueous biocidal composition. The usual concentration of surfactant in biocidal compositions for human hygiene is 15-20%. As such the composition of the present invention provides a biocidal composition having no or a comparatively reduced concentration in surfactant for a comparably high efficacy thereby presenting advantages for the environment and in terms of efficiency and costs.

The biocidal composition of the present invention has shown biocidal activity without the presence of a surfactant. However, some surfactants can be added in order to improve the efficacy of the biocidal composition of the invention. It should be noted that the concentration of surfactant is distinctly lower than the one used in prior art biocidal compositions. A surfactant which has been used is available under the trade name Surfac.UN90/90 is C_9-H alcohol ethoxylate with 9 MEO at 90.000%. Other surfactants are Surfac CDG, Surfac B4, Surfac UN65/95, Surfac TKPP. Depending upon the particular application of the composition, the concentration of the various components of the invention may vary depending upon the particular ingredient used.

However, a concentrated composition can advantageously be formulated for ease of handling and storage. The concentrate composition can then be diluted with water to obtain the following preferred concentration (% by weight) of compounds:

BAC50 35%, UN90 11%, Metso 5%, NTA 5%,

BAC50 27%, UN90 11%, Metso 5%, NTA 5% BAC50 23%, UN90 12%, Metso 5%, NTA 8%,

BAC50 11%, UN90 18%, Metso 5%, NTA 5%, or BAC50 5%, UN90 7.5%, Metso 5%, NTA 5%.

Wherein "BAC50" is a Benzalkonium Chloride 50.000% C12-C14 Alkyl Benzyl Dimethyl Ammonium Chloride from the company Univar in 50% water, "UN90" is the UN90/90 surfactant above mentioned, "Metso" is sodium metasilicate pentahydrate and "NTA" refers to the trisodic form of NTA. The same notations are used herein below:

The composition of the invention, and in particular the specific formulations above listed, can be used as a liquid disinfectant which can be used externally for the washing of human skin (for example as a spray or wipes or as a hand wash).

For such uses the composition of the invention advantageously includes wash ingredients such as: Versene (tetrasodium EDTA), Surfac CDE/G (coconut diethanolamide >90% and diθthanolamide 1 -5%), Empipearl XA 200/X ( Ci2-Ci₈ alkyl amido propyl dimethylamine betaines sodium, Ci₀-Ci₆ alkyl ethoxy sulphate), Nipagen M (methyl-4-hydroxybenzoate), Nipasol N (propyl-4-hydroxybenzoate).

The composition of the invention, and in particular the specific formulations above listed, can also be used in a wide range of applications such as a surface cleaner in wipes, sprays, foggers (automatic room sprayer), sanitizer etc, or as a fabric conditioner. It can used in hospitals, medical or dental practices, in particular to sanitize medical instruments, in nursing and care homes and in schools.

For fabric conditioner an additional surfactant such as Sepantex VK90 (alkylester ammonium isopropanol) can be advantageously used.

As a sanitizer the following additional compounds can be used: water, glycerin, Nipaguard PDU (a mixture of propylene glycol, diazolidinyl urea, methyl- 4-hydroxybenzoate and propyl-4-hydroxybenzoate) and Polysorbate 20 (polyoxyethylene (20) sorbiton monolaureate.)

The composition of the invention can be formulated to have a safe and alkaline pH of around 10-11. The pH of the invention is maintained by controlling the NTA and the Metso. A pH of 10-11 is advantageous for safe handling and storage of the product. The alkaline pH is also effective as a boost to the cleaning properties of the product.

In order that those skilled in the art can more fully appreciate the aspects of the invention, the following examples are set forth, which examples are given solely for the purposes of illustration, and should not be considered as expressing limitations unless so set forth in the appended claims.

Example 1 : Test Formulation I A biocidal composition according to the invention has been tested against various infectious organisms and it has been demonstrated that this composition is a high level, or "level 2" disinfectant. The composition tested in the following experiments has the following formulation which will be referred to hereinafter as Formulation I

- BAC 50 5 wt%,

- Un 90 7.5 wt%,

- Metso 5 wt%, and

- NTA 5 wt%. The solvent being water.

This formulation was kept at 4°C and in darkness before being diluted with hard water for the tests to take place.

The effectiveness of the formulation as described above was tested by carrying out standard suspension tests. Such tests follow a recognised approach to disinfectant validation which is the CEN (European Committees for normalisation) 216 work programme and provides a systematic approach to the testing of disinfectants. From this a number of BS EN methods have been derived for which there are clearly defined pass criteria. In particular the BS EN methods are standard suspension tests that are phase 2 /step 1 tests. This means that the test is appropriate to the intended use of the biocidal product. Suspension tests are set up with 1 part microorganism suspension, the count of microorganisms to be within defined limits, 1 part interfering substance and 8 parts biocidal agent. Contact between the biocide and microorganism/interfering substance mixture is maintained for a defined time at a defined temperature (20^QC). The biocide is then neutralised appropriately. Controls ensure correct neutralisation of the product has occurred and internally validate the test. Tests require that the biocidal agent reduces the microbiological viability by 5 Logi₀ (bacteria) or 4 Logi₀ (Fungi) or 3 Logi₀ (spores) or 3 or 4 Logi₀ (viruses). Experiment 1 - Clostridium Difficile under BS EN 1276-1997

Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas (phase 2, step 1 )

Test Method and its validation

The method used is filtration-neutralization. Neutralizer - Lecithin 3g/l, Polysorbate 80 30g/l, sodium thiosulphate 5g/l, L-histidine 1g/l, phosphate buffer 0.0025 mol/l, sterilized by autoclave.

Experimental conditions

Product diluent used: sterile synthetic hard water. Product test concentrations: 10% v/v; 20% v/v 80%v/v

Appearance product dilutions: clear

Contact time: t = 5 min + 10 s

Test temperature: 20°C ± 1°C

Interfering substance: 0.3 g/l bovine albumin Stability of mixture: No precipitation

Temperature of incubation: 37°C ± 1°C

Identification of strains: Clostridium difficile NCTC 11209.

The results are shown in Table 1

Conclusion

According to testing carried out under conditions specified in EN 1276, Formulation I possesses bactericidal activity at a concentration of 10.00% V/V after five minutes at 20°C under clean conditions (0.3 g/L bovine serum albumin) for referenced strains Clostridium difficile NCTC 11209. Table 1

Experiment 2 - Community Acquired - Methicillin Resistant Staphylococcus Aureus under BS EN 1276-1997

Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas (phase 2, step 1)

Test Method and its validation The method used is filtration-neutralization. Neutralizer - Lecithin 3g/l,

Polysorbate 80 30g/l, sodium thiosulphate 5g/l, L-histidine 1g/l, phosphate buffer 0.0025 mol/l, sterilized by autoclave.

Experimental conditions Product diluent used: sterile synthetic hard water.

Product test concentrations: 5% v/v; 10% v/v 80%v/v

Appearance product dilutions: clear

Contact time: t = 5 min ± 10 s

Test temperature: 2O⁰C ± 1°C Interfering substance: 0.3 g/l bovine albumin

Stability of mixture: No precipitation

Temperature of incubation: 37°C ± 1°C

Identification of strains: CA-MRSA SMRSARL 02.6225.E

The test results are shown in Table 2

Conditions

According to testing carried out under conditions specified in EN 1276 Formulation I possesses bactericidal activity at a concentration of 5.00% V/V after five minutes at 20°C under clean conditions (0.3 g/L bovine serum albumin) for referenced strains community acquired methicillin resistant staphylococcus aureus strain SMRSARL 02.6225. E. Table 2 en

Experiment 3 - Test Report under BS EN 1040-1997

Chemical disinfectants and antiseptics - Suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas (phase 1 )

Test Method and its validation

Experimental conditions

Product diluent used: sterile synthetic hard water.

Product test concentrations: 20% WV; 50% WV 80% WV Appearance product dilutions: clear

Contact time: t = 5 min ± 10 s

Test temperature: 20⁰C ± 1°C

Interfering substance: 0.3 g/l bovine albumin

Stability of mixture: No precipitation Temperature of incubation: 37°C ± 1 °C

Identification of strains: Pseυdomonas aeruginosa ATCC 15442

Staphylococcus aureus ATCC 6538

The results are shown in Table 3

Conclusion

According to EN 1040 (1997), Formulation I possesses bactericidal activity for the referenced strains Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus ATCC 6538 at the working concentration (20% WV as tested). In order to qualify the product as an antiseptic and/or chemical disinfectant for a defined purpose, it will be evaluated using additional standard tests which are appropriate to its intended use. Table 3

Test Organism Viable ¹ Count

Bacterial Bacterial Neutralizer Dilution-

Test Suspension toxicity neutralization

Suspension Nv (see control (Nx) control (Ny)

(See 5.4.1.4)* A.2)* (see (see A.4.1.2)*

(N) A.4.1.2)*

Pseudomonas 3.3x10⁸ 1.1 x10³ 9.0x10¹ 8.9x10¹

Aeruginosa

ATCC 15442

Staphylococcus 4.0x10⁸ 1.7 x 10³ 8.9x10¹ 1.3 x 10² aureus

ATCC 6538

For the two strains tested:

N is between 1.5 x 10⁸ and 5.0 x 10 ⁸ cfu/ml;

Nv is between 6.0 ) <10²and3x10^; cfu/ml;

Nx is equal or greater than 0.05 Nv

Ny is equal or greater than 0.05 Nv

The neutralization i s validated with I the neutralizer tested for the test concentrations of 80% V/V of product as received and for the two J strains tested.

^*The references above refer to British standard EN 1040-1997

Experiment 4 - Test Report under BS EN 1275-1997

Chemical disinfectants and antiseptics - Basic fungicidal activity (phase 1)

Test Method and its validation

Experimental conditions

Product diluent used: sterile synthetic hard water. Product test concentrations: 20% WV; 50% WV 80% WV

Appearance product dilutions: clear

Contact time: t = 5 min ± 10 s

Test temperature: 20°C ± 1°C

Temperature of incubation: 37°C ± 1 °C

Identification of strains: Candida albicans ATCC 10231

Aspergillus niger ATCC 16404 The results are shown in Table 4.

Conclusion

According to EN 1275, Formulation I possesses fungicidal activity for the referenced strains Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404 at the working concentration (20% WV as tested). In order to qualify the product as an antiseptic and/or chemical disinfectant for a defined purpose, it will be evaluated using additional standard tests which are appropriate to its intended use.

Table 4

*The references above refer to British standard EN 1275-1997

Experiment 5 - Test Report under BS EN 1276-1997

Test Method and its validation

The method used is filtration-neutralization. Neutralizer - Lecithin 3g/l, Polysorbate 80 30g/l, sodium thiosulphate 5g/l, L-histidine 1g/l, phosphate buffer 0.0025 mol/l, sterilized by autoclave. Experimental conditions

Product diluent used: sterile synthetic hard water.

Product test concentrations: 20% W/V; 50% VWV 80% WV

Appearance product dilutions: clear Contact time: t = 5 min ± 10 s

Test temperature: 2O⁰C ± 1⁰C

Interfering substance: 0.3 g/l bovine albumin

Stability of mixture: No precipitation

Temperature of incubation: 37°C ± 1 °C Identification of strains: Pseudomonas aeruginosa ATCC 15442

Escherichia coli ATCC 10536 Staphylococcus aureus ATCC 6538 Enterococcus hirae ATCC 8043

The results are shown in Table 5

Conclusion

According to testing carried out under conditions specified in EN 1276, Formulation I possesses bactericidal activity at a concentration of 20.00% V/V after five minutes at 20°C under clean conditions (0.3 g/L bovine serum albumin) for referenced strains Pseudomonas aeruginosa ATCC 15442, Escherichia coli ATCC 10536, Staphylococcus aureus ATCC 6538 and Enterococcus hirae ATCC 8043.

Table 5 ro

CO

Experiment 6 - Test Report under BS EN 1650 -1998

Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of fungicidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas (phase 2, step 1 )

Test Method and its validation

Experimental conditions

Product diluent used: sterile synthetic hard water. Product test concentrations: 20% v/v; 50% v/v 80%v/v

Appearance product dilutions: clear

Contact time: t = 5 min ± 10 s

Test temperature: 20°C ± 1°C

Temperature of incubation: 37°C ± 1°C

Identification of strains: Candida albuminv ATCC 10231.

Aspergillus niger ATCC 16404

The results are shown in Table 6

Conclusion

According to EN 1650, Formulation I possesses fungicidal activity for the referenced strains Candida albicans ATCC 10231 and Aspergilus niger ATCC 16404 at the working concentration (20% WV as tested). Table 6

Ni

Experiment 7 - Test Report under BS EN 13704:2002

Chemical disinfectants - Quantitative suspension test for the evaluation of sporicidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas (phase 2, step 1)

This test measures the efficacy of the biocidal agent against bacterial endospores. These are generally considered to be the most resistant microbial structures to biocides. Passing this test enables the biocide to be considered to be a "high level" disinfectant. Eight parts biocide are contacted with 1 part interfering substance and 1 part spore suspension of Bacillus subtilis spores at a defined concentration. The contact time is 60 minutes at 20^eC.

Test Method and its validation

Experimental conditions

Product diluent used: sterile synthetic hard water.

Product test concentrations: 20% v/v; 50% v/v; 80%v/v

Appearance product dilutions: clear

Contact time: t = 5 min ± 10 s Test temperature: 20°C ± 1 °C

Interfering substance: 0.3 g/l bovine albumin

Stability of mixture: No precipitation

Temperature of incubation: 37°C ± 1°C

Identification of strains: Bacillus subtilis ATCC 6633.

The results are shown in Table 7 Conclusion

According to EN 13704, Formulation I possesses sporicidal activity for the referenced strains Bacillus subtilis ATCC 6633 at the concentration (20% VA/ as tested).

Table 7

oo

Experiment 8 - Test Report Standard Test method for Efficacy of Antimicrobial Agents against Viruses in Suspension

Bovine Viral Diarrhoea Virus (Hepatitis C Virus surrogate)

Test Method and its validation

The method used is filtration-neutralization. Neutralizer - Dulbecco's modified Eagles medium + 5% v/v Foetal bovine serum.

Experimental conditions

Product diluent used: sterile hard water.

Product test concentrations: 2% V/V; 10% V/V; 80% V/V

Appearance product dilutions: colourless, clear product solution

Contact time: t = 5 min ± 10 s Test temperature: 2O⁰C ± 1 °C

Interfering substance: 0.6 g/l foetal bovine serum

Stability of mixture: Precipitate absent throughout the test

Temperature of incubation: 37°C ± 1°C

Identification of virus: Bovine Viral Diarrhoea Virus 1 (ATCC VR- 1422). BT cells.

The results are shown in Table 8.

Conclusion Formulation I₁ when diluted at 2.00% (V/V) in hard water, possesses virucidal activity in five minutes at 20°C under clean conditions (06 g/l protein as foetal bovine serum) for Bovine viral diarrhoea virus- (Hepatitis C virus surrogate) Table 8

Suspension test results for the efficacy of Formulation I disinfectant against Bovine Viral Diarrhoea

^* This result is due to residual cytotoxicity of the disinfectant at this concentration of disinfectant.

10 Comments:

Virucidal activity is based on a reduction in virus viability of a minimum of 4 LOGi₀. This therefore represents a minimum value. Stock BVDV: 1.48 x 10⁸

15

Residual efficacy control: minimum positive cpe for BVDV at 10^"4 dilution of Formulation 1 disinfectant after neutralisation. Experiment 9 - Test Report Standard Test method for Efficacy of Antimicrobial Agents against Viruses in Suspension

Feline Calicivirus (Human Norovirus surrogate)

Test Method and its validation

The method used is dilution-neutralization. Neutralizer - Dulbecco's modified Eagles medium + 5% v/v Foetal bovine serum.

Experimental conditions

Product diluent used: sterile hard water.

Product test concentrations: 2% V/V; 10% V/V; 80%V/V

Appearance product dilutions: colourless, clear product solution

Contact time: t = 5 min ± 10 s Test temperature: 20°C ± 1 °C

Interfering substance: 0.6 g/l foetal bovine serum

Stability of mixture: Precipitate absent throughout the test

Temperature of incubation: 37°C ± 1°C

Identification of virus: Feline calicivirus (FCV)/FEA cells (human norovirus surrogate).

The results are shown in Table 9.

Conclusion Formulation I, when diluted at 2.00% (V/V) in hard water, possesses virucidal activity in five minutes at 20°C under clean conditions (0,6 g/l protein as foetal bovine serum) for feline calicivirus (human norovirus surrogate) Table 9

Suspension test results for the efficacy of Formulation 1 Disinfectant against Feline Calicivirus

10 Comments:

Activity based on a reduction in virus viability of a minimum of 4

LOG 10-

This therefore represents a minimum value.

15 Stock FCV: 2.14 x 10⁹.

Residual efficacy control: minimum positive cytopathic effect (cpe) for

FCV at 10-4 dilution of Formulation I disinfectant after neutralisation.

Experiment 10 - Vancomycin Resistant Enterococcus under BS EN 0 1276-1997 Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas (phase 2, step 1 )

Test Method and its validation

Experimental conditions

Product diluent used - sterile synthetic hard water.

Product test concentrations: 5% VWV; 10% VWV; 80% WV Appearance product dilutions: clear

Contact time: t = 5 min ± 10 s

Test temperature: 20°C ± 1⁰C

Interfering substance: 0.3 g/l bovine albumin

Identification of strains: Vancomycin Resistant Enterococcus NCTC

12201

Conclusion According to testing carried out under conditions specified in EN 1276-1997, Formulation I possesses bactericidal activity at a concentration of 5.00% WV after five minutes at 20°C under clean conditions (0.3 g/L bovine serum albumin) for referenced strains Vancomycin Resistant Enterococcus NCTC 12201. Table 10

CO

Experiment 11 - Methicicillin Resistant Staphylococcus Aureus 15 and 16 under BSEN 1276 (1997)

Test Method and its validation The method used is filtration-neutralization. Neturalizer: Lecithin 3g/l,

Polysorbate 80 30g/l, sodium thiosulphate 5g/l, L-histidine 1g/l, phosphate buffer 0.0025mol/l, sterilized by autoclave

Experimental conditions Product diluent used: sterile synthetic hard water.

Product test concentrations: 10% VWV; 20% VWV; 80%V/V

Appearance product dilutions: clear

Contact time: t = 5 min ± 10 s

Test temperature: 20°C ± 1 °C Interfering substance: 0.3 g/l bovine albumin

Stability of mixture: No Precipitation

Temperature of incubation: 37°C ± 1°C

Identification of MRSA Strain:

EMRSA 15:CC22 (SMRL No. 00.9521. M) EMRSA 16: CC30 (SMRL No. 00.9523. R)

The results are shown in Table 11.

Although each country have their own names for MRSA, clones MLST Typing data which groups MRSA into Clonal Complexes (CC) made up of related Sequence Types (ST) is the widely adopted international nomenclature. The majority of MRSA infections worldwide are caused by MRSA clones which fall within five Clonal Complexes. A representative of each of these Clonal Complexes was used in this test. EMRSA 15 is the dominant MRSA in the UK, followed by EMRSA 16. In Scotland these two clones account for 72% and 20%, respectively, of all MRSA referred to the Scottish MRSA reference laboratory. EMRSA-16 and other clones with Clonal Complex 30 are found worldwide. They are the second commonest MRSA in the USA (9%) and are a common cause of community acquired MRSA both the USA and Australia.

According to testing carried out under condition specified in EN 1276 (1997) Formulation I possesses bactericidal activity at a concentration of 10.00% V/V after 5 minutes at 20⁰C under clean conditions (0,3 g/L bovine serum albumin) for referenced strains EMRSA 15:CC22 (SMRL No. 00.9521. M), EMRSA 16:CC30 (SMRL No. 00.9523.R).

Table 11

OO

Example 2 - Efficacy of Test Formulation 2 against Acinetobacter Baumannii under clean and dirty conditions

Test Formulation 2 (by wt %) is as follows: BAC 50 27%

Surfactant U N90 11%

Metasilicate Pentahydrate 4.5% NTA 4.5%

The test suspension is prepared and an organic load (Bovine Serum

Albumin) or interfering substance is added to the suspension at different concentrations to simulate both clean and dirty conditions. This is performed to investigate how well the agent will perform under both conditions.

Test Organisms Acinetobacter baumannii ATCC 19606

Media and Reagents

Minimal Salts Medium - MSM Buffered Peptone Water containing 5% Polysorbate 80-BUP5

Buffered peptone water -BUP

Tryptone soya broth - TSB

Tryptone soya agar - TSA

Sterile 0.45μm syringe filters Bovine Serum Albumin

Purified Water Formulation 2 was made up fresh on day of testing and diluted 10OmIs concentrated solution and 1OmIs tap water.

Preparation of Inoculum

Acinetobacter baumannii was sub cultured from a stock culture into 10ml TSB and incubated at 36°C ± 1 °C for 18-24 hours. After incubation, the culture was centrifuged at 4500rpm for 3 minutes, the supernatant discarded and the pellet resuspended in MSM.

Preparation of Bovine Serum Albumin (Interfering Substance)

Two solutions of BSA were prepared to simulate the clean and dirty conditions. For clean conditions, dissolve 0.3g BSA into 100ml purified water, and filter sterilized using a 0.45μm syringe filter. This gives a final concentration of BSA in the test procedure of 0.3g/litre. For dirty conditions dissolve 3g BSA into 100ml purified water, and filter sterilized using a 0.45μm syringe filter. This gives a final concentration of BSA in the test procedure of 3g/litre.

Procedure

Suspension Test in the Presence of Bovine Serum Albumin (BSA)

Dispense 9ml of the diluted formulation 2 into a sterile vial, and allow to equilibrate in a water bat set at 20⁰C ± 1⁰C. Add 1 ml of the filtered BSA for clean conditions. Introduce 0.1 ml of the inoculum into the test suspension and mix thoroughly. Withdraw 1 ml aliquots at 5, 15, 30 and 60 minutes and transfer to 200ml volume of BUP5 to give a 1/200 dilution. Maintain at 20⁰C ± 1⁰C for 5 minutes. After 5 minutes, remove a 1 ml aliquot from the 1/200 dilution, and use to prepare 4 further tenfold dilutions in 9ml volumes of BUP5. 1 ml aliquots from each dilution are dispensed into Petri dishes and 15-2OmI TSA added. All plates were incubated at 36°C ± 1⁰C

Control Count

To determine the number of viable organisms inoculated into the test material, a control count was carried out for the test organism. A 10ml volume of BUP was challenged with 0.1ml of the inoculum and 1 ml was transferred to a 200ml volume of BUP. Maintain at 20⁰C ± 1⁰C for 5 minutes. After 5 minutes, remove a 1 ml aliquot from the 1/200 dilution and use to prepare 4 further tenfold dilutions in 9ml volumes of BUP. Remove 1 ml from each dilution and use to prepare pour plates with TSA.

Validation

Validation is performed to ensure adequate neutralization of the active occurs at the first product dilution.

The inoculum is diluted in MSM to give a count of 1000-10000 cfu ml^'1, allow to equilibrate in a water bat set at 20⁰C ± 1⁰C. 2mls of the equilibrated inoculum is added, to give approximately 10-100 organisms per ml, to 200ml volume of BUP5 plus 1 ml of the interfering substance for clean conditions and mix thoroughly. 1 ml of the diluted cleaning solution is added to the inoculated diluent and the resulting solution is shaken to mix. The temperature is maintained at 20⁰C ± 1⁰C and 1 ml is removed after 5 minutes and again after 30 minutes and used to prepare pour plates using TSA. The procedure is repeated with a 200 ml volume of both BUP and BUP5 without product for the validation control counts.

The above test and validation method was repeated using the interfering substance for dirty conditions. Each run was then repeated a further two times for each condition. All plates were incubated at 36°± 1°C for 48 hours and the number of colony forming units were counted.

The above test and validation method was repeated using the interfering substance for dirty conditions. Each run was then repeated a further two times for each condition.

Results are shown in Tables A and B. The following acceptance criterion was a log reduction of 5 log.

Conclusion

From the results it can be seen that for the cleaning agent good biocidal activity was seen within 5 minutes against Acinetobacter baumanniπor both clean and dirty conditions, meeting the acceptance criteria as recommended in the British and European Standards BS EN 1276:1997.

Table A - Results for Cleaning Solution Testing - Clean Conditions

Table B - Results for Cleaning Solution Testing - Dirty Conditions

Example 3 - Comparative test of the combined and individual effects of the components of Formulation I

Table 12 shows the efficacy of thirteen solutions of a varied mixture of each of the chemicals used in the composition against E.Coli and Poliovirus. The aim of this experiment was to obtain a better understanding of the contribution of each of the individual raw materials to the composition and to investigate the possibility of a synergetic effect.

Table 12 shows the results of this experiment.

Table 12

- No Biocidal activity + Intermediate activity ++ Biocidai activity

The following conclusions can be taken from Table 11.

BAC 50 No effect

BAC 50 + Surfactant Bactericidal Effect

Metas (i.e. sodium metasilicate pentahydrate) Viral Effect

NTA No Effect

NTA + BAC 50 Intermediate viral activity

Results for sample 2 shows Metas to have a high level of viral activity. Results for the surfactant alone (sample 3) showed a bactericidal effect. Surprisingly, with no Metas present, (sample 7), the formulation has an intermediate viral activity with NTA + BAC 50. As expected sample 10 showed BAC 50 + Surfactant to have a bactericidal effect.

The combination of ingredients exhibits an unexpected efficacy against bacteria, viruses and spores. Even though certain individual components contained in the composition of this invention are capable of bactericidal action by themselves, the combination of these ingredients, results in a synergetic effect which is active against spores and viruses as well.

The particular advantage of compositions according to the invention such as Formulation I (Sample 13) lies in the fact that as it is water based, biodegradable, non corrosive and safe to work with in any environment, having no adverse human impact, unlike a lot of competitor products which being alcohol based are flammable. At worst they are only a minor irritant to the skin. More importantly whilst being biodegradable and water based they do show substantial biocidal activity over a broad specificity as evidenced under the tests outlined in the examples above.

Claims

1. A biocidal composition comprising a chelating agent, metasilicate and at least one anti-bacterial biocide.

2. The composition of claim 1 wherein said metasilicate is a disodium trioxosilicate.

3. The composition of claim 2 wherein said disodium trioxosilicate is disodium trioxosilicate pentahydrate.

4. The composition of any one of claims 1 to 3, wherein the chelating agent is chosen from nitrilotriacetic acid (NTA) or its derivatives.

5. The composition of any one of claims 1 to 3, wherein the chelating agent is chosen from the group consisting of ethylene glycol tetraacetic acid, diethylene triamine pentaacetic acid and their salts and esters thereof.

6. The composition as claimed in any one of the preceding claims wherein the concentration of said metasilicate ranges from 3 to 6 wt%.

7. The aqueous composition as claimed in any one of claims 1 to 5, wherein the concentration of said biocide agent ranges from 5 to 35 wt%.

8. The composition as claimed in any one of the preceding claims wherein the concentration of said chelating agent ranges from 4 to 8 wt%.

9. The composition as claimed in any one of the preceding claims wherein said biocide is chosen in the group consisting of quaternary ammonium compounds and alkyl dimethyl betaines and mixtures thereof.

10. The composition as claimed in claim 9, wherein said biocide is chosen from the group consisting of quaternary ammonium compounds.

11. The composition as claimed in any one of the preceding claims wherein said composition further comprises a surfactant.

12. The composition as claimed in claim 11 , wherein the concentration of said surfactant in the composition is less than 10 wt%.

13. The composition as claimed in claim 12, wherein said surfactant is chosen from the group consisting of Synperonic 9110, Surfactant UN90, Synperonic LF/RA 280 and Synperonic LF/RA 30.

14. The composition as claimed in any one of claims 11 to 13, wherein said surfactant is Synperonic UN90/90.

15. The composition as claimed in any one of the preceding claims, wherein said composition is an aqueous composition.

16. Use of the composition as claimed in any one of the preceding claims as a biocidal composition.

17. Method of disinfecting which comprises the step of applying a composition as claimed in any one of claims 1 to 15 to a surface and/or a body part to disinfect.