GB2378136A

GB2378136A - Disinfectant formulation and method for determining sporicidal activity

Info

Publication number: GB2378136A
Application number: GB0118833A
Authority: GB
Inventors: Thomas David White; John Richard Salvage
Original assignee: Individual
Current assignee: Individual
Priority date: 2001-08-01
Filing date: 2001-08-01
Publication date: 2003-02-05
Also published as: GB0118833D0

Abstract

A formulation suitable for use in disinfecting apparatus or areas susceptible to contamination by spores, which formulation comprises at least one aqueous alcohol and a sporicidally effective amount of at least one sporicidal agent, said at least one aqueous alcohol being present in said formulation in an amount in excess of said at least one sporicidal agent. A method for determining the activity of a sample as a sporicidal agent, which method includes: <SL> <LI>(a) permitting the sample to contact a surface having a predetermined number of spores thereon; <LI>(b) neutralising the surface so as to remove the sample from the surface; and <LI>(c) monitoring the number of spores remaining on the surface in step (b) so as to determine the activity of the sample as a sporicidal agent. </SL>

Description

Disinfectant Formulation & Method for determining Disinfectant Properties Lsin Propertie The present invention is concerned with a disinfectant formulation and, more particularly, a sporicidal disinfectant formulation for use in disinfecting apparatus or areas susceptible to contamination by spores, such as for example, hospital theatres, pharmaceutical preparation areas or the like. The invention is also concerned with a method for determining the sporicidal activity of a sample.

It is well known that alcohols have disinfectant properties and ethanol in particular has been used as a disinfectant for centuries in such applications where it is desirable or essential to exclude bacteria. In other words, alcohols work as bactericidal agents. It is also generally known that alcohols do not kill spores. Aqueous alcohols such as isopropyl alcohol (iso-propanol or IPA) and denatured ethanol, such as industrial methylated spirit (IMS) are widely used as disinfectants in hospital, pharmaceutical and veterinary (clean room) environments. Such aqueous alcohols typically have a concentration of at least 70% by volume of alcohol in water.

It is common practice to use alcohol to wash down apparatus such as medical equipment, dental instruments, containers or packaging, which are to be used in a"clean room" environment, such as a hospital or veterinary theatre, a pharmacy or biological laboratory or the like. Work areas, such as walls, surfaces (such as surfaces of work or preparation areas) and the like in such clean room environments may also be washed down or disinfected with alcohol. Such washing substantially kills bacteria present on such apparatus, surfaces or the like. However, spores already present on such apparatus or work areas are not

killed by the alcohol. This is particularly disadvantageous and may lead to human or animal infection (especially during an operation) or spoilage of drug production.

It is known to irradiate alcohol prior to the use thereof in washing medical apparatus or areas as described above, in order to kill spores present in the alcohol before irradiation. Washing such apparatus or areas with irradiated alcohol does not, however, kill spores already present on such apparatus or areas. The initial step of irradiating the alcohol is generally only beneficial because it may prevent further spores being washed onto

such apparatus or areas (thereby reducing the risk of spore Lu-L L.. L. L j -e-- contamination), but it does not lead to eradication of spores already present on such apparatus or areas.

It is, therefore, a purpose of the present invention to alleviate the above mentioned problems and in particular to provide an effective means of disinfecting apparatus or areas present in a clean room environment.

According to a first aspect of the present invention, there is provided a formulation suitable for use in disinfecting apparatus or areas susceptible to contamination by spores, which formulation comprises at least one aqueous alcohol and a sporicidally effective amount of at least one sporicidal agent, said at least one aqueous alcohol being present in said formulation in an amount in excess of said at least one sporicidal agent.

According to a second aspect of the present invention, there is provided a method of making a formulation suitable for use in disinfecting apparatus or areas susceptible to contamination by spores, which method comprises blending at

least one aqueous alcohol with at least one sporicidal agent, the at least one aqueous alcohol and at least one sporicidal agent being blended in amounts whereby a resulting formulation according to the present invention comprises the at least one aqueous alcohol being present in an amount in excess of said at least one sporicidal agent and said at least one sporicidal agent being present in a sporicidally effective amount.

According to a third aspect of the present invention, there is provided a method of disinfecting apparatus or areas susceptible to contamination by spores, which method comprises washing said apparatus or areas with a formulation according to the present invention substantially as herein before described, said formulation comprising at least one aqueous alcohol and a sporicidally effective amount of at least one sporicidal agent, said at least one aqueous alcohol being present in said formulation in an amount in excess of said sporicidal agent.

According to a fourth aspect of the present invention, there is provided use of a formulation in disinfecting apparatus or areas susceptible to contamination by spores, which formulation comprises at least one aqueous alcohol and a sporicidally effective amount of at least one sporicidal agent, said at least one aqueous alcohol being present in said formulation in an amount in excess of said sporicidal agent.

A significant advantage of a formulation according to the invention is that such a formulation can possess sporicidal properties (as well as bactericidal properties), such that the formulation can kill spores already present on apparatus or work areas prior to washing.

The wording"apparatus or areas susceptible to contamination by spores" as used herein includes in particular medical or veterinary apparatus or work surfaces. Such medical or veterinary apparatus can typically be medical (including dental) or veterinary equipment, instruments, containers, packaging or the like of the type generally employed in hospital, doctor or veterinary treatment areas, hospital or veterinary theatres, medical or veterinary laboratories, pharmacy preparation areas and the like. Such work surfaces are generally those present in medical (including dental) or veterinary work environments, such as work surfaces present in hospital, doctor or veterinary treatment areas, hospital or veterinary theatres, medical or veterinary laboratories, pharmacy preparation areas and the like. The wording "apparatus or areas susceptible to contamination by spores" as used herein may of course also include any other apparatus or areas requiring a high level of cleanliness, although a disinfectant formulation according to the present invention is particularly suitable for use in a medical or veterinary environment substantially as herein before described.

It is preferred that an aqueous alcohol employed in a formulation according to the present invention comprises a volatile alcohol, typically an alcohol having 1 to 4 carbon atoms. Typically a formulation according to the present invention comprises at least one aqueous alcohol comprising any of isopropanol, ethanol or methanol. Preferably an aqueous alcohol employed in a formulation according to the present invention comprises isopropanol or ethanol.

Typically the aqueous alcohol comprises an aqueous solution of a volatile alcohol as referred to above present at a concentration in the range of 30 to 99.9% by volume, typically 40 to 99.9% by volume.

Suitable sporicidal agents for use in a formulation according to the present invention may include, for example, chlorine dioxide, hydrogen peroxide, glutaraldehyde, formaldehyde, peracetic acid or the like, preferably in the form of aqueous solution. It is particularly preferred to use a fast acting sporicidal agent which is active in the time it takes for the aqueous alcohol to volatilise. An example of a particularly suitable sporicidal agent is a sporicidal agent comprising chlorine dioxide.

When chlorine dioxide is employed in a sporicidal agent according to the present invention, the chlorine dioxide is preferably used in conjunction with a surfactant, such as a quaternary ammonium surfactant or the like. Examples of suitable sporicidal agents containing chlorine dioxide and surfactants are disclosed in US Patent 4073888. A particularly suitable chlorine dioxide sporicidal agent for use in a formulation according to the present invention is available under the trade mark"Cryocide".

A formulation according to the present invention typically comprises the at least one sporicidal agent present in an amount of not more than about 25% by weight of the formulation. Preferably the at least one sporicidal agent is present in an amount in the range of 0. 1 to 20% by weight. Advantageously, two or more sporicidal agents may be present in a formulation according to the present invention.

It is preferred that the at least one sporicidal agent is blended with the at least one aqueous alcohol in the form of an aqueous solution. The sporicidal agent may be such as to kill nearly all spores within a pre-determined time

duration (that is, in the"contact time"). Advantageously, a sporicidal agent employed in a formulation according to the present invention can kill at least 90% (preferably at least 99%) of spores within a time of about ten minutes typically, at about room temperature (a time which is comparable to that of evaporation of the aqueous alcohol).

Advantageously, the sporicidal agent may be activated by amino acids in the cell walls of the spores being attacked.

The"contact time"of a formulation according to the present invention generally refers to the time duration in which the formulation may kill substantially all the spores. Desirably, the contact time of the formulation is less than or comparable to the drying time of the formulation as described above and is typically not more than about one minute.

The sporicidal performance of a formulation according to the present invention is dependent upon the sporicidal agent's ability to kill spores. In order to enhance sporicidal performance, it is advantageous that a sporicidal agent employed by the present invention is "active"and capable of killing substantially all spores present in selected treatment areas within the drying time of the formulation (which is typically less than about one minute).

There are a number of methods for evaluating the properties of sporicidal agents. However, known methods are disadvantageous as reproducibility is often unacceptable.

It is therefore a further aim of the present invention to provide a method for testing the sporicidal activity of a sample which is reliable, repeatable, easy to use and quantative.

According to yet a further aspect of the present invention, there is provided a method for determining the activity of a sample as a sporicidal agent, which method includes: (a) permitting said sample to contact a surface having a predetermined number of spores thereon ; (b) neutralising said surface so as to remove said sample from said surface ; and (c) monitoring the number of spores remaining on said surface in step (b) so as to determine the activity of the sample as a sporicidal agent.

The method can be used free of any interfering chemicals known or unknown. These may include hard water, BSA, surfactants or chemical products commonly found in the specific use area for a particular sporicide.

A sporicidal agent is defined as any chemical product or blend, in one or more part additions, that is said to kill spores while still in the spore form. The sporicidal activity is a level of lolo3 reduction in viable spore count after a suitable contact period under clean conditions @ 20 C.

Typically, the spores are permitted to incubate after neutralising step (b) and prior to monitoring step (c).

The incubation is typically for 24 to 72 hours (such as about 48 hours) at a temperature in the range about 35 C to 390C (such as 37 C).

The sample is typically in contact with the surface in step (a) for up to about 2 hours, preferably 1 hour.

The surface is typically neutralised in step (b) by

flushing with a wash buffer. It is further preferred that surface is further rinsed with distilled water.

It is preferred that the surface in step (a) has about 10, 000 spores on the surface. The surface is typically a membrane or the like.

The wash buffer typically includes 0. 15% soya lecithin, 1.0% Tween, 1.0% sodium thiosulphate, made up in distilled water.

Advantageously, the method may be used in clean conditions or dirty conditions. Dirty conditions can be defined as

conditions formed by the additions of interfering c or,'u-*L t.-*L on sLorme'u UY t~. Lie auu-LU-Lc) ns L-4L n +L-. e r-P e r,-' substances that may be encountered in a practical situation in the field of use of the product being tested. These may include: hard water, protein, surfactants etc. and should be defined by type and quantity at the results reporting stage. Clean conditions can be defined as conditions whereby no interfering substances are present; distilled sterile water only is used.

It is preferred that the method is performed two or more times for each sample.

Preferably a control (such as a water control) is performed for each sample.

The method typically provides: details of source, type and strain reference of test organisms; contact time for disinfectant and temperature; concentration of disinfect and type/product name; interfering substance and concentrations used plus effect

on active concentration; starting spore concentration, finishing and reduction; validation reports.

This aspect of the present invention will now be described with reference to the following examples which are by way of example only.

EXAMPLES A spore suspension was prepared as follows to establish the initial concentration of the stock spore suspension (5.5), 24 hours before the test, aseptically open 1 vial of stock culture (5.5) and serially dilute a 1ml aliquot with distilled water (5. 1). Plate out on TSA (5.2) and incubate at 370C for 24 hours +/-1 hour.

Once the concentration has been calculated dilute the remainder of the vial to a concentration of 1000 spores/ml with distilled water (5. 1). This will be the working culture used during test.

EXAMPLE 1- (Clean conditions) 1.1 Using sterile forceps place a membrane on the vacuum manifold and lock on the lid.

1.2 Add 10ml of working culture (7.1) to the membrane and slowly filter through.

1.3 Overlay the spores with 10ml of test product and leave for 1 hour +/-1 minute.

1.4 After 1 hour +/-1 minute, slowly filter the test product through the membrane.

1.5 Add 400ml of wash buffer (5.3) to the membrane, slowly filter through.

1.6 Add 200ml distilled water (5.1) to the membrane and

slowly filter through.

1.7 Remove manifold lid and using sterile forceps place membrane on TSA plate (5.2).

1.8 Incubate plate for 48 hours +/- 1 hour at 37 C+/-1 C.

1.9 Count all colonies present.

EXAMPLE 3- (Dirty Conditions) 2.1 Carry out step 1.1 and 1.2 as outlined in Example 1.

2.2 Overlay the spores with 9ml of test product and 1ml of interfering substance (dissolve 0.30g of bovine albumin serum (BSA) in 100ml distilled water).

Interfering substance concentration in test 0. 3g/l (BSA).

2.3 Carry out steps 1.4 to 1.9 EXAMPLE 3- (Water Control) A water control should be run with each test batch, steps 1.1 to 1.9 (excluding step 1.5).

The water control should show confluent growth indicating the methodology has no inhibitory effect on the test organism.

EXAMPLE 4- (Neutraliser Inhibitor Control) A water control should be run with each test batch, steps 1.1 to 1.9.

The wash buffer inhibitor control should show confluent growth indicating the neutraliser has no inhibitory effect on the test organism.

EXAMPLE 5A- (Neutraliser Inactivator Control)

A test control should be run with each test batch, steps 1.1 to 1.9 (excluding 1.2).

Remove membrane and transfer to sterile buffer peptone water and incubate overnight at 30 C.

If buffer peptone water is clear of growth after incubation reinoculate with 5ul of diluted test spore suspension (100 spores/ml) and incubate overnight @ 30 C. After incubation check for growth.

EXAMPLE 5B - (Neutraliser Inactivator Control) A test control should be run with each test batch, steps 1.1 to 1.9 (excluding 1.2). Carry out step 1.2 between 1.6 and 1.7.

The wash buffer inactivator control Test SA should show positive growth in the buffer peptone water. Test 5B should shown confluent growth both indicating the neutraliser has stopped the disinfectant activity.

Claims

CLAIMS 1. A formulation suitable for use in disinfecting apparatus or areas susceptible to contamination by spores, which formulation comprises at least one aqueous alcohol and a sporicidally effective amount of at least one sporicidal agent, said at least one aqueous alcohol being present in said formulation in an amount in excess of said at least one sporicidal agent.
2. A formulation according to claim 1, wherein the aqueous alcohol comprises a volatile alcohol.
3. A formulation according to claim 2, wherein the volatile alcohol has 1 to 4 carbon atoms.
4. A formulation according to any preceding claim wherein the aqueous alcohol comprises isopropanol, ethanol or methanol.
5. A formulation according to any preceding claim, wherein the aqueous alcohol comprises isopropanol or ethanol.
6. A formulation according to any preceding claim, wherein the aqueous alcohol comprises an aqueous solution which includes a volatile alcohol present in the range of 30 to 99% by volume of the formulation.
7. A formulation according to claim 6, wherein the volatile alcohol is present in the range 40 to 99% by volume.

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8. A formulation according to any preceding claim, wherein the sporicidal agent includes chlorine dioxide, hydrogen peroxide, glutaraldehyde, formaldehyde, peracetic acid or the like, preferably in the form of aqueous solution.
9. A formulation according to any preceding claim, wherein the sporicidal agent is a fast acting sporicidal agent which is active in the time it takes for the aqueous alcohol to volatilise.
10. A formulation according to any preceding claim, wherein the sporicidal agent is chlorine dioxide used in conjunction with a surfactant.
11. A formulation according to claim 10, wherein the surfactant is a quaternary ammonium surfactant.
12. A formulation according to any preceding claim, wherein the sporicidal agent is a chlorine dioxide sporicidal agent commercially available under the trade mark"Cryocide".
13. A formulation according to any preceding claim, wherein the sporicidal agent is present in an amount not more than about 25% by weight of the formulation, preferably in an amount in the range of 0.1 to 20% by weight.
14. A formulation according to any preceding claim which comprises two or more sporicidal agents.
15. A formulation according to any preceding claim, wherein the sporicidal agent is blended with the at least one alcohol in the form of an aqueous solution.

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16. A formulation according to any preceding claim, wherein the sporicidal agent is capable of killing at least 90% (preferably at least 99%) of spores within a time of approximately one minute, typically at about room temperature.
17. A formulation according to any preceding claim, wherein the sporicidal agent is activated by amino acids present in cell walls of the spores being attacked.
18. A formulation according to any preceding claim, wherein the sporicidal agent is capable of killing substantially all spores present in a selected treatment area within the drying time of the formulation.
19. A method of making a formulation suitable for use in disinfecting apparatus or areas susceptible to contamination by spores, which method comprises blending at least one aqueous alcohol with at least one sporicidal agent, the at least one aqueous alcohol and at least one sporicidal agent being blended in amounts whereby a resulting formulation comprises the at least one aqueous alcohol being present in an amount in excess of said at least one sporicidal agent and said at least one sporicidal agent being present in a sporicidally effective amount.
20. A method according to claim 19, wherein the aqueous alcohol comprises a volatile alcohol (preferably having 1 to 4 carbon atoms).

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21. A method according to claim 19 or 20, wherein the aqueous alcohol comprises isopropanol, ethanol or methanol (preferably isopropanol or ethanol).
22. A method according to any of claims 19 to 21, wherein the aqueous alcohol comprises an aqueous solution which includes a volatile alcohol present in the range of 30 to 99% by volume (such as 40 to 99% by volume).
23. A method according to any of claims 19 to 22, wherein the sporicidal agent includes chlorine dioxide, hydrogen peroxide, glutaraldehyde, formaldehyde, peracetic acid or the like, preferably in the form of aqueous solution.
24. A method according to any of claims 19 to 23, wherein the sporicidal agent is a fast acting sporicidal agent which is active in the time it takes for the aqueous alcohol to volatilise.
25. A method according to any of claims 19 to 24, wherein the sporicidal agent is chlorine dioxide used in conjunction with a surfactant (such as a quaternary ammonium surfactant).
26. A method according to any of claims 19 to 25, wherein the sporicidal agent is present in an amount not more than about 25% by weight of the formulation, preferably in the range 0.1 to 20% by weight.
27. A method according to any of claims 19 to 26, wherein the sporicidal agent is blended with the at least one alcohol in the form of an aqueous solution.

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28. A method of disinfecting apparatus or areas susceptible to contamination by spores, which method comprises washing said apparatus or areas with a formulation according to the present invention substantially as herein before described, said formulation comprising at least one aqueous alcohol and a sporicidally effective amount of at least one sporicidal agent, said at least one aqueous alcohol being present in said formulation in an amount in excess of said sporicidal agent.
29. Use of a formulation in disinfecting apparatus or areas susceptible to contamination by spores, which formulation comprises at least one aqueous alcohol and a sporicidally effective amount of at least one sporicidal agent, said at least one aqueous alcohol being present in said formulation in an amount of excess of said sporicidal agent.
30. A method for determining the activity of a sample as a sporicidal agent, which method includes: (a) permitting said sample to contact a surface having a predetermined number of spores thereon; (b) neutralising said surface so as to remove said sample from said surface; and (c) monitoring the number of spores remaining on said surface in step (b) so as to determine the activity of the sample as a sporicidal agent.
31. A method according to claim 30, wherein the spores are permitted to incubate after neutralising step (b) and prior to monitoring step (c).
32. A method according to claim 31, wherein the spores are

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permitted to incubate for 24 to 72 hours (such as about 48 hours).
33. A method according to claim 31 or 32, wherein the spores are permitted to incubate at a temperature in the range about 350C to 390C (such as 37 C).
34. A method according to any of claims 30 to 33, wherein the sample is in contact with the surface in step (a) for up to about 2 hours, preferably 1 hour.
35. A method according to any of claims 30 to 34, wherein the surface is typically neutralised in step (b) by flushing with a wash buffer (which typically includes 0. 15% soya lecithin, 1. 0% Tween, 1. 0% sodium thiosulphate, made up in distilled water).
36. A method according to any of claims 30 to 35, wherein the surface is rinsed with distilled water after said neutralisation in step (b).
37. A method according to any of claims 30 to 36, wherein about 10,000 spores are present on the surface.
38. A method according to any of claims 30 to 37, wherein the surface is a membrane or the like.
39. A method for determining the activity of a sample as a sporicidal agent substantially as described herein with reference to the examples.