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Publication numberWO2006012693 A1
Publication typeApplication
Application numberPCT/AU2005/001161
Publication date9 Feb 2006
Filing date4 Aug 2005
Priority date5 Aug 2004
Publication numberPCT/2005/1161, PCT/AU/2005/001161, PCT/AU/2005/01161, PCT/AU/5/001161, PCT/AU/5/01161, PCT/AU2005/001161, PCT/AU2005/01161, PCT/AU2005001161, PCT/AU200501161, PCT/AU5/001161, PCT/AU5/01161, PCT/AU5001161, PCT/AU501161, WO 2006/012693 A1, WO 2006012693 A1, WO 2006012693A1, WO-A1-2006012693, WO2006/012693A1, WO2006012693 A1, WO2006012693A1
InventorsRobert John Goldsworthy
ApplicantGreat Southern Coatings Group Pty Ltd
Export CitationBiBTeX, EndNote, RefMan
External Links: Patentscope, Espacenet
Biocidal compositions and a biocidal composition delivery system
WO 2006012693 A1
Abstract
A composition, method of application and delivery system of the composition according to the invention to deliver the biocide by sprinkler into a closed environment (such as a room) in the event of a biological attack, such as by bomb or letter or the like. The system includes a vessel (1) containing biocide and another vessel (2) containing compressed air. Each vessel is connected via a discrete plumbing conduit, (3) and (4) respectively, to a series of ejector/atomiser heads (5). Delivery of the contents of the vessels to the conduits is electrically controlled by two solenoid valves, (12) and (13) respectively. A method of manufacturing a biocidal composition using colloids and surfactants with high pH (9 - 15) to buffer the lower pH of certain active ingredients (2 - 7) which allows the surfactants and colloids to act as hyper-wetting agents and allowing the actives to be delivered more efficiently.
Claims  (OCR text may contain errors)
Claims:
1. A biocidal composition comprising: a biocide; and a surfactant
2. A biocidal composition according to claim 1 wherein the biocidal composition comprises less than 10% active constituents.
3. A biocidal composition according to claim 1 or 2 wherein the surfactant includes a colloidal surfactant,
4. A biocidal composition according to claim 3 wherein the colloidal surfactant has a pH within the range of 9 to 15,
5. A biocidal composition according to claim 1, 2, 3 or 4 wherein the composition has a small amount of active biocide.
6. A biocidal composition according to claim 5 wherein the small amount of active biocide is ghitaraldehyde.
7. A biocidal composition according to any one of the preceding claims wherein the biocidal composition comprises:
Glutaraldehyde 0%-50% of the biocide;
Octhilinone 0%-50% of the biocide; Surfactant 0%-40% of the surfactant; and
Colloids 1%-40% of the surfactant.
8. A biocidal composition according to according to any one of the preceding claims wherein the biocidal composition comprises: Glutaraldehyde 3%:5%;
Octhilinone 0-2%;
Surfactant 0%-2.5%;
Colloids l%-3%; Water 1%-3%; and
Inerts 90%-92%.
9. A method of decontaminating a surface exposed to a bio-agent such as Anthrax (Bacillus anthracis) and Golden Staph (Staphylococcus aureus), the method including: delivering to the sυrface(s) exposed to the bio-agent, a two-part biocidal composition comprising at least: in a first part a quaternary ammonium compound, octhilinone, and glutaraldehyde; and in a second part a colloidal surfactant, wherein the number of viable spores of said bio-agent treated with the biocidal composition is substantially reduced after a short exposure period to the biocidal composition, corresponding to a log reduction of about less than or equal to 7.
10. A biocidal composition effective for decontamination of surfaces exposed to a bio-agent such as Anthrax (Bacillus anthracis) and Golden Staph (Staphylococcus aureus), the composition including at least: a) glutaraldehyde, octhilinone, and a quaternary ammonium compound; and b) a surfactant wherein the composition is effective to substantially reduce spores of the bio- agent corresponding to a log reduction in excess of about 7.
11. A biocidal composition according to claim 10, wherein the surfactant is
R colloid having a pH value in the range of 9 to 15 to act as a buffer for the components in part a).
12, A biocidal composition according to claim 10 or 11, wherein the glutaraldehyde is present in an amount of less than 10%w/v of part a).
13. A delivery system for a biocidal composition comprising: a biocide reservoir/source operably connected to a plurality of spray nozzles; a compressed air reservoir/source operably connected to the plurality of spray nozzles; a first valve controlling the delivery of biocide from the S reservoir/source; a second valve controlling the delivery of compressed air from the reservoir/source; and a controller, operable to control the opening of the valves.
0 14. A delivery system according to claim 13 wherein the surfactant includes a colloidal surfactant.
15. A delivery system according to claim 13 or 14 wherein the biocidal composition comprises Less than 10% active constituents, 5
16. A delivery system according to claim 13, 14 or 15 wherein the biocidal composition comprises;
Glutaraldehyde 0%-50%;
Octbilinone 0%-5Q%; 0 Surfactant 0%-40%;
Colloids l%-40%.
17. A delivery system according to claim 13, 14 or 15 wherein the biocidal comprises: 5 Glutaraldehyde 3%-5%;
Octbilinone 0-2%;
Surfactant 0%-2.5%;
Colloids l%-3%;
Water l%-3%; 0 Inerts 90%-92%.
18. A delivery system according to any one of claims 13 to 17 wherein the system includes one or more pressure switches attached to a bomb-protection film which activates the controller when the film is stretched by the blast of a bomb or similar device.
19. A delivery system according to claim any one of claims 13 to 18 wherein the activation of the controller also acts to close down any air- conditioning plant associated with the system.
20. A delivery system according to claim any one of claims 13 to 19 wherein the system includes a manual switch to activate the controller.
21. A delivery system according to claim any one of claims 13 to 20 wherein the system includes a manual mechanical override device configured to activate a third valve and a fourth valve which allows the delivery of the biocide and compressed air, respectively, to the plurality of spray nozzles,
22. A biocidal composition having a two-part formulation that, in use, is diluted with water or other inert ingredients at a ratio of 1 : 1 : 18 (one part A to one part B to 18 parts diluent) wherein part A consists of the active biocides and part B consists of surfactants and colloidal agents.
23. A biocidal composition according to claim 22 wherein part A comprises a mix of 80% - 100% glutaraldehyde with 0% - 20% octhilinone.
24. A biocidal composition according to claim 22 or 23 wherein part B comprises a mix of 50% colloidal surfactant with 50% water.
25. A method of manufacturing a biocidal composition for use as including the steps of: mixing part A comprising a mix of 80% - 100% glutaraldehyde with 0% - 20% octhilinone; mixing part B comprising a mix of 50% colloidal surfactant with 50% water; mix 1 part of part A with equal part of part B with 18 parts of inert ingredient, e.g. water.
26. A method of manufacturing a biocidal composition of claim 25 wherein the step of mixing of part A is undertaken for at least 30 seconds,
27. A method of manufacturing a biocidal composition of claim 25 or 26 wherein the step of mixing of part B is undertaken by adding water then adding the surfactant and mixing gently for at least 20 seconds,
28. A method of manufacturing a biocidal composition of claim 25 or 26 or 27 wherein the step of mixing the inert(s) with part A and with part B, comprises adding 18 litres of water, then 1 litre of Part A, then 1 litre of Part B, and mixing/stirring well for at least S seconds,
29. A method of manufacturing a biocidal composition, of any one of claims 25 to 28 wherein the method uses colloids and surfactants with high pH (9 - 15) to buffer the lower pH of certain active ingredients (2 - 7) which allows the surfactants aαd colloids to act as hyper-wetting agents and allowing the actives to be delivered more efficiently.
30. A delivery system for a biocidal composition substantially as hereinbefore described with reference to the drawing.
Description  (OCR text may contain errors)

Biocidal compositions and a biocidal composition delivery system

Field of Invention: This invention relates to biological decontamination. In. particulate the invention relates to a biological decontamiaator which is effective against bio-agents such, as Anthrax (Bacillus anthracis) and Golden Staph (Staphylococcus aureus). The field of use could be expanded to include chemical decontamination and the product is also fungicidal, virucidal, sporicidal & tuberculocidal

Background of the Invention!

There are many other products in the field. However most products on the market can take up to 12 hours or more to be effective against anthrax and other sporicidal bacteria. Some products use actives, such as chlorine, which are corrosive and therefore make the overall product corrosive. Some products ate not based on bio¬ degradable materials therefore the products are non-biodegradable and noπ- environmentally friendly. Some products are only available in a "ready to use" form, which makes them difficult to transport and store. Still further some products contain large amounts of active ingredients such as 50%+ and therefore can be highly toxic. Other products will not penetrate porous surfaces and thus, e.g., will not be able to travel through soil. Some products are not water based, they are solvent based or surfactant based. Some products are not practical either due to their delivery system or other machinery needed for the product to work.

Therefore there is a need for an effective, relatively safe and environmentally friendly bioeidal formula that is effective against Anthrax and other bio-agents, that is easy to transport and store, easy to use and is relatively safe.

Spme products can only decontaminate one particle form of contaminate e.g, bacteria, chemical agents or spores. The present invention seeks to meet this need or at least to provide the public with a viable alternative Summary of the Invention:

In accordance with the invention there is provided a biocidal composition comprising; a biocide; and a surfactant

The invention also provides a delivery system for a biocidal composition comprising: a biocide reservoir/source operably connected to a plurality of spray nozzles; a compressed air reservoir/source operably connected to the plurality of spray nozzles; a first valve cootrolling the delivery of biocide from the reservoir/source; a second valve controlling the delivery of compressed air from the reservoir/source; and a controller, operable to control the opening of the valves.

The surfactant can include a colloidal surfactant.

It is preferred that the biocidal composition comprises:

Glutaraldehyde 0%-50%;

Octhilinone 0%-50%;

Surfactant 0%-40%;

Colloids l%-40%.

It is further preferred that the composition comprises:

Glutaraldehyde 3%-5%;

Octhilinoήe 0-2%;

Surfactant Q%-2.5%;

Colloids l%-3%;

Water l%-3%;

Inerts 90%~92%.

Also the invention provides a system that includes one or more pressure switches attached to bomb-protection film which activate the controller when the film is stretched by the blast of a bomb or similar device. Preferably, when the controller is activated it also acts to close down any air- conditioning plant that may be associated with the system.

Preferably, the system includes a manual switch to activate the controller.

In a further preferred embodiment, the system includes a manual mechanical override device configured to activate a third valve and a fourth valve which allows the delivery of the biocide and compressed air, respectively, to the plurality of spray nozzles,

A biocidal composition according to the invention is a two-part formulation that, in use, is diluted with water or other inert ingredients at a ratio of 1 : 1 : 18 (one part A to one part B to IS parts diluent). Part A consists of the active biocides and Part B consists of surfactants and colloidal agents.

Part A - Mix 80% - 100% glutaraldehyde with 0% - 20% octbilinone. (This should be mixed for at least 30 seconds.)

Part B - Mix 50% Colloidal Surfactant with 50% water. (Mixing procedure is to add water then add the surfactant and mix gently for at least 20 seconds.) Final Composition - Mix 1 Part A with 1 Part B with 18 parts of inert ingredient, e.g. water. The procedure for mixing is inert(s) + Part A + Part B, then mix/stir well; (e.g. in a 2OL container add IS litres of water, then 1 litre of Part A, then 1 litre of Part B. then mix/stir well for at least 5 seconds),

The technology of this composition is based on using colloids and surfactants with high pH (9 - 15) to buffer the lower pH of certain active ingredients (2 - 7), This also allows the surfactants and colloids to act as hyper-wetting agents, which allows the actives to be delivered more efficiently.

Traditional practice has been to increase the percentage of active to increase the effectiveness of a formulation, whereas the present invention is based on using small percentages of actives and delivering them more efficiently to increase their biocidal effectiveness,

Without wishing to be limited to the theory, the inventor believes that the present composition is effective because it uses a colloidal surfactant (CS) as a delivering agent and a small amount of active biocide (e.g. glutaraldehyde). The CS in the formula breaks down the protein shells around sporicidal bacteria, which then. allows the active to more effectively kill the cell; this works the same way against the bio-agents. Because the CS is effective at delivering the active, relatively small amounts of active can be used while still achieving excellent results. The CS is also useful to spread the formula as a travelling agent through porous surfaces; e.g. dust, dirt or fabric and like materials.

Other formulations rely on their corrosive content to be effective by corroding the bio-agents (sporicidal bacteria) or, alternatively, use relatively large amounts of active to toxically poison the bio-agents. However, we believe that they cannot achieve the same travelling results, kill times and safety as the present invention.

The advantages of the formula/product include that it can be effective against Anthrax and other sporicidal bacteria in 30 mine. It is effective against chemical contaminates and the product also has fungicidal, virucidal, sporicidal & tuberculocidal properties, In particular the product is not corrosive and is 100% based on bio-degradable materials.

The product can be provided in a concentrate form which can be diluted with any inert ingredients, such as water and ami-freeze. This allows easy transportation and storage of the product. Therefore the product is not highly toxic and does not contain large amounts of active ingredients.

The product has travelling agents to allow it to break surface tension, and therefore it can be effective on soil and similar porous surfaces. In one form the product can be water based. Brief Description of the Drawings:

Specific embodiments in accordance with this invention will now be described by way of example only with reference to the accompanying drawings wherein;

Figure 1 is a schematic layout of a delivery system according to the invention.

Detailed Description of preferred embodiments:

Shown schematically in Figure 1, is a system for the delivery of a biocidal composition according to the invention. The system is adapted to deliver the biσcide by sprinkler into a closed environment (such as a room) in the event of a biological attack, such as by bomb or letter or the like.

The system includes a vessel (1) containing biocide and another vessel (2) containing compressed air. Each vessel is connected via a discrete plumbing conduit, (3) and (4) respectively, to a series of ejector/atomiser heads (5). Delivery of the contents of the vessels to the conduits is electrically controlled by two solenoid valves, (12) and (13) respectively.

The solenoid valves are connected by an electric circuit (6) via aft electronic control box (9), The control box and solenoids are powered by an AC electric source (7) with a battery back-up power supply (8).

The control box is activated by one or more pressure switches (10) attached to bomb protection film, which are triggered by the stretching of the film that occurs in response to the blast of a bomb. Toe control box also can be activated manually (such as when a suspicious parcel is opened) by a forced manual switch (11).

A non-electrical forced mechanical lever (14) is provided to operate mechanical valves (15) and (16) so that the system can be activated in the case of failure of the electric/electronic control. Upon activation of the system, atomised biocide is dispersed by the ejector heads and the room's air-conditioning (if present) is shut-down to stop air movement interfering with the dispersal of the biocide throughout the room.

Examples:

Specific examples of the invention are given in the attached two appendices, in which:

Appendix A is a report of the effectiveness of a biocidal composition ("GSC-2000") in decontaminating biological agents; and

Appendix B is a report of testing of GSC-2000 as a decontaminant of Bacillus sρp. spores.

Finally it is to be understood that various alterations, modifications and or additions may be incorporated into the various constructions and arrangements of parts without departing from the spirit and ambit of the invention as defined in the following claims.

Appendix A: Effectiveness of GSC-2000

PAGE

TABLE UST ϋ

SECTION

1 EXECUTIVE DIGEST

1.1 SUMMARY I

1.2 TEST OBJECTIVE 1

1.3 TESTING AUTHORITY 1

1.4 TEST CONCEPT 2

1.5 SYSTEM DESCRIPTION 2

2 DETERMINATION OF FINDINGS

2.1 RECEIPT INSPECTION 3

2.2 OUTPUT OF AEROSOLIZED GSC 2000 PROM THE HYDRO-FORCE PUREMIST ULV FOGGER SPRAY NOZZLE 3

2.3 KILLING EFFECT OF BACTERIAL SPORES BY GSC 2000 SOLUTION 4

2.4 CONCLUSIONS 9

3 APPENDICES.

A TEST CRITERIA 1

B REFERENCES . 1

C ABBREVIATIONS . 1

TABLE LIST

PAGE Effect of liquid GSC-2000 on various sizes of dried BG spots: Effectiveness of Great Southern Coatings (GSC-2000) in Decontaminating Biological

Agents 5 Effect of various duration of exposure to GSC-2000 on dried BG spots:

Effectiveness of Great Southern Coatings (GSO2000) in

Decontaminating Biological Agents , 5 Effect of addition excess GSC-2000 on dried BG spores: Effectiveness of

Great Southern Coatings (GSC-2000) in Decontaminating Biological

Agents • 6 Effect of liquid GSC-2000 on Bacillus anthracis (Ames) spores;

Effectiveness of Great Southern Coatings (GSC-2000) in

Decontaminating Biological Agents 7 Effect of GSC-2000 sprayed on the Surface Containing Dried BG:

Effectiveness of Great Southern Coatings (GSC'2000) in

Decontaminating Biological Agents 7 Effect of GSC sprayed on the surface containing two strains of dried

Bacillus anthracis (ames and vollum), Effectiveness of Great Southern

Coatings (GSC-2000) in Decontaminating Biological Agents,..* 9

SECTION 1. EXECUTIVE DIGEST

1.1 SUMMARY

1.1.1 Background a. The U.S. Marine Corps Chemical/Biobgipal Incident Response Force (CBERJF) has the mission of responding to domestic use of chemical or biological warfare agents by terrorists, and supporting civilian agencies during subsequent decontamination and treatment of victims and decontamination of affected areas. A decontaminant that destroys chemical and biological agents, yet will not harm the victims or the environment, is desirable. b, GSC-2000 which has been developed by Great Southern Coatings (Erina, Australia, NSW) is a two part solution (part A and B) which is a potential biological decontaminant. The objective of this test is to determine the effectiveness of aerosolized GSC-2000 as a sterilent against two strains of Bacillus anthracis (BA) spores and a non-pathogenic simulant [Bacillus sublilte (BG)] spores. o. Optimum decontamination conditions were established for the GSC-2000, which was dis- pensed by the Hydro-Force (Salt Lake City, Utah) PureMist ™ Fogger, Data obtained from simulants were used for tests performed on BA.

1.1.2 Test Findings a. Although GSC-2000 was effective against dried Bacillus spores in solution, its effective¬ ness a* a fog was hampered by its fast evaporation on surfaces. b. It was noticeable that the fog generation by the Hydro-Force PureMist™ ULV fogger was able to wet the area contaminated with dried BA spores, it was not capable to produce a signifi¬ cant sporicjdal effect.

1.2 TEST OBJECfIVE

The test objective is to determine the effectiveness of aerosolized GSC-2000 as a sterilent against two strains of BA spores and non-pathogenic BG spores.

1.J TESTING AUTHORITY

On 19 March 2002, U.S. Army Developmental Test Command, Aberdeen Proving Ground, Maryland, issued a test execution directive tasking the West Desert Test Center (WDTC)3 U.S. Army Dugway Proving Ground (DPG), Utah, to conduct the ECASOL test in support of U.S. Marine Corps Systems Command (USMCSC), Quautko, Virginia (Reference 1). Test on GSC- 2000 was included in this project to explore the effect of this decontaminant's potential use. 1.4 TEST CONCEPT

GSC-20QQ was sprayed on to surfaces with a known amount of biological agent and then sampled to determine the decontamination effect.

1.5 SYSTEM DESCRIPTION a. GSC 2000 is sold in two concentrated parts, Part A and Part B. The dilution rate for GSC 2000 is 1 Part A to 1 Part B to 18 parts water. This cleaning component consists of a broad- spectrum quaternary ammonia compound that acts as, an antimicrobial agent OSC-2000 is a strong alkaline solution having a pH of 10.27. b. The Hydro-Force PureMist™ fogger is a commercially available spray system that is compact and portable. The PureMist fogger is capable of 95 percent of its output to particles of 20 microns with the ability to increase to a heavy setting with output of 95 percent below 60 mi¬ crons. The unit has an adjustable flow control valve, corrosion-proof supply tank, a flexible 32- inch directional base and a 2-stage, variable output motor.

1.5.1 Background

GSC 2000 is a proprietary, two part component which works by breaking the textile surface tension allowing the antimicrobial component to carry through and decontaminate the textile.

SECTION 2. DETERMINATION OF FINDINGS

2.1 RECEIPT INSPECTION

2.1.1 Objective

Determine if all test items arrived in suitable condition for testing.

2.1.2 Criteria None.

2.1.3 Inspections Procedure a. All items were checked to ensure that they matched shipping documents. b. All items were inspected to ensure that no damage had occurred during shipping.

2.1.4 Inspection Findings

AU items were found to be in suitable condition for testing.

2.1.5 Technical Analysis None.

2.2 OUTPUT OF AEROSOLIZED GSC 2000 FROM THE HYDRO-FORCE PUREMIST™ ULV FOGGER SPRAY NOZZLE

2.2.1 Objective

To determine the output of aerosolized GSC 2000 from the spray nozzle.

2.2.2 Criteria None

2-2.3 Test Procedure a. The tank of the sprayer was filled with tap water. b. The nozzle was held towards the front and moved from left to right during the spray for a set time. One 'round' was considered to be the time taken for the nozzle to reach from left to right, o. The nozzle was inserted loosely into a polyester garbage bag to collect the mist while al¬ lowing the air to escape. d. The bag was weighed before and after every 4 rounds (4 min) of mist application, 2,2.4 Results

The average weight (replicate of 4 rounds) of water that was delivered from the nozzle was 49 grams per round. From this it was determined that 49 ml liquid is delivered in each round of application.

2.3 KILLING EFFECT OF BACTERIAL SPORES BY GSC 2000 SOLUTION

2.3.1 Objective

The sterilant effect of GSC-2000 on BG spores was measured.

2.3.2 Criteria

A reduction in the spore growth at 6-log or more was considered as an effective sterilant.

2.3.3 Effect of liquid GSC-2000 on various sizes of dried BG spots

2.3.3.1 Procedure a. Various amounts of BG slurry (Table 1) were dried on to 24-well plates at room tem¬ perature. b. One mL of QSC was added (per well) and left at room temperature for 30 minutes. c. The decontaminant was neutralized after 30 minutes by adding an equal volume of con- centrated phosphate-buffered saline (10 fold concentration) to adjust the pB to 7.25. The content of each well was transferred to 10-ml tubes. d. The solutions were then appropriately diluted and applied on TSA plates to determine the growth of BG colonies. e. The colonies were.counted after 24 hours of incubation at 35° C.

2.3.3.2 Results

Data shown in Table 1 demonstrates that GSC-2000 killed the dried BG spores during the 30-min contact time. A minimum of 7-log reduction is observed in this trial. Table 1. Effect of liquid GSC-2000 on various sizes of dried Bacillus subtilis spots: Effective- ness of Great Southern Coatin s GSC-20QO in Decontaminatin Biolo ical A ents

crul ter

^milliliter

'colony forming uπiL.

*The values expressed are a mean of triplicate determinations.

2,3.4 Effect of various duration of exposure to GSC-2000 on dried BG spots 2.3.4.1 Procedure a. Table 2 represents trials of 50 μL BG slurry dried on glass surface -with 100 μL of GSC- 2000 added to the spot b. The samples were neutralized and transferred to tubes at the time intervals indicated in Table 2 and plated after dilution.

Table 2, Effect of various duration of exposure to GSC-2000 on dried Bacillus subtilis spots: Effectiveness of Great Southern Coatings (GSC-2000) in Decontaminating Biological

"colony form ing units

2.3.4.2 Results

The GSC-2000 (100 μL) evaporated within 30 minutes, Any exposure time beyond 30 min- utes has no enhanced effect. Exposure of 20 minutes is not adequate to have a killing effect on the spores as seen from other trials. This shows that the amount of GSC kept in contact with the spot is important

2.3.5 Effect of addition of GSC-2000 on wet BG spores 2.3,5.1 Procedure a. Aliquotes of 500 μL of GSC-2000 (or water in control tubes) was taken in tubes, b. To each tube, 50 μL BG slurry was added and kept at room temperature for 30 minutes. c. The contents were then neutralized with concentrated phosphate buffered saline (10 fold) and diluted with water 10 times, Appropriate aliquots were plated on agar plates and colonies counted after 24 hr incubation,

Table 3, Effect of addition of GSC-2000 on wet BG spores: Effectiveness of Great Southern Coatin s GSC-2000 in Decontaminatin Biolo ical A ents,

"Average values of triplicate assays. bcolony forming units

2.3.5.2 Results

Results in Table 3 show that GSC-2000 efficiently killed the BG in solution , 2.3.6 Effect of liquid G$C200Q on Bacillus anthracis Y Amesi spoτes 2.3.6,1 Procedure a. A slurry made of BA (Ames) spores was dried on glass plates or painted metal plates (500L slurry) with 250 μL GSC placed on the spot (Table 4), Each sample was exposed forl5 min. Table 4. Effect of liquid GSC-20Q0 on Bacillus anthracis (Ames) spores: Effectiveness of Great Southern Coatin s GSC-2Q00 in Decontaminatin Biolo ical A ents

Average o tr plicate sample., "colony forming units

2,3.6.2 Results a. Table 4 shows the differences between two surfaces, metal and glass. b. There is a potential difference in the extraction from the painted surface when it is treated with the highly alkaline GSC-2000. It is possible that the alkalinity introduces changes on the paint resulting in poor extraction of the spores into the solution.

2.3.7 Effect of GSC-200Q_sprayed on the Surface Containing Dried BG 2.3.7.1 Procedure a. Both metal and Glass surfaces were spotted with 100 μL of BG. GSC-2000 was taken in HydroForce PureMist™ ULV Sprayer. The output of the sprayer was adjusted to 50 ml per min¬ ute. b. The sprayer was held at 3 feet away from the surfaces. The decon was sprayed towards the surfaces for 1 minute in a swaying motion. Five minutes later, the spray was repeated for one more mhrute. The surfaces were left at room temperature for 30 minutes, Samples were swabbed and transferred to tubes and neutralized to stop the reaction, Appropriate dilutions were made to plate the samples on TSA plates.

Table 5. Effect of GSC-2000 sprayed on the Surface Containing Dried BG: Effectiveness of Great Southern Coatin s GSC-2000 in Decontaminatin Biolo ical A ents.

'Average of triplicate samples. bcolony forming units. 2.3.7.2 Results a. Although the plan was to make three applications Dfthe spray, the high output of the in- strument nearly flooded the sample surfaces. b. A five-log reduction was seen in both the surfaces under the experimental conditions.

2,3.8 Effect of GSC sprayed on the surface containingjwo strains of dried Bacillus anthracis fames and vollum)

2.3.8.1 Procedure

a. Painted metal surfaces were spotted with 50 μL or 100 μL of the specified agent. b. The spots were allowed to dry at room temperature. c. GSC -2000 was filled in a HydroForce PureMist™ ULV Sprayer. d. The decontaminant was sprayed with an output of 50 ml per minute. e. The sprayer was held at 3 feet away from the surfaces and sprayed towards the surfaces for 1 minute in a swaying motion. With 5 minutes of waiting period between each round of spray, a total amount of 500 ml was delivered in the 25 square feet area. The spraying was suffi- cient to keep the surfaces wet for 60 minutes. f. After 60 minutes of exposure, the samples were taken into concentrated phosphate buff¬ ered saline (ten-fold) solution and dilutions were made prior to plating on TSA plates. g. Simultaneously BG spots were treated the same way. h. The values given are average of triplicate determinations, i. The surface used was painted metal.

'BctctUwi subtilis

2.3.8.2 Results a. The effect of GSC on BG and BA (ames) suggests that the log reduction due to the expo¬ sure to the decontamination does not exceed 3-4 logs even under the extreme concentration of the spray (almost wetting the surface with the fog, close to dripping, using the sprayer). b. BA (vollum) seems to be susceptible to decontamination by approximately 6 logs under the seme conditions.

2,4 CONCLUSIONS a. The results in this report show that:

1. GSC-2000 is capable of killing BG spores when exposed in wet slurry or dry spots.

2. Dried droplets (25-200 μL) of BG slurry (3-4X108 CFU) dried on surfaces were killed by GSC-2000.

3. When small droplets (100 μL) of liquid GSC-2000 were added on top of dried BG spots, there was a substantial killing effect, although total kill was never noticed. An exposure of a minimum 20 minutes was necessary to see noticeable effect. GSC evaporation caused lack of effect.

4. Addition of excess amount of GSC-2000 caused caused total kill of the BG spores upto 8 log.

5. Dried Bacillus anthracis ( ames) spore spots were vulnerable to GSC -2000 causing 7 log reduction when spotted on painted metal plates. On the otherhand the reduction was leas when the surface was glass. 6. When GSC-2000 was sprayed on Bacillus anthracis (ames) spots dried on metal or glass surfaces, a noticeable 5 log reduction was elicited -within 30 minutes.

7. When aerosolized GSC-2000 was sprayed on dried BA spores ( ames, vollum) and BG sporicidal effect was compared, volhim strain was more vulnerable to GSC-2000. b. Based on the above findings, it may be concluded that:

1. Although the spoticidal effect of GSC-2000 is demonstrated in this work, applicabil¬ ity of its use as a fog may not be a feasible method if total decontam iπation is expected.

2. Results of the experiments using wet BG spores in this work demonstrate its spori- cidal effect. However, penetration of the decon into the dry spot is limited with limited amount of the decon coming in contact with the spot. Tiny droplets may not be effective due to the evaporation of the decon.

3. The use of HydroForce PureMist ULV Sprayer may not be applicable for delivering fog of GSC-2000 as it tends to clogg up the nozzle. It was noticed that GSC -2000 solution pro- duces suspended materials sufficient enough to clogg the nozzle of HydroForce PureMist ULV Sprayer with continuous usage. Several experiments had to be discontinued due to the abrupt stop of the spray corning out of the nozzle.

4. Therefore it is yet to be determined if attempt has to be made to decant the solution prior to addition of the sprayer tank. c. We recommend that GSO2Q00 spray may be a capable decontam inant against loosely at¬ tached spores on interior surfaces, More work is needed to prove tms potential effect.

SECTION 3. APPENDICES APPENDIX A. TEST CRITERIA

APPENDIX B. REFERENCES

I , Memorandum, U.S. Army Developmental Test Command (DTC), Aberdeen Proving Ground (APG), Maryland, 19 March 2002, subject: Test Execution Directive: Electro- Chemically Activated Solution (ECASOL) Fogger Trial, Test Support to U.S. Marine Corps Sys¬ tems Command (USMCS), DTC Project No. 8-CO-480-000-007.

APPENDIX C. ABBREVIATIONS BA - Bacillus anihracis BG - Bacillus svbtilis

CBIRF - U.S. Marine Corps Chemical/Biological Incident Response Force ofu - colony form ing unit DPG - U.S. Army Dugway Proving Ground ECASOL- electro-chemically activated solution EtOH - etbano) FAC - free available chlorine FEM - flow-through electrolytic module QSC - Great Southern Coatings rfiM - miiltinole tnS - millisecond NaCl - sodium chloride

RSCECAT - Russian Scientific Center for Electrochemical Activation Technique TSA - tryptic soy agar

USMCSC -U.S. Marine Corps System Command WDTC - West Desert Test Center

APPENDIX B

Testing of Decontaminants Against Bacillus spp, Spores

EXECUTIVE SUMMARY

The objective of this study was to evaluate the effectiveness of decoπtam kation formulations to inactivate Bacillus anthracis Ames spores in solution, These formulations included Great Southern Coatings (GSC) 2000 in which the active component (glutaraldehyde) was present or absent Two formulations from Great Southern Cαatings were provided and tested. The first Jot received ms designated as GSC 2000-1 -Lot 1 (no glutaraldehyde present) and GSC 2000-2-Lot 1 (glutaraldehyde present). The second lot of the formulation containing glutaraldehyde was designated GSC 2000-2-Lot 2. High Test Hypochlorite (HTH) at a concentration of 5% was used as a positive control. Suspension testing with GSC 2000 showed that GSC 2000-2-Lot 1 and 5% HTH decreased the number of viable B, anthracis Ames spores after 30 minutes by 3,40 and >7.21 log reductions, respectively, The GSC 2000-1-Lot 1 did not reduce the viability of B. antbracis Ames spores after 30 minutes. Following 30 minutes of suspension testing, GSC 2000-2-Lot 2 formulation and 5% HTH completely neutralized viable M. anthracis Arties spores, corresponding to log reductions in viable spores of >8.09. When compared to a glutaraldehyde reference, chemical analysis of all GSC 2000 formulations using direct-infusion eleotrospray mass spectrometry (ESI-MS) revealed the GSC 2000-2-Lot I tested contained little to no glutaraldehyde, while the GSC 2000-2-Lot 2 tested contained an abundance of glutaraldehyde.. TABLE OF CONTENTS

Page

EXECUTIVE SUMMARY i

TABLE OF CONTENTS ii

LIST OF FIGURES iii

LIST OF TABLES iv

1.0 INTRODUCTION 1

2.0 MATERIALS AND METHODS 1

2.1 Decontamination Reagents 1

2.2 Biological Agents 2

2.3 Suspension Testing of Decontaminants 2

2.3.1 Testing 2

2.3.2 Plate Enumeration 2

2.3.3 Analytical Analysis 3

3.0 RESULTS 4

3.1 Suspension Testing 4

3.2 Analytical Analysis 7

4.0 DISCUSSION 11

5.0 ACKNOWLEDGEMENTS 12

6.0 REFERENCES 12

LIST OF FIGURES

Figure L Suspension Testing with B. anthracis Ames Spores 5

Figure 2. Part A of GSC 2000 Formulations 6

Figure 3. ESl-MS Spectrum of Glutarβldebyde 7

Figure 4, ESI-MS/MS Spectrum of Glutaraldehyde 8

Figure 5. ESI-MS Spectrum of GSC 2000-1-Lot 1 8

Figure 6. ESI-MS/MS Spectrum of m/z 102 from GSC 2000-1-Lot 1 9

Figure 7. ESI-MS/MS Spectrum of m/z 214 from GSC 2000-1-Lot 1 9

Figure 8. ESI-MS Spectrum of GSC 2000-2-Lot 1 9

Figure 9. ESI-MS/MS Spectrum of m/z 102 from GSC 2000-2-Lot 1 10

Figure 10. ESI-MS/MS Spectrum of m/z 2)4 from GSC 2000-2-Lot 1 10

Figure 11, ESI-MS/MS Spectrum of GSC 2000-2-Lot 2 10

Figure 1.2, ESΪ-MS/MS Spectrum of m/z 101 from GSC 2000-2-Lot 2 I l

LIST OF TABL ES

Page

Table 1 , Bacillus anthracis Ames Spore Suspension Testing (GSC 2000-1 -Lot 1; GSC

2000-2-Lot 1 ; 5% HTH) , 5

Table 2, Bacillus anthracis Ames Spore Suspension Testing (GSC 2000-2-Lot 2; 5%

HTH) 7

1.0 INTRODUCTION

The mixture GSC 2000 from Great Southern Coatings (GSC) has been reported to be a potential decontammant. GSC 2000 solution consists of two active ingredients, a quaternary ammonium compound and an active surface agent, Part A contains benzalkonium chloride, octhilinone, and glutaraldehyde. Part B is a surfactant that was developed by GSC and is a proprietary item. This study tested GSC 2000 formulations with and without the presence of glutaraldehyde. MARCORSYSCOM has requested the Battelle Medical Research and Evaluation Facility perform decontamination testing with Bacillus amhracis Ames spores, The objective of this study was to evaluate the efficacy of these products as well as 5% HTH to neutralize B, amhracis Ames spores within 30 minutes in suspension testing.

2,0 MATERIALS AND METHODS

This study was performed under MREF Protocol No, 389.

2.1 Decontamination Reagents

The decontamination solutions were prepared according to the instructions of each manufacturer and used within one hour of testing. Initially, two formulations from Great Southern Coatings were provided and tested, which were designated as GSC 2000- 1-Lot 1 (no glutaraldehyde present) and GSC 2000-2-Lot 1 (glutaraldehyde present). A second lot of the formulation containing glutaraldehyde was also provided in this study by Great Southern Coatings, and was designated GSC 2000-2-Lot 2. All GSC 2000 formulations were mixed as instructed by the manufacturer, which consisted of combining 1;1 :18 ratios of Part A, Part B, and sterile water.

2.2 Biological Agents

The biological agent used for testing was Bacillus anthracis Ames strain spores produced according to Battelle SOP Number MRJEF. X-074. Spore stocks were enumerated and diluted in sterile water to an approximate concentration of 1 x 109 colony forming units (CFU)/mL.

2.3 Suspension Testing of Decontaminant s

2.3.1 Testing

Each decontaminant was tested in triplicate. Testing was conducted following MREF Method No. 92/Microbiology. Tests were conducted by combining 100 μL of the stock 1 x 109 CFU/mL (1 x 108 total CFU) B. anthracis spores with 1.0 mL of test material (GSC 2000 Formulations 1 or 2, or 5% HTH) in a 2.0 mL tube. Phosphate buffered saline (PBS) was used as a "no decon" control A spore-free (blank) tube was run in parallel with each test material as a control for any bacterial growth other than B. anthracis. The test material or control was allowed to mix for 30 minutes at room temperature on a rotating platform mixer. The entire contents of the tube were centrifuged at 20,800 x g for 5 minutes. The supernatant was removed, the pellet resuspended in 1.0 mL of PBS, and centrifuged at 20,800 x g for 5 minutes. The supernatant was removed and the spore pellet was resuspended In 1.0 rnL sterile PBS. The spore suspension was serially diluted through 1 O-7 in sterile PBS. One hundred microliters of each serial dilution was plated onto tryptio soy agar (TSA) plates in triplicate, allowed to dry, incubated overnight at 37°C, and enumerated.

2.3.2 Plate Enumeration

The plates were enumerated as described in Battelle SOP Number MREF. X-054, Only plates having 25-250 colonies were considered in determining the plate count, with the exception of samples where low colony counts were observed due to the decontamination process. For colony counts below 25 colonies, enumeration data were calculated folio-wing Battelle SOP Number MREF. X-1 12. The colony forming units (CFU)ZmL was determined by multiplying the average number of colonies per plate by the reciprocal of the dilution.

Enumeration of the spore stock and treated samples was conducted in parallel. To determine decontaminant efficacy, the reduction in spore number was expressed as the log N/N' where N is the number of viable spores recovered in the PBS control and N" is the remaining number of viable spores after decontamination. Data were expressed as mean ± standard deviation (SD).

2.3.3 Analytical Analysis

Direct-infusion electrøspray mass spectrometry (ESI-MS) was used to confirm the presence and/or absence of glutaraldehyde in Part A ofthe GSC 2000-ϊ-Lαt 1, GSC 2000-2-Lot 1, and GSC 2000-2-Lot 2 formulations. The testing was conducted according to Battelle SOP Number MRER III-034, "Standard Operating Procedure (SOP) for the General Use, Calibration, and Maintenance of Mass Spectrometer Systems," A Micromass Quattro II tandem mass spectrometer was used to analyze three formulations of decontaminants and compare the spectra obtained to the spectrum of a glutaraldehyde standard. Glutaraldehyde was obtained as a 25°/* aqueous solution (Sigma, St. Louis, MO; Lot 042K53001 Exp 5/05). A 2,5 μg/mL solution of gtøtaraidehyde was prepared in 50/50 water/acetonitrile with 2 inM formic acid and 2 mλf ammonium formate solvent, and analyzed by direct infusion positive ion eleotrosprøy ionization mass spectrometry (BSI-MS) and ESI-tandem mass spectrometry (ESI-MSMS). To prepare this solution, 10 μL of die 25% glutaraldehyde was diluted up to 10 roL wiώi the water/acetonitrUe solvent This solution was diluted farther by taking 100 μL ofthe primary solution and diluting up to 10 mL with solvent. ESΪ-MS produced protonated precursor ions and ESI- MS/MS provided for the collection of fragment ion spectra of selected precursor ions.

For mass spectrometer analysis of Part A from GSC 2000-1 -Lot 1 and GSC 2000- 2-Lot 1, approximately 56 mg of sample was diluted up to 10 mL with the same solvent used to prepare the glutaraldehyde standard. Each ofthe samples were diluted by taking 150 μL of the 5.6 mg/mL GSC 20004-Lot 1 and GSC 2000-2-Lot 1 samples and diluting to 10 mL with solvent prior to ESI-MS analysis. For analysis of sample GSC 2000-2-Lot 2» 10 μL of the liquid was diluted υp to 10 mL with the water/acetonitrile solvent. This solution was diluted further by taking 100 μL of the primary solution and diluting to 10 mL with solvent This second diluted solution was used for analysis by mass spectrometry,

3.0 RESULTS

3.1 Suspension Testing

During the first round of testing, GSC 2000-1-Lot 1, GSC 2000-2-Lot I, and 5% High Test Hypochlorite were evaluated for neutralization effectiveness against Bacillus anthracis Ames spores, using PBS as the control. The GSC 2000-1-Lot 1 and GSC 2000-2-Lot 1 formulations were prepared as instructed by the vendor (see Section 2.1). GSC 2000-1-Lot 1 Fart A was a pale yellow mixture with a creamy texture; GSC 2000-2- Lot 1 Part A was similar, but a darker brown color. Part B for GSC 2000-1-Lot 1 and GSC 2000-2-Lot 1 were identical - a clear, straw yellow, high-viscous mixture. When mixed, GSC 2000-1 -Lot 1 went into solution and was slightly opaque with some foaming, probably due to the surfactant from Part B. GSC 2000-2-Lαt 1 did not go entirely into solution and was slightly opaque with some foaming, probably due Io the surfactant from Part B. Fof testing of GSC 2000-l-Lot 1. GSC 2000-2-Lot 1, 5% High Test Hypochlorite, and PBS, the spore pellets were rapidly suspended in each fluid during the 30 minute decontamination procedure (Figure 1).

Figure 1. Suspension Testing with Bt antkt acts Ames Spores. Note the B. anthracis Ames spores mix well in the decontaminant (5% HTH).

Table 1 shows the calculated inoculum, total spores, and log reduction of Bacillus anthrack Ames spores exposed to each test material. One-hundred microliters of a 9.80 x 106 CFWmL spore suspension was used as the inoculum, which corresponds to an inoculum concentration of 9,80 x 107 CFU. Spores were exposed to each test material for 30 minutes and log reductions for the test materials were calculated based upon the total viable spores treated with PBS. When compared to the PBS controls, both GSC 2000-2- Lot 1 and 5% HTH were effective in Teducing viable B, anthracis Ames spores having log reductions of 3.40 and ≥7.21, respectively. No decreases in viable S, anthracis Ames spores were observed when exposed to GSC 2000-1-Lot 1,

TaT)Ie 1. Bacillus anthracis Ames Spore Suspension Testing (GSC 20QO-I-LOt 1; GSC 2000-2-Lot 1 5% HTH .

Data are expressed as mean ± SD.

During the second round of testing, The GSC 2000-2-Lot 2 was a new mixture received at the Battelle MREF; therefore, GSC 2000-2-Lot 2 should be considered separate from GSC 2000-2-Lot 1 that was tested previously (Figure 2) The GSC 2000- 2-Lot 2 was prepared as instructed by the vendor. GSC 2000-2-Lot 2 Part A was a clear, yellow-brown mixture with an odor characteristic of glutaraldehyde, GSC 2000-2-Lot 2 Part B was a clear, straw yellow, high-viscous mixture. When mixed, GSC 2000-2-Lot 2 went into solution and was clear with some foaming, probably due to the surfactant from Part B,

Figure 2. Part A of GSC 2000 Formulations, (A) GCS 2000-1-Lot: 1; (B) GSC 2000- 2-Lot 1} (C) GSC 2000-2-Lot 2.

Table 2 shows the calculated inoculum, total spores, and iog reduction of Bacillus amhracis Ames spores exposed to the revised GSC 2000-2-Lot 2, 5% High Test Hypochlorite;, and PBS in suspension. One-hundred microliters of a 1.06 x 109 CFWmL spore suspension was used as the inoculum, which corresponds to an inoculum concentration of 1.06 x 108 CFLT. Spores were exposed to each test material for 30 minutes and log reductions for the test materials were calculated based upon the total viable spores treated with PBS, When compared to the PBS controls, GSC 2000-2-Lot 2, and 5% HTH were effective in reducing viable B anlhracis Ames spores where both exhibited log reductions of ≥8.09.

3.2 Analytical Analysis

Glutaraldehyde has a molecular weight of 100, and in the ESI source the glutaraldehyde is protonated to form a precursor ion with amassxharge ratio (m/z) of 101. The ESI-MS spectrum of glutaraldehyde shows the presence of a molecular species having an m/z of 101.33 (Figure 3). Fragmenting the m/z 101 precursor ion results in fragments of rn/z 83, 73, $9, 57, 55, 4I1 and 31 (Figure 4).

Figure 3. ESI-MS Spectrum of Glutaraldehyde,

Figure 4. ESI-MSMS Spectrum of Glutaraldehyde.

The ESI-MS spectra of GSC 200(M»Lot 1 showed an abundance of precursor ions at m/z 102 and 214 were present with little to no presence of m/z 101, indicating little to no glutaraldehyde present in this sample (Figure 5). ESI-MS/MS spectra were obtained from m/z 102 and 214 and are shown in Figures 6 and 7, respectively. The even mass precursor ions may indicate the presence of an odd number of nitrogens (e.g. amines), which would be consistent with the presence of the quaternary ammonium compound in GSC 2000. All three of these spectra match the ESI-MS and ESI-MS/MS spectra obtained from the GSC 2000-2-Lot 1 sample.

Figure 6. ESI-MS/MS Spectrum of m/z 102 from GSC 2000-1-tot 1.

Figure 7, ESI-MS/MS Spectrum of m/z 214 from GSC 2000-1-Lot 1.

For analysis of GSC 2000-2-Lot 1, a high abundance of precursor ions with mlz of 102 and 214 were present with a low abundance of m/z 301, indicating little Xa no glutaraldehyde present in this sample (Figure 8), Figure 9 shows the ESI-MS/MS spectrum for the m/z 102 species and Figure 10 shows the ESWviSMS spectrum for the m/z 214 species,

Figure 9. ESI-MSMS Spectrum of m/z 102 from GSC 2000-2-Lot 1.

Figure 10. ESI-M$/M(S Spectrum of m/z 214 from GSC 2000-2-Lot 1,

For ESI-MS analysis of sample GSC 2000-2-Lot 2, there were a number of observed precursor ions that were not present in GSC 2000-2-Lot 1 (Figure 11). An abundant peak at mtz 101 was present in this sample. This MS/MS spectrum matched the ES1-MS/MS spectrum of the glutaraldehyde standard, indicating that the GSC 2000-2- Lot 2 sample contains glutaraldehyde (Figure 12).

Figure 12, ESI-MS/MS Spectrum of m/z 101 from GSC 2000-2-Lot 2.

4.0 DISCUSSION

Suspension testing with GSC 2000-2-Lot 1 (containing glutaraldehyde) decreased the number of viable B. cmthracis Ames spores by a 3,4 log reduction. However, 5% HTH resulted in a≥7.21 log reduction, Analytical analysis of Part A from GSC 2000-2- Lot 1 by ESI-MS revealed a low abundance of m/z 101, indicating little to no glutataldehyde present in this sample. The GSC 2000-1 -Lot 1 (no glutaraldehyde) did not reduce the number of viable B. anthracis Ames spores after 30 minutes. ESI-MS of Part A from GSC 2000-1 -Lot 1 revealed no glutaraldehyde present.

A second round of testing with GSC 2000-2-Lot 2 (containing glutaraldehyde) was conducted. Following 30 minutes of suspension testing, both the GSC 2000-2-Lot 2 and 5% HTH completely neutralized viable B. antkracis Ames spores, corresponding to log reductions in viable spores of ≥8.09. Chemical analysis of Part A from GSC 2000-2- Lot 2 by ESI-MS revealed a spectrum containing a number of precursor ions that were not present in Part A of the GSC 2000-2-Lot 1 sample. Furthermore, an abundant peak at m/z 101 was present in Part A of the GSC 2000-2-Lot 2 sample, which was similar to the m/z 101 observed in the glutaraldehyde standard. The MS/MS spectrum of the rn/z 101 peak in Part A of GSC 2000-2-Lot 2 was similar to the ESI-MS/MS spectrum of the glutaraldehyde standard, indicating that Part A of the GSC 2000-2-Lot 2 sample contained glutaraldehyde.

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