Description:Field of the Invention:
The current invention relates to a surface disinfecting agent for medical instruments. More particularly, the current invention relates to a surface disinfecting solution having three phases.

Background of the Invention:
The instruments and materials used in health establishments are frequently exposed to contamination with microorganisms. Certain surgical, dental and medical instruments required high-level disinfection and disinfection of these instruments prevents cross-infection, disinfection can reduce soils and organic residues consequently, it reduces occupational infection risk. Disinfection is performed by immersing contaminated surgical, dental and medical instruments containing organic residues, microorganisms, and other contaminants in a disinfection solution, with the aim of eliminating or reducing the quantity of microorganisms.

Peracetic acid is considered as a potent biocide, even at low concentrations of 0.0001% to 0.2%. Peracetic acid has the advantages of remaining effective even in the presence of organic residues, and it decomposes into nontoxic and non-mutagenic substances (acetic acid and oxygen) and provides excellent disinfection in a short period. Peracetic acid removes surface contaminants (primarily protein) on medical instruments. Peracetic acid inactivates gram-positive and gram-negative bacteria, fungi, Bacterial spores and yeasts.

Peracetic acid, generally using some concentrations of acetic acid and hydrogen peroxide has been shown to be a good biocide, but it possesses a very strong odour, especially in the concentrate. For general use applications, it is desirable to have a product with low odour. Thus, it is desirable to obtain a low odour peracetic acid antimicrobial formulation that is highly effective.

An exemplary Peracetic acid generating formulation was covered in WO2000053011 (WO ‘011). The formulation of WO ‘011 comprises a liquid base generating oxygen, a liquid generator of acetyl radicals comprising N-acetylcaprolactam solubilized in isopropanol. However, it was observed that the overall stability of peracetic acid generated in system of WO ‘011 is very low. As a result, the contact time required for achieving bacterial disinfection is approximately 5 minutes, whereas the activation time is around 30minutes. Further, the product of WO ‘011 has one more major disadvantage, that the N-acetylcaprolactam is not in fact stable during the time in solution. The lifetime of the acetyl radical generator is only 12 months.
One more instance could be found in US20030211169 (US ‘169). The disclosure of US ‘169 discusses a system for generation of peracetic acid in range of 0.001 – 10.000% just before use by mixing an activator with a parent mixture. Said parent solution comprises at least one acetylated compound in range of 0.001-13.00 % w/w, wherein the acetylated compound is selected from, N, N', N?, N'?-tetraacetyl glycoluryl and tetraacetyl ethylene diamine (TAED); glacial or concentrated acetic acid. The said parent solution further comprises an alcohol, a buffer solution which maintains pH of the solution in range of 3.50-7.00 at 20° C. The parent solution further comprises an anionic surfactant, at least one sequestrant or chelating agent, a wetting agent and wherein said activator is a peroxide compound. The system of US ‘169 uses alcohol as solvent. The addition of solvent increases both downstream and upstream cost

However, both the above approaches use a two-phase system, namely an activator solution and a parent solution or peracetic acid generating solution. As a result of which the pH of the solution is not necessarily maintained in optimum range of 7 to 8 and for optimum peracetic acid generation, the system needs to be maintained in neutral pH range.

Further, it was observed that peracetic acid generated in a two-phase system does not remain stable for a longer time and also the contact time for achieving effective cleaning is long. Furthermore, the two-phase system requires an inefficiently longer activation time, of the order of 30mins.

Therefore, it an endeavour of current inventors to provide a system for disinfection of surfaces which overcomes the disadvantages of the prior art.

Objects of invention:
In view of the above, the main object of the current invention is to provide such a system for disinfection of surfaces which remains stable overtime.

It is yet another object of invention to provide a system which produces extemporaneously organic peracetic acid, which is stable for longer period of time.

Summary of the Invention:
In view of above objects, the primary aspect of the current invention is to provide a system of three liquid components or phases for effective generation of peracetic acid.

The three liquid system is easy to handle and transport

The system in accordance with above aspect is stable over a longer period of time.

Further, said system extemporaneously produces organic peracetic acid which is stable up to 2 months within an optimized range.

The system in accordance with above aspect can be directly used for disinfection of surfaces.

In another aspect, the system of the invention substantially reduces the surface contact time to achieve substantial disinfection.

In another aspect, the present invention provides a method for in-situ generation of peracetic acid.

Brief Description of Drawings:
Fig. 1 illustrates the schematic diagram for the process for in-situ preparation of peracetic acid by using 3(three) distinct phases.

Details Description of the Drawings:
The Fig. 1 illustrates the process for in-situ preparation of peracetic acid using 3(three) distinct phases.

As illustrated in Fig. 1, the 3 phases comprise, namely, Phase 1 which is an oxygen-generating base comprising hydrogen peroxide; Phase 2 which is a pH Modifier phase and Phase 3, which is Acetyl group generating phase.

Said Phase 1, essentially comprise of Hydrogen peroxide (H2O2) as main component. The phase 1 further comprises of an anticorrosive agent; a chelating agent; a surface-active agent; a supportive corrosion inhibitor and a vehicle.

Said Phase 2 comprises of a pH modifier, which is preferably a triamine or a tertiary amine. Said Phase 2 may further comprise a buffering agent selected from a carbonate or a phosphate. Said Phase 2 can be diluted as required during the process.

Said Phase 3 comprises of an acetyl group generating compound, which can be acetic acid, acetaldehyde, N-acetylcaprolactam.

Each of the individual phases have been observed to stay stable for up to 24 months.

For in-situ generation of peracetic acid, firstly, the Phase 2 is mixed with Phase 1 liquid, preferably in Phase 2:Phase 1 ratio in range of 0.01:50 to 5:500. The said mixing step can do by mechanical stirring; manual stirring and the pH enhancement are carried out within 0 to 60 seconds. The pH increase in the aqueous solution of hydrogen peroxide results in reaction rate increased and rapid production of peracetic acid. The pH of the mixed solution of Phase 2 and Phase 1 is maintained in range of pH 7 to 8 (from initial 1 to 4).

Second step of the process is addition of phase 3 (acetyl group generating phase) to mixture of phase-1 &phase-2. Mixing ratio and mixing time of second step has also optimized to produce 0.08% to 0.2% peracetic acid. The said second step mixing can be done by mechanical stirring or manual stirring.

Mixing time of second step is 5 mins to 15 mins, most preferably 5 mins to 10 mins, mixing ratio of phase 1 and mixture of phase 1 & 2 is 0.01:50 to 5:500, most preferably in between 0.5:200.

The activated/in-situ preparation is clear to slightly yellowish coloured liquid solution with characteristic odour, which having pH range between 3 to 5 and specific gravity in between 0.900 to 1.200.

Details Description of Invention:
In order to provide a clear and consistent understanding of the terms used in the present specification, a number of definitions are provided below. Moreover, unless defined otherwise, all technical and scientific terms as used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains.

The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may not only mean “one”, but also encompasses the meaning of “one or more”, “at least one”, and “one or more than one”. Similarly, the word “another” may mean at least a second or more.

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

Described herein is a novel three-phase system for extemporaneous generation of peracetic acid and a process for the rapid and immediate production of peracetic acid ranging from 0.08% to 0.5%.

In an embodiment, the three-phase system comprises of:
(a) an oxygen-generating aqueous phase (Phase 1);
(b) a pH modifier phase (Phase 2), and
(c) an acetyl group generating phase (Phase 3).

The three distinct phases are designed for immediate in-situ generation of peracetic acid after mixing together. The system of the invention is stable for substantially long period of time.

In accordance with above embodiment, the oxygen-generating aqueous phase comprises of an oxygen-donor compound. Said oxygen-donor compound is, preferably, hydrogen peroxide (H¬2O2).

The oxygen-generating aqueous phase further comprises other ingredients which include,
(i) an anticorrosive agent;
(ii) a chelating agent;
(iii) a surface-active agent;
(iv) a supportive stabiliser , and
(v) a vehicle.

In accordance with above embodiment, the anticorrosive agent is a heterocyclic compound, preferably the heterocyclic compound is a triazole, most preferably the triazoles is a benzotriazole.

The chelating agent is an organic phosphonic acid. Preferably, the organic phosphonic acid is Hydroxyethanediphosphonic acid (HEDP) (also known as Etidronic acid) or salt thereof. More preferably, the HEDP is selected from, but not limited to, 1-hydroxyethane-1, 1-diphosphonic acid, tetrasodium salt of 1-hydroxyethane-1, 1-diphosphonic acid, powder of sodium salt of 1-hydroxyethane-1, 1-diphosphonic acid, powder of 1-hydroxyethane-1, 1-diphosphonic acid. The said chelating agent also functions as a stabiliser.

The surface-active agent is a non-ionic surface-active agent. The non-ionic surface-active agent is selected form a natural fatty alcohol or a fatty alcohol ethoxylate. Preferably, the non-ionic surface-active agent is a C12-14 fatty alcohol ethoxylate.

The supportive stabiliser is selected from ethylene glycol, glycerine, propylene glycol, 1, 3-propanediol (trimethylene glycol), and polyethylene glycol 200 and polyethylene glycol 400.

The oxygen generating aqueous phase contains water as a vehicle. Preferably the vehicle is selected from, but not limited to, purified water, demineralized water, tap water, sterile water or distilled water.

In a preferred embodiment, the oxygen-generating aqueous phase comprises of:
an oxygen-donor compound in range of 5% to 15%w/w;
an anticorrosive agent in range of 0.001% to 0.5%w/w;
a chelating agent in range of 0.01% to 5%w/w;
a surface-active agent in range of 0.01% to 5%w/w;
a supportive corrosion inhibitor in range of 0.01% to 10%w/w, and
a vehicle.

The physical state of the oxygen-generating aqueous phase varies from liquid to semi solid. Said oxygen-generating aqueous phase can be clear colourless to hazy. pH of said phase is between 0.1 and 7.

In accordance with above embodiment, the pH modifier phase comprises of a triamine selected from 2,2,6,6-tetramethylpiperidine, 2,2,5,5-tetramethylpyrrolidine, 2,6-dimethylpiperidine, 2,5-dimethylpyrrolidine, N-ethylpyrrolidine and N-ethylpiperidine, N,N,N-diisopropylethylamine and N,N,N-triethylamine and preferably 2,2',2''-trihydroxytriethylamine. Preferably, the triamine is 2,2',2''-trihydroxytriethylamine.

The said tertiary amine is in dilution form with water at 10% to 80%, also can be in dilution form in mixture with water and combinations of buffering agent from 10% to 80%. The buffering agent can be a phosphate or a carbonate.

In a preferred embodiment, the pH modifier phase comprises of:
a triamine;
a solvent in range of 10% to 80%, and
a buffering agent in range of 0.001% to 0.5%.

In accordance with above embodiment, the acetyl group generating phase comprises of an acetyl group-donor compound. Said acetyl group-donor compound is selected form, but not limited to, acetic acid, acetaldehyde, N-acetylcaprolactam and acetic anhydride.

In next embodiment, the invention provides a method for rapid generation of peracetic acid, preferably in range of 0.08% to 0.5%.

Accordingly, the method comprises steps of mixing the three phases which includes first step of addition of pH modifier phase (phase 2) to the oxygen generating aqueous phase (phase 1). The ratio of Phase 1 to Phase 2 is optimized to achieve pH 7 to 8 (from initially 1 to 4) and the mixing ratio could be in between 0.01:50 to 5:500. The said mixing step can be done by mechanical stirring or manual stirring and the pH enhancement are carried out within 0 to 60 seconds. The increase in pH of the aqueous solution of hydrogen peroxide results in increased reaction rate and rapid production of peracetic acid.

Second step of the process is addition of acetyl group generating phase (phase 3) to mixture of phase 1 and phase 2. Mixing ratio and mixing time of second step is optimized to produce 0.08% to 0.5% peracetic acid. The said second step mixing can be done by mechanical stirring or manual stirring.
Mixing time of second step is 5 mins to 15 mins, most preferably 5 mins to 10 mins, mixing ratio of phase 1 and mixture of phases 1 & 2 is 0.01:50 to 5:500, most preferably in between 0.5:200.

The activated/in-situ preparation is clear to slightly yellowish coloured liquid solution with characteristic odour, which has pH in range of 3 to 5 and specific gravity in range of 0.900 to 1.200.

The system of the present invention demonstrates excellent microbicidal activity against bacteria, spores, yeast and fungi within 1 minute while marketed product has 5 mins contact time. The present invention exhibited excellent microbicidal efficacy due to presence of fatty alcohol ethoxylate as surfactant employed in the formulation.

Produced peracetic acid is stable and ready for direct use as an antimicrobial agent, in process of disinfection and / or chemical, manual sterilization, i.e. through immersion in basin, and automated sterilization, by recirculation in washing machines for endoscopes or surgical instruments or even thermosensitive instrumentation, and / or environmental surfaces, especially in the medical-surgical field.

Further examples of suitable application of this system and preparation include disinfection of equipment for food processing, disinfection of areas for the breeding of animals, getting rid of moulds in storage warehouses, bleaching of textiles and garments, descaling and simultaneous disinfection of internal circuits of haemodialysis generators, of distribution systems of food and beverages and others where a whitening, oxidizing, descaling, disinfecting and sterilizing effect is required.

EXAMPLE
Several examples are set forth below to further illustrate the nature of the invention and the manner of carrying it out. However, the invention should not be considered as being limited to the details thereof.

Example 1:
Table 1:
Phase 1: Oxygen generating Phase %w/w Quantity in gm
Hydrogen Peroxide
HEDP Acid
Benzotriazole
C12-14 fatty alcohol ethoxylate
Glycerine
Water 7.5%
0.1%
0.05%
0.1%
1%
QS to 100 gm 1000 gm
Phase 2: pH modifier phase
Triethanolamine
Water 30%
QS to 100 gm 6.3 gm
Phase 3: Acetyl group generating phase
N-acetylcaprolactam 100% 20 gm

In-situ preparation of peracetic acid was done by using design mentioned in Table 1. The oxygen generating phase was prepared at 7.5% by weight by diluting 150 gm of 50% hydrogen peroxide and other ingredients with distilled water as needed to 1000gm and the pH was 2 to 4. pH modifier phase, that is, triethanolamine was diluted with distilled water and the pH of the solution was maintained at 11 to 12. Acetyl generating phase, that is, N-acetylcaprolactam was as such used in this design.

After five minutes of mixing time, 0.1105% w/w peracetic acid was generated. It is clear colourless solution with characteristic odour and pH between 3 and 5. Physicochemical of in-situ preparation was observed and it was found within specific limit till 60 days. 30% concentration of triethanolamine did not affect the peracetic generation and its stability.
Example 2
Table 2
Phase 1: Oxygen generating Phase %w/w Quantity in gm
Hydrogen Peroxide
HEDP Acid
Benzotriazole
C12-14 fatty alcohol ethoxylate
Glycerine
Water 7.5%
0.1%
0.05%
0.1%
1%
QS to 100 gm 1000 gm
Phase 2: pH modifier phase
Triethanolamine
Water 50%
QS to 100 gm 4.5 gm
Phase 3: Acetyl group generating phase
N-acetylcaprolactum 100% 20 gm

In Example 2, oxygen generating phase and acetyl group generating phase are same while concentration of pH modifier has changed. 50% diluted triethanolamine was used in this example and its pH was between 11 and 12. The oxygen generating phase was prepared at 7.5% by weight by diluting 150 gm of 50% hydrogen peroxide and other ingredients with distilled water as needed to 1000 gm.

After mixing 0.1151% w/w of peracetic acid was generated, pH was between 3 and 5. The generated peracetic acid is s also remained stable over 60 days. As compare with example 1 less quantity of pH modifier phase is required for pH enhancement (to achieve 7 to 8). The concentration change in pH modifier did not affect peracetic acid generation and its stability.

Example 3
Table 3
Phase 1: Oxygen generating Phase %w/w Quantity in gm
Hydrogen Peroxide
HEDP Acid
Benzotriazole
C12-14 fatty alcohol ethoxylate
Glycerine
Water 7.5%
0.1%
0.05%
0.1%
1%
QS to 100 gm 1000 gm
Phase 2: : pH modifier phase
Triethanolamine
Water 70%
QS to 100 gm 3.9 gm
Phase 3: Acetyl group generating phase
N-acetylcaprolactam 100% 20 gm

Continuation with example 1 and example 2, In Example 3, oxygen generating phase and acetyl group generating phase are same while 70% triethanolamine was used as phase 2. pH of 70% triethanolamine was observed to be in range of 11 to 12. To achieve required pH (7 to 8) 3.9 gm of pH modifier phase consumed which is lesser than the previous examples.

After mixing for 5 minutes, 0.1188% peracetic acid was generated and it was stable till 60 days. pH of the in-situ preparation was between 3 to 5.

From Example 1, Example 2 and Example 3, it was observed that initial generation of peracetic acid is significantly increased as increase in concentration of triethanolamine.

Example 4
Table 4
Phase 1: Oxygen generating Phase %w/w Quantity in gm
Hydrogen Peroxide
HEDP Acid
Benzotriazole
C12-14 fatty alcohol ethoxylate
Glycerine
Water 7.5%
0.1%
0.05%
0.1%
1%
QS to 100 gm 1000 gm
Phase 2: pH modifier phase
Triethanolamine 100% 2.4 gm
Phase 3: Acetyl group generating phase
N-acetylcaprolactum 100% 20 gm

From the observation of previous examples, as such triethanolamine was used in example 4. Oxygen generating phase was prepared as per previous examples while acetyl group generating phase was also same. 2.4 gm of triethanolamine was consumed to achieve pH 7 to 8.

After mixing 0.1320% w/w of peracetic acid was generated within 5 mins and it was stable till 60 days. pH of the in-situ preparation was between 3 to 5. When as such triethanolamine was used as pH modifier phase higher quantity of peracetic acid was generated.

Example 1 to 4 explains, role of pH modifier’s concentration which has a significant impact on Peracetic acid generation.

Example 5
Table 5
Phase 1: Oxygen generating Phase %w/w Quantity in gm
Hydrogen Peroxide
HEDP Acid
Benzotriazole
C12-14 fatty alcohol ethoxylate
Glycerine
Water 7.5%
0.1%
0.05%
0.1%
1%
QS to 100 gm 1000 gm
Phase 2: pH modifier phase
Triethanolamine
Dipotassium Hydrogen Phosphate
Potassium Dihydrogen Phosphate 50%
1%
0.02% 4.4 gm
Phase 3: Acetyl group generating phase
N-acetylcaprolactum 100% 20 gm

In this example, oxygen generating phase is prepared by diluting 150 gm 50% hydrogen peroxide and dissolving other ingredients to the distilled water up to 1000 gm. pH of oxygen generating phase is between 2 and 4. Quantity of acetyl group generating phase is same as previous examples. pH modifier phase is prepared by using 50% of triethanolamine, 1% of Dipotassium Hydrogen Phosphate, 0.02% of Potassium Dihydrogen Phosphate and remaining quantity of distilled water. pH of the said phase was between 11 and 12.

For pH enhancement of oxygen generating phase, 4.4 gm of pH modifier phase was consumed. The said quantity of pH modifier phase is almost equal to example 2 (4.5 gm). An in-situ preparation of example 5 showed an increased quantity of peracetic acid (0.1402% w/w). The generated peracetic acid is stable over 60 days and its pH was found between 3 and 5. Addition of buffering agents in pH modifier phase was gradually increased the peracetic acid generation.

Figure 1 represents stability of peracetic acid in the in-situ activated batches while pH stability has described in Figure 2 for 60 days.

Example 6
Table 6
Phase 1: Oxygen generating Phase %w/w Quantity in gm
Hydrogen Peroxide
HEDP Acid
Benzotriazole
C12-14 fatty alcohol ethoxylate
Glycerine
Water 4%
0.1%
0.05%
0.1%
1%
QS to 100 gm 1000 gm
Phase 2: pH modifier phase
Triethanolamine 100% 2.4 gm
Phase 3: Acetyl group generating phase
N-acetylcaprolactam 100% 20 gm

In this example, oxygen generating phase of hydrogen peroxide at 4% by weight, it was prepared by diluting 80 gm of 50% hydrogen peroxide, dissolving other ingredients with distilled water needed to 1000 gm batch. pH of prepared phase is between 2 to 4. As such triethanolamine is used as pH modifier phase and acetyl generating phase is as such N-acetyl caprolactam.

Activation is performed as per the design and 5 minutes of mixing time. Reduction of hydrogen peroxide quantity showed an impact on peracetic generation i.e. 0.0689% w/w of peracetic acid was generated.

Example 7
Table 7
Phase 1: Oxygen generating Phase %w/w Quantity in gm
Hydrogen Peroxide
HEDP Acid
Benzotriazole
C12-14 fatty alcohol ethoxylate
Glycerine
Water 15%
0.1%
0.05%
0.1%
1%
QS to 100 gm 1000 gm
Phase 2: pH modifier phase
Triethanolamine 100% 2.4 gm
Phase 3: Acetyl group generating phase
N-acetylcaprolactam 100% 20 gm

In continuation with Example 6, Hydrogen peroxide quantity was increased in the example 7. An oxygen generating phase was prepared by diluting 200 gm of 50% hydrogen peroxide, dissolving other ingredients in distilled water needed to 1000 gm of batch. pH of the phase was between 2 and 4. As such triethanolamine and N-acetylcaprolactam were used.

Increase in concentration of hydrogen peroxide leads to increase in peracetic acid generation i.e., 0.5108% w/w after 5 minutes of mixing time. It can be expressed that hydrogen peroxide concentration has significant effect on peracetic acid generation.

Example 8
Table 8:
Phase 1: Oxygen generating Phase %w/w Quantity in gm
Hydrogen Peroxide
HEDP Acid
Benzotriazole
C12-14 fatty alcohol ethoxylate
Glycerine
Water 7.5%
0.1%
0.05%
0.05%
1%
QS to 100 gm 1000 gm
Phase 2: pH modifier phase
Triethanolamine 100% 2.4 gm
Phase 3: Acetyl group generating phase
N-acetylcaprolactum 100% 20gm

In this example, Oxygen generating phase was prepared by diluting 150 gm of 50% hydrogen peroxide and dissolving other ingredients to the distilled water needed for 1000 gm batch. The quantity of surface active agent has reduced in this example. pH of oxygen generating phase is between 2 and 4. As such triethanolamine and N-acetylcaprolactam has used as pH modifier phase and acetyl group generating phase.

After execution of Example 8, 0.0602% w/w of peracetic acid generated in 5 minutes of mixing time, while 0.0829% w/w in 10 minutes. Surface active agent reduces the surface tension between acetyl group generating phase and oxygen generating phase which resulting in quick reaction between acetyl donor and oxygen donor and it leads to rapid peracetic acid generation. In accordance with this Example 8, the concentration of surface active agent has reduced and it leads to delay in time required for peracetic acid generation.

Example 9
Table 9
Phase 1: Oxygen generating Phase %w/w Quantity in gm
Hydrogen Peroxide
HEDP Acid
Benzotriazole
C12-14 fatty alcohol ethoxylate
Glycerine
Water 7.5%
0.1%
0.05%
0.2%
1%
QS to 100 gm 1000 gm
Phase 2: pH modifier phase
Triethanolamine 100% 2.4 gm
Phase 3: Oxygen generating Phase
N-acetylcaprolactum 100% 20 gm

The quantity of surface active agent was increased in this example, hydrogen peroxide and ingredients other than surface active agent were same as Example 8. pH of oxygen generating phase was between 2 and 4. As such triethanolamine and N-acetylcaprolactam was used as pH modifier phase and acetyl group generating phase, respectively.

Surface active agent plays an important role in peracetic acid generation this was proved in this example. After mixing for 5 minutes, 0.1517% of peracetic acid was generated and it remained stable up to 60 days as well as pH was also in 3 to 5 range.

Example 8 and Example 9 demonstrate the role of surface active agent into the formulation, peracetic acid generation accelerates as surface active agent concentration is increased and vice versa.

Example 10:
Anti-microbial Efficacy test of the in-situ preparation:
Data obtained from anti-microbial tests, it was clearly observed that peracetic acid of 0.08% w/w to 0.15% w/w extensively exhibited bactericidal, sporicidal, yeasticidal, fungicidal and mycobicidal activity.

To determine bactericidal activity of the activated in-situ preparation, was tested against Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 8739).

Table 10: Reduction (Log10-) in Pseudomonas aeruginosa
Test Microbe Contact time Reduction (Log10-)
Pseudomonas aeruginosa
(ATCC 9027) 30 sec 7.11
45 sec 7.72
1 min 8.41

Table 11: Reduction (Log10-) in Staphylococcus aureus
Test Microbe Contact time Reduction (Log10-)
Staphylococcus aureus
(ATCC 6538) 30 sec 7.29
45 sec 7.47
1 min 8.47

Table 12: Reduction (Log10-) in E Escherichia coli
Test Microbe Contact time Reduction (Log10-)
Escherichia coli
(ATCC 8739). 30 sec 7.24
45 sec 7.72
1 min 8.41

The bactericidal efficacy of an in-situ preparation was confirmed, an activated in-situ preparation showed >7 log reduction in 30 seconds and 40 seconds contact time while it has showed >8 log reduction for 1 min contact time when tested against Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 8739).

Table 13: Reduction (Log10-) in Bacillus subtilis
Test Microbe Contact time Reduction (Log10-)
Bacillus subtilis
(ATCC 6633) 30 sec 6.74
45 sec 7.18
1 min 8.18

Bacterial spores are a highly resistant cell type formed when certain members of the Firmicutes, e.g. Bacillus and Clostridium spp., encounter environmental stress, commonly nutrient starvation.

The bacterial spore structure differs markedly from that of the vegetative cell; differences which confer upon the spore remarkable resistance to many environmental stresses, including extremes of temperature, radiation and chemical assault.

These heightened resistance properties also confer upon the spore significant resistance to disinfection and sterilization procedures, relative to their vegetative cell counter parts.

Spores of Bacillus subtilis (ATCC 6633) were tested to assess their susceptibility to peracetic acid with concentration of 0.08% to 0.15%. Table No.13 exhibited excellent sporicidal activity. An activated in-situ preparation exhibited > 6 log reduction in 30 seconds, >7 log reduction in 40 seconds and >8 log reduction in 1 min contact time respectively.

Table 14: Reduction (Log10-) in Candia albicans
Test Microbe Contact time Reduction (Log10-)
Candida albicans
(ATCC 10231) 30 sec 8.04
45 sec 8.04
1 min 8.04

If a product is active against Candida albicans, the product is classed as yeasticidal, meaning it is sufficiently active against all yeasts. If the disinfectant is active against both C. albicans and A. brasiliensis, it is labelled as fungicidal.

Table 14 describes yeasticidal activity of the activated in-situ preparation against Candia albicans (ATCC 10231). An activated in-situ preparation showed >8 log reduction in 30 seconds, 45 seconds and 1 min contact time

Table 15: Reduction (Log10-) in Aspergillus brasiliensis
Test Microbe Contact time Reduction (Log10-)
Aspergillus brasiliensis (ATCC 16404) 30 sec 6.78
45 sec 6.78
1 min 6.78

Fungicidal activity of the in situ product was checked against Aspergillus brasiliensis (ATCC 16404). Results are described in table 15. An activated in-situ preparation exhibited excellent fungicidal properties; 6.78 log reductions are obtained within 1 min. Peracetic acid with 0.08% to 0.15% has successfully showed a fungicidal activity.

To determine the efficacy of the system of invention against Mycobacteria, the activated in-situ preparation, was tested against Mycobacterium terrae (ATCC15755). It was observed that the system of the invention showed an effective reduction in Log value of Mycobacteria in 1min.

Table 16: (Log10-) in Mycobacterium terrae
Test Microbe Contact time Reduction (Log10-)
Mycobacterium terrae (ATCC15755) 5 min <5.0

Comparative Example 1:
Physicochemical as well as microbicidal properties of Anioxyde 1000 LD were checked. A market sample has recommended 30 mins activation time after mixing of Generator (Hydrogen Peroxide Phase) and activator (Acetylcaprolactam phase). As per the claims of Anioxyde 1000 LD, 0.1% to 0.2% of peracetic acid will generate after 30 min of mixing and it will be stable up to only 14 days, also the recommended contact time for anti-microbial efficacy is 5 mins.

Activation of Anioxyde 1000 LD was performed by using recommended ratios of Generator and Activator. After 30 minutes of activation, clear pink coloured solution was observed and pH was approximately 6.00. Activated solution was analysed to determine generated peracetic acid. The peracetic acid content was observed 0.1974% w/v.

The bactericidal activity of Anioxyde 1000 LD was tested against Escherichia. Coli (ATCC 87390), Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC 6538) as per recommended contact time point i.e. 5 mins. The product showed > 5 log reductions against Escherichia Coli (ATCC 87390), Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC 6538).

Anioxyde 1000 LD was tested against Aspergillus. brasiliensis (ATCC 16404) Candida Albicans (ATCC 10231) for 5 mins of contact time. The product showed > 5 log reduction against Aspergillus brasiliensis (ATCC 16404) and Candida Albicans (ATCC 10231) contact time (ATCC 10231). Bacillus subtilis (ATCC 6633) was used to check sporicidal activity of Anioxyde 100 LD and it > 5 Log reduction in 5 minutes contact time.

Market product has recommended 30 minutes are required for activation of peracetic acid ranging from 0.1% w/v to 0.5% w/v. In concern with activation time, the present invention discloses the method for the rapid and immediate production of peracetic acid ranging from 0.08% to 0.5% in 5 minutes of activation time.

The present invention showed an excellent microbicidal activity against bacteria, spores, yeast and fungi within 1 minute while marketed product has recommending 5 mins contact time.
, Claims:
1. A system for in-situ generation of peracetic acid comprising three distinct phases, wherein the three phases comprise:
(a) an oxygen-generating aqueous phase or Phase 1;
(b) a pH modifier phase or Phase 2, and
(c) an acetyl group generating phase or Phase 3,
wherein, the system is stable for longer period of time and generates peracetic acid in range of 0.08% to 0.2%.

2. The system as claimed in Claim 1, wherein the oxygen-generating aqueous phase or Phase 1 comprises of:
(i) an anticorrosive agent;
(ii) a chelating agent;
(iii) a surface-active agent;
(iv) a supportive corrosion inhibitor, and
(v) a vehicle.

3. The system as claimed in Claim 2, wherein the anticorrosive agent is present in range of 0.001% to 0.5%.

4. The system as claimed in Claim 3, wherein the anticorrosive agent is selected from group consisting of a heterocyclic compounds.

5. The system as claimed in Claim 4, wherein the heterocyclic compounds is a triazole.

6. The system as claimed in Claim 2, wherein the chelating agent is present in range of 0.01% to 5%.

7. The system as claimed in Claim 6, wherein the chelating agent is an organic phosphonic acid.

8. The system as claimed in Claim 7, wherein the organic phosphonic acid Hydroxyethane diphosphonic acid or a salt thereof.

9. The system as claimed in Claim 8, wherein the Hydroxyethane diphosphonic acid is selected from 1-Hydroxyethane-1, 1-diphosphonic acid, Tetrasodium salt of 1-hydroxyethane-1, 1-diphosphonic acid, Powder of sodium salt of 1-hydroxyethane-1, 1-diphosphonic acid, Powder of 1-hydroxyethane-1, 1-diphosphonic acid.

10. The system as claimed in Claim 2, wherein the surface-active agent is present in range of 0.01% to 5%.

11. The system as claimed in Claim 10, wherein the surface-active agent is non-ionic surface active agent.

12. The system as claimed in Claim 11, wherein the non-ionic surface-active agent is natural fatty alcohols or natural fatty alcohols ethoxylate.

13. The system as claimed in Claim 2, wherein the supportive corrosion inhibitor is present in range of 0.1% to 10%.

14. The system as claimed in Claim 13, wherein the supportive corrosion inhibitor is selected from ethylene glycol, glycerol, propylene glycol, 1, 3-propanediol (trimethylene glycol), and polyethylene glycol 200 and polyethylene glycol 400.

15. The system as claimed in Claim 1, wherein the pH modifier phase or Phase 2 comprises of a triamine.

16. The system as claimed in Claim 15, wherein the concentration of triamine is in range of 50% - 100%.

17. The system as claimed in Claim 15, wherein the triamine is selected from group consisting of selected from 2,2,6,6-tetramethylpiperidine, 2,2,5,5-tetramethylpyrrolidine, 2,6-dimethylpiperidine, 2,5-dimethylpyrrolidine, N-ethylpyrrolidine and N-ethylpiperidine, N,N,N-diisopropylethylamine and N,N,N-triethylamineand preferably 2,2',2''-trihydroxytriethylamine.

18. The system as claimed in claim 1, wherein the pH modifier phase is diluted from 10% to 80% with a solvent.

19. The system as claimed in Claim 18, wherein the solvent is selected from purified water, demineralized water, tap water, sterile water, distilled water.

20. The system as claimed in claim 15, wherein the pH modifier phase, optionally, further comprises a combination of buffering agents.

21. The system as claimed in claim 20, wherein the buffering agent is selected from phosphates and carbonates.

22. The system as claimed in Claim 21, wherein the buffering agent is present in range of 0.001% to 0.5%

23. The system as claimed in claim 1, wherein the acetyl group generating phase comprises of acetyl group donor compound selected from acetic acid, acetaldehyde, N-acetylcaprolactam, acetic anhydride.

24. The system as claimed in Claim 1, wherein said Phase 1, Phase 2 and Phase 3 are stable for 24 months, individually.

25. A method for in-situ generation of peracetic acid in a three-phase system as claimed in Claim 1, wherein the process comprises of:
(a) adding the pH modifier phase or phase 3 of claim 1 to oxygen-generating aqueous phase or Phase 1 of claim 1, and
(b) adding the acetyl group generating phase or Phase 3 of claim 1 in the solution of step (a) to generate peracetic acid in-situ.

26. The method as claimed in Claim 25, wherein the method produces 0.08% to 0.2% w/w of peracetic acid.

27. The method as claimed in Claim 26, wherein the produced peracetic acid remains stable for up to 60 days.

28. The method as claimed in Claim 25, wherein the ratio of Phase 2:Phase 1 is in range of 0.01:50 and 5:500.

29. The method as claimed in Claim 25, wherein the ratio of Phase 2:Phase 1 is 0.5:200.

30. The method as claimed in Claim 25, wherein Mixing time of the step (b) is 5 min to 15 mins, most preferably 5 min to 10 mins.

31. The method as claimed in Claim 25, wherein the peracetic acid generated in a three-phase system as claimed in Claim 1 shows >7 log reduction of bacteria in 30 seconds contact time.

32. The method as claimed in Claim 25, wherein the peracetic acid generated in a three-phase system as claimed in Claim 1 shows > 6 log reduction of bacterial spores in 30 seconds.

33. The method as claimed in Claim 25, wherein the peracetic acid generated in a three-phase system as claimed in Claim 1 shows >8 log reduction of fungus in 30 seconds.

34. The method as claimed in Claim 25, wherein the peracetic acid generated in a three-phase system as claimed in Claim 1 shows >8 log reduction of fungus in 30 seconds.

35. The method as claimed in Claim 25, wherein the peracetic acid generated in a three-phase system as claimed in Claim 1 shows <5 log reduction in mycobacterial load in 5mins.