IL97883A - Stable antimicrobial glutaraldehyde system - Google Patents

Description

STABLE ANTIMICROBIAL GLUTARALDEHYDE SYSTEM STABLE ANTIMICROBIAL GLUTARALDEHYDE SYSTEM BACKGROUND OF THE INVENTIO Due to the complexity of the cidal mechanisms responsible for the antimicrobial ac lfin nf glutaraldehyde solutions, it has heretofore been impossible to prepare high-level concentrates fuo to 25* /v) of these solutions which could be diluted with potable Wftt-.er fr»r eubeequ nt ueo by the oona mcir. The only concentrated glutaraldehyde solution ever known to be marketed for direct dilution by the user was an acid solution (pH between 2.7 and 3) with a maximum glutaraldehyde content of 10%. Because this acid solution could not contain sodium nitrite as an anti-corrosion chemical, it was corrosive, and so this glutaraldehyde concentrate had very limited use and could not be used on delicate medical instruments (fiberoptics, etc. ) .

To be commercially useful as a surface disinfectant, a glutaraldehyde. concentrate must be stable at room temperature over a period of at least 12 to 24 months. As an example, during this 1 to 2 year period the variation in concentration of active ingredients in a nominal 20% w/v glutaraldehyde solution should not vary greater than 0.25% w/v per year. In addition to the stability problem of the glutaraldehyde per ee, there is also a problem with pH stability of the concentrate. Both alkaline and acid glutaraldehyde solutions when stored at room temperature drift toward lower pH. Without an buffer capacity, the pH of a 20% glutaraldehyde concentrate, as well as the pH of a corresponding 1:10 dilution, would drop rapidly. It will be seen from Table 1 that if the lowest acceptable pH for a commercial glutaraldehyde disinfectant is 5.6, a 20% unbuffered concentrate would be usable for only about one month. The unbuffered 1:10 dilution from this concentrate would be usable for only 21 days. An acceptable drop in pH for -a commercial—glutaraldehyde 97 - 2 - disinfectant, in concentrate or diluted form, should not be more than half a pH unit over a two-year (24 month) period.

Variations of pH in aqueous glutaraldehyde solutions may also affect the cidal activity of these solutions. This had been demonstrated by Paul M. Borick (Adv. Appl. Microb., 10:291-312, 1968) testing the sporicidal efficacy of a 2% aqueous glutaraldehyde solution against spores of B. subtilis. Table II reproduces the results of this author. In 1972, R. M. G. Boucher, et al, (Proc West Pharmacol. Soc, 16:282-288, 1973) provided a theoretical explanation for the microbial influence of pH in aqueous glutaraldehyde compositions. An aqueous glutaraldehyde composition contains a small amount of the pure aldehyde molecule called the monomer. This monomer is always present in equilibrium with larger, more complex molecules call hydrates (see R. M. G. Boucher, Respiratory Care, Nov. 78, Vol. 23, No. 11, 1063-1071). Hydrates result from the condensation or polymerization of the small monomers into larger agglomerates. In any aldehyde solution, an equilibrium is quickly established between the small monomers and the large polymers. The monomer is the primary cidal agent, i.e., the "killer molecule", in aqueous aldehyde solutions. The cidal efficacy of any aldehyde-containing formula seems to be directly related to the number of monomer molecules present at the time of use of the solution. As shown in Figure 1, in aqueous alkaline solutions, the monomer (OHC-CH2-CH2-CH2-CHO) polymerizes into polymers of the type II, III, and IV.

The formation of these type polymers is irreversible and the polymers cannot return to the active monomer form even by heating or ultrasonation. Aging glutaraldehyde in the alkaline pH range greatly accelerates the formation of irreversible polymers. Glutaraldehyde monomers to the contrary, have a slower rate of polymerization in the acid range (see Rasmussen K. E. , et al, Histochemistry, 38:19, 1974). Furthermore, they are in equilibrium with type V polymers whose formation are reversible. This may explain why acid glutaraldehyde solutions have a far longer biocidal shelf life than alkaline solutions. There is also supporting evidence from the work of T. J. Munton (J. Appl. Bact. , 33:410-619, 1970) and D. Hopwood (Histochemie, 17:151, 1968) that the enhanced properties of glutaraldehyde over other monofunctlonal aldehydes (such as methanal) are related to its capacity to react with two amines and thereby cross-link. The reactive amine species is NH2 and not NH3+, thus the cidal expression has a pH relationship. Since acetals and hydrates are more stable at higher pH values, the effective concentration of CHO is reduced in this region which likely explains why cidal efficacy is greatly reduced above pH 9. on the other hand, if the pH is reduced, the concentration of CHO will remain high but the concentration of NH will decrease through conversion into NH3+. A theoretical optimum for cidal activity should therefore, be on the slightly acid side of neutrality. For this reason, the formula object of the present invention targets a final pH of approximately 6·3· \ SUMMARY OP THE INVENTION The present invention relates to an antimicrobial chemical system comprising components which, when mixed and diluted with water in easy and convenient fashion, remain chemically effective for up to two years. The invention involves a disinfection system comprising the pre-packaging in separate containers of specifically buffered glutaraldehyde concentrate having a predetermined pH which, on mixing with a second solution of sodium nitrite and sodium hydroxide and diluting with water, provides both a sporicidal or disinfecting solution which resists decay over time.

One object of this invention was to manufacture an , aqueous glutaraldehyde system, so concentrated (in the 20% to 25% w/v range) that it remained effective with a 12- to 2 -month minimum shelf life when stored at room temperature. Said concentrate could be diluted with 10 to 40 times its volume with potable water and would still provide a stable solution with a pH of 6.30 ± 0.1 and a maximum decay in dialdehyde content' of 0.25% w/v or less per year. The choice of a slightly acid pH for commercial glutaraldehyde disinfectants is the result of a balance of the corrosive action of the components needed to extend the shelf life of these solutions. As the pH is lowered, the shelf life increases. For instance, a fresh 2.24% glutaraldehyde solution with a 3.3 pH has been shown (Ontario Research Foundation report dated May 1, 1974) to contain 2.16%· glutaraldehyde after two years of storage in polyethylene bottles, i.e. a net loss of pH of ,08. However, despite metals passivation, glutaraldehyde compositions with low pH values are far more corrosive than those near neutrality. A pH value about 6.3 seems to provide minimum aggressiveness to metals and plastics while also providing satisfactory shelf stability at room temperature.

Another object of the present invention was to provide a new stable system having a concentrated solution which will enable the consumer to use concentrates up to two-years old (with a glutaraldehyde content up to 25% w/v) to prepare non-corrosive sterilizing and disinfecting solutions by direct dilution with potable water. These solutions, one* diluted, will contain sodium nitrite as an anti-corrosive agent and will also work at a safe pH range closer to neutrality.

Yet another object of this invention is to Provide a system of disinfectant and sporicidal solutions ^"which are, themselves, sufficiently buffered and anti- corrosive such as to be useful in preparing sterilizing solutions useful in manufacturing, delicate medical and surgical instruments and apparatus.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the aldehyde monomer (0HC-CH2- CH2-CH2-CHO) in equilibrium with various polymers having cidal activity and comprised of the monomer; Figure 2 is a time-related-stability graph of the glutaraldehyde concentrate of varying buffering. ; Table I shows the pH variations of a 2% and 20% glutaraldehyde stored at room temperature over a period of time? Table II ehowe the euir ivaVci 1 ϊ +-.y of B. BUbtilis spores in varying pH environments of glutaraldehyde.

Table III is a result-oriented chart of buffered ylutairaldehyde ecnoentrato and the e££«cta of th* -in-iti'al pH? Table IV and V are tables of sporicidal activity of glutaraldehyde made from one- and two-year old concentrates Table VI is sporicidal activity data made from aged glutaraldehyde; and Table VII records the bactericidal and fungicidal activity of an aged glutaraldehyde concentrate.

DETAILED DESCRIPTION OF THE INVENTION The above-described biocidal mechanisms clearly show the importance of pH when one tries to achieve stability and reproducible cidal efficacy data in preparing oominoraial glutaraldehyde r.nmpoaitions . One of the main goals of the present invention was to dilute other than fresh, i.e. a one- or two-year old concentrate of glutaraldehyde with potable water and to automatically end up with a sterilizing/disinfecting solution at the right pH. This was implemented through an object of the present invention/ i.e. the unique packaging and the system.

In 1976, there was introduced in the American market a highly-successful 2.3% w/v glutaraldehyde sporicidal solution (U.S. Patent No. 3,968,250), which was buffered about pH 6.3 and contained both non-ionic surfactants as cidal boosters and sodium nitrite salts as anti-corrosion agents. However, simply making a concentrate ten times stronger than this 2.3% glutaraldehyde did not work because the nitrite reacted with the other constituents. Further, the usable life of such a preparation was found to be only a few days. Therefore, it was one object of this invention to separate the nitrite from the glutaraldehyde solution. The object product of this invention would then consist of two bottles: a larger bottle (400 ml, for instance) of glutaraldehyde concentrate and a smaller bottle (60 ml, for instance) of nitrite solution. The end user will then mix the content of the two bottles together and dilute with water to make a gallon (or four liters) of ready-to-use sterilizing solution.

It was found, howver, that simply by keeping the nitrite separate did not provide the usual and expected results because the buffered concentrate without the nitrite had too high an apparent pH. For instance, one lot (0388), see Figure 2, was prepared with the phosphate buffer, but without the nitrite. The initial pH of the 1:10 dilution was 6,31. The rate of decay for this lot (0388) was 6.4g/l00 ml per year, while the rate of decay for an unbuffered concentrate (lot 0211) was only 0.26g/l00 ml per year (see Figure 2). This high decay rate was not acceptable for a commercial formula wherein the product could be expected to be stored for · an extended period of time before use, such as in warehouses, laboratories, and on shelves.

To resolve this problem, the relationship between the pH of the buffered concentrate and stability was first investigated. The concentration of buffer phosphate salt in today's commerci l 2.3% w/v solution is 0.0429 moles/liter. Therefore, a tenfold concentrate with 0.429 moles of monobasic potassium phosphate per liter was prepared. The pH of this 400 ml solution was 4.29, Four other 400 ml samples of this concentrate with pH ranging from 3.91 to 5.26 were also prepared (see Table III) . The glutaraldehyde concentration of these solutions wae then assayed for up to 150 days. The solution with the highest initial pH of 5.26 had a rate of decay or decrease of 1.5 g% glutaraldehyde per year. While this seemed excessive, the solution with an initial pH of 4.96 had a decay rate of only 0.22 g% glutaraldehyde per year, which is a rate very close to what is observed with the unbuffered concentrate. Solutions with initial pH values less than 4.96 did not have significantly lower decay rates. However, as a matter of practical importance, the lower the pH of the concentrate, the more difficult it is to obtain the correct pH after dilution. Therefore, a pH of about 4.95 seemed to be optimal for the buffered concentrate.

When the glutaraldehyde concentrate is mixed with the contents of the nitrite solution containing bottle and then diluted to make the ready-to-use 2.3% solution, the final- pM must be 6.30 + 0.05. Therefore, while a sufficient amount of sodium hydroxide must be added to the nitrite solution, it it* important that tne amount or sodium hydroxide not be so great that the final pH would be too dependent on the volume of nitrite solution. Specifically, the tolerance on filling a 60 ml bottle for example could easily be + 2 ml. Therefore, the' nitrite solution should not have so much base than an error of 2 ml will produce more than a slight change in the final pH. It was determined experimentally that the nitrite solution should contain 24. Og of sodium hydroxide/1. When 60.0 ml, for example, of this solution is added to 400 ml, for example, of concentrate buffered at pH 4.95 with 0.429 molar phosphate, the resulting solution has a pH of 6.30 ± 0.05. An error of addition of two ml of nitrite produces a pH change in the final solution of only 0.02 pH units.

The nitrite solution prepared in this manner is quite stable. A solution similar to the one described was prepared in one molar sodium hydroxide. The stability of the solution was followed for 127 days in a polyethylene container. The rate of change of the nitrite was about a 2% increase per year (increases in concentration are due to the loss of water through the walls of the polyethylene) . This change in nitrite content is acceptable.

What follows hereafter is a brief description of the methods and equipment used to develop the inventive concept in the preparation, use and packaging of a long-life glutaraldehyde concentrate.

All pH measurements were made at 25 'C using a Kruger and Eckels model 133 pH meter to three decimal places and rounded to two places. The glass electrode was of the extended range type, and the reference electrodes were either the standard calomel electrode or the standard silver/silver chloride type. Buffers to standardize the meter were prepared from ACS grade chemicals using the system proposed by the National Bureau of Standards.

The hydroxylamine hydrochloride method of assay of the glutaraldehyde was a modification of the method official in the United States Pharmacopeia, twenty-first revision (official Monographs USP.XX) . The difference was that the pH meter was used instead of the specified dye to determine the endpoint. This allowed more precise results than would have been otherwise possible. Where possible, all chemicals were ACS grade or better. The 0.5N sulfuric acid was standardized by the official method in the Pharmacopeia with a primary standard of sodium carbonate.

The assay of the nitrite was done by titration with O.IN potassium permanganate and O.IN sodium oxalate. The O.IN sodium oxalate was prepared from a primary standard grade of salt and used to standardize the permanganate.

Packaging A Commercial stable Concentrate The packaging object of this invention consists, for instance, of two containers of suitable material such as polyethylene or similar plastic and preferably bottles. The larger container or bottle contains the buffered glutaraldehyde concentrate with the surfactant and maskant. The smaller container or bottle contains the anti-corrosion salt with sodium hydroxide. For example, to prepare one gallon of 2.3% sterilizing solution, one would pour the entire contents of the sodium nitrite solution bottle contained preferably in 60 ml into a one-gallon empty container. The entire contents of the buffered glutaraldehyde concentrate in a correspondingly related container of about 400 ml and enough potable water (3-1/2 quarts) would also be added to the one-gallon container, resulting in one gallon of ready-to-use 2.3% sterilizing solution. It has been unexpectedly found that these concentrations, dilutions and packages are critically balanced and useful.

To prepare four gallons of disinfecting solution (0.57% glutaraldehyde) one would pour the entire contents of the sodium nitrite solution (60 ml) into an empty four- to five-gallon container. The entire contents of the buffered glutaraldehyde concentrate (400 ml) and 97883/2 - 10 - enough potable water (15-1/2 quarts) would then be added to the four- to five-gallon container to produce four gallons of ready-to-use 0.57% disinfecting solution.

In a preferred formula, the buffered concentrate (23.26% glutaraldehyde) would contain, for instance, 56.58g monobasic potassium phosphate/1 and 1.953g of dibasic sodium phosphate/1. The pH of this buffered glutaraldehyde concentrate is adjusted to a pH of 4.95 + 0.05 with a strong base (i.e., sodium hydroxide) or a strong acid (i.e., hydrochloric acid). The nitrite solution contains for instance, 83.3g of sodium nitrite/1 with 24. Og of sodium hydroxide/1. The latter could vary slightly for different lots of chemicals.

Having described a typical composition of a long-life, i.e., a minimum of about 12-24 months, glutaraldehyde concentrate packaging, there is now given several examples of the cidal efficacy of "ready-to-use" solutions made from one- and two-year old concentrates. These examples are given primarily for the purpose of illustration and should not be construed as limiting the invention to the details given.

EXAMPLES Example 1 After one year storage on the shelf, 400 ml of glutaraldehyde concentrate was assayed with the wet hydroxylamine hydrochloride procedure. The initial content of glutaraldehyde was 23.26% w/v, and the content after one year was 23.04%. The glutaraldehyde concentrate was buffered with a combination of monobasic potassium phosphate (KH2P04) with anhydrous dibasic sodium phosphate (Na2HP04) . It also contained 2.28% of a non-ionic surfactant (Tergitol 15-S-12 from Union Carbide) and 0.073% of a lemon oil maskant fragrance. This concentrate was mixed with 60 ml of a nitrite solution (83.3 g/1) containing some sodium hydroxide (24.0 g/1) , and the resulting mix was adjusted to a volume of one gallon by adding 3-1/2 quarts (i.e., 3325 ml) of potable water.

This fresh solution (2.3%) made from a one-year old concentrate packaging successfully passed a full AOAC sporicidal efficacy test as described in the Official Method of Analysis of the AOAC, 14th edition, chapter 4, 1985. The test was conducted at 20° C with a solution having a 6.4 + 0.1 pH. Results of the test are abstracted in Table IV, Example 2 After two years storage on the shelf, 400 ml of glutaraldehyde concentrate was assayed with the wet hydroxylamine hydrochloride method. The initial content of glutaraldehyde was 23.26% w/v and the content after two years was 22.82%. The buffered glutaraldehyde concentrate also contained 2.28% of a non-ionic surfactant (an ethoxylate of isomeric linear alcohol manufactured by Union Carbide) and 0.073% of a lemon maskant fragrance (type 5A6 manufactured by IFF) , This concentrate was mixed with 60 ml of a nitrite solution (83.3 g/1) containing some sodium hydroxide (24.0 g/l) and adjusted with potable water (3325 ml) to produce one gallon of sterilizing solution) . The final pH of the diluted sterilizing solution made from the two-year old concentrate was 6.3 ± o.i.

This fresh solution (2.28%) made from a two-year old concentrate successfully passed a full AOAC sporicidal efficacy test as described in the Official Methods of Analysis of the AOAC, 14th edition, chapter 4, 1985. This test was conducted at 20" c and the results are reported in Table V.

Example ? Three two-year old concentrate packagings (each having two containers with 400 ml of glutaraldehyde + 60 ml nitrite solution, respectively) were used to produce three gallons of fresh sterilizing solutions. The chemical composition of these samples was identical to the one described in the preceding Example 2. From these three gallons of freshly prepared 2.28% glutaraldehyde solution, 2-1/2 gallons was used to conduct a 30-day reuse test with a complete set of inhalation equipment according to the latest or 1984 EPA protocol. The average glutaraldehyde content at the start of the experiment was 2.25%. After 30-day reuse, the stressed solution, which also contained 2% bovine serum, had a glutaraldehyde content of 1.59%. The initial pH of the 2-1/2 gallon solution was 6.36, while the pH of the 30-day stressed solution was 6.29. The sporicidal activity of this stressed 30-day solution was evaluated with the AOAC method. Results of these tests are given in Table VI.

Example 4 After two years storage on the shelf, 400 ml of glutaraldehyde concentrate was assayed with the wet hydroxy1amine hydrochloride procedure. The initial content of the glutaraldehyde concentrate before aging was 23.30% w/v. After 24 months the glutaraldehyde content decreased down to 22,86%. To prepare four gallons of disinfectant solution (1:40 of the glutaraldehyde concentrate) the contents of the 60 ml sodium nitrite bottle (83.3g of nitrite/1 + 24.0g of NaOH/1) were mixed with the contents of the 400 ml glutaraldehyde concentrate, and sufficient potable water added (15-1/2 quarts or 14,860 ml) to obtain four gallons of disinfectant. 2-1/2 gallons of this disinfecting solution was used to conduct a 21-day reuse test with a complete set of iniiaiation tnerapy equipment according to EPA requirements. The average glutaraldehyde content at the start of the experiments was 0.60% . At the end of the 21-day reuse, the stressed solution, which also contained 2% bovine serum, had a glutaraldehyde content of 0.25%. The bactericidal and fungicidal activities of this stressed solution were evaluated with the AOAC use dilution test with an exposure time of 10 minutes at 20 'C. As can be seen from the data in Table VII, the 21-day reuse disinfection test was successful.

It is to be realized that only preferred embodiments of the invention have been disclosed and that numerous modifications, substitutions, and alterations are all permissible without departing from the spirit and scope of the invention as defined in the following claims.

TABLE I Variation of pH in Unbuffered Glutaraldehyda Solutions Stored at Room Temperature Storage Time 20% 2% in Dava Concentrate (1:10 Dilution) 0 6.53 6.47 21 5.78 5.61 35 5.65 5.48 48 - 5,41 132 5.17 5.00 414 4.63 4.47 TABLE II Ability «*£ lacilloi subtilis Spores To Survive ZZ A-fneoos Glutaxraldehyde Solutions at Dl_ffcc Initial PH 4.66 Initial PH 4.96 0 days 23,33 g% 0 days 23.26 g% 84 days 23.22 g% 117 days 23.21 g% 117 days 23.24 g% 150 days 23.16 g% 150 days 23.28 g% dc/dt - -0.16 g%/yr, dc/dt - -0.22 g%/yr.

Initial pH 5.26 0 days 23.28 g% 52 days 23.13 g% 84 days 22.98 g% 118 days 22.81 g% 150 days 22.69 g% dc/dt » -1.5 g%/yr.

- TABLE IV Spbricidal Activity of A Fresh (2.3%) Glutaraldehyde Solution Made From A One-Year Old Concentrate No. of Positive Carriers/Total Type of qarrt.er Type of Spore No. of Car iers Porcelain Cylinder B. Subtilis (ATCC 19659) 0/240 Suture Loop B. SUbtiliS (ATCC 19659) 0/240 Porcelain Cylinder CI. Sporogenes (ATCC 3584) 0/240 Suture Loop el. sporogenes (ATCC 3584) 0/240 TABLE V Sporicidal Activity of A Fresh (2.2%) Glutaraldehyde Solution Made From A Two-Year Old Concentrate No. of Positive Carriers/Total Type of Carrier Type of Spore No. of Carriers Porcelain Cylinder B. Subtil id (ΑΦΓ.Γ. IQfiB!Q) 0/240 Suture Loop B. Subtilis (ATCC 19659) . 0/240 Porcelain Cylinder Cl, Sporogenes (ATCC 3584) 0/240 Suture Loop CI. Sporogenes (ATCC 3584) 0/240 TABLE VI Spcricidal Activity of A 30-Day Reuse (1.59%) Glutaraldehyde Solution adA Prnw & Two-Yoa* Old ConcciiLJL. a La No. of Positive Carriers/Total Type of Carrier Tvoe of Spore No. of Carriers Porcelain Cylinder B. SUbtilis (ATCC 19659) 0/60 Suture Loop B. SUbtilis (ATCC 19659) 0/60 Porcelain cylinder CI. Sporogenes (ATCC 3584) 0/60 Suture Loop CI. Sporogenes (ATCC 3584) 0/60 TABLE VII Bactericidal and Fungicidal Activity of A 21-Day Reuse (0.25%) Glutaraldehyde Solution Made From A Two-Year Old Concentrate No. of No. of Positive Test Organisms Carriers Carriers Salmonella Choleraesius 60 1 ATCC 10708 Staphylococcus Aureus 60 o ATCC 6538 Pseudomonas Aeruginosa 60 0 ATCC 15442 Klebsiella Pneumoniae 10 0 ATCC 13883 Escherichia Coli 10 0 ATCC 25922 Candida Albicans 10 0 MDA 336 Trichophyton Mentagrophytes 10 0 ATCC 9533

Claims (12)

97883/2 C L A I M S

1. An antimicrobial disinfection system comprising: a container containing a buffered glutaraldehyde concentrate solution capable of being diluted with water to provide a sporicidal or disinfecting solution; and a container containing a solution of an anti-corrosion solution comprising sodium hydroxide and sodium nitrite.

2. A system according to claim 1 wherein said glutaraldehyde concentrate containing container further comprises buffers surfactants and fragrances.

3. A system according to claim 1 wherein said glutaraldehyde concentrate contains about 20 to about 25% w/v glutaraldehyde .

4. A system according to claim 2 wherein said glutaraldehyde concentrate contains a buffering mixture of monobasic potassium and dibasic sodium phosphate salts sufficient to effect a pH of between about 4.0 and 4.96.

5. A system according to claim 2 wherein said glutaraldehyde concentrate comprises not more than about 2.5% w/v of non-ionic surfactants.

6. A system according to claim 2 wherein said fragrances comprise not more than about 0.08% citrus maskant fragrance. 14 - 15 -

7. A system in accordance with Claim 1 wherein said anti-corrosion solution contains between about 80 to about 85 grams of sodium nitrite/liter in combination with about 20 to about 25 grams of sodium hydroxide/liter.

8. A system in accordance with Claim 1 wherein the buffered glutaraldehyde concentrate is contained in a container in a volume of about 6 to about 10 times greater than the volume of the anti-corrosion solution.

9. A system in accordance with claim l in which, for a final volume of about 3760 ml of disinfecting or sporicidal solution, the glutaraldehyde is present in a concentration of about 2,3% w/v at a pH of about 6.3 ± 0.1 after mixing the contents of both containers and adding about 3325 ml of potable water.

10. A system in accordance with Claim 1 sufficient to produce a final volume of about 15000 ml of disinfecting solution having a glutaraldehyde concentration of about 0.57% w/v at a pH of about 6.4 ± 0.1 after mixing the contents of both containers and adding about 14680 ml of potable water,

11. The system in accordance with claim 9 in which the containers comprise. solutions aged up to about two years.

12. The system in accordance with claim 10 in which the containers comprise solutions aged up to about two years. ADVOCATE, PATENT ATTORNEY Ρ.Ο,Β. 23008, TEL-AVIV, 61 230, ISRAEL