FIELD OF INVENTION:
The present invention relates to improvement of plasticized polyvinyl chloride formulations more preferably to the improvement of permeability and leachability of plasticized polyvinyl chloride formulations useful in containers for storing blood and blood components.
PRIOR ART:
The Collection And Storage Of Whole Blood And Red Blood Concentrate:
Poly vinyl chloride (PVC) plasticised with di-(2- ethyl hexyl) phthalate is the most preferred material for the manufacture of blood bags for the collection of blood, blood component separation, and storage and has been in regular use for more than 45 years. PVC material can withstand a wide range of stresses during blood bag manufacture and use such as, steam sterilization, centrifugation at high G forces and storage at low temperatures. Various other phthalate plasticisers have also been used in place of di,(2-ethyl hexyl phthalate) such as di,n- octyl phthalate (DOP), di, iso octyl phthalate (DIOP) , di.nonyl phthalate (DNP), di, iso nonyl phthalate (DINP) and di, iso decyl phthalate (DIDP).
Phthalate esters are not covalently bonded to PVC and so could migrate out from the containers made using PVC plasticised with phthalate plasticisers. Due to the very low solubility of phthalates in water, aqueous medical solutions extract very little plasticiser from containers or tubing. When lipids, lipoproteins and albumin containing fluids such as blood is stored in such containers, the leaching of plasticiser is much more and depends on the property of the fluid, storage time, storage temperature and the plasticiser type.
It was reported (in 1970) that di,(2- ethyl hexyl) phthalate from blood bags can leach into stored human blood. This finding triggered a spate of studies on the subject during the subsequent 20 years, many of which showed deleterious and harmful effects.

Concerns have been expressed over the possible adverse effects of Di,(2-ethyl hexyl) phthalate leached into blood products and the potential toxicological hazards. - Many of these deleterious findings, however, were disputed by many subsequent • studies. This was followed by a series of excellent reviews and critical assessment of the existing knowledge by Governmental and Regulatory Agencies which presented a realistic view of the application of phthalate plasticised PVC and its limitations.
The present position is that di,(2-ethyl hexyl) phthalate is not considered harmful even to highly exposed people, those who undergo medical procedures such as regular haemodialysis or extra corporeal membrane oxygenation. It has also been appreciated that di,(2- ethyl hexyl) phthalate imparts a variety of important physical characteristics that are critical to the function of medical devices and eliminating di,(2 ethyl hexyl) phthalate in these products could cause harm. Subsequently, based on various studies done during the period 2000-2007, the Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR) came to the conclusion that there is still some risk involved in the use of di,(2-ethyl hexyl) phthalate plasticised Poly vinyl chloride containers for neonates and other high risk groups.
In Europe, DEHP is the only plasticiser recommended by the European Pharmacopea for containers for the storage of Blood and Blood products. There is however, great pressure from European countries to replace DEHP at the earliest.
Concerted research efforts are under way to find alternatives to PVC, to develop modifications to PVC and for the development and use of better plasticisers with less leaching and toxicity. These efforts have led to the development of various alternatives to PVC such as multi component polymer blends and alloys, cross linked themoplastic elastomers, laminates, co-extruded multi layered structures, copolyesters, ethylene vinyl acetate and thermo plastic polyurethanes. Improved PVC materials is another area of development such as the use of PVC of higher molecular weight, cross linking of PVC, alloying PVC with other polymers and control of the polymer structure. Yet another area of activity is the development of new type of plasticisers by the hydrogenation of various phthalates. Among such products, di, isononyl cyclohexane dicarboxylate (DINCH) is a promising plasticiser with very desirable characteristics, low migration properties and very low toxicity profile.

Various non DEHP plasticisers have been studied with a view to replacing DEHP in - blood bags. Some typical examples are: di, iso decyl phthalate (DIDP), di, iso nonyl phthalate (DINP), di,(2- ethyl hexyl) terephthalate (DEHT), di, (2-ethyl hexyl) adipate (DEHA), tri, (2-ethyl hexyl) trimellitate (TEHTM), various citrate plasticisers and more particularly n-butyryl, tri n-hexyl citrate (BTHC). Of these TEHTM is remarkable in that it has extremely low leaching characteristics. PVC containers plasticised with TEHTM are well suited for the storage of platelet concentrates. BTHC is remarkable in that this plasticiser is suitable for making PVC containers for the storage of whole blood, red cell concentrates and also for the storage of platelet concentrates.
Phthalates have the remarkable property of protecting the cell membrane of the red blood cells from becoming brittle. The membranes of animal cells are mainly composed of phospho lipids, glyco lipids and cholesterol and is present as phospho lipid bi layers consisting of hydrophobic and hydrophilic ends . During storage for more than 21 days, the membrane starts losing lipids and tend to become brittle. Phthalates, particularly DEHP can get into the cell membrane and stabilize them and maintain their viability. In consequence blood can be stored for longer periods for up to 42 days. This is a great advantage provided by many phthalates and a few plasticisers like BTHC. The compatibility of plasticisers with PVC is attributed to the presence of di ester or tri ester groups in the plasticisers. The combined presence of an ester group and on aromatic ring is also believed to be very useful. The size and structure of the alkyl group helps to determine the volatility and gelation temperature of the plasticiser.
There has been a constant search to develop plasticisers which are non-toxic and have lesser leaching characteristics. One such area of development is the hydrogenation of phthalates such as di,(iso pentyl) phthalate, di,(2-ethyl hexyl) phthalate, di,(iso heptyl) phthalate, di,(nonyl) phthalate, di,(iso nonyl) phthalate, di, (iso decyl) phthalate, and so on to the corresponding cyclohexane carboxylic acid di esters. In general, these cyclohexane carboxylic esters have less leaching, improved cold flex properties, have viscosity depressant properties, improve processibility and provide improved gelation properties as compared to PVC plasticised with the corresponding phthalates. Among cyclohexane carboxylic acid

esters, the di, isononyl cyclohexane dicarboxylate (DINCH) has very low teachability and very low toxicity profile. This plasticiser is presently manufactured in large - quantities by M/S. BASF and hence its price is expected to come down in future as new end uses develop. It is presently being used in children's toys, enteral feeding sets, cling films, sports goods, indoor flooring , rain coats and water tanks. Other possible applications are as containers for aqueous parenteral solutions and CAPD bags.
We have studied the use of the plasticiser di, iso nonyl cyclohexane Carboxylate (DINCH) as plasticiser for making blood bags for the storage of blood and red cell concentrates and for the storage of platelet concentrates. . By proper selection of the proportion of stabilizers, lubricants and other additives we could make blood bags meeting the physical, chemical and biological requirements of blood bags. These bags were then used to collect blood , which was separated into components and stored under appropriate specified conditions. The red cell concentrates were examined regularly for biochemical parameters for up to 42 days. We observed some indications of change particularly in the haemolysis of red cells and the leaching of potassium into the serum.
These changes continued up to 42 days of storage. The level of change of these parameters, however, did not proceed beyond acceptable limits but were indicative of weakening of red blood cell membrane. When blends of DEHP or BTHC were used the
red cell membrane was better protected indicating that the plasticisers which leached into the plasma reduced the development of brittleness of the cell membranes by substituting the lipids lost by the membranes.
When TEHTM [Tri,(2-ethyl hexyl)mellitate] was used as the sole plasticiser for blood bags, there was no protection of the cell membranes ( because of the extremely low leaching of the plasticiser and so red cells could be preserved only for around 21 days. However when a blend of DEHP and TEHTM was used as the plasticiser the preservation of RBC improved and was around 42 days when a 1:1 blend of these plasticisers was used.

A very interesting observation made by us was that when Dioctyl adipate was used as the plasticiser, there was very little protection to RBC membranes eventhough this plasticiser had much higher leaching into blood plasma. Similarly di,(2-ethyl hexyl) terephthalate used as plasticiser gave very little protection to RBC membranes. These observations indicate that it is not only the quantity of the plasticiser which leached in to the blood plasma but also the structure of the plasticiser was critical. The structures having a hydrophobic head such as an aromatic ring and a hydrophilic tail/tails such as di or tri esters are similar to the phospho lipids, glyco lipids, and cholesterol present in RBC membranes as phospho lipid bilayers.
The structure of DINCH is similar to that of DEHP and other phthalates with the difference that DINCH has a cyclohexane ring instead of the aromatic ring of phthalates. The cyclohexane ring is much less hydrophobic than the benzene ring. This possibly accounts for the fact that DINCH cannot effectively replace the lipids lost from the RBC membranes.
Our main interest is to develop non DEHP blood bags for the collection and storage of whole blood and red blood cell concentrates. Plasticisers such as DEHA, TEHTM and DEHTP are not suitable as seen from published literature and our studies.
BTHC is a good plasticiser which has been shown to be effective for the storage of whole blood, RBC and platelet concentrates. However it has short comings as follows:
1. BTHC plasticised sheets have a none too pleasant odour.
2. BTHC is expensive compared to DEHP.
3. BTHC plasticised PVC sheets have much higher permeability to oxygen and carbondioxide than what is needed for the storage of RBC which uses a predominantly anaerobic mechanism to get its energy.
DINCH plasticiser has a better smell, is not expensive and sheets made with this plasticiser has permeabilities to oxygen and carbondioxide which is almost similar to DEHP.

We therefore made detailed studies to use blends of DINCH and BTHC as plasticiser to make blood bags suitable for the storage of whole blood and red cell ' concentrates.
The proportion of DINCH in the BTHC-DINCH blend is very important. In the case of DEHP and BTHC the leachability to soapy water( which is very close to the leachability into blood plasma) varies from 2-2.5% by weight for sheets formulated for main blood bag requirements. The leachability from DINCH plasticised sheets is in the range 1-1.5% . DINCH, because of its affinity to BTHC holds back the leaching of the latter. In consequence the quantity of leached BTHC would come down markedly in blends. However at least 1% of leached BTHC has to be available to enable the stabilization of red cell membranes when blood or red cell concentrates are stored.
Our studies showed that the preservation of red cells improved when the proportion of BTHC in the BTHC-DINCH blend was above 50 percent and more preferably above 60 percent as indicated by better maintanence of pH, RBC haemolysis and level of K+ in the plasma. DINCH has very good solubility for BTHC and this contributes to reduce the leaching of BTHC from the blend.
An embodiment of the present invention provides for the use of a blend of the plasticiser DINCH with BTHC for plasticising PVC for the manufacture of containers for the storage of whole blood and / or red cell concentrates prepared therefrom, in which the proportion of BTHC in the BTHC-DINCH blend is above 50 percent and more preferably above 60 percent.
The Collection And Storage Of Platelets:
There has been lot of efforts recently to increase the recovery and quality of platelets from collected blood. These efforts have resulted in the development of the pooled buffy coat system which has many advantages as compared o the platelet rich plsma (PRP) method.
The recovery of platelets by the PRP method is 60-75% whereas by the buffy coat method as applied to individual blood units (450ml) is in the range of 40-60%.

However if buffy coats are processed after pooling, the recovery increases to 60 -75%.
The WBC contamination of platelets by the PRP method is 1-2 per 1000 platelets. For the buffy coat method, for individual units of blood, the WBC contamination is 1to2 per 10,000 platelets and is lowest for pooled platelets recovered by the buffy coat method using automatic component separators, at 1 to 2 units per 100,000 platelets.
Platelets get 85% of their energy from aerobic metabolism in which glucose, free fatty acids and amino acids are used. Another 15% of the energy of platelets are derived from glycolysis in which glucose is converted to lactate which acidifies the medium. The conversion of glucose by the oxidative mechanism is around 18 times more efficient than anaerobic glycolysis. These mechanisms highlight the fact that when the containers in which platelets are stored allow sufficient oxygen to get in to provide high oxygen partial pressure, the oxidative mechanism prevails. When the containers do not allow sufficient oxygen to get in, the aerobic metabolism shifts to anaerobic glycolysis resulting in the increased production of lactate and decrease in pH.
The permeability to carbondioxide of the containers has a very critical role to play. When the oxidative pathway is predominant, the carbondioxide formed gets converted to bicarbonate which acts as a buffer in the plasma. Thus the permeability of the container should allow a good part of the carbondioxide formed to escape, but if the escape is too high as would happen when the carbondioxide permeability of the container s too high, the production of bicarbonate would be compromised and the pH would tend to increase beyond 7.2 with disastrous results.
When the quantity of stored platelets are high as in the case of pooled platelets prepared by the buffy coat method, the oxygen and carbondioxide permeabilities of the containers become very critical and consequently difficult to achieve.
The sizes of the containers for the storage of pooled platelets by the buffy coat method vary depending on the application from 600ml to 1200ml, one litre being the

generally prefered volume keeping in view the ease of handling and the necessity to keep the platelets agitated in platelet agitators at 22-23°c. In general, a pool of 6 ' platelet units should be sufficient per adult which may be increased to 10 if required. The requirements of container volume, total surface area and permeabilities to oxygen and carbondioxide defines the container parameters.
Plasticisers for pvc containers for the storage of platelet concentrates:
DEHP plasticised containers can preserve the viability of platelets for up to six days, but the aggregation characteristics are compromised beyond three days. BTHC plasticised containers also behave similarly. A very distinct characteristic of BTHC plasticised PVC containers is that the permeabilities to oxygen and carbondioxide can attain very high levels. The high carbondioxide permeability of BTHC plasticised containers could result in the development of high pH levels in stored platelet concentrates when the concentration of platelets are low. In the case of DEHP and BTHC, the leachability of the plasticiser possibly accounts for compromising the aggregation characteristics of platelets.
The object of the present invention was to make use of the high levels of oxygen and carbondioxide permeabilities possible with the use of BTHC as plasticiser for PVC and at the same time reduce the leachability of BTHC by blending with the use of a non toxic, less teachable non phthalate plasticiser. Such a blend of plasticisers would provide a container which
1) has low leaching.
2) has high permeability to oxygen and carbondioxide so as to be suitable for the storage of larger quantities of platelets as obtained from 6 to 10 units of blood.
3) Would bring down the carbondioxide permeability to acceptable limits.
4) Would reduce the unpleasant odour of BTHC.
Tri octyl trimellitate (TEHTM) is a plasticiser with extremely low leachability and has a proven record as a reliable plasticiser for PVC containers for the storage of platelets. The permeabilities to oxygen and carbondioxide could be increased by using higher levels of this plasticiser, but this would make the containers very

pliable and soft and compromise its mechanical properties. Another factor to be considered is that TEHTM is invariably contaminated with DEHP because of the ' processing steps involved in its manufacture.
Di,n-decyl phthalate is a very low leaching plasticiser developed by M/s Terumo Corporation, Japan. Bags made using this plasticiser has 1/100 th the leachability of DEHP plasticised bags. It is also less toxic and less mutagenic. The plasticiser, however contains DEHP and its availability is limited.
DINCH
DINCH is 1,2,- cyclohexane dicarboxylic acid, diiso nonyl ester which was developed by M/s BASF. It has an excellent toxicological profile, no carcinogenicity, low migration characteristics and has a wide range of regulatory approvals. Our Patent Application 254/CHE/2006 covers the use of DINCH plasticised PVC containers for the storage of platelet concentrates.
DINCH plasticised platelet storage bags of 400-450 ml capacity are very well suited for the storage of platelets collected from one unit of blood (450ml). The notable advantage with this plasticiser is that the containers being DEHP free, the contamination of platelets with DEHP is eliminated. Further, the undesirable suppression of the aggregation characteristics of platelets is also avoided.
DINCH plasticised PVC bags have limited permeability to oxygen and carbondioxide . Further increase is possible if high levels of DINCH is used as plasticiser which would however adversely affect the mechanical strength of the containers.
The blending of BTHC with DINCH provides a number of advantages for making platelet storage bags and pooling bags. Blending of BTHC and DINCH would enable bags to be made with a range of permeability levels suited to specific end uses. BTHC has a pronounced typical odour which can be brought down in blends . Platelet storage bags plasticised with BTHC have a negative effect on the aggregability of stored platelets. Incorporation of DINCH would reduce the leaching of BTHC and thereby reduce the deleterious effect on platelet aggregation. In

addition, this procedure would help to bring down the cost of blood bags. There are also advantages such as improved low temperature resistance and better mechanical strength in the case of blends.
An embodiment of the present invention therefore provides for the use of a blend of the plasticiser DINCH with BTHC for plasticising PVC for the manufacture of containers for the storage of platelet rich plasma or platelet concentrates.
The proportion of BTHC in blends is very important. Up to 20 per cent BTHC In BTHC-DINCH blends is well suited for the storage of platelet concentrates recovered from 450ml of whole blood when the initial platelet count is at a higher level of 10-12 X10A10 per 450ml of blood. Up to 80% BTHC in blends is needed for the storage of platelet concentrates recovered from larger quantities of blood such as pooled platelet concentrates separated from several units of blood of 450ml each. The tendency for the deterioration of platelet aggregation at higher levels of BTHC in the BTHC-DINCH blends is a constraint.
The formulation of PVC compound for the collection and storage of whole blood and components separated therefrom such as red blood cell concentrates and platelet concentrates is similar except that the levels of the plasticiser used and the proportion of BTHC in the BTHC-DINCH blend is different.
The formulation of the PVC compound consists of
1. PVC of K value as determined using 1.0% cyclohexananone -1000
parts by weight.
2. A plasticiser system consisting of a blend, of BTHC and DINCH-
300-700parts by weight.
3. An epoxidised vegetable oil rich in linoleic acid and containing not less than 6% by weight of oxirane oxygen-30 to 100 parts by weight. This component may be selected from epoxidised soya bean oil or linseed oil or mixtures therof.
4. A non toxic stabilizer system consisting essentially of calcium and zinc salts of fatty acids containing 8 to 18 carbon atoms - 5 to 15 parts. This component may be selected from calcium palmitate, calcium

stearate, zinc octanoate, zinc laurate , zinc myristate,zinc palmitate or zinc stearate of blends of the above shich may be dispersed in an epoxidised vegetable oil Proprietory preparations containing the components described above may also be used.
5. Up to 40 parts by weight of an ester having the structural formula CH3. (CH2) n COOR where n is 10 to 16 and R is an alkyl group containing 4 to 10 carbon atoms.This component may be butyl stearate, octyl stearate, nonyl stearate or mixtures there of.
6. Up to 15 parts by weight of a saturated aliphatic hydrocarbon having 10 to 20 carbon atoms preferably containing branched side chains and having an average molecular weight in the range 200 to 300. This component may be medical grade liquid paraffin.
7. The plasticiser system for containers for blood and red cell concentrates consist of a blend of BTHC and DINCH in which the BTHC content is at least 50 percent by weight.The quantity of plasticiser required is 40 to 50 parts per 100 parts by weight of PVC.
8. The plasticiser system for containers for the storage of platelet concentrates consist of a blend of BTHC and DINCH in which the BTHC content was 20 to 80 percent by weight. The quantity of plasticiser to be used is in the range 50 to 80parts per 100 parts by weight of PVC depending upon the quantity of platelets to be stored in the container.
The permeabilities to Oxygen & Carbondioxide of the containers for the storage of blood products is related to the composition, particularly the plasticiser level , thickness of sheet and the total internal surface area of the containers.The optimum thickness of the sheets used as found in our trials was in the range 32 to 42 mm to meet the requirements of tensile strength , elongation , tear strength and to withstand centrifugation forces of up to 5000 g .
This application for a patent relates to the use of blends of n-butyryl, tri n-hexyl citrate (BTHC) and 1,2- Cyclohexane di carboxylic acid, di isononyl ester (DINCH) as plasticiser for polyvinyl chloride (PVC) for making di,(2ethylhexyl)phthalate (DOP) free containers for the storage of whole blood, and components separated therefrom such as red cell concentrates, platelet rich plasma and platelet concentrates.

1) A process for improving the permeability and teachability of DINCH plasticized polyvinyl chloride formulation (D) consisting of a composition (C) the said composition preparation for making storage containers, characterized in blending a DINCH plasticizer (A) with another plasticizer such as n-butyryl, tri n-hexyl citrate (BTHC) (B) to form a resultant improved DINCH plasticizer composition (C).
2) The formulation (D) as claimed in claim 1, further comprising :

a) PVC (E) of K value ( in 1% cyclohexanone) K 67 to K76 in the proportion of 1000 parts by weight,
b) a blend of said improved DINCH with BTHC to form improved DINCH plasticized polyvinyl chloride (C) in the proportion of 300 to 700 parts by weight,
c) an epoxidised vegetable oil rich in linolic acid containing not less than 6% of oxirane oxygen, in the proportion of 30 to 100 parts by weight,
d) a non toxic stabilizer of calcium and zinc salts of fatty acids containing 8 to 18 carbon atoms in the proportion of 5-15 parts by weight,
e) an ester having the structural formula CH3(CH2)n-COOR where n is 10 to 16 and R is an alkyl group containing 4 to 10 carbon atoms in the proportion of upto 40 parts by weight, and
f) a saturated aliphatic hydrocarbon having 10 to 20 carbon atoms preferably containing branched side chains and having an average molecular weight in the range 200 to 300 and in the proportion of 15 parts by weight.

3) The formulation as claimed in claim 2, wherein the epoxidised vegetable oil of Claim 2 (c) is selected from epoxidised soybean oil, linseed oil or mixtures thereof.
4) The formulation as claimed in claim 2, wherein the stabilizer of claim 2 (d) is calcium palmitate, calcium stearate, zinc octonoate, zinc laurate, zinc myristate, zinc palmitate or zinc stearate or blends thereof.
5) The formulation as claimed in claim 2, wherein the ester of claim 2 (e) is butyl stearate, octyl stearate, nonyl stearate or mixtures thereof.
6) The formulation as claimed in claim 2, wherein the aliphatic hydrocarbon of claim 2 (f) is a medical grade liquid paraffin.
7) The formulation as claimed in claim 2 for making storage containers adapted for storage of whole blood and red blood cell concentrates, wherein the blend of Claim 2 (b) is a blend of DINCH and BTHC, such that the BTHC forms 50 - 100% of the blend by weight, and more preferably 60 to 100 % of the blend by weight.
8) The formulation as claimed in claim 2 and claim 7, wherein the proportion of PVC of claim 2(a) and blend composition of claim 2(b) characterized such that the blend (C) of claim 2(b) is 40 to 50 parts per 100 parts by weight of the PVC (E) of claim 2(a).
9) The formulation as claimed in claim 2 for making storage containers adapted for storage of pooled platelet concentrates is a blend of DINCH and BTHC such that the BTHC forms preferably 20 - 80% of the blend by weight.
10) The formulation as claimed in claim 2 and claim 9, wherein the proportion of PVC of claim 2(a) and blend composition of claim 2(b) characterized such that the blend (C) of claim 2(b) is 50 to 80 parts per 100 parts by weight of PVC (E) of claim 2(a).

11) A improved DINCH plasticized polyvinyl chloride formulation (D) consisting of a composition (C) the said composition preparation for making storage containers, characterized in a blend of a DINCH plasticizer (A) with another plasticizer such as n-butyryl, tri n-hexyl citrate (BTHC) (B) forming a resultant improved DINCH plasticizer composition (C).