Description:Title of Invention
Composition and Method of Preparation for
Oxidized Regenerated Cellulose Powder as Haemostat

Field of invention
The present invention pertains to the technical field of medical materials, specifically to a composition and method for preparing haemostatic powder containing oxidized regenerated cellulose that ensures quick haemotasis and stable clot formation.
Background of the invention
Hemorrhage, the sudden and severe leakage of blood due to blood vessel disruption, is a leading cause of death from injuries globally, accounting for over 35% of pre-hospital deaths and about 40% of deaths within 24 hours of injury. Managing bleeding from vital organs like the kidney, brain, or liver, as well as controlling persistent oozing over broad surfaces, presents a time consuming and challenging aspect of surgical procedures. Conventional methods like suturing or ligation often prove insufficient, leading to surgical delays and frustration.
Traditionally, surgical haemotats, including gauze pads impregnated with agents like ferric chloride or thrombin, have been employed to halt bleeding. However, their removal risks disrupting blood clots and causing re-bleeding, highlighting the need for a straightforward yet effective solution for managing broad surface bleeding.
In order to overcome the above problems, various absorbable topical haemotats have been developed for controlling excessive bleeding. The most commonly used materials include oxidized cellulose, oxidized regenerated cellulose (ORC), gelatin, collagen, chitin, and chitosan. Each of these materials has unique properties that make them suitable for specific types of bleeding and surgical scenarios. For instance, ORC is biodegradable and can be absorbed by the body, making them ideal for internal use where removal post-surgery is not feasible.
Oxidized cellulose (OC) was initially employed as a haemostatic agent in 1942, and by 1960, oxidized regenerated cellulose (ORC) was developed. Derived from plant-based cellulose, ORC undergoes oxidation and regeneration, resulting in a biocompatible and bioabsorbable material widely used in medical applications, with haemostasis being a primary focus. The oxidization process increases the material's ability to promote blood clotting and absorb fluids. This makes oxycellulose and ORC highly effective in various surgical procedures, including neurosurgery, abdominal surgery, cardiovascular surgery, thoracic surgery, head and neck tumor operations, pelvic surgery, and procedures involving the skin and subcutaneous tissues. ORC facilitates haemostasis likely through the activation of the intrinsic coagulation pathway and also creating a gel-like layer (matrix) that holds clot in place and triggers vasoconstriction by the low pH of the ORC.
Commercially available haemotatic agents based on oxidized cellulose have been designed to cater to various surgical needs. Products like Surgicel, Oxycel, Pahacel, CuraCel, Gelita-Cel have become standard tools in many surgical procedures. These products are available in various forms, including powders, non-woven fabrics, knitted materials and fibril, allowing surgeons to choose the most appropriate form for each specific application. Among these, haemotatic powder specifically ORC in powder form, has gained popularity among clinicians due to its efficacy, ease of administration, versatility, and potential to reduce the need for invasive interventions.
Grinding techniques have also been explored to improve the performance of haemotatic materials. U.S. Patent No. 6,627,749 discloses methods for grinding oxycellulose using pestle and mortar or ball mills. This process aims to create finer fibres, which can enhance the material's surface area and improve its ability to absorb fluids and promote clotting. Such advancements are essential for developing next-generation haemotatic agents that are more efficient and effective.
The patent CN104321085B pertains to oxidized regenerated cellulose (ORC) haemotatic powder with specific particle characteristics, including a mean aspect ratio ranging from about 1 to about 18. The present novel invention encompasses a method for producing this haemotatic material, which involves compacting ORC powder using equipment such as ball milling. Additionally, it covers the application of this haemotatic powder to wounds for patient treatment.
Research also explores the mechanical processing of cellulose fibres to enhance their properties. For instance, the study by Howsmon and Marchessault (1959) investigated the effects of ball milling on cellulose fibres, examining how mechanical processing influences cellulose structure and reactivity. Such studies are crucial as they provide insights into how the physical manipulation of cellulose can impact its haemotatic properties, leading to the development of more effective haemotatic materials.
The present invention, titled "Composition and Method of Preparation for Oxidized Regenerated Cellulose Powder as Haemostat," involves a series of carefully designed processing steps that transform ORC fabric into a highly effective haemotatic powder agent. The present novel composition uses dual particle sizes, with larger aggregates providing rapid initial haemostasis and smaller aggregates offering sustained support, ensuring comprehensive coverage of bleeding sites. Is also eligible to be delivered via a sterile applicator for accessibility in hard-to-reach surgical areas, the ORC powder enhances haemotatic efficacy by providing a scaffold for platelet adhesion and aggregation. This approach aims to reduce surgical delays and improve patient outcomes, representing a significant advancement in surgical haemotasis.
Objectives of Invention
• Principal objective of the present invention is to develop a method for producing Oxidized Regenerated Cellulose (ORC) powder with enhanced haemotatic properties, thereby ensuring rapid and stable clot formation (e.g. sustained haemotasis) during surgical procedures.
• Another objective of the present novel invention is to develop a haemostat capable of effectively controlling bleeding over broad surfaces.
• Another objective of the present novel invention is to optimize process variables to achieve a wide range of granule dimensions, from fine to coarse of ORC powder, making it suitable for various surgical applications.
• Another objective of the present novel invention is to provide ORC Powder with particle size between 0 to 1000 microns, 0 to 500 is designed for rapid haemotasis and powder with coarser particle sizes, ranging from 500 to 1000 microns is intended to provide a prolonged haemostatic effect at the anatomical site.
• Further objective of the present novel invention is to prepare a haemotatic agent with a natural composition that is plant-based, user-friendly, cost-effective, eco-friendly, and biocompatible, thereby improving its efficacy in achieving rapid and efficient haemotasis.
• Another objective of the present novel invention is to enhance the structural integrity and stability of the ORC powder by incorporating various binding agents.
• Another objective of the present novel invention is to improve the flowability of ORC powder at the anatomical site through the addition of glidants.
• Another objective of the present novel invention is to prepare a haemostatic powder which can be absorbed substantially within 7 to 14 days and completely absorbed in body within 28 days.
• Further objective of the present novel invention is to prepare a haemostatic powder which is non-irritant, non-sensitizer, non-toxic, non-cytotoxic, non-mutagenic, non-haemolytic and non-pyrogenic.
• Another objective of the present novel invention is to prepare a haemostatic powder which has in-vitro bacteriostatic and bactericidal properties with the effect on growth and multiplication of gram positive and gram-negative organisms.
• Further objective of the present novel invention is to facilitate the application of ORC powder using a sterile applicator in hard-to-reach surgical areas.

Summary
The present invention Composition and Method of Preparation for Oxidized Regenerated Cellulose Powder as Haemostat pertains to a method for fabricating an oxidized regenerated cellulose (ORC) haemotatic powder composition, resulting in a superior product with enhanced performance and uniformity. The present novel invention involves a series of carefully designed processing steps that transform ORC fabric into a highly effective haemotatic powder agent. The key aspects of the present novel invention include optimizing particle size distribution, enhancing handling properties, improving structural integrity, and ensuring product sterility and stability. Traditional haemotatic agents often face challenges in effectively reaching and sealing bleeding sites, especially in complex or deep wounds. The present novel invention addresses these challenges by introducing unique approach with wide spectrum of granule dimensions. The present invention provides a novel haemotatic composition comprising two distinct particle sizes that enhances the haemotatic performance by utilizing larger and smaller aggregates that descend at different rates, ensuring comprehensive coverage of the bleeding site. The larger aggregates descend more swiftly, providing rapid initial haemotasis, while the smaller aggregates sink more slowly and disintegrate during their descent, offering sustained haemotatic support. The present novel ORC powder using a sterile applicator, making it accessible for use in hard-to-reach surgical areas.
List of Drawings
Figure 1: Flowchart
Figure 2: In-vitro Antibacterial study (where, 1: non-oxidized cellulose; 2: Surgicel powder, 3: ORC powder)
Figure 3: Product performance test result’s comparision in graphical presentation
Figure 4: Photomicrographs of subcutaneous implantation sit during 4-week study period. Substantial absorption of ORC powder haemostat and Surgicel observed at 7-day post-implantation and completely absorbed at 28 days post-implantation.
List of Components
1. Oxidized Regenerated Cellulose (ORC) Fabric
2. Wetting/Binding Agent
3. Oxidized Regenerated Cellulose (ORC) Powder
101. Grinding of ORC Fabric
102. Pressurized Molding Process
103. Drying
104. Ball milling
105. Size Separation & Powder Filling
106. Packaging & Sterilization
Detailed Description of the Invention
Outlined in detail below are the procedural steps that comprise the formulation process of the present novel Composition and Method for Preparation of Oxidized Regenerated Cellulose Powder (3) as Haemostat with quick haemotasis and stable clot formation.
Oxidized Regenerated Cellulose (ORC) fabric (1) selected for the powder preparation, should be complied to specifications outlined in the United States Pharmacopoeia (USP) and the results obtained are detailed in the below table (1). The ORC fabric (1) selected for the present invention is original variant, however it can be prepared from other variants of ORC fabric such as knit, fibril, or non-woven prepared with standard validated procedures.

Table 1: Analytical results of an ORC fabric selected for present invention as per USP
Sr. No. Test Acceptance criteria Result
1. Identification Flocculent White precipitate must be formed Complies
2. Loss on Drying NMT 15 % 3.88 %
3. pH 1.0 to 3.0 2.68
4. Residue on Ignition NMT 0.15 % 0.04 %
5. Limit of Nitrogen NMT 0.5 % 0.22 %
6. Limit of Formaldehyde NMT 0.5% 0.0058 %
7. Assay Carboxyl group (COOH): 18.0 to 24.0% 21.1 %

PROCESS VARIANT 1:
Grinding of ORC Fabric (101):
The grinding process for oxidized regenerated cellulose (ORC) comprises mechanically reducing the size of the ORC fabric (1) into smaller particles or fine powder. This process increases the material's surface area, thereby enhancing its suitability for subsequent processing steps.
• Cut the ORC fabric (1) into uniform sizes to make it suitable for grinding.
• The ORC fabric (1) is grounded using a grinding machine for duration of 10 to 20 minutes until a fine powder is produced.
• The grinding machine is operated with a motor speed exceeding 5000 r.p.m. to ensure proper grinding efficiency.
Pressurized Molding Process (102):
The pressurized molding process involves passing the ORC powder (3) through a molding tool under pressure to achieve a desired uniform shape.
• Wetting/Binding agent (2) (0.9% Sodium Chloride solution) is utilized as a wetting/Binding agent (2) to moisturize the ORC powder (3) for the pressurized molding process. The mixture of ORC powder and 0.9% Sodium Chloride solution is evenly churned until uniform mixing is achieved. The mass ratio of wetting/Binding agent (2) to ORC powder selected for this variant is 1:16.
• The uniformly mixed powder is then pressurised to form a cylindrical structure.

Drying (103):
Drying process removes moisture from materials by applying heat, airflow, or other means to reduce moisture content to desired levels. Methods include air drying, oven drying, vacuum drying, or lyophilization, selected based on material properties and intended use.
• The pressurized molded material should undergo a drying process in a vacuum dryer set to a temperature of 35°C ± 5°C. This process should last for a duration of 12 to 16 hours. This controlled vacuum drying process effectively removes moisture from the material while maintaining its structure and performance characteristics.
Ball Milling (104):
Ball milling is a process that involves controlled size reduction of material to achieve granules of specific size using spherical grinding balls, typically steel or ceramic balls, within a rotating cylindrical vessel.
As the cylindrical vessel (mill) rotates, the grinding balls are lifted up the wall of the vessel due to centrifugal force. Once they reach a certain height, gravity causes them to fall, imparting impact forces on the material particles. Simultaneously, as the balls roll and cascade within the vessel, they exert shear and compressive forces on the material particles, causing size reduction through attrition.
The balls in the mill impact dried ORC material, reducing its size into spherical shape.
• In this invention, previously dried material is fed into the mill for size reduction with steel balls.
• The machine is run at a speed of 50-100 r.p.m for 4 to 6 hours.
Size Separation & Powder Filling (105):
Size separation involves passing a material through a predefined mesh or perforated surface to separate particles based on the required size. This process is utilized to achieve a uniform particle size distribution, remove unwanted larger particles or impurities, and classify particles into different size fractions.
• In this invention, an automatic vibro separator/sifter is employed to achieve the desired particle size by utilizing vibrational energy. A vibro separator operates on the principle of gyratory motion and designed to separate materials into different size fractions.
• Particles with sizes ranging from 0 to 1000 microns are separated using vibro separator. The separated ORC powder (3) is filled into a Micro Enema bellow bottle according to the required weight, and the opening of the bellow bottle is closed with the Cannula.
• Here, powder with wide range of particle size selected for the separation. Powder with small particle sizes typically between 0 to 500 micron is utilized for rapid haemotasis across broad anatomical areas. In contrast, powder with larger particle sizes with 500 to 1000 micron is designed to interact with blood, providing a prolonged haemotatic effect at the anatomical site.
Packaging & Sterilization (106):
• Pack the bellow bottles in sterile barrier system in compliance with ISO 11607 series requirement under sterile conditions to maintain product specification until its shelf life.
• Employ gamma radiation for sterilization in compliance with ISO 11137 standard requirement, ensuring the elimination of microbial contaminants while preserving the integrity of the product.
PROCESS VARIANT 2:
Process variant 2 is prepared with a method and steps mentioned in the process variant 1 with notable changes in the process variable parameters and constituent concentration as mentioned below.
Pressurized Molding Process (102):
• In this invention, grinded ORC powder mixed with a wetting agent (2) 0.9% sodium chloride solution with mass ratio of wetting (2) to ORC powder 1:10.
• The uniformly mixed powder is then pressurised to form a cylindrical structure.
Ball Milling (104):
• In this variant, previously dried material is fed into the mill for size reduction with steel balls.
• The machine is set to run at a speed of 50-100 r.p.m for 12 to 14 hours.
Size Separation & Powder Filling (105):
• In this invention, particles with sizes ranging from 0 to 500 microns are separated using vibro separator. The separated ORC powder (3) is filled into a Micro Enema bellow bottle according to the required weight, and the opening of the bellow bottle is closed with the Cannula.
PROCESS VARIANT 3:
Process variant 3 is prepared with a method and steps mentioned in the process variant 1 with notable changes in the process variable parameters and constituent concentration as mentioned below.
Pressurized Molding Process (102):
• In this invention, grinded ORC powder mixed with a wetting agent (2) 0.9% sodium chloride solution with mass ratio of wetting agent to ORC powder 1:3.
• The uniformly mixed powder is then pressurised to form a cylindrical structure.
Ball Milling (104):
• In this variant, previously dried material is fed into the mill for size reduction with steel balls.
• The machine is set to run at a speed of 50-100 r.p.m for 3 to 6 hours.
Size Separation & Powder Filling (105):
• In this invention, particles with sizes ranging from 500 to 1000 microns are separated using vibro separator. The separated ORC powder (3) is filled into a Micro Enema bellow bottle according to the required weight, and the opening of the bellow bottle is closed with the Cannula.
PHYSICOCHEMICAL ANALYSIS OF THE INVENTED MATERIAL:
Important analytical parameter mentioned in the USP and other practical aspect of the ORC powder has been evaluated for the invented product performance.
1. COOH Assay: Assay of carboxylic acid content (quantitative analysis) within the ORC powder; has been analyzed as per procedure mentioned in the USP.
Procedure:
o Place the Sample (1 g) in a conical flask, add 10 mL of 0.5 N sodium hydroxide, swirl to dissolve, and add 100 mL of water.
o Immediately titrate with Titrant to a phenolphthalein endpoint. Perform a blank determination, and note the difference in volumes required.
o Each mL of the difference in volumes of 0.1 N hydrochloric acid consumed is equivalent to 4.50 mg of carboxyl groups (COOH).
o Acceptance criteria: 18.0%–24.0% on the dried basis

2. Angle of repose: Angle of repose has been analyzed as per procedure mentioned in the USP General Chapter <1174>. The angle of repose is a measure used to describe the flowability of a powder. It is defined as the maximum angle between the surface of a pile of powder and the horizontal plane at which the powder remains stable without sliding.
The angle of repose parameter is crucial for determining the flowability of powders within bellow bottles. Powders with favourable flow properties reduce the need for manual intervention, such as tapping or shaking the bottle.
Procedure:
o Form the angle of repose on a fixed base with a retaining lip to retain a layer of powder on the base. The base should be free of vibration. Vary the height of the funnel to carefully build up a symmetrical cone of powder.
o The funnel height should be maintained approximately 2–4 cm from the top of the powder pile as it is being formed in order to minimize the impact of falling powder on the tip of the cone.
o Determine the angle of repose by measuring the height of the cone of powder and calculating the angle of repose, α, from the following equation;
tan (α) = height/0.5 × base
o And the flowability of the powder can be defined as per below table (2):
Table 2: Flow property and angle of repose co-relation
Flow Property Angle of Repose (degrees)
Excellent 25–30
Good 31–35
Fair 36–40
Passable 41–45
Poor 46–55
Very poor 56–65
Very, very poor >66

3. Particle size distribution: Distribution of the particle size is measured by Dynamic Light Scattering as per ISO standard 22412:2017 and USP General Chapter <430>.
Procedure:
o The sample of the ORC Powder shall be dispersed in a liquid medium of Deionized Water.
o Place the measurement cell containing the sample in the sample holder, and wait until temperature equilibrium is reached between the sample and the sample holder.
o It is recommended to measure and maintain the temperature to within ±0.3˚C between both the sample and the sample holder. Perform a preliminary measurement of the sample, and set the particle concentration within the appropriate range.
o Confirm that no significant settling has occurred in the sample at the end of the measurement.
4. Tapped Density: Tapped Density of the ORC is measured as per USP General Chapter <616>.
Procedure:
o Fill 100 gm ORC powder sample in 100mL measuring cylinder.
o Carry out 250 taps per min from height 3 ± 0.2 mm two times and measure the volume to the nearest graduated unit. The difference between two measurements should be less than or equal to 1 mL.
o Calculate the tapped density (g/mL) using the formula mass/volume.

Table 3(A): Physicochemical Analysis Result of each Process Variant
Variant COOH Assay (%) Angle of repose (°)/
Flow property Average Particle size distribution (micron) Tapped Density (g/mL)
1 22.3 29/ Excellent 535 ± 5 0.50 ± 0.1
2 21.09 36/ Fair 260 ± 5 0.42 ± 0.1
3 22.5 36/ Fair 830 ± 5 0.84 ± 0.1

5. In-vitro Antibacterial study:
The experiment was conducted in such a manner as to determine the formation of inhibition zone using the Agar overlay. Bottom of Agar overlay; TSA medium was used. TSA medium was prepared in the same manner to make the upper surface; the pre-cultured strain was inoculated to the medium after cooling to 45-50°C. Inoculation concentration is 1 mL (1-5x10^8 cfu/mL) in 150 mL medium. The TSA medium inoculated bacteria are poured 30ml on bottom. It is placed to solidify at room temperature for 0.5 to 1 hours. Put the prepared test sample placed on a solidified medium; carefully press this sample using tweezers a glass rod to ensure good contact with the medium. By culturing 12 to 24 hours at the appropriate temperature in the strains, it confirms that the formation of the inhibition zone as shown in figure 2.
Conclusion:
In order to verify the antimicrobial effect of ORC powder, formation of zone of inhibition was confirmed. ORC powder, prepared through the oxidation reaction of regenerated cellulose, was found to have an antibacterial effect. This antibacterial effect is attributed to the low pH of ORC powder, which is generated by the oxidation of cellulose and has been reported to inhibit the growth of microorganisms.
For a comprehensive assessment, the comparison done with the present novel formulation is shown in Figure 2.
EXPERIMENT 1:
To provide a strong binding between powder particles; which ultimately results in haemostasis for longer duration.
In this experiment; chopped ORC powder (3) is bind with binding agent (2) such as glycerol, hydroxypropyl cellulose (HPC), methyl cellulose (MC), hydroxypropyl methylcellulose for the molding (102). This experiment explores alternative binding agents used for the molding of ORC powder (3). Glycerol, a highly versatile and effective binder, is utilized in pharmaceutical and medical device formulations to improve the structural integrity and stability of the product. It functions as a plasticizer, humectant, and solvent. The non-toxic and biocompatible nature of glycerol further emphasizes its suitability and wide-ranging application in products intended for human consumption and use.
Hydroxypropyl cellulose (HPC), methyl cellulose (MC), and hydroxypropyl methylcellulose (HPMC) are indispensable binders in pharmaceutical and medical devices, imparting excellent mechanical strength, prolonged stability, and controlled release properties to the product. Moreover, they enhance the bioavailability and efficacy of the final pharmaceutical product while being safe for human consumption.
As per process variant 1 ORC powder (3) has been prepared with alteration in binding agent (2) as mentioned above with powder to liquid ratio between 1:3 to 1:8.
EXPERIMENT 2:
In this experiment, to increase the flowability of the final ORC powder during the application with bellow bottle; glidants are mixed with ORC powder.
Granulated ORC powder was mixed with glidants at ball milling stage (106) in process variant 1. Glidants are used to enhance the flowability during application at anatomical site when packed in Micro Enema bellow bottles. The effectiveness of glidants such as starch, chitosan, and calcium citrate in improving powder movement and preventing clumping was evaluated.
Starch, chitosan, and calcium citrate serve as glidants, reducing friction between spherical particles, thereby enhancing flowability and ensuring uniform dispensing of granules at anatomical site. Additionally, starch and calcium citrate, can function as disintegrants, binders, and viscosity enhancers in the final formulation. Chitosan, due to its biocompatibility, biodegradability, and film-forming ability, is an attractive excipient. Furthermore, chitosan exhibits antimicrobial properties and can act as an antimicrobial agent.
Glidants are mixed with ORC powder in concentration of not more than 10% w/w. Single or in combination of mentioned glidants are mixed with ORC powder in said concentration.
EXPERIMENT 3:
In this experiment, alternative of the pressure molded equipment is explored like roller compactor.
A roller compactor is a specialized mechanical device, to compress powders and dry granulations into denser, uniform sheets, flakes or granules. It consists of two counter-rotating rolls (rollers) with a specific gap between them, through which the material passes. The rollers exert high pressure on the material, compacting it and reducing its porosity, thereby increasing its density. Roller compactors are designed to improve the flow properties, handling, and uniformity of powders, making them suitable for subsequent processing.
As per process variant 1, grinded powder with grinding machine directly fed into the roller compactor with space between the roller on 0.3 to 0.8 mm.
Two counter-rotating rolls have a X shape cross section lining to create a granule shape.
Then, the granules are transferred to the ball milling and further processed.
EXPERIMENT 4:
In this experiment, ORC powder is prepared without undergoing the pressurized molding process to assess the effectiveness of the newly developed method. The ground ORC powder is directly transferred to a ball mill and processed according to variant 1 of the procedure. A comparison is then conducted between the results obtained from this method and those achieved novel approach.
Table 3(B): Physicochemical Analysis Result of each Examples
Example COOH Assay (%) Angle of repose (°)/
Flow property Average Particle size distribution (micron) Tapped Density (g/mL)
1 20.4 33/ Good 585 ± 5 0.49 ± 0.1
2 21.05 25/ Excellent 518 ± 5 0.58 ± 0.1
3 21.3 31/ Good 690 ± 5 0.61 ± 0.1
4 22.1 43/ Passable 295 ± 5 0.71 ± 0.1

Figure 3 illustrates a comprehensive comparison of performance test results for the oxidized regenerated cellulose powder (3). Notably, among the variants tested, the novel process variant 1 showcased exceptional superiority in COOH assay, angle of repose and particle size distribution. This remarkable performance underscores the substantial potential of novel process variant 1, positioning it as a standout candidate for advancing the product's overall efficacy and market competitiveness.
To assess the safety and efficacy of the present novel Oxidized regenerated cellulose powder (3) produced via presently disclosed novel process variant 1, a comprehensive series of biocompatibility assessments has been conducted, encompassing the following methodologies:
1. Intracutaneous Reactivity Test
The test article, ORC powder haemostat, was evaluated for the potential to cause irritation following intracutaneous injection in rabbits was based on ISO 10993-10. Intracutaneous reactivity test of ORC powder (3) was conducted in New Zealand white rabbits. The test article was extracted in 0.9% sodium chloride USP solution (SC) and sesame oil, NF (SO). 0.2 mL dose of the appropriate test article extract was injected intracutaneously into five separate sites on the right side of the back of each of three animals. Similarly, the extract vehicle alone (control) was injected on the left side of the back of each animal. The injection sites wore observed immediately after injection. Observations far erythema and edema were conducted at 24, 48, and 72 hr after injection. The test article met the requirements of test for irritation and skin sensitization.
2. Maximization sensitization study
The test article, ORC powder haemostat, was evaluated for the potential to cause delayed dermal contact sensitization in guinea pig maximization test. This study was conducted based on the requirements of ISO 10993-10, Biological evaluation of medical devices-Part 10: Tests for irritation and skin sensitization. The test article was extracted in 0.9% sodium chloride USP and sesame oil, NF. Each extract was intradermally injected and occlusively patched to ten test guinea pigs (per extract). The extraction vehicle was similarly injected and ooclusively patched to five control guinea pigs (per vehicle). Following recovery period, the test and control animals received challenge patch of the appropriate test article extract and the vehicle control. The test article extracts showed no evidence of causing delayed dermal contact sensitization in the guinea pig. The test article was not considered a sensitizer in the guinea pig maximization test.
3. Systemic toxicity study
The test article, ORC powder haemostat, was surgically implanted in the subcutaneous tissue of the rat to evaluate potential systemic toxicity and local tissue response at the implantation site. A separate group of animals was similarly implanted to serve as the control group. Six male and six female animals were randomly assigned to either the test or control group. Animal were observed daily for overt signs of toxicity. Detailed clinical examinations were conducted weekly. Animals were weighed prior to implantation and at weekly intervals. At 4 weeks, the animals were euthanized and blood samples collected for haematology and clinical chemistry analysis. A necropsy was conducted, selected organs were collected and weighed, and implantation sites were excised and examined macroscopically. A microscopic evaluation of the implantation sites and collected organs was conducted. Clinical observations, body weights, necropsy results, organ weights, organ/body weight ratios, and organ/brain weight ratios were similar between test and control groups. There were no changes in hematology of clinical chemistry values considered related in with the test article. Microscopic evaluation of collected organs revealed no evidence of treatment related response. Microscopic evaluation of the implantation sites indicated no significant difference in the local tissue response between the control and test articles. There was no evidence of systemic toxicity from the test article following subcutaneous implantation in the rat. Local macroscopic tissue reaction at the test article implantation sites was similar between the test and control groups. Microscopically, the test article was classified as non-irritant as compared to the control article.
4. 4-weeks Muscle Implantation Test
The test article, ORC powder haemostat, was implanted in muscle tissue of the rabbit to evaluate the local tissue response in accordance with ISO 10993-6, Biological evaluation of medical devices Part 6, Tests for local effects after implantation. Sterile implant test articles and sponsor control articles (Surgicel) were aseptically prepared. Negative control articles were sterilized by steam. The test article, sponsor provided control and negative control were intramuscularly implanted and animals were euthanized 4 weeks Later. Muscle tissues were excised and the implant sites examined macroscopically. A microscopic evaluation of representative implant sites from each animal was conducted to further define any tissue response. The macroscopic reaction was not significant as compared to the sponsor provided control article and not significant as compared to the negative control article. Microscopically, the test article was classified as non-irritant as compared to the control and slight irritant as compared to the negative control article.
5. Modified cytotoxicity test
The test article, ORC powder and sponsor provided control (SPC), Surgicel were both evaluated separately to determine the potential for cytotoxicity. This study was conducted based on the requirements of ISO 10993-5: Biological evaluation of medical devices Part 5: Tests for in- vitro cytotoxicity. A single preparation of the test article and SPC article were extracted in single strength Minimum Essential Medium (1X MEM) at 37 °C for 24 hours. The reagent control was similarly extracted. Triplicate wells were dosed with 0.1 mL of the test article extract on filter disc (test filter disc) and triplicate wells were dosed with 0.1 mL of the SPC extract on filter disc (SPC filter disc) with the extract sides against the agarose layers. Triplicate wells were dosed with 0.1 ml of the reagent control placed on filter disc to serve as the reagent control/filter disc control. Triplicate wells were dosed with 1 cm length of high-density polyethylene as negative control. Triplicate wells were dosed with 1cm × 1cm portion of latex as positive control. Each was placed on an agarose surface directly overlaying a subconfluent monolayer of L-929 mouse fibroblast cells. After incubating at 37°C in the presence of 5% CO2 for 24-26 hours. the cultures were examined macroscopically and microscopically for any abnormal cell morphology and cell lysis. The test article extract and sponsor provided control article extract on the filter discs showed no evidence of causing cell lysis or toxicity, both were grades of 0. The test article extract and sponsor provided control extract met the requirements of the test since their grades were less than grade (mild reactivity).
6. Genotoxicity Test- Bacterial Reverse Mutation Assay
A bacterial reverse mutation assay was conducted to evaluate whether dimethyl sulfoxide (DMSO) extract and saline extract of ORC powder haemostat would induce reverse mutations at the histidine locus of the Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 or at the tryptophan locus of Escherichia coli tester strain WP2uvrA. The assay was conducted in the presence and absence of metabolic activation.
Tubes containing molten top agar were inoculated with culture from one of the five tester strains, along with the DMSO or saline extract. An aliquot of sterile water for injection or rat liver S9 homogenate, providing metabolic activation was added. The mixture was poured across triplicate plates. Parallel testing was conducted with negative control {extraction vehicle alone) and positive controls. The mean number of revertants for the test extract plates was compared to the mean number of revertants of the negative control plates for each of the five tester strains. The DMSO and saline test article extracts were considered to be non-mutagenic to Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and to E. coli tester strain WP2uvrA.
7. Genotoxicity Test-Chromosomal Aberration
The test article, ORC powder haemostat, was extracted in dimethyl sulfoxide (DMSO) and McCoy’s medium. A chromosomal aberration study was conducted to determine whether the extract would cause genotoxicity in Chinese hamster ovary (CHO-WBL) cells in the presence and absence of S9 metabolic activation.
The DMSO test article extract was diluted 1:100 with McCoy's 5A complete medium prior to dosing the cells. The serum free McCoy’s medium extract was supplemented to 10% with fetal bovine serum prior to dosing the cell. A monolayer of CHO-WBL cells was exposed to the test article extract in duplicate and in the presence and absence of S9 metabolic activation. Parallel testing was also conducted with a corresponding negative and positive control. The extraction vehicle without the test article served as the negative control. Cells were exposed for 4 hours with and without metabolic activation, and for 20 hours without metabolic activation.
Due to toxicity found in the serum free McCoy's 4-hour treatment in the presence of metabolic activation, the following dilutions of test article extract were prepared, using the extraction vehicle, to determine a suitable dilution for the definitive study through a dose range finding assay: 50%, 25%, 12.5% and 6.25%. These four dilutions of the test article extract were prepared in duplicate cultures using the same method as the original study. The highest dilution showing an acceptable level of toxicity (50%) were selected for evaluation, the 100% test extract (undiluted) already demonstrated severe toxicity and was not included as part of the dose range finding study.
Due to a potential mutagenic response for the serum free McCoy's test article extract in the presence and absence of metabolic activation, a confirmatory study was conducted. confirmatory study was conducted using the same method as original study. The following test article extract dilutions were used for the confirmatory study: 100% undiluted, 50%, 25% and 12.5% for the 4 and 20-hour treatments in the absence of metabolic activation and 25%, 12.5%, 6.25% and 3.125% for the 4-hour treatment in the presence of metabolic activation. The 200% dilution without metabolic activation and 50% dilution with metabolic activation treatment were not performed due to an error in the test article preparation. The test article dilutions were prepared using fresh McCoy's 5A complete medium.
The serum free McCoy's medium test article extract did not produce biologically significant increase in chromosome aberrations as compared to the negatives control in the presence or absence of S9 metabolic activation confirmatory assay. The DMSO test article extract did not produce a statistically significant increase in chromosome aberrations as compared to the negative control in the presence or absence of S9 metabolic activation. The test article was not considered to be mutagenic.
8. Material-mediated pyrogenicity test
The test article, ORC powder haemostat was extracted in sterile, non-pyrogenic 0.9% sodium chloride solution. The test extract was evaluated in the rabbit for material mediated pyrogenicity. The test was conducted based on USP, General Chapter <151>, Pyrogen Test. The procedure is recommended in IS0-10993-11, Biological evaluation of medical devices Part 11: Tests for systemic toxicity. A single dose of 10 mL/kg was intravenously injected via the marginal ear vein into each of three animals. Rectal temperatures were measured and recorded prior to injection and at 30-minute intervals between 1 and 3 hours after injection. The total rise of rabbit temperatures during the 3-hour observation period was within acceptable USP limits. The test article was judged as non-pyrogenic.

9. Hemolysis study
The test article, ORC powder haemotat, was evaluated for the potential to cause hemolysis according to procedures based on ASTM F756, Standard Practice for Assessment of Hemolytic Properties of Materials and IS0 10993-4, Biological evaluation of medical devices-Part 4: Selection of tests for interactions with blood. Anticoagulated whole rabbit blood was pooled, diluted, and added to tubes with CMPF-PBS test article extract. A sponsor provided control, negative control, positive control, and blank were prepared in the same manner. Following incubation for at least hours at 37°C, the tubes were centrifuged, and each supernatant collected. The supernatant was mixed with Drabkin's reagent and the resulting solution was analysed using a spectrophotometer at a wavelength of 540 nm.
The hemolytic index for the test article extract was 0.6%. The test article extract was non-hemolytic. The hemolytic index for the sponsor provided control article extract was 0.9%. ‘The sponsor provided control extract was non-hemolytic.
10. 4 week-Systemic toxicity following subcutaneous implantation
The test article, ORC powder haemotat, was surgically implanted in the subcutaneous tissue of the rat to evaluate potential systemic toxicity and local tissue response at the implantation site. A separate group of animals was similarly implanted to serve as the control group. Six male and six female animals were randomly assigned to either the test or control group. Animal were observed daily for overt signs of toxicity. Detailed clinical examinations were conducted weekly. Animals were weighed prior to implantation and at weekly intervals. At 4 weeks, the animals were euthanized and blood samples collected for haematology and clinical chemistry analysis. A necropsy was conducted, selected organs were collected and weighed, and implantation sites were excised and examined macroscopically. A microscopic evaluation of the implantation sites and collected organs was conducted. Clinical observations, body weights, necropsy results, organ weights, organ/body weight ratios, and organ/brain weight ratios were similar between test and control groups. There were no changes in hematology or clinical chemistry values considered related to treatment with the test article. Microscopic evaluation of collected organs revealed no evidence of treatment related response. Microscopic evaluation of the implantation sites indicated no significant difference in the local tissue response between the control and test articles. There was no evidence of systemic toxicity from the test article following subcutaneous implantation in the rat. Local macroscopic tissue reaction at the test article implantation sites was similar between the test and control groups. Microscopically, the test article was classified as non-irritant as compared to the control article.
11. Chemical Characterization
The Solvent and Extraction Condition Verification study was conducted to evaluate the test article to determine and document the compatibility of solvents and extraction conditions for chemical characterization testing of ORC Powder.
Test article evaluation was accomplished by selecting extraction solvents and extraction conditions that were at least as aggressive as the conditions of clinical use. This test article evaluation determined and documented the compatibility of clinically relevant extraction solvents and extraction conditions that could be used in subsequent analytical analyses, without causing apparent test article degradation. Degradation was based upon visual inspection of the test article (swelling, melting, deformation, discoloration, cracking) as well as evaluation of Non-Volatile Residue (NVR) and/or the presence of polymer in the extract by Fourier Transform Infrared Spectroscopy (FTIR). NVR was determined on all extracts.
Choice of solvents and extraction conditions depend on the test article composition, where in or on the body the test article is used and for how long it is used. The ISO 10993 series recommends the use of a polar (e.g., purified water or physiological 0.9% saline) and non-polar (e.g., hexane) extraction vehicle, and in some cases also recommends use of a semi-polar (e.g., isopropyl alcohol, ethyl alcohol, alcohol/water mixtures) extraction vehicle, in the conduct of an ISO 10993-18 chemical characterization program.
Based on the test article composition, where in the body the test article is used, and how long it is used, the following solvents were selected for use:
Polarity Vehicle
Polar Purified Water
Semi-polar Isopropyl Alcohol, Methanol, Acetonitrile
Non-Polar Heptane, Cyclohexane, Isooctane

Following which, this chemical characterization study was performed to identify and quantitate the extractables and/or leachables that may be released from the test article. This study was conducted based on guidance provided in ISO 10993-18, Biological evaluation of medical devices – Part 18: Chemical characterization of medical device materials within a risk management process.
The test article, ORC Powder, was extracted under exhaustive conditions in Acetonitrile and Cyclohexane. The resulting extracts were analyzed by Gas Chromatography – Mass Spectrometry (GC-MS) for Semi-Volatile Organic Compounds (SVOC), and Ultra Performance Liquid Chromatography – Ultraviolet Spectrometry – Mass Spectrometry (UPLC-UV-MS) for Non-Volatile Organic Compounds (NVOC).
The test article extraction in purified water resulted in significant visible change to the test article, accompanied by elevated non-volatile residues. The observed physical change, in conjunction with elevated NVR results, suggests poor solvent compatibility with the test article. The test article extractions in acetonitrile and cyclohexane did not exhibit similar results, were most compatible and were confirmed by the sponsor to be acceptable for use in chemical characterization testing in alignment with ISO 10993-18.
Sr. No. Type of Study/Test
Article Species
and
Strain Method of
Administration Test
duration Reference Result
1. Intracutaneous
reactivity test New Zealand white rabbits Intracutaneous 3 days ISO 10993-10 Non-
Irritant
2. Maximization sensitization
study Guinea
pig Intradermal administration of injection initially followed by topical patch exposure and challenge patch exposure 16 days ISO 10993-10 Non-
Sensitizer
3. Systemic toxicity study Mus musculus Hla®:(ICR) CVF® strain Mice Intravenously and intraperitoneal 3 days ISO 10993-11 Non-
Toxic
4. 4-weeks Muscle Implantation Test New Zealand white rabbits Subcutaneous 28 days ISO 10993-6 Non-
Irritant
5. Modified Cytotoxicity
Study L-929 mouse fibroblast cells Agarose Overlay method 24-26 hrs (1 day) ISO 10993-5 Non-
Cytotoxic
6. Genotoxicity Test- Bacterial Reverse Mutation Assay Salmonella Typhimunum and
Escherichia coli Plate incorporation method after Pre-incubation method 2 days ISO 10993-3 & OECD 471 Non-mutagenic
7. Genotoxicity Test-Chromosomal Aberration Chinese hamster ovary (CHO-WBL) cells In-vitro 3 days OECD 473 Non-mutagenic
8. 4 week-Systemic toxicity following subcutaneous implantation Rat (Rattus norvegicus) Hla®:(SD) CVF® strain Subcutaneous 28 days ISO 10993-6 & ISO 10993-11 Non-
Irritant
9. Material Mediated
Pyrogenicity test New Zealand white rabbits Intravenous route 3 hrs ISO 10993-11 and USP General Chapter <151> Pyrogen Test Non-
Pyrogenic
10. Hemolysis study New Zealand white rabbits used for blood extraction In-vitro testing test article extract with blood NA ISO-10993-4 & ASTM F756 Non-hemolytic
11. Chemical characterization - - - ISO-10993-18 Non extractables and leachables

12. In- vitro Haemostatic evaluation
An in vitro whole blood clotting test was conducted to assess the haemotatic performance of the powder samples. 2 mL of whole blood was mixed with powder sample in polypropylene tube. To each tube, 200 μL of re-calcified whole blood was added to each sample, followed by the addition of 20 μL of 0.2 M CaCl₂ to initiate coagulation. The tubes were incubated at 37°C and the clotting time was monitored by tilting the tube to a 45° angle every 20 seconds until a firm clot was visually detected. A control tube, without the sample, was also included in the experiment. After the predetermined incubation periods, 10 mL of deionized (DI) water was carefully added to each tube to release any unbound blood without disturbing the clot. The red blood cells (RBCs) that did not clot were lysed using DI water, leading to the release of hemoglobin. The degree of pigment release was observed, which is generally inversely proportional to the clotting degree of the blood clots. This test was performed according to the method outlined by Wang et al. (Wang, Qinghua, et al. "Rapid haemotatic biomaterial from a natural bath sponge skeleton." Marine Drugs 19.4 (2021): 220).

Sr. No. Description Time to haemostasis (Sec)
1 ORC Powder 42 s
2 Control Sample 192 s

13. Biodegradability of ORC powder haemostat in Rat
The study aimed to determine the absorption time of ORC powder haemostat from the implantation site in male Sprague Dawley rats. Rats (200-250g) were acclimatized, anesthetized, and surgically implanted with either the test article (ORC powder) or reference article (Surgicel) in the dorsal subcutis. A total of 18 samples (n=9 for each article) were used, and the rats were observed for up to 28 days. At intervals of 7, 14, and 28 days, three rats were euthanized, and the implant sites were examined histologically as shown in figure 4.
Results showed that substantial absorption of the test and reference article were overserved at day 7 and they are completely absorbed at 28 days post-implantation. There was no appreciable local tissue reaction to either article implanted in subcutaneous skin area during the study period.
, Claims:Claims:
I/We Claim,
1. A Composition and Method of Preparation for Oxidised regenerated cellulose powder as Haemostat which is non-irritant, non-sensitizer, non-toxic, non-cytotoxic, non-mutagenic, non-haemolytic and non-pyrogenic, comprising:
An Oxidized Regenerated Cellulose (ORC) Fabric (1);
A Wetting/Binding Agent (2)
Wherein the ORC fabric (1) is cut into uniform sizes and grinding of the ORC fabric (101) using a high-speed grinding machine (5000 rpm) for 10-20 minutes to achieve fine powder;
Wherein the said powder undergoes pressurized molding (102) process with the wetting/binding agent (2) to achieve a uniform shape, followed by drying process (103) using a vacuum dryer at a temperature range of 30°C- 40°C for 12-16 hrs;
wherein the pressurized molded material undergoes to ball milling process (104) at a speed of 50-100 rpm for 3-14 hrs using spherical grinding balls;
wherein for Size separation and powder filling (105), the particles with sizes less than 1000 microns are separated using vibro separator and then the ORC powder (3) is filled into a Micro Enema bellow bottle; and
wherein for Packaging and Sterilization (106), the said ORC powder (3) is packed in sterile barrier system and further sterilized with gamma irradiation.
2. The Composition and Method of Preparation for Oxidised Regenerated Cellulose Powder as Haemostat as claimed in claim 1, wherein the wetting/binding agent (3) is selected from (including but not limited to) 0.9% Sodium Chloride Solution, glycerol, hydroxypropyl cellulose (HPC), methyl cellulose (MC), hydroxypropyl methylcellulose in the ratio of 1:3 to 1:16.
3. The Composition and Method of Preparation for Oxidised Regenerated Cellulose Powder as Haemostat as claimed in claim 1, wherein glidant is selected either or in any combination from (including but not limited to) starch, chitosan, and calcium citrate, which can be mixed with ORC powder (3) granules in the ration of up to 10% w/w.
4. The Composition and Method of Preparation for Oxidised Regenerated Cellulose Powder as Haemostat as claimed in claim 1, wherein the particles of said ORC powder (3) have tap density in the range of 0.42 to 0.84 g/mL.
5. The Composition and Method of Preparation for Oxidised Regenerated Cellulose Powder as Haemostat as claimed in claim 1, wherein the said ORC powder (3) is absorbed substantially within 7 to 14 days and absorbed completely in body within 28 days.
6. The Composition and Method of Preparation for Oxidised Regenerated Cellulose Powder as Haemostat as claimed in claim 1, wherein the said ORC powder (3) has an angle of repose in the range of 25-30° ensuring excellent flowability of powder.
7. The Composition and Method of Preparation for Oxidised Regenerated Cellulose Powder as Haemostat as claimed in claim 1, wherein the ORC powder (3) has in-vitro bacteriostatic and bactericidal properties with the effect on growth and multiplication of gram positive and gram-negative organisms.
8. The Composition and Method of Preparation for Oxidised Regenerated Cellulose Powder as Haemostat as claimed in claim 1, wherein the said ORC powder (3) can be applied using sterile applicator in hard-to-reach surgical areas, and is capable of effectively controlling bleeding over broad surfaces.
9. The Composition and Method of Preparation for Oxidised Regenerated Cellulose Powder as Haemostat as claimed in claim 1, wherein for pressurized molding process (102) pressurized molding equipment and/or roller compactor is used.