Detailed data for every experiment and additional information regarding materials and methods are presented in Supplement 1. Cell lines were purchased from ATCC, reagents for cell culture were purchased from Thermo Fisher Scientific, Poland, and all chemical compounds were purchased from Sigma, Poland unless otherwise indicated.
Extraction
Extractions were performed according to ISO 10993-12 [6]. Extraction media for each experiment were chosen based on the appropriate ISO norm for the study. Briefly, the extractions were prepared by incubating the test material with a suitable extraction medium at 50±1°C for 72±2 hours for AMES, ICP MS, GC-MS, and LC-MS studies or at 37±1°C for 72±2 hours for other studies unless otherwise indicated. The extraction volume was derived from Table 1 – Standard surface areas and extract liquid volumes, ISO 10993-12 and determined at 0,2 g/mL [6]. The extracts were not centrifuged, filtered, or otherwise altered prior to dosing. The extract was clear without the presence of any particulates. The extracts were used within 24h of preparation. Since the material absorbs vehicles, the extraction vehicle that each 0,1 g of material absorbs was determined. During the extraction, the additional volume of the extraction vehicle was added to the mixture.
Chemical characterization
Per ISO 10993-18, a semi-quantitative analysis of VOC (volatile organic compound) in product and SVOC (semi-volatile organic compound) in 4SEAL® Hemostatic powder water extract was performed [7]. QP2010 Ultra gas chromatograph and QP-5000 mass spectrometer (Shimadzu) were used for analysis. In addition, quantitative analysis of elements concentration in 4SEAL® Hemostatic powder water extract was performed using NexION 300D (Perkin Elmer). As per ICH Q3D (R1), the concentration of the following elements was examined: Cd, Pb, As, Hg, Co, V, Ni, Tl, Au, Pd, Ir, Os, Rh, Ru, Se, Ag, Pt, Li, Sb, Ba, Mo, Cu, Sn, Cr [8].
For the analysis of VOCs and SVOCs, the Analytical Evaluation Threshold (AET) was calculated according to the following formula:
where:
A – number of devices extracted
B – volume of the extract
C – clinical exposure to medical device per day
DBT – the dose based threshold (TTC)
UF – uncertainty factor
Assuming an uncertainty factor of 2, ten 5 g bottles of 4SEAL® Hemostatic powder per patient usage, and Threshold of Toxicological Concert of 120 µg/day, the AET was calculated to be 0,240 µg/mL.
For the analysis of each element concentration, Total Element Exposure (TEE) was calculated assuming device patient usage of 50 g per day. TEE was calculated as follows:
Cytotoxicity
Cytotoxicity was evaluated quantitatively using the MTT method based on the ISO 10993-5 and ISO 10993-12 [6,9]. Briefly, 4SEAL® Hemostatic powder was extracted in single strength MEM at 37±1 °C for 24±1 hours. Following the extraction, quadruplicate monolayers of L-929 mouse fibroblast cells were dosed with: 100 %, 50 %, 33 %, and 25 % extracts and incubated at 37±1°C, 5±1% CO2, 95% humidity for 24±1 hours. Following the incubation, 50 μL of the MTT solution, prepared just before use, were dispended in each well and incubated for 120 minutes at 37±1°C, 5±1% CO2, 95% humidity. Following the incubation, MTT solution was replaced with 100 μL isopropanol and incubated for 10 min in 37±1°C, 5±1% CO2, 95% humidity. Finally, the optical density was measured by absorption at 570 nm (650 nm reference). The percent viability was determined from the blanks
Genotoxicity
Extraction of 4SEAL® Hemostatic powder for genotoxicity studies.
The amount of extractables was assessed by a pre-experiment "Determination of Extractables" according to ISO 10993-3 [10]. Based on the results, Method C – extraction according to ISO 10993-12 was chosen [6]. The extraction was conducted using an appropriate extraction vehicle.
Mouse Lymphoma Assay - MLA
Based on the ISO 10993-3, ISO 10993-12, ISO 10993-33, and OECD Test No 490, the 4SEAL® Hemostatic powder genotoxicity was evaluated using Mouse Lymphoma Assay [6,10–12]. In short, mycoplasma-free L5178Y TK+/-3.7.2C cells were cultured (37±1°C, 5±1% CO2, 95% humidity) in the F10 medium to the sufficient number and cleansed using THMG for 1 day and then THG for 2 days. The cleansed cells were then used in the experiment. 6 * 105 (for 4h treatment) or 4 * 105 (for 24h treatment) cells were exposed to the 10 ml of the 100% sample extract (worst case scenario), appropriate positive control, or negative control. The cells treatment was performed for 4h with and without the presence of 1% liver Aroclor-induced S9 fraction and for 24h without S9 fraction in 37±1°C, 5±1% CO2, 95% humidity. For each condition, duplicate test sample, duplicate negative control, and one positive control were prepared. After the 4h treatment, the cells were centrifuged and washed twice with fresh medium and then resuspended in the 20 ml of the F10 medium. After additional 20 hours, the cells were counted and resuspended in a fresh F10 medium at the concertation of 2 * 105. The cells were incubated for 24h (37±1°C, 5±1% CO2, 95% humidity) and recounted. After the 24h treatment, the cells were counted, washed twice, and resuspended in F10 fresh medium at the concertation of 2 * 105. The cells were incubated for 24h (37±1°C, 5±1% CO2, 95% humidity) and recounted. The number of cells counted was used to calculate total suspension growth (TSG) according to ISO 10993-33. After the expression period, the cell's relative plating efficiency (RPE; percentage plating efficiency of the test group in relation to the negative control) was determined by seeding a statistical number of 1,6 cells/well in two 96-well plates in F20 medium. The cells were incubated for 14 days at 37±1°C in the humidified atmosphere in the presence of 5% CO2. Analysis of results was based on the number of cultures without cell growth compared to the total number of cultures seeded. Relative suspension growth and relative total growth (RSG and RTG; RTG = RSG x RPE / 100) of the treated cell cultures were calculated according to the ISO 10993-33. Additionally, cultures were seeded in a selective medium. Cells from each experimental group were seeded in four 96-well plates at a density of 2000 cells/well in 200 µl selective F20 medium with TFT. The plates were scored after an incubation period of 14 days at 37±1°C in the humidified atmosphere in the presence of 5% CO2. Small colonies were counted separately. Small colonies are defined as less than a quarter of the diameter of the well, while large colonies are more than a quarter of the diameter of the well. The mutation frequencies were calculated from the data obtained from cultures used for the plaiting efficiency (cultures with non-selective medium) and those used for selection (cultures with selective medium) according to the following formula:
Bacterial Reverse Mutation Test - AMES
Genotoxicity of 4SEAL® Hemostatic powder was evaluated using commercially available Bacterial Reverse Mutation Test AMES Penta 2 (Xenometrix) according to ISO 10993-3, ISO 10993-12, ISO 10993-33, and OECD Test No. 471 [6,10,11,13].
Bacteria were exposed to the 25 µl of full strength extracts of the test material as well as positive (Table 1) and negative controls for 135 minutes in a medium containing sufficient histidine (S. typhimurium) or tryptophan (E. coli) to support approximately two cell divisions. The volume of extract added was based on the ISO 10993-33 and kit manufacturers' documentation. After exposure, the cultures were diluted in a pH indicator medium lacking histidine or tryptophan and aliquoted into 48 wells of a 384-well plate. After two days, cells that have undergone reversion to amino acid prototrophy grow into colonies. Bacterial metabolism reduces the pH of the medium, changing the color of that well. The number of wells containing revertant colonies were counted for each group (test article and positive control) and compared to a solvent (negative) control. Samples were prepared in triplicate to allow for statistical analysis of the data. The mutagenic potential of samples was assessed directly and in the presence of 4,5% of liver Aroclor-induced S9 fraction. Baseline, fold increase over baseline value, and binomial B-value were calculated using an excel spreadsheet provided by the manufacturer. The baseline is calculated as a mean plus standard deviation of the negative control. Fold increase over baseline is calculated by dividing the mean number of positive wells for a sample by the baseline value. The binomial B-value indicates the probability that spontaneous mutation events occur. For example, a binomial B-value ≥ 0,99 indicates that chances are ≤ 1% that this Result is due to spontaneous mutation. If both fold increase ≥ 2 and binomial B-value ≥ 0,99 occur for a test sample in specific conditions (strain, +/- S9 fraction), it should be considered mutagenic.
Table 1. Positive controls list for AMES test.
Strain
|
Substance
|
Without S9 fraction
|
With S9 fraction
|
Salmonella typhimurium TA98
|
2-nitrofluorene (2-NF)
|
2-aminoanthracene (2-AA)
|
Salmonella typhimurium TA100
|
4-nitroquinoline N-oxide (4-NQO)
|
2-AA
|
Salmonella typhimurium TA1535
|
N4-aminocytidine (N4-ACT)
|
2-AA
|
Salmonella typhimurium TA1537
|
9-aminoacridine (9-AA)
|
2-AA
|
E.coli WP2 uvrA[pKM101]
|
4-NQO
|
2-aminofluorene (2-AF)
|
Endotoxins
Endotoxins were measured using Pierce Chromogenic Endotoxin Quant K, which is in regard to 85. Bacterial Endotoxin Test, U.S. Pharmacopoeia [14]. The 4SEAL® Hemostatic powder was extracted in water for injection using an extraction ratio of 0,2 g/mL. According to the manufacturer's instruction, the standard curve was prepared (R2=0,9887) and is shown in Figure 2. Internal validation of the experiment was performed by spiking the samples with 0,05 EU/mL of endotoxin. The unspiked sample and the spiked sample were assayed to determine the respective endotoxin concentrations. For the test to be valid, the difference between the two calculated endotoxin values should equal the known (0,5 EU/mL) concentration of the spike ±25%.
Sensitization
The sensitization potential of the 4SEAL® Hemostatic powder was analyzed according to the ISO 10993-10 using the Local Lymph Node Assay (LLNA) [15]. Briefly, 4SEAL® Hemostatic powder was extracted using Acetone: Olive Oil 4:1. 15 adult, albino, healthy house mice (Mus musculus) of BALB/c strain were randomly assigned to solvent control, positive control, or study group. The test samples, solvents control, and positive control were applied to the dorsal side of both ears of designated mice at a dose of 25 µl / day for three consecutive days. 48h after the last extract application, mice were injected with 0.5 mL of BrdU (10 mg/mL) in phosphate-buffered saline (PBS) solution intra-peritoneally. 24 h after BrdU injection, animals were sacrificed, and auricular lymph nodes were harvested. A single-cell suspension of lymph node cells (LNC) was prepared from each mouse by gentle mechanical disaggregation through a disposable µ70 nylon cell strainer. In each case, the target volume of the LNC suspension was adjusted to 15 mL. The incorporation of BrdU was measured using the Colorimetric BrdU Cell Proliferation ELISA Kit (Abcam) according to the manufacturer's recommendations. 50 µL of each LNC was transferred in triplicate into a 96-well cell culture plate. PBS was used as a blank control. The colored reaction product was quantified using the µQuant spectrophotometer with dual-wavelength of 450/550 nm. BrdU labeling index was calculated according to the formula:
BrdU labelling index = (ABSem – ABS blankem) – (ABSref – ABS blankref)
em - emission wavelength; ref - reference wavelength.
For the test sample and positive control Sensitization Index (SI) was calculated according to the formula:
For the test to be valid, the SI of positive control (PC) must be higher than 2.
Intracutaneous reactivity
The study was conducted according to ISO 10993-10 [15]. The test article was extracted using Sodium Chloride and Cottonseed Oil as described above. Before the treatment, the fur on the animal's back on both sides of the spinal column was closely clipped over a sufficiently large test area, avoiding mechanical irritation and trauma. Then, 0,2 ml of the polar (Sodium Chloride) and non-polar (Cottonseed Oil) extracts were injected intracutaneously at five sites on one side of each rabbit. Similarly, 0.2 ml of the polar and non-polar solvent controls were injected intracutaneously on five sites of the contralateral side of each rabbit. The animals were observed immediately after injection, 24±2, 48±2, and 72±2 hours after the treatment to evaluate the signs of local reaction. Injection sites were examined for evidence of any tissue reaction such as erythema, oedema, and eschar. Tested and control sites were scored according to the Table 2 below.
Table 2. The grading system for intracutaneous (intradermal) reactions.
Reaction
|
Numerical grading
|
Erythema and eschar formation
|
No erythema
|
0
|
Very slight erythema (barely perceptible)
|
1
|
Well defined erythema
|
2
|
Moderate erythema
|
3
|
Severe erythema (beet redness) to eschar formation preventing grading of erythema
|
4
|
Oedema formation
|
No oedema
|
0
|
Very slight oedema (barely perceptible)
|
1
|
Well defined oedema (edges of area well defined by define raising)
|
2
|
Moderate oedema (edges raised approximately 1 mm)
|
3
|
Severe oedema (raised more than 1 mm and extended beyond exposure area)
|
4
|
After the 72±2 h grading, all erythema grades plus oedema grades (at 24±2 h, 48±2 h, and 72±2 h) are separately summed for each test sample or blank for each animal. To calculate the score of a test sample or blank on each animal, divide each totals by 15 (3 scoring time points x 5 test or blank sample injection sites). To determine the overall mean score for each test sample and each corresponding blank, add the scores for the three animals and divide them by three. The final test sample score is obtained by subtracting the blank score from the test sample score. The acceptance criteria are met if the final test score is 1.0 or less.
Acute Systemic Toxicity
The study was conducted according to ISO 10993-10 [15]. Four groups of 5 animals were injected with 50 ml/kg of Sodium Chloride extract, Cottonseed Oil extract, the polar and non-polar solvent controls. Polar and non-polar extracts and solvents controls were injected intraperitoneal. Animals underwent a clinical examination and were weighted 24±2 h, 48±2 h, 72±2 h after injection. 72±2 h after injection, animals were euthanized.
Subacute toxicity combined with implantation
Based on ISO 10993-6 and ISO 10993-11, 4SEAL® Hemostatic powder was evaluated for subacute toxicity combined with implantation using Starsil® Hemostat as reference material [16,17]. Cannulas from peripheral venous access catheters were divided into 10 mm pieces. Pieces were divided into two groups – the control and test group. Each piece from the control group was filled with approx. 0,3 g of Starsil® Hemostat while each piece from the test group was filled with 0,3 g of 4SEAL® Hemostatic powder. Before animal treatment, the fur on the animal's back was closely clipped over a sufficiently large test area, avoiding mechanical irritation and trauma. Place of implantation was disinfected by iodine solution. Surgery was performed under general anesthesia using isoflurane when animals received analgesic – subcutaneously injected butorphanol (2 mg/kg). An incision was made on the skin in a paraspinal line. Implants were placed on both flanks of the animal at equal intervals, in separate pockets in subcutaneous tissue. Eight pieces per rat of test or control article were implanted. Control and test materials had contact with surrounding tissue only in the base of cylindrical implants. Wounds were closed using non-resorbable threads. After implantation, each animal was injected subcutaneous meloxicam (1 mg/kg). Animals were housed separately for a week until wounds were healed. For 3 days after implantation, each animal was injected subcutaneous meloxicam (1 mg/kg). Animal's weight was observed 1, 2, 3, 7, 14, 21, 28 days after implantation. On the 27th day of the experiment, urine samples were collected. After 28 days, animals were premedicated with Ketamine/Xylazine (100 mg/kg – Ketamine, 10 mg/kg – Xylazine) to collect blood samples and then killed by CO2. Routine hematology and clinical chemistry were conducted on all animals at the end of the exposure period. Animals were anesthetized with Ketamine/Xylazine, and blood was drawn into tubes with K2-EDTA for hematology and heparin for clinical chemistry. Hemoglobin, PCV, RBC, reticulocytes, thrombocytes, and total WBC were determined with a hematology analyzer (Scil, Germany). Plasma concentrations of glucose, ALP, ALAT, ASAT, GGT, total protein, albumin, urea, creatinine, total bilirubin, total cholesterol, triglycerides, phospholipids, Ca2+, Na+, K+, Cl - and inorganic phosphate were determined using a biochemical analyzer (Fujifilm, Poland).
According to 4SEAL® Hemostatic powder Instruction For Use, maximal patient exposition to the hemostatic powder is 50g. Statistical human weight is 60 kg. Every animal was implanted with 8 implants containing 0,3g of hemostatic powder each. The dose of test articles for a single rat was more than 10x of the maximal human dose.
Gross necropsy
After animal euthanasia, a gross necropsy was performed on all animals. The following organs were weighed (paired organs together) after dissection: adrenals, brain, lungs, heart, kidneys, liver, ovaries, spleen, testes. The organ-to-body weight ratios (relative organ weights) were calculated from the rats' absolute organ weights and the terminal body weight.
Samples of the weighed organs and the colon, lymph nodes, skin, lungs, mammary gland, peripheral nerve (sciatic), esophagus, parathyroid, pituitary, prostate, rectum, small intestines (duodenum, ileum, jejunum), sternum with bone marrow, stomach, thyroid, trachea with bronchi, urinary bladder, uterus, vagina, places of implantation and all gross lesions were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. Histopathologic analysis from organs: brain, lungs, heart, liver, kidneys, adrenals, ovaries/testis, sternum, muscle, the skin was conducted on 5 μm sections of paraffin-embedded tissues, stained with hematoxylin and eosin, of the preserved organs from two representative animals per sex from control and test group by light microscopy. Each place of subcutaneous implantation was examined under a microscope and evaluated based on the guidelines provided in Table 3.
Table 3. Guidelines of histological evaluation system of place of implantation – tissue response
Histologic feature
|
Score
|
0
|
1
|
2
|
3
|
4
|
Inflammatory cell type/response — Polymorphonuclear cells — Lymphocytes
|
0
|
Rare, 1 to 5/hpf a
|
Rare,5 to 10/hpf a
|
Moderate infiltrate
|
Marked infiltrate
|
Plasma cells
|
Macrophages/gitter cells
|
Multinucleated giant cells MGC)
|
0
|
Rare, 1 to 2/hpf
|
Rare, 3 to 5/hpf
|
Necrosis
|
0
|
Minimal
|
Mild
|
Moderate infiltrate
|
Marked
|
Neovascularization
|
0
|
Minimal capillary proliferation, focal, 1 to 3 buds
|
Groups of 4 to 7 capillaries with supporting fibroblastic structures
|
Broad band of capillaries with supporting fibroblastic structures
|
Extensive band of capillaries with supporting fibroblastic structures
|
Fibrosis
|
0
|
Narrow band
|
Moderately thick band
|
Thick band
|
Extensive band
|
Astrocytosis/fatty infiltration
|
a hpf=high-powered (400x) field.
|
Pyrogenicity
Rabbit selection
Rabbits used for the study were submitted to a negative pyrogen test within 14 days preceding the test (with a rest period of a minimum of 3 days following the negative pyrogen test).
Determination of the Initial Temperature
Previously weighted rabbits were placed in a restrainer, and a thermometric rectal probe was inserted at not less than 7.5 cm but not more than 9 cm. The temperature of each rabbit was recorded every 30 minutes for 90 minutes before injection. The rabbits which showed a temperature variation two successive readings higher than 0.2 ̊C during the initial temperature determination or which showed a temperature higher than 39.6 ̊C or lower than 38.2 ̊C were not injected. The initial temperature of each rabbit was determined as the mean of two temperatures recorded at intervals of 30 minutes before the injection. In the group, the difference between the tree initial temperatures did not exceed 1 ̊C.
Rabbit Injection and Follow up
After extraction, the tested solution was warmed to about 38,5 ̊C and injected intravenously via the marginal ear vein at a dose of 10 ml/kg of body weight. The temperature of each rabbit was recorded every 30 minutes for 3 hours after injection. The maximum rise (compared to the initial temperature) of each rabbit was determined at the end of the test. Acceptance criteria for the test are presented in Table 4.
Table 4. Criteria of acceptation for pyrogenicity test.
Number of rabbits
|
Product passes if the summary response does not exceed
|
Product fails if the summary response exceeds
|
3
|
1.15 ̊C
|
2.65 ̊C
|
6
|
2.80 ̊C
|
4.30 ̊C
|
9
|
4.45 ̊C
|
5.95 ̊C
|
12
|
6.60 ̊C
|
6.60 ̊C
|