SUPLASYN Solution for injection Ref.[50640] Active ingredients: Hyaluronate sodium

Distribution

Suplasyn is detected in the synovial fluid for 2 hours and in the articular cartilage for 6 hours. The cartilage is the most firm point of the detection. The hyaluronic acid is kept in the synovial fluid for 4-5 days.

The following preclinical studies have been completed.

Subchronic (14-Day) Subcutaneous Study in Non-Swiss Mice

12 mice (6 male, 6 female) were injected subcutaneously with 25mL of Suplasyn per kg of body weight 5 times over a 14 day period. Control mice were injected with Phosphate Buffered Saline. Mice were observed for signs of toxicity immediately after each injection and once a day for 14 days. Body weight data was collected at Day 0, 7 and 14. Gross necropsy was performed after euthanasia on day 14. Blood samples were analysed for RBC, HGB, HCT, WBC, white blood cell differential and platelet count and clinical chemistry analysis, Select tissues were processed for histopathological evaluation.

No abnormal clinical signs were noted in any of animals. No abnormal findings were noted at necropsy for test and control animals. There were no indications that body weight changes resulted from environmental conditions or toxic substances. There were no significant findings in the haematology, clinical chemistry or histopathology data that were attributable to the Suplasyn.

ISO Acute Systemic Injection Test

5 mice were injected intraperitoneally with 50mL of Suplasyn per kg of body weight. Control mice were injected with Phosphate Buffered Saline. Body weight were recorded on the day of dosing and at 72±2 hours post-injection. Observations for mortality and signs of pharmacological and/or toxicological effects were made immediately postinjection and at 4, 24, 48 and 72 hours post-injection. None of the animals on study were observed with abnormal clinical signs indicative of toxicity during the 72 hours test period. All were alive at the end of the 72 hour test duration and body weight loss was not more than 2 grams over the course of the study.

In vitro Cytotoxicity Test

The toxicity of the product to mammalian cells was evaluated by allowing Suplasyn to diffuse through an agarose barrier and contact cultured cells. L-929 Mouse Fibroblast cells were used. No cytotoxic effects were observed with the Suplasyn.

In vitro Genotoxiclogy Testing

The mutagenic potential of the Suplasyn was evaluated by measuring its ability to induce back mutations at selected loci of five strains of Salmonella typhimurium in the presence and absence of microsomal enzymes. Suplasyn did not cause an increase in point mutations, exchanges or deletions and was therefore considered non-mutagenic in this essay.

Twelve mice were injected intraperitoneally with Suplasyn to determine whether it would cause genotoxic changes in chromosomes or the mitotic apparatus of murine polychromatic erythrocytes. Animals were observed immediately following injection and daily for general health. On day 4 blood was collected and polychromatic erythrocytes were evaluated for the presence of micronuclei and the frequency of micronucleated reticulocytes was determined. Suplasyn did not cause cellular toxicity. Positive and negative controls performed as expected and all animals appeared clinically normal throughout the duration of the study.

A preliminary toxicity study was conducted to determine the dose range for the mutagenicity assay. A 70% concentration of Suplasyn was exposed to the mouse lymphoma L5178Y/TK+/- cell line in the presence and absence of S9 metabolic activation system. Cells were cloned after a two day expression period. Fourteen days post cloning the number of mutant colonies was determined and compare to the number of mutant colonies that arose spontaneously from untreated cells. The 70% Suplasyn dilution was considered non-mutagenic. Positive and negative controls performed as expected.

ISO Intracutaneous Reactivity Test

3 rabbits received five sequesntial 0.2 mL intracutaneous injections along either side of the dorsal mid-line with Suplasyn on one side and the control (Phosphate Buffered Saline) on the other. Irritation reactions were scored over a 72 hour period for each rabbit and a Primary Irritation Response was calculated.

None of the animals showed abnormal clinical signs. The Primary Irritation Index scores were negligible therefore Suplasyn was considered a non-irritant.

ISO Guinea Pig Maximization Sensitization Test

Ten guinea pigs were injected with 0.1 mL of Suplasyn and Freund’s Complete Adjuvant (FCA). One week after injection, animals were topically patched with test material for 48 hours. After a two-week rest, animals were topically patched in an untreated area for 24 hours. 5 control animals received Phosphate Buffered Saline and FCA. Dermal patch sites were observed for erythema and edema 24, 48 and 72 hours after patch removal.

None of the test or control animals were observed to have a sensitization response.