Subcutaneous LD₅₀ in mice  

The LD₅₀ in mice by subcutaneous injection was obtained during the screening phase tests by the method of Lorcke in 15 male mice in comparison with bupivacaine and mepivacaine. LD₅₀ values were found to be: 

• IQB-9302.HCl: 89 mg/kg >
• Bupivacaine.HCl: 74 mg/kg (literature value : 80 mg/kg)
• Mepivacaine.HCl: 294 mg/kg (literature value: 280 mg/kg)

The LD₅₀ in rats by intravenous administration was obtained for racemic IQB-9302, L-IQB-9302 and D-IQB-9302 in comparison with bupivacaine in groups of 10 male and female Wistar rats according to the standard protocol 

LD₅₀ values were found to be: 

• Racemic IQB-9302 in males:5.1 mg/kg
• Racemic IQB-9302 in females: 4.9 mg/kg
• Bupivacaine in males: 5.2 mg/kg
• Bupivacaine in females:4.8 mg/kg

Acute subcutaneous toxicity in beagle dogs (TPS-6161B-502-410-98) 

One male and one female adult beagle dogs were dosed once subcutaneously with 2.5, 5.0, 10.0, 20.0, and 40.0 mg IQB-9302 /kg body weight. Doses were administered  every other day (Monday, Wednesday, Friday). The test article was dissolved in 0.9% Saline for Injection, USP at concentrations that allowed the appropriate dosage to be delivered in a volume of 5 mL/kg body weight. To avoid pressure necrosis at the injection sites, dose volumes were divided and administered at different sites on the back such that no more than 10 mL was, injected at any one site. Subsequent doses were increased based on results of the previous dosage. Body weights obtained just prior to the first dose and weekly thereafter were used to determine the appropriate dosage to be administered. Each dog was observed for clinical effects hourly for up to 6 hours following dosing and a minimum of twice daily throughout the evaluation. 

Results 

No clinical signs or evidence of adverse local or systemic toxicity were  noted in either dog at dosages of 2.5, 5.0, 10.0, and 20.0 mg IQB-9302/kg. Both dogs were found  
dead 1 hour following administration of the 40.0 mg/kg dosage. No clinical signs were noted prior to death. Gross necropsy examination of both dogs revealed white foam in the stomach, red tinged foam in and around the mouth, mottled light and dark areas on the spleen, a red thymus, and mottled light and dark areas on all lung lobes. One dog also had a white plaque on the medial margin of the left diaphragmatic lung lobe. All injection sites from both dogs had red subcutaneous tissue with a gelatinous material present. In addition to the injection sites, the spleen, thymus, and lungs of each dog were collected, saved in 10% neutral buffered formalin and processed for histopathologic evaluation by a board certified veterinary pathologist.  

Microscopic findings were consístent with acute death and included injection site haemorrhage, oedema and leukocyte infiltrate; thymus congestion and haemorrhage; splenic depletion; and lung  pneumonitis with or without hemorrhage for both dogs. 

In summary, the test article, IQB-9302, produced no local or systemic indications of toxicity when given subcutaneously to adult beagle dogs at dosages of 2.5, 5.0,  
10.0, and 20.0 mg/kg. Both dogs died within 1 hour of the subcutaneous administration of IQB-9302 at a dosage of 40.0 mg/kg. Based on the results of this study, the maximum tolerated dose of IQB-9302 when given subcutaneously to adult beagle dogs can be estimated to be more than 20.0 mg/kg but less than 40.0 mg/kg. 
 
 

Acute intravenous toxicity in beagle dogs (TPS-616A-501-510-98) 
 
One male and one female adult beagle dogs were dosed intravenously with 1.25, 2.5, 4.0, 5.0, and 6.0 mg of the test article IQB-9302/kg body weight. The test article was dissolved in 0.9% Saline for Injection, USP at concentrations that allowed the appropriate dose to be delivered in a volume of 5 mL/kg body weight. All doses were delivered at an infusion rate of 3 mL/minute. Dosages were increased and administered after a minimum washout period of 24 hours between doses until a maximum tolerated non-lethal dose was determined. Body weights obtained just prior to the first dose and weekly thereafter were used to determine the appropriate dosage to be administered. Each dog was observed for clinical effects hourly for 6 hours following dosing and a minimum of twice daily throughout the evaluation. 

Results 

There were no clinical indications of toxicity when IQB-9302 was administered at 1.25 and 2.50 mg/kg. Muscle twitching during dosing and ataxia after dosing were noted in both dogs at dosages of 4.0 and 5.0 mg/kg. Emesis was also noted in the male dog after administration of both the 4.0 and 5.0 mg/kg dosages while salivation was noted in the female dog at the 5.0 mg/kg dose level. At 6.0 mg IQB-9302/kg, muscle twitching during the infusion, emesis after dosing and marked ataxia for approximately 10 minutes following dosing were noted in the male dog.  
When the 6.0 mg IQB-9302/kg dosage was administered to the female dog muscle twitching was observed during the infusion, emesis occurred following infusion, and a seizure began at the end of the infusion. The seizure lasted approximately 5 mínutes and the dog remained recumbent for an additional 30 minutes. Because of the nature and duration of the seizure in the female dog, dosages above 6.0 mg/kg were not administered. 

In summary, the intravenous administration of the test article IQB-9302 at dosages up to 6.0 mg/kg body weight produced muscle twitching, salivation, emesis, and seizures. Based on the results of this study, the no-observable-effects-level for IQB-9302 delivered in a dose volume of 5 mL/kg at a rate of 3 mL/minute is 2.50 mg/kg while the maximum tolerated  non-lethal dose is estimated to be 6.0 mg/kg.