The study of acute toxicity of the topical combination with glucosamine and ketoprofen in the form of a cream-gel

Safety of new drugs is the most important criteria for the study, and it is determined by the analysis of acute toxicity. When determining acute toxicity the adverse effects of drugs are described in their single use or multiple administrations in a short period of time. Aim. To determine the toxicity class of a new topical combination containing 5.0 % glucosamine hydrochloride and 2.0 % in the form of a cream-gel or cutaneous application and intragastric introduction. Materials and methods. The studies were carried out in accordance with EC Directive 86/609 ЕЕС. The cutaneous application of the combination was in the range of doses from 43 to 22600 mg/kg. The object studied was used in the dose range from 500 to 5000 mg/kg in intragastric introduction. The behavior of animals and their survival were observed within 14 days. Results. During the observation after cutaneous application there was no mortality, changes in the common life cycle of rats and the skin irritation. In intragastric introduction of the combination its LD50 was higher than 5000 mg/kg. Conclusions. Therefore, the study of acute toxicity of the combination has shown the absence of mortality in rats at the maximum allowable doses for cutaneous application and intragastric introduction. This fact indicates a low toxicity of the G/K cream-gel, and it gives the possibility to refer the combination studied to the category of relatively harmless substances.

The aim of the study was to determine the toxicity class of a new topical combination containing 5.0 % glucosamine hydrochloride and 2.0 % in the form of a cream-gel (G/K cream-gel) for cutaneous application and intragastric administration.

Materials and methods
The G/K cream-gel combination studied was tested for toxicological properties.In the study 39 white rats aged 4-5 months were used.According to the standard health and safety regulations the experimental animals were kept on the appropriate food diet in the vivarium at the Central Research Laboratory of the National University of Pharmacy certified by the State Enterprise "Centre for Drug Evaluation and Research at the Ministry of Health of Ukraine" as a base for research in experimental pharmacology [4].The studies were conducted in accordance with EC Directive 86/609 ЕЕС of November, 24, 1986 in compliance with laws, acts and regulations of the EU countries as to protection of animals used for experimentation and other scientific purposes [5,6].
The Bioethics Commission of the National University of Pharmacy did not find violations of ethical norms in the studies conducted (No. 12 of 12.17.2014).The combination studied has a form for external use (a cream-gel), the first indicators of its acute toxicity were determined by cutaneous application in 15 rats of both sexes weighing 180-200 g.The study was carried out according to the guidelines [6].
The hair on the side of the rat's body was removed 24 hours before application to the skin of the study object.It was shaven in sizes correlated with the dose of application (from 10 % to 50 %) [7,8].The dosing of the object was conducted according to the guidelines from 43 to 22600 mg/kg [6].Within 14 days there was an observation about the general condition and behavior of animals, their survival, food intake, skin, reflex excitability [6,7].
According to the standards of Laboratory Practice systematic studies of acute toxicity are necessary even for drugs that have topical dosage forms [6].
The study of acute toxicity of the G/K cream-gel was conducted on 24 outbred white rats of both sexes weighing 150-180 g when introducing intragastrically according to the method of Pastushenko et al. [6,9].The study object was introduced in the dose range from 500 to 5000 mg/kg (500, 1000, 3000, 5000 mg/kg) [10].It is the dose threshold up to 5000 mg/kg that is considered reasonable for intragastric introduction in determination of systemic acute toxicity.As a solvent a vegetable oil was used with the ratio of 1 : 1 to the combination studied [11,12].Each experimental group contained 6 animals.Within two weeks all groups of animals were monitored.The intoxication of animals was criterion for acute toxicity assessment, namely death of animals, their general condition, changes in the body weight recorded on the 3rd, 7th and 14th day of the study.

Results and discussion
The results of the study of acute toxicity of the object are presented in Tab. 1 and indicate no mortality in all experimental groups.During 2 weeks observation in cutaneous application there were no changes in the common life cycle of rats, their behavior and appetite.In addition, no irritations or rash on the skin of rats were detected.
The results of the study of systemic acute toxicity according to Pastushenko method [13] are presented in Tab. 2.
The data indicate the absence of mortality in rats after intragastric introduction of the object studied in the range of doses from 500 to 5000 mg/kg within 14 days.
During the observation there were no changes in behavior, appetite, skin and visible mucous membranes, signs of pathological processes in breathing, motor activity, tone of muscles, the cardiovascular system, etc., in all animals studied.No disorders in diuresis and the state of the gastrointestinal tract were observed.
However, introduction of high doses of the G/K cream-gel combination from 3000 mg/kg within the first days of the study caused slight weakness and fatigue.The animals of this group were less active.Somewhere darkening of the stool was noted.But these symptoms disappeared before the 7-day of study, all general indicators returned to the normal level.During the experiment dynamics of changes in the body weight was determined as an important indicator of intoxication (Tab.3).
At the beginning of the study all animals were weighed on an empty stomach, their average indicators in different groups ranged from 164.3 to 170.5 g.On the third day of the study a significant reduction of the body weight was observed only in the dose of 5000 mg/kg, it was 158.3 ± 4.5 g.
Weighing of animals in all experimental groups on the 7-th day showed the tendency for normaliza-tion of the body weight.On the 14-th day of the study the indicators of all groups were close to their start values, and the group with the dose of 1000 mg/kg even slightly exceeded them.Taking into account the absence of mortality in animals in all experimental groups it can be argued that the LD 50 value of the object studied was higher than 5000 mg/kg in intragastric introduction.Notes: 1 -significant differences compared to the body weight (b.w.) before introduction (p <0.05); n -the total number of animals in the study.

Table 2 Mortality rates in rats when studying systemic acute toxicity of the G/K cream-gel combination (n=24)
Note. n -the total number of animals in the study