REPORT

On the experimental preclinical study of the toxic properties, local irritant action, elements of mutagenic action, elements of reproductive and immunotoxicity of Lithium Ascorbate following repeated administration to rats

Saint Petersburg 

2022STUDY CHARACTERISTICS

PCS code – 6/21-2.

PCS code – 12/18-2

Drug developer – Normopharm LLC, 655750, Republic of Khakassia, Abaza, Lenin Str., 3, office 1n.

Organization that arranges the preclinical study – Normopharm LLC, 655750, Republic of Khakassia, Abaza, Lenin Str., 3, office 1n.

Executor – FSBI Golikov Scientific and Clinical Center of Toxicology FMBA of Russia, Russia, 192019, Saint Petersburg, Bekheterev Str., 1.

The experimental part was performed in the period of July 20, 2021 to April, 20 2022 inclusive.

LIST OF EXECUTORS

The work was provided by: M.A. Anisimova, M.M. Belyaeva, A.A. Vataeva, E.V. Zarenkov, T.V. Kashina, A.A. Kozlov, A.V. Shultz.

ABBREVIATIONS AND ACRONYMS

REGULATORY STANDARDS

The studies were carried out in FSBI Golikov Scientific and Clinical Center of Toxicology of the Federal Medical and Biological Agency of Russia in accordance with the regulatory documentation:

• Federal Law № 61-FZ of 12.04.2010 “On the Circulation of Medicines”;

• Decree of the of the Council of the Eurasian Economic Commission dated 03.11.2016№ 81 “On Approval of the Rules of Good Laboratory Practice of the Eurasian Economic Union in Circulation of Medicines”;

• Decree of the Chief State Sanitary Physician of the Russian Federation dated 28.01.2021 № 4 on approval of sanitary rules and standards SanPiN 3.3686-21 “Sanitary and epidemiological requirements for the prevention of infectious diseases”;

• GOST 33044-2014 dated 01.08.2015 Principles of Good Laboratory Practice GLP (OECD Guide 1:1998 OECD Principles of Good Laboratory Practice);

• GOST 32519-2013 dated 01.08.2014 Testing of chemicals of human hazard. Chronic toxicity studies (OECD Test № 452:2008, IDT);

• OECD Test № 452 “Chronic Toxicity Studies”;

• Mironov, A.N. Guidelines for conducting preclinical studies of drugs. Part One / Ed. by A.N. Mironov. Moscow, Publishing House Grif & K, 2012. – 944 p.;

• “Guidelines for studies of general toxic effect of pharmacological substances" (in book “Guidelines for conducting preclinical studies of drugs”, Moscow, Medicine, 2012);

• ICH Topic S4 Duration of Chronic Toxicity Testing in Animals (Rodent and Non Rodent Toxicity Testing);

• Mammalian Erythrocyte Micronucleus Test No. 474 OECD guideline for thе testing of chemicals (Adopted:29 July 2016);

• GOST 7.32-2017 “System of Standards for Information, Library and Publishing. R&D report. Structure and Rules of Design”.

• Decree of the Board of the Eurasian Economic Commission dated November 26, 2019 № 202 “On Approval of the Guidelines for Preclinical Safety Studies for the purpose of conducting Clinical Trials and Drug Registration”.

 ABSTRACT

Report 137 p., 95 tables, 112 figs., 20 sources, 3 annexes.

The report presents the results of an experimental study of the toxic properties, local irritant effect, elements of mutagenic action, elements of reproductive and immunotoxicity of Lithium Ascorbate, Normopharm LLC, Russia, (international non-proprietary name - lithium ascorbate) following repeated intragastric administration to experimental rats.

The study was carried out for submission to the Ministry of Health in order to obtain approval of conducting clinical trials or registration of Lithium Ascorbate by Normopharm LLC, Russia.

The drug developer – Normopharm LLC, 655750, Republic of Khakassia, Abaza, Lenin Str., 3, office 1n.

The organization that arranges the preclinical study – Normopharm LLC, 655750, Republic of Khakassia, Abaza, Lenin Str., 3, office 1n.

The study was carried out in the Laboratory of Drug Toxicology of the Federal State Budgetary Institution “Golikov Scientific and Clinical Center for Toxicology of the Federal Medical and Biological Agency” in the period from 03.06.2021 to 16.05.2022 (PCS 6/21-2).

Outbred rats of both sexes, aged 3 months, were used as a test system. The study drug and control substance were administered daily intragastrically through a tube for 180 days. The following doses of lithium ascorbate were used in the chronic toxicity study: 10 mg/kg, 100 mg/kg and 500 mg/kg.

During the study, survival, clinical picture of intoxication, dynamics of body weight, water and feed consumption, physiological values, hematological and biochemical values were evaluated; macroscopic and microscopic examination of internal organs; the mass coefficients of organs were compared; local irritant action was examined, elements of mutagenic action, reproductive and immunotoxicity were studied.

As a result of the study, it was found that drug Lithium Ascorbate in a dose of 10 mg/kg did not affect the appearance and general condition of males and females. The animals did not differ in appearance and behavior from the control group. No gender differences were recorded. The clinical condition of the animals was satisfactory throughout the experiment. There were no signs of clinical intoxication in any of the animals in this group. 

Male rats treated with the study drug in a dose of 100 mg/kg also did not have any abnormalities in the clinical picture, as well as most females of the same group.

In the groups of male and female rats treated with lithium ascorbate in a dose of 500 mg/kg, polyuria was reported during the daily clinical examination, which persisted for 3-4 weeks. One week after the start of administration of the study drug in the indicated dose, males also experienced a decrease in motor activity, muscle mass and decreased turgor. These symptoms persisted in male rats for 2 weeks. One month after the start of administration and until the end of the study, no abnormalities in the clinical picture were recorded in male rats treated with lithium ascorbate in a dose of 500 mg/kg. 

In female rats, deaths were reported in the groups treated with the study drug in doses of 100 mg/kg and 500 mg/kg. 

Water and feed intake in rats of both sexes treated with study drug lithium ascorbate in doses of 10 mg/kg and 100 mg/kg following repeated intragastric administration did not differ from the control group.

Polydipsia was observed in rats of both sexes treated with study drug Lithium Ascorbate in a dose of 500 mg/kg following repeated intragastric administration, due to which water intake was increased compared to the control group. Feed intake in male and female rats treated with lithium ascorbate in a dose of 500 mg/kg did not differ from the control group.

There were statistically significant differences in body weight and absolute body weight gain in male and female rats treated with lithium ascorbate in doses of 100 mg/kg and 500 mg/kg compared to the control group 2 weeks after the start of the study. Data analysis using Parametric One Way Anova established statistically significant differences in the values of body weight and absolute body weight gain of males treated with the drug at weeks 17, 18 and 19 of the study, the average values of the parameters were lower by 10-20% compared to the control groups. There were no other statistically significant differences in the body weight of the rats.

In general, the body weight of male and female rats treated with the study drug in three doses tended to increase throughout the study period in both the control and experimental groups. 

Analysis of the data for male and female rats showed no statistically significant differences from the control group in the Open Field test scores, as well as blood pressure values between the groups that received the study drug in three doses.

After the end of the 180-day intragastric administration of lithium ascorbate in three doses, no statistically significant differences from the control group in the ECG parameters of male and female rats were recorded.

Analysis of ECG parameters of male rats showed statistically significant differences between the group receiving lithium ascorbate in a dose of 10 mg/kg and the control group one month after the end of administration. The level of the P wave in the group treated with lithium ascorbate in a dose of 10 mg/kg was on average 33% higher compared to the control.

The study showed random statistically significant changes in hematological parameters of blood in female rats after administration of study drug Lithium Ascorbate compared to the control group. After 180 days of intragastric administration of lithium ascorbate in a dose of 10 mg/kg, 100 mg/kg and 500 mg/kg, a statistically significant decrease in the red blood count and, consequently, hemoglobin, as well as hematocrit levels was observed in male rats. One month after the end of the 180-day intragastric administration of lithium ascorbate in doses of 100 mg/kg, male rats had a statistically significant increase in hemoglobin and hematocrit levels within the reference values.

The study showed significant changes in the biochemical parameters of the blood serum of laboratory animals administered with Lithium Ascorbate in doses of 10 mg/kg, 50 mg/kg and 500 mg/kg.

No immunotoxic or mutagenic effects were reported following repeated administration of lithium ascorbate.

Morphometric and histological examination of the reproductive organs of male and female rats did not show any statistically significant differences in the values of the experimental groups compared to the control group.

The mass coefficients of the internal organs of experimental female rats after administration of study drug Lithium Ascorbate did not differ significantly from the control.

Analysis of the mass coefficients of liver and kidneys of male rats treated with Lithium Ascorbate one month after the end of administration using Tukey HSD established statistically significant differences. The statistically significant changes in the studied parameters were not general for animals of different groups and, rather, were of an individual nature. No other statistically significant differences were recorded.

The study drug did not cause any pronounced pathomorphological changes, which indicates good tolerability and relative safety. There were no differences between the studied doses of the drug.

No local irritant action was reported.

The results of the toxicity study of Lithium Ascorbate, Normopharm LLC, Russia (international non-proprietary name – lithium ascorbate, allow us to recommend the drug for obtaining approval for clinical trials or registration in the Russian Federation.

All study materials, including the study protocol, source data and the final report will be stored in the archive of FSBI Golikov Scientific and Clinical Center of Toxicology of FMBA of Russia (office 233, building 2) for 15 years from the end of the study. Samples of test and standard objects and/or their retention samples, as well as histological slides should be stored in archive during three cycles of inspections by regulatory authorities.