UDC 615.21: 615.076.9 APPROVED

General Director CJSC “Saint Petersburg Institute of Pharmacy” Doctor of Medical Sciences, Professor V.G. Makarov [Signature] 13.09.2021

It is carried out in accordance with the GLP OECD Principles

STUDY REPORT

Study of the toxic properties of substance Lithium Ascorbate following a single intragastric administration to sexually mature mice

(final)

Study Leader [Signature] A.V. Popova

Leningrad Region
2021

ABSTRACT

Keywords: Lithium ascorbate, acute toxicity, mice, intragastric administration, single administration

The report is presented in 242 pages, including 15 tables and 31 figures.

STUDY DATES

LIST OF EXECUTORS

STUDY DECLARATION OF GLP COMPLIANCE FROM THE STUDY LEADER

This study was carried out in accordance with the standard operating procedures of the institution, and the Study Plan № 1 mutually agreed with the Sponsor (Normopharm LLC, Russia).

The study complies with the GLP requirements, except for the fact that identity, purity and stability of the test object were not determined. The values were determined by the Sponsor by standard methods without regards to GLP requirements.

There were no deviations from the Study Plan that would affect data interpretability or the scientific integrity and results of the study.

I, the undersigned, hereby confirm that I take overall responsibility for the technical conduct of the study; analysis, interpretation, documentation and presentation of results, as well as archiving of the study-related materials.

The objectives set out in the study plan have been achieved. There were no unforeseen circumstances that could affect the quality or integrity of the study.

This report presents the results reliably. I am fully responsible for the accuracy of the data obtained, as well as the confidentiality of the preclinical study.

I guarantee that after the study completion, the study plan, the final report, source data and all relevant documentation will be transferred to the archives of the research institution.

Study leader

STATEMENT BY THE MANAGEMENT OF THE RESEARCH INSTITUTION ON THE PROVISION OF RESOURCES TO CONDUCT THE STUDY IN ACCORDANCE WITH THE GLP PRINCIPLES, REGULATORY REQUIREMENTS AND STANDARDS FOR THE ETHICAL HANDLING OF ANIMALS

The management of the research institution shall ensure that the following has been provided for the proper conduct of the study:

• availability of a sufficient number of qualified and experienced personnel with a clear understanding of their responsibilities, as confirmed by training data;

• equipping the research institution with the necessary equipment, facilities and materials;

• availability of a quality service responsible for the quality assurance system;

• availability of approved standard operating procedures, as well as access to them by all personnel involved in the conduct of the study;

• appointment of a study leader in accordance with the established procedure, who has qualifications appropriate to the study objectives;

• interaction of the study leader, employees of quality service and personnel involved in the study.

Compliance with the Principles of Good Laboratory Practice

This study was carried out in accordance with the principles of GLP OECD (GOST 33044-2014 “Principles of Good Laboratory Practice”; Decree of the EEC Council № 81 “On Approval of the Rules of Good Laboratory Practice of the Eurasian Economic Union in Circulation of Medicines” dated 03.11.2016). The manipulations were performed in accordance with the standard operating procedures of the institution and the Study plan.

Regulatory Compliance

The design of this study was based on the selection of the study goal, in accordance with the regulatory legal acts and guidelines laid down in “Regulatory Standards” section of this Report.

Compliance with the Standards of Ethical Handling of Animals

This study was reviewed at a meeting of the Bioethics Commission for compliance of the draft study with the “Three R’s” principles and Directive 2010/63/EU. The study was approved for conduct (№ BEC 1.28/21 dated 18.06, 2021, 9 persons voted).

General Director of CJSC “Saint Petersburg Institute of Pharmacy”

V.G. Makarov                [Signature]               13.09.2021

/Name/             /Signature/ /Date

STATEMENT OF THE QUALITY SERVICE ON CONDUCTING AND DOCUMENTING THE INSPECTION OF THE KEY STAGES OF THE STUDY

The Quality Service conducted and documented all stages of the study inspection. The results were reported to the study leader and the management of the research institution.

The study was inspected to ensure that the procedures and manipulations performed were in accordance with the standard operating procedures of the institution, the study plan, and the regulatory requirements of the Good Laboratory Practice standards.

The final study report was reviewed by a quality officer and found to be an accurate statement of the data obtained and the procedures applied. The results presented in the final report accurately and fully reflect the data obtained during the study.

The conclusion of the quality service on this study is an Annex to the final report.

S.S. Sapynov                 [Signature]             13.09.2021

/Name/             /Signature/ /Date/

TABLE OF CONTENTS

LIST OF ABBREVIATIONS AND ACRONYMS 15

INTRODUCTION 16

STUDY GOAL AND OBJECTIVES 17

1. MATERIALS AND METHODS 18

   1. Test object 18

   2. Control substance 18

   1. Method and duration of administration 22

   2. Selection and calculation of doses 22

   3. Dosing procedure 22

   1. Study design 23

   2. Feed deprivation 23

   3. Body weight recording 23

   4. Recording of the timing of development of intoxication and clinical examination of animals.. 24

   5. Euthanasia 25

   6. Pathomorphological examination 25

   7. Evaluation of local irritant action 27

   8. Data analysis 28

   9. Study quality assurance and control 28

1. Study objects 18

2. Animals 18

3. Method of administration and selection of doses 22

4. Methodology 23

2. STUDY RESULTS 29

   1. Results of pathomorphological examination of animals with unplanned necropsy 31

   2. Results of pathomorphological examination of aimals in routine necropsy …………….…31

   3. Mass coefficients of internal organs of experimental animals 31

   4. Results of the evaluation of local irritant action (LIA) 32

1. Toxicometry 29

2. Effect of single intragastric administration of the test object on the body weight of animals…………………………………………………………………………………….………………   30

3. Pathomorphological examination data 31

FINDINGS 34

CONCLUSION 35

TABLES AND FIGURES 36

DATA ARCHIVING 50

REGULATORY DOCUMENTS 51

REFERENCES 52

APPENDIX A 54

APPENDIX B 61

ANNEX C 88

ANNEX D 91

ANNEX E 95

ANNEX F 100

ANNEX G 101

ANNEX H………………………………………………………………………………….…………………….184

ANNEX I…………………………………………………………………………………………………………239

LIST OF ABBREVIATIONS AND ACRONYMS

In this R&D report, the following abbreviations and acronyms are used:

INTRODUCTION

The test object is Lithium Ascorbate, a substance (Normopharm LLC).

Lithium ascorbate is a highly absorbable and low-toxic organic lithium salt [1]. Lithium salts are widely used as normothymics in various affective disorders [2]. Lithium ions have a significant effect on the homeostasis of acetylcholine, enkephalins, catecholamines, serotonin, and other neurotransmitters [3]. Lithium (primarily as lithium carbonate) for the treatment of bipolar disorder or inhibition [4, 5] is used in doses in the hundreds of milligrams. Such doses may lead to severe adverse effects during therapy (renal pathology, teratogenesis). Compared to lithium carbonate therapy, lithium ascorbate has also shown efficacy in ultra-low doses [1].

This study aimed at evaluating the toxic properties and local irritant action of substance Lithium Ascorbate following a single intragastric administration to sexually mature mice is part of the complex of preclinical studies required for the registration of the drug in the Russian Federation (RF) [6, 7].

The study was carried out with the engagement of employees of the necessary departments [Appendix A], the approved study plan, Amendments № 1 to the Study Plan [Appendix B] and approved by the bioethics commission [Appendix C].

The information obtained in the study did not duplicate the results of previous studies.

STUDY GOAL AND OBJECTIVES

Study goal:

Study of the toxic properties of substance Lithium Ascorbate (Normopharm LLC) following a single intragastric administration into sexually mature mice.

Study objectives:

1. study of toxic properties of the test object with analysis of the clinical picture of intoxication; 

2. evaluation of local tolerability;

3. determination of the mean lethal dose (LD50) of the test object.
   

4. Materials and methods

   1. Test object

1. Study objects

Table 1.1.1.1 - Test object

1.1.2 Control substance

Table 1.1.2.1 - Control substance

The documents of the pharmacist service are given in Appendix D.

The research institution has not carried out any identification, purity and stability studies of the test object. These values were determined by the Sponsor according to standard methods. The Sponsor of the study is responsible for the reliability of the submitted data on the identification, purity and stability of the test object.

1.2 Animals

Group allocation:                    To exclude the influence of the investigator’s preferences on the

formation of experimental groups, animals were selected with the method of modified block randomization [8]. To do this, all animals submitted to the study were randomly placed in the cages of the randomization block (the number of cages of the randomization unit is a multiple of the number of groups in the experiment). Then, using a random number generator, a list of data was obtained, containing the numbers of the cages with animals and the corresponding numbers of the groups where the animals were subsequently placed [9] [Annex F].

The animals were kept under standard conditions in accordance with Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes [10].

Method of administration and selection of doses

1. Method and duration of administration

The test object was administered to the animals intragastrically (i/g), since this method is an analogue of the oral one, which is to be used in clinical practice. In the study of acute toxicity, the test object and the control substance were administered once (fractionally) with further observation of the animals for 14 days [11].

1. Selection and calculation of doses

In accordance with the Sponsor’s recommendations, the object was administered in the following doses: 4000 mg/kg, 5000 mg/kg, 6000 mg/kg, 7000 mg/kg and 8000 mg/kg.

The experiment was carried out in stages and began with the administration of the object in a dose of 6000 mg/kg. After the administration of the dose, 3 lethal outcomes were recorded, in due to that, it was decided to administer the following doses: 5000 mg/kg and 7000 mg/kg. After the administration of these doses, doses of 4000 mg/kg and 8000 mg/kg were tested for a more accurate determination of the LD50 value.

The control substance (water for injections) was administered to the animals of group № 1 once, fractionally, in an amount equivalent to the volume of the maximum dose of the test object.

2. Dosing procedure

The test object (as a suspension) and the control substance were administered to the animals once intragastrically, fractionally, using a tube.

The test object was administered to all groups of animals in the same volume - 32 ml/kg. Concentrations of the test object are presented in Table 1.3.3.2.

Table 1.3.3.2 – Dosing volumes of the test object

The test object was administered to the animals once, using special probes and syringes. The test object was administered in fractions, in two equal portions, with an interval between doses of no more than 30 minutes, one dosing volume was 16 ml/kg.

4. Methodology

1. Study design

The total number of animals involved in the experiment was 60 outbred mice (30 males and 30 females).

The characteristics of the experimental groups and the design of the experiment are presented in Tables 1.4.1.1 and 1.4.1.2.

Table 1.4.1.1 - Characteristics of experimental groups

Table 1.4.1.2 - Manipulation schedule

1. Feed deprivation

The animals were deprived of feed for 4 hours before administration, body weight recording and euthanasia. Water was given ad libitum throughout the experiment.

2. Body weight recording

Body weight was recorded in the morning hours immediately before administration, then on days 2, 8 and 15 of the experiment. Body weight data on day 15 of the experiment were used to calculate the percentage ratio of the mass of internal organs to body weight. Source data are presented in source charts [Appendix G].

2. The procedure of weighing mice was carried out on an electronic balance Vibra AJ-1200CE (Shinko Denshi, Japan). The maximum weighing limit is 1200 g, the minimum weighing limit is 0.5 g. Calibration mark is 0.1 g. Accuracy class is 2 [Appendix B].

   1. Recording of the timing of the development of intoxication

1. Recording of the timing of the development of intoxication and clinical examination of animals

The animals were continuously monitored prior to administration, for 30 minutes after administration of the last portion of the test object in fractions, then hourly for 4 hours, then after 24 hours, and then daily for 15 days.

The following was registered:

• Behavior: distress/agitation;

• Response to stimuli: decreased/increased;

• Skin: redness / paleness / cyanosis / jaundice;

• Mucous membranes: redness/pallor/cyanosis/jaundice;

• Discharge: from the eyes / from the nose / from the anus / from the urethra;

• Muscle tone: decrease/increase;

• Motor coordination disorders: ataxia/hyperkinesis;

• Dyspnoea;

• Death.

1. Clinical examination

The animals were clinically examined prior to administration, then on days 2, 8 and 14 of the experiment. A detailed examination of the animal was performed in the cage, in the hands and in the open area. The manifestation and severity, where acceptable, of signs of intoxication were noted.

1. Examination in the cage:

• Behavior: normal/distress/agitation;

• Attitude towards other animals: normal/ aggression.

2. Examination when picking up an animal:

• Response to stimuli: normal/decreased/increased;

• Body condition: normal / dystrophic/ obese;

• Muscle tone: normal/decreased/increased;

• Hair: normal (smooth, shiny) / ruffled / hair loss / dull / dirty / discoloration.

• Skin:

• Turgor: normal/reduced;

• Color: normal / redness / paleness / cyanosis / jaundice / hemorrhage;

• Integrity: normal (not impaired)/abrasions/cracks/wounds;

• Palpable masses.

• Mucous membranes:

• Color: normal / redness / paleness / cyanosis / jaundice;

Impaired integrity.

• Eyes: normal/exophthalmos (bulging eyes)/impaired integrity/discharge;

• Nasal cavity: normal / serous discharge / purulent discharge / bloody discharge;

• Oral cavity: normal/drooling.

3. Outdoor examination:

• Position of the body in space: normal / forced lying down / forced wandering in a circle / forced movement forward and backward / forced desire to lie on one side;

• Impaired motor coordination: normal/ataxia/hyperkinesis;

• Breathing Type: normal / thoracic / abdominal / dyspnea;

• Bowel movements: normal / diarrhea / presence of blood in the stool / change in stool color;

• Urination: normal/discoloration.

1. Euthanasia

On day 15 of the experiment, the animals were euthanized with CO2 followed by exsanguination of the heart cavities (lungs, heart, brain). In accordance with Directive 2010/63/EU of the European Parliament and of the Council of the European Union on the protection of animals used for scientific purposes of 22 September 2010 [10], this type of euthanasia of animals is accompanied by a minimum of pain, suffering and distress and is carried out by competent personnel. 

1. Pathomorphological examination

All experimental animals underwent pathomorphological examination.

The post-mortem examination included a macroscopic assessment of the animals euthanized according to the schedule, as well as a pathomorphological examination of the corpses of animals that died during the experiment [13].

The pathomorphological examination included necropsy and macroscopic examination. Necropsy was performed under the direct supervision of a pathologist. After euthanasia, the animals were carefully examined for external pathological signs. A study of the condition of the thoracic and abdominal cavity and a macroscopic examination of the internal organs were carried out. Similar studies were carried out on dead animals with the completion of a necropsy map [Appendix I].

Organs extracted during necropsy were weighed, and paired organs were weighed together. The value was used to calculate the percentage ratio of organ mass to body weight.

The procedure of weighing the internal organs was carried out on the electronic scale “Adventurer” RV 214 (OHAUS, China). The maximum weighing limit is 210 g, the minimum weighing limit is 0.001 g. Calibration is 0.001 g. Accuracy class is 1 [Appendix B].

List of organs to be weighed:

• Heart

• Lungs with trachea

• Thymus

• Liver

• Spleen

• Kidneys

• Cerebrum

• Testes

Organ Collection

In necropsy, the organs (organ fragments) listed below were taken and recorded in 10% pH-neutral formalin.

The organs have been archived and will be handed over to the Client upon request.

• Lungs with trachea

• Heart

• Thymus

• Liver

• Kidneys

• Adrenal glands

• Spleen

• Stomach

• Small intestine

• Large intestine

• Cerebrum

• Oesophagus

• Testes/ovaries

Histology

Histological examination was carried out if macroscopic changes were found in the internal organs during necropsy, including animals that died on the first day after the administration of the test object (only the tissue of the altered organ was examined to clarify the diagnosis). For histological examination, stomachs were taken from 2 males and 5 females, as well as intestinal fragments from one female.

For histological examination, the material was fixed in a 10% solution of neutral formalin for 24 hours, after which it was embedded into paraffin according to the generally accepted method [13]. Then sections with a thickness of 5-7 μm were made, which were stained with hematoxylin and eosin. The analysis of histological specimens was carried out using a light-optical microscope Axio Scope A1 ZEIZZ (Carl Zeiss MicroImaging GmbH, Germany) at a magnification of 50, 100, 400, 1000.

Microphotography was carried out using a digital camera AxioCam ICc1 (Carl Zeiss MicroImaging GmbH, Germany) and ZEN 2012 software.

7. Evaluation of local irritant effect

To evaluate the local irritant effect of the test object during the necropsy procedure, the condition of the gastrointestinal tract organs during necropsy of animals was visually evaluated. If abnormalities were found during macroscopic examination, a routine histological examination of these organs and tissues was carried out.

8. Data analysis

Descriptive statistics were applied to all data: the data were checked for compliance with the law of normal distribution using the Shapiro-Wilk’s W test. In the case of a normal distribution, the mean value and the standard error of the mean were calculated, which, together with the value of n (number of cases), are presented in the summary tables. In cases where the data did not conform to the normal distribution law, the median and quartile range were calculated. For evaluation of data with signs of normal distribution was used univariate analysis of variance (ANOVA), if a significant influence of the studied factor was found, subsequent intergroup comparisons (post hoc analysis) were carried out using Tukey's test analysis. To compare the two groups, the Student’s test was used. For the analysis of data that do not conform the normal distribution law, the Kruskal-Wallis test was used, followed by the use of the nonparametric method of average ranks for multiple comparisons in case of a significant influence of the factor under study. To compare the two groups, the Mann-Whitney U-test was used. Differences were determined at the significance level of p<0.05.

Statistical analysis was performed using licensed software Statistica 10.0 (StatSoft, USA).

The LD50 was calculated using the Bliss-Prozorovsky method [14].

9. Study quality assurance and control

The quality service of the research institution carried out the following [Appendix K]: 

- review of the study plan

• checking the study schedule

• incoming audit of the preclinical study

• audit of the experimental part of the study

• checking the chronology of the study and the completeness of the study report

• verification of the final study report.

2 Study results 

The study of toxic properties with an evaluation of the local irritant effect of lithium ascorbate, substance (Normopharm LLC, Russia), was carried out following a single fractional intragastric administration into males and females of outbred mice. The data obtained during the experiment are presented in full in the source charts [Appendix F].

1. Toxicometry

During the experiment, the death of 14 males and 10 females was recorded. Lethal effects based on the results of 48 hours of observation are presented in Table 2.1.1, mortality over 14 days of the experiment is presented in Table 2.1.2. The death of animals was recorded in all groups, except for group № 1 (control).

In the groups of animals treated with 4000 mg/kg, 5000 mg/kg, 7000 mg/kg, and 8000 mg/kg, mortality was mostly delayed (more than 48 hours after administration of the test object), data are presented in Tables 2.1.3 and 2.1.4.

LD50 in males following intravenous administration was 6140±319 mg/kg, in females 6920±426 mg/kg. The test object following intravenous administration was assigned to the 4th class of low-toxic substances according to the GOST classification 12.1.007-76 (LD50 >5000 mg/kg for intragastric administration) [16] and to the 5th hazard class according to the GHS OECD classification (LD50 >5000 mg/kg) [17]. 

1. Picture of intoxication. Clinical examination

Signs of intoxication were present after administration of the substance in the entire dose range studied.

The dose is 4000 mg/kg. On the 1st day of observation, the picture of intoxication in animals was manifested in ruffled hair and diarrhea, inhibition of various degrees of severity was seen in some animals. The frequency of signs on this day is presented in Table 2.1.1.1. The most intensive signs of intoxication in the survived animals were observed 3-4 hours after the administration of the object, in the following days the condition of the animals returned to normal.

Dose 5000 mg/kg. Males had inhibition of the condition, ruffled hair, and most animals had diarrhea. In females, only diarrhea was noted. Signs of intoxication resolved within the first hour after administration (Table 2.1.1.2).

Dose 6000 mg/kg. On day 1, intoxication manifested itself in inhibition of the general condition of various degrees of severity, ruffled hair and diarrhea in most animals. The maximum signs of intoxication were observed 2-3 hours after administration of the object (Table 2.1.1.3). On the second day, signs of intoxication were present only in a few animals.

Dose 7000 mg/kg. In the first hour after administration, inhibition of the general condition in various degrees of severity, ruffled hair and diarrhea were observed in most animals. Further, there was a gradual increase in the severity of inhibition, in a single case ptosis, ataxia, tremor was seen (Table 2.1.1.4). In the survived animals, the condition returned to normal by the second day of observation.

Dose 8000 mg/kg. In all animals, inhibition, ruffled hair, and diarrhea were observed within 30 minutes after administration. The toxic effects intensified to the point of death of the animals. Before death, most animals had a decrease in response to stimuli, forced lying down, pale skin, in some cases ataxia, hyperkinesis and discharge from the eyes. The condition of the only survived female returned to normal on the second day after administration (Table 2.1.1.5).

2. Effect of single intragastric administration of the test object on the body weight of animals

Tables 2.2.1 and 2.2.2 present the body weights of male and female mice at intragastric administration of the test object.

The data corresponded to the normal distribution law. Univariate analysis of variance showed the effect of the “group” factor on the initial body weight of male and female mice (ANOVA, p<0.05). Subsequent intergroup comparison using the Tukey test did not establish the presence of significant intergroup differences on day 1 of the experiment.

On the second day of the experiment, the effect of the “group” factor on the body weight of males (ANOVA, p<0.05) was established: in animals that received the substance in a dose of 5000 mg/kg, body weight was statistically significantly lower than in the control (Table 2.2.1). A decrease in body weight on day 2 of the experiment relative to the initial values was also observed in the groups that received the substance in doses of 4000 mg/kg and 8000 mg/kg (statistically significant relative to the baseline when compared by the Student’s test for dependent variables). In females on day 2 of the experiment, the effect of the “group” factor was not shown (p>0.05). On days 8 and 15 of the experiment, there was no significant effect of the “group” factor on the body weight of males and females (p>0.05).

Thus, on day 2 of the experiment, the survived males in the groups receiving the substance in doses of 4000 mg/kg, 5000 mg/kg and 8000 mg/kg showed a slight (no more than 12% of the baseline) decrease in body weight. Throughout the further period, in the survived males, there were no differences in body weight from the control. In females, the effect of the substance on body weight was not established throughout the experiment.

3. Pathomorphological examination data

1. Results of pathomorphological examination of animals with unplanned necropsy

In all dead animals of groups № 3 and № 4, as well as in some dead animals of groups № 2, № 5, № 6, cerebral edema and plethora of its membranes were found. In all dead animals of group 5, as well as in many dead animals of groups № 2-№ 6, edema and plethora of the lungs and plethora of internal organs were also observed (Figure 2.3.1.1-2.3.1.4). Several animals of groups № 4, № 5 and № 6 had isolated hemorrhages in the lungs (Figure 2.3.1.5-2.3.1.7).

The immediate cause of death of all the dead animals, except mouse 3.3, was acute heart failure. The cause of death of male mouse 3.3 was acute post-hemorrhagic anemia secondary to gastrointestinal bleeding.

In all groups treated with the test object, marked gastrointestinal changes were also found (see section 2.3.4).

2. Results of pathomorphological examination of animals during planned necropsy

No pathological changes were found in the internal organs (Table 2.3.2.1, Figures 2.3.2.1 to 2.3.2.6), except for the female mouse 4.6, which was found to have erosive-ulcerative mucosal lesions at the administration site (see 2.3.4).

3. Mass coefficients of internal organs of experimental animals

Tables 2.3.3.1 and 2.3.3.2 show the mass coefficients of the internal organs of experimental animals.

The data corresponded to the normal distribution law. A one-factor analysis of variance shown the effect of the “group” factor on the mass coefficients of the liver and lungs of male and female mice (ANOVA, p<0.05).In male mice received the object in a dose of 5000 mg/kg, as well as in females who received the object in a dose of 4000 mg/kg, a statistically significant increase in mass lung coefficients was shown compared to the control. A tendency to increase lung mass coefficients was observed in the survived female of group 6 (dose 8000 mg/kg). A statistically significant increase in liver mass coefficients relative to the control group was also found in males who received the test object in doses of 5000 mg/kg and 6000 mg/kg, and in females who received the object in a dose of 5000 mg/kg.  The tendency to the increase of mass coefficients of liver was observed both in the male mouse survived after administration of dose 7000 mg/kg, and the survived female mice that received the substance in doses of 6000 mg/kg and above.

4. Results of evaluation of a local irritant action (MAD)

Routine necropsy

In a female mouse 4.6 that received the test object in a dose of 6000 mg/kg, erosive-ulcerative mucosal lesions were found at the administration site (stomach) (Figure 2.3.4.1). Histological examination of female mouse 4.6 shown erosions of the gastric mucosa (Figure 2.3.4.2).

In the remaining euthanized animals, no macroscopically visible changes were found at the administration site (Figure 2.3.4.3, Table 2.3.4.1).

Unplanned necropsy

Female mouse 2.9 that received the test object in a dose of 4000 mg/kg, as well as male mouse 3.3 who received the test object in a dose of 5000 mg/kg, had erosive-ulcerative mucosal lesions at the administration site (stomach) (Figures 2.3.4.4-2.3.4.6), and female 2.9 also had hemorrhages, the mucosa was loose. Histological examination of female mouse 2.9 showed erosions of the gastric mucosa (Figure 2.3.4.5). In isolated cases, the stomach contents contained blood (Figure 2.3.4.6).

Several animals administered with the object in doses of 5000, 6000, 7000 and 8000 mg/kg had hemorrhages in the gastric mucosa (Figure 2.3.4.7-2.3.4.9).

Examination of the carcasses of animals of groups № 2, № 5 and № 6 showed an abundant accumulation of watery contents in the intestines (Figure 2.3.4.10).

Since the macroscopic picture was the same among the dead mice, in which the intestines were found to be overflowing with abundant aqueous contents, the specialist  in pathomorphology took a typical specimen of altered tissues from mouse 4.10, which received the test object in a dose of 6000 mg/kg, for subsequent histological examination in order to determine the nature of microscopic changes in the epithelium and superficial epithelial necrosis (Figure 2.3.4.11).

Histological examination of the stomach of female mouse 4.10 also showed erosions of the gastric mucosa (Figure 2.3.4.12).

The mucous membrane of male mice 5.3 and 5.4 was loose, hyperemic, and hemorrhages were found within (Figure 2.3.4.13). In female mouse 5.9, erosive-ulcerative mucosal lesions were found at the administration site (stomach) (Figure 2.3.4.14). Histological examination of female mouse 5.9 showed an ulcer of the gastric mucosa (Figure 2.3.4.15).

In two animals of group 5 (7000 mg/kg), as well as in six animals of group 6 (8000 mg/kg), gastric overflow with abundant accumulation of liquid contents was found.

Male mouse 3.3 had abundant blood accumulation in the intestinal lumen (Figure 2.3.4.16). Males 6.2 and 6.5 had abundant accumulation of dense feces in the loops of the large intestine (coprostasis) (Figure 2.3.4.17).

Several animals of groups 2, 5 and 6 had flatulence (Figure 2.3.4.18).

FINDINGS

The study of the toxic properties of test object Lithium Ascorbate, substance (Normopharm LLC, Russia) following a single intragastric administration to sexually mature mice concluded the following:

1. The picture of intoxication (inhibition of the general condition of various severity, ruffled hair, diarrhea) was manifested immediately after administration. The maximum intoxication in the survived animals was in the first 3-4 hours of observation. The severity and frequency of signs of intoxication increased with increasing dose of the object. Mortality was predominantly delayed, with death occurring 3-5 days after administration.

2. In unplanned necropsia, signs of local irritation (erosive-ulcerative lesions of the gastric mucosa, hemorrhages in the gastric mucosa, flatulence, desquamation of the intestinal epithelium, accumulation of blood in the intestinal lumen) were shown in all groups that received the test object. In routine necropsia, signs of local irritation (erosive-ulcerative mucosal lesions) were found only in the group that received the object in a dose of 6000 mg/kg.

3. LD50 in males - 6140±319 mg/kg, in females - 6920±426 mg/kg. The test object during intragastric administration is assigned to the 4th class of low-toxic substances according to the GOST classification 12.1.007-76 (LD50 > 5000 mg/kg for intragastric administration) and to the 5th hazard class according to the GHS OECD classification (LD50 > 5000 mg/kg).  
   

CONCLUSION

All study activity to investigate object of Lithium Ascorbate, substance (Normopharm LLC, Russia) was planned and implemented in strict accordance with the requirements of the Ministry of Health of the Russian Federation and international standards in preclinical studies of the safety of pharmacological agents - the GLP system (Good Laboratory Practice) [18].

The picture of intoxication was observed when the object was administered in all doses. LD50 in males following intragastric administration is 6140±319 mg/kg, in females 6920±426. The test object for intragastric administration is assigned to the 4th class of low-toxic substances according to the GOST classification 12.1.007-76 (LD50>5000 mg/kg for intragastric administration) and to the 5th hazard class according to the GHS OECD classification (LD50 > 5000 mg/kg).  

Signs of local irritant action, such as erosive-ulcerative lesions of the gastric mucosa, hemorrhages in the gastric mucosa, flatulence, desquamation of the intestinal epithelium, accumulation of blood in the intestinal lumen were found in all doses of the test object, mainly in dead animals.