DC 615.21: 615.076.9 APPROVED

General Director

CJSC “Saint Petersburg Institute of Pharmacy”

Doctor of Medical Sciences, Professor

[Signature] V.G. Makarov

24.11.2021 

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 rats 

(final)

Study Leader  

[Signature] A.V. Popova

Leningrad Region
2021

ABSTRACT

o

The pattern of intoxication (depression of condition of various severity, diarrhea, ruffled hair, bloody discharge from the nose and eyes) and death of animals were observed when the object was administered in doses of 4000 mg/kg, 5000 mg/kg, 6000 mg/kg and 7000 mg/kg.

When lithium ascorbate was administered in a dose of 3000 mg/kg, no intoxication was observed, no deaths were recorded. A dose of 3000 mg/kg may be considered as the maximum tolerated dose.

Death was predominantly delayed (days 3-5 after administration). The cause of the death of one female in group 4 (dose 6000 mg/kg) was acute post-hemorrhagic anemia, in remaining animals (groups № 2-5) - acute heart failure.

On day 8 of the experiment, the survived males in the groups that received the substance in the dose range of 4000 - 6000 mg/kg, as well as the females that received the substance in the dose of 5000 mg/kg, had a decrease in body weight relative to the control group. Further, during the observation period, no differences in body weight were found in the survived animals compared to the control. Signs of LIA were found at all doses in dead animals. LD50 in males following intragastric administration is 4810±386 mg/kg, in females 5070±161 mg/kg.

The test object when administered intravenously to males is assigned to the 3rd class of moderately toxic substances according to the classification GOST 12.1.007-76 (151 mg/kg<LD50<5000 mg/kg for intragastric administration and to the 4th hazard class according to the GHS OECD classification (2000 mg/kg<LD50<5000 mg/kg). According to LD50, when administered intravenously to females, the test object is assigned to the 4th class of low-toxic substances according to the classification GOST 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).

Keywords: Lithium ascorbate, acute toxicity, rats, intragastric administration

The report is presented on 256 pages, including 14 tables and 45 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 № 2 mutually agreed with the Sponsor (Normopharm LLC, Russia).

The study complies with the GLP requirements, except for the fact that no identification, purity and stability studies 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

A.V. Popova

/Name/

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]             21.09.2021

/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. Study objects 18

1. Test object 18

2. Control substance 18

2. Animals 18

3. Method of administration and dose selection 22

1. Method and duration of administration 22

2. Selection and calculation of doses 22

3. Dosing procedure 22

4. Methodology 23

1. Study design 23

2. Feed deprivation 23

3. Body weight recording 24

4. Euthanasia 25

5. Pathomorphological examination 26

6. Evaluation of a local irritant action 27

7. Data analysis 28

8. Study assurance and quality control 28

2. STUDY RESULTS ………………………………………………………………………………………….29

1. Toxicometry 29

1. Lethality 29

2. Picture of intoxication. Clinical examination 29

2. Effect of a single intravenous administration of the test object ob body weight of animals ………………… …30

3. Pathomorphological examination data …..31

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

2. Results of pathomorphological examination of animals with planned necropsy ..…32

2.3.3 Mass coefficients of internal organs of experimental animals …………………..…..32

2.3.4 Results of evaluation of local tolerability (LIA)………………………………..….32

FINDINGS 34

CONCLUSION 35

TABLES AND FIGURES 36

DATA ARCHIVING 55

REGULATORY DOCUMENTS 56

REFERENCES 57

ANNEX А 59

ANNEX B 66

ANNEX C 99

ANNEX D 102

ANNEX E 106

ANNEX F 112

ANNEX J 114

ANNEX G 197

ANNEX I  ………………………………………………………………………………………………………..252

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 rats, 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 to sexually mature rats.

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.
   

1. Materials and methods

1. Study objects

1. Test object

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 identity, 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 identity, purity and stability of the test object.

1.2 Animals

certificate:

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].

 

Ammonia and carbon

3. 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].

2. Selection and calculation of doses

In this study, in accordance with the Sponsor’s recommendation, the test object was to be 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 object administration in a dose of 6000 mg/kg. After the administration of the test object, lethal outcomes were observed, based on which the doses of 5000 mg/kg and 7000 mg/kg were selected for the next dosing. Administration of a dose of 7000 mg/kg led to the death of 100% of the animals in the group. Based on these data, the range of studied doses was changed for the calculation of LD50: instead of the planned dose of 8000 mg/kg, a dose of 3000 mg/kg was administered to male and female rats (in accordance with Amendment № 1 of 03.08.2021) [Appendix B]. Administration of a dose of 3000 mg/kg was not accompanied by the death of animals.

Thus, male and female rats were administered once intragastrically, in fractions, with doses of 3000 mg/kg, 4000 mg/kg, 5000 mg/kg, 6000 mg/kg, 7000 mg/kg.

The control substance was administered into the animals of group № 1 once, fractionally, in an amount equivalent to the dosing volume of the test object.

3. Dosing procedure

The test object (as a suspension) and the control substance were administered into 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 in different groups are presented in Table 1.3.3.1.

Table 1.3.3.1 – Dosing volumes of the test object

Remark - a - the dose is indicated on the basis of Amendment № 1 dated 03.08.2021.

The test object was administered into animals once, fractionally, in two equal parts, using special probes and syringes, with an interval between injections of no more than 30 minutes.

4. Methodology

1. Study design

The total number of animals involved in the experiment is 60 Wistar rats (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

Remark  - a - the dose is indicated on the basis of Amendment № 1 dated 03.08.2021.

Table 1.4.1.2 - Manipulation schedule

2. Feed deprivation

The animals were deprived of food 16 hours before administration, body weight recording and euthanasia. Water was given ad libitum throughout the experiment.

3. 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 primary maps [Appendix G].

The procedure of weighing rats 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].

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

1. Recording of the timing of the development of intoxication

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 recorded:

• Behavior: distress/agitation;

• Response to stimuli: decrease/increase;

• 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.

2. Clinical examination

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

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.

5. Euthanasia

On day 15 of the experiment, the animals were euthanized with CO2 followed by exsanguination from the heart cavities. In accordance with Directive 2010/63/EU of the European Parliament and of the Council 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.

6. Pathomorphological examination

The post-mortem examination included a macroscopic examination of the animals euthanized according to the schedule, as well as a pathomorphological examination of the corpses of animals that died during the experiment [12].

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. The evaluation of the condition of the thoracic and abdominal cavity and a macroscopic examination of the internal organs were carried out. Similar examinations were carried out on dead animals with the completion of a necropsy chart [Appendix I].

Organs extracted during necropsy were weighed, and paired organs were weighed together. This 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” model RV 214 (OHAUS, China). The maximum weighing limit is 210 g, the minimum weighing limit is 0.001 g. Calibration mark is 0.001 g. Accuracy class is 1 [Appendix B].

List of organs to be weighed:

• Heart

• Lungs with trachea

• Thymus

• Liver

• Spleen

• Kidneys

• Adrenal glands

• Cerebrum

• Testes/ovaries

Organ Collection

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

The organs have been archived and will be handed over to the Sponsor 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 if macroscopic changes were found in 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). Stomachs and intestinal fragments from several animals were taken for histological examination.

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 action

To evaluate the local irritant action of the test object during the necropsy procedure, the condition of the gastrointestinal tract organs 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 was applied to all data: the data were checked for compliance with the normal distribution law using the Shapiro-Wilk’s W test. For data with normal distribution, the mean value and the standard error of the mean were calculated, which, together with the value of n (number of observations), are presented in the summary tables. To evaluate data with signs of normal distribution, univariate analysis of variance was used (ANOVA), if a significant effect of the studied factor was found, subsequent intergroup comparisons (post hoc analysis) were carried out using Tukey’s test analysis. 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 action of lithium ascorbate, substance (Normopharm LLC, Russia), was carried out following a single fractional intragastric administration into male and female outbred rats. The data obtained during the experiment are presented in full in the source charts [Appendix G].

1. Toxicometry

1. Lethality

During the experiment, the death of 13 males and 12 females was recorded. Lethal effects based on the results of 48 hours of observation are presented in Table 2.1.1.1, Lethality for 14 days of the experiment is presented in Table 2.1.1.2. Animal lethality was recorded in all groups except group 1 (control) and group 6 (dose 3000 mg/kg).

In the groups of animals that received the test object in doses of 4000 mg/kg, 5000 mg/kg, 6000 mg/kg and 7000 mg/kg, Lethality was delayed (48 hours after the administration of the test object), data on the timing of animal death are presented in Tables 2.1.1.3 and 2.1.1.4.

LD50 in males - 4810±386 mg/kg, in females 5070±161 mg/kg. 

The test object when administered intragastrically to males is assigned to the 3rd class of moderately toxic substances according to the GOST classification 12.1.007-76 (151 mg/kg<LD50<5000 mg/kg for intragastric administration) [15] and to the 4th hazard class according to the GHS OECD classification (2000 mg/kg<LD50<5000 mg/kg) [16].

According to intragastric LD50 in females, the test object 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).

2. Picture of intoxication. Clinical examination

Dose 3000 mg/kg. No deaths or signs of intoxication were recorded throughout the experiment.

Dose 4000 mg/kg. On day 1 of observation, the animals developed diarrhea, which ended by the second day of observation. From the second day of observation, the males showed inhibition of the general condition of various degrees of severity, ruffled hair. In isolated cases, ataxia, nasal discharge, tremor was observed. Subsequently, the severity of intoxication increased.

The maximum intoxication occurred on day 3 of observation. In females, the severity of toxic effects was less intensive, only diarrhea was noted on the first day after administration, in the following days the animals were in a satisfactory condition. The incidence of maximum signs of intoxication (day 3) is presented in Table 2.1.2.1.

Dose 5000 mg/kg. In the first 24 hours after administration, only diarrhea was observed in the animals, which resolved by the third day of observation. From the second day of observation, inhibition of condition of various severity, ruffled hair, discharge from the eyes and nose, tremor, and ataxia were noted in the animals. Maximum intoxication occurred on day 3 after administration of the object (Table 2.1.2.2). In survived animals, signs of intoxication persisted up to 5 days after administration of the test object.

Dose 6000 mg/kg. In the first 4 hours of observation, the picture of intoxication manifested itself in ruffled hair and diarrhea, diarrhea disappeared within the first 48 hours. Most intensive signs of intoxication were observed on days 3-4 of observation. In males, intoxication manifested itself in inhibition of the general condition of various degrees of severity, ruffled hair, discharge from the nose and eyes. In females, the picture of intoxication was more pronounced, tremor, decreased response to stimuli, ataxia, and ptosis in a single case (Table 2.1.2.3).

Dose 7000 mg/kg. Intoxication manifested itself in inhibition of the condition of various degrees of severity, ruffled hair, discharge from the nose and eyes, ataxia, tremor, and decreased response to stimuli. Maximum intoxication in animals was observed in the first 48 hours after administration (Table 2.1.2.4). By day 5, the lethality rate reached 100%.

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

Tables 2.2.1 and 2.2.2 provide data on the body weights of male and female rats when the test object is administered intragastrically. The data corresponded to the normal distribution law. Univariate analysis of variance did not show the effect of the “group” factor on the initial body weight of males (ANOVA, p>0.05), but showed the effect of the “group” factor on the initial body weight of females (ANOVA, p<0.05). Subsequent intergroup comparison using the Tukey test did not show significant intergroup differences (Tukey test, p>0.05). On the second day of the experiment, the effect of the “group” factor on the body weight of females (ANOVA, p<0.05) was established. Subsequent intergroup comparison using the Tukey test did not show significant intergroup differences (Tukey test, p>0.05). In males on day 2 of the experiment, the influence of the “group” factor was not shown. On day 8 of the experiment, the effect of the “group” factor on the body weight of males and females (ANOVA, p<0.05) was established: in males who received the substance in doses of 4000 mg/kg and 6000 mg/kg, the body weight was statistically significantly lower than in the control (Table 2.2.1). In the survived males of the 5000 mg/kg group, body weight was also reduced relative to the control (Table 2.2.1). A significant decrease in body weight on day 8 of the experiment relative to the control group was also observed in females that received the substance in a dose of 5000 mg/kg (ANOVA, p<0.05).

On day 15 of the experiment, there was no significant effect of the “group” factor on the body weight of males and females. Overall, the survived animals showed physiological weight gain relative to baseline by day 15.

3. Pathomorphological examination data

1. Results of pathomorphological examination of animals with unplanned necropsy

In all dead animals of groups № 2 and № 3, as well as in most dead animals of groups № 4 and № 5, cerebral edema and plethora of its membranes were found (Table 2.3.1.1, Figures 2.3.1.1-2.3.1.4). Most of the dead animals of groups №№ 3-5 had plethoric internal organs. Pulmonary edema was found in all groups (Figures 2.3.1.5-2.3.1.8) treated with the test object except for group 6 (dose 3000 mg/kg). Isolated lung hemorrhages were observed (Figure 2.3.1.9) One male from group 5 (dose 7000 mg/kg) had a single abscess in the lungs (Figure 2.3.1.10).

One female from group 4 that received the test object in a dose of 6000 mg/kg, had yellowish kidneys and liver. Histological examination of the kidney of this animal showed plethora of the organ. Histological examination of the liver showed plethora, as well as foci of vacuolation of hepatocytes (most likely accumulation of glycogen), which can be considered a variant of the normal (Figure 2.3.1.11).

Changes in the gastrointestinal organs were also found in the dead animals (see the section “Results of the evaluation of local irritant action (LIA)”).

The immediate cause of death in one female rat from group 4 (dose 6000 mg/kg) was acute post-hemorrhagic anemia (see the section ”Results of the evaluation of local irritant action (LIA)”). The cause of death of remaining dead animals from groups №№ 2-5 was acute heart failure.

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 2.3.2.10).

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 showed the effect of the “group” factor on the mass coefficients of the female spleen (ANOVA, p<0.05). In female rats received the object in a dose of 5000 mg/kg, a statistically significant increase in mass coefficients of the spleen compared to the control was shownконтролем.

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

Routine necropsy

No pathological changes in the internal organs were found in routine necropsy (Table 2.3.4.1, Figures 2.3.4.1-2.3.4.5). Planned necropsy in group 5 (dose 7000 mg/kg) was not performed due to the death of all animals in this group.

Unplanned necropsy

Erosions of the glandular mucosa were found at the injection site (stomach) of a male rat from group 2 and two animals from group 3 (male 3.2 and female 3.7), which received the test object in doses of 4000 mg/kg and 5000 mg/kg, respectively (Figures 2.3.4.6-2.3.4.8). Histological examination of the stomach of male rat 3.2 and female rat 3.7 showed superficial erosions of the glandular mucosa (Figure 2.3.4.9). Male rat 3.2 also had an ulcer in the mucosa of the non-glandular portion (Figure 2.3.4.10).

In female rat 3.7, irregular mucosal congestion was found at the injection site (stomach) (Figure 2.3.4.11), as well as hemorrhages in the small and large intestines (Figure 2.3.4.12). Histological examination of the small intestine of female rat 3.7 showed villous atrophy and vacuolation of epithelial cells (Figure 2.3.4.13), and large intestine showed diffuse mucosal epithelial necrosis, submucosal edema, and subepithelial hemorrhages (Figure 2.3.4.14).

Hemorrhages were found at the injection site (stomach) in two animals from group 4 that received the test object in a dose of 6000 mg/kg, as well as in one female from group 5 that received the test object in a dose of 7000 mg/kg (Figures 2.3.4.15 and 2.3.4.16).

In two animals from group 4, as well as in two animals from group 5, irregular plethora of the mucous membrane was found at the injection site (stomach) (Figures 2.3.4.17 and 2.3.4.18), and the mucous membrane was loose. In a 4.8 female rat, the gastric mucosa was also loose.

In male 4.2, hemorrhages were found in the small and large intestines, while in females 4.7 and 4.10, hemorrhages were found only in the large intestine (Figure 2.3.4.19). At the same time, in the female rat 4.7, the intestinal lumen contained a large amount of blood.

Histological examination of the large intestine of male rat 4.2 and female rat 4.7 showed diffuse mucosal epithelial necrosis, submucosal edema, and massive hemorrhages (Figure 2.3.4.20). Male rat 4.2 also showed marked inflammatory infiltration and mucosal necrosis. Histological examination of the intestine of female rat 4.10 was not performed, but given the similar macroscopic picture, it can be assumed that the changes in the intestine were similar.

Male rats 5.1, 5.2, 5.3, 5.4 and female rats 5.10 also had large intestinal hemorrhages (Figure 2.3.4.21). In the intestinal lumen of male rats 5.2, 5.3, 5.4, 5.5 and female rats 5.6, 5.7, 5.8, the contents were watery (Figure 2.3.4.22). In female rats 5.6, 5.7, 5.8, similar watery contents were found in the stomach.

Histological examination of the large intestine of male rat 5.1 showed diffuse mucosal epithelial necrosis, inflammatory infiltration in areas of necrosis, submucosal edema, and massive hemorrhages (Figure 2.3.4.23). Histological examination of the intestines of male rats 5.2, 5.3, 5.4 and female rat 5.10 was not performed, but given the similar macroscopic picture, it can be assumed that the changes were similar.

Flatulence was found in most animals given the object in doses of 5000, 6000, 7000 mg/kg (Figure 2.3.4.24).

FINDINGS

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

1. The picture of intoxication (inhibition of the general condition of various severity, ruffled hair, diarrhea) was manifested immediately after administration of the substance in doses of 4000 mg/kg and higher. The maximum intoxication in the survived animals was on the 3rd day of observation. The severity and frequency of signs of intoxication increased with increasing dose of the object. Lethality was predominantly delayed, with death occurring on days 3-5 after administration.

2. In unplanned necropsia, signs of local irritation (erosive-ulcerative lesions of the gastric mucosa, hemorrhages in the gastric mucosa, flatulence, hemorrhages in the intestinal mucosa, necrosis of the colonic mucosa) were shown in the groups that received the test object in doses of 4000 mg/kg, 5000 mg/kg, 6000 mg/kg and 7000 mg/kg.

3. A dose of 3000 mg/kg can be considered as the maximum tolerated dose, as no animal deaths have been observed.

4. LD50 in males with intragastric injection - 4810±386 mg/kg, in females 5070±161 mg/kg. The tested object when administered intragastrically to males is assigned to the 3rd class of moderately toxic substances according to the GOST classification 12.1.007-76 (151 mg/kg<LD50<5000 mg/kg for intragastric administration and to the 4th hazard class according to the GHS OECD classification (2000 mg/kg<LD50<5000 mg/kg). According to LD50, when administered intragastrically to females, the test object is classified 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 substance Lithium Ascorbate (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) [17].

The picture of intoxication was observed when the object was administered in doses of 4000 mg/kg, 5000 mg/kg, 6000 mg/kg and 7000 mg/kg. LD50 in males following intragastric administration - 4810±386 mg/kg, in females 5070±161 mg/kg. The test object when administered intragastrically to males is assigned to the 3rd class of moderately toxic substances according to the GOST classification 12.1.007-76 (151 mg/kg<LD50<5000 mg/kg for intragastric administration and to the 4th hazard class according to the GHS classification OECD (2000 mg/kg<LD50<5000 mg/kg).  According to the LD50, when administered intragastrically to females, the test object 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 irritation, such as hemorrhages in the gastric mucosa, flatulence, hemorrhages in the intestinal mucosa, necrosis of the colonic mucosa, watery contents in the stomach and intestines were found in all doses of the test object, mainly in dead animals.

A dose of 3000 mg/kg can be considered as the maximum tolerated dose, as no animal deaths have been observed.