UDC 615.214: 615.076.9 APPROVED

Director of JSC “NPO HOUSE OF PHARMACY”, Doctor of Medical Sciences M.N. Makarova [Signature] 13.09.2021

It is carried out in accordance with the GLP OECD Principles

STUDY REPORT

Study of the allergenic properties of substance Lithium Ascorbate

(final)

E.A. Roshchina Study Leader  Date, signature

Leningrad Region, 2021

ABSTRACT

“Active Cutaneous Anaphylaxis Reaction” Test in Guinea Pigs
Sensitization Stage

Remark

1 Number of guinea pigs (n) - 10 males/group.

Challenge stage

Remarks

1 Number of guinea pigs (n) - 10 males/group;

2 20 minutes after the administration of the test objects, animals of all groups were injected intravenously with 0.5 ml of a 1% solution of Evans blue dye;

3 Euthanasia of animals 30 minutes after administration of the solution of Evans blue dye.

“Delayed-type hypersensitivity reaction” in mice

Sensitization Stage

Remark

1 Number of mice (n) - 10 males/group.

CONCLUSION

Study code: 1.21/21, Active cutaneous anaphylaxis test, test system: White guinea pigs, 10 males/group, sensitization - subcutaneous (once) and intramuscular (twice), challenge - intradermal

Keywords: Lithium ascorbate, sensitization, active cutaneous anaphylaxis, delayed-type hypersensitivity, guinea pigs, mice.

The report is presented on 169 pages, including 4 figures and 10 tables.

STUDY DATES

LIST OF EXECUTORS

E.A. Roshchina
 (abstract, introduction, ab.1 materials and methods, ab.2 study results, FINDINGS)

Head of the Pharmacist Service

N.A. Aleshanova
(ab. cl. 1.1 test objects)

A.V. Vasiliev
(ab.cl. 1.2 animals)

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 and Amendment № 1 to the Study Plan 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 carried out by the research institution.

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, the Study Plan, and Amendment № 1 to the Study Plan, mutually agreed between the Sponsor and the Research Institution.

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 1.21/21 dated May 12, 2021, 10 persons voted).

Director of JSC “NPO “HOUSE OF PHARMACY”

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.

Quality Service officer

TABLE OF CONTENTS

LIST OF ABBREVIATIONS AND ACRONYMS 14

INTRODUCTION 15

1 Materials and methods 17

1.1 Study objects 17

1.1.1 Test Object 17

1.1.2 Control Substance and vehicle for the tst object 17

1.1.3 Sensitization agent (HRT test) 17

1.3 Method of administration and dose selection 22

1.3.1 Method and duration of administration 22

1.3.2 Selection and calculation of doses at the sensitization stage 22

1.3.3 Selection and calculation of doses to be administered at the challenge stage 23

1.3.4 Dosing procedure 24

1.4 Methodology 25

1.4.1 Study design 25

1.4.1.1 Active cutaneous anaphylaxis reaction test in guinea pigs 25

1.4.1.2 “Delayed-type hypersensitivity reaction” in mice 26

1.4.2 Recording of animal body weight 28

1.4.3 Clinical observation of animals 28

1.4.4                Feed deprivation 31

1.4.5                Euthanasia 31

1.4.6 Data analysis 32

1.4.7 Quality control 29

2 Study results 33

2.1 Results of the “active cutaneous anaphylaxis reaction” test 33

2.2 Results of the “delayed-type hypersensitivity reaction” test 33

2.3 Body weight of animals 34

2.4 Results of clinical observation 34

FINDINGS 35

LIST OF ABBREVIATIONS AND ACRONYMS

In this study report, the following abbreviations and acronyms are used:

INTRODUCTION

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

Organic lithium salts are most often used as neuroprotective agents for various affective disorders and vascular cognitive disorders [1, 2]. There are data on the efficacy of lithium not only in endogenous mental disorders, but also in patients with organic psychoses, epilepsy and various psychopathies. In experiments, the neuroprotective effect of lithium salt preparations was established in various models of cerebral ischemia [3]. However, the mechanism of action of lithium salts is not well understood. At the same time, tests on rats have shown a low cumulative effect and low toxicity of lithium ascorbate [4]. In this regard, the study of lithium preparations, in particular the complex of lithium and ascorbic acid, remains relevant. It has been established that this salt has anti-stress and antidepressant activity [5].

This study aimed at studying the allergenic properties of the test object substance Lithium Ascorbate (Normopharm LLC, Russia), is regulated by the requirements of regulatory documents [6-9] and is part of a set of preclinical studies necessary for the product registration in the Russian Federation and within the Eurasian Economic Union.

Allergenic properties are understood as the ability of any substance to cause a hypersensitivity condition (hypersensitivity, sensitization) when administered into the body [6, 10]. The pathogenesis of the most severe allergic complications developing in type 1 hypersensitivity is based on the activation of Th2 helper cells and the production of cytokines IL-4, IL-5, and IL-13, followed by the synthesis of IgE antibodies with a high affinity for mast cells and basophils [6, 11]. The antigen interacts with mast cell-fixed IgE antibodies, which leads to cell activation and secretion of allergy mediators (histamine, serotonin, etc.) [2].

The mechanism by which an allergic reaction can develop depends on many factors - the nature of antigen, dose, route of administration, frequency and duration of administration, presence of adjuvants, selection of animals, physicochemical structure of a pharmacological substance, the ability to combine with proteins in the body, etc.

“Immediate” type allergic reactions develop rapidly, within a few minutes (1-20 minutes), and their mechanism involves the antigen-antibody reaction in tissues and liquid tissue media.

“Delayed” type allergic reactions are reactions between the antigen and sensitized T-lymphocytes with the subsequent development (after 24-48 hours) of allergic inflammation. To enhance the formation of antibodies at the sensitization stage, FCA (Freund’s complete adjuvant) is used.

The “immediate” and “delayed” types correspond to different stages of allergic reactions: immunological, pathochemical, and pathophysiological.

The study was carried out with the engagement of employees of the necessary departments to [Annex A], in accordance with the approved Study Plan and Amendment № 1 to the Study Plan [Annex B], reviewed by the bioethics commission and approved for [Annex C].

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

STUDY GOAL AND OBJECTIVES

Study goal:

Evaluation of allergenic properties of test substance Lithium Ascorbate (Normopharm LLC, Russia).

Study objectives:

1. Analysis of the ability of the test object to induce a local anaphylaxis reaction in guinea pigs in the “active cutaneous anaphylaxis reaction” test;

2. Analysis of the ability of the test object to induce delayed-type allergic reactions in outbred mice in the “delayed-type hypersensitivity reaction” test.
   

1. Materials and methods

1. Study objects

1. Test Object 

Name: Lithium Ascorbate

INN: Ascorbate lithium.

Manufacturer: Normopharm LLC, Russia.

Study Object Code: T-1.21/21.

Form: Substance.

Pharmacotherapeutic group: Normothymics (thymoisoleptics).

Batch: not provided by the Sponsor (Study report № 120a/21 Annex D).

Manufacturing date: 15.12.2020.

Expiry date: 15.12.2022.

Storage conditions: In a place protected from light at a temperature of +2°C to +8°C. It may be stored for a short period of 3-5 days at room temperature.

2. Control Substance and Vehicle for Test Object

Name: 0.9% Sodium Chloride saline solution 

Manufacturer: Gematek LLC, Russia.

Study Object Code: M-1.21/21.

Dosage form: solution for infusion.

Batch: 20450221 / 21290421.

Manufacturing date: 28.02.2021 / 22.04.2021.

Expiry date: 31.04.2024 / 31.03.2024.

Storage conditions: At a temperature not above 30°C.

3. Sensitization agent (HRT test)

Name: FCA (Freund’s Complete Adjuvant).

Manufacturer: Sigma Aldrich, USA.

Form: Cell suspension.

Catalogue number: F5881-10ML.

Lot: SLCC3348.

Manufacturing date: 28.02.2021 / 22.04.2021.

Expiry date: 08.2024.

Storage conditions: at a temperature of 2 to 8°C.

The documents of the pharmacist service are given in Annex D. The research institution did not carry out identification, purity and stability studies of the test object. These values were determined by the Sponsor according to standard methods. The study sponsor is responsible for the reliability of the submitted data on the identity, purity and stability of the test object.

1.2 Animals

Rationale for the selection of animal species: In this study, as the test system, animals of one sex were used 

The number of animals, 10 male guinea pigs, 10 male mice in the sensitization/challenge groups, and 2 male guinea pigs and 3 male mice in the challenge dose groups, was sufficient to evaluate the nature and frequency of reported effects. At the same time, the number of animals was minimal from the point of view of ethical principles.

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 [14].

3. Method of administration and dose selection

             1.3.1 Method and duration of administration

In accordance with the Guidelines for Preclinical Studies of Drugs, the following administration scheme is used to investigate the allergenic properties of the test object: the first injection is made subcutaneously into the withers area, the next two intramuscularly into the thigh area [6].

Thus, during the sensitization stage in the “active cutaneous anaphylaxis reaction” test, the test object and the control substance were administered to guinea pigs subcutaneously and intramuscularly. In the “delayed-type hypersensitivity reaction” test, at the sensitization stage, the test object and the control substance were injected into mice intradermally

1.3.2 Selection and calculation of doses at the sensitization stage

When investigating the sensitizing properties of the test objects in accordance with the Guidelines for Preclinical Studies of Drugs, it is optimal to study at least two dose levels - the therapeutic dose and a dose a level higher [6].

In accordance with the Sponsor’s recommendations, the following doses were used in the experiment: HTD, 5HTD, 10HTD, which corresponds to 10 mg/kg, 50 mg/kg and 100 mg/kg for humans. Taking into account the conversion factors [16], the studied doses for guinea pigs were:

10 mg/kg (HTD) x 37 (conversion factor for a 60 kg person) / 8 (conversion factor for guinea pig) ≈ 46.3 mg/kg.

50 mg/kg (HTD) = 232 mg/kg.

100 mg/kg (10HTD) = 463 mg/kg.

Taking into account the conversion factors [16], the studied doses for mice were:

10 mg/kg (HTD) x 37 (conversion factor for a 60 kg human) / 3 (conversion factor for a mouse) ≈ 123.3 mg/kg.

50 mg/kg (5HTD) = 617 mg/kg.

100 mg/kg (10HTD) = 1234 mg/kg.

Based on Amendment № 1 to Study Plan № 1 dated July 27, 2021 [Annex B], the originally planned doses for the “delayed-type hypersensitivity reaction” test were reduced, since according to the results of the test sample preparation (report № 109.21 dated July 16, 2021 [Annex D]), the solubility of substance Lithium Ascorbate in 0.9% sodium chloride solution was 250 mg/ml or 7.5 mg in 30 μl,  which corresponds to a dose of 250 mg/kg (with an average mouse body weight of 30 g and a planned dosing volume of 30 μl of the substance solution + 30 μl of FCA). Therefore, the following doses of substance lithium ascorbate were studied in mice in the “delayed-type hypersensitivity reaction” test: 61.7 mg/kg (1/2HTD), 123.3 mg/kg (HTD) and 250 mg/kg (2HTD). Since the doses are calculated per kg of animal body weight, and the average body weight of a mouse is 30 g, solutions of the test object with a concentration of 61.7 mg/ml (dose - 1.8 mg/mouse), 123.3 mg/ml (dose - 3.7 mg/mouse) and 250 mg/ml (dose - 7.5 mg/mouse) were used for administration. The dosing volume for one mouse was fixed at 60 μL/mouse.

The control substance was administered to the animals in an amount equivalent to the dosing volume of the maximum dose of the test object.

3.  Selection and calculation of doses to be administered at the challenge stage

The challenge concentration of the test object was selected based on the results of the search for the challenge dose in “active cutaneous anaphylaxis” and “delayed-type hypersensitivity reaction” tests.

Search of the challenge dose in the active cutaneous anaphylaxis reaction test

On day 15 day of the experiment, the following concentrations of the test object were investigated on intact male guinea pigs: 10 mg/mL, 1 mg/mL, and 0.1 mg/mL (Table 1.3.3.1). The test object was injected intradermally at a rate of 50 μL/animal. In order to control reactivity, a single intradermal injection of saline solution in the amount of 50 μl/animal was made in another area of the skin.

Table 1.3.3.1 - Characteristics of experimental groups for the search of the challenge dose in the “active cutaneous anaphylaxis reaction” test in male guinea pigs

According to the study results, it was found that none of the studied concentrations of the test object caused skin irritation 24 hours after intradermal administration.

Search of the challenge dose in the delayed-type hypersensitivity reaction test

On day 2 of the study, the following concentrations of the test object were investigated on intact male mice: 10 mg/mL, 1 mg/mL, and 0.1 mg/mL (Table 1.3.3.2). The test object in the studied concentrations was injected under the plantar aponeurosis of the right pelvic extremity (the “experimental” extremity) in the amount of 40 μl/animal. In order to control the reactivity under plantar aponeurosis of the left pelvic extremity (the “control” extremity), a single injection of saline solution in the amount of 40 μl/animal was made.

According to the study results, it was found that none of the studied concentrations of the test object and saline had an irritating effect and did not lead to the development of dermal edema of the “experimental” extremity 24 hours after administration.

Table 1.3.3.2 - Characterization of experimental groups for the search of a challenge dose in the “delayed-type hypersensitivity reaction” test in male mice

Thus, a maximum test concentration of 10 mg/ml was used for challenge injections in the “active cutaneous anaphylaxis” and “delayed-type hypersensitivity reaction” tests.

4. Dosing procedure

At the sensitization stage:

• “Active cutaneous anaphylaxis reaction” test. The test object and the control substance were administered into guinea pigs for three days: the first injection subcutaneously (in the withers area), then on days 3 and 5 of the experiment - intramuscularly in the thigh area using a three-component syringe with a needle diameter of 26 G. Volumes for subcutaneous and intramuscular injection did not exceed the maximum allowable doses for this type of animal, which was no more than 1.4 ml per animal.

• Delayed-type hypersensitivity reaction test. The test object and the control substance in FCA suspension (Freund’s complete adjuvant) were administered to mice once intradermally at the base of the tail in a volume of 60 μL/animal - a dose equivalent to 10 mM solution in FCA in a ratio of 1:1 - using a three-component syringe with a needle diameter of 26 G. Volumes for intradermal injection did not exceed the maximum allowable doses, which was no more than 60 μL/animal.

At the challenge stage:

• “Active cutaneous anaphylaxis reaction” test. The test object in a maximum concentration that does not have a local irritant action - 10 mg/ml was administered into guinea pigs intradermally in the back area (the skin area was previously shaved) in a volume of 50 μl/animal using a three-component syringe with a needle diameter of 26 G. 20 minutes after the injection of the test objects to the animals, using an intravenous catheter and a three-component syringe with a needle diameter of 26 G,  0.5 ml of 1% solution of Evans blue dye was administered.

• Delayed-type hypersensitivity reaction test. The test object in a maximum concentration that does not have a local irritant effect - 10 mg/ml was administered under the plantar aponeurosis of the right pelvic extremity в объеме 40 μL/animal in a volume of 40 μl/animal using a three-component syringe with a needle diameter of 26 G.

1.4 Methodology

1. Study design

1. Active cutaneous anaphylaxis reaction test in guinea pigs 

Sensitization

The test object and control substance were administered into guinea pigs three times: on days 1, 3 and 5 of the experiment. In the following days (from the 6th to the 14th) the animals were observed.

        The characteristics of the experimental groups and the schedule of the study are presented in Tables 1.4.1.1.1 and 1.4.1.1.2.

Table 1.4.1.1.1 – Characteristics of the experimental groups in “Active cutaneous anaphylaxis reaction test” in guinea pigs

Table 1.4.1.1.2 – Study schedule in the “active cutaneous anaphylaxis reaction” test in guinea pigs

Search of the challenge dose

On day 15 of the experiment, the challenge dose of the test object was searched for on 6 male intact guinea pigs (groups №№ 5-7) not involved in the main experiment. When searching for the challenge dose, the following concentrations of the test object were examined: 10 mg/ml, 1 mg/ml, 0.1 mg/ml. In order to control reactivity in another area of the skin, a single intradermal injection of saline in the amount of 50 μl/animal was performed. Previously, the hair was shaved in the back area, where intradermal injections were performed. The test object concentration of 10 mg/mL, which did not cause skin irritation 24 hours after intradermal administration, was taken as the challenge dose. The selection of the challenge dose is described in Section 1.3.3 “Selection and calculation of doses to be administered at the challenge stage”.

Challenge Stage

In the period from day 1 to day 18 of the experiment, the animals of groups №№ 1-4 were monitored daily. On day 18 of the experiment, these animals were administered once intradermally with a solution of the test object in a concentration that did not have a local irritant effect - 10 mg/ml. In order to control the reactivity of the test object and exclude the factor of injection into another area of the skin, 50 μl of saline solution was administered once intradermally. Previously, the hair was shaved in the back area, where the drug was administered intradermally.

20 minutes after the administration of the test objects, animals of all groups were administered intravenously with 0.5 ml of a 1% solution of Evans blue dye to distribute the dye through the systemic circulation to organs and tissues and stain the injection sites.

30 minutes after the injection of the blue Evans dye solution, the animals were euthanized, a patch of skin was cut out at the site of intradermal injections, and the size of the blue spot on the inside of the skin flap at the injection site was determined. A positive reaction was not found when the size of the blue spot exceeded 6 mm in diameter [6].

2. “Delayed-type hypersensitivity reaction” in mice 

Sensitization

The test object and the control substance in a solution of FCA suspension (Freund’s complete adjuvant) were administered into mice once intradermally into the base of the tail in a volume of 60 μl/animal using a three-component syringe with a needle diameter of 26 G in a ratio of 1:1. In the following days (from the 2nd to the 5th) the animals were observed.

The characteristics of the experimental groups and the study schedule are presented in Tables 1.4.1.2.1 and 1.4.1.2.2.

Table 1.4.1.2.1 - Characteristics of experimental groups in the “delayed-type hypersensitivity reaction” test in mice

Table 1.4.1.2.2 - Study schedule in the “delayed-type hypersensitivity reaction” test in mice

The search for the challenge dose of the test object was carried out on day 2 of the experiment on 12 male intact mice (groups №№ 12-15) not involved in the main experiment. When searching for a challenge dose, the following concentrations of the test object were examined: 10 mg/ml, 1 mg/ml, 0.1 mg/ml. In order to control reactivity under plantar aponeurosis of the left pelvic extremity (“control” extremity), a single injection of saline solution in the amount of 40 μl was performed. Group № 15 was administered with the control substance as a negative control according to the same scheme. The allowable dose was concentration 10 mg/ml, which did not cause swelling of the tissues of the “tested” extremity 24 hours after administration. The degree of edema was evaluate using the formula (1):               Wtr - Wc 

                                    RI =    Wc                            (1)   

where RI – DTR response index, Wtr – weight of the “test” extremity (right pelvic extremity), Wc – weight of the “control” extremity (left pelvic extremity).

The selection of the challenge dose is described in Section 1.3.3 “Selection and calculation of doses to be administered at the challenge stage”.

Challenge stage

In the period from day 1 to day 7 of the experiment, the animals of groups №№ 8-11 were subjected to daily clinical observation. To detect sensitization in experimental animals, on day 6 day of the experiment, a single challenge dose of the test object in the amount of 40 μl (the “experimental” extremity) in a concentration of 10 mg/ml was administered under the plantar aponeurosis of the right pelvic extremity, and 40 μl of saline (the “control” extremity) was administered under the plantar aponeurosis of the left pelvic extremity.

24 hours after the administration of the challenge dose, the animals were euthanized with CO2 followed by cervical dislocation, the pelvic extremities were cut off at the level of the tarzotibial joint, weighed and the response index (RI) was determined according to formula (1).

The procedure of weighing the pelvic extremities of mice was carried out on an electronic scale “Adventurer” 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 [Annex B].

2. Recording of animal body weight

The body weight of guinea pigs was recorded before the first administration of the study objects in order to determine the volume of administration. The weight of the mice was fixed to fulfill the condition of deviation from the mean for all groups by no more than 20%. [Annex G].

The procedure of weighing guinea pigs 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 [Annex B].

The procedure of weighing mice was carried out on an 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 [Annex B].

3. Clinical observation of animals

General clinical observations of the animals were carried out daily. A detailed examination of the animal was carried out in the cage, in the hands and in the open area [Annex G]. The following was recorded:

• Animal behavior in a cage: normal /distress / excitement / aggression

• Animal behavior in the open area: impaired coordination of movements: ataxia/hyperkinesis;

• Response to stimuli: normal/decreased/increased;

• Muscle tone: decreased/increased;

• Skin: normal / discoloration / hemorrhage / impaired integrity / palpable masses / decreased turgor;

• Eyes and conjunctiva: normal / impaired integrity / ptosis of the eyelid / exophthalmos / presence of discharge / discoloration of the conjunctiva;

• Nasal mucosa: normal/discharge/discoloration/impaired integrity;

• Oral cavity and oral mucosa: normal / drooling / discoloration;

• Injection site: normal/swelling at injection site/redness at injection site/palpable masses;

• Death.

1. Feed deprivation

Guinea pigs and mice were not restricted in food and water throughout the experiment.

1. Euthanasia

Guinea pigs of groups №№ 5-7 used for the search of the challenge dose, were returned to the veterinary service.

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 [12], guinea pigs were euthanized by overdosing with an anesthetic followed by exsanguination from the heart cavities. The mice were euthanized with CO2 followed by cervical dislocation. These types of animal euthanasia are accompanied by a minimum of pain, suffering and distress and are carried out by competent employees.

4. Data analysis

Descriptive statistics were applied to all the data: the data were checked against the normal distribution law using the Shapiro-Wilk’s W test. For the data corresponding to the normal distribution law, the mean value (M) and the standard error of the mean (SEM) were calculated, which, together with the value of n (number of cases), are presented in the summary tables. For data that do not conform to the normal distribution law, the median (Me) and quartile range (Q1; Q3), which, together with the value of n (number of cases), are presented in the summary tables. To evaluate data with signs of normal distribution, univariate analysis of variance (ANOVA) was used, followed by the post hoc Tukey’s test). For the analysis of data that does not conform to the normal distribution law, as well as expressed as a percentage, the Kruskal-Wallis 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).

5. Quality control

The quality service of the research institution performed:

• review of the study design; 

• checking the study plan - schedule;

• incoming audit of a preclinical study;

• audit of the experimental part of the study;

• checking the chronology of the study and the completeness of the R&D report;

• verification of the final R&D report.

The results of the inspection are presented in the conclusion of the quality service [Annex I].

2 Study results

1. Results of the “active cutaneous anaphylaxis reaction” test

Search of the challenge dose

Taking into account the skin reaction 24 hours after intradermal injection of the test object in concentrations of 10 mg/mL, 1 mg/mL, and 0.1 mg/mL, it was found that none of the tested concentrations had a local irritant action 24 hours after intradermal administration. Thus, a test object concentration of 10 mg/mL was used as a challenge dose.

Challenge stage

There was no reaction to the test objects regardless of the group (Figure 14). A spot was not found at the injection site of the study object, and therefore the reaction was taken as 0. No statistical analyses were performed.

Thus, in the groups of animals sensitized with test substance Lithium Ascorbate (Normopharm LLC, Russia) in 3 doses (corresponding to HTD, 5HTD, 10HTD), after intradermal administration of the challenge dose, no signs of an allergic reaction as an active cutaneous anaphylaxis were recorded.

2. Results of the “delayed-type hypersensitivity reaction” test

Search of the challenge dose

Based on the mass values of the left and right extremities, the values of the response index were calculated, according to formula (1). Analysis of the data using the Kruskel-Wallis test showed no statistically significant differences between the experimental groups of male mice (p>0.05, Table 1). Thus, the maximum concentration of the test object of 10 mg/ml was selected as the challenge dose for the “delayed-type hypersensitivity reaction” test.

Challenge stage

When analyzing the data using the Kruskel-Wallis test, the statistically significant influence of the group factor was not determined, but the obtained value of p=0.055 borders on the statistical significance of the results. Therefore, a cross-group analysis of the data was carried out using the Mann-Whitney test, which showed a statistically significant increase in the response index in male mice treated with the test object in a dose of 123.3 mg/kg compared to male mice treated with the test object in a dose of 250 mg/kg and the control substance (p<0.05, table 2). There were no statistically significant differences between the maximum dose group (250 mg/kg) and the control group, so the observed changes can be considered clinically insignificant.

Thus, in the groups of animals sensitized with test substance Lithium Ascorbate (Normopharm LLC, Russia) in 3 doses (corresponding to 1/2HTD, HTD, 2HTD), after the administration of the challenge dose under plantar aponeurosis of the right pelvic extremity, no signs of an allergic reaction of the delayed hypersensitivity type were found.

3. Body weight of animals

The body weight of guinea pigs was recorded immediately before the first injection of the study objects in order to determine the volume of administration. The weight of the mice was fixed to fulfill the condition of deviation from the mean for all groups by no more than 20%. The data corresponded to the normal distribution law. The weight of guinea pigs and mice before the experiment did not differ between the corresponding groups (p>0.05, Tables 3 and 4).

4. Results of clinical observation

3. The examination of male guinea pigs and mice in the cage, in the hands and in the open area did not show any abnormalities in clinical signs, appearance and behavior of the experimental animals in comparison with the animals that received the control substance.
   

FINDINGS

Based on the results of the study of the allergenic properties of substance Lithium Ascorbate (Normopharm LLC, Russia), the following FINDINGS can be drawn:

1. In the analysis of the ability of the test object in doses of 46.3 mg/kg (HTD), 232 mg/kg (5HTD) and 463 mg/kg (10HTD) to cause a local anaphylaxis reaction in guinea pigs, no cases of an “immediate” type of reaction were recorded.

2. In the analysis of the ability of the test object in doses of 61.7 mg/kg (1/2HTD), 123.3 mg/kg (HTD) and 250 mg/kg (2HTD) to cause a delayed-type allergic reaction in mice, no cases of a “delayed” type reaction were recorded.