A series of toxicity studies are required to reveal the possible toxic reactions of drugs before clinical application and to ensure the safety and effectiveness of clinical use. Local toxicity test is an integral part of toxicology research, which is designed to study the toxic reactions caused by local (via eyes, ears, mouth, nose, respiratory tract, joint cavity, skin, muscle, mucous membrane tissue, etc.) administration of drugs, mainly including irritation test, allergy test, hemolysis, and skin toxicity tests.
Many alternative methods have emerged for local toxicity tests on animals, such as in vitro tests and computer simulations, to assess the toxicity of chemical substances. In general, topical toxicity testing of drugs is required for dosage forms such as ointments, drops, lotions, scalp patches, sprays, and topical powders. The local toxicity test mainly includes the following:
1. Allergy test
The allergy test observes animals' systemic or local allergic reactions after injection. There are four types of allergic reactions, including：
（1）Rapid type I mediated by immunoglobulin E (IgE)
（2）Cytotoxic type or cytolytic type II mediated by IgG
（3）Immune complex type or vasculitis type III mediated by IgG and IgM
（4）Late-type or type IV mediated by T lymphocytes.
1.1 Skin allergy test
Cutaneous hypersensitivity is a skin reaction associated with an immunological response after administering a test substance to the skin. The skin anaphylaxis test is to observe and detect whether a systemic or local allergic reaction occurs after the initial skin contact with the test substance and then the stimulated contact with the test substance. At the same time, the guinea pig Buehler test (BT) and guinea-pig maximization test (GPMT) need to be considered.
1.2 active systemic anaphylaxis (ASA)
The "Technical Guidelines for the Study of Chemical Drug Irritation, Allergenicity, and Hemolytic Properties" stipulate that drugs that are usually administered topically to exert systemic effects (such as injectables and transdermal absorption agents) need to be investigated for type Ⅰ allergic reactions, such as active systemic anaphylaxis (ASA).
To explore and establish the method of type I allergy test in rats based on blood biomarker detection, some researchers first conducted the development of the rat systemic allergic reaction symptom table and allergenicity assessment criteria, then the detection of serum indexes in sensitized rats, and then the observation of the performance of allergic reaction symptoms in rats after excitation, etc. Finally, it was found that the serum IgE level after sensitization in the rat allergy test method has a high correlation with the allergic reaction symptoms and can be used as a blood biomarker to evaluate the sensitization of the tested drugs.
1.3 Active cutaneous anaphylaxis (ACA)
Cutaneous anaphylaxis induced in animal models can be divided into active and passive anaphylaxis. In the active cutaneous anaphylaxis (ACA) model, mice are sensitized by receiving fractionated doses of the allergen, whereas in the passive cutaneous anaphylaxis (PCA) model, the animals are passively sensitized by receiving serum from other mice that were previously actively sensitized [1].
1.4 Passive cutaneous anaphylaxis (PCA)
Passive cutaneous anaphylaxis (PCA) is a susceptible method for detecting antibodies or antigens using local allergic reactions caused by antibodies that bind to the same or different animal tissues. Passive cutaneous anaphylaxis is a relatively sensitive method for testing specific antibody titers and investigating whether drugs induce type I allergic reactions. IgE is an essential medium for mediating type I hypersensitivity, and detecting IgE levels in serum or body fluid is of great significance in clarifying the drug-induced type I hypersensitivity reaction and understanding the mechanism of drug-induced allergic reaction.
The serum of sensitized animals is rich in IgE antibodies. After intradermal injection into normal guinea pigs, the Fc end of IgE binds to specific receptors on the surface of skin mast cells to form an IgE complex, which passively sensitizes normal animals. When sensitizing reagents are injected intravenously, the combination of antigen and antibody will cause mast cells to degranulate and release allergenic media such as histamine, thereby increasing the permeability of local blood vessels in the skin. The range of skin blue staining at this place can be used to know the size of the change in vascular permeability, which can reflect the degree of skin allergic reaction induced by the sensitizing agent[2].
Figure 1 Effects of asarone injection on the formation of blue spots in the skin of guinea pigs in passive anaphylaxis test （1×）[2]
Allergy is the most common and severe adverse reaction to injectables. The type of allergy test should be determined according to the characteristics of the drug, clinical indications, mode of administration, mechanism of occurrence and influencing factors, etc.
2. Irritant test
The irritant test mainly observes whether the blood vessels, muscles, skin, and other parts of the animal cause inflammatory lesions such as redness, congestion, exudation, degeneration, or necrosis after contact with the preparation when the drug is administered according to the proposed clinical route, and also observes the general state, behavior and signs of the animal to make a comprehensive evaluation of the local and overall.
2.1 Acute eye irritation test
Such as an acute eye irritation test aimed at confirming and evaluating whether the test product has an irritating or corrosive effect on the eyes of mammals and its degree to provide the basis for its production and use in safety protection.
2.2 Skin irritation test
Skin irritation refers to the reversible inflammatory lesions on the skin after the drug is administered through the skin. If the drug site produces irreversible tissue damage is called corrosive. The skin irritation test observes whether the animal's skin causes local reactions such as redness, congestion, and exudation after contact with the test substance. The skin irritation test can be divided into acute and multiple skin irritation tests. It has always been predicted by animal testing after the irritation of the subject on human skin, and skin irritation has three kinds primary, allergic, painful, etc.
2.3 Vascular irritation test
Intravenous administration is often used for drugs with low bioavailability but needing rapid action, acute patients, and patients who cannot swallow due to coma. Intravenous and intramuscular administration allows the drug to enter the blood circulation directly and act rapidly. However, the high concentration of drug solution will directly contact the inner wall of blood vessels during intravenous administration, which may cause vascular endothelial damage and trigger adverse reactions such as redness, swelling, heat and pain, and pruritus.
The vascular irritation test is one of the main tests for evaluating intravenous and intramuscular drug injections safely. The standard vascular irritation evaluation methods are mainly animal and in vitro cell evaluation tests.
2.4 Intramuscular irritation test
Observe the muscle inflammation reaction at the drug administration site after intramuscular injection of the test substance in animals. The test drug should be consistent with the clinical application of the preparation.
2.5 Nasal drops and inhalant irritation test
Observe the inflammatory response of the respiratory mucosa after the test drug is given through the respiratory tract. The test drug should be consistent with the clinical application of the preparation, generally liquid or spray.
2.6 Rectal irritation test, etc.
Observe the inflammatory response of rectal mucosa after the animals are given the test substance through the rectum. The test substance should be consistent with the clinical application of the preparation, generally as a suppository, liquid, or cream.
In toxicological research, do irritation test should be combined with the specific circumstances of the species and the specific problems found in the study, using reasonable methods to provide more valuable test results and improve the clinical predictability of the test.
Toxicological research is a science that applies the principles and methods of toxicology to evaluate the safety and clarify the mechanisms of toxicity of drugs based on their physical and chemical properties to reduce the risk of drugs to human health. Medicilon is a pharmaceutical R&D outsourcing company with expertise in drug metabolism, pharmacokinetics, pharmacodynamic studies, and toxicology. We provide our clients with high-quality data and fast turnaround time to support various drug development, preclinical and clinical studies.
3. Hemolysis test
Some drugs have toxic side effects that cause changes in the red blood cell membrane and even lead to red blood cell rupture, which can be life-threatening in severe cases. For example, injections made from herbal medicines often contain saponins, which can cause red blood cells to rupture and lyse, producing hemolysis.
The hemolytic test can observe whether the drug can cause a hemolytic reaction and whether the drug has the effect of causing erythrocyte aggregation. The hemolytic test includes in vivo/in vitro hemolysis test.
Hemolytic reactions include immune hemolysis and non-immune hemolysis. Immune hemolysis is caused by antibodies produced by the drug through the immune response and is a type II and type III allergic reaction. Non-immune hemolysis includes oxidative hemolysis caused by drugs as triggering factors and hemolysis and erythrocyte coagulation caused by changes in blood homeostasis due to drug agents.
Therefore, through the observation of hemolysis tests, the safety of specific herbal preparations can be determined, especially for intravenous injection of drugs.
4. Skin toxicity test
The skin toxicity test is to observe the toxic reactions of intact and damaged skin of animals exposed to the test substance within a short time. The experimental principle is that when the skin is exposed to the test substance, the stimulating substances in the test substance stimulate the nerve endings of the skin to cause vasodilatation and exudation and produce the corresponding reversible inflammatory reaction. The intensity of the irritation of the test substance is evaluated by recording the score according to the strength of the response.
The toxicity test of skin administration includes:
• Acute toxicity test of skin administration.
• Long-term toxicity test of skin administration.
• Skin irritation test.
• Skin sensitization test.
• Phototoxicity test.
（1） Acute toxicity test for dermal administration
Acute toxicity test for dermal drug administration is a test for toxic reactions that occur within a short time after a single exposure to the test drug on intact or broken skin of the animal. The dose design of the acute toxicity test for dermal administration should have at least three dose groups. The dose design should be sufficient to reflect the test drug's dose-toxicity effect relationship. The group spacing should be determined according to the efficacy dose and pre-test results, usually 0.65~0.85.
（2） Long-term toxicity test for dermal administration
To fully understand the toxicological effects of the drug, after obtaining preliminary toxicological information from acute dermal toxicity tests, topical medications require further long-term toxicity tests.
Long-term toxicity tests should be set up in at least three dose groups. In principle, the high dose should cause the animals to produce noticeable toxic reactions or even individual animal death; the low amount should, in direction, be higher than the equivalent dose of the animal pharmacodynamic experiments and not cause the animals to appear toxic reactions; between the high dose and low dose to set up a medium dose, animals can appear mild poisonous reactions.
Another excipient control group, if necessary, set up a blank control group. A separate broken skin group should be established if the subject is likely to come into contact with broken skin during the proposed clinical administration.
If the subject is likely to produce severe skin irritation, it should be administered at lower concentrations while maintaining the administered dose. If the quantity of the issue exceeds the adequate amount by more than 20 times, and the animals still do not show noticeable toxic reactions and death, only the high-dose group can be set up for the limited test.
（3） Skin photoallergy and phototoxicity test
Photoallergic reactions are allergy-like reactions caused by exposure to sunlight after local or systemic exposure to certain drugs or chemicals. Photoallergic reactions include induction and excitation phases clinically manifested as acute urticaria, delayed-onset herpes, or persistent eczema, and symptoms of allergic reactions are often produced on the skin in areas not irradiated by sunlight.
Phototoxic reactions are the most common of the photosensitivity reactions and are dose-dependent, with clinical manifestations similar to those of sunburn.
The phototoxic reaction is a toxic skin reaction caused by UV light exposure after skin or systemic exposure to chemical substances. The UV energy absorbed by the drug is released into the skin causing skin damage. UV-B usually causes it and is occasionally associated with UV-A.
Drug development is mainly concerned with efficacy and safety. Toxicological studies can identify safety windows, toxic target organs, adverse reactions, etc., and provide data for predicting negative human responses.
Reference:
[1] Mountford AP, Fisher A, Wilson RA. The profile of IgG1 and IgG2a antibody responses in mice exposed to Schistosoma mansoni. Parasite Immunol (1994) 16:521–7. doi: 10.1111/j.1365-3024.1994.tb00306.x
[2] Chen Huaiguang, Yang Canbang, Chen Xiaorui, Et al. Passive cutaneous anaphylaxis of Asarum injection and its effect on serum IgE. DOI: 10.16809/j.cnki.2096-3653.2019110101
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