TCM and Natural Drug Immunotoxicity (Hypersensitivity, Phototoxic Reactions)

TCM and Natural Drug Immunotoxicity (Hypersensitivity, Phototoxic Reactions)
Guidelines for Research Technology
(Draft 2)

March 18, 002

TCM and Natural Drug Immunotoxicity (Hypersensitivity, Phototoxic Reactions)
Guidelines for Research Technology

[Overview]
     1. Definition
Immunotoxicology is a key subdiscipline of toxicology focused on investigating the adverse effects and mechanisms by which exogenous compounds impact the immune systems of organisms, including humans and laboratory animals. The damage caused to the immune system by these compounds falls into two categories: immunosuppression, which involves widespread suppression of immune function leading to increased susceptibility to infections and higher tumor incidence; and immunoenhancement, characterized by an excessive immune response that may include immunogenicity, hypersensitivity reactions (allergic responses), autoimmune reactions, and other adverse immune stimulations.
An allergic reaction, also known as hypersensitivity, is an abnormal or pathological immune response triggered when the body is re-exposed to the same antigen. Based on the interaction between antigens and antibodies or cells, and whether complement is involved, allergic reactions are classified into four types: Type I, II, III, and IV. Among these, Type I is the most well-understood. Most current allergy testing methods are designed based on different mechanisms of Type I hypersensitivity.
Phototoxic reactions are a special type of IV hypersensitivity reaction. They occur when photosensitizing substances in drugs—administered locally or systemically—interact with light, causing adverse skin reactions after exposure.
Traditional Chinese medicine and natural drugs are exogenous substances that may also act as allergens, triggering allergic reactions in the body.
Because the immune system is highly active and sensitive to exogenous substances, but each chemical has a specific target organ, it is unnecessary to blindly monitor all test articles for immunotoxicity. Instead, focus selectively on certain test articles, especially those with the immune system as their target organ. Drugs used for specific indications may affect the human immune system due to their unique mechanisms of action. Additionally, immunotoxicity evaluation should be conducted for known immunomodulators or drugs that show significant effects on the immune system during preliminary safety assessments. For other drugs requiring additional immunotoxicity evaluation, consider adding immune system injury indicators to long-term toxicity studies.
    II. Background
Starting in the late 70s, advanced countries such as those in Europe and the United States began researching the toxicity of drugs on the immune system. Since the 90s, immunotoxicity has gradually become a key component of drug safety evaluation. As traditional Chinese medicine (TCM) expands globally, many high-tech innovations are now widely applied in the development of new TCM drugs, with research increasingly aligned with international standards for new chemical entities. As an exogenous chemical substance, TCM can cause varying degrees of toxic effects on the immune system once it enters the body. During the development of new TCM drugs, studying their impact on the immune system and underlying mechanisms is a critical part of preclinical safety assessment. Historically, safety evaluations of new TCM drugs rarely focused on or tested the immune system, leading to few reported instances of immunotoxicity. However, since changes in immune function can signal potential adverse drug reactions and the immune system is highly sensitive to toxins, it is essential to develop standardized evaluation methods during new drug assessments. These methods should enable the detection of immunotoxicity and associated functional immune alterations within routine safety studies.
Conventional wisdom holds that Traditional Chinese Medicine (TCM) is mild and safe. However, in recent years, with the increasing number of TCM products and new formulations—including a rise in injectables and an expansion of their clinical use—the incidence of adverse reactions, particularly hypersensitivity reactions, has been climbing annually. The following factors may be primary causes of TCM-induced hypersensitivity: (1) Inherent characteristics of TCM: TCM ingredients are complex and can be categorized by physiological activity into active compounds, auxiliary components, inactive substances, tissue materials, and impurities. Among these are allergens such as animal and plant proteins, peptides, polysaccharides, and other macromolecules. These possess both immunogenicity and antigenicity, acting as complete antigens that directly stimulate the immune system to mount an immune response, generating antibodies or sensitized lymphocytes, ultimately triggering hypersensitivity. Some small-molecule chemicals act as haptens; upon entering the body, they bind to proteins to form complete antigens, leading to sensitization. (2) Allergens in TCM and its preparations: Beyond the drug's own components, additives, solvents, stabilizers, colorants, diluents, and impurities generated during manufacturing can all serve as allergens. Additionally, impurities formed through oxidation, reduction, decomposition, or polymerization of the drug itself can induce allergies, triggering various types of hypersensitivity reactions. (3) Compound TCM preparations: These formulations contain multiple different drugs mixed together to create decoctions, injectables, pills, topical agents, etc. Drug components may interact with one another even outside the body, and further interactions can occur during digestion, absorption, and metabolic conversion within the body. These interactions undoubtedly contribute to the risk of hypersensitivity reactions. Given this, research into the immunotoxicity of TCM—especially hypersensitivity—is a critical component of preclinical safety evaluation.
    III. Scope of Application
This technical guidance applies to immunotoxicity studies of Chinese medicines and natural drugs intended for human use or already on the market. These refer to non-oral formulations, including Chinese medicine injections and other locally administered preparations.
  IV. Key Content
This technical guidance focuses on hypersensitivity testing and photoallergy testing within immunotoxicity evaluation.
   [Basic Content]
  I. Basic Principles
Immunotoxicity studies must comply with the Good Laboratory Practice (GLP) regulations.
When designing experiments, adhere to the principles of randomization, control, and replication.
Immune toxicity studies should be conducted through standardized testing that reflects the principles of comprehensiveness and integration. This requires applying case-specific analysis based on general safety evaluation practices, while accounting for the test article's unique characteristics. Key factors to consider include pharmaceutical properties, pharmacodynamics, other toxicology findings (including long-term and acute toxicity), and intended clinical use. Studies must employ scientifically sound and rational methodologies, followed by a comprehensive analysis and evaluation of results to achieve the objectives of immune toxicity assessment.
  II. Allergy Test
(I) Issues to Consider During Testing
The specific sensitization studies required for traditional Chinese medicine and natural drugs should be determined based on the drug's characteristics, clinical indications, and route of administration.
Drugs intended for systemic effects via local administration (e.g., injectables and transdermal products) require evaluation for Type I hypersensitivity. For example, injectables must undergo active systemic anaphylaxis testing and passive cutaneous anaphylaxis testing, while transdermal products require active cutaneous sensitization testing.
Type II and III hypersensitivity reactions can be monitored during long-term toxicity studies by selecting relevant indicators, such as animal signs, general observations, and assessments of immune system damage.
Transdermal drug delivery systems (including those designed for systemic or local effects) shall undergo Type IV hypersensitivity testing.
The selection of specific test methods should be based on the route of administration, mechanisms of allergic reactions, influencing factors, and clinical significance. Examples include skin sensitization tests (active), systemic active sensitization tests, skin passive sensitization tests, Mouse Ear Swelling Test (MEST), Local Lymph Node Assay (LLNA) in rodents, Buehler method, Guinea Pig Maximization Test (GPMT), and others. Alternative detection methods may also be used, provided their rationale is justified and the specific methods and procedures are clearly described.
All allergy tests must include positive and negative controls. Multiple doses may be tested to identify a dose with no allergic reaction, which can guide the starting dose for desensitization in clinical settings. This approach also helps prevent false-negative results due to excessively low doses and aids in determining whether a positive result is caused by a strong irritant response.
The test article in the allergy study must adequately represent both the clinical trial material and the marketed drug. Therefore, the test article should be a sample that is manufactured using a stable process and meets the quality standards for clinical use. Typically, pilot-scale samples are used, with documentation of the test article's name, source, lot number, potency (or specification), storage conditions, and preparation method. If pilot-scale samples are not used, a valid justification must be provided. In cases where administration volume or route constraints preclude the use of finished products, the active pharmaceutical ingredient (API) or extract may be used instead. The vehicle or excipients employed in the study must include their lot number, specification, and manufacturer.

(II) Type I Hypersensitivity Test
1 Definition
Type I hypersensitivity, also known as immediate hypersensitivity, occurs when a drug molecule acts as an allergen. Upon entering the body, it triggers the immune system to produce specific IgE antibodies. These IgE antibodies bind to mast cells and basophils, sensitizing them. When the same allergen re-enters the body, it binds to the IgE antibodies on the surface of these cells, triggering an antigen-antibody reaction. This causes degranulation of mast cells and basophils, releasing bioactive mediators that act on various tissues and organs, leading to diverse pathophysiological responses. Clinical manifestations include anaphylactic shock, bronchial asthma, allergic rhinitis, and gastrointestinal or cutaneous allergic reactions.
Type I hypersensitivity is typically evaluated using skin active anaphylaxis (ACA), systemic active anaphylaxis (ASA), and skin passive anaphylaxis (PCA) tests. For inhaled drugs, respiratory sensitivity testing is commonly employed.
2 Active Cutaneous Anaphylaxis (ACA)
Skin sensitization is a skin reaction resulting from the immunological transmission of a test substance. An allergic state may occur if an animal is challenged with the test substance at least 1 weeks after its initial exposure.
This study aims to observe the immune system's cutaneous response following repeated skin exposure to the test substance, specifically determining whether sensitization occurs and assessing its severity.
3 Active Systemic Anaphylaxis (ASA)
When a drug acts as an antigen or hapten upon initial exposure, it triggers the body to produce specific antibodies (IgE). Upon re-exposure, the drug binds with these antibodies to form immune complexes that stimulate mast cells and basophils to release active mediators. This can lead to local edema, nose rubbing, piloerection, dyspnea, suffocation, spasms, and even shock or death.
The purpose of this study is to observe allergic reactions induced by the test substance in animals after administration.
Allergic reactions to Traditional Chinese Medicine (TCM) injections are common clinically, often manifesting as systemic allergic responses or even severe adverse events such as anaphylactic shock. Therefore, conducting active systemic allergy testing for TCM injections is particularly significant.
4 Passive Cutaneous Anaphylaxis (PCA)
The passive cutaneous anaphylaxis (PCA) test is a sensitive method for measuring specific antibody titers. Serum from animals sensitized with the test substance, which contains abundant IgE antibodies, is injected intradermally into normal animals. The Fc region of the IgE binds to specific receptors on mast cells in the skin, forming IgE complexes and sensitizing the mast cells. Upon antigen challenge, the antigen binds to the Fab region of the IgE on the mast cell surface, triggering a conformational change in the IgE molecule. This causes mast cell degranulation and the release of allergic mediators such as histamine and slow-reacting substances. As a result, local vascular permeability increases. When Evans blue dye is administered intravenously alongside the antigen, it leaks into the sensitized skin area, causing localized blue staining. The extent or intensity of the blue discoloration indicates the degree of vascular permeability change and thus the severity of the cutaneous allergic reaction.
5. Antibody titer determination
IgE is the primary cause of type I hypersensitivity reactions. Measuring IgE levels in serum or body fluids is crucial for determining whether a type I hypersensitivity reaction exists and for understanding the mechanisms behind drug-induced hypersensitivity.
If the allergy test is positive or suspiciously positive, or if the substance has potential sensitizing properties, this test is recommended.
6 Test Method
See the test method in the Appendix.
(III) Type II and III Hypersensitivity
Type II hypersensitivity, also known as cytotoxic or cytolysis-type reactions. Drug molecules bind to cell membranes (typically blood cells) after entering the body, triggering the immune system to produce specific antibodies. The primary antibodies involved are IgG and IgM. This reaction is characterized by antibodies binding directly to antigens on target cell membranes, leading to cell lysis. Clinical manifestations include drug-induced hemolytic anemia, granulocytopenia, and thrombocytopenic purpura.
Type III hypersensitivity, also known as immune complex-mediated or vasculitic reactions, occurs when drug molecules enter the body and stimulate the immune system to produce specific antibodies (IgG, IgM). When antigens and antibodies bind in a certain ratio, they form immune complexes that deposit on the basement membranes of tissue blood vessels. This triggers vascular damage and inflammation. Common manifestations include serum sickness, allergic glomerulonephritis, and systemic lupus erythematosus-like reactions.
No standard preclinical trials exist for prediction.
If pharmacology and toxicology study results suggest potential Type II or III hypersensitivity reactions, further relevant studies are recommended.
Refer to the test methods in the appendix.
(IV) Type IV Hypersensitivity Test
Also known as delayed-type. The drug directly acts on T lymphocytes, sensitizing them. Upon re-exposure to the same drug, these sensitized lymphocytes are triggered to release mediators, causing tissue damage. This reaction occurs without antibody involvement and develops slowly, typically manifesting clinically 48–72 hours after re-exposure to the antigen. Common manifestations include drug eruptions, contact dermatitis, and exfoliative dermatitis.
Refer to the test methods in the appendix.
    III. Photopatch Test (Photoallergy Test)
Photosensitivity reactions are adverse skin responses to light exposure following medication. They include phototoxic reactions and photoallergic (photohypersensitivity) reactions, both caused by photosensitizing substances in the test item. However, the two types differ in mechanism, experimental methods, clinical presentation, and significance.
Photosensitivity reactions can be triggered by both topical and systemic administration, not limited to topical use alone. Therefore, in principle, any drug that distributes to the skin should undergo phototoxicity testing regardless of the route of administration. If a test article's chemical structure or certain components (including the drug and excipients) are documented in the literature as having photosensitizing properties, or if its structure resembles known photosensitizers, phototoxicity testing is recommended for traditional Chinese medicine preparations with reported or suspected photosensitizing effects.
The purpose of this study is to observe whether the test article causes a photoallergic reaction upon skin contact or application followed by light exposure.
(I) Photopatch Test
Phototoxicity is an acquired, immune-mediated skin reaction triggered by light exposure. It occurs when a photosensitizing agent is absorbed through the skin or circulates to the skin, where it interacts with light in the epidermal cell layer. Upon absorbing light energy, the drug becomes activated and binds with skin proteins as a hapten, forming a drug-protein complex (full antigen). This complex is then presented by Langerhans cells in the epidermis to immune-competent cells, triggering an allergic response. Phototoxicity is classified as a Type IV (delayed-type) hypersensitivity reaction. The onset is relatively slow and involves a latent period. Typically, 5–10 days of continuous medication and sun exposure are required to induce a photoreaction. Upon re-exposure, a photoreaction can occur within 24–48 hours of combined drug administration and sunlight exposure.
Refer to the test methods in the appendix.
(II) Considerations for Phototropic Metamorphosis Testing
1: Animal Selection
White guinea pigs or domestic rabbits are recommended as the first choice.
2 Selection of Irradiation Light Source and Wavelength
Xenon lamps, quartz lamps, or mixed light sources (including UVA and UVB) or pure UV sources (UVA) may be used. The wavelength range should cover medium- to long-wave UV 280–40 nm. Since drug sensitivity varies with structure, select the specific wavelength ranges sensitive to each test substance for testing. Optimal irradiation dose, light intensity, exposure duration, and distance should be determined through preliminary trials.
3 Test Article
Requirements for the test article are the same as those for the sensitization study.
Use clinical drug concentrations (or equivalent doses). For photoallergy challenge testing, use appropriately diluted concentrations to rule out phototoxicity.
4, Positive Light Sensitivity Control
For photodermatitis, salicylanilide bromide may be used.
5 Dosing Time
Before irradiation, ensure that the test and control substances have sufficient time to penetrate the stratum corneum and interact with epidermal cells; 60 minutes is generally recommended.
6. Results Statement
All major stages of the photosensitivity test shall be documented and represented by photographs.
7: Test System
A comprehensive phototoxicity evaluation system is the foundation for assessing drug-induced photosensitivity. A well-designed system should integrate in vitro photochemical models, in vitro biological models, and animal models based on the mechanism of phototoxicity. In the photochemical model phase, exposure to UVA at different wavelengths is used to identify the most sensitive wavelength for inducing a phototoxic reaction, providing a basis for subsequent biological evaluation.

   4. Data Analysis and Evaluation
(I) The experimental methodology must be described in detail, including group allocation, dosing regimens, number of animals, and frequency of administration. Allergic reactions should be thoroughly documented, covering clinical manifestations, duration, recovery time, animal mortality, and histopathological findings. Supporting photographs must be provided to facilitate result analysis and interpretation. In the case of animal death during a passive systemic anaphylaxis (PSA) test, necropsy is required; if significant lesions are observed, histopathological examination must be performed.
(2) Based on test results, describe the occurrence and severity of reactions at different doses (or concentrations) using a scoring system. Analyze the dose-response and time-course relationships, as well as reversibility, to determine whether observed changes are related to the test substance. Identify the allergic reaction dose, safe dose, and safety margin.
(3) Conduct a comprehensive analysis and overall evaluation by integrating pharmacodynamic data with other toxicology findings. Do not uncritically extrapolate animal results to humans, and do not ignore or selectively exclude animals exhibiting toxic reactions. Objectively report the toxicity of the test article. Based on the trial results, provide clinical implications and safety precautions for use.
(4) The immune response triggered in the host is a complex and continuously evolving process. Therefore, studies on the potential immunotoxicity of traditional Chinese medicine (TCM) should be as comprehensive and in-depth as possible. Researchers must not rely on isolated or limited experimental results to assess the overall immunotoxicity of the test drug. Instead, they should select appropriate animal models based on the type and characteristics of the immune response, along with the physiological traits of different species, to obtain comprehensive and objective data. Only after a thorough evaluation of all results can a valid conclusion be drawn regarding whether a new TCM product exhibits immunotoxicity.
(5) Any immunotoxic effects identified during preclinical toxicology studies (e.g., acute toxicity, long-term toxicity, teratogenicity, carcinogenicity studies) must be evaluated.
(VI) The relationship between immunosuppression and tumors is complex and controversial. In most cases, an increased tumor incidence observed in long-term toxicity studies may be linked to genotoxicity, hormonal effects, or other established mechanisms. However, for certain test articles, carcinogenic effects may not be readily detected in nonclinical studies; in such instances, potential immunosuppressive activity should be considered.
   V. Frequently Asked Questions and Troubleshooting
(1) A wide range of adverse reactions can occur during the clinical use of traditional Chinese medicine (TCM) injections. These include hypersensitivity reactions such as: Type I (e.g., anaphylactic shock, bronchial asthma, allergic rhinitis, gastrointestinal and cutaneous reactions), Type II (e.g., hemolytic anemia, granulocytopenia, thrombocytopenic purpura), Type III (e.g., serum sickness, post-streptococcal glomerulonephritis, systemic lupus erythematosus), and Type IV (e.g., contact dermatitis, eczematous reactions, transplant rejection). Therefore, in addition to Type I hypersensitivity testing, tests for Types II–IV should be conducted as needed.
(2) In immunotoxicity evaluation, special attention should be paid to the effects of dosage and administration rate on the occurrence of allergic reactions in animals. It is essential to distinguish between true allergic reactions and pseudoallergic (anaphylatoid) reactions. While pseudoallergic reactions exhibit characteristics of immediate hypersensitivity, they occur independently of immune mechanisms, without involvement of antigen-antibody interactions. Analysis of adverse reaction data for traditional Chinese medicines (TCM) shows that up to 40%–66% meet clinical diagnostic criteria for allergic reactions. However, further investigation reveals that many of these so-called "allergic reactions" occur after the first dose, in patients with no prior exposure history and thus no opportunity for sensitization. Such Type I-like reactions are often triggered during initial TCM injection administration when drug concentration is too high or infusion speed is too rapid. Given their potential risks, this issue warrants serious attention, and related studies should be strengthened in preclinical safety evaluations.
(3) Adverse reactions can occur with all types of Chinese medicine preparations. Parenteral administration, including intravenous and intramuscular injections, is particularly prone to triggering allergic reactions. Topical Chinese medicines may also cause allergic or irritant responses, such as contact dermatitis, drug eruptions, or other adverse reactions. Therefore, attention must be paid not only to the immunotoxicity of Chinese medicine injections but also to other forms of Chinese medicine preparations.
(4) Selection of Test Animals: The high rate of false-negative results in preclinical injection safety assessments for allergic reactions, coupled with the inconsistency observed in clinical adverse event reports, necessitates a re-evaluation of the role and value of preclinical allergenicity testing. We recommend conducting exploratory studies to develop assessment methods that fully reflect the safety profile of injections. Historically, guinea pigs have been selected for allergenicity testing due to their perceived sensitivity to sensitizing agents. For long-term toxicity studies of traditional Chinese medicine injections in dogs, close monitoring for related sensitization reactions is advised.
(5) Traditional Chinese medicine injections should be administered alone. Avoid combining them with multiple drugs whenever possible. If clinical protocols propose combination therapy, sufficient safety data from formulation studies must demonstrate the safety of such combinations.
(6) Strengthen research on the in vivo metabolism of key Chinese herbal ingredients to characterize the antigenicity of their metabolites and metabolic products, laying the foundation for in vitro detection of severe allergic reactions. Additionally, prioritize experimental studies on interactions between Chinese herbs and cross-reactivity allergies.
(7) Extrapolating results from test animals to humans in immunotoxicity assessments presents many challenges, leading to the following limitations of animal data in safety evaluations: (1) Animal studies are typically conducted at high doses and for short durations, creating difficulties when extrapolating from high-to-low doses and short-term to long-term exposure. (2) Significant species differences exist between animals and humans in genetic background, tissue structure, and physiological function. (3) In animal studies, immune function assays usually use cells derived from immune organs (e.g., spleen, thymus, lymph nodes), which can effectively predict potential effects on the immune system. However, human immune function testing primarily relies on peripheral blood cells; lymphocytes in peripheral blood account for only 1% of total lymphocytes. Beyond this numerical difference, cells from different sources are at distinct developmental stages and exhibit varying responses, resulting in dissimilar findings.
In summary, although challenges remain in immunotoxicity testing for safety assessments, it is essential to strengthen immunotoxicity evaluation in the toxicology studies of new drugs to ensure a comprehensive safety assessment. Furthermore, due to the following limitations in human immunotoxicity research, animal-based immunotoxicity evaluation remains critical. (1) Detecting impaired immune function in humans requires prolonged observation periods, and low exposure doses make it difficult to establish dose-response relationships. (2) Human immunotoxicity studies lack specific biomarkers and often rely on non-specific indicators, necessitating large sample sizes. (3) There are no established normal or reference values for interpreting immune function test results. Even when results fall within reference ranges, significant deviations from control groups may occur; how should these be interpreted? The relationship with disease onset also requires further investigation. (4) Non-invasive methods for human immunotoxicity testing are currently unavailable.
   VI. Selection of Test Items for Traditional Chinese Medicine and Natural Drugs in Different Dosage Forms
(1) Intravenous preparations: Conduct systemic allergy tests, passive skin sensitization tests, and, when necessary, antibody titer assays and Type II, III, and IV hypersensitivity tests.
(II) Injectable for Intramuscular Use: Must undergo systemic allergy testing and passive cutaneous anaphylaxis testing.
(3) Topical Administration Preparations: Skin sensitization testing is required. Phototoxicity testing should be conducted if necessary.
(4) Hypersensitivity reactions of traditional Chinese medicines (TCMs) and natural drugs are closely linked to formulation types. Even with identical active ingredients, different TCM formulations may exhibit significantly varying hypersensitivity profiles. For drugs where preliminary hypersensitivity studies indicate potential sensitization risk, comparative studies against marketed formulations administered via the same route must be conducted to ensure safety and efficacy in clinical use.
(5) Test items may be adjusted based on the drug's specific characteristics. For formulations requiring assessment of irritation and hemolysis, refer to the relevant guidelines for local irritation and hemolysis.
   [References]
1. Toxicity testing of dermatological preparations. "Guidelines for Research on New Chinese Medicinal Products," Drug Administration, Ministry of Health of the People's Republic of China, 1993 year 209-212.
2. Toxicity testing of dermatological preparations. Edited by Yuan Bojun and Wang Zhiqiao. "Preclinical Safety Evaluation and Practice of New Drugs." Military Medical Science Press, Beijing 152
3. Preclinical Toxicology Testing Methods. Edited by Xu Shuyun. "Methods in Pharmacological Experiments" (3rd Edition) 226
5. Principles and Methods of Toxicology. Fourth edition, edited by A. Wallace Hayes, Taylor & Francis, Philadelphia. 2001.
6.FDA; Guidance for Industry: Immunotoxicology Evaluation of Investigational New Drugs
7. Gong Yifen. Sensitivity and Predictive Analysis of Immunotoxicity Detection Indicators. Journal of Hygiene Research. 1995, 24(Special Issue): 12-14
8. Xue B, Gong YF. Immunotoxicity and safety evaluation. Journal of Hygiene Toxicology. 1989, 3(1): 53
9. Qiao C, Li Y. Common methods and their evaluation in immunotoxicology. Foreign Medical Sciences Section of Hygiene 1987, 6: 329
10 Wang Hongxing. Observations on allergic reactions to traditional Chinese medicine injections using beagle dogs. Pharmacology and Clinical Applications of Traditional Chinese Medicine. 2002, 18(2): 8
11. Zhang Jianying, Qi Pidong, Yang Ping. A preliminary study on allergic reactions caused by traditional Chinese medicine injections. Clinical Pharmacy. 2002, 11(2):64
12. Lai Yuhong, Chen Hao'an, Yang Weirong. Research on hypersensitivity reactions to Chinese medicine injections needs urgent strengthening. New Drugs and Clinical Pharmacology of Traditional Chinese Medicine. 2002, 13(5): 324-326
13. Wang Q, Zhang YC, Xie LJ, et al. Literature analysis of adverse drug reactions of traditional Chinese medicine in China from 1990 to 1999. China Pharmacy. 2000, 11(5): 226
14 Chen Lin, Jia Zhansheng, Lin Yongxin, et al. Significance of serum IgE level detection and skin prick test in the diagnosis of type I hypersensitivity reactions. Journal of the Fourth Military Medical University. 2001, 22(4): 344-346.

  [Appendix]
  I. Active Skin Allergy Test
(I) Animal Selection
Guinea pigs are recommended. Shave the fur on both sides of the back prior to administration; the shaved area should be at least 3 × 3 cm².
(II) Test Article
The test article should match the formulation intended for human use, containing active ingredients along with excipients or penetration enhancers. It must be prepared using a stable manufacturing process, and details such as the supplier, batch number, content (or concentration), and preparation method must be clearly documented. If the test article is a cream or liquid, it generally should not be diluted. For solid powders, an appropriate amount of water or excipient should be added to ensure uniform mixing and adequate skin contact. When using excipients, their potential impact on the transdermal absorption of the test article must be considered.
(3) Comparison Settings
A positive control group and a negative or vehicle control group should be included. The positive control may use 2, 1-chloro-2,4-dinitrobenzene (sensitization concentration of 1% and challenge concentration of 0.1%).
(IV) Scope of Administration and Duration of Stay
During the sensitization contact phase of the test substance, ensure adequate skin contact duration (6 hours) and coverage area.
(V) Allergenicity
On days 0, 7, and 14, apply the test substance locally using the same method. On day 14 after the final sensitization dose, during the challenge phase, reapply the test substance to the depilated area. Approximately 6 hours later, observe skin allergic reactions for 72 hours and score them according to the skin allergic reaction scoring criteria.
(VI) Results Description
Describe the experimental methods in detail. Record the mean scores for each group at each time point as shown in Table 1, and closely monitor animals for signs of severe systemic allergic reactions, such as asthma, loss of balance, or shock.
(VII) Result Evaluation
Based on the differences in skin reactions between the test and control groups, determine the nature of the substance's sensitizing effect. Then, calculate the sensitization incidence rate using Table 2 (number of animals showing skin erythema, edema, or systemic allergic reaction divided by the total number of tested substances).
  Scoring criteria for skin allergic reaction severity in Table 1

  Skin Allergic Reaction Score

No erythema 0

Mild erythema, barely visible 1

Moderate erythema, clearly visible 2

Severe erythema 3

Erythematous macules progressing to mild eschar formation 4

Edema 0

No edema 18

Mild edema, barely visible 1

Moderate edema, clearly visible (raised above surrounding skin) 2

Severe edema, skin elevation of 1 mm, well-defined borders 3

Severe edema with skin elevation >1 mm, blistering, or ulceration 4

Maximum total score: 8

No erythema 0

Table 2 Skin Sensitization Evaluation Criteria

              Sensitization Incidence (%) | Skin Sensitization Assessment

0-10 Hypoallergenic

11-30 Mildly Allergenic

61-80 Highly Allergenic

81-100 Extremely Allergenic

   II. Active Systemic Sensitization Test
(I) Animal Selection
Guinea pigs are commonly used. Guinea pigs that have undergone allergy testing should not be reused.
(II) Test Article
For most test articles, the substance itself can be used directly as the sensitizing solution. For certain products, a semi-hydrolyzed form of the raw material or a mixture of the test article and adjuvant 1:1 may be used as the sensitizing agent to enhance test sensitivity.
(3) Sensitization
Sensitization is typically achieved using methods that readily induce antibody production, such as subcutaneous, intraperitoneal, or intramuscular injections. For example, guinea pigs may receive 0 mL of the test substance via intramuscular injection every other day for a total of 3 to 5 doses.
(4) Dose Settings
Typically, two dose groups are established: the low-dose group uses the maximum intended clinical dose, while the high-dose group is several times higher.
(5) Attack 19
Divide into two groups; administer a single challenge on days 14–21 after the initial injection. The standard route is intravenous, using 2–3 times the sensitizing dose, and monitor for signs of an allergic reaction post-injection. Other routes may lead to desensitization.
(6) Reference Settings
The study should include a positive control group and a vehicle control group. The positive control agent may be bovine serum albumin or ovalbumin at 1-5mg/ mg, or other known sensitizing substances.
(VII) Result Evaluation
Observe allergic reaction symptoms based on table 3.
Following the challenge injection, if allergic reaction symptoms are observed, administer the test substance intravenously at the challenge dose to 2 healthy, non-sensitized guinea pigs. Monitor for similar allergic-like symptoms induced by the drug to provide reference data for result interpretation.
If hypersensitivity reactions occur, dose-response studies may be conducted as needed to identify a non-sensitizing dose, which can guide the starting dose for desensitization therapy.
(VIII) Judgment Criteria
Assess the severity of allergic reactions based on the criteria for Table 4: — No symptoms; + Restlessness, trembling, nose rubbing, sneezing, urination, rapid breathing, etc.; ++ In addition to the above symptoms, difficulty breathing and motor impairment; +++ In addition to the above symptoms, respiratory failure leading to convulsions (reversible); ++++ Death.

 Table 3 Allergic Reaction Symptoms

0 Normal 7 Tachypnea 14 Ataxia

1 Restless 8 Urination 15 Jump

2 Shivering 9 Defecating 16 Panting

3 Shivering 10 Crying 17 Convulsing

4 Scratch nose 11 Difficulty breathing 18 Turn sideways

5 Sneezing 12 Rhonchi 19 Cheyne-Stokes respiration

6 Cough 13 Purpura 20 Death

Table 4: Criteria for Whole-Body Sensitization Evaluation

0 - Allergy test negative

1-4 symptoms + weak positive allergic reaction

1-10 Symptoms: Allergy Positive

1-19 Symptoms: +++ Strong Positive Allergic Reaction

20 ++++ Extremely Strong Positive Allergic Reaction

    III. Passive Skin Allergy Test
(I) Selection of Test Animals
Rats are the most common animals used for PCA reactions, though mice are also employed. Guinea pigs may be used depending on experimental requirements. When selecting an animal model, it is important to note that PCA reactions are IgE-mediated.
(II) Experimental Grouping
Establish negative control, positive control, and 2 dose groups for the test substance. The negative control group shall receive an equivalent volume of vehicle, while the positive control group shall be administered ovalbumin, trichosanthes root powder, or a known sensitizing positive agent.
(3) Sensitization Routes and Methods
Follow the intended clinical route of administration. Perform sensitization once every other day for a total of 3 to 5 times. Collect blood approximately 10 days after the final sensitization to prepare antiserum.
(4) Selection of Test Methods
Rat homologous passive cutaneous anaphylaxis (PCA) tests, mouse PCA tests, or mouse heterologous ear swelling tests may be used.
(V) Test Operation
Dilute the anti-sera containing IgE from each group with normal saline to various dilution factors (typically 1:10-1:50). Intradermally inject the corresponding anti-serum for each group into the depilated skin areas on both sides of the spine in animals. After 24 or 48 hours, challenge the animals with an antigenic stimulus by intravenous injection of a dose equivalent to the sensitizing dose (containing 0.5-1% Evans blue dye). Sacrifice the animals after 30 minutes, excise the back skin, and measure the diameter of the blue reaction spots on the inner surface of the skin; simultaneously capture photographs. Alternatively, use colorimetric analysis.
(VI) Evaluation Methods
Two methods are available: one uses the highest dilution titer yielding a positive reaction as the indicator, while the other employs a fixed serum dilution with spot size or optical density as the indicator. A spot diameter of 5 mm or greater is considered positive. Provide a photograph of the blue spots.
(VII) Determination of Blue Reaction Spots
The size of pigmented spots can be determined using either the direct measurement method or the colorimetric method. In the direct measurement method, the average diameter of blue spots from both the control and treated groups is measured to calculate the percentage change. In the colorimetric method, the blue spot sections are cut out, minced, and soaked in an acetone-saline mixture (7:3) with 5–6 mL added. After overnight incubation, the samples are centrifuged, and absorbance is measured at a wavelength of 610 nm. The average absorbance values from both groups are then used to calculate the percentage change.
  IV. Type III Hypersensitivity
Antigens that induce Type III hypersensitivity must persist to facilitate binding with corresponding antibodies and the formation of immune complexes. Evaluation can be performed using the Arthus reaction test in rabbits (most susceptible) or rats. For the rabbit model, administer 1 ml of the test substance or positive control ovalbumin mixed with Freund's Complete Adjuvant via intramuscular injection weekly for 4 doses. On day 10 following the final injection, shave the dorsal area and perform an intradermal injection of the test substance or 1% ovalbumin at multiple sites, injecting 0.02 ml per site. Observe the maximum diameter and severity of local skin redness and swelling at each site at 2, 3, 4, 6, 8, 12, and 24 hours post-antigen challenge, then calculate the averages. Reaction severity shall be analyzed and graded according to established criteria.
The route and timing of administration for the test article are determined by its pharmacodynamics, planned clinical use, and other relevant characteristics.
   V. Type IV Hypersensitivity Test
(I) Buehler Analysis and Guinea Pig Maximization Test (GPMT)
Induction doses are administered intradermally or topically to test animals. After an induction period of 10–14 days, when the immune response develops, a challenge dose is given to assess whether an allergic reaction has occurred. Skin reactions and their severity during both the induction and challenge phases must be compared against each other and against the sham-treated group.
1 Experimental Animals
Prefer young adult guinea pigs. The number and sex of animals depend on the selected test method; both sexes may be used. If females are selected, use those that have not given birth and are not pregnant. For the Buehler test, the test group must include at least 20 animals and the control group at least 10. For the GPMT test, the test group must include at least 10 animals and the control group at least 5.
2 Comparison
Recommended positive controls include benzothiazole, benzocaine, dinitrochlorobenzene, 331 epoxy resin, and others. To ensure that the elicitation response is due to sensitization rather than irritation, a vehicle control group should be included. The selected vehicle must not interfere with or alter the interpretation of the test results.
3: Dosage
Depends on the selected method. In the Buehler test, the sensitization dose should be high enough to produce mild irritation; the challenge dose is the highest non-irritating dose. In the GPMT test, the sensitization dose should be high enough to cause mild-to-moderate skin irritation while being well tolerated systemically; the challenge dose is the highest non-irritating dose.
4 Results Observation
Skin reactions shall be graded and assessed at the time points determined by the methodology, typically 24 and 48 hours. For abnormal reactions, the timing shall be adjusted accordingly. The animal's body weight shall be recorded at both the start and end of the study.
5 Test Steps
5.1 Buehler test was induced by applying occlusive patches topically on days 0, 6-8, and 13-15. On day 27-28, a 6-hour challenge patch was applied to the untreated flank. Results were read 10 and 11 hours after patch removal. If results are inconclusive, re-challenge after one week using either the original or a new control group.
5.2 The GMPT test uses intradermal administration with or without an adjuvant for induction. A challenge dose is administered 20–22 days after local induction, which occurs 5–8 days later. Results are observed 24 and 48 hours after the challenge. As in the Buehler test, if results are inconclusive, re-challenge after one week. If only 10 animals were initially selected and results remain unclear, add 10 additional test animals and 5 control animals.
6 Data Analysis
Organize test data in tabular format, including skin reactions for each animal and the induction/elicitation times as determined by the method. The table should include at least erythema and edema grading data and records of any abnormal reactions. Provide the proportion of sensitized animals per group and the severity (mild, moderate, severe) of sensitization for each animal.
(II) Local Lymph Node Assay (LLNA) for Rodents
Test substances that cause skin sensitization can stimulate lymphocyte proliferation in the draining lymph nodes. Under appropriate conditions, this proliferation is dose-dependent. This test provides an objective, quantitative assessment of the sensitizing potential of the test substance.
1, Test Animals
Select female CBA/Ca or CBA/J mice aged 8–12 weeks (nulliparous and non-pregnant), matched by age (ideally within one week).
2. Preparation of the Test Article
Solid test articles should be dissolved and diluted in an appropriate solvent or vehicle prior to administration. Liquid test articles may be used directly or diluted before use.
3 Solvent Selection
Select the vehicle based on the test item's maximum concentration. Recommended vehicles include acetone/olive oil (4:1 v/v), N,N-dimethylformamide, butanone, propylene glycol, dimethyl sulfoxide, and other suitable solvents. The selected vehicle must not interfere with the evaluation of the test results, and its maximum concentration should be sufficient to achieve the maximum skin exposure concentration of the substance. For hydrophilic substances, ensure the vehicle wets the skin effectively and does not evaporate immediately.
4 Comparison
Positive and negative controls should be included. The positive control must demonstrate method feasibility, producing a positive result (Stimulus Index [SI] ≥ 3) at an appropriate exposure level. The SI is defined as the ratio of the amount of test article 3H-thymidine or 125-iododeoxyuridine (125IU) incorporated into lymph nodes in the treated group versus the vehicle control group. Cinnamaldehyde and 2-mercaptobenzothiazole are preferred positive controls; other suitable alternatives may also be used, but ethyl aminobenzoate should be avoided whenever possible. Positive controls must be dissolved in a solvent known to elicit a consistent response (e.g., acetone/olive oil) to ensure a sustained reaction. If a non-standard solvent is selected, it must first undergo validation via an LLNA before evaluating results.
5 Test Method
At least three doses should be established. Dose selection may be based on information such as toxicity, solubility, irritation potential, structure-activity relationships, and other experimental results. To avoid false negatives, the highest feasible concentration should be used. The maximum concentration should avoid overt systemic toxicity and excessive local irritation. When LLNA results are negative, the positive control must show a stimulation index (SI) greater than 3 relative to the negative control.
6 Experimental Steps
On day 1, apply 25 μl of the test substance to the dorsal surface of each mouse's ear. Continue this treatment for 3 consecutive days. No treatment is administered on days 4 and 5. On day 6, inject 250 μl of PBS containing either 20 μCi of tritiated thymidine or 2 μCi of 125 IU and 10-5-fluorodeoxyuridine into the tail vein of each mouse. After 5 hours, harvest the auricular lymph nodes from each mouse, place them in PBS solution, and filter through a 200-mesh stainless steel screen to prepare a single-cell suspension of lymph node cells (LNC). Wash the cells with PBS 2 times. Precipitate DNA by incubating in 5% trichloroacetic acid at 4°C for 18 hours. For the tritiated thymidine assay: disperse the precipitate in 1mlTCA, transfer it to 10 ml of scintillation fluid, and measure using a beta-scintillation counter; results are expressed as disintegrations per minute (DPM) per mouse. For the 125 IU method: transfer the precipitate directly to a gamma counter and measure; results are also expressed as DPM per mouse.
7 Results Observation
Observe at least once daily for local irritation and systemic toxicity. Weigh before dosing and at necropsy to aid in assessing systemic toxicity.
8 Reviews and Calculation Results
Lymph node cell proliferation response is expressed as the rate of radioactive decay per minute per animal, with background values subtracted. Calculate the mean and standard deviation for each group. The final result is reported as the Stimulation Index (SI), calculated as: SI = mean decay rate of the test group / mean decay rate of the solvent control group. In addition to evaluating the SI ratio, perform statistical analysis on the dose–response relationship (e.g., ANOVA) and conduct paired comparisons for SI.
9 Data Analysis
A positive LLNA result is indicated when the stimulation index (SI) of the test group is ≥3 at least at one concentration, suggesting the substance is a skin sensitizer. However, SI magnitude alone is not the sole criterion; individual data should undergo statistical analysis for a comprehensive evaluation. Key factors influencing the assessment include SI determination, statistical methods, strength of dose-response relationships, chemical toxicity, solubility, vehicle consistency, and positive control performance.
Intense irritation can cause false positives in LLNA tests by inducing significant lymphocyte proliferation. However, because the dose-response range for irritation-induced proliferation is narrow, the dose-response relationship can help distinguish strong irritant responses. Combined with assessment of ear swelling, this approach can differentiate weak sensitizers from strong irritants. Detailed reporting of the experimental methods should include a table listing disintegrations per minute (DPM) per mouse, stimulation index (SI) values, and their means and standard deviations for each group, along with toxicity symptoms, to clearly demonstrate the dose-response relationship.

6. Photopatch Test
(I) Determination of Minimal Erythema Dose (MED)
Within the hair-deprived area of the animal's spine, under fixed light source and distance conditions, determine the minimal erythema dose (MED) using cumulative irradiation with long-wave ultraviolet light in graded steps.
(II) Sensitization
Inject 1.1 mL of complete Freund's adjuvant at each of the four corners of the depilated area (2 × 4 cm²). Apply a 20% sodium dodecyl sulfate solution to the depilated area, then apply the test substance to this site. After a specified duration, wipe away any residual drug from the surface. The positive control may be chosen as N-(2-hydroxybenzoyl)aniline bromide.
(3) Irradiation
Irradiate the drug-applied area with ultraviolet light at a wavelength of 280-400 nm until significant erythema is observed. Repeat the application of sodium dodecyl sulfate and the test substance, followed by irradiation of the treated area, every other day for a total of 5 times.
(4) Stimulate
Reapply the test substance to the prepared skin area 2 weeks after the last sensitization. The concentration should be lower than the sensitizing concentration to avoid irritation and phototoxic reactions. Prior to irradiation, allow sufficient time for both the test and control substances to penetrate the stratum corneum and interact with epidermal cells; typically, 60 minutes is appropriate. Irradiate using sub-erythemal doses of UV light ranging from 1/2 to 2/3 MED, ensuring no erythema is induced.
(5) Result Interpretation
Observe skin reactions within 1-72 hours after irradiation. Any individual who experiences multiple exposures to the test substance followed by skin inflammation or systemic reactions after UV exposure can be considered to have a photoallergic reaction caused by a photosensitizing agent in the test substance.
    [Drafting Notes]
    I. Importance and Significance of Drafting This Guideline
In recent years, the number of traditional Chinese medicines (TCMs) and their new formulations has increased, and their scope of use has expanded. Consequently, adverse reactions and drug-induced diseases associated with TCMs have also risen annually. Among these, hypersensitivity reactions account for approximately 70%. These reactions are diverse, with skin allergies being the most common and anaphylactic shock being the most severe. However, the significance of TCM-induced hypersensitivity in adverse events is not solely due to its proportion but because, like antibiotic- and chemical drug-induced hypersensitivities, they are difficult to predict, can be life-threatening, and may even lead to death. Therefore, it is crucial to prioritize safety evaluations of TCM preparations, especially TCM injections—including single-ingredient, compound, and animal/plant protein-containing injectables. Furthermore, insufficient experimental data exist regarding the use of herbal medicines in modern formulations, particularly injections. Key areas lacking in-depth basic research include identification of active and inactive components, chemical analysis, pharmacological profiles, toxicity, sensitization potential, and interactions with other ingredients. As a result, several challenges remain before rational application can be fully achieved.
Therefore, this guideline aims to clarify the purpose, significance, importance, and necessity of conducting immunotoxicity studies, particularly allergy tests (hypersensitivity tests), and to guide the discovery or identification of adverse reactions to new traditional Chinese medicine drugs through more rational and objective testing methods, thereby supporting safe clinical use.
   II. Establishment of Evaluation Indicators and Systems for Immunotoxicity
To improve immunotoxicity testing, we need methods that are sensitive, specific, reliable, and simple. Many countries have established national immunotoxicity testing protocols, but these continue to evolve. China should continuously refine its evaluation methods based on ongoing research developments. Determining the appropriate evaluation systems and indicators requires further discussion. In addition to whole-body immunotoxicity testing, local immune damage—such as in the lungs, gastrointestinal tract, liver, kidneys, and skin—must be carefully evaluated. This will enable a comprehensive assessment of how exogenous compounds affect both systemic and local immune systems after entering the body through various routes.
    III. Issues Regarding Strengthening Basic Research on Hypersensitivity Reactions to Traditional Chinese Medicine Injections
Due to the complex composition of Traditional Chinese Medicine (TCM), unclear pharmacological mechanisms, inconsistent injection manufacturing processes, and quality standards that cannot fully control all internal components, current safety tests (e.g., pyrogenicity, hemolysis, abnormal toxicity) rarely include animal sensitization testing. Therefore, for drugs with potential safety risks, it is necessary to evaluate the feasibility and necessity of mandating animal sensitization testing in quality standards and implementing batch testing. Given the significant discrepancy between high negative results in preclinical animal allergy studies and high positive clinical outcomes—driven by substantial species differences that limit the extrapolation of animal data to humans—what specific foundational research requirements should be established? Furthermore, considering the frequent occurrence of adverse reactions, particularly hypersensitivity, associated with TCM injections characterized by rapid onset and severe consequences, a feasibility study on dosage form selection is required. Specifically, for formulations transitioning from traditional oral TCM to intravenous administration, it must be clearly demonstrated whether the injectable form offers superior efficacy and safety compared to oral preparations, especially intravenous infusions.
 IV. Selecting the Appropriate Hypersensitivity Assay
The need for Type I, II, III, and IV hypersensitivity testing should be determined based on the specific characteristics of each drug. For example, traditional Chinese medicine (TCM) compound injections containing animal or plant proteins should undergo all four types of hypersensitivity tests, with antibody titer measurements performed as necessary. Regarding where to conduct these tests, it is recommended that Type II and III hypersensitivity studies be included in long-term toxicity studies, while Type IV hypersensitivity testing should be performed for topically administered drugs when required.
  V. Selection of Animal Species for Passive Cutaneous Allergy Tests
Two types of antibodies mediate the PCA reaction in various animals: IgG-class antibodies, which have low affinity for tissue cells and disappear from the local skin within approximately 10 hours after injection, primarily mediating a 2-4 hour PCA reaction; and IgE antibodies, which exhibit strong affinity for tissue cells, persist for at least 2 days after injection, and mediate a 24-72 hour PCA reaction. Extrinsic bronchial asthma is an IgE-mediated immediate hypersensitivity reaction. Similarly, the animal 24-72 hour PCA reaction is also mediated by IgE-class antibodies, indicating a close resemblance between the two.
Previous studies often used guinea pigs for passive cutaneous anaphylaxis (PCA) tests. However, current literature notes that the PCA reaction in guinea pigs is primarily mediated by IgG, so their use is not recommended; rats, mice, or rabbits are preferred instead. Since guinea pigs can also produce IgE antibodies, if they are used, the impact of the timing of IgE production on the test results must be carefully considered.

[Author]

Research Group on the "Technical Guidelines for Investigating Immunotoxicity (Hypersensitivity and Phototoxic Reactions) of Traditional Chinese Medicines and Natural Drugs"