Local Irritation and Hemolysis Tests for Traditional Chinese Medicine and Natural Drugs

Local Irritation and Hemolysis Tests for Traditional Chinese Medicine and Natural Drugs
Research Technical Guidelines
(Draft 2)

March 18, 002

[Overview]

1. Definition and Significance
Local irritation and hemolysis in Traditional Chinese Medicine (TCM) and natural medicines refer to the toxic effects on the administration site and the whole body following non-oral administration (including active ingredients and excipients) via skin, mucous membranes, body cavities, muscle, or blood vessels through absorption or injection. These effects include irritation of the blood vessels, muscles, and mucous membranes, as well as extravascular or intravascular hemolysis and red blood cell aggregation. This assessment is part of preclinical safety evaluation.
The physicochemical properties of Traditional Chinese Medicine (TCM) and natural medicine preparations—including active ingredients or components, in vivo metabolites, excipients, related impurities, and the formulation itself (e.g., pH, osmotic pressure)—can induce local toxicity or hemolysis. To guide rational clinical use, inform stakeholders about potential toxic reactions and their severity, establish safe dosages or concentrations, define therapeutic ranges, identify monitoring indicators for clinical studies, and outline antidotes or rescue measures, local toxicity testing is required for TCM and natural medicine preparations intended for topical application.
2. Background and Objectives
Safety and efficacy are critical in clinical medication. Traditionally, Chinese medicine was considered gentle and safe. However, this view is limited. With rapid advances in medicine and pharmaceutical technology, research into Chinese medicine has deepened, leading to new formulations and administration routes that differ significantly from traditional oral use. These changes alter drug pharmacokinetics and introduce safety concerns not seen with conventional preparations. Consequently, while therapeutic effects are achieved, adverse reactions in clinical practice have increased. As national pharmacovigilance systems improve, reports of adverse reactions—especially for Chinese medicine injections—are becoming increasingly common.
Furthermore, the complex composition of traditional Chinese medicine (TCM)—including active ingredients, new compounds formed through herb combinations, in vivo metabolites, impurities and excipients from manufacturing processes, and physicochemical properties of formulations—can all contribute to toxicity at the site of administration. Since local safety directly affects patient compliance and, ultimately, clinical efficacy, there is growing emphasis on the safety of novel TCM drugs administered via local routes (e.g., skin, mucous membranes, body cavities) and injection. Consequently, rigorous safety evaluation has become increasingly critical.
Given the above, this guideline aims to clarify the purpose, significance, and importance of conducting preclinical local administration toxicity studies; outline the basic principles, technical requirements, and existing challenges in safety evaluation of new traditional Chinese medicines (TCMs); and emphasize the need for case-by-case analysis tailored to each drug's characteristics. By integrating pharmaceutical, pharmacodynamic, other toxicological, and clinical application data through rational and objective study designs, this guideline seeks to identify, reveal, or discover toxicity reactions associated with local administration of TCMs and natural medicines, predict potential toxicities in clinical use, and guide appropriate clinical medication practices.
   3. Scope of Application
This technical guidance applies to non-oral Chinese medicines and natural drugs, including injectable preparations and topical formulations. It covers the basic principles for toxicity assessment of local administration, as well as content on irritation and hemolysis studies.

[Basic Content]

   1. Core Principles
Special toxicity studies for local administration must comply with the Good Laboratory Practice (GLP) regulations.
When designing experiments, adhere to the principles of randomization, control, and replication.
Based on general principles of safety evaluation, apply case-specific analysis that integrates the unique characteristics of the test article. Consider all relevant data—including pharmaceutical properties, pharmacodynamics, other toxicology findings, and intended clinical use—to reflect a holistic and comprehensive approach. Conduct standardized trials only after confirming that the methods are scientifically sound and reasonable. Then, perform a thorough analysis of the results to reach an overall safety assessment for the drug.
  II. Local Irritation Test
(I) Definition
Irritancy refers to reversible changes at the site of administration following the delivery of non-oral drug formulations. If irreversible tissue damage occurs at the administration site, it is termed corrosivity. Local irritation testing evaluates whether contact with the test substance causes local reactions such as redness, swelling, congestion, exudation, degeneration, or necrosis in animal blood vessels, muscles, skin, and mucous membranes.
(II) Common Issues to Consider in Testing
1 Purpose of the Test
Objectively evaluate potential drug toxicity.
2 Test Article
The test article must adequately represent the clinical trial investigational product and the marketed drug. Therefore, it should be a sample prepared using a stable manufacturing process that meets the quality standards for clinical use, typically a pilot-scale batch. The following details must be specified: name, source, batch number, potency (or specification), storage conditions, and preparation method. If a pilot-scale batch is not used, sufficient justification must be provided. When administration volume or route of administration imposes limitations, the active pharmaceutical ingredient (API) or extract may be used for the study.
3: Control Drug Selection
Use the vehicle, excipient, and/or base as a negative control. The batch number, specifications, and manufacturer of the vehicle/excipient must be clearly stated. If required, a positive control may also be used. Since local irritation is closely linked to the test article formulation, different formulations containing the same active ingredient may exhibit markedly different levels of local irritation. In the event of toxic reactions, comparative studies with marketed products should be conducted to ensure the safety of clinical drug use.
4, Species Selection
Select animals with skin and mucosal reactions similar to humans; common choices include rabbits and guinea pigs. The choice of species should be based on the indicators to be observed and the rationale for the model. Typically, one animal species is used per test type for evaluation.
5 Dose Selection
Multiple formulations with different concentrations can be tested, including those intended for clinical studies, to support dose selection for clinical safety. Dose adjustments can be made by altering the dosing frequency. For topical semi-solid formulations, increasing the application thickness should not be used to increase the applied dose if the application area remains unchanged.
6, Route of Administration
Generally align with the clinical route of administration; specify any exceptions.
7 Site of administration
Select sites similar to clinical application sites, considering surrounding tissues or areas potentially exposed to the investigational product. The test site should generally match the intended therapeutic target organ or location (e.g., skin for topical administration, eyes for ophthalmic administration), but may differ from the intended non-therapeutic target tissue (e.g., transdermal absorption patches, intravenous formulations). When designing dose levels and dosing frequencies, account for the anatomical and physiological characteristics of the animal model's administration site. Ensure effective exposure duration of the test substance at the administration site and describe the methods used. For single-dose irritation testing, a semi-occlusive patch method using gauze as the patch material is recommended; secure with a semi-occlusive dressing provided there is no irritating effect.
8 Determination of Dosing Frequency and Timing
The dosing frequency and timing for animals should be determined based on the proposed clinical application, i.e., whether multiple or single doses are to be administered according to the intended clinical use of the test article. However, the general dosing period shall not exceed 4 weeks.
9 Observation Metrics
9.1 Visual Observation: Provide a detailed description of the animal's general condition, behavior, clinical signs, and local reactions to administration. Local reactions should be scored, including severity and extent of erythema, edema, and congestion.
9.2 Histopathological Examination: Local tissue histopathology must be prioritized. Emphasis should be placed on selecting and documenting specific lesion sites, overall scoring, semi-quantitative analysis and interpretation of results, and providing corresponding histopathological images.
9.3 Systemic Toxicity Observation: When conducting local toxicity studies, also monitor systemic responses following local drug absorption.
9.4 Reversibility Observation If moderate to severe local irritation occurs, monitor the recovery period after discontinuation to assess the resolution of toxic reactions.
10 Test Report
Coverage should be comprehensive, including the test item name, physicochemical properties, pH, preparation method, and dosing regimen; animal species, sex, number, and weight range; strain and source (with certificate number and grade). Results of irritation responses at each observation point must be presented in a table. Any toxic effects other than irritation should also be described in detail. Conclusions must be specific and based on the findings.
11, Other
The reliability of extrapolating results from animal studies to humans is limited due to the irritancy test. White rabbits are generally more sensitive to irritating substances than humans. If similar results are obtained using other animal strains, the reliability of extrapolation from animals to humans will increase.
(3) Test Method
See Appendix.

3. Hemolysis Test
(I) Definition
Hemolysis tests evaluate whether a test substance causes hemolysis or red blood cell aggregation.
Hemolytic reactions caused by drug formulations can be classified into two types: immune-mediated and non-immune hemolysis. Immune hemolysis results from antibody production triggered by an immune response to the drug, corresponding to Type II and III hypersensitivity reactions. Non-immune hemolysis involves direct drug-induced hemolysis and red blood cell agglutination.
Certain Chinese medicine injections may cause hemolysis when directly administered into the bloodstream due to the presence of hemolytic components or physical, chemical, and biological factors. Some formulations containing impurities can induce local pain and swelling upon injection; once introduced into the blood vessels, they may trigger red blood cell aggregation, leading to circulatory dysfunction and other adverse reactions. Additionally, the complex composition of Chinese medicine preparations can provoke immune-mediated hemolysis. Therefore, all injectable products and other drug formulations that may induce either immune or non-immune hemolytic reactions must undergo hemolysis testing.
(2) Considerations for the Trial
Due to the complexity of hemolysis mechanisms, no comprehensive preclinical hemolysis assay currently exists. For decades, the determination of hemolytic properties in Traditional Chinese Medicine (TCM) injections has relied on the conventional in vitro hemolysis test using test tubes (see Appendix). This method relies on visual inspection to assess hemolysis, resulting in significant variability. It is also susceptible to interference from colored TCM injections and cannot quantify the specific number of lysed red blood cells.
To accurately assess the hemolytic activity of Traditional Chinese Medicine (TCM) injections, it is recommended to supplement standard methods with additional techniques such as spectrophotometry, in vitro red blood cell counting, or in vivo red blood cell counting and analysis. While the former two methods are more complex and subject to multiple influencing factors compared to standard protocols, they serve as valuable auxiliary tools when standard results are inconclusive or further verification is required, enabling a more precise determination of injection-induced hemolysis.
In long-term toxicity studies, consider adding or including hemolysis assessments (e.g., changes in red blood cell count and reticulocyte levels, bilirubin, urinary protein, nephritis, splenic congestion, and bone marrow morphology). If a test article shows positive results for hemolysis in vitro, conducting an in vivo hemolysis study can not only determine the potential for drug-induced hemolysis but also provide additional insights through other measurements, such as elevated reticulocytes, presence of hemoglobinuria, increased spherocytes, and evidence of hemolytic anemia in bone marrow examinations.
(3) Test Method
See Appendix.
   4. Data Analysis and Evaluation
(1) Provide a detailed description of the test methodology, including group allocation, procedures, dosage and frequency of administration, and number of animals. Report results in detail, such as signs of toxicity, duration of toxic effects, recovery status and timeline, number of deaths, and histopathological findings. Include histopathology images to facilitate result analysis and interpretation.
(2) Based on test results, describe the occurrence and severity of specific responses at different doses (or concentrations) using a scoring system. Analyze the dose-response and potential time-response relationships, including reversibility, to determine whether observed data changes are related to the test substance. Identify the toxic dose, safe dose, and safety margin. When interpreting results, distinguish between mechanical effects of administration or purely physicochemical properties of the test substance and their actual toxic or pharmacological effects to ensure accurate understanding of the findings.
(3) Based on local toxicity reactions, pathological findings, and systemic toxicity observations, identify the target organ(s) of toxicity or organs/tissues potentially involved.
(4) In hemolysis tests, if red blood cell agglutination occurs, determine whether it is true or false agglutination. If suspicious hemolysis is observed in vitro, use alternative methods to further evaluate and confirm or rule out the test substance's hemolytic effect.
(5) Emphasize histopathological examination and quantitative analysis of results: Local tissue pathology during irritancy testing should be prioritized, with pathological changes assessed using semi-quantitative methods.
(VI) Conduct a comprehensive analysis and overall evaluation by integrating pharmacodynamic data with other toxicology study results. Do not extrapolate animal findings directly to humans without proper analysis, and do not omit or selectively exclude animals exhibiting toxic responses. Objectively report the toxicity of the test substance. Based on the study outcomes, provide clinical implications and key precautions for human use.
    V. Common Issues and Solutions
(1) Topical TCM dosage forms are diverse, including patches (rubber-based, hydrocolloid, transdermal), plasters (black and white), gels, ointments (creams, pastes), liniments, lotions, aerosols or sprays (for respiratory inhalation, or skin/mucosa/cavity administration), washes (for skin application, cavity washing, or rinsing), film-forming solutions, nasal preparations (nasal drops, sprays, washes, ointments, powders), and ocular preparations (eye drops, ophthalmic ointments, powders). When conducting local irritation studies, ensuring accurate dosing concentrations and determining the effective residence time at the site of application are critical for a correct safety assessment.
(2) As new technologies are increasingly adopted, traditional Chinese medicine (TCM) topical preparations and injectables now incorporate more excipients, such as permeation enhancers, solubilizers, antioxidants, emulsifiers, suspending agents, pH adjusters, and osmotic regulators. Additionally, different formulations may include various bases. Therefore, when conducting local irritation studies, it is essential to isolate the effects of these excipients and bases. If any excipient or base itself exhibits significant local irritation, further investigation is required to re-evaluate and appropriately select alternatives, thereby enhancing the safety of TCM topical applications. Moreover, for TCM topical products containing toxic herbs combined with permeation enhancers, special attention must be paid to potential local and systemic toxicity.
(3) Vascular irritation from traditional Chinese medicine (TCM) injections directly affects patient adherence and the successful implementation of treatment regimens; therefore, evaluating vascular irritation holds significant value for guiding clinical use. However, single or repeated dosing in preclinical animal studies often fails to reveal vascular irritation. In clinical practice, some drugs may show no irritation after a single dose but exhibit significant irritation upon multiple administrations at certain concentrations. This suggests that both drug concentration and dosing frequency are closely linked to vascular irritation. We recommend that preclinical safety evaluations focus on selecting appropriate test substance concentrations and dosing frequencies, as well as monitoring the duration of local vascular residence. These measures aim to enhance the sensitivity of preclinical animal safety assessments and their correlation with human clinical outcomes, thereby providing guidance for rational and accurate clinical use.
(4) Preclinical and clinical studies reveal a discrepancy: while most preclinical in vitro hemolysis assays yield negative results, numerous positive cases have been reported during clinical use. To address this inconsistency, we recommend that preclinical studies employ conventional methods alongside additional assays. Specifically, long-term toxicity studies (via intravenous administration) should include or closely monitor relevant indicators to comprehensively evaluate the hemolytic potential of Chinese herbal injections and improve their correlation with human clinical outcomes.
(5) Post-marketing data on some Chinese medicine injections show significant variations in hemolysis rates among different batches of the same product from the same manufacturer. These differences may be attributed to factors such as the degree of impurity removal during production, the amount of additives used, and the low solubility of certain Chinese medicines in water, combined with variability in content due to different origins, growing seasons, and plant varieties. Therefore, it is recommended to conduct hemolysis testing on at least 3 batches of制剂 samples during preclinical studies.
(6) When performing spectrophotometric tests, pay attention to the effects of centrifugation speed and temperature on test results. Some drug molecules and excipients may collide with red blood cells at high speeds during centrifugation, which can lead to non-drug-induced hemolysis. Additionally, since different TCM injections vary in color and intensity, if their color interferes with the maximum absorption of hemoglobin, take steps to exclude non-drug factors.

VI. Selection of Test Items for Traditional Chinese Medicine and Natural Drug Products in Different Dosage Forms
(1) Intravenous injections: Must undergo vascular irritation, muscle irritation, and hemolysis tests.
(2) Injectable preparations for intramuscular use: Must undergo tests for vascular irritation, muscle irritation, and hemolysis.
(3) Topical preparations: Skin irritation testing is required; eye irritation testing should be conducted when necessary.
(4) Nasal Drops: Nasal mucosa irritation testing shall be conducted.
(5) Oral preparations: Must undergo oral or nasal mucosa irritation testing (e.g., for sprays).
(VI) Ophthalmic preparations: Eye irritation testing shall be conducted.
(VII) Rectal and vaginal preparations: Must undergo relevant mucosal irritation tests.
(VIII) Test items may be adjusted based on the specific characteristics of the drug. For formulations requiring assessment of immunotoxicity (e.g., hypersensitivity), refer to the Technical Guidance Principles for Immunotoxicity Studies of Traditional Chinese Medicine and Natural Drugs.
   [References]
1. Toxicity tests for dermatological preparations and mucosal preparations. "Guidelines for the Research of New Chinese Medicines", Drug Administration, Ministry of Health of the People's Republic of China 1993, 209, 213
2. Toxicity testing of dermal drugs. Yuan Bojun, Wang Zhiqiao. "Preclinical Safety Evaluation and Practice of New Drugs". Beijing: Military Medical Sciences Press. 1997 year 152
3. Other toxicity tests. Edited by Xu Shuyun. "Pharmacological Experimental Methods", 3rd Edition. Beijing: People's Medical Publishing House, 2002 year 234.
5. Zhou Yanwen, He Shuhua, Mo Wuning. Comparison of 3 methods for determining hemolysis degree of traditional Chinese medicine injections. Chinese Journal of Traditional Chinese Medicine. 2002, 27(6): 465-467
6 Qin Baixuan, Yin Yi. A new method for precise determination of hemolytic effects of Chinese herbal injections. Chinese Journal of Traditional Chinese Medicine. 1990, 15(9):32
7. Xu Guiyan, Chen Yuzhong, Zhang Yajie. Improvement of the in vitro hemolysis test method for Shuanghuanglian injection. Heilongjiang Medical Journal 2001, 24(5): 29
8. Meiyun Zhong, Hang Xiao, Lingfang Tang. Vascular irritation and hemolysis tests of captopril. Strait Pharmaceutical Journal. 1994, 6(2): 20-21
9. Wang Xiaochun, Yan Minying, and Yang Jianchun. Comparative study on in vitro hemolysis of compound Danshen injection from 6 manufacturers. Chinese Pharmaceutical Affairs. 2000, 14(5): 343.
10. Principles and Methods of Toxicology. Fourth Edition, edited by A. Wallace Hayes, Taylor & Francis, Philadelphia. 2001.
11. EMEA; Note for Guidance on Non-clinical Local Tolerance Testing of Medicinal Products.
12. FDA: Guidance for Industry on Botanical Drug Products
13. FDA; Guidance for Industry: Labeling Guidance for OTC Topical Drug Products for the Treatment of Vaginal Yeast Infections (Vulvovaginal Candidiasis)
14.FDA;Guidance for Industry: Bacterial Vaginosis — Developing Antimicrobial Drugs for Treatment.

    [Appendix]
   I. Local Irritation Test Method
(I) Vascular Irritation Test
Rabbits are the preferred test animals. Use saline or vehicle as the negative control. Inject into the marginal ear vein and monitor local vascular and surrounding tissue responses at various time points after administration, such as hyperemia and edema. The dosing duration should be determined based on the intended clinical use of the drug; for repeated dosing, the total period generally should not exceed 7 days. Perform multiple gross examinations of the animals and injection sites, along with microscopic evaluations, 48 to 96 hours post-administration. At the conclusion of the observation period, conduct histopathological examination of the injection site vasculature and surrounding tissues, and provide pathological images.
It is recommended to concurrently evaluate the test article's vascular irritation in dogs during long-term intravenous toxicity studies, including gross examination and histopathological analysis. Detailed reporting of findings should be provided along with corresponding histopathology images.
(II) Muscle Irritation Test
The guinea pig is the preferred test animal, though rats may also be used. A negative control group shall be established using normal saline or vehicle. Under aseptic conditions, a specified amount of the test article shall be injected into each quadriceps muscle on both sides of the animals. The local muscle reaction following administration—such as hyperemia, swelling, degeneration, and necrosis—shall be monitored at various time points. Animals shall be sacrificed 48 hours post-injection; the quadriceps muscles shall then be dissected, longitudinally incised, and examined for injection-site irritation. Reaction grades shall be calculated according to Table 1, followed by histopathological examination of the local tissue, with pathological images provided.
  Table 1 Muscle Stimulation Response Grading Standards
Stimulus-response                                           Reaction Level

No significant changes 0

Mild congestion, within 0.5×1.0 cm. 1

Moderate congestion, extending over 0.5×1.0 cm or more. 2

Severe congestion with muscle degeneration 3

Necrosis observed with brown degeneration. 4

Extensive necrosis 5

Calculate the total of 4 quadriceps response grades from the table above. If the difference between the highest and lowest quadriceps response grades exceeds 2, repeat the test using 2 additional animals.
(3) Skin Irritation Test
Rabbits are the preferred test animals, though guinea pigs may also be used. A vehicle control group must be included. This study should simultaneously evaluate irritation on both intact and abraded skin. For abraded skin tests, abrasion can be performed using sandpaper, followed by making a "cross-hatch" incision (to the point of bleeding) before administering the test substance. The back is the recommended site for testing. After depilating the back skin, apply the test substance and observe local skin reactions at various time points post-dosing, such as erythema, edema, scaling, crusting, or other lesions. Also assess for pigmentation changes, petechiae, roughness, or thinning of the skin. Record the onset and resolution times of these reactions and evaluate their severity. The duration of each application and whether occlusion is required should be determined based on the intended clinical use; however, the minimum application time shall be 4-6 hours. For multiple dosing, administer consecutively at the same site. If severe adverse reactions occur during repeat dosing, the application site may be changed, but observation must continue. There is no strict limit on the trial duration; it should be determined based on changes observed at 24, 48, and 72 hours post-dosing. If changes persist, extended observation beyond these time points is required. At the conclusion of the study, perform histopathological examination of the local tissue and provide pathological images.
Result Evaluation: (1) Single-dose skin irritation test: Calculate the mean score of skin reactions for each test substance and vehicle group at every observation time point. The Primary Irritation Index is obtained by subtracting the mean vehicle skin reaction score from the mean test substance skin reaction score, with irritation intensity assessed according to Table 3. (2) Repeated-dose skin irritation test: First calculate the mean primary irritation scores for each group at every observation time point, then compute the cumulative irritation index (sum of mean irritation scores per animal over the observation period) for each animal, with irritation intensity assessed according to Table 3.
Table 2. Skin Irritation Response Scoring Criteria

           Stimulus-response                                                Score

           Erythema (1)

No erythema 0

Mild erythema (barely visible) 1

Moderate erythema (clearly visible) 2

Severe erythema 3

Edema (2)

No edema 0

Mild edema (barely visible) 1

Moderate edema (distinct swelling) 2

Severe edema (skin elevation 1 mm, well-defined contour) 3

Severe edema (skin elevation >1 mm with expansion) 5

Maximum Total Score 8

 Table 3. Skin Irritation Intensity Evaluation Criteria

              Average Score                                             Review

0 - <0.5> Non-irritating

0.5 - <2> Mild irritant

2.0 - <6> Moderate Irritant

6.0 - 8.0 Strong Irritant

(4) Mucosal Irritation Test
1: Ocular Irritation Test
The eye irritation test evaluates the irritancy of drugs intended for ocular and other mucosal use. The test design, including type and scope, depends on the nature of ocular exposure to the test substance.
Rabbits are typically used. The dosing regimen (single or multiple administrations) should reflect the intended clinical application of the test article, and a vehicle control group must be included. In each animal, one eye is treated while the other serves as the control. Apply 0 mL of the test article to each eye via instillation or 0 g via topical application, ensuring adequate contact between the drug and the ocular surface. The dosing duration should align with the proposed clinical use; for repeated dosing, the period generally should not exceed 4 weeks. The observation period must be sufficient to detect both reversible and irreversible effects. Examination should cover the periorbital region, eyelids, conjunctiva, nictitating membrane, cornea, and iris. Assess corneal, iris, and conjunctival reactions at 30, 60, 90, 120min, 24, 48, and 72 hours post-dosing using binocular magnifiers, slit lamps, and in vivo microscopes, scoring responses according to Table 3. Additionally, evaluate signs of irritation such as conjunctival hyperemia, edema, photophobia, and lacrimation, and assign scores accordingly. Calculate the total score by summing individual animal scores, then divide by the number of animals to determine the degree of irritation based on Table 4.
Table 3 Eye Irritation Response Scoring Criteria

             Eye irritation response                                            Score

           I: Cornea

Turbid (based on the densest area) (A)

No turbidity 0

Scattered or diffuse opacities; iris is clearly visible 1

Semi-transparent area is easy to distinguish; iris is blurry 2

Corneal opacity; iris not visible 4

       Corneal Damage Extent (B)

             <1/4                                                       1

           1/4~<1/2                                                     2

           1/2~<3/4                                                     3

            3/4~1                                                       4

A×B×5 Theoretical Maximum 80

II: Iris (A)

Normal 0

Markedly deepened folds, congestion, and swelling; mild pericorneal hyperemia; pupil still reacts to light 1

Bleeding / Visible necrosis / No response to light (or any of these) 2

A × 5 | Theoretical Maximum: 20

         III: Conjunctiva

 Congestion (of palpebral and bulbar conjunctiva) (A)

Normal vasculature 0

Vascular congestion appears bright red 1

Vessels appear congested and deep red; difficult to distinguish 2

               Edema (B)

No edema 0

Mild edema (including eyelids) 1

Obvious edema with partial eyelid eversion 2

Swollen eyelids nearly closed 3

Periorbital edema with eyelid closure exceeding 50% 4

           Discharge (C)

No discharge 1

Minor discharge 2

Moisture or sticking of the eyelids and eyelashes due to discharge 3

Wet or sticky discharge in the entire eye area 4

(A+B+C) × 2 — Theoretical Maximum: 20

Cumulative score of corneal, iris, and conjunctival reactions = I + II + III = 110 (maximum score)

Table 4 Grading Criteria for Ocular Irritation Responses

            Rate               Stimulus Level                Kay and Calanda Review

0-0.5 Nonirritating

0.5-2.5 Mildly Irritating Practically Nonirritating

2 Rectal Irritation Test
Rabbits or dogs are typically selected. The dosing volume may be based on the proposed human therapeutic volume or the maximum tolerable dose for different animal species. Dosing frequency is determined by clinical application, generally 1-2 times daily for at least 7 days. Each administration should maintain mucosal contact for at least 2-4 hours; the anus may be temporarily sealed if necessary. Observations include: the anal region and anal sphincter, clinical signs post-dosing (e.g., pain symptoms), fecal characteristics (e.g., blood, mucus), animal mortality and necropsy findings post-dosing, local tissue reactions (e.g., congestion, edema), and histopathological examination of the perianal mucosa.
3 Vaginal Irritation Test
Typically, rats, rabbits, or dogs are selected. The dosing volume can be based on the proposed human therapeutic volume or the maximum dose for different animal species. The dosing frequency depends on clinical application and is typically 1 to 2 times per day for at least 7 days. Each dose must remain in contact with the mucosa for at least 4 hours. Observation parameters include vaginal site, clinical signs (e.g., pain), and vaginal discharge (e.g., blood, mucus). Additionally, monitor for animal death post-dosing and conduct necropsy. Assess local tissue for congestion, edema, or other abnormalities, and perform histopathological examination of the vaginal cavity and reproductive system.
4 Nasal Drop and Inhalation Irritation Tests
Rabbits, guinea pigs, or rats may be used. The test substance is instilled or sprayed onto the animals to ensure contact with the mucosa for at least 4 hours. After administration, monitor the animals for systemic effects (e.g., respiratory, circulatory, and central nervous system changes) and local irritation symptoms (e.g., asthma, coughing, vomiting, asphyxiation). Sacrifice the animals 24 hours after a single dose or 24 hours after cessation of multiple doses. Examine the respiratory tract tissues (nose, pharynx, trachea, bronchi) for signs of congestion and swelling, and perform histopathological examination.
5: Oral medications, otic drops, and other irritancy tests
Following the above protocol, administer the test article via the oral cavity or external auditory canal to evaluate its effects on the oral and laryngeal mucosa, as well as on the external auditory canal and tympanic membrane. For oral administration, golden hamsters are recommended to assess irritation of the buccal mucosa.
(V) Phototoxicity Test
Phototoxicity refers to skin damage caused by the release of absorbed UV energy from a drug within the skin. It is a toxic skin reaction triggered after contact or application of a chemical substance followed by exposure to ultraviolet radiation. Phototoxicity is the most common adverse reaction in photosensitivity. It is dose-dependent and clinically resembles sunburn, presenting with erythema, edema, pruritus, and pigmentation. Severe cases may result in local necrosis, ulceration, or epidermal sloughing. Once triggered, the reaction occurs rapidly, typically within 24 hours or less of light exposure.
1, Test Animals
Healthy albino or hairless guinea pigs 300-500g, at least 14 animals, half male and half female.
2, Trial Grouping
Guinea pigs were randomly divided into two groups by weight and sex: the control group with 4 animals and the experimental group with 10 animals, with equal numbers of males and females. The control group was not exposed to light after administration of the test substance and positive control drug, whereas the experimental group was exposed to light after administration.
3 Medications and Dosages
Positive control agents may include 8-methoxsalen. The dosing regimen should be determined based on the test article's solubility, required concentrations for the study, physicochemical properties, and local and systemic tolerability in animals, with justification provided. Typically, two concentration levels are selected; the lower concentration corresponds to the clinical dosage.
4 Method of Administration
The back hair removal area was divided into four regions, each receiving a dose of 0.025ml g/cm². The dosing details are as follows:

Group Body Site Treatment Light Irradiation

Control Group 1 Solvent or Excipient No

2 Test Article Concentration 1 No

3               Test Article Concentration 2            No

4 Positive Control No

Trial Group 1 Vehicle or Excipient Yes

2               Test Article Concentration 1            Yes

3 Test Article Concentration 2 Yes

4 Positive Control Yes

Immediately after dosing, place the animal in a specialized restraint device to limit movement. Before irradiation, ensure sufficient time has elapsed for the test item and control substance to penetrate the stratum corneum and interact with epidermal cells; 60 minutes is generally appropriate. At an appropriate time after dosing (typically 0.5-4 hours), remove any residual test item or vehicle from the treated site of control animals using warm water or a non-irritating suitable solvent, then return the animals to their cages. Residual test item or vehicle on the treated sites of test group animals should be removed after irradiation.
5: Lighting Method
Experiments should be conducted using wavelength ranges to which the test substance is sensitive. Preliminary trials are required first to determine the irradiation dose, light intensity, exposure duration, and distance from the light source. During testing, animals are secured on a light platform with their heads covered, then exposed to an appropriate light source (e.g., water-cooled xenon lamp) for a suitable duration. The wavelength range is generally considered to be 280-450 nm, with an exposure time of 0.5-2 hours. Light intensity should be appropriate, and measurements must be taken both before and after irradiation.
6 Check Project
Weigh participants before and after the trial. Observe skin reactions at 24 and 48 hours, document with photos, and record observations. Record skin reaction scores for each time point according to Table 7.

Table 7: Grading Criteria for Skin Reaction Severity

Skin Reaction Status Score

No erythema 0

Mild erythema 1

Moderate erythema 2

Severe erythema (with or without edema) 3

7. Result Assessment and Evaluation
Compared with the control group: if all control group scores are less than 1, a test group score ≥ 1 is considered positive. If any control group score is 1 or higher, a test group score exceeding the maximum control score is considered positive. The positivity rate can then be calculated.
III. Hemolysis Assay Method
(I) Standard In Vitro Tube Method (Visual Observation)
Typically, a red blood cell suspension is prepared using blood from rabbits, sheep, dogs, or humans. The test substance is added at various doses (see Table 5), with distilled water and physiological saline serving as positive and negative controls, respectively. After incubation, hemolysis or agglutination reactions are observed. Observations are conducted for at least 1 hours at 37°C, or for 2-3 hours. A transparent red solution indicates hemolysis, while the presence of brownish-red or reddish-brown flocculent precipitate indicates red blood cell aggregation.
Table 5. In Vitro Hemolysis Test

            ML                                    Test Tube ID

                                        1      2      3     4     5     6     7

          2% Red Blood Cell Suspension               2.5    2.5    2.5    2.5   2.5   2.5   2.5

             Saline                 2.0    2.1    2.2    2.3    2.4  2.5

             Distilled water                                                          2.5

             Test Article                  0.5    0.4    0.3    0.2    0.1