Guiding Principles for Acute Toxicity Studies of Traditional Chinese Medicine and Natural Drugs

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Guideline ID: [Z] GPT2-1

Guiding Principles for Acute Toxicity Studies of Traditional Chinese Medicine and Natural Drugs
(Draft 2)

March 18, 2004

Guiding Principles for Acute Toxicity Studies of Traditional Chinese Medicine and Natural Drugs

1. Overview
Acute toxicity refers to toxic reactions observed in animals shortly after a single or multiple exposures (within 24 hours) to a test substance at a specified dose. Its purpose is to provide reference data for new drug development.
The "Guidelines for the Research of New Traditional Chinese Medicine Drugs" issued by the Ministry of Health of the People's Republic of China in 1993 and the "Technical Requirements for Pharmacology and Toxicology Studies of New Traditional Chinese Medicine Drugs" issued by the State Drug Administration in 1999 have played a positive role in standardizing acute toxicity testing for TCMs and advancing their research and development in China. However, with continuous improvements in new drug research methodologies and deeper insights into TCMs and natural medicines, these requirements are increasingly showing their limitations.
Most traditional Chinese medicines (TCM) and natural drugs have mild effects, and the rational combination of ingredients in TCM compound preparations may reduce toxicity. Most new TCM and natural drug formulations are derived from ancient prescriptions, hospital-compounded preparations, or clinical experience-based formulas, providing a solid foundation for clinical application. However, modern TCM and natural drug preparations often incorporate advanced technologies and novel theories. Compared with traditional forms, their material basis and administration methods may differ significantly. Some of these changes yield unknown outcomes; particularly when certain component concentrations increase substantially, pharmacological effects may intensify, and toxic reactions may become more severe. Therefore, conducting acute toxicity studies is essential for TCM and natural drugs.
This guideline was developed based on the characteristics of traditional Chinese medicines and natural drugs, in alignment with international requirements for drug safety evaluation and the current status of drug safety research in China.
This guidance applies to acute toxicity studies of traditional Chinese medicines and natural drugs.
The landscape of traditional Chinese medicine (TCM) and natural drugs is complex. The content discussed herein cannot encompass all practical aspects of acute toxicity studies for TCM and natural drugs. When conducting such studies, the principle of "analyzing specific issues in a specific manner" should be followed.
II. Basic Principles of Acute Toxicity Studies:
(1) Test Management
Acute toxicity studies must comply with Good Laboratory Practice (GLP) for non-clinical drug research.
(II) Experimental Design
Experimental design must adhere to the core principles of randomization, control, and replication.

 III. Basic Content of Acute Toxicity Studies
(I) Investigational Product
The test article in acute toxicity studies should adequately represent the clinical trial material and marketed drug. Therefore, it is recommended that the test article be prepared using a stable manufacturing process and meet the quality standards for clinical use.pilot testingSamples, with details on the test article's name, source, lot number, potency (or specification), storage conditions, and preparation method. If pilot-scale samples are not used, provide a valid justification. If dosing volume or administration route constraints apply, the test may be conducted using the active pharmaceutical ingredient (API) or extract.
Solvent or Excipient: Select an appropriate solvent or excipient.
    (II)Laboratory animals
Mammalian species should generally be used with an equal sex ratio. If clinical use involves only one sex, animals of that corresponding sex may be used. Rats and mice are commonly employed as experimental animals; other species may also be used. When non-rodent species are more suitable for the study, they should be prioritized. Rodents must comply withNational Experimental Animal Standard Grade IIand above grade requirements; non-rodent animals shall comply withNational Standard for Laboratory Animals Class Iand above grade requirements. Tests are typically conducted using healthy adult animals. If the test article is intended for use in children, juvenile animals should be considered. The initial animal dose should not exceed or fall below 20% of the average body weight.
    (3)Experimental Groups
In addition to appropriate dose groups for the test article, a blank (and/or negative) control group should be included.
    (IV)Route of Administration
Different routes of administration result in variations in the absorption rate, speed, and exposure of the test substance. To maximize the observation of acute toxic effects in animals, multiple routes may be used in acute toxicity studies. Typically, two routes are employed, with at least one matching the intended clinical route. If the intended clinical route is not used, a clear justification must be provided. When administering via gavage, the animal should be fasted.
    (5)Dosage Volume: For oral gavage, the volume per dose should generally not exceed 20ml/kg for rats and 40ml/kg for mice. Dosage volumes for other species and administration routes should be determined based on relevant literature and specific experimental conditions.
    (VI)Observation Period: Typically 7 to 14 days. If adverse reactions develop slowly, extend the observation period accordingly. If the observation period is less than 7 days, provide a detailed justification.
Generally, animal responses should be observed in detail within 4 hours after administration, followed by twice-daily observations (morning and afternoon).
    (VII)Observation parameters include animal body weight changes, food intake, appearance, behavior, secretions, and excreta. Record all deaths, signs of poisoning, onset time, severity, duration, and reversibility of toxic reactions for every animal. Perform gross necropsy on moribund or deceased animals promptly; conduct gross necropsy on remaining animals after the observation period ends. If organ volume, color, texture, or other abnormalities are detected, perform histopathological examination on the affected organs.
    (VIII)Result Processing and Analysis
1 Analyze the incidence and severity of each observed adverse reaction across different doses, considering factors such as onset time, severity, and duration. Based on the observations, summarize the findings to assess the dose-response relationship and temporal trends for each reaction.
2. Refer to Appendix 1 to identify the organs, tissues, or systems potentially involved in each observed reaction.
3 Based on gross anatomical lesions visible to the naked eye and results from histopathological examinations, preliminarily identify potential toxic target organs. Histopathological reports must be signed by the responsible pathologist and include photographs of affected tissues.
4 Based on the incidence of various reactions and animal mortality across different dose groups, assess the test article's tolerance in animals, including the no-observed-adverse-effect level (NOAEL), lowest-observed-adverse-effect level (LOAEL), maximum tolerated dose (MTD), and minimum lethal dose, to preliminarily determine the drug's safety margin.
5. Describe the calculation and statistical methods used, and provide justification for their appropriateness.
6 Based on acute toxicity study results, identify key considerations for other safety studies, clinical trials, and quality control. Integrate findings from additional safety, efficacy, and quality assessments to weigh risks and benefits and evaluate the development potential of the test article.

IV. General Requirements for Acute Toxicity Testing of Traditional Chinese Medicine and Natural Drugs Under Different Circumstances
Due to the unique characteristics of traditional Chinese medicines and natural drugs, trials may be conducted in accordance with the following requirements. If these requirements are not followed, a detailed justification must be provided.
1. New effective components and preparations extracted from traditional Chinese medicine (TCM) or natural drugs that have not been marketed domestically; preparations made from medicinal substances derived from plants, animals, minerals, etc., that have not been marketed domestically; preparations made from new medicinal parts of TCM materials that have not been marketed domestically; preparations made from new effective parts extracted from TCM or natural drugs that have not been marketed domestically; and injectable preparations made from TCM or natural drugs that have not been marketed domestically.
In such cases, given the significant changes in the underlying material compared to traditional Chinese medicine or limited historical usage, acute toxicity should be evaluated comprehensively using two administration routes and both rodent and non-rodent animal models. Results must then be analyzed.
If the clinical route of administration is non-intravenous, an intravenous route is recommended for another administration to fully assess toxicological responses. If intravenous administration is not feasible due to formulation constraints or other reasons, provide a detailed justification. In such cases, toxicity assessment may be conducted using an alternative administration route or solely the intended clinical route.
Intravenous administration may be assessed solely using the clinical route for toxicity evaluation.
2. Compound preparations of traditional Chinese medicines and natural drugs, not yet marketed in China, for non-injectable administration.
If the formulation aligns with traditional Chinese medicine (TCM) theory and has established clinical use, a single animal species is generally sufficient for acute toxicity assessment via the intended clinical route of administration.
If the compound preparation is a natural medicine, acute toxicity shall be observed using both rodents and non-rodent species via the intended clinical route of administration.
   If the formulation contains herbal drugs, active ingredients, or chemical substances as described above, acute toxicity interaction studies must be conducted on these pharmaceutical substances.。
3. Formulations that change the route of administration for drugs already marketed in China (excluding changes from non-injectable to injectable forms).
A single animal species may be used to compare the toxic responses before and after changing the route of administration.
4. Chinese medicines or natural medicine compound preparations that change the dosage form of drugs already marketed in China or alter the manufacturing process without changing the route of administration.
If the manufacturing process undergoes a qualitative change, it is recommended to conduct an acute toxicity study in one animal species via the intended clinical route of administration.
5. Variants with new indications or therapeutic functions.
If extending the treatment duration or increasing the dosage is required, determine whether acute toxicity studies are needed and define their scope based on the original submission documents and prescription composition.
Traditional Chinese Medicine (TCM) and natural drugs present complex scenarios; appropriate test methods should be selected based on the characteristics of the test article, with proper analysis of the results. Appendix I outlines "General Research Methods for Acute Toxicity Tests," and Appendix II details "General Observations from Acute Toxicity Tests and Potential Organs, Tissues, and Systems Involved." These appendices are provided for researchers' reference when selecting test methods and interpreting results.
V. Appendix

Appendix I: General Research Methods for Acute Toxicity Tests

LD method:
The severity of drug toxicity can be expressed using the lethal dose in animals, as physiological indicators of life and death are more distinct, objective, and easier to measure than other metrics. When determining the lethal dose, it is also essential to closely observe signs of poisoning before animal death.
The determination of lethal dose primarily includes the minimum lethal dose (LD5), median lethal dose (LD50), and maximum lethal dose (LD95). Since LD50 is relatively easy to determine, it is generally used for lethal dose assessment.
    Maximum Tolerated Dose Study: The Maximum Tolerated Dose (MTD) is the highest dose that animals can tolerate without causing death. From a safety assessment perspective, observing and analyzing abnormal reactions and pathological processes in experimental animals may be more toxicologically significant than using mortality as the sole endpoint.
    Maximum Dose Tolerance Study:Maximum dose refers to the highest dosage administered in a single dose or multiple doses within 24 hours (2-3 times). The maximum dose test involves administering the maximum allowable dose to experimental animals under reasonable dosing concentrations and volumes, then observing any reactions. Many traditional Chinese medicines and natural drugs have low toxicity; if the single-dose amount is far greater than the equivalent dose from pharmacodynamic studies, further observation at higher doses may not be necessary.
Other methods
    Fixed-dose methodFixed-dose procedure, Approximate Lethal Dose (ALD), limit test, acute toxic class method, and up-and-down method.

[Appendix II: General Observations from Acute Toxicity Tests and Potential Organs, Tissues, and Systems Involved

 

	B.	Lethargy: Animals appear drowsy but can be easily roused to resume normal activity.	Sleep center of the CNS.
	C.	Normal reflexes absent; righting reflex absent.	CNS, Sensory, Neuromuscular
	D.	Paralysis: Normal and pain reflexes are absent.	CNS, sensory.
	E.	Tonic convulsion: Posture remains unchanged regardless of placement.	CNS, Sensory, Neuromuscular, Autonomic
	F.	Ataxia: Inability to control and coordinate movement while walking, without spasm, local paralysis, or stiffness.	CNS, sensory, autonomic
	G.	Abnormal movements: spasms, toe-walking, pacing, fidgeting, crouching.	CNS, Sensory, Neuromuscular
	H.	Prone: Remain still, abdomen against the floor.	CNS, Sensory, Neuromuscular
	I.	Tremors: Shaking or trembling of the limbs and entire body.	Neuromuscular, CNS
	J.	Myokymia: involuntary muscle movements in the back, shoulders, hind limbs, and feet.	neuromuscular, CNS, autonomic nervous system
III. Convulsions: Involuntary, often rhythmic contractions of voluntary muscles or seizure-like activity.	A.	Clonic convulsion: Alternating spasms of muscle contraction and relaxation.	CNS, respiratory failure, neuromuscular, autonomic nervous system
	B.	Tonic convulsion: sustained muscle contraction followed by rigid extension of the hind limbs.	CNS, respiratory failure, neuromuscular, autonomic nervous system
	C.	Tonic-clonic seizure: Alternating phases of stiffening and jerking.	CNS, respiratory failure, neuromuscular, autonomic nervous system
	D.	Syncope convulsion: typically clonic seizures accompanied by gasping and cyanosis.	CNS, respiratory failure, neuromuscular, autonomic nervous system
	E.	Opisthotonos: A tonic seizure characterized by the back arching and head tilting backward.	CNS, respiratory failure, neuromuscular, autonomic nervous system
IV. Reflection	A.	Corneal eyelid closure: Contact with the cornea causes the eyelid to close.	Sensory, Neuromuscular
	B.	Basic reflex: Lightly tapping the inner side of the outer ear causes it to twitch.	Sensory, Neuromuscular
	C.	Positive Righting Reflex: Righting Reflex	CNS, Sensory, Neuromuscular
	D.	Stretch reflex: The ability to retract the hindlimbs when they fall off an edge.	Sensory, Neuromuscular
	E.	Light reflex (pupillary light reflex); pupil constriction upon exposure to light.	Sensory, Neuromuscular, Autonomic
	F.	Startle reflex: Response to external stimuli (e.g., touch, noise).	Sensory, Neuromuscular
V. Indications for Eye Examination	A.	Tearing: Excessive, clear, or colored tears	Autonomic
	B.	Miosis: Pupillary constriction regardless of light exposure.	Autonomic
	C.	Mydriasis: Pupil dilation regardless of light conditions.	Autonomic
	D.	Proptosis	Autonomic
	E.	Ptosis: Ptosis, animals fail to recover after stimulation.	Autonomic
	F.	Hemolacria: Tears appear red.	autonomic nervous system, hemorrhage, infection
	G.	Drooping upper eyelids	Autonomic
	H.	Corneal opacity, iritis, conjunctivitis	Eye irritant
VI. Cardiovascular Indications	A.	Bradycardia	Autonomic dysfunction and impaired cardiopulmonary function
	B.	Tachycardia		Autonomic dysfunction and impaired cardiopulmonary function
	C.	Vasodilation: Redness of the skin, tail, tongue, ears, paw pads, and conjunctiva; scrotal redness; elevated body temperature.		Autonomic nervous system, CNS, increased cardiac output, high ambient temperature
	D.	Vasoconstriction: Pale skin, low body temperature.		Autonomic nervous system, CNS, decreased cardiac output, low ambient temperature
	E.	Arrhythmia: Irregular heartbeat		CNS, autonomic nervous system, reduced cardiopulmonary function, myocardial ischemia

	E
VII. Saliva Production	A.	Excessive Saliva Production: Moisture Around the Mouth		Autonomic
VIII. Piloerection	A.	contraction of arrector pili muscles		Autonomic
IX. Anesthesia	A.	Reduced responsiveness to noxious stimuli (e.g., hot plate)		Sensory, CNS
X. Muscle Tone	A.	Decreased tone: generalized reduction in muscle tone		Autonomic
	B.	Increased tone: generalized increase in muscle tone		Autonomic
XI. Gastrointestinal Indications
Bowel movement	A.	Solid, dry, small quantity	Autonomic Nervous System, Constipation, Gastrointestinal Motility
	B.	Decreased mobility, watery stool	Autonomic nervous system, dysentery, gastrointestinal motility
Vomiting	A.	Vomiting or nausea	Sensory, CNS, Autonomic Nervous System (Rats: No Emesis)
Polyuria	A.	Red urine	Kidney injury
	B.	urinary incontinence	Autonomous Sensory Meridian Response
VII. Skin	A.	Edema: Swelling caused by fluid accumulation in tissues.	Irritation, kidney failure, tissue damage, prolonged immobility
	B.	Erythema: Redness of the skin	Irritation, inflammation, sensitization

 

VI. References

1. Yuan Bojun, Wang Zhiqiao, Preclinical Safety Evaluation and Practice of New Drugs, Military Medical Science Press, 1997
2. Guidance for Industry: Single-Dose Acute Toxicity Testing for Pharmaceuticals. FDA, 1996.
3 Xu Shuyun, Pharmacological Experimental Methods, People's Medical Publishing House, 2002
4. Chen Qi (Ed.), Methods in Pharmacological Research of Chinese Materia Medica, People's Medical Publishing House, 1993
5. Acute Toxicity and Eye Irritancy. In: A.Wallace Hays (ed.), Principles and Methods of Toxicology, 4th ed., 2001: 853-916.

VII. Drafting Notes
   1. Background
The "Guidelines for the Research of New Traditional Chinese Medicine (TCM) Drugs" issued by the Ministry of Health of the People's Republic of China in 1993 and the "Technical Requirements for Pharmacological and Toxicological Studies on New TCM Drugs" issued by the State Drug Administration in 1999 have played a positive role in standardizing acute toxicity testing for TCM and advancing its research and development in China. However, with technological advancements and evolving understanding of acute toxicity, coupled with rapid progress in the development of new TCM and natural medicine drugs, these original guidelines and technical requirements no longer adequately reflect current realities. Revision is therefore essential. Furthermore, as China joined the WTO and global interest in TCM has grown, aligning TCM standards with international expectations has become critical for its global expansion. This revised guideline was developed based on the original framework, incorporating the unique characteristics of TCM and natural medicines, while integrating international drug safety evaluation standards and China's current landscape in drug safety research.
On 2003-11-29, the entire research group met in Guangzhou to discuss the first draft of the "Technical Guidelines for Acute Toxicity Studies." We reached a general consensus on the key issues raised by the working team and provided suggestions for revision. Based on feedback from experts, we revised the initial draft into this version and are submitting it again for discussion by both the research team members and the broader community. We welcome your extensive input to help us further refine and improve these guidelines.
2. Guidance on Key Focus Areas for Acute Toxicity Studies
Unlike previous guidelines, this version no longer emphasizes specific LD500 values or the multiple of the intended clinical dose. Instead, it highlights that the focus of acute toxicity studies should be on toxic reactions following administration and underscores the importance of comprehensive result analysis. Given the research-oriented nature of this guideline, specific testing methods are not included in the main text to prevent these studies from becoming rigid, formulaic exercises. Developers are encouraged to select appropriate methods based on the characteristics of the test substance.
3. Description of Test Sample
To ensure that acute toxicity studies accurately represent the investigational product for clinical trials and marketed drugs, and to genuinely reflect toxicity profiles in clinical use and post-marketing settings, experts have reached a consensus recommending the use of pilot-scale samples. These samples must be produced using a stable manufacturing process and meet the quality standards applicable for clinical testing. The term "pilot-scale sample" refers specifically to the standardized materials used in formal acute toxicity studies submitted for regulatory approval, not to preliminary acute toxicity screening samples generated during earlier research phases.
4. Description of requirements for test animals
The 1990 guidelines did not specify animal grade requirements for acute toxicity studies. Should this guidance document now include specifications for the number of animals? Internationally, SPF-grade animals are generally required. After discussion, it was concluded that with technological advancements, safety evaluation standards for new traditional Chinese medicines should also be elevated. We recommend that rodents meet National Standard Grade II or higher, while non-rodents meet National Standard Grade I or higher. Is this requirement feasible? We welcome broad feedback and suggestions.
5. Instructions on fasting time for oral administration
During the first research group meeting, experts noted that the fasting duration for mice and rats specified in the original guideline (12–16 hours) is too long. However, should specific limits be set (e.g., a minimum or maximum) to facilitate experimental procedures? The current technical guidance does not prescribe exact values but only states that animals must be "fasted."
6. Explanation of Maximum Dose Study
The original Guidelines and Technical Requirements mandate that acute toxicity studies determine the animal LD500. When this cannot be measured, a maximum dose study may be conducted. However, for traditional Chinese medicines (TCM) and natural drugs, acute toxicity is often difficult to manifest. Even if death occurs at high doses, these doses may differ significantly from the effective or intended clinical doses, rendering their toxicological implications less relevant for other safety studies and clinical trials. Therefore, it is recommended that animal lethal doses and maximum tolerated doses not be mandatory test items. Instead, maximum dose studies should serve as the assessment method for TCM and natural drug acute toxicity. The appropriate dose level for maximum dose studies warrants further discussion.
5. Guidance on using two animal species for acute toxicity testing of traditional Chinese medicine compound preparations.
The new "Interim Measures for Registration Administration" does not explicitly require this. However, unlike Chinese herbal compound preparations, natural medicine compound preparations may be formulated without adhering to traditional Chinese medicine (TCM) theory. Given the lack of corresponding clinical application basis for such drugs, it is recommended that acute toxicity studies be conducted using two animal species.
6. The trial protocol specifies only "appropriate doses" without defining exact amounts. Since the primary objective is to establish an appropriate safety margin, characterize the nature of toxic reactions, identify target organs for toxicity, and determine causes of death under selected doses, investigators should consider relevant factors such as indications, the toxicity level of the investigational product, and its purity.
7 About References
This guideline was developed by reviewing domestic and international literature. However, the content was synthesized and adapted to reflect the characteristics, development trends, and challenges in research and evaluation of traditional Chinese medicines and natural drugs. Since much of the material is not directly excerpted from any single guideline, references are listed only at the end rather than cited within specific sections.

8. Authors
Research Group for the "Guidance Principles on Acute Toxicity Studies of Traditional Chinese Medicine and Natural Drugs"