Research Guidelines For Evaluating The Safety And Efficacy Of Herbal Medicines: The research guidelines for evaluating the safety and efficacy of herbal medicines are intended to facilitate the work of research scientists and clinicians in this field and to furnish some reference points for the governmental, industrial, and non-profit organizations that provide financial support for their work. It is hoped that these guidelines will be found general enough to enable each Member State to modify them to meet its own specific needs. It must be emphasized that these guidelines are offered as a summary of scientific standards governing various aspects of the study of herbal medicines. As such, they may be useful to the regulatory authorities who control the sale of these products and the governmental agencies and medical authorities who supervise their use in the health care system.
The research guidelines for evaluating the safety and efficacy of herbal medicines include the following:
- Guidelines for quality specifications of plant materials and preparations.
- Guidelines for pharmacodynamics and general pharmacological studies of herbal medicines.
- Guidelines for toxicity investigation of herbal medicines.
(A) Guidelines for quality specifications of plant materials and preparations: To ensure the reliability and repeatability of research on herbal medicines, the identity, and quality of the plant material or preparation must be determined and stipulated according to the following headings:
(a) Information for Fresh, Dried, and Processed Plant Materials:
(i) Name and characteristics:
- Name of the plant material in Latin, native languages and English whenever applicable.
- The scientific name of the plant concerning the authors and the family to which it belongs.
- Part of the plant used and its condition (such as; fresh aerial parts, dried root, and rhizome, sliced or decorticated).
- Time and method of collection, preliminary preparation, and drying. If the material has been processed, the method of processing (such as steamed, stir-baked, and carbonized) should be indicated.
- A brief description of the distribution and habitat of the plant, growing wild or cultivated (including possible pesticide used). If more than one plant species is concerned, their differences should be indicated. Drawings or photographs of the plants should be provided.
- Characterizing of the plant materials, which may also be the biologically or therapeutically active principle, should be quantified and described with their structural formulae, particularly if they are uncommon. For processed plant material, changes in the quantities of these characterizing compounds should be described.
(ii) Quality specifications:
Authenticity: A description of the macroscopic, microscopic, and sensory characteristics of the plant should be provided including; drawings or photographs if possible. A description should be provided of the physical or chemical tests done to identify the plant substances and a chromatogram of the active fraction or characterizing compound should be provided. If this is not possible, it should be sufficient to identify a characteristic mixture of substances (“fingerprint”) of the plant material.
Purity: Limits of foreign organic matter (such as; stem fragments in the leaves or leaflets, leaf fragments in the flowers, etc.) and foreign mineral matter (such as; sand and soil adhering to the plant material) should be specified and ash determinations should be provided.
Assay: A physical, chemical or biological assay of any known or active fractions should be described and the biological activity of the plant materials expressed in terms of this essay along with an acceptable range for the assay results.
Packaging, labeling, and storage: The conditions for packaging. labeling and storage should all be recorded.
(b) Information for medicinal preparations of plant materials: Among the medicinal preparations; now widely used are powders, granules, pills, extracts, tablets, and injections. Traditional powders and pills are made of powdered plant materials, tablets, granules, ointments, and newer types of pills are mostly made of extracts, injections are made of purified extracts or pure active constituents isolated from the plant material. There are also certain medicinal preparations made of both powdered plant materials and extracts.
(i) Name and formula of the product:
- Name in Latin, English, and native languages.
- Formula including; the name of each ingredient and the quantities used for 1000 gm or 1000 ml of the product. Any excipient used should be specified.
- Method of preparation to make 1000 gm or 1000 ml of the product. The description of the method should include details of any process such as; solvent used, time and temperature of extraction and concentration, as well as the process used to reduce the level of microbial contamination.
- The active constituents should be stated and their structural formulae are given. Any chemical or pharmacological incompatibility should be mentioned.
(ii) Quality specifications:
Authenticity: A description of macroscopic and sensory characteristics should be given and if powdered plant materials are used as ingredients, their microscopic characteristics should be described together with drawings or pictures. Physical or chemical identification tests should be described and thin layer chromatographic procedures for characterizing compounds should be described. A drawing or photograph of the chromatogram should be included. For compound preparations, the most important ingredients including the use of a “finger-print” should be obtained by either thin layer chromatography or high-performance liquid chromatography.
Purity: Limit tests for heavy metals in extracts and the test for freedom from methanol in alcoholic preparations should be specified. Limit tests for contaminants such as; micro-organisms, mycotoxins, and pesticides may be needed.
Assay: The content of biologically or therapeutically active constituents, particularly those which influence the efficacy of the product should be determined and an acceptable range specified. For herbal mixtures, the most characterizing compounds possible should be assayed.
Tests related to the form of the preparation for both non-clinical and clinical tests should follow any available regulatory requirements of the Member States or the WHO guidelines.
Packaging, labeling, and storage: The conditions for packaging, labeling, and storage should all be recorded.
(B) Guidelines for Pharmacodynamics and General Pharmacological Studies of Herbal Medicines: Herbal medicines have various pharmacological effects. The appropriate methods for evaluating the particular herbal medicine tested should be applied. The guidelines present basic concepts and principles which should be of utmost concern.
Species: Appropriate animals may include; mice, rats, guinea pigs, rabbits, cats, dogs, etc. Characteristics of the animals such as; strain, sex, age, and holding conditions should be specified.
Disease model: Disease models can be made by treating animals with certain chemicals or other modalities. E.g.: immunologically depressed mice can be made by treating them with an immunosuppressive agent, such as; cyclophosphamide. Such animals can be used to evaluate the immunostimulating activity of a test medicine.
Animals with genetic defects can also be useful. E.g.: The autoimmune mouse (NZB W/F1, MRL/1) and the hypertensive rat (SHR), etc.
For the study of those herbal medicines which are used under the principles of traditional medicine, animal models may need to be established according to those principles.
Test assays can use:
- Whole animals
- Isolated organs and tissues.
- Blood and its components
- Ex vivo and tissue culture cells
- Subcellular constituents
Special attention should be given to the sensitivity, reproducibility and general acceptance of the test animals or test systems selected.
An examination of the literature may help to select the species and test systems considered to be most predictive of clinical results and therefore provide the most useful information.
Route of administration: Since oral dosage forms of herbal medicines are usually used clinically, the oral route of administration is ordinarily the most suitable for use with test animals. Additional routes may be used to approximate the intended route of administration in man.
Frequency of administration: Usually, doses selected for a study should be established through a dose-response relationship but since such relationships often cannot be demonstrated with herbal medicines in whole animals, it may be sufficient to select one or more doses that provide the desired effect. The selection of doses for animal studies should be by regular clinical doses.
Control group: All studies must include; a negative (vehicle only) control group of animals and if possible, a positive control group, that is a group of animals in which the effect of a drug known to be positive is examined.
(C) Guidelines for toxicity investigation of herbal medicines: These guidelines are intended to indicate the standard methods of non-clinical toxicological studies related to evaluating the safety of herbal medicines.
(a) Acute toxicity test:
Animal species: At least two species can be used, one of them to be selected from rodents and the other from non-rodents.
Sex: In at least one of the species, males and females should be used.
Number of animals: In the case of rodents, each group should consist of at least five animals per sex. In the case of non-rodents, each group should consist of at least two animals per sex.
Route of administration: Ordinarily, the oral route is sufficient as this is the normal route of clinical administration. However, some regulatory agencies suggest in addition a parenteral route of administration.
In cases where it is proposed to administer the herbal preparation to a human subject by the parenteral route, it may be sufficient to use this route alone for animal testing.
Dose levels: A sufficient number of dose levels should be used in rodents to determine the approximate lethal dose in non-rodents, sufficient dose levels should be used for the observation of overt toxic signs.
Frequency of administration: The test substance should be administered in one or more doses during 24 hours.
Observation: Toxic signs and the severity, onset, progression, and reversibility of the signs should be observed and recorded about dose and time. As a general rule, the animals should be observed for at least seven to fourteen days.
Animals dying during the observation period, as well as rodents surviving to the end of the observation period, should be autopsied.
If necessary, a histopathological examination should be conducted on any organ or tissue showing macroscopic changes at autopsy.
(b) Long-term toxicity test:
Animal species: Many regulatory agencies require that at least two species be used, one a rodent and the other a non-rodent.
Sex: Normally, the same number of male and female animals should be used.
Number of animals: In the case of rodents, each group should consist of at least ten males and ten females. In the case of non-rodents, each group should consist of at least three males and three females.
Route of administration: Normally, the expected clinical route of administration should be used.
Administration period: The period of administration of the test substance to animals will depend on the expected period of clinical use. The period of administration of the toxicity study may vary from country to country, according to its regulations.
The following table reflects the commonly-used ranges of administration periods:
Table.1: Ranges of Administration Periods
|The Expected Period of Clinical Use
|Administration Period for the Toxicity Study
|Single administration or repeated administration for less than one week
|2 weeks to 1 month
|Repeated administration, between one week to four weeks
|4 weeks to 3 months
|Repeated administration, between one to six months
|3 to 6 months
|Long-term repeated administration for more than six months
|9 to 12 months
As a rule, the test substance should be administered seven days a week. Administration periods for the toxicity study must be recorded in each result.
Dose levels: Groups receiving at least three different dose levels should be used.
One dose level should not cause toxic changes (no-effect dose) and one dose level that produces overt toxic effects should be included. Within this range, the addition of at least one more dose may enhance the possibility of observing a dose-response relationship for toxic manifestations. All studies should include a vehicle control group of test animals.
Observations and examinations: Observations and examinations should be performed on the following items (from i to vi):
(i) General signs, body weight, and food and water intake: For all experimental animals, the general signs should be observed daily and body weight and food intake should be measured periodically. If useful, water intake should also be determined. The frequency of measurements should normally be as follows:
- Bodyweight: Before the start of drug administration, at least once a week for the first three months of the administration, and at least once every four weeks thereafter.
- Food intake: Before the start of drug administration, at least once a week for the first three months of the administration and at least once every four weeks thereafter. If the test substance is administered mixed in the food, the intake should be measured once a week.
(ii) Haematological examination: For rodents, blood samples should be taken before the autopsy. For non-rodents, blood samples should be taken before the start of drug administration, at least once during the administration period (for studies of longer than one month), and before autopsy. For both hematological and blood chemistry examinations, it is desirable to include as many parameters as possible.
(iii) Renal and hepatic function tests: Since the liver and kidneys are the usual organs of metabolism and excretion, they are easily affected by potentially toxic agents their functions should be monitored in long-term toxicity studies.
For rodents, a fixed number of animals from each group should be selected and urinalysis should be performed before the start of drug administration and at least once during the administration period.
(iv) Other function tests: ECG and visual, auditory tests should be performed. For rodents, the ophthalmological examination should be performed on a fixed number of animals from each group at least once during the administration period. For non-rodents, the examination should be performed on all animals before the start of drug administration and at least once during the period of administration.
(v) All moribund animals should be sacrificed rather than allowed to die. Before sacrifice, clinical observations should be recorded and blood samples collected for hematological and blood chemical analysis. At autopsy, a macroscopic examination of organs and tissues and measurement of organ weights should be recorded. A full histopathological examination should be performed in an attempt to characterize the nature (severity or degree) of all toxic changes.
(vi) Animals found dead during the examination should be autopsied as soon as possible. A macroscopic examination should be made of organs and tissues. In addition, where possible, organ weight measurements and histopathological examinations should be performed in an attempt to identify the cause of death and the nature (severity or degree) of the toxic changes present.
All survivors should be autopsied at the end of the administration period or of the recovery period after taking blood samples for hematological (including, blood chemistry) examinations. Organs and tissues should be examined macroscopically and organ weights should be measured. Histopathological examination of the organs and tissues of animals receiving lower dosage should also be performed, if changes are found on gross or microscopic examination of their organs and tissues of these animals, or if the highest dose group reveals significant changes. On the other hand, histopathological examination of all rodents will further improve the chances of detecting toxicity.
Recovery from toxicity: To investigate the recovery from toxic changes, animals that are allowed to live for varying lengths of time after cessation of the period of administration of the test substance, should be examined.
(c) Local Toxicity test:
Skin sensitization test: Dermatological preparations to be tested.
- Solid preparations: To be prepared by wetting the preparation with water or a suitable solvent to provide a uniform application.
- Semi-solid preparations: To be tested as undiluted preparations.
- Liquid preparations: To be tested as undiluted preparations. However, an aerosol agent can be diluted if necessary.
Experimental animals: Use a species with high susceptibility Guinea pigs are considered the most suitable experimental animals.
Test methods (in alphabetical order):
- Adjuvant and patch test
- Buehler test
- Draize test
- Freund’s complete adjuvant test
- Maximization test
- Open epicutaneous test
- Optimization test
- Split adjuvant test
Evaluation of test results: The skin reaction of each animal should be evaluated according to the assessment standard of the particular test method used.
Other local toxicity tests: Other local toxicity tests may be conducted if the herbal medicine is intended for such use i.e. vaginal, rectal, respiratory, etc. irritations tests.
(d) Special toxicity tests:
- Mutagenic test: Test methods include:
(1) Reverse Mutation Test in Bacteria:
Strains: Salmonella typhimurium TA 1535, TA 1537, TA 98, and TA 100, and Escherichia coli WP2 over A, are the most commonly used bacteria.
Dose levels: At least five dose levels should be employed.
Control groups: A solvent group should normally serve as a negative control. Authentic mutagens that require 59 (9000 gm supernatant) mixture, as well as those which do not require 59 mixture, should be employed as positive control groups.
Metabolic activation: Tests in the presence of 59 mixtures should also be performed.
Test methods: Either a preincubation method or a plate incorporation method should be used.
Presentation of results: The actual number and mean value of revertants should be presented in tables.
(2) Chromosomal Aberration Test with Mammalian Cells in Culture:
Cells: Primary or established cell lines of mammalian cells in culture should be used.
Dose levels: At least three dose levels should be employed.
Control groups: Solvent group should serve as a negative control. A substance is known to cause chromosomal aberrations should be employed as a positive control.
Metabolic activation: Tests should also be performed with a suitable method of metabolic activation (such as 59 mix).
- Chromosomal preparations should be made at an appropriate time after treatment.
- At least two plates should be used for each dose level. The examination should be made for chromosomal structural aberrations and polyploid cells on 100 metaphase cells per plate.
Presentation of results: The relative frequency of cells with chromosomal aberrations and the frequency of chromosomal aberrations per cell should be presented in tables.
(3) Micronucleus Test with Rodents:
Animals: Male mice should normally be used.
Number of animals: Each group should consist of at least five animals.
Route of administration: Administration should be intraperitoneal or via the expected clinical route.
Dose levels: At least three dose groups should be employed.
Control groups: As a general rule, a solvent group should serve as a negative control. A positive control group should receive a substance known to induce micronuclei.
Frequency of administration: Single or repeated administration may be employed.
- Animals should be sacrificed at an appropriate time after administration of the test substance and bone marrow smears prepared.
- Normally, observation should be made of the incidence of micronuclei in 1000 polychromatic erythrocytes per animal. The relative frequency of polychromatic. erythrocytes and total erythrocytes should also be calculated.
Presentation of results: The incidence of polychromatic erythrocytes with micro-nuclei and the frequency of polychromatic erythrocytes per total erythrocytes should be presented in tables.
Carcinogenic test: Experimental animals:
- Species and strains of the animals should be selected in consideration of such factors as; resistance against infectious disease, life span, spontaneous tumor incidence, and sensitivity to known carcinogens.
- Animals of the same species and strain should be used for preliminary and full-scale carcinogenicity studies with the same test substance.
(i) Preliminary carcinogenicity study:
This study is performed to set the dose levels for the full-scale carcinogenicity study. However, if sufficiently reliable data are available, some or all of the following studies may be omitted.
- Single-dose toxicity studies: These studies are performed on a small number of animals to determine the highest dose to be used in the following repeated dose studies.
- Repeated dose toxicity studies: These studies are performed to determine the highest dose to be used in the full-scale carcinogenicity study.
- Animals: At least two species of animals of both sexes should be used. It is desirable to initiate studies with normal animals of the same age, but not more than six weeks in rodents.
- Number of animals: Each group should contain about ten males and ten females.
- Route of administration: The same route of administration should be used for the full-scale carcinogenicity study.
- Dose levels: At least three dose groups and a control group should be established for each sex.
- Administration period: The administration period should be 90 days with the dose usually administered seven days a week. However, if the test substance has delayed toxicity or a cumulative effect, the administration for a longer period may be necessary.
- For all animals in each group, the general signs should be observed daily and body. weight measured at least once a week.
- Autopsy and gross observations on organs and tissues should be performed on dead animals on each occasion and on surviving animals at the end of the administration period. Organs and tissues with gross changes should be examined histopathologically.
- Results: The dose in the preliminary carcinogenicity study that inhibits body weight gain by less than 10% in comparison with the control and causes neither death due to toxic effects nor remarkable changes in the general signs and laboratory examination findings of the animals. This is the highest dose to be used in the full-scale carcinogenicity study.
- The highest dose should be set for each species and sex,
(ii) Full-scale carcinogenicity study:
- Animals: At least two species of animals of both sexes should be employed. It is desirable to use animals with normal growth of the same age, up to the age of so weeks.
- Number of animals: Each group should comprise at least 50 males and 50 females Allocation of the animals to each group should be made with the proper random sampling method based on body weight, etc.
- Route of administration: The expected route of clinical application should be used if possible.
- Dose levels: At least three dose groups and a control group should be employed for each sex.
- A negative control group should be included.
- If various vehicles or emulsifiers are required to administer the test substance, the negative control group should receive such vehicles or emulsifiers alone. It is also desirable to establish an untreated control group.
- Administration period: The administration period should last from 24 to 30 months for rats and from 18 to 24 months for mice and hamsters, with administration normally performed seven days a week.
- Experimental period: Studies should be terminated from one to three months after the administration of the test substance has been terminated. However, the maximum experimental period should be 30 months for rats and 24 months for mice and hamsters. When cumulative mortality reaches 75% in either the lowest dose group or in the control group of either sex, the survivors of that sex should be sacrificed and the study terminated.
- All animals of each group should be observed daily for general signs and body weight should be measured at least once a week during the first three months of administration of the test substance and at least once every four weeks thereafter.
- Animals that died during the experimental period should be autopsied immediately and macroscopic and histopathological examinations should be made of organs and tissues.
- Animals that appear to be moribund during the experimental period should be isolated or sacrificed and autopsied immediately and organs and tissues should be examined macroscopically and histopathologically. At the time of sacrifice, blood samples should be taken to measure red and white blood cells as well as to prepare smear specimens. The smear specimens should be examined in cases suggestive of blood disorders such as anemia or pathology of lymph nodes, liver, or spleen.
- At the end of the study, the survivors should be autopsied immediately, the organs and tissues of all animals in each group should be examined macroscopically. The histopathological examination should be performed on animals in the highest dose group and the control group.
Evaluation of results: A test substance is considered to be positive for carcinogenicity when any of the following types of response has been observed in the carcinogenicity study:
- Development of tumors of a type not seen in the control group.
- Development of tumors was seen with greater frequency in the test group, compared with the control group.
- Greater varieties of organs and tissues are involved in tumor development in the test group, compared with the control group.
- Earlier development of tumors in the test group, though in the absence of any significant difference in the incidence of tumors between the test group and the control group.
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