Research Guidelines for Evaluating the Safety and Efficacy of Herbal Medicines
(1993; 94 pages)
Table of Contents
View the documentFOREWORD
Open this folder and view contents1. INTRODUCTION
Open this folder and view contents2. GENERAL CONSIDERATIONS IN HERBAL MEDICINE RESEARCH
Open this folder and view contents3. RESEARCH STUDIES
Close this folder4. USING THE GUIDELINES
Open this folder and view contentsA. GUIDELINES FOR QUALITY SPECIFICATIONS OF PLANT MATERIALS AND PREPARATIONS
Open this folder and view contentsB. GUIDELINES FOR PHARMACODYNAMIC AND GENERAL PHARMACOLOGICAL STUDIES OF HERBAL MEDICINES
Close this folderC. GUIDELINES FOR TOXICITY INVESTIGATION OF HERBAL MEDICINES
View the documentACUTE TOXICITY TEST
View the documentLONG-TERM TOXICITY TEST
View the documentLOCAL TOXICITY TEST
View the documentSPECIAL TOXICITY TESTS
Open this folder and view contentsANNEXES
View the documentNOTES
View the documentBIBLIOGRAPHY
View the documentSELECTED WHO PUBLICATIONS OF RELATED INTEREST
 

SPECIAL TOXICITY TESTS

Mutagenicity test

Test methods

I. Reverse mutation test in bacteria

1. Strains:

Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli WP2 uvr A, are the most commonly used bacteria.*


* See Notes.


2. Dose levels:

At least five dose levels should be employed.



3. Control groups:

A solvent group should normally serve as a negative control. Authentic mutagens which require S9 (9000 g supernatant) mixture, as well as those which do not require S9 mixture, should be employed as positive control groups.



4. Metabolic activation:

Tests in the presence of S9 mixture should also be performed.



5. Test methods:

Either a preincubation method or a plate incorporation method should be used.



6. Presentation of results:

The actual number and mean value of revertants should be presented in tables.

II. Chromosomal aberration test with mammalian cells in culture

1. Cells:

Primary or established cell lines of mammalian cells in culture should be used.



2. Dose levels:

At least three dose levels should be employed.



3. Control groups:

Normally a solvent group should serve as a negative control. A substance known to cause chromosomal aberrations should be employed as a positive control.



4. Metabolic activation:

Tests should also be performed with a suitable method of metabolic activation (such as, S9 mix)



5. Experimental procedure:

a. Chromosomal preparations should be made at an appropriate time after treatment.

b. At least two plates should be used for each dose level. Examination should be made for chromosomal structural aberrations and polyploid cells on 100 metaphase cells per plate.



6. Presentation of results:

The relative frequency of cells with chromosomal aberrations and the frequency of chromosomal aberrations per cell should be presented in tables.

III. Micronucleus test with rodents

1. Animals:

Male mice should normally be used.



2. Number of animals:

Each group should consist of at least five animals.



3. Route of administration:

Administration should be intraperitoneal or via the expected clinical route.



4. Dose levels.

At least three dose groups should be employed.



5. 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.



6. Frequency of administration:

Single or repeated administration may be employed.



7. Experimental procedure:

a. Animals should be sacrificed at an appropriate time after administration of the test substance, and bone marrow smears prepared.

b. 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.



8. 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.

Carcinogenicity test

Experimental animals

1. Species and strains of the animals should be selected in consideration of such factors as resistance against infectious disease, life span, spontaneous tumour incidence, and sensitivity to known carcinogens.

2. Animals of the same species and strain should be used for preliminary and full-scale carcinogenicity studies with the same test substance.

Experimental method

1. 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.

(1) Single dose toxicity studies

These studies are performed on a small number of animals in order to determine the highest dose to be used in the following repeated dose studies.



(2) Repeated dose toxicity studies

These studies are performed in order to determine the highest dose to be used in the full-scale carcinogenicity study



a. 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 no more than six weeks in roder(...).



b. Number of animals:

Each group should contain about ten males and ten females.



c. Route of administration:

The same route of administration should be used as for the full-scale carcinogenicity study.



d. Dose levels:

At least three dose groups and a control group should be established for each sex.



e. 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, administration for a longer period may be necessary.



f. Experimental procedure:

1. For all animals in each group, the general signs should be observed daily and body weight measured at least once a week.

2. 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.



g. Results:

1. 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.

2. It is desirable that the highest dose should be set for each species and sex.

2. Full-scale carcinogenicity study

a. 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 six weeks.



b. 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.



c. Route of administration:

The expected route of clinical application should be used, if possible.



d. Dose levels:

At least three dose groups and a control group should be employed for each sex.



e. Control group:

i. A negative control group should be included.

ii. 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.



f. 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.



g. 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.



h. Experimental procedure:

i. 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 ad ministration of the test substance and at least once every four weeks thereafter.

ii. Animals that died during the experimental period should be autopsied immediately and macroscopic and histopathological examinations should be made of organs and tissues.

iii. 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 anaemia or pathology of lymph nodes, liver or spleen.

iv. 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. Histopathological examination should be performed on all animals in the highest dose group and the control group. If the incidence of neoplastic lesions between organs and tissues of the highest dose group and the control group are found to differ, the relevant organs and tissues of all animals in other dose groups should be examined histopathologically and blood examined as in (iii) above.

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:

1. Development of tumours of a type not seen in the control group.

2. Development of tumours seen with greater frequency in the test group, compared with the control group.

3. Greater varieties of organs and tissues are involved in tumour development in the test group, compared with the control group.

4. Earlier development of tumours in the test group, though in the absence of any significant difference in the incidence of tumours between the test group and the control group.

Reproductive and development toxicity test

Experimental animals

1. Species and strains should be selected in consideration of reproductive and developmental information such as fertility, incidence of spontaneous malformation, and susceptibility to substances known to affect reproduction and development.

2. It is desirable to select species and strains with a low incidence of spontaneous malformations.

3. It is desirable that animals used in studies referred to as Segment I, II and III studies be of the same strain and species.

Experimental methods

1. Segment I. Study on administration of the test substance prior to and in the early stages of pregnancy.

a. Animals:

At least one species of animal of both sexes such as rats or mice should be used.



b. Number of animals:

In the case of rats or mice, each group should consist of at least 20 males and 20 females.



c. Route of administration:

The route of administration ordinarily will be the expected clinical route of administration.



d. Dose levels:

Groups with three different doses plus a control group should be employed.



e. Control group:

i. A negative control group should be employed. A positive or a comparative control group is desirable.

ii. When vehicles or emulsifiers are required for the administration of the test substance, a negative control group should normally receive such vehicles or emulsifiers alone. A positive control group should receive a substance known to have potent reproductive and developmental toxicity, and a comparative control group should receive a drug with a similar chemical structure or pharmacological effects as the tested drug.



f. Administration period:

When rats or mice are used, males at least 40 days of age should be dosed daily for 60 days or more before mating, and administration should be continued until successful copulation. Sexually mature females should be dosed daily for at least 14 days before mating, during mating and after successful copulation until the beginning of organogenesis.



g. Experimental procedure:

i. During the experimental period, mortality should be recorded, general signs noted and body weights and food intake should be measured.

ii. A treated male and a treated female should be housed together and observed daily for confirmation of successful copulation.

iii. The mating period between the male and female pairs should be about two weeks. If necessary, a treated male and a non-treated female, or a treated female and a non-treated male should be housed together and observed daily for confirmation of successful copulation.

iv. After successful copulation, females should be autopsied at term, and examined for the number of corpora lutea, successful pregnancies and mortality of fetuses. Additionally, a gross examination of the organs and tissues for all dams should be made.

v. Males used for mating and females without successful copulation should be autopsied at an appropriate time, and gross observation on organs and tissues should be made.

2. Segment II. Study on administration of the test substance during the period of organogenesis.

a. Animals:

Females of at least one species of rodent and a non-rodent such as rabbits should be used.



b. Number of animals:

Each group should consist of at least 30 animals for rats or mice and at least 12 animals for rabbits.



c. Route of administration:

The route of administration should ordinarily be that expected clinically.



d. Dose levels:

At least three different dosage groups plus a control group should be employed.



e. Control group:

i. A negative control group is necessary and a positive or a comparative control group is generally desirable.

ii. When vehicles or emulsifiers are required for the administration of the test substance, a negative control group should normally receive such emulsifiers alone. A positive control group should receive a substance known to have potent reproductive and developmental toxicity and a comparative control group should receive a drug with a similar chemical structure or pharmacological effects.



f. Experimental procedure

i. During the experimental period, mortality, general signs, body weights and food intake should be measured for all dams.

ii. In the case of rodents such as rats or mice, approximately 2/3 of the dams in each group, and in the case of non-rodents such as rabbits, all the dams in each group should be autopsied at term. They should be examined for successful pregnancy and mortality of fetuses. Body weight measurement and morphological examinations should be made on live fetuses. Gross observations on organs and tissues should be made for dams.

iii. For rats or mice, etc., the remaining approximately 1/3 of the dams should be allowed to deliver their offspring. Dams should be examined for abnormality on delivery.

iv. Litter size, mortality, sex and external changes of neonates should be examined, and body weights should be measured.

v. Offspring should be examined for growth and development, appearance of specific signs, reproductive performance, etc. Growth and development should be recorded and morphological, functional and behavioural examinations should be made. Reproductive performance of the offspring, that is, the ability to establish pregnancy, should be examined. If necessary, observation for a longer period should be made.

vi. At an appropriate time, autopsy and gross observation of the organs and tissues of treated dams should be made on treated dams. If necessary, an examination of the second litters should be done.

3. Segment III. Study on administration of the test substance during the perinatal and lactation periods

a. Animals:

At least one species of female animals such as rats or mice should be used. Species should be selected from among those used in the study of administration of the test substance during organogenesis specified in the segment II study.



b. Number of animals:

Each group should consist of at least 20 animals for rats or mice.



c. Route of administration:

The route of administration should be the expected clinical route as a rule.



d. Dose levels:

At least three dose groups plus a control group should be employed.



e. Control group:

i. A negative group should be employed. A positive or a comparative control group may be employed, if necessary.

ii. When vehicles or emulsifiers are required for administration of the test substance, a negative control group should normally receive such vehicles or emulsifiers alone. A positive control group should receive a substance known to have potent reproductive and developmental toxicity and a comparative control group should receive a drug with a similar chemical structure or pharmacological effects.



f. Administration period:

i. During the experimental period, all the dams in each group should be examined for mortality and general signs and body weights and food intake should be measured.

ii. All the dams in each group should be allowed to deliver and nurse their offspring. Dams should be examined for abnormality on delivery.

iii. Litter size, mortality, sex and external changes of neonates should be examined, and body weights should be measured.

iv. Offspring should be examined for growth and development, appearance of specific signs, reproductive performance, etc. For observation of growth and development, morphological, functional and behavioural examinations should be made. Reproductive performance of offspring should be examined on the basis of establishment of pregnancy. If necessary, observation for a longer period should be made.

v. At an appropriate time, autopsy and gross observations on organs and tissues should be made on treated dams. If necessary, an examination of the second litters should be done.

Analysis of results

1. The results obtained should be presented in the form of tables and figures with discussion of the results. For presentation, summary tables which give an overview of the results of all groups should be prepared. In addition, appendix tables which provide data for individual animals in each group should be prepared for reference.

2. For statistical analysis of the data obtained before weaning, it is desirable that the litter, instead of the individual fetus or offspring, serve as the unit for analysis.

3. The discussion should address the no-effect dose level of the test substance concerned with the reproduction of the parent animals and development of the next generation. It is desirable to compare the reproductive and developmental toxicity with that of similar drugs.

 

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