10.1 OBJECTIVE The objective of the present investigation was to perform the preliminary work to evaluate the acute oral toxicity of the developed herbal liquid oral formulations. 1. Scoparia dulcis Linn. herbal formulation 2. Achyranthes aspera Linn. herbal formulation 3. Poly herbal formulation 10.2 INTRODUCTION The herbal medicine developed consisting of herbal extracts as well as modern pharmaceutical excipients, should posses green signal for their safety. Hence preliminary work was performed for their safety assessment of acute oral toxicity. Herbal medicine is popular among the rural and urban community in India. The reason for the popularity and acceptability being the belief, that all natural products are safe. Despite the popular use of herbal preparations for livestock disorders, there is limited scientific data available regarding safety aspects of these remedies (Aniagu et al, 2005). Published literature indicates the possible adverse effects and drug-herb interactions on use of herbal remedies (Brinker, 2000). With regard to the safety evaluation of herbal medicines, the regulatory requirements in different countries seem not to be uniform. Several countries have developed their own national regulations for traditional medicines (World Health Organization, 199). Assessment of acute oral toxicity of a test substance forms the primary step in the characterization of its toxic potential. Different test methods have been devised by the Organization for Economic Co-operation and Development (OECD) for acute toxicity evaluation (Organization for Economic Co-Operation and Development, 2001a; 2001b; 2001c). Taking into account this background, the present study was aimed at evaluation of safety of some herbal preparations on acute oral exposure as per OECD guideline 20-Fixed dose procedure that utilizes non-lethal endpoints to determine the toxicity of the test material. 10.3 LITERATURE REVIEW Acute toxicity is usually defined as the adverse changes occurring immediately or a short time following a single or short period of exposure to a substance or substances or as adverse effects occurring within a short time administration of a single dose of a 26
substance or multiple doses given within 2 hr. An adverse effect is "any effect that results in functional impairment and/or biochemical lesions that may affect the performance of the whole organism or that reduce the organ's ability to respond to an additional challenge (Rhodes et al, 1993). Several cases of adverse effects of herbal drugs have been reported in developed countries during the last few years, which are allegedly caused by taking herbal products or traditional medicines prescribed by the practitioners of indigenous systems of medicine. These products may be contaminated with excessive banned pesticides, microbial contaminants, heavy metals and chemical toxins which cause various deformities like congenital paralysis, sensori-neural defects, liver and kidney damage etc. These contaminants may be related to the source of herbal drugs, if these are grown under contaminated environment. Chemical toxins may come from unfavorable post harvest techniques/ wrong storage conditions or chemical treatment during the storage period etc. Some of these environmental factors may be controlled by implementing good source; good agricultural practices and standard operating procedures (SOP) for producing good quality herbal products (Vartika Rai and Shanta Mehrotra, 2005). Toxicology is the fundamental science of poisons. A poison is generally considered to be any substance that can cause severe injury or death as a result of physicochemical interaction with living tissues (John et al, 1996). Toxicology, like medicine, is both science and an art. The science of toxicology is defined as observational and data gathering phase, whereas the art of toxicology consists of utilization of data to predict the outcome of exposure in human and animal population (Michael, 1996). Many scientists have advocated changes in the emphasis of acute toxicity testing. To date there is a general consensus among toxicologists in academia, industry, and government that a change in the emphasis of acute toxicity testing is needed. The value of a precise LD 50, except for highly toxic substances, is being deemphasized and the focus is now on obtaining as much information as possible on toxic manifestations and mechanism of toxicity with least number of experimental animals. Undoubtedly, such information will be more useful than the LD 50 to physicians in treating overexposure. Even then, the emphasis of dose - response and 27
development of signs of toxicity remain the basis of the science of toxicology (Lorke, 193; Muller, 192; Schutz, 192). Lethal dose 50 is defined as the statistically derived dose that, when administered in an acute toxicity test, is expected to cause death in 50% of the treated animals in a given period (Oliver, 196). At present the following chemical labeling and classification of acute systemic toxicity based on oral LD 50 values are recommended by the Organization for Economic Co-operation and Development (OECD): very toxic, < 5 mg/kg body weight; toxic, > 5 < 50 mg/kg; harmful, >50<500 mg/kg; and no label, > 500 < 2000 mg/kg. The absolute LD 50 value for a compound varies among different laboratories, and these variations have been attributed to differences in e.g., protocol details, animal strains, caging, and test-chemical source.ld 50 and alternative methods for testing of acute toxicity have been discussed extensively in different international forums during the last two decades (Oliver, 196; Clark, 1991; Fielder, 1995; Lindgren, 193; OECD 197, 1992, 1996). Toxicity is defined as the harmful effect of a chemical or a drug on a living organism (Chan, 199). The Organization for Economic Cooperation and Development (OECD) defines acute toxicity as "the adverse effects occurring within a short time of (oral) administration of a single dose of a substance or multiple doses given within 2 hours." All chemicals can produce toxicity under some experimental conditions, for instance, if a sufficiently large dose is administered. It is therefore, misleading to conduct acute toxicity studies at unreasonably high dose levels for the sake of demonstrating lethality and / or toxicity, which may be irrelevant to the use of compound itself. An extremely high dose of a practically nontoxic compound for example, can cause gastrointestinal blockage, which in turn can result in gastrointestinal tract dysfunction. Toxicity in such a case is not related to the intrinsic characteristic of the test substance, since effect manifested is a direct result of the physical blockage caused by the biologically inert substance. There must be a point, however, at which an investigator may conclude that a test substance is practically 2
nontoxic or nonlethal after an acute exposure. This test limit for oral toxicity generally is considered to be 05.0 g / kg body weight. If no mortality is observed at this dose level, a higher dose level generally is not necessary (Chan, 199). Areas of toxicology The professional activities of toxicologists fall into three major categories: descriptive, mechanistic and regulatory. A descriptive toxicologist is concerned directly with toxicity testing, which provides information for safety evaluation and regulatory requirements. A mechanistic toxicologist is concerned with identifying and understanding the mechanisms by which chemicals exert toxic effect on living organisms (John et al 1996; Michael, et al 1996). There are several specialized areas of toxicology like forensic, clinical and environmental toxicology (David, 1996). Forensic toxicology is a hybrid of analytical chemistry and fundamental toxicological principles. It is concerned primarily with the medico legal aspects of the harmful effects of chemicals on humans and animals. The expertise of forensic toxicologists is invoked primarily to aid in establishing the cause of death and in determining its circumstances in a postmortem investigation. Toxic dose: One could define a poison as any agent capable of producing a deleterious response in a biological system, seriously injuring function or producing death. This is however not a useful working definition that (Michael et al, 1996). A chemical agent does not produce toxic effects in biological system unless that agent or its metabolic breakdown (biotransformation) products reach appropriate sites in the body at a concentration and for a length of time sufficient to produce a toxic manifestation(david, 1996). Limit test: The limit test, described in OECD guideline 01, is a restricted version of an acute toxicity test and is used after a range finding study or a literature search has made it likely that the chemical is of low toxicity. Several measures have been taken to reduce the number of animals needed for a complete study. The limit test ideally involves the use of three groups (three dose levels; the highest dose limit is set to 2 g/kg and is the initial starting dose) of five animals of a single sex, with assessment of toxicity in the second sex in a separate study. In this second study five animals are 29
used at a single dose level to establish the absence of any significant sex difference in toxicity (OECD, 1992). 10. MATERIALS, METHODS USED IN THE STUDY: Scoparia dulcis Linn. herbal formulation, Achyranthes aspera herbal formulation & Poly herbal formulation, Test animals (Albino Swiss mice). Acute toxicity study on Scoparia dulcis Linn. formulation. An acute toxicity study was carried out on Scoparia dulcis Linn. formulation using mice as the experimental model. The study was carried out to assess the acute toxicity of the herbal formulation on oral administration. Study protocol is given below. Acute toxicity study on Scoparia dulcis Linn. formulation STUDY PROTOCOL Name of the study - Acute oral toxicity study Test material - 1) Scoparia dulcis Linn.herbal formulation 2) Achyranthes aspera Linn.herbal formulation. 3) Poly herbal formulation Animal model - Albino Swiss Mice Animals procured from - Haffkine Biopharmaceutical Ltd., Parel, Mumbai Sex - Male and Female Weight of animals - Between 35 to 55 g No. of dose groups - Three groups Animals per group - 3 male and 3 female Route of administration - Intragastric administration with help of gavage No. Dose volume - 0.2, 0., 0. ml per animal Total volume - Not more than 2ml with distilled water. Vehicle for administration - Distilled water. 250
No. of administrations - Single Concentration of dose -, and ml/kg body weight Study duration - Acclimatization for 1 days, one day drug administration and 1 days observation period. Parameters observed - Cage side observations, daily food and water intake, daily body weight and mortality record. Animal Maintenance - The animals were housed in polyurethane cages. The cages were provided with rice husk bedding and were cleaned daily. The animals were provided with drinking water and libitum and were fed on commercially available Mice feed supplied by AMRUT FEED. The specifications of the feed are listed below. Crude Protein Ether Extractive Crude Fiber Ash 20-21 % minimum 0-05 % minimum 0 % maximum 0 % maximum Calcium 1.2 % Phosphorus 0.6 % minimum NFE 5 % ME Kcal / Kg 3600 Pellet Size 12 mm The feed was enriched with stabilized vitamins such as Vit. A and D 3, Vit. B 12, Thiamine, Riboflavin, Folic acid and supplemented with all minerals and microelements. Measured quantities of water and feed were supplied daily in each cage. The consumption of water and food was estimated from the amount of water remaining in feeding bottles and from the amount of feed remaining in the feed hopper. 251
10.5 RESULTS Observations: Mortality: The treated animals were observed for mortality twice daily till the completion of the study period. Mortality is the main criteria in assessing the acute toxicity (LD 50 ) of any drug. There was no mortality recorded even at the highest dose level i.e. ml / kg body weight. Clinical signs: Observations of clinical signs were made at 10 min, 1, 2, and 6 h after dosing and once daily for 1 days at approximately same time. Assessment of the behavior of animals was carried out by general observations of each animal on a daily basis from the stage of dosing to the end of the study. Cage-side observations included daily recording of condition of the fur; damaged areas of skin; subcutaneous swellings or lumps (the size, shape and consistency); areas of tenderness; abdominal distension; eyes - for dullness, discharges, opacities, pupil diameter, ptosis (drooping of upper eyelid), the color and consistency of the faeces, wetness or soiling of the perimenum, condition of teeth, breathing abnormalities, gait, etc. Any changes or abnormalities recorded could be an indication of toxicity. The test animals at all dose levels showed no significant changes in behavior before and after the administration of an oral dose of Scoparia dulcis Linn. formulation. Table 10.1 shows the dosage regime. Table 10.2 shows the observations for the parameters studied. Table 10.12 shows the mortality record. Body weight: Body weight is an important factor to monitor the health of an animal. Loss of body weight is frequently the first indicator of the onset of an adverse effect. A dose, which causes 10 % or more reduction in the body weight, is considered to be a toxic dose. It is considered to be the dose, which produces minimum toxic effect, irrespective of whether or not it is accompanied by any other changes. All the animals from treated groups did not show any significant decrease in body weights for all the 1 days as compared with the 0 day values, indicating no signs of toxicity. The variation in body weight changes of male and female and the data is given in Table 10.. Food and water consumption: There was no significant change in food and water intake of the test animals at all dose levels. The data for food and water consumption is given in tables respectively. 252
Gross pathology and histopathology: After the observation study the animals were not sacrificed, complete study was not performed to identify the gross pathological changes. 10.6 CONCLUSION To conclude, acute oral toxicity testing of the mice did not cause deaths, no toxicity signs were observed from the cage side or any significant gross pathological changes upto the dose level of ml / kg body weight were observed. The overall weight gain was found to be normal in treated animal and hence the tested products are labeled unclassified in the hazard category according to Globally Harmonized System and can be considered relatively safe. Table 10.1. Acute toxicity study of Scoparia dulcis Linn. formulation dosage regime. Group Sex No. of Animals used Dose ml/ kg body Weight 1 2 3 Male 3.0 Female 3.0 Male 3.0 Female 3.0 Male 3.0 Female 3.0 253
Table 10.2. Acute toxicity study of Scoparia dulcis Linn. formulation based on cage side observations S No. Parameters Observations 1. Condition of the fur Normal 2. Skin Normal 3. Subcutaneous swellings Nil. Abdominal distension Nil 5. Eyes - dullness Nil 6. Eyes - opacities Nil 7. Pupil diameter Normal. Ptosis Nil 9. Colour and consistency of the faeces Normal 10. Wetness or soiling of the perimenum Nil 11. Condition of teeth Normal 12. Breathing abnormalities Nil 13. Gait Normal Table 10.3. Acute toxicity study of Scoparia dulcis Linn. formulation based on ml/kg body Weight Sex mortality record. Mortality up to 1 days Scoparia Achyranthes Poly herbal dulcis Linn. aspera Linn. formulation formulation formulation Male 0 / 3 0 / 3 0 / 3 Female 0 / 3 0 / 3 0 / 3 Male 0 / 3 0 / 3 0 / 3 Female 0 / 3 0 / 3 0 / 3 Male 0 / 3 0 / 3 0 / 3 Female 0 / 3 0 / 3 0 / 3 25
Table 10.. Acute toxicity study of Scoparia dulcis Linn. formulation based on daily body weight record (in grams) Weight of Mice Days 1 7 1 Male 51 5 55 Female 35 35 37 Male 6 Female 35 3 39 Male 5 56 5 Female 50 53 Table 10.5. Acute toxicity study of Scoparia dulcis Linn. formulation daily food intake record (in grams) Weight of food intake Days 1 7 1 Male 1 19 Female 1 13 12 Male 15 22 17 Female 13 1 Male 17 20 23 Female 11 10 12 255
Table 10.6 Acute toxicity study of Scoparia dulcis Linn.formulation based on daily water intake record (in cm 3 ) Water in take Record Days 1 7 1 Male 20 Female 1 12 1 Male 1 15 19 Female 1 1 1 Male 22 1 2 Female 15 13 12 Table 10.7. Acute toxicity study of Achyranthes aspera Linn. formulation based on daily body weight record (in grams) Weight of Mice Days 1 7 1 Male 59 61 62 Female 3 5 Male 7 50 Female 3 3 39 Male 59 60 63 Female 53 55 55 256
Table 10.. Acute toxicity study of Achyranthes aspera Linn. formulation based on daily food intake record (in grams) Weight of Food intake Days 1 7 1 Male 19 15 19 Female 11 15 Male 20 1 Female 12 Male 1 20 23 Female 15 21 Table 10.9. Acute toxicity study of Achyranthes aspera Linn. formulation based on daily water intake record (in cm 3) Water intake Record Days 1 7 1 Male 1 20 Female 1 12 1 Male 1 19 Female 1 1 Male 19 22 Female 15 15 12 257
Table 10.10. Acute toxicity study of poly herbal formulation based on daily body weight record (in grams) Weight of Mice Days 1 7 1 Male 62 65 66 Female 50 53 Male 51 52 5 Female 7 9 50 Male 61 63 66 Female 56 57 60 Table 10.11. Acute toxicity study of poly herbal formulation based on daily food intake record ( in grams) Weight of Food intake Days 1 7 1 Male 1 15 19 Female 17 1 15 Male 17 19 Female 17 1 1 Male 1 1 17 Female 13 25
Table 10.12. Acute toxicity study of poly herbal formulation based on daily water intake record (in cm 3 ) Water in take Record Days 1 7 1 Male 1 15 20 Female 15 12 1 Male 1 Female 17 1 Male 19 1 Female 1 19 15 10.6 DISCUSSION OF RESULTS IN LIGHT OF OTHERS WORK There are no reports on toxicity studies of Achyranthes aspera Linn. herbal formulation, Scoparia dulcis Linn. herbal formulation and poly herbal formulation. In the present study the acute toxicity (Table 10.3) mortality is found nil, hence the formulations can be considered safe for oral administration. Daily body weight remained unchanged (Table 10.). The daily food and water intake were not considerably reduced or increased by the dosing (Table 10.5 & 10.6), hence the formulations can be considered safe. 10.7 CONCLUSION From the above preliminary studies it can be concluded that the three Herbal formulations developed can be labeled unclassified in the hazard category according to Globally Harmonized System and can be considered relatively safe. 10. FUTURE PROSPECTS After the observation period the animal are to be scarified, and complete histological studies to be performed. The above studies are insufficient, hence complete toxicity studies need to be perform to determine the efficiency of the formulations. 259
10.9 REFERENCES Aniagu SO, Nwinyi FC, Akumka DD, Ajoku G.A and Dzarma S. Afr. J. Biotechnol., 2005; : 72-7. Brinker F.N.D. The Toxicology of Botanical Medicines. 3rd Edn., Eclectic Medical Publications, Sandy, Oregon, ISBN: 13091, 2000;13-1. Chan P.K. and Hayes A.W. Principles and Methods of Toxicology; Third Edition, edited by A. Wallace Hayes. Raven Press, New York, 199; 579-67. Clark DG, Fielder R, Joseph C, Gardner J, Smith M. The future for acute oral toxicity testing. In: Animals and Alternatives in Toxicology (Balls M, Bridges J, Southee J, eds).london:macmillan Academic and Professional Ltd.1991;1-21. David L. Eaton and Curtis D. Klassen, Casarett and Doull s Toxicology - The Basic Science of Poison, Ed. Curtis D. Klassen, International edition, McGrath-Hill Health Professions Division, 5 th edition. 1996;13. European Agency for the Evaluation of Medicinal Products, 197. Single dose toxicity. 3BS1a. EMEA, London, UK. http://www.emea.europa.eu/pdfs/human/swp/3bs1a en.pdf Fielder RJ. The fixed dose procedure as an alternative to the classical LD50 test: acceptance by the EEC and OECD. In: Alternative Methods in Toxicology and the Life Sciences. New York: Mary Ann Liebert., 1995; 11: 269-27. John H. Duffus, Fundamental Toxicology for Chemists, Ed. John H. Duffus and Horward G. J. Worth, Royal Society of Chemistry.,1996; 1-5. Lindgren P, Thelestam M, Lindquist NG, eds. First CFN symposium on LD50 and possible alternatives. Acta Pharmacol Toxicol., 193; 52. Lorke D. Arch. Toxicol., 193; 5: 275-27. Marlene Cimons., Nature Medicine., 2001; 7: 1077. Michael A. Gallo, Casarett and Doull s Toxicology - The Basic Science of Poison, Ed. Curtis D. Klassen, International edition, McGrath-Hill Health Professions Division, 5 th edition. 1996; 3-5. 260
Muller H and Kley H.P. Arch. Toxicol., 192; 51: 19-196. OECD guidelines for testing of chemicals, OECD, Paris. 191. OECD. OECD Guideline for Testing of Chemicals, No 01: Acute Oral Toxicity. Paris:Organisation for Economic Cooperation and Development, 197. OECD. OECD Guideline for Testing of Chemicals, No 20: Acute Oral Toxicity- Fixed Dose Method. Paris:Organisation for Economic Co-operation and Development, 1992. OECD. OECD Guideline for Testing of Chemicals, No 23: Acute Oral Toxicity- Acute Toxic Class Method. Paris: Organisation for Economic Co-operation and Development, 1996. Oliver JA. Opportunities for using fewer animals in acute toxicity studies. In: Chemicals Testing and Animal Welfare. Solna, Sweden:The National Chemicals Inspectorate, 1 19-12:196. Organization for Economic Co-operation and Development, OECD guideline for testing of chemicals: Guideline 20, Acute oral toxicity- Fixed dose procedure, OECD, Paris, 2001a.France. http://iccvam.niehs.nih.gov/suppdocs/feddocs/oecd/oecd_gl20.pdf Organization for Economic Co-operation and Development, OECD guideline for testing of chemicals: Guideline 23, Acute oral toxicity- Acute toxic class method, OECD, Paris, France,2001b.http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/OECD/OECD_GL23. pdf Organization for Economic Co-operation and Development, OECD guideline for testing of chemicals: Guideline 25, Acute oral toxicity- Up-and-Down procedure, OECD, Paris, France, 2001c. http://www.oecd.org/dataoecd/17/51/1937.pdf Rhodes C, Thomas M, Athis J. Principles of testing for acute toxic effects. In: General and Applied Toxicology. Vol 1 (Ballantyne B, Marrs T, Turner P, eds). New York: Stockton Press, 9-7; 1993. Schutz E., and Fuchs H. Arch. Toxicol., 192; 51 : 197-220. 261
Seth P. K, Jaffery F. N and Khanna V. K. Toxicology Ind. J. Pharmacol., 2000; 32: S13 - S151. The truth behind herbal drugs, Express pharma -31 March 2006. Vartika Rai And Shanta Mehrotra., Toxic Contaminants in Herbal Drugs 2005; 11 (). World Health Organization, Regulatory situation of herbal medicines: A worldwide review. WHO/TRM/9.1. 199. http://whqlibdoc.who.int/hq/199/who_trm_9. 1.pdf. 262