INTRODUCTION
Medicinal plants, for several centuries, have been widely used as a primary
source of prevention and control of livestock diseases (Sharma and Singh,
1989). The global trend towards the increased demand and use of herbal
medicine in the animal healthcare sector has resulted primarily due to
the increasing cost of livestock maintenance, stressful transit for seeking
professional veterinary care and the introduction of new technology in
the production of plant-based therapeutics (Hoareau and DaSilva, 1999;
Sikarwar, 1997). In 2004, the global market for natural animal healthcare
products was estimated to be around $ 444 million (Natesh, 2007), while
in India, the total market of veterinary products was approximately Rs.
1500 crores in which about Rs. 225 crores was the share of herbal products
during the same period (DBT, 2004).
Despite their growing popularity as naturally safe with pharmacologically
active principles (Mabeku et al., 2007), only little information
is available regarding the regulations on quality and requirements for
demonstrating efficacy and safety of herbs and herbal veterinary products.
Non-existence of detailed regulations, especially in many developing countries,
has been a matter of concern for the sustainable and appropriate use of
medicinal plants and their products in veterinary health care (ICS-UNIDO,
2007). This necessitates the generation of adequate safety data in approved
model systems proposed by the regulatory agencies (Agaie et al.,
2007; Bulder and Noordam, 2008; Jegede et al., 2006).
In the safety evaluation of a test substance, determination of acute
oral toxicity is generally the initial step and provides information on
health hazards that may arise from an acute exposure by the oral route.
Data from such acute studies can serve as a basis for classification and
labeling of the test substance and establishing the dosage regimen of
repeated dose and other related studies. It may also provide early information
on the mode of toxic action of a substance (Rispin et al., 2002).
The present study was carried out to assess certain popular veterinary
products for their acute toxic potential by fixed dose procedure adopted
by the Organisation for Economic Co-operation and Development (OECD).
This approach avoids using death of animals as an endpoint and relies
instead on the observation of clear signs of toxicity after administration
of test material at a series of fixed dose levels. The method also renders
information on the hazardous properties and allows the substance to be
ranked and classified according to the Globally Harmonised System (GHS).
MATERIALS AND METHODS
Test materials: A total number of five herbal veterinary products
(M/s Natural Remedies Pvt. Ltd., Bangalore, India) were evaluated for
acute oral toxicity study. The products viz. Becknor® for
anti-diarrhoeal, Hygest™ to prevent ketosis, Zigbo®
to optimize liver functions, Involon™ as an uterine tonic and Zigup™
syrup for its growth promoting effects are recommended for their favourable
gastro-intestinal and reproductive effects in livestock and pet animals.
Experimental animals: Female albino Wistar rats of 8 to 12 weeks
age were chosen for the study. The animals were received from Central
Animal Facility, Research and Development Centre, Natural Remedies Pvt.
Ltd., Bangalore, India. They were kept in individual polypropylene cages
provided with clean bedding of rice husk. They were acclimatized for five
days prior to dosing under standard housing conditions (temperature: 25
± 2 °C, relative humidity: between 30 and 70% with optimal
air changes per hour and 12 h each ofdark and light cycle) and provided
with standard pelleted feed and U.V. treated water ad libitum.
Study design: Healthy adult female rats were used in this experiment.
Animals were randomly assigned to the cages and each animal was identified
by cage card number and individual picric acid marking on fur. The females
were nulliparous and non-pregnant. Typically, one animal each at 2000
and 5000 mg kg-1 body weight and four animals at 5000 mg kg-1
body weight were selected, respectively, for the sighting and main studies
for products evaluated by main test, whereas a total of six female rats
at the dose level of 5000 mg kg-1 body weight were assigned
for products tested under limit test. Consideration of limit test for
products was mainly based on certain previous information available about
the test materials. The rats were deprived of feed overnight before and
3 h after the administration of the test substance. Water was not withheld
during this period. Demineralised water was used as vehicle for dosing
of test substance.
The concentration of the test substance was varied so as to maintain
the dose volume constant at 10 or 20 mL kg-1 body weight as
per the nature of the product. The doses were prepared fresh on the day
of dosing.
In-life clinical observations: The treated animals were observed
for mortality (twice daily) and clinical signs were recorded to note the
onset, duration and reversal (if any) of toxic effects at 10, 30 min,
1, 2, 4 and 6 h after the administration of the test substance and once
daily thereafter for 14 days. The routine cage side observations included
changes in skin and fur, eyes and mucous membrane and also respiratory,
autonomic and central nervous systems and somatomotor activity and behaviour
pattern. The behavioural profile studied included alertness, visual placing,
stereotypy, passivity, grooming, vocalization, irritability, spontaneous
activity, reactivity and touch response whereas neurological observations
such as straub response, tremor, convulsions, staggering gait, limb tone,
grip strength, corneal reflex and pinna reflex were taken into consideration.
The criteria for autonomic profile included findings on pupil size, palpebral
opening, exophthalmos, salivation, piloerection and skin colour. Miscellaneous
signs like arching of the back, alopecia, wound, nasal discharge, lacrimation
and loose stool were also recorded during the observations. The clinical
signs were graded by a scoring system wherein scores for normal, abnormal,
subnormal and supernormal responses were assigned as 4, 0, <4 and >4,
respectively and the maximum score for any response was assigned as 8.
Body weight: Body weight data of individual animals were recorded
following the period of fasting on the day of dosing, weekly thereafter
and at termination on day 15. Weekly changes in body weight gain were
calculated and recorded.
Gross pathology and histopathology: All the rats in the study,
dying during the observation period, sacrificed moribund for humane reasons
or sacrificed terminally were subjected to a complete necropsy and the
gross pathological changes, if any, were recorded. Histopathology examination
of organs and tissues was considered in case of evidence of any gross
pathology findings.
RESULTS
Reported here are the results of acute toxicity studies on selected herbal
veterinary formulations. The reference data on scores for exhaustive profile
of clinical signs and mean body weight of individual animals in the main
study are not presented here for brevity of the article.
Becknor®: Animals treated at the dose level of
5000 mg kg-1 body weight in the sighting study and main study
survived throughout the study period and did not show any major abnormal
clinical signs following dosing and during the observation period of 14
days, post treatment. Body weight and percent body weight gain in dosed
rats after 7 and 14 days of treatment was found to be increased during
the sighting study (Table 1) and main study (Table
2). Macroscopic examination of animals sacrificed at termination revealed
no abnormalities.
| Table 1: |
Effect of herbal products on body weight and percent
body weight gain in rats (Sighting study) |
 |
| Table 2: |
Effect of herbal products on mean body weight and percent
body weight gain in rats (Main study) |
 |
Hygest™: Neither mortality nor any abnormal clinical
signs were observed in rats treated at the dose level of 5000 mg kg-1
body weight in the sighting and main studies, respectively. Table
1 and 2 show the effect of Hygest™ on
body weight and percent weight gain after single oral administration in
female rats. Overall, the percent body weight gain during the complete
14 day observation period was found to be normal in all the animals except
for one female rat each in sighting study and main study showed reduction
in body weight gain during the 14 day observation period. Gross pathology
of animals sacrificed at termination revealed no abnormalities.
Zigbo®: Treatment related death or toxic effects
were not noticed in animals upto the dose level of 5000 mg kg-1
body weight during the experiment. Two animals in the main study showed
reduced body weight gain during the first week, but regained during the
second week and revealed comparable body weight gain at the end of the
study period. The average body weight and percent weight gain of treated
animals was found to be normal (Table 1, 2).
No significant abnormalities were noticed during the postmortem examination
of treated rats.
Involon™: The treatment with Involon™ was found to be
non lethal at and upto the dose level of 5000 mg kg-1 body
weight as the treated animals were alive throughout the study period and
did not show any adverse clinical signs following dosing and after the
period of 14 days, post treatment. Table 1 and 2
show that the treatment with Involon™ did not reveal any adverse
effect on body weight gain in female rats. Autopsy of treated animals
did not show any abnormalities during terminal sacrifice.
Zigup™ syrup: No mortality was recorded in experimental animals
treated with Zigup™ syrup upto 5000 mg kg-1 body weight
in the sighting and main studies. The observations on clinical signs after
test substance administration did not show any significant effects throughout
the study period. Zigup™ syrup treatment did not reveal any major
adverse effect on the body weight gain except for the treated female rats
in the main study group, which showed reduction in body weight gain during
the second week of observation period (Table 1, 2).
On necropsy, no major gross pathological changes were observed in any
of the treated rats.
DISCUSSION
Continued exploration of lesser known medicinal plants provides excellent
leads for development of new therapeutics (Lee, 2005). As more pharmacological
and clinical information on medicinal plants become available, the toxicological
database of these agents also grows increasingly more refined (Francis,
2000). Like their synthetic counterparts, toxicological studies must be
performed for herbal preparations to validate their safety (Becker et
al., 2007). Unfortunately, there is limited scientific evidence reported
pertinent to safety of phytopreparations to back up the continued therapeutic
application of these remedies (Aniagu et al., 2005).
Many official guidelines have been developed by various regulatory agencies
for toxicity testing. These guidelines relate to the use of botanical
products as medicinal preparations and provide standard methods for toxicological
studies to assess the safety of medicinal products. Not all tests are
necessarily performed for each herbal product and the need for each toxicity
test should be evaluated depending on the availability of published literature
related to safety, efficacy, established and traditional claims and intended
uses (Schilter et al., 2003).
Single dose studies are conducted to define the extent of toxicity in
the absence of other data. Information on acute toxicity will usually
be required for botanical preparations if they contained biologically
active principles (Schilter et al., 2003). Commenting on the disadvantages
of acute toxicity testing quoted by various researchers as a parameter
for assessing safety of a substance, Aniagu et al. (2005) supports
the useful information that could be obtained from such studies in certain
occasions. Apart from these, the fixed dose method also provides the minimum
lethal dose data of the polyherbal products.
The experimental protocols recommended by different authorities worldwide
place a great deal of emphasis on effects of test substance on mortality
of treated animals (Rispin et al., 2002). As per fixed dose procedure,
the selection of appropriate dose levels during acute toxicity testing
and final classification of the test material depends on lethality/evident
toxicity signs observed during the experiment. In the present study on
polyherbal veterinary preparations, neither incidence of mortality nor
animals found in a moribund condition was recorded at the treated dose
levels throughout the observation period.
From the long-term traditional folk use and clinical applications, it
is fairly understood that most herbal plants or products derived from
them have an excellent safety record (Bhattacharjee, 1998; Agaie et
al., 2007; Michael, 2007). Nevertheless, critical investigation
of suspected adverse effects forms the important prerequisite in safety
appraisal of herbal veterinary formulations in view of product stewardship
in regulatory programmes across the world (Michael, 2007). The battery
of clinical signs investigated during the acute oral toxicity studies
can strengthen the foundation of knowledge on toxicity and safety issues
of the substance under study. The findings of the current studies did
not reveal any major abnormal behavioural and or clinical signs relevant
to the screened products at the employed dose levels. The behavioural,
neurological and autonomic parameters recorded in terms of graded scores
in the experimental animals immediately after the administration of the
herbal formulations and once daily thereafter for 14 days were well within
the normal levels.
The negative influence of toxic compounds on body weight of the laboratory
animal species is recognized and well documented in published literature
(Aniagu et al., 2005; Joshi et al., 2007; Gorniak et
al., 2003). The toxic nature of the administered product is generally
correlated with its ability to produce a 10% or more decrement in body
weight or growth rate of the selected test animals (Schilter et al.,
2003). From the results of the current study, the overall percent body
weight gain in rats treated with the herbal products was found to be normal
at the end of 14 days observation period. A temporary reduction in weight
gain after the administration of few products can best be explained by
the altered physiological processes in test animals for transient period
due to exaggerated dosages of tested products.
Postmortem toxicology of treated animals is customarily recommended in
the adopted guidelines for acute toxicity testing (Dadarkar et al.,
2007). The gross pathological finding for each animal is genuinely considered
as potential source of information on the target organ/system and the
toxic nature of the chosen test substance. Necropsy examination conducted
at the termination of 14 day observation study on individual veterinary
product was normal and did not show any significant treatment related
macroscopic changes of organs or other structures.
In conclusion, acute oral toxicity testing of screened herbal veterinary
products did not produce mortality, toxicity signs or any significant
pathological changes upto the dose level of 5000 mg kg-1 body
weight and an overall normal body weight gain was observed in all the
treated female rats and hence resulted in tested products being labelled
unclassified in the hazard category according to Globally Harmonised System.
ACKNOWLEDGMENTS
The authors are thankful to Sri. R.K. Agarwal, Chairman, M/s Natural
Remedies Pvt. Ltd., Banglore, India for his constant encouragement and
support in completing this research successfully.
