Here is a list of toxicological tests done on animals: 1. Behavioural Responses (Tests)2. Analytic Tests 3. Functional Tests 4. Potentiation and Synergism 5. Pathological and Histological Examinations 6. Other Toxicity Tests.
The pharmacological response/toxicological response to a drug/toxicant, which manifests itself through changes in the behaviour patterns of the test animal, is known as behavioural response.
Mc Namara (1976) compiled a list of toxic signs and the systems and organs affected as a guide to facilitate toxicological observations in animals (Table 16.1):
The observation period should be sufficiently long so that delayed effects, including death, would not be missed. The period is usually 4-14 days but may be even longer.
Gross autopsies should be performed on all animals that have died as well as at least some of the survivors, especially those that are morbid at the time of termination of the experiment. Autopsy can provide useful information on the target organ, especially when death does not occur shortly after the dosing. Histopathological examination of selected organs and tissue may also be indicated.
Different drugs produce different types of behavioural and psychological alterations in the subject when administered e.g.:
i. Reserpine produces suicidal tendencies.
ii. Amphetamine produces confusion and inability to concentrate.
iii. Glucocorticoids in certain amount may cause euphoria (feeling of well-being), restlessness and psychological upsettings.
iv. Heroin causes a fixed intention — whatsoever a person thinks about.
There is a large body of information on behavioural toxicology, resulting from a widespread feeling that behaviour is a subtle and sensitive indicator of toxicity. However, this view has been questioned, for example by Norton (1980), who stated- “Scientific data supporting this view are not only scanty but the available evidences often flatly contradict this assumption. It is hoped that the improved testing procedures would increase the sensitivity and utility of this approach in neurotoxicology and this branch will undoubtedly grow.”
Analytic test includes the analysis of various toxicants. At present, several toxicants are in use and the number is increasing steadily every year. For some, analytic data are meagre while for others more than one analytic methods are available. Rapid advances in analytic chemistry, instrumentation, and techniques are being speedily evolved for identification of toxicants, their metabolites and degradation products from food and biological materials. In addition, modifications are often made in analytic method for their improvements.
There are various analytic methods frequently used in chemical analyses of environmental toxicants:
i. Neutron Activation Analysis (NAA)
ii. Anodic Stripping Voltammetry (ASV)
iii. Atomic Absorption Spectrophotometry (AAS)
iv. Optical and Emission Spectroscopy (OES)
v. Classical Polarography
vi. Potentiometry with Ion-Selective electrodes
vii. Gas Chromatography
viii. Gas Chromatography/Mass Spectrometry (GC/MS)
ix. High Performance (Pressure) Liquid Chromatography (HPLC)
x. X-ray fluorescence
xi. Fourier Transform Infrared Spectrometry
xii. Immuno Assays
In general, three basic types of test may be recommended for the precise assessment of toxic effects:
i. Morphological tests — adopted to evaluate morphological changes;
ii. Biochemical tests — adopted to evaluate biochemical changes;
iii. Functional tests — adopted to evaluate functional changes.
Morphological changes refer to gross and microscopic alterations in the morphology of the tissues. Some of these changes, e.g., necrosis and neoplasis are, however, irreversible and serious while functional changes usually represent reversible changes in the functions of target organs. Kidney and liver function tests (e.g., rate of excretion of dyes) are commonly utilized in toxicological studies.
Several investigators have attempted to show that functional changes might be detected earlier in animals exposed to lower dose than those with morphologic changes. The evidence collected in certain hepatic and renal tests, however, does not support this assumption. Nevertheless, more recent findings indicate that functional tests are more sensitive and are also valuable in following the progress of effects on target organs in long-term studies in animals including humans.
Biochemical effects usually refer to those without apparent morphological changes. An example of such effects is the elevation of serum transminases, which is associated usually with toxic effects on the liver but occasionally with myocardial infarction and muscle injury. Certain biochemical effects lead to changes in functions like the toxic manifestations associated with cholinesterase inhibition following exposure to organophosphate and carbamate insecticides.
From the functional test viewpoint, toxicants do not affect all organs to the same extent. The reason is, however, not always apparent. For example, methoxyflurane is metabolized to release F–. This takes place in the liver, but the target of toxicity are kidneys. Presumably, for some reasons, the kidney is more sensitive than the liver.
Potentiation in toxicology is used to describe the situation in which the toxicity of a chemical substance on an organ is markedly increased by another chemical substance that alone has no toxic effect on that organ. For example — isopropanol has no effect on the liver, but it can increase considerably the hepatotoxicity of carbon tetrachloride (CCl4).
Another known example of potentiation is administration of scopolamine and morphine. These drugs are sedative in action. The simultaneous administration of these two drugs produce more sedation than the sum of sedation produced by the two drugs, if given at different times.
Thus, the administration of two drugs simultaneously to exert a greater response than the sum of their independent responses may be termed ‘potentiation’. In simple words, when one toxicant potentiates the toxicity of the other toxicant, the phenomenon is referred to as potentiation and can be mathematically represented as:
According to the definition of potentiation
C > A + B
Hewitt et al (1982) studied that the potentiation is medicated through a variety of routes e.g., increasing the bio-activation of CCl4, favouring the formation of phosgene over CCl3 depicting hepatic glutathione and enhancing the susceptibility of subcellular organelles.
There are other chemicals, such as antimetabolites, that exert their toxicity through interference with metabolic pathways. Phalloidin acts via attachment to membrane receptors.
A number of chemically related compounds, e.g. chloroform, tetrachloroethane, and carbon tetra-bromide, as well as phosphorus, appear to act in similar ways.
The potentiation tests may be performed very easily. Certain fraction of lethal doses of two toxicants having similar mode of action may be exposed to test animals and a parameter may be selected on the basis of their mode of action as a measure of toxic effect.
Similarly, the sum fraction of both toxicants may be applied together and the same parameter may be again measured to quantify the toxic effect produced by those toxicants.
i. Application of DDT with C6H6 (benzene) potentiates its toxic action on insect pest
DDT + Benzene → More toxicity
ii. Use of alcohol with barbiturate potentiates its toxic action
C2H5OH + Barbiturate → More toxicity
iii. Use of morphine with heroin potentiates its toxic action
Morphine + Heroin → More toxicity
The chemical compounds that produce greater effects in combination than when they act individually are termed synergists and the phenomenon as synergism.
(a) Many drugs produce greater effect in combination viz. – (i) use of filixmas with castor oil raises action of the former against tapeworm, (ii) Adrenaline, when applied with Procaine, becomes more effective.
(b) Most widely used synergist of pesticides is pyrethrin with piperonylbutoxide. The latter increases pyrethrin toxicity ten-fold.
After the administration of a toxicant or exposure to environmental toxicants, the affected organs show various pathological and histological alterations. Histological and pathological examinations in combined form constitute histopathological examination.
For example, first of all, the gross morphology of the liver of an animal viz., rat, is used to study the effect of any chemical/toxicant. The colour and appearance of target tissue by toxicant in comparison to control group often indicate the nature of toxicity, such as fatty liver or cirrhosis. The net weight is often a very sensitive indicator of toxic effect on the liver.
Histopathological examinations can reveal the site, extent and morphological nature of specific tissue lesions viz. renal lesions, hepatic lesions etc.
Sharrat and Frazer (1963) reported that histopathological examinations in certain cases, viz. kidney toxicology, prove to be more sensitive than the functional tests.
Histological examinations may be of two types:
(a) Light Microscopy; and
(b) Electron Microscopy.
(a) Light Microscopy:
Light microscopy of any target organ, viz. liver, can reveal a variety of histological abnormalities e.g.:
(i) Fatty changes
(iv) Hyperplastic nodules
(vi) Sinusoidal congestion
(vii) Cellular vacuolations etc.
(b) Electron Microscopy:
Electron microscopy proves to be much sensitive than light microscopy. It is useful in assessing ultrastructural changes in the cells, such as mitochondria, lysosomes and other organelles. So much so, it is even useful in the detection of density of various cell organelles like mitochondria and lysosomes.
Observations of subcellular changes, along with the biochemical findings, are often useful in depicting the mechanism of action of toxicants.
Certain histopathological studies on specific tissues of animals made by various authors under different toxicants have been summarized in Table 16.2:
Although pathological and histological examination in toxicology have been found useful in assessing the extent of damage in various tissues, yet a variety of pathotoxicological data may also be useful indicators of immune functions. For example, thymic atrophy appears to be a very sensitive indicator of immunotoxicity. Paucity of lymphoid follicles and germinal centers in the spleen is indicative of B-cell deficiency, whereas T-cell deficiency is characterized by lymphoid hypoplasia in the paracortical areas.
Important pathotoxicological assessments are:
(i) Haematology Profile—hemoglobin, erythrocyte count, leukocyte count, differential cell count.
(ii) Clinical Chemistry — creatine phospho- kinase (CPK), creatinine acid and alkaline phosphatase, lactic dehydrogenase (LDH), cholinesterase,
(iii) Serum Proteins — albumin, globulin, albumin/globulin ratio.
(iv) Weights — body, spleen, thymus, liver, kidney, and
(v) Histology — liver, thymus, lung, kidney, heart, spleen.
A. Teratogenic Tests:
Teratogenesis is the formation of congenital defects (Greek teras = monster). This type of illness has been known for decades and is an important cause of morbidity and mortality among newborns. Out of 320 human pregnancies reviewed by Murphy (1928), 14 cases of children with small head circumference and mental retardation have been noticed. Their mothers were, however, exposed to therapeutic radiation in the first trimester.
A new era in teratology was initiated as a result of the clinical use of thalidomide, a sedative hypnotic.
Numerous drugs, food additives, pesticides, environmental contaminants and radiation etc. cause potential teratogenicity.
The process of gamete formation is termed gametogenesis — development in males is spermatogenesis, while in females it is called oogenesis.
Chemobiokinetical studies reveal that toxicants may interfere with the various events of the reproductive cycle of test animal. Toxicants may act directly on the reproductive system or indirectly via certain endocrine glands. Before toxicants act directly they must reach the target organs in sufficiently high concentration.
This concentration may be higher or lower than that in the blood. For example, with DDT, the concentration is almost 80 times higher in the ovary than in the plasma. Fabro (1978) observed that a number of other substances may penetrate the oocyte, oviduct, uterine fluid, and the blastocyst.
Toxicity of xenobiotics on the testis is influenced by the blood-testis barrier (BTB) and by the activities of certain enzymes. BTB is a complex multicellular system composed of both myoid cells surrounding the seminiferous tubules and several barriers. The penetration rate of chemicals (toxicants) into the testis is proportional to their molecular weights and their partition coefficients.
Testis contains both activating and detoxicating enzyme systems. These two enzyme systems are capable of, respectively, increasing and decreasing the toxicities of toxicants. Furthermore, there is an efficient DNA repair system in the premeiotic spermatogenic cells, but there is none in spermatids nor in spermatozoa. Mutations can, therefore, be induced by electrophilic substances.
Various toxicants have been reported to adversely affect the spermatogenesis and cause testicular atrophy as shown in Table 16.3:
Hypotensive drugs such as guanethidine (like thalidomide) can induce non-pregnancy by interfering with seminal emission rather than by any direct effect on the fetus.
A variety of chemicals may be toxic to female reproductive functions. Certain toxicants affect the oocyte, others (e.g., Haloperidol) prevents implantation or affect the development and growth of the conceptus, e.g., nicotine and DDT lower fetal weight in rabbits. Some toxicants, e.g. spironolactone, have various effects on reproductive functions, such as interference with ovulation and implantation and retardation of development of the sex organs of the offspring.
Shukla J. P. and Pandey K. (1983,1984,1985) have made extensive studies of certain metallic toxicants like arsenic on the reproductive cycling (testicular and ovarian cycle) of freshwater teleost and observed impaired reproductive development in testis and ovary during different phases.
In general, rats or mice are the animals of choice for reproductive tests. A minimum of 20 females and 10 males are placed in each of three dose groups along with a control group. The doses are so selected that the high dose will produce some minimal toxic signs but will not result in a mortality greater than 10%, i.e., LD10 for a specified period. The low dose will not induce any observable effect.
Prior to breeding, the males are dosed throughout the period of spermatogenesis (60-80 days) and the females are dosed throughout the development of the ova (16 days). Dosing of the females is continued through the gestation and lactation periods. The test chemical (toxicant) should be administered by a route that most closely resembles the human exposure condition.
To determine the potential effect of the chemical on the reproductive function of the offsprings (F1), a second generation offsprings (F2) is bred and reared to reproductive immunity.
The adult animals are observed for the body weight, food consumption, general appearance, oestrus cycle, and mating behaviour. Also, the fertility and nesting and nursing behaviours are noted. Half of the maternal rats are killed on day 13 of gestation for examination of corpora lutea, implantations, and resorptions.
The other half of the maternal rats are allowed to deliver their pups. The pups are examined for litter size, number of the stillborn, sex distribution, and congenital anomalies. In addition, the viability and pup weight are recorded at birth, on day 4, and at weaning, and preferable also on day 12 or 14. Auditory and visual functions and behaviour are also examined for subtle congenital defects.
An appropriate number of males and females are randomly selected from all generations and examined histopathologically, especially with respect to the reproductive organs.
From the data described, the following reproductive indices can be derived:
i. Fertility Index:
The percentage of matings resulting in pregnancy.
ii. Gestation Index:
The percentage of pregnancies resulting in the birth of live litters.
iii. Viability Index:
The percentage of live pups that survive 4 days or longer.
iv. Lactations Index:
The percentage of pups alive on day 4, that survive 21 days, or longer.
v. Mating index (the percentage of oestrus cycles that had matings).
vi. Male fertility index (the percentage of males exposed to fertile non-pregnant females that resulted in pregnancies).
vii. Female fertility index (the percentage of females exposed to fertile males that resulted in pregnancies), and
viii. 12 or 14 days’ survival indices.
Multi-generation reproductive study can reveal a variety of deleterious effects on the reproductive functions. However, non-specific response (e.g., non-pregnancy) is common. It may follow unusual routes of administering the chemical, such as inhalation, topical application to the eye or nose, and parental administration. Excessive handling may also disturb the normal reproductive function.
The pathological examination may reveal suppression of spermatogenesis, advanced stages of follicle atresia, and ovulatory or meiotic failure.
In order to save time and expenses, the males of the parent generation (F0) used in the multi-generation study can be utilized in a dominant lethal test, and a second litter of the first generation offsprings (F1b) may be bred to study teratogenicity.
The effects revealed in multi-generational reproduction studies outlined above may result from paternal, maternal, or fetal exposure. Thus additional tests are often conducted to establish the cause of the effect.
A few such reproductive tests are described:
i. Prenatal and Postnatal Studies:
Such studies are often conducted to determine the effects on late development of the offspring. The procedure is essentially the same as that of the multi-generation study, except that only the females are treated and their treatment covers only the last third of gestation and throughout, and lactation. The litter data, as described above, are noticed and evaluated.
ii. Analysis of the Semen:
It is useful in epidemiologic and animal studies. The sperm count and their motility, survival and morphology often provide information on deleterious effects on testis.
iii. The Profused Male Reproductive Tracts Tests:
The profused male reproductive tracts have been found as a useful model in studying the effects of toxicants on the secretion and accumulation of androgens.
iv. In Vitro Study:
To study the deleterious effects of toxicants, Brackett (1978) described a procedure using in vitro study of deleterious effects by observing the development of fertilized ova.
The eye, being complex in structure, is a spherical body and is covered chiefly by three coats or layers of tissues – the sclera, the choroid and the retina. The sclerotic coat mainly consist of fibrous tissues. The choroids coat consists of pigments and blood vessels, and the retina is composed of the nerve fibers, cells, and special receptors. They are actually nontransparent, however, light is admitted through the front of the eye, where the three coats are replaced by a number of tissues, notably the cornea and the lens.
The cornea is formed by the modification of the sclera. It consists of a relatively thick stroma and is covered, in front, by an epithelium, consisting of several layers of cells and Bowman’s membranes and, behind, by Descimet’s membrane and an endothelium. The cornea and the front portion of the sclera as well as the inside of the eyelids are covered by a thin layer of conjunctiva.
The lens is formed of transparent fibers enclosed in the lens capsule. It is suspended by the ciliary zonule to the ciliary body and its curvature is adjustable by the contraction and relaxation of the ciliary muscle.
The space between the lens and the cornea is filled with the aqueous humor. Also, in this space and just in front of the lens, is the iris. The iris is heavily pigmented and rich in blood vessels. The iris has a central opening — the pupil.
The retina is a photosensitive structure that responds to light stimuli. It consists of several layers. The outermost is a pigmented epithelium. Next to it are the retinal rods and cones, which are the light- responsive neural structures. They are linked to the ganglion cells via the bipolar cells. The axons from the ganglion cells converge and exit from the eye at the optic papilla.
Due to the diverse physiological nature and spatial relations, these ocular structures may exhibit a variety of effects as a consequence of exposure to toxicants.
The ophthalmoscope is used in assessing the deleterious effects of xenobiotics on different parts of the retina. The examination is usually intended to trace the existence of edema, hyperaemia, atrophy of the optic disk, pigmentation, or the state of blood vessel. Alterations in the vitreous humor, lens, aqueous humor, iris and cornea can also be traced.
Effect on the visual field can be readily determined in the humans only but not in the laboratory animals, except the non-human primates. It may reveal the loss of peripheral vision resulting from exposure to any xenobiotic.
Procedures involving instrumentations, viz., electro-oculography, and visually evoked responses are also useful in ophthalmic toxicity experimentation.
Light microscopy may also reveal the site of action of toxicants on the eye. Electron microscopy may reveal ultra-structural changes and the biochemical studies can elucidate the mechanism of toxic effects.
For example, choroquine has been observed with light microscopy to cause a thickening of the pigment epithelium, followed by migration of the pigment to the outer-nuclear layer, and, finally, total atrophy of the photoreceptors. Electron microscopy showed mitochondrial swelling and disorganisation of the endoplasmic reticulum in the photoreceptor inner segment.
Biochemical studies revealed inhibition of many enzymatic reactions, especially those related to protein metabolism of the pigment epithelium.
Eye irritation tests are widely used to assess the ocular irritancy of the xenobiotics. In general, the albino rabbits are the animals of choice. Despite the variability of the results, the tests in rabbit have proved useful in predicting eye irritation in humans.
A large number of animal experimentations and clinical studies indicate that there is fair correlation between humans and animals in their reactions to xenobiotics with respect to cataract formation and retinopathy.
Effects on the eye can be examined after topical application of the toxicants. In certain cases, systemic administration can also result in adverse ocular changes.
Mainly albino rats are used to determine the ocular irritancy of ophthalmic medications and other chemicals that might come in contact with the eyes. Dogs and non-human primates, viz. rhesus monkeys, may also be used as test animal.
For testing the ocular irritancy, the test described by Draize and Kelley (1952) has been accepted as standard procedure.
It specifies the use of nine rabbits. Into one eye of each rabbit is instilled 0.1 ml of the test material. In three of the nine rabbits, the test material is washed with 20 ml of lukewarm water 2 seconds after the instillation, and in three other rabbits, the washing is done after a 4-second delay. In the remaining three rabbits, the eyes are left unwashed. The ocular reactions are read with the unaided eye or with the aid of a hand slip lamp at 24, 48 and 72 hours and at 4 and 7 days after treatment.
The reactions of the conjunctiva (redness, chemosis, and discharge), cornea (the degree and extent of opacity), and iris (congestion, swelling and circumcorneal injection) are scored according to a specified scale. A series of coloured pictures — as a guide for grading eye irritation by the U.S. Food and Drug Administration (1965) — has also been used.
Further, in 1980, Griffith et al recommended following points to be taken into account in conducting eye irritation tests:
1. A 0.01 ml dose, or its weight equivalent of solids and powders, be applied directly to the central corneal surface of at least six eyes without subsequent rinsing or manipulation of the eyelids.
2. Evaluation of the irritancy may be based on the median duration for the eyes to return to normal, instead of using a scoring system based on the type and extent on the deleterious effects produced by toxicants.
In 1983, U.S.C.P.S.C. (United States Consumer Product Safety Commission) reported the need of six albino rabbits for each test chemical substance. For test liquids, 0.1 ml is used, and for solids and pastes, 100 mg. The test substance is instilled into one eye of each rabbit without washing. Ocular examinations are made after 24, 48 and 72 hours. A rabbit is considered as having positive reaction if the eye shows ulceration or opacity of the cornea, inflammation of the iris or swelling of the conjunctiva with partial aversion of the eyelids.