Oral ingestion studies

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Under normal situations, the oral route is usually the means by which toxicants from food enter organisms. Through industrial exposures or other rare situations, toxicants from foods may enter organisms by inhalation as a dust or vapor suspension in the air or percutaneous (dermal) penetration, respectively. The toxicologist selects the experimental route to test the bioactivity of a chemical based on the common route of exposure for the substance. Selection of the route of exposure is important because the toxicant encounters various barriers during absorption, distribution, biotransformation, and elimination or excretion. The route of administration can influence the quantitative toxicological response to a bioactive compound, such as altering the slope and position on the dose-response curve.

The oral route may be viewed as providing entry of a bioactive substance to the body through a tube in the body, starting at the mouth and ending at the anus. As such, the tube is essentially exterior to the body fluids, because the gastrointestinal surface is a barrier to the contents of the tract, similar to the skin being a barrier. Therefore, ingested chemicals can impact systemically only after absorption through the gastrointestinal tract. Usually, food stays for too short a time in the mouth and esophagus to allow appreciable chemical absorption.

The bioactivity of a toxicant ingested varies with the frequency, presence of food, and the makeup of the food, such as amount of purified sugar, fiber, high protein, or high fat. The different pH conditions of the gastrointestinal tract affect the ionization of weak organic acids and bases. Following absorption in the gastrointestinal tract, the bioactive chemical is translocated to either the lymphatic system or the portal circulation. The portal circulation directs the chemicals to the liver, and many of the chemicals are excreted by the liver as bile. Because the bile empties back into the intestine, a cycle involving translocation of the chemical from the gastrointestinal tract to the liver and via the bile back to the intestine occurs. This cycle is known as enterohepatic circulation. Ingestion of chemicals from the gastrointestinal tract and enterohepatic cycle exposes the liver to those concentrations of the agent that would not be obtained by other exposure routes, such as intravenous or inhalation routes (first-pass effect). Thus, the hepatotoxicant is expected to be more toxic following oral ingestion on repeated situations but less hazardous when administered by other routes. A major function of the liver is to detoxify many natural and human-made chemicals that enter the body. The liver is able to handle many substances because of its large surface area and the high blood flow rate through this organ. The high blood flow to the liver provides nourishment and allows tissue repair and regeneration if there is no irreversible damage.

Toxicologists employ three types of ingestion toxicity studies: acute, sub-chronic, and chronic. Ingestion toxicity studies are differentiated by the length of time for which the test substance is administered via gavage, in the feed, or in the drinking water.

Acute Toxicity Testing

Essentially all chemicals of biological interest undergo acute toxicity testing. The purpose of the test is to determine the order of lethality of the substance. Lethality information can be used to give a quantitative measure of acute toxicity (LD50) for comparison with other substances and to give a dose-ranging guidance for other tests. Acute toxicity testing represents the first line of toxicity testing for toxicity assessment, particularly in the absence of data from long-term studies. In addition, the acute toxicity test is useful for determining the symptoms consequent to administering the toxicant or to identify clinical manifestations of the acute toxicity. In pharmacology, these tests have been referred to as screening for the physiological

TABLE 4.2

Objectives of Acute Toxicity Testing

• Define intrinsic toxicity

• Predict hazard to nontarget species or toxicity to target species

• Identify target organs

• Provide information for risk assessment of acute exposure

• Provide information for the design and selection of dose levels for prolonged studies

• Provide valuable information for clinicians to predict, diagnose, and prescribe treatment

• Be useful for regulatory classification and labeling

• Develop clues on mechanism of toxicity basis of toxicity. Careful evaluations of the symptoms produced by administering a bioactive chemical can provide significant information to help characterize the toxicant's mode of action, i.e., neurological, cardiovascular, respiratory, or others. Table 4.2 shows the objectives of acute toxicity testing.

Thus, the objectives of acute toxicity testing are to define the intrinsic toxicity of the substance, predict hazards to nontarget species or toxicity to target species, identify target organs, and provide information for risk assessment of acute exposure. In addition, the information derived from acute toxicity studies can be useful to design long-term studies, such as predicting, diagnosing, and prescribing treatment; providing information to support classification and labeling by regulatory authorities; and providing clues used by research to determine the underlying mechanisms of toxicity.

The test material is administered by gavage (gastric intubation) once or as a single bolus dose to test animals, and the animals are kept under observation for 2 weeks (14 days). Gastric gavage involves intubating the animal with a dosing tube (plastic or stainless steel) into the mouth and down the throat, through the esophagus into the stomach. Alternatively, in the bolus dose, a series of small doses may be administered during a 24-h period. Often, adverse effects are noticed within a short time, e.g., 24 h. The goal is to test multiple levels in groups with at least two species. Number of animal deaths, duration between the administration of the test material and death, and various symptoms before death and observed changes at necropsy are recorded. Thereafter, a second test may be performed in which the test substance is incorporated in the diet at various levels and includes at least one level found toxic in the prior test using at least two species.

If the substance does not demonstrate acute toxicity at levels that are comparable to expected normal intakes of the substance by populations, testing proceeds to the next stage.

Toxicology Screen

The basic premise of toxicological or pharmacological screening is not to allow true biological activity to go undetected. Screening has been a useful tool for pharmacologists to identify potentially useful therapeutical compounds. The use of toxicity screens during experimental determination of the acute toxicity of a compound in rodents can be extremely valuable to assess the site and mechanisms of action of a toxicant.

Screening involves scanning and evaluation. Scanning involves a test or a group of tests that permit the detection of physiological activity and is referred to as the multidimensional screening-evaluative procedure, blind screen, or the "Hippocratic screen." The Hippocratic screen for detecting physiological activity is analogous to a physician's Hippocratic diagnosis, a clinical situation in which the physician views the symptomatology of the patient. A patient's gross physical appearance, muscle tone, coordination, and even mental attitude are integrated with the physician's subjective impressions and clinical measurements, e.g., urinalysis or blood analysis. Diagnosis of a disease by this procedure can be rapid and provide clues, which can be verified by additional clinical and medical tests. Likewise, scanning can be followed by evaluation that involves other more refined or specialized tests designed to better remove the uncertainty of scanning tests. Thus, if the results from a scanning test suggest neurological toxicity, a more specific neurofunction test may be used in the evaluation.

Expensive equipment is not required for the Hippocratic screen or scanning tests; however, attentiveness and training to be unbiased is important. Because individuals differ in their abilities to observe, use of standardized work sheets with blanks to be filled in sequentially for either positive or negative observations is critical. Procedures are largely observational, qualitative, and use semiquantitative techniques utilizing standardized work sheets by trained researchers. The etiology of these procedures is that toxic compounds do not create new activity; these compounds can only modify existing physiological systems. Overall, compounds act by the basic mechanisms of stimulation, inhibition, or irritation of a biological system, or their combination. Irritation is an undesirable nonspecific type of response characterized eventually by necrosis if concentration is sufficiently high enough.

The Hippocratic screen is the initial screen, general in nature but carefully standardized to give reliable and reproducible results. Usually, the amount of test material needed for the scanning is less and can be either chemically pure or grossly crude material. Scanning can be a useful tool as crude material extracts during chemical fractionation can be followed pharmacologically.

Table 4.3 gives a standardized work sheet for the Hippocratic screen. A work sheet must be used for each animal and dose tested. Numbers used to fill in a blank are the actual measurements (rectal temperature, body weight), and subjective ratings are indicated by + and 0 (neutral) values. For example, decrease of motor activity is rated in the following manner: 0, animal is quiet, occasionally moves spontaneously; +, does not move spontaneously, but when handled moves rapidly; ++, when handled moves slowly; +++, when handled moves very sluggishly; ++++, when handled does not move at all. Observations for the Hippocratic screen or scanning may be elicited during experimental determination of the acute toxicity of a compound, including initial rough dose-range-finding and subsequent experiments to narrow the range of effective doses for measurement of lethality and to establish the dose-response curve for lethality.

TABLE 4.3

Hippocratic Work Sheet: Observational Examination of Animals

Date:

Source of Sample:

Dosage:

Test Animal:

Animal Identification:

Weight:

Tested By:

Symptom

Ataxia

Loss of righting reflex

Analgesia

Anesthesia

Loss of corneal reflex Loss of pinnal reflex Screen grip loss Paralysis, neck Paralysis, hind legs Paralysis, front legs Decrease in respiratory rate Decrease in respiratory depth Dyspnea

Cheyne-Stokes respiration

Notebook Number:/Page: Project title:

Test Concentration: Sex:

Time of Fasting: Volume of Sample: Evaluated By:

Dosage Vehicle Animal Source:

Time of Intubation:

Response"

Control +5 min +10 min +15 min +30 min +60 min +2 h +4 h +6 h +24 h +2 d +4 d +7 d

Decreased Motor Activity

Fine body tremors Coarse body tremors Startle sensitivity Clonic convulsions Tonic convulsion Fasciculation

Increase in respiratory rate

Increase in respiratory depth

Enopthalmus

Exopthalmus

Palpebral ptosis

Pupil size (mm)

Nystagmus

Lacrimation

Chromodacryorrhea

Skin blanching (ears)

Hyperemia

Cyanosis

Metachrosis

Salivation

Rales

Tail erection Tail lashing Tail grasping Pilomotor erection Robichaud test Micturition Diarrhea

Increased Motor Activity

TABLE 4.3 (Continued)

Hippocratic Work Sheet: Observational Examination of Animals

Responseb

Symptom" Control +5 min +10 min +15 min +30 min +60 min +2 h +4 h +6 h +24 h +2 d +4 d +7 d

Priapism/colpectasia Abdominal griping Temperature (Rectal, °C) Body weight (g) Head tap, aggressive Head tap, passive Head tap, fearful Body grasp, aggressive Body grasp, passive Body grasp, fearful Circling motions Status positions Excess curiosity

Notes and other symptoms: Death and autopsy notes:

a Subjective or quantitative intensity of the response after administration of the dose.

Dose-Range-Finding and Dose-Response Curve for Lethality

Methodical and rigid standardized procedures for the standard components of the screen are essential to produce reproducible and meaningful results.

Animals and Sample Administration

Nonfasted young albino rats weighing between 150 and 250 g are used, with doses assigned to both sexes randomly. Food and water are taken away for a period of 2 h after oral administration and returned subsequently, allowing ad libitum feeding. Test samples are dissolved or suspended in an aqueous vehicle or 0.25% agar. In situations where oily substances resist uniform dispersion by trituration, vegetable oils may be used; however, caution is recommended because of the potential of the solvent vehicle to interact with the test sample or produce its own physiological effects. The dosage volume should be constant; 5 ml/kg eliminates handling errors or aberrant effects due to too much or too little volume. Initially, one or two rats are used at each dosage level, ending with a minimum of five rats to an optimum of ten rats per dosage level. Approximately 200 mg of a pure chemical or 2 g of crude material is needed to complete the evaluation.

The goal of the acute toxicity study is to determine one lethal dose, one dose that is ineffective, and at least three log doses in between the lethal and ineffective dosages. The initial dosage given to the first animal is 100 mg/kg and depending on the effects seen in the first animal, the second animal receives either 10 mg/kg or 1 g/kg. For example, a lethal observation at 100 mg/kg results in reducing the dose to 10 mg/kg and no effect results in increasing the dose to 1 g/kg. Once the ineffective dose and the lethal dose are determined, the experimenter narrows down the range by dosing an animal with a log dose between the extremes. This scheme is continued until the dose elicits some symptoms but is neither ineffective nor lethal. The aim is to have one lethal dose, one ineffective dose, and three log doses in between that exhibit dose-response symptoms. Therefore, each injection is based on the result of the preceding animal. After each animal is injected, observations given in Table 4.3 are made at prescribed time intervals. Observations of the animals are made by placing the animal in a rink (65 x 54 x 8 cm3) with normal animals to make comparisons. The listed format for observations facilitates evaluation; however, observations need not be determined in the order listed.

Autopsy is done immediately after an animal dies acutely from the effects of the test substance. Particular attention is given to whether death is due to cardiac or respiratory arrest. Whether the heart is in systole or diastole if the heart has stopped and whether the auricles are beating in normal rhythm should be noted. The color of abdominal wall, kidneys, liver, uterus, testes, and lungs; the degree of intestinal motility; dilatation or constriction of mesenteric vessels; and any unusual changes should be described.

It is extremely important that an acute toxicity test be standardized as much as possible. Table 4.4 summarizes factors that are likely to cause variations in the LD50 values if serious consideration is not given during standardization. Use of subjective evaluations can introduce considerable amount of human variation if not controlled.

TABLE 4.4

Potential Factors Causing Variation in LD50 Values

Species Health

Strain Nutrition

Age Gastrointestinal content

Weight Route of administration

Sex Housing

Temperature Time of day Season

Human error

The acute toxicity data aid in designing subchronic toxicity tests. Researchers and regulatory people often review the data from acute toxicity testing in order to derive a starting point by which to set guidelines and design further testing. The information in the absence of long-term studies may help find ways to proceed for future work, give safety information, and help decide whether to proceed with the development of the food products or additive or to discontinue further research.

During the last decade, because of animal rights activism or humanitarian reasons, the issue of using alternative methods to animal testing for toxicity has been debated. The outcomes of such debates have been the recognition of the need to reduce the number of animals used in testing, refining existing testing methods to minimize suffering and pain of the animals, and setting a goal to develop replacement non-animal-based methodologies. Several in vitro strategies have been suggested to replace whole-animal toxicity testing, but, to date, none has been accepted by regulatory agencies. Thus, replacement of whole animals with test-tube methods for toxicity testing is more of a goal than a reality.

Subchronic Toxicity Testing

Experimental parameters for subchronic toxicity testing are based on the results of acute toxicity testing. Subchronic testing is usually for a prolonged period, ranging from several months to perhaps a year. The length of the testing period gives time to evaluate the possible cumulative effects of the substance on tissues or metabolic systems, or both. Dietary exposures of 90 days and for two test animal species are common subchronic testing protocols. Animals are monitored for changes in physical appearance, behavior, body weight, food intake, and excretion. Blood, urine, and excreta are routinely obtained and tested for hematological, urine and fecal alteration, and biochemical changes. Other tests may be used to measure specific functions (hepatic, eye, renal, gastrointestinal, and blood pressure and body temperature). Table 4.5 lists some commonly used analytical and functional tests used in conjunction with prolonged toxicity studies. Most tests are performed on the test species at 1- to 2-week intervals.

Observations are made on the animals, such as measuring animal activity levels, response to stimuli, general behaviors (eating and grooming), morbidity, and death. Other parameters include heart rate, blood pressure, nervous system function, and other tests required to answer specific questions, depending on the research design

TABLE 4.5

Prolonged Toxicity Tests: Clinical and Functional Evaluations

Blood Chemistry and Hematology

Urine

Function Tests

Electrolytes Calcium Carbon dioxide pH

Specific gravity Total protein

Liver function tests Kidney filtration tests RBC hemolysis

Serum glutamate (pyruvate/oxalacetic transaminase) Sediment

Serum alkaline phosphatase Serum protein electrophoresis Fasting blood sugar

Glucose Ketones Bilirubin

Blood urea nitrogen

Total serum protein

Total serum bilirubin

Serum albumin

RBC and WBC

Leukocyte, differential count

Hematocrit and sedimentation rates

Hemoglobin protocol. Because of limited time and resources, it is not uncommon for investigators to limit their observations to only those required by regulatory authorities.

At the completion of the study or when any animal becomes ill or moribund, all animals undergo necropsy, and tissues are examined for pathological changes and organ and major glandular weights are obtained. Table 4.6 lists common patho-

TABLE 4.6

Prolonged Toxicity Tests: Histology and Pathology

Weight

Histology

Total body weight

Heart

Liver

Thyroid

Spleen

Adrenals

Testes and epididymis

Liver Heart Adrenals

Gastrointestinal tract

Spleen

Ovary

Mesenteric lymph nodes

Pituitary

Thyroid

Kidneys

Pancreas

Urinary bladder

Testes

All tissue lesions logical and histological parameters measured in prolonged toxicity testing. The preparation of tissue slides is vital to assess pathological changes in tissues, such as lesions and other gross abnormalities that might occur. Histology is an integral part of the process of evaluating toxicity in tissue, and pathological interpretations are integrated into the final reports of findings for submission to publication, study sponsors, or review by regulatory authorities. These slides are typically archived and stored for possible review by researchers, contact research laboratories, sponsoring companies, and regulatory authorities.

The subchronic toxicity study usually involves the use of four groups of test animals, including a control group. Animals range from 10 to 20 per group per gender. The test diet is prepared by blending the test substance with a commercial stock or purified diet on a weight-to-weight basis. Caution is taken to ensure the stability of the test substance, which may require refrigeration of prepared test diets before feeding animals.

It is important that some form of toxic effect is exhibited in the high-dose test group. If, during the course of testing, the animals assigned to the high-dose group fail to develop signs of toxicity, the dose has to be adjusted such that some form of significant toxicity is exhibited.

The subchronic testing regimen seeks information about chronic effects but not necessarily with respect to cancer formation. These studies are often used to derive a dose regimen for long-term studies. Important information can be derived regarding effects on the target organ and the bioaccumulation of the test substance in the tissue. Such information can be useful to derive the no observable effect level (NOEL), discussed in Chapter 6.

Guidelines from regulatory agencies cover the spectrum of potential routes of exposure to toxic substances. For example, one may conduct tests based on other potential exposure routes, such as a 30-day dermal test or a 30- to 90-day inhalation test for compounds that may come in contact with the skin or be inhaled, respectively.

A company can investigate a compound more thoroughly than what may be required by regulatory agencies; however, the regulatory agency requirements must be satisfied so that the test substance qualifies for consideration by the regulatory agency with jurisdiction over its development and use.

Chronic Toxicity Testing

Chronic toxicity testing refers to long-term studies over the major duration of the animal's life span. The objective of such testing is to look for adverse effects that are not evident in subchronic testing, such as the propensity for carcinogenesis. This type of study is most valuable in terms of elucidating the biochemical mechanisms responsible for toxicity. The potential toxic effects from structure and function of the compound can be discovered and confirmed by results obtained through chronic toxicity testing.

Testing for cancer-producing substances usually requires 50 animals of each gender per dose level. Choosing the number required for the test group has been debated. The theoretical sizes of test groups based on the number required to determine toxicity at a frequency of 1 in 20 for a level of significance of 0.05 and

0.001 are 58 and 134 animals, respectively. The sizes increase for a frequency of 1 in 100 for a level of significance of 0.05 and 0.001 to 295 and 670 animals, respectively. High numbers of animals are needed to ensure adequate survivors at the end of the lifetime study to detect small percentage changes in histopathology and statistically evaluate the data.

In the past, large doses of the substances were used to reduce the group sizes as dictated by statistical theory; however, because test organisms respond differently to high and low doses, high-dose protocols are less commonly used. High doses of a substance are likely to produce toxic effects because the substance overburdens various systems of an organism that would normally dispose of at low doses. Systems include absorption via gastrointestinal tract, excretion by kidneys, hepatic metabolism, and DNA repair, which may be highly sensitive to concentration, particularly saturable substrate levels.

Chronic testing is costly and rats and mice are widely used to keep costs down. Besides cost, the advantage of using mice and rats as test species is the well-established volume of knowledge available on these species. Strain-specific species of rodents can be selected for susceptibility or sensitivity to organ-specific carcinogens or toxicity. Thus, if a substance is a carcinogen, the effect can be better shown in a particular stain sensitive to such compounds.

If a substance is found to be a carcinogen, then it is likely to be rejected as a substance for use in food. Thus, the chronic toxicity test is the final step of the overall risk assessment of a substance. Extra testing beyond chronic toxicity testing occurs in unexpected situations, e.g., when there are concerns about faulty data or inadequate test design.

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