Chapter 7 General Toxicity StudiesGeneral ConsiderationsAcute≤24-h (single or repeated administrations)Subchronic≥3-m in rats or 1-2y in dogs (up to 10% of life span, repeated administrations) Chronic18-m in mice, 24-m in rats, or 7-10y in dogs and monkeys (entire/major life-span) Part 1 Acute Toxicity StudiesBasic Conception•One or more routes of administration, and one or more species of laboratory animals are used.•Within 24 h of exposure duration•14 d for observation, to make sure all possible toxicity can be assessed.•Toxic effects: Mortality, signs of intoxication, behavioral modifications, morbidity, etc.Objectives and Parameters of Studies1.ObjectivesQuantitative estimation of acute toxicity (LD50); target organs and otherclinical signs; reversibility; a basis of dosing for further studies.monly Used ParametersMedianlethal dose (LD50), median lethal concentration (LC50, for inhalation), with 95% confidence limits, values affected by many factors related to theexperimental conditions (p185).Experimental Design1.Selection of Animal Species•Mouse and rat most often used, suitable for both inhalation and oral toxicity test (preference is rat, but mouse is more economical).•At least 5 d for acclimatizing the animals to the lab environment, at least 5 animals at each sex per dose level, females should be nulliparous andnon-pregnant2.Route of Administration•The main routes for xenobiotics to enter the body: GI tract (ingestion), lungs (inhalation), and skin (dermal).•By ingestion: in diet, in drinking water, by gastric gavage, or by gelatin capsules.•By inhalation: using inhalation exposure chambers (static or dynamic)•Dermal administration:•By i.p., i.m., i.v., s.c., intradermal injection3.Dosage and Experimental Groups•Four or more doses are required for a classic LD50 determination.•At lease 3 doses “effective”•Low≥ 10•Middle≈ 50•High≤ 90•To improve the precision of LD50 (for a narrow confidence range)a.By increasing the number of animals per doseb.By decreasing the ratio between successive doses (ordinarily 1.2—1.5)4.Observation and Examination•Duration of observation, typically 14 d, but not fixed to this length of time.Extended observation should be done when necessary , especially delayeddeath is expected.•Careful clinical examination, at least once each day, particular attention to tremors, convulsion, salivation, diarrhea, lethargy, sleep and coma; Time ofdeath (precise); necropsy done for dead animals and at the end ofobservation.Alternative Methods of Acute Toxicity Tests•In order to reduce the number of test animals or refine procedures to make exposures less stressful to animals, alternative methods of acute toxicity tests have been explored, and several of them have been adopted by OECD (Organization for Economic Cooperation and Development).1.Fixed Dose Procedure•Its main study involves non-lethal effect s.• A sighting study is a preparation of the main study, searching for an appropriate dose for the latter.•Sighting study starts from a fixed dose, taken from the following levels, 5, 50, 300, 2000, and rarely 5000 mg/kg, according to the related literature aboutthe chemical.•Five animals per sex are required for a main study.• A dose producing some signs of toxicity without severe toxic effects or death is expected from the main study.•The tested chemical is then classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS).2.Acute Toxic Class Method• A stepwise procedure with 3 animals in the first step.• A group of animals receive one of the following doses: 5, 50, 300, 2000 mg/kg.•Depending on the resultant mortality, in the next step experiment three or more animals may receive the same or higher or lower dose.•2-4 steps may be carried out before allowed for judgment of acute toxicity.•Biometric evaluation on the obtained data to find a proximate LD50 value, then classification of a substance is done according to GHS.3.Up-and Down Procedure•Sequential dose, one animal at each step (1.75, 5.5, 17.5, 55,175, 550, 2000, 5000 mg/kg)•Dosage depending on survival/death of the previously dosed animal•Providing a point estimate of lethality and confidence limit.•Both lethal and non-lethal endpoints•EPA has developed a computer program to simplify the designation and calculation of LD50 and its confidence limit.Evaluation of Acute ToxicityPart 2 Local Toxicity Studies•Part of acute toxicity studies•Significant for identification of health risk before the onset of systemic response, removal of exposed people from the exposure situation, and provision of prompt treatment of the intoxication─ “Alarming” signals.1.Dermal Irritation/Corrosion Test•Dermal irritation: inflammatory damage, reversible•Dermal corrosion: irreversible damageWaived in the following situations:•Strongly acidic or alkaline substances;•LD50 < 200mg/kg by dermal route;•Known not to produce skin irritation up to 2000mg/kg.•Albino rabbit recommended as preferred species•Fur removed•Treated with 0.5 ml or 0.5 g of a tested compound, usually for 4 h.•Examined for signs of erythema and edema within 30-60 min, and at 24, 48, and 72 h after pad removal.•Observation duration permitting evaluation of reversible and irreversible effects (≤14 d).2.Skin Sensitization Test•Designed to test the ability of chemicals to illicit immune reactions•Two types of tests: with or without adjuvant(Freund’s Complete Adjuvant)•Guinea pig is the preferred species.•Removal of hair; exposure by intradermal injection or epidermal application(0.2 ml of liquid or 0.2 g of solid)•Two to three weeks as the induction phase, then challenged with a nonirritating dose, and development of erythema is evaluated.3.Acute Eye Irritation/Corrosion Test•The test substance is applied to the anterior surface of the eye.•Eye irritation is reversible within 21 d, eye corrosion is irreversible.•Substances that are strongly acidic or alkaline, and that have been proven to induce strong skin irritation/corrosion, are waived from this test.•The albino rabbit is the preferable species.•Healthy young adult rabbits (3 for each test)•Test substance placed in the conjunctival sac of one eye (0.1 ml of liquid and0.1g of solid), the other served as the control.•The eyes should be washed 30s or 24h after instillation of the test substance.•Examination of ocular reaction: conjunctiva (hyperemia and edema), cornea (turbidity), and iris (hyperemia and hemorrhage), at 1, 24, 48, 72 h and 4 and7 d.Part 3 Subchronic and Chronic Studies•Exposure to the tested compound at levels lower than the lethal level and for longer periods of time, like subchronic and chronic toxicicity studies, is more representative of the realistic situations of human exposure to zenobiotics.Basic Concept•Subchronic toxicity refers to the adverse effects observed after exposure of animals to the test substance for a part of the life-span (within 10%).•Chronic toxicity represents cumulative damage to specific organ systems.•Bioaccumulation is a prerequisite for the toxic effects observed in these long-term toxicity studies.Purpose of the Test and Parameter•Subchronic toxicity study: to establish a no observable adverse effect level (NOAEL) and characterization of target organs；to provide the data neededto predict doses for chronic toxicity studies.•Chronic toxicity study: to establish NOAEL in chronic exposure conditions, very important for risk assessment.Subchronic Toxicity Experimental Design1.Species and Number of Animals•Two species: rat and dog•Route of administration: oral or another as appropriate•Number of animals: 10-20 rats and 4-6 dogs per sex per group•Interim necropsy: If necessary, should be extra numbered.•Age: Just after weaning (within 6-8 w for rodents)2.Route of Administration and Duration•Route of administration: should approximate normal human exposure•Oral route: for food ingredients and some drugs•Dermal, inhalation, non-GI routes: for industrial & agricultural products and drugs•Exposure duration: not invariable, but the most common is 90d in rats and 1-2y in dogs.3.Dosage and Experimental Groups• A minimum of three dose levels plus a concurrent control group.•The high dose should produce toxicity but fatalities ≤ 10%•The low dose produces no apparent toxic effects•The intermediate dose high enough for minimal toxic effects4.Observations and Examinations•General ObservationsAt least once a day (body weight, food consumption, changes in fur, respiratory or cardiovascular distress, motor and behavioral abnormalities)Body weight and food consumption measured at least weeklyHematology and blood chemistry at least three times (before, in the middle and at the end of experiment)•Pathological ExaminationComplete gross necropsy for all test animals (external surfaces, cranial, thoracic and abdominal cavities, and main internal organs)Full histopathology on preserved organs and tissues in at least all animals in the control and the high dose group; when necessary, extended to other groups. Part 4 Chronic Toxicity Experimental Design1.Species and Number of Animals•Selection of animals: very similar to subchronic test, usually two species, one is a rodent (rat), the other is a non-rodent (dog or primate).•Number of animals: 20 rats for each sex each dose group and concurrent control group; a minimum of 4 non-rodents for each sex each group.2.Route of Administration and Duration•Route of Administration: The same as in subchronic toxicity test•Chronic exposures: Usually 2y in rats and 7-10y in dogs.3.Dosage and Administration Groups• A minimum of three dose levels•High dose: inducing toxic response but no fatalities•Low dose: no biologically significant toxic response•Intermediate dose: with minimal toxic effects in test animals4.Observation and Examinations•The same as for subchronic toxicity studies•The positive indicators observed in subchronic toxicity test should be paid more attention.Part 5 Evaluation of Subchronic and Chronic Toxicity•Dose-effect relationship, dose-response relationship•Target organs•Severity of lesions•Indication of further investigation•Determination of NOAEL, one of the parameters used in establishment of ADI and MAC in humans。