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INTRODUCTION 2 TEST GUIDELINE NO. 407 Repeated Dose 28-Day Oral Toxicity Study in Rodents. Adopted in 1981 & revised in 1995


PRINCIPLE OF TEST 3 The test substance is orally administered daily in graduated doses to several groups of experimental animals, one dose level per group for a period of 28 days. During the period of administration the animals are observed closely, each day for signs of toxicity. Animals that die or are euthanised during the test are necropsied and at the conclusion of the test surviving animals are euthanised and necropsied .


PRINCIPLE CONT… 4 A 28 day study provides information on the effects of repeated oral exposure. It can also provide information on the selection of concentrations for longer term studies. The data derived from using the TG should allow for the characterization of the test substance toxicity, for an indication of the dose response relationship and the determination of the No-Observed Adverse Effect level(NOAEL)


DESCRIPTION OF THE METHOD 5 A. Selection of animals species preferred rodent species is the rat other rodent species may be used . But a detailed justification should be given . weight variation of animals used should be minimal and not exceed ± 20% of the mean weight of each sex.


DESCRIPTION CONT… 6 B. Housing and feeding T emperature in the experimental animal room should be 22°C (± 3°C ). Relative Humidity: 30% - 70% Lighting should be artificial, the photoperiod being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water . For group caging, no more than five animals should be housed per cage.


DESCRIPTION CONT… 7 C. Preparation of animals Healthy young adult animals are randomly assigned to the control and treatment groups . Cages should be arranged in such a way that possible effects due to cage placement are minimized. The animals are identified uniquely and kept in their cages for at least five days prior to the start of the treatment study to allow for acclimatisation to the laboratory conditions.


DESCRIPTION CONT… 8 D. Preparation of doses The test compound is administered by gavage or via the diet or drinking water. The method of oral administration is dependent on the purpose of the study, and the physical/chemical/ toxico -kinetic properties of the test material. Where necessary, the test substance is dissolved or suspended in a suitable vehicle.


PROCEDURE 9 A. Number and sex of animals At least 10 animals (five female and five male) should be used at each dose level. If interim euthanasia are planned, the number should be increased by the number of animals scheduled to be euthanised before the completion of the study.


PROCEDURE CONT… 10 B. Dosage at least three test groups and a control group should be used. there are no suitable data available, a range finding study (animals of the same strain and source) may be performed to aid the determination of the doses to be used . If a vehicle is used in administering the test substance, the control group should receive the vehicle in the highest volume used.


PROCEDURE CONT… 11 The highest dose level should be chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and no-observed-adverse effects at the lowest dose level (NOAEL).


PROCEDURE CONT… 12 C. Administration of doses The animals are dosed with test substance daily 7 days each week for a period of 28 days . The volume should not exceed 1 ml/100g body weight except in the case of aqueous solutions where 2 ml/100 g body weight may be used.


OBSERVATION 13 General clinical observations should be made at least once a day. At least twice daily, all animals are observed for morbidity and mortality . In the fourth exposure week sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli ) assessment of grip strength and motor activity assessment should be conducted.


OBSERVATION CONT… 14 Functional observations conducted in the fourth exposure week may be omitted when the study is conducted as a preliminary study to a subsequent subchronic (90-day) study. At necropsy, the oestrus cycle of all females could be determined (optional) by taking vaginal smears. These observations will provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of estrogen sensitive tissues

Body weight and food/water consumption :

Body weight and food/water consumption 15 All animals should be weighed at least once a week . Measurements of food consumption should be made at least weekly. If the test substance is administered via the drinking water, water consumption should also be measured at least weekly.

Haematology :

Haematology 16 H aematocrit , Haemoglobin concentrations, Erythrocyte count, Reticulocytes , Total and differential leucocyte count , P latelet count M easure of blood clotting time/potential.

Rat Hematologic Reference Ranges:

Rat Hematologic Reference Ranges 17 RBC - 6.76-9.75 x 10 6 /mm 3 PCV- 37.6-50.6% WBC - 6.6-12.6 x 10 3 /mm 3 Hemoglobin - 11.5-16.1 g/ dL Neutrophils - 1.77-3.38 x 10 3 /mm 3 Lymphocytes- 4.78-9.12 x 10 3 /mm 3 Eosinophils-0.03-0.08 x 10 3 /mm 3 Monocytes-0.01-0.04 x 10 3 /mm 3 Basopnils-0.00-0.03 x 10 3 /mm 3 Platelets-150-460 x 10 3 / mL

Clinical biochemistry :

Clinical biochemistry 18 T o investigate major toxic effects in tissues and, specifically, effects on kidney and liver. Investigations of plasma or serum shall include sodium, potassium, glucose, total cholesterol, urea, creatinine , total protein and albumin, at least two enzymes indicative of hepatocellular effects (such as alanin aminotransferase , aspartate aminotransferase , alkaline phosphatase , γ- glutamyl trans-peptidase and glutamate dehydrogenase ), and bile acids.

Rat Biochemical Reference Ranges :

Rat Biochemical Reference Ranges 19 Total protein 5.6-7.6 g/ dL Albumin 3.8-4.8 g/ dL Glucose 50-135 mg/ dL BUN 15-21 mg/ dL Creatinine 0.2-0.8 mg/ dL Sodium 143-156 mEq /L Potassium 5.4-7 mEq /L Chloride 100-110 mEq /L Phosphorous 3.11-11 mg/ dL Calcium 5.3-13 mg/ dL

Rat Biochemical Reference Ranges cont.. :

Rat Biochemical Reference Ranges cont.. 20 ALT- 17.5-30.2 U/L AST -45.7-80.8 U/L Alkaline phos-56.8-128 U/L Cholesterol- 40-130 mg/ dL Total bilirubin-0.2-0.55 mg/ dL Amylase-128-313 SU/ dL BUN = blood urea nitrogen ALT = alanine aminotransferase AST = aspartate aminotransferase


PATHOLOGY 21 A. Gross Necropsy The liver, kidneys, adrenals, testes, epididymides , prostate + seminal vesicles with coagulating glands as a whole, thymus, spleen, brain and heart of all animals should be trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon as possible after dissection to avoid drying.

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22 B. Histopathology Full histopathology should be carried out on the preserved organs and tissues of all animals in the control and high dose groups.


DATA 23 all data should be summarised in tabular form showing for each test group. The number of animals at the start of the test. The number of animals found dead during the test or euthanised for humane reasons and the time of any death or euthanasia. The number showing signs of toxicity. A description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects.

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24 Comparisons of the effect along a dose range should avoid the use of multiple t-tests.

Test report :

Test report 25 Test substance physical nature, purity and physicochemical properties; identification data. Vehicles justification for choice of vehicle, if other than water.

Test report Cont…:

Test report Cont… 26 Test animals Species/ Strain used; Number, sex & age of animals; source, housing conditions, diet, etc.; individual weights of animals at the start of the test. justification for species if not rat

Test report Cont…:

Test report Cont… 27 Test conditions: rationale for dose level selection; details of test substance formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation; details of the administration of the test substance; conversion from diet/drinking water test substance concentration ( ppm ) to the actual dose (mg/kg body weight/day), if applicable; details of food and water quality. Optional endpoints investigated list of optional endpoints investigated

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