2 acute toxicity studies

Question 1

what are in vivo toxicity studies and what is the point of them?

Answer 1

-scientific study of adverse effects that occur in animals due to a chemical substance (how strong is effect, is it debilitating, etc)
-to observe and report symptoms, mechanisms, detection and treatment of toxic substances

Question 2

how long should we wait to see toxicological effects?

Answer 2

Question 3

what are single dose/acute toxicity studies? what is the point of them?

Answer 3

-administration of a single dose or multiple doses (more rare) within 24 hours
-different doses in different animals, and effect is observed for 14 days
-to acquire data on the dose-effect relationship of a new drug
-regulatory body requires the acute toxicity test report for labelling and classification of substances for human use
-goal is to find LD50

Question 4

what are the main goals for single dose studies?

Answer 4

-to determine LD50 during short-term exposure
-to investigate the potential for acute toxicity in humans
-provides insight into target organs and toxic mode of action (toxicokinetics)
-provide data on signs expected in cases of acute overdose (extrapolate warning signs from animals)
-time course of drug-induced clinical observations (hourly time course)
-to establish dose levels for subsequent repeated-dose studies (no effect to death)
-species differences in toxicity (usually use rodents)

Question 5

what are the initial considerations before testing (on animals)?

Answer 5

-identify the chemical structure of the test substance
-physio-chemical properties (solubility) (if IV, needs to be soluble in saline and water)
-results in any other in vitro and/or in vivo toxicity tests on the substance
-toxicological data in structurally related substances or similar mixtures

Question 6

what are the two tests in acute oral toxicity?

Answer 6

-limit test
-main test

-to save number of animals
-not the only acute toxicity study; just more drugs are taken orally

Question 7

what is the limit test?

Answer 7

-dose one animal at the test dose (at 2000mg/kg or 5000 mg/kg)
-if animal dies, conduct the main test to determine the LD50
-if the animal survives, dose four additional animals sequentially so that a total of five animals are tested

Question 8

when is the LD50 greater than the test dose? (in the limit test)

Answer 8

-the LD50 is greater than the test dose (2000 mg/kg) when three or more animals survive

Question 9

when is the LD50 lesser than the test dose? (limit test)

Answer 9

-the LD50 is less than the test dose (2000 mg/kg) when three or more animals die

Question 10

what is the main test?

Answer 10

-single animals are dosed in sequence usually at 48hr intervals
-starting dose is selected based on previous data (if available)
-the first animal is dosed a step below the best preliminary estimate of the LD50
-if the animal survives, the second animal receives a higher dose
-if the first animal dies or appears moribund (severly debilitating), the second animal receives a lower dose

Question 11

what is the dose progression factor?

Answer 11

the dose progression factor should be chosen to be the antilog of: 1/(the estimated slope of the dose-response curve)

-factor is to see how much increase the dose is for testing

Question 12

what is the default of the dose progression factor?

Answer 12

-dose progression should remain constant throughout testing
-when there is no information on the slope of the substance to be tested, a dose progression factor of 3.2 is used (corresponds to a slope of 2)
-using the default progression factor, doses would be selected from the sequence 1.75 (very toxic), 5.5, 17.5, 55, 175, 550, 2000mg/kg

Question 13

what is the reversal process?

Answer 13

a combination of stopping criteria is used to keep the number of animals low (6-15 animals)
-at least four animals have followed the first reversal (when the response changes from one outcome to another)

Question 14

what is the stopping criteria to ensure the number of animals low (6-15 animals)?

Answer 14

-3 consecutive animals survive at the upper bound (highest concentration, usually 2000 mg/kg or 5000 mg/kg-low toxicity compounds)
-at least four animals have followed the first reversal
-five reversal occur in any six consecutive animals tested (e.g. OXOXOX)

Question 15

what are done with the moribund animals?

Answer 15

moribund animals killed for humane reasons are considered in the same way as animals that died on test
-if an animal unexpectedly dies late in the study, stop dosing and observe all animals to see if they also die during a similar observation period
-if the above is true, it is appropriate to start the study again beginning at least two steps below the lowest dose with deaths (and increasing the observation period)

Question 16

what are the animal models for acute oral toxicity?

Answer 16

-rodent species, preferably female rats (easy to translate to humans due to similar toxicokinetics)
-females should be nulliparous and non-pregnant (more sensitive so therefore we are conservative)
-between 8 and 12 weeks old (avoid hormone cycles)
-weight should fall in an interval within +-20% of the mean initial weight of any previously dosed animals

Question 17

what is the housing and feeding conditions in the acute oral toxicity?

Answer 17

-room should be 22 degrees celcius (+-3), humidity 50-60%
-artificial lighting: 12 hour light, 12 hour dark
-at 48 hours after dosing, animals may be returned to group housing
-conventional rodent laboratory diets with an unlimited supply of drinking water (need to keep in mind the food that can alter absorption by altering pH of GI tract)

Question 18

what is the preparation of the doses of acute oral toxicity?

Answer 18

-constant volume over the range of doses to be tested
-maximum volume of liquid depends on the size of the test animal
-rodents: 1 ml/100g of body weight; or 2ml/100g of body weight (if aqueous solution)
-vehicle: preferably water (solvent we are using to dissolve)
-doses should be prepared shortly prior to administration (if stability is not known)

Question 19

what is the administration of doses in acute oral toxicity?

Answer 19

-single dose by gavage using a stomach tube or a suitable intubation cannula
-animals should be fasted prior to dosing (rats, overnight; mice, 3-4 hours)
-animals should be weighed prior to dosing
-after dosing, food withheld for a further 3-4 hours (rats), 1-2 hours (mice)

Question 20

what are the observations taken in the acute oral toxicity test?

Answer 20

-the first 30 minutes after dosing (at least once);
-periodically during the first 24 hours (special attention during the first 4 hours)
-daily thereafter, for a total of 14 days
-duration of the observation should not be fixed rigidly

-body weight: at least weekly after dosing and at the end of the test

-time course of onset of signs of toxicity and whether these were reversible for each animal

pathology: all animals should be subjected to gross necroscopy (target organ identification, histopathology)

Question 21

what are the organs and what happens to them in acute toxicity tests?

Answer 21

Question 22

what organs and tissues do we test that may be retained at necroscopy?

Answer 22