Acute Toxicity Testing

Principles of Acute Toxicology

• Median lethal dose (LD50)
• Historically used for the standardization of digitalis extracts, insulin, and
diphtheria toxin.
• High precision LD50 values can only be established with a large number of
animals.
Principles of Acute Toxicology
• Median lethal dose (LD50)
• extraneous factors that affect the precision includes, among other factors,
sex, species, strain, age, diet, nutritional status, general health conditions,
animal husbandry, experimental procedures, route of administration, stress,
dosage formulation (vehicle), and intra- and interlaboratory variations.
Definition of Acute Toxicity
• harmful effect of a chemical or a drug on a living organism.

• Based on Organization for Economic Cooperation and Development (OECD)

• acute toxicity is “the adverse effects occurring within a short time of (oral)
administration of a single dose of a substance or multiple doses given within 24
hours.”
Dose-Response Relationship
• Two types of Data
• Quantal response – all or none; happens or does not happen
• e.g. mortality, pharmacotoxic signs
• Graded response – determined quantitatively
• e.g. enzyme activity, protein concentration, body weight, feed consumption, and
electrolyte concentration are quantitative parameters.
LD50 and Its Determination
• dose of a compound that causes 50% mortality in a population.
Or
• “statistically derived single dose of a substance that can be
expected to cause death in 50% of the animals” - OECD
Determination of LD50
• Methods
• Normal population assumption – graphic procedures like probit analysis
• normal population assumption-free methods like Thompson’s moving average
interpolation
Acute Toxicity Testing
• Objectives:
• define the intrinsic toxicity of the chemical
• predict hazard to nontarget species or toxicity to target species
• determine the most susceptible species
• identify target organs
• provide information for risk assessment of acute exposure to the chemical
• provide information for the design and selection of dose levels for
prolonged studies
• important and practical of all, provide valuable information for clinicians in
the prediction, diagnosis, and treatment for acute overexposure
(poisoning) to chemicals.
Acute Toxicity Testing
• Objectives:
• provide valuable information for clinicians in the prediction,
diagnosis, and treatment for acute overexposure (poisoning) to
chemicals  most important and practical of all
Acute Toxicity Testing
• Acute toxicity is not equivalent to the LD 50, and that the LD50 is not
an absolute biological constant to be equated, as some investigators
have, with such chemical constants as pH, pKa, melting point, and
solubility.
Acute Oral Toxicity
• Classical Method
• Fixed Dose
• Acute Toxic Class
• Up and Down Procedure
Classical Method
• Principle:
• test material, undiluted or diluted with the appropriate solvent or suspending
vehicle, is given to several groups of animals by gavage with a feeding needle
or by gastric intubation.
• A vehicle control group is included if needed, but generally this group is not
necessary if the toxicity of the vehicle is
known.
• Clinical signs, morbidity, and mortality are observed at specific intervals.
Classical Method
• Principle:
• Animals that die or become extremely moribund during the study are
subjected to necropsies.
• At the conclusion of the study’s observation period, surviving animals are
killed and necropsied.
• Tissues may be saved for histopathological examination to facilitate the
understanding of the acute toxicity of the compound.
Classical Method
• Number and Sex
• Ideal three animals per dose level
• Literature surveys have shown that when there are sensitivity differences
between the sexes, females, in general, are more sensitive.
• Dose Levels
• Three dose levels generally are suffiient.
• Selected dose levels should bracket the expected LD50 value with at least one
dose level higher than the expected LD50but not causing 100% mortality, and
one dose level below the expected LD50 value but not causing 0% mortality,
when the probit analysis method is applied to estimate the LD50.
Classical Method
• Dosages
• test substance should be dissolved or sus pended in a suitable vehicle,
preferably in water, saline, or an aqueous suspension such as 0.5%
methylcellulose in water.
• If a test substance cannot be dissolved or suspended in an aqueous medium
to form a homogeneous dosage preparation, corn oil or another solvent can
be used
• If the toxicity of the vehicle is not known, a vehicle control group should be
included in the test.
• The animals in the vehicle control group should receive the same volume of
vehicle given to animals in the highest dose group.
Classical Method
• Dosage
• The maximum dose volume in rodents should not exceed 10 mL/kg body
weight for nonaqueous vehicles or 20 mL/kg body weight for aqueous
solution or suspension. In any event, for scientific and humane reasons the
dose volume should be as small as possible.
Fixed-dose Procedure (test limit)
• traditional test limit for acute oral toxicity was considered to be 5.0
g/kg body weight
• nowadays a test limit of 2.0 g/kg body weight is being used
• Modified dosing includes 5, 50, 500, and 2000 mg/kg.
Acute Toxic Class Method
• described by Roll et al. and based on the assumption that using a
minimum number of animals in a stepwise procedure will provide
enough information on the acute toxicity of a substance to allow
classification according to the most commonly used classification
schemes.
Acute Toxic Class Method
• Three animals of one sex are used for each step;while normally
females are used (considered to be generally slightly more sensitive,
either sex can be used.
Acute Toxic Class Method
• The initial dose is selected from one of the following fixed-dose levels:
5, 50, 300, or 2000 mg/kg body weight and should be chosen to
produce some mortality.
• If existing information suggests that mortality is unlikely at the 2000
mg/kg dose, then a limit test at that level may be conducted with
three animals of each sex.
Up-and-Down Procedure
• one of the more modern methods of estimating the LD50, is based on
the maximum likelihood method.
• Follows a stepwise procedure.
• Animals are dosed one at a time at a minimum of 48 h intervals, with
the fist animal receiving a dose just below the estimated LD50.
Up-and-Down Procedure
• If the fist animal dies, the next one receives a lower dose. The spacing
of doses generally is adjusted by a factor of 3.2 (default factor
corresponding to a dose progression of one half log unit) up or down
depending on the outcome of the previous animal.
Up-and-Down Procedure
• The OECD test guideline also contains the provision for a limit test
that uses a maximum of five animals.
• This test is used when there is information suggesting that the test
material has a low potential to be toxic, in this study a test dose of
2000 mg/kg or, as required, 5000 mg/kg is used.
Acute Dermal Toxicity
• Objective: To assess the adverse effects resulting from a single dermal
application of a test substance.
• The acute dermal test also provides the initial toxicity data for:
• regulatory purposes
• Labeling
• Classification
• Transportation
• subsequent subchronic and chronic dermal toxicity studies.
Principle
• Test material is applied dermally, undiluted or diluted with the
appropriate solvent, in graduated doses to several groups of animals.
• A vehicle control group is included if needed, but generally this group
is not necessary if the toxicity of the vehicle is known.
Animals
• The three most commonly used animal species for this type of test
are:
• Rabbits
• Rats
• guinea pigs
• At the start of the study, healthy animals between 8 and 12 weeks old
with a range of weight variation not exceeding ±20% of the
appropriate mean value should be used.
Animals
• The animals should be housed individually in a controlled
environment.
• Quarantine, acclimatization, and randomization are as described in
earlier text for acute oral toxicity studies.
• The back of the animal or a band around the trunk should be clipped
free of hair.
Animals
• When clipping the hair, care must be taken not to abrade the skin.
• If abraded skin is called for, a needle may be used, but care must be
taken not to damage the dermis.
• Fasting animals overnight is not necessary.
• Generally, 10 animals per dose level (5/sex) are suffiient to allow for
an acceptable estimation of the dermal LD50.
• Females used in the study should be nulliparous and not pregnant.
Dose Levels
• Higher doses do not need to be tested when a test substance at 2000
mg/kg considered the limit dose, has not produced test substance-
related mortality.
• Administration of additional test substance would only be applied on top of
the test substance layer already present.
• layering may form a physical barrier to prevent further absorption of the
test substance from the application site.
Dose Levels
• While a control group generally is not needed, a vehicle
control group should be included in the study if the toxicity
of the vehicle is not known.
• Its influence on dermal penetration of the test substance should be
fully established prior to the study.
Preparation of dosage and dosing Procedure
• test substance should be applied uniformly to approximately 10% of
the body surface of the animal (e.g., 4 cm × 5 cm for rats, 12 cm × 14
cm for rabbits, 7 cm × 10 cm for guinea pigs).
Preparation of dosage and dosing Procedure
• Liquid test substances generally are applied undiluted.
• If the test substance is a solid, it should be pulverized, weighed,
placed on a plastic sheet or porous gauze dressing, moistened so as to
form a paste with normal saline (one part test substance for one part
saline) or an appropriate solvent, and then spread evenly on the
closely clipped skin to ensure uniform contact with the skin.
Exposure Period and Removal of Cuff
• Almost all testing guidelines call for 24 h continuous exposure.
• Upon completion of the exposure, the cuff is removed and the
application site is gently wiped with a paper towel soaked with saline,
water, or any appropriate solvent to remove residual test substance
remaining on the application site.
Observation Period
• recommended minimum observation period is 14 days.
• Skin irritation should be assessed according to a scoring system
such as the one described by Draize et al.
Acute Inhalational Toxicity
• Objective: To evaluate the toxic potential of a test material that may
be inhaled, such as a gas, a volatile substance, or an aerosol.
• Acute inhalation test provides the:
• Initial toxicity data for regulatory purposes
• Labeling
• Classifiation
• Transportation
• Subsequent subchronic and
chronic dermal toxicity studies
Principle
• Several groups of animals are exposed to a fixed concentration of test
material by inhalation for a short period of time, one concentration
per group.
• While whole body exposure inhalation data are accepted by
regulatory agencies, it is recommended that nose-only or head-only
exposure be used as this minimizes oral exposure from animals licking
the compound off their fur.
Animals
• Preferred species: Rat
• At the start of the study, healthy animals between 8 and 12 weeks
old with a range of weight variation within and between test
groups should not exceed ±20% of the mean weight.
• The animals should be housed individually in a controlled
environment.
• Quarantine, acclimatization, and randomization are as
described in earlier text for acute oral toxicity studies.
Animals
• At least 10 animals (5/sex) per concentration level are recommended
in most regulatory guidelines giving sufficient numbers to allow for an
acceptable estimation of the inhalation LC50.
Environmental Conditions
• Inhalation equipment used should be able to sustain a dynamic
airflow of 12–15 air changes per hour, ensure adequate oxygen
content of 19
• If a whole body chamber is used, individual housing must be used and
the total volume of test animals should not exceed 5% of the volume
of the test chamber.
• Temperature and relative humidity need to be monitored
continuously and should be maintained at 22°C ± 2°C and 30%–70%,
respectively.
Dose levels
• In general, three concentration levels should be used and
spaced to produce a concentration–response curve in order
to permit an acceptable determination of the LC50.
• Animals are usually exposed for a period of 4 h.
• EPA guidelines recommend that the mass median aerodynamic
diameter (MMAD) particle size range should be between 1 and 4 μm
(particle size distribution that permits deposition throughout the
respiratory tract).
Test Limit
• EPA guidelines recommend that the mass median aerodynamic
diameter (MMAD) particle size range should be between 1 and 4 μm
(particle size distribution that permits deposition throughout the
respiratory tract).