Determination of toxicity

Toxicity can be measured by the effect the substance has on an organism, a

tissue or a cell.

Individuals will respond differently to the same dose of a substance because of

a number of factors including their gender, age and body weight. Therefore a
population-level measure of toxicity is often used.

The probability of an outcome for a population is then related to a given

individual in a population
Importance of testing toxicity

 Rapid industrial and chemical development introduces many new, toxicologically

undefined substances into the global environment and poses new challenges for
toxicology as a scientific discipline.
 Toxicological testing is used to determine the toxicity of the chemicals we use or are
exposed to, and to give information about the potency of their toxic effects.
 It gives information about industrial chemicals, pharmaceuticals and natural
products, and Knowing whether a chemical can cause cancer, allergic reactions or
abnormalities in unborn children is important to human health,
 to know the biochemical mechanisms of the toxicity,
IMPORTANCE

 To reveal potential carcinogenic effects and to perceive adverse

effects.
 The testing also helps in evaluating poisoning potency of various
drugs.
 It also helps in determining whether a particular food additive is safe
for consumption by the general public
 Toxicological testing includes reproductive testing, developmental
testing and genotoxic testing in the human environment.
Acute toxicity testing
 Acute toxicity testing is carried out to determine the effect of a single dose on a particular animal
species.

 Acute toxicity testing is carried out with two different animal species (one rodent and one nonrodent).

 In acute toxicological testing, the investigational product is administered at different dose levels, and
the effect is observed for 14 days.

 All mortalities caused by the investigational product during the experimental period are recorded and
morphological, biochemical, pathological, and histological changes in the dead animals are
investigated.

 Acute toxicity testing permits the 50% lethal dose (LD50) of the investigational product to be

determined.
Acute toxicity testing
• The information for an LD50 must include the substance, the
route of entry and the animal species. For example, table
salt has an oral LD50 of 3 gm/kg in rats. Paracetamol has an
oral LD50 of 1.944 gm/kg in rats
• The LD50 was used as an indicator of acute toxicity
previously. The determination of the LD50 involves large
numbers of animals, and the mortality ratio is high. Because
of these limitations, modified methods were developed .
Acute toxicity testing cont.,--

•Advantages

• This is a simple method to apply and does not require statistical calculations of

the lethal concentrations.

• It takes a short period of time to carry out the test and come up with the results.

• It is cheap to execute since it does not involve use of expensive equipment

Disadvantages
• Use of this method contributes to destruction of biodiversity by killing the

animals used as specimen. It also does not lead to accurate identification of the

toxin concentration (Hunt, et al, 2018).

Fixed-dose procedure

• In 1992, the fixed-dose procedure (FDP) was proposed as an alternative

test to LD50. It uses fewer animals, and there is less pain and suffering. In this
procedure, the test substance is given at one of four fixed-dose levels (5, 50,
500 and 2000 milligrams per kilogram) to five male and five female rats.
• When a dose produces clear signs of toxicity but no death is identified, the
chemical is then classified at that level
•Advantages
• It is an easy method to use since fixed-dose concentrations are utilized.
The method is also relatively cheap to apply and uses fewer number of animals
compared to the lethal dose procedure (Hunt, et al, 2018).
•Disadvantages
• It contributes to the death or possible maiming of the animals used as
 Parts per million

• Some chemicals can cause toxicity at very low doses, so it is important to

understand how low doses compare to one another. Parts per million (ppm), parts
per billion (ppb) and parts per trillion (ppt) are the most commonly used terms to
describe very small amounts of substances.

They are measures of concentration – the amount of one substance in a larger

amount of another substance.
Scientists use these measurements when measuring a toxic chemical in a lake or
toxins in the air such as greenhouse gases
Parts per million Cont.,--

•Advantages

• This technique is applicable in detecting very small concentrations of toxins hence it

can be used for trace analysis. It is also relatively accurate and its findings are reliable

• Disadvantages

• This technique requires training to be able to use the equipment that generate these

minute concentrations. The person conducting toxicology test using this method should

further be trained on how to statistically calculate the concentrations (Tan, & Schirmer,

2017).
Sub chronic Toxicity Testing

•It provides information about some chronic effects, target organs and
the possible accumulation of the substance in the tissues.
•It is performed over a period of several months to a year, and the testing
parameters are determined according to the results of acute testing.
•The study usually involves four groups of test animals, including a
control group.
•The number of the animals in each group is usually from 10 to 20 for
each gender, and usually involves two test animal species.

•The test substance is blended with the purified diet, ensuring the
stability of the test substance.

•.
Sub chronic toxicity test
• The physical appearance, behavior, body weight, food intake and
excretion of the animals are monitored regularly.
• Biological fluids are collected for hematological and biochemical
tests.
• Specific hepatic, eye and gastrointestinal functions are monitored and
blood pressure and body temperature are measured at regular time
intervals.
• Sub chronic testing usually reveals cumulative effects of the examined
substance in tissues and on metabolic systems.
• Upon demonstration of animal illness, or upon the completion of the
study, a necroscopy is performed and organs and tissues are examined
for evidence of pathological changes
1.2.3 Chronic Toxicity

•The main goal of chronic toxicity testing is to elucidate the biochemical

mechanisms of the toxicity, to reveal potential carcinogenic effects and to perceive
adverse effects that are not clearly evident in sub chronic testing.
•In this long-term study, a larger group of experimental animals is employed and
lower doses of the test substance are applied for longer periods.
•The study seeks evidence of long-term effects and the doses are carefully calibrated
so as not to overburden the organism’s capacity for absorption, transportation,
biotransformation and excretion.
•Since the aim of chronic toxicity study is to explain and define mechanisms by
which adverse effects arise, chronic toxic effects of the free substances are
undesirable and are avoided by adjusting the dose.
•The number of test animals in a single dose-level group is usually 50, but may be
higher if a higher level of confidence is desired
Mice Bioassay
•A mice bioassay is using a mouse to determine how safe a food is. An extract is made from
the food and injected directly into a mouse, and if the mouse survives, the food is safe, and
if the mouse dies, the food is unsafe.

• But it doesn’t tell us why the food is unsafe, at what level the food might be unsafe, and it
is subject to the mouse dying for other reasons which are not related to food safety.
Sometimes it can miss toxins, and also sometimes the test is positive but there’s no toxin in
the food. Gastric intubation (gavage), an artificial means of substance introduction, has
advantages with respect to dosing exactness and reduced variation.

•The gavage is performed after the animals have fasted in order to avoid mixing chemical
test substances with stomach contents. the chemical dose should not exceed 5% of the test
animal’s diet.
•Gastric intubation (gavage), an artificial means of substance introduction, has
advantages with respect to dosing exactness and reduced variation.

•The gavage is performed after the animals have fasted in order to avoid mixing
chemical test substances with stomach contents. the chemical dose should not
exceed 5% of the test animal’s diet.
Shell fish cont.,---
• Housing of the animals also is very important, as the material
composition of their cages, residue from wood chips, isolation and
other parameters, have been shown to affect test results by affecting
enzyme and hormone levels, which in turn influence biotransformation
processes.
• A mouse bioassay, it just gives you very simple information to tell you
whether or not there’s potentially a toxin there. The mouse bioassay is
a functional assay that detects biologically active toxin.
• The assay requires a three-part approach: toxin screening, toxin titer,
and finally toxin neutralization using monovalent antitoxins. The
process requires two days of analysis at each step.
Bioassay shellfish

•Advantages
 The method is economical, amenable to frequent handling and
lacking in the vomiting reflex
 The animals metabolize and secretes a substance in the same manner
as humans do thus suitable for use in experiments activity of a
substance in humans.
 Use of younger animals provides the advantage of obtaining faster
results due to their higher rate of metabolism, as well as their being
cheaper to purchase and house.
 Examines the results of different routes of exposure under controlled
conditions.
•Disadvantages
 Metabolic differences between humans and surrogate test animals can
result in erroneous conclusions.
 differences in genetic pool, lifespan, susceptibility, metabolic
pathways and other parameters of organic life, present serious
limitations when extrapolating the results of animal testing to human
populations.
Using liquid chromatography-mass spectrometry

• In 2000, the Cawthon Institute in Nelson designed a way to test for toxicity levels in

shellfish using liquid chromatography-mass spectrometry (LC-MS).

• Using LC-MS, the actual toxin can be identified (unless it’s a new toxin that hasn’t

been identified before) as well as the level of its toxicity.

• This test means that toxins can be detected and monitored using chemical procedures

instead of mice.

• As a result of this method, mice would only be needed to detect a new toxin – which

would be rare. Legislation has been passed that, from 2012, all countries in the European

Union use LC-MS to detect known toxins and measure toxicity levels in shellfish
Advantages and disadvantages

•Advantages

• This technique has the advantage of producing very accurate results if the samples are

accurately taken. It also does not need the use of life animals hence helps in saving the

lives of these animals

•Disadvantages

• The use of the liquid chromatography –mass spectrometry technique is a technical

method that requires expert training on how to use the equipment to take readings and

interpret them. Additionally the initial cost of purchasing and installing this equipment is

quite high.
• These two disadvantages make the application of this technique limited to only a few
In silico approaches

•The method use computer (in silico) modelling using structure-activity

relationships (SAR). In SAR, the chemical structure and other physico-
chemical properties of the substance are used to predict its
toxicokinetic. Based on available toxicity databases, a predicted
threshold of toxicological concern can be assigned to the chemical.

•Silico method refer to methods or prediction using computational

approaches.
Silico method cont.,---
•The silico methods include databases, quantitative structure-activity
relationships, pharmacophores, homology models and other molecular
modeling approaches, machine learning, data mining, network analysis
tools and data analysis tools that use a computer

•The silico method is very useful in the absence of other toxicological

data, but are based on data from other chemicals.
Advantage of silico method

 They can make fast predictions for a large set of compounds in a

high-throughput mode.
 They make their prediction based on the structure of a compound
even before it has been synthesized
 Such models have seen frequent use in the discovery and
optimization of novel molecules with affinity to a target, the
clarification of absorption, distribution, metabolism, excretion and
toxicity properties as well as physicochemical characterization.
Disadvantages

 The approaches are complex to use and expensive especially for the developing
countries
 The approaches have a long computational time
 In some cases, settings may produce inaccurate predictions for optimal growth
and production fluxes.
 Does not supply the function of amplification for the predicted genes for any
finer performance