Measuring Toxicity and Assessing Risk

Introduction
Toxicology is the scientific study of poisons and their adverse effects on living organisms. While
virtually any substance can be toxic in high enough quantities (including water), toxicology
primarily investigates substances that can cause harm at relatively low doses. Understanding the
relative toxicity of various substances is crucial for multiple applications, including drug
development and environmental risk assessment. This chapter outlines the methods toxicologists
use to measure toxicity, focusing on the dose-response relationship, laboratory animal testing,
statistical analyses for quantifying toxicity, and the application of toxicity data in risk assessment.

Chemistry of Toxicants
A thorough understanding of the chemistry of toxicants is essential, as it significantly influences
their solubility, reactivity, and overall behavior in biological systems. Key aspects include:

• Solubility and Reactivity: The chemical properties of a substance determine how it

interacts with biological systems, including how it is absorbed, distributed, metabolized,
and excreted.
• Administration Vehicles: Knowledge of the chemical nature of a toxicant guides the
choice of vehicle (e.g., solvent or medium) used for its administration during testing.

• Biotransformation: Understanding the chemical structure aids in predicting potential

metabolic pathways and the products of biotransformation, which can influence toxicity.
• Small Molecules vs. Large Compounds:

• For small molecules, toxicity can often be assessed through straightforward

chemical analyses, such as determining the octanol/water partition coefficient to
evaluate lipid solubility.

• The emergence of large polymeric compounds (e.g., proteins, nucleic acids)

presents challenges in toxicity assessment due to their complex structures and
behaviors.

Toxicity Testing Methods

Toxicity testing encompasses a variety of methods aimed at assessing the potential harmful effects of
substances. These methods can be broadly categorized into structural analysis, in vitro testing,
and animal bioassays.

1. Structural Analysis
 > Chemical Structure Observation: Analyzing the chemical structure of a compound can
provide insights into its potential behavior within biological systems.

> Structure-Activity Relationship (SAR): This concept involves comparing the chemical
structure of a compound to similar compounds with known toxicity. By understanding the
relationship between chemical structure and toxicity, researchers can predict potential toxic
effects and prioritize chemicals for further testing.

2. In Vitro Testing
> Definition: "In vitro" means "in glass," referring to experiments conducted outside of a
living organism, typically in controlled laboratory settings such as test tubes or petri dishes.

> Applications:

o Cell Cultures: Used to identify carcinogens and assess cellular responses to

toxicants.
o Isolated Organ Testing: Perfused organs (e.g., liver or kidney) can be used to
study the effects of toxicants in a more complex biological context.

> Limitations: While in vitro methods provide valuable insights into mechanisms of toxicity,
they are inherently limited in their ability to replicate the complexities of whole living
organisms.

3. Animal Bioassays
> Most Effective Model: Animal bioassays are currently the most effective method for
gaining comprehensive insights into the potential effects of chemicals on human health and
ecological systems.

> Species Selection:

o The choice of animal species depends on the desired endpoints of the study. For
research focused on human health, species that share physiological similarities with
humans are preferred.

o For ecological health studies, key species within ecosystems are selected to
understand broader environmental impacts.

o Smaller and simpler model organisms (e.g., rodents) are often chosen for their ease
of handling and lower costs in laboratory settings.

> Transgenic Models:

o These are genetically modified organisms designed to study specific aspects of

human diseases.
 o Knockout Animals: A specific gene is deliberately disabled to study its effects.
Various knockout mouse lines are available, each with different genes knocked out,
allowing researchers to investigate the role of specific genes in toxicity and disease
processes.

Testing in Animals
> Purpose and Importance:
o Animal testing provides valuable data on the mode of action of substances and
identifies affected physiological systems, organs, and tissues.

> Regulatory Guidelines:

o Various regulatory agencies, such as the FDA and EPA, have developed specific
protocols for toxicity testing, but these protocols generally share common
characteristics.
> Considerations in Animal Testing:
o Sex and Age: It's important to test both male and female organisms, and testing
across different ages maybe necessary.

o Humane Treatment: All animals must be treated humanely, with consistent

handling across both treated and control groups.

> Animal Identification and Housing:

o Animals may be tagged for identification using methods such as numbered metal
ear tags or electronic transponding implants.

o Housing conditions must be clean, comfortable, and provide adequate food and
water.
o Animals are typically housed in conventional box-type cages, but specialized cages
(e.g., metabolism cages) may be used to collect and measure urine and feces
accurately.

> Monitoring and Observations:

o Body Weight: Measured daily or periodically to assess health and growth.

o Behavioral Observations: Comparing behaviors of treated animals with control

groups to identify any changes.
 o Symptom Monitoring: Close observation for symptoms of poisoning, such as
tremors or convulsions, and timing of symptom onset, which can provide insights
into the mechanism of toxicity.

Post-Exposure Procedures:
o Necropsy: After the exposure period, animals are sacrificed for necropsy, where
treated and control animals are dissected.
o Organ Examination: Organs are weighed and examined for gross morphological
and physiological toxic effects.

o Histopathological Analysis: Tissue samples are sliced using a microtome and

examined under a microscope for any abnormalities in cells or tissues
(histopathology).

o Biochemical Analysis: Tissue samples may also be analyzed for biochemical

indicators of pathology to further assess the impact of the substance.

FACTORS TO BE CONSIDERED IN PLANNING TOXICITY

TESTING
There are several questions that must be answered in designing laboratory studies to determine the
toxicity of a chemical substance. Among these are

Through what physiological route does exposure occur (in other words,
how does the substance enter the body)?
How much of the substance is necessary to produce toxicity?
Over what period of time does exposure occur?
1. Routes of Exposure in Toxicity Testing
Oral Toxicity:
Primary concern for substances that may be ingested, such as pesticides, food
additives, or oral drugs.
Dosing methods:
. Peroral/Per Os: Administered through the mouth.

. Food/Water Addition: Substance maybe mixed directly into the animal's

food or water.
. Gavage: Dissolved in a suitable vehicle (water, vegetable oil, etc.) and
introduced directly into the esophagus or stomach using a curved needle-
like tube.
 Dermal Administration:
Relevant for substances handled by workers (e.g., paints, inks, dyes) or cosmetics
applied to the skin.
Method:
. Test substance is painted onto the skin.
. Covered with a gauze patch secured with tape.
. Plastic wrap is used to prevent ingestion of the substance.

Respiratory Administration:
Important for testing industrial solvents or cosmetics in aerosol sprays.
Involves exposure through the respiratory system.

Injection Methods:
Direct injection of substances using a syringe and needle.
Types of injections:
. Intraperitoneal (IP): Injected into the body cavity.
. Intramuscular (IM): Injected into a large muscle of the hind leg.
. Subcutaneous (SC): Injected just beneath the skin.
. Intravenous (IV): Injected directly into a large vein.

2. Determining Responses to Varying Doses of a Substance

Terminology:
Dose: Refers to the total amount of a chemical administered (e.g., expressed in
milligrams).

Dosage: Refers to the amount of a chemical administered per unit body weight (e.g.,
milligrams per kilogram).

Standardization:
Dosages allow for better standardization and comparison of effects across individuals and
species with varying body sizes.

Exposure Measurement:
 > In respiratory exposures, levels are typically measured by the concentration of the
substance in the air (e.g., parts per million).

Quantitative Toxicology:
> Involves administering the substance in an ordered series of doses to test animals.
Independent Variable: The dose is controlled by the toxicologist.
Dependent Variable: The response of the animals, which is influenced by the dose applied.

Response Measurement:
> Responses can be scored and related to the dose to establish a dose-response relationship.

3. Timing of Exposure in Toxicity Testing

> Acute Toxicity:
o Defined as toxicity resulting from a single exposure to a substance.

o Typically involves administering a single dose to animals and observing them for
up to 14 days.

o Example: LD50 test, which measures the median lethal dose.

> Subacute Toxicity:

o Involves repeated or continuous exposure to substances over a period not exceeding
14 days.
o Assesses the response to shorter-term exposure.

> Subchronic Toxicity:

o Involves repeated or continuous exposure over a period of 90 days.
o Provides insight into the effects of longer-term exposure.

> Chronic Toxicity:

o Refers to repeated or continuous exposures lasting more than 90 days.
o Focuses on long-term effects and potential cumulative toxicity.

> Maximum Tolerated Dose (MTD):

o Animals are often exposed to the MTD, defined as the highest dose that does not
cause death or incapacitating symptoms.
o High doses are used to ensure that chronic toxicity can be observed.

Considerations for High Doses:

o While high doses may reveal toxicity, effects observed at these levels could result
from severe physical damage or mitogenesis (cell regeneration due to cell death).

o Such effects may not accurately predict toxicity at lower doses, highlighting the
importance of dose selection in toxicity testing.

LD50 (Median Lethal Dose) Experiment Testing

Purpose:
Measure acute toxicity of substances.

Determine the predicted dose at which 50% of individuals in a treated population

would be killed.
Methodology:
Groups of uniform test animals are exposed to a geometric series of doses under
controlled conditions.
The abbreviation LD50 (lethal dose 50) is commonly used.

Test Subjects:
White Norway rats (Rattus norwegicus) are the standard laboratory
animals used.

Dosage Units:
Expressed in milligrams of active ingredient per kilogram of body
weight (mg/kg).

Dose Selection:
The highest dose is chosen to ensure that 90% or more of the animals in that group
will be killed.
Determined from:
. Previous results with chemically similar substances.
. Pilot range-finding experiments with a smaller number of animals.

Dose Administration:
Serial dilutions create a gradient of responses over four or five doses.
 > At least 10 animals (ideally 5 males and 5 females) are exposed to each of the six
doses.
> Always includes a negative control group exposed only to the vehicle.

> Test Substance:

> Typically applied as the technical grade (approximately 95% purity).
> May require additional tests with analytical grade (greater than 99% purity).

> Purity can be verified through gas chromatography or high-performance liquid

chromatography.

> Vehicle and Route of Administration:

> Chosen based on the physical properties of the test substance (solubility,
melting/boiling point, vapor pressure, color, and odor).

> Dosing Procedure:

> Animals of similar weight are selected and fasted overnight.
> Assigned to treatment groups using random number tables.

> Doses are applied starting with the negative control (vehicle only) and then
increasing doses of the test substance.

> Ensures no contamination of lower doses with higher dose residues.

Analysis of Susceptibility and Tolerance of LD50

> Within any population, there is a small proportion of individuals who are very susceptible
and another small proportion who are very tolerant to the same dose of a poison.

Variability in response can stem from:

> Experimental Error: Factors like precision in dose administration, environmental
conditions, and the physical condition of the animal (e.g., fasting, handling).
> Genetic Variability: True heterogeneity arises from genetic differences in physiological
characteristics among individuals.
Response Distribution:
> Variation in response is illustrated as long tails in the dose versus response histogram.
> If individual tolerances are normally distributed:
• Values are symmetrical around the mean.
 • Approximately 68% of values fall within one standard deviation of the
mean, and 98% within two standard deviations.

A sigmoid curve typically describes the accumulated percentage mortality plotted against the
logarithm of the dose, indicating increasing mortality with higher doses.

Mortality and Response Uniformity:

Mortality rates are related to the uniformity of response within the animal population.

Slope of Response:
A slope of 2.0 suggests that about 68% of the population responds to a 10-fold range in doses
centered around the median lethal dose.
A slope of 6.0 indicates that over 99.6% of the population would respond to a 10-fold increase in
doses around the median lethal dose.

Alternative Tests for Toxicity

The median lethal dose (LD50) is a crucial value for toxicologists but has faced criticism due to
the high number of animals required for rigorous estimation.

Up-and-Down Method:
> A substitute method that requires fewer animals and provides a less precise estimate
of LD50.
> Originally developed for optimizing mixtures of explosive materials with fewer
trials and reduced waste and hazard.
 Procedure of the Up-and-Down Method:
> One animal is exposed to a specific dose of the substance.
> If the animal survives, the next animal is exposed to a higher dose.
> This process continues with increasing doses until mortality is observed.
> After mortality, the next animal is given a lower dose, and the pattern continues
until an equilibrium is established.

Advantages:
> Can estimate the population median lethal dose with accuracy similar to full-scale
LD50 experiments using more than 60 animals, but with fewer than 10 animals.

Limitations:
> Variability of response cannot be estimated using this method.
> Requires additional time to wait for the outcome of one animal before determining
the dose for the next, although this can be mitigated by reducing the observation
period.

Overall Utility:
> Despite its limitations, the up-and-down method is sufficient for routine
comparisons of the toxicity of poisons while significantly reducing the number of
animals used in testing.

Categories of Toxicity
> A ranking system based on median lethal dose (LD50) has been established:
> Extremely Toxic: Median lethal dose (LD50) < 1 mg/kg.
> Highly Toxic: LD50 < 50 mg/kg.
> Moderately Toxic: LD50 < 500 mg/kg.
> Slightly Toxic: LD50 > 500 mg/kg and up to approximately 5 g/kg (approaching
practical dosing limits).

No Observed Adverse Effect Levels (NOAEL):

> NOAELis defined as the highest dosage tested where the measured response is not
significantly different from the control group.
> Lowest Observed Adverse Effect Level (LOAEL):
• The lowest dosage tested where the response is significantly different from the
control.
• Limitations of this approach include the need for careful dosage selection to
determine the threshold between no effect and effect.
 Mixtures of Poisons
Mixtures can exhibit increased or decreased toxicity compared to individual
components:
Synergism: Increased toxicity due to interactions between components (e.g., one
component interferes with the elimination of another).
Antagonism: Reduced toxicity when one component enhances the
inactivation of another, leading to higher concentrations of a less toxic
metabolite.
Evaluation of Mixtures:
Empirical testing is necessary to assess interactions in mixtures of drugs, pesticides,
industrial chemicals, etc.
If one component is nontoxic, it can be administered at high concentrations
alongside varying doses of the toxic component to test for interactions.
A significant difference in the dosage-mortality line indicates an
interaction.
If both components are toxic, testing becomes more complex:
Prepare a mixture at median lethal doses of each component.
Dilutions are administered, and the observed dosage-mortality line is compared to
a predicted line based on the individual components'mortalities

Toxicity and Hazard

The term "hazard" describes the actual risk of poisoning from a
substance.
An estimate of toxicity does not directly equate to an estimate of
hazard.
Toxicity is just one variable in predicting the hazard associated with a
substance.

• Factors Influencing Hazard:

The potential level of human exposure is a significant variable in hazard
assessment.
Exposure predictions depend on factors such as:
. Concentration of the substance.
. Circumstances of use.

• Reducing Hazard:
Although intrinsic toxicity is an unchangeable property of a substance, the hazard
can be minimized by reducing exposure risk.
• Examples of Hazard Reduction:
• Childproof Packaging:
 > Nonprescription drugs are packaged in childproof containers to limit access
and reduce poisoning risks.
• Pesticide Application:
> Pesticides are prepared in dissolvable polymer bags with pre-measured
quantities, allowing applicators to add them to sprayer tanks without
opening the bags.
> This innovation reduces the risk of exposure to concentrated pesticides by
eliminating the need for measuring and mixing.

Role of Laboratory Testing in Hazard Estimation

> Toxicological data from laboratory studies are used by regulatory agencies to estimate
hazards to human health from specific toxicants.
> Extrapolation from animal data to humans and from higher to lower exposure levels
presents challenges, but laboratory studies remain valuable for hazard estimation.

Use of Statistics in Toxicological Studies:

> Statistics aid in experimental design and interpretation of toxicological data.
> Key aspects of experimental design include:
• Randomization of subjects.
• Choice of sample size.

Descriptive Statistics:
> Useful for summarizing large toxicological data sets.
> Examples include:
> Mean.
> Standard deviation (SD).
> Standard error of the mean (SEM), calculated as: [ SEM = \frac{SD}{\sqrt{N}} ] (where
N is the number of data points).

Analyzing Differences and Trends:

> Parametric statistics are used for normally distributed continuous data (e.g., weight,
volume).
> Nonparametric statistics are used for non-normally distributed or discrete data.

Tests include:
> Student's t-test (parametric).
> Mann-Whitney U-test (nonparametric).
> p-values indicate the probability that observed differences are due to random chance:
> Ap-value ≤ 0.05 is considered statistically significant.
 Comparing Multiple Groups:
> Analysis of variance (ANOVA) tests can compare multiple groups, available in both
parametric and nonparametric forms.
> If ANOVA indicates significant differences, post hoc tests can identify specific differences
between groups.

Trend Analysis:
> Linear regression analysis determines relationships between variables (e.g., dose and
response).
> The correlation coefficient (r²) measures how well data fit the hypothesized linear
relationship.

Challenges in Extrapolation:
> Extrapolating from laboratory studies to real-world situations is complex and often
controversial.
> Species differences exist between laboratory animals and humans.
> High doses used in laboratory tests may not reflect actual low human exposure levels.
Data interpretation must be approached with caution.

Epidemiological Data in Risk Estimation

Laboratory data to assess risk:
> Examines the relationship between toxicant exposure (accidental or voluntary) and:
> Disease incidence: rate of new disease cases in a population.
> Disease prevalence: number of existing disease cases at a specific time.

Drawbacks of Epidemiological Studies:

> Variability in genetic and environmental factors complicates the interpretation of results.
> Difficult to determine if differences in disease incidence/prevalence are due to the factor
being tested or confounding factors.
> Estimating exposure levels can be challenging, especially for past exposures.

Maximizing Reliability of Results:

> Exposed and control groups are matched for potential confounding factors (e.g., age, sex,
lifestyle, working conditions).
> Larger participant numbers improve detection of small differences between groups.

Meta-Analysis:
 > A technique that combines results from different studies to enhance statistical power.
> Helps determine differences between exposed and control groups regarding disease
development.
> Quality of meta-analysis depends on the quality of the underlying data.
> Disagreements exist on the selection of studies for inclusion.
> Published data maybe incomplete; bias may exist as negative results are less likely to be
published.

Important Considerations:
> Correlation does not imply causation.
> A positive association between a risk factor and disease does not confirm that the risk factor
causes the disease.

Risk Assessment and Risk Management

Risk assessment and risk management are critical processes in evaluating and regulating
potentially toxic substances. These processes help balance the potential hazards posed by
chemicals against their benefits, guiding regulatory decisions to protect public health and the
environment.
Risk Assessment
The National Academy of Sciences/National Research Council outlined a structured approach to
risk assessment in their 1983 report. The process consists of four main components:

1. Hazard Identification:
• This step involves determining whether a substance poses a potential health hazard.
It includes reviewing scientific literature, conducting studies, and evaluating data
on the substance's toxicological effects.
2. Dose-Response Evaluation:

• This component quantifies the relationship between the dose of a substance and the
magnitude of its toxic effect. It helps establish the threshold levels at which adverse
effects may occur and is essential for understanding how different doses affect
health outcomes.
3. Exposure Assessment:

• In this phase, potential exposure levels to the substance are estimated. This includes
assessing the routes of exposure (e.g., inhalation, ingestion, dermal contact),
duration, frequency, and the population that may be exposed. It aims to estimate
how much of the substance individuals or populations are likely to encounter.
 4. Risk Characterization:
• This final step integrates the information from the previous three components to
estimate the effects on the exposed population. Risk characterization often presents
the likelihood of adverse health outcomes, such as the probability of developing a
specific disease over a lifetime of exposure at a given level.

Risk Management
Following risk assessment, the process of risk management takes place. This involves making
regulatory decisions regarding health risks associated with toxic substances. Key aspects of risk
management include:
• Integration of Multiple Factors:

• Risk management considers not only the results of the risk assessment but also
political, social, and economic factors. This holistic approach ensures that decisions
are well-rounded and take into account various stakeholder perspectives.
• Regulatory Agencies:

• Several government agencies are involved in risk management, each focusing on

different aspects of public health and safety:

• Occupational Safety and Health Administration (OSHA): Regulates

workplace safety and health hazards.

• Food and Drug Administration (FDA): Oversees the safety of food,

drugs, and cosmetics.

• Environmental Protection Agency (EPA): Responsible for protecting

human health and the environment by regulating pollutants and hazardous
substances.

thanks
sidrasabir