What is acute toxicity and what is the need of acute toxicity testing?

Acute systemic toxicity evaluates the adverse effects that occur following exposure of
organisms to a single or multiple doses of a test substance within 24 hours by a known route (oral,
dermal or inhalation).
Acute toxicity tests can provide preliminary information on the toxic nature of a material for
which no other toxicology information is available. Such information can be used to:

• deal with cases of accidental ingestion of a large amount of the material (e.g., for poison
control information);
• determine possible target organs that should be scrutinized and/or special tests that should be
conducted in repeated-dose toxicity tests;
• select doses for short-term and sub-chronic toxicity tests when no other toxicology
information is available.

In most acute toxicity tests, each test animal is administered a single (relatively high) dose of
the test substance, observed for 1 or 2 weeks for signs of treatment-related effects, then necropsied.
Some acute toxicity tests ( LD50 test) are designed to determine the mean lethal dose of the test
substance.

Explain OECD guidelines for chronic toxicity.

ANS.
OECD guidelines for chronic toxicity (452)
Introduction:
The three main routes of administration used in chronic toxicity studies are oral, dermal
and inhalation. While long-term chronic toxicity studies involving exposure via the dermal or
inhalation routes may also be necessary for human health risk assessment.
The objectives of chronic toxicity studies covered by the test guideline include:
The identification of the hazardous properties of a chemical,
The identification of target organs
Characterisation of the dose: response relationship
Identification of a no-observed-adverse-effect level(NOAEL) or point of departure for
establishment of a Benchmark Dose (BMD)
The predication of chronic toxicity effects at human exposure levels
Provision of data to test hypothesis regarding mode of action

Principle of the test:

The test substance is administered daily in graduated doses to several groups of
experimental animals for a period of 12 months, although longer or shorter durations may also be
chose. The test substance is normally administered by the oral route although testing by inhalation or
dermal route may also include one or more interim kills, e.g. at 3 and 6 months, and additional
groups of animals may be included to accommodate this. During the period of administration the
animals are observed closely for signs of toxicity. Animals which die or are killed during the test are
necropsied and, at the conclusion of the test, surviving animals are also killed and necropsied.

Description of the method:

Selection of animal species
In this guideline, the preferred rodent species is the rat, although other rodent species, e.g., the mouse,
may be used. Rats and mice have been preferred experimental models because of their relatively short
life span, their widespread use in pharmacological and toxicological studies, their susceptibility to
tumour indication, and the availability of sufficiently characterised strains.
Housing and feeding conditions
Animals may be housed individually, or be caged in small groups of the same sex. The temperature
in the experimental animal room should be 22℃(± 3℃). Although the relative humidity should be at
least 30% and preferably not exceeded 70% other than during room cleaning, the aim should be 50-
60%. Lighting should be artificial, the being 12 hours light, 12 hours dark. For feeding, conventional
laboratory diets may be used with an unlimited supply of drinking water. The diet should meet all
nutritional requirements. Dietary contaminants like pesticide residues, organic pollutants and
phytoestrogens, heavy metals and micotoxins should be as low as possible. Analytical information
on the nutrient and dietary contaminant levels should be generated periodically and included in the
final report.

Preparation of Animals
Healthy animals, acclimated to laboratory conditions for at least 7 days and not been subjected to
previous experimental procedures should be used. In the case of rodents, dosing of animals should
begin after weaning and acclimatisation before the animals 8 weeks old. The test animal should be
characterized as to species, strain, source, sex, weight and age. At the commencement of study, the
weight variation of animals used should be minimal and not exceed ± 20 % of mean weight of all the
animals within the study. Animals should be randomly assigned to the control and treatment groups.
Each animal should be assigned a unique identification number.

Procedure
1. Number and Sex of Animals:
At least 20 animals per sex per group should be used at each dose level. It is possible to increase the
statistical power of the key estimates by allocating animals and equally to the different dose levels.

2. Provision for interim kills, satellite groups and sentinel animals:

Provision for interim kills at six months to provide information on progression of toxicological
changes and mechanistic information.
Satellite groups to monitor the reversibility of any toxicological changes and additional 5
sentinel animals to monitor the disease status.
If interim kills are inclusion of satellite and sentinel groups or planned number of animals
included in the study designed should be increased by the number of animals scheduled to be
killed.
These animals undergoes same observations, including body weight, food/water
consumptions, haematological and clinical biochemistry measurements and pathological
investigations as the animals in chronic toxicity phase of the main study.

3. Dose groups and Dosage

Atleast three dose levels and a concurrent control should be used. Dose levels will generally be based
on the results of short-term repeated dose or range finding studies and should take into account any
existing toxicological and toxico-kinetic data available for the test substance.
The highest dose level should be chosen to identify the principal target organ and toxic effects
while avoiding morbidity or death.
The top dose should not exceed 1000mg/kg body weight/day
The dose level spacing should be designed to demonstrate a dose-response and to establish a
no observed adverse-effect level (NOAEL). Dose level spacing of two or three fold intervals
are frequently optimal for setting the descending dose levels.
Preparation of doses and administration of test substances
1. The route and method of administration is dependent on the purpose of the study, the physical and
chemical properties of the test substance, its bioavailability and the predominant route and method of
exposure in humans.
2. The test substance is dissolved in suitable vehicle.
3. For substances administered via diet or drinking water it is important to ensure that the quantities
of test substance do not interfere with normal nutrition or water balance. In long-term toxicity studies,
the concentration of the chemical in feed should not normally exceeed an upper limit of 5% of the
total diet. Dietary concentration of chemical in mg/kg or ppm in terms of animal’s body weight.
4. In case of oral or dermal administration, the animals are dosed with the test substance daily
normally for period of 1 year.
5. Dosing by inhalation route is carried out 6 hours per day, 5 days per week.
6. By gavage: through a stomach tube or intubation cannula, a single dose is administered once daily.
The dose volume should not exceed 2 ml/100g body weight.

Duration of the Study

12 month chronic toxicity study is recommended by Test Guideline
For mechanistic purposes can be shorter (6 mos or 9 mos) and long term (18 or 24 mos)

Observations
Neurobehavioural signs observed at the beginning and end of each day
Changes in skin, fur, eyes and mucous membranes, occurrences of secretions and
excretionsand autonomic activity, changes in gait, posture, bizarre behaviour should be
recorded.
Auditory, visual and proprrioceptive stimuli assessment and motor activity assessment
conducted at 3,6, 9 and 12 months

Body weight, food/water consumption and food efficiency

All animals should be weighed at the start of treatment, atleast once a week for the first 13 weeks
and atleast monthly thereafter. Measurements of food consumption and water consumption should
be measured atleast weekly for first 13 weeks & monthly thereafter.

Hematology and Clinical Biochemistry

In studies involving rodents, hematological examinations should be carried out in atleast 10
male and 10 female animals per group, at 3, 6 and 12 months.
The following hematological parameters are investigated:
Total and differential leukocyte count,
erythrocyte count,
platelet count,
haemoglobin concentration,
hematocrit(packed cell volume),
mean corpuscular volume (MCV),
mean corpuscular haemoglobin (MCH),
mean corpuscular haemoglobin concentration (MCHC),
prothrombin time.
The clinical biochemistry parameters are investigated: glucose, urea, creatinine, total
protein, albumin, calcium, sodium, potassium, total cholesterol, alanine
aminotransferase, glutamate dehydrogenase, aminotransferase, total bile acids, alkaline
phosphatase
Pathology
Gross necropsy and histopathology are performed.
Individual animal data should be provided for all parameters.
Organ weights are collected from all animals.
ICH S4 has also provided safety guidelines for “Duration of chronic toxicity testing in
animals (Rodents and Non Rodents Toxicity testing).

What is mutagenicity? Describe in detail the different tests for mutagenicity.

ANS.
Mutagenicity
Mutagenicity refers to induction of permanent transmissible changes in the structure of genetic
material of cells.
Chemical or physical impacts can result in a fixed alteration in the genetic material which lead to
lethal or heritable defects and may involve single gene or block genes.

Mutagens or are the agents which give rise to to these genetic changes and cause increased occurrence
of mutations.

Genotoxicity is a process that alters the structure, information content it or segregation of DNA.

Types of mutagens
Mutagens of two types
-direct acting mutagens( active without metabolic conversion)
-promutagens (require metabolic activation)

The nature of mutagen

- chemical compounds for ionization radiation
- base analogues get inserted into the DNA strand during replication
-cause structural changes that lead to miscopying of the template strand

Ames test for gene mutation in bacteria

Salmonella typhimurium that carry a mutation in genes involved in amino acid histidine is used.

An auxotropic strain of Salmonella typhimurium medium survives only in medium with

histidine. After treatment with mutagen some auxotropic cells turn into normal ones that synthesise
histidine and survivein a normal medium. Suspension of histidine requiring strain of Salmonella
typhimurium has been created with a mixture of rat liver enzymes on agar lacking histidine. A Disc
of filter paper impregnated with 10 ug of 2- amino fluorine, a known carcinogen is placed on agar.
The change of growth phenotype indicates mutagenic response.

Test for chromosomal aberrations in mammalian cells in vitro

The purpose of in vitro chromosomal aberration test it is to identify agents that cause structural
chromosomal aberrations in cultured mammalian cells. Cell cultures are exposed to the test substance
during about 1.5 normal cell cycle lengths. After exposure, cells or treated with metaphase arresting
substance, harvested and stained. Metaphase cells or analysed microscopically for the presence of
chromosomal aberrations.

In Vitro genetic assay

Mammalian cells designed to detect induction of mutations at specific locker such as those coding
for the enzymes hypoxanthine guanine phosphoribosyl transferase or thymidine kinase (mouse
lymphoma thymidine kinase.

Micronucleus assay
It detects damage to chromosomes or spindle Apparatus of the cells and involves microscopic
examination of cytological preparations of polychromatic erythrocytes obtained from bone marrow
of animals. Precursor cells divide after exposure to test substance resulting in formation of smaller
micronucleus. The micronuclei in the binucleated cells are scored using microscopy indicates
mutagenic response.

Comet assay
It uses single cell gel electrophoresis, a sensitive technique for detection of DNA damage. The cells
are embedded in agarose on microscopical slide and is lysed with detergent and high Salt to form
nucleoid containing supercoiled loops of DNA link to nuclear matrix. Electrophoresis at high pH
results in Migration of DNA fragments out from nucleus to form the tail of a comet like structure.
Supercoiling is lost when loops contains break and become free to extend towards anode. Staining of
gel is done to calculate fluorescence to determine extent of DNA damage.