Regulatory Toxicology Studies

  1. Acute Toxicity
  2. Subacute Toxicity (Repeated Dose)
  3. Subchronic And Chronic Exposure
  4. Chronic Exposure
  5. Drug Disposition/Pharmacokinetics (ADME)
  6. In Vitro Permeation Studies
  7. In Vivo Absorption Studies
  8. Irritation And Sensitization
  9. Immunotoxicity
  10. Reproductive Toxicity
  11. Genotoxicity/Mutagenicity
  12. Adjuvant Safety And Immunogenicity
  13. Safety Pharmacology

Acute Toxicity

Acute Toxicity Study Objectives

  • To determine the Median Lethal Dose (LD50) after a single dose administered through one or more routes, one of which is the intended route of administration in humans.
  • To determine Maximum Tolerated Dose (MTD) and No Observable Effect Level (NOEL).
  • To identify potential target organs for toxicity, determine reversibility of toxicity, and identify parameters for clinical monitoring.
  • To help select doses for repeated-dose toxicity tests.

Duration
A few days to 2 weeks after a single dose

Test System/Animal System
2 species required. Mice, rats, sometimes rabbits or dogs.

Dose Administration

  • Oral (by gavage or with food)
  • Subcutaneous
  • Intraperitoneal
  • Intradermal
  • Inhalation
  • Intranasal
  • Topical (epicutaneous)
  • Intravenous

Parameters

  • Mortality
  • Clinical pathology
  • Gross necropsy
  • Weight change
  • Signs of toxicity

Estimated Cost
$-$$ depending on species, dose range, controls, and methods of analysis

Subacute Toxicity (Repeated Dose)

Subacute Toxicity Study Objectives

  • To determine toxicity after repeated administration of the test material.
  • To help establish doses for subchronic studies.

Duration
14 days

Test System/Animal System
2 species required. Mice, rats,  rabbits, guinea pigs, dogs.

Dose Administration
3 to 4 doses given by the same routes as previous toxicity tests.

Parameters

  • Mortality
  • Signs of toxicity
  • Pathology and histopathology
  • Weight change
  • Clinical pathology

Estimated Cost

$$

Subchronic And Chronic Exposure

Subchronic and Chronic Toxicity Study Objectives

  • To establish a “no observable effect level” (NOEL)
  • To characterize dose-response relationships following repeated doses
  • To identify and characterize specific organs affected after repeated administration
  • To predict a reasonable and appropriate dose for chronic exposure studies (maximum tolerated dose or MTD)

Duration
Commonly 90 days, but varies from 2 weeks to 6 months or up to 10% of species’ lifespan.

Test System/Animal System
2 species required. Rodents, dogs.

Dose Administration  
At least 3 doses given by the same routes as previous toxicity tests; the lowest producing no apparent toxicity and the highest producing toxicity but less than or equal to 10%  mortality.

Parameters

  • Mortality
  • Weight change
  • Signs of toxicity
  • Clinical pathology
  • Pathology and histopathology

Estimated Cost  
$$-$$$ depending on analytical methods required.

Chronic Exposure

Chronic Toxicity Study Objectives

  • To evaluate the cumulative toxicity of chemicals.
  • To assess carcinogenic potential.

Duration
Rodents – 6 to 24 months; non-rodents – 12 months or longer or up to 10% of species’ lifespan. Length depends on intended period of human exposure.

Test System/Animal System
2 species required. Rodents, dogs.

Dose Administration
As in subacute/ subchronic toxicity studies.

Parameters

  • Mortality
  • Pathology and histopathology
  • Weight change
  • Clinical pathology of all animals (mortalities and survivors)

Estimated Cost
$$$ depending on analytical methods required.

Drug Disposition/Pharmacokinetics (ADME)

Drug Disposition Study Objective

  • Evaluate the bioavailability, tissue distribution, active metabolite formation, and elimination of test materials.

Duration
<48 hours

Test System/Animal System
Rat, Dog, Swine, Primate.

Dose Administration
Various

Parameters

  • AUC (Area Under Curve)
  • Cmax (max concentration in blood)
  • Tmax (Time Cmax is reached)

Estimated Cost
$-$$

In Vitro Permeation Studies

In Vitro Permeation Study Objective

  • To study the effect of test materials on skin metabolism, or the effect of skin metabolism on xenobiotics

Duration
<48 hours

Test System/Animal System
Excised skin, mucosa, or other biological membranes cultured on specialized diffusion cells.

Parameters

  • Evaporation rate
  • Quantitation of deposition in different skin layers
  • Degree of percutaneous penetration

Estimated Cost
$-$$

In Vivo Absorption Studies

In Vivo Absorption Study Objective 

  • To study percutaneous absorption of drugs or environmental contaminants.

Duration
48 – 72 hours

Test System/Animal System
Small rodents (guinea pigs), swine.

Dose Administration
Subcutaneous, topical (skin on torso), intravaginal

Parameters
Absorption rate (quantitation in blood, urine, feces, vaginal fluids, and tissue samples)

Estimated Cost

$

Irritation And Sensitization – Local Tolerance Studies

Irritation and Sensitization Study Objective

  • To determine the potential of a test material to provoke ocular irritation, dermal irritation, or sensitization.

Duration
Irritation – one hour to three weeks after a single topical or corneal administration. Sensitization – intradermal or topical induction doses followed by topical challenges with a non-irritating dose (6 – 8 weeks total).

Test System/Animal System
Rodents,  rabbits

Dose Administration

  • Single patch administration
  • Multiple doses over 2—4 weeks
  • Topical (epicutaneous), intradermal, or corneal

Parameters

  • Degree of pruritis, erythema, edema, papules, and vesicles
  • Corneal irritation, swelling, or injury
  • Microscopic integrity of corneal endothelium
  • Other features of the eye (conjuctive, cornea, iris, lens, anterior portion of vitreous humor)

Estimated Cost
$-$$ depending on duration and animal system used.

Immunotoxicity

Immunotoxicity Study Objective

  • To determine the potential of a test material to induce immune suppression or immune enhancement.

Duration
Subacute (14 days) or sub¬chronic (90 days) exposure

Test System/Animal System
Rodents

Dose Administration
Repeated doses administered as in subacute/subchronic toxicity studies

Parameters

Level I

  • Hematology
  • Histopathology or lymphoid organs
  • Quantity of T- and B-cells (cellularity of lymphoid organs)
  • Blastogenesis (mitogen responsiveness; mixed lymphocyte reaction)
  • Quantitation and funciton of natural killer cells
  • Macrophage function
  • Cytokine production

Level II

  • Kinetics of antibody production to T-dependent antigens
  • Quantity of IgM/IgG-producing (plaque-forming) cells
  • Delayed hypersensitivity responses to known sensitizers
  • Immune response to infectious agents (e.g., Listeria, Streptococcus)
  • Immune response to transplantable tumors

Estimated Cost
$-$$$ depending on protocol.

Reproductive Toxicity

Reproductive Toxicity Study Objectives

  • To determine potential adverse effects of a test material on mammalian gametogenesis, fetal organogenesis, and neonatal development.
  • To determine potential adverse effects on delivery, lactation, neonatal survival and vitality.

Duration

Varies depending upon end point.

Segment I

  • Preconception
  • Treatment during gameto­genesis (females, 60 days; males, 15 days), before mating; pregnant females treated throughout gestation, parturition, until weaning.

Segment II

  • Preimplanation
  • Treatment during gestation (rodents:  day 6 – day 15; rabbits: day 6 – day 18)

Segment III

  • Perinatal to Postnatal
  • Treatment through at least 15 days of gestation and 21 days of lactation.
  • All Segments
  • Data collection and evalua­tion are often done through two or more generations.

Test System/Animal System
Rodents, rabbits.

Dose Administration
As in subchronic/chronic exposure studies

Parameters

  • Preconception
  • Mating behavior
  • Preimplantation and fertilization rates
  • Integrity and quantity of sperm and egg cells
  • Post Conception
  • Maternal weight gain
  • Time from conception to delivery
  • Litter size
  • Number of corpora lutea and implanted fetuses
  • Fetal mortality and viability
  • Placental weight
  • Pup weight and crown-rump length
  • Postnatal
  • Problems at parturition
  • Maternal-newborn relationship; maternal ability to rear young
  • Postnatal growth; time of occurrence of developmental landmarks
  • 21-day survival of young
  • Functional parameters after 21 days

Estimated Cost
$$-$$$ depending on duration and animal system used.

Genotoxicity/Mutagenicity

Genotoxicity Study Objective

  • To assess the potential of a test material to induce genetic damage in vivo

Duration

  • Rodents: 12 – 72 hours, depending on the assay (excluding quantitation of results)
  • TK+/- mouse lymphoma or CHO HGPRT mutation assay: 14 – 18 days
  • Bacterial mutagenesis assay: 48 hours

Test System/Animal System
Rodents, mammalian-cells, bacteria.

Dose Administration
Rodents: Intraperitoneal (preferred), or expected route of human exposure. Dosing schedule similar to acute/subacute studies

Parameters

Rodents

  • Frequency ratio of micronucleated polychromatic erythrocytes (MPCE) to normochromatic erythrocytes (NCE) in mouse bone marrow.
  • Bone marrow mitotic index
  • Total number, types, and frequency of metaphase chromosome aberrations
  • Frequency of damaged cells
  • Severity of damage in cells (average number of aberrations per cell)
  • Unscheduled DNA synthesis (UDS) in primary rat hepatocyte cultures

Mammalian Cells

  • Total growth of cells in culture
  • Relative cloning efficiency
  • Frequency of chromosome damage

Bacterial Cells

  • Frequency of revertant colonies on selective medium

Estimated Cost
$-$$

Adjuvant Safety And Immunogenicity

Adjuvant Safety and Immunogenicity Study Objectives

  • To evaluate the safety and immunogenicity of the test substance.

Pacific BioLabs can also do the following procedures in in vivo (rabbits or rodents) or in vitro systems, as required:

  • Evaluation of immunization efficacy by viral or bacterial challenge
  • Peptide conjugation
  • Polyclonal antibody production
  • Monoclonal antibody production
  • Development of ELISA assays

Duration
30 to 60 days

Test System/Animal System
Rabbits

Dose Administration
Desired volume by intramuscular injection

Parameters

  • Body weight
  • Hematology
  • Serum chemistry
  • Specific serum antibody levels
  • Degree of swelling at injection site
  • Skin reactions at injection site
  • Slit lamp eye examination
  • Pathology and histopathology

Estimated Cost
$-$$

Safety Pharmacology

Safety Pharmacology Study Objectives

To evaluate the effects of drugs or biomateri­als on the function of vital organ systems, such as the cardiovascular and central ner­vous systems. These effects must be evaluated before human exposure, in separate studies or as additions to toxicity studies. Safety tests in animal systems are often preceded by in vitro tests to evaluate biological and pharmacological activity at the cellular level. Standard safety pharmacology studies (as commonly conducted for pharmaceuticals) are not generally required for biotech­nology-derived products. These studies define expected and unexpected pharmacological effects of the test material, especially on parameters associated with desired clinical activity.

Duration
Variable – depending on the test system

Test System/Animal System
In Vivo: selected pharmacologically relevant species; more than one species indicated for cases with no previous data or when species relevance is difficult to determine.

In Vitro: cell lines derived from relevant animal species; may predict the choice of species most appropriate for in vivo studies.

Dose Administration
Variable, depending on the expected effective dose range and test system.

Parameters
In Vivo:

  • Safety studies may be required for the following systems and specific target organs:
  • Respiratory (lungs and bronchi)
  • Gastrointestinal and hepatic (esophagus, stomach, intestines, liver)
  • Renal (kidney)
  • Cardiovascular (heart and blood vessels)
  • Blood
  • Endocrine (thyroid and other endocrine glands)
  • Nervous/neurobehavioral (CNS and behavior)

In Vitro:

  • Biological activity at the cellular level may be assessed in terms of:
  • Receptor occupancy
  • Receptor affinity
  • Binding and transport kinetics
  • Production and or secretion of specific proteins in response to test material (for example, antibody production in response to test antigen)
  • Other pharmacological effects on cellular function.
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