Sub-chronic & Chronic Toxicity (Repeat Dose Toxicity Studies)

28 Days Repeat Dose Toxicity- Rodents (Sub-Acute) (OECD 407)

The test item is orally (by gavage) administered daily in graduated doses to several groups (at least 4) of experimental animals (mostly rodents – rats/mice), one dose level per group (minimum 5M+5F per group) for a period of 28 days. During the period of administration, the animals are observed closely, each day for signs of toxicity. Animals that die or are euthanised during the test are necropsied and at the conclusion of the test surviving animals are euthanised and necropsied. A 28-day study provides information on the effects of repeated oral exposure and can indicate the need for further longer-term studies. It also provides information on the selection of concentrations for longer term studies. The data derived from this study allow for the characterization of the test substance toxicity, for an indication of the dose response relationship and the determination of the No-Observed Adverse Effect Level (NOAEL).

90 Days Repeat Dose Toxicity- Rodents (Sub-Chronic) (OECD 408)

The test substance is orally (by gavage / feed) administered daily in graduated doses to several groups (at least 4) of experimental animals, one dose level per group (minimum 10M+10F per group) for a period of 90 days. During the period of administration, the animals are observed closely for signs of toxicity. Animals which die or are humanely killed during the test are necropsied and at the conclusion of the test, remaining animals are also humanely killed and necropsied after the full dosing period. The observations or parameters includes mortality, clinical signs, body weights, behavioural observations, clinical pathology, gross pathology, histopathology, etc.

180 Days Repeat Dose Toxicity – Rodents (Chronic) (OECD 452)

The chronic toxicity study provides information on the possible health hazards likely to arise from repeated exposure over a considerable part of the lifespan of the species (rodents) used. The study provides information on the toxic effects of the substance, indicate target organs and the possibility of accumulation. It can also provide an estimate of the no-observed-adverse effect level which can be used for establishing safety criteria for human exposure.

The test substance is administered daily in graduated doses (control + 3) to several groups (20M+20F per group) of experimental animals (mostly rats), normally for a period of 12 months (any duration between 6 months to 12 months).  The test item is normally administered by the oral route although testing by the dermal or other route may also be appropriate. The study design 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 killed and necropsied. Study observations include clinical signs, body weights, regular detailed observations (haematological, urinalysis, clinical chemistry), behavioural observations, clinical pathology, gross pathology, histopathology etc.

Carcinogenicity Studies (OECD 451)

Not yet offered. Will be coming up soon.

Combined Chronic Toxicity / Carcinogenicity (OECD 453)

Not yet offered. Will be coming up soon.

Neurotoxicity Studies – Rodents

Neurotoxicity Studies – Rodents (OECD 424)

This neurotoxicity Test Guideline is designed to detect major neurobehavioral and neuropathological effects in adult rodents.

The test item is administered by the oral route across a range of doses (control+3 doses) to several groups (10M+10F in each group) of laboratory rodents (rats).  An acute (single) or repeated doses are normally required, and the dosing regimen may be 28 days, sub-chronic (90 days) or chronic (1 year or longer). The animals are tested to allow the detection or the characterization of behavioural and/or neurological abnormalities.  A range of behaviours that could be affected by neurotoxicants (functional observations) is assessed during each observation period.  The study paraments include daily clinical signs, measurements (weighing, food /water consumption), functional tests, detailed clinical observations and ophthalmology, haematology, clinical biochemistry and histopathology). At the end of the test, a subset of animals of each sex from each group are perfused in situ and sections of the brain, spinal cord, and peripheral nerves are prepared and examined.

Genotoxicity Studies (In-Vivo)

In-vivo Micronucleus Test (OECD 474)

The purpose of the micronucleus test is to identify substances that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes.

Animals (mostly mice, rats can also be used) are exposed to the test substance by an appropriate route (usually oral gavage).  If bone marrow is used, the animals are sacrificed at appropriate times after treatment, the bone marrow extracted, prepared and stained.  When peripheral blood is used, the blood is collected at appropriate times after treatment and smear preparations are made and stained.  Preparations are analysed for the presence of micronuclei. The frequency of micronucleated immature (polychromatic) erythrocytes is the principal endpoint.  The number of mature (normochromatic) erythrocytes in the peripheral blood that contain micronuclei among a given number of mature erythrocytes can also be used as the endpoint of the assay when animals are treated continuously for 4 weeks or more.

In-vivo Chromosomal Aberration Assay in Bone Marrow Cells (OECD 475)

The mammalian In-vivo chromosomal aberration test is used for the detection of structural chromosome aberrations induced by test compounds in bone marrow cells of animals, usually rodents (mostly rats, mice can also be used).

Animals (mostly rats) are exposed to the test chemical by an appropriate route (oral by gavage) of exposure and are humanely euthanized at an appropriate time after treatment. Prior to euthanasia, animals are treated with a metaphase-arresting agent (e.g., colchicine or Colcemid®). Chromosome preparations are then made from the bone marrow cells and stained, and metaphase cells are analysed for chromosomal aberrations. The mitotic index, the number of metaphase cells scored, the number of aberrations per metaphase cell and the percentage of cells with structural chromosomal aberration(s) are evaluated for each animal. Different types of structural chromosomal aberrations are listed with their numbers and frequencies for treated and control groups.

Local Lymph Node Assay (LLNA) in Mice: followed by BrdU-ELISA (OECD 429)

The LLNA provides an alternative method for identifying potential skin sensitizing test items. The assay is of equal merit and may be employed as an alternative in which positive and negative results generally no longer require further confirmation.

In the LLNA assay sensitizers induce proliferation of lymphocytes in the lymph nodes draining the site of test substance application. This proliferation is proportional to the dose and to the potency of the applied allergen and provides a simple means of obtaining a quantitative measurement of sensitization. The method is based on the use of In-vivo radioactive labelling to measure an increased number of proliferating cells in the draining auricular lymph nodes.

CBA/Ca or CBA/J mice are used. Minimum of 4 animals per group, at least three doses, plus positive and concurrent negative (vehicle) control groups.

In-vivo Mammalian Alkaline Comet Assay (OECD 489)

The In-vivo alkaline single cell gel electrophoresis assay, also called alkaline Comet Assay is a method measuring DNA strand breaks in eukaryotic cells.

Each treated group is composed of a minimum of 5 animals of one sex (or of each sex as appropriate). A positive and a vehicle control group are also used. Administration of the treatment consists of daily doses over duration of 2 days or more, ensuring the test chemical reaches the target tissue which can be the liver, the kidney or other tissues if justified.

Tissues of interest are dissected and single cells/nuclei suspensions are prepared and embedded in agarose on slides. Cells/nuclei are treated with lysis buffer to remove cellular and/or nuclear membranes. The nuclear DNA in the agar is then subjected to electrophoresis at high pH. This results in structures resembling comets which by using suitable fluorescent stain, can be observed by fluorescent microscopy. Based on their size DNA fragments migrate away from the head to the tail, and the intensity of the comet tail relative to the total intensity (head plus tail) reflects the amount of DNA breakage.

Pharmaceuticals

Single Dose Studies and MTD Studies 

The Single dose or Acute Toxicity studies of drug candidate in at least two species like rats and mice, by oral (or any other therapeutic route like IM/IP/SC) and IV route.

In MTD studies, rats are usually administered increasing doses of the test item until the highest possible dose is reached, or until severe toxicity or death occurs. MTD studies are conducted to determine the highest dose of a drug or chemical that can be given to rats without causing significant toxicity or death and these studies help to identify the potential toxic effects of drug or chemical and provide guidance for determining safe dosages for human trials. 

Repeated Dose Studies

Repeat dose toxicity studies of different durations like 7-Day or 14-Day Dose Range Finding studies or 28-Day or 90-Day toxicity studies in species like rats and mice by different routes of administration like oral (by gavage), IM/IV/SC routes, etc. The repeat dose toxicity studies of 28-Days or 90-Days generally have high dose and control reversal groups of 7-day or 28-Days respectively for any delayed effects, reversibility or irreversible toxic effects. Many a times, these repeat dose toxicity studies have satellite groups for Toxicokinetics parameters like Cmax, Tmax, AUC, VD, half-life, etc.

Disease Models (Therapeutic Areas)

Diabetes and Obesity Models (Metabolic Diseases)

1. Streptozotocin-induced diabetes in rats or mice: This model involves the injection of streptozotocin, a chemical that damages pancreatic beta cells and causes insulin deficiency, leading to hyperglycaemia.
2. High-fat diet-induced diabetes in rodents: This model involves feeding rodents a high-fat diet to induce obesity, insulin resistance, and hyperglycaemia.
3. Other models including transgenic animal models as per the specific requirements of clients.

Inflammatory diseases / conditions (COPD /Asthma)

1. Ovalbumin-induced asthma model: This model involves the sensitization of rodents to ovalbumin, a protein found in egg whites, and subsequent challenge with ovalbumin, which leads to airway inflammation and hyperreactivity.
2. Lipopolysaccharide (LPS)-induced model: This model involves the administration of LPS, a bacterial cell wall component, which leads to airway inflammation and mucus production.
3. Other models as per the requirement of clients or sponsor.

CNS Models

1. Stroke: The middle cerebral artery occlusion (MCAO) model in rodents is a commonly used model of stroke. This model involves occlusion of the middle cerebral artery, leading to cerebral ischemia and infarction.
2. Multiple sclerosis: The experimental autoimmune encephalomyelitis (EAE) model in rodents is commonly used to study multiple sclerosis. EAE is induced by injecting myelin proteins or peptides, which leads to an autoimmune response and inflammation in the CNS, resulting in demyelination and neurological deficits.
3. Parkinson’s disease: Rodent models of Parkinson’s disease are typically created by administering neurotoxins such as 6-hydroxydopamine or rotenone, which selectively destroy dopaminergic neurons in the substantia nigra, leading to motor deficits such as tremors and rigidity or MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced animal models of Parkinson’s disease.
4. Other CNS models using mazes like radial arm maze, elevated plus maze, etc.

Psoriasis and other Skin conditions

1. Imiquimod-induced psoriasis model: Imiquimod is a TLR7 agonist that can induce psoriasis-like skin inflammation in mice. Imiquimod is typically applied topically to the skin, leading to keratinocyte hyperproliferation, immune cell infiltration, and cytokine release, resulting in skin lesions that resemble human psoriasis.
2. Allergic contact dermatitis model: In this model, rodents are sensitized to a hapten, such as dinitrofluorobenzene (DNFB), and then challenged with the same hapten on the skin. This leads to an inflammatory reaction and skin lesions that resemble human allergic contact dermatitis.
3. Atopic dermatitis model: Several animal models are used to study atopic dermatitis, including NC/Nga mice, which spontaneously develop a dermatitis-like skin condition, and mice that are sensitized to ovalbumin, which leads to an allergic skin reaction when challenged with ovalbumin.
4. Wound healing models: Animal models of wound healing are used to study the mechanisms of skin repair and test potential therapies for promoting wound healing. The most common models include excisional and incisional wound models in mice and rats.

Oncology, etc.

1. 1. Xenograft models: In this model, human tumor cells or tissues are implanted into immunocompromised mice, such as nude or SCID mice. This model allows for the study of tumor growth, invasion, and metastasis, as well as for testing potential cancer therapies.
   2. Carcinogen-induced models: In these models, rodents are exposed to chemical or physical carcinogens, such as UV radiation or tobacco smoke, to induce cancer development. This model allows for the study of cancer initiation and the effects of environmental factors on cancer development.

Spontaneous models: Some strains of mice and rats spontaneously develop tumors, such as mammary tumors or sarcomas, which can be used to study the mechanisms of tumor development and progression.

Safety Pharmacology Studies

FOB / Irwin Test

1. FOB test involves a series of observations of the animal’s behaviour and responses to stimuli, including reflexes, posture, gait, and autonomic functions such as respiration and body temperature. The FOB test is used to detect changes in neurological function and identify potential adverse effects of drugs or chemicals on the nervous system.
2. The Irwin Test is a comprehensive battery of behavioural tests that evaluates the general health, neurological function, and behaviour of rodents. The Irwin Test includes a series of observations of the animal’s behaviour, including assessments of general activity, grip strength, sensory function, and reflexes. The test can also assess the effects of drugs or chemicals on the autonomic nervous system, including changes in body temperature, blood pressure, and heart rate.

The differences between the two tests are subtle. The procedures used are essentially the same, but when considering neurotoxicology, the FOB is often conducted using GLP guidelines, with more animals being used per group, and doses that are low enough to determine a no effect level and high enough to induce marked nervous system behaviors.

In-Vitro Tox Studies

Cytotoxicity assays are among the first in-vitro bioassay methods used to predict toxicity of substances to various tissues. In-vitro cytotoxicity testing provides a crucial means for safety assessment and screening, and for ranking compounds.

MTT Assay: Measures metabolic activity of cells as an indicator of cell viability

The MTT assay is a widely used method for evaluating the cytotoxicity of drugs or other treatments in cell culture experiments. It is also used to assess cell proliferation, migration, and differentiation. MTT (3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay is a colorimetric assay commonly used to measure cell viability and proliferation in cell culture experiments and measures mitochondrial dehydrogenase enzyme, which coverts MTT into formazan products that can be detected using a spectrophotometer. The amount of formazan produced is directly proportional to the number of viable cells present in the culture.

LDH Assay: Measures the release of lactate dehydrogenase (LDH) as an indicator of cell damage or death.

LDH assays are commonly used in cell culture experiments to measure cell death or damage caused by various treatments, such as drugs or toxins. LDH (Lactate dehydrogenase) assay is a biochemical assay commonly used to measure the activity of LDH in biological samples, such as cell culture supernatants or tissue homogenates. LDH is an enzyme that catalyses the conversion of lactate to pyruvate and its activity is commonly used as a marker of cell damage or death. The assay typically uses a colorimetric or fluorometric substrate that reacts with LDH, producing a signal that can be measured using spectrophotometer or fluorometer.

The experiment will start after receipt of signed protocol and the test substance. A draft report will be ready for review within 4-6 weeks of the experiment start date.

In-Vitro Genotox Studies

Bacterial Reverse Mutation Test (AMES) (OECD 471)

The bacterial reverse mutation test uses amino-acid requiring at least five strains of Salmonella typhimurium and Escherichia coli to detect point mutations by base substitutions or frameshifts. The principle of this bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid.

Suspensions of bacterial cells are exposed to the test substance (liquid or solid) in the presence and in the absence of an exogenous metabolic activation system (S9 fraction). At least five different analysable concentrations of the test substance are used. The recommended maximum test concentration for soluble non-cytotoxic substances is 5 mg/plate or 5 ml/plate. There are two methods: the plate incorporation method and the preincubation method. For both techniques, after two or three days of incubation at 37°C, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates.

In-Vitro Mammalian Cell Micronucleus Test (OECD 487)

The In-vitro micronucleus (MNvit) test is a genotoxicity test for the detection of micronuclei (MN) in the cytoplasm of interphase cells. Micronuclei may originate from acentric chromosome fragments (i.e., lacking a centromere), or whole chromosomes that are unable to migrate to the poles during the anaphase stage of cell division. Therefore, the MNvit test is an In-vitro method that provides a comprehensive basis for investigating chromosome damaging potential In-vitro because both aneugens and clastogens can be detected in cells that have undergone cell division during or after exposure to the test chemical. Micronuclei represent damage that has been transmitted to daughter cells, whereas chromosome aberrations scored in metaphase cells may not be transmitted. In either case, the changes may not be compatible with cell survival.

During or after exposure to the test chemical, the cells are grown for a period sufficient to allow chromosome damage or other effects on cell cycle/cell division to lead to the formation of micronuclei in interphase cells. For induction of aneuploidy, the test chemical should ordinarily be present during mitosis. Harvested and stained interphase cells are analysed for the presence of micronuclei. Ideally, micronuclei should only be scored in those cells that have completed mitosis during exposure to the test chemical or during the post-treatment period, if one is used. For all protocols, it is important to demonstrate that cell proliferation has occurred in both the control and treated cultures, and the extent of test chemical-induced cytotoxicity or cytostasis are assessed in all of the cultures that are scored for micronuclei.

In-Vitro Mammalian Chromosome Aberration Test (OECD 473)

The purpose of the In-vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian somatic cells. Structural aberrations may be of two types: chromosome or chromatid.

The In-vitro chromosome aberration test may employ cultures of established cell lines, cell strains or primary cell cultures. Cell cultures are exposed to the test substance (liquid or solid) both with and without metabolic activation during about 1.5 normal cell cycle lengths. At least three analysable concentrations of the test substance are used. At each concentration duplicate cultures are normally used. At predetermined intervals after exposure of cell cultures to the test substance, the cells are treated with a metaphase-arresting substance (Colchicine), harvested, stained. Metaphase cells are analysed microscopically for the presence of chromosome aberrations (chromatid-type and chromosome-type aberrations).

In-Vitro Mammalian Cell Gene Mutation Test (OECD 476)

The In-vitro mammalian cell gene mutation test is used to detect gene mutations induced by chemical substances. In this test, the used genetic endpoints measure mutation at hypoxanthine-guanine phosphoribosyl transferase (HPRT), and at a transgene of xanthineguanine phosphoribosyl transferase (XPRT). The HPRT and XPRT mutation tests detect different spectra of genetic events.

Cells in suspension or monolayer culture are exposed to, at least four analysable concentrations of the test substance, both with and without metabolic activation, for a suitable period of time. They are subcultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection. Cytotoxicity is usually determined by measuring the relative cloning efficiency (survival) or relative total growth of the cultures after the treatment period. The treated cultures are maintained in growth medium for a sufficient period of time, characteristic of each selected locus and cell type, to allow near-optimal phenotypic expression of induced mutations. Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the cloning efficiency (viability). After a suitable incubation time, colonies are counted.

In-Vitro Mouse Lymphoma Assay (OECD 490)

The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced by chemical substances. This TG includes two distinct in vitro mammalian gene mutation assays requiring two specific tk heterozygous cells lines: L5178Y tk+/-3.7.2C cells for the mouse lymphoma assay (MLA) and TK6 tk+/- cells for the TK6 assay. Genetic events detected using the tk locus include both gene mutations and chromosomal events.

Cells in suspension or monolayer culture are exposed to, at least four analysable concentrations of the test substance, both with and without metabolic activation, for a suitable period of time. They are subcultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection. Cytotoxicity is usually determined by measuring the relative cloning efficiency (survival) or relative total growth of the cultures after the treatment period. The treated cultures are maintained in growth medium for a sufficient period of time, characteristic of each selected locus and cell type, to allow near-optimal phenotypic expression of induced mutations. Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the cloning efficiency (viability). After a suitable incubation time, colonies are counted.

In-Vitro Alternatives to Animal Studies 

Bovine Corneal Opacity & Permeability Test (BCOP) (OECD 437)

The Bovine Corneal Opacity and Permeability test method (BCOP) is an in vitro test method that can be used to identify chemicals (substances or mixtures) as either 1) causing “serious eye damage” (category 1 of the Globally Harmonised System for the Classification and Labelling of chemicals (GHS)), or 2) not requiring classification for eye irritation or serious eye damage according to the GHS.

The BCOP uses isolated corneas from the eyes of cattle slaughtered for commercial purposes, thus avoiding the use of laboratory animals. Each treatment group (test chemical, negative/positive controls) consists of a minimum of three eyes where the cornea has been excised and mounted to a holder. Depending on the physical nature and chemical characteristics of the test chemical, different methods can be used for its application since the critical factor is ensuring that the test chemical adequately covers the epithelial surface. Toxic effects to the cornea are measured as opacity and permeability, which when combined gives an Irritancy Score (IVIS or LIS, depending on the device) for each treatment group. A chemical that induces an IVIS ≥ 55.1, or an LIS>30 and OD490 > 2.5, or LIS>30 and lux/7 > 145, is defined as a category 1 (“causing serious eye damage” according to the GHS); a chemical that induces an IVIS≤ 3 or an LIS≤ 30 is considered as not requiring classification for eye irritation or serious eye damage according to the GHS.

Isolated Chicken Eye Test (OECD 438)

The Isolated Chicken Eye (ICE) test method is an in vitro test method that can be used to classify substances as causing serious eye damages (UN GHS Category 1) or as not requiring classification (UN GHS No category). The ICE method uses eyes collected from chickens obtained from slaughterhouses where they are killed for human consumption, thus eliminating the need for laboratory animals. The eye is enucleated and mounted in an eye holder with the cornea positioned horizontally. The test substance and negative/positive controls are applied to the cornea. Toxic effects to the cornea are measured by a qualitative assessment of opacity, a qualitative assessment of damage to epithelium based on fluorescein retention, a quantitative measurement of increased thickness (swelling), and a qualitative evaluation of macroscopic morphological damage to the surface. The endpoints are evaluated separately to generate an ICE class for each endpoint, which are then combined to generate an Irritancy Classification for each test substance. Optionally, histopathology of the eye can be evaluated to improve the predictivity of the test for chemicals causing serious eye damage.

In-Vitro Ocular Irritation Test (EpiOcular) (OECD 491 /492)

OECD 491:

This Test Guideline describes a cytotoxicity-based In-vitro assay that is performed on a confluent monolayer of Statens Seruminstitut Rabbit Cornea (SIRC) cells, cultured on a 96-well polycarbonate microplate. After five-minute exposure to a test chemical, the cytotoxicity is quantitatively measured as the relative viability of SIRC cells using the MTT assay. Decreased cell viability is used to predict potential adverse effects leading to ocular damage. Cell viability is assessed by the quantitative measurement, after extraction from the cells, of blue formazan salt produced by the living cells by enzymatic conversion of the vital dye MTT, also known as Thiazolyl Blue Tetrazolium Bromide. The obtained cell viability is compared to the solvent control (relative viability) and used to estimate the potential eye hazard of the test chemical. A test chemical is classified as UN GHS Category 1 when both the 5% and 0.05% concentrations result in a cell viability smaller than or equal to (≤) 70%. Conversely, a chemical is predicted as UN GHS No Category when both 5% and 0.05% concentrations result in a cell viability higher than (>) 70%.

OECD 492:

This Test Guideline describes an In-vitro procedure allowing the identification of chemicals (substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS. It makes use of reconstructed human cornea-like epithelium (RhCE) which closely mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The test evaluates the ability of a test chemical to induce cytotoxicity in a RhCE tissue construct, as measured by the MTT assay. Coloured chemicals can also be tested by use of an HPLC procedure. RhCE tissue viability following exposure to a test chemical is measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues. The viability of the RhCE tissue is determined in comparison to tissues treated with the negative control substance (% viability), and is then used to predict the eye hazard potential of the test chemical. Chemicals not requiring classification and labelling according to UN GHS are identified as those that do not decrease tissue viability below a defined threshold (i.e., tissue viability > 60%, for UN GHS No Category).

In-Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method (OECD 439)

This Test Guideline describes an In-vitro procedure that may be used for the hazard identification of irritant chemicals (substances and mixtures) in accordance with the UN Globally Harmonized System of Classification and Labelling (GHS) Category 2.  It is based on reconstructed human epidermis (RhE), which in its overall design closely mimics the biochemical and physiological properties of the upper parts of the human skin. Cell viability is measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively measured after extraction from tissues. Irritant test substances are identified by their ability to decrease cell viability below defined threshold levels (below or equal to 50% for UN GHS Category 2). Coloured chemicals can also be tested by use of an HPLC procedure. There are three validated test methods that adhere to this Test Guideline. Depending on the regulatory framework and the classification system in use, this procedure may be used to determine the skin irritancy of test substances as a stand-alone replacement test for in vivo skin irritation testing, or as a partial replacement test, within a tiered testing strategy.

In-Vitro Dermal Absorption (Episkin /Epiderm) /Human Cadaver Skin /Porcine Skin /Cornea (OECD 428)

This Test method has been designed to provide information on absorption of a test substance, (ideally radiolabelled), applied to the surface of a skin sample separating the two chambers (a donor chamber and a receptor chamber) of a diffusion cell. Static and flow-through diffusion cells are both acceptable.

Skin from human or animal sources can be used. Although viable skin is preferred, non-viable skin can also be used. The skin has been shown to have the capability to metabolise some chemicals during percutaneous absorption. In this case, metabolites of the test chemical may be analysed by appropriate methods. Normally more than one concentration of the test substance is used in typical formulations, spanning the realistic range of potential human exposures. The application should mimic human exposure, normally 1-5 mg/cm² of skin for a solid and up to 10 µl/cm² for liquids. The temperature must be constant because it affects the passive diffusion of chemicals. The absorption of a test substance during a given time period (normally 24h) is measured by analysis of the receptor fluid and the distribution of the test substance chemical in the test system and the absorption profile with time should be presented.

Local Lymph Node Assay (LLNA): BrdU-ELISA (OECD 429)

The LLNA provides an alternative method for identifying potential skin sensitizing test items. The assay is of equal merit and may be employed as an alternative in which positive and negative results generally no longer require further confirmation.

In the LLNA assay sensitizers induce proliferation of lymphocytes in the lymph nodes draining the site of test substance application. This proliferation is proportional to the dose and to the potency of the applied allergen and provides a simple means of obtaining a quantitative measurement of sensitization. The method is based on the use of in vivo radioactive labelling to measure an increased number of proliferating cells in the draining auricular lymph nodes.

CBA/Ca or CBA/J mice are used. Minimum of 4 animals per group, at least three doses, plus positive and concurrent negative (vehicle) control groups.

In-Vitro 3T3 NRU Phototoxicity (OECD 432)

This Test Guideline describes a method to evaluate photo-cytotoxicity by the relative reduction in viability of cells exposed to the chemical in the presence versus absence of light.

Balb/c 3T3 cells are maintained in culture for 24 h for formation of monolayers. Two 96-well plates are pre-incubated with eight different concentrations of the test substance for 1 h. Thereafter one of the two plates is exposed to the highest non-cytotoxic irradiation dose whereas the other plate is kept in the dark. Cytotoxicity in this test is expressed as a concentration-dependent reduction of the uptake of the Vital dye Neutral Red (NR) when measured 24 hours after treatment with the test chemical and irradiation. NR penetrates cell membranes by non-diffusion, accumulating in lysosomes. Alterations of the cell surface of the sensitive lysosomal membrane leads to lysosomal fragility and other changes that gradually become irreversible. Such changes result in a decreased uptake and binding of NR. It is thus possible to distinguish between viable, damaged or dead cells. To predict the phototoxic potential, the concentration responses obtained in the presence and in the absence of irradiation are compared, usually at the IC50 level, i.e., the concentration reducing cell viability to 50 % compared to the untreated controls.

Academic and Research Projects

• Research Collaborations with different organizations

Please connect with us for any collaborative research or regulatory works. We encourage collaboration of any and all sorts possible for better and optimal utilization of scientific expertise, manpower, facilities and infrastructure of all collaborating organizations /labs.

• Students Research for PG/Ph.D. Programs

We offer our facilities, infrastructure and resources to the students for their PG/PhD works, in case their institutes /organizations do not have any of the facility or resource required for their research work. This is not for profit work for us and we extend our support to the students at a very reasonable cost (only to recover our expenses), so that students can complete their research projects. Please connect for your specific requirements.

Biological evaluation of medical devices

We also conduct and offer hemocompatibility and biocompatibility studies for various medical devices

Cytotoxicity (ISO 10993-5)

ISO 10993-5:2009 Biological evaluation of medical devices Part 5: Tests for in vitro cytotoxicity

Testing for cytotoxicity is a good first step toward ensuring the biocompatibility of a medical device. Cytotoxicity testing is a rapid, standardized, sensitive, and inexpensive means to determine whether a material contains significant quantities of biologically harmful extractables. The high sensitivity of the tests is due to the isolation of the test cells in cultures and the absence of the protective mechanisms that assist cells within the body.

Cytotoxicity test methods are useful for screening materials that may be used in medical devices because they serve to separate reactive from nonreactive materials, providing predictive evidence of material biocompatibility. A negative result indicates that a material is free of harmful extractables or has an insufficient quantity of them to cause acute effects under exaggerated conditions with isolated cells.

Sensitization (ISO 10993-10)

ISO 10993-10:2010 Biological evaluation of medical devices Part 10: Tests for irritation and skin sensitization

Biological evaluation of medical devices — Part 10: Tests for skin sensitization specifies procedures for assessing devices and their constituent materials for their potential to induce skin sensitization as possible contact hazards from chemicals released from medical devices. It is the basic document for the selection and conduct of tests enabling evaluation of irritation and dermal sensitization responses relevant to safety of medical materials and devices. This includes: a) pretest considerations for irritation, including in silico and In-vitro methods for dermal exposure; b) details of In-vivo (irritation and sensitization) test procedures; c) key factors for the interpretation of the results. Sensitization can at the moment only be determined by an In-vivo assay. This can be accomplished by using the local lymph node assay (LLNA) in mice, the occluded patch test in guinea pigs or the guinea pig maximization test (GPMT).

1. Guinea Pig Maximization Test (GPMT)
2. Local Lymph Node Assay (LLNA)

Haemocompatibility (ISO 10993-4)

ISO 10993-4:2002/Amd 1:2006 Biological evaluation of medical devices Part 4: Selection of tests for interactions with blood

1. Haemolysis
2. Complement Activation
3. Thrombogenicity

It is critical that any medical device having contact with blood be hemocompatible. If a medical device upsets a balance or improperly activates a blood component, it can present a danger to the patient. ISO 10993-4 provides a structured test-selection system that manufacturers can use to customize a blood compatibility analysis to their devices, thus ensuring patient safety.

It includes In-vitro complement activation (immunology), hemolysis (hematology), and partial thromboplastin time (coagulation) tests. Such In-vitro test methods are usually quicker and less costly than In-vivo methods and do not require the use of animals. Complement activation is the most relevant immunology test for devices exposed to circulating blood. An increase in a downstream complement component over baseline levels indicates activation of the complement cascade. Acceptable complement activation limits have not been established, but comparative data are valuable.

ASTM F 756, a standardized ASTM hemolysis test method, is available for determining the hemolytic potential of a device or material. These In-vitro tests involve a quantitative measurement of plasma hemoglobin. An increase in plasma hemoglobin correlates with lysis of red blood cells, thereby indicating hemolytic activity of the material exposed to the cells. Such testing is frequently performed using rabbit blood.

A device’s effects on blood coagulation may be measured In-vitro by determining the rate of clot formation or the partial thromboplastin time of plasma exposed to the biomaterial or device during an incubation period. The reaction of white blood cells to materials can also be used as an effective hematology test.

Thrombosis may be addressed by performing either an In-vivo or ex vivo test. An evaluation of the thrombogenic potential of a device typically involves placing the device in a simulated clinical setting for a period of time, then removing the device and evaluating the extent of thrombus formation on or in it. The use of an appropriate control article is essential to the interpretation of results in these tests.

Pyrogenicity (ISO 10993-11)

ISO 10993-11:2006 Biological evaluation of medical devices Part 11: Tests for systemic toxicity

Rabbit Pyrogen Test

Systemic toxicity studies address the effects of chemicals that migrate from a device into a patient, where they may exert adverse effects on vital organs.

Some compounds derived from chemical or bacterial sources are capable of causing a fever when present in the body at sufficiently high doses. Called pyrogens, these compounds can be detected by the rabbit pyrogen test. This test consists of preparing fluid extracts of the test materials, administering the extracts intravenously to rabbits, and monitoring the animals’ rectal temperatures over the course of several hours. A significant rise in temperature indicates the presence of pyrogens.

Implantation (ISO 10993-6)

ISO 10993-6:2016 Biological evaluation of medical devices Part 6: Tests for local effects after implantation

ISO 10993-6:2016 specifies test methods for the assessment of the local effects after implantation of biomaterials that are

– solid and non-absorbable,

– non-solid, such as porous materials, liquids, gels, pastes, and particulates, and

– degradable and/or absorbable, which may be solid or non-solid.

intended for use in medical devices.

The test sample is implanted into a site (intramuscular /subcutaneous) and animal species (rats / rabbits) appropriate for the evaluation of the biological safety of the material. The local effects are evaluated by a comparison of the tissue response caused by a test sample to that caused by control materials used in medical devices whose clinical acceptability and biocompatibility characteristics have been established. The objective of the test methods is to characterize the history and evolution of the tissue response after implantation of a medical device /biomaterial including final integration or absorption /degradation of the material. In particular for degradable /absorbable materials, the degradation characteristics of the material and the resulting tissue response are determined.

Genotoxicity (ISO 10993-3)

ISO 10993-3:2014 Biological evaluation of medical devices Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity

1. Bacterial Reverse Mutation Test OECD 471
2. In-Vitro Mammalian Cell Gene Mutation Test OECD 476
3. In-Vitro Mammalian Chromosome Aberration Test OECD 473
4. In-Vitro Mammalian Cell Micronucleus Test OECD 487
5. In-vivo Mammalian Erythrocyte Micronucleus Test OECD 474
6. In-vivo Mammalian Bone Marrow Chromosome Aberration Test OECD 475

There are three major types of genotoxic effects: gene mutations, chromosomal aberrations, and DNA effects. Because no single In-vitro assay is capable of detecting all three types, a battery of tests is recommended. Gene mutation and chromosomal aberration tests detect actual lesions in the DNA molecule, while DNA effects tests detect events that may lead to cell damage. In-vitro tests in each category can be conducted using microorganisms or mammalian cells.

Classical In-vitro and In-vivo tests can be used to evaluate the genotoxicity of medical device materials. In all cases, adverse or equivocal findings warrant further investigation. Confirmation testing by dose-response relationship is the standard course of action. In addition, a presumptive positive finding in an In-vitro assay can be confirmed by conducting an alternative In-vivo model.

Acceptable results from a battery of genotoxicity tests will not only go a long way toward ensuring the safety of a proposed biomaterial; in some cases, such data can justify not pursuing In-vivo carcinogenicity studies, particularly if there is existing information about the lack of genotoxicity of the material in question.

Routes of Administrations employed:

Depending on the type of study, the objective of the study, the duration and the species used and regulatory requirements, the routes of administration employed for in-vivo studies are mainly

• oral,
• intravenous and
• dermal routes

Although along with the above regular routes, other routes like

• subcutaneous,
• intradermal,
• intraperitoneal,
• ocular,
• intraarticular,
• intrathecal or
• intracranial

are also employed (as and when needed).

We have expertise (trained and experienced manpower /staff) and know-how available for the above routes of administrations.

Guidelines followed:

Most of our studies are as per the OECD Guidelines but at the same time we also use other guidelines like ICH, ISO 17025, OPPTS (now OCSPP), CDSCO, CIB&RC, Indian/US/British Pharmacopoeia, etc.

The selection of the guideline depends on the clients /sponsor (Pharmaceutical / Agrochemical / Biotech / Medical Device or other Companies, etc.), the test material / substance / item (Pharmaceutical / Agrochemical / Biotech product / Medical Device /Industrial chemicals, etc.), region where data is to be submitted (India / APAC / EU / NA /Latin America / ROW, etc.).

At Trans-Genica, we strictly follow following regulatory processes

• All the experiments are conducted in accordance with CCSEA, (Committee for Control and Supervision of Experiments on Animals) an Indian Governmental body managing ethical animal’s research.
• Trans-Genica is committed to strictly follow 4R Principles on Experimental Animals – Replacement, Refinement, Reduction and Rehabilitation. We are committed to make sure all our animal colonies are looked after properly as per the standard ethical guidelines.
• All our experiments are governed through Institutional Animal Ethic Committee (IAEC).
• At Trans-Genica, all our scientists working with animal models and industry experienced veterinarian ensures compliance of Institutional Animal Welfare policies.