Animal Toxicology studies as per the CDSCO Guidance

General Principles:

Toxicity studies should comply with the norms of Good Laboratory Practices (GLP). Briefly, these studies should be performed by suitably trained and qualified staff employing properly calibrated and standardized equipment of adequate size and capacity. Studies should be done as per written protocols with modifications (if any) verifiable retrospectively. Standard operating procedures (SOPs) should be followed for all managerial and laboratory tasks related to these studies. Test substances and test systems (in-vitro or in-vivo) should be properly characterised and standardized. All documents belonging to each study, including its approved protocol, raw data, draft report, final report, and histology slides and paraffin tissue blocks should be preserved for a minimum of five years after marketing of the drug.

1. Systemic toxicity studies
   • Single dose toxicity studies: These studies (see Table 1) should be carried out in 2 rodent species (mice and rats) using the same route as intended for humans. In addition, unless the intended route of administration in humans is only intravenous, at least one more route should be used in one of the species to ensure systemic absorption of the drug. This route should depend on the nature of the drug. A limit of 2g/kg (or 10 times the normal dose that is intended in humans, whichever is higher) is recommended for oral dosing. Animals should be observed for 14 days after the drug administration, and minimum Lethal Dose (MLD) and Maximum Tolerated Dose (MTD) should be established. If possible, the target organ of toxicity should also be determined. Mortality should be observed for up to seven days after parenteral administration and up to 14 days after oral administration. Symptoms, signs and mode of death should be reported, with appropriate macroscopic and microscopic findings where necessary. LD10 and LD50 should be reported preferably with 95 percent confidence limits. If LD50 cannot be determined, reasons for the same should be stated.
   • Repeated dose systemic toxicity studies: These studies (see Table 1) should be carried out in at least two mammalian species, of which one should be a non-rodent. Dose ranging studies should precede the 14-, 28-, 90- or 180- day toxicity studies. Duration of the final systematic toxicity study will depend on the duration, therapeutic indication and scale of the proposed clinical trial. If a species is known to metabolise the drug in the same way as humans, it should be preferred for toxicity studies.
   • In repeated-dose toxicity studies the drug should be administered seven days a week by the route intended for clinical use. The number of animals required for these studies, i.e. the minimum number of animals on which data should be available.
   • Wherever applicable, a control group of animals given the vehicle alone should be included, and three other groups should be given graded d oses of the drug. The highest dose should produce observable toxicity; the lowest dose should not cause observable toxicity, but should be comparable to the intended therapeutic dose in humans or a multiple of it. To make allowance for the sensitivity of the species the intermediate dose should cause some symptoms, but not gross toxicity or death, and should be placed logarithmically between the other two doses.
2. Notes:
   • Single dose toxicity study: Each group should contain at least five animals of either sex. At least four graded doses should be given. Animals should be exposed to the test substance in a single bolus or by continuous infusion or several doses within 24 hours. Animals should be observed for 14 days. Signs of intoxication, effect on body weight, gross pathological changes should be reported. It is desirable to include histopathology of grossly affected organs, if any.
   • Dose Ranging Study: Objectives of this study include the identification of target organ of toxicity and establishment of Maximum Tolerated Dose (MTD) for subsequent studies.
   • Rodents: —Study should be performed in one rodent species (preferably rat) by the proposed clinical route of administration. At least four graded doses including control should be given, and each dose group as well as the vehicle control should consist of a minimum of five animals of each sex. Animals should be exposed to the test substance daily for 10 consecutive days. Highest dose should be the maximum tolerated dose of single-dose study. Animals should be observed daily for signs of intoxication (general appearance, activity and behaviour etc), and periodically for the body weight and laboratory parameters. Gross examination of viscera and microscopic examination of affected organs should be done.
   • Non-rodents: One male and one female are to be taken for ascending Phase Maximum Tolerated Dose (MTD) study. Dosing should start after initial recording of cage-side and laboratory parameters. Starting dose may be three to five times the extrapolated effective dose or Maximum Tolerated Dose (MTD) (whichever is less), and dose escalation in suitable steps should be done every third day after drawing the samples for laboratory parameters. Dose should be lowered appropriately when clinical or laboratory evidence of toxicity are observed. Administration of test substance should then continue for 10 days at the well-tolerated dose level following which, samples for laboratory parameters should be taken. Sacrifice, autopsy and microscopic examination of affected tissues should be performed as in the case of rodents.
   • 14-18 Days Repeated dose toxicity Studies: One rodent (6-10 / sex / group) and one non-rodent (2-3 / sex / group) species are needed. Daily dosing by proposed clinical route at three dose levels should be done with highest dose having observable toxicity, mid dose between high and low dose, and low dose. The doses should preferably be multiples of the effective dose and free from toxicity. Observation parameters should include cage side observations, body weight changes, food or water intake, blood biochemistry, haematology, and gross and microscopic studies of all viscera and tissues.
   • 90 Days repeated dose toxicity studies: One rodent (15-30 / sex/group) and one non-rodent (4- 6/sex/group) species are needed. Daily dosing by proposed clinical route at three graded dose levels should be done. In addition to the control a “high-dose-reversal” group and its control group should also be included. Parameters should include signs of intoxication (general appearance, activity and behaviour etc), body weight, food intake, blood biochemical parameters, haematological values, urine analysis, organ weights, gross and microscopic study of viscera and tissues. Half the animals in “reversal” groups (treated and control) should be sacrificed after 14 days of stopping the treatment. The remaining animals should be sacrificed after 28 days of stopping the treatment or after the recovery of signs or clinical pathological changes – whichever comes later, and evaluated for the parameters used for the main study.
   • 180 Day repeated dose toxicity studies: One rodent(15-30/sex/group)and one non-rodent (4-6/sex/group) species are needed. At least four groups, including control, should be taken. Daily dosing by proposed clinical route at three graded dose levels should be done. Parameters should include signs of intoxication, body weight, food intake, blood biochemistry, haematology, urine analysis, organ weights, gross and microscopic examination of organs and tissues.
3. Male Fertility study: One rodent species (preferably rat) should be used. Dose selection should be done from the results of the previous 14 days or 28 days toxicity study in rat. Three dose groups, the highest one showing minimal toxicity in systemic studies, and a control group should be taken. Each group should consist of six adult male animals. Animals should be treated with the test substance by the intended route of clinical use for minimum 28 days and maximum 70 days before they are paired with female animals of proven fertility in a ratio of 1:2 for mating. Drug treatment of the male animals should continue during pairing. Pairing should be continued till the detection of vaginal plug or 10 days, whichever is earlier. Females getting thus pregnant should be examined for their fertility index after day 13 of gestation. All the male animals should be sacrificed at the end of the study. Weights of each testis and epididymis should be separately recorded. Sperms from one epididymis should be examined for their motility and morphology. The other epididymis and both testis should be examined for their histology.
4. Female reproduction and development toxicity studies: These studies need to be carried out for all drugs proposed to be studied or used in women of child bearing age. Segment I, II and III studies (see below) are to be performed in albino mice or rats, and segment II study should include albino rabbits also as a second test species. On the occasion, when the test article is not compatible with the rabbit (e.g. antibiotics which are effective against gram positive, anaerobic organisms and protozoas) the Segment II data in the mouse may be substituted.
   • Female fertility study: The study should be done in one rodent species (rat preferred). The drug should be administered to both males and females, beginning a sufficient number of days (28 days in males and 14 days in females) before mating. Drug treatment should continue during mating and, subsequently, during the gestation period. Three graded doses should be used, the highest dose (usually the Maximum Tolerated Dose (MTD) obtained from previous systemic toxicity studies) should not affect general health of the parent animals. At least 15 males and 15 females should be used per dose group. Control and the treated groups should be of similar size. The route of administration should be the same as intended for therapeutic use. Dams should be allowed to litter and their medication should be continued till the weaning of pups. Observations on body weight, food intake, clinical signs of intoxication, mating behaviour, progress of gestation or parturition periods, length of gestation, parturition, postpartum health and gross pathology (and histopathology of affected organs) of dams should be recorded. The pups from both treated and control groups should be observed for general signs of intoxication, sex-wise distribution in different treatment groups, body weight, growth parameters, survival, gross examination, and autopsy. Histopathology of affected organs should be done. abnormalities and the other half for visceral abnormalities. Observation parameters should include: (Dams) signs of intoxication, effect on body weight, effect on food intake, examination of uterus, ovaries and uterine contents, number of corpora lutea, implantation sites, resorptions (if any); and for the foetuses, the total number, gender, body length, weight and gross or visceral or skeletal abnormalities, if any.
   • Perinatal study: This study is specially recommended if the drug is to be given to pregnant or nursing mothers for long periods or where there are indications of possible adverse effects on foetal development. One rodent species (preferably rat) is needed. Dosing at levels comparable to multiples of human dose should be done by the intended clinical route. At least four groups (including control), each consisting of 15 dams should be used. The drug should be administered throughout the last trimester of pregnancy (from day 15 of gestation) and then the dose that causes low foetal loss should be continued throughout lactation and weaning. Dams should then be sacrificed and examined as described below. One male and one female from each litter of F1 generation (total 15 males and 15 females in each group) should be selected at weaning and treated with vehicle or test substance (at the dose levels described above) throughout their periods of growth to sexual maturity, pairing, gestation, parturition and lactation. Mating performance and fertility of F1 generation should thus be evaluated to obtain the F2 generation whose growth parameters should be monitored till weaning. The criteria of evaluation should be the same as described earlier. Animals should be sacrificed at the end of the study and the observation parameters should include (Dams) body weight, food intake, general signs of intoxication, progress of gestation or parturition periods and gross pathology (if any); and for pups, the clinical signs, sex-wise distribution in dose groups, body weight, growth parameters, gross examination, survival and autopsy (if needed)
   • and where necessary, histopathology.
5. Local Toxicity: These studies are required when the new drug is proposed to be used by some special route (other than oral) in humans. The drug should be applied to an appropriate site (e.g., skin or vaginal mucous membrane) to determine local effects in a suitable species. Typical study designs for these studies should include three dose levels and untreated or vehicle control, preferably use of two species, and increasing group size with increase in duration of treatment. Where dosing is restricted due to anatomical or humane reasons, or the drug concentration cannot be increased beyond a certain level due to the problems of solubility, pH or tonicity, a clear statement to this effect should be given. If the drug is absorbed from the site of application, appropriate systemic toxicity studies will also be required.
6. Dermal toxicity: The study may be done in rabbit and rat. The initial toxicity study shall be carried out by non-animal alternative tests as given in Organisation for Economic Cooperation and Development Guidelines. In rabbit and rat studies, daily topical (dermal) application of test substance in its clinical dosage form should be done; Test material should be applied on shaved skin covering not less than 10% of the total body surface area. Porous gauze dressing should be used to hold liquid material in place. Formulations with different concentrations (at least 3) of test substance, several fold higher than the clinical dosage form should be used. Period of application may vary from seven to 90 days depending on the clinical duration of use. Where skin irritation is grossly visible in the initial studies, a recovery group should be included in the subsequent repeated-dose study. Local signs (erythema, oedema and eschar formation) as well as histological examination of sites of application should be used for evaluation of results.
7. Photo allergy or dermal photo toxicity: It should be tested by Armstrong or Harber test in guinea pig. This test should be done if the drug or a metabolite is related to an agent causing photosensitivity or the nature of action suggests such a potential (e.g., drugs to be used in treatment of leucoderma). Pretest in eight animals should screen four concentrations (patch application for two hours 115 min.) with and without UV exposure (10 J/cm2). Observations recorded at 24 and 48 hours should be used to ascertain highest non-irritant dose. Main test should be performed with 10 test animals and five controls. Induction with the dose selected from pretest should use 0.3 ml/patch for 2 hour 115 min. followed by 10 J/ cm2 of UV exposure. This should be repeated on
8. day 0, 2,4,7,9 and 11 of the test. Animals should be challenged with the same concentration of test substance between day 20 to 24 of the test with a similar 2-hour application followed by exposure to 10 J/cm2 of UV light. Examination and grading of erythema and oedema formation at the challenge sites should be done 24 and 48 hours after the challenge. A positive control like musk ambrett or psoralin should be used.
9. Vaginal toxicity test: —Study is to be done in rabbit or dog. Test substance should be applied topically (vaginal mucosa) in the form of pessary, cream or ointment. Six to ten animals per dose group should be taken. Higher concentrations or several daily applications of test substance should be done to achieve multiples of daily human dose. The minimum duration of drug treatment is seven days (more according to clinical use), subject to a maximum of 30 days. Observation parameters should include swelling, closure of in troit us and histopathology of vaginal wall.
10. Rectal tolerance test: For all preparations meant for rectal administration this test may be performed in rabbits or dogs. Six to ten animals per dose group should be taken. Formulation in volume comparable to human dose (or the maximum possible volume) should be applied once or several times daily, per rectally, to achieve administration of multiples of daily human dose. The minimum duration of application is even days (more according to clinical use), subject to a maximum of 30 days. Size of suppositories may be smaller, but the drug content should be several fold higher than the proposed human dose. Observation parameters should include clinical signs (sliding on backside), signs of pain, blood or mucus in faeces, condition of anal region or sphincter, gross and (if required) histological examination of rectal mucosa.
11. Parenteral drugs: For products meant for intravenous or intramuscular or subcutaneous or intradermal injection the sites of injection in systemic toxicity studies should be specially examined grossly and microscopically. If needed, reversibility of adverse effects may be determined on a case to case basis.
12. Ocular toxicity study: These studies should be carried out in two species, one of which should be the albino rabbit which has a sufficiently large conjunctival sac. Direct delivery of drug onto the cornea in case of animals having small conjunctival sacs should be ensured. Liquids, ointments, gels or soft contact lenses (saturated with drug) should be used. Initial single dose application should be done to decide the exposure concentrations for repeated-dose studies and the need to include a recovery group. Such initial toxicity studies shall be carried out by nonanimal alternative tests as given in Organisation for Economic Cooperation and Development Guidelines. Duration of the final study will depend on the proposed length of human exposure subject to a maximum of 90 days. At least two different concentrations exceeding the human dose should be used for demonstrating the margin of safety. In acute studies, one eye should be used for drug administration and the other kept as control. A separate control group should be included in repeated-dose studies. Slit-lamp examination should be done to detect the changes in cornea, iris and aqueous humor. Fluorescent dyes (sodium fluorescein, 0.25 to 1.0%) should be used for detecting the defects in surface epithelium of cornea and conjunctiva. Changes in intra-ocular tension should be monitored by a tonometer. Histological examination of eyes should be done at the end of the study after fixation in Davidson’s or Zenker’s fluid.
13. Inhalation toxicity studies: The studies are to be undertaken in one rodent and one non-rodent species using the formulation that is to be eventually proposed to be marketed. Acute, subacute and chronic toxicity studies should be performed according to the intended duration of human exposure. Standard systemic toxicity study designs (described above) should be used. Gases and vapours should be given in whole body exposure chambers; aerosols are to be given by nose-only method. Exposure time and concentrations of test substance (limit dose of 5mg/l) should be adjusted to ensure exposure at levels comparable to multiples of intended human exposure. Three dose groups and a control (plus vehicle control, if needed) are required. Duration of exposure may vary subject to a maximum of 6 hours per day and five days a week. Food and water should be withdrawn during the period of exposure to test substance. Temperature, humidity and flow rate of exposure chamber should be recorded and reported. Evidence of exposure with test substance of particle size of 4 micron (especially for aerosols) with not less than 25% being 1 micron should be provided. Effects on respiratory rate, findings of bronchial lavage fluid examination, histological examination of respiratory passages and lung tissue should be included along with the regular parameters of systemic toxicity studies or assessment of margin of safety.
14. Allergic or Hypersensitivity: —Standard tests include guinea pig maximization test (GPMT) and local lymph node assay (LLNA) in mouse. Any one of the two may be done.
15. Guinea pig maximization test: The test is to be performed in two steps; first, determination of maximum non-irritant and minimum irritant doses, and second, the main test. The initial study will also have two components. To determine the intradermal induction dose, four dose levels should be tested by the same route in a batch of four male and four female animals (2 of each sex should be given Freund’s adjuvant). The minimum irritant dose should be used for induction. Similarly, a topical minimum irritant dose should be determined for challenge. This should be established in two males and two females. A minimum of six male and six female animals per group should be used in the main study. One test and one control group should be used. It is preferable to have one more positive control group. Intradermal induction (day 1) coupled with topical challenge (day 21) should be done. If there is no response, re-challenge should be done 7 to 30 days after the primary challenge. Erythema and oedema (individual animal scores as well as maximization grading) should be used as evaluation criteria.
16. Local lymph node assay— Mice used in this test should be of the same sex, either only males or only females. Drug treatment is to be given on ear skin. Three graded doses, the highest being maximum nonirritant dose plus vehicle control should be used. A minimum of 6 mice per group should be used. Test material should be applied on ear skin on three consecutive days and on day 5, the draining auricular lymph nodes should be dissected out 5 hours after i.v H-thymidine or bromo-deoxyuridine (BrdU). Increase in H-thymidine or BrdU incorporation should be used as the criterion for evaluation of results.
17. Genotoxicity: Genotoxic compounds, in the absence of other data, shall be presumed to be transspecies carcinogens, implying a hazard to humans. Such compounds need not be subjected to long-term carcinogenicity studies. However, if such a drug is intended to be administered for chronic illnesses or otherwise over a long period of time – a chronic toxicity study (up to one year) may be necessary to detect early tumorigenic effects. Genotoxicity tests are invitro and in-vivo tests conducted to detect compounds which induce genetic damage directly or indirectly. These tests should enable a hazard identification with respect to damage to De-oxy Ribonucleic Acid (DNA) and its fixation.