SDM COLLEGE OF AYURVEDA AND HOSPITAL, HASSAN DEPARTMENT OF AGADA TANTRA

CHRONIC TOXICITY STUDY METHODS

PRESENTED BY-Dr. GOPENDRA CHANDRA KAMALPG 1ST YEAR SCHOLAR

GUIDED BY-Dr. NAVEEN K.ASST. PROFESSOR

CONTENTS- 1.TOXICITY- GENERAL 2.CHRONIC TOXICITY-INTRODUCTION 3. OECD GUIDELINE -4521. OBJECTIVES2. INITIAL CONSIDERATIONS3. PRINCIPLE4. DESCRIPTION OF METHODS5. OBSERVATION6. TEST REPORT AYUSH GUIDELINE -170 10. DISCUSSION 11. CONCLUSION

“All substances are poisons, there is none which is not a poison. The right dose differentiates a poison from a remedy.” (Paracelsus, 1493-1541)

Toxicology:It is the study of adverse effects of chemical and physical agents and the degree to which a substance can harm human or animals.

TOXICITY- GENERAL

Benefit –risk ratio can be calculated

Prediction of therapeutic index

A toxicity test is designed to generate data concerning the adverse effects of a substance on human , animal health or environment.

Why toxicity study should be done?

Studies should comply with GLP.

Performed by trained and qualified staff.

Use of standardized and calibrated equipment.

SOP’s followed in laboratory tasks.

All documents should be preserved for minimum 5 years .

PRINCIPLES OF TOXICITY STUDIES

1.ACUTE TOXICITY TESTS (Single dose or multiple dose within 24 hours)

2.SUB-ACUTE TOXICITY TESTS (Daily dose : 14-28 days)

3.SUB-CHRONIC TOXICITY TESTS (Daily dose: up to 90 days)

4.CHRONIC TOXICITY TESTS (Daily dose: up to 12 months)

TYPES OF TOXICITY STUDIES

• International Council on Hormonisation(ICH)

• Organisation for Economic Co-operation and Development(OECD)

• Food and Drug Administration (FDA)

• European Medicines Agency (EMEA)

• AYUSH Guideline 170

GUIDELINES FOLLOWED

• Chronic toxicity test determines toxicity for a substantial portion of a subject’s life.

• According to GHS (GLOBALLY HARMONIZED SYSTEM), it is defined as “ Specific TARGET ORGAN / SYSTEMIC TOXICITY, arising from a REPEATED EXPOSURE up to 12 MONTHS”

• Conducted with a minimum of one rodent and one non-rodent species. • The test compound is administered over more than 90 days, and the

animals are observed periodically.

.

CHRONIC TOXICITY STUDY- INTRODUCTION

OECD TEST GUIDELINE: 452 The original Guideline 452 was adopted in 1981.

The updating of TG 452 has been carried out in parallel with

revisions of the TG 451- Carcinogenicity Studies

and

TG 453- Combined Chronic Toxicity/Carcinogenicity studies

OBJECTIVES

1. Identification of the hazardous properties of a chemical.

2. Identification of target organs.

3. Characterisation of the dose:response relationship.

4. Identification of a no-observed-adverse-effect level (NOAEL).

5. Prediction of chronic toxicity effects at human exposure levels.

6. Provision of data to test hypotheses regarding mode of action

PRINCIPLE 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 chosen.

The test substance is normally administered by the oral route , inhalation or dermal route.

The study design may also include one or more interim kills. 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.

INITIAL CONSIDERATIONS

the identity, chemical structure, and physico-chemical properties of the test substance

any information on the mode of action results of any in vitro or in vivo toxicity tests potential for human exposure available (Q)SAR data and toxicological data available toxicokinetic data

The determination of chronic toxicity may be carried out after initial information on toxicity has been obtained from repeated dose 28-day and/or 90-day toxicity tests.

DESCRIPTION OF METHODSELECTION OF ANIMAL SPECIES RODENTS AND NON-RODENTS are selected to be the test animals. Animals , with a clearly known ORIGIN, SPECIES and BREED

should be used Preferred rodent is rat Smaller species should be avoided Young, healthy adult animals should be used Females should be non-pregnant Weight variation of animals should be minimal (not greater than 20%

of mean weight of each sex)

Acclimatization of animalsi.Period – 7 days (Recording of body weight and food intake twice in a week)ii.Urine qualitative test (Ames multiple sticks)iii.Fecal consistency (Filter paper technique)

PREPARATION

Healthy animals, which have been acclimated to laboratory conditions for at least 7 days

AND

have not been subjected to previous experimental procedures, should be used.

In the case of rodents, dosing of the animals should begin as soon as possible after weaning and acclimatisation and preferably before the animals are 8 weeks old.

Animals should be randomly assigned to the control and treatment groups

Each animal should be assigned a unique identification number, and permanently marked with this number by tattooing, microchip implant, or other suitable method.

HOUSING

Animals may be housed individually, or be caged in small groups of the same sex.

Cages should be arranged in such a way that possible effects due to cage placement are minimised.

Temperature should be 22◦C (± 3◦C). Relative humidity should be 30% - 70% Lighting should be artificial, the sequence being 12 hours light, 12 hours

dark.

FEEDING conventional laboratory diets may be used with an unlimited supply of

drinking water.

Analytical information on the nutrient and dietary contaminant levels and Drinking water should be generated periodically.

The choice of diet may be influenced by the need

1. to ensure a suitable admixture of a test substance

2. and to meet the nutritional requirements. The concentration of the chemical in the feed should not normally exceed an

upper limit of 5% of the total diet.

PROCEDURENUMBER AND SEX OF ANIMALS Animals of both sexes should be used.

A sufficient number of animals should be used so that at the end of the study enough animals in every group are available for thorough biological and statistical evaluation.

Normally, at least 20 animals per sex per group should be used at each dose level.

In studies involving mice, additional animals may be needed in each dose group to conduct all required haematological determination.

INTERIM KILLS, SATELLITE GROUPS, SENTINEL GROUP

The study may make provision for interim kills, e.g. at 6 months, to provide information on progression of toxicological changes and mechanistic information.

Satellite groups may also be included to monitor the reversibility of any toxicological changes induced by the chemical under investigation.

An additional group of sentinel animals (typically 5 animals per sex) may also be included for monitoring of disease status.

DOSE GROUP AND DOSAGE

At least three dose levels and a concurrent control should be used, the highest dose level should be chosen to identify the principal target organs and toxic effects .

The top dose should not exceed 1000 mg/kg body weight/day. Dose level spacing should be designed to demonstrate a dose:response and to establish a

no-observed adverse-effect level (NOAEL). The dose level spacing selected will depend on the characteristics of the test substance,

but 2-4 fold intervals are frequently optimal, and maximum upto 6-10 fold.

The control group shall be an untreated group or a vehicle-control group . Animals in the control group should be handled in an identical manner to those in the test groups.

DOSE PREPARATION AND ADMINISTRATION

FOR ORAL TOXICITY STUDIES Test substances may be administered via the diet , drinking water, oral administration in capsules or by gavage, normally in a vehicle.

It depends on1. the physical and chemical characteristics of the test substance

2. the predominant oral route of exposure in humans

oral gavage should normally be selected only1. for those agents for which a bolus dose administration reasonably represents potential human exposure

2. when sufficient dietary concentrations cannot be achieved due to e.g. physical, chemical properties of the test substance, or to its palatability in the diet.

The test substance is administered in the diet –1. as a constant dietary concentration (mg/kg diet)- monitored on a cage basis at least weekly

2. as a constant dose level in terms of the animal body weight- adjusted regularly based on anticipated food consumption and body weight of the animals

The test substance is normally incorporated at a fixed concentration in the drinking water- (in mg/ml water)

Dose ( mg/kg body weight per day)- based on anticipated water consumption of the animals.

Doses ( mg/ml water)- water consumption must be monitored on a cage basis at least weekly . The maximum volume of solution that can be given by gavage in one dose

depends on the size of the test animal.

For rodents, the volume ordinarily should not exceed 1 ml/100 g body weight, except in the case of aqueous solutions where 2 ml/100g body weight may be used.

If the gavage vehicle is oil, the use of a low-fat diet should be considered, and the volume administered should normally not exceed 0.5 ml/100 g body weight/day.

For larger animals, e.g. dogs, the test substance may be administered in capsules, dissolved or suspended in a suitable vehicle.

Order of choice for test vehicle is: AQUEOUS SOLUTION> EMULSION IN OIL (Corn oil) > Solution in other vehicles

For solution in other vehicles , analyze the toxicity level of the vehicle. Determine stability of the test substance . While administering test substance with food , monitor:

i. Homogeneity

ii. Additive concentration

iii. Safety of test substance

For dermal toxicity studies: - Animals are treated with the test substance for at least 6 hrs./ day (daily) Test substance should be applied uniformly over an area approx. 10% of

body surface For highly toxic substances , surface area may be less Area should be covered with a thin and uniform film Between applications , the test substance is held in contact with skin using a

gauze dressing/ non-irritating tape Cover test site in order to retain gauze dressing Ensure that the animal cannot ingest the test substance inhalation route Dosing is carried out for 6 hours per day, 5 days per week. Either as a gas, a vapour, or a liquid or solid aerosol

DURATION OF STUDY Test Guideline primarily is designed as a 12 month chronic toxicity

study the study design also allows for and can be applied to either shorter

(e.g. 6 or 9 months) or longer (e.g., 18 or 24 months) duration studies, depending on the

requirements of particular regulatory regimes .

OBSERVATIONS

Observation period should be about a year Observations can be classified as :

A. GENERAL CONDITION AND DEATH RATE: -Made once a day ,at the same time each day -Consider peak levels of anticipated effects after dosing -Record health condition of animals -Observe all animals for MORBIDITY and MORTALITY at least TWICE

DAILY

B. WEIGHT,FOOD AND WATER INTAKE ,FOOD INTAKE EFFICIENCY:

-Weigh all animals once a week-Measure rate of food concentrations once a week -If test substance is given via water, measure water consumption weekly

C. HAEMATOLOGICAL TESTS:• In rodents, haematological examinations should be carried out in at least 10

male and 10 female animals per group, at 3, 6, and 12 months• In mice, satellite animals may be required .• In non-rodent studies, samples will be taken from smaller numbers of animal

- blood samples collected(at the end of test period,/ prior to killing animals, or as a part of it),under appropriate conditions-Observed for the following values:i. Hematocritii. Hb conc.iii. Erythrocyte countiv. TLC v. DLCvi. Platelet countvii.Measure of blood clotting time

D. BLOOD BIOCHEMISTRY TEST:Used to investigate:a.Toxic effects in tissuesb.Toxic effects in organs

-Observe for the following serum values:i. Sodiumii. Potassiumiii. Glucoseiv. Ureav. BUNvi. Creatininevii.Total protein and albuminviii.More than 2 enzymes that indicate hepatocellular effects (AST,

ALT,ALP,Sorbitol dehydrogenase)

.

. E. URINE TEST:-Timely urine vol. collection-Check for urine:i. Appearanceii. Vol.iii.Specific gravityiv. Ph.v. Proteinvi.Glucose and blood cells

F. PATHOLOGY TESTS:-Involves :i. G/Eii. M/E• Subject all animals in study to a full, detailed, gross necropsy,

that includes careful examination of:a.Ext. surface of bodyb.All orificesc.Cranial, thoracic and abdominal cavities, and their contents.

• Adherent tissue from liver, heart, kidney, uterus, testis, epididymis, ovaries, adrenals etc. be removed.

• Determine wet weight after dissection to avoid drying

The minimum histopathological examinations should be: • all tissues from the high dose and control groups • all tissues from animals dying or killed during the study

• all tissues showing macroscopic abnormalities

• target tissues, or tissues which showed treatment-related changes in the high dose group, from all animals in all other dose groups

• in the case of paired organs, e.g., kidney, adrenal, both organs should be examined.

TEST REPORT -Test report must include the following info:

1. Test substance

2. Vehicle (if appropriate)

3. Test animals

4. Test conditions :

* Rationale for dose level selection

*Details of test substance formulation

*Details of administration of test substance

*Actual doses and conversion factor from diet/ drinking water

*Details of food and water quality

5.Results: *Body wt. *Body wt. changes *Food consumption and water concentration *Toxic response data by sex and dose levels , including toxicity signs *Nature, severity and duration of clinical observations (reversible / not) *Ophthalmic exam. results *Sensory and motor activities *Terminal body wt. ,organ wt. and organ body weight ratios *Necropsy findings *Detailed description of all pathologic findings *Statistical results presentation

6. Result discussion7 Conclusion

AYUSH GUIDELINE 170 FOR ASU DRUGS Gazette Notification issued under the Drugs & Cosmetics Rule 1945

170.GUIDELINES FOR PRE-CLINICAL SAFETY EVALUATION FOR AYURVEDA, SIDDHA AND UNANI DRUGS AND OTHER TRADITIONAL MEDICINE IN INDIA SUB-CHRONIC TOXICITY TESTS Duration: - Sub - chronic toxicity studies for 2 -

12 weeks . Five rats (two non rodents, if required) of each sex should be used for each dose level. The expected clinical route of administration should be used.  At least three different dose levels should be used - therapeutic dose, one higher

than that which produces overt toxicity and one sub-therapeutic dose. All studies should include a vehicle control group of test animals.

General signs, body weight and food and water intake: - For all experimental animals, the general signs should be observed daily and body weight and food intake should be measured periodically. If useful, water intake should also be determined.

For rats, blood samples should be taken before autopsy. For non-rodents, bloods samples should be taken before the start of drug administration, at least one during the administration period (for studies of longer than one month), and before autopsy.

Renal and hepatic functions tests should be done. ECG, visual and auditory tests should be performed, if relevant. Animals found dead during the examination should be autopsied as soon as

possible. Look out for Cause of death and the nature of the toxic changes present. Moribund animals should be sacrificed rather than allowed to die after all the

relevant observations and investigations done.

All surviving animals should be autopsied at the end of the administration period or of the recovery period .

Organs and tissues should be examined macroscopically and organ weights measured.

Histopathological examinations of the organs and tissues of animals receiving lower dosage should also be performed, if changes are found on gross or macroscopic examination or if the highest dose group reveals significant change.

In order to investigate the recovery from toxic changes, animals that are allowed to live for varying lengths of time after cessation of the period of administration of the test medicine, should be examined.

ARTICLES

AYU | Jan-Mar 2013 | Vol 34 | Issue 1 119

An approval for the study was obtained from Institutional Animal Ethics Committee. A total of 80 Swiss albino mice of either sex with body weight ranging from 28 to 30 g were obtained. Mice were acclimatized for 7 days. Temperature and relative humidity were maintained at 25  ±  1°C

and 40‑70%, respectively and illumination of12 hours light and 12 hours dark. Mice were individually housed in polypropylene cages (27 × 19 × 14 cm) with lids and rice husk

bedding. Pelleted rodent diet was provided along with deionized water using plastic nozzle bottles ad libitum.

The animals were weighed and randomly divided into four groups of 10 animals/sex Chronic toxicity study was conducted by single daily administration of test drug @ 7.8

mg/kg i.e. Therapeutically Equivalent Dose (TED), 39 mg/kg (TED  ×  5), and 78 mg/kg (TED  ×  10) body weight along with vehicle control for 90 consecutive days.

The test drug was given as suspension in vehicle [honey mixed with water (2:3)] by gavage, and control receiving vehicle.

All animals were observed for morbidity and mortality twice daily. General clinical observations were made twice a day at the same time throughout study. The animals were observed for changes in skin, fur, eyes, mucus membrane, occurrence of secretions, and excretions. For neurological examination, the animals were taken outside the cage in a standard arena. The behavior of the animals was recorded. Body weights and feed consumption of each animal were recorded at the start of study and thereafter at weekly intervals.haematological and histopathological examinations were performed.

J Toxicol Environ Health B Crit Rev. 2010 Feb; 13(0): 51–138.

doi:  10.1080/10937404.2010.483176

TOXICITY TESTING IN THE 21ST CENTURY: A VISION AND A STRATEGYDaniel Krewski et al. Present technique is under severe pressure to meet the demands such as.. To Test large numbers of existing chemicals, many of which lack basic toxicity data. To Test the large number of new chemicals and novel materials, such as nanomaterials. To Evaluate potential adverse effects with respect to all critical endpoints and life stages. To Evaluate potential toxicity in the most vulnerable members of the human population. To Minimize animal use. To Reduce the cost and time TO Acquire detailed mechanistic and tissue-dosimetry data (human extrapolation)

Research is poised to take advantage of the revolutions in biology and biotechnology. Advances could transform toxicity testing from a system based on whole-animal testing to one founded primarily on in vitro methods that evaluate changes in biologic processes using cells, cell lines, or cellular components, preferably of human origin. Suggested TIERED STUDY METHOD .

DISCUSSION

This duration is chosen to be sufficiently long to allow any effects of cumulative toxicity to become manifest, without the confounding effects of geriatric changes.

Smaller species can lead to problems, due to increased technical challenges of dissecting smaller organs.

Dose levels will generally be based on the results of shorter-term repeated dose toxicity study.

Consideration should be given to carrying out a combined chronic toxicity and carcinogenicity study (TG 453), rather than separate execution of a chronic toxicity study (TG 452) and carcinogenicity study (TG 451).

CONTD..

Rats and mice have been preferred experimental models because of their relatively short life span, their widespread use in pharmacological and toxicological studies.

The chronic toxicity study should be carried out in animals from the same strain and source as those used in preliminary toxicity studies of shorter duration.

CONCLUSION

The chronic toxicity study provides information on the possible health hazards likely to arise from repeated exposure over a considerable part of the entire lifespan .

The study will provide information on the toxic effects of

the substance, indicate target organs and the possibility of accumulation.

REFERENCES Draft OECD guidelines for testing of chemicals, Test guideline 452: Chronic

Toxicity Studies, Draft’s Consultant’s proposal, V. 8, Nov 2008 Test No. 453: Combined Chronic Toxicity/Carcinogenicity Studies:OECD

Guidelines for the Testing of Chemicals, Section 4: Health Effects:Published on September 07, 2009

Considerations on the applicability of OECD TG 453 to whole food/feed testing, European Food Safety Authority, European Food Safety Authority (EFSA), Parma, Italy, EFSA Journal 2013;11(7):3347

OECD Draft Guidance document no. 116 on the design and conduct of chronic toxicity and carcinogenicity studies, supporting tg 451, 452, 453 table of contents.

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