SOP FOR CONDUCTING PRECLINICAL ACUTE ORAL TOXICITY IN RODENTS BY OECD TG-420

1.0 OBJECTIVE

1.1 To lay down a standard procedure to be followed for conducting acute oral toxicity in rodents by OECD Test Guideline-420 (OECD TG-420).

2.0 SCOPE

2.1 This Standard Operating Procedure (SOP) shall be applicable for conducting acute oral toxicity in rodents by OECD Test Guideline-420 (OECD TG-420).

3.0 RESPONSIBILITY

3.1 Head- Animal House Facility

3.2 Study Director

3.3 All Personnel involved in Study

4.0 DEFINITIONS

4.1 Nil

5.0 PROCEDURE

5.1 Pre-Experimental Activities

5.1.1 IAEC approval for conducting oral acute toxicity of test item shall be taken as per the currently running version of SOP…………… The study shall be initiated after getting approval from the IAEC committee for the conduct of the experiment.

5.1.2 Detailed Study Plan for the study shall be prepared by following OECD Test Guideline 420.

5.1.3 Study personnel shall be given study-specific training if any (if required) as per the currently running version of SOP……………...

5.1.4 After getting approval of the study plan from the sponsor, the required number of data recording sheets (DRS) formats shall be issued from QAD.

5.1.5 Animal requisition shall be given to animal house facility and animals shall be issued as per the currently running version of SOP……………..

5.1.6 After issue animals shall be acclimatized to standard laboratory conditions for a period of at least 5 days as per the currently running version of SOP……………...

5.1.7 After completion of the acclimatization period animals shall be randomized and grouped by the following SOP………………..

5.1.8 Numbering and identification of animals shall be carried by following the currently running version of SOP………………...

5.1.9 If the test item needs to be formulated (prepared) / diluted in any vehicle then dose calculation and formulation of the test item shall be carried out as per the sponsor’s instruction / recommended procedure.

5.2 Experimental Activities

5.2.1 Administration of doses

5.2.1.1 The test item is administered in a single dose by gavage using a stomach tube by following the currently running version of SOP……………….. In the unusual circumstance that a single dose is not possible, the dose shall be given in smaller fractions over a period not exceeding 24 hours.

5.2.1.2 Animals shall be fasted prior to dosing (e.g. with the rat, food but not water shall be withheld overnight; with the mouse, food but not water shall be withheld for 3-4 hours).

5.2.1.3 Following the period of fasting, the animals shall be weighed and the calculated amount of test substance shall be administered.

5.2.1.4 After the substance has been administered, food may be withheld for a further 3-4 hours in rats or 1-2 hours in mice.

5.2.1.5 Where a dose is administered infractions over a period of time, food and water shall be provided to animals depending on the length of the period.

5.2.2 Sighting study

5.2.2.1 The purpose of the sighting study is to allow the selection of the appropriate starting dose for the main study. The test substance is administered to single animals in a sequential manner following the flow charts below:

5.2.2.2 The sighting study is completed when a decision on the starting dose for the main study is made (or if a death is seen at the lowest fixed dose).

5.2.2.3 The starting dose for the sighting study shall be selected from the fixed-dose levels (as mentioned in OECD TG-420) of 5, 50, 300, and 2000 mg/kg as a dose expected to produce evident toxicity based, when possible, on evidence from in vivo and in vitro data from the same chemical and from structurally related chemicals. In the absence of such information, the starting dose will be 300 mg/kg.

5.2.2.4 A period of at least 24 hours shall be allowed between the dosing of each animal. All animals shall be observed for at least 14 days.

5.2.2.5 Exceptionally, and only when justified by the sponsor, the use of an additional upper fixed dose level of 5000 mg/kg shall be considered as per Annex 4 of OECD guideline-420.

5.2.2.6 In cases where an animal tested at the lowest fixed dose level (5mg/kg) in the sighting study dies, the normal procedure is to terminate the study. However, if further confirmation of the classification is required, an optional supplementary procedure may be conducted, as follows. A second animal is dosed at 5mg/kg. If this second animal dies, then GHS Category 1 will be confirmed and the study will be immediately terminated. If the second animal survives, then a maximum of three additional animals will be dosed at 5mg/kg. Because there will be a high risk of mortality, these animals shall be dosed in a sequential manner to protect animal welfare.

5.2.2.7 If a second death occurs, the dosing sequence will be immediately terminated and no further animals will be dosed.

5.2.3 Main study

5.2.3.1 A total of five animals of one sex shall be used for each dose level investigated. The five animals will be made up of one animal from the sighting study dosed at the selected dose level together with an additional four animals (except, unusually, if a dose level used on the main study was not included in the sighting study).

5.2.3.2 The time interval between dosing at each level will be determined by the onset, duration, and severity of toxic signs. Treatment of animals at the next dose shall be delayed until one is confident of survival of the previously dosed animals.

The action to be taken following testing at the starting dose level is indicated by the flow charts below:

5.2.4 Limit test

5.2.4.1 The limit test is primarily used in situations where the study director has information (may be provided by sponsor) indicating that the test material is likely to be nontoxic, i.e., having toxicity only above regulatory limit doses.

5.2.4.2 In those situations, where there is little or no information about its toxicity, or in which the test material is expected to be toxic, the main test should be performed.

5.2.4.3 Using the normal procedure, a sighting study starting dose of 2000 mg/kg (or exceptionally 5000mg/kg) followed by dosing of a further four animals at this level serves as a limit test for this guideline.

5.2.5 Observations

5.2.5.1 Clinical signs of toxicity after dosing shall be recorded as per current running version of SOP……………. and data shall be recorded in associated format to the SOP. All animals shall be observed individually after dosing at least once during the first 30 minutes, periodically during the first 24 hours, with special attention given during the first 4 hours, and daily thereafter, for a total of 14 days, except where they need to be removed from the study and humanely killed for animal welfare reasons or are found dead. However, the duration of observation will not be fixed rigidly. It will be determined by the toxic reactions, time of onset, and length of the recovery period, and may thus be extended when considered necessary.

5.2.5.2 Animals shall be observed daily during the observation period for mortality as per SOP/AHF/057 and observation shall be recorded in associated format to SOP.

5.2.5.3 Individual weights of animals shall be determined shortly before the test substance is administered and at least weekly thereafter. Bodyweight shall be recorded as per the currently running version of SOP………… and data shall be recorded in associated format to SOP. At the end of the test surviving animals shall be weighed and then humanely killed.

5.2.5.4 All test animals (including those that die during the test or are removed from the study for animal welfare reasons) shall be subjected to a gross necropsy as per the currently running version of SOP………….. and all gross pathological changes shall be recorded in associated format to SOP.

5.2.5.5 Microscopic examination of organs showing evidence of gross pathology in animals surviving 24 or more hours after the initial dosing may also be considered because it may yield useful information.

5.2.5.6 If, microscopic examination is indicated, same shall be carried out by authorized histopathology laboratory.

5.2.5.7 All observations shall be systematically recorded, with individual records being maintained for each animal.

5.2.5.8 Additional observations will be necessary if the animals continue to display signs of toxicity. Observations will include changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic, and central nervous systems, and somatomotor activity and behavior pattern. Attention will be directed to observations of tremors, convulsions, salivation, diarrhea, lethargy, sleep, and coma.

5.2.5.9 Animals found in a moribund condition and animals showing severe pain or enduring signs of severe distress shall be humanely killed. When animals are killed for humane reasons or found dead, the time of death shall be recorded as precisely as possible.

5.3 Data and Reporting

5.3.1 Individual animal data shall be provided. Additionally, all data shall be summarized in tabular form, showing for each test group the number of animals used, the number of animals displaying signs of toxicity, the number of animals found dead during the test or killed for humane reasons, time of death of individual animals, a description and the time course of toxic effects and reversibility, and necropsy findings.

5.3.2 Statistical analysis, if required shall be carried out by using a licensed version of Graphpad instant 3.01 or by Graphpad 5.01 by following its user manual.

5.4 Test report

5.4.1 Draft test report shall be prepared and shall include the following information, as appropriate:

Test substance	:	Certificate of analysis of Test Item Vehicle (if appropriate): justification for the choice of vehicle, if other than water.
Test animals	:	Species/strain of animals used Number, age, and sex of animals (including, where appropriate, a rationale for use of males instead of females) Source, housing conditions, diet etc.
Test conditions	:	Details of the test substance formulation, including details of the physical form of the material administered Details of the administration of the test substance including dosing volumes and time of dosing Details of food and water quality (including diet type/source, water) The rationale for the selection of the starting dose
Results	:	Tabulation of response data and dose level for each animal (i.e. animals showing signs of toxicity including mortality, nature, severity, and duration of effects) Tabulation of body weight/body weight changes Individual weights of animals at the day of dosing, in weekly intervals thereafter, and at time of death or sacrifice Date and time of death if prior to scheduled sacrifice Time course of onset of signs of toxicity and whether these were reversible for each animal Necropsy findings and histopathological findings for each animal, if available.
Discussion and interpretation of results	:	Discussion and interpretation of results shall be mentioned clearly
Conclusions	:	The study shall be concluded as appropriate

5.5 Archive

5.5.1 After the approval of the draft study report from the sponsor, the study file and all related materials / raw data shall be achieved.

5.6 Flow chart for conducting Acute Oral toxicity in rodents (By OECD TG-420)

5.7 Checklist of SOPs required for conducting acute oral toxicity in rodents by OECD TG-420

SOPs required	SOP No.	Title of SOP
Pre-experimental activities	SOP…………	Standard Operating Procedure for Institutional Animal Ethics Committee meeting and obtaining protocol approval for experimentation on animals
	SOP…………	Standard Operating Procedure for personnel training for animal care and use
	SOP…………	Standard Operating Procedure for the supply of laboratory animals to studies
	SOP…………	Standard Operating Procedure for acclimatization of laboratory animals
	SOP…………	Standard Operating Procedure for  randomization of experimental animals
	SOP…………	Standard Operating Procedure for animal numbering and identification procedure for laboratory animals
experimental activities	SOP…………	Standard Operating Procedure for weighing of laboratory animals
	SOP…………	Standard Operating Procedure for  administration of test item by oral, parenteral, and topical route in laboratory animals
	SOP…………	Standard Operating Procedure for observation of clinical signs in experimental animals
	SOP…………	Standard Operating Procedure for the handling of animal morbidity and mortality in Animal House Facility
	SOP…………	Standard Operating Procedure for gross necropsy and collection, weighing & fixation of organs/tissues in a laboratory animals

6.0 ENCLOSURES

6.1 Nil

7.0 ABBREVIATIONS

7.1 SOP: Standard Operating Procedure

7.2 AHF: Animal House Facility

7.3 DRS: Data Recording sheets

7.4 GHS: Global Harmonized System of Classification and Labeling of Chemicals

7.5 OECD: Organization for Economic Cooperation and Development

7.6 QAD: Department of Quality Assurance

8.0 REFERENCES

8.1 OECD- Test Guideline (TG)- 420

9.0 REVISION HISTORY

Sr. No.	Change Control No.	Reason for change
1.	CC………………	Change of Format of SOP

                                                      

           END OF DOCUMENTS

You may like to read these links:   

1. List of All SOPs and Documents for In-vivo Laboratory

2. List of All SOPs and Documents for Study Protocol

3. List of Pharmacological Study Models with details about disease Inducing agent, Standard or Reference Drug, and related parameters.

4. List of OECD Guidelines or Toxicological Studies

5. List of Pharmacopeial Tests Quality Control Tests or Batch Testing Parameters (In-vivo)

6. Check Out the Trick and Tips with Some Trouble Shooting Points While Conducting in OECD 407 and 408

7. List of Guidelines Used for Medical Devices Testing 

8. What are Bradford Hill's criteria for any chemical identification?

9. Animal  Facility Design - Small Laboratory Animals (Rat, Mice, Rabbit, Guinea Pig)

10. List of Chemicals Needed for In-vivo Laboratory

11. OECD Test Guidelines 425 detailedOutlines| AOT425StatPgm Software Installation LD50 Calculation

12. List of Guidelines for Toxicology Animal Studies 

13.  Checklist for Toxicology (OECD-407) study preparation for Histopathology

SHARE

Owner

Hi. I’m Writer of Researchsop.com. ’ ’ Please share these SOPs to all concern pharma people for their development. I like to fullfill the need of curious people. These things inspire me to make things looks better.

• 
• 
• 
• 
•