ANNEX 6

REVISED GUIDELINES FOR RESEARCH IN TRANSGENIC PLANTS &

GUIDELINES FOR TOXICITY AND ALLERGENICITY EVALUATION OF TRANSGENIC SEEDS, PLANTS AND PLANT PARTS, 1998

Department of Biotechnology, Ministry of Science and Technology, Govt. of India

 

1. INTRODUCTION

 

The revised present document is meant for the researchers in the country who are involved in recombinant DNA research on plants. Earlier the Department of Biotechnology in January 1990 issued a compendium of guidelines under the title Recombinant DNA Safety Guidelines. A revision was made in 1994 under the title Revised Guidelines for Safety in Biotechnology. The current guidelines have been developed in the light of enormous progress that has been made in recombinant DNA research and its widespread use in developing improved microbial strains, cell lines and transgenic plants for commercial exploitation.

 

2. COVERAGE OF THE REVISED GUIDELINES

 

The current guidelines cover areas of recombinant DNA research on plants including the development of transgenic plants and their growth in soil for molecular and field evaluation. The guidelines also deal with import and shipment of genetically modified plants for research use only.

 

3. STATUTORY BODIES DEALING WITH THE RECOMBINANT DNA WORK

 

In accordance with the Notification No. GSIR 1037 (E) dated 5th December, 1989 of the Ministry of Environment & Forests which empowers the Review Committee on Genetic Manipulation (RCGM) to bring out manuals of guidelines specifying procedure for regulatory process with respect to activities involving genetically engineered organisms in research use and applications including industry with a view to ensuring environmental safety, the present changes in the procedures are being made. These changes are made reiterating the powers conferred on the RCGM to lay down procedures restricting or prohibiting production, sale, importation and use of genetically engineered organisms or cells as are mentioned in the attached schedule of the above mentioned notifications.

 

A. IBSC (Institutional Biosafety Committee)

 

i.         The IBSC is the nodal point for interaction within an Institute/ University/commercial organisation involved in r-DNA research for the implementation of the recombinant DNA guidelines. As such, in the first instance, it is necessary that the organisations intending to carry out research activities involving genetic manipulation of microorganisms, plants or animals should constitute their IBSC in accordance with the procedures in vogue and as informed to the public through the above notification. All recombinant research carried out by the organisation should have a designated Principal Investigator (P.I.). It would be the duty of the P.I. to apprise its IBSC about the nature of the experiments being carried out. Depending upon the category of the experiments as narrated on in the present guidelines the P.I. Can inform the IBSC about the recombinant experiments, seek permission of IBSC before starting the experiments or seek the permission of the RCGM through its IBSC in cases where the risks involved in the experiments are considered to be of higher magnitude having the potential of polluting/endangering the environment, the biosphere, the eco system, the animals and the human beings.

 

The Department of Biotechnology in January 1990 enumerates the duties of the IBSC in pages 15-16 of the original Recombinant DNA Safety Guidelines prepared.

 

B. RCGM (Review Committee on Genetic Manipulation)

 

i.         The RCGM is functioning in the Department of Biotechnology to monitor the safety-related aspects of ongoing research projects involving genetically engineered organisms.

ii.        The RCGM shall include representatives of a) Department of Biotechnology; b) Indian Council of Medical Research; c) Indian Council of Agricultural Research; d) Council of Scientific and Industrial Research; and e) others experts in their individual capacity. RCGM may appoint subgroups to monitor specific projects.

iii.      The RCGM would review all the reports of all approved on-going research projects involving high-risk category and controlled field experiments.

iv.      The RCGM or its constituted subgroups shall visit the site of experimental facilities periodically, where projects with biohazard potential are being pursued and also at a time prior to the commencement of the activity to ensure that adequate safety measures have been taken as per the guidelines.

v.       The RCGM would issue the clearance for import/export of etiologic agents and vectors, transgenic germplasms including transformed calli, seeds and plant parts for research use only.

vi.      The RCGM shall meet at least twice in a year.

vii.    For research in recombinant DNA work-involving risks categorised as category-III and above in this revised document the permission of the RCGM through the Department of Biotechnology must be obtained by the P.I. Before conducting the research work.

viii.   RCGM can authorise applicants (P.I.s) to conduct limited field trails in multi locations in the country. The design of the trial experiments is either provided by the RCGM or it may approve the protocol designed by the P.I. The protocol will seek answers related to animal and human health. Data should also be generated on economic advantage of the transgenics over the existing varieties.

ix.       RCGM can, if required, direct the applicants to generate toxicity, allergenicity and any other relevant data on transgenic materials in appropriate systems. RCGM may design or approve a protocol for conducting experiments to seek answers to the above.

x.        The RCGM can put such conditions as would be required to generate long term environmental safety data from the applicants seeking release of transgenic plants into the open environment, and who have complied with initial safety evaluation.

xi.       RCGM can approve applications for generating research information on transgenic plants. Such information may be generated in contained green house as well as in very small plots, as research needs to be conducted in such environment for seeking answers to specific environmental safety issues emanating from the use of transgenic plants. The small experimental trials should be limited to a total area of 20 acres in multi-locations in one crop season. In one location where the experiment is conducted with transgenic plants, the land used should not be more than one acre. Any experiment beyond the above limits in one crop seasons would require the approval of the Genetical Engineering Approval Committee (GEAC).

 

4. CATEGORIES OF GENETIC ENGINEERING EXPERIMENTS ON PLANTS AND THEIR NOTIFICATIONS

 

A. CATEGORY I, routine recombinant DNA experiments

 

This category includes routine cloning of defined genes, defined non-coding stretches of DNA and open reading frames in defined genes in E. coli or other bacterial and fungal hosts which are GENERALLY CONSIDERED AS SAFE (GRAS) to human, animals and plants. A list of such microorganisms will be prepared by the RCGM and shall be made available to the P.I. on request.

 

This category involves experiments in the lab in contained environment and includes the following:

 

i.         Routine cloning of defined DNA fragments of microbial, animal and plant origin in GRAS organisms.

ii.        Transfer of defined cloned genes into Agrobacterium;

iii.      Use of defined reporter genes to study transient expression in plant cells to study genetic transformation conditions;

iv.      Molecular analysis of transgenic plants grown in-vitro.

 

Categories I experiment need only intimation to the IBSC in the prescribed proforma (available with the RCGM secretariat).

B. CATEGORY II

 

This category includes lab and green house/net house experiments in contained environment where defined DNA fragments non-pathogenic to human and animals are used for genetic transformation of plants, both model species and crop species and the plants are grown in the green house/net house for molecular and phenotypic evaluation.

 

This category includes the experiments described below :

 

1.        Transgenics with constitutive, tissue specific and chimeric promoters used for experimenting expression of defined DNA fragments.

2.        Marker genes extensively used in genetic transformation of plants in lab and green house/net house experiments.

3.        Lab and green house/net house experiments with plants with herbicide resistance conferring genes;

4.        Lab and green house/net house experiments with plants using heterologous genes which confer resistance to biotic and abiotic stresses (i.e. genes like chalcone synthase, heat shock proteins, chitinase, protease inhibitors etc);

5.        Lab and green house/net house experiments with genes from plants, animals and microbial sources that would confer resistance to plant pathogens.

6.        Lab and green house/net house experiments with transgenics with genes for the production of antibodies.

7.        Green house/net house experiments with transgenics with transposable elements for gene tagging in crop species.

 

Permission for performing Category II experiments will be provided by IBSC. The decision of the IBSC would be intimated to the RCGM before execution of the experiments and RCGM would put this information on record.

 

C. CATEGORY III & ABOVE

 

This category pertains to high risk experiments where the escape of transgenic traits into the open environment could cause significant alterations in the biosphere, the ecosystem, the plants and animals by dispersing new genetic traits, the effects of which can not be judged precisely. All experiments conducted in green house and open field conditions not belonging to the above Category II types, would fall under Category III risks. Such experiments could be conducted only after clearance from RCGM and notified by the Department of Biotechnology:

 

5. CONTAINMENTS

 

Different levels of containment are prescribed for the three different categories of rDNA experiments.

 

1.        Category I experiment should be performed using routine good laboratory practices (See Appendix I for details)

2.        For Category II experiments dealing with evaluation of transgenics in green house/net house, the designs for the contained facility shall be as described in Appendix II. The transgenic experiments of Category II risks will have to be carried out in green house/net house, the specification of which is significantly stringent to ensure arrest of transgenes within the contained facility.

3.        For Category III experiments in green house/net house, the later needs to be designed as indicated broadly in Appendix II. The specifications of the green house/net house have been designed to ensure near complete isolation of the facilities from the open environment; care has also been taken to prevent the entry of insects into the green house/net house facility.

 

For limited field experiments in the open environment, the RCGM would provide for and/or would approve the design of the experimental field plots.

 

6. MONITORING AND EVALUATION MECHANISMS FOR GREEN HOUSE/ NET HOUSE EXPERIMENTS AND LIMITED FIELD TRIALS IN THE OPEN ENVIRONMENT

 

The RCGM can bring out manuals of Guidelines specifying procedures for regulatory process with respect to activities involving genetically engineered organisms in research and applications to ensure environmental safety. To monitor, over a period of time, the impact of transgenic plants on the environment, a special Monitoring cum Evaluation Committee of the following constitution will be set up by the RCGM. The Committee shall have the following constitution.

 

a)       Chairman of the Committee : Secretary, DBT & Secretary, DARE shall
jointly discuss and elect a leader of the
committee.

b)       Eminent Plant Biotechnologists : To be nominated by RCGM, 3-4 Nos.

c)       Seed Technologies : To be nominated by ICAR, 2-3 Nos.

d)       Plant Breeders : To be nominated by ICAR, upto 2 Nos.

e)       Plant Ecologists/Environmentalists : To be nominated by RCGM, upto 2 Nos.

f)        Nominee of NBPGR : To be nominated by ICAR.

g)       Nominee of MOE&F : To be nominated by the Chairman, GEAC

h)       Member-Secretary : Member-Secretary, RCGM

 

This committee will undertake field visits at the experimental site/s. The committee shall be guided by the RCGM on the design of field experiments and on the preparation of formats for collecting scientific information on plants in green house/net conditions as well as in limited field trials. Based on the on-the-spot situation the committee can suggest remedial measures to adjust the original trial design and assist the RCGM in collecting, consolidating and analysing the field data for evaluating the environmental risks emanating from the transgenic plants. This committee shall also collect or cause to collect the information on the comparative agronomic advantages of the transgenic plants. From time to time, the committee shall advise the RCGM on the risks and benefits from the use of the transgenic plants put into evaluation. Trials will be done for at least one year with minimum four replications and ten locations in the agroecological zone for which the material is intended. The biological advantage of transgenic will have to be clearly enumerated by the applicant, the Institution, the University or the Industry. The latter would recommend those transgenics, which would be found to be environmentally safe and economically viably by the RCGM, to the Genetic Engineering Approval Committee for consideration for release into the environment.

 

7. BIOSAFETY ASPECTS OF THE TRANSGENIC PLANTS

 

Experiments are designed to systematically identify the hazards, to access to risks and to take step to manage the risks by applying logically valid strategies, to systematically identify the hazards and to assess the risks; the information on the following aspects would be required to be generated.

 

I.         Characteristics of the donor organisms providing the target nucleic acids. These may include the following:

 

1.        Name of the donor organism with its identification characteristics with relevant reference to published information if any.

2.        Pathogenically and toxicity characteristics to plants and animals.

3.        Allergenicity characteristics to human alongwith of the allergenic substances, wherever possible.

4.        The geographical origin of the organisms, its distribution pattern and survival mechanisms.

5.        The method of transfer of its genetic materials to other organisms.

 

II. Characteristics of the vectors used : These may include the following :

 

1.        The origin, identity and habitat of the vectors used.

2.        The sequence, frequency of mobilisation, specificity and marker genes if any, present in the vectors.

3.        The abilities of the vectors to get established in other hosts; the hosts are also to be specified.

 

III. Characteristics of the transgenic inserts : These may include the following :

 

1.        The specific functions coded by the inserted nucleic stretches including the marker gene inserts.

2.        The expression of the nucleic acid products and their activities/properties.

3.        The toxicity of the expression products on the host plant, if any.

4.        The toxicity and allergenicity of the nucleic acid products to human and animals.

 

IV. Characteristics of the transgenic plants : These may include the following :

 

1.        Methods of detection of the transgenic plant in the environment.

2.        Methods of detection and characterization of the escaped transgenic traits in the environment.

3.        Toxicity and pathogenicity of the transgenic plants and their fruits to other plants in the ecosystem and the environment.

4.        Possibility of and the extent of transgenic pollen escape and pollen transfer to wild near relatives, and the consequences to the environment.

5.        Pathogenicity, toxicity and allergenicity of the transgenic plants and their fruits to human and animals.

 

Information on many of the above questions may already be available. Many questions may however be required to be investigated and answers found out, for which appropriate new experiments would have to be designed to gather data. For generating toxicity and allergenicity data, standard protocols devised by international agencies could be used. The Indian national toxicological laboratory like the Industrial Toxicology Research Centre, Lucknow could be consulted to generate appropriate protocol for these purposes.

 

For minimizing the risk arising from the limited release of transgenic plants, the following may be taken into consideration :

 

1.        Special separation for isolation, for preventing reproduction/fertilisation and seed setting.

2.        Biological prevention of flowering by making use of sterility properties etc.

3.        Human intervention for the removal of reproductive structures of flowers.

4.        Controlling the reproductive structures of transgenic plants like the seeds and the plant propagules from unaccounted spread.

5.        Controlling and destroying volunteer plants from the experimental field.

6.        To take into account the proximity to human activity in case the transgenic plants have allergenic properties to human and animals.

7.        Appropriate training of field personnel responsible for handling the transgenic plants.

8.        Plans for handling unexpected events.

9.        Documentation of previous published information, if any, including any documented evidence of effects of release to ecosystem.

 

Thorough comparison with national checks for productivity and susceptibility/resistance to biotic and abiotic stresses will have to be made.

 

All the information as above are to be documented in the form of a document which would be called the registration document.

 

8. IMPORT AND SHIPMENT OF TRANSGENIC GERMPLASM FOR RESEARCH PURPOSES:

 

Clearance for import of transgenic material, for research purposes would be provided by the RCGM. The RCGM will issue an import certificate after looking into the documents related to the safety of the material and the national need. The RCGM will take into consideration the facilities available with the importer for in-soil tests on the transgenic material. The importer of a transgenic material may import the material accompanied by an appropriate phyto-sanitary certificate issued by the authority of the country of export, and such import may be routed through the Director, NBPGR on the basis of the import permit issued by the DBT, based on the recommendations of the RCGM. The import certificate would be cancelled if NBPGR would not provide the phyto-sanitory certificate. NBPGR will provide information on the time that is required for phyto-sanitary evaluation. These evaluations will be done in a time-bound manner in presence of the agents of the institutes or the commercial organisations that are importing the material, if they so desire. Parts of the seed material will be kept at NBPGR in double lock system in the presence of the importer. This lot of seed will act as a source material in case of any legal dispute.

 

APPENDIX I

 

GOOD LABORATORY PRACTICES

 

1.        Use a pipettor for all the solution transfers. No mouth pipetting.

2.        Plug pipettes with cotton.

3.        Do not blow infectious material out of pippetes.

4.        Do not prepare mixtures of infectious material by bubbling expiratory air through the liquid with a pipette.

5.        Before and after infecting an animal, swab the site of injection with a disinfectant.

6.        Sterilise discarded pipettes and syringes in pan where they were first placed after use.

7.        Before centrifuging, inspects tubes for cracks. Inspect the inside of the Turin cup for rough walls caused by erosion or adhering matter. Carefully remove all bits of glass from the rubber cushion.

8.        Use of centrifuge tunnion cups with screw caps or equivalent.

9.        Avoid decanting centrifuge tubes; if you must do so, wipe off the outer rim with a disinfectant. Avoid filling the tube to the point that the rim ever becomes wet with culture.

10.     Sterilise all contaminated material before discarding.

11.     Periodically, clean out deep-freeze and dry-ice chests in which cultures are stored to remove broken ampules or tubes. Use rubber glovers and respiratory protection during the cleaning.

12.     Avoid smoking, eating and drinking in the laboratory.

13.     Do not reuse plasticware that has been used for PCR, recombinant DNA work and plant transformation work.

14.     Sterilise all the plasticware before discarding it.

15.     Burn all the transgenic material in an incinerator after observations have been taken.

 

APPENDIX II

 

MODEL PLAN FOR THE CONSTRUCTION OF
A GREEN HOUSE/NET HOUSE FOR EXPERIMENTS USING TRANSGENIC PLANTS

 

Frame Structure : The structure should be made from galvanised mild steel designed to with stand wind loading of not less than 100 km/hour. The method of affixing the polythene film cover to the frame should be strong enough to with stand similar wind velocities. The base may be constructed with bricks and cement or with any durable structure up to a height of 1.5 to 2 feet from the ground level so as to isolate the land inside the framed structure from the outside land.

 

Optimum size of unit : The recommended minimum size of the unit would be 1000 to 1500 cubic meters. In dimensions each such unit may be 30 meters long 13 meters wide and having and under the gutter height of about 3 to 4.5 meters from the base. The plan view as well as the side view of a multi span unit with double door entry recommended for an optimum size unit is enclosed along with this appendix-II. It is recommended that all the green house structures should have double door entry as indicated in the enclosed drawings, and the span of the area for the double door entry can be kept as 5 to 6 meters in length and about 3 meters in width along with height maintained commensurate with the main structure of the unit. The main entrance may be optionally be provided with an air curtain. The outer door shall be only one panel of flush door opening inside the buffer area and the inside doors may be more than one (two sliding doors have been shown in the drawing). In case sliding doors are not installed, the inside doors should be of one panel each, opening inside the buffer area only. The entry wall can be utilised for housing the suction fans as shown in the drawing while the opposite wall can be mounted with evaporation pads (shown in the drawing). The optimum sized unit recommended above would provide a growing area of about 350 sq. meters, allowing 10% for path ways. This unit have a Volume of about 1100 - 1200 cubic meters. Such a unit would be able to maintain a stable temperature, the desired humidity with adequate and ample air circulation.

 

Plastic film covering : It is recommended that the area covering the frame should be of 200 micron (800 gauge) thickness, UV stabilised polymer film. Such materials are expected to have a life span of 4 to years. All coverings should be double film covering on all surfaces to give double UV filtration and a more stable temperature control. The roof covers are likely to be inflated by the action of blower fans, thus maintaining a cavity throughout the unit. In addition to its suggested that an internal separation wall can be constructed to bifurcate the spans if there are more than one, which can be done by fixing the plastic films to the securing rails. With in the whole unit facilities can thus be provided for separate crop studies.

 

Fan, Pad system and Filter screens : An evaporative cooling system will be required to enable the maintenance of stable temperature gradient from the site of evaporating pad to the suction end. The surface are of the cooling unit will depend upon the overall all size of the structure. If the unit exceeds 30 meters in length then the temperature variation through out the length of unit may be such than an even temperature may not be maintainable even with the introduction of turbo circulation fans. The dimensions of the evaporation pad required to obtain a temperature 15 degree centigrade below ambient for a give volume of green house can be calculated from the following approximate equation.

 

Pad area (P) = Length X Width X Height, the whole divided by 94.85

Where P is in sq. Meter area.

 

As an example it is stated that a unit having the dimensions of 30 meters X 13 meters X 3 meters requires a pad area of not less than 12.35 Sq. meters. As most pad units are constructed to order, it is expected that it would not be difficult to have the pad areas of correct size.

All external surfaces of the pad should have filter screens of at least a 40 X 30 mesh net covering made from durable plastic material.

 

The fans required for a unit of above dimensions, to be housed at the other end of the unit should be about 61 centimeters (24 inch) in diameter with low noise and high C.u. ft./min (CFM) air circulation capacity, with four numbers to be installed per unit. It is recommended that motors with 1.5 H.P. with three phase may be installed which is slightly over designed but which is expected to have more life span and there fore substantial saving on replacements. Compromises can be made by installing 1 H.P. three phase motors, but this may need more maintenance. The fan units should have 40 X 40 mesh durable plastic screen fitted to the out side of the external surface. Each motor unit can be connected to one semi automatic temperature controlled which should shut down the fan as and when the temperature drops below the required levels. Such designs are available in the market.

 

Blower fans are required to be fitted on the each roof section which will inflate the top roof sheet. These fans must also to be fitted with 40 X 40 mesh durable plastic screen on the induction side to prevent any pollen evacuation. As these fans are expected to be constantly in operation it is recommended that these should be fitted with bearings and not with bust type.

 

It is essential to have circulation fans within the green house to ensure that a uniform temperature is maintained though out the growing area. The number and the positioning will however depend upon the external conditions and therefore may vary from place to place. The manufacturer may be consulted for selecting the correct number.

 

Irrigation : Full over head irrigation systems are available and can be installed. In smaller houses it would be advisable to carryout the watering manually as regulation of humidity is difficult to maintain through over head irrigation system because any extra watering will increase the humidity level. In line feeding units can be installed to take care of the nutrient requirements of the plants. A water tank needed to supply water to the pads and irrigation may be installed slightly below the ground level to avoid direct influence by sun or solar heat. The water will therefore remain cool.

 

Proposed positioning : The location and the orientation of the unit is of significant importance. The fans should not be positioned in a manner that they below directly to wards the plants. Electricity and water are continuously required. Therefore these must be positioned within a reasonable reach of the unit to keep costs down. The area selected for the unit must be flat, and as far as possible leveled to accommodate the unit plus approximately 2 metros off around the outside. It would be useful to provide a drainage system around the unit at suitable lower levels to enable the drainage of extra water. A suitable drain off area is also recommended to enable the extra water running off from the gutters; the drain off area may be more than 10 meters away from the unit.

 

Views showing the Different aspects of Playhouse/Greenhouse : Five diagrams showing schematically one recommended unit of the dimension 30 meters X 13 meters X 3 meters (Length X Breadth X Gutter height, excluding the dome height) are appended at enclosures I to V. The installers can install units bigger than the one suggested above. However, they have to ensure that all the safety precautions namely, installation of double doors, use of durable structures for the framework, use of at least 200 micron (800 gauge) plastic films in double coverings are used in the construction. Further, all the outlets would have to be secured by applying 40X40 mesh durable plastic coverings as indicated above.

 

 

 

 

 

 

 

 

 

 

 

 


ACUTE ORAL TOXICITY TESTS OF TRANSGENIC SEED IN RAT

 

Adoption: OECD 401

 

Application and limitation of tests: Acute oral toxicity is the adverse effects occurring within a short time of oral administration of a single dose of a test chemical or multiple doses given within 24 hrs. It is the initial step to find out median lethal dose (LD50) value which serve as basis for classification and labelling of the compound. It also forms a basis for selection of dose for subchronic studies. It will provide information on target organs toxicity after single exposure.

 

Principle: The test compound is administered orally by gavage in numerical doses to groups of animals, one dose per group. Signs of toxicity and death of animals are observed during 14 days observation period. The dead animals are necropsied during and the surviving animals are sacrificed and necropsied after the 14 days observation period for gross pathology. Vital tissues of moribund and sacrificed animals are put for histopathological studies, clinical biochemistry and haematological examination.

 

Description of the test Procedure

 

Animals: Healthy animals kept under standard animal husbandry conditions are used. At least 10 animals (male/female) are dosed. The weight variation of animals does not exceed 5-10g.

 

Animals maintenance: Animals are acclimatized to the experimental animal room having temperature 75 2 F, humidity 30-70% and 12 : 12 hrs light dark condition. Animals are caged with maximum of 2 animals in each polypropylene cages. Standard animal diet and water and libitum is given to animals.

 

Preparation of dose: Test sample i.e. fine powder of transgenic seed dissolved/suspended in groundnut oil is administered to rats fasted overnight. The volume does not exceed 1 ml/100 g body weight. At least four doses of the test sample spaced in geometrical factor are selected. The treatment schedule is as given below.

 

Group 1 - Control (normal diet)

Group 2 - Non transgenic seed

Group 3 - Transgenic seed

 

Limit test dose: If a test sample at 5000 mg/kg weight produces no mortality, then other doses are not essential.

 

Observations: The dosed animals are observed twice daily for 14 days to record the signs of poisoning and death of animals. The signs of poisoning include tremor, convulsion, salivation, diarrhoea, lethargy sleep, coma, dyspnea, nasal bleeding etc. the time of death of animals is recorded. the body weight, food and water intake is recorded daily and monitored weekly. All the animals (moribund/live) are sacrificed after 14 days and examinated for gross and histopathological changes, clinical biochemistry and haemotological examinations.

 

Haematology: Haemtology is carried out in oxalated blood using standard methods of Wintrole and Landsberg 1935 and Kolmer et. al. 1951. Blood is analysed for WBC, RBC, Hb differential leucocytes, clotting and prothrombin time and ESR.

 

Clinical Enzymes: Serum and blood are analysed for :

 

1.        Glutamic oxaloacetic transaminase (GOT),

2.        Glumatic pyruvic transaminase (GPT),

3.        Alakline phosphatase (Orthophosphoric monoester hydroxylase ALP),

4.        Bilirubin

5.        Blood glucose

6.        Blood urea nitrogen, (BUN),

7.        Non protein nitrogen, (NPN) by the method of Wootton (1982),

8.        Acetylcholinesterase (AchE) by the method of Hesterin 1949 and

9.        Protein by the method of Lowry et. al. 1951;

10.     Serum histamine level.

 


Calculations: LD50 values and its range are calculated by the procedure of Weil 1952 and toxicity rating is done by Gleasons et. al. 1969. All observed are recorded and calculated by appropriate methods. the statistical evaluation is done by Fishers student t test. The results are summerised in tabular form.

 

References

1.     Weil, C.S., tables for convenient calculation of median effective dose (LD or ED) and instruction in their use. Biometrics, 8, 249, 1952.

2.     Gleason, M.N. Gosseling, R.E., Hodge, H.C. and Smith, R.P. Clinical toxicology of commercial products. Acute poisoning 3rd ed. Williams and Williams, Baltimore, Maryland.

 

REPORT ON ACUTE ORAL TOXICITY

 

Test Animal : Rats

Sex : Male/Female

Test Sample : Solid, Liquid, any other

Nature of vehicle : Dist. water, peanut oil, corn oil, any other

Date of experiment started :

Date of experiment terminated :

 

LD50_____________mg/kg; Range______________to____________mg/kg

 

Dosage (mg/kg)

Animals
Died/Dosed

Death

Symptoms of toxicity

1. Control

 

 

 

2.

 

 

 

3.

 

 

 

 

Statistical Method

Gross Pathology

Observations

Conclusions

Toxcity Rating

 

REPORT ON ACUTE ORAL TOXICITY

 

Test animal : Rat

Test Chemical : Solid, Liquid, any other

Nature of vehicle : Solid, Liquid , any other

Nature of vehicle : Dist. water, peanut oil, corn oil, any other

Date of expt. Started :

Date of expt. terminated :

 

FOOD (G) WATER (ml) INTAKE OF MALE or FEMALE ANIMALS EXPOSED TO ...................... .............................. FOR 14 DAYS.

Dosage

Days

(mg/kg/day)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

MALE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1. Control

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FEMALE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

 

 

 

 

 

 


REPORT ON ACUTE ORAL TOXICITY

 

 

Test animal : Rat

Test Chemical : Solid, Liquid, any other

Nature of vehicle : Dist, water, peanut oil, corn oil, any other

Date of expt. started :

Date of expt. terminated :

 

RELATIVE ORGAN WEIGHT OF MALE or FEMALE ANIMALS EXPOSED TO ........... FOR 14 DAYS

 

Dosage
(mg/kg/day)

Liver

Kidney

Adrenalm

Heart

Spleen

Brain

Pituitary

Testis

Epididymis

Cervix

Vagina

Ovary

Uterus

MALE

 

 

 

 

 

 

 

 

 

 

 

 

 

1. Control

 

 

 

 

 

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

 

 

 

 

 

FEMALE

 

 

 

 

 

 

 

 

 

 

 

 

 

1.

 

 

 

 

 

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

 

 

 

 

 

4.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

* (Organ weight)/Body weight) x 100


REPORT ON ACUTE ORAL TOXICITY

 

 

Test Animal : Rat

Test Chemical : Solid, Liquid, any other

Nature of Vehicle : Dist. water, Peanut oil, corn oil, any other

Date of expt. started :

Date of expt. terminated :

 

BLOOD PICTURE OF MALE or FEMALE ANIMALS EXPOSED TO ......... FOR 14 DAYS

 

Dosage

RBC

WBC

Hb

PVC platelet

Differential Leucocyte Count (%)

(mg/kg/day)

(x10 mm)

(x10 mm)

 

 

Neutrophils

Lymphocytes

Monocytes

Eosinophils

MALE

 

 

 

 

 

 

 

 

1. Control

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

4.

 

 

 

 

 

 

 

 

FEMALE

 

 

 

 

 

 

 

 

1.

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

4.

 

 

 

 

 

 

 

 

 


REPORT ON ACUTE ORAL TOXICITY

 

 

Test Animal : Rat

Test Chemical : Solid, Liquid, any other

Nature of Vehicle : Dist. water, Peanut oil, corn oil, any other

Date of expt. started :

Date of expt. terminated :

 

BIOCHEMICAL CHANGES IN MALE or FEMALE ANIMALS EXPOSED TO ................. FOR 14 DAYS

 

Dosage (mg/kg/day)

Blood Sugar

Alk. Phos.

Protein

GOT

GPT

 

 

Liver

Serum

Liver

Serum

Liver

Serum

Liver

Serum

MALE

 

 

 

 

 

 

 

 

 

1. Control

 

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

 

4.

 

 

 

 

 

 

 

 

 

FEMALE

 

 

 

 

 

 

 

 

 

1. Control

 

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

 

4.

 

 

 

 

 

 

 

 

 

 

 


SUBCHRONIC (90 DAYS) ORAL TOXICITY TEST OF
TRANSGENIC SEED IN RAT

 

Adoption: OECD 408

 

Application and limitation of test: Subchronic oral toxicity is the adverse effect occuring as a result of repeated daily oral dosing of a chemical to the animals. In the evaluation of toxic characteristics of chemical the subchronic oral toxicity provides information on possible health hazards due to repeated exposure over a limited period ot time. It will provide the information on target organ and the possibility of cumulation and for the selection of dose level for chronic studies.

 

Principle: The test sample is orally administered in three doses to animals for a period of 90 days. The animals are observed for any signs of toxicity and death during the period of exposure. Vital tissues of moribund and sacrificed animals are put for histopathological studies. Clinical biochemistry and haematological examination are also made.

 

Description of the test Procedure: Rat is the preferred rodent model for subchronic oral toxicity studies. Health animals kept standard animal husbandry conditions are used. At least 20 animals of 6-8 weeks old are used per group for three dose levels. The weight of the animals does not vary + 20 g.

 

Animal maintenance: Animals are acclimatized to the experimental animal room having temperature (75+2 F), humidity (30-70%) and 12 : 12 hr light : dark conditions. Animals are given commercial feed and water ad libitum.

 

Preparation of dose: Test sample i.e. fine powder of transgenic seed dissolved/suspended in peanut oil is orally administered by gavage to animals consequently (5 days/week) for 90 days. The selection of the dose is made on the basis of acute toxicity studies of the test sample. At least three dose level, one maximum, one minimum and one intermediate are used. Consideration is given that the highest dose may result toxic effects without causing excessive lethality and lowest dose may not produce any toxic effects. A group of vehicle control is also used.

 

Limit test dose: If a test at one dose level of at least 1000 mg/kg body weight (but expected human exposure may indicate the need for a higher dose level), using the procedures described for this study, produces no observable toxic effects, then a full study using three dose levels may not be considered necessary. The treatment schedule is given below :

 

Group 1 - Control

Group 2 - Non transgenic seed

Group 3 - Transgenic seed

 

Observations: Animals are observed once daily to record the signs of poisoning, like tremor, convulsion, diarrhoea, lethargy, dyspnea and nasal bleeding etc. The time of death is also recorded. The body weight, food, and water intake is recorded daily and monitored weekly. At the end of 90 days animals are weighed sacrificed.

 

Pathology: The liver, kidney, gonads, adrenals, spleen and brain are weighed immediately after autopsy. All animals are subjected for gross pathological changes. The vital organs like liver, kidney, brain, gonads, spleen, adrenal, thyroid, lungs, heart, stomach, duodenum, jejunum, colon, uterus, prostate are fixed in formal saline solution and tissues embeded in parafin wax and section cut at 6 um on rotary microtome. The prepared slides are then stained in haematoxylin eosin for microscopic examinations.

 

Haematology: Haematology is carried out in oxalated blood using standard methods of Wintrobe and Landsberg 1935 and Kolmer et. al. 1951. Blood is analysed for WBC, RBC, Hb differential leucocytes, clotting and prothrombin time and ESR. Immunoglobulin profile (IGM, IGA, IGE).

 


Clinical Enzymes: Serum and blood are analysed for

 

1.        Glumatic oxaloacetic transminase (GOT),

2.        Glumatic pyruvic transaminase (GPT),

3.        Alakline phosphatase (Orthophosphoric monoester hydroxylase ALP),

4.        Billirubin

5.        Blood glucose

6.        Blood urea nitrogen, (BUN)

7.        Non protein nitrogen, (NPN) by the method of Wooton (1982),

8.        Acetylcholinesterase (AchE) by the method of Hestrin 1949 and

9.        Protein by the method of Lowry et. al. 1951.

10.     Serum histamine level.

 

Calculation and evaluation of data: All observed data are recorded and calculated by appropriate methods. The statistical evaluation is done by Fishers student t test 1950. The results are summerised in tabular form.

 

References

1.        Wintrobe, M. and Landsberg, J.W. A standard technique for blood sedimentation test. American J. Med. Sci. 189, 102, 1935.

2.        Kolmer, K.A. Spaulding, E.H. and Robinson, H.W. Approved laboratory techniques Ved Scientific Book Agency Calcutta, India, 1951.

3.        Wootton, I.D.P. Microanalysis in Medical Biochemistry Sixty Edition, Churchill Ltd., London, 1982.

4.        Hestrin, S.H. The reaction of Acetylcholine and other carboxylic acid derivatives with hydroxyl amine and its analytical applications J. Biol. Chem. 180, 249, 1949.

5.        Lowry, O.H. Rosenburgh, N.J. Farr, A.L. and Randall, R.J. Protein measurement with the Folin Phenol reagent J. Biol. Chem. 193, 265, 1951.

6.        Fisher, R.A. Statistical methods for research workers 11th edition Edinburgh Oliver and Boyd, 1950.

 

REPORT ON SUBCHRONIC ORAL TOXICITY

 

Test animal : Rat

Test Chemical : Solid, Liquid, any other

Nature of vehicle : Solid, Liquid , any other

Nature of vehicle : Dist. water, peanut oil, corn oil, any other

Date of expt. started :

Date of expt. terminated :

 

FOOD (G) WATER (ml) INTAKE OF MALE or FEMALE ANIMALS EXPOSED TO ............FOR 14 DAYS.

Dosage

 

 

 

 

 

 

Days

 

 

 

 

 

 

 

(mg/kg/day)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

MALE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1. Control

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FEMALE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4.

 

 

 

 

 

 

 

 

 

 

 

 

 

 


 

REPORT ON SUBCHRONIC ORAL TOXICITY

 

 

Test animal : Rat

Test Chemical : Solid, Liquid, any other

Nature of Vehicle : Dist. water, peanut oil, corn, oil, any other

Date of expt. started :

Date of expt. terminated :

 

RELATIVE ORGAN WEIGHT OF MALE or FEMALE ANIMALS EXPOSED TO ........ FOR 13 WEEKS

 

Dosage
(mg/kg/day)

Liver

Kidney

Adrenalm

Heart

Spleen

Brain

Pituitary

Testis

Epididymis

Cervix

Vagina

Ovary

Uterus

MALE

 

 

 

 

 

 

 

 

 

 

 

 

 

1. Control

 

 

 

 

 

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

 

 

 

 

 

4.

 

 

 

 

 

 

 

 

 

 

 

 

 

FEMALE

 

 

 

 

 

 

 

 

 

 

 

 

 

1.

 

 

 

 

 

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

 

 

 

 

 

4.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

* (Organ weight / Body weight) x 100


REPORT ON SUBCHRONIC ORAL TOXICITY

 

 

Test Animal : Rat

Test Chemical : Solid, Liquid, any other

Nature of vehicle : Dist. water, peanut oil, corn oil, any other

Date of expt. started :

Date of expt. terminated

 

BLOOD PICTURE OF MALE or FEMALE ANIMALS EXPOSED TO ....................... FOR 13 WEEKS

 

Dosage

RBC

WBC

Hb

PVC platelet

Differential Leucocyte Count (%)

(mg/kg/day)

(x10 mm)

(x10 mm)

 

 

Neutrophils

Lymphocytes

Monocytes

Eosinophils

MALE

 

 

 

 

 

 

 

 

1. Control

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

4.

 

 

 

 

 

 

 

 

FEMALE

 

 

 

 

 

 

 

 

1.

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

4.

 

 

 

 

 

 

 

 

 

 


REPORT ON SUBCHRONIC ORAL TOXICITY

 

 

Test animal : Rat

Test Chemical : Solid, Liquid, any other

Nature of vehicle : Dist. Water, Peanut oil, corn oil, any other

Date of expt. started :

Date of expt. terminated :

 

BIOCHEMICAL CHANGES IN MALE or FEMALE ANIMALS EXPOSED TO .......................... FOR 13 WEEKS

 

Dosage (mg/kg/day)

Blood Sugar

Alk. Phos.

Protein

GOT

GPT

 

 

Liver

Serum

Liver

Serum

Liver

Serum

Liver

Serum

MALE

 

 

 

 

 

 

 

 

 

1. Control

 

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

 

4.

 

 

 

 

 

 

 

 

 

FEMALE

 

 

 

 

 

 

 

 

 

1. Control

 

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

 

4.

 

 

 

 

 

 

 

 

 

 

 


PRIMARY SKIN IRRITATION TEST OF TRANSGENIC SEED IN RABBIT

 

Application: OECD 404

 

Application and limitation of test: The assessment and evaluation of the toxic characterestics of a substances, determination of the irritant effects on the skin of mammals is an important initial step. Information derived from the test serves to indicate the existance of possible hazard likely to arise from exposure of the skin to the test substance.

 

Principle: The substances to be tested is applied in a single dose to the skin of several experimental animals, each animal serving as its own control. The degree of irritation is read and scored at specified itervals andis further described to provide a complete evaluation of the effects. The duration of the study should be sufficient to evaluate fully the reversibility or irreversibility of the effects observed.

 

Description of the test procedure

 

Animals: Animals are acclimatized to the experimental animal room having temperature 75+2F, humidity 30-70% and 12 : 12 hrs. light dark cycle. Animals are caged with maximum of two animals in each cage. Standard animal diet and water at libitum.

 

Preparation of dose and limit test dose: Test sample i.e. transgenic seed at a dose of 0.5 ml of liquid or 0.5g of solid is applied to the test side. The treatment schedule is given below :

 

Group 1 - Control

Group 2 - Non transgenic seed

Group 3 - Transgenic seed

 

Observations: Animals are observed for signs of erythema and oedema and the responses scored at 30-60 minutes, and then at 24, 48, 72 hours and 7 and 14 days after patch removal. Dermal irritation is scored and recorded as per the grades given in the table below.

 

References

1.        Draise, J.H. The Appraisal of Chemicals in Foods, Drugs, and cosmetics pp. 46-48. Association of Food and Drug Officials of United Statesm, Austin, Texax 1959.

2.        Draise, J.H. Appraisal of the Safety of chemicals in Foods, Drugs and Cosmetics; pp 46-59. Association of Food and Drugs official of the United States, Topeka, kanasas 1965.

 

EVALUATION OF SKIN REACTION

 

Erythema and Eschar Formation Value

 

No erythema 0

Very slight erythema (barely perceptible)y 1

Well-defined erythema 2

Moderate severe erythema 3

Severe erythema (beet redness) to slight eschar formation

(injuries in depth) 4

Maximum possible - 4

Oedema Formation

No oedema 0

Very slight oedema (barely perceptible) 1

Slight oedema (edges of area well defined by definite raising) 2

Moderate oedema (raised approximately 1 millimetre) 3

Severe oedema (raised more than 1 millimetre) and extending

beyond area of exposure 4

 

Maximum possible - 4


IRRITATION TO MUCOUS MEMBRANE TEST OF
TRANSGENIC SEED IN FEMALE RABBIT

 

Adoption : OECD 405

 

Application and Limitation of test: In the assessment and evaluation of the toxic characteristics of a substance, determination of the irritant effect on the mucous membrane of the rabbit is an important step. Information derived from this study serves to indicate the existence of possible hazards likely to arise from exposure on the mucous membrane to the test substance.

 

Principle: The substance is tested is applied in a single dose to the mucous membrane of the experimental animals. Simultaneous animals in the control group are also tkaen. The degree of irritation is read and scored at specific itervals. The complete evaluation is then described. The duration of the study is sufficient to evaluate fully the dermal irritation.

 

Description of the test procedure: Healthy adult animals at least 3 in number are used in both experimental and control groups. Animals are kept in the experiment in the experimental animal room having temperature (752F), humidity (30-70%), and 12 : 12 light : dark conditions. Animals are fed conventional laboratory diet and water ad libitum.

 

A dose of 0.1 ml. of liquid or 0.1 gm of solid or semisolid is applied to the upper vault of the vagina. Exposure duration is 4 hrs. Longer exposure may be indicated under certain conditions. At the end of the exposure period residual subtance is removed where practicable using water or appropriate solvent without disturbing the epidermis. The treatment schedule is given below :

 

Group 1 - Control

Group 2 - Non transgenic seed

Group 3 - Transgenic seed

 

Observation: Observation period is not fixed but is sufficient to evaluate fully the effects of the test substance. Normally it need exceed 14 days after application. Animals are examined for signs of erythema and oedema and the responses scored at 30-60 minutes, 24, 48, 72 hrs and then at 7 and 14 days. Mucous membrane irritation is scored and recorded as per the grades given in the table below:

 

References:

1.        Draise, J.H. The approval of chemical in Food, Drug and cosmetics pp, 46-48. Association of Food Officials of United States, Austin Texas 1959.

2.      Draise, J.H. Appraisal of the Safety of chemicals in Foods, Drugs and Cosmetics ; pp, 46-59. Association of Food and Drugs officials of the United States, Topeka, kanasas 1965.

 

EVALUATION OF SKIN REACTION

 

Erythema and Eschar Formation Value

 

No erythema 0

Very slight erythema (barely perceptible) 1

Well-defined erythema 2

Moderate severe erythema 3

Severe erythema (beet redness)y to slight eschar formation

(injuries in depth) 4

Maximum possible - 4

Oedema Formation

 

No oedema 0

Very slight oedema (barely perceptible) 1

Slight oedema (edges of area well defined by definite raising) 2

Moderate oedema (raised approximately 1 millimetre) 3

Severe oedema (raised more than 1 millimetre) and extending

beyond area of exposure 4

Maximum possible - 4


SKIN SENSITIZATION TEST OF TRANSGENIC SEED IN GUINEA PIGS

 

Adoption : OECD 406

 

Application and Limitation of Test: In the assessment and evaluation of the toxic characeristics of a substance, determination of its potential to provoke skin sensitization reaction (allergic dermatitis) is important. Information derived from skin sensitization serves to identify the possible hazards to a population exposed to the substance.

 

Principle: After initial exposure to a test substance the animals are subsequently subjected for 9 injections, then a challange exposure to establish a hypersensitive state. Sensitization is determined by examining the reaction to the challenge exposure.

 

Description of test procedure: The guinea pigs are the generally recommended species. A sufficient number of animals are used. Animals are keipt in experimental animal room having temperature (752F), humidity 30-70% and 12 : 12 light : dark condition. Animals are fed on conventional laboratory diet and water ad libitum. It is essential that guinea pigs receive an adequate amount of ascorbic acid. A treatment and a control groups are simultaneously taken. Animals are clipped off at a dorsal side for the area of 6 x 6 cm. The test substance 0.05ml is adminstered intradermally as a initial dose. There after nine subsequent injections are given intradermally on every alternate days. After giving a rest period of 15 days a booster dose of 0.5 ml is injected. The treatment schedule is given below :

 

Group 1 - Control

Group 2 - Non transgenic seed

Group 3 - Transgenic seed

 

Observation: Scoring of skin reaction was performed on day 2 and then 24 hours after each injection. On day 36 and 37 animals are shaved again to check the intensity of erythema or edaema. With administration of booster dose, skin sensitization reaction was observed. The subsequently spreaded to longer area of the skin and resulted in necrosis at site of injection. Scored reaction are recorded in form of table.

 

References

Draize, J.H., Food Drug Cosmets. Law J. 10, 722, 1955.

 

EVALUATION OF SKIN REACTION

 

 

Erythema and Eschar Formation Value

 

No erythema 0

Very slight erythema (barely perceptible) 1

Well-defined erythema 2

Moderate severe erythema 3

Severe erythema (beet redness) to slight eschar formation

(injuries in depth) 4

 

Maximum possible - 4

 

Oedema Formation

 

No oedema 0

Very slight oedema (barely perceptible) 1

Slight oedema (edges of area well defined by definite raising) 2

Moderate oedema (raised approximately 1 millimetre) 3

Severe oedema (raised more than 1 millimetre) and extending

beyond area of exposure 4

 

Maximum possible - 4


SUBCHRONIC ORAL TOXICITY-GOATS-90 DAYS STUDY FOR

GENETICALLY ENGINEERED SEEDS

 

OBJECTIVE: The objective of this study is to compare the whole/someness of engineered seeds with control seeds and control seeds lines will be administered to the goats through the diet for 90 days.

 

MATERIAL AND METHODS: The methods, species of animals and the route of administration described in this protocol are based up on standard OECD guidelines No. 408 (1993). This procedure deals with handling, maintaining and other procedures to be followed while dealing with feeding studies with goats. In order to maintain even distribution, the goats will be provided a number, based on random selection.

 

The test material will be administered in the diet. This route of administration was selected because it represents the most likely route of exposure of goat species in their natural habitat.

 

The test substance will be property identified as per the detailed specification provided by the sponsor.

 

Treatment Groups: A group of 12 goats (6 males and 6 females) will be assigned to each group by the indiscriminate draw to each of the treatment and control group. All goats will be uniquely identifiable with an identification mark on the body and/or with a number plate around their neck.

 

The test will complete feeding of the goats for 90 days regularly with concentrate of which 12.5% will be test seed and the concentrate itself will be 10% of the total feed i.e. concentrate and green grass. The consumption range of the feed will be pre-determined.

 

Each group is fed for 90 days and observed. An additional control group will be fed normal diet which will not contain cotton seeds throughout the test period.

 

Duration of the study: All animals in the treatment groups will get Indian hybrid control cotton seeds in diet during acclimation.

 

Analysis will be initiated during this period itself viz., feed consumption, weight gain etc. This will facilitate statistical analysis.

 

Pilot study will be done before acclimation to assess the consumption of cotton seeds. Parameters like feed consumption, weigh gain etc. will also be assessed for this group.

 

The study will be divided as under.

 

1.        Acclimation : From receipt of the animals till the initiation of the study (a minimum duration of 15 days)

2.        Exposure : 90 day

 

Test animals: Goat husbandry is generally associated with agriculture in Indian rural set ups. The availability of standard genetically defined goats and dietary and husbandry conditions, also make goats ideal in the Indian context and safety data on this ruminant model will be appropriate.

 

All goats will be 12 months old and healthy at the initiation of the study. The body weight will range between 15 and 18 kg. Each treatment and control group will have 12 animals. The Barbari goats will be obtained from the State Animal Husbandary Departments. All the animals will be acclimated to their pens and facilities from the time of receipt until the initiation of the study.

 

ANIMAL CARE AND FACILITY

 

Animal Species: Goat - The Indian Barberi breed

Source: State Animal Husbandry Departments

Number of animals: Twelve animals (6 males and 3 females) per group

Age and weight: Age of the animals will be 12 months and the weight between 15 and kg.

 

Acclimation: The animals will undergo an acclimation for a period of not less than 15 days prior to the actual studies. The goats will be given anti-helminth drugs and also drugs for treatments for ectoparasites before the initiation of the study. All animals in the treatment groups will get Indian hybrid control cotton in diet during acclimation and group will not be given any cotton but will have groundnut cake instead in its diet.

 

Animals Identification: Each animal will be numbered accordingly with the help of a tag around the neck.

 

Housing and animal care: Goats will be housed individually in a well constructed, cemented pens and maintained under strict hygienic conditions of veterinary care.

 

Housing and animals care: Goats will be housed individually in a well constructed, cemented pens and maintained under strict hygienic conditions of veterinary care.

 

Food and Water: Each animal will be allowed access to food for the whole day. Clean drinking water will be provided ad libitum. Feed consisting of wheat bran, gram, salt, minerals, salt, minerals, cotton seeds and grass will form the daily diet of the goats.

 

The test will comprise feeding of the goats for 90 days regularly with concentrate of which 12.5% will be cotton seed and the concentrate itself will be 10% of the total feed i.e. concentrate and green grass. The consumption range of the feed will be predetermined.

 

Bedding: No bedding will be used; instead the floor will be made of rough cement/concrete to avoid slipping of goats while walking or standing.

 

Exercise: Though the goats do not need any strenuous exercise, they will however, be allowed to go out of their pens in an open field for about 2-3 hours each day but ensuring that they do not eat any other foliage. The area of their movement would be devoid of any vegetarian but water will be provided during this period of their routine.

 

Animal diet: The test diet will be prepared by blending the test substance directly with the ration. Blending is normally done with a blender. Unless otherwise specified, the diets will be prepared every day. The diets will be provided to the goats from day 0 of the 90 day exposure period. Every batch of concentrate will be analysed and relevant record will be maintained. Cotton seed will be added to the concentrate everyday to avoid the concentrate going rancid because of the presence of cotton oil, if the concentrate is blended with cotton seed and stored. the ingredients will be purchased in bulk and made available for mixing; but the mixing and blending of the constituents will be done daily. The feed ingredients will be maintained in a dry and clean room to avoid attack by fungus. The test material will be crushed and mixed with the feed. The analysis of the feed will be for the followng parameters : Crude protein, fat, acid detergent fiber, neutral detergent fiber, Calcium, phosphorus, Magnesiium, Sodium, Potassium, Copper; Zinc, Manganese, Iron, Vitamin A, Vitamin D, Vitamin E. The analysis will be done on the mix and the raw ingredients. Also the mix will be randomly analysed once a week.

 

Housing and environmental conditions: Goats will be housed in properly constructed pens. Each pen measuring 1.5 sq. mt. per goat, allowing proper movement to the animals. The floor of the pen would be constructed of concrete and the wall of bricks. The roof will be made of corrugated sheet. Al initiation of the study, each pen will hold a single goat and goat will be identifiable by a number. During the test, the temperature in the housing will be 25-300C approximatel. If necessary, air cooler will be provided to maintain the specified temperature. Relative humidity will be recorded at 24 hour interval. The goats will be provided a 16 hour light and 8 hour dark photoperiods during the test. Housing and animal husbandry practices will be followed as mentioned by Devendra and McLeroy 1982.

 

EXPERIMENTAL DESIGNS:

 

Design: The study will be conducted as a randomised block design in which goats will be distributed randomly in different treatment groups evenly consisting of a single goat as a replicate.

 

The study would have atleast three following groups

1.        Genetically engineered cotton line

2.        Indian hybrid cotton line

3.        Control group - Normal diet without cotton seeds but ground nut, instead.

 

OBSERVATIONS: All the animals will be observed daily for morbidity, mortality and clinical signs.

 

Daily observations: The general health of all the animals will be monitored daily and relevant records will be maintained. Any adverse observation will be documented. Animals found moribund or dead during the study period will be necropsied to the extent necessary to determine the probable cause.

 

Body weight and temperature: Body weights will be measured weekly at a predetermined time along with their health status. A chart of weekly temperature will also be maintained.

 

Body weight/feed consumption: Individual body weights will be taken at the initiation of the experiment, during the exposure period and at the end of the exposure period. Average feed consumption for individual animal will be maintained for the entire period. Determination of feed consumption and body weight will continue, if the study period is extended. Daily feed offered and refused will be measure for the concentrate and grass.

 

Feed intake: Goats will have access to the experimental feed (concentrate) from 9 a.m. to 12 p.m. each day.

 

Necropsy and Pathological examinations: Goats found moribund or dead during the study period will be necropsied to the extent necessary to determine the probable reason. Any gross lesions observed at necropsy will be processed for histopathological examinations.

 

Hematological observations: Following parameters would be assessed:

         Total RBC count

         Total WBC count

         Differential leucotyic count.

         Haemoglobin concentration

         Clotting time ESR immunoglobulin profile

 

Clinical biochemistry: The following parameters will be analysed :

         Total Serum protein

         Glucose

         Blood urea

         Nitrogen

         Non-protein

          Nitrogen

         Bilrubin

         Histamine

         GOT

         GPT

          Alkaline phosphatase

          LDh

NECROPSY: All the animals are sacrificed on day 91. Goats are sacrificed by administration of a saturated solution of magnesium sulphate intravenously and the autopsy is carried out as the standard procedure by the veterinary pathologist of the study.

 

Organ weights: The gross lesions in the organ are noted and weights of the following organs are recorded:

         Adrenals,

         Heart,

         Liver;

         Gonads (testes and ovaries),

         Brain,

         Kidneys,

         Spleen

 

Histopathological examinations: Following organs are preserved in 10% buffered formalin:

         Adrenals

         Kidneys

         Testes

         Liver

         Thymus

         Lungs

         Colon

         Spleen

         Spleen

         Ovaries

         Stomach (all 4 compartments)

         Heart

         Small instetine

 

 

Histopathological examinations of these organs will only be conduced if gross lesions are noted.

 

The tissues are subjected to dehydration procedure and processed in a tissue processor through different grades of alcohol and cleared and chloroform. They are embedded in paraffin wax, sectioned at 7 to 10 microns and stained with Haematoxylin-Eosin.

DISPOSAL: The carcas will be mutilated by using Calcium bydroxide and buried deep ensuring that these are not removed by men or other animals like dogs and jackals.

 

REFERENCES:

1.        OECD (1982). Guidelines for testing of chemicals Section 4, Health effects (No. 407-409) Organisation of European Cooperation and Development, Paris.

2.        Schalm, O.W. (1969). Veterinary Hematologoy, Lea and Febiger, Philadalphia.

3.        Devendra C. and McLeroy, G.B. (1982). Goat and Sheep Production in the tropics. Intermediate Tropical Agricultural Series, Longman, London.


ACUTE ORAL TOXICITY TEST OF TRANSGENIC VEGETABLES IN RAT

 

Adoption: OECD 401

 

Application and limitation of tests: Acute oral toxicity is the adverse effects occuring within a short time of oral administration of a single dose of a test chemical or multiple doses given within 24 hrs. It is the initial step to find out median lethal dose (LD50) value which serve as basis for selection of dose for subchronic studies. It also forms a basis for selection of dose for subchronic studies. It will provide information on target organ toxicity after single exposure.

 

Principle: The test compound is administered orally by gavage in numerical doses to groups of animals, one dose per group. Signs of toxicity and death of animals are observed during 14 days observation period. The dead animals are necropsied during and the surviving animals are sacrificed and necropsied after the 14 days observation period for gross and histopathological studies, clinical biochemistry and haemotological examinations.

 

Description of the test Procedure

 

Animals: Health rats kept under standard animal husbandry conditions are used. At least 10 animals (male/female) are dosed. The weight variation of animal does not exceed 5-10g.

 

Animals maintenance: Animals are acclimatized to the experimental animal room having temperature 75 2F, humidity 30-70% and 12 : 12 hrs. light dark condition. Animals are caged with maximum of 2 animals in each polypropylene cages. Standard animal diet and water ad libitum is given to animals.

 

Preparation of dose: Test sample i.e. concentrated paste or cryogenic dehydrated powder of transgenic vegetables dissolved/suspended in groundnut oild is administered to rat fasted overnight. The volume does not exceed 1 ml/100 g body weight. At least four doses of the test sample spaced in geometrical factor are selected. The treatment schedule of short term toxicity is given below:

 

Group 1 - Control (normal diet)

Group 2 - Non transgenic vegetables

Group 3 - Transgenic vegetables.

 

Limit test dose: If a test sample at 5000 mg/kg body weight produces no mortality, then other doses are not essential.

 

Observations: The dosed animals are observed twice daily for 14 days to record the signs of poisoning and death of animals. The signs of poisoning include tremor, convulsion, salivation, diarrhoea, lethargy, sleep, coma, dyspnea, nasal bleeding etc. The time of death of animals is recorded. The body weight, food and water intake is recorded daily and monitored weekly. All the animals (moribund/live) are sacrificed after 14 days and examined for gross and histopathological changes, clinical biochemistry and haematological examination.

 

Pathology: The liver, kidney, gonads, adrenals, spleen and brain are weighed immediately after autopsy. All animals are subjected for gross pathological changes. The vital organs like liver, kindey, brain, gonads, spleen, adrenal, thyroid, lungs, heart, stomach, duodenum, jejunum, colon, uterus, prostate are fixed in formal saline solution and tissues embeded in parafin wax and section cut at 6 um on rotary microtome. The prepared slides are then stained in haematoxylin eosin for microscopic examinations.

 

Haematology: Haematology is carried out in oxalated blood using standard methods of Wintrobe and Landsberg 1935 and Kolmer et. al. 1951. Blood is analysed for WBC, RBC, Hb differential leucocytes, clotting and prothrombin time and ESR.

 

Clinical Enzymes: Serum and blood are analysed for:

 

1.        Glumatic oxalocetic treatment (GOT)

2.        Glumatic pyruvic transminase (GPT)

3.        Alakline phosphatase (Orthophosphoric monoester hydroxylase ALP)

4.        Bilirubin

5.        Blood glucose

6.        Blood urea nitrogen, (BUN)

7.        Non protein nitrogen, (NPN) by the method of Wootton (1982)

8.        Acetylcholinesterase (AchE) by the method of Hestrin 1949 and

9.        Protein by the method of Lowry et. al. 1951.

Calculations: LD50 values and its range are calculated by the procedure of Weil 1952 and toxicity rating is done by Gleasons et. al. 1969. All observed data are recorded and calculated by appropriate methods. The statistical evaluation is done by Fishers student t test. The results are summerised in tabular form.

 

References

1.        Weil, C.S., tables for convenient calculation of median effective dose (LD or ED) and instruction in their use. Biometrics, 8, 249, 1952.

2.        Gleason, M.N., Gosselin, R.E., Hodge, H.C. and Smith, R.P. Clinical toxicology of commercial products. Acute poisoning 3rd ed. Willams and Williams, Baltimore, Maryland.

 

REPORT ON ACUTE ORAL TOXICITY

 

Test Animal

 

Rats Sex : Male/Female

 

Test Sample

 

Solid Liquid any other

 

Nature of vehicle : dist. Water, peanut oil, corn oil, any other

Date of experiment started :

Date of experiment terminated :

 

LD50 _____________ mg/kg; Range ___________ to ____________ mg/kg

 

Dosage
(mg/kg)

Animals
Died/Dosed

Death

Symptoms of toxicity

1. Control

 

 

 

2.

 

 

 

3.

 

 

 

4.

 

 

 

 

Statistical Method

Gross Pathology

Observations

Conclusions

Toxicity Rating


REPORT ON ACUTE ORAL TOXICITY

 

Test animal : Rat

Test chemical : Solid, Liquid, any other

Nature of vehicle : Dist. Water, peanut oil, corn oil, any other

Date of expt. Started :

Date of expt. terminated :

 

FOOD (G) WATER (ml) INTAKE OF MALE or FEMALE ANIMALS EXPOSED TO .................... ........................ FOR 14 DAYS

 

Dosage

Days

(mg/kg/day)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

 

MALE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1. Control

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FEMALE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


REPORT ON ACUTE ORAL TOXITY

 

 

Test animal : Rat

Test Chemical : Solid, Liquid, any other

Nature of vehicle : Dist. Water, Peanut oil, corn oil, any other

Date of expt. started :

Date of expt. terminated :

 

RELATIVE ORGAN WEIGHT OF MALE or FEMALE ANIMALS EXPOSED TO ........................... FOR 14 DAYS

 

Dosage
(mg/kg/day)

Liver

Kidney

Adrenalm

Heart

Spleen

Brain

Pituitary

Testis

Epididymis

Cervix

Vagina

Ovary

Uterus

MALE

 

 

 

 

 

 

 

 

 

 

 

 

 

1. Control

 

 

 

 

 

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

 

 

 

 

 

4.

 

 

 

 

 

 

 

 

 

 

 

 

 

FEMALE

 

 

 

 

 

 

 

 

 

 

 

 

 

1.

 

 

 

 

 

 

 

 

 

 

 

 

 

2.

 

 

 

 

 

 

 

 

 

 

 

 

 

3.

 

 

 

 

 

 

 

 

 

 

 

 

 

4.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

* (Organ weight / Body weight) x 100


REPORT ON ACUTE ORAL TOXICITY

 

 

Test Animal : Rat

Test Chemical : Solid, Liquid, any other

Nature of vehicle : Dist. water, peanut oil, corn oil, any other

Date of expt. started :

Date of expt. terminated :

 

BLOOD PICTURE OF MALE or FEMALE ANIMALS EXPOSED TO .................. FOR 14 DAYS

 

 

Dosage

RBC

WBC

Hb