Form B (per rule 8(a)* for Submission of Research Protocol (s)

Application for Permission for Animal Experiments

Application to be submitted to the CPCSEA, New Delhi after approval of Institutional

Animal Ethics Committee (IAEC)

Section -I

Name and address of the establishment Department of Pharmaceutical Engineering and

1. Technology, Banaras Hindu University, Varanasi-
221 005
Registration number and date of Regd. No. 2123/GO/Re/S/21/CPCSEA
2. registration.

Name, address, and registration Central Animal House, Registration Number:

number of breeder from which animals
3. acquired (or to be acquired) for
experiments mentioned in parts B &C
Place where the animals are presently
4. kept (or proposed to be kept).
Place where the experiment is to be
performed (Please provide CPCSEA
5. Reg. Number)
Date and Duration of the experiment.
6.
2123/GO/Re/S/21/CPCSEA
18/03/2021
Animal House, Department of Pharmaceutical
Engineering and Technology, Indian
Institute of Technology (Banaras Hindu
University).
Department of Pharmaceutical Engineering and
Technology, Indian Institute of
Technology (Banaras Hindu University)
2123/GO/Re/S/21/CPCSEA.
6 months

Type of research involved (Basic

Research / Educational/ Regulatory/ Basic Research
7. Contract Research)

Name & Signature:

Dr. Jairam Meena

Designation of Investigator: Assistant professor
Date:
Place: IIT (BHU), Varanasi, India
Section-II

Protocol form for research proposals to be submitted to the Institutional Animal

Ethics Committee/CPCSEA, for new experiments or extensions of ongoing
experiments using animals.

1. Project/Dissertation/Thesis Title: Assessment of formulated PLG-based iron-oxide

nanoparticles decorated with CCP for the selective delivery of paclitaxel in
melanoma mouse model.

2. Principal Investigator/Research Guide/Advisor:

a. Name: - Dr. Jairam Meena

b. Designation: - Assistant professor
c. Dept/Div/Lab: - Pharmaceutical Engineering and Technology
d. Telephone No.: - 8826415221
e. E-mail Id: - jairam.phe@itbhu.ac.in
f. Experience in Lab animal experimentation: - more than 10 years

3. List all individuals authorized to conduct procedures under this proposal.

a. Name: - Mr. Hitesh Chandpa

b. Designation: - Ph.D. Student
c. Department: - Pharmaceutical Engineering and Technology
d. Telephone No.: - 7984833905
e. E-mail Id: - hiteshhchandpa.rs.phe22@itbhu.ac.in
f. Experience in Lab animal experimentation: - 4 years

g. Name: - Mr. Mayur Aher

h. Designation: - M.Pharm Student
i. Department: - Pharmaceutical Engineering and Technology
j. Telephone No.: - 8767237183
k. E-mail Id: - ahermayursomnath.phe22@itbhu.ac.in
l. Experience in Lab animal experimentation: - 1 year

m. Name: - Mr. Shovan Naskar

n. Designation: - M.Pharm Student
o. Department: - Pharmaceutical Engineering and Technology
p. Telephone No.: - 8420380660
q. E-mail Id:- shovannaskar.phe22@itbhu.ac.in
r. Experience in Lab animal experimentation: - 1 year
 s. Name: - Mr. Madhav Gopulwad
t. Designation: - M.Pharm Student
u. Department: - Pharmaceutical Engineering and Technology
v. Telephone No.: - 9923906636
w.E-mail Id: - gopulwadmadhavhanmant.phe22@itbhu.ac.in
x. Experience in Lab animal experimentation: - 1 year

y. Name: - Ms. Shalini Agrawal

z. Designation: - M.Pharm Student
aa. Department: - Pharmaceutical Engineering and Technology
bb. Telephone No.: - 8529419164
cc. E-mail Id: - shaliniagarwal.phe22@itbhu.ac.in
dd. Experience in Lab animal experimentation: - 1 year

4. Funding Source / Proposed Funding Source with complete address (Please attach the
proof)
NA

5. Duration of the animal experiment:- 6 months

a. Date of initiation (Proposed): -

b. Date of completion (Proposed): -

6. Describe details of study plan to justify the use of animals (Enclose Annexure)
7. Animals required
a. Species and Strain: - BALB/C mice
b. Age and Weight: - 6-8 weeks
c. Gender: - Either sex
d. Number to be used (Year-wise break ups and total figures needed to be
given in tabular form)
Total number of animals required: - 32

Group No. of animals

Disease control 8
PLG-IONF-CPP 8
Paclitaxel 8
(Positive control)
Normal control (NC) 8
 e. Number of days each animal will be housed.
3 months
8. Rationale for animal usage
a. Why is animal usage necessary for these studies?
This study requires estimation and validation of efficacy of Paclitaxel
nanoformulation, which can only be done in animals.
Also, the studies in animals are more reliable for the correlation of data to
humans.
b. Whether a similar study has been conducted on in vitro models? If yes,
describe the leading points to justify the requirement of animal experiment.
NO
c. Why are the particular species selected?
Mice are sensitive to immune response and for cancer studies mice are
preferred animals.
d. Why is the estimated number of animals essential?
To overcome bias due to any error, the number of animals used is considered
the minimum required to allow meaningful biological and statistical
evaluation of the data.
e. Are similar experiments conducted in the past in your establishment?
NO
f. If yes, justify why new experiment is required?
NA
g. Have similar experiments been conducted by any other organization in
same or other in-vivo models? If yes, enclose the reference.
NA
9. Describe the procedures in detail:

a. Describe all invasive and potentially stressful non-invasive procedures that

animals will be subjected to in the course of the experiments:
b. Furnish details of injection schedule Substances: Paclitaxel, Paclitaxel based
iron oxide nanoformulation, Paclitaxel PLG-iron oxide-CPP
nanoformulation

c. Doses: 10 mg/kg
Sites: Subcutaneos, I.V
Volumes: 100 µl
Blood withdrawal Details:
Volume: 0.1ml
Sites: Retro-orbital sinus
d. Radiation (dosage and schedules): NA
10. Does the protocol prohibit use of anesthetic or analgesic for the conduct of
painful procedures? If yes, justify.
NO
11. Will survival surgery be done? NO

If yes, the following to be described.

a. List and describe all surgical procedures (including methods of asepsis)

b. Names, qualifications and experience levels of personals involved.
c. Describe post-operative care
d. Justify if major survival surgery is to be performed more than once on a
single animal.

12. Describe post-experimentation procedures.

a. Scope for Reuse: NO

b. Rehabilitation (Name and Address, where the animals are proposed to be
rehabilitated): NA

c. Describe method of Euthanasia. : CO2 Chamber

d. Method of carcass disposal after euthanasia. : All the biohazard will be
taken away by Biohazard Company, from department of Pharmaceutical
Engineering and Technology, IIT (BHU), Varanasi.

13. Describe animal transportation methods if extra-institutional transport is

envisaged.
As per CCSEA guideline in air conditioned vehicle.

14. Use of hazardous agents (use of recombinant DNA-based agents or potential

human pathogens requires documented approval of the Institutional Biosafety
Committee (IBC). For each category, the agents and the biosafety level required,
appropriate therapeutic measures and the mode of disposal of contaminated food,
animal wastes and carcasses must be identified).
If, your project involved use of any of the below mentioned agents, attach copy of
the approval certificates of the respective agencies:

(a) Radio nucleotides (AERB)

(b) Microorganisms/Biological infectious Agents (IBSC)
(c) Recombinant DNA(RCGM)
(d) Any other Hazardous Chemical/Drugs
 Investigator’s declaration.

1. I certify that the research proposal submitted is not unnecessarily

duplicative of previously reported research.

2. I certify that I am qualified and have experience in the

experimentation on animals.

3. For procedures listed under item 10, I certify that I have reviewed
the pertinent scientific literature and have found no valid alternative
to any procedure described herein which may cause less pain or
distress.

4. I will obtain approval from the IAEC/CCSEA before initiating any

changes in this study.

5. I certify that performance of experiment will be initiated only upon

review and approval of scientific intent by appropriate expert body
(Institutional Scientific Advisory Committee/funding agency/other
body).

6. I certify that I will submit appropriate certification of review and

concurrence for studies mentioned in point14.

7. I shall maintain all the records as per format (Form D) and submit to
Institutional Animal Ethics Committee (IAEC).

8. I certify that, I will not initiate the study before approval from IAEC/
CPCSEA received in writing. Further, I certify that I will follow the
recommendations of IAEC/CCSEA.

9. I certify that I will ensure the rehabilitation policies are adopted

(wherever required).

Signature

Name of Investigator
Date:
 Certificate
This is to certify that the project entitled “Assessment of formulated PLG-based iron-
oxide nanoparticles decorated with CCPs for the selective delivery of paclitaxel in
melanoma mouse model.” submitted by Mr. Mayur Aher under the supervision of Dr.
Jairam Meena has been approved by the IAEC having
IAEC approval No…………………………….

Authorized by Name Signature Date

Chairman: ……………………. ……………… ………………..

Member Secretary: ……………………. ……………… ………………..

Main Nominee of ……………………. ……………… ………………..

CPCSEA:

(Kindly make sure that minutes of the meeting duly signed by all the participants
are maintained by Office)
 ANNEXURE

Aim and Objectives:

1) Formulation and characterization of Paclitaxel PLG (poly(lactic-co-glycolic) acid)
based iron oxide nanoparticles decorated with CPPs.
2) In-vivo evaluation of nanoformulation.

Study plan:

Group No. of Dosing

animals
Disease 8 B16F10 cell line (1 × 105 cells/mice) S.C.
control
PLG- 8 B16F10 cell line (1 × 105 cells/mice) S.C. +
IONF-CPP Paclitaxel PLG- iron oxide-CPP nanoformulation
(10mg/kg) I.V.
Paclitaxel 8 B16F10 cell line (1 × 105 cells/mice) S.C. +
(Positive Paclitaxel (10mg/kg) I.V.
control)
Normal 8 Saline (100 µl) I.V.
control
(NC)

Procedure:
 Total 32 BALB/C mice, aged 6 to 8 weeks will be used with the approval of the
Institutional Animal Ethics Committee.
 Out of 32, 24 animals will be injected with B16F10 melanoma cell line (1 × 10 5
cells/mice) subcutaneously in flank.
 After 5 to 10 days palpable tumor should appear and it will be allowed to grow
upto 30-40 mm3, Tumor size in mm3 will be estimated from the formula (w2 ×
l)/2.
 Tumor-bearing animals will be divided into 3 groups ( PLG-IONF-CPP,
Paclitaxel and Disease control) with 6-8 animals in each group.
 Normal control and disease control group will receive IV saline.
 Other 2 groups will receive Paclitaxel and its nanoformulations at the dose of
10mg/kg by tail vein for every 2 days upto 14 days.
 Throughout the experiment body weight of animals will be recorded every day.
 On 15th day 0.1 ml blood will be collected from retro-orbital sinus which will be
used for biochemical parameters estimation and animals will be sacrificed and
tumor will be isolated for histopathology.