REPORT Title MAMMALIAN (MICE) ERYTHROCYTE … · 2018-11-14 · : 0.5 ml 7.4.4. Duration of experiment : 3 days 7.4.5. Route : Oral : 7.5. Test System Mice. 7.6. Test groups : S

TEENA BIOLABS PVT. LTD Study No. : TFRCRDMNT03032015 (MOUSE).

Name of the Test item : POLY HERBAL FORMULATION

Page 1 of 28

Registration No. 177/PO/cb/99/CPCSEA

Ministry of Environment and Forest, Government of India.

REPORT

Title

MAMMALIAN (MICE) ERYTHROCYTE MICRONUCLEUS TEST

ON

POLY HERBAL FORMULATION

Sponsor

M/s. SRI SRI SRI VEMAYASWAMYAYURVEDA OUSHADHI,

Chollaveedu (V), Prakasam (Dist.)

A.P., INDIA.

Performing Laboratory

TEENA BIOLABS PVT. LTD.,

Pharmacology Division

Plot No.41, S.V. Co-operative Indl. Estate,

Bachupally (V), Quthbullapur (M),

R.R. (Dist.), Telangana State,

INDIA.

Study No. : TFRCRDMNT03032015 Date: 16th

Mar. 2015.

Total Number of pages: 28

105 & 106, Sree Rama Towers, Kukatpally, Hyderabad – 500072, Telangana State,

INDIA. Tel.: +91 040 23068027 Tel. Fax : + 91 040 23065808,

Email : [email protected]. Website : www.teenabiolabs.com.

PHARMACOLOGY DIVISION

mailto:[email protected]



Page 2 of 28

2.0 STUDY INFORMATION

STUDY NUMBER : TFRCRDMNT01022015.

STUDY TITLE : Mammalian (mice) erythrocyte micronucleus test of Poly

herbal formulation in mice.

SPONSOR : M/s. SRI SRI SRI VEMAYASWAMYAYURVEDA

OUSHADHI,


A.P., INDIA.

TESTING FACILITY : M/s.TEENA BIOLABS PVT. LTD.,



R.R. (Dist.), Telangana State,

INDIA.

DATE OF SUBMISSION

OF FINAL REPORT

: 16

th Mar. 2015.



Page 3 of 28

3.0 CONTENTS

S.NO TITLE PAGE NO.

1. PROJECT TITLE……………………………………………………………..

……..

1

2. STUDY INFORMATION……………………………………………………

………

2

3. CONTENTS…………………………………………………………………..

.

……..

3

4. STATEMENT OF COMPLIANCE…………………………………………..

……..

4

5. STUDY PERSONNEL, FACILITY AND ACCREDITATION……………..

….....

5

6. ABSTRACT…………………………………………………………………..

……..

6

7. SUMMARY…………………………………………………………………..

……..

7-8

8. ABBREVIATIONS………………………………………………………......

……..

9

9. GENERAL INFORMATION………………………………………………..

……..

10

10. MATERIALS AND METHODS……………………………………………..

……..

11-13

11. EXPERIMENTAL OBSERVATIONS……………………………………….

……..

14-15

12. RESULTS………………………………………………………………….....

……..

16

13. CONCLUSION……………………………………………………………….

.

……..

17

14. APPENDICES INDEX……………………………………………………….

……..

18

15. PHOTOGRAPHS……………………………………………………………..

.

……..

24

16. REFERENCES………………………………………………………………..

……..

28



Page 4 of 28

4.0 STATEMENT OF COMPLIANCE

Test Item : POLY HERBAL FORMULATION

The objective of this Mammalian (Mice) erythrocyte micronucleus test in mice was to

assess the mutagenic hazard profile when administered orally in a constant volume of 0.5

ml/animal on initial day of the experiment. The study was carried out in full compliance with

the guidelines laid down in “OECD Guidelines for the Testing of Chemicals, Section-4, Test

No. 474 and Experiments on Animals” (CPCSEA, M/s. Teena Biolabs Registration No.

177/PO/cb/99/CPCSEA).

Dr. C.SRIDEVI

_______________ _____________________ _________________

Study Director Signature Date



Page 5 of 28

5.0 STUDY PERSONNEL, FACILITY AND ACCREDITATION

I TEST FACILITY MANAGEMENT : K.Anjaiah.

II STUDY DIRECTOR : Dr.Sridevi

III STUDY PERSONNEL : V.A Nageswara Rao

: B.Raju

: D.Sandhya Rani

: M.Sunitha

1 CLINICAL CHEMIST : Md.Ghouse

2 HISTO-TECHNICIAN : Sanoj kumar yadav.

3 PATHOLOGIST : Dr. K.Pradip

4 BIOSTATISTICIAN : V.A Nageswara Rao

IV TEST FACILITY : M/s. TEENA BIOLABS PVT. LTD.

V SPONSOR : M/s. SRI SRI SRI

VEMAYASWAMYAYURVEDA

OUSHADHI,

VI ACCREDITATION : ISO 9001:2008 Certified

: NABL-ISO/IEC 17025:2005

VII APPROVALS : CPCSEA Approved

: DCGI Approved

: DCA Approved



Page 6 of 28

6.0 ABSTRACT

MAMMALIAN ERYTHROCYTE MICRONUCLEUS TEST OF POLY HERBAL

FORMULATION IN MICE BY ORAL ROUTE

The present investigation in mice is to evaluate the mutagenic hazard profile of Poly herbal

formulation manufactured by M/s. SRI SRI SRI VEMAYASWAMYAYURVEDA

OUSHADHI, Hyderabad. The study design involves four groups consisting of 5 animals per

group (5 males). The vehicle control (VC) group received D.M water where as Positive control

(PC), Poly herbal formulation-low (500 mg/kg) and Poly herbal formulation-high (1000

mg/kg) group animals have received test item at various dose levels. The test item has been

administered orally in a constant volume 0.5 ml/animal once on first day of the experiment.

Conclusion:

Poly herbal formulation did not induce mutagenicity in the polychromatic erythrocytes of

the bone marrow of male mice treated up to a nominal dose of 500 mg/kg and 1000

mg/kg..

Statistically no significant increase in micronuclei was observed in the 500 mg/kg and

1000 mg/kg following initial slide analysis. There was also no evidence of a dose-

related effect, or bone marrow toxicity.



Page 7 of 28

7.0 SUMMARY

7.1.

Title : Mammalian (mice) Erythrocyte Micronucleus Test of

Poly herbal formulation in mice by oral route

.

7.2.

Objective : To assess the mutagenic hazard profile of Poly herbal

formulation in mice.

7.3. Sponsor : M/s. SRI SRI SRI VEMAYASWAMYAYURVEDA

OUSHADHI.

7.4. Test item details

7.4.1. Test item name : Poly herbal formulation

7.4.2. Appearance : Brownish colored powder.

7.4.3. Dosage

regimen(Clinical) : 0.5 ml

7.4.4. Duration of

experiment : 3 days

7.4.5. Route : Oral

7.5.

Test System : Mice.

7.6.

Test groups :

S. No Study

Type Group details Dose

Duration

of

exposure

Study

period

1

Mammalian

Erythrocyte

Micronucleus

Test

Vehicle control -

Single dose

on day 1 of

experiment

10 days

2 Positive control 40 mg/kg

3 Poly herbal formulation-

low 500 mg/kg

4

Poly herbal formulation-

high 1000 mg/kg

7.7. Methodology

The animals were acclimatized to the laboratory conditions and exposed to the test item by

through oral route. The animals were observed twice daily for 3 days for any symptoms of

toxicity after exposure to the test item. All the animals were sacrificed on 4th

day.



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7.8. Observations

The animals were observed for symptoms of toxicity, behavioral changes and changes related

to cage side activity. Particularly animals were observed for lethality, if any, during the

observation period of 3 days. Body weight changes and feed intake changes were monitored

for all the animals during the experimental period.

7.9 Data retention

All raw data, documentation, records, specimens, slides, and final reports generated as a

result of this study were kept confidential and archived by the archiving In-charge at archives

room of the main building of M/s. Teena Biolabs Pvt. Ltd. The retention duration of these

records and the excess test material provided by the sponsor kept under appropriate storage

conditions will be for five years after the release of the final report.



Page 9 of 28

8.0 ABBREVIATIONS

PCE = Polychromatic erythrocytes

NCE = Normo chromatic erythrocytes

MNPCE = Micro nucleated polychromatic erythrocytes

OECD = Organisation for Economic Co-operation and Development

CPCSEA = Committee for the Purpose of Control and Supervision on

Experiments on animals

Dl = Decilitre

Gm = Gram

IU = International unit

Kg = Kilogram

Mg = Milligram

mL = Millilitre

µl = Micro litre

SD = Standard deviation

TI = Test item

VC = Vehicle control



Page 10 of 28

9.0 GENERAL INFORMATION

STUDY NUMBER : TBLRDMNT03032015.

SPONSOR : M/s. SRI SRI SRI VEMAYASWAMYAYURVEDA

OUSHADHI,


A.P., INDIA.

TEST FACILITY : M/s. TEENA BIOLABS PVT. LTD.

R&D Division



R.R. (Dist).

Telangana State, INDIA.

STUDY CENTRE : Animal House (Biological Testing Lab)

IAEC APPROVAL DATE : 26.02.2015

RECEIPT OF TEST ITEM : 01.03.2015

RECEIPT OF ANIMALS : 01.03.2015

STUDY INITIATION : 06.03.2015

FIRST DAY OF DRUG

EXPOSURE : 06.03.2015

NECROPSY : 10.03.2015

RAW DATA COMPLETION : 13.03.2015

SUBMISSION OF FINAL

REPORT : 16.03.2015



Page 11 of 28

10.0 MATERIALS AND METHODS

10.1. TEST ITEM

10.1.1. Test item details:

Poly herbal formulation supplied by the sponsor was used. The certificate of

analysis was provided by the Sponsor is given in Appendix 3. The test

item information and certificate of analysis provided by the Sponsor are

considered an adequate description of the characterization and purity of the

test item. 10.1.2. Test item preparation:

Dosing preparations were freshly prepared prior to each dosing occasion by

formulating Poly herbal formulation (low & high concentration) in D.M water

and The test item was administrated in a standard volume of 0.5 ml/animal

orally.

10.1.3 Control preparation

The vehicle control group consisted of animals dosed with D.M water using

the same dosing regimen and dose volume used for the test item treated

animals.

10.1.4 Positive control preparation

The positive control, Cyclophosphamide (CPA, Sigma-Aldrich) was

freshly prepared in saline and administered once only via oral gavage.

Dose volume

(mL)

Concentration of CPA

solution (mg/mL)

Concentration of CPA

solution (mg/kg)

0.5 0.2 40.0

10.2. TEST SYSTEM

10.2.1. Age & weight of animals:

Mice 6-8 weeks of age, at the commencement of treatment, the weight

variation of the animals will not exceed ± 20 % of the mean body weight in

each sex and group.

10.2.2. Source of animals:

M/s. Teena Biolabs Pvt Ltd.



Page 12 of 28

10.2.3. Rationale for the selection of Mice as test system:

Mice are one of the recommended species as test system for the conduct of

toxicity studies. They have been widely used throughout the industry for the

evaluation of product safety. Extensive data available for comparative purpose.

10.2.4. Route of administration and reason for choice:

Oral route has been chosen because it is the proposed route for administration

to humans. The dosage can be accurately administered. The oral route is one of

the proposed routes for toxicity testing.

10.2.5. Randomization and numbering of animals:

All the animals were acclimatized to the laboratory conditions for a minimum

period of five days, after acclimatization all the animals were randomized and

assigned to four groups of 20 animals consisting 5 males.

Group Number of

animals

Animal No.

Male

Vehicle control 5 Males M 101 – 105

Positive control 5 Males M 201 – 205

Poly herbal formulation-low

(500 mg/kg ) 5 Males M 301 – 305

Poly herbal formulation-

high (1000 mg/kg ) 5 Males M 401 – 405

10.2.6. Animal husbandry:

The animals were housed in polypropylene cages as group wise and sex wise in

experimental room. Animal rooms were maintained under standard animal

husbandry conditions. The temperature of the animal room was maintained at

22±30C and relative humidity 30 to 70%. The photoperiodicity was 12 hours

light and 12 hours dark.



Page 13 of 28

10.2.7. Feed and water:

The animals were fed on pelleted feed containing standard composition of all

macro and micronutrients (Appendix–III), purified water collected through aqua

guard was provided to animals ad libitum.

10.2.8. Identification of animals:

Animals were identified by body marking. Each cage was identified with

individual cage tags by study number, animal numbers, dose, group, route,

species, sex and experiment starting date.

10.2.9. Route of administration:

The test item was administered by oral route.

10.3. RANDOMIZATION

Randomization ensured the allocation of treatments to animals/groups was independent of

their characteristics and were similar in all the groups. It was also taken care while

randomization, base variables were homogenized and were allotted to different groups.

The need for randomization applies not only to the allocation of the animals to the

different (control as well as treatment) groups but also to anything that can materially

affect the recorded response.

10.4. STATISTICAL ANALYSIS

The results of data on weight gain, food intake, haematology, biochemistry and various

organ weights of animals will be interpreted and expressed as Mean ± SEM. Statistical

analysis shall be performed with one way analysis of variance (ANOVA) followed by

Dunnet’s multiple comparison test by using Graphpad prism software.

P ≤ 0.05 will be considered to be statistically significant

5.



Page 14 of 28

11.0 EXPERIMENTALOBSERVATIONS

11.1 Post dosing observations

All animals were observed daily for signs of ill health. In the

Micronucleus Experiment post-dosing observations were

performed immediately after each dose administration, at least twice

in the 4 hours following each dose. An individual record was

maintained of the clinical condition of all Micronucleus animals dosed

in the study.

11.2 Food intake

The food intake profile was recorded after completion of the

experimental period.

11.3 body weight

The body weight of each animal was recorded at the beginning of

experiment and after completion of the study period.

11.4 Bone marrow sampling and slide preparation

Vehicle control-treated mice were sampled in groups, 36 hours after the

dose administration. The animals will be fasted overnight and euthanized

by carbon-di-oxide.

Both femurs from each animal were exposed, removed, cleaned of

adherent tissue and the ends removed from the shanks. Using a syringe

and needle, bone marrows were flushed from the marrow cavity with 1mL

normal saline into appropriately labelled centrifuge tubes.

A further 1 mL of normal saline was added to the tubes, which were

then centrifuged at 2000rpm for approximately 10 minutes; the serum was

aspirated to leave one or two drops and the cell pellet. The pellet was

mixed into this small volume of serum in each tube by using a Pasteur

pipette, and from each tube one drop of suspension was placed on the end

of each slide labelled with the appropriate study number, sampling time,



Page 15 of 28

sex, date of preparation and animal number (details where appropriate).A

smear was made from the drop by drawing the end of a clean slide

along the labelled slide.

Slides were allowed to air-dry and then fixed for 5 minutes in

absolute methanol before being stained. Slides were stained

immediately. Staining was performed on the same day as slide

preparation. Prior to staining any stored slides were fixed again for 5

minutes in absolute methanol. After rinsing several times in distilled

water, slides were stained for 10 minutes in filtered Giemsa stain

diluted in distilled water (1:4 (v/v)). Slides were rinsed and allowed to

dry before clearing in xylene for 3 minutes. When dry, slides was

mounted with coverslips and stored at room temperature.

11.5 Slide analysis

Slides from the CPA-treated mice were initially checked to ensure the

system was operating satisfactorily. The slides from all groups were

arranged in numerical order by sex and sampling time.

Initially the relative proportions of polychromatic erythrocytes (PCE),

seen as pale blue or blue/grey enucleate cells, and normochromatic

erythrocytes (NCE), seen as yellow/orange-stained enucleate cells, was

determined until a total of at least 1000 cells where possible (PCE +

NCE) had been analysed. Counting continued until at least 2000 PCE

per animal where possible had been examined. All PCE

containing micronuclei observed during these two phases of counting

was recorded.

A further 2000 PCE per animal was examined from reserve slides for all

dose groups to clarify the initial result obtained.



Page 16 of 28

12.0 RESULTS

The total numbers of PCE, NCE, and MN PCE counted for each animal,

together with calculated %PCE and % MN PCE values presented in Appendix-I.

Poly herbal formulation-low (500 mg/kg) and Poly herbal formulation-High (1000 mg/kg)

were Non-significant when compared with Vehicle control.

Statistical analysis of Poly herbal formulation is presented in Appendix-II.



Page 17 of 28

13.0 CONCLUSION

Poly herbal formulation did not induce mutagenicity in the polychromatic erythrocytes

of the bone marrow of male mice treated up to a nominal dose of Poly herbal

formulation-Low (500 mg/kg) and Poly herbal formulation-High (1000 mg/kg).

Statistically no significant increase in micronuclei was observed in the high dose

group, following initial slide analysis. There was also no evidence of a dose-

related effect, or bone marrow toxicity.



Page 18 of 28

14.0 APPENDICES INDEX

S.NO. TITLE PAGE NO.

I INDIVIDUAL ANIMAL OBSERVATION DATA……………. 19

II SUMMARY OF MICRONUCLEUS DATA…………………… 20

III STANDARD COMPOSITION OF FEED……………………… 21-23



Page 19 of 28

APPENDIX-I

INDIVIDUAL ANIMAL OBSERVATION DATA

Group

Animal

Number

PCE

count

NCE

count % PCE

Total

CE

count

MN

PCE

% MN

PCE

Negative

control

1 382 618 38.2 1000 3 0.3

2 421 579 42.1 1000 2 0.2

3 418 582 41.8 1000 3 0.3

4 512 488 51.2 1000 0 0

5 486 514 48.6 1000 1 0.1

Poly herbal

formulation

-Low Dose

6 422 578 42.2 1000 3 0.3

7 352 648 45.2 1000 4 0.4

8 362 638 36.2 1000 2 0.2

9 453 547 45.3 1000 1 0.1

10 398 602 39.8 1000 2 0.2

Poly herbal

formulation

- High Dose

11 338 662 33.8 1000 3 0.3

12 469 531 46.9 1000 4 0.4

13 332 668 33.2 1000 3 0.3

14 457 543 45.7 1000 2 0.2

15 494 506 49.6 1000 3 0.3

Positive

Control

16 487 513 48.7 1000 22 2.2

17 498 502 49.8 1000 34 3.4

18 512 488 51.2 1000 23 2.3

19 495 505 49.5 1000 18 1.8

20 534 466 53.4 1000 28 2.8

PCE – Polychromatic erythrocytes NCE – Normo chromatic erythrocytes MNPCE – Micro

nucleated polychromatic erythrocytes



Page 20 of 28

APPENDIX-II

SUMMARY OF MICRONUCLEUS DATA

STATISTICAL ANALYSIS OF POLY HERBAL FORMULATION

Treatment

group Duration Sex % PCE [mean]

Group mean %

Micro nucleated

PCE +SD

Significance

-Ve control 36 hrs Male 44.38 0.18 ± 0.13 -

Low dose 500

mg/kg 36 hrs Male 41.74 0.24 ± 0.11

Non

significant

High Dose 1000

mg/kg 36 hrs Male 41.84 0.30± 0.07

Non

significant

+ ve control 36 hrs Male 50.52 2.5 ± 0.55 Significant



Page 21 of 28

APPENDIX-III

STANDARD COMPOSITION OF FEED

Diet composition for mice

a Wheat flour 22.5%

b Roasted Bengal gram 60.0%

c Skim milk powder 5.0%

d Casein 4.0%

e Refined ground nut oil 4.0%

f Salt mixture 4.0%

g Vitamin mixture 0.5%



Page 22 of 28

Vitamin mixture composition

1 (dl) – a Tocopherol Acetate (E)

50% dry powder 12.0 g

2 Menadione (K) 0.15 g

3 Thiamine (B1) 1.2 g

4 Riboflavin (B2) 0.5 g

5 Pyridoxine (B6) 0.6 g

6 Niacin 1.0 g

7 Pantothenic acid (calcium salt) 1.2 g

8 Cynacobalamine (B12) 0.5 mg

9 Folic acid 0.1 g

10 Para Amino Benzoic Acid (PABA) 10.0 g

11 Biotin 40.0 mg

12 Inositol 10.0 g

13 Choline chloride 100.0 g

Total vitamins put together 176.76 g

Starch 323.34 g

500.00 g

i.e., 500 g of vitamin mixture is used for every 100 kg of the diet prepared.



Page 23 of 28

Salt mixture composition

Mineral Per 100 kg diet (g)

1 Dicalcium phosphate 1250.00

2 Calcium carbonate (CaCO3) 555.00

3 Sodium chloride (NaCl) 300.00

4 Magnesium sulphate (MgSO47H2O) 229.20

5 Ferrous sulphate (FeSO47H2O) 50.00

6 Magnesium sulphate (MgSO4H2O) 16.04

7 Potassium iodide (KI) 1.00

8 Zinc sulphate (ZnSO47H2O) 2.192

9 Copper sulphate (CuSO45H2O) 1.908

10 Cobalt chloride (CoCl26H2O) 0.012

All minerals together 2405.35 g

Starch 1594.65 g

4000.00 g



Page 24 of 28

15.0 PHOTOGRAPHS INDEX

S.NO. TITLES PAGE NO.

1. Microscopic Observation of Negative control group ………………………….… 27

2. Microscopic Observation of Positive control ………………………………… 28

3. Microscopic Observation of Poly herbal formulation High dose Group ………… 29



Page 25 of 28

1.0 PHOTOGRAPH

MICROSCOPIC OBSERVATION OF NEGATIVE CONTROL GROUP

Black arrow – Arrow indicate Normo chromatic erythrocytes



Page 26 of 28

2.0 PHOTOGRAPH

MICROSCOPIC OBSERVATION OF POSITIVE CONTROL

BlackArrow - indicates Micro nucleated – polychromatic erythrocytes



Page 27 of 28

3.0 PHOTOGRAPH

MICROSCOPIC OBSERVATION OF POLY HERBAL FORMULATION HIGH DOSE

GROUP

Black arrow – Arrow indicate Normo chromatic erythrocytes



Page 28 of 28

16.0 REFERENCES

Good Laboratory Practice (GLP):World Health Organisation, Second Edition.

CPCSEA Guidelines for Laboratory Animal Facility, Government of India.

OECD (1997) “Genetic Toxicology: Mammalian Erythrocyte Micronucleus Test”.

In OECD Guidelines for the testing of chemicals. OECD Paris, Test Guideline 474

European Agency for the Evaluation of Medicinal Products. (1995) ICH Topic S 2 A.

“Genotoxicity: Guidance on Specific Aspects of Genotoxicity Tests for

Pharmaceuticals”. ICH Harmonised Tripartite Guideline.

Documents

REPORT Title MAMMALIAN (MICE) ERYTHROCYTE … · 2018-11-14 · : 0.5 ml 7.4.4. Duration of experiment : 3 days 7.4.5. Route : Oral : 7.5. Test System Mice. 7.6. Test groups : S