DOSSIER FOR REGISTRATION OF - vanankrpharmas.comvanankrpharmas.com/sample-dossier.pdf · DOSSIER...

DOSSIER FOR REGISTRATION OF

ARTEMETHER + LUMEFANTRINE

TABLET

MANUFACTURED BY

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TABLE OF CONTENT

Sr. No. CONTENTS Page No.

I

SECTION-1

a) Name of product

b) Promotional category

II SECTION-2

Indications

III

SECTION-3

Presentation & Packaging

Detailed information about packaging material

Specification of packaging material

IV SECTION-4

Name and quality of each ingredients

V SECTION-5

Chemical name & structural formula of active ingredients

VI

SECTION-6

Method of Manufacture

Manufacturing process details

Process validation report

VII

SECTION-7

Route and conditions of administration

VIII SECTION-8

Dosage form

IX SECTION-9

Side effects

X SECTION-10

Contraindication

XI SECTION-11

Adverse reactions

XII SECTION-12

Antidote in the event of over dosage

XIII SECTION-13

Teratogenecity

XIV

SECTION-14

Analytical Method of each Ingredient, Chemical or

Microbiological

Specification & analytical method of raw material (Active

& Inactive)

Specification & analytical method of finished product

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Certificate of analysis for finished product

In-process controls

XV SECTION-15

Shelf life & stability data

XVI SECTION-16

Toxicological data

XVII SECTION-17

Clinical data

XVIII SECTION-18

Bioavailability / Bioequivalence

XIX

SECTION-19

a) Certificate or license to from the country of origin.

b) Certificate of pharmaceutical product (COPP)

c) Free sale certificate (FSC)

XX

SECTION-20

a) Sample of the product to be registered

b) Leaflets accompanying the product to be registered

c) Labeling information

XXI

SECTION-21

a) Documentary evidence if the product has been registered in

the country of origin (Product License)

b) Indicate if the product is actually on sale in the country of

origin

XXII

SECTION-22

Name of Countries in which the Product is being Marketed /

Registered

XXIII PUBLISHED LITERATURE SECTION

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SECTION-1

a) NAME OF PRODUCT

b) PROMOTIONAL CATEGORY

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a) NAME OF PRODUCT

Brand Name: XYZ

Generic Name: Artemether + Lumefantrine Tablet

b) PROMOTIONAL CATEGORY: Prescription only Medicine

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SECTION-2

INDICATION

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Indication

LEMALAR is indicated for the treatment of acute uncomplicated Plasmodium

falciparum malaria in adult, children and infants of 5 kg and above.

Consideration should be given to official guidance regarding the appropriate use of

antimalarial agents.

This medication is used to treat malaria in adults and children. The two ingredients in this

medication belong to a class of drugs known as antimalarial. Malaria is an infection caused

by mosquito bites received while traveling or living in regions of the world where malaria

is common. Malaria parasites enter the body, and live in body tissues such as

red blood cells or the liver.

This medication is used to kill the malaria parasites living inside red blood cells. In some

cases, you may need to take a different medication (such as primaquine) to kill the malaria

parasites living in the liver. Both treatments may be needed for a complete cure and to

avoid the return of infection (relapse). This product is not used to prevent malaria.

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SECTION-3

PRESENTATION & PACKAGING



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Pack Style: 6 tablets are packed in a one blister strip, such a 1 blister strips are packed in a

mono carton.

The materials of construction of the primary packaging material are safe to be used. The

above mentioned packaging materials are checked / tested as per In-House specification.


PARAMETERS

SPECIFICTION

1. PRINTED ALUMINIUM

FOIL

DESCRIPTION Silver coloured Background having Black coloured

Printing.

SIZE Width: 186±2 mm

Thickness: 0.03±0.005 mm

G.S.M. 70 ± 5g/m²

2. PLAIN FOIL (ALU-ALU)

DESCRIPTION Plain Alu-Alu foil.

SIZE Width: 188±2mm

Thickness: 0.15±0.01mm

G.S.M. 250 ± 20gm/m²

3. LEAFLET

DESCRIPTION White Background having Black colored printing.

SIZE Length:175±5 mm

Width: 130±5 mm

G.S.M. 60 ± 5gm/m²

4. CARTON

DESCRIPTION White Background having Black, Green, Orange &

Purple coloured printing.

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SIZE Length:107±2mm

Width:23±2

mm Height:44±2

mm

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SECTION-4

NAME & QUALITY OF EACH INGREDIENT

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QUALITATIVE AND QUANTITATIVE COMPOSITION

Product Name: XYZ

Generic Name: ARTEMETHER + LUMEFANTRINE TABLET

Composition:

Each uncoated Table Contains:

Artemether 80 mg

Lumefantrine 480 mg

Excepient Q.S

BATCH COMPOSITION

Batch Size: 100,000 Tablets

S.No. Ingredients Spec. Claim Overages

Qty/Tab in mg

Qty.

/Batch

1. Artemether InH 80 mg ---- 80.0 mg 8.0kg

2. Lumefantrine InH 480 mg ---- 480.0 48.00 kg

3. Microcrystalline

cellulose

BP 85.2 8.52

4. Croscarmellose sodium BP 112.0 11.2

5. Colloidal Silicon

dioxide BP 20.0

2.00

6. Microcrystalline

cellulose BP 40.0 4.00

7. Polysorbate 80 BP 2.0 0.200

8. Microcrystalline



dioxide BP 12.0 1.2

10. Magnesium stearate 40.0 4.000

Average weight of un coated tablet: 984.0 mg + 5%

Overages are not added

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SECTION-5

CHEMICAL NAME & STRUCTURAL FORMULA OF

ACTIVE INGREDIENT

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CHEMICAL NAME AND STRUCTURAL FORMULA OF ACTIVE INGREDIENTS

ARTEMETHER

1

Active

Ingredient

: Artemether

2 Chemical

formula :

(1R,4S,5R,8S,9R,10S,12R,13R)-10-methoxy-1,5,9-

trimethyl-11,14,15,16-

tetraoxatetracyclo[10.3.1.0^{4,13}.0^{8,13}]hexadecane

3 Structural

Formula :

4

CAS Number

: 71963-77-4

5

Molecular

Formula

: C16H26O5

6

Molecular

weight

: 298.3746

7 Action and use : Antimalarial agent

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LUMFANTRINE

1

Active

Ingredient

: Lumefantrine

2 Chemical

formula :

2-(dibutylamino)-1-[(9Z)-2,7-dichloro-9-[(4-

chlorophenyl)methylidene]-9H-fluoren-4-yl]ethan-1-ol

3 Structural

Formula :

4

CAS Number

: 82186-77-4

5

Molecular

Formula

: C30H32Cl3NO

6

Molecular

weight

: 528.94

7 Action and use : Antimalarial agent

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SECTION-6

METHOD OF MANUFACTURE



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METHOD OF MANUFACTURE


Product : XYZ

Generic Name : Artemether +Lumefantrine Tablet

Composition : Each uncoated Tablet Contains:

Artemether 80 mg

Lumefantrine 480 mg

Excepient Q.S

Batch Size : 100,000 Tablets

Shelf Life : 36 Month

BATCH COMPOSITION

Batch Size: 100,000 Tablets

S.No. Ingredients Spec. Claim Overages

Qty/Tab in mg

Qty.

/Batch

1. Artemether InH 80 mg ---- 80.0 mg 8.0kg

2. Lumefantrine InH 480 mg ---- 480.0 48.00 kg

3. Microcrystalline


4. Croscarmellose sodium BP 112.0 11.2


dioxide BP 20.0

2.00

6. Microcrystalline


7. Polysorbate 80 BP 2.0 0.200

8. Microcrystalline



dioxide BP 12.0 1.2

10. Magnesium stearate BP 40.0 4.000

Average weight of uncoated tablet: 984.0 mg + 5%

Overages are not added

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MANUFACTURING PROCESS OF

XYZ

(Artemether +Lumefantrine Tablet)

FLOW CHART FOR THE MANUFACTURING PROCESS

WEIGHING SHIFTING MIXING

PACKING QUALITY CONTROL

SIFTING

COMPRESSION

LUBRICATION

GRANULATION

FINAL DRYING

SEMI DRYING

CHECKING

R.M.

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Manufacturing process

MANUFACTURING INSTRUCTIONS

I. Good Manufacturing Practices:

To ensure a quality production, all current manufacturing practices should be followed

such as:

1. Area and Equipments

The area should be free from unwanted material as well as material from the last batch.

The equipments to be used should be labelled for product batch no. and date o prior to use.

The equipments to be used must bear a ―clear equipment‘ tag and wash water analysis

report releasing the equipment in case of product changes over.

II. Personnel

All personnel should be of good health and should practice good sanitation habits.

Persons engaged in the manufacture, processing, packing or holding of drug product should

bear protective apparent such as head, face, hand and arm covering necessary to protect the

product from contamination.

III. Raw Material & Packing Material

All ingredients and packing material must be tested for conformance to written

specifications.

Weight and volume of the ingredients should be checked by the authorized persons.

IV. Production and Process Control

Production record must be complete and accurate reflecting all the procedure and process

adopted during production

Batch should be fabricated strictly as per the written procedure and any deviation in the

process should be approved by Q.A.

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BATCH MANUFACTURING PROCEDURE

PREPARATORY WORK

1. CLEANING:

(a) Clean thoroughly all the equipments Trolleys, Sieves, Mass Mixer, Multi Mill, Sifter,

Fluid Bed Drier, and Compression Machine & Coating Pan as per cleaning protocols of

equipments. Intimate the Quality Control Dept. for sampling of washed water.

(b) Manufacturing should be started on the receipt of approval from the Q.C. dept.

2. SPECIAL PRECAUTION IF ANY:

(a) The batch should not be started if Q.C.Dept. not approved the cleaning of equipment.

(b) Humidity should be maintained 40% to 50% during compression.

(c) The persons working in the manufacturing area must wear clean dresses, hand gloves,

mask, cap / headgear, footwear.

(d) The manufacturing area including the roof, wall, floor, windows, doors, AHU-grills &

electrical fixtures must be free from dust.

(e) The utensils, equipments and machines directly in contact with the raw materials, must

be strictly cleaned and labeled with ‗Ready for Use and certificate should be issued by

Quality Control Department.

(f) Chewing, eating & drinking must not be allowed in manufacturing area.

(g) The personal working in manufacturing area must have sound health without any

contagious disease.

(h) Exposure of the material to the atmosphere should be kept minimum.

3. RESPONSIBILITY:

1. Production : Manufacturing Supervisor / Production Chemist

2. In-process Quality Control: Quality Control & Quality Assurance Chemist

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GOOD MANUFACTURING PRACTICES

To ensure a quality production, all current manufacturing practices should be

followed such as:

A. Area and Equipments

The area should be free from unwanted material as well as material from the last batch.

The equipments to be used should be labeled for product batch no. and date prior to use.

The equipments to be used must bear a ‗Ready for Use‘ label and wash water analysis

report releasing the equipment in case of product change over.

B. Personnel

All personnel should be of good health and should practice good sanitation habits.

Persons engaged in the manufacture, processing, and packing or holding of drug product

should wear protective apron such as head, face, hand and arm covering necessary to

protect the product from contamination.

C. Raw Material & Packing Material

All ingredients and packing material must be tested for conformance to written

specifications.

The authorized persons should check weight and volume of the ingredients.

D. Production and Process Control

Production record must be complete and accurate reflecting all the procedure and process

adopted during production.

Batch should be fabricated strictly as per the written procedure and any deviation in the

process should be approved by Q.A.

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LIST OF EQUIPMENTS / MACHINERY REQUIRED

List of Machines and Equipments Required In Manufacturing

NAME OF THE

MACHINES

MAKE CAPACITY CODE NO

Weighing Balance Sansui 100 kg WB/G/001

Mechanical sifter Bectochem 30 inch Dia Tab/G/002

Mass mixer Fabebia 200 Kg Tab/G/003

Octagonal Blender Bectochem 250 Kg Tab/G/009

Rotary Compression 27 Stn. Riddhi 3.2 Lac /shift Tab/G/017

Weighing balance Sansui 150g IPQC/TG/001

DT Apparatus Tanco 1X6 tubes IPQC/TG/002

Friability Test Apparatus Tanco N.A IPQC/TG/003

Hardness apparatus Multitech N.A IPQC/TG/004

Vernier Caliper Mitutoyo N.A IPQC/TG/005

Stop Watch Kadio N.A IPQC/TG/007

Neocota Neomachine 36 Inches Tab/G/022

Colloidal Mill Cadmach N.A Tab/G/023

Mechanical stirrer Remi N.A Tab/G/024

Peristaltic Pump Electrolab N.A Tab/G/025

Alu-Alu Blister packing PGL 4.5 lac /shift Pac/G/004

Conveyor Belt Fabavia 12 feet Pac/G/009

Tab inspection belt Fabavia Standard Pac/G/013

Weighing Balance Sansui 100kg WB/G/006

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IN-PROCESS CONTROL TEST AND DATA

A. In-process checks for mixed powder:

Uniformity of Mixing: By assay of active ingredients from different places.

B. In-process checks for lubricated granules:

Assay: ± 10% of the label claim

Moisture content: By IR moisture balance (NMT 2.0%)

C. In-process checks for uncoated tablets:

Draw samples at regular intervals during compression and check for:

Average weight: At regular intervals (984.0 mg + 5.0%)

Uniformity of weight: + 5.0% of average weight.

Hardness: At regular intervals (NLT 3 kg/cm2)

Friability: At regular intervals (Not more than 1%)

D. In-Process Control during Packing:

a. The material issue for packing is checked & the inspector puts his signature on the batch

card.

b. Batch coding & other over printing like Mfg., Exp. etc. is checked on strips & cartons

etc.

c. Blister packing machine temperature, pressure etc. are also checked.

d. Pocket cuts; smudged printing, faulty strip cutting, bridge rupture, missing tablet etc. are

checked.

e. Contents of the cartons, less number of strips, absence of pack inserts etc. is checked at

random on the packing line itself.

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PRODUCTION PROCESS

1. WEIGHING AND CHECKING:

Check the weights of all the raw materials as per the formulation sheet also check the AR

No. of all the materials as per the issuance sheet.

2. SIFTING:

Sift Artemether, Lumefantrine, Magnesium stearate BP, Microcrystalline cellulose,

Polysorbate 80 BP, colloidal silicon dioxide, Carmellose sodium through sifter using 80 #

sieve in In process container.

Transfer the contents from IPC to the Rapid Mixer Granulator.

3. LUBRICATION AND BLENDING:

Then add Magnesium stearate, colloidal silicon dioxide to the above mixture and blend for

a further 2 minutes.

4. UNLOADING:

Unload the lubricated granules into clean, dry polythene lined suitable containers. Weigh

and Record the wt. of lubricated granules. Label the containers with regard to:

Batch No.,

Product Name,

Container No.,

Quantity of the materials,

Total weight of lubricated granules should be as per flow sheet. Send the Lubricated

granules for Q.C. Dept for testing.

5. COMPRESSION:

Compress the lubricated granules only after reauthorization of QA, if there is undue time

lag between lubrication and further processing.

1. Get the line clearance for equipments & machinery from Q.A chemist.

2. Get the line clearance for compression area from Q.A chemist.

3. Compress the granules using 27 Station punching machine.

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4. Following parameters should be observed during compression.

Parameter Name Permissible Norms Frequency

Description White colour, round uncoated tablet,

plain on both sides.

At the time of start

up.

Average Weight 984.0 mg + 5.0% After an interval of

30 minutes

Disintegration test NMT 15 minutes Initially and after

an interval of 2

hours.

Friability NMT 1.0% After an interval of

30 minutes

Hardness NLT 3.0kg/cm² After an interval of

30 minutes

5. Store the compressed tablets in well-closed airtight container properly labeled in 25+

2°C area.

6. Check & record weight of compressed tablets.

7. Give bulk analysis request sheet along with batch card to Q.A. Only on getting release

report from Q.A department the batch should be taken for coating.

6. INSPECTION OF CORE TABLETS

Collect the compressed tablets in a previously weighed plastic container containing double

polythene bags. Label the container & weigh it. Keep the container in quarantine room.

Inform Quality Control Department to collect the sample for analysis for following

parameters:

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Parameter Name Permissible Norms

Description White colour, round uncoated tablet, plain on both

sides.

Average Weight 984.0 mg + 5.0%

Disintegration test NMT 15 minutes

Friability NMT 1.0%

Hardness NLT 3.0kg/cm²

Assay

Each uncoated tablet contain:

Artemether

Lumefantrine

Claim Limit

80 mg 90 % - 120%

72.0 mg -96.0 mg

480 mg 90% - 120%

432.0 mg to 576.0 mg.

Check the weight, thickness & hardness after every half an hour & record the same. Check

Friability & Disintegration Time after every 2 hour & record the same.

Tablets to be inspected for physical defect such as capping, cracked, black-spot, etc.

Tablet with black-spot or any suspected foreign matter should be destroyed.

Collect the inspected tablets in a previously weighed plastic container containing double

Polythene bags. Label the container & weigh it. Keep the container in quarantine room.

9. PACKING

Packing operations should be started on receipt of approval from Quality Control

Department. Packing to be done after visual inspection in blister strip pack as per Standard

Operating Procedure.

10. CONTROL SAMPLE:

Collect a sample of 17 blister strips at random from the packing, for control sample & keep

this sample for a period of additional 12 months to expiry.

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11. YIELD:

Count the number of packs & record in the BPR.

12. RELEASE:

Quality Assurance Department shall inspect & verify the packed material as per the

standard norms. They shall order the Release of Goods in writing to the Finished Goods

Store.

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Kindly Refer the Annexure I

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SECTION-7

ROUTE & CONDITIONS OF ADMINISTRATION

SECTION-8

DOSAGE FORM

SECTION-9

SIDE EFFECT

SECTION-10

CONTRA-INDICATION

SECTION-11

ADVERSE REACTIONS

SECTION-12

ANTIDOTE IN THE EVENT OF OVER DOSAGE

SECTION-13

TERATOGENECITY

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Route & Conditions of Administration

Route of Administration: Oral

Dosage Form

Tablet

Side Effects

The safety of Lemalar has been evaluated in 20 clinical trials with more than 3500 patients.

A total of 1810 adults and adolescents above 12 years of age as well as 1788 infants and

children of 12 years of age and below have received in clinical trials.

Adverse reactions reported from clinical studies and post-marketing experience are listed

below according to system organ class.

Adverse reactions are ranked under headings of frequency using the MedDRA frequency

convention:

Very common (≥1/10)

Common (≥1/100 to <1/10)

Uncommon (≥1/1,000 to <1/100)

Rare (≥1/10,000 to <1/1,000)

Very rare (<1/10,000)

Not known (cannot be estimated from available data).

Table 1 Frequency of Undesirable effects

Adults and

adolescents above

12 years of age

Infants and children of 12 years of age

and below (incidence estimates)

Immune system disorders

Hypersensitivity Not known Rare

Metabolism and nutrition disorders

Decreased appetite Very common Very common (16.8 %)

Psychiatric disorders

Sleep disorders Very common Common (6.4 %)

Insomnia Common Uncommon

Nervous system disorders

Headache Very common Very common (17.1 %)

Dizziness Very common Common (5.5 %)

Paraesthesia Common --

Ataxia, Hypoaesthesia Uncommon --

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Somnolence Uncommon Uncommon

Clonus Common Uncommon

Cardiac disorders

Palpitations Very common Common (1.8 %)

Electrocardiogram QT

prolonged

Common Common (5.3 %)

Respiratory, thoracic and mediastinal disorders

Cough Common Very common (22.7 %)

Gastrointestinal disorders

Vomiting Very common Very common (20.2 %)

Abdominal pain Very common Very common (12.1 %)

Nausea Very common Common (6.5 %)

Diarrhoea Common Common (8.4 %)

Hepatobiliary disorders

Liver function tests

increased

Uncommon Common (4.1 %)

Skin and subcutaneous tissue disorders

Rash Common Common (2.7 %)

Pruritus Common Uncommon

Urticaria Uncommon Uncommon

Angioedema* Not known Not known

Musculoskeletal and connective tissue disorders

Arthralgia Very common Common (2.1 %)

Myalgia Very common Common (2.2 %)

General disorders and administration site conditions

Asthenia Very common Common (5.2 %)

Fatigue Very common Common (9.2 %)

Gait disturbance Common --

*: These adverse reactions were reported during post-marketing experience. Because these

spontaneously reported events are from a population of uncertain size, it is difficult to

estimate their frequency.

Contraindications

Lemalar is contraindicated in:

Patients with known hypersensitivity to the active substances or to any of the excipients.

Patients with severe malaria according to WHO definition*.

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patients who are taking any drug which is metabolised by the cytochrome enzyme

CYP2D6 (e.g. metoprolol, imipramine, amitriptyline, clomipramine).

patients with a family history of sudden death or of congenital prolongation of the QTc

interval on electrocardiograms, or with any other clinical condition known to prolong the

QTc interval.

Patients taking drugs that are known to prolong the QTc interval (proarrhythmic). These

drugs include:

Antiarrhythmic of classes IA and III,

Narcoleptics, ant depressive agents,

Certain antibiotics including some agents of the following classes: macrolides,

fluoroquinolones, imidazole and triazole antifungal agents,

Certain non-sedating antihistamines (terfenadine, astemizole),

Over Dosage

In cases of suspected overdosage symptomatic and supportive therapy should be given as

appropriate, which should include ECG and blood potassium monitoring.

Teratogenicity

Carcinogenesis, Mutagenesis, Impairment of Fertility

General toxicity

The main changes observed in repeat-dose toxicity studies were associated with the

expected pharmacological action on erythrocytes, accompanied by responsive secondary

haematopoiesis.

Neurotoxicity

Studies in dogs and rats have shown that intramuscular injections of Artemether resulted in

brain lesions. Changes observed mainly in brainstem nuclei included chromatolysis,

eosinophilic cytoplasmic granulation, spheroids, apoptosis and dark neurons. Lesions were

observed in rats dosed with Artemether at 25 mg/kg for 7 or 14 days and dogs dosed at 20

mg/kg for 8 days or longer, but lesions were not observed after shorter courses of drug or

after oral dosing.

The estimated Artemether 24 h AUC after 7 days of dosing at the no observed effect level

(10 mg/kg/day given intramuscularly) is approximately 7-fold greater than the estimated

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Artemether 24 h AUC in humans on day 1 of the standard 3-day oral treatment regimen;

oral exposure in humans decreases on subsequent days, thus the exposure margin increases.

Dogs dosed orally with 143 mg/kg Artemether showed a statistically measureable effect on

the hearing threshold at 20 dB.

This dose is equivalent to about 29 times the highest Artemether clinical dose (160 mg/day)

based on body surface area comparisons. Most nervous system disorder adverse events in

the studies of the 6-dose regimen were mild in intensity and resolved by the end of the

study.

Mutagenicity

No evidence of mutagenicity was detected in in vitro or in vivo tests with an artemether:

lumefantrine combination (consisting of 1 part artemether:6 parts Lumefantrine). In the

micronucleus test myelotoxicity was seen at all dose levels (500, 1,000 and 2,000 mg/kg),

but recovery was almost complete 48 hours after dosing.

Carcinogenicity

Carcinogenicity studies with the artemether: lumefantrine combination was not conducted.

Reproductive toxicity studies

Reproductive toxicity studies performed with the artemether: lumefantrine combination

caused maternal toxicity and increased post-implantation loss in rats and rabbits at doses

≥50 mg/kg/day (corresponding to approximately 7 mg/kg/day Artemether) and 175

mg/kg/day (corresponding to 25 mg/kg/day Artemether) respectively. These effects were

not observed at lower doses.

Lumefantrine alone caused no sign of reproductive or development toxicity at doses up to

1,000 mg/kg/day in rats and rabbits.

Embryotoxicity has been observed in rat and rabbit reproductive toxicity studies conducted

with Artemether, a derivative of artemisinin. Artemisinin (e.g. artesunate) are known to be

embryotoxic.

Artemether caused increases in post-implantation loss and teratogenicity (characterised as a

low incidence of cardiovascular and skeletal malformations) in rats at 19.4 mg/kg, and in

rabbits at 30 mg/kg. Maternal toxicity was also observed in rabbits at 30 mg/kg/day. No

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other adverse effects were observed at lower doses in rabbits. The no observed effect dose

was 3 mg/kg/day in rats and 25 mg/kg/day in rabbits.

The embryotoxic Artemether dose, 20 mg/kg/day in the rat, yields Artemether and dihydro

artemisinin exposures similar to those achieved in humans.

Artesunate, a structurally related compound, also caused increases in post-implantation loss

and teratogenicity (low incidence of cardiovascular and skeletal malformations) in rats at 6

mg/kg and in the lowest dose tested in the rabbits, 5 mg/kg/day.

Fertility

After artemether:lumefantrine administration for 10 weeks in males and 2 weeks in

females, reduced fertility occurred at 1000 mg/kg/day where altered sperm motility,

abnormal sperm, reduced epididymal sperm count, increased testes weight, and

embryotoxicity and other reproductive effects (decreased implants and viable embryos,

increased preimplantation loss) were also observed. General toxicity was observed in males

and females at doses ≥ 300 mg/kg/day. The no adverse effect level for fertility was 300

mg/kg/day. The relevance to this finding in humans is unknown.

Juvenile toxicity studies

A specific study to investigate the neurotoxicity of Artemether in juvenile rats involved

oral administration of Artemether during four different dosing intervals, at doses of 30 or

80 mg/kg/day on post partum days 7 to 13, and at doses of 30 or 120 mg/kg/day on post

partum days 14 to 21, 22 to 28, or 29 to 36.

Mortality, clinical signs and reductions in body weight parameters occurred most notably

during the first two dosing intervals. Despite the systemic toxicity noted, there were no

effects of Artemether on any of the functional tests performed and there was no evidence of

a direct neurotoxic effect of orally administered Artemether on the brain of juvenile rats.

Juvenile studies in the rat indicate that very young animals (aged 7-21 days) are more

sensitive to Artemether than adult animals.

There is no difference in sensitivity in slightly older (3-5 weeks of age) animals following

13 weeks of Artemether/Lumefantrine administration. Consistent with the later data,

clinical studies have established the safety of Artemether and Lumefantrine administration

in patients weighing 5 kg and above.

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Cardiovascular Safety Pharmacology

In toxicity studies in dogs at doses >600 mg/kg/day only, there was some evidence of

prolongation of the QTc interval (safety margin of 1.3-fold to 2.2-fold for Artemether using

calculated free Cmax), at higher doses than intended for use in man. In an in vitro assay of

HERG channels stably expressed in HEK293 cells, Lumefantrine and the main metabolite

desbutyl-lumefantrine showed some inhibitory potential in one of the currents responsible

for cardiac repolarisation. The potency was lower than the other antimalarial drugs tested.

From the estimated IC50 values, the order of potency of HERG current block was

halofantrine (IC50 = 0.04 μM) >chloroquine (2.5 μM) >mefloquine 2.6 μM) >desbutyl-

lumefantrine (5.5 μM) >Lumefantrine (8.1 μM).

Additional studies were performed to evaluate the in vitro effects of Artemether and its

active metabolite, dihydro artemisinin, on the HERG current. At concentrations that

produced significant inhibition, the safety margins for Artemether and dihydro artemisinin

are greater than 100 if they are estimated using the total therapeutic concentration at Cmax

or greater than 1000 if they are estimated using the calculated free Cmax. Based on the

available non-clinical data, a potential for QTc prolongation in the human cannot be

discounted. For effects in the human.

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SECTION-14

ANALYTICAL METHOD OF EACH INGREDIENT,

CHEMICAL OR MICROBIOLOGICAL

Specification & analytical method of raw material

(Active & Inactive)



In-process controls

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Specification & analytical method of raw material

(Active & Inactive)

SPECIFICATION OF ACTIVE

ARTEMETHER

Reference Protocol: InHouse

S. NO. TEST SPECIFICATION

1.0 Description White or almost white, crystalline powder.

2.0 Solubility Soluble in water,

3.0 Identification

A. By IR

In the assay the principle peak in the

chromatogram obtained with the test

solution corresponded to the peak in the

chromatogram obtained with the reference

solution.

4.0 pH 2.0 to 4.0.

5.0 Water 5.0 % to 8.0 %

6.0 Related compound

NMT 2.0 %

7.0 Assay 98.0 per cent to 102.0 per cent (anhydrous

substance).

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METHOD OF ANALYSIS OF ACTIVE SUBSTANCE

ARTEMETHER

Reference Protocol: In House

DEFINITION

(1R,4S,5R,8S,9R,10S,12R,13R)-10-methoxy-1,5,9-trimethyl-11,14,15,16-

tetraoxatetracyclo[10.3.1.0^{4,13}.0^{8,13}]hexadecane

Content

98.0 per cent to 102.0 per cent (anhydrous substance).

CHARACTERS

Appearance

White or almost white, crystalline powder.

Solubility

Freely soluble in water.

IDENTIFICATION

First identification A,

A. Infrared absorption spectrophotometry.

Comparison Artemether CRS

A. Dissolve about 20 mg in 10 ml of water R, add 1 ml of dilute acetic acid R and 1.6 ml of

1 M sodium hydroxide, heat on a water-bath for 30 min and allow cooling. Add 5 ml of

dilute sodium hydroxide solution R, 10 ml of potassium ferricyanide solution R and 10 ml

of butanol R and shake vigorously for 2 min. The upper alcoholic layer shows an intense

light-blue fluorescence, especially in ultraviolet light at 365 nm. Repeat the test using 0.9

ml of 1 M sodium hydroxide and 0.2 g of sodium sulphite R instead of 1.6 ml of 1 M

sodium hydroxide. Practically no fluorescence is seen.

TESTS

Solution S

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Dissolve 2.5 g in distilled water R and dilute to 25 ml with the same solvent.

pH

2.0 to 4.0.

Dilute 2.5 ml of solution S to 10 ml with water R.

Water

Maximum 5.0 % to 8.0 %, determined on 0.40 g

Related Compound

Liquid chromatography

Solution A Add 5 volumes of glacial acetic acid R to 95 volumes of water R and mix.

Test solution Dissolve 0.35 g of the substance to be examined in 15.0 ml of solution A

and dilute to 100.0 ml with water R.

Reference solution (a) Dissolve 5 mg of the substance to be examined and 5 mg of

Artemether impurity E CRS in 4 ml of solution A and dilute to 25.0 ml with water R.

Dilute 5.0 ml of the solution to 25.0 ml with water R.

Reference solution (b) Dilute 1.0 ml of the test solution to 50.0 ml with water R. Dilute

5.0 ml of this solution to 25.0 ml with water R.

Column:

Size: l = 0.25 m, Ø = 4.0 mm,

Stationary phase: spherical end-capped octadecylsilyl silica gel for chromatography R (5

µm) with a specific surface area of 350 m2/g and a pore size of 10 nm,

Temperature: 45 °C.

Mobile phase:

Mobile phase A: 3.764 g/l solution of sodium hexanesulphonate R adjusted to pH 3.1 with

phosphoric acid R, mobile phase B: methanol R2,

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Flow rate 1.0 ml/min.

Detection Spectrophotometer at 248 nm

Injection 25 µl

Relative retention with reference to Artemether (retention time = about 30 min): impurity A

= about 0.3; impurity B = about 0.9; impurity C = about 1.2.

System suitability Reference solution (a):

Resolution: minimum 1.6 between the peaks due to impurity E and to Zoledronic acid.

Limits:

Not More Than; 2.0%

ASSAY

Dissolve 0.110 g in 5 ml of anhydrous formic acid R and add 50 ml of acetic anhydride R.

Titrate immediately with 0.1 M perchloric acid, determining the end-point

potentiometrically (2.2.20) and carrying out the titration within 2 min. carry out a blank

titration.

1 ml of 0.1 M perchloric acid is equivalent to 12.98 mg of C16H26O5

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SPECIFICATIONS OF LUMFANTRINE



1.0 Description White or almost white, crystalline powder.

2.0 Solubility Soluble in Methanol

3.0 Identification

By HPLC

In the assay, the principle peak in the

chromatogram obtained with the test

solution corresponds to the peak in the

chromatogram obtained with the reference

solution.

4.0 Loss on drying NMT 0.2% w/w

5.0 Related compound NMT 2.0%

6.0 Assay

By HPLC (on dried

basis)

98.0 per cent to 102.0 per cent (anhydrous

substance).

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METHOD OF ANALYSIS OF LUMFANTRINE


DEFINITION

2-(dibutylamino)-1-[(9Z)-2,7-dichloro-9-[(4-chlorophenyl)methylidene]-9H-fluoren-4-

yl]ethan-1-ol.

Content

98.0 per cent to 102.0 per cent (anhydrous substance).

CHARACTERS

Appearance

White or almost white, crystalline powder.

Solubility

Soluble in methanol.

IDENTIFICATION

First identification A,

D. Examine the chromatograms obtained in the test for related substances. The retention

time and size of the principal peak in the chromatogram obtained with test solution (b) are

approximately the same as those of the principal peak in the chromatogram obtained with

reference solution (b).

TESTS

Solution S

Dissolve 2.5 g in distilled water R and dilute to 25 ml with the same solvent.

Loss on drying

Maximum: 2.0% w/w

Not more than 0.2 per cent, determined on 1.000 g by drying in an oven in vacuo at 70 °C

for 3 h.

Related Compound

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Liquid chromatography

Solution A Add 5 volumes of glacial acetic acid R to 95 volumes of water R and mix.

Test solution Dissolve 0.35 g of the substance to be examined in 15.0 ml of solution A

and dilute to 100.0 ml with water R.

Reference solution (a) Dissolve 5 mg of the substance to be examined and 5 mg of

Lumfantrine impurity E CRS in 4 ml of solution A and dilute to 25.0 ml with water R.

Dilute 5.0 ml of the solution to 25.0 ml with water R.

Reference solution (b) Dilute 1.0 ml of the test solution to 50.0 ml with water R. Dilute

5.0 ml of this solution to 25.0 ml with water R.

Column:

Size: l = 0.25 m, Ø = 4.0 mm,

Stationary phase: spherical end-capped octadecylsilyl silica gel for chromatography R (5

µm) with a specific surface area of 350 m2/g and a pore size of 10 nm,

Temperature: 45 °C.

Mobile phase:

Mobile phase A: 3.764 g/l solution of sodium hexanesulphonate R adjusted to pH 3.1 with

phosphoric acid R, mobile phase B: methanol R2,

Flow rate 1.0 ml/min.

Detection Spectrophotometer at 248 nm

Injection 25 µl

Relative retention with reference to Lumfantrine (retention time = about 30 min): impurity

A = about 0.3; impurity B = about 0.9; impurity C = about 1.2.

System suitability Reference solution (a):

Resolution: minimum 1.6 between the peaks due to impurity E and to Lumfantrine.

Limits:

Not More Than; 2.0%

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ASSAY

Dissolve 0.110 g in 5 ml of anhydrous formic acid R and add 50 ml of acetic anhydride R.

Titrate immediately with 0.1 M perchloric acid, determining the end-point

potentiometrically (2.2.20) and carrying out the titration within 2 min. carry out a blank

titration.

1 ml of 0.1 M perchloric acid is equivalent to 12.98 mg of C30H32Cl3NO

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SPECIFICATION OF MAGNESIUM STEARATE

Reference Protocol: BP


1.0 Description White, very fine, light powder, greasy to

the touch

2.0 Solubility Practically insoluble in water and in

anhydrous ethanol.

3.0 Identification

a). Solution S

b). Acid Value

c). By Gas chromatography

d). Test for Magnesium

Freezing point : not lower than 53 °C.

195 to 210

The retention time of the peaks

corresponding to stearic acid and

palmitic acid in the chromatogram of

the test solution should correspond to

those in the chromatogram of the system

suitability solution / reference solution,

as obtained in the test for ―Fatty acid

composition‖.

A white, crystalline precipitate should

form.

4.0 Acidity or alkalinity Not more than 0.05 mL of 0.01 M

Hydrochloric acid or 0.01 M Sodium

hydroxide is required to change the

colour of the indicator.

5.0 Chloride NMT 0.1 %

6.0 Sulphate NMT 1.0 %

7.0 Cadmium NMT 3.0 ppm

8.0 Lead NMT 10.0 ppm

9.0 Nickel NMT 5.0 ppm

10.0 Loss on Drying NMT 6.0 %

11.0 Microbial contamination

Total aerobic microbial count

Total yeast mould count

Escherichia coli

Salmonella

NMT 103

cfu/gm

NMT 102

cfu/gm

Absent/g.

Absent/g.

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12.0

Assay (On dried basis)

- Magnesium

- Stearic Acid

- The sum of the stearic acid and

Palmitic acid

NLT 4.0% and NMT 5.0 % of

Magnesium

Not less than 40 %

Not less than 90 %

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METHOD OF ANALYSIS OF MAGNESIUM STEARATE


DESCRIPTION:

Spread the sample over the white paper sheet. Observe the colour visually.

Specification: White, very fine, light powder, greasy to the touch

SOLUBILITY:

Practically insoluble in water and in anhydrous ethanol.

IDENTIFICATION

A. By Solution S

The residue obtained in the preparation of solution S (see Tests) has a freezing point

(2.2.18) not lower than 53 °C.

Chemical & Reagents:

Ether (Peroxide free)

Nitric acid

Procedure:

Mix 5 g of sample with 50 ml of ether, 20 ml of dilute nitric acid, and 20 ml of water in a

round bottom flask. Connect the flask to a reflux condenser, and heat under reflux until

dissolution is complete. Allow to cool, and transfer the contents of the flask to a separator.

Shake, allow the layers to separate, and transfer the aqueous layers to a flask. Extract the

ether layers with two 4 ml portions of water, and add these aqueous extract to the main

aqueous extract. Wash the aqueous extract with 15 ml of ether; collect the aqueous extract

in a 50 ml volumetric flask, dilute with water to volume and mix (Solution S).

B. By Acid Value

Acid value of the fatty acids (2.5.1) is 195 to 210, determined on 0.200 g of the residue

obtained in the preparation of solution S dissolved in 25 ml of the prescribed mixture of

solvents.

C. By gas chromatography

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Carry out the procedure as described in the test for ―Fatty acid composition‖.

The retention time of the peaks corresponding to stearic acid and palmitic acid in the

chromatogram of the test solution should correspond to those in the chromatogram of the

system suitability solution / reference solution, as obtained in the test for ―Fatty acid

composition‖.

D. Magnesium test:


Concentrated Ammonia (13.5M)

Ammonium chloride

Disodium hydrogen orthophosphate (Dodecahydrate)

Sample preparation: Use solution S as such.

Procedure:

To 1 ml of sample preparation add 1 ml of dilute ammonia, mix. To this add 1 ml of

ammonium chloride solution, mix. Further add 1 ml of disodium hydrogen orthophosphate

solution, mix.

Observation:

On addition of dilute ammonia a white precipitate should form that should dissolve on

addition of ammonium chloride solution.

On addition of disodium hydrogen orthophosphate, a white crystalline precipitate should

form.

ACIDITY OR ALKALINITY:

Chemicals & Reagents:

Sodium hydroxide

Hydrochloric acid (35% w/w)

Bromothymol blue

Alcohol

Water

Procedure:

Transfer 1 g to a 100-ml beaker, add 20 ml of carbon dioxide-free water, boil on a steam

bath for 1 minute with continuous shaking, cool, and filter. Add 0.05 ml of bromothymol

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blue TS to 10 ml of the filtrate. Not more than 0.5 ml of 0.01 N hydrochloric acid or 0.01 N

sodium hydroxide is required to change the color of the indicator.

CHLORIDE:

Instruments & Apparatus:

Nessler cylinder / Colour comparison tube


Nitric acid

Silver nitrate

Sodium chloride

Water

Sample preparation:

Dilute 0.5 mL of solution S to 15 mL with water and mix.

PROCEDURE:

Transfer 15 mL of each of the sample preparation and chloride standard solution to two

separate test tubes and add 1 mL of dilute nitric acid to each of them. Pour the mixture as a

single addition into two separate Nessler cylinders containing 1 mL of silver nitrate

solution, mix. Allow to stand for 5 min protected from light. Examine both sample

preparation and standard preparation laterally against a black background. Any opalescence

in the sample preparation should not be more intense than that in the chloride standard

solution.

SULPHATES:




Glacial acetic acid

Barium chloride

Dipotassium sulphate

Ethanol

Distilled Water

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Preparation of Standard solutions:

Sulphate standard solution R1 (10 ppm SO4):

Dissolve 0.181 g of dipotassium sulphate in 100 mL ethanol (30%), mix. Dilute 1 mL of

this solution to 100 mL with ethanol (30%), mix. Prepare this solution immediately before

use.

Sulphate standard solution (10 ppm SO4):

Dissolve 0.181 g of dipotassium sulphate in 100 mL water, mix. Dilute 1 mL of this

solution to 100 mL with water and mix. Prepare this solution immediately before use.

Sample preparation:

Dilute 0.3 mL of the solution S to 15 mL with water and mix.

Procedure:

Add 3 mL of barium chloride solution to 4.5 mL of sulphate standard solution R1. Shake

and allow standing for 1 min. Take 2.5 mL of this solution in two different Nessler

cylinders. To one Nessler cylinder add 15 mL of sulphate standard solution and to the other

add 15 mL of sample preparation. Add 0.5 mL of acetic acid to each of the Nessler

cylinder. Mix. Allow to stand for 5 min.

Observation:

Any opalescence in the sample preparation should not be more intense than that in sulphate

standard solution (10 ppm SO4).

CADMIUM:

Instruments & Appatatus:

Atomic absorption spectrometry


Hydrochloric acid

Nitric acid

Preparation of solution:

Test solution:

Place 50.0 mg of the substance to be examined in a polytetrafluoroethylene digestion

bomb and add 0.5 ml of a mixture of 1 volume of hydrochloric acid and 5 volumes of

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cadmium- and lead-free nitric acid . Allow to digest at 170 °C for 5 h. Allow to cool.

Dissolve the residue in water and dilute to 5.0 ml with the same solvent.

Reference solutions:

Prepare the reference solutions using cadmium standard solution (10 ppm Cd), diluted as

necessary with a 1 per cent V/V solution of hydrochloric acid.

Procedure:

Add to at least 3 similar volumetric flasks equal volumes of the solution of the substance to

be examined (test solution) prepared as prescribed. Add to all but one of the flasks

progressively larger volumes of a reference solution containing a known concentration of

the element to be determined to produce a series of solutions containing increasing

concentrations of that element known to give responses in the linear part of the curve.

Dilute the contents of each flask to volume with solvent.

Introduce each of the solutions into the instrument at least 3 times and record the steady

reading. Rinse the apparatus with solvent each time and ascertain that the reading returns to

its initial blank value.

If a furnace is being used, it is fired between readings.

Calculate the linear equation of the graph using least-squares fit, and derive from it the

concentration of the element to be determined in the test solution.

Alternatively, plot on a graph the mean of readings against the added quantity of the

element to be determined.

Extrapolate the line joining the points on the graph until it meets the concentration axis.

The distance between this point and the intersection of the axes represents the


If a solid sampling technique is required, full details of the procedure to be followed are

provided in the monograph.

Measure the absorbance at 228.8 nm, using a cadmium hollow-cathode lamp as a source of

radiation and a graphite furnace as atomic generator.

LEAD:



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Hydrochloric acid

Nitric acid


Test solution:






Prepare the reference solutions using lead standard solution (10 ppm Pb) R, diluted as

necessary with water R.

Procedure:














element to be determined. Extrapolate the line joining the points on the graph until it meets

the concentration axis. The distance between this point and the intersection of the axes

represents the concentration of the element to be determined in the test solution.



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Measure the absorbance at 283.3 nm, using a lead hollow-cathode lamp as a source of

radiation and a graphite furnace as atomic generator, depending on the apparatus the line at

217.0 nm may be used.

NICKEL:




Hydrochloric acid

Nitric acid


Test solution:






Prepare the reference solutions using nickel standard solution (10 ppm Ni) R, diluted as

necessary with water R.

Procedure:











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element to be determined. Extrapolate the line joining the points on the graph until it meets

the concentration axis. The distance between this point and the intersection of the axes

represents the concentration of the element to be determined in the test solution.



Measure the absorbance at 232.0 nm, using a nickel hollow-cathode lamp as a source of

radiation and a graphite furnace as atomic generator.

LOSS ON DRYING:

Instrumentats & Apparatus:

Oven

Desiccator

Stoppered glass bottle

Procedure:

Weigh accurately about 1.0 g of the sample and transfer it to a stoppered weighing bottle of

weight (W1) previously dried at 100°-105°C for 30 min. Note down the weight of the

stoppered weighing bottle with the sample (W2). Distribute the sample evenly by sidewise

shaking to a height of 5 mm. Place the weighing bottle uncovered in an oven at 105°C till a

constant weight is obtained. (Note: Keep the stopper of the bottle by the side of the bottle

in the oven.)

Once constant weight is achieved, open the oven and immediately place the stopper on the

weighing bottle.

Place the stoppered weighing bottle in a desiccator to reduce to room temperature.

Determine the weight of weighing bottle with the contents. Note down the weight

(W3).Calculate loss on drying with the following formula.

Calculation:

(W2 - W3)

Loss on Drying (%w/w) = ---------------- X 100

(W2 - W1)

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Where,

W1 = Weight of the empty stoppered weighing bottle

W2 = Weight of stoppered weighing bottle with sample

W3 = Weight of stoppered weighing bottle with sample, after

drying.

MICROBIAL CONTAMINATION:

Instruments:

Weighing Balance

Autoclave

Hot air oven

pH meter, Incubator

Refrigerator Water

Sampling bottle

Reagents & Media:

Phosphate buffer pH 7.2

IPA

Soybean casein digest medium/Nutrient broth/Fluid lactose medium

Pre-treatment of the sample:

Suspend 10 g or dilute 10 ml of the preparation being examined, unless otherwise

specified, in sterile buffered 0.9% sodium chloride- 0.1% peptone solution pH 7/ Fluid

lactose medium/ Soya bean casein digest medium/ Fluid casein digest soya lecithin

polysorbate 20 medium and adjust the volume to 100 ml with the same medium. A suitable

surface active agent such as 0.1% w/v of the polysorbate 80 may be added to assist the

suspension of the poorly wettable substance.

Total Aerobic Microbial Count:

Dissolve 10 gm of the sample in 90 ml of sterile buffered 0.9% sodium chloride/

0.1%peptone solution pH 7 / Fluid lactose medium/ Soya bean casein digest medium/ Fluid

casein digest soya lecithin polysorbate 20 medium. For liquid sample take 10 ml of sample

and dissolve in 90 ml of sterile buffered 0.9% sodium chloride/0.1%peptone solution pH 7

/ Fluid lactose medium/ Soya bean casein digest medium/ Fluid casein digest soya lecithin

polysorbate 20 medium.

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For viscous sample that cannot be pipetted easily at its initial dilution, dilute the sample

until so that it can be pipetted. Dilute the sample so that the 1ml sample expected to yield

30-300 CFU/plate.

Mix and take required sample perform as per requirement by following method as below:

Pour plate method:

(a) Aseptically add 1 ml of sample in sterile Petri plate in duplicate. Pour 15-20 ml of

molten Soyabean casein digest agar medium or any other suitable for bacteria and

Sabouraud dextrose agar with antibiotic for fungi at not more than 45°C. This will serve as

a product control. Gently swirl the plate for uniform mixing of content. Allow the plates to

solidify at room temp.

(b) Aseptically add 15-20 ml of Soyabean casein digest agar medium or any other suitable

and Sabouraud dextrose agar with chloramphenicol in sterile Petri plate this will serve as a

negative control. Allow the plates to solidify at room temperature.

(c) Incubate the SCDA plates for bacteria in inverted position at 30°C to 35°C for 4 days

and Sabouraud dextrose agar plate with antibiotics for fungi at 20°C to 25°C for 5 days.

(d) Following the incubation, examine the plates for growth, count the number of colonies,

and express the results as number of colony forming unit (cfu).If no microbial growth is

observed express the result representing 1:10 dilution ―as less than 10 microorganism per g

or per ml‖. No growth should be observed in negative control.

Test for Escherichia coli:

Enrichment: Incubate the same testtube used in total viable aerobic count at 30- 350C for

18-48 hours. Examine the medium for growth (turbidity in medium).

Primary test:

Add 1.0 ml of the enrichment culture to a tube containing 5 ml of MacConkey broth

containing Durham‘s tube. Incubate this at 35 - 37 ºC for 48 hrs. If the contents of the tube

show gas and acid carry out the secondary test.

Secondary test:

Add 1 ml of the tubes containing (a) 5 ml MacConkey broth, and (b) 5 ml of peptone water.

Incubate in water bath or incubator for 35 ºC - 37ºC for 24 hrs. Examine the tube (a) for

acid and gas, for indole. To test for indole add 0.5 ml of Kovac‘s reagent, shake well, and

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allow to stand for one minute. Examine the tube for characteristic growth as per Table-1. If

no characteristic growth observed Escherichia Coli is absent in the sample.

Alternative test :

Take loop full of culture from enrichment medium and streak on MacConkey agar Plate.

Incubate the plate at 35-37 ºC. After incubation observe the plates, Examine the tube for

characteristic growth as per Table-1, If no characteristic growth observed the Escherichia

Coli is absent in the sample.

Take loopfull of culture from enrichment medium and streak on Levin eosin methylene

blue agar plate. Incubate the plate at35 - 37 ºC. After incubation observe the plates,

Examine the plate for characteristic growth as per Table-1, If no characteristic growth

observed Escherichia Coli is absent in the sample.

Test for Salmonella

Primary test :

Add 1.0 ml of the enrichment culture to each of the two tubes containing (a) 10ml of

selenite F broth (b) tetrathionate-bile brilliant green broth. Incubate at 35-37 ºC for 48

hours. From each of these two cultures, streak on at least two of the following media

Bismuth sulphite agar, Brilliant green agar, Deoxycholate citrate agar, Xylose-lysine-

deoxycholate. Incubate the plates at 35-37 ºC for 18-24 hours. Examine the medium for

characteristic growth as per Table- 1, If any colonies conforming to description in Table 2

carry out the secondary test

Secondary test :

Take a loopfull of culture showing the characteristics given in table 1 on triple sugar iron

agar by streaking on the surface of the slant and stab culture with the same inoculating

needle. At the same time inoculate a tube of urea broth. Incubate at 35-37 ºC for 18 to 24

hours. Examine the medium for characteristic growth as Table-1, If no characteristic

growth observed, the Salmonella is absent in the sample.

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ASSAY

Magnesium


Autotitrator


Ammonium chloride

Ammonia (13.5 M)

Xylenol orange

Sodium edetate

Sodium chloride

Hydrochloric acid (35 % w/w)

Butanol

Ethanol

Sodium hydroxide

Mordant black 11

Procedure:

Transfer about 500 mg of sample, accurately weighed, to a 250 mL conical flask. Add 50

mL of a mixture of butanol and ethanol (1:1 v/v), 5 mL of ammonia, 3 mL of ammonium

chloride buffer solution (pH 10.0), 30 mL of 0.1 M sodium edetate and 15 mg of mordant

black 11 triturate and mix. Heat to 45°C-50°C until the solution is clear and titrate with 0.1

M zinc sulfate. Perform a blank determination, and make any necessary correction.

End-point: Blue to violet

Calculation:

Factor:

Each mL of 0.1 M sodium edetate is equivalent to 2.431 mg of Mg.

Assay TR X M1 X 2.431 X 100 X 100

(As Mg, %w/w on dried basis) = ------------------------------------------------

W X 0.1 X (100 – LOD)

Where,

TR = Difference in volume of 0.1 M zinc sulfate required for the

sample titration and blank titration, in mL.

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M1 = Actual molarity of zinc sulphate.

W = Weight of the sample taken, in mg.

LOD = Loss on drying for the sample, in %w/w.

Stearic acid and Palmitic acid (BY GC):


The Gas Chromatograph equipped with flame-ionization detector and Headspace sampler.


n-Heptane

Sodium chloride

14 % Boron trifluoride solution

Preparation of solutions:

Blank solution:

n-Heptane is used as blank solution.

System suitability solution /reference solution:

Transfer about 50 mg each of Stearic Acid reference standard / working standard / certified

material and Palmitic Acid reference standard / working standard / certified material to a

small conical flask fitted with a suitable reflux condenser.

Add 5 ml of a solution prepared by dissolving 14 g of boron trifluoride in methanol to

make 100 ml, swirl to mix, and reflux for 10 min until the solids have dissolved. Add 4 ml

of chromatographic n-heptane through the condenser, and reflux for 10 min. Cool, add 20

ml of saturated sodium chloride solution, shake, and allow the layers to separate. Pass the

n-heptane layer through 0.1 g of anhydrous sodium sulfate (previously washed with

chromatographic n-heptane) into a suitable flask. Transfer 1 ml of this solution to a 10 ml

volumetric flask, dilute with chromatographic n-heptane to volume, and mix.

Sample solution:

Transfer about 100 mg of sample, accurately weighed, to a small conical flask fitted with a

suitable reflux condenser, and proceed as directed for System suitability solution, beginning

with "Add 5.0 ml of a solution prepared by dissolving."

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Chromatographic Condition Instrument : GC , Auto sampler

Column : G16 (DB wax), 30 m X 0.32 mm ID, film thickness

0.5 µm (Fused silica analytical column coated with

polyethylene glycol, average molecular weight

15000)

Detector : FID

Carrier gas : Helium

Carrier velocity : 50 cm / sec. (as per USP)

: 2.4 ml /min (As per Ph.Eur.)

Column temp.

: Hold at 70°C for 2 min then raise @ 5°C / min

up to 240°C hold for 5 min.

Equilibration time : 0.5 min

Injector temp. : 220°C

Detector temp : 260°C (FID)

Split mode : Splitless

Detector Attenuation : 1

Auto sampler parameters

Cycle : GC injection

Syringe : 10µL

Sample volume : 1.0 µL

Air volume : 2.0 µL

Pre cleaning solvent 1 : 4

Pre cleaning solvent 2 : 2

Pre cleaning sample 1 : 2

Fill volume : 5.0 µL

Fill speed : 5 µl /s

Fill strokes : 5

Injection to : GC inj 1

Injection speed : 100µl /s

Pre injection delay : 0 ms

Post injection delay : 4s

Post cleaning solvent 1 : 4

Post cleaning solvent 2 : 2

Evaluation of system suitability:

Inject 1 µL of a blank solution and record the chromatogram.

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Inject 1 µL of system suitability preparation/ reference solution (six replicates) using the

GC parameters and record the chromatogram. It should comply with the system suitability

criteria as mentioned.

The relative retention times are about 0.86 for methyl palmitate and 1.0 for methyl Stearate.

The resolution, R, between the methyl palmitate and methyl stearate peaks is not less than 5.

The relative standard deviation of the peak area responses for the palmitate and stearate

peaks for

replicate injections is not greater than 6.0 %

The relative standard deviation of the peak area response ratio of the palmitate to stearate

peaks from these replicate injections is not more than 1.0 %.

Procedure:

Inject 1 µL of sample preparation in duplicate and run the chromatograph. Record the

chromatograms and measure the peak responses.

Calculations:

Stearic acid in the fatty acid fraction of magnesium stearate = ASS / ATS X 100

Palmitic acid in the fatty acid fraction of magnesium stearate = ASP / ATS X 100

Where,

ASS = Average area of the stearate peak obtained in the chromatogram of

sample preparation.

ASP = Average area of the palmitate peak obtained in the chromatogram

of sample preparation.

ATS = The sum of the area(s) of all the fatty acid ester peaks in the

chromatogram of sample preparation.

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SPECIFICATION OF

CROSCARMELLOSE SODIUM



1.0 Description White or greyish-white powder.

2.0 Solubility Practically insoluble in acetone, in

anhydrous ethanol and in toluene.

3.0 Identification:

A. By absorptive test

B. Colour test

C. Sodium test

Croscarmellose sodium should absorb the

methylene blue and settle as a blue, fibrous

mass.

A reddish-violet color should develop at

the interface.

A dense white precipitate should form.

4.0 pH (1% w/v suspension in

water)

5.0 to 7.0

5.0 Sodium Chloride and Sodium

Glycolate

The sum of the percentages of sodium

chloride and sodium glycolate should not

be more than 0.5% (dreid substance )

6.0 Water-soluble substances Not more than 10%w/w

7.0 Heavy Metals Not more than 10 ppm

8.0 Loss on drying Not more than 10% w/w

9.0 Sulphated Ash Between 14.0% and 28.0 % w/w,


Total aerobic microbial count:

Total combined molds and

yeast:

Escherichia coli:

Not more than 1000 cfu/g.

Not more than 100 cfu/g.

Should be absent per gm

11. Settling volume 10ml to 30 ml

12. Degree on substitution 0.60 to 0.85 (on dried basis)

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METHOD OF ANALYSIS OF

CROSCARMELLOSE SODIUM


DESCRIPTION:


Specification: White or greyish-white powder.

SOLUBILITY

Practically insoluble in acetone, in anhydrous ethanol and in toluene.

IDENTIFICATION

A. By Absorption test:


Methylene blue

Procedure:

Weigh accurately 1 g of sample and mix with 100 ml of methylene blue solution (4 ppm).

Stir the mixture and allow it to settle.

Observation:

The sample should absorb the methylene blue and settle as a blue, fibrous mass.

B. Color test:


Sulfuric acid

Methanol

α-naphthol

Sample preparation:

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Dissolve 1 gram of sample in 50 ml water.

Procedure:

Transfer 1 ml of sample preparation to a test tube. Add 1 ml of water and 0.05 ml of α-

naphthol. Incline the test tube and carefully add 2 ml of sulfuric acid down the side so that

it forms a lower layer.A reddish-violet color should develop at the interface.

C. Reaction of Sodium:


Potassium pyrroantimonate

Potassium carbonate

water

Procedure:

Dissolve 0.1 g of the substance to be examined in 2 ml of water. Add 2 ml of a 150 g/l

solution of potassium carbonate and heat to boiling. No precipitate is formed. Add 4 ml of

potassium pyrroantimonate solution and heat to boiling. Allow to cool in iced water and if

necessary rub the inside of the test-tube with a glass rod. A dense white precipitate is

formed.

pH:

Instruments& Apparatus:

pH meter

Sample preparation:

Shake 1 g with 100 ml of carbon dioxide-free water for 5 min.

Procedure:

Transfer the sample preparation to a 50 ml beaker and adjust the temperature to 20ºC to

25°C. Dip the electrode of the pH meter in the sample and operate the instrument as per

current SOP. Read the indicated pH value.

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SODIUM CHLORIDE AND SODIUM GLYCOLATE:

Sodium chloride:


Potentiometer

Silver-based indicator electrode

Double- junction reference electrode


Silver nitrate

Hydrogen peroxide

Nitric acid

Potassium nitrate

Methanol

Eosin Y

Procedure:

Weigh accurately and transfer about 5 g of sample to a 250 ml beaker.

Add 50 ml of water and 5 ml of 30 % w/v hydrogen peroxide, heat on a steam bath for 20

min, stirring occasionally to ensure hydration. Cool, add 100 ml of water and add 10 ml of

nitric acid and titrate with 0.05 M silver nitrate VS, determining the endpoint

potentiometrically, using a suitable silver-based indicator electrode and a double junction

reference electrode, containing a 10 % potassium nitrate in the outer jacket and a standard

filling solution in the inner jacket and stirring constantly.

Calculation:

TR X N X 2.922

Sodium chloride content (% w/w) = ----------------------------

(100 – LOD) X W

Where,

N = Actual normality of silver nitrate.

W = Weight of the sample taken, in g.

TR = Volume of 0.05 M silver nitrate, in ml.

2.922 = Equivalence factor for NaCl.

Sodium Glycolate:


UV spectrophotometer

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Sulfuric acid

Glacial acetic acid

Acetone

Sodium chloride

Glycolic acid

2,7-dihydroxynaphthalene

Standard stock solution:

Weigh accurately and transfer about 100 mg of glycolic acid, previously dried in a

dessicator at room temperature overnight, to a 100 ml volumetric flask. Dissolve in and

dilute with water to volume, mix.

Standard preparation (1):

Transfer 1 ml standard stock solution to a 100 ml volumetric flask. Add 5 ml of water and5

ml of glacial acetic acid, mix. Dilute to volume with acetone, mix.


Transfer 2 ml standard stock solution to a 100 ml volumetric flask. Add 5 ml of water and

5 ml of glacial acetic acid, mix. Dilute to volume with acetone, mix.







Blank:

Transfer 5 ml each of glacial acetic acid and water to a 100 ml volumetric flask. Dilute to

volume with acetone and mix.

Sample preparation:

Weigh accurately and transfer about 500 mg of sample to a 100 ml beaker. Moisten

thoroughly with 5 ml of glacial acetic acid, followed by 5 ml of water, and stir with a glass

rod to ensure proper hydration (usually about 15 min). Slowly add 50 ml of acetone while

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stirring, and then add 1 g of sodium chloride and stir for several min to ensure complete

precipitation of the carboxymethylcellulose. Filter through a soft, open-textured paper,

previously wetted with a small amount of acetone, and collect the filtrate in a 100 ml

volumetric flask. Use an additional 30 ml of acetone to facilitate the transfer of the solids

and to wash the filter cake, then dilute with acetone to volume, and mix.

Procedure:

Transfer 2 ml of each blank, sample preparation, standard preparation (1), (2), (3) and (4)

to separate 25 ml volumetric flasks. Place the uncovered flasks in a boiling water bath for

20 min, accurately timed, to remove the acetone, remove from the bath and cool. Add to

each flask add 5 ml of 2,7-dihydroxynaphthalene TS, mix, add an additional 15 ml, and

again mix. Cover the mouth of each flask with a small piece of aluminum foil. Place the

flasks upright in a boiling water bath for 20 min, then remove from the bath, cool, dilute

with sulfuric acid to volume, and mix.

Operate the UV visible spectrophotometer as per current SOP. Determine the absorbance

of each solution at 540 nm against blank. Prepare a standard curve using the absorbances

obtained from the standard preparation (1), (2), (3) and (4).

Calculation:

10 X 1.29 X w

Sodium glycolate content (% w/w) = ----------------------------

(100 – LOD) X W

Where,

w = Weight of glycolic acid in the sample, obtained from the

standard curve and absorbance of sample, in mg.

W = Weight of sample taken, in g.

LOD = Loss on drying of sample, in % w/w.

12.9 = Factor for converting glycolic acid to sodium glycolate.

Total (%w/w) = Sodium chloride content (%w/w) + Sodium glycolate content (% w/w)

WATER-SOLUBLE SUBSTANCES:


Weighing Balance

Centrifuge

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Vaccume Pump

Hot Plate

Oven

Desiccator

Procedure:

Weigh accurately about 10 g of the sample (W2) and disperse in 800 ml of water,

accurately measured and stir for 1 min every 10 min during the first 30 min. Allow to stand

for an additional one hour and centrifuge, if necessary. Decant about 200 ml of the aqueous

slurry onto a rapid-filtering filter paper in a vacuum filtration funnel, apply vacuum, and

collect about 150 ml of the filtrate. Pour the filtrate into a previously weighed 250 ml

beaker (W) and calculate the weight, in g, of the filtrate (W3), by difference. Concentrate

on a hot plate to a small volume, but not to dryness. Dry at 105 º C for 4 h, cool in a

dessicator and weigh. Calculate the weight in g, of residue (W1), by difference.

Calculations:

Wt of Residue in g X 800 X 100

Water - soluble materials (% w/w) = ----------------------------------------------

Wt of sample in g X 150

HEAVY METALS:



Crucible (Platinum, Quartz, Porcelain or Silica)

Water bath


Strong hydrogen peroxide solution

Sulphuric acid

Phenolphthalein


Ammonia (13.5 M)

Glacial acetic acid

Sodium hydroxide

Thioacetamide

Glycerol (85%)

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Preparation of Solutions:

Lead standard solution (0.1 %):

Dissolve 0.400 g of lead (II) nitrate in sufficient water to produce 250 ml. Mix.

Lead standard solution (100 ppm):

Dilute 1 volume of lead standard solution (0.1%) to 10 volumes with water immediately

before use. Mix.

Standard lead solution (10 ppm):

Dilute 1 volume of lead standard solution (100 ppm) to 10 volumes with water immediately

before use. Mix.

Lead Standard Solution (1 ppm Pb)

Dilute 1 volume of lead standard solution (10 ppm Pb) to 10 volumes with water

immediately before use.

Sample preparation:

To the residue obtained in the determination of the sulphated ash add 1 ml of hydrochloric

acid and evaporate on a water-bath. Take up the residue in 20 ml of water.

12 ml of the prescribed aqueous solution of the substance to be examined.

Standard preparation:

Prepare as described for the sample preparation, using 2 ml of lead standard solution (1

ppm Pb) instead of the sample to be examined.

Monitor preparation:

Prepare as described for the sample preparation, adding to the sample to be examined 2 ml

of lead standard solution.

Blank preparation:

Prepare as described for the sample preparation, omitting the sample to be examined.

Procedure:

To each of the tube containing sample preparation, standard preparation, monitor

preparation and blank preparation add 2 ml of buffer solution pH 3.5 and 1.2 ml of

thioacetamide reagent. Mix immediately and dilute to 50 ml with water, mix. Examine the

solutions vertically against a white background after 2 min.

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Observation:

Any brown colour in the sample preparation should not be more intense than that in

standard preparation.

Note: The test is invalid if the reference solution does not show a slight brown colour

compared to the blank solution or if the monitor solution is not comparable with the

reference solution.

If the result is difficult to judge, filter the solutions through a membrane filter (pore size 3

µm; without the prefilter). Carry out the filtration slowly and uniformly, applying moderate

and constant pressure to the piston. Compare the spots on the filters obtained with the

different solutions.

LOSS ON DRYING:


Oven

Desiccator

Weighing Balance

Procedure:

Weigh accurately about 1 g of the sample and transfer it to a stoppered weighing bottle of

weight (W1) previously dried at 100°C- 105°C for 30 min. Note down the weight of the

stoppered weighing bottle with the sample (W2). Distribute the sample evenly by sidewise

shaking to a height of 5 mm. Place the weighing bottle uncovered in an oven at 100°C-

105°C for 6 h. (Note: Keep the stopper of the bottle by the side of the bottle in the oven.)

After 6 h open the oven and immediately place the stopper on the weighing bottle.


Determine the weight of weighing bottle with the contents. Note down the weight (W3).

Calculate loss on drying with the following formula.

Calculation:

(W2 - W3)

Loss on Drying = ---------------- X 100

(% w/w) (W2 - W1)

Where,

W1 = Weight of the empty stoppered weighing bottle.

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W2 = Weight of stoppered weighing bottle with sample.

W3 = Weight of stoppered weighing bottle with sample, after drying.

SULPHATED ASH:


Muffle Furnace

Desiccator

Crucible (Silica/ Platinum/ Porcelain or Quartz)


Sulfuric acid

Procedure:

Ignite a crucible at 600 ± 50°C for 30 min, allow to cool in a desiccator for 15 min and

weigh as (W1). Place about 1 g of sample into crucible and weigh (W2). Moisten the sample

with small amount (usually 1 ml) equal mixture of sulphuric acid and water. Heat, gently at

first at a temperature as low as practicable until substance is thoroughly charred on a

electric bunsen, cool, then moisten the residue with small equal mixture of sulphuric acid

and water (usually 1 ml), heat gently until white fumes are no longer evolved and transfer it

into Muffle furnace, and ignite at 600 ± 50°C until the residue is completely incinerated.

Cool it in a desiccator, weigh the crucible (W3) and calculate the weight of residue. If the

amount of residue so obtained exceeds the limit specified, continue moistening the residue

with equal amount of mixture of sulphuric acid and water , heating and ignition until two

consecutive weighing do not differ by more than 0.5 mg per g of the test sample taken.

Calculation:

(W3 - W1)

Sulphated ash (%w/w) = ----------------- X 100

(W2 - W1)

Where,

W1 = Weight of the empty crucible.

W2 = Weight of crucible + test sample.

W3 = Weight of the crucible + residue.

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Microbial contamination:


Weighing Balance

Autoclave

Hot air oven

pH meter, Incubator

Refrigerator Water

Sampling bottle

Reagents & Media:


IPA









Total aerobic microbial count:






polysorbate 20 medium.

For viscous sample that cannot be pipetted easily at its initial dilution, dilute the sample

until so that it can be pipetted. Dilute the sample so that the 1ml sample expected to yield

30 -300 cfu/plate.


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Pour plate method:


















Primary test




Secondary test :






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Alternative test:


Incubate the plate at 35-37 ºC.

After incubation observe the plates, Examine the tube for characteristic growth as per

Table-1, If no characteristic growth observed the Escherichia Coli is absent in the

sample.Take loopfull of culture from enrichment medium and streak on Levin eosin

methylene blue agar plate. Incubate the plate at35 - 37 ºC. After incubation observe the

plates, Examine the plate for characteristic growth as per Table-1, If no characteristic

growth observed Escherichia Coli is absent in the sample.

SETTLING VOLUME

10ml to 30 ml

DEGREE ON SUSTITUTION

0.60 to 0.85 (on dried basis)

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SPECIFICATION OF MICROCRYSTALLINE CELLULOSE (PH 112)



1.0 Description White or almost white, fine or granular powder.

2.0 Solubility Practically insoluble in water, in acetone, in

anhydrous ethanol, in toluene, in dilute acids and

in a 50 g/l solution of sodium hydroxide.

3.0 Identification

A. Colour test:

B. Degree of polymerization:

A. Should comply

B. Not more than 350

4.0 Solubility

(In Ammoniacal Solution of

Copper Tetrammine):

Should comply

5.0 pH: 5.0 to 7.5

6.0 Conductivity Not more than 75 µS/cm

7.0 Ether-soluble substances Not more than 0.05 %

8.0 Water-soluble substances Not more than 0.25 %

9.0 Heavy metals Not more than 10 ppm

10.0 Loss on drying Not more than 3.0 %

11.0 Sulphated ash Not more than 0.1 %


Total viable aerobic count:

Fungi:

Escherichia coli:

Salmonella:

Pseudomonas aeruginosa :

Staphylococcus aureus:

NMT 103 cfu/g.

NMT 102 cfu/g.

Should be absent

Should be absent

Should be absent

Should be absent

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METHOD OF ANALYSIS OF MICROCRYSTALLINE CELLULOSE (PH 112)


DESCRIPTION:


Specification: White or almost white, fine or granular powder.

SOLUBILITY:

Practically insoluble in water, in acetone, in anhydrous ethanol, in toluene, in dilute acids

and in a 50 g/l solution of sodium hydroxide.

IDENTIFICATION

A. Colour test:


Iodine

Zinc chloride

Procedure:

Place about 10 mg of sample on a watch glass, and disperse in 2 ml of iodinated zinc

chloride solution. Observe for the colour change. The sample takes on a violet-blue color.

B. Degree of polymerization:

Instrument & apparatus

Viscometer


Cupriethylenediamine hydroxide solution

Procedure:

Transfer about 1.3 g of microcrystalline cellulose, to a 125 ml conical flask. Add 25 ml of

water and 25 ml of cupriethylenediamine hydroxide solution. Immediately purge the solution

with nitrogen, insert the stopper, and shake until completely dissolved. Transfer an

appropriate volume of solution to a suitable capillary viscometer. Equilibrate the solution at

25 ± 0.1°C for atleast 5 min. Record the flow time (t1), in sec between the 2 marks on the

viscometer. Calculate the kinematic viscosity, (v1) of the solution using the following

formula:

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Kinematic viscosity, (v1) = t1 (k1)

Where,

k1 = Viscometer constant

t1 = Time required to flow between the two marks, in sec.

Dilute a suitable volume of cupriethylenediamine hydroxide solution with an equal volume of

water and measure the flow time (t2) using a suitable capillary viscometer. Calculate the kinematic

viscosity (v2) of the solvent using the following formula.

Kinematic viscosity, (v2) = t2 (k2)

Where,

k2 = Viscometer constant

t2 = Time required to flow between the two marks, in sec.

Determine the relative viscosity, rel, of the Microcrystalline Cellulose using the following

formula:

Relative viscosity rel = (v1) / (v2)

Where,

v1 = Kinematic viscosity of microcrystalline cellulose solution

v2 = Kinematic viscosity of 0.5 M cupriethylenediamine hydroxide solution

Determine the intrinsic viscosity, []c, by interpolation, using the Intrinsic Viscosity Table in the

Reference Tables section (Ph.Eur.– 5.7). Calculate the degree of polymerization, P, using the

formula:

Degree of polymerization (P) = (95) []c / m [(100 – b) / 100]

Where,

m = The mass, in g, of the substance to be examined.

b = Loss on drying as %w/w.

Solubility (In Ammoniacal Solution of Copper Tetrammine):


Copper (II) Sulphate

Sodium hydroxide

Concentrated ammonia (13.5 M)

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Ammoniacal solution of copper tetrammine

Procedure:

To about 50 mg of sample, add 10 ml of ammoniacal solution of copper tetrammine. Shake

vigorously for 1 min and place in a constant temperature device, maintained at a

temperature of 25.0°C ± 0.5°C for 15 min. If the substance is not completely dissolved,

repeat the shaking for 1 min and place the tube in the constant temperature device for 15

min. It dissolves completely, leaving no residue.

pH:


pH meter

Balance

Sample preparation:

Shake about 5 g of sample with 40 ml of water for 20 min, and centrifuge. Use the

supernatant solution for pH determination.

Procedure:

Transfer the sample preparation into a 50 ml clean and dry beaker. Dip the glass electrode in the

sample preparation. Ensure that the bulb of the electrode is completely dipping in the sample

preparation. Operate the instrument as per current SOP and note the pH of the sample

preparation.

Conductivity:


Conductivity meter

Balance

Procedure:

Accurately weigh about 5 g of sample and shake with 40 ml of water for 20 min, and

centrifuge. Operate the instrument as per current SOP. Measure the conductivity of the

supernatant after a stable reading is obtained, and measure the conductivity of the water

used to prepare the test solution.

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The conductivity of the supernatant does not exceed the conductivity of the water by more

than 75 S cm-1

.

Ether soluble substances:


Oven

Desiccator

Chromatographic column


Ether (Peroxide-free)

Procedure:

Prepare a chromatographic column, having internal diameter of about 20 mm, by placing a

plug of non-adsorbent cotton at the bottom of the column. Accurately weigh about 10 g of

sample (W) and transfer it to a chromatographic column. Pass 50 ml of peroxide-free ether

through the column. Collect the eluate into the clean and dry evaporating dish, which is

previously weighed as (W1). Evaporate the eluate with the aid of a current of air in a fume

hood. After all the ether has evaporated, dry the residue at 105°C for 30 min, cool in a

desiccator, and weigh as (W2). Carry out the blank determination using 50 ml of peroxide

free ether. Evaporate the ether with the aid of a current of air in a fume hood. After all the

ether has evaporated, dry the residue at 105°C for 30 min, cool in a desiccator, and weigh as

(W3).

Calculations:

Ether soluble substances (X, mg / 10 g) = (W2 – W1) S (W3– W1) B

X

Ether soluble substances (%w/w) = ------ X 100

W

Where,

X = Ether soluble substances, in mg / 10 g.

W = Weight of sample taken, in mg.

(W2 – W1) S = Weight of the residue of the sample, in mg.

(W3 – W1) B = Weight of the residue of the blank, in mg.

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WATER SOLUBLE SUBSTANCES:


Vacuum pump

Oven

Desiccator

Filter paper

Procedure:

Accurately weigh about 5 g of the sample (W) and shake with about 80 ml of water for 10

min. Filter through, a filter paper with the aid of vacuum into a tared flask (W1). Evaporate

the filtrate to dryness without charring, and dry at 105°C for 1 h. Note down the weight of

the flask with the residue as (W2).

Carry out the blank determination using 80 ml of water. Evaporate the water to dryness and

dry at 105°C for 1 h. Note down the weight of the flask with residue as (W3).

Calculations:

Water soluble substances (X, mg / 5 g) = (W2 – W1) S (W3 – W1) B

X

Water soluble substances (%w/w) = ------ X 100

W

Where,

X = Water soluble substances, in mg / 5 g

W = Weight of sample taken, in mg

(W2 – W1) S = Weight of the residue of the sample, in mg.

(W3 – W1) B = Weight of the residue of the blank, in mg.

HEAVY METALS:


Muffle furnace




Magnesium Sulphate

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Sulphuric acid

Phenolphthalein


Ammonia 13.5 M

Glacial acetic acid

Sodium hydroxide

Thioacetamide

Glycerol (85%)





before use. Mix.



before use. Mix.

Sample solution:

In a silica crucible, mix thoroughly 2 g of the substance to be examined with 4 ml of

magnesium sulphate solution, mix using a fine glass rod. Heat cautiously. Evaporate to

dryness on a water bath. Progressively heat to ignition and continue heating until an almost

white or at most greyish residue is obtained. Carry out the ignition at a temperature not

exceeding 800°C. allow to cool.

Moisten the residue with a few drops of dilute sulphuric acid. Evaporate, ignite again and

allow to cool. The total period of ignition must not exceed 2 h. Take up the residue in 2

quantities, each of 5 ml, of dilute hydrochloric acid. Add 0.1 ml of phenolphthalein

solution and then concentrated ammonia until a pink colour is obtained. Cool, add glacial

acetic acid until the solution is decolorized and add 0.5 ml in excess. Filter if necessary and

wash the filter. Dilute to 20 ml with water.

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Reference solution:

Prepare as described for the sample solution, using 2 ml of lead standard solution (10 ppm

Pb), instead of the substance to be examined. To 10 ml of the solution obtained add 2 ml of

the sample solution.

Monitor solution:

Prepare as described for the sample solution, adding to the substance to be examined 2 ml

of lead standard solution (10 ppm Pb). To 10 ml of the solution obtained add 2 ml of the

sample solution.

Blank solution:

Prepare a mixture of 10 ml of water and 2 ml of the sample solution.

Procedure:

To 12 ml of each solution, add 2 ml of buffer solution pH 3.5. Mix. Add 1.2 ml of

thioacetamide reagent. Mix immediately. Examine the solutions after 2 min.

Observation:

The substance to be examined complies with the test if any brown colour in the test

solution is not more intense than that in the reference solution.


µm; without the pre-filter). Carry out the filtration slowly and uniformly, applying

moderate and constant pressure to the piston. Compare the spots on the filters obtained

with the different solutions.

LOSS ON DRYING:


Oven

Desiccator

Stoppered weighing bottle

Procedure:

Weigh accurately about 1.0 g of the sample and transfer it to a stoppered weighing bottle of

weight (W1) previously dried at 105°C for 30 min. Note down the weight of the stoppered

weighing bottle with the sample (W2). Distribute the sample evenly by sidewise shaking to

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a height of 5 mm. Place the weighing bottle uncovered in an oven at 105°C for 3 h. (Note:

Keep the stopper of the bottle by the side of the bottle in the oven.)

After 3 h, open the oven and immediately place the stopper on the weighing bottle.


Determine the weight of weighing bottle with the contents. Note down the weight

(W3).Calculate loss on drying with the following formula.

Calculation:

(W2 - W3)

Loss on Drying (%w/w) = ---------------- X 100

(W2 - W1)

Where,

W1 = Weight of the empty stoppered weighing bottle.

W2 = Weight of stoppered weighing bottle with sample.

W3 = Weight of stoppered weighing bottle with sample, after drying.

SULPHATED ASH:


Muffle furnace

Desiccator



Sulphuric acid (96% w/w)

Desiccant (Silica gel or other suitable desiccant)

Procedure:

Ignite a crucible at 600 ± 50°C for 30 min, allow to cool in a desiccator for 15 min and

weigh as (W1) Place about 1g of sample into crucible and weigh (W2). Moisten the sample

with small amount (usually 1 ml) of sulphuric acid. Heat gently, first at a temperature as

low as practicable until substance is thoroughly charred on a electric bunsen, cool, then

moisten the residue with small amount of sulphuric acid (usually 1 ml), heat gently until

white fumes are no longer evolved and transfer it into Muffle furnace, and ignite at 600 ±

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50°C until the residue is completely incinerated. Cool it in a desiccator, weigh the crucible

(W3) and calculate the weight of residue. If the amount of residue so obtained exceeds the

limit specified, continue moistening the residue with sulphuric acid, heating and ignition

until two consecutive weighing do not differ by more than 0.5 mg per g of the test sample

taken.

Calculation:

(W3 - W1)

Sulphated ash (%w/w) = ----------------- X 100

(W2 - W1)

Where,




MICROBIAL CONTAMINATION:

Instruments:

Weighing Balance

Autoclave

Hot air oven

pH meter, Incubator

Refrigerator Water

Sampling bottle

Reagents & Media:


IPA 70 %


Sabouraud chloramphenicol agar

MacConkey broth purple

Selenite F broth

Tetrathionate bilr brilliant green broth

Cetrimide agar

Vogel johnson agar

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Mannitol salt agar








Total Aerobic Microbial Count:






polysorbate 20 medium. For viscous sample that cannot be pipetted easily at its initial

dilution, dilute the sample until so that it can be pipetted. Dilute the sample so that the 1ml

sample expected to yield 30 -300 CFU/plate.


Pour plate method:











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Primary test:




Secondary test:






Alternative test:


Incubate the plate at 35-37 ºC. After incubation observe the plates, Examine the tube for

characteristic growth as per Table-1, If no characteristic growth observed the Escherichia

Coli is absent in the sample.Take loopfull of culture from enrichment medium and streak

on Levin eosin methylene blue agar plate. Incubate the plate at35 - 37 ºC. After incubation

observe the plates, Examine the plate for characteristic growth as per Table-1, If no

characteristic growth observed Escherichia Coli is absent in the sample.

Test for Salmonella:

Primary test:

Add 1.0 ml of the enrichment culture to each of the two tubes containing (a) 10ml of

selenite F broth (b) tetrathionate-bile brilliant green broth. Incubate at 35-37 ºC for 48

hours. From each of these two cultures, streak on at least two of the following media

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Bismuth sulphite agar, Brilliant green agar, Deoxycholate citrate agar, Xylose-lysine-

deoxycholate. Incubate the plates at 35-37 ºC for 18-24 hours. Examine the medium for

characteristic growth as per Table- 1, If any colonies conforming to description in Table 2

carry out the secondary test.

Secondary test:

Take a loopfull of culture showing the characteristics given in table 1 on triple sugar iron

agar by streaking on the surface of the slant and stab culture with the same inoculating

needle. At the same time inoculate a tube of urea broth. Incubate at 35-37 ºC for 18 to 24

hours.

Examine the medium for characteristic growth as Table-1, if no characteristic growth

observed, the Salmonella is absent in the sample.

Pseudomonas aeruginosa:

Primary test:

If growth is observed then streak the portion of the medium on the surface of sterile

Cetrimide agar medium. incubate the plate AT 35-37 ºc for 24 hrs., examine the plate for

characteristic growth as per table-1, if no characteristic growth observed pseudomonas

aeruginosa is absent in the sample, and if any colonies present carry out the secondary test.

Secondary test:

by means of an inoculating loop, subculture the suspected colonies from the Cetrimide agar

on the surface of each of pseudomonas agar for flourescin and pseudomonas agar for

pyocyanin medium plate, and incubate at 35-37 ºc FOR 18-72 hours. examine the medium

for characteristic growth as table-1, if no characteristic growth observed the pseudomonas

aeruginosa is absent in the sample, and if present perform the

Oxidase Test:

Smear suspected colonies on N, N-dimethyle-p-phenylenediamine dihydrochloride

strip/disk. If there is no development of a pink colour changing to purple colour within 10

seconds the Pseudomonas aeruginosa is absent in the sample.

Staphylococcus aureus:

Primary test:

For Staphylococcus aureus subculture by streaking a portion from the enrichment medium

on the surface of Vogel-Johnson Agar / Mannitol-Salt Agar/ Baird parker plates, and

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incubate at 35-370C for 18-72 hours. Examine the medium for characteristic growth as per

Table-1, if no characteristic growth observed the Staphylococcus aureus is absent in the

sample and if present carry out the secondary test.

Secondary test:

Coagulase Test: Transfer representative suspected colonies from the Agar surface of any of

the media used in Primary Test to tube that contains 0.5ml of Mammalian Plasma,

preferably rabbit or horse and incubate in a water bath at 370C, examine the tube at 3 hours

and subsequently at suitable intervals upto 24hours for coagulation. If no coagulation in

any degree is observed the Staphylococcus aureus is absent in the sample but if coagulation

occurs indicates presence of Staphylococcus aureus.

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SPECIFICATIONS OF COLLOIDAL ANHYDROUS SILICA



1.0 Description A light, fine, white or almost white,

amorphous powder, with a particle size of

about 15 nm.

2.0 Solubility Practically insoluble in water and in mineral

acids except hydrofluoric acid. It dissolves in

hot solutions of alkali hydroxides.

3.0 Identification Should comply

4.0 pH (1g. in 30 ml of water) Between 3.5 and 5.5

5.0 Chlorides Not more than 250 ppm

6.0 Heavy Metals Not more than 25 ppm

7.0 Loss on Ignition

Not more than 5.0 %

8.0 Assay

(As SiO2 on ignited basis)

Not less than 99.0 % and not more than 100.5

%

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METHOD OF ANALYSIS OF COLLOIDAL ANHYDROUS SILICA


DESCRIPTION:


Specification: A light, fine, white or almost white, amorphous powder, with a particle size

of about 15 nm.

SOLUBILITY:

Practically insoluble in water and in mineral acids except hydrofluoric acid. It dissolves in

hot solutions of alkali hydroxides.

IDENTIFICATION:

1. By IR


Weighing Balance

Hot Plate


Sodium fluoride

Sulphuric Acid

Procedure:

Mix 20 mg of the substance to be examined in a platinum crucible by means of a copper

wire with about 10 mg of sodium fluoride and a few drops of sulphuric acid to give thin

slurry. Cover the crucible with a thin, transparent plate of plastic under which a drop of

water is suspended and warm gently. Within a short time a white ring is rapidly formed

around the drop of water.

PH:


pH meter

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Sample preparation:

Shake 1.0 g of sample with 30 ml of carbon dioxide-free water.

Procedure:

Transfer the sample preparation to a 50 ml beaker and adjust the temperature to 20 ºC to

25°C. Dip the electrode of the pH meter in the sample and operate the instrument as per

current SOP. Read the indicated pH value.

CHLORIDES:


Nitric acid

Silver nitrate

Sodium chloride

Water

Chloride standard solution (5 ppm):

Dissolve 82.4 mg of sodium chloride in 100 mL water, mix. Dilute 1 mL of this solution to

100 mL with water. Prepare this solution immediately before use.

Sample preparation:

To 1.0 g of sample add a mixture of 20 ml of dilute nitric acid and 30 ml of water and heat

on a water-bath for 15 min, shaking frequently. Dilute to 50 ml with water if necessary,

filter and cool. 10 ml of the filtrate diluted to 15 ml with water.

Procedure:

Transfer 15 mL of each of the sample preparation and chloride standard solution to two

separate test tubes and add 1 mL of dilute nitric acid to each of them. Pour the mixture as a

single addition into two separate Nessler cylinders containing 1 mL of silver nitrate

solution, mix. Allow to stand for 5 min protected from light. Examine both sample

preparation and standard preparation laterally against a black background. Any opalescence

in the sample preparation should not be more intense than that in the chloride standard

solution.

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HEAVY METALS


Nessler cylinder

Platinum Crucible


Strong hydrogen peroxide solution

Sulphuric acid

Phenolphthalein

1M Hydrochloric acid

Dilute Ammonia

Glacial acetic acid

Sodium hydroxide

Thioacetamide

Glycerol

Preparation of Solutions:





before use and mix.

Standard lead solution (10 ppm):


before use and mix.

Lead Standard Solution (1 ppm Pb):

Dilute 1 volume of lead standard solution (10 ppm Pb) to 10 volumes with water

immediately before use.

Sample preparation:

Suspend 2.5 g of sample in sufficient water to produce semi-fluid slurry. Dry at 140 °C.

When the dried substance is white, break up the mass with a glass rod. Add 25 ml of 1 M

hydrochloric acid and boil gently for 5 min, stirring frequently with the glass rod.

Centrifuge for 20 min and filter the supernatant liquid through a membrane filter. To the

residue in the centrifuge tube add 3 ml of dilute hydrochloric acid and 9 ml of water and

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boil. Centrifuge for 20 min and filter the supernatant liquid through the same membrane

filter. Wash the residue with small quantities of water, combine the filtrates and washings

and dilute to 50 ml with water. To 20 ml of the solution add 50 mg of ascorbic acid and 1

ml of concentrated ammonia. Neutralise with dilute ammonia. Dilute to 25 ml with water.

12 ml of the prescribed aqueous solution of the substance to be examined.

Standard preparation:

Prepare as described for the sample preparation, using 2 ml of lead standard solution (1

ppm Pb) instead of the sample to be examined.

Monitor preparation:

Prepare as described for the sample preparation, adding to the sample to be examined 2 ml

of lead standard solution.

Blank preparation:

Prepare as described for the sample preparation, omitting the sample to be examined.

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Procedure:

To each of the tube containing sample preparation, standard preparation, monitor

preparation and blank preparation add 2 ml of buffer solution pH 3.5 and 1.2 ml of

thioacetamide reagent. Mix immediately and dilute to 50 ml with water, mix. Examine the

solutions vertically against a white background after 2 min. Any brown colour in the

sample preparation should not be more intense than that in standard preparation.

Note: The test is invalid if the reference solution does not show a slight brown colour

compared to the blank solution or if the monitor solution is not comparable with the

reference solution.


µm; without the prefilter). Carry out the filtration slowly and uniformly, applying moderate

and constant pressure to the piston. Compare the spots on the filters obtained with the

different solutions.

LOSS ON IGNITION:


Muffle Furnace

Desiccator

Crucible (Platinum)

Procedure:

Ignite a platinum crucible at 900 °C for 30 min, allow to cool in a desiccator for 15 min

and weigh as (W1). Place about 0.200 g of sample into crucible and weigh (W2). Transfer

it into Muffle furnace, and ignite at 900 °C for 2 h. Cool it in a desiccator, weigh the

crucible (W3) and calculate the weight of residue.

Calculation:

(W3 - W1)

Residue on ignition (%w/w) = ----------------- X 100

(W2 - W1)

Where,




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ASSAY:


Muffle Furnace

Desiccator

Crucible ( Platinum)

Hot Plate


Sulfuric acid

Alcohol

Hydrofluoric acid

Procedure:

To the residue obtained in the test for loss on ignition add 0.2 ml of sulphuric acid and

sufficient alcohol to moisten the residue completely. Add 6 ml of hydrofluoric acid and

evaporate to dryness on a hot-plate at 95 °C to 105 °C, taking care to avoid loss from

sputtering. Wash down the sides of the dish with 6 ml of hydrofluoric acid and evaporate to

dryness. Ignite at 900 °C, allow to cool in a desiccator and weigh.

The difference between the mass of the final residue and the mass of the residue obtained

in the test for loss on ignition gives the amount of SiO2 in the quantity of the substance to

be examined used.

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SPECIFICATIONS OF POLYSORBATE-80



1.0 Description Oily, yellowish or brownish-yellow, clear or

slightly opalescent liquid.

2.0 Solubility Dispersible in water, in anhydrous ethanol, in

ethyl acetate and in methanol, practically

insoluble in fatty oils and in liquid paraffin.

3.0 Relative density

About 1.10.

4.0 Viscosity

About 400 mPa·s at 25 °C.

5.0 Identification

By IR

Hydroxyl value (see Tests).

Saponification value

Composition of fatty acids.

Test for solution

Should be complies

Should be complies

Should be complies

Should be complies

Should be complies

6.0 Acid Value Not more than 2

7.0 Hydroxyl value 65 to 80

8.0 Peroxide value

Maximum 10.0.

9.0 Saponification value 45 to 55, determined on 4.0 g.

10.0 Composition of fatty acids

—myristic acid: maximum 5.0 per cent,

—palmitic acid: maximum 16.0 per cent,

—palmitoleic acid: maximum 8.0 per cent,

—stearic acid: maximum 6.0 per cent,

—oleic acid: minimum 58.0 per cent,

—linoleic acid: maximum 18.0 per cent,

—linolenic acid: maximum 4.0 per cent,

11.0 Ethylene oxide and dioxan Maximum 1 ppm

12.0 Heavy metals Maximum 10 ppm.

13.0 Water Maximum 3.0 per cent

14.0 Total ash Maximum 0.25 per cent

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METHOD OF ANALYSIS POLYSORBATE-80


DEFINITION

Mixture of partial esters of fatty acids, mainly Oleic acid (0799), with sorbitol and its

anhydrides ethoxylated with approximately 20 moles of ethylene oxide for each mole of

sorbitol and sorbitol anhydrides.

CHARACTERS

Appearance

Oily, yellowish or brownish-yellow, clear or slightly opalescent liquid.

Solubility

Dispersible in water, in anhydrous ethanol, in ethyl acetate and in methanol, practically

insoluble in fatty oils and in liquid paraffin.

Relative density

About 1.10.

Viscosity

About 400 mPa·s at 25 °C.

IDENTIFICATION

First identification: A, D.

Second identification: B, C, D, E.

A. Infrared absorption spectrophotometry (2.2.24).

Comparison:

Ph. Eur. reference spectrum of polysorbate 80.

B. Hydroxyl value (see Tests).

C. Saponification value (see Tests).

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D. Composition of fatty acids (see Tests).

E. Dissolve 0.1 g in 5 ml of methylene chloride R. Add 0.1 g of potassium thiocyanate R

and 0.1 g of cobalt nitrate R. Stir with a glass rod. The solution becomes blue.

TESTS

Acid value (2.5.1)

Maximum 2.0.

Dissolve 5.0 g in 50 ml of the prescribed mixture of solvents.

Hydroxyl value (2.5.3, Method A)

65 to 80.

Peroxide value

Maximum 10.0.

Introduce 10.0 g into a 100 ml beaker, dissolve with glacial acetic acid R and dilute to 20

ml with the same solvent. Add 1 ml of saturated potassium iodide solution R and allow to

stand for 1 min. Add 50 ml of carbon dioxide-free water R and a magnetic stirring bar.

Titrate with 0.01 M sodium thiosulphate, determining the end-point potentiometrically

(2.2.20). Carry out a blank titration.

Determine the peroxide value using the following expression:

n1 = volume of 0.01 M sodium thiosulphate required for the substance to be examined, in

millilitres,

n2 = volume of 0.01 M sodium thiosulphate required for the blank, in millilitres,

M = molarity of the sodium thiosulphate solution, in moles per litre,

m = mass of substance to be examined, in grams

Saponification value (2.5.6)

45 to 55, determined on 4.0 g.

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Use 30.0 ml of 0.5 M alcoholic potassium hydroxide, heat under reflux for 60 min and

add 50 ml of anhydrous ethanol R before carrying out the titration.

Composition of fatty acids

Gas chromatography (2.4.22, Method C). Use the mixture of calibrating substances in

Table 2.4.22.-3.

Column:

—material: fused silica,

—size: l = 30 m, Ø = 0.32 mm,

—stationary phase: macrogol 20 000 R (film thickness 0.5 µm).

Carrier gas helium for chromatography R.

Linear velocity 50 cm/s.

Temperature:

Detection Flame ionisation.

Injection 1 µl.

Composition of the fatty acid fraction of the substance:

—myristic acid: maximum 5.0 per cent,

—palmitic acid: maximum 16.0 per cent,

—palmitoleic acid: maximum 8.0 per cent,

—stearic acid: maximum 6.0 per cent,

—oleic acid: minimum 58.0 per cent,

—linoleic acid: maximum 18.0 per cent,

—linolenic acid: maximum 4.0 per cent,

Ethylene oxide and dioxan (2.4.25, Method A)

Maximum 1 ppm of ethylene oxide and maximum 10 ppm of dioxan.

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Heavy metals (2.4.8)

Maximum 10 ppm.

2.0 g complies with test C. Prepare the reference solution using 2 ml of lead standard

solution (10 ppm Pb) R.

Water (2.5.12)

Maximum 3.0 per cent, determined on 1.00 g.

Total ash (2.4.16)

Maximum 0.25 per cent, determined on 2.0 g.

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FINISHED PRODUCT TESTING SPECIFICATION

Product name :

XYZ

Generic name : Artemether + Lumefantrine Tablet

Composition : Each uncoated Table Contains:

Artemether 80 mg

Lumefantrine 480 mg

Excepient Q.S

S.

No.

Tests Specification References

1. Description White colour, round uncoated tablet, plain

on both sides.

In House

2. Identification By TLC: Positive for Artemether and Lumfantrine

In House

3. Average Weight 984.0 mg + 5% In House

4. Uniformity of Weight + 5% BP

5. Disintegration Time Not more than 15 minutes BP

6. Related Substances Individual impurity NMT 1.0 %

Total impurities NMT 2.0 %

In House

7. Assay

Artemether

Lumefantrine

Claim Limit

Each uncoated tablet contain

80 mg 90 % - 120%

72.0 mg -96.0 mg

480 mg 90% - 120%

432.0 mg to 576.0 mg.

In House

8. Microbiological Purity

Total bacterial count:

Total yeast and mould count:

Pathogens-

E. coli & Salmonellae

NMT: 1000 cfu/gm.

NMT: 100cfu/gm.

Should be absent/gm.

BP

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METHOD OF ANALYSIS FOR FINISHED PRODUCT

Artemether + Lumefantrine Tablet

Reference protocol: In House

DESCRIPTION:

White colour, round uncoated tablet, plain on both sides.

IDENTIFICATION:

Carry out Thin Layer Chromatography using silica gel GF254 as a coating substance and a

mixture of 75 volume of Toluene, 25 volume of methanol & 0.25 volume of Ammonia as

mobile phase. Apply separately to the plate 10 microlitre each of three solutions prepared

in methanol.

Test solution: Weigh tablet powder equivalent to 80mg of Artemether & 480mg of

Lumfantrine in 25ml flask & suspend in 20ml of methanol, sonicate for five minutes, make

up to 25ml with methanol, filter.

Standard solution: (1) Dissolve 20mg of Artemether RS in 25ml of methanol.

Standard solution: (2) Dissolve 100mg of Lumfantrine RS in 25ml of methanol.

Procedure: After removal of the plate, allow it to dry in air. Examine in UV light at

254nm.The spots obtained by standard solution no. (1) & (2) corresponds to the spots

obtained by test solution-Positive for Artemether & Lumfantrine

AVERAGE WEIGHT:

Weigh 20 tablets selected at random and calculate the average weight by following

equation:

Average Wt. = Wt. of 20 tablets

20

Limits: 984.0 mg + 5%

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UNIFORMITY OF WEIGHT:

Weigh accurately 20 tablets and calculate the average weight. Then weigh 20 tablets

individually. Find out the tablet having highest weight and the tablet having lowest weight

in the above 20 tablets.

Calculate the maximum & minimum deviation as follows.

(WH -A) x 100

Maximum deviation (+ve) = ——————-

A

(A - WL) x 100

Minimum deviation (-ve) = ———————

A

Where,

A: Average weight of tablets

WH: Highest weight of tablets

WL: Lowest weight of tablets

Limits: Not more than two of the individual weights deviate from average weight by more

than 5% and none deviate by more than 10%.

DISINTEGRATION TIME:

Introduce one tablet into each of six tubes of the rigid basket rack assembly supporting six

cylinders, and a disc to each tube. Suspend the assembly in the beaker containing water at

15°C to 25°C, and operate the apparatus for 3 minutes. Remove the assembly from the

liquid and examine the tubes. The tablets pass the test if all six have disintegrated. If the

tablets fail to comply because of adherence to the discs, repeat the test on a further six

tablets omitting the discs. The tablets comply with the test if all six have disintegrated.

Limits: Not more than 15 minutes.

RELATED SUBSTANCES: (By HPLC)

Procedure:

Carry out the method for High Performance Liquid Chromatography (HPLC), using the

following solutions:

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For Artemether and Lumfantrine:

Buffer solution - Dissolve 408 mg of potassium dihydrogen orthophosphate in 150 ml of

water; adjust pH 7.5 with 10 %w/v sodium hydroxide.

Mobile Phase- To the above prepared buffer solution mix 350 ml of acetonitrile. Filter and

degassed mixture

Diluent- Prepare a mixture of water and acetonitrile (70:30)

Standard Preparation-

Artemether: Transfer about 100 mg of Artemether WS, accurately weighed, to a 50 ml

volumetric flask, add 2 ml of water, sonicate for 30 minutes and dilute to volume with

diluent and mix.

Lumfantrine: Transfer about 20 mg of Lumfantrine WS, accurately weighed, to a 50 ml

volumetric flask, add 2 ml of water, sonicate for 30 minutes and dilute to volume with

diluent and mix.

Assay Preparation: Mix the contents of 20 capsules. Weigh powder equivalent to 20 mg

of Lumfantrine to a 50 ml volumetric flask. Add 2 ml of water sonicate for 30 minutes and

dilute to volume with diluent, mix and filter through whatman filter paper # 42.

Chromatographic System: The liquid chromatography is equipped with a 215 nm

detector and a C-8 (Column (Symmetry): 250mm x 4.6mm, 5µm, Make: Water‘s). The

flow rate is about 1.5 ml per minute.

Chromatograph the standard preparation, and record the peak responses as directed under

procedure; the relative standard deviation for replicate injection is not more than 2.0%.

Procedure:

Separately inject equal volumes (about 20l) of the Standard preparation and the Assay

preparation into the Chromatograph, record the chromatograms, and measure the areas for

all peaks, except to disregard the solvent peak. Calculate the % impurity contents in the test

sample using the following equations.

Maximum single impurity = Area of Max. Single impurity in sample solution x 100

Total Area of principal peaks in standard solution (A & B)

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Total Impurities = Area of total impurities in sample solution x 100

Total Area of principal peaks in standard solution (A & B)

Limits:

Individual impurities are not more than 1.0%.

Total impurities are not more than 2.0 %.

ASSAY:

A. FOR Artemether:

Standard Preparation: Dissolve 50 mg of Artemether RS in 50 ml of methanol, and

dilute 2 ml to 100 ml with methanol.

Test Preparation: Weigh and powdered 20 tablets. Weigh accurately powder equivalent to

50 mg of Artemether in a 50 ml volumetric flask & add 30 ml of methanol, shake for 15

minutes & make volume up to 50 ml with methanol, centrifuge & dilute 2 ml to 100 ml

with methanol.

Measure the absorbances of Standard & Test solutions maximum at about 292nm against

methanol as blank.

Calculations: Calculate the content of Artemether by the following equation.

Test Absorbance x Wt. of Std. x Potency x Avg. Wt.

Std. Absorbance x Wt. of Test x 100

Limits: 90.0 % to 110.0 % of Artemether per tablet.

B. FOR Lumfantrine:

Standard Preparation: Weigh & dissolve accurately 100 mg of Lumfantrine RS in 100 ml

with chloroform & mix. Take 20ml for titration in stopper flask.

Procedure: Weigh accurately powder sample equivalent to 20 mg of Lumfantrine in

stopper flask add 20ml chloroform & shake, add 10 ml of 1N sulphuric acid, add 0.1 ml of

dimethyl yellow indicator. Shake & titrate with 0.004M Sodium Lauryl Sulphate to

permanent pink colour in chloroform layer.

Repeat the same procedure for standard preparation using 20 ml of standard solution.

Calculations: Calculate the content of Lumfantrine by the following equation.

Volume used for test x Wt. of Std. x 20 x Potency x Avg. Wt.

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Volume used for std. x 100 x Wt. of test. x 100

Limits: 90.0 % to 110.0 % of Lumfantrine.

MICROBIOLOGICAL PURITY:

Limits:

1) Total bacterial count should not be more than 1000cfu/gm.

2) Total mould and yeast count should not be more than 100cfu/gm.

3) Pathogens- E. coli & Salmonellae should be absent/gm.

Method for bacterial count: Aseptically transfer 10gm of sample in 100 ml of sterile

normal saline tube. Allow the sample to disperse and mix well. Transfer 1 ml aliquot into

two sterile petriplates. Add 20 ml of sterile soyabean casein digest agar medium (cool up to

about 40°C) in both the petri-plates. Mix the contents of petriplates by rotating the plates

and allow medium to solidify. Incubate all the plates at 35° to 37°C for 3 days in inverted

position. Following incubation, count the no of colonies and calculate the total bacterial

count per gm as follows:

No of colonies observed x 100

10

Combined method for yeast & mould counts: Aseptically transfer 1 ml of above aliquot

into two sterile petriplates. Add 20 ml of sterile subourard dextrose agar medium (cool up

to 40°C) in both the petriplates. Mix the content of petri-plates by rotating the plate and

allow the medium to solidify. Incubate all the plates at 20°C to 25°C for 5 days in inverted

position. Following incubation, count the no of colonies and calculate the total fungal count

per gm as follows:

No of colonies observed x 100

10

Observation: After incubation period count the number of colonies from each plate and

find out average. Express the results as number of organisms per gm or ml by multiplying

average number of colonies with dilution factor.

Note: Carry out the positive and negative control simultaneously to confirm the validity of

test.

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Pathogens Testing:

Test for presence of E. coli:

Filter 40 ml of Solution A through a 0.45 µ membrane filter. Wash the filter paper with

100 ml of 0.1N NaOH solution and with 6x50 ml quantities 0.1% peptone solution with 1%

tween 80. Place the membrane filter in 50 ml Nutrient broth, and incubate at 37°C ± 1°C

for 24 hours.

Primary Test:

Add 1.0 ml of this enrichment culture to a tube containing 5 ml of MacConkey‘s broth with

Durham‘s tube. Incubate at 37°C ± 1°C for 48 hours. If the contents of the tube show acid

and gas, carry out the secondary test.

Secondary Test:

Add 0.1 ml of this content of the tube showing acid & gas to each of two tubes containing

(a) 5 ml of MacConkey‘s broth and (b) 5 ml of peptone water.

Incubate at 44°C ± 0.5°C for 24 hours & examine tube (a) for acid and gas and tube (b) for

indole.

To test for indole, add 0.5 ml of Kovac‘s reagent, shake well and allow to stand for one

minute, if a red colour is produced in the reagent layer, indole is present. The presence of

acid and gas and of indole in the secondary test indicates the presence of Escherichia coli.

Carry out a control test by repeating the primary and secondary tests using 0.1 ml of a 24-

hour-old broth culture of Escherichia coli. (NCTC 9002), for inoculation of tubes (a) and

(b). Test is invalid if the results do not indicate that control contains E. coli.

Test for presence of Salmonella:

Filter 40 ml of Solution A through a 0.45 µ membrane filter. Wash the filter paper with

100 ml of 0.1 N NaOH solution and with 6x50 ml quantities of 0.1% peptone solution with

1% tween 80. Place the membrane filter in 100 ml Nutrient broth and incubate at 37°C ±

1°C for 24 hours. Add one ml of enrichment culture to each of two tubes containing (a) 10

ml of Selenite F broth and (b) 10 ml of tetrathionate bile brilliant green broth and incubate

at 37° ± 1°C for 48 hours.

From each of these two cultures inoculate any two plates containing a layer of 1] Brilliant

green agar 2] Deoxycholate citrate agar and 3] Bismuth sulfite agar. Incubate the plates at

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37°C ± 1°C for 24 hours. If any colonies conforming to the description in the table I are

produced, carry out the secondary test.

Secondary Test:

Subculture any colonies showing the characteristics in Table I in triple sugar iron agar, by

first inoculating the surface of the slope and then making the stab culture with the same

loop and at the same time inoculate a tube of urea broth. Incubate at 37°C ± 1°C for 24

hours.

The formation of acid and gas in the stab culture (with or without concomitant blackening)

and the absence of acidity from the surface growth in the triple sugar iron agar, together

with absence of red colour in the urea broth, indicate the presence of Salmonella.

Carry out a control test by repeating the primary and secondary tests using 0.1 ml of a 24-

hour-old broth culture of Salmonella abony (NCTC 6017), for inoculation of tubes (a) and

(b). The test is invalid if the results do not indicate that control contains Salmonella.

Medium Description of Colony

Brilliant green agar Small transparent and colourless or opaque, pinkish

or white (frequently surrounded by pink or red zone)

Deoxycholate citrate agar Colourless and opaque, with or without black center.

Bismuth sulfite agar Black or green

Packing:

Blister strip of 6 tablets packed in a unit carton with leaflet.

Shelf life:

36 months from the month of manufacturing.

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CERTIFICATE OF ANALYSIS OF FINISHED PRODUCT

The Drugs & Cosmetics Act 1940 & the rules there under

Product Name XYZ

A.R.No. 0125/13

Generic Name Artemether + Lumefantrine Tablet

Reference No. STP.QC/STP/F/15

9-00

Batch No. GMH0125 Batch Size 1.0 lac Sample Taken on 20.04.2013

Mfg. Date 04.2013 Exp. Date 03.2016 Sample Qty. 40 tablet

Mfg. Lic. No. MNB/06/373/MB/06/374

S.

No.

Tests Specification Result


on both sides.

Complies


Complies

3. Average Weight 984.0 mg + 5% 989.47 mg

4. Uniformity of Weight + 5% Complies

5. Disintegration Time Not more than 15 minutes 3 min 5 sec



0.28%

1.49%

7. Assay

Artemether

Lumefantrine

Claim Limit


80 mg 90 % - 120%

72.0 mg -96.0 mg

480 mg 90% - 120%

432.0 mg to 576.0 mg.

98.76%

99.48%




Pathogens-


NMT: 1000 cfu/gm.

NMT: 100cfu/gm.


Complies

Opinion In the opinion of the undersigned, the sample referred to above is

of standard quality

Date

20.04.2013

Analyst Manger quality control

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Product Name XYZ

A.R.No. 0126/13



9-00



Mfg. Lic. No. MNB/06/373/MB/06/374

S.

No.



on both sides.

Complies


Complies






0.29%

1.48%

7. Assay

Artemether

Lumefantrine

Claim Limit


80 mg 90 % - 120%

72.0 mg -96.0 mg

480 mg 90% - 120%

432.0 mg to 576.0 mg.

98.77%

99.49%




Pathogens-


NMT: 1000 cfu/gm.

NMT: 100cfu/gm.


Complies


of standard quality

Date

21.04.2013


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Product Name XYZ

A.R.No. 0127/13



9-00



Mfg. Lic. No. MNB/06/373/MB/06/374

S.

No.



on both sides.

Complies


Complies






0.32%

1.54%

7. Assay

Artemether

Lumefantrine

Claim Limit


80 mg 90 % - 120%

72.0 mg -96.0 mg

480 mg 90% - 120%

432.0 mg to 576.0 mg.

98.79%

99.42%




Pathogens-


NMT: 1000 cfu/gm.

NMT: 100cfu/gm.


Complies


of standard quality

Date

22.04.2013


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In-process controls

IN-PROCESS CONTROL TEST AND DATA

A. In-process checks for mixed powder:

Uniformity of Mixing: By assay of active ingredients from different places.

B. In-process checks for lubricated granules:

Assay: ± 10% of the label claim

Moisture content: By IR moisture balance (NMT 2.0%)

C. In-process checks for uncoated tablets:

Draw samples at regular intervals during compression and check for:

Average weight: At regular intervals (984.0 mg + 5.0%)

Uniformity of weight: + 5.0% of average weight.

Hardness: At regular intervals (NLT 3 kg/cm2)

Friability: At regular intervals (Not more than 1%)

D. In-Process Control during Packing:

a. The material issue for packing is checked & the inspector puts his signature on the batch

card.

b. Batch coding & other over printing like Mfg., Exp. etc. is checked on strips & cartons

etc.

c. Blister packing machine temperature, pressure etc. are also checked.

d. Pocket cuts; smudged printing, faulty strip cutting, bridge rupture, missing tablet etc. are

checked.

e. Contents of the cartons, less number of strips, absence of pack inserts etc. is checked at

random on the packing line itself.

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SECTION-15

SHELF LIFE & STABILITY DATA

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SHELF LIFE AND STABILITY DATA

Shelf Life: 36 Months

Stability Data: Attached

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SECTION-16

TOXICOLOGICAL DATA

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Toxicology Data

General toxicity

The main changes observed in repeat-dose toxicity studies were associated with the

expected pharmacological action on erythrocytes, accompanied by responsive secondary

haematopoiesis.

Neurotoxicity

Studies in dogs and rats have shown that intramuscular injections of artemether resulted in

brain lesions. Changes observed mainly in brainstem nuclei included chromatolysis,

eosinophilic cytoplasmic granulation, spheroids, apoptosis and dark neurons. Lesions were

observed in rats dosed with artemether at 25 mg/kg for 7 or 14 days and dogs dosed at 20

mg/kg for 8 days or longer, but lesions were not observed after shorter courses of drug or

after oral dosing.

The estimated artemether 24 h AUC after 7 days of dosing at the no observed effect level

(10 mg/kg/day given intramuscularly) is approximately 7-fold greater than the estimated

artemether 24 h AUC in humans on day 1 of the standard 3-day oral treatment regimen;

oral exposure in humans decreases on subsequent days, thus the exposure margin increases.

Dogs dosed orally with 143 mg/kg artemether showed a statistically measureable effect on

the hearing threshold at 20 dB.

This dose is equivalent to about 29 times the highest artemether clinical dose (160 mg/day)

based on body surface area comparisons. Most nervous system disorder adverse events in

the studies of the 6-dose regimen were mild in intensity and resolved by the end of the

study.

Mutagenicity

No evidence of mutagenicity was detected in in vitro or in vivo tests with an artemether:

lumefantrine combination (consisting of 1 part artemether: 6 parts Lumefantrine). In the

micronucleus test myelotoxicity was seen at all dose levels (500, 1,000 and 2,000 mg/kg),

but recovery was almost complete 48 hours after dosing.

Carcinogenicity

Carcinogenicity studies with the artemether: lumefantrine combination was not conducted.

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Reproductive toxicity studies

Reproductive toxicity studies performed with the artemether: lumefantrine combination

caused maternal toxicity and increased post-implantation loss in rats and rabbits at doses

≥50 mg/kg/day (corresponding to approximately 7 mg/kg/day artemether) and 175

mg/kg/day (corresponding to 25 mg/kg/day artemether) respectively. These effects were

not observed at lower doses.

Lumefantrine alone caused no sign of reproductive or development toxicity at doses up to

1,000 mg/kg/day in rats and rabbits.

Embryotoxicity has been observed in rat and rabbit reproductive toxicity studies conducted

with artemether, a derivative of artemisinin. Artemisinin (e.g. artesunate) are known to be

embryotoxic.

Artemether caused increases in post-implantation loss and teratogenicity (characterised as a

low incidence of cardiovascular and skeletal malformations) in rats at 19.4 mg/kg, and in

rabbits at 30 mg/kg. Maternal toxicity was also observed in rabbits at 30 mg/kg/day. No

other adverse effects were observed at lower doses in rabbits. The no observed effect dose

was 3 mg/kg/day in rats and 25 mg/kg/day in rabbits.

The embryotoxic artemether dose, 20 mg/kg/day in the rat, yields artemether and dihydro

artemisinin exposures similar to those achieved in humans.

Artesunate, a structurally related compound, also caused increases in post-implantation loss

and teratogenicity (low incidence of cardiovascular and skeletal malformations) in rats at 6

mg/kg and in the lowest dose tested in the rabbits, 5 mg/kg/day.

Fertility

After artemether-Lumefantrine administration for 10 weeks in males and 2 weeks in

females, reduced fertility occurred at 1000 mg/kg/day where altered sperm motility,

abnormal sperm, reduced epididymal sperm count, increased testes weight, and

embryotoxicity and other reproductive effects (decreased implants and viable embryos,

increased preimplantation loss) were also observed. General toxicity was observed in males

and females at doses ≥ 300 mg/kg/day. The no adverse effect level for fertility was 300

mg/kg/day. The relevance to this finding in humans is unknown.

Juvenile toxicity studies

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A specific study to investigate the neurotoxicity of artemether in juvenile rats involved oral

administration of artemether during four different dosing intervals, at doses of 30 or 80

mg/kg/day on post partum days 7 to 13, and at doses of 30 or 120 mg/kg/day on post

partum days 14 to 21, 22 to 28, or 29 to 36. Mortality, clinical signs and reductions in body

weight parameters occurred most notably during the first two dosing intervals. Despite the

systemic toxicity noted, there were no effects of artemether on any of the functional tests

performed and there was no evidence of a direct neurotoxic effect of orally administered

artemether on the brain of juvenile rats.

Juvenile studies in the rat indicate that very young animals (aged 7-21 days) are more

sensitive to artemether than adult animals. There is no difference in sensitivity in slightly

older (3-5 weeks of age) animals following 13 weeks of artemether/Lumefantrine

administration. Consistent with the later data, clinical studies have established the safety of

artemether and Lumefantrine administration in patients weighing 5 kg and above.

Cardiovascular Safety Pharmacology

In toxicity studies in dogs at doses >600 mg/kg/day only, there was some evidence of

prolongation of the QTc interval (safety margin of 1.3-fold to 2.2-fold for artemether using

calculated free Cmax), at higher doses than intended for use in man. In an in vitro assay of

HERG channels stably expressed in HEK293 cells, Lumefantrine and the main metabolite

desbutyl-lumefantrine showed some inhibitory potential in one of the currents responsible

for cardiac repolarisation. The potency was lower than the other antimalarial drugs tested.

From the estimated IC50 values, the order of potency of HERG current block was

halofantrine (IC50 = 0.04 μM) >chloroquine (2.5 μM) >mefloquine 2.6 μM) >desbutyl-

lumefantrine (5.5 μM) >Lumefantrine (8.1 μM).

Additional studies were performed to evaluate the in vitro effects of artemether and its

active metabolite, dihydro artemisinin, on the HERG current. At concentrations that

produced significant inhibition, the safety margins for artemether and dihydro artemisinin

are greater than 100 if they are estimated using the total therapeutic concentration at Cmax

or greater than 1000 if they are estimated using the calculated free Cmax. Based on the

available non-clinical data, a potential for QTc prolongation in the human cannot be

discounted.

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SECTION-17

CLINICAL DATA

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CLINICAL DATA:

Clinical Efficacy of Artemether-Lumefantrine in Congolese Children with Acute

Uncomplicated Falciparum Malaria in Brazzaville.

The Republic of the Congo adopted artemisinin-based combination therapies (ACTs) in

2006: artesunate-amodiaquine and artemether-Lumefantrine as the first-line and second-

line drugs, respectively.

The baseline efficacy of artemether-Lumefantrine was evaluated between March and July

2006 in Brazzaville, the capital city of Congo. Seventy-seven children aged between 6

months and 10 years were enrolled in a nonrandomized study. The children were treated

under supervision with 6 doses of artemether-Lumefantrine and followed up for 28 days in

accordance with the 2003 World Health Organization guideline. Pretreatment (i.e., day 0)

and recrudescent Plasmodium falciparum isolates between day 14 and day 28 were

compared by the polymerase chain reaction to distinguish between true recrudescence and

reinfection. The overall cure rate on day 28 was 96.9% after PCR correction. Reported

adverse effects included pruritus and dizziness. Artemether-Lumefantrine was highly

efficacious in Brazzaville.

Approximately 30% of the Congolese population reside in Brazzaville, the capital city. The

epidemiology of malaria in the city of Brazzaville is heterogeneous. Depending on the

district, malaria transmission is low or intense. In general, malaria is meso- to hypoendemic

in the city centre and hyper endemic in the periphery. In terms of malaria burden, there are

twice as many malaria-infected patients consulting health centres in the periphery, as

compared with health centres in the city centre. Surveys conducted in the main hospital in

Brazzaville have shown that malaria is the first cause of admission in the department of

paediatrics, mostly in children aged less than 4 years old.

Due to the high levels of clinical resistance to chloroquine, amodiaquine, and

sulphadoxine-pyrimethamine, the Congolese Ministry of Public Health changed the

national antimalarial drug policy in 2006. Two artemisinin-based combination therapies

(ACTs) were adopted: artesunate-amodiaquine and artemether-Lumefantrine for the first-

line and second-line treatment of uncomplicated malaria, respectively. Before the drug

policy change, only a single clinical study on the efficacy of artesunate-amodiaquine and

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artemether-Lumefantrine had been conducted in a rural area in Congo. The present

nonrandomized study was conducted between March and July 2006 to provide the baseline

data of artemether-Lumefantrine efficacy in an urban area where the majority of the

Congolese population reside.

The study was conducted in Tenrikyo health centre located in Makélékélé district, which is

in the southern part of Brazzaville. The patients consulting the health centre reside in either

the neighboring district of Bacongo (low transmission city centre) or the district of

Makélékélé itself (high transmission peripheral area).

Febrile children aged between 6 months old and 10 years old were enrolled after a written

informed consent was obtained from the parents or the legal guardian. The inclusion

criteria were as follows: P. falciparummonoinfection with parasitaemia between 2,000 and

200,000 asexual parasites/μL of blood, axillary temperature >37.5°C, haematocrit >15%,

absence of concomitant febrile illness, and easy accessibility of the residence for home

visits. Febrile patients who received antimalarial drugs, mostly non-ACTs, prior to

consultation were also enrolled. Patients with signs and symptoms of severe malaria were

excluded.

Based on the recommendation of the drug manufacturer, the following numbers of

artemether-Lumefantrine (Coartem, Novartis Pharma) tablets were administered under

supervision, for a total of six doses: 1 tablet per dose for 5–14 kg body weight, 2 tablets per

dose for 15–24 kg body weight, 3 tablets per dose for 25–34 kg body weight, and 4 tablets

per dose for ≥35 kg body weight.

For small children, the tablets were crushed and mixed with milk before administration.

After the initial dose (on day 0), the patients were observed for one hour for possible

vomiting and were discharged. If the patient vomited during the observation period,

another dose of artemether-Lumefantrine was administered. If vomiting occurred again, the

patient was withdrawn from the study and treated with parenteral quinine.

The second dose (on day 0) was given 8 hours after the initial dose at home under

supervision. The patients returned to the health centre in the morning of day 1 and day 2 for

the third and fifth doses. The fourth (evening of day 1) and sixth (evening of day 2) doses

were given at home by the research team. The study protocol and written consent forms (in

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French, English, and local dialects) were reviewed and approved by the Congolese

Ministry of Health and WHO Secretariat Committee on Research Involving Human

Subjects (SCRIHS).

Finger prick capillary blood was collected to prepare Giemsa-stained thick blood films,

measure the packed cell volume in a capillary tube, and store parasite DNA on Whatman

3 MM filter paper. Parasite density was determined by counting the number of asexual

parasites against 200 leukocytes and expressed as the number of asexual parasites/μL of

blood, assuming a leukocyte density of 8,000 per μL. In case of hyperparasitaemia, the

parasite count was stopped after reaching 500 asexual parasites even if 200 leukocytes had

not been reached.

The patients were followed up for 28 days, according to the WHO protocol. The body

temperature was measured and clinical examination was performed during each visit (i.e.,

before each of the 6 doses for the first 3 days (day 0, day 1, and day 2), then on days 3, 7,

14, 21, and 28). Blood smears were examined during each visit on days 2, 3, 7, 14, 21, and

28. Body temperature and parasite density were measured on any other day during an

unscheduled visit if the patient was febrile during the 28-day period. Recrudescent

parasites between day 14 and day 28 were collected and stored on Whatman 3MM filter

paper.

Parasite DNA was extracted using QI AMp DNA blood mini kit (Qiagen GmbH, Hilden,

Germany) according to the manufacturer‘s instructions. Paired samples (day 0 and

recrudescent parasites on or after day 14) were genotyped by analysing the highly

polymorphic loci, the block 2 of merozoite surface protein-1 (msp-1), and the central

region of merozoite surface protein-2 (msp-2), as previously described. Samples from

patients responding with early treatment failure (i.e., on or before day 3) were not analysed

by PCR and were considered as recrudescent or persistent parasitaemia. Paired samples

were initially genotyped using msp-2 locus.

If different bands were found, the reappearance of parasites was considered to be due to

reinfection. If the msp-2 bands were similar, msp-1 locus was further compared in paired

samples.

Before PCR adjustment, clinical outcomes were classified as early treatment failure (ETF),

late clinical failure (LCF), late parasitological failure (LPF), and adequate clinical and

parasitological response (ACPR). ETF was defined as (i) the development of danger signs

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or severe malaria on day 1, day 2, or day 3 in the presence of parasitaemia, (ii) parasitaemia

on day 2 > initial parasitaemia on day 0, (iii) presence of parasitaemia on day 3 with fever,

or (iv) parasitaemia on day 3 ≥ 25% of initial parasitaemia on day 0. LCF was defined as

(i) the development of danger signs or severe malaria after day 3 in the presence of

parasitaemia or (ii) presence of parasitaemia and fever on any day between day 4 and day

28, without previously meeting any of the criteria of ETF. LPF was defined as the presence

of parasitaemia on day 28 in the absence of fever, without previously meeting any of the

criteria of ETF or LCF. ACPR was defined as the absence of parasitaemia on day 28, with

or without fever, without previously meeting any of the criteria of ETF, LCF, or LPF. PCR

allowed further classification of late failures (LCF and LPF) into true recrudescence

(persistence or reappearance of the same isolates as those present on day 0) and new

infection (appearance of a new isolate, absent on day 0).

Due to the lack of previous data on the efficacy of artemether-Lumefantrine in Brazzaville,

the minimum sample size was determined to be 50 patients. Clinical and parasitological

data were analysed using the pre-programmed Excel spreadsheet provided by the

Department of Global Malaria Programme, WHO (Geneva, Switzerland). Patients who

withdrew from the study and those lost to follow up during the 28-day period were

excluded from further analysis (per protocol analysis), and the proportions of ETF, LCF,

LPF, and ACPR were calculated.

The treatment failure rate was defined as the number of patients responding with ETF,

LCF, or LPF divided by the total number of included patients who completed the 28-day

follow-up. Statistical analysis was performed using Epi-info version 6.04 (Centres for

Disease Control and Prevention, Atlanta, GA).

From March to July 2006, there were 1,355 febrile patients consulting the Tenrikyo health

centre. Of 1,355 patients, 285 (21.0%) received antimalarial drugs before consultation,

mostly due to self-medication: chloroquine (115 patients), quinine (62), amodiaquine (32),

sulphadoxine-pyrimethamine (32), artemisinin derivatives (32), ACT (10), and halofantrine

(2). Of 1,355 febrile patients, 313 (23.1%) had positive thick smears and 204 (15.0%) were

aged <10 years old. Seventy-seven febrile patients aged ≤10 years old were eligible and

enrolled. Among these 77 eligible patients, 14 (18.2%) received an antimalarial drug (self-

medication) prior to enrollment. The geometric mean parasite density (95% confidence

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intervals (95% CI)) was 33,300 (28,200–38,500) asexual parasites/μL. The characteristics

of these 77 patients are summarized in Table 1.

Two patients with uncomplicated malaria associated with parasitaemia >200,000 asexual

parasites/μL were recruited after the approval of the medical staff. During the follow-up, 4

children were excluded (1 for repeated vomiting, 1 for the development of pneumonia, and

2 for protocol violation (1 received erythromycin and another self-medicated with an

antimalarial drug)) and 4 were lost to follow up.

Table 1: Baseline characteristics of patients before treatment with artemether-

Lumefantrine.

Number of patients 77

Age (years), mean ± SD (range) 4.5 ± 2.4 (8 months–10 years)

Number of patients <5 years old 42 (54.5%)

Number of patients aged 5–10 years old 35 (45.5%)

Weight (kg), mean ± SD (range) 15.9 ± 5.4 (8–28)

Sex ratio (F/M) 39/38 (1.03)

Number of patients who took an antimalarial drug

before inclusion 14 (18.2%)*

Body temperature, mean ± SD (range) 38.1 ± 0.8 (36.0–40.3)**

Parasite density (asexual parasites/L)

Overall geometric mean (range), (95% CI) 33,300 (2,450–381,000),

[28,200–38,500]

Number of patients with parasite density > 200,000

asexual parasites/μL 2 (2.8%)

Geometric mean (range), (95% CI) in patients <5

years old

35,000 (3,480–213,000)

[14,100–55,200]

Geometric mean (range) (95% CI) in patients 5–10 33,700 (2,450–381,000)

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years old [8,990–58,400]

Haematocrit (%) mean (range), (95% CI) 32.6 (20–40) [31.6–33.8]

SD: standard deviation; 95% CI: 95% confidence intervals; *eight patients received

chloroquine; 6 received quinine; **two patients had fever within 24 hr before consultation

but were no longer febrile at the time of consultation.

The therapeutic efficacy of artemether-Lumefantrine is summarized in Table 2. After PCR

adjustment, 62 of 64 patients (96.9% (95% CI, 89.2–99.6%)) responded with ACPR on day

28. If reinfection () is considered as ACPR, 67 of 69 patients (97.1% (95% CI, 89.9–

99.6%)) had ACPR on day 28.

Fever and parasite clearance was rapid. The mean (±SD) body temperatures were °C on

day 0 (before treatment), °C on day 1 (24 hr after the first dose), °C on day 2, and °C on

day 3. On day 2, 5 patients were still febrile and only 3 had positive smears at low

parasitaemia (53–161 asexual parasites/μL). On day 3, 3 patients were still febrile and none

had a positive blood smear.

None of the patients, including 3 patients presenting gametocytaemia on day 0, had

gametocytaemia between day 2 and day 28.

One patient had an aggravation of signs and symptoms with repeated vomiting and asthenia

despite a decrease of parasitaemia from 119,380 asexual parasites/μL on day 0 (axillary

temperature, 40.3°C) to 50,000 asexual parasites/μL on day 1 (axillary temperature, 38°C).

This clinical outcome was considered as ETF, and the child was referred to the district

hospital for parenteral treatment with quinine on day 2, according to the national guidelines

for the management of severe and complicated malaria.

The following adverse effects were reported by the patients aged >5 years old themselves

or parents between day 0 and day 7: asthenia (30%), diarrhea (18%), abdominal pain

(12%), vomiting (12%), headache (12%), skin rash (9%), dizziness (3%), and anorexia

(3%). On day 3, 3% of patients reported skin rash, abdominal pain, diarrhoea, and vomiting

and 6% reported asthenia. From day 3 to day 7, 3% of patients had diarrhoea and asthenia.

None of these adverse effects was reported beyond day 7. No severe adverse effect was

observed during the 28-day follow up period.

http://www.hindawi.com/journals/mrt/2012/749479/tab2/

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This nonrandomized study on artemether-Lumefantrine efficacy is the first trial conducted

in Brazzaville. The results of the present study demonstrated its high efficacy and are in

agreement with other studies conducted in African countries. Its high efficacy was, in

particular, in agreement with the results reported from countries sharing common borders

with Congo, that is, Angola and Cameroon.

Elsewhere in Sub-Saharan African countries, the cure rate (i.e., the proportion of ACPR)

on day 28 after artemether-Lumefantrine treatment was reported to be >95.5% (a single

study in Malawi showed a cure rate >93%). Moreover, in our study, most reported adverse

effects were mild and were difficult to attribute to malaria infection itself or to drug intake,

with the exception of skin rash and dizziness.

Artemether-Lumefantrine paediatric formulations (syrup, dispersible tablet) have become

available in more recent years. These formulations are much more convenient than tablets

that had to be crushed and mixed with milk to treat small children in the present study.

Moreover, for unsupervised treatment, these novel formulations are expected to increase

patient compliance and possibly improve drug tolerance in children, as compared with

crushed tablets.

At the time when the present study was conducted, artemether-Lumefantrine combination

was one of the most expensive antimalarial drugs sold in Congolese pharmacies (US$10

for 24 adult tablets). This is the main reason why this ACT was not used for self-

medication. Since 2008, artemether-Lumefantrine has been available free of charge in the

public sectors as second-line antimalarial drug, as in the health centre where the present

study was performed, and an increasing number of malaria-infected Congolese patients is

expected to be treated with this ACT.

The drug is still available at approximately the same cost as in 2006, that is, US$ 10 for 24

tablets, in private pharmacies. The dispersible paediatric formulation costs US$ 2 for 12

tablets. These costs are far above the goal of less than US$ 1 per treatment, considered to

be an affordable cost for the majority of African patients.

Further clinical studies comparing the efficacy of artesunate-amodiaquine and artemether-

Lumefantrine, which are current drugs of choice for the treatment of uncomplicated

falciparum malaria, in different epidemiological strata in Congo, including rural and urban

endemic areas, are required to monitor their efficacy in the country.

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Drug Interactions

Interaction with drugs that are known to prolong the QTc interval

Lemalar is contraindicated with concomitant use of drugs (they may cause prolonged QTc

interval and Torsade de Pointes) such as: antiarrhythmic of classes IA and III, narcoleptics

and antidepressant agents, certain antibiotics including some agents of the following

classes: macrolides, fluoroquinolones, imidazole, and triazole antifungal agents, certain

non-sedating antihistaminic (terfenadine, astemizole), cisapride, flecainide.

Interaction with drugs metabolized by CYP2D6

Lumefantrine was found to inhibit CYP2D6 in vitro. This may be of particular clinical

relevance for compounds with a low therapeutic index. Co-administration of Lemalar with

drugs that are metabolised by this iso-enzyme is contraindicated (e.g. narcoleptics,

metoprolol, and tricyclic antidepressants such as imipramine, amitriptyline, clomipramine)

is contraindicated.

Interaction with strong inducers of CYP3A4 such as rifampin

Oral administration of rifampin (600 mg daily), a strong CYP3A4 inducer, with Lemalar

Tablets (6-dose regimen over 3 days) in six HIV-1 and tuberculosis confected adults

without malaria resulted in significant decreases in exposure to artemether (89%), DHA

(85%) and Lumefantrine (68%) when compared to exposure values after Lemalar alone.

Concomitant use of strong inducers of CYP3A4 such as rifampin, carbamazepine,

phenytoin, St. John's Wort is contraindicated with Lemalar.

Inducers should not be administered at least one month after Lemalar t administration,

unless critical to use as judged by the prescriber.

Concomitant use not recommended

Interaction with other antimalarial drugs

Data on safety and efficacy are limited, and Lemalar should therefore not be given

concurrently with other antimalarial unless there is no other treatment option.

If Lemalar is given following administration of mefloquine or quinine, close monitoring of

food intake (for mefloquine) or of the ECG (for quinine) is advised. The long elimination

half-life of Lumefantrine must be taken into account when administering quinine in

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patients previously treated with Lemalar. In patients previously treated with halofantrine,

Lemalar should not be administered earlier than one month after the last halofantrine dose.

Mefloquine

A drug interaction study with Lemalar in man involved administration of a 6-dose regimen

over 60 hours in healthy volunteers which was commenced at 12 hours after completion of

a 3-dose regimen of mefloquine or placebo. Plasma mefloquine concentrations from the

time of addition of Lemalar were not affected compared with a group which received

mefloquine followed by placebo.

Pre-treatment with mefloquine had no effect on plasma concentrations of artemether or the

artemether/dihydro artemisinin ratio but there was a significant reduction in plasma levels

of Lumefantrine, possibly due to lower absorption secondary to a mefloquine-induced

decrease in bile production. Patients should be encouraged to eat at dosing times to

compensate for the decrease in bioavailability.

Quinine

A drug interaction study in healthy male volunteers showed that the plasma concentrations

of Lumefantrine and quinine were not affected when i.v. quinine (10 mg/kg BW over 2

hours) was given sequentially 2 hours after the last (sixth) dose of Lemalar (so as to

produce concurrent plasma peak levels of Lumefantrine and quinine). Plasma

concentrations of artemether and dihydro artemisinin (DHA) appeared to be lower. In this

study, administration of Lemalar to 14 subjects had no effect on QTc interval. Infusion of

quinine alone in 14 other subjects caused a transient prolongation of QTc interval, which

was consistent with the known cardiotoxicity of quinine. This effect was slightly, but

significantly, greater when quinine was infused after Lemalar in 14 additional subjects. It

would thus appear that the inherent risk of QTc prolongation associated with i.v. quinine

was enhanced by prior administration of Lemalar.

Concomitant use requiring caution

Interactions affecting the use of Lemalar

Interaction with CYP3A4 inhibitors

Both artemether and Lumefantrine are metabolised predominantly by the cytochrome

enzyme CYP3A4, but do not inhibit this enzyme at therapeutic concentrations.

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Ketoconazole

The concurrent oral administration of ketoconazole with Lemalar led to a modest increase

(≤ 2-fold) in artemether, DHA, and Lumefantrine exposure in healthy adult subjects. This

increase in exposure to the antimalarial combination was not associated with increased side

effects or changes in electrocardiographic parameters. Based on this study, dose adjustment

of Lemalar is considered unnecessary in falciparum malaria patients when administered in

association with ketoconazole or other potent CYP3A4 inhibitors.

Lemalar should be used cautiously with drugs that inhibit CYP3A4 and are contraindicated

with drugs which additionally are known to prolong QTc (see Section 4.3

Contraindications), due to potential for increased concentrations of Lumefantrine which

could lead to QT prolongation.

Grapefruit juice

Administration of artemether with grapefruit juice in healthy adult subjects resulted in an

approximately two fold increase in systemic exposure to the parent drug. Grapefruit juice

should be used cautiously during Lemalar treatment.

Interaction with weak to moderate inducers of CYP3A4

When Lemalar is co-administered with moderate inducers of CYP3A4, it may result in

decreased concentrations of artemether and/or Lumefantrine and loss of antimalarial

efficacy.

Interaction with anti-retroviral drugs such as protease inhibitors and non-nucleoside reverse

transcriptase inhibitors

Both artemether and Lumefantrine are metabolized by CYP3A4. Anti-retroviral drugs

(ARTs), such as protease inhibitors and non-nucleoside reverse transcriptase inhibitors, are

known to have variable patterns of inhibition, induction or competition for CYP3A4.

Lemalar should be used cautiously in patients on ARTs since decreased artemether, DHA,

and/or Lumefantrine concentrations may result in a decrease of antimalarial efficacy of

Lemalar, and increased Lumefantrine concentrations may cause QT prolongation.

Lopinavir/ ritonavir

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In a clinical study in healthy volunteers, lopinavir/ritonavir decreased the systemic

exposures to artemether and DHA by approximately 40% but increased the exposure to

Lumefantrine by approximately 2.3- fold. Exposures to lopinavir/ritonavir were not

significantly affected by concomitant use of Lemalar.

Nevirapine

In a clinical study in HIV-infected adults, nevirapine significantly reduced the median

Cmax and AUC of artemether by approximately 61% and 72%, respectively and reduced

the median Cmax and AUC of dihydro artemisinin by approximately 45% and 37%,

respectively. Lumefantrine Cmax and AUC were non-significantly reduced by nevirapine.

Artemether/Lumefantrine reduced the median Cmax and AUC of nevirapine by

approximately 43% and 46% respectively.

Efavirenz

Efavirenz decreased the exposures to artemether, DHA, and Lumefantrine by

approximately 50%, 45%, and 20%, respectively. Exposures to efavirenz were not

significantly affected by concomitant use of Lemalar.

Interactions resulting in effects of Lemalar on other drugs

Interaction with drugs metabolized by CYP450 enzymes

When Lemalar is co-administered with substrates of CYP3A4 it may result in decreased

concentrations of the substrate and potential loss of substrate efficacy. Studies in humans

have demonstrated that artemisinin have some capacity to induce CYP3A4 and CYP2C19

and inhibit CYP2D6 and CYP1A2. Although the magnitude of the changes was generally

low it is possible that these effects could alter the therapeutic response of drugs that are

predominantly metabolised by these enzymes.

Interaction with hormonal contraceptives

In vitro, the metabolism of ethinyl estradiol and levonorgestrel was not induced by

artemether, DHA, or Lumefantrine. However, artemether has been reported to weakly

induce, in humans, the activity of CYP2C19, CYP2B6, and CYP3A. Therefore, Lemalar

may potentially reduce the effectiveness of hormonal contraceptives. Patients using oral,

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transdermal patch, or other systemic hormonal contraceptives should be advised to use an

additional no hormonal method of birth control for about one month.

Drug-food/drink interactions

Lemalar should be taken with food or drinks rich in fat such as milk as the absorption of

both artemether and Lumefantrine is increased.

Grapefruit juice should be used cautiously during Lemalar treatment.

ADVERSE REACTIONS

The safety of Lemalar has been evaluated in 20 clinical trials with more than 3500 patients.

A total of 1810 adults and adolescents above 12 years of age as well as 1788 infants and

children of 12 years of age and below have received in clinical trials.

Adverse reactions reported from clinical studies and post-marketing experience are listed

below according to system organ class.

Adverse reactions are ranked under headings of frequency using the MedDRA frequency

convention:

Very common (≥1/10)

Common (≥1/100 to <1/10)

Uncommon (≥1/1,000 to <1/100)

Rare (≥1/10,000 to <1/1,000)

Very rare (<1/10,000)

Not known (cannot be estimated from available data).

Table 1 Frequency of Undesirable effects

Adults and

adolescents above

12 years of age

Infants and children of 12 years of age

and below (incidence estimates)

Immune system disorders

Hypersensitivity Not known Rare

Metabolism and nutrition disorders

Decreased appetite Very common Very common (16.8 %)

Psychiatric disorders

Sleep disorders Very common Common (6.4 %)

Insomnia Common Uncommon

Nervous system disorders

Headache Very common Very common (17.1 %)

http://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=f1e72f44-561a-4905-ab29-15ef83eac928

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Dizziness Very common Common (5.5 %)

Paraesthesia Common --

Ataxia, Hypoaesthesia Uncommon --

Somnolence Uncommon Uncommon

Clonus Common Uncommon

Cardiac disorders

Palpitations Very common Common (1.8 %)

Electrocardiogram QT

prolonged

Common Common (5.3 %)

Respiratory, thoracic and mediastinal disorders

Cough Common Very common (22.7 %)

Gastrointestinal disorders

Vomiting Very common Very common (20.2 %)

Abdominal pain Very common Very common (12.1 %)

Nausea Very common Common (6.5 %)

Diarrhoea Common Common (8.4 %)

Hepatobiliary disorders

Liver function tests

increased

Uncommon Common (4.1 %)

Skin and subcutaneous tissue disorders

Rash Common Common (2.7 %)

Pruritus Common Uncommon

Urticaria Uncommon Uncommon

Angioedema* Not known Not known

Musculoskeletal and connective tissue disorders

Arthralgia Very common Common (2.1 %)

Myalgia Very common Common (2.2 %)

General disorders and administration site conditions

Asthenia Very common Common (5.2 %)

Fatigue Very common Common (9.2 %)

Gait disturbance Common --

*: These adverse reactions were reported during post-marketing experience. Because these

spontaneously reported events are from a population of uncertain size, it is difficult to

estimate their frequency.

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OVERDOSAGE

In cases of suspected overdosage symptomatic and supportive therapy should be given as

appropriate, which should include ECG and blood potassium monitoring.

DOSAGE AND ADMINISTRATION

Tablets for oral administration.

To increase absorption, Lemalar should be taken with food or a milky drink (see section

5.2). If patients are unable to tolerate food, Lemalar should be administered, but the

systemic exposure may be reduced. Patients who vomit within 1 hour of taking the

medication should repeat the dose.

For administration to small children and infants, the tablet/s may be crushed.

Adults and children weighing 35 kg and above

For patients 12 years of age and above and 35 kg body weight and above, a course of

treatment comprises six doses of four tablets i.e. total of 24 tablets, given over a period of

60 hours as follows: the first dose of four tablets, given at the time of initial diagnosis,

should be followed by five further doses of four tablets given at 8, 24, 36, 48 and 60 hours

thereafter.

Children and infants weighing 5 kg to less than 35 kg

A six-dose regimen is recommended with 1 to 3 tablets per dose, depending on

bodyweight:

5 to less than 15 kg bodyweight: the first dose of one tablet, given at the time of initial

diagnosis, should be followed by five further doses of one tablet given at 8, 24, 36, 48 and

60 hours thereafter.

15 to less than 25 kg bodyweight: the first dose of two tablets, given at the time of initial

diagnosis, should be followed by five further doses of two tablets given at 8, 24, 36, 48 and

60 hours thereafter.

25 to less than 35 kg bodyweight: the first dose of three tablets, given at the time of initial

diagnosis, should be followed by five further doses of three tablets given at 8, 24, 36, 48

and 60 hours thereafter.



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5.1 Pharmacodynamic properties.

Pharmacotherapeutic group: antimalarial, blood schizontocide, ATC code: P01 BF01.

Pharmacodynamic effects

Lemalar comprises a fixed ratio of 1:6 parts of artemether and Lumefantrine, respectively.

The site of antiparasitic action of both components is the food vacuole of the malarial

parasite, where they are thought to interfere with the conversion of haem, a toxic

intermediate produced during haemoglobin breakdown, to the nontoxic haemozoin, malaria

pigment. Lumefantrine is thought to interfere with the polymerisation process, while

artemether generates reactive metabolites as a result of the interaction between its peroxide

bridge and haem iron. Both artemether and Lumefantrine have a secondary action

involving inhibition of nucleic acid- and protein synthesis within the malarial parasite.

Treatment of Acute Uncomplicated P. falciparum Malaria

The efficacy of Lemalar Tablets was evaluated for the treatment of acute, uncomplicated

malaria (defined as symptomatic P. falciparum malaria without signs and symptoms of

severe malaria or evidence of vital organ dysfunction) in five 6-dose regimen studies and

one study comparing the 6-dose regimen with the 4-dose regimen. Baseline parasite density

ranged from 500/μL - 200,000/μL (0.01% to 4% parasitaemia) in the majority of patients.

Studies were conducted in otherwise healthy, partially immune or non-immune adults and

children (≥5kg body weight) with uncomplicated malaria in Thailand, sub-Saharan Africa,

Europe, and South America.

Efficacy endpoints consisted of:

• 28-day cure rate, proportion of patients with clearance of asexual parasites within 7 days

without recrudescence by day 28

• parasite clearance time (PCT), defined as time from first dose until first total and

continued disappearance of asexual parasite which continues for a further 48 hours

• fever clearance time (FCT), defined as time from first dose until the first time body

temperature fell below 37.5°C and remained below 37.5°C for at least a further 48 hours

(only for patients with temperature >37.5°C at baseline)

The modified intent to treat (mITT) population includes all patients with malaria diagnosis

confirmation who received at least one dose of study drug. Evaluable patients generally are

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all patients who had a day 7 and a day 28 parasitological assessment or experienced

treatment failure by day 28. The results are presented in the table below:

Table 2 Clinical efficacy results

Study No. Age Polymerase

chain reaction

(PCR)-corrected

28-day cure

rate1 n/N (%) in

evaluable

patients

Median FCT2

[25th

,

75th

percentile]

Median PCT2

[25th

,

75th

percentile]

Year/ Study

location

A0254 3-62 years 93/96 (96.9) n

3=59

35 hours [20, 46]

n=118

44 hours [22, 47]

1996-97

Thailand

A026 2-63 years 130/133 (97.7) n3=87

22 hours [19, 44]

NA 1997-98

Thailand

A028 12-71 years 148/154 (96.1) n3=76

29 hours [8, 51]

n=164

29 hours [18, 40]

1998-99

Thailand

A2401 16-66 years 119/124 (96.0) n3=100

37 hours [18, 44]

n=162

42 hours [34, 63]

2001-05

Europe,

Columbia

A2403 2 months-9

years

289/299 (96.7) n3=309

8 hours [8, 24]

n=310

24 hours [24, 36]

2002-03

3 countries in

Africa

B2303CT

3 months-12

years

403/419 (96.2) n3=323

8 hours [8, 23]

n=452

35 hours [24, 36]

2006-07

5 countries in

Africa

B2303DT

3 months-12

years

394/416 (94.7) n3=311

8 hours [8, 24]

n=446

34 hours [24, 36]

2006-07

5 countries in

Africa 1 Efficacy cure rate based on blood smear microscopy

2 mITT population

3 For patients who had a body temperature >37.5°C at baseline only

4Only the 6-dose regimen over 60 hours group data is presented

CT – Lemalar tablets administered as crushed tablets

DT – Lemalar Dispersible tablets

Lemalar is not indicated for, and has not been evaluated in, the treatment of malaria due

to P. vivax, P. malariae or P. ovale, although some patients in clinical studies had co-

infection with P. falciparum and P. vivax at baseline. Lemalar is active against blood stages

of Plasmodium vivax, but is not active against hypnozoites.

Paediatric population

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Two studies have been conducted

Study A2403 was conducted in Africa in 310 infants and children aged 2 months to 9

years, weighing 5 kg to 25 kg, with an axillary temperature ≥37.5°C. Results of 28-day

cure rate (PCR-corrected), median parasite clearance time (PCT), and fever clearance time

(FCT) are reported in table 3 below.

Study B2303 was conducted in Africa in 452 infants and children, aged 3 months to 12

years, weighing 5 kg to <35 kg, with fever (≥37.5°C axillary or ≥38°C rectally) or history

of fever in the preceding 24 hours. This study compared crushed tablets and dispersible

tablets. Results of 28-day cure rate (PCR-corrected), median parasite clearance time (PCT),

and fever clearance time (FCT) for crushed tablets are reported in table 3 below.

Table 3 Clinical efficacy by weight for pediatric studies

Study No.

Weight category

Median PCT1

[25th

, 75th

percentile]

PCR-corrected 28-day cure

rate2

n/N (%) in evaluable

patients

Study A2403

5 - <10 kg

10 - <15 kg

15 -25 kg

24 hours [24, 36]

35 hours [24, 36]

24 hours [24, 36]

145/149 (97.3)

103/107 (96.3)

41/43 (95.3)

Study B2303CT

5 - <10 kg

10 - <15 kg

15 -<25 kg

25-35 kg

36 hours [24, 36]

35 hours [24, 36]

35 hours [24, 36]

26 hours [24, 36]

65/69 (94.2)

174/179 (97.2)

134/140 (95.7)

30/31 (96.8)

1 mITT population

2 Efficacy cure rate based on blood smear microscopy

CT Lemalar tablets administered as crushed tablets

QT/QTc Prolongation:

Adults and children with malaria

For information on the risk of QT/QTc prolongation in patients.

Healthy adults

In a healthy adult volunteer parallel group study including a placebo and moxifloxacin

control group (n=42 per group), the administration of the six dose regimen of Lemalar was

associated with prolongation of QTcF.

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The mean changes from baseline at 68, 72, 96, and 108 hours post first dose were 7.45,

7.29, 6.12 and 6.84 msec, respectively. At 156 and 168 hours after first dose, the changes

from baseline for QTcF had no difference from zero. No subject had a >30 msec increase

from baseline nor an absolute increase to >500 msec. Moxifloxacin control was associated

with a QTcF increase as compared to placebo for 12 hours after the single dose with a

maximal change at 1 hour after dose of 14.1 msec.

In the adult/adolescent population included in clinical trials, 8 patients (0.8%) receiving

Lemalar experienced a QTcB >500 msec and 3 patients (0.4%) a QTcF >500 msec.

Prolongation of QTcF interval >30 msec was observed in 36% of patients.

In clinical trials conducted in children with the 6-dose regimen, no patient had post-

baseline QTcF >500 msec whereas 29.4% had QTcF increase from baseline >30 msec and

5.1% >60 msec. In clinical trials conducted in adults and adolescents with the 6-dose

regimen, post-baseline QTcF prolongation of >500 msec was reported in 0.2% of patients,

whereas QTcF increase from baseline >30 msec was reported in 33.9% and >60 msec in

6.2% of patients.

In the infant/children population included in clinical trials, 3 patients (0.2%) experienced a

QTcB >500 msec. No patient had QTcF >500 msec. Prolongation of QTcF intervals >30

msec was observed in 34% of children weighing 5-10 kg, 31% of children weighing 10-15

kg and 24% of children weighing 15-25 kg, and 32% of children weighing 25-35 kg.

5.2 Pharmacokinetic properties

Pharmacokinetic characterisation of Lemalar is limited by the lack of an intravenous

formulation, and the very high inter-and intra-subject variability of artemether and

Lumefantrine plasma concentrations and derived pharmacokinetic parameters (AUC,

Cmax).

Absorption

Artemether is absorbed fairly rapidly and dihydro artemisinin, the active metabolite of

artemether, appears rapidly in the systemic circulation with peak plasma concentrations of

both compounds reached about 2 hours after dosing. Mean Cmax and AUC values of

artemether ranged between 60.0-104 ng/mL and 146-338 ng·h/mL, respectively, in fed

healthy adults after a single dose of Lemalar, 80 mg artemether/480 mg Lumefantrine.

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Mean Cmax and AUC values of dihydro artemisinin ranged between 49.7-104 ng/mL and

169-308 ng·h/mL, respectively.

Absorption of Lumefantrine, a highly lipophilic compound, starts after a lag-time of up to 2

hours, with peak plasma concentration (mean between 5.10-9.80 µg/mL) about 6-8 hours

after dosing. Mean AUC values of Lumefantrine ranged between 108 and 243 µg·h/mL.

Food enhances the absorption of both artemether and Lumefantrine: in healthy volunteers

the relative bioavailability of artemether was increased more than two-fold and that of

Lumefantrine sixteen-fold compared with fasted conditions when Lemalar was taken after

a high-fat meal.

Food has also been shown to increase the absorption of Lumefantrine in patients with

malaria, although to a lesser extent (approximately two-fold), most probably due to the

lower fat content of the food ingested by acutely ill patients. The food interaction data

indicate that absorption of Lumefantrine under fasted conditions is very poor (assuming

100% absorption after a high-fat meal, the amount absorbed under fasted conditions would

be <10% of the dose). Patients should therefore be encouraged to take the medication with

a normal diet as soon as food can be tolerated.

Distribution

Artemether and Lumefantrine are both highly bound to human serum proteins in

vitro (95.4% and 99.7%, respectively). Dihydro artemisinin is also bound to human serum

proteins (47-76%).

Metabolism

Artemether is rapidly and extensively metabolised (substantial first-pass metabolism)

both in vitro and in humans. Human liver microsomes metabolise artemether to the

biologically active main metabolite dihydro artemisinin (demethylation), predominantly

through the isoenzymes CYP3A4/5. This metabolite has also been detected in humans in

vivo.

Dihydro artemisinin is further converted to inactive metabolites.

The pharmacokinetics of artemether in adults is time-dependent. During repeated

administration of Lemalar, plasma artemether levels decreased significantly, while levels of

the active metabolite (dihydro artemisinin) increased, although not to a statistically

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significant degree. The ratio of day 3/day 1 AUC for artemether was between 0.19 and

0.44, and was between 1.06 and 2.50 for dihydro artemisinin.

This suggests that there was induction of the enzyme responsible for the metabolism of

artemether. Artemether and dihydro artemisinin were reported to have a mild inducing

effect on CYP3A4 activity.

Lumefantrine is N-debutylated, mainly by CYP3A4, in human liver microsomes. In vivo in

animals (dogs and rats), glucuronidation of Lumefantrine takes place directly and after

oxidative biotransformation. In humans, the exposure to Lumefantrine increases with

repeated administration of Lemalar over the 3-day treatment period, consistent with the

slow elimination of the compound.

Systemic exposure to the metabolite desbutyl-lumefantrine, for which the in

vitro antiparasitic effect is 5 to 8 fold higher than that for Lumefantrine, was less than 1%

of the exposure to the parent drug. Desbutyl-lumefantrine data is not available specifically

for an African population. In vitro, Lumefantrine significantly inhibits the activity of

CYP2D6 at therapeutic plasma concentrations.

Elimination

Artemether and dihydro artemisinin are rapidly cleared from plasma with a terminal half-

life of about 2 hours. Lumefantrine is eliminated very slowly with an elimination half-life

of 2 to 6 days. Demographic characteristics such as sex and weight appear to have no

clinically relevant effects on the pharmacokinetics of Lemalar.

Limited urinary excretion data are available for humans. In 16 healthy volunteers, neither

Lumefantrine nor artemether was found in urine after administration of Lemalar, and only

traces of dihydro artemisinin were detected (urinary excretion of dihydro artemisinin

amounted to less than 0.01% of the artemether dose).

In animals (rats and dogs), no unchanged artemether was detected in faeces and urine due

to its rapid and extensive first-pass metabolism, but numerous metabolites (partly

identified) have been detected in faeces, bile and urine. Lumefantrine was excreted

unchanged in faeces and with traces only in urine. Metabolites of Lumefantrine were

eliminated in bile/faeces.

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Dose proportionality

No specific dose proportionality studies were performed. Limited data suggest a dose-

proportional increase of systemic exposure to Lumefantrine when doubling the Lemalar

dose. No conclusive data is available for artemether.

Bioavailability/bioequivalence studies

Systemic exposure to Lumefantrine, artemether and dihydro artemisinin was similar

following administration of Lemalar as dispersible tablets and crushed tablets in healthy

adults.

Systemic exposure to Lumefantrine was similar following administration of Lemalar

dispersible tablets and intact tablets in healthy adults. However, exposure to artemether and

dihydro artemisinin was significantly lower (by 20-35%) for the dispersible than for the

intact tablet. These findings are not considered to be clinically relevant for the use of the

dispersible tablets in the paediatric population since adequate efficacy of Lemalar

dispersible tablets was demonstrated in this population. The dispersible tablet is not

recommended for use in adults.

Special populations

No specific pharmacokinetic studies have been performed in elderly patients. However,

there is no information suggesting that the dosage in patients over 65 years of age should

be different than in younger adults.

In paediatric malaria patients, mean Cmax (CV%) of artemether (observed after first dose

of Lemalar) were 223 (139%), 198 (90%) and 174 ng/mL (83%) for body weight groups 5-

<15, 15-<25 and 25-<35 kg, respectively, compared to 186 ng/mL (67%) in adult malaria

patients. The associated mean Cmax of DHA were 54.7 (108%), 79.8 (101%) and 65.3

ng/mL (36%), respectively compared to 101 ng/mL (57%) in adult malaria patients. AUC

of Lumefantrine (population mean, covering the six doses of Lemalar) were 577, 699 and

1150 µg•h/mL for paediatric malaria patients in body weight groups 5-<15, 15-<25 and 25-

<35 kg, respectively, compared to a mean AUC of 758 µg•h/mL (87%) in adult malaria

patients. The elimination half-lives of artemether and Lumefantrine in children are

unknown.

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No specific pharmacokinetic studies have been performed either in patients with hepatic or

renal insufficiency or elderly patients. The primary clearance mechanism of both

artemether and Lumefantrine may be affected in patients with hepatic impairment. In

patients with severe hepatic impairment, a clinically significant increase of exposure to

artemether and Lumefantrine and/or their metabolites cannot be ruled out. Therefore

caution should be exercised in dosing patients with severe hepatic impairment. Based on

the pharmacokinetic data in 16 healthy subjects showing no or insignificant renal excretion

of Lumefantrine, artemether and dihydro artemisinin, no dose adjustment for the use of

Lemalar in patients with renal impairment is advised.

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SECTION-18

BIOAVAILABILITY / BIOEQUIVALENCE

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BIOAVAILABILITY / BIOEQUIVALENCE

Not Required

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SECTION-19

a) CERTIFICATE OR LICENSE TO FROM THE COUNTRY OF ORIGIN

b) CERTIFICATE OF PHARMACEUTICAL PRODUCT

c) FREE SALE CERTIFICATE (FSC)

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a) CERTIFICATE OR LICENSE TO FROM THE COUNTRY OF

ORIGIN

Applicable

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b) CERTIFICATE OF PHARMACEUTICAL PRODUCT

Applicable

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c) FREE SALE CERTIFICATE (FSC)

Applicable

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SECTION-20

a) SAMPLES OF THE PRODUCT TO BE REGISTERED

b) LEAFLETS ACCOMPANYING THE PRODUCT TO BE

REGISTERED

c) LABELING INFORMATION

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a. SAMPLES OF THE PRODUCT TO BE REGISTERED

Send along with dossier.

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b. LEAFLETS ACCOMPANYING THE PRODUCT TO BE

REGISTERED

-----

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a) LABELING INFORMATION

Applicable

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SECTION-21

a. Documentary evidence if the product has been registered in

the country of origin (Product License)

b. Indicate if the product is actually on sale in the country of

origin

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a. Documentary evidence if the product has been registered in the

country of origin (Product License)

APPLIABLE

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a. Indicate if the product is actually on sale in the country of origin

----------

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SECTION-22

Name of Countries in which the Product is being Marketed /

Registered

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Name of Countries in which the Product is being Marketed/ Registered

S. No. Country Name Registration Status

1. ---- ----

2. ---- ----

3. ---- ----

4. ---- ----

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SECTION 23

PUBLISHED LITERATURE SECTION

J. F. Trape and A. Zoulani, ―Malaria and urbanization in Central Africa: the example of

Brazzaville. Part III: relationships between urbanization and the intensity of malaria

transmission,‖ Transactions of the Royal Society of Tropical Medicine and Hygiene, vol.

81, no. 2, pp. 19–25, 1987.

J. F. Trape and A. Zoulani, ―Malaria and urbanization in Central Africa: the example of

Brazzaville. Part II: results of entomological surveys and epidemiological

analysis,‖ Transactions of the Royal Society of Tropical Medicine and Hygiene, vol. 81,

no. 2, pp. 10–18, 1987.

M. Ndounga, P. N. Casimiro, V. Miakassissa-Mpassi, D. Loumouamou, F. Ntoumi, and L.

K. Basco, ―Malaria in health centres in the southern districts of Brazzaville,

Congo,‖ Bulletin de la Societe de Pathologie Exotique, vol. 101, no. 4, pp. 329–335, 2008.

G. Moyen, S. Nzingoula, J. C. Mowandza-Ndinga, J. L. Nkoua, A. B. Mpemba, and V.

Fourcarde, ―Paludisme de l‘enfant dans un service de pédiatrie à Brazzaville—à propos de

1073 observations,‖MéDecine D‘Afrique Noire, vol. 40, no. 3, pp. 177–181, 1993.

J. R. Mabiala-Babela, P. B. Makoumbou, A. Mbika-Cardorelle, J. B. Tsiba, and P. Senga,

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