This document discusses procedures for evaluating parenteral products, including sterility testing, clarity testing, leakage testing, and potency testing. It focuses on sterility testing methods such as membrane filtration and direct inoculation. Key steps include incubating samples in culture media to check for microbial growth, which would indicate a failed sterility test. The document also covers pyrogen (fever-causing substance) testing using rabbits, where an intravenous injection is given and temperature changes are monitored; a large temperature increase would mean a failed pyrogen test. Sample sizes, incubation times, interpretation of results, and pass/fail criteria are outlined for these evaluation methods.

1. BY RAHUL DEV
SMCP MOHNA

2. SARASWATI MODERN COLLEGE OF PHARMACY
MOHNA , FARIDABAD

3. EVALUATION OF PARENTERALS
STERILIT TEST
CLARITY TEST
LEAKAGE TEST
PROGEN TEST
ASSAY

4. STERILITY TEST
 TEST FOR STERILITY IS DONE BY DETECTING THE
PRESENCE OF VIABLE FORMS OF
BACTERIA,FUNGI AND YEAST.
 STERILITY TEST MUST BE CARRIED OUT UNDER
STRICT ASEPTIC CONDITIONS IN ORDER TO
AVOID ACCIDENTAL CONTAMINATION OF
PRODUCT
 TESTS
 1. MEMBRANE FILTERATION METHOD
 2. DIRECT INOCULATION METHOD

5. PRINCIPLE
IF BACTERIA OR FUNGI ARE PLACED IN A
MEDIUM WHICH PROVIDES NUTRITIVE
MATERIAL AND WATER,AND KEPT AT A
FAVOURABLE TEMPERATURE, THE
ORGANISM WILL GROW AND THEIR
PRESENCE CAN BE INDICATED BY
TURBIDITY IN CLEAR MEDIUM.

6. SAMLE SIZE
NO. OF ITEMS IN THE BATCH NO OF ITEMS RECOMMENDED TO
BE TESTED
NOT MORE THAN 100 CONTAINERS 10% OR 4 WHICHEVER IS GREATER
MORE THAN 100 BUT NOT MORE
THAN 500
10 CONTAINERS
MORE THAN 500 CONTAINERS 2% OR 20 CONTAINERS WHICHEVER
IS LESS

7. QUNATITY OF PRODUCT TO BE USED (LIQUID)
QUANTITY OF EACH CONTAINER QUANTITY TO BE USED FOR EACH
CULTURE MEDIUM
LESS THAN 1 ML WHOLE CONTENTS OF A
CONTAINER
1 ML OR MORE BUT LESS THAN 40
ML
HALF OF CONTENTS OF A
CONTAINER
40 ML OR MORE BUT LESS THAN 100
ML
20 ML
100 ML OR MORE 10% OF CONTENT BUT NOT LESS
THAN 20 ML

8. QUNATITY OF PRODUCT TO BE USED
(SOLID)
QUANTITY OF EACH CONTAINER QUANTITY TO BE USED FOR EACH
CULTURE MEDIUM
LESS THAN 50 MG THE WHOLE CONTENT
50 MG OR MORE BUT LESS THAN 300
MG
HALF OF CONTENT OF A
CONTAINER
300 MG OR MORE 100 MG

9. MEMBRANE FILTERATION METHOD
 USED IN FOLLOWING CASES
1. OIL OR OILY PREPARATION
2. AN OINTMENT THAT CAN BE PUT INTO SOLUTION
3. A NON BACTERIOSTATIC SOLID NOT READILY
SOLUBLE IN CULTURE MEDIUM
4. A SOLUBLE POWDER OR A LIQUID THAT
POSSESSES BACTERIOSTATIC AND FUNGISTATIC
PROPERTIES
5. LIQUID PRODUCTS WHERE VOLUME IN A
CONTAINER IS 100 ML OR MORE

10. MEMBRANE FILTERATION METHOD
A. 1. FILTER THE SAMPLE THROUGH MEMBRANE FILTER
(POROSITY 0.45 MICRON, DIAMETER APPROX. 47 mm)
 2.REMOVE THE MEMBRANE ASEPTICALLY FROM METALLIC
HOLDER
 3. DEVIDE MEMBRANE IN TWO HALVES
 4. TRANSFER 1St HALF INTO 100 ml OF CULTURE MEDIA
MEANT FOR FUGNI AND INCUBATE AT 20-25 ⁰C FOR NOT LESS
THAN SEVEN DAYS
 5. OTHER HALF IS TRANSFERRED INTO 100 ml OF FLUID
THIOGLYCOLLATE MEDIUM AND INCUBATED AT 30-35 ⁰C FOR
NOT LESS THAN 7 DAYS.
 6. OBSERVE THE GROWTH IN THE MEDIA.

11. DIRECT INOCULATION METHOD
 1. SPECIFIC QUANTITY OF SAMPLE IS DRAWN
FROM CONTAINER ASETICALLY.
 2. TRANSFER THE SAMLE INTO VESSEL OF
CULTURE MEDIA.
 3. MIX THE LIQUID , INCUBATE FOR NOT LESS
THAN 14 DAYS.
 4. OBSERVE THE GROWTH IN THE MEDIUM.

12. OBSERVATION AND RESULT
OBSERVATION RESULT
NO EVIDENCE OF GROWTH PASSES TEST FOR STERILITY
EVIDENCE OF GROWTH RE-TESTING USING SAME NO.OF SAMPLE
,VOLUME
NO EVIDENCE OF GROWTH PASSES TEST FOR STERILITY
EVIDENCE OF GROWTH ISOLATE AND IDENTY THE ORGANISM
1. NOT READILY DISTIGUISHABLE FROM
THOSE GROWING IN CONTAINER
RESERVED IN FIRST TEST--- FAILS TEST
2. READILY DISTINGUISHABLE– SECOND
RETEST ON TWICE NO. OF SAMPLES
NO EVIDENCE OF GROWTH
EVIDENCE OF GROWTH
PASSES TEST FOR STERILITY
FAILS TEST FOR STERILITY

13. PYROGEN TEST
 PYROGEN TEST IS DONE TO CHECK PRESENCE OR
ABSENCE OF PYROGEN IN ALL AQUEOUS
PARENTERAL PREPARATIONS.
 PRINCIPAL—THE TEST INVOLVES THE
MEASUREMENT OF RISE IN BODY TEMPERATURE
OF RABBIT FOLLOWING I.V INJECTION OF A
STERILE SOLUTION.
 MATERIAL ---TEMPERATURE RECORDING DEVICE,
GLASS WARES, SYRINGES AND NEEDLES,
RETAINING BOXES FOR RABBITS

14. REQUIREMENTS
 BOX- ANIMALS ARE RETAINED ONLY BY LOOSLY FITTING –NECK
–STOCK AND REST OF THE BODY REMAINS RELATIVELY FREE SO
THAT RABBITS MAY SIT IN A NORMAL POSITION.
 THE ANIMALS MUST BE PUT 1 HOUR BEFORE THE TEST.
 ROON TEMP. WHERE TEST IS CARRIED OUT IS WITHIN THE
RANGE OF 3 ⁰C OF THE LIVING QUARTERS OF THE RABBIT.
 WITHHOLD THE FOOD FROM THE ANIMALSOVERNIGHT AND
UNTIL THE TEST IS COMPLETED
 WITHHOLD WATER DURING TEST.
 3 HEALTHY RABBITS OF EITHER SEX,EACH WEIGHING NOT LESS
THAN 1.5 KG FED ON COMPLETE BALANCED DIET
 RABBITS NOT SHOWING ANY LOSS OF WEIGHT DURING THE
WEEK PROCEEDING THE TEST.
 DO NOT USE RABBIT
 (A)HAVING TEMP. HIGHER THAN 39.8⁰C.
 (B) SHOWING TEMP. VARIATION GREATER THAN 0.2⁰C BETWEEN
TWO SUCCESIVE READINGS

15. METHOD
 1. TEST IS CARRIED OUT IN AN AIR CONDITIONED ROOM
 2.DISSOLVE SAMPLE ,OR DILUTE WITH, A PYROGEN FREE
SALINE
 3.WARM THE LIQUID TO APPROX. 38.5⁰C BEFORE
INJECTION
 4.AMOUNT OF SAMPLE INJECTED ACCORDING TO
MONOGRAPH.
 5. VOLUME OF INJECTION IS NOT LESS THAN 0.5 ml AND
NOT MORE THAN 10 ml PER KG OF BODY WEIGHT.
 6. CLINICAL THERMOMETER OF SENSING PROBE IS
INSERTED INTO THE RECTUM OF RABBIT FOR
RECORDING TEMPERATURE.
 7. TWO NORMAL READINGS OF RECTAL TEMP. ARE TAKEN
PRIOR TO TEST INJECTION AT AN INTERVAL OF 30 MIN
AND ITS MEAN IS CALCULATED, WHICH IS INITIAL TEMP.
OF RABBIT.

16. METHOD
 8. TEST SOLUTION IS INJECTED SLOWLY
THROUGH AN EAR VEIN
 9 RECORD THE TEMP. OF EACH RABBIT IN AN
INTERVAL OF 30 MINUTE FOR THREE HOURS
AFTER INJECTION.
 10 . THE DIFFERENCE BETWEEN THE INITIAL
TEMPERATURE AND MAXIMUM TEMPERATURE
RECORDED IS TAKEN TO BE ITS RESPONSE.
 11. WHEN TEMPERATURE DIFFERENCE IS
NEGATIVE , RESULT IS COUNTED AS ZERO
RESPONSE.

17. INTERPRETATION OF RESULT
OBSERVATION RESULT
SUM OF RESPONSES OF 3 RABBITS NOT EXCEED 1.4 ⁰C
RESPONSE OF INDIVIDUAL RABBIT IS LESS THAN 0.6 ⁰C
PASS
SUM OF RESPONSES OF 3 RABBITS EXCEEDS 1.4 ⁰C
RESPONSE OF INDIVIDUAL RABBIT IS 0.6 ⁰C OR MORE
RE TEST ON 5
OTHER RABBITS
NOT MORE THAN 3 RABBITS OUT OF 8 SHOW INDIVIDUAL
RESPONSE OF 0.6⁰C OR MORE
SUM OF RESPONSES OF GROUP OF 8 RABBITS NOT EXCEED
3.7⁰ C
PASS

18. THANK YOU