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STUDY PLAN

STUDY TITLE
DETERMINATION OF MICROORGANISMS RECOVERY RATE TO VERIFY THE
EFFICIENCY OF THE TEST METHOD AND TO ESTABLISH A CORRECTION FACTOR
FOR PRODUCT - 20 mm FLIP-OFF SEAL; GREEN (GN-16).

TEST GUIDELINE (S): ISO 11737-1:2018

STUDY NO.: RRCF-G0008-21P

STUDY CODE: RRCF

SPONSOR
Autofits Packaging Pvt. Ltd.
D-3, M.I.D.C., Malegaon Ind. Area,
Sinnar, Nashik - 422113.
Maharashtra, India.
www.autofits.com

SERVICE PROVIDER
Medzus Healthcare Private Limited
Office-7, Ganga Heights,
Pathardi Road,
Nashik, 422010,
MH, India.
www.medzus.co.in

TEST FACILITY
Synergy Sterilisation (M) Sdn Bhd,
a STERIS Company (512058-V)
Plot 203, Kuala Ketil Industrial Park,
09300, Kuala Ketil, Kedah,
Malaysia.
www.steris-ast.com

Page 1 of 9
CONFIDENTIAL
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TABLE OF CONTENTS

1. STUDY DETAILS....................................................................................................... 3

1.1 Overview .................................................................................................................. 3

1.2 Study Responsibilities............................................................................................. 3
1.3 Proposed Study Schedule*...................................................................................... 3
1.4 Sponsor Details ........................................................................................................ 3

2. QUALITY ASSURANCE............................................................................................. 4

3. STUDY COMPLIANCE .............................................................................................. 4

4. STUDY GUIDELINES ................................................................................................ 4

5. SAFETY PRECAUTIONS ........................................................................................... 4

6. OBJECTIVE OF STUDY ............................................................................................. 4

7. PRINCIPLE OF STUDY ............................................................................................. 4

8. MATERIALS .............................................................................................................. 5

8.1 Test Item Information ............................................................................................. 5

8.2 Equipments & Material ........................................................................................... 5
8.3 Environmental Test Conditions ............................................................................. 5
8.4 Sample Size .............................................................................................................. 5

9. TEST PROCEDURE ................................................................................................... 5

10. EVALUATION OF RESULTS ..................................................................................... 6

11. AMENDMENTS AND DEVIATIONS ......................................................................... 6

12. STUDY PLAN AND REPORT DISTRIBUTION ......................................................... 6

13. ARCHIVING .............................................................................................................. 7

14. STUDY REPORT ....................................................................................................... 7

15. STUDY PLAN APPROVAL ........................................................................................ 8

Annexure 1. Test Item Information Sheet ......................................................................... 9

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1. STUDY DETAILS
1.1 Overview
Study Title : Determination of microorganisms recovery rate to verify
the efficiency of the test method and to establish a
correction factor for product - 20 mm flip-off seal; Green
(GN-16).

Study Number : RRCF-G0008-21P

Study Code : RRCF

Sponsor : Autofits Packaging Pvt. Ltd.

D-3, M.I.D.C., Malegaon Ind. Area,
Sinnar, Nashik - 422113.
Maharashtra, India.

Service Provider : Medzus Healthcare Private Limited

Office-7, Ganga Heights,
Pathardi Road,
Nashik, 422010,
MH, India.

Test Facility : Synergy Sterilisation (M) Sdn Bhd,

a STERIS Company (512058-V)
Plot 203, Kuala Ketil Industrial Park,
09300, Kuala Ketil, Kedah,
Malaysia.

1.2 Study Responsibilities

Technical : Mr.
Analyst-I : Mr.
Analyst-II : Mr.
QA : Mr.

1.3 Proposed Study Schedule*

Study Initiation Date : XX/XX/XXXX
Experiment Start Date : XX/XX/XXXX
Result Recording Date : XX/XX/XXXX
Experiment Completion Date : XX/XX/XXXX
* Actual date will be mentioned in Final Report.

1.4 Sponsor Details

Sponsor Representative
: Mr. Suveeth Kumar
Autofits Packaging Pvt. Ltd.
D-3, M.I.D.C., Malegaon Ind. Area,
Sinnar, Nashik - 422113.
Maharashtra, India.

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Monitoring Scientist : Mrs. Leena More
Autofits Packaging Pvt. Ltd.
D-3, M.I.D.C., Malegaon Ind. Area,
Sinnar, Nashik - 422113.
Maharashtra, India.
2. QUALITY ASSURANCE
The Quality Assurance Unit personnel of Medzus Healthcare Private Limited will
inspect at different phases of the study and reviews the draft and final study
plans, draft and final reports to assure the integrity of study in compliance with
ISO/IEC 17025:2017.

3. STUDY COMPLIANCE
This study will be conducted in compliance with Quality Management System as
per ISO/IEC 17025:2017.

4. STUDY GUIDELINES
This study will follow below guidelines-
 ISO 11737-1:2018- Sterilization of medical devices - Microbiological methods
- Part 1: Determination of a population of microorganisms on products
 EN ISO 11737-1:2006- Sterilization of medical devices - Microbiological
methods - Part 1: Determination of a population of microorganisms on
products (ISO 11737-1:2006) EN ISO 11737-1:2006/AC: 2009.

5. SAFETY PRECAUTIONS
All necessary safety measures will be taken care during the conduct of the study.
When required, personnel involved in the study shall wear gloves, head cap and
facemask in addition to the protective clothing to ensure adequate personnel
health and safety.

6. OBJECTIVE OF STUDY
The objective this study is to determine the bacterial & fungal recovery rate to
verify the efficiency of the test method and to establish a correction factor for
product - 20 mm flip-off seal; Green (GN-16).

7. PRINCIPLE OF STUDY
The material and the geometry of a medical product have a major influence on
the efficiency of the test method. Therefore, a one-time validation of the test
method should be carried out for each product.
For this purpose, the product is inoculating with a known number of a known
test organism and then test with clearly define parameters. At the end of the test,
the CFUs are count and compare with the number applied. This ratio is the
recovery rate. This is then use to calculate a correction factor which will calculate
into all subsequent bioburden determinations on this product.

In accordance with the ISO 11737-1 standard, the recovery rate should be at
least 50 %, otherwise the parameters of the test method must be change and test
again. If the recovery rate reached a satisfactory value, all bioburden
determinations on this product must be carried out with exactly the same
parameters as during this test.
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8. MATERIALS
8.1 Test Item Information
The test item information provided by the sponsor as per Test Item information
Sheet (TIIS) is furnished as below:
Name of the Test Item : 20 mm flip-off seal; Green (GN-16)
Test item Code by Sponsor : NA
Name to be used in Report : 20 mm flip-off seal; Green (GN-16)
Test Item Code by Test Facility : NA
Batch No./Lot No. : 279,08,20 (B)
Date of Manufacture : 07/05/2021
Date of Expiry : 06/05/2025
Storage Conditions : Ambient (21 to 29°C)

The Sponsor is responsible for authenticity of the test item and no further
characterization of test item will be performed at Medzus Healthcare Private
Limited. Test item information provided by the sponsor in Test Item Information
Sheet.

8.2 Equipments & Material

No. Item
1 Laminar Air Flow
2 Colony Counter
3 Incubators
4 Sabouraud Soyabean Casein Digest Agar (SCDA)
5 Peptone Water, 0.5% tween 80
6 B subtilis -Subculture

8.3 Environmental Test Conditions

Tests will perform at a temperature within the range of 15 °C to 25 °C and at a
relative humidity between 25 % and 65 %.

Environment for Recovery Rate: According to ISO 11737-1, Recovery Rate will
conduct under aseptic conditions in controlled environments as indicated in ISO
14644, such as under a laminar air flow in an assigned room.

8.4 Sample Size

5 Numbers of 20 mm flip-off seal; Green (GN-16), will use for gathering the
recovery rate.

9. TEST PROCEDURE
Tests shall performed as per-
A. Calculation of Spore Concentration:
a. Calculate of the known amount spore to be place on product (Approx. 100
CFU spores of B. subtilis) by serial dilution of culture and direct plating.

B. Placing the Spore on Product:

a. Once the spore concentration is known (Approx. 100 CFU spores of B.
subtilis) then place the known spore on sterile product.

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b. Aseptically place 1 mL of approx. 100 CFU spores of B. subtilis (obtained
from serial dilution) on sterile products separately.

C. Method for recovery:

a. After placing the known amount of spore (B subtilis ) on sterile product,
allow it to dry for few minutes then transfer the products into stomaching
bag containing previously sterilized (at 1210C) 300 ml 0.1% Peptone
water with 0.5% tween 80 (Diluting Fluid).
b. Shake the sample preparation for 30 minutes at 250 rpm.
c. Filter the 300 ml eluents (product + B.subtilis) through 0.45 µm cellulose
nitrate membrane filter and place the membrane filter (i.e eluent with
B.subtilis) onto sterile pre-incubated SCDA plates. Incubate plates at 30-
35°C.
d. Similarly, Negative control plates SCDA incubates at above condition.
e. Negative control plate should not show any CFU.
f. On completion of incubation period count the number of colonies
recovered for B. subtilis on SCDA.

D. Calculating the Recovery Rate:

Determine the recovery rate by following formula
𝑹𝒆𝒄𝒐𝒗𝒆𝒓𝒆𝒅 𝑪𝑭𝑼
𝑹𝒆𝒄𝒐𝒗𝒆𝒓𝒚 𝒓𝒂𝒕𝒆 = 𝒙 𝟏𝟎𝟎
𝑪𝑭𝑼 𝑷𝒍𝒂𝒄𝒆𝒅 𝒐𝒏 𝑷𝒓𝒐𝒅𝒖𝒄𝒕

g. Calculate the average of recovery rate.

E. Calculating the Correction Factor:

Calculate the correction factor as below-
𝟏
𝑪𝒐𝒓𝒓𝒆𝒄𝒕𝒊𝒐𝒏 𝑭𝒂𝒄𝒕𝒐𝒓 =
𝑹𝒆𝒄𝒐𝒗𝒆𝒓𝒚 𝒓𝒂𝒕𝒆 𝒂𝒔 𝒂 𝒅𝒆𝒄𝒊𝒎𝒂𝒍

10. EVALUATION OF RESULTS

When tested in accordance with ISO 11737-1: 2018; the recovery rate should be
at least 50 %,

11. AMENDMENTS AND DEVIATIONS

The study will be conducted as per the mutually agreed and approved study plan
with the sponsor. The amendments / deviations if any occur during study period
will be mentioned in the study report.

12. STUDY PLAN AND REPORT DISTRIBUTION

The original Study Plan and Study Report will be distributed between Sponsor
and Test Facility as follows:
Study Plan
a) Copy No. 1/2 – Sponsor.
b) Copy No. 2/2 – Archives,
Study Report
a) Copy No. 1/2 – Sponsor.
b) Copy No. 2/2 – Archives,

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13. ARCHIVING
All study related records, study plan, and final report will be maintained in the
archives of Medzus Healthcare Pvt. Ltd. for 1 year from the date of the
completion of the study, all the records will be handled according to
ISO/IEC17025;2017 Management System. After the completion of archiving
period, the test facility management will co-ordinate with the sponsor for further
course of action on archived material.

14. STUDY REPORT

Final study report will include, but not limited to the following information:
 Study Schedule
 Quality Assurance Statement
 Identification of Study
 Test Item details
 Test System details
 Materials and test methods
 Result and Conclusion
 Incubation Time
 Incubation Temperature
 Observed CFU
 Calculated Recovery Rate/Ratio
 Calculated Correction Factor

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15. STUDY PLAN APPROVAL
The Study Plan for the Study No: RRCF-G0008-21P, entitled – “Determination of
microorganisms recovery rate to verify the efficiency of the test method and to
establish a correction factor for product - 20 mm flip-off seal; Green (GN-16)” has
been mutually agreed and approved.

For Medzus Healthcare Private Limited (Service Provider):

Mr. Rahul R. Patil.

_____________
Technical Manager
(Sign & Date)

Mrs. Neha Patil.

______________
QA-Manager
(Sign & Date)

Ms. Madhuri Patil.

______________
QC-Manager
(Sign & Date)

For Autofits Packaging Pvt. Ltd.:

Mr. Suveeth Kumar _____________

Sponsor Representative (Sign & Date)

Mrs. Leena More _____________

Monitoring Scientist (Sign & Date)

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Annexure 1. Test Item Information Sheet

RRCF-G0008-21P Study Plan Page 9 of 9