MICLAB 150 Initial Investigation of Out of Specification (OOS) Results in Microbiological Laboratory

Standard Operating Procedure
Title: Initial Investigation of Out of Specification (OOS) Results in Microbiological Laboratory
Department Micro Laboratory Document no MICLAB 145

Initial Investigation of Out of Specification (OOS) Results in Microbiological
Title
Laboratory
Prepared by: Date: Supersedes:
Checked by: Date: Date Issued:
Approved by: Date: Review Date:
1.0 DOCUMENT OWNER

Laboratory/Quality Manager
2.0 PURPOSE
To establish a procedure for the investigation of initial out-of-specification (OOS) or questionable

results which have been generated for the product or material being tested.
3.0 SCOPE
3.1 This procedure applies to test results generated for raw materials, in-process materials, stability and
finished products tested for medicinal products for commercial sale.
3.2 This procedure applies to test results generated for release, stability, in-process and retained samples
associated with a complaint investigation.
3.3 This procedure does not apply to results generated by testing not described in 3.1 or 3.2 or out of
specification results for process water and environmental samples.
3.4 An investigation for out-of-specification or questionable results must be initiated when an analysis
has been performed on the sample in question.
3.5 Instances of out-of-specification or questionable results, where assignable cause is not readily
apparent shall be investigated in accordance with the requirements described in this procedure.
3.6 If assignable cause is readily apparent, the occurrence is documented using the Laboratory
Investigation Report (LIR) form, as applicable.
3.7 All LIR’s must be completed and fully approved within 30 business days of the discovery of the
initial OOS or questionable result. If laboratory investigation goes beyond 30 business days, an
interim status report must be issued to the QA Manager.
3.8 All LIR’s should be reviewed once per year to determine if trends are observed that require further
action
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4.0 RESPONSIBILITY \ BUSINESS RULES
4.1 Quality Assurance Manager is responsible for:

- Reviewing laboratory investigations and considering in batch/lot release decisions.
- Approval re-sampling programs of materials for use in the laboratory investigation.
- Issuing Action reports to appropriate personnel when deemed necessary.
- Evaluate the need for investigations where confirmed out-of-specification results occur and
ensuring they are completed.
- Approving laboratory investigations on completion.
4.2 Laboratory Manager is responsible for:

- Ensuring that this SOP is understood and adhered to by all laboratory personnel.
- Establishing and maintaining a log of all laboratory reports.
- Where appropriate notifying the QA Manager to instigate further investigation where
confirmed out-of-specification results are produced.
- Reports trends of Microbiology Laboratory Investigations to the QA Manager on a periodic
basis.
- Complete final review of the Laboratory Investigation Report.
- Review LIR’s once a year, observe trends and take action if applicable.
4.3 Microbiology Team Leader is responsible for:

- Reviewing and approving the applicable sections for the laboratory investigation report.
- Approving investigation measurements and re-testing plans.
- Ensuring laboratory investigation reports, all results and laboratory records are achieved.
- Responsible for closing out LIR in database for tracking and trending purposes.
- Responsible for circulating the Alert Reports.
- Assisting the Laboratory Analyst in the completion of the Microbiology Laboratory
Investigation Report.
4.4 Microbiological Analyst:

- Immediately notifying the Microbiology Team Leader or Laboratory Manager of any out-of-
specification or questionable result.
- Initiating and assisting in the completion of the Laboratory Investigation Report.
- Including all data generated in the laboratory investigation report.

- Immediately notifying the Microbiology Team Leader or the Lab Manager of any
microbiological test result which does not meet specification or which fits the description of a
questionable test result.
5.0 PROCEDURE
5.1 Discovery of out-of-specification or questionable result.
5.1.1 Upon discovery of any OOS or Questionable Result, the Lab Analyst shall immediately,
before initiating any repeat testing, or retesting take the following actions:
• report the results to the Lab Team Leader or Lab Manager;

• document findings;
• document obvious errors (e.g., spilling of solutions or incomplete transfer of sample or
standard), if known;
• retain all original samples and test preparations, such as sample solutions, standard
solutions, glassware, microbiological culture media, and reagents used in the analysis
and subsequent investigation, to the extent possible, until the initial investigation is
completed; and
• retain samples and associated test preparations, when possible, in a manner that
ensures their integrity (e.g., refrigerated) until the test article receives a final
disposition.
Note: The Lab Team Leader shall determine if obvious errors (e.g., calculation or dilution
errors) exist that might have caused the OOS or Questionable Result. If an obvious
error is found, the OOS or Questionable Result is considered an invalidated result
and no further investigation is required, unless the error would impact other testing.
If obvious errors are not found, the Lab Team Leader shall initiate a further
investigation.
5.1.2 The analyst ensures that an entry into the laboratory investigation database (online) or if
this database is not available, an entry into the log book (manual system) is made and that
a unique number is assigned to the laboratory investigation report. At this time, the analyst
and team leader or designate completes section 1 (general information) of the laboratory
investigation report. In cases where more than one lot of material is investigated for the
same problem, only one investigation report is required. All affected lots must be clearly
listed.
5.1.3 All Laboratory Investigation Reports are assigned a unique number. In cases where the
online system is used, the number is assigned automatically by the database system and is
denoted by the following format:
• (YYYYMMXXX, where YYYY and MM are representative of the computer system

date’s year and month respectively and XXX is uniquely allocated a sequential
number applied based on the month and year.

In cases where a manual system is used, the number consists of a two-letter prefix (LI)
followed by an eight-digit identification number. The format of the Laboratory
Investigation Report number will be as follows:
• The first two digits of the identification number are the Department Number for the
Laboratory in which the investigation was conducted. The department numbers are as
follows: 10 = Analytical Services, 23 = Quality Control & Microbiology, 41 =
Research & Development.
• The second two digits of the identification number indicate the year in which the
Laboratory Investigation was conducted.
• The remaining four digits are assigned sequentially from the Laboratory Investigation
log book for that laboratory with the numbering system starting anew at the beginning
of the year.
5.2 Initial Investigation.

5.2.1 Within one business day, the analyst and the microbiologist must initiate a Microbiological
Laboratory Investigation, completing the following sections of the Laboratory Investigation
Report.
Section I: Purpose of Investigation

Section II: Reason For Investigation
Section III: Initial Investigation
Section IV: Findings/Conclusions from initial investigation
All sections must be completed with appropriate detail to fully describe steps taken. If a section
is considered not applicable to the investigation, i.e. assignable cause is readily apparent, mark
the section "Not Applicable". Justification must be provided in Section IX: Overall conclusion
from III, IV, V (if applicable), VI, VII.
5.2.2 After opening the LIR, the initial investigation to determine assignable cause begins when the
Micro Team Leader holds discussions with the Lab Analyst and completes the LIR Form
Section I “General Information”. Section I shall include a clear and concise description of the
reason for the investigation. In cases where more than one lot of material is investigated for the
same problem, only one LIR is required. All affected lots must be clearly listed. The initial
investigation also includes but is not limited to examinations of the following (LIR Form
Section II, “Review of Testing Parameters”):
• Test records (raw data and/or laboratory notebooks);
• Test procedures used;
• Calculations;
• Dilution schemes;
• Instrumentation or equipment functionality;
• Reference Standards, reagents, and media used;
• Sample preparations; and
• Personnel training and qualifications.

5.2.3 If, in the Micro Team Leader’s judgement, assignable cause is identified for an OOS or
questionable result, the original result is invalidated. The assignable cause shall be documented
in the LIR Form Section III “Initial Investigation” and Section IV “Findings/Conclusions from
Initial Investigation” within one business day of discovery of the result. Testing is then
repeated to generate valid results.
5.2.3.1 The analyst completes Section VII of the Laboratory Investigation Report: Retest
Protocol. The Microbiology Team Leader approves the retest protocol. Retest is
conducted as per the approved protocol.
The OOS or Questionable Result may be accepted as valid following the initial
investigation with no further retesting.
If there is no readily apparent assignable cause the investigational measurement

protocol (IMP) (section V of the laboratory investigation report) is prepared and
signed by the team leader or designate.
The purpose of conducting investigational measurement is to determine an

assignable cause. This section should only be completed when a suitable protocol is
decided. Data generated during and investigational measurement is for investigation
only and shall not be used as a valid test result.
5.2.3.2 On the Laboratory Investigation Report, the Micro Team Leader delegate/
completes Section VIII: Results of Retest, Section IX: Overall Conclusions and if
corrective action is required, Section X: Corrective Action Plan.
The potential impact on other samples tested during the initial testing must be
evaluated when an assignable cause is found. All related test results must be
assessed for validity. A statement documenting the assessment and the decision
must be included on the LIR Form Section III “Review of Testing Parameters”, or
Section IV “Findings/Conclusions from Initial Investigation”.
5.2.3.3 If, in the Lab Manager’s judgment, no readily apparent assignable cause is
determined by the initial investigation, it may be necessary to issue an Alert report
to notify others of the situation. The Lab Manager shall determine whether an alert
report is needed and notify the QA Manager that an alert report is needed. The QA
Manager shall ensure that such reports are issued within one business day following
the completion of Section IV of the LIR form.
5.3 Assignable Cause.
5.3.1 Test Results, whether In- or Out-of-Specification, that are obtained under the following
conditions must be invalidated and the test repeated. These are examples of the classifications
that are included on the LIR Form to document types of assignable causes:
• Sample – The original sample was contaminated or insufficient in quantity;

• Method/Documentation – Unclear test method for Standard Operating Procedure directions
which resulted in incorrect test execution;
• Analyst Error – Examples include, but are not limited to:

- incorrect sample quantity used;

- sample, sample solution, or standard spills;
- dilution errors; or
- improper test procedure; and
• Instrument/Mechanical/System Malfunction – eg incubators.
5.4 Repeating the test (Assignable Cause Identified).
All tests that were invalidated by establishment of an assignable cause are repeated. The repeat test
result(s) shall replace the original invalidated results only. The original sample preparation(s) shall be
used for this testing if:
• there is sufficient quantity of sample remaining to repeat the test;

• the assignable cause was not due to sample preparation;
• the sample preparations are stable and maintained under proper environmental conditions; and
• the sample or the sample preparations are not inadvertently contaminated.
5.4.1 LIR form Section VII Retest Protocol and Section VIII results of retest shall be used to
document the repeat tests and the results obtained. The repeat /retest protocol shall be approved
by Micro Team Leader prior to any repeat testing.
5.4.2 Microbial Limits Testing may be repeated for OOS or Questionable Results, using a 25gram
sample for solids or 25 mL for liquids.
5.5 Retesting (No Assignable Cause Identified).
5.5.1 A Retest Protocol – LIR Form Section VII “Repeat /Retest Protocol” shall be approved by the
Lab Team Leader prior to any retesting.
5.5.2 The retest protocol must be based on the specific problem identified, the history of the product,
method, and batch/lot, any applicable compendial requirements, and must delineate the number
of retests to be performed.
5.5.3 Where sample is available, retesting must be executed using the same sample set that was the
source of the original OOS or Questionable Result, unless there is scientific rationale for not
using the same sample. The rationale must be documented.
5.5.4 A control lot may be used to verify the accuracy of the analyses. The retest plan must specify
acceptance criteria for the control lot.
5.6 Resample.
5.6.1 If evidence is provided that the original sample was contaminated, the material is re-sampled
per approved sampling procedures and approval is required by QA Manager. If the approved
sampling procedure is found to be the cause, an investigation must be initiated to assess the
impact of the defective sampling technique on other batches.

5.6.2 Obtaining additional stability samples is permitted for confirmatory purposes. However, when
possible, the sample that was the source of the initial OOS or Questionable Result must also be
included in the retest plan.
5.6.3 All stability samples from the same packaging lot and stored under identical conditions are
considered equivalent for the resample purposes.
5.7 Evaluation of results.
5.7.1 If an assignable cause is associated with the original OOS or Questionable Result, the original
result is invalidated and the impact of the assignable cause on other samples in the test must be
determined.
5.7.2 Data from the original sample shall be retained in the test record, invalidated, and not included
in the batch disposition decision when an assignable cause has been established.
5.7.3 If no assignable cause is found to be associated with the original OOS, and retesting is
performed, all test results shall be documented on the LIR, forwarded to the QA Manager, and
considered in batch/lot release decisions.
5.7.4 If the test method is shown by investigation to be in question, a general review of the method
must be conducted, and required corrective action taken. Corrective action is to be recorded on
the LIR form.
5.8 Reporting Results.
5.8.1 Invalidated OOS Results shall not be averaged with retest results for reporting purposes.
5.8.2 LIR Form Section VIII “Repeat/Retest Results” shall be used to report results [i.e., the value
determined to be the final valid results (e.g., the retest results, or the confirmed initial OOS)].
5.8.3 Upon completion of the Laboratory Investigation for initial OOS results, the Laboratory
Manager determines if the QA Manager needs to issue an Alert report.
5.8.4 A confirmed OOS microbiological test result shall result in rejection of the test article.
5.8.5 In the event of a confirmed OOS Result, it may be necessary to issue an alert report to other
departments within the site to notify them of the situation. The QA Manager shall ensure that
such reports are issued within one business day following confirmation of an OOS Result.
The QA Manager shall also evaluate the need for an investigation into the source of the
confirmed OOS result and ensure that such investigations are completed.
5.8.6 The QA Manager shall inform the Senior Management Team (QO Manager, Production
Manager and the Operations Director) which shall determine if the issuance of any reports
responsible for notifying the Area Quality Review Team (A-QRT) in the event of any proposed
market Action (eg. Product Recall) or stop distribution notice.
5.9 Closing the Laboratory Investigation.

5.9.1 LIR Form Section IX “Overall Conclusions”, and if relevant, Section X “Corrective Action
Plan,” shall be completed by the Lab Team Leader.
5.9.2 The Lab Manager and the QA Manager, using LIR Form Section XI “Laboratory Investigation
Report Approval,” shall approve the completed LIR.
5.9.3 All LIRs must be completed and fully approved within 30 business days of the discovery of the
initial OOS or Questionable Result. If the Laboratory Investigation will go beyond 30 business
days, an interim status report must be issued by the Lab Manager to the QA Manager
containing at least the following:
• The date of discovery of the OOS or Questionable Result,

• Summary of the LIR general information,
• Reason for the delay,
• Current status of the investigation, and
• Estimated date for completion of the LIR.
5.9.4 The Approved LIR shall be:

• considered part of the batch record;
• distributed as per Site procedures; and
• retained by the QA according to record retention policies.
5.9.5 LIR will be reviewed by the QA and QC Manager at least once per year to determine if trends
are observed that require further corrective action.

LI Process Flowchart
OOS or Questionable Laboratory Team Leader NOTE 1: At any time during the LI, QA
Result Detected Notified and LIR Raised if Manager may issue Alerts to principals
required potentially affected
NOTE 2: Following Initial Investigation, the

OOS or Questionable Result may be
SEEK ASSIGNABLE CAUSE (AC)
accepted as valid with no further
investigational measurement or testing.
Review for obvious errors
AC Found? Yes Take Corrective Action
No
Conduct Initial Investigation

Declare Original
Results invalid
AC Found? Yes
Repeat tests to replace

No initial result
Is Resample needed? No Retesting – Establish Retest Protocol

and conduct retest
Yes Report all test results

to the QA Manager
OOS Confirmed? No
Obtain QA manager
approval for resampling Yes
Complete & Close

QA Manager LIR Form
• issues all required Alerts; and
• initiates any further required investigations

6.0 DEFINITIONS / ACRONYMS
6.1 Microbiological Laboratory Investigation:

The means by which the laboratory personnel document and investigate test results that do not
conform to a predetermined specification or are questionable. The information is documented in a
Microbiological Laboratory Investigation Report. The investigation shall be initiated when a
questionable result or an Out of Specification (OOS) result has been identified.
6.2 Assignable Cause:

The documented and scientifically justified explanation of the reason for an out of specification or
questionable result. This is determined only after conducting a Microbiological Laboratory
Investigation.
6.3 Assessment of Microbiological Contamination:

In situations where raw material or finished product specifications are not clear as to whether an
organism is objectionable, the significance of the micro -organism shall be assessed relative to the
end product use, the nature of the product, and the potential hazard to the user.
6.3.1 The significance of the micro organism(s) isolated must be assessed encompassing the
below listed criteria:
• The end product use.
• Nature of the finished product.
• Potential hazard to the user.
• Route of administration.
• Ability of the organism to degrade the product.
• Capacity of the product to support growth of the organism.
6.4 Questionable Result:

Unusual or aberrant test results which do not involve an out of specification result. Examples
of questionable results in a Microbiology Laboratory include:
6.4.1 A pour plate method for determining the total aerobic microbial count using
duplicate plates exhibited colonies on one plate in numbers that suggested the
duplicate plate may not have contained the sample inoculum preparation.
6.4.2 Microbial Identification System identifies an organism for which an expected

identification is not supported by the initial Gram stain morphology and
confirmatory testing, or the probability of the identification based on the statistical
confidence in the identification may be considered questionable and in need of
further evaluation.

6.4.3 When serial dilutions are plated, higher number of CFU's are recovered from higher
dilutions.
6.4.4 Micro-organism recovery is obtained on selective medium but no growth is

obtained on non-selective medium, both coming from the same broth preparation.
6.5 Resample:
A new sample obtained from previously sampled batch.
6.6 Retest:
Additional testing on the original test sample. Re-testing is only appropriate after a
Microbiological Laboratory Investigation has been completed, and where an assignable cause
has been identified for an OOS result during a Microbiological Laboratory Investigation.
6.7 Out of Specification Result:

A single determination of a test sample that does not conform to established specification.
6.8 Analyst Error

Situations where OOS or questionable data are the result of a failure to comply with the
approved procedure or an error during the analysis. Examples include the following; improper
test procedure, contamination through the use of non-sterile equipment, media, diluents etc.
7.0 RELATED DOCUMENTS
Form 690 – Laboratory Investigation Report Form
8.0 SUMMARY OF CHANGES
Version # Revision History

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Standard Operating Procedure

Title: Initial Investigation of Out of Specification (OOS) Results in Microbiological Laboratory

Department Micro Laboratory Document no MICLAB 145

1.0 DOCUMENT OWNER

To establish a procedure for the investigation of initial out-of-specification (OOS) or questionable

Copyright©www.gmpsop.com. All rights reserved

4.0 RESPONSIBILITY \ BUSINESS RULES

4.1 Quality Assurance Manager is responsible for:

4.2 Laboratory Manager is responsible for:

4.3 Microbiology Team Leader is responsible for:

4.4 Microbiological Analyst:

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5.1 Discovery of out-of-specification or questionable result.

• report the results to the Lab Team Leader or Lab Manager;

• (YYYYMMXXX, where YYYY and MM are representative of the computer system

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5.2 Initial Investigation.

Section I: Purpose of Investigation

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If there is no readily apparent assignable cause the investigational measurement

The purpose of conducting investigational measurement is to determine an

5.3 Assignable Cause.

• Sample – The original sample was contaminated or insufficient in quantity;

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- incorrect sample quantity used;

5.4 Repeating the test (Assignable Cause Identified).

• there is sufficient quantity of sample remaining to repeat the test;

5.5 Retesting (No Assignable Cause Identified).

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5.7 Evaluation of results.

5.8 Reporting Results.

5.9 Closing the Laboratory Investigation.

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• The date of discovery of the OOS or Questionable Result,

5.9.4 The Approved LIR shall be:

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NOTE 2: Following Initial Investigation, the

AC Found? Yes Take Corrective Action

Conduct Initial Investigation

Repeat tests to replace

Is Resample needed? No Retesting – Establish Retest Protocol

Yes Report all test results

Complete & Close

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6.0 DEFINITIONS / ACRONYMS

6.1 Microbiological Laboratory Investigation:

6.2 Assignable Cause:

6.3 Assessment of Microbiological Contamination:

• The end product use.

• Nature of the finished product.

• Potential hazard to the user.

• Ability of the organism to degrade the product.

• Capacity of the product to support growth of the organism.

6.4 Questionable Result:

6.4.2 Microbial Identification System identifies an organism for which an expected

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6.4.4 Micro-organism recovery is obtained on selective medium but no growth is

6.7 Out of Specification Result:

6.8 Analyst Error

7.0 RELATED DOCUMENTS

Form 690 – Laboratory Investigation Report Form

8.0 SUMMARY OF CHANGES

Version # Revision History

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