ICU M ED IC A L SU P P O RT ING INF O R M AT IO N

ChemoClave™ Closed System Transfer Device (CSTD)
Microbial Ingress Study
Report of a study commissioned by ICU Medical, Inc. and conducted by AAIPharma Services Corp.

Background
The ChemoClave CSTD is a needlefree closed system transfer device (CSTD) that meets the definition recognized by USP<800> as “a drug transfer device that mechanically prohibits the transfer of environmental contaminants into the systemand the escape of the hazardous drug or vapor concentrations outside the system.”1,2 To validate a CSTD’s ability to“mechanically prohibit the transfer of environmental contaminants into the system,” microbial ingress test methods havebeen developed and commissioned by ICU Medical, Inc.
The US Food and Drug Administration (FDA) has issued a guidance document related to microbial ingress testing as partof the premarket clearance 510(k) process for intravascular administration sets.3 This guidance document serves to assist
manufacturers in the development of appropriate test methods, types of microorganisms to consider in the study, andsample sizing. Microbial ingress test results are also included in the documentation submitted to the FDA to obtain 510(k)clearance. The ChemoClave Closed System Drug Transfer Device was cleared under K173477 with the following Indications
for Use: The ChemoClave CSTD mechanically prohibits the transfer of environmental contaminants, including bacterial
and airborne contaminants, into the system and the escape of drug or vapor concentrations outside the system.4

Introduction
The purpose of this microbial ingress study was to evaluate the ChemoClave CSTD’s ability to prevent microbial
contamination of the system during a worst-case, simulated use test model. The test model included the artificial
contamination of the ChemoClave CSTD, followed by a decontamination process and multiple activations over a 7-day
use life.
This white paper describes the methods including the types of challenge organisms used, the microbial recovery methods,
sample size, controls, procedures including the inoculation methods, the environmental conditions, the validation
methods, and the rationales used for these choices.5Methods
Organisms (Four Bacterial Strains)
The organisms chosen for this study include 2 gram-negative and 2 gram-positive organisms, which represent normal
microbiological flora found in a clinical setting. The target concentration of 1-5x103 represents a higher-than-expected
microbial contamination load during worst-case clinical use.
> Staphylococcus aureus (ATCC 6538)> Pseudomonas aeruginosa (ATCC 9027)> Staphylococcus epidermidis (ATCC 12228)> Klebsiella pneumoniae (ATCC 4352)

Microbial Recovery Study
The microbial recovery study is designed to accurately estimate the number of microorganisms present in the test solutionto demonstrate a method to confirm microbial inoculum levels. This study was done following the below methods. TheChemoClave CSTD septum was inoculated in duplicate with an appropriate volume of inoculum to yield 1-5x103 colonyforming units (CFUs) of each test organism listed in the report under Organisms, and then allowed to dry for 1 minute atambient conditions. After drying, the Clave™ needlefree connector was placed in 10 mL of sterile 0.9% sodium chloride
and was mixed using a vortex mixer. The positive control was prepared by inoculating a 10 mL volume of 0.9% sodium
chloride with the same inoculum volume used to inoculate the port, followed by 1 mL aliquots being plated to trypticase
soy agar (TSA) and incubated at 30–35°C for 1–2 days.
After incubation, the lab performed the colony counts to determine the percent recovery as
Percent recovery =CFU recovery fluid
CFU inoculum count

X 100

The acceptance criteria of the percent recovery must be greater than 70%. As seen in Table 1, all microbial recovery testing
meets this criterion.
Table 1. Microbial Recovery Studies Results

Device Recovery
Average (CFU)Positive Control
Recovery Average(CFU)

Percent
Recovery

Meets
Criteria

Staphylococcus aureus*

280

290

97%

Yes

Staphylococcus epidermidis

132

183

72%

Yes

Pseudomonas aeruginosa

188

230

82%

Yes

Klebsiella pneumoniae

150

151

99%

Yes

Test Organism* Results are estimated; number of colonies recovered was greater than 250 CFU/plate

Sample Size
The study evaluated 6 replicate ChemoClave CSTD septa, which were inoculated with 4 separate organisms and accessed
14 times. The sample size represents the worst-case clinical use and is in alignment of the FDA’s requirements for themicrobial recovery of medical devices.

Inoculum Concentration
1-5x103 colony-forming units (CFUs) for each activation

Procedure
Each ChemoClave CSTD septum was inoculated with the test organisms before each activation. The ChemoClave CSTD
was then disinfected with a 70% isopropyl alcohol (IPA) prep pad using an aggressive circular motion for not less than 3
seconds and allowed to dry. The ChemoClave CSTD was activated using the Spiros™ closed male luer syringe assembly,
where 10 cc of normal saline was transferred through the ChemoClave CSTD, and the transferred saline was collected andfiltered through a 0.45 µ m cellulose nitrate filter. The filter was rinsed with 100 mL of 0.9% saline, transferred to a solidified
plate of TSA, incubated for a minimum of 48 hours at 30–35 °C, and examined for growth.
This process was repeated for a total of 14 times using artificial contaminations, followed by 2 activations each day,for 7 days. The total number of artificial contamination and fluid transfer events was 14.

Positive Controls
The positive controls were performed in duplicate for each organism by performing the activation and testing procedure
above without the 70% IPA disinfection step.

Negative Controls
Negative controls were performed following the same procedure as the test samples with the omission of the microbial
inoculation.

Results
Results for the microbial ingress are shown in Tables 2–5. There was no microbial recovery on the negative controls.
Table 2. Staphylococcus Aureus Microbial Ingress Results

Activation

Replicate (CFU/Filter)

Table 3. Staphylococcus Epidermidis Microbial Ingress Results

123456

Inoculum
Verification(CFU/Device)

Activation

Replicate (CFU/Filter)
123456Inoculum
Verification(CFU/Device)

DAY 1120000001.7 X 103

DAY 1120000002.4 X 103

DAY 234000000

1.8 X 103

DAY 2340000002.2 X 103

DAY 3

560000001.1 X 103

DAY 3

56000000

1.2 X 103

DAY 4

78000000

1.2 X 103

DAY 4

780000002.2 X 103

DAY 5

9100000001.1 X 103

DAY 5

910000000

1.3 X 103

DAY 611120000001.4 X 103

DAY 61112000000

1.5 X 103

DAY 713140000002.2 X 103

DAY 71314000000

1.2 X 103

Positive Control Average (CFU/Filter)

24

Table 4. Pseudomonas Aeruginosa Microbial Ingress Results

Activation

Replicate (CFU/Filter)

Positive Control Average (CFU/Filter)

61

Table 5. Klebsiella Pneumoniae Microbial Ingress Results

123456Inoculum
Verification(CFU/Device)

Activation

Replicate (CFU/Filter)
123456Inoculum
Verification(CFU/Device)

DAY 1120000002.2 X 103

DAY 1120000002.0 X 103

DAY 2340000001.4 X 103

DAY 2340000001.1 X 103

DAY 3

560000001.2 X 103

DAY 3

560000001.4 X 103

DAY 4780000001.1 X 103

DAY 4780000002.3 X 103

DAY 5

9100000001.3 X 103

DAY 5

9100000002.4 X 103

DAY 611120000001.6 X 103

DAY 611120000002.2 X 103

DAY 713140000001.5 X 103

DAY 713140000002.2 X 103

Positive Control Average (CFU/Filter)

106

Positive Control Average (CFU/Filter)

22

Conclusion
Based on the results of the microbial ingress testing as reported above, it is determined that the ChemoClave CSTD meets
established criteria for microbial ingress. As part of the devices 510(k) clearance, data was provided to demonstrate theChemoClave system’s ability to prohibit the transfer of environmental contaminants, including bacterial and airborne
contaminants, into the system as reflected in the devices Indications for Use statement.

References
1.

The United States Pharmacopeial Convention. USP General Chapter <800> Hazardous Drugs – Handling in Healthcare Settings. Chapter 14:Administering. 20172.

National Institute for Occupational Safety and Health (NIOSH): A Performance Test Protocol for Closed System Transfer Devices Used During
Pharmacy Compounding and Administration of Hazardous Drugs. NIOSH Docket Number 288-A

3.

Guidance for Industry and FDA Staff: Intravascular Administration Sets Premarket Notification Submissions [510(k)], US Department of Healthand Human Services, Food and Drug Administration, 2008, Rockville, MD. http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm070850.pdf

4.

ICU Medical ChemoClave 510(k) Summary K173477

5.

AAIPharma, Microbial Ingress Study on ChemoClave, September 2013

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