Film Data Package for GIBCO® Liquid Media Bags - Invitrogen

Film Data Package for 

GIBCO ® 

Liquid Media Bags

Table of Contents 

Preface 

Film Technical Data 

Film Technical Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .05 

Biological Testing 

USP Class VI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .07 

ISO Intramuscular Implantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .08 

ISO Systemic Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .09 

ISO Intracutaneous Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 

Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 

Hemolysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 

Kligman Maximation Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18 

Physicochemical Testing 

United States Pharmacopoeia Conformity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 

European Pharmacopoeia Conformity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21 

Japanese Pharmacopoeia Conformity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22 

Thai Industrial Standard Conformity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23 

Endotoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24 

Leachable Study 

Leachable Summary Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26 

Leachable Study Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27 

Animal Origin Free Conformity 

Animal Origin Free Conformity Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40 

©2010 Invitrogen Corporation. All rights reserved. These products may be covered by one or more Limited Use Label Licenses (see Invitrogen catalog or www.invitrogen.com). By use of these products you accept the 

terms and conditions of all applicable Limited Use Label Licenses. For research use only. Not intended for any animal or human therapeutic or diagnostic use, unless otherwise stated. CO11506-0210 

www.invitrogen.com 

2

Throughout the basic research and industrial sectors, the name GIBCO® is synonymous with quality sera, media, and cell culture reagents. 

This reputation has been built on the understanding that when a company can consistently deliver quality products that reduce uncer- 

tainty, trust is the inevitable result. GIBCO® products embody the high performance and proven reliability that allow scientists to focus 

their attention on more important matters than troubleshooting, providing a stable foundation for successful discovery. 

The GIBCO® film—A better material for better 

media delivery 

While the media bag films we currently use are fully validated 

and acceptable, there is a growing need for a clearer, multi-layer, 

single-web film that can be converted into a wide range of bag 

sizes—from a researcher’s bench top to the needs of the larg- 

est industrial user. Invitrogen has found such a film—the GIBCO® 

film—that can be used for all sizes of media bags, greatly simpli- 

fying your validation process. Additionally, it is superior to other 

commercially available films in many ways, including gas trans- 

mission rates, leachable/extractable properties, and clarity. 

Technical overview of GIBCO® film 

Film construction 

Many media bag films are dependent upon the size of bag 

requested; bags with less than fifty liters capacity typically have 

an EVA contact layer, while bags that are hundred liters or larger 

are most often constructed with a polyethylene contact layer. 

The GIBCO® film is a very clear, Film multilayered, structure for the single-web 3-ply film film 

PA* 

20 µm 

with a polyethylene contact surface that Outer can layer be made into a 

VLDPE 

70 µm 

LDPE 

3-ply 

EVOH Gas barrier layer 80 µm 

evaluated). In addition, the film exhibits exceptional gas barrier 

LDPE 

Total thickness = 320 µm 

Figure AL 1—GIBCO® = Adhesive layer; film structure. EVOH = Ethyl Employing vinyl alcohol; single-ply construction and a ULDPE con- 

LDPE = Low-density polyethylene; PA = Polyamide, 

tact PE layer, = Polyethylene; GIBCO® ULDPE film = Ultra can low-density be used for all sizes of media bags. 



1-ply 

VLDPE 

PE 

AL 

EVOH 

AL 

ULDPE 

Contact layer 70 µm 

physical properties of our film are summarized in Table 1. 

Total thickness = 240 µm 

polyethylene; VLDPE = Very low-density polyethylene 

Preface 

The GIBCO® film is a single-ply material with a ULDPE contact 

layer (Figure 1), and is suitable for use in all sizes of media bags. 

Physical properties 

wide range of bag sizes (1L–1,000 L; other custom sizes can be 

properties with very low extractable and leachable results. The 

Film structure for single-web film (9101) 

Outer layer 

Adhesive layer 

Gas barrier layer 

Adhesive layer 

50 µm 

10 µm 

20 µm 

10 µm 

Contact layer 230 µm 

*PA is only present on sizes greater than 200 liters 

Table 1—Technical summary of GIBCO® film properties. 

Physical Property* Dimension 

Value 

(before/after gamma sterilization † ) Procedure 

Haze % 5/5 ASTM D-1003 

Clarity % 98/98 ASTM D-1003 

Transmittance % 93/93 ASTM D-1003 

Tensile strength at break MPa 14/13 ASTM D-882 

Elongation at break % 280/300 ASTM D-882 

Elastic modulus MPa 370350 ASTM D-882 

Break at cold temperature °C < –45/< –45 ISO 8570 

Density g/cm3 0.9 ASTM D-792 

Water vapor transmission rate ‡ 

g/(m3•day) 0.35/0.32 ASTM F-1249 

Oxygen permeability ‡ 

cm3/(m2•day•atm)

Testing the GIBCO® film 

In order to establish its suitability as a media bag material, the 

GIBCO® film has been extensively and rigorously tested for a 

wide range of properties that impact its performance in research 

and bioproduction applications: 

Biological suitability tests are designed to determine the 

biological reactivity of mammalian cell cultures and biologi- 

cal response of animals. A material’s biological acceptability is 

determined by certifying that a film meets certain industry rec- 

ognized standards such as EP and USP. USP Class VI certification 

is widely known and achieved through a series of Systemic Injec- 

tion, Implantation, and Intracutaneous tests; additionally, Elution 

tests are often included as part of the biological testing. 

Physicochemical tests are designed to chemically charac- 

terize the primary contact surfaces and its extracts. These are 

typically characterized through an industry recognized set of 

standards such as the USP , “Physicochemical Tests – Plas- 

tics”. The USP standard helps characterize the type of extracts 

through the use of non-volatile residue, residue on ignition, 

heavy metals and buffering capacity. These tests actively/aggres- 

sively try to pull chemicals from the test subject. 



Chemical compatibility is the interaction of a solution, 

medium or reagent with that of a packaging system. Under- 

standing the interaction between the formulation and packag- 

ing system enables the product to be successfully stored and 

shipped without compromising either one. 

Leachability testing is a passive way of determining which 

chemicals migrate from the film to the medium within the con- 

tainment system. This test is conducted in real-time, under nor- 

mal processing and environmental conditions. 

Animal origin material refers to any animal originated 

component or additive that may have been introduced into a 

packaging system through either the resin production or the 

manufacturing conversion processes. The films used for Invitro- 

gen’s products are animal origin–free. 

Globally recognized compendia testing is performed on 

the packaging materials. These compendia include (but are not 

limited to) EP, USP, and JP. The use of industry-recognized stan- 

dard testing minimizes excursions in test methods from vendors 

and manufacturers alike. 

The results of these tests are described in detail in the 

remainder of this document. 


4

Film Technical Data

Compound: GIBCO® 

Product: INFUFLEX BARRIER 

Main polymers: PE, EVOH 

Main application areas: Biotechnology applications, high oxygen barrier 

Physical property* Dimension 

Value 

Before/after gamma sterilization 



Procedure 

Haze % 5 / 5 ASTM D-1003 

Clarity % 98 / 98 ASTM D-1003 

Transmittance % 93 / 93 ASTM D-1003 

Tensile strength at break MPa 14 / 13 ASTM D-882 

Elongation at break % 280 / 300 ASTM D-882 

Elastic Modulus Mpa 370 / 350 ASTM D-882 

Break at cold temperature °C below -45 / below -45 ISO 8570 

Density g/cm3 0.9 ASTM D-792 

Water vapour transmission rate** g/(m2.day) 0.35 / 0.32 ASTM F-1249 

Oxygen Permeability** cm3/(m2.day.atm) < 0.05 / < 0.05 ASTM D-3985 

Carbondioxide Permeability** cm3/(m2.day.atm) < 0.2 / < 0.2 ASTM F-2476 

Film thickness: 0.325 mm 

Film width: 850 mm 

Recommended sealing method: Heat sealing 

Recommended sterilization method: Gamma 

Formulation characteristics: • Multi layer structure with inert PE 

fluid contact layer 

• Low extractables/leachables 

• No animal derived ingredients 

*Gauge test film 0.325 mm 

Gamma sterilization dose 25 kGy except **50 kGy 

Film Technical Data 

This technical information consists of typical product data and should not be used as a specification 2007/09 

The data contained in this document are provided in good faith for the sake of general information. It is deemed to be accurate at the time of going into press. The purchaser 

or user is required to verify with our technical services whether any specific application is appropriate. Freedom of possession, use or marketing under intellectual property 

rights or legal provisions or regulations, whether national or local, must be duly considered before use. Under no circumstances should any Solmed film, tubing or compound 

be used in any cosmetic, reconstructive or any other temporary or permanent bodily implant application. 


6

Biological Testing

Technical initiation: 11/30/2006 

Technical completion: 12/7/2006 

Report date: 12/11/2006 

Project number: 06-5591-G1 

Test Article Compound GIBCO® gamma gesteriliseerd 

Lot/Batch # 634PN 

Study Class VI Test − USP 

Ratio 120cm2/20 mL 

Vehicles 

USP 0.9% Sodium Chloride for Injection (NaCl), Cottonseed Oil (CSO), 1 in 20 Ethanol in NaCl (EtOH), and Polyethylene 

Glycol 400 (PEG) 

Extraction conditions 50 ± 2 °C for 72 ± 2 hours 

Comments None 

References 

The study was conducted based upon the following references: 

USP 29, NF 24, 2006. Biological Reactivity Tests, In Vivo. 

Draize Scale for Scoring Skin Reactions, Draize, J.H. “Dermal Tox- 

icity”, Appraisal of the Safety of Chemicals in Foods, Drugs and 

Cosmetics − Dermal Toxicity, pp. 49−52. Association of Food and 

Drug Officials of the United States, Topeka, Kansas, 1965. 

ISO/IEC 17025, 2005, General Requirements for the Compe- 

tence of Testing and Calibration Laboratories. 

General procedure 

The extraction conditions were performed as stated above. The 

test article extracts and corresponding blanks were injected sys- 

temically and intracutaneously in mice and rabbits, respectively. 

The injections were in the amounts and routes set forth by USP, 

including the further dilution of the extracts prepared with PEG. 

The animals were observed for signs of toxicity and skin reactiv- 

ity for up to 72 hours post treatment. In addition, the test article 

was implanted into the paravertebral muscles of rabbits for 7 

days and observed macroscopically for signs of hemorrhage, 

necrosis, discoloration, encapsulation, and infection. 

Results 

None of the mice injected with the test article extracts exhibited 

any signs of toxicity in the Systemic Injection Test. In addition, 

none of the rabbits injected intracutaneously with the test article 

extracts exhibited any signs of erythema, edema or clinical tox- 

icity. In both the Systemic and Intracutaneous Tests the controls 

were normal through 72 hours. Also, the implant sites exhibited no 

significant signs of hemorrhage, necrosis, discoloration, encapsu- 

lation, or infection compared with the control sites. 

Conclusion 

The test article meets the requirements of the guidelines for the 

Biological Test for Plastics, Class VI − 50°C. 



USP Class VI 

Test results 


8

2 Week Implantation 

Project Number: 06-5174-G3 

Test Article: Compound GIBCO®, gamma-gesteriliseerd 

The relative size of the involved area was scored by assessing the 

width of the area from the implant/tissue interface to unaffected 

areas which have the characteristics of normal tissue and normal 

vascularity. Relative size of the involved area was scored using 

the following scale: 

0 = 0 mm No site 

0.5 = up to 0.5 mm Very slight 

1 = 0.6 − 1.0 mm Mild 

2 = 1.1 − 2.0 mm Moderate 

3 = > 2.0 mm Marked 

For each implanted site, a total score is determined. The average 

score of the test sites for each animal is compared to the average 

score of the control sites for that animal. The average difference 

between test and controls for all animals is then calculated and 

the initial Bioreactivity Rating is assigned as follows: 

0 − 1.5 No Reaction* 

> 1.5 − 3.5 Mild Reaction 

> 3.5 − 6.0 Moderate Reaction 

> 6.0 

* A negative calculation is reported as zero (0). 

Marked Reaction 

ISO Intramuscular Implantation 

Test results 

Results 

Macroscopic 

Macroscopic evaluation of the test article implant sites indicated 

no significant signs of inflammation, encapsulation, hemorrhage, 

necrosis, or discoloration at the 2 week time period. 

Microscopic 

Microscopic evaluation of the test article implant sites indicated 

no significant signs of inflammation, fibrosis, hemorrhage, necro- 

sis, or degeneration as compared to the control article sites. The 

Bioreactivity Rating for the 2 week time period (average of three 

animals) was 0.1, indicating no reaction as compared to the con- 

trol implant sites. 

Conclusion 

The test article was implanted in the paravertebral muscle tis- 

sue of New Zealand White rabbits for a period of 2 weeks. The 

results indicate that the test article does not demonstrate any 

remarkable difference as compared to the control implant sites 

(Bioreactivity Rating of 0.1) when implanted for 2 weeks. 




9





Test Article Compound GIBCO®, gamma-gesteriliseerd 

Lot/Batch # 634PP 

Study Systemic Injection Test − ISO 

Ratio 6 cm2/1 mL 

Vehicle USP 0.9% Sodium Chloride for Injection (NaCl) and Cottonseed Oil (CSO) 

Extraction Conditions 70 ± 2 °C for 24 ± 2 hours 

Comments None 

References 

The study was conducted based upon the following refer- 

ences: ISO 10993−11, 1993, Biological Evaluation of Medical 

Devices − Part 11: Tests for Systemic Toxicity. ISO 10993−12, 2002, 

Biological Evaluation of Medical Devices − Part 12: Sample Prepa- 

ration and Reference Materials. 



General Procedure 

The Systemic Injection Study is designed to screen solutions 

and test article extracts for potential toxic effects as a result of 

a single−dose systemic injection in mice. The extraction condi- 

tions were performed as stated above. The test article extracts 

were injected intravenously (NaCl) and intraperitoneally (CSO) 



ISO-Systemic Injection 

Test results 

at 50 mL per kg, in groups of five mice. Similarly, groups of five 

mice were injected with the control articles (vehicles). The ani- 

mals were observed for signs of biological reactivity for 72 hours 

post inoculation. 

Results 

None of the animals injected with the test article extracts or the 

control articles (vehicles) exhibited any signs of toxicity through 

the observation period. 

Conclusion 

The animals treated with the test article extracts did not exhibit 

biological reactions greater than the controls. Therefore, the test 

article meets the requirements of the ISO 10993−11 guidelines 

for the Systemic Injection Test. 


10







Study Intracutaneous Injection Test − ISO 


Vehicles USP 0.9% Sodium Chloride for Injection (NaCl) and Cottonseed Oil (CSO) 


Comments None 

References 


ISO 10993−10, 2002, Biological Evaluation of Medical Devices − 

Part 10: Tests for Irritation and Delayed−Type Hypersensitivity. ISO 

10993−12, 2002, Biological Evaluation of Medical Devices − Part 

12: Sample Preparation and Reference Materials. 



General Procedure 

The Intracutaneous Test is designed to evaluate local responses 

to the extracts of test articles, following intracutaneous injection 

into rabbits. The extraction conditions were performed as stated 

above. Control extracts were prepared, in a similar manner, with 

each extracting medium. Two rabbits were injected intracutane- 

ously, using one side of the animal for one test article extract 

and the other side for the other extract, at 0.2 mL per site. The 

injected sites were examined immediately after injection and at 

24, 48, and 72 hours post inoculation for gross evidence of tissue 

reaction such as erythema, edema, and necrosis. Observations 

were scored according to the Classification System for Scoring 

Intracutaneous Injection 

Test results 

Skin Reactions and included all clinical signs. All average ery- 

thema and edema scores for the test and control sites at 24, 48, 

and 72 hours were totaled separately and divided by 12 (2 ani- 

mals × 3 scoring periods × 2 scoring categories) to determine the 

overall mean score for the test article versus the corresponding 

control article. The requirements of the test are met if the differ- 

ence of the mean reaction score (erythema/edema) for the test 

article and the control article is 1.0 or less. 

Results 

Both of the test animals increased in weight. None of the animals 

exhibited overt signs of toxicity at any of the observation points. 

The requirements of the test were met because the difference 

of the mean reaction score for the test article and control article 

was 0.0. 

Conclusion 

The test article meets the requirements of the Intracutaneous 

Test, ISO 10993−10 guidelines using extracts prepared with NaCl 

and CSO. 




11

Conclusion 

GIBCO® batch 729XC, meets the requirements described in the Solmed Cytotoxicity Test Method. 



Cytotoxicity 

Test results 

Nature of control Cytotoxicity 

Result + 

Lower limit 0 

Upper limit 2+ 

We herewith represent that at the time of delivery to the customer, the material will conform to the specification in our brochure and databases and that the material has been 

tested in accordance with the methods set forth on this Certificate of Analysis and has achieved the results disclosed herein. We make no other warranty, express or implied, 

and we hereby disclaim any warranty that the material : (i) is fit for any particular purpose or intended use by the customer, or (ii) is in compliance with any application law, 

regulation or ordinance. 


12

Hemolysis − Human Blood − ISO 


Test Article: GIBCO® gamma-gesteriliseerd 

Toxikon final GLP report: 06-5174-G5 

Test Article GIBCO® gamma-gesteriliseerd 

Study Hemolysis - Human blood - ISO 

Compliance 

21 CFR, Part 58 

Good Laboratory Practice for Non−Clinical Laboratory Studies 

Final Report Date November 3, 2006 



Hemolysis 

Test results 


13




Table of contents 

Title Page 

Table of Contents 

Study Summary 

Quality Assurance Statement 

Study Director Signature and Verification Dates 

1.0 Purpose 

2.0 References 

3.0 Compliance 

4.0 Identification of Test and Control Articles 

5.0 Identification of Test System 

6.0 Justification of Test System and Route of Administration 

7.0 Experimental Design and Dosage 

8.0 Evaluation Criteria 

9.0 Results 

10.0 Conclusion 

11.0 Records 




14




Study summary 

The test article, GIBCO® gamma-gesteriliseerd, exhibited 0.0% 

hemolysis. The test article is considered non−hemolytic under 

the experimental conditions employed. 

Quality assurance statement 

This study was conducted in compliance with U.S. Food and 

Drug Administration regulations set forth in 21 CFR, Part 58. 

Inspections Date of inspection Date reported study director Date reported management 

Addition of blood 10/24/06 10/24/06 10/24/06 

Raw data 11/03/06 11/03/06 11/03/06 

Final report 11/03/06 11/03/06 11/03/06 



The sections of the regulations not performed by or under 

the direction of Toxikon Corporation, exempt from this Good 

Laboratory Practice Statement, included characterization and 

stability of the test article and its mixture with carriers, 21 CFR, 

Parts 58.105 and 58.113. 

The Quality Assurance Unit conducted inspections on the following dates. The findings were reported to the Study Director and to 

Toxikon’s Management. 

Study director signature and verification dates 

This study meets the technical requirements of the protocol. The study also meets with the requirements of the Good Laboratory Prac- 

tice Regulations, 21 CFR, Part 58, with the exemptions as stated in the Quality Assurance Statement. 

Verification dates: 

Protocol Effective Date: 10/12/06 

Test Article Receipt: 10/17/06 

Project Log Date: 10/20/06 

Technical Initiation: 10/24/06 

Technical Completion: 10/24/06 


15




1.0 Purpose 

The purpose of the study was to assess the hemolytic activity of 

the test article in direct contact with human blood. 

2.0 References 


2.1 ISO 10993−4, 2002, Biological Evaluation of Medical 

Devices − Part 4: Selection of Tests for Interactions with 

Blood, as amended 2006. 

2.2 Autian Method, ATTP−I, Material Sciences Toxicology 

Laboratories, University of Tennessee Center for the 

Health Sciences, Memphis, TN, April 18, 1977. 

2.3 Hemolysis − Rabbit Blood, Evaluation of Hemodialyz- 

ers and Dialysis Membranes, DHEW Publication # (NIH) 

77−1294, pg. 213, 1977. 

2.4 ISO 10993−12, 2002, Biological Evaluation of Medical 

Devices − Part 12: Sample Preparation and Reference 

Materials. 

2.5 ISO/IEC 17025, 2005, General Requirements for the 

Competence of Testing and Calibration Laboratories. 

3.0 Compliance 

The study conformed to the current FDA 21 CFR, Part 58 − Good 

Laboratory Practice for Non−Clinical Laboratory Studies. 

4.0 Identification of test and control articles 

The Sponsor supplied the following information on a test requisi- 

tion form or other correspondence, wherever applicable (exclud- 

ing confidential or trade secret information). The Sponsor was 

responsible for all test article characterization data as specified 

in the GLP regulations. 

4.1 Test Article: 



Test Article Name: GIBCO® gamma-gesteriliseerd 

CAS/Code #: Not Supplied by Sponsor (N/S) 

Lot/Batch #: 634PP 

Physical State: N/S 

Color: N/S 

Expiration Date: N/S 

Density: N/S 

Stability: N/S 

Solubility: N/S 

pH: N/S 

Storage Conditions: Room Temperature 

Safety Precautions: Standard Laboratory Safety 

Precautions 

4.2 Control Articles (Toxikon Supplied): 

4.2.1 Negative Control Article Name: USP 0.9% Sodium 

Chloride for Injection (NaCl) 

Toxikon QC #: CSC-06-09-003-GT 

Physical State: Liquid 

Color: Colorless 

Stability: Stable at Room Temperature 



Precautions 

4.2.2 Positive Control Article Name: Sterile Water for 

Injection (SWFI) 

Toxikon QC #: CSC-06-08-036-CC 

Physical State: Liquid 

Color: Colorless 

Stability: Stable at Room Temperature 



Precautions 


16




5.0 Identification Of Test System 

Human blood was utilized in evaluating the effect of a test arti- 

cle on the cellular components of blood when in direct contact. 

Human blood was obtained from donors at Toxikon Corporation, 

Bedford, MA. 

6.0 Justification Of Test System And Route Of 

Administration 

6.1 The system for the determination of hemolytic activity 

of a test article, when in direct contact with human 

blood, is recommended in the DHEW Publication, 

Evaluation of Hemodialyzers and Dialysis Membranes. 

The guidelines have no alternative methods. 

6.2 The system for the determination of hemolytic 

activity of a test article when in direct contact with 

human blood is designed to simulate the conditions 

of the use of the test article. Evaluation of blood 

compatibility was a significant screening test because 

elevated plasma hemoglobin levels reflect red blood 

cell lysis upon direct contact with material and devices 

and is a requirement to assess effects on blood, per 

ISO 10993−4 guidelines. 

6.3 The test article was administered in vitro, directly 

to the test system. The route of administration was 

recommended by the references in Section 2.0. 

7.0 Experimental Design And Dosage 

7.1 Pre−Dose Procedure: 

Blood Sample: 



Fresh, whole human blood was collected into EDTA− 

coated vacutainer tubes and was utilized within 

one hour of collection. It was diluted sufficiently in 

NaCl until 0.2 mL was hemolyzed in 10 mL of SWFI 

and the spectrophotometric reading at 545 nm was 

approximately 1.0 Absorbance unit (A). 

7.2 Dose Administration: 

7.2.1 Test Article: 

The test article was added to each of three test vials 

containing NaCl at a ratio of 0.5 g test article per 10 

mL of NaCl. 

7.2.2 Positive Control Article: 

A volume of 10 mL of SWFI was added to each positive 

control vial. 

7.2.3 Negative Control Article: 

A volume of 10 mL of NaCl was added to each negative 

control vial. 

7.2.4 Replication: 

The test article as well as the positive and negative 

controls were tested in triplicate. 

7.3 Post−Dose Procedure: 

7.3.1 All vials were incubated in a 37 ± 2 °C water bath for 

30 minutes. 

7.3.2 Diluted human blood was added to all vials at a ratio 

of 0.2 mL blood per 10 mL article. 

7.3.3 All vials were then incubated in a 37 ± 2 °C water 

bath for 60 minutes. After incubation, the vials were 

centrifuged for five minutes at approximately 500 x g. 

7.3.4 Absorbance of each supernatant was determined 

against a NaCl blank at 545 nm. 


17




8.0 Evaluation Criteria 

8.1 The average absorbance values are used to determine 

the percent (%) Hemolysis of the test article. 

% Hemolysis = Ave. Abs. of Test Article − Ave. Abs. of 

Negative Control × 100 

Ave. Abs. of Positive Control − Ave. Abs. of Negative 

Control 

8.2 The test article is considered hemolytic if it causes 

greater than 5% hemolysis. 

8.3 If deemed necessary by the Study Director, a retest 

is performed using fresh blood from the same donor 

and a new test article. 

8.4 The study and its design employ methodology to 

9.0 Results 

minimize uncertainty of measurement and control of 

bias for data collection and analysis. 

Test Article Tube #1 Absorbance = 0.011 



Positive Control Article Tube #1 Absorbance = 1.003 



Negative Control Article Tube #1 Absorbance = 0.015 



Average Test Article Absorbance = 0.013 

Average Positive Control Article Absorbance = 1.004 

Average Negative Control Article Absorbance = 0.014 

Hemolysis = 0.0% 

10.0 Conclusion 

The test article, GIBCO® gamma-gesteriliseerd, exhibited 0.0% 

hemolysis. The test article is considered non−hemolytic under 

the experimental conditions employed. 

11.0 Records 



11.1 Original raw data are archived at Toxikon Corporation. 

11.2 A copy of the final report and any report amendments 

is archived at Toxikon Corporation. 

11.3 The original final report, and a copy of any protocol 

amendments or deviations, is forwarded to the 

Sponsor. 

11.4 All unused test article shall be disposed of by Toxikon. 


18

Technical Initiation: 10/31/2006 

Technical Completion: 11/27/2006 

Report Date: 2/27/2007 




Study Kligman Maximization Test - ISO 


Vehicle USP 0.9% Sodium Chloride for Injection (NaCl) and Cottonseed Oil (CSO) 


Comments None 

References 



Part 10: Tests for Irritation and Delayed−Type Hypersensitivity. 


Part 12: Sample Preparation and Reference Materials. Zhai, H. and 

H.I. Maibach, eds. Dermatotoxicology. 6th edition. Boca Raton: 

CRC Press, 2004. 729−732. Magnusson, B. and A.M. Kligman. “The 

Identification of Contact Allergens by Animal Assay. The Guinea 

Pig Maximization Test.” J. Invest. Dermatol. 52 (1969): 268−276. 

Magnusson, B. and A.M. Kligman, Allergic Contact Dermatitis in 

the Guinea Pig. Identification of Contact Allergens. Springfield, 

IL.: Thomas, 1970. 



General procedure 

The purpose of the study was to detect the allergenic potential 

of a test article. Hartley guinea pigs, 20 experimental, 10 nega- 

tive control, and 5 positive control, were used for this study. The 

test article was extracted at the conditions specified above. The 

Induction Phase (Day 0) was conducted by intradermally inject- 

ing the test article extracts or controls. The Topical Application 



Kligman Maximization 

Test results 

Phase (Day 7) was conducted by applying the test article extract 

or control article for 48 hours, at the site of the intradermal injec- 

tions. The 24 hour Challenge Phase was performed on Day 23. 

Test and control animals were scored for erythema and edema 

according to the Classification System for Scoring Skin Reactions 

at 24, 48, and 72 hours post Challenge Phase. The study and its 

design employed methodology to minimize uncertainty of mea- 

surement and control or bias for data collection and analysis. 

Results 

The skin sites that were exposed to the test article extracts and 

negative control showed no signs of erythema or edema. The 

skin sites exposed to the positive control showed the expected 

signs of erythema and edema. 

Conclusion 

The skin treated with the test article extracts exhibited no reac- 

tion to the challenge (0% sensitization). Therefore, as defined by 

the scoring system of Kligman, this is a Grade I reaction and the 

test article extracts (as prepared) are classified as having weak 

allergenic potential. A Grade I sensitization rate is not considered 

significant. 


19

Physicochemical Testing

Title 

Tests according toUSP28/NF20, Edition 2006, General Chapter 

, page 2657-2658, on Solmed® Formulation GIBCO® inner- 

layer gamma radiated 25 kGy 

Background 

General Chapter USP CONTAINERS describes several 

chemical tests. The laboratory of Renolit Nederland BV has per- 

formed the tests according to European Pharmacopoeia 5th edi- 

tion, supplement 2005, 3.1.6 and specific according to USP 28/ 

NF20, Edition 2006, General Chapter , page 2657-2658 for 

Buffering Capacity and Novolatile Residu.. This combination of 

test results was performed to show compliance with all USP 661 

specifications for plastic containers. 

Methods of analysis 

See USP28/NF20, Edition 2006, General Chapter , page 

2657-2658 

Conclusion 

The tested formulation meets the test-requirements as described 

in USP28/NF20, Edition 2006, General Chapter CONTAIN- 

ERS, Physico-Chemical Tests – Plastics. 

Test Result Specification 

Buffer Capacity 0.10 ml Difference between volume sample and blank

Product tested: Compound GIBCO® 

Batch: 200609A-Innerlayer gamma radiated 25kGy 

Sample code: 2007020315 

Quality System: ISO9001/ISO13485 

Test Method 

See European Pharmacopoeia 5th edition 3.1.5, 2005 Material 

based on Polyethylene for containers and closures for prepara- 

tion and for parenteral and ophthalmic use 

Analysis realized by 

Quality Control Laboratory Renolit Nederland BV 

Conclusion 



EP Conformity 

Test results 

Renolit Nederland B.V compound GIBCO® batch 200609A-Inner- 

layer gamma irradiated 25kGy meets the relevant test require- 

ments described in the European Pharmacopoeia 5th edition. 

Monograph 3.1.5, 2005 

Nature of Control Results Specification 

Characters Pass Pass 

Identifiction A-I.R. spectrophotometry Pass Pass 

B-additives present Pass Pass 

C-Titanium-dioxide (1) Pass Pass 

Test Appearance of solution (2) Pass Pass 

Acidity 0.34 ≤1.5 ml NaOH 

Alkalinity 0.39 ≤1.0 ml HCI 

UV absorbance (220-340nm) 0.15 ≤0.20 abs 

Reducing substances 0.35 ≤0.5 ml KMnO4 

Substances soluble in hexane Not relevant Due to composition of product 

Extractable aluminum (1) Pass ≤1.0 ppm 

Extractable chromium (1) Pass ≤0.05 ppm 

Extractable titanium (1) Pass ≤1.0 ppm 

Extractable vanadium (1) Pass ≤0.01 ppm 

Extractable zirconium (1) Pass ≤0.1 ppm 

Extractable zinc (1) Pass ≤1.0 ppm 

Extractable heavy metals (1) Pass ≤2.0 ppm 

Sulphated ash 0.00 ≤1.0 % 

max. 3 antioxidants Pass Pass 

Product Tested: Compound 910 

Batch: 6061514ffh inner layer 


Test Method 

See Japanese Pharmacopoeia XIV, 2001Test method for plastic 

containers 



Conclusion 



JP Conformity 

Test results 

Renolit Nederland BV compound GIBCO® batch 6061514ffh inner 

layer meets the test requirement described in the Japanese 

Pharmacopoeia XIV, 2001 test methods for plastic containers 

Test Lower limit Upper limit Result Unit 

Cytotoxicity 0 2+ + - 

Cadmium (1) 0 1 0 ppm 

Lead (1) 0 10 < 1 ppm 

Tin (1) 0 5 1 ppm 

Heavy metals (1) Pass - 

Residue on ignition 0.00 0.10 0.01 % 

Residue on evaporation 0.0 0.5 0.0 mg/20ml 

pH shift -1.5 1.5 0.4 - 

Reducing substances 0.0 1.0 0.0 ml KMnO4 

UV 220-240nm 0.00 0.08 0.03 abs 

UV 241-350nm 0.00 0.05 0.02 abs 

1. ICP is used for analytical reasons 

2. Active cells toxicity test on Human Embryonal cells 

3. ISO 10993-5, University of Amstradam 


23

Product Tested: Compound 910 

Batch: 634PD inner layer gamma irradiated 25kGy 


Quality System: ISO9001/ISO13485 

Test Method 

See Thai Industrial Standard Tis531-2536 (1993) 

Plastic containers for sterile pharmaceutical products 



Conclusion 



Thai Conformity 

Test Results 

Renolit Nederland BV compound GIBCO® batch 634PD inner layer 

gamma irradiated 25kGy meets the test requirement described 

in the Thai Industrial Standard, Plastic containers for sterile phar- 

maceutical products. 

Test Lower limit Upper limit Result Unit 

Cytotoxicity 0 2+ + - 

Cadmium (1) 0 1 0 ppm 

Lead (1) 0 10 < 1 ppm 

Tin (1) 0 5 1 ppm 

Heavy metals (1) Pass - 

Residue on ignition 0.00 0.10 0.01 % 

Residue on evaporation 0.0 0.5 0.0 mg/20ml 

Zn in extract 0.0 0.5 < 0.02 ppm 

pH shift -1.5 1.5 0.4 - 

Reducing substances 0.0 1.0 0.0 ml KMnO4 

UV 220-240nm 0.00 0.08 0.03 abs 

UV 241-350nm 0.00 0.05 0.02 abs 

1. ICP is used for analytical reasons 

2. Active cells toxicity test on Human Embryonal cells 

3. ISO 10993-5, University of Amsterdam 


24

Project Number TE 06674 

Study Number 06-B2171-N1 

Report Date: 19/10/2007 



Endotoxin 

Test results 

Study: Amebocyte Lysate Test-Kinetic-QCL Method 

Test: Item GIBCO® gamma gesteriliseerd Influflex 

Lot #: 634PP 

Reference: Base don ‘USP 29-NF 24, 2006, ‘Bacterial Endotoxin Test’ and ‘Guidelines on Validation of the LAL test as an End Product Endotoxin Test 

for Human and Animal Perenteral Drugs, Biological Products and Medical Devices, FDA December 1987 

Procedure 

A bag with a surface area of 60cm2 was made from a test article, 

filled with 20ml of LAL Reagent water and extracted for 1hr. A 

standard curve of endotoxin (Cambrex brand) was prepared 

with concentrations of 0.005,0.05, 0.5,5 and 50EU/ml. A positive 

product control was prepared to give a final concentration of 

0.5EU/ml. LAL Reagent water served as a negative control. The 

microtiter plate was preincubated in the plate reader at 37+-1C 

for ≥10 minutes.After incubation kinetic QCL-Reagent (0.1ml) was 

added to each well and the absorbance of each well at 405nm 

was read every 150 seconds for a total of 40 data points or until 

the concentration reaches 0.2 absorbance units. The Kinetic QCL 

reader uses the initial reading of each well as its own blank. The 

absolute value of the correlation coefficient (r) must be ≥0.980 in 

order for the test to be valid. 

Results 

Dilution 1:1 EU/ml

Leachable Study

Leve ls 




Summary Table 

WFI WFI – pH 3 WFI – pH 11 

Extractant 

3M NaCl 96% Ethanol 1% Tween 80 10% DMSO 

2 – 10 

ppm 

1,3-di-tertbutylbenzene 

(5.3 ppm) 

1 – 2 

ppm 

TOC TOC 

C8 –alkenes 

(

91 Days Leachables Study On Gamma Irradiated Articles Made 

Of Solmed® Polyethylene Based Materials 

Solmed® materials used: 

→ Infuflex GIBCO® 

→ Tubeflex 4101 

→ Granuflex 4301 

Normative basis: 

→ ISO 10993-17, and -18 [Chemical Characterization of Medical Devices] 

→ European Pharmacopoeia, 5th Edition (2005 + supplements). 




Test report 


28

Contents 

I. Summary 

Aggregated Table of Findings 

II. Introduction 

A. Study Outline 

A.1.Samples Description 

A.2.Extraction Fluids 

A.3.Analytical methods and chemical entities aimed for 

A.3.1. Acidity / Alkalinity 

A.3.2. Conductivity 

A.3.3. Total organic Carbon 

A.3.4. Metal Ions – ICP-OES 

A.3.5. Acetate/Formate – Ion Chromatography 

A.3.6. Volatile organic compounds / Headspace GC/MS 

A.3.7. Semi-volatile organic compounds / GC/MS 

A.3.8. Non-volatile organic compounds / LC/MS 

A.3.9. Derivatization GC/MS 

A.4 .Identification 

A.5. Quantification 

B. Results / Findings 

B.1. Water for Injection 

B.2. Phosphate buffer solution pH 3 

B.3. Phosphate buffer solution pH 11 

B.4. Sodium Chloride 3M 

B.5. Ethanol 96% 

B.6. 1% Tween 80 

B.7. 10% DMSO 

C. Conclusions 





29

Chemical Characterization Of Solmed® Polyethylene Based Products (Ix GIBCO® – Tx 4101 – Gx 4301) 

After Gamma Irradiation 

I. Summary 

Preparation 

In co-operation with a well-known bags maker articles (empty 

bags) made of Solmed® Infuflex GIBCO® film, Tubeflex 4101 tub- 

ing and Granuflex 4301 injection moulding compound were 

manufactured, gamma sterilized (25 kGy) and sent to Toxikon 

Europe to be filled with one of 7 contact fluids: 

→ Water for Injection (WFI), 

→ PBS(saline) pH3, 

→ PBS (saline) pH 11, 

→ NaCl 3M, 

→ Ethanol 96%, 

→ 1% Tween 80, 

→ 10% DMSO). 

The filled bags were climatized at 40ºC-75% Relative Humidity 

and samples were taken at T=0, T= 30 days, T=91 days. The bags 

were filled according to a surface to content ratio of 2cm2/mL 

GX 4301 material has been examined in the WFI test only. 



Various analytical methods were used, aiming for different 

groeps of molecules, e.g. ICP-AES for metals, Headspace Gaschro- 

matography/Mass Spectrography for volatiles, etc. 

All analytical assays were performed against a blank (in 

glass container). 

Results 

Examples of molecules found are reaction products of antioxi- 

dants, oligomers from the Polyethylene articles, and their reac- 

tion products. 

As expected, the Ethanol, Tween and DMSO contact fluids 

showed the most extractables, but the highest concentration of 

any extractable found in this study was approx. 5.3 ppm for 1,3- 

Di-tert-butylbenzene (a reaction product coming from certain 

phenolic antioxidants. 

Based on the findings from this study we can say, that the 

30 days and the 91 days test results only revealed extractable 

chemical entities that were expected, with most concentrations 

in the ppb – 1 ppm range. 


30


Levels 

WFI PBS – pH 3 PBS – pH 11 

Extractant 

3M NaCl 96% Ethanol 1% Tween 80 10% DMSO 

2 – 10 

ppm 

1,3-di-tertbutylbenzene 

(5.3 ppm) 

1 – 2 

TOC 

TOC 

C8 –alkenes 

ppm 

(1.3 ppm C/L) (1.1 ppm C/L) 

(

II. 91 Days Leachables Study Of Gamma Irradiated 

Articles Made Of Solmed® Formulations 

GIBCO® – 4101 - 4301. 

Introduction 

Solmed® plastic products are being used worldwide for the 

manufacture of medical devices and pharmaeutical packaging 

such as infusion bags, blood bags, biotech reactors, etc. 

An extractables study has been performed on the Solmed® 

newly developed family of Polyethylene based products aimed 

for use in biotechnological processes: 

→ Infuflex GIBCO® film 

→ Tubeflex 4101 tubing 

→ Granuflex 4301 injection moulding compound 

This study has been set up with the aim to provide our customers 

and the final users of systems made of Infuflex GIBCO®, Tubeflex 

4101 and Granuflex 4301 with information regarding the extract- 

ables coming from these materials at well defined and rigorous 

conditions with standard extractants (contact fluids). 

It remains the responsibility of our customer, or the end 

user to make sure that articles made of these materials are suited 

for the intended purpose or use and are in compliance with any 

applicable law, regulation or ordinance. 

A. Study Outline 

A.1. Samples Description 

In co-operation with an external bag making company bags 

with a regular design were welded, comprising one small bore 

filling tube. Tubes were closed with a metal clamp. 



Some bags contained a small quantity of Granuflex 4301 

(GX 4301), calculated for the relative mass on a 200 Liter bag with 

a port or stopper made of GX 4301. 

Adequate numbers of these bags were gamma sterilized 

(dose 26-32 kGy) and sent to the external laboratory, Toxikon 

Europe N.V. (Toxikon). 

After filling with the respective contact fluids the bags were 

climatized at 40ºC and 75% RH for 91 days. Samples for analysis 

were taken at T= 0 days, 30 days, 91 days. Blanks were used, with 

glass bottles as containers. 

The GX 4301 is considered a very clean compound without 

additives. As such the material has been included in the WFI test 

for comparison purposes. 

A.2. Extraction or Contact Fluids 

In co-operation with Toxikon the following contact fluids were 

chosen: 

1. WFI (Water for Injection, pH 7) 

2. Phosphate Buffer Saline solution pH 3 

3. Phosphate Buffer Saline solution pH 11 

4. NaCl 3 M in WFI 

5. 96% Ethanol 

6. 1% Tween 80 in WFI 

7. 10% DMSO in WFI 

A.3. Analytical methods and Chemical Entities aimed for 

Various analytical methods were used: 

A.3.1. Acidity / Alkalinity – pH measurement 

Detection of extractables that could change the pH of the fluid. 


32

A.3.2. Conductivity 

Detection of molecules that could conduct electric current 

through the fluid, mostly inorganic ions. 

A.3.3. Total Organic Carbon (TOC) 

Measure of the Total of organic components leaching into the 

contact fluid. This is the sum mass balance of all other analytical 

methods aimed at discovering organic molecules. 

The method first purges inorganic carbon from the sample, 

then converts the remaining carbon into Carbon dioxide ( CO2 ); 

measurement with Infrared Absorption Spectroscopy 

A.3.4. Metal ions – ICP-OES 

Metals may come from e.g. the catalysts used for the polymer- 

ization processes of the polyolefins used to be blended into the 

Solmed products. They may also come from certain additives 

used in these polymers. 

Metals are best analysed using Atomic / Optical Emission 

Spectroscopy with an inductively coupled plasma (ICP-AES or 

ICP-OES). 

For the list of metals specifically aimed for, see Annex C. 

A.3.5. Acetate/Formate - Ion Chromatography 

Acetates and Formates can be found in small quantities every- 

where in plastic products, either coming from raw materials 

used, or being the smallest degradation particles form organic 

molecules. The most sensitive method to analyse their presence 

is the use their different polarity and therefore their different 

affinity to polar adsorbents (ion chromatography). 

A.3.6. Volatile Organic Compounds – Headspace GC/MS 

Volatile organic molecules may come from a host of sources, 

such as monomers and oligomers, residual solvents from various 

production steps, additives, residues form polymer treatment, 

degradation products. 



Volatile molecules can be analysed by means of headspace 

Gas Chromatography coupled with e.g. Mass spectrograph 

A.3.7. Semi-volatile Organic Compounds – GC/MS 

Many compounds are not volatile enough to be analyzed by 

Headspace GC/MS but are still volatile enough to be analyzed 

by “standard” GC/MS. These compounds may comprise solvents 

with higher boiling points, lubricants, plasticizers, antioxidants 

and many others. 

A.3.8. Non-volatile Organic Compounds - Liquid Chromatography LC/MS 

If the molecules cannot be properly analysed in their gaseous 

state a different form of chromatography is used, dissolving the 

compound in a liquid mobile phase: Liquid chromatography, 

again coupled with a Mass Spectrograph. 

Typically, compounds such as the widely used phenolic 

antioxidant scan be analysed by means of this method. 

A.3.9. Derivatization GC/MS 

Some groups of organic compounds, e.g. organic acids need to 

be treated differently compared with Par. A.3.7. for generating a 

suffcient signal in the GC/MS assay. 

Derivatization comprises treatment with BF3 and Butanol. 

This method esp. Shows the presence of fatty acids, e.g. palmitic 

acid, stearic acid. 

A.4. Identification 

The Mass Spectrometer as detector coupled onto a GC or LC 

analytical device makes use of a huge library of molecules ( > 


33

190,000) and tries to match the found signal with the most prob- 

able chemical entity in the library. 

A.5. Quantification (see also e.g. European Pharmacopoeia, Chapter 2.2.46) 

All analytical methods have their limitations and, although today 

IC = Identified Compound (“100%” fit) 

MPC = Most probable Compound (>80% fit) 

TIC = Tentatively Identified Compound (>50% fit) 

we can really find low quantities of molecules, we have to take 

into account the following: 

any analytical system base line is not a 

Noise 

straight line, when magnification factor is 

high enough 

does the analytical signal emerge from the 

Signal to noise ratio 

base line noise? 

Detection limit signal to noise ratio is 3 

Quantitation limit signal to noise ratio is 3x detection limit 

specification for “impurity” in monograph 

Disregard limit 

of pharmaceutical raw material (active 

ingredient or excipient). 

10% of the analytical response of the inter- 

Reporting limit 

nal standard used. 



Furthermore, the analytical system chosen must be suit- 

able, i.e. peak shape, retention times, must comply with certain 

specifications. For “in-line” suitability an internal standard is used. 


34

B. Results / Findings 

The following paragraphs present the analytical results (in ppm, where applicable) per Extraction or Contact Fluid. 

B.1. Water For Injection (WFI) 

If at all extracted compounds were quantifiable, their concentration was found to be in the micrograms per Liter range (ppb). 

Analysis Unit/ concentration 

Ions ppm 

Metals ppm 

Compound / 

Chemical entity 

Blank after 91 days Bag after 91 days 

Acetate Traces 0.09 0.09 

Unknown (level 

unknown) 

- traces - 

Ca (0.53, Mg (0.045) 

in 30days bag => 

artefacts 

Si 0.06 

Na 0.02 

Si 0.08 

Na 0.03 



Bag after 91 days, containing 

GX 4301 

Si 0.02 

Na 0.01 

Volatile organic comp ppm 

2-methyl-1-propene 

Hexanal 

- 

- 

0.008 

< 0.005 

0.012 

< 0.005 

2-Octanone - 0.01 < 0.01 

Semi-volatile organic 

compounds 

ppm 

2,4-Di-tert-butylphenol7,9-Di-tert-butyl-1- 

- 0.01 0.10 

oxaspiro(4,5)deca-6,9diene-2,8-dione Pentaerythrityl 

- 0.01 < 0.01 

Non-volatile organic 

compounds 

ppm 

tetratkis[3-(3,5-di-tertbutyl-4-hydroxyphenyl)propionate] 

Myristic, Palmitic, 

- - 0.002 

Organic acids/ ppb ppm 

Stearic acid, other, all 

blow detection limit 

- - - 

Acidity/alkalinity pH 6.59 6.38 5.64- 

Conductivity 

mS/cm at 25ºC 0.0015 0.0044 0.0042 

Total organic carbon (mg C/L) 0.06 1.33 1.11 

- = not detected 

Traces = reported concentration is < quantification limit for this compound, or not quantifiable 


35

B.2. Phosphate Buffer Saline solution - pH 3 

No metals found in comparison with blank (glass bottle). 

Analysis Unit/concentration 

Compound / 



Ions Ppm Acetate 

Traces 

< 0.60 

0.70 

Volatile organic comp. Ppm - - - 

Hexanal - 0.02 

3-Methyl-3-heptanol - 0.02 


compounds 

Ppm 

2,4-Di-tert-butylphenol - 0.10 

Squalene - - 

Diisobutyl phthalate - 0.014 

Non-volatile organic compounds 

ppm - - - 

Organic acids Ppm - - - 

Acidity/alkalinity pH 3.46 3.51 

Conductivity mS/cm at 25ºC 18.0 17.9 

Total organic carbon/ mg C / Liter < 0.5 1.1 

- not detected 

B.3. Phosphate Buffer Saline solution - pH 11 

Analysis Unit/Concentration 

Compound / 



Ions Ppm Acetate 0.60 0.80 

Metals Ppm Aluminium (traces) < 0.002 0.07 

Volatile organic comp. Ppm 

2-methyl-1-propene 

2-octanone 

- 

- 

0.015 

< 0.05 

2-Octanone < 0.010 


compounds 

Ppm 

2,4-Di-tert-butylphenol 

7,9-Di-tert-butyl-1-oxaspiro 

- 0.092 

(4,5)deca-6,9-diene-2,8dione 

Pentaerythrityl tetratkis[3- 

- 0.016 


Ppm 

(3,5-di-tert-butyl-4-hydroxyphenyl)propionate] 

Tris(2,4-di-tert-butylphenyl) 

phosphite (oxidated) 

- 

- 

0.008 

0.008 

Organic acids Ppm Stearic acid - < 0.06 

Acidity/alkalinity pH 10.88 11.27 

Conductivity/ mS/cm at 25ºC 17.1 17.4 

Total organic carbon mg C / Liter < 0.5 0.5 

- not detected 




36

B.4. Sodium Chloride 3 Molar in WFI 

Analysis Unit/concentration 

Ions Ppm 

Metals Ppm Mg, Si and Sr traces 



compounds 


Compound / 



Acetate < 0.20 < 0.20 

Formate < 0.80 < 0.80 

Mg 0.02 

Si 0.07 

Sr

B.6. 1% Tween 80 in WFI 

As expected, this contact fluid shows more extractables than the water-based contact fluids. Tween extraction results in a more complex 

matrix, giving less clean chromatograms. 

Analysis Unit/Concentration 

Compound / 




Blank after 91 

days 

Bag after 91 

days 

Ions Ppm 

Acetate 

Formate 

5.5 

3.7 

3.9 

< 3.0 

Metals Ppm Traces of Kalium, some metals not detectable due to complex matrix K 0.06 K 0.08 

2-methyl-1-propene - 0.04 

2-Methylpentane - 0.01 

3-Methylpentane - 0.02 

Methylcyclopentane - 0.12 

Cyclohexane - 0.03 

3-Methylheptane - 0.06 


Alkene - 0.01 

1-Octene - 0.33 

C8-Alkenes - 0.25 

Isododecane - 0.02 

2-Octanone - 0.01 

1,3-Di-tert-butylbenzene - 0.04 

Unknown (Main masses 41, 55,67,85) 0.01 


compounds 

Ppm 

1,3-Di-tert-butylbenzene 

complex matrix => no ID of other extractables possible 

- 0.74 

Non-volatile organic 

comp. 

Ppm No reportable compounds - - 

- not detected / Δ- =slight decrease / Δ+ =slight increase 

Traces = reported concentration is ≤ quantification limit for this compound 


38

B.7. 10% DMSO in WFI 

As expected, this contact fluid shows more extractables than the water-based contact fluids. DMSO 10% extraction results in a more 

complex matrix, giving less clean chromatograms 

C. Conclusions 

Bags made of Solmed Infuflex GIBCO®, Tubeflex 4101 and Granuflex 4301 (WFI only), after gamma irradiation (25kGy) were filled with 7 

different contact fluids. The analysis after 91 days, revealed low concentrations of extractables as compared with a blank. 

The results as shown in the tables under B.1.1. – 7. have been aggregated in the Table of Findings, hereafter. 

Most findings are in the concentration range up to 1 ppm. 

Virtually all identified extracted chemical entities are explanable as either oligomers from the polymers used, or degradation products 

from the antioxidants used. 

Based on the findings from this study we can say, that the 30 days and the 91 days test results only revealed extractable chemical entities 

that were expected, with most concentrations in the ppb – 1 ppm range. 




39

Animal Origin Free 

Conformity Statement

Statement 

On the absence of components of animal origin in solmed® infuflex GIBCO®, tubeflex 4101 and granuflex 4301 products 

We have received written information from our raw materials suppliers regarding the presence of components of animal origin, c.q. the 

TSE risk status of the raw materials used for the production of our Solmed® Infuflex (IX) GIBCO®, Tubeflex (TX) 4101 and Granuflex (GX) 

4301 formulations. 

Our raw materials suppliers state, that their products used by RENOLIT Nederland B.V. for the production of the abovementioned 

Solmed® IX GIBCO®, TX 4101 and GX 4301 Products do not contain any intentionally introduced components of animal origin. 

Furthermore, RENOLIT Nederland B.V. states, that no components of animal origin are added to these raw materials in the IX GIBCO®, 

TX 4101 or GX 4301 compounding and production processes. 



Animal Origin Free 

Conformity statement 


41

Film Data Package for GIBCO® Liquid Media Bags - Invitrogen

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