FluidTechnologySolutions

Full validation guideBioprene

Executive Summary
This is the full validation guide for Bioprene tubing. It contains
additional hyperlinks to the test reports. The additional
hyperlinks provided in section 8 page 7 will provide detailed
test procedures and test results.

Contents

1 Introduction
2 Conditions of use
3 Chemical compatibility
4 Materials, manufacturing and regulatory compliance statements
a.
b.
c.
d.
e.
f.
g.

Materials of construction
Manufacturing environment
Country of origin
Compliance declaration summary
REACH legislation
RoHS
Storage conditions

5 Compendial and non compendial testing
a.
b.
c.
d.
e.
f.
g.
h.
i.
j.
k.
l.

Summary table
USP <87>
USP <88>
USP <85>
ISO 10993-4
ISO 10993-5
ISO 10993-6
ISO 10993-10 - Kligman Maximisation Test
ISO 10993-10 - Intracutaneous Injection Test
ISO 10993-11
USP <381>
USP <788>

6 Extractables
7 Conclusions
8 Hyperlinks to test reports2

1. Introduction
Bioprene® is a high performance USP Class VI validated
thermoplastic elastomer tube manufactured from a USP
Class VI raw material in an ISO 14644-1 Class 7 cleanroom
and operating within an ISO9001 quality system.
Manufacturing is located at Watson-Marlow Ltd, Bickland
Water Rd, Falmouth, UK, TR11 4RU.
Bioprene tubing has the following key features:•

Meets USP Class VI requirements•

Meets FDA regulations CFR 177.2600 for contact withaqueous food•Made from NSF51 food compliant raw material•Long peristaltic life•

Broad chemical compatibility•

Opaque to visible and UV light•

Low extractables; no phthalates added, no latex used

2. Conditions of Use
Bioprene can be sterilised by EtO, autoclave, and gamma
irradiation up to 35 kGY.

3 Chemical compatibility
A general guide on chemical compatibility of Bioprene
tubing can be found on the Watson-Marlow Fluid
Technology Solutions website. wmfts.com/chemical

3

4	Regulation compliance statements andmanufacturing conditions

4e Storage conditions
To maintain the performance of the components
throughout their lifecycle, they should be stored ina cool, dry environment away from direct sunlightwithout exposure to chemicals and not subjected
to stress. Normal warehouse conditions of 5C−40C(40F−86F) are acceptable. Wherever possible, original
packaging should be maintained. Stock should berotated on a first in, first out (FIFO) basis.

4a Country of Origin
Bioprene is manufactured at Watson-Marlow Ltd,
Bickland Water Rd, Falmouth, UK, TR11 4RU.

4b REACH legislation
All raw materials, compounds used in the
manufacturing process and the final Bioprene tubing
comply with the REACH regulations.

The performance of any component beyond its use
by date, or which has not been stored according
to the recommendations outlined above, cannot beassured.

4c RoHSIn compliance with the restriction of hazardous
substances (RoHS) directives, no listed substances
are used in the manufacture of Bioprene tubing.

5. Compendial and Non compendial testing
5a Summary table

4d Working temperature range

Table 1 contains a summary of all the compendial
testing and qualifications that Bioprene has been
evaluated for. Full test methods and results areavailable on request. All test work was performedon tubing samples and this was used to represent
the Bioprene product family as the components aremanufactured using the same material at the same
manufacturing site.

Bioprene tubing can be used in a pump from +5C to+80C. In static applications the working temperature
range can be extended. The brittle temperature is<-60C.

Table 1: List of compliance statements for Bioprene tubing and substances not present in themanufacture of Bioprene
Named substance/compliance statement

Raw material

Manufacturing process

Final productAnimal Derived Content–––

Melamine–––

Phthalates–––

Bisphenol A (BPA)–––Latex–––Presence of heavy materials–––Allergens–––‘–’ denotes not present or not added

Table 1: Summary list of Compendial and non-compendial testing

4TestResult

USP <87> Biological Reactivity tests, In Vitro

Pass

USP <88> Biological Reactivity tests, In Vivo

Pass

USP <85> Bacterial EndoToxins Test

Pass

ISO 10993-4 Hemolysis

Pass

ISO 10993–5 Biological evaluation of medical devices - part 5: tests for In Vitro cytotoxicity

Pass

ISO 10993–6 Short term Intramuscular Implantation Test

Pass

ISO 10993–10 Kligman Maximisation Test

Pass

ISO 10993–10 guidelines for Intracutaneous Injection Test

Pass

ISO 10993–11 guidelines for Systemic Injection Test

Pass

USP <381> Elastomeric closures for injections - physiochemical tests

Pass

5b USP <87> Biological Reactivity tests, 		In Vitro
USP 87 determines the biological reactivity of a cell
culture in response to a given test article. Samples ofBioprene tubing were tested in accordance with USP
35, NF 30, <87>, Biological reactivity tests, In Vitro.
Extracts, positive control (rubber) and negative control
articles were prepared at 37C (98.6F) for 48 hours.
Biological reactivity was rated on a scale ranging from
Grade 0 (no reactivity) to Grade 4 (severe reactivity).
Results: No reactivity was exhibited by the cell
cultures when exposed to Bioprene tubing.
Therefore, they passed the requirements of USP
87 biological reactivity tests and have no cytotoxic
potential.

5c USP <88> Biological Reactivity tests, 		In Vivo
USP Class VI Plastics Test assesses the potential
toxicity of a given test article systemically,
intracutaneously and through implantation. Samples
of Bioprene tubing were tested in accordance with
USP 35, NF30 <88>, Biological reactivity tests,
In Vivo. This included the immersion of the test
articles in the following solutions: USP 0.9% sodium
chloride, cottonseed oil, 1 in 20 ethanol in NaCl andpolyethylene glycol 400 at 70C (250F) for 24 hours.
Results: Bioprene tubing extracts and implants
showed no toxicity. Therefore, they passed therequirements of USP <88> biological reactivity
tests.

compared to the endotoxin concentration andreaction time of the sample extract.
Results: Bioprene extracts had an EU/ml value of<0.009 EU/cm² which is comparable to the levelsof endotoxin observed in water for injection.

5e ISO 10993-4 Hemolysis
The hemolysis test assesses the potential for indirectcontact of a given sample with blood to cause therupture of erythrocytes (red blood cells).
Bioprene were tested in accordance with ISO 109934, Biological Evaluation of Medical Devices – Part 4:
Selection of Tests for Interactions with Blood.
Results: Per ISO 10993-4, Bioprene is considered
non-hemolytic

5f ISO 10993–5 Biological evaluation of 		
medical devices - part 5: tests for In Vitrocytotoxicity
The biological reactivity of a cell culture, in response
to extracts from Bioprene tubing was determined.
The maintenance medium on the cell cultures wasreplaced by extracts of Bioprene tubing or controlarticle.
The cell cultures were incubated for 48 hours at
37±1C (98.6F±33.8F).
Results: Bioprene tubing showed no signs ofcytotoxic potential. Therefore, they passed therequirements of ISO 10993–5.

5d USP <85> Bacterial EndoToxins Test
USP Limulus amebocyte lysate (LAL) bacterial
entoxin assay Endotoxins are lipopolysaccharide
complexes in gram negative bacterial cell walls. The
limulus ameobocyte lysate (LAL) Kinetic Chromogenic
assay is used to determine the quantitative level ofendotoxin present within our products. Bioprene
tubing was tested in accordance to the requirementsof USP 85. Bioprene (27.6.0 cm² surface area) wasextracted in 50mL of LAL reagent water at room
temperature for 60 minutes. LAL-lysate was added
to each sample well on an automated microtiter plate
and then transferred to a plate reader where theabsorbance of each well was read at 405 nm and37 C at set timepoints. A standard curve wasgenerated using software based on reaction time
versus endotoxin concentration which was then5g ISO 10993–6 Short term Intramuscular 		
Implantation TestThe purpose of this test is to evaluate the solidmaterial in direct contact with living tissue.
Bioprene tubing and the negative control plastics
were tested. The test sites were examined for
encapsulation, necrosis, haemorrhage anddiscolouration macroscopically.
Results: Bioprene tubing did not demonstrate any
remarkable difference as compared to the control
implant sites when in contact with tissue for 2weeks.

55h ISO 10993–10 Kligman Maximisation Test

5j

The purpose of this test is to detect the allergenic
potential of a test article.

The purpose of the systemic injection study isdesigned to screen test articles extracts for potential
toxic effects as a result of a single dose systemic
injection. Bioprene is extracted using 0.9% sodium
chloride for injection, cottonseed oil, 1 in 20 ethanol in
NaCl or polyethylene glycol 400 at 70C for 24 hour.

Bioprene was tested in accordance with ISO 1099310. Samples of Bioprene were extracted in USP
0.9% NaCl for injection and cottonseed oil at 70Cfor 24 hours and then injected intradermally. After
two weeks, an additional topical application wasintroduced to the site of intradermal injections.

Results: Bioprene tubing meet the requirements of
ISO 10993–11 guidelines for the systemic injection
test toxicity.

Results: The sites that were exposed to the test
articles and negative control showed no signsof erythema or edema. Therefore, Bioprene isdeemed not to contain any allergy.

5k5i

USP <381> Elastomeric closures for 		
injections - physiochemical tests
Extracts of Bioprene were prepared according to
the requirements of USP 32, NF 27, Chapter 381 asdirected under physiochemical tests. The results ofthe tests are summarised in Table 2.

ISO 10993–10 guidelines for 			
Intracutaneous Injection Test
The intracutaneous test is designed to evaluate local
responses to the extracts of Bioprene tubing following
intracutaneous injection. Sample was extracted using
0.9% sodium chloride for injection, cottonseed oil, 1
in 20 ethanol in NaCl or polyethylene glycol 400 at
70C for 24 hour.
Results: Bioprene tubing meet the requirementsof ISO10993–10 guidelines for the intracutaneousinjection test.

ISO 10993–11 guidelines for Systemic 		
Injection Test

Results: Based on the evaluation criteria mentionedbelow, Bioprene tubing meets the requirements ofthe USP <381> section physiochemical tests.

5l

USP <788> Particulate testing/			
microscopic particulate count analysis
testThis test is used to determine a level of particulates
measuring 10 micron (µm) or less and 25 micron(µm) that may be present in any given drug product.
A length of Bioprene tubing was filled with low

Table 2: USP <381> Summary
Test

Test Result

Evaluation Criteria (Type One Closures)

ResultAppearance of Solution:
Turbidity

0.032 NTU

≤ 6 NTU For Type 1 Closure

PassAppearance of Solution:
ColorColour less intense than Colour Colour is less intense than colour standard
Standard(Eu. Ph. GY5 Colour Standard)

PassAcidity or Alkalinity< 0.05 mL NaOH Blue colour developed≤ 0.3 mL NaOH 0.01 N to produce ablue colour

Pass

Heavy Metals

Colour is less intense thancolour in the Standard

Colour less intense than Standard Solution 2ppm Lead

Pass

Reducing Substances

0.1 mL≤ 3.0 mL For Type 1 Closure

Pass

Absorbance

0.024 A.U

≤ 0.20 A.U For Type 1 Closure

Pass

Extractable Zinc

0.067 ppm ZN≤ 5 ppm ZnPassAmmonium

Colour is less intense thancolour in the Standard

Colour is less intense than 2 ppm ammonium
Standard Solution

Pass

Volatile Sulfides

No black stain visible

Any Stain is less intense than the Control Solution

Pass6

particulate water and shaken 20 times. The extraction
fluid was then recovered and the particles weremeasured using light obscuration.

The extracts were evaluated for the elemental impurities
listed in the ICH Q3D and USP <232> guidelines.
Results: These studies have shown that there
were extractables indicative of the materialsof construction. WMFTS can provide further
information and assistance in the evaluation of
extractable data for risk assessment purposes.

Results: Extracts from a Bioprene tube contained
571 particles > 10µm and 16 particles > 25µm

6. Extractables
Bioprene tubing connectors were subjected to extraction
in multiple solvents at controlled temperatures. The solvent
extracts were then analysed using high pressure liquid
chromatography—diode array detector-mass spectrometry(HPLC-DAD/MS), Direct injection Gas Chromatography—
Mass spectrometry (DI-GC/MS), Headspace Gas
Chromatography—Mass spectrometry (HS-GC/MS) andInductively Coupled plasma—Mass Spectrometry (ICP/MS). HPLC-DAD/MS is used to detect the presenceof non-volatile and UV active extractables. DI-GC/MSidentifies if there are any semi volatile compounds present
in the extracts whilst volatile extractables can be detectedusing HS-GC/MS. Potential elemental impurities can beidentified by ICP-MS.

7. Conclusions
Bioprene has been shown to pass several compendialand ISO testing, as summarised in this guide. For further
information with full compliance statements and the test
reports, please contact your WMFTS representative.

8. Hyperlinks to test reports
Please see table below.

Table 3: Sample Preparation Summary
Test ProcedureHall Sample ID

Extraction Solutions

Extraction Method

WaterHeadspace GC-MS
Direct Injection GC-MS
LC-DAD-MS (ESI)

H80017 and Negative Control

50% Ethanol(aq)0.1M Phosphoric acid

Incubation, 40°C for 21 days

0.5N NaOH

Table 4: Summary of Extraction Conditions
Component

Number ofComponents

Extraction Volume (mL)

Reporting Threshold(µg/mL)

Reporting Threshold(µg/cm2)

Bioprene Tubing

1 x 125 cm60

0.1

0.02Section Number

Title of test

Link to the report (Click hyperlink to open)

5b,5f

USP <87> Biological Reactivity tests, In Vitro
ISO 10993–5 Biological evaluation of medicaldevices - part 5: tests for In Vitro cytotoxicity

USP 87/ISO 10993-5

5c,5g,5i,5j

USP <88> Biological Reactivity tests, In Vivo
ISO 10993–10 guidelines for IntracutaneousInjection Test
USP Class VI & ISO 10993 parts 6, 10 & 11
ISO 10993–11 guidelines for Systemic Injection Test
ISO 10993–6 Short term Intramuscular Implantation
Test

5dUSP <85> Bacterial Endotoxins Test

USP 855eISO 10993-4 HemolysisISO 10993-4

5hISO 10993–10 Kligman Maximisation Test

ISO 10993-10 ( Kligman Maximisation)

5k

USP <381> Elastomeric closures for injections physiochemical tests

USP 3815l

USP <788> Particulate testing/microscopic
particulate count analysis test

USP <788>

6

Extractables testing

Extractables Study
Extractables - Tubing Component Analysis

7FluidTechnologySolutions

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Disclaimer: The information contained in this document is believed to be correct but BioPure
Technology Limited accepts no liability for any errors it contains and reserves the right to alterspecifications without notice. Tri-Clamp is a registered trademark of Alfa Laval Corporate AB.
Q-Clamp is used under licence.

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