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3. MATERIAL AND METHODS

Plastics are being increasingly utilized for the storage and delivery of life

saving fluids. Although, virgin polymer generally considered being safe due

to its inertness, however, the plastic products could be harmful due to

migration of certain chemical additives like plasticizers, stabilizers,

pigments and unreacted monomers into the stored commodity. Therefore,

plastic products intended for storage and delivery of life saving fluids must

be evaluated for their safety. In this regards, different countries have laid

down various safety assessment tests and guidelines for the suitability and

quality assessment of the plastics.

Indian climatic conditions are quite different in comparison to the western

countries. The guidelines provided by the Bureau of Indian Standards, New

Delhi has recommended the test for Global migration Residue for safety

assessment of plastics. Several International regulatory agencies have also

recommended tests for the presence of heavy metals, UV absorbing

materials and oxidizable materials in the plastics used in biomedical devices

as well as food packaging.

In order to assess the risk of human exposure to leachable plastic additives

from plastic products, physicochemical and biological studies were

conducted using standard procedures laid down by BIS and OECD.

It is desirable to study the physicochemical nature of the leachable. The

leachates of the samples were prepared in different simulated solvents under

varying temperature conditions and then analyzed. The leachates are

examined for change in physical state. Chemical tests are used for the

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determination of global migration residue, oxidizable materials, UV

absorbing materials, heavy metals and styrene monomers.

Cytotoxicity assays were carried out in cultured L929 cells by exposing

them to various leachates of plastic biomedical devices for different time

intervals. The assays selected in the study were: determination of cell

growth, survival rate, inhibition of cell growth, cfa and MTT assay. The

control sets were the untreated batches of cells run simultaneously under

identical conditions. For the purpose, plastic medical devices i.e. IV

infusion sets, DNS bottles, RL bottles and Ryles tube were procured from

Lucknow and processed in the laboratory. The sample details are as under:

Table 3.1 Types of plastic medical devices

S. No. Type of sample Brand Name Code

A.

Intravenous

Infusion sets

1. RMS infusion set, Ramsons Juniors India

IV-1

2. JVS Super Delux Quality. S. K. Trading

Corporation

IV-2

3. JVS infusion set, S. K. Trading Corporation

IV-3

4. Dispocath, infusion set, Trinity Health Care

IV-4

5. TMS, Extrasuper, Trinity Health Care

IV-5

6. Infusion set premium, Protec

IV-6

7. Infusion set, poly Medicure Limited

IV-7

1. Albert David limited D-1

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B. DNS bottles 2. Claris Lifesciences limited

D-2

3. Axa Parenterals Limited D-3

C.

RL bottles

1. Albert David limited RL-1

2. Claris Lifesciences limited

RL-2

3. Axa Parenterals Limited RL-3

4. Nirlife Healthcare RL-4

5. Aishwarya Life Sciences RL-5

D.

Ryles tube

1. Romolene, Romsons Scientific and Surgical

Industries Private

Limited

S-1

2. La-Med, Lakhani Medicare Private

Limited

S-2

3. Ryles Tube, life line systems Private Limited

S-3

4. Polymed Ryles Tube, Poly medicure Limited

S-4

Equipments used

1. Hot air oven capable of maintaining temperature 1C.

2. Heating mantle with temperature regulator.

3. Electronic balance with sensitivity of 0.001 mg.

4. UV - Visible Spectrophotometer.

5. Atomic Absorption Spectrophotometer.

6. Laboratory glassware and silica crucible.

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Chemicals used

Acetic acid, ethanol, sodium chloride, sodium carbonate, potassium

permanganate, potassium iodide, sodium thiosulphate, starch, sulphuric

acid, nitric acid were procured of AR grade of highest purity available. All

the chemicals and reagents used during the course of the study were of

reputed brands.

Simulating conditions and solvents

Plastic biomedical devices were washed thoroughly with sterilized double

distilled water prior the leaching. Double distilled water, Ethanol (8% v/v in

double distilled water), Acetic Acid (3% v/v in double distilled water),

Sodium Chloride (0.9% w/v in double distilled water) and Sodium

Carbonate (5% w/v in double distilled water) were used as the simulating

solvents (IS 9845). Plastic biomedical devices were exposed in 100 ml of

either of simulating solvents in sterile beakers at a ratio of 2 ml/cm2. The

samples were kept at 41C for 72 h (refrigerated conditions), 252C for

24 h (ambient conditions) and 602C for 2 hours (elevated conditions). 1, 2

Parallel sets having simulating solvents only will also be run under identical

conditions and will serve as basal control. One set of leachates were

lyophilized and makeup again the same volume in serum free culture

medium and were considered as stock test solution in cytotoxicity studies.

Typically, lyophilization methods include freeze-drying a liquid solution or

suspension to provide a dry residue containing a high concentration of the

dissolved or suspended compounds. Typically, aqueous solutions are used

in lyophilization, although mixed aqueous/solvent solutions, and other

liquid solutions, may be used. Lyophilization was carried out in lyophilizer.

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In the lyophilization, vials are individually attached to the ports of a drying

chamber. The product was frozen in a freezer. The prefrozen product is

quickly attached to the drying chamber to prevent warming. The vacuum

must be created in the product container quickly, and the operator relies on

evaporative cooling to maintain the low temperature of the product. Since

the vessels are attached to the manifold individually, each vial has a direct

path to the collector. Several vessels can be accommodated on a manifold

system allowing drying of different products at the same time, in different

sized vessels, with a variety of closure systems. Since the products and their

volumes may differ, each vessel can be removed from the manifold

separately as its drying is completed. The close proximity to the collector

also creates an environment that maximizes drying efficiency. Cytotoxicity

studies were carried out using leachates in serum free medium itself.

Endotoxin detection was carried out by gel clot technique using LAL

reagent.

3.1 Physicochemical tests

Physical properties

The plastic biomedical devices were examined for the changes in physical

state, color, texture, turbidity and pH of the simulating solvents on the

completion of respective simulations.

Statistical Analysis

Data were analyzed by one-way analysis of variance (ANOVA) and

Students t test were employed to assess the significance of variations

between the pH of control and samples using a computer based software,

GraphPad Prism 5. A p value less than 0.05 is considered as significant.

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BIS and international standards for plastic medical devices

Physicochemical test for plastic (USP-400280)

pH determination (IS 3025)

Global migration residue (IS-9845)

Oxidizable materials, UV absorbing materials (Indian

Pharmacopoeia1996)

Styrene analysis (IS: 10142)

Heavy metals (USP-400220)

Biosafety analysis (ISO 10993)

LAL test (USP )

3.1.1 pH Determination

The pH was measured using a digital pH

meter, model number LT-11,

Labtronics at 25C. Standard solutions of pH 4.0 and 7.0 (Qualigens Fine

Chemicals), 9.2 (Fisher Scientific) were used for calibration of the pH meter

(IS 3025).

3.1.2 Global Migration Residue

The overall migration of chemical additives which includes the inorganic

compounds, heavy metals, phthalates, organo-metallic compounds and other

additives which are not volatile up to 95 C. The test has been

recommended by various national and international regulatory agencies and

is of importance since some of the additives are toxic. Following the

simulation, leaching solvents were kept for evaporation till dryness in

constant pre-weighed silica crucible in the oven maintained at constant

temperature (90C) for 24 hours and the crucible were weighed again. The

difference in the weight obtained was taken as the measure of the global

migration residue expressed as mg/100 ml of the simulants. The test was

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performed in triplicates. Migration residues should not be more than 5

mg/100 ml of extract (IS 9845).

Data were analyzed by one-way analysis of variance (ANOVA) and

Dunnetts Multiple Comparison test and No Post Test were employed to

assess the significance of variations between the control and samples using

a computer based software, GraphPad Prism 5. A p value less than 0.05 is

considered as significant.

3.1.3 Oxidizable matters

Oxidizable materials are also known as antioxidants, which protect the

plastics by reacting with the atmospheric oxygen. Commonly used

oxidizable matters are organophosphite and derivatives of phenols. The test

has been recommended by various national and international regulatory

agencies and is essential as some of these chemicals are toxic in nature. Due

to migration of these compounds, the durability of plastics may be

decreased and the consumers will also be at risk to the other leachable toxic

chemicals. Oxidizable matters were measured by titration of plastic extract

and corresponding blank against sodium thiosulphate. The extract (20 ml) is

taken in an Erlenmeyer flask and 20 ml of 0.01 N KMnO4 and 1.0 ml 2N

H2SO4 is added and the mixture is boiled for 3 minutes. The solution is

cooled and 0.1 gm of KI and 5 drop of starch solution are added and finally

titrated with 0.01 N sodium thiosulphate solutions till pink color

disappeared. A blank was also titrated in parallel. The difference in the

volume of 0.01 N sodium thiosulphate consumed in titration of leachate and

blank gave the measure of oxidizable matters (Indian Pharmacopoeia1996).

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3.1.4 UV absorbing materials

UV absorbing materials are the substances which give characteristic

absorption peak in UV region. The commonly used UV absorbing materials

are derivatives of benzophenones, benzotriazoles, salicylates, acrylates,

organonickels and amines which are added during the synthesis of plastic to

protect them from degradation from sunlight and fluorescent light. The test

is essential as some of these compounds are toxic. Following the simulation,

leaching solvents were processed for the estimation of migration of UV

absorbing materials from the plastic biomedical devices. The samples were

scanned between 220-400 nm. The results were expressed as the difference

in optical density (OD) obtained from the leachates and blank (Indian

Pharmacopoeia1996).

3.1.5 Estimation of heavy metals

Plastic biomedical devices were washed thoroughly with sterilized double

distilled water prior to leaching. Aseptic dried plastic biomedical devices

were cut into small pieces of 1 cm2 and immersed in 100 ml of either of

simulating solvent viz.1, 2

double distilled water, ethanol (8%), acetic Acid

(3%), sodium Chloride (0.9%) and sodium Carbonate (5%) at 252C for

24h (ambient conditions) and 602C for 2h (elevated conditions). Parallel

sets having simulating solvents only were also run under identical

conditions and were served as basal control. The leachates will be taken in

flask and digested with concentrated Nitric acid and the volume of digested

samples will be made upto 10 ml using 1% Nitric acid. The digested

samples were analyzed for metal content, (Cd, Cr, Cu, Fe, Mn, Ni, Pb and

Zn) using a Perkin- Elmer atomic absorption spectrophotometer. 3

Heavy

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metal analysis were performed according to USP-400220 guidelines by

Atomic Absorption Spectrophotometer.

3.1.6 Estimation of un-reacted monomer (Styrene)

The leachates collected above, were extracted with DCM and analyzed by

GC-ECD. The GLC method is equally efficient as High Performance Liquid

Chromatography (HPLC) for the determination of styrene monomer. The

un-reacted styrene monomer leached out from biomedical devices, was

estimated using GLC Clarus- 500 (Perkin Elmer) equipped with electron

capture detector (ECD) (IS: 10142).

3.2 Biosafety analysis (ISO 10993)

3.2.1 Fibroblast culture

L929, an adherent type mouse fibroblast derived from CH3 mouse (ATCC

No. 1) were used in all the experiments were originally procured from

National Centre for Cell Sciences (NCCS), Pune, India. These cells were

maintained at In Vitro Toxicology Laboratory, Industrial Toxicology

Research Centre, Lucknow, India. Monolayer culture (passage number 6

15) were grown in minimal essential medium (MEM) containing 12% fetal

calf serum (FCS) (Boehringer Mannheim, Germany), vitamins, essential

and nonessential amino acids, penicillin (100 unit/ml) and streptomycin

(100 g/ ml). The cells were grown at 37C in an atmosphere of 5% CO2

with more than 95% humidity.

3.2.2 Exposure of fibroblast (L929) with leachates

Aseptic dried plastic biomedical devices were cut in to small pieces of 1

cm2

and immersed in simulating solvent, i.e., serum-free MEM at 50C for

72 h. 1, 2

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Cells were seeded in 3.5-cm multidishes in serum- enriched MEM for 48 h.

Under these conditions cells were at the beginning of their exponential

phase of growth, which were continued for about 80 h in control cells. After

48 h, cells were washed thrice with SFM and subsequently incubated with

simulated leachates, i.e., serum-free MEM containing plastic leachates for 1

h at 37C in CO2 incubator.4 Cells in serum-free MEM only were processed

under identical conditions and served as the control. After 1 h of incubation,

control and treated cells were washed three times with serum-free MEM and

were processed for determination of cell growth, survival rate, inhibition of

cell growth, cfa and MTT assay.

3.2.3 Determination of cell growth

Following washing, the cells were maintained up to 96 h in serum-enriched

medium after which cells were again washed three times with SFM,

trypsinized and resuspended in SFM. Cell numbers were determined in a

Coulter Counter at a time interval of 12 h for 96 h of reincubation.

3.2.4 Determination of survival rate

Cell viability can be assessed directly through the presence of cytoplasmic

esterases that cleave moieties from a lipid-soluble non fluorescent probe to

yield a fluorescent product. The product is charged and thus is retained

within the cell if membrane function is intact. Hence, viable cells are bright

and nonviable cells are dim or non fluorescent. Typical probes include

fluorescein diacetate (FDA, described here), carboxyfluorescein, and

calcein. Variations in uptake or retention of the dye among individual cells

or under different conditions affect the efficacy of particular probes.

Cell survival rate were determined following the method of Rotman and

Papermaster5

using fluorescence diacetate (FDA). Fifty thousand cells were

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seeded in petri dishes (5 cm diameter). Three days later, at sub confluence,

the cells were exposed to leachates as described earlier. Subsequently, the

cells were incubated with 2 ml freshly dissolved FDA (30g/ml in buffered

saline) for 2 min at 37 C. The cells were then trypsinized and at least 300

cells/dish were counted under fluorescence microscope, where living cells

(defined as cells with membrane integrity and metabolic ability to convert

FDA to fluorescein) show fluorescence in contrast to dead cells. The

survival rate was calculated from the ratio of the number of living cells to

the total cell number.

3.2.5 Inhibition of cell growth

Cells (30,000) were seeded in petri dishes of 5 cm diameter. Two days later,

under the logarithmic phase of growth, the cells were treated with the

leachates as described earlier. After treatment and repeated washing, one set

of control and treated cells were harvested and measured immediately (for

protein contents), whereas the other portion were allowed to grow until the

cells of control reached subconfluence (two to three doubling in 3545 h).

The cells were then washed three times with PBS and air dried. The protein

content of cells in all the dishes were measured according to the method of

Lowry et al, 6 as modified by Oyama and Eagle.

7 Growth rate were

calculated from the ratio of protein content of the cells at the end of growth

period and the protein content directly after treatment [growth=ln1, (protein

end/protein beginning):ln2]. Growth inhibition is expressed as percentage of

growth rate of treated compared to untreated cells.

3.2.6 Determination of cfa

Culture cells (50,000) were maintained in petri dishes (5 cm diameter) for 3

days until subconfluence were reached. After the exposure of leachates, the

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cells were washed thrice, trypsinized and seeded on five dishes at a density

of 2000 cells/dish. Following reincubation for about 14 days the cultures

were fixed stained with Giemsa stain and colonies were counted.8

3.2.7 MTT Assay

MTT (3-4, 5-dimethyl thiazol-2-yl) 2, 5 diphenyl tetrazolium bromide), a

pale yellow substrate is converted into farmazone, a violet compound by the

activity of succinate dehydrogenase of mitochondria. Since the conversion

takes place in living cells, the amount of formazan produced is directly

correlated with the number of viable cells present.

The MTT assay were done following the protocol of Pandey et al (2006).9

Cells (1 104 per well) were seeded in poly-L-lysine pre-coated 96-well

culture plates and allowed to adhere for 24 h in CO2 incubator at 37C. The

medium were then replaced with the serum free medium containing

different concentrations of leachates. The cells were exposed to leachates

for different time intervals. Following this, tetrazolium bromide salt

(5 mg/ml of stock in PBS) was added in the plate (10 l/well in 100 l of

cell suspension) and the plates were incubated for 4 h. The reaction mixture

was carefully taken out and 200 l of DMSO were added to each well and

pipetting up and down several times unless the content got homogenized.

The plates were kept on rocker shaker for 10 min at room temperature and

then read at 550 nm using multiwell microplate reader (Synergy HT, Bio-

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Tek, USA). Parallel sets without leachates were also run under identical

conditions and served as basal controls.

3.2.8 Bacterial Endotoxin detection by gel clot technique

using Limulus Amoebocyte Lysate (LAL) reagent, (USP )

Nowadays, the most popular method for bacterial endotoxin quantitation in

medical devices is the Limulus assay. The LAL tests are very sensitive and

have a broad measurement range from 1 pg/ml10 ng/ml. In 1977, this test

was accepted and recommended by the United States Food and Drug

Administration (FDA) as the standard assay for bacterial endotoxin

detection. According to FDA guidelines, an endotoxin concentration in the

sample is expressed in endotoxin units (EUs), which describe the biological

activity of endotoxins. The EU is standardized against the defined reference

material, i.e. reference standard endotoxin (RSE). As stated by the FDA,

both RSEs, i.e. 100 pg of the Escherichia coli EC-5 and 120 pg of the E.

coli O111:B4 have activity equal to 1 EU.

In the gel-clot Limulus test, extracting medical devices using LAL Reagent

Water in the pyrogen-free tubes. After 60 minutes of incubation at 37C in a

water bath, the tubes are removed from the incubator and observed for clot

formation after inverting them 180 degrees. A positive result is the

formation of a solid gel-clot at the bottom of the reaction tube that

withstands inversion without breaking. The concentration of endotoxin in

examined sample is determined by the highest extract dilution at which

coagulation is still observed. A series of RSE dilutions (usually starting

from 1 EU/ml) is used to determine the sensitivity, which is the lowest

endotoxin concentration forming a clot. The gel-clot test is the simplest

among all Limulus assays and requires minimal laboratory equipment.

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References

1. Northup SJ, (1999), Safety evaluation of medical devices: U.S. food and

drug administration and international standards organization

guidelines, Int J Toxicol, 18: 275283.

2. ISO 10993-5, Biological evaluation of medical devices: Part 5. Tests for

cytotoxicity in vitro methods, 1992.

3. Srivastava SP, Saxena AK, Seth PK, (1984), Safety evaluation of some of

the commonly used plastic materials in India, Indian J Environ

Health, 26 (4): 346354.

4. Jacobi H, Krieger G, Witte I, (1995), Characterization and applicability

of a cytotoxicity assay determining growth inhibition after a 1 hour

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(5): 751756.

5. Rotmann JJ, Papermaster BW, (1966), Membrane properties of living

mammalian cells as studied by enzymatic hydrolysis of fluorogenic

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6. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ, (1951), Protein

measurement with the Folin phenol reagent, J Biol Chem, 193: 265

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7. Oyama VI, Eagle H, (1956), Measurement of cell growth in tissue

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environmental chemicals, Toxicol In Vitro, 9 (3): 327331.

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