**3.3.1 Cytotoxicity**

Definitions of cytotoxicity vary, depending on the nature of the study and whether cells are killed or simply have their metabolism altered. Cytotoxicity is the toxicological effect that a substance can cause *in vitro*, at cellular level (Freshney, 2000).

As defined in ISO 10993, "the numerous methods used and end-points measured in cytotoxicity determination can be grouped into categories of evaluation type, like assessments of cell damage by morphological means; measurements of cell damage; measurements of cell growth and measurements of specific aspects of cellular metabolism" [IOS, 2010]. The cells can be exposed to the samples or their extracts.

## **3.3.2 The protocols**

#### **Cell culture**

Chinese hamster ovary cell line (CHO-k1) was standardized for cytotoxicity and genotoxicity tests. Cells were maintained in RPMI medium supplemented with antibiotics and antimycotics (100 units/mL penicillin, 100 µg/mL streptomycin and 0.025 µg/mL amphotericin), 2mM glutamine, and 10% calf serum, at 37o C in a humidified 5% CO2 atmosphere until they reached confluence. For subculturing and for experiments, cells were harvested using 0.05% trypsin and 0.02% EDTA in phosphate-buffered saline, pH 7.4.

#### **Extract preparation**

Samples of packaging, before and after sterilization, were submitted to this assay. The samples were immersed separately in RPMI culture medium at a final concentration of 1 cm2 / mL and left in an incubator at 37oC for 72 hours to fulfil the extraction condition. The first concentration was sterilized by filtration and the subsequent dilutions were performed in sterile RPMI medium at a ratio of 1:2.

### **Cytotoxicity test**

A colorimetric method that uses the tetrazolium compound MTS was used to determine the number of viable cells in proliferation (Cory et al, 1991). 96-well microplates were prepared with 50 µL of extract diluted from 100 to 6.25% in RPMI medium in quadruplicates. The positive control was a phenol solution (0.5% v/v) as 100% concentration and the negative control was a high density polyethylene (HDPE) extract. The 100% concentration was the non-extract well A suspension of CHO-k1 (from second to fourth passages after thawing) with 6 x 104 cell/mL was prepared and 50 µL/well was pipetted into the microplates. The microplates were incubated for 72 hours at 37oC in a humidified 5% CO2 atmosphere. Blank and controls of the cells were also prepared. Cell viability was measured by adding 20 µL of MTS/PMS (20:1) solution to the humidified 5% CO2 incubator, followed by incubation for 2 hours at 37oC. The microplates were read in a spectrophotometer reader at 490 nm.

Cell viability was calculated by the equation:

84 Current Frontiers in Cryopreservation

The specific limit of monomer migration of hexamethylenediamine established in Resolution 105/99 of the National Health Surveillance Agency – ANVISA of 19 May 1999 is 2.4 mg/kg of simulant. The values for specific migration of hexamethylenediamine found in the analysis of the 42 µm film copolyamide, under the analytical conditions used, were

Assay performed in accordance with Standard ISO 10993-5: Biological evaluation of medical devices – Part 5: Cytotoxicity Assays: *in vitro* methods in samples of coextruded

Definitions of cytotoxicity vary, depending on the nature of the study and whether cells are killed or simply have their metabolism altered. Cytotoxicity is the toxicological effect that a

As defined in ISO 10993, "the numerous methods used and end-points measured in cytotoxicity determination can be grouped into categories of evaluation type, like assessments of cell damage by morphological means; measurements of cell damage; measurements of cell growth and measurements of specific aspects of cellular metabolism"

Chinese hamster ovary cell line (CHO-k1) was standardized for cytotoxicity and genotoxicity tests. Cells were maintained in RPMI medium supplemented with antibiotics and antimycotics (100 units/mL penicillin, 100 µg/mL streptomycin and 0.025 µg/mL amphotericin), 2mM glutamine, and 10% calf serum, at 37o C in a humidified 5% CO2 atmosphere until they reached confluence. For subculturing and for experiments, cells were harvested using 0.05% trypsin and 0.02% EDTA in phosphate-buffered saline, pH 7.4.

Samples of packaging, before and after sterilization, were submitted to this assay. The samples were immersed separately in RPMI culture medium at a final concentration of 1 cm2 / mL and left in an incubator at 37oC for 72 hours to fulfil the extraction condition. The first concentration was sterilized by filtration and the subsequent dilutions were performed

A colorimetric method that uses the tetrazolium compound MTS was used to determine the number of viable cells in proliferation (Cory et al, 1991). 96-well microplates were prepared with 50 µL of extract diluted from 100 to 6.25% in RPMI medium in quadruplicates. The positive control was a phenol solution (0.5% v/v) as 100% concentration and the negative control was a high density polyethylene (HDPE) extract. The 100% concentration was the

substance can cause *in vitro*, at cellular level (Freshney, 2000).

[IOS, 2010]. The cells can be exposed to the samples or their extracts.

below the established limit.

**3.3 Cytotoxicity assay** 

plastic film.

**3.3.1 Cytotoxicity** 

**3.3.2 The protocols** 

**Extract preparation** 

**Cytotoxicity test** 

in sterile RPMI medium at a ratio of 1:2.

**Cell culture** 

$$\text{CV\%} = \frac{\text{ODsample}}{\text{OD nonzero} \times 100} \times 100$$

Where: CV% = cell viability, OD sample = optical density at 490 nm of the extract dilution, OD non extract = optical density at 490 nm of the well without extract.

The results consider the following parameters:

a. Controls (positive and negative)

Positive control: 0.5% Phenol solution

Negative control: HDPE (high-density polyethylene) extract.

	- Positive control: material which, when tested according to Standard ISO 10993-5, promotes a cytotoxic response.
	- Negative control: material which, when tested according to Standard ISO 10993-5, does not promote a cytotoxic response.
	- IC50(%): cytotoxicity index 50%, concentration of extract that kills 50% of the viable cell population.

The results of the assay showed that the samples of packaging material, before and after sterilization by ethylene oxide (EtO), resulted in viability of over 90%, and therefore do not become cytotoxic.
