**3.4 Sterility assay and ethylene oxide residues**

The samples were classified according to their position (0 to 5a; 0 to 5b) during exposure to EtO, for the penetration analysis. The outermost or surface position corresponds to the number 5, and the innermost position to the number 0. (Figure 6)

Sterility tests were carried out through the analyses of two biological indicators (bi 3M - ATTEST –TM Bacillus atrophaeus and Terragene – Bionova BT40 - Bacillus atrophaeus). Incubation time was 48 h at a temperature of 35°C ± 1.5.

Samples of packaging were also submitted to direct incubation for 7 days with TSB liquid culture at a temperature of 35°C ± 1.5. The methodology used is in accordance with the Brazilian Pharmacopoeia. The analyses of the three sterility tests (biological indicator Bionova BT40, 3Mattest and direct incubation) confirm the sterility of the packaging material after being submitted to ethylene oxide gas.

Validation of Primary Packaging for Cryopreserved Musculoskeletal Tissues 87

The analysis of ethylene oxide residues was performed by the Gas Chromatography test, determining the levels of Ethylene chlorohydrin and Ethylene glycol. The data are shown in

ETO

ETCH

ETG

Note. Maximum limits according to the Brazilian legislation. [ ETO up to 25/ ETCH up to 25/ETG up to

Analyses of the packaging used in this study demonstrated that it is a good option for

Our experience with assays to validate coextruded polyethylene and polyamide plastic film shows that the mechanical properties of this material are not altered by cryopreservation and sterilization. Penetration resistance of the thermal seal remained unaltered after all the

We found a loss of barrier due to increased oxygen permeability of around 10% after sterilization and cryopreservation, which can be explained by the humidification of the polyamide. However, this slight alteration in oxygen permeability does not compromise the

In relation to total migration, we did not observe any alterations in the assays, i.e. once again, sterilization and cryopreservation did not lead to monomer migration at levels above

Fig. 9. Residues (ETO, ETCH, ETG) found in the samples submitted to sterilization.

5A 4A 3A 2A 1A 0 1B 2B 3B 4B 5B

processes carried out in a tissue bank, such as sterilization and cryopreservation.

inner vacuum of the packaging, and does not place at risk the tissue packaged in it.

250]

0

5

10

15

20

25

30

35

40

**4. Final considerations** 

cryopreservation of tissues at a temperature of – 80ºC.

**Figure 9** and show that the levels are within the limits accepted by our legislation.

Fig. 6. Samples of packaging positioned from 0 to 5 for EtO penetration analysis.

Fig. 7 and 8. Performance of the sterility test by direct incubation in samples submitted to ethylene oxide.

The analysis of ethylene oxide residues was performed by the Gas Chromatography test, determining the levels of Ethylene chlorohydrin and Ethylene glycol. The data are shown in **Figure 9** and show that the levels are within the limits accepted by our legislation.

Note. Maximum limits according to the Brazilian legislation. [ ETO up to 25/ ETCH up to 25/ETG up to 250]

Fig. 9. Residues (ETO, ETCH, ETG) found in the samples submitted to sterilization.
