**3.2 Physicomechanical and barrier properties tests**

#### **A) Characterization of coex film in relation to its thickness and water vapor transmission rate (WVTR)**

The thickness of each layer of plastic material in the film sample was determined through images captured by a Metaval inverted microscope operating at a magnification of 200x, using the image analysis system Axio Vision (Zeiss®). The cross-section of the sample was obtained using a Leica microtome, model RM2245, with section thickness set to 40 μm. To facilitate visualization of the barrier layer, 2% iodine solution was used as a contrasting agent. Five cross-section samples of the material were obtained; five measurements were performed for each sample, totalling 25 thickness measurements. The test was carried out at a temperature of around 23°C, after packaging of the sample in a controlled environment at 23°C ± 2°C and (50 ± 3)% relative humidity for a minimum period of 48 hours.

The water vapor transmission rate was determined using a MOCON PERMATRAN-W 3/31 device, following the procedure described in regulation ASTM F1249-06 - Standard test methods for water vapor transmission rate through plastic film and sheeting using a modulated infrared sensor. In this test, the water vapor that passes through the film is carried to the infrared sensor by ultra-dry nitrogen flow. The sensor measures the fraction of energy absorbed by the water vapor, and emits an electrical signal with amplitude proportional to the concentration of water vapor. The range of this signal is compared with that of the signal produced by the water vapor that passes through a calibration film with a known water vapour transmission rate. The effective permeation area of each sample was 50cm2. The assay was performed at 38oC/100%RH and in this condition, the calibration standard showed water vapour transmission rate of 4.54g water.m-2.day-1. The water vapor transmission rate of the sample was corrected for the condition 38oC/90%RH, multiplying the results by a factor of 0.9.

The total thickness of each layer of coextruded film is shown in Table 1. Figure 10 shows an example of a cross-section image of the sample obtained for the assay.

Validation of Primary Packaging for Cryopreserved Musculoskeletal Tissues 73

Table 2. Water vapor transmission rate (WVTR) of the coextruded plastic film at

The WVTR depends on the thickness of the LLDPE layers of the coex film (80µm).

samples for a minimum period of 48 hours in this environment.

The penetration resistance of the coextruded film was determined based on Standard ASTM F 1306-90 (2008) e1 - Standard test method for slow rate penetration resistance of flexible barrier films and laminates, on an Instron 5500R universal testing machine, using load cells of 100 N. The speed of penetration, performed with a spherical-tipped metal probe with diameter of approximately 3.2 mm, was 25 mm/min. The penetration was performed from the inner surface to the outer surface of the material. The test was conducted in an environment of 23°C ± 2°C and (50 ± 3)% relative humidity, after leaving the packaged

The evaluation of penetration resistance was carried out on samples in their original or nonfrozen condition (0) and 30, 60, 120 and 150 days after freezing at -80 ◦C. The data are

> **Evaluation periods (days) 0 30 60 90 120 150**

**Mean** 12.1 ab 12.6 bc 12.9 c 12.6 bc 11.1 d 11.8 a **VI** 11.5–12.7 11.8– 13.5 11.9– 14.2 11.2– 14.1 9.4 – 12.3 10.9– 12.4 **CV (%)** 3.1 3.6 4.7 6.9 9.1 3.7

**Mean** 9.4 a 11.0 b 10.0 c 9.8 c 8.3 d 9.0 e **VI** 9.2 – 9.8 10.7– 11.4 9.5 – 10.5 8.9 – 10.6 7.4 – 9.1 8.2 – 9.5 **CV (%)** 2.3 2.3 2.6 5.2 5.5 3.7

**Mean** 70 ad 76 b 78 b 73 ab 62 c 69 d **VI** 64 – 74 70 – 84 72 – 85 64 – 86 52 – 73 62 – 73 **CV (%)** 4.6 5.5 5.4 8.6 11 5.6

a,b,c,d: for an analyzed property, mean values on the same line accompanied by the same superscript letter did not show any difference between them in the least significant difference (LSD) test, at a level

Table 3. Penetration resistance of the sample during storage at -80°C.

Coextruded film LLDPE / LLDPE / PA / LLDPE / PA / LLDPE / LLDPE

VI – variation interval; CV – coefficient of variation

38C/90%RH – Permatran-W 3/31 method.

Values relate to four measurements

**B) Penetration resistance** 

shown in Table 3 and Figure 3.

Values relate to 10 measurements: 1 N = 0.102 kgf VI/CV: Variation interval/Coefficient of variation

**Penetration resistance** 

**Force at break (N)** 

**Deformation at break (mm)** 

> **Energy at break (mJ)**

of error of 5%.

**Sample WVTR (g water . m-2.day-1)** 

**Mean VI CV (%)** 

3.30 3.2 – 3.4 4.4


Values for 25 measurements

A-G: Text for visualization in Figure 10

Table 1. Total thickness of each layer of sample of coextruded plastic film.

Thus, by optical microscopy, it was observed that it is a coextruded material with seven layers, with two intermediate layers of PA of approximately 6µm each. The other LLDPE layers totalled 80µm.

Fig. 2. Example of cross-section image of the sample obtained with the microscope operating at a magnification of 200x.

The characterization of the film in relation to water vapor transmission rate is shown in Table 2.


Values relate to four measurements

72 Current Frontiers in Cryopreservation

**Total** 91.1 90.0 – 92.9 0.9 **LLDPE (A)** 27.3 26.2 – 28.5 2.4 **LLDPE blend (B)** 7.6 7.0 – 8.0 3.8

**PA (C)** 5.6 5.1 – 5.9 4.8

**PA (C)** 5.6 5.0 – 6.0 5.6

Thus, by optical microscopy, it was observed that it is a coextruded material with seven layers, with two intermediate layers of PA of approximately 6µm each. The other LLDPE

Fig. 2. Example of cross-section image of the sample obtained with the microscope operating

The characterization of the film in relation to water vapor transmission rate is shown in

**LLDPE blend (D)** 6.8 5.3 – 7.8 11

**LLDPE blend (F)** 7.7 7.1 – 8.6 5.9 **LLDPE (G)** 31.0 30.1 – 32.1 1.9

Table 1. Total thickness of each layer of sample of coextruded plastic film.

**Thickness (µm)** 

**Mean Variation Interval Coefficient of variation (%)** 

**Determinations** 

Values for 25 measurements

layers totalled 80µm.

at a magnification of 200x.

Table 2.

A-G: Text for visualization in Figure 10

VI – variation interval; CV – coefficient of variation

Table 2. Water vapor transmission rate (WVTR) of the coextruded plastic film at 38C/90%RH – Permatran-W 3/31 method.

The WVTR depends on the thickness of the LLDPE layers of the coex film (80µm).

#### **B) Penetration resistance**

The penetration resistance of the coextruded film was determined based on Standard ASTM F 1306-90 (2008) e1 - Standard test method for slow rate penetration resistance of flexible barrier films and laminates, on an Instron 5500R universal testing machine, using load cells of 100 N. The speed of penetration, performed with a spherical-tipped metal probe with diameter of approximately 3.2 mm, was 25 mm/min. The penetration was performed from the inner surface to the outer surface of the material. The test was conducted in an environment of 23°C ± 2°C and (50 ± 3)% relative humidity, after leaving the packaged samples for a minimum period of 48 hours in this environment.

The evaluation of penetration resistance was carried out on samples in their original or nonfrozen condition (0) and 30, 60, 120 and 150 days after freezing at -80 ◦C. The data are shown in Table 3 and Figure 3.


Values relate to 10 measurements: 1 N = 0.102 kgf

VI/CV: Variation interval/Coefficient of variation

a,b,c,d: for an analyzed property, mean values on the same line accompanied by the same superscript letter did not show any difference between them in the least significant difference (LSD) test, at a level of error of 5%.

Table 3. Penetration resistance of the sample during storage at -80°C.

Validation of Primary Packaging for Cryopreserved Musculoskeletal Tissues 75

3.41 – 3.83

5.20

Fig. 4. Maximum seal strength during the period of storage at -80°C ( days).

The oxygen transmission rate at humid was determined by the coulometric method, according to the procedure described in ASTM F 1927 - Standard test method for determination of oxygen gas transmission rate, permeability and permeance at controlled relative humidity through barrier materials using a coulometric detector, on MOCON OXTRAN device, model 2/20, operating with pure oxygen as permanent gas. The tests were carried out at 23oC to 75%RH, with the samples packaged for 88 to 112 hours in a temperature-controlled room at 25ºC and 75%RH. The effective area of permeation of each sample was 50cm2. The result obtained was corrected for 1 atm of partial pressure gradient

a,b: for a seal type, mean values on the same line accompanied by the same superscript letter did not show any difference between them in the least significant difference (LSD) test, at a level of error of 5%.

**Evaluation periods (days) 0 30 60 90 120 150** 

> 3.34 – 3.97

> 4.31 – 5.95

3.43 – 3.96

4.68 – 5.54

3.12 – 3.78

4.48 – 5.44

**Mean** 3.60 ab 3.65 b 3.60 ab 3.73 b 3.73 b 3.44 a

3.38 – 3.80

**CV (%)** 4.4 **4.0 4.4 5.7 4.5** 7.0

**Mean** - 4.69 a 4.75 a 5.07 a 5.09 a 4.84 a

3.20 – 5.85

**CV (%)** - 9.6 15 9.6 5.1 6.6

**Maximum seal strength (kgf/25.4 mm)** 

**VI** 3.36 –

3.95

**VI** - 3.65 –

Values relate to ten determinations: 1 kgf/25.4 mm = 386.1 N/m

VI/CV: Variation interval/Coefficient of variation

Table 4. Seal strength during storage at -80°C.

**D) Oxygen transmission rate** 

of oxygen.

**Side seal** 

**Top seal** 

Fig. 3. Penetration resistance of the sample during the period of storage at -80°C ( days).

The results shown in Table 3 and Figure 3 demonstrate that the process of sterilization and packaging of human bone, and storage for 150 days at -80°C, did not alter the penetration properties of the coextruded film. Variations were observed in the results of the three properties evaluated, including statistical differences in some periods/properties, but as these variations were small, without any clearly-defined trend, no alteration is expected in penetration resistance of the film during packaging and storage of human tissue for a period of 150 days at -80 °C.

#### **C) Seal strength**

The seal strength of the packaging was determined according to ASTM F 88/F 88M-09 - Standard test method for seal strength of flexible barrier materials. Samples of 25.4 mm in width were submitted to tensile test in a 5500R Instron universal testing machine, operating with load cells of 50N and 100N, at a speed of 300mm/min. The distance between the fixing clamps and the sample was 25mm. The test was conducted in an environment of 23 °C ± 2 °C and (50 ± 3)% relative humidity, after leaving the pre-prepared, packaged samples in this same environment for at least 48 hours.

The strength of the side seal is different from that of the top seal, because the top seal of the packaging was made after the bone tissue packaging process, while the side seals were made by the package manufacturer.

Despite this, there was no statistical difference between the results obtained for both seals. Thus, the sterilization and packaging process of human tissue, and storage for 150 days at - 80°C did not alter the seal strength.


Values relate to ten determinations: 1 kgf/25.4 mm = 386.1 N/m

VI/CV: Variation interval/Coefficient of variation

74 Current Frontiers in Cryopreservation

Fig. 3. Penetration resistance of the sample during the period of storage at -80°C ( days).

of 150 days at -80 °C.

same environment for at least 48 hours.

made by the package manufacturer.

80°C did not alter the seal strength.

**C) Seal strength** 

The results shown in Table 3 and Figure 3 demonstrate that the process of sterilization and packaging of human bone, and storage for 150 days at -80°C, did not alter the penetration properties of the coextruded film. Variations were observed in the results of the three properties evaluated, including statistical differences in some periods/properties, but as these variations were small, without any clearly-defined trend, no alteration is expected in penetration resistance of the film during packaging and storage of human tissue for a period

The seal strength of the packaging was determined according to ASTM F 88/F 88M-09 - Standard test method for seal strength of flexible barrier materials. Samples of 25.4 mm in width were submitted to tensile test in a 5500R Instron universal testing machine, operating with load cells of 50N and 100N, at a speed of 300mm/min. The distance between the fixing clamps and the sample was 25mm. The test was conducted in an environment of 23 °C ± 2 °C and (50 ± 3)% relative humidity, after leaving the pre-prepared, packaged samples in this

The strength of the side seal is different from that of the top seal, because the top seal of the packaging was made after the bone tissue packaging process, while the side seals were

Despite this, there was no statistical difference between the results obtained for both seals. Thus, the sterilization and packaging process of human tissue, and storage for 150 days at -

a,b: for a seal type, mean values on the same line accompanied by the same superscript letter did not show any difference between them in the least significant difference (LSD) test, at a level of error of 5%.

Table 4. Seal strength during storage at -80°C.

Fig. 4. Maximum seal strength during the period of storage at -80°C ( days).

#### **D) Oxygen transmission rate**

The oxygen transmission rate at humid was determined by the coulometric method, according to the procedure described in ASTM F 1927 - Standard test method for determination of oxygen gas transmission rate, permeability and permeance at controlled relative humidity through barrier materials using a coulometric detector, on MOCON OXTRAN device, model 2/20, operating with pure oxygen as permanent gas. The tests were carried out at 23oC to 75%RH, with the samples packaged for 88 to 112 hours in a temperature-controlled room at 25ºC and 75%RH. The effective area of permeation of each sample was 50cm2. The result obtained was corrected for 1 atm of partial pressure gradient of oxygen.

Validation of Primary Packaging for Cryopreserved Musculoskeletal Tissues 77

The evaluations of overall migration were performed according to Resolution 51 of 26 November 2010 published by *Agência Nacional de Vigilância Sanitária do Ministério da Saúde* (National Agency of Sanitary Surveillance of Health Ministry) in Diário Oficial da União (official journal of the Brazilian Government) on 30 November 2010. This Resolution

The methodology to quantify the overall migration was according to the method of the European Standard EN 1186-1: materials and articles in contact with foodstuffs. Plastics. Part 1: guide to the selection of conditions and test methods for overall migration, EN 1186- 3: Materials and articles in contact with foodstuffs. Plastics. Test methods for overall migration into aqueous food simulants by total immersion and EN 1186-14: materials and articles in contact with foodstuffs. Plastics. Part 1: Part 14: test methods for "substitute tests" for overall migration from plastics intended to come into contact with fatty foodstuffs using test media isooctane and 95% ethanol and consists of the sample contact with extraction solutions in certain periods and temperatures that simulate their actual condition of use. The residues of overall migration were determined by the weight difference after the contact and evaporation of the solutions through an analytical scale with 0.01mg of accuracy. The

> **Model Solution Contact condition**  Ultra purified water 40oC/10 days

The results of the overall migration tests performed on the transparent coextruded plastic film, obtained using the model solutions and the specific contact conditions are shown in Table 7.

20oC/48 hours 8.0 Before EtO 1.21 0.64 0.50(2) – 1.85

Table 7. Residues of overall migration obtained for the transparent coextruded plastic film,

**Sample Mean Standard** 

**Deviation** 

Before EtO 0.72 0.21 0.50(2) - 1.00 After EtO 0.51 0.02 0.50(2) - 0.55

Before EtO 0.54 0.09 0.50(2) – 0.67 After EtO 0.92 0.19 0.72 – 1.12

After EtO 1.50 0.72 0.50(2) – 2.05

**Variation interval**

water at 3% (w/v) 40oC/10 days Isooctane 40oC/10 days

internalizes Mercosul Technical Regulation GMC 32/10.

sample was evaluated under the contact conditions shown in Table 6.

Table 6. Conditions of time and temperature used in the overall migration.

**Maximum limit of overall migration**

8.0

8.0

before and after the application of EtO, in mg/dm2 (1).

(2) Limit of quantification of the method in the analytical conditions used.

Acetic acid solution in Ultra purified

Model Solution/Contact Condition

> Ultra purified water/ 40oC/10 days

3% Acetic acid solution (w/v)/ 40oC/10 days

Isooctane/

(3) Not applicable.

(1) Result of four determinations.

**E) Overall Migration** 


values relate to two determinations

VI – variation interval; CV – coefficient of variation

a,b: for a seal type, mean values on the same line accompanied by the same superscript letter did not show any difference between them in the least significant difference (LSD) test, at a level of error of 5%.

0 10 20 30 40 50 60 70 80 90 100 110 120 0 20 40 60 80 100 120 140 160 **Storage days at -80ºC O2TR mL (STP).m-2.dia-1 at 23°C/75%RH**

Table 5. Oxygen transmission rates (O2TR) during storage at -80°C.

Fig. 5. Oxygen transmission rates (O2TR) during storage at -80°C.

The results of the oxygen permeability rates shown in Table 5 and Figure 5 indicate a mean increase of 10% in permeability after the process of sterilization, vacuum packaging of the bone, and storage at -80°C, which was observed after 30 days of storage. This tendency to increase, probably due to humidification of the PA, led to a small loss of barrier which was maintained throughout the storage period of 120 days. Meanwhile, in the analysis of samples from 30 days of storage at -80°C, a high oxygen transmission rate of was observed, which was not expected, and as we did not have any more stored samples, it was not possible to re-evaluate this result. This higher O2TR may be the result of some variation in thickness of the PA in the samples evaluated in this period. In any case, the level of oxygen transmission rate of the film did not lead to loss of vacuum in the samples stored in the Tissue Bank for 150 days at -80°C.

#### **E) Overall Migration**

76 Current Frontiers in Cryopreservation

**Mean** 69.75 a 75.95 a 74.97 a 82.57 a 76.98 a 113.67 b

74.48- 75.46

**CV (%)** 2.3 7.8 0.9 5.5 5.8 10.7

a,b: for a seal type, mean values on the same line accompanied by the same superscript letter did not show any difference between them in the least significant difference (LSD) test, at a level of error of 5%.

71.77- 80.14

Table 5. Oxygen transmission rates (O2TR) during storage at -80°C.

Fig. 5. Oxygen transmission rates (O2TR) during storage at -80°C.

The results of the oxygen permeability rates shown in Table 5 and Figure 5 indicate a mean increase of 10% in permeability after the process of sterilization, vacuum packaging of the bone, and storage at -80°C, which was observed after 30 days of storage. This tendency to increase, probably due to humidification of the PA, led to a small loss of barrier which was maintained throughout the storage period of 120 days. Meanwhile, in the analysis of samples from 30 days of storage at -80°C, a high oxygen transmission rate of was observed, which was not expected, and as we did not have any more stored samples, it was not possible to re-evaluate this result. This higher O2TR may be the result of some variation in thickness of the PA in the samples evaluated in this period. In any case, the level of oxygen transmission rate of the film did not lead to loss of vacuum in the samples stored in the

0 20 40 60 80 100 120 140 160 **Storage days at -80ºC**

**Evaluation periods (days)** 

**0 30 60 90 120 150** 

79.34- 85.80 73.83- 80.13 105.05- 122.29

**O2TR**  mL (STP).m-2.day-1 at 23°C/75%RH

**VI** 68.63–

values relate to two determinations

Tissue Bank for 150 days at -80°C.

**O2TR mL (STP).m-2.dia-1 at 23°C/75%RH**

70.87

VI – variation interval; CV – coefficient of variation

The evaluations of overall migration were performed according to Resolution 51 of 26 November 2010 published by *Agência Nacional de Vigilância Sanitária do Ministério da Saúde* (National Agency of Sanitary Surveillance of Health Ministry) in Diário Oficial da União (official journal of the Brazilian Government) on 30 November 2010. This Resolution internalizes Mercosul Technical Regulation GMC 32/10.

The methodology to quantify the overall migration was according to the method of the European Standard EN 1186-1: materials and articles in contact with foodstuffs. Plastics. Part 1: guide to the selection of conditions and test methods for overall migration, EN 1186- 3: Materials and articles in contact with foodstuffs. Plastics. Test methods for overall migration into aqueous food simulants by total immersion and EN 1186-14: materials and articles in contact with foodstuffs. Plastics. Part 1: Part 14: test methods for "substitute tests" for overall migration from plastics intended to come into contact with fatty foodstuffs using test media isooctane and 95% ethanol and consists of the sample contact with extraction solutions in certain periods and temperatures that simulate their actual condition of use. The residues of overall migration were determined by the weight difference after the contact and evaporation of the solutions through an analytical scale with 0.01mg of accuracy. The sample was evaluated under the contact conditions shown in Table 6.


Table 6. Conditions of time and temperature used in the overall migration.

The results of the overall migration tests performed on the transparent coextruded plastic film, obtained using the model solutions and the specific contact conditions are shown in Table 7.


(1) Result of four determinations.

(2) Limit of quantification of the method in the analytical conditions used.

(3) Not applicable.

Table 7. Residues of overall migration obtained for the transparent coextruded plastic film, before and after the application of EtO, in mg/dm2 (1).

Validation of Primary Packaging for Cryopreserved Musculoskeletal Tissues 79

(mL) 10.0 mL Before EtO 0.5(2) (3) (3)

(mg) 15 mg Before EtO 1.0(2) (3) (3)

The tests acidity or alkalinity, sulfated ash, absorbance, extractable aluminum, chromium, titanium, vanadium, zinc, zirconium and extractables from heavy metals, expressed as lead were conducted based in the methodology described in the European Pharmacopoeia, Chapters "3.1.3 Polyolefines", "3.1.4 Polyethylene without Additives for Containers for Parenteral Preparations and for Ophthalmic Preparations" and "3.1.5 Polyethylene with Additives for Containers for Parenteral Preparations and for

The methodology for quantification of extractables aluminum, chromium, titanium, vanadium, zinc and zirconium involved contact of 100 grams of sample with a solution of 0.1 M hydrochloric acid for one hour at the reflux temperature. After treating the sample, the metals contents were quantified by atomic emission spectrometry induced by plasma, with an optical detector, in a Perkin Elmer equipment, model OPTIMA 2000DV, using

The same procedure described for quantification of extractable aluminum, chromium, titanium, vanadium, zinc and zirconium was used. The method for quantification of extractable lead was used instead of the colorimetric method of determination of heavy metals established by the European Pharmacopoeia, which is based on color comparison between the extracting solution and a solution of lead at a concentration of 2.5 mg/kg.

The method for quantification of alkalinity or acidity and absorbance involved contact of 12.5 grams of sample with 250 mL of deionized water for five hours at the reflux

After the extraction time, both the extracting solution in contact with the samples and a

blank solution (reference) were assessed as to the following tests:

(2) Corresponds to the limit of quantification of the method in the analytical conditions used.

**Extractables of aluminium, chromium, titanium, vanadium, zinc and zirconium** 

**USP Sample Mean Standard** 

**deviation** 

After EtO 0.5(2) (3) (3)

After EtO 1.0(2) (3) (3)

Before EtO 0.05(2) (3) (3) After EtO 0.05(2) (3) (3)

**Variation interval** 

**Limit based on** 

1 mg/kg (ppm)

Table 8. Physicochemical assays of the analyzed samples(1). **E 2.) Assays According to the European Pharmacopoeia** 

**Physicochemical Assay** 

Buffering capacity

Non-volatile residue:

Heavy Metals (as lead) (mg/kg (ppm))

(3) Values no applicable

(1) Results of three determinations.

Ophthalmic Preparations".

appropriate calibration curves for the analyses. **Extractables of Heavy Metals, expressed as lead** 

**Alkalinity or Acidity, and Absorbance** 

temperature.

The maximum limit of overall migration provided by Resolution nº105/99 is of 8mg of residue per dm2 of contact plastic material, with an analytical tolerance of 10%. Therefore 8.8mg/dm2 is the maximum tolerable value.

The overall migration values found in the samples analyzed, in the analytical conditions used, were below the established limit. There was no statistical significance with sterilization with ethylene oxide (EtO).

#### **E 1.) Physicochemical tests – According to USP 33**

The physicochemical tests were conducted based in the methodology describe in the Chapter <661> Containers – Plastics - Physicochemical Tests of the **United States Pharmacopeia (USP 33)**.

In accordance to the United States Pharmacopeia, physicochemical tests are designed to determine physical and chemical properties of plastic materials. The extracts methodology consists of the sample contact with a extraction solution (deionized water) at 70ºC during 24 hours, maintaining the ratio area / volume of 120 cm2 total surface area of plastic material for each 20 mL of extraction solution.

The analyzed sample was received in the form of the film cut into strips with dimensions of 5.0 cm long, 0.3 cm wide and thickness less than 0.1 cm. In this case, the thickness of the material to determine the total area was not considered and was maintained the ratio of 120 cm2 for each 20 mL of extraction solution. Water was used as extraction solution.

After the contact, the extraction solution and blank reagent were analyzed by the following tests:

**Buffering Capacity:** Titrate 20 mL of the extraction solution potentiometrically to a pH of 7.0, using 0.01 N sodium hydroxide. Treat a 20.0 mL portion of the blank reagent similarly. The difference between the two volumes can not be greater than 10.0 mL.

**Nonvolatile Residue:** 50 mL of the extraction solution were evaporated on a hot plate, after the residue was dried at 105ºC for 1 hour on a oven and finally the nonvolatile residue was weighted through an analytical balance with 0.01mg of accuracy. Treat a 50.0 mL portion of the blank reagent similarly. The difference between the two volumes can not be greater than 15.0 mg.

**Residue on Ignition:** in the residues obtained in nonvolatile residues test, add sulfuric acid and burn on the muffle furnace until constant weight. Treat the blank reagent similarly. The difference between the two volumes should not be greater than 5.0 mg. It is not necessary to perform this test when the nonvolatile residue test result does not exceed 5.0 mg.

**Heavy Metal, as lead:** an aliquot of extraction solution has been transferred to a volumetric flask and acidified with nitric acid and the volume was completed with the extraction solution. After treating the sample, the lead content was quantified by atomic emission spectrometry induced by plasma, with an optical detector, in a Perkin Elmer equipment, model OPTIMA 2000DV, using appropriate calibration curves for the analyses. This test was conducted in replacement to the heavy metal test stablished by American Pharmacopeia, whose result is expressed as lead (and is based on the colour comparison among the test solution and a solution of lead with concentration of 1.0 mg / kg).


(1) Results of three determinations.

(2) Corresponds to the limit of quantification of the method in the analytical conditions used.

(3) Values no applicable

78 Current Frontiers in Cryopreservation

The maximum limit of overall migration provided by Resolution nº105/99 is of 8mg of residue per dm2 of contact plastic material, with an analytical tolerance of 10%. Therefore

The overall migration values found in the samples analyzed, in the analytical conditions used, were below the established limit. There was no statistical significance with

The physicochemical tests were conducted based in the methodology describe in the Chapter <661> Containers – Plastics - Physicochemical Tests of the **United States** 

In accordance to the United States Pharmacopeia, physicochemical tests are designed to determine physical and chemical properties of plastic materials. The extracts methodology consists of the sample contact with a extraction solution (deionized water) at 70ºC during 24 hours, maintaining the ratio area / volume of 120 cm2 total surface area of plastic material

The analyzed sample was received in the form of the film cut into strips with dimensions of 5.0 cm long, 0.3 cm wide and thickness less than 0.1 cm. In this case, the thickness of the material to determine the total area was not considered and was maintained the ratio of 120

After the contact, the extraction solution and blank reagent were analyzed by the following

**Buffering Capacity:** Titrate 20 mL of the extraction solution potentiometrically to a pH of 7.0, using 0.01 N sodium hydroxide. Treat a 20.0 mL portion of the blank reagent similarly.

**Nonvolatile Residue:** 50 mL of the extraction solution were evaporated on a hot plate, after the residue was dried at 105ºC for 1 hour on a oven and finally the nonvolatile residue was weighted through an analytical balance with 0.01mg of accuracy. Treat a 50.0 mL portion of the blank reagent similarly. The difference between the two volumes can not be greater than

**Residue on Ignition:** in the residues obtained in nonvolatile residues test, add sulfuric acid and burn on the muffle furnace until constant weight. Treat the blank reagent similarly. The difference between the two volumes should not be greater than 5.0 mg. It is not necessary to

**Heavy Metal, as lead:** an aliquot of extraction solution has been transferred to a volumetric flask and acidified with nitric acid and the volume was completed with the extraction solution. After treating the sample, the lead content was quantified by atomic emission spectrometry induced by plasma, with an optical detector, in a Perkin Elmer equipment, model OPTIMA 2000DV, using appropriate calibration curves for the analyses. This test was conducted in replacement to the heavy metal test stablished by American Pharmacopeia, whose result is expressed as lead (and is based on the colour comparison

perform this test when the nonvolatile residue test result does not exceed 5.0 mg.

among the test solution and a solution of lead with concentration of 1.0 mg / kg).

cm2 for each 20 mL of extraction solution. Water was used as extraction solution.

The difference between the two volumes can not be greater than 10.0 mL.

8.8mg/dm2 is the maximum tolerable value.

**E 1.) Physicochemical tests – According to USP 33** 

sterilization with ethylene oxide (EtO).

for each 20 mL of extraction solution.

**Pharmacopeia (USP 33)**.

tests:

15.0 mg.

Table 8. Physicochemical assays of the analyzed samples(1).

#### **E 2.) Assays According to the European Pharmacopoeia**

The tests acidity or alkalinity, sulfated ash, absorbance, extractable aluminum, chromium, titanium, vanadium, zinc, zirconium and extractables from heavy metals, expressed as lead were conducted based in the methodology described in the European Pharmacopoeia, Chapters "3.1.3 Polyolefines", "3.1.4 Polyethylene without Additives for Containers for Parenteral Preparations and for Ophthalmic Preparations" and "3.1.5 Polyethylene with Additives for Containers for Parenteral Preparations and for Ophthalmic Preparations".

#### **Extractables of aluminium, chromium, titanium, vanadium, zinc and zirconium**

The methodology for quantification of extractables aluminum, chromium, titanium, vanadium, zinc and zirconium involved contact of 100 grams of sample with a solution of 0.1 M hydrochloric acid for one hour at the reflux temperature. After treating the sample, the metals contents were quantified by atomic emission spectrometry induced by plasma, with an optical detector, in a Perkin Elmer equipment, model OPTIMA 2000DV, using appropriate calibration curves for the analyses.

#### **Extractables of Heavy Metals, expressed as lead**

The same procedure described for quantification of extractable aluminum, chromium, titanium, vanadium, zinc and zirconium was used. The method for quantification of extractable lead was used instead of the colorimetric method of determination of heavy metals established by the European Pharmacopoeia, which is based on color comparison between the extracting solution and a solution of lead at a concentration of 2.5 mg/kg.

#### **Alkalinity or Acidity, and Absorbance**

The method for quantification of alkalinity or acidity and absorbance involved contact of 12.5 grams of sample with 250 mL of deionized water for five hours at the reflux temperature.

After the extraction time, both the extracting solution in contact with the samples and a blank solution (reference) were assessed as to the following tests:

Validation of Primary Packaging for Cryopreserved Musculoskeletal Tissues 81

**Sample Mean Standard** 

Before EtO 0.1(2) (3) (3)

After EtO 0.1(2) (3) (3)

Before EtO 0.5(2) (3) (3) After EtO 0.5(2) (3) (3)

Before EtO 0.23 0.01 0.22 – 0.23 After EtO 0.24 0.04 0.22 – 0.26

Before EtO 0.29 0.01 0.28 – 0.30 After EtO 0.25 0.12 0.11 – 0.33

**Deviation** 

**Variation interval** 

**Limit based on European Pharmacopeia 6.0**

2.5mg/kg (ppm)

1.5 mL de sodium hydroxide 0.01 M

(2) Corresponds to the limit of quantification of the method in the analytical conditions used.

Table 10. Results for heavy metals, expressed as lead, acidity, absorbance and ash of the

The lead values, volume of 0.1 M sodium hydroxide used, and sulphated ash values found in the samples analyzed were below the maximum limits established in the European Pharmacopeia 6.0. In relation to absorbance, the values obtained in the two samples analyzed, before and after the application of sterilization with ethylene oxide (EtO), were

A038-2/11 – Packaging for human tissue – Final Report 17/20 This means that some substance of the coextruded film may have migrated to the extraction solution in contact

It should be emphasized that the methodology of the American and European Pharmacopeias apply to single-layer packaging, and that substances from internal layers of the film analyzed

The extruded film before and after sterilization with ethylene oxide was evaluated in relation to specific migration of 1-octene and ε-caprolactam. The evaluations of specific migrations of 1-octene, ε-caprolactam and hexamethylenediamine were carried out

The quantification of specific migration of 1-octene was evaluated based on Standard **CEN/ TS 13130-26**: materials and articles in contact with foodstuffs – Plastics substances subject to

may have been extracted, slightly increasing the absorbance of the extraction solution.

with the sample (deionized water), a fact that requires further investigation.

**Physicochemical Assay** 

Extractables of Heavy Metals, expressed as lead (ppm)

Acidity (mL of NaOH 0.01 M)

Absorbance

Sulphated Ash

UA- Unit of Absorbance.

(3) Values no applicable

samples analyzed (1).

**E 3.) Specific migration** 

limitation – Part 26:

according to the Brazilian legislation. **E 3.1) Specific migration of 1-octene** 

(1) Results of three determinations.

(UA) 0.2 UA

(4) Varies according to the presence of additives

(%) 0.02% and 1.00%(4)

slightly higher than the maximum limits established.

**Alkalinity and Acidity:** measurement of pH in a Micronal pHmeter, model B 474 and titration of 100 mL of extracting solution with sodium hydroxide 0.01M or hydrochloric acid 0.01M up to pH 7.0.

**Absorbance:** absorbance of the extracting solution was measured in the 220 nm to 340 nm wavelength range, using a quartz cuvette with 10 mm pathlenght in a UV / VIS spectrophotometer, using an Analytik Jena instrument, model Specord 210.

#### **Sulphated Ash**

Sulfated ash were assessed per requirements in European Pharmacopoeia 6.0, Chapter 2.4.14 - Sulfated Ash. The method for quantification of sulfated ash consisted in weighing 5.00 g 0.01 g of sample on an analytical scale with 10-5 g resolution and incineration at a temperature of 600 °C 20 ºC using a Milestone microwave heating furnace, model Pyro. After incinerating the sample, the ash were determined gravimetrically, using an analytical scale with 10-5 g resolution. There was not need to use sulfuric acid.

#### **Extractable aluminum, chromium, titanium, vanadium, zinc and zirconium**

The results of the extractable aluminum, chromium, titanium, vanadium, zinc and zirconium tests for the analyzed sample are shown in Table 9.


(1) Result of four determinations.

(2) Corresponds to the limit of quantification of the equipment in the analytical conditions used.

(3) Not applicable.

Table 9. Extractables of the metals aluminum (Al), chromium (Cr), titanium (Ti), vanadium (V), zinc (Zn) and zirconium (Zr), in mg/kg(1).

#### **Extractable Heavy Metal, Expressed as Lead**

The results of the extractable heavy metals, expressed as lead, acidity, absorbance and sulphated ash tests, for the analyzed sample are shown in Table 10.


UA- Unit of Absorbance.

80 Current Frontiers in Cryopreservation

**Alkalinity and Acidity:** measurement of pH in a Micronal pHmeter, model B 474 and titration of 100 mL of extracting solution with sodium hydroxide 0.01M or hydrochloric acid

**Absorbance:** absorbance of the extracting solution was measured in the 220 nm to 340 nm wavelength range, using a quartz cuvette with 10 mm pathlenght in a UV / VIS

Sulfated ash were assessed per requirements in European Pharmacopoeia 6.0, Chapter 2.4.14 - Sulfated Ash. The method for quantification of sulfated ash consisted in weighing 5.00 g 0.01 g of sample on an analytical scale with 10-5 g resolution and incineration at a temperature of 600 °C 20 ºC using a Milestone microwave heating furnace, model Pyro. After incinerating the sample, the ash were determined gravimetrically, using an analytical

The results of the extractable aluminum, chromium, titanium, vanadium, zinc and

Al 1.0 Before EtO 0.71 0.08 0.62 – 0.78

Cr 0.05 Before EtO 0.03 0.00 0.02 – 0.03

(2) Corresponds to the limit of quantification of the equipment in the analytical conditions used.

Table 9. Extractables of the metals aluminum (Al), chromium (Cr), titanium (Ti), vanadium

The results of the extractable heavy metals, expressed as lead, acidity, absorbance and

**Sample Mean Standard** 

**Deviation** 

After EtO 0.74 0.06 0.70 – 0.81

After EtO 0.33 0.05 0.27 – 0.37

Before EtO 0.10(2) (3) (3) After EtO 0.10(2) (3) (3)

Before EtO 0.10(2) (3) (3) After EtO 0.10(2) (3) (3)

Before EtO 0.05(2) (3) (3) After EtO 0.05(2) (3) (3)

Before EtO 0.05 0.00 0.05 – 0.06 After EtO 0.05 0.01 0.05 – 0.06

**Variation interval** 

spectrophotometer, using an Analytik Jena instrument, model Specord 210.

scale with 10-5 g resolution. There was not need to use sulfuric acid.

zirconium tests for the analyzed sample are shown in Table 9.

**Limit based on European Pharmacopeia 6.0** 

**Extractable aluminum, chromium, titanium, vanadium, zinc and zirconium** 

0.01M up to pH 7.0.

**Sulphated Ash** 

**Extractables** 

Ti 1.0

V 0.10

Zn 1.0

Zr 0.10

(V), zinc (Zn) and zirconium (Zr), in mg/kg(1). **Extractable Heavy Metal, Expressed as Lead** 

sulphated ash tests, for the analyzed sample are shown in Table 10.

(1) Result of four determinations.

(3) Not applicable.

(1) Results of three determinations.

(2) Corresponds to the limit of quantification of the method in the analytical conditions used.

(3) Values no applicable

(4) Varies according to the presence of additives

Table 10. Results for heavy metals, expressed as lead, acidity, absorbance and ash of the samples analyzed (1).

The lead values, volume of 0.1 M sodium hydroxide used, and sulphated ash values found in the samples analyzed were below the maximum limits established in the European Pharmacopeia 6.0. In relation to absorbance, the values obtained in the two samples analyzed, before and after the application of sterilization with ethylene oxide (EtO), were slightly higher than the maximum limits established.

A038-2/11 – Packaging for human tissue – Final Report 17/20 This means that some substance of the coextruded film may have migrated to the extraction solution in contact with the sample (deionized water), a fact that requires further investigation.

It should be emphasized that the methodology of the American and European Pharmacopeias apply to single-layer packaging, and that substances from internal layers of the film analyzed may have been extracted, slightly increasing the absorbance of the extraction solution.

#### **E 3.) Specific migration**

The extruded film before and after sterilization with ethylene oxide was evaluated in relation to specific migration of 1-octene and ε-caprolactam. The evaluations of specific migrations of 1-octene, ε-caprolactam and hexamethylenediamine were carried out according to the Brazilian legislation.

#### **E 3.1) Specific migration of 1-octene**

The quantification of specific migration of 1-octene was evaluated based on Standard **CEN/ TS 13130-26**: materials and articles in contact with foodstuffs – Plastics substances subject to limitation – Part 26:

Validation of Primary Packaging for Cryopreserved Musculoskeletal Tissues 83

for the ultrapure water simulants and 3% acetic acid solution (m/v) in ultrapure water after

Table 12. Specific migration of -caprolactam obtained for transparent coextruded plastic

The quantification of specific hexamethylenediamine migration was evaluated based on Standard **CEN/ TS 13130-21**: materials and articles in contact with foodstuffs - Plastics substances subject to limitation – Part 21: Determination of ethylenediamine and hexamethylenediamine in food simulants. The sample of hexamethylenediamine, adipic acid e caprolactam copolyamide in the form of a film, was placed in contact with the simulants, obeying an area:volume ratio of 600 cm2 to 1000 mL. The sample was evaluated

> **Maximum limit of specific migration of**  hexamethylenediamine

**Sample Mean** (1) **Standard** 

Before EtO 2.4 0.3 1.9 - 2.7 After EtO 1.7 0.2 1.3 - 1.9

Before EtO 2.7 0.1 2.6 - 2.8

After EtO 2.2 0.3 1.8 - 2.6

Before EtO 3.4 0.4 3.0 - 4.0 After EtO 3.2 0.2 2.9 - 3.5

**Mean** (1) **Standard** 

2,4 1.2(2) (3) (3)

2,4 1.1(2) (3) (3)

2,4 2.0(2) (3) (3)

**Deviation** 

**Variation Interval**

**Deviation** 

**Variation Interval**

**Maximum limit of specific migration of**  -caprolactam

15

film, before and after the application of EtO, in mg/dm2.

**E 3.3) Specific migration of hexamethylenediamine** 

under the contact conditions shown in Table 13.

(2) Quantification Limit of the method under the analytical conditions.

Table 13. Specific migration of hexamethylenediamine obtained for a 42 m film

sterilization with ethylene oxide.

40oC/10 days <sup>15</sup>

40oC/10 days <sup>15</sup>

(1) Result of three determinations.

Simulants/Contact Condition

Ultra purified water/ 100 ºC/30 minutes + 40 oC/10 days

3% Acetic acid solution in ultra purified water (w/v)/ 100 ºC/30 minutes + 40 oC/10 days

Olive oil/ 100 ºC/30 minutes + 40 oC/10 days

(1) Result of three determinations.

copolyamide, in mg/kg.

(3) Not applicable.

Simulants/Contact Condition

Ultra purified water/

3% Acetic acid solution in ultra purified water (w/v)/ 40oC/10 days

Olive oil/

Determination of 1-octene and tetrahydrofuran in food simulants, and consists of contact of the sample with solutions of extraction with times and temperatures that simulate its real condition of use.

The internal sides of the samples were placed in contact with the simulants, obeying an area:volume ratio of 600 cm2 to 1000 mL. The same was evaluated in the contact conditions shown in Table 11.


(1) Result of three determinations.

(2) Quantification Limit of the method under the analytical conditions.

(3) Not applicable.

Table 11. Specific migration of 1-octene obtained for transparent coextruded plastic film, before and after the application of EtO, in mg/dm2.

The specific limit of monomer migration of 1-octene established in Resolution 105/99 of the National

Health Surveillance Agency – ANVISA of 19 May 1999 is 15 mg/kg of simulant. The values for specific migration of 1-octene found in the samples analyzed, in the analytical conditions used, were below the established limit. Sterilization with ethylene oxide (EtO) did not affect the monomer migration potential of 1-octene.

#### **E 3.2) Specific migration of ε-caprolactam**

The quantification of specific ε-caprolactam was evaluated based on Standard **CEN/ TS 13130-16**: materials and articles in contact with foodstuffs – Plastics substances subject to limitation - Part 16: Determination of caprolactam and caprolactam salt in food simulants.

The internal surfaces of the samples were placed in contact with the simulants, obeying an area:volume ratio of 600 cm2/1000 mL. The samples were evaluated under the contact conditions shown in Table 12.

The specific limit of monomer migration of ε-caprolactam established in Resolution 105/99 of the National Health Surveillance Agency – ANVISA of 19 May 1999 is 15 mg/kg of simulant. The values for specific migration of ε-caprolactam found in the samples analyzed, in the analytical conditions used, were below the established limit. Sterilization with ethylene oxide (EtO) did not affect the potential monomer migration of ε-caprolactam for the fatty simulant, but was significantly lower (probability of 95% confidence – Tukey Test)


for the ultrapure water simulants and 3% acetic acid solution (m/v) in ultrapure water after sterilization with ethylene oxide.

(1) Result of three determinations.

82 Current Frontiers in Cryopreservation

Determination of 1-octene and tetrahydrofuran in food simulants, and consists of contact of the sample with solutions of extraction with times and temperatures that simulate its real

The internal sides of the samples were placed in contact with the simulants, obeying an area:volume ratio of 600 cm2 to 1000 mL. The same was evaluated in the contact conditions

40oC/10 days <sup>15</sup>Before EtO <sup>≤</sup>2.8(2) (3) (3)

40oC/10 days <sup>15</sup>Before EtO 8.7(2) (3) (3)

Table 11. Specific migration of 1-octene obtained for transparent coextruded plastic film,

The specific limit of monomer migration of 1-octene established in Resolution 105/99 of the

Health Surveillance Agency – ANVISA of 19 May 1999 is 15 mg/kg of simulant. The values for specific migration of 1-octene found in the samples analyzed, in the analytical conditions used, were below the established limit. Sterilization with ethylene oxide (EtO) did not affect

The quantification of specific ε-caprolactam was evaluated based on Standard **CEN/ TS 13130-16**: materials and articles in contact with foodstuffs – Plastics substances subject to limitation - Part 16: Determination of caprolactam and caprolactam salt in food simulants. The internal surfaces of the samples were placed in contact with the simulants, obeying an area:volume ratio of 600 cm2/1000 mL. The samples were evaluated under the contact

The specific limit of monomer migration of ε-caprolactam established in Resolution 105/99 of the National Health Surveillance Agency – ANVISA of 19 May 1999 is 15 mg/kg of simulant. The values for specific migration of ε-caprolactam found in the samples analyzed, in the analytical conditions used, were below the established limit. Sterilization with ethylene oxide (EtO) did not affect the potential monomer migration of ε-caprolactam for the fatty simulant, but was significantly lower (probability of 95% confidence – Tukey Test)

**Sample Mean (1)**

**Standar d Deviatio n** 

After EtO ≤2.8(2) (3) (3)

Before EtO 1.6(2) (3) (3)

After EtO 1.6(2) (3) (3)

After EtO 8.7(2) (3) (3)

**Variation Interval** 

**Maximum limit of specific migration of 1-octene** 

15

(2) Quantification Limit of the method under the analytical conditions.

before and after the application of EtO, in mg/dm2.

the monomer migration potential of 1-octene. **E 3.2) Specific migration of ε-caprolactam** 

condition of use.

shown in Table 11.

**Simulants/Contact Condition** 

Ultra purified water/

3% Acetic acid solution in ultra purified water (w/v)/ 40oC/10 days

Olive oil/

(3) Not applicable.

National

(1) Result of three determinations.

conditions shown in Table 12.

Table 12. Specific migration of -caprolactam obtained for transparent coextruded plastic film, before and after the application of EtO, in mg/dm2.

#### **E 3.3) Specific migration of hexamethylenediamine**

The quantification of specific hexamethylenediamine migration was evaluated based on Standard **CEN/ TS 13130-21**: materials and articles in contact with foodstuffs - Plastics substances subject to limitation – Part 21: Determination of ethylenediamine and hexamethylenediamine in food simulants. The sample of hexamethylenediamine, adipic acid e caprolactam copolyamide in the form of a film, was placed in contact with the simulants, obeying an area:volume ratio of 600 cm2 to 1000 mL. The sample was evaluated under the contact conditions shown in Table 13.


(1) Result of three determinations.

(2) Quantification Limit of the method under the analytical conditions.

(3) Not applicable.

Table 13. Specific migration of hexamethylenediamine obtained for a 42 m film copolyamide, in mg/kg.

Validation of Primary Packaging for Cryopreserved Musculoskeletal Tissues 85

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

> ODsample CV%= ×100 ODnonextract

Where: CV% = cell viability, OD sample = optical density at 490 nm of the extract dilution,

Positive control: material which, when tested according to Standard ISO 10993-5,

Negative control: material which, when tested according to Standard ISO 10993-5,

IC50(%): cytotoxicity index 50%, concentration of extract that kills 50% of the viable

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

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

Sterility tests were carried out through the analyses of two biological indicators (bi 3M - ATTEST –TM Bacillus atrophaeus and Terragene – Bionova BT40 - Bacillus atrophaeus).

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

hours at 37oC. The microplates were read in a spectrophotometer reader at 490 nm.

OD non extract = optical density at 490 nm of the well without extract.

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

does not promote a cytotoxic response.

Cell viability was calculated by the equation:

The results consider the following parameters:

b. Observations: definitions of some terms.

promotes a cytotoxic response.

**3.4 Sterility assay and ethylene oxide residues** 

number 5, and the innermost position to the number 0. (Figure 6)

Incubation time was 48 h at a temperature of 35°C ± 1.5.

material after being submitted to ethylene oxide gas.

a. Controls (positive and negative) Positive control: 0.5% Phenol solution

cell population.

become cytotoxic.

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 below the established limit.
