Evaluation of the contamination resistance of impregnated wipes

Evaluation of the contamination resistance of impregnated wipes

1. Objective

The aim of the present instructions is to describe a method for evaluating the resistance to microbial contamination of impregnated wipes.

1. Scope

This method applies to products such as cosmetic wipes, baby wipes and other similar impregnated nonwovens.

1. Principle

The test consists of artificial contamination of the wipes followed by counts of surviving microorganisms after different contact times.

Nota : On request, the contamination may be done a second time after the first inoculation

1. Reference strains

• Staphylococcus aureus ATCC 6538 (calibrated strain 108)
• Pseudomonas aeruginosa ATCC 9027 (calibrated strain 108)
• Escherichia coli ATCC 8739 (calibrated strain 108)
• Candida albicans ATCC 10231 (calibrated strain 108)
• Burkholderia cepacia ATCC 25416 (calibrated strain 108)
• Aspergillus niger ATCC 16404 (calibrated strain 108)

1. Procedure

1. Microbiological examination of the product before artificial contamination

Sample aseptically a wipe and introduce it into a sterile stomacher bag. Add 90ml of neutralizing media and shake at least 1 min. Take 1 ml and introduce it into a Petri dish. Add 15 - 20 ml of nutrient media and incubate for 5 days at 30°C.

1. Preparation of microbial suspension

Sample aseptically a strain ball and re-hydrate it into 1.1 ml of re-hydratation fluid. This mean of between 0.7 and 1.5 x 108 cfu of the strain.

Dilute each bacterial strain in order to have a test suspension with 107 ufc /ml.

Dilute Yeast strain in order to have a test suspension with 107 ufc /ml.

Dilute Mold strain in order to have a test suspension with 106 ufc /ml.

From this suspension, make successive dilutions to obtain between 100 and 300 cfu per ml and transfer 1 ml of this dilution into a Petri dish. Add 15 to 20 ml of agar and incubate for 5 days at 30°C (control strain and enumeration N)

1. Preparation of the test sample

Clean the packaging (1 per test strain + 1) using a single-use paper soaked with alcohol and place it immediately under a laminar flow hood.

1. Determination of the average weight of a wipe

Withdraw from the same package, at least 2 wipes. Weighed separately and calculate the average weight.

1. Artificial contamination of wipes

Use 1 pack of wipes for each test strain. Take off 10 wipes aseptically using sterile gloves and tongs and place them in a 140 mm diameter Petri dish.

Wipes in the bottom of the 140 mm Dish are aseptically inoculated successively with 80μl of calibrated inoculum (6.2) by 8 spots of 10 µl and placed in the lid of the box by stacking them on top of each other.
Then reposition the wipes in their original packaging and locate the side of the package inoculated (first wipe) by making a mark on the packaging.

Nota : Use different sterile gloves for each strain. Ensure sealing of the package before storing step.

1. Validation of neutralization

Use 1 wipe for each test strain.

Take a wipe and place it in a sterile stomacher bag. Add 90ml +/ - 2 ml of neutralizing diluent and mix it in the stomacher for 1 mn.

Wait for an appropriate time to let the neutralizing agent work.

Inoculate the suspension with 80µl of the strain. Then make serial dilution and count the numbers of surviving bacteria.

To validate the neutralization, the decimal logarithm reduction obtained must be less than 1.

Log (number of theoretical initial cfu/wipe) - log (number of cfu enumerated on validation point/ wipe) < 1

1. Storage of wipes

The samples thus prepared are stored at 24°C +/- 1°C.
In the absence of additional information, they will be positioned horizontally.

1. Control points

In the general case, the controls will be carried out according to the instructions of the European Pharmacopoeia for the products of local application either T0, 48h, 7 days, 14 days, 21 days and 28 days.

Nota : On request, the control points may be done with different times.