Products fo hygienic and surgical handrub and handwash - Bactericidal activity (phase 2/step 1) (EN 12054)

Scope

This European Standard specifies a test method and requirements for the minimum of handwash and handrub products for postcontamination treatment of hands or for surgical hand disinfection that are intended to be used with water (handwash) or without water (handrub).

Requirements

The product, when tested in accordance with the test protocol described above, shall demonstrate a certain reduction in viable counts for each of the following test organism: Pseudomonas aeruginosa, Escherichia coli, Enterococcus hirae and Staphylococcus aureus.

The following specific requirements shall be met by the product according to its application.

Hygienic handrub: The product shall demonstrate a reduction in viable count from between 1 x 10⁷ cfu/ml to 3 x 10⁷ cfu/ml to no more than 3 x 10² cfu/ml at 1 min. In addition if the manufacturer recommends a shorter exposure time than 1 min, the product shall demonstrate a similar reduction in viable count at 30 s.

Hygienic handwash: The product shall demonstrate a reduction in viable count from between 1 x 10⁷ cfu/ml to 3 x 10⁷ cfu/ml to no more than 3 x 10⁴ cfu/ml at 1 min. In addition if the manufacturer recommends a shorter exposure time than 1 min, the product shall demonstrate a similar reduction in viable count at 30 s.

Surgical handrub: The product shall demonstrate a reduction in viable count from between 1 x 10⁷ cfu/ml to 3 x 10⁷ cfu/ml to no more than 3 x 10² cfu/ml at 5 min. In addition if the manufacturer recommends a shorter exposure time than 5 min, the product shall demonstrate a similar reduction in viable count at 3 min.

Surgical handwash: The product shall demonstrate a reduction in viable count from between 1 x 10⁷ cfu/ml to 3 x 10⁷ cfu/ml to no more than 3 x 10⁴ cfu/ml at 5 min. In addition if the manufacturer recommends a shorter exposure time than 5 min, the product shall demonstrate a similar reduction in viable count at 3 min.

Test Method

The bactericidal activity is evaluated using the following six strains:

Pseudomonas aeruginosa ATCC 15442

Staphylococcus aureus ATCC 6538

Escherichia coli ATCC 10538

Enterococcus hirae ATCC 8043

Candida albicans ATCC 10231

Proteus mirabilis ATCC 12453

In order to prepare the working culture of bacterial strains, subculture from the stock culture by streaking onto TSA (bacteria) or MEA (Candida) agar and incubate overnight. Make a second subculture and incubate as earlier. The second subculture is the working culture. Bacterial test suspensions are prepared by taking 10 ml of diluent (Tryptone Sodium Chloride solution or MEA culture media) in a 100 ml flask with 5g of glass beads. From the working culture a loopfull of microbial cells is transferred into the diluent and suspended. Flask is shaken 3 minutes using a mechanical shaker. The number of cells in the suspension is adjusted to 1x10⁸ cfu/ml to 3x10⁸ cfu/ml using diluent by turbidimetry. Always check the cell count by dilution method.

The handrub products are used undiluted and handwash products as 55% (V/V) dilutions in standard hard water.

Dilution-neutralization method is the method of choice. Prior to testing all reagents are equilibrated to the test temperature of 20^oC using the water bath.

Hygienic or surgical handrub:

Pipette 9 ml of the test products in to container of suitable capacity . Add 1ml bacterial suspension containing 1x10⁸ cfu/ml to 3x10⁸ cfu/ml. Immediately start the stopwatch, mix and place the container in the water bath at 20^oC. The activity of the product shall be determined for a contact time 30 sec + 5s for hygienic and 3min+5s surgical handrub products. At the end of the contact time, pipette 1ml of the test mixture into a tube containing 8ml neutralizer (30g/l polysorbate 80 + 3g/l lecithine)

and 1ml of water. Mix and incubate in the water bath for minute. After neutralization take a 1ml sample and its 10-¹ dilution in duplicate and transfer on TSA plates. Incubate the plates in 36 ^oC for 24 hours. Count the plates and determine the number of colony forming units for each plate.

In this test we also used inhibitory organic material by request of the manufacturer. For hygienic handrub the inhibitory material was 0.3% BSA+ 0.3% sheep red blood cells and for surgical handrub 0.03% BSA.

Results

For each test organism record the number of cfu/ml in the bactericidal test suspension (N) and after the test procedure for bactericidal activity of the product (N_a).

N x 10^-1

Reduction in viability = N_a

Conclusion

The following specific requirements shall be met by the product according to its application.

Hygienic handrub: The product shall demonstrate a reduction in viable count from between 1 x 10⁷ cfu/ml to 3 x 10⁷ cfu/ml to no more than 3 x 10² cfu/ml at 1 min. In addition if the manufacturer recommends a shorter exposure time than 1 min, the product shall demonstrate a similar reduction in viable count at 30 s.

Surgical handrub: The product shall demonstrate a reduction in viable count from between 1 x 10⁷ cfu/ml to 3 x 10⁷ cfu/ml to no more than 3 x 10² cfu/ml at 5 min. In addition if the manufacturer recommends a shorter exposure time than 5 min, the product shall demonstrate a similar reduction in viable count at 3 min.

Test report

European Standard EN 12054 (modified by request of manufacturer)

Test laboratory: Laboratory of Hygiene and Microbiology, Dept. of Public Health, University of Helsinki

Name of the product: Hygisoft

Batch number:

Manufacturer: SoftProtector Oy

Date of delivery: January 2005

Storage conditions: room temperature

Test method and validation: Dilution-neutralization method

Polysorbate 80+lecithin neutralizer

Period of analysis: week 3 2005

Product test concentrations: 0.2 and 0.5% in standard hard water

Test temperature: 20^{o +} 1 C

Contact times: 1 min

Temperature of incubation: 37^{o +} 1 C

Test organisms	viable	cfu/ml	counts
	Bacterial test suspension	Hygisoft 0.2%	Hygisoft 0.5%	microbial reduction log
Pseudomonas aeruginosa	8.6 x 10^7	<1.5 x 10^2	<1.5 x 10^2	>1.5 x 10^5
Staphylococcus aureus	1.32 x 10^8	<1.5 x 10^2	<1.5 x 10^2	>1.5 x 10^5
Enterococcus hirae	1.9 x 10^8	<1.5 x 10^2	<1.5 x 10^2	>1.5 x 10^5
Escherichia coli	1.24 x 10^8	<1.5 x 10^2	<1.5 x 10^2	>1.5 x 10^5

Conclusion:

According to EN 12054 the product tested possess bacterical activity for the referenced microbial strains at 1 min contact time required for hygienic hand disinfection products according to the requirements.

Helsinki 24.1.2005

Kirsi Laitinen, PhD clinical microbiologist

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