Technical Product Information

Validation Report for AccuClean Advanced

(Neogen item 9960)
Revision November, 2017

TECHNICAL PRODUCT INFORMATION

Table of Contents

Introduction............................................................................................................................2
Robustness Testing.................................................................................................................2
Beta Site Testing..........................................................................................................................5
Commodity Testing......................................................................................................................6
Conclusion..................................................................................................................................7

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AccuClean Advanced Validation Report

INTRODUCTION
AccuClean Advanced has been designed as a simple, 10-second visual test to assist in determining the cleanliness of food contact surfaces
and equipment by detecting protein residues left on surfaces after cleaning. This validation report details the findings of the experimental
evaluation designed to validate the test parameters and establish the performance characteristics for the suitability of testing surfaces
using AccuClean Advanced.
Operator 1
Lot 1 Lot 2
ROBUSTNESS TESTING 1 2 3 4 5 1 2 3 4 5
Purpose: To validate lot-to-lot and operator-to-operator ruggedness
of the AccuClean Advanced Rapid Protein Residue Test. 0 μg - - - - - - - - - -
5 μg + + + + + + + + + +

DESIGN SUMMARY 10 μg + + + + + + + + + +
Two lots of AccuClean Advanced were evaluated for ruggedness 20 μg + + + + + + + + + +
by three operators. Three sampling methods were used to test 50 μg + + + + + + + + + +
the performance of the test kits: Bovine Serum Albumin (BSA)
protein solution applied directly to sampling sponges, BSA solution 100 μg + + + + + + + + + +
swabbed from polyethylene surfaces, and food matrix slurry 200 μg + + + + + + + + + +
swabbed from stainless steel and polyethylene surfaces. A brief
summary of each portion follows:
Operator 2
Lot 1 Lot 2
Part I: LOD by Direct Titration – Design Summary
1 2 3 4 5 1 2 3 4 5
Solutions of BSA in filter-sterilized Type 1 water were produced so
that a 20 μL aliquot of each solution contained a target quantity of 0 μg - - - - - - - - - -
BSA (0 μg, 5 μg, 10 μg, 20 μg, 50 μg, 100 μg, and 200 μg) to be 5 μg + + + + + + + + + +
delivered directly to the sampler sponge via pipette. Each operator
tested five replicates of each level with each kit lot and recorded 10 μg + + + + + + + + + +
the qualitative Clean/Dirty result in Table 1. 20 μg + + + + + + + + + +
50 μg + + + + + + + + + +
Part I: LOD by Direct Titration – Conclusion 100 μg + + + + + + + + + +
Based on the results shown in Table 1, the level of protein detection 200 μg + + + + + + + + + +
was 5 µg for direct addition of BSA to the sampler sponge. There
were no false positive results for samplers where sterile water was
added to the sampler sponge and all the samplers tested with 5 µg Operator 3
to 200 µg of protein deposited on the sampler sponge were positive Lot 1 Lot 2
(dirty) for all 3 operators with both lots of samplers.
1 2 3 4 5 1 2 3 4 5
0 μg - - - - - - - - - -
5 μg + + + + + + + + + +
10 μg + + + + + + + + + +
20 μg + + + + + + + + + +
50 μg + + + + + + + + + +
100 μg + + + + + + + + + +
200 μg + + + + + + + + + +

Table 1: Direct Titration Results. “Result” is either - (Clean) or + (Dirty) as

specified in the kit insert.

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 AccuClean Advanced Validation Report

Part II: BSA Recovered from Surfaces – Design Summary

Polyethylene surfaces (approximately 4" square) were inoculated with BSA solution to reach a target quantity (0 μg, 100 μg, 200 μg, 400
μg and 1,000 μg) of delivered BSA per surface. Each inoculation was spread evenly across the surface and allowed to dry for one hour.
Operators then sampled each surface by swabbing according to the kit insert. Each operator tested five replicate surfaces of each inoculation
level with each kit lot on two separate days and recorded the qualitative Clean/Dirty results in Table 2.

Operator 1 Operator 2 Operator 3

Lot 1 Lot 2 Lot 1 Lot 2 Lot 1 Lot 2
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
Day 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
0 μg
Day 2 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Day 1 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
100 μg
Day 2 + + + + + + + + + + + + + + + + + + + + + - + + - + + + + +
Day 1 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
200 μg
Day 2 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
Day 1 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
400 μg
Day 2 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
Day 1 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
1000 μg
Day 2 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Table 2: BSA Surface Sampling Results. Result is either - (Clean) or + (Dirty) as specified in the kit insert.
Indicates the result was different than expected

Part II: BSA Recovered from Surfaces – Conclusion

Table 2 shows there were no false positive results where sterile water was added to the surface and sampled. All of the surfaces prepared
with 100 µg to 1000 µg of protein deposited on the surface were positive (dirty) for all 3 operators and both lots of samplers on both days,
with the exception of two of the 60 surfaces prepared with 100 µg of protein. The effect was intermittent and was only observed by a
single operator on a single day.

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AccuClean Advanced Validation Report

Part III: Detection of Food from Surfaces – Design Summary

Greek yogurt was prepared as a 1:10 slurry by stomaching 5g of the food sample with 45 mL sterile Type 1 water until thoroughly
blended. Polyethylene and Stainless Steel surfaces (approximately 4” square) were inoculated with 50 μL of food slurry (50 μL sterile
water aliquots were also tested as a negative control). Each inoculation was spread evenly across the surface and allowed to dry for one
hour. Operators sampled each surface by swabbing according to the kit insert. Each operator tested five replicates of the food matrix/
negative control on each surface type with each kit lot on two separate days and recorded the qualitative Clean/Dirty results in Table 3.

Day One
Operator 1 Operator 2 Operator 3
Plastic Stainless Steel Plastic Stainless Steel Plastic Stainless Steel
(-) Yogurt (-) Yogurt (-) Yogurt (-) Yogurt (-) Yogurt (-) Yogurt
1 - + - + - + - + - + - +
2 - + - + - + - + - + - +
Lot 1 3 - + - + - + - + - + - +
4 - + - + - + - + - + - +
5 - + - + - + - + - + - -
1 - + - + - + - + - + - +
2 - + - + - + - + - + - +
Lot 2 3 - + - + - + - + - + - +
4 - + - + - + - + - + - +
5 - + - + - + - + - + - +

Day Two
Operator 1 Operator 2 Operator 3
Plastic Stainless Steel Plastic Stainless Steel Plastic Stainless Steel
(-) Yogurt (-) Yogurt (-) Yogurt (-) Yogurt (-) Yogurt (-) Yogurt
1 - + - + - + - + - + - +
2 - + - + - + - + - + - +
Lot 1 3 - + - + - + - + - + - +
4 - + - + - + - + - + - +
5 - + - + - + - + - + - +
1 - + - + - + - + - + - +
2 - + - + - + - + - + - +
Lot 2 3 - + - + - + - + - + - +
4 - + - + - + - + - + - +
5 - + - + - + - + - + - +

Table 3: Food Surface Sampling Results. Result is either - (Clean) or + (Dirty) as specified in the kit insert.
Indicates the result was different than expected

Part III: Detection of Food from Surfaces – Conclusion

Data in Table 3 shows there were no false positive results where sterile water was added to the surface and sampled. Of the 120
surfaces prepared with 50 µL of 1:10 diluted yogurt dried on the surface, 119 of the surfaces were positive (dirty) for all three operators
and both lots of samplers on both days. Overall accuracy achieved for this portion of the study was >99%.

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 AccuClean Advanced Validation Report

BETA SITE TESTING

Purpose: To validate operator-to-operator ruggedness, ease of use and clarity of kit instructions of the AccuClean Advanced Rapid Protein
Residue Test.

DESIGN SUMMARY
A single lot of AccuClean Advanced was evaluated by 10 different locations (Beta Sites) with 2 operators performing the testing
at each location on two separate days. 5 vials labeled A, B, C, D and E were provided as well as a set of fixed volume, single use
50 µL pipettes. The vial contents were unknown to the operators. Operators were instructed to dispense 50 µL of each solution evenly
across a clean surface, allow the surface to dry for one hour and sample each surface according to kit instructions. The same procedure
was performed by two operators at each location on two separate days. Results are recorded in Table 4.

Operator One Operator Two

A B C D E A B C D E
0 μg 500 μg 100 μg 250 μg 50 μg 0 μg 500 μg 100 μg 250 μg 50 μg
Day 1 - + + + + - + + + +
Site A
Day 2 - + + + + - + + + +
Day 1 - + + + + + + + + +
Site B
Day 2 - + + + + - + + + +
Day 1 - + + - - - + + + +
Site C
Day 2 - + + + + - + + + +
Day 1 - + + + + - + + + +
Site D
Day 2 - + + + + - + + + +
Day 1 - + + + + - + + + +
Site E
Day 2 - + + + + - + + + +
Day 1 - + + + + - + + + +
Site F
Day 2 - + + + + - + + + +
Day 1 - + + + + - + + + +
Site G
Day 2 - + + + + - + + + +
Day 1 - + + + + - + + + +
Site H
Day 2 - + + + + - + + + +
Day 1 - + + + + - * + + +
Site I
Day 2 - + + + + - + + + +
Day 1 - + + + + - + + + +
Site J
Day 2 - + - + + - + + + +

Results Key
Table 4: Beta Site testing results. Result is either - (Clean) or + (Dirty) as specified in the kit insert.
“-” Clean No color change
*Test was reported by the operator as invalid and was not included in the results
“+” Dirty Any color change
Indicates the result was different than expected
Beta Site Testing – Conclusion
Results in Table 5 show one false positive was obtained by Operator Two from Site B on day 1, but not day 2 of testing. A false positive is
a surface indicated as dirty when the expected result was clean. There were no false positive results for any other operator at the other
beta-site locations. Of 200 data points collected by 10 sites with two operators over two days, 98% accuracy was achieved. Anomalies
can be attributed to consistency with which samples were applied to the surface.

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AccuClean Advanced Validation Report

COMMODITY TESTING
Purpose: To determine the ability of the AccuClean Advanced Rapid Protein Residue Test to detect protein residues from various commodities
compared to competitor assays.

DESIGN SUMMARY
Various food commodities were tested to determine device response with the AccuClean Advanced device alongside two competitive
devices. Each commodity was prepared as a 1:5 slurry by adding 1.0g of the food sample to 4.0 mL sterile Type 1 water and vortexing
until thoroughly blended. For each commodity, 50 µL of the slurry was dispensed onto a clean, stainless steel surface and allowed to dry
for one hour. Operators sampled each surface by swabbing according to the kit insert for each kit. Each operator tested five replicates of
the food matrix with each kit. Results were recorded in Table 4 as the percentage of tests determined to be positive for protein detection
according to the kit inserts.

Commodity AccuClean Advanced Competitor A Competitor B

Ham 100% Detection 100% Detection 80% Detection
Tuna 100% Detection 100% Detection 80% Detection
Pasteurized Milk 100% Detection 100% Detection 100% Detection
UHT Milk 100% Detection 100% Detection 80% Detection
Asparagus (frozen) 100% Detection 100% Detection 100% Detection
Broccoli (frozen) 100% Detection 100% Detection 100% Detection
Peanuts (shelled) 100% Detection 100% Detection 100% Detection
Peanuts (whole) 100% Detection 100% Detection 100% Detection
Peas, black eyed (frozen) 100% Detection 100% Detection 100% Detection
Noodles, egg (cooked) 100% Detection 100% Detection No Detection
Green Beans (frozen) 100% Detection 100% Detection 20% Detection
Macaroni 100% Detection 100% Detection 20% Detection
Bread, white 100% Detection 100% Detection 20% Detection
Potato, with skin (baked) 100% Detection 80% Detection 100% Detection
Cantaloupe 100% Detection 100% Detection 80% Detection
Celery 100% Detection No Detection No Detection
Strawberry (fresh) 100% Detection 100% Detection 80% Detection
Rice, boiled 100% Detection 100% Detection 40% Detection
Carrots, cooked 100% Detection 100% Detection 20% Detection
Banana 100% Detection 100% Detection 100% Detection
Raisin, seedless 60% Detection 100% Detection 60% Detection
Blueberry 100% Detection 100% Detection 100% Detection
Apple Juice No Detection 100% Detection 100% Detection
Apple, Red
100% Detection 100% Detection 80% Detection
Delicious, peeled

Table 5: AccuClean Advanced and competitor results for detection of various commodities diluted 1:5 with sterile water, deposited and dried on stainless
steel surfaces.

Commodity Testing – Conclusion

Data in Table 5 showed AccuClean Advanced Rapid Protein Residue test detected all the commodities listed, with the exception of seedless
raisin and apple juice dried from a 1:5 dilution of slurried commodity. This is due to lack of protein content. Competitive chemistries are known
to cross-react with sugars and other reducing substances, causing false positive results. Significantly more commodities were detected
using AccuClean Advanced than Competitor B and the same number of commodities were detected as Competitor A. Both competitor assays
required 10 min. or longer for detection as indicated in the use instructions. This is compared to 10 seconds with AccuClean Advanced.

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 AccuClean Advanced Validation Report

ACCUCLEAN ADVANCED VALIDATION STUDY CONCLUSION

AccuClean Advanced delivers industry – leading speed and sensitivity for determining the cleanliness of food contact surfaces and equipment.
AccuClean Advanced is a simple, 10-second visual test that reveals detected protein residue through an easy-to-interpret color change.
Overall results from this validation report indicate AccuClean Advanced provides sensitive, reliable and fast detection of protein residues
on surfaces from a variety of food commodities.

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© Neogen Corporation, 2017. Neogen and AccuClean are registered trademarks of Neogen Corporation, Lansing, Michigan. FD1106-1117
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