CERTIFICATION. Certificate No. The AOAC Research Institute hereby certifies that the performance of the test kit known as: Hygiena UltraSnap ATP Kit

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1 CERTIFICATION AOAC Performne Teste SM Certifite No The AOAC Reserh Institute herey ertifies tht the performne of the test kit known s: Hygien UltrSnp ATP Kit mnufture y Hygien LLC 941 Aveni Aso Cmrillo, Cliforni USA This metho hs een evlute in the AOAC Performne Teste Methos SM Progrm, n foun to perform s stte y the mnufturer ontingent to the omments ontine in the mnusript. This ertifite mens tht n AOAC Certifition Mrk Liense Agreement hs een exeute whih uthorizes the mnufturer to isply the AOAC Performne Teste SM ertifition mrk long with the sttement "THIS METHOD'S PERFORMANCE WAS REVIEWED BY AOAC RESEARCH INSTITUTE AND WAS FOUND TO PERFORM TO THE MANUFACTURER'S SPECIFICATIONS" on the ove mentione metho for perio of one lenr yer from the te of this ertifite (Otoer 18, 2018 Deemer 31, 2019). Renewl my e grnte t the en of one yer uner the rules stte in the liensing greement. Sott Cotes, Senior Diretor Signture for AOAC Reserh Institute Otoer 18, 2018 Dte 2275 Reserh Blv., Ste. 300, Rokville, Mryln, USA Telephone: Fx: Internet e mil: ori@o.org * Worl Wie We Site:

2 METHOD AUTHORS Rihr To, Mthew Smith, Pul Meighn, Mrtin Ester SUBMITTING COMPANY Hygien Interntionl 40 Om Ro Guilfor, Surrey, GU2 7YG KIT NAME(S) UltrSnp Surfe ATP CATALOG NUMBERS INDEPENDENT LABORATORY Cmpen BRI Centenry Hll Cooper s Hill R Rehill, RH1 4HY AOAC EXPERTS AND PEER REVIEWERS Mrk Crter 1, Mihel Brosky 2, Jmes Agin 3 1 MC2E, INC, New Hope, PA, USA 2 Brosky Consultnts, Thornhill, Ontrio, Cn 3 Q Lortories, In. Cininnti, OH, USA APPLICABILITY OF METHOD Aenosine triphosphte (ATP) Mtries (10 x 10 m) stinless steel surfes ontminte y rw lm, rey to et uk wrp, ornge, juie, yogurt, n oughnut resiues Performne lims The UltrSnp Surfe ATP test ws proven to e effetive t eteting the presene of ATP on stinless steel surfes eing representtive of foo proessing n mnufturing filities. Liner regression etermine tht the limit of etetion (LOD) of UltrSnp sws in the Ensure luminometer ws 0.82 femtomoles of ATP. ORIGINAL CERTIFICATION DATE Otoer 18, 2018 METHOD MODIFICATION RECORD NONE Uner this AOAC Performne Teste SM Liense Numer, this metho is istriute y: NONE CERTIFICATION RENEWAL RECORD New Renewl 2018 SUMMARY OF MODIFICATION NONE Uner this AOAC Performne Teste SM Liense Numer, this metho is istriute s: NONE PRINCIPLE OF THE METHOD (1) UltrSnp Surfe ATP Test is self ontine evie use with Hygien luminometers. The test evie n luminometer is system use for monitoring the hygieni sttus of surfes on proessing equipment n other environments in wie rnge of inustries. The system mesures enosine triphosphte (ATP), the universl energy moleule foun in ll niml, plnt, teril, yest, n fungl ells. Prout resiues from orgni mtter left on surfes ontin ATP. After proper lening, ll soures of ATP shoul e signifintly reue. When smple is ollete, ny ATP is mixe with the unique liqui stle Luiferse/Luiferin regent in the UltrSnp test evie, light is emitte in iret proportion to the onentrtion of ATP present in the smple. The EnSURE luminometer mesures generte light n reports results in Reltive Light Units (RLU). RLU results provie informtion on the level of ontmintion within seons. The higher the RLU numer, the more ATP present, n the irtier the surfe. It is importnt to note tht UltrSnp is esigne to etet invisile/tre mounts of resiue. Overloing the sw with physil mtter y swing visily irty surfe will inhiit the ioluminesent retion n proue non proportionl results. To use the test, remove the UltrSnp sw evie from the tue n sw 10x10 m surfe. For optiml swing: 1. Do not touh sw or insie of smple evie with fingers 2. Rotte sw while olleting smple to mximize smple olletion on sw tip 3. Apply suffiient pressure to rete flex in sw shft 4. Sw in rissross pttern vertilly, horizontlly, n in oth igonl iretions. Reple the sw evie into the tue, snp the ul k n forth, then squeeze the ul to expel the liqui regents to the ottom of the tue. The tivte evie is then inserte into n EnSURE luminometer to mesure the RLU output. Any ATP present on the swe surfe will ontriute to the RLU result.

3 DISCUSSION OF THE VALIDATION STUDY (1) For n ATP test to funtion well s rpi hygiene monitoring tool it must e sensitive to pure nlyte, e ple of eteting ATP from n in the presene of foo n miroil spoilge n must give proportionl reprouile results. The pure nlyte testing of UltrSnp emonstrte exellent sensitivity with LODs of 2.27RLU (Tle 1) n 0.77RLU (Tle 13), from the metho eveloper n inepenent l stuies respetively. These RLU LODs orrespon to 0.82 n 0.23 femtomoles of ATP onverte using liner regression nlysis. The RLU signl proue n urte ose response to the ATP onentrtion (R 2 vlue = ), t levels tht re key to tionle pss/ution/fil results in hygiene monitoring pplitions. The results from foo mtrix testing revel tht UltrSnp is ple of eteting ATP from, n in the presene of, surfe soil y foo n everges. Detetion of ATP ws possile t ilutions of 1:1000 or lower for ll mtrixes teste (Tles 3 & 4). This sensitivity to low levels of foo/everge resiue shows tht ATP hygiene monitoring is fr more urte hek of lening performne thn simply looking for irty surfe. The five mtrixes teste emonstrte the ility of UltrSnp to perform well ross ro rnge of inustril environments. The results from miroil mtrix testing vlite the foo testing t y showing tht UltrSnp n suessfully reover n mesure miroil ATP from grm positive teri (B. sutilis ry LOD = 30,694 CFU), grm negtive teri (P. eruginos ry LOD = 74,615 CFU), n yest (S. erevisie ry LOD = 1012 CFU). In eh se there is liner ose response with orreltions of >85% for ll orgnisms teste in oth wet n ry onitions. An importnt point to rw from oth the foo n miroil mtrix t is the suessful etetion of ll mtrixes fter rying. Although the signl reovery is mrginlly lower from rie surfes ompre to wet surfes, there is still lrge mount of ATP signl from foo n miroil ATP reovere in ll ses. This emonstrtes tht there is not rmti erese in ATP vilility ue to ATP instility on surfes or when expose to rying. ATP remins stle when rie on surfes (3) n will not simply eome negtive over time when left without lening. To get negtive result y surfe ATP hygiene monitoring the ATP must e physilly remove y iligent lening, sene of regulr well performe lening will le to positive RLU results from surfes monitore y ATP testing regrless of the inustril lotion. In oth the foo n miroil mtrix t sets from surfe testing, some results were ientifie s outliers y the Grus test n remove from the nlysis. The experimentl setup introues vrious soures of vrition tht re likely to use the oserve outliers. There n e vrition from smple homogeniztion, smple spreing, smple rying, smple swing, non smple ATP from the environment, n iffering environmentl onitions uring rying tht ll ftor into the RLU results. Three ifferent lsses of ommonly use inustril snitizers were teste for inhiition of the ATP signl from ULS evies. The quternry mmonium snitizer inrese the RLU vlue y %. The other two snitizers oth use reution in RLU vlue; i nioni surftnt y 7 48% n pereti i y 15 48%. In the experimentl esign followe for this testing the snitizers were not rinse from the surfes efore smpling. In rel worl situtions snitizers woul e rinse from surfes efore testing, s result we woul expet to see reue effet from ll snitizers teste here in rel worl situtions. Seletivity t shows tht ULS is highly seletive for ATP over other similr nuleoties n not suseptile to ompetitive inhiition. Out of 12 ifferent non ATP nlogues, only ATP n ADP give ny signl t ll t 2500fmoles (44 & 42 RLUs respetively <2% of the ATP signl). In the presene of 100x onentrtion of ll 12 non ATP nlytes ULS urtely mesures 25 fmoles of ATP (Tle 9). Prout onsisteny n stility ws emonstrte through testing three thes of ULS sws mnufture over perio of 6 months. There ws no sttistil ifferene in the reore RLU from 0, 10, n 100 fmoles of ATP ross the three ifferent ULS thes. Instrument Vrition ws teste y using three ifferent EnSURE luminometers to mesure ULS RLU with three ifferent levels of ATP. There ws sttistilly signifint vrition in the kgroun reings from the three instruments, lthough this is lrgely ue to the numer of zero vlues otine. This uses the vrition (rnging from 0 3 RLU) to pper very lrge in omprison to the very low verges tht re lose to 0 (0.6, 1, n 2.8 RLU respetively). There ws no sttistil ifferene oserve etween the three instruments when reing UltrSnp evies ontining 10 n 100 fmoles of ATP. Roustness testing ws rrie out y ltering the temperture of ULS evies n the time etween tivting the ULS evie n reing it in the EnSure luminometer. Ftoril ANOVA ws performe on the t from the three ATP onentrtion levels teste (0, 10, 100 fmoles ATP). There ws no sttistilly signifint ifferene in the kgroun reings when ULS evies were time n temperture use. However, there ws sttistilly signifint ifferene etween RLU reings in oth the 10 fmole n 100 fmole ATP t sets. In oth ses it ws seen tht RLU signl inreses with time n temperture, with the lrgest effet oming from the temperture use of ULS evies. These results show the importne of using the ULS test oring to the kit insert proeure.

4 Tle 1: RLU mesurement of pure nlyte ATP e to UltrSnp evies n re in n Ensure luminometer. Results re given s rw, men, s r, RSDr, n LOD vlues. (1) Replites RLU, t Applie ATP (femtomoles) Men RLU s r RSDr 86.1% 39.7% 6.73% 23.6% 19.9% 17.1% 17.8% LOD e 2.27RLU ATP quntity e to sw tip. Averge RLU from 10 replites per ATP level. s r lulte from 10 replites per ATP level. RSDr lulte from 10 replites per ATP level. e LOD. lulte using regression nlysis of RLU ginst ATP.

5 Tle 2: Interpolte pure nlyte ATP onentrtions mesure using UltrSnp in n EnSure luminometer. Corresponing RLU vlues tken from Tle 1, were onverte into femtomoles of ATP using the line eqution y = x , generte from Figure 2 ove. (1) ATP (femtomoles) Replites Men RLU s r RSDr 9.94% 10.7% 4.07% 19.3% 18.1% 16.8% 17.6% Interpolte ATP onentrtion. Averge ATP (femtomoles) from 10 theoretil replites per ATP level. s r lulte from 10 preite replites per RLU level. RSDr lulte from 10 preite replites per RLU level.

6 Tle 3: Replite RLU, men RLU, Sr, n RSDr vlues of UltrSnp Surfe ATP metho use on wet foo mtrixes. All RLU vlues mesure in n EnSURE luminometer. A negtive ontrol onsisting of sterile nlyte free wter ws use. (1) Dilution Replite Men Stn. Dev. RSDr wter NA e Lm Leg Stek 5 2 e e NA f wter Pink Iing Doughnut wter e Strwerry Yogurt e e wter e Ornge Juie (smooth) e wter e Duk Wrp e Dilution of the foo mtrix teste. The men result of 10 wet replite oupons per ilution. s r lulte from 10 wet replite oupons per ilution. f Co effiient of vrine perentge lulte from 10 wet replite oupons per ilution. e Exlue ue to Grus test. f NA = Not pplile.

7 Tle 4: Replite RLU, men RLU, s r, n RSDr vlues of UltrSnp Surfe ATP metho use on ry foo mtrixes. All RLU vlues mesure in n EnSure luminometer. A negtive ontrol onsisting of sterile nlyte free wter ws use. (1) Dilution Replite Men Stn. Dev. RSDr wter Lm Leg Stek e e wter e Pink Iing Doughnut wter e e e Strwerry Yogurt e wter 1 9 e Ornge Juie (smooth) e e e e wter e Duk Wrp e e The ilution of the foo mtrix teste. The men result of 10 ry replite oupons per ilution. Sr lulte from 10 ry replite oupons per ilution. Coeffiient of vrine perentge lulte from 10 ry replite oupons per ilution. e Exlue ue to Grus test. f NA = Not pplile (the reing ws inorretly tken).

8 Men RLU Tle 9: A tle showing the verge RLU of thirteen pure nlytes, plus negtive ontrol, nlyte free wter t high onentrtions (2500 fmol) with n without ATP present (25 fmol). Five replites were teste for eh onition, for eh nlyte. (1) Arevition Nme RLU t 2500 fmol ompoun, 0 fmol ATP RLU t 2500 fmol ompoun, 25 fmol ATP NA nlyte free wter 2 30 ATP Aenosine 5' triphosphte soium slt hyrte ATP 2' eoxyenosine 5' triphosphte soium slt UTP Uriine 5' triphosphte trisoium slt 1 37 GTP Gunosine 5' triphosphte soium slt 3 39 TTP Thymiine 5' triphosphte soium slt 2 36 UTP 2' Deoxyuriine 5' triphosphte soium slt 1 33 CTP Cytiine 5' triphosphte 1 35 GTP 2' eoxygunosine 5' triphosphte trisoium slt 1 31 ITP Inosine 5' triphosphte trisoium slt 1 34 IMP 2' eoxyinosine 5' monophosphte soium slt 1 34 CTP 2' eoxyytiine 5' triphosphte isoium slt 1 35 ADP enosine iphosphte (teril origin) AMP enosine monophosphte 1 31 Not pplile. Tle 13: RLU mesurement of pure nlyte ATP e to UltrSnp evies n re in n Ensure luminometer. Results re given s rw, men, s r, RSDr, n LOD vlues. (1) Replites RLU, t Applie ATP (femtomoles) s r RSDr LOD e 0.77RLU ATP quntity e to sw tip. Averge RLU from 10 replites per ATP level. Sr lulte from 10 replites per ATP level. RSDr lulte from 10 replites per ATP level. e LOD. lulte using regression nlysis of RLU ginst ATP.

9 Tle 14: Interpolte pure nlyte ATP onentrtions mesure using UltrSnp in n EnSure luminometer. Corresponing RLU vlues tken from Tle 13, were onverte into femtomoles of ATP using the line eqution y = 0.88x , generte from Figure 7 ove. (1) ATP (femtomoles) Replites Men RLU s r RSDr ATP quntity e to sw tip. Averge RLU from 10 replites per ATP level. Sr lulte from 10 replites per ATP level. RSDr lulte from 10 replites per ATP level. Tle 15: Replite RLU, men RLU, Sr, n RSDr vlues of UltrSnp Surfe ATP metho use on wet foo mtrixes. All RLU vlues mesure in n EnSure luminometer. A negtive ontrol onsisting of sterile nlyte free wter ws use. (1) Wet Foo Mtrix RLU Trget Rnge Replite Men Stn. Dev RSDr Ornge juie (smooth) Duk wrp e e The men result of 10 wet replite oupons per ilution. Sr lulte from 10 wet replite oupons per ilution Co effiient of vrine perentge lulte from 10 wet replite oupons per ilution. Exlue ue to Grus test.

10 Tle 16: Replite RLU, men RLU, s r, n RSDr vlues of UltrSnp Surfe ATP metho use on ry foo mtrixes. All RLU vlues mesure in n EnSure luminometer. A negtive ontrol onsisting of sterile nlyte free wter ws use. (1) Dry Foo Mtrix RLU Trget Rnge Replite Men Stn. Dev. RSDr Ornge juie (smooth) Duk wrp The men result of 10 wet replite oupons per ilution. Sr lulte from 10 wet replite oupons per ilution. Co effiient of vrine perentge lulte from 10 wet replite oupons per ilution. Exlue ue to Grus test. The men result of 10 wet replite oupons per ilution. REFERENCES CITED 1. To, R., Smith, M., Meighn, P., n Ester, M., Vlition of the UltrSnp Surfe ATP Test n EnSURE Luminometer for ATP Hygiene Monitoring on Stinless Steel Surfes, AOAC Performne Teste SM ertifition numer AOAC Reserh Institute Performne Teste Metho SM Poliies n Proeures, (esse My, 2017) 3. Alf, M.J. et l. (2015) Aenosine tri phosphte (ATP) se lening monitoring in helth re: how rpily oes environmentl ATP eteriorte? Journl of Hospitl Infetion, Volume 90, Issue 1, 59 65