Use of reliable bioassays to detect potential hazard of food contact materials extracts to ensure quality, safety and innovation of paperboards

HAL Id: hal-01985967

https://hal-agrosup-dijon.archives-ouvertes.fr/hal-01985967

Submitted on 18 Jan 2019

HAL is a multi-disciplinary open access

archive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Use of reliable bioassays to detect potential hazard of food contact materials extracts to ensure quality, safety

and innovation of paperboards

Emilie Souton, Isabelle Séverin, Marie-Christine Chagnon

To cite this version:

Emilie Souton, Isabelle Séverin, Marie-Christine Chagnon. Use of reliable bioassays to detect potential hazard of food contact materials extracts to ensure quality, safety and innovation of paperboards. 51st Congress of the European Societies of Toxicology (EUROTOX), Sep 2015, Porto, Portugal. 51st Congress of the European Societies of Toxicology (EUROTOX), 238 (2, supplément), pp.S348, 2015, Toxicology Letters. �hal-01985967�

s"

Use$of$reliable$bioassays$to$detect$poten2al$hazard$of$food$

contact$materials$extracts$to$ensure$quality,$safety$and$

innova2on$of$paperboards$

E.$Souton,$$I.$Séverin,$$M.$C.$Chagnon$

INSERM'UMR'U866'+'Université'de'Bourgogne'+'Agrosup'Dijon,'bâ@ment'Epicure'

1,'esplanade'Erasme'21000'Dijon"

Food$contact$materials$(FCM)$represent$a$major$economic$issue$and$also$a$field$of$innova2on.$Food$packaging$produc2on$must$meet$manufacturing$good$prac2ces,$safety$for$human$health$and$thus$be$in$

compliance$with$1935/2004$european$regula2ons.$Its$third$ar2cle$specifies$that$FCM$do$not$transfer$their$cons2tuents$to$food$in$quan22es$which$could$endanger$human$health$under$normal$or$foreseeable$

condi2ons$of$use.$Indeed,$these$materials$are$not$inert$and,$in$addi2on$to$started$substances,$nonRinten2onnally$added$substances$(NIAS)$are$able$to$migrate$from$FCM$into$food.$NIAS$can$be$contaminants$from$

recycled$materials,$impuri2es,$new$substances$occured$during$the$packaging$produc2on$chain,$synthe2c$residues$etc.,$and$they$could$represent$great$part$of$migra2ng$substances$(Grob$et$al.,$2006$;$Skejevrak$et$

al.,$2005).$The$European$Regula2on$No$10/2011$on$plas2c$materials,$mul2layers$and$ar2cles$intended$to$come$into$contact$with$food$is$the$first$Commission$regula2on$that$regulates$NIAS,$but$there$are$no$

available$guidelines$to$assess$the$risk$of$NIAS.$Furthermore,$NIAS$may$also$be$present$in$other$materials,$such$as$paper$and$boards.$Because$of$the$complex$nature$of$FCM,$it$is$per2nent$to$evaluate$the$poten2al$

toxicity$of$FCM$as$a$whole$and$then$to$take$into$account$any$«$cocktail$effect$».$The$objec2ves$of$this$study$are$to$provide$to$packaging$and$food$companies$scien2fic$relevant$tools$combining$physicochemical$

and$toxicological$strategies$based$on$bioassays$applied$to$FCM$extracts,$especially$paper$and$boards.$Three$toxicological$endpoints$must$be$checked$as$relevant$for$low$dose$exposure$:$cytotoxicity,$genotoxicity$

and$endocrine$disrup2on.$

Aqeous"f"

Figure'5':'3_{H+urindine"uptake"kine4cs"on"HepG2"cells"a9er"20"h"} exposure"to"all"extracts"of"produc4on"chain.' .'

! 

$Sensi2ve$bioassay$detec2ng$and$quan2fying$DNA$primary$damages.$

Comet$assay$

Figure'3':''Genotoxicity"evalua4on"on"HepG2"cells" a9er"20h"exposure"to"all"extracts"of"the"produc4on" chain."Three"independent"experiments";"two+two" comparison"of"samples"with"Wilcoxon+Mann" Whitney"test,"**"p<0,01" " " " " " " " " " "

Discussion/Conclusion$

" "_{T+$→""Metyl"methanesulfonate""C} 2H6O3S"(50"µM)"" Principle$:"Detec4ng"single"and"double"strand"breaks"of"DNA,"and"alkali+labile"sites"by" electrophoresis"in"alkaline"condi4on"(pH"="13)"a9er"staining"of""DNA"with"a"fluorescent" intercala4ng"agent"2_{"(propidium"iodide)}3"_"

Resazurin$assay$

Figure'5:'Number'of'revertant'colonies'of'TA98'and'TA100'strains'aOer'axposure'to'three' concentra@ons'of'samples'of'the'produc@on'chain,'with'or'without'metabolic'ac@va@on'S9."One" experiment"performed;"three"replicates"by"condi4on."Posi4ves"controls:"TA98"without"S9"T+:"2+ nitrofluorene;"TA98"with"S9"T+:"benzo(a)pyrene;"TA100"without"S9"T+:"azide"sodium;"TA100"with"S9"T +:"2+aminoanthracene' Principle:"Colorimetric"and"fluorimetric" assay"based"on"metaboliza4on"of" resazurine"by"mitochondria""in"viable"cells."" " "T$+"→"dodecylsulfate"sodium"SDS"3%" Figure'1':'Sampling"sta4ons"along"the"produ4on"chain"(red"circles).""

! 

"Sensi2ve$bioassay$to$quan2fy$cell$

viability.$

$

Applica2on$to$paper$and$boards$FCM$$

Evalua4on"of"in'vitro"toxicity"and"iden4fica4on"of"the"cri4cal"sta4ons"along"the"produc4on"chain"of"a"packaging"industry.""

Significant$increase$of$DNA$damages$of$samples$of$the$produc2on$

chain$in$comparison$to$nega2ve$control$but$no$genotoxicity$at$the$

end$of$the$process.$

+  None"of"the"samples"were"significantly"cytotoxic"for"HepG2"cells."

Pro

to

co

l$o

f$ext

ra

c2o

n"

Perspec2ves$

+  Posi4ve" effect" detected" with" Comet" assay" and" nega4ve" effect" with"

micronucleus"assay"maybe"due"to"DNA"repara4on."

+  Need" to" establish" a" dose" effect" using" Comet" assay" and" micronucleus"

assay."

+  Comet"FPG"could"be"performed"to"detect"oxidized"DNA"bases"

+  Poten4al"oxidizing"effect"of"samples"of"the"produc4on"chain"could"be"

checked"with"DCFDA"assay."

+  Check,"using"a"chemical"analysis,"which"substances"could"be"responsible"

of"the"hazardous"effects"

+  Check" the" endocrine" disrup4on" effect" as" an" other" toxicological"

endpoint"

Star2ng$product$ _Tub$ Grinder$ Frontbox$ End$product$

Micronucleus$assay$

Ames$test$

TA98%&S9 Samples((µM) Nu mb er (o f(( re ve rt an ts T(7( S.P.(10S.P_S.P.(50.(100Tub(10Tub(50_Tub(100 Grind er(10 Grind er(50 Grind

er(100FB.(10FB_FB.(100.(50_{E.P.(10E.P.(50E.P}.(100T(+(

0 10 20 30 40 50 200 400 600 800 1000 T"#" S.P."10S.P_S.P."50."100Tub"10Tu_Tub"50b"100 Grind er"10 Grind er"50 Grind

er"100FB."10FB_FB."100."50_{E.P."10E.P."50E.P}."100T"+"

0 50 100 150 200 1000 1200 1400 1600 1800 2000 Nu mb er "o f"" re ve rt an ts

TA100%+S9

Samples"(µM) TA98%&S9 Samples((µM) Nu mb er (o f(( re ve rt an ts T(7( S.P.(10S.P_S.P.(50.(100Tub(10Tu_Tub(50b(100 Grind er(10 Grind er(50 Grind

er(100FB.(10FB_FB.(100.(50_{E.P.(10E.P.(50E.P}.(100T(+(

0 10 20 30 40 50 200 400 600 800 1000 T"#" S.P."10S.P_S.P."50."100Tub"10Tu_Tub"50b"100 Grind er"10 Grind er"50 Grind

er"100FB."10FB_FB."100."50_{E.P."10E.P."50E.P}."100T"+"

0 50 100 150 200 400 600 800 1000 Nu mb er "o f"" re ve rt an ts

TA100%&S9

Samples"(µM) Samples$(μM)$

! 

$Reverse$muta2on$assay$performed$to$evaluate$poten2al$mutagenic$proper2es$of$tested$substances$

on$a$bacterial$model$(Salmonella$Typhimurium).$$

Principle$:"Detect"ponctual"muta4ons"(subs4tu4on,"addi4on,"dele4on"of"one"or"several"DNA"bases)."The"growth"of"strains"of"S." Typhimurium"used"is"dependent"of"his4dine"amino"acid"(here"TA98"and"TA100)."Spontaneous"reversion"occurs"allowing"the"revertants" to"grow"on"a"media"without"his4dine;"mutagenic"substances"cause"an"increase"in"the"number"of"revertant"colonies"rela4ve"to"the" background"level."

FCM$

migra2on$

FOOD$

RISK$

ASSESSMENT$

Chemical$

analysis$

Characterized$

molecules

$

molecules

Unknown$

$

Low$dose$effect?

$

“Cocktail”$effect?

IAS$$$R$$$NIAS

$

$

1)$Migra2on$

2)$Extrac2on$

Biological$

analysis$

Iden2fica2on$

of$NIAS?$

Exposure$

In#vitro#assays:$global$approach$

He pG2$ Ca co 2$ He La $9903$

HAZARD$

ASSESSMENT$

Figure'2':'Percentage"of"viability"of"HepG2"cells" a9er"20"h"exposure"to"all"extracts"of"the" produc4on"chain";"three"independent" experiments.'

Unaltered#DNA## Fragmented#DNA##

TR$ T+$ 2'independent'experiments'were' performed'and'the'results'were' averaged'and'expressed'as'a'score.' '_{Formula'score'='(comets'class'0'x'} 0)'+'(comets'class'1'x'1)'+'(comets' class'2'x'2)'+'(comets'class'3'x'3)'+' (comets'class'4'x'4)."

! 

"Sensi2ve$bioassay$to$detect$aneugenic$and$clastogenic$ac2vity$of$tested$substances.$

$ Principle$:"Detec4ng"presence"of"micronucleus"in"cells"having"made"a"mitosis"and" blocked"in"anaphase"because"of"the"adjonc4on"of"Cytochalasine"B."Micronuclei"can"be" due"to"acentric"chromosomes"or"chromosomes"not"capable"of"migrate"to"cell"poles.""

Samples$$ Preserva2on$before$extrac2on$ Direct$extrac2on$ Preserva2on$aker$extrac2on$ Before$using$

Tub"(c°"="1,5g/mL)" _4°C" 1)  centri."103_g+30min.""

2)"recovery"of"supernatant" +"20°C"" (glass"flacons)" Steriliza4on"by"filtra4on" Frontbox"(FB)(c°"="1,5g/mL)" Grinder"(c°=1,5/5"g/mL)" 4°C" 1)"small"squares"2x1cm." "2)"addi4on"of"water."" 3)"orbital"shaking"24h"+"23°C.""" 4)"recovery"of"extracted"water" "" + 20°C"" (glass"flacons)" "" "" Steriliza4on"by"filtra4on""" Star2ng$product$(S.P.)$ (c°=1,5/8""g/mL)" Room"temperature"in" aluminium"foil" End$product$(E.P.)$ (c°=1,5/10"g/mL)" "End$product$(Norm)$$ (c°="0,04g/mL)" Room"temperature"in"" aluminium"foil" Norm"AFNOR"NF"EN"645""(c°="10g"/"250mL)"" + 20°C" "(glass"flacons)" "" Steriliza4on"by"filtra4on"" "" Aq eo us $ fo rm " Non$ aqe ous $ fo rm " Nor m "

Cytotoxicity$

Genotoxicity$

+  Ames"test:"None"of"the"tested"samples"were"significantly"mutagenic"

on"both"strains"of"Salmonella"Typhimurium"used."

+  Comet"assay:"Posi4ve"effect"detected"all"along"the"produc4on"chain""

excepted"for"the"end"product."

+  Micronucleus" assay:" Cytotoxic" effect" observed" a9er" long" 4me"

exposure"to"star4ng"product"but"no"cytotoxic"effect"of"other"samples"

of" the" chain." No" posi4ve" effect" of" all" tested" samples" regarding" the"

number"of"micronuclei."

Star%n g(prod uct S.P.(1/2 Tu b Grind er Frontbox End(pr oduc t Posi%v e(ctrl 0 2 4 6 8 10 0 20 40 60 80 100

%"

m

icr

on

uc

le

i

%"

cyt

oto

xic

ity

55%"cytotoxicity" Figure'4':''Genotoxicity"evalua4on"on"HepG2"cells"a9er" long"4me"exposure"to"all"extracts"of"the"produc4on" chain."One"experiment." S.P."1/2"(star4ng"product"two"fold"diluted)"analysed" because"of"the"high"cytotoxicity"of"star4ng"product" (69%"cytotoxicity)." " "_{T+$→""Vinblas4ne"(10"µM)"}

Vi

ab

ilit

y(

(%

)

Nega% ve'ct rl Star%n g'prod uct _Tub Grind er Frontbox End'pr oduc t Norm Posi%v e'ctrl 0 50 100

Decrease$of$cell$viability$aker$

treatment$with$star2ng$product$

and$tub,$but$no$cytotoxic$effect$

of$other$samples,$in$comparison$

to$nega2ve$control.$

Picture'of'cells'stained'with' orange'acrydine'and' observa@on'of'cells'with' microscope.' 1st_{'cell'coun@ng'+>'} percentage'of'cytostase' 2nd_{'coun@ng:'percentage'of'} micronuclei''" Binucleated#cell#

with#a#micronucleus#Mononucleated#cell#

No$cytotoxic$effect$(%$cytotoxicity$>$55%)$of$samples$excepted$for$the$

star2ng$product.$

No$increase$of$the$number$of$micronuclei$in$comparison$with$the$

nega2ve$control.$

No$mutagenic$

effect$on$both$

strains$of$all$

samples$of$the$

produc2on$chain.$

Mutagenic'substances'are'able'to' cause'an'increase'of'the'number'of' revertant'colonies'rela@ve'to'the' background'level." Background'level'of'revertant'colonies."