Review Article Ethoxyquin: An Antioxidant Used in Animal Feeddownloads.hindawi.com/journals/ijfs/2013/585931.pdf · Review Article Ethoxyquin: An Antioxidant Used in Animal Feed

Hindawi Publishing CorporationInternational Journal of Food ScienceVolume 2013 Article ID 585931 12 pageshttpdxdoiorg1011552013585931

Review ArticleEthoxyquin An Antioxidant Used in Animal Feed

Alina BBaszczyk1 Aleksandra Augustyniak1 and Janusz Skolimowski2

1 Department of General Genetics Molecular Biology and Plant Biotechnology Faculty of Biology andEnvironmental Protection University of Łodz Banacha 1216 90-237 Łodz Poland

2Department of Organic Chemistry Faculty of Chemistry University of Łodz Tamka 12 91-403 Łodz Poland

Correspondence should be addressed to Alina Błaszczyk ablaszbiolunilodzpl

Received 15 January 2013 Accepted 2 April 2013

Academic Editor Angel Medina-Vaya

Copyright copy 2013 Alina Błaszczyk et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Ethoxyquin (EQ 6-ethoxy-12-dihydro-224-trimethylquinoline) is widely used in animal feed in order to protect it against lipidperoxidation EQ cannot be used in any food for human consumption (except spices eg chili) but it can pass from feed tofarmed fish poultry and eggs so human beings can be exposed to this antioxidant The manufacturer Monsanto Company (USA)performed a series of tests on ethoxyquin which showed its safety Nevertheless some harmful effects in animals and peopleoccupationally exposed to it were observed in 1980rsquos which resulted in the new studies undertaken to reevaluate its toxicity Herewe present the characteristics of the compound and results of the research concerning for example products of its metabolismand oxidation or searching for new antioxidants on the EQ backbone

1 Introduction

During storage of animal feed many different processes mayoccur which alter their initial natural proprieties First of alllipids undergo peroxidation the process during which theyare deteriorated in a free radical autocatalytic oxidation chainreaction with atmospheric oxygen Lipid autooxidation is acascade phenomenon ensuring continuous delivery of freeradicals which initiate continuous peroxidationThis processresults in food rancidity which manifests itself as the changeof taste scent and color as well as decrease in shelf life of theproduct Natural or synthetic antioxidants are usually used toslow down or stop lipid peroxidation and in consequence topreserve freshness of the product Many natural antioxidantssuch as tocopherols vitamin C flavonoids for a shortperiod may be effective in food preserving but in manycases such protection is not sufficient Therefore syntheticantioxidants are widely used among which BHT (butylatedhydroxytoluene) BHA (butylated hydroxyanisole) and EQ(ethoxyquin) are the most frequent However some effects ofsynthetic antioxidants are not always beneficial for our healthAntioxidants such as BHA or BHT have been widely usedfor many years to preserve freshness flavor and colour offoods and animal feeds as well as to improve the stability of

pharmaceuticals and cosmeticsThere aremany controversiesabout the use of these two antioxidants in foods Someexperimental studies have reported that both BHT andBHA have tumour-promoting activity [1 2] On the otherhand there were reports on anticarcinogenic properties ofthese antioxidants when they are used at low concentrations[3] Human exposures are at least 1000-fold below thoseassociated with any neoplastic actions in laboratory animalsthus it is assumed that they are not harmful for human beings[3 4]

The third compound EQ is one of the best known feedantioxidants for domestic animal and fish Its unquestionableadvantage is its high antioxidant capacity and low productioncosts However some of the authors have suggested that itis responsible for a wide range of health-related problemsin dogs as well as in humans [5ndash9] Due to the increaseduse of this antioxidant it was nominated by FDA (US Foodand Drug Administration) for carcinogenicity testing [10]The tests were carried out byMonsanto Company (USA) EQproducer and after that in 1977 FDA requested for optionallowering of the maximum level of EQ in complete dog foodsfrom allowed 150 ppm (0015) to 75 ppm (00075) At thesame time new studies were started by the Pet Food Instituteto determine whether even lower EQ levels (between 30 and

2 International Journal of Food Science

60 ppm) would provide antioxidant protection for dog food[11]

Ethoxyquin is also known as Santoquin SantoflexQuinol It was originally developed in rubber industry toprevent rubber from cracking due to oxidation of isoprene[12] The Monsanto Company (USA) taking into account itshigh antioxidant efficiency and stability as well as low costsof synthesis refined it later for use as a preservative in animalfeeds because it protects against lipid peroxidation andstabilizes fat soluble vitamins (A E) Presently ethoxyquin isused primarily as an antioxidant in canned pet food and infeed intended for farmed fish or poultry

The use of ethoxyquin is not permitted in foods intendedfor human except preserving powdered paprika and chilicolour and using it as an antiscald agent in pears and apples(inhibition of ldquobrown spotsrdquo development)However becauseEQ is used as a feed antioxidant it can be also found in otherproducts intended for human consumption like fishmeal fishoils and other oils fats and meat (Table 1) An acceptabledaily intake (ADI) of EQ for human (0ndash0005mg kgminus1 bw)based on the results obtained from studies on dogs wasestablished in 1998 [13 14]

This paper presents characteristics of ethoxyquin withregard to its properties metabolism toxicity possible car-cinogenicity and antioxidant activity

2 Physical and Chemical Properties ofEthoxyquin (EQ)

For the first time EQ was synthesized in 1921 by Knoevenagel[15]The synthesis was based on condensation of aniline withmolecules of acetone or its analogues [15 16] Błaszczyk etal [16] synthesized EQ from p-phenetidine (4-ethoxyaniline)and diacetone alcohol in the presence of p-toluenesulfonicacid or iodine Pure ethoxyquin (EQ 6-ethoxy-12-dihydro-224-trimethylquinoline CAS number 91-53-2 Figure 1) isa light yellow liquid but it changes color to brown if itis exposed to oxygen [12] It also tends to polymerize onexposure to light and air The scent of EQ is described asmercaptan like As a nonpolar substance EQ is soluble onlyin organic solvents Some of the additional properties of EQare presented in Table 2

3 Biotransformation of EQ

31 Animals Ethoxyquin is rapidly absorbed from gastroin-testinal tract of laboratory animals like rats and mice Peakblood concentration of the compound is observed within1 h Distribution of EQ in animal body is similar when itis administered orally and intravenously Small amounts ofparent EQ were detected in liver kidney and adipose tissueand fish muscles [20ndash24] It is excreted predominantly asmetabolites via urine Metabolism of EQ was studied in ratsmice dogs chickens and fish as well as in plants [13 21 25]It is not fully described but some metabolites were identified(Table 3) The most important EQ metabolites observed inrat urine and bile result from O-deethylation at position 6-Cand then conjugation with sulphate or glucuronide residues

Table 1 Permitted amounts of EQ in different products approvedby FDAlowast

Product Dose [ppm]Animal feed 150Spices 150Uncooked fat of meat from animals (except poultry) 5Uncooked liver and fat of poultry 3Uncooked muscle meat of animals 05Poultry eggs 05Milk 0Pear 3lowastAccording to the Code of Federal Regulations (CFR) Title 21 Parts 573380573400 172140

The other metabolic pathways include hydroxylation andglucuronidation at position 8-C deethylation at 6-C andepoxidation between positions 3-C and 4-C [21] The mainmetabolites observed may be different depending on ani-mal species In mice mainly glucuronide metabolites weredetected while in rats those result from conjugation of EQwith sulfate

In the studies of Bohne et al [23 26] parent EQ dem-ethylated EQ (DEQ) quinone imine (QI) and EQ dimer(EQDM) were observed in salmonid fish after long-termdietary exposure to EQ It was in agreement with the resultsobtained earlier by Skaare and Roald [27] EQ is consideredas a model inducer of phase II enzymes involved in themetabolism of xenobiotics but influence of EQ on phase Ienzyme gene transcript levels was also observed [28 29]Thekey role in mediating phase I reactions (eg oxidation orreduction) producingmore hydrophilic compounds is playedby the CYP (cytochrome P450) enzyme family Bohne et al[28 29] observed the alteration of CYP3A gene expressionan increase in the amount of CYP3A transcripts was detectedin salmon after feeding them with the diet containing EQ atthe highest dose used (1800mg kgminus1) The authors speculatethat EQ may regulate CYP3 gene expression by interactionfor example with pregnane X nuclear receptor (PXR) whosefunction is to sense the presence of toxic xenobiotics andin response enhance the expression of proteins involvedin their detoxification On the other hand CYP1A1 geneexpression which was described as an exposure biomarkerto both endogenous and exogenous compounds [30] wasnot increased after dietary exposure of salmonid fish to EQand during the depuration period a trend toward down-regulation was noted [28 29] Such an effect was observeddespite the increase in the expression of AhR mRNA (AhRcytosolic transcription factor responsible for changes in genetranscription) This effect can be explained in several waysFor example the parent EQ may bind CYP1A1 proteinand as a result may inhibit the gene expression and activ-ity of protein [31] Hepatic antioxidant response elements(ARE) or AhR repressor (AhRR) together with basic-helix-loop-helix-PAS (Per-AhRARNT-Sim homology sequence)of transcription factor usually associated with each other toform heterodimers (AhRARNT or AhRRARNT) may be

International Journal of Food Science 3

Table 2 Physical and chemical properties of ethoxyquin

Properties of EQMolecular formula C14H19NOMolecular mass 21734 [gmolminus1]IUPAC name 12-dihydro-224-trimethylquinolin-6-yl ethyl etherCAS name 6-ethoxy-12-dihydro-224-trimethylquinolineChemical class QuinolineMelting point 0∘Ca

Boiling point 123ndash125∘C at 2mmHga

Solubility Insoluble in water soluble in fat and animal and plant oils and soluble in ethanol methanol DMSO andDMF

Vapor density 748 (AIR = 1)a

Stability Stable under ordinary conditions storage temp 0ndash6∘CSpectral properties Index of refraction 119899

119863

1569ndash1672 at 25∘Ca

Max absorption 354 nm Intense mass spectral peaks 202mz (100) 108mz (53) 174mz (48) and137mz (36)a13C NMR in CDCl3 EQ (8 solution) 120575

119862

in ppm 1512 (EQ-C-6) 1376 (EQ-C-4) 1295 (EQ-C-3) 1285(EQ-C-9) 1228 (EQ-C-10) 1146 (EQ-C-8) 1136 (EQ-C-7) 1111 (EQ-C-5) 642 (EQ-CH2-O) 516(EQ-C-2) 304 (EQ-CH3-4) 185 (EQ-CH3-4) 150 (EQ-CH3-CH2-O-)

b

aAccording to the National Library of Medicine HSDB Database (last revision date 2003) [18]bAccording to Błaszczyk and Skolimowski [19]DMSO dimethyl sulfoxide DMF NN-dimethylformamide

also involved in the CYP1A1 downregulation process Theseheterodimers can influence gene expression by binding AREsequences in the gene promoter regions [32]

However EQ as other phenolic antioxidants first ofall causes induction of phase II xenobiotic-metabolizingenzymes Bohne et al [28 29] observed elevated dose-relateduridine diphosphate glucuronosyl-transferase (UDPGT)mRNA expression after dietary exposure to EQ As UDPGTreacts with the compounds that have the hydroxyl group(-OH) parent EQ cannot be the potential substrate forglucuronidation only its metabolites for example DEQ(6-hydroxy-224-trimethyl-12-dihydroquinoline Table 3)the metabolite identified by Berdikova Bohne et al [26] inAtlantic salmon Changes in the expression of glutathioneS-transferase (GST) gene were also observed after feedinganimals with EQ containing feed The alterations in GSTactivity caused by EQ were documented in Atlantic salmon[28 29] in rodents [33 34] and in nonhuman primates[35] In addition to UDPGT and GST some other enzymesare involved in phase II metabolism of EQ for exampleNADP(H) quinone oxidoreductase and epoxide hydrolase[36]

The expression pattern of both phase I and II enzymesinvolved in EQmetabolismmay vary in different animals andshould be considered in relation to the ratio of parent EQand its metabolites (first of all DEQ QI and EQDM) in theliver [29 37] The research concerning this issue is currentlyin progress

32 Plants Ethoxyquin is also registered as an antioxidant tocontrol scald (browning) in apples and pears The EQ plantmetabolitesdegradation products were detected and it was

shown that in general they are different from those observedin animals (Table 3) In pears treated with ring-labeled[14C]ethoxyquin the following compoundswere detectedNndashN and CndashN dimers demethylethoxyquin (DMEQ) dehy-drodemethylethoxyquin (DHMEQ) and dihydroethoxyquin(DHEQ) [14 25] It was shown that ethoxyquin was rapidlydegradated or metabolized but itself it was not translocatedinto the pulp of fruit where the residues were detected (lessthan 05 of total radioactive residue was EQ) Toxicity ofEQ metabolites MEQ DHMEQ and DHEQ was studied indogs (oral administration single doses of 50 to 200mg kgminus1bw) and it was found that they did not show any significanttoxicity In the report of Gupta and Boobis [14] the rankorder of toxic potency for the plant metabolites and EQ isMEQltEQltDHEQltDHMEQ (the least toxic first) MEQDHMEQ and DHEQ were also evaluated for genotoxicity inin vitro and in vivo tests The compounds did not cause genemutations in Salmonella typhimurium and Escherichia colistrains but they induced chromosomal aberrations orandendoreduplication in Chinese hamster ovary cells On theother hand plant metabolitesdegradation products did notexhibit genotoxic potential in vivo ADI intake for humans forMEQ DHMEQ and DHEQ was estimated at the same levelas for EQ (0ndash0005mg kgminus1 bw)

4 Antioxidant Activity of EQ

EQ possesses high-antioxidative activity It is very efficient inprotecting lipidswhich are present in food against oxidization[38 39] Specifically it is used to retard oxidation of carotenexanthophylls and vitamins (like vitamins A or E) In animalstreated with ethoxyquin three times higher level of vitamins


Table 3 Metabolitedegradation products of EQ detected in different organisms

EQ metabolitedegradation product Organism

NH

CH3

OO

O

CH3

CH3

SOH

12-Dihydro-6-hydroxy-224-trimethylquinoline sulphate Rats1

NH

CH3O

CH3

CH3

Glu

12-Dihydro-6-hydroxy-224-trimethylquinoline glucuronide Mice1

CH2

CH3

CH3

OH

NH

S OOO

OH

1234-Tetrahydro-36-dihydroxy-4-metylene-22-dimethylquinoline sulphate Rats1

EtCH3

NH

O

OGlu

CH3

CH3

12-Dihydro-6-ethoxy-8-hydroxy-224-trimethylquinoline glucuronide Rats1Mice1

CH3

N

O

SGCH3

CH3

8-(S-glutathionyl)-224-trimethylquinol-6-one Rats1Mice1

Et

CH3

CH3

OH

NH

SO

GH3C

1234-Tetrahydro-6-ethoxy-3-hydroxy-4-(S-glutathionyl)-224-trimethylquinoline

Rats1Mice1

N

CH3

CH3

CH3

O

26-Dihydro-224-trimethyl-6-quinolone (quinone imine (QI)) Rats1Atlantic salmon2

CH3

CH3

CH3

HO

NH

6-Hydroxy-224-trimethyl-12-dihydroquinoline (deethylated EQ (DEQ)) Atlantic salmon2

EtCH3

N

O

H3C

CH3

CH3

H3C

H3CEtO

NHlowast

181015840-Di(12-dihydro-6-ethoxy-224-trimethylquinoline) or661015840-diethoxy-222101584021015840441015840-hexamethyl-1101584021015840-dihydro-2H-181015840-biquinoline

(ethoxyquin dimer (EQDM))lowastInstead of the hydrogen atom there is a methyl group it gives methyl C-N

ethoxyquin dimer661015840-Diethoxy-11015840222101584021015840441015840-heptamethyl-1101584021015840-dihydro-2H-181015840-biquinoline

Atlantic salmon2

Pears34


Table 3 Continued


EtCH3

N

N

O

CH3

CH3

Et OCH3

CH3

CH3

N-N Ethoxyquin dimer111015840-di(12-dihydro-6-ethoxy-224-trimethylquinoline) or

661015840-diethoxy-222101584021015840441015840-hexamethyl-2H21015840H-111015840-biquinolinePears34

EtCH3

N

O

CH3

CH3

CH3

6-Ethoxy-1224-tetramethyl-12-dihydroquinolinemethylethoxyquin (MEQ) Pears34

EtCH3

N

O

CH3

6-Ethoxy-24-dimethylquinoline dehydrodemethylethoxyquin (DHMEQ) Pears34

EtCH3

N

O

CH3

CH3H

6-Ethoxy-224-trimethyl-1234-tetrahydroquinoline dihydroethoxyquin(DHEQ) Pears34

EtCH3

N

O

CH3H

6-Ethoxy-24-dimethyl-12-dihydroquinolinedemethylethoxyquin (DMEQ) Pears34

Glu glucuronide G glutathione Et ethyl group (C2H5)1Burka et al [21] 2Berdikova Bohne et al [26] 3Gupta and Boobis [14] 4JMPR [25]

A and E in blood plasma was observed [40] This findingsuggests that an organism is using EQ instead of naturalantioxidants High efficiency of this antioxidant results notonly from chemical features of EQ itself but also from thefact that products of its oxidation also possess antioxidativeproperties [12 39 41]

Studies on EQ antioxidant properties were performedby Taimr [39] with the use of alkylperoxyls and it wasshown that the reaction rate of EQ with them is veryhigh In the presence of high oxygen concentrations EQreacts with alkylperoxyl molecule to form aminyl radical (6-ethoxy-224-trimethyl-12-dihydroquinolin-1-yl) which sub-sequently may enter various pathways In nonoxidizing con-ditions it can be stabilized both by the loss of methyl groupand aromatization of heterocycle to form 24-dimethyl-12-dihydroquinoline (dehydrodemethylethoxyquin (DHMEQ))and through dimerization to form EQ dimer (EQDM) [3942] On the other hand in an oxidizing medium other

molecules can be formed for example 26-dihydro-224-trimethyl-6-quinolone (QI) or nitroxide radical (6-ethoxy-224-trimethyl-12-dihydroquinolin-N-oxyl) which is also astrong antioxidant [39 42]

Products of EQ oxidation were detected by differentauthors in fish oil and meal [22 43ndash45] According toHe and Ackman [46] the following oxidization productsof EQ dominate in fish meal and fish feed 26-dihydro-224-trimethyl-6-quinolone (QI) and 181015840-di(12-dihydro-6-ethoxy-224-trimethylquinoline) (EQDM) At high storagetemperature neither QI nor EQDM accumulates how-ever another product of EQ oxidation 24-dimethyl-6-ethoxyquinoline is stable [41] As it was pointed out earlierethoxyquin oxidization products also possess antioxidativeproperties The EQDM and QI show 69 and 80 of EQefficacy respectively (studies on fish meal) [12] On the otherhand in the studies of Thorisson et al [45] quinone imine(QI) and EQ nitroxide were also powerful antioxidants while


Ethoxyquin

NH

CH3

OH3C

CH3

CH3

6-Ethoxy-12-dihydro-224-trimethylquinoline

Hydroxyquin Hydroquin

NH

CH3

CH3

CH3

HO

224-Trimethyl-12-dihydroquinolin-6-ol

CH3

CH3

CH3

NH

H

224-Trimethyl-12-dihydroquinoline

CH3

CH3

CH3

NHH3C

2247-Tetramethyl-1234-

tetrahydroquinoline

H3C

H3C

H3C

CH3

CH3

NH

HHO

6-Ethoxy-2257-tetramethyl-

1234-tetrahydroquinoline

Figure 1 Chemical structure of ethoxyquin (EQ) and of some new compounds synthesized on ethoxyquin backbone with promisingantioxidant properties (according to de Koning [12] Dorey et al [15] and Błaszczyk and Skolimowski [17])

EQDM themain product of EQoxidation showed little or noantioxidant behavior

Antioxidant activity of EQ was also demonstrated inexperiments performed both in vivo and in vitro Antimu-tagenic effect of this antioxidant was observed in mice ratsand Chinese hamsters treated with cyclophosphamide anagent widely used in cancer chemotherapy [47ndash49] Duringcyclophosphamide bioactivation reactive oxygen species areformed which can cause damage of genetic material [5051] EQ reduced the number of chromosome aberrationsmicronuclei and dominant lethal mutations induced by theanticancer drug [47ndash49] There were also some reports thatEQ can modify carcinogenic response to different carcino-gens [35 52 53] EQ given to Fischer 344 rats in dietcompletely prevented the formation of aflatoxin B1-inducedpreneoplastic liver lesions [52 53]

In in vitro experiments with human lymphocytes antiox-idant activity of EQ was observed in the comet assay (themethod used to detect single- and double-strand DNAbreaks cross-links and alkaline labile sites) and in micronu-cleus test (the method for the detection of micronucleiinduced by clastogens or aneugens) EQ used at the con-centrations ranging from 1 120583M to 10 120583M protected humanlymphocytes against DNA damage caused by hydrogen per-oxide (H

2

O2

10 120583M) [17] This antioxidant also reduced thenumber ofmicronuclei caused byH

2

O2

used at concentrationof 75120583MHowever the significant reductionwas evident only

in the case of lower EQ concentrations (5120583M 10 120583M)with noeffect at higher concentration [17]

5 Adverse Effects of EQ In Vivo andIn Vitro Studies

Different phenolic antioxidants may be used in animal feedsuch as BHA (butylated hydroxyanisole) BHT (butylatedhydroxytoluene) and the most efficacious EQ The levels ofthe antioxidants in finished feed should not be higher than150 ppm for EQ and 200 ppm for BHT and BHA (US FoodandDrugAdministration permissions)The fact that efficientantioxidants work optimally when they are used at low con-centrations is their remarkable characteristic On the otherhand when antioxidants are used at high concentrations theyact as prooxidants The impact of these compounds dependson their concentration as well as on other factors suchas metal-reducing potential chelating behaviour solubilityand pH The effect of antioxidants on living organisms alsodepends on their bioavailability and stability in tissues [5455] Phenolic antioxidants under favorable conditions maybe converted to phenoxyl radicals with prooxidant activity[55] It was shown that dissolved EQ may exist partly in thefree radical form which it was also detected in the compounditself [56]Therefore ethoxyquin nitroxidewhich is producedby EQ oxidation similarly as other nitroxide molecules


Table 4Harmful effects of EQobserved after its oral administrationin different animals or in humans (contact exposure)

The harmful effect Animals

Loss of weight Marmosets1 rats2 dogs2 mice2rabbits3

Changes in liver Marmosets1 rats14 dogs2 mice2broiler chickens2 tilapia5

Changes in kidney Marmosets1 rats267 dogs2 broilerchickens28

Changes in alimentary duct Marmosets1 dogs2 mice2 broilerchickens28

Changes in urinary bladder Rats2

Anemia Marmosets1

Changes in mitochondria Rats2

Lethargy Rabbits3

Colored urine skin or fur Dogs2 rats2

Increase in mortality Broiler chickens28

Effect on immunity Tilapia5

Condition factor the finalbody weight in relation tobody length of fish

Large yellow croaker9

Allergy (contact exposure) Humans1011121McIntosh et al [58] 2Drewhurst [13] 3Little [10]4Ito et al [59] 5Yamashitaet al [60] 6Neal et al [61] 7Manson et al [53] 8Leong and Brown [62]9Wang et al [63] 10Rodrıguez-Trabado et al [9] 11 Brandao [5] 12Savini etal [6]

(eg tempol) may also show prooxidative properties [57]Formation of free oxygen species as a result of using too highEQ concentrations can cause adverse health effects in animalsfed with EQ containing feed or in people consuming meatfrom farmed animals for example different fishes

The studies on EQ prooxidant activity and toxicity associ-atedwith it were performed both in vivo and in vitro Dogs aremost susceptible to the harmful effects of EQ and first reportsof such effects were received by FDA in 1988 The symptomsobserved by dog owners and veterinarians were liver kidneythyroid and reproductive dysfunction teratogenic and car-cinogenic effects allergic reactions and a host of skin andhair abnormalities [7] According to the studies on dogs andlaboratory animals it was shown that ethoxyquin had littleacute toxicity except when it is administered parenterallyValues of LD

50

for EQ are 1700mg kgminus1 bw (rats oralgavage) gt2000mg kgminus1 bw (rats dermal treatment 24 h)sim900mg kgminus1 bw (mice intraperitoneal administration) andsim180mg kgminus1 bw (mice intravenous administration) [13]Despite species differences in the majority of animals treatedwith EQ at the concentrations higher than those permitted inanimal feed the same characteristic symptoms and patholo-gies appeared such as weight loss liver and kidney damagealterations of alimentary duct (Table 4) The concentrationof 100 ppm (equivalent to 25mg kgminus1 bw per day) wasconsidered to be a minimal-effect level for clinical signs oftoxicity and liver effects in dogs themost susceptible animals[13 14]

Detrimental effects of EQ were also seen when theexperiments were performed at the cell metabolism levelHernandez et al [64] andReyes et al [65] analysed the impactof EQon themetabolic pathways of rat renal and hepatic cellsas well as on mitochondria and submitochondrial particlesobtained from bovine heart and kidney They observedinfluence of EQ on energy processes in cells EQ inhibitedrenal Na+ K+-ATPase activity involved in ion transport [64]The authors suggested that EQ interacted with site I of themitochondrial respiratory chain and it resulted in inhibitionof oxygen consumption in the mitochondria of kidney andliver cells when glucose was a respiratory substrateThe effectwas dose dependent

More than 30 years ago when EQ began to be morecommonly used in animal feed research started to assess itsmutagenicity with the use of Ames test which is performedon different Salmonella typhimurium strainsThe results wereequivocal as some results were negative [66ndash68] but thepositive effects were also observed [69 70] It was alsoshown that EQ enhanced the mutagenic activity of DMBA(321015840-dimethyl-4aminobiphenyl) a compound having car-cinogenic properties [70] Ethoxyquin was reported to bothenhance and inhibit genetic changes induced by knowncarcinogens on the other hand it can also lead to cancerin exposed animals Manson et al [53] observed in Fischer344 rats that EQ caused severe damage in kidney Manyhyperplastic and putative preneoplastic tubules were foundwhich suggested that EQ may be exerting a carcinogeniceffect Similar effects were observed earlier by Ito et al [59] inrelation not only to the kidney but also to the urinary bladder

Possible carcinogenicity of EQ is probably connectedwith its prooxidant activity and induction of reactive oxygenradicals which cause DNA damage DNA damage is usuallyrepaired by cellular repair system but if it is severe or thereare too many lesions this leads to programmed cell death(apoptosis) Sometimes however the programmed cell deathpathway is damaged so when the defense mechanisms failthere is no way to stop a cell from becoming a cancer cellSome in vitro studies showed both cytotoxic effects of EQleading to cell apoptosis or necrosis and damage of geneticmaterial at DNA or chromosome levels Cytotoxic effects ofpure EQ (puritygt 97) were studied in vitro with the use ofhuman lymphocytesThe IC

50

value (the concentration caus-ing 50 growth inhibition) for EQ determined after 72-hourtreatment of the cells in the MTT assay was 009mM [71]This antioxidant significantly reduced viability of lympho-cytes detected with trypan blue exclusion method after 24-hour treatment at the concentrations of 025 and 05mM (celldivisions were stimulated by phytohemagglutinin (PHA))[19] or of 005mM and higher when 1-hour treatment wasperformed [72] EQ-induced apoptosis by observed in invitro cultured human lymphocytes starting from 005mMconcentration and the detected number of apoptotic cellsdepended on the treatment time [71] Ethoxyquin caused alsoDNA damage in the comet assay [72] however most lesionscould be repaired by cellular DNA repair systems [73] On theother hand the results obtained with the use of chromosomeaberration test showed that unrepairedDNAdamage inducedby EQ could lead to permanent changes in genetic material


[16 74] Błaszczyk et al [16] and Gille et al [74] showedthat this antioxidant induced chromosome aberrations suchas breaks dicentrics atypical translocated chromosomes orchromatid exchanges in human lymphocytes and Chinesehamster ovary cells These aberrations are known to haveserious biological consequences [75]

6 Analogues and Derivatives of EQ

Because of adverse health effects caused by EQ it is reasonableto search for new antioxidants as effective in scavenging freeradicals as EQ which produce no such problems In thepaper of de Koning [12] nine analogues of EQ prepared tocompare their antioxidant efficacy with that of the parentchemical are presentedThe compounds have been tested in arefined fish oil and subsequently some of the most promisingones have been also tested in fish meal It was noted thatthe results obtained in fish oil were not always the sameas in fish meal for example hydroxyquin (12-dihydro-6-hydroxy-224-trimethylquinoline Figure 1) was 35 times aseffective as EQ in fish oil while only 34 of its efficacy wasobserved in fish meal In the case of another compoundmdashhydroquin (12-dihydro-224-trimethylquinoline Figure 1)antioxidant efficacy in relation to EQ was 101 in fish oiland 52 in fish meal Despite the lower efficiency of thiscompound in fish meal the author stated that hydroquincan compete with EQ as an antioxidant of choice [12] Thereason is that preparation of hydroquin based on anilineand acetone is more cost-effective than that of EQ whoseproduction requires p-phenetidine (more expensive thananiline) Hydroquin was earlier patented as an antioxidantin animal feeds in 1997 [76] The 2-year dermal researchwith the use of F344N rats and B6C3F1 mice conductedunder the National Toxicology Program [77] showed that thecompound was not carcinogenic but the studies performedby Sitarek and Sapota [78] showed its teratogenic properties

In 2000 Dorey et al [15] presented the report concerningthe synthesis and biological properties of a new class ofantioxidants based on the EQ backbone The studies wereperformed to search for new quinolinic derivatives withradical scavenging activity potential candidates for centralnervous system protection EQ is not suitable for that as ithas been shown to exhibit significant hypothermic effectprobably as a result of an inhibition of electron transportin the mitochondrial respiratory chain [65] Dorey et al[15] synthesized and studied many 12-dihydro and 1234-tetrahydroquinolines and then selected for further evalu-ation a group of antioxidants (5 compounds) with highradical scavenging capacities relatively low toxicity andmoderate hypothermia The compounds belonging to thegroup of 1234-tetrahydroquinolines (eg 6-ethoxy-2257-tetramethyl-1234-tetrahydroquinoline characterized withthe lowest toxicity and high radical scavenger capacity)are structurally similar to 2247-tetramethyl-1234-tetrahy-droquinoline synthesized and tested in our laboratory(Figure 1) [17] The latter compound also had promisingfeatures its antioxidant activity was comparable to thatof EQ but its cytotoxicity and genotoxicity studied with

Table 5 Comparison of different activities of ethoxyquin andits derivatives based on the data presented by Błaszczyk andSkolimowski [19 71 80ndash82] and Błaszczyk et al [79]

Activitity Activity from the lowest (left) to the highest(right) one

Cytotoxicity EQ-F lt EQ-HCl lt EQ-C lt EQ-S lt EQ-T lt EQ-Qlt EQ-R lt EQ-H lt EQ

Genotoxicity EQ-F lt EQ-C lt EQ-H lt EQ-HCl lt EQ-Q ltEQ-T lt EQ-S lt EQ-R lt EQ

Antioxidantactivity

EQ-F lt EQ-HCl lt EQ-R lt EQ-T lt EQ-S lt EQ-Clt EQ-Q lt EQ lt EQ-H

EQ-F ethoxyquin phosphate EQ-HCl ethoxyquin hydrochloride EQ-Cethoxyquin L-ascorbate EQ-S ethoxyquin salicylate EQ-T ethoxyquin saltof Trolox C EQ-Q ethoxyquin complex with quercetin EQ-R ethoxyquincomplex with rutin EQ-H ethoxyquin 119899-hexanoate EQ ethoxyquin

the use of human lymphocytes in vitro were significantlylower We believe that this chemical is worth of detailedstudies to confirm its usefulness as a food preservativeSome other EQ derivatives and salts were also studied forcytotoxicity genotoxicity and antioxidant activity namelythe complexes of ethoxyquin with flavonoids (rutin orquercetin) ethoxyquin hydrochloride ethoxyquin phos-phate ethoxyquin L-ascorbate ethoxyquin n-hexanoateethoxyquin salicylate and ethoxyquin salt of Trolox C [1979ndash82] The biological properties of the compounds wereanalysedwith the use ofMTT TUNEL and trypan blue stain-ing methods (cytotoxicity testing) comet assay (genotoxicitytesting) and micronucleus test (mutagenicity testing) Fromamong the compounds tested ethoxyquin phosphate (EQ-F) was the least toxic (Table 5)mdashits cytotoxic and genotoxicactivities in comparison with those of EQ were reducedpositively (IC

50

= 08mM versus 009mM for EQ) [71] Onthe other hand antioxidant activity of EQ-F was observedbut it was the lowest of the tested compounds [82]The studiesshowed that all the tested compounds were less toxic tohuman lymphocytes than EQ and the antioxidant activity offour of them (ethoxyquin n-hexanoate ethoxyquin complexwith quercetin ethoxyquin L-ascorbate and ethoxyquinsalicylate) was comparable with that of EQ [79ndash82] Theresults obtained indicate that their use as antioxidants maybe considered

7 Food Safety Aspect

EQ safety has been under consideration for many yearsThe level of this antioxidant in animal feeds should not behigher than 150 ppm (US Food and Drug Administrationpermissions) The approved uses of ethoxyquin in animalfeeds are addressed in theCode of Federal Regulations (CFR)Title 21 Parts 573380 and 573400 and established tolerancesare in Part 172140 On the one hand the observed adversehealth effects (firstly in dogs) could be caused by the factthat the animals ate a lot of feed containing EQ but onthe other hand it could also be the result of its excessiveamounts in the feed Ethoxyquin is added to animal feedeither directly or indirectly as a component of an ingredientFrom time to time FDA reminds industry about labeling and


safe use requirements for ethoxyquin but if it is added at theingredient level this is not always indicated

Another important safety issue is the presence of EQoxidation and EQ metabolism products in animal feed orin foods prepared from farmed animal meat de Koning[12] described main products of EQ oxidation which can beobserved in stored feeds or in fish meal EQ dimer (EQDM181015840-di(12-dihydro-6-ethoxy-224-trimethylquinoline) andquinone imine (QI 26-dihydro-224-trimethyl-6-quin-olone) Both compounds were shown to be potent antioxi-dants but they can also have detrimental effect especially sobecause the half-life of the dimer was considerably greaterthan that of EQ [26 28] In the recent studies no adversetoxicological effects of EQDM in terms of kidney and liverfunction were observed in in vivo experiments with F344rats exposed for 90 days to the compound [37] On theother hand Augustyniak et al [83] showed that EQDMsimilarly as EQ was cytotoxic and genotoxic to humanlymphocytes Toxicity of QI has not been studied yet but theresults obtained by the authors indirectly indicated that thecompound could be cytotoxic to human cells

The levels of the parent compound (EQ) in meat offarmed animals are usually lower than MRL (MaximumResidue Level) [20 84] but EQ oxidation products areusually not controlled It was shown that EQDM and otherEQ residues can be present in different animal tissues [2324 26 28 37] In the studies of Bohne et al [23] inwhich Atlantic salmons were fed for 12 weeks with the feedcontaining this antioxidant four compounds were identifiedin their muscles parent EQ (6-ethoxy-12-dihydro-224-trimethylquinoline) deethylated EQ (6-hydroxy-224-trim-ethyl-12-dihydroquinoline) quinone imine (26-dihydro-224-trimethyl-6-quinolone QI) and EQ dimer (181015840-di(12-dihydro-6-ethoxy-224-trimethylquinoline EQDM) It wasalso shown that the concentration of EQ in fish muscle wasproportional to the duration of exposure and the level ofEQ in the feed [23] The same linear increase was seen forEQDM the main metabolite of EQ and the sum of EQ andEQDM Bohne et al [23] found that the level of EQ andits metabolites in fish muscle could be predicted from thelevel of dietary EQ and then controlled but because it isnot the only factor which may affect the levels of EQ andits metabolites in the salmon tissue (others for example arefish size and age) the concentration of EQ and EQDM infish ready for consumptionmay be higher than that observedin their studies In their experiments it was shown thatthe elimination of EQ from salmon was concurrent withsignificant increase in the level of EQDM and they concludedthat mandatory 14 days of depuration were not sufficient forelimination of EQ residuesmdashit is mainly because EQDM ischaracterized by the considerably longer half-life than that ofEQ Moreover EQDM accounted for 99 of the sum of thetwo compounds (EQandEQDM) and its toxicological effectsin animals and humans are unknown EQ and EQ dimerwere also detected in similar amounts not only in Atlanticsalmon but also in other commercially important species offarmed fishes (halibut rainbow trout) by Lundebye et al [24]They found that in Atlantic salmon halibut and rainbowtrout the concentration of EQDM was more than 10-fold

higher than that of EQThe authors estimated that consumerexposure to EQ from a single portion (300 g) of skinned-fillets of different species of farmed fish could amount up to15 of the ADI In the light of data concerning the presenceof EQDM in the body of farmed fishesand providing that EQdimer was included in the ADI the EQ and EQDM intakefrom a single portion of Atlantic salmon would be close toADI [24] Farmed fish is probably themajor source of EQ andits residues for European consumers (its use as a food additiveis forbidden) In our opinion however both fish and otherfarmed animals for example chickens should be controlledfor the presence of not only EQ but also EQDM its mainoxidation product

8 Conclusion

Ethoxyquin has been used as an antioxidant in animal feedfor several decades anddespite the search for new compoundsthat could be used as free radical scavengers it is still themosteffective antioxidant The negative health effects in domesticanimals fed with EQ containing feed were observed someyears ago but the presence of its approved doses should notbe hazardous Toxicity andmutagenicity of EQwere observedin in vivo and in vitro studies showing its potential harmfuleffects This makes it very important to label all products andingredients to which EQ is added and to comply with therecommended doses Additionally the results of the studieson products of EQ oxidation especially EQDM detected infarmed animal tissues indicate that it should be under controland some regulations should be introduced

Conflict of Interests

The authors declare that there is no conflict of interests

References

[1] H Babich ldquoButylated hydroxytoluene (BHT) a reviewrdquo Envi-ronmental Research vol 29 no 1 pp 1ndash29 1982

[2] R Kahl ldquoSynthetic antioxidants biochemical actions and inter-ference with radiation toxic compounds chemical mutagensand chemical carcinogensrdquo Toxicology vol 33 no 3-4 pp 185ndash228 1984

[3] G M Williams M J Iatropoulos and J Whysner ldquoSafetyassessment of butylated hydroxyanisole and butylated hydrox-ytoluene as antioxidant food additivesrdquo Food and ChemicalToxicology vol 37 no 9-10 pp 1027ndash1038 1999

[4] A A M Botterweck H Verhagen R A Goldbohm JKleinjans and P A van den Brandt ldquoIntake of butylatedhydroxyanisole and butylated hydroxytoluene and stomachcancer risk results from analyses in the Netherlands cohortstudyrdquo Food and Chemical Toxicology vol 38 no 7 pp 599ndash605 2000

[5] F M Brandao ldquoContact dermatitis to ethoxyquinrdquo ContactDermatitis vol 9 no 3 article 240 1983

[6] C Savini R Morelli E Piancastelli and S Restani ldquoContactdermatitis due to ethoxyquinrdquo Contact Dermatitis vol 21 no5 pp 342ndash343 1989

[7] D A Dzanis ldquoSafety of ethoxyquin in dog foodsrdquo Journal ofNutrition vol 121 no 11 pp S163ndash164 1991


[8] K Alanko R Jolanki T Estlander and L Kanerva ldquoOccu-pational rsquomultivitamin allergyrsquo caused by the antioxidantethoxyquinrdquo Contact Dermatitis vol 39 no 5 pp 263ndash2641998

[9] A Rodrıguez-Trabado J Miro I Balague and R GuspildquoHypersensitivity to the antioxidant ethoxyquinrdquoActas Dermo-Sifiliograficas vol 98 no 8 p 580 2007

[10] A D Little ldquoEthoxyquin national toxicology programexecutive summary of safety and toxicity informationrdquoChemical Committee Draft Report Ethoxyquin CAS Number91-53-2 1990 httpntpniehsnihgovntphtdocschem back-groundexsumpdfethoxyquin 508pdf

[11] US Food and Drug Administration Animal and VeterinaryldquoFDA requests that ethoxyquin levels be reduced in dogfoodsrdquo 1997 httpwwwfdagovAnimalVeterinaryNewsEv-entsCVMUpdatesucm127828htm

[12] A J de Koning ldquoThe antioxidant ethoxyquin and its analoguesa reviewrdquo International Journal of Food Properties vol 5 no 2pp 451ndash461 2002

[13] I Drewhurst ldquoEthoxyquin JMPR Evaluationsrdquo 1998 httpwwwinchemorgdocumentsjmprjmpmonov098pr09htm

[14] P K Gupta and A Boobis ldquoEthoxyquin (Addendum)rdquo 2005httpwwwinchemorgdocumentsjmprjmpmonov2005pr-10pdf

[15] G Dorey B Lockhart P Lestage and P Casara ldquoNew quino-linic derivatives as centrally active antioxidantsrdquo Bioorganic ampMedicinal Chemistry Letters vol 10 no 9 pp 935ndash939 2000

[16] A Błaszczyk R Osiecka and J Skolimowski ldquoInductionof chromosome aberrations in cultured human lymphocytestreated with ethoxyquinrdquo Mutation Research vol 542 no 1-2pp 117ndash128 2003

[17] A Błaszczyk and J Skolimowski ldquoComparative analysis of cyto-toxic genotoxic and antioxidant effects of 2247-tetramethyl-1234-tetrahydroquinoline and ethoxyquin on human lympho-cytesrdquoChemico-Biological Interactions vol 162 no 1 pp 70ndash802006

[18] National Library of Medicine HSDB Database ldquoEthoxyquinCASRN 91-53-2rdquo 2003 httptoxnetnlmnihgovcgi-binsissearchrdbs+hsdbterm+DOCNO+400

[19] A Błaszczyk and J Skolimowski ldquoSynthesis and studies onantioxidants ethoxyquin (eq) and its derivativesrdquoActa PoloniaePharmaceuticamdashDrug Research vol 62 no 2 pp 111ndash115 2005

[20] A Hobson Frohock ldquoResidues of ethoxyquin in poultry tissuesand eggsrdquo Journal of the Science of Food and Agriculture vol 33no 12 pp 1269ndash1274 1982

[21] L T Burka J M Sanders and H B Matthews ldquoComparativemetabolism and disposition of ethoxyquin in rat and mouse IIMetabolismrdquo Xenobiotica vol 26 no 6 pp 597ndash611 1996

[22] P He and R G Ackman ldquoResidues of ethoxyquin andethoxyquin dimer in ocean-farmed salmonids determined byhigh-pressure liquid chromatographyrdquo Journal of Food Sciencevol 65 no 8 pp 1312ndash1314 2000

[23] V J B Bohne A K Lundebye and K Hamre ldquoAccumulationand depuration of the synthetic antioxidant ethoxyquin in themuscle of Atlantic salmon (Salmo salar L)rdquo Food and ChemicalToxicology vol 46 no 5 pp 1834ndash1843 2008

[24] A K Lundebye H Hove A Mage V J B Bohne and KHamre ldquoLevels of synthetic antioxidants (ethoxyquin butylatedhydroxytoluene and butylated hydroxyanisole) in fish feed andcommercially farmed fishrdquo Food Additives and Contaminants AChemistry Analysis Control Exposure and Risk Assessment vol27 no 12 pp 1652ndash1657 2010

[25] JMPR ldquoPesticide residues in foodmdash2005 Report of the JointMeeting of the FAO Panel of Experts on Pesticide Residuesin Food and the Environment and the WHO Core Assess-ment Group on Pesticide Residuesrdquo JMPR Report Ethoxyquin035 2005 httpwwwfaoorgagAGPAGPPPesticidJMPRDownload99 eva14Ethoxyquinpdf

[26] V J Berdikova Bohne H Hove and K Hamre ldquoSimul-taneous quantitative determination of the synthetic antioxi-dant ethoxyquin and its major metabolite in atlantic salmon(Salmo salar L) ethoxyquin dimer by reversed-phase high-performance liquid chromatography with fluorescence detec-tionrdquo Journal of AOAC International vol 90 no 2 pp 587ndash5972007

[27] J U Skaare and S O Roald ldquoEthoxyquin (EMQ) residues inAtlantic salmon measured by fluorimetry and gas chromatog-raphy (GLC)rdquo Nordisk Veterinaermedicin vol 29 no 4-5 pp232ndash236 1977

[28] V J B Bohne K Hamre and A Arukwe ldquoHepatic biotrans-formation and metabolite profile during a 2-week depurationperiod in Atlantic Salmon fed graded levels of the syntheticantioxidant ethoxyquinrdquo Toxicological Sciences vol 93 no 1pp 11ndash21 2006

[29] V J B Bohne K Hamre and A Arukwe ldquoHepatic metabolismphase I and II biotransformation enzymes in Atlantic salmon(Salmo Salar L) during a 12 week feeding period with gradedlevels of the synthetic antioxidant ethoxyquinrdquo Food andChemical Toxicology vol 45 no 5 pp 733ndash746 2007

[30] T D Bucheli and K Fent ldquoInduction of cytochrome P450as a biomarker for environmental contamination in aquaticecosystemsrdquo Critical Reviews in Environmental Science andTechnology vol 25 no 3 pp 201ndash268 1995

[31] A Arukwe L Forlin and A Goksoslashyr ldquoXenobiotic and steroidbiotransformation enzymes in Atlantic salmon (Salmo salar)liver treated with an estrogenic compound 4-nonylphenolrdquoEnvironmental Toxicology and Chemistry vol 16 no 12 pp2576ndash2583 1997

[32] T M Buetler E P Gallagher C Wang D L Stahl J D Hayesand D L Eaton ldquoInduction of phase I and phase II drug-detabolizing enzyme mRNA protein and activity by BHAethoxyquin and oltiprazrdquo Toxicology and Applied Pharmacol-ogy vol 135 no 1 pp 45ndash57 1995

[33] J D Hayes D J Pulford E M Ellis et al ldquoRegulation of ratglutathione S-transferaseA5 by cancer chemopreventive agentsmechanisms of inducible resistance to aflatoxin B1rdquo Chemico-Biological Interactions vol 111-112 pp 51ndash67 1998

[34] K L Henson G Stauffer and E P Gallagher ldquoInductionof glutathione S-transferase activity and protein expressionin brown bullhead(Ameiurus nebulosus) liver by ethoxyquinrdquoToxicological Sciences vol 62 no 1 pp 54ndash60 2001

[35] T K Bammler D H Slone and D L Eaton ldquoEffects of dietaryoltipraz and ethoxyquin on aflatoxin B1 biotransformation innon-human primatesrdquo Toxicological Sciences vol 54 no 1 pp30ndash41 2000

[36] R Kahl ldquoElevation of hepatic epoxide hydratase activity byethoxyquin is due to increased synthesis of the enzymerdquoBiochemical and Biophysical Research Communications vol 95no 1 pp 163ndash169 1980

[37] R Oslashrnsrud A Arukwe V Bohne N Pavlikova and A KLundebye ldquoInvestigations on the metabolism and potentiallyadverse effects of ethoxyquin dimer a major metabolite of thesynthetic antioxidant ethoxyquin in salmon musclerdquo Journal ofFood Protection vol 74 pp 1574ndash1580 2011


[38] L Taimr M Prusikova and J Pospisil ldquoAntioxidants andstabilizers 113 Oxidation-products of the antidegradantethoxyquinrdquo Angewandte Makromolekulare Chemie vol 190pp 53ndash65 1991

[39] L Taimr ldquoStudy of the mechanism of the antioxidant action ofethoxyquinrdquoAngewandteMakromolekulare Chemie vol 217 pp119ndash128 1994

[40] C Lauridsen K Jakobsen and T K Hansen ldquoThe influence ofdietary ethoxyquin on the vitamin E status in broilersrdquo Archivfur Tierernahrung vol 47 no 3 pp 245ndash254 1995

[41] D R Brannegan Analysis of ethoxyquin and its oxidation prod-ucts using supercritical fluid extraction and high performanceliquid chromatography with chemiluminescent nitrogen detec-tion [thesis] Faculty of the Virginia Polytechnic Institute andState University in Partial Fulfillment of the Requirements ofthe Degree of Master of Science in Chemistry 2000 httpscholarlibvteduthesesavailableetd-03302000-20440044

[42] L Taimr M Smelhausova and M Prusikova ldquoThe reactionof 1-cyano-1-methylethyl radical with antidegradant ethoxyquinand its aminyl and nitroxide derivativesrdquo Angewandte Makro-molekulare Chemie vol 206 pp 199ndash207 1993

[43] A J de Koning and G van der Merwe ldquoDetermination ofethoxyquin and two of its oxidation products in fish meal bygas chromatographyrdquoThe Analyst vol 117 no 10 pp 1571ndash15761992

[44] P He and R G Ackman ldquoHPLC determination of ethoxyquinand its major oxidation products in fresh and stored fish mealsand fish feedsrdquo Journal of the Science of Food and Agriculturevol 80 pp 10ndash16 2000

[45] S Thorisson F Gunstone and R Hardy ldquoThe antioxidantproperties of ethoxyquin and of some of its oxidation productsin fish oil and mealrdquo Journal of the American Oil ChemistsrsquoSociety vol 69 pp 806ndash809 1992

[46] P He andR G Ackman ldquoPurification of ethoxyquin and its twooxidation productsrdquo Journal of Agricultural and FoodChemistryvol 48 no 8 pp 3069ndash3071 2000

[47] H W Renner ldquoAntimutagenic effect of an antioxidant inmammalsrdquoMutation Research vol 135 no 2 pp 125ndash129 1984

[48] H W Renner and M Knoll ldquoAntimutagenic effects on malegerm cells ofmicerdquoMutation Research vol 140 no 2-3 pp 127ndash129 1984

[49] D Guyonnet C Belloir M Suschetet M H Siess and AM LeBon ldquoAntimutagenic activity of organosulfur compounds fromAllium is associated with phase II enzyme inductionrdquoMutationResearch vol 495 no 1-2 pp 135ndash145 2001

[50] A Stankiewicz E Skrzydlewska and M Makieła ldquoEffectsof amifostine on liver oxidative stress caused by cyclophos-phamide administration to ratsrdquo Drug Metabolism and DrugInteractions vol 19 no 2 pp 67ndash82 2002

[51] S Ray B Pandit S D Ray S Das and S ChakrabortyldquoCyclophosphamide induced lipid peroxidation and changesin cholesterol content protective role of reduced glutathionerdquoInternational Journal of PharmTech Research vol 2 no 1 pp704ndash718 2010

[52] J R P Cabral and G E Neal ldquoThe inhibitory effects ofethoxyquin on the carcinogenic action of aflatoxin B1 in ratsrdquoCancer Letters vol 19 no 2 pp 125ndash132 1983

[53] MMManson J A Green andH E Driver ldquoEthoxyquin aloneinduces preneoplastic changes in rat kidney whilst preventinginduction of such lesions in liver by aflatoxin B1rdquo Carcinogene-sis vol 8 no 5 pp 723ndash728 1987

[54] E A Decker ldquoPhenolics prooxidants or antioxidantsrdquo Nutri-tion Reviews vol 55 no 11 pp 396ndash398 1997

[55] Y Sakihama M F Cohen S C Grace and H Yamasaki ldquoPlantphenolic antioxidant and prooxidant activities phenolics-induced oxidative damage mediated by metals in plantsrdquoToxicology vol 177 no 1 pp 67ndash80 2002

[56] J U Skaare and T Henriksen ldquoFree-radical formation inantioxidant ethoxyquinrdquo Journal of the Science of Food andAgriculture vol 26 pp 1647ndash1654 1975

[57] C S Wilcox and A Pearlman ldquoChemistry and antihyperten-sive effects of tempol and other nitroxidesrdquo PharmacologicalReviews vol 60 no 4 pp 418ndash469 2008

[58] G H McIntosh J S Charnock P H Phillips and G JBaxter ldquoAcute intoxication of marmosets and rats fed highconcentrations of the dietary antioxidant rdquoethoxyquin 66rdquordquoAustralian Veterinary Journal vol 63 no 11 pp 385ndash386 1986

[59] N Ito S Fukushima and H Tsuda ldquoCarcinogenicity andmodification of the carcinogenic response by BHA BHT andother antioxidantsrdquo Critical Reviews in Toxicology vol 15 no 2pp 109ndash150 1985

[60] Y Yamashita T Katagiri N Pirarat K Futami M Endo andM Maita ldquoThe synthetic antioxidant ethoxyquin adverselyaffects immunity in tilapia (Oreochromis niloticus)rdquoAquacultureNutrition vol 15 no 2 pp 144ndash151 2009

[61] G E Neal D J Judah G G Hard and N Ito ldquoDifferences inethoxyquin nephrotoxicity betweenmale and female F344 ratsrdquoFood and Chemical Toxicology vol 41 no 2 pp 193ndash200 2003

[62] V Y Leong and T P Brown ldquoToxicosis in broiler chicks dueto excess dietary ethoxyquinrdquo Avian Diseases vol 36 no 4 pp1102ndash1106 1992

[63] J Wang Q Ai K Mai et al ldquoEffects of dietary ethoxyquinon growth performance and body composition of large yellowcroaker Pseudosciaena croceardquo Aquaculture vol 306 no 1ndash4pp 80ndash84 2010

[64] M E Hernandez J L Reyes C Gomez-Lojero M S Sayave-dra and E Melendez ldquoInhibition of the renal uptake of p-aminohippurate and tetraethylammonium by the antioxidantethoxyquin in the ratrdquo Food and Chemical Toxicology vol 31no 5 pp 363ndash367 1993

[65] J L Reyes M Elisabeth Hernandez E Melendez and CGomez-Lojero ldquoInhibitory effect of the antioxidant ethoxyquinon electron transport in the mitochondrial respiratory chainrdquoBiochemical Pharmacology vol 49 no 3 pp 283ndash289 1995

[66] P E Joner ldquoButylhydroxyanisol (BHA) butylhydroxytoluene(BHT) and ethoxyquin (EMQ) tested for mutagenicityrdquo ActaVeterinaria Scandinavica vol 18 no 2 pp 187ndash193 1977

[67] T Ohta MMoriya Y Kaneda et al ldquoMutagenicity screening offeed additives in the microbial systemrdquoMutation Research vol77 no 1 pp 21ndash30 1980

[68] E Zeiger ldquoMutagenicity of chemicals added to foodsrdquoMutationResearch vol 290 no 1 pp 53ndash61 1993

[69] A Rannug U Rannug and C Ramel ldquoGenotoxic effects ofadditives in synthetic elastomers with special consideration tothe mechanism of action of thiurams and dithiocarbamatesrdquoProgress in Clinical and Biological Research vol 141 pp 407ndash4191984

[70] B S Reddy D Hanson L Mathews and C Sharma ldquoEffectof micronutrients antioxidants and related compounds on themutagenicity of 32rsquo-dimethyl-4-aminobiphenyl a colon andbreast carcinogenrdquo Food and Chemical Toxicology vol 21 no2 pp 129ndash132 1983


[71] A Błaszczyk and J Skolimowski ldquoApoptosis and cytotoxicitycaused by ethoxyquin and two of its saltsrdquo Cellular and Molec-ular Biology Letters vol 10 no 1 pp 15ndash21 2005

[72] A Błaszczyk ldquoDNA damage induced by ethoxyquin in humanperipheral lymphocytesrdquo Toxicology Letters vol 163 no 1 pp77ndash83 2006

[73] J J Skolimowski B Cieslinska M Zak R Osiecka and ABłaszczyk ldquoModulation of ethoxyquin genotoxicity by free radi-cal scavengers andDNAdamage repair in human lymphocytesrdquoToxicology Letters vol 193 no 2 pp 194ndash199 2010

[74] J J PGille P PasmanCGM vanBerkel andH Joenje ldquoEffectof antioxidants on hyperoxia-induced chromosomal breakagein Chinese hamster ovary cells protection by carnosinerdquoMutagenesis vol 6 no 4 pp 313ndash318 1991

[75] T H Rabbitts ldquoChromosomal translocations in human cancerrdquoNature vol 372 no 6502 pp 143ndash149 1994

[76] A J de Koning ldquoAn antioxidant for fish mealrdquo Republic ofSouth Africa Patent 970894 1997

[77] National Toxicology Program (NTP) Toxicology andCarcinogenesis Studies of 12-Dihydro-224-Trimethylquinoline(CAS no 147-47-7) in F344N Rats and B6C3DF

1

mice (DermalStudies) and the InitiationPromotion (Dermal Study) inFemale Sencar Mice Technical Report Series no 456US Department of Health and Human Services 1997httpntpniehsnihgovntphtdocsLT rptstr456pdf

[78] K Sitarek and A Sapota ldquoMaternal-fetal distribution andprenatal toxicity of 224-trimethyl-12-dihydroquinoline in theratrdquo Birth Defects Research Part BmdashDevelopmental and Repro-ductive Toxicology vol 68 no 4 pp 375ndash382 2003

[79] A Błaszczyk J Skolimowski and A Materac ldquoGenotoxic andantioxidant activities of ethoxyquin salts evaluated by the cometassayrdquo Chemico-Biological Interactions vol 162 no 3 pp 268ndash273 2006

[80] A Błaszczyk and J Skolimowski ldquoApoptosis and cytotoxicitycaused by ethoxyquin salts in human lymphocytes in vitrordquoFood Chemistry vol 105 no 3 pp 1159ndash1163 2007

[81] A Błaszczyk and J Skolimowski ldquoEvaluation of the genotoxicand antioxidant effects of two novel feed additives (ethoxyquincomplexes with flavonoids) by the comet assay and micronu-cleus testrdquo Food Additives and Contaminants vol 24 no 6 pp553ndash560 2007

[82] A Błaszczyk and J Skolimowski ldquoPreparation of ethoxyquinsalts and their genotoxic and antioxidant effects on humanlymphocytesrdquo Arkivoc vol 2007 no 6 pp 217ndash229 2007

[83] A Augustyniak A Niezgoda J Skolimowski R Kontek andA Błaszczyk ldquoCytotoxicity and genotoxicity of ethoxyquindimersrdquo Bromatologia i Chemia Toksykologiczna vol 45 pp228ndash234 2012

[84] Y Aoki A Kotani N Miyazawa et al ldquoDetermination ofethoxyquin by high-performance liquid chromatography withfluorescence detection and its application to the survey ofresidues in food products of animal originrdquo Journal of AOACInternational vol 93 no 1 pp 277ndash283 2010

Submit your manuscripts athttpwwwhindawicom

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Microbiology


60 ppm) would provide antioxidant protection for dog food[11]

Ethoxyquin is also known as Santoquin SantoflexQuinol It was originally developed in rubber industry toprevent rubber from cracking due to oxidation of isoprene[12] The Monsanto Company (USA) taking into account itshigh antioxidant efficiency and stability as well as low costsof synthesis refined it later for use as a preservative in animalfeeds because it protects against lipid peroxidation andstabilizes fat soluble vitamins (A E) Presently ethoxyquin isused primarily as an antioxidant in canned pet food and infeed intended for farmed fish or poultry

The use of ethoxyquin is not permitted in foods intendedfor human except preserving powdered paprika and chilicolour and using it as an antiscald agent in pears and apples(inhibition of ldquobrown spotsrdquo development)However becauseEQ is used as a feed antioxidant it can be also found in otherproducts intended for human consumption like fishmeal fishoils and other oils fats and meat (Table 1) An acceptabledaily intake (ADI) of EQ for human (0ndash0005mg kgminus1 bw)based on the results obtained from studies on dogs wasestablished in 1998 [13 14]

This paper presents characteristics of ethoxyquin withregard to its properties metabolism toxicity possible car-cinogenicity and antioxidant activity

2 Physical and Chemical Properties ofEthoxyquin (EQ)

For the first time EQ was synthesized in 1921 by Knoevenagel[15]The synthesis was based on condensation of aniline withmolecules of acetone or its analogues [15 16] Błaszczyk etal [16] synthesized EQ from p-phenetidine (4-ethoxyaniline)and diacetone alcohol in the presence of p-toluenesulfonicacid or iodine Pure ethoxyquin (EQ 6-ethoxy-12-dihydro-224-trimethylquinoline CAS number 91-53-2 Figure 1) isa light yellow liquid but it changes color to brown if itis exposed to oxygen [12] It also tends to polymerize onexposure to light and air The scent of EQ is described asmercaptan like As a nonpolar substance EQ is soluble onlyin organic solvents Some of the additional properties of EQare presented in Table 2

3 Biotransformation of EQ

31 Animals Ethoxyquin is rapidly absorbed from gastroin-testinal tract of laboratory animals like rats and mice Peakblood concentration of the compound is observed within1 h Distribution of EQ in animal body is similar when itis administered orally and intravenously Small amounts ofparent EQ were detected in liver kidney and adipose tissueand fish muscles [20ndash24] It is excreted predominantly asmetabolites via urine Metabolism of EQ was studied in ratsmice dogs chickens and fish as well as in plants [13 21 25]It is not fully described but some metabolites were identified(Table 3) The most important EQ metabolites observed inrat urine and bile result from O-deethylation at position 6-Cand then conjugation with sulphate or glucuronide residues

Table 1 Permitted amounts of EQ in different products approvedby FDAlowast

Product Dose [ppm]Animal feed 150Spices 150Uncooked fat of meat from animals (except poultry) 5Uncooked liver and fat of poultry 3Uncooked muscle meat of animals 05Poultry eggs 05Milk 0Pear 3lowastAccording to the Code of Federal Regulations (CFR) Title 21 Parts 573380573400 172140

The other metabolic pathways include hydroxylation andglucuronidation at position 8-C deethylation at 6-C andepoxidation between positions 3-C and 4-C [21] The mainmetabolites observed may be different depending on ani-mal species In mice mainly glucuronide metabolites weredetected while in rats those result from conjugation of EQwith sulfate

In the studies of Bohne et al [23 26] parent EQ dem-ethylated EQ (DEQ) quinone imine (QI) and EQ dimer(EQDM) were observed in salmonid fish after long-termdietary exposure to EQ It was in agreement with the resultsobtained earlier by Skaare and Roald [27] EQ is consideredas a model inducer of phase II enzymes involved in themetabolism of xenobiotics but influence of EQ on phase Ienzyme gene transcript levels was also observed [28 29]Thekey role in mediating phase I reactions (eg oxidation orreduction) producingmore hydrophilic compounds is playedby the CYP (cytochrome P450) enzyme family Bohne et al[28 29] observed the alteration of CYP3A gene expressionan increase in the amount of CYP3A transcripts was detectedin salmon after feeding them with the diet containing EQ atthe highest dose used (1800mg kgminus1) The authors speculatethat EQ may regulate CYP3 gene expression by interactionfor example with pregnane X nuclear receptor (PXR) whosefunction is to sense the presence of toxic xenobiotics andin response enhance the expression of proteins involvedin their detoxification On the other hand CYP1A1 geneexpression which was described as an exposure biomarkerto both endogenous and exogenous compounds [30] wasnot increased after dietary exposure of salmonid fish to EQand during the depuration period a trend toward down-regulation was noted [28 29] Such an effect was observeddespite the increase in the expression of AhR mRNA (AhRcytosolic transcription factor responsible for changes in genetranscription) This effect can be explained in several waysFor example the parent EQ may bind CYP1A1 proteinand as a result may inhibit the gene expression and activ-ity of protein [31] Hepatic antioxidant response elements(ARE) or AhR repressor (AhRR) together with basic-helix-loop-helix-PAS (Per-AhRARNT-Sim homology sequence)of transcription factor usually associated with each other toform heterodimers (AhRARNT or AhRRARNT) may be








119863



119862


b












NH

CH3

OO

O

CH3

CH3

SOH


NH

CH3O

CH3

CH3

Glu


CH2

CH3

CH3

OH

NH

S OOO

OH


EtCH3

NH

O

OGlu

CH3

CH3


CH3

N

O

SGCH3

CH3


Et

CH3

CH3

OH

NH

SO

GH3C


Rats1Mice1

N

CH3

CH3

CH3

O


CH3

CH3

CH3

HO

NH


EtCH3

N

O

H3C

CH3

CH3

H3C

H3CEtO

NHlowast




Atlantic salmon2

Pears34


Table 3 Continued


EtCH3

N

N

O

CH3

CH3

Et OCH3

CH3

CH3



EtCH3

N

O

CH3

CH3

CH3


EtCH3

N

O

CH3


EtCH3

N

O

CH3

CH3H


EtCH3

N

O

CH3H








Ethoxyquin

NH

CH3

OH3C

CH3

CH3



NH

CH3

CH3

CH3

HO


CH3

CH3

CH3

NH

H


CH3

CH3

CH3

NHH3C


tetrahydroquinoline

H3C

H3C

H3C

CH3

CH3

NH

HHO







2

O2


2

O2













Anemia Marmosets1


Lethargy Rabbits3









50





50











Antioxidantactivity




50









8 Conclusion




References

















































































1
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








119863



119862


b












NH

CH3

OO

O

CH3

CH3

SOH


NH

CH3O

CH3

CH3

Glu


CH2

CH3

CH3

OH

NH

S OOO

OH


EtCH3

NH

O

OGlu

CH3

CH3


CH3

N

O

SGCH3

CH3


Et

CH3

CH3

OH

NH

SO

GH3C


Rats1Mice1

N

CH3

CH3

CH3

O


CH3

CH3

CH3

HO

NH


EtCH3

N

O

H3C

CH3

CH3

H3C

H3CEtO

NHlowast




Atlantic salmon2

Pears34


Table 3 Continued


EtCH3

N

N

O

CH3

CH3

Et OCH3

CH3

CH3



EtCH3

N

O

CH3

CH3

CH3


EtCH3

N

O

CH3


EtCH3

N

O

CH3

CH3H


EtCH3

N

O

CH3H








Ethoxyquin

NH

CH3

OH3C

CH3

CH3



NH

CH3

CH3

CH3

HO


CH3

CH3

CH3

NH

H


CH3

CH3

CH3

NHH3C


tetrahydroquinoline

H3C

H3C

H3C

CH3

CH3

NH

HHO







2

O2


2

O2













Anemia Marmosets1


Lethargy Rabbits3









50





50











Antioxidantactivity




50









8 Conclusion




References

















































































1
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




NH

CH3

OO

O

CH3

CH3

SOH


NH

CH3O

CH3

CH3

Glu


CH2

CH3

CH3

OH

NH

S OOO

OH


EtCH3

NH

O

OGlu

CH3

CH3


CH3

N

O

SGCH3

CH3


Et

CH3

CH3

OH

NH

SO

GH3C


Rats1Mice1

N

CH3

CH3

CH3

O


CH3

CH3

CH3

HO

NH


EtCH3

N

O

H3C

CH3

CH3

H3C

H3CEtO

NHlowast




Atlantic salmon2

Pears34


Table 3 Continued


EtCH3

N

N

O

CH3

CH3

Et OCH3

CH3

CH3



EtCH3

N

O

CH3

CH3

CH3


EtCH3

N

O

CH3


EtCH3

N

O

CH3

CH3H


EtCH3

N

O

CH3H








Ethoxyquin

NH

CH3

OH3C

CH3

CH3



NH

CH3

CH3

CH3

HO


CH3

CH3

CH3

NH

H


CH3

CH3

CH3

NHH3C


tetrahydroquinoline

H3C

H3C

H3C

CH3

CH3

NH

HHO







2

O2


2

O2













Anemia Marmosets1


Lethargy Rabbits3









50





50











Antioxidantactivity




50









8 Conclusion




References

















































































1
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 3 Continued


EtCH3

N

N

O

CH3

CH3

Et OCH3

CH3

CH3



EtCH3

N

O

CH3

CH3

CH3


EtCH3

N

O

CH3


EtCH3

N

O

CH3

CH3H


EtCH3

N

O

CH3H








Ethoxyquin

NH

CH3

OH3C

CH3

CH3



NH

CH3

CH3

CH3

HO


CH3

CH3

CH3

NH

H


CH3

CH3

CH3

NHH3C


tetrahydroquinoline

H3C

H3C

H3C

CH3

CH3

NH

HHO







2

O2


2

O2













Anemia Marmosets1


Lethargy Rabbits3









50





50











Antioxidantactivity




50









8 Conclusion




References

















































































1
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Ethoxyquin

NH

CH3

OH3C

CH3

CH3



NH

CH3

CH3

CH3

HO


CH3

CH3

CH3

NH

H


CH3

CH3

CH3

NHH3C


tetrahydroquinoline

H3C

H3C

H3C

CH3

CH3

NH

HHO







2

O2


2

O2













Anemia Marmosets1


Lethargy Rabbits3









50





50











Antioxidantactivity




50









8 Conclusion




References

















































































1
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









Anemia Marmosets1


Lethargy Rabbits3









50





50











Antioxidantactivity




50









8 Conclusion




References

















































































1
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










Antioxidantactivity




50









8 Conclusion




References

















































































1
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






8 Conclusion




References

















































































1
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










































































1
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











































1
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









1
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Review Article Ethoxyquin: An Antioxidant Used in Animal Feeddownloads.hindawi.com/journals/ijfs/2013/585931.pdf · Review Article Ethoxyquin: An Antioxidant Used in Animal Feed