Zeaxanthin: Review of Toxicological Data and Acceptable Daily Intake

Review ArticleZeaxanthin Review of Toxicological Data andAcceptable Daily Intake

James A Edwards

NIC-RDHN Toxicology and Kinetics DSM Nutritional Products Ltd Wurmisweg 576 4303 Kaiseraugst Switzerland

Correspondence should be addressed to James A Edwards james-aedwardsdsmcom

Received 29 October 2015 Accepted 6 December 2015

Academic Editor Joan E Roberts

Copyright copy 2016 James A Edwards This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Zeaxanthin is a nutritional carotenoid with a considerable amount of safety data based on regulatory studies which form thebasis of its safety evaluation Subchronic OECD guideline studies with mice and rats receiving beadlet formulations of highpurity synthetic zeaxanthin in the diet at dosages up to 1000mgkg body weight (bw)day and in dogs at over 400mgkg bwdayproduced no adverse effects or histopathological changes In developmental toxicity studies therewas no evidence of fetal toxicity orteratogenicity in rats or rabbits at dosages up to 1000 or 400mgkg bwday respectively Formulated zeaxanthin was not mutagenicor clastogenic in a series of in vitro and in vivo tests for genotoxicity A 52-week chronic oral study in Cynomolgusmonkeys at dosesof 02 and 20mgkg bwday mainly designed to assess accumulation and effects in primate eyes showed no adverse effects In a rattwo-generation study the NOAEL was 150mgkg bwday In 2012 this dosage was used by EFSA (NDA Panel) in association witha 200-fold safety factor to propose an Acceptable Daily Intake equivalent to 53mgday for a 70 kg adult The requested use level of2mgday was ratified by the EU Commission

1 Introduction

Zeaxanthin (3 31015840-dihydroxy-120573-carotene CAS number 144-68-3) is a nutritional carotenoid in a category referred toas xanthophylls Zeaxanthin is structurally closely similar tolutein The intake of both carotenoids in the human dietis regarded as healthy with these components reflecting anadequate intake of fruit and vegetables

Lutein as a human dietary supplement is often obtainedas an extract from Tagetes (marigold) and the extract alwayscontains some zeaxanthin Zeaxanthin itself on the otherhand tends to be produced from both biological sources andin a highly pure form synthetically The predominant zeax-anthin stereoisomer in nature and consequently in the dietis the 3R 3R1015840-stereoisomer which is also the predominantstereoisomer of synthetic zeaxanthin (Figure 1)

In normal human food sources lutein ismore abundantlypresent than zeaxanthin for example in spinach but thereare other food sources with a relatively higher contentof zeaxanthin such as egg yolk corn (maize) or orangepepper [1 2] The usual dietary ratio of lutein zeaxanthin isapproximately 5 1 (Table 1) [3 4]

A closely related stereoisomer that is rarer than 3R 31015840Rzeaxanthin isomer in nature is the 3R 31015840S stereoisomercommonly referred to asmeso-zeaxanthinThis stereoisomerlike lutein and zeaxanthin is found in the human maculaand its source has been determined in primates fed with azeaxanthin-free diet to be derived from lutein [5 6]

In addition to being generally healthy and acting asantioxidants a specific protective activity exists in the eyesof primates In the primate eye in the center of the retinaan area known as the macula lutea is visible as a yellowspot due to the accumulation of the macular xanthophyllsThe presence of the xanthophyll carotenoids in the humanappears to be physiologically significant the concentrationof xanthophylls in the macula is the highest concentrationfound everywhere in the primate body Furthermore basedon filtration of potentially damaging light and quenchingof photochemically induced reactive oxygen species it isbelieved [7 8] that via these mechanisms lutein zeaxanthinand meso-zeaxanthin may contribute to reducing the riskof Age-Related Macular Degeneration (AMD) a leadingcause of irreversible loss of vision observed in western coun-tries

Hindawi Publishing CorporationJournal of OphthalmologyVolume 2016 Article ID 3690140 15 pageshttpdxdoiorg10115520163690140

2 Journal of Ophthalmology

HO

OH

OH

HO

OH

HO

OH

HO

3R 3998400R 6998400R-lutein

3S 3998400S-zeaxanthin

3R 3998400S-zeaxanthin (meso-zeaxanthin)

3R 3998400R-zeaxanthin

Figure 1 Structures for optical isomers of all-trans zeaxanthin and lutein

Table 1 Average intake of lutein and zeaxanthin by age group(Mohamedshah et al 1999) [3]

Age group Lutein(120583gday)

Zeaxanthin(120583gday)

Lutein Zeaxanthinratio

20ndash29 745 178 42 130ndash39 896 174 51 140ndash49 920 187 49 150ndash59 1053 182 58 160ndash69 1056 170 62 170+ 990 170 58 1

There is a considerable amount of safety data for zeax-anthin based mainly on routine regulatory studies with highpurity synthetic zeaxanthin manufactured by DSM Ltd(previously manufactured by F Hoffmann-La Roche Ltd)A series of in vitro and in vivo tests for genotoxicity havebeen undertaken as well as subchronic safety studies (13weeks in duration) by dietary exposure at high dosage levelsin mice rats and dogs Developmental toxicity studies havebeen undertaken in rats and rabbits a two-generation study

was performed on the rat and a chronic study of 52 weeksrsquoduration was performed on Cynomolgus monkeys ADME(absorption distribution metabolism and excretion) studieshave been undertaken These studies with zeaxanthin arereviewed here

Potentially data for certain closely related substancesmay have relevance or should be taken into considerationin the safety evaluation of zeaxanthin The inclusion of aferret study with the related xanthophyll 120573-cryptoxanthinon a read-across basis to address the question if zeaxanthinconsumption might have an adverse impact on cigarettesmokers is described Reference is also made to knownstudies with lutein and meso-zeaxanthin

Safety data from human intervention studies in whichsynthetic zeaxanthin has been supplemented of which theAREDSII study is by far the largest is also considered andthe apparent safe level of intake from these studies comparedwith that derived from the animal studies

The safety data for lutein have been evaluated by theEuropean Food Safety Authority (EFSA) Due to the closesimilarity of lutein and zeaxanthin it is probable that thetoxicology for the pure substances is very similar althoughit has to be remembered that within the eye a highly specific

Journal of Ophthalmology 3

Table 2 List of genotoxicity repeat dose and reproductive safety studies conducted with DSM-manufactured synthetic zeaxanthin based oninternational regulatory study designs

Safety studies Formulationnominal Concentration or dosage Result

ConcentrationGenotoxicity in vitro

Ames S typhimuriummutation assay Crystalline 0 24ndash1500 120583gplate NegativeGene mutation in V79 cells Crystalline 0 1ndash16 120583gmL NegativeUnscheduled DNA Synthesis (UDS) in rathepatocytes Crystalline 01ndash16120583gmL Negative

Human lymphocytes Crystalline 0 60 and 120120583gmL NegativeDose (mgkg bwday)

Genotoxicity assays in vivoMouse micronucleus 10 beadlet 0 445 89 and 178 Negative

Subchronic and chronic13-week oral (admix) in mice 10 beadlet 0 0 250 500 and 1000 NOAEL high dose13-week oral (admix) in rats 10 beadlet 0 0 250 500 and 1000 NOAEL high dose

13-week oral (feed cubes) in dogs 10 beadlet 0 123 204 and 422 males 0 104 238 and 442females NOAEL high dose

1-year oral (gavage) in monkeyszeaxanthin or lutein 10 beadlet 0 02 and 20 for zeaxanthin or lutein NOAEL high dose

Reproductive studiesTeratology oral (admix) in rats 10 beadlet 0 250 500 and 1000 NOAEL high doseTeratology oral (gavage) in rabbits Crystalline in oil 0 100 200 and 400 NOAEL high doseTwo-generation (admix) in rats 10 beadlet 0 0 50 150 and 500 NOAEL inter dose

biological stereoisomeric differentiation may occur Many ofthe analytical methods used in the past did not differentiatezeaxanthin and lutein such that the information on thedifferential occurrence of lutein and zeaxanthin in fruitsand vegetables for many years was incomplete The JointFAO (Food and Agriculture Organization of the UnitedNations)WHO (World Health Organization) Expert Com-mittee on Food Additives (JECFA) in 2006 [9] in their safetyevaluation of lutein and zeaxanthin defined a ldquogrouprdquo ADI(Acceptable Daily Intake) for lutein and zeaxanthin of 0ndash2mgkg bwday covering both substances

The toxicity of compounds can often be strongly influ-enced by their purity If coming from a natural source theother natural components or contaminants (eg pesticides)need to be taken into consideration If coming from chemicalsynthesis a representative batch of typical purity containingthe synthetic by-products that might be present needs tobe tested In the regulatory studies for zeaxanthin presentedhere coming from DSM Nutritional Products Ltd the zeax-anthin tested was in accord with the manufacturerrsquos purityspecification of at least 98 zeaxanthin (gt96 all-trans lt2cis) This high purity substance is marketed in a formulationdesigned to provide stability against oxidation and enhancebioavailability

The DSM studies reported here have been undertakenby a number of toxicologists and safety specialists over anumber of years and are described mainly in summary formThe individual study reports describe the results in detail Inthe regulatory processes to obtain approval for human use

the detailed reports are supplied to the respective regulatoryauthority

2 Methods Regulatory Animal Safety Studieswith Synthetic Zeaxanthin

An extensive array of conventional toxicology studies hasbeen undertaken with DSM synthetic zeaxanthin (Ro 01-9509) The studies undertaken by DSM were for the purposeof assessing safety from oral intake or for worker safetypurposes and are listed in Table 2

The toxicology studies undertaken byDSMwere predom-inantly undertaken using recognized international regulatoryguidelines and in particular respectiveOrganization for Eco-nomic Cooperation and Development (OECD) guidelinesThe OECD guideline stipulates for each study design howthe study should be performed with a detailed range ofstudy design requirements such as numbers of replicates oranimals the concentrations or dosages that are appropriate toensure sufficiently stringent testing details of the endpointsthat should be investigated and guidance on the evaluationof the data obtained

In the case of the two-generation rat study the keyregulatory study for the overall safety assessment the studywas designed to meet the known requirements of the OECD416 guidelines (22 January 2001) and the US FDA Centerfor Food Safety and Applied Nutrition Redbook 2000Toxicological Principles for the Safety of Food IngredientsIVC9a Guidelines for Reproduction Studies (20 July 2000)


Additionally the studies were undertaken following theprinciples of Good Laboratory Practice (GLP) In the caseof the two-generation rat study the study was conductedin accordance with the OCD GLP guideline and UK GLPguidelines as the study was conducted in UK Analysesundertaken to confirm the stability and concentrations ofzeaxanthin in the treated diet as well as analyses undertakento assess the concentration of zeaxanthin in plasma and liversamples were performed at the DSM test site in Switzerlandand performed in accordance with Swiss ordinance on GLP

Additionally in vivo studies were undertaken followingthe local national requirements on animal housing andanimal welfare requirements such as in the UK the HomeOffice ldquocode of practice for the housing and care of animalsused in scientific proceduresrdquo

Importantly the evaluation of zeaxanthin involved aspecial study on Cynomolgus monkeys which included arange of specific endpoints to investigate safety to the primateand human eye The Cynomolgus monkey has been shownto be an excellent model to investigate the induction anddose dependency of canthaxanthin crystal formation in theretina [10ndash13] Using similar procedures as described in thesepublications the study included indirect ophthalmoscopicexaminations performed using the Bonnoskop and directophthalmoscope and a contact lens biomicroscope Addi-tional evaluations were performed using the ophthalmic slitlamp biomicroscope in combination with wide-field cornealcontact fundus lenses Electroretinography (ERG)was under-taken at intervals during the study Terminal eye pathologyinvolved evaluation of whole-mounts of retinas from theright eyes by microscopic investigation with light or confocalmicroscopy Maculas were investigated under the polariza-tion microscope and semiquantitative analysis of inclusionswas performed Routine histopathological investigation ofparaffin sections from retinal periphery was performed andzeaxanthin and lutein in the retina and lens were measuredanalytically by High-Performance Liquid Chromatography(HPLC)

3 Results

31 Genotoxicity Studies

311 S typhimurium Mutagenicity (Ames) Test OECD 471Crystalline zeaxanthin was evaluated for mutagenic activityin theAmes assay using the plate incorporation and the prein-cubation method Seven Salmonella typhimurium standardtester strains were employed (TA1535 TA1537 TA1538 TA97TA98 TA100 and TA102) with and without an exogenousmetabolic activating enzyme system (S9-mix) derived fromlivers of phenobarbital120573-naphthoflavone treated male ratsDue to the strong precipitation of the test compound in theaqueous medium 1500 120583gplate was chosen as the highestdose level There was no increase of the numbers of mutantsin any of the tester strains while the positive controls verifiedthe sensitivity of the strains and the activity of the S9-mix [14]

In one very early laboratory batch of pure crystallinezeaxanthin a positive result was found in theAmes test It wasdetermined that pure zeaxanthin is not mutagenic however

degradation products formed during exposure of crystallinezeaxanthin to air and light were considered responsible forthe mutagenic activity [15] In addition it was determinedthat components in the beadlet formulation scavenged themutagenic activity of degraded crystalline zeaxanthin thusfurther protecting against the occurrence of mutagenicactivity The beadlet formulation for the marketed productincludes the antioxidants (ascorbyl palmitate sodium ascor-bate and dl-120572-tocopherol) which prevent degradation

Crystalline zeaxanthin that had been kept in storagebeyond the maximum shelf life was subsequently assessed ina non-GLP Ames test [16] The purpose of this study was toconfirm the absence of mutagenic activity of crystalline zeax-anthin Five Salmonella typhimurium tester strains (TA1535TA97 TA98 TA100 and TA102) were employed with andwithout metabolic activation (S9-mix) No relevant increasein the number of revertant colonies was apparent and it wasconcluded that neither zeaxanthin nor any of the metabolitesformed by the metabolic activation system was mutagenic inthe Ames test

312 Gene Mutation Assay in V79HGPRT Cells OECD 476In the gene mutation assay in cultured mammalian cellszeaxanthin was tested for its ability to induce gene muta-tions at the HGPRT (Hypoxanthine Guanine PhosphoribosylTransferase) locus in the established cell line V79 derivedfrom Chinese hamster lung cells Treatment with 1120583g to16 120583gmL (0002ndash003mmolL) did not induce mutations to6-thioguanine resistance in V79 cells in vitro neither in theabsence nor in the presence of a rat liver activation system[17]

313 Unscheduled DNA Synthesis (UDS) Assay OECD 482The ability of zeaxanthin to induce DNA damage was testedby the Unscheduled DNA Synthesis assay (UDS test) asmeasured by the incorporation of radiolabeled nucleotidesinto nonreplicated DNA of freshly isolated rat hepatocytesA 20-hour exposure to 1 120583g to 16 120583g zeaxanthin per mL didnot induce DNA repair synthesis in primary cultures of rathepatocytes [18]

314 Chromosome Analysis of Human Peripheral Lympho-cytes OECD 473 The potential in vitro clastogenic activityof zeaxanthin was assessed using human peripheral bloodlymphocytes as target cells in the presence and absenceof rat liver activating enzyme system (S9-mix) Under theexperimental conditions described neither zeaxanthin norany of its metabolites induced chromosomal aberrations inhuman peripheral blood lymphocytes [19]

315 MouseMicronucleus Assay OECD 474 Zeaxanthin wastested in the in vivomicronucleus assay in mice Zeaxanthin10 beadlet formulation was administered orally at doselevels of 445 890 and 178mgkg of zeaxanthin 30 and 6hours prior to sacrificeThere was no increase ofmicronucleithus it was concluded that under the conditions of the studyzeaxanthin did not induce chromosome breaks or mitoticnondisjunctions in mouse bone marrow cells at doses up to178mgkg of zeaxanthin [20]


32 Short-Term Toxicity Studies

321 Acute Safety Studies Pre-OECD Guideline Similaritiesto Guideline OECD 401 Acute studies with zeaxanthin wereperformed in rats and mice Zeaxanthin has a low order ofacute toxicity All mice and rats survived a single oral dose ofup to 4000mgkg in rats and 8000mgkg in mice The LD

50

values in rats andmice therefore were greater than 4000 and8000mgkg body weight respectively [21]

322 Guinea Pig Skin Sensitization Test OECD 406 Anoptimization test (according to Maurer) was performed withzeaxanthin in albino guinea pigs of both sexes No signsof skin irritation or sensitization were observed [22] Asubsequent maximization test in albino guinea pigs based onOECD guideline 406 was also negative [23]

323 Rabbit Irritation Test OECD 405 The primary eyeirritation potential of zeaxanthin was studied in young adultrabbits [24] The risk that an accidental or occasional ocularexposure to zeaxanthin could cause injury to the eye in manwas considered to be low

33 General Toxicology Studies

331 Subchronic Safety Studies 13-week subchronic toxicitystudies have been performed with synthetic zeaxanthin inthree species mouse rat and dog Preliminary studies (5-and 10-day studies) were conducted beforehand to ensureappropriate selection of dosages for the main studies

332 13-Week Study in Mice Similar to OECD 408 A 13-week oral safety study was performed in mice with a 93beadlet formulation of zeaxanthin administered as a feedadmixture Groups of 10 male and 10 female mice weretreated with 0 250 500 and 1000mgkg body weightday(mgkg bwday) of zeaxanthin The placebo beadlets wereadded to the diet so that all 4 groups received similar amountsof beadlets There was no treatment-related hematology orclinical chemistry findings No discoloration of adipose tissueor other findings were observed at necropsy and there wereno histopathological effects attributable to zeaxanthin or thebeadlet formulations The No Observed Adverse Effect Level(NOAEL) of zeaxanthin was gt1000mgkg bwday in mice[25]

In line with the respective OECD guideline proceduresformice the study did not include ophthalmoscopy althoughhistopathology of the eyes was undertaken

333 13-Week Study in Rats OECD 408 An original 13-weekoral safety study was conducted in rats with a 93 beadletformulation of zeaxanthin administered as a feed admixtureGroups of 16 male and 16 female rats were treated with 0250 500 and 1000mgbwday of zeaxanthinTheNOAEL forzeaxanthin was gt1000mgkg bwday in rats [26]

Due to a change in manufacturing process a second 13-week oral safety study in rats was performed with a 10beadlet formulation of zeaxanthin from an updated processGroups of 16 male and 16 female rats were treated with doses

of 0 250 500 and 1000mgkg bwday of zeaxanthin as adietary admixture [27] All groups received similar amountsof beadlets by adjusting the diet with control beadletsThere was no effect of treatment on food intake and bodyweight Yellow-orange discoloration of the feces was seenin all zeaxanthin-treated rats especially at the high doseNo treatment-related changes in hematological and clinicalchemistry parameters were observed Urine pH values wereslightly decreased in male rats of all dose groups In linewith the respective OECD guideline the study includedophthalmoscopic evaluations About 20ndash30 minutes prior toexamination amydriatic was instilled into each eye of controland high-dose animals The examinations were made using aldquoKEELERrdquo Fison binocular ophthalmoscope There were notreatment-related changes

At necropsy a slight orange discoloration of the adiposetissue was reported in all treated animals however this wasnot considered an adverse effect but due to color of thetest compound There were no treatment-related changesin organ weights or histopathological findings Under theconditions of this study the NOAEL in this second rat studywas again gt1000mgkg bwday

334 13-Week Dog Study Similar to OECD 409 A 13-weeksafety study in dogs was conducted with a 94 beadletformulation of zeaxanthin Zeaxanthin beadlets were incor-porated into feed pellets and fed to groups of 3 male and3 female beagle dogs to achieve a dose of zeaxanthin of 0123 204 and 422mgkg bwday (males) and 0 104 238 and442mgkg bwday (females) This corresponds to test articleconcentrations in feed of 0 4 8 and 16 respectivelyControl beadlets were added so that the amount of beadletspresent in the feed cubes was similar for all groups

No treatment-related toxicity was observed throughoutthe study The test article was found to strongly discolorand to slightly soften the feces particularly in the high-dose group Ophthalmoscopic evaluations were undertakenat day and at the end of week 13 Following induction ofmydriasis eyes including cornea chambers lens and retinaof all dogs were examined using a fundus-camera KOWARC-2 The central parts of the retina (generally includingthe optic disc) were recorded on an Ektachrome-X film Notreatment-related findings were reported Urinalysis as wellas hematological and serum clinical chemistry investigationsshowed no treatment-related effects At necropsy male dogsfrom the mid- and high-dose groups showed slight to mod-erate discoloration (yellow to reddish) in the adipose tissuewhich was considered not an adverse effect and probablyreflected presence of zeaxanthin There were no treatment-related histopathological findings The NOAEL in this studywas gt422mgkg bwday [28]

34 Reproductive Safety Studies

341 Developmental Toxicity Study in Rats OECD 414 Ina developmental toxicity study in rats zeaxanthin (10beadlet formulation) was administered at doses of 0 250500 and 1000mgkg bwday orally as a feed admixture fromday 7 through day 16 of gestation [29] A subgroup was


Table 3 Effects on reproduction data in the zeaxanthin two-generation study in rats [31]

Nominal dosage (mgkg bwday) 0 0(placebo) 50 150 500

F1generationAdults mating index 1000 1000 960 960 793 lowast119865Mean number of pupsborn 102 109 107 107 97alive day 4 postpartum(before culling) 100 109 105 105 97

P generation pup weight gainDays 4ndash7 postpartum 623 620 642 616 593Days 1ndash21 postpartum 7747 7533 7809 7550 7253 lowastlowast119869

F1generation pup weight gainDays 4ndash7 postpartum 644 628 605 613 581 lowast119869Days 1ndash21 postpartum 7389 7451 7135 7249 6950

119865 = Cochran-Armitage and Fisherrsquos exact test119869 = dose response test Kruskal-Wallis Terpstra-Jonckheere and Wilcoxonlowast119901 lt 005 lowastlowast119901 lt 001

Caesarian-sectioned on day 21 of gestation and a rearingsubgroup was allowed to deliver naturally and was observedup to day 23 of lactation There was no indication of anyembryotoxic or teratogenic action of zeaxanthin in any of thetreated groups The rearing subgroup showed no indicationof any functional abnormalities in the treated groups It wasconcluded that under the conditions of this study zeaxanthinwas neither embryotoxic nor teratogenic in rats at doses up to1000mgkg bwday

342 Developmental Toxicity Study in Rabbits OECD 414In a developmental toxicity study in rabbits zeaxanthin wasadministered at doses of 0 100 200 and 400mgkg bwdayorally in rapeseed oil from day 7 through day 19 of gesta-tion Rabbits were Caesarian-sectioned on day 30 of ges-tation [30] No deaths or signs of maternal toxicity wereobserved in the treated groups There was no indicationof any embryotoxic or teratogenic action of zeaxanthin inthe treated groups There were some isolated malformationsamong the groups including controls but there was noevidence of any treatment-related effect It was concludedthat under the conditions of this study zeaxanthin wasneither embryotoxic nor teratogenic in rabbits at doses up to400mgkg bwday

343 Two-Generation Study in Rats OECD 416 A two-generation study was performed with synthetic zeaxan-thin in rats [31] Multigeneration studies involve exposureto test compounds beginning before mating continuingduring mating and throughout gestation and lactationuntil weaning and cover all reproductive life phases overtwo generations There was a range of developmental andbehavioral testing in addition to reproduction endpointsOphthalmologic examination is not part of the OECD 416guideline study requirements but testing on the young rats

included confirmation of the pupillary reflex response andcorneal tactile response

In the two-generation study DSM-manufactured zeax-anthin was administered in the diet at nominal doses of0 (control diet) 0 (placebo beadlet control) 50 150 and500mgkg bwday active ingredient by admixture of 10WSbeadlets to the feed Although zeaxanthin in a 13-week toxi-city study in the rat was well tolerated at 1000mgkg bwdayto achieve a dose level of 1000mgkgday the beadlet con-centration in the diet approached 20 The high-dose level500mgkg bwday was selected on the basis of avoidingpotential nutritional imbalance due to the high beadletcontent in the diet over the duration of a two-generationstudy Two control groups received either the control dietonly or placebo beadlets incorporated in the diet at the sameconcentrations as the zeaxanthin beadlets in the high-dosegroup The parental (P) generation females were allowedto litter and rear their offspring to weaning Young wererandomly selected from each group to form the filial (F

1)

generationAdministration of 500mgkg bwday zeaxanthin active

ingredient to rats for two successive generations producedmarginal adult toxicity in terms of slightly lower food intakeduring the lactation period of the P generation a slightlylower body weight gain during the gestation period of the F

1

generation and a possible slight adverse effect on fertility ofthe F1generation with a lower mating index (mating index is

the number of females with determined copulationsnumberof oestrous cycles required for their insemination times 100) andslightly fewer pupswere born (Table 3) At this dosage in bothgenerations percentage of pup growth during lactation wasalso slightly lower than in the control groups (Table 3)

Samples of plasma and liver from the P and F1generation

adults and pups were analysed for zeaxanthin exposuremonitoring The analysis results showed that exposure was


Table 4 Plasma and liver concentration of zeaxanthin in adults andweanlings in the two-generation study in rats [31]

Zeaxanthin concentration (120583L or 120583kg) at nominal dosage(mgkg bwday)

Dosage 0 0 50 150 500P generationPlasma 120583LAdults

Male mdash mdash 24 47 111Female mdash mdash 19 29 71

Weanlings mdash mdash 61 127 353Liver 120583kgAdults

Male 14 mdash 599 1121 2581Female 4 5 1147 1992 3159

Weanlings mdash mdash 4015 10892 23836F1 generationPlasma 120583LAdults



Male mdash mdash 114 645 1689Female 11 mdash 1077 1382 3785

Weanlings mdash 11 4313 7904 21611

essentially similar in the P and F1generations Higher plasma

concentrations and notably higher liver concentrations wereobserved in the weanling pups at day 21 postpartum com-pared to the adults of the corresponding treatment group(Table 4) Exposure increased with increasing dose althoughit was not proportional to doseThe higher exposure in the 21-day-old pups probably reflects intake through the maternalmilk and that they had already started eating the treateddiet provided to the mothers The relative concentrationsin the liver in comparison to concentrations in the plasmawere notably higher in both adults and weanlings (Table 4)suggesting these are accumulation in the liver However thehigh accumulation in the liver of weanlings (3 weeks of age)clearly diminished during subsequent rearing and as adults ofthe F1generation showed similar tissue concentrations to the

P generation which was exposed from 6 weeks of ageIn conclusion the NOAEL was defined as the nominal

dosage of 150mg zeaxanthinkg bwday

35 Chronic Rodent Carcinogenicity Studies No carcino-genicity studies are available for zeaxanthin or for luteinGenotoxicity studies were negative and histological exami-nations of tissues from repeat dose toxicity studies have notshown any preneoplastic effects or indeed no indication ofhistological effects at all

There is data for two carotenoids (canthaxanthin andastaxanthin) showing that chronic administration over twoyears in rats but not mice induces liver toxicity These twocarotenoids induce liver enzyme including cytochrome P450enzyme CYP1A in the rat [32 33] although not in the mouse[34] and not in vitro in human hepatocytes [35] Howeverlutein did not affect phase-I or phase-II liver enzyme activitiesin the rat [32] Due to its close isomeric relationship to luteinit is considered unlikely that zeaxanthin is a liver enzymeinducer

In 2010 EFSA applied an additional safety factor of 2 intheADI calculations for lutein [36] and in 2012 for zeaxanthin[37] to take account of the absence of chronic rodent studies

36 ADME (Absorption Distribution Metabolism and Excre-tion) Studies Metabolism studies or ADME studies are auseful component of safety testing as the provided informa-tion on themetabolism and kinetics of a substance and whenhuman data is available on human relevance

361 Balance Study in Rats A distribution study with (14C)-zeaxanthin was performed in male rats after a pretreatmentfeeding with zeaxanthin-poor or zeaxanthin-enriched diet(0001 in feed) and subsequent single dose administrationof (14C)-zeaxanthin in a liposomal preparation One dayafter dosing approximately two-thirds of the administeredradioactivity was excreted in feces and urine and approxi-mately 13 of the administered radioactivitywas present in thebody and GI-tract The pattern of distribution in the tissuesand excretion was similar for rats prefed with zeaxanthin-poor and those fed with zeaxanthin-enriched diet After 1week less than 1 of the administered radioactivity was inthe body and the digestive tract The amount of radioactivityabsorbed and excreted in the urine tended to be lower for ani-mals fed with the zeaxanthin-poor diet It was concluded thatthe radioactivity from (14C)-zeaxanthin is rapidly depletedfrom the body and the GI-tract of rats [38]

362 Distribution Study in Rats A study was performedto investigate zeaxanthin distribution in rats fed with azeaxanthin 5 beadlet formulation-enriched diet Male ratsreceived a diet containing 10mg or 100mg zeaxanthinkg feed(approximately 08mg or 8mgkg bwday) for five weeks Adose-dependent accumulation of zeaxanthin was found invarious tissues with the highest concentrations in the smallintestine and spleen followed by liver fat and adrenal glandsThe thyroid gland and the eye levels were below the levels ofdetection There was a marked decrease of zeaxanthin con-centration during a subsequent 5-week reversibility period[39]

363 Radioactivity in Expired Air Mass Balance StudyIn balance studies with a liposomal preparation of (14C)-zeaxanthin in male rats about 1 of the administered dosethat is about 4 to 7 of the absorbed dose wasmeasured inthe expired air during the first 24 hours after administrationContribution of respiration in the excretion of radioactivitywas considerably higher in the case of zeaxanthin when


CMO2

Zeaxanthin

CMO2

HO

O

H

OHO

HO

O

H

O

O

H

OHO

HO

910

10998400

9998400

HO

OH9

1010998400

9998400

120573-cryptoxanthin

3-OH-120573-ionone

3-OH-120573-apo-10998400-carotenal


120573-ionone

3-OH-120573-ionone

120573-apo-10998400-carotenal

Figure 2 Xanthophyll carotenoids 120573-cryptoxanthin and zeaxanthin and themetabolites fromCMO2 cleavage in ferrets (adapted fromMeinet al 2011) [42]

compared to previous studies with astaxanthin and canthax-anthin Absorption (biliary excretion not considered) variedfrom around 10 to around 20 [38 40]

364 Metabolite Studies It is known that 120573-carotene ismetabolised by central cleavage by the enzyme 120573 120573-carotene-15 151015840-monooxygenase (CMO1) On the other hand thenonprovitamin A xanthophylls lutein and zeaxanthin aremetabolised preferentially by eccentric cleavage by carotene-9 10-monooxygenase (CMO2) alternatively known as 120573-carotene oxygenase 2 (BCO2) 120573-Cryptoxanthin is alsometabolised eccentrically through CMO2 which has beenidentified in humans mice and ferrets [41]

The metabolite pathways of lutein zeaxanthin and 120573-cryptoxanthin have been published in 2011 [42] In thispublication the production of apocarotenoids from CMO2metabolism in ferrets was shown for all of these threexanthophylls All three are cleaved at the 9 10 position aswell as at 91015840 101015840 This gives rise to four metabolites forboth lutein and 120573-cryptoxanthin Zeaxanthin however issymmetrical such that there are only two metabolites 3-OH-120573-apo-101015840-carotenal and 3-OH-120573-ionone Both of thesemetabolites are derived from eccentric cleavage of luteinand 120573-cryptoxanthin (Figure 2) As zeaxanthin has the samering structure at each end of the molecule the same twometabolites are produced irrespective of whether cleavageoccurs at the 9 10 position or the 91015840 101015840 position

In addition to cleavage reaction products there is evi-dence of a common metabolite from both lutein and zeax-anthin from noncleavage metabolism In a human studywith zeaxanthin the metabolite all-E-31015840-dehydro-lutein wasformed under normal dietary conditions all-E-31015840-dehydro-lutein is predominantly formed from other sources mostlikely from lutein rather than from dietary zeaxanthin[43]

Further using chiral-phase HPLC two diastereoisomers(3R 61015840R)-31015840-dehydro-lutein and (3R 61015840S)-31015840-dehydro-lutein

were identified and shown to be common metabolites oflutein and zeaxanthin in rhesus monkeys [5]

37 Special Toxicological Studies

371 One-Year Chronic Study in Monkeys Safety studiesare not normally undertaken in monkeys at least not fornutritional substances However a known profile of humanresponse that has been observed in the past in humans withhigh intake of the carotenoid canthaxanthin is accumulationin the eye and for the so-called ldquocanthaxanthin retinopathyrdquo[10] Therefore a chronic study was undertaken in monkeyswith the purpose to assess the chronic safety of zeaxanthinand lutein in primates and to determine the potential forcrystal formation in the retina There are morphologicaldifferences in the structure of the eye between rodents andprimates and further investigation in primates was consid-ered important There is no specific OECD guideline for thestudy design undertaken which was designed taking intoaccount general requirements for primate safety studies

The chronic study performed in Cynomolgus monkeyswas 52 weeks in duration and was an investigation forzeaxanthin and also for lutein (each with separate groups)10 beadlet formulations of both substances were used Oraldoses of 02 and 20mgkg bwday of zeaxanthin or luteinwere given respectively by gavage to groups of 2 male and2 female monkeys For both 20mgkg bwday groups oneadditional male and female were sacrificed after 6 months oftreatment Normal toxicological endpoints were included aswell as specific endpoints for the eyes

All monkeys survived the treatment period There wereno clinical signs of toxicity and there was no effect oftreatment on overall mean body weight gain or group meanfood intake At the high dose of zeaxanthin orange-yellowcoloration of the feces was observed during the treatmentperiod and at necropsy yellow discoloration of adiposetissue was observed These were considered as coloration


changes from the presence of the test compound andwere notconsidered an adverse effect There were no changes in ECGor blood pressure data considered to be related to zeaxanthintreatmentThere were no treatment-related changes in urinehematological and serum clinical chemistry parameters Atnecropsy there were no abnormal gross findings or organweight change There were no treatment-related histopatho-logical findings

A comprehensive examination of the eyes of treatedmonkeys was performed which included ophthalmoscopyand biomicroscopy examinations fundus photography andelectroretinography (ERG) Postmortem examinations of theretina of the right eye included macroscopic inspectionmicroscopic pathology under polarized and bright lightfor peripheral retina and macula confocal microscopy ofmacula and histopathological examination of the peripheralretina A determination of lutein and zeaxanthin in retina andlens of the left eye was performed byHPLCThese proceduresand results are described in more detail

Ophthalmic Examinations Ophthalmic examinations wereperformed on the monkeys by two independent examin-ers Indirect ophthalmoscopic examinations were performedusing the Bonnoskop and direct ophthalmoscope and acontact lens biomicroscopeOverall based on the ophthalmicexamination findings it was concluded that there wereno adverse findings that were considered to be related totreatment and there was no evidence for crystalline depositsin the retina of treated monkeys [44 45]

Additional evaluations were performed using the oph-thalmic slit lamp biomicroscope in combination with wide-field corneal contact fundus lenses The results of theseexaminations showed that there were no crystalline depositsor inclusions similar to those that have been seen in humansor in Cynomolgus monkeys ingesting high dosages of thecarotenoid canthaxanthin There were some retinal findingsoften seen in the human- and nonhuman-primate retinahowever none of these were considered to be related to treat-ment [44 45] The polarising structures that were observedwere found not only in the zeaxanthin- and lutein-treatedmonkeys but also in the control monkeys The implicationof the special eye examinations included into the 52-weekmonkey study is considered to be that even for high intakezeaxanthin or lutein consumers there is no indication thatcrystalline deposits could develop in the retina as wasseen in man and monkeys with high-dose canthaxanthinsupplementation

Electroretinography (ERG) ERGwas performed in all animalsonce predose and during weeks 25 to 26 weeks 38 to 39and weeks 51 to 52 of treatment There were no treatment-related effects in electroretinograms which is considered asensitive procedure to detect early signs of generalized retinaldegeneration [46]

Eye Pathology Whole-mounts of retinas from the righteyes were used for microscopic investigations with light orconfocalmicroscopyMaculas were punched out with a 7mmtrephine before mounting them on slides and the peripheral

remaining parts of the retinas were flat-mounted and inves-tigated under the polarization microscope separately Semi-quantitative analysis of inclusions was performed by screen-ing the flat-mounted retinas of the right eyes under polarizedlight using a Zeiss Axioplan In addition all maculas wereinvestigated using a confocal microscopic system Routinehistopathology of paraffin sections from retinal peripherywas performed [44 45 47] The routine histopathologicalinvestigation of paraffin sections from retinal periphery didnot show any differences between treated or control animals

It was concluded that there were no treatment-relatedadverse changes in the eyes noted under the conditions ofthis study Polarizing inclusions were observed in the maculaof monkeys which were not related to zeaxanthin nor luteintreatment The incidence and grade of the inclusions inthe maculas of the monkeys were not treatment or doserelatedThe inclusions clearly differed from crystals observedafter long-term treatment at high doses of canthaxanthinIn the case of canthaxanthin crystals were strongly dose-dependent occurred predominantly in the peripheral retinaand exhibited crystalloid morphology and larger size [12] Incontrast inclusions in the current study were restricted tothe fovea were very small and showed no typical crystallinemorphologyThe nature of the observed polarizing structuresremains unknown Since they were also observed in controlanimals with a naturally yellow macula a physiologicalfunction may be hypothesized [48]

Zeaxanthin and Lutein Determinations in Retina and Lens byHPLC Determination of lutein and zeaxanthin in retina andlens was made using HPLC Treatment with lutein resulted ina dose-related increase of lutein in central retina peripheralretina and lens In addition after treatment with luteinat both dose levels elevated amounts of zeaxanthin wereobserved in the central retina This finding may be due tothe residual zeaxanthin content in the lutein test articleZeaxanthin levels in peripheral retina and lens were similarto those observed in the placebo group [49]

Treatment with zeaxanthin resulted in a dose-relatedincrease of zeaxanthin in the peripheral retina In centralretina and lens zeaxanthin content was markedly increasedin animals of the high-dose group Levels in the low dosegroup were comparable to those determined in the placebogroup In animals treated with zeaxanthin lutein content wasin the same order of magnitude as in the placebo group [49]

Variability of individual animal lutein and zeaxanthincontent was considerable in all tissues investigated for bothsexes and at all dose levels including the placebo groupConsidering the variability there was no significant dif-ference between sexes In addition no relevant differencewas observed in animals sacrificed in week 26 and animalssacrificed at the end of the treatment period This suggeststhat steady state conditions were reached before week 26 inall eye segments investigated

Overall there were no clinical and no morphologicalevidence for treatment-related adverse changes in the eyes ofCynomolgus monkeys during or after 52 weeks of treatmentwith zeaxanthin or lutein both as a 10 beadlet formulationSpecifically there was no evidence for crystal formation in


the eyes of treated monkeys The NOAEL for lutein and forzeaxanthin was the highest dosage 20mgkg bwday

372 Other Monkey Studies There is a published studywith female rhesus macaques (5group) exposed to 10mgkg bwday of lutein supplements providing 934mg luteinand 066mg zeaxanthin 10mgkg bwday of zeaxanthin sup-plements or supplements of a combination of lutein andzeaxanthin (each at 05mgkg bwday) for 12 months [50]After 12 months one control animal two lutein-treatedanimals two zeaxanthin-treated animals and all lutein andzeaxanthin combined-treated animals were killed The otheranimals were kept under observation for six additionalmonths without receiving further supplementation and werethen killed Plasma and ocular carotenoid analyses fundusphotography and retina histopathology were performed onthe animals

Supplementation of female rhesus macaques with934mg luteinkg bwday or 10mg zeaxanthinkg bwday for12 months resulted in 32-fold and 37-fold increases inthe mean concentrations of lutein and 40-fold and 43-fold increases in the mean concentrations of zeaxanthinin plasma and retina respectively Supplementation ofmonkeys with lutein or zeaxanthin for one year at a doseof approximately 10mgkg bwday did not cause oculartoxicity and had no effect on biomarkers associated withnephrotoxicity

38 Inhalation Study in Ferrets No carcinogenic hazard isexpected from direct intake of zeaxanthin or lutein Therehas been a question as to whether these xanthophylls mightexacerbate the risk of lung tumors in heavy smokers as wasindicated to occur in two human intervention studies withhigh dosages of 120573-carotene [51 52] It has been establishedthat this exacerbating influence of 120573-carotene could bemimicked in the ferret [53] a species selected on the basisof metabolic considerations and certain similarities to manFerrets show a weak central (CMO1) cleavage of 120573-carotenein a similar way to humans In contrast rats show a muchstronger CMO1 activity and a greater propensity to centrallysplit beta-carotene which raised doubts about the relevanceof the rat as a suitable human model

This concern of a possible adverse influence in com-bination with smoking can potentially be addressed forzeaxanthin using a published study in ferrets treated with 120573-cryptoxanthin and exposed to cigarette smoke [54] Zeaxan-thin itself has not been tested in the ferretmodel Structurallyzeaxanthin is closely related to120573-cryptoxanthin As describedpreviously the metabolites of zeaxanthin central cleavage 3-OH-120573-apo-101015840-carotenal and 3-OH-120573-ionone (Figure 2) arealso metabolites of 120573-cryptoxanthin central cleavage Fromthis overlap of CMO2 metabolites and as CMO1 in thelung of man and the ferret is not the predominant cleavageenzyme data from the 120573-cryptoxanthin study can contributeto zeaxanthin evaluation on a ldquoread-acrossrdquo basis

In this 120573-cryptoxanthin study both the low and highdose lowered the incidence of cigarette smoke-induced lungsquamous metaplasia The reduction was significant for thehigh dose (119901 = 0015 16 ferrets affected) and was marginally

significant for the low dose (119901 = 006 26 ferrets affected)compared to the control (66 ferrets affected) Further theexpression of proinflammatory markers TNF120572 (expressionof which was tremendously increased in smoke exposedferret lungs) and of NF-120581B was lowered by 120573-cryptoxanthinadministration with stronger beneficial effects for high-dose120573-cryptoxanthin than for the low-dose 120573-cryptoxanthin

However the usefulness of this read-across approachwas limited by the dose selection in the 120573-cryptoxanthinferret study The dosages of 120573-cryptoxanthin used (75 120583gkgand 375 120583gkg bwday) were based on equivalence to anaverage American intake of 104 120583gday (approximately15 120583gkg bwday for a 70 kg person) increased by a factorof 5 and 25 and not by a factor of at least 100 as is usualin toxicological safety testing Also from a read-acrossperspective only half of the CMO2 metabolites formedfrom 120573-cryptoxanthin would be theoretically common tothose from zeaxanthin So ignoring any possible kineticdifferences 375 120583gkg bwday possibly only correspondsto 1875 120583gkg bwday in terms of zeaxanthin dosage or13mgday for a 70 kg adult The relative ldquointernalrdquo humandose could be even lower if systemic carotenoid absorptionin the ferret is lower than in man as indicated by the authors[54]

So from this study with 120573-cryptoxanthin in ferrets it isconsidered that zeaxanthin supplementation at low intakesis unlikely to exacerbate the occurrence of lung cancer andmight even have a protective effect against the occurrence ofsquamous metaplasia However due to the low dosages of 120573-cryptoxanthin used the extent to which the dosage-relatedinfluences might extend to higher intakes of 120573-cryptoxanthinor intakes of zeaxanthin above 13mgday is unclear

4 Summary and Discussion

A series of well-conducted safety studies are available andprovide a good basis for a safety assessment of zeaxanthinAcute studies in rats and mice show a low order of acutetoxicity with LD

50values greater than 4000 and 8000mgkg

respectively Subchronic safety studies demonstrated thatrepeated intakes of high oral doses up to 1000mgkg bwdayin rat and mouse and 400mgkg bwday in the dog arewell tolerated systemically The macroscopic observation ofyellow discoloration of the adipose tissue which can beattributed to the presence of the zeaxanthin indicates thatthere was systemic exposure in these studies and this hasbeen analytically confirmed by analysis of plasma and liversamples in the two-generation rat study Despite the systemicexposure and high dosages no target organ toxicity wasidentified in the subchronic studies during the in-life phaseor by pathologicalhistopathological evaluation

In developmental toxicity studies there was no evi-dence of maternal toxicity fetal toxicity or teratogenicity intreated rats or rabbits at doses up to 1000mgkgday and400mgkgday respectively

For zeaxanthin there is no chronic study in rodentsas is also the case for lutein but there is a two-generationstudyThis study design involves exposure to test compoundsbeginning before mating continuing during mating and


throughout gestation and lactation until weaning and coversall reproductive life phases over two generations The newversion of the study design finalized in 2001 introduceda range of additional end-points focused on detection offine disturbances of reproductive function and fertility Suchstudies can sometimes give a lower NOAEL than respectivesubchronic toxicity studies in a similar way that the NOAELsfrom chronic studies in general are lower than in correspond-ing subchronic studies Indeed on the basis of the dosagesfor which data was available the NOAEL from the rat two-generation study with zeaxanthin (150mgkg bwday) was afactor of 67 down from the NOAEL in the subchronic ratstudy

Potentially the high systemic exposure observed in younganimals during the lactation phase of the two-generationstudy from a combination of intake from maternal milk anddirect feeding may have contributed to an effect of treatmentoccurring at a lower nominal dosage than in the other safetystudies Irrespective of the reason the lowest dosage fromrepeat dose toxicity studies was the intermediate dosage of150mg zeaxanthinkg bwday

ADME studies in the rat showed that zeaxanthin wasrapidly but incompletely absorbed after oral administrationfollowing a single dose of 14C-zeaxanthin and there is a widebodily distribution with clear deposition in fatty tissuesreflecting the lipophilic nature of zeaxanthin In addition tospecific ADME studies important information on the poten-tial to bioaccumulate can be obtained from samples takenduring the course of the toxicology studies As referred to inthe two-generation study there is evidence of accumula-tion in the liver in comparison to concentrations in theplasma

Metabolite studies have shown there is eccentric CMO2cleavage of zeaxanthin and other xanthophylls but for zeaxan-thin being symmetric only two rather than four metabolitesare expected Both CMO2 cleavage metabolites of zeaxanthinoccur as cleavage metabolites of lutein and 120573-cryptoxanthin

Genotoxicity studies are important studies to indicateif there is interaction with DNA When unformulated purecrystalline zeaxanthin is exposed to air and light there maybe a potential for mutagenic breakdown products to occurHowever DSM formulated zeaxanthin contains antioxidantsthat prevent the degradation of zeaxanthin No mutagenicitywas observed in the Ames test with zeaxanthin or crystallinezeaxanthin retained beyond the shelf life or in culturesof V79 at the HGPRT locus No evidence of UnscheduledDNA Synthesis was detected in rat hepatocytes up to thehighest dose tested There was no evidence of clastogenicpotential with or withoutmetabolic activation from tests withperipheral blood lymphocytes at doses In the in vivo mousemicronucleus test there was no evidence for mutagenicity orclastogenicity It is concluded that there is no evidence formutagenicity or clastogenicity with formulated zeaxanthinunder appropriate conditions of use

Based on the wide range of genotoxicity studies with noindication of DNA damage and the absence of any indicationof preneoplastic organ changes in repeat dose toxicity studiesand the absence of clear liver enzyme induction effect for

lutein no carcinogenic hazard is expected from direct intakeof zeaxanthin

The question as to whether these xanthophylls mightexacerbate the incidence of lung tumors in heavy smokersas was demonstrated to occur for high dosages of 120573-carotenein two human intervention studies [51 52] has only beenpartly addressed by using published studies in ferrets forthis related xanthophyll The ferret model with exposure tocigarette smoke has been positively validated for 120573-carotene[53] and the metabolite overlap has enabled theoretical use ofa study with 120573-cryptoxanthin to support the safety of zeax-anthin on a read-across basis Although the study showeda protective effect of 120573-cryptoxanthin against pulmonarysquamous metaplasia the potential applicability of this datafor zeaxanthin intake was considered to be limited due to thelow 120573-cryptoxanthin dosages that were used in the study

Besides this study in ferrets a pooled analysis of sevencohort studies demonstrated that the association betweenintake of xanthophylls (lutein or lutein plus zeaxanthin)and the risk of lung cancer was negative in smokers andnonsmoking subjects [55]

In general the structural similarity of the xanthophyllcompounds might be considered sufficient to enable theprinciples of read-across where there are safety data gapsas has been done with the 120573-cryptoxanthin data in ferretsHowever it appears to be the case that in the humanand primate eye there is a notable specificity in biologicaldifferentiation between the xanthophyll isomers [56] Withthis being the case there is the need for caution in carryingacross information from one of these related substances toanother at least in respect to the primate eye

The animal safety data for lutein and meso-zeaxanthinis notably less than what is available for zeaxanthin EFSAhas reviewed the available data for lutein [36] For meso-zeaxanthin there is published safety information [57] Ingenotoxicity studies reported by Xu et al there was noevidence of genotoxicity which is consistent with the datafor zeaxanthin The NOAEL from their 13-week rat toxicitystudy was 300mgkg bwday with clear adverse effects inthe liver being reported at the higher dosages of 600 and1200mgkg bwday This is in contrast to the subchronicsafety data for synthetic zeaxanthin where higher NOAELswere obtained with no indication of liver toxicity No pub-lished regulatory 13-week rat study with 120573-cryptoxanthincould be located although there is ADME data in the ratfollowing chronic oral intake [58]

There is a publication reporting toxicology studies fora lutein and zeaxanthin concentrate from marigold flow-ers (Tagetes erecta L) with a minimum 80 carotenoidcontent [59] In the subchronic study Wistar rats wereadministered the concentrate at dose levels of 0 4 40 and400mgkg bwday (gavage) for 13 weeks with no toxicolog-ically significant treatment-related changes The dosage interms of zeaxanthin at the high dose can be calculated to be216mgkg bwday (taking 75 of the carotenoid content tobe zeaxanthin)

In a recent publication of safety studies of the zeaxanthinconcentrate OmniXan RR-zeaxanthin 65 enriched productobtained from paprika [60] there was no indication of


genotoxicity In the 13-week rat toxicity study the highestdosage in terms of concentrate was 400mgkg bwday andthis was considered the NOAEL

A known profile of human response that has beenobserved in the past in humans with a high intake of thecarotenoid canthaxanthin is accumulation in the eye andfor so-called ldquocanthaxanthin retinopathyrdquo The accumulationin the eyes however was not found to be functionallyharmful and gradually reversible following discontinuationof consumption Nevertheless this accumulation is regardedas undesirable and has been evaluated as an adverse effect byEFSA [61]

A chronic study with synthetic zeaxanthin in Cynomol-gus monkeys an animal model used to investigate theinduction and dose dependency of canthaxanthin crystalformation in the retina has been undertaken involving acomprehensive battery of ocular testing as well as usualtoxicological endpoints Overall there were no clinical andno morphological evidence for treatment-related adversechanges in the eyes and specifically no evidence for crystalformation in the eyes of treated monkeys

In the safety evaluation of dietary substances includingnutritional substances being consumed at higher intakes thantraditionally occurs human data needs to be kept in mindas it becomes available A number of human interventionstudies have been undertaken or are in progress with respectto investigating the protective function for zeaxanthin andlutein in the eye These studies indicate good systemictolerance of zeaxanthin At the upper end of the dosage rangewere a studywith a dose of up to 20mgday for up to 6months[62] and a study with 8mgday for a year both withoutevidence of adverse effects A further study has been morerecently reported in which 24 subjects were supplementedwith 20mgday of zeaxanthin over 4 months without anyadverse effects [63 64]

41 ADI (Acceptable Daily Intake) In the 13-week subchronictoxicity studies the NOAEL in all cases was the highestdosage investigated namely 1000mgkg bwday in themouseand rat and at least 422mgkg bwday in the dogA traditionalapproach of a 100-fold safety factor in conjunction with thelowest relevantNOAEL from the safety studies would be usedto derive the ADI

For zeaxanthin the lowest NOAEL from a standardregulatory study was 150mg zeaxanthinkg bwday in thetwo-generation study in rats This NOAEL is at least a factorof 67 lower than the NOAEL in the 13-week rat study(gt1000mgkg bwday) In their evaluation of the safety ofsynthetic zeaxanthin as a Novel Food the EFSA Panel onDietetic Products Nutrition and Allergies (NDA) [37] usedthe 150mgkg bwday NOAELwith a 200-fold safety factor todefine an ADI of 075mgkg bwday or 53mgday for a 70 kgadult (70 kg is the new default human weight used by EFSA)Use of the lowest NOAEL for ADI calculations as was doneby the NDA Scientific Panel is the traditional precautionaryapproach used in safety evaluationTheNDA Scientific Panelstated that a daily intake of 53mg for a person with a bodyweight of 70 kg does not raise safety concerns and that the use

level of 2mgday requested by the applicant was confirmed assafe

In the case of lutein the EFSA Panel on Food Additivesand Nutrient Sources added to Food (ANS) in their reevalua-tion [36] introduced an additional safety factor of 2 (makinga total 200-fold safety factor) due to the absence of chronicstudies or a multigeneration reproductive toxicity studyAdditional factors taken into account were that the other data(reproductive studies and genotoxicity data) did not indicatea cause for concern and that lutein is a normal constituent ofthe dietThe highest dose tested for lutein in a comprehensive13-week rat toxicity study was 200mgkg bwday and this wasthe NOAEL The EFSA ANS Panel applied a 200-fold factorto this NOAEL giving an ADI of 1mgkg bwday or 60mgluteinday for an adult

As a passing comment application of the 200-fold factorto the rat zeaxanthin subchronic data would give a 5-foldhigher ADI than for lutein due to the higher dosages usedand the higher NOAELs that were established for zeaxanthin

In the United States synthetic zeaxanthin is marketedsince 2002 under the Generally Regarded as Safe legislationbased on DSM safety studies available at the time with uselevel in foods and beverages of 025mgserving

The use level of zeaxanthin of 2mgday proposed by theapplicant was ratified by the European Union (EU) Commis-sion in 2013 [65] However this upper use level is much lowerthan the safe level (53mgday) defined for zeaxanthin bythe NDA Scientific Panel Potentially therefore the currentlyapproved level for synthetic zeaxanthin in Europe could beset as a higher level Probably this could be closer to the ADIcalculated by the NDA PanelThis ADI (53mgday) is similarto the ADI of 60mgday currently defined by EFSA for lutein[36]

5 Conclusion

Zeaxanthin was negative formutagenic and clastogenic activ-ity in a comprehensive battery of in vitro and in vivo testsfor genotoxicity Based on these studies it is concluded thatthere is no evidence for mutagenicity or clastogenicity withformulated zeaxanthin under appropriate conditions of use

In repeat dose toxicity studies in the rat mouse anddog synthetic zeaxanthin was well tolerated at high dosageswith no indication of target organ toxicity or preneoplasticorgan changes Taken together these data indicate thatno carcinogenic hazard is expected from direct intake ofzeaxanthin A study in primate did not indicate any evidenceof ocular toxicity or excessive accumulation A publishedstudy in ferret provides limited support for the absence ofany stimulating effect of zeaxanthin consumption on theincidence of lung cancer in heavy smokers

The regulatory study that gave rise to the lowest overallNOAEL of 150mg zeaxanthinkg bwday was a comprehen-sive two-generation study in the rat In their evaluationof the safety of synthetic zeaxanthin as a Novel Food theEFSA NDA Scientific Panel [37] applied a 200-fold safetyfactor to this NOAEL to define an ADI of 075mgkg bwdayor 53mgday for a 70 kg adult The EU in 2013 [65] for-mally approved upper use levels of 2mgday (equivalent to


003mgkg bwday) as this was the use level proposed by theapplicant

Information from human intervention studies also sup-ports that an intake higher than 2mgday is safe and anintake level of 20mgday for up to 6 months was withoutadverse effect

Disclosure

The studies referred to in this publication are principally thework of many dedicated toxicologists and safety specialistsover a number of years and are not the work of the author

Conflict of Interests

The author is an employee of DSMNutritional Products Ltdwhich manufactures synthetic zeaxanthin

Acknowledgment

Dr Wolfgang Schalch is thanked for his help in editing thispaper

References

[1] A Perry H Rasmussen and E J Johnson ldquoXanthophyll (luteinzeaxanthin) content in fruits vegetables and corn and eggproductsrdquo Journal of Food Composition and Analysis vol 22 no1 pp 9ndash15 2009

[2] O Sommerburg J E E Keunen A C Bird and F J G Mvan Kuijk ldquoFruits and vegetables that are sources for luteinand zeaxanthin the macular pigment in human eyesrdquo BritishJournal of Ophthalmology vol 82 no 8 pp 907ndash910 1998

[3] F Y Mohamedshah J S Douglass M M Amann and JT Heimbach ldquoDietary intakes of lutein-zeaxanthin and totalcarotenoids among Americans age 50 and aboverdquo The FASEBJournal vol 13 no 4 part 1 abstract 44117 p A554 1999

[4] JM SeddonUAAjani RD Sperduto et al ldquoDietary caroten-oids vitamins A C and E and advanced age-related maculardegenerationrdquo Journal of the AmericanMedical Association vol272 no 18 pp 1413ndash1420 1994

[5] G I Albert U Hoeller J Schierle M Neuringer E J JohnsonandW Schalch ldquoMetabolism of lutein and zeaxanthin in rhesusmonkeys identification of (3R61015840R)- and (3R61015840S)-31015840-dehydro-lutein as common metabolites and comparison to humansrdquoComparative Biochemistry and Physiology B Biochemistry andMolecular Biology vol 151 no 1 pp 70ndash78 2008

[6] E J Johnson M Neuringer R M Russell W Schalch andD M Snodderly ldquoNutritional manipulation of primate retinasIII Effects of lutein or zeaxanthin supplementation on adiposetissue and retina of xanthophyll-free monkeysrdquo InvestigativeOphthalmology and Visual Science vol 46 no 2 pp 692ndash7022005

[7] W Schalch ldquoCarotenoids in the retinamdasha review of theirpossible role in preventing or limiting damage caused by lightand oxygenrdquo in Free Radicals and Aging I Emerit and BChance Eds vol 62 of EXS pp 280ndash298 Birkhauser BaselSwitzerland 1992

[8] W Schalch P Dayhaw-Barker and F M Barker ldquoThe caro-tenoids of the human retinardquo in Nutritional and Environmental

Influences on the Eye A Taylor Ed pp 215ndash250 CRC PressBoca Raton Fla USA 1999

[9] JECFA ldquoSafety evaluation of certain food additivesrdquo in Proceed-ings of the 63rd Meeting of the Joint FAOWHO Expert Com-mittee on Food Additives (JECFA rsquo06) vol 54 of InternationalProgramme on Chemical Safety World Health OrganizationWorld Health Organization 2006

[10] G B Arden and F M Barker ldquoCanthaxanthin and the eye acritical ocular and toxicological assessmentrdquo Journal of Toxicol-ogy Cutaneous and Ocular Toxicology vol 10 pp 115ndash155 1991

[11] R Goralczyk ldquoHistological aspects of primate ocular toxicitywith special emphasis on canthaxanthin-induced retinopathy inan animal model with cynomolgus monkeysrdquo in Towards NewHorizons in Primate Toxicology R Korte and G F WeinbauerEds pp 159ndash174 Waxmann Munster 2002

[12] R Goralczyk F M Barker S Buser H Liechti and J BauschldquoDose dependency of canthaxanthin crystals in monkey retinaand spatial distribution of its metabolitesrdquo Investigative Oph-thalmology andVisual Science vol 41 no 6 pp 1513ndash1522 2000

[13] R Goralczyk S Buser J Bausch W Bee U Zuhlke andF M Barker ldquoOccurrence of birefringent retinal inclusionsin cynomolgus monkeys after high doses of canthaxanthinrdquoInvestigative Ophthalmology and Visual Science vol 38 no 3pp 741ndash752 1997

[14] E Gocke ldquoMutagenicity evaluation of zeaxanthin Ro 01-9509007 in the Salmonellamicrosome assay (Ames test)rdquoDSM Report B-0153207 1987

[15] E GockeDecay of (3R 31015840R)-Zeaxanthin Internal Communica-tion Hoffmann-La Roche Basel Switzerland 1987

[16] E Gocke ldquoRo 01-9509007 bacterial reverse mutation test(Ames test)rdquo DSM Report 1015664 2004

[17] R Strobel ldquoGene mutation assay in cultured mammalian cellswith Ro 01 9509007 (All-Trans-Zeaxanthin) (V79HGPRTTest)rdquo DSM Report B-0153078 1986

[18] R Strobel ldquoUnscheduled DNA synthesis assay with thecarotenoid Ro 01-9509007 (all-trans-Zeaxanthin) using pri-mary cultures of rat hepatocytesrdquo DSM Report B-0153081 1987

[19] R Strobel and A Bonhoff ldquoChromosome analysis ofhuman peripheral blood lymphocytes exposed in vitro tothe carotenoid Ro 01-9509007 (all-trans-(3R 31015840R) zeaxanthin)in the presence and absence of a rat liver activation systemrdquoDSM Report B-0153083 1987

[20] F Galandere ldquoMutagenicity studies with Ro 01-9509 in mam-malian systems 1 The micronucleus test in the mouserdquo DSMReport B-0090156 1980

[21] H P Baechtold ldquoAcute toxicity studies with zeaxanthin andits precursorsrdquo Interoffice Memorandum 7100 Hoffmann-LaRoche Basel Switzerland 1977

[22] G Klecak and H Geleick ldquoDetermination of allergenicity ofcolorants used in products of the pharmaceutical cosmetic andnutrition industries in Guinea pigsrdquo DSM Report B-00765381977

[23] M Csato and G Arcelin ldquoRo 01-9509000 (zeaxanthin) studyof skin sensitization in albino Guinea pigs maximization-testrdquoDSM Report B-0171910 2000

[24] M Csato and G Arcelin ldquoPrimary eye irritation study inrabbitsrdquo DSM Report B-0171911 2000

[25] R Ettlin A Steiger and H Hummler ldquoTolerance study withRo 01-9509007 (zeaxanthin as 10 water soluble beadlets)administered orally as a feed admixture to mice over 13 weeksrdquoDSM Report B-0093153 1980


[26] R Ettlin A Steiger and H Hummler ldquoTolerance study withRo 01-9509007 (zeaxanthin as 10 water soluble beadlets)administered orally as a feed admixture to rats over 13 weeksrdquoDSM Report B-0093152 1980

[27] S Buser ldquoA 13-week toxicity study with Ro 01-9509013 in therat po (feed admix)rdquo DSM Report B-0105657 1985

[28] R A Ettlin ldquo13Week tolerance study of Ro 01-9509013 admin-istered orally in capsules to dogsrdquo DSMReport B-0105639 1985

[29] A Kistler ldquoEmbryotoxicity and teratogenicity study in rats withoral administration (feed admix) of Ro 01-9509 zeaxanthinSegment II-teratological study with postnatal evaluationrdquo DSMReport B-0104974 1984

[30] A Kistler ldquoEmbryotoxicity and teratogenicity study in rabbitswith oral administration of Ro 01-9509 zeaxanthin Segment II-teratological studyrdquo DSM Report B-0104954 1983

[31] J Edwards S Clode J Schierle and N Decker ldquoZeaxanthin10 WS beadlets (Ro 01 9509) two generation oral (dietaryadministration) reproduction toxicity study in the ratrdquo DSMReport No-2500072 2006

[32] S Gradelet P Astorg J Leclerc J Chevalier M-F Vernevautand M-H Siess ldquoEffects of canthaxanthin astaxanthinlycopene and lutein on liver xenobiotic-metabolizing enzymesin the ratrdquo Xenobiotica vol 26 no 1 pp 49ndash63 1996

[33] E Wolz H Liechti B Notter G Oesterhelt and A KistlerldquoCharacterization of metabolites of astaxanthin in primary cul-tures of rat hepatocytesrdquo Drug Metabolism and Disposition vol27 no 4 pp 456ndash462 1999

[34] P Astorg S Gradelet J Leclerc and M-H Siess ldquoEffects ofprovitamin A or non-provitamin A carotenoids on liverxenobiotic-metabolizing enzymes in micerdquo Nutrition and Can-cer vol 27 no 3 pp 245ndash249 1997

[35] A Kistler H Liechti L Pichard et al ldquoMetabolism and CYP-inducer properties of astaxanthin in man and primary humanhepatocytesrdquo Archives of Toxicology vol 75 no 11 pp 665ndash6752002

[36] EFSA ANS Panel ldquoScientific opinion on the re-evaluation oflutein (E 161b) as a food additiverdquo EFSA Journal vol 8 no 7article 1678 2010

[37] EFSANDA Panel ldquoStatement on the safety of synthetic zeaxan-thin as an ingredient in food supplementsrdquo EFSA Journal vol10 no 10 article 2891 2012

[38] D Glatzle J Bausch and F Ringenbach ldquoRadioactivity inexpired air during zeaxanthin balance studies compared toprevious findings for canthaxanthin and astaxanthin with ratsrdquoDSM Report B-0106789 1999

[39] J Bausch D Glatzle M Bruchlen and H Liechti ldquoZeaxanthindistribution study in ratsrdquo DSM Report B 0106776 1999

[40] D Glatzle J Bausch S Moalli F Ringenbach and U MatterldquoZeaxanthin balance studiesrdquo DSM Report B-0106788 1999

[41] K-Q Hu C Liu H Ernst N I Krinsky R M Russell and X-D Wang ldquoThe biochemical characterization of ferret carotene-91015840101015840-monooxygenase catalyzing cleavage of carotenoids invitro and in vivordquo Journal of Biological Chemistry vol 281 no28 pp 19327ndash19338 2006

[42] J RMein G G Dolnikowski H Ernst RM Russell andX-DWang ldquoEnzymatic formation of apo-carotenoids from the xan-thophyll carotenoids lutein zeaxanthin and120573-cryptoxanthin byferret carotene-91015840 101015840-monooxygenaserdquo Archives of Biochem-istry and Biophysics vol 506 no 1 pp 109ndash121 2011

[43] D Hartmann P A Thurmann V Spitzer W Schalch BManner and W Cohn ldquoPlasma kinetics of zeaxanthin and 31015840-dehydro-lutein after multiple oral doses of synthetic zeaxan-thinrdquo The American Journal of Clinical Nutrition vol 79 no 3pp 410ndash417 2004

[44] F Pfannkuch E Wolz C P Aebischer J Schierle B Nigge-mann and U Zuhlke ldquoRo 01-9509 (zeaxanthin 10) and Ro 15-3971 (lutein 10) combined 52-week oral (gavage) pilot toxicitystudy with two carotenoids in the cynomolgus monkeyrdquo DSMReport and Subsequent Report Addenda B-0171423 2000

[45] F Pfannkuch ldquoRo 01-9509 (zeaxanthin 10) and Ro 15-3971(lutein 10) combined 52-week oral (gavage) pilot toxicitystudy with two carotenoids in the cynomolgus monkey (Rocheresearch report No B-0171423) comprehensive overview on eyeexaminationsrdquo DSM Report 1004238 2001

[46] E Zrenner ldquoAdditional evaluation of electroretinography(ERG) and expert commentaryrdquo Roche Addendum 12 2000Regulatory Document 1003501 2000

[47] R Goralczyk F Barker O Froescheis et al ldquoOcular safety oflutein and zeaxanthin in a long-term study in cynomolgousmonkeysrdquo Investigative OphthalmologyampVisual Science vol 43no 12 p 2546 2002 ARVOMeeting Abstracts

[48] R Goralczyk ldquoRo 01-9509 (zeaxanthin 10) and Ro 15-3971(lutein 10) combined 52-week oral (gavage) pilot toxicitystudy with two carotenoids in the cynomolgus monkey pathol-ogy report on eyes amendment to final report no 11rdquo DSMReport 1003526 2000

[49] O Froescheis B Rossi and J Bausch ldquoDetermination oflutein and zeaxanthin in retina and lens by HPLCrdquo RegulatoryDocument 1003501 Hoffmann-La Roche 2000 AddendumNo12 in Amendment to Final Report No 1

[50] F Khachik E London F F DeMoura et al ldquoChronic ingestionof (3R31015840R61015840R)-lutein and (3R31015840R)-zeaxanthin in the femalerhesus macaquerdquo Investigative Ophthalmology amp Visual Sciencevol 47 no 12 pp 5476ndash5486 2006

[51] ATBC Study Group ldquoThe effect of vitamin E and beta caroteneon the incidence of lung cancer and other cancers in malesmokersrdquo The New England Journal of Medicine vol 330 no15 pp 1029ndash1035 1994

[52] G S Omenn G E GoodmanM DThornquist et al ldquoChemo-prevention of lung cancer the beta-carotene and Retinol Effi-cacy Trial (CARET) in high-risk smokers and asbestos-exposedworkersrdquo IARC Scientific Publications vol 136 pp 67ndash85 1996

[53] C Liu X-D Wang R T Bronson D E Smith N I Krinskyand R M Russell ldquoEffects of physiological versus pharmaco-logical 120573-carotene supplementation on cell proliferation andhistopathological changes in the lungs of cigarette smoke-exposed ferretsrdquo Carcinogenesis vol 21 no 12 pp 2245ndash22532000

[54] C Liu R T Bronson R M Russell and X-D Wang ldquo120573-Cryptoxanthin supplementation prevents cigarette smoke-induced lung inflammation oxidative damage and squamousmetaplasia in ferretsrdquo Cancer Prevention Research vol 4 no 8pp 1255ndash1266 2011

[55] S Mannisto S A Smith-Warner D Spiegelman et al ldquoDietarycarotenoids and risk of lung cancer in a pooled analysis ofseven cohort studiesrdquo Cancer Epidemiology Biomarkers andPrevention vol 13 no 1 pp 40ndash48 2004

[56] R A Bone J T Landrum L M Friedes et al ldquoDistributionof lutein and zeaxanthin stereoisomers in the human retinardquoExperimental Eye Research vol 64 no 2 pp 211ndash218 1997


[57] X Xu L Zhang B Shao X Sun C-T Ho and S Li ldquoSafetyevaluation of meso-zeaxanthinrdquo Food Control vol 32 no 2 pp678ndash686 2013

[58] M Sugiura K Ogawa and M Yano ldquoAbsorption storage anddistribution of 120573-cryptoxanthin in rat after chronic adminis-tration of satsuma mandarin (Citrus unshiu MARC) juicerdquoBiological and Pharmaceutical Bulletin vol 36 no 1 pp 147ndash151 2013

[59] R Ravikrishnan S Rusia G Ilamurugan et al ldquoSafety assess-ment of lutein and zeaxanthin (Lutemax 2020) subchronic tox-icity and mutagenicity studiesrdquo Food and Chemical Toxicologyvol 49 no 11 pp 2841ndash2848 2011

[60] K B Ravi K R R Reddy J Shankaranarayanan J V Deshpan-de V Juturu and M G Soni ldquoSafety evaluation of zeaxanthinconcentrate (OmniXan) acute subchronic toxicity and muta-genicity studiesrdquo Food and Chemical Toxicology vol 72 pp 30ndash39 2014

[61] EFSA ANS Panel ldquoScientific opinion on the re-evaluation ofcanthaxanthin (E 161 g) as a food additiverdquo EFSA Journal vol8 no 10 article 1852 2010

[62] J van de Kraats M J Kanis S W Genders and D van NorrenldquoLutein and zeaxanthin measured separately in the livinghuman retina with fundus reflectometryrdquo Investigative Ophthal-mology and Visual Science vol 49 no 12 pp 5568ndash5573 2008

[63] G Forma G Carboni B J Jennings and A IannacconeldquoMeasures of macular function after dietary supplementationwith zeaxanthin (Zx)rdquo Investigative Ophthalmology amp VisualScience vol 52 no 6 p 3637 2011 ARVOMeeting Abstracts

[64] G Carboni G Forma B J Jennings and A LannacconeldquoEffects of zeaxanthin (Zx) supplementation on macular pig-ment optical density (MPOD)rdquo Investigative Ophthalmologyamp Visual Science vol 52 abstract 3622 2011 ARVO MeetingAbstracts

[65] EU Commission ldquoCommission implementing decision of 22January 2013 authorising the placing on themarket of syntheticzeaxanthin as a novel food ingredient under regulation (EC)No25897 of the European Parliament and of the CouncilrdquoOfficialJournal of the European Union 2412013 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


HO

OH

OH

HO

OH

HO

OH

HO

3R 3998400R 6998400R-lutein

3S 3998400S-zeaxanthin

3R 3998400S-zeaxanthin (meso-zeaxanthin)

3R 3998400R-zeaxanthin

Figure 1 Structures for optical isomers of all-trans zeaxanthin and lutein

Table 1 Average intake of lutein and zeaxanthin by age group(Mohamedshah et al 1999) [3]

Age group Lutein(120583gday)

Zeaxanthin(120583gday)

Lutein Zeaxanthinratio

20ndash29 745 178 42 130ndash39 896 174 51 140ndash49 920 187 49 150ndash59 1053 182 58 160ndash69 1056 170 62 170+ 990 170 58 1

There is a considerable amount of safety data for zeax-anthin based mainly on routine regulatory studies with highpurity synthetic zeaxanthin manufactured by DSM Ltd(previously manufactured by F Hoffmann-La Roche Ltd)A series of in vitro and in vivo tests for genotoxicity havebeen undertaken as well as subchronic safety studies (13weeks in duration) by dietary exposure at high dosage levelsin mice rats and dogs Developmental toxicity studies havebeen undertaken in rats and rabbits a two-generation study

was performed on the rat and a chronic study of 52 weeksrsquoduration was performed on Cynomolgus monkeys ADME(absorption distribution metabolism and excretion) studieshave been undertaken These studies with zeaxanthin arereviewed here

Potentially data for certain closely related substancesmay have relevance or should be taken into considerationin the safety evaluation of zeaxanthin The inclusion of aferret study with the related xanthophyll 120573-cryptoxanthinon a read-across basis to address the question if zeaxanthinconsumption might have an adverse impact on cigarettesmokers is described Reference is also made to knownstudies with lutein and meso-zeaxanthin

Safety data from human intervention studies in whichsynthetic zeaxanthin has been supplemented of which theAREDSII study is by far the largest is also considered andthe apparent safe level of intake from these studies comparedwith that derived from the animal studies

The safety data for lutein have been evaluated by theEuropean Food Safety Authority (EFSA) Due to the closesimilarity of lutein and zeaxanthin it is probable that thetoxicology for the pure substances is very similar althoughit has to be remembered that within the eye a highly specific
























3 Results













50




























1)



1





























CMO2

Zeaxanthin

CMO2

HO

O

H

OHO

HO

O

H

O

O

H

OHO

HO

910

10998400

9998400

HO

OH9

1010998400

9998400


3-OH-120573-ionone



120573-ionone

3-OH-120573-ionone




































































5 Conclusion







Disclosure




Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































3 Results













50




























1)



1





























CMO2

Zeaxanthin

CMO2

HO

O

H

OHO

HO

O

H

O

O

H

OHO

HO

910

10998400

9998400

HO

OH9

1010998400

9998400


3-OH-120573-ionone



120573-ionone

3-OH-120573-ionone




































































5 Conclusion







Disclosure




Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Results













50




























1)



1





























CMO2

Zeaxanthin

CMO2

HO

O

H

OHO

HO

O

H

O

O

H

OHO

HO

910

10998400

9998400

HO

OH9

1010998400

9998400


3-OH-120573-ionone



120573-ionone

3-OH-120573-ionone




































































5 Conclusion







Disclosure




Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















50




























1)



1





























CMO2

Zeaxanthin

CMO2

HO

O

H

OHO

HO

O

H

O

O

H

OHO

HO

910

10998400

9998400

HO

OH9

1010998400

9998400


3-OH-120573-ionone



120573-ionone

3-OH-120573-ionone




































































5 Conclusion







Disclosure




Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























1)



1





























CMO2

Zeaxanthin

CMO2

HO

O

H

OHO

HO

O

H

O

O

H

OHO

HO

910

10998400

9998400

HO

OH9

1010998400

9998400


3-OH-120573-ionone



120573-ionone

3-OH-120573-ionone




































































5 Conclusion







Disclosure




Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































CMO2

Zeaxanthin

CMO2

HO

O

H

OHO

HO

O

H

O

O

H

OHO

HO

910

10998400

9998400

HO

OH9

1010998400

9998400


3-OH-120573-ionone



120573-ionone

3-OH-120573-ionone




































































5 Conclusion







Disclosure




Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CMO2

Zeaxanthin

CMO2

HO

O

H

OHO

HO

O

H

O

O

H

OHO

HO

910

10998400

9998400

HO

OH9

1010998400

9998400


3-OH-120573-ionone



120573-ionone

3-OH-120573-ionone




































































5 Conclusion







Disclosure




Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































































5 Conclusion







Disclosure




Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























































5 Conclusion







Disclosure




Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































5 Conclusion







Disclosure




Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























5 Conclusion







Disclosure




Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Disclosure




Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Zeaxanthin: Review of Toxicological Data and Acceptable Daily Intake