Biodegradable chitosan-sodium alginate-oleic acid nanocarrier promotes bioavailability and target delivery of lutein in rat model with no toxicity.

Efficient delivery of macular carotenoid lutein to target retinal tissue is possible with enhanced intestinal uptake remains a major challenge owing to the polarity, sensitivity to light, heat and solubility. In this study, to overcome such constraints, biodegradable polymers chitosan-sodium alginate-oleic acid based nano-carrier loaded with lutein (LNCs) was prepared and safety efficacy was examined in vivo. Acute-toxicity of LNCs (0.1, 1, 10 and 100 mg/kg body weight) revealed that the LD50 of LNCs was higher than 100 mg/kg body weight. In subacute-toxicity of LNCs (1 and 10 mg/kg body weight) revealed no mortality with no morphological and clinical changes in rats. Histology, haematology and biochemical analysis of urine and plasma confirmed no toxicity of LNCs compared to control. Post-prandial plasma and tissue (retina) levels of lutein from LNCs were higher. Results demonstrate increased bioavailability of lutein from LNCs with no toxicity suggests applications in food and pharma.

Cytoprotective effects of carotenoids-rich extract from Lycium barbarum L. on the beauvericin-induced cytotoxicity on Caco-2 cells.

In this work, the cytotoxicity of Beauvericin (BEA), lutein (LUT), zeaxanthin (ZEAX) and goji berries extract (GBE) rich in carotenoids, was investigated, as well as cytoprotective effects of these carotenoids against BEA induced-cytotoxicity on Caco-2 cells. Cytotoxicity was carried out using MTT and protein content (PC) assays during 24 and 48 h of exposure. Only BEA showed cytotoxic effect obtaining a reduction in cell proliferation range from 6.5 to 92.8%. Simultaneous combination of LUT and ZEAX with BEA slightly increased cell proliferation compared to BEA tested alone. LUT, ZEAX and GBE showed cytoprotective effects against cytotoxicity induced by BEA on Caco-2 cells. Pre-treatment assays showed the highest cytoprotection effect at the highest dose of BEA assayed (2.5 µM) in 29%, 31% and 35% for LUT, ZEAX and LUT + ZEAX, respectively; GBE showed a cytoprotection of 20%, for the same dose of BEA. The interaction between LUT, ZEAX and BEA studied by means of CI-isobologram method showed a synergism and antagaonism effect for all the combinations tested. These findings highlight that food containing high level of carotenoids, as goji berries, could contribute to reduce the toxicological risk that natural contaminant as BEA mycotoxin in diet can produce to the humans.

Lutein derived from marigold (Tagetes erecta) petals triggers ROS generation and activates Bax and caspase-3 mediated apoptosis of human cervical carcinoma (HeLa) cells.

The present investigation was designed to determine molecular and cellular events involved in anticancer properties of lutein derived from marigold (Tagetes erecta) petals using Human cervical carcinoma (HeLa) cell lines. In vitro experiments demonstrated that lutein at concentration of 10 µM significantly inhibited proliferation of HeLa cells by up to 62.85% after 24 h of treatment and 84.85% after 48 h of treatment. In addition, lutein inhibited proliferation of HeLa cells in a dose-dependent manner by inducing apoptosis. Lutein-treated HeLa cells also showed enhanced accumulation of reactive oxygen species (ROS) correlated with significant downregulation of Bcl-2 (anti-apoptotic) expression and upregulation of Bax (pro-apoptotic) expression. Furthermore, lutein mediated activation of caspase-3 and imbalance between Bax and Bcl-2 expression, causing significant loss of mitochondrial membrane potential of HeLa cells. TUNEL assays revealed significant damage of nuclei DNA in lutein-treated HeLa cells, demonstrating a critical role of lutein in the final stage of apoptosis. Taken together, the results indicate that lutein-induced apoptosis of HeLa cells occurs through enhanced ROS production, interaction with mitochondrial factors, and upregulation of caspase-3-mediated pathway, leading to fragmentation of nuclei DNA. Therefore, lutein could be potentially useful as a chemotherapeutic and/or chemopreventive biomolecule against Human cervical carcinoma.

Investigating retinal toxicity of a lutein-based dye in a model of isolated and perfused bovine retina.

PURPOSE: Retidyne™ is a new lutein-based dye for internal limiting membrane staining. It uses the intrinsic staining characteristics of lutein which is already known to act as an antioxidant and blue-light filter in the human retina. We investigated retinal tolerance to different staining times measured by the electroretinogram (ERG) of an isolated and perfused retina whole mount. METHODS: For functionality, testing bovine retinas were prepared and perfused with an oxygen saturated standard solution and the ERG was recorded until stable b-wave amplitudes were reached. Then the perfusion was stopped and Retidyne™ was applied directly onto the retinal surface for exposure times of 60 or 120 s. After restarting the perfusion with standard solution, the ERG amplitudes were monitored for 75 min. To investigate the effects on photoreceptor function alone, 1 mM asparate was added to block b-waves. RESULTS: For an exposure time of 60 s amplitudes of a- and b-waves remained stable throughout the experiment. Exposure times of 120 s caused an initial drop of amplitudes that reached statistical significance only for a-waves (a, - 21%, p = 0.047; b, - 14%, p = 0.052). This effect was only seen during the first minutes of the washout and the ERG recovered completely. CONCLUSIONS: In the model of isolated and perfused bovine retina, Retidyne™ showed a good safety profile for common intraoperatively used staining times. An initial toxic effect regarding the transient drop of amplitudes cannot be ruled out but the effect might also be explained by the partial blockage of the flashlight due to a more intense staining effect at the beginning of the washout.

Phytoglycogen to increase lutein solubility and its permeation through Caco-2 monolayer.

Increasing the solubility of poorly water-soluble bioactives is essential to enhancing their bioavailability. The objective of this study was to evaluate the impact of phytoglycogen (PG) on water solubility of lutein and its transepithelial permeation across Caco-2 cell monolayers. Solid PG-LT complexes were prepared by combining an acetone solution of LT with an aqueous solution of PG under sonication, followed by centrifugation and vacuum drying of the supernatant as well as a further purification procedure. X-ray powder diffraction and differential scanning calorimetry showed negligible crystalline structure of LT in the PG-LT complex tested. The maximal water solubility of LT (130.65µg/mL) occurred at the PG/LT combination ratio of 53.3/1, which was significantly higher than that of LT alone (0.56µg/mL). Remarkably, LT, when complexed with PG, exhibited much-enhanced permeation through Caco-2 monolayer than LT alone, suggesting a potential role of PG to improve LT bioavailability. This study indicated that PG could be applied for the delivery of LT and possibly other hydrophobic ingredients.

Biocompatible lutein-polymer-lipid nanocapsules: Acute and subacute toxicity and bioavailability in mice.

Lutein-poly-(lactic-co-glycolic acid) (PLGA)-phospholipid (PL) nanocapsules were prepared (henceforth referred as lutein nanocapsules) and studied for acute, subacute oral toxicity and bioavailability of lutein in mice. Prior to examining the safety of lutein nanocapsules, particle size, zeta potential, surface morphology and interaction between lutein, PLGA and PL were studied. In acute study, mice were gavaged with a single dose of lutein nanocapsules at 0.1, 1, 10 and 100mg/kg body weight (BW) and examined for 2weeks, while in subacute study, daily mice were gavaged with a dose of 1 and 10mg/kg BW for 4weeks. Results revealed that mean size and zeta value of lutein nanocapsules were 140nm and -44mV, respectively. Acute and subacute toxicity studies did not show any mortality or treatment related adverse effect in clinical observations, ophthalmic examinations, body and organ weights. No toxicity related findings were observed in hematology, histopathology and other blood and tissue clinical chemistry parameters. In subacute study, no observed adverse effect level (NOAEL) of lutein nanocapsules was found to be at a dose of 10mg/kg BW. Feeding lutein nanocapsules resulted in a significant (p<0.01) increase in lutein level in plasma and tissue compared to the control group. Lutein nanocapsules did not cause toxicity in mice. However, human trials are warranted.

In vitro and in vivo effects of lutein against cisplatin-induced ototoxicity.

INTRODUCTION: Cisplatin is a commonly prescribed drug that produces ototoxicity as a side effect. Lutein is a carotenoid with antioxidant and anti-inflammatory properties previously tested for eye, heart and skin diseases but not evaluated to date in ear diseases. AIM: To evaluate the protective effects of lutein on HEI-OC1 auditory cell line and in a Wistar rat model of cisplatin ototoxicity. MATERIALS AND METHODS: In vitro study: Culture HEI-OC1 cells were exposed to lutein (2.5-100 µM) and to 25 µM cisplatin for 24h. In vivo study: Twenty eight female Wistar rats were randomized into three groups. Group A (n=8) received intratympanic lutein (0.03 mL) (1mg/mL) in the right ear and saline solution in the left one to determine the toxicity of lutein. Group B (n=8) received also intraperitoneal cisplatin (10mg/kg) to test the efficacy of lutein against cisplatin ototoxicity. Group C (n=12) received intratympanic lutein (0.03 mL) (1mg/mL) to quantify lutein in cochlear fluids (30 min, 1h and 5 days after treatment). Hearing function was evaluated by means of Auditory Steady-State Responses before the procedure and 5 days after (groups A and B). Morphological changes were studied by confocal laser scanning microscopy. RESULTS: In vitro study: Lutein significantly reduced the cisplatin-induced cytotoxicity in the HEI-OC1 cells when they were pre-treated with lutein concentrations of 60 and 80 µM. In vivo study: Intratympanic lutein (1mg/mL) application showed no ototoxic effects. However it did not achieve protective effect against cisplatin-induced ototoxicity in Wistar rats. CONCLUSIONS: Although lutein has shown beneficial effects in other pathologies, the present study only obtained protection against cisplatin ototoxicity in culture cells, but not in the in vivo model. The large molecule size, the low dose administered, and restriction to diffusion in the inner ear could account for this negative result.

Lutein and cataract: from bench to bedside.

Cataract is one of the most important leading causes of blindness in the world. Extensive research showed that oxidative stress may play an important role in the initiation and progression of a cataract and other age-related eye diseases. Extra-generation of reactive oxygen and nitrogen species in the eye tissue has been shown as one of the most important risk factors for cataracts and other age-related eye diseases. With respect to this, it can be hypothesized that dietary antioxidants may be useful in the prevention and/or mitigation of cataract. Lutein is an important xanthophyll which is widely found in different vegetables such as spinach, kale and carrots as well as some other foods such as eggs. Lutein is concentrated in the macula and suppresses the oxidative stress in the eye tissues. A plethora of literature has shown that increased lutein consumption has a close correlation with reduction in the incidence of cataract. Despite this general information, there is a negligible number of review articles considering the beneficial effects of lutein on cataracts and age-related eye diseases. The present review is aimed at discussing the role of oxidative stress in the initiation and progression of a cataract and the possible beneficial effects of lutein in maintaining retinal health and fighting cataract. We also provide a perspective on the chemistry, sources, bioavailability and safety of lutein.

Dye solutions based on lutein and zeaxanthin: in vitro and in vivo analysis of ocular toxicity profiles.

PURPOSE: To study the safety profile of Lutein/Zeaxanthin(L/Z)-based natural dye solutions in in vitro and in vivo models. MATERIAL AND METHODS: In vitro cytotoxicity and cellular growth experiments were carried out on ARPE-19 and human corneal epithelial (HCE) cell lines using different L/Z-based dye solutions, either alone or in association with brilliant blue (BB) or trypan blue (TB). Light and transmission electron microscopy studies were performed seven days after intravitreal injection of dye solutions in rabbits. Electroretinogram (ERG) recordings were taken at baseline and before histopathology. RESULTS: In vitro cytotoxicity assays demonstrated that the different L/Z-based solutions (from 0.3 to 2%), either alone or in association with BB (0.025%) or TB (0.04%), did not significantly alter mitochondrial activity (≤15%) in the cell lines tested. In addition, in vitro cell growth was inhibited by up to 60% depending on the dye solution, and in direct proportion to the concentration assayed. There was no evidence of structural alterations in the neurosensory retina, retinal pigment epithelium (RPE), or choriocapillaris-choroidal complex. b-Wave ERG records showed no significant differences (±15.2%) in comparison with baseline. CONCLUSIONS: L/Z-based dye solutions demonstrated a safe profile in in vitro and in vivo models, and may be a useful tool for staining intraocular structures.

Acute and subacute toxicity assessment of lutein in lutein-deficient mice.

Dietary lutein consumption is lower than the actual recommended allowances to prevent macular degeneration; thus dietary lutein supplements have been recommended. This study aimed to investigate potential adverse effect of lutein from Tagetes erecta in lutein-deficient (LD) male mice. Preliminary acute toxicity study revealed that the LD50 exceeded the highest dose of 10000 mg/kg BW. In a subacute study, male mice were gavaged with 0, 100, 1000 mg/kg BW/day for a period of 4 wk. Plasma lutein levels increased dose dependently (P < 0.01) after acute and subacute feeding of lutein in LD mice. Compared to the control (peanut oil without lutein) group, no treatment-related toxicologically significant effects of lutein were prominent in clinical observation, ophthalmic examinations, body, and organ weights. Further, no toxicologically significant findings were eminent in hematological, histopathological, and other clinical chemistry parameters. In the oral subacute toxicity study, the no-observed-adverse-effect level (NOAEL) for lutein in LD mice was determined as 1000 mg/kg/day, the highest dose tested.

Use of lutein and zeaxanthin alone or combined with Brilliant Blue to identify intraocular structures intraoperatively.

PURPOSE: To determine whether a natural dye solution based on lutein and zeaxanthin alone or combined with Brilliant Blue stains and facilitates peeling of intraocular membranes in human eyes. METHODS: In this study of 60 cadaveric eyes, open-sky vitrectomy including posterior hyaloid detachment was performed. Different lutein and zeaxanthin concentrations (0.01-20%) were tested alone or combined with different Brilliant Blue concentrations (0.0125-0.025%) in the corneal endothelium, corneal epithelium, anterior and posterior capsule, vitreous cavity through the macula including the posterior hyaloid, and internal limiting membrane. The various dye solutions were in contact with the intraocular membranes for <1 minute and then were removed by mechanical aspiration or membrane peeling initiated and completed with intraocular forceps. The specimens were examined by light and electron transmission microscopy. RESULTS: Contact between lutein and zeaxanthin and the retinal, lens, and vitreous surface resulted in orange and greenish staining of the intraocular membranes, which facilitated surgical steps in all eyes. Lutein and zeaxanthin alone was useful for vitreous identification and lutein and zeaxanthin combined with Brilliant Blue had strong affinity for internal limiting membrane and anterior capsule. Light microscopy confirmed internal limiting membrane removal in all eyes tested. No dye solutions remained in the eyes after the membrane removal. CONCLUSION: A natural dye solution based on lutein and zeaxanthin alone or combined with Brilliant Blue efficiently stained the anterior capsule, vitreous, and internal limiting membrane in human cadaveric eyes and may be a useful tool for vitreoretinal or cataract surgery.

Lutein improves antioxidant defense in vivo and protects against DNA damage and chromosome instability induced by cisplatin.

Lutein (LT) is the second most prevalent carotenoid in human serum, and it is abundantly present in dark, leafy green vegetables. The objectives of this study were to evaluate the genotoxicity and mutagenicity of LT, and its protective effects in vivo against DNA damage and chromosome instability induced by cisplatin (cDDP). For this purpose, we used the comet assay and micronucleus (MN) test, and we evaluated the antioxidant effects of LT by determination of enzymatic (catalase-CAT) and non-enzymatic (reduced glutathione-GSH) activity. Mice were divided into six groups: cDDP, mineral oil (OM), LT groups and LT + cDDP groups. To perform the MN test on peripheral blood (PB) cells, blood samples were collected before the first treatment (T0), and 36 h (T1) and 14 days (T2) after the first treatment. To perform the comet assay, blood samples were collected 4 h after the first and the last treatment. Oxidative capacity was analyzed in total blood that was collected 24 h after the last treatment, when bone marrow (BM) sample was also collected for the MN test. No genotoxic or mutagenic effects of LT were observed for the doses evaluated. We did find that this carotenoid was able to reduce the formation of crosslinks and chromosome instability induced by cDDP. No differences were observed in CAT levels, and LT treatment increased GSH levels compared with a negative control group, reinforcing the role of this carotenoid as an antioxidant.

Genotoxic effects of carotenoid breakdown products in human retinal pigment epithelial cells.

PURPOSE: To investigate the genotoxic effects of lutein (LBP) and beta -carotene breakdown products (beta -apo-8-carotenal, BA8C) and the preventive role of GSH in human retinal pigment epithelial cells (ARPE-19). METHODS: LBP- and BA8C-induced DNA damage in human retinal pigment epithelial cells (ARPE-19) was determined by comet assay. The DNA damage was quantified by the image analysis system using Comet Score software. ARPE-19 cell viability was determined by CellTiter 96 AQ(ueous) one-solution cell proliferation assay kit. Intracellular GSH levels were measured by Ellman's reagent. RESULTS: Incubation of serum-starved ARPE-19 cells with LBP and BA8C caused significant DNA damage in a dose- and time-dependent manner. The DNA damage and cell death incurred by LBP and BA8C were significantly prevented by N-acetylcysteine (NAC) but not by alpha -tocopherol + ascorbic acid (T + AA). Furthermore, BSO-induced GSH depletion in ARPE-19 cells caused a significant elevation in LBP- and BA8C-induced DNA damage, whereas increased GSH levels in ARPE-19 cells prevented it. CONCLUSIONS: Our results suggest that breakdown products of dietary carotenoids could be genotoxic in ARPE-19 cells. LBP-induced genotoxic effects could worsen oxidative stress. The intracellular GSH pool in ARPE-19 cells might play a critical role in carotenoid breakdown products-induced genotoxicity.

Phototoxicity and cytotoxicity of fullerol in human lens epithelial cells.

The water-soluble, hydroxylated fullerene [fullerol, nano-C60(OH)22-26] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have assessed fullerol's potential ocular toxicity by measuring its cytotoxicity and phototoxicity induced by UVA and visible light in vitro with human lens epithelial cells (HLE B-3). Accumulation of nano-C60(OH)22-26 in the cells was confirmed spectrophotometrically at 405 nm and cell viability estimated using MTS and LDH assays. Fullerol was cytotoxic to HLE B-3 cells maintained in the dark at concentrations higher than 20 microM. Exposure to either UVA or visible light in the presence of >5 microM fullerol-induced phototoxic damage. When cells were pretreated with non-toxic antioxidants: 20 microM lutein, 1 mM N-acetyl cysteine, or 1 mM l-ascorbic acid prior to irradiation, only the singlet oxygen quencher-lutein significantly protected against fullerol photodamage. Apoptosis was observed in lens cells treated with fullerol whether or not the cells were irradiated, in the order UVA>visible light>dark. Dynamic light scattering (DLS) showed that in the presence of the endogenous lens protein alpha-crystallin, large aggregates of fullerol were reduced. In conclusion, fullerol is both cytotoxic and phototoxic to human lens epithelial cells. Although the acute toxicity of water-soluble nano-C60(OH)22-26 is low, these compounds are retained in the body for long periods, raising concern for their chronic toxic effect. Before fullerols are used to deliver drugs to the eye, they should be tested for photo- and cytotoxicity in vivo.

Toxicity profile of lutein and lutein ester isolated from marigold flowers (Tagetes erecta).

Lutein is a carotenoid with antioxidant properties and is commonly present in many fruits, vegetables, and egg yolk. Lutein affords protection against the development of the two common eye diseases of aging: cataract and macular degeneration. As the dietary lutein concentration is much lower compared to the actual requirement to reduce macular degeneration, supplementation of lutein is under consideration. There are very few data on the toxicity of lutein. In the present study, the authors have evaluated the short-term and long-term toxicity profile of lutein and its esterified form isolated from marigold flowers (Tagetes erecta) in young adult male and female Wistar rats. Lutein and its ester form administered orally at doses of 4, 40, and 400 mg/kg body weight for 4 weeks for short-term toxicity study and 13 weeks for a subchronic toxicity study did not produced any mortality, change in body weight, food consumption pattern, organ weight, and other adverse side reactions. Administration of lutein and ester form did not alter the hepatic and renal function, and did not produce any change in the hematological parameters and in lipid profile. Histopathological analysis of the organs supported the nontoxicity of lutein and its ester form.

Chronic ingestion of (3R,3'R,6'R)-lutein and (3R,3'R)-zeaxanthin in the female rhesus macaque.

PURPOSE: To investigate how supplementation of the monkey's diet with high doses of lutein (L), zeaxanthin (Z), or a combination of the two affects the plasma levels and ocular tissue deposition of these carotenoids and their metabolites over time and to determine whether these high doses can cause ocular toxicity. METHODS: Eighteen female rhesus monkeys were divided into groups of control (n = 3 control), L-treated (n = 5, 9.34 mg lutein/kg and 0.66 mg zeaxanthin/kg), Z-treated (n = 5, 10 mg zeaxanthin/kg), and L/Z-treated (n = 5, lutein and zeaxanthin, each 0.5 mg/kg). After 12 months of daily supplementation, one control animal, two L-treated animals, two Z-treated animals, and all the L/Z-treated animals were killed. The rest of the monkeys were killed after an additional six months without supplementation. Plasma and ocular tissue carotenoid analyses, fundus photography, and retina histopathology were performed on the animals. RESULTS: Supplementation of monkeys with L and/or Z increased the mean plasma and ocular tissue concentrations of these carotenoids and their metabolites. The mean levels of L and Z in the retinas of the L- and Z-treated animals after 1 year increased significantly over baseline. High dose supplementation of monkeys with L or Z did not cause ocular toxicity and had no effect on biomarkers associated with kidney toxicity. CONCLUSIONS: The mean levels of L and Z in plasma and ocular tissues of the rhesus monkeys increase with supplementation and in most cases correlate with the levels of their metabolites. Supplementation of monkeys with L or Z at high doses, or their combination does not cause ocular toxicity.

Antioxidant activity, mutagenicity/anti-mutagenicity, and clastogenicity/anti-clastogenicity of lutein from marigold flowers.

High dietary intake of lutein has been associated with risk reduction of many chronic diseases, including age-related macular degeneration (AMD), cancer, and cardiovascular diseases. Lutein in food is generally regarded as safe. However, information on the toxicological and beneficial effect of lutein at higher doses is limited. In this study, large amount of lutein was extracted and purified from marigold flower (Tagetes erecta L.). The antioxidant activity of lutein was examined by using the photochemiluminescence (PCL) assay and the beta-carotene-linoleic acid model system (beta-CLAMS). Lutein showed a greater antioxidant activity than the other two common carotenoids, beta-carotene and lycopene. The mutagenicity and anti-mutagenicity of lutein at 334, 668 and 1335 microg/plate were examined using the standard Ames test in the presence and absence of S9 mix. Lutein was not only found to be non-mutagenic at all doses, but it showed an anti-mutagenic effect in a dose-dependent manner. Similar results were found in a chromosome aberration test using Chinese hamster ovary cells for the evaluation of clastogenicity and anti-clastogenicity of lutein at 66.8, 133.5 and 267.0 mg/L. Our findings provided scientific evidence for the safe use and health beneficial effects of lutein.

The science behind lutein.

In humans, as in plants, the xanthophyll lutein is believed to function in two important ways: first as a filter of high energy blue light, and second as an antioxidant that quenches and scavenges photo induced reactive oxygen species (ROS). Evidence suggests that lutein consumption is inversely related to eye diseases such as age-related macular degeneration (AMD) and cataracts. This is supported by the finding that lutein (and a stereo isomer, zeaxanthin) are deposited in the lens and the macula lutea, an area of the retina responsible for central and high acuity vision. Human intervention studies show that lutein supplementation results in increased macular pigment and improved vision in patients with AMD and other ocular diseases. Lutein may also serve to protect skin from UV-induced damage and may help reduce the risk of cardiovascular disease. Crystalline lutein is readily absorbed from foods and from dietary supplements whereas, to enter the bloodstream, lutein esters require prior de-esterification by intestinal enzymes. Unlike the hydrocarbon carotenoids which are mainly found in the LDL fraction, xanthophylls like lutein and zeaxanthin are incorporated into both HDL and LDL. Today, lutein can be obtained from the diet in several different ways, including via supplements, and most recently in functional foods. Animal toxicology studies have been performed to established lutein's safety as a nutrient. These studies have contributed to the classification of purified crystalline lutein as generally recognized as safe (GRAS). The achievement of GRAS status for purified crystalline lutein allows for the addition of this form into several food and beverage applications. This achievement speaks directly to the quality and safety of purified lutein.

An innovative approach to the determination of safety for a dietary ingredient derived from a new source: case study using a crystalline lutein product.

Lutein and zeaxanthin are antioxidant carotenoids that occur naturally in the diet. A new source of these carotenoids, a crystalline lutein product, is an extract from the marigold flower (Tagetes erecta) that contains approximately 86% by weight of the carotenoids lutein and zeaxanthin. The safety of consumption of a crystalline lutein product used as an ingredient in food is determined by evaluating the safety of ingestion of the whole product, as well as safety of ingestion of the major constituents, lutein and zeaxanthin. The approach to evaluating the safety of increased lutein and zeaxanthin intake from consumption of crystalline lutein product is based on an evaluation of the incremental increase this ingestion will produce in lutein and zeaxanthin and in total carotenoids, compared to background exposure. In addition, bioavailability of lutein+zeaxanthin from crystalline lutein product, when added to food, is determined and used to adjust the estimated daily intake of lutein and zeaxanthin from this new source and standardize it to the bioavailable dose of these carotenoids from food sources. The proposed level of ingestion of lutein and zeaxanthin from the crystalline lutein product would increase intake of lutein zeaxanthin in the generally vegetable-poor American diet to a level comparable to the mean intake of individuals consuming the recommended number of servings of vegetables per day and is therefore determined to be both prudent and safe. Safety of consumption of the whole product is determined by evaluating the source of the product, production process, nature and quantity of impurities, and product specifications. Corroboration of safety is provided by animal toxicology studies of the crystalline lutein product, as well as human and epidemiologic studies of lutein and zeaxanthin intake. It can be concluded that crystalline lutein is a safe and GRAS source of lutein for its proposed uses in food.