Gras notice 000394: spirulina (anthrospira platensis)

ORIGINAL SUBMISSION Spirulina platensis GRAS self affirmation 7-1-2011 Executive Summary
The objective of this Generally Recognized as Safe (GRAS) determination is to summarize the available safety information on Spirulina platensis, which is used as an ingredient in foods and beverages. We, the undersigned expert panel members, Susan Cho, Ph.D., Joanne Slavin, Ph.D., and George C. Fahey, Jr., Ph.D., have individually and collectively critically evaluated the materials summarized in the Spirulina platensis GRAS report. We conclude that Spirulina platensis is safe and GRAS for its intended use in food. There is broad-based and widely disseminated knowledge concerning the chemistry and health benefits of Spirulina platensis in both human and animals. Pursuant to 21 CFR § 170.30, this GRAS determination for Spirulina platensis is based on scientific procedures. There are no indications of significant adverse effects related to Spirulina platensis in the publicly available literature, and the manufacturing process of Spirulina platensis does not employ any treatments with organic solvents. In the United States, Spirulina platensis has been already recognized as a GRAS substance since 2003 (FDA, GRN 000127). Since that time, several toxicity and human clinical studies have been published to report higher values of safe intake levels than the previously reported. This GRAS notice captures the findings from recent studies. Documentation qualifying a substance as GRAS has been compiled. Such documentation includes technical evidence and common knowledge of safety, as recognized by qualified experts (the Expert Panel). Technical evidence of safety includes the chemical identity of the substance, the method of manufacture, analytical data on composition and specifications, safety data from animal and human clinical studies, and nutritional benefits from animal and human clinical studies. Spirulina platensis belongs to the "practically nontoxic" category, according to a toxicity rating chart. Intended use includes granola bars, cereal bars, protein bars and power bars, meal replacements and mixes, sports beverages, energy drinks, energy soft drinks, fruit juices, low calorie fruit and vegetable juice drinks, low fat soy milk, and medical foods. As described in GRN 000127, Spirulina platensis is not intended for use in meat or poultry-containing products or as a coloring agent. Assuming that 100% of the product will be used at the maximum levels under the intended use, the 90th percentile intakes from the intended use by users of one or more foods are 13.5 g/d (208 mg/kg BW/d) for the population aged 1 year and above (combining males and females), 15.5 g/d (241 mg/kg BW/d) for males, and 7.5 g/d (139 mg/kg BW/d) for females (Table 3). After adjustments for market shares, the 90th percentile intakes by users of one or more foods are 1.35 g/d (20.8 mg/kg BW/d) for the population combining males and females, 1.55 g/d (or 24.1 mg/kg BW/d) for males, and 0.75 g/d (13.9 mg/kg BW/d) for females. Spirulina platensis GRAS self affirmation 7-1-2011 Even if all of the products are used at the maximum levels, exposure estimate levels are much lower than the no-observed-adverse-effect level (NOAEL) values (10,000 mg/kg BW/d) that have been found from toxicity studies in animals and a proven safe intake level of 4,132 mg/kg BW/d which has been found from human clinical trials. Therefore, the proposed use of Spirulina platensis is not only safe within the terms of the Federal Food, Drug, and Cosmetic Act (meeting the standard of reasonable certainty of no harm), but it is also GRAS according to Title 21 Code of Federal Regulations (21 CFR) because of this consensus among experts. Susan Cho, Ph.D. President, NutraSource, Inc., Clarksville, MD 21029 Signature: _ Date: _ Joanne Slavin, Ph.D., R.D. Professor, University of Minnesota, St. Paul, MN 55108 Signature: _ Date: _ George C. Fahey, Jr., Ph.D. Professor Emeritus, University of Illinois, Urbana, IL 61801 Signature: _ Date: _ Spirulina platensis GRAS self affirmation 7-1-2011 I. Identity of Substance
A. Common or trade name: Spirulina, organic Spirulina, Spirulina platensis,
Arthrospira, or Arthrospira platensis.

B. Standards of identity: We note that an ingredient that is lawfully added to food
products may be used in a standardized food only if it is permitted by the applicable
standard of identity that is located in Title 21 of the Code of Federal Regulations.
C. Background
Spirulina (Arthrospira), a class of cyanobacteria, is a free-floating filamentous
microalgae which is capable of photosynthesis (Ciferri and Tiboni, 1985; Komarek et al.,
2009; Sapp, 2005 ). Spirulina naturally grows in high-salt alkaline water reservoirs in
subtropical and tropical areas including Hawaii, Mexico, Asia, and Central Africa
(Gershwin and Belay, 2008). Among the Spirulina species, Spirulina platensis
(Arthrospira platensis), Spirulina maxima (Arthrospira maxima), and Spirulina fusiformis
(Arthrospira fusiformis) have been most intensively studied (Gershwin and Belay, 2008;
Karkos et al., 2008; Khan et al., 2005).
The nutritional value of Spirulina is well recognized with its exceptionally high content of
protein (60–70% by dry weight), vitamins, minerals, essential fatty acids, and other
nutrients (Annapurna et al., 1991; Deng et al., 2010; McCarty, 2010). The 2008 FAO
position paper describes Spirulina as follows: "An easily digestible high (c. 60%) protein
product with high levels of beta-carotene, vitamin B12, iron and trace minerals, and the
rare essential fatty acid y-linolenic acid [also called gamma-linolenic acid (GLA), or
omega-6".
Over the history of safe use of Spirulina, it has been generally recognized as safe (GRAS) for human consumption. Human clinical studies and animal studies over the past several decades support such notion. Thus, FDA had no question on the GRAS notice (GRN 127) of Spirulina platensis (FDA, 2003). D. General properties of Spirulina platensis
Spirulina is a free-flowing, dark blue-green powder with a mild seaweed smell, produced by spray drying the biomass of the cyanobacterium, Arthrospira platensis (Dillon et al. 1995). It is not readily soluble in water or solvents, but it forms a suspension when mixed with water. E. Manufacturing Process
E.1. Conventional Spirulina
1. Selection and culture preparation of Spirulina seed in the lab: The certified culture obtained from the Academy of Sciences of China is cultured in the lab in media using ultra-high purity fertilizer in triangular flasks and glass bottles. These Spirulina platensis GRAS self affirmation 7-1-2011 are cultivated at 18-38○C. 2. Cultivation in Spirulina farming pool: The cultured Spirulina seed is cultivated in large ponds, which contains food grade fertilizers (like sodium bicarbonate, potassium chloride, potassium dihydrogen phosphate, and salt), water, and carbon dioxide. The Spirulina is harvested after a series of QC tests are performed including microscopic testing. 3. Spray drying: The harvested Spirulina then is dried using a spray drying system after washing with water to bring the pH to neutral from alkaline. The Spirulina droplets are sprayed into the chamber from the top of the tower to flash evaporate the water. 4. Packaging: The Spirulina powder then is packaged. E.2. Organic Spirulina 1. For organic Spirulina, Spirulina seeds are cultivated at 18-38○C with ultra-high purity fertilizer in triangular flasks and glass bottles. 2. The cultured Spirulina seed is cultivated in large ponds, which contains organic fertilizers (non-allergenic soy dregs, non-allergenic peanut dreg, humic acid, natural potassium ore powder, and CO2), water, and carbon dioxide. 3. The Spirulina is harvested after a series of QC tests are performed including microscopic testing. 4. The harvested Spirulina then is dried using a spray drying system after washing with water to bring the pH to neutral from alkaline. The Spirulina droplets are sprayed into the chamber from the top of the tower to flash evaporate the water. 5. The Spirulina powder then is packaged. Spirulina platensis GRAS self affirmation 7-1-2011 F. Specifications

Tables 1a and 1b lists specifications of conventional and organic Spirulina.
Table 1a. Specifications of conventional Spirulina powder Product Name: Spirulina Powder (conventional) Arthrospira platensis Carrier/Preservatives: Country of Origin: Appearance Visual FTIR/scientific method Complies to standard Mild like sea weed AOAC 973.03 (TQ-106) 100% through 80 mesh Paper chromatography Crude phycocyanin ICP/MS AOAC 993.14 ICP/MS AOAC 993.14 ICP/MS AOAC 993.14 Informative, test annually or every 5 lots ICP/MS AOAC 993.14 Informative, test annually or every 5 lots SW846/7196A (EPA) Informative, test annually or every 5 lots Microbiology Aerobic plate count < 100,000 cfu/g USP33, NF28, 2010 Salmonella USP33, NF28, 2010 Staphylococcus aureus USP33, NF28, 2010 Yeast/mold FDA-BAM, Informative, test annually or every 5 lots AOAC 991.31 (HPLC)/ < 20 ppb, test annually or every 5 lots AOAC 990.34 (AFLA-20 Cup Test Kit) *This is a natural product and there could be color and taste variations from lot to lot due to crop fluctuations from harvest to another. Spirulina platensis GRAS self affirmation 7-1-2011 Table 1b. Specifications of organic Spirulina powder Product Name: Spirulina Powder - Organic Botanical Name: Arthrospira platensis Product Code: Carrier: None Country of Origin: China Fine dark green powder FTIR/scientific method Complies to standard UV Spectrophotometry 11-14 mg/g, test semi-annually Carotenoids AOAC 4.0-5.5 mg/g, test semi-annually AOAC 973.03 (TQ-106) 100% pass on 80 Mesh Crude phycocyanin UV Spectrophotometry 12-19%, test semi-annually ICP/MS AOAC 993.14 ICP/MS AOAC 993.14 ICP/MS AOAC 993.14 Informative, test annually (ppm) ICP/MS AOAC 993.14 Informative, test annually (ppm) SW846/7196A (EPA) Informative, test annually (ppm) Aerobic plate count < 100,000 cfu/g USP33, NF28, 2010 Salmonella USP33, NF28, 2010 Staphylococcus aureus USP33, NF28, 2010 FDA-BAM 7th ed. AOAC 991.31 (HPLC)/ < 20 ppb, test annually AOAC 990.34 (AFLA-20 Cup Test Kit) *This is a natural product and there may be color and taste variations from lot to lot due to crop fluctuations from harvest to harvest. Quality control process After harvest, the products, product was washed several times, and then spray dried at high temperature. End product control: every lot of product is tested for microbial, heavy Spirulina platensis GRAS self affirmation 7-1-2011 metal, and foreign material contamination. The product must pass evaluation before it is released. An important quality control issue surrounding production of cyanobacteria is
the possibility of inadvertently harvesting other cyanobacteria containing cyanotoxins.
This is a risk when harvesting algae from natural bodies of water containing mixed
populations of phytoplankton, but is unlikely to be a problem with the tightly
controlled Arthrospira platensis monocultures utilized by the notifier. However,
Spirulina is periodically assayed for microcystin and nodularin toxins by ELISA
analysis using in-house testing as well as independent testing.
II. Natural occurrence and exposure to Spirulina platensis
A. Use of Spirulina as a foodstuff
Spirulina has been safely consumed as a food ingredient in Mexico and Central Africa for the past four centuries and is currently used as a dietary supplement and a novel food ingredient, especially among Asians, including Asian-Americans (Ciferri and Tiboni, 1985). B. Intended use
Table 2 presents primary applications of Spirulina platensis that include granola bars, cereal bars, protein bars and power bars, meal replacements and mixes, sports beverages, energy drinks, energy soft drinks, lime juice, blackberry juice, grape juice, low calorie fruit and vegetable juice drinks, low fat soy milk, and medical foods. As described in GRN 000127, Spirulina platensis is not intended for use in meat or poultry-containing products or as a coloring agent. Table 2. Intended use of Spirulina platensis Proposed food use Granola bars, cereal bars, protein bars, and power 30 bars Meal replacement and mixes Sports beverages Energy soft drinks Fruit juices, such as lime juice, blackberry juice, grape juice; low calorie fruit and vegetable juice drinks Low fat soy milk Spirulina platensis GRAS self affirmation 7-1-2011 C. Probable consumption of Spirulina platensis
Using food intake data reported in the 2005-2008 NHANES, exposure levels to Spirulina
platensis that will result from the intended uses were estimated. Results of the exposure
estimates under the intended use of Spirulina platensis in foods for the U.S. population
ages 1 y and older are presented in Tables 3 and 4.
The first set of estimates is based on the assumption that all the products in each food
category will be used at the maximum intake levels under the intended use although it is
far from a realistic situation. The 90th percentile intakes from the intended use by users
of one or more foods are 13.5 g/d (208 mg/kg BW/d) for the population aged 1 y and
above (combining males and females), 15.5 g/d (241 mg/kg BW/d) for males and 7.5 g/d
(139 mg/kg BW/d) for females (Table 3).
The second set of estimates is based on the market adjustment which assumes that
10% of the products in each food category will be used at the maximum intake levels
under the intended use (Table 4). From a marketing perspective, an assumption that
10% of the product will be used at the maximum levels for each food category is a
highly optimistic projection. It is not possible to use all the foods under the intended use.
Also, wastage and other losses should be considered. The 90th percentile intakes by
users of one or more foods are 1.35 g/d (20.8 mg/kg BW/d) for the population
combining males and females, 1.55 g/d (or 24.1 mg/kg BW/d) for males, and 0.75 g/d
(13.9 mg/kg BW/d) for females.
These estimated daily exposure levels are far lower than the no-observed-adverse-
effect level (NOAEL) values (S. platensis, 10,000 mg/kg BW/d, the maximum levels
tested; other Spirulina species, 30,000 mg/kg BW/d) that have been found from various
toxicity studies in rats. Also, these levels are far below the safe use level of 4,132 mg/kg
BW/d that has been found from human clinical studies (Simpore et al., 2005, 2006).
Medical foods have not been included in the exposure estimate of the general
population since those foods will be used by a small segment of special population.
 
Spirulina platensis GRAS self affirmation 7-1-2011 Table 3a. Exposure estimate for Spirulina platensis intakes assuming 100% of products will be used at the maximal levels under the intended use, g/d. population Mean SE 11.85 5.62 0.21 13.45 0.89 13.56 7.41 0.35 15.46 0.87 females 8915 0.35 0.020 10.24 3.41 0.11 7.49 0.43 10.24 2.85 0.29 5.61 1.12 14.08 4.15 0.32 9.15 0.46 16.79 7.74 0.58 14.64 0.90 15.46 4.60 0.29 10.97 0.68 10.88 5.64 0.28 13.74 0.90 males 4824 0.97 0.087 12.88 7.55 0.47 16.30 1.36 Table 3b. Exposure estimate for Spirulina platensis intakes assuming 100% of products will be used at the maximal levels under the intended use, mg/kg BW/d. population Mean SE 13-19, females 1419 Spirulina platensis GRAS self affirmation 7-1-2011
Table 4a. Exposure estimate for Spirulina platensis intakes after the market share
adjustment (assuming that 10% of the products in each food category will be used at
the maximum intake levels under the intended use), g/d
population Mean SE Table 4b. Exposure estimate for Spirulina platensis intakes after the market share adjustment, mg/kg BW/d population Mean SE 17635 1.09 0.062 11.85 9.20 0.32 20.8 0.91 8720 1.52 0.100 13.56 11.19 females 8915 0.69 0.042 10.24 6.70 0.31 13.9 1.04 1521 2.25 0.327 10.24 22.14 3324 1.85 0.176 14.08 13.17 2851 2.03 0.230 16.79 12.11 males 1432 2.83 0.418 18.11 15.60 1419 1.20 0.147 15.46 7.85 0.45 16.5 1.79 9939 0.77 0.054 10.88 7.03 0.31 15.8 1.39 males 4824 1.18 0.010 12.88 9.11 0.55 20.2 1.94 Spirulina platensis GRAS self affirmation 7-1-2011 III. Basis for GRAS determination
A. Current regulatory status.
Spirulina platensis has been classified as a GRAS substance by the United States Food
and Drug Administration (GRN 000127; FDA, 2003).
B. Intended technical effects
Spirulina platensis can be used as an ingredient in foods and beverages as a food
ingredient. Spirulina platensis is not intended for use as a food colorant.
C. Review of safety data
 
1. Animal Studies of Spirulina
Safety evaluations on Spirulina sp. have included acute, subchronic, chronic,
teratogenic, mutagenic, carcinogenic, and multiple generation effects. No signs of
toxicity were noted in any of these studies. The NOAELs appear to be over 10,000
mg/kg BW/d for Spirulina platensis and 30% in the diet for Spirulina sp (corresponding
to 30,000 mg/kg BW/d), the maximum levels tested (Tables 5 - 7). Overall, Spirulina has
high NOAEL values.
1.1. Acute Toxicity Tests (Table 5)
Dried Spirulina at the dosage of 10,000 mg/kg BW showed no toxic effects. Some
authors recommend accompanying this test with a histological analysis similar to the
one conducted with phycocyanin, an active component of Spirulina. The LD50 value for
phycocyanin was found to be >5,000 mg/kg (Naidu et al., 1999). Since the 1990s, these high LD50 values (over 5,000 mg/kg BW) belong to the "practically nontoxic" category, according to a toxicity rating chart (Atlug, 2003). 1.1.1. Acute toxicity of S. platensis in mice Hutadilok-Towatana et al. (2008) tested acute effects of Spirulina platensis. Mice that were fed with Spirulina platensis at the dose of 30 g/kg BW fresh algae or 10 g/kg BW dried algae did not have any signs of toxicity during 7 d of observation. There were no obvious differences between treatment and the control groups. The gross examinations of internal organs revealed no pathological abnormalities. These results suggest that Spirulina platensis is not toxic to mice after exposure to a high dose (dried algae, 10,000 mg kg BW/d) for 7 d (Hutadilok-Towatana et al., 2008). Krishnakumari et al. (1981) reported that oral treatment with 800 mg/kg Spirulina platensis produced no toxic effects on BW, organs, and histological parameters. Also, application of 2,000 mg/kg onto the skin of albino rats did not elicit an allergic skin reaction (Krishnakumari et al., 1981). Spirulina platensis GRAS self affirmation 7-1-2011 Table 5. Acute toxicity studies of Spirulina platensis Species Dose No skin allergic Krishnakumari BW dried algae application 1.2. Subchronic Toxicity Studies (Table 6)
The consumption of dried Spirulina at doses up to 300 mg/kg/d for 12 wk did not show
any adverse effects in rats (Hutadilok-Towatana et al., 2008; Krishnakumari et al.,
1981)


Table 6. Subchronic toxicity studies of Spirulina platensis

Species Dose
Dried algae, up 12 wk Fresh algae, up 12 wk 1.2.1. Subchronic toxicity studies of Spirulina platensis by Hutadilok-Towatana et al. Two separate experiments were performed to evaluate subchronic toxicity of fresh or dried forms of Spirulina platensis. In each experiment, four groups of six Sprague-Dawley male and female rats were given fresh (up to 1,200 mg/kg BW/d) or dried algae (up to 120 mg/kg BW/d) for 12 wk. The consumption of algae showed no effect on behavior, food and water intake, growth or health status of these animals during the course of this investigation. There were no significant differences in serum clinical chemistry values and all values remained within the normal ranges (Casey and King, 1980; Angkhasirisap et al., 2002) and no treatment effects were noted. Spirulina platensis GRAS self affirmation 7-1-2011 There were no significant differences in BW gains, final BW, or fecal characteristics of rats among all treatment and control groups. Such findings indicate that Spirulina platensis did not alter protein, carbohydrate, or fat utilization, or gastrointestinal effects in rats. To determine if rats treated with fresh Spirulina platensis had any intravascular effects and bone marrow activity, Hutadilok-Towatana et al. (2008) performed hematological examinations. The average hematocrit values in all female groups appear to be lower than those in the males. However, feeding the rats with fresh algae significantly improved hematological parameters. At autopsy, macroscopic observation of the organs did not show any abnormality in their gross appearances or weights due to the consumption of Spirulina platensis. There were no changes in morphology and no unusual lesions in the gastrointestinal tissues exposed to Spirulina platensis. In addition, post-mortem examination found no abnormalities in the gross findings. The results demonstrated that short-term and long-term consumption of Spirulina platensis, up to high feeding levels, did not produce any adverse effects in experimental animals. 1.2.2. Subchronic toxicity of Spirulina platensis by Becker et al (1980). Becker et al. (1980) demonstrated that feeding Spirulina platensis at 300 mg/kg BW/d did not cause any undesirable effects to the experimental animals. 1.2.3. Subchronic toxicity of other types of Spirulina As shown in Table 7, subchronic toxicity studies of other types of Spirulina (either unidentified species or S.maxima) did not show any treatment-associated effects on their BW gains or final weights in experimental animals. There were no significant differences among the treated and control groups of the same sex. Such findings thus indicated that Spirulina, in general, did not alter protein, carbohydrate, or fat utilization in rats and mice (Salazar et al., 1998). The NOAEL appears to be over 30% Spirulina (or 30,000 mg/kg BW/d) in the diet, the maximum level tested in those studies. Spirulina platensis GRAS self affirmation 7-1-2011 Table 7. Subchronic toxicity studies of other sources of Spirulina (unidentified species or S. maxima) Animals Concentration Duration Results NOAEL, 30% in the diet. Spirulina group had insignificantly lower weight gains species); 10, compared to a casein control group. All rats survived the experimental period in apparently good health. All organs were normal macroscopically and microscopically. Rats Spirulina-rich No histological abnormalities were found in Bourges et several organs examined. Tolerance was at the final conc. of 36 and 48% of protein No obvious toxicity signs. species) at the conc. of 14.25% protein NOAEL, 30% in the diet. Abnormal findings were not detected in growth and species); 10, external appearance of the whole body or in the shape, weight, and histological findings of organs. Hematological tests such as Hb and SGPT showed some statistical differences S.maxima; 10, 13 wk for NOAEL, 30% in the diet. No toxic effects on food consumption, BW, hematology, clinical chemistry, urine analyses, organ weights, histology or renal function tests to up to 30,000 mg/kg BW/d) S.maxima; 10, NOAEL, 30% in the diet. No effects on mouse behavior, food and water intake, growth, or survival. Hematology, clinical chemistry, and histopathology values did not reveal differences compared to the control animals. **Modified from Chamorro et al., 2008; Hb=hemoglobin; SGPT= serum glutamic pyruvic
transaminase

Spirulina platensis GRAS self affirmation 7-1-2011 1.3. Chronic Toxicity Tests
Chamorro et al. (1988b) performed chronic toxicity tests of Spirulina maxima (10, 20, and 30% in diet) for 86 wk to assess the cumulative toxicity and carcinogenic
potential of Spirulina. The survival of animals treated with the Spirulina was equal or
slightly higher than that of soy-fed controls. No adverse effects on hematology, urine,
macroscopic or histopathological findings, or serum biochemistry were observed.
Spirulina at the concentration of 30% in diet did not impact palatability and did not
induce diminished food consumption (Chamorro et al., 1988b). The NOAEL was
determined to be 30% in the diet.
1.4. Reproductive Toxicity Tests (Table 8)
1.4.1. Reproductive Performance of Spirulina platensis
Kapoor and Mehta (1993, 1998) studied the effects of Spirulina platensis on the
outcome of pregnancy. As shown in Table 8, treatment with Spirulina platensis at the
final concentration of 22% protein was not associated with any adverse effects in any
measure of reproductive performance, including food intake, weight gain, numbers of
pups, total litter weight, mean birth weight, or hematological status.
Table 8. Reproductive toxicity studies showing no adverse effects of Spirulina platensis
Animals Dose
S. platensis Fertility and pregnancy; food intake, weight gain, numbers of pups, total litter (1993) wt, and mean birth wt. S. platensis Hematological status during pregnancy and conc. of 22% and lactation Growth, egg production, egg quality, fertility, hatchability and the growth (1990) of the F1 generation of dams In the study of Kapoor and Mehta (1993), pregnant rats were fed 5 different kinds of diets providing 22% protein (casein, Spirulina, wheat gluten, Spirulina + wheat gluten, Spirulina without additional vitamins and minerals) during the period of pregnancy. The outcome of pregnancy was assessed from litter and dams' weight and litter size. Maternal weight gain was found to be maximum with Spirulina + wheat gluten and least with the wheat gluten diet. Litter size was higher with Spirulina diets than casein and wheat gluten diets. There no significant differences in food intake and pup's birth weight among the groups. No adverse effects of Spirulina were noted. Authors concluded that Spirulina appeared to be a good dietary supplement during pregnancy. Spirulina platensis GRAS self affirmation 7-1-2011
Kapoor and Mehta (1993b) also studied the effects of Spirulina platensis on
hematological status in rats during pregnancy and lactation. As shown in Table 8, diets
containing Spirulina alone or in combination with wheat gluten resulted in significantly
higher iron storage and hemoglobin (Hb) content compared to casein and wheat gluten
diets during the first half of pregnancy and lactation. The values for serum iron and iron
binding capacity remained unchanged with different diets. Spirulina appears to be
effective in improving the iron status of rats during pregnancy and lactation.

1.4.2. Effects of S. platensis on reproductive performance of Japanese quail
Ross and Dominy (1990) studied the effects of S. platensis (0, 1.5, 3.0, 6.0, and 12.0%
of diet for 33 wk) on growth, egg production, egg quality, fertility, hatchability, and the
growth of the F1 generation of dams fed Spirulina in Japanese quail. There were no
significant differences due to the Spirulina content in any of the parameters studied,
except for yolk color (which increased with each succeeding level of Spirulina; 12% S.
platensis, 2.2 vs. control, 4.9 Roche scale score, p<0.05) and for fertility (which was
higher for all Spirulina treatments versus the control; 12% S. platensis, 95.1 vs. control,
86.8%, p<0.05).
These data are consistent with findings from Spirulina maxima studies. Spirulina maxima was not associated with any adverse effects in any measure of reproductive performance and fetus developmental markers except for isolated results with no toxicological significance (Chamorro et al., 1985, 1988c, 1989b, 1997; Romeo-Manilla et al., 2008; Salazar et al., 1996, 1997). Spirulina, even at a 30% dietary concentration, did not result in reproductive toxicity. Teratogenic studies showed that Spirulina given during different gestation periods did not affect embryo development or produce embryo resorption in rats and mice. 1.4.3. Effects of other types of Spirulina (S. maxima) on reproductive performance In addition, two multi-generational studies showed that Spirulina given to three generations over a period of approximately 2 years showed no adverse effects from Spirulina on fertility or gestation or any other parameters (Table 9; Chamorro et al., 1985, 1988c). It is noteworthy that F3 generation rats that were maintained for 13 wk on the same Spirulina diets as their parents' had no adverse effects on food consumption, BW, hematology, clinical chemistry, urine analyses, organ weights, histology or renal function tests (Chamorro et al., 1988c). Spirulina platensis GRAS self affirmation 7-1-2011 Table 9. Reproductive toxicity studies showing no adverse effects of Spirulina maxima* Animals Dose S. maxima Fertility and pregnancy; the Salazar et number of live or dead pups at birth, survival rate, or weaning rate. mating to 3 wk after delivery S. maxima Fertility and pregnancy; the Chamorro number of corpora lutea, total implantations, or number of live or dead fetuses. The number of Mice Unidentified teratogenicity; external, to 500 mg/kg to gestation visceral and skeletal abnormalities of fetus S. maxima Teratogenicity; maternal was given on and fetal wt. and no embryo resorptions of S. maxima Peri- and post-natal development; pregnancy and litter, BW, and survival S. maxima Multigenerational; fertility, gestation, size of litters, or fetus viability, and subchronic toxicity parameters of F3 generation S.maxima; 13 wk for F3 NOAEL, 30% in the diet. No toxic effects on food consumption, BW, hematology, clinical chemistry, urine analyses, organ weights, histology or renal function tests *Modified from Chamorro et al., 2008. Spirulina platensis GRAS self affirmation 7-1-2011

1.5. Genetic
Toxicity
Effects of Spirulina on genotoxicity have been studied in rats and mice as well as in bacteria (Table 10). There are reports that Spirulina platensis and other types of Spirulina exerted antigenotoxic effects in various animal models (Qishen et al., 1989; Zhang et al., 2001). For example, Spirulina platensis (12 mg/kg) increased the level of red cells, white cells, and hemoglobin in blood and nucleated cells in bone marrow in dogs (Qishen et al., 1989). Polysaccharide of Spirulina platensis reduced hematopoietic damage induced by injection of cyclophosphamide (CTX) and 60Co-gamma irradiation in mice and dogs (Zhang et al., 2001). Spirulina platensis (30, 60 mg/kg) increased the concentration of the white cells in blood and nucleated cells and DNA in bone marrow in mice but had no effects on red cells and hemoglobin. Other types of Spirulina also showed protection against genetoxicity induced by cisplatin and urethane (Prekumar et al., 2004) as well as by cyclophosphamide and/or mitomycin-C (Table 10; Chamorro et al., 2006; Prekumar et al., 2001). Models have been developed that allow for the detection of potential damage induced in germ cells at a specific stage of development, to be expressed through weekly mating, or of the potential damage induced in germ cells at various stages of development, to be expressed during the first week of mating, respectively. In both cases, Spirulina maxima showed no genotoxicity in mice and rats (Chamorro and Salazar, 1989, 1995; Salazar and Chamorro, 1990). Urine of animals fed Spirulina for 4 mo showed no mutagenicity when the Ames test was conducted with five strains of S. typhimurium and Schizosaccharomyces pombe (Bizzi et al., 1980). Spirulina platensis GRAS self affirmation 7-1-2011 Table 10. Antitoxic effects of Spirulina against genotoxicity* Animals Dose and duration Polysaccharide of Gamma-radiation-induced damage Zhang et S. platensis, 30 and in hematopoietic system; The level al., 2001 of the white cells in blood and nucleated cells, as well as DNA in bone marrow in mice. The level of the red cells, white cells, and hemoglobin in blood as well as nucleated cells in bone marrow in dogs. S. platensis extract (1- The micronucleus test; mouse 5 mg/kg) given orally bone marrow polychromatic erythrocytes against gamma-radiation injury. S. fusiformis (250, The micronucleus; inhibition of 500, and 1000 mg/kg) genotoxicity and reduced lipid peroxidation induced by cisplatin and urethane S. fusiformis (250, Micronucleus test; chromosomal damage and lipid peroxidation induced by cyclophosphamide and mitomycin-C. S. maxima given Antimutagenic effect against orally (200, 400, or cyclophosphamide, evaluated by the dominant-lethal test. *Modified from Chamorro et al., 2008. 1.6. Antioxidant and Antitoxic Effects of Spirulina

Numerous studies have demonstrated antioxidant effects of Spirulina platensis (Bhat
and Madyastha, 2001; Chu et al., 2010; Dartsch, 2008; Kim et al., 2010). Other types of
Spirulina also showed antioxidant effects (unidentified species, S. maxima, or S.
fusformis (Kuhad et al., 2006; Thaakur and Jyothi, 2007; Wu et al., 2005).
Antioxidant properties of Spirulina platensis may partly contribute to antitoxic effects against nephrotoxicity, cardiotoxicity, ovary toxicity, and metal-induced toxicity as well as antiviral and immune-enhancing effects (Tables 11-13; Avdagic et al. 2008; Grawish et al., 2010; Karadeniz et al., 2008; Karaka et al., 2007; Khan et al., 2005, 2006; Kumar et al., 2009; Lu et al., 2010; Simsek et al., 2009; Zaccaro et al., 2004). No studies reported any side effects of Spirulina platensis; in these studies, dosages up to 1,000 mg/kg BW/d were not associated with any adverse effects. Spirulina platensis GRAS self affirmation 7-1-2011 Other types of Spirulina also showed similar antitoxic effects (Ble-Castillo et al., 2002; Gonzalez de Rivera et al., 1993; Haque and Gilani, 2005; Kuhad et al., 2006; Mohan et al., 2006; Torres-Duran et al., 1998; Vadiraja et al., 1998). C-phycocyanin, a potent antioxidant which has the ability to scavenge free radicals, is known as an active component in Spirulina (Bhat and Madyastha, 2000; Deng and Chow, 2010; Hsiao et al., 2005). Table 11. Antitoxic effects of Spirulina platensis Animals Methods Gentamicin-induced acute tubular Gentamicin-induced nephrotoxicity Karadeniz Cyclosporine-induced nephrotoxicity, as Khan et al., measured by MDA rise in plasma and kidney tissues, isometric vacuolization and interstitial widening. S.platensis DMBA-induced carcinogenicity measured by pathological alterations mg/d, 24 wk (immunohistochemical) Doxorubicin-induced cardiotoxicity, measured by lipid peroxidation, antioxidant enzymes, total antioxidant activity, and histopathology of heart 2,6, 9% (or up Acetaminophen-induced liver injuries, to 1,350 mg/kg examined by hepatic malonaldehyde, BW/d) in diet, serum conc. of GOT, GPT, and IL-8 Ovary toxicity; lead-induced alterations in the number of mast cells in the cortex 2007 and medulla of rat ovaries during the oestrus cycle. S.platensis, 28 Hematological damage induced by ultraviolet (UVC)-irradiation Normalization of collagen-induced arthritis, examined by the joint histopathology, serum chemistry, lipid profile, and lipid peroxidation. Rat, female 300 mg/kg Cadmium- or lead-induced toxicity, examined by hematological values Spirulina platensis GRAS self affirmation 7-1-2011 Table 12. Effects of Spirulina platensis on immune function and Measurements Reference Macrophage function 14, 35, 42 d Release of histamine from mast cells Antibody production; macrophage functions, phagocytes and IL-1 in diet, 46 or 67 d Table 13. Antiviral activities of Spirulina platensis Animals Dose S. platensis extract, Antiviral activity, measured by Ayehunie et al., HIV-1 replication in human T cell 1998 line S. platensis extract, Gram positive and gram negative Ozdemir et al., 20-30 ul of 100 bacteria and Candida albicans 2. Human Feeding Programs for Nutritional Rehabilitation
Spirulina has been used as a health food supplement for malnourished children and adults in clinical studies since the early 1970's (Deng et al., 2010; Habib et al., 2008; Seshadriet al., 1993). Because of its unusual high nutritional values, several Spirulina feeding programs were administered in various countries. Examples include the following: 1. The Intergovernmental Institution for the use of Micro-algae Spirulina Against Malnutrition (IIMSAM) was launched in the mid-1970's to promote Spirulina as a high nutritional food to fight against starvation and malnutrition in the world (Habib et al., 2008). 2. India had a one year feeding program of Spirulina (1 g/d) with 5,000 pre- school children. A Spiulina-supplemented diet reduced the occurrence of "Bitof's spot", a symptom of vitamin A deficiency, from 80% to 10% (Seshadriet al., 1993). Spirulina platensis GRAS self affirmation 7-1-2011 3. Due to its concentrated nutrition, Spirulina was recommended for astronauts by National Aeronautics and Space Administration (Deng et al., 2010).
3. Human Clinical Studies
Recent human clinical trials have demonstrated various health benefits in malnourished
young children, healthy volunteers, or subjects with non-insulin dependent diabetes
mellitus (NIDDM), allergic rhinitis, or hypercholesterolemia with no side effects (Table
14). Studies on nutrition rehabilitation support various nutrition feeding programs
involving Spirulina. For example, Simpore et al. (2009) assessed the impact of an
elementary integrator composed of Spiruline (Spirulina platensis, 10 g/d, corresponding
to 1,666 mg/kg BW/d) and Misola (millet, soja, and peanut) on the nutritional status of
undernourished children. Five hundred fifty (550) undernourished children (severe
marasma, 57 marasma of medium severity, and 38 kwashiorkor plus marasma) of less
than 5 years old were given one of 4 types of diets for 8 wk: Misola (731 ± 7 kcal/day),
Spiruline plus traditional meals (748 ± 6 kcal/day), Spiruline plus Misola (767 ± 5
kcal/day), and traditional meals (722 ± 8 kcal/day; the control group). Weight gain was
observed in all the groups; 20 g/d in the Misola group, 25 g/d in the Spiruline plus
traditional meals group, 34 g/d in the Misola plus Spiruline group and 15 g/d in the
control group. However, Spiruline plus Misola appeared to have synergic effects on the
nutrition rehabilitation better than traditional meals. (i.e., the simple addition of protein
and energy intake). These pre/post differences within groups were statistically
significant considering the differences in the nutritional status changes across the
groups, but this difference was less significant in the control group. No side effects were
reported related to daily consumption of 10 g (1,666 mg/kg BW) Spirulina platensis.
Another Spirulina supplementation study (8 wk) with undernourished HIV-infected and HIV-negative children resulted in similar outcomes (Simpore et al., 2005). Rehabilitation with Spirulina platensis showed an average weight gain of 15 g/d (corresponding to 2,479 mg/kg BW/d) and 25 g/d (corresponding to 4,132 mg/kg BW/d) in HIV-infected and HIV-negative children, respectively. The level of anemia decreased during the study in all children, but recuperation was less efficient among HIV-infected children: 81.8% in HIV-negative and 63.6% in HIV-infected. These studies support the historic usage of Spirulina for improving the nutritional status of malnourished population. Other demonstrated benefits of Spirulina platensis include hypocholesterolemic and hypoglycemic effects, antioxidant and immunity effects, and reduction of skeletal muscle damage during exercise (Table 14; Baicus and Baicus, 2007; Cingi et al., 2008; Kalafati et al., 2009; Kim and Kim, 2005; Lee et al., 2008; Lobner et al., 2008; Lu et al., 2006; Park and Kim, 2005; Park et al., 2008; Selmi et al., 2011). No studies reported side effects of Spirulina platensis. Spirulina platensis GRAS self affirmation 7-1-2011 Table 14. Human clinical studies demonstrating beneficial or no adverse effects of Spirulina platensis Duration Measurement endpoints 550 undernourished Nutritional rehabilitation children aged 6-60 mo including correction of BW 2006 (median age 15.3 mo) to ave. of 1,666 166 malnourished Nutritional rehabilitation; children, HIV-infected and HIV-negative to ave. of 2,479 hematological parameters including anemia 12 healthy elderly Serum lipid profiles cholesterolemic and normocholesterolemic subjects) 51 elderly women with Serum lipid profiles and hypercholesterolemia Apolipoprotein B, and IL-6 concentration, and IL-6 production by peripheral blood lymphocyte. 37 patients with Serum lipid profiles, blood Lee et al., 2008 pressure, IL-6 levels, and plasma malondialdehyde 78 healthy elderly Serum lipid profiles, IL-2, volunteers aged 60-87 IL-6, superoxide Improvement of idiopathic Baicus and chronic fatigue 150 allergic rhinitis Improvement of allergic patients aged 19-49 y 11 healthy males extracts from S. platensis, 200 mg/d 9 moderately trained Ergogenic and antioxidant Kalafati et al., effects during exercise 16 college students Skeletal muscle damage under exercise-induced oxidative stress 40 elderly aged 50 or Immune functions (indoleamine 2,3- dioxygenase activity) and serum hematology Dosages=dosages of dried Spirulina platensis powder unless noted otherwise. Spirulina platensis GRAS self affirmation 7-1-2011 Also, human clinical trials on other types of Spirulina (unidentified species, S. maxima, or S. fusiformis) reported similar benefits with no side effects (Table 15; Becker et al., 1986; Branger et al., 2003; Cingi et al., 2008; Ferreira-Hermosillo et al., 2010; Juarez-Oropeza et al., 2009; Kamalpreet et al., 2008; Kaur et al., 2008; Mani et al., 2000; Mathew et al., 1995; Nielson et al., 2010; Nikaya et al., 1988; Parikh et al., 2008; Ramamoorthy and Premakumari, 1996; Samuels et al., 2002; Torres-Duran et al., 2007). Taken together, human studies showed that Spirulina platensis is safe for human consumption and daily consumption of approximately 4,132 mg/kg BW/d did not result in any adverse effects, even in infants and young children. Also, no member of the Genus Spirulina is known to be toxic. Spirulina platensis GRAS self affirmation 7-1-2011 Table 15. Human clinical studies demonstrating beneficial or no adverse effects of other types of Spirulina Spirulina (unidentified Infant malnutrition species), 5 g/d (or 1,000 mg/kg BW/d) 19 overweight subjects Spirulina (unidentified species), 7.2 g/d 30 healthy volunteers Spirulina (unidentified Serum lipid profiles 15 Patients with NIDDM Spirulina (unidentified Blood sugar, lipid, and Mani et al., 2000 species) tablets, 2 g/d glycated serum protein levels 25 patients with NIDDM SUNOVA spirulina Serum lipid profiles (unidentified species) and the level of apolipoprotein A1. 60 patients with NIDDM SUNOVA spirulina Blood lipid and sugar Kaur et al., 2008 (unidentified species) 23 Patients with Spirulina (unidentified Serum lipid profiles nephritic syndrome species) capsules, 36 allergic rhinitis Spirulina (unidentified Cingi et al., 2008 patients aged 18-55 y allergic rhinitis, as measured by cytokines 12 healthy volunteers Spirulina (unidentified Killing K562 tumor species), 0.4 g/d cells by natural killer 60 Patients with type 2 SUNOVA Spirulina Serum lipid profiles diabetes aged 40-60 y capsules(unidentified and both fasting and species), 1 or 2 g/d postprandial blood glucose levels 36 Healthy volunteers S. maxima, 4.5 g/d Serum lipid profiles and blood pressures 36 Healthy volunteers S. maxima, 4.5 g/d Vascular reactivity 30 Patients with S. fusiformis, 2 or 4 g/d 3 mo Plasma lipid profiles ischaemic heart disease and Premakumari, 1996 60 Patients with oral S. fusiformis, 1g/d 3 Patients with non- S. maxima, 4.5 g/d Blood lipid profiles and Ferreira- alcoholic fatty liver Hermosillo et al., aminotransferase Dosages=dosages of dried Spirulina powder unless noted otherwise. Spirulina platensis GRAS self affirmation 7-1-2011 4. Allergy
Rare cases of side-effects (gastrointestinal discomfort) may happen with excessive intakes (over 100 g/d). 5. Potential contamination
Anatoxin-a, a cyanotoxin with acute neurotoxicity, was detected in 3 of the 39 cyanobacterial samples (Rellan et al., 2009). It was thus recommended that quality control of cyanobacterial food supplements including Spirulina was required to avoid potential adverse effects in animals and humans. IV. Conclusions
This GRAS determination for Spirulina platensis is based upon scientific procedures. There is abundant literature describing the composition and safety of Spirulina platensis. Also, numerous human and animal studies examined the health benefits of Spirulina platensis. There are no reports of safety concerns in any of the studies. RFI utilizes a HACCP-controlled manufacturing process and rigorously tests its final production batches to verify adherence to quality control specifications. The information/data provided by RFI in this report and supplemented by the publicly available literature/toxicity data on Spirulina platensis provide a sufficient basis for an assessment of the safety of Spirulina platensis for the proposed use as an ingredient in foods and beverages, when prepared according to appropriate specifications and used according to GMP. Key findings are summarized here: 1. Spirulina platensis is well characterized and free from chemical and microbial 2. Manufacturing processes of Spirulina platensis have been safely used for many years in the food industry. 3. The safety and nutritional benefits of Spirulina platensis are well established by human clinical trials and animal studies. There are no indications of significant adverse effects related to Spirulina platensis consumption in the publicly available literature. Spirulina platensis and its active component, phycocyanin, are classified into the "practically nontoxic" category. Subchronic toxicity and reproductive toxicity studies showed that consumption of dried Spirulina platensis up to 1,200 mg/kg BW/d, the maximum level tested, had no adverse effects. Human studies showed that daily consumption of 4,132 mg/kg BW of Spirulina platensis did not result in any adverse effects even in infants and young children. 4. Spirulina platensis has a long history of safe use in foods. Spirulina platensis is of natural biological origin and has been widely consumed for its nutrient properties, without known detrimental effect. Spirulina platensis GRAS self affirmation 7-1-2011 Therefore, not only is the proposed use of Spirulina platensis in foods and beverages
safe within the terms of the Federal Food, Drug, and Cosmetic Act (meeting the
standard of reasonable certainty of no harm), but because of this consensus among
experts, it is also GRAS.
V. Discussion of information inconsistent with GRAS determination
We are not aware of information that would be inconsistent with a finding that the
proposed use of Spirulina platensis preparations in foods and beverages, meeting
appropriate specifications and used according to GMP, is GRAS.

VI. Availability of Information
The data and information that serve as the basis for this GRAS Notification will be
available for review and copying at reasonable times at the offices of:
RFI
300 Corporate Drive, Suite 14
Blouvelt, NY 10913
TEL: 800-962-7663
Spirulina platensis GRAS self affirmation 7-1-2011 References

Al-Batshan HA, Al-Mufarrej SI, Al-Homaidan AA, Qureshi MA. Enhancement of
chicken macrophage phagocytic function and nitrite production by dietary
Spirulina platensis. Immunopharmacol Immunotoxicol. 2001;23:281-289.
Altug T. Introduction to toxicology and food. CRC Press, Boca Raton, FL. 2003.
Annapurna VV, Deosthale YG, Bamji MS. Spirulina as a source of vitamin A.
Plant Foods Hum Nutr. 1991;41:125-134.
Avdagić N, Cosović E, Nakas-Ićindić E, Mornjaković Z, Zaciragić A, Hadzović-Dzuvo A.
Spirulina platensis protects against renal injury in rats with gentamicin-induced acute
tubular necrosis. Bosnian J Basic Med Sci. 2008;8:331-336.
Ayehunie S, Belay A, Baba TW, Ruprecht RM. Inhibition of HIV-1 replication by an
aqueous extract of Spirulina platensis (Arthrospira platensis). J Acquir Immune Defic
Syndr Hum Retrovirol. 1998;18:7-12.
Baicus C, Baicus A. Spirulina did not ameliorate idiopathic chronic fatigue in four N-of-1
randomized controlled trials. Phytother Res. 2007;21:570-573.
Becker EW. Comparative toxicology studies with algae in India, Thailand, and Peru. In
Algae biomass production and use. Shelef G, Soeder CJ (Eds). Elsevier, Nothe Holland,
Amsterdam, The Netherlands. 1980 pp 565-574.
Becker EW, Venkataraman IV, Production and utilization of the blue green alga,
Spirulina in India. Biomass. 1984;4:105.
Becker EW, Jakber B, Luft D, Schmulling RM. Clinical and biochemical
evaluations of the algae Spirulina with regard to its application in the treatment of
obesity. A double blind crossover study. Nutr Rep Int. 1986;33:565-574.
Bhat VB, Madyastha KM. C-phycocyanin: A potent peroxyl radical scavenger in
vivo and in vitro. Biochem Biophys Res Commun. 2000;275:20-25.
Bizzi A. Trattamenti prolungati nel ratto con diete connten Spirulina. Cited in Chamorro
et al. 2008.
Blé-Castillo JL, Rodríguez-Hernández A, Miranda-Zamora R, Juárez-Oropeza MA,
Díaz-Zagoya JC. Arthrospira maxima prevents the acute fatty liver induced by the
administration of simvastatin, ethanol and a hypercholesterolemic diet to mice.
Life Sci. 2002;70:2665-2673.
Spirulina platensis GRAS self affirmation 7-1-2011 Boudene, C., E. Collas, and C. Jenkins. Recherche et dosage de divers toxiques minereaux dans les algues spirulines de differentes origins, et evaluation de la toxicite a long terme chez le rat d'un lot algues spirulines de provenance Mexicaine. Ann Nutr Alim. 1976;30: 577-588. Branger B, Cadudal JL, Delobel M, Ouoba H, Yameogo P, Ouedraogo D, Guerin D, Valea A, Zombre C, Ancel P; personnels des CREN. [Spiruline as a food supplement in case of infant malnutrition in Burkina-Faso]. Arch Pediatr. 2003;10:424-431. French. Casey JD, King DJ. Clinical chemical values for some common laboratory animals. Clin Chem. 1980;26:1877-1879. Chamorro G. Etude toxicologique de l' alga Spirulina plante pilote productrice de proteins Report. UNIDO 1980;10;387. Cited in Chamorro et al. 2008. Chamorro GA, Herrera G, Salazar M, Salazar S, Ulloa V. Subchronic toxicity study in rats fed Spirulina. J Pharm Belg. 1988a;43:29-36. Chamorro GA, Herrera G, Salazar M, Salazar S, Ulloa V. Short-term toxicity study of Spirulina in F3b generation rats. J Toxicol Clin Exp. 1988b;8:163-167. Chamorro G, Salazar M. Dominant lethal assay of Spirulina maxima in male CD-1 mice after short-term and prolonged-term feeding. J Food Protection. 1989;52:125-127. Chamorro G, Salazar M. [Teratogenic study of Spirulina in rats]. Arch Latinoam Nutr. 1989;39:641-649. Chamorro G, Salazar M. [Teratogenic study of Spirulina in rats]. Arch Latinoam Nutr. 1990;40:86-94. Chamorro G, Salazar M, Favila L, Bourges H. [Pharmacology and toxicology of Spirulina alga]. Rev Invest Clin. 1996;48:389-99. Chamorro G, Salazar S, Favila-Castillo L, Steele C, Salazar M. Reproductive and peri- and postnatal evaluation of Spirulina maxima in mice. J Appl Physiol.1997; 9:107-112. Chamorro G. Antimutagenic effect of Arthrospira (Spirulina) maxima evaluated by the dominant-lethal test in mice. 2006. Cited in Chamorro et al. 2008. Chamorro-Cevallos G, Barron BL, Vazquez-Sanchez J. Toxicologic studies and antitoxic properties of Spirulina. In Spirulina un human nutrition and health, Gershwin ME and Belay A (Eds), CRC Press, Boca Raton, FL. 2008, pp 27-50. Spirulina platensis GRAS self affirmation 7-1-2011 Chu WL, Lim YW, Radhakrishnan AK, Lim PE. Protective effect of aqueous extract from Spirulina platensis against cell death induced by free radicals. BMC Complement Altern Med. 2010;10:53. Ciferri O, Tiboni O. The biochemistry and industrial potential of Spirulina. Ann Rev Microbiol. 1985;39:503–526. Cingi C, Conk-Dalay M, Cakli H, Bal C. The effects of spirulina on allergic rhinitis. Eur Arch Otorhinolaryngol. 2008;265:1219-1223. Dartsch PC. Antioxidant potential of selected Spirulina platensis preparations. Phytother Res. 2008;22:627-633. Deng R, Chow TJ. Hypolipidemic, antioxidant, and antiinflammatory activities of microalgae Spirulina. Cardiovasc Ther. 2010;28:e33-45. Dillon JC, Phuc AP, Dubacq JP. Nutritional value of the alga Spirulina. World Rev Nutr Diet. 1995;77:32-46. Ferreira-Hermosillo A, Torres-Duran PV, Juarez-Oropeza MA. Hepatoprotective effects of Spirulina maxima in patients with non-alcoholic fatty liver disease: A case series. J Med Case Reports. 2010;4:103. FAO. FAO Fisheries and Aquaculture Circular No. 1034. A review on culture, production and use of spirulina as food for human and feeds for domestic animals and fish. 2008. Available from ftp://ftp.fao.org/docrep/fao/011/i0424e/i0424e00.pdf FDA. GRN 000127. Spirulina, the dried biomass of Arthrospira platensis. 2003 Available from: tttp://www.accessdata.fda.gov/scripts/fcn/fcnDetailNavigation.cfm?rpt=grasListing&id=127 FDA. U.S. Code of Federal Regulations (CFR). Title 21-Food and Drugs (Food and Drug Administration). U.S. Government Printing Office (GPO), Washington, DC. 2008. Available from: http://www.access. po.qov/c i-in/cfrassemble.cqi?title= 008[S2e1e Table for CFR sections]. González de Rivera C, Miranda-Zamora R, Díaz-Zagoya JC, Juárez-Oropeza MA. Preventive effect of Spirulina maxima on the fatty liver induced by a fructose-rich diet in the rat, a preliminary report. Life Sci. 1993;53:57-61. Grawish ME, Zaher AR, Gaafar AI, Nasif WA. Long-term effect of Spirulina platensis extract on DMBA-induced hamster buccal pouch carcinogenesis (immunohistochemical study). Med Oncol. 2010;27:20-28. Spirulina platensis GRAS self affirmation 7-1-2011 Gershwin ME, Belay A (editors). Spirulina in human nutrition and health. CRC Press, Boca Raton, FL. 2008. Habib MAB, Parvin M, Huntington TC, Hasan MR. A review on culture, production, and use of Spirulina as food for humans and feeds for domestic animals and fish. FAO Fisheries and Aquaculture Circular No.034, 2008. Halidou Doudou M, Degbey H, Daouda H, Leveque A, Donnen P, Hennart P, Dramaix-Wilmet M. [The effect of spiruline during nutritional rehabilitation: Systematic review]. Rev Epidemiol Sante Publique. 2008;56:425-431. Haque SE, Gilani KM. Effect of ambroxol, spirulina and vitamin-E in naphthalene induced cataract in female rats. Indian J Physiol Pharmacol. 2005;49:57-64. Hayashi O, Hirahashi T, Katoh T, Miyajima H, Hirano T, Okuwaki Y. Class specific influence of dietary Spirulina platensis on antibody production in mice. J Nutr Sci Vitaminol (Tokyo). 1998;44:841-851. Hayashi O, Katoh T, Okuwaki Y. Enhancement of antibody production in mice by dietary Spirulina platensis. J Nutr Sci Vitaminol (Tokyo). 1994; 40:431-441. Hirahashi T, Matsumoto M, Hazeki K, Saeki Y, Ui M, Seya T. Activation of the human innate immune system by Spirulina: Augmentation of interferon production and NK cytotoxicity by oral administration of hot water extract of Spirulina platensis. Int Immunopharmacol. 2002;2:423-434. Hsiao G, Chou PH, Shen MY, Chou DS, Lin CH, Sheu JR. C-phycocyanin, a very potent and novel platelet aggregation inhibitor from Spirulina platensis. J Agric Food Chem. 2005;53:7734-7740. Hutadilok-Towatana N, Reanmongkol W, Satitit S, Panichayupakaranant P, Ritthisunthorn P. A subchronic toxicity study of Spirulina platensis. Food Sci Technol Res. 2008;14:351-358. Iwata K, Inayama T, Kato T. Effects of Spirulina platensis on plasma lipoprotein lipase activity in fructose-induced hyperlipidemic rats. J Nutr Sci Vitaminol (Tokyo). 1990;36:165-171. Juárez-Oropeza MA, Mascher D, Torres-Durán PV, Farias JM, Paredes-Carbajal MC. Effects of dietary Spirulina on vascular reactivity. J Med Food. 2009;12:15-20. Kalafati M, Jamurtas AZ, Nikolaidis MG, Paschalis V, Theodorou AA, Sakellariou GK, Koutedakis Y, Kouretas D. Ergogenic and antioxidant effects of spirulina supplementation in humans. Med Sci Sports Exerc. 2010;42:142-51. Spirulina platensis GRAS self affirmation 7-1-2011 Kamalpreet K, Rajbir S, Kiran G. Effect of supplementation of Spirulina on blood glucose and lipid profile of the non-insulin dependent diabetic male subjects. J Dairying, Foods Home Sci. 2008;27:3-4. Kapoor R, Mehta U. Effect of supplementation of blue green alga (Spirulina) on outcome of pregnancy in rats. Plant Foods Hum Nutr. 1993;43:29-35. Kapoor R, Mehta U. Supplementary effect of spirulina on hematological status of rats during pregnancy and lactation. Plant Foods Hum Nutr. 1998;52:315-324. Karadeniz A, Yildirim A, Simsek N, Kalkan Y, Celebi F. Spirulina platensis protects against gentamicin-induced nephrotoxicity in rats. Phytother Res. 2008;22:1506–1510. Karkos PD, Leong SC, Karkos CD, Sivaji N, Assimakopoulos DA. Spirulina in clinical practice: Evidence-based human applications. Evid Based Complement Alternat Med. 2008;eCAM:1–4. Khan Z, Bhadouria P, Bisen PS. Nutritional and therapeutic potential of Spirulina. Curr Pharm Biotechnol. 2005;6:373–379. Khan M, Shobha JC, Mohan IK, Naidu MU, Sundaram C, Singh S, Kuppusamy P, Kutala VK. Protective effect of Spirulina against doxorubicin-induced cardiotoxicity. Phytother Res. 2005;19:1030-1037. Khan M, Shobha JC, Mohan IK, Rao Naidu MU, Prayag A, Kutala VK. Spirulina attenuates cyclosporine-induced nephrotoxicity in rats. J Appl Toxicol. 2006;26:444-451. Kim HM, Lee EH, Cho HH, Moon YH. Inhibitory effect of mast cell-mediated immediate-type allergic reactions in rats by spirulina. Biochem Pharmacol. 1998;55:1071-1076. Kim MH, Kim WY. The change of lipid metabolism and immune function caused by antioxidant material in the hypercholesterolemic elderly women in Korea. Korean J Nutr. 2005;38:67-75. Kim MY, Cheong SH, Lee JH, Kim MJ, Sok DE, Kim MR. Spirulina improves antioxidant status by reducing oxidative stress in rabbits fed a high-cholesterol diet. J Med Food. 2010;13:420-426. Kom´arek J, Hauer T. CyanoDB.cz—On-line database of cyanobacterial genera. Worldwide electronic publication, Univ. of South Bohemia and Inst of Botany AS CR 2009; http://www.cyanodb.cz. Spirulina platensis GRAS self affirmation 7-1-2011 Krishnakumari M. K., H. P. Ramesh, and L. V. Venkataraman. Food safety evaluation: Acute oral and dermal effects of the algae Scenedesmus acutus and Spirulznaplatenszs on albino rats. J Food Protect. 1981;44: 934-935. Kuhad A, Tirkey N, Pilkhwal S, Chopra K. Effect of Spirulina, a blue green algae, on gentamicin-induced oxidative stress and renal dysfunction in rats. Fundam Clin Pharmacol. 2006;20:121-128. Kumar N, Singh S, Patro N, Patro I. Evaluation of protective efficacy of Spirulina platensis against collagen-induced arthritis in rats. Inflammopharmacology. 2009;17:181-190. Lee EH, Park JE, Choi YJ, Huh KB, Kim WY. A randomized study to establish the effects of spirulina in type 2 diabetes mellitus patients. Nutr Res Pract. 2008;2:295-300. Li B, Zhang X, Gao M, Chu X. Effects of CD59 on antitumoral activities of phycocyanin from Spirulina platensis. Biomed Pharmacother. 2005;59:551-560. Løbner M, Walsted A, Larsen R, Bendtzen K, Nielsen CH. Enhancement of human adaptive immune responses by administration of a high-molecular-weight polysaccharide extract from the cyanobacterium Arthrospira platensis. J Med Food. 2008;11:313-322. Lu HK, Hsieh CC, Hsu JJ, Yang YK, Chou HN. Preventive effects of Spirulina platensis on skeletal muscle damage under exercise-induced oxidative stress. Eur J Appl Physiol. 2006;98:220-226. Lu J, Ren DF, Wang JZ, Sanada H, Egashira Y. Protection by dietary Spirulina platensis against D-galactosamine--and acetaminophen-induced liver injuries. Br J Nutr. 2010;103:1573-1576. Mani UV, Desai S, Iyer U. Studies on the long-term effect of Spirulina supplementation on serum lipid profile and glycated proteins in NIDDM patients. J Nutraceut Funct Med Foods. 2000;2:25–32. Mao TK, Van de Water J, Gershwin ME. Effects of a Spirulina-based dietary supplement on cytokine production from allergic rhinitis patients. J Med Food. 2005;8:27-30. Mathew B, Sankaranarayanan R, Nair PP, Varghese C, Somanathan T, Amma BP, Amma NS, Nair MK. Evaluation of chemoprevention of oral cancer with Spirulina fusiformis. Nutr Cancer. 1995;24:197-202. Mazokopakis EE, Karefilakis CM, Tsartsalis AN, Milkas AN, Ganotakis ES. Spirulina platensis GRAS self affirmation 7-1-2011 Acute rhabdomyolysis caused by Spirulina (Arthrospira platensis). Phytomedicine. 2008;15:525-527. McCarty MF. Potential utility of full-spectrum antioxidant therapy, citrulline, and dietary nitrate in the management of sickle cell disease. Med Hypotheses. 2010;74:1055-1058. Misbahuddin M, Islam AZ, Khandker S, Ifthaker-Al-Mahmud, Islam N, Anjumanara. Efficacy of spirulina extract plus zinc in patients of chronic arsenic poisoning: a randomized placebo-controlled study. Clin Toxicol. 2006;44:135-141. Mohan IK, Khan M, Shobha JC, Naidu MU, Prayag A, Kuppusamy P, Kutala VK. Protection against cisplatin-induced nephrotoxicity by Spirulina in rats. Cancer Chemother Pharmacol. 2006;58:802-808. Naidu KA. Toxicity assessment of phycocyanin-a blue colorant from blue-green alga Spirulina platensis. Food Biotechnol. 1999; 13:51-66. Nakaya N, Homa Y, Goto Y. Cholesterol lowering effect of Spirulina. Nutr Rep Int. 1988;37:1329-1337. Nielsen CH, Balachandran P, Christensen O, Pugh ND, Tamta H, Sufka KJ, Wu X, Walsted A, Schjørring-Thyssen M, Enevold C, Pasco DS. Enhancement of natural killer cell activity in healthy subjects by Immulina®, a Spirulina extract enriched for Braun-type lipoproteins. Planta Med. 2010;76:1802-1808. Ozdemir G, Karabay NU, Dalay MC, Pazarbasi B. Antibacterial activity of volatile component and various extracts of Spirulina platensis. Phytother Res. 2004;18:754-757. Parikh P, Mani U, Iyer U. Role of Spirulina in the control of glycemia and lipidemia in type 2 diabetes mellitus. J Med Food. 2001;4:193-199. Park JY, Kim WY. The effect of Spirulina on lipid metabolism, antioxidant capacity and immune function in Korean elderly. Korean J Nutr. 2003;36:287–297. Park HJ, Lee YJ, Ryu HK, Kim MH, Chung HW, Kim WY. A randomized double-blind, placebo-controlled study to establish the effects of Spirulina in elderly Koreans. Ann Nutr Metab. 2008;52:322-328. Premkumar K, Abraham SK, Santhiya ST, Ramesh A. Protective effect of Spirulina fusiformis on chemical-induced genotoxicity in mice. Fitoterapia. 2004;75:24-31. Premkumar K, Pachiappan A, Abraham SK, Santhiya ST, Gopinath PM, Ramesh A. Effect of Spirulina fusiformis on cyclophosphamide and mitomycin-C induced genotoxicity and oxidative stress in mice. Fitoterapia. 2001;72:906-911. Spirulina platensis GRAS self affirmation 7-1-2011 Price JA, Isanny C, Shelvlin D. Inhibition of mast cells by algae. J Med Food. 2002;5:205-210. Qishen P, Guo BJ, Kolman A. Radioprotective effect of extract from Spirulina platensis in mouse bone marrow cells studied by using the micronucleus test. Toxicol Lett. 1989;48:165-169. Ramamoorthy A, Premakumari S. Effect of supplementation of Spirulina on hypercholesterolemic patients. J Food Sci Technol. 1996;33:124-128. Rellán S, Osswald J, Saker M, Gago-Martinez A, Vasconcelos V. First detection of anatoxin-a in human and animal dietary supplements containing cyanobacteria. Food Chem Toxicol. 2009;47:2189-2195. Ross E, Dominy W. The nutritional value of dehydrated, blue-green algae (Spirulina platensis) for poultry. Poult Sci. 1990;69:794-800. Salazar M, Chamorro GA, Salazar S, Steele CE. Effect of Spirulina maxima consumption on reproduction and peri- and postnatal development in rats. Food Chem Toxicol. 1996;34:353-359. Salazar M, Martínez E, Madrigal E, Ruiz LE, Chamorro GA. Subchronic toxicity study in mice fed Spirulina maxima. J Ethnopharmacol. 1998;62:235-241. Samuels R, Mani UV, Iyer UM, Nayak US. Hypocholesterolemic effect of spirulina in patients with hyperlipidemic nephrotic syndrome. J Med Food. 2002;5:91-96. Sapp J. The prokaryote-eukaryote dichotomy: Meanings and mythology. Microbiol Mol Biol Rev. 2005;69:292–305. Selmi C, Leung PS, Fischer L, German B, Yang CY, Kenny TP, Cysewski GR, Gershwin ME. The effects of Spirulina on anemia and immune function in senior citizens. Cell Mol Immunol. 2011 Jan 31. [Epub ahead of print] Seshadri, C. V. Large scale nutritional supplementation with Spirulina alga. All India Coordinated Project on Spirulina. Shri Amm Murugappa Chettiar Research Center (MCRC) Madras, India. 1993. Simpore J, Kabore F, Zongo F, Dansou D, Bere A, Pignatelli S, Biondi DM, Ruberto G, Musumeci S. Nutrition rehabilitation of undernourished children utilizing Spiruline and Misola. Nutr J. 2006;5:3-7. Simpore J, Zongo F, Kabore F, Dansou D, Bere A, Nikiema JB, Pignatelli S, Biondi DM, Ruberto G, Musumeci S. Nutrition rehabilitation of HIV-infected and HIV-negative undernourished children utilizing spirulina. Ann Nutr Metab. 2005;49:373-380. Spirulina platensis GRAS self affirmation 7-1-2011 Simsek N, Karadeniz A, Kalkan Y, Keles ON, Unal B. Spirulina platensis feeding inhibited the lead- and cadmium-induced anemia and leucopenia in rats. J Hazard Mater. 2009;164:1304-1309. Thaakur SR, Jyothi B. Effect of Spirulina maxima on the haloperidol induced tardive dyskinesia and oxidative stress in rats. J Neural Transm. 2007;114:1217-1225. Torres-Durán PV, Miranda-Zamora R, Paredes-Carbajal MC, Mascher D, Díaz-Zagoya JC, Juárez-Oropeza MA. Spirulina maxima prevents induction of fatty liver by carbon tetrachloride in the rat. Biochem Mol Biol Int. 1998;44:787-793. Torres-Duran PV, Ferreira-Hermosillo A, Juarez-Oropeza MA. Antihyperlipemic and antihypertensive effects of Spirulina maxima in an open sample of Mexican population: A preliminary report. Lipids Health Dis. 2007;6:1–8. Vadiraja BB, Gaikwad NW, Madyastha KM. Hepatoprotective effect of C-phycocyanin: Protection for carbon tetrachloride and R-(+)-pulegone-mediated hepatotoxicty in rats. Biochem Biophys Res Commun. 1998;249:428-431. Vonshak A. (editor). Spirulina platensis (Arthrospira): Physiology, Cell-Biology and Biotechnology. Taylor & Francis, London. 1997. Wu LC, Ho JA, Shieh MC, Lu IW. Antioxidant and antiproliferative activities of Spirulina and Chlorella water extracts. J Agric Food Chem. 2005;53:4207-4212. Zaccaro M. Efecto de la ingesta de Spirulina platensis en ratones expuestos a estres ambiental. 2004. Cited in Chamorro et al. 2008. Zhang HQ, Lin AP, Sun Y, Deng YM. Chemo- and radio-protective effects of polysaccharide of Spirulina platensis on hemopoietic system of mice and dogs. Acta Pharmacol Sin. 2001;22:1121-1124. Pages 000182 - 001301 have been removed in accordance with copyright laws. Please see pages 000034-000042 for a list of the references that have been removed.

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