Absorption, Distribution and Excretion
Rats were fed a diet containing 5.19% of Allura Red. It was observed that 0.1% and 29% of the intact dye was excreted in the urine and feces respectively. In later studies, rats and dogs were pretreated daily with nonradioactive Allura Red. Subsequently, the animals were dosed with the 35S labelled compound and studied for up to 72 hours for excretion and distribution patterns of the color. Both species showed limited absorption of the compound with the major route of excretion being via the feces. In the dog 92-95% of the recovered radioactivity appeared in the feces within 72 hours while in the rat 76-92% of the recovered radioactivity appeared in the feces within this time period. Urinary recoveries of the color in rats and dogs, respectively varied between 5.7 and 19.8% and 2.7 and 3.6%. After sacrifice, significant retention of radioactivity was located in the intestinal contents of both species and in the washed intestines of the rats. This was thought to be due to adhesion of the compound to the intestinal wall, since the total carcass and viscera of these animals contained <0.4% of the administered dose.

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Metabolism / Metabolites
Several metabolites, possibly resulting from azo-reduction in the gastrointestinal tract (two identified as aromatic amines, p-cresidine sulfonic acid being the major one), were also found in the feces and urine. Finally, significant retention in the washed intestines of rat was observed, probably due to adhesion to the intestinal wall.
Cresidinesulfonic acid was found to be the major metabolite of Allura Red in the urine of these two species, whereas the parent compound was not measurable. In addition, two other unidentifiable metabolites were found in the urine of the rats. In the rat fecal extracts, cresidinesulfonic acid was a major metabolite along with two unknowns and the parent compound. The dog fecal sample revealed an identical metabolite pattern as seen in the rat, and in addition, a third unknown was discovered. One of the urinary unknowns demonstrated an Rf value which was identical to that of the one of the fecal unknowns suggesting that they were one and the same. The other unknowns exhibited distinctive Rf values which indicated that these metabolites were different. It has been postulated that azo reduction by gut flora of the dye will yield the two components of the parent compound: 2-methoxy-5-methyl-aniline-4-sulfonic acid (cresidine-4-sulfonic acid) and 1-amino-2-naphthol-6-sulfonic acid. It appears that negligible quantities of intact Red are absorbed and excreted in the urine, and that the major portion of the color is excreted as metabolites in the feces.

Interactions
In this study, the color and its alumina lake were applied to the subjects volvar forearms (200 subjects) as an aqueous solution for 10 alternate days, for 24-hr periods, followed by a 14-day rest period. Challenge batches were then applied under occlusion to fresh skin sites on the subjects scapular backs for 24 hours. The color did not produce either irritation or allergic responses during the induction phase nor contact dermatitis in the challenge period. ... Allura Red and its lake were evaluated on sites under occlusion for five 48-hr, alternate-day periods. These sites had been previously irradiated for 5 min with Xenon light which had been filtered through a window-glass equivalent to limit the exposure to non-erythema-producing, long-wave radiation. A 10-day rest period followed this induction exposure, and then the color was applied to fresh skin sites, irradiated for 5 min with Xenon and subsequently removed and the sites were evaluated. Allura Red was shown not to produce photosensitization on the 25 subjects studied.
In 2006, the Korea Food and Drug Administration reported that combinations of dietary colors such as allura red AC, tartrazine, sunset yellow FCF, amaranth, and brilliant blue FCF are widely used in food manufacturing. Although individual tar food colors are controlled based on acceptable daily intake (ADI), there is no apparent information available for how combinations of these additives affect food safety. In the current study, the potencies of single and combination use of /dyes/ were examined on neural progenitor cell (NPC) toxicity, a biomarker for developmental stage, and neurogenesis, indicative of adult central nervous system (CNS) functions. /allura red AC/ and /amaranth/ reduced NPC proliferation and viability in mouse multipotent NPC, in the developing CNS model. Among several combinations tested in mouse model, combination of /tartrazine/ and /brilliant blue FCF/ at 1000-fold higher than average daily intake in Korea significantly decreased numbers of newly generated cells in adult mouse hippocampus, indicating potent adverse actions on hippocampal neurogenesis. However, other combinations including /allura red AC/ and /amaranth/ did not affect adult hippocampal neurogenesis in the dentate gyrus. Evidence indicates that single and combination use of most tar food colors may be safe with respect to risk using developmental NPC and adult hippocampal neurogenesis...

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Non-Human Toxicity Values
LD50 Dog oral (male) > 5,000 mg/kg bw[EPA/Office of Pollution Prevention and Toxics; High Production Volume (HPV) Challenge Program's Robust Summaries and Test Plans for: 2-Naphthalenesulfonic acid, 6-hydroxy-5-
LD50 Rat oral >10,000 mg/kg bw[EPA/Office of Pollution Prevention and Toxics; High Production Volume (HPV) Challenge Program's Robust Summaries and Test Plans for: 2-Naphthalenesulfonic acid, 6-hydroxy-5-
LD50 Rabbit dermal 10,000 mg/kg bw

[1]. Characterisation of Interaction Between Food Colourant Allura Red AC and Human Serum Albumin: Multispectroscopic Analyses and Docking Simulations. Food Chem. 2015 Mar 1;170:423-9.

Allura red AC is a naphthalenesulfonic acid.

C18H14N2NA2O8S2

496.4219

495.998

25956-17-6

33258

Brown to reddish brown solid powder

300ºC

5.247

1

10

3

32

809

0

CEZCCHQBSQPRMU-UHFFFAOYSA-L

InChI=1S/C18H16N2O8S2.2Na/c1-10-7-14(16(28-2)9-17(10)30(25,26)27)19-20-18-13-5-4-12(29(22,23)24)8-11(13)3-6-15(18)21;;/h3-9,21H,1-2H3,(H,22,23,24)(H,25,26,27);;/q;2*+1/p-2

disodium;6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfonatophenyl)diazenyl]naphthalene-2-sulfonate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~100 mg/mL (~201.44 mM)
DMSO : ~62.5 mg/mL (~125.90 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)