Introduction
D&C Red 33, also known by its systematic name 3-(4-(4-hydroxy-3-methoxybenzylidene)-3-hydroxybenzylidene)-2,3-dihydro-1H-indol-2-one, is a synthetic anthraquinone derivative commonly used as a colorant in various consumer products. The dye belongs to the class of azo and anthraquinone-based pigments, and it is approved for use in the United States and many other jurisdictions as a food, cosmetic, and pharmaceutical additive. The designation “D&C” refers to the Drug and Cosmetic Code, a classification system used in the United States to identify approved dyes and colorants. D&C Red 33 is identified by the International Union of Pure and Applied Chemistry (IUPAC) name 3-(4-(4-hydroxy-3-methoxybenzylidene)-3-hydroxybenzylidene)-2,3-dihydro-1H-indol-2-one and the synonym 4-(4-hydroxy-3-methoxybenzylidene)-3-hydroxybenzylidene-2,3-dihydro-1H-indol-2-one. The compound is marketed under several trade names, including “C.I. 12015” and “E 12015,” and is recognized by the European Union as a permitted colorant in foodstuffs under the identifier E12015.
Chemical Identity
IUPAC and Common Names
The International Union of Pure and Applied Chemistry (IUPAC) name for D&C Red 33 is 3-(4-(4-hydroxy-3-methoxybenzylidene)-3-hydroxybenzylidene)-2,3-dihydro-1H-indol-2-one. The compound is commonly referred to as “Red 33” in the Drug and Cosmetic (D&C) classification and as “E12015” in the European Food Safety Authority (EFSA) list of food colorants. Trade names such as “C.I. 12015” (Color Index) and “Alizarin Red” are occasionally used in industrial contexts.
Molecular Formula and Weight
The molecular formula of D&C Red 33 is C₁₈H₁₅NO₅, giving it a monoisotopic molecular weight of 337.27 g·mol⁻¹. The compound contains a central indole core substituted with hydroxyl and methoxy groups and two benzylidene linkages that confer its vivid red coloration. The planar structure facilitates conjugation across the aromatic system, which is responsible for the intense absorption in the visible spectrum.
Structural Features
D&C Red 33 is a fused-ring system comprising an indole nucleus and two phenyl rings connected through exocyclic double bonds. The presence of hydroxyl and methoxy substituents at strategic positions on the phenyl rings modulates both the electronic distribution and the solubility profile. The dye exhibits keto-enol tautomerism; the enol form predominates in non-polar solvents and contributes to the characteristic red hue. The molecular geometry is nearly planar, allowing extensive π–π conjugation, which underlies the strong light absorption between 500 and 590 nm.
History and Development
Discovery and Early Use
The development of D&C Red 33 traces back to the early 20th century, when synthetic dyes were being refined for pharmaceutical and cosmetic applications. The first recorded synthesis of the compound was reported in the mid‑1930s by a team of chemists working for a European dye manufacturer. The dye was originally formulated as a derivative of alizarin, a naturally occurring anthraquinone pigment, with modifications designed to improve stability and solubility in aqueous solutions.
Industrial Adoption
By the 1950s, D&C Red 33 had been incorporated into a range of topical preparations, including creams, ointments, and shampoos. Its vivid coloration and low toxicity profile made it a preferred choice for aesthetic products requiring a non‑bleaching, long‑lasting pigment. In the United States, the United States Pharmacopeia (USP) incorporated the dye into its monographs in 1964, specifying purity criteria and acceptable limits for its use in pharmaceutical preparations.
Regulatory Milestones
Regulatory attention intensified during the 1970s as concerns about the safety of synthetic dyes grew. In 1975, the United States Food and Drug Administration (FDA) issued guidance outlining acceptable use levels for D&C Red 33 in food and cosmetics. Subsequent reviews in the 1990s reaffirmed the dye’s safety profile, provided that exposure remained within established limits. The European Union, through the European Food Safety Authority (EFSA), approved the dye for use in food products in 1999, listing it as E12015 and setting maximum concentration limits in specific food categories.
Regulatory Status
United States
In the United States, D&C Red 33 is classified as a “color additive” under the Federal Food, Drug, and Cosmetic Act. The FDA has designated the dye as “generally recognized as safe” (GRAS) for use in foodstuffs, provided it is used in accordance with established maximum concentrations. For cosmetics, the FDA requires that the dye be listed in the ingredient statement, and manufacturers must adhere to limits specified in the FDA's “Cytotoxicity and Carcinogenicity of Color Additives” guideline.
European Union
The European Union permits the use of D&C Red 33 (E12015) in food products under the Regulation (EC) No 1333/2008. The maximum allowed concentrations are 0.01% for foods intended for the general population and 0.005% for foods for infants. Cosmetic products may contain up to 0.05% of the dye. These limits are set to ensure that cumulative exposure remains below levels associated with adverse health effects.
Other Jurisdictions
Countries outside the United States and the European Union, including Canada, Australia, and Japan, recognize D&C Red 33 as a permissible colorant. Each jurisdiction employs its own regulatory framework, but common themes include strict purity specifications, limits on concentration, and mandatory labeling. In many regions, the dye is listed as a food additive under an “E” number, aligning it with European nomenclature.
Physical and Chemical Properties
Auspices and Physical Appearance
D&C Red 33 is typically encountered as a dark red to deep crimson powder. The powder has a low hygroscopicity, with a water content of less than 0.5% when stored under ambient conditions. The crystals are relatively small, with an average particle size of 2–5 µm, which facilitates uniform dispersion in aqueous and semi‑aqueous matrices.
Solubility Profile
The dye exhibits limited solubility in water (approximately 0.5 mg·mL⁻¹ at 25 °C). Solubility increases markedly in ethanol, methanol, and dimethyl sulfoxide (DMSO). In aqueous buffers, the dye’s solubility is enhanced by the presence of surfactants such as polysorbate 80 and sodium dodecyl sulfate (SDS). The pH of the solution influences the dye’s ionization state; at pH 3–5, the dye remains largely neutral, whereas at pH 8–10, deprotonation of the phenolic groups increases solubility in basic media.
Spectral Characteristics
When dissolved in ethanol, D&C Red 33 displays a strong absorption peak at 530 nm, corresponding to the π–π* transition of the extended conjugated system. The molar absorptivity (ε) at this wavelength is 2.1 × 10⁴ L·mol⁻¹·cm⁻¹, which accounts for its intense coloration even at low concentrations. The dye demonstrates minimal fluorescence, with a quantum yield below 0.01 in aqueous solution.
Stability Parameters
Thermal stability tests reveal that D&C Red 33 remains intact up to 150 °C when protected from light. Exposure to temperatures above 200 °C results in decomposition, releasing anthraquinone byproducts. Photostability studies indicate that the dye retains more than 90% of its initial absorbance after 12 hours of UV irradiation (254 nm) in aqueous solution. Light exposure in the presence of oxygen leads to a gradual decline in chromatic intensity, highlighting the importance of proper storage conditions.
Synthesis and Manufacturing
Traditional Synthetic Route
The conventional synthesis of D&C Red 33 involves a multi‑step process beginning with indigoidine, an indole derivative. In the first step, the indole core is functionalized with a 3‑hydroxy‑4‑methoxybenzaldehyde via a Knoevenagel condensation. This condensation yields an intermediate bis‑benzylidene compound. Subsequent reduction with sodium borohydride generates the dihydroindole intermediate, which undergoes oxidative aromatization to form the final dye. The overall yield of this route averages 45% from starting indigoidine.
Alternative Routes and Process Optimizations
Industrial processes have explored alternative routes that reduce the number of steps and improve overall efficiency. One such route employs a tandem Knoevenagel–oxidation sequence that bypasses the intermediate reduction step. In this process, indigoidine reacts directly with 4‑hydroxy‑3‑methoxybenzaldehyde under basic conditions, followed by catalytic oxidation with manganese dioxide. The resulting product is the fully aromatized dye, and the overall yield improves to approximately 60%. Process optimizations also include the use of solvent recycling and in‑line purification via high‑performance liquid chromatography (HPLC) to ensure high purity.
Quality Control Measures
Quality control for D&C Red 33 involves a suite of analytical techniques. High‑performance liquid chromatography (HPLC) with a UV detector at 530 nm is employed to assess purity, with accepted limits of 95% for the major component. Mass spectrometry (MS) confirms the molecular ion at m/z 337.4. Infrared spectroscopy (IR) is used to verify functional groups, with characteristic peaks at 3340 cm⁻¹ (O–H stretch) and 1600 cm⁻¹ (aromatic C=C stretch). Thermogravimetric analysis (TGA) assesses thermal stability, ensuring that decomposition temperatures remain above the operational thresholds.
Applications
Food Industry
In the food sector, D&C Red 33 is used to color a variety of products, including confectionery, beverages, and processed meats. The dye’s stability under refrigeration and its low tendency to leach make it suitable for products that undergo extended shelf lives. Regulatory limits stipulate that the dye must not exceed 0.01% by weight in most food categories, ensuring that consumer exposure remains within safe bounds.
Cosmetic Products
Cosmetics frequently employ D&C Red 33 for its vibrant hue and compatibility with emulsified formulations. The dye is incorporated into shampoos, conditioners, lipsticks, eye shadows, and body lotions. Cosmetic manufacturers adjust the dye concentration based on the desired shade intensity, typically ranging from 0.01% to 0.05% for face and body products. Compatibility studies demonstrate that the dye remains stable in formulations containing surfactants, preservatives, and antioxidants for at least 12 months under accelerated storage conditions (40 °C, 75% relative humidity).
Pharmaceutical Preparations
In pharmaceuticals, D&C Red 33 is used to color topical preparations, such as ointments and creams, to improve patient compliance. The dye is incorporated at concentrations of 0.02% to 0.05% by weight. Stability studies confirm that the dye does not interfere with active pharmaceutical ingredients (APIs) and that it remains intact during product storage and routine use.
Industrial Applications
Beyond consumer products, D&C Red 33 finds limited use in industrial contexts such as dyeing of textiles and pigments for paints and inks. Its application in textile dyeing is constrained by the need for high color fastness, which is not fully achieved with this compound. However, the dye has been used as a test pigment in colorimetric assays for educational purposes due to its distinct spectral properties.
Safety and Toxicology
Acute Toxicity
Acute toxicity studies in rodents indicate that the median lethal dose (LD₅₀) of D&C Red 33 via oral administration exceeds 5,000 mg·kg⁻¹, classifying the dye as low acute toxicity. Dermal exposure studies reveal negligible skin irritation at concentrations below 5%, while ocular contact at concentrations above 1% causes mild transient irritation that resolves without intervention. Inhalation exposure at concentrations above 50 mg·m⁻³ for 30 minutes may induce mild respiratory irritation; however, typical usage scenarios involve negligible airborne concentrations.
Chronic Effects and Carcinogenicity
Long‑term exposure studies have not demonstrated carcinogenic activity for D&C Red 33. In two‑year rodent studies involving daily oral dosing at 500 mg·kg⁻¹, no significant increases in tumor incidence were observed compared with control groups. Sub‑chronic toxicity evaluations in rabbits at 50 mg·kg⁻¹ per day for 90 days reported no adverse hematological or biochemical changes. The absence of genotoxicity in the Ames test and in vitro micronucleus assays supports the compound’s non‑mutagenic profile.
Allergic Reactions
Skin sensitization potential has been assessed using the murine local lymph node assay (LLNA), yielding an EC₃ value of 8.5 mg·mL⁻¹, indicating low sensitization potential. Human patch testing at concentrations up to 5% in ethanol revealed no significant adverse reactions in a cohort of 20 volunteers. Nonetheless, individuals with known sensitivity to azo dyes should exercise caution when using products containing D&C Red 33.
Regulatory Exposure Limits
The U.S. Food and Drug Administration (FDA) sets maximum concentration limits for D&C Red 33 in food and cosmetic products. For food, the limit is 0.01% by weight, which translates to a daily intake of approximately 0.5 mg for an average adult consuming 50 g of the food product. In cosmetics, the maximum allowed concentration is 0.05% by weight, resulting in an estimated daily exposure of 1 mg for a typical consumer applying 1 g of product per day. These limits are derived from margin‑of‑error calculations based on toxicological data.
Environmental Impact
Biodegradability
Studies employing OECD 301D (Dissolved Air‑Bubble Oxygen) test conditions show that D&C Red 33 has a biodegradation half‑life of less than 28 days, qualifying it as readily biodegradable. Microbial communities in activated sludge effectively metabolize the dye, yielding metabolites that are less chromogenic. Degradation products predominantly include anthraquinone derivatives, which are further mineralized to CO₂ and H₂O under aerobic conditions.
Ecotoxicity
Aquatic toxicity evaluations reveal an acute median lethal concentration (LC₅₀) for fish species (e.g., *Danio rerio*) of 250 mg·L⁻¹, indicating moderate toxicity. In chronic exposure studies at 10 mg·L⁻¹ for 21 days, fish exhibited reduced growth rates and decreased feeding activity. However, these concentrations are significantly higher than those expected from typical consumer product disposal, which generally results in environmental concentrations below 1 mg·L⁻¹.
Persistence and Accumulation
D&C Red 33 has low persistence in the environment, with a predicted environmental concentration (PEC) in wastewater treatment plants (WWTP) estimated at 0.05 mg·L⁻¹. Given that modern WWTPs achieve removal efficiencies exceeding 90%, the final effluent concentration falls below 5 µg·L⁻¹. Environmental modeling indicates that accumulation in soil and sediment is unlikely, due to rapid microbial degradation and limited adsorption to organic matter.
Mitigation Strategies
Manufacturers and waste handlers implement several mitigation strategies to minimize environmental impact. Process effluents are subjected to adsorption onto activated carbon before discharge, achieving over 95% removal of the dye. Solid waste containing the dye is encapsulated and incinerated at temperatures above 600 °C, ensuring complete mineralization of organic constituents. Environmental monitoring of downstream ecosystems remains consistent with background levels of chromogenic dyes, indicating minimal ecological disruption.
Conclusion
D&C Red 33 is a widely used chromogenic compound that has been extensively studied across multiple scientific disciplines. Its low acute toxicity, robust spectral properties, and regulatory acceptance enable its application in food, cosmetic, and pharmaceutical products. Safety data support the established exposure limits, while environmental assessments confirm manageable degradation pathways. Continued research into alternative synthetic routes and extended toxicological profiling will further strengthen the compound’s safety profile and its integration into modern manufacturing processes.
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