Pathway for FDA Approval of Biosynthetic Food Colors: A Case Study of Beetroot Red

FDA recently approved a color additive petition (CAP 4C0326) submitted by Phytolon, allowing beetroot red to be used as a color additive exempt from batch certification in human food. The FDA accepted the petition on March 12, 2024, and issued a final rule in the Federal Register on February 6, 2026. A new regulation, 21 CFR § 73.39 Beetroot red (under 21 CFR Part 73), was established. The rule will take effect 45 days after publication, i.e., on March 23, 2026.

Definition and Scope of Use

Beetroot red is a red-purple powder or liquid produced via controlled fermentation using a non-pathogenic, non-toxigenic strain of Saccharomyces cerevisiae. This strain has been genetically engineered to express genes involved in betalain biosynthesis derived from plants in the order Caryophyllales (e.g., Beta vulgaris L. var. rubra). Betalains are the primary coloring components of this additive.

Beetroot red is intended for use in coloring human food in compliance with current good manufacturing practice (cGMP). It is not permitted for use in products regulated by the United States Department of Agriculture, infant formula, or foods with standards of identity established under Section 401 of the FD&C Act, unless such standards explicitly allow its use.

Specifications

The FDA has established strict specifications for beetroot red, including defined limits for heavy metals such as lead, arsenic, mercury, and cadmium. The product must also be free from other impurities that can be avoided under good manufacturing practice (GMP).

Manufacturing Process

• Strain construction: Genetically engineering Saccharomyces cerevisiae to express betalain biosynthesis genes from beetroot and related plants.
• Controlled fermentation: Using the modified strain to produce betalains.
• Separation and purification: Removing the production yeast from the fermentation broth.
• Formulation: Concentrating into liquid form or drying into powder form.

Dietary Exposure Assessment

The petitioner initially estimated dietary exposure to betalains for consumers. The FDA further conducted a comprehensive dietary exposure assessment covering both powder and liquid forms of beetroot red. Since it is intended to replace beetroot powder (21 CFR 73.40), it is not expected to increase dietary exposure to betalains.

Safety Assessment

The petitioner applied a weight-of-evidence approach to evaluate safety under intended use conditions:

1. History of Safe Consumption

Betalains have a long history of safe consumption in natural dietary sources such as beetroot and dragon fruit. The FDA approved beetroot powder containing betalains as a color additive in 1967 (21 CFR 73.40), with no maximum daily intake limit established due to lack of safety concerns.

Saccharomyces cerevisiae has a long history of safe use in food, and the specific engineered strain is non-toxic and non-pathogenic.

2. Toxicological Studies

(1)Studies conducted by the petitioner:

• Ames test, in vitro mammalian chromosomal aberration test, in vivo micronucleus test, Pig-a gene mutation assay, and next-generation sequencing-based mutagenicity assessment in liver/stomach/intestine tissues all indicated no mutagenic or genotoxic effects.
• A 90-day subchronic oral toxicity study in rats identified a NOAEL at the highest dose level (5%): males: 3,581 mg/kg bw/day; females: 4,055 mg/kg bw/day.

(2)Published literature

Summaries of acute, subchronic, and chronic toxicity studies in animals, as well as human clinical studies on betalains and related substances (e.g., beetroot juice powder, betanin), further support safety.

3. Allergenicity Assessment

Bioinformatics analysis showed no significant similarity between introduced protein sequences and known allergens, indicating no evidence of allergenic risk.

The FDA concluded that beetroot red is safe for use as a color additive in human food under the intended conditions and meets the requirements for exemption from batch certification.

Some commenters objected to the name “beetroot red,” arguing that the colorant is not directly extracted from beetroot and suggesting “betanin” instead. The FDA responded that: The colorant is produced through expression of beetroot-derived genes, making “beetroot red” an appropriate name. Although betanin is the primary pigment, the additive contains other pigments and non-color components; therefore, “betanin” is not suitable as a name.

Additional Information: Approved Biosynthetic Colors

Note: Komagataella phaffii was formerly known as Pichia pastoris; both refer to the same yeast species.

If you need any assistance or have any questions, please get in touch with us via service@cirs-group.com.