Progress 06/01/18 to 07/31/19

Outputs
Target Audience:As a small business our target audience is potential customers, in this case food color and consumer product manufacturers. Food color additives require pre-market approval, so our audience also includes the Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration. Changes/Problems:A 15-day toxicology study in mice was completed prior to initiating the 90-day study in rats to estimate appropriate dosage levels. What opportunities for training and professional development has the project provided?Dr. Tanedjeu Kemgang participated in the webinar Overview of U.S. Intellectual Property Rights and the SBIR/STTR Stakeholders, September 25, 2018. How have the results been disseminated to communities of interest?Results of this research have been disseminated at quarterly meetings of the International Association of Color Manufacturers (IACM) andat the Global Color Conference IACM 2018, The Future of Food Color, November 6-8 in Arlington, VA,at the IFT 2019 Annual Meeting and Food Expo, June 2-5 in New Orleans, LA, as well as, directly to potential customers. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. Conduct sub-chronic toxicological tests of AvoColor® necessary for preparing a Color Additive Petition for submission to the FDA Center for Food Safety and Applied Nutrition AvoColor® is a red-orange semi-purified avocado (Persea americana, Lauraceae) seed extract with potential application as a natural food color additive. The safety of avocado seed extracts has been previously examined, but these studies have used a variety of extracts and focused on oral bolus dosing rather than dietary administration. In order to better understand the potential usefulness of AvoColor® as a food color additive, we conducted a 15-d sub-chronic study in male and female CD-1 mice using the dietary route of administration. AvoColor® was well-tolerated in the diet at levels of up to 5% w/w. No significant effect of AvoColor® on rate of body weight gain or final relative organ weights for the heart, liver, kidneys, lung, or spleen was observed. Mice treated with 5% AvoColor® had significantly lower blood urea nitrogen levels than the control or other treatment groups. Histopathological analysis showed that mice treated with 5% AvoColor®, the extreme dose, had mild inflammation in the kidney compared to control, but no differences were observed in other histopathological markers of kidney, liver, or small intestinal toxicity. In summary, our results demonstrate that AvoColor® at doses that exceed expected use levels by up to 50 times are well-tolerated. While these results support the feasibility of using AvoColor® as a food color additive, additional sub-chronic safety studies in rodents are needed to confirm our results.A 90-day sub-chronic toxicology study in Sprague-Dawley rats has been initiated withIntegrated Laboratory Services, Inc. Objective 2. Develop analytical tests and gather analytical data necessary for preparation of a color additive petition to FDA. We have developed an extraction method and HPLC-UV/VS analytical method for analysis of AvoColor® in solid matrices such as foods and biological samples (e.g. feces). The method employs theaflavin as an internal standard. A 100 g composite sample of AvoColor® was submitted to Microbac Laboratories, Inc. (Warrendale, PA) for microbial analyses. Using Methods CMMEF 5th Chs. 21, 8 and 9, the counts were less than the reporting limit of 10 cfu/g, 100 cfu/g, and 10 cfu/g for yeast and mold, total aerobic plate count, and coliforms, respectively. Samples of three batches of AvoColor® were also submitted to Microbac Laboratories, Inc. for heavy metal and pesticide analyses. Using Method AOAC 2007.01 Multi Residue Pesticide Screen for the presence of 206 different pesticide residues all were less than 10 ppb (reporting limit). The content of arsenic, lead, cadmium and mercury were all below 0.06 ppm by Method SW-846 6020B. Objective 3. Demonstrate proof-of-concept to employ large-scale vacuum filtration chromatography in the preparation of AvoColor®. We scaled the process from "kitchen" scale to a small pilot-plant scale, meaning, with the probable exception of grinding operations, we are operating with the same type of equipment that will be used as we scale production. We successfully developed an agitated adsorption/desorption process based on dry column vacuum chromatography. This adsorption/desorption process had significantly greater productivity than column chromatography (shorter times for adsorption and desorption with higher flow rates) and produced product of equal or better purity.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ziegler, GR. Innovations in Natural Food Colors. AIChE Food Innovation and Engineering (FOODIE) Conference, December 2-4, Napa, CA.

Progress 06/01/18 to 05/31/19

Outputs
Target Audience:As a small business our target audience is potential customers, in this case food color and consumer product manufacturers. Food color additives require pre-market approval, so our audience also includes the Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration. Changes/Problems:A 15-day toxicology study in mice was completed prior to initiating the 90-day study in rats. What opportunities for training and professional development has the project provided?Dr. Tanedjeu Kemgang participated in the webinar Overview of U.S. Intellectual Property Rights and the SBIR/STTR Stakeholders, September 25, 2018. How have the results been disseminated to communities of interest?Results of this research have been disseminated at quarterly meetings of the International Association of Color Manufacturers (IACM) and at the Global Color Conference IACM 2018, The Future of Food Color, November 6-8 in Arlington, VA, as well as, directly to potential customers. What do you plan to do during the next reporting period to accomplish the goals?Complete a 90-day sub-chronic toxicology study in Sprague-Dawley rats at Integrated Laboratory Services, Inc.

Impacts
What was accomplished under these goals? Objective 1. Conduct sub-chronic toxicological tests of AvoColor® necessary for preparing a Color Additive Petition for submission to the FDA Center for Food Safety and Applied Nutrition AvoColor® is a red-orange semi-purified avocado (Persea americana, Lauraceae) seed extract with potential application as a natural food color additive. The safety of avocado seed extracts has been previously examined, but these studies have used a variety of extracts and focused on oral bolus dosing rather than dietary administration. In order to better understand the potential usefulness of AvoColor® as a food color additive, we conducted a 15-d subacute study in male and female CD-1 mice using the dietary route of administration. AvoColor® was well-tolerated in the diet at levels of up to 5% w/w. No significant effect of AvoColor® on rate of body weight gain or final relative organ weights for the heart, liver, kidneys, lung, or spleen was observed. Mice treated with 5% AvoColor® had significantly lower blood urea nitrogen levels than the control or other treatment groups. Histopathological analysis showed that mice treated with 5% AvoColor®, the extreme dose, had mild inflammation in the kidney compared to control, but no differences were observed in other histopathological markers of kidney, liver, or small intestinal toxicity. In summary, our results demonstrate that AvoColor® at doses that exceed expected use levels by up to 50 times are well-tolerated. While these results support the feasibility of using AvoColor® as a food color additive, additional sub-chronic safety studies in rodents are needed to confirm our results. Objective 2. Develop analytical tests and gather analytical data necessary for preparation of a color additive petition to FDA. We have developed an extraction method and HPLC-UV/VS analytical method for analysis of AvoColor® in solid matrices such as foods and biological samples (e.g. feces). The method employs theaflavin as an internal standard.A 100 g composite sample of AvoColor® was submitted to Microbac Laboratories, Inc. (Warrendale, PA) for microbial analyses. Using Methods CMMEF 5th Chs. 21, 8 and 9, the counts were less than the reporting limit of 10 cfu/g, 100 cfu/g, and 10 cfu/g for yeast and mold, total aerobic plate count, and coliforms, respectively. Samples of three batches of AvoColor® were also submitted to Microbac Laboratories, Inc. for heavy metal and pesticide analyses. Using Method AOAC 2007.01 Multi Residue Pesticide Screen for the presence of 206 different pesticide residues all were less than 10 ppb (reporting limit). The content of arsenic, lead, cadmium and mercury were all below 0.06 ppm by Method SW-846 6020B. Objective 3. Demonstrate proof-of-concept to employ large-scale vacuum filtration chromatography in the preparation of AvoColor® During this Phase I period, we scaled the process from "kitchen" scale to a small pilot-plant scale, meaning, with the probable exception of grinding operations, we are operating with the same type of equipment that will be used as we scale production. We successfully developed an agitated adsorption/desorption process based on dry column vacuum chromatography. This adsorption/desorption process had significantly greater productivity than column chromatography (shorter times for adsorption and desorption with higher flow rates) and produced product of equal or better purity.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ziegler, GR. Innovations in Natural Food Colors. AIChE Food Innovation and Engineering (FOODIE) Conference, December 2-4, Napa, CA.