Heptadecanoic acid

Safety evaluation of odd-chain fatty acid algal oil

Ray A. Matulka a,*, Lauren A. Howell a, B. Pratyusha Chennupati b, J. Teresa Bock b
a Burdock Group Consultants, Orlando, FL, 32814, USA
b Heliae Development LLC, Gilbert, AZ, 85297, USA

Abstract

In the food industry, most fatty acid-rich oils are primarily composed of saturated even-chain fatty acids. However, saturated odd-chain fatty acids are potentially a beneficial alternative to other saturated fatty acid- containing oils. In this communication, we examine the safety of odd-chain fatty acid (OCFA) algal oil, a microalgal-sourced oil composed primarily of the saturated odd-chain fatty acids pentadecanoic acid and hep- tadecanoic acid. OCFA algal oil was assessed for toXicity in a 14-day palatability study and comprehensive 13- week dietary study at inclusion levels of 5%, 10%, and 15% in the diet, utilizing a DHA-rich algal oil as a comparator control. No adverse effects attributed to the consumption of OCFA algal oil were observed in either study. Therefore, we report a No Observable Adverse Effect Level (NOAEL) of 150,000 ppm (15% in the diet), equivalent to an OCFA algal oil intake of 7553.9 and 8387.7 mg/kg bw/day for male and female rats, respec- tively. The genotoXic potential of OCFA algal oil was also examined in an in vitro bacterial reverse mutation assay and in vivo mammalian bone marrow chromosome aberration test. OCFA algal oil was non-mutagenic in Sal- monella typhimurium and Escherichia coli test strains and did not exhibit clastogenicity in vivo.

1. Introduction

Assessments of dietary fatty acid consumption typically focus on the intake of saturated even-chain fatty acids (ECFAs), which have been associated with an elevated risk of heart disease at high intake levels (Mensink, 2016). The U.S. Dietary Guidelines for Americans (2020–2025) suggests limiting saturated fat intake to less than 10% of calories per day and replacing them with unsaturated (mainly poly- unsaturated) fatty acids (USDA/HHS, 2020). The majority of fatty acid intake is comprised of even-numbered fatty acids, while odd-numbered fatty acids are found in the diet in low levels (<1%), primarily from dairy sources, as odd-numbered fatty acids can make up approXimately 2.0–3.0% of the fatty acid content of whole ruminant milk (CDC, 2019; Devle et al., 2012). Even-numbered fatty acids are metabolized by beta-oXidation to form acetyl-CoA units, while odd-numbered fatty acids, while also going through beta-oXidation, end with propionyl-CoA, which must go through additional steps to be converted to succinyl CoA and enter the citric acid cycle (Gotoh et al., 2008; Nelson and CoX, 2004). This alteration in metabolism may have an impact on overall energy production or utilization. Consumption of saturated odd-chain fatty acids (OCFAs), specifically pentadecanoic acid (C15:0) and hep- tadecanoic acid (C17:0), has been associated with several health benefits, including the modulation of glucose metabolism and inflam- matory pathways (Kurotani et al., 2017; Venn-Watson et al., 2020). Currently, new sources of saturated OCFAs are being researched to better evaluate the potential advantages of consuming these fatty acid molecules, for the potential addition to products that replace dairy in the diet to provide a source of these OCFAs. OCFA algal oil is a saturated odd-chain fatty acid-rich oil extracted from the microalgae Aurantichytrium acetophilum, a unicellular eukary- otic protist of the Thraustochytrid clade that was originally harvested off the coast of the United States and may also be found in other ocean waters and sediments, as Thraustochytrids are typically isolated from marine habitats (Ganuza et al., 2019). OCFA algal oil contains ≥95% triacylglycerols (TAGs), consisting of 40–60% saturated OCFAs (pre- dominately pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0)) and 40–60% ECFAs, which includes approXimately 25% ECFA content of the omega-3 fatty acid docosahexaenoic acid (DHA). The proposed use for OCFA algal oil is as a nutritive ingredient that provides a dietary source of both saturated odd-chain fatty acids and poly- unsaturated even-chain fatty acids. Evaluation of the scientific literature failed to locate any published studies that evaluated the safety of satu- rated OCFA-rich oils produced by A. acetophilum. Therefore, to deter- mine if repeated ingestion of high intake levels of TAGs composed of saturated OCFAs produced from A. acetophilum in the diet induced toXicological effects in a rat model, a comprehensive 13-week dietary study in rats was conducted that evaluated clinical observations, serum, hematological, pathological, and urinary endpoints, a standardized preclinical study that is typically utilized to support the evaluation of the safety of potential food ingredients. Further, the mutagenic and clasto- genic potential of OCFA algal oil was evaluated utilizing the in vitro bacterial reverse mutation assay and in vivo mammalian bone marrow chromosome aberration test, respectively. 2. Materials and methods 2.1. Test materials The OFCA algal oil ingredient is an off-white to amber semi-solid butter at room temperature comprised of triacylglycerols, composed primarily of saturated odd-chain fatty acids (predominately pentade- canoic acid (C15:0) and heptadecanoic acid (C17:0)) and even-chain fatty acids that include DHA at approXimately 25% of the fatty acid content, extracted from the microalgae Aurantiochytrium acetophilum (internal designation number HS399). The stability and homogeneity of OCFA algal oil was confirmed per Good Laboratory Practices (GLP) and ICH Guideline Q2(R1): Validation of Analytical Procedures: Text and Methodology (data not shown). Levels of mycotoXins, heavy metals, and microbiological contaminants were confirmed to be within company specifications for OCFA algal oil (data not shown). The DHA algae oil comparator control is a blend of Palm Fruit Oil and a refined algal oil known as DHA ORIGINS® 400 (Certificates of Analysis available by request) to approXimate the amount of DHA in OCFA algal oil. The palm fruit and algal oils are blended together to produce the 15% DHA algal oil utilized herein, yielding a fatty acid content as follows: C16:0 (27.1%), C18:1n9 (32.3%), C18:2n6 (7.2%), C22:5n6 (4.7%), and
C22:6n3 (23.9%).

2.2. Experimental design

2.2.1. 14-Day palatability study

Male and female CRL Sprague-Dawley CD IGS rats were obtained from Charles River Laboratory, Inc. Rats were seven to eight weeks of age at study initiation, with variations in weight not to exceed 20% of the mean weight for each sex. Animals were individually housed under a 12-h light/dark cycle at a temperature of 18–20 ◦C and relative humidity of 10–54%. Food and water were provided ad libitum. Rats were accli- mated for seven days prior to study initiation and randomized per standard operating procedures of the testing laboratory, Product Safety Labs (PSL). PSL is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and certified in the appropriate care of all live experimental animals. PSL maintains current staff training, ensuring animals were handled humanely during the experimental phase of this study, in compliance with the recommen- dations of the Guide for the Care and Use of Laboratory Animals from the National Research Council (NRC, 2011).

CRL Sprague-Dawley CD IGS rats (5/sex/group) were administered a basal rodent diet (supplied by Research Diets, Inc., New Brunswick, NJ) formulated with 16% soybean oil (referred to as the 0% negative con- trol) or OCFA algal oil at nominal concentrations of 5%, 10%, or 15% (50,000, 100,000, or 150,000 ppm) for 14 consecutive days, yielding a targeted daily dose of OCFA algal oil of 4167, 8333, or 12,500 mg/kg bw/day, respectively. Rat laboratory diets range in fat content; nutrient requirements for fat by laboratory rats is estimated at 5% of the diet (National Research Council, 1995), but may range in much higher concentrations, up to 21% fat in the rat diet to mimic a U.S. Western diet (Research Diets, 2021), which would be considered an obese diet in rats (Buettner et al., 2006). Therefore, a diet range was chosen to provide the nutritive requirements for rats while approaching fat content similar to a Western diet without reaching levels to cause obesity. DHA content of the OCFA algal oil was taken into account to reflect maximum DHA intake by humans at 3000 mg/person/day (GRN#137, 2004), as per U.S.

Food and Drug Administration (FDA, 2007) 21 CFR 184.1472. Mor- tality was assessed at least twice per day, and cage-side observations were performed once daily. Detailed clinical evaluations of the skin, fur, eyes, mucous membranes, gait, posture, behavior, and occurrence of secretions, excretions, seizures, stereotypies (e.g., excessive grooming or repetitive circling), or autonomic activity (e.g., lacrimation, piloer- ection, pupil size, and unusual respiratory patterns) were performed on Day 0 prior to treatment and at weekly intervals until the end of the study period. Bodyweight and food consumption were measured at least two times during the acclimation period, on Day 0 prior to study initi- ation, and on Days 3, 7, 10, and 14 post-initiation. Food efficiency was calculated by dividing the mean daily bodyweight gain by the mean daily food consumption for each animal. All animals (including de- cedents) were subjected to a full necropsy to include examination of the external surface of the body, all orifices, and the thoracic, abdominal, and cranial cavities and their contents.
Blood was collected via the vena cava under isoflurane anesthesia prior to euthanasia at the termination of the study for hematological and clinical chemistry analysis. Hematological analysis included assess- ments of red blood cell (erythrocyte) count (RBC), hemoglobin con- centration, hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin con- centration (MCHC), red cell distribution width (RDW), absolute reticu- locyte count (ARET), platelet count, total white blood cell count (WBC), absolute reticulocytes (ARET), absolute neutrophils (ANEU), absolute lymphocytes (ALYM), absolute monocytes (AMON), absolute eosino- phils (AEOS), absolute basophils (ABAS), absolute large unstained cells (ALUC), differential leukocyte count, and blood smears (if necessary), in addition to assessments of coagulation to include prothrombin time (PT) and activated partial thromboplastin time (APTT). Clinical chemistry parameters included serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH), alkaline phosphatase (ALKP), total bilirubin (BILI), urea nitrogen (BUN), blood creatinine (CREA), albumin (ALB), globulin (GLOB), triglycerides (TRIG), total cholesterol, fasting glucose, total serum protein, calcium, inorganic phosphorus, sodium, potassium, and chloride. All animals (including decedents) were subjected to a full necropsy to include ex- amination of the external surface of the body, all orifices, and the thoracic, abdominal, and cranial cavities and their contents. The brain, liver, thymus, adrenals, kidneys, testes, heart, epididymides, spleen, pituitary gland, uterus, ovaries with oviducts, thyroid/parathyroid, and prostate and seminal vesicles with coagulating gland were weighed to determine treatment-related effects on organ weight. The 14-day palatability study was performed in compliance with the guidelines set forth in OECD Guideline 407 (OECD, 2008; modified from a 28-day termination to a 14-day termination) and consistent with FDA Redbook 2000 guidelines.

2.2.2. 13-Week dietary study

Male and female CRL Sprague-Dawley CD IGS rats were obtained from Charles River Laboratory, Inc. Rats were seven to eight weeks of age at study initiation, with variations in weight not to exceed 20% of the mean weight for each sex. Animals were individually housed under a 12-h light/dark cycle at a temperature of 20–23 ◦C and relative humidity of 50–62%. Food and water were provided ad libitum. Rats were accli- mated for siX days prior to study initiation and randomized per standard operating procedures of the testing laboratory, PSL. PSL is accredited by AAALAC and certified in the appropriate care of all live experimental animals. PSL maintains current staff training, ensuring animals were handled humanely during the experimental phase of this study, in compliance with the recommendations of the Guide for the Care and Use of Laboratory Animals from the National Research Council (NRC, 2011).

CRL Sprague-Dawley CD IGS rats (10/sex/group) were administered a basal rodent diet (supplied by Research Diets, Inc., New Brunswick, NJ) formulated with 16% soybean oil (referred to as the 0% negative control), 15% DHA oil (comparator control), or OCFA algal oil at nominal concentrations of 5%, 10%, or 15% (50,000, 100,000, or 150,000 ppm) for 90 consecutive days, yielding a targeted daily dose of OCFA algal oil of 3125, 6250, or 9375 mg/kg bw/day, respectively, the same nominal concentrations utilized in the 14-day palatability study. Diet characterization of the different oils in the feed to provide nutri- tional equivalence among groups is provided, ensuring comparable fat, protein and carbohydrate content across dose groups (Supplemental Table 1). Mortality was assessed at least twice per day, and cage-side observations were performed once daily. Ophthalmological evalua- tions (focal illumination, slit lamp biomicroscopy, and indirect oph- thalmoloscopy) were performed during the acclimation period and on Day 86 of the dosing period. Detailed clinical evaluations of the skin, fur, eyes, mucous membranes, gait, posture, behavior, and occurrence of secretions, excretions, seizures, stereotypies (e.g., excessive grooming or repetitive circling), or autonomic activity (e.g., lacrimation, piloer- ection, pupil size, and unusual respiratory patterns) were performed on Day 0 prior to treatment and at weekly intervals until the end of the study period. Bodyweight and food consumption were measured at least two times during the acclimation period, on Day 0 prior to study initi- ation, and at weekly intervals until the end of the study period. Food efficiency was calculated by dividing the mean daily bodyweight gain by the mean daily food consumption. Urine was collected at the termination of the study for urinalysis, to include microscopic examination of urine sediment and assessments of clarity, quality, color, volume, pH, bilirubin, ketones, blood, specific gravity, total protein, urobilinogen, and glucose.

Blood was collected via the sublingual vein or vena cava/abdominal aorta under isoflurane anesthesia prior to euthanasia at the termination of the study for hematological and clinical chemistry analysis. Hema- tological analysis included assessments of red blood cell (erythrocyte) count (RBC), hemoglobin concentration, hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distri- bution width (RDW), platelet count, total white blood cell count (WBC), absolute reticulocytes (ARET), absolute neutrophils (ANEU), absolute lymphocytes (ALYM), absolute monocytes (AMON), absolute eosino- phils (AEOS), absolute basophils (ABAS), absolute large unstained cells (ALUC), differential leukocyte count, and blood smears (if necessary), in addition to assessments of coagulation that included prothrombin time (PT) and activated partial thromboplastin time (APTT). Clinical chem- istry parameters included serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH), alkaline phosphatase (ALKP), total bilirubin (BILI), urea nitrogen (BUN), high-density lipoprotein (HDL), low-density lipoprotein (LDL), blood creatinine (CREA), albumin (ALB), globulin (GLOB), triglycerides (TRIG), total cholesterol, fasting glucose, total serum protein, calcium, inorganic phosphorus, sodium, potassium, and chloride, in addition to analysis of the thyroid hormones triiodothyronine (T3), thyroXine (T4), and thyroXine stimulating hormone (TSH).

All animals (including decedents) were subjected to a full necropsy and evaluation of organ weight as described above at the end of the in- life phase, in addition to histopathological analysis to include exami- nation of the kidneys, eyes, skin, spleen, skeletal muscle, urinary bladder, sternum, femur, sciatic nerve, mammary gland, bone marrow (from femur and sternum), Harderian gland, optic nerve, aorta, heart, brain (including medulla/pons, cerebellar, and cerebral cortex), salivary glands (sublingual, submandibular, and parotid), spinal cord (cervical, mid-thoracic, and lumbar), lymph nodes (mandibular and mesenteric), and organs of the respiratory (lungs, larynx, pharynx, nose, nasal tur- binates, and trachea), endocrine (pituitary gland, thyroid, parathyroid, pancreas (with islets), thymus, and adrenals), reproductive (ovaries, oviducts, vagina, uterus, cerviX, epididymides, testes, prostate, and seminal vesicles), and gastrointestinal (esophagus, colon, stomach, cecum, duodenum, jejunum, ileum with Peyer’s patches, pancreas (with islets), liver, and rectum) systems. The 13-week dietary study was per- formed in compliance with GLP and the guidelines set forth in OECD Guideline 408 (OECD, 2018) and consistent with FDA Redbook 2000 guidelines.

2.2.3. Bacterial reverse mutation assay

Utilizing the plate incorporation method, Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain WP2 uvrA (obtained from Molecular ToXicology Inc.) were incubated with OCFA algal oil in DMSO at concentrations of 1.25, 3.0, 12.5, 30.0,
125, 300, 1250, or 3000 μg/plate, in the presence or absence of metabolic activation [using chemically induced rat liver S9 miX (Molecular ToXicology, Inc, Boone, NC)]. Sodium azide (15 μg/mL; TA100 and TA1535), ICR 191 acridine (10 μg/mL; TA1537), daunomycin (6 μg/mL; TA98), and methyl methanesulfonate (25 μL/mL; WP2 uvrA) were used as positive controls in the absence of metabolic activation. In the pres- ence of metabolic activation, 2-aminoanthracene (100 μg/mL) served as a positive control in all strains. DMSO served as the negative (vehicle) control under both conditions of metabolic activation. An additional confirmatory test was performed with OCFA algal oil at concentrations of 1.58, 5.0, 15.8, 50, 158, 500, 1580, or 5000 μg/plate, utilizing the method of concentration spacing of the plate incorporation test. After incubation, the number of colonies per plate was counted manually and/ or with the aid of a Colony-Doc-It™ plate reader, and the mean and standard deviation was calculated in triplicate for each strain and metabolic state. The mutation factor (MF) was calculated by dividing the mean revertant colony count by the mean revertant colony count for the respective vehicle control group. A significant increase in mutagenicity was gauged by a resultant MF 2 for TA98, TA100, and WP2 uvrA and MF 3 for TA1535 and TA1537, with mean values out of range of historical controls, occurring in a dose-dependent or reproducible manner. The bacterial reverse mutation test was performed in compli- ance with GLP and the standards outlined in FDA Redbook 2000 and ICH S2 (R1) Guidance on GenotoXicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use (2012) guidelines.

2.2.4. In vivo mammalian bone marrow chromosome aberration test

Male and female Wistar (Crl:WI (Han)) rats were obtained from Charles River Laboratories, Inc. Rats were siX to ten weeks of age at study initiation, with variations in weight not to exceed 20% of the mean weight for each sex. Following acclimation and randomization, two to three animals of identical sex were housed per cage under a 12-h light/dark cycle at a temperature of 22 3 ◦C and relative humidity of
55 10%, with at least 10 air changes per hour. Food and water were provided ad libitum. The animal testing site, BSL Bioservice (Munich), is accredited by AAALAC, and BSL Bioservice followed the principles of the OECD Guidance Document on the Recognition, Assessment, and Use of Clinical Signs as Humane Endpoints for EXperimental Animals Used in Safety Evaluation (OECD, 2002). The study was reviewed and accepted by local authorities, per German animal protection laws, and was sub- jected to the Ethical Review Process and authorized by the Bavarian animal welfare administration.

Wistar (Crl:WI (Han)) rats (5/sex/group) were gavaged with a single oral dose of corn oil (vehicle control; 10 mL/kg bw) or OCFA algal oil at dose levels of 400, 800, 1500, or 2000 mg/kg bw. An additional group of rats received a single intraperitoneal injection of cyclophosphamide in physiological saline (10 mL/kg bw; 40 mg/kg bw) as a positive control. Animals were sacrificed at 24 (all groups) or 48 (negative control group and 1500 and 2000 mg/kg bw/day groups only) hours post- administration, following an intraperitoneal injection with the meta- phase arresting agent colchicine 4 h prior to sacrifice. Femurs were removed and flushed with potassium chloride solution to remove bone marrow cells. The cells were fiXed with methanol: glacial acetic acid, and slides were prepared and stained with Giemsa. Slides were exam- ined for the presence of polyploid cells, and the frequency of chromo- some aberrations (including gaps, breaks, fragments, deletions, exchanges, and chromosomal disintegrations) was analyzed in 200 well spread metaphases per animal. As a measure of cytotoXicity, the mitotic index was measured in at least 1000 cells per animal. The in vivo mammalian bone marrow chromosome aberration test was performed in compliance with GLP and per the guidelines set forth in OECD Guideline 475 (OECD, 2016).

2.3. statistical analysis

2.3.1. 14-Day palatability study and 13-week dietary study

For the 14-day palatability study, data analysis was performed using Provantis® version 9, Tables and Statistics, Instem LSS, Staffordshire UK; and Pristima® version 7, Statistical Analysis. For the 13-week di- etary study, data analysis was performed using Provantis® version 10, Tables and Statistics, Instem LSS, Staffordshire UK. For both repeated dose oral toXicity studies, statistical analysis was performed between each group and the 0% control group, and statistical significance was determined at a probability value of P < 0.05. Statistically significant alterations for in-life endpoints (e.g., bodyweight, food consumption, and food efficiency) were determined via two-way analysis of variance (ANOVA), with significant alterations further analyzed via post hoc multiple comparisons tests (e.g., Dunnett’s test). Organ weight data was evaluated for homogeneity of variances and normality by Bartlett’s test. Where homogeneous variances and normal distribution were observed, data was compared using a one-way ANOVA. When one-way ANOVA was significant, a multiple comparisons test (e.g., Dunnett’s test) was applied. Where variance was considered significantly different, the data was analyzed via a nonparametric method (e.g., Kruskal-Wallis non- parametric analysis of variance). When the non-parametric analysis of variance was determined significant, a Dunn’s test was utilized to compare data. Clinical pathology data was preliminarily analyzed via Barlett’s test for homogeneity and Shapiro-Wilk test for normality. Where homogeneity and normality were not significant, data was compared using a one-way ANOVA followed by a Dunnett’s test. When these preliminary tests were significant, a log transformation was applied to the data. If the log transformation failed to achieve normality and variance homogeneity, the data was analyzed via a non-parametric method (e.g., Kruskal-Wallis non-parametric analysis of variance). When the non-parametric analysis of variance was significant, a Dunn’s test was utilized to compare data. 2.3.2. Bacterial reverse mutation assay The mean values and standard deviations for all quantitative data was calculated by Product Safety Labs. 2.3.3. In vivo mammalian bone marrow chromosome aberration test Data analysis was performed using GraphPad Prism version 6.0. The non-parametric Mann-Whitney test was utilized to determine the sta- tistical significance of chromosomal aberrations compared to the cor- responding negative controls, in the absence of chromosomal gaps. Dose-related trends in chromosomal damage were analyzed using the χ2 test. Statistical significance was determined at the 5% level (P < 0.05). 3. Results and discussion 3.1. 14-Day palatability study The 14-day palatability study was performed to evaluate the palat- ability and general toXicity of OCFA algal oil in rats following 14 days of dietary administration. All rats survived the study, and no significant changes in bodyweight (Supplemental Table 2), bodyweight gain (Supplemental Table 3), food consumption (Supplemental Table 4), or food efficiency (Supplemental Table 5) were observed in male or female rats. The average administered dose of OCFA algal oil was calculated as 4574.0, 8990.7, and 13314.4 mg/kg/day for males and 4085.1, 7987.8, and 11389.4 mg/kg/day for females in the 5%, 10%, and 15% OCFA algal oil groups, respectively. A single incidence of bilateral, brown ocular discharge was observed in a single 5% OCFA algal oil male on days 12–14, accompanied by lacrimation on day 14, and a single 5% OCFA algal oil female presented with a damaged ear at the end of the cortical hyperplasia was most notable in the zona fasciculata, which was notably thicker with higher numbers of slightly enlarged cells with an increased amount of finely vacuolated cytoplasm, often accompanied by the presence of small numbers of mitotic figures. However, due to the lack of concomitant focal pre-neoplastic or neoplastic findings or negative effects on the function or life span of the affected animals, minimal diffuse cortical hyperplasia in females is not adverse in nature, may be due to stress from various causes (Hoenerhoff et al., 2015b) and thus unrelated to OCFA algal oil administration. Microscopic analysis of the liver (Supplemental Table 8) identified an increased incidence of minimal hepatocellular hypertrophy in males in the 15% OCFA algal oil group and females in the 10% and 15% OCFA algal oil groups and 15% DHA comparator control group. The minimal hepatocellular hypertrophy was primarily restricted to the centrilobular regions of the liver, with affected cells presenting with an increase in size and cytoplasmic volume. In females, these observations correlated with an increase in absolute liver weight and relative liver-to-body and liver- to-brain weights. However, no concomitant alterations in liver enzymes typical of an indication of toXicity (e.g., ALT, AST, SDH) were detected upon serum clinical chemistry analysis in the female dose groups, while a slight but significant (P < 0.05) increase in serum AST was reported in the 15% OCFA algal oil male dose group (102.3 14.3 U/L), when compared to the control group (82.6 30.6). However, the increased value was still within historical control ranges for this strain of rat (77.0–110.00 U/L) (Clifford and Giknis, 2006). An international panel of experts convened by the European Society of ToXicologic Pathology (Hall et al., 2012) conducted a workshop and concluded “that hepato- megaly as a consequence of hepatocellular hypertrophy without histo- logic or clinical pathology alterations indicative of liver toXicity was considered an adaptive and a non-adverse reaction.” Therefore, cen- trilobular liver hypertrophy is considered an adaptive response and unrelated to consumption of OCFA algal oil. 3.3. Bacterial reverse mutation assay No treatment- or dose-related increases in the number of revertant colonies (Table 5) or mutation factor (Table 6) were observed in Sal- monella typhimurium strains TA98, TA100, TA1535, or TA1537, or Escherichia coli strain WP2 uvrA upon incubation with OCFA algal oil up to 3000 μg/plate, in the presence or absence of metabolic activation. These results were reproducible in an additional confirmatory test performed with OCFA algal oil up to 5000 μg/plate (Supplemental Table 9 and Supplemental Table 10). In all bacterial tester strains, pre- cipitation was reported at the highest dose levels of 3000 (main test) and 5000 (confirmatory test) μg/plate, but no signs of contamination or toXicity were observed. The mean revertant colony counts of all strains treated with DMSO (vehicle) were within range of historical controls (Mortelmans and Zeiger, 2000; Gatehouse, 2012), and the positive controls yielded a substantial increase in revertant colony count. 3.4. In vivo chromosomal aberration assay Administration of a single oral dose of OCFA algal oil for 24 or 48 h did not affect the incidence of chromosomal aberrations in the bone marrow of rats of either sex (Table 7). No polyploid cells were detected in any animal, and no notable in- crease in the frequency of aberrant cells was observed in male or female rats treated with OCFA algal oil at dose levels of 400, 800, 1500, or 2000 mg/kg bw for 24 or 48 h, with all mean values within the range of the concurrent negative controls or historical values. A non-parametric Mann-Whitney test and a χ2 test were performed to validate these observations and confirmed that no statistically significant increase in the number of aberrant cells was present in male or female rats upon treatment with OCFA algal oil. Further, the mitotic index was assessed as a measure of cytotoXicity (Table 7), and no notable change in the mitotic index was observed in the 400 and 800 mg/kg bw groups following 24 h of OCFA algal oil exposure. In 1500 mg/kg bw females and 2000 mg/kg bw males, the mitotic index was significantly increased, compared to the concurrent negative control groups, upon administration of OCFA algal from rats orally administered OCFA algal oil in an acute dose up to 2000 mg/kg bw/day. Together, these data indicate no safety concern for the consumption of OCFA algal oil in rats up to 15% in the diet and contribute to the potential use of OCFA algal oil as a potential beneficial alternative to other saturated fatty-acid rich oils utilized in the food industry. Author contributions BPC and TB helped prepare the protocol, assisted in monitoring the studies, and contributed to the preparation of the manuscript. LH assisted in manuscript preparation. RM aided in study initiation and monitoring, evaluated the results, and assisted in manuscript dietary toXicity study, no adverse effects were observed in rats consuming OCFA algal oil at dietary inclusion levels of 5%, 10%, or 15% (50,000, 100,000, or 150,000 ppm) for 13 consecutive weeks. There- fore, under the conditions of this study and based on the toXicological endpoints evaluated, the NOAEL for OCFA algal oil was determined to be 150,000 ppm (15% in the diet), the highest dose evaluated, equiva- lent to dietary intake levels of at least 7553.9 and 8387.7 mg/kg bw/day in male and female rats, respectively. In addition, an in vitro bacterial reverse mutation assay and in vivo mammalian bone marrow chromo- some aberration test were performed to examine the genotoXic potential of OCFA algal oil. OCFA algal oil was non-mutagenic in Salmonella typhimurium and Escherichia coli test strains at concentrations up to 5000 μg/plate and did not exhibit clastogenicity in vivo in bone marrow cells preparation. Funding The studies and manuscript preparation were funded by Heliae Development, LLC. CRediT authorship contribution statement Ray A. Matulka: Conceptualization, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Supervision. Lauren A. Howell: Formal analysis, Investigation, Writing – original draft, Writing – review & editing. B. Pratyusha Chennupati: Resources, SD, standard deviation; CPA, cyclophosphamide; OCFA, odd-chain fatty acid; MTD, maximum tolerated dose. †EXcluding chromosomal gaps; *P < 0.05 vs. corresponding control group; **P < 0.01 vs. corresponding control group. #For each study group, 200 metaphases were counted per animals, unless noted as follows. a Four mice (200 metaphases/rat), one rat (173 metaphases). b Two rats (25), one rat (40), one rat (14), one rat (20). c One rat (50), one rat (60), one rat (26), one rat (33), one rat (45). d Four rats (200), one rat (103). e Four rats (200), one rat (147). f Four rats (200), one rat (68). g Two rats (200), one rat (66), one rat (71), one rat (70). h Four rats (200), one rat 178. Writing – review & editing, Project administration. J. Teresa Bock: Conceptualization, Supervision, Writing – review & editing, Project administration, Funding acquisition. Declaration of competing interest The authors declare the following financial interests/personal re- lationships which may be considered as potential competing interests: BPC and TB are employees of Heliae Development, LLC. RM and LH, employees of the consulting firm Burdock Group that was engaged by and who received payment from Heliae Development, LLC., were compensated for study monitoring and manuscript preparation. Acknowledgements The 14-day palatability study, 13-week dietary study, and bacterial reverse mutation assay were conducted at Product Safety Labs in Day- ton, New Jersey, US. Clinical pathology evaluation was conducted at Eurofins Advinus, Ltd, Bengaluru, India. Histopathologic examination for the 13-week dietary study was conducted at Histo-Scientific Research Laboratories, Mount Jackson, VA. The chromosomal aberra- tion study was conducted at Eurofins BioPharma Product Testing Munich GmbH in Planegg, Germany. 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