In vitro and in vivo toxicological evaluation of 6′-Sialyllactose sodium salt produced by a novel method

Abstract

6′-Sialyllactose (6′-SL) sodium salt is the sodium salt of a naturally occurring trisaccharide found in dairy products and human milk. 6′-SL sodium salt formulations produced by different methods have been the subject of other safety studies and are approved for use in food in the United States and other countries. Because the manufacturing process can influence the toxicological profile of a substance, studies were undertaken to assess the safety of 6′-SL sodium salt produced by a novel manufacturing process. The results of genetic toxicity tests (bacterial reverse mutation assay, an in vitro mammalian chromosome aberration test, and an in vivo micronucleus test in mice), and acute and 26-week repeated dose oral toxicity studies in rats for this novel 6′-SL sodium salt are reported herein. All studies were conducted according to Good Laboratory Practice (GLP) and the respective guidelines from the Korean Ministry of Food and Drug Safety (MFDS). The substance showed no evidence of genotoxicity in all three assays that were conducted to address this endpoint and was not acutely toxic to rats at doses up to 6000 mg/kg body weight (bw). The no observed adverse effect level (NOAEL) for toxicity in the 26-week study in both male and female rats was 6000 mg/kg bw/day, the highest dose administered. This new 6′-SL sodium salt is safe in rats at a higher level and for a longer period of time than previously studied in either rats or pigs, demonstrating a more robust safety profile.

Graphical Abstract

Highlights

• •6'-SL sodium salt was prepared by a method that has not been previously described.
• •It is not genotoxic in the Ames, chromosomal aberration, or micronucleus tests.
• •The acute oral LD₅₀ value in rats is > 6000 mg/kg bw of 6′-SL sodium salt.
• •The 26-week oral NOAEL for the substance is 6000 mg/kg bw/day in rats of both sexes.

1. Introduction

6'-Sialyllactose (6′-SL) is a nondigestible oligosaccharide found in human milk that is comprised of N-acetylneuraminic acid (also known as sialic acid) and lactose. Because 6′-SL is not readily digestible, it acts as a metabolic substrate for intestinal bacteria, helping to establish and maintain a healthy gut microbiome in infants, children and adults [1], [10], [2]. Other purported benefits of 6′-SL include improvement in cognition, muscle health and exercise performance [13], [14], [5]. According to Soyyılmaz et al. [17], the mean concentration of 6′-SL in breast milk of mothers throughout nursing ranges from approximately 300 – 710 mg/L, with higher amounts reported in colostrum and transitional stage milk (6 – 14 days) than later stage milk (> 90 days). The amount of 6′- SL in breast milk is considerably higher than the amount in bovine milk (3.3 – 10.4 mg/L) or reconstituted infant formula (3.6 – 5.1 mg/L) [4]. One of the strategies used to make infant formula more like human milk is to add 6′- SL as 6′-SL sodium salt.

Over the past 10 years, various manufacturers have obtained approval for use of 6′-SL sodium salt formulations in infant formula and conventional food in several countries. Because each manufacturer produces this substance by a different manufacturing process, each 6′- SL sodium salt formulation is unique. Although some manufacturers have demonstrated safety of their specific 6′- SL sodium salt ingredient in standard toxicity tests [15], [6], it is possible that a different manufacturing process could introduce substances that could adversely affect the toxicological profile. While it may be possible to demonstrate safety of a new formulation through a comparison approach with a formulation that has been previously tested, the best way to show safety is through direct testing.

The results of genetic toxicity tests and acute and 26-week repeated dose oral toxicity studies in rats for the safety evaluation of 6′-SL sodium salt produced by a new process are described in this publication. Similar tests are typically conducted for candidate food ingredients; however, in this case, the duration of the repeated dose oral toxicity study in rats exceeds the generally recommended 13-week study period. The synthesis of 6′-SL sodium salt employs a multi-enzyme reaction utilizing polyphosphate kinase (PPK), CMP-N-acetylneuraminic acid synthetase, N-acetyl-D-glucosamine-2-epimerase, N-acetylneuraminic aldolase, and α2,6-sialyltransferase. These enzymes were derived from a β-D-galactosidase-deficient strain of E. coli K-12 ∆lacZ, which originates from the non-pathogenic E. coli K-12 lineage. This production method differs from that reported by Gurung et al. [6] in its use of a nonmetallic phosphate donor and PPK as an enzyme, instead of acetate kinase (ACK).

2. Materials and methods

2.1. Testing laboratory and test substance

All studies were conducted according to Good Laboratory Practice (GLP) at H&H BIO Hoseo Univ. GLP Center, Asan-si, Republic of Korea except for the chronic toxicity study, which was conducted according to GLP at DT&CRO Co., Ltd., Republic of Korea. The test material used in all studies was 6′-Sialyllactose sodium salt (lot number BO08Vl19–01 (purity 96.88 w/w% anhydrous)) for all studies except for the chronic toxicity study with lot numbers BO08Vc11–01, BO08Vh12–01, and BO08Vl19–01 (purity 97.50, 96.91 and 96.88 w/w% anhydrous, respectively).

2.2. Bacterial Reverse Mutation Test (Ames Test)

The bacterial reverse mutation test was performed according to the “Guideline for Toxicological Test” Genotoxicity Test (Bacterial Reverse Mutation Test), Korean Ministry of Food and Drug Safety, Notification No. 2022–18 (March 2, 2022). The following strains were used in preliminary and main studies: Salmonella (S.) typhimurium TA98, TA100, TA1535, TA1537, and Escherichia (E.) coli strain WP2 uvrA (sourced from Molecular Toxicology, Inc.). The tests used the plate incorporation method, in the presence and absence of metabolic activation from phenobarbital/5,6-benzoflavone induced male rat liver S9 mix (also from Molecular Toxicology, Inc.). A preliminary study showed that the test material was dissolved in water for injection at the desired concentration. Therefore, this solvent was used as the vehicle, as well as the negative control. The positive controls in the absence of S9 mix were sodium azide (15 µg/mL) for TA100 and TA1535, 9-aminoacridine hydrochloride monohydrate (800 µg/mL) for TA1537, 2-nitrofluorene (5 µg/mL) for TA98, and 4-nitroquinolone N-oxide (5 µg/mL) for WP2 uvrA. In the presence of S9, the positive controls were 2-aminoanthracene (10, 20, 20 or 100 µg/mL) for TA100, TA1535, TA1537 and WP2 uvrA (respectively) and benzo[a]pyrene (10 µg/mL) for TA98. All positive controls were prepared in dimethyl sulfoxide (DMSO), except for sodium azide, which was prepared in water for injection.

In the initial test, the following materials were mixed and poured over a minimal agar plate: 100 µL of each test substance, negative (vehicle) control, or positive control, 500 µL of S9 mix or sodium phosphate buffer, 100 µL of bacterial suspension and 2 mL of molten top agar containing trace amounts of histidine or tryptophan. Concentrations of the test material were 313, 625, 1250, 2500, and 5000 µg/plate. Each plate was prepared in duplicate. After the agar gelled, the plates were inverted and incubated at 37°C for 48 h. Based on the results of the initial test, two main tests were subsequently conducted using the same procedure, with each plate prepared in triplicate.

Following incubation, colony counts and evidence of precipitation were assessed manually, but growth inhibition of the background lawn was assessed using a microscope. Results were considered positive if the number of revertant colonies for any strain at one or more concentrations were increased relative to the negative control, or if there was a reproducible dose-dependent increase in the number of colonies. The assay was considered acceptable if: the cell concentration was at least 1 × 10⁹ cells/mL, results for negative control cultures were within the range of historical control data, the number of revertant colonies in the positive control cultures was increased compared to negative control cultures, and contamination did not interfere with the results.

2.3. In Vitro chromosome aberration assay

The in vitro chromosome aberration test was performed according to the “Guideline for Toxicological Test” Genotoxicity Test (In vitro Mammalian Chromosome Aberration Test), Korean Ministry of Food and Drug Safety, Notification No. 2022–18 (March 2, 2022). The Chinese Hamster Lung (CHL/IU) cells used in this assay were supplied from American Type Culture Collection. The cells were cultured at 37°C (5 % CO₂) in a Minimum Essential Medium (MEM) containing 10 % fetal bovine serum, 1 % penicillin/streptomycin, and 1 % glutamine. Cells used in the study were 8 passages or less.

The cells were exposed to test or control materials both with and without the addition of an S9 activation system. Water for injection served as the vehicle for the test material and the negative control. The positive controls in the absence and presence of S9 mix were mitomycin C (0.2 µg/mL) and benzo[a]pyrene (20 µg/mL), respectively. The S9 system was sourced from livers of male Sprague-Dawley (SD) rats induced by phenobarbital and 5,6-benzoflavone (Molecular Toxicology, Inc.). In treatments including S9, a Cofactor mix from Genogen Co., Ltd was added to cell cultures.

In a preliminary study, CHL/IU cells (1 × 10⁴/mL) were precultured in 60 mm plates containing 4.0 or 4.5 mL of culture medium for 3 days. After counting, 0.5 mL of S9 mix was added to the plates containing 4.0 mL of medium, and the cells were treated with 0.5 mL of negative or positive control solutions or serially diluted test material to provide final concentrations of 7.81, 15.6, 31.3, 62.5, 125, 250, or 500 µg/mL/plate. Two sets of plates without S9 mix were prepared: one set was incubated for 6 h, followed by medium replacement and further incubation for 18 h (short term treatment) and the other set was incubated for 24 h (long term treatment). Plates containing S9 mix were incubated under the short-term treatment scenario. Osmotic pressure and pH were checked before and after culture to determine if changes in these variables occurred. At the end of the experiment, cells were released from culture by adding 0.05 % trypsin-EDTA solution, diluted in culture medium and counted using a hemocytometer to determine the percentage of dead cells for each test condition.

Based on the results of the study, the dose levels chosen for the main study were 125, 250 and 500 µg/mL/plate. The main study was conducted under the same conditions as the preliminary study, but duplicate cultures were prepared for short- or long-term treatments. At approximately 22 h of culture, colcemid (0.2 µg/mL) was added to the cells. At 24 h, cells were washed with phosphate-buffered saline, released from culture, and counted as previously described to determine the percentage of dead cells. Cells were pelleted using a centrifuge, resuspended in 3 mL of 75 mM KCl (37 ºC), maintained at 37 ºC for 30 min, and treated with fixative (3:1 methanol: acetic acid). These cells were pelleted using a centrifuge and resuspended in fixative. The centrifuge procedure was repeated, and the cells were dropped onto slides (two per plate). After drying, the cells were stained with 5 % Giemsa for 20 min, washed with distilled water, and dried. One slide per test condition (300 metaphase cells) was checked for structural and numerical chromosome aberrations under magnification (1000x) in a blinded manner. Gaps were recorded but were not included as structural aberrations or in the total frequency of chromosome aberrations. Results were considered positive if the frequency of structural aberrations increased at one or more concentrations of the test material compared to the concurrent negative control or historical negative control results, or if there was a dose-dependent increase in the frequency of chromosome aberrations. The assay was considered acceptable if: the results for negative control cultures were within the range of historical control data, the number of revertant colonies in the positive control cultures was increased compared to negative control cultures and was within the historical range of positive control cultures, at least three doses could be evaluated, and contamination did not interfere with the results.

2.4. In Vivo micronucleus assay

The in vivo micronucleus assay was performed according to the “Guideline for Toxicological Test” Genotoxicity Test (In vivo micronucleus test using rodent hematoblast), Korean Ministry of Food and Drug Safety, Notification No. 2022–18 (March 2, 2022). The study was approved by the Animal Experiment Ethics Committee of the laboratory that conducted the study (Approval No. IACUC-M24009) in accordance with the “Animal Protection Act” No. 19486 (partially amended on June 20, 2023). CrlOri:CD1 (ICR), Specific Pathogen Free (SPF) mice from Orient Bio Inc., Seongnam, Republic of Korea, were acclimated for at least 6 days and were 8 weeks old when assigned to each study. The mice were supplied with Teklad Certified Irradiated Global 18 % Protein Rodent Diet (2918 C, Envigo, USA) and drinking water ad libitum. They were housed in polycarbonate cages containing radiation-sterilized bedding (LAS bedding P3, Germany) by group and sex (no more than 5/cage) under a 12-hour light/dark cycle in a room maintained at 19.4 – 25.0ºC and 43.1 – 69.7 % humidity, with more than 10 air changes/hour. The animals were uniquely identified by an indelible marking on the tail, and cages were individually identified by an animal ID card.

In a preliminary study, groups of mice (n = 3/sex/group) were dosed with 500, 1000, or 2000 mg/kg body weight (bw) of test material dissolved in water for injection by oral gavage, once a day for two days. The volume administered at each time was 10 mL/kg bw. Both sexes tolerated these doses and did not exhibit any abnormal clinical signs. Based on these findings, the main study was conducted with the same doses, in male mice only. In the main study, negative (water for injection) and positive control (80 mg/kg bw cyclophosphamide monohydrate, CPA) groups were included, and each group contained five animals. The negative control and test substance groups were treated by oral gavage once a day for two days, and the positive control animals were given CPA 24 h before bone marrow collection. All animals were observed for clinical signs at appropriate times, and body weights were recorded before dosing and prior to harvesting of bone marrow cells. Animals were euthanized 24 h after their last dose using CO₂. Bone marrow cells were collected from the dissected femur of each animal and suspended onto clean, dry slides in duplicate. The slides were air-dried, fixed with methanol for 5 min, and stained with 5 % Giemsa for approximately 20 min. The stained slides were washed with buffer and a 0.004 % citric acid solution, air dried, mounted and covered with coverslips. One slide from each animal was coded and observed under a microscope at 1000x magnification. Four thousand polychromatic erythrocytes (PCE) were evaluated to determine the incidence of micronucleated polychromatic erythrocytes (MNPCE). To detect possible bone marrow toxicity, the ratio of polychromatic (immature) to mature erythrocytes was determined from 500 total erythrocytes per animal. The test substance was judged positive if there was a dose dependent increase in MNPCE, the results were outside the range of the historical negative control data, or if there was a statistically significant increase in the frequency of MNPCE in any test group compared with the concurrent negative control. The study was considered valid and acceptable if: the results for the negative control group were within the range of historical control data, the number of MNPCE in the positive control group was increased compared to the negative control group and within the historical range of the positive control group, and at least three doses were evaluated, and the number of cells for analysis was appropriate.

2.5. Acute oral toxicity study

The acute oral toxicity study was performed according to the “Guideline for Toxicological Test” Single Dose Toxicity Study, Korean Ministry of Food and Drug Safety, Notification No. 2022–18 (March 2, 2022). The study was approved by the Animal Experiment Ethics Committee of the laboratory that conducted the study (Approval No. IACUC-R24044) in accordance with the “Animal Protection Act” No. 19486 (partially amended on June 20, 2023). Sprague-Dawley SPF rats from SAMTAKO BIO KOREA (105, Seorang-ro, Osan-si, Gyeonggi-do, Republic of Korea) were acclimated for at least 6 days and were 8 weeks old when assigned to the study. The rats were supplied with Teklad Certified Irradiated Global 18 % Protein Rodent Diet (2918 C, Envigo, USA) and drinking water ad libitum. They were housed in polycarbonate cages containing sterilized bedding (LASvendi P3, Germany) by group and sex (no more than 5 per cage) under a 12-hour light/dark cycle in a room maintained at 19.2 – 25.0ºC and 30.3 – 69.8 % humidity, with more than 10 air changes/hour. The group assignment was performed by randomly selecting animals close to average body weight the day before administration. The animals were uniquely identified by an indelible marking on the tail, and cages were individually identified by an animal ID card. Groups of rats (n = 5/sex/group) which had been fasted overnight (more than 12 h), were dosed once with water for injection (control) or 1500, 3000, or 6000 mg/kg bw test material dissolved in water for injection by oral gavage. Feed was provided 3 h post-dosing. All animals were observed for mortality, general condition, and clinical signs (time, onset, severity, and recovery) at least once for 30 min after dosing and at 1, 2, 3, and 4 h after dosing on Day 0 and once a day thereafter for 14 days (Days 1–14). Body weights were recorded once on animal acquisition, group assignment, prior to dosing (Day 0), on Days 1, 3, 7, and 14 (Day of necropsy). On Day 14, all surviving animals were anesthetized with CO₂ gas, exsanguinated from the abdominal aorta, and examined grossly. Statistical analyses were planned but not performed because no dead animals were observed.

2.6. Chronic (26-week) oral toxicity study

The 26-week oral toxicity study was performed according to the “Guideline for Toxicological Test” Standards for Toxicity Studies of Drugs, as per the Korean Ministry of Food and Drug Safety, Notification No. 2022–18 (March 2, 2022) and ICH Harmonised Tripartite Guideline-M3 (R2) (June 11, 2009). The study was approved by the Institutional Animal Care and Use Committee (IACUC) of the laboratory that conducted the study (Approval No.: 220177), in accordance with the “Animal Protection Act” No. 16977 (revised on Feb. 11, 2020) and the ‘Guide for the Care and Use of Laboratory Animals’ (revised Jun. 10, 2022, No. 18969). The rat strain, source and diet were the same as described above for the acute toxicity study. Diet and drinking water were supplied ad libitum except during urine collection and the night before blood collection/necropsy when food was withdrawn. The animals were housed in stainless wire mesh cages (one per cage during the study) under a 12-hour light/dark cycle in a room maintained at 19 – 25ºC and 30 – 70 % humidity, with 10–15 air changes/hour. The animals were 5 weeks old at receipt and 6 weeks old at initiation of treatment. The animals were uniquely identified by an indelible marking on the tail and a color-coded cage card was placed on each cage to describe the group and dose levels. For the toxicity study, animals were uniformly allocated to study groups as follows: 15 animals/sex/group for Groups 1 and 4, and 10 animals/sex/group for Groups 2 and 3. Five of the animals/sex for Groups 1 and 4 served as recovery animals, which were kept for an additional 4 weeks without treatment after the main study was completed. Animals in Group 1 received water for injection (control), while those in Groups 2, 3 and 4 received 1500, 3000 or 6000 mg/kg bw of test material/day, dissolved in water for injection by oral gavage. Additional groups of animals (3 animals/sex for Group 1 and 6 animals/sex for Groups 2–4) participated in a toxicokinetics study. The dose volume was 10 mL/kg bw. The dosing formulations were confirmed to be homogenous, stable and within acceptance criteria for concentration.

All animals in the toxicity and toxicokinetics studies were observed once daily for clinical signs and twice daily for mortality and moribundity, and body weights were recorded at appropriate intervals. The remaining endpoints described here were measured on toxicity study animals only: food consumption (appropriate intervals), ophthalmology and urinalysis (5/sex/group at Week 26 and all recovery group animals at week 4), and clinical pathology, hematology, and coagulation (all toxicity study animals). Parameters measured are shown in tables in the results section and supplemental materials. All animals were anesthetized with isoflurane and exsanguinated from the abdominal aorta on Day 183 and Day 211 for the main and recovery groups, respectively. Complete gross postmortem examinations were performed on all animals and the following organs were weighed: brain, thymus, heart, liver, kidneys (paired), epididymides (paired), prostate, uterus, pituitary gland, spleen, adrenal glands (paired), testes (paired), ovary (paired) and seminal vesicle with coagulating gland. The thyroid gland was weighed together with the parathyroid gland after fixation in 10 % neutral buffered formalin. All other organs stated above, as well as the aorta, femur (with bone marrow), skeletal muscle (thigh), tongue, Harderian gland, sciatic nerve, sternum (with bone marrow), inguinal mammary gland (with skin), lungs (with bronchi), vagina, esophagus, stomach, duodenum, ileum, jejunum, colon, cecum, rectum, pancreas, submandibular lymph node, trachea, urinary bladder, mesenteric lymph node, salivary gland (submandibular, sublingual and parotid gland), and any other organs with gross lesions were fixed in the same fixative, except for the testes, the eyes with the optic nerve and thoracic spinal cord, which were preserved in Davidson fixative. Organs or tissues from all animals in Groups 1 and 4 (as well as for lower dose groups if changes were observed in Group 4), and from macroscopic lesions in Group 2 were examined.

Blood collection from the jugular vein and plasma separation for the toxicokinetic analysis were performed on Days 1, 92 and 182 at various times after dosing (times are shown in Table 5). Following blood sample collection, animals of the toxicokinetic group were euthanized. Analyses of the circulating concentrations of the test substance in plasma were performed using a validated internal analytical method. The following parameters were calculated using WinNonlin (Ver. 8.3.5, Pharsight-A Certara, U.S.A.): Area under the plasma concentration-time curve (AUC_0–24), maximum plasma concentration (C_max), Time to reach C_max (T_max) and half-life (t_1/2).

Table 5.

Toxicokinetic Results in the 26-Week Toxicity Study of 6′-SL Sodium Salt.

Parameter	1500 mg/kg bw/day			3000 mg/kg bw/day			6000 mg/kg bw/day
	Day 1	Day 92	Day 182	Day 1	Day 92	Day 182	Day 1	Day 92	Day 182
Males
AUC0–24(ng•hr /mL)	11,588.36	2704.60	5254.00	20,011.17	4790.23	6587.29	101,872.41	47,204.10	35,135.84
Cmax(ng/mL)	4143.00	539.13	1253.70	5850.00	1461.27	1653.37	34,040.00	30,180.57	20,469.37
Tmax(hr)	0.5	1.0	1.0	0.5	1.0	0.5	0.5	0.5	0.5
t1/2(hr)	2.32	4.92	2.84	1.51	4.18	3.47	8.21	2.62	1.92
Females
AUC0–24(ng•hr/mL)	10,225.06	1864.58	1810.72	17,144.64	8155.31	3877.74	70,212.52	39,232.07	18,002.96
Cmax(ng/mL)	3130.83	688.67	629.43	4765.03	2002.73	949.77	28,128.23	23,349.47	18,965.67
Tmax(hr)	1.0	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
t1/2(hr)	1.77	1.20	1.31	2.95	3.15	3.21	2.51	3.87	2.38

AUC_0–24 – Area under the curve for first 24 hr; bw – Body weight; C_max – Area under the plasma concentration-time curve for the first 24 hr; hr – Hour(s); kg – Kilogram; mg – Milligrams; mL – Milliliter; ng – Nanogram(s); T_max – Time to reach C_max; t_1/2 – half-life

2.7. Statistical analysis

For the genetic toxicity studies, SPSS (IBM® SPSS Statistics, ver. 18) was used to analyze the data, and statistical significance between treatment and control groups was evaluated at p < 0.05 using a Fisher’s exact test or analysis of variance (ANOVA). In the acute and 26-week repeat dose oral toxicity studies, continuous data from the main study were subjected to statistical analysis using the SAS Program (version 9.4, SAS Institute Inc., U.S.A.). For the main study groups, these data were analyzed using Bartlett’s test for homogeneity of variance at p < 0.05. A one-way ANOVA was employed on homogeneous data, and a Dunnett’s test was applied for multiple comparisons. The Kruskal-Wallis test was employed on heterogeneous data, and a Steel test was applied for multiple comparisons. For the recovery groups in the 26-week study, these data were analyzed using the Folded F-test for homogeneity of variance at p < 0.05. When the data were homogeneous, a Student-t test was conducted, and when the variances were heterogeneous, an Aspin-Welch t-test was conducted. Statistical significance was reported at p < 0.05 and p < 0.01 (two-tailed).

3. Results

3.1. Mutagenicity Assay

Under the conditions of the study, 6′-SL sodium salt was not mutagenic in the evaluated strains (Table 1). None of the tested concentrations were toxic or precipitated in the culture medium, and contamination was not noted. The positive and negative controls produced the expected responses, verifying the validity of the assay.

Table 1.

Bacterial Reverse Mutation Assay of 6′-SL Sodium Salt: Mean Number of Revertant Colonies per Plate.

Concentration (µg/plate)	TA98		TA100		TA1535		TA1537		WP2uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Experiment 1
0a	19	14	186	190	10	10	8	9	38	42
313	20	16	187	192	10	10	9	9	38	41
625	19	17	188	191	10	11	7	9	39	40
1250	20	14	187	197	10	10	8	9	37	38
2500	16	14	190	190	8	10	8	11	37	40
5000	20	15	189	192	7	11	8	9	39	42
Positive control	112b	149c	535d	989e	857d	370e	190 f	336e	649 g	251e
Experiment 2
0a	22	30	172	179	8	10	6	7	40	38
313	24	29	173	174	8	9	5	8	41	32
625	26	28	184	169	8	11	5	8	39	32
1250	25	28	177	169	10	10	5	7	41	32
2500	25	28	178	173	9	9	6	8	40	34
5000	22	27	177	186	8	11	6	7	38	35
Positive control	126b	171c	764d	1141e	768d	380e	467 f	338e	607 g	327e
Historical negative control range (-S9)	11 – 47		73 – 189		4 – 24		2 – 22		20 – 69
Historical negative control range (+S9)	10 – 53		79 – 196		2 – 38		2 – 28		20 – 69

Substances were tested using the plate incorporation method. Results are presented as the mean of 3 plates per experimental condition and Experiment 2 is a duplication of Experiment 1 (same test method). No precipitation or inhibition of the bacterial lawn was observed in any of the plates. S9 - Rat liver preparation that contains metabolizing enzymes; µg - micrograms

^aWater for injection (vehicle);

^b2-Nitrofluorene;

^cBenzo[a]pyrene;

^dSodium azide;

^e2-aminoanthracene;

^f9-aminoacridine;

^g4-nitroquinoline-1-oxide

3.2. In vitro chromosomal aberration assay

Under the conditions of the study, numerical observations were not observed in any culture. In the short-term (6 h) experiment with metabolic activation, no chromosomal aberrations were observed in either control cultures or cultures exposed to 6′-SL sodium salt (Table 2). In both the short-term (6 h) experiment without metabolic activation and the long-term (24 h) experiment, there were no differences in the frequency of chromosomal aberrations (gap- or gap+) between cultures exposed to any concentration of 6′-SL sodium salt and the negative control (water for injection). No precipitation of the test material, contamination or cytotoxicity was observed under any test condition. The responses of positive controls B[a]P and MMC were statistically significant compared to their respective negative controls in both the short- and long-term cultures; however, the response of the positive control MMC in long-term cultures without metabolic activation was slightly below the historical control range. Therefore, the long-term study without metabolic activation did not meet the validity requirement that the result for the positive control should be within the historical range.

Table 2.

Results of in vitro Chromosomal Aberration Study of 6′-SL Sodium Salt.

S9 mix/hr.	Substance	Dose (µg/mL)	Cells Analyzed	Cells with Structural Aberrations
				ctg	ctb	cte	csg	csb	cse	frg/ othersa	Total (%)
											gap-		gap+
+ / 6 hr.	Water for injection	0	150	0	0	0	0	0	0	0	0	0.0	0	0.0
			150	0	0	0	0	0	0	0
	6'-SL sodium salt	125	150	0	0	0	0	0	0	0	0	0.0	0	0.0
			150	0	0	0	0	0	0	0
		250	150	0	0	0	0	0	0	0	0	0.0	0	0.0
			150	0	0	0	0	0	0	0
		500	150	0	0	0	0	0	0	0	0	0.0	0	0.0
			150	0	0	0	0	0	0	0
	B[a]P	20	150	8	15	26	0	0	2	0	59	19.7*	63	21.0*
			150	13	19	32	0	0	2	0
Historical Control Range (B[a]P, %)											8.8–30.2		NS
- / 6 hr.	Water for injection	0	150	0	0	0	0	0	0	0	1	0.3	1	0.3
			150	0	1	0	0	0	0	0
	6'-SL sodium salt	125	150	0	0	0	0	0	0	0	0	0.0	0	0.0
			150	0	0	0	0	0	0	0
		250	150	0	0	0	0	0	0	0	0	0.0	0	0.0
			150	0	0	0	0	0	0	0
		500	150	1	1	0	0	0	0	0	1	0.3	2	0.7
			150	0	0	0	0	0	0	0
	MMC	0.2	150	4	27	26	0	0	2	0	62	20.7*	67	22.3*
			150	11	3	41	0	0	2	0
Historical Control Range (MMC, %)											13.0–25.8		NS
- / 24 hr.	Water for injection	0	150	0	0	0	0	0	0	0	0	0.0	0	0.0
			150	0	0	0	0	0	0	0
	6'-SL sodium salt	125	150	0	0	0	0	0	0	0	0	0.0	1	0.3
			150	0	0	0	1	0	0	0
		250	150	0	0	0	0	0	0	0	0	0.0	0	0.0
			150	0	0	0	0	0	0	0
		500	150	1	0	0	1	0	0	0	0	0.0	2	0.7
			150	0	0	0	0	0	0	0
	MMC	0.2	150	7	10	46	0	0	0	0	76	25.3*	83	27.7*
			150	10	12	41	0	0	1	0
Historical Control Range (MMC, %)											26.9–44.4		NS

B[a]P – Benzo[a]pyrene; mL – Milliliter; MMC – Mitomycin C; frg – fragmentation; csb – chromosome break; cse – chromosome exchange; csg – chromosome gap; ctb – chromatid break; cte – chromatid exchange; ctg – chromatid gap; NS – Not specified; S9 – rat liver preparation that contains metabolizing enzymes; µg – micrograms

*Significant difference from negative control (water) by Fisher’s exact test (p < 0.05).

^aOthers were excluded from the number of cells with chromosomal aberrations.

3.3. In vivo micronucleus assay

No abnormal clinical signs were noted in mice given 6′-SL sodium salt and there was no effect of the material on body weight. As shown in Table 3, 6′-SL sodium salt tested negative for micronucleus induction in PCE from the bone marrow of mice. The average micronucleus frequency in mice administered 6′-SL sodium salt was not statistically different from that of the concurrent vehicle control at any dose, and no dose-response relationship was observed for the findings. The test was valid because the negative and positive control substances produced responses within historical control ranges, the positive control induced a statistically significant increase in micronucleus frequency compared to the vehicle control, and the number of cells evaluated was appropriate.

Table 3.

Results of in vivo Micronucleus Study of 6′-SL Sodium Salt.

Substance	Dose (mg/kg)	Time to Sample Collection	Animal ID	PCE	NCE	PCE / (PCE+NCE)	MNPCE / 4000 PCE
Water for injection (negative control)	0	24 hr	1101	241	259	0.482	2
			1102	251	249	0.502	0
			1103	226	274	0.452	2
			1104	240	260	0.480	2
			1105	255	245	0.510	2
			Mean	-	-	0.485	1.6
			SD	-	-	0.023	0.89
Historical control values (Range)						0.438–0.553	0–3
6'-SL sodium salt	500	24 hr	1201	243	257	0.486	0
			1202	244	256	0.488	1
			1203	246	254	0.492	3
			1204	234	266	0.468	2
			1205	238	262	0.476	0
			Mean	-	-	0.482	1.2
			SD	-	-	0.010	1.30
	1000	24 hr	1301	237	263	0.474	1
			1302	237	263	0.474	1
			1303	256	244	0.512	0
			1304	232	268	0.464	1
			1305	259	241	0.518	4
			Mean	-	-	0.488	1.4
			SD	-	-	0.025	1.52
	2000	24 hr	1401	232	268	0.464	1
			1402	231	269	0.462	0
			1403	230	270	0.460	1
			1404	223	277	0.446	2
			1405	247	253	0.494	0
			Mean	-	-	0.465	0.8
			SD	-	-	0.018	0.84
Cyclophosphamide monohydrate (positive control)	80	24 hr	1501	252	248	0.504	171
			1502	230	270	0.460	157
			1503	229	271	0.458	159
			1504	237	263	0.474	167
			1505	221	279	0.442	163
			Mean	-	-	0.468	163.4*
			SD	-	-	0.023	5.73
Historical control values (Range)						0.429–0.561	157–231

hr – hours; kg – Kilogram; mg – Milligrams; kg – Kilogram; MNPCE – Micronucleated polychromatic erythrocyte(s); NCE – Normochromatic erythrocyte(s); PCE – Polychromatic erythrocyte(s); SD – Standard deviation

* Significantly different from negative control (water) by Fisher’s exact test (p < 0.05)

3.4. Acute oral toxicity study

None of the rats administered 1500, 3000 or 6000 mg/kg bw of 6′-SL sodium salt died, and no abnormal clinical signs were noted during the 14-day observation period. Further, there was no effect of 6′-SL sodium salt on body weight or gross pathology. Based on the findings, the oral median lethal dose (LD₅₀) for 6′-SL sodium salt is greater than 6000 mg/kg bw, and doses of 1500, 3000 and 6000 mg/kg bw/day were chosen for use in the chronic toxicity study.

3.5. Chronic oral toxicity study

None of the animals in the study died, and no ocular abnormalities were noted in the ophthalmological examinations. Soft stools were noted in up to 6 males and 7 females in the highest dose group during the first 14 days of the study (Supplemental Tables 1 and 2). Sporadic incidences of soft stools (1–3 per sex) were noted from Days 15–107 and were zero after this time. Sporadic incidences of diarrhea were reported in 1–3 high-dose males from Day 39–112, after which they were not reported in males. Sporadic incidences of diarrhea were also reported in 3–4 high-dose females on Days 2 and 3 of the study but were rarely reported from Days 4–105 and not at all after this period in this sex. These findings were considered by the study investigators to be related to the test material but not adverse due to their transient nature and the lack of an effect of the test material on related parameters. A nodule/mass in the left axillary region was observed in one female in the control group on Days 141–182, and a nodule/mass in the left abdominal region was observed in one female in the 1500 mg/kg bw/day group on Days 113–182. As a result of histopathological examination, the masses were confirmed to be adenomas in the mammary gland and skin. These changes were not considered to be test substance-related, because they were observed in the control and low-dose groups. During the recovery period, no abnormal clinical signs were observed in males and females in the control and 6000 mg/kg bw/day groups.

There was no effect of any dose of 6′-SL sodium salt on the body weight of males or females (Fig. 1, Fig. 2), although slight decreases in food consumption were noted in males and females given 6000 mg/kg bw/day of 6′-SL sodium salt at several different time points in the study (Supplemental Table 3). Therefore, the decreases in food consumption are not considered to be toxicologically relevant. There was a dose-dependent increase in urine volume in main study males and females, with the values in females given 3000 or 6000 mg/kg bw/day being statistically significantly increased compared to controls (Supplemental Table 4).The increase in urine volume in females resolved after 4 weeks of non-treatment but did not resolve in males; however, the statistically significant increase in urine volume in high-dose recovery group males versus the corresponding control group may be an artifact of the low urine volume in the control group. The increase in urine volume appears to be related to the test material but is not adverse based on results for other study parameters. Other changes observed in urinalysis, such as increased incidences of cloudy urine or ketones in the urine of treated males, do not appear to be related to the test material because they are not dose dependent.

Fig. 1.

Body Weights of Male Rats in the 26-Week Toxicity Study of 6′-SL Sodium Salt.

Fig. 2.

Body Weights of Female Rats in the 26-Week Toxicity Study of 6′-SL Sodium Salt.

There was no effect of the test material on any hematology parameter in the main study animals (Supplemental Table 5). Although some statistically significant differences were noted in high-dose recovery animals versus controls (decreased hemoglobin, hematocrit, and lymphocytes and increased neutrophils in males, and prothrombin time in females), the differences were slight and do not appear to be due to the administration of test material because values for these variables were not affected in the main study animals. Results of clinical chemistry analyses in animals given 6 ´ -SL sodium salt were also unremarkable (Table 4).

Table 4.

Clinical Chemistry Results in the 26-Week Toxicity Study of 6′-SL Sodium Salt.

Parameter	Main Study Groups (mean ± SD)				Recovery Groups (mean ± SD)
	Control	1500 mg/kg bw/day	3000 mg/kg bw/day	6000 mg/kg bw/day	Control	6000 mg/kg bw/day
Males
AST (U/L)	77.1 ± 15.1	76.9 ± 24.2	69.5 ± 10.1	72.1 ± 8.5	89.7 ± 24.0	76.0 ± 17.3
ALT (U/L)	36.5 ± 10.3	34.1 ± 4.2	33.1 ± 8,1	33.2 ± 8.9	46.7 ± 11.7	38.9 ± 11.0
ALP (U/L)	241.5 ± 54.6	201.1 ± 44.1	214.2 ± 65.5	220.2 ± 61.9	295.9 ± 56.6	240.0 ± 26.6
GGT(U/L)	0.18 ± 0.23	0.36 ± 0.23	0.25 ± 0.19	0.21 ± 0.15	0.31 ± 0.44	0.28 ± 0.45
Bilirubin (mg/dL)	0.07 ± 0.03	0.03 ± 0.02*	0.05 ± 0.02	0.05 ± 0.02	0.06 ± 0.03	0.06 ± 0.04
TC (mg/dL)	142 ± 38	151 ± 47	129 ± 22	113 ± 25	149 ± 26	205 ± 72
TG (mg/dL)	80 ± 56	105 ± 46	89 ± 49	70 ± 44	103 ± 34	138 ± 76
Glucose (mg/dL)	156 ± 7	158 ± 13	159 ± 20	161 ± 14	178 ± 16	173 ± 10
BUN (mg/dL)	16.1 ± 1.6	14.9 ± 1.7	16.1 ± 1.8	17.0 ± 1.6	16.4 ± 1.1	19.4 ± 6.3
Creatinine (mg/dL)	0.51 ± 0.05	0.46 ± 0.05	0.48 ± 0.04	0.49 ± 0.04	0.47 ± 0.02	0.53 ± 0.13
TP (g/dL)	6.5 ± 0.2	6.6 ± 0.2	6.5 ± 0.2	6.4 ± 0.2	6.5 ± 0.2	6.4 ± 0.2
Albumin (g/dL)	2.3 ± 0.1	2.4 ± 0.1	2.4 ± 0.1	2.4 ± 0.1	2.3 ± 0.1	2.2 ± 0.2
IP (mg/dL)	5.36 ± 0.28	5.33 ± 0.47	5.45 ± 0.47	5.53 ± 0.35	5.39 ± 0.10	5.75 ± 0.25*
Calcium (mg/dL)	9.9 ± 0.3	10.2 ± 0.2*	10.2 ± 0.3*	10.2 ± 0.3	10.1 ± 0.4	10.4 ± 0.2
Sodium (mM)	143 ± 1	142 ± 1	142 ± 1*	141 ± 1**	143 ± 1	143 ± 1
Potassium (mM)	4.8 ± 0.2	4.8 ± 0.3	4.8 ± 0.3	4.6 ± 0.4	4.7 ± 0.2	4.6 ± 0.3
Chloride (mM)	103 ± 2	103 ± 1	102 ± 2	100 ± 2**	102 ± 1	101 ± 1
A/G ratio	0.56 ± 0.04	0.56 ± 0.05	0.57 ± 0.05	0.59 ± 0.03	0.56 ± 0.01	0.51 ± 0.04
Females
AST (U/L)	88.8 ± 11.9	95.0 ± 17.0	82.0 ± 24.0	73.4 ± 14.1	103.0 ± 44.6	184.5 ± 132.9
ALT (U/L)	30.9 ± 6.2	44.0 ± 33.2	27.0 ± 7.5	31.1 ± 8.0	44.3 ± 16.0	68.2 ± 35.9
ALP (U/L)	142.4 ± 32.7	154.6 ± 50.8	132.5 ± 49.5	125.1 ± 27.8	132.6 ± 25.8	107.5 ± 17.9
GGT(U/L)	0.56 ± 0.42	0.46 ± 0.21	0.57 ± 0.36	0.48 ± 0.41	0.39 ± 0.22	0.26 ± 0.22
Bilirubin (mg/dL)	0.09 ± 0.03	0.08 ± 0.03	0.08 ± 0.04	0.08 ± 0.02	0.18 ± 0.06	0.14 ± 0.04
TC (mg/dL)	105 ± 16	108 ± 29	109 ± 23	110 ± 31	100 ± 20	133 ± 27
TG (mg/dL)	21 ± 6	29 ± 14	42 ± 28	34 ± 25	24 ± 7	37 ± 14
Glucose (mg/dL)	148 ± 18	143 ± 11	140 ± 16	140 ± 9	161 ± 16	162 ± 9
BUN (mg/dL)	14.8 ± 1.9	14.7 ± 1.4	15.8 ± 2.4	15.3 ± 2.1	16.6 ± 2.5	17.2 ± 1.5
Creatinine (mg/dL)	0.48 ± 0.05	0.48 ± 0.04	0.48 ± 0.03	0.47 ± 0.05	0.47 ± 0.04	0.46 ± 0.04
TP (g/dL)	6.7 ± 0.6	6.4 ± 0.3	6.4 ± 0.3	6.3 ± 0.4	6.3 ± 0.4	6.8 ± 0.4
Albumin (g/dL)	2.9 ± 0.3	2.7 ± 0.2	2.7 ± 0.2	2.6 ± 0.2**	2.6 ± 0.2	2.9 ± 0.3
IP (mg/dL)	4.55 ± 0.49	4.62 ± 0.34	4.88 ± 0.31	5.11 ± 0.52**	4.65 ± 0.51	4.64 ± 0.36
Calcium (mg/dL)	9.5 ± 0.4	9.6 ± 0.3	9.7 ± 0.4	9.7 ± 0.3	9.8 ± 0.2	10.3 ± 0.4
Sodium (mM)	142 ± 1	142 ± 1	143 ± 1	142 ± 2	142 ± 1	141 ± 1
Potassium (mM)	4.6 ± 0.3	4.5 ± 0.2	4.5 ± 0.3	4.3 ± 0.5	4.5 ± 0.3	4.6 ± 0.3
Chloride (mM)	103 ± 1	103 ± 2	103 ± 2	102 ± 3	103 ± 1	101 ± 1
A/G ratio	0.75 ± 0.05	0.72 ± 0.04	0.71 ± 0.05	0.69 ± 0.05	0.72 ± 0.03	0.75 ± 0.08

A/G – Albumin/globulin; ALP – Alkaline phosphatase; ALT – Alanine aminotransferase; AST – Aspartate aminotransferase; BUN – Blood urea nitrogen; bw – Body weight; dL – Deciliter: g – Grams; GGT – Gamma-glutamyl transferase; IP – Inorganic phosphorous; kg – Kilogram; L – Liter; mg – Milligrams; mM – Millimolar; SD – Standard deviation; TC – Total cholesterol; TG – Triglycerides; TP – Total protein; U – Units

^*Significantly different from control by Dunnett’s t-test (p < 0.05) ** (p < 0.01).

There was no effect of the test substance on absolute organ weights (Supplemental Table 6) or organ weights relative to body weight (Supplemental Table 7). Although decreased seminal vesicle weights (both absolute and relative) were noted in mid-dose males and decreased relative pituitary weights were noted in low- and mid-dose males, they were not dose-dependent. Further, while absolute brain weights of treated recovery group males and heart (absolute), liver (both absolute and relative), kidney (both absolute and relative), and ovary (relative) weights of treated recovery group females were statistically different from controls, these findings were not observed in main study males or females and are therefore considered unrelated to the administration to the test substance. Further, none of the changes that were observed were accompanied by changes in organ pathology.

At necropsy, the only notable gross pathological changes were a mass in the axillary or abdominal region of one female each in the control group and the 1500 mg/kg bw/day group, and black foci in the glandular stomach of three males in the 6000 mg/kg bw/day group. Each mass in the females was confirmed to be an adenoma in the histopathological evaluation and was considered spontaneous and incidental. Upon histological examination, the black foci in the glandular stomach in the high-dose males were identified as erosion/ulcer. The investigators did not consider the erosion/ulcer to be toxicologically relevant because the severity was graded as minimal (n = 2, animal numbers 1403 and 1406) to mild (n = 1, animal number 1405), and the finding was not observed in high-dose recovery group males. Further, although these high-dose male animals also experienced bouts of loose stools or diarrhea early on in the study (see Supplemental Table 1), their frequency and duration were similar to those of other high-dose animals that had normal gastrointestinal pathology. The animals that exhibited erosion/ulcer of the glandular stomach also had normal food consumption and weight gain during the study. Because minimal erosion/ulceration of the glandular stomach was also observed in one main study control female, the lesion does not appear to have been caused by administration of the test material.

The results of the toxicokinetic analysis are shown in Table 5. The systemic exposure of the high-dose group on Days 92 and 182 was much higher than the dose ratio of 1:4. With respect to each dose, the mean AUC_0–24 ratios were increased to 1:1.7:8.8 for males and 1:1.7:6.9 for females on Day 1, and to 1:1.8:17.5 for males and 1:4.4:21.0 for females on Day 92, and to 1:1.3:6.7 for males and 1:2.1:9.9 for females on Day 182. The mean C_max ratios were increased to 1:1.4:8.2 for males and 1:1.5:9.0 for females on Day 1, and to 1:3.1:56.0 for males and 1:2.9:33.9 for females on Day 92, and to 1:1.3:16.3 for males and 1:1.5:30.1 for females on Day 182. When comparing the results of Days 92 and 182 with Day 1, the mean AUC_0–24 ratio of Day 92/Day 1 was from 0.2 to 0.6-times and Day 182/Day 1 was from 0.2 to 0.5-times, showing that bioaccumulation did not occur over time. When comparing the results of males and females, the mean AUC_0–24 ratio of males to females was from 0.6 to 1.5-times on Days 1 and 92, demonstrating no meaningful sex difference. However, on Day 182, this ratio was 1.7–2.9-times greater in males than females. The T_max ranged from 0.5 to 1.0 h in both sexes. The t_1/2 generally ranged from 1.20 to 3.87 h in both sexes, except for the Day 1 value of 8.21 h for the 6000 mg/kg bw/day male group, which also had a higher AUC _0–24 value than any other dose group.

4. Discussion

The results of the studies described herein show that a new 6′-SL sodium salt produced by a modification of the procedure described by Gurung et al. [6] is not genotoxic and has a high acute oral LD₅₀ value in the SD rats (> 6000 mg/kg bw). The no observed adverse effect level (NOAEL) in the 26-week study in both male and female SD rats was 6000 mg/kg bw/day, the highest dose administered. The safety of 6′-SL sodium salt in rats and piglets has been previously studied, with NOAELs of 4000 or 5000 mg/kg bw/day reported for 90–91-day studies in rats and 360 mg/kg bw/day for 21-day studies in piglets [12], [15], [6], [7]. The current study indicates that this new 6′-SL sodium salt is safe in rats at a higher level and for a longer period than previously studied in either rats or pigs, demonstrating a more robust safety profile [12], [15], [6], [7].

The maximum tolerated dose for repeated administration of 6′-SL sodium salt to rats was 6000 mg/kg bw/day as demonstrated by the occurrence of diarrhea or loose stools in some of the animals receiving this dose during the early stages of this study. These findings appear to be related to the administration of the test material but are not adverse due to their transient nature and the lack of an effect on body weight and other parameters. The fact that diarrhea or loose stools was observed in high-dose animals is not surprising given that 6′-SL is not readily digestible and can draw water into the intestine based on osmotic effects [3], [8]. Although minimal (n = 2) to mild (n = 1) erosion/ulceration of the glandular stomach was observed in 3 males in the 6000 mg/kg/day group, this does not appear to be the cause of the diarrhea or loose stools because high-dose animals that had normal stomach pathology also had bouts of diarrhea or loose stools which stopped within a similar time frame. Further, erosion/ulceration of the glandular stomach was not observed in males allowed to recover from the high dose for 4 weeks or in females administered this dose, and one control female also exhibited the same type of lesion. It is possible that the lesion was caused by local trauma from the gavage procedure [9].

Other findings that may have been related to administration of 6′-SL sodium salt were decreased food consumption in male and female animals administered 6000 mg/kg bw/day and increased urinary volume in females provided 3000 or 6000 mg/kg bw/day. The decrease in food consumption is not adverse because there was no effect of the substance on body weight. The fact that animals provided 6000 mg/kg bw/day 6′-SL sodium salt ate less food but maintained body weight is consistent with the fact that that 6′-SL sodium salt is fermented by intestinal microbiota to produce short chain fatty acids, which can be used as sources of energy by internal cells after absorption [1], [11]. It is possible that the increased urinary volume was the result of increased water consumption due to the test material containing sodium; however, this cannot be confirmed because water consumption was not measured in the study. Based on a molecular weight of 655.5 g/mol [16], 6′-SL sodium salt contains 3.5 % sodium (assuming sodium has a molecular weight of 23 g/mol). At 6000 mg/kg bw/day, the rats were exposed to 210 mg/kg bw/day sodium from the test material, plus sodium in the diet. However, there was no effect of daily administration of 6′-SL sodium salt at 6000 mg/kg bw/day on the plasma sodium level of the rats, indicating that the rats were able to eliminate the additional amount of sodium provided by the test material.

The results of the toxicokinetic study show that some 6′-SL is absorbed by rats after daily administration of 1500, 3000 or 6000 mg/kg bw/day 6′-SL sodium salt for up to 181 days. Regardless of dose, the mean AUC_0–24 did not increase over time, showing no potential for bioaccumulation with repeated dosing. Further, the results of AUC_0–24 measurements were similar between males and females early in the study but were 1.7–2.9-times greater in males than females on Day 182, regardless of dose. This suggests that over time, males developed a greater potential to absorb or retain 6 ´ -SL than females. However, in general, there were no meaningful differences in t_1/2 between sexes, indicating similar potential for elimination between males and females. The notable exception is for Day 1, when the 6000 mg/kg bw/day male group had a t_1/2 value of 8.21 h. This suggests that elimination mechanisms were saturated after the first dose in this group but adapted by 92 days of dosing. There were no differences in the NOAEL between males and females in this study, indicating that the slight differences in pharmacokinetics between males and females had no bearing on the safety of 6′-SL sodium salt.

In contrast to our toxicokinetic study, results of a study performed by Vazquez et al. [18] show that when 6′-SL is administered to adult female Sprague-Dawley rats at single oral doses of 200, 1000 or 3750 mg/kg bw, plasma levels at 5 h (the last time point measured) are similar to those at 30 min. Although 6′-SL was not analyzed in plasma after 5 h in this study, it is clear that the t_1/2 of 6′-SL is greater than 5 h. In our study, the maximum t_1/2 in female rats was 3.87 h (the value obtained for the 6000 mg/kg bw/day group at Day 92). The reasons for the different results aren’t clear but may be related to different detection methods or test materials. Nonetheless, results of our study mitigate the concern for bioaccumulation of 6′-SL with repeated dosing of 6′-SL sodium salt that could be interpreted from the results of the Vazquez et al. [18] study.

5. Conclusion

To conclude, results of the studies described in this publication show that 6′-SL sodium salt produced by a novel process is not genotoxic and has a very good safety profile in rats. Specifically, this substance is not genotoxic in Ames, in vitro chromosomal aberration, or in vivo micronucleus tests, is well tolerated in an acute toxicity study in rats up to 6000 mg/kg bw, and the NOAEL is determined to be 6000 mg/kg bw/day in male and female rats, the highest dose tested in a 26-week repeated dose oral toxicity study. Results of these studies support the safety of this substance in humans at a higher level than previously determined for 6′-SL sodium salt produced by other methods.

CRediT authorship contribution statement

Rit B. Gurung: Writing – review & editing, Supervision. Laurie C. Dolan: Writing – review & editing, Writing – original draft. Hiroe Go: Writing – review & editing, Supervision, Project administration. Benjamin G. Arceneaux: Writing – review & editing, Writing – original draft. Eun Jung Park: Writing – review & editing, Supervision, Project administration.

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: GeneChem Inc. reports financial support was provided by Ministry of SMEs and Startups, Republic of Korea (Grant No. S3122086). Rit B. Gurung reports a relationship with GeneChem Inc. that includes: employment. Hiroe Go reports a relationship with GeneChem Inc. that includes: employment. Eun Jung Park reports a relationship with GeneChem Inc. that includes: employment. Laurie C. Dolan reports a relationship with GRAS Associates, LLC that includes: employment. Benjamin C. Arceneaux reports a relationship with GRAS Associates, LLC that includes: employment. No other additional relationships or activities to declare! If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

Supplementary material

Data availability

Data will be made available on request.