bw: body weight; CSO: Chondroitin Sulfate Oligosaccharide; DMSO: Dimethyl Sulfoxide; ELISA: Enzyme-Linked Immunosorbent Assay; HD: High Dose; kDa: kiloDaltons; kg: kilogram; LD: Low Dose; MD: Middle Dose; mg: milligram; mL: milliliter; NOAEL: No Observed Adverse Effect Level; OECD: Organisation of Economic Co-operation and Development; PCE: Polychromatic Erythrocytes; TSH: Thyroid-Stimulating Hormone; T3: Thyroxine; T4: Triiodothyronine; µg: microgram; VC: Vehicle Control

Chondroitin sulfate belongs to the class of heteropolysaccharides known as glycosaminoglycans which are components of the extracellular matrix of connective tissues [1]. Glycosaminoglycans have long, linear repeating disaccharide units of amino sugar and uronic acid or galactose [2]. Chondroitin sulfates are unbranched chains of varying lengths of repeating units of N-acetyl-D-galactosamine and D-glucuronic acid.

Chondroitin sulfate can be extracted from bovine, porcine, marine, chicken, and other sources and varies in the degree and position of sulfation and molecular weight based on the source, age of the source, and tissue of origin [3]. Shark cartilage chondroitin sulfate is a polymer of repeating heterodimers of β-glucuronic and N-acetyl-β-d-galactosamine sulfated in the 4-position (>60%) and in the 6-position (>30%). Chondroitin sulfates from terrestrial animal cartilage are either non-sulfated or 4-mono-sulfated (>60%). The molecular weights of naturally occurring chondroitin sulfates range from 10 to 100 kiloDaltons (kDa) depending on the source [4]. Shark and skate chondroitin sulfates have molecular weights in the range of 50 – 70 kDa. Chondroitin sulfates from terrestrial animals have molecular weights ranging from 14 – 26 kDa [3]. Chondroitin sulfates in dietary supplements have a lower molecular weight (approximately 16.9 kDa) [5,6]. Chondroitin is widely used for its anti-inflammatory, anticoagulant, antioxidant, and antitumor activity. No information exists regarding how much is ingested as a part of the normal diet or how much is consumed as a dietary supplement [7].

It has been reported that lower molecular weight CS undergoes faster and more complete absorption than higher molecular weight CS [8-10]. The bioactivity varies based on source, size, degree of polymerization, and sulfation pattern. However, variability is not often addressed in studies on chondroitin which treat all chondroitins as a uniform class.

Chondroitin sulfate oligosaccharide (CSO), trade name "LetopCS", is prepared by the enzymatic hydrolysis of shark cartilage chondroitin sulfate by chondroitin sulfate oligosaccharide lyase, a bacterial lyase. CSO appears to be most closely related to depolymerized shark cartilage CS, which has a molecular weight range of 5 – 10 kDa and a ratio of 4-sulfate to 6-sulfate of 1:3 [11], and Mythocondro®. Mythocondro® is produced via thermo-acid hydrolysis of the capsular polysaccharide naturally produced by a specific strain of E. coli followed by chemical sulfation [12]. Mythocondro® is monosulfated primarily on the 6-carbon position and to a lesser extent on the 4-carbon position. Mythocondro has been described as shark-like based on its sulfation pattern and charge density.

Although the safety of chondroitin has been evaluated in clinical and toxicological studies and is used extensively as a dietary supplement, no such evidence has been published for CSO. The current study reflects the first toxicology studies conducted on CSO. This battery of toxicological studies was conducted in accordance with international guidelines: mutagenicity (bacterial reverse mutation and in vitro mammalian cell gene mutation tests, genotoxicity (in vivo mammalian erythrocyte micronucleus test and sub-chronic oral toxicity in Sprague Dawley rats.

Test article

The test article was chondroitin sulfate oligosaccharide also known as "LetopCS" as determined by high-performance liquid chromatography (Nanjing Letop Biotechnology Co., Ltd.) to be 101.9% by weight. CSO is sulfated at the C-4 and the C-6 position; the most common forms are monosulfated at C-4 (>30%) and C-6 (>40%) or disulfated at C-2,6 (> 15% with a minor amount unsulfated (<10%). CSO is a mixture of disaccharides (>60%) and tetrasaccharides (>29%), with an average molecular weight of 1 kDa. The test article was stored at 21 to 29 degrees Celsius in a tightly sealed container away from moisture and direct sunlight.

In vitro studies on CSO

Bacterial reverse mutation test:

The bacterial reverse mutation test was conducted to investigate the genotoxicity of CSO. The study adhered to the Organisation of Economic Co-operation and Development (OECD 471) guideline [13] and OECD Good Laboratory Practice (GLP) compliant procedures ENV/MC/CHEM (98)17 (as revised in 1997) and adopted by decision of the OECD Council [C(97)186/Final] [14]. Salmonella typhimurium strains TA1537, TA1535, TA98, TA100, and E. coli strain WP2 uvrA pKM101 were used in the study.

A cytotoxicity assessment was conducted using the plate incorporation method in strains TA100 and E. coli WP2 uvrA pKM101 in the presence and absence of metabolic activation with S9 fraction (liver microsomal homogenate) at doses of 16, 50, 160, 500, 1600, and 5000 µg/plate.

Following the cytotoxicity study, CSO was tested at 0, 50, 160, 500, 1600, and 5000 µg/plate, both in the absence and presence of S9 metabolic activation and using both the plate incorporation and pre-incubation methods. For both the plate incorporation and the preincubation methods, the positive control in the presence of metabolic activation was 2- aminoanthracene. The positive controls in the absence of metabolic activation were sodium azide (TA 1535 and TA 100), 4-nitro-o-phenylenediamine (TA 1537 and TA 98), and methyl methane sulfonate (E. coli). Reverse osmosis water was the vehicle control and there was no negative control. Testing was conducted in triplicate.

For the plate incorporation method, CSO or control, a bacterial culture, and top agar were mixed. S9 mix was added for the study conducted in the presence of metabolic activation and sodium phosphate buffer was added for the study conducted in the absence of metabolic activation and the mixture was poured onto agar plates. After the contents of the plate solidified, the plates were inverted and incubated at 37 ± 2°C for 48 hours. For the preincubation method, the CSO or bacterial culture were mixed with the appropriate controls and S9 or sodium phosphate buffer were combined and mixed for 20 minutes. The top agar (maintained at 45 ± 2°C) was mixed and then added to selective agar plates. After the plate contents were solidified, they were inverted for 65 hours and 40 minutes at 37 ± 2°C.

On completion of the incubation period, the number of colonies was counted manually and the mean numbers of revertant counts were determined for each plate. A result was considered positive if there was at least a two-fold increase in the number of revertant colonies in a plate compared with the negative control for strains TA 98, TA 100, and E. coli WP2 uvrA pKM101 and a more than three-fold increase for strains TA 1535 and TA 1537 compared with vehicle control. Dose-dependent increases were categorized as biologically relevant if the threshold was exceeded at greater than one concentration, for example, if there was a concentration-related increase over the range that was tested as well as a reproducible increase at one or more concentrations in the number of revertant colonies per plate for at least one strain in the presence or absence of metabolic activation. The positive controls showed the requisite fold increase in the number of revertants compared with the vehicle control which shows the sensitivity of the assay and validates the assay procedures. Controls elicited the number of mean revertants that was in the historical range for control data for the assay. A microscope was used to assess precipitation or clearing or thinning of the background lawn.

Mammalian cell gene mutation assay on L5178Y mouse lymphoma cells TK^+/-: The thymidine kinase assay using the mouse lymphoma cell line L5178Y was conducted to study the ability of CSO to induce gene mutations in L5178 TK^+/- cells. The study adhered to OECD Test No. 490: In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene [15]. L5178Y TK^+/- cells were cleaned to decrease the likelihood that mutations in TK^+/- would occur spontaneously by incubating them for one day with a culture medium containing 3.0 µg/mL thymidine, 5.0 µg/mL hypoxanthine, 0.1 µg/mL methotrexate and 7.5 µg/mL glycine. The solubility of CSO was tested to identify the appropriate vehicle for the test item and the precipitation and pH of the highest concentration were determined to select the highest dose to be used in the cytotoxicity study. CSO was tested for cytotoxicity at doses of 0.0625, 0.125, 0.25, 0.5, 1, and 2 mg/mL based on guidance provided in OECD [16].

A two-phase dose range-finding study was conducted. The short-term exposure phase was three hours long and was conducted in the presence and absence of metabolic activation with S9. The long-term exposure phase was conducted only in the absence of metabolic activation and lasted for 24 hours. CSO was dissolved in DMSO. Concentrations of 0.0625, 0.125, 0.25, 0.5, 1, and 2 mg/mL of CSO were tested in duplicate using 6 x 10⁶ cells/5 mL culture. The negative control was reverse osmosis water. The positive controls in the presence and absence of metabolic activation with S9 were 5 µg/mL cyclophosphamide monohydrate and 10 µg/mL methyl methanesulfonate, respectively. The mutant frequency was determined. An inverted microscope was used to count the number of wells containing colonies for the plates for assessing mutant frequency and viability. For the assessment of mutant frequency, colonies were classified as either small (≤¼ of well diameter) or large (colony is > ¼ of well diameter) for each dose group. Small colonies were considered to be produced primarily by rearrangement and large colonies were considered to be produced primarily by point mutations. The percent relative suspension growth was determined by multiplying the day 1-fold increase and the day 2-fold increase in cell numbers. Morphology was described as small, compact, large, total, or partially diffuse. The relative total growth was determined by diving the respective RSG by the respective percent cloning efficiency. A positive result was a concentration-related or reproducible increase in the mutant frequency; however, the biological relevance of the results was the primary consideration, with statistical methods used also to assess the study results.

In vivo mammalian erythrocyte micronucleus assay

The ability of CSO to induce the formation of micronuclei in the bone marrow erythrocytes of Swiss Webster mice [17] was investigated in a study that adhered to OECD Test No. 474: Mammalian Erythrocyte Micronucleus Test (OECD, 2016a). In a dose range-finding study, Swiss Webster mice that were seven to eight weeks old at the beginning of dosing were administered two doses of 0 (vehicle control, water), 500, 1000, or 2000 mg/kg of CSO (n=3 animals/sex/dose group) 24 hours apart via gavage. The positive control was 40 mg/kg bw cyclophosphamide monohydrate. Cage side observations for clinical signs were conducted before dosing and at 1, 2, 3, 4, and 24 hours post-dosing. Detailed observations for clinical signs include handling response, skin, fur, mucous membranes, breathing patterns, ears, eyes, excretions, oral cavity, ventral-posterior side of the body, posture, gait, behaviors, and any injury wound or swelling on the final day of acclimatization. The rats were observed for mortality and morbidity two times each day except holidays when they were observed at least once daily. Body weights of the vehicle control and CSO groups were measured when the animals were received at the facility, at the time they were randomized, on study days 1, 2, and 3, and before they were killed. For the positive control group, body weights were measured before administration of the positive control and at the time of necropsy. No mortality or clinical signs were noted and no differences between the ratio of polychromatic erythrocytes to total erythrocytes for the control and CSO groups were noted. The maximum tolerated dose was 2,000 mg/kg bw.

Based on the results of the dose range-finding study, the main study was conducted as a limit test with a maximum dose of 2,000 mg/kg bw of CSO. The animals were administered, via gavage, two doses of 2,000 mg/kg of CSO or vehicle control with a 24-hour interval between doses (n= 5 animals/sex/dose group). The positive control was administered on day 2, 22 hours before the animals were killed. Bone marrow was collected 22 hours after the final dose was administered.

The animals were euthanized via carbon dioxide asphyxiation. Bone marrow cells were acquired 22 hours after the final treatment. The bone marrow was flushed out using fetal bovine serum and the cell suspension was centrifuged at 1,500 rpm for 10 minutes. The supernatant was removed, and the pellet was spread on a slide, air-dried, and stained with May-Grunwald and Giemsa stains. Two slides were analyzed for each rat. A cytotoxic effect was defined as one in which the ratio of immature or polychromatic erythrocytes compared with total (polychromatic and normal erythrocytes were counted by counting a minimum of 500 erythrocytes for each animal in the dose range finding experiment and the main experiment.

For the micronucleus counts, a 100x oil immersion objective was used and a minimum of 4,000 PCEs were analyzed for each animal. The dose was considered clastogenic if a minimum of one CSO dosing group showed a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared with the vehicle control group. The biological relevance of the results was the main consideration. Slides for the main study were coded but those for the dose range-finding study were not.

Animal study

Ninety-day repeated dose oral toxicity study: The toxicological potential of CSO when administered by gavage was studied in a 90-day oral toxicity study with a 28-day recovery period [18]. Following an acclimatization period of 5 to 6 days, rats were assessed using cage-side observations and were physically examined before being selected for the study. In the main study, 7- to 8-week-old female nulliparous and male Sprague Dawley rats without any visible signs of illness were randomly assigned based on body weight to one of four dose groups with 10 rats/sex/dose group: 0 (control group), 1,000 (low dose or LD group), 2,000 (middle dose or MD group), or 4,000 (high dose or HD group ) mg/kg bw/day of CSO for 90 consecutive days and they were killed on day 91. Two other groups of rats including 5 rats/sex/dose group were treated with 0 (control recovery group (CR)) or 4,000 mg/kg bw/day of CSO (high dose recovery (HDR) group) for 90 consecutive days. Rats in the CR and HDR groups were kept alive for an additional 28 days during which there was no administration of CSO or control. The dose volume was 10 mL/kg bw based on the most recent body weight. Animals were killed by carbon dioxide asphyxiation.

Mortality and morbidity observations were conducted two times per day except during holidays when these assessments were conducted once per day. Clinical signs were observed from the cage side once daily. Detailed clinical examinations were performed outside of the cage once per week starting on the day the animals were randomized and on seven through day 70 and then on days 76, 84, and 90 as well as on days 96, 103, 110, and 117 for the recovery groups. The observations included changes in the skin, fur, eyes, or mucous membranes, occurrence of secretions and excretions and autonomic activity, such as lacrimation, piloerection, pupil size, unusual respiratory pattern; changes in gait, posture, response to handling, clonic or tonic movements, stereotypic behaviors such as excessive grooming and repetitive circling, and bizarre behavior. Body weights were measured before the animals were randomly assigned to groups and before dosing on day one, weekly through day 85, and then on day 90 for the main study. Additional measurements of body weight were made for the recovery group on days 97, 104, 111, and 118. Terminal fasting body weights were also measured on days 91 and 119 for the main group and the recovery group, respectively. Feed consumption was measured for one-week intervals through day 85 and then for days 85 to 90 for the main group with additional food consumption measurements for one-week intervals starting with day 90 through day 118 for the recovery groups.

Functional observational batteries, including observations in the home cage, during handling, and in the open field and measurement of sensory reactivity, foot splay, and grip strength were performed on day 86 for the main study groups and on day 114 for the recovery groups. Ophthalmological examinations were conducted following randomization and during the last week of administration for the main group for the main and recovery study control and high dose groups following induction of mydriasis by adding one or two drops of 1% tropicamide to the eye 5 minutes before the examination was conducted.

Animals were fasted overnight with free access to water, and blood samples were collected for hematology and clinical chemistry analyses via the retro-orbital sinus under isofluorane anesthesia immediately before the animals were killed. Bone marrow smears from the femur were fixed in methanol and stained using Giemsa stain. Blood smears were performed. Hematology analyses included red blood cells, mean corpuscular volume, red cell distribution width, hematocrit, platelet count, mean platelet volume, white blood cells, hemoglobin concentration, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, prothrombin time, activated partial thromboplastin time, neutrophils, lymphocytes, monocytes, eosinophils, basophils, relative reticulocytes count, and absolute reticulocyte count. Clinical chemistry analyses including albumin, albumin globulin ratio, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase blood urea nitrogen, calcium, creatinine, glucose, phosphorus, total bilirubin, total protein, globulin, triglyceride, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, sodium, potassium, chloride, and urea. Animals in the main group were placed in metabolic cages on days 90 and 118 for the main study and recovery groups for urine collection. The volume, color, and clarity of the urine were determined. Urinalysis was conducted to assess the pH, specific gravity, blood, bilirubin, urobilinogen, ketones, protein, nitrite, glucose, and leucocyte concentrations. Vaginal smears were performed for all females on the day of necropsy to determine the stage of the estrus cycle of all female rats.

Terminal body weights were measured immediately before killing the animals via carbon dioxide asphyxiation. Gross pathological analysis was performed, which included an examination of the external surface of the body, orifices, and cranial, thoracic, and abdominal cavities, and a detailed internal examination of the viscera was conducted. Organs and tissues including adrenal, aorta, bone (sternum) with bone marrow, bone marrow smear (femur), brain (cerebrum, cerebellum, midbrain), caecum, colon, duodenum, epididymides, esophagus, eyes, heart, ileum with Peyer’s patches, jejunum, kidneys, liver, lungs, mesenteric and auxiliary lymph nodes, peripheral nerve (sciatic nerve), ovaries and oviducts, pancreas, prostate and seminal vesicles with coagulation glands, rectum, skeletal muscle, skin with mammary glands, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid and parathyroid, trachea, urinary bladder, uterus with cervix, vagina, pituitary, salivary glands. All gross lesions were examined. Histopathological analyses of all the above tissues were conducted for all control and high dose animals in the main and recovery groups. The uteruses of three rats also underwent microbiological examinations. The weights of organs including the liver, kidneys, adrenal, testes, epididymides, uterus, ovaries, thymus, spleen, lung, brain, pituitary, prostate, and seminal vesicles with coagulation glands, and heart were determined. Organs that were paired were weighed at the same time. An ELISA method was used to estimate triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH).

Statistical analyses

Systat statistical software version 13 was used to determine the mean and the standard deviation of the number of revertants in the bacterial reverse mutagenicity assay. In the thymidine kinase assay, the ANNOVA, and Dunnett’s test Two-Side method of Systat version No. 13 software were used to analyze data. Comparisons were made between the number of mutant colonies for cells treated with CSO compared with those treated with vehicle control. A trend was considered to be significant if the probability value was less than 0.05. Both biological relevance and statistical significance were considered. For the micronucleus assay, the statistical analysis was conducted with Systat statistical software version 13 and statistical significance was assessed using the non-parametric Mann-Whitney test.

In the 90-day animal study continuous data were analyzed with SYSTAT version 13, Basic statistics, homogeneity of variance by Bartlett’s Test, ANOVA, Dunnett’s two-side tests for equal variance and Dunnett T3 for unequal variance were performed for the CSO dosing groups in the main study in comparison to the control group. t-tests were conducted to compare the control recovery and high dose recovery groups and the level of significance was 5% (p<0.05).

In vitro studies

Reverse mutagenesis (Ames) test: There was no increase in the mean number of revertants for bacteria treated with CSO in the presence or absence of metabolic activation compared to the vehicle control for TA 98, TA 100, TA 1535, TA 1537, or E. coli WP2 uvrA pKM101 for the plate incorporation or preincubation methods. The results of studies conducted with negative and positive controls elicited the expected results and showed that the assay was valid. CSO was concluded to be non-mutagenic under the tested conditions.

Mammalian cell gene mutation assay on L5178Y mouse lymphoma TK^+/- cells: CSO was not associated with any statistically or biologically significant increases in the numbers of mutants using the short-term exposure method in the presence or absence of metabolic activation or the long-term exposure method when compared with the negative control. For the cells treated with CSO, there was no increase in mutant frequency that was greater than that of the concurrent vehicle control. Increases in mutant frequencies were observed for the positive controls, cyclophosphamide, in the presence of S9 and methane methanesulfonate in the absence of S9 both showed increases which indicated that the test system was efficient and that the experimental conditions were valid. CSO did not induce gene mutations under the conditions of this assay.

In vivo mammalian erythrocyte micronucleus assay

In the dose-range finding and the main assay, all animals were healthy and there were no deaths or clinical signs observed before or following dosing. There was no difference in body weight across treatment groups. Neither the dose range-finding study nor the main study revealed any differences in the percent reduction in the ratios of polychromatic erythrocytes to total erythrocytes, indicating a lack of substantial cytotoxicity to bone marrow cells. The ratios of polychromatic erythrocytes to total erythrocytes in the test article groups were not significantly different from the vehicle control group. The was no significant difference in the frequency of micronucleated erythrocytes for the negative control group compared with the CSO groups. The mean percent of PCE to TE was 3.67% in males and 3.46% in females compared with the vehicle control group. There was no biologically significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) compared with the vehicle control group. The observed mean percent of MNPCE was 0.03% in males and 0.02% in females. The positive control resulted in a significant increase in the frequency of micronuclei, confirming the validity of the assay. CSO does not induce micronuclei in mouse bone marrow cells and is considered non-mutagenic.

Animal study

Ninety-day repeated dose oral toxicity study: No animals died during the study and no clinical signs were observed. The functional observation battery revealed some significant differences between groups in rearing, foot splay, and grip strength but these differences were not considered to be treatment-related because they were not dose-dependent or were due to higher group mean values for these parameters in the control groups. For the main study control HD groups there was a significant difference that was not considered to be substantial enough to be considered treatment-related.

There were transient increases in body weight and body weight gain for animals in the main study when compared with the control group but not the control and high dose recovery groups (Figure 1). There was no dose-response relationship and no correlation with feed consumption. There was a transient decrease in feed consumption for males in the middle dose group. These results were considered incidental and not related to the treatment. No ophthalmologic abnormalities were observed in the control and HD main study groups.

No hematological differences were observed for male rats in the main study treatment groups, or the recovery group compared to their respective control groups (Table 1).

Female LD rats had a reduction in the absolute reticulocyte counts compared with control females. There was also a reduction in the platelet count for HDR females compared with CR females which was due to a higher group mean platelet count for one CR female and was not considered treatment-related (Table 2). An increase in the prothrombin time for HDR females compared with CR females was not considered substantial enough to be treatment related. Blood and bone marrow smears revealed no morphological abnormalities, and the bone marrow smear revealed no cellular abnormalities.

No differences in clinical chemistry parameters for the main study females treated with CSO compared with control group females (Table 3). There was a reduction in alkaline phosphatase and an increase in chloride in HDR group females compared with CR group females.

In males, there was an increase in albumin and total protein in MD group males compared with control group males and a decrease in calcium and an increase in sodium in HD group males compared with control group males (Table 4). Males in the HDR group showed a reduction in glucose and an increase in phosphorus compared with CR group males. These changes were considered biological variations and were not toxicologically significant. Urinalysis revealed no differences in the parameters measured. There were no gross pathological abnormalities related to treatment. A distended uterus with watery contents was observed in one female rat in the control, LD, and MD groups on day 91 but these were considered physiological and incidental findings.

There were no significant differences in organ weights observed for HDR males compared with CR males (Table 5). Some differences in organ weights were observed such as a reduction in absolute epididymis weight for the MD group males and prostate with seminal vesicle and coagulation glands for MD and HD male groups compared with the control group. There were no significant differences in relative organ weight for the LD, MD, or HD groups compared with the control group or for the HDR group compared with the CR group (Table 6). Increases in the absolute weights of ovaries were noted for LD females and in brain weight for HD females compared with the control group females (Table 7). In addition, reductions in absolute adrenal thymus and heart were reported for HDR females compared with CR females. No significant differences in relative organ weights between the HDR and CR female groups were reported; however, there was a significant difference in the relative ovary weight for the HD female group compared with the control group (Table 8).

In general, there were no correlated histopathological findings. Histopathological differences were considered incidental or spontaneous background changes typical for the species and strain and were not thought to be test-article related.

An increase in TSH in HD males on day 91 compared with control was considered incidental and unrelated to treatment because there were no changes in T3 and T4 and no correlative histopathology.

A series of in vitro and animal toxicity studies on CSO did not produce evidence of toxicity at the doses tested. CSO did not induce gene mutations and is not mutagenic in bacterial reverse mutation, in vitro mammalian cell gene mutation, and in vivo mammalian erythrocyte micronucleus assays.

No mortality was observed in a 90-day repeated-dose oral toxicity study with a 28-day recovery period in Sprague-Dawley rats (10/sex/dose group) receiving 0, 1,000, 2,000, or 4,000 mg/kg/day (control, low dose, middle dose, and high dose groups, respectively) or in the additional the recovery groups receiving 0 or 4,000 mg/kg/day during the test period with a 28-day no treatment follow-up. Transient increases in body weight and body weight gain were not dose-dependent or correlated with feed consumption. Hematological and clinical chemistry differences were not dose-dependent, were not observed in both sexes, lacked correlating changes in other parameters, were of small magnitude, not consistently observed, and were, therefore, not considered to be of toxicological significance. Variations in organ weight did not correlate with histopathological findings and were considered incidental and not test-article related. The NOAEL was 4,000 mg/kg bw/day, the highest dose tested.

The toxicity profile of CSO was similar to that observed in in vitro and in vivo studies on Mythocondro®, an ichthyic-like chondroitin chemically sulfated derivative of a non-sulfated chondroitin backbone produced by E. coli [10]. Mythocondro® is compositionally similar to CSO. No evidence of Mythocondro® genotoxicity was observed in bacterial reverse mutation, in vitro mammalian cell gene mutation, and in vivo mammalian erythrocyte micronucleus assays.

No mortality or morbidity was observed in a subchronic, repeated-dose oral toxicity study of Mythocondro® administered to Sprague Dawley rats at 0, 250, 500, or 1,000 mg/kg bw/day with 28-day recovery groups of 0 and 1,000 mg/kg/day. No test article-related changes were observed in body weight, body weight gain, or feed consumption. Minor differences from control were observed in various hematological and clinical chemistry parameters but these were not observed in both sexes, lacked correlating changes in other parameters, and of small magnitude, and were considered incidental and not of toxicological relevance. Slight decreases in relative and absolute organ weights in the liver, brain, and spleen were without histopathological relevance and considered incidental. The authors estimated a NOAEL of 1,000 mg/kg bw for Mythocondro®, the highest dose tested.

Although there are several pharmacokinetic studies in humans and rats on depolymerized shark cartilage chondroitin sulfate, none of these studies reported on safety-related parameters [11,19-21]. Various systematic reviews and meta-analyses of clinical trials have concluded that chondroitin sulfates derived from all sources are well tolerated with rare occurrences of adverse events [22-29]. The dosage in these trials has commonly ranged from 400 – 1200 mg/day and the duration of treatment has been as long as three years. There is nothing in the results of this study to indicate that CSO will pose any greater risk than chondroitin sulfates derived from any other sources.

The preparation of the manuscript was funded by Nanjing Letop Biotechnology Co., Ltd. The sponsor was involved in the planning of the studies but had no role in the data collection, analysis, interpretation of results, or writing of the report.

The studies were conducted by Vedic Lifesciences, Pvt. Ltd, Mumbai, India. All studies were sponsored by Nanjing Letop Biotechnology Co., Ltd., Nanjing, China.

None.