Mouse Micronucleus Research Articles

Cytotoxicity and genotoxicity assessment of silver nanoparticles in mouse

Silver nanoparticles (AgNPs) are among the most commercially used nanomaterials and their toxicity and genotoxicity are controversial. Although many in vitro studies have been conducted to evaluate the genotoxicity of AgNPs, in vivo genotoxicity studies on the nanomaterials are limited. Given the unique physicochemical properties and complex pharmacokinetics behavior of nanoparticles (NPs), in vivo genotoxicity assessment of AgNPs is badly needed. In this study, the clastogenicity and mutagenicity of AgNPs with different sizes and coatings were evaluated using mouse micronucleus (MN) assay, Pig-a assay and Comet assay. Five 7-week-old male B6C3F1 mice per group were treated with 5 nm polyvinylpyrrolidone (PVP)-coated AgNPs at a single dose of 0.5, 1.0, 2.5, 5.0, 10.0 or 20.0 mg/kg body weight (bw) via intravenous injection for both the MN and Pig-a assays; or with 15–100 nm PVP- or 10–80 nm silicon-coated AgNPs at a single or 3-day repeated dose of 25.0 mg/kg bw for the MN assay and Comet assay in mouse liver. Inductively coupled plasma mass spectrometry (ICP-MS) and transmission electron microscopy (TEM) analyses indicated that AgNPs reached the testing tissues (bone marrow for the MN and Pig-a assays and liver for the Comet assay). Although there was a reduction of reticulocytes in the PVP-coated AgNPs-treated animals, indicating cytotoxicity of the AgNPs, none of the treatments resulted in a significant increase of either mutant frequencies in the Pig-a gene or the percent of micronucleated reticulocyte over the concurrent controls. However, both the PVP- and silicon-coated AgNPs induced oxidative DNA damage in mouse liver. These results demonstrate that the AgNPs can reach mouse bone marrow and liver, and generate cytotoxicity to the reticulocytes and oxidative DNA damage to the liver.

Antigenotoxicity of Roupala montana extract in the mouse micronucleus and comet assays

Roupala montana Aubl. (Proteaceae) is a typical savannah species and native to tropical South America that has a moderate mortality for adult forms of Schistossoma mansoni. Because this species has been little studied, the aim of this investigation was to evaluate the influence of R. montana extract on DNA damage induced by methyl methanesulfonate (MMS) in peripheral blood cells and liver of Swiss mice using the micronucleus and comet assay, respectively. R. montana dichloromethane extract was prepared from a stock solution (0.5 mg/mL) in 5% dimethyl sulfoxide in water. Animals received a single dose of different concentrations of R. montana (5, 10 and 20 mg/kg body weight) by gavage (0.5 mL/animal). For antigenotoxicity assessment, different concentrations of R. montana were administered simultaneously with MMS diluted in water (40 mg/kg, intraperitoneally; 0.3 mL/animal). Peripheral blood and hepatocyte samples were obtained 48 and 24 h after treatment, respectively. Results showed that R. montana administered alone indicated the absence of genotoxicity in the mouse micronucleus or comet assay. On the other hand, administration of different doses of R. montana concomitantly with MMS led to a significant reduction in frequency of micronucleated polychromatic erythrocytes and DNA damage, when compared to the group treated only with MMS. Further, for the micronucleus assay, the gradual increase of R. montana concentration led to a proportional increase in the reduction of genotoxicity induced by MMS, indicating a dose-response relationship.

Safety evaluation of a thermolysin enzyme produced from Geobacillus stearothermophilus

Thermolysin is a zinc metalloprotease that has potential uses in the food industry. The safety of thermolysin has not been demonstrated before, and therefore a series of standard toxicological tests to assess its potential toxicity was undertaken. The thermolysin used in this study was derived from the thermophilic bacterium Geobacillus stearothermophilus, which had undergone chemical mutagenesis to generate strains with increased thermolysin production. Acute toxicity studies in rats and mice showed that thermolysin powder is not acutely toxic with an oral LD₅₀ of more than 18,000 mg/kg (2520 mg/kg thermolysin protein) in rats and more than 24,000 mg/kg (3360 mg/kg protein) in mice. Subchronic feeding studies in rats for 91 days at doses up to 1000 mg/kg (390 mg/kg protein) revealed no significant differences between treated and non-treated groups and a No Observed Effect Level (NOEL) of 1000 mg/kg (390 mg/kg protein) per day was established. Results from genotoxicity tests such as in vitro chromosomal aberration assay and in vivo mouse micronucleus were negative. Allergenicity sequence analysis revealed no evidence suggesting that thermolysin is an allergen. The data presented in this study support the conclusion that thermolysin is safe for use in food production.

INVESTIGATION ON THE ANTI-GENOTOXIC EFFECT OF OCIMUM SANCTUM IN FLUORIDE INDUCED GENOTOXICITY

The present study was designed to investigate the anti - genotoxic effect of Ocimum sanctum on fluoride induced genotoxicity and its impact on oxidative stress. Exposure to fluoride can mainly occur through drinking water when the levels far exceed th e permissible limit. Fluorosis is a serious problem the world over resulting in damage to DNA. Micronuclei assessment from bone marrow and peripheral blood was used in the present study to assess the damage to DNA. Sodium fluoride in a single dose (30 mg/kg, i.p.) was used to induce micronuclei in albino mice. Treatment with the aqueous extract of Ocimum sanctum was initiated in the single dose study (100, 400 & 800 mg/kg) and as a time cours e for 1 day, 3 days and 7 days (100 mg/kg) . 24 h after injecting sodium fluori de, the animals were sacrificed and m icronuclei were determined from smears prepared from bone marrow and peripheral blood. The antioxidant impact of the e xtract was determined using ferric ion reducing capaci ty of plasma and thiobarbituric acid reactive substances as a measure of lipid peroxidation. All doses were capable of p reventing the formation of micronuclei but 100 mg/kg of the aqueous extract was most efficacious as a single dose and in the time course study. The beneficial effect of Ocimum sanctum is possibly due to the synergistic action of constituents like polyphenols, triterpenoids, and flavonoids.

Comparative assessment of redox-sensitive biomarkers due to acacia honey and sodium arsenite administration in vivo

Apart from their nutritional properties, foods are basically health promoting or disease preventing. Typical example of such type of foods is honey. Sodium arsenite has been implicated in covalent interactions with the thiol groups in proteins, thereby affecting their structure–function relationship. This study was undertaken to investigate on the comparative assessment of redox-sensitive biomarkers from brain, blood serum and liver tissues as well as genotoxic effects in bone marrow cells of male Wister albino rats due to acacia honey and sodium arsenite administration in vivo. Male Wister albino in four groups of five rats, each were administered with distilled water, acacia honey (20 %), sodium arsenite (5 mg/kg body weight), acacia honey and sodium arsenite daily for 1 week. They were killed using 60 mg/kg sodium pentothal. Blood serum, brain and liver tissues were used for the assessment of GSH level, catalase, SOD activities, protein content and lipid peroxidation. The degree of clastogenicity was assessed using the mouse micronucleus assay in bone marrow cells. Sodium arsenite significantly (p < 0.05) suppresses the GSH level, SOD and catalase activities with simultaneous induction of lipid peroxidation (MDA) and clastogenic effects. Acacia honey was able to increased (p < 0.05) GSH level SOD and catalase activities with concomitant anti-clastogenic effects due to reduction in micronuclei. Acacia honey mitigates sodium arsenite induced-oxidative stress and clastogenicity in male Wister albino rats with its antioxidant properties more pronounced in liver tissues.

Assessment of the genotoxicity and antigenotoxicity of (+)-usnic acid in V79 cells and Swiss mice by the micronucleus and comet assays

Usnic acid is one of the most common and abundant metabolites found in various lichen genera, which are important sources of biologically active compounds. The aim of this study was to evaluate the genotoxic and antigenotoxic potential of (+)-usnic acid (UA) by the micronucleus and comet assays in V79 cell cultures and Swiss mice. For assessment of genotoxicity, V79 cells were treated with 15, 30, 60, and 120μg/mL UA, established based on clonogenic efficiency cytotoxic assay. Swiss mice were treated with UA doses of 25, 50, 100, and 200mg/kg body weight. The same concentrations of UA were combined with methyl methanesulfonate (MMS) for evaluation of antigenotoxicity. The in vitro results demonstrated that UA induced DNA damage at concentrations of 60 and 120μg/mL in the comet assay. However, no genotoxic effect was observed in the micronucleus test using V79 cells at the concentrations tested. No genotoxic effects were observed for the different UA treatments in in vivo test system. Combined administration of UA and MMS significantly reduced the frequencies of micronuclei and DNA damage in vitro and in vivo when compared to treatment with MMS alone. Although the mechanisms underlying the protective effect of UA are not completely understood, the antioxidant activity of this metabolite may explain its protective effect against MMS-induced genotoxicity.

In Vivo Protective Activity of Styrax camporum Hydroalcoholic Extract against Genotoxicity Induced by Doxorubicin and Methyl Methanesulfonate in the Micronucleus and Comet Assays

Styrax camporum Pohl is a tall shrub or a tree with small white flowers, which grows in the states of São Paulo and Minas Gerais and is popularly used for the treatment of gastroduodenal diseases. Considering this last fact, the aim of this study was to evaluate the genotoxic potential of S. camporum hydroalcoholic extract and its influence on genotoxicity induced by doxorubicin and methyl methanesulfonate in Swiss mice using the micronucleus and comet assays, respectively. The animals were treated by gavage with different doses of the extract (250, 500, and 1000 mg/kg body weight). For antigenotoxicity assessment, different doses of the S. camporum extract were administered simultaneously with doxorubicin (micronucleus test; 15 mg/kg) and methanesulfonate (comet assay; 40 mg/kg). The results showed that the S. camporum extract itself was not genotoxic in the mouse micronucleus or comet assay. The number of micronucleated polychromatic erythrocytes was significantly lower in animals treated with the S. camporum extract and doxorubicin when compared to animals treated only with doxorubicin. In the comet assay, the S. camporum extract, at the doses tested, significantly reduced the extent of DNA damage in liver cells induced by methanesulfonate. The putative activity of the active compounds of S. camporum extract may explain the effect of this plant on genotoxicity induced by doxorubicin and methanesulfonate.

Influence of counting methodology on erythrocyte ratios in the mouse micronucleus test

The mammalian erythrocyte micronucleus test is widely used to investigate the potential interaction of a test substance with chromosomes or mitotic apparatus of replicating erythroblasts. In addition to the primary endpoint, micronucleated erythrocyte frequency, the proportion of immature erythrocytes is measured to assess the influence of treatment on erythropoiesis. The guideline recommendation for an acceptable limit of the immature erythrocyte fraction of not < 20% of the controls was based on traditional scoring methods that consider RNA content. Flow-based sample analysis (e.g., MicroFlow®) characterizes a subpopulation of RNA-containing reticulocytes (RETs) based on CD71 (transferrin receptor) expression. As CD71+ cells represent a younger cohort of RETs, we hypothesized that this subpopulation may be more responsive than the RNA+ fraction for acute exposures. This study evaluated RET population in the peripheral blood of two strains of mice treated by oral gavage with three clastogens (cyclophosphamide, N-ethyl-N-nitrosourea, and methyl methanesulfonate). Although CD71+ frequencies correlated with RNA-based counts, the relative treatment-related reductions were substantially greater. Accordingly, when using the flow cytometry-based CD71+ values for scoring RETs in an acute treatment design, it is suggested that a target value ≥ 5% CD71+ reticulocytes (i.e., 95% depression in reticulocytes proportion) be considered as acceptable for a valid assay.

Evaluation of the Toxicity of Intravenous Delivery of Auroshell Particles (Gold–Silica Nanoshells)

Gold nanoshells (155 nm in diameter with a coating of polyethylene glycol 5000) were evaluated for preclinical biocompatibility, toxicity, and biodistribution as part of a program to develop an injectable device for use in the photothermal ablation of tumors. The evaluation started with a complete good laboratory practice (GLP) compliant International Organization for Standardization (ISO)-10993 biocompatibility program, including cytotoxicity, pyrogenicity (US Pharmacopeia [USP] method in the rabbit), genotoxicity (bacterial mutagenicity, chromosomal aberration assay in Chinese hamster ovary cells, and in vivo mouse micronucleus), in vitro hemolysis, intracutaneous reactivity in the rabbit, sensitization (in the guinea pig maximization assay), and USP/ISO acute systemic toxicity in the mouse. There was no indication of toxicity in any of the studies. Subsequently, nanoshells were evaluated in vivo by intravenous (iv) infusion using a trehalose/water solution in a series of studies in mice, Sprague-Dawley rats, and Beagle dogs to assess toxicity for time durations of up to 404 days. Over the course of 14 GLP studies, the gold nanoshells were well tolerated and, when injected iv, no toxicities or bioincompatibilities were identified.

Mouse micronucleus assay as a surrogate to assess genotoxic potential of arsenic at its human reference dose

Exposure to high contents of arsenic (a genotoxic carcinogen) in humans through drinking water is one of the most serious concerns in many parts of the world including India. The United States Environmental Protection Agency (USEPA) has recommended a permissible limit of daily exposure in humans to arsenic as its reference dose (0.3μgkg−1d−1) with almost no likelihood of any adverse effect. The present work was a quantitative assessment of the genotoxic potential of arsenic at the exposure level of its human reference dose through micronucleus (MN) assay in mice. The animals were exposed to various doses of arsenic through oral gavaging for 15 consecutive days. Significant increases in the frequency of micronucleated erythrocytes were observed in mice upon exposure to arsenic which occurred even at its human reference dose and in a dose-dependent manner. The study of the genotoxic potential of arsenic in humans at lower exposure levels (including its human reference dose) is, therefore, highly desirable for risk assessment and hazard identification.

Scientific Opinion on the safety evaluation of the substance, 1,3‐bis(isocyanatomethyl)benzene, CAS No. 3634–83‐1 for use in food contact materials

This scientific opinion of EFSA deals with the risk assessment of the monomer 1,3-bis(isocyanatomethyl) benzene, CAS No. 3634–83-1, FCM Substance No 988. In water and gastric fluid simulant, immediate and complete hydrolysis of the substance to 1,3-benzenedimethanamine occurs. The toxicity of 1,3-bis(isocyanatomethyl)benzene was tested in three in vitro genotoxicity tests, with and without metabolic activation and in an in vivo micronucleus assay in rats and was considered as non-genotoxic in vivo. In addition, its hydrolysis product, 1,3-benzenedimethanamine, was tested in two in vitro tests, in the presence and absence of a metabolising system, and one in vivo mouse micronucleus assay. The tests were negative. No concern for genotoxicity in vivo is raised by 1,3-bis(isocyanatomethyl)benzene and its hydrolysis product 1,3-benzenedimethanamine. The CEF Panel concluded that that the use of the substance 1,3-bis(isocyanatomethyl)benzene as co-monomer in the manufacture of a middle layer coating in a multilayer film does not raise safety concern if the migration of its hydrolysis product, 1,3-benzenedimethanamine, does not exceed 0.05 mg/kg food.

Scientific Opinion on the safety evaluation of the substance, 2‐phenyl‐3, 3‐bis(4‐hydroxyphenyl)phthalimidine, CAS No. 6607–41‐6, for use in food contact materials

This scientific opinion of EFSA deals with the risk assessment of the substance 2-phenyl-3, 3-bis(4-hydroxyphenyl)phthalimidine with the CAS No. 6607–41-6 and the FCM substance No 872, for use as a co-monomer for manufacturing polycarbonate polymers intended to be used for single and repeated contacts with all kinds of foodstuffs without restriction in time and temperature. Based on three in vitro genotoxicity tests and a mouse micronucleus test in vivo on the substance, the Panel considered that there is no concern with respect to genotoxicity of the substance. The CEF Panel concluded that there is no safety concern for the consumer if 2-phenyl-3, 3-bis(4-hydroxyphenyl)phthalimidine is used as a co-monomer in polycarbonate copolymer and its migration does not exceed 0.05 mg/kg food. Potential migration of aniline, an impurity of the substance, should be in compliance with the restriction set in the Commission Regulation (EU) No 10/2011, i.e. not detectable.

Phytochemical composition and toxicity of an antioxidant extract from Pimpinella brachycarpa (Kom.) Nakai

Pimpinella brachycarpa (Kom.) Nakai (PB) is one of the most favored edible greens grown in Asian regions. In our previously study, we found PB extract had antioxidant effects in vitro. In the present study, an EtOAc soluble extract (PBet) was isolated from PB. Then the antioxidant properties at cellular level, phytochemical composition and toxicity of PBet were examined. The results indicated that PBet (0.5–2mg/mL) could protect Bel-7404 cells from H2O2 induced cell damage through scavenging of intracellular ROS. Moreover, myristic acid, 24ζ-methyl-5α-lanosta-25-one, β-sitosterol, pregnenolone and β-daucosterol were firstly isolated from PB. In addition, PBet (0.75g/kg BW, ig) had no acute toxicity and it (0.03–0.12g/kg BW, ig, 7 d) could not influence the rate of bone marrow polychromatic erythrocytes micronucleus and chromosome aberration in KM mice. All above findings suggested that PBet could be considered as a safe functional food with antioxidant activities.

In vivo protective effect of Copaifera langsdorffii hydroalcoholic extract on micronuclei induction by doxorubicin

Copaifera lansdorffii Desf. is known as 'copaíba', 'copaiva' or 'paú-de-óleo', and is found in part of Brazil. The present study was undertaken to evaluate the genotoxic potential of C. langsdorffii leaf hydroalcoholic extract (CLE) and its influence on the genotoxicity induced by the chemotherapeutic agent doxorubicin (DXR) using the Swiss mouse peripheral blood micronucleus test. HPLC analysis of CLE using two monolithic columns linked in series allowed quantification of two major flavonoid heterosides, quercitrin and afzelin. Animals were treated with CLE by gavage at doses of 10, 20, 40 and 80 mg kg(-1) body weight per day, each for 20 days. Peripheral blood samples were collected at 24 and 48 h, and 7, 15 and 21 days after the beginning of the treatment. For the antigenotoxicity evaluation, the animals treated with different concentrations of CLE received DXR (15 mg kg(-1) body weight, intraperitoneal) at day 20. The peripheral blood samples were collected 24 and 48 h after the treatment with DXR. The results demonstrated that CLE itself was not genotoxic in the mouse micronucleus assay. In animals treated with CLE and DXR, the number of micronucleus was significantly decreased compared with animals receiving DXR alone. The putative antioxidant activity of one or more of the active compounds of CLE may explain the effect of this plant on DXR genotoxicity.

The In Vitro and In Vivo Genotoxicity of Benzocaine: A Brief Communication

Benzocaine has a long history of use in human medicine. However, benzocaine also has been used in aquaculture with finfish for more than 40 years for sedating fish for marking, transport, surgery, and so on, although benzocaine does not have a current Food and Drug Administration (FDA) approval for this application in the United States. As part of a FDA approval for use as an animal drug, the genotoxicity of benzocaine was evaluated in the in vitro bacterial reverse mutation assay and the forward mutation assay and in vivo in the mouse micronucleus assay. These studies were conducted in compliance with Good Laboratory Practice regulations and according to Veterinary International Conference on Harmonisation guidelines. Based on the results of these studies, benzocaine was determined not to be genotoxic.

Evaluation of the Genotoxicity of Extracts of Houttuynia cordata Thunb.

The present study was conducted to evaluate the activity of methanol extracts from Houttuynia cordata Thunb. (HC) in a reverse mutation assay in Salmonella typhimurium, and a chromosome aberration assay in the Chinese hamster ovary (CHO) cell line and to evaluate its effect on the occurrence of polychromatic erythrocytes in mice. In the reverse mutation assay using Salmonella typhimurium TA98, TA100, TA1535, and TA1537 and Escherichia coli WP2urvA(-), methanol extracts of HC (5, 2.5, 1.25, 0.62, or 0.312 mg/plate) did not induce reverse mutations in the presence or absence of an S9 metabolic activation mixture. In the chromosome aberration test using CHO cells, methanol extracts (1.25, 2.5 or 5 μg/ml) caused a few incidences of structural and numerical aberrations, in both of absence or presence of an S9 metabolic activation mixture, but in comparison with the positive control group, these incidences were not significantly increased. In the mouse micronucleus test, no significant increases in the occurrence of micronucleated polychromatic erythrocytes were observed in male ICR mice that were orally administered methanol extracts of HC at doses of 2.0, 1.0, or 0.5 g/kg. From these results, we concluded that the methanol extracts of HC did not induce harmful effects on genes in bacteria, a mammalian cell system or in mouse bone marrow cells. Thus, HC's use for health promotion and/or a sick remedy for humans may be safe.

Safety Study of Culinary-Medicinal Royal Sun Agaricus, Agaricus brasiliensis S. Wasser et al. KA21 (Higher Basidiomycetes) Assessed by Prokaryotic as well as Eukaryotic Systems

As part of the safety evaluation of culinary-medicinal Royal Sun Agaricus, Agaricus brasiliensis KA21, for human consumption, we performed the bacterial reverse mutation test, the mouse micronucleus test, and mouse lymphoma test using A. brasiliensis KA21 as the test substance. The reverse mutation test utilized five bacterial strains, including Salmonella typhimurium TA100, TA1535, TA98, and TA1537, and Escherichia coli WP2 uvrAO. For the micronucleus test we used mice. For the mouse lymphoma test, we used one of the most commonly used mammalian cell mutagenesis systems; the L5178YTK +/- mouse lymphoma-TK assay detects the mutations at the thymidine kinase locus caused by base-pair changes, frameshift, and small deletions. All the tests were conducted according to the guidelines for genotoxicity testing of drugs by the Ministry of Health, Labor, and Welfare, Japan. In the bacterial reverse mutation test, no toxicity was observed up to a dose of 5,000 μg/plate. In the mouse micronucleus test, no toxicity was noted up to a dose of 1 g/kg body weight. In the mouse lymphoma test, frequency of the mutation was equal both in the presence or absence of KA21. Supporting the long history of human consumption of A. brasiliensis, the data shown in this study strongly indicate the safety of this mushroom.

Evaluation of mutagenic effect of two antiobesity drugs on mice’s genetic materials

Obesity is a complex, multifactorial disease that, similarly to high blood pressure and diabetes, frequently requires pharmacological treatment with long-term use, suggesting that pattern of use could increase the rates of genetic damage. Among antiobesity drugs, meridia and orlistat act with completely different mechanisms of action. This study aimed to evaluate the mutagenic effect of meridia and orlistat on genetic material of mice by cytogenetic analysis, which included the micronucleus test and chromosomal aberration assay at two doses comparable to propose human therapeutic and double therapeutic doses. Results revealed that the total number of structural chromosomal aberrations in bone marrow cells, with gap, was significantly increased for the two drugs at therapeutic doses. The structural chromosomal aberrations involved breaks, gaps, deletions and fragments, and centric fusion. Chromosomal deletions and fragments were the most frequently increased types of structural chromosomal aberrations. At double therapeutic doses, the treated animals showed a high significant increase of total structural chromosomal aberrations with and without gaps for the two drugs. The frequency of micronucleus in mice treated with therapeutic doses was significantly increased for both drugs. The treated animals at double therapeutic doses showed a positive response for both drugs. In conclusion, treatment with these two drugs at therapeutic doses should be taken under precaution and contraindicated at double therapeutic doses, because the cytogenetic analysis of meridia and orlistat showed an adverse effect on genetic materials at therapeutic doses and a mutagenic effect at double therapeutic doses.

Toxicological assessment of tridecafluorohexylethyl methacrylate (6:2 FTMAC)

The toxicity of tridecafluorohexylethyl methacrylate (6:2 FTMAC), an acrylic monomer used in producing polymeric substances, was evaluated. 6:2 FTMAC has low acute oral and dermal toxicity (LD50 > 5000 mg/kg), was not a skin or eye irritant, and did not demonstrate skin sensitization potential in a local lymph node assay (LLNA). 6:2 FTMAC was not mutagenic in the bacterial reverse mutation (Ames) test or in the mouse lymphoma assay. 6:2 FTMAC induced structural aberrations in human peripheral blood lymphocytes in vitro in the absence of metabolic activation but not in the presence of S9 metabolic activation. No numerical aberrations were detected under any testing condition. Also, no increase occurred in structural or numerical chromosomal aberrations in an in vivo mouse micronucleus assay in 6:2 FTMAC treated animals compared to controls. 6:2 FTMAC was administered at 0, 100, 500 and 1000 mg/kg/day via gavage to male and female SD rats for 14 days. No test substance-related effects on mortality, clinical signs, body weights, nutritional parameters, or clinical pathology were observed at any dose. Test substance-related increases in liver weights in males and females at all dose levels and thyroid and kidney weights in 500 and 1000 mg/kg/day males were noted. While there was no histopathological correlate for thyroid and kidney weight changes, minimal hypertrophy was noted in liver in males and females at 1000 mg/kg/day group. The changes noted in teeth (altered mineralization; retention of basophilic material) and femur (increased mineralization) in all treated groups were not associated with clinical signs or microscopic changes and were likely related to free fluoride formed from 6:2 FTMAC metabolism. Plasma (3–4-fold) and urine (30–50-fold) fluoride was higher in treated groups versus controls. Therefore, the changes noted in organ weights, teeth, femur, plasma or urine were not considered adverse. In the repeated dose toxicity study, the no-observed-adverse-effect-level (NOAEL) was 1000 mg/kg/day. Based on mean measured concentrations, the 96-h LC50 in fathead minnow was >14.5 mg/L and the 72-h EC50 in Pseudokirchneriella subcapitata was >24.6 mg/L, while the 48-h EC50 in Daphnia magna, based on nominal concentrations, was >120 mg/L. Overall, 6:2 FTMAC is considered to have low toxicity potential based on these studies.

Genotoxicity testing of low molecular weight fucoidan from brown seaweeds

Fucoidan extracts from brown seaweed have anticoagulant, antithrombotic, and antiviral activities. Low molecular weight fucoidan (LMF) obtained by acid hydrolysis of high molecular weight fucoidan showed more favorable bioactivity. Despite extensive work on LMF bioactivities, detailed studies on the genotoxicity of LMF have not been conducted. As part of a safety evaluation, the potential genotoxicity of LMF was evaluated using a standard battery of tests (bacterial reverse mutation assay, chromosomal aberrations assay, and mouse micronucleus assay). The LMF was determined not to be genotoxic under the conditions of the reverse mutation assay, chromosomal aberrations assay, or mouse micronucleus assay. In a reverse mutation assay using four Salmonella typhimurium strains and Escherichia coli, LMF did not increase the number of revertant colonies in any tester strain regardless of metabolic activation by S9 mix, and did not cause chromosomal aberration in short tests with the S9 mix or in the continuous (24h) test. A bone marrow micronucleus test in ICR mice dosed by oral gavage at doses up to 2000mg/kg body weight/day showed no significant or dose-dependent increases in the frequency of micronucleated polychromatic erythrocytes. Use of LMF is presently expected to be safe, as anticipated intake is small compared to doses administered in the genotoxicity assays and may prove to be a useful bioactive agent after further toxicity research.