Mouse Micronucleus Assay Research Articles

Safety assessment of a novel ingredient for removable chewing gum

Rev7™ is an indigestible gum polymer used for the manufacturing of chewing gum. It allows for the formulation of chewing gum with low adhesion; thus can be readily removed from surfaces such as sidewalks, clothing, carpets and furniture. In a toxicological safety assessment, Rev7™ was found to be non-mutagenic in the AMES assay. The highest concentration tested in a mouse lymphoma thymidine kinase locus gene mutation assay induced a slight but biologically relevant increase in mutations under non-metabolic activation conditions after 24h. Because of this finding, a mouse micronucleus assay was performed, and the test article was found to be negative for inducing chromosomal damage. A 28-day repeated oral toxicity study resulted in a NOAEL of 80,000ppm; the highest concentration tested. Rev7™ was found to be free from contaminants such as heavy metals, monomers, and solvents. Lastly, Rev7™ did not demonstrate skin-sensitizing properties in the murine local lymph node assay.

Evaluation of genotoxicity of Yanhuanglian dehydrocavidine (YHL-DC) in vitro and in vivo

It is reported that dehydrocavidine (DC), the main component of a traditional Chinese medicine, Yanhuanglian (YHL), can protect hepatic tissue against HBV and HAV impairment. As part of a safety evaluation on YHL-DC for use in the treatment of HBV, the present study evaluated the potential genotoxicity of YHL-DC by using the standard battery of tests (i.e., bacterial reverse mutation, chromosomal aberrations, and mouse micronucleus assays) recommended by the State Food and Drug Administration of China. The results showed that YHL-DC was not genotoxic under the conditions of the reverse mutation, chromosomal aberrations, and mouse micronucleus assay conditions. The anticipated clinical dose should be smaller than the doses used in the genotoxicity assays. With confirmation from further toxicity studies, YHL-DC would hopefully prove to be a useful anti-HBV agent.

Nonclinical Safety Assessment of the Histone Deacetylase Inhibitor Vorinostat

Vorinostat (SAHA, Zolinza), a histone deacetylase inhibitor, is assessed in nonclinical studies to support its approval for cutaneous T-cell lymphoma. Vorinostat is weakly mutagenic in the Ames assay; is clastogenic in rodent (ie, CHO) cells but not in normal human lymphocytes; and is weakly positive in an in vivo mouse micronucleus assay. No effects are observed on potassium ion currents in the hERG assay up to 300 microM (safety margin approximately 300-fold the approximately 1 microM serum concentration associated with the 400 mg/d maximum recommended human dose. No rat respiratory or central nervous system effects are found at 150 mg/kg (>2-fold maximum recommended human dose). No cardiovascular effects, including effects on QTc interval, are observed after a single oral dose (150 mg/kg) in dogs. Vorinostat is orally dosed daily in rats (controls, 20, 50, or 150 mg/kg/d) and dogs (controls, 60, 80, or 100/125/160 mg/kg/d) for 26 weeks with a 4-week recovery. Rat vorinostat-related adverse findings are decreased food consumption, weight loss, and hematologic changes; a no observed adverse effects level is not established. In dogs, adverse effects are primarily gastrointestinal; the no observed adverse effects level is 60 mg/kg/d (approximately 6-fold maximum recommended human dose). Toxicities are reversible and can be monitored in the clinic.

Safety studies of LP20 powder produced from heat-killed Lactobacillus plantarum L-137

The safety of LP20 and its prototype, a powder, with potential use in food, produced from a mixture of dextrin and heat-killed Lactobacillus plantarum L-137, was assessed in an acute study in mice, and in an in vitro bacterial reverse mutation assay, an in vitro chromosome aberration assay, and an in vivo mouse micronucleus assay. LP20 prototype was not acutely toxic when administered to male and female Slc:ICR mice by single gavage at 2000 mg/kg bw. Dosing was not associated with mortality, clinical signs, changes in bodyweight, or macroscopic abnormalities. The LD 50 in mice was greater than 2000 mg/kg bw. There was no evidence of genotoxicity of LP20 in the Ames assay (0–5000 μg/plate) or in the in vitro chromosome aberration assay with Chinese hamster lung fibroblasts (0–5000 μg/mL). Administration of two consecutive daily doses of 500, 1000, or 2000 mg/kg bw by gavage to male Crlj:CD-1 mice was not associated with an increased incidence of micronuclei and did not alter the ratio of polychromatic to normochromatic erythrocytes. These studies show that LP20 powder is not acutely toxic and is without genotoxic activity both in vitro and in vivo.

Evaluation of chemopreventive activity of glutamine by the comet and the micronucleus assay in mice's peripheral blood

This research has evaluated the effects of enteral supplementation of glutamine in clastogens and genotoxic damages caused by the acute administration of cisplatin. For this, it was utilized Swiss mice distributed in eight experimental groups: control, cisplatin, glutamine, in three different doses and the combination of these with cisplatin. The results show that the glutamine was present in neither genotoxic nor mutagenic activity. When in association with glutamine and cisplatin, in simultaneous treatment, it was verified the frequency decreased of micronuclei and comets. The damage reduction percentages to the micronucleus ranged from 95.4 to 91.8% after 24 h of administration of these compounds and 76.7 to 56.8% after 48 h. In the same time the damage reduction percentages to the comet test ranged from 117.0 to 115.0%. The results suggest that glutamine is capable of preventing genotoxic and mutagenic damage according to the experimental design proposed.

Safety assessment of a proprietary preparation of a novel Probiotic, Bacillus coagulans, as a food ingredient

It has been demonstrated that some strains of Bacillus coagulans can survive extremes of heat, acidity of the stomach, and bile acids, to which commonly consumed probiotics are susceptible. A toxicological safety assessment was performed on a proprietary preparation of B. coagulans – GanedenBC30™ – a novel probiotic. Seven toxicologic studies were conducted and included: in vitro bacterial reverse mutation assay; in vitro chromosomal aberration assay; micronucleus assay in mice; acute and 90day subchronic repeated oral toxicity studies were conducted in Wistar Crl:(WI) BR rats; acute eye and skin irritation studies were conducted in rabbits.The results of this toxicological safety assessment indicate that GanedenBC30™B. coagulans does not demonstrate mutagenic, clastogenic, or genotoxic effects. Furthermore, the results of the acute and 90-day subchronic oral toxicity studies in rats resulted in the conclusion of a NOAEL greater than 1000mg/kg per day. Since the concentration of the cell mass used in the 90-day study was 1.36×1011 CFUs/g, this corresponds to 95.2×1011 CFUs for a 70kg human and since the suggested human dose is in the range of 100×106 to 3×109 CFUs, this gives a safety factor ranging from 3173 to 95,200 times. Based upon scientific procedures and supported by history of use, GanedenBC30™ is considered safe for chronic human consumption.

An assessment of the carcinogenic potential of ezetimibe using nonclinical data in a weight-of-evidence approach

Ezetimibe blocks intestinal absorption of sterols via interaction with the Neimann-Pick C1-Like 1 (NPC1L1) transporter and is approved for use in the treatment of primary hyperlipidemia (heterozygous familial and non-familial), homozygous familial hypercholesterolemia, and homozygous sitosterolemia. A recently completed randomized clinical trial [simvastatin and ezetimibe in aortic stenosis (SEAS)] testing the effectiveness of Vytorin™ (a combination of simvastatin and ezetimibe) in patients with aortic stenosis reported an unexpected safety finding: an increase in overall cancer incidence and cancer-associated mortality (all types) in the treated groups relative to the placebo control. A subsequent meta-analysis utilizing a much larger database from two ongoing clinical trials indicated that the observed findings in the SEAS trial were likely due to chance and not a true drug-induced effect. Nonetheless, it has been suggested by various commentators on the SEAS trial that ezetimibe may be carcinogenic. The extensive nonclinical database for ezetimibe was used to test the hypothesis that ezetimibe may be a direct or indirect carcinogen. Using two different in silico approaches, ezetimibe showed no structural alerts for genetic toxicity or carcinogenicity. Ezetimibe was not genotoxic in two reverse mutation assays, one in vitro clastogenicity assay, and two mouse micronucleus assays. No evidence of proliferative lesions was observed in three species in studies of 1–12 months in duration. Ezetimibe was not carcinogenic in standard 2-year bioassays in mice and rats. Additionally, in these 2-year bioassays, no drug-related non-neoplastic lesions were noted. The absence of drug-induced non-neoplastic or proliferative lesions in these studies indicates that ezetimibe treatment was not associated with findings characteristic of carcinogens (i.e., DNA reactivity or cell proliferation) Administration of pharmacologic doses of ezetimibe to mice did not alter hepatic expression patterns of genes associated with apoptosis, cell proliferation, or epithelial–mesenchymal transition. No evidence of drug-induced tumors was observed in mice in which the molecular target of ezetimibe (NPC 1L1) was knocked out over the life span of the animal. In conclusion, the nonclinical data do not support the proposed hypothesis based on the single observation from the SEAS trial and, rather, support the conclusion that ezetimibe does not represent a carcinogenic hazard to humans using this drug in a therapeutic setting.

Lack of genotoxic effect of food dyes amaranth, sunset yellow and tartrazine and their metabolites in the gut micronucleus assay in mice

The food dyes amaranth, sunset yellow and tartrazine were administered twice, at 24 h intervals, by oral gavage to mice and assessed in the in vivo gut micronucleus test for genotoxic effects (frequency of micronucleated cells) and toxicity (apoptotic and mitotic cells). The concentrations of each compound and their main metabolites (sulfanilic acid and naphthionic acid) were measured in faeces during a 24-h period after single oral administrations of the food dyes to mice. Parent dye compounds and their main aromatic amine metabolites were detected in significant amounts in the environment of colonic cells. Acute oral exposure to food dye additives amaranth, sunset yellow and tartrazine did not induce genotoxic effect in the micronucleus gut assay in mice at doses up to 2000 mg/kg b.w. Food dyes administration increased the mitotic cells at all dose levels when compared to controls. These results suggest that the transient DNA damages previously observed in the colon of mice treated by amaranth and tartrazine by the in vivo comet assay [Sasaki, Y.F., Kawaguchi, S., Kamaya, A., Ohshita, M., Kabasawa, K., Iwama, K., Taniguchi, K., Tsuda, S., 2002. The comet assay with 8 mouse organs: results with 39 currently used food additives. Mutat. Res. 519, 103–119] are unable to be fixed in stable genotoxic lesions and might be partly explained by local cytotoxicity of the dyes.

Genotoxicity testing of aqueous extracts of Mahwangyounpae-tang, a polyherbal formula

Mahwangyounpae-tang (MT), consisting of 22 types of herbal extracts has been used for thousands of years in Korean traditional medicine for the oral treatment of respiratory diseases including asthma. As part of a safety evaluation of MT extracts for use in asthma, the potential genotoxicity of an aqueous MT extract was evaluated using the standard battery of tests (bacterial reverse mutation assay; chromosomal aberrations assay; mouse micronucleus assay) recommended by Korea Food and Drug Administration (KFDA). The MT extract was determined not to be genotoxic under the conditions of the reverse mutation assay, chromosomal aberrations assay and mouse micronucleus assay. Use of MT is presently expected to be safe, as anticipated intake is small compared to the doses administered in the genotoxicity assays and may, after further toxicity research, prove to be a useful anti-asthma agent.

Developmental and transplacental genotoxicology: Fluconazole

Over the last 40 years mankind has been facing new types of radiochemical environmental settings with every decade. During the last decade, biomonitoring was additionally focused on assessing associations between environmental exposure(s) and both early and late biological effects in children. Despite efforts to control and avoid child exposure to genotoxic agents the incidence of childhood cancers is increasing. Some cancers in adulthood may be the consequence of a multi-step process which starts with intrauterine and childhood exposure. This highlights the importance of a comprehensive interpretation of multiple health effects, especially considering recent studies suggesting that most health disorders are related to DNA changes. When exposed to genotoxic agents, a developing organism (fetus or child) is constantly being forced to reorganize into new equilibriums in order to adjust to a xenobiotic environment. In addition, the influence of sex hormones on radiochemical sensitivity is still unknown. For this reason special attention should be paid to puberty. The results of recent studies on animal models and follow up studies on children after nuclear accidents show long-lasting cytogenetic damage even after low dose exposures and their transgenerational persistance. To evaluate age-related difference and transplacental genotoxic potency fluconazole (FC) was investigated by in vivo micronucleus (MN) assay in adult mice, young mice and in transplacentally exposed newborn pups. Compared to the baseline values, FC caused no detectable genome damage in adult animals, but there was a significant increase in MN frequency in young animals and in newborn pups. Our study thus exemplifies an age-related chemosensitivity, and argues that cancer-promoting disturbances of complex prenatal developmental mechanisms and maturation during childhood require a new approach using systems biology.

Acute, subchronic and genotoxicity studies conducted with Oligonol, an oligomerized polyphenol formulated from lychee and green tea extracts

Oligonol is a phenolic product derived from lychee fruit extract and green tea extract, containing catechin-type monomers and oligomers of proanthocyanidins, produced by a manufacturing process which converts polyphenol polymers into oligomers. The safety of Oligonol was assessed in acute and subchronic studies and genotoxicity assays. In a single dose acute study of Oligonol, male and female rats were administered 2000 mg/kg body weight (bw) Oligonol in water by gavage. Oligonol caused no adverse effects and body weight gain and food consumption were within normal range, thus the LD 50 of Oligonol was determined to be greater than 2000 mg/kg. A 90 day subchronic study (100, 300 and 1000 mg/kg bw/day, oral gavage) in male and female rats reported no significant adverse effects in food consumption, body weight, mortality, clinical chemistry, haematology, gross pathology and histopathology. Similarly, no adverse effects were observed in mice fed diets providing 2, 20 or 200 mg/kg bw Oligonol or 200 mg/kg bw lychee polyphenol for 90 days. Oligonol did not show any potential to induce gene mutations in reverse mutation tests using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA strains. Oligonol did not induce chromosomal aberrations in cultured Chinese hamster lung cells, but it showed increased polyploidy. In a micronucleus assay in mice, Oligonol did not induce any micronuclei or suppress bone marrow, indicating it does not cause chromosome aberrations. The results from these safety studies and previous reports support the safety of Oligonol for human consumption.

Studies of the Toxicological Potential of Capsinoids: V. Genotoxicity Studies of Dihydrocapsiate

A series of studies was performed to evaluate the safety of dihydrocapsiate (4-hydroxy-3-methoxybenzyl 8-methylnonanoate; CAS no. 205687-03-2). This study evaluated the potential genotoxicity of this compound using a variety of in vitro and in vivo test systems, including bacterial reverse mutation test, chromosomal aberration test, micronucleus test, gene mutation assay with transgenic rats, and single-cell gel (SCG) assay (Comet assay). In vitro tests (bacterial reverse mutation test and chromosomal aberration test) produced positive results in the absence of metabolic activation, but negative results in the presence of metabolic activation. The in vivo gene mutation assay (with transgenic rats) produced negative results, as did the in vivo mouse micronucleus assay, which failed to induce micronucleated polychromatic erythrocytes. Although the rat SCG assay produced statistically significant increases in the Olive tail moment and % tail DNA of the liver and intestine in the 2000 mg/kg group (compared with the negative-control group), a number of factors caused the authors to question the validity of these findings. Taken together, these results suggest that dihydrocapsiate has a low or extremely low likelihood of inducing genotoxicity.

Evaluation of the genotoxic potential of three phenyltetrahydropyridinyl butylazole-derived sigma-receptor ligand drug candidates

Three structurally related phenyltetrahydropyridinyl butylazole (PTHPB)-derived drug candidates with sigma receptor-binding properties were evaluated for genotoxic potential in the ICH standard battery of genetic toxicology assays. These comprised an Ames test, a mouse-lymphoma assay, and a mouse bone-marrow micronucleus test. The maximum test concentrations in the in vitro assays were determined by the solubility and/or the cytotoxicity of the compounds. In the mouse micronucleus assay, the compounds were administered orally at three levels up to the maximum tolerated dose (MTD). Negative results were obtained for all three drug candidates in the Ames test and in the mouse-lymphoma assay, both in the absence or presence of metabolic activation. In the mouse micronucleus test, there was no effect on the frequency of micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after oral administration of any of the three test compounds, at any dose level or sampling time (24 and 48 h). Administration of all three compounds at the MTD induced a clear decrease in mouse body-temperature of 3.1–4.8 °C below normal; the temperature returned to normal within 8 h of dose administration. The produced mild hypothermia and absence of micronucleus induction was in contrast to the induction of MNPCE secondary to marked hypothermia reported for a structurally similar PTHPB-derived sigma-receptor ligand, the antipsychotic compound E-5842. The results obtained in the current series of studies suggest that exposure to the three tested PTHPB-derived drug candidates would not pose a genotoxic risk under clinical conditions.

Genotoxicity studies on green tea catechin

The beneficial effects of tea catechins are well documented. We evaluated the genotoxic potential of a green tea catechin preparation using established genotoxicity assays, including a bacterial reverse mutation assay (Ames test), a chromosomal aberration assay in cultured Chinese hamster lung cells (CHL/IU), a mouse lymphoma L5178Y/tk assay, and a bone marrow micronucleus (MN) assay in ICR CD mice and SD rats. No significant increases in the number of revertant colonies were observed in the Ames test, but positive responses were observed in two in vitro assays: the chromosomal aberration assay and mouse lymphoma L5178/tk assay. However, the in vivo study demonstrated no significant increase in micronucleated polychromatic erythrocytes (MNPCE) in the bone marrow of both ICR CD mice and SD rats administered a high dose of the green tea catechin preparation up to 2000mg/kg. Combined with favorable epidemiological information suggesting a chemopreventive effect of tea catechins on carcinogenesis, we conclude that green tea catechin presents no significant genotoxic concern under the anticipated conditions of use. These results are consistent with other genotoxicity studies of tea catechins, which show minimal, if any, genotoxic potential.

Evaluation of the genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) in a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay. Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 μg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 μg/ml for 3 and 20 h in the absence of S9, and for 3 h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 μg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 μg/kg, the last dose being administered 2 h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays. TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.

No evidence of genotoxic effect in vivo of the phytosterol oxidation products triols and epoxides

Phytosterols (PS) are naturally occurring compounds present in food products of plant origin. Due to reported positive health effects, some food products are also enriched with PS. In the same way as cholesterol is oxidised, PS also oxidise to a variety of oxidation products (POPs). The biological effects and safety aspects of POPs are still unclear. This study investigated whether POPs are genotoxic in vivo, using a flow cytometer-based micronucleus assay in mice. The highest dose of oxidation products administered was 67 mg/kg b.w. for epoxides and 9.4 mg/kg b.w. for triols. Synthesised and separated triols and epoxides from a mixture of sitosterol and campesterol were investigated. The frequency of micronucleated polychromatic erythrocytes (fMNPCE) in POP-exposed mice did not significantly differ from the control values in either of two experiments performed. The flow cytometer-based method also allows for restriction of the analysis to micronuclei with a high DNA content, indicating an aneugenic potency. Even with this approach, there was no significant increase in the frequency of micronucleated erythrocytes among POP-treated mice compared with control mice. Furthermore, no significant deviation in cell proliferation rate (%PCE) was observed.

Genotoxic evaluation of morphine, buprenorphine, pentazocine, and noscapine by micronucleus and comet assay in albino mice

<b>Objectives:</b> The present study was planned to explore the genotoxicity of morphine, buprenorphine, pentazocine, and noscapine.<br><b> Materials and Methods:</b> Bone marrow micronucleus assay and single cell gel electrophoresis assay were employed after 24 h (single dose) and 72 h (three doses) of treatment with clinically equivalent doses of opioids in albino mice. Percentage of micronucleated polychromatic erythrocytes and comet tail length were determined and the results were analyzed by one-way ANOVA and Student's 't' test.<br><b> Results:</b> Only morphine and noscapine showed significant (<i>P</i> < 0.01) increase in both the number of micronucleated polychromatic erythrocytes and comet tail lengths in acute (24-h) as well as subacute (72-h) studies.<br><b> Conclusions:</b> These results clearly indicate the genotoxic potential of morphine and noscapine at the clinically equivalent doses.

Furan is not genotoxic in the micronucleus assay in vivo or in vitro

Furan, a potential human carcinogen, is formed during heat-treatment of food. Previous studies of the genotoxicity of furan have given disparate results. Hence, there is a need for complementary data to clarify the mechanism behind the carcinogenicity of furan. In this study, we have used the flow cytometer-based micronucleus assay in mice and the cytokinesis-block micronucleus assay in human lymphocytes to investigate the genotoxic potential of furan. Three in vivo experiments were performed: intraperitoneal or subcutaneous injection of furan in male Balb/C mice (0–300 and 0–275 mg/kg body weight, respectively) and intraperitoneal injection of male CBA mice (0 and 225 mg/kg body weight). No increased level of micronucleated erythrocytes was detected in any of the in vivo experiments. In the in vitro setup, human lymphocytes from two donors were treated with furan in concentrations from 0 to 100 mM, either with or without metabolic activation (liver homogenate from rat). In parity with the in vivo results there was no significant increase in the frequency of micronucleated cells here either. As neither the in vivo nor the in vitro studies disclose any significant increase in the micronucleus frequency after treatment with furan, our results support that the carcinogenicity of furan is caused by a non-genotoxic mechanism.

Safety evaluation of α-lipoic acid (ALA)

The safety of the antioxidant α-lipoic acid (racemic form) (ALA), also called thioctic acid (CAS RN 1077-28-7) was assessed in acute and subchronic toxicity studies as well as in in vitro and in vivo mutagenicity/genotoxicity studies. ALA was not acutely toxic to rats (LD 50 > 2000 mg/kg bw, OECD method 425). Administration of 31.6 or 61.9 mg ALA/kg bw/day for 4 weeks to male/female Wistar rats did not show any adverse effects. Specifically, there was no significant difference between control and treated animals at 31.6 or 61.9 mg ALA/kg bw with regard to body weight gain, feed consumption, animal behaviour, or haematological and clinical chemistry parameters. Only the high-dose of 121 mg ALA/kg bw was associated with slight alterations in liver enzymes as well as histopathological effects on the liver and mammary gland. ALA did not possess any mutagenic activity in the Ames assays conducted with various bacterial strains of Salmonella typhimurium. Moreover, there was no evidence of genotoxic activity in a mouse micronucleus assay. The results of these studies support the safety of ALA. The no-observed-adverse-effect level (NOAEL) is considered to be 61.9 mg/kg bw/day.

Genotoxicity testing of a Salacia oblonga extract

Salacia oblonga has been used for thousands of years in Ayurvedic medicine for the oral treatment of diabetes. The root extract has been shown to inhibit the activity of intestinal α-glucosidases, therefore S. oblonga holds potential as a natural method to mitigate the blood glucose response for people with diabetes. As part of a safety evaluation of novel ingredients for use in blood glucose control, the potential genotoxicity of a S. oblonga root extract (SOE) was evaluated using the standard battery of tests (reverse mutation assay; chromosomal aberrations assay; mouse micronucleus assay) recommended by US Food and Drug Administration (FDA) for food ingredients. SOE was determined not to be genotoxic under the conditions of the reverse mutation assay and mouse micronucleus assay, and weakly positive for the chromosomal aberrations assay. A reproducible, although weak, positive chromosomal aberrations response in human lymphocytes is of concern and further toxicity research is recommended. Use of SOE is presently expected to be safe, as anticipated intake is small compared to the doses administered in the genotoxicity assays and may, after further toxicity research, may prove be a useful ingredient in foodstuffs.