Two generation reproduction and teratogenicity studies of feeding cyadox in Wistar rats

To investigate the teratogenic potential and reproductive toxicity of cyadox, a growth promoting agent, Wistar rats (F₀) were fed with diets containing cyadox (0, 50, 150 and 2500 mg/kg) or olaquindox (150 mg/kg), approximately equivalent to cyadox 5, 15, 250 or olaquindox 15 mg/kg b.w./day across two generations. Half of the pregnant rats (F₀, F_1b) were subjected to caesarean section on gestational day 20 for teratogenic examination and the other half produced pups F_1a and F_2a, respectively. At the 250 mg/kg b.w./day cyadox group, body weights of F_1b pregnant rats and F_2a on day 21 after birth decreased; fetal body lengths and tail lengths decreased; the number of fetal resorptions increased significantly; litter weights, number of viable fetuses decreased; number of embryo resorptions increased significantly; number of liveborn F_1a, F_1b and F_2a decreased. No macroscopic or microscopic change of any significance was found in the reproductive organs. Significant increases in the incidence of cervial ribs or lumbar ribs in F_2a pups and significant increases of relative organ weight of testis and epididymis in F_1b were observed at the 250 mg/kg b.w./day cyadox group. The NOAEL for reproduction/development of cyadox for rats was estimated to be 150 mg/kg diet, which was equivalent to approximately 15 mg/kg b.w./day.     

Evaluation of tolerable levels of dietary quercetin for exerting its antioxidative effect in high cholesterol-fed rats

The tolerable level of dietary quercetin for exerting its antioxidative effect was evaluated in high cholesterol-fed rats, using quercetin-containing diets (31–1260 mg quercetin/kg body weight/day) and onion diets (19–94 mg quercetin aglycone equivalent/kg body weight/day), from the viewpoint of a safety assessment. After feeding for 4 weeks, the urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) levels of the quercetin-containing diet groups fed more than 157 mg quercetin/kg body weight/day were higher than the group fed a quercetin-free diet, although the plasma quercetin metabolite levels and plasma antioxidative activity were elevated depending on the amounts of quercetin or onion diet intake. No significant effect on body weight gain by quercetin-containing diets or onion diets was observed. However, ratios of the liver and kidney weights to the body weight were significantly increased in the quercetin-containing diet groups fed more than 314 mg and 157 mg quercetin/kg body weight/day, respectively, and in the onion diet groups fed more than 47 mg quercetin aglycone equivalent/kg body weight/day. These results indicated that the tolerable level for dietary quercetin for exerting its antioxidative effect was between 126 and 157 mg/kg/day for the quercetin diet and between 19 and 34 mg/kg/day for the onion diet.     

Safety assessment of heat-sterilized green tea catechin preparation: A 6-month repeat-dose study in rats

Evidence suggests that the purported health benefits associated with green tea consumption are related to tea catechins. In the present study, potential adverse effects of a standardized heat-sterilized green tea catechin (GTC-H) preparation was investigated following gavage administration to rats at doses of 0, 120, 400, 1200 mg/kg/day for 6 months. A decaffeinated high-dose group (1200 mg/kg/day) (GTC–HDC), was included for comparison. A possibly test article-related clinical finding of intermittent increased activity was noted in the 400 and 1200 mg/kg/day GTC-H groups, but was not considered to be adverse. Lower body weight gains without any decrease in food consumption were noted in the high-dose (1200 mg/kg/day)-treated GTC-H and GTC–HDC females. In the high-dose male GTC-H group, a lower total motor activity count for the 60-min session was noted prior to dosing at the study week 25 evaluations compared to the control group. Similar changes were not observed in the GTC–HDC group. Based on the results of this study, the no-observed-adverse-effect level (NOAEL) for GTC-H was 1200 mg/kg/day for males, the highest dose tested, and 400 mg/kg/day for females based on reduced body weight gains. The NOAEL for GTC–HDC was 1200 mg/kg/day for males and could not be determined in females.     

A 90-day oral (dietary) toxicity study of the 2R,4R-isomer of monatin salt in Sprague–Dawley rats

The root bark of Sclerochitin ilicifolius contains an intensely sweet substance analytically identified as isomers of 2-hydroxy-2-(indol-3-ylmethyl)-4-aminoglutaric acid and generically coined “monatin.” Groups of male and female Crl:CD(SD) rats were fed 0 (control), 5000, 10,000, 20,000 or 35,000 ppm R,R-monatin salt in the diet for 90 days. There were no toxicologically relevant clinical or histopathological findings in any of the test article-treated groups. Significantly lower cumulative body weight gains were noted in the 35,000 ppm group. Mean body weights in the 35,000 ppm group males and females were 7% and 12% lower, respectively, than the control group at study week 13. In the absence of other observations associated with systemic toxicity and lower food consumption, the magnitude of the body weight difference in the 35,000 ppm group females relative to the control group exceeded 10%, which indicated attainment of a maximum tolerated dose (MTD) level. Based on the results of this study, and conservatively assuming the body weight observations at the MTD to be indicative of an adverse effect, the dietary no-observed-adverse-effect level (NOAEL) of R,R-monatin salt for 90 days was 20,000 ppm in female rats (approximately 1544 mg/kg bw/day) and 35,000 ppm in male rats (approximately 2368 mg/kg bw/day).     

Twenty-eight days repeated oral dose toxicity study of gemifloxacin in Wistar albino rats

The purpose of this study was to investigate the potential toxicity of gemifloxacin by 28-day repeated oral dose in Wistar albino rats. The test article, was administered daily by gavage to male and female rats at dose levels of 0, 50, 100, 200 mg/kg/day. At the end of treatment period, 12 rats/sex/group was sacrificed, while six extra rats/sex in the vehicle control and highest dose groups sacrificed after 14 days recovery period. During the treatment and recovery periods, clinical signs, mortality, body weights, food and water consumption, ophthalmoscopy, urinalysis, phototoxicity, hematology, serum biochemistry, synovial fluid biochemistry, electrocardiogram (ECG), gross findings, organ weights, microscopic examination of synovial fluid, and histopathology were examined. Hematological and serum biochemical investigations revealed a dose-dependent increase in the total white blood cell (WBC), total bilirubin (T-BIL), glucose (GLU), alanine aminotransferase (ALT) and significant decreases in total protein (TP) were observed in both sexes at the same dose, at the end of treatment period, but the levels returned toward normal during the recovery period. Histopathology of talar joint showed that erosion of the articular surface of that joint in both sexes at the end of treatment period at the dose level of 200 mg/kg/day. Degenerative changes in tendinocytes were observed in Achilles tendon of both sexes at the high dose level at the end of treatment period. In histopathological study shows partial effacement of liver architecture and focal ulceration in gastric mucosa at the high dose level at the end of treatment period. Based on these results, it was concluded that 28 days repeated oral dose of gemifloxacin caused increases in the liver weight, WBC count, T-BIL, glucose level, ALT, decreasing the TP, cause chronic hepatitis and acute gastritis, erosion of the articular surface of joint and histopathologic changes in Achilles tendon in rats at the dose level of 200 mg/kg/day.     

Toxicologic evaluation of DHA-rich algal oil: Genotoxicity,acute and subchronic toxicity in rats

DHA-rich algal oil ONC-T18, tested in a battery of in vitro and in vivo genotoxicity tests, did not show mutagenic or genotoxic potential. The acute oral LD50 in rats has been estimated to be greater than 5000 mg/kg of body weight. In a 90-day subchronic dietary study, administration of DHA-rich algal oil at concentrations of 0, 10,000, 25,000, and 50,000 ppm in the diet for 13 weeks did not produce any significant toxicologic manifestations. The algal oil test article was well tolerated as evidenced by the absence of major treatment-related changes in the general condition and appearance of the rats, neurobehavioral endpoints, growth, feed and water intake, ophthalmoscopic examinations, routine hematology and clinical chemistry parameters, urinalysis, or necropsy findings. The no observed adverse effect level (NOAEL) was the highest level fed of 50,000 ppm which is equivalent to 3,305 and 3,679 mg/kg bw/day, for male and female rats, respectively. The studies were conducted as part of an investigation to examine the safety of DHA-rich algal oil. The results confirm that it possesses a toxicity profile similar to other currently marketed algal oils and support the safety of DHA-rich algal oil for its proposed use in food.     

Results of a 90-day safety assessment study in mice fed a glucan produced by Agrobacterium sp. ZX09

Salecan is a novel water-soluble glucan produced by Agrobacterium sp. ZX09. It has potential application as a food additive with a unique chemical composition and excellent physicochemical properties. The objective of this study was to investigate the acute and subchronic toxicity of Salecan. The oral LD50 of Salecan in ICR mice was greater than 3000 mg/kg body weight. In the subchronic study, ICR mice (10/sex/group) were fed diets containing 0%, 1.0%, 2.5% and 5.0% of Salecan (weight/weight) for 13 weeks. Based on the results from the subchronic study, the overall health, body weight gain, food consumption and clinical pathology parameters were comparable between the groups feed Salecan and the control. No dose-related effects were observed in the treated animals. The only exception was the observation that blood glucose in female mice fed Salecan was lower than in the control group. In addition, the fecal matter from Salecan fed mice exhibited increased water content versus the control animals. The no observed adverse effect level (NOAEL) of 14478 mg/kg body weight/day was determined. The results from this study support the conclusion that Salecan is non-toxic at the levels tested and does not pose a risk to human health when used in food.     

Toxicity evaluation of the aqueous extract of the rhizome of Elephantorrhiza elephantina (Burch.) Skeels. (Fabaceae), in rats

Elephantorrhiza elephantina root extract has been used as a traditional remedy for a wide range of ailments both in humans and livestock. As part of the safety assessment of the extract, acute, sub-acute and chronic toxicity tests were conducted by the oral route in rats. Male and female rats were divided into four groups consisting of five rats each and given doses of 200–1600 mg/kg bwt, 200–800 mg/kg bwt and 50–400 mg/kg bwt in acute (1 day), sub-acute (14 days) and chronic toxicity (35 days), respectively. During the experiment, no deaths were observed in any groups and there were no remarkable changes in general appearance, as well as in food and water consumption. Significant (P < 0.05) changes were however noted in body weights, haematological and serum biochemical parameters between the control and treated groups. Histopathological changes were also noted in kidneys, lungs, liver and spleen of rats receiving high doses. Based on these findings, it can be inferred that the plant has some potential toxicity at certain dose levels; therefore caution has to be taken when using E. elephantina for medicinal purposes.     

A chronic toxicity study of cyadox in Wistar rats

To investigate the chronic toxicity of cyadox, a growth promoting agent, five groups of Wistar rats (30 rats/group/sex) were fed with the diets containing cyadox (0, 100, 400 and 2000 mg/kg) or olaquindox (400 mg/kg) for 78weeks. There were significant decreases in body weights in both genders during most of the study period in 2000 mg/kg cyadox and 400 mg/kg olaquindox rats. Significant decreases in serum alkaline aminotransferase in the 2000 mg/kg cyadox rats at weeks 26, 52 and 78 were observed. Relative weights of liver and kidney were significantly increased in 2000 mg/kg cyadox and 400 mg/kg olaquindox rats at weeks 26, 52 and 78. A significant increase in relative brain and heart weights in 2000 mg/kg cyadox males was observed. The histopathological examinations revealed that 2000 mg/kg cyadox diet or 400 mg/kg olaquindox diet could induce proliferation of bile canaliculi in the portal area of liver and swelling and fatty degeneration of the proximal renal tubular epithelial cells in kidneys. In conclusion, the target organs of cyadox for rats were liver and kidney. The no-observed-adverse-effect level of cyadox in this study was estimated to be 400 mg/kg diet.     

Oral toxicological studies of black soybean (Glycine max) hull extract: Acute studies in rats and mice, and chronic studies in mice

Black soybean (Glycine max) has been used for traditional medicine and food in Asian countries, but safety of its hull has not been studied. We conducted acute and chronic oral toxicity studies. For the acute study, an extract of black soybean hull (BE; 2.5 g/kg body weight) was administered singly by intragastric intubation to Sprague–Dawley rats and C57BL/6 mice. There was no death or significant decrease in body weight in rats and mice, and the oral LD₅₀ of BE was >2.5 g/kg body weight. In the chronic study, BE was administered at dietary levels of 0% (control), 2.0%, and 5.0% to male and female C57BL/6 mice for 26 weeks. No mortality or toxicologically significant clinical changes were observed through the experimental period. Although body weights, as well as abdominal fat, blood levels of triglyceride and total cholesterol in 5.0% males were significantly lower than that in control and 2.0% groups, these changes were considered not to be adverse. Hematology and histopathological observation revealed no toxicologically significant changes. The no-observed adverse-effect-level of BE was estimated to be 5.0% in the diet (5074.1 mg/kg body weight/day for males and 7617.9 mg/kg body weight/day for females).     

Safety assessment of ellagic acid, a food additive, in a subchronic toxicity study using F344 rats.

Ellagic acid is a phenolic acid compound, used as a food additive for its antioxidative properties. Because of its chemical characteristics, use is also to be expected in cosmetics. The present 90-day subchronic toxicity study was performed in F344 rats at dose levels of 0, 1.25, 2.5 and 5% in powdered basal diet, with actual doses of 9.4, 19.1, 39.1 g/kg b.w., respectively, in males, and 10.1, 20.1, 42.3 g/kg b.w. in females. No mortality or treatment-related clinical signs were observed throughout the experimental period. Body weight gain was significantly reduced from weeks 3 (5% group), 6 (2.5% group) and 7 (1.25% group) to the end of the experiment (except week 8 in the lowest group) in the treated females, the final body weights being decreased in the 5% (92.5%), 2.5% (94.2%) and 1.25% (94.8%) treated groups as compared to the control. Changes in MCV and serum AST, ALP, Ca, Cl and P were sporadically observed, but these were not considered to be treatment-related alterations. There were no obvious histopathological changes in any of the groups. The no-observed-effect level (NOEL) was estimated to be 5% (3011 mg/kg b.w./day) for males and the no-observed-adverse-effect level (NOAEL) and NOEL in females were estimated to be 5% (3254 mg/kg b.w./day) and <1.25% (778 mg/kg b.w./day), respectively.     

Lack of chronic toxicity or carcinogenicity of dietary N-acetylglucosamine in F344 rats

Chronic toxicity and carcinogenicity of N-acetylglucosamine (GlcNAc) were examined in male and female F344 rats. GlcNAc was given in the diet at levels of 0%, 1.25%, 2.5% or 5% to groups of 10 rats of each sex for 52 weeks in the chronic toxicity study and 0%, 2.5% or 5% to groups of 50 rats of each sex for 104 weeks in the carcinogenicity study. GlcNAc exerted no toxic effects with regard to clinical signs, mortality, hematology, serum biochemistry and histopathological assessment. Slight suppression of body weight gain was observed at more than 2.5%, but this appeared to be due to slight reduction of caloric intake with the high concentration of test compound, rather than any toxicity. Thus, it was concluded that GlcNAc has neither toxic nor carcinogenic effects in F344 rats, the no observed adverse effect levels (NOAEL) estimated from the chronic toxicity study being 5% in both sexes, equivalent to 2323 and 2545 mg/kg/day in males and females, respectively.     

Two generation reproduction and teratogenicity studies of feeding quinocetone fed to Wistar rats

To investigate the reproductive toxicity and teratogenic potential of quinocetone, a growth promoting agent, Wistar rats were fed different diets containing 0, 50, 300 and 1800 mg/kg quinocetone or 300 mg/kg olaquindox. Groups of 15 males and 30 females (F₀) were fed through a 10-week prebreed period as well as during mating, gestation, parturition and lactation. At weaning, 12 males and 24 females of F₁ generation weanlings per group were selected randomly as parents for F₂ generation. Selected F₁ weanlings were exposed to the same diet and treatment as their parents. At the highest quinocetone group, body weights in F₀ and F₁ rats, fetal body weight on day 21 after birth and number of viable fetuses in F₀ and F₁ generation significantly decreased. In teratogenicity study, groups of 12 males and 24 females were fed with the same diets through a 12-week prebreed period and matting periods. Pregnant rats were subjected to cesarean section on GD 20 for teratogenic examination. At the highest quinocetone group, body weights and feed efficiency, fetal body lengths, tail lengths, litter weights and number of viable fetuses significantly decreased. The NOAEL for reproduction/development of quinocetone for rats was estimated to be 300 mg/kg diet.     

A 90-day subchronic toxicity study of nivalenol, a trichothecene mycotoxin, in F344 rats.

A subchronic toxicity study of nivalenol (NIV), a trichothecene mycotoxin, was conducted in male and female F344 rats fed diet containing 0, 6.25, 25 or 100 ppm concentration for 90 days. Decrease of body weight and loose stools were observed at 100 ppm in both sexes from the start of the experiment, and body weight reduction was also observed at 25 ppm in males from week 6. At necropsy, many organs demonstrated reduced absolute weights at 100 ppm in both sexes, mostly due to the reduction in the body growth, with reduction of relative thymus weight also being evident in females. Hematologically, decrease of the white blood cell count was found at 100 ppm in males and from 6.25 ppm in females. In addition, decreased platelet counts in both sexes, red blood cell counts in males, and the hemoglobin concentration in females were detected at 100 ppm. Histopathologically, treatment-related changes were predominantly observed in the hematopoietic and immune organs and the anterior pituitary in both sexes and female reproductive organs at 100 ppm, such as thymic atrophy, hypocellularity in the bone marrow, diffuse hypertrophy of basophilic cells with increase of castration cells in the anterior pituitary, and increase of ovarian atretic follicles. Based on the hematological data, the no-observed-adverse-effect level of NIV was determined to be less than 6.25 ppm (0.4 mg/kg body weight/day for both males and females).     

A 90-day ad libitum administration toxicity study of oligoglucosamine in F344 rats.

A 90-day ad libitum administration toxicity study of oligoglucosamine (OG) was carried out using F344 rats of both sexes. The animals were divided into four groups of 20 animals each, 10 of each sex, and fed a diet containing 0, 0.04, 0.2 or 1.0 (w/w)% OG. During the administration period, no animals of either sex died or exhibited abnormal signs in the 0.04% OG and 0.2% OG groups. In the 1% OG group, in both sexes, erythema and swelling of the snout and forelimbs and loss of fur in the forelimbs were observed. On macroscopic observation, emaciation, swelling of the snout, auricles and forelimbs and alopecia of the forelimbs were also observed in 2-3 males of the 1% OG group. It was suggested that these topical abnormalities might be due to dermal responses to OG adhering to the skin and fur, which are easily soiled with saliva during grooming. In the animals of the 1% OG group, food consumption decreased, resulting in body weight gain being suppressed. This was found concomitantly with the abnormal findings mentioned above. Thus, feeding difficulties due to the topical lesions on the snout and forelimbs were thought to affect body weight. In hematology, platelet count, lymphocyte count and differential neutrophil count increased in males of the 1% OG group. These changes might be related to the dermal inflammation. Abnormalities in urinalysis and blood chemistry, as well as a small thymus, small spleen, dark spots or areas on the glandular stomach mucosa, pale Harderian glands and small testes in histopathology, were also observed in males in the 1% OG group. Whether or not all these changes were related only to the malnutrition remains to be elucidated. From these results, OG gave rise to no adverse effects in rats up to the dose level of 0.2 (w/w)%. Thus, the no observed adverse effect level was determined to be 0.2 (w/w)% for rats of either sex (124.0mg/kg/day in males, 142.0mg/kg/day in females).     

Toxicologic evaluations of DHA-rich algal oil in rats: Developmental toxicity study and 3-month dietary toxicity study with an in utero exposure phase

DHA-rich algal oil ONC-T18, tested for subchronic, reproductive, and developmental toxicity in the rat, did not produce any significant toxicologic manifestations. Based on the absence of maternal or developmental toxicity at any dosage level, a dosage level of 2000 mg/kg/day was considered to be the no observed adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when DHA-rich algal oil was administered orally by gavage to pregnant Crl:CD(SD) rats during gestation days 6–19. In a dietary combined one-generation/90-day reproductive toxicity study in rats, the NOAEL for F0 male and female and F1 male systemic toxicity was considered to be 50,000 ppm (highest concentration administered) and 25,000 ppm for F1 female systemic toxicity (higher mean body weight, body weight gain, and food consumption). F0 reproductive performance values, estrous cycle length, gestation length, or the process of parturition, and the numbers of former implantation sites and unaccounted-for sites were unaffected by algal oil exposure. Postnatal survival and developmental parameters in the F1 generation were unaffected by algal oil exposure at all dietary concentrations. There were no neurotoxic effects noted at any algal oil exposure level. The results support the safety of DHA-rich algal oil for its proposed use in food.     

The sub-chronic oral toxicity of 1,3,5-trimethylbenzene in Sprague–Dawley rats

The systemic toxicity of a trimethylbenzene isomer and constituent of C9 aromatic solvents (1,3,5-trimethylbenzene, 135-TMB) was studied in Sprague–Dawley rats following a 90-day oral gavage exposure to 0, 50, 200 and 600 mg/kg/day. No statistically significant effects on body weight, body weight gain or food consumption were observed at study termination. Treatment-related changes in clinical chemistry parameters at the end of the 90-day dosing period were limited to small, but statistically significant, increases in phosphorus levels in high dose males and females. Liver enlargement in high dose male/female rats was considered an adaptive response as this was reversible and was not associated with histopathological lesions or increased liver enzyme markers indicative of liver damage. Kidney weight changes were limited to a small, but statistically significant, increase in relative weights in high dose males. This was not associated with histopathological lesions and thus not considered toxicologically relevant. Overall, the No-Observed-Adverse-Effect-Level (NOAEL) was the highest concentration tested (600 mg/kg/day). The results of the present study are relevant for assessing the risk of trimethylbenzenes through the oral route of exposure and provide a basis for the development of provisional screening values for trimethylbenzene isomers while avoiding the uncertainty associated with route-to-route extrapolation.     

Acute and subchronic toxicity studies of pyrroloquinoline quinone (PQQ) disodium salt (BioPQQ™) in rats

The potential use of pyrroloquinoline quinone disodium salt (BioPQQ™), as a supplemental food ingredient, was evaluated in a range of oral toxicity studies in rats including an acute study, a 14-day preliminary and a 28-day repeated-dose study, and a 13-week subchronic study. The median lethal dose of BioPQQ™ was shown to be 1000–2000 mg/kg body weight (bw) in male and 500–1000 mg/kg bw in female rats. In the 14-day study, high doses of BioPQQ™ resulted in increases in relative kidney weights with associated histopathology in female rats only, while a follow-up 28-day study in female animals resulted in increases in urinary protein and crystals. These findings were reversible, and resolved during the recovery period. In the 13-week study, a number of clinical chemistry findings and histopathological changes were noted, which were deemed to be of no toxicological significance, as the levels were within the historical control range, were not dose-dependent, occurred at a similar frequency in control groups, or only occurred in the control group. Based on these findings, a no-observed-adverse-effect level of 100 mg/kg bw/day was determined for BioPQQ™ in rats, the highest dose tested in the 13-week study.     

Safety assessment of a new multivitamin

A newly created multivitamin possesses many protective health functions. To investigate its safety when applied in medical treatment and when used as a food supplement, we studied its acute oral toxicity and 13-week oral toxicity in mice. The results showed that the oral lethal dose, 50% (LD₅₀) of the biomass of the multivitamin in mice was greater than 2492 mg/kg body weight (BW) and that poisoned mice recovered within 72 h. The no observed effect level (NOEL) of long-term consumption was more than 249.3 mg/kg BW for haematological parameters, clinical chemistry parameters, histopathological examination of organs, food consumption, BW, ratio of organ weight to BW and other physiological parameters and conditions. Therefore, we conclude that dosages of up to 249.3 mg/kg BW/day of this multivitamin do not cause chronic toxicity in animals. Administration of this multivitamin may even improve the resistance of animals to negative environmental factors and may be safe for long-term consumption to enhance the health of individuals in accordance with the prescribed dosage (1.4 ∼ 4.2 mg/kg BW/day).     

Benzocaine-induced methemoglobinemia in an acute-exposure rat model

Tricaine methanesulfonate, a sedative for temporarily immobilizing fish, has a 21-day withdrawal time. Benzocaine has been proposed as an alternative sedative because a withdrawal period may not be required. Since benzocaine is known to induce methemoglobinemia, the potential for orally administered benzocaine to induce methemoglobin was assessed in rats. Sprague-Dawley rats were given a single gavage administration of 64 mg benzocaine hydrochloride per kg bw and then euthanized at intervals up to 120 min. Plasma levels of benzocaine were relatively low at all times, whereas methemoglobin peaked at 24 min. Additional rats were orally gavaged with 0-1024 mg benzocaine hydrochloride per kg bw and euthanized after 24 min. Plasma levels of benzocaine increased from 0.01 μM at 2 mg per kg bw to 2.9 μM at 1024 mg per kg bw. Methemoglobin levels did not differ from controls at doses up to 32 mg per kg bw in females and 64 mg per kg bw in males, whereupon the value increased to ∼80% at 1024 mg per kg bw. These data were used to estimate the potential impact of benzocaine residues in fish and suggest that the consumption of fish treated with benzocaine hydrochloride will not cause methemoglobinemia in humans.