A study of the teratogenic potential of caffeine ingested in drinking-water.

Caffeine dissolved in drinking-water was available ad lib. to Osborne-Mendel rats at dose levels of 0, 0.007, 0.018, 0.036, 0.07, 0.10, 0.15 or 0.20% during days 0-20 of gestation. The corresponding daily caffeine intakes were 0, 10.1, 27.4, 50.7, 86.6, 115.8, 160.9 and 204.5 mg/kg body weight. Dosages of 160.9 and 204.5 mg/kg were associated with decreased implantation efficiency, increased resorptions and decreased mean numbers of viable foetuses. Numbers of runts were significantly increased after dosages of 115.8-204.5 mg/kg/day. Foetal body weight and length were decreased and oedematous foetuses were increased at dosages of 86.6-204.5 mg/kg/day. Contrary to results seen after gavage studies, caffeine available ad lib. in drinking-water produced no dose-related gross anomalies. Only two animals with missing or hypoplastic nails were produced, both in the 160.9-mg/kg group. Sternebral ossification deficiencies were increased at all dose levels except 10.1 mg/kg/day. Skeletal ossification deficiencies were increased in a dose-related manner at the four highest dose levels. Caffeine given by water bottle produced ossification deficiencies similar to those seen after intubation, but at higher dosages.

Developmental toxicity of sodium fluoride measured during multiple generations.

Sodium fluoride (NaF) has been used to fluoridate drinking water in the United States since the mid 1940s. Because of the lack of reliable studies on the multigeneration effects of the compound, NaF (0, 25, 100, 175 or 250 ppm in drinking water) was given to rats continuously during three generations. Parental (F0) generation rats were treated for 10 weeks and mated within groups. At gestation day 20, caesarean sections were performed and eight F0 females per group and their litters (F1) were observed for implant status, fetal weight and length, sex and morphological development. The remaining F0 females (29-32 per group) were allowed to litter. F1 offspring (36 of each sex per group) were mated within groups, and caesarean sections were performed at gestation day 20. The F1 females and their litters (F2) were observed for implant status, fetal weight and length, sex and morphological development. In addition, F2 fetuses were evaluated for internal (soft-tissue) and skeletal development. Decreased fluid consumption for F0 and F1 dams at 175 and 250 ppm was attributed to decreased palatability of the solution. No dose-related effects in feed consumption or mean body weight gain were observed in either F0 or F1 females. Numbers of corpora lutea, implants, viable fetuses and fetal morphological development were similar in all groups. No dose-related anomalies in internal organs were observed in F2 fetuses. Ossification of the hyoid bone of F2 fetuses was significantly decreased at 250 ppm. Because of the decreased ossification of the hyoid bone, 250 ppm is considered the effect level.

Developmental effects of serum from flaxseed-fed rats on cultured rat embryos.

Gestation day 9.5 rat embryos were cultured for 45 h in serum obtained from pregnant rats that had been fed throughout gestation with either a control diet (based on the AIN-93 formulation), a diet supplemented with flaxseed (20% or 40%, w/w), or a diet supplemented with de-fatted flaxseed ("flaxseed meal", 13 or 26%, w/w). The embryos were fixed in neutral formalin at the end of culture. Overall growth and development was assessed, and the presence of abnormalities was noted. A significant inhibition of growth (as determined by crown-rump length) relative to control was observed in embryos cultured in serum from rats fed the 20% flaxseed diet. The incidence of spontaneous heart inversions was increased significantly in the embryos cultured in serum from the 20% flaxseed and 26% flaxseed meal fed rats. The incidence of flexion defects was increased significantly in embryos cultured in serum from 20% flaxseed-fed rats. The lack of an apparent dose response in any of the statistically significant effects suggests that the observed anomalies were chance occurrences unrelated to the treatment group from which serum was obtained. It is therefore concluded that diets high in flaxseed or flaxseed meal do not result in serum factors that are directly embryotoxic to organogenesis-staged rat embryos. This finding is consistent with the findings of a parallel in vivo rat teratology study where no significant embryotoxicity attributable to flaxseed exposure was observed.

Developmental toxicity of sodium fluoride in rats.

Despite the chronic exposure of the US population to fluoridated drinking water since the 1940s, existing studies have been judged inadequate to determine any potential reproductive or developmental hazard. This study was conducted to determine the effects of sodium fluoride (NaF) on foetal development. Sperm-positive female rats were given 0, 10, 25, 100, 175 or 250 ppm NaF daily throughout gestation. They were dosed by drinking water to mimic human exposure to fluoridated water. No dose-related behavioural changes or maternal clinical signs were noted. Fluid consumption by females in the 175- and 250-ppm groups was significantly less than that of the control females. Because of this decreased fluid consumption, the daily amount of NaF ingested (0, 1.4, 3.9, 15.6, 24.7 and 25.1 mg/kg body weight) was less than expected at the two high levels. Feed consumption decreased significantly at 250 ppm, and body weights of pregnant females reflected feed consumption trends. The mean number of viable foetuses per female in all treated groups was similar to that of the control group. The significant decrease in the mean number of implants per litter in the 250-ppm group is probably linked to the lower mean number of corpora lutea in this group. The occurrence of in utero deaths was similar in the control and treated groups. Foetal growth (in terms of foetal body weight and crown-rump length) was not affected by NaF, despite the fact that the dams in the 250-ppm group ate significantly less feed and drank significantly less fluid. There was no dose-related increase in the number of external anomalies in foetuses due to NaF ingestion. At the doses given, NaF had no effect on the development of specific bones, including sternebrae. A significant increase was seen in the average number of foetuses with three or more skeletal variations in the 250-ppm group; the number of litters with foetuses with three or more skeletal variations was increased in the 250-ppm group also, but the increase was not significant. There was no dose-related effect of NaF on the incidence of soft tissue variations.

Multigenerational evaluation of sodium fluoride in rats.

Since the mid 1940s, fluoride has been added to tap water in American communities in an effort to reduce the incidence of dental caries in the population. When the levels of fluoride in drinking water were tested and set, water was the only measurable source of fluoride for most communities. Now, adults and children ingest fluoride with foods and beverages prepared with fluoridated water, and they are exposed to fluoride-containing dental products. As a result, exposure to fluoride is greater than had been anticipated. In the early 1990s, the existing reproductive studies were reviewed in several reports and were considered to be inadequate to determine potential reproductive or developmental hazards. The effects of sodium fluoride ingestion at 0, 25, 100, 175 or 250 ppm in drinking water measured in rats throughout three generations are reported here. Feed and fluid consumption, body weights and clinical signs were recorded at regular intervals. Decreased fluid consumption observed at 175 and 250 ppm was attributed to decreased palatability and did not affect reproduction. No cumulative effects were observed in the three generations. Mating, fertility and survival indices were not affected. Organ-to-body-weight ratios and organ-to-brain weight ratios were not affected. Sodium fluoride up to 250 ppm did not affect reproduction in rats.

Effects of fumonisin B1 in pregnant rats. Part 2.

The developmental toxicity of purified fumonisin B1 (FB1), a mycotoxin from the common corn fungus Fusarium moniliforme, was examined in Charles River rats. Pregnant rats were dosed orally on gestation days 3-16 at 0, 6.25, 12.5, 25 or 50 mg FB1/kg body weight/day. FB1 was not teratogenic at the doses tested. At 50 mg/kg, maternal toxicity (inappetence, emaciation, lethargy, death, resorption of entire litters) and foetal toxicity (increased number of late deaths, decreased foetal body weight, decreased crown rump length, increased incidence of hydrocephalus, increased incidence of skeletal anomalies) were seen. The foetal toxicity observed at 50 mg/kg may be related to maternal toxicity. Histopathological evaluation of tissues from dams of control and all treated groups revealed dose-related toxic changes in kidney and liver tissues. Acute toxic tubular nephrosis was seen in kidneys from all treated groups. Hepatocellular cytoplasmic alteration and individual cellular necrosis of the liver was seen in the two high-dose groups. Sphinganine (Sa) and sphingosine (So) were measured in day-17 adult and foetal tissues. Dose related increases in Sa/So ratios were seen in maternal liver, kidney, serum and brain, but there was no effect on foetal liver, kidney and brain. These data suggest that FB1 does not cross the placenta and further suggest that the observed foetal toxicity is a secondary response to maternal toxicity.

Effects of fumonisin B1 in pregnant rats.

Fumonisin B1 (FB1), the major mycotoxin from Fusarium moniliforme, has been implicated as a causative agent in several animal and human diseases. Despite animal toxicity studies and human epidemiological studies of FB1, knowledge of its reproductive effects is scarce. In this study, one of a series of proposed studies that will allow extrapolation to humans, pregnant rats were given oral doses of 0, 1.875, 3.75, 7.5 or 15 mg FB1/kg on gestation days 3 16. Caesarean sections were performed on day 17 or 20, and maternal condition, implantation efficiency, foetal viability and foetal development were measured. Dose-related decreases in overall feed consumption and body weight gain were seen, but only the feed consumption decrease at 15 mg/kg, and the decreased body weight gain at 15 mg/kg on days 0-17 were statistically significant. Foetal body weights at day 17 were similar in control and treated groups; but in day-20 foetuses, female weight and crown-rump length were significantly decreased at 15 mg/kg. FB1 was not teratogenic at the doses tested, and no dose-related effects were seen in either skeletal or soft-tissue development. In day-17 animals, maternal and foetal brain, liver and kidney tissues, and maternal serum were preserved to study the levels of sphinganine (Sa), sphingosine (So), and the Sa/So ratios. Dose-related increases were seen in Sa/So ratios in maternal livers, kidneys and serum. Sa/So ratios of maternal brains were not affected, nor were those of foetal kidneys, livers or brains.

Developmental toxicity of Orange B given to rats in drinking water.

Orange B, a pyrazolone dye used to color frankfurter and sausage casings, was given in distilled drinking water to pregnant Osborne-Mendel rats throughout gestation. Assessed on the basis of fluid consumption, the dose levels of 0, 0.05, 0.1, 0.2, and 0.4% corresponded to daily Orange B consumption of 0, 67.5, 129.6, 266.6, and 532.3 mg/kg body weight, respectively. On gestation day 20, the females were euthanized and cesarean sections were performed. Throughout gestation, the treated animals consumed less fluid than did the controls, but the decreases were not dose-related. Feed consumption and maternal weight gain were not affected. No dose-related changes were seen in maternal clinical findings, implantations, fetal viability, or fetal size (weight and length). No compound-related effects were seen in sternebral development. Ossification of the interparietal bones was reduced at some dose levels, but the decreases were considered random because of absence of dose response. No dose-related effect was seen in the incidence of skeletal variations in fetuses or in the number of litters containing fetuses with skeletal variations. Skeletal development, as measured by the average number of ossified vertebrae, was similar in all groups. Soft-tissue development was not affected by dose levels of 0.05 to 0.2%. In animals treated with 0.4% Orange B, significant increases were seen in the incidence of hydroureters (severe and moderate), in the average numbers of fetuses with at least one and at least two soft-tissue variations per litter, and in the percentage of litters containing fetuses with at least two soft-tissue variations.

High pressure liquid chromatographic determination of sodium saccharin, sodium benzoate, and caffeine in soda beverages: collaborative study.

A collaborative study for the determination of sodium saccharin, sodium benzoate, and caffeine in 3 types of soda beverage, cola, grape, and lemon-lime, has been completed using reverse phase high pressure liquid chromatography with a muC18 column and acetic acid mobile phase. Recoveries for sodium saccharin were 98.6, 98.0, and 99.1%; for sodium benzoate, 100.6, 102.6, and 100.6%; and for caffeine, 100.8, 101.4, and 101.1%, respectively. The method has been adopted as official first action.

Teratogenic potential of FD&C Red No. 3 when given by gavage.

FD&C Red No. 3 (erythrosine) is a commonly used food additive. As part of a series of studies on the potential fetal developmental effects of food colors, FD&C Red No. 3 was administered by gavage to pregnant Osborne-Mendel rats at daily dose levels of 15, 30, 100, 200, 400, or 800 mg/kg on days 0-19 of gestation. Control animals were given distilled water by gavage. On gestation day 20, the animals were euthanized and cesarean sections were performed. During the entire treatment period, feed consumption by the animals given 400 mg/kg doses was increased significantly; the increases in the animals given 30 or 800 mg/kg were of borderline significance. The only significant increase in maternal weight gain, on days 0-7 in the animals given 30 mg/kg, was considered a random occurrence. No dose-related changes were seen in maternal clinical findings, implantations, fetal viability, or fetal size (weight and length). No fetal terata were seen, and neither skeletal nor visceral development was affected. FD&C Red No. 3 was neither fetotoxic nor teratogenic at 800 mg/kg when given by gavage.

Teratological evaluation of FD&C Red no. 2 -a collaborative government-industry study. II. FDA's study.

Because of recent studies indicating possible embryolethality and teratogenicity of FD&C Red No. 2, and ad hoc committee was convened by the Food and Drug Administration to consider these questions. The committee suggested a collaborative study by three laboratories [Food and Drug Administration (FDA), Industrial Bio-Test Laboratories (IBT), and National Center for Toxicological Research (NCTR)] in which Red No. 2 was given at 200 mg/kg body weight, by gavage during days 0-19, 6-15, or 7-9 of gestation. FD&C Red No. 2 was also given at the same dose level via water bottle. Appropriate controls were utilized. FDA used Osborne-Mendel strain rats, IBT used Charles River, and NCTR used both strains. No significant increases in skeletal or visceral abnormalities were seen. No significant increase in resorptions was seen in the Osborne-Mendel strain, but the Charles River strain at IBT showed a significant increase in litters with two or more resorptions after dams had been given 200 mg/kg at 0-19 days of gestation. The NCTR study on the Charles River strain also showed an increase in the same parameter for the same dose level and in addition showed a significant increase in the percentage of resorptions per litter. It was concluded that because of the inherent variation and the absence of an increase in abnormalities or other indications of embryotoxicity, there is reason to doubt that this effect is either biologically significant or reproducible.

Teratological evaluation of FD&C red no.2-a collaborative government-industry study. I Introduction, experimental materials, and procedures.

Because of recent studies indicating possible embryolethality and teratogenicity of FD&C Red. No 2, and ad hoc committee was convened by the Food and Drug Administration to consider these questions. The committee suggested a collaborative study by three laboratories [Food and Drug Administration (FDA), Industrial Bio-Test Laboratories (IBT), and National Center for Toxicological Research (NCTR)] in which Red No. 2 was given at 200 mg/kg body weight, by gavage during days 0-19, 6-15, or 7-9 of gestation. FD*C Red No. 2 was also given at the same dose level via water bottle. Appropriate controls were utilized. FDA used Osborne-Mendel strain rats, IBT used Charles River strain also showed an increase in the same parameter for the same dose level and in addition showed a significant increase in the percentage of resorptions per litter. It was concluded that because of the inherent variation and the absence of an increase in abnormalities or other indications of embryotoxicity, there is reason to doubt that this effect is either biologically significant or reproducible.

Developmental toxicity of orange B when given to rats by gavage.

The pyrazolone dye Orange B was given by gavage to pregnant Osborne-Mendel rats throughout gestation. Dose levels of 0, 15, 30, 100, 200, 400, or 700 mg/kg body weight were given daily. On gestation day 20, the females were killed and cesarean sections were performed. Feed consumption and maternal weight gain were not affected. No dose-related changes were seen in maternal clinical findings, implantations, fetal viability, or fetal size (weight and length). No compound-related effects were seen in sternebral development. No dose-related effect was seen in the incidence of skeletal variations in fetuses or in the number of litters containing fetuses with skeletal variations. Skeletal development, as measured by the average number of ossified vertebrae, was similar in all groups. No compound-related effects were seen in soft-tissue development.

Teratological evaluation of FD&C Red no. 2-a collaborative government -industry study. V. Combined findings and discussion.

Because of recent studies indicating possible embryolethality and teratogenicity of FD&C Red No. 2, anad hoc committee was convened by the Food and Drug Administration to consider these questions. The committee suggested a collaborative study by three laboratories [Food and Drug Administration (FDA), Industrail Bio- Test Laboratories (IBT), and National Center for Toxicological Research (NCTR)] in which Red No. 2 was given at 200 mg/kg body weight, by gavage during days 0-19, 6-15, or 7-9 of gestation. FD&C Red No. 2 was also given at the same dose level via water bottle. Appropriate controls were utilized. FDA used Osborne-Mendel strain rats, IBT used Charles River, and NCTR used both strains. No significant increases in skeletal or visceral abnormalities were seen. No significant increase in resorptions was seen in the Osborne-Mendel strain, but the Charles River strain at IBT showed a significant increase in litters with two or more resorptions after dams had been given 200 mg/kg at 0-19 days of gestation. The NCTR study on the Charles River strain also showed an increase in the same parameter for the same dose level and in addition showed a significant increase in the percentage of resorptions per litter. It was concluded that because of the inherent variation and the absence of an increase in abnormalities of other indications of embryotoxicity, there is reason to doubt that this effect is either biologically significant or reproducible.

Intermediate-duration toxicity study of patulin in rats.

The toxicity of patulin was studied in two generations of Sprague-Dawley rats over a period of approximately 10 months. Patulin in 1 mM citrate buffer was administered by gavage to FO generation rats at a dose level of 0, 1.5, 7.5, or 15.0 mg/kg a body weight five times a week for 10-14 wk; females were treated seven times a week during pregnancy. High mortality and insufficient progeny in the groups given 7.5 and 15.0 mg/kg made it impossible to continue those two regimens into the second generation. The study was continued for 20-23 wk with F1A generation animals given 1.5 mg/kg and controls. The only lesion found at necropsy that could be attributed to patulin administration was gaseous distention of the gastrointestinal tract, which was probably the result of the antibiotic effect of this mycotoxin on the normal intestinal flora. A decreased weight gain in male rats of the FO generation was dose-related. An impairment in growth rates of F1A and F2A progeny of both sexes was statistically significant at the 1.5 mg/kg dose level. Fetuses taken from patulin-treated females on day 20 of pregnancy were noticeably smaller than controls fetuses and the difference was significant for F2A males. No other teratological abnormalities related to patulin dosing at the 1.5 mg/kg level were observed consistently in either F1A or F2A fetuses. Patulin did not appear to produce dominant lethal effects at dose levels up to 15.0 mg/kg when given by gavage to the males five times a week for 10 or 11 wk.