Charles J. Kensler

Evaluation of the teratogenic potential of fresh-brewed coffee and caffeine in the rat☆☆☆

Dilutions of fresh-brewed coffee, at 12.5, 25, and 50% resulting in caffeine intakes of approximately 9, 19, and 38 mg/kg/day, respectively, were consumed by female rats as their sole beverage for 5 weeks prior to mating, throughout gestation and, in representative animals, until Day 27 after parturition. Other rats received a daily dose of 30 mg/kg of caffeine or 125 mg/kg of aspirin (the positive control) by intubation, or 30 mg/kg of caffeine in the water. None of these regimes interfered with the normal behavior, growth, patterns of eating or drinking, or reproductive performance. In fetuses from aspirin-treated rats the incidence of bowed fibula and shortened humerus was 4.3 and 2.7%, respectively, and in the caffeine-intubated group the incidence of absence of the supraoccipital bone was 5.8%; none of these abnormalities was observed in H2O controls. No dose-related teratogenic effects were observed in the coffee-treated groups or the caffeine-in-water group. There was an apparent slight delay in ossification in fetuses of all treated groups, some kidney-pelvis underdevelopment in the 25 and 50% coffee, caffeine-in-water, and aspirin groups, and slightly lower organ weight in the 50% coffee and aspirin groups. Cleft palate, which could not be detected either by an increased mortality rate among F1 pups at Day 38 or grossly in those permitted to mature, was more common in fetuses of coffee-consuming groups and the aspirin group than in the water controls but in the coffee groups the incidence was inversely related to coffee and caffeine dosage. Teratogenic differences between the groups given caffeine by gavage or via the drinking water were not detected but a lower number of pups/litter was recorded for the latter group and lower liver weights and lung weights were observed in the gavage and drinking water groups, respectively. However, both groups differed from the H2O control group in the incidence of cryptorchism (generally unilateral with the testes partially descended) and focally irregular (presumably delayed) ossification of ribs and vertebrae. Mating was slightly delayed in the F1 offspring of all “treated” groups except the 12.5% coffee group. This did not appear to be solely related to the test materials since a similar finding was observed in their parents before any treatment. However, the data collected during the current study are insulfficient to determine the etiology or the significance, if any, of this observation.

Exposure of four generations of mice to caffeine in drinking water

A population of CD-1 mice has been bred for four generations with continuous ingestion of caffeine. Three caffeine levels (and a control) were used with intakes averaging 4–5, 12–18 and 25–39 mg/kg/day (25–39 mg/kg/day is equivalent to 19–30 cups of coffee in man). At each level 20 pairs of animals were bred in each generation, the F1 matings being divided between brother-sister matings and out-crosses. F2s and F3s were out-crossed. There were no consistent, dose-related effects of caffeine on fertility, age at sexual maturity, mean litter size, weight of offspring at weaning, sex ratio or fetal abnormalities. A few pairs at the highest caffeine level failed to produce litters, but only in the F1 brother-sister and in the F3 matings. Litters in the caffeine-treated series showed a somewhat higher incidence of underweight animals in the F2 and the F3 generations, but not in a dose-related way. A difference in sex ratios (males slightly predominating) in the caffeine-treated groups compared to the control groups is related to an apparent deficit of male births in the control groups rather than to an effect of caffeine. In this study 3824 caffeine-exposed offspring (352 litters) were examined at birth for birth defects and 1365 of these (147 litters) were reexamined at weaning age. The only defect observed was in one litter (of F2 offspring from out-cross parents receiving the middle dose of caffeine) with a digital abnormality. This low frequency of abnormal offspring is consistent with control group experience. Further litters from the same parental pair continued on caffeine showed no abnormalities. These studies in the mouse, thus support the safety of caffeine in relatively high doses during reproduction.

Mucus production and ciliary transport activity. In vivo studies using the chicken.

A new technique for studying in vivo mucus transport and mucus production has been developed using the chicken. This technique is based on the observation that the trachea is easily exteriorized (into the chicken’s mouth) and experiments conducted on an in situ tissue. This procedure may be repeated without any apparent harm to the chicken. Exposing animate to smoke from regular or carbon filter cigarettes once a day for 32 days does not appear to produce permanent impairment in ciliary function. Mucus output was markedly increased during the period of smoke exposure. These in vivo studies indicate that gas-phase components (particularly hydrogen cyanide [HCN] and acrolein) are of major importance in the inhibition of ciliary transport and that appropriate filters can significantly reduce this inhibitory effect, but not the increase in mucus production observed during smoking.

Restoration of tracheal mucosa and ciliary particle transport activity after mechanical denudation in the chicken

Abstract A new technique was developed to study the regeneration of respiratory epithelium in situ. The restoration of the epithelium of mechanically denuded chicken tracheas was characterized by measurements of ciliary particle transport activity and by counting ciliated cells, mucous cells and mucous glands. Results show that although more than 90% of the epithelial cells were destroyed as a result of the mechanical denudation process, most of the restoration of ciliary function and regeneration of the epithelium in the chicken occurred by 14 days. Complete restoration of the epithelium to the predenudation condition occurred before 29 days. This method, we believe, will be of value in studying the effects of potentially toxic or beneficial agents in the form of dusts, gases, aerosols and drugs on replacement of respiratory epithelium. In addition, the impairment of pulmonary clearance, together with the presence of large numbers of cells in an active phase of cellular division, may provide a milieu for the study of carcinogenesis induced or influenced by airborne chemical agents.

Cigarette Smoke: Charcoal Filters Reduce Components That Inhibit Growth of Cultured Human Cells

Water-soluble components of total cigarette smoke inhibit cell growth and protein synthesis by the KB line of human cells. The cytotoxic components were in both the gas phase and the particulate phase of smoke. Conventional filters of cellulose acetate reduced cytotoxicity of the particulate phase in proportion to the weight of particles trapped, that is, they did not alter the specific activity of the particulate phase. Appropriately designed filters containing activated charcoal granules selectively reduced cytotoxic components in cigarette smoke which would have appeared in both phases, although the reduction, as anticipated, occurred to a greater extent in the gas phase.

Current status of human safety and environmental aspects of fluorescent whitening agents used in detergents in the United States

Fluorescent whitening agents (FWAs) are incorporated into household detergents at approximately 0.5% dry weight in order to maintain fabric whiteness during laundering. Seven different FWAs are currently used in American detergents produced by the major manufacturers. In 1974, 11 million 1b of these seven compounds were used in detergent manufacture and had the potential for appearing in laundry effluent. As such, FWAs can be identified routinely in sewage. The concentration in various natural waters and in fish caught in polluted waters is commonly on the order of 10 ppb. Only trace quantities have been found in drinking waters. All seven compounds show a low fish toxicity, low biodegradability, and a low potential for bioconcentration. Considerable information is available on the mammalian toxicity of FWAs. Tests of oral toxicity including mutagenicity, teratogenicity, and carcinogenicity which have been completed using high daily doses do not indicate any need for concern. Studies of effects resulting ...

Unusual metabolism of caffeine in the squirrel monkey

Abstract The chronic toxicity of caffeine observed with the squirrel monkey appears to be related to the long plasma half-life of caffeine in this species. A half-life of 11 hr was found following the administration of 5 mg/kg compared to 2.4 hr in the rhesus monkey (5 hr or less have previously been reported for the mouse, dog and man). The methylxanthines found in the tissues and urine of the squirrel monkey following caffeine administration were the same as those reported for other species. No difference in the metabolism of caffeine by a squirrel monkey showing a toxic response to 25 mg/kg/day and a monkey tolerating this dose could be determined. The squirrel monkey appears to have a unique deficit in its ability to catabolize caffeine to metabolites which can be effectively excreted.

Genetic tests in mice of caffeine alone and in combination with mutagens

The possible mutagenicity of caffeine has been studied in mice by the dominant-lethal method, in three experiments. (I) Male mice were given caffeine in drinking water for 8 weeks at 3.6, 13.4, 49, and 122 mg/kg/day (comparable to human consumption of 2.8–95 cups of coffee per day). Subsequent mating of each of six males from each group to five females per week for 8 weeks showed no significant increase in dominantlethal mutations (embryonic deaths) whether expressed as early deaths per pregnant female or as mutation index. Although males consuming the two higher levels of caffeine produced fewer pregnancies, litter sizes of females giving birth were not reduced. (II) Single ip injections of caffeine (15 mg/kg) were given to groups of male mice prior to, subsequent to, and immediately at the time of receiving X-rays (100 r). Each of five males from each group was mated to five females per week for 7 weeks. Embryonic deaths did not show any enhancing effect of caffeine on the mutagenicity produced by the irradiation. (III) Three groups of male mice ingested caffeine in water for 16 weeks at levels of 0, 4 and 13 mg/kg/day. Subgroups of five from each group were given either: no further treatment, a single dose of triethylene melamine at 0.2 mg/kg, or 100 r of X-ray, and mated for 7 weeks as above. Fertility and litter size were not affected by the caffeine pretreatment, nor did it modify the induction of dominant-lethal mutations by triethylene melamine or X-rays. Litter sizes showed no significant preimplantation losses in any experiment. Thus, under the conditions described herein and at the doses employed (higher than human exposure), there was no evidence for the mutagenicity of caffeine or the inhibition of DNA repair mechanisms in these mammalian systems.

A new system for quantitatively exposing laboratory animals by direct inhalation: delivery of cigarette smoke.

A system for quantitatively introducing cigarette smoke into the respiratory tract of spontaneously breathing laboratory animals has been developed. At the present time, its use is restricted to animals either with tracheotomies or capable of mouth breathing. The volume, frequency, and duration of cigarette puffing is programmed and not determined by the animal’s respiratory activity. Smoke is loaded into a holding tube and inhaled during normal inhalation. To prevent anoxia, animals are allowed fresh air between puffs. The system can be automated for simultaneously exposing four to eight animals. Except for modest loss of particulate material, the composition of smoke does not appear to be altered materially.

Use of the Nonimmersed in Vitro Chicken Tracheal Preparation for the Study of Ciliary Transport Activity Cigarette Smoke and Related Components

A bioassay has been developed and used to quantitatively characterize the effects of cigarette smoke on ciliary function. Diluting smoke from 50% (40 ml) to 3% (2.5 ml) increases the number of four-second exposures required for 90% to 100% inhibition from 8 to 80 (or more) puffs. A high degree of correlation and notable similarity in slope of dose-response curves was found for regular cigarettes, cellulose acetate filter cigarettes, and cigarettes with filters of cellulose acetate with activated carbon. A 50% reduction (dose required to inhibit particle transport rate to 50% of control rate [ED50]) in transport activity was observed with 8 ml (one to ten dilution) of nonfiltered cigarette smoke after eight exposures of four seconds each, delivered at one-minute intervals. The ED50 for hydrogen cyanide under similar conditions was attained at 10μg per puff or at concentrations lower than those reported in smoke. Recovery from HCN was more rapid than after cigarette smoke. Smoke from cigarettes with filters...