Nicotine contributes nearly all the alkaloid content of tobacco plants. Historically, human exposures were associated with smoking, chewing, or sniffing various forms of tobacco, and abundant literature details the adverse effects of chronic exposures to these tobacco products. Less attention has been given, however, to understanding the specific role nicotine plays in these adverse outcomes. Although recognized to possess potent pharmacological and addictive properties, nicotine is now delivered in electronic cigarettes and is a common ingredient in over-the-counter products designed to reduce or stop smoking, including gum and dermal patches.
Nicotine bitartrate dihydrate (NBD) salt was selected for rodent toxicity studies because it is used in nicotine-containing consumer products and is more stable than pure freebase (-)-nicotine. Additionally, no significant differences were observed between the salt and freebase forms in the evaluation of toxicokinetic parameters following oral exposure of rats. Following palatability testing, Sprague Dawley rat dams were exposed to NBD in drinking water ad libitum from gestation day (GD) 6 through lactation day (LD) 21. Upon weaning, pups were exposed to the same drinking water concentrations as their respective dams for 4 weeks or 3 months. Young adult male and female Swiss mice also were exposed to NBD in drinking water ad libitum for 4 weeks or 3 months.
Two-week Palatability Studies in Rats and Mice: Initial studies were conducted to evaluate the palatability of NBD to Sprague Dawley rats and Swiss mice exposed to 0, 6.25, 12.5, 25, 50, or 100 mg nicotine/L (mg/L) in drinking water for 14 days. In rats, the highest two exposure concentrations (50 and 100 mg/L) were considered unpalatable due to severely reduced body weight and water consumption for both sexes. In mice, water consumption was significantly decreased at 100 mg/L for both sexes, although it remained within animal welfare guidelines and was therefore selected as the highest exposure concentration for mice in the 4-week study.
Perinatal and Four-week Dose Range-finding Study in Rats: Beginning on GD 6, groups of F0 time-mated female rats were exposed to NBD in drinking water throughout gestation and lactation at exposure concentrations of 0, 1.56, 3.12, 6.25, 12.5, or 25 mg/L. Groups of 10 F1 rats per sex continued on study after weaning and were provided drinking water with the same respective NBD concentrations as their dams for 4 additional weeks. Although feed and water consumption by dams was reduced at the higher NBD concentrations, there were no significant effects of NBD exposure on pregnancy status, gestation, or parturition. However, during late lactation, a cluster of pup mortality was observed in exposed groups at NBD concentrations ≥3.12 mg/L. At necropsy, pups terminated early often presented with gross lesions in the intestine, which correlated histopathologically with moderate diffuse inflammation. In total, 42 pups died in the exposed groups at NBD concentrations ≥3.12 mg/L, whereas no mortality was observed in the control groups and in the group exposed to 1.56 mg/L during the same period (postnatal days [PNDs] 19–24). Therefore, exposure was suspended in all exposed groups approximately 4 days before weaning, after which the remaining pups survived to study termination. After resumption of initial exposures following weaning, no mortality occurred during the remainder of the study.
During the 4-week exposure period, body weights of male and female F1 rats in the 12.5 and 25 mg/L groups were decreased by >10% relative to the control groups. Water consumption was less than that of control rats for most exposed groups, with the 25 mg/L groups consuming on average <60% (females) and <70% (males) as much water as respective control groups. Although there were some organ weight changes, no histopathological correlation with those changes was observed.
While not significant, mild peritubular/focal kidney inflammation was observed in exposed males and females, as was minimal intestine inflammation in females and degenerative lesions of the testes in males in the 25 mg/L groups compared to control rats, the only groups other than early-death pups examined for histopathology.
Perinatal and Three-month Study in Rats: Beginning on GD 6, groups of F0 dams were exposed to NBD in drinking water throughout gestation at exposure concentrations of 0, 1.56, 3.12, 6.25, 12.5, or 25 mg/L. During lactation, all exposed groups were administered 1.56 mg/L to avoid the late-lactation pup mortality observed in the perinatal and 4-week study. After weaning, groups of 10 F1 rats/sex/exposure group were continued on study and were given drinking water containing the same respective concentrations as their dams for an additional 3 months.
There were no significant effects of NBD exposures on pregnancy status, gestation, or parturition, although water consumption by F0 dams in the 25 mg/L group was below that of the control dams, and at GD 21 was 58% of that of the control group. No abnormal pup mortality occurred preweaning, and no mortality occurred postweaning. During lactation, body weights of male and female pups in most exposed groups were somewhat lower than the control pups. At the end of the 3-month study, body weights of male and female rats were again within 10% of control groups, although water consumption by the 25 mg/L groups was approximately 60% of that of the control groups.
Histopathological evaluation showed that minimal to mild inflammation in the kidney was significantly increased in males at 12.5 mg/L. While not significant, minimal inflammation of the intestine was observed in greater numbers in some exposed groups of males and females. The incidence of minimal chronic reactive mandibular lymphadenitis was significantly increased in males at 25 mg/L. A statistically significant increased trend of the total number of degenerative lesions of the testes and animal bearing lesions were observed in males. Degenerative lesions of the testes, a rare finding in this strain at this age, were observed only in the 12.5 and 25 mg/L groups, whereas no degenerative lesions were observed in the control groups and at the lowest exposures. No increases in lesions with clear toxicological significance were attributed to the NBD exposures.
Four-week Dose Range-finding Study in Mice: Groups of 10 male and 10 female mice were exposed to NBD in drinking water for 4 weeks at exposure concentrations of 0, 6.25, 12.5, 25, 50, or 100 mg/L. Additional groups of 10 male and 10 female mice were exposed to 0 or 100 mg/L in drinking water and then monitored for a 2-week recovery period after exposure. All exposure concentrations were well tolerated with no reductions in body weight or feed and water consumption. There were no significant differences in organ weights. Histopathological evaluation of mice exposed to the highest concentration (100 mg/L) showed higher incidences of minimal focal inflammation in the kidney in males and Harderian gland in females, although the incidences were not significant. Exposed female mice also had higher, nonsignificant incidences of minimal focal necrosis with inflammation in the liver compared to control mice.
Three-month Study in Mice: Groups of 15 male and 15 female mice were exposed to NBD in drinking water for 3 months at exposure concentrations of 6.25, 12.5, 25, or 50 mg/L, and groups of 30 male and 30 female mice were exposed to 0 or 100 mg/L. No mortality was observed during the 3-month study. All exposure concentrations were well tolerated, with slightly lower water consumption by the highest exposed group (100 mg/L). During histopathological evaluation, minimal inflammation was diagnosed in several organs in exposed and control mice; however, none of these lesions were considered of toxicological significance.
Summary: Under the conditions of these studies, other than reductions in feed and water consumption and resulting lower body weight gains at the higher NBD exposure concentrations, there were no clinical findings considered to be of clear toxicological significance for the NBD exposures in Sprague Dawley rats or Swiss mice. However, there was a high rate of mortality of rat pups exposed to NBD at concentrations ≥3.12 mg/L during the late-lactation phase of the perinatal and 4-week study that was unexpected and may be related to inhibition of normal cholinergic neural development by prenatal nicotine exposures, whereas no mortality was observed in the control groups and in the group exposed to 1.56 mg/L during the same period. This finding prompted a change in the design of the perinatal and 3-month study in rats to provide all groups of exposed lactating dams the same lowest NBD exposure concentration (1.56 mg/L), which effectively prevented pup mortalities during this apparently vulnerable developmental period. There were no histopathological findings considered to be of clear toxicological significance.
Synonyms: nicotine tartrate dihydrate; (S)-3-(1-methyl-2-pyrrolidinyl)pyridine (2R,3R)-2,3-dihydroxybutanedioate dihydrate
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