Abstract
Salcaprozate sodium (SNAC) (sodium 8-((2-hydroxybenzoyl) amino) octanoate, CAS RN 203787-91-1) is classified as an oral absorption promoter and may be a useful means for improving the absorption of certain nutrients and pharmaceutical agents. Presented herein is a subset of data from a larger study evaluating the potential effects of SNAC on the gestation, parturition, lactation, maternal behavior, and offspring development of rats. Pregnant Crl:CD BR VAF/Plus female rats (F0; n = 25) received SNAC at 1000 mg/kg/d orally (gavage) from implantation through lactation and weaning. F1 pups were exposed in utero and potentially through maternal milk; observations continued through sexual maturity. The study concluded with Caesarean sectioning of F1 dams for litter observations and fetal evaluations. No deaths, abortions, premature deliveries, or gross lesions occurred in (F0) dams. Excess salivation, red perivaginal substance, and slight reductions in body weights, body weight gains, and/or feed intake were noted in late gestation/early lactation. SNAC was associated with a prolonged gestation period, leading to a greater number of dams with stillborn pups, higher number of stillborn pups, and reduced live litter size. Offspring body weights/gains, feed consumption, age of sexual maturation, mating, fertility, behavioral parameters, and organ weights at necropsy were unaffected by SNAC. No gross external changes were observed in F1 or F2 pups. In summary, SNAC administered orally at 1000 mg/kg/d to pregnant rats from gestation to weaning resulted in a slight decrease in maternal body weights (−3.8%) and prolonged gestation, along with an increase in stillbirths, but had no effects on growth and development in surviving offspring.
Salcaprozate sodium (SNAC) (sodium 8-((2-hydroxybenzoyl)amino) octanoate, CAS RN 203787-91-1) is classified as an oral absorption promoter. Although several clinical investigations have explored the potential therapeutic applications of SNAC as a delivery agent for oral forms of heparin and insulin, 1–6 limited information about the nonclinical safety of SNAC itself was found in the published scientific literature. In a published review article on the development of an oral heparin formulation, 7 the results of SNAC toxicological studies are given, but the item provides no details regarding their conduct.
The present report summarizes the findings of a study 8 that examined the effects of SNAC on the gestation, parturition, lactation, maternal behavior, and offspring development of rats. In that study, SNAC was administered to pregnant rats at 0, 500, 750, and 1000 mg/kg/d with 5000 USP units of heparin sodium/d. The study, sponsored by Emisphere Technologies, Inc (Cedar Knolls, New Jersey), involved extensive evaluations and conformed to International Conference on Harmonization (ICH) Harmonized Tripartite Guideline stages C (implantation) through F (lactation and weaning) of the reproductive process. 9 To detect any potential delayed adverse effects resulting from prenatal and perinatal SNAC exposure, observations were made until F1 generation rats reached sexual maturity and F1 females became pregnant. Only the findings from groups treated with SNAC alone or the vehicle (deionized water) are included in the present report because the presence of heparin in other groups might be confounding.
Materials and Methods
Study Design
The study was based on the requirements of the US Food and Drug Administration (FDA) Guideline on Detection of Toxicity to Reproduction for Medicinal Products 10 and was in compliance with good laboratory practices regulations (GLPs) of the US FDA (21 CFR Part 58); the Japanese Ministry of Agriculture, Forestry and Fisheries (Japanese MAFF, 1997); and the European Economic Community (ECC) (1989). Animal housing conditions were in compliance with the Guide for the Care and Use of Laboratory Animals. 11
This study lasted approximately 5 months.
Test Substance, Dose Selection, and Dosing
SNAC (lot no. E414-47S [EM 0074], 93.2% purity) was supplied by Emisphere Technologies, Inc and stored at room temperature with desiccant. Information concerning the identity, composition, strength, and activity of SNAC was established before initiation of this study. Stability of the prepared formulation at concentrations bracketing the range to be used in this study was established during the course of a dosage range study in rats (Argus Research Laboratories, Inc, Horsham, Pennsylvania). Dosing solutions of SNAC were considered to be within an acceptable range if they were between 90.0% and 110.0% of the stated concentration.
An oral dose of 1000 mg/kg body weight/d is used in the Organization for Economic Cooperation and Development (OECD) and European Union (EU) guidelines as a default maximum dose in limit tests for studies on reproductive toxicity. 12
Animals received the vehicle (deionized water) or SNAC at 1000 mg per kg of body weight per day (1000 mg/kg/d) by oral gavage. The dosage was adjusted daily, based on the most recently obtained rat body weights. Dosing solutions were prepared at least once weekly, and SNAC was considered 100% active for the purpose of dose calculations. Prepared formulations were stored refrigerated (approximately 35–46°F). F0 generation females received the vehicle or SNAC once daily from day 7 of gestation (DG 7) through day 20 of lactation (DL 20). F1 pups weaned on DL 21 were selected for continued evaluation. F1 generation animals did not receive SNAC directly but potentially would have been exposed to SNAC in utero and/or via maternal milk during lactation.
Animals
F0 Generation
Male and female Crl:CD BR VAF/Plus (Sprague-Dawley) rats were obtained from Charles River Laboratories, Inc (Portage, Michigan); male rats were used only for the purposes of breeding and were not considered part of the test system. Upon arrival, animals were randomly assigned to individual housing units. Healthy, mated female rats were divided into 25/group, based on a body weight stratification design. Body weights ranged from 211 to 233 g at study start.
F1 Generation
F1 generation rats were born at the testing facility. Litters were not culled during the lactation period. The pups were not individually identified during lactation; all preweaning parameters were evaluated in terms of the litter.
At weaning (DL 21), a table of random units was used to select 25 weanling rats per sex per group for continued evaluation. At least 1 male pup and 1 female pup per litter, when possible, were selected.
Housing
Study rooms were maintained at a positive airflow relative to a hallway, a minimum of 10 changes per hour of HEPA-filtered 100% fresh air, and a 12-hour light/dark cycle. Room temperature and humidity were recorded constantly throughout the study and were targeted at 64 to 79°F and 30% to 70%, respectively.
F0 generation rats were housed individually in stainless steel, wire-bottomed cages, except during the cohabitation and postpartum periods. During cohabitation, each pair (1 male, 1 female) was housed in the male’s cage. Beginning no later than DG 20, F0 generation female rats were individually housed in nesting boxes. Each dam and delivered litter was housed in a common nesting box until weaning of the offspring.
After weaning, F1 generation rats were housed individually before cohabitation, in pairs (1 male rat, 1 female) during cohabitation and individually thereafter. The same type of caging was used as for the F0 generation.
Throughout the study, animals received Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) and water (reverse osmosis processed) ad libitum. Chlorine was added to the processed water as a bacteriostat. There were no contaminants present in the water, feed, or bedding at levels that would be expected to affect the outcome of the study.
Mating Procedures
At approximately 10 weeks of age, each healthy virgin F0 female rat was placed into cohabitation with a breeder male rat. Female rats with evidence of a copulatory plug or spermatozoa in a vaginal smear were considered pregnant (DG 0) and were moved to individual housing.
At approximately 12 weeks of age, F1 generation rats were mated for 3 weeks, based on a table of random units (sibling matings excluded). As with the F0 generation, F1 females with evidence of having mated were moved to individual housing. F1 females that did not mate within the first 2 weeks were assigned alternate males.
Clinical Observations
Animals were examined at least once per day for viability, clinical signs, and general health. F0 generation females were examined immediately before and 30 to 90 minutes after dosing for abortions, premature deliveries, and deaths. Reproductive and litter parameters measured in F0 generation females included clinical signs during parturition, duration of gestation (DG 0 to the day the first pup was delivered), length of parturition (time of delivery of last pup minus the time of delivery of the first pup divided by n – 1 pup in each litter, measured during the light cycle), litter sizes (defined as all pups delivered), and pup viability. Maternal behavior on lactation days (DL) 1, 4, 7, 14, and 21 was recorded, as were pup body weights. During the 21-day lactation (postpartum) period, F1 pups were counted daily and examined twice daily for viability. Pups found dead were removed from the nesting box and underwent necropsy, unless precluded by autolysis or cannibalization. For pups that appeared stillborn or died before the initial litter viability examination, a determination of the vital status at birth was made by removing and immersing the lungs in water. Pups with lungs that sank were considered stillborn, whereas pups with lungs that floated were considered to have been liveborn and died shortly after birth.
F1 females were monitored for sexual maturation (ie, vaginal patency) on postpartum days 28 to 36, and F1 males were monitored on postpartum days 39 to 51 for preputial separation.
Body Weight and Food Consumption
The body weights of F0 generation dams were recorded weekly during the acclimation period, on DG 0, daily during the dosing period, and at sacrifice. Feed consumption values were recorded on DGs 0, 7, 10, 12, 15, 18, 20, and 25 (if necessary) and DLs 1, 4, 7, 10, and 14. Because pups begin to consume maternal feed by DL 14, feed consumption values were not recorded after DL 14.
F1 generation body weights and feed consumption values were recorded weekly during the postweaning period, on DGs 0, 7, 10, 14, 17, and 20 (females) and at sacrifice (males). Feed consumption values were not recorded during the cohabitation period.
Behavioral Testing
Passive Avoidance
Beginning on days 23 to 25 after birth, F1 generation rats (1/sex/litter) underwent a passive avoidance test for learning and short- and long-term memory retention.
The apparatus consisted of a 2-compartment chamber with hinged Plexiglas lids. One compartment was fitted with a bright light and Plexiglas floor, whereas the other was fitted with a grid floor to which a brief (1-second) pulse of mild electric current (1 mA) could be delivered. The 2 compartments were separated by a sliding door. On each test trial, the rat was placed into the “bright” compartment; the sliding door was opened, and the light was turned on. The rat was allowed to explore the apparatus until it entered the “dark” compartment. The sliding door was then immediately closed; the light was turned off, and the brief pulse of current was delivered to the grid floor. The rat was then removed from the apparatus and placed into a holding cage for 30 seconds before the start of the next trial. Trials were repeated until the rat remained in the “bright” compartment for 60 seconds on 2 consecutive trials (the criterion for learning) or until 15 trials had been completed. The latency to enter the dark compartment or the maximum 60-second interval was recorded for each trial.
Each rat was tested twice. The test sessions were separated by a 1-week interval, and the criterion was the same for both days of testing.
M Maze
Beginning on approximately day 70 after birth, F1 generation rats (1/sex/litter) were tested in a water-filled M maze for overt coordination, swimming ability, learning, and memory. Each rat was tested in a watertight, 16-gauge stainless steel, modified M maze. The maze was filled with water to a depth of approximately 9 inches, and the water was monitored for temperature (range, 21 ± 1°C).
On each test trial, the rat was placed into the starting position (base of the M maze stem farthest from the 2 arms) and required to swim to 1 of the 2 goals of the M maze to be removed from the water. On the first trial, the rat was required to enter both arms of the maze before being removed from the water. The initial arm chosen on trial 1 was designated the incorrect goal during the remaining trials. Rats that failed to choose the correct goal within 60 seconds in any given trial were guided to the correct goal and were then removed from the water. Each trial was separated by an intertrial period (15 seconds). Each rat was required to reach a criterion of 5 consecutive, errorless trials to terminate the session. The maximum number of trials in any session was 15. Latency (measured in seconds) to choose the correct goal or the maximum 60-second interval was recorded for each trial, as was the number of errors (incorrect turns in the maze) during each trial.
Each rat was tested twice. The test sessions were separated by a 1-week interval, and the correct goal and the criterion were the same for both test sessions.
Necropsy Observations and Organ Weights
Female rats that were found dead underwent examination to determine the cause of death, pregnancy status, and uterine contents. Full necropsies with examination of the thoracic, abdominal, and pelvic viscera were performed on all animals found dead or sacrificed. Select tissues, including any gross lesions found, were preserved in an appropriate fixative such as neutral buffered 10% formalin (NBF) or Bouin’s solution.
F0 Generation Rats and F1 Generation Litters
F0 dams were sacrificed by carbon dioxide asphyxiation at the end of the 21-day postpartum period. The number and distribution of implantation sites were recorded. Rats that did not deliver a litter were sacrificed on DG 25 and examined for gross lesions. Uteri were stained with 10% ammonium sulfide to confirm the absence of implantation sites. 13 Dams with no surviving pups were sacrificed and necropsied after the last pup was found dead, missing, or presumed cannibalized.
F1 pups culled on DL 21 were sacrificed and examined for gross lesions; pups with gross lesions were preserved in NBF. Necropsy of these pups included a single cross section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. Pups found dead on DL 1 were evaluated for vital status at birth as previously described. Pups with gross lesions were preserved in Bouin’s solution for possible future evaluation. Pups identified to have gross lesions on DLs 2 to 4 were also preserved in Bouin’s solution for possible future evaluation. Gross lesions identified at necropsy of pups that died on DLs 5 to 21 were preserved in NBF.
F1 Generation Rats and F2 Generation Litters
F1 generation males were sacrificed by carbon dioxide asphyxiation after completion of the 21-day cohabitation period and successful pregnancy evaluation at Caesarean sectioning of the respective paired F1 female. Necropsy and tissue preservation were performed as described previously. The testes and epididymides were excised, weighed, and fixed.
F1 generation females without a confirmed mating date were sacrificed on an estimated DG 20. All other F1 females were sacrificed by carbon dioxide asphyxiation on DG 20. Caesarean sectioning was performed, and the number and distribution of corpora lutea, implantation sites, live and dead fetuses (a live fetus was one that responded to stimuli; a dead fetus was defined as a term fetus that did not respond to stimuli and was not markedly autolyzed; dead fetuses demonstrating marked to extreme autolysis were considered to be late resorptions), and early and late resorptions (a conceptus was defined as a late resorption if it was grossly evident that organogenesis had occurred; if this was not the case, the conceptus was defined as an early resorption) were measured. Uteri of apparently nonpregnant rats were stained with 10% ammonium sulfide to confirm the absence of implantation sites. All placentas were examined for size, color, and shape.
Live F2 generation fetuses were sacrificed by an intraperitoneal injection of euthanasia solution (Beuthanasia-D Special, Schering-Plough Animal Health, Boxmeer, The Netherlands). Each fetus was weighed and examined for sex and gross external alterations. Fetuses were tagged with identification for possible future evaluation. Approximately half of the fetuses in each litter were retained in Bouin’s solution; the remaining fetuses were retained in alcohol.
Statistical Analyses
Statistical analyses were performed with a Unisys 5000/95 computer.
For all statistical analyses, a P value less than or equal to .05 or .001 was considered significant.
Clinical observation incidence data, as well as other proportion data, were analyzed as contingency tables using the variance test for homogeneity of the binomial distribution. 14
Body weights, body weight changes, feed consumption data, durations of gestation and delivery, litter averages for pup and fetal body weights and percent male fetuses or pups, mortality, cumulative survival, number of trials, trial latencies and errors per trial in passive avoidance and water maze testing, and parameters involving continuous data were analyzed using Bartlett’s test of homogeneity of variances 15 and the analysis of variance 14 when appropriate (ie, Bartlett’s test was not significant, P > .05). If analysis of variance was significant (P ≤ .05), Dunnett’s test 16 was used to identify the statistical significance between groups. If the analysis of variance was not appropriate (ie, Bartlett’s test was significant, P ≤ .05), the Kruskal-Wallis test 15 was used when 75% or fewer ties were present; when more than 75% ties were present, Fisher’s exact test 17 was used.
In cases where the Kruskal-Wallis test was statistically significant (P ≤ .05), Dunn’s method of multiple comparisons 18 was used to identify the statistical significance between groups.
All other natural delivery data involving discrete data were evaluated using the Kruskal-Wallis test procedures 15 previously described.
Results
Clinical signs noted in SNAC-treated (F0) dams during late gestation and early lactation consisted of excess salivation (in 13/24 dams vs 1/24 dams in control) and red perivaginal substance (in 9/24 dams vs 1/24 dams in control). As shown in Table 1, dam body weights were slightly lower than control throughout gestation, reaching statistical significance (P ≤ .01) only on gestation day (DG) 20. However, maternal body weights were only a maximum of −3.8% of mean control values on DG 20. Body weights were also slightly lower during lactation, but the differences were not statistically significant (Table 2). SNAC-treated dams had significantly (P ≤ .01) smaller body weight gains (days 0–20) than vehicle control dams but only during gestation. Some minor reductions in feed consumption were also observed during gestation (−5.8% of control values from DG 7–20) but were not overall statistically significant. Dosages of SNAC as high as 1000mg/kg/d did not affect maternal feed consumption values during lactation (data not shown).
Table 3 depicts F0 fertility and natural delivery data. The duration of gestation was slightly but significantly longer among dams receiving SNAC (23.4 days vs 22.7 days with dosing vehicle). The number of dams with stillborn pups (11 vs 5) and the incidence of stillbirths (24 vs 9) were significantly higher in the SNAC-treated group than in the group receiving the dosing vehicle. This resulted in a reduced albeit not statistically significant live litter size (13.4 vs 14.1 pups; Table 4).
Exposure to SNAC in utero and potentially during lactation resulted in significant decreases in pup survival and litter size among the F1 generation preweanlings compared with the control group. After weaning on lactation day 21, there were no statistically significant effects on offspring body weights, body weight gains, or feed consumption (data not shown).
Tables 5, 6, and 7 show the sexual maturation, mating, and fertility of the F1 generation male and female rats. Dosages as high as 1000 mg/kg/d of SNAC administered to the F0 generation dams did not affect the day of vaginal patency or the day of preputial separation in the F1 generation rats. There were also no significant differences in mating and fertility parameters. All mating and fertility parameters (days in cohabitation, rats that mated, fertility index, rats with confirmed mating dates during the first and second weeks of cohabitation, and rats pregnant per rats in cohabitation) for both sexes were unaffected by dosages of SNAC as high as 1000 mg/kg/d.
There were no biologically important differences in the values for learning, short-term retention, long-term retention, or response inhibition in the F1 generation male and female rats, as evaluated by performance in a passive avoidance paradigm. No statistically significant differences occurred in the number of trial latencies or number of rats that failed to learn.
No biologically important or dosage-dependent differences occurred in water maze performance of the F1 generation male and female rats regarding learning, short-term retention, long-term retention, or response inhibition.
No necropsy observations that occurred in the F1 generation male rats were considered related to treatment because the incidences were not dosage dependent and/or the observation commonly occurs in this strain of rat. The only necropsy observation in the F1 generation female rats was multiple raised tan areas on both kidneys in one 1000-mg/kg/d SNAC group rat. This was a single event unrelated to administration of the test article to the F0 generation dam.
Terminal body weights, the weights of the epididymides and testes, and the ratios of these organs to the terminal body weight of the F1 generation male rats were unaffected by administration of SNAC to the F0 generation dams. F1 generation Caesarean sectioning observations (Table 8) were based on 24 pregnant rats in each dosage group. Total resorptions (early + late) were increased, albeit not statistically significantly, in the SNAC-exposed F1 generation dams compared with the control dams (1.4 vs 0.5). No other Caesarean sectioning or litter parameters were affected by dosages of SNAC as high as 1000 mg/kg/d. The litter averages for corpora lutea, implantations, litter sizes, live fetuses, early and late resorptions, fetal body weights, percent resorbed conceptuses, and percent live male fetuses were comparable between the 2 dosage groups and did not significantly differ (Table 9). All values were within the historical ranges of the testing facility.
One dam had a litter consisting of only resorbed conceptuses, but there were no dead fetuses in any other litter. All placentas appeared normal.
Fetal evaluations of F2 generation offspring were based on 342 and 306 live, DG 20 Caesarean-delivered fetuses in the vehicle control and SNAC-exposed groups, respectively. Each fetus was examined for gross external alterations. The results of F2 fetal gross external examinations are summarized in Table 10. SNAC exposure was not associated with adverse effects on any of the measured parameters.
Discussion
The purpose of this article is to provide development and reproductive toxicity data on SNAC, an oral absorption promoter intended for improving the absorption of certain nutrients and pharmaceutical agents from the gastrointestinal tract. SNAC was tested at multiple dose levels alone and in combination with heparin. However, because the presence of heparin might be confounding to an evaluation of the safety of SNAC itself, only data from the vehicle control and 1000-mg/kg/d SNAC-alone groups are presented.
Oral administration of 1000 mg/kg/d SNAC to pregnant female rats resulted in excess salivation, red perivaginal substance, and slight reductions in food consumption and body weights during late gestation and early lactation. The slight but statistically significant differences in maternal weights between the control and SNAC-exposed groups were seen only on DG 20 (363.8 vs 349.8 g, respectively) and may have been due to the difference in litter size (14.1 vs 13.4 pups delivered, respectively) because maternal body weights did not differ significantly on lactation day 1 (265.1 vs 257.5, respectively).
In this study, SNAC was associated with a prolonged gestation period, a greater number of dams with stillborn pups, a higher number of stillborn pups, and reduced live litter size. Prolonged gestation is often seen coupled with these adverse reproductive effects. It has been produced in animal studies and may be seen in human pregnancies. 19–23 The current dose is equivalent to the default maximum dose in limit tests for studies on reproductive toxicity used in OECD and EU guidelines.
There were no apparent effects on the growth and development of surviving offspring, including adverse clinical observations of body weights, body weight gains, feed consumption, age at sexual maturation, mating, fertility, learning, memory and behavioral parameters, and male reproductive organ weights. No gross external changes from SNAC exposure were observed in F1 or F2 generation offspring. Smaller litter sizes were noted in the F1 and F2 generation pregnancies, but all were within the historical control of the testing facility.
In summary, oral administration of SNAC at 1000 mg/kg/d to pregnant rats from DG 7 through lactation resulted in signs of maternal toxicity but had no effects on the growth and development of surviving offspring.
Footnotes
Tables
Acknowledgements
The authors acknowledge the assistance of the staff of Cantox Health Sciences International in the preparation of this manuscript.