N'(Hydroxymethyl)-acrylamide (HACR) was evaluated for reproductive toxicity, neurotoxicity, and dominant lethal effects using a modified Reproductive Assessment by Continuous Breeding (RACB) Protocol in CD-1 (Swiss) mice. HACR, previously reported to have both reproductive and neural toxicity, was administered at relatively low doses over the course of the study. Our goal was to assess the feasibility of measuring neurotoxic and dominant lethal effects during the protocol and to separate the onset of these toxicities from those for reproduction. In addition. we added measures of neurotoxicity during development of the F1 generation.
During the continuous breeding phase, exposure to HACR at doses ranging from 11.1±0.57 mg/kg/day in the low-dose group males (60 ppm in water), to 113.4±15.86 mg/kg day in the high-dose group females (360 ppm in water), for 27 weeks of cohabitation, significantly affected measures of reproductive competence, including a 26% decrease in the number of live pups per litter at 360 ppm and an increased inter-birth interval at all doses. However, the mean number of litters per pair. the proportion of pups born alive, or adjusted live pup weight were not affected. In a modified dominant lethal test. HACR-treated F0 males were mated to nontreated females. Significantly more early resorptions were detected at 180 ppm (~30 to 40 mg/kg/day) and higher, and fewer pups were born at 360 ppm (~80 mg/kg/day), indicating an effect of HACR on male reproduction.
However, the crossover mating trial indicated no significant affect of 360 ppm HACR on any indices of fertility or mating and did not clearly indicate the affected sex, even though the number of pups per litter was reduced by 1.5 pups on the average when sires were HACR treated (control x control, 10.6±1.0 vs. HACR male x control female, 9.1±0.9). Clinically, no dose-related signs were noted in F0 HACR- treated mice. F0 body weight and feed consumption during the initial weeks of HACR exposure were not adversely affected by treatment.
HACR produced only slight changes in grip strength: in forelimbs of females after 12 weeks (360 ppm) and 27 weeks (180 and 360 ppm) of treatment. Trends towards reduced grip strength were also observed in the hind limbs of females and forelimbs of males after 12 weeks of dosing. At necropsy of F0 adults, HACR (360 ppm) caused a 14% decrease in testicular weight from controls, a 7% reduction in the total number of spermatids, and a 14% reduction in the concentration of epididymal spermatozoa. No other dose-related trends in reproductive or somatic organ or body weights were noted for males and none for females. Estrual cycle characteristics were normal. No treatment-related reproductive, neural, or somatic organ histopathology was observed in either sex.
In the second generation ( F1) growth phase. HACR at dose levels of greater than or equal to 60 ppm in water did not adversely affect preweaning growth or survival of the F1 generation. However, after weaning, dose-related decreases in body weight occurred in the face of increased water and feed consumption between 82±10 and 110±10 days of age in the F1 generation, more so in males than in females. Calculated exposure averaged between 15.6±0.61 and 188.8±11.3 mg/kg/day for adult F1 males and females at 60 and 360 ppm. respectively. No treatment related clinical signs were noted.
In the F1 mating trial, HACR at greater than or equal to 60 ppm had slight effects on the reproductive competence of the F1 generation. HACR caused a 18% to 55% reduction in the number of live F2 pups per litter at greater than or equal to 60 ppm and reduced dam weight by 5% to 9% at greater than or equal to 60 ppm. HACR clearly decreased forelimb and hind limb grip strength of males and hind limbs of females for Weeks 3 to 10 at most doses, with the forelimb of females being affected only at Week 10. There were significant treatment affects on male body weight, so that any decreases in reproductive organ weight when adjusted for body size were not significant. The exception was the seminal vesicles at 360 ppm, which were relatively smaller than for controls. Sperm parameters significantly affected by HACR were limited to the epididymal spermatozoa concentrations at 360 ppm (reduced by 13%): Spermatid counts (per mg of testicular weight) were non- significantly decreased (8%), and testicular histopathology was not markedly different from controls. In F1 females, terminal body weight was not reduced, and HACR had no affect on reproductive organ weight, vaginal cytology, or histopathology.
In summary, exposure to HACR in water at dose levels as high as 360 ppm (~115 mg/kg/day), for up to 27 weeks, resulted in moderate reproductive toxicity--decreased pups per litter at 360 ppm in F0 mice and at 60, 180, and 360 ppm in F1 mice, longer inter-litter interval in F0 females at 60, 180, and 360 ppm, and increased postimplantation loss (dominant lethal effect at 180 and 360 ppm) for untreated females mated to F0 males, in the absence of any clear-cut systemic toxicity in F0 mice. F1 mice had lowered body weights and increased liver and kidney/adrenal weight at all doses. Testicular toxicity was present in F0 animals at 360 ppm as indicated by decreased weight. Histopathology was not evident. F1 animals were more susceptible than F0 animals to the neuromuscular effects of HACR at all doses: HACR consistently decreased grip strength in both limbs of both sexes in F1 animals during post-weaning development. as opposed to sporadic effects in the hind limbs and forelimbs of F0 animals: more often in females. Neurotoxicity and litter effects were present in both F0 and F1 animals at doses lower than for currently or previously described testicular effects. The F1 generation, exposed from the time of gametogenesis. was more susceptible to neurotoxic and reproductive effects than were the F0 animals. We were not able to separate reproductive effects from testicular toxicity, although early resorptions occurred at a lower dose (180 ppm) than decreased testicular weight (360 ppm). Testicular effects were observed in F0 males at doses that did not cause neurotoxicity.
Thus, these data indicate that HACR does affect reproductive performance of either F0 or F1 mice under the conditions used in Tasks 2 through 4 of this study (at doses less than or equal to 120 mg/kg/day). HACR had dominant lethal effects, which reduced live litter size by 26% in the F0 mice and by as much as 55% for the F1 mice at the high dose. The reduced number of pups per litter was equivalent in pairs fed HACR to those after exposed males were mated to nontreated females in the dominant lethal test. Thus, the dominant lethal effects may account for all of the F0 reproductive toxicity.