The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program. The findings may not have been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009. For more information, see the Explanation of Levels of Evidence for Developmental Toxicity. The findings and conclusions for this study should not be construed to represent the views of NTP or the U.S. Government.
In a previous study, Sprague-Dawley (CD ) rats were exposed to boric acid in the diet [0.1%, 0.2% or 0.4% on gestational days 0 to 20, or 0.8% on gd 6 to 15] resulting in average intakes of 78,163, 330 and 539 mg BORA/kg/day. Evaluation on gd 20 revealed fetal body weight reduction at greater than or equal to 0.1 % BORA, increased malformation incidence at greater than or equal to O.2% BORA, and increased prenatal mortality at 0.8% BORA (Heindel et al., 1992a). Among the anomalies observed, enlarged lateral ventricle(s) of the brain occurred in 0% of control fetuses, but in 11 % and 35% of fetuses examined in the 0.4% and 0.8% BORA groups, accompanied by severe reductions in fetal body weight (63% and 46% of control weight, respectively). Hydrocephaly was found in only one fetus (0.7%) in the 0.8% BORA group, and in no fetuses from the other groups (Heindel et al, 1992a). The present study was designed to determine (1) whether the induction of ventricular enlargement could be experimentally repeated following exposure to BORA on gd 6 to 15, (2) whether the incidence would exhibit a dose-response relationship at exposure concentrations of 0.4% to 0.8% BORA in the diet, and (3) whether the incidence and severity of ventricular enlargement would change during postnatal life.
Thus, in the present study, BORA (0.4%, 0.5%, 0.6% or 0.8% in the diet) was provided to timed-mated CD rats (42-76/group) from gd 6 to 15. All dams were monitored at regular intervals throughout gestation for body weight, clinical condition, food intake and water intake. In Phase I (teratology evaluation), dams were terminated and the uterine contents evaluated on gd 20. In Phase II, dams were allowed to deliver and rear their litters until postnatal day 21. In both phases, offspring were evaluated for post implantation mortality, body weight and morphological development of the head (gross morphology of external and internal structures). Phase I (and Phase II) dams ingested average doses of 299 (283), 361 (368), 432 (434), or 549 (562) mg BORA/kg/day in the low- through high-dose groups, respectively.
Maternal effects were generally consistent with the results of the earlier study. No maternal deaths occurred, but all BORA-exposed groups exhibited decreased body weight gain during the treatment period as a whole (gd 6 to 15) and at the end of gestation (gd 18 to 20). Reduced gravid uterine weight contributed to maternal weight gain deficits, so that neither corrected maternal weight gain (Phase I), nor maternal body weight on pnd 0 (Phase II) differed among groups. Maternal body weight deficits observed in the 0.5%-0.8% BORA group were indicative of residual maternal effects during lactation, especially at the high dose. Food intake decreased across groups during treatment. In Phase I, only the high dose was significantly below controls, but all groups were below controls in Phase II (the high dose reduction in Phase II did not reach statistical significance, apparently due to the smaller number of dams, but did appear to be biologically relevant). After treatment ended, maternal food and water intake increased in all BORA-exposed groups (gd 15 to 20). Maternal relative liver weight was increased at 0.8% BORA on gd 20, and decreased at greater than or equal to 0.6% BORA on pnd 21. Relative kidney weight was increased at greater than or equal to 0.5% BORA on gd 20, with no treatment- related effects on pnd 21.
Post implantation mortality (i.e., resorption, late fetal death and postnatal death) was increased at greater than or equal to 0.6% BORA on gd 20 (4%, 4%, 5%, 9% and 25% for control through high-dose groups), and at all BORA exposures by pnd 21 (9%,17%, 30%, 64% and 97%). The 0.8% BORA group was eliminated from Phase 11 (postnatal evaluation) in the second study replicate due to excessive offspring mortality. It is noteworthy that approximately 1%, 5%,17%, 45% and 67% of live bom pups in the control through high-dose groups were missing between pnd 0 and 14, presumably due to cannibalism by their own dams. This substantially reduced the numbers of BORA-exposed pups available for examination of internal head structures, and may have biased the incidence of related findings. Offspring body weight was decreased in all BORA-exposed groups on gd 20 (79%, 70%, 62% and 51 % of control weight for the low- to high-dose groups) and on pnd 0 (94%, 87%, 76% and 66% of control weight). On pnd 14 and 21, the average weight of offspring in the 0.4-0.6% BORA groups was comparable to controls (96-107% of control weight per group). Posthoc analysis of body weight (pnd 4, 7, 14 and 21) for the subset of pups surviving to pnd 21 supported the interpretation that pups actually recovered from intrauterine growth retardation, that is, recovery was not due simply to attrition of low weight offspring. However, persistent weight deficits were still noted on pnd 21 (76% of control weight) at 0.8% BORA.
Craniofacial (external) malformations were observed in 28% of high-dose fetuses on gd 20 and 17% of high-dose pups on pnd 21, as compared to 0% for controls at both ages. As in the earlier study, anophthalmia and microphthalmia were the most frequently observed craniofacial malformations. Examination of head sections revealed a high background incidence of minimal ventricular enlargement on gd 20 (77% of control fetuses), with a dramatic decrease in ELV by pnd 21 (2% of control pups). On gd 20, ELV incidence was increased at 0.4%-0.6% BORA (92-95%), but not in the 0.8% group (80%), and no treatment-related effects were observed on pnd 21. Furthermore, covariate analysis in this study indicated that lower fetal body weight was associated with higher incidence and greater severity of ELV. After adjusting for body weight effects, there were no significant dose-related effects of BORA on ELV incidence or severity, and incidence of ELV was elevated only at 0.5% BORA ELV incidence and severity did not covary with pup body weight on pnd 21. Since the incidence of this finding has increased in control CD rat fetuses in our laboratory in recent years, the high incidence observed in this study is most likely due to a shift in incidence for the experimental population as a whole. Based upon the high control incidence of ELV on gd 20, and the low incidence on pnd 21, ELV on gd 20 (e.g., minimal separation of the lateral ventricular walls) appears to represent a normal stage of CNS development for the population of rats examined in this study.
In contrast to ELV, hydrocephaly, which is characterized by more extensive separation of the ventricular walls and generally accompanied by loss or compression of CNS tissue, was not observed in any control fetuses or pups from this study, but was found in 2%, 1 %, 5% and 15% of BORA-exposed fetuses (low to high dose) on gd 20, in 5 and 9% of the premature postnatal deaths examined in the 0.6% and 0.8% BORA groups, and in 0.3% of pups at 0.4% BORA and 2% of pups at 0.6% BORA on pnd 21. Covariate analysis in this study indicated that lower fetal body weight was associated with higher incidence and greater severity of hydrocephaly. After adjusting for fetal body weight effects, there remained a significant dose-related effect of treatment upon hydrocephaly incidence and severity for all BORA groups. Covariate analysis indicated that hydrocephaly severity, but not incidence, covaried with pup body weight on pnd 21. There were dose-related increasing trends, and increased incidence and severity of hydrocephaly at 0.4% and 0.6% BORA on pnd 21 relative to the control group.
In the earlier study, 0. 1 % BORA (gd 0 to 20) was the lowest observed adverse effect level for reduced fetal body weight. Exposure to 0.4-0.8% BORA (gd 6 to 15) in this study was also associated with dose-related intrauterine growth retardation. Complete recovery from growth deficits was observed prior to the end of lactation at 0.4-0.6% BORA, but not at 0.8% BORA. The LOAEL for post implantation mortality was 0.8% BORA In the earlier study (Heindel et al., 1992a), but was 0.4% BORA in this study when postnatal mortality was taken into consideration. After adjusting for fetal body weight by covariate analysis, the incidence of ELV showed no significant dose-response relationship, but the incidence of hydrocephaly was increased at all doses. These results support the interpretation that BORA exposure during organogenesis adversely affects CNS development in the rat independent of its effect upon fetal growth. The LOAEL for adverse CNS effects in this study was 0.4% BORA in the diet, consistent with the outcome of the prior investigation in which CNS findings were observed at 0.4% (gd 0 to 20) and 0.8% (gd 6 to 15), but not at less than or equal to 0.2% BORA (gd 0 to 20).