FUNDAMENTAL AND APPLIED TOXICOLOGY 34, 1 7 6 - 1 8 7 (1996)
ARTICLE NO. 0188
The Developmental Toxicity of Boric Acid in Rabbits1
CATHERINE J. PRICE,* 2 MELISSA C. MARR,* CHRISTINA B. MYERS,* JOHN C. SEELY,!
3
JERROLD J. HEINDEL,^ AND BERNARD A. ScHWETzt'
*Chemistry and Life Sciences, Center for Life Sciences and Toxicology, Research Triangle Institute, P.O. Box 12194, Research Triangle Park,
North Carolina 27709-2194; \PATHCO, Inc., P.O. Box 12796, Research Triangle Park, North Carolina 27709; and
tDevelopmental and Reproductive Toxicology Group, National Toxicology Program, National Institute
of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709
Received October 20, 1995; accepted August 14, 1996
of controls. The most prevalent cardiovascular malformation (in-
The Developmental Toxicity of Boric Acid in Rabbits.
PRICE,
terventricular septal defect) was observed in 57% of high-dose
C. J., MARR, M. C, MYERS, C. B., SEELY, J. C , HEINDEL, J. J., fetuses compared to 0.6% among controls. At 250 mg/kg/day, averAND SCHWETZ, B. A. (1996). Fundam. Appl. Toxicol. 34,176-187. age fetal body weight/litter was 92% of the average control weight
Boric acid (BA), an ingredient of many pharmaceutical, cosmetic, and pesticide products, was previously shown to induce
reproductive and developmental toxicity in laboratory rodents. In
this study, BA (0, 62.5, 125, or 250 mg/kg/day, po) was administered on Gestational Days (GD) 6-19 to New Zealand White
rabbits (18-23 pregnant/group). Maternal body weight, food consumption, and clinical condition were monitored at regular intervals throughout gestation. At termination (GD 30), the numbers
of uterine implantations, resorptions, dead fetuses, and live fetuses
were determined. Fetuses were weighed, and live fetuses examined
for external, visceral, and skeletal defects. Maternal food intake
decreased during treatment at 250 mg/kg/day and increased at
s= 125 mg/kg/day after treatment. Maternal body weight (GD 9 30), weight gain during treatment, gravid uterine weight, and
number of ovarian corpora lutea decreased at 250 mg/kg/day. In
contrast, maternal corrected gestational weight gain increased at
s= 125 mg/kg/day. Maternal liver weight was not affected. Relative
(but not absolute) maternal kidney weight increased at 250 mg/
kg/day, and microscopic evaluation revealed no treatment-related
renal pathology. At 250 mg/kg/day, prenatal mortality was increased (90% resorptions/litter vs 6% for controls), the proportion
of pregnant females with no live fetuses was increased (73% vs
0%), and live litter size was reduced (2.3 fetuses/litter vs 8.8). As
a result, there were only 14 live fetuses (6 live litters) available for
evaluation in the high-dose group, compared to 153-175 live fetuses (18-23 live litters) in the other groups. The percentage malformed fetuses/litter was increased at 250 mg/kg/day, primarily
due to cardiovascular defects in 72% of high-dose fetuses vs 3%
The U.S. Government's right to retain a nonexclusive royalty-free license
in and to the copyright covering this paper, for governmental purposes, is
acknowledged.
1
Presented in part at the 31 st Annual Meeting of the Society of Toxicology, Seattle, WA, February 23-27, 1992 (Price el ai, 1992), and at the
International Symposium on the Health Effects of Boron and Its Compounds, Irvine, CA, September 16-17, 1992 (Heindel et al., 1994).
2
To whom all correspondence should be addressed.
3
Present address: National Center for Toxicological Research (NCTR),
HFT-1, 3900 NCTR Road, Jefferson, AR 72079-9502.
0272-0590/96 $18.00
Copyright © 1996 by the Society of Toxicology.
All rights of reproduction in any form reserved.
(not statistically significant). In summary, no definitive maternal
or developmental toxicity was observed at 62.5 or 125 mg/kg/day
BA. Mild maternal effects and severe developmental toxicity were
Observed at 250 mg/kg/day.
© 1996 Society of Toxicology.
Boric acid (BA) occurs naturally and is also produced
commercially from kernite ore (Woods, 1994; Rainer, 1994).
Boron-containing compounds are used in the manufacture
of many consumer products, primarily glass and ceramics,
and also as weatherproofing and fireproofing agents, fertilizers, and herbicides. BA powder (99%) is used as an insecticide (U.S. EPA, 1993), and BA is also a constituent of many
cosmetic and medical products intended for human use, antibacterial and antifungal products for horticultural use, and
veterinary products (Windholz et al, 1983; Siegel and Wason, 1986; PDR, 1982, 1991).
BA is toxic to humans and animals at sufficiently high
doses following oral, systemic, or dermal exposure (Roe ex
al, 1972; Weir and Fisher, 1972; Pinto et al, 1978; Arena,
1979; Fail et al, 1991; Treinen and Chapin, 1991; Ku et
al, 1991, 1993a,b, 1994). Due to its widespread use, human
poisoning by accidental ingestion or dermal contact has been
reported (Arena, 1979). BA is distributed throughout the
body and excreted in the urine (Draize and Kelley, 1959;
Ku et al, 1991).
A single dose of boric acid (3000 mg/kg/day, po) on the
first day of pregnancy disrupted pregnancy in mice, such that
94% of the embryos failed to undergo blastulation. Lower
percentages of embryos were affected at 500-1000 mg/kg/
day (Strongina et al, 1970, as cited in Beyer et al, 1983).
Despite this initial evidence that high-dose exposure to BA
could disrupt embryogenesis in mammals, there remained a
lack of developmental toxicity data (Barlow and Sullivan,
1982) until the following series of studies was initiated.
Time-mated Sprague-Dawley-derived (CD) rats were
176
DEVELOPMENTAL TOXICITY OF BORIC ACID
given BA in the diet (0.1, 0.2, or 0.4% on GD 0 to 20 or
0.8% on GD 6 to 15) yielding average intakes of 78, 163,
330, or 539 mg BA/kg body wt/day, respectively (Heindel
et al., 1992). Maternal effects included altered food and/or
water intake at 5=0.2% BA, increased relative maternal liver
and kidney weights at 5=0.2% BA, decreased gravid uterine
weight at 5=0.4% BA, decreased weight gain during treatment and gestation at 5=0.4% BA, and increased corrected
body weight gain at 0.4% BA. The percentage malformed
fetuses/litter was increased at 0.2, 0.4, and 0.8% BA (8, 50,
and 73% malformed compared to 2% in controls). Additional
detail regarding morphological findings in rats is presented
under Discussion. Prenatal mortality was increased at 0.8%
BA (39% of implanted conceptuses vs 4% in controls). Fetal
body weight was reduced in all groups (94, 87, 63, and 46%
of control weight, respectively) (Heindel et al., 1992). The
reduction of fetal body weight at 0.1% BA in the diet (76
mg/kg/day) was confirmed, and the no observable adverse
effect level (NOAEL) for developmental toxicity in the rat
was established at 0.075% BA in the diet (55 mg/kg/day)
from GD 0 to 20 (Price et al., 1995b, 1996).
CD-I mice fed 0.1, 0.2, or 0.4% BA in the diet on GD 0
to 17 ingested considerably higher doses (248, 452, or 1003
mg/kg/day) than rats fed the same concentrations (Heindel
et al., 1992). Mouse dams exhibited mild renal lesions at
5=0.2% BA, increased water intake and kidney weights
(0.4% BA), and decreased weight gain during treatment
(0.4% B A). Reduction of fetal body weight was dose dependent (94, 89, and 66% of controls), but statistically significant only at 5=0.2% BA. At 0.4% BA, resorptions/litter (19%
vs 6% for controls) and malformed fetuses/litter (9% vs
3% for controls) were increased (Heindel et al., 1992). For
additional detail regarding morphological findings in mice,
see Discussion.
Based upon the widespread use of BA, a developmental
toxicity study of BA in a nonrodent species [as recommended
by EPA and FDA (Kimmel and Price, 1990)] appeared to be
warranted. The present study was designed to evaluate maternal
condition as well as embryo/fetal growth, viability, and morphological development following administration of BA by gavage
to timed-pregnant rabbits during major organogenesis (GD 6 19). Maternal and developmental endpoints were comparable
to previous developmental toxicity studies of BA in mice and
rats (Heindel et al, 1992).
MATERIALS AND METHODS4
Animals and husbandry. The experimental animals were female New
Zealand White rabbits3 approximately 5 months of age. Male rabbits5 of the
4
This study was conducted in accordance with the Food and Drug Administration's Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (FDA, 1988). Copies of the final study report (NTP, 1991)
are available for a fee from the National Technical Information Service,
U.S. Department of Commerce, 5285 Port Royal Road, Springfield, VA
22161.
5
Hazleton Research Products, Inc., Denver, PA.
177
same strain were maintained as breeding stock. Rabbits were individually
identified by eartags. Following a 14-day quarantine, ovulation was induced
by injection of Pregnyl6 (0.1 ml/kg, iv) immediately prior to artificial insemination (Bredderman, 1964; Hafez, 1970) on GD 0. The study was performed
in two replicates with two consecutive breeding days within each replicate
and 34 days between replicates. Females (30/group) were assigned to treatments by stratified randomization so that body weight on GD 0 did not
differ among groups within either replicate. The body weight range for
individual females on GD 0 was 2690 to 4380 g.
Inseminated females were individually housed in stainless steel cages
with mesh flooring.7 Feed8 and deionized/filtered water were available ad
libitum throughout gestation. Environmental conditions were monitored and
controlled by computer.9 Lights were on from 0700 to 1900 hr for females
and from 0700 to 2100 hr for males. Mean temperature was 65°F (range,
64-79°F) and mean relative humidity was 56% (range, 47-89%).
Chemical. Boric acid9 (BA) was determined to be 99% pure by infrared
spectroscopy, and aqueous formulations were stable at room temperature
in a 28-day stability study.10
Treatment. Artificially inseminated New Zealand White does were administered BA (62.5, 125, or 250 mg/kg) or vehicle (distilled/deionized
water) once daily on the mornings of GD 6-19. Treatments were administered by gavage (po) using a rubber catheter" fitted with a syringe adapter12
and attached to a syringe of appropriate volume. Due to the limited solubility
of BA in water (55 mg/ml), the dose volume was 5 ml/kg body wt adjusted
daily.
Assays (UV/VIS spectrometry) conducted prior to dosing indicated that
all formulations were within a range of 94-106% of the theoretical concentrations. Formulations were used within the period of demonstrated stability.
Dose selection. In a previous investigation, all rabbits died following
oral administration of 850-1000 mg BA/kg/day for 4 consecutive days. At
800 mg BA/kg, symptoms included anorexia, weight loss, and diarrhea.
Minor symptoms (unspecified) were noted at 600-700 mg BA/kg/day, and
no toxic signs were reported at =s500 mg BA/kg/day (Draize and Kelley,
1959). Since published data from rabbits exposed orally to BA were quite
limited, preliminary toxicity data were collected in order to select an appropriate dose range for the developmental toxicity study.
In the preliminary toxicity study, nonpregnant female rabbits (2/group)
were dosed with BA (0 or 275 mg/kg/day, po) using a dose volume of 5
ml/kg in distilled/deionized water. No deaths or major clinical symptoms
occurred following 14 consecutive days of exposure to 275 mg/kg/day.
However, average food and water intake (g/animal/day) were depressed
during BA exposure (29 and 26% of controls for food and water, respectively). Average weight loss during exposure was —0.05 kg for control
females and - 1 . 0 kg for BA females.
Based upon these findings, 250 mg BA/kg/day (GD 6-19) in distilled/
deionized water (5 ml/kg) was selected as the high dose for the developmental toxicity study. It was assumed that this dose would produce some
maternal toxicity while allowing survival of all treated females through
scheduled necropsy (GD 30). Two lower doses (125 and 62.5 mg/kg/day)
6
Organon, Inc., West Orange, NJ.
Hoeltge, Inc., Cincinnati, OH.
8
Purina Certified Rabbit Chow (No. 5322), Ralston Purina Co., St. Louis,
MO.
9
Barber Coleman Network Supervisor System, Diversified Environmental Control, Inc., Greensboro, NC.
i0
The test chemical. (boric acid; CAS No. 10043-35-3) was obtained
from Fisher Scientific Co. (Lot No. 872703) under NIEHS/NTP Contract
NO1-ES-45061. Purity determinations and dose formulation studies were
also conducted under that contract.
" Nelaton, 18FR, American Hospital Supply.
12
Popper and Sons, New Hyde Park, NY.
7
178
PRICE ET AL.
were selected as one-half and one-fourth of the high dose, respectively,
with the assumption that the lowest dose would be at or near the NOAEL
for does.
Evaluations.
Inseminated females were weighed on GD 0, 6-19, 25,
and 30. Females were observed daily during and after treatment for clinical
signs of toxicity. Maternal food consumption was measured at 2- to 3-day
intervals throughout gestation. Surviving females were terminated on GD
30 by injection of Beuthanasia-D Special13 into the marginal ear vein. The
maternal liver, kidneys, and intact uterus were weighed and corpora lutea
were counted. Uteri which presented no visible implantation sites were
stained with ammonium sulfide (10%) to detect very early resorptions (Salewski, 1964). Maternal kidneys were bisected, fixed in 10% neutral buffered Formalin, and subsequently sectioned, stained with hematoxylin/eosin,
and evaluated histologically.
Live fetuses were dissected from the uterus and euthanized with an ip
injection of T-61 Euthanasia Solution14 or sodium pentobarbital.15 They
were weighed, examined for external morphological abnormalities, including cleft palate, and dissected for visceral examination and determination
of sex by a fresh tissue dissection technique (Staples, 1974; Stuckhardt and
Poppe, 1984). Half of the fetuses were decapitated after dissection; the
heads were fixed in Bouin's solution and then examined by a freehand
sectioning technique (Wilson, 1965). All fetal carcasses were skinned,
cleared, and stained with Alcian blue/Alizarin red S and examined for
skeletal malformations and variations (Kimmel and Trammell, 1981; Marr
etai, 1988).
Statistics. The doe or litter was considered the experimental unit for
all statistical analyses. General Linear Models (GLM) procedures were
applied for the analyses of variance (ANOVA) of maternal and fetal parameters (SAS Institute, 1989a,b, 1990a,b,c). Prior to GLM analysis, an arcsinesquare root transformation was performed on all litter-derived percentage
data (Snedecor and Cochran, 1967) and Bartlett's test for homogeneity of
variance was performed on all data to be analyzed by ANOVA (Winer,
1962). GLM analysis determined the significance of dose-response relationships and the significance of dose effects, replicate effects, and dose x
replicate interactions. When ANOVA revealed a significant (p < 0.05)
dose effect, Dunnett's multiple comparison test (Dunnett, 1955, 1964) compared BA-exposed groups to control groups. One-tailed tests were used for
all pairwise comparisons except maternal body and organ weights and
fetal body weight. Nominal scale measures were analyzed by a x2 t e s t f° r
independence and by a test for linear trend on proportions. When a x2
test showed significant groupwise differences, a one-tailed Fisher's exact
probability test was used for pairwise comparisons of control and BA
groups.
RESULTS
Maternal effects. Two maternal deaths occurred (one
doe at 62.5 mg/kg/day on GD 25; one doe at 125 mg/kg/
day on GD 22), but necropsy did not reveal a definitive
cause of death. The condition of the dam at the low dose
was suggestive of a gavage error affecting the respiratory
system, and the dam at the mid dose had evidence of gastric
lesions. Since there was no dose-related incidence of maternal deaths, and no deaths in the high-dose group, there appears to be no connection between maternal death and BA
exposure in this study. Among 120 females assigned to this
13
Schering Corp., Kenilworth, NJ.
American Hoechst Corp., Somerville, NJ.
15
Barber Veterinary Supply, Inc., Fayetteville, NC.
14
study, 22 (3-7 does/group) were removed for cause as follows: 3 for gavage accidents, 1 for accidental injury, 3 for
abortion between GD 20 and 23, and 15 for deviation from
ad libitum access to food and/or water (these cases were
generally due to the animal dislodging the food hopper or
water bottle from the cage or digging the food out of the
food hopper). Among the remaining does, the following percentages were confirmed pregnant by uterine examination
on GD 30 for the control through high-dose groups: 75%
(18/24), 88% (23/26), 87% (20/23), and 96% (22/23).
Pregnant does did not exhibit clinical symptoms attributable to BA toxicity except for vaginal bleeding (i.e., fresh
or dried blood in the cage pan, on the legs, in the urine, or
near the vagina). The incidence of bleeding was noteworthy
at 250 mg/kg/day (2-11 does/day on GD 19-30), and all
does with this symptom had no live fetuses on GD 30. Bleeding was not observed in any control females, and in only
one female/group at 62.5 and 125 mg/kg/day (Days 20 and
22, respectively; 5 - 7 live fetuses/litter at term).
At 250 mg/kg/day, maternal food consumption was decreased during the first 10 days of treatment (GD 6 to 15),
was comparable among groups during the final days of
treatment (GD 15 to 19), was increased during for the
period immediately following treatment (GD 19 to 25),
and was increased in both the 125 and 250 mg/kg/day
groups relative to controls during the final days of gestation (GD 25 to 30) (Fig. 1; Table 1). Maternal body weight
(GD 9-30), weight change during treatment, and gravid
uterine weight were each decreased at 250 mg/kg/day (Fig.
2; Table 1). Maternal weight change throughout gestation
was greater than that of controls at 125 mg/kg/day. Corrected maternal weight change was increased at both 125
and 250 mg/kg/day (Table 1). Maternal relative liver
weight was comparable among groups (Table 1). Relative
kidney weight was increased at 250 mg/kg/day, but appeared to be secondary to decreased maternal body weight
since absolute kidney weight (data not shown) was comparable across groups. Furthermore, microscopic evaluation
of maternal kidney sections failed to provide evidence for
any BA-induced renal toxicity (Table 1).
Embryo/fetal effects. No definitive evidence of developmental toxicity was observed following exposure of pregnant
does to either 62.5 or 125 mg/kg/day BA during the period
of major organogenesis (GD 6-19) (Tables 2 and 3). At 250
mg/kg/day, developmental toxicity included a high average
rate of resorptions and a high percentage of does with complete prenatal loss (Table 2). In contrast, the incidence of
late fetal deaths was low in all groups (=s2.8%/litter) and
showed no systematic relationship to BA exposure (data not
shown). Average fetal body weight/litter was 92% of controls at the high dose, but this difference did not reach statistical significance, in part due to the small sample size for
this parameter (only 6 litters survived to GD 30 at the high
dose, compared to 18-23 litters in the other groups).
179
DEVELOPMENTAL TOXICITY OF BORIC ACID
Control
62.5 mg/kg
60
125mg/kg
250 mg/kg
50
40
30
20
10
0to6
6 to 9
9 to 12
12 to 15
15 to 19
19 to 25
25 to 30
Gestation Period
FIG. 1. Maternal food consumption (mean ± SEM). *Signifies a significant difference (p < 0.05) from the control group during the same period of
measurement.
The overall incidence of malformed fetuses/litter was increased at 250 mg/kg/day BA, but not at 62.5 or 125 mg/
kg/day (Table 2). Even though there was an unusually high
background incidence of cleft sternum (Table 3), this did
not in any way obscure the identification of treatment-related
developmental effects in this study. For reference, when cleft
sternum was included in the calculations, the overall incidence of malformed fetuses was 25.2% (40/159), 28.6% (50/
175), 34.6% (53/153), and 78.6% (11/14) for the control
through high-dose groups, respectively. When cleft sternum
was excluded from the calculations, the overall incidence of
malformed fetuses was 8.8% (14/159), 9.7% (17/175),
11.8% (18/153), and 78.6% (11/14). When malformations
were analyzed by general class, the percentage fetuses/litter
with external or visceral malformations was increased at the
high dose, but the incidence of skeletal malformations was
comparable to that of controls (Table 2).
External malformations were observed with the following
incidence among individual fetuses in the control through
high-dose groups, respectively: 0.6% (1/159), 1.1% (2/175),
0.7% (1/153), and 14.3% (2/14) (Table 3). Although the
overall incidence of external malformations was increased
at the high dose of BA, distinctive dose-response patterns
for individual malformations were not observed (Table 3).
Two fetuses were found to have multiple anatomical defects
(major and minor), but in the absence of a dose-response
relationship. Thus, one fetus in the control group displayed
a domed head, low-set ears, cleft palate, microglossia, enlarged gall bladder, clubbed limb with no underlying bone
change, and bilateral full rib on lumbar I. Another fetus in
the mid-dose group showed a similar range of defects—
domed head, low-set ears, cleft palate, micrognathia, microglossia, and clubbed limb with no underlying bone
change.
The incidence of fetuses with visceral malformations was
8.2, 6.3, 7.8, and 78.6% in the control through high-dose
groups (Table 3). Malformations of the cardiovascular (CV)
system (great vessels and heart) were observed with the
greatest frequency, and their incidence was significantly increased at the high dose (Table 2). CV malformations with
an elevated incidence included interventricular septal defect
in 0.6, 1.7, 1.3, and 57% of the fetuses examined (control
through high-dose, respectively); enlarged aorta in 0, 0.6,
0.7, and 36% of fetuses examined; papillary muscle malformations in 3, 2, 4, and 14% of fetuses examined; and double
outlet right ventricle (pulmonary artery and aorta both arising
from the right ventricle) in 0, 0, 0, and 14% of fetuses
examined (Table 3). The gallbladder was the only other
180
PRICE ET AL.
TABLE 1
Maternal Toxicity in New Zealand White Rabbits Exposed to Boric Acid on Gestational Days 6 through 19
Boric acid (mg/kg/day, po)
No. pregnant at euthanization
No. of live litters
No. of live fetuses
Maternal wt. change (g)**
Treatment period (GD 6 to 19)
Gestation period (GD 0 to 30)
Corrected gestation wt. gain"
Gravid uterine wt (g)°
Maternal liver wt**
(% body wt)
Maternal kidney wt**
(% body wt)
Renal pathology^
Relative food consumption (g/kg/day)"
Pretreatment (GD 0 to 6)
Treatment (GD 6 to 19)
Posttreatment
GD 19 to 25
GD 25 to 30
93
357
-205
562
0
62.5
125
250
18
18
159
23
23
175
20
20
153
22
6
14
±
±
±
±
30*
69*
78*
40*
132
493
-52
504
±
±
±
±
40
51
63
26
97
543
40
502
±
±
±
±
51
63**
57**
28
-137
226
165
62
±
±
±
±
42**
35
40**
18**
2.59 ± 0.13
2.80 ± 0.09
2.78 ± 0.08
2.87 ± 0.09
0.46 ± 0.02
0/18
0.46 ± 0.01
2/23
0.47 ± 0.01
0/20
0.51 ± 0.01**
1/22
48.1 ± 1.7
38.8 ± 1.7*
48.0 ± 1.8
40.0 ± 2.0
48.9 ± 2.4
38.7 ± 2.3
46.4 ± 1.5
26.6 ± 2.2**
36.9 ± 2.5*
24.5 ± 3.0*
37.0 ± 2.6
30.9 ± 2.1
40.0 ± 3.1
33.9 ± 1.9**
44.9 ± 2.2
41.9 ± 1.6**
° Includes all dams pregnant at euthanization; mean ± SEM.
* Maternal gestational and corrected weight gains and relative organ weights were calculated using body weight at the time of euthanization.
c
Weight gain during gestation minus gravid uterine weight.
"* Renal tubular regeneration (minimal). In addition, one dam in the low-dose group showed minimal mineralization of the renal tubules; one dam in
the mid-dose group showed mild, multiple subcapsular cysts.
* p < 0.05, linear trend.
** p < 0.05, Dunnett's test.
visceral organ which displayed changes in malformation incidence potentially associated with BA exposure. The incidence of enlarged gallbladder decreased with increasing
dose, while the incidence of agenesis increased (Table 3).
The incidence of fetuses with skeletal malformations (all
types) was comparable across treatment groups, i.e., 19, 22,
29, and 29%. The incidence of skeletal malformations among
study controls (19%) was noticeably higher than that for
historical controls (36/912 or 4%) from the same species/
strain in our laboratory (see NTP, 1991). This was due primarily to the high incidence of cleft sternum which is presumed to occur due to abnormal midline fusion of the procartilagenous sternal bands. This defect was found in 18% (28/
159) of the control fetuses in this study vs 2% (18/912)
among historical controls. In subsequently conducted studies
using the same species/strain in our laboratory, only 0.31%
(4/1280) of control rabbit fetuses exhibited cleft sternum.
The unusually high incidence of cleft sternum in this study
thus represents a transient phenomenon. Although the reasons for this sudden change in background incidence remain
obscure, it is clear that the incidence of this defect was not
related to BA exposure.
Only two individual skeletal malformations appeared to
show an increased incidence in BA-exposed animals (Table
3): fused sternebrae occurred with an incidence of 1.3, 1.7,
0, and 7%, and fused ribs occurred with an incidence of 0,
0, 1.3, and 7% of fetuses examined. At the high dose, each
of these findings occurred in only one fetus and the affected
fetuses came from separate litters (i.e., neither defect occurred in more than one high-dose fetus). Thus, the association of fused sternebrae or fused rib with BA exposure was
considered equivocal.
The percentage fetuses/litter with anatomical variations
was not significantly elevated above controls in any BAexposed group (Table 2). The only external variation noted
was clubbed limb (without underlying bone change) which
never occurred in more than one fetus per group (Table 4).
A wide variety of visceral variations was observed and the
two which appeared to show a dose-related incidence involved the same organs that exhibited BA-related malformations. An abnormal number of cardiac papillary muscles was
observed in 5, 6, 5, and 50% of fetuses examined, and small
gallbladder was observed in 3, 5, 5, and 14% of fetuses
examined (Table 4). Only two skeletal variations were noted
and neither of them showed a clear dose-response relationship (Table 4). Misaligned sternebrae occurred in 1.26, 1.14,
181
DEVELOPMENTAL TOXICITY OF BORIC ACID
-•-
Control
- • - 62.5 mg/kg
4200
-±-
125 mg/kg
-•-
250 mg/kg
r
4000
S 38OO
O)
|
3600
m 3400
3200
3000
0
6
9
12
15
Day of Gestation
19
25
30
FIG. 2. Maternal body weight (mean ± SEM). *Signifies a significant difference (p < 0.05) from the control group on the same day.
0.65, and 0% of fetuses examined for the control through
high-dose groups, respectively. Full extra rib on lumbar I
(bilateral or unilateral) occurred in 57, 39, 30, and 43% of
fetuses, and rudimentary extra rib on lumbar I (bilateral or
unilateral) occurred in 3, 4, 2, and 14% of fetuses examined.
Post hoc statistical analysis for individual variations or types
of variations was performed only for cardiovascular findings
since these showed the only robust association with BA
exposure in the present study (Table 2).
DISCUSSION
Developmental and reproductive toxicity studies of BA in
laboratory animals have demonstrated disruption of embryo/
fetal development as well as decreased fertility due to disruption of spermatogenesis following BA exposure (Weir and
Fisher, 1972; Heindel et al, 1992; Fail et al, 1991; Treinen
and Chapin, 1991; Ku et al, 1991, 1993a,b, 1994; Price et
al, 1995a,b, 1996). Thus, the finding of developmental toxicity in rabbits exposed to BA during major organogenesis
extends previously reported findings in other laboratory species. Developmental and reproductive toxicity studies in laboratory animals have played a pivotal role in recent risk
assessments for boron compounds (ECETOC, 1995; IEHR,
1995; Murray, 1995; Smallwood et al, 1995). The empirical
identification of a NOAEL (Price et al, 1996) and the statistical derivation of a benchmark dose based on developmental
effects in rats (Allen et al, 1996) have been of particular
importance in this context since the rat is the most sensitive
mammalian species evaluated to date. Rather than attempt to
recapitulate the comprehensive reviews presented elsewhere,
this discussion is focused on interpretation of the present
study in rabbits and a comparison of these results with previously reported studies in rats and mice.
In order to determine whether decreased maternal food
intake contributed to adverse developmental outcome at the
high dose of BA, our results were compared to those previously reported following feed restriction. Prenatal growth,
viability, and morphological development of New Zealand
White rabbits (17 mated/group) were not affected by severe
food restriction during major organogenesis (15 g/day, GD
7-13, and 25 g/day, GD 14-19) when compared to controls
fed ad libitum throughout gestation (250 g of feed/day, GD
0-29; Parker et al, 1986). In contrast, pregnant New
Zealand White rabbits (6-10/group) fed a rationed diet of
150, 60, or 20 g of feed/day (GD 6-20) exhibited increased
prenatal mortality (15% at 60 g/day and 46% at 20 g/day,
compared to 8% for ad libitum controls consuming 195—
248 g of feed/day). At 20 g/day, fetal body weight was
63% of ad libitum controls, but the incidence of external
malformations was not affected (visceral and skeletal examinations were not conducted) (Matsuzawa et al, 1981). In
another study, New Zealand White rabbits fed 50 or 15 g/
day (GD 6 to 18) had 14 and 16% resorptions/litter vs 8%
for controls which consumed 150 g/day (Clark et al, 1986).
Average fetal body weight was reduced (95 and 87% of
182
PRICE ET AL.
TABLE 2
Developmental Toxicity in New Zealand White Rabbits Following Maternal Exposure
to Boric Acid on Gestational Days 6 through 19
Boric acid (mg/kg/day, po)
No. implantation sites per litter"
% Resorptions per litter"
% Litters with one or more
resorptions
% Litters with 100% resorptions
No. live fetuses per litter*
Average fetal body wt (g) per litter*
All malformations
% Fetuses per litter*
%Litters
External malformations
% Fetuses per litter*
% Litters
Skeletal malformations
% Fetuses per litter*
% Litters
Visceral malformations
% Fetuses per litter*
% Litters
Cardiovascular (CV) malformations
% Fetuses per litter*
% Litters
All variations
% Fetuses per litter*
% Litters
Cardiovascular variations
% Fetuses per litter*
% Litters
0
62.5
125
250
9.5 ± 0.8
6.3 ± 2.4*
8.4 ± 0.6
5.9 ± 1.9
8.3 ± 0.5
7.7 ± 2.1
8.6 ± 0.7
89.9 ± 5.0**
39
0
45
0
8.8 ± 0.8*
44.8 ± 1.5
7.6 ± 0.6
46.5 ± 1.4
7.7 ± 0.5
45.7 ± 1.2
95***
73***
2.3 ± 0.8**
41.1 ± 2.7
25.5 ± 5.8*
26.1 ± 3.8
30.4 ± 6.3
80.6 ± 16.3**
39
0
72
78
75
0.8 ± 0.8*
1.4 ± 1.0
1.0 ± 1.0
6
9
5
83
11.1 ± 8.2**
33
19.9 ± 5.4
19.9 ± 4.0
24.3 ± 6.4
38.9 ± 20.0
61
65
55
50
5.9 ± 2.0
7.4 ± 2.0
35
45
2.7 ± 1.6*
3.1 ± 1.5
4.2 ± 1.3
17*
22
35
7.3 ± 1.9*
50
80.6 ± 16.3**
83
72.2 ± 16.5**
83***
67.7 ± 7.2
54.8 ± 5.1
40.4 ± 5.2
86.1 ± 9.0
94
100
90
100
5.7 ± 1.8
7.2 ± 2.5
35
35
10.6 ± 5.5*
44
63.9 ± 17.4**
83
" Includes all dams pregnant at euthanization; litter size is number of implantation sites per dam; mean ± SEM.
* Includes only dams with live fetuses; litter size is number of live fetuses per dam; mean ± SEM.
* p < 0.05, linear trend.
** p < 0.05, Dunnett's test.
*** p < 0.05, Fisher's exact test.
control weight, respectively), and an increased incidence of
fetal malformations, including omphalocele, clubbed forefeet, and sternebral anomalies, was noted at 15 g/day. By
comparison, rabbits in the high-dose group (this study) consumed 94 g of food/day during treatment and exhibited 90%
resorptions/litter (controls consumed 150 g/day and exhibited 6% resorptions/litter). Thus, the control group resorption
rate was consistent with the other studies reported, but the
incidence of prenatal mortality was disproportionately high
in BA-exposed rabbits (this study) relative to rabbits with
even greater restriction of food intake (Parker et ai, 1986;
Matsuzawa et ai, 1981; Clark et ai, 1986). Average fetal
body weights in BA-exposed groups (this study) were statistically comparable to controls, and the types of malformations (primarily cardiovascular) were dissimilar to those reported after feed restriction in other rabbit studies. Thus,
decreased maternal food intake may have been a contributing
factor, but cannot be solely responsible for the range and
severity of adverse developmental effects observed at the
high dose of BA.
The background incidence of cleft sternum was unusually
high in this study, but was not influenced by BA treatment,
per se. Furthermore, cleft sternum was most often observed
as an isolated defect and was not consistently associated
with other morphological anomalies in individual fetuses.
Cleft sternum is a midline defect related specifically to the
process of fusion between the procartilagenous sternal bands
(Ramirez-Solis et ai, 1993). In mice, the expression of cleft
sternum has a genetic basis (Ramirez-Solis et ai, 1993), and
its induction by chemical agents, such as ethylene oxide,
occurs during the early zygotic period (Polifka et ai, 1996).
Based on these considerations, as well as the transient nature
183
DEVELOPMENTAL TOXICITY OF BORIC ACID
TABLE 3
Morphological Abnormalities Observed in New Zealand White Rabbit Fetuses Following Maternal Exposure
to Boric Acid on Gestational Days 6 through 19: Listing by Defect Type"
Boric acid (mg/kg/day, po)
Number examined
Fetuses Examined*
Litters Examined"
Any malformations
Fetuses with any malformations''
Litters with any malformations'
External malformations
No. of fetuses with external malformations''
No. of litters with external malformations'
Dome-shaped head
Ears lower than normal
Micrognathia
Facial cleft
Agenesis of the upper lip
Cleft lip
Microglossia
Cleft palate
Spina bifida
Short tail
Skeletal malformations
No. of fetuses with skeletal malformations''
No. of litters with skeletal malformations'
Cleft sternum
Fused sternebrae
Floating (detached) extra rib: lumbar I. right side
Fused ribs
Visceral malformations
No. of fetuses with visceral malformations''
No. of litters with visceral malformations'
Enlarged lateral ventricle of the brain: left
Abnormal papillary muscle
Agenesis of all in right ventricle
Bifurcated, left ventricle
Bifurcated, right ventricle
Small, right ventricle
Abnormal tricuspid valve: solid septum
Agenesis of the subclavian artery: right
Common truncus
Enlarged aorta
Enlarged heart
Interventricular septal defect
Pulmonary artery and aorta arise from right ventricle
Transposition of aorta, and pulmonary artery
Agenesis of the gall bladder
Enlarged gall bladder
Agenesis of the spleen
Small pulmonary artery
62.5
125
250
(159)
(18)
(175)
(23)
(153)
(20)
(14)
(6)
40
13
50
18
53
15
11
5
1
1
2
2
2
2
i
l
1
30
11
28
2
39
15
36
3
44
11
40
1
5
13
9
1
12
9
I
11
5
1
1
2
2
1
1
1
1
1
2
1
3
1
I
" The incidence of individual defects is expressed as the number of individual fetuses exhibiting that defect. Thus, a single fetus may be represented
more than once in listing individual defects.
* Only live fetuses were examined.
c
Fetuses with one or more malformations.
d
Includes only litters with live fetuses.
' Litters with one or more malformed fetuses.
184
PRICE ET AL.
TABLE 4
Morphological Variations Observed in New Zealand White Rabbit Fetuses Following Maternal Exposure
to Boric Acid on Gestational Days 6 through 19: Listing by Defect Type"
Boric acid (mg/kg/day, po)
Number examined
No. of fetuses examined*
No. of litters examined"
Any variations
No. of fetuses with any variations'*
No. of litters with any variations'
External variations
No. of fetuses with external variations'*
No. of litters with external variations'
Clubbed limb (without bone change)
Skeletal variations
No. of fetuses with skeletal variations'*
No. of litters with skeletal variations'
Misaligned sternebrae
Rib on lumbar I
Full (bilateral, left or right)
Rudimentary (bilateral, left or right)
Visceral variations
No. of fetuses with visceral variations'*
No. of litters with visceral variations'
Abnormal number of papillary muscles
Agenesis of the innominate artery
Left auricular flap of heart { normal size
White mucous-like material in stomach
Liver-like tissue on gall bladder
Small gall bladder
Mottled spleen
Small spleen
Pale mottled liver
Yellow liver
White spot on left lateral liver lobe
Blood-filled kdiney capsule: right
Red area on left kidney
White spot on kidney
62.5
125
250
(159)
(18)
(175)
(23)
(153)
(20)
(14)
(6)
107
17
96
23
64
18
11
6
0
0
1
1
97
16
2
75
21
2
50
17
1
91
5
68
7
46
3
28
12
39
18
10
1
1
15
4
2
9
9
1
24
13
8
1
1
1
5
8
10
1
1
° The incidence of individual variations is expressed as the number of individual fetuses exhibiting that defect. Thus, a single fetus may be represented
more than once in listing individual defects.
b
Only live fetuses were examined.
c
Fetuses with one or more variations.
d
Includes only litters with live fetuses.
' Litters with one or more fetuses with variations.
of the increase observed in our laboratory, it appears likely
that the high background incidence of cleft sternum in this
study represents a genetically determined phenomenon.
Three mammalian species (rat, mouse, and rabbit) have
now been evaluated for developmental toxicity of BA at
exposure levels which did not cause maternal mortality. A
brief comparison of results across studies is provided below
(see also Heindel et al., 1994), recognizing that differences
in experimental design, as well as inherent differences in
response across species, may have contributed to the relative
outcomes. Although there was overlap in the dose ranges
across species, underlying kinetics undoubtedly differed due
to differences in the method of administration. Rodents were
given BA in feed throughout gestation (GD 0 to 20, rats;
GD 0 to 17, mice) or only during organogenesis (GD 6 to
15, rats; Heindel et al, 1992; Price et al, 1995a, 1996). Rats
given BA in the diet have been shown to reach steady-state
blood and tissue levels of boron by the fourth day of exposure (Treinen and Chapin, 1991). In contrast, rabbits in this
study were given an aqueous solution of BA by gavage
DEVELOPMENTAL TOXICITY OF BORIC ACID
once daily during major organogenesis (GD 6-19). Further
studies are needed to determine whether critical internal exposure parameters are based on area under the curve (AUC)
or peak blood and tissue concentrations.
Based upon a comparison of lowest observed adverse effect levels (LOAELs) for developmental toxicity, the rat
conceptus was the most sensitive, showing a significant reduction of body weight («94% of control weight) at daily
intakes of 2=76 mg/kg/day (Heindel et al, 1992; Price et al,
1996). In rabbits (this study), fetal weight reduction (92%
of control weight) at the high dose did not reach statistical
significance possibly due to the small number of litters surviving at this dose. Thus, the developmental LOAEL in rabbits was defined by increased prenatal mortality and malformations at 250 mg/kg/day. The developmental LOAEL in
mice (452 mg/kg/day) was based on fetal weight reduction
(89% of control weight) (Heindel et al, 1992). NOAELs for
developmental toxicity in these studies were 55 mg/kg/day
for rats (Price et al., 1996), 125 mg/kg/day for rabbits (this
study), and 248 mg/kg/day for mice (Heindel et al., 1992).
Susceptibility to BA-induced prenatal mortality showed
differences across species despite comparable control values ( 4 - 6 % of implants resorbed/litter). With regard to
prenatal mortality, rabbits were the most sensitive and
most severely affected species, experiencing 90% resorptions/litter at 250 mg/kg/day (GD 6-19). The extent of
prenatal mortality in rats depended upon both the dose
and the period of administration. Rats showed 36% resorptions at 539 mg/kg/day during major organogenesis (GD
6 to 15; Heindel et al., 1992), but 76% resorptions at 617
mg/kg/day throughout gestation (GD 0 to 20; NTP pilot
study). In contrast, mice were relatively resistant showing
only 20% resorptions/litter at 1003 mg/kg/day throughout
gestation (GD 0 to 17; Heindel et al., 1992).
As with other developmental endpoints, the induction of
malformations by BA also showed species-specific characteristics. In rabbits, the cardiovascular system was clearly a
target for morphological defects; 72% of fetuses/litter at 250
mg/kg/day had at least one major cardiovascular malformation (predominantly, interventricular septal defect) compared
to only 3% among controls. Because of the high rate of
prenatal mortality (90%) at the high dose in rabbits, additional studies using lower doses and/or shorter exposure periods would be useful in characterizing the disruption of cardiovascular morphogenesis. Unlike the rat and mouse, development of the rabbit skeletal system appeared to be relatively
insensitive to disruption following BA exposure.
In rats, the incidence of major malformations was elevated
at 163, 330, and 539 mg/kg/day BA (8, 50, and 73% malformed/litter) against a control value of 2% malformed
(Heindel et al., 1992). A wide variety of malformations was
associated with BA exposure in the rat, including curly and/
or short tail, anophthalmia, microphthalmia, displacement of
185
the eye, cleft sternum, and fused ribs. Other effects on skeletal development included an increased incidence of wavy
rib, agenesis or shortening of rib XIII, and a decreased incidence of extra rib on lumbar I (Heindel et al., 1992; Price
et al., 1996). Axial skeletal defects have subsequently been
examined in another laboratory following high oral doses of
BA (500 mg/kg, bid, on single or multiple days of gestation)
in order to characterize critical periods of susceptibility and
to provide the foundation for mechanistic research (Narotsky
et al., 1995, 1996). Cardiovascular malformations in rats
were qualitatively similar to those observed in rabbits (i.e.,
double outlet right ventricle and interventricular septal defect), and the incidence was elevated at the highest BA dose
(5% of fetuses vs 0% for vehicle controls). At doses above
280 mg/kg/day, enlargement of the lateral ventricles in rats
has been demonstrated following BA exposure in the diet
from GD 0 to 20 or GD 6 to 15 (Heindel et al, 1992; Price
et al., 1995a). This morphological finding occurred in the
presence of, but was not entirely accounted for by, severe
intrauterine growth retardation (Price et al., 1995a).
The mouse was least sensitive to BA-induction of malformations as well as other developmental effects. The maximum response achieved in mice was 9% malformed fetuses/
litter at 1003 mg/kg/day against a control value of 3%. The
most apparent treatment-related morphological changes involved deficient rib development at the thoracolumbar junction, i.e., increased incidence of short rib XIII (classified as
a malformation in that study) and decreased incidence of
extra rib(s) at lumbar I (classified as a variation). Cardiovascular malformations showed a low incidence («sO.4%) in all
groups, and the incidence was not related to BA exposure
in mice (Heindel et al., 1992).
Despite the relative resistance of mice to BA-induced developmental toxicity, the dams were susceptible to induction
of renal lesions (Heindel et al., 1992). Pregnant mice exhibited a dose-related incidence of renal tubular dilatation/regeneration, while pregnant rats (Heindel et al., 1992) and
rabbits (this study) failed to show treatment-related microscopic renal lesions at doses that were clearly toxic to the
developing conceptus. By inference, maternal renal pathology is not a prerequisite for induction of developmental
toxicity after BA exposure.
To summarize this study, decreased food intake and vaginal bleeding associated with pregnancy loss were the principal manifestations of maternal toxicity in New Zealand
White rabbits exposed to 250 mg/kg/day BA on GD 6-19.
The same dose was associated with severe developmental
toxicity, including a high rate of prenatal mortality and malformations. Development of the cardiovascular system was
particularly sensitive to disruption. At 125 mg/kg/day, increased maternal food intake and weight gain were not considered adverse. Increased food intake was noted in all three
species at one or more doses (Heindel et al., 1994), and
186
PRICE ET AL.
some of these increases were attributable to posttreatment
rebound. However, further studies are needed to determine
the cause of increased food intake when it is not preceded
by a decrease during the treatment period. No definitive
evidence of developmental toxicity was observed at 125 mg/
kg/day. The low dose (62.5 mg/kg/day) was clearly nontoxic
to both the maternal animal and the developing conceptus.
Thus, the maternal and developmental NOAELs for rabbits
were considered to be 125 mg/kg/day.
ACKNOWLEDGMENTS
These studies were conducted at Research Triangle Institute (RTI), Research Triangle Park, North Carolina, under contract to the National Toxicology Program and the National Institute of Environmental Health Sciences
(NTP/NIEHS Contract NO1-ES-95255). Pathology support was provided
by John C. Seely, D.V.M., PATHCO, Inc. (Research Triangle Park, NC)
under a subcontract. The authors express their appreciation to the following
RTI personnel who contributed to the completion of this investigation: Ms.
Doris J. Smith, Ms. Gwendolyn McNeill, Dr. Lawrence E. Myers, Dr.
Donald B. Feldman, Dr. Brian M. Sadler, Mr. William P. Ross, Ms. Kimberly K. Rhodes, Ms. Frieda S. Gerling, Ms. Vicki I. Wilson, Ms. Deanna
L. Gibson, Ms. Kimberly E. Prince, Ms. Lawson B. Pelletier, Mr. M.
Michael Veselica, Mr. Randy A. Price, Mr. Donald L. Hubbard, and Ms.
Rachel M. Johnson. We also thank Dr. Hudson K. Bates for assistance in
the graphic presentation of study data and Ms. Virginia M. Young for
secretarial assistance.
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