FUNDAMENTAL
AND
APPLIED
TOXICOLOGY
19,527-537
(1992)
Subchronic Toxicity of Barium Chloride Dihydrate Administered to Rats and Mice in the Drinking Water D. D. DIETZ, M. R. ELWELL, W. E. DAVIS, JR.,* AND E. F. MEIRHENRY* Division of Toxicology Research and Testing, National Institute of Environmental Health Sciences, National Toxicology Program, Research Triangle Park. North Carolina 27709; and *Toxicology Laboratory. SRI International, Menlo Park, California 94025 Received October 23, 199 I; accepted May 6, 1992
Subchronic Toxicity of Barium Chloride Dihydrate Administered to Rats and Mice in the Drinking Water. DIETZ, D. D., M. R., DAVIS, W. E., JR., AND MEIRHENRY, E. F. (1992). Fundam. Appl. Toxicol. 19, 527-537.
ELWELL,
Barium Chloride dihydrate (BaCI,. 2HZO) was given for 92 days to B6C3F1mice and Fischer 344/N rats in their drinking water at levels of 0, 125, 500, 1000, 2000, and 4000 ppm. The no-effect level for this study was2000 ppm BaCl, . 2H20 in the drinking water. At 4000 ppm, daily consumption for mice was 436 to 562 mg/kg barium, up to four times more chemical than rats. Mortality ranged from 60 to 70% in mice and from 10 to 30% in rats in the 4000 ppm groups. Deaths in mice were associated with a treatment-related renal toxicity. Renal lesions in rats were much lessseverethan in miceand did not contribute to the treatment-related deaths seen in the high dose group. Body weights of both speciesand sexesin the 4000 ppm groups were lower than controls at 92 days. Male and female rats in treated groupsexhibited higher serumphosphorusthan controls. Serum sodium, potassium,and calcium levels in rats were unchangedby barium treatment, aswere hematologicalvalues. In both species at 4000 ppm, motor activity, grip strength, and thermal sensitivity were marginally affected. Theseeffects were probably secondary changesresulting from BaCl, toxicity observedat this doselevel. In a mating trial, no anatomical effects on offspring of rats or mice were seen.Rats receiving 4000 ppm exhibited marginal reductions in pup weights. No effects were seenon reproductive indices. o 1992 Society of Toxicology.
Barium occurs in the earth’s crust at an estimated level of 450 ppm and in some drinking water supplies the concentration of barium may exceed 20 ppm (McCauley and Washington, 1983). Elevated barium concentrations in drinking water derived from deep rock and drift wells have been described as a naturally occurring problem (Wones et al., 1990; Calabrese, 1977). The U.S. Environmental Protection Agency (199 1) recently established a revised maximum contaminant level (enforceable federal standard) of 2 ppm barium for public drinking water. Other exposures to barium chloride occur during its use in the manufacture of pigments, in aluminum refining, in sugar refining, in tanning and finishing leather, and as a pesticide.
The toxicity of barium salts is a function of their aqueous solubility (Borzelleca et al., 1988; Syed and Hosain, 1972). Barium sulfate is water insoluble; this nontoxic salt is used in radiology as a radioopaque material (Nielsen, 1986). By contrast, water-soluble salts, such as carbonate, chloride, and acetate, produce a variety of acute toxic effects in humans and experimental animals involving the cardiovascular, gastrointestinal, nervous and hematopoietic systems, and the skeletal muscles (Stokinger, 198 1). The most characteristic toxic signs resulting from barium ingestion involve an intense stimulation of smooth, striated, and cardiac muscle (Brenniman et al., 198 1). Prolonged exposures to barium are reported to produce muscle weakness (Diengott et al., 1964; Phelan and Hagley, 1984; Stokinger, 198 1). Relatively little is known of the toxicological effects resulting from long-term or continuous exposures to soluble barium salts. Schroeder and Mitchener (1975a,b) found no evidence of toxicity in rats and mice exposed during their life span to 5 ppm of barium acetate in the drinking water. Other investigators found no changes in body weight, appearance, and selected organ weights and morphology in rats given 1, 10, and 100 ppm BaCl* for up to 16 months in their drinking water. They did find cardiomyopathy expressed by alterations in physiologic and metabolic parameters at 100 ppm and increased blood pressure in treated animals at 1 month (100 ppm) and 8 months (10 ppm) after the start of exposure. Because the cardiovascular actions of barium mimic those of calcium and digitalis, it has been suggested that Ba+2 acts as a Ca+2 agonist in these tissues (Perry et al., 1989; Brenniman et al., 198 1; Shanbaky et al., 1978). Recent studies have demonstrated that the cardiotoxic properties of barium chloride are blocked by the calcium antagonist verapamil (Mattila ef al., 1986). Tardiff et al. (1980) conducted a 13-week study with BaC12 administered in the drinking water of rats at concentrations ranging from 10 to 250 ppm. Except for a decrease in the relative weight of the adrenals at the 250 ppm level, no adverse effects were found. Izraelson (1967) performed a study in which guinea pigs were given 50 mg/kg BaC12 repeatedly for 30 days by oral administration. He noted treatment-related anemia, leukopenia, and degen-
527 Copyright Ail rights
0272-0590192 $5.00 0 1992 by the Society of Toxicology. of reproduction in any form reserved.
528
DIETZ ET AL.
eration of hepatocytes, myocardial cells, and renal convoluted tubule cells. Several case studies of human barium poisoning have shown that water-soluble barium salts induce hypokalemia and that toxic symptoms involving the cardiovascular system and skeletal muscles (stimulation followed by weakness) are reversed by potassium therapy (Diengott cr al., 1964; Phelan and Hagley, 1984). Roza and Berman (197 1) demonstrated that BaCI, produces hypokalemia in dogs and that most of the cardiotoxic effects of barium were reversed by the administration of potassium. Hypertension was the only effect not responding to treatment. Although the acute toxicity and long-term cardiovascular effects of ingested barium have been extensively described there is continuing concern for the lack of adequate information to evaluate the safety of continuous low level exposures to barium that occur via the drinking water. Thus, Wones et al. (1990) recently reported that “the data on the health effects of drinking water barium are incomplete, mixed, and inadequate.” The importance of this problem is underlined by the fact that the barium content of drinking water in several geographical areas exceed the current drinking water standard of 2 ppm barium (Wones et al., 1990; Calabrese, 1977; Brenniman et al., 1981; EPA, 1991). The present studies were, therefore, initiated in rats and mice to help address the issue of safe levels of barium in the drinking water. The study design was comprehensive in order to establish a maximum-tolerated dose and minimally effective doses using a variety of toxic endpoints. The exposure concentrations were several fold greater than normally encountered in the drinking water and ranged from a level (250 ppm) that did not produce toxicity in a previous 13-week drinking water study (Tardiff et al., 1980) to a level (4000 ppm) expected to produce adverse effects. Borzelleca et al. ( 1988) recently studied the effects of repeated oral exposures (gavage) of rats to barium chloride and demonstrated that large oral doses (300 mg/kg/day, 10 days) induced only marginal effects involving the ovaries (reduced ovary/brain weight ratio) and blood chemistry (reduced BUN). A large range of doses was, therefore, studied to establish a maximum-tolerated dose (MTD). A large dose range was also necessary due to the level of uncertainty regarding the extent of gastrointestinal absorption of barium, which has been estimated to range from 5 to 90% the ingested dose (Wones et ul., 1990; McCauley and Washington, 1983; Clary and Tardiff, 1974; Taylor et al.. 1962). The variability in gastrointestinal absorption of barium is reported to be dependent on a variety of factors including age and the degree of fasting prior to ingestion. There is no information on differences in absorption among species. Due to reports by Ridgeway and Karnofsky ( 1952) indicating the teratogenic effects of barium in chicks, and by Ayre and LeGuerrier (1967), showing possible precancerous effects of barium on uterine cells, we conducted preliminary studies to investigate the ability of barium
to effect male and female reproductive and developmental endpoints. Finally, neurobehavioral endpoints were measured due to reports that barium affects the central nervous system (tremors, convulsions, antinociceptive effects, and microscopic degenerative changes) (Stokinger, 198 1; Welch et al., 1983). MATERIALS
AND METHODS
Clremicul. BaC12* 2H20 (Lot 123 120) was obtained from Baker Chemical Co. (Phillipsburg. NJ) and analyzed by EDTA titration and found to be 99.5% pure. Solutions were made weekly in l9-liter quantities by dissolving weighed portions of the chemical in glass-distilled water. Dosage analyses performed on all levels before and after use, and at the beginning and midway through the test period, indicated that the concentrations were within 1 to 6% of the theoretical concentrations. Animals. Male and female 32day-old Fischer-344/N rats and B6C3F, mice were obtained from Simonsen Laboratories (Gilroy, CA). The animals were quarantined for 10 to 11 days after arrival, and representatives were necropsied to verify that they were grossly free of disease. Animals were randomly assigned to groups of 10 per dose level after weight-sorting them by sex.During both the quarantine and test phases. the animals were housed five per cage in drawer-type polycarbonate cages. The shelves supporting the cages were covered with filter sheets. The bedding (Ab-Sorb-Dri, Lab Products, Rochelle Park. NJ), cages. and water bottles were changed twice a week. feeders once a week, and racks and filters every other week. During changing the racks were rotated clockwise and the cages, which were in columns by dose. were rotated by moving the bottom cage to the top. Fluorescent lighting in the animal room was on for 12 hr (0630 to 1830) and off for 12 hr. Filtered fresh air ( 13.5 room vol/hr) was supplied directly to and removed from the animal room. The temperature range in the room was 21 to 24°C. The animals were fed a diet of NIH-07 pellets (Ziegler Brothers, Gardners. PA) and dosed or undosed water on an ad libitum basis for 92 consecutive days. Treatment and ubservutions. Drinking water contained O.l25,5CKl. 1000. 2000. and 4000 ppm BaClz +2Hz0 and exposures were continuous throughout both the rat and mouse studies. All animals were observed twice daily for clinical signs of toxicity. Body weights were determined weekly and cage water consumptions were measured twice weekly. Serum electrolyte determinations. Blood for serum electrolyte determinations was taken by heart puncture from sodium pentobarbital-anesthetized rats before they were terminated for necropsy. Serum sodium and potassium were measured by flame ion emission using a Coleman Ca-5 I flame photometer (Perkin-Elmer, Oakbrook, IL). Calcium and phosphorus were measured using a Gemeni miniature centrifugal analyzer (ElectroNucleonics. Inc., Fairfield, NJ) in conjunction with a Gemeni loader. Histopathology. All animals were examined for gross lesions and their tissues were fixed in neutral-buffered 10% formalin. The brain. liver, right kidney, lung, thymus. right testis, heart, and adrenals were weighed before fixation. Complete histologic exams were performed on 30 or more tissues from animals of the 4000 ppm and the control groups. Tissues were trimmed, embedded in paraffin. sectioned to 6 brn thickness and stained with hematoxylin and eosin for microscopic examination. Because histopathologic changes were observed in several tissues (thymus, spleen, kidneys, and lymph nodes) from rats and mice in the 4000 ppm group. these tissues were examined from lower dose animals to determine a no-effect level. Behavioral tests. The behavioral test battery to which both rats and mice were subjected consisted of (I ) undifferentiated motor activity, (2) forelimb and hindlimb grip strengths, (3) thermal sensitivity to a 55°C water bath, (4) startle response to acoustic and air-puff stimuli, and (5) hindlimb foot splay. These tests were performed on each animal at 0. 45 to 48, and
TOXICITY
OF BaC&. 2H20 IN MICE AND RATS
91 days of exposure and were performed sequentially in the above order. The tests were performed using established methods (Tests 1, 3, and 4 see Pryor et al., 1983; Test 2, Meyer et al., 1979; Test 5, Edwards and Parker, 1977). For the activity measurements. the movements of each animal were monitored electronically for 30 min. Grip strength and foot splay results were averaged after three trials while thermal sensitivity and startle response tests were conducted one time. The startle response test was repeated up to two times if no response was attained on the first trial. Reproductive and fertility assessment. The mating trials and fertility cytological evaluations were performed on separate groups of rats and mice than used in the core study. Only four dose levels of Bat&. 2H20 were tested: 0, 1000, 2000, and 4000 ppm in rats and 0, 500, 1000, and 2000 ppm in mice. Each group contained 20 animals of each sex. After 60 days of exposure, the males were placed in individual cages and one female receiving the same dose level (but exposed for 30 days) was cohabited with each male for up to 1 week. Each morning following a day of cohabitation. each female was examined for the presence of a vaginal plug (mice) or microscopic evidence of sperm in a vaginal swab (rats). When evidence of mating was found, the female was separated from the male: after mating determinations were made on the eighth day of cohabitation, all remaining pairs were separated. Females were weighed when evidence of mating was found and on the day of parturition. Live offspring were weighed, counted, and examined on Day 0 (day of birth) and again on Day 5. Dead pups were recovered from the nest and examined for external abnormalities. All females were terminated on Days 96 and 97; the vagina, cervix, oviducts, and ovaries were grossly examined and the implantation sites in the uteri were counted. An evaluation of sperm morphology, density, and motility, male reproductive organ weights, and vaginal cytology among treated and control groups were performed according to previously described methods (Morrissey et al.. 1988). Stafistical analyses. Each parameter for which individual values were available was subjected to a linear least squares regression over the dose levels and the direction of the slope and the p value indicating the significance of the deviation of the slope from 0 was determined. Group means and standard deviations or standard errors were calculated for continuous variables. The multiple comparison procedure of Dunnett (1955) was employed for pairwise comparisons of these variables between dosed groups and controls. Fisher’s exact test was used to make pairwise comparisons of discrete variables between dosed groups and controls and the Cochran-Armitage test was used to assessthe significance of dose-related trends (Armitage, 197 I ; Cart et al. 1979). Temporal and dose-related variations were evaluated using a repeated measures analysis of variance (Winer. 197 1). When a collection of measurements were made on each animal, a multivariate analysis of variance (Morrison, 1976) was used to test for the simultaneous equality of measurements across dose levels. RESULTS
Fluid Consumption and Barium Intake Water consumption was decreased slightly in the mice and even more so in rats at some dose levels. For example, mice in the 4000 ppm groups consumed 85% ofthe amounts of water consumed by the controls, whereas rats consumed approximately 70%. The total intake of barium for the 13 weeks and the dosages based on the average daily water consumption and the animals average body weight at the start and completion of the study are shown in Table 1. Mortality and Body Weights Six of 10 male and 7 of 10 female mice in the 4000 ppm groups died during the study (Table 2). Mouse mortalities
529
were initially observed on the thirteenth day of the study. Three of 10 male and 1 of 10 female rats in the 4000 ppm groups died during the last week of the study. Body weights of both sexes and species in the 4000 ppm groups were significantly (p < 0.05) lower than controls. Clinical Signs Except for signs of weight loss in rats and mice and a hunched posture in mice from the 4000 ppm groups, no clinical signs of toxicity were observed in either species. Organ Weights Although there were several statistically significant organ weight changes involving a variety of organs in both species, there were only a few changes considered biologically significant and reflective of barium toxicity. These changes as shown in Table 3 involved the liver, kidney, and thymus. Treatment-related depressed liver weights were observed among rats drinking 4000 ppm and among mice drinking 2000 ppm or greater barium chloride. Absolute kidney weights were depressed among high-dose mice and elevated in the 1000 and 4000 ppm female rats. Relative kidney weights were elevated among treated rats (males, 4000 ppm; females, 1000 ppm or greater) and mice (4000 ppm). Thymus weights were depressed among high-dose (4000 ppm) female rats and mice and male mice who became moribund or died early in the study. Serum Electrolyte Determinations The results of the serum electrolyte determinations in rats are given in Table 4. Similar analyses in mice were not performed. In the male rats, there was a significant elevation in phosphorus in the 1000,2000, and 4000 ppm groups compared with the controls. Calcium, sodium, and potassium were unaffected by BaC12. 2H20 administration. In the females, a significant elevation in phosphorus was seen in the 500, 1000, 2000, and 4000 ppm groups similar to that observed in males. Calcium, sodium, and potassium levels in the treated groups were unaffected. These results were generally similar to those for the males. Histopathologic
Findings
Treatment-related lesions associated with the barium chloride toxicity were present in the kidneys of rats and mice (Table 5). In mice, a mild to marked toxic nephrosis was seen in all high-dose males and females. In the mildly affected kidneys, this lesion was characterized by tubular dilatation that was most prominent in the outer stripe of the outer medulla and extended into medullary rays toward the capsular surface. Dilated tubules contained pale eosinophilic finely granular casts and yellow, refractile crystals. Although tubular cell regeneration was present in foci of tubular di-
DIETZ ET AL.
530
TABLE 1 Per Animal Intake of Barium Given in Drinking Water for 13 Weeks to Mice and Rats
Dose level’ (mm)
Sex
Average daily water consumption (ml)
Total water consumption (ml)
Barium dose bWk/day) Initial
Final
Cumulative barium intake 0-w)
Mice 0 125 500
1000 2000 4000
M F M F M F M F M F M F
6.1 4.8 6.1 4.8 5.8 4.8 5.5 4.4 5.7 4.1 5.2 4.1
561 442 616 442 534 442 506 405 524 377 478 377
0 0
0 0
18.6 17.0 63.9 69.9 122.1 127.5 247.4 233.9 469.4 475.1
12.4 41.9 48.0 82.9 83.0 164.7 165.8 436.2 562.0
0 0
0 0
12.3 10.5 49.5 43.8 94.0 83.1
4.3 5.8 17.0 23.3 32.9 45.4
11.5
0 0 50.8 36.3 176.2 145.9 334.0 267.3 691.7 497.6 1261.9 995.3
Rats 0 125 500
1000 2000 4000
M F M F M F M F M F M F
23.1 16.2 21.8 16.5 22.2 16.2 20.4
15.5 18.7 13.6 16.7
10.8
2125 1490 2006 1518 2042 1490 1877 1426
1720 12.51 1536 994
162.9 147.0 282.3 222.1
61.1 80.9 120.7 136.4
0 0 165.7 125.4 673.9 49 1.7 1238.8 941.2 2270.4 1651.3 4055.0 2624.2
’ Refers to ppm BaCl, . 2H20.
latation, many tubules were decreased in diameter and lined by closely packed epithelial cells with a scant amount of basophilic stained cytoplasm (Fig. 1). Irregular depressions or indentations of the renal capsule were present over these focal areas of collapsed or atrophic tubules. In more severely affected kidneys there was an increased amount of fibrous connective tissue between tubules: most of the outer medulla and cortex consisted of dilated, collapsed, or regenerative tubules. Aggregates of pale yellow-staining crystals were present in tubule lumens at the junction of inner and outer stripe of the outer medulla; crystals were also present in tubules of the cortex and inner medulla. Intratubular crystals were strongly birefi-ingent when examined by polarized light and varied in shape from irregular rounded to elongated and had a smooth or rough, granular surface. Lymphoid depletion was seen in the spleen, thymus, and lymph nodes of early death mice. The kidney changes in rats (Fig. 2) were much less severe than in mice and were limited to a few foci of dilated tubules in the outer medulla or medullary rays. Tubular cell regeneration, casts, and crystals were not a fea-
ture of the renal lesions in rats. Lymphoid depletion was also present in the spleen and thymus of the early death rats. There were no treatment-related histopathologic effects in the brain or other tissues of rats or mice. In one female rat administered 4000 ppm BaC12, there was moderate degeneration of the myocardium. This lesion was not clearly related to treatment; the morphologic features were similar to those seen in the spontaneously occurring cardiomyopathy (minimal to mild severity) which was present in most treated and control male and female rats in this study. Behavioral l@ects Compared to their controls, rats and mice exposed to 2000 ppm BaC12 or lower did not show any consistent changes in behavioral indices (motor activity, fore- and hindlimb grip strength, and thermal sensitivity). Marginal although significant behavioral effects were noted at the 4000 ppm level in rats and mice. These changes were probably a result of the overall BaC12 toxicity observed at the 4000 ppm dose level.
TOXICITY
BaCl*.
2H20
IN
MICE
AND
531
RATS
TABLE 2 and Body Weights of Animals Treated with Barium Chloride Dihydrate for 13 Weeks
Mortality Dose level (mm)
OF
Number dead/total
Sex
Final body weight
Body weight gain k)
k * SD)
Weight difference” v@)
-
Mice 0
M F
O/IO o/10
12.4 10.3
38.1 -t 1.9 29.5 f 2.6
125
M F M F M F M F M F
l/IO o/10 O/IO o/10 O/IO o/10 O/IO o/10 6110 7110
12.3 8.7 12.7 8.5 10.8 9.7 12.0 1.9 1.9 -3.0
37.7 28.5 38.2 27.8 36.1 29.1 37.9 27.6 26.8 16.4
* f f f + f * f + k
4.4 2.5 3.1 3.3 3.4 3.2 3.3 3.2 4.9’ 3.4’
0
M F
o/10 O/IO
210 86
125
M F M F M F M F M F
o/10 o/10 O/IO o/10 O/IO O/IO O/IO O/l0 3/10 l/IO
222 86 234 87 220 83 211 82 174 64
347.7 189.6 352.4 197.1 359.6 190.7 342.2 186.9 339.9 185.6 307.1 173.3
+ -t f + + f i + zk f f f
25.4 8.2 24.4 12.7 19.8 10.8 16.1 10.0 17.4 10.9 13.6b 15.26
500 1000 2000 4000
-1.0 -3.4 -0.3 -5.8 -5.2 -1.4 -0.5 -6.4 -29.7 -44.4
Rats
500 1000 2000 4000
’ Weight difference = weight b Significantly different from
test group - weight control group + weight control control value of the same sex (t test, p < 0.05).
The behavioral effects observed at the 4000 ppm level are as follows: Decreased undifferentiated motor activity in male mice on Days 45-48, female mice on Day 9 1, and female rats on Day 9 1; weaker grip strength in male mice (forelimb) on Days 45-48 and 9 1 and in female mice (fore- and hindlimb) on Days 90 and 9 1; decreased thermal sensitivity as measured by increased tail flick latency in female mice on Day 90. No significant or dose-related effects were seen in the startle response to acoustic and air-puff stimuli or the hindlimb foot splay in either species. Reproductive and Fertility
Evaluation
In the mice, the pregnancy rates were 55% in the controls and ranged from 55 to 70% in the treated groups. Live litters were produced by all pregnant mice, and necropsy revealed no evidence of pregnancy in mice that did not produce viable offspring. The average length of gestation of the control and test mice ranged from 18.5 to 18.9 days. There was no evi-
group
1.3 3.9 3.4 0.6 -1.6 -1.4 -2.2 -2.1 -11.7 -8.6
X 100.
dence of maternal toxicity in the treated mice: maternal weight gain during pregnancy was comparable to controls for all groups. A statistically significant (p < 0.05) reduction was seen in the average live litter size on Days 0 and 5 for mice in the 1000 ppm dose group compared with controls (Day 0, 10.7 + 0.40 pups compared to 7.9 -t 1.02 pups; Day 5, 10.8 ? 0.38 pups compared to 7.7 4 0.97 pups; mean f SEM). The significance of this is unclear, because the average live litter size in the high-dose group (2000 ppm) approximated that of controls. Few pups were found dead at birth in any group, and survival from birth to Postpartum Day 5 ranged from 98 to 100%. No external anomalies were noted in any of the offspring, and there were no statistical differences in live pup body weights. Although the pregnancy rates for the rat studies (from 40% in controls to 65% in the 4000 ppm group) were below the generally accepted norms for reproduction studies, this problem was not corrected by remating due to restrictions
DIETZ
532 Significant Organ Weight Changes” Following
ET
AL
TABLE 3 13-Weeks of Exposure to Barium Chloride in the Drinking Barium
Orean Male mouse Liver Absolute Relative Kidney Absolute Relative Thymus Absolute Relative Body weight Female mouse Liver Absolute Relative Kidney Absolute Relative Thymus Absolute Relative Body weight Male rat Liver Absolute Relative Kidney Absolute Relative Body weight Female rat Liver Absolute Relative Kidney Absolute Relative Thymus Absolute Relative Body weight
125
Control
500
chloride
concentration
Water
(ppm)
1000
2000
4000
2062 + 120 54.2 +_ 3.10
2011 f 776 53.9 + 2.81 b
1892 + 37 49.7 f 1.10
1716 + 64 47.7 f 1.69
1714 + 53 45.3 -t 0.94
1088 +- 86”** 40.8 + 0.98e,*
306 + 8.2 8.04 + 0.15
301 5 12.26 8.06 f o.44b
302 + 10.3 7.95 +- 0.34
286 + 10.4 7.94 + 0.22
291 + 9.5 7.71 + 0.25
235 iz 12.6’,** 8.98 + 0.89’
44.2 +- 3.13 1.16 +- 0.08 38.1 +_ 0.61
50.8 + 5.29’ 1.33 i 0.116 37.8 2 l.49b
46.6 +_ 4.06 1.22 f 0.10 38.2 f 0.99
40.4 +_ 3.12 1.11 20.09 36.1 + 1.09
41.7 + 4.32 1.09 + 0.09 37.9 * 1.04
21.5 +_ 7.44e~* 0.75 r 0.22’1 26.8 -r- 2.46”*
1502 ?I 52 50.9 +. 1.09
1446 5 69 50.6 2 1.40
1375 + 14 49.3 f 1.43
1334 k 53 45.8 + 0.91*
1196 + 37** 43.5 -t 0.91**
753 rt l26”** 45.5 it 2.30d
181 +- 4.1 6.15 + 0.14
183 k 7.5 6.42 + 0.18
180 i 5.6 6.51 + 0.20
188 + 5.9 6.50 zk 0.21
182 zk 7.9 6.61 5 0.22
143 f 28.5d 8.61 + 0.65”**
51.2 k 3.72 1.94 * 0.11 29.5 + 0.83
51.1 f 2.98 1.80f0.11 28.5 f 0.80
46.5 + 2.73 1.68 k 0.09 27.8 f 1.05
52.2 f 3.12 1.79 f 0.09 29.1 + 1.00
47.1 + 1.98 1.72 i: 0.07 27.6 2 1.00
8.33 ‘- 5.36d,** 0.46 +- 0.25d,** 16.4 + 1.98”*
11956 t 439 34.3 t_ 0.65
12033 k 462 34.1 -+ 0.12
12839 _+ 252 35.7 t- 0.36
12572 f 493 36.7 ? 1.17
11549” 324 33.9 + 0.48
1061 + 35 3.05 f 0.05 348 f 9.0
1044 % 31 2.96 2 0.04 352 + 7.1
1097 It 22 3.05 A 0.05 360 + 6.3
1092 + 22 3.19 f 0.03 342 f 5.1
1064 t 25 3.13 -t 0.05 340 I 5.5
1050 + 37’ 3.42 + O.lO’,** 304 f 4.8’,*
5944 + 156 31.3 + 0.58
6439 2 143 32.7 + 0.40
6083 f 98 31.9 f 0.44
5899 + 192 31.5 f 0.68
5858 k 137 31.6 2 0.47
5024 5 173b+* 29.0 t 0.59 ‘,*
570 t 11 3.01 * 0.04
609 k 22 3.09 * 0.09
591 t9 3.10 + 0.04
602 + 13 3.22 f 0.04*
635 zk 14** 3.42 -c 0.05**
620 + 18’ 3.59 f 0.09”**
237 +_ 9.9 1.25 k 0.05 190 +- 2.6
234 t 7.7 1.19 + 0.04 197 2 4.0
254 f 12.1 1.34 + 0.07 191 + 3.4
253 f 13.9 1.35 k 0.07 187 t 3.2
229 -+ 20.2 1.24 t 0.11 186 ?I 3.5
185 + 19.1b.* 1.05 f O.lOb 173 + 5.1&*
a Mean k standard error (absolute ’ Nine animals were examined. ’ Seven animals were examined. d Four animals were examined. ‘Three animals were examined. * p < 0.05. **p
AND
APPLIED
TOXICOLOGY
19,527-537
(1992)
Subchronic Toxicity of Barium Chloride Dihydrate Administered to Rats and Mice in the Drinking Water D. D. DIETZ, M. R. ELWELL, W. E. DAVIS, JR.,* AND E. F. MEIRHENRY* Division of Toxicology Research and Testing, National Institute of Environmental Health Sciences, National Toxicology Program, Research Triangle Park. North Carolina 27709; and *Toxicology Laboratory. SRI International, Menlo Park, California 94025 Received October 23, 199 I; accepted May 6, 1992
Subchronic Toxicity of Barium Chloride Dihydrate Administered to Rats and Mice in the Drinking Water. DIETZ, D. D., M. R., DAVIS, W. E., JR., AND MEIRHENRY, E. F. (1992). Fundam. Appl. Toxicol. 19, 527-537.
ELWELL,
Barium Chloride dihydrate (BaCI,. 2HZO) was given for 92 days to B6C3F1mice and Fischer 344/N rats in their drinking water at levels of 0, 125, 500, 1000, 2000, and 4000 ppm. The no-effect level for this study was2000 ppm BaCl, . 2H20 in the drinking water. At 4000 ppm, daily consumption for mice was 436 to 562 mg/kg barium, up to four times more chemical than rats. Mortality ranged from 60 to 70% in mice and from 10 to 30% in rats in the 4000 ppm groups. Deaths in mice were associated with a treatment-related renal toxicity. Renal lesions in rats were much lessseverethan in miceand did not contribute to the treatment-related deaths seen in the high dose group. Body weights of both speciesand sexesin the 4000 ppm groups were lower than controls at 92 days. Male and female rats in treated groupsexhibited higher serumphosphorusthan controls. Serum sodium, potassium,and calcium levels in rats were unchangedby barium treatment, aswere hematologicalvalues. In both species at 4000 ppm, motor activity, grip strength, and thermal sensitivity were marginally affected. Theseeffects were probably secondary changesresulting from BaCl, toxicity observedat this doselevel. In a mating trial, no anatomical effects on offspring of rats or mice were seen.Rats receiving 4000 ppm exhibited marginal reductions in pup weights. No effects were seenon reproductive indices. o 1992 Society of Toxicology.
Barium occurs in the earth’s crust at an estimated level of 450 ppm and in some drinking water supplies the concentration of barium may exceed 20 ppm (McCauley and Washington, 1983). Elevated barium concentrations in drinking water derived from deep rock and drift wells have been described as a naturally occurring problem (Wones et al., 1990; Calabrese, 1977). The U.S. Environmental Protection Agency (199 1) recently established a revised maximum contaminant level (enforceable federal standard) of 2 ppm barium for public drinking water. Other exposures to barium chloride occur during its use in the manufacture of pigments, in aluminum refining, in sugar refining, in tanning and finishing leather, and as a pesticide.
The toxicity of barium salts is a function of their aqueous solubility (Borzelleca et al., 1988; Syed and Hosain, 1972). Barium sulfate is water insoluble; this nontoxic salt is used in radiology as a radioopaque material (Nielsen, 1986). By contrast, water-soluble salts, such as carbonate, chloride, and acetate, produce a variety of acute toxic effects in humans and experimental animals involving the cardiovascular, gastrointestinal, nervous and hematopoietic systems, and the skeletal muscles (Stokinger, 198 1). The most characteristic toxic signs resulting from barium ingestion involve an intense stimulation of smooth, striated, and cardiac muscle (Brenniman et al., 198 1). Prolonged exposures to barium are reported to produce muscle weakness (Diengott et al., 1964; Phelan and Hagley, 1984; Stokinger, 198 1). Relatively little is known of the toxicological effects resulting from long-term or continuous exposures to soluble barium salts. Schroeder and Mitchener (1975a,b) found no evidence of toxicity in rats and mice exposed during their life span to 5 ppm of barium acetate in the drinking water. Other investigators found no changes in body weight, appearance, and selected organ weights and morphology in rats given 1, 10, and 100 ppm BaCl* for up to 16 months in their drinking water. They did find cardiomyopathy expressed by alterations in physiologic and metabolic parameters at 100 ppm and increased blood pressure in treated animals at 1 month (100 ppm) and 8 months (10 ppm) after the start of exposure. Because the cardiovascular actions of barium mimic those of calcium and digitalis, it has been suggested that Ba+2 acts as a Ca+2 agonist in these tissues (Perry et al., 1989; Brenniman et al., 198 1; Shanbaky et al., 1978). Recent studies have demonstrated that the cardiotoxic properties of barium chloride are blocked by the calcium antagonist verapamil (Mattila ef al., 1986). Tardiff et al. (1980) conducted a 13-week study with BaC12 administered in the drinking water of rats at concentrations ranging from 10 to 250 ppm. Except for a decrease in the relative weight of the adrenals at the 250 ppm level, no adverse effects were found. Izraelson (1967) performed a study in which guinea pigs were given 50 mg/kg BaC12 repeatedly for 30 days by oral administration. He noted treatment-related anemia, leukopenia, and degen-
527 Copyright Ail rights
0272-0590192 $5.00 0 1992 by the Society of Toxicology. of reproduction in any form reserved.
528
DIETZ ET AL.
eration of hepatocytes, myocardial cells, and renal convoluted tubule cells. Several case studies of human barium poisoning have shown that water-soluble barium salts induce hypokalemia and that toxic symptoms involving the cardiovascular system and skeletal muscles (stimulation followed by weakness) are reversed by potassium therapy (Diengott cr al., 1964; Phelan and Hagley, 1984). Roza and Berman (197 1) demonstrated that BaCI, produces hypokalemia in dogs and that most of the cardiotoxic effects of barium were reversed by the administration of potassium. Hypertension was the only effect not responding to treatment. Although the acute toxicity and long-term cardiovascular effects of ingested barium have been extensively described there is continuing concern for the lack of adequate information to evaluate the safety of continuous low level exposures to barium that occur via the drinking water. Thus, Wones et al. (1990) recently reported that “the data on the health effects of drinking water barium are incomplete, mixed, and inadequate.” The importance of this problem is underlined by the fact that the barium content of drinking water in several geographical areas exceed the current drinking water standard of 2 ppm barium (Wones et al., 1990; Calabrese, 1977; Brenniman et al., 1981; EPA, 1991). The present studies were, therefore, initiated in rats and mice to help address the issue of safe levels of barium in the drinking water. The study design was comprehensive in order to establish a maximum-tolerated dose and minimally effective doses using a variety of toxic endpoints. The exposure concentrations were several fold greater than normally encountered in the drinking water and ranged from a level (250 ppm) that did not produce toxicity in a previous 13-week drinking water study (Tardiff et al., 1980) to a level (4000 ppm) expected to produce adverse effects. Borzelleca et al. ( 1988) recently studied the effects of repeated oral exposures (gavage) of rats to barium chloride and demonstrated that large oral doses (300 mg/kg/day, 10 days) induced only marginal effects involving the ovaries (reduced ovary/brain weight ratio) and blood chemistry (reduced BUN). A large range of doses was, therefore, studied to establish a maximum-tolerated dose (MTD). A large dose range was also necessary due to the level of uncertainty regarding the extent of gastrointestinal absorption of barium, which has been estimated to range from 5 to 90% the ingested dose (Wones et ul., 1990; McCauley and Washington, 1983; Clary and Tardiff, 1974; Taylor et al.. 1962). The variability in gastrointestinal absorption of barium is reported to be dependent on a variety of factors including age and the degree of fasting prior to ingestion. There is no information on differences in absorption among species. Due to reports by Ridgeway and Karnofsky ( 1952) indicating the teratogenic effects of barium in chicks, and by Ayre and LeGuerrier (1967), showing possible precancerous effects of barium on uterine cells, we conducted preliminary studies to investigate the ability of barium
to effect male and female reproductive and developmental endpoints. Finally, neurobehavioral endpoints were measured due to reports that barium affects the central nervous system (tremors, convulsions, antinociceptive effects, and microscopic degenerative changes) (Stokinger, 198 1; Welch et al., 1983). MATERIALS
AND METHODS
Clremicul. BaC12* 2H20 (Lot 123 120) was obtained from Baker Chemical Co. (Phillipsburg. NJ) and analyzed by EDTA titration and found to be 99.5% pure. Solutions were made weekly in l9-liter quantities by dissolving weighed portions of the chemical in glass-distilled water. Dosage analyses performed on all levels before and after use, and at the beginning and midway through the test period, indicated that the concentrations were within 1 to 6% of the theoretical concentrations. Animals. Male and female 32day-old Fischer-344/N rats and B6C3F, mice were obtained from Simonsen Laboratories (Gilroy, CA). The animals were quarantined for 10 to 11 days after arrival, and representatives were necropsied to verify that they were grossly free of disease. Animals were randomly assigned to groups of 10 per dose level after weight-sorting them by sex.During both the quarantine and test phases. the animals were housed five per cage in drawer-type polycarbonate cages. The shelves supporting the cages were covered with filter sheets. The bedding (Ab-Sorb-Dri, Lab Products, Rochelle Park. NJ), cages. and water bottles were changed twice a week. feeders once a week, and racks and filters every other week. During changing the racks were rotated clockwise and the cages, which were in columns by dose. were rotated by moving the bottom cage to the top. Fluorescent lighting in the animal room was on for 12 hr (0630 to 1830) and off for 12 hr. Filtered fresh air ( 13.5 room vol/hr) was supplied directly to and removed from the animal room. The temperature range in the room was 21 to 24°C. The animals were fed a diet of NIH-07 pellets (Ziegler Brothers, Gardners. PA) and dosed or undosed water on an ad libitum basis for 92 consecutive days. Treatment and ubservutions. Drinking water contained O.l25,5CKl. 1000. 2000. and 4000 ppm BaClz +2Hz0 and exposures were continuous throughout both the rat and mouse studies. All animals were observed twice daily for clinical signs of toxicity. Body weights were determined weekly and cage water consumptions were measured twice weekly. Serum electrolyte determinations. Blood for serum electrolyte determinations was taken by heart puncture from sodium pentobarbital-anesthetized rats before they were terminated for necropsy. Serum sodium and potassium were measured by flame ion emission using a Coleman Ca-5 I flame photometer (Perkin-Elmer, Oakbrook, IL). Calcium and phosphorus were measured using a Gemeni miniature centrifugal analyzer (ElectroNucleonics. Inc., Fairfield, NJ) in conjunction with a Gemeni loader. Histopathology. All animals were examined for gross lesions and their tissues were fixed in neutral-buffered 10% formalin. The brain. liver, right kidney, lung, thymus. right testis, heart, and adrenals were weighed before fixation. Complete histologic exams were performed on 30 or more tissues from animals of the 4000 ppm and the control groups. Tissues were trimmed, embedded in paraffin. sectioned to 6 brn thickness and stained with hematoxylin and eosin for microscopic examination. Because histopathologic changes were observed in several tissues (thymus, spleen, kidneys, and lymph nodes) from rats and mice in the 4000 ppm group. these tissues were examined from lower dose animals to determine a no-effect level. Behavioral tests. The behavioral test battery to which both rats and mice were subjected consisted of (I ) undifferentiated motor activity, (2) forelimb and hindlimb grip strengths, (3) thermal sensitivity to a 55°C water bath, (4) startle response to acoustic and air-puff stimuli, and (5) hindlimb foot splay. These tests were performed on each animal at 0. 45 to 48, and
TOXICITY
OF BaC&. 2H20 IN MICE AND RATS
91 days of exposure and were performed sequentially in the above order. The tests were performed using established methods (Tests 1, 3, and 4 see Pryor et al., 1983; Test 2, Meyer et al., 1979; Test 5, Edwards and Parker, 1977). For the activity measurements. the movements of each animal were monitored electronically for 30 min. Grip strength and foot splay results were averaged after three trials while thermal sensitivity and startle response tests were conducted one time. The startle response test was repeated up to two times if no response was attained on the first trial. Reproductive and fertility assessment. The mating trials and fertility cytological evaluations were performed on separate groups of rats and mice than used in the core study. Only four dose levels of Bat&. 2H20 were tested: 0, 1000, 2000, and 4000 ppm in rats and 0, 500, 1000, and 2000 ppm in mice. Each group contained 20 animals of each sex. After 60 days of exposure, the males were placed in individual cages and one female receiving the same dose level (but exposed for 30 days) was cohabited with each male for up to 1 week. Each morning following a day of cohabitation. each female was examined for the presence of a vaginal plug (mice) or microscopic evidence of sperm in a vaginal swab (rats). When evidence of mating was found, the female was separated from the male: after mating determinations were made on the eighth day of cohabitation, all remaining pairs were separated. Females were weighed when evidence of mating was found and on the day of parturition. Live offspring were weighed, counted, and examined on Day 0 (day of birth) and again on Day 5. Dead pups were recovered from the nest and examined for external abnormalities. All females were terminated on Days 96 and 97; the vagina, cervix, oviducts, and ovaries were grossly examined and the implantation sites in the uteri were counted. An evaluation of sperm morphology, density, and motility, male reproductive organ weights, and vaginal cytology among treated and control groups were performed according to previously described methods (Morrissey et al.. 1988). Stafistical analyses. Each parameter for which individual values were available was subjected to a linear least squares regression over the dose levels and the direction of the slope and the p value indicating the significance of the deviation of the slope from 0 was determined. Group means and standard deviations or standard errors were calculated for continuous variables. The multiple comparison procedure of Dunnett (1955) was employed for pairwise comparisons of these variables between dosed groups and controls. Fisher’s exact test was used to make pairwise comparisons of discrete variables between dosed groups and controls and the Cochran-Armitage test was used to assessthe significance of dose-related trends (Armitage, 197 I ; Cart et al. 1979). Temporal and dose-related variations were evaluated using a repeated measures analysis of variance (Winer. 197 1). When a collection of measurements were made on each animal, a multivariate analysis of variance (Morrison, 1976) was used to test for the simultaneous equality of measurements across dose levels. RESULTS
Fluid Consumption and Barium Intake Water consumption was decreased slightly in the mice and even more so in rats at some dose levels. For example, mice in the 4000 ppm groups consumed 85% ofthe amounts of water consumed by the controls, whereas rats consumed approximately 70%. The total intake of barium for the 13 weeks and the dosages based on the average daily water consumption and the animals average body weight at the start and completion of the study are shown in Table 1. Mortality and Body Weights Six of 10 male and 7 of 10 female mice in the 4000 ppm groups died during the study (Table 2). Mouse mortalities
529
were initially observed on the thirteenth day of the study. Three of 10 male and 1 of 10 female rats in the 4000 ppm groups died during the last week of the study. Body weights of both sexes and species in the 4000 ppm groups were significantly (p < 0.05) lower than controls. Clinical Signs Except for signs of weight loss in rats and mice and a hunched posture in mice from the 4000 ppm groups, no clinical signs of toxicity were observed in either species. Organ Weights Although there were several statistically significant organ weight changes involving a variety of organs in both species, there were only a few changes considered biologically significant and reflective of barium toxicity. These changes as shown in Table 3 involved the liver, kidney, and thymus. Treatment-related depressed liver weights were observed among rats drinking 4000 ppm and among mice drinking 2000 ppm or greater barium chloride. Absolute kidney weights were depressed among high-dose mice and elevated in the 1000 and 4000 ppm female rats. Relative kidney weights were elevated among treated rats (males, 4000 ppm; females, 1000 ppm or greater) and mice (4000 ppm). Thymus weights were depressed among high-dose (4000 ppm) female rats and mice and male mice who became moribund or died early in the study. Serum Electrolyte Determinations The results of the serum electrolyte determinations in rats are given in Table 4. Similar analyses in mice were not performed. In the male rats, there was a significant elevation in phosphorus in the 1000,2000, and 4000 ppm groups compared with the controls. Calcium, sodium, and potassium were unaffected by BaC12. 2H20 administration. In the females, a significant elevation in phosphorus was seen in the 500, 1000, 2000, and 4000 ppm groups similar to that observed in males. Calcium, sodium, and potassium levels in the treated groups were unaffected. These results were generally similar to those for the males. Histopathologic
Findings
Treatment-related lesions associated with the barium chloride toxicity were present in the kidneys of rats and mice (Table 5). In mice, a mild to marked toxic nephrosis was seen in all high-dose males and females. In the mildly affected kidneys, this lesion was characterized by tubular dilatation that was most prominent in the outer stripe of the outer medulla and extended into medullary rays toward the capsular surface. Dilated tubules contained pale eosinophilic finely granular casts and yellow, refractile crystals. Although tubular cell regeneration was present in foci of tubular di-
DIETZ ET AL.
530
TABLE 1 Per Animal Intake of Barium Given in Drinking Water for 13 Weeks to Mice and Rats
Dose level’ (mm)
Sex
Average daily water consumption (ml)
Total water consumption (ml)
Barium dose bWk/day) Initial
Final
Cumulative barium intake 0-w)
Mice 0 125 500
1000 2000 4000
M F M F M F M F M F M F
6.1 4.8 6.1 4.8 5.8 4.8 5.5 4.4 5.7 4.1 5.2 4.1
561 442 616 442 534 442 506 405 524 377 478 377
0 0
0 0
18.6 17.0 63.9 69.9 122.1 127.5 247.4 233.9 469.4 475.1
12.4 41.9 48.0 82.9 83.0 164.7 165.8 436.2 562.0
0 0
0 0
12.3 10.5 49.5 43.8 94.0 83.1
4.3 5.8 17.0 23.3 32.9 45.4
11.5
0 0 50.8 36.3 176.2 145.9 334.0 267.3 691.7 497.6 1261.9 995.3
Rats 0 125 500
1000 2000 4000
M F M F M F M F M F M F
23.1 16.2 21.8 16.5 22.2 16.2 20.4
15.5 18.7 13.6 16.7
10.8
2125 1490 2006 1518 2042 1490 1877 1426
1720 12.51 1536 994
162.9 147.0 282.3 222.1
61.1 80.9 120.7 136.4
0 0 165.7 125.4 673.9 49 1.7 1238.8 941.2 2270.4 1651.3 4055.0 2624.2
’ Refers to ppm BaCl, . 2H20.
latation, many tubules were decreased in diameter and lined by closely packed epithelial cells with a scant amount of basophilic stained cytoplasm (Fig. 1). Irregular depressions or indentations of the renal capsule were present over these focal areas of collapsed or atrophic tubules. In more severely affected kidneys there was an increased amount of fibrous connective tissue between tubules: most of the outer medulla and cortex consisted of dilated, collapsed, or regenerative tubules. Aggregates of pale yellow-staining crystals were present in tubule lumens at the junction of inner and outer stripe of the outer medulla; crystals were also present in tubules of the cortex and inner medulla. Intratubular crystals were strongly birefi-ingent when examined by polarized light and varied in shape from irregular rounded to elongated and had a smooth or rough, granular surface. Lymphoid depletion was seen in the spleen, thymus, and lymph nodes of early death mice. The kidney changes in rats (Fig. 2) were much less severe than in mice and were limited to a few foci of dilated tubules in the outer medulla or medullary rays. Tubular cell regeneration, casts, and crystals were not a fea-
ture of the renal lesions in rats. Lymphoid depletion was also present in the spleen and thymus of the early death rats. There were no treatment-related histopathologic effects in the brain or other tissues of rats or mice. In one female rat administered 4000 ppm BaC12, there was moderate degeneration of the myocardium. This lesion was not clearly related to treatment; the morphologic features were similar to those seen in the spontaneously occurring cardiomyopathy (minimal to mild severity) which was present in most treated and control male and female rats in this study. Behavioral l@ects Compared to their controls, rats and mice exposed to 2000 ppm BaC12 or lower did not show any consistent changes in behavioral indices (motor activity, fore- and hindlimb grip strength, and thermal sensitivity). Marginal although significant behavioral effects were noted at the 4000 ppm level in rats and mice. These changes were probably a result of the overall BaC12 toxicity observed at the 4000 ppm dose level.
TOXICITY
BaCl*.
2H20
IN
MICE
AND
531
RATS
TABLE 2 and Body Weights of Animals Treated with Barium Chloride Dihydrate for 13 Weeks
Mortality Dose level (mm)
OF
Number dead/total
Sex
Final body weight
Body weight gain k)
k * SD)
Weight difference” v@)
-
Mice 0
M F
O/IO o/10
12.4 10.3
38.1 -t 1.9 29.5 f 2.6
125
M F M F M F M F M F
l/IO o/10 O/IO o/10 O/IO o/10 O/IO o/10 6110 7110
12.3 8.7 12.7 8.5 10.8 9.7 12.0 1.9 1.9 -3.0
37.7 28.5 38.2 27.8 36.1 29.1 37.9 27.6 26.8 16.4
* f f f + f * f + k
4.4 2.5 3.1 3.3 3.4 3.2 3.3 3.2 4.9’ 3.4’
0
M F
o/10 O/IO
210 86
125
M F M F M F M F M F
o/10 o/10 O/IO o/10 O/IO O/IO O/IO O/l0 3/10 l/IO
222 86 234 87 220 83 211 82 174 64
347.7 189.6 352.4 197.1 359.6 190.7 342.2 186.9 339.9 185.6 307.1 173.3
+ -t f + + f i + zk f f f
25.4 8.2 24.4 12.7 19.8 10.8 16.1 10.0 17.4 10.9 13.6b 15.26
500 1000 2000 4000
-1.0 -3.4 -0.3 -5.8 -5.2 -1.4 -0.5 -6.4 -29.7 -44.4
Rats
500 1000 2000 4000
’ Weight difference = weight b Significantly different from
test group - weight control group + weight control control value of the same sex (t test, p < 0.05).
The behavioral effects observed at the 4000 ppm level are as follows: Decreased undifferentiated motor activity in male mice on Days 45-48, female mice on Day 9 1, and female rats on Day 9 1; weaker grip strength in male mice (forelimb) on Days 45-48 and 9 1 and in female mice (fore- and hindlimb) on Days 90 and 9 1; decreased thermal sensitivity as measured by increased tail flick latency in female mice on Day 90. No significant or dose-related effects were seen in the startle response to acoustic and air-puff stimuli or the hindlimb foot splay in either species. Reproductive and Fertility
Evaluation
In the mice, the pregnancy rates were 55% in the controls and ranged from 55 to 70% in the treated groups. Live litters were produced by all pregnant mice, and necropsy revealed no evidence of pregnancy in mice that did not produce viable offspring. The average length of gestation of the control and test mice ranged from 18.5 to 18.9 days. There was no evi-
group
1.3 3.9 3.4 0.6 -1.6 -1.4 -2.2 -2.1 -11.7 -8.6
X 100.
dence of maternal toxicity in the treated mice: maternal weight gain during pregnancy was comparable to controls for all groups. A statistically significant (p < 0.05) reduction was seen in the average live litter size on Days 0 and 5 for mice in the 1000 ppm dose group compared with controls (Day 0, 10.7 + 0.40 pups compared to 7.9 -t 1.02 pups; Day 5, 10.8 ? 0.38 pups compared to 7.7 4 0.97 pups; mean f SEM). The significance of this is unclear, because the average live litter size in the high-dose group (2000 ppm) approximated that of controls. Few pups were found dead at birth in any group, and survival from birth to Postpartum Day 5 ranged from 98 to 100%. No external anomalies were noted in any of the offspring, and there were no statistical differences in live pup body weights. Although the pregnancy rates for the rat studies (from 40% in controls to 65% in the 4000 ppm group) were below the generally accepted norms for reproduction studies, this problem was not corrected by remating due to restrictions
DIETZ
532 Significant Organ Weight Changes” Following
ET
AL
TABLE 3 13-Weeks of Exposure to Barium Chloride in the Drinking Barium
Orean Male mouse Liver Absolute Relative Kidney Absolute Relative Thymus Absolute Relative Body weight Female mouse Liver Absolute Relative Kidney Absolute Relative Thymus Absolute Relative Body weight Male rat Liver Absolute Relative Kidney Absolute Relative Body weight Female rat Liver Absolute Relative Kidney Absolute Relative Thymus Absolute Relative Body weight
125
Control
500
chloride
concentration
Water
(ppm)
1000
2000
4000
2062 + 120 54.2 +_ 3.10
2011 f 776 53.9 + 2.81 b
1892 + 37 49.7 f 1.10
1716 + 64 47.7 f 1.69
1714 + 53 45.3 -t 0.94
1088 +- 86”** 40.8 + 0.98e,*
306 + 8.2 8.04 + 0.15
301 5 12.26 8.06 f o.44b
302 + 10.3 7.95 +- 0.34
286 + 10.4 7.94 + 0.22
291 + 9.5 7.71 + 0.25
235 iz 12.6’,** 8.98 + 0.89’
44.2 +- 3.13 1.16 +- 0.08 38.1 +_ 0.61
50.8 + 5.29’ 1.33 i 0.116 37.8 2 l.49b
46.6 +_ 4.06 1.22 f 0.10 38.2 f 0.99
40.4 +_ 3.12 1.11 20.09 36.1 + 1.09
41.7 + 4.32 1.09 + 0.09 37.9 * 1.04
21.5 +_ 7.44e~* 0.75 r 0.22’1 26.8 -r- 2.46”*
1502 ?I 52 50.9 +. 1.09
1446 5 69 50.6 2 1.40
1375 + 14 49.3 f 1.43
1334 k 53 45.8 + 0.91*
1196 + 37** 43.5 -t 0.91**
753 rt l26”** 45.5 it 2.30d
181 +- 4.1 6.15 + 0.14
183 k 7.5 6.42 + 0.18
180 i 5.6 6.51 + 0.20
188 + 5.9 6.50 zk 0.21
182 zk 7.9 6.61 5 0.22
143 f 28.5d 8.61 + 0.65”**
51.2 k 3.72 1.94 * 0.11 29.5 + 0.83
51.1 f 2.98 1.80f0.11 28.5 f 0.80
46.5 + 2.73 1.68 k 0.09 27.8 f 1.05
52.2 f 3.12 1.79 f 0.09 29.1 + 1.00
47.1 + 1.98 1.72 i: 0.07 27.6 2 1.00
8.33 ‘- 5.36d,** 0.46 +- 0.25d,** 16.4 + 1.98”*
11956 t 439 34.3 t_ 0.65
12033 k 462 34.1 -+ 0.12
12839 _+ 252 35.7 t- 0.36
12572 f 493 36.7 ? 1.17
11549” 324 33.9 + 0.48
1061 + 35 3.05 f 0.05 348 f 9.0
1044 % 31 2.96 2 0.04 352 + 7.1
1097 It 22 3.05 A 0.05 360 + 6.3
1092 + 22 3.19 f 0.03 342 f 5.1
1064 t 25 3.13 -t 0.05 340 I 5.5
1050 + 37’ 3.42 + O.lO’,** 304 f 4.8’,*
5944 + 156 31.3 + 0.58
6439 2 143 32.7 + 0.40
6083 f 98 31.9 f 0.44
5899 + 192 31.5 f 0.68
5858 k 137 31.6 2 0.47
5024 5 173b+* 29.0 t 0.59 ‘,*
570 t 11 3.01 * 0.04
609 k 22 3.09 * 0.09
591 t9 3.10 + 0.04
602 + 13 3.22 f 0.04*
635 zk 14** 3.42 -c 0.05**
620 + 18’ 3.59 f 0.09”**
237 +_ 9.9 1.25 k 0.05 190 +- 2.6
234 t 7.7 1.19 + 0.04 197 2 4.0
254 f 12.1 1.34 + 0.07 191 + 3.4
253 f 13.9 1.35 k 0.07 187 t 3.2
229 -+ 20.2 1.24 t 0.11 186 ?I 3.5
185 + 19.1b.* 1.05 f O.lOb 173 + 5.1&*
a Mean k standard error (absolute ’ Nine animals were examined. ’ Seven animals were examined. d Four animals were examined. ‘Three animals were examined. * p < 0.05. **p
