Boron in drinking-water
Background document for development of WHO Guidelines for Drinking-water Quality
Effects on experimental animals and in vitro test systems
The oral LD50 values for boric acid or borax in mice and rats are in the range of about 400–700 mg of boron per kg of body weight (Pfeiffer et al., 1945; Weir & Fisher, 1972). Oral LD50 values in the range of 250–350 mg of boron per kg of body weight for boric acid or borax exposure have been reported for guinea-pigs, dogs, rabbits, and cats (Pfeiffer et al., 1945; Verbitskaya, 1975). Signs of acute toxicity for both borax and boric acid in animals given single large doses orally include depression, ataxia, convulsions, and death; kidney degeneration and testicular atrophy are also observed (Larsen, 1988).
In a 13-week study, mice (10 per sex per dose) were fed diets containing boric acid at approximately 0, 34, 70, 141, 281, or 563 mg of boron per kg of body weight per day. At the two highest doses, increased mortality was seen. Degeneration or atrophy of the seminiferous tubules was observed at 141 mg of boron per kg of body weight per day. In all dose groups, extramedullary haematopoiesis of the spleen of minimal to mild severity was seen (NTP, 1987).
In a study in which borax was given in the diet to male Sprague-Dawley rats (18 per dose) at concentrations of 0, 500, 1000, or 2000 mg of boron per kg of feed (approximately equal to 0, 30, 60, or 125 mg of boron per kg of body weight per day) for 30 or 60 days, body weights were not consistently affected by treatment. Organ weights were not affected by 500 mg of boron per kg of feed; at 1000 and 2000 mg of boron per kg of feed, absolute liver weights were significantly lower after 60 days, and epididymal weights were significantly lower (37.6% and 34.8%, respectively) after 60 days, but not after 30 days. Weights of prostate, spleen, kidney, heart, and lung were not changed at any dose (Lee et al., 1978).
In a 90-day study in rats (10 per sex per dose) receiving 0, 2.6, 8.8, 26, 88, or 260 mg of boron per kg of body weight per day in the diet as boric acid or borax, all animals at the highest dose died within 3–6 weeks (Weir & Fisher, 1972). In animals receiving 88 mg of boron per kg of body weight per day, body weights in males and females were reduced; absolute organ weights, including the liver, spleen, kidneys, brain, adrenals, and ovaries, were also significantly decreased in this group. Organ-to-body-weight ratios for the adrenals and kidneys were significantly increased, but relative weights of the liver and ovaries were decreased. A pronounced reduction in testicular weights in males in the 88 mg of boron per kg of body weight per day group was also observed.
Boric acid or borax was also fed to beagle dogs for 90 days or for 2 years. In the 90-day boric acid study (weight-normalized doses of 0, 0.44, 4.4, or 44 mg of boron per kg of body weight per day; five animals per sex per dose), testis weight was significantly lower than controls in the middle and upper dose groups (reduced by 25% and 40%, respectively). Although testicular microscopic structure was not detectably abnormal in the controls and middle dose group, four of five dogs in the high-dose group had complete atrophy, and the remaining high-dose dog had one-third of tubules showing some abnormality. In the borax study, testis weights in the low-, middle-, and high-dose groups were 80%, 85%, and 50% of controls, respectively; only the last was significantly different from controls. No mention was made of the testicular microscopic structure of the controls or low-dose animals; middle-dose animals were not detectably altered (aside from the considerable fixation-induced artifact in the outer third of the tissue), whereas four of five high-dose dogs had complete testicular atrophy, and the remaining high-dose dog had "partial" atrophy. No other clinical or microscopic signs of toxicity were reported in any animals (Weir & Fisher, 1972).
In the 2-year study, the dogs (four per sex per dose) received the boric acid or borax in the diet at weight-normalized doses of 0, 1.5, 2.9, or 8.8 mg of boron per kg of body weight per day. An additional group received 29 mg of boron per kg of body weight per day for 38 weeks. Testicular atrophy was observed in two test dogs receiving borax at 26 weeks and in the two and one dogs, respectively, killed after 26 or 38 weeks of boric acid consumption. The authors stated that boric acid caused testicular degeneration in dogs, including spermatogenic arrest and atrophy of the seminiferous epithelium. The study was terminated at 38 weeks. In these studies, the number of dogs was small and variable (one or two dogs at each of three time points) and inadequate to allow statistical analysis. All three treated dogs had widespread and marked atrophy in 25–40% of the seminiferous tubules. A common control group was used for both the borax and boric acid studies. Testicular lesions occurred in the controls (one of four controls had slight to severe seminiferous tubular atrophy, another had moderate to severe atrophy, whereas a third had a detectable but insignificant reduction in spermatogenesis and 5% atrophic seminiferous tubules) (Weir & Fisher, 1972). These studies were conducted before the advent of Good Laboratory Practices (GLPs). Confidence in these studies is low, and they were considered not suitable for inclusion into the risk assessment because of 1) small and variable numbers of dogs, 2) variable background lesions in controls leading to uncertainty of the strength of the response to treatment, 3) lack of GLPs, and 4) other, more recent studies of greater scientific quality with findings at similar intake levels of boron (Ku et al., 1993; Price et al., 1996a).
A 2-year study in mice (50 per sex per dose) receiving approximately 0, 275, or 550 mg of boric acid per kg of body weight per day (0, 48, or 96 mg of boron per kg of body weight per day) in the diet (NTP, 1987; Dieter, 1994) demonstrated that body weights were 10–17% lower in high-dose males after 32 weeks and in high-dose females after 52 weeks. Increased mortality rates were statistically significant in males, with significant lesions in male mice appearing in the testes and no significant non-neoplastic lesions in female mice.
In a 2-year study, rats (35 per sex per dose) were administered weight-normalized boron doses of 0, 5.9, 18, or 59 mg/kg of body weight per day in the diet (Weir & Fisher, 1972). High-dose animals had coarse hair coats, scaly tails, hunched posture, swollen and desquamated pads of the paws, abnormally long toenails, shrunken scrotum, inflamed eyelids, and bloody eye discharge. The haematocrit and haemoglobin levels were significantly lower than controls, the absolute and relative weights of the testes were significantly lower, and relative weights of the brain and thyroid gland were higher than in controls. In animals in the mid- and low-dose groups, no significant effects on general appearance, behaviour, growth, food consumption, haematology, serum chemistry, or histopathology were observed.
Reproductive and developmental toxicity
Short- and long-term oral exposures to boric acid or borax in laboratory animals have demonstrated that the male reproductive tract is a consistent target of toxicity. Testicular lesions have been observed in rats, mice, and dogs administered boric acid or borax in food or drinking-water (Truhaut et al., 1964; Weir & Fisher, 1972; Green et al., 1973; Lee et al., 1978; NTP, 1987; Ku et al., 1993). The first clinical indication of testicular toxicity in dogs is shrunken scrota observed during treatment; significant decreases in absolute and relative testicular weight are also reported. After subchronic exposure, the histopathological effects range from inhibited spermiation (sperm release) to degeneration of the seminiferous tubules with variable loss of germ cells to complete absence of germ cells, resulting in atrophy and transient or irreversible loss of fertility, but not of mating behaviour.
In time–response and dose–response reproductive studies (Linder et al., 1990), adult male Sprague-Dawley rats were administered two doses in one day, with a total dose of 0 or 350 mg of boron per kg of body weight in the time–response experiment (animals were sacrificed at 2, 14, 28, or 57 days post-treatment) and a total dose of 0, 44, 87, 175, or 350 mg of boron per kg of body weight in the dose–response experiment (animals were sacrificed after 14 days). Adverse effects on spermiation, epididymal sperm morphology, and caput sperm reserves were observed during histopathological examinations of the testes and epididymis. The NOAEL for male reproductive effects in the dose–response study was 87 mg of boron per kg of body weight per day.
In a multi-generation study, doses of 0, 117, 350, or 1170 mg of boron per kg of feed (as borax or boric acid) were administered to male and female rats (Weir & Fisher, 1972). At the highest dose, rats were found to be sterile, males showed atrophied testes in which spermatozoa were absent, and females showed decreased ovulation. The NOAEL in this study was 350 mg of boron per kg of feed, equivalent to 17.5 mg of boron per kg of body weight per day.
To investigate the development of testicular lesions, boric acid was fed at 61 mg of boron per kg of body weight per day to male F344 rats; sacrifice of six treated and four control rats was conducted at intervals from 4 to 28 days. At 28 days, there was significant loss of spermatocytes and spermatids from all tubules in exposed rats, and basal serum testosterone levels were significantly decreased from 4 days on (Treinen & Chapin, 1991). In another study, the activities of enzymes found primarily in spermatogenic cells were decreased, and enzyme activities associated with premeiotic spermatogenic cells were significantly increased in rats exposed to 60 or 125 mg of boron per kg of body weight per day for 60 days (Lee et al., 1978). Mean plasma follicle-stimulating hormone levels were significantly elevated in a dose-dependent manner in all treatment groups (30, 60, or 125 mg of boron per kg of body weight per day) in this study after 60-day exposures.
Reversibility of testicular lesions was evaluated by Ku et al. (1993) in an experiment in which F344 rats were dosed at 0, 3000, 4500, 6000, or 9000 mg of boric acid per kg of feed (equivalent to 0, 26, 39, 52, or 78 mg of boron per kg of body weight per day) for 9 weeks and assessed for recovery up to 32 weeks post-treatment. Inhibited spermiation was exhibited at 3000 and 4500 mg of boric acid per kg of feed (5.6 µg of boron per mg of tissue), whereas inhibited spermiation progressed to atrophy at 6000 and 9000 mg of boric acid per kg of feed (11.9 µg of boron per mg of testes); there was no boron accumulation in the testes to levels greater than those found in the blood during the 9-week period. After treatment, serum and testis boron levels in all dose groups fell to background levels. Inhibited spermiation at 4500 mg of boric acid per kg of feed was reversed by 16 weeks post-treatment, but focal atrophy, which did not recover up to 32 weeks post-treatment, was detected.
Developmental toxicity has been demonstrated experimentally in rats, mice, and rabbits (NTP, 1990; Heindel et al., 1992; Price et al., 1996b). Rats were fed a diet containing 0, 14, 29, or 58 mg of boron per kg of body weight per day as boric acid on gestation days 0–20 (Heindel et al., 1992). An additional group of rats received boric acid at 94 mg of boron per kg of body weight per day on gestation days 6–15 only. Average fetal body weight per litter was significantly reduced in a dose-related manner in all treated groups compared with controls. The percentage of malformed fetuses per litter and the percentage of litters with at least one malformed fetus were significantly increased at =29 mg of boron per kg of body weight per day. Malformations consisted primarily of anomalies of the eyes, the central nervous system (CNS), the cardiovascular system, and the axial skeleton. The most common malformations were enlargement of lateral ventricles in the brain and agenesis or shortening of rib XIII. The LOAEL of 14 mg of boron per kg of body weight per day (the lowest dose tested) for rats occurred in the absence of maternal toxicity; a NOAEL was not found in this study.
Price et al. (1996a) did a follow-up to the Heindel et al. (1992) study in Sprague-Dawley (CD) rats to determine a NOAEL for fetal body-weight reduction and to determine whether the offspring would recover from prenatally reduced body weight during postnatal development. Boric acid was administered in the diet to CD rats on gestation days 0–20. Dams were terminated and uterine contents examined on gestation day 20. The intake of boric acid was 0, 3.3, 6.3, 9.6, 13, or 25 mg of boron per kg of body weight per day. Fetal body weights were 99, 98, 97, 94, and 88% of controls for the low- to high-dose groups, respectively. Incidences of short rib XIII (a malformation) or wavy rib (a variation) were increased in the 13 and 25 mg of boron per kg of body weight per day dose groups relative to control litters. There was a decreased incidence of rudimentary extra rib on lumbar 1 (a variation) in the high-dose group that was deemed biologically but not statistically significant. The NOAEL in this study was 9.6 mg of boron per kg of body weight per day, based on a decrease in fetal body weight at the next higher dose.
Developmental toxicity and teratogenicity of boric acid in mice at 0, 43, 79, or 175 mg of boron per kg of body weight per day in the diet were investigated (Heindel et al., 1992). There was a significant dose-related decrease in average fetal body weight per litter at 79 and 175 mg of boron per kg of body weight per day. In offspring of mice exposed to 79 or 175 mg of boron per kg of body weight per day during gestation days 0–20, there was an increased incidence of skeletal (rib) malformations. These changes occurred at doses for which there were also signs of maternal toxicity (increased kidney weight and pathology); the LOAEL for developmental effects (decreased fetal body weight per litter) was 79 mg of boron per kg of body weight per day, and the NOAEL was 43 mg of boron per kg of body weight per day.
Developmental toxicity and teratogenicity of boric acid in rabbits were investigated by Price et al. (1996b) at doses of 0, 11, 22, or 44 mg of boron per kg of body weight per day, given by gavage. Frank developmental effects in rabbits exposed to 44 mg of boron per kg of body weight per day included a high rate of prenatal mortality, an increased number of pregnant females with no live fetuses, and fewer live fetuses per live litter on day 30. At the high dose, malformed live fetuses per litter increased significantly, primarily because of the incidence of fetuses with cardiovascular defects, the most prevalent of which was interventricular septal defect. Skeletal variations observed were extra rib on lumbar 1 and misaligned sternebra. The NOAEL for maternal and developmental effects was 22 mg of boron per kg of body weight per day.
Mutagenicity and related end-points
The mutagenic activity of boric acid was examined in the Salmonella typhimurium and mouse lymphoma assays, with negative results. No induction of sister chromatid exchange or chromosomal aberrations was observed in Chinese hamster ovary cells (NTP, 1987). Sodium borate did not cause gene mutations in the S. typhimurium preincubation assay (Benson et al., 1984). Borax was not mutagenic in cell transformation assays with Chinese hamster cells, mouse embryo cells, and human fibroblasts (Landolph, 1985).
Tumour incidence was not enhanced in studies in which B6C3F1 mice received 0, 2500, or 5000 mg of boric acid per kg of feed for 103 weeks (NTP, 1987) and Sprague-Dawley rats received diets containing 0, 117, 350, or 1170 mg of boron per kg of feed (as borax or boric acid) for 2 years (Weir & Fisher, 1972).