Warren W. Ku

Testicular toxicity of boric acid (BA): Relationship of dose to lesion development and recovery in the F344 rat

High-dose boric acid (BA) produces testicular lesions in adult rats, characterized by inhibited spermiation followed by atrophy. The present study addressed whether inhibited spermiation can be separated from atrophy based on dose, compared testis boron (B) dosimetry to lesion development, determined how inhibited spermiation was reflected by common reproductive endpoints, and examined reversibility of the testicular lesions. Rats were fed 3000, 4500, 6000, or 9000 ppm BA for up to 9 weeks and examined. Recovery was assessed for up to 32 weeks post treatment. Inhibited spermiation could be separated from atrophy based on dose (inhibited spermiation: 3000/4500 ppm; atrophy: 6000/9000 ppm), with each lesion aspect expressed at different threshold testis B concentrations (inhibited spermiation: 5.6 micrograms B/g and atrophy: 11.9 micrograms B/g) with no B accumulation during the 9-week exposure. These data suggest that separate mechanisms may be operating for these lesion aspects based on testis B concentration and that B dose rate was important for testicular toxicity. Inhibited spermiation was most reliably reflected by informed testicular histology, with the more severe cases decreasing epididymal sperm count to levels that could affect fertility. After treatment, serum and testis B levels in all dose groups rapidly fell to background levels at the earliest time points evaluated (7 days and 8 weeks posttreatment, respectively). The severely inhibited spermiation at 4500 ppm was resolved by 16 weeks posttreatment, but areas of focal atrophy were detected that did not recover posttreatment. Also, no signs of recovery from atrophy were observed (6000 and 9000 ppm). Atrophic tubules contained a normal complement of spermatogonia (2.6 to 2.9 germ cells/100 Sertoli cells), with occasional dividing and degenerating germ cells. Elevations in serum FSH and LH levels suggested an intact hormonal response to the atrophy. In summary, 1) the different aspects of the BA-induced testicular lesion can be separated using different doses, 2) inhibited spermiation does not necessarily proceed to atrophy, and 3) there is no recovery from the atrophy despite the absence of testis B after treatment. The ability to separate inhibited spermiation from atrophy based on dose and testis B dosimetry will be useful in evaluating possible mechanisms. Furthermore, the presence of dividing spermatogonia during long-term BA-induced atrophy suggests that this model should be useful for identifying critical components involved in the reinitiation of spermatogenesis.

Tissue disposition of boron in male Fischer rats

Boric acid (H3BO3), an inorganic acid with widespread commercial use and consumer exposure, impairs fertility in male rodents at dose levels lower than those required to cause other adverse effects. Previous studies found a testicular lesion in adult Fischer rats fed 9000 ppm boric acid (1575 ppm boron) and slightly reduced basal serum testosterone levels. A CNS-mediated hormonal component to this lesion was suggested. Detailed data on the tissue disposition of boron in the rat, including accessory sex organs and the brain, are lacking. This study examined the tissue disposition of boron in reproductive, accessory sex organs, and other selected tissues in adult male Fischer rats fed 9000 ppm boric acid to determine if selective accumulation of boron in reproductive tissues, accessory sex organs, and/or the brain might correlate with and explain the apparent selective testicular toxicity. Adult male Fischer rats were fed 9000 ppm boric acid for up to 7 days. Animals were killed at 1, 2, 3, 4, and 7 days after the start of exposure. Plasma and excised tissues were heat-digested in acid and analyzed for boron by inductively coupled argon plasma emission spectrometry (ICAP). With the exception of adrenal glands, control boron levels in all tissues examined were below 4 micrograms/g. There was a rapid increase in plasma and tissue boron 1 day after the start of exposure (range 2- to 20-fold), with the exception of adipose tissue. With the exception of bone and adipose tissue, all soft tissues examined, including the testis, epididymis, accessory sex organs, hypothalamus, and rest of brain, appeared to reach steady-state boron levels (range 12-30 micrograms/g) by 3-4 days. Bone boron levels continued to increase up to the termination at 7 days (40-50 micrograms/g by Day 7). Bone attained the greatest concentration of boron (2- to 3-fold over plasma levels) while levels in adipose tissue were 20% of plasma levels during the 7-day exposure period. All other tissues appeared to show no appreciable accumulation of boron over plasma levels. The data suggest that neither the apparent selective testicular toxicity nor the slight CNS hormonal effect associated with boric acid exposure can be explained on the basis of selective accumulation of boron in the testis or brain/hypothalamus, respectively. Thus, the testicular toxicity is likely the result of certain biological processes that are unique to the testis and which are targets of boron exposure.

The Effects of Dietary Boron on Bone Strength in Rats

Previous studies from our laboratory found that when boric acid (BA) was administered in the diet to rats, boron levels in bone were approximately fourfold greater than serum levels. The current studies were undertaken to determine if these elevations produced adverse effects on several bone-related measures, including serum electrolyte levels, bone structure, and bone strength. Data from two studies are presented: in the first study, young adult male rats consumed a powdered diet containing 0, 3000, 4500, 6000, or 9000 ppm BA for 9 weeks. Endpoints were serum calcium, phosphorous, potassium, and chloride, as well as blood and bone boron concentrations ([B]) measured weekly during the 9-week exposure period, and at 8, 16, 24, and 32 weeks after the end of exposure. In the second study, the male and female young adult rats diet contained 0, 200, 1000, 3000, or 9000 ppm BA for 12 weeks; endpoints measured weekly were serum levels of calcium, phosphorous, and magnesium, bone [B], and bone structure (humerus) and strength (tibia, femur, and lumbar vertebrae). In treated rats, calcium was reduced in the first study but not the second. Serum phosphorous was reduced in both studies; potassium was unchanged, chloride was increased by 1%, and magnesium was reduced in all BA-exposed groups in the second study, to a maximal 19% reduction. Bone [B] was consistently increased in all treated groups, to concentrations approximately fourfold those of serum. After cessation of exposure, serum and urinary boron concentrations dropped to within control values within a week. However, even 32 weeks after the end of exposure, bone [B] remained threefold greater than controls. Male tibia and femur resistance to bending was unchanged. However, vertebral strength in compression was significantly increased by 5-10% in all dose groups (200 to 9000 ppm). The pattern was substantially similar in females. Only the humerus was examined by light microscopy and was found to be unchanged at any level of BA consumption. These data show that, despite a reduction in some serum electrolyte levels, BA consumption increased vertebral resistance to crush force, without detectably altering the microscopic structure of the humerus or the resistance of femur and tibia to a bending load. This increase in compression resistance occurred at exposure levels substantially below those that were previously reported to be reproductively toxic.

The effects of dietary boric acid on bone strength in rats

The effects of dietary boron (B) (from boric acid [BA]) on bone strength were evaluated using male F344 rats. B was administered by dietary admixture of BA to NIH-07 feed at concentrations of 200, 1000, 3000, and 9000 ppm. The latter two levels were found in previous studies to be reproductively toxic to both males and the developing fetus. The first two levels are below and just at, respectively, the levels for producing fetal malformations, and are below the dose required to produce male reproductive toxicity.Resistance to destructive testing was measured on femora, tibiae, and lumbar vertebrae. Although femur and tibia resistance to bending force were not affected by any amount of dietary B, vertebral resistance to a crushing force was increased by ≈10%, at all dose levels (200-9000 ppm). These data show that even levels of BA that are not reproductively toxic can affect the strength of the axial skeleton in rats.

THE EFFECTS OF BORIC ACID (BA) ON TESTICULAR CELLS IN CULTURE

High-dose boric acid (BA) exposure produces testicular lesions in adult rats characterized by inhibited spermiation that may progress to nonrecoverable atrophy. The mechanism for the testicular toxicity of BA is unknown. To examine possible direct effects, the present study evaluated selected aspects of various testicular cell culture systems after in vitro BA exposure. Specifically, the hallmarks of the BA testicular toxicity were addressed: the mild hormone effect, the initial inhibition of spermiation, and atrophy. No effect of BA on the steroidogenic function of isolated Leydig cells was observed, supporting the contention of a CNS-mediated rather than a direct hormone effect. Since increased testicular cyclic AMP (cAMP) produces inhibited spermiation, and a role for the serine proteases plasminogen activators (PAs) in spermiation has been proposed, we evaluated both Sertoli cell cAMP accumulation in Sertoli-germ cell cocultures and the stage-specific secretion of PA activity in cultured seminiferous tubules after in vitro BA exposure, respectively. The results showed that the inhibited spermiation is not due to BA effects on either process. To address the atrophy, we evaluated BA effects in Sertoli-germ cell cocultures on 1) morphology/germ cell attachment, which might identify a target cell; 2) Sertoli cell energy metabolism, because lactate, secreted by Sertoli cells, is a preferred energy source for germ cells; and 3) DNA/RNA synthesis, because germ cells synthesize DNA/RNA and BA impairs nucleic acid synthesis in liver and may do so in testis. Despite the absence of overt morphologic changes and germ cell loss, the most sensitive in vitro endpoint was DNA synthesis of mitotic/meiotic germ cells, with energy metabolism in Sertoli or germ cells affected to a lesser extent. A re-evaluation of testis sections from rats exposed to BA revealed a decrease in the early germ cell/Sertoli cell ratio prior to atrophy. Thus, although the mechanism for the inhibited spermiation is still undefined and is the subject of future work, these combined studies revealed some changes offering a plausible explanation for the atrophy aspect of the BA testicular lesion.

Spermatocyte toxicity of 2-methoxyethanol in vivo and in vitro: Requirement for an intact seminiferous tubule structure for germ cell degeneration.

Cultures of isolated testicular cells are widely used for evaluating mechanisms of action of direct-acting testicular toxicants. However, for testicular cells, the expression of toxicity in vitro is frequently different from that found in vivo. 2-Methoxyethanol (ME) produces testicular lesions in rats which are characterized by pachytene spermatocyte degeneration 24 hr after dosing. As part of a study to evaluate mechanisms of male germ cell toxicants, we compared the morphological aspects of spermatocyte toxicity after in vivo exposure to ME with those found in various testicular cell culture systems after in vitro exposure to the active ME metabolite, 2-methoxyacetic acid (MAA). Immature rats were used because they respond to in vivo ME treatment in the same way as adults, but their testes are relatively enriched in pachytene spermatocytes. 24-day-old rats were given a single dose of 250 mg ME/kg body weight by gavage and the testes were evaluated 24 hr after dosing. In vitro, cultured seminiferous tubules, Sertoli-germ cell co-cultures, and enriched mixed germ cells, all prepared from 24-day-old rats, were evaluated after 24-hr in vitro exposure to 5 mm MAA (a level of MAA found after ME exposure in vivo). Testes from ME-exposed rats showed the expected pachytene spermatocyte degeneration 24 hr after dosing. Similar changes were observed in cultured seminiferous tubules after 24 hr of exposure to MAA in vitro. However, without the intact seminiferous tubule structure in vitro, the expression of MAA spermatocyte toxicity was different. In conventional Sertoli-germ cell co-cultures, spermatocyte detachment from the Sertoli cell monolayer occurred as expected, although no significant morphological degeneration was observed in these detached germ cells. Similarly, no increase in degenerating spermatocytes was noted in isolated enriched mixed germ cells after in vitro MAA exposure. Instead, toxicity in these germ cell fractions was expressed only by increased uptake of trypan blue dye, revealing an increase in plasma membrane permeability. In summary, this in vivo/in vitro comparison of the spermatocyte toxicity of ME/MAA showed that the expression of toxicity is different in the different culture architectures and that an intact seminiferous tubule structure is required for full expression of the morphological degeneration produced by ME/MAA, and suggests the usefulness of culture seminiferous tubules in future mechanistic studies.

A simple orchidometric method for the preliminary assessment of maturity status in male cynomolgus monkeys (Macaca fascicularis) used for nonclinical safety studies.

INTRODUCTION The identification and use of mature male non-human primates in nonclinical toxicology studies could be important for evaluating candidate drugs for which the profile of toxicity may differ depending on sexual maturity. This investigation sought to establish operational criteria to complement the current standard of histological evaluation for defining sexual maturity in male cynomolgus monkeys (Macaca fascicularis) used for toxicology studies, and to identify a practical non-invasive measure to select mature males for study. METHOD Retrospectively, the relationships between body weight, testicular weight and testis histology were established in control males (n=126) used in previous toxicology studies. Prospectively, testicular volumes were measured in-life by orchidometry using comparative scrotal palpation (n=23 males used for study), then compared to testicular weights measured at necropsy. RESULTS Consistent with previous literature, a weak relationship was observed between body weight and testicular weight. There was, however, a very good relationship between testicular weight and histological maturation level, which was based upon microscopic examination of testes, epididymides and prostates. Orchidometric measurement of testicular volume was found to be a reasonable predictor of testicular weight and served to rapidly select sexually mature males for study, and a total testicular volume (left and right combined) of >20 ml correlated with the histological appearance of maturity. CONCLUSION Based upon this preliminary exploratory study, the initial simple measurement of testicular volume by orchidometry may provide a non-invasive alternative approach for assessing the sexual maturity of male cynomolgus monkeys in research colonies or during toxicology studies that will require more thorough validation.

Acceptability of Low Levels of Genotoxic Impurities in New Drug Substances

The Safety Working Party (SWP) of the European Committee for Proprietary Medicinal Products (CPMP) published a draft “Position paper on the limits for genotoxic impurities” in December 2002. Genotoxic impurities was a topic selected for the joint Drug Information Association (DIA)/European Medicines Agency (EMEA) meeting that was organised in London on October 27–28, 2003, to facilitate the exchange of opinion and perspective between industry and regulatory scientists. Scientific and regulatory updates were presented and discussed in the light of case studies, which are described in this article. The four cases span a range of different scenarios that can be encountered in development: (i) candidate for life-threatening indication — an alkylating reagent used in synthesis is an impurity in the active substance; (ii) late-stage candidate for non-life-threatening chronic indication — a route change leads to a new intermediate becoming a potential impurity — as it is an isolated intermediate, worker safety data is generated and needs to be risk managed; (iii) late-stage candidate for non-life-threatening chronic indication — a mutagenic and structural-alerting starting material — the commercial route confers excellent purging; and (iv) early-stage candidate for non-life-threatening chronic indication — considers strategic approaches for impurities with structural-alerting functionality under different scenarios (dependent upon toxicological data available, daily dosing regimen, route of delivery etc.).The outcomes of the discussions of the cases at the DIA/EMEA Workshop are presented in a separate article in this issue of the journal.

Acceptability of Low Levels of Genotoxic Impurities in New Drug Substances: Case Reports

The Safety Working Party (SWP) of the European Committee for Proprietary Medicinal Products (CPMP) published a draft Position paper on the limits for genotoxic impurities in December 2002. Genotoxic impurities was a topic selected for the joint Drug Information Association (DIA)/European Medicines Agency (EMEA) meeting that was organised in London on October 27-28, 2003, to facilitate the exchange of opinion and perspective between industry and regulatory scientists. Scientific and regulatory updates were presented and discussed in the light of case studies, which are described in this article. The four cases span a range of different scenarios that can be encountered in development: (i) candidate for life-threatening indication - an alkylating reagent used in synthesis is an impurity in the active substance; (ii) late-stage candidate for non-life-threatening chronic indication - a route change leads to a new intermediate becoming a potential impurity - as it is an isolated intermediate, worker safety data is generated and needs to be risk managed; (iii) late-stage candidate for non-life-threatening chronic indication - a mutagenic and structural-alerting starting material - the commercial route confers excellent purging; and (iv) early-stage candidate for non-life-threatening chronic indication - considers strategic approaches for impurities with structural-alerting functionality under different scenarios (dependent upon toxicological data available, daily dosing regimen, route of delivery etc.). The outcomes of the discussions of the cases at the DIA/EMEA Workshop are presented in a separate article in this issue of the journal.