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The testicular toxicity of dichloroacetic acid (DCA), a disinfection byproduct of drinking water, was evaluated in adult male rats given both single and multiple (up to 14 d) oral doses. Delayed spermiation and altered resorption of residual bodies were observed in rats given single doses of 1500 and 3000 mg/kg; these effects persisted to varying degrees on post-treatment days 2, 14, and 28. Delayed spermiation and formation of atypical residual bodies also were observed on days 2, 5, 9, and 14 in rats dosed daily with 1440, 480, 160, and 54 mg/kg. Distorted sperm heads and acrosomes were observed in step 15 spermatids after 14 doses of 480 and 1440 mg/kg. Decreases in the percentage of motile sperm occurred after 9 doses of 480 and 1440 mg/kg and 14 doses of 160 mg/kg. Increased numbers of fused epididymal sperm were observed on days 5, 9, and 14 in rats dosed with 1440, 480, and 160 mg/kg, respectively; other morphologic abnormalities occurred at 160 mg/kg and higher. On day 14, a significant decrease in epididymis weight was observed at 480 and 1440 mg/kg, and epididymal sperm count was decreased at 160 mg/kg and higher. These studies demonstrate that the testicular toxicity induced by DCA are similar to those produced by the analogue, dibromoacetic acid. However, the testicular toxicity of DCA is less severe at equal molar concentrations. Moreover, the DCA-induced testicular lesions occur with greater potency as the duration of dosing increases, indicating the importance of using low-dose subchronic exposures to assess the health risk of prevalent disinfection byproducts.

The present report details histopathologic changes in the testis and epididymis of rats gavaged daily for 2 to 79 d with a by-product of water disinfection, dibromoacetic acid (DBAA). On treatment day 2 abnormal retention of Step 19 spermatids was observed in animals given the highest dosage of 250 mg/kg. Additional changes on day 5 included the fusion of mature spermatids and the presence of atypical residual bodies (ARB) in the epithelium and lumen of Stage X-XII seminiferous tubules. By day 9, ARB were seen in most stages of the seminiferous epithelial cycle and in the caput epididymidis. On day 16 distorted sperm heads were recognized in Step 12, and older spermatids, and luminal cytoplasmic debris was found throughout the epididymis. On day 31, there was vacuolation of the Sertoli cell cytoplasm, extensive retention of Step 19 spermatids near the lumen of Stage IX and X tubules, and vesiculation of the acrosomes of late spermatids. Marked atrophy of the seminiferous tubules was present 6 months after 42 doses of 250 mg/kg. ARB and retention of Step 19 spermatids were observed after 31 and 79 doses of 50 mg/kg and increased retention of Step 19 spermatids was seen in several rats dosed with 10 mg/kg. No abnormalities were detected at the dosage of 2 mg/kg. The changes suggest that the testicular effects of DBAA are sequelae to structural and/or functional changes in the Sertoli cell.

Epididymal sperm counts, a common measurement in male reproductive toxicity studies, are routinely determined using a hemacytometer. Recently, computer assisted methods for automated sperm counts have been developed. In the present study we evaluated an automated system, the TOX IVOS (Hamilton Thorne Research, Beverly, MA) HTM-IDENT option, that utilizes a DNA-specific stain and fluorescence illumination to identify sperm for enumeration. Cauda and caput epididymal sperm counts were determined in 48 adult male Sprague-Dawley rats, using both the hemacytometer and HTM-IDENT. The mean hemacytometer and HTM-IDENT counts (+/- SD) were 250 +/- 43 and 254 +/- 52 million, respectively, for cauda sperm, and 123 +/- 13 and 127 +/- 18 million, respectively, for caput sperm. The average coefficient of variation using the hemacytometer was 13.8% as compared to 17.3% for the HTM-IDENT. Comparison of the machine count and a visual count from the Display Statics screen of the HTM-IDENT indicated that when two or more sperm heads touched or overlapped, the machine counted them as one. Manual (visual) and machine counts when compared over a range of nine concentrations from 3.7 to 47.8 million/mL differed by 4 to 12% at the lowest to highest concentration. The concentration of epididymal sperm samples used in comparing the two counting methods ranged from 5.8 to 17.7 million/mL. Therefore, the HTM-IDENT undercounting error attributable to sperm heads touching was less than 6%. Overall the data indicate good agreement between the HTM-IDENT and the hemacytometer counts. Furthermore, both counting time and technician fatigue were markedly reduced. Thus the HTM-IDENT option improves the efficiency of epididymal sperm counting without loss of precision.

To determine if anesthetic agents used in laboratory animal euthanasia affected sperm motion parameters, rats (n = 10 per group) were euthanized by one of 5 different methods: decapitation alone, or decapitation following either ether, halothane, or Nembutal anesthesia, or CO2 asphyxiation. Sperm were collected from the distal cauda epididymis, diluted, and videotaped for computer-aided sperm analysis (CASA; HTM-2030, Hamilton-Thorn Research, Beverly, MA). The percentage of motile sperm (MOT), their straight-line velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), linear index (LINX), and linearity (LIN) were measured on ≥ 200 motile sperm per sample. No significant differences in any of these 6 motion parameters were found among the treatment groups. Thus, none of these 5 methods of euthanasia affect sperm motion as assessed by CASA methods, making them equally suitable for use in reproductive toxicology studies.

Chlorine and bromine can react with natural organic substances in source waters to form haloacetic acids, major disinfection by-products of water chlorination. Several toxic effects including testicular damage have been attributed to the chloroacetic acids but little information is available on the bromine analogues. In this report we present the results of acute toxicity and acute spermatotoxicity studies of monobromoacetic acid (MBAA) and dibromoacetic acid (DBAA). In adult male rats the acute oral toxicity of MBAA was 10-fold that of DBAA (LD50 177 vs 1737 mg/kg). No reproductive-related endpoints were affected in rats given a single dose of 100 mg MBAA/kg or 14 daily doses of 25 mg MBAA/kg/day. In rats dosed with DBAA, serum testosterone fell to 17% of control 2 days after a single dose of 1250 mg/kg but returned to control levels by Day 14. Marked effects on sperm motion were seen on post-treatment Days 14 and 28. Degenerative flagellar changes in cauda sperm were present on Day 14 while abnormal sperm head shapes and flagellar degeneration were observed in both caput and cauda sperm on Day 28. Histopathology indicated altered spermiation at all time-points as evidenced by retention of Step 19 spermatids beyond Stage VIII of the cycle of the seminiferous epithelium. Disorganization, distortion, and degeneration of late spermatids were also observed. On Day 14 structures resembling residual bodies were rarely seen in the testis but were numerous in the epididymis. Caput sperm counts were decreased on Day 2 and cauda sperm counts were decreased on Days 14 and 28.(ABSTRACT TRUNCATED AT 250 WORDS)

Optimization of the Hamilton-Thorn Computerized Sperm Motility Analysis System for Use with Rat Spermatozoa in Toxicological Studies. Slott, V. L., Suarez, J. D., Poss, P. M., Linder, R. E., Strader, L. F., and Perreault, S. D. (1993). Fundam. Appl. Toxicol. 21, 298-307. To optimize the Hamilton-Thorn Motility Analyzer (HTM; Hamilton-Thorn Research, Beverly, MA) for use in reproductive toxicology studies with rat spermatozoa, the accuracy and precision of the instrument were assessed under a variety of instrument settings. Videotapes of both fast- and slow-swimming sperm were analyzed repeatedly to obtain data across a range of sperm velocities as might be encountered as a consequence of exposure to reproductive toxicants. Acquisition rates were varied across the HTM menu choices (30, 19, 10, or 7 frames/sec) as were the number of frames analyzed (5 to 20) at each framing rate. For fast-swimming samples (mean straight-line velocity (VSL) ∼130 μm/sec) generally good agreement between computer-assisted sperm analysis (CASA) and manually obtained data was found for percentage of motile sperm and straight-line velocity; i.e., CASA values were within 10% of manual values for most frame/rate combinations. The accuracy of these measures held true over a wide range of sperm concentrations and percentage motilities. However, CASA measures were less accurate for sperm samples of lower velocities (mean VSL ∼50 μm/sec and mean VSL ∼30 μm/sec) in that the velocity of very slow sperm was overestimated (particularly at 30 frames/sec). A software change (6.5R) and performing analyses at 19 instead of 30 frames/sec improved straight-line accuracy for the slow sperm and enhanced the discrimination between fast (presumably control) and slow (presumably treated) sperm samples. These data show that this motility analyzer could be successfully configured to evaluate rodent sperm samples. The use of such CASA systems in toxicology studies will provide valuable information that may improve human reproductive risk assessment.

Adult male Sprague-Dawley rats (100 days of age) were given single oral doses of the fungicide benomyl (methyl 1-(butylcarbamoyl)-2- benzimidazolecarbamate) in dosages ranging from 25 to 800 mg/kg body weight. The testis and excurrent ducts were examined at 2 and 70 days posttreatment to determine the chemical effects on spermatogenesis and the epididymis. Primary effects seen at 2 days postexposure were testicular swelling and occlusions of the efferent ductules. Mean testis weight peaked with 400 mg/kg. Premature release of germ cells (sloughing), detected even with the lowest dosage (25 mg/kg), was the most sensitive short-term response to benomyl. Sloughing was found primarily in Stage VII of the spermatogenic cycle at the lower dosages, but at higher dosages sloughing extended into all stages except for Stages VIII-XI. Occlusions of the efferent ductules of the testis were dose-dependent and correlated with testis weight. Sperm and sloughed germ cells were compacted in the ductal lumen of occluded ductules, which were surrounded by two to four layers of polymorphonuclear leukocytes and other inflammatory cells. Although the caput epididymidis contained sloughed germ cells and appeared swollen, evidence of permanent occlusion was not found. The long-term (70 days) effects of benomyl were decreased testis weight (at 400 mg/kg), dose-dependent increases in seminiferous tubular atrophy, and increases in the number of reproductive tracts containing occluded efferent ductules. Fibrosis, granulomas, and abnormal growth were long-term consequences of occluded ductules, which were present 100% of the time in testes containing 26-100% seminiferous tubular atrophy. Only a few testes were found to be completely regressed. Occlusion of efferent ductules and disruption of the seminiferous epithelium by sloughing of germ cells are overlapping dose-dependent mechanisms responsible for short- and long-term effects of benomyl on the rat testis.

We are developing a data base that will allow us to select endpoints that would be useful in the detection of reproductive toxicity in a multigenerational test. In this effort, carbendazim (MBC), a known reproductive toxicant, was administered to male and female rats from weaning, through puberty, gestation, and lactation. A similar study was conducted with hamsters. In rats, MBC was administered at 0, 50, 100, 200, or 400 mg/kg/day. Hamsters were dosed at 0 or 400 mg/kg/day. In the parent (P0) generation, landmarks of puberty were measured. In females, estrous cyclicity, litter size, the number of implants, organ weights, and histology were assessed. Our assessment of the male rat included organ weights, testicular and epididymal sperm counts, a quantitative measure of sperm motility, sperm morphology, testicular histology, and endocrine measures. The growth, viability, and reproductive function of the offspring (F1) were observed during a 4-month period of continuous breeding. In the P0 of both species. MBC did not alter pubertal development, growth, or viability. The reproductive potential of the rats treated with MBC at 200 and 400 mg/kg/day was reduced due to effects on sperm production and fetal viability. In the male rat, MBC treatment markedly altered sperm morphology, testicular and epididymal weights, and sperm numbers and testicular histology. Fertility, sperm motility, and hormonal levels were altered, primarily in the males with very low sperm counts. The ability to conceive did not appear to involve a female factor. In P0 female rats, MBC administration caused postimplantation losses in the high-dosage groups and a few malformed rat pups were found in the litters from the 100 and 200 treatment groups. MBC was less toxic to the hamster than the rat. The only reproductive effects induced by MBC treatment were on sperm measures. Fertility of the P0 generation and fetal and neonatal (F1) viability were not decreased by MBC administration. In the male rat, testis weight, sperm numbers in the cauda epididymis and testis and sperm morphology were sensitive to the effects of MBC. In females, counting implantation scars at necropsy was useful, as this information allowed us to confirm pregnancy and identify postimplantation losses induced by MBC administration.

These studies evaluated the reproductive response and the possible influence of testicular maturation on the reproductive parameters, in male rats treated with 1,3-dinitrobenzene (m-DNB). Young adult male rats (75 or 105 days of age) were given a single oral dose of 0, 8, 16, 24, 32, or 48 mg/kg of m-DNB and killed at 14 days post-treatment. Mortality and neurotoxicity were observed at 48 mg/kg, but only in the older animals. Epididymis weight, testicular sperm head counts, cauda sperm reserves, and sperm morphology were affected at 16 and 24 mg/kg and higher in the older and younger animals, respectively. Testis weight and sperm motility were affected at 24 mg/kg and higher in both age groups. Histologic changes included maturation depletion of mid and late spermatids at 16 mg/kg and higher, atrophy of a few to many seminiferous tubules at 24 mg/kg and higher, and immature germ cells in the epididymis. The movement and/or mixing of luminal elements in the epididymis appeared to be influenced by severe testicular effects. In separate groups given only the 48 mg/kg dosage, fertilizing ability was lost by 5-6 weeks post-treatment and several animals failed to recover in 5 months. In the breeder males, minimal to extensive degrees of seminiferous tubule atrophy and sloughed germ cells in the epididymis were still present after 175 days. The studies indicated that the lowest dosage to produce reproductive changes was 16 mg/kg with a no-effect level of 8 mg/kg. A few animals suffered protracted or permanent reproductive damage. Since the older animals were more susceptible to both the general and the reproductive toxicity of m-DNB, the less severe reproductive changes in the younger animals cannot be attributed solely to maturational differences in the testis.

Recently, we demonstrated with short-duration tests that dibromoacetic acid (DBAA), a commonly occurring by-product of water disinfection, alters sperm morphology and motility in the male rat. These results suggested that the effects of DBAA on sperm quality were likely to compromise reproductive competence of the male rat early in subchronic exposure. The present studies were undertaken to investigate the dose response and time course of alterations in fertility and sperm quality. Proven breeder male rats were gavaged daily with 0, 2, 10, 50, or 250 mg DBAA/kg for up to 79 days; interim and terminal measurements of sperm quality and reproductive outcome were made. Because of the known neurotoxicity of the analogue, dichloroacetic acid, both natural breeding and artificial inseminations were evaluated in untreated females to distinguish between possible behavioral and spermatogenic effects. DBAA compromised male fertility during the second treatment week in naturally bred rats dosed with 250 mg/kg. The early antifertility effect appeared to be the result of behavioral changes since females artificially inseminated with sperm collected on Day 9 successfully produced offspring. However, sperm morphology and motility also were rapidly affected by DBAA treatment so that no offspring via natural insemination and only one litter via artificial insemination were produced subsequent to Day 15. Through 31 days, substantial effects on sperm motility, sperm morphology, and epididymal sperm numbers were observed, but there was no demonstrable effect on serum testosterone or sperm production. Because severe toxicity developed in the group given 250 mg/kg, exposure of these animals was prematurely terminated after 42 doses and their recovery was monitored through a 6-month posttreatment period; decreased testis weights and only limited recovery of reproductive performance were observed. Exposure to 50 mg/kg resulted in moderate changes in sperm morphology and motility and moderate decreases in epididymal sperm counts in rats dosed for 31 or 79 days. However, these males remained fertile, litter size was unaffected, and no paternally mediated developmental defects were noted in their offspring. No effects on sperm quality were detected at dosages of 2 or 10 mg/kg. However, compared to controls, naturally bred DBAA-treated rats tended to have fewer inseminations, fewer copulatory plugs, and fewer multiple litters, suggesting that DBAA may have altered mating behavior at dosages as low as 10 mg/kg.

Halogenated acetic acids are major disinfection by-products of water chlorination and ozonation. Limited data in experimental animals indicate that repeated doses of dichloroacetic acid (DCA) or single doses of dibromoacetic acid (DBAA) cause testicular damage. In the present study, spermatotoxic effects were investigated in rats given oral doses of 0, 10, 30, 90, or 270 mg DBAA/kg/day for 14 days. In rats dosed with 270 mg/kg/day, there were marked effects on epididymal sperm motility and morphology including the flagellar fusion of 2 or more sperm. Testis weight, epididymis weight, and testicular sperm head counts were mildly reduced relative to control, whereas epididymal sperm counts were substantially decreased. Histologic changes in the tests included retention of Step 19 spermatids in Stages IX to XII, abnormal development of late spermatids, and the formation of atypical structures resembling residual bodies that were observed predominantly in Stages X to XIV and I of the cycle of the seminiferous epithelium. At the dosage of 90 mg/kg/day, effects on spermiation, spermatid development, epididymal sperm counts, sperm motility, and sperm morphology were less severe than at the higher dosage. Reduced caput sperm counts and mild effects on spermiation also occurred at 30 and 10 mg/kg/day. These studies indicate that subchronic exposure to DBAA has the potential to affect reproductive outcome in the rat. Compared to previous studies of DCA (12), DBAA, on a molar basis, appears to be a stronger testicular toxicant than the dichloro analogue.

Multiple endpoints of spermatotoxicity in short duration tests (1-5 days exposure; 2.5-week assay interval) were investigated in a number of chemicals reported to produce minimal to severe reproductive effects when administered subchronically. Six of these chemicals (boric acid, dinoseb, 2,5-hexanedione, methoxychlor, metronidazole, ornidazole) produced substantial spermatotoxicity after 1 to 5 doses. Spermatotoxic effects of chlordimeform were equivocal while p,p'-DDT, n-hexane, and sodium chlorite were judged negative. Four chemicals with known acute effects (benomyl, busulfan, ethylene glycol monomethyl ether, nitrobenzene) elicited expected histopathologic responses after a single dose. Testicular histology, testicular sperm head counts, cauda sperm counts, sperm morphology, and sperm velocity proved to be the most toxicologically sensitive endpoints in one or more of the studies, but histopathology of the testis and epididymis was the most consistent indicator of reproductive damage. The percentage of motile sperm and sperm concentration in the epididymal fluid were the least sensitive measurements. The data suggested that most chemicals with the potential to produce moderate to severe sperm damage are detectable with a short duration test. Complementary multiple endpoints enhanced the interpretation of results, often identified cellular targets, and provided insight on possible mechanisms. Specific responses were often similar to specific effects reported for subchronic exposures. A short duration test could be of value as a screen in structure-activity studies or to set priorities for chemicals requiring further evaluation. As a supplement to breeding studies, the data generated in the short test could also be used to enhance the design and interpretation of the longer tests.

Adult male rats were dosed orally on d 0 with 0 or 2000 mg/kg of boric acid and killed on posttreatment d 2, 14, 28, and 57, or dosed with 0, 250, 500, 1000, or 2000 mg/kg of boric acid and killed on posttreatment d 14. At d 14, atypical structures that appeared to be enlarged irregular cytoplasmic lobes of Step 19 spermatids were observed in Stage VIII seminiferous tubules of rats dosed with 1000 and 2000 mg/kg. Abnormal retention of Step 19 spermatids and residual bodies was also observed in Stage IX-XIII tubules of these rats. The retained spermatids and residual bodies were seen in both the luminal and basal regions of the epithelium. A substantial increase in the testicular sperm head count occurred in animals dosed with 2000 mg/kg. Abnormal caput epididymal sperm morphology and reduced caput epididymal sperm reserves were observed at 1000 mg/kg and higher. Serum LH, FSH, TSH, and prolactin values were not affected at any dosage. At d 28, rats dosed with 2000 mg/kg exhibited continued retention of Step 19 spermatids into Stage X, abnormal caput and cauda sperm morphology, and decreased percentages of motile cauda spermatozoa with reduced straight-line swimming velocities. By d 57 substantial recovery was apparent; some retention of Step 19 spermatids into Stage X tubules was still present in two out of six rats but the sperm parameters were comparable to controls. The study indicated that acute oral exposure to boric acid adversely affected spermiation and sperm quality in the adult male rat. At the dosages used the effects appeared reversible. The no-effect level was 500 mg/kg.

Sherman strain female rats (200) were fed 100 ppm of polychlorinated biphenyl (Aroclor 1260) for apporximately 21 months, and 200 female rats were kept as controls. The rats were killed when 23 months old. Twenty-six of 184 experimental animals and 1 of 173 controls had hepatocellular carcinomas. None of the controls but 146 of 184 experimental rats had neoplastic nodules in their livers, and areas of hepatocellular alteration were noted in 28 of 173 controls and 182 of 184 experimental animals. Thus the polychlorinated biphenyl Aroclor 1260, when fed in the diet, had a hepatocarcinogenic effect in these rats. The incidence of tumors in other organs did not differ appreciably between the experimental and control groups.

The determination that a chemical poses a reproductive risk to man typically relies upon fertility studies using rodents. However, fertility in rodents is often difficult to disrupt and more sensitive indicators of reproductive function should be included in the risk assessment process. The present discussion compares the sensitivity of fertility to other endpoints following exposure to known reproductive toxicants. In our studies rats were dosed from weaning through puberty, gestation, and lactation. The reproductive function of the male, the female, and the offspring was assessed. The effects of methoxychlor, carbendazim (MBC), dibutyl phthalate (DBP), and lindane are discussed and compared to fertility. For each compound a ratio (SR = sensitivity ratio) of the lowest effect level (LEL) for infertility or reduced fecundity to the LEL for the most sensitive physiologic endpoint was calculated. The SR should be large when a compound produces effects over a wide range of doses, but should equal unity when the dose-response curve is very steep. For methoxychlor, which blocked implantation, pubertal landmarks and estrous cyclicity provided rapid and sensitive indicators of the subsequent reproductive failure. The SR = 8 (100/12) for methoxychlor using data from females. In contrast, DBP and MBC directed altered testicular function, and for these compounds, sperm and testicular measures provided sensitive indicators of toxicity. The SR for MBC was 2 (100/50), while DBP had a SR of 1 (500/500). In the lindane study, fertility was not reduced but most of the pups (F1) died shortly after birth. The SR for lindane is equal to 0.5 (10/20). At 20 mg/kg the treated females were larger and their estrous cycles were erratic. The data from these studies indicate that no single endpoint will consistently be the most sensitive indicator of reproductive toxicity. Studies must include a number of well validated endpoints that provide a comprehensive assessment of the entire reproductive system of the male and female.

LD50 values were determined for 57 pesticides administered by the oral or dermal route to adult male and female Sherman rats. Thirty-six of the chemicals were also tested by the oral route in one sex of weanlings. Nine pesticides tested by the oral route (bufencarb, cacodylic acid, dialifor, deltamethrin, dicamba, diquat, quintozene, phoxim, pyrazon) and four tested by the dermal route (bufencarb, chlordimeform, dichlofenthion, leptophos) were more toxic to females than to males whereas famphur and 2,4,5-T (oral route) were less toxic to females. Eighteen of the test chemicals were more toxic to the adult than to the weanling and four compounds (leptophos, methidathion, pyrazon, and sulfoxide) were more toxic to the weanling. In additional studies the variability of the LD50 value over a 1-year period was examined for two typical insecticides. Six consecutive bimonthly oral LD50 determinations for parathion and DDT in adults of both sexes indicated that the LD50 values were little affected by the time of year that the tests were done.

In the present study rats were dosed from weaning, through puberty and gestation, to Day 15 of lactation with methoxychlor at 25, 50, 100, or 200 mg/kg/day. Morphological landmarks of puberty were measured, including the ages at vaginal opening, first estrus, and first estrous cycle in females and at preputial separation in males. In the female, estrous cyclicity, fertility, litter size, number of implantation sites, organ weights, and ovarian and uterine histology were also measured. The viability of the offspring (F1) and their fertility were evaluated using a continuous breeding protocol. Males were necropsied after breeding, the reproductive organs were weighed, and the cauda epididymal sperm counts were determined. One testis was used for histopathology, while the other was used to quantify interstitial fluid (IF) content, IF testosterone concentration, and testicular sperm production. Testosterone and androgen-binding protein were measured in the caput epididymis, and sperm motility and morphology were evaluated from a caudal sample. The serum and pituitary were saved for hormonal determinations. Methoxychlor accelerated the age at vaginal opening and first estrus, and the vaginal smears were cornified. Growth was retarded at 100 and 200 mg/kg/day and fertility was reduced when the females were bred with untreated or similarly treated males. In the highest-dose group, the mated females went from constant estrus into pseudopregnancy following mating, but they had no implants. In males, methoxychlor treatment markedly reduced growth, seminal vesicle weight, cauda epididymal weight, caudal sperm content, and pituitary weight. Puberty was delayed in the two highest-dosage groups. Testicular sperm measures were much less affected than caudal measures. Testis weight and histology were slightly affected, and testicular sperm production, sperm morphology, and motility were unaffected. Endocrine function of the testes and pituitary was altered by methoxychlor administration. Leydig cell testosterone production, in response to human chorionic gonadotropin challenge, was reduced and pituitary levels of prolactin, thyroid-stimulating hormone (TSH), and follicle-stimulating hormone (FSH) were altered. In contrast, serum levels of prolactin, FSH, and luteinizing hormone were unaffected. Serum TSH was reduced by 50% of control at 100 and 200 mg/kg/day, while pituitary levels were increased. Gonadotropin-releasing hormone concentration in the mediobasal hypothalamus was also elevated. In spite of the many reproductive alterations, the fertility of treated males was not reduced when they were mated with untreated females.(ABSTRACT TRUNCATED AT 400 WORDS)

Female Sprague-Dawley CD rats were fed 0, 60, 80, 100, 120 and 140 ppm hexachlorobenzene (HCB) continuously in the diet and 2 successive litters raised. These doses were selected to range from approximately the no observable effect level to lethality in suckling offspring of treated dams. In the F1a generation, the 21-day mortality was 9.2, 19.8, 30.0, 45.4, 93.1 and 92.6% in offspring of dms fed 0, 60, 80, 100, 120 and 140 ppm HCB, respectively. In the F1b generation, a similar mortality of 18.5, 21.5, 19.5, 45, 100, and 94.1% was observed at these 5 dose levels, respectively. The neonatal lethality observed was related to both maternal dose of HCB and the cumulative lactational exposure. Clinical signs of maternal toxicity were not observed and fertility and fecundity were unaffected. In the lungs of HCB treated dams, increased numbers f intraalveolar foamy histiocytes and hypertrophy and proliferation of the lining endothelial cells of pulmonary venules were observed. These microscopic findings of pulmonary effects of HCB confirmed findings of this laboratory.

Electron-capture-gas chromatographic (EC-GC) methods for the determination of chlorinated phenol metabolites of hexachlorobenzene (HCB) and pentachlorophenol (PCP) in urine are presented. After extraction the sample was reacted with diazomethane to produce the methyl ether of each metabolite prior to determination by EC-GC. An acid alumina column was used for cleanup and separation of methylated phenols into groups. Average recoveries of greater than 80% were obtained from urine fortified with known amounts of the phenol metabolites under investigation. A level of 1 ppb1 was established as minimum detection limit for each phenol metabolite. Previously unreported urinary metabolites of HCB and PCP were found as a result of a rat feeding study. Levels of chlorinated phenol residues from (a) human general population and (b) a worker occupationally exposed to PCP are also included.

m-Dinitrobenzene (m-DNB)-induced testicular atrophy has been attributed to a direct effect upon the germinal epithelium. However, such degenerative changes in the germinal epithelium should induce shifts in the testicular hormonal milieu, which would in turn alter the hypothalamic-pituitary gonadal axis in general. This study evaluated the endocrine status of male rats (killed 3 hr, 24 hr, 1 week, and 2 weeks) following a single oral dose of m-DNB (32 mg m-DNB/kg). Serum and pituitary leuteinizing hormone, follicle-stimulating hormone (FSH), and protactin and hypothalamic gonadotropin-releasing hormone (GnRH) concentrations were determined. Testosterone and androgen-binding protein concentrations in serum, interstitial fluid, seminiferous tubule fluid, and caput epididymis were also determined. In vitro basal and hCG-stimulated testosterone release was determined in the decapsulated testis. Results of the present study indicate that pituitary hormone concentrations and hypothalamic GnRH were unaffected after a single oral dose of m-DNB. Serum FSH was elevated at 2 weeks. There was a transient decrease in serum testosterone at 24 hr, which returned to control values at 1 and 2 weeks. Interstitial fluid, seminiferous tubule fluid, and caput epididymal testosterone concentrations were increased at 1 and 2 weeks. Basal testosterone release in vitro was increased at 2 weeks, while hCG-stimulated testosterone release was increased at 1 and 2 weeks. Androgen-binding protein concentrations in serum and interstitial fluid were increased at 1 and 2 weeks. Androgen-binding protein was increased at 24 hr and 1 week in seminiferous tubule fluid, but returned to control concentrations by 2 weeks. However, the total tubular content of androgen-binding protein was dramatically decreased at 2 weeks. Androgen-binding protein in the caput epididymis was unaltered following m-DNB treatment. These data demonstrate that m-DNB exerts a direct effect on the testes and not through alterations in hypothalamic and pituitary control of gonadal function.

A method is presented for the analysis of trace amounts of dinitroaniline herbicides in tissue and excreta. The method employs extraction of the tissue or excreta with organic solvent, clean up by liquid/liquid partitioning or silica gel chromatography, and ultimate analysis by gas chromatography using electron capture detection. Recoveries of greater than 80% were noted over the range of concentration 0.1 to 1.0 microgram/g.

The concentrations of hexachlorobenzene (HCB) in adipose tissue were similar for Fo and Fib generations in rats fed 20 ppm HCB until 45 weeks of age. Nulliparous females receiving treatment equivalent to the HCB-treated F0 generation rapidly accumulated HCB in their fat and, by 13 weeks of age, the residue values coalesced with values in 13-week-old F1a females which had received additional HCB via the placenta and milk. Between 13 and 30 weeks of age, steady-state storage was approached and no significant increase in HCB concentrations occurred through 65 weeks of age. Postlactation dams, that nursed average size second litters, had considerably lower concentrations of HCB in their fat than dams weaning no pups, suggesting substantial redistribution and/or elimination of maternal stores during lactation.

The hepatic effects of technical and pure grade pentachlorophenol were investigated in female rats fed 20, 100 and 500 ppm of each for 8 months. Technical pentachlorophenol was contaminated with 8 ppm hexa-, 520 ppm hepta-, and 1380 ppm octachlorodibenzodioxtns and with 4 ppm tetra-, 42 ppm penta-, 90 ppm hexa-, 1500 ppm hepta- and 200 ppm octachlorodibenzofurans; pure pentachlorophenol contained less than 0.1 ppm of each of these contaminants. Technical pentachlorophenol produced hepatic porphyria and increased hepatic aryl hydrocarbon hydroxylase activity, glucuronyl transferase activity, liver weight, cytochrome P-450 and microsomal heme, but not N-demethylase activity. The peak of the CO-difference spectrum of cytochrome P-450 was shifted to 448 nm, and there was a dramatic increase in the 455-430 ratios of the ethyl isocyanide difference spectrum. The enzyme changes were observed at 20 ppm of technical pentachlorophenol. Porphyria occurred at 100 and 500 ppm. Pure pentachlorophenol had no significant effect on aryl hydrocarbon hydroxylase activity, liver weight, cytochrome P-450, microsomal heme, the ethyl isocyanide difference spectrum or N-demethylase activity at any dose level, but did increase glucuronyl transferase at 500 ppm. In contrast, both pure and technical pentachlorophenol decreased body weight gain comparably at 500 ppm. It is concluded that technical pentachlorophenol produces a number of liver changes which cannot be attributed to pentachlorophenol itself, but are consistent with the effects of biologically active chlorinated dibenzo-p-dioxins and dibenzofurans.

In a two-generation reproduction study, rats were fed dietary levels of 0, 20, 100, and 500 ppm of 2,4 dichlorophenyl-p-nitrophenyl ether (TOK) (1.8 to 1.1 mg/kg/ day, 9.2 to 5.2 mg/kg/day, and 46 to 26 mg/kg/day, respectively). The survival of the offspring was not affected at the 0 and 20 ppm dietary levels. At the 100 ppm dietary level the survival of the offspring to weaning was reduced and at the 500 ppm dietary level no offspring survived the neonatal period in the two breedings of the first generation. In further studies rats were dosed during pregnancy either by stomach tube or by adding TOK to the diet. The reduction in the number of surviving litters was caused by poorly developed lungs in the exposed groups. Technical TOK had the same effect as 99% pure TOK. Furthermore, 2,7-dichlorodibenzodioxin, which may be a contaminant of TOK, does not affect the maturation process of the lungs when given at a dose of 0.04 mg/kg/day on days 7 to 15 of gestation.

Adult female Sherman strain rats were fed 500 ppm paraquat (1,1-dimethyl-4,4-bipyridylium dichloride) in their diet. Four poisoned as well as three control rats were sacrificed 1, 3, 5, 7 and 11 wk after onset of exposure. Two well-developed paraquat lung lesions were encountered. The alveoli of these lesions contained a very prominent amorphous material which was highly osmiophilic when examined with the electron microscope. It consisted of concentrically arranged whorls which were surrounded by or next to less electrondense lattice-like material. Additional ultrastructural findings in those and other lungs consisted of edematous and vacuolated membranous pneumocytes (type I) as well as endothelial cells. The granular pneumocytes were increased in 3 of 20 lungs of exposed animals.

Male and female Sherman strain rats were fed polychlorinated biphenyls Aroclor 1260 and Aroclor 1254 at 0, 20, 100, 500 and 1,000 ppm in their diet. Rats received the dietary levels for eight months. Light microscopic changes consisted of hypertrophy of the liver cells, inclusions in the cytoplasm, brown pigment in Kupffer cells, lipid accumulation, and, at the higher dietary levels, adenofibrosis. Ultrastructural changes of the livers of exposed animals consisted of an increase in smooth endoplasmic reticulum and atypical mitochondria. Lipid vacuoles were occasionally surrounded by concentric membranes. The epithelial component of adenofibrosis consisted of goblet cells and cells that resembled the epithelium which lines the bile ducts. In general, the effect of Aroclor 1254 on the liver was more pronounced than that of Aroclor 1260.

Adult male rats were fed different dosage levels of DDT, dieldrin, or combination of the two. After eight weeks, hexobarbital sleeping time was determined in some of these rats. Livers of the other rats were examined with the light microscope and electron microscope. Hexobarbital sleeping time was significantly reduced in all treated rats compared to that of controls, but no difference was found between the various treated groups. Liver weights of all treated groups were significantly higher than those of the controls, and livers of rats fed the combination of DDT and dieldrin weighed significantly more than those rats given DDT or dieldrin alone. Morphological changes, including an increase in smooth endoplasmic reticulum and atypical mitochondria, were observed in all treated groups and were more pronounced in rats given both DDT and dieldrin.

The single oral LD50 of hexachlorophene in adult male Sherman strain rats was 66 mg/kg, in females 56 mg/kg; in famale weanling rats the single oral LD50 was 120 mg/kg. The acute dermal toxicity was low. The iv LD50 in adult male rats was 7.5 mg/kg. Rats fed 500 ppm of hexachlorophene for 98 days developed leg weakness in 12–19 days. At sacrifice, after 98 days' exposure, status spongiosus was present throughout the white matter of the brain. Rats fed 100 ppm of hexachlorophene for 98 days did not develop leg weakness, but in 50% of the brains focal areas of status spongiosus were observed in the white matter. Rats fed 20 and 100 ppm of hexachlorophene for 258 days did not develop leg weakness. The brains of rats fed 20 ppm were normal, while the brains of some rats fed 100 ppm showed focal vacuolization of the white matter. One hundred parts per million reduced the survival of pups in the F 1 generation. This was statistically significant in the F 1b generation. The F 2 generation litters tended to be smaller. Reproduction was normal in rats fed 20 ppm or given hexachlorophene by oral intubation at doses as high as 10 mg/kg/day during day 7 to day 15 of pregnancy. Dermal application of 12 mg/kg/day of hexachlorophene for 30 days produced no microscopic changes in the brain and produced only erythema and desquamation of the treated skin. Doses of 24 and 48 mg/kg/day produced ulceration of the treated skin and status spongiosus of the entire white matter of the brain in some rats.

Several environmental contaminants, notably dibromochloropropane (Whorton et al. 1977) and kepone (Taylor et al. 1978; Cannon et al. 1978) have been implicated in sperm deficiencies among occupationally exposed males. These incidents emphasize the need for adequate testing of chemicals for effects on the male reproductive system. Although important in clinical diagnosis, the evaluation of sperm motility has not been used extensively as a tool in chemical toxicology, particularly in commonly used small laboratory species. Measurements of sperm motility parameters are complicated by several variables including sample manipulation, temperature, cell concentration, time factors and the method of quantitation. In the present study, a simple objective procedure to estimate the proportion of motile epididymal spermatozoa in the rat has been developed. Emphasis is given to details of handling the sperm sample to minimize intersample variation due to external factors. Quantitation of progressively motile spermatozoa is achieved simply by standard manual and electronic blood cell counting techniques. The difficulty in visual tracking of multiple motile spermatozoa is circumvented by counting only nonmotile cells. An index of motility is calculated from the difference in the nonmotile and total counts, each made on separate aliquots of a sperm suspension. The procedure described is an elaboration of a technique used by Mason and Thompson, 1977. The modified technique is demonstrated in rats given selected doses of dinoseb (2-sec___-butyl 4,6-dinitrophenol), a testicular toxicant in the rat (Linder et al. 1982) and ~-chlorohydrin (3-chloro-l,2-propanediol) whose antimotility effects have been documented in several species (Jones 1983).

This study determined the quantitative and qualitative histopathologic effects of a single oral dose of 1,3-dinitrobenzene (48 mg/kg) on the rat testis from 1 to 175 days postexposure. The testis was damaged severely by hour 24, as evidenced by increased numbers of regressive seminiferous tubules that exhibited degenerating pachytene spermatocytes, chromatin margination in spermatids, giant cells, deformed spermatid heads, retained spermatids, and reduced numbers of meiotic figures. The major effects during the first 48 hours posttreatment were degeneration or exfoliation of pachytene spermatocytes and round spermatids and the retention of step 19 spermatids. These regressive effects continued until 24 days, after which the tubules either recovered or became atrophic. At the end of the study (175 days), three males were normal, one had regressed testicles, and three males had atrophic tubules (15 to 45%). Several cellular abnormalities were common throughout the period. In addition, the frequency of the stages of spermatogenesis was altered, an indication of a disturbance in the kinetics of spermatogenesis. 1,3-Dinitrobenzene produced profound and specific lesions in the seminiferous tubules, and recovery was slow and incomplete. Atrophic tubules seemed to form if the normal cellular associations were not reestablished within 24 days, possibly due to the inability of Sertoli cells to reorganize the synchrony of germ cell development.

Adult male rats were fed diets containing 0, 5, 15, and 30 ppm chlordecone for 90 d and then either bred to untreated females or sacrificed for terminal studies. Chlordecone residues in liver, fat, and serum were determined in the treated males. Reproductive performance was unaffected, and no histologic changes in the male sex organs could be attributed to chlordecone treatment. Reversible decreases in the motility and viability of epididymal spermatozoa and decreased sperm reserves in the cauda epididymidis were observed in rats fed 15 or 30 ppm. No effect on sperm morphology or on sperm concentration in epididymal fluid was detected. Chlordecone accumulation in tissues was linearly related to dietary levels, with the highest chlordecone concentration occurring in the liver.

Tri-o-cresyl phosphate (TOCP) and O-ethyl O-(4-cyanophenyl) phenylphosphonothioate (cyanofenphos, Surecide) were found to be delayed neurotoxicants. They were administered to chickens by gavage at 100 and 30 mg/kg . d for 15 d, respectively. In CD-1 mice neither TOCP nor cyanophenphos induced any of the usually recognized clinical symptoms of neuropathy when administered daily by gavage at 262 or 31.25 mg/kg . d for 30 d, respectively. In the chickens, TOCP and cyanofenphos produced about 98 and 90% in vivo inhibition of brain neurotoxic esterase (NTE) activity. In the mice, 24 h after the last daily dose, TOCP and cyanofenphos produced only about 50 and 40% in vivo inhibition of the brain NTE activity. Parathion [O,O-diethyl O-(4-nitrophenyl) phosphorothioate], at 2 or 6.75 mg/kg . d for 15 or 30 d, did not induce neuropathy in either chicken or mice and produced no significant in vivo inhibition of brain NTE activity at the end of the dosing regimen. The specific activity of NTE in control chicken brain crude homogenate was much higher than that in mouse brain homogenate. These results suggest that the differences between chickens and mice in susceptibility to neurotoxic organophosphates may be attributed to (1) inhibitor specificity of NTE forms in the brain in these two different animal species and/or (2) inability of the active metabolites of these neurotoxic compounds to reach the site of action.

Diets containing the herbicide dinoseb (2-sec- butyl 4,6-dinitrophenol) were fed to adult male Sherman rats for 11 weeks. One-half the survivors were killed for terminal studies during the eleventh week and the remainder bred to untreated females during posttreatment, and then killed for terminal studies. Interim sacrifices were made in groups fed 0 and 300 ppm. In rats fed 300 ppm, differential classification of spermatozoa from the cauda epididymidis indicated 90% of the cells were atypical by 20 days of treatment. By 30 days, bizarre and amorphous forms were observed and epididymal sperm counts were decreased. Histologic changes in the testes included abnormal spermatozoa and spermatids and multinucleated spermatogenic cells at 20 and 30 days and severe damage to spermatogenic cells by 50 days. Dietary levels of 225 and 300 ppm produced marked oligospermia and extensive loss of spermatogenic cells in rats fed dinoseb for 11 weeks. Evidence of necrotic spermatogenic cells was seen in some tubules, and in many tubules, only Sertoli cells remained. Reproductive failure occurred at 225 and 300 ppm, although mating behavior and libido were unaffected. There was little or no remission of these effects during the 16-week post-treatment period. Decreased epididymal sperm counts, atypical epididymal spermatozoa, and minimal testicular changes were seen in rats fed 150 ppm, but reproduction was unaffected and the anomalies were reversible. No effects were detected in animals fed 75 ppm.

The early histologic lesions and sequential changes in the development of mirex-induced cataracts were studied in the offspring of lactating female Sherman rats given oral doses of mirex for 5 consecutive days post partum. The earliest histologic change, seen at neonatal day 7, was slight swelling of individual cortical lens fibers. At day 9, many swollen but intact fibers were observed, and at days 11 and 13, extensive degeneration and necrosis appeared throughout the cortex of the affected lenses.

Dietary concentrations of 0, 20, 100, and 500 ppm of technical grade pentachlorophenol were fed to male and female Sherman strain rats for 8 months. The same experiment using purified pentachlorophenol was carried out. The food consumption was measured in all rats during the second week of exposure and for one week every 6 weeks thereafter. An autopsy was performed on all rats at the end of the experiment. The brain, lungs, spleen, liver, kidneys, heart, and testes were weighed and examined grossly and microscopically in all rats fed purified pentachlorophenol, all female rats fed technical pentachlorophenol, and in the male rats fed the highest dose of technical pentachlorophenol and the controls. Only the kidneys and livers were examined microscopically in the male rats fed 20 and 100 ppm of technical pentachlorophenol. Although the food intake was comparable, male and female rats fed 500 ppm of technical and male rats fed 500 ppm of purified pentachlorophenol gained less weight. The livers of the male and female rats fed 500 ppm technical pentachlorophenol weighed significantly more than those of the controls. The kidneys of all male rats fed purified pentachlorophenol weighed significantly more than those of the controls; however, there was no dose-related increase. No morphological changes were seen in the kidneys. At the 500-ppm dietary concentrations, technical pentachlorophenol produced a severe effect in the liver of female rats characterized by vacuolation of the hepatocytes, an increase in fibroblasts and other mononuclear cells within sinusoids, bile duct proliferation, periportal fibrosis, degenerated liver cells, increased mitotic figures, and an accumulation of brown pigment in macrophages and in Kupffer cells. In male rats at the 100- or 500-ppm dietary concentrations of technical pentachlorophenol, the predominant lesion consisted of enlarged pleomorphic hepatocytes which had foamy cytoplasm or cytoplasm with large vacuoles. The walls of the hepatic central veins of the livers in animals of both sexes were thickened. At the 100-ppm dietary concentrations similar but less pronounced effects were observed in the livers. Only mild alterations were noted at the 20-ppm dietary concentration. Purified pentachlorophenol caused slightly enlarged liver cells with occasional eosinophilic cytoplasmic inclusions at 500 ppm but no alterations were observed in the livers of rats fed the 100- and 20-ppm dietary concentrations. The results suggest that most of the toxicity associated with feeding technical grade pentachlorophenol to rats at these dietary concentrations stems from toxic contaminants rather than from pentachlorophenol.

A method was developed to selectively deplete glutathione (GSH) in a single rat testis. Using intratesticular injections of a mixture of two GSH-depleting agents, diethylmaleate and buthionine sulfoximine, testicular GSH levels were decreased to 33-54% of control 2 hr after injection and remained suppressed for 24 hr. GSH levels in the contralateral testis and liver were not affected by this treatment. Comparisons between GSH-depleted and vehicle-injected (contralateral) testes, evaluated 2 weeks later, showed that although testis and epididymal weights and cauda epididymal sperm reserves were slightly reduced (to greater than or equal to 90% of controls), no changes were seen in testicular spermatid counts or in the morphology or motility of cauda epididymal sperm. An increase in histologically abnormal tubules localized to the injection site occurred in some GSH-depleted testes; however, the proportion of normal tubules containing step 19 spermatids was not affected. Thus, intratesticular injections of GSH-depleting agents selectively lowered GSH levels in the treated testis, with minimal adverse effects. This protocol can now be applied to investigate specific roles of GSH in the testes, particularly with regard to the possible modulation of the effects of testicular toxicants.

Weanling male Sprague-Dawley rats were gavaged 5 d/wk with 1,3-dinitrobenzene (m-DNB) at dosages of 0, 0.75, 1.5, 3.0, and 6.0 mg/kg X d. Males were bred to untreated females during treatment wk 10 and were killed during treatment wk 12. Although males dosed with 3 mg/kg X d inseminated the females and evidence of mating was observed in males dosed with 6 mg/kg X d, none of the males in these groups sired litters. Diminished sperm production (reduced testicular sperm head counts), decreased cauda epididymal sperm reserves, nonmotile spermatozoa, atypical sperm morphology, decreased weights of the testes and epididymides, seminiferous tubular atrophy, and incomplete spermatogenesis were also observed in these groups. Sperm production was also decreased in males dosed with 1.5 mg/kg X d. Changes in the spleen included increased weight at dosages of 1.5 mg/kg X d or higher and splenic hemosiderosis, which ranged from slight in rats treated with 0.75 mg/kg X d to moderately severe in those dosed with 6 mg/kg X d. The data indicate that m-DNB is a potent testicular toxicant in the male rat, capable of producing extensive damage to reproductive tissues and reproductive failure. Limited data on four rats that received 6 mg/kg X d and were allowed a 5-mo posttreatment recovery period suggested that the testicular effects are at least partially reversible.

Proven-breeder 102-d-old male Wistar rats were gavaged daily with 0, 1, 5, 15, or 45 mg/kg.d benomyl. The animals were bred to untreated females after 62 d and killed after 76-79 d for evaluation of selected male reproductive end points. Minimal to moderate changes were observed in rats dosed with 45 mg/kg.d; these included decreased testis and epididymis weight, reduced cauda sperm reserves, decreased sperm production, increased numbers of decapitated spermatozoa, and increased numbers of seminiferous tubules containing multinucleated giant cells. Reproductive performance, seminal vesicle and prostate weight, sperm motility, serum luteinizing hormone, follicle-stimulating hormone, prolactin, and androgen binding protein were not affected by any of the dosages tested. Based on these end points, the no-effect level was 15 mg/kg.d.

Reproduction, pathology and acute toxicity were studied in Sherman rats exposed to the polychlorinated biphenyls, Aroclor 1254 and Aroclor 1260. Rats exposed to Aroclor 1254 at dietary levels of 20 ppm or more had fewer pups per litter than the controls in the F 1b and F 2 generations. The 100 ppm exposure level of Aroclor 1254 increased mortality in the F 1b offspring and markedly decreased mating performance of the F 1b adults. The 500 ppm dietary level of Aroclor 1260 reduced litter size and decreased survival in the F 1 litters. Dietary levels of 5 ppm Aroclor 1254 and 100 ppm Aroclor 1260 had no effect on reproduction in rats exposed through two generations. Liver weights were increased in 21-day-old F 1 male weanlings at the 1 ppm level of Aroclor 1254 and in either sex of F 1 and F 2 weanlings at 5 ppm or higher levels of both Aroclor 1254 and 1260. Histological changes in the liver and increased liver weights were observed in adult rats exposed to the higher levels. Pregnant rats given Aroclor 1254 at the rate of 100 mg/kg/day on days 7–15 of gestation produced grossly normal litters, but only 30·1% of the pups survived to weaning. Reproduction and pup survival were not affected at dosage rates of 50 mg. Aroclor 1254/kg/day or 100 mg Aroclor 1260/kg/day. Oral LD 50 values in 3–4-wk-old male rats were 1295 and 1315 mg/kg for Aroclors 1254 and 1260, respectively. The iv LD 50 for Aroclor 1254 in adult females was 358 mg/kg.

Groups of eight adult male rats were given a single oral dose of 0 or 48 mg/kg of 1,3-dinitrobenzene and sacrificed at 1, 2, 4, 8, 16, 24, 32, 72, and 175 days posttreatment. The groups killed at 175 days were bred to untreated females during weeks 3, 4, 6, 9, 13, and 24. Decreased testis weight and testicular sperm numbers were observed by day 4; decreased cauda sperm reserves and epididymis weight occurred by day 8 and day 16, respectively. Reduced numbers of motile spermatozoa and abnormal sperm morphology were seen in spermatozoa from the cauda epididymidis by day 16. Fertilizing ability, as indicated by the presence of two pronuclei and a sperm tail in eggs flushed from the oviducts of inseminated females, was slightly reduced by week 4 and declined to zero by week 6. Group means for reproductive organ weights, sperm production, and sperm reserves failed to return to control levels although some individual animals approached full recovery. Normal fertilizing ability was restored in most animals by week 13, but two of seven remained infertile. Occlusion of some efferent ductules was observed in three of seven animals at 175 days. This study indicates that 1,3-dinitrobenzene is a potent testicular toxicant in the rat, capable of producing marked testicular damage, infertility, and possibly sterility from a single exposure.

THE insecticide ‘Isolan’ (1-isopropyl-3-methyl-5-pyrazolyl dimethylcarbamate) is more toxic to rats by the dermal than by the oral route1. This finding is interesting, because the opposite is true of most compounds. It was not known whether the unexpected difference in the toxicity of ‘Isolan’ by the two routes was caused by detoxification in the intestinal lumen, poor absorption from the gastrointestinal tract, detoxification in the liver, unusually efficient percutaneous absorption, or some other mechanism.

A single oral dose of 4080 mg/kg and a single dermal dose of 2500 mg/kg of amitrole, the highest doses given, were not lethal to adult rats. Dietary levels of 0, 1000, 500, 100 and 25 ppm amitrole were fed to groups of 10 male and 10 or 12 female rats. The dietary levels of 1000 and 500 ppm were fed for 107–110 days, the dietary levels of 100 and 25 ppm for 240–247 days to the F0 generation and for 141 days to the F1b generation. At the dietary levels of 1000 and 500 ppm the pups that were born were smaller and had atropic thymuses and spleens indicative of runt disease. Most of them had died with-in a week after weaning. Reproduction was not affected at the lower dietary levels. Hyperplasia of the thyroid was seen in all rats fed dietary levels of 100 ppm or greater. In the 25 ppm group 4 10 F0 and 4 10 F1b females and 6 10 F1b males, but none of the F0 males developed hyperplasia of the thyroid.

Fifty male Sherman strain rats were fed 500 ppm of a polychlorinated biphenyl (PCB) (Aroclor 1254) for six months. Five each were killed zero, one, two, three, four, six, eight, and ten months after exposure to Aroclor had ceased. The livers of these rats were examined by light and electron microscopy. Liver lesions persisted although exposure to PCBs ceased. Ten months after exposure ceased, 1,192 ppm PCBs were still present in the rats’ adipose tissue and 22.65 ppm in the rat livers. Aroclor patterns found in the tissues by electron capture gas chromatography differed from patterns of dietary Aroclors. Mass spectral analysis of liver and adipose tissue revealed three major Aroclor components with masses of 324, 358, and 392. These contained isotopic clusters indicative of the presence of C15, C16, and C17, respectively.