Reproductive toxicity and lack of dominant lethal effects of 2,4-dinitrotoluene in the male rat

Adult male Sprague Dawley rats were gavaged with 2,4-dinitrotoluene (2,4-DNT) dissolved in corn oil at 0, 60, 180, or 240 mg/kg/day for five days. A single oral dose (0.5 mg/kg) of triethylenemelamine was used as a positive control. Induction of dominant lethal events was scored on the basis of early fetal deaths. At the two lower doses, no consistent changes were observed in the numbers of pre-implantation losses, implantation sites, or living or non-living fetuses. The highest dose of 2,4-DNT tested resulted in a marked decrease in the numbers of sperm-positive females (determined by microscopic examination of vaginal smears for sperm) and pregnant females. These two effects diminished in the latter weeks of mating. The low number of pregnant females at the highest dose made meaningful statistical evaluations difficult. The results indicate that 2,4-DNT does not cause dominant lethal mutations but does adversely affect reproductive performance.     

Metabolism and excretion of 2,4-dinitrotoluene in male and female Fischer 344 rats after different doses

Female Fischer 344 rats are less susceptible to the hepatocarcinogenic effects of 2,4-dinitrotoluene (2,4-DNT) than males. This study is a comparison of the metabolism and excretion of 2,4-DNT in male and female rats after oral doses of 10, 35, or 100 mg of 14C-2,4-DNT per kg. The major route of elimination of 14C after all doses was the urine. 4-(N-Acetyl)amino-2-nitrobenzoic acid (4Ac2NBAcid), 2,4-dinitrobenzoic acid (2,4-DNMBAcid), 2-amino-4-nitrobenzoic acid (2A4NBAcid), and 2,4-dinitrobenzyl alcohol glucuronide (2,4-DNBAlcG) were identified in urine of rats. These four compounds accounted for greater than 85% of the radioactivity excreted in urine. Female rats excreted a significantly greater percentage of the dose in the urine as 2,4-DNBAlcG at doses of 10 or 35 mg/kg when compared to males. Both sexes showed dose-dependent changes in urinary excretion of 2,4-DNT metabolites. Males excreted a smaller percentage of the dose as 2,4-DNBAcid at 100 mg/kg than at 10 or 35 mg/kg. Females excreted less of the dose as 2,4-DNBAcid and 2,4-DNBAlcG at 100 mg/kg than at 10 or 35 mg/kg. The only sex difference in 2,4-DNT metabolism or excretion of sufficient magnitude to account for the sex difference in susceptibility to the hepatocarcinogenic effects of 2,4-DNT was the greater percentage of 2,4-DNT excreted as 2,4-DNBAlcG by female rats at 10 or 35 mg/kg.     

Metabolism and excretion of 2,4-[14C]dinitrotoluene in conventional and axenic Fischer-344 rats

Comparisons of in vitro reduction of 2,4-dinitrotoluene (2,4-DNT) by cecal microflora and liver have indicated that microflora may play a large role in the in vivo metabolism of 2,4-DNT to reduced metabolites. Furthermore, reduction of 2,4-DNT by cecal microflora produces nitroso and, presumably, hydroxylamino intermediates which may account for the toxic actions of 2,4-DNT, including hepatocarcinogenesis. This study examines the metabolism, excretion, and hepatic covalent binding of 2,4-DNT in conventional, DNT-fed, and axenic Fischer-344 rats in order to define more precisely the role of DNT pretreatment and intestinal microflora in the disposition and toxicity of 2,4-DNT. No differences in 2,4-DNT disposition were produced by 30 days of feeding DNT (35 mg/kg/day) in the diet of male or female rats. Axenic males and females excreted less of a dose of 2,4-DNT in the urine than did conventional animals, and half-times for excretion of 4-(N-acetyl)amino-2-nitrobenzoic acid (4NAC2NBA), 2,4-dinitrobenzoic acid, 2-amino-4-nitrobenzoic acid (2A4NBA), and 2,4-dinitrobenzyl alcohol glucuronide were longer in axenic males than in conventional males. In axenic females half-times for excretion of only 4NAC2NBA and 2A4NBA were longer than in conventional females. Amounts of 4NAC2NBA and 2A4NBA excreted by axenic animals were 1/10th to 1/5th those excreted by conventional animals. Hepatic covalent binding was decreased by half in axenic animals. These data suggest that intestinal microflora play a major role in the appearance of reduced urinary metabolites and of covalently bound material after 2,4-DNT administration.     

Distribution,elimination, and test for carcinogenicity of 2,4-dinitrotoluene in strain a mice

2,4-Dinitrotoluene (2,4-DNT) was examined for its ability to produce lung tumors in strain A mice. Its tissue distribution and elimination in this mouse strain were also determined. At total doses of 600, 1500, or 3000 mg/kg, 2,4-DNT did not produce an increase in lung adenomas when compared to controls. The urine was found to be the major route of elimination, with 52.5, 60.1, and 70.0% of ip doses of 1, 10, and 100 mg/kg, respectively, excreted within 4 hr after administration. The distribution of 2,4-DNT in various tissues (blood, liver, kidneys, lungs, adipose tissue, small and large intestine) showed no evidence for preferential uptake or retention at any of the doses tested. At 0.25 to 4 hr after administration, the lungs contained 0.05 to 0.2% of the dose. Terminal half-lives of radioactive meterial in liver (1.1 to 1.7 hr) and kidney (0.9 to 1.4 hr) were not related to dose. At all doses (1, 10, and 100 mg/kg), rapid and extensive metabolism of 2,4-DNT by liver and small intestine was observed, as judged by the low amounts of 2,4-DNT (less than 13% of the total ³H/tissue) that could be re-isolated from these tissues. Blood and lungs contained much higher levels of unchanged 2,4-DNT and, in most cases, the extent of 2,4-DNT metabolism was similar in these two tissues, suggesting that the lung is not an active site of 2,4-DNT metabolism. It is concluded that 2,4-DNT is not carcinogenic in the strain A mouse lung tumor bioassay and that ip administered 2,4-DNT is rapidly eliminated in the urine. The liver and the small intestine are major sites of 2,4-DNT metabolism, while the lungs are relatively inactive in this respect.     

Tissue distribution of 2,4-dinitrotoluene and its metabolites in male and female Fischer-344 rats

The hepatocarcinogen, dinitrotoluene (DNT), produces a lower incidence of hepatocellular carcinoma in female rats than in male rats. This study compares the elimination of radiolabeled 2,4-DNT in male rats at three dosages and the elimination of 2,4-DNT in male and female rats at a dose of 100 mg/kg. Terminal half-lives of total radioactivity in various tissues were similar after doses of 10 or 35 mg/kg in male rats, but were shorter after a dose of 100 mg/kg in plasma, red blood cells, liver, and kidney. Although terminal half-lives for total radioactivity were similar in male and female rats after a dose of 100 mg/kg, female liver contained concentrations of radioactivity which were one-half those found in males. The tissues of both sexes contained parent compound, 2,4-dinitrobenzoic acid, and 2-amino-4-nitrobenzoic acid. No evidence for the presence of the hepatocarcinogen, 2,4-diaminotoluene, was found. The difference between nanomole equivalents of 2,4-DNT and its metabolites found by specific assay and nanomole equivalents found by radioactivity measurements was two to five times larger in female rats than in males. The data suggest that dose-dependent changes occur in the elimination and distribution of 2,4-DNT at high doses and that female rats metabolize 2,4-DNT to unidentified metabolites to a greater extent than males. Furthermore, the lack of evidence for the formation of 2,4-diaminotoluene from 2,4-DNT suggests that the hepatocarcinogenicity of 2,4-DNT is not due to its conversion to 2,4-diaminotoluene.     

Mutagenic evaluations of two rubber accelerators

Because of evidence of mutagenic activity in certain short-term in vitro assays (unpublished data) N-oxydiethylene thiocarbamyl-N-oxydiethylene sulfenamide (OTOS) and N-oxydiethylene-2-benzothiazole sulfenamide (OBTS) were studied to determine their potential to induce dominant lethal mutations. For 56 consecutive days OTOS (0, 6.25, 12.5, or 25 mg/kg) or OBTS (0, 125, 250, or 500 mg/kg) was administered by gavage to groups of male Sprague-Dawley rats, while the negative control received corn oil. Each male then was mated with two virgin females in each of two successive 1-week periods. A positive control group was given triethylenemelamine (TEM, 0.25 mg/kg) in a single ip dose 1 day prior to mating. A significant depression in body weight gain (24.8%) was observed at the highest dose for OTOS (25 mg/kg). No effect on body weight was noted for OBTS at doses as high as 500 mg/kg. Although sequalae were observed in the OBTS top-dose animals during the test period, none were ascribed clearly to the test material. Similar pregnancy rates (70 to 100%) were observed for the concurrent negative and positive controls and for all test groups of both chemicals. In the TEM controls for both compounds, the number of implantation sites and preimplantation losses were significantly decreased, and the number of early fetal deaths per pregnant female were significantly elevated. However, both OTOS and OBTS failed to produce dominant lethal mutations. The absence of a response emphasized the difficulty in using in vitro and in vivo mutagenic assays as predictors of mutagenesis in man.     

Biliary excretion and enterohepatic circulation of 2,4-dinitrotoluene metabolites in Fischer-344 rats

Technical grade dinitrotoluene (DNT) is hepatocarcinogenic when fed to rats. DNT is oxidatively metabolized by hepatic enzymes and reductively metabolized by rat intestinal microflora in vitro. The objectives of the present studies were to determine the importance of bile as a route of excretion for DNT metabolites and to investigate the role of enterohepatic circulation in the metabolism of DNT. The common bile ducts of male and female F-344 rats were cannulated with an uninterrupted cannula at the hepatic and ileal ends. After 24 hr, male rats were given a po dose of 35, 63, or 100 mg 2,4-[¹⁴C]DNT/kg; female rats received 35 mg 2,4-[¹⁴C]DNT/kg. Immediately prior to dosing, the cannula was snipped and bile was allowed to collect in a glass reservoir, surgically implanted in the peritoneal cavity, which could be sampled externally. In males, excretion of ¹⁴C in bile was linearly related to dose. From 9.2 to 29.2 μmol eq of [¹⁴C]DNT (approximately 25% of the dose) appeared in bile within 24 hr. Females dosed with 35 mg/kg excreted only 18% of the dose in the bile. Over 90% of the radioactivity in the bile was the glucuronide conjugate of 2,4-dinitrobenzyl alcohol (DNBAlc-G). In comparison to control rats, in which bile flow to the small intestine was uninterrupted, collection of bile decreased the amount of ¹⁴C excreted in urine. In both males and females most of the 2,4-DNT dose excreted in the urine was in the form of the oxidized metabolites DNBAlc-G and 2,4-dinitrobenzoic acid. These results indicate that bile is an important route of excretion for 2,4-DNT metabolites and that metabolites excreted in the bile can be reabsorbed from the gut.     

An in vivo assay of the mutagenic potential of imidacloprid using sperm head abnormality test and dominant lethal test

Objective: To assess the mutagenic effects of imidacloprid in germ cells of Swiss albino male mice by sperm head abnormality (SHA) assay and dominant lethal test (DLT). Methods: Swiss albino mice were exposed to imidacloprid (22, 11 and 5.5?mg/kg/day) along with 3% gum acacia as vehicle control through oral route for 7, 14 and 28 days for SHA assay and for 28 days for DLT. The epididymal sperm smear in 1% eosin stain was analyzed for SHAs. In DLT, male mice were allowed to mate with females after 1, 3 and 6 weeks of end of pesticide treatment. The uterine contents of the sacrificed females were observed for live and dead implants. The analysis of test and control groups data was done by one way ANOVA at p?Results: Exposure of all dose levels of imidacloprid (22, 11 and 5.5?mg/kg/day) for seven days did not induce significant SHAs while they induced significant SHAs compared with the control group following exposure for 14 and 28 days. The analysis of uterine content revealed a significant increase in the number of dead implants/female compared with the vehicle control in only those females which were mated with male mice after six weeks of treatment of highest dose level of imidacloprid. The dominant lethal mutations were observed only at spermatogonial stage. Conclusions: Long-term exposure of pesticide generated SHAs even at lowest dose level (5.5?mg/kg/day for 14 days) and mutagenic effects at spermatogonial stage at highest dose level (22?mg/kg/day for 28 days).     

Metabolism and DNA binding of 2,6-dinitrotoluene in Fischer-344 rats and A/J mice

2,6-Dinitrotoluene (2,6-DNT) is a potent hepatocarcinogen in Fischer-344 rats, while its 2,4-isomer is believed to be noncarcinogenic. Neither 2,6-DNT nor 2,4-DNT is carcinogenic in the strain A mouse lung tumor bioassay. To explore the possible reasons for these differences in tumor responses, we have studied the in vitro metabolism and DNA binding of 2,6-DNT in cultured hepatocytes of the Fischer-344 rat and the A/J mouse, and have also investigated the in vivo DNA binding of 2,6-DNT and 2,4-DNT in these two species. In vitro metabolism of 2,6-DNT by rat and mouse hepatocytes was similar and resulted mainly in the formation of 2,6-dinitrobenzyl alcohol, either unconjugated or as a glucuronide (57.5 to 85.5% of the total per fraction), with smaller amounts of polar, acidic metabolites (8.4 to 38.7%) and minor amounts (1.2 to 5.3%) of 2-amino-6-nitrotoluene. Anaerobic metabolism of 2,6-DNT by an extract of rat or mouse cecal contents resulted mainly in the formation of 2-amino-6-nitrotoluene and 2-(N-acetylamino)-6-nitrotoluene, and minor amounts of 2,6-diaminotoluene. Ip administration of 2,6-DNT or 2,4-DNT (150 mg/kg each) to Fischer-344 rats resulted, after 24 hr, in covalent binding to DNA of the liver (131.1 to 259.9 pmol 2,6-DNT/mg DNA; 215.4 to 226.8 pmol 2,4-DNT/mg DNA), and lower binding to DNA of the lungs and the intestine (14.9 to 22.7 pmol 2,6-DNT/mg DNA; 45.0 to 75.0 pmol 2,4-DNT/mg DNA). Similar treatment of A/J mice resulted in lower binding in the liver (25.9 to 31.9 pmol 2,6-DNT/mg DNA; 42.6 to 58.9 pmol 2,4-DNT/mg DNA), no detectable binding of 2,6-DNT in extrahepatic tissues and low amounts of binding of 2,4-DNT to lung and intestinal DNA (9.7 to 39.0 pmol/mg DNA). In vitro binding of 2,6-DNT to DNA of cultured hepatocytes from both A/J mice and Fischer-344 rats required prior metabolism of 2,6-DNT by the respective extracts from cecal contents. DNA binding was non-detectable in hepatocytes incubated with 2,6-DNT only. It is concluded that binding of 2,6-DNT to liver DNA requires its prior reductive metabolism, probably by intestinal microorganisms, and that the higher binding of 2,6-DNT in the Fischer-344 rat than in the A/J mouse may, in part, be responsible for the high susceptibility of the Fischer-344 rat to 2,6-DNT carcinogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Acute, Pharmacokinetic, and Subchronic Toxicological Sudies of 2,4-Dichlorophenoxyacetic Acid

The single-dose oral LD₅₀ values in Fischer 344 rats for technical-grade2,4-dichlorophenoxyacetic acid (2,4-D), es ters, and salts rangedfrom 553 mg/kg (isobutyl ester in females) to 1090 mg/kg (dimethylaminesalt in males). The LDH values for the acid, esters, or salts,when expressed as acid equivalents, were consistent which suggeststhat the acute toxicity was due to 2,4-D per se. Acute dermalLD₅₀ values in rabbits for the acid, esters, and salts weregreater than 2000 mg/kg. Overall, these results indicate thatthe acute oral and dermal toxicity of 2,4-D are low. Pharmacokineticswere evaluated in male Fischer 344 rats given single oral dosesof 10, 25, 50, 100, or ISO mg 2,4- [¹⁴CJD/kg The amount of 2,4-Din the plasma, kidney, and urine 6 hr postdosing indicated thatthe urinary elimination of 2,4-D was saturated in male ratsgiven oral doses in excess of 50 mg/ kg. Subchronic dietarystudies in male and female Fischer 344 rats used dose levelsof 0, 15, 60, 100, or 150 mg/kg/thy of purified or technical-grade2,4-D acid for 13 weeks. Body weight gains were decreased forboth sexes at the higher dose levels of purified and technical-grade2,4-D acid. Kidney weights were increased in all treated malerats and in females given the higher three dose levels of purified2,4-D. Treatment-related cytoplasmic alterations were presentin the renal proximal tubules of most rats given 60mg/kg/thyand higher of purified or technical-grade 2,4- D; a few femalesgiven 15 mg/kg/thy also had slight alterations in the cytoplasmof the proximal tubules. A dose-related degenerative changewas identified in the descending proximal renal tubules of allmale rats given the highest three dose levels of either testmaterial and some given 15 mg/kg/thy. Dose levels of 100 or150 mg/kg/thy of either compound for both sexes produced minimalswelling and increased staining homogeneity in the liver cellsand were associated with a slight elevation of liver weightand serum glutamic pyruvic transaminase activity. Higher doselevels of technical-grade and purified 2,4-D decreased totalserum tetraiodothyronine levels in female rats, however, themorphology of the thyroid gland was normal. The no-observed-effectlevel (NOEL) was less than 15 mg/kg/day for both purified andtechnical-grade 2,4-D acid.     

A new approach to dominant lethal testing

Male weanling rats were given triethylenemelamine (TEM) at 0.025, 0.050, or 0.075 mg/kg and male adult rats were given TEM at 0.050 mg/kg, all by gavage 5 days/week for 10 weeks. At the end of the treatment period, the animals were immediately cohoused with two females each during Week 1 and with two other females during Week 2. The females were sacrificed 14 days from the midweek of cohousing, and the numbers of corpora lutea and live and dead implantations were counted. Statistical analyses of the number of dead implantations per pregnant female and of the number of females with one or more dead implantations or with two or more dead implantations indicated significant increases in all indexes for the adults and a more variable response on the part of the weanlings. The results from this study indicate that there is no loss of pertinent information by employing this approach except that of stage sensitivity. It reduces the amount of data usually required and assesses the more significant dominant lethal effect, that of postimplantation loss.     

A study of the effect of (2"R)-4'-O-tetrahydropyranyladriamycin, a new antitumor antibiotic, on reproduction. II. Its teratogenicity in rats and rabbits

This paper describes the embryotoxicity and teratogenic effects of (2"R)-4'-O-Tetrahydropyranyladriamycin (THP). The drug was administered intravenously to female rats at 0.01, 0.03, 0.1 or 0.3 mg/kg daily from day 7 to day 17 of pregnancy and to female rabbits at 0.01, 0.05 or 0.1 mg/kg daily from day 6 to day 18 of pregnancy. Results were summarized as follows. Rats THP, at the highest dose of 0.3 mg/kg, decreased body weight gains of pregnant females. This dose caused a decrease in body weights of fetuses, tendencies to increase the rate of death of fetuses or of resorption, an increase in the number of lumbar vertebrae and a delayed ossification of forelimbs of fetuses. Other parameters were not affected by THP at any dose levels. At any dose levels, THP did not produce external, visceral or skeletal malformations in the offspring (F1), nor did it affect the development, physiological functions, behavior, mating, fertility or pregnancy of the offspring. However, at the highest dose level, THP decreased the weight of testes of the offspring. The results suggest that the maximum "no effect" dose level of THP to pregnant females and offspring is 0.1 mg/kg/day intravenously. Rabbits The highest dose of THP, 0.1 mg/kg, decreased the consumption of food and water by pregnant females, but at any dose levels, it did not affect their body weight gain. THP did not cause teratological effects such as external malformation or visceral and skeletal anomalies in the fetuses at any dose levels tested. The results suggest that the maximum "no effect" dose of THP is 0.05 mg/kg/day intravenously to pregnant females and above 0.1 mg/kg/day intravenously to fetuses.     

Dominant lethal effects of nocodazole in germ cells of male mice

The ability of the anticancer drug, nocodazole, to induce dominant lethal mutations in male germ cells was investigated by the in vivo dominant lethal test. Mice were treated with single doses of 15, 30 and 60 mg/kg nocodazole. These males were mated at weekly intervals to virgin females for 6 weeks. Nocodazole clearly induced dominant lethal mutations in the early spermatid stage with the highest tested dose. Mice treated with 60 mg/kg nocodazole showed an additional peak of dominant lethal induction in mature spermatozoa during the first week matings after treatment. The percentage sperm count and sperm motility were significantly decreased after treatment of males with 30 and 60 mg/kg nocodazole. Moreover, the middle and highest doses of nocodazole significantly increased the percentage of abnormal sperm. Our study provides evidence that nocodazole is a germ cell mutagen. Marked alteration in the spermiogram analysis after nocodazole treatment possibly confirms that nocodazole has a significant effect on sperm maturation and development during storage and transit. The demonstrated mutagenicity profile of nocodazole may support further development of effective chemotherapy with less mutagenicity. Moreover, the cancer patients and medical personnel exposed to this drug chemotherapy may stand a higher risk for abnormal reproductive outcomes.     

Developmental Toxicity Studies of Methyl Ethyl Ketoxime (MEKO) in Rats and Rabbits

Abstract

The developmental toxicity of methyl ethyl ketoxime (MEKO), an industrial antioxidant used primarily as an antiskinning agent in alkyd paint, was investigated in rats and rabbits. Following preliminary dose range finding studies, groups of 25 pregnant rats or 18 pregnant rabbits were dosed by gavage with aqueous solutions of MEKO at 0, 60, 200, or 600 mg/kg (rats) or 0, 8, 14, 24, or 40 mg/kg (rabbits) on gestation days 6–15 or 6–18, respectively. In rats, dose-dependent clinical signs of maternal toxicity including reduced body weight gains were noted at 200 and 600 mg/kg. At 60 mg/kg and above enlarged spleens were observed at necropsy. The preliminary study found methemoglobin formation and reticulocytosis indicative of anemia at these dose levels. No treatment-related gestational effects, malformations or developmental variations were observed in the rats. In rabbits, 3 females aborted and 8 females were found dead at 40 mg/kg between gestation days 11 and 24. Clinical signs of maternal toxicity were present in surviving doses at this dose level. Body weight gains were reduced at 24 and 40 mg/kg. The preliminary study indicated maternal hematological effects in the rabbits similar to the rats at dose levels as low as 10 mg/kg. MEKO was not considered to have produced any treatment-related gestational effects, malformations or developmental variations in the rabbit at dose levels at or below 24 mg/kg. Because of excessive maternal mortality and abortions at the 40 mg/kg dose level, only 6 rabbits produced litters. The severe maternal toxicity and limited number of litters precluded a full assessment of developmental toxicity at 40 mg/kg. Nonetheless, MEKO did not appear to be teratogenic to the rabbit at this dose level.     

Dominant lethal assay of chlordecone and its distribution in the male reproductive tissues of the rat

Male rats received 3.6 or 11.4 mg/kg/day of chlordecone orally for 5 days. Some statistically significant events were seen in the reproductive data of females mated to males receiving chlordecone. However, these events did not follow a consistent pattern and do not suggest the conclusion that chlordecone causes dominant lethal effects. Male rats received a single oral dose (40 mg/kg) of chlordecone and were killed at 1, 2, 3, 5, 7, 14 or 21 days. Chlordecone was distributed throughout the reproductive tract. The descending order of concentration was seminal vesicular fluid > prostate > vas deferens > seminal vesicle > unwashed sperm > washed sperm. It is concluded that chlordecone is well distributed throughout the reproductive tract of the male rat, appears in the ejaculate, and does not appear to produce dominant lethal effects.     

Failure of hexachlorobenzene to induce dominant lethal mutations in the rat.

Three groups of 10 male rats each were gavaged with 0, 70, or 221 mg hexachlorobenzene (HCB)/kg daily for 5 consecutive days. Ten other male rats received a single oral dose of 0.5 mg triethylenemelamine (TEM)/kg. Both compounds were dissolved in corn oil. Following the last administration, each male rat was allowed to mate with two naive, nulliparous females per week for 14 consecutive weeks. Females were sacrificed on Day 14 of gestation. Their ovaries and uteri were exposed and examined for numbers of corpora lutea and total, living and dead implantations. Mating and fertility frequencies were calculated. TEM elicited typical dominant lethal events. HCB did not. Dose-dependent decreases in the numbers of females inseminated and impregnated were observed.     

In vivo and in vitro metabolism of 2,4-dinitrotoluene in strain A mice

The elimination and metabolism of a single dose (100 mg/kg) of 2,4-dinitrotoluene (2,4-DNT) in A/J mice were examined. After intraperitoneal administration, elimination was rapid, with 70% of the dose appearing in the urine within 4 hr. Four hours after oral administration, only 28.5% of the dose was excreted in the urine, which increased to 66% after 8 hr. Elimination via the feces was minimal (less than 2.1% of the dose) in both cases. From 0.5 to 4 hr after intraperitoneal administration, 3.6 to 8.8% of the urinary metabolites was unconjugated while 2.4 to 8.8% was present in the glucuronide fraction. After oral administration these amounts were 5.5 to 6.8% and 20.5 to 28.2% respectively. After both intraperitoneal and oral administration, no unchanged 2,4-DNT could be detected in the urine, and 2,4-dinitrobenzyl alcohol (2,4-DNBAlc) represented the most abundant identifiable neutral metabolite. Small amounts of 2,4-diaminotoluene, 2-amino-4-nitrobenzyl alcohol, 2-(N-acetyl)amino-4-nitrotoluene, 4-amino-2-nitrotoluene (4A2NT), and 2-amino-4-nitrotoluene (2A4NT) were also present. In almost all cases the largest proportion of metabolites represented unknowns, some of which exhibited the chromatographic properties of carboxylic acid metabolites. Metabolism of 2,4-DNT by liver and lung microsomes yielded mainly 2,4-DNBAlc with lower amounts of 4A2NT and 2A4NT, and their formation was dependent on the presence of oxygen and NADPH. Pretreatment of the animals with 2,3,7,8-tetrachlorodibenzo-p-dioxin resulted in increased yields of all three metabolites. Aerobic metabolism of 2,4-DNT by explants of the small intestine, large intestine, or by cecal contents yielded 2,4-DNBAlc, 2A4NT, 4A2NT and 4-(N-acetyl)amino-2-nitrotoluene (4Ac2NT). The proportion of reduced metabolites (2A4NT, 4A2NT, and 4Ac2NT) was much higher in these systems than with liver or lung microsomes and their formation by small intestine and cecal contents was enhanced several-fold under anaerobic conditions, while that of 2,4-DNBAlc was abolished. It is concluded that 2,4-DNT metabolism in the A/J mouse is rapid and complete and that the major neutral urinary metabolite is 2,4-DNBAlc. Minor amounts of reduced or partially reduced products appear to be formed mainly in the intestine, with a major role by its microflora.     

Hepatic microsomal metabolism and covalent binding of 2,4-dinitrotoluene

The effects of 2,4-dinitrotoluene (2,4-DNT) on xenobiotic metabolizing enzymes and the hepatic metabolism and covalent binding of this compound to microsomal proteins in vitro were studied. Male Fischer-344 rats received po doses of DNT daily for 5 days at 14, 35, and 70 mg/kg/day. Hepatic oxygen-insensitive cytosolic azoreductase activity was increased and microsomal nitroreductase was decreased by DNT treatments. A small but significant increase in liver/body weight ratio and in hepatic cytochromes P-450 and b₅ occurred in the absence of changes in microsomal biphenyl hydroxylase or aryl hydrocarbon hydroxylase activities. The patterns of in vitro microsomal metabolism of DNT were dependent on oxygen tension: under aerobic conditions, 2,4-dinitrobenzyl alcohol (DNBAlc) was the major metabolite whereas under anaerobic conditions no DNBAlc was detected; 2-amino-4-nitrotoluene (2A4NT) and 4-amino-2-nitrotoluene (4A2NT) were the major metabolites. Pretreatment of rats with phenobarbital or Aroclor 1254 increased the metabolism of 2,4-DNT to DNBAlc by six- to sevenfold. Metabolism to the alcohol was inhibited by SKF-525A. These data suggested that oxidative metabolism of 2,4-DNT to DNBAlc was mediated by cytochrome P-450-dependent mixed-function oxidases. Covalent binding studies showed that a maximum of only 7 pmol of 2,4-DNT-derived radioactivity was bound per milligram of microsomal protein per hour; this binding was increased to 1.0 nmol bound/mg protein/hr in microsomes from phenobarbital of Aroclor 1254-pretreated rats. It is concluded that 2,4-DNT treatment had little effect on the activity of some hepatic xenobiotic metabolizing enzymes and was readily metabolized by liver preparations in vitro. The pathways of in vitro metabolism were dependent on oxygen tension. This in vitro metabolism produced mostly polar metabolites which did not bind appreciably to microsomal macromolecules.     

Dominant lethal study of the antiandrogen alpha, alpha, alpha-trifluoro-2-methyl-4'-nitro-m-propionotoluidide: a comparison of statistical methods

The antiandrogen α,α,α-trifluoro-2-methyl-4′-nitro-m-propionotoluidide (SCH 13521) was given orally in a single dose of 500 or 1000 mg/kg to adult male rats. Triethylenemelamine (TEM) was given ip in a single dose of 0.25 or 0.50 mg/kg as a positive control. Placebo was given po or ip as a negative control. Males were mated sequentially with 2 untreated virgin females each week for 8 weeks. Near mid-pregnancy, the number of implantation sites and fetal deaths were measured. For comparative purposes, 4 methods of statistical analysis were used to evaluate genetic damage; with one of these, fetal deaths were expressed as a function of the sire. Compared with values from the negative controls, females that were mated with males from the 4 treatment groups had fewer implantation sites after the first posttreatment mating period. The results of the 4 analyses did not indicate that treatment with SCH 13521 induced dominant lethal mutations, although 3 of the 10 rats given 1000 mg/kg died. In contrast, data from the analyses indicated that TEM induced lethal mutations and, by inference, primarily affected mitotic and postmeiotic spermatocytes.     

Dominant lethal testing of theobromine in rats

Theobromine (TBR) 3,7-dimethylxanthine, the major purine alkaloid present in chocolate and cocoa products, was evaluated for dominant lethality in male Sprague-Dawley rats after administration of subacute oral doses of 0, 50, 150 and 450 mg/kg. Dominant lethal mutations were assessed weekly for a 10-week period by killing the females 13 days after the midweek of presumptive mating with treated males and counting total implants (living and dead), corpora lutea and number of pregnant females. Dead implants per total implants and preimplantation loss per total corpora lutea were evaluated statistically.

No significant dose-dependent effects were observed in the % dead implants of preimplantation loss throughout the study; pregnancy rate averaged 94%. These results indicated no induction of dominant lethal mutations or adverse effects on pregnancy rate after TBR doses equivalent to 25–225 times the maximum human consumption level.