Tirumuru V. Reddy

ASSESSMENT OF ENVIRONMENTAL HAZARDS OF 1,3,5-TRINITROBENZENE

The remedial investigation/feasibility studies conducted at certain Army installations showed a need to clean up contaminated sites, where high levels of ammunition chemicals such as 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), 1,3-dinitrobenzene (DNB), and their degradation products/metabolites were detected in surface soil and groundwater. TNB is a photodegradation product of TNT; it is not easily degraded, and persists in the environment. The toxicity data on TNB are scanty. Hence the U.S. Environmental Protection Agency in 1988 (U.S. EPA, 1997) developed a reference dose (RfD) for TNB (0.00005 mg/kg/d for chronic toxicity) based on the toxicity of DNB, which is structurally similar to TNB. Since then we have completed acute, subacute, subchronic, chronic, reproductive, and developmental toxicity studies and toxicokinetics studies. We have reviewed the mammalian toxicity data for TNB and have determined the no observed adverse effect levels (NOAEL) and low observed adverse effect levels (LOAEL) for subchronic, chronic, reproductive, and developmental toxicity. Based on the newly determined NOAEL and LOAEL values, we have now developed a new RfD for TNB (0.03 mg/kg/d), based on the chronic toxic effects on hematology and histopathological changes in testes and kidney.

Separation and quantitation of nitrobenzenes and their reduction products nitroanilines and phenylenediamines by reversed-phase high-performance liquid chromatography

Abstract A reversed-phase high-performance liquid chromatographic method for the separation and quantitation of a mixture consisting of nitrobenzene, dinitrobenzene isomers, 1,3,5-trinitrobenzene and their reduction products: aniline, nitroanilines and phenylenediamines has been developed. The method is sensitive and highly reproducible. The mixture is resolved on a Zorbax C 8 column with 0.1% triethylamine and methanol as the mobile phase. The detection limits for individual chemicals at 254 nm are in the range of 25–50 ng.

Fourteen‐day Toxicity Study of 1,3,5‐Trinitrobenzene in Fischer 344 Rats

Toxic effects of 1,3,5‐trinitrobenzene (TNB) in male and female rats were evaluated by feeding powdered certified laboratory chow diet supplemented with varied concentrations of TNB (0, 50, 200, 400, 800 and 1200 mg kg−1 diet) for 14 days. Food intake by female rats in 400, 800 and 1200 mg TNB diet groups was reduced and resulted in a significant decrease in absolute body weights (BW). Food and water consumption by male rats in high‐dose groups (800 and 1200 mg TNB kg−1 diet) was also reduced and resulted in a significant decrease in body weight. The calculated average TNB intake (from 1200 mg TNB kg−1 diet) was 92 mg kg−1 BW day−1 for male rats and 80 mg kg−1 BW day−1 for females. A decrease in testicular weight in males and an increase in spleen weight of both sexes in high‐dose groups was noted. In addition, histopathological examinationsrevealed that the susceptible organs for TNB toxicity were kidney (hyaline droplets), spleen (extramedullary hematopoiesis), brain (hemorrhage, malacia and gliosis) and testes (seminiferous tubular degeneration). Hematology and clinical chemistry studies indicated a decrease in red blood cell count and hematocrit, a decrease in alkaline phosphatase, an increase in Heinz bodies and increased methemoglobin concentration as compared to controls in both sexes. A lowest observed adverse effect level of 4.41 mg TNB kg−1 BW day−1 was established based on the findings of this study.

Gas Chromatographic and Mass Spectrometric Determination of Hemoglobin Adducts of 1,3-Dinitrobenzene and 1,3,5-Trinitrobenzene in Shrew Cryptotis Parva

1,3-Dinitrobenzene (DNB) and 1,3,5-trinitrobenzene (TNB) are used primarily in explosive compositions and munitions and have been detected as environmental contaminants of surface waters as well as ground waters near production waste disposal sites. Hemoglobin (Hb) adducts have recently been proposed as biological markers of exposure assessment for various environmental compounds, including nitroaromatics. In the present study, we have investigated the formation of DNB and TNB hemoglobin adducts in vivo and in vitro in the blood of shrew (Cryptotis parva). DNB and TNB hemoglobin adducts were detected by gas chromatography/mass spectrometry (GC/MS) after either basic (0.1 N NaOH) or acid (2 N HCl) hydrolysis followed by organic solvent extraction and derivatization of the corresponding amines. The levels of DNB-Hb adducts detected after basic hydrolysis (238.7 & pm; 50.2 pg/mg Hb) are higher than the corresponding levels detected after acid hydrolysis (52.5 & pm; 16.2 pg/mg Hb). For the TNB-Hb the levels after acid hydrolysis (132.2 & pm; 37.8 pg/mg Hb) are higher than the levels detected after basic hydrolysis (44.7 & pm; 15.3 pg-mg Hb). These results demonstrate the effectiveness of the hemoglobin adduct model for monitoring exposure to nitroaromatics.

Toxicity of Tetryl (N-Methyl-N,2,4,6–Tetranitroaniline) in F344 Rats

The toxicity of tetryl (N-methyl-N,2,4,6–tetranitroaniline) in male and female F344 rats was evaluated after adminstration in the diet for 14 or 90 days. The 14–day study diet concentrations used were 0, 500, 1250, 2000, 2500, and 5000 ppm; the 90–day study diet concentrations were 0, 200, 1000, and 3000 ppm tetryl in the diet. The calculated average daily tetryl intake was 32.1, 82.5, 130.3, 178.9, and 374.4 mg/kg body weight (BW) for females and 31.8, 80.0, 121.0, 170.5, and 349.7 mg/kg BW for males in the 14–day study. For the 90–day studies, the daily tetryl intake was 14.2, 68.8, and 199.0 mg/kg BW for females and 13.0, 62.4, and 179.6 mg/kg BW for males. In the 14–day study, there was a significant decrease in body weights (males), whereas relative(organ/body weight) liver and spleen (females), and kidney (males) weights were significantly increased in the 5000–ppm dose group. Hematological effects observed were decreased hemoglobin and hematocrit and an increased number of reticulocyts in females (2000 to 5000 ppm). Methemoglobin levels in males (2000 to 5000 ppm) and females (5000 ppm) and total blood protein and albumin levels in all groups of males and females (except 500 ppm) were significantly increased. Histopathological changes were observed in kidneys (deposition of cytoplasmic droplets) of all dose groups of male rats. In the sub–chronic (90–day) study, feed intake was reduced in all dose groups, but a significant decrease in terminal body weights was observed in females (1000 and 3000 ppm) and males (3000 ppm). An increase in the relative liver, kidney (1000–3000 ppm), and spleen (3000 ppm) weights were noted in both sexes. The hemoglobin content and red blood cell count were decreased whereas the reticulocyte count was elevated (3000 ppm) in both sexes at 45 and 90 days. Methemoglobin levels were increased in both sexes (1000 and 3000 ppm). Histopathologicalchanges were noted in the spleen (pigment deposition and erythroid cell hyperplasia) of both sexes (3000 ppm) and kidneys (tubular degeneration and cytoplasmic droplets containing alpha-2-micro globulin) of male rats (1000 to 3000 ppm). A no observed adverse effect level (NOAEL) for both sexes was 13 mg/kg BW/day was determined.

Subchronic Toxicity of 1,3,5-Trinitrobenzene in Fischer 344 Rats

The subchronic toxicity of 1,3,5-trinitrobenzene (TNB) in male and female Fischer 344 rats was evaluated by feeding a powdered certified laboratory diet containing 0, 66.7, 400 and 800 mg TNBl kg diet for 90 days. The calculated average TNB intake was 4.29, 24.70, and 49.28 mg/kg body weight (BW)day for females and 3.91, 22.73, and 44.16 mg/ kg BWI day for males. Food intake in the 400 and 800 mg/kg diet dose groups of both sexes was decreased throughout the study and resulted in a significant decrease in absolute body weights. A significant decrease in relative testicular weights and a significant increase in the relative liver weight were observed in male rats receiving 400 or 800 mg TNB/kg diet. A significant increase in the relative spleen weights of both sexes receiving 400 or 800 mg TNB diet was noted. The relative liver weight was also increased only in female rats maintained on the 800 mg TNB diet. Histopathological examinations revealed that the susceptible organs for TNB toxicity were kidney (hyaline droplets) in all male dose groups and testes (seminiferous tubular degeneration) in rats receiving 400 and 800 mg TNB diet groups. The spleen was also affected (extramedullary hematopoiesis) in both sexes in the 400 and 800 mg dose groups. Hematological studies at both 45 (data not given) and 90 days in the 400 and 800 mg dose groups indicated decreased values for red blood cell counts and hemoglobin content, while reticulocytes and methemoglobin levels were increased. Clinical chemistry parameters were unaffected. Based on kidney toxicity and hematological effects, a Low Observed Adverse Effect Level (LOAEL) of 3.91 mg/kg BWI day was suggested for subchronic toxicity studies on TNB.

Macromolecular adduction by trichloroacetonitrile in the Fischer 344 rat following oral gavage

Male Fisher 344 rats were administered 1- or 2-[14C]trichloroacetonitrile (TCAN) by oral gavage. DNA was isolated from the liver, kidneys and stomach and several protein fractions (globin, albumin and globulins) were isolated from blood. TCAN binds to both the DNA and the blood proteins in a dose-related manner. More radiolabel was associated with the DNA when the carbon at C2 position was labeled, than at C1 position. However, the position of the radiolabel did not influence the levels of radioactivity associated with the blood proteins. The stomach exhibited the highest level of DNA binding, followed in order by the liver and kidney. TCAN binding level was higher in DNA isolated from rats killed at 24 h than at 4 h after administration. In contrast, the three blood proteins showed similar binding levels, regardless of the exposure time. Radioactivity associated with DNA was not incorporated into the nitrogen bases (i.e. via de novo synthesis) and a covalent binding index (mumol chemical bound/mol nucleotide phosphate per mmol/kg body wt. of chemical administered) of 30-120 was observed for various tissues. Most of the radioactivity (60-80%) associated with globin could be released and separated from the protein by the treatment with concentrated ammonium hydroxide and precipitation of protein by organic solvent. Three peaks were observed in the HPLC elution profiles of the radioactivity released from the globin. Trichloroacetic acid co-eluted with one of these released products (peak II), however, the chemical identity of the material under the major peak (peak III) and peak I are still uncharacterized.