3.2.1.1. Death

In a retrospective cohort mortality study of 457 munitions workers who were exposed to either 2,4-DNT or Tg-DNT at two geographically different U.S. manufacturing plants, significant increases in death rates due to ischemic heart disease and residual diseases of the circulatory system were found (standardized mortality ratios [SMRs] of 126 and 143; 95% confidence intervals [CIs] of 65–234 and 112–179, respectively) (Levine et al. 1986a). Residual diseases of the circulatory system include congestive heart failure, cardiac arrest, and arteriosclerosis. The workers had been exposed to unreported concentrations of either 2,4-DNT (98% pure) or Tg-DNT for periods ranging from 30 days to >5 years (Levine et al. 1986a). Cigarette smoking was not taken into account in this study, but the study authors suggested that it may not have been a risk factor because mortality from lung cancer was less than expected. Among workers at both plants, there appeared to be a latency period of >15 years for a significant increase in mortality due to ischemic heart disease. There also appeared to be a relationship between heart disease and the intensity of exposure to DNTs. No statistical increase was found in death due to cancer, either from malignant neoplasms as a whole or from individual cancers, although the statistical power of the study was insufficient to detect anything but gross changes in the death rate due to cancer.

The Levine et al. (1986a) retrospective cohort mortality study was limited by small cohort size, and thus, the study had diminished power to detect an effect. As a result, the finding of elevated mortality from heart disease among workers in two plants from different parts of the United States linked only by exposure to DNTs is unusual. Workers in the United States generally have lower rates of heart disease than the general population because of the “healthy worker effect.” At both plants, mortality from ischemic heart disease during the first 15 years following cohort entry was less than expected, and mortality increased only in later years. Suggestive, but not significant, is evidence of a relationship between heart disease and duration and intensity of exposure, also reported by Levine et al. (1986a). No studies were identified with respect to mortality after inhalation exposure of humans to 2,3-, 2,5-, 2,6-, or 3,5-DNT.

In an acute-duration study, no mortality was observed in male or female F344 rats (5/sex/group) exposed nose-only to 2,6-DNT as a vapor at 26 mg/m³ for 6 hours and observed 14 days after dosing (CMA 1991). In the same study, groups of male and female rats exposed to 2,6-DNT at 0, 196, 473, or 694 mg/m³ as an aerosol exhibited mortality at ≥196 mg/m³. Two of five males (and no females) died at 196 mg/m³; all males and three of five females died at 694 mg/m³. Rats that died showed evidence of lung congestion and had increased relative lung weights compared to controls. An LC₅₀ of 0.43 mg/L was identified in rats, with LC₅₀ values for males and females of 0.24 and 0.66 mg/L, respectively.

No studies were located regarding death in animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 3,4-, or 3,5-DNT.

3.2.1.2. Systemic Effects

No studies were located regarding dermal, ocular, endocrine, or body weight effects in humans or animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. No studies were located regarding respiratory effects in humans after inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT or in animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 3,4-, or 3,5-DNT.

Respiratory Effects. No studies were identified with respect to respiratory effects after inhalation exposure of humans to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

In an acute-duration study, groups of male and female rats were exposed to 2,6-DNT at 0, 196, 473, or 694 mg/m³ as an aerosol for 6 hours and observed 14 days after dosing (CMA 1991). Rats exposed to 2,6-DNT as an aerosol at ≥196 mg/m³ exhibited signs of respiratory distress (exaggerated respiratory movements) for several days following exposure; recovery from this effect occurred by day 5 of the observation period. Rats that died showed evidence of lung congestion and had increased relative lung weights compared to controls.

No studies were identified with respect to respiratory effects after inhalation exposure of animals to 2,3-, 2,4-, 2,5-, 3,4-, or 3,5-DNT.

Cardiovascular Effects. No studies were identified with respect to cardiovascular effects after inhalation exposure of humans to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Levine et al. (1986a) reported a significant increase in deaths from diseases of the circulatory system in workers involved in the manufacture and processing of 2,4-DNT and/or Tg-DNT. The preponderance of circulatory disease deaths was due to ischemic heart disease. The SMR was significantly elevated for ischemic heart disease when compared to the U.S. male population and to persons living in the area. When workers were divided by exposure duration, the significant increase in cardiovascular deaths was only observed in workers exposed for at least 5 months. Although the study did not control for cigarette smoking, a known risk factor for heart disease, the investigators noted that the study did not find significant increases in lung cancer or disease deaths.

No studies were located regarding cardiovascular effects in animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Gastrointestinal Effects. Vomiting and nausea were mentioned as health complaints in a survey of male workers involved in the production of smokeless gunpowders during World War II (McGee et al. 1947). The exposure concentrations of 2,4-DNT were not specified. The investigators noted that prior to improving industrial hygiene practices, a much higher incidence of gastrointestinal symptoms was found. Since exposure to other compounds cannot be ruled out, attribution of these symptoms to DNT cannot be verified.

No studies were identified with respect to gastrointestinal effects after inhalation exposure of humans to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. No studies were located regarding gastrointestinal effects in animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Hematological Effects. Several hematological effects, including anemia and cyanosis, were found in male workers employed by a munitions factory during World War II (McGee et al. 1947). In some cases, there were increases in leukocyte count, which may be related to prolonged exposure to DNT. The study authors presumed that the exposure concentrations to 2,4-DNT were relatively high because of the relatively primitive industrial hygiene practices at that time. Although 36 of 154 workers were anemic in the earlier study and 73 of 714 workers were anemic in the follow-up study, no control groups were used as a basis for comparison. Because of possible exposure to other compounds, lack of work histories, lack of exposure monitoring, lack of a control population, and small cohort size, the results obtained are equivocal and may be best used as qualitative descriptions of symptoms. Marked cyanosis and other incapacitating symptoms were reported after exposure to unspecified concentrations of Tg-DNT in a study of French workers in a DNT production plant during World War I (Perkins 1919). It is assumed that workers were exposed to high concentrations of Tg-DNT via both inhalation and dermal pathways, since the processes described involved direct handling of large amounts of Tg-DNT without protective equipment.

No studies were identified with respect to hematological effects after inhalation exposure of humans to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

In an acute-duration study, male and female F344 rats (5/sex/group) were exposed to 2,6-DNT as a vapor at 0 or 26 mg/m³ or as an aerosol at 0, 196, 473, or 694 mg/m³ for 6 hours and observed 14 days after dosing (CMA 1991). Blood was analyzed for serum methemoglobin concentration 24 hours prior to dosing and 1, 24, and 48 hours and 7 days after dosing. Although the study authors noted slight increases in serum methemoglobin in 2,6-DNT-exposed animals in the first 24 hours after exposure, these changes were not clearly dose-related and were not statistically significantly different from control animals. No other hematological end points were evaluated.

No studies were located regarding hematological effects in animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 3,4-, or 3,5-DNT.

Musculoskeletal Effects. Joint pain, especially in the knees, and other incapacitating symptoms were found in unspecified numbers of French workers in a plant that produced DNTs during World War I (Perkins 1919). No exposure concentrations were reported, but it is assumed that they were high because of the direct handling of large amounts of Tg-DNT without protective equipment, which also suggests that the workers were exposed dermally. However, because exposure to other compounds cannot be ruled out and no control data are available, caution must be used when interpreting these results.

No studies were located regarding musculoskeletal effects humans after inhalation exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT or in animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Hepatic Effects. A study of 714 workers at a munitions plant found that 29 experienced liver tenderness (McGee et al. 1947). Other factors, such as alcohol consumption, may account for these results which should be viewed with caution because of the lack of control data, lack of information on exposure concentrations, and possible multiple chemical exposure. Medical surveys of 52 male workers exposed to Tg-DNT in a chemical plant that manufactured toluenediamine (TDA) revealed no differences in hepatic blood chemistry profiles (NIOSH 1982). Air samples contained concentrations ranging from 0.026 to 0.890 mg/m³ Tg-DNT (mean 0.207 mg/m³).

No studies were located regarding hepatic effects in humans after inhalation exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT, or in animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Renal Effects. No effects were observed on either of the renal parameters (blood urea nitrogen [BUN], creatinine) monitored in blood chemistry in a medical survey of 52 male workers exposed to Tg-DNT in a chemical plant that manufactured TDA (NIOSH 1982). Exposure concentrations in air samples taken for this study ranged from 0.026 to 0.890 mg/m³ Tg-DNT (mean 0.207 mg/m³). The study was limited by a small exposure population and lack of historical individual exposure monitoring.

Evidence of tubular and/or glomerular damage (alterations in urinary protein excretion patterns) was found among a cohort of approximately 160 workers at a copper mine exposed to Tg-DNT explosives (Brüning et al. 2001). Dividing the workers into exposure categories resulted in a significant dose-related trend for the incidence of tubular and/or glomerular damage among the workers without renal cell cancer or urothelial cancer. Approximately 80% of the 25 workers in the very high exposure category had evidence of renal damage. Dose-related increases in the excretion of α1-microglobulins and glutathione-S-transferase-α were also observed suggesting proximal tubule damage. The lack of effect on glutathione-S-transferase-π levels suggested a lack of damage to the distal tubules.

No studies were located regarding renal effects in humans or animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

3.2.1.3. Immunological and Lymphoreticular Effects

No studies were located regarding immunological or lymphoreticular effects in humans or animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

3.2.1.4. Neurological Effects

Dizziness and headache were reported by Perkins (1919) in a study of French workers exposed to Tg-DNT at a production plant during World War I. Although no exposure concentrations were reported, it is assumed that the workers were exposed to high concentrations of Tg-DNT via both inhalation and dermal pathways, since the manufacturing processes required workers to handle large amounts of Tg-DNT without protective equipment. Exposure to chemicals other than DNT in this environment could not be ruled out. Health effects of munitions workers exposed to unspecified levels of what was presumed to be 2,4-DNT were studied by McGee et al. (1947). Neurological signs reported by these workers included headache, dizziness, and pain, numbness, and tingling in the extremities. The 2,4-DNT exposure concentrations were not specified, but were considered by these authors to be relatively high as a result of the lack of safety practices.

No studies were located regarding neurological effects in humans after inhalation exposure to 2,3-, 2,5-, 2,6-, or 3,5-DNT or in animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

3.2.1.5. Reproductive Effects

Studies of men occupationally exposed to Tg-DNT at DNT and TDA plants showed no significant differences in sperm counts or morphology, follicle stimulating hormone (FSH) levels, or incidence of miscarriage in their wives compared to controls (Hamill et al. 1982; NIOSH 1982). In the NIOSH (1982) study, Tg-DNT concentrations ranged from 0.026 to 0.890 mg/m³ (mean 0.207 mg/m³). Interpretation of these studies is somewhat confounded by the lack of distinction between DNT and TDA exposure and the lack of information regarding exposure concentration in the Hamill et al. (1982) study. The limitations of these studies are similar (small exposure populations and the lack of individual exposure monitoring) and limit the ability of the studies to detect adverse effects.

No significant effects on the fertility of workers occupationally exposed to Tg-DNT have been found in several studies (Hamill et al. 1982; Levine et al. 1985a; NIOSH 1982). However, Levine et al. (1985a) estimate that only a 50–70% reduction in fertility could have been detected in the worker population that they studied.

One study by the CDC (1981) noted that sperm counts were decreased by >50% in workers in a Kentucky chemical plant exposed to DNTs and TDA compared to workers not exposed to these chemicals. The study was limited because of multiple chemical exposures and the small numbers of workers examined. Thirty workers participated in the study: 9 currently exposed, 12 previously exposed, and 9 with no history of exposure to DNTs/TDA.

No studies were located regarding reproductive effects in humans or animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

3.2.1.6. Developmental Effects

No studies were located regarding developmental effects in humans or animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

3.2.1.7. Cancer

The mortality of a cohort of 4,989 men who worked at least 5 months in a munitions facility was analyzed to determine whether DNT exposure was associated with an increased risk of cancer of the liver and biliary tract (Stayner et al. 1993). Workers were considered exposed if they had worked at least 1 day on a job with probable exposure to DNT. In this study, a significant increase in hepatobiliary cancer mortality (standard rate ratio [SRR]=3.88, 95% CI 1.04–14.41) was observed among DNT-exposed workers compared to non-exposed control workers. However, no significant changes were noted when compared to the U.S. population, the SRR for hepatobiliary cancer being 2.67 (95% CI 0.98–5.83; p=0.052). No quantitative data were available on the DNT exposure of these men. This study is limited by the small numbers of hepatobiliary cancer cases, small numbers of workers with long term exposure to DNT, and possible exposure of the workers to other chemicals. However, no significant increases in mortality from malignant neoplasms as a group or from particular cancers (liver, lung, gallbladder, kidney, and connective tissues) were observed in workers occupationally exposed to 2,4-DNT and/or Tg-DNT (Levine et al. 1986b). Exposures were not quantified and the cohort was small. The study authors estimated that an 8-fold increase in liver and gallbladder cancer in exposed workers would be necessary in order to be detected at the p=0.05 level; thus, the statistical analysis was not strong enough to detect small increases in cancer.

Among 500 workers in an underground copper mine where Tg-DNT-containing explosives were extensively used, increases, as compared to the German national cancer registry, in the incidence of renal cell cancer and urothelial cancer incidence were observed (Brüning et al. 1999). However, when the workers were divided by exposure categories, no apparent relationship between renal cell cancer incidence and exposure category was found. In contrast, four of the six workers with urothelial cancer cases were in the high exposure group (one was in the lowest group and the other was in the highest exposure group). The genotype distribution of several polymorphic xenobiotic enzymes, including N-acetyltransferase 2 and glutathione-S-transferases M1 (GSTM1) and T1 (GSTT1), was examined in the 14 workers with renal cell cancer and 6 workers with urothelial cancer. The genotype distribution among the renal cell cancer cases did not differ from the normal German population. Similarly, the distribution of GSTM1 and GSTT1 genotypes for the urothelial cancer cases was similar to the German population.

However, all of the urothelial cancer cases were found to be “slow acetylators” compared to a 58% distribution in the German population.

Harth et al. (2005) reported three cases of urothelial carcinoma of the urinary bladder among 60 workers exposed to Tg-DNT at a German explosive manufacturing facility. The investigators noted that this cancer rate was 15.9 times higher than the expected incidence in the federal state.

Seidler et al. (2014a) examined the cancer incidence through 2005 in a cohort of 16,441 male copper miners who worked between 1953 and 1990 in one of two German mines that used Tg-DNT explosives. The mean duration of employment was 6.8 years. Compared with the cancer incidence in the general population of the region in Germany where the mines were located (Saxony-Anhalt), the cancer incidences in the miners were not significantly increased for any cancer other than lung cancer (standardized incidence ratio [SIR] = 1.29, 95% CI 1.13–1.46). Moderate, but not statistically significant, increases in the incidences of kidney and bladder cancer (SIRs of 1.25 and 1.41 for kidney and bladder cancer, respectively, among workers with >20 years of employment with exposure to Tg-DNT) were seen when the data were stratified by duration of employment with Tg-DNT exposure. A follow-up case-cohort study of this group, with follow-up through 2006, compared the Tg-DNT exposure history (by job exposure matrix) of 109 renal cancer cases with that of 999 randomly chosen cohort members (Seidler et al. 2014b). The Cox proportional hazard ratio (HR) was elevated, but not significantly, for high inhalation exposure to Tg-DNT (HR 1.36, 95% CI 0.84–2.21). When combined across medium and high exposure categories for both dermal and inhalation exposure, the HR for renal cancer was significantly increased (HR 2.12, 95% CI 1.03–4.37).

No studies were located regarding cancer in humans following inhalation exposure to 2,3-, 2,5-, 3,4-, or 3,5-DNT or in animals following inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

3.2.2.1. Death

No studies were located regarding death in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

2,4-DNT is lethal to experimental animals after oral administration. Animals generally developed cyanosis and ataxia after dosing. In general, rats are more sensitive than mice to the lethal effects of 2,4-DNT. The LD₅₀ values that have been determined for rats after gavage dosing with 2,4-DNT range from 270 to 650 mg/kg (U.S. Army 1975, 1978a; Vernot et al. 1977); in mice, LD₅₀ values were reported to be between 1,340 and 1,954 mg/kg after 2,4-DNT administration (U.S. Army 1975, 1978a; Vernot et al. 1977). In female Sprague-Dawley rats (5/group) administered a single dose of 2,4-DNT (in 5% v/v DMSO in corn oil) via gavage at 398 mg/kg and observed for 24 or 48 hours after dosing, two of five animals and one of five animals died within 24 and 48 hours, respectively (Deng et al. 2011). No deaths occurred in rats administered 2,4-DNT at 5, 50, 99, or 198 mg/kg. In a dominant lethal study by Lane et al. (1985), 8 of 15 male Sprague-Dawley rats died after receiving five daily doses of 240 mg/kg 2,4-DNT. No deaths were reported when male and female Sprague-Dawley rats were fed 78 or 82 mg/kg/day 2,4-DNT, respectively, in the diet for 14 days (McGown et al. 1983). When male Sprague-Dawley rats were given gavage doses of 284 mg/kg/day for 14 days, all six exposed rats died (Lent et al. 2012a; USAPHC 2011b).

Death has been reported after intermediate- and chronic-duration exposure to 2,4-DNT in numerous studies. One of eight male and eight of eight female CD rats died after 3–13 weeks of ingesting 2,4-DNT in the diet (Lee et al. 1985; U.S. Army 1978b). Concentrations in the feed causing these deaths were equivalent to doses of 93 and 145 mg/kg/day in males and females, respectively. Death has also been reported in rodents fed concentrations equivalent to doses of 371–413 mg/kg 2,4-DNT in the diet for up to 6 months (Hong et al. 1985; Kozuka et al. 1979; U.S. Army 1978b). No treatment-related deaths were reported in rats fed up to 16.5 mg/kg/day 2,4-DNT or mice fed up to 28.5 mg/kg/day 2,4-DNT for 4 weeks, or in rats fed up to 22 mg/kg/day 2,4-DNT or mice fed up to 76 mg/kg/day 2,4-DNT for 78 weeks (NCI 1978). In a 3-generation reproductive study, there appeared to be an increased incidence of death among F₀ dams during parturition after receiving 45.3 mg/kg/day 2,4-DNT in the diet for 6 months (U.S. Army 1979). These deaths were associated with prolonged parturition, hemorrhage, and placental retention. However, because these effects were also seen to a lesser extent in control animals, it may be that the effects of 2,4-DNT simply enhanced effects caused by the advancing age of the dams (U.S. Army 1979).

In a 13-week study, some dogs fed 25 mg/kg/day became moribund after ≥22 days and had to be terminated, whereas no treatment-related deaths were reported in dogs fed 5 mg/kg/day (Ellis et al. 1985; U.S. Army 1978b). In addition to severe weight loss, severe neurological effects and histopathological changes were found in these animals, including vacuolization and focal gliosis in the cerebellum and perivascular hemorrhages in the cerebellum and brain stem, as well as peripheral neuropathy, testicular degeneration, and biliary hyperplasia. In a 24-month study of dogs, the administration of 10 mg/kg/day 2,4-DNT by capsule caused death within 6 months, but no deaths were reported at 1.5 mg/kg/day; clinical signs prior to death were similar to those reported in the 13-week study (Ellis et al. 1985; U.S. Army 1979). Decreased longevity was reported in 1–2-year studies of CD rats at average daily intakes as low as 3.9 mg/kg/day (males) and 5.1 mg/kg/day (females), and of CD-1 mice at 898 mg/kg/day (Hong et al. 1985; Lee et al. 1985; U.S. Army 1978b, 1979).

The experimental data are more limited for 2,6-DNT than for 2,4-DNT. After administration of 2,6-DNT, LD₅₀ values have been reported to range from 180 to 795 mg/kg in rats and from 621 to 807 mg/kg in mice (U.S. Army 1975, 1978a; Vernot et al. 1977). Complete lethality was reported in female Sprague-Dawley rats (5/group) administered a single dose of 2,6-DNT via gavage (in 5% v/v DMSO in corn oil) at 398 mg/kg (Deng et al. 2011). No deaths occurred in animals administered 5–199 mg/kg and observed for 2 days after dosing. Mortality was reported in a dose-range finding study of male rats given oral doses of 200 mg/kg/day 2,6-DNT in corn oil for 3 days; no rats died at 100 mg/kg/day (Rothfuss et al. 2010). The maximum tolerated dose (MTD) of 2,6-DNT corresponding to 100% survival of A/J mice after 6 doses over a 2-week period was 250 mg/kg (Schut et al. 1983). No male Sprague-Dawley rats died when groups of six rats were given 14 consecutive daily gavage doses up to 134 mg/kg/day 2,6-DNT (Lent et al. 2012a; USAPHC 2011d).

All six male rats exposed to oral doses of 33 mg/kg/day 2,6-DNT in corn oil for 29 consecutive days survived the treatment (Rothfuss et al. 2010). Intermediate-duration studies have shown an increase in mortality of mice and dogs after 2,6-DNT administration. After feeding 51 mg/kg/day 2,6-DNT to male Swiss albino mice in the diet for up to 13 weeks, 8 of 16 of these animals died; 6 of 16 females fed 55 mg/kg/day 2,6-DNT also died (U.S. Army 1976). No treatment-related deaths were reported when rats were fed up to 155 mg/kg/day 2,6-DNT for the same duration (U.S. Army 1976). Two of eight dogs treated with 20 mg/kg 2,6-DNT by capsule died in a 13-week study (U.S. Army 1976). Thus, dogs seem to be the most sensitive of the three species to intermediate-duration oral 2,6-DNT exposure.

Vernot et al. (1977) reported LD₅₀ values for 2,3- DNT of 1,120 mg/kg/day in male Sprague-Dawley rats and 1,070 mg/kg/day in male CF-1 mice. No cause of death or additional information was reported. Exposure to 550 mg/kg/day 2,3-DNT via gavage for 14 days resulted in the deaths of 6/6 male Sprague-Dawley rats; no deaths occurred at doses of ≤275 mg/kg/day (Lent et al. 2012a; USAPHC 2011a).

For 2,5-DNT, LD₅₀ values of 710 and 1,230 mg/kg/day, respectively, were reported in male Sprague-Dawley rats and male CF-1 mice (Vernot et al. 1977); no additional information was provided. One rat in a group of six male Sprague-Dawley rats died after 14 consecutive daily gavage doses of 308 mg/kg/day 2,5-DNT (Lent et al. 2012a; USAPHC 2011c).

Lent et al. (2012a; USAPHC 2011e, 2011f) provide the only acute-duration lethality data for 3,4- and 3,5-DNT. When groups of six male Sprague-Dawley rats received gavage doses of 3,4-DNT up to 227 mg/kg/day for 14 days, there were no deaths. In contrast, 3,5-DNT was lethal to 1/6 rats at a dose of 39 mg/kg/day and to all rats at doses of 77 and 155 mg/kg/day.

Administration of up to 150 mg/kg/day Tg-DNT for 14 days was lethal to 6 of 13 pregnant F344 rats when administered by gavage during gestation (Jones-Price et al. 1982), yet this same concentration of Tg-DNT fed in the diet for 30 days did not kill any of the same strain of rats in another study (Hazleton Laboratories 1977). Decreased survival was found in CDF rats fed 35 mg/kg/day Tg-DNT for 52 weeks or 14 mg/kg/day Tg-DNT for 104 weeks (Hazleton Laboratories 1982).

All LOAEL values from each reliable study for death in each species and duration category for 2,3-, 2,4-DNT, 2,5-, 2,6-, 3,4-, and 3,5-DNT are recorded in Tables 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, respectively, and plotted in Figures 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, respectively.

Table 3-1

Levels of Significant Exposure to 2,3-Dinitrotoluene - Oral.

Figure 3-1

Levels of Significant Exposure to 2,3-Dinitrotoluene - Oral. Acute (≤14 days)

Table 3-2

Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral.

Figure 3-2

Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral.

Table 3-3

Levels of Significant Exposure to 2,5-Dinitrotoluene - Oral.

Figure 3-3

Levels of Significant Exposure to 2,5-Dinitrotoluene - Oral. Acute (≤14 days)

Table 3-4

Levels of Significant Exposure to 2,6-Dinitrotoluene - Oral.

Figure 3-4

Levels of Significant Exposure to 2,6-Dinitrotoluene - Oral.

Table 3-5

Levels of Significant Exposure to 3,4-Dinitrotoluene - Oral.

Figure 3-5

Levels of Significant Exposure to 3,4-Dinitrotoluene - Oral. Acute (≤14 days)

Table 3-6

Levels of Significant Exposure to 3,5-Dinitrotoluene - Oral.

Figure 3-6

Levels of Significant Exposure to 3,5-Dinitrotoluene - Oral. Acute (≤14 days)

3.2.2.2. Systemic Effects

The systemic effects observed after oral exposure of humans and animals to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT are discussed below. No studies were located regarding musculoskeletal effects in humans or animals after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

All LOAEL values from each reliable study for systemic effects in each species and duration category for 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-DNT are recorded in Tables 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, respectively, and plotted in Figures 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, respectively.

Respiratory Effects. No studies were located regarding respiratory effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

No histopathological effects on the lungs were found when Sprague-Dawley rats were fed 261 mg/kg/day (males) or 273 mg/kg/day (females) 2,4-DNT for 14 days (McGown et al. 1983).

No respiratory system effects were observed when CDF rats were fed 14 mg/kg/day Tg-DNT for 2 years (Hazleton Laboratories 1982). Histopathological examination of the lungs and respiratory tract tissues of rats exposed to 14 mg/kg/day Tg-DNT for 2 years or 35 mg/kg/day for 1 year did not reveal any abnormalities (Hazleton Laboratories 1982).

No studies were located regarding respiratory effects in animals after oral exposures to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Cardiovascular Effects. No studies were located regarding cardiovascular effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Neither changes in heart weight nor microscopic lesions in the heart were observed in male Sprague-Dawley rats receiving gavage doses up to 275 mg/kg/day 2,3-DNT for 14 days (Lent et al. 2012a; USAPHC 2011a).

No histopathological effects on the cardiovascular system were found after Sprague-Dawley rats received 261 mg/kg/day (males) or 273 mg/kg/day (females) 2,4-DNT in the diet for 14 days (McGown et al. 1983). Neither changes in heart weight nor microscopic lesions in the heart were observed in male Sprague-Dawley rats receiving gavage doses up to 142 mg/kg/day 2,4-DNT for 14 days (Lent et al. 2012a; USAPHC 2011b).

Increased absolute and relative heart weight, along with mild-to-moderate fibrosis in 4/5 animals and trace-to-moderate inflammation in 2/5 animals, were noted in male rats receiving 308 mg/kg/day 2,5-DNT via gavage for 14 days (Lent et al. 2012a; USAPHC 2011c).

One of six male Sprague-Dawley rats exposed to 227 mg/kg/day 3,4-DNT (the highest dose tested) exhibited cardiac lesions consisting of mild myocardial fibrosis, inflammation, and necrosis; heart weight was not affected at this dose or at the lower doses tested (Lent et al. 2012a; USAPHC 2011e). Exposure to 14 days of gavage doses up to 39 mg/kg/day 3,5-DNT did not result in changes in heart weight or microscopic lesions in the hearts of male Sprague-Dawley rats (Lent et al. 2012a; USAPHC 2011f).

At the 26-week interim sacrifice in a 104-week study in which CDF rats were fed 0, 3.5, 14, or 35 mg/kg/day Tg-DNT in the diet, an increased incidence and severity of myocarditis was noted in males at 35 mg/kg/day (Hazleton Laboratories 1982). It was believed that this spontaneous inflammatory condition was exacerbated by ingestion of Tg-DNT in the high-dose animals. Although this condition was also observed at the 55-week sacrifice, it was not observed at 52 or 104 weeks.

Gastrointestinal Effects. No studies were located regarding gastrointestinal effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

There were no histopathological effects on the gastrointestinal tract of Sprague-Dawley rats fed 261 mg/kg/day (males) or 273 mg/kg/day (females) 2,4-DNT in the diet for 14 days (McGown et al. 1983).

Treatment of rats with up to 35 mg/kg/day Tg-DNT for up to 1 year or 14 mg/kg/day for up to 2 years did not cause any histopathological changes in the gastrointestinal tract (Hazleton Laboratories 1982).

No studies were located regarding gastrointestinal effects in animals after oral exposures to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Hematological Effects. No studies were located regarding hematological effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Hematological effects were noted in virtually all animal studies of oral exposure to DNTs in which circulating blood was examined. The most common findings were methemoglobinemia, anemia, reticulocytosis, and an increase in Heinz bodies. The hematological effects are caused by oxidation of the iron in hemoglobin, producing methemoglobin. Heinz bodies are granules in erythrocytes that are believed to result from denatured hemoglobin. Reticulocytosis, a finding in many animals in these studies, is caused by the increased production of immature erythrocytes (red blood cells) and is seen as a compensatory mechanism in anemia resulting from exposure to 2,4- and 2,6-DNT. This hematotoxic syndrome is a common effect of exposure to aromatic amines and most organic and inorganic nitrates, and it has been implicated for many oxidizing agents (Smith 1996; U.S. Army 1979).

When male Sprague-Dawley rats were treated for 14 days with gavage doses of 275 mg/kg/day 2,3-DNT, hematological effects included extramedullary hematopoiesis and lymphoid hyperplasia of the spleen and lymphoid depletion (Lent et al. 2012a; USAPHC 2011a).

Female Sprague-Dawley rats (5/group) administered 2,4-DNT via gavage (in 5% v/v DMSO in corn oil) and observed for 24 or 48 hours after dosing showed evidence of erythrocytosis, as indicated by significant increases in hemoglobin, hematocrit, and/or erythrocyte and granulocyte counts, at doses ≥99 mg/kg (Deng et al. 2011). Relative to controls, erythrocyte and granulocyte counts were increased by 11 and 552% at 99 mg/kg after 24 hours; elevations in hemoglobin (27–31%) and hematocrit (29–33%) were only statistically significant in rats treated at 198 or 398 mg/kg and evaluated at 48 hours. No significant changes in hematological end points were observed in rats treated with 2,4-DNT at 5 or 50 mg/kg and evaluated 24 or 48 hours after dosing. Development of erythrocytosis 48 hours following a single exposure to 2,4-DNT may be a secondary effect of dehydration, rather than a direct effect on the hematological system; however, no information on drinking water consumption was reported in this study. Slight cyanosis was observed in rats administered 60 mg/kg 2,4-DNT by gavage for 5 days (Lane et al. 1985). No changes in hematological parameters were found in Sprague-Dawley rats fed 261 mg/kg/day (males) or 273 mg/kg/day (females) 2,4-DNT in the diet for 14 days (McGown et al. 1983). In male rats receiving 2,4-DNT by gavage for 14 days, red blood cell count was significantly decreased at 142 mg/kg/day, while extramedullary hematopoiesis and lymphoid hyperplasia of spleen were seen at doses ≥71 mg/kg/day (Lent et al. 2012a; USAPHC 2011b). Hematocrit, percent reticulocytes, and relative spleen weight were not different from controls in C57Bl/6N mice exposed to 134 mg/kg/day 2,4-DNT via gavage for 14 days (Wilbanks et al. 2014). Kozuka et al. (1979) found methemoglobin concentrations increased to 7 times those of controls in the blood of rats fed a time-weighted average (TWA) dose of 371 mg/kg/day 2,4-DNT in the diet for 6 months. Anemia was observed in a 13-week feeding study in which male and female CD rats were fed 266 and 145 mg/kg/day, respectively, in the diet; milder effects, such as reticulocytosis and hemosiderosis or abnormal pigment in the spleen, were found at 93 and 108 mg/kg/day in males and females, respectively (Lee et al. 1985; U.S. Army 1978b). No hematological effects were observed in males and females administered 34 and 38 mg/kg/day, respectively. Mild anemia (as indicated by decreases in erythrocyte count, hematocrit, or hemoglobin concentration) and concurrent reticulocytosis were also observed in male and female CD-1 mice administered 413 and 468 mg/kg/day 2,4-DNT, respectively, in the diet for 13 weeks (Hong et al. 1985; U.S. Army 1978b). Anemia, accompanied by the presence of Heinz bodies, was observed in beagle dogs given 25 mg/kg/day 2,4-DNT in capsules (Ellis et al. 1985; U.S. Army 1978b).

As part of a 2-year study in Beagle dogs (6/sex/group) exposed to 2,4-DNT at doses of 0.2, 1.5, or 10 mg/kg/day, effects on hematological parameters were evaluated after 3, 6, and 9 months of treatment (U.S. Army 1979). At these intermediate-duration timepoints, hematological effects consistent with development of methemoglobinemia, anemia, and compensatory hematopoiesis (including decreased hemoglobin, hematocrit, and erythrocyte counts, and increased serum methemoglobin and reticulocyte counts) were observed in beagle dogs administered oral 2,4-DNT at 1.5 or 10 mg/kg/day. In female dogs administered 10 mg/kg/day, statistically significant decreases in erythrocyte count, hematocrit, and hemoglobin, a statistically significant increase in reticulocyte count, and the presence of Heinz bodies in serum were observed. Similar hematological effects were observed in female dogs administered 0.2 and 1.5 mg/kg/day, although effects did not reach statistical significance, most likely because the power of the study to detect statistically significant changes was compromised by the small number of dogs per treatment group. However, a clinically significant increase in methemoglobin levels of 225% was observed in female dogs administered 1.5 mg/kg/day; no significant hematological effects were observed at 0.2 mg/kg/day. Although effects at all time points were qualitatively similar, hematological changes observed after 9 months of exposure were more consistent and pronounced than those observed at the 3- and 6-month time periods.

Chronic studies of animals administered 2,4-DNT provide data that strengthen the weight-of-evidence supporting hematological effects. In a 24-month study, hematological effects consistent with development of methemoglobinemia, anemia, and compensatory hematopoiesis (including decreased hemoglobin, hematocrit, and erythrocyte counts, and increased serum methemoglobin and reticulocyte counts) were observed in beagle dogs (6/sex/group) administered oral 2,4-DNT at 1.5 or 10 mg/kg/day for 12 months of continuous dosing (U.S. Army 1979). No significant hematological effects were observed in dogs administered 2,4-DNT at 0.2 mg/kg/day. After treatment for 18 or 24 months, only slight or no anemia, near normal reticulocyte levels, no Heinz bodies, and minimal amounts of methemoglobin were detected, likely reflective of an adaptive response (Ellis et al. 1985; U.S. Army 1978b).

In a 2-year study (with a 1-year interim sacrifice) in which CD rats were fed 0.6, 3.9, or 34.5 mg/kg/day (males) or 0.7, 5.1, or 45.3 mg/kg/day (females) 2,4-DNT, significant decreases in red blood cell count were found in mid-dose males compared to controls, and anemia (as indicated by further reductions in red blood cell count, decreased hematocrit, decreased hemoglobin, and a compensatory increase in reticulocytes) was found in high-dose animals after 1 year (Lee et al. 1985; U.S. Army 1978b, 1979). No changes in methemoglobin or Heinz bodies were found. CD-1 mice that were administered 14, 95, or 898 mg/kg/day 2,4-DNT in the diet for 24 months were found to be anemic (as shown by significant reductions in erythrocytes and hemoglobin) at the high concentration, with compensatory increases in reticulocytes (Hong et al. 1985; U.S. Army 1979).

Dark spleen and mild-to-moderate extramedullary hematopoiesis were observed in male Sprague-Dawley rats treated for 14 days with gavage doses ≥39 mg/kg/day 2,5-DNT (Lent et al. 2012a; USAPHC 2011c). Dose-related hematology changes were noted, including decreased red blood cells at ≥77 mg/kg/day; increased mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) at ≥154 mg/kg/day; and increased red blood cell distribution width (at 77 and 154 mg/kg/day, but not at 308 mg/kg/day). Total white blood cell count was increased at 308 mg/kg/day, resulting from a nonsignificant increase in lymphocytes; in addition, the percent of eosinophils was decreased at this dose (Lent et al. 2012a; USAPHC 2011c).

Female Sprague-Dawley rats (5/group) administered 199 mg/kg 2,6-DNT as a single dose via gavage (in 5% v/v DMSO in corn oil) showed evidence of erythrocytosis, as indicated by statistically significant increases in serum hemoglobin (45%), hematocrit (41%), and erythrocyte (61%) and granulocyte (11-fold) counts 48 hours after dosing (Deng et al. 2011). Increased numbers of reticulocytes (44% higher than controls), which were associated with mature erythrocytes containing Heinz bodies, were also observed in rats administered 199 mg/kg and evaluated at 24 hours. No significant changes in hematological end points were observed in rats administered 2,6-DNT at 5–99 mg/kg and evaluated 24 or 48 hours after dosing. Development of erythrocytosis 48 hours following a single exposure to 2,6-DNT may be a secondary effect of dehydration, rather than a direct effect on the hematological system; however, no information on drinking water consumption was reported in this study. Lent et al. (2012a; USAPHC 2011d) reported hematology changes consisting of significantly reduced hemoglobin and hematocrit, as well as increased neutrophils, increased monocytes, and increased percentages of neutrophils and lymphocytes, in male rats exposed by gavage to 134 mg/kg/day of 2,6-DNT. Nonsignificant decreases in hemoglobin and hematocrit were seen at all doses in this study (4–134 mg/kg/day). As part of a 13-week study in Beagle dogs (4/sex/group) exposed to 2,6-DNT at doses of 4, 20, or 100 mg/kg/day, effects on hematological parameters were evaluated after 2 weeks of treatment (U.S. Army 1976). Dogs treated at 20 mg/kg/day showed a statistically significant decrease in erythrocyte count (16%) and a significant increase in mean cell hemoglobin (5%) after dosing for 2 weeks. At 100 mg/kg/day, more pronounced changes consistent with development of methemoglobinemia, anemia, and compensatory hematopoiesis were observed; dogs showed statistically significant reductions in erythrocyte count, hematocrit, and hemoglobin and an increase in reticulocyte count after 2 weeks of continuous dosing (U.S. Army 1976).

Subchronic administration (13 weeks) of 2,6-DNT in dogs and rats provide data that strengthen the weight-of-evidence supporting hematological effects. Significant hematological effects were observed in beagle dogs (4/sex/group) after administration of 2,6-DNT at 20 and 100 mg/kg/day, but not 4 mg/kg/day (U.S. Army 1976). Dogs treated at 20 mg/kg/day showed a statistically significant decrease in erythrocyte count (12%) after dosing for 4 weeks. At 100 mg/kg/day, more pronounced changes consistent with development of methemoglobinemia, anemia, and compensatory hematopoiesis were observed; dogs showed statistically significant reductions in erythrocyte count, hematocrit, and hemoglobin and an increase in reticulocyte count after 4 weeks of continuous dosing. Histopathological evaluation of the spleen showed an increased incidence of extramedullary erythropoiesis in dogs treated with 2,6-DNT at 4, 20, or 100 mg/kg/day for 4 or 13 weeks; this effect is an adaptive response to 2,6-DNT-induced methemoglobinemia and anemia.

Subchronic (13-week) administration of 2,6-DNT in CD rats induced changes in hematological parameters (measured at 4, 8, and 13 weeks) indicative of anemia and compensatory hematopoiesis (including significant decreases in erythrocytes, hematocrit, and hemoglobin and increased reticulocytes) at the highest tested dose (145 and 155 mg/kg/day for male and female rats, respectively) only; these effects were most pronounced after treatment for 4 weeks (U.S. Army 1976). No significant hematological changes were observed at 7 and 35 mg/kg/day (males) or 7 and 37 mg/kg/day (females). However, histopathological effects (extramedullary hematopoiesis and/or splenic hemosiderosis), indicative of an adaptive response to anemia and compensatory erythropoiesis, were observed in male and female rats administered 2,6-DNT at doses ≥7 mg/kg/day. Although histopathological effects (extramedullary hematopoiesis) were observed in CD-1 mice administered 2,6-DNT at ≥51 mg/kg/day (but not 11 mg/kg/day) for 4 or 13 weeks, no statistically significant changes in hematological parameters were seen at levels up to 289 mg/kg/day (males) or 299 mg/kg/day (females). The study authors indicated that some blood samples clotted, making hematological analyses impossible to perform. The small number of animals evaluated likely contributed to the identification of histopathological findings of the spleen in the apparent absence of 2,6-DNT-induced hematological effects. The 2,6-DNT isomer was not tested for hematological end points in studies of chronic duration.

Fourteen-day gavage exposure of male Sprague-Dawley rats to 3,4-DNT resulted in reduced hemoglobin and hematocrit at all doses, with statistically significant reductions at doses of 14, 28, and 113 mg/kg/day; however, the authors indicated that both parameters remained within reference ranges at all doses (Lent et al. 2012a; USAPHC 2011e). Splenic lesions, of trace-to-mild severity, were observed at doses ≥57 mg/kg/day, including extramedullary hematopoiesis and lymphoid hyperplasia.

In the corresponding 14-day rat study of 3,5-DNT, Lent et al. (2012a; USAPHC 2011f) observed no changes in hematology parameters and no splenic lesions at doses up to 39 mg/kg/day.

Hematological changes consistent with those observed in anemia were found in pregnant F344 rats administered 100 mg/kg Tg-DNT by gavage during gestation days 7–20 (Jones-Price et al. 1982). Administration of Tg-DNT to rats in the diet for 4 weeks (Hazleton Laboratories 1977) or 26 weeks (Hazleton Laboratories 1982) resulted in dose- and duration-related adverse effects on hematological parameters. In the 4-week study at 37.5 mg/kg/day, significant increases in reticulocytes and percentage of Heinz bodies were noted in both sexes and significant increases in methemoglobin levels were found in females; anemia was observed at 100 mg/kg/day in both sexes (Hazleton Laboratories 1977). Spleens of rats fed 150 mg/kg Tg-DNT for 30 days in the diet were altered in appearance; these alterations included discoloration, enlargement, and surface irregularity (Hazleton Laboratories 1977). An increased incidence of extramedullary hematopoiesis was noted in the splenic red pulp of male, but not female, rats fed 35 mg/kg/day Tg-DNT in the diet for 52 weeks (Hazleton Laboratories 1982). In rats sacrificed after 26 weeks in a 24-month study, no effects on hematological parameters were observed at 14 mg/kg/day Tg-DNT. However, at 35 mg/kg/day, there were increases in reticulocytes and methemoglobin and decreases in red blood cells along with hemosiderosis and extramedullary hematopoiesis in males, and increases in MCV in females (Hazleton Laboratories 1982). After 1 year, slight-to-moderate myeloid and erythroid hyperplasia was noted in the bone marrow of most male rats treated with 35 mg/kg/day Tg-DNT (Hazleton Laboratories 1982). In a 24-month study in which Tg-DNT was administered to rats in the diet, anemia was observed at 14 mg/kg/day in males but not in females; the NOAEL for this effect in males was 3.5 mg/kg/day Tg-DNT (Hazleton Laboratories 1982).

The consistent observation of adverse hematological effects following exposure of laboratory animals to DNTs indicates that the blood is a primary target of DNT toxicity. As noted previously and shown in Table 3-7, the various DNT isomers elicited isomer-specific differences in selected hematological parameters following 14 days of gavage treatment of male Sprague-Dawley rats (Lent et al. 2012a; USAPHC 2011a, 2011b, 2011c, 2011d, 2011e, 2011f). The 2,4- and 2,5-DNT isomers were the only ones causing decreases in red blood cell counts, with 2,5-DNT being more potent than 2,4-DNT. Increased spleen weight was observed at similar concentrations of 2,4-, 2,5-, and 2,6-DNT. Splenic lesions were observed after acute exposure to all DNT isomers except 3,5-DNT; the available data suggest that the 2,5-DNT isomer is more potent than the other isomers.

Musculoskeletal Effects. No studies were located regarding musculoskeletal effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, 3,5-, or Tg-DNT or in animals after oral exposure to 2,3-, 2,5-, 2,6-, 3,4-, 3,5-, or Tg-DNT. Swim-to-exhaustion time was decreased by 82% (relative to controls) in C57Bl/6N mice exposed to 134 mg/kg/day 2,4-DNT via gavage for 14 days (Wilbanks et al. 2014).

Hepatic Effects. No studies were located regarding hepatic effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

The hepatotoxic effects of DNTs have been consistently observed in animals. The liver appears to be a target organ of DNT toxicity, particularly when administered to rats, but hepatotoxic effects have also been observed in mice and dogs. Hepatic effects of DNTs include liver discoloration and inflammation, alteration of hepatocytes, proliferation of bile duct epithelium, and hyperplastic foci. However, as discussed in Section 3.2.2.7 (Cancer), 2,4-, 2,6-, and Tg-DNT have been shown to induce hepatocellular carcinoma following chronic-duration oral exposure. Thus, hepatic effects observed at less-than-chronic exposure durations or at lower doses may represent early stages of progressive development to hepatic cancer.

In male Sprague-Dawley rats receiving gavage doses of 2,3-DNT for 14 days, mean relative (but not absolute) liver weight was increased at 275 mg/kg/day, but this increase was likely attributable to lower body weight at this dose (Lent et al. 2012a; USAPHC 2011a). Dose-related clinical chemistry changes were not observed, and no histopathology lesions were seen in the liver.

Table 3-7

NOAELs and LOAELs for Hematological Effects Following 14-Day Gavage Dosing of Male Sprague-Dawley Rats with Individual DNT Isomers.

In female Sprague-Dawley rats (5/group) administered 2,4-DNT via gavage (in 5% v/v DMSO in corn oil) and observed for 48 hours, significantly decreased levels of serum albumin (13–51% lower than controls) were observed at doses ≥99 mg/kg (Deng et al. 2011). Although relative liver weight was also significantly increased at 99 mg/kg, this effect was not observed at 198 or 398 mg/kg. Hepatic sinusoid congestion was observed in rats administered 398 mg/kg 2,4-DNT in the absence of other histopathological effects. Significantly increased (14% higher than controls) relative liver weight was observed in wild type C57Bl/6N mice exposed to 134 mg/kg/day 2,4-DNT via gavage for 14 days; serum triglycerides and glucose levels were not affected (Wilbanks et al. 2014). Increased blood cholesterol was found in male and female Sprague-Dawley rats fed 78 or 82 mg/kg/day 2,4-DNT, respectively, in the diet for 14 days, and increased alanine aminotransferase levels were found in males (McGown et al. 1983). Blood glucose levels trended upward in all male and female groups in this study, but were increased significantly only in females fed 273 mg/kg/day. Trace-to-mild single cell necrosis occurred in the livers of male Sprague-Dawley rats receiving gavage doses of 18, 36, 71, and 142 mg/kg/day 2,4-DNT; vehicle controls and rats exposed to 9 mg/kg/day did not exhibit this effect (Lent et al. 2012a; USAPHC 2011b). In addition, the authors observed trace apoptosis in the livers of 1/6 rats in each of the 36 and 71 mg/kg/day groups, and increases in glycogen deposition at ≥36 mg/kg/day. No clinical chemistry changes were seen at any dose.

Oral administration of 2,4-DNT for 13 weeks to rats (266 or 145 mg/kg/day in males and females, respectively) and dogs (25 mg/kg/day) did not result in liver toxicity (Ellis et al. 1985; U.S. Army 1978b). After 26 weeks of treatment, rats fed 27 mg/kg/day in the diet had significant increases in epoxide hydrolase (EH) activity, which is sometimes considered to be a phenotypic marker of neoplastic nodules; however, hepatocellular lesions did not develop in these animals when treatment was carried through 52 weeks (Leonard et al. 1987). Mild hepatocellular dysplasia was observed in mice fed 137 mg/kg/day (males) or 468 mg/kg/day (females) of 2,4-DNT for 13 weeks (Hong et al. 1985; U.S. Army 1978b).

Hepatic effects have also been observed in laboratory animals following chronic-duration oral exposure to DNTs. However, these effects are often observed in conjunction with the development of hepatocellular carcinoma and may represent precancerous changes. For example, dietary exposure of male rats to 0.6 mg/kg/day 2,4-DNT induced “hepatocellular” alterations; however, neoplastic nodules were noted at this dose level and higher as well (Lee et al. 1985). Hepatocellular degeneration and vacuolation accompanied by acidophilic foci and occasional basophilic foci of cellular alteration were found in F344 rats fed 27 mg/kg/day 2,4-DNT for 52 weeks (Leonard et al. 1987). The incidences of focal areas of alteration were less in the 2,4-DNT-treated rats than they were in rats similarly treated with 2,6-DNT or Tg-DNT. Wistar rats fed a TWA dose of 371 mg/kg/day 2,4-DNT in the diet for 6 months had increased relative liver weights, formation of puruloid matter, and increased levels of serum glutamic-oxaloacetic transaminase (SGOT), lactate dehydrogenase (LDH), alkaline and acid phosphatase, triglycerides, and blood glucose levels compared to controls (Kozuka et al. 1979). In this study, the levels of serum albumin and the albumin/globulin ratios were decreased. Hepatocellular dysplasia was found in male and female CD-1 mice fed 14 or 898 mg/kg/day 2,4-DNT, respectively, for 24 months (Hong et al. 1985; U.S. Army 1979). Administration of 10 mg/kg/day 2,4-DNT for 24 months resulted in biliary hyperplasia in dogs; this effect was not seen in dogs administered 1.5 mg/kg/day (Ellis et al. 1985; U.S. Army 1979).

When 2,5-DNT was administered to male Sprague-Dawley rats via gavage for 2 weeks at doses up to 308 mg/kg/day, there were no treatment-related adverse effects on serum liver enzyme activity, liver weight, or liver histology (Lent et al. 2012a; USAPHC 2011c).

Increased ALT activity and histopathological changes to the liver were observed in individual female Sprague-Dawley rats administered 50 and 99 mg/kg 2,6-DNT (in 5% DMSO in corn oil) by gavage and observed for 48 hours (Deng et al. 2011). Serum ALT activity was significantly increased 7-fold compared to controls in rats administered 50 and 99 mg/kg 2,6-DNT, but no increase in serum ALT level was observed at lower dose levels (5–25 mg/kg). Histopathological changes to the liver observed in rats administered 2,6-DNT at 50 or 99 mg/kg included congested sinusoids with sloughed hepatocytes and segmented neutrophils, disorganized midzonal regions characterized by infiltration of erythrocytes and hepatocytes with pyknotic nuclei and microvesiculated cytoplasm, and apoptotic hepatocytes (severity of effects not specified). ALT levels were similar to controls and hepatocytes were mostly undamaged in rats administered 2,6-DNT at 199 mg/kg, although evidence of sinusoid congestion, occasional erythrocyte infiltration (with no signs of necrosis), and enlarged nuclei in several hepatocytes were noted. Three consecutive daily oral doses of 100 mg/kg/day 2,6-DNT resulted in mild diffuse hepatocellular hypertrophy in male Sprague-Dawley rats (Rothfuss et al. 2010). After 2 weeks of gavage doses of 68 or 134 mg/kg/day 2,6-DNT, male rats exhibited significant increases in serum ALT and aspartate aminotransferase (AST), as well as a nonsignificant but dose-related increase in alkaline phosphatase (ALP) (Lent et al. 2012a; USAPHC 2011d). Liver lesions consisting of hepatocellular and oval cell hyperplasia and hepatocellular hypertrophy occurred at doses ≥35 mg/kg/day. Additional lesions (increases in mitotic activity, single cell necrosis, and karyocytomegaly) occurred at higher doses of 68 and 134 mg/kg/day (Lent et al. 2012a; USAPHC 2011d).

Intermediate duration exposure to 2,6-DNT also results in liver toxicity. After oral exposure to 33 mg/kg/day 2,6-DNT for 29 consecutive days, male rat livers exhibited hepatocellular hypertrophy, single cell necrosis, bile duct hyperplasia, and focal hepatocellular vacuolation, in the absence of changes in serum liver enzyme levels (Rothfuss et al. 2010). Six weeks of dietary consumption of 7 mg/kg/day 2,6-DNT caused a 380% increase in EH levels in rats but did not increase the level of DT-diaphorase (DTD) (Leonard et al. 1987). In the same study, both of these enzymes were elevated after 6 weeks of treatment with 14 mg/kg/day of 2,6-DNT. Dosing of rats, mice, and dogs with 2,6-DNT for 13 weeks resulted in liver toxicity (U.S. Army 1976). Bile duct hyperplasia was observed in rats fed 35 mg/kg/day and mice fed 51 mg/kg/day 2,6-DNT for 13 weeks (U.S. Army 1976). Liver degeneration and bile duct hyperplasia were observed in dogs dosed with 20 mg/kg/day 2,6-DNT but were not seen in dogs dosed with 4 mg/kg/day (U.S. Army 1976). After 52 weeks of treatment with 7 mg/kg/day 2,6-DNT, hepatocellular degeneration and vacuolation accompanied by acidophilic and basophilic foci of cellular alteration were found in F344 rats (Leonard et al. 1987).

Gavage doses up to 227 mg/kg/day 3,4-DNT did not alter clinical chemistry or liver histology in male Sprague-Dawley rats (Lent et al. 2012a; USAPHC 2011e). Significant increases in relative, but not absolute liver weight were noted at 113 and 227 mg/kg/day, but were likely attributable to reduced body weights at these doses.

In the 14-day gavage study of rats by Lent et al. (2012a; USAPHC 2011f), no exposure-related changes in serum liver enzymes, liver weight, or histopathology were seen at doses up to 39 mg/kg/day of 3,5-DNT.

Irregular liver surfaces were found in male F344 rats fed 37.5 mg/kg/day Tg-DNT for 30 days (Hazleton Laboratories 1977). Hepatocytic necrosis, nonsupportive pericholangitis, and periportal megalocytosis were found in CDF rats fed 14 mg/kg/day for 26 weeks, and when treatment of these animals was extended to 2 years, slight-to-severe biliary cirrhosis was found in males (Hazleton Laboratories 1982). It has been suggested that this latter lesion may be a precursor to cholangiocarcinoma (Hazleton Laboratories 1982). Hepatocytic degeneration and acidophilic and basophilic foci of cellular alteration were observed in F344 rats fed 35 mg/kg/day Tg-DNT in the diet for 52 weeks (Leonard et al. 1987). When administration of Tg-DNT was continued for 24 months, liver discoloration resulted at 3.5 mg/kg/day and liver nodules and malignancies at 14 mg/kg/day (Hazleton Laboratories 1982).

As shown in Table 3-8, 2,4- and 2,6-DNT were the only isomers that elicited hepatic effects in Sprague-Dawley rats following 14 days of gavage treatment (Lent et al. 2012a; USAPHC 2011a, 2011b, 2011c, 2011d, 2011e, 2011f). The 2,4-DNT isomer caused increased liver weight (LOAEL=142 mg/kg/day) and increased incidences of single cell necrosis and glycogen deposition (LOAEL=36 mg/kg/day). The 2,6-DNT isomer caused increased serum liver enzyme activity (LOAEL=68 mg/kg/day) and increased incidences of hepatocellular hypertrophy and hyperplasia and oval cell hyperplasia (LOAEL=35 mg/kg/day).

Table 3-8

NOAELs and LOAELs for Hepatic Effects Following 14-Day Gavage Dosing of Male Sprague-Dawley Rats with Individual DNT Isomers.

Renal Effects. No studies were located regarding renal effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Exposure to 2,3-DNT by daily gavage for 14 days did not result in serum chemistry changes indicative of renal toxicity, or changes in kidney weight in male rats; however, increased incidences of renal tubular dilatation (2/6 rats) and renal lymphocytic infiltration (4/6) were observed (Lent et al. 2012a; USAPHC 2011a).

Sprague-Dawley rats (5/group) administered 2,4-DNT (in 5% DMSO in corn oil) via gavage at doses up to 398 mg/kg and evaluated at 24 or 48 hours showed no effects on levels of serum creatinine or urea (Deng et al. 2011). Hyaline droplet accumulation in the epithelium of the proximal convoluted tubule was found in both sexes of Sprague-Dawley rats after they were administered 78, 104, 165, or 261 mg/kg/day 2,4-DNT (males) or 82, 109, 173, or 273 mg/kg/day 2,4-DNT (females) in the diet (McGown et al. 1983). Although this effect was observed at all concentrations, there was no dose response evident. The kidney was not a target organ of 2,4-DNT in male rats exposed via gavage for 14 days at doses up to 142 mg/kg/day; serum chemistry and kidney weight were not affected by exposure, nor were there histopathology findings in the kidney (Lent et al. 2012a; USAPHC 2011b). Oral administration of 2,4-DNT to mice (413 mg/kg/day), rats (145 mg/kg/day), and dogs (25 mg/kg/day) for 13 weeks did not result in significant adverse effects in the kidney (Hong et al. 1985; U.S. Army 1978b). Treatment of the same species for 24 months resulted in renal dysplasia in male mice at a dose of 14 mg/kg/day of 2,4-DNT, but no renal effects were observed in rats or dogs dosed with 34.5 mg/kg/day or 10 mg/kg/day, respectively (U.S. Army 1979). Adverse effects in the kidneys of mice included cystic dysplasia in the tubular epithelium, atypical epithelium lining the cysts, and a variety of tumors (Hong et al. 1985). These effects were more pronounced in male mice than in female mice.

Exposure to 2,5-DNT for 14 days at gavage doses up to 308 mg/kg/day did not result in kidney effects in male rats, as assessed by serum chemistry, kidney weight, and histopathology (Lent et al. 2012a; USAPHC 2011c).

Renal tubular degeneration and proximal tubule degeneration were observed in male rats exposed by gavage to 2,6-DNT at 134 mg/kg/day (the highest dose tested) for 14 days (Lent et al. 2012a; USAPHC 2011d).

Female Sprague-Dawley rats (5/group) administered 2,6-DNT (in 5% DMSO in corn oil) via gavage at doses up to 199 mg/kg showed no effects on levels of serum creatinine or urea at 24 or 48 hours (Deng et al. 2011). Dosing of dogs with 20 mg/kg/day 2,6-DNT for 13 weeks resulted in dilated tubules, foci of inflammation, and degeneration of the kidney (U.S. Army 1976). No treatment-related effects on the kidney were found when rats were fed 2,6-DNT for 13 weeks (U.S. Army 1976). The severe renal effects observed after 2,4-DNT administration in mice were not observed when mice were fed 289 mg/kg/day 2,6-DNT for 13 weeks (U.S. Army 1976). However, the renal toxicity of 2,4-DNT in mice was observed only after chronic administration. Chronic studies of 2,6-DNT have not been performed in mice.

After 26 or 52 weeks of dietary consumption of 35 mg/kg/day Tg-DNT, BUN levels were significantly increased in CDF rats (Hazleton Laboratories 1982). Exacerbation of chronic interstitial nephritis that was also observed in controls was observed at 14 mg/kg/day Tg-DNT in a chronic study in rats (Hazleton Laboratories 1982).

Renal effects were observed in male rats exposed to 3,4-DNT by gavage for 14 consecutive days (Lent et al. 2012a; USAPHC 2011e). These effects consisted of proximal tubule degeneration, renal tubule basophilia, and lymphocytic infiltration at the highest dose tested (227 mg/kg/day); however, renal histopathology was not examined at lower doses.

In the corresponding study of 3,5-DNT (Lent et al. 2012a; USAPHC 2011f), no effects on kidney weight, kidney histopathology, or serum parameters indicative of renal toxicity were seen in male rats exposed by gavage to doses up to 39 mg/kg/day for 14 days.

The kidney does not appear to be a sensitive target of DNT toxicity for all species tested. Severe renal effects were observed only in CD-1 mice fed 14 mg/kg/day 2,4-DNT for 24 months, and less severe renal effects were observed in dogs administered 20 mg/kg/day 2,6-DNT for 13 weeks.

In the 14-day gavage studies that compared the effects of the various DNT isomers on the kidneys of male Sprague-Dawley rats (Lent et al. 2012a; USAPHC 2011a, 2011b, 2011c, 2011d, 2011e, 2011f), only the 2,3-, 2,6-, and 3,4-DNT elicited adverse effects. Increased kidney weight accompanied by increased incidences of histopathologic lesions (trace tubular dilatation and lymphocytic infiltration) were observed for 2,3-DNT (LOAEL=275 mg/kg/day). Increased kidney weight (LOAEL=68 mg/kg/day) and increased incidences of proximal tubule degeneration and renal tubular basophilia were observed for 2,6-DNT (LOAEL=134 mg/kg/day). Increased incidences of proximal tubule degeneration, renal tubule basophilia, and lymphocytic infiltration were noted for 3,4-DNT (LOAEL=227 mg/kg/day).

Endocrine Effects. No studies were located regarding endocrine effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Administration of 2,4-DNT in the diet for 14 days, at 78 mg/kg/day for males or 82 mg/kg/day for females did not cause any histopathological changes in adrenal, pituitary, or thyroid glands of Sprague-Dawley rats (McGown et al. 1983).

No histopathological effects on adrenal, pituitary, or thyroid glands were found in rats treated with 14 mg/kg/day Tg-DNT for up to 2 years or 35 mg/kg/day for 1 year (Hazleton Laboratories 1982). Increases in the incidence and severity of parathyroid hyperplasia (males) and increases in the incidence and severity of fatty metamorphosis and vascular ectasia (males and females) were found in rats fed 14 mg/kg/day Tg-DNT in the diet in a chronic study (Hazleton Laboratories 1982).

No studies were located regarding endocrine effects in animals after oral exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Dermal Effects. No studies were located regarding dermal effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Concentrations of up to 261 mg/kg/day 2,4-DNT for males or 273 mg/kg/day 2,4-DNT for females administered in the diet for 14 days to Sprague-Dawley rats caused no histopathological changes in their skin (McGown et al. 1983).

No effects were found on the skin of rats treated for up to 2 years with 14 mg/kg/day Tg-DNT or up to 1 year with 35 mg/kg/day Tg-DNT (Hazleton Laboratories 1982).

No studies were located regarding dermal effects in animals after oral exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Ocular Effects. No studies were located regarding ocular effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

The eyes of male and female Sprague-Dawley rats administered up to 261 or 273 mg/kg/day 2,4-DNT, respectively, in the diet for 14 days did not exhibit any alterations upon histopathological examination (McGown et al. 1983).

No effects were found on the eyes of rats treated for up to 2 years with 14 mg/kg/day Tg-DNT in feed or up to 1 year with 35 mg/kg/day Tg-DNT in feed (Hazleton Laboratories 1982).

No studies were located regarding ocular effects in animals after oral exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Body Weight Effects. No studies were located regarding body weight effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Adverse effects on body weight and body weight gain in rats, mice, and dogs were observed after oral administration of 2,4-DNT, 2,6-DNT, and Tg-DNT, and in rats exposed to 3,4-DNT. In most of these studies, a concurrent decrease in food consumption was also observed. Because exposure resulted from intake of the test article in feed in most of these studies, it is possible that some of the body weight changes resulted from unpalatability.

Body weight was not altered in male rats receiving doses up to 275 mg/kg/day 2,3-DNT by gavage for 14 days (Lent et al. 2012a; USAPHC 2011a).

Adverse effects on body weight, including body weight loss, have been reported after almost all acute-, intermediate-, and chronic-duration oral administration of 2,4-DNT (Bloch et al. 1988; Deng et al. 2011; Ellis et al. 1985; Hazleton Laboratories 1982; Hong et al. 1985; Kozuka et al. 1979; Lane et al. 1985; Lee et al. 1985; Lent et al. 2012a; USAPHC 2011b; Leonard et al. 1987; McGown et al. 1983; NCI 1978; U.S. Army 1978b, 1979). Female Sprague-Dawley rats (5/group) administered 2,4-DNT (in 5% DMSO in corn oil) via gavage at 398 mg/kg showed decreased body weight gain (1–4 g compared to 17 g for controls) 48 hours after dosing (Deng et al. 2011). In another acute study, rats dosed by gavage with 240 mg/kg 2,4-DNT for 5 days lost weight (Lane et al. 1985). Lent et al. (2012a; USAPHC 2011b) reported a 12% decrease in body weight (relative to controls) in male rats given 2,4-DNT by gavage for 14 days at a dose of 142 mg/kg/day. Wilbanks et al. (2014) observed greater body weight loss (0.96 versus 0.35 g), compared with untreated controls, in wild type C57Bl/6N mice exposed to 134 mg/kg/day 2,4-DNT via gavage for 14 days. In general, there were losses of 10–40% in body weight in acute-, intermediate-, and chronic-duration studies in rats. After 6 months, decreases in body weight gain were noted in rats fed 27 mg/kg/day 2,4-DNT (Leonard et al. 1987), and a 25% decrease in body weight was seen in rats fed 34.5 mg/kg/day (Lee et al. 1985; U.S. Army 1978b, 1979). This reduction in body weight gain tended to become more pronounced when 2,4-DNT was continued for periods of 1–2 years (Leonard et al. 1987). Body weight was decreased 25% in rats that received 20 mg/kg/day 2,4-DNT in the diet for 78 weeks; the NOAEL in this study was 8 mg/kg/day (NCI 1978). Mice showed similar decreases in body weight after intermediate- (13 weeks) and chronic-duration (78–104 weeks) exposure, but the doses of the test article needed to evoke this effect were considerably higher than in rats (Hong et al. 1985; NCI 1978; U.S. Army 1978b). An 18–24% decrease in body weight was seen in rats receiving 72–76 mg/kg/day 2,4-DNT in the diet for 78 weeks (NCI 1978).

In a 14-day study of 2,5-DNT, body weight was not altered in male rats receiving doses up to 308 mg/kg/day (Lent et al. 2012a; USAPHC 2011c).

Female Sprague-Dawley rats (5/group) administered 2,6-DNT (in 5% DMSO in corn oil) via gavage at 50 or 99 mg/kg gained less weight than control animals over the 48-hour observation period (1–4 g compared to 17 g for controls); rats from the same study administered 2,6-DNT at 199 mg/kg lost weight over the course of 48 hours (Deng et al. 2011). Body weight reductions of at least 10% were observed in male rats receiving gavage doses ≥35 mg/kg/day for 14 days, although the reductions were statistically significant only at the highest dose of 134 mg/kg/day (Lent et al. 2012a; USAPHC 2011d). Reduced body weight was reported in male Sprague-Dawley rats exposed to 29 consecutive daily oral doses of 33 mg/kg/day 2,6-DNT (Rothfuss et al. 2010). Administration of 2,6-DNT also caused decreased body weight gain or body weight loss in rats, mice, and dogs at doses ranging from 14 to 145 mg/kg/day in intermediate-duration studies (U.S. Army 1976). Treatment with 7 mg/kg/day 2,6-DNT for 52 weeks decreased body weight in rats by 18% (Leonard et al. 1987).

A 10% reduction in terminal body weight was seen in male rats exposed to 227 mg/kg/day 3,4-DNT by gavage for 14 days; however, the difference from control weight was not statistically significant (Lent et al. 2012a; USAPHC 2011e).

Body weight was not altered in male rats receiving gavage doses up to 39 mg/kg/day 3,5-DNT for 14 days (Lent et al. 2012a; USAPHC 2011f).

A 29% decrease in absolute maternal weight gain was observed in dams fed 14 mg/kg/day Tg-DNT for 14 days during gestation (Jones-Price et al. 1982). Decreased body weight or decreased body weight gain was reported in rats at levels as low as 14 mg/kg/day Tg-DNT in intermediate- or chronic-duration studies (Hazleton Laboratories 1982). Other intermediate- and chronic-duration studies also confirmed these body weight effects (Hazleton Laboratories 1977; Leonard et al. 1987; NCI 1978).

Metabolic Effects. No studies were located regarding metabolic effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Female Sprague-Dawley rats (5/group) administered 2,4-DNT (at 198 or 398 mg/kg) or 2,6-DNT (at 199 mg/kg) via gavage (in 5% DMSO in corn oil) showed small but statistically significant reductions in sodium levels in the serum (4–11% lower than controls) 24 and/or 48 hours after dosing (Deng et al. 2011). Rats treated with 2,4-DNT at 398 mg/kg also had elevated levels of blood glucose (increased 2.6-fold) 48 hours after dosing.

No studies were located regarding metabolic effects in animals after oral exposure to 2,3-, 2,5-, 3,4-, or 3,5-DNT.

3.2.2.3. Immunological and Lymphoreticular Effects

No studies were located regarding immunological and lymphoreticular effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, or 3,5-DNT.

Testing for immunological effects of DNTs is limited. Acute (14-day) exposure of male rats to 2,3-DNT resulted in lymphoid hyperplasia of the spleen and lymphoid depletion at gavage doses of 275 mg/kg/day (Lent et al. 2012a; USAPHC 2011a). In the corresponding study of 2,4-DNT, lymphoid hyperplasia was seen at doses ≥35 mg/kg/day, and lymphoid depletion was seen at 134 mg/kg/day (Lent et al. 2012a; USAPHC 2011b). No changes in serum concentrations of IgE were observed in rats and dogs administered 2,4-DNT at levels up to 206 and 25 mg/kg/day, respectively, for 13 weeks (Ellis et al. 1985; Lee et al. 1985; U.S. Army 1978b). In these studies, the rats received the test article in feed, while the dogs received it in capsules. No histopathological changes were found in the spleen or thymus of Sprague-Dawley male rats fed 78 mg/kg/day 2,4-DNT or female rats fed 82 mg/kg/day 2,4-DNT in the diet for 14 days (McGown et al. 1983).

Administration of 2,6-DNT to dogs (up to 100 mg/kg) and rats (up to 145 mg/kg/day) for 13 weeks resulted in no observable changes in IgE serum concentrations (U.S. Army 1976). IgE is the antibody associated with allergic or hypersensitive reactions, and so it may be expected that the human sensitizing potential of 2,4- and 2,6-DNT would be low. Involution of the thymus was noted when dogs were administered 100 mg/kg 2,6-DNT, but was not noted when they were administered 20 mg/kg 2,6-DNT by capsule for 13 weeks (U.S. Army 1976).

Exposure to 3,4-DNT by gavage for 14 days induced lymphoid hyperplasia of the spleen in male rats exposed to doses ≥57 mg/kg/day (Lent et al. 2012a; USAPHC 2011e).

All LOAEL values from each reliable study for immunological/lymphoreticular effects in each species and duration category for 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-DNT are recorded in Tables 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, respectively, and plotted in Figures 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, respectively.

No studies were located regarding immunological and lymphoreticular effects in animals after oral exposure to 2,5- or 3,5-DNT, although Lent et al. (2012a; USAPHC 2011c, 2011f) did not observe splenic lymphoid lesions in rats exposed to these two compounds for 14 days.

3.2.2.4. Neurological Effects

No studies were located regarding neurological effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Neurotoxicity appears to be a characteristic syndrome of subchronic and chronic DNT poisoning of animals. Neurotoxic symptoms, of decreased severity compared to dogs, were observed in mice and rats at doses higher than neurotoxic doses in dogs; however, the test article was administered in feed to rodents and in capsules to dogs.

Acute (14-day) oral exposure of male Sprague-Dawley rats to 2,3-DNT at doses up to 275 mg/kg/day or 2,4-DNT at doses up to 142 mg/kg/day did not lead to any clinical signs of neurotoxicity, nor was brain weight affected (Lent et al. 2012a; USAPHC 2011a).

In a subchronic study, beagle dogs (4/sex/group) administered 2,4-DNT at 1 or 5 mg/kg/day showed no signs of behavioral changes or clinical signs of neurotoxicity. Neurotoxicity was observed at 25 mg/kg/day, with signs of neurotoxicity (loss of hind leg control) first observed in a female dog on day 12 of treatment. Three additional male dogs showed similar signs (signs not specified) on day 14 of treatment. All dogs administered 25 mg/kg/day showed signs of neurotoxicity after treatment for 12–22 days. The onset and severity of toxic signs reportedly varied among dogs within the same treatment group; some dogs were moribund at the same time that others began experiencing symptoms. In individual dogs, symptom severity varied over time, with no duration-related pattern of severity. The specific neurotoxic effects in dogs affected within the first 14 days of treatment were not specified. The NOAEL for the neurotoxicity observed after 12 days was 5 mg/kg/day. No histopathological changes were found in the brain or spinal cord of male and female Sprague-Dawley rats fed 2,4-DNT for 14 days in the diet at doses of 78 and 82 mg/kg/day, respectively (McGown et al. 1983).

Neurotoxicity has been reported in laboratory animals after intermediate- or chronic-duration exposure to 2,4-DNT with symptoms ranging from tremors, convulsions, and ataxia to paralysis. These effects were observed in 13-week studies of rats and dogs. Administration of 93 mg/kg/day 2,4-DNT in the diet for 13 weeks caused demyelinization in the cerebellum and brain stem of 1 male rat, while at 266 mg/kg/day, some rats exhibited a widespread or stiff-legged gait that did not progress to the rigid paralysis observed in dogs (Lee et al. 1985; U.S. Army 1978b). After 3 months of being fed 2,4-DNT at 0.5% in the diet (an estimated dose of 350 mg/kg/day based on body weight and feed consumption data provided by the study authors), Wistar rats exhibited humpback and jerky incoordination (Kozuka et al. 1979). Dogs that were administered 25 mg/kg/day 2,4-DNT in capsules for 13 weeks began to show neurotoxic effects within 2 months; these effects included incoordination, abnormal gait, rigid paralysis of the hind legs, eventually progressing to paralysis up to the neck (Ellis et al. 1985; U.S. Army 1978b). No neurological signs were observed in mice fed 413 mg/kg/day in males or 468 mg/kg/day in females 2,4-DNT in the diet for 13 weeks (Hong et al. 1985; U.S. Army 1978b).

An abnormal gait was also observed in chronic studies of laboratory animals fed 2,4-DNT. The characteristic widespread and stiff-legged gait was observed after feeding 34.5 or 45.3 mg/kg/day 2,4-DNT to male and female rats, respectively, for up to 2 years (Lee et al. 1985; U.S. Army 1978b, 1979). This stiff-legged gait and hyperactive behavior were also noted in mice fed 898 mg/kg/day in the diet for 24 months but were not observed in mice at 95 mg/kg/day (Ellis et al. 1985; U.S. Army 1979). In dogs dosed at 1.5 mg/kg/day 2,4-DNT in a 2-year study, one of six dogs showed intermittent loss of hindquarter control (Ellis et al. 1985; U.S. Army 1979). Central nervous system lesions were identified in high-dose (10 mg/kg/day) dogs in this study and included vacuolization, hypertrophy, endothelial mitosis, and focal gliosis in the cerebellum, as well as some perivascular hemorrhage in the cerebellum and brain stem (Ellis et al. 1985; U.S. Army 1979).

Male rats exposed for 14 days by gavage to 2,5-DNT at doses up to 308 mg/kg/day or 2,6-DNT at doses up to 134 mg/kg/day did not display clinical signs of neurotoxicity or alterations in brain weight (Lent et al. 2012a; USAPHC 2011c).

Dogs dosed with 20 or 100 mg/kg/day of 2,6-DNT for 13 weeks exhibited dose-related neurotoxic symptoms that included muscular incoordination, weakness, tremors, and paralysis (U.S. Army 1976). Rats and mice dosed at 145 and 289 mg/kg/day of 2,6-DNT, respectively, for 13 weeks did not display neurotoxic symptoms (U.S. Army 1976).

Clinical signs of neurotoxicity consisting of facial twitching, hypoactivity, and staring were noted in male rats exposed by gavage to 227 mg/kg/day 3,4-DNT for 14 days (Lent et al. 2012a; USAPHC 2011e).

Male rats exposed to ≥39 mg/kg/day 3,5-DNT by gavage for 14 days were reported to exhibit neurological signs progressing from facial twitching to paralysis of the forelimbs (Lent et al. 2012a; USAPHC 2011f). However, all rats exposed to higher doses died or were sacrificed moribund, as did one rat exposed to 39 mg/kg/day. It is not clear whether these signs were seen only in the animals that were moribund or if some neurological signs were seen in animals that survived treatment. Animals in the 39 mg/kg/day group also showed a 30% increase (compared with controls) in brain weight relative to body weight, as well as mild or moderate inflammatory infiltrates in the brain (Lent et al. 2012a; USAPHC 2011f).

Administration of 150 mg/kg/day Tg-DNT to F344 dams during gestation days 7–20 caused hind limb weakness in 7 of 13 animals (Jones-Price et al. 1982). No clinical signs of neurotoxicity or histopathological changes were found in rats fed up to 35 mg/kg/day Tg-DNT in the diet for 26 or 52 weeks (Hazleton Laboratories 1982).

In the 14-day gavage studies that compared the effects of the various DNT isomers on neurological end points in male Sprague-Dawley rats (Lent et al. 2012a; USAPHC 2011a, 2011b, 2011c, 2011d, 2011e, 2011f), adverse effects were elicited only by 3,4-DNT (facial twitching, hypoactivity, staring; LOAEL=227 mg/kg/day) and 3,5-DNT (facial twitching and paralysis, inflammatory infiltrates in the brain; LOAEL=39 mg/kg/day).

All LOAEL values from each reliable study for neurological effects in each species and duration category for 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-DNT are recorded in Tables 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, respectively, and plotted in Figures 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, respectively.

3.2.2.5. Reproductive Effects

No studies were located regarding reproductive effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Studies in laboratory animals have shown that oral exposure to 2,4-DNT can result in adverse effects on reproduction, as shown by decreased fertility and the development of lesions of the male and female reproductive tracts. The male reproductive system seems to be particularly sensitive; observed effects include decreased sperm production, testicular atrophy, changes in Sertoli cell morphology, and degenerated seminiferous tubules (Bloch et al. 1988; Kozuka et al. 1979; Lane et al. 1985; McGown et al. 1983; U.S. Army 1976, 1978b, 1979). In the female reproductive system, ovarian atrophy and dysfunction were observed (U.S. Army 1979).

Fourteen-day exposure to 2,3-DNT at doses up to 275 mg/kg/day did not result in alterations in testes or epididymides weights in male Sprague-Dawley rats exposed by gavage (Lent et al. 2012a; USAPHC 2011a).

The effects on the male reproductive system have been reported in studies of brief durations. Decreased fertility was noted in male rats dosed with 180 mg/kg 2,4-DNT for 5 days; no dominant lethal effect was observed at this dose (Lane et al. 1985). Decreased absolute and relative testes weight, seminiferous tubular degeneration, multinucleated giant cell formation, and interstitial atrophy of the testes were noted in male rats after 14 days of gavage doses of 142 mg/kg/day 2,4-DNT (Lent et al. 2012a; USAPHC 2011b). Sprague-Dawley rats administered 104, 165, or 261 mg/kg/day 2,4-DNT in the diet for 14 days exhibited oligospermia with degenerative changes, such as syncytial cell formation and focal spermatic granuloma, in a dose-related manner (McGown et al. 1983). A dose of 78 mg/kg/day 2,4-DNT caused a decrease in the thickness of spermatogenic cell layers. No histopathological changes were found in the reproductive organs of females in this study (McGown et al. 1983). Although no changes were found in sperm morphology of male mice that were administered 250 mg/kg/day 2,4-DNT for 2 days, significant decreases in fertile matings of these animals were observed during weeks 2, 3, and 6 post-treatment (Soares and Lock 1980). However, sperm morphology was examined at 8 weeks post-treatment, so it is possible that a toxic effect was selective for specific types of sperm cells.

In intermediate studies of 2,4-DNT, serious effects on the male reproductive system have been observed in numerous animal studies. In a series of three dominant lethal studies using male rats for 13 weeks, 45 mg/kg/day 2,4-DNT in the diet caused severe atrophy and degeneration of the seminiferous tubules, resulting in decreased fertility, although no dominant lethal effect was observed (U.S. Army 1979). Another study using CD rats found that spermatogenesis was impaired after 4 weeks of feeding 93 mg/kg/day 2,4-DNT in the diet and had completely ceased after 13 weeks (Lee et al. 1985; U.S. Army 1978b). This effect was not reversible after a 4-week post-treatment period. Higher concentrations of 2,4-DNT were needed to cause these effects in mice. Testicular atrophy and aspermatogenesis occurred in CD-1 mice fed 413 mg/kg/day 2,4-DNT for 13 weeks (Hong et al. 1985; U.S. Army 1978b) and rats fed a TWA dose of 371 mg/kg/day 2,4-DNT for 6 months in feed (Kozuka et al. 1979). Decreased fertility was observed after male mice were treated with 1,032 mg/kg/day, but not 295 mg/kg/day, 2,4-DNT in the feed for 4 weeks in a dominant lethal study (U.S. Army 1978b). The decreased fertility was not observed in mice fed 295 mg/kg/day (U.S. Army 1978b). The testicular atrophy was considered to be due to a direct toxic effect on spermatogenic cells. Mild-to-severe testicular degeneration with decreased spermatogenesis has also been observed in dogs administered 25 mg/kg 2,4-DNT in capsules for 13 weeks (Ellis et al. 1985; U.S. Army 1978b). No testicular effects were found at 5 mg/kg in the study.

Histopathological examination of the testes after treatment with 2,4-DNT revealed changes, which suggest specific causes for the male infertility observed in animal studies. Dose-related changes in sperm cell morphology were found in Sprague-Dawley rats fed 76.7 or 153.4 mg/kg/day 2,4-DNT in the diet for 3 weeks (Bloch et al. 1988). At the low dose, vacuolation and lipid accumulation were noted in Sertoli cells; multinucleated spermatid and irregularities of the basal lamina were also found. These changes were limited and variable with most samples, demonstrating patchy damage. More extensive degenerative changes in both spermatocytes and spermatids were found at the high dose as well as ultrastructural changes in Sertoli cells; epididymal sperm counts were decreased 63%. The high-dose animals also had increased levels of serum luteinizing hormone (LH) and FSH but not testosterone (Bloch et al. 1988).

Chronic-duration studies in laboratory animals have also demonstrated both male and female reproductive effects. Male CD-1 mice fed 14 mg/kg/day 2,4-DNT for 12 months showed atrophy of the testes and decreased spermatogenesis (Hong et al. 1985). Female mice fed 898 mg/kg/day 2,4-DNT in this study had ovarian atrophy with non-functioning follicles, with a NOAEL of 95 mg/kg/day (Hong et al. 1985). Male CD rats that received 34 mg/kg/day 2,4-DNT in the diet for 12 months showed an increased incidence of seminiferous tubule atrophy compared to controls (100% affected at the high dose versus 0% of controls), with a NOAEL of 3.9 mg/kg/day (Lee et al. 1985; U.S. Army 1978b, 1979). No adverse reproductive effects were found in dogs fed 10 mg/kg/day 2,4-DNT for 24 months (Ellis et al. 1985; U.S. Army 1979).

A three-generation reproductive toxicity study was performed in rats fed 2,4-DNT for up to 6 months before mating of original prenatal animals (U.S. Army 1979). Effects on neonatal viability were observed at the highest concentration of 2,4-DNT used, 40 mg/kg/day. Reductions in neonatal viability became more severe with successive litters within each generation, such that no second litters were produced by the second generation of high-dose animals, which were fed 34.5 mg/kg/day (male) or 45.3 mg/kg/day (female) 2,4-DNT. Decreased fetal viability was attributed to maternal neglect and maternal death during parturition. Decreases in the number of fetal implants were attributed to the adverse impact of 2,4-DNT on sperm production.

No effects on testes or epididymides weights were observed in male rats receiving 308 mg/kg/day 2,5-DNT by gavage for 14 days (Lent et al. 2012a; USAPHC 2011c).

In contrast, seminiferous tubular degeneration, multinucleated giant cell formation, and interstitial atrophy of the testes were reported in rats after gavage doses ≥68 mg/kg/day 2,6-DNT for 14 days (Lent et al. 2012a; USAPHC 2011d). At the highest dose (134 mg/kg/day 2,6-DNT), these effects were accompanied by marked decreases (49–57% compared with controls) in absolute and relative testes weight (Lent et al. 2012a; USAPHC 2011d).

In studies of rats, mice, and dogs dosed with 2,6-DNT for 13 weeks (U.S. Army 1976), decreased spermatogenesis was observed in male mice administered 51 mg/kg/day, but normal spermatogenesis was observed in animals dosed with 11 mg/kg/day. Testicular atrophy was reported in rats administered 35 mg/kg/day 2,6-DNT, and no effects were observed in rats dosed with 7 mg/kg/day (U.S. Army 1976). Dogs dosed with 20 and 100 mg/kg/day had testicular degeneration, but no effects were observed in dogs dosed with 4 mg/kg/day (U.S. Army 1976).

3,4-DNT doses up to 227 mg/kg/day administered by gavage for 14 days to male Sprague-Dawley rats did not result in any changes in testicular or epididymides weights (Lent et al. 2012a; USAPHC 2011e).

Exposure to 3,5-DNT, in contrast, resulted in significant reductions in absolute and relative testes weights (≥40% lower than controls), as well as seminiferous tubular degeneration and multinucleated giant cell formation in the testes of male rats after exposure to doses of 19 and 39 mg/kg/day for 14 days (Lent et al. 2012a; USAPHC 2011f).

Based upon the testicular effects observed after administration of 2,4-, 2,6-, and 3,5-DNT it is not surprising that these effects are found after treatment with Tg-DNT. Testicular degeneration was found in male rats fed 35 mg/kg/day Tg-DNT for 26 weeks, but since the finding was unilateral, the relationship to treatment may be considered equivocal (Hazleton Laboratories 1982). When treatment with this concentration was carried through 52 weeks, however, bilateral mild-to-severe testicular degeneration and hypospermatogenesis were observed (Hazleton Laboratories 1982). No changes were found in the fertility or sperm morphology of male mice that received 250 mg/kg Tg-DNT by gavage for 2 days in a dominant lethal study (Soares and Lock 1980).

In the 14-day gavage studies that compared the effects of the various DNT isomers on reproductive end points in male Sprague-Dawley rats (Lent et al. 2012a; USAPHC 2011a, 2011b, 2011c, 2011d, 2011e, 2011f), adverse effects were elicited only by 2,4-DNT (decreased weights of testis and epididymis, tubular degeneration, multinucleated giant cell formation, and interstitial atrophy of the testes; LOAEL=142 mg/kg/day), 2,6-DNT (decreased testis weight at a LOAEL of 134 mg/kg/day; tubular degeneration, multinucleated giant cell formation, and interstitial atrophy of the testes at a LOAEL of 68 mg/kg/day), and 3,5-DNT (small testes, significantly reduced testes weight, tubular degeneration and multinucleated giant cell formation in the testes; LOAEL=19 mg/kg/day).

The highest NOAEL values and all reliable LOAEL values for reproductive effects in each species and duration category for 2,3-, 2,4-, 2,5-, 2,6, 3,4-, and 3,5-DNT are recorded in Tables 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, respectively, and plotted in Figures 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, respectively.

3.2.2.6. Developmental Effects

No studies were located regarding developmental effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. However, developmental toxicity from DNTs could potentially occur because exposure to any substance that depletes the amount of oxygen available to developing fetal tissues may have adverse consequences. U.S. Army (1979) conducted a 3-generation reproductive study in which 2,4-DNT was administered to male and female rats at doses up to 34.5 and 45.3 mg/kg/day, respectively. Normal birth weights, liveborn index, and weight at weaning were observed. Decreases in pup viability at 45.3 mg/kg/day in this study resulted from maternal neglect and a high incidence of maternal death during parturition; these decreases did not appear to result from pup defects since no anomalies were detected in offspring from any generation. These effects were not observed in animals fed 5.1 mg/kg/day 2,4-DNT (U.S. Army 1979).

Tg-DNT was administered by gavage to pregnant rats for 14 days during gestation, and pups were evaluated for developmental toxicity either at gestation day 20 or postpartum day 60 (Jones-Price et al. 1982). Adverse effects on hematologic parameters and altered organ weights were observed in both dams and fetuses when dams were administered 100 or 150 mg/kg/day. However, the fetal toxicity was not dose related. A decrease in relative liver weight was observed, however, in the postpartum pups at the low dose of 14 mg/kg/day; this dose is considered to be a LOAEL. Dose-related effects on postnatal development were not observed in pups when dams were administered 35 or 75 mg/kg/day. Transient and statistically significant signs of neurotoxicity, which were not dose-related, included delayed eye opening and cliff avoidance when dams were treated with 35 or 75 mg/kg/day. No evidence of toxicity was found in pups at postpartum day 60 of the postnatal study.

No studies were located regarding developmental effects in animals after oral exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

The highest NOAEL values and all reliable LOAEL values for developmental effects in each species and duration category for 2,4-DNT are recorded in Table 3-1 and plotted in Figure 3-1.

3.2.2.7. Cancer

No studies were located regarding cancer in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

The carcinogenic activity of DNTs has been extensively studied in typical chronic bioassays and in some less-than-lifetime studies. 2,4-DNT produced renal tumors in male mice and was hepatocarcinogenic in rats. 2,6-DNT and Tg-DNT are potent hepatocarcinogens in rats (Lee et al. 1985; U.S. Army 1978b, 1979).

2,4-DNT (98% 2,4-DNT, 2% 2,6-DNT) produced renal tumors (76%) in male CD-1 mice fed 95 mg/kg/day for 2 years (U.S. Army 1979). A statistically significant increase in renal tumors in female mice was not observed. A National Cancer Institute (NCI) bioassay (NCI 1978) of 2,4-DNT (95% 2,4-DNT, the other components not specified) did not detect a carcinogenic effect in mice dosed with 72 mg/kg/day for 78 weeks. The NCI bioassay used the C57BL/6N strain of mouse, lower doses, and a shorter treatment schedule than did U.S. Army (1979).

Hepatocellular carcinoma were significantly increased in male CD rats fed 34.5 mg/kg/day 2,4-DNT and in females fed 45.3 mg/kg/day 2,4-DNT for 2 years (U.S. Army 1979). The tumor response in females was higher than in the males. Two other studies of rats in which malignancies were not observed used the F344 strain, lower doses, and shorter exposure durations than did U.S. Army (1979): 10 mg/kg/day for 78 weeks (NCI 1978) and 27 mg/kg/day for 52 weeks (Leonard et al. 1987). NCI (1978) reported significant increases in subcutaneous tissue fibroma in male rats at 7.5–8 mg/kg/day and mammary gland fibroadenomas in female rats at 22 mg/kg/day. U.S. Army (1979) found significant increases in subcutaneous tissue fibromas in male rats at 34.5 mg/kg/day and mammary gland fibroadenomas in female rats at 45.3 mg/kg/day; these were benign tumors.

2,4-DNT was not found to be carcinogenic in the Strain A/J mouse pulmonary tumor bioassay when 250 mg/kg was administered by gavage twice a week for 12 weeks (Stoner et al. 1984). 2,4-DNT was a hepatic tumor promoter, but not a tumor initiator, using in vivo hepatic initiation-promotion protocols (Leonard et al. 1986).

2,6-DNT administered for 1 year at 7 and 14 mg/kg/day produced hepatocellular carcinomas in 85% and 100%, respectively, of male F344 rats (Leonard et al. 1987). Pulmonary metastases of hepatocytic origin were also observed. Both tumor-initiating and tumor-promoting activities of 2,6-DNT in rat liver were reported (Leonard et al. 1983, 1986; Mirsalis and Butterworth 1982). 2,6-DNT was not found to be a lung carcinogen in the Strain A/J mouse pulmonary tumor bioassay when 250 mg/kg was administered by gavage twice a week for 12 weeks (Schut et al. 1983; Stoner et al. 1984).

The effect of diet-induced changes in gut microflora on the hepatocarcinogenicity of 2,6-DNT was studied in male F344 rats (Goldsworthy et al. 1986). Groups of the rats were placed on one of three diets containing 2,6-DNT at doses of 0, 0.6–0.7, or 3–3.5 mg/kg/day. Ten animals from each group were sacrificed at 3, 6, and 12 months, and their livers were evaluated histopathologically. The diets used were NIH-07, an open formula cereal-based diet high in pectin content; AIN-76A, a purified pectin-free diet; or AP, which is AIN-76A supplemented with 5% pectin. The number and size of γ-glutamyl transpeptidase-staining foci in the liver increased in a dose- and time-related manner in animals given 2,6-DNT in the NIH-07 diet. Hepatocellular carcinomas and neoplastic nodules were observed only in rats fed NIH-07 containing 2,6-DNT. No tumor was observed in rats receiving the control diets or 2,6-DNT in the AIN-76 diet with or without pectin. This finding suggested that pectin did not influence the tumor outcome of the experiment. Unidentified contaminants in cereal-based diets may influence liver foci and tumor production in the rat liver during carcinogen treatment.

Tg-DNT provided positive hepatocarcinogenic results in two bioassays of less-than-lifetime duration. In a 52-week study of male rats dosed with 35 mg/kg/day of Tg-DNT, Leonard et al. (1987) observed a 47% increase in hepatocellular carcinoma; cholangiocarcinomas were also found in 10% of rats treated with 35 mg/kg/day Tg-DNT in the Leonard et al. (1987) study. Hazleton Laboratories (1982) reported that dietary administration of 35 mg/kg/day Tg-DNT to rats for 55 weeks resulted in an increased incidence (100% in males and 55% in females) of hepatocellular carcinoma; this lesion was found in some animals treated at this level for 26 weeks. The administration of 3.5 mg/kg/day Tg-DNT for 104 weeks caused hepatocellular carcinoma in 9 of 70 males compared to 1 of 61 controls. Mammary fibroadenoma and subcutaneous fibroma were also found in both sexes at 3.5 mg/kg/day after 104 weeks (Hazleton Laboratories 1982). Administration of 14 mg/kg/day Tg-DNT for 104 weeks caused cholangiocarcinomas and parathyroid adenomas in males and hepatocellular carcinomas and hepatocholangiocarcinomas in females (Hazleton Laboratories 1982).

Tg-DNT contains about 76% 2,4-DNT and 19% 2,6-DNT, as well as small amounts of other isomers. Rats that received 35 mg/kg/day in the Leonard et al. (1987) and Hazleton Laboratories (1982) studies were provided approximately 28 and 7 mg/kg/day of 2,4- and 2,6-DNT, respectively. This dose of 2,6-DNT in Tg-DNT bioassays is equivalent to the low dose of 2,6-DNT administered to rats by Leonard et al. (1987) that produced hepatocellular carcinomas.

In hepatic tumor initiation-promotion protocols, Tg-DNT was reported to have tumor-promoting and tumor-initiating activity (Leonard et al. 1983, 1986; Mirsalis and Butterworth 1982). The results of the initiation-promotion protocols for 2,4-, 2,6-, and Tg-DNT indicate that 2,6-DNT is a complete hepatocarcinogen and is primarily responsible for the carcinogenic activity of Tg-DNT.

No studies were located regarding cancer in animals after oral exposure to 2,3-, 2,5-, 3,4-, or 3,5-DNT.

3.2.3.1. Death

No studies were located regarding mortality in humans associated with dermal exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

One study was located that examined death among humans exposed to DNTs. A retrospective mortality study of munitions workers exposed to either 2,4-DNT or Tg-DNT revealed an increased death rate due to ischemic heart disease and residual diseases of the circulatory system in the exposed cohort (Levine et al. 1986a, 1986b). Exposure concentrations to 2,4-DNT or Tg-DNT were not reported. The residual diseases included cardiac arrest and arteriosclerosis. Exposure levels were not reported, and the study is further limited by the small cohort size and concurrent inhalation exposure of the workers.

No studies were located regarding death in animals after dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

3.2.3.2. Systemic Effects

No studies were located regarding respiratory, renal, body weight, or endocrine effects in humans or animals after dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Gastrointestinal Effects. No studies were located regarding gastrointestinal effects in humans associated with dermal exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Gastrointestinal complaints of munitions workers exposed to either 2,4-DNT or Tg-DNT included nausea and vomiting (McGee et al. 1947). These workers also presumably inhaled DNT in the occupational setting. Exposure concentrations to 2,4-DNT or Tg-DNT were not reported.

No studies were located regarding gastrointestinal effects in animals after dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Cardiovascular Effects. No studies were identified with respect to cardiovascular effects after dermal exposure of humans to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Levine et al. (1986a) reported a significant increase in heart disease mortality in workers involved in the manufacture and processing of 2,4-DNT and/or Tg-DNT.

No studies were located regarding cardiovascular effects in animals after dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Hematological Effects. No studies were located regarding hematological effects in humans associated with dermal exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Hematological effects, such as anemia and cyanosis (which can be indicative of anemia), have been found in men employed at munitions factories (McGee et al. 1947; Perkins 1919). These workers were exposed to either 2,4-DNT or Tg-DNT. Because these studies lacked worker histories, exposure data, and reported on small cohorts, the results are equivocal and are best used to qualitatively describe symptoms. In addition, the workers probably received their primary exposure via the inhalation pathway.

No studies were located regarding hematological effects in animals after dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Musculoskeletal Effects. No studies were located regarding musculoskeletal effects in humans associated with dermal exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Muscle weakness and joint pain have been reported by munitions workers after occupational exposure to unspecified concentrations of 2,4-DNT or Tg-DNT (McGee et al. 1947; Perkins 1919).

In the Perkins (1919) study, joint pain and other incapacitating symptoms were noted following exposure to what were presumed to be very high concentrations of Tg-DNT since the processes described required direct handling without protective equipment. In both of these studies, however, no exposure data were available; exposure to other compounds may have occurred, and concomitant exposure via inhalation was also likely.

No studies were located regarding musculoskeletal effects in animals after dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Hepatic Effects. No studies were located regarding hepatic effects in humans associated with dermal exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

In a study of male munitions workers exposed to unspecified concentrations of 2,4-DNT, 29 of 714 workers displayed tenderness of the liver (McGee et al. 1947). No other clinical evaluation was performed that might provide further insight into the significance of this finding. These workers were also exposed to DNTs via inhalation.

No studies were located regarding hepatic effects in animals after dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Dermal Effects. No studies were located regarding dermal effects in humans associated with dermal exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

A study of dermal effects of topical exposure to 2,4-DNT in workers employed by a munitions factory during World War II reported that 32 of 714 workers complained of dermatitis (McGee et al. 1947). Exposure levels were not quantified in this study.

Both 2,4- and 2,6-DNT were shown to be mild primary dermal irritants in rabbits (U.S. Army 1975, 1978a).

No studies were located regarding dermal effects in animals after dermal exposure to 2,3-, 2,5-, 3,4-, or 3,5-DNT.

Ocular Effects. No studies were located regarding ocular effects in humans after dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

No ocular irritation was found in rabbits in a primary eye irritation test using unspecified concentrations of 2,4- or 2,6-DNT (U.S. Army 1975, 1978a). However, mild eye irritations were reported in rabbits treated with 2,4-DNT (Ford 1981).

No studies were located regarding ocular effects in animals after dermal exposure to 2,3-, 2,5-, 3,4-, or 3,5-DNT.

3.2.3.3. Immunological and Lymphoreticular Effects

No studies were located regarding immunological and lymphoreticular effects in humans after dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

In dermal sensitization tests, 2 of 10 guinea pigs exhibited mild sensitization to 2,6-DNT, but no sensitization was evident when 2,4-DNT was tested (U.S. Army 1975, 1978a).

No studies were located regarding immunological and lymphoreticular effects in animals after dermal exposure to 2,3-, 2,5-, 3,4-, or 3,5-DNT.

3.2.3.4. Neurological Effects

No studies were located regarding neurological effects in humans after dermal exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Various neurological symptoms, including headache, vertigo, and pain or numbness in the extremities, have been reported in surveys of munitions workers exposed to unspecified concentrations of 2,4-DNT (McGee et al. 1947). Although it is assumed that some dermal exposure to 2,4-DNT occurred in these workers, inhalation was the probable primary route of exposure.

No studies were located regarding neurological effects in animals after dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

3.2.3.5. Reproductive Effects

No significant effects on fertility were observed in workers occupationally exposed to Tg-DNT (Levine et al. 1985a). However, Levine et al. estimated that only a 50–70% reduction in fertility could have been detected in the worker population that they studied.

No studies were located regarding reproductive effects in animals after dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

3.2.3.6. Developmental Effects

No studies were located regarding developmental effects in humans or animals after dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

3.2.3.7. Cancer

In a case-cohort study of German copper miners with exposure to Tg-DNT (see Section 3.2.1 for additional detail), Seidler et al. (2014b) observed elevated Cox proportional HRs for renal cancer among workers with medium or high dermal exposure to Tg-DNT (HR 2.73, 95% CI 1.0–7.42 for medium exposure and HR 1.81, 95% CI 0.75–4.33 for high exposure). When combined across medium and high exposure categories for both dermal and inhalation exposure, the HR for renal cancer was significantly increased (HR 2.12, 95% CI 1.03–4.37).

No studies were located regarding cancer in humans or animals after dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

3.4.1.1. Inhalation Exposure

No studies were located regarding absorption of inhaled of 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT in humans or laboratory animals. However, detection of DNT metabolites in the urine of workers at DNT manufacturing plants provides evidence that DNT may be absorbed following presumed inhalation exposure (Levine et al. 1985b; Turner 1986; Woollen et al. 1985).

3.4.1.2. Oral Exposure

Rickert et al. (1983) suggested that the rapid disappearance of radioactivity from the first quarter of the small intestine of rats following the oral administration of uniformly [¹⁴C]-ring-labeled 2,4- or 2,6-DNT indicates rapid and fairly complete absorption.

Excretion data and observed systemic effects indicate that DNTs are absorbed following oral administration to experimental animals. Several strains of rats, New Zealand rabbits, beagle dogs, and Rhesus monkeys excreted 55–90% of the radioactivity from orally-administered radiolabeled DNTs in the urine, primarily within the first 24 hours (Long and Rickert 1982; Rickert and Long 1981; U.S. Army 1978b). In mice, most of the radioactivity from ³H-labeled 2,6-DNT was excreted in the urine (about 50% in 8 hours) (Schut et al. 1983), whereas most of the radioactivity from ¹⁴C-labeled 2,4-DNT administered to mice was excreted in the feces, and only about 10% was excreted in the urine (U.S. Army 1978b). Increased fecal excretion could be due to reduced absorption or to greater excretion via the bile.

Studies on absorption of 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT following oral exposure of humans or animals were not identified.

3.4.1.3. Dermal Exposure

There were no in vivo data available specifically on the absorption of 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT via the dermal route of exposure. Two studies of occupational exposure to Tg-DNT have suggested that dermal absorption can be a significant route of entry for DNT in humans since the levels of urinary metabolites of 2,4- and 2,6-DNT in loaders and operators at a DNT manufacturing plant exceeded those that would have resulted from the inhaled concentrations (Levine et al. 1985b; Woollen et al. 1985). An in vitro study examined the dermal absorption of 2,4- and 2,6-DNT in soil or acetone using excised pigskin (Reifenrath et al. (2002). When in acetone, 36 and 24% of the 2,4- and 2,6-DNT, respectively, was absorbed. The absorption of radiolabel from soil was less; 15 or 16%, respectively, was absorbed from a low carbon soil and 5.4 and 3.8%, respectively, was absorbed from a high carbon soil.

3.4.2.1. Inhalation Exposure

No studies were located regarding distribution in humans or animals following inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

3.4.2.2. Oral Exposure

The tissue distribution of 2,4-DNT and its metabolites was studied by Rickert and Long (1980). 2,4-DNT was administered orally to male and female rats at doses of 10, 35, or 100 mg of ¹⁴C-labeled 2,4-DNT/kg. When distribution is studied solely by detecting a radioisotope label, it is the labeled atom(s) that are being followed, and this label may be part of either the parent DNT molecule or a metabolite. Peak concentrations of radioactivity in plasma, red blood cells, liver, and kidney were proportional to dose. Levels in liver and kidney were 5–10 times higher than those in plasma or red blood cells. Levels of radioactivity in other tissues were lower than those in plasma. The only clear differences between males and females were the higher retention of radioactivity in red blood cells of females and the concentration of radioactivity in livers of females, which was only half that found in males. In addition, concentrations of 2,4-DNT in male kidneys peaked at 4–8 hours and were 3–10 times higher than the concentrations in female kidneys, which peaked 1 hour after the dose.

Rickert et al. (1983) observed that hepatic concentrations of radioactivity in male rats increased in two stages, with the first peak occurring 1–2 hours and a second peak occurring 8–12 hours after an oral dose of 10 or 35 mg/kg of radiolabeled 2,4- or 2,6-DNT. The second peak was followed by a gradual decline up to 16 days and was thought to be the result of enterohepatic cycling.

In mice administered ³H-labeled 2,6-DNT, the distribution of the label was similar in the blood, liver, kidneys, lungs, and small and large intestines at 8 hours after administration, with very low levels detected in the brain, lungs, heart, and spleen (Schut et al. 1983).

In a radioisotope labeling study in dogs and monkeys, total 2,4-DNT and its metabolites recovered in blood and other tissues were approximately 3.6% (dogs) and 2.2% (monkeys) of the administered dose (U.S. Army 1978b). Relative to blood concentrations, the liver had the highest levels of 2,4-DNT or metabolites. Detectable levels of 2,4-DNT or metabolites were also found in the kidney and in skeletal muscle (U.S. Army 1975, 1978b).

No studies were located regarding distribution in humans or animals following inhalation exposure to 2,3-, 2,5-, 3,4-, or 3,5-DNT.

3.4.2.3. Dermal Exposure

No studies were located regarding distribution in humans or animals following dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

3.4.4.1. Inhalation Exposure

In occupational settings, in addition to inhalation, some oral and dermal exposure can occur. The elimination of DNTs in the urine of workers exposed to Tg-DNT has been studied by several investigators (Levine et al. 1985b; Turner et al. 1985; Woollen et al. 1985).

Woollen et al. (1985) observed that the highest rates of excretion of 2,4-dinitrobenzoic acid occurred near the end of the work shift. The half-life for urinary excretion of 2,4-dinitrobenzoic acid was calculated to be 2–5 hours. This estimate appears to be the initial phase of a biphasic elimination profile since even 3 days after the last exposure, detectable levels of 2,4-dinitrobenzoic acid were present in urine.

Turner et al. (1985) determined the metabolic profiles in workers exposed to DNTs. The half-life for excretion of DNT metabolites in urine ranged from 0.8 to 4.5 hours. The half-lives for 2,4-dinitrobenzoic acid and 2,4-dinitrobenzyl alcohol glucuronide tended to be shorter than those for the metabolites that resulted from both oxidative and reductive metabolism.

No studies investigating elimination and excretion of 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT following inhalation exposure of humans or animals were located.

3.4.4.2. Oral Exposure

No studies were located regarding excretion in humans following oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Schut et al. (1983) reported that in mice, urine was the main route of elimination of ³H-labeled 2,6-DNT, with about 50% excreted after 8 hours. U.S. Army (1978b) observed that most of the radioactivity from ¹⁴C-labeled 2,4-DNT administered to mice was excreted in the feces and only about 10% in the urine. Differences between these two studies could be due, in part, to the use of different species of mice.

Male and female rats excreted 55–90% of the radioactivity from ¹⁴C-2,4-DNT or ¹⁴C-2,6-DNT in the urine, and 15–30% in the feces, within 72 hours after dosing (Long and Rickert 1982; Rickert and Long 1981). With 2,4-DNT, the females excreted a greater percentage of the dose in the urine as 2,4-dinitrobenzylalcohol glucuronide than did the males (except at the highest dose), but with 2,6-DNT, no sex-related difference in urinary excretion was seen.

In experiments with bile duct-cannulated rats, male rats excreted 25% of the radioactivity from ¹⁴C-2,4-DNT into the bile over a 36-hour period, whereas female rats excreted 18% (Medinsky and Dent 1983). Biliary excretion of radioactivity was linearly related to dose in males; females were evaluated only at one dose. Biliary excretion of radioactivity was virtually complete within 24 hours for males and 12 hours for females. Mean half-times of biliary excretion ranged from 3.3 to 5.3 hours. Urinary excretion was also significant, with greater amounts of radioactivity excreted in the urine of rats from which bile was not collected (60–90% of the dose) than in the urine of rats from which bile was collected (20–60% of the dose). This finding indicates that biliary metabolites were absorbed from the intestines (enterohepatic cycling). Whether or not bile was collected, female rats excreted more radioactivity in urine than did male rats. Greater than 90% of the urinary excretion of labeled metabolites appeared in urine collected during the first 24 hours. At the end of 36 hours, only 0.02–0.05% of the radioactivity was detectable in the livers; 20–60% of this was covalently bound.

No studies investigating elimination and excretion of 2,3-, 2,5-, 3,4-, or 3,5-DNT following oral exposure of humans or animals were located.

3.4.4.3. Dermal Exposure

There are no kinetic data in humans in which the route of exposure was specifically dermal. Occupational exposure studies available for Tg-DNT involved multiple routes of exposure (Levine et al. 1985b; Turner et al. 1985; Woollen et al. 1985). The major routes of exposure in these studies were considered to be inhalation and dermal. The results were discussed previously in Section 3.4.4.1.

No studies were located regarding excretion in animals following dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT.

Biomarkers of Exposure and Effect

Exposure. A rapid, accurate method for determining exposure to DNTs has been developed using spectrophotometric analysis of complexes of primary arylamines, which result from the reduction of DNTs and its metabolites (Smith et al. 1995).

Effect. Epidemiological studies that correlate quantitative estimates of exposure with disease outcomes would be useful. Studies that identify subtle physiological changes, such as altered blood chemistry indices, associated with a particular disease state are not available.

A disease registry is not currently available. The development of a registry of exposures and diseases would provide a useful reference tool for assessing the variations in exposure concentrations and health effects from, for example, geography, season, regulatory actions, presence of hazardous waste landfills, or manufacturing and use facilities. These assessments, in turn, would provide a better understanding of the needs for some types of research or data acquisition based on the current exposure concentrations.

Absorption, Distribution, Metabolism, and Excretion. The toxicokinetics of 2,4- and 2,6-DNT in rats by the oral route have been extensively studied. That Tg-DNT is absorbed and excreted in the urine by humans in an occupational setting (where the main routes of absorption are considered to be inhalation and dermal) has also been documented. There are no data available in animals on the toxicokinetics of DNTs by the dermal or inhalation routes. Toxicokinetics studies in rats administered the test materials by the inhalation and dermal routes would be critical in understanding possible differences in the toxicity of DNTs by different routes of administration. The main routes of exposure of humans are dermal and inhalation. Understanding the possible differences in toxicity in animals by different routes would be valuable in determining the significance of findings in humans who may be exposed by inhalation or dermal routes.

Comparative Toxicokinetics. Absorption and excretion studies in several species indicate that there are considerable differences between mice and the other species evaluated. More detailed study of the metabolism of DNTs by mice, including the role of biliary excretion and enterohepatic cycling, would assist in understanding why the metabolism in mice is different from other species and which species may be the most appropriate model for evaluating hazards and risks to humans.

Methods for Reducing Toxic Effects. The most important method for reducing the toxic effects of DNTs is removal of the person from the area of exposure. Skin and eyes should be rinsed copiously (Bronstein and Currance 1994), although absorption through the skin has not been adequately examined. Gastric lavage, with subsequent administration of activated charcoal, and cathartics may be of some benefit in reducing peak absorption after oral exposure to DNTs. Methylene blue treatment is used with patients presenting with serious methemoglobinemia (Bentur and Keyes 2004). No additional studies are considered necessary at this time to examine further methods for reducing body burden of DNTs. Further studies on supportive therapy after DNT exposure, such as the use of hemodialysis, forced diuresis, hyperbaric oxygen, or hemoperfusion might be useful.

Children’s Susceptibility. Data needs relating to both prenatal and childhood exposures, and developmental effects expressed either prenatally or during childhood, are discussed in detail in the Developmental Toxicity subsection above.

There is inadequate experimental evidence to evaluate if the pharmacokinetics of DNTs are different in children. There are no studies on whether DNTs or their active metabolites can cross the placenta or be excreted in breast milk, so it cannot be determined if fetuses may be exposed in utero or if infants may be exposed via breast milk ingestion. There are also no data to show if DNTs and their metabolites are stored in maternal tissues and thus might be later mobilized during gestation or lactation; however, DNTs and their metabolites are not likely to be stored because of their low octanol-water partition coefficient.

There is little experimental evidence to evaluate whether the metabolism of DNTs or their mechanisms of action are different in children. As discussed in Section 3.7, newborns are highly sensitive to the methemoglobin-generating effect of DNTs because of their deficiency in methemoglobin reductase (Gruener 1976), which reduces methemoglobin back to hemoglobin. In addition, newborns have a high concentration of fetal hemoglobin in their erythrocytes. It will be useful to determine if fetal hemoglobin is more sensitive to the methemoglobin-generating effect of DNTs. It will also be helpful to have data on the metabolism and mechanism of action of DNTs on children to determine if children are more vulnerable than adults to health effects from exposure to DNTs, as some enzymes involved in DNT metabolism are known to have developmental regulation. There are no biomarkers of exposure or effect that have been validated in children or in adults exposed as children. There are no data to determine whether there are any interactions with other chemicals unique to children, or whether interactions observed in adults also occur in children. Although DNTs are shown to be genotoxic, it is not known if parental exposure to DNT may affect children via parental germ cells, or if DNTs may indirectly affect the fetus during maternal exposure.

Child health data needs relating to exposure are discussed in Section 6.8.1, Identification of Data Needs: Exposures of Children.