Dinitrotoluene

Carcinogenicity

Administrative data

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Apr 1977 - 26 Apr 1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability as cited in OECD SIDS Dinitrotoluene (isomers mixture) CAS No.: 25321-14-6, 2004. Comparable to guideline study with minor deviations: Haematological examinations were performed on 10 rats/sex of all groups instead of 20.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

No haematological examinations performed at 3 months. For remaining examinations, 10 rats/sex of all groups instead of 20 were used.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dinitrotoluene

EC Number:

246-836-1

EC Name:

Dinitrotoluene

Cas Number:

25321-14-6

Molecular formula:

C7H6N2O4

IUPAC Name:

Reaction mass of 1-methyl-2,4-dinitrobenzene and 2-methyl-1,3-dinitrobenzene

Details on test material:

- Name of test material (as cited in study report): dinitrotoluene
- Physical state: light brown solid
- Analytical purity: assumed to be 100%

Test animals

Species:

rat

Strain:

other: CD-F (Fischer 344 albino)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts
- Age at study initiation: 4 - 5 weeks
- Weight at study initiation: 106 - 208 g (male), 95 - 157 g (female)
- Housing: in groups of three per polycarbonate cage; bedding consisted of heat-treated hardwood chips.
- Diet (e.g. ad libitum): Purina Laboratory Chow, ad libitum
- Water (e.g. ad libitum): tap water; ad libitum


IN-LIFE DATES: From: 18 Apr 1977 To: 03 May 1979

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: diet

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Dinitrotoluene was melted in a water bath, dissolved in 1% acetone as a dispersing agent, added to the basal diet (Purina Laboratory Chow) on a weight-per-weight basis, and thoroughly mixed in a twin-shell blender for one minute per kilogram of feed to provide the appropriate dietary levels.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

gas chromatography of diet samples

Duration of treatment / exposure:

26, 52, (55; termination of high dose animals) and 104 weeks

Frequency of treatment:

continuously

Post exposure period:

no

Doses / concentrations

Remarks:

Doses / Concentrations:
3.5, 14.0 and 35 mg/kg bw/d
Basis:
nominal in diet

No. of animals per sex per dose:

130 (interim sacrifice at week 26 and 52: 10 rats/sex/dose; at week 78: 20 rats/sex/dose; high dose: sacrifice of all surviving rats at week 55; terminal sacrifice: at week 104 all surviving rats)

Control animals:

yes, plain diet

Details on study design:

Compound consumption was calculated based on target dose levels (weekly from weeks 1-14, biweekly through week 26, and monthly through week 104).

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly from weeks 1-14, biweekly through week 26, and monthly through week 104


BODY WEIGHT: Yes
- Time schedule for examinations: weekly from weeks 1-14, biweekly through week 26, and monthly through week 104


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Both eyes of each animal to be sacrified at weeks 26, 52, 78 and at termination.
- Dose groups that were examined: all animals


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at weeks 26, 52, 78 and at termination
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: 10 animals of each sex and group at weeks 26 and 52; 20 animals of each sex and group at 78 and 104 weeks
- Parameters checked: hematocrit, hemoglobin and methemoglobin levels, erythrocytes, reticulocytes, Heinz bodies, total leucocyte and differential leucocyte counts, mean corpuscular hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin concentration


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at weeks 26, 52, 78 and at termination
- Animals fasted: Yes
- How many animals: 10 animals of each sex and group at weeks 26 and 52; 20 animals of each sex and group at 78 and 104 weeks
- Parameters checked: alkaline phosphatase, urea nitrogen, serum glutamicpyruvic transaminase


URINALYSIS: Yes
- Time schedule for collection of urine: at weeks 26, 52, 78 and at termination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: pH, specific gravity, ketones, total protein, occult blood andmicroscopic examination of the sediment


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all relevant organs
organ weights: brain, heart, liver, kidneys, lungs, and testes with epididymides and ovaries
HISTOPATHOLOGY: Yes, all relevant organs
Examination of: brain, (cerebellum, cerebrum, brain stem), thoracic spinal cord, pituitary, thyroid, parathyroid, adrenal, heart, aorta, lung, spleen, liver, kidney, stomach, small intestine (duodenum, jejunum, ileum), large intestine (upper and lower colon, rectum), pancreas, ovary with oviduct, uterus, prostate, thymus, esophagus, trachea, nasal turbinate, adipose tissue, submaxillary salivary gland, lymph nodes (mesenteric and thoracic), urinary bladder, thigh skeletal muscle with sciatic nerve, bone marrow (sternum), skin (flank), mammary gland, eyes, testes with epididymides and unusual lesions
Interim sacrifice at week 26 and 52: 10 rats/sex/dose; at week 78: 20 rats/sex/dose; high dose: sacrifice of all surviving rats at week 55; terminal sacrifice: at week 104 all surviving rats.

Statistics:

Bartlett's test for homogeneity of variance and the one-way classification analysis of variance (ANOVA) were performed. If significant results were obtained from both, a multiple pair-wise comparison procedure was used to compare the group mean values. If a significant result was not obtained from Bartlett's test, but was obtained from ANOVA, Scheffe's multiple pair-wise comparison procedure was used to compare the group mean values. All analyses were evaluated at the 5.0% probability level.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
- Mortality: 10 control males, 12 control females, 14 low-dose males and 15 low-dose females, 48 mid-dose males, 9 mid-dose females, 8 high-dose males, and 2 high-dose females died during the study. 9 control males, 11 control females, 6 lowdose males, 14 low-dose females, 19 mid-dose males, 13 mid-dose females, and 1 high-dose male were sacrificed in extremis. Statistical analysis revealed a significantly lower survival rate in the high-dose males at week 55 and in the mid-dose males at termination.
- Clinical signs: Treatment-related observations consisted of a hunched, thin, and/or bloated appearance. Incidental clinical signs, noted during theone hundred and four week study at comparable incidences among the control and treated animals were: bloody crusts around the eye(s) and nose; cloudy, squinted, pale, small or lacrimating eyes(s); wheezing; soft feces; stains on the fur; rough hair coat; localized alopecia; localized sores; and head tilt. An increase in the number of pal pable nodules and/or tissue masses was noted in the low- and mid-dose animals beginning at week 66, with particularly higher incidences in the mid-dose males through termination.

BODY WEIGHT AND WEIGHT GAIN
during the first fifty weeks of the study: a statistically significant dose-related decrease in body weight gain of the males (and females treated at all levels when compared to the appropriate control animals. A dose-related decrease in mean body weight values was noted in the treated animals at low- and mid-dose levels when compared to the appropriate control animals during weeks 54 through 104. During weeks 86 to 104, male mean body weights in the control and treated groups were noted to drop. A definitive cause for this is unknown; however, it is not an uncommon finding in the laboratory in the latter stages of a long term study when the population is declining.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption: Food consumption during the first 50 weeks of the study was statistically significantly lower in all treated males than in the control males. Food consumption values for the treated males were comparable to those of the control males during weeks 54 through 104. Analysis of food consumption of the females revealed a dose-related and statistically significant decrease in the values for the mid- and high-dose rats when compared to the values from the control group. No statistically significant differences were noted in the low-dose females during the first fifty weeks of the study. Mean food consumption values of the treated males were comparable to the control males during weeks 54 through 104, while a dose-related decrease in the food consumption for the females was noted during weeks 54 through 78, but values were comparable during weeks 82 through 104.
- Compound intake: the overall mean consumption for the low-dose group was 3.388 mg/kg bw/day for males and 3.379 mg/kg bw/day for females. For the middose-group was 13.451 mg/kg bw/day for males and 13.633 mg/kg bw/day for females, respectively.

OPHTHALMOSCOPIC EXAMINATION
- 3.5 mg/kg bw/d: Ophthalmological examinations conducted prior to the scheduled sacrifice revealed synechia in the left eye of 1 low-dos female. Incidental findings at week 52 which were noted sporadically in the control and treated groups upon ophthalmoscopic examination prior to the scheduled sacrifice at week 52 consisted of focal corneal dystrophy, cataract, focal corneal cloudiness, red discharge, corneal dystrophy, and pale eyes.
- 14 mg/kg bw/d: Ophthalmological examinations conducted prior to the scheduled sacrifice revealed synechia in the left eye of 1 mid-dose female. Synechia in both eyes was observed in 1 mid-dose male. At week 26, focal corneal opacity was observed in 1 mid-dose male and 1 mid-dose female. Incidental findings at week 52 which were noted sporadically in the control and treated groups upon ophthalmoscopic examination prior to the scheduled sacrifice at week 52 consisted of focal corneal dystrophy, cataract, focal corneal cloudiness, red discharge, corneal dystrophy, and pale eyes.
- 35 mg/kg bw/day: Ophthalmological examinations conducted prior to the scheduled sacrifice revealed synechia in the left eye of 1 one high-dose female, and 1 high-dose male. At week 26, focal corneal opacity was observed in 1 high-dose female.
Incidental findings at week 52 which were noted sporadically in the control and treated groups upon ophthalmoscopic examination prior to the scheduled sacrifice at week 52 consisted of focal corneal dystrophy, cataract, focal corneal cloudiness, red discharge, corneal dystrophy, and pale eyes.
Incidental findings in week 72 noted sporadically in the control an d treated groups at week 78 consisted of focal corneal dystrophy, red discharge, slight cloudines, panus, and cataracts. The low- and mid-dose animals also exhibited pale eyes. Ophthalmological examinatio-as conducted prior to scheduled terminal at week 104 sacrifice revealed cataracts, focal and diffuse cornea dystrophy, red discharge, synechia, pannus, diffuse retinal dystrophy, reduced vascularity of the retina, iritis, keratitis, corneal vascularization, and thickening of the lens or cornea.
HAEMATOLOGY
- 3.5 mg/kg bw/d: At week 78, statistically significantly higher than control hematocrit values for the low-dose males, were noted. The leukocyte counts for the low-dose females were statistically significantly higher than control. Evaluation of hematology values also revealed statistically significantly higher reticulocyte counts in the low-dose males when compared to the control. Methemoglobin values were statistically significantly lower than control in the low-dose females.
- 14 mg/kg bw/d: group mean values at week 26 which were statistically significantly higher than those of the control group included the leukocyte count in the mid-dose males. Group mean values at week 52 which were statistically significantly lower than those of the control group included the hemoglobin concentration in the high-dose males, the erythrocyte count in the mid- and high-dose males and the MCHC in the high-dose males. Group mean values which were statistically significantly higher than those of the control group included MCH in the mid-dose males. The hematocrit, methemoglobin, and Heinz body values were unremarkable. Analyses at week 55 revealed markedly increased erythrocyte morphology changes which were also noted at week 52. At week 78, statistically significantly lower than control hematocrit values were noted in the mid-dose males were detected. Statistically significantly lower than control erythrocyte counts were noted in the mid-dose males. The leukocyte counts for the mid-dose females were statistically significantly higher than control. Evaluation of hematology values also revealed statistically significantly higher reticulocyte counts in the mid-dose males when compared to the control. Methemoglobin values were statistically significantly higher than control in the mid-dose females. Evaluation for hematology at week 104 revealed a statistically significantly higher than control reticulocyte value for the mid-dose males. Statistically significantly lower than control values were noted in the hematocrit and hemoglobin values and the erythrocyte count in the mid-dose males.
- 35 mg/kg bw/d: group mean values at week 26 which were statistically significantly higher than those of the control group included the reticulocyte count and methemoglobin level in the high-dose males, and the mean corpuscular volume in the high-dose males and females. The erythrocyte count was decreased in high-dose animals, while the decrease was statistically significant in the males only. Methemoglobin levels in all treated females were significantly lower than values obtained from control animals. Mean corpuscular hemoglobin concentrations were slightly higher in the high-dose animals than the controls, but the difference was not statistically significant. Values for Heinz bodies, hematocrit, hemoglobin, and mean corpuscular hemoglobin were unremarkable. Group mean values at week 52 which were statistically significantly lower than those of the control group included the hemoglobin concentration in the high-dose males, the erythrocyte count in the high-dose males and the MCHC in the high-dose males. Group mean values which were statistically significantly higher than those of the control group included reticulocyte counts, white blood cell counts, and MCV in the high-dose males and MCH in the high-dose males. The hematocrit, methemoglobin, and Heinz body values were unremarkable. A treatment-related trend of increased erythrocyte morphology changes was noted in the high-dose males. These changes included hypochromasia, poikilocytes, polychromatophilia, nucleated red blood cells, anisocytosis, and target cells. Analyses at week 55 revealed markedly increased erythrocyte morphology changes which were also noted at week 52. In addition to those noted at Week 52,
several bizarre-shaped erythrocytes including helmet cells an d red cell fragments were observed in seventeen of the twenty high-dose males. Less severe morphology changes were also observed in the high-dose females.

CLINICAL CHEMISTRY
- 3.5 mg/kg bw/d: At week 26, blood urea nitrogen levels in the low-dose males were significantly lower than levels for the respective control groups. When compared with values obtained for control males, serum glutamic pyruvic transaminase and alkaline phosphatase in the low-dose males were statistically significantly lower.
At week 55, serum glutamic pyruvic transaminase, alkaline phosphatase and blood urea nitrogen values were elevated in the males. Alkaline phosphatase values were decreased in the females. Analysis at week 78 showed that blood urea nitrogen, serum glutamic pyruvic transaminase, and alkaline phosphatase values were statistically significantly lower than control in the low-dose males, low-dose females, and low-dose females, respectively. At week 104, alkaline phosphatase levels were found to be statistically significantly lower in the low-dose males and females. In addition, statistically significantly lower blood urea nitrogen and serum glutamic pyruvic transaminase levels were noted in the low-dose males and low-dose females, respectively.
- 14 mg/kg bw/d:, When compared with values obtained for control males at week 26, serum glutamic pyruvic transaminase and alkaline phosphatase in the mid-dose males were statistically significantly lower.
At week 52, alkaline phosphatase values were statistically significantly lower in the mid-dose females when compared to the control group value. At week 55, serum glutamic pyruvic transaminase, alkaline phosphatase and blood urea nitrogen values were elevated in the males. Alkaline phosphatase values were decreased in the females. Analysis at week 78 showed that alkaline phosphatase values were statistically significantly lower than control in the mid-dose females. A higher mean serum glutamic pyruvic transaminase value for the mid-dose males was noted and indicates active hepatic parenchymal damage. At week 104, statistically significantly higher serum glutamic pyruvic transaminase and blood urea nitrogen levels were noted in the mid-dose males, while alkaline phosphatase levels were found to be statistically significantly lower in mid-dose females.
- 35 mg/kg bw/d: At week 26, blood urea nitrogen levels in the high-dose males and females were statistically significantly higher than levels for the respective control groups. At week 52, serum glutamic pyruvic transaminase, alkaline phosphatase, and blood urea nitrogen values were statistically significantly higher in the high-dose males than in the controls. Alkaline phosphatase values were statistically significantly lower in the high-dose females when compared to the control group value. The elevated serum glutamic pyruvic transaminase values for the high-dose males indicate active hepatic parenchymal damage. The elevated alkaline phosphatase values for the high-dose males indicate cholestasis, while the elevated blood urea nitrogen values in these animals indicate s slight renal impairment
At week 55, serum glutamic pyruvic transaminase, alkaline phosphatase and blood urea nitrogen values were elevated in the males. Alkaline phosphatase values were decreased in the females.
URINALYSIS
Weeks 26, 52, 78, and 104: The results of urinalysis were not remarkable in all dose groups

ORGAN WEIGHTS
- 3.5 mg/kg bw/d: Analyses of organ weight data at weeks 26, 52 and 78 revealed decreased mean body weights in the treated males and females when compared to controls, apparent treatment-related increases (statistically significant) in the absolute and relative liver weights of the low-dose males and females at weeks 78 and 104 (relative liver weight was not significant for the low-dose females at week 78). The absolute and relative testes weights of the low-dose males were statistically significantly higher than control at weeks 78 and 104. Other statistically significantly higher than control values included relative brain weights in the low-dose males and females at week 104; relative heart weights of low-dose males and females at week 104; the relative kidney weights of the low-dose females at week 104; relative ovary weights in the low-dose females at week 104. Statistically significantly lower than control values included the absolute lung weight in the low-dose males at week 52; the absolute heart weight in the low-dose females at week 52; the relative heart weight in the low-dose females at week 52;
- 14 mg/kg bw/d: Analyses of organ weight data at weeks 26, 52 and 78 revealed decreased mean body weights in the treated males and females when compared to controls, apparent treatment-related increases (statistically significant) in the absolute and relative liver weights of the mid-dose males and females at weeks 26 and 52, and the mid-dose males and females at weeks 78 and 104. The relative testicular weights of the mid-dose males were statistically significantly higher than control at weeks 78 and 104. Other statistically significantly higher than control values included relative brain weights in the mid-dose males at weeks 52 and 78, and in the mid-dose males and females at week 104; relative lung weights in the mid-dose males at weeks 78 and 104 and the mid-dose females at week 104; relative heart weights of the mid-dose males and females at week 104; the absolute kidney weights of the mid-dose males at week 78, and the mid-dose males and females at week 104; the relative kidney weights of the mid-dose males at weeks 52, 78, and 104 and the mid-dose females at weeks 26 and 104; relative ovary weights in the mid-dose females at week 104. Statistically significantly lower than control values included the absolute lung weight in the mid-dose males at week 26 and the mid-dose females at weeks 52 and 78; the absolute heart weight in the mid-dose males at weeks 26 and 104, and the mid-dose females at week 52;
- 35 mg/kg bw/d: Analyses of organ weight data at weeks 26, 52 and 78 revealed decreased mean body weights in the treated males and females when compared to controls, apparent treatment-related increases (statistically significant) in the absolute and relative liver weights of the high-dose males and females at weeks 26 and 52. The absolute testes weights of the high-dose males at week 52 were significantly lower than control. Other statistically significantly higher than control values included relative brain weights in the high-dose males and females at weeks 26, 52, and 78; relative lung weights in the high-dose males at weeks 26, 52, and 78 and the high-dose females at week 26; relative heart weights of the high-dose males at weeks 26, 52, and 78 and the high-dose females at week 26; the absolute kidney weights of the high-dose males at week 78; the relative kidney weights of the high-dose males and females at weeks 26, 52, and 78; Statistically significantly lower than control values included the absolute lung weight high-dose males at week 52; the absolute heart weight in the high-dose females at week 52; the absolute testes weight in the high-dose males at week 52

GROSS PATHOLOGY
Necropsy of the animals sacrificed by design at weeks 26, 52, 78, and 104 revealed gross alterations of the liver which were attributable to the administration of dinitrotoluene. These alterations included white and/or yellow foci in the livers of the low-, mid-, and high-dose males and females, and an increased incidence of nodules and/or tissue masses in the mid-dose males and females at weeks 78 and 104. In addition, nodules on the lungs were observed in three males at week 52, and in one female at week 52. Gross pathology findings in the animals found dead or sacrificed in a moribund condition through week 104 included foci in the livers of the mid- and high-dose males and females and abnormally small testes in the mid- and high-dose males.

HISTOPATHOLOGY: NON-NEOPLASTIC AND NEOPLASTIC
3.5 mg/kg bw/d:
- Liver: by week 26, periportal hyperbasophilia of hepatocytes was prominent in two females from the low-dose group. Hepatic neoplasms were not observed at week 52 in controls or low-dose males and females. Other treatment-related lesions occurred in the liver in nearly all animals in the mid- and high-dose groups and in the low-dose males. These lesions consisted of foci/areas of cell alteration, hyperbasophilia and megalocytosis of hepatocytes, and vacuolation and necrosis of individual hepatocytes. By week 78, neoplastic nodules were observed in one male and two females. Foci/areas of cellular alteration were observed in nineteen males and eighteen females, and one control male and eight control females. Other treatment-related lesions occurred in the liver, and consisted of cystic degeneration, necrosis of individual hepacocytes, fatty metamorphosis and occasional necrosis. After 104 weeks, hepatocellular carcinomas were observed in the livers of nine males and one control male. Neoplastic nodules were observed in the livers of eleven males and twelve females, and nine control males and five control females. Other treatment-related lesions occurred in the livers of treated animals and consisted principally of an increased incidence or severity of fatty metamorphosis, necrosis, cystic degeneration, megalocytosis, and foci/areas of cell alteration.
14 mg/kg bw/d:
- Liver: at week 26, hepatotoxicity was observed to a less degree in the mid-dose animals. This consisted of degenerative, reactive, proliferative change affecting hepatocyte and bile duct epithelium. Minimal to moderately severe nonsuppurative pericholangitis was evident in some mid-dose females. Necrosis of individual hepatocytes was prevalent in males. Foci or areas of cell alteration were recognized most frequently and mid-dose animals. Periportal megalocytosis of hepatocytes was recognized in several males. Periportal hyperbasophilia of hepatocytes was prominent in several animals from the mid-dose group.
By week 52, three mid-dose males had hepatocellular carcinomas. These neoplasms were often multinodular involving much of the liver; the histologic appearance varied from well-differentiated to poorly-differentiated tumors. Neoplastic nodules were also observed in the livers of four males. One male had biliary hyperplasia with atypia of the bile duct epithelium. Hepatic neoplasms were not observed in controls, mid-dose females, or low-dose males and females. Other treatment-related lesions occurred in the liver in nearly all animals. These lesions consisted of foci/areas of cell alteration, hyperbasophilia and megalocytosis of hepatocytes, and vacuolation and necrosis of individual hepatocytes. At week 78, hepatocellular carcinomas were observed in nineteen males, neoplastic nodules were observed in eleven males and ten females. Foci/areas of cellular alteration were observed in nineteen males and twenty females, and one control male and eight control females. In addition, cholangiocarcinomas were recognized in four males. Two males from this group also had biliary hyperplasia with atypia of ductal epithelium. Other treatment-related lesions occurred in the liver, and consisted of cystic degeneration, necrosis of individual hepacocytes, fatty metamorphosis and occasional necrosis. After 104 weeks, hepatocellular carcinomas were observed in the livers of twenty-one males (seven of which metastisized to the lung) and forty females (one with
pulmonary metastasis). Neoplastic nodules were observed in the livers of fifteen males and fifty-three femalesand nine control males and five control females. In addition, one female had hepatocholangiocarcinomas, and two males had cholangiox carcinomas. Two males had biliary hyperplasia with atypia of the ductal epithelium, and ten males from this group had slight to severe biliary cirrhosis. Other treatment-related lesions occurred in the livers of treated animals and consisted principally of an increased incidence or severity of fatty metamorphosis, necrosis, cystic degeneration, megalocytosis, and foci/areas of cell alteration.
- Kidney: by week 26, male and female rats had an increased incidence and amount of tubular pigment when compared to reference control animals. An increase in the severity of chronic interstitial nephritis was observed in mid-dose males and females after 78 weeks. After 104 weeks, an increase in the severity of chronic interstitial nephritis occurred in the kidneys of mid-dose males and, to a lesser degree, the mid-dose females
- Bone marrow: Slightly increased proliferation of hematopoietic cells occurred in the sternal marrow of mid-dose animals but this change was not evident in the splenic red pulp.
- Testes: by week 78, all control and mid-dose males had interstitial cell tumors which were usually large, multiple, and bilateral and destroyed most of the testicular tissue.
- Parathyroid: after 104 weeks, an increase in the severity and incidence of parathyroid hyperplasia was observed in mid-dose animals, and two mid-dose males had parathyroid adenomas.
- Adrenal gland: in the adrenal gland, an increased incidence and severity of fatty metamorphosis and vascular ectasia were observed in mid-dose males and females after 104 weeks.

35 mg/kg bw/d:
- Heart: by week 26, an increase in incidence and severity of chronic myocarditis was observed in the high-dose males. Minimal to moderate chronic myocarditis was noted in all high-dose males, while four animals from the reference control had minimal chronic myocarditis. The incidence and severity of this change was comparable in females in the high-dose group and controls. This probably represents a treatment-related exacerbation of spontaneous disease. At week 55, an increased incidence and severity of chronic myocarditis was observed in the male rats
- Spleen: an increased amount of hemosiderin and extramedullary hematopoiesis, indicative of increased cell turnover, was recognized in the high-dose animals, particularly in the males. By week 52, an increased incidence and degree of extramedullary hematopoiesis occurred in the splenic red pulp of the males. Byweek 55, moderate to moderately severe ryhemosiderosis and extramedullary hematopoiesis occurred in the splenic red pulp of all males. The incidence and severity of these changes appeared essentially comparable in treated rats and control female rats observed at week 52.
-Liver: at week 26, hepatotoxicity was observed in the high-dose animals. This consisted of degenerative, reactive, proliferative change affecting hepatocyte and bile duct epithelium. Minimal to moderately severe nonsuppurative pericholangitis was evident especially in the high-dose males and, to a lesser degree, in the high-dose females. Necrosis of individual hepatocytes was prevalent in males. Foci or areas of cell alteration were recognized most frequently in the high-dose males. Vacuolated hepatocytes were occasionally recognized in animals from the high-dose group. Slight to moderately severe biliary hyperplasia, periportal fibrosis and necrosis of bile duct epithelium were present in high-dose males. Periportal megalocytosis of hepatocytes was recognized in several males. Periportal hyperbasophilia of hepatocytes was prominent in the high-dose females. This specific change was not observed in the high-dose males, probably due to a masking effect by the more severe changes which were noted. Two females from the high-dose group also showed nuclear polymorphism consisting of enlarged and hyperchromatic or vesiculated nuclei, some showing clumping of chromatin. Two male rats receiving the high-dose had hepatocellular carcinomas. Primary hepatic neoplasms were not observed in the control group, and in conjunction with the liver alterations previously discussed, these are considered to be induced by dinitrotoluene. An increased incidence of microgranulomas in the hepatic parenchyma of experimental animals was observed. This is considered a spontaneous change since they occurred with some frequency in control rats even though this specific control group had an unusually low incidence.
By week 52, all ten high-dose males and four high-dose females had hepatocellular carcinomas. These neoplasms were often multinodular involving much of the liver; the histologic appearance varied from well-differentiated to poorly-differentiated tumors. Neoplastic nodules were also observed in the livers of three males and eight females. Cholangiocarcinomas were observed in two males, and two males and had biliary hyperplasia with atypia of the bile duct epithelium. Hepatic neoplasms were not observed in controls. Other treatment-related lesions occurred in the liver in nearly all animals in the high-dose groups. These lesions consisted of foci/areas of cell alteration, hyperbasophilia and megalocytosis of hepatocytes, and vacuolation and necrosis of individual hepatocytes. At week 55, Hepatocellular carcinomas occurred in all twenty males and in eleven female rats. Hepatic neoplastic nodules were observed in five males and twelve females. In addition, two male rats had hepatocellular-cholangiocarcinomas and three males had cholangiocarcinomas. Biliary hyperplasia with atypia of bile duct epithelium was also observed in two males.
Evidence of hepatotoxicity occurred in all animals and consisted of foci/areas of cell alteration, hyperbasophilia and megalocytosis of hepatocytes, foci of cystic degeneration, vacuolation and necrosis of individual hepatocytes and fatty metamorphosis.

- Kidney: at week 26, a slightly increased incidence of chronic interstitial nephritis was noted in the high-dose males. Male and female rats had an increased incidence and amount of tubular pigment when compared to reference control animals. By week 52, an increased severity of chronic interstitial nephritis was observed in the high-dose males which was accompanied by a slight increase in the amount of tubular pigment. By week 55, the incidence and severity of chronic interstitial nephritis and amount of renal tubular pigment was increased as compared to control animals at week 52 in male rats and, to a lesser degree, in female rats.
- Testes: at week 26, three males had moderately severe testicular degeneration while none were observed in control males. This was a unilateral change in each case and may represent a spontaneous lesion. At week 52, Moderate to moderately severe testicular degeneration and hypospermatogenesis occurred in nearly all of the high-dose males and in one control male. Ath the 55 weeks necropsy, slight to moderately severe testicular degeneration and/or hypospermatogenesis was observed in fifteen animals at risk while one control male had this lesion. Also an early onset of interstitial cell tumors was observed occurring in nine treated males while only two occurred in the control male group.
- Lymph node: by week 26, female animals from the control and high-dose group had minimal to moderately severe proliferation of fixed reticuloendothelial cells principally in the mesenteric lymph node. This change appeared essentially comparable in incidence and severity in control and experimental groups. This represents an unusual change not previously observed in control animals; however, since both groups were essentially equally affected, the significance of this change is unclear. The majority of female rats from the control and high-dose groups and approximately one-half of the high-dose males had multiple foci of proliferating fixed reticuloendothelial cells in lymph nodes at week 52, principally the mesenteric lymph node. The severity of this change was slightly increased in the high-dose females compared to reference controls, but this change was not observed in the male control group. In lymph nodes, especially mesenteric nodes, an increase in the incidence and/or severity of foci of proliferating reticuloendothelial cells was observed in nearly allexperimental animals after 55 weeks. This change was not observed in male controls.
- Bone marrow: slight to moderate hyperplasia (myeloid and erythroid) or erythroid hyperplasia was observed in the majority of the males. By week 55, slight to moderate hyperplasia of sternal bone marrow was present in nine treated male rats while this change was not observed in the control males.
- Pancreas: By week 52, an equivocally increased amount of interstitial pigment was observed in the high-dose males. Increased amounts of pancreatic interstitial pigment occurred in male rats after 55 weeks.

Also, an increased incidence of relatively common benign neoplasms was observed in experimental animals and a wide variety of spontaneous lesions was observed in all groups and appeared to be unrelated to experimental regimen.

Effect levels

Dose descriptor:

LOAEL

Effect level:

3.5 mg/kg bw/day (nominal)

Sex:

male/female

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Any other information on results incl. tables

Body weights:

	Male				Female
Week	Control	3.5 mg/kg bw/d	14 mg/kg bw/d	35 mg/kg bw/d	Control	3.5 mg/kg bw/d	14 mg/kg bw/d	35 mg/kg bw/d
26	328 ± 23	311 ± 14*	297 ± 31*	253 ± 13*	184 ± 8	182 ± 12*	182 ± 13*	158 ± 6*
52	363 ± 22	338 ± 21	287 ± 21*	245 ± 14*	214 ± 14	207 ± 15	190 ± 12*	169 ± 11*
78	374 ± 71	358 ± 15*	286 20*	-	260 ± 15	239 ± 12*	201 ± 12*	-
104	352 ± 29	333 ± 21*	258 ± 31*	-	352 ± 23	333 ± 21	257 ± 30*	-

*p<0.05

Organ weights:

		Male				Female
	Organ	Control	3.5 mg/kg bw/d	14 mg/kg bw/d	35 mg/kg bw/d	Control	3.5 mg/kg bw/d	14 mg/kg bw/d	35 mg/kg bw/d
Week 26	Brain	1.97 ± 0.05	1.96 ± 0.06	1.95 ± 0.05	1.95 ± 0.04	1.85 ± 0.07	1.84 ± 0.06	1.80 ± 0.03	1.80 ± 0.04
	Lung	1.36 ± 0.1	1.26 ± 0.09	1.23 ± 0.08*	1.25 ± 0.07	0.97 ± 0.09	0.91 ± 0.06	0.96 ± 0.08	0.93 ± 0.07
	Heart	1.04 ± 0.08	0.99 ± 0.1	0.9 ± 0.09*	0.94 ± 0.1	0.66 ± 0.04	0.66 ± 0.08	0.68 ± 0.05	0.64 ± 0.06
	Liver	8.78 ± 0.87	8.80 ± 0.56	9.87 ± 0.8*	13.19 ± 1.01	4.81 ± 0.17	4.98 ± 0.35	5.89 ± 0.44*	7.26 ± 0.56*
	Kidney	2.25 ± 0.2	2.14 ± 0.13	2.17 ± 0.19	2.38 ± 0.1	1.27 ± 0.05	1.32 ± 0.08	1.34 ± 0.08	1.35 ± 0.08
	Testes	4.32 ± 0.24	4.18 ± 0.24	3.96 ± 0.27	4.21 ± 0.40	-	-	-	-
	Ovaries					0.074 ± 0.016	0.074 ± 0.016	0.079 ± 0.013	0.075 ± 0.011
Week 52	Brain	2.13 ± 0.16	2.01 ± 0.08	1.59 ± 0.07	2.00 ± 0.13	1.54 ± 0.16	1.85 ± 0.06	1.83 ± 0.08	1.85 ± 0.07
	Lung	1.51 ± 0.15	1.35 ± 0.17	1.36 ± 0.17	1.44 v 0.26	1.14 ± 0.2	0.98 ± 0.09*	0.95 ± 0.08*	0.96 ± 0.05*
	Heart	1.07 ± 0.09	1.13 ± 0.1	1.02 ± 0.09	1.11 ± 0.27	0.92 ± 0.18	0.70 ± 0.06*	0.73 ± 0.06*	0.78 ± 0.06*
	Liver	9.11 ± 0.66	10.15 ± 0.54	15.46 ± 1.23*	20.73 ± 7.86*	5.74 ± 0.3	5.80 ± 0.5	7.06 ± 0.41*	8.90 ± 0.61*
	Kidney	2.54 ± 0.21	2.43 ± 0.15	2.61 ± 0.16	2.84 ± 0.43	1.55 ± 0.11	1.48 ± 0.08	1.44 ± 0.15	1.60 ± 0.07
	Testes	5.14 ± 0.62	4.52 ± 0.29	4.89 ± 0.42	3.93 ± 1.09*
	Ovaries					0.095 ± 0.016	0.103 ± 0.026	0.141 ± 0.135	0.097 ± 0.022
Week 79	Brain	2.10 ± 0.11	2.12 ± 0.07	2.02 ± 0.12	-	2.10 ± 0.16	2.03 ± 0.19	1.87 ± 0.13*	-
	Lung	1.37 ± 0.12	1.49 ± 0.29	1.36 ± 0.12	-	1.23 ± 0.15	1.17 ± 0.19	1.02 ± 0.18*	-
	Heart	1.09 ± 0.12	1.08 ± 0.09	1.14 ± 0.18	-	0.98 ± 0.16	0.89 ± 0.19	0.87 ± 0.12	-
	Liver	9.42 ± 0.60	11.36 ± 0.96*	18.58 ± 3.81*	-	6.64 ± 0.45	6.73 ± 0.75	9.07 ± 0.48*	-
	Kidney	2.49 ± 0.13	2.67 ± 0.15*	2.94 ± 0.27*	-	1.86 ± 0.19	1.74 ± 0.19	1.92 ± 0.63	-
	Testes	5.43 ± 1.16	6.61 ± 1.07*	5.71 ± 1.19	-	-	-	-	-
	Ovaries	-	-	-	-	0.22 ± 0.45	0.31 ± 0.41	0.12 ± 0.02	-
Week 55 (terminal sacrifice)	Brain	-	-	-	1.87 ± 0.08	-	-	-	1.84 ± 0.11
	Lung	-	-	-	1.56 ± 0.84	-	-	-	1.04 ± 0.18
	Heart	-	-	-	1.00 ± 0.10	-	-	-	0.75 ± 0.06
	Liver	-	-	-	29.88 ± 5.77	-	-	-	9.92 ± 1.21
	Kidney	-	-	-	0.64 ± 1.15	-	-	-	1.63 ± 0.14
	Testes	-	-	-	3.86 ± 1.23	-	-	-	-
	Ovaries	-	-	-	-	-	-	-	0.099 ± 0.055
Week 104 (terminal sacrifice)	Brain	2.08 ± 0.81	2.09 ± 0.11	2.05 ± 0.10	-	1.82 ± 0.18	1.81 ± 0.88	1.51 ± 0.12	-
	Lung	1.71 ± 0.36	1.72 ± 0.36	1.51 ± 0.47	-	1.39 ± 0.48	1.21 ± 0.14	1.29 ± 0.80	-
	Heart	1.26 ± 0.16	1.128 ± 0.12	0.988 ± 0.15*	-	0.92 ± 0.12	0.93 ± 0.10	0.89 ± 0.09	-
	Liver	10.62 ± 1.78	12.13 ± 1.6*	17.25 ± 2.1*	-	7.46 ± 1.07	8.78 ± 1.18*	12.3 ± 1.79	-
	Kidney	2.84 ± 0.4	2.80 ± 0.2	3.13 ± 0.40*	-	1.88 ± 0.22	1.88 ± 0.16	2.39 ± 0.27*
	Testes	7.58 ± 1.86	8.60 ± 1.91*	7.54 ± 3.31	-	-	-	-	-
	Ovaries	-	-	-	-	0.115 ± 0.026	0.114 ± 0.034	0.165 ± 0.235	-

*p<0.05

Methemoglobin content (%):

	Male				Female
Week	Control	3.5 mg/kg bw/d	14 mg/kg bw/d	35 mg/kg bw/d	Control	3.5 mg/kg bw/d	14 mg/kg bw/d	35 mg/kg bw/d
Week 26	0.36 ± 0.36	0.97 ± 0.71	0.74 ± 0.55	1.80 ± 1.18*	2.85 ± 0.72	1.89 ± 0.80*	1.02 ± 0.66*	1.48 ± 0.48*
Week 52	0.87 ± 0.56	1.37 ± 0.61	1.49 ± 0.96	1.55 ± 0.92	1.74 ± 1.13	2.48 ± 1.33	1.02 ± 0.75	1.09 ± 0.76
Week 55	-	-	-	1.42 ± 1.33	-	-	-	1.77 ± 191
Week78	1.77 ± 1.16	1.56 ± 0.46	1.31 ± 101	-	1.65 ± 1.18	0.70 ± 0.33*	3.08 ± 0.93*	-
Week 104	1.16 ± 1.01	1.32 ± 0.99	1.46 ± 0.86	-	1.31 ± 0.93	0.97 ± 0.81	1.58 ± 0.90	-

*p<0.05

Other blood parameter:

- 35 mg (52 or 55 week exposure): increase in serum GPT (m/f), blood urea nitrogen and alkaline phosphatase (m), decreased hemoglobin content (m), erythrocyte count (m), and treatment related morphological changes in erythrocytes; increased reticulocyte (m) and leucocyte (m) count, mean corpuscular volume (m, females only up to week 26), and mean corpuscular hemoglobin (m); methemoglobin was increased in males and decreased in females at week 26; increased absolute liver weights (by m: 215%; f: 55%) and relative liver weights (by m: 270%; f: 96%); absolute kidney weights increased by about 12% in males; decreases in relative weights of other organs in line with body weight reduction; hepatotoxic changes (m/f) in nearly all animals, foci/areas of cell alteration, hyperbasophilia and megalocytosis of hepatocytes, vacuolation and necrosis of individual hepatocytes; exacerbation of chronic interstitial nephritis and spontaneous cardiomyopathy, increased red cell turnover (hemosiderosis, extramedullary hematopoiesis), increased interstitial pigment in pancreas; testicular degeneration (in 15/20 animals) plus hypospermatogenesis (in 14/20)

- 14 mg (104 week exposure): decrease in hematocrite (m), hemoglobine content (m), and erythrocyte count (m); increase in reticulocyte (m), leucocyte (m/f) count only at week 52 and 78; methemoglobin was decreased in females at week 26 and increased at week 78; significant increase in serum GPT (m/f) and blood urea nitrogen (m) and decrease in serum phosphatase (f); absolute liver weights (m: 62%; f: 65%) and relative liver weights increased; absolute kidney weights increased by about 11% in males and 16% in females; absolute weights of ovaries enhanced by 57%, however, no dose dependent effect; hepatotoxic changes (m/f in nearly all animals, foci/areas of cell alteration, hyperbasophilia and megalocytosis of hepatocytes, vacuolation and necrosis of individual hepatocytes), exacerbation of chronic interstitial nephritis and degenerative changes in the adrenal glands

Histopathological incidences:

- Control: Liver: hepatocellular carcinomas (male, 1/120; female, 0/120), neoplastic nodules (male, 8/120; female, 5/120); mammary glands: fibroadenomas (male, 6/91; female, 16/90); at 52 weeks all 10 sacrificed control animals showed interstitial cell tumours of the testes, at 104 weeks male animals had interstitial cell tumours of the testes (82/101)

- 3.5 mg/kg bw/d (104 week exposure): Liver: hepatocellular carcinomas (male, 10/130; female, 1/129), neoplastic nodules (male, 14/130; female, 15/129); testes: interstitial cell tumours (1/1)

- 14 mg/kg bw/d (104 week exposure): Liver: hepatocellular carcinomas (male, 98/128; female, 45/130), neoplastic nodules (male, 64/128; female, 74/130), cholangiocarcinomas (male, 15/128; female, 0/130), skin: subcutaneous fibromas (male, 44/52; female, 11/15) and fibrosarcomas (male, 10/52; female, 4/15); mammary gland: fibroadenomas (male, 22/79; female, 29/91); testes: interstitial cell tumours (104/108)

- 35 mg/kg bw/d (52 or 55 week sacrifice): Liver: hepatocellular carcinomas (male, 32/40; female, 15/40), neoplastic nodules (male, 8/40; female, 9/49), hepatocellular cholangiocarcinomas (male, 3/40; female, 15/40); testes: interstitial cell tumours (13/40); frequent occurrence of multiple tumours

Under the test conditions used, the test substance is carcinogenic.