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carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Nov 1983 - 22 Jan 1993
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (limited list of tissues (only target organs) examined histologically)

Data source

Referenceopen allclose all

Reference Type:
study report
Report Date:
Reference Type:
Carcinogenicity and toxcity of inhaled nitrobenzene in B6C3F1 mice and F344 and CD rats.
Cattley, R.C. et al.
Bibliographic source:
Fundam. Appl. Toxicol. 22, 328-340

Materials and methods

Test guideline
equivalent or similar to
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
limited list of tissues examined histologically
GLP compliance:

Test material

Details on test material:
- Name of test material (as cited in study report): nitrobenzene
- Supplier: First Chemical
- Analytical purity: nominal > 99.8%; actually determined: 99.977%
- Purity test date: 29 Nov 1983
- Impurities: 0.010% 2-nitrotoluene, 0.006% 4-nitrotoluene, 1,3-dinitrobenzene, 0.001% unspecified

Test animals

Details on test animals and environmental conditions:
- Source: Charles River Breeding Laboratories, Kingston, NY
- Age at study initiation: ca. 62 days
- Weight at study initiation: ca. 268 ± 11.07 g
- Housing: three per cage in stainless steel wire cages
- Diet (e.g. ad libitum): NIH-07 pellet diet; ad libitum
- Water (e.g. ad libitum): filtered, deionized water; ad libitum
- Acclimation period: 16 days

- Temperature (°C): 20 - 25
- Humidity (%): 40 - 60
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 Nov 1983 To: 12 Dec 1985

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
other: HEPA-filtered air
Details on exposure:
- Exposure apparatus: 8 m3 stainless steel and glass inhalation chambers with dynamic flow mode.
- Generation system of test atmosphere: A stainless steel J-tube wrapped with heat tape and filled with glass beads, FMI pump or Harvard syringe pump and nitrobenzene reservoir. The exposure atmosphere was produced by evaporating nitrobenzene from the surface of warm glass spheres and diluting the vapour and zero-air carrier gas into the total chamber airflow.
- Method of holding animals in test chamber: animals were in their cages
- Temperature, humidity, pressure in air chamber: 20 - 25 °C; 40 - 60%
- Air flow rate: 2.2 m3/min
- Air change rate: 16/h
- Treatment of exhaust air: filtering with three charcoal canisters in series

- Brief description of analytical method used: on line GC analysis with a flame ionization detection
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Daily GC sampling of chamber exposure atmospheres throughout the course of the study
Duration of treatment / exposure:
107 weeks with a total of 505 exposure days; interim sacrifice after 60 w
Frequency of treatment:
6 h/d, 5 d/w
Post exposure period:
Doses / concentrationsopen allclose all
Doses / Concentrations:
0.005, 0.025 and 0.125 mg/L
nominal conc.
Doses / Concentrations:
5, 25 and 125 mg/m3
nominal conc.
Doses / Concentrations:
1, 5 and 25 ppm
nominal conc.
No. of animals per sex per dose:
50 for the final necropsy; 10 for the interim sacrifice
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were chosen based on the results of a 90-day inhalation study conducted in mice and rats.


Observations and examinations performed and frequency:

- Time schedule: twice daily

- Time schedule for examinations: weekly during the first 13 weeks and biweekly thereafter


- Time schedule for collection of blood: at interim sacrifice and at terminal sacrifice; all animals that died in between
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: at interim sacrifice (10 animals/group) and at terminal sacrifice (20 animals/group)
- Parameters checked: white blood count, red blood count, hemoglobin, hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, red cell distribution width, platelets- thrombocytes, platelet hematocrit, mean platelet volume, platelet distribution width and differential counts

- Time schedule for collection of blood: at interim sacrifice and at terminal sacrifice; all animals that died in between
- Animals fasted: Yes
- How many animals: at interim sacrifice (10 animals/group) and at terminal sacrifice (20 animals/group)
- Parameters checked: glucose, blood urea nitrogen, alkaline phosphatase, albumin, total bilirubin, serum glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminsae, calcium, inorganic phosphorous, gamma-glutamyl transpeptidase, determination of methemoglobin, sodium, potassium, chloride


Sacrifice and pathology:
organ weights of spleen, liver, brain and kidney
various organs and gross lesions
brain, parathyroid, stomach, cecum, lymph nodes, heart, kidney, prostate, oviducts, harderian gland, sciatic nerve, nose, spinal cord, larynx, duodenum, colon, aorta, adrenal glands, testes, uterus, skin, bone, pituitary gland, trachea, jejunum, pancreas, thymus, tongue, lungs, epididymis, urinary bladder, mammary glands, thyroid glands, esophagus, ileum, salivary gland, spleen, liver, seminal vesicle, ovaries, eyes and optic nerves, skeletal muscle, ear canal and gross lesions
If possible, calculation were performed using the SAS System.
survival data: Kaplan-Meier, Cox and Tarone; incidence data: Dinse and Haseman and Gart et al., Fisher Exact test; continuous variables: Dunnett, Shirley, Dunn and Jonckheere. A significance level of 0.05 was used.

Results and discussion

Results of examinations

Details on results:
- Mortality: nitrobenzene exposure did not alter survival of the animals
- Clinical signs: In 1 ppm male CD rats, the incidence of overgrown teeth was increased by +243%, although incidence was not affected by exposure to 5 or 25 ppm nitrobenzene.

A significant decrease by -2% in body weights of the 25 ppm exposed group during the 31-60 week period was the only significant body weight effect noted in male CD rats.

At the time of the interim sacrifice, a significant decrease in platelet distribution width was present in all groups of exposed male CD rats (1 ppm: -3%; 5 ppm: -3%; 25 ppm: -5%). In 25 ppm exposed male CD rats, a significant decrease of -6% in red blood cell count was present at the interim sacrifice. A significant negative trend was seen in the MCHC with a decrease in 5 ppm (-1%) and 25 ppm (-5%) exposed animals. The MCV was significantly increased by +7% in male CD rats in the 25 ppm exposed group at the interim sacrifice. A significant decrease of -2% in MCHC was seen in the male CD rats at the final sacrifice. A significant increase was noted in methemoglobin levels in all exposed groups at the interim sacrifice (1 ppm: +246%; 5 ppm: +427%; 25 ppm: 396%), and in the 25 ppm exposed group at the final sacrifice (+67%). Male CD rats exposed to 25 ppm nitrobenzene also had significant increases in presence of macrocytes (4/10 at interim sacrifice compared to 0/0 in control), Howell-Jolly bodies (+300% at interim and +100% at final sacrifice) and polychromasia (+800% at interim sacrifice) in blood smears. Male CD rats exposed to 25 ppm also had significantly -51% decreased eosinophil counts at the final sacrifice. All other examined parameters did not show significant changes.

At final sacrifice significant slight elevations in ALT (+72%), AST (+83%), and GGT (+67%) were noted in male CD rats exposed to 25 ppm nitrobenzene. A significant trend toward elevated phosphorus levels was noted for interim sacrifice animals but was not present at final sacrifice. Five ppm and 25 ppm concentration exposed animals in the interim sacrifice group had slightly elevated phosphorus as compared to controls (5 ppm: +32%; 25 ppm: +30%). Significant elevated potassium was noted in high concentration exposed CD rats at the final sacrifice (+11%), and a significant trend toward decreased sodium was noted at both interim and final sacrifice, although these changes were slight. Male CD rats had significant decreases in sodium following nitrobenzene exposure at the interim (1 ppm: -1%; 5 ppm: -3%; 25 ppm: -1%) and final (5 ppm: -1%; 25 ppm: -1%) sacrifices.
All other examined parameters did not show significant changes.

A significant relative (liver to body weight) increase by +18% in hepatic weight was found in male rats exposed to 25 ppm nitrobenzene at the interim sacrifice, but no increases in absolute or relative organ weight changes were found in final sacrifice animals.

Few gross changes were found at the interim sacrifice time, but unscheduled deaths and final sacrifice control and nitrobenzene exposed animals had numerous renal changes associated with chronic progressive nephropathy.

A) Interim sacrifice animals:
- Spleen: Splenic lesions consisted of an increase in the incidence of splenic congestion in exposed rats of all groups and an increase in the severity of pigmentation (hemosiderin-laden macrophages within the red pulp) in male rats exposed to nitrobenzene . A slight increase in the amount of extramedullary hematopoiesis was observed.
- Liver: treatment-related lesions in the liver of interim sacrifice animals consisted of centrilobular hypertrophy of hepatocytes (hepatocytomegaly) and pigment deposition in Kupffer cells of 5 ppm and 25 ppm exposed group male rats, as compared to controls.
- Nose: the incidence of CD rats with nasal epithelial hyperplasia at level 1 was increased in nitrobenzene exposed groups. Increased pigmentation in the mucosa and submucosa of the olfactory epithelium in nose levels 3 and 4 was observed in nearly all control and nitrobenzene exposed CD rats, but lesion severity was increased in association with nitrobenzene exposure in a concentration-dependent manner. The pigmentation consisted of small amounts of brown granular pigment primarily within macrophages in the olfactory submucosa.

B) Final sacrifice:
- Spleen: exposure-related changes consisted of a significant increase in the incidence of sinusoidal congestion at all nitrobenzene concentrations (control: 25/63; 1 ppm: 43/67; 5 ppm: 44/69; 25 ppm: 38/65). A minor exposure-related increase in the severity of splenic extramedullary hematopoiesis (control: 58/63; 1 ppm: 56/67; 5 ppm: 61/96; 25 ppm: 60/65) and degree of pigmentation (pigment-laden macrophages in the red pulp) was noted (control: 59/63; 1 ppm: 58/67; 5 ppm: 67/69; 25 ppm: 65/65).
- Liver: at final sacrifice, there was a significantly increased incidence of hepatocellular adenoma in male rats exposed to 25 ppm nitrobenzene (+568%). There was also a significant increase in the incidence of 25 ppm nitrobenzene exposed male rats with hepatocellular carcinoma, but the incidence of hepatocellular carcinoma in this group or other nitrobenzene exposure groups was not different from controls. Multiple hepatocellular adenomas were present in two 25 ppm exposed rats, and multiple hepatocellular carcinomas were present in one 25 ppm exposure concentration group animal. The hepatocellular adenomas consisted of well differentiated hepatocytes arranged in sheets or irregular cords. They were sharply demarcated and compressed surrounding parenchyma. Hepatocellular carcinomas, in contrast, were generally larger and more irregular than adenomas and were comprised of pleomorphic cells arranged in sheets or disorganized cords.
The majority of the liver changes observed in treated animals at the final sacrifice consisted of non-neoplastic findings. These included significant increases in incidences of centrilobular hypertrophy in 5 ppm (+320%) and 25 ppm (+1160%) exposed animals (hepatocytomegaly), pigmentation of Kupffer cells in all exposed concentration groups, spongiosis hepatis in 25 ppm exposed animals (+65%), and a nonsignificant but increased incidence of eosinophilic foci in the 25 ppm exposed animals (+67%; p = 0.086). Centrilobular hypertrophy consisted of enlarged hepatocytes with increased granular eosinophilic cytoplasm radiating around central veins. Intracytoplasmic brown, granular pigment, thought to be hemosiderin, was noted in Kupffer cells. Eosinophilic cell foci were characterized by well-circumscribed areas of enlarged hepatocytes with an increased amount of eosinophilic cytoplasm.These foci had little disorganization of hepatic cords and only minimal peripheral compression. Hepatic cords within foci were contiguous with normal surrounding hepatic parenchyma . Changes diagnosed as spongiosis hepatis were characterized by foci of multilocular cysts filled with finely granular acidophilic cytoplasm. Occasionally these lesions were noted within eosinophilic foci.
- Testis and Epididymis: testicular atrophy was present in both control and nitrobenzene exposed male rats. There was a significant positive trend in the incidence of bilateral testicular atrophy in exposed rats, and the increases in incidence of this lesion in 25 ppm exposed (significant, +223%) and 5 ppm exposed (+77%) rats. These lesions varied in severity from minimal to severe. Animals with minimal to slight degenerative changes had scattered seminiferous tubules which had undergone degeneration. Severely affected animals had tubules which were lined by Sertoli cells and which had a general absence of germinal epithelium and no evidence of spermatogenesis. Their testes were ususally smaller in size and had generalized involvement of most seminiferous tubules. Animals with moderate to severe bilateral testicular atrophy had little evidence of active spermatogenesis which was reflected by a significant increase by +307% in the incidence of bilateral hypospermia observed in the epididymis of 25 ppm exposed rats.
- Kidney: relatively severe chronic nephropathy was noted in both the control and nitrobenzene exposed male CD rats in this study, with only a slight increase in severity of this change noted in 25 ppm exposed animals. However, the incidence of secondary lesions associated with severe nephropathy were increased in 25 ppm exposed animals (+6%), suggesting a true compound-related effect. These changes included an increase in the severity grade of parathyroid hyperplasia, and increases in the incidence of fibrous osteodystrophy and various soft tissue mineralizations.
- Nose: changes in the nose at final sacrifice consisted of inflammation in the anterior nasal passages. These lesions consisted of increases in severity and the incidence of suppurative exudate (1 ppm: +56%; 5 ppm: +33%; 25 ppm: +126%), subacute inflammation (1 ppm: +12%; 5 ppm: +8%; 25 ppm: +38%), and mucosal epithelial hyperplasia (1 ppm: +270%; 5 ppm: +195%; 25 ppm: +288%) in exposed as compared to control rats. A significant increase in the incidence and severity of brown pigment in the submucosa of the olfactory region in section 3 of 5 and 25 ppm exposed rats (+43%). The severity of this lesion was also greater in level 4 of rats in these exposure groups.

Various other proliferative, inflammatory, and degenerative changes were noted which were not deemed to be exposure concentration related . The incidence, appearance, and distribution of these lesions were similar to those known to occur as spontaneous lesions of aging male CD rats (Lang, 1992).

Effect levels

open allclose all
Dose descriptor:
Effect level:
0.005 mg/L air (nominal)
Basis for effect level:
other: methemoglobin formation, non-neoplastic lesions e.g. chronic toxic liver damage (centrilobular hypertrophy)
Remarks on result:
other: Effect type: toxicity (migrated information)
Dose descriptor:
Effect level:
0.005 mg/L air (nominal)
Basis for effect level:
other: no significant tumour response
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

Any other information on results incl. tables

Mean body weight:

Day 0 Weeks 1-15 Weeks 16-30 Weeks 31-60 Weeks>60
control 322.0 444.4 590.0 670.1 710.1
1 ppm 321.4 447.2 588.1 686.3 722.2
5 ppm 324.7 442.8 585.8 662.2 714.2
25 ppm 323.0 435.6 563.4 656.6* 701.9


Organ weights:

control 1 ppm 5 ppm 25 ppm
Interim sacrifice Organ weight (g) Spleen - - - -
Liver 19.5 ± 1.3 18.6 ± 1.6 19.8 ± 1.2 21.6 ± 0.8
Brain 2.2 ± 0.04 2.3 ± 0.04 2.2 ± 0.03 2.2 ± 0.05
Kidney 4.0 ± 0.2 3.8 ± 0.2 4.8 ± 0.6 4.3 ± 0.07
Relative organ weights (%) Spleen
Liver 2.7 2.8 3 3.2*
Brain 0.3 0.4 0.4 0.3
Kidney 0.6 0.6 0.7 0.8
Final sacrifice Organ weight (g) Spleen 1.4 ± 0.8 1.4 ± 0.1 1.4 ± 0.1 1.7 ± 0.2
Liver 19.5 ± 0.8 18.4 ± 0.8 17.8 ± 0.7 20.1 ± 1.1
Brain 2.2 ± 0.02 2.2 ± 0.03 2.3 ± 0.02 2.1 ± 0.03
Kidney 6.0 ± 0.5 5.7 ± 0.4 4.9 ± 0.2 5.1 ± 0.3
Relative organ weights (%) Spleen 0.2 0.2 0.2 0.3
Liver 2.9 2.8 2.8 3.2
Brain 0.3 0.3 0.4 0.4
Kidney 0.9 0.9 0.8 0.8


Histopathological findings:

Tissue control 1 ppm 5 ppm 25 ppm
Spleen extramedullary hematopoesis 58/63 56/67 61/69 60/65
pigmentation 59/63 58/67 67/69 65/65
congestion 25/63 43/67 44/69 38/65
Liver hepatocellular carcinoma 2/63 0/67 2/70 2/65
hepatocellular carcinoma, multiple - - - 1/65
hepatocellular adenoma 1/63 1/67 2/70 7/65
hepatocellular adenoma, multiple - - - 2/65
centrilobular hypertrophy 3/63 1/67 14/70 39/65
Kupffer cell pigmentation - 6/67 10/70 42/65
eosinophile cell foci 11/63 3/67 8/70 19/65
spongiosis hepatis 25/63 25/67 25/70 37/65
Testes unilateral atrophy 6/62 9/66 10/70 5/61
bilateral atrophy 11/62 17/66 22/70 35/61
interstitial cell tumor 3/62 6/66 7/70 4/61
Epididymis unilateral hypospermia 5/60 6/65 5/67 2/59
bilateral hypospermia 8/60 13/65 15/67 32/59
Kidney chronic nephropathy 54/63 60/67 63/70 59/65
tubular hyperplasia 3/63 1/67 5/70 6/65
tubular adenoma 2/63 0/67 2/70 0/65
tubular carcinoma 0/63 1/67 0/70 0/65
tubular adenoma or carcinoma 2/63 1/67 2/70 0/65
Thyroid follicular cell adenoma 2/63 4/64 2/68 3/64
follicular cell adenocarcinoma 4/63 1/64 1/68 2/64
follicular cell adenoma or adenocarcinoma 5/63 5/64 3/68 5/64
Parathyroid hyperplasia 13/61 9/64 14/68 19/63
Stomach glandular mucosa: mineralization 5/63 4/23 7/22 12/65
Pancreas Artery: mineralization 3/61 1/12 0/6 8/63
Aorta mineralization 4/63 3/3 3/3 12/65
Tongue Artery: mineralization 3/62 - - 9/64
Lungs alveolar mineralization 3/63 4/54 5/52 7/65
Thyroid follicular cell hyperplasia 2/63 2/64 1/68 4/64
Nose pigment deposition, olfactory epithelium 42/63 49/64 60/66 58/61
Multiple mononuclear cell leukemia 1/63 4/67 0/70 0/65

Applicant's summary and conclusion