4-nitrotoluene

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

other information

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

(no post exposure period, max humidity > 70%, max Temp > 25°C, limited chemical chemistry, not all recommended organs were weighed, no post exposure group)

GLP compliance:

yes

Remarks:

FDA Good Laboratory Practices regulation (21 CFR 58)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-nitrotoluene

EC Number:

202-808-0

EC Name:

4-nitrotoluene

Cas Number:

99-99-0

Molecular formula:

C7H7NO2

IUPAC Name:

1-methyl-4-nitrobenzene

Constituent 2

Reference substance name:

2-nitrotoluene

EC Number:

201-853-3

EC Name:

2-nitrotoluene

Cas Number:

88-72-2

Molecular formula:

C7H7NO2

IUPAC Name:

1-methyl-2-nitrobenzene

Details on test material:

- Name of test material (as cited in study report): p-nitrotoluene
- Analytical purity: >96%
- Impurities: < 1% (mostly m- and o-nitrotoluene)
- Storage: room temperature, protected from light
- Stability: reanalysis performed at approx. 4 months intervals indicated that the test substance was stable under the storage conditions chosen
- Other:-source: Aldrich Chemical Co. (Milwaukee, WI, USA)

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY, USA)
- Age at study initiation: 6-8 weeks of age
- Mean weight range at study initiation: 20.7-21.6 (male), 17.9-18.1 (female)
- Housing: 5/cage (polycarbonate cages)
- Diet: Control groups received NIH-07 Open formula feed (Zeigler Brothers, Inc., Gardners, PA, USA) ad libitum; treated groups received NIH-07 Open formula feed mixed with the appropriate concentration of o-nitrotoluene, ad libitum.
- Water: ad libitum
- Acclimation period: 10-15 days
- Other: 5 viral screens performed at the study start and termination indicated no positive antibody titer


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 - 26.1
- Humidity (%): 32-90%
- Air changes (per hr): 16-29
- Photoperiod (hrs dark / hrs light): 12 hrs dark /12 light

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 625, 1250, 2500, 5000, or 10000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based on the results of a 14-day preliminary study
- Rationale for animal assignment: Animals were weighed and randomized using a computer program
- Post-exposure period: no

Examinations

Observations and examinations performed and frequency:

CMORTALITY: Yes
Time schedule for check: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week

BODY WEIGHT: Yes
- Time schedule for examinations: recorded at study start and weekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for determination: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood and serum samples were collected from the retroorbital sinus using heparinezed microcapillary tube, after 1 week, 3 weeks, and at the end of the 13-week studies
- Anaesthetic used for blood collection: Yes (70% CO2/30% O2)
- Animals fasted: No
- Parameters checked: For hematologic analyses, samples were collected in plastic tubes containing potassium EDTA. Automated analyses were performed using a Coulter S-Plus IV (Hialeah, FL) and included erythrocyte, leukocyte, and platelet counts, hematocrit (HCT), hemoglobin (HGB) concentration, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). Leukocyte differentials and morphologic evaluations of blood cells were determined from blood smears stained with Wright´s stain. Reticulocytes were stained by mixing equal amounts of blood and new methylene blue and incubating the preparation for 20 minutes. Smears made from these preparations were examined microscopically for determination of reticulocyte counts.
Methemoglobin concentrations were measured using a Co-Oximeter 482 (Instrumentation
Laboratories, Lexington MA).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 1 week, 3 weeks, and at the end of the 13-week studies.
- Parameters checked: alanine aminotransferase (ALT), alkaline phosphatase (AP), creatine kinase (CK), and concentrations of total protein, albumin, urea nitrogen (UN), and creatinine, SDH and concentrations of total bile acids

REPRODUCTIVE SYSTEM EVALUATIONS: Yes
- Time schedule for examinations: For the 12 days prior to sacrifice, females were subject to vaginal lavage with saline. The relative preponderance of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were used to identify the stages of the estrual cycle. Sperm motility was evaluated at necropsy
- Test groups examined: 0, 2500, 5000, and 10000 ppm dose groups
- Parameters examined: Sperm morphology and vaginal cytology (SMVC), sperm motility, spermatid head count
- Methods: as described by Morrissey et al. (1988) Fundam. Appl. Toxicol. 11, 343-358

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Time schedule: at conclusion of the feeding phase
- Number of animals: complete necropsies were performed on all (surviving) animals. Organs and tissues were examined for gross lesions
- Organs weighed: heart, right kidney, liver, lung, right testis, and thymus

HISTOPATHOLOGY: Yes
- Complete necropsy performed on all animals. Protocol-required tissues examined in all control animals, all early death animals, and all animals in the highest dose group with 60% survivors.
- Tissues examined: gross lesions, tissue masses or suspect tumors and regional lymph nodes, skin, mandibular and mesenteric lymph nodes, mammary glands with adjacent skin, salivary glands, thigh muscle, ileum, colon, cecum, rectum, liver, femur (to include diaphysis with marrow cavity and epiphysis), thymus, trachea, lungs and bronchi, heart, thyroid, parathyroids, esophagus, stomach, duodenum, jejunum, pancreas, spleen, kidneys, adrenal glands, urinary bladder, seminal vesicles, prostate, testes, epididymides, ovaries, uterus, nasal cavity and nasal turbinates, brain with stem, pituitary, preputial or clitoral glands.

Other examinations:

The kidneys of male rats were evaluated for alpha-2u globulin accumulation. To determine total protein and alpha-2u-globulin in the kidney, the entire right kidney from each male rat was homogenized with twice the organ volume of buffered saline (pH 7.2) at 4°C then stored at -20°C until analysis. At that time, kidney homogenates were thawed, then centrifuged at 2000 RPM for 10 minutes. Total protein content in the supernatant was determined by the bicinchoninic acid assay (Kit No. BCA-1, Sigma, St. Louis, MO, USA; Smith et al., 1985. Anal. Biochm. 150, 76-85).
The amount of alpha -2u-globulin in the supernatant was determined by an enzyme-linked immunosorbant assay (ELISA) as described by Charbonneau et al. (1987) Toxicol. Appl. Pharmacol. 91, 171-181.
The standard alpha -2uglobulin and the antibody (a mouse immunoglobulin G raised toward rat alpha -2u-globulin) for ELISA were provided by Dr. S. Borghoff (Chemical Industry Institute of Toxicology, Research Triangle Park, NC, USA). The second antibody (anti-mouse IgG), conjugated with alkaline phosphatase, was obtained from Sigma Co. (St. Louis, MO; USA). Results were expressed as the ratio of alpha-2u-globulin to total protein in the supernatant. These assays were run parallel with the alpha -2u-globulin assays previously reported for p-chloro- alpha-¿alpha- alpha -trifluorotoluene (NTP, 1991.Toxicity Report Series No. 14. Research Triangle Park, NC: National Toxicology Program.

Statistics:

See (any other informations on materials and methods incl. tables)

Results and discussion

Results of examinations

Details on results:

All animals survived to the end of the studies. Body weight gains of dosed male and female mice were decreased relative to controls in the 2 highest dose groups with p-nitrotoluene feed consumption also was decreased in these groups. There were no clinical signs attributed to administration of the nitrotoluenes.

At necropsy, relative liver weights showed dose-related increases in all groups of males and females. There were no gross or microscopic treatment-related lesions seen in either males or females given p-nitrotoluene. p-Nitrotoluene had no adverse effects on measured reproductive parameters

Target system / organ toxicity

Critical effects observed:

not specified