3-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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

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:

3-nitrotoluene

EC Number:

202-728-6

EC Name:

3-nitrotoluene

Cas Number:

99-08-1

Molecular formula:

C7H7NO2

IUPAC Name:

3-nitrotoluene

Details on test material:

- Name of test material (as cited in study report): m-nitrotoluene
- Analytical purity: >96%
- Impurities: < 1% (mostly o- and p-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: Eastman Kodak Co. (Rochester, NY, USA)

Test animals

Species:

rat

Strain:

other: F344/N

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: 119-125 (male), 95-98 (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 m-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)

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:

MORTALITY: 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.

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 after exposure to m-nitrotoluene.
Body weight gains of rats receiving m-nitrotoluene were reduced in groups given diet containing 5000 or 10000 ppm. Feed consumption was less in the groups receiving the 10000 ppm of the m-nitrotoluene compared to controls. There were no other clinical signs of toxicity attributed to m-nitrotoluene.


One week of treatment with m-nitrotoluene produced mild increases in erythrocyte count, HGB concentration (males only), and HCT in male and female rats in the highest dose group.
Other relevant findings were mild decreases in reticulocyte and platelet counts and activity of AP and mild increases in concentrations of UN, creatinine, and albumin.
After 3 weeks of treatment with m-nitrotoluene, erythrocyte count, HGB concentration, and HCT were decreased in male rats in most dose groups and in female rats in the highest dose group.
Increases in reticulocyte, nucleated erythrocyte, and platelet (males only) counts and methemoglobin concentrations also were present in the highest dose groups of both sexes. Mild increases in lymphocyte counts were seen in high dose male and female rats; high dose females had increases in leukocyte counts. Serum biochemical effects in male rats consisted of minimal decreases in concentrations of UN; increases in concentrations of creatinine were noted in male and female rats. Additionally, a mild increase in activity of ALT occurred in female rats in the 3 highest dose groups.
At the end of 13 weeks, erythrocyte counts, HGB concentration, and HCT were decreased in female rats in the highest 1 or 2 dose groups; in male rats, only erythrocyte counts were decreased in the highest treatment group. Increases occurred in MCV, MCH, reticulocyte and platelet counts, and methemoglobin concentrations in rats of both sexes in high dose groups (5000 and 10000 ppm). Biochemical changes in serum were limited to mild to moderate increases in bile acid concentrations in male and female rats in the 5000 and/or 10000 ppm groups.
At necropsy, the only treatment-related gross lesions were seen in the 10000 ppm group of male rats. In 4/10 rats, the testes and epididymes were smaller than in controls. Relative liver weights were moderately increased in males and females in the highest dose group, and relative kidney weights were increased in the top 2 dose groups in both sexes. The relative testis weight was substantially less than controls in the 10000 ppm group.
There was a treatment-related hyaline droplet nephropathy in the kidney of male rats. This was characterized by the presence of eosinophilic protein droplets in the renal tubular epithelium and tubule lumen. The droplets were irregular-shaped and increased in size and number as compared to the protein "resorption droplets" typically present in the kidney of control male rats.
Hyaline droplet nephropathy was graded a minimal severity in all dosed groups, but the number of protein droplets increased with dose. Necrosis and increased regeneration of the renal tubular epithelium, granular casts, and focal mineralization were not associated with the hyaline droplet nephropathy in this study. A no-effect-level for the hyaline droplet nephropathy was not achieved.
An increase in hemosiderin pigment and congestion of the spleen was observed in treated male and female rats when compared to controls; both were of minimal to mild severity.
Congestion was diagnosed when the vascular spaces of the red pulp were distended with erythrocytes; increased hemosiderin pigment was diagnosed when the brown (iron-positive) pigment in macrophages of the red pulp exceeded the amount normally seen in the spleen of control rats.
All male rats from the 10000 ppm group displayed mild to moderate degeneration of the testis, characterized by a reduction of germ cells and mature spermatids in the seminiferous tubules; cellular debris was present in the ducts of the epididymis. This testicular lesion was accompanied by a reduction in epididymal sperm count and concentration. Among females there was a dose-related decrease in the length of estrous and an increase in the period of diestrus. The length of the estrous cycle increased while the number of cycling animals diminished. There were no gross or histopathologic effects on the uterus or ovaries in this 13-week study.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Weight gain, Feed and Compound Consumption of Male and Female F344/N Rats in the 13 Week Dosed Feed Studies on m-Nitrotoluene

 

		Mean BodyWeights(grams)
Nominal dose in feed (ppm)	Survival	Initial	Final	Change	Final weight relative to control (%)	Feedconsumption(g/day)	Estimated chemical consumed (mg/kgbw/d)
Female
0	10/10	98	194	96		10.8	0
625	10/10	95	199	104	103	10.7	48
1250	10/10	96	194	98	100	10.3	87
2500	10/10	94	195	101	101	10.2	172
5000	10/10	95	177	82	91	9.4	336
10000	10/10	97	166	69	86	8.4	638
Male
0	10/10	119	346	227		16.3	0
625	10/10	123	354	231	103	16.5	46
1250	10/10	119	342	223	99	16.1	86
2500	10/10	123	353	230	102	16.3	171
5000	10/10	125	338	213	98	15.8	342
10000	10/10	123	281	158	81	13.4	661

 

Table 2: Lesions in F344/N Rats Receiving m-Nitrotoluene for 13 Weeks

Male F344 rats
Dose (ppm)		0	625	1250	2500	5000	1000
No. of animals / dose group		10	10	10	10	10	10
Organ	Finding	Incidence
Kidney	neuropathy,hyalinedroplets	0	9  (1.0)	10 (1.0)	10 (1.0)	10 (1.0)	10 (1.0)
Spleen	hemosiderin pigment	0	1 (1.0)	0	2 (1.0)	5 (1.0)	10 (1.4)
	congestion	1 (1.0)	0	0	1 (1.0)	0	9 (1.0)
	Female F344 rats
Spleen	Hemosiderin pigment	1 (1.0)	9 (1.1)	10 (1.1)	10 (1.2)	8 (1.5)	10 (1.2)
	Congestion	0	0	0	0	2 (1.0)	9 (1.0)

Incidence and severity score ( ) based on a scale of 1 to 4; 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. Severity scores are averages based on the number of animals with lesions from groups of 10