2-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 1

Reference Type:

publication

Title:

Unnamed

Year:

1992

Reference 2

Reference Type:

secondary source

Title:

European Union Risk Assessment Report - 2-Nitrotoluene

Author:

European Commission - European Chemicals Bureau

Year:

2008

Bibliographic source:

Office for Official Publications of the European Communities

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:

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): o-nitrotoluene
- Analytical purity: >96%
- Impurities: < 1% (mostly m- 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: 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:

MORTALITY
- All animals survived until the end of the study

BODY WEIGHT AND WEIGHT GAIN
- Body weight gains of males and females given diets containing o-nitrotoluene were reduced in a dose-related fashion (See table 1 below for figures)

FOOD CONSUMPTION AND COMPOUND INTAKE
- Feed consumption was less in the groups receiving 10000 ppm of o-nitrotoluene attaining approx. 30.9 % reduction in males compared to control and approx. 18.2% reduction in females (See table 1 for figures)

HAEMATOLOGY AND CLINICAL CHEMISTRY (percentage increase or decrease indicated below is always statistically significantly different in comparison to the control animals)
AFTER 1 WEEK
- Increases in erythrocyte (7.2%), leukocyte (lymphocyte) (35.9%), and platelet counts (14.3%), HGB (6.1%) and methemoglobin concentrations (122%) in male rats of 10000 ppm dose group; methemoglobin (56%), platelets (9.2%), and leukocytes (lymphocytes) (21%) also were increased at 5000 ppm. Reticulocyte counts were decreased in male rats in the 2 highest dose groups (21% and 26.1%, respectively). In female rats, effects were confined to increases in methemoglobin concentration (71.9%) and platelet counts (20 and 25%) in animals of the highest and 2 highest dose groups, respectively. Biochemical changes in serum at this time included dose dependent decreases in concentrations of total protein (6.1-18%) and albumin (6-18%) in male rats in all treatment groups and in female rats in the 2 highest dose groups.

AFTER 3 WEEKS
- In both sexes, mild decreases in erythrocyte count (5.0-6.8%), hemoglobin concentration (4.4-10.5%), MCHC (1.7-2.3%), and HCT (4.5-5.4%) (effects in male in the highest and in females in the 2 highest dose groups) and moderate increases in platelet (6.4-56.3%), nucleated erythrocyte (male only with a 620% increase over controls), and leukocyte (lymphocyte) counts (22.6-53.8%) and methemoglobin concentrations (61.6-496.0% in the 3 to 4 highest dose groups). Biochemical findings in serum included decreases in concentrations of total protein (4.5-21.8%) and albumin (6.3-19.6%) and activities of AP (5.1-31.6%, all dose groups of both sexes). Serum concentrations of bile acids were increased in male rats in the highest dose group (145% over controls).

AFTER 13 WEEKS
- Mild decreases in erythrocyte count (1.7-12.9%), HGB concentration (5.5-8.1%), MCHC (1.7-2.3%), and HCT (5.5-7.9%) were present in both sexes in the highest 2 or 3 dose groups. Mild increases in MCV (0.9-6.0%), MCH (1.6-2.8%), platelet (6.1%-32%), reticulocyte (39%-84%), and leukocyte (lymphocyte) counts (15.7%-68%), and methemoglobin concentrations (97%-351%) were associated with these changes in many animals of the same dose groups. In male rats, there were mild increases in concentrations of total protein (5.8% over controls) in the highest treatment group and albumin (2-10%) in all dose groups, while in female rats, total protein was slightly reduced in the 2 highest treatment groups [11.5% (5000 ppm), 8.5% (10000ppm)] and albumin was also reduced in the 2 highest treatment groups [4.5% (5000 ppm), 6.0% (10000ppm)]. In animals of both sexes, mild to moderate increases occurred in concentrations of bile acids in the 5000 ppm and 10000 ppm dose groups. Male: 217% and 285% over controls, respectively; Females: 49% and 83.4% over controls, respectively. Additionally, in male rats only, there were mild increases in activities of ALT [23.6% (10000 ppm)] and SDH [30%, 23%, and 38% (2500, 5000, and 10000 ppm)].

ORGAN WEIGHTS (percetage increase or decrease indicated below is always in comparison to the control animals)
LIVER
- Absolute liver weights were increased in a dose dependent fashion in male rats with statistical significance in the 2500, 5000 and 10000 ppm dose groups (14, 21.7 and 25.6% over controls, respectively). Relative liver weights were significantly increased at all dose groups; in males by 8.8, 15.6, 31.3, 70, 124% over controls for the 625, 1250, 2500, 5000 and 10000 ppm dose groups, respectively (p<= 0.01) and in females by 9.1, 15.8, 6.0, 20.8 and 41.6% over controls for the 625, 1250, 2500, 5000 and 10000 ppm dose groups, respectively (p<= 0.01 and 0.05).

KIDNEY
The relative weight of the kidney was increased in dosed animals of both sexes, more so in males than females. Relative kidney weights were increased (over controls) in male rats with statistical significance in the 2500, 5000 and 10000 ppm dose groups (10.2, 25.2, and 53.8% over controls, respectively). Relative kidney weights were increased (over controls) in female rats with statistical significance in the 1250, 2500, 5000 and 10000 ppm dose groups (5.9, 5.6, 14.7 and 20.1%, respectively). Absolute kidney weights were reduced in male rats in the 5000 and 10000 ppm dose groups by 9.9% and 13.5%, respectively. In female rats absolute kidney weights were significantly reduced in 3 highest doses (2500, 5000 and 10000 ppm) by 7.6, 5, and 7.5%, respectively

TESTES
Absolute testis weights at 5000 and 10000 ppm dose groups experienced a statistically significant reduction by 71.7% and 36.6%, respectively. Relative testis weight in the 10000 ppm dose group was reduced by 34.5% compared to controls (p<= 0.01). Absolute epididymal weights were markedly lower than those of controls in animals of the 2500, 5000, and 10000 ppm groups by 20, 44 and 76% , respectively.

The organ-to-body-weight ratios of several organs including heart, lungs, and thymus were higher than controls in several dosed groups, which was attributed to the lower body weights of animals in these groups, and the normally higher organ-weight-to-body-weight ratios in smaller animals.

GROSS PATHOLOGY
Chemical related gross lesions were observed in the liver, testis, and spleen. In all males from the 10000 ppm exposure group, the liver appeared larger and the testes was smaller than in controls; alterations in the color (pale, mottled focus) of the liver and testis were also observed. In 5 males from the highest exposure group, the spleen appeared darker and/or slightly thicker than in controls.

HISTOPATHOLOGY: NON-NEOPLASTIC (see table 2 for more details)
LIVER
- Hepatic lesions [cytoplasmic vacuolization (25/50 versus 0/10 in controls), oval cell hyperplasia (22/50 versus 0/10 in controls), and minimal to mild inflammation (38/50 versus 5/10 in controls) occurred only in males at dose levels of 2500 ppm and higher. The relationship of the inflammation to treatment was less clear than for the other hepatic lesions. The incidence of inflammation was slightly increased at the higher exposures, however the severity was similar among all dosed and control groups

KIDNEY
In both sexes, an accumulation of brown pigment globules in the cytoplasm of the tubular epithelium of the renal cortex was seen from 2500 ppm onwards in females (23/30 versus 0/10 in controls) and from 5000 ppm in males (11/20 versus 0/10 in controls). Hyaline droplet nephropathy was present in groups of male rats fed diets containing 1250 ppm or more o-nitrotoluene (35/40 versus 0/10 in controls). ELISA results indicated that the hyaline droplets were as a result of the accumulation of alpha-2u globulin, increased in male rats from of 2500 ppm or higher dose groups.

SPLEEN
There was a minimal to mild increase in haematopoiesis in the spleen of male rats from 2500 ppm (26/30 versus 0/10 in controls). Also, minimal to mild haemosiderin deposition (iron positive) (27/30 versus 0/10 in controls), was seen from 2500 ppm. Mild increase in methaemoglobin concentration in male and female rats in the two highest dose groups. There was a mild capsular fibrosis of the spleen in 9/10 male rats of the highest dose group. Minimal hematopoiesis in female rats in the 5000 ppm (1/10) and 10000 ppm dose groups (10/10). Hemoseridin pigmentation was increased in the 2500 ppm and above at an incidence of 24/30 versus 0/10 in controls. There was a mild capsular fibrosis of the spleen in 2/10 female rats of the highest dose group

REPRODUCTIVE ORGANS
MALES
In animals of the 5000 and 10000 ppm dose group, a decrease in epididymal sperm motility (6.5% and 73.1%, respectively versus 78% in control) and epididymal sperm concentration (179 million and 14 million, respectively versus 417 million/g caudal epididymal tissue) and in testicular spermatid count/ 1E +05 ml (70.1 and 11.9, respectively versus 91.5) was seen in rats from the 5000 and 10000 ppm groups. Testicular degeneration was seen in rats from the 5000 (10/10 animals, moderate) and 10000 ppm groups (10/10 animals, marked).
FEMALES
There were no histopathologic, treatment-related effects in the uterus or ovaries in any group. Only effect seen was an increase in estrous cycle length in the females of the highest dose group (10000 ppm, ca 6.63 days versus 4.90 in controls), with only 4/10 animals having a measurable estrous cycle

HISTOPATHOLOGY: NEOPLASTIC
Treatment-related tumours occurred in male rats as demonstrated by the occurrence of mesothelial cell hyperplasia and mesothelioma. Mesotheliomas of the tunica vaginalis were observed in 3/10 male rats at 5000 ppm, and mesothelial cell hyperplasia was observed in 2/10 male rats at 10000 ppm. Metastatic foci were not observed.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

		MeanBodyWeight (grams)
Dose (ppm) infeed	Survival	Initial	Final	Change	Final Weight Relative to Controls (%)	Average Feed Consumption	Estimated Chemical Consumed
Male B6C3F1 Mice
0	10/10	21.0	33.3	13.3		4.5	0
675	10/10	21.6	34.5	12.9	104	4.5	104
1250	10/10	21.1	32.9	11.8	99	4.7	223
2500	10/10	20.9	32.8	11.9	99	4.3	415
5000	10/10	21.1	29.2	8.1	88	3.8	773
10000	10/10	20.7	25.9	5.2	78	3.4	1536
Female B6C3F1 Mice
0	10/10	18.1	32.8	14.7		5.3	0
675	10/10	18.3	34.0	15.7	104	5.1	132
1250	10/10	17.9	33.4	15.5	101	5.2	268
2500	10/10	18.1	32.1	14.0	98	5.3	542
5000	10/10	18.1	29.5	11.4	90	4.7	1007
10000	10/10	18.1	22.9	4.8	70	3.4	1712

Table1.Survival Weight Gain, and Feed and Compound Consumption of Male B6C3F1 Mice in the 13- Week Dosed Feed Studies of o-Nitrotoluene.

	0 ppm	625 ppm	1250 ppm	2500 ppm	5000 ppm	10000 ppm
Male
Nose olfactory epithelium, degeneration/metaplasia	0	0	1 (1.0)	2 (1.0)	10 (2.0)	10 (3.0)
Female
Nose olfactory epithelium, degeneration/metaplasia	0	0	2 (1.5)	9 (1.0)	10 (1.9)	10 (2.9)

 

Table2. Lesions in B6C3F1 Mice Receiving o-Nitrotoluene for 13 Weeksa

a 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.