Nitrotoluene

Endpoint summary

Description of key information

There is no repeated dose toxicity study available for nitrotoluene (CAS 1321-12-6), but a read-across can be made from 4-nitrotoluene (CAS 99-99-0).

In a 13-week feeding study with rats, similar to OECD TG 408 and compliant to GLP, 4-nitrotoluene caused hematopoiesis and hemosiderin pigment accumulation in the spleen of both sexes at all dose levels tested. Methemoglobinemia was noted at study end in the 13 week study at 10,000 ppm (male: approximately 723 mg/kg bw/day, female: approximately 680 mg/kg bw/day). At high and systemically toxic exposure levels, testicular degeneration was found in the males, and lengthened estrous cycles in the females. In male rats, alpha-2u-globulin nephropathy was observed in all dosed groups. This effect is species specific and therefore of no relevance for humans.

A NOAEL for systemic toxicity could not be derived. A LOAEL of 625 ppm (corresponding to 42 mg/kg bw/day in males and 44 mg/kg bw/day in females) was determined based on effects on the spleen.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

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

Specific details on test material used for the study:

- 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)

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: 101 - 113g (male), 88 - 93g (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

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Dose / conc.:

625 ppm

Remarks:

nominal in diet; (male: 42 mg/kg bw/d; female: 44 mg/kg bw/d)

Dose / conc.:

1 250 ppm

Remarks:

nominal in diet; (male: 82 mg/kg bw/d; female: 82 mg/kg bw/d)

Dose / conc.:

2 500 ppm

Remarks:

nominal in diet; (male: 165 mg/kg bw/d; female: 164 mg/kg bw/d)

Dose / conc.:

5 000 ppm

Remarks:

nominal in diet; (male: 342 mg/kg bw/d; female: 335 mg/kg bw/d)

Dose / conc.:

10 000 ppm

Remarks:

nominal in diet; (male: 723 mg/kg bw/d; female: 680 mg/kg bw/d)

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

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.

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)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were related to treatment.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the end of the studies with the p-nitrotoluene.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gains of rats receiving p-nitrotoluene were reduced in groups given diet containing 5000 or 10000 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption was less in the groups receiving the 10000 ppm of the nitrotoluenes compared to controls, and this effect was most evident in male rats receiving feed containing p-nitrotoluene. There were no other clinical signs of toxicity attributed to the nitrotoluenes.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

One week of treatment with p-nitrotoluene produced mild increases in erythrocyte count, HGB concentration, and HCT in male and female rats in the 5000 and/or 10000 ppm dose groups.
Reticulocyte counts were decreased in rats in the highest dose groups of both sexes; platelet counts were decreased in the 3 highest dose groups of female rats; methaemoglobin concentrations were not affected.
After 3 weeks of treatment, there were mild decreases in erythrocyte counts in male rats and in HGB concentrations in female rats, both in high dose groups. In male rats there were increases in MCV and MCH and, in animals of both sexes, in counts of nucleated erythrocytes.
At the end of 13 weeks, erythrocyte count, HGB concentration, and HCT (females only) were decreased in rats in multiple dose groups. Additionally, there were increases in MCV, reticulocyte count (female rats only), methaemoglobin concentration, and numbers of nucleated erythrocytes.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

After one week of treatment with p-nitrotoluene , the activity of ALT in serum was increased in female rats in the 2 highest dose groups.
After 3 weeks of treatment, biochemical findings were limited to animals in the top dose groups and included mild increases in activity of ALT in male and female rats and in concentrations of bile acids in female rats.
At the end of 13 weeks, there were minimal increases in serum UN and creatinine and mild increases in concentrations of bile acids in male rats in the 10000 ppm dose group. Total protein concentrations decreased in male and female rats in multiple dose groups.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At necropsy, the relative liver and kidney weights of males at the 2 highest doses, and females at the highest dose, were mildly increased compared to controls. Unlike with the o- and m-nitrotoluenes, relative testis weights were unchanged in dosed animals in the p-nitrotoluene studies. Potential treatment-related gross lesions were limited to 2 males from the 10000 ppm exposure group which had small testes.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, treatment-related lesions were found in the kidney, spleen, and testis of rats.
In male rats, there was a dose-related increase in hyaline droplet nephropathy, karyomegaly, and pigment in the kidney. As described in previous sections, hyaline droplet nephropathy was characterized by the presence of an increase in the number and size of abnormally shaped eosinophilic protein droplets within the cytoplasm and lumen of renal tubules. These droplets stained intensely eosinophilic with the Mallory-Heidenhain stain for protein. Measurement of alpha-2u-globulin content of the kidney confirmed an increased accumulation of this protein in dosed male rats. Karyomegaly was present in the proximal tubular epithelium of dosed male and female rats. This minimal to mild nuclear enlargement was most prominent in the 5000 ppm and 10000 ppm groups. A yellow to brown pigment also was present in the cytoplasm of renal tubule cells of dosed male and female rats; this pigment, possibly lipofuscin, was PAS-positive; it did not stain with acid-fast, iron, or bile stains.
A retrospective examination was performed on kidney sections of male and female rats from all dose groups in the 14-day study of p-nitrotoluene. Hyaline droplet nephropathy was present in all dose groups of male rats but was most prominent in the 1250, 2500, and 5000 ppm groups.
The reason for the decreased prominence of hyaline droplets in the 10000 and the 20000 ppm groups relative to that seen in the lower doses could not be determined. It is possible the marked decreased body weight gain or actual body weight loss which occurred during 14-day study in these 2 high-dose groups may have resulted in decreased production of alpha-2u globulin by the liver and a reduction in the amount available for resorption by the kidney. There was no evidence of necrosis and regeneration of tubular epithelium; granular casts or focal mineralization were not present in the 14-day study. Karyomegaly and pigment observed in the 13-week study were not present in the kidney of male or female rats from the 14-day study.
Also, in the 13-week study for male and female rats, there was an increase in hematopoiesis, hemosiderin pigment, and congestion compared to that which is typically seen in the spleen of control rats. Degeneration of the testis (minimal to mild) was seen in high-dose male rats; this was characterized by the absence of spermatogenesis, decreased number of germinal epithelial cells, and the presence of syncytial giant cells (degenerate spermatids) in a few seminiferous tubules, usually at the periphery of the testis. Epididymal sperm concentration and testicular spermatid head count were reduced in high dose males but to a lesser extent than with the o- or m-nitrotoluenes. Among females, 9/10 in the 10000 ppm group did not have a discernible estrous cycle. There were no gross or histopathologic changes in the uterus or ovaries at the end of this 13-week study.

Dose descriptor:

LOAEL

Effect level:

42 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

42 mg/kg bw/day (actual dose received)

System:

other: spleen, testes, liver

Organ:

liver

spleen

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

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

 

		Mean Body Weights (grams)
Nominal dose in feed (ppm)	Survival	Initial	Final	Change	Final weight relative to control (%)	Feed consumption (g/day)	Estimated chemical consumed (mg/kgbw/d)
Male
0	10/10	136	350	214		16.4	0
625	10/10	145	349	204	100	15.9	42
1250	10/10	130	342	212	98	15.6	82
2500	10/10	133	340	207	97	15.8	165
5000	10/10	137	315	178	90	15.4	342
10000	10/10	138	253	115	72	14.1	723
Female
0	10/10	110	199	89		10.9	0
625	10/10	108	199	91	100	10.6	44
1250	10/10	116	200	84	101	10.7	82
2500	10/10	111	193	82	97	10.5	164
5000	10/10	106	182	76	91	10.0	335
10000	10/10	109	173	64	87	9.7	680

 

Table 2: Lesions in F344/N Rats receiving of p-Nitrotoluene for 13 Weeks

Male F344 rats
Dose (ppm)		0	625	1250	2500	5000	10000
No. of animals / dose group		10	10	10	10	10	10
Organ	Finding	Incidence
Kidney	nephropathy, hyaline droplet	0	10 (1.0)	10 (1.0)	10 (1.0)	10 (2.0)	10 (2.0)
	kariomegaly	0	0	3 (1.0)	5 (1.0)	10 (2.0)	10 (2.0
	pigment	0	0	0	0	0	10 (1.0)
Spleen	Hematopoiesis	0	6 (1.0)	9 (1.0)	10 (1.1)	10 (1.2)	10 (2.2)
	Hemosiderin pigment	0	6 (1.0)	8 (1.0)	10 (1.1)	9 (1.3)	10 (2.4)
	congestion	0	8 (1.0)	10 (1.0)	9 (1.0)	10 (1.0)	10 (2.0)
Testis	Degeneration	0	0	1 (2.0)	0	1 (2.0)	4 (1.80)
-
	Female F344 rats
Kidney	Kariomegaly	0	10 (1.0)	10 (1.0)	10 (2.0)	10 (2.0)	10 (2.0)
	Pigment	0	10 (1.0)	10 (1.0)	10 (1.0)	10 (2.0)	10 (2.0)
Spleen	Hematopoiesis	0	4 (1.0)	4 (1.7)	5 (1.2)	9 (1.2)	10 (1.8)
	Hemosiderin pigment	0	5(1.0)	6 (1.7)	10 (1.6)	10 (1.9)	10 (2.0)
	CapsularFibrosis	0	4 (1.0)	6 (1.0)	10 (1.0)	10 (1.2)	10 (2.0)

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

 

Conclusions:

In the 13 week feeding study with rats, 4-nitrotoluene caused hematopoiesis and hemosiderin pigment accumulation in the spleen of both sexes at all dose levels tested. Methemoglobinemia was noted at study end in the 13 week study at 10,000 ppm (male: approximately 723 mg/kg bw/day, female: approximately 680 mg/kg bw/day). At high and systemically toxic exposure levels, testicular degeneration was found in the males, and lengthened estrous cycles in the females. In male rats, alpha-2u-globulin nephropathy was observed in all dosed groups. This effect is species specific and therefore of no relevance for humans.
A NOAEL for systemic toxicity could not be derived. A LOAEL of 625 ppm (corresponding to 42 mg/kg bw/day in males and 44 mg/kg bw/day in females) was determined based on effects on the spleen.

Executive summary:

In the 13-week feeding studies with rats, there were no effects on survival and decreased body weights were only observed at the higher dose levels ( ≥ 5,000 ppm, corresponding to 342 mg/kg bw/day). In male rats, alpha-2u-globulin nephropathy was seen in all dosed groups, i.e. at ≥ 625 ppm, corresponding to 42 mg/kg bw/day. This type of nephropathy is species and gender specific and therefore not of

relevance for humans.

In the spleen, most of exposed male and female rats had increases in the incidences of hematopoiesis, hemosiderin deposition and/or congestions at ≥ 625 ppm, corresponding to 42 mg/kg bw/day in males and 44 mg/kg bw/day in females. High, systemically toxic doses induced degeneration of the testis in males (at 10,000 ppm, corresponding to 723 mg/kg bw/day) , and an increase in the length of the estrous cycle in females (at 10,000 ppm, corresponding to 680 mg/kg bw/day).

Thus, a NOAEL for systemic toxicity could not be derived, The LOAEL is 625 ppm (corresponding to 42 mg/kg bw/day in males and 44 mg/kg bw/day in females).

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

There is no experimental test data available for nitrotoluene (CAS 1321-12-6), but a read-across can be made from its components. Nitrotoluene (CAS 1321-12-6) is a mixture of mainly 4-nitrotoluene (CAS 99-99-0) and/or 2-nitrotoluene (CAS 88-72-2). In addition, the mixture is containing small amounts of 3-nitrotoluene (CAS 99-08-1). A wealth of data is existing for the hazard assessment of 2- and 4-nitrotoluene, which can be used for the classification of nitrotoluene (CAS 1321-12-6). Key data and classification are derived from the isomer with the most critical hazard identified for each specific end point. The available experimental test data are considered reliable and suitable for the classification of nitrotoluene (CAS 1321-12-6) under Regulation 1272/2008.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

LOAEL

Effect level:

42 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

42 mg/kg bw/day (actual dose received)

System:

other: liver, spleen, testes

Organ:

liver

spleen

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

42 mg/kg bw/day

Study duration:

subchronic

Species:

rat

System:

other: spleen, kidney, testes

Organ:

kidney

spleen

testes

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Mononitrotoluene is a mixture of mainly 4-nitrotoluene (CAS 99-99-0) and/or 2 -nitrotoluene (CAS 88-72-2). In addition the mixture is containing small amounts of 3-nitrotoluene (CAS 99-08-1). A wealth of data is existing for the hazard assessment of 2- and4-nitrotoluene. Key data and classification is derived from the isomer with the most critical hazard identified for each specific end point. For completeness key data of the other isomer is added as supporting information.

Regarding repeated dose toxicity classification is driven by 4-nitrotoluene.

Conclusion:

In the OECD SIDS Initial Assessment Report the key data on repeated dose toxicity was summarized as following:

In 13 week and 2 year feeding studies with rats, 4-nitrotoluene caused hematopoiesis and hemosiderin pigment accumulation in the spleen of both sexes at all dose levels tested (NTP 1992, 2002). Methemoglobinemia was noted at study end in the 13 week study at 10,000 ppm (male: approximately 723 mg/kg bw/day, female: approximately 680 mg/kg bw/day). At high and systemically toxic exposure levels, testicular degeneration was found in the males, and lengthened estrous cycles in the females. In male rats, alpha 2µ-globulin nephropathy was observed in all dosed groups. This effect is species specific and therefore of no relevance for humans (LOAEL: 625 ppm, corresponding to approximately 42 mg/kg bw/day, based on splenic toxicity).

Qualitatively the effects observed in 13 week and 2 year feeding studies with rats and 2-nitrotoluene were similar to those observed with 4 -nitrotoluene (increase in liver weight, spleen lesions) although at somehow higher effect levels (NTP 1992, 2002).

Repeated dose toxicity of 4-nitrotoluene:

In the the 13-week feeding studies on rats with 4-nitrotoluene, which was conducted similar to OECD 408 ( no post exposure period, max humidity > 70%, max Temp > 25°C, limited chemical chemistry, not all recommended organs were weighed, no post exposure group) and GLP-compliant, there were no effects on survival and decreased body weights were only observed at the higher dose levels ( ≥ 5,000 ppm, corresponding to 342 mg/kg bw/day). In male rats, alpha-2u-globulin nephropathy was seen in all dosed groups, i.e. at ≥ 625 ppm, corresponding to 42 mg/kg bw/day. This type of nephropathy is species and gender specific and therefore not of relevance for humans. In the spleen, most of exposed male and female rats had increases in the incidences of hematopoiesis, hemosiderin deposition and/or congestions at ≥ 625 ppm, corresponding to 42 mg/kg bw/day in males and 44 mg/kg bw/day in females. High, systemically toxic doses induced degeneration of the testis in males (at 10,000 ppm, corresponding to 723 mg/kg bw/day) , and an increase in the length of the estrous cycle in females (at 10,000 ppm, corresponding to 680 mg/kg bw/day). Thus, a NOAEL for systemic toxicity could not be derived, The LOAEL is 625 ppm

(corresponding to 42 mg/kg bw/day in males and 44 mg/kg bw/day in females).

No relevant substance related lesions were seen in mice in 13 week feeding studies with 4 -nitrotoluene. The NOAEL based on body weight reduction was 2500 ppm (approximately 439 mg/kg bw/day).

In the 2-year combined chronic toxicity and carcinogenicity studies in rats with 4 -nitrotoluene, hematological data and clinical chemistry data were not reported and could therefore not be considered for the NOAEL or LOAEL. Alpha-2u-Globulin nephropathy was found in all exposed male dose groups, and there were increased incidences of mild hematopoietic cell proliferation and pigmentation in the spleen of exposed male and female animals at ≥ 1,250 ppm, corresponding to 55 mg/kg bw/day in males, and to 60 mg/kg bw/day in females. In livers of males and females, increased incidences of various types of altered cell foci were found at ≥ 2,500 ppm (corresponding to 110 mg/kg bw/day in males, and to 125 mg/kg bw/day in females). Testicular degeneration was seen in high-dose male rats (at 5,000 ppm, corresponding to 240 mg/kg bw/day). In the corresponding study with mice, hematological data and clinical chemistry data were also not reported and could therefore not be considered for the NOAEL or LOAEL. Male and female mice showed an increase in the incidence of alveolar bronchiolar epithelialization (without evidence of a viral infection) at ≥ 1,250 ppm (corresponding to 170 mg/kg bw/day in males and 155 mg/kg bw/day in females), and males an increase in the incidence of hepatocyte syncytial alterations at 5,000 ppm (690 mg/kg bw/day). Thus a NOAEL for systemic toxicity could not be derived neither for rats nor for mice, The LOAEL for both species is 1,250 ppm (corresponding to 55 mg/kg bw/day in male and 60 mg/kg bw/day in female rats and 170 mg/kg bw/day in male and 155 mg/kg bw/day in female mice, respectively).

Repeated dose toxicity of 2-nitrotoluene:

In a 13-week tudy performed with 2-nitrotoluene in essence according to OECD guideline 408 with deviations (no post exposure period, max humidity > 70%, max Temp > 25°C, limited chemical chemistry, not all recommended organs were weighed, no post exposure group) and GLP compliant, F344/N rats (10 animals/sex/group, 6 weeks of age) were administered 0, 625, 1250, 2500, 5000 and 10000 ppm of 2-nitrotoluene in the diet for 13 weeks after acclimation periods of 10-15 days. Toxic effects were most severe in male rats. o-nitrotoluene caused depressed body weight gain at doses of 2500 ppm and above, and toxic effects to the liver, spleen, and kidney at dietary concentrations of 1250 ppm and above. Even at the lower dose of 625 ppm, liver weights were increased. Changes in clinical chemistry parameters, indicating liver toxicity, were observed in male rats at dosage levels of 2500 ppm and above. More importantly,o-nitrotoluene caused chemically-related tumors in male rats after only 13 weeks of dosing, as demonstrated by the occurrence of mesothelial cell hyperplasia and mesothelioma. Among female rats, o-nitrotoluene caused body weight effects at dosage levels of 2500 ppm and above, and kidney and spleen lesions at levels of 2500 ppm and above.O-Nitrotoluene impaired testicular function of the rat, as shown microscopically and by measurement of sperm density, motility, and number; o-nitrotoluene also increased the length of the estrous cycle in rats. Although some of these effects may have been due in small part to decreased body weight gains, the severity of the effects indicates that the o-nitrotoluene are likely directly toxic to the reproductive system. These results show that the o-nitrotoluene has the potential to cause injury to the kidney, spleen and/or hematopoietic, and reproductive system in rodents, o-Nitrotoluene was carcinogenic in male rats in 13-week studies, based on the occurrence of mesotheliomas and mesothelial hyperplasia in dosed groups. A NOAEL of 625 ppm (corresponding to 45 mg/kg bw/day in males and 44 mg/kg bw/day in females) was determined based on the occurence of splenic capsular fibrosis seen in male rats from 1250 ppm (89 mg/kg bw/d).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. There is no repeated dose toxicity study available for nitrotoluene (CAS 1321-12-6), but a read-across can be made from 4-nitrotoluene (CAS 99-99-0). 4-nitrotoluene caused treatment-related adverse effects to testes, spleen and liver as well as Methemoglobinemia at high systemic doses.

Furthermore, 4-nitrotoluene (CAS 99-99-0) is included in Annex VI of Regulation (EC) No. 1272/2008 with the following classification:

• STOT RE Cat. 2 (H373)

Therefore, nitrotoluene (CAS 1321 -12 -6) is also classified for STOT RE Cat. 2 (H373: May cause damage to organs <testes, spleen, liver>) under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.