Nitrotoluene

Carcinogenicity

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 2-nitrotoluene (CAS 88-72-2).

In a GLP-compliant study similar to OECD TG 451, there was clear evidence of carcinogenic activity of 2-nitrotoluene in rats, based on increased incidences of malignant mesothelioma, subcutaneous skin neoplasms, mammary gland fibroadenoma and liver neoplasms in males and increased incidences of subcutaneous skin neoplasms and mammary gland fibroadenoma in females (NTP, 2002).

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to Guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Remarks:

(no differential blood counts of the highest dose and no blood smears performed)

GLP compliance:

yes

Specific details on test material used for the study:

Name of test material (as cited in study report): o-nitrotoluene
- Analytical purity: 99%
- Physical state: yellow-green liquid
- Lot: 8056-58-05RTI
- Stability: stable as a bulk chemical for at least 2 weeks when stored protected from light at temperatures up to 25° C
- Storage: the bulk chemical was stored in amber glass bottles inside metal cans at room temperature. Regular monitoring of the stability detected no degradation of the bulk chemical
- Other: Source: Aldrich Chemical Company (Milwaukee, WI, USA)

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6-7 wks
- Housing: male (3/cage), female (5/cage), uring urine collection, animals were housed singly
- Diet: ad libitum, NTP-2000 open formula meal/pelleted diet (Zeigler Brothers, Inc., Gardners, PA). Animals initially received nonirradiated feed for about 5 months and irradiated feed thereafter.
- Water: ad libitum, tap water (Birmingham, AL, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI),
- Acclimation period: 12-14 days (parasitic and gross evaluation for disease performed on randomly chosen rats (5/sex) revealed no infections)

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Method: Gas chromatography
- Number of formulations tested: 210
- Time schedule for analysis: every 8 to 12 wks
- Result: 202 of 210 possessed concentration of 90-115% of target concentration. The concentrations in animal room samples analyzed after 3 days of use in the feeders ranged from 75% to 94% of the target concentrations. The remaining 8 formulations were remixed and had concentrations within 90-115% of target concentration

Duration of treatment / exposure:

- Core study: 105 weeks
- Stop exposure study: 13 wks, then animals received normal diet from week 14 till natural death or sacrifice

Frequency of treatment:

daily

Dose / conc.:

625 ppm (nominal)

Remarks:

Core study;
approx. 25 mg/kg bw/d for males and 30 mg/kg bw/d for females

Dose / conc.:

1 250 ppm (nominal)

Remarks:

Core study;
approx. 50 mg/kg bw/d for males and 60 mg/kg bw/d for females

Dose / conc.:

2 000 ppm (nominal)

Remarks:

Core study;
approx. 90 mg/kg bw/d for males and 100 mg/kg bw/d for females

Dose / conc.:

2 000 ppm (nominal)

Remarks:

Stop exposure study;
approx. 125 mg/kg bw/d

Dose / conc.:

5 000 ppm (nominal)

Remarks:

Stop exposure study;
approx. 315 mg/kg bw/d

No. of animals per sex per dose:

CORE STUDY
- Dosed animals: 60/sex/dose
- Control: male; 60 animals + 10 animals for interim sacrifice at 13 weeks, female; 60 animals
STOP EXPOSURE STUDY
- Test substance: 60 animals + 10 animals for interim sacrifice at 13 weeks
- Control: see core study

Control animals:

yes, plain diet

Details on study design:

Dose selection rationale: The exposure concentrations were selected based on the findings from a previous 13-week studies (NTP 1992)
- Rationale for animal assignment: Animals were distributed randomly into groups of approximately equal initial mean body weights

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: every 4 wks

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed initially, during week 4, and every 4 weeks thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: every 4 weeks
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

URINALYSIS: Yes
- Time schedule for collection of urine: at 2 weeks and 3, 12, and 18 months.
- Metabolism cages used for collection of urine: Yes
- No. of animals: 5/sex/dose, by random selection
- Duration of collection: 24h urine
- Animals fasted: no data
- Parameters examined: urine volume and creatinine, o-nitrobenzoic acid, o-nitrobenzylmercapturic acid, and o-aminobenzoic acid concentrations (HPLC)

Sacrifice and pathology:

SACRIFICE
- Method: CO2 asphyxiation
- Time schedule: Ten male rats in the 0 ppm group and the 2000 and 5000 ppm stop-exposure groups were designated for interim evaluation at 3 months. The animals in the core study were sacrificed after 104 weeks

GROSS PATHOLOGY
- Gross pathology (all organs) and microscopic examinations were performed on all animals
- Organs weighed: At 3 months interim evaluation (male rats); the heart, right kidney, liver, lungs, right testis, and thymus weights were taken (organ weights were not taken at end of 2 yrs

HISTOPATHOLOGY
- Organs examined: Complete histopathology was performed on all animal. In addition to gross lesions and tissue masses, the following tissues were
examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland (except male mice), nose, ovary, pancreas, pancreatic islets, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.

Statistics:

- Probability of survival: product-limit procedure of Kaplan and Meier
- Possible dose-related effects on survival: Cox's (1972) method for testing two groups for equality and Tarone's (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided
- Neoplasm and nonneoplastic incidences prevalence: The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997), continuity-corrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are one sided. For determination of significance of low incidences, the fisher exact test was employed
- Analysis of continuous variables: variables with normal distribution (from historical data); parametric multiple comparison procedures of Dunnett (1955) and Williams for pairwise comparisons between exposed and control groups. Fisher's least significant difference test (following initial ANOVA) was used for pairwise comparisons of urinary biomakers. Average severity values were analyzed for significance with the Mann Whitney U test

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings included large subcutaneous masses in the torso, head, and appendages of exposed males and females; the numbers of these masses generally increased with exposure concentration. Males in both 2000 ppm groups and the 5000 ppm stop-exposure groups had small ears and thin tails (causes unknown). The presence of this unusual clinical finding in both stop-exposure and core study groups suggests that the initial insult occurred during the first 3 months of o-nitrotoluene exposure.

Mortality:

mortality observed, treatment-related

Description (incidence):

Survival (before end of study):

STOP EXPOSURE GROUP
- 2000 ppm: male; 11/60 survivors
- 5000 ppm: male 0/60 survivors

CORE STUDY
- 0 ppm: 39/60 and 47/60 survivors in males and females, respectively
- 625 ppm: 18/60 and 47/60 survivors in males and females, respectively
- 1250 ppm: 3/60 and 39/60 survivors in males and females, respectively
- 2000 ppm: 0/60 and 33/60 survivors in males and females, respectively

Survival of 625 ppm core study and 2000 ppm stop-exposure males and of 2000 ppm females was significantly less than that of the controls.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of all exposed groups of males except of the 625 ppm group were generally less than those of the controls throughout the study with decreases at the end of the study of 2.4%, 5.5% and 10% compared to controls for 625, 1250 and 2000 ppm, respectively. Mean body weights of 2,000 ppm females were less than those of the controls during year 2 of the study reaching 6.3% below controls. Mean body weights of males in the stop exposure group were reduced by 6% and 18 % below controls for animals in the 2000 and 5000 ppm group, respectively.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Feed consumption by exposed males and females was similar to that of the controls throughout the study.

Dietary concentrations of 625, 1250, or 2000 ppm resulted in average daily doses of approximately 25, 50, or 90 mg o-nitrotoluene/kg body weight in core study males and 30, 60, or 100 mg/kg in females. Dietary concentrations of 2,000 or 5,000 ppm in stop-exposure males resulted in average daily doses of approximately 125 or 315 mg/kg for the first 3 months of the study

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Three urinary metabolites were followed during the study as biomarkers of exposure. The ratios of o-nitrobenzoic acid to creatinine and of o-nitrobenzylmercapturic acid to creatinine determined at 2 weeks and at 3, 12, and 18 months were linearly related to exposure concentration in males and females. The ratio of o-aminobenzoic acid to creatinine was not related to exposure concentration.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Exposure to o-nitrotoluene caused increased incidences of nonneoplastic lesions of the testes (males), mammary gland (females only), liver, bone marrow, spleen, lung, and mandibular lymph node (females only) in male and female rats.

The incidences of mammary gland hyperplasia were significantly increased in 625 and 1250 ppm females.

Nonneoplastic lesions of the liver) included eosinophilic, mixed cell, and clear cell foci in exposed groups of males and females and mixed cell infiltrate in exposed males and basophilic focus in exposed females.

The incidences of alveolar/bronchiolar hyperplasia were significantly increased in most exposed groups of males and females.

The incidences of nonneoplastic lesions of the bone marrow, kidney, mediastinal and mandibular lymph nodes, nose, pancreatic islets, pituitary gland, preputial and clitoral glands, salivary gland, and spleen were increased in exposed groups of males and/or females at 3 months and/or 2 years.

Incidences of moderate hyperplasia of bone marrow cells were increased in exposed groups of males after 2 years in the stop exposure groups [37/60** (2000 ppm) and 33/60** (5000 ppm) vs 2/60 (controls) and in the core study groups [25/60** (625 ppm), 43/60** (1250 ppm) and 45/60** (2000 ppm) vs 2/60 (controls)]. Incidences of moderate hyperplasia of bone marrow cells were also increased in exposed groups of females after 2 years in the core study groups [15/60** (1250 ppm) and 24/60** (2000 ppm) vs 2/60 (controls)].

Increased incidences of mild to moderate hyperplasia of the mandibular lymph node were seen after 2 years in groups of females exposed to 2000 ppm o-nitrotoluene (15/60** vs 3/60 in controls).

At the 3 months interim evaluation, mild degeneration of the kidney, characterized by hyaline droplets was seen in 5000 ppm stop-exposure males (10/10** vs. 1/10 in control).

At 3 months, mild to moderate olfactory epithelial degeneration (atrophy, vacuolization and disruption) of the nose occurred in the nasal cavity of stop-exposure males in both 2000 ppm and 5000 ppm dose groups (4/10* and 10/10**, respectively vs 0/10 in controls).

Minimal to mild hyperplasia of the pancreatic islets occurred in 5000 ppm stop-exposure males at 3 months (8/10** vs 0/10 in controls) and 2 years (10/60 ** vs 2/60) and was characterized by a greater number and size of islets (up to 500 µm in diameter). Mild pancreatic islet pigmentation was observed at 2 years in 5000 ppm stop-exposure males (11/60** vs 1/60 in controls).

Incidences of mild to moderate cytoplasmic alteration (hypertrophy, increased eosinophilia, and cytoplasmic vacuolization) of the pituitary gland (pars distalis) were significantly increased in the 2000 ppm core study (4/60* vs 1/59 in controls) and 5000 ppm stop-exposure (42/60** vs 1/59 in controls) male groups at 2 years.

The incidences of moderate pigmentation of the spleen were increased in 2000 ppm core-study males (16/60** vs 9/60 in controls) and females (46/60* vs 36/60 in controls). Incidences of hematopoietic cell proliferation of spleen were increased in exposed groups of males at 2 years [33/60** (625 ppm), 38/60** (1250 ppm) and 47/60** (2000 ppm) vs 7/60 (controls)] and females at 2 years [38/60** (625 ppm), 48/60** (1250 ppm) and 48/60** (2000 ppm) vs 22/60 (controls)]. In males of the stop exposure group, mild splenic hematopoietic cell proliferation was also seen after 2 years with incidences of 36/60** (2000 ppm) and 35/60 (5000 ppm) vs 7/60 (controls).

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The incidences of malignant mesothelioma in male rats occurred with positive trends in both the core and stopexposure studies and were significantly greater in exposed groups than in the controls. Incidences of subcutaneous skin neoplasms (fibroma, fibrosarcoma, and lipoma) were increased in exposed groups of males, while the incidences of fibroma or fibrosarcoma (combined) were increased in exposed females. In all exposed groups of males and females except 2,000 ppm core study males, the incidences of mammary gland fibroadenoma were significantly increased. The incidences of mammary gland hyperplasia were significantly increased in 625 and 1,250 ppm females.

Increased incidences of mesothelioma, skin neoplasms, and mammary gland fibroadenoma in the stop-exposure males indicated that 3 months of dosing were sufficient to produce a carcinogenic effect.

Liver weights of 5,000 ppm stop-exposure males were significantly greater than those of the controls at 3 months. The incidences of hepatocellular adenoma in 2,000 ppm core study males and females and of hepatocellular adenoma or carcinoma (combined) in 2,000 ppm core study and 5,000 ppm stop-exposure males were significantly increased. Cholangiocarcinoma occurred in three 5,000 ppm stop-exposure males, and a single hepatocholangiocarcinoma occurred in a 625 ppm male and in a 2,000 ppm core study male. Nonneoplastic lesions of the liver included eosinophilic, mixed cell, and clear cell foci in exposed groups of males and females and mixed cell infiltrate in exposed males and basophilic focus in exposed females.

The incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased in 5,000 ppm stop-exposure males, as were alveolar/bronchiolar hyperplasia in most exposed groups of males and females.

The incidences of hematopoietic cell proliferation of the spleen and of hyperplasia of the mandibular lymph node (females) and bone marrow were increased in exposed groups of males at 3 months and/or 2 years and in exposed groups of females at 2 years.

The incidences of mononuclear cell leukemia were significantly decreased in all groups of males exposed to 1,250 ppm or greater and in all exposed groups of females; the incidence of testicular interstitial cell adenoma was significantly decreased in 5,000 ppm stopexposure males.

Dose descriptor:

LOAEL

Effect level:

<= 25 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Table 1: Summary of the main neoplastic lesions in the rat carcinogenicity study (2-year evaluation)a,b,c

 

 

	0ppm	625ppm	1250ppm	2000ppm	2000ppm (stop-exposure)	5000ppm (stop-exposure)
Male Rats
Average Daily Dose (mg/kg)	0	25	50	90	125	315
Body weights			Less than controls	Less than controls	Less than controls	Less than controls
Survival	39/60	18/60	3/60	0/60	11/60	0/60
Mesothelium Malignant Mesothelioma	2/60	20/60	29/60	44/60	44/60	54/60
Skin (Subcutaneous)
Lipoma	0/60	4/60	13/60	13/60	10/60	12/60
Fibroma	5/60	46/60	52/60	59/60	45/60	52/60
Fibrosarcoma	0/60	7/60	17/60	20/60	8/60	12/60
Fibroma or Fibrosarcoma	5/60	47/60	55/60	59/60	47/60	53/60
Mammary Gland
Fibroadenoma	0/60	7/60	10/60	2/60	13/60	20/60
Liver
Hepatocellular Adenoma	2/60	3/60	3/60	7/60	3/60	4/60
Hepatocellular Adenoma or Carcinoma	3/60	3/60	3/60	8/60	3/60	6/60
Cholangiocarcinoma	0/60	0/60	0/60	0/60	0/60	3/60
Hepatocholangiocarcinoma	0/60	1/60	0/60	1/60	0/60	0/60
Lung
Alveolar/bronchiolarAdenoma	1/60	5/60	1/60	2/60	3/60	8/60
Alveolar/bronchiolar adenoma or Carcinoma	2/60	5/60	1/60	2/60	3/60	11/60
Female Rats
Average Daily Dose (mg/kg)	0	30	60	100
Bodyweights				Lessthan controls
Survival	47/60	47/60	39/60	33/60
Skin (Subcutaneous)c
Fibroma	3/59	3/60	18/60	19/60
Fibroma or Fibrosarcoma	3/60	3/60	21/60	22/60
MammaryGland
Fibroadenoma	23/60	47/60	52/60	56/60
Liver
HepatocellularAdenoma	1/60	0/59	1/60	6/60

a NTP, 2002

b Most treated animals (all exposed groups of males and the high exposure group of females) died before getting 2 years old (in contrast to the controls). Thus, the necropsy results for the treated animals refer to the time-points when they actually died. Early deaths among treated animals were due to the development ofneoplasms. For males, malignantmesotheliomaswere the reason for early deaths.

cIncidences ofneoplasmsare given as number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for liver and lung; for other tissues, denominator is number of animalsnecropsied.

 

Table 2: Summary of the main non-neoplastic lesions in the rat carcinogenicity study (2-year evaluation)a,b,c

 

	0ppm	625ppm	1250ppm	2000ppm	2000ppm (stop-exposure)	5000ppm (stop-exposure)
Male Rats
Average Daily Dose (mg/kg)	0	25	50	90	125	315
Body weights			Less than controls	Less than controls	Less than controls	Less than controls
Survival	39/60	18/60	3/60	0/60	11/60	0/60
Liver
Eosinophilic focus	7/60	18/60	29/60	24/60	15/60	13/60
Mixed cell focus	5/60	7/60	12/60	6/60	12/60	8/60
Clear cell focus	29/60	29/60	34/60	31/60	30/60	34/60
Mixed cell cellular infiltration	1/60	5/60	11/60	20/60	15/60	33/60
Bone marrow
Hyperplasia	2/60	25/60	43/60	45/60	37/60	33/60
Spleen
Hematopoietic cell proliferation	7/60	33/60	38/60	47/60	36/60	35/60
Lung
Alveola repithelial hyperplasia	2/60	8/60	3/60	7/60	15/60	29/60
FemaleRats
Average Daily Dose (mg/kg)	0	30	60	100
Bodyweights				Lessthan controls
Survival	47/60	47/60	39/60	33/60
Mammary gland
Hyperplasia	14/60	36/60	3060	19/60
Liver
Eosinophilic focus	5/60	12/59	25/60	32/60
Mixed cell focus	6/60	9/59	11/60	28/60
Clear cell focus	16/60	30/59	28/60	33/60
Basophilic focus	51/60	56/59	60/60	54/60
Bone marrow
Hyperplasia	2/60	7/60	15/60	24/60
Spleen
Hematopoietic cell proliferation	22/60	38/59	48/60	48/59
Lung
Alveolar epithelial hyperplasia	6/60	14/60	16/60	9/60
Lymph node (mandibular)
Lymphoid hyperplasia	3/60	5/60	6/59	15/59

 

aNTP, 2002

bMost treated animals died before getting 2 years old (in contrast to the controls). Thus, the necropsy results for the

treated animals refer to the time-points when they actually died.

cIncidences of non-neoplasticlesions are given as number of non-neoplasticlesions-bearing animals/number of

animalsexamined. Denominator is number of animals examined microscopically for liver and lung; for other tissues,

denominatoris number of animalsnecropsied.

 

Conclusions:

Under the conditions of this study, there was clear evidence of carcinogenic activity of o-nitrotoluene in male rats based on increased incidences of malignant mesothelioma, subcutaneous skin neoplasms, mammary gland fibroadenoma, and liver neoplasms. The increased incidences of lung neoplasms in male rats were also considered to be exposure related.
There was clear evidence of carcinogenic activity of o-nitrotoluene in female rats based on increased incidences of subcutaneous skin neoplasms and mammary gland fibroadenoma. The increased incidence of hepatocellular adenoma in female rats was also considered to be exposure related.
Exposure to o-nitrotoluene caused increased incidences of nonneoplastic lesions of the mammary gland (females only), liver, bone marrow, spleen, lung, and mandibular lymph node (females only) in male and female rats.
Decreased incidences of mononuclear cell leukemia occurred in exposed groups of rats; the incidence of testicular interstitial cell adenoma was decreased in exposed male rats.

Executive summary:

In the core study, performed in essence according to OECD guideline 451 and GLP compliant, F344/N rats (60 animals/sex/group, 6-7 weeks old) were administered 0 (females only), 625, 1250 and 2000 ppm (25, 50 and 90 mg/kg b.w. in males; 0, 30, 60 and 100 mg/kg b.w. in females) of 2-nitrotoluene (>99% pure) in the diet for 105 weeks after acclimation periods of 12-14 days; in a 3-month stop-exposure study, groups of 70 males were fed diets containing 0, 2000 and 5000 ppm (0, 125 and 315 mg/kg b.w.) for 13 weeks followed by undosed feed for the remainder of the study, 10 males from each stop exposure and control group were sacrificed at 3 months.

All 2000 ppm core study, all 5000 ppm stopexposure and all but three core study 1250 ppm male rats died before the end of the studies.Survival of 625 ppm core study and 2000 ppm stop-exposure males and of 2000 ppm females was significantly less than that of the controls. Mean body weights of all exposed groups of males except the 625 ppm group were generally less than those of the controls throughout the study. Mean body weights of 2000 ppm females were less than those of the controls during year 2 of the study. Feed consumption by exposed groups was similar to that by the controls throughout the study. Clinical findings included large subcutaneous masses in the torso, head and appendages of exposed males and females, which increased in number with exposure; males in both 2000 ppm groups and the 5000 ppm stop-exposure had small ears and thin tails.

Mesothelium: At the 3-month interim sacrifice, no mesotheliomas or mesothelial hyperplasia were observed in exposed males. However, as time progressed, chemical-induced mesotheliomas ocurred in all exposed male groups, including the stop-exposure groups. The incidences of malignant mesothelioma were significantly greater in exposed groups than in controls and exceeded the 2-year historical control ranges. Mesotheliomas were mainly associated with the tunica vaginalis of the testis or epididymis.

Skin: Significant increases in the incidences of subcutaneous fibroma, fibrosarcoma, fibroma or fibrosarcoma (combined) and lipoma in all exposed groups of males and of fibroma and fibroma or fibrosarcoma (combined) in 1250 and 2000 ppm females. These increases occurred with positive trends and exceeded the 2-year historical control ranges.

Mammary gland: In all exposed groups of males and females except 2000 ppm core study males, the incidences of fibroadenoma were significantly greater than those in the controls and exceeded the 2-year historical control ranges. The incidences of hyperplasia, a precursor of fibroadenoma, were significantly increased in 625 and 1250 ppm females.

Liver: Liver weights of 5000 ppm stop-exposure males were significantly greater than those of the controls at 3 months. The incidences of hepatocellular adenoma in 2000 ppm core study males and females and of hepatocellular adenoma or carcinoma (combined) in 2000 ppm core study and 5000 ppm stop-exposure males were significantly greater than those in the controls and generally exceeded the 2-year historical control ranges. Cholangiocarcinoma occurred in three 5000 ppm stop-exposure males, and a single hepatocholangiocarcinoma occurred in a 625 ppm male and in a 2000 ppm core study male. In exposed groups there was an increase of several nonneoplastic lesions such as eosinophilic, mixed cell and clear cell foci in both sexes, mixed cell infiltrate in males, and basophilic focus in females.

Lung: Incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased in 5000 ppm stop-exposure males and exceeded the 2-year historical control ranges, as were alveolar epithelial hyperplasia in most exposed groups of both sexes.

Haematopoietic system: The incidences of hyperlasia in the bone marrow (all exposed males, 1250 and 2000 ppm exposed females) and of haematopoietic cell proliferation in the spleen (all exposed groups) and of hyperplasia of the mandibular lymph node (2000 ppm females) were significantly increased.

Blood: The incidences of mononuclear cell leukemia were significantly decreased in all groups of males exposed to 1250 ppm or greater and in all exposed groups of females and were less than the 2-year historical control ranges. Decreased incidences of leukemia was most likely related to the splenic toxicity.

Testis: The incidence of testicular interstitial cell adenoma was significantly decreased in 5000 ppm stop-exposure males, likely related to testicular toxicity.

Under the conditions of this study, there was clear evidence of carcinogenic activity of o-nitrotoluene in male rats based on increased incidences of malignant mesothelioma, subcutaneous skin neoplasms, mammary gland fibroadenoma, and liver neoplasms. The increased incidences of lung neoplasms in male rats were also considered to be exposure related.

There was clear evidence of carcinogenic activity of o-nitrotoluene in female rats based on increased incidences of subcutaneous skin neoplasms and mammary gland fibroadenoma. The increased incidence of hepatocellular adenoma in female rats was also considered to be exposure related.

Exposure to o-nitrotoluene caused increased incidences of nonneoplastic lesions of the mammary gland (females only), liver, bone marrow, spleen, lung, and mandibular lymph node (females only) in male and female rats.

Decreased incidences of mononuclear cell leukemia occurred in exposed groups of rats; the incidence of testicular interstitial cell adenoma was decreased in exposed male rats.

Reference 2

Endpoint:

carcinogenicity: 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:

<= 25 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

25 mg/kg bw/day

Study duration:

chronic

Species:

rat

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

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 carcinogenicity study available for nitrotoluene (CAS 1321-12-6), but a read-across can be made from 2-nitrotoluene (CAS 88-72-2).

There was clear evidence of carcinogenic activity of 2 -nitrotoluene in a carcinogenicity study in rats based on increased incidences of malignant mesothelioma, subcutaneous skin neoplasms, mammary gland fibroadenoma and liver neoplasms in males and increased incidences of subcutaneous skin neoplasms and mammary gland fibroadenoma in females.

 

Furthermore, 2-nitrotoluene (CAS 88-72-2) is included in Annex VI of Regulation (EC) No. 1272/2008 with the following classification:

• Carc. Cat. 1B (H350)

Therefore, nitrotoluene (CAS 1321 -12 -6) is also classified for Carc. Cat. 1B (H350: May cause cancer.) under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.

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- and 4-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 carcinogenicity classification is driven by 2-nitrotoluene.

Data on Carcionogenicity of 2-NT is summarised in the EU-RA:

Only a long-term feed carcinogenicity study in rodents was available (NTP, 2002), performed in essence according to OECD guideline 451 and GLP compliant, and therefore considered adequate for risk assessment. There was clear evidence of carcinogenic activity of 2-nitrotoluene in rats, based on increased incidences of malignant mesothelioma, subcutaneous skin neoplasms, mammary gland fibroadenoma and liver neoplasms in males and increased incidences of subcutaneous skin neoplasms and mammary gland fibroadenoma in females. The increased incidences of lung neoplasms in males and of hepatocellular adenoma in females were also considered to be exposure related. Malignant mesotheliomas occurred with incidences of 33%, 48% and 73% in the 625, 1250 and 2000 ppm core study male rat groups, respectively. The incidences of malignant mesotheliomas were 73% and 90% in the 2000 and 5000 ppm stop-exposure male rat groups. The incidence of mesothelioma was higher in the 2000 stop-exposure group than in the 625 ppm even though the latter group received approximately 50% more total exposure to 2-nitrotoluene. The incidences of mesotheliomas were similar in the 2000 ppm core study and stop-exposure groups of male rats. Thus, critical events leading to mesothelioma occurred early in the study, and this damage was irreversible. The molecular patogenesis of mesotheliomas is not well understood. Decreased incidences of mononuclear cell leukaemia and of testicular interstitial cell adenoma in exposed groups were related to splenic and testicular toxicity, respectively.

There was clear evidence of carcinogenic activity of 2-nitrotoluene in male and female mice based on increased incidences of hemangiosarcoma, carcinoma of the large intestine (cecum), and hepatocellular neoplasms (females only because males died early due to the development of hemangiosarcomas). The occurrence of p53 or ß-catenin mutations in 2-nitrotoluene induced hemangiosarcomas, but not in spontaneous hemangiosarcomas, suggest that the pathways leading to 2-nitrotoluene-induced cancer differ from the pathways in spontaneous hemangiosarcomas.

No 2-nitrotoluene epidemiology studies on carcinogenesis have been reported in the literature. However, according to Ward et al., 1991 (cited in NTP, 2002) excess cancers have been found in workers exposed to a related chemical, o-toluidine. In summary, there is a good evidence of an increase in tumour incidence at multiple sites in both rats and mice. There is also evidence that time to onset is very short. These observations are consistent with genotoxic aetiology, which is consistent with the findings from the genotoxicity studies.

Data on Carcionogenicity of 4-NT is summarised in the OECD-SIDS:

Under the conditions of 2-year feed studies, there was equivocal evidence of carcinogenic activity of 4-nitrotoluene in male rats based on the increased incidences of subcutaneous skin neoplasms (NTP 2002). There was some evidence of carcinogenic activity in female rats based on increased incidences of clitoral gland neoplasms. There was equivocal evidence of carcinogenic activity in male mice based on increased incidences of alveolar/bronchiolar neoplasms. There was no evidence of carcinogenic activity in female mice exposed to 1,250, 2,500, or 5,000 ppm (approximately 155, 315, or 660 mg/kg bw/day).