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Description of key information

Target organs of repeated dose toxicity in rats and mice are red blood, liver, kidney and spleen with methemogobinaemia as the most sensitive parameter. In rats exposed by inhalation, the respiratory epithelium in the nose was also a target organ. The repeated dose toxicity inhalation studies in rats and mice resulted in the LOAEC of 7 mg/m3 in rats and a NOAEL of 28.8 mg/m3 in mice. In the 2-year chronic carcinogenicity study, the lowest dose level of 4 mg/kg bw/day in rats and 11-14 mg/kg bw/day in mice was considered to be a LOAEL for toxicity. Chronic oral exposure to 2-chloronitrobenzene in feed induced hepatocellular and renal cell tumours in rats and hepatocellular tumours in mice. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Dose descriptor:
LOAEL
4 mg/kg bw/day
Study duration:
chronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Dose descriptor:
LOAEC
7 mg/m³
Study duration:
subchronic
Species:
rat

Mode of Action Analysis / Human Relevance Framework

Additional information

Inhalation

The repeated dose toxicity was examined in male and female Fischer 344/N rats and in male and female B6C3F1 mice for a period of 13 weeks via whole body inhalation of vapor (NTP 1993). During exposure rats and mice were observed twice daily and were weighed at the start of the study, weekly thereafter and at necropsy. Clinical observations were recorded weekly. After cessation of exposure, complete necropsies were performed on all animals. Histopathologic evaluations, especially on target organs identified (kidney, liver, nasal cavity, and spleen (rats); liver and spleen (mice)) and on reproductive organs were performed on all animals in the control and the highest exposure groups and on all animals that died early. Target organs identified were also examined in all lower exposure groups. Groups of 10 male and 10 female rats were exposed to 0, 1.1, 2.3, 4.5, 9, 18 ppm (approx. 0, 7, 14.7, 28.8, 57.6, or 115.2 mg/m3), 6 hours per day, 5 days per week over a period of 13 weeks. Additional 10 male and 10 female rats per group were exposed for clinical pathology studies at d 1 (only methaemoglobin - data not shown), d 4, and d 23 consisting of hematology and clinical chemistry evaluations. Animals in the base study were evaluated at the end of the study. There were no clear clinical signs of toxicity. All rats survived till the end of the study. Body weight gain was similar to the respective controls. At necropsy, males of the 18 ppm group had significant increased spleen (absol. and rel.) and from 9 ppm increased right kidney (rel.) weights. Absolute liver weights were increased from 1.1 ppm and the relative liver weight from 2.3 ppm. In males exposed to 18 ppm, abs. and rel. lung weights were significant decreased. 2/10 males in the 18 ppm group showed darkened spleen. Histopathologic evaluation of the kidney showed tubule pigments from 4.5 ppm and tubule regeneration from 1.1 ppm. In the liver, cytoplasmic basophilia was noted from 9 ppm. Spleenic congestion was observed in all exposed and in the control male rats with dose-dependent slight increase in severity. Females, at necropsy, had increased right kidney (absol. and rel.) in the 18 ppm-group and increased absolute liver weights from 2.3 ppm and increased relative liver weights from 4.5 ppm. Significant increased spleen weights (absol. and rel.) were noted from 4.5 ppm. 1/10 females in the 18 ppm group showed darkened spleen. Histopathologic evaluation yielded in the kidney tubule pigment and cytoplasmic basophilia of the liver from 9 ppm. Spleenic congestion was noted in exposed and in the control females with dose-dependent slightly increased incidences. Hyperplasia of the nasal cavity respiratory epithelium in all exposed male and female rats was considered as a toxic effect due to 1-chloro-2-nitrobenzene exposure. Concentration-related increase in methaemoglobinaemia (males: significant from 1.1 ppm at d 23 and from 2.3 ppm at all time points with max. of 1.14 g/dl at 18 ppm; females: significant from 1.1 ppm at week 13 and from 2.3 ppm at all time points with max. of 1.04 g/dl at 18 ppm; data from d1 not shown) and oxidative damage to red blood cells occurred from the first days of exposure (males: significant at 1.1 ppm (d23), at 4.5 ppm (week 13), at 9 ppm (d4, week13), at 18 ppm (at all time points) when compared to the control values at the respective time point; females: significant in every exposure group at week 13 when compared to the control values at the respective time point). Decrease in haematokrit, haemoglobin and increase in leukocytes predominantly in the highest dose groups of male and female rats was recorded. The beginning regeneration could be recognized in the increase in reticulocyte count at all dose groups of male and female rats at week 13. Serum activities of alanine aminotransferase and sorbitol dehydrogenase were mildly increased in different male and female exposure groups at various time points. A NOAEL was not achieved, the LOAEL is 1.1 ppm (7 mg/m³).

Male and female mice were exposed to 0, 1.1, 2.3 , 9, 4.5, 18 ppm, 6 hours per day, 5 days per week over a period of 13 weeks. There were no clinical signs of toxicity. 2/10 male mice exposed to 18 ppm died. In females from 1.1 ppm body weight gain was greater than in the concurrent control females; in males, body weight gain was similar to the respective control. Exposed mice had treatment-related increased liver and kidney weights (males: abs. and rel. right kidney weights, rel. liver weights sign. increased from 2.3 ppm, abs. liver weights from 9 ppm; females: abs. right kidney weight from 2.3 ppm, abs. liver weights in all exposed groups, rel. liver weight from 9ppm) . Pale discoloration in the liver was noted in 6/10 males and 1/10 females in the 18 ppm group. The spleen was grossly enlarged in 3 females in the 9 ppm group and 4 females in the 18 ppm group. Hepatocellular necrosis, cytomegaly, mineralization and chronic inflammation were seen in the liver, primarily in mice in the 18 ppm group but also in the 9 ppm-group. In addition, increased haematopoetic activity of the spleen was seen in both sexes of mice, particularily in females at 9 ppm and greater. The NOAEL for histopathologic injury is 4.5 ppm (28.8 mg/m³).

Oral

The repeated dose toxicity was also examined in a subacute feeding study with B6C3F1 mice according OECD Guideline 407 (Bayer 1991, 1993). The objective of the study was to recognize possible prae-neoplastic lesions by means of enzyme histochemistry. 12 mice/sex/dose received 0, 50, 500, 5000 ppm 1-chloro-2-nitrobenzene for 5 weeks. Additional 6 mice/sex/dose were used for interim kill and examination after one week of treatment. The calculated substance intake was 0, 16, 167, 1120 mg 1-chloro-2-nitrobenzene/kg bw/day for males and 0, 24, 220, 1310 mg/kg bw/day for females. Except of one male in the lowest dose group, no animal died during treatment. No clinical signs of toxicity up to and including 500 ppm were observed. At 5000 ppm narrowed palpebral fissures and corneal opacity in males were reported. From 5000 ppm reduced body weight gain and reduced food intake in both sexes and additionally in females from 500 ppm.

From 5000 ppm in both sexes reduced number of erythrocytes (change in morphology: anisocytosis, poikilocytosis and polychromasie), haematokrit- and haemoglobin-content and increased bilirubin-, methaemoglobin-(f: 2.8 %; m:1,7 %) MCV-, MCH- and MCHC-values. Increased spleen weights, dark red discoloration of the spleen and increased haemosiderin deposition could be seen. No treatment related changes in the kidneys were observed. From 500 ppm increase in cholesterol content in the blood, increased liver weights (differences of up to 89 % were noted in females) accompanied by hypertrophy of the centrolobular hepatocytes. From 5000 ppm gross changes in the liver, increase in the activity of ASAT and ALAT and alkaline phosphatase (male) was noted. In males, blood-urea was decreased. Additional investigations demonstrate from 500 ppm increase in liver enzyme activities (EOD, ALD, EH, GSH-T,GLU-T) and disturbance of carbohydrate metabolism (decreased gluconeogenesis and glycogen, activated pentose phosphate cycle (at 5000 ppm), increased glycolysis (at 5000 ppm)). At 5000 ppm males showed decreased testis weight without histopathological changes. No other treatment-related functional disturbances or impairment of other organs were observed. Thus, the NOAEL of 50 ppm (16 mg/kg bw/day for males and 24 mg/kg bw/day for females) could be derived.

The GLP-compliant 13 -week feeding studies were conducted according to OECD guideline 408 with rats and mice (Matsumoto, 2006). Groups of 10 male and female F344 /Crj rats and Crj:BDF1 mice received the test substance in dose levels of 63, 250, 10 00, 2000 and 4000 ppm (rats) and 78, 313, 1250, 2500 and 5000 ppm (mice) in feed for 13 weeks. Blood for hematological and clinical chemistry examinations was collected from all surviving animals before termination after overnight fasting. All animals were subjected to gross pathological and histopathological examinations.

In the rat study, the average daily intakes were 3.5, 13.8, 57.5, 119.5 and 234.1 mg/kg bw/day for males and 4.0, 15.5, 63.9, 133.3 and 249.3 mg/kg bw/day in females. The terminal body weights of the highest dose treated male and female rats were significantly decreased by 10% or more, compared with the respective controls. The most sensitive, significant hematological change was a decrease in RBC and hemoglobin for the female rats fed at the lowest dose level. Serum total bilirubin was significantly increased in the male rats fed 1000 ppm and above and in female rats fed 2000 and 4000 ppm. AST was significantly increased from 2000 ppm in male rats and at 4000 ppm in female rats. ALT was significantly increased from 1000 ppm in male rats and from 2000 ppm in female rats.

The relative spleen and liver weights of the rats of both sexes were linearly increased with an increase in dietary concentration of o-CNB.

A significantly increased incidence of erythropoiesis in the bone marrow was noted in the male and female rats fed 2000 and 4000 ppm. The incidence of congestion and hemosiderin deposition in the spleen was significantly increased in the male and female rats fed 250 ppm and above, and the incidence of increased extramedullary hematopoiesis in the spleen was increased in the male and female rats fed 1000 ppm and above. Capsule hyperplasia was observed in the male and female rats fed 2000 and 4000 ppm o-CNB. The affected capsule was characterized by focal or multifocal fibrous thickening, outward expansion of the capsule and the incorporation of hematopoietic cells.

The incidence of hemosiderin deposition in Kupffer's cells was significantly increased in the male and female rats fed 1000 ppm and above.

A significantly increased incidence of centrilobular hypertrophy of hepatocytes was noted in the male rats fed 2000 and 4000 ppm and in female rats fed 4000 ppm. The incidence of single cell necrosis was significantly increased in the male and female rats fed 1000 ppm and above. The incidence of hydropic degeneration of the centrilobular hepatocytes, which was characterized by the ballooning of hepatocytes enriched with watery materials, was significantly increased in male rats fed 1000 ppm and above and female rats fed 2000 and 4000 ppm.

Based on the decrease in RBC and hemoglobin for the female rats fed at the lowest dose level, the lowest dose of 4 mg/kg bw/day was considered to be a LOAEL for females. For males, the lowest dose level of 3.5 mg/kg bw/day was considered to be a NOAEL, based on the decreased hemoglobin, congestion of spleen and deposit of hemosiderin in spleen in males at the next dose level. The authors also used the benchmark dose approach by using all sets of the experimental data to delineate a dose-response relationship. The BMDL10 value for the hematotoxic endpoint was 1.03 mg/kg bw/day for rats.

In the mouse study, the average daily intakes were 10.4, 43.6, 170.4, 345.1 and 684.1 mg/kg bw/day for males and 12.2, 49.5, 196.5, 400.3 and 762.5 mg/kg bw/day for females. The most sensitive, significant hematological change was a decrease in MCV for the female mice fed 313 ppm.

Serum total bilirubin was significantly increased in the female mice fed 5000 ppm. ALT was significantly increased from 2500 ppm in male mice and from 1250 ppm in female mice. The relative spleen and liver weights of mice of both sexes were linearly increased with an increase in dietary concentration of o-CNB. The incidence of congestion and increased extramedullary hematopoiesis in the spleen was significantly increased in male and female mice fed 1250 ppm and above. The incidence of hemosiderin deposition in the spleen was significantly increased from 313 ppm in both sexes. The incidence of hemosiderin deposition in Kupffer's cells was significantly increased in the male and female mice fed 1250 ppm and above. A significantly increased incidence of centrilobular hypertrophy of hepatocytes was noted in male mice fed 313 ppm and above and in the female mice fed 1250 ppm and above. The incidence of nuclear enlargement with atypia of centrilobular hepatocytes, which was characterized by cell enlargement, varying nuclear size and shape and coarse chromatin in the nucleus was significantly increased in the male mice fed 313 ppm and above and in the female mice fed 1250 ppm and above.

The lowest dose level equal to 10.4 mg/kg bw/day in males and 12.2 mg/kg bw/day in females was considered to be a NOAEL, based on the increased MCV in female mice, hemosiderin deposit in spleen in both sexes, and centrilobular hypertrophy and nuclear centrilobular enlargement with atypia in the liver in male mice at the next dose level.

In addition, GLP-compliant OECD guideline 451 carcinogenicity studies with rats and mice are available, which are reported in the section on carcinogenicity. Groups of 50 male and female rats and 50 male and female mice received the test substance in diet at dose levels of 80, 400 and 2000 ppm (rats) and 100, 500 and 2500 ppm (mice) for 2 years. Animals were observed for clinical signs and mortality, changes in body weights and body weight gains and food consumption. Prior to termination, blood was collected for hematological and clinical chemistry investigation from all surviving animals after overnight fasting. Animals were sacrificed at the end of the 2 -year period and subjected to gross pathological and histopathological examinations.

In the rat study, the average daily intake of the test substance was 4, 19 and 99 mg/kg bw/day for males and 4, 22 and 117 mg/kg bw/day for females in low, mid- and high-dose groups, respectively. The survival rates of 2000 ppm fed males was significantly lowered after the 76th week, and all of them died before the end of the 2-year administration period. However, no significant diference in the survival rate was found between any of the other o-CNB fed male groups and the control. The terminal survival rate was 80% for the male control, 80% for the low-dose, 78% for the mid-dose and 0% for the high dose males. The markedly decreased survival rate in the high dose males group was attributed to the increased number of non-neoplastic deaths, because 47 out of 50 high-dose males died of chronic progressive nephropathy (CPN) before the end of the 2-year administration period. On the other hand, there was no significant difference in the survival rate between any of the o-CNB-fed female groups and female controls. The terminal survival rate was 82% for the female control, 84% for the low-dose group, 90% for the mid-dose group and 78% for the high-dose females.

The body weights of high-dose males were markedly suppressed after the 20th week. The markedly decreased growth rate was due to the development of severe CPN. The body weights of high-dose fed females were decreased by 10% at the 78th week and by 18% at the end of the 2-year period, compared with the respective female controls. The mid-dose males exhibited significantly decreased terminal body weight by 10% compared with the control, while the mid-dose females did not. There was no difference in the terminal body weight between any of the low-dose male and female groups and the respective controls.

There were no significant differences in food consumption between any of the o-CNB fed groups or either sex and the respective control except for the high-dose males.

A significant decrease in red blood cell count along with a concurrent increase in reticulocyte count occurred only in high-dose females. Hemoglobin and mean corpuscular hemoglobin were significantly decreased in mid-dose males and in mid- and high-dose females. Haematocrit was significantly decreased in mid-dose males and high-dose females. Mean corpuscular volume was significantly decreased in mid-dose males. The methemoglobin level was significantly increased in mid-dose males and in mid- and high-dose females. Platelets were significantly increased in mid-dose males and mid- and high-dose females.

Total bilirubin and ALT were increased in the high-dose females. γ-GTP was increased in the low- and mid-dose males and in mid and high-dose females. LDH was decreased in mid-dose males and in mid- and high-dose females. Creatinine was increased in mid-dose males and in high-dose females. Urea nitrogen was increased in mid-dose males and in mid- and high dose females.

Relative liver weight was significantly increased in the low and mid-dose males, and in mid- and high-dose females. Relative spleen weight was significantly increased in the high-dose females. Relative kidney weight was significantly increased in the mid-dose males and in mid- and high-dose females.

CPN was the most prevalent non-neoplastic lesion found in the present study, which caused deaths in 47 males out of high-dose group and 3 males out of the mid-dose group before the end of the 2-year period, and severely affected the 43 survivng males of mid-dose group. The incidences and severities of CPN were higher in males than in females. The severity of CPN was increased dose-dependently in both males and females, and the severe CPN was characterized by glomerular sclerosis or atrophy without the normal parenchymal tissue of kidneys. In addition, cortical mineralization and urothelial hyperplasia occurred predominantly in the treated males. The deposit of brown pigment in the proximal tubules was observed in the proximal tubules of both mid-and high-dose males and females. The brown pigment was observed as dark brown droplets in the H&E staining specimen, and was stained negatively with Berlin blue.

Single cell necrosis and centrilobular hydropic degeneration were observed in both high dose males and females, whereas fatty change occurred in high-dose males. Deposit of brown pigment occurred only in the liver of both high-dose males and females. The brown pigment was observed as dark brown droplets in the H&E staining specimen and was stained negatively with Berlin blue.

In the spleen of the treated rats of both sexes, capsule hyperplasia, angiectasis, engorgement of erythrocytes, increased extramedullary hematopoiesis and hemosiderin deposition were noted as signs of hematotoxicity.

The incidences of hepatocellular adenomas and carcinomas in both males and females were increased in a dose-related manner, as indicated by a significant positive trend by Peto's test. A statistically increased incidence of hepatocellular adenomas was noted only in the high-dose females. The incidence of hepatocelluar adenomas in the mid-dose males (14%) exceeded the maximum incidence of the JBRC historical control data for male hepatocellular adenomas (7 cases in 400 male rats with a maximum incidence of 6% in a single study). The incidences of hepatocellular carcinomas in the mid-dose males and high-dose females (6% and 8%, respectively) exceeded the respective maximum incidence of the JBRC historical control data for hepatocellular carcinomas (null cases in 400 male and female rats). One of the four hepatocellular carcinomas in high-dose females metastasized to the lung. The incidence of acidophilic cell foci was significantly increased in the mid-dose males and in mid- and high-dose females. The incidence of basophilic cell foci and spongiosis hepatis were significantly increased in the mid-dose males, while the incidence of clear cell foci was significantly increased in the high-dose females. Altered cell foci including acidophilic cell foci, basophilic cell foci and clear cell foci were proliferative in nature, and classified as preneoplastic lesion developing to liver tumour.

The incidences of renal cell adenomas (4%) in the high-dose females and renal cell carcinomas (8%) in the high-dose males exceeded the maximum tumour incidence of the JBRC historical control data for female renall cell adenomas (1 case in 400 female rats with a maximum incidence of 2% in a single study) and for male renal cell carcinomas (0 cases in 400 male rats), respectively. The increased incidences of atypical tubule hyperplasias at the proximal tubule as a proliferative and preneoplastic lesion were noted in both high-dose males and females.

Based on the results of the study, the dose level of 4 mg/kg bw/day was considered a NOAEL for carcinogenicity in male and female rats based on the increased incidence of preneoplastic lesions (acidophilic (male, female) and basophilic (male) foci) and increased incidence (not statistically significant, but exceeding historical control range) of hepatocellular adenomas (male) and hepatocellular carcinomas (males) at the next dose level. The lowest dose level of 4 mg/kg bw/day was also considered a LOAEL for toxicity, based on increased liver weight and increased γ-GTP in males, and increased incidence of chronic progressive nephropathy in females at all dose levels, and an increased incidence of increased extramedullary hematopoiesis in low-dose males.

In the mice study, the average daily intake of the substance was 11, 54 and 329 mg/kg bw/day for males and 14, 69 and 396 mg/kg bw/day for females. The survival rates of the high- and mid-dose males were significantly lowered at the 73rd and 92nd weeks, respectively, and thereafter. The survival rate of low-dose males was not significantly lowered compared with that of the male control group. The terminal survival rate was 70% for the male control, 70% for the low-dose males, 34% for the mid-dose males and 16% for the high-dose males. The markedly lowered survival rates of high- and mid-dose males were causally related to the increased death rate due to malignant liver tumours before the end of the 2-year period. The high-dose females exhibited a significantly lowered survival rate after the 71st week which was attributed to the increased death rate due to malignant liver tumours before the end of the 2-year period. The death rate due to liver tumours occurring before the end of the 2-year period was increased in both dose-and time-dependent manner. There was no significant difference in the survival rate between any of the mid- and low-dose females and the female control. The terminal survival rate was 58% for the female control, 68% for the low-dose females, 52% for the mid-dose females and 10% for the high-dose females. The body weights of mid- and high-dose males were significantly decreased by 22% and 40% compared with the male control, respectively. The mid- and high-dose females exhibited significantly decreased terminal body weights by 12% and 29% compared with the female control, respectively. There was no significant difference in growth rate between any of the low-dose groups of both sexes and the respective control. There were no significant differences in food consumption between any of the o-CNB fed groups or either sex and the respective control. There was no significant change in any erythrocyte parameter except for the significantly increased reticulocyte count, which occurred in both the males and females of the mid- and high dose groups. AST, ALT, LDH, γ-GTP and total bilirubin were significantly increased in both the males and females of the mid- and high-dose groups. Urea nitrogen was increased only in high-dose females. The relative weights of the liver, spleen and kidneys were significantly increased in both the males and females of mid-and high-dose groups, except for the spleen weight in high-dose females.

As non-neoplastic hepatic lesions, centrilobular hypertrophy and nuclear enlargement of the hepatocytes were observed in males at all dose levels and in mid- and high-dose females. The incidences of hemosiderin deposition in the kidney were significantly increased in mid- and high-dose males and in high-dose females. Significantly increased incidences of hemosiderin in the spleen were noted in all treated groups of males and in mid- and high-dose females. The incidences of increased extramedullary hematopoiesis in the spleen were significantly increased in both mid- and high-dose males and females.

The incidences of hepatocellular adenomas and carcinomas, and hepatoblastomas were increased dose-dependently in both sexes, as indicated by a significant positive trend by Peto's test. The incidences of hepatocellular adenomas were significantly increased in all treated males and females groups, while the incidences of hepatocellular carcinomas were significantly increased in the high-dose males and mid- and high-dose females. The incidences of hepatoblastomas were significantly increased in both the mid- and high-dose males and females. In addition, the incidence of hepatoblastomas in the low-dose males exceeded the maximum incidence of the JBRC historical control data for hepatoblastomas (3 cases in 348 males with a maximum incidence of 2% in a single study). More than half of the total malignant liver tumours metastasized to the lung, bone marrow, peritoneum and pancreas.

Based on the results of the study, the lowest dose level of 11 -14 mg/kg bw/day was considered to be a LOAEL for carcinogenicity, based on the increased incidence of hepatocellular adenomas in all treated groups, and increased incidence of hepatoblastomas (not statistically significant, but exceeding historical control range) in low-dose males. The lowest dose level of 11 -14 mg/kg was also considered a LOAEL for toxicity, based on increased incidences of centrilobular hepatocellular hypertrophy and deposit of hemosiderin in the spleen in males.

Justification for classification or non-classification

Based on the hematological effects and the effect on the nasal respiratory epithelium observed in rats in the 90 -day inhalation toxicity study at concentration levels from 7 mg/m3, and on the hematological effects in the 90-day oral study at dose levels from 4 mg/kg bw/day, 1 -chloro-2 -nitrobenzene should be classified as STOT RE Category 1, H372 according to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. According to EU Directive 67/548/EEC, as the observed effects are observed at dose levels more than 10 -fold lower than the limit values of 50 mg/kg bw/day and 250 mg/m3, it should be classified as T, R48/23/25.