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Carcinogenicity

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Description of key information

1-chloro-2-nitrobenzene induced tumours in the liver and kidney of rats and in the liver of mice in the 2-year GLP-compliant OECD Guideline 451 study. The NOAEL for carcinogenicity was 4 mg/kg bw/day for rats, while for mice the lowest dose level of 11-14 mg/kg bw/day was considered to be a LOAEL for carcinogenicity. 

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion
Dose descriptor:
NOAEL
4 mg/kg bw/day

Additional information

Carcinogenicity of 1 -chloro-2 -nitrobenzene was studied in rats and mice in a GLP-compliant OECD guideline 451 study (Matsumoto, 2011). 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 rate 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 withblue.

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 renal 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 2 -year mouse 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.

In the study of Weisburger, 1978, 25 male CD rats/group were given 1-chloro-2-nitrobenzene in the diet for 18 months (50 % of MTD, MTD): 0, 1000, 2000 mg/kg diet (approx. 0, 75, 150 mg/kg bw/day). After 6 months of treatment, dosage was reduced to 500, 1000 mg/kg diet (approx. 37.5, 75 mg/kg bw/day), because body weight gain was reduced by 10 % when compared to the control group or deaths occurred from toxicity (no further information). Reduced doses were given for the remaining 12 months. Following the 6-month-observation period, necropsy was performed and male rats with tumours were recorded: 1/22 in the simultaneous control group (pooled control: 14/111) and 7/22 resp 1/19 in the low resp. the high dose group. These tumours of the low dose group usually included pituitary adenomas along with either a stomach papilloma, a tumour of the adrenals, a thyroid adenocarcinoma, a lymphosarcoma, a bile duct carcinoma or a subcutaneous fibroma.

25 male and female CD1 HaM/ICR mice/group were given 1-chloro-2-nitrobenzene in the diet for 18 months (50 % of MTD, MTD): 0, 3000, 6000 mg/kg diet (approx. 0, 450, 900 mg/kg bw/day). After 8 months of treatment dosage was reduced to 1500, 3000 mg/kg diet (approx. 225, 450 mg/kg bw/day) which was given for the remaining 10 months (see above). Following the 3-month observation period, necropsy was performed and mice with tumours were recorded: 3/18 (m), 0/20 (f) in the simultaneous control group (pooled control: (m) 7/99, (f) 1/102) and 7/17 (m), 5/22 (f) resp 3/16 (m), 5/19 (f) in the low resp. the high dose group, identified as hepatocellular carcinomas.

The objective of a subacute feeding study with B6C3F1 mice was to recognize possible preneoplastic lesions by means of enzyme histochemistry. 12 mice/sex/dose received 0, 50, 500, 5000 ppm 1-chloro-2-nitrobenzene in the diet 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/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. The 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)). These marked changes in the carbohydrate metabolism were evaluated as possible promotion activity of 1-chloro-2-nitrobenzene (Bayer 1991, 1993).

Justification for classification or non-classification

Based on the ability of 1 -chloro-2 -nitrobenzene to induce hepatic tumours in two different species and renal tumours in the rat, it should be classified as Carc Cat. 2, R45 according to Directive 67/548/EEC and Carc 1B, H350 according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.