PREFACE The tests were contracted and supported by the Ministry of Labour of Japan. The tests were conducted by Japan Bioassay Laboratory (JBL) and th
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1 Summary of Feeding Carcinogenicity Study of 1-Chloro-2,4-Dinitrobenzene in BDF 1 Mice August 1992 Japan Bioassay Laboratory Japan Industrial Safety and Health Association
2 PREFACE The tests were contracted and supported by the Ministry of Labour of Japan. The tests were conducted by Japan Bioassay Laboratory (JBL) and the report was prepared by JBL and peer reviewed by outside expert pathologist. Complete report was submitted to Ministry of Labour of Japan on August 19, This English Summary was translated by JBL from Japanese complete report.
3 (Study No.0096) Summary of Feed Carcinogenicity Study of 1-Chloro-2,4-Dinitrobenzene in BDF 1 Mice Purpose, materials and methods 1-Chloro-2,4-dinitrobenzene (CDNB : CAS No ) is a yellow crystal with a melting point of 51 C. It is insoluble in water and soluble in ether, benzene, carbon disulfide, and hot ethanol. The carcinogenicity and chronic toxicity of CDNB were examined in Crj:BDF 1 mice. Groups of test animals were administered CDNB in their diets for 2 years (104 weeks). Each group consisted of either 50 male or 50 female mice. The dietary concentrations of CDNB were 0, 320, 800 or 2000 ppm (w/w). Both sexes were administered each concentration of CDNB. The highest dose level was chosen so as not to exceed the maximum tolerated dose (MTD), based on both growth rate and toxicity in a previous 13-week toxicity study. The identity of the CDNB used in these experiments was confirmed by mass spectrometry. It was analyzed by infrared spectrometry, ultraviolet spectrometry and gas chromatography before and after its use to affirm its stability. The concentrations of CDNB in the diet were determined by gas chromatography at the time of preparation and on the 7th day after preparation while stored at room temperature. The animals were observed daily for clinical signs and mortality. Body weight and food consumption were measured once a week for the first 14 weeks and every 2 weeks thereafter. Animals found dead, or in a moribund state, or surviving to the end of the 2-year administration period underwent complete necropsy. Urinalysis was performed near the end of the administration period. Hematology and blood biochemistry analysis were performed at the terminal necropsy: surviving animals were fasted overnight and bled under deep ether anesthesia. Organs and tissues were removed, weighed and examined for macroscopic lesions at necropsy. The organs and tissues were then fixed and embedded in paraffin. Five μm thick tissue sections were prepared and stained with hematoxylin and eosin and examined microscopically. Incidences of neoplastic lesions were statistically analyzed by Fisher s exact test. Positive dose-response trends of CDNB induction of neoplastic lesions were analyzed by Peto s test. Incidences of non-neoplastic lesions and urinalysis were analyzed by the Chi-square test. Changes in body weight, food consumption, hematological and blood biochemical parameters, and organ weights were analyzed by Dunnett s test. The present study was conducted with reference to the Organisation for Economic Co-operation and Development 1
4 (Study No.0096) (OECD) Good Laboratory Practice and the OECD Guideline for Testing of Chemicals 451 Carcinogenicity Studies. Results No significant differences in survival rates were found between any of the groups administered CDNB and their respective controls. Yellow urine and yellow coloration of the fur were observed in all the CDNB-fed groups of both sexes after the 75th week of administration. Body weights were decreased in first half of the administration period in males fed 2000 ppm CDNB and throughout most of the administration period in females fed 2000 ppm CDNB. Food consumption by the 2000 ppm-fed males and females decreased in approximate accordance with the decrease in body weight. Hemoglobin concentration, hematocrit value and mean corpuscular hemoglobin were decreased in females fed 2000 ppm CDNB. Plasma levels of glutamic oxaloacetic transaminase (GOT), lactate dehydrogenase and creatine phosphokinase were increased in males fed 2000 ppm and GOT and chlorine were increased in females fed 2000 ppm CDNB. There were no CDNB related changes in urinalysis parameters or organ weights in any of the CDNB administered groups. No significant increases in the incidence of neoplastic or tumor-related lesions were found in any of the CDNB-administered groups of either sex. Induction of several non-neoplastic lesions in the CDNB-administered groups was noted: The incidences of hemosiderin deposition and extramedullary hematopoiesis in the spleen were significantly increased in 2000 ppm CDNB fed males and hemosiderin deposition in the spleen was significantly increased in 2000 ppm CDNB fed females. Deposition of pigment in the thyroid was significantly increased in both males and females fed 800 and 2000 ppm CDNB. Hyperplasia in the forestomach was significantly increased in males fed 2000 ppm CDNB and hyperplasia in the glandular stomach was significantly decreased in females fed 2000 ppm CDNB. Conclusions There was no evidence for carcinogenicity of 1-chloro-2,4-dinitrobenzene in male or female mice. 2
5 (Study No. 0096) Incidences of selected neoplastic lesions of male mice in the 2-year feed carcinogenicity study of 1-chloro-2,4-dinitrobenzene Dose (ppm) Number of examined animals benign tumor lung bronchiolar-alveolar adenoma liver hepatocellular adenoma Harderian gland adenoma malignant tumor lung bronchiolar-alveolar carcinoma lymph node malignant lymphoma spleen malignant lymphoma hemangioendothelioma liver hepatocellular carcinoma 7 1 * 9 3 histiocytic sarcoma hemangioendothelioma Peto test Cochran- Armitage test Incidences of selected neoplastic lesions of female mice in the 2-year feed carcinogenicity study of 1-chloro-2,4-dinitrobenzene Dose (ppm) Number of examined animals benign tumor lung bronchiolar-alveolar adenoma liver hepatocellular adenoma pituitary adenoma malignant tumor lung bronchiolar-alveolar carcinoma lymph node malignant lymphoma uterus histiocytic sarcoma Peto test Cochran- Armitage test. Significant difference * : p 0.05 ** : p 0.01 (Fisher test) : p 0.05 increase : p 0.01 increase (Peto, Cochran-Armitage test) : p 0.05 decrease : p 0.01 decrease (Cochran-Armitage test) 3
6 (Study No.0096) SELECTED TABLES TABLE 27 TABLE 28 TABLE 29 TABLE 30 SURVIVAL ANIMAL NUMBERS AND BODY WEIGHT CHANGES IN MALE MOUSE (TWO-YEAR STUDIES) SURVIVAL ANIMAL NUMBERS AND BODY WEIGHT CHANGES IN FEMALE MOUSE (TWO-YEAR STUDIES) FOOD CONSUMPTION IN MALE MOUSE (TWO-YEAR STUDIES) FOOD CONSUMPTION IN FEMALE MOUSE (TWO-YEAR STUDIES) TABLE 31 CLINICAL OBSERVATION (104W-SUMMARY) - MICE - TABLE 32 CAUSE OF DEATH : MOUSE 4
7 5
8 5~-7 6
9 ~.3 9~-7 10~-7 7
10 ~t. 8
11 9
12 10
13 (Study No.0096) SELECTED FIGURES FIGURE 8 SURVIVAL ANIMAL RATE : MOUSE MALE (TWO-YEAR STUDIES) FIGURE 9 SURVIVAL ANIMAL RATE : MOUSE FEMALE (TWO-YEAR STUDIES) FIGURE 10 BODY WEIGHT CHANGES : MOUSE MALE (TWO-YEAR STUDIES) FIGURE 11 BODY WEIGHT CHANGES : MOUSE FEMALE (TWO-YEAR STUDIES) FIGURE 12 FOOD CONSUMPTION : MOUSE MALE (TWO-YEAR STUDIES) FIGURE 13 FOOD CONSUMPTION : MOUSE FEMALE (TWO-YEAR STUDIES) 11
14 12
15 13
16 14
17 (Study No.0096) PHOTOGRAPHS PHOTOGRAPH 6 THYROID, DEPOSIT OF PIGMENNT:A 104-WEEK STUDY, MOUSE, MALE, 2000ppm, ANIMAL No (H. E., X150) PHOTOGRAPH 7 STOMACH, HYPERPLASIA;FORESTOMACH:A 104-WEEK STUDY, MOUSE, MALE, 2000ppm, ANIMAL No (H. E., X60) 15
18 16
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