Regulatory Toxicology and Pharmacology 70 (2014) 535–544
Contents lists available at ScienceDirect
Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph
Carcinogenicity of metamifop, a novel herbicide, in Wistar rats following oral administration for 104 weeks Hyun-Ji Choi a,b, Hyun-Kyu Park a,b, Woori Jo a,b, Jae-Eun Ryu a,b, Sungwoong Jang a,b, Hee-Kyung Gu c, Kyung-Sung Kim c, Bong Jin Chung c, Woo-Chan Son a,b,⇑ a b c
Asan Institute for Life Sciences, Asan Medical Center, Seoul 138-736, Republic of Korea Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Republic of Korea Dongbu Farm Hannong, Daejeon 305-708, Republic of Korea
a r t i c l e
i n f o
Article history: Received 4 June 2014 Available online 4 September 2014 Keywords: Metamifop Herbicide Carcinogenicity study Rats Aryloxyphenoxypropionic acid
a b s t r a c t Metamifop is a novel herbicide with as yet undetermined properties. To assess its carcinogenicity, metamifop was mixed into standard rodent chow and fed to male and female Wistar rats at doses of 10, 100 and 750 ppm for 104 weeks. The viability/mortality of these rats was not affected by treatment with metamifop. Treatment had no significant effects on clinical parameters, and food consumption. Males and females fed 750 ppm of metamifop for 104 weeks showed decreases in body weight and body weight gain. Histopathological examination revealed that treatment with metamifop reduced nonneoplastic findings (chronic progressive nephropathy, tubular basophilia, tubular casts, glomerulosclerosis, basophilic and clear cell foci, senile atrophy, and mesothelial hyperplasia) and reduced neoplastic findings (thymoma, pituitary adenoma, and mammary fibroadenoma and adenocarcinoma in females, and mesenteric lymph node hemangioma in males) compared with control groups. Benign granulosa cell tumors were increased in a dose-dependent manner. As metamifop did not show any genotoxic potential, and there was no correlation between ovarian cancer and increased gonadal hormone levels in humans, the granulosa cell tumors observed in female rats fed a high dose of metamifop were considered not relevant to humans. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
Metamifop (2-[4-[(6-Chloro-2-benzoxazolyl)oxy]phenoxy]-N(2-fluorophenyl)-N-methylpropanamide, Mol. Formula: C23H18ClFN2O4, CAS. RN.: 256412-89-2) is a novel agricultural product, an aryloxyphenoxypropionic acid herbicide, shown to control weeds when spread onto rice fields at 100–200 g/ha. Metamifop had acute oral and percutaneous LD50 values in rats > 2000 mg/kg, suggesting low acute oral toxicity (Tomlin, 2004). Metamifop acts by inhibiting the enzyme acetyl-CoA carboxylase (ACCase), which catalyzes the first committed step in de novo fatty acid biosynthesis; this interrupts lipid biosynthesis, ultimately killing the plant (Kobek et al., 1988; Secor and Cseke, 1988). To determine its carcinogenicity, male and female Wistar rats were fed metamifop for 104 weeks, at doses of 10, 100 and 750 ppm admixed in standard rat chow.
2.1. Test material and formulation analysis
⇑ Corresponding author at: Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, Republic of Korea. Fax: +82 2 3010 8163. E-mail address: [email protected] (W.-C. Son). http://dx.doi.org/10.1016/j.yrtph.2014.08.011 0273-2300/Ó 2014 Elsevier Inc. All rights reserved.
Metamifop was synthesized by Dongbu Farm Hannong Co., Ltd. (Seoul, Korea) and admixed into pelleted rodent diet. Metamifop was weighed and mixed with microgranulated feed. Water was added to make pellets. The pellets were dried with air for about 48 h before storage. Feed for the group 1 was prepared in the same way but without metamifop. The pellets were prepared approximately once a week until week 14 and at 2-week intervals thereafter. Feed preparations were stored at room temperature (17–23 °C) in disposable paper bags. The concentration and homogeneity of metamifop in the feed preparations was determined in the first mix. The concentration and homogeneity were determined every three months. The homogeneity of each diet preparation was investigated by analyzing samples collected from three different regions of the pellet drying shelves (top, middle and bottom). Twenty grams of each diet sample was homogenized with 100 mL of acetonitrile/water (75 + 25, v/v). After sedimentation, the supernatant was taken,
536
H.-J. Choi et al. / Regulatory Toxicology and Pharmacology 70 (2014) 535–544
filtered through a 0.45 lm Acrodisk filter (Pall Life Sciences, MI) and analyzed by HPLC (High Performance Liquid Chromatography). Storage stability samples were collected after storage of the diet preparations at room temperature (about 20 °C) for 7, 14, and 21 days. Then, the latter samples were analyzed. Three samples were collected for each concentration and analyzed in the same manner. 2.2. Animal husbandry and maintenance Five-week-old male and female outbred Wistar rats were obtained from a specific pathogen-free colony at RCC Ltd, Animal Breeding and Biotechnology Division (Fullinsdorf, Switzerland). The mean weight of the males was 95 g (range, 77–110 g), and the mean weight of the females was 79 g (range 65–92 g). The rats were housed in individual cages after compound administration. Rats were fed a pelleted standard Kliba 3433 rat maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst, Switzerland) ad libitum, except for a one-night fast prior to sacrifice. Rats were allowed community tap water from Itingen ad libitum. 2.3. Experimental design and rationale for dose selection Metamifop was administered orally due to the expected route of human exposure. Metamifop was admixed into pelleted rodent diet at constant concentrations of 10, 100 and 750 ppm, resulting in daily intake over the 104 week treatment period of 0.42, 4.18, and 34.60 mg/kg body weight, respectively, for males and 0.52, 5.17, and 41.80 mg/kg body weight, respectively, for females. Fifty male and fifty female rats were fed each dose of metamifop, whereas a control group was fed untreated pellets (Table 1). 2.4. In-life phase examination Animals were visually inspected at least once per day, with detailed physical examinations performed weekly on each animal to monitor any superficial palpable nodules. Morbidity, mortality, and nodule location, size, consistency and time of first observation were recorded. Rats were weighed at the beginning of the study, every week for the first 13 weeks, every two weeks thereafter, and at the end of the study. The weight of food supplied to each cage and the weight remaining were recorded every week for the first 13 weeks and every two weeks thereafter; these records were used to calculate the mean weekly consumption per animal. Blood samples were collected under isoflurane anesthesia from the retroorbital plexus after weeks 78 and 104. The animals were not allowed to fast before blood sampling. Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. At the end of the study, all animals were anesthetized with isoflurane, weighed, and sacrificed.
Table 1 Composition of test groups. Group
Gender
Number of animals
Dosage (ppm)
Control
Male Female
50 50
0
T1
Male Female
50 50
10
T2
Male Female
50 50
100
T3
Male Female
50 50
750
2.5. Gross pathology and histopathology All macroscopic abnormalities were recorded. All animals surviving to the end of the observation period and all moribund animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination. Full macroscopic examinations of the tissues were performed in accordance with the OECD Test Guideline 453 for the testing of chemicals (OECD, 1981), EPA Health effects testing guidelines, OPPTS 870.4300 (EPA, 1998), the guidelines related to study reports for the registration application of pesticide, No. 12 NohSan 8147 (Jap. Ministry of Agriculture, Forestry and Fisheries, 2000), and Directive 88/302/EEC, Part B (European Commission, 1987). During necropsy, the adrenal glands, heart, ovaries, uterus, brain, kidneys, spleen, epididymides, liver, and testes were weighed. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution, unless otherwise specified: adrenal glands, aorta, auricles, bones (sternum, femur including joint), bone marrow (femur, sternum), brain (medulla/pons, cerebral and cerebellar cortex), cecum, colon, duodenum, epididymides (fixed in Bouin’s solution), esophagus, eyes w/optic nerve (fixed in Davidson’s solution), Harderian gland (fixed in Davidson’s solution), head (remaining), heart, ileum, jejunum, kidneys, larynx, lacrimal gland (exorbital), liver, lung (filled with formalin at necropsy), lymph nodes (mesenteric, mandibular, mammary gland area), nasal cavity, ovaries, pancreas, pharynx, pituitary gland, prostate gland, rectum, salivary glands (mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, midthoracic, lumbar), spleen, stomach, testes (fixed in Bouin’s solution), thymus, thyroid including the parathyroid gland (if possible), tongue, trachea, urinary bladder (filled with formalin at necropsy), uterus with cervix, vagina, Zymbal’s gland, gross lesions and tissue masses. 2.6. Quality assurance and test guidelines Quality assurance inspections and audits (undertaken at RCC Ltd) were conducted at each critical phase of the study (formulation analysis, dosing, necropsy, and hematology) and appropriately reported according to standard operating procedures. All experimental results were promptly recorded and provided to the appropriate management personnel. 2.7. Statistical analysis 2.7.1. Survival analysis Survival analysis was calculated according to the Cox regression model. Survival rate curves were plotted using the Kaplan–Meier method and compared using the log-rank test. 2.7.2. Tumor analysis Neoplastic lesions were analyzed as described using tests for positive trend as a function of dose rates (Peto et al., 1980). 2.7.3. Other examinations Normally distributed variables were compared using the Dunnett’s test, based on a pooled variance estimate, whereas non-normally distributed variables were compared using the Steel-test. Quantitative laboratory data were analyzed by oneway analysis of variance (ANOVA) when the variances were considered homogeneous, followed by Dunnett’s test, or by the non-parameteric Kruskal–Wallis test, followed by Dunn’s test if the variances were considered heterogeneous (p 6 0.05). Major non-neoplastic findings were analyzed according to the method of Cochran Armitage.
H.-J. Choi et al. / Regulatory Toxicology and Pharmacology 70 (2014) 535–544
537
3. Results
3.3. General clinical signs
3.1. Analytical chemistry
The incidence of all findings was within the expected range for Wistar rats over 104 weeks, suggesting that mortality was not associated with toxicology.
The content of samples analyzed within this study was within 80–120% of the corresponding nominal concentrations. Almost every sample showed a homogeneous distribution of metamifop in the diet because single recovery results deviated
Contents lists available at ScienceDirect
Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph
Carcinogenicity of metamifop, a novel herbicide, in Wistar rats following oral administration for 104 weeks Hyun-Ji Choi a,b, Hyun-Kyu Park a,b, Woori Jo a,b, Jae-Eun Ryu a,b, Sungwoong Jang a,b, Hee-Kyung Gu c, Kyung-Sung Kim c, Bong Jin Chung c, Woo-Chan Son a,b,⇑ a b c
Asan Institute for Life Sciences, Asan Medical Center, Seoul 138-736, Republic of Korea Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Republic of Korea Dongbu Farm Hannong, Daejeon 305-708, Republic of Korea
a r t i c l e
i n f o
Article history: Received 4 June 2014 Available online 4 September 2014 Keywords: Metamifop Herbicide Carcinogenicity study Rats Aryloxyphenoxypropionic acid
a b s t r a c t Metamifop is a novel herbicide with as yet undetermined properties. To assess its carcinogenicity, metamifop was mixed into standard rodent chow and fed to male and female Wistar rats at doses of 10, 100 and 750 ppm for 104 weeks. The viability/mortality of these rats was not affected by treatment with metamifop. Treatment had no significant effects on clinical parameters, and food consumption. Males and females fed 750 ppm of metamifop for 104 weeks showed decreases in body weight and body weight gain. Histopathological examination revealed that treatment with metamifop reduced nonneoplastic findings (chronic progressive nephropathy, tubular basophilia, tubular casts, glomerulosclerosis, basophilic and clear cell foci, senile atrophy, and mesothelial hyperplasia) and reduced neoplastic findings (thymoma, pituitary adenoma, and mammary fibroadenoma and adenocarcinoma in females, and mesenteric lymph node hemangioma in males) compared with control groups. Benign granulosa cell tumors were increased in a dose-dependent manner. As metamifop did not show any genotoxic potential, and there was no correlation between ovarian cancer and increased gonadal hormone levels in humans, the granulosa cell tumors observed in female rats fed a high dose of metamifop were considered not relevant to humans. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
Metamifop (2-[4-[(6-Chloro-2-benzoxazolyl)oxy]phenoxy]-N(2-fluorophenyl)-N-methylpropanamide, Mol. Formula: C23H18ClFN2O4, CAS. RN.: 256412-89-2) is a novel agricultural product, an aryloxyphenoxypropionic acid herbicide, shown to control weeds when spread onto rice fields at 100–200 g/ha. Metamifop had acute oral and percutaneous LD50 values in rats > 2000 mg/kg, suggesting low acute oral toxicity (Tomlin, 2004). Metamifop acts by inhibiting the enzyme acetyl-CoA carboxylase (ACCase), which catalyzes the first committed step in de novo fatty acid biosynthesis; this interrupts lipid biosynthesis, ultimately killing the plant (Kobek et al., 1988; Secor and Cseke, 1988). To determine its carcinogenicity, male and female Wistar rats were fed metamifop for 104 weeks, at doses of 10, 100 and 750 ppm admixed in standard rat chow.
2.1. Test material and formulation analysis
⇑ Corresponding author at: Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, Republic of Korea. Fax: +82 2 3010 8163. E-mail address: [email protected] (W.-C. Son). http://dx.doi.org/10.1016/j.yrtph.2014.08.011 0273-2300/Ó 2014 Elsevier Inc. All rights reserved.
Metamifop was synthesized by Dongbu Farm Hannong Co., Ltd. (Seoul, Korea) and admixed into pelleted rodent diet. Metamifop was weighed and mixed with microgranulated feed. Water was added to make pellets. The pellets were dried with air for about 48 h before storage. Feed for the group 1 was prepared in the same way but without metamifop. The pellets were prepared approximately once a week until week 14 and at 2-week intervals thereafter. Feed preparations were stored at room temperature (17–23 °C) in disposable paper bags. The concentration and homogeneity of metamifop in the feed preparations was determined in the first mix. The concentration and homogeneity were determined every three months. The homogeneity of each diet preparation was investigated by analyzing samples collected from three different regions of the pellet drying shelves (top, middle and bottom). Twenty grams of each diet sample was homogenized with 100 mL of acetonitrile/water (75 + 25, v/v). After sedimentation, the supernatant was taken,
536
H.-J. Choi et al. / Regulatory Toxicology and Pharmacology 70 (2014) 535–544
filtered through a 0.45 lm Acrodisk filter (Pall Life Sciences, MI) and analyzed by HPLC (High Performance Liquid Chromatography). Storage stability samples were collected after storage of the diet preparations at room temperature (about 20 °C) for 7, 14, and 21 days. Then, the latter samples were analyzed. Three samples were collected for each concentration and analyzed in the same manner. 2.2. Animal husbandry and maintenance Five-week-old male and female outbred Wistar rats were obtained from a specific pathogen-free colony at RCC Ltd, Animal Breeding and Biotechnology Division (Fullinsdorf, Switzerland). The mean weight of the males was 95 g (range, 77–110 g), and the mean weight of the females was 79 g (range 65–92 g). The rats were housed in individual cages after compound administration. Rats were fed a pelleted standard Kliba 3433 rat maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst, Switzerland) ad libitum, except for a one-night fast prior to sacrifice. Rats were allowed community tap water from Itingen ad libitum. 2.3. Experimental design and rationale for dose selection Metamifop was administered orally due to the expected route of human exposure. Metamifop was admixed into pelleted rodent diet at constant concentrations of 10, 100 and 750 ppm, resulting in daily intake over the 104 week treatment period of 0.42, 4.18, and 34.60 mg/kg body weight, respectively, for males and 0.52, 5.17, and 41.80 mg/kg body weight, respectively, for females. Fifty male and fifty female rats were fed each dose of metamifop, whereas a control group was fed untreated pellets (Table 1). 2.4. In-life phase examination Animals were visually inspected at least once per day, with detailed physical examinations performed weekly on each animal to monitor any superficial palpable nodules. Morbidity, mortality, and nodule location, size, consistency and time of first observation were recorded. Rats were weighed at the beginning of the study, every week for the first 13 weeks, every two weeks thereafter, and at the end of the study. The weight of food supplied to each cage and the weight remaining were recorded every week for the first 13 weeks and every two weeks thereafter; these records were used to calculate the mean weekly consumption per animal. Blood samples were collected under isoflurane anesthesia from the retroorbital plexus after weeks 78 and 104. The animals were not allowed to fast before blood sampling. Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. At the end of the study, all animals were anesthetized with isoflurane, weighed, and sacrificed.
Table 1 Composition of test groups. Group
Gender
Number of animals
Dosage (ppm)
Control
Male Female
50 50
0
T1
Male Female
50 50
10
T2
Male Female
50 50
100
T3
Male Female
50 50
750
2.5. Gross pathology and histopathology All macroscopic abnormalities were recorded. All animals surviving to the end of the observation period and all moribund animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination. Full macroscopic examinations of the tissues were performed in accordance with the OECD Test Guideline 453 for the testing of chemicals (OECD, 1981), EPA Health effects testing guidelines, OPPTS 870.4300 (EPA, 1998), the guidelines related to study reports for the registration application of pesticide, No. 12 NohSan 8147 (Jap. Ministry of Agriculture, Forestry and Fisheries, 2000), and Directive 88/302/EEC, Part B (European Commission, 1987). During necropsy, the adrenal glands, heart, ovaries, uterus, brain, kidneys, spleen, epididymides, liver, and testes were weighed. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution, unless otherwise specified: adrenal glands, aorta, auricles, bones (sternum, femur including joint), bone marrow (femur, sternum), brain (medulla/pons, cerebral and cerebellar cortex), cecum, colon, duodenum, epididymides (fixed in Bouin’s solution), esophagus, eyes w/optic nerve (fixed in Davidson’s solution), Harderian gland (fixed in Davidson’s solution), head (remaining), heart, ileum, jejunum, kidneys, larynx, lacrimal gland (exorbital), liver, lung (filled with formalin at necropsy), lymph nodes (mesenteric, mandibular, mammary gland area), nasal cavity, ovaries, pancreas, pharynx, pituitary gland, prostate gland, rectum, salivary glands (mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, midthoracic, lumbar), spleen, stomach, testes (fixed in Bouin’s solution), thymus, thyroid including the parathyroid gland (if possible), tongue, trachea, urinary bladder (filled with formalin at necropsy), uterus with cervix, vagina, Zymbal’s gland, gross lesions and tissue masses. 2.6. Quality assurance and test guidelines Quality assurance inspections and audits (undertaken at RCC Ltd) were conducted at each critical phase of the study (formulation analysis, dosing, necropsy, and hematology) and appropriately reported according to standard operating procedures. All experimental results were promptly recorded and provided to the appropriate management personnel. 2.7. Statistical analysis 2.7.1. Survival analysis Survival analysis was calculated according to the Cox regression model. Survival rate curves were plotted using the Kaplan–Meier method and compared using the log-rank test. 2.7.2. Tumor analysis Neoplastic lesions were analyzed as described using tests for positive trend as a function of dose rates (Peto et al., 1980). 2.7.3. Other examinations Normally distributed variables were compared using the Dunnett’s test, based on a pooled variance estimate, whereas non-normally distributed variables were compared using the Steel-test. Quantitative laboratory data were analyzed by oneway analysis of variance (ANOVA) when the variances were considered homogeneous, followed by Dunnett’s test, or by the non-parameteric Kruskal–Wallis test, followed by Dunn’s test if the variances were considered heterogeneous (p 6 0.05). Major non-neoplastic findings were analyzed according to the method of Cochran Armitage.
H.-J. Choi et al. / Regulatory Toxicology and Pharmacology 70 (2014) 535–544
537
3. Results
3.3. General clinical signs
3.1. Analytical chemistry
The incidence of all findings was within the expected range for Wistar rats over 104 weeks, suggesting that mortality was not associated with toxicology.
The content of samples analyzed within this study was within 80–120% of the corresponding nominal concentrations. Almost every sample showed a homogeneous distribution of metamifop in the diet because single recovery results deviated
