DIMETHOMORPH. First draft prepared by Jürg Zarn 1 and Maria Tasheva 2

Transcription

1 DIMETHMRPH First draft prepared by Jürg Zarn 1 and Maria Tasheva 2 1 Food Toxicology Section, Swiss Federal ffice of Public Health, Zurich, Switzerland; and 2 National Center of Public Health Protection, Sofia, Bulgaria Explanation Evaluation for acceptable daily intake Biochemical aspects Absorption, distribution, metabolism and excretion Effects on enzymes and other biochemical parameters Toxicological studies Acute toxicity (a) ral administration (b) Dermal application (c) Exposure by inhalation (d) Dermal and ocular irritation (e) Dermal sensitization Short-term studies of toxicity Long-term studies of toxicity and carcinogenicity Genotoxicity Reproductive toxicity (a) Multigeneration studies (b) Developmental toxicity Special studies: pharmacology (a) Anticonvulsive activity in mice (b) Potentiation of hexobarbiton sleeping time in mice (c) Alteration of body temperature in mice (d) Spontaneus motor activity in mice (e) Tail flick test for analgesia (f) Effect on the Irwin test in mice (g) Charcoal meal transit times in the rat small intestines (h) Effect on motility of uterine smooth muscle in rats (i) Assessment of potential anti-inflammatory activity in rats (j) Cardiovascular, respiratory and nictitating membrane alterations in cat (k) Effect on the spontaneous motility of isolated rabbit ileum (l) Effect on the isolated guinea-pig ileum and action on four agonists (m) Surface anaesthetic activity in the guinea- pig bservations in humans Comments References DIMETHMRPH JMPR 2007

2 274 Explanation Dimethomorph is a cinnamic acid derivative for which the chemical name is (E,Z)-4-[3-(4- chlorophenyl)-3-(3,4-dimethoxyphenyl)acryloyl]morpholine or (EZ)-4-[3-(4-chlorophenyl)-3-(3,4- dimethoxyphenyl)-1-oxo-2-propenyl]morpholine, according to International Union of Pure and Applied Chemistry (IUPAC) and Chemical Abstract Service (CAS) nomenclatures respectively (CAS No ). Dimethomorph is a mixture of E and Z isomers in the ratio of approximately 1 : 1. Dimethomorph is a fungicide that disrupts fungal cell-wall formation. Fungicidal activity is primarily associated with the Z isomer. Dimethomorph has not been evaluated previously by the JMPR and was reviewed at the present Meeting at the request of the Codex Committee on Pesticide Residues (CCPR). All pivotal studies with dimethomorph were certified as complying with good laboratory practice (GLP). Evaluation for acceptable daily intake 1. Biochemical aspects 1.1 Absorption, distribution, metabolism and excretion Absorption, distribution, metabolism and excretion of [ 14 C]dimethomorph was studied in male and female Sprague-Dawley CD rats. [ 14 C]Dimethomorph labelled uniformly on the chlorophenyl ring (radiochemical purity, 98.5%) had a specific activity of GBq/mmol and the E : Z isomer ratio was : Non-labelled dimethomorph had a purity of 99.2% and the isomer ratio was virtually 50 : 50. Figure 1. E- and Z-isomers of dimethomorph N N Cl Cl (Z)- (E)- Table 1. Dosing regimen for a study in rats treated with [ 14 C]dimethomorph Group No. of rats of each sex Dose (mg/kg bw) Remark A Single dose, expired air B 5 10 Single dose C 5 10 Repeated doses of non-labelled dimethomorph for 14 days, followed by a single radiolabelled dose D Single dose E 5 10 Repeated doses of radiolabelled dimethomorph for 7 consecutive days From Schluter (1990) DIMETHMRPH JMPR 2007

3 275 Five different treatment groups were investigated (Table 1). The study complied with GLP. Within 24 h no radioactivity was found in the expired air of animals in group A. In the group receiving a single lower dose (group B), the group receiving a single higher dose (group D), and the group receiving pre-treatment with non-radiolabelled dimethomorph (group C), nearly 90% of the radioactivity administered was excreted via the faeces within 3 days (Table 2). While only marginal differences in faecal excretion were observed in males and females, urinary excretion was 6 7% in males and 10 16% in females. Residual radioactivity in the carcass was low, ranging from 0.08% to 0.43% of the administered dose. ne male and one female in group E were sacrificed 1, 6, 24, 48 or 120 h after the last administration of radiolabelled dimethomorph and organs and tissues were analysed for radiolabel. Radioactivity at a concentration of greater than 0.01% of the administered dose after 1 day was only found in the liver, the muscles, fat and the gastrointestinal tract (GIT). Except for liver and GIT, residual radioactivity was less than 1% of the administered dose after 1 day. Therefore, only liver, kidney and fat of rats in groups B, D, and C were investigated for radioactivity on day 7 after treatment (Table 3). From the results for groups B, C, D and E, no potential for accumulation was evident. Table 2. Excretion patterns in rats treated with [ 14 C]dimethomorph (groups B, C and D) Group Day Excretion (% of administered dose) Males Females Faeces Urine Carcass Faeces Urine Carcass B Total (7 days) ± ± ± ± ± ± 0.03 D Total (7 days) ± ± ± ± ± ± 0.03 C Total (7 days) ± ± ± ± ± ± 0.03 From Schluter (1990) Table 3. Recovery of radioactivity in selected organs of rats treated with [ 14 C]dimethomorph Group Radioactivity on day 7 after treatment (% of administered dose) Males Females Liver Kidney Fat Liver Kidney Fat B D C From Schluter (1990) DIMETHMRPH JMPR 2007

4 276 Profiles for urinary and faecal metabolites were not substantially different between sexes. In urine samples for group D, major metabolites were demethylated products in position 3 and 4 in the dimethoxyphenyl moiety and small amounts of unchanged dimethomorph as well as oxo metabolites of the morpholine ring. In the faeces of rats at the lower dose (groups B and C), 5 9% of the administered dose was recovered as demethylation products and another 5% was unchanged dimethomorph (see Figure 2). In rats receiving the higher dose (group D), dimethomorph accounted for up to 50% of the administered dose, with traces of demethylation products present (Schluter, 1990). The absorption, distribution, metabolism and excretion of [ 14 C]dimethomorph via bile was studied in bile-duct cannulated male and female Sprague-Dawley CD rats. [ 14 C]Dimethomorph labelled uniformly at the chlorophenyl ring (radiochemical purity, 99.7%) had a specific activity of GBq/mmol and the E : Z isomer ratio was 57.9 : Non-radiolabelled dimethomorph was of 99.2% purity and the isomer ratio E : Z was 49.5 : Rats were dosed by oral gavage as deszcribed in Table 4. The study complied with GLP. In males and females at the lower dose, nearly 100% of the administered dose was absorbed and 92 95% was excreted within 48 h via the bile (Table 5). At the higher dose, absorption is saturated and, especially in females, large amounts of the dose (44.1%) remained in the GIT. Biliary excretion had a half-life of approximately 3 h at the lower dose and was saturated at the higher dose, with half-lives of 11 h for males and approximately 6 h for females. In the bile of rats at the lower and higher doses, more than 10 metabolite fractions were identified, most of them being glucuronidated. Quantitatively, the most important metabolites in all treated groups were the demethylated compounds Z 67 and Z 69 (Figure 2) (Van Dijk, 1990). In a supplementary study, dimethomorph metabolites in urine samples from rats of group B from the previous study (Schluter, 1990) were further characterized. The study complied with GLP. As the major pathway, the demethylation of one methoxy group at either position 3 or 4 was identified with formation of conjugates (Figure 2). A second pathway included the oxidative degradation of the morpholine ring moiety (Schluter, 1991). Table 4. Dosing regimen for bile-duct cannulated rats treated with [ 14 C]dimethomorph Group No. of males/females Dose (mg/kg bw) 1 Four males 10 2 Three females 10 3 Three males Three females 500 From Van Dijk (1990) Table 5. Excretion and half-lives of biliary excretion in bile-duct cannulated rats treated with [ 14 C]dimethomorph Dose (mg/kg bw) Excretion after 48 h (% of administered dose) Males Females Faeces Urine Bile t 1/2 bile (h) Faeces Urine Bile t 1/2 bile (h) ± ± ± ± ± ± ± ± ± ± ± ± 15.7 a 5.7 From Van Dijk (1990) a 44.1% in the gastrointestinal tract and 10.1% in residual carcass. DIMETHMRPH JMPR 2007

5 277 To investigate the significance of the label position for the outcome of studies of excretion and metabolism, five male and five female Sprague-Dawley CD rats were given dimethomorph labelled uniformly on either the chlorophenyl ring or the morpholine ring as a single dose at 500 mg/kg bw by oral gavage. Chlorophenyl-ring labelled test compound (radiochemical purity, > 98%) had a specific activity of GBq/mmol and the E : Z isomer ratio was 45.3% E and 54.7% Z. Morpholine-ring labeled test compound (radiochemical purity, > 98%) had a specific activity of GBq/mmol and the E : Z isomer ratio was close to 50 : 50 (mixed from separated isomers). The study complied with GLP. The excretion patterns of dimethomorph labelled at one of two different positions did not exhibit significant differences (Table 6). Most of the radioactivity was excreted within 3 days after administration, with faecal excretion being the major elimination pathway accounting for approximately 80% in females and approximately 90% in males. Urinary excretion was approximately 5% in males and approximately double (10%) in females. No difference in metabolite profiles between the two test compounds was identified, although an additional very minor metabolic pathway as cleavage of the amide bond was found with the chlorophenyl ring-labelled test compound (Schluter, 1993). To further characterize the faecal metabolites, the metabolism and excretion of [ 14 C]dimethomorph was investigated in 10 male and 10 female Wistar rats (BRL-HAN) given a single dose at 50 mg/kg bw by gavage. Uniformly chlorophenyl-ring labelled [ 14 C]dimethomorph (radiochemical purity, 99%) had a specific activity of GBq/mmol and the E : Z isomer ratio was 51 : 49. Nonradiolabelled dimethomorph was of 99.1% purity and the isomer ratio E : Z was 48 : 52. The study complied with GLP. Again, faecal excretion was predominant while urinary excretion was less than 20% of the administered dose (Table 7). When compared with studies in Sprague-Dawley rats, rates of urinary excretion seemed to be higher in male Wistar rats (17.1% of the administered dose). In metabolite analyses, apart from the known degradation products (see Figure 2) an additional metabolite (Z 98) was identified (Schluter & Grahl, 1994). To investigate the pharmacokinetic features of dimethomorph, groups of four male and four female Sprague-Dawley rats (Crl:CD BR) were given [ 14 C]dimethomorph as single oral gavage doses at 10 mg/kg bw or 500 mg/kg bw, respectively. Test compound was uniformly chlorophenyl-ring labelled [ 14 C]dimethomorph (radiochemical purity, 96%) with a specific activity of GBq/mmol Table 6. Excretion patterns in rats given dimethomorph labelled with 14 C in two different positions Position of radiolabel Day Excretion (% of administered dose) Males Females Faeces Urine Total Faeces Urine Total Chlorophenyl ring NR NR NR NR NR NR Total ± ± ± ± ± ± 2.92 Morpholine ring NR NR From Schluter (1993) NR, not reported NR NR NR NR Total ± ± ± ± ± ± 1.80 DIMETHMRPH JMPR 2007

6 278 Table 7. Excretion pattern in rats treated with [ 14 C]dimethomorph labelled on the chlorophenyl ring Day Excretion (% of administered dose) Males Females Faeces Urine Faeces Urine Total ± ± 3.7 From Schluter & Grahl (1994) Figure 2. Proposed metabolic pathways for dimethomorph in the rat H Cl Z 67 N Cl N WL Cl N H H H Cl Z 69 N Cl N Dimethomorph Cl N CUR 7586 Cl NH CUR 7117 H Z43 H Cl N WL H H Cl N H CUR 7216 H Cl NH 2 Z 98 Cl DIMETHMRPH JMPR 2007

7 279 and the E : Z isomer ratio was approximately 50 : 50 after dilution with non-radiolabelled dimethomorph. Non-labelled dimethomorph was 98.8% chemically pure. Another four males and four females served as a control group and were treated with vehicle only. Blood samples at different time intervals from 0.25 h to 144 h after dosing were collected and analysed for radioactivity. The study complied with GLP. In the groups receiving the lower dose, values for both sexes were comparable; t max was reached at < 3h, terminal t 1/2 was h and AUC 0 at μg h/g (Table 8). In the groups receiving the higher dose, increased c max and AUC 0 values were found in females and this correlated with a prolonged half-life. Total radioactive residues (TRR) in plasma and erythrocytes 168 h after treatment were below the limit of detection (LD) in both rats of groups at the lower dose and in the plasma of rats in groups receiving the higher dose (LD at lower dose, ppm; LD at higher dose, ppm). In the groups at the higher dose, TRR in erythrocytes were 0.75 ppm and ppm in male and female rats, respectively (Afzal & Wu, 1995a). To investigate the tissue distribution of dimethomorph, groups of nine male and nine female Sprague-Dawley rats (Crl:CD BR) were given [ 14 C]dimethomorph as single oral doses at 10 mg/kg bw or 500 mg/kg bw by gavage. The test compound was uniformly chlorophenyl-ring labelled [ 14 C] dimethomorph (radiochemical purity, 96%) and had a specific activity of GBq/mmol and the E : Z isomer ratio was approximately 50 : 50 after dilution with non-radiolabelled dimethomorph (purity, 98.8%). Another three males and three females served as a control group and were treated with vehicle only. At t max, 24 h and 168 h after dosing, three males and three females from each group were killed and tissues collected for TRR analysis. T max values were derived from a previous study (Afzal & Wu, 1995a) considering the lower bounds of t max ranges in actual dosing groups; for females at the lower dose, t max was set at 1.5 h; for males at the lower dose at 0.5 h; and for males and females at the higher dose at 8 h. The study complied with GLP. No signs of toxicity were recorded in any group. Highest TRR levels in all dosed groups at t max and 24 h after dosing were found in the GIT and contents, followed by the carcass and liver (Table 9 and Table 10). In liver, maximal TRR values were 1.05% of the administered dose. In all other organs, low levels of less than 0.1% were observed even at t max and they were significantly depleted within 24 h in the groups at the lower dose. In the groups at the higher dose, a certain delay in depletion from organs within 24 h was observed when compared with the groups at the lower dose. Levels at t max and 24 h were all less than 0.1% of the administered dose. At 168 h, liver contained the highest levels of residues, with a range of % TRR of the administered dose; in all other tissues, TRR was virtually negligible (Afzal & Wu, 1995b). To investigate the dermal absorption of dimethomorph, groups of four male Sprague-Dawley CD rats were exposed dermally to [ 14 C]dimethomorph at a dose of 7.73 or mg/kg bw for 8 h. The test compound was uniformly chlorophenyl-ring labelled [ 14 C]dimethomorph (radiochemical purity, 96%; purity, 98.8%) and had a specific activity of GBq/mmol and the E : Z isomer ratio was approximately 45 : 55. The chemical purity of dimethomorph before labelling was 97.6%. The study complied with GLP. In the group at the lower dose, 4.75% of the applied dose was absorbed, based on the sum of radioactivity recovered 168 h after topical application in urine, faeces, cage wash, carcass, blood and untreated skin. In the group at the higher dose, 1.2% of the applied dose was absorbed (Bounds, 1995). 1.2 Effects on enzymes and other biochemical parameters No information was available. DIMETHMRPH JMPR 2007

8 280 Table 9. Tissue distribution of radiolabel in groups of rats receiving [ 14 C]dimethomorph at a lower dose of 10 mg/kg bw Tissue Recovery (% of the total radioactive residues in the administered dose) t max 24 h 168 h Males Females Males Females Males Females GIT contents GIT Carcass Liver Kidneys Pancreas Plasma Erythrocytes Heart Lungs Fat Muscle Spleen Testes Bone Brain Thymus Adrenals Bone marrow Pituitary Thyroid varies Uterus Total From Bounds (1995) GIT, gastrointestinal tract. 2. Toxicological studies 2.1 Acute toxicity (a) ral administration Groups of five male and five female overnight fasted CD rats were given dimethomorph (purity not stated) as a single dose at 3200, 4000 or 5000 mg/kg bw in 0.1% Tween 80 by oral gavage. The study complied with GLP. Mortalities occurred between 24 h and 48 h after dosing. ne female died in the group at the lower dose, three males and four females died in the group at the intermediate dose, and three males and all females died in the group at the higher dose. Median lethality for males was at 4300 mg/kg bw, for females at 3500 mg/kg bw, and for both sexes combined, 3900 mg/kg bw. Animals found DIMETHMRPH JMPR 2007

9 281 Table 10. Tissue distribution of radiolabel in groups of rats receiving [ 14 C]dimethomorph at a higher dose of 10 mg/kg bw Tissue Recovery (% of the total radioactive residues in the administered dose) t max 24 h 168 h Males Females Males Females Males Females GIT contents GIT Carcass Liver Kidneys Erythrocytes Plasma Lungs Fat Heart Pancreas Testes Bone Muscle Spleen Thymus Adrenals Bone marrow Brain Pituitary Thyroid varies Uterus Total From Bounds (1995) GIT, gastrointestinal tract. dead had congested lungs and pallor of the liver, kidneys and spleen. Signs of toxicity were observed 30 min after dosing, including hunched posture, pilo-erection, abnormal gait, lethargy, decreased respiratory rates. Recovery was not observed before 10 days after dosing. Surviving animals showed no treatment-related pathological findings (Kynoch, 1985). To investigate the possible differences in isomer-specific toxicity in Wistar rats, groups of five male and five female overnight fasted rats were given the dimethomorph Z-isomer (purity not stated) as a single doses at 0 or 5000 mg/kg bw in 0.1% Tween 80 by oral gavage. Rats were observed for 15 days. The study complied with GLP. The only finding in the treated rats was pale faeces in the first 2 days after dosing. No signs of toxicity and no mortalities were recorded within the 2 weeks of observation. Therefore, the median lethal dose (LD 50 ) was greater than 5000 mg/kg bw for males and female (Heusener & Jacobs, 1987a). DIMETHMRPH JMPR 2007

10 282 In an additional study on possible isomer-specific acute toxicity in Wistar rats, groups of five male and five female overnight fasted rats were given dimethomorph E-isomer (purity not stated) as a single dose of 0, 4000 or 5000 mg/kg bw in 0.1% Tween 80 by oral gavage. Rats were observed for 15 days. The study complied with GLP. In all dosed groups, signs of toxicity started within 1 h after dosing and included (among others) posture and locomotion disturbances, salivation, blood-crusted snouts and haemorrhagic lacrimation. Mortalities were observed between study day 2 and day 7; in the group at the lower dose, four females and no males died; in the group at the higher dose, two females and four males died. Findings in rats that died were restricted to the GIT and included erosions. In rats sacrificed at study termination, congested lungs and opaque eyes were found. The LD 50 for the E-isomer was calculated to be 4715 mg/kg bw in males and 4754 mg/kg bw in females, in both sexes combined, 4472 mg/kg bw (Heusener & Jacobs, 1987b). (b) Dermal application To investigate the dermal toxicity of the dimethomorph Z-isomer in Wistar rats, groups of five males and five females were exposed dermally to the Z-isomer (purity, 98.7%) at a dose of 0 or 5000 mg/kg bw for 24 h and then observed for 15 days. The study complied with GLP. No signs of toxicity and no mortalities were observed. Therefore, the LD 50 was greater than 5000 mg/kg bw in males and females (Heusener & Eberstein, 1985). Dermal toxicity was investigated in a second study in Fischer 344 rats. Groups of five males and five females exposed dermally to dimethomorph (purity, 98.5%; E : Z, 46 : 54) at a dose of 2000 mg/kg bw for 24 h and then observed for 14 days. The study complied with GLP. No signs of toxicity and no mortalities were observed. Therefore, the LD 50 was greater than 2000 mg/kg bw in males and females (Gardner, 1989) (c) Exposure by inhalation Groups of five male and five female Wistar rats were exposed by inhalation to dimethomorph Z-isomer (purity, 98.7%) at a concentration of 4.24 mg/l (the highest concentration attainable) in a whole-body chamber for 4 h. 56% of particles had a mean aerodynamic diameter of 5.5 µm or less. Animals were then observed for 14 consecutive days. The study complied with GLP. All rats survived till study termination. During the exposure, the rats showed changes in posture and respiratory patterns. Noticeable breathing was resolved after 5 days. Within the first 2 3 days, reduced feed intake and reduced body-weight gain was found. Therefore, the median lethal concentration (LC 50 ) of the Z-isomer was greater than 4.24 mg/l for males and females (Jackson & Hardy, 1986). (d) Dermal and ocular irritation The dermal and ocular irritation potential of dimethomorph was investigated in New Zealand White (NZW) rabbits. The test compound was dimethomorph (purity, 98.5%; E : Z, 46 : 54). For dermal irritation, three rabbits (two males, one female) were exposed to 500 mg of test compound for 4 h under semi-occlusive conditions and observed for another 72 h after removal. For ocular irritation, three animals (one male, two females) were given 50 mg of test compound in a volume of 0.1 ml into the conjunctival sac of one eye. The eyes were not rinsed. The study complied with GLP. In the test for dermal irritation, no signs of an irritating potential were recorded. In the test for ocular irritation, all rabbits showed reddened conjunctivae and slight chemosis. These signs resolved within 4 h after dosing (Gardner, 1989). DIMETHMRPH JMPR 2007

11 283 (e) Dermal sensitization The dermal sensitization potential of dimethomorph (purity, 98.0%) was investigated in male Crl (HA)BR guinea-pigs via the maximization test of Magnusson & Kligman. In two pre-test studies, four males received 0.1 ml of dimethomorph intradermally at a concentration of 1%, 5%, 10%, 15%, and 25% in mineral oil and another four males received dimethomorph topically at a concentration of 1%, 5%, 10%, and 25% in petrolatum occluded for 24 h by patches. All guinea-pigs in both groups received all doses and were evaluated for skin irritation 24 h and 48 h after application. In the group treated by intradermal injection, all doses induced dose-dependent skin irritations that were moderate-intense at 5%. In the group treated by topical application, no irritation was observed at any dose. For the main study, the concentrations of 5% for intradermal injection and 25% for topical application were selected. Twenty males were induced intradermally with 5% dimethomorph in mineral oil and Freund complete adjuvant and then on day 9 topically with 25% dimethomorph in petrolatum. n day 23, the guinea-pigs were challenged by topical application of 25% dimethomorph in petrolatum. The negative-control group consisted of 20 males. The study complied with GLP. No skin irritation was recorded in any of the challenged or control guinea-pigs and the Meeting concluded that dimethomorph has no skin sensitization potential (Glaza, 1997) Table 11. Results of studies of acute toxicity with dimethomorph Isomer Species Strain Sex Route LD 50 (mg/kg bw) LC 50 (mg/l) Purity (%) Reference E : Z Rat CD Male ral 4300 NS Kynoch (1985) E : Z CD Female ral 3500 NS Kynoch (1985) Z Wistar Males and females ral > 5000 NS Heusener & Jacobs (1987a) E Wistar Male ral 4715 NS Heusener & Jacobs (1987b) E Wistar Female ral 4754 NS Heusener & Jacobs (1987b) E : Z Wistar Males and females E : Z Fischer Males and females E : Z Wistar Males and females E : Z Rabbit NZW Males and females E : Z Rabbit NZW Males and females Dermal > Heusener & Eberstein (1985) Dermal > Gardner (1989) Inhalation > Jackson & Hardy (1986) Dermal Not irritating 98.5 Gardner (1989) cular Not irritating 98.5 Gardner (1989) E : Z Guinea-pig Crl (HA)BR Male Dermal No sensitization 98.0 Glaza (1997) NS, not stated; NZW, New Zealand White. DIMETHMRPH JMPR 2007

12 Short-term studies of toxicity Mouse In a 6-week dose range-finding study, 10 male and 10 female CD-1 mice were initially fed diets containing dimethomorph (purity, 96.6%) at a concentration of 0, 300, 800, or 2000 ppm. Since there were no signs of toxicity in any groups, the doses were increased at treatment day 23 from 300 ppm to ppm in males and 8000 ppm in females, and from 2000 ppm to 5000 ppm in males and 4000 ppm in females for the remaining study period. The intended 4-week exposure was prolonged to 6 weeks. Clinical signs were recorded twice per day and body weight, feed and water consumption each week. At the end of the study, investigations of gross pathology were carried out and histopathology on livers was performed. The study complied with GLP. There were no clinical signs of toxicity and body-weight development was unaffected in all groups. The only finding was a dose-related increase in relative liver weights, with no concomitant histological finding (Table 12). n the basis of the pronounced liver weight increases in the groups at the intermediate and the higher dose, the NAEL was 800 ppm, equal to 184 mg/kg bw per day (Perry, 1986). Rats In a 4-week dose range-finding feeding study, groups of five male and five female Sprague- Dawley rats were fed diets containing dimethomorph (purity, 99%) at a concentration of 0, 200, 1000, or 5000 ppm, equal to 0, 15.8, 80.9, and mg/kg bw per day in males and 0, 17.5, 81.1, mg/kg bw per day in females. Rats were examined twice per day for general health, moribundity and mortality, and detailed clinical examinations were performed daily. Feed and water consumption and body-weight development were recorded regularly. At the end of the study, haematology, clinical chemistry, and urine analysis were performed and organ weights and gross pathological changes were recorded. No statement on GLP compliance was provided. In the groups receiving the highest dose, severe signs of toxicity were recorded as loose faeces, brown staining of the perinasal and perigenital regions, swollen abdomen, hunched posture, pilo-erection, emaciation, lethargy, bad grooming and unsteady gait. ne male and two females died during the study and the remaining rats were killed before termination owing to severely impaired general health in week 4 after sampling for laboratory investigations. Males gained weight only minimally and females did not (Table 13). Statistical significant increases in neutrophils and platelets were recorded in males and females at the highest dose. In males, the only statistical significant change in clinical chemistry was an increase in urea nitrogen in the group at the highest dose. In Table 12. Body-weight adjusted increases in liver weight in mice fed diets containing dimethomorph for 6 weeks Sex Body-weight increase (% of control) Dietary concentration (ppm) /5000 (males) 2000/4000 (females) Males 10 18** 33*** Females 15** 30*** 40*** From Perry (1986) ** Significantly different from controls, p < 0.01 *** Significantly different from controls, p < /10000 (males) 300/8000(females) DIMETHMRPH JMPR 2007

13 285 females at the highest dose, total protein and creatinine concentrations were decreased and urea nitrogen, potassium and phosphorus concentrations were increased. Urea nitrogen was also increased in females at the intermediate dose. In both sexes receiving the highest dose, urine volumes were significantly increased and protein concentrations were decreased in males three out of five males did not excrete any protein in the urine. Similarly to rats found dead/killed in a moribund condition before termination that showed thickened and congested large and small intestines, rats at the highest dose at termination showed thickened stomachs and males also showed thickened ileums. Males at the highest dose had empty seminal vesicles. Absolute pituitary weights of males at the intermediate (9 ± 2.2 mg) and highest dose (9 ± 1.0 mg), and of females at the highest dose (7 ± 0.6 mg) were reduced relative to controls (controls: males, 14 ± 3.6 mg; females, 14 ± 2.1), as was the absolute uterus weight of females at the highest dose. In the group at the highest dose, absolute weights of adrenals were increased in males and relative liver weights were increased in females. No histopathology was performed on organs. n the basis of decreases in absolute pituitary weight in males at 1000 ppm, the NAEL was 200 ppm, equal to 15.8 mg/kg bw per day (Warren, 1985). In a second 4-week dose range-finding feeding study, groups of 10 male and 10 female Sprague-Dawley rats were fed diets containing dimethomorph at a concentration of 0, 2000, 3000, or 4000 ppm, equal to approximately 0, 200, 290, and 370 mg/kg bw per day in males and 0, 220, 300, 420 mg/kg bw per day in females (purity, 96.6%). The rats were examined twice per day for general health, moribundity and mortality, and weekly detailed clinical examinations were performed. Feed and water consumption and body-weight development were recorded regularly. At the end of the study, organ weights were recorded and gross pathology performed. Additionally, histopathology on livers was performed. The study complied with GLP. All rats survived until scheduled death. At the highest dose, males and females showed piloerection and swollen abdomens and perigenital staining were observed in females. In all dosed females and in males at the highest dose, statistically significant decreases in body-weight gains were recorded (Table 14). Feed consumption was reduced by 16% in males and 26% in females at the highest dose. Two males at the highest dose and two females at the intermediate dose and six females at the highest dose showed distended intestines, in some instances with thickened mucosa and often filled with fluids or gelatinous material. Body-weight adjusted organ weights were only significantly changed for livers of rats at 2000 ppm (males, 4%; females, 14%), at 3000 ppm (males, 10%; females, 25%) and at 4000 ppm (males, 13%; females, 36%). Increased dose-related incidences of liver hypertrophy were found in males at the intermediate and highest dose and in females at the lowest and intermediate dose and only in one female at the highest dose. In this group of females, at the highest dosehypertrophy may have been masked by vacuolation in the other females of this group. n the basis of the body-weight and liver findings, the NAEL was less than 2000 ppm, equal to approximately 200 mg/kg bw per day (Scott, 1986). Table 13. Body-weight gain and feed consumption in rats fed diets containing dimethomorph for 4 weeks Dietary concentration (ppm) Dose (mg/kg bw) Body-weight gain (g), weeks 0 4 Feed consumption (% of control) Males Females Males Females Males Females From Warren (1985) DIMETHMRPH JMPR 2007

14 286 To investigate the contribution of the E and Z isomers to the toxicity of dimethomorph, two 4-week studies in rats treated by oral gavage were performed with both isomers. Gavage administration was chosen to reduce exposure of the test compounds to light, since photoisomerization is known to occur between the E- and Z-isomers of dimethomorph. In the first 4-week study, groups of seven male and seven female Fischer 344 rats received dimethomorph E-isomer (purity, 99.5%) at a dose of 0, 10, 100, or 750 mg/kg bw per day. During the 4 weeks of the study, no changes in the composition of the test compound were observed. Clinical examinations were performed twice per day, feed consumption and body-weight development were recorded weekly. At the end of the study, haematology and clinical chemistry parameters were investigated and organ weights (brain, heart, liver, kidneys, spleen, adrenals, testes) were recorded and gross pathology performed. Histopathology was performed for rats in the control group and at the highest dose for adrenals, heart, intestines, liver (also for rats at the lowest and intermediate dose), kidneys, pituitary, spleen and stomach. The study complied with GLP. There were no mortalities in the study. A minimal increase in body-weight gain was observed in males at the highest dose coinciding with a minimal increase in feed intake. In rats at the highest dose there were some statistical significant haematological changes, including reduced leukocyte counts in males, and mildly reduced haemoglobin and increased platelet counts and some changes in plateletassociated parameters in males and females. Statistical significant changes in clinical chemistry for males and females at the highest dose included increased concentrations of protein, urea, bilirubin, cholesterol, calcium, creatinine, and phosphate. In males at the intermediate and highest dose, there was also an increase in urea concentration, while females at the intermediate and at the highest dose showed an increased albumin concentration and increased activity of gamma-glutamyltransferase. Significant decreases in spleen weight were observed in males at the highest dose, and in males at the intermediate and the highest dose liver and adrenal weights were also increased. In females at the highest dose, liver weights were increased, as were adrenal weights, although this was not statistically significant (Table 15). Macroscopic and histological changes found were caecal enlargements, liver enlargements, liver dark discolorations and liver fatty vacuolations in both sexes. In males, most findings were present in groups at the intermediate dose and the highest dose, but in females mostly only at the highest dose ( Table 16). Table 14. Body weight and body-weight gain in rats fed diets containing dimethomorph for 4 weeks Dietary concentration (ppm) Body weight (g) Body-weight gain Males Week 0 Week 1 Week 2 Week 3 Week 4 (g) % of control * *** 264*** 292*** 312*** Females * 165** 179** 190** ** 170*** 172*** *** 151*** 154*** 155*** From Scott (1986) * p < 0.05; ** p < 0.01; *** p < DIMETHMRPH JMPR 2007

15 287 The NAEL was 10 mg/kg bw per day on the basis of several changes in the liver in males and females at 100 mg/kg bw per day (Esdaile, 1991a). In the second 4-week study, groups of seven male and seven female Fischer 344 rats received dimethomorph Z-isomer (purity, 95.6%) at a dose of 0, 10, 100, or 750 mg/kg bw per day. During the 4 weeks of the study, no changes in the composition of the test compound were observed. Clinical examinations were performed twice per day, feed consumption and body-weight development were recorded weekly. At the end of the study, haematology and clinical chemistry parameters were investigated and organ weights (brain, heart, liver, kidneys, spleen, adrenals, testes) were recorded and gross pathology performed. Histopathology was performed for rats in the control group and in the group at the highest dose, for the adrenals, brain, heart, intestines, liver (also for rats at the lowest and intermediate dose), kidneys, pituitary, spleen and stomach. The study complied with GLP. All rats survived till study termination and body-weight gain was not affected. Minimal haematological changes in males and females in the groups at the intermediate and the highest dose showed no dose response relationship and were therefore judged not to be treatment-related. Treatment-related changes in clinical chemistry in both sexes comprised increased plasma protein concentrations in the groups at the intermediate and highest dose, while bilirubin concentrations were increased in males at the intermediate and highest dose and in females only at the highest dose. In the Table 15. rgan weights a in rats given dimethomorph E-isomer by gavage for 4 weeks rgan Males rgan weight (g) Dose (mg/kg bw per day) Spleen ** Liver * 8.14** Adrenals * 0.044** Females Liver ** Adrenals From Esdaile (1991a) a rgan weights were adjusted for terminal body weight. * p 0.05; **, p Table 16. Macroscopic and histological changes in organs of rats given dimethomorph E-isomer by gavage for 4 weeks Change Dose (mg/kg bw per day) Males Females Caecal enlargement Liver enlargement Liver, dark discoloration Liver, patchy midzonal cytoplasmic lipid vacuolation From Esdaile (1991a) DIMETHMRPH JMPR 2007

16 288 groups at the highest dose, females had increased cholesterol and males increased calcium and phosphate concentrations. Body-weight adjusted liver weights were increased in both sexes at 100 and 750 mg/kg bw (Table 17). As with the E-isomer, caecal and liver enlargements, liver discoloration and hepatocyte lipid vacuolation were observed, all effects starting at 100 mg/kg bw (Table 18). The NAEL was 10 mg/kg bw per day on the basis of several changes in the liver in males and females at 100 mg/kg bw per day (Esdaile, 1991b). For E- and Z-isomers of dimethomorph, no obvious differences in toxicity profile or potency were evident. In a 13-week feeding study, groups of 10 male and 10 female Sprague-Dawley rats were fed diets containing dimethomorph (purity, 98.7) at a concentration of 0, 40, 200, or 1000 ppm (equal to 0, 2.9, 14.2, 73 mg/kg bw per day in males and 0, 3.2, 15.8, 82 mg/kg bw per day in females). Additionally, recovery groups of 10 male and 10 female rats were fed diets containing dimethomorph at a concentration of 0 or 1000 ppm for 13 weeks and were then given control feed for an additional 4 weeks to evaluate the reversibility of any effects induced during treatment with dimethomorph. Daily cage-side observations, daily/weekly detailed clinical examinations were performed and ophthalmology at the end of the study in the control group and in the group at the highest dose. Body weights and feed consumption were recorded weekly and at the end of the study, haematology, clinical chemistry and urine-analysis parameters were analysed, and a gross necropsy with extensive histopathological examination of tissues and organ weights was conducted. The study complied with GLP. There were no mortalities in the study and body-weight gain and feed consumption were comparable within all groups and sexes. Minimal changes in haematology parameters were noted in males and most of them were reversible in the recovery group (Table 19). Mildly decreased activity of alanine aminotransferase in males and minimally increased calcium and decreased chloride Table 17. Liver weights a in rats given dimethomorph Z-isomer by gavage for 4 weeks Sex Liver weight (g) Dose (mg/kg bw per day) Males ** 7.97** Females ** 5.81** From Esdaile (1991b) a Adjusted for terminal body weight. ** p Table 18. Macroscopic and histological changes in organs of rats given dimethomorph Z-isomer by gavage for 4 weeks Change Dose (mg/kg bw per day) Males Females Caecal enlargement Liver enlargements Liver, dark discoloration Liver, patchy midzonal cytoplasmic lipid vacuolation From Esdaile (1991b) DIMETHMRPH JMPR 2007

17 289 concentrations in females were found in rats at the highest dose only. Mild changes in a few bodyweight adjusted organ weights did not follow a dose response relationship and were therefore judged to be incidental. nly the relative liver weights in females at the highest dose were higher than in controls and showed a dose response relationship (12.3 g at 1000 ppm and 10.5 g in the controls). Although less pronounced, the same was found in the recovery groups (11.6 g at 1000 ppm and 10.7 g in the controls). n histopathology in males at the highest dose, an increased incidence (six out of ten) of vacuolation of adrenal cells in the zona fasciculata was observed (two out of ten in controls, zero out of ten in other groups) and focal vascular congestion in the mucosa of ileum was found in both sexes (two out of ten in males and three out of ten in females, one out of ten in males in the control group and zero out of ten in all other groups). The NAEL was 200 ppm, equal to 14.2 mg/kg bw per day, on the basis of liver-weight increases in females at 1000 ppm (Ruckman, 1987). Table 19. Changes in haematology parameters in male rats fed diets containing dimethomorph for 13 weeks Parameter Study groups Recovery groups Dietary concentration (ppm) Packed cell volume a (%) * 48 45*** Erythrocytes 10 6 /mm * Mean cell haemoglobin concentration (%) * Lymphocytes 10 3 /mm ** From Ruckman (1987) a Erythrocyte volume fraction. * p 0.05; ** p 0.01; *** p Table 20. Histological findings in the ileum of rats and mice fed diets containing dimethomorph Finding Dietary concentration (ppm) Males (n = 10) Females (n = 10) Rats (Scott, 1986) Dilatation Inflammation Serosa monocytes infiltrates Mucosal hyperplasia Fiber separation in muscle layer Villous atrophy Dietary concentration (ppm) Males (n = 10) Females (n = 10) / / / /8000 Mice (Perry, 1986) Dilatation Villous atrophy From Perry (1987) and Scott (1986) DIMETHMRPH JMPR 2007

18 290 The ileums from all mice in the 6-week dose range-finding study (Perry, 1986) and all rats in the 4-week dose range-finding study (Scott, 1986) were examined histologically, this organ seemed to be a target of toxicity of dimethomorph in the rat. The study complied with GLP. Table 20 presents histological findings in the ileum of rats and mice. For both species, the ileum is a target of toxicity attributable to dimethomorph. Rats were more sensitive to changes in the ileum than were mice, and female rats were more sensitive than male rats. This difference between the sexes was not evident in mice (Perry, 1987). Dog In the first part of a two-part study to determine the maximum tolerated dose in beagle dogs, one male and one female were fed diets containing dimethomorph (purity, 96.6%) at a concentration of 1000 ppm for the first 7 days, then at 750 ppm for 7 days, then at 900 ppm for 7 days and finally at 1200 ppm for 7 days. In the second part, one male and one female received diet containing dimethomorph at a concentration of 1200 ppm for 14 consecutive days. The study complied with GLP. In the first part, clinical signs in the male were emesis, subdued behaviour and increased micturition on a few occasions. No signs of toxicity were seen in the female. In the second part, the male showed occasionally body tremors. In both parts of the study, no marked effects on body-weight gain were recorded and there wre no evident changes in clinical chemistry, urine analysis and macroscopic lesions in organs. The maximum tolerated dose was greater than 1200 ppm (Greenough & Goburdhun, 1986a). In a 13-week feeding study, groups of four male and four female beagle dogs were fed 400 g of diet containing dimethomorph (purity, 96.6%) at a concentration of 0, 150, 450, or 1350 ppm (equal to 0, 6, 15, 43 mg/kg bw per day) each day. The dogs were examined daily for general health, moribundity and mortality, and weekly detailed clinical examinations were performed. Feed consumption and body-weight development were recorded regularly. Before treatment and in the final week of treatment, detailed ophthalmic examinations were performed. At the beginning, at week 6 and at the end of the study, haematological and clinical chemistry and urine analysis parameters were analysed; at the end of the study, organ weights were recorded and gross pathology and histopathology were performed. The study complied with GLP. There were no mortalities in the study and the only treatment-related clinical signs were increased incidences of lip-licking and subdued behaviour in males and females at 1350 ppm most frequently observed from week 4 on and usually within 2 h after feeding. A very slight body-weight reduction in females at 1350 ppm coincided with a minor decrease in feed intake in these dogs; bodyweight development and feed intake in all other groups were not affected. There were no significant haematological changes in any group. In all dogs receiving dimethomorph, an increase in alkaline phosphatase activity was observed at 6 and 13 weeks and this became statistically significant for males at the highest dose only at 6 and 13 weeks. Alkaline phosphatase activity in these dogs was nearly doubled. No other biochemistry parameters were changed. In males at 1350 ppm, body-weight adjusted thymus weights were increased and prostate weights were decreased, and in females a significant increase in liver weights was recorded (Table 21). Histopathologically, only in the prostate were treatment-related findings recorded. All males at 1350 ppm had increased fibrosis in the prostate and prostatitis was found in two males. Prostatitis was also diagnosed in one male at 150 ppm. The NAEL was 450 ppm, equal to 15 mg/kg bw per day, on the basis of weight and histological changes in organs and behavioural findings at 1350 ppm (Greenough & Goburdhun, 1986b). In a 52-week feeding study, groups of four male and four female beagle dogs were fed 400 g of diet containing dimethomorph (purity, 96.6%) at a concentration of 0, 150, 450, or 1350 ppm (equal to 0, 5, 15.2, and 44.8 mg/kg bw per day) each day. The dogs were examined daily for general DIMETHMRPH JMPR 2007

19 291 health, moribundity and mortality, and weekly detailed clinical examinations were performed. Feed consumption and body-weight development were recorded regularly. Before treatment, at week 26 and in the final week of treatment, detailed ophthalmic examinations were performed. At the beginning, at weeks 13 and 26 and at the end of the study, haematological and clinical chemistry and urine analysis parameters were analysed. At the end of the study, organ weights were recorded and gross pathology and histopathology examinations were performed. The study complied with GLP. There were no mortalities in the study and no dose-related changes in body-weight gain and feed consumption were recorded. In haematology and urine analysis, no significant changes were found at any dose. Alkaline phoshatase activity was increased slightly but statistically significantly in week 13 and then nearly doubled at week 26 and 51 in males at 1350 ppm; the same effect was somewhat more pronounced in females at 1350 ppm. There were no other treatment-related changes in biochemical parameters. rgan-weight analysis (Table 22) showed significant liver-weight increases in males and females at the highest dose and testes-weight increases in all groups receiving dimethomorph, these effects becoming statistically significant at the intermediate and the highest dose. In the testes, no histological changes were observed. Although statistically not significant, prostate weights were decreased in dogs at 1350 ppm. Histopathologically, three out of four males at 1350 ppm had mildly increased hepatic lipid while none did in the other groups; two out of four females at 1350 and 450 ppm showed this effect as well as one out of four in the control group and none in the group at 150 ppm. A very slight increase in severity of prostate interstitial fibrosis was observed at the highest dose. All other organs investigated histologically were not different from controls. The NAEL was 450 ppm, equal to 15.2 mg/kg bw per day, on the basis of changes in the weights of the liver, prostate and testes and prostate fibrosis at 1350 ppm (Greenough & Goburdhun, 1988). 2.3 Long-term studies of toxicity and carcinogenicity Mouse In a 24-month study of toxicity and carcinogenicity, groups of 50 male and 50 female CD-1 mice were fed diets containing dimethomorph (purity, 96.6%) adjusted to provide doses of 0, 10, 100 and 1000 mg/kg bw per day. Adjustment of dietary concentrations was weekly for the first 13 weeks and every 4 weeks thereafter. In a satellite study, four males and six females served as a control group and 15 males and 15 females were fed diets adjusted to provide dimethomorph at doses of 1000 mg/kg bw per day. In this satellite study, eight males and eight females at the highest dose and all mice in Table 21. Mean relative organ weights of dogs fed diets containing dimethomorph for 13 weeks rgan Males Mean relative weight (% of body weight, ± SD) Dietary concentration (ppm) Thymus ± ± ± ± * Prostate ± ± ± ± ** Females Liver ± ± ± ± 0.510** From Greenough & Goburdhun (1986b) * p < 0.05; ** p < 0.01 SD, standard deviation. DIMETHMRPH JMPR 2007