DAMINOZIDE EXPLANATION Daminozide was considered by the Joint Meeting in 1977 and 1983 (Annex 1, FAO/WHO 1978a, 1984). The following information was required before an ADI could be considered: 1. adequate data to assess the carcinogenicity of daminozide; 2. an adequate teratology study; and 3. information on biotransformation in animals. Additional data are reviewed in this monograph addendum. EVALUATION FOR ACCEPTABLE DAILY INTAKE BIOLOGICAL DATA Biochemical aspects Absorption, distribution and excretion Rat Groups of two male Sprague-Dawley rats/dose group were given a single oral dose of 30, 300 or 3000 µg of [14C-methyl]daminozide by gavage. One additional male served as a control. The primary route of excretion of radioactivity was the feces: 63-86% (dose-related) of the dose was recovered in feces in 48 hours. At all doses, about 10% of the administered radioactivity was recovered in urine. Excretion as CO2 accounted for 2-16% of the dose and was inversely dose-related. Tissue levels at 48 hours accounted for 1.9-8.8% of the dose and was inversely dose-related indicating that tissue accumulation is unlikely. Excretion was rapid: 74-87% of the dose within 12 hours and 91-96% within 24 hours (Milad et al. 1984). In another study in which 14C-daminozide was applied dermally to 24-28 male Sprague-Dawley rats/group at doses of 0.05, 0.5 or 5 mg, 98.6-100% of the administered dose was recovered from the site of treatment after up to 24 hours exposure. At 5 mg, about 0.75% of the dose was recovered in the carcass after a 24-hour exposure period. Carcass residues were negligible at shorter exposure times and at the lower doses (Chadwick & Silveira, 1987). Guinea pig Groups of 15 male Hartley guinea pigs were given 2 ml oral doses of aqueous solutions of [14CH3]-daminozide at concentrations of about 13 or 135 ppm. At both dose levels about 10% of the dose was recovered in urine mainly in the first 12 hours after dosing. Fecal excretion accounted for about 13% of the low dose and 25% of the high dose, again mainly in the first 12 hours after dosing. CO2 was collected from two animals/dose level and accounted for 46% of the low dose and 13% of the high dose radioactivity. GI tract and contents had high levels of radioactivity at 6 hours but only 1.5% of the dose at 48 hours. Liver levels were 4-5% of the dose level, declining slightly from 6 to 48 hours. Blood, lung and kidney levels accounted for less than 1% of the dose at all intervals. Total recovery in this study was low (Novak & Ambrose, 1983a). Groups of 15 male Hartley guinea pigs were given 2 ml oral doses of aqueous solutions of 14C-UDMH at about 5 or 150 ppm (actually 5.6 or 186 ppm). About 16-19% was recovered in urine within 48 hours with most recovered in the first 4 hours. Fecal excretion accounted for 6% of the low dose and 12% of the high dose and was recovered mainly in the first 24 hours. CO2 was collected from two animals/dose level and accounted for about 40% of the dose at both dose levels. GI tract and contents contained about 16% of the dose at 2 hours but 2-4% of the dose at 48 hours. Liver levels were 17-19% of the dose at 2 hours declining to 10% of the dose at 48 hours. Blood levels were about 3% at 2 hours and 1% at 48 hours (Novak et al. 1983b). Autoradiography of the stomach and duodenum from these animals indicated that in the stomach radiolabel was localized in epithelial cells and in the duodenum in the lower mucosa. The muscularis layer in both tissues contained very little radioactivity (Lengen & Frederick, 1984). Biotransformation Tissue analyses indicated that, of the administered dose, unchanged daminozide comprised 0-30%, dimethyl hydrazine 0-40%, 1,1-dimethylhydrazine 0-3%, and unidentified metabolites 1-30%, suggesting that 1,1-dimethylhydrazine is a transient metabolite (Frederick et al. 1984a,b). Toxicological studies Short-term study Dog Groups of 6 male and 6 female beagle dogs were given diets containing 0, 300, 3000 or 7500 ppm of daminozide (99% pure) for one year. Clinical observations, body weight and food consumption were recorded throughout the study. Physical examinations were made every 3 months and ophthalmoscopic examinations pre-test and at termination. Blood samples were taken at 0, 6 and 12 months for analyses of hematological and clinical chemistry parameters. Urine samples were collected at the same intervals. One female dog at 7500 ppm died with acute hemorrhagic enteritis on Day 336. The males at 3000 and 7500 ppm had body weights 5% and 5-8%, respectively, higher than controls by the end of the study. The biological significance of these findings is doubtful. There were no other apparent treatment-related effects at any dose level. One female at 7500 ppm had a renal cell adenoma but no other kidney effects were noted in any other animal. There were a few difficulties in the conduct of the study; achieving homogeneity of mixing of the diet at the low dose level and observations of convulsions in a few of the dogs including one control. However, the NOAEL (considering the body weight effects to be treatment-related but the renal cell adenoma incidental) was 7500 ppm (equal to 193-209 mg/kg bw/day (males/females) (Johnson et al. 1988c). Long-term/carcinogenicity studies Rats Groups of 50 male and 50 female Charles River CD-1 mice were given diets containing 0, 300, 3000, 6000 or 10000 ppm daminozide (99.8% pure) for 105 weeks. Clinical observations, body weight gain and food consumption were measured throughout the study. Blood samples were taken from 10/sex/group for hematological studies at 12, 18 and 24 months. All survivors to 105 weeks were sacrificed. All animals which died or were sacrificed were given a complete post-mortem examination. Sections of kidney, liver and lungs from all animals were examined for histopathology. Sections of about 37 other tissues/organs were examined for the sacrificed control and high dose animals, and all animals which died or were sacrificed in extremis from all groups. Mortality was slightly higher in the high dose males than in the other groups: 50% mortality was observed at week 85 in the high dose males compared to weeks 96-103 for other groups of males and weeks 99-105 for all groups of females. The high dose females had slightly lower bodyweights (approx. 5%) than controls during weeks 1-7 but all groups were similar for the rest of the study. RBC counts were lower than controls in the high dose males at 18 months and the high dose females at 24 months. Platelet counts were very variable; mean values were lower than controls in females at 3000, 6000, and 10000 ppm but no pattern was obvious in males. In males at 6000 and 10000 ppm there were increased incidences of brown pigment (probably hemosiderin and bile pigments) in the liver. Brown pigment was observed in the adrenal cortex of both sexes at 10000 ppm and increased extramedullary hematopoiesis in spleen was observed in males at 10000 ppm. Lymph node congestion was increased in both sexes at 10000 ppm and in males at 6000 ppm. There were high incidences of lung tumours in all groups. The incidences of adenomas were slightly higher in treated groups but there was no dose relationship observed. Hemagiomas and hemangiosarcomas were observed in all groups of animals, chiefly in liver in males and liver and uterus in females. The incidences of hemangiosarcomas at any site was slightly higher in the 10000 ppm males and females than controls (8 vs 4 in both cases). However, control incidences were higher than in any other treated groups (0-2/group). The earliest observation of an hemangiosarcoma (at death of animal) was 477 days in control males, 415 days in males at 10000 ppm, 530 days in control females and 585 days in females at 10000 ppm. Mean day of death of animals dying because of hemangiosarcomas was generally similar. The increased incidence of hemangiosarcomas was not statistically significant and, therefore, it is concluded that no carcinogenic effect was demonstrated. The NOAEL for non-neoplastic effects in this study was 3000 ppm (equal to 396 mg/kg bw/day) (Johnson et al. 1988a). Rats Groups of 60 male and 60 female Charles River Fischer 344 rats/dose level were given diets containing 0, 100, 500, 5000 or 10000 ppm daminozide (99.8% pure and containing 29 ppm UDMH) for 105 weeks. Clinical observations, body weights and food consumption were measured throughout the study. Ophthalmoscopic examinations were made pre-test and at 12 and 24 months. Blood samples were taken from the same 10 rats/sex/group when possible at 6, 12, 18 and 24 months for hematological and clinical chemistry analyses. Urine was collected during the fasts preceding blood sampling. At 12 months 10 rats/sex/group were sacrificed; the survivors among the remaining animals were sacrificed at 24 months. From these animals organ weights were recorded for adrenal, brain, heart, kidney, liver, ovary and testes. All animals which died or were sacrificed were given a complete post-mortem examination and samples of about 36 tissues/organs were saved. All tissues from all control and high dose animals and all animals which died were examined for histopathology. Liver, lung, kidney, ovary and gross lesions were examined for low and mid-dose animals. In this study there was considerable variability in the weekly concentrations achieved but the diets were within ± 20% of the target. Overall mean concentrations were very close to the nominal concentrations. There was no effect on survival in this study. Survival at 105 weeks was 46-72% in males (highest in the high dose group) and 70-78% in females. There appeared to be a slight increase in number of males with withdrawn testes at 10000 ppm during the later weeks of the study. In females at 10000 ppm heart/body weight ratio was higher than controls The only other non-neoplastic pathological change which may have been related to treatment was slightly increased incidence of atrophy of the ovary in all treated groups. There were no apparent treatment-related increases in tumour incidences. Although some ovarian atrophy appeared to occur at all dose levels, the degree was slight at dose levels of 5000 ppm and less. Therefore, 5000 ppm (equal to 243 mg/kg bw/day) appeared to be a NOAEL in this study (Johnson et al. 1988b). Reproduction study Rat In a two-generation reproduction study, groups of 25 male and 25 female Charles River Crl:CD(SD)BR rats were given diets containing 0, 100, 1000 or 10000 ppm daminozide (99.3% pure) during growth, mating, gestation and lactation for one litter per generation. The rats were mated on a one to one basis after 10 weeks of dietary exposure to produce the F1 litters. From these litters 25 rats/sex/group were selected to be parents for the F2 litters. In both generations females given 10000 ppm consumed more food than controls during the pre-mating stages of the study. There was a slight tendency toward increased food intakes in the F0 males at 10000 ppm but not in the F1 males. The F1 males had reduced body weight gains in he latter part of the study period. Fertility was not affected but there was some indication of an increase in the pre-coital interval in the 10000 ppm F1 - F2 mating in the 10000 ppm group. Pup weight at weaning was slightly lower than in other groups in the F1 10000 ppm pups. No pathological lesions were observed. The possible effects, although slight, showed some consistency and in the absence of additional litters, a conservative NOAEL of 1000 ppm (equivalent to about 50 mg/kg bw/day) was established (MacKenzie et al. 1987). Special studies on mutagenicity UDMH was negative in three of four in vitro mutagenicity studies. in the fourth a weakly positive result was obtained but there was some concern about the methodology used since HC1 was used as the diluant. An initial forward gene mutation assay gave equivocal results but on repetition the assay was determined to be negative (Table 1). TABLE 1. RESULTS OF MUTAGENICITY ASSAYS ON UDMH CONCENTRATION TEST SYSTEM TEST ORGANISM OF UDMH RESULTS REFERENCE Ames testa Salmonella typhimurium 25-5000 ug/plate Negative Stankowski & Naismith, 1986 TA1535, TA1537, TA1538 TA98, TA100 Chromosome aberration Chinese hamster ovary cells 5-5000 ug/ml Weakly San Sebastien & Naismith, analysisa (CHO-K1-BH4, lot #A-12) positiveb 1986 DNA repair test Rat hepatocytes 8.3-250 ug/ml Negative Barfknecht & Naismith, 1986 Forward gene mutation Chinese hamster ovary cells 50-1000 ug/ml Equivocal Stankowski & Naismith, 1987 assaya (CHO-K1-BH4) (study) repeated below) Forward gene mutation Chinese hamster ovary cells 50-1000 ug/ml Negative Stankowski, 1988 a,c (CHO-K1-BH4) a With and without metabolic activation. b The appropriateness of the methodology is questions. HC1 was used as the diluant. c A repeat confirmatory study to replace the study above. Special study on teratogenicity Rabbit In a range-finding study, groups of 5 female virgin Dutch-Belted rabbits were inseminated artificially. Daminozide (99% pure) was administered by gavage at a dose of 0, 100, 300, 1000, 2000 or 3000 mg/kg bw/day Days 7-29 of gestation. Day of insemination was designated Day 0 of gestation. Clinical observations and body weights were recorded during gestation. On Day 28 the females were sacrificed and uteri examined. At doses of 1000, 2000, and 3000 mg/kg bw/day all females died by Day 25, 15 and 9, respectively. Soft stool and/or diarrhea were noted prior to death in these animals and also in 3/5 and 4/5 females at 100 and 300 mg/kg bw/day, respectively. One dam at 300 mg/kg bw/day delivered on Day 28 prior to sacrifice. One female at 300 mg/kg bw/day had non-viable fetuses as well as viable fetuses in utero (Schardein et al. 1985a). As a result of the above study the main study was done with doses of daminozide of 0, 50, 150, or 300 mg/kg bw/day. Groups of 16 artificially inseminated virgin female Dutch Belted rabbits were dosed by gavage Days 7 through 19 of gestation (Day of insemination = Day 0 of gestation). Clinical observations, body weights and food consumption were recorded throughout the study. On Day 28 all surviving females were sacrificed and the ovary and uterus of each examined. All fetuses were weighed and examined for external, visceral and skeletal malformations. One rabbit at 300 mg/kg bw/day died Day 12. Diarrhea and absence of stool were noted only in some females at 150 and 300 mg/kg bw/stool. There were no apparent effects on reproductive performance. There was no evidence of embryotoxicity or teratogenicity. Because of the deaths observed in the range-finding study, the death at 300 mg/kg bw/day might be treatment-related although it occurred earlier than would be expected. A conservative NOAEL for maternal toxicity would be 150 mg/kg bw/day. Only some stool changes were noted at this level. The NOAEL for teratogenicity was 300 mg/kg bw/day (Schardein et al. 1985b). COMMENTS When administered orally, daminozide was rapidly excreted by rats and guinea pigs. Absorption appeared to be dose-related with larger proportions of the dose being excreted in feces at higher dose levels. In rats, the primary route of excretion was in the feces, with lesser amounts in urine and as expired CO2 and tissue analysis indicated biotransformation to unsymmetrical dimethylhydrazine (UDMH, 1,1-dimethylhydrazine) and then to 1,1-dimethylhydrazone. Radiolabelled UDMH administered orally to guinea pigs was excreted primarily as CO2. The concentration in the liver tended to be high, with 10% of the dose remaining in this organ after 2 hours. The NOAEL in a one-year feeding study with daminozide to dogs was 7500 ppm (equal to 200 mg/kg bw/day). In a 2-year feeding study in mice, a level of 10000 ppm dietary daminozide (containing 29 ppm UDMH) resulted in the accumulation of brown pigment in the liver. Although the incidence of pulmonary adenomas was slightly higher in all treated groups than in controls, it was neither statistically significant (except for males at 6000 ppm) nor dose-related. The incidence of hepatic and uterine hemangiosarcomas were increased in males and females respectively at 10000 ppm, but these increases were also not statistically significant. The Meeting therefore concluded that daminozide was not oncogenic in mice. The NOAEL was 3000 ppm (equal to 396 mg/kg bw/day). Daminozide (containing 29 ppm UDMH) was not oncogenic in a 2-year feeding study in rats. The NOAEL was 5000 ppm (equal to 243 mg/kg bw/day) based on ovarian atrophy. Daminozide was not embryotoxic or teratogenic in rabbits at 300 mg/kg bw/day, although maternal toxicity was observed at doses above 150 mg/kg bw/day. In a 2 generation, 1 litter per generation reproduction study in rats, the NOAEL was 1000 ppm in the diet (equivalent to 59 mg/kg bw/day). UDMH was weakly positive in a chromosomal aberration assay but not in other mutagenicity assays. The Meeting was informed that carcinogenicity bioassays of UDMH are in progress. UDMH, which is a contaminant and degradation product of daminozide, occurs as a trace component of daminozide residues in food (see Residues Section). Since the daminozide used in the carcinogenicity bioassays was contaminated with UDMH, and because daminozide is partially transformed to UDMH in vivo, the Meeting concluded that both compounds had been adequately bioassayed, and therefore the available data are relevant to the assessment of daminozide and its residues. TOXICOLOGICAL EVALUATION Level causing no toxicological effect Mouse: 3000 ppm in the diet, equal to 396 mg/kg bw/day Rat: 1000 ppm in the diet, equivalent to 50 mg/kg bw/day (based on reproduction) Dog: 7500 ppm in the diet, equal to 200 mg/kg bw/day. Estimate of acceptable daily intake for humans 0-0.5 mg/kg bw * Studies which will provide information valuable to the continued evaluation of the compound 1. Results of ongoing carcinogenicity bioassays of UDMH in rats and mice. 2. Quantitative data on the conversion of daminozide to UDMH in experimental animals. 3. Observations in humans. * Daminozide containing less than 30 ppm UDMH. REFERENCES Barfnecht, T.R. & Naismith, R.W. (1986) Rat Hepatocyte Primary Culture/DNA Repair Test - UDMH. Unpublished Report PH 311-UN-001-86 from Pharmakon Research International, Inc. Submitted to WHO by Uniroyal, Inc. Chadwick, M. & Silveira, D.M. (1987) Dermal absorption of alar in rats. Unpublished Report from Arthur D. Little, Inc. Submitted to WHO by Uniroyal, Inc. Frederick, C.B., Tortora, N.J., Lengen, M.R., Fitzpatrick, K.C., Sullivan, E.A. & Dzialo, D.G. (1984a) Analysis of the residues of daminozide in treated guinea pigs. Uniroyal Chemical Project. No. 8308. Submitted to WHO by Uniroyal, Inc. Frederick, C.B., Abdel-Kader, M.H., Peterson, G.S., Lengen, M.R. & Tortora, N.J. (1984b) Structure analysis of the principal residues in the urine of daminozide-dosed guinea pigs. Uniroyal Chemical Project. No. 8308B. Submitted to WHO by Uniroyal, Inc. Johnson, D.E., Frith, C.H., Laughlin, K.A. & Blair, M. (1988a) Alar technical (Daminozide) - Two-year oncogenicity study in mice. Unpublished Report No. IRDC 399-054 from International Research and Development Corporation. Submitted to WHO by Uniroyal, Inc. Johnson, D.E., Rajasekaran, C.H., Frith, C.H., Laughlin, K.A. & Blair, M. (1988b) Alar technical (Daminozide) - Two-year oncogenicity study in rats. Unpublished Report No. IRDC 399-055 from International Research and Development Corporation. Submitted to WHO by Uniroyal, Inc. Johnson, D.E., Rajasekaran, C.H., Blair, L.S. & Blair, M. One-year dietary toxicity study in dogs - Alar technical. Unpublished Report No. IRDC 399-066 from International Research and Development Corporation. Submitted to WHO by Uniroyal, Inc. Longen, M.R. & Frederick, C.B. (1984) Distribution of radioactivity in stomach and duodenum from guinea pigs dosed with [14C-CH3]- 1,1-dimethylhydrazine, Uniroyal Chemical Co. Project No. 8433. Unpublished report submitted by Uniroyal, Inc. MacKenzie, K.M. (1987) Two-generation reproduction study with Alar in rats (one litter per generation). Final report. Unpublished Report No. (HLA) from Hazleton Laboratories America, Inc. Submitted to WHO by Uniroyal, Inc. Milad, G., Gottfried, G.J. & Dillon, F. (1984) Balance study of 14C-Labelled daminozide in the rat. Unpublished Report from Biospherics, Inc., Rockland, Md. Submitted to WHO by Uniroyal, Inc. Novak, R.A. & Ambrose, S.M. (1983a) Daminozide distribution and excretion study in guinea pigs. Unpublished Report No. 2308B from Borriston Laboratories, Inc. Submitted to WHO by Uniroyal, Inc. Novak, R.A. & Ambrose, S.M. (1983b) 1,1-dimethyl hydrazine. Bioavailability study in guinea pigs. Unpublished Report No. 2308A from Borriston Laboratories, Inc. Submitted to WHO by Uniroyal, Inc. San Sebastian, J.R. & Naismith, R.W. (1986) In vitro chromosome aberration analysis in Chinese hamster ovary (CHO) cells-UDMH. Unpublished Report No. PH 320-UN-002-86 from Pharmakon Research International, Inc. Submitted to WHO by Uniroyal, Inc. Schardein, J.L., Aldridge, D. & Blair, M. (1985a) Alar range finding teratology study in rabbits. Unpublished Report No. 399-056 from International Research and Development Corporation (IRDC). Submitted to WHO by Uniroyal, Inc. Schardein, J.L., Arnold, K.S. & Blair, M. (1985b) Alar teratology study in rabbits. Unpublished Report No. 399-059 from International Research and Development Corporation (IRDC). Submitted to WHO by Uniroyal, Inc. Stankowski, L.F. & Naismith, R.W. (1986) Ames/ Salmonella plate incorporation assay - UDMH. Unpublished Report PH 301-UN-005-86 from Pharmakon Research International, Inc. Submitted to WHO by Uniroyal, Inc. Stankowski, L.F. (1988) Unsymmetrical dimethylhydrazine (UDMH) - CHO/HPRT mammalian cell forward gene mutationa assay. Unpublished Report PH 314-UN-001-88 from Pharmakon Research International, Inc. Submitted to WHO by Uniroyal, Inc. Stankowski, L.F. & Naismith, R.W. (1987) CHO/HPRT mammalian cell forward gene mutation assay - UMDH. Unpublished Report PH 314-UN-001-86 from Pharmakon Research International, Inc. Submitted to WHO by Uniroyal, Inc.
See Also: Toxicological Abbreviations Daminozide (Pesticide residues in food: 1977 evaluations) Daminozide (Pesticide residues in food: 1983 evaluations) Daminozide (Pesticide residues in food: 1991 evaluations Part II Toxicology)