ZIRAM First draft prepared by J.-J. Larsen, Institute of Toxicology, National Food Agency, Ministry of Health, Soborg, Denmark Explanation Evaluation for acceptable daily intake Biochemical aspects Absorption, distribution, and excretion Biotransformation Toxicological studies Acute toxicity Short-term toxicity Long-term toxicity and carcinogenicity Reproductive toxicity Developmental toxicity Genotoxicity Special studies Dermal and ocular irritation and dermal sensitization Neurotoxicity Observations in humans Comments Toxicological evaluation References Explanation Ziram was evaluated for toxicological effects by the Joint Meeting in 1965, 1967, 1970, 1974, 1977, and 1980 (Annex 1, references 4, 8, 14, 22, 28, and 34). A temporary ADI of 0-0.025 mg/kg bw for ziram or ziram in combination with other dimethyldithiocarbamates was allocated in 1967, on the basis of the NOAEL in a one-year study in dogs. This temporary ADI was lowered to 0-0.005 mg/kg bw in 1974. A group ADI of 0-0.02 mg/kg bw for ferbam and ziram was allocated in 1977 and confirmed in 1980. The compound was reviewed by the present Meeting within the CCPR periodic review programme. This monograph summarizes the data received since the previous evaluation and contains relevant summaries from the previous monograph and monograph addenda on ziram (Annex I, references 4, 9, 15, 23, 29, and 35). Evaluation for acceptable daily intake 1. Biochemical aspects (a) Absorption, distribution and excretion A group of 71 rats were given a single oral dose of 15 mg/kg bw; 14 daily doses of 15 mg/kg bw nonradiolabeled ziram (purity, 99%) followed by a single dose of 14C-ziram (radiochemical purity, 96.9%) on day 15; or a single dose of 352 mg/kg bw 14C-ziram. Urine, faeces, expired carbon dioxide, and volatile organic compounds were collected from all animals 0-4, 4-8, 8-12, and 12-24 h after treatment. The rats were killed seven days after administration of the radiolabelled dose. The mean recovery of radiolabel was 79-92% of the total dose administered. Most of the radiolabel was eliminated within 24 h after administration of the low dose and within 48 h of the high dose. Most radiolabel (36-53%) was found in expired air, with 17-35% in urine and 9-18% in faeces. The amounts retained in tissues and carcass represented 1-2% of the total dose. The highest residue levels were found in blood, liver, kidney, heart, lungs, spleen, and thyroid gland. No apparent sex-related differences were observed in the elimination or distribution of 14C-ziram (Cheng, 1991a). The extent of absorption of 14C-ziram was studied in 74 rats given single dermal doses of 0, 1.1, 12, or 91 mg per animal (0.086, 0.95, or 7.25 mg/cm2). Urine, faeces, blood, skin at the application site, and the carcass were collected from four animals killed 0.5, 1, 2, 4, 10, or 24 h after initiation of exposure to ziram. A mean of 74-101% of the dose was recovered. Most of the radiolabel (68-99%) was washed off the skin at the application site; only 16% remained on or in the skin. Less than 0.3% of the administered radiolabel was retained in the carcass and was eliminated in the excreta within 24 h after exposure. The mean amount absorbed (sum of radiolabel in urine, carcass, and skin at the test site) by 24 h was 29% of the administered dose in animals at 1.1 mg, 31% in those at 12 mg, and 5% for those at 91 mg, indicating non-linear dermal absorption (Cheng, 1991b). (b) Biotransformation Three lactating goats weighing about 40 kg were fed 14C-ziram for six consecutive days at concentrations of 0 or 300 ppm, equal to 0 or 12.5 mg/kg bw per day. All milk, urine, and faeces were collected for radioanalysis. The animals were killed 6 h after the last dose and the organs were removed. The concentrations of radiolabel in the milk appeared to reach a plateau by day 3, at 0.6-1.8 ppm. In the edible tissues, the concentrations of radiolabel were 22-28 ppm in the liver, 3 ppm in the kidneys, and < 1 ppm in fat and muscle. The total amounts of radiolabel excreted during the test period were 42-61% of the administered dose in faeces, 3% in urine, and 0.28-0.51% in milk. Dithiocarbamates represented maxima of 28 and 10% of the total radioactive residues in urine and liver, respectively, and undetectable amounts in other tissues. N-Nitrosodimethylamine residues (detection limit, 1 ppb) were not found. Radiolabelled lactose and casein were isolated from milk and radiolabelled urea from urine, indicating that the metabolism of ziram in goats occurs at least in part via a single-carbon pathway, which results in extensive radiolabelling of natural products. The author concluded that the bound and extractable radioactivity in milk and tissues was derived largely from radiolabelled natural products rather than from discrete dithiocarbamate metabolites (Bodden, 1993). The expected metabolic pathways of ziram, on the basis of data on other dimethyldithocarbamates, are shown in Figure 1.2. Toxicological studies (a) Acute toxicity The acute toxicity of ziram is summarized in Table 1. (b) Short-term toxicity Mice Ziram (purity 97.6%) was administered orally to C57B1 and A mice at doses of 0 or 75 mg/kg bw twice a week (equivalent to 20 mg/kg bw per day) for 2.5 months. A positive control group was exposed in the same way to urethane. The presence of adenomas in the lungs and the liver was studied after the end of the exposure at intervals of 1.5 months for six months; adenomas were found only in the lungs. In the A mice, adenomas occurred in 100% of animals given urethane, 51% of those given ziram, and 43% of the controls. The differences were not statistically significant. The first adenomas were seen after three months. In C57B1 mice, adenomas occurred in 26% of mice given urethane, 7.4% of those given ziram, and none of the controls. The results were statistically significant according to Student's t test ( t = 2.08; p = 0.05) but not according to the chi-squared test (chi-squared = 2.18; p = 0.15) (Khicenko & Chernev, 1968; Annex 1, reference 15). In a range-finding study, groups of five male and five female Crl.CD(ICR)BR mice were fed ziram (purity, 89.5%) at concentrations of 0, 3000, 4000, or 5000 ppm, equal to 0, 370, 510, or 740 mg/kg bw per day for males and 0, 420, 660, or 780 mg/kg bw per day for females, for four weeks. The body weight, food intake, and efficiency of food use were significantly reduced at all doses. The brain and heart weights were reduced in all female mice, and the heart weight was also reduced in males at 5000 ppm. There was no NOAEL (Chambers et al., 1992a). In another preliminary study, groups of 10 male and 10 female Crl:CD-1 mice were fed ziram (purity, 98.7%) at concentrations of 0, 100, 300, 900, or 2700 ppm, equal to 0, 15, 44, 120, or 360 mg/kg bw per day for males and 0, 19, 49, 130, or 410 mg/kg bw per day for females, for 13 weeks. Clinical signs, body weight, food and water consumption, and efficiency of food use were recorded. On completion of treatment, all mice were sacrificed for macroscopic and microscopic examination. Over week 1, a reduction was noted in food consumption and use in animals of each sex and in the body weight of males and the body-weight gain of females at 2700 ppm. Lower weight gain was also noted during the study in males and females at 900 ppm. A lower spleen weight was noted in animals of each sex at 900 and 2700 ppm and in females at 300 ppm. Uterine weight was reduced at 2700 ppm. An increased incidence of hyperkeratosis of the non-glandular epithelium Table 1. Acute toxicity of ziram in experimental animals Species Sex Route Purity LD50 (mg/kg bw) Reference or LC50 (mg/litre) Rat Male Oral 98.5 380 Liggett & Allan (1989a) Female 270 Rat Male Inhalation (4 h) 98.5 0.08 Jackson & Hardy (1989) Female 0.06 Rat Male Inhalation (4 h) 98.4 0.18 Blagden (1991) Female 0.08 Rabbit NR Oral NR 400 Annex I, reference 4 Rabbit Male Dermal 98.5 > 2000 Liggett & Allen (1989b) Female > 2000 NR, not reported in the stomach was seen among mice given 900 or 2700 ppm. Epithelial hyperplasia of the limiting ridge in the stomach was also seen at 2700 ppm. Contraction of the spleen and extramedullary haematopoiesis in the spleen were observed in mice at 900 and 2700 ppm. Corpora lutea were absent in one mouse at 900 ppm, and recent corpora lutea were absent in two mice at 2700 ppm and in one at 900 ppm. The uteri of some of these mice showed changes consistent with the ovarian changes, i.e. cellular stroma and reduced luminal diameter. These changes were not seen at 0, 100, or 300 ppm. The NOAEL was 100 ppm, equal to 15 mg/kg bw per day, on the basis of decreased spleen weight at doses > 300 ppm (Powell et al., 1993). Rats Groups of 10 male and 10 female weanling rats were fed ziram (purity unspecified) at concentrations of 0, 100, 500, 2500, or 5000 ppm (equivalent to 0, 10, 50, 250, or 500 mg/kg bw per day) for 28 days. Growth retardation was marked in animals at 2500 and 5000 ppm; a slight retardation was also seen in those at 500 ppm, but growth was normal in those at 100 ppm. Except for slight anaemia at 2500 and 5000 ppm, the haematological findings were normal and no significant histopathological changes were seen in any of the animals. The NOAEL was 100 ppm, equivalent to 10 mg/kg bw per day, on the basis of growth retardation at doses > 500 ppm (Annex I, reference 4). In a separate experiment, 10 male and 10 female rats were maintained for 28 days on a diet containing 0 or 2500 ppm ziram. No thyroid abnormalities were seen (Annex I, reference 4). Groups of 15 male and 15 female Wistar rats were given ziram (purity, 98%) by gavage at doses of 0, 3, 15, or 100 mg/kg bw per day for four weeks. Food consumption, efficiency of food use, and body weights were reduced significantly in males at the highest dose and in females at 15 and 100 mg/kg bw per day. End-points of haematology and clinical chemistry did not show clear dose-dependent changes, except for an increase in aspartate transaminase and alkaline phosphatase activities and blood urea nitrogen values at the highest dose. The liver weight was increased in males at this dose, and degenerative changes in the liver and kidneys were seen in animals of each sex. Degenerative liver changes were also seen in males and females at 15 mg/kg bw per day. The NOAEL was 3 mg/kg bw per day, on the basis of decreased body weight, food consumption, and food use and degenerative liver changes at doses > 15 mg/kg bw per day (Dickhaus & Heisler, 1980). In a range-finding study, groups of five male and five female Crl:CD(SD)BR rats were fed ziram (purity, 98.5%) at concentrations of 0, 500, 1000, or 2000 ppm (equal to 0, 46, 90 or 170 mg/kg bw per day for males and 0, 46, 85, or 170 mg/kg bw per day for females) for four weeks. The body weight, food intake, and efficiency of food use were significantly reduced in animals of each sex at all doses. In males, the absolute weights of the liver, pituitary, and testes were reduced at all doses. The absolute weights of the brain in animals of each sex and of the uterus were also reduced at the highest dose. There was no NOAEL (Chambers et al., 1992b). In another range-finding study, groups of 10 male and 10 female Crl:CD(SD)BR rats were fed ziram (purity, 98.7%) at concentrations of 0, 100, 300, or 1000 ppm (equal to 0, 7.4, 21, or 68 mg/kg bw per day for males and 0, 8.8, 24, or 77 mg/kg bw per day bw for females) for 13 weeks. The body-weight gain, food intake, and food use were dose-dependently reduced at 300 and 1000 ppm. Brain weight and spleen weight were increased and a higher incidence of hair loss was noted at 300 and 1000 ppm. Epithelial hyperplasia of the nonglandular stomach was seen at 1000 ppm. The NOAEL was 100 ppm, equal to 7.4 mg/kg bw per day, on the basis of decreased body-weight gain, food intake, and food use and increased brain and spleen weights and hair loss at doses > 300 ppm (Powell et al., 1992). Rabbits Ziram (purity 98.5%) was applied to the intact skin of groups of five male and five female New Zealand white rabbits, weighing 2.2-2.6 kg, daily for 21 consecutive days at doses of 0, 100, 300, or 1000 mg/kg bw per day. The test substance was moistened with distilled water and maintained on the backs of the rabbits for 6 h each day, after which the dressings were removed and the treated skin washed with tap-water at 30-40°C and gently blotted dry. No dermal reaction to the treatment was observed at any dose. Significant losses in body weight or low body-weight gain and reduced food consumption were recorded for female rabbits receiving 1000 mg/kg bw. The number of lymphocytes was reduced in both males and females at 1000 mg/kg bw, and alanine and aspartate transaminase activities were increased at 300 and 1000 mg/kg bw. At the highest dose, significantly increased levels of bilirubin were found in females and of cholesterol in animals of each sex. The NOAEL was 300 mg/kg bw per day, on the basis of decreased body weight, body-weight gain, food consumption, and number of lymphocytes and increased bilirubin and cholesterol levels at 1000 mg/kg bw per day (Edwards et al., 1989). Dogs In a range-finding study, single male and female beagle dogs were fed ziram (purity, 89.5%) at concentrations of 1000, 2000, or 5000 ppm (equal to 38, 78, or 260 mg/kg bw per day for males and 28, 91 or 270 mg/kg bw per day for females) for four weeks; there was no control group. Both dogs at 5000 ppm and the female at 2000 ppm were killed for humane reasons due to loss of body weight and deteriorated condition. Convulsive episodes of up to 5-min duration were seen in females at 2000 ppm. Body-weight loss was seen in both animals at 5000 ppm and in the female at 1000 ppm. Food intake and efficiency of food use were reduced in females at all doses and in males at 5000 ppm. Increased liver weight in comparison with historical controls in the laboratory was seen in males at all doses. No histopathological examinations were performed. There was no NOAEL (McLean et al., 1992a). In another range-finding study, groups of four male and four female beagle dogs were fed ziram (purity, 98.5%) in the diet at concentrations of 0, 100, 300, or 1000 ppm (equal to 0, 4.1, 12, or 42 mg/kg bw per day for males and 0, 4.3, 13, or 41 mg/kg bw per day for females) for 13 weeks. One of the four dogs at 1000 ppm suffered a seizure and was killed for humane reasons, and another had tremors. Animals at this dose showed reduced body weight and food intake; lowered erythrocyte count, packed cell volume, and haemoglobin; and elevated circulating reticulocyte counts, with associated slight polychromasia, hypochromasia, and anisocytosis. Activated partial thromboplastin time was increased in male dogs at 300 or 100 ppm. Alkaline phosphatase activity was elevated in females at 1000 ppm, and the cholesterol level was elevated in females at 300 and 1000 ppm. The albumin concentration was decreased and that of globulin increased in some dogs at all doses during week 13. The liver weight was increased in male and female dogs at 1000 ppm and in one dog at 300 ppm. Focal myocardial necrosis with acute inflammation and haemorrhage were seen in the dog at 1000 ppm that was killed for humane reasons. Focal necrosis in the liver with loss of cells and dilated sinusoids were seen in one dog at 1000 ppm, and focal necrosis was also seen in a dog receiving 300 ppm. Minimal amounts of pigment in Kupffer cells were observed in two dogs at 300 ppm and in three dogs at 1000 ppm. The NOAEL was 100 ppm, equal to 4.1 mg/kg bw per day, on the basis of increased liver weight, focal liver necrosis, pigment in Kupffer cells, increased activated partial thromboplastin time, and an elevated cholesterol level at doses > 300 ppm (McLean et al., 1992b). Groups of four male and four female beagle dogs were fed ziram (purity, 98.5%) in the diet to give concentrations of 0, 50, 180, or 700/500 ppm (equal to 0, 1.6, 6.6, or 17 mg/kg bw per day for males and 0, 1.9, 6.7, or 21 mg/kg bw per day for females) for one year. The highest dose was reduced from 700 to 500 ppm in week 12 due to treatment-related deaths. All animals were killed and necropsied after one year. Body-weight gain was reduced in females at 180 and 500 ppm. Alanine transaminase, alkaline phosphatase, and, occasionally, aspartate transaminase activities were elevated in males receiving 180 or 500 ppm; and the liver weights of males receiving 500 ppm were increased. Reduced albumin values and raised cholesterol levels were found for male dogs receiving 500 ppm. In the liver, some foci of degenerated hepatocytes were seen in a female at 180 ppm and in a number of dogs at 500 ppm, single-cell necrosis was seen in one male dog at 500 ppm, and inflammatory-cell infiltration around the central veins and branches of the hepatic veins was seen in a proportion of these dogs. An increase in centrilobular fibrocytes was seen in one male dog at 180 ppm and three males at 500 ppm. Aggregates of pigmented Kupffer cells were observed in the livers of dogs of each sex in a dose-related incidence, including one male and one female at 50 ppm. An increased incidence and degree of pigmented macrophages were seen in the spleens of male dogs at 180 and 500 ppm. The NOAEL was 50 ppm, equal to 1.6 mg/kg bw per day, on the basis of decreased body-weight gain, degenerated hepatocytes, and increases in centrilobular fibrocytes, spleen macrophages, alanine transaminase and alkaline phosphatase activities, and pigmented Kupffer cells at doses > 180 ppm (Smith et al., 1993). (c) Long-term toxicity and carcinogenicity Mice Groups of 49 or 50 male and female B6C3F1 mice were fed ziram (purity, 89%; containing 6.5% thiram, 2% other zinc salts, and 2% unidentified additional impurities) at concentrations of 0, 600, or 1200 ppm (equal to 0, 120, or 200 mg/kg bw per day for males and 0, 130, or 250 mg/kg bw per day for females) for 103 weeks. All animals were observed daily for clinical signs, and body weight and food consumption were recorded monthly. At termination, necropsies were performed on all animals, and selected organs and tissues from most animals were examined microscopically. The survival of treated mice was not adversely affected. The body-weight gain of male mice at 600 and 1200 ppm and of female mice at 600 ppm was reduced, and food consumption was reduced in both males and females at 1200 ppm. The incidence of alveolar or bronchiolar adenomas was statistical significantly increased in female mice (2/50 controls, 5/49 at the low dose, 10/50 at the high dose), and the combined incidence of alveolar and bronchiolar adenomas and carcinomas in female mice showed a statistically significant, positive trend. The incidence in males at 1200 ppm was significantly higher than that in the controls (4/50 controls, 6/49 at the low dose, 11/50 at the high dose). Pulmonary adenomatous hyperplasia consistent with chronic Sendai viral infection was observed in control and treated animals. The authors concluded that any interpretation of the increase in lung tumours is complicated by the presence of intercurrent viral infection (National Toxicology Program, 1983). Groups of 50 male and 50 female Crl:CD-1(ICR)BR mice were fed ziram (purity, 98.7%) in the diet to give concentrations of 0, 25, 75, 225, or 675 ppm (equal to 0, 3, 9, 27, or 82 mg/kg bw per day for males and 0, 4, 11, 33, or 95 mg/kg bw per day for females) for 80 weeks. The concentrations of ziram given to the groups at 25 and 75 ppm were greater than the nominal concentration in order to compensate for losses during storage. Statistically significant reductions in body-weight gain and food intake were seen in males and females receiving 225 or 675 ppm. Dose-related, statistically significant decreases in the absolute brain weight of males receiving 225 or 675 ppm and the brain weight adjusted for final body weight of females receiving 75, 225, or 675 ppm were noted. Macroscopic examination of all animals that died or were killed at termination revealed increased incidences of reduced adipose tissue in males that died, irregular cortical scarring of the kidneys in males at terminal sacrifice, brown kidneys in males, and roughened and white forestomachs in females, all receiving the highest dose. An increased incidence of centrilobular and/or generalized hepatocellular enlargement was seen in all treated groups, and an increased incidence of urinary bladder epithelial hyperplasia was seen in males and females receiving 675 ppm and in males at 225 ppm. No treatment- related neoplastic effects were seen. The NOAEL was 25 ppm, equal to 3 mg/kg bw per day, on the basis of reduced brain weight and hepatocellular enlargement at doses > 75 ppm (Powell et al., 1994a). Rats Groups of 25 male and 25 female weanling rats were fed ziram at concentrations of 0, 25, 250, or 2500 ppm (equivalent to 0, 1.3, 13, or 125 mg/kg bw per day) for two years. The growth rate and life span were normal in all groups. Neurological changes were observed at 2500 ppm, but no cystic lesions were seen in the brain post mortem. No neurological changes were seen at the lower doses. In some males, the testes were atrophied and there was a slight indication of thyroid hyperplasia, notably at 2500 ppm. There was no increase in tumour incidence in the treated animals (Annex I, reference 4). Groups of 50 Fischer 344/N rats were fed ziram (purity, 89%; containing 6.5% thiram, 2% other zinc salts, and 2% unidentified additional impurities) at concentrations of 0, 300, or 600 ppm (equal to 0, 11, or 22 mg/kg bw per day for males and 0, 13, or 26 mg/kg bw per day for females) for 103 weeks. All animals were observed daily for clinical signs, and body weights and food consumption were recorded monthly. At termination, necropsies were performed on all animals, and selected organs and tissues from most animals were examined microscopically. The survival, body weight, and food consumption of treated rats were not adversely affected. C-Cell carcinomas of the thyroid occurred in male rats, with a statistically significant, positive trend ( p < 0.01); the incidence among animals at 600 ppm was significantly greater than that in controls (0/50 controls, 2/49 at the low dose, 7/49 at the high dose). The combined incidence of C-cell adenomas and carcinomas in males also showed a statistically significant, positive trend (4/50 controls, 9/49 at the low dose, 12/49 at the high dose). No significant histopathological changes were found in the follicular cells. There was no NOAEL, since the combined incidence of C-cell adenomas and carcinomas of the thyroid in males showed a statistically significant, positive trend at 300 ppm, the lowest dose tested (National Toxicology Program, 1983). In a study of toxicity, involving groups of 20 CD(SD)BR rats of each sex, and carcinogenicity, involving groups of 50 rats of each sex, the animals were fed diets containing ziram (purity, 98.7%) providing concentrations of 0, 60, 180, or 540 ppm (equal to 0, 3.0/2.5, 9.1/7.7, or 27/24 mg/kg bw per day for males and 0, 3.9/3.4, 12/10, or 38/35 mg/kg bw per day for females), for 12 or 24 months. Clinical signs, body weight, and food consumption were observed weekly, and ophthalmoscopic, haematological, biochemical, and urinary investigations were performed at certain intervals. At termination, the organ weights, haematological parameters, and the results of clinical chemical, urinary, and macroscopic and microscopic examinations were recorded. Body-weight gain, food intake, and food conversion ratios were reduced in a dose-dependent fashion in male and female rats at 180 and 540 ppm. The erythrocyte count was reduced dose-dependently in animals of each sex at 60, 180, and 540 ppm, and a dose-dependent decrease in thromboplastin time was observed in males at all three doses. At 180 and 540 ppm, packed cell volume and haemoglobin were reduced in females and thromboplastin time in males. Tri-iodothyronine, thyroxine, albumin, total protein, and calcium levels were reduced and that of blood urea nitrogen was increased at 180 and 540 ppm in animals of each sex. Haemangiomata in the mesenteric lymph nodes, hypertrophy with vacuolation of the adrenals, and C-cell hyperplasia of the thyroids were observed in males at 540 ppm. In females, cortical cystic degeneration of the adrenals was seen at 540 ppm. Dose-dependent adipose replacement, narrowing of the myofibres in skeletal muscle, haemosiderosis in the spleen, adipose tissue replacement in the pancreas, prominent ultimobranchial cysts in the thyroids, and epithelial hyperplasia and subepithelial oedema in the nonglandular part of the stomach were observed in animals at 60, 180, or 540 ppm. There was no NOAEL, since decreased erythrocyte counts and thromboplastin time, haemosiderosis in the spleen, ultimobranchial cysts in the thyroids, and epithelial hyperplasia in the stomach were seen at 60 ppm, the lowest dose tested (Powell et al., 1994b). (d) Reproductive toxicity Mice Groups of 10 CH3 or AK male mice were given ziram (purity unspecified) by gavage at concentrations of 0 or 0.2 mg% (equivalent to 0 or 0.02 mg/kg bw per day) for 21 days before mating with untreated females of the same strain. The males were then killed for histopathological examination. Some of the female mice were killed on day 17 of gestation in order to examine the numbers of corpora lutea, nidation places, and dead and live embryos; the other females were kept alive to breed. Newborn mice were immediately sacrificed and stained with alizarin for examination of skeletal abnormalities. Ziram increased the incidence of sterility: only 20% of the C3H and 80% of the AK females, but all of the controls, were fertilized. The mean number of embryos was reduced from 11.1 in controls to 7.5 in the CH3 mice and from 11.2 in controls to 9.1 in the AK mice. The calculated embryonic mortality was 4.5% in the CH3 strain and 6.3% in the AK strain (Abbota's formula). When the numbers of corpora lutea and of live and resorbed embryos were included, embryonic mortality rates of 27% for C3H mice and 32% AK mice were found (method of dominant lethals). Analysis of the testes showed atrophy of the seminiferous tubules and impaired spermatogenesis. Examination of meiotic chromosomes in diakinesis showed the presence of tetravalent and univalent chromosomes and frequent pyknosis, demonstrating the clastogenic effect of ziram on future gametes. The offspring of both CH3 and AK strains had anomalies of the spine, cyphosis, scoliosis, sternum ossification failure, branching ribs (some of which were incompletely ossified), assymetry of the cranium, microcephalus, and eviscerations. The bone dimensions of the CH3 mice were greater than those of the controls. There was no NOAEL for male fertility, because of decreased fertility, atrophy of seminiferous tubules, and impaired spermatogenesis, or for developmental toxicity, because anomalies of the spine, cyphosis, scoliosis, and sternum ossification failure were seen in the offspring at 0.02 mg/kg bw per day, the lowest dose tested (Cilievici et al., 1983). In the absence of important information on the design of the study, this report was not considered in establishing the ADI. Groups of six Swiss albino male mice, aged 12-14 weeks, received ziram (purity unspecified), dissolved in 5% dimethyl sulfoxide, intraperitoneally as single doses of 0, 50, or 100 mg/kg bw or repeated doses of 25 mg/kg bw per day for five days. The mice were killed one month after the treatment. The epididymides were isolated, minced in 4 ml NaCl, and dispersed by slow aspiration. The tissue particles were filtered out, and smears were made on clean, dry slides for microscopy. A minimum of 2000 sperm was scored per animal, and the abnormalities were classified. The frequency of abnormal sperm was 1.6% in the controls, 5.6% in animals at 50 mg/kg bw, 8.2% in those at 100 mg/kg bw, and 8.4% following repeated doses of 25 mg/kg bw per day. The abnormalities included sperm with acrosomes bent upwards, acrosomes bent downwards, and without acrosomes. Many head abnormalities, including double heads, banana heads, amorphous heads, microcephaly, and macrocephaly, were found, and a high frequency of coiled sperm was seen. The abnormalities observed were considered to be due either to changes in the genes responsible for spermatogenesis or to changes in differentiation during gene expression involving post-transcriptional stages and thereafter in the translation of the genetic message. There was no NOAEL, as sperm abnormalities were seen at 25 mg/kg bw per day, the lowest dose tested (Hemavathi & Rahiman, 1993). Rats In a two-generation study of reproductive and developmental neurotoxicity, groups of 30 male and 30 female Crl:CD BR Sprague- Dawley rats were fed ziram at concentrations of 0, 72, 210, or 540 ppm, equal to 0, 3, 10, or 25 mg/kg bw per day for males and 0, 5, 13, or 32 mg/kg bw per day for females. The parental animals received the diets from about six weeks of age for the F0 generation and on day 22 postnatally for the F1 generation, for at least 70 days before mating and throughout all subsequent phases of the study until termination of the generation. All animals were observed twice daily for appearance and behaviour. Body weights and food consumption were recorded at appropriate intervals. All females were allowed to deliver and rear their pups to weaning on lactation day 21. Offspring from the pairing of the F0 animals (30 pups of each sex per group) were selected to constitute the F1 generation. Thirty F2 pups of each sex per group were selected for testing of developmental landmarks and behaviour, neuropathological examination, and brain weight measurement. Surplus F1 pups were killed and necropsied on postnatal day 28, and surplus F2 pups were killed and necropsied on postnatal day 22. The F0 and F1 parental animals and selected F2 pups that were not allocated for measurements underwent detailed gross necropsy; and the weights of the brain, kidneys, liver, pituitary gland, ovaries, or testes and epididymides (F0 and F1 only) were recorded. Designated tissues from the controls and from F0 and F1 parental animals at 540 ppm and F2 pups selected for neuropathological evaluation were evaluated for histopathological changes. Reproductive parameters (fertility, mating, and days between pairing and coitus, gestation, and parturition) in the F0 and F1 generations were not adversely affected by concentrations of 72, 210, or 540 ppm ziram. All F0 and F1 parental animals survived to the scheduled necropsies, and no adverse clinical signs attributable to treatment were observed. The mean body weights and body-weight gains of F0 males at 540 ppm were reduced early in the treatment period, and the mean body weights and body-weight gains of F1 males at 540 ppm were generally reduced. The mean body weights and body-weight gains of F0 and F1 females at 540 ppm were generally reduced before breeding, during gestation and lactation, and after weaning. No adverse effects were seen on the body weights or body-weight gains of animals in either generation at 72 or 210 ppm. The food consumption of F0 and F1 males at 540 ppm was generally reduced throughout each generation. The food consumption of F0 and F1 females at 540 ppm was reduced before breeding, during gestation and lactation, and after weaning of the F0 and F2 generations. No adverse effects on food consumption were observed in males or females given ziram at concentrations of 72 and 210 ppm in either generation. No treatment-related, macroscopic internal changes were found in treated F0 or F1 animals, and no adverse effects on organ weights were observed at any dose. No microscopic lesions attributable to treatment were observed in tissues from animals at 540 ppm on histopathological examination. Microscopic examination of gross lesions seen at the scheduled necropsies of animals at 540 ppm did not reveal any adverse effects. The mean body weights of F1 and F2 pups in the litters of animals at 540 ppm were slightly reduced (usually statistically significantly) during lactation and throughout the remainder of the study until postnatal day 70 (selected F2 pups). The F1 and F2 pup sex ratios, live litter sizes, number of dead pups on lactation day 0, viability indices, general physical condition, and brain weight (selected F2 pups) were not adversely affected by parental treatment at any concentration. The findings at necropsy of F1 and F2 pups that died or were killed at the scheduled postnatal necropsies did not suggest any correlation with parental treatment. Various indicators of physical and functional development and behavioural responses in the selected F2 pups were comparable to those in controls. Neuropathological examinations on postnatal days 11 and 70 showed no gross or microscopic treatment-related lesions in the F2 pups. The NOAEL for maternal toxicity was 210 ppm, equal to 10 mg/kg bw per day, on the basis of reduced food consumption and body-weight gain; that for neonatal toxicity was 210 ppm, equal to 10 mg/kg bw per day, on the basis of reduced body weight; and that for reproductive and developmental neurotoxicity was 540 ppm, equal to 25 mg/kg bw per day (Nemec, 1996). (e) Developmental toxicity Rats In a preliminary study, groups of 10 pregnant Crl:CD(SD)BR rats were given ziram (purity, 98.9%) by gavage at doses of 0, 5, 20, or 80 mg/kg bw per day on days 6-15 of gestation. Dams were observed for clinical symptoms, body weight, and food and water consumption, and were killed on day 20 and examined for congenital abnormalities and macroscopic pathological changes in organs. The ovaries and uteri were examined to determine the number of corpora lutea, the number and distribution of live young, and the number of early and late embryo-fetal deaths and fetal abnormalities. Live young were examined externally and weighed; no further examinations were done. Dams showed body-weight loss up to day 8, decreased food intake, and increased water intake at doses of 5, 20, and 80 mg/kg bw per day. Hair loss was observed in dams at 20 and 80 mg/kg bw per day. Salivation and increased postimplantation losses were seen in dams at 80 mg/kg bw per day. Reduced litter and fetal weights were seen at 20 and 80 mg/kg bw per day. The dose range was considered to be too high for the main study (Smith et al., 1990). Groups of 25 pregnant Crl:CD(SD)BR rats were given ziram (purity, 98.9%) by gavage at doses of 0, 1, 4, 16, or 64 mg/kg bw per day on days 6-15 of gestation and were then observed for clinical symptoms, body weight, and food and water consumption. On day 20, the animals were killed and examined for congenital abnormalities and macroscopic pathological changes in organs. The ovaries and uteri were examined to determine the number of corpora lutea, the number and distribution of live young, and the number of early and late embryo-fetal deaths and fetal abnormalities. Live young were examined externally and weighed. One-half of the fetuses were subsequently examined for visceral and the other half for skeletal abnormalities. Dams showed dose-related body-weight loss up to day 8, followed by retarded weight gain throughout treatment; decreased food consumption and increased water consumption and salivation were seen at 16 and 64 mg/kg bw per day. Pre- and postimplantation losses were unaffected by treatment. At 4 mg/kg bw per day, only marginally increased water consumption was observed during treatment. Significantly lower litter and fetal weights were seen at 64 mg/kg bw per day; litter size and the incidences of skeletal and visceral anomalies were unaffected by treatment. The NOAELs were 4 mg/kg bw per day for maternal toxicity, on the basis of decreased body weight and food intake, increased water intake, and salivation at doses > 16 mg/kg bw per day, and 16 mg/kg bw per day for developmental toxicity, on the basis of decreased litter and fetal weight at 64 mg/kg bw per day. No teratogenicity was seen (Smith et al., 1990). Rabbits In a preliminary study, groups of five pregnant New Zealand white rabbits were given ziram (purity, 98.0%) by gavage at doses of 0, 5, 10, or 20 mg/kg bw per day on days 7-19 of gestation. All animals were observed for clinical symptoms, body weight, and food consumption. On day 28, the animals were killed, dissected, and examined macroscopically for abnormalities. The ovaries and uteri were removed and examined to determine the gravid uterine weight, number of corpora lutea, and the distribution of live young and of early and late embryo-fetal deaths. Live young were examined externally for abnormalities and weighed. Two animals at 20 mg/kg bw per day died, and one aborted. Treatment had no effect on the clinical condition or findings at necropsy for animals that survived to term. Marked reductions in weight gain and food intake, particularly at the start of treatment, were observed in animals at 20 mg/kg bw per day. Slight, temporary reductions in weight gain and food intake were also observed at 10 mg/kg bw per day. At 20 mg/kg bw per day, postimplantation losses were increased and litter size, litter weight, and fetal weight were reduced. Because maternal toxicity was observed at 20 mg/kg bw per day, the high dose for the main study was < 20 mg/kg bw per day (Barker, 1985). Groups of 16 pregnant New Zealand white rabbits were given ziram (purity, 98.1%) by gavage at doses of 0, 3, 7.5, or 15 mg/kg bw per day on days 7-19 of gestation. All animals were observed for clinical symptoms, body weight, and food consumption. On day 28, the animals were killed, dissected, and examined macroscopically for abnormalities. The ovaries and uteri were removed and examined to determine the gravid uterine weight, the number of corpora lutea, and the distribution of live young and of early and late embryo-fetal deaths. Live fetuses were weighed and examined for abnormalities before alizarin staining. Significant reductions in maternal body-weight gain and food intake were observed from the start of treatment at 15 mg/kg bw per day; slight reductions in weight gain and food intake were also observed during the early part of the treatment period in animals at 7.5 mg/kg bw per day. At 15 mg/kg bw per day, postimplantation loss was slightly increased and litter size, litter weight, fetal weight, and the crown-rump length were reduced. There was no teratogenic effect. The NOAEL for maternal and developmental toxicity was 7.5 mg/kg bw per day, on the basis of decreased body-weight gain and food intake, increased postimplantation loss, and reduced litter size, litter weight, fetal weight, and crown-rump length at 15 mg/kg bw per day (Barker, 1986). (f) Genotoxicity The results of studies on the genotoxicity of ziram are summarized in Table 2. Ziram is mutagenic in bacteria. It induced chromosomal aberrations in some, but not all, studies with cultured mammalian cells and did not induce unscheduled DNA synthesis in hepatocytes. In vivo, ziram induced DNA single-strand breaks in the livers of rats but not mice. Chromosomal aberrations were not induced in mice in vivo in bone-marrow cells or spermatogonia, and micronuclei were not induced in bone-marrow cells or peripheral erythrocytes. Studies for clastogenicity have not been conducted in rats in vivo. Consequently, the Meeting was unable to reach a conclusion about the genotoxicity of ziram. Table 2. Results tests for the genotoxicity of ziram End-point Test system Concentration Purity Results Reference (%) In vitro Reverse mutation S. typhimurium < 100 µg/plate NR Positivea Hedenstedt TA98, TA100, TA1535, at 10, 50, et al. (1979) TA1537, TA1538 100 µg/ plate Reverse mutation S. typhimurium < 50 µg/plate 98.5 Positiveb Jones et al. TA98, TA100, TA1535, at 15, 50 (1990) TA1537, TA1538 µg/plate Reverse mutation S. typhimurium < 90 µg/plate 98.5 Positivea Crebelli et al. TA98, TA100, TA102, TA1537, at 7.5-90 (1992) TA1950, TA1975, TA1535 µg/plate Reverse mutation S. typhimurium 15-90 µg/plate NR Positiveb Franekic et al. TA98, TA100, TA102, (1994) TA104, TA1535, TA1538 Chromosomal Saccharomyces 5-50 µg/ml NR Negative Franekic et al. malsegregation cerevisiae (1994) Cell division Shallot root-tip 0.5-50 µg/ml NR Positive Franekic et al. cells (1994) Chromosomal aberration Chinese hamster < 1.5 µg/ml 98.5 Negative Brooker & ovary cells Akhurst (1989) Chromosomal aberration Chinese hamster < 1.75 µg/ml 86.2 Positivea Gulati et al. ovary cells (1989) Table 2. (Cont'd) End-point Test system Concentration Purity Results Reference (%) Chromosomal aberration Chinese hamster < 10 µg/ml 98.5 Positiveb Mosesso et al. ovary cells (1994) Chromosomal aberration Chinese hamster < 2.15 µg/ml 98.5 Positiveb Mosesso et al. epithelial liver cells (1994) Unscheduled DNA synthesis Rat hepatocytes < 100 µg/ml 98.5 Negative Proudlock (1989) In vivo Chromosomal aberration Male and female NMRI 40, 120, 400 mg/kg bw 98.4 Negative Völkner (1992a) mice, bone-marrow cells Chromosomal aberration Male NMRI mice, germ cells 20, 67, 200 mg/kg bw 98.4 Negative Völkner (1992b) Micronucleus formation Male and female Crl:CD-1 3.8, 11, 34, 101 mg/kg bw 98.8 Negative Proudlock & mice, erythrocytes Taylor (1992) Micronucleus formation Male and female B6C3F1 2.5, 5.0, 10, 20 mg/kg bw 98.5 Negative Crebelli et al. mice, bone-marrow cells (1992) Single-strand DNA breaks Male Swiss mice, 13, 25, 50, 100 mg/kg bw NR Negative Scarabelli et al. liver cells (1993) Single-strand DNA breaks Male Wistar rats, 13, 25, 50, 100 mg/kg bw NR Positive Scarabelli et al. liver cells (1993) NR, Not reported a In the presence or absence of metabolic activation b In the presence of metabolic activation (g) Special studies (i) Dermal and ocular irritation and dermal sensitization Rabbits The irritancy of ziram to the skin was tested in New Zealand white rabbits. About 24 h before application of the test substance, hair was removed from the dorsal lumbar region. Ziram (purity, 98.5%) was applied at a dose of 0.5 g under a 2.5-cm2 gauze pad moistened with 0.5 ml distilled water to one skin site on each of six animals, and each treatment site was occluded with an elastic adhesive dressing for 4 h. The animals were not restrained during the exposure and were returned to their cages. At the end of treatment, the semiocclusive dressing and gauze pads were removed and the treatment sites were washed with water to remove any residual test substance. The treated skin was examined for erythema and oedema on days 1, 2, 3, and 4. None of the animals showed a response to treatment (Liggett & McRae, 1990a). The irritancy of ziram to the eye was tested in New Zealand white rabbits. The eyes of the animals were examined before instillation of 80 mg ziram (purity, 89.5%) in 0.1 ml into the lower everted lid of one eye. Ocular lesions were graded and scored 1 and 24 h after instillation. As the first rabbit had a severe ocular reaction (irridal inflammation and opacity), the study was terminated on humane grounds (Liggett & McRae, 1990b). Guinea-pigs The skin sensitizing potential of ziram (purity, 99.0%) was examined in 30 adult female Dunkin-Hartley guinea-pigs. After induction by epicutaneous applications of the test substance (25% w/w in corn oil) and challenge with 10, 5, or 1% (w/w) in corn oil, six guinea-pigs showed a positive skin response (grade 2 or more) to the 10% concentration. Five of these animals also reacted positively to the 5% dose and three to the 1% concentration. No positive reactions were observed in the control animals. Thus, a sensitization rate of 30% was obtained. The author considered that ziram has moderate sensitizing properties (Daamen, 1988). (ii) Neurotoxicity Rats The neurotoxicity of repeated doses of ziram was studied in groups of 10 Crl:CD(SD)BR rats fed diets containing ziram (purity, 97.9%) providing concentrations of 0, 72, 210, or 540 ppm, equal to 0, 5, 14, or 34 mg/kg bw per day for males and 0, 6, 16, or 40 mg/kg bw per day for females, for 91 days. Five animals of each sex per group were allocated for evaluation of neurotoxic esterase and five for evaluation of neuropathological changes. Viability, clinical signs, body weight, and food consumption were recorded for all animals. The results of a functional observational battery (home cage, handling, open field, sensorimotor, neuromuscular, and physiological observations) and of tests for total and ambulatory motor activity were recorded for all animals during week 3, 7, and 12 of exposure. The body weights and food consumption of male and female rats at 540 ppm were reduced, but no treatment-related effects indicative of neurotoxicity were apparent. Brain neurotoxic esterase activity was 47% lower than that in controls in males and 38% lower in females at 540 ppm. The NOAEL for brain neurotoxic esterase inhibition was 210 ppm, equal to 14 mg/kg bw per day (Nemec, 1993). The neurotoxicity of a single dose of ziram was studied in groups of 12-16 Crl:CD(SD)BR rats given single doses of 0, 15, 300, or 600 mg/kg bw ziram (purity, 97.8%). Viability, clinical signs, and body weight were recorded for all animals. The results of a functional observational battery (home cage, handling, open field, sensorimotor, neuromuscular, and physiological observations) and tests for total and ambulatory motor activity were recorded for all animals before and 4 h after treatment and on days 7 and 14. Five controls and five animals at 600 mg/kg bw were selected for evaluation of neuropathological changes. Four males and seven females at 600 mg/kg bw and one female at 300 mg/kg bw died. At these doses, body weight was reduced, and several treatment-related clinical signs (e.g. cyanosis, hypothermia, enophthalmus, unkempt appearance, gait alterations, hypoactivity, ptosis, and abdomal respiration) were observed. Remarkable differences from the controls were seen in the functional observational battery for animals at 300 and 600 mg/kg bw. Most of the responses were observed 4 h after treatment (e.g. altered posture, palpebra closure, and faecal consistency) or daily during the first week after dosing (e.g. lacrimation, salivation, changes in fur appearance, altered palpebral closure, changes in respiratory rate, and altered gait). Ambulatory and total motor activity counts were reduced in males and females at 300 and 600 mg/kg bw. No treatment-related microscopic lesions were observed in the central and peripheral nervous tissues examined. The NOAEL for neurotoxicity was 15 mg/kg bw, on the basis of mortality, reduced body weight, ambulatory, and total motor activity, and clinical signs at doses > 300 mg/kg bw (Lamb, 1994). 3. Observations in humans The ability of ziram to induce chromosomal aberrations in peripheral leukocytes was studied in four men and five women who had been exposed for three to five years to ziram at a concentration of 2-4 mg/m3 air. The control group consisted of three women and one man. The exposed group had 5.9% cells with aberrations and the controls, 0.75% (Pilinskaya, 1970; Annex I, reference 15). Comments In experiments with 14C-labelled ziram in rats, elimination was essentially complete within 48 h. Excretion occurred mainly in expired air, urine, and faeces. Less than 2% of the administered dose remained in the tissues. The biotransformation of ziram has not been studied in rodents. In goats, it is metabolized at least in part via a single-carbon pathway, which results in extensive radiolabelling of natural products. The primary effects of short- and long-term treatment with ziram in mice, rats, and dogs were on the liver, thyroid gland, and testis. The hepatic effects were increased liver weight, degeneration, and focal-cell necrosis. Effects in the thyroid were C-cell hyperplasia and carcinomas, and that on the testes was sterility. Ziram had moderate acute oral toxicity in rats and rabbits (LD50 = 200-400 mg/kg bw). WHO has classified ziram as 'slightly hazardous' (WHO, 1996). In a four-week study of toxicity in mice given dietary concentrations of 0, 3000, 4000, or 5000 ppm, an NOAEL was not identified. Reductions in body weight, food intake, efficiency of food use, and brain and heart weight occurred at all doses. In a 13-week study of toxicity in mice given dietary concentrations of 0, 100, 300, 900, or 2700 ppm, the NOAEL was 100 ppm, equal to 15 mg/kg bw per day, on the basis of lowered spleen weight at higher doses. At 900 and 2700 ppm, the number of corpora lutea was reduced, which was consistent with cellular changes in the uterus. In two four-week studies of toxicity in rats either given diets providing doses of 0, 100, 500, 2500, or 5000 ppm or treated by gavage with 0, 3, 15 or 100 mg/kg bw per day, the NOAEL was 3 mg/kg bw, on the basis of degenerative liver changes. At 100 mg/kg bw, degenerative changes in the kidneys and reductions in body weight, food intake, efficiency of food use, and absolute weights of the liver, pituitary, testes, brain, and uterus were seen. In a 13-week study of toxicity in which rats received dietary levels of 0, 100, 300, or 1000 ppm, the NOAEL was 100 ppm, equal to 7.4 mg/kg bw per day, on the basis of reduced body-weight gain, food intake, and food use and increased brain and spleen weights at higher doses. In a four-week study of toxicity in dogs given diets providing doses of 0, 1000, 2000, or 5000 ppm, an NOAEL was not identified. Increased liver weight occurred at all doses. At 2000 ppm, convulsive episodes were observed. In a 13-week study of toxicity in dogs given diets providing 0, 100, 300, or 1000 ppm, the NOAEL was 100 ppm, equal to 4.1 mg/kg bw per day, on the basis of increased liver weight, focal liver necrosis, pigment in Kupffer cells, activated partial thromboplastin time, and elevated cholesterol level at higher doses. In a one-year study of toxicity in which dogs were fed diets providing doses of 0, 50, 180, or 500 ppm, the NOAEL was 50 ppm, equal to 1.6 mg/kg bw per day, on the basis of reductions in body-weight gain, degeneration of hepatocytes, and increased activity of alanine and aspartate aminotransferases and alkaline phosphatase at doses > 185 ppm. At 500 ppm, single liver-cell necrosis was observed, and the liver weight and cholesterol values were increased; albumin values were reduced. Inflammatory cell infiltration around the hepatic vein and its branches and aggregates of pigmented Kupffer cells were observed in the liver. Two long-term studies of toxicity and carcinogenicity in mice have been reported. One was considered inadequate for evaluating the carcinogenicity of ziram. In the other, mice were given diets providing doses of 0, 25, 75, 220, or 680 ppm for 80 weeks. The NOAEL was 25 ppm, equal to 3 mg/kg bw per day, on the basis of reduced brain weight at doses > 75 ppm. There was no evidence of carcinogenicity. In a two-year study of toxicity and carcinogenicity in rats at dietary concentrations of 0, 25, 250, or 2500 ppm, the NOAEL was 250 ppm, equivalent to 12 mg/kg bw per day, on the basis of testicular atrophy and thyroid hyperplasia at 2500 ppm. There was no evidence of carcinogenicity. In a two-year study of toxicity and carcinogenicity in Fischer 344 rats with dietary concentrations of 0, 300, or 600 ppm, an NOAEL was not identified, since the combined incidence of C-cell adenoma and carcinoma of the thyroid in males showed a positive trend. This finding was considered to represent an extension of the known toxicity of the compound to the thyroid, to which the rat is particularly sensitive, and not to indicate carcinogenic potential for humans. In a study of toxicity and carcinogenicity in CD rats treated with 0, 60, 180, or 540 ppm in the diet for 12-24 months, an NOAEL was not identified because dose-related changes in organ weights and histopathological and haematological changes were observed at 60 ppm, equal to 2.5 mg/kg per day. Other effects included reduced body weight, erythrocyte count, and triiodothyronine and thyroxine activity. Cysts in the thyroids, epithelial hyperplasia, hypertrophy with vacuolation, cortical cystic degeneration of the adrenals, and C-cell hyperplasia of the thyroid were also demonstrated. The tumour incidence was not increased. In a study of sperm quality in mice treated intraperitoneally with ziram at a single dose of 0, 50, or 100 mg/kg bw or repeated doses of 25 mg/kg bw per day for five days, severe morphological abnormalities were observed. The frequency of abnormal sperm was 1.6% in the controls, 5.6% at 50 mg/kg bw, 8.2% at 100 mg/kg bw, and 8.4% after repeated doses of 25 mg/kg bw. In a two-generation study of reproductive toxicity and developmental neurotoxicity, rats were fed ziram at concentrations of 0, 72, 210, or 540 ppm. The NOAEL for maternal toxicity was 210 ppm, equal to 10 mg/kg bw per day, on the basis of reduced food consumption and body-weight gain at 540 ppm. The NOAEL for neonatal toxicity was 210 ppm, equal to 10 mg/kg bw per day, on the basis of reduced body weight gain at 540 ppm. The NOAEL for reproductive toxicity and developmental neurotoxicity was 540 ppm, equal to 25 mg/kg bw per day. In a study of developmental toxicity, rats were given ziram at 0, 1, 4, 16, or 64 mg/kg bw per day on days 6-15 of gestation. The NOAEL for maternal toxicity was 4 mg/kg bw per day for maternal toxicity, on the basis of decreased body-weight gain and food intake and increased water intake and salivation at doses > 16 mg/kg bw per day. The NOAEL for developmental toxicity was 16 mg/kg bw per day, on the basis of decreased litter weight and fetal weight at 64 mg/kg bw per day. No teratogenicity was seen. In a study of developmental toxicity in rabbits given ziram at doses of 0, 3, 7.5, or 15 mg/kg bw per day on days 7-19 of gestation, the NOAEL for maternal toxicity and developmental toxicity was 7.5 mg/kg bw per day, on the basis of decreased body-weight gain and food intake in the dams and post-implantation loss, reduced litter size, litter weight, fetal weight, and crown-rump length at 15 mg/kg bw per day. There was no evidence of teratogenicity. Ziram is mutagenic in bacteria. It induced chromosomal aberrations in some, but not all, studies with cultured mammalian cells but did not induce unscheduled DNA synthesis in hepatocytes. In vivo, ziram induced single-strand breaks in DNA in the livers of rats but not mice. Chromosomal aberrations were not induced in mice in vivo in bone-marrow cells or spermatogonia, and micronuclei were not induced in bone-marrow cells or peripheral erythrocytes. Studies for clastogenicity have not been conducted in rats in vivo. In an old study of nine workers exposed for three to five years to ziram at a concentration of 2-4 mg/m3 air, the percentage of peripheral leukocytes with chromosomal aberrations was 5.9%; in a control group, the percentage was 0.75%. The Meeting was unable to reach a conclusion about the genotoxicity of ziram. Ziram caused severe ocular irritation but no dermal irritation in rabbits and moderate skin sensitization in guinea-pigs. In two studies of neurotoxicity in rats treated with single doses of 0, 15, 300, or 600 mg/kg bw or 0, 72, 210, or 540 ppm for 91 days, behavioural effects indicative of neurotoxicity were apparent after the single high doses but not after repeated dosing at a lower level. The NOAEL was 210 ppm, equal to 14 mg/kg bw per day, on the basis of reduced body weight and food consumption and inhibition of brain neurotoxic esterase activity at 540 ppm. An ADI of 0-0.003 mg/kg bw was established on the basis of long-term toxicity in the rat. In this study, effects were seen at all doses, the LOAEL being 60 ppm, equal to 2.5 mg/kg bw per day. In view of the absence of an NOAEL, a safety factor of 1000 was used. The NOAEL of 1.6 mg/kg bw per day observed in a long-term study of toxicity in dogs supported this ADI, which served as the basis for the group ADI that was established for ziram alone or in combination with ferbam. Toxicological evaluation Levels that cause no toxic effect Mouse: 25 ppm, equal to 3 mg/kg bw per day (80-week study of toxicity and carcinogenicity) Rat: NOAEL could not be determined: lowest effective dose, 60 ppm, equal to 2.5 mg/kg bw per day (12-24-month study of toxicity, various effects) 100 ppm, equal to 7.4 mg/kg bw per day (13-week study of toxicity) 250 ppm, equivalent to 12 mg/kg bw per day per day (two-year study of toxicity and carcinogenicity) 210 ppm, equal to 10 mg/kg bw per day (maternal toxicity in a study of reproductive toxicity) Rabbit: 7.5 mg/kg bw per day (maternal toxicity and embryotoxicity in a study of developmental toxicity) Dog: 50 ppm, equal to 1.6 mg/kg bw per day (one-year study of toxicity) 100 ppm, equal to 4.1 mg/kg bw per day (13-week study of toxicity) Estimate of acceptable daily intake for humans 0-0.003 mg/kg bw (for ferbam and ziram) Studies that would provide information useful for continued evaluation of the compound 1. Further studies on long-term toxicity in the rat 2. Further studies on genotoxicity in rats 3. Further studies on male reproductive toxicity 4. Further observations in humans Toxicological criteria for estimating guidance values for dietary and non-dietary exposure to ziram Exposure Relevant route, study type, species Results, remarks Short-term (1-7 days) Oral, toxicity, rat LD50 = 270 mg/kg bw Inhalation, toxicity, rat LC50 = 0.06 mg/litre Dermal, irritation, rabbit Not irritating Ocular, irritation, rabbit Severely irritating Dermal, sensitization, guinea-pig Moderately sensitizing Medium-term (1-26 weeks) Repeated oral, 13 weeks, toxicity, mouse NOAEL = 15 mg/kg bw per day, decreased spleen weight Repeated oral, 4 weeks, toxicity, rat NOAEL = 3 mg/kg bw per day, reduced body weight and food consumption, degenerative hepatic changes Repeated oral, 13 weeks, toxicity, rat NOAEL = 7.4 mg per kg bw per day, reduced body weight and food consumption, increased brain and spleen weight Repeated oral, 13 weeks, toxicity, dog NOAEL = 4.1 mg/kg bw per day, hepatic toxicity Repeated oral, reproductive toxicity and NOAEL = 25 mg/kg bw per day, reproductive toxicity developmental neurotoxicity, rat and development neurotoxicity NOAEL = 10 mg/kg bw per day, maternal and neonatal toxicity (reduced body weight) Repeated oral, developmental toxicity, rat NOAEL= 16 mg/kg bw per day, developmental toxicity (reduced fetal weight) NOAEL = 4 mg/kg bw per day, maternal toxicity (reduced body weight) Repeated oral, developmental toxicity, NOAEL = 7.5 mg/kg bw per day, developmental and rabbit maternal toxicity (reduced fetal and maternal weight) Repeated oral, neurotoxicity, rat NOAEL = 14 mg/kg bw per day, inhibition of neuropathy target esterase activity Long-term (> 1 year) Repeated oral, 18 months, toxicity, mouse NOAEL = 3 mg/kg bw per day, reduced brain weight and hepatic toxicity Repeated oral, 2 years, toxicity and No NOAEL; LOAEL = 2.5 mg/kg bw per day, haematological carcinogenicity, rat toxicity and toxic effects on the thyroid Repeated oral, 1 year, toxicity, dog NOAEL = 1.6 mg/kg bw per day, reduced body weight and hepatic toxicity References Barker, L. 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(1989) Analysis of metaphase chromosomes obtained from CHO cells cultured in vitro and treated with ziram. Unpublished report No. 7/89675 from Huntingdon Research Centre Ltd, Cambs, United Kingdom. Submitted to WHO by UCB Chemicals, Brussels, Belgium. Chambers, P.R., Cary, R.J., Gibson, W.A. & Gopinath, C. (1992a) Ziram. Palatability study in mice for 4 weeks. Unpublished report No. ZIR 3/89720 from Huntingdon Research Centre Ltd, Cambs, United Kingdom. Submitted to WHO by UCB Chemicals, Brussels, Belgium. Chambers, P.R., Cary, R.J., Gibson, W.A. & Gopinath, C. (1992b) Ziram. Palatability study in rats for 4 weeks. Unpublished report No. ZIR 2/89719 from Huntingdon Research Centre Ltd, Cambs, United Kingdom. Submitted to WHO by UCB Chemicals, Brussels, Belgium. Cheng, T. (1991a) Metabolism of ziram in rats. Unpublished report No. HLA 6225-106 from Hazleton Laboratories America, Inc., WI, USA. Submitted to WHO by UCB Chemicals, Brussels, Belgium. Cheng, T. 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See Also: Toxicological Abbreviations Ziram (ICSC) Ziram (FAO Meeting Report PL/1965/10/1) Ziram (FAO/PL:1967/M/11/1) Ziram (IARC Summary & Evaluation, Volume 53, 1991)