ETHEPHON First draft prepared by J-J. Larsen National Food Agency of Denmark, Soborg, Denmark EXPLANATION Ethephon was evaluated at the 1978 JMPR, but an ADI was not allocated since the available toxicological data were insufficient (Annex 1, reference 30). It is a plant growth regulator that acts by release of ethylene, influencing directly several physiological processes such as ripening and maturation and stimulating the production of endogenous ethylene. Since the compound in high concentration (> 87%) is a waxy solid and difficult to handle, a technical product that contains 71% ethephon and 21% water is marketed and also used for most toxicological studies. Although ethephon is a dibasic phosphonic acid, its commercial formulation exhibits some anticholinesterase activity. EVALUATION FOR ACCEPTABLE DAILY INTAKE BIOLOGICAL DATA Biochemical aspects Absorption, distribution, and excretion Rats The absorption, distribution, metabolism, and elimination of 14C-ethephon was studied by single and multiple dose regimens in male and female Crl:CD(SD)BR rats. Unlabelled ethephon (purity 96.1%) and 14C-ethephon (radiochemical purity > 96%,) dissolved in methanol, soluted with a 0.9% (w/v) sodium chloride, and adjusted to a pH of 3-4 with lactic acid were used. The compound was administered orally to 5 male and 5 female rats in a single dose of 50 or 1000 mg/kg bw (dose volume 5 ml/kg bw, radioactivity 10 µCi/g), multiple oral doses of 50 mg/kg bw (non-radiolabelled ethephon administered daily for 14 days followed 24 hours later by a single dose of 14C-ethephon), or intravenously to 5 male and 5 female rats in a dose of 50 mg/kg bw (dose volume 1 ml/kg bw, radioactivity 60 µCi/g). Urine and faeces were collected in individual all-glass metabolism cages for 7 days. Expired radioactivity was captured by a mercuric perchlorate and 2-ethoxy ethanol: ethanolamine-containing trapping system. Urine, faeces, and volatile trap solutions were collected at intervals up to 120 hours after dosing, after which the rats were sacrificed and tissues taken at necropsy for radioactivity measurement. In a parallel pharmacokinetic study using a single dose of 50 or 1000 mg/kg bw, blood was sampled from the lateral tail vein at intervals up to 168 hours and the concentration of radioactivity in whole blood was determined. Blood levels of radioactivity (50 or 1000 mg/kg bw) peaked at 0.5-2 hours post-dose and declined exponentially with levels near background by 12-24 hours post-dose. Following a single oral dose of 14C-ethephon (50 mg/kg bw), about 90% of the administered radioactivity was recovered, principally in urine (55%), expired air (20%), and faeces (5%) during 120 hours after dosing. Most of the dose was recovered within 24 hours post-dose. Pretreatment with non- radiolabelled ethephon did not significantly alter tissue residues at 120 hours post-dose over those animals receiving a single administration only. Following a single oral dose of 14C-ethephon (1000 mg/kg bw) about 86% of the administered radioactivity was recovered, principally in urine (48%), expired air (19%), and faeces (6%) at 120 hours post-dose. Most of the dose was recovered within 24 hours post-dose. No major sex difference in pharmacokinetics was observed (Savage, 1990). Dogs The disposition of ethephon was studied in 3 male beagle dogs. The animals, weighing 6.1-6.3 kg, were placed for 72 hours acclimatization in individual metabolism cages the week before dosing. A single oral dose of 180 mg/kg bw made up of 1.5 ml of a methanolic 14C-ethephon solution (362.6 µCi/5 ml) and 2.0 ml of a 5 normal citric acid non-radiolabelled ethephon solution (purity 88%) was given in a 4 ml gelatin capsule. Ethylene and carbon dioxide were collected from the expired air by a carbon dioxide and ethylene-trapping system, and blood samples, urine, and faeces were collected at intervals for scintillation counting. Blood samples were also used for determination of RBC and plasma cholinesterase levels. The animals were sacrificed 72 hours after dosing and tissues removed for scintillation counting. Two of the three dogs vomited 15-30 min after dosing thus questioning the dosing. About 35% of the dose appeared in the urine of two dogs within 24 hours after dosing. The radioactivity in urine dropped within 72 hours to levels below 1%. In one dog receiving the full dosage, 4, 0.1, and 0.03% of the dose was found in faeces at 24, 48, and 72 hours after dosing, respectively. In this dog, 30% of the dose was recovered as ethylene in the expired air and only traces of 14CO2 were recovered. Total selected organs retained at sacrifice a maximum of 0.25% of the administered dose. Peak plasma and blood cell concentrations were observed 2 hours after dosing. Only traces were observed after 22 hours. Plasma cholinesterase levels were reduced at 2 hours with recovery starting within a few hours. Erythrocyte cholinesterase levels responded more slowly with signs of recovery at 72 hours (Stephen & Walker, 1971). Biotransformation Rats The mono- and di-sodium salts of ethephon, and some unidentified metabolites were present in urine and faeces of male and female Crl:CD(SD)BR rats dosed with single (50 or 1000 mg/kg bw) or multiple dosages (50 mg/kg bw) of 14C-ethephon (Savage, 1990). Ethylene was eliminated in the expired air. No significant sex, route or dose differences in metabolism were observed (Hardy et al., 1990). Dogs The urine of 3 beagle dogs was examined by thin layer and gas chromatography following a single oral dose of 180 mg/kg bw 14C- ethephon made up of 1.5 ml of a methanolic 14C-ethephon solution (362.6 µCi/5 ml) and 2.0 ml of a 5 normal citric acid non- radiolabelled ethephon solution (purity 88%) (Stephen & Walker, 1971). Ethephon was partly excreted unchanged in the urine and also metabolized to ethylene and eliminated in the expired air (Stephen & Stanovick 1971). Effects on enzymes and other biochemical parameters In beagle dogs dosed with 180 mg/kg bw ethephon, plasma cholinesterase activity was inhibited at 2 hours with recovery starting within a few hours. Erythrocyte cholinesterase levels responded more slowly with signs of recovery at 72 hours. Very sparse information on the test procedures, including time schedule for sampling and measurements, was available (Stephen & Walker, 1971). Toxicological studies Acute toxicity studies Ethephon has a low oral acute toxicity in mice, rats, and rabbits. The LD50 or LC50 values are given in Table 1. WHO has classified ethephon as unlikely to represent acute hazard in normal use (WHO, 1992). Table 1. Acute toxicity of ethephon Species Sex Route Purity LD50 LC50 Reference (%) (mg/kg bw) (mg/m3) Mouse M oral 90 1920 Holsing, 1969 Rat M oral 71 3730 Myers, 1984 Rat M oral 71 2210 Myers, 1984 Rat F inh. (4 h) 72 4500 Nachreiner & Klonne, 1989 Rat F inh. (4h) 72 4500 Nachreiner & Klonne, 1989 Rabbit F oral 90 > 560 Weatherholtz, 1989 Rabbit M dermal 71 1700 Meyers, 1989 Rabbit F dermal 71 1400 Meyers, 1989 Short-term toxicity studies Mice A 4-week dietary study in mice was carried out with ethephon. After 2 weeks' acclimatization, groups of 10 CD-1 mice/sex received in the diet 0, 30, 100, 300, 1000, or 3000 ppm ethephon (purity 71%), equal to 0, 5.3, 18, 51, 181, or 546 mg/kg bw/day in males and 0, 6.5, 22, 69, 210, or 635 mg/kg bw/day in females. An additional 5 animals/sex were included in the 0, 300, and 1000 ppm groups for 2- week cholinesterase activity determinations. The mice were 7 weeks of age at initiation of dosing. Stability and homogeneity of the test substance in the diet was not examined. No effects were observed in the incidence of clinical signs or mortality, food consumption, body weight, organ weights or ASAT, SDH, ALP, ALT or brain ChE activity at any dose level. Biologically significant (> 20%) inhibition of plasma ChE activity was observed at 300 ppm (18- 22% in males and 23-29% in females), 1000 ppm (43-44% in males and 46-49% in females) and 3000 ppm (63% in males and 55% in females). Significant inhibition of RBC was observed at 1000 ppm (29-34% in males and 38-41% in females) and 3000 ppm (62% in males and 56% in females). A statistically significant decrease (26%) in SDH was seen in the female high-dose group, and a statistically significant increase (24%) was seen in ALP in males of the high-dose group. Based on erythrocyte cholinesterase inhibition, the NOAEL was 300 ppm equal to 51 and 69 mg/kg bw/day in male and female mice, respectively (Miller & Troup, 1986a). In order to establish an MTD for ethephon in mice, a 4-week dietary study was carried out. After 2 weeks' acclimatization, groups of 15 CD-1 mice/sex received in the diet 0, 3000, 10 000, 25 000, or 50 000 ppm ethephon (purity 71%), equal to 0, 530, 1800, 4500, or 10 000 mg/kg bw/day in males and 0, 630, 2200, 5900, or 15 000 mg/kg bw/day in females. No effects were observed in the incidence of clinical signs or mortality. Food consumption reduction in the 50 000 ppm group at week 1 and body-weight reductions in the same group throughout the study for both sexes were; attributed primarily to initial aversion to the test diets. For male mice, organ weight changes at 28 days were consistent with the decreased body weights. In females, a larger than expected reduction in spleen weight, which was reflected in a decreased spleen to final body weight and spleen to brain weight ratios, was observed in the 50 000 ppm group. The significance of this finding could not be determined. Dose-related inhibition of plasma (54-82%) and erythrocyte (54-92%) ChE activity was observed in both sexes at 2 and 4 weeks. Brain ChE activity was not inhibited. Based on the finding that only plasma and erythrocyte ChE activities were inhibited and only minimal additional biological responses were observed, an initial high dose of 50 000 ppm was established for oncogenicity testing of ethephon in mice (Miller & Troup, 1986b). Rats A 4-week dietary toxicity study was carried out with ethephon in rats. After 2 weeks' acclimatization, groups of 10 Sprague-Dawley CD rats/sex received in the diet 0, 625, 1250, 2500, 5000, or 10 000 ppm ethephon (purity 71%), equal to 0, 52, 106, 214, 430, or 830 mg/kg bw/day in males and 0, 59, 120, 251, 487, or 980 mg/kg bw/day in females. An additional 5 animals/sex were included in the 0, 1250, and 2500 ppm groups for 2-week cholinesterase activity determinations. No clinical signs of toxicity and no statistically significant effect on food consumption, body weight, haematology, or clinical chemistry were observed in either sex at any dosage level. Inhibition of plasma ChE activity was 21-27% at 1250 ppm, 16-18% at 2500 ppm, 30% at 5000 ppm and 35% at 10 000 ppm in males and 29% at 625 ppm, 44-50% at 1250 ppm, 45-50% at 2500 ppm, 49% at 5000 ppm and 63% at 10 000 ppm in females. The inhibition of erythrocyte ChE activity was 16-22% at 1250 ppm, 36-41% at 2500 ppm, 58% at 5000 ppm and 73% at 10 000 ppm in males and 14-35% at 1250 ppm, 32-50% at 2500 ppm, 67% at 5000 ppm and 78% at 10 000 ppm in females. Brain ChE activity was not inhibited at any dose level. The NOAEL for ethephon in this study, based on erythrocyte ChE inhibition, was 625 ppm, equal to 52 and 59 mg/kg bw/day in males and females, respectively (Miller & Troup, 1986c). In order to establish an MTD for ethephon in rats, a 4-week dietary toxicity study was carried out. After 2 weeks' acclimatization, groups of 15 Sprague-Dawley CD rats/sex received in the diet 0, 10 000, 25 000, or 50 000, ppm ethephon (purity 71%), equal to 0, 962, 2300, or 4673 mg/kg bw/day in males and 0, 996, 2488, or 4900 mg/kg bw/day in females. Initial aversion to the test diets, resulting in decreased body weight was observed in both sexes in the 25 000 and 50 000 ppm groups. Inhibition of erythrocyte ChE activity (69-91%) and plasma ChE activity (23-74%) was cumulative and dose-related in both sexes. Brain ChE activity was not inhibited at any dose level. Diarrhoea, associated with cholinesterase inhibition, was observed from day 10 in the 50 000 ppm group. Based on the rapid development of diarrhoea in the 50 000 ppm group, an initial high dose of 30 000 ppm was established for chronic toxicity and oncogenicity testing of ethephon in rats (Miller & Troup, 1986d). Rabbits A 21-day toxicity test was carried out with dermal application of ethephon in rabbits. Groups of 5-10 male and female adult New Zeeland white rabbits weighing 2.0-2.9 kg, received five days a week, for three weeks, on the abdominal skin clipped free of hair 0, 300, or 600 mg/kg bw/day ethephon (purity 39.5%, stability and compound verification data not given). After 6-8 hours' exposure, the abdomen was washed with water. The general appearance, behaviour, body weight, clinical chemistry, signs of dermal irritation, gross pathology and histopathology were studied. No systemic toxicity of ethephon was demonstrated at the two dose levels apart from a severe dermal irritation characterized by subepidermal fibrosis, acanthosis, hyperkeratosis, and ulceration of the epidermis (Holsing, 1969). Dogs A one-year dietary toxicity study in dogs was carried out with ethephon. After 10 weeks' acclimatization, groups of 5 beagle dogs/sex received in the diet 0, 100, 300, 1000, or 2000 ppm ethephon (purity 71%), equal to 0, 2.8, 8.1, 27, or 54 mg/kg bw/day in males and 0, 2.6, 8.4, 30, or 50 mg/kg bw/day in females. Mean values for homogeneity and stability assays were within acceptable ranges. The methodology for diet analyses of ethephon was initially unsatisfactory and was changed during the study. During the first 10 weeks of dosing, examples of great variation (e.g., 260% and 60%) between duplicate samples were seen. These initial problems were solved. After week 11, all diets analyzed were generally within 10% of the target levels. There was a higher incidence of soft stools in the ethephon-treated groups, and there was a significant decrease in terminal body weights, absolute and relative spleen weight (spleen- to-body-weight ratio, and spleen-to-brain weight ratio), absolute heart and thyroid/parathyroids weights for the 2000 ppm males when compared to control values. For the 2000 ppm females, a significant decrease in relative spleen weight and a significant increase in kidney/brain weight ratio was seen. Based on the findings of soft stools and changes in body weight and spleen weight, the NOAEL was 1000 ppm, equal to 27 mg/kg bw/day in males and 30 mg/kg bw/day in females (Hamada, 1989). In a 2-year study, groups of 6 beagle dogs/sex received in the diet 0, 30, 300, or 1500 ppm ethephon (purity 75.6%), equal to 0, 0.86, 7.6, or 42 mg/kg bw/day in males and 0, 0.86, 8.4, or 47.8 mg/kg bw/day in females. Treatment-related findings included an increased incidence of soft stools in the 300 and 1500 ppm groups. Statistically significant erythrocyte ChE inhibition occurred in the 300 ppm group (42-56% in males and 36-56% in females) and in the 1500 ppm group (68-79% in males and 59-79% in females), and a statistically significant plasma ChE inhibition occurred in the 30 ppm (22-31% in males and 24-38% in females), 300 ppm (46-62% in males and 51-58% in females), and 1500 ppm (45-67% in males and 59-66% in females) groups. In treated groups, brain ChE values ranged from 3.7-52% above the control values. Compound-related gross pathology findings included thickened stomach and intestinal walls in dogs from all groups and compound-related histopathology consisted of smooth muscle hypertrophy in the stomach and small intestine in the 300 and 1500 ppm groups. Based on the inhibition of erythrocyte ChE activity and smooth muscle hypertrophy in the stomach and small intestine, the NOAEL in this study was 30 ppm, equal to 0.86 mg/kg bw in both males and females (Reno & Voelker, 1977). Long-term toxicity/carcinogenicity studies Mice Male and female Swiss Albino mice (85/sex/group) were fed diets containing ethephon (purity 75%) at concentrations of 0, 30, 300, or 1000 ppm for 78 weeks, equivalent to 0, 4.5, 45, or 150 mg/kg bw/day. Statistically significant decreases in survival were noted among 300 and 1000 ppm males. The increased mortality was considered related to genitourinary infections, dermatitis and haematopoietic tumours and not compound-related. Ethephon caused a dose-related inhibition of plasma ChE activity in both sexes (9-34% in the 300 ppm group, and 37-64% in the 1000 ppm group). Similar results were observed in erythrocyte ChE activity measurements (11-56% at 300 ppm and 13-51% at 1000 ppm). Ethephon treatment did not cause biologically significant inhibition of brain ChE at any dietary concentration (0-9%). The incidences of haematopoietic tumours were 4, 3, 13, and 11% in males and 22, 18, 39 and 29% in females at 0, 30, 300, and 1000 ppm, respectively. The major contributing factor to the increase of 39% in the 300 ppm females was the incidence of thymic lymphoma involving the thymus itself (incidence of 5, 5, 19, and 13% at dose levels of 0, 30, 300, and 1000 ppm, respectively). Since the incidence of haematopoietic tumours was not significantly increased in females at the 1000 ppm level, the increased incidence in the 300 ppm females was considered as random variation and unrelated to ethephon administration. Based on inhibition of erythrocyte ChE activity, the NOAEL was 30 ppm, equivalent to 4.5 mg/kg bw/day. There was no evidence of carcinogenicity (Voss & Becci, 1985). Male and female CD-1 mice (70/sex/group) were fed diets containing ethephon (purity 71.6%) at concentrations of 0, 100, 1000, or 10 000 ppm for 78 weeks, equal to 0, 14, 140, or 1480 mg/kg bw/day (males) and 0, 17, 170, or 1780 mg/kg bw/day (females). An additional group (70 males and 70 females), that received 50 000 ppm, were sacrificed in the first week of the study due to extensive morbidity and mortality after several days of dosing. An interim sacrifice (20 mice/sex per group) was performed at 52 weeks of treatment. Data on homogeneity and stability were acceptable. Concentration verifications of the diets gave analytical values ranging from 88-111% of nominal concentrations. No treatment-related changes were observed in the incidence of abnormal clinical signs, mortality, food consumption, efficiency of food utilization, water consumption, or incidence of ophthalmic findings during the study. Body weight and weight gain for males were not affected throughout the study. No altered haematology measurements, serum chemistry measurements (except ChE inhibition), organ weights, gross pathological observations, or histopathological observations were related to ethephon exposure at the 52-week or 78-week evaluation periods. Potentially treatment-related findings included depressed body weights and weight gains in females from the 10 000 ppm dose group that were evident starting at approximately 39 weeks. In addition, decreased urine pH, presumably resulting from the highly acidic nature of the test material, was observed in male mice from the 10 000 ppm group at 52 and 77 weeks and from the 1000 ppm group at 77 weeks. No other effects on urinalysis measurements were observed in either sex at any dose level, and the decrease in pH was not considered to be a toxic effect of ethephon treatment. Ethephon was determined to be a potent inhibitor of plasma ChE activity with dose-related inhibition (35-41 and 65-71% in the 1000 and 10 000 ppm male dose groups, respectively, and 18-24, 41-61, and 74-76% in the 100, 1000, and 10 000 ppm female dose groups, respectively). Similar results were observed in erythrocyte ChE activity measurements (35-36 and 70-72% in the 1000 and 10 000 ppm male dose groups, respectively, and 21-36 and 60-74% in the 1000 and 10 000 ppm female dose groups, respectively). This inhibition was, however, not considered to be detrimental to the animals based on the lack of other findings consistent with ChE inhibition. Ethephon treatment did not cause biologically significant inhibition of brain ChE at any dietary concentration used in this study (4-18%). Ethephon was not considered to be carcinogenic since no neoplasms resulted from the treatment. Two tumour types in males and two types in females were observed at a frequency greater than 5% (males: hepatocellular adenoma 6/69, 10/69, 4/70, 8/70 and lung adenoma 2/69, 5/70, 14/70, 6/70; females: thymic region lymphosarcoma 1/68, 2/19, 3/16, 5/68 and lung adenoma 7/70, 4/69, 7/69, 7/70 at dose levels of 0, 100, 1000, or 10 000 ppm, respectively). Only lung adenomas incidences in the 1000 ppm males were statistically increased over the controls. Based on no dose relation, common occurrence of lung adenomas in this mouse strain and that the adenomas were neither larger nor tended toward malignancy more than in other groups, it was concluded that the increase in lung adenomas in males was not treatment-related. Based on inhibition of erythrocyte ChE activity, the NOAEL was 100 ppm equal to 14 and 17 mg/kg bw/day in males and females, respectively (Miller, 1988). Rats Male and female Sprague-Dawley CD rats (55/sex/group), received diets containing ethephon (purity 75.6%) at concentrations of 0, 30, 300, or 3000 ppm for 104 weeks, equal to 0, 1.2, 12.5, or 129 mg/kg bw/day (males) and 0, 1.6, 16, or 171 mg/kg bw/day (females). Cholinesterase activity was consistently inhibited in plasma (22-31% at 30 ppm, 46-62% at 300 ppm and 45-67% at 3000 ppm in males and 29-38% at 30 ppm, 51-58% at 300 ppm and 59-66% at 3000 ppm in females). Similar results were obtained on erythrocytes (42-56% at 300 ppm and 68-80% at 3000 ppm in males and 47-56% at 300 ppm and 59-79% at 3000 ppm in females). Brain cholinesterase activity was not affected by ethephon (0-10%). Evaluation of all other parameters did not reveal any findings which could be attributed to the test material. Ethephon was not found to be carcinogenic in doses up to 3000 ppm equal to 129 and 171 mg/kg bw/day in males and females, Based on inhibition of erythrocyte ChE activity, the NOAEL was 30 ppm equal to 1.2 and 1.6 mg/kg bw/day in males and females, respectively (Reno et al., 1978). Four groups of 90-100 Sprague-Dawley CD rats/sex/dose were fed diets containing ethephon (purity 71%) at concentrations of 0, 300, 3000, 10 000, or 30 000 ppm for 97 weeks (males) or 104 weeks (females), equal to 0, 13, 131, 446, or 1420 mg/kg bw/day (males) and 0, 16, 161, 543, or 1790 mg/kg bw/day (females). The additional 10 animals/sex in the 0, 10 000, and 30 000 ppm groups were maintained on control diet for 4 weeks following a 12-month exposure period to determine recovery of altered measurements. Data on homogeneity and stability of the diet were within acceptable ranges. Treatment-related alterations were observed at dietary concentrations of 10 000 and 30 000 ppm. These changes included an increased incidence of semi-solid faeces (males, 30 000 ppm), depressed body weight and weight gain, decreased efficiency of food utilization, decreased serum glucose (30 000 ppm group only), dose- related alterations in urinalysis measurements, particularly decreased urine pH (attributed to the highly acidic nature of the test material), and an increase in absolute (15% in females, 30 000 ppm) and relative kidney weight (31% in females, 30 000 ppm). For animals treated with 3000 ppm ethephon or lower, no signs of toxicity were seen. Plasma and erythrocyte ChE activities were statistically and dose-dependently inhibited at all dose levels. In plasma from males, the inhibition ranged from 17-44, 27-47, 38-67, and 45-56% at 300, 3000, 10 000, and 30 000 ppm, respectively, and in plasma from females from 15-27, 37-59, 47-65, and 56-72% at 300, 3000, 10 000, and 30 000 ppm, respectively. In erythrocytes from males, the inhibition ranged from 4-10, 39-47, 65-81, and 83-87% at 300, 3000, 10 000, and 30 000 ppm, respectively, and in erythrocytes from females from 9-19, 43-63, 72-79, and 82-86% at 300, 3000, 10 000, and 30 000 ppm, respectively. Inhibition of brain cholinesterase activity (< 9%) was not considered biologically significant. Carcinogenicity was not demonstrated. Animals from the 10 000 and 30 000 ppm dose groups, maintained under control conditions for 4 weeks following 52-week exposure, showed recovery of all altered measurements. Complete recovery to control values was, however, not observed for body weight and plasma and erythrocyte ChE activity. Based on decreased body weight, weight gain, efficiency of food utilization, and increased absolute and relative kidney weight, the author concluded that the NOAEL was 3000 ppm, equal to 131 and 161 mg/kg bw/day in males and females, respectively. The ChE inhibition was not considered detrimental to the animals based on the lack of other findings in both sexes consistent with ChE inhibition (Miller, 1989). However, based on inhibition of erythrocyte ChE activity, the Meeting concluded that the NOAEL was 300 ppm, equal to 13 and 16 mg/kg bw/day in males and females, respectively. Reproduction studies Rats Male and female Crl:CD(SD)BR Sprague-Dawley weanling rats (28/sex/group in the F0 generation) were exposed for 10 weeks to ethephon (purity 71.3%) at dietary concentrations of 0, 300, 3000, or 30 000 ppm, equal to 0, 22, 220, or 2260 mg/kg bw/day in F0 males and 0, 25, 260, or 2570 mg/kg bw/day in F0 females and 0, 20, 200, or 2220 mg/kg bw/day in F1b males and 0, 24, 245, or 2520 mg/kg bw/day in F1b females. The animals were randomly paired within dose groups for a 3-week mating period to produce the F1a generation. Exposures continued through mating, gestation, parturition and lactation. At weaning on lactational day 21, 28 F1a weanlings/sex/group were randomly selected to produce the F2 generation and were exposed to the same dietary concentration of ethephon as their parents for 10 weeks. In addition, 10 F1a weanlings/sex/dose were randomly selected for necropsy and examination of gross lesions. At least 10 days after weaning of the F1a litters, the F0 parents were paired again for 3 weeks to produce the F1b generation. Exposures continued through mating, gestation, parturition and lactation. After the F1b pups were weaned, the F0 parental animals were necropsied and high-dose and control animals examined for histopathologic lesions. At the time of weaning of the F1b litters, the decision was made to use the F1b generation to produce the F2 generation and the F1a animals (who had completed a 10-week exposure) were euthanized and discarded. Twenty-eight F1b weanlings/sex per group were randomly selected and exposed to the same dietary concentrations of ethephon as their parents for 10 weeks. In addition, 10 randomly selected F1b weanlings/sex/dose were necropsied and examined for gross lesions. After their pre-breed exposure, F1b parental animals were paired as described above to produce the F2a and F2b generations. Mating, gestation, lactation, necropsy of the F1b parents and selected F2a and F2b weanlings were performed as described above except that no F2 animals were selected as parents. Non-selected F1a, F1b, F2a and F2b pups were euthanized and discarded after the necropsy of their selected littermates. During the 10-week pre-breed exposure, F0 males and females at 30 000 ppm exhibited consistently reduction of body weight and weight gain and increased incidence of loose faeces. Females at 3000 ppm showed reduced food consumption during the 1st, 3rd and 6th weeks of exposure. At the F0 breed to produce F1a litters, gestational parameters were unaffected by treatment. Significant reductions in gestational body weights and lactational body weights were observed at 30 000 ppm. The F1a litters exhibited reduced body weights per litter at 30 000 ppm. During the second breeding of the F0 animals to produce F1b litters, gestational and lactational body weights were reduced at 30 000 ppm. An increased number of stillborn pups was observed at 30 000 ppm. F1b pup body weights per litter were reduced at 30 000 ppm and F1b pup deaths were increased at 3000 and 30 000 ppm. There were no treatment-related lesions observed in the necropsy of F1a and F1b pups which died during lactation, of randomly selected F1a and F1b pups or of F0 adults. There were also no treatment-related lesions observed in the histopathologic examination of selected organs from high-dose and control F0 adults. While terminal body weight was reduced in F0 males and females at 30 000 ppm, absolute organ weights were unaffected by treatment. Brain weight relative to body weight was increased for F0 males and females at 30 000 ppm. During the 10-week exposure of the F1a animals, males at 3000 and 30 000 ppm exhibited reductions in body weight, weight gain and food consumption. F1a females at 300, 3000, and 30 000 ppm exhibited significantly reduced body weight throughout the 10-week exposure, and significantly reduced body-weight gain at 3000 and 30 000 ppm. Both males and females at 30 000 ppm exhibited increased incidences of loose faeces. Since the F1b generation was selected to produce the F2 generation, selected F1b animals were also subjected to a 10-week pre-breed exposure and F1a animals were euthanized and discarded after their 10-week exposure. During this period, F1b males and females at 30 000 ppm exhibited reduced body weight, weight gain and food consumption as well as increased incidences of loose faeces. Weight gain was reduced in F1b males and females at 3000 ppm as well. In F1b males, an increased incidence of loose faeces was observed at 3000 ppm. At the F1b breeding to produce F2a litters, gestational parameters were unaffected by treatment. Maternal gestational body weight and lactational body weight at 30 000 ppm was reduced. F2a pup body weights per litter were reduced at 30 000 ppm. Perinatal deaths and lactational survival were unaffected by treatment. During the second breeding of the F1b animals to produce F2b litters, maternal F1b gestational body weight and lactational body weight at 30 000 ppm were reduced. The number of stillborn F2b pups and perinatal deaths were increased at 30 000 ppm. F2b pup body weights per litter were reduced at 3000 and 30 000 ppm. There were no treatment-related lesions observed in the necropsy of F2a and F2b pups which died during lactation, of randomly selected F2a and F2b pups (10/sex/dose) or of F1b adults. There were also no treatment-related lesions observed in the histopathologic examination of selected organs from high-dose and control F1b adults. Terminal body weight was reduced in F1b males and females at 30 000 ppm. Brain weight relative to body weight and ovaries/testes weight relative to body weight were increased for F1b males and females at 30 000 ppm; brain weight relative to body weight was also increased in F1b parental adults at 3000 ppm. Based on reduced food intake, body weight and body weight gain, the NOAEL for maternal toxicity and for filial toxicity was 300 ppm, equal to 22 and 25 mg/kg bw/day in males and females, respectively. There was no adverse effect on reproduction at the highest dose tested of 30 000 ppm, equal to 2220 and 2520 mg/kg bw/day in males and females, respectively (Neeper-Bradley & Tyl, 1990). Special studies on delayed cutaneous hypersensitivity Evaluation of the delayed cutaneous hypersensitivity of ethephon (purity 72%) was performed in young adult albino Dunkin- Hartley guinea-pigs (10 animals/sex/group), allocated in one test article-control group (induction with vehicle and challenge with test article), one treated group (induction and challenge with test article) and one positive control group (induction and challenge with DNCB). Induction was performed by 3 series of 2 x 0.1 ml intradermal injections (Freund's complete adjuvant (50% V/V in water), ethephon (0.75 V/V in water), and a 1:1 mixture of the two solutions), or by topical occlusive route for 48 hours with 0.5 ml of ethephon (50% V/V in water). During challenge, the topical occlusive application was performed with 0.5 ml ethephon (35% V/V in water) or 0.5 ml of 1-chloro-2,4-dinitrobenzene (DNCB) (0.05% W/W in 1.2 propylene glycol). The cutaneous macroscopic examinations were performed according to the scale of Magnusson & Kligman after 24 and 48 hours followed by histopathological examinations. Doubtful macroscopic reactions were observed in 15 out of 20 ethephon-treated animals. Histopathological examination of these lesions showed images of orthoergic irritation in 9 animals. No reactions of cutaneous sensitization were observed in the 20 ethephon-treated guinea-pigs but the lesions of orthoergic irritation noted in 9 of them may hide possible reactions of cutaneous sensitization (Clement, 1989). Special studies on delayed neurotoxicity Groups of 10 white Vantress chickens received by intubation 1000 mg/kg bw/day ethephon (purity 88%) on days 1 through 5, or 1000 mg ethephon/kg bw on day 1 and thereafter 500 mg/kg bw/day on days 2 through 10. Two positive control groups received 60 mg/kg bw/day tri-o-cresyl phosphate. Two negative control groups received 300 mg/kg bw/day of olive oil. Fifteen out of 40 animals died in the test groups and 8 out of 20 in the positive control groups. Only the deaths which occurred in the positive control group were considered to be due to neurotoxicity. No clinical signs of neurotoxicity and no gross pathology was observed in any of the necropsied chickens. Microscopic examination showed no cytopathologic changes in the spinal cord or sciatic nerve of the animals receiving ethephon. Administration of tri-o-cresyl phosphate caused clinical signs of neurotoxicity and some spinal axonal dystrophy in 10 chickens and sciatic neuropathy in one chicken. The Meeting concluded that there was no evidence of delayed neurotoxicity at doses up to 1500 mg/kg bw/day (Weatherholtz & Shott, 1970). Groups of 15-30 white leghorn chickens received a single oral dose of 0, 3160, or 3850 mg/kg bw ethephon (purity 71%). A positive control group received 500 mg/kg bw tri-o-tolyl phosphate orally. Twenty-one days following dosing, all surviving birds (21) were treated orally with a single dose of 2370 mg/kg bw ethephon. Twenty-eight of thirty birds in the 3850 mg/kg group were found dead within 24 hours after the first dose. One additional mortality was recorded in this group on test day 8. Ten of thirty birds in the 3160 mg/kg group were found dead within 24 hours after the first dose. One bird from this group was found dead within 48 hours after the second dose. Signs of lethargy and anorexia were present following dosing. Complete recovery of all surviving birds was seen during both 21-day test periods. There were no signs of locomotor disturbances or other clinical signs of delayed neurotoxicity among any of the ethephon-treated chickens during the 42-day test period. Ethephon-treated birds exhibited decreased food intake and body weight loss during the test period. The positive control birds lost weight and exhibited behavioural signs of neurotoxicity by day 9 of the investigation. All positive control birds were sacrificed in extremis on test day 17 or 18. Gross pathologic examination of birds found dead within 24 hours after dosing revealed diffuse red discoloration with severe dilation of the vessels in the intestinal tract and diffuse light grey discoloration with transparent gel circumscribing the crop area in all birds. Histopathology of neural tissues from the ethephon-treated birds revealed no changes. Treatment-related lesions were noted with respect to the positive control birds. No evidence of delayed neurotoxicity was observed in this study at 3160 mg/kg bw. The 3850 mg/kg bw dose could not be evaluated since at this high level only one bird survived the observation period (Fletcher, 1983). Special studies on embryotoxicity/teratogenicity Sexually mature, virgin female Charles River COBS(R) CD rats were acclimated for 10 days before being mated with males of the same strain at an age of 15 weeks. Groups of pregnant rats (25 rats/group) were dosed orally by garage (10 ml 0.5% aqueous Methocel(R) suspension/kg bw) with 0, 200, 600, or 1800 mg/kg bw/day ethephon (purity not given) on days 6 through 19 of gestation. There were no biologically significant differences in maternal appearance, behaviour and body-weight gain, and there were no signs of fetotoxicity or teratogenicity in the 200 and 600 mg/kg bw/day group when compared to the control group. At 1800 mg/kg bw/day, 14 of the 25 dams died during the treatment period and only nine litters with viable fetuses were available for evaluation. The NOAEL was 600 mg/kg bw/day, based on maternal toxicity (Rodwell, 1980). Male and female Crl:CD(SD)BR rats were used for breeding. Mated females (25/group) were treated with ethephon (purity 71.7%) by oral gavage at 0, 125, 250, or 500 mg/kg bw/day on days 6 through 115 of gestation. Survival rates were 100% at all dose levels and there were no test material-related clinical observations. There were no statistically significant differences in body weights of treated animals when compared with those of controls and no test material- related parental necropsy observations. The pregnancy rates were 92%, 84%, 88%, and 88% at 0, 125, 250, and 500 mg/kg bw/day, respectively. There were no significant differences in the number of corpora lutea or implantations, implantation efficiency, the number or percentage of live or resorbed fetuses, or mean fetal weights and no test material-related fetal external, soft tissue, or skeletal abnormalities. There was a significant increase in the total number of fetuses at 125 and 250 mg/kg bw/day with skeletal abnormalities such as reduced vertebral arches; however, there was no statistical difference in the percent of total fetal skeletal abnormalities. This observation was not considered toxicologically important because there was no dose-response and no increase in the total number or percent of fetuses in the high dose. The NOAEL for maternal toxicity was 500 mg/kg bw/day. There was no teratogenicity at the dose levels tested (Henwood, 1989). Rabbits Ethephon (purity unknown) at 0, 50, 100, or 250 mg/kg bw/day in water was administered orally by intubation to groups of 17 pregnant New Zeeland white rabbits from day 6 through day 19 of gestation. An increased incidence of inactive animals was noted in the maternal 250 mg/kg bw/day group during the treatment and post- treatment phases. Lower food consumption values during the treatment and a significantly lower maternal survival rate were also noted in this group. No other significant findings were noted in the maternal animals. The mean number of resorptions was higher at 100 and 250 mg/kg bw/day and on a per litter basis, a significantly higher incidence of resorption was noted in the 250 mg/kg bw/day animals. The mean number of live fetuses and fetal viability was lower at 100 and 250 mg/kg bw/day. However, no statistically significant differences were noted. All other fetal data were comparable between the control and treated groups. The NOAEL for maternal and embryo/fetotoxicity was 50 mg/kg bw/day based on the lower survival rate and higher resorptions. There were no teratogenic effects (Weatherholtz et al., 1981). Ethephon (purity 72.2%) was administered by oral gavage to pregnant New Zeeland white rabbits on days 7 through 19 of gestation at dosages of 0, 63, 125, or 250 mg/kg bw/day. The control animals received deionized water. The percent of females surviving to the scheduled necropsy was 95, 91, 95 and 14%, for the control, 63, 125, and 250 mg/kg bw/day group, respectively. Test material-related clinical observations were seen for 17/22 females at 250 mg/kg bw/day including ataxia and prostration. There were no test material-related clinical observations at 63 or 125 mg/kg bw/day. At 250 mg/kg bw/day, but not at the two lower dose levels, the body weight and weight gain were significantly lower than those of controls. Parental necropsy findings, such as erosions in the stomach, were more frequent among the 250 mg/kg bw/day does (8 does compared with 2 control does). Pregnancy rates were 95, 95, 91, and 82% for the control, 63, 125, and 250 mg/kg group, respectively. Post-implantation loss and the percent of early resorptions were considerably higher and the percent of live fetuses was lower at 250 mg/kg bw/day. There were no statistical differences in fetal body weight and no test material- related fetal external, soft tissue, or skeletal variations or malformations. The NOAEL for maternal and embryo/fetotoxicity was 125 mg/kg bw/day. There were no teratogenic effects (Henwood, 1990). Special studies on genotoxicity Ethephon was not considered genotoxic since it was negative in several in vitro and in vivo test systems except when tested in the Ames test with Salmonella typhimurium, strain TA1535. Data are shown in Table 2. Special studies on skin irritation In a skin irritation test, 0.5 ml of an aqueous ethephon (70% active ingredient) solution was placed on one intact site of the dorsal area of the trunk of each of 6 New Zeeland white rabbits, clipped a few days before dosing and trimmed just before application of the test material. Contact periods were 1 or 4 hours. Ethephon was found to be corrosive after the 4-hour contact period since spots of necrosis and edema were observed in 4 rabbits and contact erythema in 6 rabbits. After the 1-hour procedure, erythema, but no edema or necrosis, was observed (Myers, 1983). Special studies on skin sensitization Groups of Hartley-derived albino guinea-pigs (5/sex), received a patch containing 0.4 ml 25% w/v ethephon (72% active ingredient) water solution. The patch was applied to each animal and occluded for 6 hours once weekly for three consecutive weeks. Dinitrochlorobenzene in 100% alcohol was used as the control. Following a 2-week rest period, the guinea-pigs were topically challenged with ethephon (10% w/v in distilled water). No evidence of contact sensitization was observed following challenge with ethephon (Rush, 1989). Observations in humans A preliminary dose range study with ethephon was carried out in 2 human volunteers. They received 5.4-120 rag/day ethephon (purity 88%) in propylene glycol (by oral capsule, in 3 divided doses, one immediately after each meal) over a 7-week period. The approximate dosage ranged from 0.06-1.25 mg/kg bw/day. Table 2. Results of genotoxicity assays on ethephon Test system Test object Concentration Purity Results Reference of ethephon In vitro Ames test (with and Salmonella typhimurium 0.1-50 72.3% Jagannath, 1987 without metabolic TA1535 µg/plate +(±S9) activation) TA1537 -(±S9) TA1538 -(±S9) TA98 -(±S9) TA100 -(+S9) DNA repair test Rat hepatocyte primary 10-1000 71.0% - Barfknecht et al., culture µg/ml - 1984 Unscheduled DNA Rat hepatocyte 25-1000 71.3% - Cifone, 1988 synthesis assay µg/ml Chromosomal aberration Chinese hamster ovary 502-2010 71.3% - Murli, 1988 (± activat.) cell µg/ml CHO/HGPRT mutation Mammalian CHO-KI- 500-2500 71.0% - Godek et al., assay (+ activat.) BH4 cells µg/ml 1983 CHO/HGPRT mutation Mammalian CHO-KI- 166-5000 71,0% - Godek et al., assay (+ activat.) BH4 cells µg/ml 1984 CHO/HGPRT mutation Mammalian CHO-KI- 500-2600 72.3% - Young, 1988 assay (+ activat.) BH4 cells µg/ml Table 2 (contd) Test system Test object Concentration Purity Results Reference of ethephon In vivo Micronucleus test Mice 200 mg/kg >90% - Sorg et al., 1981 bw Dominant lethal Rats 250-1000 ? - Naismith & mg/kg bw (>707) Matthews, 1979 The compound had no inhibitory activity on human plasma or erythrocyte ChE activity. No persistent side effects were observed during the course of the study. However, transient, subjective feelings of urinary urgency were experienced by both volunteers. A slight elevation in serum GPT was also observed from the 26-46th day of study. Laboratory studies performed two weeks following the last compound administration gave test results within normal biological limits (Reese, 1971). Sixteen human volunteers in good health received orally by capsule 0 (3 males and 3 females) or approximately 120 mg/day (5 males and 5 females) of ethephon (divided into 3 doses) for 28 days (purity 10% in a powdered formulation), followed by a 14-day dose- free period. Based on average substance consumption and average body weight for the subjects, the approximate dosage was 1.5 mg/kg bw/day in makes and 2.2 mg/kg bw/day in females. The results indicated that the compound had no inhibitory effect on human plasma or erythrocyte cholinesterase activity. No persistent side effects were observed during the course of the study. However, transient subjective complaints of urinary urgency, sudden "onset of diarrhoea", effect on appetite and dyspepsia were recorded. Laboratory studies performed during the study and two weeks following the last dose of test material were, in general, within normal biological limits. Based on clinical symptoms, a NOAEL could not be determined (Reese, 1972). Thirty human volunteers in good health received orally by capsule, without knowing to which group they were assigned, 0 (6 males and 4 females) or 0.5 mg/kg bw/day (10 males and 10 females) of ethephon (purity 2.5% in a powdered formulation), divided into 3 doses, for 16 days, followed by a 29-day dose-free period. Plasma cholinesterase activity was significantly inhibited and in a reversible manner (54-62% of pre-dose levels). No gross symptoms nor changes in haematology, clinical chemistry, or urinalysis were associated with the test substance administration. The NOAEL was 0.5 mg/kg bw/day in both males and females, based on lack of inhibition of erythrocyte cholinesterase (Weir, 1977a). Twenty human volunteers in good health received orally by capsule, without knowing to which group they were assigned, 0 (3 males and 3 females), 0.17 (3 males and 4 females) or 0.33 mg/kg bw/day (4 males and 3 females) of ethephon (purity 22% in a powdered formulation), divided into 3 doses, for 22 days, followed by a 14- day dose-free period. Plasma ChE activity was significantly and irreversibly inhibited (59-74% of pre-dose values). No gross symptoms nor changes in haematology, clinical chemistry, or urinalysis were associated with the test substance administration. The NOAEL was 0.33 mg/kg bw/day in both males and females, based on lack of inhibition of erythrocyte cholinesterase (Weir, 1977b). COMMENTS Following oral administration of ethephon to rats, about 90% of the administered radioactivity was recovered, principally in urine (50%), expired air (19%), and faeces (6%) during 120 hours post- dose. Most of the dose was recovered within 24 hours post-dose. After oral administration of ethephon to dogs, radioactivity was found in urine (40%), expired air (30%) and faeces (5%). Total body retention was 1%. Peak plasma and red blood cell concentrations were observed 2 hours after dosing. Only traces were observed after 22 hours. After oral administration to rats, ethephon was excreted in urine and faeces as the mono- and disodium salts and some unidentified metabolites and metabolized to ethylene and eliminated in the expired air. In dogs, ethephon is partly metabolized to ethylene and eliminated in expired air and also excreted unchanged in the urine. In dogs dosed orally with ethephon, plasma cholinesterase activity was inhibited at 2 hours with recovery starting within a few hours. Erythrocyte cholinesterase levels responded more slowly with signs of recovery at 72 hours. Ethephon has a low oral acute toxicity in mice, rats, and rabbits. WHO has classified ethephon as unlikely to represent acute hazard in normal use. Ethephon is corrosive to the skin of rabbits. In a four-week study in mice at dietary concentrations of 0, 30, 100, 300, 1000 or 3000 ppm, the NOAEL was 300 ppm (equal to 51 mg/kg bw/day), based on inhibition of erythrocyte cholinesterase activity. In a four-week study in rats at dietary concentrations of 0, 625, 1250, 2500, 5000 or 10 000 ppm, the NOAEL was 625 ppm (equal to 52 mg/kg bw/day), based on inhibition of erythrocyte cholinesterase activity. In a one-year study in dogs at dietary concentrations of 0, 100, 300, 1000 or 2000 ppm, the NOAEL was 1000 ppm (equal to 27 mg/kg bw/day), based on soft stools and changes in body and spleen weight. However, cholinesterase activities were not determined. In a two-year study in dogs at dietary concentrations of 0, 30, 300, or 1500 ppm, the NOAEL was 30 ppm (equal to 0.86 mg/kg bw/day), based on inhibition of erythrocyte cholinesterase activity and smooth muscle hypertrophy in the stomach and small intestine. In two 78-week studies in mice at dietary concentrations of 0, 30, 100, 300, 1000 or 10 000 ppm, the NOAEL was 100 ppm (equal to 14 mg/kg bw/day), based on inhibition of erythrocyte cholinesterase activity. There was no evidence of carcinogenicity. In two 104-week studies in rats at dietary concentrations of 0, 30, 300, 3000, 10 000 or 30 000 ppm, the NOAEL was 30 ppm (equal to 1.2 mg/kg bw/day), based on inhibition of erythrocyte cholinesterase activity. There was no evidence of carcinogenicity. Brain cholinesterase was not depressed in any study. In a two-generation reproduction study in rats at dietary concentrations of 0, 300, 3000 or 30 000 ppm, the NOAEL for maternal and filial toxicity was 300 ppm (equal to 22 mg/kg bw/day), based on reduced food intake, body weight and weight gain. There was no adverse effect on reproduction. In two studies in hens for delayed neurotoxicity, no evidence of delayed neurotoxicity was observed. In two oral teratogenicity studies in rats at dose levels of 0, 125, 200, 250, 500, 600, or 1800 mg/kg bw/day, the NOAEL was 600 mg/kg bw/day, based on maternal toxicity. There were no teratogenic effects. In two teratogenicity studies in rabbits at oral dose levels of 0, 50, 63, 100, 125, or 250 mg/kg bw/day, the NOAEL was 50 mg/kg bw/day, based on maternal and embryo/fetotoxicity. There were no teratogenic effects. After reviewing the in vitro and in vivo genotoxicity data, the Meeting concluded that there was no evidence of genotoxicity. In 16 male and female human volunteers treated orally with 0 or 120 mg/day (divided into 3 doses) of ethephon (approximately 1.5 and 2.2 mg/kg bw/day in males and females, respectively) for 28 consecutive days, no significant inhibitory effect on human plasma or erythrocyte cholinesterase activity was observed. Subjective complaints of urinary urgency, sudden onset of diarrhoea, effect on appetite and dyspepsia were recorded. Based on clinical symptoms, a NOAEL could not be determined. In 30 male and female human volunteers treated orally with 0 or 0.5 mg/kg bw/day of ethephon (divided into 3 doses) for 16 consecutive days, plasma cholinesterase activity was inhibited but recovered within the recovery period of 29 days. The NOAEL was 0.5 mg/kg bw/day in both males and females, based upon the lack of inhibition of erythrocyte cholinesterase. In 20 male and female human volunteers receiving 0, 0.17 or 0.33 mg/kg bw/day of ethephon orally (divided into 3 doses) for 22 consecutive days, plasma cholinesterase activity was inhibited and did not recover within the recovery period of 14 days. The NOAEL was 0.33 mg/kg bw/day in both males and females, based upon the lack of inhibition of erythrocyte cholinesterase. An ADI of 0-0.05 mg/kg bw was established, based on the NOAEL in the 16-day study in humans of 0.5 mg/kg bw/day, using a 10-fold safety factor. TOXICOLOGICAL EVALUATION Level causing no toxicological effect Mouse: 100 ppm, equal to 14 mg/kg bw/day (78-week study) Rat: 30 ppm, equal to 1.2 mg/kg bw/day (104-week study) Dog: 30 ppm, equal to 0.86 mg/kg bw/day (two-year study) Humans: 0.5 mg/kg bw/day (16-day study). Estimate of acceptable daily intake for humans 0-0.05 mg/kg bw. Studies which will provide information valuable in the continued evaluation of the compound The Meeting noted that ethephon is a dibasic phosphonic acid and therefore not able to phosphorylate hydrolases at the serine residue. However, in vivo data showed inhibition of plasma and erythrocyte, but not brain, cholinesterase. Neither data with the pure compound nor in vitro studies were available. The Meeting considered that these effects on cholinesterases need clarification and recommended re-evaluation of the compound in 1995. Further observations in humans. REFERENCES Barfknecht, T.R., Naismith, R.W. & Matthews, R.J. (1984). Rat hepatocyte primary culture/DNA repair test. Unpublished report No. PH 311-UC-002-84 from Pharmacon Research International, Inc., Waverly, Pennsylvania 18471, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Cifone, M.A. (1988). Mutagenicity test on ethephon in the rat primary hepatocyte unscheduled DNA synthesis assay. Unpublished Report No. 10065-0-447 from Hazleton Laboratories America, Inc., 5516 Nicholson Lane, Suite 400, Kensington, Maryland 20895, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Clement, C. (1989). Test to evaluate the sensitizing potential in the guinea-pig. Guinea-pig maximization test. Unpublished report No. 903326 from Hazleton France, Les Oncins, B.P. 118 -69210 L'Arbresle, France. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Fletcher, D.W. (1983). 42-Day neurotoxicity study with ethephon in mature white leghorn chickens. Unpublished and unnumbered report (project No. 83 DN 102) from Bio-life Associates, Ltd. Route 3, Box 156, Neilisville, Wisconsin 54456, USA. Submitted to WHO by Rhône- Poulenc, Secteur Agro, Lyon, France. Godek, E.G., Naismith, R.W. & Matthews, R.J. (1983). CHO/HGPRT, mammalian cell forward gene mutation assay. Unpublished report No. PH-314-UC-003-83 from Pharmacon Research International, Inc., Waverly, Pennsylvania 18471, USA. Submitted to WHO by Rhône-Poulenc Secteur Agro, 14, 20, Rue Pierre Baizet, BP 9163 - 69263 Lyon Cedex 09, France. Godek, E.G., Naismith, R.W. & Matthews, R.J. (1984). CHO/HGPRT, mammalian cell forward gene mutation assay. Unpublished report No. PH-314-UC-001-84 from Pharmakon Research International, Inc., Waverly, Pennsylvania 18471, USA. Submitted to WHO by Rhône-Poulenc Secteur Agro, 14, 20, Rue Pierre Baizet, BP 9163 - 69263 Lyon Cedex 09, France. Hamada, N.N. (1989). One-year oral toxicity study in Beagle dogs with ethephon(R). Unpublished and unnumbered report (Project No. HLA 400-722) from Hazleton Laboratories America Inc., 9200 Leesburg Turnpike, Vienna, Virginia 22180, USA. Submitted to WHO by Rhône- Poulenc, Secteur Agro, Lyon, France. Hardy, I.A.J., Chem, C., Marshall, I.R. & Outram, J.R. (1990). Plant growth regulators: ethephon. Spectroscopic identification of metabolites from a 14C-ethephon ADME study in the rat. Unpublished report No. D. Ag. 1523 from Rhône-Poulenc Agriculture Limited, Fyfied Road, Ongar, Essex, United Kingdom. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Henwood, S.M. (1989). Teratology study with ethephon technical-base 250 in rats. Unpublished and unnumbered report (Project No. HLA 6224-125) from Hazleton Laboratories America Inc., 3301 Kinsman Boulevard., P.O. Box 7545, Madison, Wisconsin 53707, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Henwood, S.M. (1990). Teratology study with ethephon technical-base 250 in rabbits. Unpublished and unnumbered report (Project No. HLA 6224-158) from Hazleton Laboratories America Inc., 3301 Kinsman Boulevard., P.O. Box 7545, Madison, Wisconsin 53707, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Holsing, G.C. (1969). Acute oral - Mice, five compounds. Unpublished and unnumbered report (Project No. 141-197) from Hazleton Laboratories, Inc., TRW Life Sciences Center, P.O. Box 30, Falls Church, Virginia 22046, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Holsing, G.C. (1969). Three-week repeated dermal application - rabbits. Unpublished and unnumbered report (Project No. 141-171) from Hazleton Laboratories, Inc., TRW Life Sciences Center, P.O. Box 30, Falls Church, Virginia 22046, USA. Submitted to WHO by Rhône- Poulenc, Secteur Agro, Lyon, France. Jagannath, D.R. (1987). Mutagenicity test on ethephon Base 250 in the Ames Salmonella/microsome reverse mutation assay. Unpublished report No. 10065-0-401 from Hazleton Laboratories America, Inc., 5518 Nicholson Lane, Suite 400, Kensington, Maryland 20895, USA. Submitted to WHO by Rhône-Poulenc Secteur Agro, 14, 20, Rue Pierre Baizet, BP 9163 - 69263 Lyon Cedex 09, France. Miller, J.P. Van & Troup, C.M. (1986a). Twenty-eight day dietary toxicity study with ethephon in mice. Unpublished report No. 48-139 from Bushy Run Research Center, R.D. 4, Mellon Road, Export, Pennsylvania 15632, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Miller, J.P. Van & Troup, C.M. (1986b). Twenty-eight day dietary toxicity study with ethephon in mice study II. Unpublished report No. 49-4 from Busily Run Research Center, R.D. 4, Mellon Road, Export, Pennsylvania 15632, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Miller, J.P. Van & Troup, C.M. (1986c). Twenty-eight day dietary toxicity study with ethephon in rats. Unpublished report No. 48-123 from Bushy Run Research Center, R.D. 4, Mellon Road, Export, Pennsylvania 15632, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Miller, J.P. Van & Troup, C.M. (1986d). Twenty-eight day dietary toxicity study with ethephon in rats Study No. II. Unpublished report No. 49-3 from Bushy Run Research Center, R.D. 4, Mellon Road, Export, Pennsylvania 15632, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Miller, J.P. Van (1988). Lifetime dietary oncogenicity study with ethephon in albino mice. Unpublished report No. 51-502 from Bushy Run Research Center, R.D. 4, Mellon Road, Export, Pennsylvania 15632, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Miller, J.P. Van (1989). Lifetime dietary combined chronic toxicity and oncogenicity study with ethephon in albino rats. Unpublished report No. 51-501 from Bushy Run Research Center, R.D. 4, Mellon Road, Export, Pennsylvania 15632, USA. Submitted to WHO by Rhône- Poulenc, Secteur Agro, Lyon, France. Murli, H. (1988). Mutagenicity test on ethephon base 250 in an in vitro cytogenetic assay measuring chromosomal aberration frequencies in Chinese hamster ovary (CHO) cells. Unpublished report No. 10065-0-437 from Hazleton Laboratories America, Inc., 5516 Nicholson Lane, Suite 400, Kensington, Maryland 20895, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Myers, R.C. (1983). Ethephon base 250, primary skin irritancy (D.O.T.). Unpublished report No. 46-11 from Bushy Run Research Center, R.D. 4, Mellon Road, Export, Pennsylvania 15632, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Myers, R.C. (1984). Ethephon base 250, acute peroral toxicity study. Unpublished report No. 47-49 from Bushy Run Research Center, R.D. 4, Mellon Road, Export, Pennsylvania 15632, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Myers, R.C. (1989). Ethephon base 250, acute percutaneous toxicity study. Unpublished report No. 46-122 from Bushy Run Research Center, R.D. 4, Mellon Road, Pennsylvania 15632, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Nachreiner, D.J. & Klonne, D.R. (1989). Ethephon base 250, acute aerosol inhalation toxicity test in rats. Unpublished report No. 52- 580 from Bushy Run Research Center, R.D. 4, Mellon Road, Export, Pennsylvania 15632, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Naismith, R.W. & Matthews, R.J. (1979). Dominant lethal study. Unpublished report No. 56375 from Pharmakon Laboratories, 1140 Quincy Avenue, Scranton, Pennsylvania 18510, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Neeper-Bradley, T.L. & Tyl, R.W. (1990). Two-generation reproduction study in CD albino rats exposed to ethephon by dietary inclusion. Unpublished report No. 51-539 from Bushy Run Research Center, 6702 Mellon Road, Export, Pennsylvania 15632-8902, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Reno, F.E & Voelker, R.W. (1977). A two-year dietary study in dogs, ethrel. Unpublished and unnumbered report (Project No. 141-260) from Hazleton Laboratories America Inc., 9200 Leesburg Turnpike, Vienna, Virginia 22180, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Reno, F.E, Serota, D.G., & Voelker R.W. (1978). 104-Week chronic toxicity study in rats. Unpublished and unnumbered report (Project No. 141-263) from Hazleton Laboratories America Inc., 9200 Leesburg Turnpike, Vienna, Virginia 22180, USA. Submitted to WHO by Rhône- Poulenc, Secteur Agro, Lyon, France. Reese, W.H. (1971). Preliminary dose range study in two human volunteers. Unpublished and unnumbered report (project No. 1223) from Bionetics Research Laboratories, Division of Litton Industries, Bionetics Research Laboratories, Inc. 7300 Pearl Street, Bethesda, Maryland 20014, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Reese, W.H. (1972). Evaluation of ethrel in human volunteers. Unpublished and unnumbered report (project No. 7223) from Bionetics Research Laboratories, Division of Litton Industries, Bionetics Research Laboratories, Inc. 7300 Pearl Street, Bethesda, Maryland 20014, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Rodwell, D.E. (1980). Teratology study in rats. Unpublished report No. 369-042 from International Research and Developmental Corporation, Mattawan, Michigan 49071, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Rush, R.E. (1989). Dermal sensitization study in guinea-pigs with Base A-250. Unpublished and unnumbered report (study No. SLS 3147.44) from Springborn Laboratories, Inc. Mammalian Toxicology Division, 553 North Broadway, Spencerville, Ohio 45887, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Savage, E.A. (1990). 14C-ethephon: absorption, distribution, metabolism, and excretion in the rat. Unpublished report No. 68/103 & P89/366 from Hazleton UK, North Yorkshire HG3 1PY, United Kingdom. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Sorg, R.M., Naismith, R.W. & Matthews, R.J. (1981). Genetic toxicology micronucleus test (MNT). Unpublished report No. PH 309A- UC-001-81 from Pharmakon Laboratories, Waverly, Pennsylvania 18471, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Stephen, W. & Walker D. (1971). Metabolism of 14C-ethephon in the dog. Unpublished and unnumbered report from Hazleton Laboratories America Inc., 9200 Leesburg Turnpike, Vienna, Virginia 22180, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Stephen, W. & Stanovick, R.P. (1971). Identification of 14C- ethephon metabolites in the dog. Unpublished and unnumbered report from Hazleton Laboratories America Inc., 9200 Leesburg Turnpike, Vienna, Virginia 22180, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Voss, K.A. & Becci, P.J. (1985). 78-Week oncogenic evaluation in Swiss albino mice. Unpublished and unnumbered report (study no. 5754) from Food and Drug Research Laboratories, Inc, Route 17C, P.O. Box 107, Waverly, NY 14892-0107 607 565-8131, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Weatherholtz, W.M. (1980). Acute oral toxicity study in rabbits. Unpublished and unnumbered report (Project No. 400-630) from Hazleton Laboratories America, Inc., 9200 Leesburg Turnpike, Vienna, Virginia 22180, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Weatherholtz, W.M. & Shott, L.D. (1970). Neurotoxicity Study - Hens, Ethrel, formulated, Etrel, technical. Unpublished and unnumbered report (Project No. 141-218) from Hazleton Laboratories, Inc., TRW Life Sciences Center, P.O. Box 30, Falls Church, Virginia 22046, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Weatherholtz, W.M, Wolfe, G.W. & Durloo, R.S. (1981). Teratology study in rabbits, technical ethephon. Unpublished and unnumbered report (Project No. 400-635) from Hazleton Laboratories America, Inc., 9200 Leesburg Turnpike, Vienna, Virginia 22180, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Weir, R.J. (1977a). Evaluation of ethephon in human volunteers. Unpublished and unnumbered report (project No. 2416) from Litton Bionetics, Inc. 5516 Nicholson Lane, Kensington, Maryland 20795, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. Weir, R.J. (1977b). Evaluation of ethephon in human volunteers. Unpublished and unnumbered report (project No. 2476) from Litton Bionetics, Inc. 5516 Nicholson Lane, Kensington, Maryland 20795, USA. Submitted to WHO by Rhône-Poulenc, Secteur Agro, Lyon, France. WHO (1992). The WHO recommended classification of pesticides by hazard and guidelines to classification 1992-1993 (WHO/PCS/92.14). Available from the International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland. Young, R.R. (1988). Mutagenicity test on ethephon base 250 in the CHO/HGPRT forward mutation assay. Unpublished report No. 10065-0-435 from Hazleton Laboratories America, Inc. 5516 Nicholson Lane, Suite 400, Kensington, Maryland 20895, USA. Submitted to WHO by Rhône- Poulenc, Secteur Agro, Lyon, France.
See Also: Toxicological Abbreviations Ethephon (Pesticide residues in food: 1977 evaluations) Ethephon (Pesticide residues in food: 1978 evaluations) Ethephon (Pesticide residues in food: 1983 evaluations) Ethephon (JMPR Evaluations 2002 Part II Toxicological)