PESTICIDE RESIDUES IN FOOD - 1997 Sponsored jointly by FAO and WHO with the support of the International Programme on Chemical Safety (IPCS) TOXICOLOGICAL AND ENVIRONMENTAL EVALUATIONS 1994 Joint meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group Lyon 22 September - 1 October 1997 The summaries and evaluations contained in this book are, in most cases, based on unpublished proprietary data submitted for the purpose of the JMPR assessment. A registration authority should not grant a registration on the basis of an evaluation unless it has first received authorization for such use from the owner who submitted the data for JMPR review or has received the data on which the summaries are based, either from the owner of the data or from a second party that has obtained permission from the owner of the data for this purpose. AMINOMETHYLPHOSPHONIC ACID (AMPA) First draft prepared by E. van Apeldoorn and P.H. van Hoeven National Institute of Public Health and Environment, Bilthoven, The Netherlands 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 Genotoxicity Reproductive toxicity Multigeneration reproductive toxicity Developmental toxicity Special studies: Dermal and ocular irritation Toxicological profiles of AMPA and glyphosate Comments Toxicological evaluation References Explanation Glyphosate was evaluated toxicologically by the 1986 JMPR, which allocated an ADI of 0-0.3 mg/kg bw (Annex I, reference 47). The primary degradation product of glyphosate in plants, soil, and water is aminomethylphosphonic acid (AMPA), whose chemical structure is very similar to that of glyphosate (Figure 1). AMPA itself has no commercial use. On the basis of the low residual levels of AMPA in crops which are susceptible to glyphosate, the 1986 Joint Meeting concluded that AMPA could be omitted from the residue in recommendations for MRLs, but recent supervised trials on the application of glyphosate to genetically modified crops have shown AMPA to be the main residue. As residues of AMPA may therefore be of toxicological concern, the compound was evaluated by the present Meeting. Evaluation for acceptable daily intake 1. Biochemical aspects (a) Absorption, distribution, and excretion Eight male Wistar rats weighing about 150 g were fasted for 4 h and then given a single oral dose of 6.7 mg/kg bw 14C-AMPA by gavage. Urine, faeces, and expired carbon dioxide (in 1 N NaOH) were analysed for radiolabel 12, 24, 48, 72, 96, and 120 h after treatment (see Table 1). After 120 h, a blood sample was taken and the animals were killed; cage washes and tissues (see Table 2) were then analysed for radiolabel. AMPA was moderately absorbed from the gut, as demonstrated by excretion of 13% of the radiolabel in urine within 12 h, 18% within 24 h, and 20% within 120 h. Consequently, the major route of excretion is the faeces: > 50% of the administered radiolabel was excreted within 24 h and 74% by 120 h. In tissues, 0.06% of the radiolabel was recovered after 120 h; < 0.1% was recovered as expired CO2 within 24 h. Minor residues were found in liver, kidneys, and muscle (Colvin et al., 1973). Bone, bone-marrow, and eyes, which contain relatively high residues of radiolabel (0.2-0.6% of the dose in comparison with 0.02-0.05% in other tissues) after oral administration of 14C-glyphosate to rats (Howe et al., 1988; Ridley & Mirly, 1988), were not analysed in the study of Colvin et al. (1973). (b) Biotransformation Two groups of five Wistar rats of each sex received 10 mg/kg bw 14C-glyphosate orally, one group after preconditioning with unlabelled glyphosate. Only 0.2-0.4% of the radiolabel recovered in the excreta appeared to be AMPA (Howe et al., 1988). Table 1. Recovery of radiolabel after a single oral dose of 6.7 mg/kg bw 14C-AMPA Matrix Time after treatment (h) 0-12 12-24 24-48 48-72 72-96 96-120 0-120 Urine 13 4.5 1.5 0.66 02.3 0.05 20 Faeces 5.9 47 19 0.91 0.12 0.06 73 CO2 0.06 0.01 0 0 0 0 0.07 Cage wash 0.13 Tissues 0.06 Total 19 52 21 1.6 0.35 0.11 94 Table 2. Radiolabelled AMPA in tissues 120 h after a single oral dose of 6.7 mg/kg bw Tissue % of dose AMPA equivalent (µg/kg fresh weight) Liver 0.01 6 Kidney <0.01 6 Muscle 0.02 3 Fat 0.01 4 Gut 0.02 8 Spleen <0.01 4 Heart <0.01 4 Brain <0.01 1 Testis <0.01 3 Blood <0.01 3 Colvin et al. (1973) showed that most orally absorbed 14C-APMA is excreted as unchanged compound in the urine of rats, as measured by thin-layer and gas-liquid chromatography and nuclear magnetic resonance and mass spectral analyses. The fact that < 0.1% of the administered activity was expired as carbon dioxide also demonstrated that only very small amounts of the absorbed material were metabolized. 2. Toxicological studies (a) Acute toxicity Groups of two male and three female Sprague-Dawley rats were given AMPA (purity unspecified) as a 40% solution in corn oil at one of four doses by gavage and observed for seven days. Deaths occurred at a dose of 8300 mg/kg bw after one to two days. Reduced appetite and activity, increasing weakness, slight diarrhoea, collapse, and death were observed. At autopsy, slight liver discolouration and acute gastrointestinal inflammation were seen; the viscera appeared normal macroscopically (Birch, 1973). (b) Short-term toxicity Rats In a 14-day range-finding study available only in summary, groups of five male and five female rats received AMPA at 0, 1000, 2000, or 4000 mg/kg bw per day in the diet. Moderately reduced body-weight gain and food consumption (no further details) were seen at 4000 mg/kg bw per day. No other effects were observed. The NOAEL was 2000 mg/kg bw per day (Goldenthal, 1978). In a 13-week study of toxicity, groups of 20 male and 20 female weanling Charles River CD rats (aged four weeks) received AMPA (purity, 99.9%) at 0, 400, 1200, or 4800 mg/kg bw per day in the diet. The animals were observed for death and signs of overt toxicity twice a day on seven days a week. Body weights and food consumption were monitored weekly. Haematological, biochemical, and urinary parameters were measured in 10 rats of each sex per group after 45 and 88 days; the baseline values for these parameters were measured in an additional 10 male and 10 female rats that were sacrificed for this purpose. At the end of the study, the absolute and relative (to body weight) weights of the liver, kidneys, testes/ovaries, heart, and brain were determined in each animal, and macroscopy was performed. About 30 tissues from controls and rats at the highest dose and liver, kidneys, heart, urinary bladder, and tissues with gross lesions from all rats at 400 and 1200 mg/kg bw per day were examined microscopically. One male and five female rats at 4800 mg/kg bw per day were found dead after blood collection on day 45, and one female rat in the control group and two female rats at 1200 mg/kg bw per day were dead after blood collection on day 88. One male control rat found moribund after observation of soft stools, distended abdomen, and morbidity was killed at week 12. Body-weight gain was significantly decreased in males and females at 4800 mg/kg bw per day (difference for females at termination, < 10%) and in males at 1200 mg/kg bw per day (difference, < 10%). The food consumption of treated males was lower than that of control males, but the differences were not statistically signifcant. No treatment-related changes in haematological parameters were observed after 45 or 88 days. Biochemical analyses showed increased lactic dehydrogenase activity in males and females at 4800 mg/kg bw per day, which was statistically significant for males after 45 and 88 days and for females after 88 days only. Males at 1200 mg/kg bw per day also showed statistically significant increases in lactic dehydrogenase activity after 88 days; females at 400 or 1200 mg/kg bw per day showed significant increases in the activity of this enzyme after 88 days, but the biological significance of these observations is questionable in view of the magnitude of the increases and the low control values in females. Glucose levels were significantly decreased in males and females at 4800 mg/kg bw per day after 88 days in comparison with control values, but the levels in animals at 400 or 1200 mg/kg bw per day were significantly increased after 45 days. At 4800 mg/kg bw per day, males and females showed increased activity of aspartate aminotransferase, which was significant in females after 45 days and in both males and females after 88 days, and males showed increased serum cholesterol levels, which were significant after both 45 and 88 days. The occasionally significant changes in other biochemical parameters (albumin and bilirubin) did not show a dose-response relationship and were considered not to be biologically significant. Urinalysis revealed significant decreases in pH and increased calcium oxalate crystals in males at 4800 mg/kg bw per day after 88 days and in females after 45 and 88 days. The weights of several organs showed significant changes from control values, but the absence of any dose-response relationship or of morphological changes in these organs make their biological significance questionable. Macroscopy revealed no treatment-related changes. Microscopy showed dose-related increases in the incidence and severity of irritation of the mucosal and submucosal layers of the urinary tract in males and females at 1200 or 4800 mg/kg bw per day, corresponding to hyperplasia of the urinary bladder, which was more marked in males than in females. Epithelial hyperplasia in the pelvic section was seen in the kidneys of several rats at 4800 mg/kg bw per day, and some of the hyperplastic epithelial cells contained a hyaline-like cytoplasmic material. The NOAEL was 400 mg/kg bw per day (Estes et al., 1979). Dogs In a one-month range-finding study, groups of two male and two female beagle dogs (aged about six months and weighing 7.3-9.8 kg for males and 5.9-8.4 kg for females) received AMPA (purity, 94.4%) at 0, 10, 30, 100, 300, or 1000 mg/kg bw per day orally in gelatin capsules. The animals were observed for mortality, moribundity, and signs of toxicity twice daily and for detailed signs of toxicity once weekly. Body weight and food consumption were determined weekly. Haematological and biochemical analyses were carried out in all dogs at termination; no urinalysis was performed. The absolute and relative weights of the liver, kidneys, spleen, heart, brain, thyroid, adrenals, and testes of all dogs were determined. Macroscopy was performed on all animals, but no microscopy was done. No unscheduled death occurred. Diarrhoea was seen in both males and one female dog at 1000 mg/kg bw per day (total, 18 incidents), and emesis was observed in both females and one male dog at the same dose (four incidents). No effects on body-weight gain or food consumption were seen. Males and females at 1000 mg/kg bw per day had decreased haemoglobin and haematocrit values (significant in females), decreased erythrocyte counts (significant in males and females), and increased reticulocyte counts (significant in females). In addition, an increase in mean corpuscular haemoglobin concentration was seen in males at 1000 mg/kg bw per day (significant) and 300 mg/kg bw per day, but with no dose-response relationship. Females at 300 mg/kg bw per day had significantly decreased haemoglobin and haematocrit values and a nonsignificant decrease in reticulocyte count. Activated partial prothrombin times were significantly decreased in females at all doses, but these decreases were considered not to be biologically significant because of their small size and the lack of a clear dose-response relationship. No effects on biochemical parameters were seen. Organ weights were unchanged, and macroscopy revealed no treatment-related abnormalities. No adverse effects were seen at 100 mg/kg bw per day. There was no NOAEL owing to the limited number of animals. The authors stated that no adverse effects were observed at 300 mg/kg bw per day in males and 100 mg/kg bw per day in females (Stout, 1991). Groups of five male and five female beagle dogs (males weighing 8.1-12 kg and females, 6.8-11 kg) received AMPA (purity, 87.8%) at doses of 0, 10, 30 100, or 300 mg/kg bw per day orally in gelatin capsules for 91-92 days. The dogs were observed twice daily for mortality, moribundity, and overt signs of toxicity. Ophthalmoscopy was conducted on all dogs before the study and at week 12. Body weights were recorded weekly and food consumption daily. Haematological, biochemical, and urinary analyses were carried out in all dogs before the study and during weeks 6 and 13. The absolute and relative weights of the liver, kidneys, brain, thyroid, adrenals, and ovaries/testes of all dogs were determined. All dogs were examined macroscopically, and about 40 tissues and all gross lesions were examined microscopically. Treatment had no effect on survival, and ophthalmoscopy revealed no abnormalities. Several clinical signs were seen at all doses and in controls, but there was generally no clear dose-response relationship. Only the incidence of scabbed, reddened, and/or swollen ears in males at 300 mg/kg bw per day was increased; in females at this dose, the incidence of these symptoms was below that in controls, the latter being the same as that in males at 300 mg/kg bw per day. The incidence of soft stools varied from group to group, with considerable individual variability; no dose-response relationship was seen. No significant treatment-related effects on body-weight gain or food consumption were observed. Haematology showed occasionally significant decreases in erythrocyte count, haemoglobin and haematocrit values, mean corpuscular volume, mean corpuscular haemoglobin, or mean corpuscular haemoglobin concentration at low doses. No relationship with dose or time was seen, and these changes were considered not to be biologically significant. No treatment-related effects on biochemical or urinary parameters or organ weights were seen, and there were no macroscopic or microscopic changes. The NOAEL was 300 mg/kg bw per day, the highest dose tested (Tompkins, 1991). (c) Long-term toxicity and carcinogenicity No long-term toxicity studies have been performed with AMPA itself, but two-year toxicity studies are available in mice and rats treated with technical-grade glyphosate contaminated at a low level with AMPA (WHO, 1994). In a two-year study of toxicity and carcinogenicity in which technical-grade glyphosate (AMPA content not specified) was given to mice at dietary levels of 0, 0.1, 0.5, or 3%, no treatment-related increase in tumour incidence was seen. The NOAEL was 0.5%, equal to 810 mg/kg bw per day (Bio/Dynamics Inc., 1983). In a 26-month study of toxicity and carcinogenicity in which technical-grade glyphosate (AMPA content not specified) was given to rats at dietary levels of 0.006, 0.02, or 0.06%, equal to about 3, 10, or 32 mg/kg bw per day, no treatment-related increase in tumour incidence was seen. The NOAEL was 32 mg/kg bw per day, the highest dose tested (Bio/Dynamics Inc., 1981a). In a two-year study of toxicity and carcinogenicity, rats were given technical-grade glyphosate containing 0.68% AMPAin the diet to provide doses of 0.2, 0.8, or 2% glyphosate, equivalent to 100, 400, or 1000 mg/kg bw per day. The diets thus contained 14, 54, and 135 mg of AMPA per kg, corresponding to 0.69, 2.8, and 7.2 mg/kg bw per day. No increase in tumour incidence was seen. At the highest dose, female body-weight gain was reduced, and cataracts were found in males. The NOAEL was 0.8% in the diet, equivalent to 400 mg/kg bw per day glyphosate and 2.8 mg/kg bw per day AMPA (Monsanto 1990a; Stout & Ruecker, 1990). (d) Genotoxicity The results of tests for the genotoxicity of AMPA are shown in Table 3. In the test for micronucleus formation, groups of 15 male and 15 female mice were used. Five animals of each sex per dose were killed 24, 48, and 72 h after treatment. Toxicity was observed at 500 and 1000 mg/kg bw, as demonstrated by weight loss and listlessness. No significant decrease in mean polychromatic erythrocytes:total erythrocytes was seen at any dose. At 100 mg/kg bw, a significant increase in mean nucleated polychromatic erythrocyte frequency was seen after 72 h in females only; however, the increased frequency was within the range in historical vehicle controls in the same laboratory, and the frequency was not increased in males at 100 mg/kg bw or in males or females at higher doses. The increase in mean nucleated polychromatic erythrocyte frequency in females at 100 mg/kg bw was thus considered not to be related to treatment (Kier & Stegeman, 1993). (e) Reproductive toxicity (i) Multigeneration reproductive toxicity No multigeneration studies of reproductive toxicity have been performed with AMPA itself, but two studies were reported in rats treated with technical-grade glyphosate contaminated at low levels with AMPA (WHO, 1994). In a three-generation study of reproductive toxicity, rats were given diets providing technical-grade glyphosate (AMPA content not specified) at doses of 0, 3, 10, or 30 mg/kg bw per day. The only effect was an increased incidence of unilateral renal tubular dilatation in male F3b pups at 30 mg/kg bw per day (6/10 versus 0/10 in controls); F1 and F2 pups were not examined (Bio/Dynamics Inc., 1981b). In a two-generation study of reproductive toxicity, groups of 30 male and 30 female Sprague-Dawley rats received diets containing technical-grade glyphosate with 0.61% AMPA (Reyna, 1990), providing 0, 0.2, 1, or 3% glyphosate and 0, 12, 61, or 180 ppm AMPA. Parental animals receiving 3% glyphosate had soft stools, decreased body weights, and slightly decreased litter sizes; pup weights were decreased on days 14 and 21 of lactation. Parental and pup body weights were also slightly decreased after treatment with 1.0%. The NOAEL was 1.0% in the diet, corresponding to 740 mg/kg bw per day glyphosate and 3 mg/kg bw per day AMPA (Monsanto, 1990b). The renal changes seen in the three-generation study were thus not reproduced; however, the numbers of pups examined histologically was limited in both studies. (ii) Developmental toxicity In a range-finding study, groups of eight pregnant Sprague-Dawley rats received AMPA (purity, 94.4%) in corn oil at doses of 0, 125, 250, 500, 750, or 1000 mg/kg bw by gavage daily on days 6-15 of gestation. All animals were killed on day 20 of gestation. Clinical observations, body weights, net body-weight changes, and gravid uterine weights of the dams were recorded, and the weights of the liver, kidney, and spleen were determined. Uteri and ovaries were examined, and the numbers of fetuses, early and late resorptions, implantation sites, and corpora lutea were recorded. Fetuses were Table 3. Results of tests for the genotoxicity of AMPA End-point Test system Concentration Purity Results Reference (%) In vitro Reverse mutationa S. typhimurium 10-5000 µg/plate 99 Negativeb Shirasu et al. (1980) TA98, TA100, in distilled water TA1535, TA1537, TA1538, and E. coli WP2 DNA repairc B. subtilis H17, 20-2000 µg/disc 99 Negatived Shirasu et al. (1980) M45 (rec+/-) in distilled water Unscheduled DNA Primary hepatocytes 5-5000 µg/ml in 94.4 Negative Bakke (1991) synthesise from male Fischer culture medium 344 rats In vivo Micronucleus CD-1 mice Single intraperitoneal 94.4 Negative Kier & Stegeman formulationf injection of 100, 500, (1993) or 1000 mg/kg bw in corn oil a No independent duplicate study but duplicate plates at each concentration; distilled water used as solvent; solvent and positive controls included b With and without metabolic activation c No independent duplicate study; distilled water used as solvent; solvent and positive controls included d Only without metabolic activation e Cytotoxicity at 3800 and 5000 µg/ml f No characterization of test or control substances or their concentrations, homogeneity in carrier, or stability of test and control substances neat and after mixing with carrier weighed, sexed, and examined externally for developmental abnormalities and variations. No effect on survival or body or organ weights was seen in the dams. Greyish faeces were observed infrequently in animals at doses > 125 mg/kg bw per day. The predominant findings 1 h after treatment were red staining around the nose in one or two animals per treated group and a low incidence of salivation in those at 250, 500, 750, or 1000 mg/kg bw per day. In the absence of a clear dose-response relationship and the limited findings, they did not appear to be treatment-related. Fetuses had no treatment-related effects (Holson, 1991a). Groups of 25 pregnant Sprague-Dawley rats received AMPA (purity, 94.4%) in corn oil at doses of 0, 150, 400, or 1000 mg/kg bw by gavage, daily on days 6-15 of gestation. All animals were killed on day 20 of gestation. The dams were observed twice daily for moribundity and mortality, and signs of toxicity about 1 h after treatment and clinical observations on days 0-20 of gestation were recorded. The body weights and food consumption of the dams were determined on gestation days 0, 6, 9, 12, 16, and 20. Gravid uterine weights and net body-weight changes were recorded, and the liver, kidney, and spleen of all dams were weighed. Uteri and ovaries were examined, and the numbers of fetuses, early and late resorptions, implantation sites, and corpora lutea were recorded. Fetuses were weighed, sexed, and examined for external, visceral, and skeletal abnormalities or variations. Maternal survival was not affected. Dams at 400 or 1000 mg/kg bw per day had dose-related increased incidences of mucoid faeces, hair loss, and soft stools. Dams at 150 mg/kg bw per day had only an increased incidence of soft stools. The total occurrence of soft stools/no. of animals was 49/15 in controls, 89/19 in dams at 150 mg/kg bw per day, 102/22 at 400 mg/kg bw per day, and 135/23 at 1000 mg/kg bw per day. At the highest dose, the dams had short periods of slightly decreased body-weight gain and food consumption. No effect was seen on the weights of the liver, kidney, spleen, or gravid uterus. Fetal survival was not affected. A slight but significant decrease in mean fetal body weight was seen at 1000 mg/kg bw per day. No further developmental effect was seen, and there were no teratogenic effects. Because the increased incidence of soft stools at 150 mg/kg bw per day was not accompanied by possibly associated effects such as hair loss and mucoid faeces, this dose is the NOAEL for maternal toxicity. The NOAEL for developmental toxicity was 400 mg/kg bw per day, as the authors stated that the only possible maternal responses at this dose were mucoid faeces, soft stools, and hair loss (Holson, 1991b). (f) Special studies: Dermal and ocular irritation Two male and one female albino rabbits (strain not specified) received an application of 0.5 g finely ground AMPA moistened with water on the clipped intact skin (6.5 cm2), which was kept under cover for 24 h. Observations were made after 1, 24, 48, 72, 120, and 168 h. No skin irritation was seen (Birch, 1973). Only a limited report was available. One male and two female albino rabbits (strain not specified) received an application of 0.1 g finely ground AMPA into one eye. Both the control and the treated eye were rinsed with warm isotonic saline after 24 h. Observations were made after 1, 24, 48, 72, 120, and 168 h and scored according to Draize. Slight erythema, very slight oedema, and copious discharge were seen after 10 min and after 1 h, and slight-to-moderate erythema, a moderate discharge containing a white exudate, and no oedema were seen after 24 h. After 72 h, slight erythema was seen in one animal, but without oedema or discharge. After 120 and 168 h, the treated eyes were normal. The average scores were 10 out of 110 after 1 h and 7.3, 2.6, and 0.6 after 24, 48, and 72 h, respectively. AMPA was considered by the author to be a slight ocular irritant; however, the average score after 24, 48, and 72 h was 3.5, indicating that AMPA is not an ocular irritant (Birch, 1973). In the limited report, separate scores were not given for conjunctivae, iris, and cornea. 3. Toxicological profiles of AMPA and glyphosate The results of toxicological studies of the metabolite AMPA and of its parent compound glyphosate, as previously evaluated (Annex 1, reference 47; WHO, 1994), are compared in Table 4. AMPA was no more toxic than glyphosate. Similar effects were often found,e xcept for the lesions in the salivary gland seen in the 90-day studies of toxicity and carcinogenicity with glyphosate in mice nad rats from the US national Toxicology Program and the cataracts induced by glyphosate in a two-year toxicity study in rats. Such effects were not seen in other studies with glyphosate or AMPA. Comments After oral administration of AMPA to rats, 20% of the dose was absorbed and excreted unmetabolized in the urine within 120 h (17% of the dose within 24 h), and 73% of the dose was eliminated in the faeces. Only 0.07% of the dose was excreted as expired carbon dioxide within 24 h, and 0.06% of the dose was recovered from tissues after 120 h. Minor amounts (1-6 µg/kg) were found in tissues after 120 h. AMPA is slightly hazardous to rats given a single oral dose, with an LD50 of 8300 mg/kg bw (WHO, 1996). In a 90-day study of toxicity, rats received AMPA in the diet at 0, 400, 1200, or 4800 mg/kg bw per day. A significant, dose-related decrease in body-weight gain was seen in males at the two highest doses and in females at the highest dose. The two highest doses also resulted in significantly increased lactate dehydrogenase activity, whereas aspartate aminotransferase activity and cholesterol levels were significantly increased only at the highest dose. Urinalysis showed a significant decrease in urinary pH and increased amounts of calcium oxalate crystals in the urine of animals at the highest dose. Table 4. Toxicity of AMPA and technical-grade glyphosate Type of study AMPA Glyphosatea Metabolism 20% absorption after oral exposure; excreted 36% absorption after oral exposure; unchanged in urine; only 0.07% expired as essentially no biotransformation (0.2- CO2; minor residues in tissues 0.4% AMPA) Acute oral toxicity LD50 = 8300 mg/kg bw LD50 > 5000 mg/kg bw 90-day toxicity, diet, mice NOAEL = 10 000 ppm, equal to 1890 me/kg bw. Effects at LOAEL of 50 000 ppm: decreased growth, increased brain, heart, and kidney weights 90-day toxicity, diet, mice NOAEL = 3125 ppm, equal to 505 mg/ kg bw. Effects at LOAEL of 6250 ppm: lesions of salivary gland 90-day toxicity, diet, rats NOAEL = 400 mg/kg bw. Effects at LOAEL No effect at highest dose of 20 000 ppm, of 1200 mg.kg bw: biochemical changes, equal to 1300 mg/kg bw changes in urinary bladder. At 4800 mg/kg bw, also renal changes 90-day toxicity, diet, rats NOAEL < 3100 ppm, equal to < 200 mg/ kg bw; lesions of salivary gland 90-day toxicity, diet, rats No effect at highest dose of 12 500 ppm, equal to 1300 mg/kg bw 90-day toxicity, gelatin capsule, dogs No effect at highest dose of 300 mg/kg bw 52-week toxicity, gelatin capsule, dogs No effect at highest dose of 500 mg/kg bw Table 4. (continued) Type of study AMPA Glyphosatea Two-year toxicity and carcinogenicity, NOAEL = 5000 ppm, equal to 810 mg/ diet, mice kg bw. Effects at LOAEL of 30 000 ppm: decreased growth, changes in urinary bladder and kidneys Two-year toxicity and carcinogenicity, No effect at highest dose of 600 ppm, diet, rats equal to 32 mg/kg bw; slight decrease in growth considered not to be relevant Two-year toxicity and carcinogenicity, NOAEL = 8000 ppm, equivalent to 400 diet, rats mg/kg bw. Effects at LOAEL of 20 000 ppm: decreased growth and cataracts. Technical-grade glyphosate containing 0.68% AMPA Three-generation reproductive Increased incidence (6/10 versus 1/10 in toxicity, diet, rats controls) of unilateral renal tubular dilatation in F3b pups at highest dose of 30 mg/kg bw; not seen in more recent study (below) Two-generation reproductive NOAEL = 10 000 ppm, equivalent to toxicity, diet, rats 500 mg/kg bw. Effects at LOAEL of 30 000 ppm: soft stools, decreased weights of parental animals and pups. Technical-grade glyphosate containing 0.61% AMPA Developmental toxicity, rats NOAEL for maternal toxicity = 150 mg/kg NOAEL = 1000 mg/kg bw. Effects at bw; NOAEL for developmental toxicity = 400 LOAEL of 3500 mg/kg bw: deaths, mg/kg bw. At 400 mg/kg bw, increased clinical signs, decreased growth of dams, incidences of soft stools, mucoid faeces, and resorptions, decreased implantations and hair loss in dams. At 1000 mg/kg bw, slight visible fetuses, decreased ossification of decrease in fetal body weight fetal sternebrae; no fetal malformations Table 4. (continued) Type of study AMPA Glyphosatea Developmental toxicity, rabbits NOAEL = 175 mg/kg bw. Effects at LOAEL of 350 mg/kg bw: diarrhoea, soft stools, and nasal discharge in dams Genotoxicity Reverse mutation, S. typhimurium Negative Negative Reverse mutation, E. coli Negative Negative DNA repair, B. subtilis rec Negative Negative Gene mutation, Chinese hamster ovary Negative cells Unscheduled DNA synthesis, rat Negative Negative hepatocytes Chromosomal aberration in vivo, rat Negative bone marrow Micronucleus formation in vivo, Negative Negative mouse bone marrow Dominant lethal mutation in vivo, Negative mice Recessive sex-linked lethal mutation, Negative Drosophila melanogaster a From WHO (1994) and Annex I, reference 47 Dose-related irritation of the mucosal and submucosal layers of the urinary tract, corresponding to hyperplasia of the urinary bladder, was seen in rats at 1200 and 4800 mg/kg bw per day, the effect being more marked in males than in females. In addition, epithelial hyperplasia in the renal pelvis was observed at the highest dose. The NOAEL was 400 mg/kg bw per day. In a 90-day study of toxicity in dogs receiving AMPA at 0, 10, 30, 100, or 300 mg/kg bw per day in gelatin capsules, no statistically significant treatment-related changes were observed. The NOAEL was thus the highest dose, 300 mg/kg bw per day. It should be noted that in a one-month range-finding study with groups of only two male and two female dogs, changes in some haematological parameters (e.g. decreased haemoglobin, packed cell volume, and erythrocyte counts) were seen in animals at 300 or 1000 mg/kg bw per day. These effects were not reproduced in the 90-day study. No indication of genotoxic activity was seen in studies of gene mutation in bacteria, of DNA repair in bacteria and mammalian cells in vitro, or of micronucleus formation in vivo. No assays for gene mutation were performed in mammalian cells in vitro, but the structural similarity of AMPA to glyphosate and the lack of genotoxicity of glyphosate, including in an assay for gene mutation in mammalian cells in vitro, indicate that such an assay with AMPA would be redundant. In a study of developmental toxicity, rats received AMPA at 0, 150, 400, or 1000 mg/kg bw per day in corn oil by gavage. Dose-related increases in the incidences of soft stools, mucoid faeces, and hair loss were seen in dams at the two higher doses. Dams at the highest dose also had short periods of decreased body-weight gain and food consumption. Fetal body weight was decreased at 1000 mg/kg bw per day. No teratogenic effects were observed. Dams at 150 mg/kg bw per day also had an increased incidence of soft stools; however, in the absence of any associated effects, such as hair loss or mucoid faeces, the Meeting considered this dose to be the NOAEL for maternal toxicity. The NOAEL for developmental toxicity was 400 mg/kg bw per day. AMPA did not induce dermal or ocular irritation in rabbits. No long-term study of the toxicity or carcinogenicity of AMPA has been carried out, but in the more recent of two such studies with technical-grade glyphosate in rats at dietary levels of 0.2, 0.8, or 2%, the AMPA content of the test compound was given, namely 0.68%. At the highest dose of 2% glyphosate in the diet, females showed decreased body-weight gain and males showed an increased incidence of degenerative lenticular changes. The NOAEL for technical-grade glyphosate was 0.8% in the diet, corresponding to 400 mg/kg bw per day for glyphosate and 2.7 mg/kg bw per day for AMPA. No increase in tumour incidence was seen in this study. No multigeneration study of the reproductive toxicity of AMPA has been reported, but in a recent two-generation study in rats with technical-grade glyphosate at dietary levels of 0.2, 1, or 3%, the test compound contained 0.61% AMPA. At the highest dose, soft stools, decreased parental body weights, slightly decreased litter sizes, and decreased pup weights were observed. The NOAEL was 1% in the diet, corresponding to 740 mg/kg bw per day glyphosate and 4.5 mg/kg bw per day AMPA. Glyphosate and AMPA have very similar chemical structures. Studies of the metabolism of glyphosate in experimental animals indicate that essentially none is biotransformed into AMPA. Toxicological data on the metabolite are therefore essential for risk assessment. The Meeting compared the toxicity profile of AMPA with that of glyphosate and concluded that the major targets of the toxicity of AMPA had been investigated. The results showed little toxicity. The Meeting concluded that the two compounds have similar toxicological profiles and concluded that a full database on AMPA is unnecessary. AMPA was considered to be of no greater toxicological concern than its parent compound. The Meeting established a group ADI for AMPA alone or in combination with glyphosate of 0-0.3 mg/kg bw on the basis of the 26-month study of toxicity in rats fed technical-grade glyphosate, using a safety factor of 100. Since the last JMPR evaluation fof glyphosate or toxicity in 1986, new data have become available, some of which are evaluated in WHO (1994). The Meeting recommended that glyphosate be re-evaluated by the JMPR. Toxicological evaluation Levels that cause no toxic effect AMPA Rat: 400 mg/kg bw per day (90-day study of toxicity) 150 mg/kg bw per day (maternal toxicity in a study of developmental toxicity) 400 mg/kg bw per day (fetal toxicity in a study of developmental toxicity) Dog: 300 mg/kg bw per day (highest dose in 90-day study of toxicity) Glyphosate (from Annex I, reference 47): Mouse: 0.5% in the diet, equal to 810 mg/kg bw per day (two- year study of toxicity and carcinogenicity) Rat: 31 mg/kg bw per day (26-month study of toxicity and carcinogenicity) Dog: 500 mg/kg bw per day (one-year study of toxicity) Estimate of acceptable daily intake for humans 0-0.3 mg/kg bw (sum of glyphosate and AMPA) Toxicological criteria for setting guidance values for dietary and non-dietary exposure to aminomethylphosphonic acid (AMPA) Human exposure Relevant route, study type, species Results/remarks Short-term Oral toxicity, rat LD50 = 8300 mg/kg bw (1-7 days) Skin irritation, rabbit Not irritating Eye irritation, rabbit Not irritating Skin sensitization No data Medium-term Repeated oral, 90 days, toxicity, rat NOAEL = 400 mg/kg bw per day: urinary tract changes (1-26 weeks) Repeated oral, developmental toxicity, rat NOAEL = 150 mg/kg bw per day: maternal toxicity NOAEL = 400 mg/kg bw per day: developmental toxicity Repeated oral, reproductive toxicity No data Long-term Repeated oral, toxicity No data (> 1 year) References Bakke, J.P. (1991) Evaluation of the potential of AMPA to induce unscheduled DNA synthesis in the in vitro hepatocyte DNA repair assay using the male F-344 rat. Unpublished study No. 2495-V01-91 from SRI International, Menlo Park, California, USA (SRI Project LSC-2495; Monsanto study No. SR-91-234, dated 4 December 1991). Submitted to WHO by Monsanto, Agricultural Group, St Louis, Missouri, USA. Bio/Dynamics Inc. (1981a) A lifetime feeding study of glyphosate (Roundup technical) in rats (Project N0. 410/77 [BDN-77-416]). Bio/Dynamics Inc., Division of Biology and Safety Evaluation, East Millstone, New Jersey, USA. Submitted to WHO by Monsanto, St Louis, Missouri, USA. Bio/Dynamics Inc. (1981b) A three generation reproduction study in rats with glyphosate (Project No. 77-2063 [BDN-77-147]). Final report. Bio/Dynamics Inc., Division of Biology and Safety Evaluation, East Millstone, New Jersey, USA. Submitted to WHO by Monsanto, St Louis, Missouri, USA. Bio/Dynamics Inc. (1983) A chronic feeding study of glyphosate (Roundup technical) in mice (Project No. 77-2061 [BDN-77-420]). Bio/Dynamics Inc., Division of Biology and Safety Evaluation, East Millstone, New Jersey, USA. Submitted to WHO by Monsanto, St Louis, Missouri, USA. Birch, M.D. (1973) Toxicological investigation of CP 50435 Lot: XHD-16. Unpublished report from Younger Laboratories Inc., St Louis, Missouri, USA, dated 7 March 1973. Submitted to WHO by Monsanto, Agricultural Group, St Louis, Missouri, USA. Colvin, L.B., Moran, S.J. & Miller, J.A. (1973) Final Report on CP 67573 residue and metabolism. Part 11: The metabolism of aminomethylphosphonic acid-14C (CP 50435-14C) in the laboratory rat. Job No. 9-23-760.06-7863. Unpublished report from Monsanto Commercial Products Co., Agricultural Research Report No. 303 dated August 1973. Submitted to WHO by Monsanto, Agricultural Group, St Louis, Missouri, USA. Estes, F.L., Jefferson, N.D., Blair, M. & Goldenthal, E.I. (1979) 90-Day subacute rat toxicity study (IRD-78-174). Test article: CP 50435. Unpublished report from International Research and Development Corporation, Mattawan, Michigan, USA, dated 15 August 1979. Submitted to WHO by Monsanto, Agricultural Group, St Louis, Missouri, USA. Goldenthal, E.I. (1978) CP 50435 (aminomethyl phosphonic acid) fourteen day rat feeding study. Unpublished report No. IRD-77-319 from Monsanto. Submitted to WHO by Monsanto, St Louis, Missouri, USA. Holson, J.F. (1991a) A dose range-finding developmental toxicity study of AMPA in rats. Final report. Unpublished report from WIL Research Laboratories Inc., Ashland, Ohio, USA. WIL Project No.: WIL-50146. Sponsor No.: WI-90-247, dated 21 May 1991. Submitted to WHO by Monsanto, Agricultural Group, St Louis, Missouri, USA. Holson, J.F. (1991b) A developmental toxicity study of AMPA in rats. Final report. Unpublished report from WIL Research Laboratories Inc., Ashland, Ohio, USA. WIL Project No.: WIL-50159. Sponsor No.: WI-90-266, dated 6 August 1991. Submitted to WHO by Monsanto, Agricultural Group, St Louis, Missouri, USA. Howe, R.K., Chott, R.C. & McClanahan, R.H. (1988) Volume 2: Metabolism of glyphosate in Sprague-Dawley rats. Part II. Identification, characterization, and quantitation of glyphosate and its metabolites after intravenous and oral administration. Unpublished report from Monsanto Co., St Louis, Missouri, USA. Laboratory project no. MSL-7206, dated February 1988. Submitted to WHO by Monsanto, Agricultural Group, St Louis, Missouri, USA. Kier, L.D. & Stegeman, S.D. (1993) Final report. Mouse micronucleus study of AMPA. Unpublished report from Environmental Health Laboratory of Monsanto Co., St Louis, Missouri, USA. EHL Study Number: 90170. Sponsor Project Number: ML-90-404. Submitted to WHO by Monsanto, Agricultural Group, St Louis, Missouri, USA. Monsanto (1990a) Chronic study of glyphosate administered in feed to albino rats (Project No. MSL-10495). Monsanto Environmental Health Laboratory, St Louis, Missouri, USA. Unpublished report submitted to WHO by Monsanto, St Louis, Missouri, USA. Monsanto (1990b) Two generation reproduction feeding study with glyphosate in Sprague-Dawley rats (Study No. MSL-10387). Monsanto Environmental Health Laboratory, St Louis, Missouri, USA. Unpublished report submitted to WHO by Monsanto, St Louis, Missouri, USA. Reyna, M.S. (1990) Two generation reproduction study with glyphosate in Sprague-Dawley rats. Unpublished Report no. ML-88-106. Submitted to WHO by Monsanto, St Louis, Missouri, USA. Ridley, W.P. & Mirly (1988) Volume 1: The metabolism of glyphosate in Sprague-Dawley rats. Part I. Excretion and tissue distribution of glyphosate and its metabolites following intravenous and oral administration. Unpublished report MSL-7215 from Monsanto Co., Environmental Health Laboratory, St Louis, Missouri, USA. Submitted to WHO by Monsanto, Agricultural Group, St Louis, Missouri, USA. Shirasu, Y., Moriya, M. & Ohta, T. (1980) CP 50435: Microbial mutagenicity study. Unpublished report from the Institute of Environmental Toxicology Kodaira, Tokyo, Japan, dated November 1980. Submitted to WHO by Monsanto, Agricultural Group, St Louis, Missouri, USA. Stout, L.D. (1991) One month study of AMPA administered by capsule to beagle dogs. Unpublished report from Environmental Health Laboratory of Monsanto Co., St Louis, Missouri, USA. Study Number 90074. Project Number: ML-90-186. Submitted to WHO by Monsanto, Agricultural Group, St Louis, Missouri, USA. Stout, L.D. & Ruecker, F.A. (1990) Chronic study of glyphosate administered in feed to albino rats. Unpublished report no. ML-90-186. Submitted to WHO by Monsanto, St Louis, Missouri, USA. Tompkins, E.C. (1991) 90-Day oral (capsule) toxicity study in dogs with AMPA. Final report. Unpublished report from WIL Research Laboratories Inc., Ashland, Ohio, USA. Project No.: WIL-50173. Sponsor No.: WI-90-354, dated 16 July 1991. Submitted to WHO by Monsanto, Agricultural Group, St Louis, Missouri, USA. WHO (1994) Environmental Health Criteria 159. Glyphosate. Geneva, International Programme on Chemical Safety. WHO (1996) The WHO Recommended Classification of Pesticides by Hazard and Guidelines to Classification 1996-1997 (WHO/PCS/96.3), Geneva, International Programme on Chemical Safety.
See Also: Toxicological Abbreviations