TOXICOLOGY
Guazatine was first evaluated by the Joint Meeting in 1978, when an ADI of 0-0.03 mg/kg bw was established on the basis of an NOAEL of 200 ppm (equivalent to 3 mg/kg bw per day) in a two-year dietary study in dogs. The pesticide was reviewed at the present Meeting within the CCPR periodic review programme. New data on its absorption and metabolism, the toxicity of repeated doses in mice and dogs, its long-term toxicity in rats and mice, genotoxicity, reproductive toxicity, and developmental toxicity were assessed. Data on the pesticide l,1-iminodi(octamethylene)diguanidine, with the common name iminoctadine (BSI, draft ISO), which constitutes about 1.5% of the guazatine mixture, were also considered.
Guazatine is a preparation of the triacetates of dimeric and trimeric guanidated octane-1,8-diyldiamine which also contains a range of oligomers and reaction products. The Meeting was concerned that the production controls and specifications for guazatine were inadequate. The quoted purity of 70% is based on normalization to a component which comprises approximately 1.5% of the mixture and provides no control over the levels of the other components. Some data were provided to show that the composition of the batch used in the key toxicity studies was similar to that of other batches produced at the same time, 1990-91, but there were no data to confirm that the batches used in the studies of toxicity were representative of those currently produced.
Some components of guazatine were absorbed by rats to a limited extent after oral administration of the 14C-labelled material and then excreted rapidly. Within 24 h, faecal elimination represented 85-94% of the dose, with 3-6% in the urine and < 1% in exhaled air. The highest levels of radiolabel were found in the kidney and liver, with evidence that the salivary, pituitary and thyroid glands may also contain significant amounts of residue. A study involving treatment with 14 doses of 2 mg/kg bw showed limited potential accumulation in the liver and kidney. The results of a study by intravenous injection showed that some components of guazatine may be secreted back into the gastrointestinal tract via the stomach and salivary glands. The metabolism of guazatine has not been fully characterized, but successive conversion of the terminal guanidino groups to amino plays a significant role in vivo.
Guazatine produces severe local irritation, and single oral doses are of moderate toxicity, with an oral LD50 value in rats of 280 mg/kg bw. WHO has classified guazatine as moderately hazardous.
In a number of short-term studies in rats, guazatine was administered at doses of 0, 60, 200, 800/1200, or 1500/2000 ppm in the diet for 14 weeks. At doses of 60 and 200 ppm, the activity of serum alkaline phosphatase was slightly decreased, but no significant changes were seen in body-weight gain or in the results of pathological, haematological, or urinary examinations. At doses of 800 ppm and above, decreased body-weight gain, increased activities of alanine and aspartate aminotranferases, and decreased activity of alkaline phosphatase were found, together with pathological changes such as local irritation of the gut and hyperplasia of the epithelia of the parotid gland excretory ducts with mononuclear cell infiltration. Increased weights of the kidney, liver, and heart were seen without associated histopathological changes. The overall NOAEL was 200 ppm, equivalent to 10 mg/kg bw per day.
In a 13-week range-finding study, mice received guazatine at 0, 10, 50, 200, or 500 ppm. Significantly reduced body-weight gain was seen in animals of each sex at 200 ppm and above. Increased liver weights and alterations in centrilobular hepatocytes were seen in both males and females at 500 ppm. Alterations in erythrocyte parameters were seen in animals at doses of 200 ppm and above. Although no significant effects were reported at 10 or 50 ppm, an NOAEL was not identified in view of limited histological investigations in the study.
In a one-year study in dogs, guazatine was administered at 0, 25, 75, or 250 ppm. Reduced body-weight gain in females, increased alanine aminotransferase activity in animals of each sex, and increased aspartate aminotransferase activity in males were observed at a dietary concentration of 250 ppm. In females at 75 ppm, body-weight gain was reduced. The NOAEL was 25 ppm, equal to 0.8 mg/kg bw per day.
Guazatine has been tested in an adequate battery of assays for genotoxicity. The Meeting concluded that guazatine is not genotoxic.
Guazatine was not carcinogenic in two two-year studies in rats given doses of 0, 20, 60 or 200 ppm or 0, 50, 150, or 350 ppm. Non-neoplastic findings included reduced serum alanine and aspartate aminotranferase activities, salivary gland hyperplasia, and testicular atrophy at 350 ppm. In a two-year study of iminoctadine administered at 0,10, 100 or 300 ppm, there was no reported increase in tumour incidence. The overall NOAEL was 150 ppm, equal to 7 mg/kg bw per day.
In a study of carcinogenicity, mice received 0, 50, 120, or 300 ppm guazatine. The incidences of malignant tumours were increased at 120 and 300 ppm: haemangiosarcoma of the liver and spleen was seen in males at 120 and 300 ppm and hepatocellular carcinoma in females at 300 ppm. The incidence of renal-tubular tumours (adenoma and carcinoma) was increased in males receiving 300 ppm. These are rare tumour types in the mouse strain that was used, normally being seen in only 0-6% of animals. Although the absolute incidences of these tumours in guazatine-treated animals were low and not statistically significant, they were clearly greater than those in historical controls. No convincing information was available on the underlying mechanism of tumour production. The non-neoplastic effects seen in this study were increased incidences of lymphoid foci in the lung, bronchiole-associated lymphoid tissue, keratinized vaginal epithelium, and brain mineralisation in females receiving 300 ppm. Body weight gain was reduced by approximately 20% in animals of each sex receiving 300 ppm. In addition, an abstract describing a study on iminoctadine (0, 10, 100, or 300 ppm) reported a slight increase in the incidence of renal epithelial tumours in male mice receiving 300 ppm. The Meeting considered that the production of rare malignant tumours by an unknown mechanism is of great concern. The overall NOAEL for long-term administration to mice was 50 ppm, equal to 6.8 mg/kg bw per day, on the basis of increases in the incidence of haemangiosarcoma in males at 120 ppm, equal to 17 mg/kg bw day.
In a multigeneration study of reproductive toxicity in rats receiving guazatine at 0, 60, or 200 ppm, no significant effects were seen at the highest dose, equivalent to 12 mg/kg bw per day. In a two-generation study of reproductive toxicity in rats guazatine administered at 0, 50, 150, or 350 ppm did not affect reproductive performance at the highest dose, equal to 22 mg/kg bw per day.
In a study of developmental toxicity in rats, guazatine was administered at 0, 5, 10, or 20 mg/kg bw per day. The NOAEL for maternal toxicity, teratogenicity, and fetotoxicity was 20 mg/kg bw per day, the highest dose tested. In a range-finding study, significant mortality was seen at 40 mg/kg bw per day.
In a study of developmental toxicity in rabbits, guazatine was administered at 0, 2.8, 5.6, or 11 mg/kg bw per day. There were no signs of fetotoxicity or teratogenicity at the highest dose. The NOAEL was 5.6 mg/kg bw per day on the basis of marked decreases in maternal body-weight gain.
The Meeting concluded that it could not establish an ADI for guazatine owing to the inadequate information on its composition and concerns about the production of rare malignant tumours in mice.
A toxicological monograph summarizing the data received since the previous evaluation and relevant data from the previous monograph was prepared.
TOXICOLOGICAL EVALUATION
Levels that cause no toxic effect
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Mouse: |
50 ppm, equal to 6.8 mg/kg bw per day (two-year study of toxicity and carcinogenicity) |
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Rat: |
350 ppm, equal to 22 mg/kg bw per day (two-generation study of reproductive toxicity) |
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20 mg/kg bw per day (study of developmental toxicity) |
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150 ppm, equal to 7 mg/kg bw per day (two-year study of toxicity and carcinogenicity) |
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Dog: |
25 ppm, equal to 0.8 mg/kg bw per day (one-year study of toxicity) |
Studies that would provide information necessary for the continued evaluation of the compound
1. Data on the levels of individual components in batches of guazatine from recent production runs.2. Investigation of the mechanism of tumour production in mice.
3. Clarification of the extent of absorption, excretion, and metabolism of all components of guazatine.
4. Clarification as to whether the stated doses used in the studies of toxicity were expressed as free base or triacetate.
RESIDUE AND ANALYTICAL ASPECTS
Guazatine was evaluated by the JMPR in 1978 and 1980, and is now re-evaluated in the CCPR periodic review programme. It is a non-systemic contact fungicide which disturbs the membrane function of fungi. It controls a wide range of seed-borne diseases of cereals, e.g. seedling blight (fusarium spp.), glume blotch (septoria), common bunt (tilletia spp.), common root rot (helminthosporium) and smut (ustilago). On citrus fruit, guazatine is used as a bulk dip after harvest, in the packing line as a spray and in washing installations to disinfect the process water. It controls sour rot (geotrichum candidum), green mould (penicillium digitatum) and blue mould (penicillium italicum).
Guazatine is a mixture of reaction products from polyamines, comprising mainly octa-methylenediamine, iminodi(octamethylene)diamine, octamethylenebis(imino-octamethylene)diamine, and carbamonitrile. A coding system is used for the compounds that make up guazatine in which 'N' represents any amino group. Thus NN stands for H2N-(CH2)8-NH2, NNN stands for H2N-(CH2)8-NH-(CH2)8-NH2 and so on. 'G' stands for any amino group (NH or NH2) of the above which is guanidated. For example GG stands for H2N-C(NH)NH-(CH2)8-NH-C(NH)-NH2.
The fate of residues has been studied in animals, plants and soil.
Studies on rats and lactating cows showed poor absorption from the gastrointestinal tract, rapid elimination mainly in the faeces (>90%), excretion largely as the unchanged parent mixture and no accumulation in any organs, tissues or milk.
When cows were dosed daily with 0.5 mg/kg bw for 10.5 days, 93% of the administered radioactivity was recovered in the faeces as unchanged guazatine, and the low levels in plasma indicated minimal absorption. 14C in the milk and plasma, expressed as guazatine, reached plateau levels of about 0.02 and 0.015 mg/l respectively by day 3 in milk and day 6 in plasma. Following slaughter after the last dose residues of about 0.08 mg/kg were found in the liver and kidney with only very low levels in other edible tissues (<0.02 mg/kg in skeletal muscle and fat).
Adequate metabolism studies with full characterization of the metabolites in farm animals, an animal transfer study on ruminants and an analytical method for commodities of animal origin were not submitted. The Meeting was therefore unable to establish a definition of the residue of guazatine in animal products and could not estimate maximum residue levels for products of animal origin.
When wheat seeds were dressed with [14C] guazatine at 1.05 g ai/kg seed there was no difference between the total radioactive residue (TRR) levels in the harvested grain, straw or chaff from the treated and the control plots. The method of application was according to GAP.
The foliar application of [14C] guazatine to wheat at 1.1 kg ai/ha, 11 weeks before harvest, resulted in mean TRRs of 29 mg/kg guazatine equivalents in the straw, 18 mg/kg in the chaff, and 0.8 mg/kg in the grain.
When [14C] guazatine was applied to the leaf surface or the fruit of apples (brushing with 0.05 or 0.1 kg ai/hl) its translocation was extremely limited. Autoradiography showed no observable movement in the leaves or fruit and this was confirmed by quantitative determination of the TRR: 87% of the applied radioactivity was recovered from the leaves after 12 weeks, 66% from the surface and 21% from the leaf tissues (61% was identified as the parent mixture). In the fruit 62% of the TRR was located on the surface and 38% in the tissues after 12 weeks, with 81% of the TRR identified as the parent. The remainder comprised a major photodegradation product (4.5%), other extractable compounds (9.7%), and unextractable residues (5.2%).
The uptake of guazatine residues from soil by soya beans and rice plants was investigated by treating soils with 5 mg/kg of [14C] guazatine and planting soya bean and rice plants after 26 weeks. Four weeks after planting, the TRR in soya beans amounted to only 0.08% of the applied radioactivity in the aerial part and 0.12% in the whole plant. The residues expressed as guazatine equivalents on a dry weight basis were 2.8 mg/kg in the aerial part and 3.7 mg/kg in the whole plant. The pods contained 0.052 mg/kg on a dry weight basis 9 weeks after planting.
Guazatine residues taken up from flooded soil were low in the whole rice plant, which absorbed only 0.13% of the applied 14C (0.57 mg/kg on a dry weight basis) during a period of four weeks, with 0.05 % of the applied radioactivity or 0.23 mg/kg (dry weight) in the shoot.
Guazatine has been shown to be metabolized in about 100 days when applied to wheat seeds planted in soil, via deguanidation and subsequent mineralization. The test system had a substantial influence on the degradation time.
When guazatine was applied to wheat seeds which were subsequently planted in soil and the soil leached to simulate rainfall, the guazatine components were found to be associated with the seeds or the soil surrounding the seeds. The compounds that had moved from the seeds to the soil showed no tendency to migrate. Significant mineralization to carbon dioxide occurred during the leaching period.
The Meeting concluded that these studies were adequate for the use of guazatine for the seed treatment of cereals, and that no further studies on rotational crops were necessary for such uses.
The use of such a complex mixture as guazatine presents a problem in choosing a residue analytical method. It is not considered practical to attempt the determination of all the components so some alternative is necessary. Two approaches may be applicable.
1. Development of a 'total residue' method by conversion to a single compound.2. The choice of a major component as a 'marker', with the inclusion of a correction factor to give the total residue.
Many of the residue studies used the first approach, involving the hydrolysis of residues to bis(8-amino-octyl)amine (NNN) and its determination either directly or after derivatization. This method was used, e.g., for the analysis of citrus fruits, where the LODs (expressed as guazatine) were 0.05 mg/kg for finisher pulp, 0.2 mg/kg for wet peel and 1 mg/kg for dried peel. The metabolites are determined by the total residue method together with the parent material.
Better results were achieved with cereals, however, by using the marker GG (octane-1,8-diyldiguanidine, H2N-C(NH)NH-(CH2)8-NH-C(NH)-NH2, one of the major guazatine components, for quantification. This method incorporates a correction factor to allow for the fact that GG represents only 30% of the total guazatine. The homologue GG-C6 (1,6-diguanidinohexane, H2N-C(NH)NH-(CH2)6-NH-C(NH)-NH2, is used as an internal standard. The analytical method for grain and straw consists in extraction of samples fortified with the internal standard with hot 1M HCL, clean-up on a cation exchange column, derivatization with hexafluoroacetylacetone (HFAA), clean-up on basic Al2O3, and determination of the HFAA derivatives of GG and the internal standard GG-C6 by GC-MS.
Samples fortified with guazatine showed LODs of 0.05 mg/kg for cereal grains and 0.1 mg/kg for straw with recoveries of 88% and 94% respectively. The lowest fortification levels at the LOD of the marker GG were also 0.05 mg/kg for grain and 0.1 mg/kg for straw (recoveries: grain 97%, straw 82%).
The Meeting concluded that 0.05 mg/kg is a practical limit of determination for GG.
The justification for the choice of GG as representative of the total guazatine residues in cereals has been supported by the following facts.
1. Guazatine shows low uptake and translocation in cereals. This is consistent with the lack of detectable residues reported in crops after seed treatments.2. Where the material has been applied as a foliar spray on dwarf apples trees there is little evidence of significant metabolism or hence of changes in the proportions of the components of the guazatine mixture.
3. In a situation where metabolism is demonstrably occurring (see below), GG remains a significant component after 29 days.
Evidence for GG still being present under 'metabolizing' conditions comes from an aerobic soil degradation study. In this, a mixture of GG, GN and GGG was applied to seed surfaces, and the seeds were planted in soil in metabolism vessels. Most of the seeds germinated. It was possible to distinguish the seeds from the soil and extract the seeds separately up to 29 days after planting. Analysis of these extracts indicated a change in the profile of components present on the seed with GGG levels decreasing. This is consistent with the generation of 14CO2 in the study. However at day 29 GG was still the predominant single compound on the seed, despite the degradation which had been occurring at the seed surface or in the soil in contact with it.
On this basis, it is considered that GG represents a satisfactory marker compound to represent guazatine residues in seed-treated cereals.
The storage stability of analytical samples was investigated by storing analysed samples of wheat grain, ears and straw at -20°C and re-analysing them after two years. The study was not satisfactory as an unvalidated analytical method was used.
Definition of the residue. The metabolism of guazatine in animals has not been fully elucidated, and the Meeting concluded that the residue of guazatine in products of animal origin could not be satisfactorily defined.
The metabolism of guazatine in plants has also not been fully characterized. The main uses of guazatine are for the seed treatment of cereals and the post-harvest protection of citrus fruits. The Meeting concluded that the available studies were adequate only for the seed treatment of cereals. Should further uses (e.g. foliar spray or treatment of plants other than cereals) be planned in future, detailed metabolism studies would be required.
Guazatine has been determined by a total residue method based on conversion to the corresponding triamine, bis(8-amino-octyl)amine, which also occurs as a metabolite. Modern analytical methods using octane-1,8-diyldiguanidine (GG), one of the main components of guazatine, as a marker are more specific.
The Meeting concluded that the residue should be defined for enforcement purposes as "octane-1,8-diyldiguanidine" (GG). Assuming that the content of GG is 30% of the total guazatine content, the GG content should be multiplied by 3 for risk assessment purposes for commodities of plant origin.
Definition of the residue for enforcement purposes: octane-1,8-diyldiguanidine (GG), expressed as octane-1,8-diyldiguanidine.
Definition of the residue for risk assessment purposes: guazatine.
Supervised trials
Citrus fruits. Concentrations of 0.05 to 0.2 kg ai/hl water or 0.3 kg ai/hl wax are registered for post-harvest treatment.
In Australia, guazatine is registered for the post-harvest treatment of citrus fruits with 0.052 kg ai/hl. Three residue trials according to GAP (one each on oranges, mandarins and lemons) were reported and showed residues of <0.2, 0.3 and 0.5 mg/kg (calculated as guazatine) in the whole fruit.
South African GAP specifies 0.3 kg ai/hl in wax for the treatment of citrus fruits. Five trials (3 on oranges, one each on lemons and grapefruit) at the lower rate of 0.2 kg ai/hl in wax were reported. The residues in the whole fruit ranged from 0.33 to 1.8 mg/kg, calculated as guazatine. These results and the data on the validation of the method were submitted only as summaries.
After dipping oranges in water with 0.2 kg ai/hl guazatine, residues of 5.5 mg/kg were calculated in the whole fruit (2 trials). These results are inconsistent with the results found after waxing and indicate a more critical residue situation. Furthermore, no data were available on residues in small citrus fruits (e.g. mandarins) after treatment with 0.2 kg ai/hl.
The Meeting concluded that the residue data were not adequate for citrus fruits as a major crop and recommended the withdrawal of the existing CXL of 5 mg/kg.
Tomatoes and melons, except watermelons. Post-harvest uses of guazatine exist in Australia but no residue data were received.
No maximum residue level could be estimated for tomatoes, and the Meeting recommended the withdrawal of the existing CXL of 5 mg/kg for melons, except watermelon.
Pineapples and potatoes. Since no residue data or information on GAP were received, the Meeting recommended the withdrawal of the existing CXLs of 0.1* mg/kg for pineapple and potato.
Cereal grains. The use of guazatine for seed treatment is registered in many countries with application rates from 0.05 to 1.05 g ai/kg seed (mainly 0.45-0.6 g ai/kg). A total of 84 supervised trials with treatments at 0.4, 0.6, 0.8, 0.9, 1, 1.2 or 1.5 g ai/kg seed were reported to the Meeting. The samples from 61 trials carried out from 1972 to 1987 were analysed by an unvalidated analytical method and could not be used for evaluation. Valid results from 23 trials carried out in 1994/95 on wheat in France (7), Germany (6) and Italy (10) were submitted. No residues were found above the LOD of 0.05 mg/kg, calculated as guazatine.
In view of the non-systemic character and particular use pattern of guazatine as a seed treatment, the Meeting concluded that the residue in cereal grains was "essentially zero" and estimated an STMR of 0 mg/kg.
The Meeting estimated a maximum residue level of 0.05* mg/kg expressed as GG for cereal grains as a practical limit of determination.
Sugar cane. Guazatine is registered in South Africa for the treatment of plant segments before planting with a solution of 0.08 kg ai/hl water. Only two trials, not complying with GAP, were reported. Sugar cane was treated in Hawaii with solutions of 0.01 or 0.025 kg ai/hl. Residues in cane, bagasse, molasses and raw sugar were reported as 0.1 mg/kg. The report was submitted only as a summary with little information (e.g. the PHI and analytical method were not stated).
The Meeting recommended the withdrawal of the existing CXL (0.1 mg/kg).
Rape seed. The use of guazatine as a foliar spray is registered in Germany but no residue data were received. No maximum residue level could be estimated.
Straw and fodder of cereal grains. After treatment of wheat with 0.6-0.8 kg ai/kg seed the residues found in 21 trials carried out in 1994/95 in France (7), Germany (6) and Italy (8) were all <0.1 mg/kg calculated as guazatine.
As there was no residue definition for guazatine in animal products, the Meeting did not recommend an MRL for the straw and fodder of cereal grains as a feed item.
Animal products. No transfer study was carried out on ruminants, no definition of the residue in products of animal origin could be proposed, and no maximum residue levels were estimated for any animal feed items.
The Meeting concluded that there was insufficient information to estimate maximum residue levels for guazatine in products of animal origin.
No feeding or metabolism studies were reported for laying hens. As no residues occur in cereal grains after seed treatment, the Meeting concluded that further studies and the estimation of maximum residue levels for residues in poultry commodities resulting from seed treatment were not necessary.
A study of the storage stability of radiolabelled guazatine on oranges after drenching with 0.1 or 0.2 kg ai/hl showed no decrease of the residues after 50 days.
The results of commercial processing studies on citrus fruits indicate that the residues are on the peel surface. Processing factors calculated for dried peel were 4.9, 6.4, 13 and 15, mean 9.8, median 9.7, and for molasses 1, 1.7, 3.6 and 18, mean 6.1, median 2.7. There was a clear reduction of the residue during processing to pulp and juice. The analysis of fresh peel in 15 supervised trials showed ratios of the residues in the peel to those in the whole fruit ("processing factors") of 1.6 (2), 1.8, 2.5, 2.7 (2), 3.1, 3.5 (2), 3.9, 4 (2), 4.3, 6.7 and 10. with a mean of 3.7 and a median of 3.5.
Residues in the edible portions of citrus fruits were low. After treatment according to GAP, most pulp and juice samples contained guazatine residues at or about the LOD (<0.05 mg/kg) and never more than 0.13 mg/kg.
No information was provided on residues in commodities in commerce or at consumption.
The Meeting estimated the maximum residue level shown in Annex I (Part 2). As the Meeting withdrew the ADI for guazatine this is recorded only as a Guideline Level.
FURTHER WORK OR INFORMATION
Desirable
Any further evaluations for uses apart from the seed treatment of cereals would require the following data.
1. Clarification of the metabolism of all major components in ruminants.2. Animal transfer studies on ruminants including an analytical method for the determination of residues in products of animal origin.
3. Clarification of the metabolism of all major components in plants.
