# `oo~9 ~'~~-`©F'PT .:). o - oo 33 Submission For Fulfilment of Data Requirements

for Biuret Under TSCA Section 4 1. Chemical Name: Biuret 2 . CAS Number : 108-19-0 3 . Industry Sponsor : The Fertilizer Institute WilliamHerz 820First Street NE, Union Ctr Plaza Ste 430 Washington, DC 20002 Tel : (202) 515-2706 -r. tr. CJ t

9 . Date of Submission : July 2007 a - 10 . Comments : Biuret is not manufactured or processed for distribution i commerce as chemical substances per se and, relative to tl~e, ---1=--i fertilizer industry, has no commercial purpose separate fromthe =~~ , substance mixture or article ofwhich it may be a part (40CFR 710 .4(d). Biuret relies on data presented for Urea (CAS 57-13-6 ; Screening Information Data Set (SIDS) of OECD High Production Volume Chemicals Programme (1994 ; see attached documents) . Both Biuret and Urea are enzymatically hydrolyzed to ammonia and carbon dioxide in the environment and in mammalian systems. BIURET

Up. 2 BIURET 

## CONTENTS 1 IDENTITY .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . .. . . .. .. .. .. . . . . . . . . . . . .. . . . . . . . . . . . . . 5 1 .1 Identification of the Substance ... . .. .. .. .. .. .. .. .. .. . .. .. .. .. .. .. .. .. .. .. .. . .. ... .. . .. .. .. .. .. .. . ... .. . .. .. .. .. .. .. . ... . .. .. .. .. .. .. .. .. .. .. .. .. .. .. . 5 1 .2 Physico-Chemical properties . . .. .. .. .. .. . . . . . . . . . . . .. .. . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. .. . . . . . . .. . .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . .6 2 GENERAL INFORMATION ON EXPOSURE.. .. .. . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . .. .. .. .. .. .. .. .. .. . .. .. .. .. .. .. .. .. .. . .. .. .. .. .. .. .. .. .. .. .. .. . 6 2.1 Environmental Exposure and Fate. . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. .. . . .. .. .. . . .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . .. .. .. .. .. .. . . . . . . 7 2.1 .1 Sources of Environmental Exposure .. . . .. .. .. .. .. . . .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . 7 2 .1 .2 Photodegradation . . .. . . .. .. .. .. .. .. . .. .. .. .. . . .. .. .. .. . . . . . . . . . . . .. .. .. .. . . .. .. . . . . . .. .. .. .. .. .. .. . . . . . .. .. .. .. .. .. . . .. .. .. . . . . . . . . . . . .. .. .. . . . . . 7 2.1 .3 Stability in Water .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. .. . . . . . . .. . . . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. .. .. .. .. .. . . . . . 7 2.1 .4 Transport between Environmental Compartments . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. . . .. .. .. .. .. . . . . . . .. .. . . . . 7 2.1 .5 Biodegradation . . . . . . . . . .. . . .. . . . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. . . .. .. .. . . .. .. .. .. . .. .. .. .. .. .. .. .. .. . . . . . . . . . . . .. .. .. . . .. . . . . . .7 3 HUMANHEALTH HAZARDS. .. .. .. .. .. .. .. . .. .. .. .. .. .. .. .. .. . . .. .. .. ... . .. .. .. .. .. .. .. .. .. . .. .. .. .. .. .. .. .. .. . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . ... .. .. .. 8 3.1 Effects on Human Health .. . . .. .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . .. .. . . . . . . . . . . . . . 8 3.1 .1 Toxicokinetics, Metabolismand Distribution .. .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. . . . . . . 8

.. .. .. . .. .. .. .. . . . . .. . . . . . . . . . . . . . . . . . .. .. . . .. . . . . . . . . . . . .. .. .. .. .. . . . . . . . . . .. .. .. .. .. . . . . . . . . . . . . . ."""""""""""""" 9 3.1 .2 Acute Toxicity 8 3 .1 .3 Repeated Dose Toxicity . . . . . . . . . .. .. .. .. .. . . . . . . . . . . . .. .. .. .. .. .. . . . . . . . . . . .. . . . . . . .. .. . . .. . . . . . . . . . . . .. .. .~ .~ .~ .~ . . . . . . . . . . . .. .. .. .. .. .. . . . . . .'. 3.1 .4 Mutagenicity . .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. .. . . . . .. . . .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . .12 3.1 .5 Toxicity for Reproduction .. . . . .. .. .. .. .. .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. . . .. .. . . .. . . . . .. . . . . . . . . . . . . . .. .. .. . . . . . .13 4 HAZARDS TOTHE ENVIRONMENT .. .. .. . .. .. .. .. .. .. .. .. .. .. . ... .. . ... . .. .. .. .. .. .. .. .. .. . .. .. .. .. .. .. .. .. .. . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . .. .. .13 4 .1 Aquatic Effects . .. .. .. . . .. .. . . . .. .. .. . . . . .. . . . . . . . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . . . .. . . .. .. .. .. .. .. . . .. .. .. .. .. .. .. .. .. . . . . . . . . . . . .. .. . . . . . .13 5 RECOMMENDATIONS. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. . . . . . . .. .. .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. . . .. .. .. .. . . .. . . .. .. . . .. .. .. .. .. .. . . . . . . .. . . . . . .14 6 REFERENCES .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. .. .. .. .. . . .. .. .. .. . . . . . . .15p. 3 BIURET 

## Tables Table 1 Summary of physico-chemical properties .. .. . . .. .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . .. . . . .. .. .. .. . . . . .. . . . . . .. .. .. .. . . . . .. .. .. .. . . . . . . . . .. . . . 6 Table 2 Summary of acute toxicity. .. .. .. .. . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. . . . . .. .. . . .. . . . . .. . . . . . . . . . . . .. .. .. . . . . . . . . .. .. .. .. .. .. . . . . . . . . . . . .. .. .. . . . . . . . . . 9 Table 3 Summary of repeated dose toxicity .. .. .. .. .. . . . . .. .. .. .. . . .. .. .. .. . . . . . . . . . . . .. .. .. .. .. . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. . . .. .. . . .. .. .. .. .. .. . . . . .12

## Submission for Fulfilment of Data Requirements

for Biuret Under TSCA Section 4 1 IDENTITY 1 .1 Identification of the Substance CAS Number : 108-19-0 Molecular Formula: C2H5N302 Structural Formula: 

# O NH2 

# O NH---\ NHZ Molecular Weight : 103 Synonyms: Imidodicarbonic diamide Biuret Allophanamide Allophanic acid amide Allophanimidic acid Carbamylurea Dicarbamylamine Isobiuret L-101 Urea, (aminocarbonyl)- Ureidoformamidep. 5 BIURET 1 .2 Physico-Chemical properties Table 1 Summaryofphysico-chemical properties Property Value Reference Physical state Melting point 188-190°C Aldrich, 1978, 2000 Boiling point 193 °C at 1013.25 hPa Merck, 1976 Relative density 1 .467 at -5 °C Merck, 1976 Vapour pressure 1 .02E-5 mm Hg Epiwin, v3.20 Water solubility 8.777E+4 mg/L YALKOWSKY, and DANNENFELSER, 1992 Partition coefficient noctanoUwater (log value) -1 .3988 Epiwin, v3.20 2 GENERAL INFORMATION ON EXPOSURE Biuret is not a commercial product for the members of The Fertilizer Institute (TFI) ; it is formed incidentally during the production of fertilizer grade urea at high temperatures (TFI, 2007) . The existence of biuret was first identified by Wiedemann in 1848 after heating urea nitrate to 150° - 170°C . (Redemann et al . 1958) . It has long been recognized as a component of urea fertilizers (Webster et al ., 1957) . The amount ofbiuret increases with increasing temperatures and some biuret is formed even when a solution of urea in water is heated above 50° C . Current levels of biuret in fertilizer grade urea are slightly less than 1 .0% wt with a recent range of 0 .84 to 1 .04%

. Some urea fertilizer manufactures reported the manufacture and import of biuret and urea reaction products associated with the manufacture and import of urea fertilizer in TSCA Inventory Update Rule (IUR) Form U submissions including the 2002 reports for activities in 2001- EPA Form Numbers 6511017 and 65011017. In 2003, TFI, petitioned EPA for a partial reporting exemption for fertilizers and certain fertilizer materials including biuret and urea reaction products from inventory update reporting requirements proposed for 2006 and beyond. In November 2003 and December 2004, EPA issued letters to TFI concluding that biuret and urea reaction products with formaldehyde are "impurities" as defined in 40 CFR . § 720 .3 and therefore were not subject to 2006 IUR reporting. The applicable provision of the now final regulation is § 710 .50(c) which defines manufacturing a chemical substance "in a manner described in § 720 .30(g) or (h)" of the regulations as an activity for which reporting is not required. A similar regulation, § 710 .30(c), applies to 2002 IUR reporting. Some of the urea fertilizer manufacturers 2002 IUR reports included chemical substances for which it is now clear, based on EPA's November 2003 and December 2004 interpretation letters, that reporting was not required. These urea fertilizer manufacturers revised and resubmitted 2002 FormU Reports removing biuret (CAS No. 108-19-0) and urea reaction products with formaldehyde (CAS No. 68611-64-3)

. Other producers sell biuret as an FDA-approved non-protein nutrient supplement for ruminants. Biuret is considered an impurity of urea with no commercial purpose for reporting under TSCA Section 8 Inventory Update Reporting (IUR), is not manufactured or processed for distribution in commerce as a chemical substances per se and, relative to the fertilizer industry, has no commercial purpose separate from the substance mixture or article of which they may be a part (40 CFR § 710 .4(d)

.p. 6 BIURET Based on the well established mechanisms for enzymatic hydrolytic degradation in the environment and in the mammalian gut of biuret to urea (and then to ammonia and carbon dioxide), data from urea (UNEP Publications, date not specified) are used to provide additional information for biuret. As stated in the UNEP document, "urea is an important endogenous product of mammalian metabolism

. This may partly explain why it has not been rigorously studied with toxicological tests. Nevertheless, urea appears to cause little or no toxicity to most mammalian species (ruminants are an exception) and humans at reasonable dose levels. Further, The EPA has indicated that urea poses no risk concerns within the limits of the existing tolerance exemptions (http://www

.epa. ov/oppsrrdl/reregistration/urea/) , urea is approved for several human therapeutic uses (h=://216.239.51 .104/search?q=cache_p1264Z8FFIJ :www

.fda. gov/cder/ogd/rld/12154s24.pdf+FDA+category+C+urea&hl=en&ct=clnk&cd =1 & 1=us ) and is considered "Generally Recognized As Safe" (GRAS) as affirmed by the U . S. Food and Drug Administration for use in food (21 C .F .R. § 184.1923 Urea. Title 21 - Food and Drugs) . 2 .1 Environmental Exposure and Fate 2 .1 .1 Sources of Environmental Exposure 2 .1 .2 Photodegradation The overall OH Rate Constant was = 3.0000 E-12 cm3/molecule-sec with a half-life = 3.565 days (Epiwin, v3.20) . 2 .1 .3 Stability in Water Stability on water studies have not been conducted with biuret. Modeling indicates that the hydrolysis rate will be extremely slow(tl/2 > 1 year) (Epiwin, v3.20) . 2 .1 .4 Transport between Environmental Compartments The EQC Level III Fugacity model (Epiwin, v3.20) was used to evaluate the fate, transport and distribution of biuret between environmental matrices. Level III fugacity modelling, using loading rates of 1000 kg/h each for air, soil and water, shows the following percent distribution when it is released simultaneously to all three compartments: Air = 4.83E-6% ; Soil = 60.9%

; Water = 39%

; Sediment = 0

.713% . 2 .1 .5 Biodegradation Standard ready biodegradability studies have not been located for biuret. Biuret does not accumulate in soil and is mineralized within a single growing season. Biuret is not retained in soil (Orgata, year not specified) and is easily leached (Gadet et al ., 1959) . Nishihare et al . (1965) found an enzyme, biuret-hydrolyzing enzyme, capable of decomposing biuret into ammonia, carbon dioxide and urea. Jensen (1966) found several widespread species of soil fungi and bacteria capable of degrading biuret . Jensen also reported that biuret at a concentration of 100 ppmwas almost completely nitrified after two to three months. Several studies have concluded that the rate of biuret mineralization depends on soil composition, temperature, and biuret concentration (Mithyantha et al ., 1977) . Epiwin (v3.20) modeling suggests biuret is biodegradable, but may not meet the criteria for readybiodegradability.p. 7 BIURET Urea also is ultimately biodegradable in inherent biodegradability test (OECD 302 B) . In semicontinuous activated sludge (SCAS) urea is biodegraded on the average of 93-98% in 24-hour cycle. The main mode of degradation is enzymatic mineralization. In soil and water urea is expected to biodegrade fairly rapidly to ammonia and bicarbonate if temperature is not too low . Ammonia is a volatile gas in alkaline solutions. In natural waters, most ammonia appears in the form of ammonium, fromwhich nitrogen is oxidized as the result of bacterial action, forming nitrite and nitrate . According to US-EPA PCGEMS-model hydrolysis of urea is expected to be extremely slow (T1/2 > 1 years) (UNEP, date not specified) . 3 HUMAN HEALTH HAZARDS Data fromurea (UNEP Publications, date not specified) are used to provide additional information for biuret. 3.1 Effects on Human Health 3.1 .1 Toxicokinetics, Metabolismand Distribution It has been well established that in the rumen, biuret and urea are hydrolyzed by microflora enzymes to ammonia and simple carbon compounds. These hydrolysis products can then be used to synthesize amino acids. The complete hydrolysis and deamination of protein involves numerous enzymes. Biuret is decomposed by biuretase to urea, and then to ammonia and carbon dioxide. Biuret is more slowly hydrolyzed than urea (Fonnesbeck, et al, 1975), thus accounting for the lower toxicity profile of biuret (reduces the toxic accumulation of ammonia in blood plasma) . Biuret has not been shown to accumulate in rats, chickens or sheep. Urea is extremely soluble in water and oral doses are rapidly absorbed and distributed in humans. Urea is a normal human body constituent and is constantly being produced through amino acid and protein metabolismwhere urea is formed through a cyclic mechanism . 3.1 .2 Acute Toxicity The acute toxicity of biuret has been studied extensively, although most studies have been conducted in livestock fromthe 1950's to the 1970's. The reliability of the studies with biuret are generally considered to be = (4) not assignable due to the inability to access the original studies. Fonnesbeck et al (1975) provides a review of many of these studies with biuret, and some are provided in the attached dossier for biuret and in Table 2 . Biuret is of low

acute oral toxicity. The acute toxicity by urea is well delineated by the oral route. Toxicity is low in mammals other than ruminants, especially cattle, and sheep, in which the rumen micro-organisms contain urease activity and metabolize urea to ammonia at ahigh rate. In mice and rats, urea is of low toxicity even by the subcutaneous and intravenous route (UNEP, date not specified) . Examples of specific toxicity values published for urea areprovided for comparison in Table 2. Despite the assignment of RL =4, the studies provide a weight of evidence regarding the lack of acute toxicity of biuret, as well as its lower toxicity when compared to urea.p. 8 BIURET Table 2 Summary of acute toxicity Species Value (mg/kg bw) RL/Reference Biuret Sheep No effect at 4000 4/Swart and Lynch, 1972 Sheep No effect at 680 4/Mieske et al ., 1955 Sheep No effect at 6900 4/Clark et al ., 1963, 1965; Fonnesbeck et al ., 1975 Sheep Toxicity at 4700 4/Hatfield et al ., 1969; Fonnesbeck et al ., 1975 Urea Sheep Oral LD50 = 510 Not specified/FDA, 1978 Rat Oral LD50=14300 (M) ; = 15000 (F) Not specified/Sato et al ., 1978 3 .1 .3 Repeated Dose Toxicity Repeated dose toxicity studies with biuret have been conducted by the oral route. No wellconducted repeated dose toxicity studies on urea were located; oral chronic/carcinogenicity screening studies and repeated dermal exposure studies have been published for urea (LTNEP; no date specified)

. These studies are summarized below and in Table 3

. Male rats (6/group) were fed a basal diet supplemented with biuret at levels of 0, 0 .2, 0 .4, 0 .6, 0 .8 and 1 .0%for 30 days (Berry et al ., 1956; RL=2) . Animals were observed for signs of toxicity and body weights were recorded after 30 days . Based on the lack of toxicity noted in these animals a second experiment was initiated. Animals fromgroups 1 (control), 3 (0.4%) and 4 (0.6%) were used in the second experiment. The control group was continued as previously described. Biuret was added to the basal diet at the rate of 5.0%and 10.0%and fed to groups 3 and 4, respectively. This feeding experiment was continued for 140 days to observe any long range effects of a high biuret diet . On the 35th and 98th days, one randomly selected animal fromeach of the three groups was killed and subjected to gross necropsy. There were no significant differences in growth rates among the groups . The appetites were normal, all animals appeared thrifty throughout the duration of the experiment, and no illnesses or deaths occurred. There were no histological findings. Since biuret is chemically related to urea it might be expected that the reaction of the rat to biuret would be similar to its reaction to urea. Observation of a higher water intake among the biuret-fed groups was noted. Groups of ten pullets were provided one of four dietary treatments (basal, urea, biuret, and soybean) (two groups of 10 within each treatment) (Berry et al ., 1956; RL=2) . Group 1 (control) was fed a milo-soybean meal basal ration calculated to contain 14.5%crude protein. Groups 2and 3 were fed the same basal diet except 1 % of the milo of each ration was replaced with 1 % feed urea or 1 % purified biuret, respectively, which raised the calculated crude protein equivalents to 17.3% . Group 4was fed the basal diet with enough milo replaced by soybean oil meal to raise the calculated crude protein value to 17 .3% . The duration of the study was not reported. Feed consumption was about 5%lower for the biuret and urea supplemented groups than for the control and soybean oil meal supplemented groups. The body weight gain of the birds receiving the added soybean oil meal was significantly higher than that of the basal, urea and biuret groups. The inclusion of biuret or urea had no statistically significant effect on body weight gain. Three groups of 9 lambs were fed for 84 days with rations consisting of 42.5%cottonseed hulls, 10%blackstrap molasses, and 40-45%ground milo (Berry et al ., 1956; RL=2) . The various crude protein supplements were 7.5%cottonseed meal (Group 1), 1 % cottonseed meal plus 1 % urea feedurea (Group 2), and 1 %cottonseed plus 1 %crude biuret (Group 3) . All three rations werecalculated to supply equal levels of dry matter, total digestible nutrients and crude protein. In order

to prevent differences related to palatability of the rations, the total intake of each was adjusted to that of the lowest consuming group. This was accomplished by hand feeding twice daily . Daily observations ofwater intake and urination were monitored. Average weight gain was calculated at study termination. One lamb fromeach test group was killed and a gross necropsy conducted. The biuret group was the slowest, the urea group intermediate and the control group the fastest in cleaning up the rations. Therefore, feed consumption of the biuret-fed group controlled the feed intake of the other groups. Daily observations indicated greater water intake and urination for the non-protein nitrogen (NPN)-fed groups, these conditions being more apparent for the biuret group than for the urea group. An examination of the internal organs showed no changes among the lambs fromthe three treatment groups. Five groups of steers were fed a high-roughage, low-concentrate wintering ration in dry lot for 84 days (Berry et al ., 1956; RL=2) . The basal ration was made up of 36.5%Atlas silage (hay basis), 24% cottonseed hulls, 6%alfalfa leaf meal, 11% blackstrap molasses, and from 13%to 20%milo grain and 1 %minerals (bonemeal and salt) . The crude protein supplements used were 7.5% cottonseed meal (Group 1), 1 % feed-urea (Group 2), 1 % crude biuret (Group 3), 1 .5%a mixture of crude biuret and feed-urea (Group 4), and 1 .25%of a urea-fatty acid complex (Group 5) . The total intake of each group was adjusted to that of the lowest consuming group. This was accomplished by hand feeding. Each ration was calculated to supply equal amounts of dry matter, total digestible nutrients, and except for Group 4, protein equivalent . Group 4 was 1 .4%higher in crude protein equivalent . The urea-fatty acid complex was 75%urea and 25%cottonseed foots. Animals were observed for signs of toxicity and average weight gain was calculated at study termination. The average gains made by the control group and the urea group (Group 2) were not significantly different. However, when the urea was replaced with the crude biuret (Group 3), a significantly lower gain resulted. This group also consumed the least feed and therefore, restricted the intake of the other groups. When additional urea was included with the crude biuret (Group 4), the average gain was statistically significantly. Thus, simply adding urea overcame the adverse effects of biuret . Although Group 4 gained slightly more than the group fed urea alone (Group 2), the difference was not significant. The increase in daily gain may suggest that contrary to the results obtained with Group 3, the biuret nitrogen was utilized to some extent by the ruman bacteria. The group fed urea-fatty acid complex (Group 5) gained essentially the same as the urea fed group (Group 2) . Based on this single study it appears that urea fed as a urea-fatty acid complex is efficiently utilized. Biuret is an FDA-approved (Federal Register . Rules and Regulations. 21 CFR Part 573. Federal Register 68(99) :27904. 2003) NPN supplement for ruminants and a good deal of data exists for toxicity and nutritive dosages of urea/biuret combinations for these animals (Fonnesbeck et al ., 1975) . Many of these studies are summarized in the attached dossier; however, the RL of these studies are considered to be (3), as per se they do not provide useful hazard information for the purposes of this program, or (4), as sufficient details regarding methods and results were not available. Generally, plant proteins stimulate the most efficient animal growth and milk production. Animal response is satisfactory, however, when biuret and other NPNproducts provide part of the nitrogen requirements. When lowquality forage diets deficient in nitrogen were fed to ruminants, response to feed grade biuret was very good. Supplementation with biuret stimulated good response from animals receiving medium and high energy diets. Biuret is an ideal supplement because ruminant animals maybe fed more biuret than urea. The low solubility of biuret allows it to remain longer in the rumen . Biuret is hydrolyzed more slowly to ammonia and carbon dioxide in the rumen than urea and thereby eliminates toxic accumulation of ammonia in blood plasma. This is cited as the primary reason biuret is readily consumed by animals at high levels. Biuret is comparatively non-toxic even when supplying nitrogen at levels several times the requirements of the animal . This enlarges the margin of safety permitting feeding of higher concentrations of biuretp. 10 BIURET in the diet than of other commercial NPN products. Biuret exhibits no undesirable palatability characteristics. Its low solubility and slowdegradation rate reduces the hazard of self-feeding on a continuing or intermittent basis. No well-conducted repeated dose toxicity studies on urea were located (UNEP, no date specified) . Chronic toxicity and carcinogenicity screening studies in mice and rats fed with 4500, 9000 or 45000 ppm

in diet (up to about 6750 mg/kg body weight/day for mice and about 2250 mg/kg body weight/day for rats) did not uncover any treatment-related toxic syndromes in the various organs studied (Fleischman et al ., 1980) . Neither was any weight depression noted at terminal necropsy for animals of either sex or species at any dose levels. Thus the NOAELs were about 6750 mg/kg body weight/day for mice and about 2250 mg/kg body weight/day for rats. Repeated dose toxicity studies with rats by skin application over 4 weeks and 25 weeks were conducted using urea ointment at 10%, 20% and 40%

concentrations, and no consistent treatmentrelated toxic effects were found. The ointments were applied on a 20 cm2 area of the back skin but, unfortunately, the quantities of urea applied were not given. Therefore no value for NOAEL by dermal route can be determined. It can be concluded, however, that the repeated dose toxicity of urea by dermal route was low(UNEP, no date specified) .p. 11 BIURET Table 3 Summary ofrepeated dose toxicity Study Design Result RL/Reference Biuret Rats (M) ; diet: 0, 0.2, 0.4, 0.6, 0.8 and 1 .0%- 30 d NOAEL = 1% 2/ Berry et al ., 1956 Pullets; diet: 0, 1%

urea, 1% biuret, or soybean oil - NOAEL = 1% 2/ Berry et al ., 1956 exposure time not specified biuret Lambs; diet: 7 .5%cottonseed meal (Group 1), 1% NOAEL =1% biuret 2/ Berry et al ., 1956 cottonseed meal plus 1%urea feed-urea (Group 2), 1 % cottonseed plus 1%

crude biuret (Group 3) - 84 d Steers; diet: 7.5%cottonseed meal (Group 1), 1% NOAEL =1%biuret 2/ Berry et al ., 1956 feed-urea (Group 2), 1%crude biuret (Group 3), 1 .5% a mixtureofcrude biuret and feed-urea (Group 4), and 1 .25%

ofa urea-fatty acid complex (Group 5) - 84 d Urea (*) Pullets; diet : 0, 1 %

urea, 1%

biuret, or soybeanoil - NOAEL = 1% urea 2/ Berry et al ., 1956 exposure time not specified Lambs ; diet : 7.5%

cottonseed meal (Group 1), 1% NOAEL=1%urea 2/ Berry et al ., 1956 cottonseed meal plus 1%urea feed-urea (Group 2), 1% cottonseed plus 1%

crude biuret (Group 3) - 84 d Steers; diet: 7

.5% cottonseed meal (Group 1), 1% NOAEL =1%urea 2/ Berryet al ., 1956 feed-urea (Group 2), 1%crude biuret (Group 3), 1 .5% a mixture of crude biuret and feed-urea (Group 4), and 1 .25%of a urea-fatty acid complex (Group 5) - 84 days Rat or mouse; diet : 4500, 9000 or 45000 ppm - 12 m NOAEL (mouse) ca. = Not

6750 mg/kg bw/day; (rat) specified/Fleischman ca. = 250 mg/kg bw/day et al ., 1980 Rat; dermal : 10%, 20% and 40%

- 4or 25 w NOAEL not identified. Notspecified/ Not Repeated dose toxicity specified ; UNEP, date "low" notspecifed (*) The Berry et al ., 1956 studies presented in Table 2 were not included in the SIDS documents for Urea. 3.1 .4 Mutagenicity Mutagenicity studies were not located for biuret . Urea has been tested for mutagenicity in vitro and in vivo. The mechanismof genotoxicity of urea is probably non-specific (e.g. difference in osmotic pressure across the cell membrane) (UNEP, no date specified) . Similar effects are expected for biuret. In vivo Studies Urea was clastogenic for mammalian cells in vivo but the interpretation of this clastogenic effect is limited by the usage ofa single extremely high dose level (Chaurasia and Sinha, 1987) . In vitro Studies Urea has been negative in several appropriately conducted bacterial mutagenicity tests (Ishdate, et al, 1981 ; Shimizu et al ., 1985; Mortelmans et al ., 1986) . Urea caused DNAsingle strand breaks in mammalian cells in mouse lymphoma cells (Garberg et al ., 1988) but not rat hepatocytes (Sina et al ., 1984) . Urea was clastogenic in a Mouse Lymphoma TK Locus Assay ; but only at concentrations much beyond the physiological range (about 50-100 higher concentrations thanfound in human blood) (Wangenhaim . and Bolcsfoldi, 1988) . The positive genotoxic findings may depend on secondary cellular effects (e.g. difference in osmotic pressure across the cell membrane) at high concentration which might not take place under in vivo conditions. The maximally effective concentration or production of chromosome aberrations in Chinese hamster fibroblast cells was 266 mmolar, a very high concentration suggesting a low clastogenic potential (Ishidate and Odashima, 1977) . Chromosomal aberrations were detected in 20% of metaphases of Chinese hamster fibroblast cells at urea concentration of 13 g/L (216 mmolar) indicate a low clastogenic potential (Ishdate et al . 1981) . The mechanism of genotoxicity is probably non-specific (e.g. difference in osmotic pressure across the cell membrane) . 3.1 .5 Toxicity for Reproduction Standard reproductive toxicity studies have not been conducted with biuret or urea. Urea is an important endogenous product of mammalian metabolism . This may partly explain why it has not been rigorously studied with toxicological tests. Nevertheless, urea appears to cause little or no toxicity to most mammalian species (ruminants are an exception) and humans at reasonable dose levels (UNEP, no date specified) . Effects on Fertility The studies cited under repeated dose toxicity for urea did not indicate any toxic effects on the reproductive organs of mice and rats. No NOAEL can be given for the reproductive/developmental toxicity ofurea because appropriate studies are lacking (UNEP, no date specified) . Developmental Toxicity Several non-standard studies with pregnant livestock (primarily cows) have indicated that biuret has no adverse effect on weight gains of cows or nursing calves, mild production or composition (Behrens, 1971 ; Iwata et al, 1956; Fonnesbeck et al ., 1975; Raleigh, 1969) . No adequate teratogenicity/developmental toxicity studies of urea with mammals were located. According to one rat study, 50 g/kg body weight/day administered by gavage in two doses 12 hours apart for an average of 14 days did not cause outstanding (external) teratogenicity; the mean birth weight of the newborn was lower but the litter size greater. Injection of urea into the air sack of eggs shows that urea is toxic to the development of chick embryo (UNEP, no date specified) . 4 HAZARDS TOTHE ENVIRONMENT 4 .1 Aquatic Effects Acute aquatic toxicity studies were not located for biuret ; data on urea is expected to be representative of biuret based on its degradation to urea, carbon dioxide and ammonia. Urea has generally low

acute ecotoxicity to organisms. The degradation product of urea, ammonia, is known to be toxic to all vertebrates. In neutral and acidic conditions, however, ammonia exists in the form of ammonium

. Instead of toxic effects, more emphasis should be put on the indirect influence of urea to the environment via eutrophication and the pollution risk of urea to groundwater, when urea is used as a fertilizer and a deicer agent in airports. Also the long-terminfluence ofurea use e.g. soil acidification after the long-termfertilization and ammonia emissions to air can have influence to the whole ecosystemand should be considered (UNEP, no date specified) .p. 13 BIURET Acute Toxicity Test Results Urea is not acutely toxic to fish, algae or aquatic invertebrates. Examples of published data include an LC50 for 96 hours > 9100 mg/L for Barilius barna (Dodriyal and Bahuguna, 1988; a toxicity threshold for 192 hours > 10000 mg/L for Scenedesmus quadricauda (Bringmann and Kuhn, 1978) and an EC50for 24 hours > 10000mg/L for Daphnia magna (Bringmann and Kuhn, 1982) . 5 RECOMMENDATIONS Additional testing is not required for biuret . The existing or modeled data for biuret in combination with published data on urea provides a complete profile for this substance.p. 14 BIURET 

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