### 4:5 Ptl - 1~e? - e ;,1-1111 7T "_~. oo -$- _ 0,0 ~, 3

Hi Greg: David Brooks/DC/USEPA/US To Greg Schweer/DC/USEPA/US@EPA, Catherinr#s -r 10/02/2007 02:14 PM Roman/DC/USEPA/US@EPA cc Mark TownsendIDC/USEPA/US@EPA, opreskodm@ornl .gov, rossrh@ornl .gov 'y bcc Subject Review

of Biuret (CAS # 108-19-0)

We have reviewed our contractor's report and are in agreement with their evaluation of information submitted for Biuret (Cas No. 108-19-0) and its supporting substance (urea) . This report covers health and aquatic toxicity endpoints . The Health and Aquatic information were reviewed and found to be in compliance with the test guidelines specified by the test rule (71 FR 13708, March 16, 2006) . Therefore, these studies should be considered as satisfying the test rule requirements for the followingtests: Acute Mammalian toxicity Genetic toxicity (gene mutation, chromosomal aberration) Repeated-dose toxicity Reproductive toxicity Developmental toxicity and aquatic toxicity (fish, invertebrates and plants) Acute toxicity to fish, invertebrates (daphnia) and plants (algae)

Final Orphan 108-19-0 Data-RSS Adequacy Review ORNL_Oct_2 07.doc Ijust wanted to bring your attention to the metabolismassessment . The submitter argued that since biuret would enzymatically be converted to urea by microflora in the stomach ofruminant animals (e.g., cows), health concerns should be based on urea as the hydrolyzed by-product of biuret in the gut. Even though some of the same intestinal microorganisms are present in humans and other non-ruminant animals, there are no experimental data to indicate to what extent conversion of biuret to urea would occur in non-ruminant mammals, including humans. Our assessment indicates that there are inadequate data to support the submitter's metabolismclaim . Nevertheless, because urea is structurally similar to biuret and contains the same functional subsitutent groups, it is an appropriate analog to assess both health and ecological effects for biuret . Thus, our overall case in based on urea as an analog to support health and aquatic toxicity concerns for biuret . David Brooks Risk Assessment Division EPA East Room6308N (202) 564-8603Data Adequacy Reviewand Robust Study Summary Adequacy Requirements Imidodicarbonic Diamide (Biuret, CAS No. 108-19-0)



### October 2, 2007

Submitted to: High Production Volume Chemicals Branch Risk Assessment Division Office of Pollution Prevention and Toxics Environmental Protection Agency WAM : David Brooks 1200 Pennsylvania Avenue, NW Washington, DC 20460-0001 Prepared By: Toxicology and Hazard Assessment Environmental Sciences Division Oak Ridge National Laboratory Oak Ridge, TN 37830p. 3

Data and Robust Summary Adequacy Repo rt CAS No. 108-19-0 A . SUMMARYOF HUMANHEALTH AND ECOTOXICITY DATA General Comments: The first orphanTest Rule (71 FR 13708, March 16, 2005) included the following required testing for biuret : mammalian acute toxicity, gene mutation, chromosomal aberration, repeated dose toxicity, reproductive toxicity, developmental toxicity and aquatic toxicity (fish, invertebrates and plants) . Following posting ofthe Test Rule, industry (TFI) submitted on July 9, 2007, additional information, including a Submission under TSCA Section 4, an IUCLID Data Set for Biuret, and a Data Summary Document for urea. TFI has taken the position that urea is an appropriate surrogate for biuret because both compounds are hydrolyzed to ammonia and carbon dioxide in the environment and in mammalian systems. Although biuret is enzymatically converted to urea by microflora in the stomach of ruminant animals, and even though some ofthe same intestinal microorganisms are present in non-ruminants, there are no experimental data to indicate to what extent conversion to urea would occur in non-ruminant mammals, including humans. Nevertheless, because urea is structurally similar to biuret and contains the same functional subsitutent groups, it would be an appropriate surrogate for biuret . Because the SIDS submission for urea has been approved by OECD (1994) and no additional toxicity testing was required, the SIDS data for urea can also be considered adequate to satisfythe testing requirements for biuret. A-1 . Adequacy of the Data 

### Asummary of the current overall data adequacy for the biuret is given in the Table 1 . A-2 . Dose Selection and Use of Limit Dose OECD test guidelines specify the dose ranges and the limit dose for each type of test. Normally, the required dose range should, at a minimum, include a no effect dose, a minimallytoxic dose, and a clearly toxic dose. Alternately, if a substance is thought to be very low

in toxicity, only a single limit dose need be used, and if no effects are observed, then no further testing is required. The limit dose for acute oral toxicity testing is 2,000mg/kg. Forbiuret, a dose higher than the limit dose for acute oral toxicity testing was used in an oral repeated dose toxicity study in which rats were maintained for 140 days on a diet containing 10% biuret (8,000 mg/kg, based on a food factor of 0 .08) . Because no toxic effects were observed in the repeated dose study at such a high dose level, additional acute toxicity data are not needed for biuret. Nevertheless, acute toxicity studies are available for an acceptable surrogate, urea.p. 4

Data and Robust SummaryAdequacy Report CAS No. 108-19-0

Stud Information Provided table Acce Data Gap Endpoint 108-19-0 SC p 

### Health Effects Yes No Nol Acute Toxicity Yes Yes Yes Yes No Repeated-Dose Toxicity Yes Yes No No' Gene Mutations Yes Yes Chromosomal No - N 2 Aberrations Yes Yes No - No3,4 Toxicity to Reproduction Yes Yes No 

### - - No4 Developmental Toxicity Yes 47 No 

### A quatic Effects Acute Toxicity to Fish No - No 2 Yes Yes Acute Toxicity to No - No2 Daphnia Yes Yes Toxicity to Aquatic No - No2 Plants Yes Yes SC- Supporting Compound, urea (CAS No. 57-13-6) 'Repeat-dose toxicity testing indicates that the oral LDso for biuret exceeds the limit dose specified by OECDtest guidelines; therefore, acute toxicity testing is not required. ZAdequate data exist for an acceptable supporting compound, urea. 3No adverse effects observed in reproductive organs in repeated dose toxicity studies and no adverse developmental effects observed in developmental toxicity studies for an acceptable surrogate, urea. 4Although the individual developmental toxicity studies for urea were not considered to be totally adequate, based on the OECD approval of the entire SIDS submission for urea, it is assumed here that the available developmental toxicity data are adequate based on a Weight ofEvidence.

A-3 . Appropriateness of Supporting Substances Appropriate supporting chemicals are those which are structurally and functionally similar to the subject compound, and which will be metabolized in a similar manner, or be metabolized to a compound that is similar to the subject chemical . Ideally, the potential toxicity of the supporting chemical should be greater than that ofthe subject chemical to insure that potential effects are not overlooked. Asmaller molecule of

TABLE 1 . Data Ade

quac y for Biuret 108-19-0p. 5

Data and Robust Summary Adequacy Report CAS No. 108-19-0 similar structure is likely to be a better surrogate than a larger molecule because the smaller molecular is likely to have greater potential toxicity. In soil and mammalianrumen, microorganisms, such species as Homodendrum, Klebsiella, Penicillium, Pseudomonas, Ralsonia, Escherichia, Rhodococcus, Sporothrix and numerous other bacterial and fungal species, enzymatically degradebiuret by Biuret Amidohydrolase (EC3.5.1 .84) . In the reaction, the biuret undergoes hydrolysis to form urea, carbon dioxide and ammonia . The urea is subsequently hydrolyzed by urease to formtwo ammonia molecules and carbon dioxide. In ruminants, the hydrolytic action on biuret is delayed until sufficient gut bacteria capable of hydrolyzing biuret are present. No information is available on the degree that biuret is metabolized to urea in the GI tract of non-ruminant mammals. Similar kinds ofintestinal microorganismare present in the GI tract of non-ruminants, however, there are no experimental data indicating to what extent conversion to urea would occur. Even in the absence of the microbial conversion ofbiuret to urea in the intestine of nonruminant species, it can be argued that urea is an acceptable surrogate for biuret inview of its similarity in chemical structure and the likelihood that it would be similar in biological activity. Limited acute and repeated dose toxicity data (see below) suggest that urea is more toxic than biuret; therefore, using data for urea is less likely to overlook potential toxic effects of biuret . NH2 

### NH NH2 Biuret (CA

S No. 108-19-0) Urea (CAS No. 57-13-6)

A-4 . Use of Repeated Dose Studies to Support the Reproductive Toxicity Endpoint The OECD guidelines state that a one generation reproductive toxicity studyis not required if and only if no adverse effects are seen in thereproductive organs of male and female animals tested in an adequate repeated dose study, and no adverse developmental effects occur in an acceptable developmental toxicity study. If adverse effects are reported in either the developmental study or in the reproductive organs in the repeatdose studies, then an appropriate reproductive toxicity test is required. In the OECD/SIDS documentation for urea, the reproductive toxicity requirement is met based on the results ofcarcinogenicity screening studies with mice and rats and repeated dose dermal toxicity studies in rats where no toxic effects were observed in the gonads, and the lack of evidence for developmental toxicity in limited mouse and rat studies.p. 6

Data and Robust Summary Adequacy Report CAS No. 108-19-0 

### Because urea is an acceptable surrogate for biuret, these data can also be used to satisfy the reproductive and developmental toxicity endpoints for biuret . B . SPECIFIC INFORMATIONONTHE AVAILABLE TOXICITY DATA Data available for each of SIDS toxicity endpoints is evaluated below . If a study is concluded to be inadequate, a basis for that conclusion is supplied. If a study is concluded to be adequate, an explanation of the details needed to enhance the robust study summary document for the submission is provided.p. 7

Data and Robust Summary Adequacy Report CAS No. 108-19-0 B-1 . METABOLISM Test Compound: Biuret The data are: 7Adequate ® Not Adequate Summary

: In soil and mammalian rumen, microorganisms such as species of Homodendrum, Klebsiella, Penicillium, Pseudomonas, Ralsonia, Escherichia, Rhodococcus, and Sporothri, as well as numerous other bacterial and fungal species enzymatically degrade biuret by Biuret Amidohydrolase (EC 3.5.1 .84) . In the reaction, the biuret undergoes hydrolysis to formurea, carbon dioxide and ammonia. Theurea is subsequently hydrolyzed by urease to formtwo ammonia molecules and carbon dioxide. In ruminants, the hydrolytic action on biuret is delayed until sufficient gut bacteria capable of hydrolyzing biuret are present. Thereafter, biuret is used as a non-protein nitrogen (NPN) source. Conclusions: Although some of the same micro-organisms found in the GI tract of ruminants are also found in the GI tract of humans, there are insufficient data to determine to what extent biuret is metabolized to urea in the human gut. The extent that unmetabolized biuret is absorbed throughthe GI tract into the blood of non-ruminants is also unknown

; however, because of the low water solubility of biuret, such absorption is likely to be minimal . References : Cook, A .M ., Beilstein, P

., Grossenbacher, H

. and Hutter R

. Ring cleavage and degradative pathway of cyanuric acid in bacteria. Biochem J. 231(1985) 25-30

. Currier, T.A., Bohnert, D .W ., Falck, S.J ., Schauer, C .S. and Bartle, S.J . Daily and alternate-day supplementation ofurea or biuret to ruminants consuming low-quality forage: lI . Effects on site of digestion and microbial efficiency in steers. J. Anim . Sci . 82(2004) 1518-1527. Eaton R .W . and Karns, J .S. Cloning and analysis of s-triazine catabolic genes from Pseudomonas sp. Strain NRRLB-12227. J. Bact . (1991) 1215-1222. Farlin, S.D., Garrigus, U .S. and Hatfield, E.E. Changes in metabolismofbiuret during adjustment to a biuret-supplemented diet . J. Anim . Sci . 27(1968) 785-789 . Oltmeh, R

.R., Slyter, L.L., Kozak, A .S. and Williams, Jr ., E.E. Evaluation of urea, biuret, urea phosphate and uric acid as NPNsources for cattle. J . Nutr. 94(1968) 193- 202 . Sahrawat, K

.L. Mineralization of biuret nitrogen in soil . Plant and Soil 62(1981) 469- 471 .p. 8

Data and Robust Summary Adequacy Report CAS No. 108-19-0 Slyter, L.L., Oltjen, R

.R., Kern, D

.L. and Weaver, J .M

. Microbial species including ureolytic bacteria fromthe rumen of cattle fed purified diets. J . Nutr. 94(1968) 185-192 . Stamper, D

.M ., Radosevich, M

., Hallberg, K

.B., Traina, S.J . and Tuovinen, O .H . Ralstonia basilensis M91-3, a denitrifying soil bacteriumcapable of using s-triazines as nitrogen sources. Can

. J. Microbiol . 48(2002) 1089-1098 . Wheldon G

.H . and MacDonald, R

.E. Utilization ofbiuret by Pseudomonas aeruginosa. Nature 196(1962) 596-597.p. 9

Data and Robust Summary Adequacy Report CAS No. 108-19-0 B-2 . ACUTE TOXICITY Study 1: Acute oral toxicity Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) Reference: Sato, N

. et al . 1978. OyoYakuri (Pharmacometrics) 13:749-772. From OECD SIDS, 1994, as cited in IRPTCData Profile The full study is: ® Adequate 0

Not Adequate Detailed Review

: Single oral doses of 14,300-15,000mg/kg, bw, were given to male and female rats. The study was not conducted according to GLP . The general symptoms consisted mainly of sedation, loss of righting reflex and staggering gait . The oral LDso was reported to be 14,300 mg/kg for males and 15,000 mg/kg for females. The full study is adequate based on a weight of evidence and OECD's approval of the SIDS submission for urea (1994) . Robust Summary: For the robust summaries to be considered adequate, the following details need to be provided:

Test substance purity information Number of animals/sex per dose tested Test guideline followed Statistical methods Whether or not necropsy examinations were performed. Whether the animals were fasted before treatment Duration ofpost-exposure observation period Klimisch Reliability Code Flag as critical study Reference missing titlep. 10

Data and Robust Summary Adequacy Repo rt CAS No. 108-19-0 B-2 . ACUTE TOXICITY Study 2 : Acute oral toxicity Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) Reference: Sato, N

. et al . 1978. OyoYakuri (Pharmacometrics) 13:749-772. From OECD SIDS, 1994, as cited in IRPTCData Profile The full study is : ® Adequate F~ Not Adequate Detailed Review

: Single oral doses of 11,500-13,000mg/kg, bw, were given to male and female rats (strain not reported) . The study was not conducted according to GLP. The general symptoms consisted mainly of sedation, loss of righting reflex and staggering gait . The oral LD5o was reported to be 11,500 mg/kg for males and 13,000 mg/kg for females. The full study is adequate based on a weight of evidence and OECD's approval of the SIDS submission for urea (1994) . Robust Summary: For the robust summaries to be considered adequate, the following details need to be provided (Select the ones that apply or add others) :

Test substance purity information Number of animals/sex per concentration Test guideline used Statistical methods Whether or not necropsy examinations were performed. Whether the animals were fasted before treatment Duration ofpost-exposure observation period Dose-specific results. Klimisch Reliability Code Flag as critical study Reference missing titlep. 11

Data and Robust Summary Adequacy R eport CAS No. 108-19-0 B-2 . ACUTE TOXICITY - OVERALL ASSESSMENT General Comment : The only available acutetoxicity data for biuret were from studies in which sheep were "drenched (to administer a large oral dose of a liquid to an animal)" with doses up to 16 g/kg bw (IUCLIDData Set for biuret) . (NOTE: no further information is provided on the experimental protocol, but it is assumed that drenching involved a dermal exposure) . Distress was observed in animals treated with 8 g/kg and death occurred in sheep treated with 16 g/kg. The studies were given a reliability rating of 4by the submitter ; however, the submitter argues that taken together they provide a weight of evidence regarding the lack of acute toxicity of biuret . The studies however, are inadequate in terms of the species tested and the experimental protocol in that the actual oral dose levels cannot be determined. In an oral repeated dose toxicity study conducted on Sprague Dawley rats (Berry et al ., 1956, see below), dietary levels of 10% biuret for 140days produced no toxic effects. Using a food factor of 0 .08, a dietary level of 10% is equivalent to 8,000mg/kg/day . Because an oral dose of 8,000mg/kg/day is substantially above the limit dose of 2,000 mg/kg recommended in the OECD test guidelines for acute oral toxicity testing, it canbe assumed that the oral LDSO would also exceed 8,000mg/kg . Therefore, the requirement for acute toxicity testing is indirectly achieved with this study and no further acute toxicity testing is required. Furthermore, there is adequate acute toxicity data for urea which is considered an acceptable supporting chemical (see below) .p. 12

Data and Robust Summary Adequacy Report CAS No . 108-19-0 

### B-3. REPEAT-DOSE TOXICITY Study 1 : 30and 140-day dietary studies in rats Test Compound: Biuret Reference: Berry, W

.T., Riggs, J .K

., and Kunkel, H

.O . (1956) . The lack of toxicity of biuret to animals. Journal ofAnimal Sciences 15:225. As cited in the IUCLID Data Set for Biuret . The full study is: ® Adequate F~ Not Adequate Detailed Review

: Experiment 1: Male Sprague-Dawley rats (6/group) were fed a basal diet supplemented with Biuret (99 .1 % purity) at levels of 0

.2, 0 .4, 0 .6, 0 .8 and 1 .0%for 30days. Acontrol group (6 rats) was fed the basal diet without added Biuret . Animals were observed for signs of toxicity and body weights were recorded after 30days. The study did not followGLP . Experiment II : After 30 days it was evident that rats could tolerate Biuret in their diets up to 1%without any apparent toxic effects . Based on the lack of toxicity noted in animals in Experiment II, a second experiment was initiated. Animals in groups 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 six groups fed for only 30days or the three test groups that were fed for 140 days. The appetites were normal, all animals appeared thrifty throughout the duration of the experiment, and no illnesses or deaths occurred. All specimens submitted for histological examination were found to be normal . Slight changes which occurred could not be attributed to Biuret toxicity. Since Biuret is chemically related to urea it might be expected that the reaction ofthe rat body to Biuret wouldbe similar to its reaction to urea. Observation of a higher water intake among the Biuret-fed groups with no evident histological changes was noted. Although not a guideline study, the full study is adequate according to the requirements of the Final Test Rule. Effects ofVarious Levels of Biuret in the Diet on Laboratory Rats: Experiment Days on Test % Biuret N Average Gain (9m)p. 13

Data and Robust Summary Adequacy Report CAS No. 108-19-0 

### 1 30 0 .0 6 139.6 ' 0 .2 6 135.0 0 .4 6 130 .3 0 .6 6 141 .0 0 .8 6 142.4 1 .0 6 137.0 II 140 0 .0 4 206 .8 

### 5.0 4 203.1 10.0 4 198 .2

Robust Summary: For the robust summaries to be considered adequate, the following details need to be provided:

Statistical methods used Information on whether thereproductive organs were examined histologically Flag as critical studyp. 14

Data and Robust Summary Adequacy Report CAS No. 108-19-0 

### B-3. REPEAT-DOSE TOXICITY Study 2 : 12-month oral toxicity/carcinogenicity study in mice Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) Reference: Fleischman, R . W . et al . Journal of Environmental Pathology and Toxicology, 3(5/6), 149-170, (1980) . From OECD SIDS, 1994, as cited in IRPTC Data Profile. The full studyis: NAdequate 0Not Adequate Detailed Review

: A 12-month chronic toxicity and carcinogenicity screening studywas conducted on CB7B1/6 mice. Mice were fed a diet containing 0 .45%, 0 .9% or 4.5% of urea. No carcinogenic effects were reported. OECD/SIDS Comment : 5 animals per sex fromall dose groups were necropsied at the end of thetreatment period, and the gonads were histologically examined. No pathology was reported. After further 4months of followup, all survivors were killed, necropsied and the tissues were fixed . However, concerning the reproductive organs, histological tumor data on the ovary and the uterus only were given. Other sex organs may not have been histologically studied. The full study is adequate based on OECD's approval of the SIDS submission for urea (1994) . Robust Summary: For the robust summary to be considered adequate, the following details need to be provided:

Test substance purity information Number of animals/sex per dietary concentration Complete discussion of clinical observations and non-carcinogenic systemic effects including bodyweight and organ weight changes Statistical methods GLP and test guidelines followed Klimisch Reliability Code Reference titlep. 15

Data and Robust Summary Adequacy Report CAS No. 108-19-0 B-3 . REPEAT-DOSE TOXICITY Study 3: 12-month oral toxicity/carcinogenicity study in rats Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) Reference: Fleischman, R . W . et al . Journal of Environmental Pathology and Toxicology, 3(5/6), 149-170, (1980) . From OECD SIDS, 1994, as cited in IRPTCData Profile The full study is: ® Adequate R

Not Adequate Detailed Review

: A 12-month chronic toxicity and carcinogenicity screening study was conducted on male and female rats. Rats were fed a diet containing 0 .45%, 0 .9%or 4.5% of urea. No carcinogenic effects were reported. OECD/SIDS Comment : Five animals per sex fromall dose groups were necropsied at the end of the treatment period, and the gonads were examined histologically. No pathology was reported. After further 4months of followup, all survivors were killed, necropsied and the tissues were fixed. The testes, prostate and uterus were histologically examined for the occurrence oftumours. Although there was a statistically increased incidence ofinterstitial cell adenomas of the testis in the high dose group, its biological significance was deemed questionable, since the lesions may occur in 100%of controls. The full study is adequate based on OECD's approval ofthe SIDS submission for urea (1994) . Robust Summary: For the robust summaries to be considered adequate, the following details need to be provided:

Test substance purity information Number of animals/sex per dietary concentration Complete discussion of clinical observations and systemic noncarcinogenic effects including body weight and organ weight changes Statistical methods GLP and test guidelines followed Klimisch Reliability Code Reference titlep. 16

Data and Robust SummaryAdequacy Report CAS No. 108-19-0 B-3 . REPEAT-DOSE TOXICITY Study 4 : Four-week cutaneous study in rats Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) Reference: Sato, N

. et al . Oyo Yakuri (Pharmacometrics), 13, 749-772, (1977) . From OECD SIDS, 1994, as cited in IRPTC Data Profile. The full study is : ® Adequate F~ Not Adequate Detailed Review

: Urea in 10%, 20%

or 40% ointment was applied to the back skin (20 cm 2 area) of Wistar rats for 4weeks. Purity not reported. Not reported if GLP . No dose-dependent toxicity was observed. There were no consistent treatment-related effects on standard haematological parameters, clinical chemistry, organ weights or organ histopathology, including testicles, prostate, seminal vesicles, ovaries and uterus. The full study is adequate based on OECD's approval ofthe SIDS submission for urea (1994) . Robust Summary: For the robust summaries to be considered adequate, the following details need to be provided:

Test substance purity information Vehicle information Control information Number of animals/sex per dose Statistical methods GLP? Klimisch Reliability Code Reference titlep. 17

Data and Robust Summary Adequacy Report CAS No. 108-19-0 B-3 . REPEAT-DOSE TOXICITY Study 5 : Twenty-four-week cutaneous study in rats Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) Reference: Sato, N

. et al . OyoYakuri (Pharmacometrics), 13, 749-772, (1977) . From OECD SIDS, 1994, as cited in IRPTC Data Profile. The full study is : ® Adequate 0Not Adequate Detailed Review

: Urea in 10%, 20%

or 40% ointment was applied to the back skin (20 cm 2 area) of Wistar rats for 24weeks. Purity not reported. Not reported if GLP. There were no consistent treatment-related effects on standard haematological parameters, clinical chemistry, or on any organs. The full study is adequate based on OECD's approval ofthe SIDS submission for urea (1994) . Robust Summary: For the robust summaries to be considered adequate, the following details need to be provided:

Test substance purity information Vehicle information Control information 

### Number of animals/sex per dose Statistical methods GLP? Klimisch Reliability Code Reference titlep. 18

Data and Robust Summary Adequacy Report CAS No. 108-19-0 

### B-3. REPEAT-DOSE TOXICITY Study 6 : 190-day oral toxicity study in rats Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) Reference: FDA(US-Food and Drug Admin.) . Health Hazard Evaluation Determination Report, PB-288673, (1978) . From OECD SIDS, 1994, as cited in IRPTC Data Profile. The full studyis: ® Adequate F~ Not Adequate Detailed Review

: Rats were fed a diet containing 20,000-250,000 ppm urea for 190days (about 2000-25,000 mg/kg/day) . Purity ofthe test substance was unknown; also unknown if GLP . 250,000 ppm

and 20,000 ppm

were lethal in 12 days and 20-76 days, respectively. There was weight loss and suppression of sexual function at lower doses. Author's comment : "the validity ofthe study is limited by the small number of animals used (often 1 to 3 per group) and failure to report actual food intake. Extreme weight loss ofthe rats suggested that inanition was likely" . The full study is adequate based on OECD's approval of the SIDS submission for urea (1994) . Robust Summary: For the robust summaries to be considered adequate, the following details need to be provided:

Control information Number of animals/sex per dose Statistical methods GLP? Klimisch Reliability Code Reference titlep. 19

Data and Robust Summary Adequacy Report CAS No. 108-19-0 B-4 . GENETICTOXICITY (gene mutations) Study 1 : Bacterial reverse mutation study (Ames assay) Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) Reference: Ishidate, M . et al . GANN Monograph on Cancer Research, 27, 95-108, (1981) . From

OECD SIDS, 1994, as cited in IRPTCData Profile. The full study is : ® Adequate 0Not Adequate Detailed Review

: Tested on Salmonella typhimuriumstrains TA98, TA100, TA1537, with and without metabolic activation. Concentrations not reported. Negative results with and without metabolic activation. No other information provided. The full study is adequate based on OECD's approval ofthe SIDS submission for urea (1994) . For the robust summaryto be considered adequate, the following details need to be provided: Test substance purity information

Dose levels Number of replicates per concentration Solvent/Vehicle Information Positive and Negative Control Information (Positive controls named by strain and metabolic activation) Source ofmetabolic activation Incubation conditions (temperature, duration) Any deviations fromthe guideline stated Statistical Methods and Results (meannumber of revertent colonies per plate for treated and control cultures) Criteria used to evaluate a positive response Whether or not cytotoxicity was observed Results for Positive and Negative Controls Klimisch Reliability Code 

### Reference titlep. 20

Data and Robust Summary Adequacy Report CAS No. 108-19-0 B-4 . GENETICTOXICITY (gene mutations) Study 2 : Bacterial reverse mutation study (Ames assay) Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) Reference: Shimizu, H . et al . Japanese Journal of Industrial Health, 27, 400-419, (1985) . From OECD SIDS, 1994, as cited in IRPTCData Profile. The full studyis: ® Adequate 0Not Adequate Detailed Review

: Tested on Salmonella typhimuriumstrains TA98, TA100, TA1537, TA1538 and Escherichia coli WP2uvrA, with and without metabolic activation. Dose levels used: 5-5000 gg/plate. Purity 99% ; solvent water. Negative results with and without metabolic activation at dose range of 5-5000 gg/plate. No other information provided. No information as to GLPor test guideline followed. The full study is adequate based on OECD's approval of the SIDS submission for urea (1994) . 

### For the robust summary to be considered adequate, the following details need to be provided: Number of replicates per concentration

Positive and Negative Control Information (Positive controls named by strain and metabolic activation) 

### Source ofmetabolic activation Incubation conditions (temperature, duration) Statistical Methods and Results (mpannumber of revertent colonies per plate for treated and control cultures) Criteria used to evaluate a positive response Whether or not cytotoxicity was observed Klimisch Reliability Code Reference titlep. 21

Data and Robust Summary Adequacy Report CAS No . 108-19-0 B-4 . GENETICTOXICITY (gene mutations) Study 3: Bacterial reverse mutation study (Ames assay) Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) Reference: Mortelmans, K . et al . Environmental Mutagenesis, 8 Suppl . 7, 1-119, (1986) . From OECD SIDS, 1994, as cited in IRPTCData Profile. The full study is: ® Adequate F-1 Not Adequate Detailed Review

: Tested on Salmonella typhimuriumstrains TA98, TA100, and TA1537, with and without metabolic activation (rat liver and hamster liver S9 mix) . Dose levels used: 100-10,000 ~Lg/plate. Purity 99% ; solvent DMSO

. Negative results with and without metabolic activation at dose range of 100-10,000 ~tg/plate. Minimum concentration at which toxicity to bacteria was observed: 100-10000ug/plate depending on strain. No information as to GLPor test guideline followed. The full study is adequate based on OECD's approval of the SIDS submission for urea (1994) . For the robust summaries to be considered adequate, the following details need to be provided: Number of replicates per concentration



### Positive and Negative Control Information (Positive controls named by strain and metabolic activation) Incubation conditions (temperature, duration) Statistical Methods and Results (meannumber of revertent colonies per plate for treated and control cultures) Criteria used to evaluate a positive response Klimisch Reliability Code Reference titlep. 22

Data and Robust Summary Adequacy Report CAS No . 108-19-0 B-5 . GENETICTOXICITY (chromosomal aberrations) Study 1 : Chinese hamsterfibroblast (CHL) assay Test Compound: Supporting Compound; Urea (57-13-6) Reference: Ishidate, M . and Odashima, S. Mutation Research, 48, 337-354, (1977) . From OECD SIDS, 1994, as cited in IRPTCData Profile. The full study is: ® Adequate 7Not Adequate Detailed Review

: Achromosomal aberration test was conducted on Chinese hamster fibroblasts. The concentration range tested was not reported. Purity unknown. Not reported if GLP

. Vehicle: physiological saline. Positive results without metabolic activation at 266 mmolar . Aberrations in 37% of metaphases at 24 hours (chromosomal gaps, chromatid or chromosomal breaks, translocations, fragmentation) . Negative result with metabolic activation OECD/SIDS Comment : The maximally effective concentration was 266 mmolar, a very high concentration suggesting a low clastogenic potential . The full study is adequate based on OECD's approval ofthe SIDS submission for urea (1994) . Robust Summary

:For the robust summaries to be considered adequate, the following details need to be provided:



### Concentrations tested Positive and negative control information Method of slide preparation Statistical methods and results Criteria for scoring a positive response Whether or not toxicitywas observed Klimisch Reliability Code Reference titlep. 23

Data and Robust Summary Adequacy Report CAS No . 108-19-0 

### B-5. GENETIC TOXICITY (chromosomal aberrations) Study 2 : Chinese han

:sterfcbroblast (CHL) assay Test Compound: Supporting Compound; Urea (57-13-6) Reference: Ishidate, M . et al . GANN Monograph on Cancer Research, 27, 95-108, (1981) . From

OECDSIDS, 1994, as cited in IRPTC Data Profile. 

### The full study is: ® Adequate 0Not Adequate Detailed Review

: Achromosomal aberration test was conducted on Chinese hamster fibroblasts. The concentration tested was 13 g/L (urea concentration 216mmolar) . Purity not reported. Not reported if GLP. Vehicle not reported. Positive results were observed without metabolic activation. Chromosomal aberrations were detected in 20%

of metaphases at 216 mmolar concentration. OECD/SIDS Comment : chromosomal aberrations detected in 20% of metaphases at urea concentration of 13 g/L (216 mmolar) indicate a low clastogenic potential . The full study is adequate based on OECD's approval of the SIDS submission for urea (1994) . Robust Summary: For the robust summaries to be considered adequate, the following details need to be provided:

Chemical purity, vehicle used Number of replicates per concentration Positive and negative control information Statistical methods and results Criteria for scoring apositive response Whether or not toxicity was observed Klimisch Reliability Code Reference titlep. 24

Data and Robust Summary Adequacy Report CAS No . 108-19-0 B-5 . GENETICTOXICITY (chromosomal aberrations) Study 3" Mouse lymphoma T% locus assay

. Test Compound: Supporting Compound; Urea (57-13-6) Reference: Wangenhaim, J. and Bolcsfoldi, G . Mutagenesis, 3, 193-205, (1988) . From OECD SIDS, 1994, as cited in IRPTCData Profile. The full studyis: ® Adequate 0Not Adequate Detailed Review

: Concentrations of 7.9-38 g/L (132 -662 mmolar of urea) were used with and without metabolic activation on mouse lymphoma L5178Y TK+/- cells. No information on compound purity, vehicle used, or GLP. Weakly positive at very high urea concentrations 530 - 662 mmolar negative at 132 mmolar, equivalent at 265 -397 mmolar. OECD/SIDS Comment : Authors discuss the possibility that 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 full studyis adequate based on OECD's approval of the SIDS submission for urea (1994) . Robust Summary: For the robust summaries to be considered adequate, the following details need to be provided:

Chemical purity, vehicle used Number ofreplicates per concentration Positive and negative control information Statistical methods and results for each concnetration Criteria for scoring a positive response Whether or not toxicity was observed Klimisch Reliability Code Reference titlep. 25

Data and Robust Summary Adequacy Report CAS No . 108-19-0 B-5 . GENETICTOXICITY (chromosomal aberrations) Study 4 : Bone marrow

cytogenetic assay Test Compound: Supporting Compound; Urea (57-13-6) Reference: Chaurasia, O . P. and Sinha, S. P

. Cytologia, 52, 877-882, (1987) . From OECD SIDS, 1994, as cited in IRPTC Data Profile. The full study is: ® Adequate Fj Not Adequate Detailed Review

: Swiss albino mice were fed 500mg urea per animal per day in food for 5 days (dosage approaching an acute lethal dose) . Purity not reported. Bone marrow

cell, metaphases exhibited chromosome breaks, acentric fragments, translocations, gaps and constrictions at a 7-fold rate compared to controls. OECD/SIDS Comment : The interpretation of this clastogenic effect is limited by the usage of a single extremely high dose level . The full studyis adequate based on OECD's approval of the SIDS submission for urea (1994) . Robust Summary: For the robust summaries to be considered adequate, the following details need to be provided:

Chemical purity Number of replicates Positive and negative control information Statistical methods and results Criteria for scoring a positive response Whether or not toxicity was observed Klimisch Reliability Code Reference titlep. 26

Data and Robust Summary Adequacy Repo rt CAS No. 108-19-0

B-6 . REPRODUCTIVE TOXICITY (OVERALL ASSESSMENT) Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) Reference: OECD SIDS, 1994, as cited in IRPTC Data Profile. The full studyis: ® Adequate F~ Not Adequate OECD/SIDS Conclusion: Based on the carcinogenicity screening studies with mice and rats and repeated dose toxicity studies by dermal route with rats, where no toxic effects were observed in the gonads, even at thehistopathological examination, and the lack of evidence for developmental toxicity in limited mouse and rat studies, urea would not be expected to display significant potential for reproductive toxicity.p. 27

Data and Robust Summary Adequacy Report CAS No. 108-19-0 B-7. DEVELOPMENTAL TOXICITY Study 1 : Developmental screening study in rats Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) Reference: Teramoto et al . Teratology, Journal ofAbnormal Development, 23, 335-342, (1981) . FromOECD SIDS, 1994, as cited in IRPTC Data Profile. The full study is: [] Adequate ® Not Adequate Detailed Review

: Asingle dose of 2,000 mg/kg was administered orally to 10 pregnant ICRmice on day 10 of pregnancy . Not GLP

. No effects on foetal data (live/dead, sex, external defects, soft tissue and skeletal defects) . No maternal toxicity OECD/SIDS Comment : "not an adequate teratogenicity study" . Even though the study is not adequate, because OECD approved the SIDS submission for urea (1994), the developmental toxicity endpoint is considered to be satisfied based on a weight of evidence. Robust Summary: For the robust summaries to be considered adequate, the following details need to be provided:

Test substance purity information Vehicle/solvent used, if any Control information 

### Statistical Methods and Results Criteria used to evaluate a positive response Klimisch Reliability Code 

### Referencep. 28

Data and Robust Summary Adequacy Report CAS No. 108-19-0 B-7. DEVELOPMENTAL TOXICITY Study 2: Developmental screeningstudy in rats Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) Reference: Teramoto et al . Teratology, Journal ofAbnormal Development, 23, 335-342, (1981) . From

OECD SIDS, 1994, as cited in IRPTC Data Profile. The full study is : [:] Adequate ® Not Adequate Detailed Review

: Asingle dose of 2,000mg/kg was administered to 4pregnant Wistar rats on day 12 ofpregnancy . Not GLP

. 

### No effects on foetal data (live/dead, sex, external defects, soft tissue and skeletal defects) OECD/SIDS Comment : "not an adequate teratogenicity study" . The full study was inadequate according to OECD ; however, because OECD approved the SIDS submission for urea (1994), the developmental toxicity endpoint is considered to be satisfied based on a weight of evidence. Robust Summary: For the robust summaries to be considered adequate, the following details need to be provided:

Test substance purity information Vehicle/solvent used, if any Control information 

### Statistical Methods and Results Criteria used to evaluate apositive response Klimisch Reliability Code 

### Referencep. 29

Data and Robust Summary Adequacy Report CAS No. 108-19-0 B-7. DEVELOPMENTAL TOXICITY Study 3: Developmental screening study in rats Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) Reference: Seipelt, H . et al . Zeitschrift Urologie, 62, 623-627, (1969) . From OECD SIDS, 1994, as cited in IRPTC Data Profile. The full study is: 0 Adequate ®

Not Adequate Detailed Review

: Urea (50g/kg bw) was administered by gavage in 2 doses 12 hours apart to 6rats during pregnancy for an average of 14 days and the dams were allowed to delivery. Within 48 hours after delivery the pups were killed and the kidneys examined; No hypertrophy or other kidney changes were detected nor were any teratogenic effects noted. 

### No effects on foetal data (live/dead, sex, external defects, soft tissue and skeletal defects) OECD/SIDS Comment : "not a full-range teratogenicity study" . The full studywas inadequate according to OECD ; however, because OECD approved the SIDS submission for urea (1994), the developmental toxicity endpoint is considered to be satisfied based on a weight of evidence. Robust Summary: For the robust summaries to be considered adequate, the following details need to be provided:

Test substance purity information Vehicle/solvent used, if any Control information 

### Statistical Methods and Results Criteria used to evaluate a positive response Klimisch ReliabilityCode Referencep. 30

Data and Robust Summary Adequacy Report CAS No. 108-19-0 B-7. DEVELOPMENTAL TOXICITY - OVERALL ASSESSMENT Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) OECD considered the available data, in conjunction with repeat dose studies which examined the reproductive organs adequate to satisfy the SIDS reproductive toxicity requirement ; therefore, it is concluded that OECD must have considered the available developmental toxicity data adequate to meet the SIDS requirements for this endpoint based on aWeight of Evidence.p. 31

Data and Robust Summary Adequacy Report CAS No. 108-19-0 B-8 . ACUTE TOXICITY TOFISH Study 1 : 96-hr static toxicity test onfish Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) Reference: Juhnke, I . and Ludemann, D . Zeitschrift fuer Wasser und Abwasser Forschung, 11, 5, (1978) . From OECD SIDS, 1994, as cited in IRPTCData Profile. The full study is: ® Adequate 0

Not Adequate Detailed Review

: Urea was tested on golden orfe (Leuciscus idus melanotous) under static conditions. Not GLP . LCo for 48 hours > 10,000 mg/L The full study is adequate based on OECD's approval of the SIDS submission for urea (1994) . Robust Summary: For the robust summaries to be considered adequate, the following details need to be provided:

Test Substance Purity Water chemistry parameters (temperature, temperature range, dissolved oxygen level, pH, hardness) Mean length and length of fish as well as loading Photoperiod Controls used and control responses Mortality at each concentration Klimisch Reliability Code Referencep. 32

Data and Robust Summary Adequacy Report CAS No. 108-19-0 B-9 . ACUTE TOXICITY TOAQUATICINVEREBRATES Study 1 : 24-hr static test on Daphnia Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) Reference: Bringmann, G . and Kuhn, R

. Zeitschrift fuer Wasser und Abwasser Forschung, 15(1), 1-6, (1982) . From OECD SIDS, 1994, as cited in IRPTCData Profile The full study is: ® Adequate 0

Not Adequate Detailed Review

: Study conducted on Daphnia magna according to DIN 38412Teil 11 (modified) . ECSOfor 24 hours > 10,000 mg/L. The full study is adequate based on OECD's approval of the SIDS submission for urea (1994) . Robust Summary: Forthe robust summaries to be considered adequate, the following details need to be provided:

Test Substance Purity Concentrations tested Water chemistry parameters (temperature, temperature range, dissolved oxygen level, pH, hardness) Controls used and control responses Inactivation/mortality at each concentration Klimisch Reliability Code Reference titlep. 33

Data and Robust Summary Adequacy Repo rt CAS No. 108-19-0 B-10 . ACUTE TOXICITY TOAQUATIC PLANTS Study 1 : Cell multiplication inhibition test in green algae Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) Reference: FromOECD SIDS, 1994, as cited in IRPTC Data Profile The full study is: ® Adequate F-1 Not Adequate Detailed Review

: In tests conducted on green algae (Scenedesmus quadricauda), the toxicity threshold for 192 hours was > 10,000 mg/L. No other data reported. The full study is adequate based on OECD's approval of the SIDS submission for urea (1994) . Robust Summary: For the robust summaries to be considered adequate, the following details need to be provided:

Test Substance Purity Concentrations tested Water chemistry parameters (temperature, temperature range, dissolved oxygen level, pH, hardness) Photoperiod and light intensity Controls used and control responses Klimisch Reliability Code Reference