4:5 Ptl - 1~e? - e;,1-1111 7T" 

_~. 

o o -$- _ 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 following tests : 

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 

I just wanted to bring your attention to the metabolism assessment. The submitter argued that 

since biuret would enzymatically be converted to urea by microflora in the stomach of ruminant 

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 metabolism claim. 

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 Room 6308N 

(202) 564-8603 
Data Adequacy Review and 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 37830 
p. 3 

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

A. SUMMARY OF HUMAN HEALTH AND ECOTOXICITY DATA 

General Comments: The first orphan Test 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 of the 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 of the 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 

satisfy the testing requirements for biuret. 

A-1. Adequacy of the Data 

A summary 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 

minimally toxic 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 2,000 mg/kg. For biuret, 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 Summary Adequacy Report CAS No. 108-19-0 

Stud Information Provided 

Endpoint Acce table Data Gap 

108-19-0 SC 

p 

Health Effects 

Yes No Nol 

Acute Toxicity Yes Yes 

Yes Yes 

Repeated-Dose Toxicity No 

Yes Yes 

No No' Gene Mutations Yes Yes 

Chromosomal No - N 2 

Aberrations Yes Yes 

No - No 3,4 

Toxicity to Reproduction Yes Yes 

No 

- 

- No 4 Developmental Toxicity Yes 47 

No 

Aquatic Effects 

Acute Toxicity to Fish No - No 2 

Yes Yes 

Acute Toxicity to No - No2 

Daphnia Yes Yes 

Toxicity to Aquatic No - No 2 

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 OECD test guidelines; therefore, acute toxicity testing is not required. 

Z Adequate data exist for an acceptable supporting compound, urea. 

3 No 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. 

4 Although 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 of Evidence. 

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 of the subject 

chemical to insure that potential effects are not overlooked. A smaller molecule of 

TABLE 1. Data Adequacy for Biuret 108-19-0 
p. 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 mammalian rumen, microorganisms, such species as Homodendrum, 

Klebsiella, Penicillium, Pseudomonas, Ralsonia, Escherichia, Rhodococcus, Sporothrix 

and 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 form 

urea, carbon dioxide and ammonia. The urea is subsequently hydrolyzed by urease to 

form two 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 of intestinal microorganism are 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 of biuret to urea in the intestine of nonruminant species, it can be argued that urea is an acceptable surrogate for biuret in view 

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 (CAS 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 study is not 

required if and only if no adverse effects are seen in the reproductive 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 of carcinogenicity 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 INFORMATION ON THE 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: 7 Adequate ® 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 form urea, carbon dioxide and ammonia. The urea is 

subsequently hydrolyzed by urease to form two 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 through the 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 of urea 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 metabolism of biuret 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 NPN sources 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 from the 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 bacterium capable of using s-triazines as 

nitrogen sources. Can. J. Microbiol. 48(2002) 1089-1098. 

Wheldon G.H. and MacDonald, R.E. Utilization of biuret 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. Oyo Yakuri (Pharmacometrics) 13:749-772 . From 

OECD SIDS, 1994, as cited in IRPTC Data Profile 

The full study is : ® Adequate 0 Not Adequate 

Detailed Review: Single oral doses of 14,300-15,000 mg/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 of post-exposure observation period 

Klimisch Reliability Code 

Flag as critical study 

Reference missing title 
p. 10 

Data and Robust Summary Adequacy Report 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. Oyo Yakuri (Pharmacometrics) 13:749-772 . From 

OECD SIDS, 1994, as cited in IRPTC Data Profile 

The full study is : ® Adequate F~ Not Adequate 

Detailed Review : Single oral doses of 11,500-13,000 mg/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 of post-exposure observation period 

Dose-specific results. 

Klimisch Reliability Code 

Flag as critical study 

Reference missing title 
p. 11 

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

B-2. ACUTE TOXICITY - OVERALL ASSESSMENT 

General Comment: The only available acute toxicity 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 (IUCLID Data 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 4 by 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 140 days produced no toxic effects. 

Using a food factor of 0.08, a dietary level of 10% is equivalent to 8,000 mg/kg/day. 

Because an oral dose of 8,000 mg/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 can be 

assumed that the oral LDSO would also exceed 8,000 mg/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 : 30 and 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 of Animal 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 

30 days. A control group (6 rats) was fed the basal diet without added Biuret . Animals 

were observed for signs of toxicity and body weights were recorded after 30 days. The 

study did not follow GLP. 

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 from each 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 

30 days 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 of the rat body 

to Biuret would be 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 of Various 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 the reproductive organs were examined 

histologically 

Flag as critical study 
p. 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 study is : N Adequate 0 Not Adequate 

Detailed Review: A 12-month chronic toxicity and carcinogenicity screening study was 

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 from all dose groups were necropsied at the 

end of the treatment period, and the gonads were histologically examined. No pathology 

was reported . After further 4 months of follow up, 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 body weight and organ weight changes 

Statistical methods 

GLP and test guidelines followed 

Klimisch Reliability Code 

Reference title 
p. 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 IRPTC Data 

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 from all dose groups were necropsied at 

the end of the treatment period, and the gonads were examined histologically . No 

pathology was reported . After further 4 months of follow up, all survivors were killed, 

necropsied and the tissues were fixed. The testes, prostate and uterus were histologically 

examined for the occurrence of tumours . Although there was a statistically increased 

incidence of interstitial 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 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 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 title 
p. 16 

Data and Robust Summary Adequacy 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 4 weeks. 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 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 

Vehicle information 

Control information 

Number of animals/sex per dose 

Statistical methods 

GLP? 

Klimisch Reliability Code 

Reference title 
p. 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 . Oyo Yakuri (Pharmacometrics), 13, 749-772, (1977) . From 

OECD SIDS, 1994, as cited in IRPTC Data Profile. 

The full study is : ® Adequate 0 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 24 weeks. 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 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 

Vehicle information 

Control information 

Number of animals/sex per dose 

Statistical methods 

GLP? 

Klimisch Reliability Code 

Reference title 
p. 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 study is : ® Adequate F~ Not Adequate 

Detailed Review: Rats were fed a diet containing 20,000-250,000 ppm urea for 190 days 

(about 2000-25,000 mg/kg/day). Purity of the 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 of the 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 

of the 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 title 
p. 19 

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

B-4. GENETIC TOXICITY (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 IRPTC Data Profile. 

The full study is : ® Adequate 0 Not Adequate 

Detailed Review: Tested on Salmonella typhimurium strains 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 of the SIDS submission for urea 

(1994) . 

For the robust summary to 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 of metabolic activation 

Incubation conditions (temperature, duration) 

Any deviations from the guideline stated 

Statistical Methods and Results (mean number 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 title 
p. 20 

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

B-4. GENETIC TOXICITY (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 IRPTC Data Profile. 

The full study is : ® Adequate 0 Not Adequate 

Detailed Review : Tested on Salmonella typhimurium strains 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 GLP or 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 of metabolic activation 

Incubation conditions (temperature, duration) 

Statistical Methods and Results (mpan number 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 title 
p. 21 

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

B-4. GENETIC TOXICITY (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 IRPTC Data Profile. 

The full study is : ® Adequate F-1 Not Adequate 

Detailed Review : Tested on Salmonella typhimurium strains TA98, TA100, and 

TA 1537, 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: 100-10000 ug/plate depending 

on strain. No information as to GLP or 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 (mean number of revertent colonies per 

plate for treated and control cultures) 

Criteria used to evaluate a positive response 

Klimisch Reliability Code 

Reference title 
p. 22 

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

B-5. GENETIC TOXICITY (chromosomal aberrations) 

Study 1 : Chinese hamster fibroblast (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 IRPTC Data Profile. 

The full study is : ® Adequate 7 Not Adequate 

Detailed Review: A chromosomal 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 of the 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 toxicity was observed 

Klimisch Reliability Code 

Reference title 
p. 23 

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

B-5. GENETIC TOXICITY (chromosomal aberrations) 

Study 2: Chinese han:ster fcbroblast (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 OECD SIDS, 1994, as cited in IRPTC Data Profile. 

The full study is : ® Adequate 0 Not Adequate 

Detailed Review: A chromosomal aberration test was conducted on Chinese hamster 

fibroblasts. The concentration tested was 13 g/L (urea concentration 216 mmolar). 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 a positive response 

Whether or not toxicity was observed 

Klimisch Reliability Code 

Reference title 
p. 24 

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

B-5. GENETIC TOXICITY (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 IRPTC Data Profile. 

The full study is : ® Adequate 0 Not 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 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 for each concnetration 

Criteria for scoring a positive response 

Whether or not toxicity was observed 

Klimisch Reliability Code 

Reference title 
p. 25 

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

B-5. GENETIC TOXICITY (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 500 mg 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 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 

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 title 
p. 26 

Data and Robust Summary Adequacy Report 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 study is : ® 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 the histopathological 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 of Abnormal Development, 23, 335-342, 

(1981) . From OECD SIDS, 1994, as cited in IRPTC Data Profile. 

The full study is : [] Adequate ® Not Adequate 

Detailed Review: A single dose of 2,000 mg/kg was administered orally to 10 pregnant 

ICR mice 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 

Reference 
p. 28 

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

B-7. DEVELOPMENTAL TOXICITY 

Study 2 : Developmental screening study in rats 

Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) 

Reference: Teramoto et al . Teratology, Journal of Abnormal Development, 23, 335-342, 

(1981) . From OECD SIDS, 1994, as cited in IRPTC Data Profile. 

The full study is : [:] Adequate ® Not Adequate 

Detailed Review: A single dose of 2,000 mg/kg was administered to 4 pregnant Wistar 

rats on day 12 of pregnancy. 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 a positive response 

Klimisch Reliability Code 

Reference 
p. 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 (50 g/kg bw) was administered by gavage in 2 doses 12 hours 

apart to 6 rats 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 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 a positive response 

Klimisch Reliability Code 

Reference 
p. 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 a Weight of Evidence. 
p. 31 

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

B-8. ACUTE TOXICITY TO FISH 

Study 1 : 96-hr static toxicity test on fish 

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 IRPTC Data 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 

Reference 
p. 32 

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

B-9. ACUTE TOXICITY TO AQUATIC INVEREBRATES 

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 IRPTC Data Profile 

The full study is : ® Adequate 0 Not Adequate 

Detailed Review: Study conducted on Daphnia magna according to DIN 38412 Teil 11 

(modified) . ECSO for 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: 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) 

Controls used and control responses 

Inactivation/mortality at each concentration 

Klimisch Reliability Code 

Reference title 
p. 33 

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

B-10. ACUTE TOXICITY TO AQUATIC PLANTS 

Study 1: Cell multiplication inhibition test in green algae 

Test Compound: Supporting Compound; Urea (CAS No. 57-13-6) 

Reference: From OECD 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