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PRIVILEGED AND' CONFIDENTIAL ATTORNEY'S ' WORK PRODUCT Covington & Burling DRAFT' - October 15,, 1986 SUNihARY OF DATA ON UREA Abstract. Urea is~a natural component of tobacco, and it is also added to tobacco as a flavor. A cigarette to which urea has been added'may contain 2.2'5 to 5.4 mg of urea,, or les&than 1% of the cigarette by weight. Urea has been approved for use in ciigarettes in Great Britain, at lelrels up to 2%, and is permitted for use in Germany. The.exposure to:ureaifrom cigarettes is insignifi- cant compared to other exposures. Ureaiis a natural consti- tuent of many foods, andi it is producediin the body as the final degirediation product of protein metabolism. A 160' poundd adu~lt forms an average of 25 to 30 grams of urea each day. Tn contrast, if' all of the urea in a pack of cigarettes were ~ inhaled, the total urea exposure would be about 108 mg. Tests using the Salmonella/microsome assay to compare cigarette smoke condensate from reference cig,arettes to condensate from cigarettes containing 1.5, 3' or 6 times the level of urea added to a typical commercial cigarettes showOd no chang,e in biological activity. Smoke composition analyses of the giu:s phase of urea-treated,cigarettes have also indicated that the additioniof urea to cigarettes does, not resuilt in any major qualitative~changes in smoke constituents as compared' too the smoke of control cigarettes, though 10 to 20% red!uctions were observed in the amounts of organic gas phase components. Acute toxicity tests have.shown that urea may be ingested by humans at levels of 2' to 3 grams,per kilogram of body weiyht with no adverse effects~, and long term ingestion studies at high d'oses have.not ind'icated any adverse effects., Mutageni:city tests have been inconsistent, but an NTP bioaissay has not shown urea to be carcinogenic. Background. Urea (,CO(,NH,),; CAS No. 57'-13'-6), is thee d'iamide of carbonic acid. Urea is produced by the.dehydration ~O. *A of ammoniumicarbamate which is synthesized from ammonia and! k.a carbon dioxide. Urea decomposes below its boiling point. 0 N

A Urea~ is the final degradation product of protein metabolism in maniand other mammals~, elasmobranch~s, amphibia, and chelonia., Most of the, nitrogen:consur.ted in food is excretedi as urea. Urea is formed in the liver mi*ochondria through the Krebs cycle and is the major organic component excreted'in the urine. Urea is a natural con~stituent of many foods., Plants cont~ain15to15% non-proteinaceous nitrogen, much of it in the form of urea. For example, oats may contain 4.5% of their total nitrogen content as~urea, while oil meals contain about 0.251%. Urea is used as an animal food supplement, in the production of fertilizers and urea-formaldehyde resins~, andi as a coating;material for foodlpackaging and fabrics. It is also used in foods., The Select Committee on GRAS Substances concluded that the per capita urea intake in man, as a direct or indirect food additive, is less than 5 grams daily (SCOGS, 19,77)1.. Ureaihas been given therapeutically to reduce intra- ocular and intracranial pressure, to treat sickle cell anemia, as~ a topical antiseptic:, and as an ingredient in ammoniatedi :r dlentifrices!. Urea has been used as an oral diuretic for = .;,.. chronic edema, a~l:th:oug;h~ it is~ no:lo~~nger the~ treatment of „~ 1~+ choice. CJ Tobacco Use. Ureaiis used by the tobacco industry a:s~ a flavorant, andl is~~~ applied to~~ tobac~colw:ith~ calsin~g mater~ia~ls~.,

r Approximately 1.3 million pounds of urea were used by the industry in 1985, andithe current usage level of urea in the cigarette is between 2.25 mg and 5.4, mg. Using gas chroma- tographu, urea has.been identified a~s a natural conistituent in Burley tobacco (iCarugno, 1974). Regullatory Status. In 1978, the Federat_'on of American Societies for Experimental Biology published a report written by: the Select Committee on GRAS Substances (SCOGS Com- mittee) which provided an independent evaluation of the safety of urea as a direct food additive. The SCOGS Committee concluded that there was nolevidence to suggest that urea, when used at current or future reasonable levels, presents any hazard to the public when used in foods (SCOGS, 1977). Following; the SCOGS review, urea was granted GRAS status by the Food andiDrug Administration for use as a direct food' additive when used a~s a fermentation aid in the production of alcoholic beverages, in yeast-raised' bakery products, and as a formulation aid in gelatin. 21 C'.F.R. § 184'.1923'. As a~n indirect food'additive, urea is used as a cellulose softener or plaisticizer, in side seam cements f.or food containers, and in glassine and greaseproof paper for packaged'dry food's., 21 ~ . . . ^ ..+ C'.F.R. §§ 175.3'00, 176.180, 177.1200. ~J. ~ ~+. The Independent Scientific Committee on Smoking and ~ Health in. Great Britain (,the Hunter Committee) has approvedd urea for use as.a tobacco additive at a maximum level of 2%, or approximately 15 mg/cig. Urea.is permitted as aitobacco ~

! f flavorant under the German Tobacco Ord'inance because it occurs naturally in Burley tobacco. NYetabolism,., Urea is very soluble in water and is rapidly absorbed'and distributed throughout the body. In man, the normal level of urea is 26 mg urea per 100 ml blood plasma:. A 70 kg adult forms an average of 25 to 30 grams of urea eachiday which is excreted in the urine. One indication of kidney functionlis the ability of the kidney to remove urea from the blood as measured by the glomerular filtrationn capacity (Guyton, 19751. Patients with renal insufficiency frequently have levels as high ais 2'00 mg urea per 1001ml blood plasma., Therapeutic Observations. In humans, the therapeutic diose for uread administered orally as a diuretic in tYie treat- ment of chronic edema is 4,0 to 100 grams (,about 01.7 to 1.6 g!/kg)I. The recommended dose to reduce intraocular or intra- cranial pressure i&1 g/kg of body weight administered intra- venously. Dosage levels of urea at 2 to 3' g/Yg body weight have been given orally to healthy volunteers with no reported' adverse effects ('Eknoyani,. 1969)'. The most common side effects of orally administered urea are diuresis, thirst, nausea, vomiting, minor gastrointestinal disturbances and somnolence.. Intravenous administration of urea can result in headaches, mental confusion and~vasomotor symptoms. The observed side effects of urea may be due, iinn part, to high urea levels and'changes in tissue electrolytes..

- 5 - Johnson and coworkers maintained high blood ureaiconcentrations by intermittent dialysis in three patients suffering from advanced renal fail'ure. Blood!concentrations of 181 to 600 mg urea per 100i ml were maintained for periods of 7 to 910 days. When the urea concentration was kept below 100 mg per 10:0 ml, no adverse effects were noted,, although this level is 6 to 15 times greater than normal. Above 300 mg urea per 1001ml . plasma, patients exh,ibited'malalise, vomiting, bleeding, tenderness and' headaches (Johnson, 1971)1.. Acute and Subchronic Toxicity. The acute toxicity of urea appears to be relatively low, particularly amongg nonruminants (SCOGS, 1977)!. Ruminants are much more sensitive to urea poisoning than nonruminants. In the rat, the oral LD50, value is~14.3'; g/kg; the subcutaneous LD50 is 8.2 g/kg; and the intravenous LD, 50, is' 5.3' g/'kg,(RTECS, 1981). Large d'oses.of urea have beeniadtninistered intra- venouslyto patients with sickle cell anemia to shorten a sickling crisis. The total dose of urea varied from 2.6 to 6.0 g/kg injected over 10 hours in one study and over 12 to 24 diuresis,. hours in another study. The side effects included, headache, and vein irritation, but these were not considered' acute (Cooperative LTrea Trials Group:,, 1974). The toxic effects of long-term urea ingestion have been evaluated in several animal: species. Dogs which were unilaterally nephrectomized received a subcuta~neou~s injection of 3000!to 4'000 mg/kg every 8 hours for 45 days. Plasma urea

levels were mai:ntained'between 2001and 700 mg per 100 ml plasma. Measurements of hematocrit and platelet counts, electroencephalo- graphic recordings and spontaneous movement were essentially normal in the nephrectomized dbgs. The only observed effects were mild drowsiness anddiuresi&(Balestri, 1971)~. Rats fed wiith rations containing 2 to 25% urea. (about 2 to 25 g/kg/day) for periods up to,190 days exhibited weight loss and'suppression of sexual function even at the lower dosage levels. Rats.receiving 14% urea in their diet and deprived of water died' within a few d'ays but survived 20 to:76 days if waiter was aillowed' with a 20% level of urea supplement, and 1'2 days wJ:thia 25%supplement (Richet, 1951). This study used only a small number of animals in each series~ (only 1 to 3), and data on actual food intake was lackingi. Ca!rcinogenicity. Urea was evaluated in a long-termi feeding study using C57B'1'/6 mice and!Fischer 344 rats as partt of the National Toxi:coiogy: Frogrami- National Cancer Iinstitute (tVTP-NCT_) carcinogienesis bioassay program. Urea was admini- stered'at 0.45%, 01.9%, and 4.51levels in the diet for 12 months. No weight depression was~noted'for animals of either sex and species at any of the test d'oses. Male rats in the 'Z ~ middle diose level group showed decreased survival (8'9'$) L.? ~ relative to diet controls (95%'), while survival of all other k,~e ~. .. test groups remained'unaffected. There was a significant . occurrence of hematopoiietic tumors, particularly malignant lymphomas among female mice receiving the mid-dbse level..

- 7 - However, because lymphomas did~ not occur in the lowdose or high diose groups, the authors of the study concludedd that this lesioniwas of questionable biological significance. In the same study„ rats treated'withiurea Yiadia significantly higher proportion of testicular interstitial adenomas in the high dose group. The authors concluded that since this lesion may occur spontaneously in up to 100 percent of the control animals of the species, its biological significancewasq,uestionable (Fleischman, 19,80).. Several stud'ies suggest that urea can inhibit tumor formation and growth retardation in experimental animals. Urea was found to inhibit hepaitocarcinogenesis induced'by N,N-dimethyl-4-aminoazobenzene in rats (Lin, 197'3)'. Tumor growthiin rats innoculated with Walker 256 carcinosarcoma cells was inhibited by the local injection of urea (Takamura, 1977Y. Elson (1972) foundithat growth retardation of rats exposed to the smoke of high-sugar cigarettes was decreased1by the addition of g;uanid'ine-nitrate or urea:to the cigarettes.. Genotoxi:ci,ty.A~t a level of 16 mg!/ml, urea was judged to be positive in a chromosome aberration test using Chinese Hamster lung (CHL) cells. CHL cells exposed to urea had more than a 1!0$ incidence of polyploid cells and~ chromatid' aberrations as compared with the controls. As pa~rt of the same study, ureaiwais negative in the Ames reverse mutation assay using S. typhlimuirium st.raiins TA,98, TA100, and TA'1537' (Ishidate,, 19181). .

~ - 8 - In an earlier evaluation, urea induced severe chromosome fragmentation andicytotoxic effects, particularly in metaphase cells at a final concentration of 501mM. The authors concluded that chromosome fragmentation in cells exposed to high concentrations of urea maybe the nonspecifi:c effect of exposure to a high osmolality solution, rather than; the unique effect of urea on cells. The same, well-recog,nizedd effect might be seen for many other compounds at sufficiently highiconcentration levels. There was no evidience of cliromosoma~ i aberrations in cell's treated with urea at the physiological level of 11 mMi (Oppenheim, 19651. Urea was found'to be negative inithe alkaline elution/rat hepatocyte assay at the 3 mM'level. The alkaline elution/rat hepatocyte assay was designed to measure the ability of a compound'to induce DNA single-strand breaks ass a predictor of its carcimogenic/mutagenic potential (Sina, 1983Y. Pyrol'ysis Toxicology Studies. A study was performed'. in which the effect of add!ingiurea to cigarettes was evaluated by testing the resulting cigarette smoke condensate (CSC) in the Salmonellai/microsome (S/M)' assay (Ames plate incorporation assay)'. U:rea was dissolved in water and injected into Uni:ver- siity of Kentucky 1R4F Research Cigarettes at levels 1.5, 3.0 and 6.0 times those normally! used in commercial cigarettes. The cigarettes were smoked'., and the impaction-trapped CSC was tested in the S/M assay (strains TA98' andiTA100) with and

r 41 9 without metabolic activation. The results indicated that the addition of urea to cigarettes, at an-v of the levels tested, did not alter the activity of the CSC in Salmonella. Pyrolysis Chemistry Studies. Chemical analysis of smoke was carriedi out using cigarettes treated with 2% urea versus untreated controls. The blend composition1was similar to the University of Kentucky Reference 1R4F'cigarette. There were no quantitative differences in the concentration of cras phase CO, NO, HCN, andi RCHO of urea-treated'cigarettes compared withi controls:. Org,anic gas phase analysis using nitrogen phosphorus detector (NPD)' and flame ionization detection (FID), resulted, in no major qualitative differences between urea-treatted cigarettes compared with controls. There were no peaks (new or absent)' present in the gas phase of urea-treated cigarettes that were not present in the gas phase of the control ciga- rettes. However,, both the NPD and FID data indicated'a I0$ to 20% quantitative reduction in the levels of gas phase compo- nents for the urea-treated cigarettes.

rI y REFERENCES 1. Balestri, P. L., P. Rindi, M. Biagnini, Chronic Urea Intoxication in Dogs, Experientia 27 8'11-812 r'1i971y.., 2. Carugno, N., M4 Neri, G4 Lionetti, Quantitative Deter- mination of Free and Protein"Bound'~ Amino Acids~of Tobacco, Beitrag,e zur Tabakforsch:ung 7(4), 222'-227' ('1974)',., 3. Cooperative Urea Trials Group, Clinical Trials of Therapy for Sickle.Cell Va.so-occlusive Crisisy Journal of the American Medical Association 2'28' 112'0-1124 (1974). 4. Cboperative Urea Trials G'roup, Treatment of Sickle Cell C'riisi&withUrea in Invert Sugar, Journal of theAmerican~ Medical Association 228 1125-1128 (19741. 5. Eknoyan, G., S. J. Wacksman, H. T_. Gluek, J. J. Will,: Platelet Functionin Renal Failure, New Englandi Journal of Medicine 2'8'0' 677-681 (19691. 6. Elson., L. A., T'. E. Betts, Sugar Content of the Tobacco and pH of the Smoke in Relation to Lung C'ancer Risk&of Smoking, Journal of the National Cancer: Institute 48'('6) 1885-1890 (1972) . 7. Fleischman, R. WA, J. R. Baker, M. Hagopian, C. G. Wade, D. W. HaydenV E. R. Smith, J. H. Weisburger, E. K. Weisburger, Carcinogenesis Bioassay of Acetamide,. Hexanamide, Adipamid'e,. Urea, and' P-Tolylurea in Mice and 8. Rats, Journal of EnvironmentalPathology 3(5-6) 149-170 (:1H0 )' . Guyton, A. C'., Textbook of Medical Physiology (6th ed., 1981Y C . .~. Ishidate,M.„ T'. Sofuni, K. Yashikawa, Chromosomal ~ Aberration Tests in Vitro as a Primary Screening Tool for ~ Environmental Mutagens and'/or Carcinogens, GANN Mono- (N ~ graphs on Cancer Resea!rch 27 95-108 (1981). 10. Johnson, W'. J., W~ W. Hagge, R. D. Wagoner, R. P. Dinapoli, J. W. Rosewear, Effects of Urea.Loading i:n: Patients with Far-Advanced Renal Failure, Mayo Clinic Proceedings 4" 2-29 (19'7'21. ~ 11. Lin, ?'., Y. Chen, S. Horing,, T. Tung, Inhibition of Urea on the Hepatocarcinogenesis Induced by N',N-dimethyl- 4-a2ninoazobenzene in Rats, T''ai-Wan I Hsueh Hui T'sa Chih 72(5) 262-282 (1973) .