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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460

.. 21111005915

OFFICE OFPESTICIDES AND TOXIC SUBSTANCES

MEMORANDUM

SUBJECT: Kelthane: Evaluation of a rat metabolism study of dicofol

FROM:

TO:

THROUGH:

Whang Phang, Ph.D. l,..,P ~ S-{C16/f7Pharmacologist ~VN~~D !~Toxicology Branch/HED (769c)

D. Edwards, PM (12)Registration Division (767c)

Marci a van. Gernert, Ph .D. IJA hn .. rJ.lAU'. J s:f-, b lt7Hea~~d Sect1 on II I IPI·f/L&UA.OK.IVV,#I~ ~LSTheodore M. Farber, Ph.D. #ChiefToxicology Branch/HED (769c)

The registrant, Rohm & Haas Co., has submitted a rat metabolism study of p,pl­dicofol, an active ingredient of Kelthane. This study have been evaluated byDynamac Corp. and is approved by Toxicology Branch. The DER is attached.

Sinlge dose (50 mg/kg) of 14C-dicofol was orally administered to groups of rats(4/sex). After 168 hrs of dosi ng, the major route of excretio.n was found to be throughfeces for both male and females. The highest tissue residue level was in adiposetissue (30% and 80.5% of the dose in males and females, repectively), after 48 hrsof dosi ng.

Dicofol was found to be metabolized via several pathways. The major pathway appearedto be dechlorination of a non-ring chlorine atom to yield FW-152 and followed byoxidation to form dichlorobenzophenone (DCBP) and dichlorobenzoic acid (DCBA). Inthis study very little DOE was found. Apparently, the metabolism of dicofol is dif-ferent from that of DDrwFiich is O1etabolized to DOE. .

This study is acceptable as a study to demonstrate the metabolism of dicofol.

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NAIIONAL SceUlUlY lNI'QAAV"t IIVN lEO 12065}

DATA EVALUATION RECORD

KELTHAN.E

Metabolism in Rats

005915EPA: 68-02-4225

DYNAMAC No. 266-A. May 26, 1981

STUDY IDENTIFICATION: Tillman, A. M., Mazza, L. S., and Williams, M.Part I: Absorption and excretion of l4C-p,j)'-dicofol in male and femalerats. Part II: A metabolism study of 14C-p,p'-dicofol in male andfemale rats. (Unpublished study No. 31L-86-02 prepared by Rohm and Haas,Spring House, PA, and Analytical Bio-Chemistry, Columbia, MO, for Rohm andHaas, Spring House, PA; dated December 30, 1986.) Accession No. 400420.

APPROVED BY:

I. Cecil Felkner, Ph.D.Department ManagerDynamac Corporation

1

Signature:

Date:

Le.,dO~~•

5'"- ~b-gl

1.

2.

CHEMICAL: Kelthane; p,p'-dicofol; dicofol; 1, l-bis(4-chlorophenyl)-2.2,2-trichloroethanol.

TE~T MATERIAL: 1.l-Bis (4-[U_14C]Chl orophenyl) -2,2, 2-tr1ch1oroethano1([ 4C]dicofo1) with a specific activity of 26.4 mCi/mgand a radio­chemical purity of 98 percent.

3. STUDY/ACTION TYPE: Metabolism in rats.

4. STUDY IDENTIFICATION: Tillman, A.. M.. Mazza, L S.• and Williams,M. Part I: Absorption and excretion of }4C-p.p'-dicofol in maleand female rats. Part II: A metabolism study of 14C-p,p'-dicofolin male and female rats. (UnpUblished study No. 3lL-86-02 preparedby Rohm and Haas. Spring House. PAt and Analytical Bio-Chemistry,Columbia, MO. for Rohm and Haas, Spring House. PA; dated December 30.1986.') Accession No. 400420.

5. REV IEWED BY:

Charles. E. Rothwell. Ph.D.Principal ReviewerDynamac Corporation

Nicolas P. Hajjar, Ph.O.Independent ReviewerDynamac Corporation

6. APPROVED BY:

1. Cecll Felkner. Ph.D.Technical Quality ControlDynamac Corporation

W.Phang, Ph.D.EPA Reviewer

Marcia Van Gernert. Ph.D.EPA Section Head

2

Date:

Signature: :hAW ffi~Date: 5"- 2,,-8 J

Signature:~

Date: / u-/Y6 f f lT r

Signature:

Date:

7. CONCLUSIONS:

A. Groups of four male and four female Sprague-Dawley rats wereadministered single oral doses of [14C]dicOfol .at 50 mg/kg andsacrificed 48 or 168 hours later. The major route of excretionwas through the feces for all groups, accounting for 61.7 and32.2 percent of the administered dose in males and females,respectively, after 168 hours. In addition, approximately 15percent of the dose was excreted in the urine of both males andfemales after 168 hours. The highest residue levels of[14C]dicofol equivalents were observed in adipose tissue at 48hours: 200 ppm, or 30.6 percent of the dose, in males and 547 ppm,or 80.5 percent of the dose, in females. [14C] residues infat, liver, and blood decreased with time for both sexes, but thedecrease was faster in males than in females.

Urine, feces, fat, liver, and plasma were analyzed for dicofolmetabolites. Oicofol is metabolized via several pathways to forma large number of compounds (Figure 1). The major pathway appearsto be one whose mechanisms involve replacement .of a nonringchlorine atom with hydrogen (dechlorination) to yield FW-152 andsubsequent oxidation to form dichlorobenzophenone (OCBP) anddichlorobenzoic acid (OCBA). Further metabolism yields dichloro­benzil (OCBH) and various hydroxy or conjugated secondarymetabolites. Cleavage also occurs to yield p-chlorobenzoic acid(CBA), which is subsequently conjugated with glycine to yieldp-chlorohippuric acid (CHA). Apparently, the hydroxyl group ofthe trichloroethanol moiety (lone oxygen atom) of dicofol is notcleaved and, therefore, the metabolism of dicofol is totallydifferent from that of oor (Figure 2) . Spec1ficanalysis of thesetissues revealed that, at most, 0.2 percent of the [14C] resi­dues were ODE. Although this study indicates that little, ifany, formation of ODE from pure dicofol occurs, technicalKelthane may contain larger amounts of ODE and/or OOT,whichshould accumulate in fat as ODE.

B. This metabolism study is acceptable.

Items 8 through 10--see footnote 1.

11. MATERIALS AND METHODS (PROTOCOLS):

A. Materials and Methods: .

1. The test material, [14c]dicofol, was purchased from ICI,England. It had a specific activity of 26.4 mCi/g and aradi ochemi ca 1 purity of 98 percent. Cochromatographicana lysi s wi th pure standards by high-performance 1iquidchromatography (HPLC) and subsequent radioanalysis by

10nly items appropriate to this OER have been included.

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OICOFOL C'.ell..........·

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Source: The assessment of the carcinogenicity of dicofol(Kelthane), DDT, DOE, and DOD (TOE).u.S. EPA, Carcinogen Assessment Group (1986).

Figure 2. Theoretical metabolism of dicofol, and kno\ln metabolism of OOT, ODE, and 000.

The chemical structures of dicofol, DDT, and ODE are shownbelow:

ScheduledSacrifice

/

/Wt..uu

c·o'cO'·, c.-, -a-c-c.

RatNo. Sex

p,p'-dicofolo,p'-dicofol

..a{_>10ao-c-<.•CI

00'

Wt. Scheduled.uu Sacrifice

98.0 percent0.. 4 percent

RatNo. Sex

Eight male and eight female Sprague-Dawley rats, 6 to 8 weeksold, were purchased from Charles River Kingston (Stoneridge,NY). The animals were provided Purina Rodent lab Chow andwater ad libitum. After a 1-week quarantine. the animals wereacclimated to their individual metabolism cages for 3 daysprior to dosing. The rats were assigned to the followingschedule of sacrifice postdosing:

c·-c-o0IC0F0l L

l1quid scintillation cOlJnting (lSC) showed that the testmaterial contained the following compounds:

2.

12345678

Male 191Male 265Male 255Male 200Female 155Female 150Female 110Female 161

48 hrs48 hrs48 hrs48 hrs48 hrs48 hrs48 hrs48 hrs

910111213l41516

Male 235Male 175Male 175Male 185Female 155Female 155Female 178Female 170

168 hrs168 hrs168 hrs168 hrs168 hrs168 hrs168 hrs168 hrs

3. The test material was diluted with unlabeled dicofo1 (98percent pure) to 10 "ClImg and s.ubsequent1y dissolved incorn oil. Each rat was adminhtered a single oral dose of[14C]dicofol at 50 mg/kg body weight (16-132 lIei/rat) andreturned to its· metabolism cage. One rat (No. 10) died .48hours after dosing and, therefore. no samples were collectedfrom this animal.

4. Urine and feces were collected at 24. 48. 12, 96. and 168hours postadministratlon when possible. Expired air was notcollected. The liver and samples of adipose tissue wereremoved and weighed from all dosed rats after sacrifice.

6

Additionally, the kidneys were removed from· rats sacrificedat 168 hours postadministration. 8lood samples were collectedfrom all 15 rats just before sacrifice. The urfne collectionfunnels and cages were washed with water and the washes weresaved for radioassay. All samples were stored frozen at-15°C until required for analysis.

5. Al1quots of urine, plasma, cage washes, and washes from theurine collection funnel were radioassayed by lSC. Samples ofl1ver, kidney, adipose tissue, whole blood, and feces wereradioassayed by combustion and SUbsequently by LSC. Thefollowing assumptions were made for the conversion of dpm persample to percent dose recovery: 1) whole .blood and plasmaboth have a specific gravity of 1 g/ml, 2) the ....at contains65 ml of whole blood/kg body weight, and 3) total body fatweight is 7 percent of the rat's body weight at sacrifice.

6. For metabolite identification, all samples were extractedwith organic solvents to remove unbound radioactivity. Feceswere extracted twice with methanol ina Soxh1et apparatus;the extracts were concentrated and subsequently purified bySep-Pak chromatography. Urine samples were acidified withhydrochloric acid (pH 2) and extracted three times with ethylacetate; the ethyl acetate extracts were combined and co.ncen­trated. liver samples from rat Mos. 2, 4, 6, 8. 12. 14, and16 were extracted twice (combined) by homogenization inmethanol-sodium sulfate and once by homogenization inmethanol acidified with hydrochloric acid. The methanolextracts were concentrated separately and· extracted threetimes with petroleum ether and then three times with ethylacetate. Samples of adipose tissue from rat Nos. 2, 4, 6, 8,12, 14, and 16 were extracted three times with petroleumether by sonication; the extracts were combined and concen­trated. Plasma from rat Nos. 1,3.7,8,11,12. 15,and 16was mixed with 1 to 2 ml of 10 percent sodium chloride,acidified with hydrogen chloride, and extracted three timeswith n-butano1; the butanol extracts were combined andconcentrated. Ti ssues and excreta from each rat wereextracted separately. A1iquots of each extract and residuewere radioassayed to determine recoveries of [~4C].

7. Radioactive. metabolites were separated from the variousextracts by preparative TlC and by HPlC. Several metaboliteswere identified by cochromatography with analytical standardsusing TlC and HPlC and by isotope dilution experiments.Structural identification of other metabolites was accom­pli shed by mass spectrometry (MS) or gas chromatography/massspectrometry (GC/MS). DOE was specifically looked for in analiquot of the methanolic fecal extract from rat No.6 bycompari son of HPlC chromatograms of the sample and a DOEstandard.

7

\

8. Liver and adipose tissue samples from rat Nos. 1, 3, 5, 7, 9,11, 13, and 15 were sent to Analytical Bio-Chemistry,Columbia, MO, to quantitate dicofol, DDE, and FW-152 (a majordicofol metabolite) res idues. The methodology employed byAnalytical Sio-Chemistry was slightly different from thatused by Rohm and Haas, but basically involved the sainesteps. The tissues were extracted with an organic solventand the extracts were purified (by gel-permeation chroma­tography) before metabol ite identification by HPLC cochroma­tography with analytical standards. A copy of AnalyticalBio-Chemistry's methodology is included in this report asAppendix A.

B. Protocol: See Appendix B.

12. REPORTED RESULTS:

A. Recover" of Excreted Radioactivity: Approximately 85 percent ofthe [14C]-dicOfol dose was recovered in the tissues and excretaof male rats sacrificed at 48 and 168 hours and in female ratssacrificed 168 hours p.ostadministration (Table 1). The balanceof the dose for these groups may have been 1n the carcasses,which were not radioassayed. However, total recoveries of[14C] in females sacrificed at 48 hours averaged 124 percent ofthe administered dose; [14C] residues in the carcasses were notradioassayed.

B. Elimination: The major route of [14C] excretion for all groupswas the feces (Table 1).· Male rats excreted a larger percentageof the dose in the feces than did females. Urinary excretion,however, was similar for males and females at 48 hours (means of5.6 and 5. 7 percent, respectively) and at 168 hours (means of16.3 and 13.3 percent, respectively). Total excretion of [14C]ave"raged 51.5 and 40.5 percent in males and females, respectively,at 48 hours postdosing and 80.0 and 54.6 "percent in males andfemales, respectively, at 168 hours.

C. Tissue Distribution: [14C] residues found in selected tissuesat 48 and 168 hours postadministration are presented in Tables 1and 2. [14C] residues were expressed as percent of administereddose (Table 1~ or ppm (Table 2). The highest [14C] residueswere found in fat. [14C] residues in fat accounted for 30.6and 80.5 percent of the dose in males and females, respectively,48 hours "postadministration (Table 1). Mean concentrations of[14C]dlc.ofol equivalents were 200 and 547 ppm, respectively(Table 2). Levels of [HC] in liver and blood were similar inmales and females at 48 hours and were much lower than those infat. In male rats, tissue levels of [14C] at 168 hours were 80to 90 percent lower than at 48 hours. Ti ssue concentrat ions of[14C] declined more slowly in female rats; levels at 168 hourswere 60 to 70 percent of those at 48 hours. In male and female

8

TABLE 1. Mean Recoveries of ~ld1oactivity 48 and 168 Hours afterAdministration of [ C]Oicofol to Rats at 50' mg/kga

Sample

Feces

'Urined

Cage Wash

Fat

liver

Kidney.

Blood

Total

Recovery of [14C] (Percent of administered dose) after:

48 hours b 168 hourscMa 1es Fema1es Ma les ':"'Fe-ma""""l:-'"e-s

44.35±3.69 31.96±9.25 61.70±12.85 32.21±4.12

5.59±2.19 5.72±1.46 16. 33±9. 72 13.33t3.02

1.57±0.32 2.85±0.57 2.00±1.63 7.07±2.64

51.49±5.12 40.53±8.73 80.02±8.99 54.62±6.95

30.6±12.1 80.5±22.7 3.20±4.54 28.37±5.33

2.83±0.94 2.03±0.30 0.28±0.11 0.82±0.10

No sample No sample 0.02±0.01 0.16±0.08

0.17±0.38 O. 7l±0. 29 0.1l±0.04 0.34±0.10

34.2±12.2 83.3±23.0 3.62±4.S9 29.69±5.27

85.73±13.20 123.8±14.9 83.63±8.21 84.30±1.69

aAll v.alues were recalculated from individual animal data by our reviewers.

bvalues are mean ± S.D. of four animals.cValues are mean ± S.D. of three animals; male rat No. 10 died and femalerat No. 14 was excluded from these calculations as an outlier.

dlncludes urinary funnel wash.

9

TABLE 2. Tissue Concentrations (ppm) of (14C]DiCOfO~4EqUivalents48 and 16.8 Hours afte.r Administration of [ C]Dicofolto Rats at 50 mg/kg

168 hours bMal es =Fe-ma--l=-e-s

48 hoursaMa 1es ·-:F=-e-ma--,l....e-sTissue

Fat

Liver

Kidney

Blood

200.0±77.0

24.66±7.39

NO sample

5.34±2.44

546.7±202.4

22.55±5.35

No sample

5.19±2.49

20. 59±30.01

2.48±0.90

1.14±0.20

0.66±O.23

195.4±32.3

9.99±1.06

9.81±4.43

2.54±O.81

aValues are mean ± S.D. of four animals.

bva1ues are mean ± S.D. of three animals.

10

r,ls sacrificed 168 hours after dosing, mean levels of[ C]dicofol equivalents in fat were 29- and 83-fold higherthan the corresponding blood levels, respectively. .

O. Fecal Metabolites: Methanol extraction of fecal samples removedan average of 92.4±17.6 percent of the sample radioactivity.Of that amount, approximately 91.1 percent was recovered afterSep-Pak purification. Oicofo1 and six metabolites were identifiedin the fecal extracts. The structures of all identified dicofo1metabolites are presented in Figure 1. The distribution ofradioactive metabolites in the feces is shown in Table 3.Quantities of metabolites varied greatly for individual samples.as is reflected by the large standard deviations.

Parent compound accounted for only 2 to 5 percent of theradioactivity in purified fecal extracts of all four groups.Metabolite FW-152 was the major metabolite found in the purifiedextracts of 0- to 48-hour feces from males and females and the48- to 168-.hour feces of female rats. accounting for 27 to 51percent of the radioacti vi ty in those samples. OCBH accountedfor 30 and 35 percent of the [14C] in the 0- to 4~-hour and48- to 168-hour samples from males, respectively, but only 6 and13 percent of the respective samples from female rats. Otheridentified metabolites. OC8H, OH-OCBP, OH-OCBH, and OCBA-g1ycine.were present in smaller amounts.

A zone of radioactivity that cochromatographed with analyticalDOE on TlC accounted for approximately 1 to 3 percent of thefecal radioactivity. However. HPlC analysis of this zone fromthe 0- to 48-hour fecal extract from male rats showed that only0.22 percent of the extract's [14C] could come from ODE; thebulk of the zone's [14C] was not identified. Unidentifiedfecal metabolites accounted for approximately 13 to 24 percent ofthe total radioactivity. In general, nonpolar metabolites(dicofo1, OCBP, FW-152, OCBH. and the 1I00P zone) accounted for alarger percentage of the fecal radioactivity at 0 to 48 hoursthan at 48 to 168 hours.

E. Urinary Metabolites: Ethyl acetate extraction removed means of80 to 82 percent and 95 to 99 percent of tne radioactivitypresent in urine from male and female rats, respectively(Table 4). Oi~ofol and DOE were not identified in urine. Theonly nonpolar metabolite, OCBP, accounted for less than 2 percentof the urinary [14C] in each group. Most of the urinary[14C], "therefore, was associated. with polar metabolites (OCBHwas shown by other techniques to be a minor component of the"OH-OCBP/OCBH zone). One major metabolite and numerous minormetabolites in urine from each group could not be ·identified.Together. these unidentified metabolites accounted for 45 to 57percent of the extractable urinary radioa.ctivity. These unknownswere more polar than the most polar identified metabolite, CHA.As with fecal metabolites, there was a large individual variation

11

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,~~,(gdJ)~~ Tabla 3.

~)Y; Distribution ot ~tabolitea in 'eoea Extraot, rro.

(Q) Hala aDd , ...la lata .ctar a SlD8la Oral Doaa at -e.Ol00tolo .~ Male Bats . 'e.le Bata~ _ 0-1&8 hb 1&8-168 hO 0-1&8 htl "8-168 heifr~- ,

lvereae • ot ~:c Doa.a : ?T() .....35 '* 3.6961.10 '* 12.9 31.96 * 9.25 31.95 * 5.6"lvereae.ot C 1n Methanol t 92.1 * 18.8 93.1 '* 16.8 96.8 * 18.1 86.3 '* 17.0lverase. aeoy.ery Sep-Pak Purit1 95.8 '* 1."9 91.8 '* 9.11 86.6 * 8.81 86.0 '* 16.6lverase • ot C 1n Sep-PaSt. Eluent-: ~

0100tol ~J1r1?J,:23' 2.07 .. 0.90' U_ .. U3 2.62 .. 1.__-

DCBP 2.97~2~ 2.13 * 0.62 2.89 * 1.26 2.51& * 1.49b

P1I-I52 36.6 .. 25'ilt!4J.6.6 .. 2M 51.0 * 25.0 27.1.. 11.3

DCBH 29.5- .. 20.6 ~~.3 5.87 .. _.68 12.9 .. 3.23

OR-DeBP 5.01 '* 2.681 9.9~~~ 10.1 * 9.81 22.8 '* 6.53

OH-DCBH/DCB.·Sljo1na 5.91 .. 6.03 10._ .. ~~~~08 .. 3.27 6.70 .. 3.39

._.' zonal< 2.63 .. 2.16J 0.88 .. 0.38 (/~~0.6_ 1.27 .. 1.26

untdentttied (tnoludea baseltne ~;1U~+ 3-10 moor zones) 12.8,* 9.50 21.1 '* 11.9 19.1 *(/It!J6p 23.6 '* 13.9

---Moan '* atandard deViation ~~.b Total ot 10. aa.plea trca 5 ani..ls choaen aa repreaentative aa.p.lea ~12~.o Total ot 8 a-.ples t~ 3 ani..la ohoaen aa repreaentative aa~les ~d Total,ot 10 aa.ples trca 5 ant..ls ohoaen aa repreaentattve aallPles 41'• Total ot 9 aa.ples rra. 3 ani..la choaenaa repreaentative sa.plea ~~t Soao DCBP and DDD residues are included in thia data ~~.&Some DCBP residues are inoluded in thia data ~.b Soao Dioorol residues are inoluded in this data .1 Soao DCB" residues are included in this data (no separation rrca OH-DCBP on tic) ~~.J Sc.e DDT and DbD residues are included in thia data ,IE Oata ara tro- a zooa wblcb cocbrcaatesrapbaa wlth DOE but was latar &hOwe not to ba ODE. See text ~

for detaUs. ' .... '1j

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~~ Dlatrlbutlon of Kotabolltee ln UriDe Extraotf fro-~Mol. and r...l. Rata Attar a 810&1. oral Doe. of -C-Dloofol

(Q~'Hale Rata Fe le Rata

~ 0-'18 bb 118-168 bO 0-'18 b'l' _8-168 bO

• of l_C Doae:& . ~~J?;;:19 16.33 '* 9.72 5.72 * 1._6 19._0 * 12.50

• of "C 10 8tbyl &ootato 8xtraot, 8O:~~j) 81.9 * 13.8 98.9 * 32.8 95.0 * 26.6

• of "C Extraot aaO, . lj~

DCBP 1.62 * 0.92 ~~.30 1.21 * 0.116 1.28 * 0.62OR-DcaP/DCB f 2_._ * 6._ 30 23.3 * 5.9- 22.1 * 9.04OlWlaIHlaIA' 5.3'* 2.1' 5.60 ~ 3._6 * 1.88 2.46 * 1.50

DCBA _.00 * 8.01 3.78 '* 0 2.26 * 0.96 1.31 * 0.81DCBA alloine/CHAf 24.7 7.32 *3.~.2 .1 * 9.-1 10.0 * 6.24

CHA 5.11 * 3.22 6.-3 * 2.60 * 1.91 5.65 * 0.07DCBl-e!yolnei 13.9 * 1.81 10.5 * 5.96 . ~1.3- 11.1 a 3.96unknown _tabol1te 12.2 * 10.5 11.2 '* 15.9 9.')JiP~ 11.2 * 11.8

unidentified (baaeline ije;ttv;>+ 5-1081__a) 33.2 * 13.8 29.' * 12.8 38.1 * 10.r~~ 15.6

• Mean '* Standard deviation W>41 .b Total ot 9 a.-plea analyzed fro. 5 ani..la taken as representative aubsa.ples ~. ~~o Total of 9 asaples analyzed fro. 3 an~ls taken as representative subsa.ple.d Total of 9 aaapl•• a..lyzedfro- 5 OD~la tak.n as rop.....totl•• aubaaopl.a ~~~e Total of 9 sa.ples analyzed froa 3 ani..ls taken aa representative aub.a~les ~~ .f Tboa...tabolit.a were lna.parabl. on ...t tlo plataa; • reported rapre.ent. the aWl of botb c"poun •~j)&Tboa. data were son.rated fro- tlc analya08 ln wblob CHi andDCBA-slyolne were a.parabl.. ~~

o....~

c:I£uo,...:'

in the quantities of metabolites; urine samples from rat Nos. 11,12, and 14 were consistently high in unidentified metabolites.

F. Metabolites in Fat: Petroleum ether extraction of adipose tissueremoved approximately 109±36.5 (93 to 143) percent of thesample radioactivity. The majority of the radioactivity in fatwas associated with the parent compound; 83 percent in males and89 percent in females (Table 5). FW-152 was the major dicofo1metabo11 te in fat, accounti ng for 7.8 and 3.8 percent of theextractable radioactivity in males and females, respectively.Only 3.5 and 2.5 percent of the extractable [14C] in males andfema'les, respectively, was associated with 'unidentifiedmetabolites.

Similar results were obtained by Analytical 8io-Chemistry on fatsamples from the odd-numbered animals. Recoveries of extractableradioactivity ranged from 71.3 to 284 percent for individualsamples; the study authors felt the high recoveries for somesamples were due to desiccation. Purification of the extractsresulted in almost no loss of radioactivity; recoveries averaged

.96 percent. Di cofo1 accounted for 92. 8±3. 9 and 92. 3±2.9percent of the adipose (14C] in .males and females, .respectively.FW-152 accounted for 3.06±2.1 and 2.07±1.0 percent .in malesand females, respectively. DOE was detected in only one of sevensamples, from male rat No. 3, at 0.2 percent of the extractable(1 4C]. Analytical 8io-Chemistry estimated elimination halflives for dicofol and FW-152 in fat to be 30 and 24 hours,respectively.

G. Metabolites in Liver: Re'su1ts are presented in Table 6. Averagerecoveries of hepatic (14C] were 87.2 and 67.3 percent aftermethanol extraction in males and females, respectively. The bulkof the extracted (14C] (81-91 percent) partitioned intopetroleum ether. Only 13 to 19 percent partitioned into ethylacetate. Dicofol was present at less than 5 percent of extract­able hepatic radioactivity. FW-152 was the major hepaticresidue, accounting for approximately 65.3 and 74.5 percent ofthe total extractable (14C] in males and females, respectively.The ADDE ' zone from the petroleum ether extract was used in anisotope dilution experiment. After a single purification step,85 percent of the zone's (14C] did not cochromatograph withanalytical DOE.

Analytical 8io-Chemistry rep.orted similar results. Dicofolaccounted for 3. 98±3. 7 and 3.07±1 .7 percent of the extract­able hepatic radioactivity in males and females, respectively.FW-152 accounted for 81.5±18.2 and 82.7±1.2 percent in malesand females, respectively. DOE was detected in the livers of twofemale rats (Nos. 13 and 15). at 0.34. and 0.25 percent of theextractable (14C], respectively.

14

::0•

r-1»0\IoN

....•og....o......~rn/> Tabla 5"

~~O Diatrlbutlon ot Metabolites in Fat Extraots (ro.~~le and Feule Rats Atter a Slnale Oral Dose ot 1 e':'Diootol

~ Halesb Fe_leso</?J(gg/) 0-"8 h "8-168 h 0-"8 h 118-168 h

Average S ot l11e Dose: </~~ ~ 12.2 3.62 ~ ".59 83.3 ~ 23.0 22.6'* 1".9

l"eraga. "Cin pat gther Ext....ct: v9f~}'.~ 9~.6 9M ~ 1.50 101.1 ~ 11.5

l"er_ • ot I~C Extract ..0: &'1!i?3~Diootol .fi.58 89.2 * 11.32DCBP 2. 1 2.80 * 0.68FW-152 1.11 ~ . 3.83 * 2."6

..... DCBH 0.13 ~ o. '47 0.18 * 0.15U1 OH-Dmp 0."3 * 0.3Hr,CCU 0.30 * 0.29

<II-DC8H 1.31 ~ 1.95 ~ -((J> 0.93 * 1.09-ODE- zoned 0.63 ~ 1.01 0.33 * 0.591IIl1_Utled 3.53 ~ 3.~6 ~~ 2.~5 ~ h~2

~-c-c 3 _laa analysed (2 t 0-'18 h SrGdP and ltl'Olll 118-168 h croup)·r~h Total ot ~ _laa analysed (2 t aach aroup) ~~° Mean ~ Standard DeViation ~~d Data ..ea _rated trca a zena ....lch cccb......tc.raphecl ..itll DIlIl but wa. .hctm not to be BOB.· •••.•.•• axti tor data~l. 4~~

~ ~~kh~ v~

, ..., ))" ,;.I),

....0'

) )I ) , "

, );.(~'j~~,,;>\

;J~f».) Table 6

. Oi;"'~g,., taboUt.•• 1n U".r Extraot.. rroa ...1••nd , ...1•••t.. Att..... S1nal. Oral ~. or I"C..D100tol

02 ,'..... "'1•••h lt , ..1••at..-'-';;;'S~ ,"', 0....8 b ..8-168 b 0....8 b "8-168 h

' ••r y( I"C Ood: /,r' 2.8) * 0.9" 0.28 * 0.11 2.0) & 0.)0 0.69,0.28' ••r elntleOlllat.r.~:))' 86.1.27.8 90.8 1).6,25.1 60.9 a 16.1'wer-.•• ::C ln Pet. It.b.r·'~''',l~lonfl 1).6. 1.85 11.11 90.5 • 0.07 II.) •.9.26ber.... C Pet It.her I.tr.at.( ••-; 1/1 4

dleorol ' ,j:.::.. ./0, 2.97 • 1.00 ).70 a 2.• )04

DaII' • 1/ ! ,?1? " 2.00 • 0.57_ 2.21 • 1.110'''-152 <,/,1'/.'1',72.8.8.19 71."'.26DaIII v I ~,-y: 6."~ * 2.'" '.') • 0.61OH..DatP ~ Ij 2. 1* 0.96 II.)) • 2 ....OH-DaUI " ~ .08 1.115 • 0.1)DaaI"al,ol...1 '). .~., 1.55 a 1.91eDllle aone O. ;l W,..... 0.11) & 0.29unld.nUrllld 5.117. ~)l 11.61 * 1.56

b.r e 1n ItO&o Partition 1).5 * ).19 ' ....~~~? 12.5 * 2.19 16,.9 • 1.9'' ••r I C ItGAo Part.1Uon a.: ' ::v-~

dloorol 0.95 • 0.)54 q; ).804Dell' 0.60 a 0.)0 1.2) • O.n1V-152 21.9 • 10.2' ?1~ ".2 * 11.9'

16.6 a 1.1)· <::J 52."DaNI 2.15 • o.o~ V')~(l~ • 1016,oei-DQIP 2.0 * 0.0 ' l(\ ~~b • 1.12

11.9 • 1I.IOa ~d~'DOII-al,oln 5.117 • 5.61 ). ,'. i)71LCllIOII..DQNI· 5.)7 * 5.85 1. t .. ~~~'"DOll 2.11 • 0.70 ).8'$ ,) 8 ~all ).60 • ).22 2."8 • /hUllknown 2...1 * 10.) 1).5 * 5. ///••_ttl......." ... '-11 .._ .....1 '2.0. 12.0 26.6. 7.7) ~ ~

• ..... * Standard De.lation Va;j?J~It Tot..l ot ) 1......l'alld (2 trOll 0 ..... b aroup .nd I troa "'-168 b arGUlt) .....,...aont..U........1• Total ot II 1•••nal,alld (2 tree ..ob aroup) •• r.'.....ntaU.........1. p• DCIP ....1duea l_lUllIld In data (lna.par.bl. troa eUootol on tlo) v• Oaa eet ot cIIlta l_lude. DalH r••ldue. Una.par.bl. troa PM-IS2 on Uo), Dloorol 1 1_1\Mtecl In elat.a Una.,.rabl. troa '''-152 on tlo) ~• flal. data "1Id 'roe ••par.". anal,... uncI.r 1••• pol.r tlo ooMUlona ..lob ..par_tlld flf-1S2 ..... cl1oorol ~~•••

or DaHl and OH..DaIf• to _ ,.. '"'''''' , , - , , U. _.U - :-j~J2a Data 1_ trOll a aone ""lob ooobroaatOlr.pbacI wlt" DDt: IMIt • aot to DCII. S.. t ••t. tor clat.U.. WISource, CSl p.70. ~Jf

H. Metabolites in Plasma: Results are shown in Table 7. Extractionof plasma [14C] varied considerably among groups .. Less radio­activity was extractable from 48- to 168-hour samples than from0- to 48-hour samples. Extractable [14C] was associated mainlywith polar metabolites, most of which remained unidentified.

13. STUDY AUTHORS i CONCLUSIONS/QUALITY ASSURANCE MEASURES:

A. Following a single oral dose of [l4c]dicofol at 50mg/kg toma 1e and fema 1e rats, 85. 7±13. 2 and 102. 5±25.2 percent of thedose was accounted for up to 168 hours postadministration inmales and females, respectively. Feces was the major route ofexcretion. .Levels of [14C] were highest in tissues after 48hours and decreased by 168 hours after dosing. Adipose tissuecontained the highest levels of radioactivity, with concentrations1n females approximately 2.5 to 4.5 times those in males. Con­centrationsof [14C] in liver and whole blood were similar formales and females at 48 hours postdosing.

In spite of its close structural similarity to DDT, dicofol ismetabolized in rats by a unique pathway (Figure 1), quite. distinctfrom DDT metabolism (Figure 2). Dicofol is not converted toDOE. The small amounts of radioactivity asso.ciated with theIIDDE" zone are most 1ikely an artifact of the chromatographicmethods used or from the 0.01 percent DOE present in the testmaterial. Dicofol is converted to a number of polar metabolitessuch as FW-152, DCBH, OH-DCBP, CHA, and DCBA-glycine, which areexcreted through the feces and urine. The unidentified metab­olites were most likely conjugates or glucuronides of dicofolmetabolites. [14C]dicofol is eliminated from rat.s two to tentimes faster than is [14C]DDT. .

B. A signed quality assurance statement dated March 24, 1986 wasincluded for the work performed by Analytical Bio-Chemistry. Nostatement was present for the work performed by Rohm and Haas.

14·. REVIEWERS I DISCUSSION AND INTERPRETATION OF STUDY RESULTS:

This study was designed to produce samples of urine, feces, liver,fat,and blood that. contained sufficient radioactivity for metaboliteanalysis. Therefore, it does not meet guideline requirements forexcretion or tissue distribution studies. However, it is acceptableas a study to demonstrate the metabolism of dicofol.

The data on excretion and tis.sue distribution of [14C] showed largeindividual variation, with -some standard deviations exceeding thecorresponding means. Female rat No. 14, in particular, deviated fromthe other animalS, showing relatively high [14C] "levels in urineand low [14C] levels in feces. Additionally, [14C] levels intissues, especially fat and blood, were extremely low. The study

17

::uID

t-iID()

:l"::s.....()

l»~

r1»0\

•oI\)

0.29 * 0.130.11 "& 0.29

I J/,

(' 'vI'~ f':--'~>J--~\k ,

'J))) l'j 1/''0/'

.~' L;.:> Table 1 .

(,r' ((~~;A Distribution of Metabolites in Blooo Plasma {"OlD'-.._'//',.) Male and Female Rats lfte.. a Single Oral Dose or 1 C-Dicofol./ 0'.

1/., ~

v ,)«/ i)" Males-. Femalesa.._~/,yJ> 0-48 h 48-168 h 0-48 h 48-168 h

i/ / ~:1)'{/(~d1 ~ 0.38 0.11 "& 0.0"

j! ,'>1/ , .

15,~·t-,1A1~:,-· 33.8"& 8.11 15.1 "& 18.9 55.9 "& 12.4

J t~V)11.8 * 9.5~~50 * 1.41 14.3 * 1.06 18.3 ~ 0.35MO, to 3.82 0!!,,-S"&. 0.35 3.55 * 0.21 2.45 * 0.92

2.80 'l. ~/~~1.35 1.50 "& 0.99 3.55 * 1.484.55 * 0.64 3. ~. ~ 4.45 * 1.18 3.00 * 0.851.20 "& 5.52 t 5.30 "& ~(n\ 0.15"& 0.35 0.15 "& 0.21

9.20 ".65 "& 2.1~\. ~~45 * 0."9 2.25 "& 1.6331.9 ..... '-,:::,>OJfl; 1.10 9.90

29." "& 11.1 11.3 "& 8.3 ~j!~4 64.8 * 1.35'1/',;<.t:;1 r,

~1l/1J//0<I~,g:"~~,~

~~~~ "

~//'"~u-.J-J?

~4

; )I).

'.If

b Hean * Standard Deviation

e These .etabolites were insepa..able under .the conditions used

t Includes residues 'of unidentified metabolites (inseparable frOID DeBl)

d Sc.e OH-DCBP residues are included (insepa..able f ..om DCBH by t~o)

Averase S ot 14C Dose:

Averaae S ot l ..C in Extract:

lveraae S or'l"c Extract asb:DCBP/Dioorol/FW-152°DamOII-DCBP

~ OII-DCBR/CBl­CICI DCBA

DCBl-alyoineunknownW11dentitied (inol',ldes baseline

+ "-6 .inor zones)

° This data represents total residues or DCBP, dicorol and FW-152 (1naeparable by tlc)

-Total ot" saaples analyzed, 2 from eaoh aroup

authors did not discuss this point. One possible explanation couldbe that the animals h.ad diarrhea associated with cor.n oil dosing,causing a decrease in the amount of [14C]diCOfol absorbed and ofthe mixture of urine and feces in the collection funnel. Anotherless likely explanation could be that this rat metabolized theabsorbed dicofol much faster than the other rats, resulting in higherurinary versus fecal excretion of polar metabolites and lower tissuestorage of less polar compounds. This reasoning is also supported bythe results indicating that tissues and excreta from rat No. 14 wereconsistently higher in unidentified polar metabolites than the otheranimals. Consequently, results from this animal were not included inthe mean values.

The authors used sufficient analytical methods to demonstrate thestructure of the identified metabolites. Results from Rohm and Haas'laboratory agreed with those of Analytical Bio-Chemistry. Theidentified meta.bolites all had the .original dicofol oxygen intact,strongly supporting the authors' conclusion that dicofol metabolism

. is totally different from DOT metabolism. Analyses by both labora­tories of samples for residues of ODE, particularly in fat, indicatedthat traces of this compound were present in some of the samples.However, the amounts detected were extremely low and may have beendue to the ODE present in the [14C]dicofol or an artifact of thechromatography. The formation of DOE could theoretically occur bydehydration, i.e., removal of H20 from FW-152. However, for thisreaction to occur, it would require the removal of a positive hydroxylradical, an unlikely process in biological systems. Therefore, weagree with the authors that despite the structural similarity betweendicofo1 and DOT, they are metabolized by different pathways; dicofolmetaboli sm does not produce s ignHicant amounts of ODE. However, itshould be pointed out that these experiments were conducted with verypure dicofo1. The concern therefore would be that technical Ke1thanemay contain larger amounts of ODE and/or DOT as impurities; DOT isreadily metabolized to DDE, which then accumulates in fat.

Item 15--see footnote 1.

16. CBI APPENDIX: Appendix A, Analysis of liver and Fat: AnalyticalBio-Chemistry's Methodology, CBI pp. 401-40B; Appendix B, StudyProtocol, CBl pp. 1.08-113.

19

APPENDIX A

Ana 1ys is of Li ver and Fat:Analytical Bio-Chemistry1s Methodology

(C81 pp. 401-408)