Reproductive Toxicity of Di-n-butylphthalate in a Continuous BreedingProtocol in Sprague-Dawley RatsRobert N. Wine,1 Ling-Hong Li,1 Leta Hommel Barnes,2 Dushyant K. Gulati,2 and Robert E. Chapin11National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 USA;2Environmental Health Research and Testing, Lexington, KY 40503 USA

The esters of o-phthalic acid are employedextensively in the manufacture of plasticswhere they are used to impart flexibility.Since they are not covalently linked to theplastic polymer, they can leach from thematrix, providing opportunity for wide-spread exposure [see Thomas and Thomas(1) for review]. The phthalate ester di-n-butylphthalate (DBP) is used in the manu-facture of consumer products as diverse asnail polish, insect repellents, and denturebase material (2-4). DBP has recently beenfound to have clinical applications as well,selectively eliminating tumor cells frombone marrow (5,6).

DBP has been previously characterizedas a developmental and reproductive toxi-cant in the rat (7-12). Comparable oraldoses of DBP given to juvenile and adultmale rats produces a testicular lesion char-acterized by early sloughing of germ cells,vacuolization of the Sertoli cell cytoplasm,and testicular atrophy (7,10). It has alsobeen shown that the testicular toxicity ofphthalates is age dependent, with immatureanimals being more sensitive than matureanimals (13,14). However, the reproductivetoxicity following exposure during develop-ment has not been addressed. Although no

data are available specifically on the trans-fer of DBP across the placenta, studieswith other phthalate esters show that thesecompounds readily cross the placenta ofrats (15-17) and that exposure of lactatingdams to another phthalate, di (2-ethyl-hexyl) phthalate (DEHP), could lead toDEHP and mono (2-ethylhexyl) phthalate(MEHP) exposure in the suckling rat pups.Because DBP is lipid soluble, as are DEHPand MEHP, one might predict a certaindegree of lactational transfer of DBP.

Recently, Jobling et al. (18) reportedthat DBP could reduce the binding of estro-gen to the estrogen receptor and stimulatetranscriptional activity These authors calledfor further in vivo data before any firm con-clusions as to the estrogenicity of DBPcould be drawn. Review of archived chemi-cal studies generated with the NationalToxicology Program's ReproductiveAssessment by Continuous Breeding(RACB) protocol revealed an earlier studythat examined the reproductive toxicity ofDBP in Sprague-Dawley rats. If the findingsof Jobling et al. (18) are correct and DBPcan act as an estrogen, then one wouldexpect to see greater reproductive effects insecond generation animals (195) under the

RACB protocol. This is expected in theRACB design because the Fo rats are exposedonly as adults, the F1 rats are born to moth-ers that are treated during gestation and lac-tation, and the F1 animals themselves aretreated during maturation to sexual maturityand through mating. The results of this DBPRACB study are presented below; the resultsshow that structural defects are seen in thesecond generation rats that were not foundin the first.

MethodsGeneral. This study was conducted usingthe National Toxicology Program's RACBprotocol for which the detailed methodshave been previously described (20,21).Briefly, the protocol is composed of foursegments, or tasks. Task 1 is a 14-dayrange-finding study that is used to set threedose levels used in Task 2. Task 2 is the 14-week continuous breeding phase, generat-ing up to five litters per pair. Task 3 con-sists of crossover matings between treatedand control Fo animals to determine theaffected sex, and Task 4 assesses the fertilityof the last litter (F1) born during continu-ous breeding (Task 2).

Chemical. DBP was obtained fromChem Central via Midwest ResearchInstitute (Kansas City, MO, Lot/Batch # L-121 1-83/02). The purity of DBP used inpreparation of the feed formulations wasgreater than 99% as determined by gaschromatography.

Dosed feed formulations. DBP wasblended into powdered NIH-07 (ZeiglerBros., Gardeners, PA) feed on a weight-to-

Address correspondence to R.E. Chapin, NationalInstitute of Environmental Health Sciences, P.O.Box 12233, Mail Drop A2-02, Research TrianglePark, NC 27709 USA.This work was conducted under contract NO1-ES-65142 to the National Institute of EnvironmentalHealth Sciences. The authors are indebted to BradCollins and Tom Goehl for assistance in procuringand characterizing the DBP, to Jerry Heindel forsupport and encouragement, and to Paul Foster andKim Treinen for stimulating discussions. The statis-tical support was provided by Analytical SciencesInc., Durham, NC, and chemistry support was pro-vided by Midwest Research Institute and ResearchTriangle Institute under contracts NO1-ES-45060and NO1-ES-45061, respectively.Received 20 August 1996; accepted 18 September1996.

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weight basis. Animals in the control groupreceived undosed NIH-07. Each dose levelwas independently formulated and pre-pared at least every 3 weeks and storedfrozen. Dosage formulation studies indicat-ed that DBP blended with feed at a con-centration of 0.5 mg/g was stable for 3weeks when sealed at -200C and for 7 dayswhen open to air and light at room tem-perature. Thus, dosed feed was stored inthe dark and refrigerated and was changedoff the cages every week.

Animals. VAF Crl:CD BR outbredSprague-Dawley albino rats were purchasedfrom Charles River Breeding Laboratories(Portage, MI). During quarantine, represen-tative animals were sacrificed and their seraevaluated for antibodies against nine rodentviruses and Mycoplasma pulmonis (CharlesRiver Professional Services, Wilmington,MA) All sera were negative for viral antibod-ies. Animals were quarantined separately forapproximately 3 weeks for Task 1 and 2weeks for Task 2; rats were 70 days of age atthe start of both Tasks 1 and 2. Histo-pathologic evaluations were performed onrepresentative animals during quarantine tocheck for signs of infectious disease.

Male and female rats were housed twoper cage by sex during quarantine and the1-week pre-mating period on SaniChipbedding (P.J. Murphy Forest Products) insolid bottom polycarbonate cages withstainless steel wire-bar lids. Deionized waterand rodent meal feed (NIH-07 diet; ZeiglerBros.) were provided ad libitum.The ratswere subsequently housed as breeding pairsor individually at 23 ± 2°C with a 14:10-hrlight:dark cycle. All housing and procedureswere performed in accordance with theNIEHS guidelines for the humane use ofanimals in research.

Study design. DBP was administered viafeed to CD Sprague-Dawley rats. In Task 1,the dose-finding segment of the study, dosesof 0.0 (control), 0.1, 0.5, 1.0, 1.5, and 2.0%(w/w) DBP were tested using eight animalsof each sex per group. Endpoints for Task 1were dinical signs of toxicity, body weight,and food consumption.

In an optimal study, the highest doseduring Task 2 should be set so as not todepress weight gain by more than 10% orresult in greater than 10% mortality. If acompound is a reproductive toxicant, themiddle dose should elicit reproductiveeffects with little or no systemic toxicity inevidence, and the lowest dose should pro-vide a no-effect level. Based on the resultsof the Task 1 pilot (data not shown), con-centrations of 0.1, 0.5, and 1.0% DBP inthe feed were selected for Task 2.

Task 2, the continuous breeding phase,used a control group consisting of 40

male/female breeding pairs and three dosegroups with 20 breeding pairs per group.The endpoints for Task 2 were clinicalsigns of toxicity, parental body weight andaverage consumption of feed during repre-sentative weeks, fertility (the proportion ofcohabited pairs producing any live pups),the number of litters per pair, the numberof live pups per litter, the proportion ofpups born alive, the sex ratio of live pups,and the pup body weights immediatelyafter birth. The pups in the first four litterswere removed after birth, counted,weighed, sexed, and killed without necrop-sy. The last litter, born during the holdingperiod following the continuous breedingphase, was reared by the dam until weaning(day 21 after birth); at this time, treatmentof the Fl animals was initiated by the sameroute and at the same concentrations asconsumed by their parents in Task 2.These animals were used for assessment ofsecond generation fertility in Task 4.

During Task 2, if an effect on fertilitywas detected, a 1-week crossover matingtrial, Task 3, was performed to determinethe affected sex. In this study, this was per-formed after the last litter of Task 2 wasweaned. The mating trial consisted of threegroups of 20 pairs each: control males xcontrol females, control males x 1%females, and 1% males x control females.Endpoints for Task 3 were the same asTask 2, with the addition of checking forthe presence of a vaginal copulatory plug orsperm. After the litter in Task 3 had beendelivered, evaluated, and disposed of, theFo females were subject to vaginal lavagefor 12 days to evaluate estrous cyclicity; thecontrol and high dose Fo adults were thenkilled by CO2 asphyxiation and subjectedto necropsy. Necropsy endpoints wereorgan weights and histopathology, bodyweight, epididymal sperm motility (count-ed visually), sperm morphology, and spermcount. For histopathological evaluation,selected organs were examined after fixa-tion in 10% neutral buffered formalin orBouin's fixative (ovaries) and embedded inglycol methacrylate (testis) or paraffin.Sections were stained with periodicacid-Schiff (PAS) and hematoxylin (testis)or hematoxylin and eosin according tostandard procedures.

Task 4 assessed the growth and fertilityof Fl animals. The last litter from Task 2was reared, weaned, and held until matingat postnatal day 88 ± 10. During matura-tion, siblings were housed by sex at a maxi-mum of two per cage and received the samechemical treatment as their parents. Uponreaching sexual maturity, 20 each nonsib-ling F1 males and females within the sametreatment groups were housed in pairs for 7

days and then housed individually untildelivery of a litter. The endpoints for theTask 4 mating trial was the same as in Task2, with the addition of checking for thepresence of a copulatory plug and vaginalsperm. At the end of Task 4, F1 animals inall dose groups were killed by CO2 asphyxi-ation and subjected to necropsy with thesame endpoints as in the Task 3 necropsy.

Statistics. When data were expressed asa proportion, such as the fertility, mating,and pregnancy indices, the Cochran-Armitage test was used to test for a dose-related trend (22). Each dose group wascompared to the control group with a X2test (23). In Task 3, where the animalswere cross-mated and dose groups did notrepresent increasing dose levels, a X2 testfor homogeneity was used to test for anoverall difference in fertility among groupsfor pairwise comparisons.

The number of litters and the numberof live pups per litter were computed on aper-fertile-pair basis and treatment groupmeans were determined. The proportion oflive pups was defined as the number ofpups born alive divided by the total num-ber of pups produced by each pair. The sexratio was expressed as the proportion ofmale pups born alive divided by the totalnumber of pups produced by each pair. InTask 2, dose group means for these andother parameters were compared to thecontrol group with Shirley's test (24,25).When a trend was present, Jonckheere'strend test (26) was used, otherwise Dunn'stest (271 was applied. In Task 3, parame-ters were tested for overall differences usingthe Kruskal-Wallis test (28), and multiplecomparisons were made using Dunn's test.

To remove the potential effect of thenumber of pups per litter on the averagepup weight, an analysis of covariance(ANCOVA) was performed (29) using theaverage litter size, including live and deadpups, as the covariate to produce an adjust-ed live pup weight. Least squares estimatesof dose group means, adjusted for littersize, were computed and tested for overallequality using an F-test and pairwise equal-ity using Dunnett's test (30). To evaluatepotential sex differences, these analyseswere performed on males, females, andboth sexes combined.

Absolute body and organ weights andorgan weights adjusted for body weightwere analyzed by Shirley's or Dunn's test,while dose-related trends were identified byWilcoxon's test in Task 3 or Jonckheere'stest in Task 4.

Vaginal cytology data were analyzedusing a multivariate analysis of variance(ANOVA) (31) to test for the simultaneousequality of measurement across dose levels.

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Before running the ANOVA, an arcsinetransformation was performed to bring thedata into close conformance with normali-ty assumptions.

Group means, standard deviations, andstandard errors were calculated for all dataand for each sex. Jonckheere's test was per-formed to look for increasing or decreasingtrends. Significance among dose groupswas then calculated using Shirley's test if atrend was evident or Dunn's test if notrend was evident.

ResultsContinuous breeding (Task 2).Representative dosage formulations fromTask 2 were 99-106% of the target con-centrations. The average daily feed con-sumption was similar for control and DBP-dosed animals. During the first 14 days ofTask 2, body weight gain was significantlylower only in the high-dose females (2%),compared with a 7% increase for controlfemales. This resulted in significantlyreduced body weights after each litter andat necropsy for these high-dose females.During the course of the Task 2 continu-ous breeding phase, body weights werewithin 10% of the controls except for high-dose females at week 17 (the end of cohabi-tation) when they were 11% less than thecontrols. Over the duration of continuousbreeding, there were no clinical signs oftoxicity noted during the twice-daily healthsurveillance.

Based on body weights and food con-sumption, the average calculated dailyintakes of DBP for the 0.1, 0.5, and 1.0%dose groups were 52, 256, and 509 mg/kgbody weight for males and 80, 385, and794 mg/kg for females, respectively.

DBP treatment did not affect fertilityor the average number of litters per pair,but it did dose-dependently reduce thenumber of lives pups per litter (Table 1).In addition, live pup weights (bothabsolute and adjusted for litter size) weresignificantly decreased in the middle andhigh dose groups. The cumulative-days-to-litter values were similar for control andtreated animals.

Dam weights at delivery were signifi-cantly decreased at each litter of the highdose group (by <10%). During nursing ofthe final litter, dam weights were signifi-cantly decreased at postnatal day 21 in thelow dose group (by 7%), at postnatal days14 and 21 in the middle dose group (by=6%), and at all time points in the highdose group (by =10%; data not shown).

Crossover mating (Task 3). Sinceadverse effects on reproduction wereobserved in Fo rats, the crossover mating(Task 3) was performed on Fo animals to

Table 1. Reproductive performance of first generation breeding pairs

Dose groupaReproductive parameter 0% 0.1% 0.5% 1% TrendAverage litters per pair 4.8 ± 0.1 (40) 5.0 ± 0.1 (20) 4.9 ± 0.1 (19) 4.9 ± 0.1 (20) p= 0.322Live pups per litter 12.9 ± 0.2 (40) 11.9 ± 0.3 (20)* 11.0 ± 0.5 (19)* 10.7 ± 0.4 (20)* p < 0.001Absolute live pup wt (g) 5.96 ± 0.06 (40) 5.99 ± 0.06 (20) 5.74 ± 0.07 (19)* 5.38 ± 0.10 (20)* p < 0.001Adjusted live pup wt (g) 6.04 ± 0.06 (40) 5.99 ± 0.08 (20) 5.66 ± 0.08 (19)* 5.30 ± 0.08 (20)* p < 0.001

aData are mean ± SE (number of breeding pairs).*p < 0.05 when dosed groups are compared to controls.

Table 2. Mating trial to determine affected sex: fertility and reproductive performance

Treatment groupaControl male x 1% Male x Control male x Overall

Reproductive parameter control female control female 1% female differenceMating indexb 16/19 (84%) 19/20 (95%) 16/19 (84%) p= 0.487Pregnancy indexc 12/18 (67%) 18/20 (90%) 15/19 (79%) p = 0.212Fertility indexd 12/15 (80%) 18/19 (95%) 15/16 (94%) p = 0.303Live pups per litter 12.7 ± 1.3 (12) 13.4 ± 0.6 (18) 12.9 ± 0.7 (15) p = 0.934Adjusted live pup wt (g) 5.96 ± 0.16 (12) 6.16 ± 0.12 (18) 5.28 ± 0.14 (15)*' p < 0.001

aData are mean ± SE (number of breeding pairs).bNumber of females with plug/number of cohabiting pairs (% with plug).cNumber of fertile pairs/number of cohabiting pairs (% pregnant).dNumber of fertile pairs/number of females with plug (% fertile).*p<0.05 when dosed groups are compared to controls.'Treated groups differ at p<0.05.

determine the affected sex. For Task 3,control animals were randomly paired withother control or high dose (1.0%) animalsfor 7 days. Among the three groups (con-trol male x control female, 1% male x con-trol female, and control male x 1%female), there was no overall differencewith respect to mating, pregnancy, or fer-tility indices (Table 2). Live pup weightadjusted for litter size was significantlyreduced in the control male x 1% femalegroup. Treated females weighed =11% lessthan controls during Task 3 and consumed=12% less feed. There were no significantclinical signs of toxicity noted during Task3. Although two females died, one in thecontrol group and the other in the highdose group, neither of these deaths wereattributed to DBP exposure.

At the end of Task 3 (4 weeks aftercohabitation), male body weights wereunchanged, but high dose females weighed=14% less than controls. At necropsy ofthe controls and high-dose rats, organ-to-body weight ratios for the liver and kidneyswere significantly increased in both sexes inthe high dose group (Table 3). The grossstructure of the reproductive system wasunchanged by DBP consumption, andsperm studies showed that sperm concen-tration, motility, percent abnormal forms,and testicular spermatid head counts werenot affected by 1% DBP consumption.DBP caused no apparent changes in aver-age estrous cycle length (control length4.20 ± 0.07 days; n = 33) or progression

through the stages of the cycle (data notshown).

Secondgeneration fertility (Task 4). Thefinal Task 2 litters of all dose groups werereared and exposed to the same concentra-tions of DBP between weaning and necrop-sy as their parents were. Before weaning, Flpups were counted and weighed on postna-tal days 0,4, 7, 14, and 21. Survival was notaffected, but live male and female pupweights in the 1.0% dose group were signifi-cantly (10-15%) lower than controls atpostnatal days 0, 14, and 21.

During rearing (at =postnatal day 46),three F 1 males in the high dose group werefound to have small and malformed pre-puces and/or penises and were without pal-pable testes. Just prior to mating (postnatalday =80), one of these males had palpabletestes. No such abnormalities were notedfor the control males.

At 88 (±10) days of age, F1 rats werecohabited for 1 week. Feed consumptionvalues during the week of cohabitationwere =12% lower in the 1% DBP dosegroup compared to the control group.Mating, pregnancy, and fertility indices inthe 1.0% group were significantly lower:only 1 litter was born to 20 breeding pairsin the high dose group, compared with 19litters to 20 breeding pairs in the controlgroup (Table 4). While there was nochange in the numbers of live pups per lit-ter, absolute and adjusted live F2 pupweights were =6-9% lower in all treatmentgroups. The average number of days to lit-

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ter and average dam weight at delivery werenot affected by DBP consumption.

At the end of Task 4, all F1 animalswere weighed, killed, and necropsied. Thehigh dose males and females weighed=8-14 % less than their controls (Table 5).Body weights were unchanged in the mid-dle and lower dose groups. For males, onlythe 1% treatment group showed a signifi-cant increase in liver weight, while kidneyweight was increased at both the middleand high dose levels. F, females showed nosignificant change in liver or kidneyweights. F1 ventral prostate, seminal vesi-cle/coagulating gland, and right testisweights all were decreased in the 1.0%treatment group, while average ovaryweights remained unchanged (Table 5).

Epididymal sperm counts and testicularspermatid head counts were both reducedin the high dose group (Table 5).Epididymal sperm concentration values(expressed as 1,000 sperm/mg caudal tis-sue) in high dose rats were 51% those ofcontrol animals. Likewise, both the totalspermatid heads per testis, a measure of tes-ticular output, and total spermatid headper gram of testis, a measure of spermato-genic efficiency, were decreased in 1.0%DBP treated males.

Histopathologic examination of select-ed organs was performed on 10 representa-tive males from the control, 0.5%, and1.0% groups and is summarized in Table6. Three males (of 10 total) in the 0.5%dose group showed degeneration of semi-niferous tubules, compared with 1 control,and 8 males in the 1.0% group. In addi-tion, 7 of the 10 males examined in the1.0% DBP treatment group demonstratedapparent interstitial cell hyperplasia.Finally, half of the rats (5 of 10) observedin the high dose group had either underde-veloped or otherwise defective epi-didymides.

DiscussionThe results from the present RACB studyindicate that DBP is a developmental toxi-cant in that live pup weights (absolute andadjusted) were significantly decreased inboth the 0.5% and 1.0% dose groups inTask 2 (Table 1) and in control males x1.0% dosed females of Task 3 (Table 2).Decreased fetal weight is considered anearly indicator of developmental toxicity(32) and has been observed in Wistar ratstreated with 600 mg DBP/kg by gavage ongestation days 0-21 (33). Similar resultswere observed by Reel and Lawton (34) inan earlier DBP RACB study using Swissmice. Although general toxicity of DBP (asignificant increase in liver and kidneyweights in both males and females and

Table 3. Mating trial to determine affected sex: necropsy outcome

Dose groupOrgan 0% 1% DifferenceMaleAbsolute body wt 683.6 ± 7.6 (40)8 656.8 ± 15.0 (20) p<0.154Adjusted liver wt 37.6 ± 0.63 (40) 43.4 ± 1.1 (20)* p<0.001Adjusted kidney wt 6.6 ± 0.11 (40) 7.3 ± 0.18 (20)* p=0.004FemaleAbsolute body wt 379.0 ± 9.6 (38) 326.4 ± 6.5 (19)* p<0.001Adjusted liver wt 34.0 ± 0.62 (38) 38.9 ± 0.69 (19)* p<0.001Adjusted kidney wt 7.0 ± 0.10 (38) 7.6 ± 0.14 (19)* p=0.002

aMean ratio (mg/g body weight) ± SE (number of animals).*p<0.05 when dosed groups are compared to controls (Wilcoxon test).

Table 4. Mating, fertility, and reproductive performance of second generation breeding pairs

Dose groupaReproductive parameter 0% 0.1% 0.5% 1% TrendMating indexb 20/20 (100%) 19/20 (95%) 18/20 (90%) 6/20 (30%)* p<0.001Pregnancy indexc 19/20 (95%) 17/20 (85%) 17/20 (85%) 1/20 (5%)* p<0.001Fertility indexd 19/20 (95%) 17/19 (89%) 17/18 (94%) 1/6 (17%)* p<0.001Live pups per litter 14.0 ± 0.8 (19) 15.5 ± 0.4 (17) 12.8 ± 0.8 (17) 13.0' p=0.233Absolute live pup wt (g) 5.97 ± 0.11 (19) 5.60 ± 0.09 (17)* 5.60 ± 0.09 (17)* 5.00# p=0.020Adjusted live pup wt (g) 5.98 ± 0.08 (19) 5.69 ± 0.09 (17)* 5.50 ± 0.09 (17)* p<0.001

aData are mean ± SE (number of breeding pairs).bNumber of females with plug/number of cohabiting pairs (% with plug).cNumber of fertile pairs/number of cohabiting pairs (% pregnant).dNumber of fertile pairs/number of females with plug (% fertile).*p<0.05 when dosed groups are compared to controls.'Treated groups differ at p<0.05.

Table 5. Second generation male and female necropsy organ weights

Dose groupaOrgan 0% 0.1% 0.5% 1% TrendMaleBody (g) 506.0 ± 8.6 (20) 508.8 ± 14.2 (20) 496.8 ± 11.8 (20) 466.7 ± 9.8 (20)* p = 0.008Liver 40.7 ± 0.89 (20) 38.9 ± 0.68 (20) 40.2 ± 0.95 (20) 47.4 ± 0.73 (20)* p<0.001Kidneys 7.7 ± 0.11 (20) 7.9 ± 0.14 (20) 8.2 ± 0.10 (20)* 8.2 ± 0.14 (20)* p = 0.006Prostate 1.7 ± 0.07 (20) 1.6 ± 0.13 (20) 1.5 ± 0.07 (20) 1.3 ± 0.11 (20)* p = 0.022Seminal vesicles 5.0 ± 0.18 (20) 5.1 ± 0.23 (20) 5.0 ± 0.17 (20) 3.9 ± 0.30 (20)* p = 0.003Right testis (mg)b 1774.6 ± 67.0 (20) 1767.4 ± 47.8 (20) 1810.7 ± 105 (20) 1087.9 ± 125 (20)* p = 0.008

Epididymal sperm parmetersNumber 574.9 ± 37.6 (19) 575.8 ± 22.2 (20) 547.5 ± 36.9 (19) 295.1 ± 84.5 (18) p = 0.017Percent motilec 71.0 ± 1.6 (19) 72.0 ± 2.0 (20) 69.8 ± 1.5 (20) 72.4 ± 3.2 (9) p = 0.770Percent abnormald 1.0 ± 0.21 (18) 0.97 ± 0.12 (20) 1.0 ± 0.14 (19) 0.94 ± 0.12 (7) p = 0.470

Total spermatid headsper testise (x 107) 14.52 ± 0.95 (20) 16.84 ± 0.61 (20) 15.26 ± 0.99 (20) 6.69 ± 1.73 (20)'

Total spermatid headsper g testisf(x 107) 8.29 ± 0.58 (20) 9.64 ± 0.41 (20) 8.29 ± 0.54(20) 4.37 ± 0.98 (20) -

FemaleBody (g) 323.0 ± 5.1 (20) 311.6 ± 9.3 (20) 318.6 ± 8.6 (20) 281.2 ± 8.6 (20)* p<0.001Liver 37.0 ± 0.82(20) 36.3 ± 0.77 (20) 37.4 ± 0.96 (20) 37.9 ± 0.62 (20) p = 0.275Kidneys 7.4 ± 0.11 (20) 7.7 ± 0.14 (20) 7.8 ± 0.26 (20) 7.8 ± 0.14(20) p = 0.128Right ovary 0.16± 0.01 (20) 0.19± 0.01 (20) 0.18± 0.01 (20) 0.15± 0.01 (19) p = 0.250

aMean ratio (mg/g body weight) ± SE (number of animals).bNot adjusted for body weight.cDose group means and SEs are computed only from samples with at least 20 sperm.dDose group means and SEs are computed only from samples with at least 100 sperm.eX(10,000)Y, (X, average number of spermatid heads per tertiary square; Y, dilution factor).'Total spermatid heads per testis/testicular weight in grams.'Significantly different (p<0.05) from the control group.*p<0.05 when dosed groups are compared to controls.

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Table 6. Summary of incidence of histopathologiclesions (Task 4)

Treatment groupControl 0.5% 1.0%

Liver (total examined) (10) (10) (10)Hepatocellular degeneration 3a 1Focal hepatitis 6 1 4

Testis (total examined) (1O)b (10) (10)Degeneration of 1 3 8seminiferous tubules

Interstitial cell hyperplasia 1 1 7Granuloma 1

Epididymis (total examined) (10) (10) (10)Degeneration of 1 2 1epithelial cells

Interstitial epididymitis 1 1 1Focal granuloma 1Underdeveloped epididymis - 5Presence of fluid/ 1 1 2degenerated cells

Apparent sperm content 1 1 3reduction

Seminal vesicles (10) (10) (10)(total examined)

Vesticulitis - 1Inspissated secretion 1

aNumber of animals exhibiting lesion.bOne animal had variable loss of spermatogonia,spermatocytes, and spermatids and had an apparentincreased number of Sertoli and interstitial cells.

reduction in body weight in females) wasobserved in the 1.0% DBP group, therewas no difference in the body weightsbetween the control and 0.5% dose groups.This shows that pup weight was reduced inthe absence of a change in maternal weightat 0.5% DBP.

In the litters produced by the F1 adults(Task 4), decreased live pup weights(absolute and adjusted) were observed in allthe dose groups (including the 0.1% dosegroup). This was seen in the absence ofobvious general toxicity (Table 5). Theseresults suggest that the animals exposedduring development were much more sen-sitive to the developmental toxicity ofDBPthan were animals exposed only as adults.Furthermore, severe structural defects inthe male reproductive system wereobserved in the 0.5% and 1.0% dosegroups (results in Task 4 and Table 6). Thestructural defects included small and mal-formed prepuces and/or penises, underde-veloped or otherwise defective epi-didymides, and degenerate seminiferoustubules. From these data alone, we cannotdeduce whether the effects represent devel-opmental toxicity alone, if developmentalexposure renders the animals more suscep-tible to the adverse effects of DBP duringadult exposure, or both, but if DBP is anestrogenic compound (18), the former maybe more likely.

The statistically significant decreases inthe number of live pups per litter seen inall Fo dose groups during continuousbreeding (Task 2, Table 2) were not seenin the Task 3 crossover or in the secondgeneration (Tables 2 and 4, respectively),despite being of equivalent absolute magni-tude (i.e., =two pups per litter). Earlieranalyses (35) suggest that this is due in partto the differences in statistical sensitivity: inTask 2, up to five litters were generated,while Tasks 3 and 4 generated only one lit-ter. The multiple litters in Task 2 yield atest that is statistically much more powerfulthan the single litters of the Task 3 or 4mating trials. Based on this, one might pre-dict that the nonsignificant differencebetween the controls and middle dose ani-mals in Task 4 (14 vs. 12.8) would becomestatistically significant if five litters wereproduced.

While the reproductive toxicity ofDBPwas relatively mild in the Fo rats (Table 1,Table 2), the reproductive functions of F1rats were severely damaged. First, all theindices (mating, pregnancy, and fertilityindex) in F1 1.0% dose group were signifi-cantly reduced because only one litter oflive young was delivered (Table 4). Second,while the weight of the right ovary of FIfemales in all the dose groups remainednormal, the weights of ventral prostate,seminal vesicles/coagulating gland, andright testis in F1 males of the 1.0% dosegroup were markedly reduced. This mayindicate that the F1 males are more sensi-tive to DBP than F1 females, although theovary is notoriously insensitive as an indexof female reproductive toxicity (35,36).Third, histologic examination of the repro-ductive systems of F1 males showed degen-eration in seminiferous tubules togetherwith significantly fewer spermatids in testes(Table 5, 6). While the numbers of epi-didymal sperm were reduced at the highdose, morphology and visually estimatedmotility were normal in all groups; thehigh dose animals were making fewersperm, although these sperm appeared nor-mal. In contrast to the males, female rats inboth the Fo and F1 generations seemed tobe relatively resistant to the reproductivetoxicity ofDBP in terms of numbers of liveyoung and necropsy endpoints. But in theFo generation, females appeared to be moresensitive to the systemic toxicity of DBPbecause the body weight of Fo females inthe 1.0% dose group was significantlydecreased while the males at that concen-tration showed no detectable change.

Consistent with previous reports onother phthalates (13), the present dataindicate that effects of DBP exposure aregreater in animals exposed from conception

than those exposed as adults only. Becausethe F1 rats were possibly exposed duringgestation and throughout nursing and weredosed during maturation until and throughmating, we cannot specify which period ofdevelopment (e.g., which developmentaltrimester, nursing, puberty, etc.) is moresensitive to the effects of DBP. However,the male reproductive structural defectssuggest that the period of organogenesisand perhaps hormonal imprinting for thesetissues, in the prenatal and perinatal peri-ods, is the most critical time. The mecha-nisms underlying this developmental toxic-ity are still unknown. In the past, most ofthe toxicity studies on phthalates havefocused on their adverse effects on dosedjuvenile and adult animals, rather than onpups of dosed dams. Nevertheless, theseearly studies have led to the widespreadview that phthalates affect the testis pri-marily at the Sertoli cells (14,37,38).Indeed, in neonatal rat testes, significantdecreases in testis weights and Sertoli cellnumbers after five daily oral doses of 1000mg/kg DEHP have been reported byDostal et al. (39).

Jobling et al. (18) found that DBPinteracted weakly with the estrogen recep-tor in a variety of constructs. If DBPdemonstrated such an interaction in vivo,one would expect to find altered reproduc-tive development (19). The present studyfound such alterations: degenerate seminif-erous tubules with decreased sperm counts,apparent interstitial cell hyperplasia, andstructural defects in epididymides andpenises of 1.0% DBP-treated F1 rats whenthey reached maturity. These mirror simi-lar effects seen in rats and mice exposedperinatally to the strong estrogen agonistdiethylstilbestrol (DES) (40,41) and areconsistent with the possibility that theeffects seen in the present study were medi-ated through an interaction with the estro-gen receptor system. Interestingly, there arealso reports showing that some phthalateshave hormone-disrupting effects in maleadult rats (42-44).

In conclusion, the results from the pre-sent RACB study indicate that the develop-mental and reproductive toxicities of DBPare more prominent in animals exposedduring development and maturation thanin animals exposed as adults only. This isprompting an investigation of the period ofsensitivity for more of the phthalates.

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