# Reproductive Toxicity of 

# Di-n-butylphthalate in a 

# Continuous Breeding 

# Protocol in 

# Sprague-Dawley Rats

RobertN. Wine,1 

### Ling-Hong Li,1 

### Leta Hommel 

### Barnes,2 Dushyant K. 

### Gulati,2 and Robert E. 

### Chapin1

1National 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 

### employed extensively in 

### the manufacture of

### plastics where 

### they are 

### used to impart 

### flexibility. Since 

### they are not 

### covalently linked to 

### the 

### plastic polymer, 

### they can 

### leach 

### from the 

### matrix, 

### 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 as 

### nail 

### polish, insect 

### repellents, and denture base material 

### (2-4). DBP has 

### recently been 

### found to 

### have clinical 

### applications as 

### well, 

### selectively eliminating tumor 

### cells 

### from bonemarrow 

### (5,6).
DBP 

### has been 

### previously characterized

as a 

### developmental and 

### reproductive toxicant in 

### the rat 

### (7-12). 

### Comparable oral doses of DBP given to 

### juvenile and 

### adult male 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 also been 

### shown that the testicular toxicity 

### of 

### phthalates is age 

### dependent, with immature 

### animals 

### being more sensitive 

### than mature 

### animals 

### (13,14). However, 

### the 

### reproductive toxicity 

### following exposure 

### during development 

### has not 

### been 

### addressed. 

### Although no 

### data are 

### available 

### specifically on 

### the trans

### fer of DBP across 

### the 

### placenta, studies with 

### other 

### phthalate esters 

### show that these 

### compounds 

### readily cross 

### the 

### placenta of
rats 

### (15-17) and that exposure 

### oflactating dams to 

### another 

### phthalate, di 

### (2-ethylhexyl) 

### phthalate (DEHP), could 

### lead to DEHP 

### and mono 

### (2-ethylhexyl) phthalate (MEHP) exposure in

### the 

### sucklingratpups. Because DBP is 

### lipid 

### soluble, as are DEHP 

### and MEHP, one 

### might 

### predict a certain 

### degree oflactational transferofDBP. 

### Recently, 

### Jobling et 

### al. 

### (18) 

### reported that DBP 

### couldreduce the

### bindingofestrogen to the estrogen receptor 

### and stimulate 

### transcriptional activity 

### These authors 

### called for 

### further in vivo

### databefore any firm con

### clusions as to 

### the estrogenicity 

### of DBP 

### could be 

### drawn. Review 

### ofarchived chemical 

### studies 

### generated with the 

### National 

### Toxicology Program's 

### Reproductive
Assessment 

### by Continuous 

### Breeding 

### (RACB) 

### protocol revealed an 

### earlier 

### study that examined the 

### reproductive toxicity 

### of
DBP in

### Sprague-Dawleyrats. 

### Ifthe

### findings of

### Jobling et 

### al. 

### (18) are correct 

### and DBP

can act as an estrogen, 

### then one 

### would expect to see greater 

### reproductive effects in 

### second generation animals 

### (195) under the RACB protocol. This is expected in the RACB 

### designbecause 

### the

### Fo rats are 

### exposed 

### onlyas 

### adults, the 

### F1 rats are 

### born to 

### mothers 

### that are 

### treated during gestation and lactation, 

### and the F1 

### animals themselves are 

### treatedduringmaturation to 

### sexual maturity 

### and throughmating. 

### The results 

### ofthis DBP RACB 

### studyare 

### presentedbelow; 

### the results show that structural 

### defects are seen in 

### the second generation rats 

### that were not 

### found in 

### the 

### first.


### Methods
General. 

### This 

### study was 

### conducted using 

### the National 

### Toxicology Program's RACB 

### protocol for which the 

### detailed methods have 

### been previously described (20,21). 

### Briefly, the 

### protocol is 

### composed of four segments, or 

### tasks. Task 1 is a 

### 14-day range-finding study that is 

### used to set 

### three 

### dose levels used in

### Task 2. 

### Task 2 is 

### the 14- 

### week continuous 

### breeding phase, generating up to 

### five litters per pair. Task 3 consists 

### of crossover matings 

### between 

### treated and control 

### Fo animals to 

### determine the affected sex, 

### andTask 4 assesses 

### the 

### fertility of the last litter 

### (F1) born 

### during continuous 

### breeding (Task2). 

### Chemical. DBP was 

### obtained from Chem 

### Central via 

### Midwest Research Institute 

### (Kansas City, MO, 

### Lot/Batch # L121 1-83/02). 

### The purity of DBP 

### used in preparation 

### of the feed 

### formulations was greater 

### than 99% as 

### determined by gas 

### chromatography. Dosed

### feedformulations. DBP was 

### blended into 

### powdered NIH-07 

### (Zeigler Bros., 

### Gardeners, PA) 

### feed on a 

### weight-toAddress correspondence to R.E. Chapin, National Institute of Environmental Health Sciences, P.O. Box 12233, Mail Drop A2-02, Research Triangle Park, NC 27709 USA. This workwas conducted under contract NO1-ES65142 to the National Institute of Environmental Health Sciences. The authors are indebted to Brad Collins and Tom Goehl for assistance in procuring and characterizing the DBP, to Jerry Heindel for support and encouragement, and to Paul Foster and KimTreinen for stimulatingdiscussions. The statistical support was provided byAnalytical Sciences Inc., Durham, NC, andchemistry support was provided by Midwest Research Institute and Research Triangle Institute under contracts NO1-ES-45060 andNO1-ES-45061, respectively. Received 20 August 1996; accepted 18 September 1996.

Volume 105, Number 1, January 1997 

### * EnvironmentalHealth

### Perspectives 

### 102Articles

- Reproductive toxicity of

### di-n-butylphthalate

weight basis. Animals in the control group 

### received undosed NIH-07. Each dose level

was 

### independently formulated and pre

### pared at 

### least every 3 

### weeks 

### and stored frozen. Dosage 

### formulation studies indicat

### ed that DBP 

### blended with 

### feed at a concentration 

### of 0.5 mg/g was 

### stable 

### for 3 

### weeks when 

### sealed at -200C 

### and for 7 

### days when open to air 

### and light at room temperature. 

### Thus, dosed feed was 

### stored in 

### the dark and 

### refrigerated and was 

### changed offthe cages every

### week. 

### Animals. VAF 

### Crl:CD BR 

### outbred 

### Sprague-Dawley albino rats were 

### purchased from Charles River 

### Breeding Laboratories (Portage, MI). During quarantine, representative 

### animals were 

### sacrificed and their sera 

### evaluated for antibodies against nine 

### rodent viruses 

### and Mycoplasmapulmonis (Charles River 

### Professional Services, 

### Wilmington, MA) All serawere negative 

### forviral 

### antibodies. 

### Animals were 

### quarantined separately for approximately 3 

### weeks for Task 1 

### and 2 

### weeks 

### forTask2; ratswere 70 

### days ofage at 

### the start 

### of both Tasks 1 

### and 2. Histo

### pathologic evaluations were 

### performed on representative 

### animals 

### during quarantine to 

### checkforsigns 

### ofinfectious disease. Male 

### and female rats were 

### housed two per cage 

### by sex 

### during quarantine 

### and the 

### 1-week pre-mating 

### period on 

### SaniChip 

### bedding (P.J. 

### Murphy Forest 

### Products) in 

### solid bottom 

### polycarbonate cages 

### with stainless steelwire-bar 

### lids. Deionizedwater 

### and rodent meal 

### feed 

### (NIH-07 

### diet; Zeigler Bros.) were 

### provided ad 

### libitum.The rats

were 

### subsequentlyhoused as 

### breeding pairs

or 

### individually at 23 ± 

### 2°C

### with a 

### 14:10-hr light:dark

### cycle. All housing and

### procedures

were 

### performed in 

### accordance with the NIEHS 

### guidelines for the 

### humane use 

### of 

### animals in

### research. 

### Studydesign. DBP was 

### administeredvia 

### feed to CD 

### Sprague-Dawleyrats. In

### Task 1, 

### the

### dose-finding segment 

### ofthe 

### study, doses 

### of0.0 

### (control), 0.1, 0.5, 1.0, 1.5, 

### and 2.0% 

### (w/w) DBP were 

### tested using 

### eight animals 

### ofeach sexper group. 

### Endpoints forTask 1

were 

### dinical signs 

### of toxicity, 

### bodyweight, and 

### foodconsumption.
In an 

### optimal study, the 

### highest dose 

### during Task 2 

### should be set so as not to 

### depress 

### weight gain 

### by more 

### than 10% or 

### result in greater 

### than 

### 10% mortality. If a 

### compound is a 

### reproductive toxicant, the 

### middle dose should elicit 

### reproductive effects with little or no systemic toxicity in 

### evidence, and the lowest 

### dose should pro

### vide a 

### no-effect level. 

### Based on 

### the results 

### ofthe Task 1 

### pilot 

### (data not 

### shown), concentrations 

### of 0.1, 0.5, 

### and 1.0% DBP in 

### the feedwere 

### selected forTask 2. 

### Task 2, 

### the continuous 

### breedingphase, used a 

### control group 

### consisting of 40 male/female breeding pairs and three dose
groups with 20 breeding pairs per group. 

### The endpoints 

### for Task 2 were clinical signs 

### oftoxicity, 

### parental bodyweight and
average 

### consumption 

### of feed during representative weeks, 

### fertility (the proportion of 

### cohabited pairs 

### producing any live pups), the 

### number oflitters per pair, 

### the number 

### of live pups per 

### litter, the proportion 

### of
pups 

### born 

### alive, the sex ratio 

### of live pups, 

### and the pup 

### body weights immediately afterbirth. The pups in 

### the first 

### four litters

were 

### removed 

### after birth, counted, 

### weighed, sexed, and killedwithout necropsy. 

### The last 

### litter, born 

### during the holding 

### period 

### following the continuous 

### breeding phase, was 

### reared

### bythe damuntilweaning 

### (day 21 

### after 

### birth); at 

### this time, treatment 

### ofthe

### Fl animals was 

### initiated bythe same route 

### and at 

### the same concentrations as 

### consumed by their parents in 

### Task 2. These animals were 

### used for assessment 

### of 

### secondgeneration 

### fertilityin

### Task4. During 

### Task 2, if an 

### effect on 

### fertility

was 

### detected, a 

### 1-week crossover mating 

### trial, Task 3, was 

### performed to 

### determine the 

### affected sex. In 

### this study, thiswas per

### formed after the last 

### litter of Task 2 was 

### weaned. The mating 

### trial consisted ofthree
groups 

### of 20 pairs 

### each: control males x control 

### females, control males x 1% 

### females, and 1% 

### males x 

### control females. 

### Endpoints for Task 3 were 

### the same as 

### Task 2, 

### with the 

### addition of 

### checking for thepresence 

### ofa

### vaginal copulatoryplugor sperm. 

### After the litter in 

### Task 3 

### had been 

### delivered, evaluated, and 

### disposed of, the 

### Fo females were 

### subject to 

### vaginal 

### lavage for 12 

### days to 

### evaluate estrous 

### cyclicity; the 

### control and 

### high dose 

### Fo adults were 

### then 

### killed by 

### CO2 asphyxiation and 

### subjected
to necropsy. Necropsy 

### endpoints were organ 

### weights and 

### histopathology, body weight, epididymal sperm 

### motility (counted

### visually), sperm

### morphology, and sperm count. For 

### histopathological evaluation, selected organs were 

### examined after fixation in 10% 

### neutral 

### buffered formalin or 

### Bouin's 

### fixative (ovaries) 

### and embedded in 

### glycol 

### methacrylate (testis) or 

### paraffin. Sections were 

### stained with 

### periodic acid-Schiff 

### (PAS) and 

### hematoxylin (testis)

or 

### hematoxylin and eosin 

### according to 

### standard

### procedures. Task 4 

### assessed the 

### growth and 

### fertility of

### Fl animals. The last litter from Task 2

was 

### reared, 

### weaned, and held until mating
at 

### postnatal 

### day 88 ± 10. During maturation, 

### siblings were 

### housed

### by sex at a maximum

### oftwopercage 

### andreceived the same 

### chemical treatment as 

### their parents. Upon 

### reaching sexual maturity, 20 

### each nonsib

### ling F1 males 

### and females within 

### the same treatment groups were

### housed inpairs 

### for 7 

### days and then housed 

### individually until delivery of a litter. The 

### endpoints for the 

### Task 4 mating trial was the same as in

### Task 2, 

### with the addition 

### of checking for the
presence 

### of a 

### copulatory plug and vaginal
sperm. At 

### the end ofTask 4, 

### F1 animals in all 

### dose groups were 

### killed

### byCO2 

### asphyxiation 

### and 

### subjected to necropsy 

### with the

same 

### endpoints as in theTask 3 necropsy. Statistics. When data were expressed as

a proportion, 

### such as 

### the fertility, mating, 

### and pregnancy 

### indices, the CochranArmitage test was 

### used to test 

### for a 

### doserelated trend (22). Each dose group was 

### compared to 

### the control group 

### with a 

### X2 test 

### (23). In 

### Task 3, 

### where the animals

were 

### cross-mated and dose groups 

### did not represent increasing 

### dose 

### levels, a 

### X2 test 

### for 

### homogeneity was 

### used to test 

### for an 

### overall difference in 

### fertility among groups 

### forpairwise comparisons. 

### The number of litters and the number 

### oflive pups per 

### litter were 

### computed on a 

### per-fertile-pair basis and treatment group
means were 

### determined. The proportion 

### of live pups was 

### defined as 

### the number of
pups 

### born alive 

### divided 

### by the total num

### ber ofpups 

### produced byeachpair. 

### The sex ratio was 

### expressed as 

### the proportion 

### of male pups 

### born alive 

### divided by the total 

### number ofpups 

### produced by each pair. In 

### Task 2, 

### dose group means 

### for these and other parameters were 

### compared to 

### the control group 

### with 

### Shirley's test 

### (24,25). When a 

### trend was present, 

### Jonckheere's 

### trend test 

### (26) was 

### used, otherwise Dunn's test 

### (271 was 

### applied. In 

### Task 3, parameterswere 

### tested foroverall differences using 

### the Kruskal-Wallis test 

### (28), and 

### multiple comparisonswere

### made using

### Dunn's test. To remove the potential effect 

### of the 

### number 

### of pups per 

### litter on 

### the average pup 

### weight, an 

### analysis of covariance 

### (ANCOVA) was 

### performed (29) using 

### the
average 

### litter size, 

### including live and 

### dead
pups, as 

### the covariate to

### produce an 

### adjusted live pup 

### weight. Least squares estimates 

### of 

### dose group means, 

### adjusted for litter size, were 

### computed and tested for overall 

### equalityusing an F-test 

### andpairwise 

### equality using 

### Dunnett's test 

### (30). To 

### evaluate 

### potential sex 

### differences, these 

### analyses

were 

### performed on 

### males, females, and both sexes 

### combined. 

### Absolute 

### body and organ 

### weights and
organ 

### weights adjusted for 

### body weight

were 

### analyzed by Shirley's or 

### Dunn's test, 

### while 

### dose-related trends were 

### identified

### by Wilcoxon's test in 

### Task 3 or 

### Jonckheere's
test in

### Task4. 

### Vaginal cytology data were 

### analyzed using a 

### multivariate 

### analysis of variance 

### (ANOVA) (31) to test 

### for the 

### simultaneous equality ofmeasurement across dose levels.

Environmental Health

### Perspectives * Volume 

### 105, Number 1, 

### January 1997 

### 103Articles

- Wine et al.



### Before running the ANOVA, an arcsine 

### transformation was 

### performed to 

### bring 

### the data into 

### close conformance with normalityassumptions. Group means, 

### standard deviations, 

### and 

### standard errors were 

### calculated for all data 

### and for each sex. 

### Jonckheere's testwas 

### per

### formed to 

### look for increasing or 

### decreasing trends. 

### Significance among 

### dose groups
was 

### then calculated using 

### Shirley's test 

### ifa 

### trend was 

### evident or 

### Dunn's test 

### if no 

### trendwas evident.


### Results
Continuous 

### breeding (Task 2). Representative 

### dosage formulations from Task 2 were 99-106% 

### of the target concentrations. 

### The average 

### daily feed consumptionwas 

### similar forcontrol 

### and DBP

### dosed animals. During 

### the first 14 

### days of Task 2, 

### body

### weight gain was 

### significantly lower 

### only in 

### the 

### high-dose females (2%), 

### compared with a 7% increase 

### for control 

### females. This resulted in 

### significantly reduced 

### body weights after each litter 

### and
at necropsy 

### for these 

### high-dose females. During 

### the course 

### of the Task 2 continuous 

### breeding 

### phase, body weights were 

### within 10% 

### ofthe controls except 

### forhighdose females at

### week 17 

### (the endofcohabi

### tation) when 

### they were 11% 

### less than the 

### controls. Over 

### the duration of continuous 

### breeding, there were no 

### clinical signs 

### of toxicity

### noted

### during the 

### twice-dailyhealth surveillance. 

### Based on 

### body weights and food con

### sumption, the average 

### calculated 

### daily intakes 

### ofDBP 

### for the 0.1, 0.5, 

### and 1.0% 

### dose groups were 52, 

### 256, 

### and 509 

### mg/kg body weight for males 

### and 80, 385, 

### and 794 

### mg/kg 

### forfemales, 

### respectively.
DBP treatment 

### did not 

### affect 

### fertility

or 

### the average 

### number of litters per pair, 

### but it 

### did 

### dose-dependently reduce the number of lives pups per 

### litter 

### (Table 1).
In 

### addition, live pup 

### weights (both absolute and 

### adjusted for litter 

### size) were 

### significantly decreased in 

### the middle and 

### high dose groups. 

### The 

### cumulative-days-tolitter 

### values were 

### similar for control 

### and treatedanimals.
Dam 

### weights at 

### delivery were 

### signifi

### cantly decreased at 

### each litter 

### of the 

### high dose group 

### (by <10%). During nursing of the 

### final 

### litter, dam 

### weights were 

### significantlydecreased at 

### postnatal day 21 in 

### the low 

### dose group 

### (by 7%), at 

### postnatal days 14 

### and 21 in 

### the 

### middle dose group 

### (by =6%), and at 

### all time 

### points in 

### the 

### high dose group 

### (by=10%; 

### datanot 

### shown). Crossover 

### mating 

### (Task 3). Since 

### adverse effects on 

### reproduction were 

### observed in 

### Fo rats, 

### the crossover 

### mating 

### (Task 3) was 

### performed on 

### Fo animals to Table 1. Reproductive performance offirstgeneration breeding pairs

Dose groupa Reproductive parameter 0% 0.1% 0.5% 1% Trend
Average littersperpair 4.8± 0.1 (40) 5.0 ± 0.1 (20) 4.9± 0.1 (19) 4.9± 0.1 (20) p= 0.322 Livepupsperlitter 12.9 ± 0.2(40) 11.9 ± 0.3 (20)* 11.0 ± 0.5 (19)* 10.7 ± 0.4 (20)* p< 0.001 Absolute livepupwt(g) 5.96 ± 0.06 (40) 5.99 ± 0.06 (20) 5.74 ± 0.07 (19)* 5.38 ± 0.10 (20)* p< 0.001 Adjusted livepupwt (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 ofbreeding pairs). *p< 0.05when dosed groups are compared to controls.

Table2. Matingtrial todetermine affected sex: fertilityand reproductive performance

Treatmentgroupa Control male x 1% Male x Control male x Overall Reproductive parameter control female control female 1% female difference Mating indexb 16/19 (84%) 19/20 (95%) 16/19(84%) p= 0.487 Pregnancy indexc 12/18 (67%) 18/20 (90%) 15/19(79%) p= 0.212 Fertilityindexd 12/15 (80%) 18/19 (95%) 15/16 (94%) p= 0.303 Live pups perlitter 12.7 ± 1.3 (12) 13.4 ± 0.6 (18) 12.9 ± 0.7 (15) p = 0.934 Adjusted live pupwt (g) 5.96 ± 0.16 (12) 6.16 ± 0.12 (18) 5.28 ± 0.14 (15)*' p < 0.001

aData are mean ± SE (number ofbreeding pairs). 

### bNumber offemaleswithplug/number of cohabiting pairs (%withplug). 

### cNumber offertile pairs/number of cohabiting pairs 

### (% pregnant). 

### dNumber offertile pairs/number offemaleswith plug (% fertile). *p<0.05when dosed groups are compared to controls. 'Treated groups differ atp<0.05.



### determine the affected sex. For 

### Task 3, 

### control animals were 

### randomly

### pairedwith other control or 

### high 

### dose (1.0%) animals for 7 

### days. Among 

### the three groups (con

### trol male x 

### control 

### female, 1% 

### male x control 

### female, and control 

### male x 1% 

### female), there was no 

### overall 

### difference with respect to mating, pregnancy, or 

### fer

### tility indices 

### (Table 2). Live pup 

### weight 

### adjusted for litter size was 

### significantly reduced in the 

### control 

### male x 

### 1% female
group. 

### Treated 

### females 

### weighed =11% 

### less than 

### controls 

### duringTask 3 

### and consumed =12% 

### less 

### feed. There were no 

### significant clinical signs 

### oftoxicity 

### noted 

### during Task 3. 

### Although two 

### females 

### died, one in 

### the control group 

### and the other in 

### the 

### high dose group, 

### neither 

### of these 

### deaths were 

### attributed to DBP exposure. At 

### the 

### end of Task 3 

### (4 weeks after 

### cohabitation), male 

### body weights were 

### unchanged, but 

### high dose 

### females 

### weighed =14% less 

### than controls. At necropsy 

### of the controls and 

### high-dose rats, organ-to

### bodyweight ratios 

### for the 

### liver and

### kidneys

were 

### significantlyincreased in

### both sexes in 

### the 

### high dose group 

### (Table 3). The gross structure 

### of the 

### reproductive system was 

### unchanged by DBP consumption, 

### and
sperm 

### studies 

### showed that sperm concentration, 

### motility, percent 

### abnormal 

### forms, and testicular 

### spermatid head counts were not 

### affected 

### by 1% DBP consumption.
DBP 

### caused no apparent 

### changes in average estrous 

### cycle 

### length (control length 4.20 ± 0.07 

### days; n = 

### 33) or 

### progression 

### through the stages 

### of the 

### cycle (data not 

### shown). Second

### generation

## fertility (Task4). The final Task 2 

### litters 

### of

### all dose groups were 

### reared and exposed to 

### the same concentrations 

### ofDBP 

### betweenweaning 

### and necropsyas 

### their parents were. 

### Before weaning, 

### Fl
pups were 

### counted and

### weighed on 

### postnatal 

### days 0,4, 7, 

### 14, and 21. 

### Survival was not 

### affected, but live male 

### and female pup 

### weights in 

### the 1.0% 

### dose groupwere

### signifi

### cantly (10-15%) lower than 

### controls at 

### postnataldays 0, 

### 14, and 21. During rearing 

### (at 

### =postnatal day 46), three F1 

### males in 

### the 

### high dose group were 

### found to 

### have small 

### and 

### malformed prepuces 

### and/orpenises 

### andwere

### without

### palpable testes. Just prior to mating 

### (postnatal 

### day =80), one 

### of these males had 

### palpable
testes. No 

### such abnormalities were 

### noted forthe control males.
At 88 

### (±10) 

### days of age, 

### F1 rats were 

### cohabited for 1 

### week. Feed consumption 

### values 

### during the week of cohabitation

were =12% 

### lower in 

### the 1% DBP 

### dose
group 

### compared to 

### the control group. Mating, pregnancy, 

### and 

### fertility indices in 

### the 1.0% group were 

### significantly lower: 

### only 1 

### litter was 

### born to 20 

### breeding pairs in 

### the 

### high dose group, 

### compared with 19 

### litters to 20 

### breeding pairs in 

### the control
group 

### (Table 4). While there was no 

### change in 

### the 

### numbers oflive pups 

### per 

### litter, 

### absolute and 

### adjusted live F2 

### pup 

### weights were 

### =6-9% lower in

### all treatment groups. 

### The average 

### number of

### days to 

### litVolume 105, Number 1, 

### January 1997 * EnvironmentalHealth

### Perspectives 104Articles * Reproductive toxicity of

### di-n-butylphthalate

terandaverage damweight atdeliverywere not affectedbyDBP consumption. At the end of Task 4, 

### all 

### F1 animals



### were weighed, killed, and necropsied. The 

### high dose males and females 

### weighed =8-14 % less than their controls (Table 5). Bodyweights were unchanged in the mid

### dle and lower dose groups. For 

### males, only 

### the 1% treatment group showed a signifi

### cant increase in 

### liver weight, 

### while kidney 

### weight was increased at both 

### the middle and high dose levels. 

## F, females showed no significant change in liver or kidney 

### weights. F1 

### ventral prostate, 

### seminal vesi

### cle/coagulating gland, and right testis 

### weights all were decreased in 

### the 1.0% treatment group, while average ovary weights remained unchanged (Table 5). Epididymal sperm counts and testicular spermatid head counts were both reduced in the high dose group (Table 5). Epididymal sperm concentration values 

### (expressed as 

### 1,000 sperm/mg 

### caudal tissue) in high dose rats were 51% those of control animals. Likewise, both the total spermatidheads per testis, ameasure oftes

### ticular output, 

### and total 

### spermatid head per gram of testis, a measure of 

### spermatogenic efficiency, were decreased in 1.0% DBP treated males. 

### Histopathologic examination 

### of selected organs was 

### performed on 10 

### representative males from the control, 0.5%, 

### and 1.0% groups and is summarized in Table 6. Three males 

### (of 10 total) in 

### the 0.5% dose group 

### showed degeneration 

### of seminiferous tubules, compared with 1 control, and 8 males in the 1.0% group. In 

### addition, 7 of the 

### 10 males examined in the 1.0% DBP treatment group demonstrated apparent interstitial cell hyperplasia. Finally, half of the rats (5 of 10) observed in the high dose group had either underde

### veloped or 

### otherwise 

### defective epi

### didymides.


### Discussion
The results from 

### the present 

### RACB study indicate that DBP is a developmental toxicant in that live pup weights (absolute and adjusted) were 

### significantly 

### decreased in both the 0.5% and 1.0% dose groups in 

### Task 2 (Table 1) and in 

### control males x 1.0% dosed females of Task 3 (Table 2). Decreased fetal weight is considered an early indicator of 

### developmental toxicity 

### (32) and has been observed in Wistar rats 

### treated with 600 mg 

### DBP/kg by gavage on gestation 

### days 0-21 (33). 

### Similar 

### results

were observed by 

### Reel and Lawton 

### (34) in

an earlier DBP 

### RACB study 

### using Swiss mice. Although general toxicity 

### of DBP (a 

### significant increase in liver 

### and 

### kidney weights in 

### both males 

### and females 

### and Table3. Matingtrial to determine affected sex: necropsyoutcome

Dose group Organ 0% 1% Difference
Male Absolute bodywt 683.6 ± 7.6 (40)8 656.8 ± 15.0 (20) p<0.154 Adjusted liverwt 37.6 ± 0.63 (40) 43.4 ± 1.1 (20)* p<0.001 Adjusted kidneywt 6.6 ± 0.11 (40) 7.3 ± 0.18 (20)* p=0.004
Female Absolute bodywt 379.0 ± 9.6(38) 326.4 ± 6.5 (19)* p<0.001 Adjusted liverwt 34.0 ± 0.62 (38) 38.9 ± 0.69 (19)* p<0.001 Adjusted kidneywt 7.0 ± 0.10 (38) 7.6 ± 0.14 (19)* p=0.002

aMean ratio (mg/g bodyweight) ± SE (number ofanimals). *p<0.05when dosed groups are compared to controls (Wilcoxontest).

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

Dose groupa Reproductive parameter 0% 0.1% 0.5% 1% Trend Mating indexb 20/20 (100%) 19/20 (95%) 18/20 (90%) 6/20 (30%)* p<0.001 Pregnancyindexc 19/20 (95%) 17/20 (85%) 17/20 (85%) 1/20 (5%)* p<0.001 Fertilityindexd 19/20 (95%) 17/19 (89%) 17/18 (94%) 1/6 (17%)* p<0.001 Live pups per litter 14.0 ± 0.8 (19) 15.5 ± 0.4 (17) 12.8 ± 0.8 (17) 13.0' p=0.233 Absolute livepupwt (g) 5.97 ± 0.11 (19) 5.60 ± 0.09 (17)* 5.60 ± 0.09 (17)* 5.00# p=0.020 Adjusted live pupwt (g) 5.98 ± 0.08 (19) 5.69 ± 0.09 (17)* 5.50 ± 0.09(17)* p<0.001


### aData are mean ± SE (number ofbreeding pairs). 

### bNumberoffemaleswithplug/number of cohabiting pairs (%withplug). 

### cNumberoffertile pairs/number ofcohabiting pairs (% pregnant). 

### dNumberoffertile pairs/number offemaleswithplug (%fertile). *p<0.05whendosed groups are compared to controls. 

### 'Treated groups differ atp<0.05.

Table 5. Secondgeneration male and female necropsyorganweights

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

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

Total spermatidheads
pertestise (x107) 14.52 ± 0.95 (20) 16.84 ± 0.61 (20) 15.26 ± 0.99 (20) 6.69 ± 1.73

### (20)' Total spermatidheads
pergtestisf(x107) 8.29 ± 0.58 (20) 9.64 ± 0.41 (20) 8.29 ± 0.54(20) 4.37 ± 0.98(20)

-

Female Body(g) 323.0 ± 5.1 (20) 311.6 ± 9.3(20) 318.6 ± 8.6 (20) 281.2 ± 8.6(20)* p<0.001 Liver 37.0 ± 0.82(20) 36.3 ± 0.77 (20) 37.4± 0.96(20) 37.9 ± 0.62

### (20) p = 0.275 Kidneys 7.4 ± 0.11 (20) 7.7 ± 0.14(20) 7.8 ± 0.26(20) 7.8 ± 0.14

### (20) p =0.128 Rightovary 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 bodyweight) ± SE (number ofanimals). 

### bNotadjusted forbodyweight. 

### cDose group means and SEs are computed onlyfromsampleswith at least 20 sperm. dDose group means and SEs are computed onlyfrom sampleswith at least 100 sperm. 

### eX(10,000)Y, (X, average numberofspermatid heads pertertiarysquare; Y, dilutionfactor). 'Total spermatid heads pertestis/testicularweight ingrams. 

### 'Significantlydifferent (p<0.05) fromthe control group. *p<0.05whendosed groups are compared to controls.

Environmental Health Perspectives * Volume 105, Number 

### 1, January 1997 

### 105Articles * Wine et al.

Table 6. Summary of incidence of histopathologic lesions (Task4)

Treatmentgroup

Control 0.5% 1.0%
Liver(total examined) (10) (10) (10) Hepatocellulardegeneration 3a 1 Focal hepatitis 6 1 4
Testis (total examined) (1O)b (10) (10) Degenerationof 1 3 8 seminiferoustubules Interstitial cellhyperplasia 1 1 7 Granuloma 1 Epididymis (totalexamined) (10) (10) (10) Degenerationof 1 2 1 epithelial cells Interstitial epididymitis 1 1 1 Focalgranuloma 1 Underdeveloped epididymis

- 5 Presenceoffluid/ 1 1 2 degeneratedcells Apparentspermcontent 1 1 3 reduction
Seminalvesicles (10) (10) (10) (totalexamined) Vesticulitis

- 1 Inspissated secretion 1

aNumberofanimals exhibiting lesion. 

### bOne animal had variable loss of spermatogonia, spermatocytes, andspermatids andhad anapparent increasednumberofSertoli and

### interstitial cells.



### reduction in 

### bodyweight in 

### females) was 

### observed in 

### the 1.0% DBP group, 

### there

was no 

### difference in 

### the 

### body weights between the control 

### and 0.5% 

### dose groups. 

### This shows that pup

### weightwas 

### reduced in 

### the absence ofa 

### change in

### maternal 

### weight
at 0.5% DBP. In 

### the litters 

### produced 

### by the 

### F1 adults 

### (Task 4), decreased live pup 

### weights (absolute and

### adjusted) were 

### observed in

### all the 

### dose groups 

### (including the 0.1% 

### dose 

### group). This was seen in 

### the absence 

### of obvious 

### general toxicity 

### (Table 5). 

### These results suggest 

### that 

### the animals 

### exposed 

### during 

### development were 

### much more sensitive to 

### the

### developmental toxicity

### ofDBP 

### than were 

### animals 

### exposed only as 

### adults. 

### Furthermore, severe 

### structural 

### defects in 

### the male 

### reproductive system were 

### observed in 

### the 0.5% 

### and 1.0% 

### dose
groups 

### (results in

### Task 4 

### andTable 6). The 

### structural 

### defects included small and malformed prepuces 

### and/or penises, 

### underde

### veloped or 

### otherwise 

### defective epi

### didymides, and 

### degenerate seminiferous tubules. From 

### these 

### data 

### alone, we cannot 

### deduce whether 

### the 

### effects represent 

### devel

### opmental toxicity 

### alone, if

### developmental
exposure 

### renders 

### the animals more suscep

### tible to 

### the 

### adverse effects of DBP 

### during adult exposure, or 

### both, but if DBP is an estrogenic 

### compound (18), the former may be more 

### likely. The statistically significant decreases in 

### the number 

### of live pups per 

### litter seen in 

### all 

### Fo dose groups 

### during continuous 

### breeding (Task 2, 

### Table 2) were not seen in 

### the Task 3 crossover or in 

### the second generation 

### (Tables 2 

### and 4, 

### respectively), despite being

### ofequivalent 

### absolutemagni

### tude (i.e., =two pups per 

### litter). Earlier 

### analyses (35) suggest 

### that this is 

### due inpart to 

### thedifferences in

### statistical sensitivity: in 

### Task 2, up to 

### five litters were 

### generated, while Tasks 3 

### and 4 

### generated onlyone 

### litter. 

### The 

### multiple litters in 

### Task 2 

### yield a test 

### that is 

### statisticallymuchmore

### powerful than 

### the 

### single litters of the Task 3 or 4 mating

### trials. Based on 

### this, one

### mightpre

### dict that the 

### nonsignificant difference between the controls 

### and middle dose ani

### mals in

### Task 4 

### (14 vs. 

### 12.8) 

### wouldbecome 

### statistically 

### significant if 

### five litters were 

### produced. While the 

### reproductive toxicity

### ofDBP

was 

### relatively mild in 

### the 

### Fo rats 

### (Table 1, 

### Table 2), 

### the reproductive 

### functions of

## F1 rats were 

### severely damaged. First, 

### all the indices (mating, pregnancy, 

### and fertility index) in F1 1.0% 

### dose group were 

### significantly reduced because 

### only one 

### litter 

### of liveyoungwas 

### delivered (Table 4). Second, while the weight of the right ovary 

### of

## FI females in 

### all the 

### dose groups 

### remained normal, the weights of ventral prostate, 

### seminal 

### vesicles/coagulating gland, and 

### right testis in

## F1 males 

### of the 1.0% 

### dose
group were 

### markedly reduced. This may 

### indicate that 

### the 

### F1 males are more sensitive to DBP 

### than

## F1 females, although the
ovary is 

### notoriously insensitive as an 

### index of 

### female 

### reproductive toxicity 

### (35,36). Third, histologic examination 

### ofthe repro

### ductive systems 

### of

### F1 males showed 

### degeneration in 

### seminiferous tubules 

### together with 

### significantlyfewer 

### spermatids in testes 

### (Table 5, 

### 6). 

### While the numbers of epi

### didymal sperm were 

### reduced at 

### the 

### high dose, morphology and 

### visually estimated 

### motility were 

### normal in 

### all groups; 

### the 

### high dose animals were 

### making fewer
sperm, 

### although these sperm

### appeared nor

### mal. In contrast to 

### themales, female rats in 

### both the 

### Fo and 

### F1 generations 

### seemed to 

### be 

### relatively resistant to 

### the 

### reproductive toxicity

### ofDBP in terms 

### of

### numbers of

### live
young 

### and necropsy 

### endpoints. But in 

### the 

### Fo generation, 

### females 

### appeared to 

### be more sensitive to 

### the systemic 

### toxicity 

### of DBP 

### because the 

### bodyweight of

### Fo females in 

### the 1.0% 

### dose group was 

### significantly decreased while the 

### males at 

### that concentration 

### showedno

### detectable

### change. Consistent 

### with previous reports on 

### other 

### phthalates (13), the present 

### data indicate that effects of DBP exposure are greater in animals exposed from conception 

### than 

### those exposed as 

### adults only. Because 

### the F1 rats were 

### possibly exposed during gestation 

### and throughout nursing

### andwere 

### dosedduringmaturation 

### until and 

### through mating, we cannot 

### specifywhich period of development (e.g., 

### which developmental trimester, nursing, 

### puberty, etc.) is more sensitive to 

### the effects of DBP. However, 

### the male 

### reproductive structural 

### defects suggest 

### that the 

### period 

### of organogenesis 

### andperhaps hormonal imprinting 

### for these tissues, in 

### the 

### prenatal and 

### perinatal peri

### ods, is 

### the most 

### critical time. 

### The mechanisms 

### underlying this developmental toxicity are 

### still unknown. In 

### the past, most 

### of 

### the toxicity 

### studies on 

### phthalates have focused on 

### their adverse effects on 

### dosed juvenile and adult 

### animals, rather than on pups 

### of dosed dams. 

### Nevertheless, these 

### early studies have 

### led to 

### the 

### widespread view 

### that phthalates affect the testis pri

### marily at 

### the 

### Sertoli cells (14,37,38). 

### Indeed, in 

### neonatal rat testes, 

### significant decreases in testis 

### weights and Sertoli cell numbers after 

### five daily oral doses 

### of 1000 

### mg/kg DEHP 

### have 

### been reported by Dostal et

### al. 

### (39). Jobling et 

### al. (18) found that DBP 

### interacted 

### weaklywith the estrogen receptor in a variety 

### of constructs. 

### If DBP 

### demonstrated such an interaction in vivo,

one

### would expect to 

### find altered 

### reproductive 

### development 

### (19). The present 

### study found such alterations: 

### degenerate seminiferous 

### tubules with decreased sperm counts, apparent 

### interstitial 

### cell 

### hyperplasia, and structural defects in 

### epididymides and penises 

### of 1.0% 

### DBP-treated 

### F1 rats 

### when they 

### reached maturity. 

### These mirror simi

### lar effects seen in rats 

### and mice 

### exposed perinatally to 

### the strong estrogen agonist 

### diethylstilbestrol (DES) 

### (40,41) and are consistent 

### with the 

### possibility that the 

### effects seen in 

### thepresent 

### studywere

### mediated 

### through an interaction 

### with the estrogen receptor system. 

### Interestingly, there are 

### also reports 

### showing that some 

### phthalates have 

### hormone-disrupting effects in 

### male 

### adult rats 

### (42-44).
In 

### conclusion, the 

### results from the present RACB 

### studyindicate that 

### the

### developmental 

### and reproductive toxicities 

### of DBP

are more prominent in 

### animals 

### exposed during development and maturation 

### than in 

### animals 

### exposed as 

### adults 

### only. This is prompting an investigation 

### ofthe

### period of sensitivity

### formore 

### ofthe

### phthalates.

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### 106 Volume 105, Number 1, January 1997 * EnvironmentalHealthPerspectivesArticles * 

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