Reproductive Toxicity of Di-n-butylphthalate in a Continuous Breeding

Protocol in Sprague-Dawley Rats

Robert N. 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 widespread exposure [see Thomas and Thomas

(1) for review]. The phthalate ester di-nbutylphthalate (DBP) is used in the manufacture 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

bone marrow (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 characterized 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 transfer 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 of lactating

dams to another phthalate, di (2-ethylhexyl) phthalate (DEHP), could lead to

DEHP and mono (2-ethylhexyl) phthalate

(MEHP) exposure in the suckling rat pups.

Because DBP is lipid soluble, as are DEHP

and MEHP, one might predict a certain

degree of lactational transfer of DBP.

Recently, Jobling et al. (18) reported

that DBP could reduce the binding of estrogen to the estrogen receptor and stimulate

transcriptional activity These authors called

for further in vivo data before any firm conclusions as to the estrogenicity of DBP

could be drawn. Review of archived 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-Dawley rats. If the 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 design because the Fo rats are exposed

only as adults, the F1 rats are born to mothers that are treated during gestation and lactation, and the F1 animals themselves are

treated during maturation to sexual maturity

and through mating. The results of this DBP

RACB study are presented below; 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, and Task 4 assesses the fertility

of the last litter (F1) born during continuous breeding (Task 2).

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 feed formulations. 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 work was 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

Kim Treinen for stimulating discussions. The statistical support was provided by Analytical Sciences

Inc., Durham, NC, and chemistry support was provided by Midwest Research Institute and Research

Triangle Institute under contracts NO1-ES-45060

and NO1-ES-45061, respectively.

Received 20 August 1996; accepted 18 September

1996.

102 Volume 105, Number 1, January 1997 * Environmental Health Perspectives
Articles - Reproductive toxicity of di-n-butylphthalate

weight basis. Animals in the control group

received undosed NIH-07. Each dose level

was independently formulated and prepared at least every 3 weeks and stored

frozen. Dosage formulation studies indicated 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

off the 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 Mycoplasma pulmonis (Charles

River Professional Services, Wilmington,

MA) All sera were negative for viral antibodies. Animals were quarantined separately for

approximately 3 weeks for Task 1 and 2

weeks for Task 2; rats were 70 days of age at

the start of both Tasks 1 and 2. Histopathologic evaluations were performed on

representative animals during quarantine to

check for signs of infectious 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 steel wire-bar lids. Deionized water

and rodent meal feed (NIH-07 diet; Zeigler

Bros.) were provided ad libitum.The rats

were subsequently housed 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.

Study design. DBP was administered via

feed to CD Sprague-Dawley rats. In Task 1,

the dose-finding segment of the study, doses

of 0.0 (control), 0.1, 0.5, 1.0, 1.5, and 2.0%

(w/w) DBP were tested using eight animals

of each sex per group. Endpoints for Task 1

were dinical signs of toxicity, body weight,

and food consumption.

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 provide a no-effect level. Based on the results

of the Task 1 pilot (data not shown), concentrations of 0.1, 0.5, and 1.0% DBP in

the 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 dose

groups with 20 breeding pairs per group.

The endpoints for Task 2 were clinical

signs of toxicity, parental body weight and

average consumption of feed during representative weeks, fertility (the proportion of

cohabited pairs producing any live pups),

the number of litters 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

after birth. The pups in the first four litters

were removed after birth, counted,

weighed, sexed, and killed without necropsy. The last litter, born during the holding

period following the continuous breeding

phase, was reared by the dam until weaning

(day 21 after birth); at this time, treatment

of the Fl animals was initiated by the same

route and at the same concentrations as

consumed by their parents in Task 2.

These animals were used for assessment of

second generation fertility in Task 4.

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, this was performed after the last litter of Task 2 was

weaned. The mating trial consisted of three

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

the presence of a vaginal copulatory plug or

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 of two per cage and received the same

chemical treatment as their parents. Upon

reaching sexual maturity, 20 each nonsibling F1 males and females within the same

treatment groups were housed in pairs 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 of Task 4, F1 animals in

all dose groups were killed by CO2 asphyxiation and subjected to necropsy with the

same endpoints as in the Task 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

for pairwise comparisons.

The number of litters and the number

of live 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 number of pups produced by each pair. The sex

ratio was expressed as the proportion of

male pups born alive divided by the total

number of pups 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, parameters were tested for overall differences using

the Kruskal-Wallis test (28), and multiple

comparisons were 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

equality using an F-test and pairwise 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 Task 4.

Vaginal cytology data were analyzed

using a multivariate analysis of variance

(ANOVA) (31) to test for the simultaneous

equality of measurement across dose levels.

Environmental Health Perspectives * Volume 105, Number 1, January 1997 103
Articles - Wine et al.

Before running the ANOVA, an arcsine

transformation was performed to bring the

data into close conformance with normality assumptions.

Group means, standard deviations, and

standard errors were calculated for all data

and for each sex. Jonckheere's test was performed to look for increasing or decreasing

trends. Significance among dose groups

was then calculated using Shirley's test if a

trend was evident or Dunn's test if no

trend was evident.

Results

Continuous breeding (Task 2).

Representative dosage formulations from

Task 2 were 99-106% of the target concentrations. The average daily feed consumption was similar for control and DBPdosed 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% of the controls except for highdose females at week 17 (the end of cohabitation) 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-daily health

surveillance.

Based on body weights and food consumption, the average calculated daily

intakes of DBP 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 for females, 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

treated animals.

Dam weights at delivery were significantly decreased at each litter of the high

dose group (by <10%). During nursing of

the final litter, dam weights were significantly decreased 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%; data not 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 of first generation breeding pairs

Dose groupa

Reproductive parameter 0% 0.1% 0.5% 1% Trend

Average litters per pair 4.8 ± 0.1 (40) 5.0 ± 0.1 (20) 4.9 ± 0.1 (19) 4.9 ± 0.1 (20) p= 0.322

Live pups per litter 12.9 ± 0.2 (40) 11.9 ± 0.3 (20)* 11.0 ± 0.5 (19)* 10.7 ± 0.4 (20)* p < 0.001

Absolute 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 < 0.001

Adjusted 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 groupa

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

Fertility indexd 12/15 (80%) 18/19 (95%) 15/16 (94%) p = 0.303

Live pups per litter 12.7 ± 1.3 (12) 13.4 ± 0.6 (18) 12.9 ± 0.7 (15) p = 0.934

Adjusted 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 with

other control or high dose (1.0%) animals

for 7 days. Among the three groups (control 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 fertility 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 during Task 3 and consumed

=12% less feed. There were no significant

clinical signs of toxicity 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-tobody weight ratios for the liver and kidneys

were significantly increased 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).

Secondgeneration fertility (Task 4). The

final Task 2 litters of all dose groups were

reared and exposed to the same concentrations of DBP between weaning and necropsy as 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 group were significantly (10-15%) lower than controls at

postnatal days 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/or penises and were without palpable testes. Just prior to mating (postnatal

day =80), one of these males had palpable

testes. No such abnormalities were noted

for the 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 of live pups per litter, absolute and adjusted live F2 pup

weights were =6-9% lower in all treatment

groups. The average number of days to lit104 Volume 105, Number 1, January 1997 * Environmental Health Perspectives
Articles * Reproductive toxicity of di-n-butylphthalate

ter and average dam weight at delivery were

not affected by DBP 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).

Body weights were unchanged in the middle and lower dose groups. For males, only

the 1% treatment group showed a significant 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 vesicle/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

spermatid heads per testis, a measure of testicular 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 underdeveloped or otherwise defective epididymides.

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

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

Dose group

Organ 0% 1% Difference

Male

Absolute body wt 683.6 ± 7.6 (40)8 656.8 ± 15.0 (20) p<0.154

Adjusted liver wt 37.6 ± 0.63 (40) 43.4 ± 1.1 (20)* p<0.001

Adjusted kidney wt 6.6 ± 0.11 (40) 7.3 ± 0.18 (20)* p=0.004

Female

Absolute body wt 379.0 ± 9.6 (38) 326.4 ± 6.5 (19)* p<0.001

Adjusted liver wt 34.0 ± 0.62 (38) 38.9 ± 0.69 (19)* p<0.001

Adjusted 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 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

Pregnancy indexc 19/20 (95%) 17/20 (85%) 17/20 (85%) 1/20 (5%)* p<0.001

Fertility indexd 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 live pup wt (g) 5.97 ± 0.11 (19) 5.60 ± 0.09 (17)* 5.60 ± 0.09 (17)* 5.00# p=0.020

Adjusted 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 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

Seminal vesicles 5.0 ± 0.18 (20) 5.1 ± 0.23 (20) 5.0 ± 0.17 (20) 3.9 ± 0.30 (20)* p = 0.003

Right 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 parmeters

Number 574.9 ± 37.6 (19) 575.8 ± 22.2 (20) 547.5 ± 36.9 (19) 295.1 ± 84.5 (18) p = 0.017

Percent motilec 71.0 ± 1.6 (19) 72.0 ± 2.0 (20) 69.8 ± 1.5 (20) 72.4 ± 3.2 (9) p = 0.770

Percent 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 heads

per 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 heads

per g testisf(x 107) 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

Right 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.

Environmental Health Perspectives * Volume 105, Number 1, January 1997 105
Articles * Wine et al.

Table 6. Summary of incidence of histopathologic

lesions (Task 4)

Treatment group

Control 0.5% 1.0%

Liver (total examined) (10) (10) (10)

Hepatocellular degeneration 3a 1

Focal hepatitis 6 1 4

Testis (total examined) (1O)b (10) (10)

Degeneration of 1 3 8

seminiferous tubules

Interstitial cell hyperplasia 1 1 7

Granuloma 1

Epididymis (total examined) (10) (10) (10)

Degeneration of 1 2 1

epithelial cells

Interstitial epididymitis 1 1 1

Focal granuloma 1

Underdeveloped epididymis - 5

Presence of fluid/ 1 1 2

degenerated cells

Apparent sperm content 1 1 3

reduction

Seminal vesicles (10) (10) (10)

(total examined)

Vesticulitis - 1

Inspissated secretion 1

aNumber of animals exhibiting lesion.

bOne animal had variable loss of spermatogonia,

spermatocytes, and spermatids and had an apparent

increased number of Sertoli and interstitial cells.

reduction in body weight 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 weight was reduced in

the absence of a 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 of DBP

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 and Table 6). The

structural defects included small and malformed prepuces and/or penises, underdeveloped or otherwise defective epididymides, and degenerate seminiferous

tubules. From these data alone, we cannot

deduce whether the effects represent developmental toxicity alone, if developmental

exposure renders the animals more susceptible 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 of equivalent absolute magnitude (i.e., =two pups per litter). Earlier

analyses (35) suggest that this is due in part

to the differences in statistical sensitivity: in

Task 2, up to five litters were generated,

while Tasks 3 and 4 generated only one litter. The multiple litters in Task 2 yield a

test that is statistically much more powerful

than the single litters of the Task 3 or 4

mating trials. Based on this, one might predict that the nonsignificant difference

between the controls and middle dose animals in Task 4 (14 vs. 12.8) would become

statistically significant if five litters were

produced.

While the reproductive toxicity of DBP

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

live young was 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 of the reproductive systems of F1 males showed degeneration in seminiferous tubules together

with significantly fewer spermatids in testes

(Table 5, 6). While the numbers of epididymal 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 normal. In contrast to the males, female rats in

both the Fo and F1 generations seemed to

be relatively resistant to the reproductive

toxicity of DBP 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 body weight of Fo females in

the 1.0% dose group was significantly

decreased while the males at that concentration showed no 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 and were

dosed during maturation until and through

mating, we cannot specify which 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

and perhaps hormonal imprinting for these

tissues, in the prenatal and perinatal periods, 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 primarily 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 weakly with 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 similar 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 the present study were 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 study indicate 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 of the period of

sensitivity for more of the phthalates.

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