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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
three-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented and scientifically acceptable, according to GLP guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995

Materials and methods

Principles of method if other than guideline:
The effects of exposure to dibutyl phthalate on reproduction were assessed by the performance of continuous breeding studies in Sprague-Dawley rats administered dibutyl phthalate in feed. Reproductive assessment consists of four phases: dose finding, continuous breeding, identification of the affected sex, and offspring assessment.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Dibutyl phthalate was obtained from Chem Central (Kansas City, MO). The cumulative results of elemental analyses, Karl Fischer water analysis, free acid and ester hydrolysis titration, thin-layer chromatography, and gas chromatography indicated a purity of 98% or greater. Stability studies indicated that dibutyl phthalate is stable as a bulk chemical for 2 weeks when stored protected from light at temperatures up to 60° C.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Male and female VAF Crl:CD BR outbred Sprague-Dawley albino rats were used in the 2-week dose-setting and continuous breeding studies; rats were obtained from Charles River Breeding Laboratories (Portage, MI). Rats in the continuous breeding study were approximately 8 weeks old at receipt. Rats were quarantined for 2 to 3 weeks before the start of the studies. Blood samples were periodically collected from sentinel rats and were also collected from study rats; these samples were analyzed for antibody titers to rodent viruses. A single serum sample from a sentinel rat showed a positive antibody response to mouse encephalomyelitis virus; however, no clinical signs of disease were detected in the study rats.

Administration / exposure

Route of administration:
oral: feed
Duration of treatment / exposure:
119 d
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
2 week dose setting phase: 0, 1000, 5000, 10000, 15000, 20000 ppm; continuous breeding phase: 1000, 5000, 10000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
8 (2 week dose setting phase)
20 (continuous breeding phase)
Control animals:
yes

Examinations

Litter observations:
Clinical signs of toxicity, body weights, feed consumption, fertility, number of litters per pair, number of live pups per litter, proportion of pups born alive, sex ratio of live pups, and pup body weights were recorded at birth.
Postmortem examinations (parental animals):
At necropsy, sperm data were collected and the following organs were weighed: liver, kidneys, right cauda epididymis, right epididymis, right testis, right ovary , prostate gland, and seminal vesicles
Postmortem examinations (offspring):
At the end of the study, F1 rats were necropsied; organ weights (kidney, liver, right cauda epididymis, right epididymis, right testis, prostate gland, seminal vesicles, and right ovary) and body weights were determined, and sperm morphology and vaginal cytology evaluations were made for 12 days prior to necropsy.
Statistics:
For data expressed as proportions (fertility, mating, and pregnancy indexes), the Cochran-Armitage test (Armitage, 1971) was used to test for dose-related trends. Each dose group was compared to the control group with a chi-square test (Conover, 1971) or Fisher's exact test. A chi-square test for homogeneity was used to identify overall differences in fertility and for pairwise comparisons in the crossover mating trials. The number of litters and the number of live pups per litter were determined per fertile pair and then 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 number of male pups born alive divided by the total number of live pups born to each fertile pair.
Dose group means for data with skewed distributions were analyzed by the nonparametric multiple comparisons methods of Shirley, Dunn, or Mann and Whitney. Jonckheere's test or Wilcoxon's test was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test (Shirley) was more appropriate for pairwise comparisons than a test capable of detectin g departures from monotonic dose response (Dunn). In the crossover mating trials, parameters were tested for overall differences by the Kruskal-Wallis test (Kruskal and Wallis, 1952), and multiple comparisons were made with Dunn's test or the Mann-Whitney U test.
Analyses of covariance (Neter and Wasserman, 1974), with average litter size as the covariate, were performed t o remove the potential effect of number of pups per litter on average pup weight. Least-square estimates of dose group means adjusted for litter size were tested for overall equality by an F-test and for pairwise equality by Dunnett's test or a t-test; these tests were performed on males, females, and males and females combined to analyse potential sex differences.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related

Effect levels (P0)

Dose descriptor:
LOAEL
Effect level:
1 000 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related

Effect levels (F1)

Dose descriptor:
LOEC
Generation:
F1
Effect level:
10 000 ppm
Sex:
male
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

2 -Week Dose-Finding Phase

All rats survived to the end of the 2-week phase. The mean body weight gain of males in the 20,000 ppm group was 10% less than that of control males, and females exposed to 20,000 ppm lost weight. Feed consumption by males exposed to 10,000 ppm or greater was decreased during Week 1. Feed consumption by females decreased with increasing exposure concentration during Week 1, and feed consumption by females exposed to 20,000 ppm was also decreased during Week 2. No clinical signs related to dibutyl phthalate exposure were noted.

Continuous Breeding Phase One male and one female exposed to 5,000 ppm died during the continuous breeding period; the male was killed due to paralysis of undetermined etiology, and the female due to renal failure. The mean body weight of females in the 10,000 ppm group was 11% lower than that of the controls at 17 weeks (the end of the continuous breeding period). Feed consumption by exposed animals was generally similar to that by the controls.

All control and exposed pairs were fertile (Table E1). The average number of litters per pair and cumulative days to litter for exposed pairs were similar to those of the controls. The mean body weight of dams in the 10,000 ppm group was significantly lower than that of the controls at the delivery of each litter and throughout lactation of the final litter; the mean body weights of dams in the 1,000 and 5,000 ppm groups were also slightly lower than the mean body weight of control dams during lactation, and the decreases were significant on Days 14 and 21 for dams in the 5,000 ppm group and on Day 21 for dams in the 1,000 ppm group (Table E1). The number of live pups per litter generall decreased with increasing exposure concentration (Table E2). Male pup weights in three of five litters in the 5,000 and 10,000 ppm groups and female pup weights in all litters in the 10,000 ppm group were significantly lower than those of control pups. Total and adjusted live pup weights of all litters in the 10,000 ppm group and two litters in the 5,000 ppm group were significantly lower than in the controls; two other litters in the 5,000 ppm group also had lower adjusted live pup weights than the controls. The average ratio of live male pups to live pups was decreased in the first and third litters in the 10,000 ppm group. No biologically significant clinical signs of toxicity were noted.

For the final litter of pups reared to weaning, the weights of male and female pups from breeding pairs in the 10,000 ppm group were less than those of control pups through postnatal Day 21 (Table E3). The number of live pups per breeding pair and pup survival in exposed groups were similar to those of the controls.

Crossover Mating Trial

During the crossover mating trial, one control female died due to lymphoma and one female from the 10,000 ppm group died due to cardiac failure. These deaths were not attributed to exposure to dibutyl phthalate. There were no significant differences in mating, pregnancy, or fertility indexes or number of days to litter between the groups (Table E4). During the week of breeding, the mean body weight of females exposed to 10,000 ppm was significantly less than that of control dams; at delivery, the mean body weight of exposed dams remained slightly less than that of the controls. No significant clinical signs of toxicity were noted.

Relative kidney and liver weights of exposed males and females and the absolute liver weight of exposed males were significantly greater than those of the controls (Table E5). The relative right cauda epididymal weight of exposed males was also significantly increased. There were no significant differences in spermatid or epididymal spermatozoal measurements in exposed males or in estrous cycle lengths in exposed females (Table E6). These trials suggest that fertility and gametogenesis were unaffected in the F0 rats that received 10,000 ppm.

Offspring Assessment Phase

All F1 rats survived to the end of the offspring assessment phase. The mean body weights of male and female F1 rats exposed to 10,000 ppm were less than those of the controls at weaning (males, 15%; females, 7%) and remained less throughout breeding; however, mean body weights of control and exposed dams were similar at delivery (Table E7). During the week of breeding, feed consumption by rats in the 10,000 ppm group was less than that by the control group. Mating, pregnancy, and fertility indexes of rats in the 10,000 ppm group were significantly decreased, and only a single litter was produced by dams in this group. These data suggest that adverse reproductive effects were present in both male and female F1 rats receiving 10,000 ppm. Female (F2 ) pup weights, total live pup weights, and adjusted live pup weights were decreased in all exposed groups (Table E7), and the adjusted live male pup weight for litters in the 5,000 ppm group was also decreased. There were no clinical signs of toxicity in F1 rats or F2 rat pups.

At necropsy, the relative kidney weights of F1 males in the 5,000 and 10,000 ppm groups and the relative liver weight of males in the 10,000 ppm group were significantly increased (Table E8); absolute and relative right epididymal, cauda epididymal, and testis weights, prostate gland weights, and seminal vesicle weights were decreased in males exposed to 10,000 ppm. Absolute kidney, liver, and right ovary weights of females in the 10,000 ppm group were decreased. Spermatid heads per testis and per gram testis, spermatid count, and sperm concentration were significantly decreased in F1 males (Table E9). Epididymides were absent or poorly developed in 12 of 20 males in the 10,000 ppm group and in 1 of 20 males in each of the two lower exposure groups. The testes of four males exposed to 10,000 ppm and one male exposed to 5,000 ppm were atrophied. The testes of three males in the 10,000 ppm group were not descended into the scrotal sacs; four males in this group had poorly developed seminal vesicles and four had an underdeveloped prepuce or penis. No significant differences in estrous cycle length or in the percent of time spent in the various estrous stages were noted in females, although an extended estrous cycle (primarily due to a longer estrus) was suggested by the data (Table E9). These necropsy data also suggest that the reproductive system was a target of dibutyl phthalate toxicity in male and female F1 rats receiving 10,000 ppm. Delayed maturation is suggested as well in male F1 rats receiving 5,000 ppm or greater, based upon the testicular and accessory gland findings.

Applicant's summary and conclusion

Conclusions:
A multitude of effects were shown in the offspring generations.
Executive summary:

This study is part of a series of examinations performed by the National Toxicity Programm.