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Repeated dose toxicity: oral

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repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented and scientifically acceptable, according to GLP guidelines
Reason / purpose:
reference to other study

Data source

Reference Type:
study report

Materials and methods

Principles of method if other than guideline:
No guideline specified, cf. "Any other information on material and methods" for details.
GLP compliance:

Test material

Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
Dibutyl phthalate (Lot L-121 1-83) was obtained from Chem Central (Kansas City, MO). Initia lidentity and purity analyses were performed by Midwest Research Institute (MRI, Kansas City, MO).

The chemical, a colorless liquid, was identified as dibutyl phthalate by infrared, ultraviolet/visible, and nuclear magnetic resonance (NMR) spectroscopy; spectra were consistent with those expected for the structure of dibutyl phthalate, with a literature reference (Sadtler Standard Spectra), and with a previously analyzed lot of dibutyl phthalate (Lot C100682) that was not used in the current studies. The results of elemental analysis for carbon and hydrogen were in agreement with theoretical values. Karl Fischer analysis indicated 0.106 ± 0.002% water. Free acid titration indicated less tha n
0.001 mEq acid/g sample; titration of the ester group by hydrolysis followed by back titration with hydrochloric acid indicated a purity of 97.2 ± 0.3%. Thin-layer chromatography (TLC) by tw o solvent systems indicated a major spot only. Gas chromatographic analysis by two systems with a flame ionization detector (FID) indicated a major peak and three impurities with a combined area of 0.4% relative to the major peak area. Gas chromatographic major peak comparison indicated that samples from two drums of Lot L-121 1-83 were identical within the limits of experimental error ; these samples had a purity of 99.8 ± 0.3% relative to a concomitantly analyzed sample of Lot C100682. The cumulative data indicated a purity of 98% or greater for Lot L-121 1-83.

Test animals

Fischer 344
Details on test animals and environmental conditions:
Male and female F344/N rats used in the standard 13-week studies (without perinatal exposure) were obtained from Simonsen Laboratories (Gilroy , CA). Rats studies were 29 to 30 days old at receipt. Quarantine periods and ages of rats and mice at the beginning of the studies are given in Table 2. Blood samples were collected from five rats of each sex at the beginning of each study. Blood was also collected from five male and five female control rats at the end of the 13-week studies. The sera were analyzed for antibody titers to rodent viruses (Boorman et al., 1986; Rao et al., 1989). Results for all studies were negative.

Administration / exposure

Route of administration:
oral: feed
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Doses / concentrations
Doses / Concentrations:
0, 2500, 5000, 10000, 20000, 40000 ppm

No. of animals per sex per dose:
Control animals:


Observations and examinations performed and frequency:
For ALL examinations cf. Tables 2 & 3 and "any other information on materials and methods"
In the standard 13-week studies, two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which are approximately normally distributed, were analyzed using the parametric multiple comparisons procedures of Williams (1971, 1972) or Dunnett (1955). The following parameters, which typically have skewed distributions, were analyzed with the nonparametric multiple comparisons methods of Shirley (1977) or Dunn (1964): hematology, clinical chemistry, spermatid and epididymal spermatozoal data, palmitoyl-CoA oxidase activity data, zinc and testosterone concentrations, gestation length, and live pups per litter (number and percentage).
For all studies, Jonckheere's test (Jonckheere, 1954) was used to assess the significance of dose response trends and to determine whether a trend-sensitive test (Williams' or Shirley's test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose response (Dunnett's or Dunn's test). Trend-sensitive tests were used when Jonckheere's test was significant at a P-value less than 0.01.
Prior to analysis, extreme values identified by the outlier test of Dixon and Massey (1951) were examined by NTP personnel. Implausible values, extreme values from animals that were suspected of being sick due to causes other than treatment, and values that the study laboratory personne l indicated as being inadequate due to technical problems were eliminated from the analysis.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
all rats survived
no mortality observed
Description (incidence):
all rats survived
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related

Effect levels

Dose descriptor:
Effect level:
5 000 ppm
Basis for effect level:
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

All rats survived to the end of the study (Table 17). Males receiving 10,000 ppm or greater an d females receiving 20,000 or 40,000 ppm had lower final mean body weights and mean body weight gains than the respective controls (Table 17 and Figure 4). All male and female rats that received 40,000 ppm were emaciated. No other clinical signs were considered related to exposure. Feed consumption by males and females receiving 40,000 ppm was lower than that by the controls (Table 17).

Relative liver and kidney weights were significantly greater in male rats administered 5,000 ppm dibutyl phthalate or greater than in the controls (Table 18). Absolute liver weights were greater in male rats receiving 5,000, 10,000, or 20,000 ppm than in the controls. Absolute and relative liver weights and relative kidney weights were greater in females receiving 10,000 ppm or greater than in the controls. In male rats that received 20,000 or 40,000 ppm, absolute and relative testis weights were significantly less than those of the controls. Other statistically significant differences in organ weights were considered secondary to body weight changes.

Hematology and clinical chemistry results are provided in Tables 19 and 20. Treatment-related hematology changes occurred only in male rats and were characterized primarily by a minimal anemia in males receiving 5,000 ppm or greater (Table 19). This anemia was evidenced by lower Hct values, Hgb concentrations, and RBC counts in exposed males than in the controls. Hemoconcentration by dehydration was evidenced by higher albumin concentrations in exposed groups than in the controls (Table 20), and the anemia may have been more severe than the data indicate. Reticulocyte counts of exposed and control males were similar, suggesting no bone marrow response to the minimal anemia. However, MCV values were slightly greater in exposed groups than in the controls , suggesting that young cells were being released from the bone marrow maturation pool. NRBC counts in males and females in the 40,000 ppm groups were four to five times higher than control values, possibly indicating an inappropriate release of erythroid precursors from the bone marrow. The number of platelets in males receiving 5,000 ppm or greater were higher than in the controls; this would be compatible with a reactive thrombocytosis. Other differences in hematology parameters were not consistent and did not indicate a treatment-related response.

As was mentioned previously, minimally higher concentrations of albumin occurred in all treated groups of male rats (Table 20); this would be compatible with hemoconcentration by dehydration. In male rats in the 40,000 ppm group, the change was counterbalanced by a lower total protei n concentration than in the controls, suggesting that the globulin protein fraction in this group wa s lowered. The globulin fraction may have been reduced in female rats as well; total protein concentrations were lower in female rats in the 20,000 and 40,000 ppm groups than in the controls, with no changes in albumin concentration evident. Triglyceride and cholesterol concentrations were lower in exposed male and female rats than in the controls; these differences were most pronounced in the 20,000 and 40,000 ppm groups and were more pronounced in males than females. Triglyceride concentrations were more affected than cholesterol concentrations, as evidenced by the greate r percentage differences and the higher number of exposure groups affected. Cholestasis was evidenced by higher alkaline phosphatase activities and bile salt concentrations in exposed groups than in the controls; this change primarily involved males and females in the 20,000 and 40,000 ppm groups, though females in the 5,000 and 10,000 ppm groups also had similar but milder changes.

Liver palmitoyl-CoA oxidase activities were significantly higher in male and female rats receivin g 5,000 ppm or greater than in the controls (Table 21). Liver palmitoyl-CoA oxidase activities were 13fold higher in males and 32-fold higher in females administered 40,000 ppm than in the controls. The serum concentration of zinc in male rats in the 40,000 ppm group was slightly lower than that in the controls (Table 22); testis zinc was lower in males in the 20,000 and 40,000 ppm groups than in the controls. Serum testosterone was also lower in males in the 20,000 and 40,000 ppm groups than in the controls; however, testis testosterone concentrations in exposed and control rats were similar.

As in the 13-week study with perinatal exposure (Table 18), the liver and testes in rats in the standard 13-week study were identified as sites of dibutyl phthalate toxicity (Table 23). Microscopic examination revealed a liver lesion that was characterized as cytoplasmic alteration (Table 23) and that consisted of hepatocytes with a more intensely staining eosinophilic cytoplasm and fewer small, clear vacuoles than in the controls. Subsequent staining of adjacent sections with PAS, with and without diastase, confirmed this to be a decrease in the number of glycogen-containing vacuoles. Small, fine, eosinophilic granules were more commonly observed in the cytoplasm of hepatocytes from livers of rats in the 40,000 ppm groups than in the controls. The no-effect level for the cytoplasmic alterations in the liver was considered to be 5,000 ppm for males and females. In a subset of animals examined for ultrastructural changes, peroxisome proliferation was clearly evident in males and females in the 40,000 ppm groups. Electron-dense, membrane bound structures, consistent with tertiary phagolysomal bodies or lipofuscin, were also observed. Subsequent staining for lipofuscin was conducted on formalin-fixed, paraffin-embedded tissue. The presence of dark green, granular staining of the cytoplasm of hepatocytes, consistent with lipofuscin, was observed in Shmorl's-stained sections. The staining intensity increased with increasing exposure concentration, becoming more diffusely distributed, and the staining was generally more severe in males than in females. Rats that received 20,000 ppm or greater had greater staining than th e controls; in the 20,000 ppm groups, the lesion was mild and diffusely distributed in all males and was restricted to focal accumulations of increased Schmorl's-positive staining in four of five females. In all males and females receiving 40,000 ppm, the lipofuscin accumulation was diffuse and more severe, with fine to coarse Schmorl's-positive granules outlining bile canaliculi. Although the positive control tissue (cardiac muscle) stained intensely, no positive staining was detected with the AFIP method.

The testicular lesion was characterized by degeneration of the germinal epithelium (Table 23). In males exposed to 40,000 ppm, the lesion was diffuse and consisted of an almost complete loss of germinal epithelial cells in all seminiferous tubules, with no spermatogenesis occurring. The tubules were lined only with Sertoli cells, many of which contained vacuolated cytoplasm. This lesion also occurred in the 20,000 ppm group; however, in this group the atrophy was focal in distribution. Four of 10 rats in the 10,000 ppm group were also considered to have focal atrophy of seminiferous tubules. The no-effect level was considered to be 5,000 ppm. Marked hypospermia of the epididymis was present in the 20,000 and 40,000 ppm groups; the no-effect level for this lesion was determined to be 10,000 ppm.

Additional reproductive parameters were evaluated in males and females in the 0, 2,500, 10,000, and 20,000 ppm groups. Left epididymal, cauda epididymal, and testis weights, the number of spermatid heads per testis and per gram testis, spermatid count, an d epididymal spermatozoal motility and concentration were significantly lower in males in the 20,000 ppm group than in the controls. There were no significant differences in estrous cycle length or in the percentage of time spent in the various estrous stages between exposed an d control female rats.

Applicant's summary and conclusion

Several effects were shown, especially on rats receiving 5000 ppm DBP and more.
Executive summary:

This study is part of a series of examinations on DBP performed by the National Toxicology Programm from 1987 till 1989.