Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non guideline study. Available as a published report. Acceptable with restrictions.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1995

Materials and methods

Principles of method if other than guideline:
continuous breeding study
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
purchased material - analysis indicated purity of 98% or greater

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI
- Age at study initiation: 10-11 wk
- Housing: in breeding pairs
- Diet (e.g. ad libitum): NIH-07 Open Formula meal (Zeigler Brothers In., Gardners, PA), ad libitum
- Water (e.g. ad libitum): deionized water, ad libitum
- Acclimation period: 2-3 wk

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
Continuous exposure to 1000, 5000 or 10000 ppm DIBP in diet over 5 litters and 2 generations
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation (continuous breeding phase): 112 days
- Length of cohabitation (for second generation): 7 days
- Proof of pregnancy: sperm in vaginal smear
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Homogeneity: confirmed using HPLC (within 10% of nominal)
- Stability in diet: confirmed using GC-FID (stable for 1 week under animal room conditions)
Frequency of treatment:
continuous, via feed
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 1000, 5000, 1000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 52, 256, 509 mg/kg bw/d
Basis:
other: approx. amount ingested, males
Remarks:
Doses / Concentrations:
0, 80, 385, 794 mg/kg bw/d
Basis:
other: approx. amount ingested, females
No. of animals per sex per dose:
Continuous breeding study: 40 controls/sex; 20 test/sex
Two-generation phase: 20/sex
Control animals:
yes, plain diet
Details on study design:
Male and female SD rats (F0) were housed in breeding pairs for 112 days for delivery of 5 litters
- individually housed for delivery of final litters while exposure continued.

Two-generation phase performed using final litters from continuous breeding phase:
- F1 pups exposed to DIBP (same level as received by parents) until sexual maturity achieved (approx. 77 d old)
- male and female pairs mated for 7 days while receiving treatment
- F1 females allowed to deliver F2 litters

Examinations

Parental animals: Observations and examinations:
- clinical signs
- body weight and feed consumption
Oestrous cyclicity (parental animals):
Estrous cycle data collected for 12 d prior to necropsy:
- cycle length (days)
- estrous stages (as percentage of cycle)
Sperm parameters (parental animals):
Sperm data collected at necropsy:
- spermatid heads (per g testis and per testis)
- epididymal sperm motility (%), concentration (per g), abnormal sperm (%)
Litter observations:
- total and live pups per litter
- sex ratio
- pup body weight
Postmortem examinations (parental animals):
- organ weights (F0 and F1)
- histopathology of selected tissues (F0 and F1)
Statistics:
For normally distributed data, the Cochran-Armitage test used to assess any dose-trend effects present and chi-square or Fisher's exact test used for control versus treatment comparisons. Trend testing of results with skewed distributions used Jonckheere's test or Wilcoxon's test, while control versus test comaprisons were performed according to Shirley, Dunn or Mann and Whitney.
Reproductive indices:
- mating index
- pregnancy index
- fertility index
- dam weight at delivery
- days to litter

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

In the F1 high dose group, only 1 of 6 females where mating was confirmed to have occurred produced a litter.

F1 adult mating index (30% of control), pregnancy index (5% of control) and fertility index (17% of control) were significantly decreased at 10000 ppm.

F1 spermatid heads were decreased significantly at 10000 ppm (47% reduction per g testis, 54% reduction per testis); epididymal spermatozoal concentration was decreased significantly at 10000 ppm (49% reduction per g cauda).

Mild to moderate degeneration of seminiferous tubules and interstital cell hyperplasia was present in 7-8 of 10 F1 males subject to microscopic examination from the 10000 ppm, with minimal or mild changes present in 1-2 of 10 males from the 5000 ppm group (absent from the 1000 ppm group).

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

Pup weights were decreased in all groups (93% of control at 1000 and 5000 ppm; 84% of control at 10000 ppm)

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
5 000 ppm (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decreased spermatid heads in testis, decreased epididymal spermatozoal concentration
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
5 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: decreased mating index, pregnancy index and fertility index
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 ppm (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: degeneration of seminiferous tubules and interstital cell hyperplasia

Results: F2 generation

Effect levels (F2)

Dose descriptor:
LOAEL
Generation:
F2
Effect level:
1 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: decreased pup weights

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Treatment with dibutyl phthalate over two generations was associated with reduced pregnancy and fertility indices, decreased pup weights, reductions in sperm and spermatid counts and degeneration of seminiferous tubules and interstital cell hyperplasia.
Executive summary:

The effects of dibutyl phthalate on reproductive functions was examined in male and female SD rats. Animals were intially housed in breeding pairs as part of a continuous breeding study, with treatment extending to 112 days with delivery of 5 litters. A second generation was then mated using pups from the final litters of the continuous breeding phase, with animals receiving DIBP to sexual maturity (approx. 77 d old) after which they were mated. Dose levels of 0, 1000, 5000 or 10000 ppm DBP in diet were used, equivalent to received treatments of approx. 52, 256 or 509 mg/kg bw/d for males, and 80, 385 and 794 mg/kg bw/d for females. Pregnancy and fertility indices for F1 parents were statistically significantly lower for the 10000 ppm group, with live F2 pup weights significantly decreased at all dose levels. Decreased epididymal sperm count and testicular spermatid head count were apparent at 10000 ppm, with mild to moderate degeneration of seminiferous tubules and interstital cell hyperplasia present in the 5000 ppm and 10000 ppm F1 males. An overall LOAEL of 1000 ppm (52-80 mg/kg bw/d) was obtained for effects on pup weight, with a NOAEL of 1000 ppm (52 mg/kg bw/d) for microscopically detected changes in male gonadal tissue.