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Toxicological information

Toxicity to reproduction: other studies

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Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Published literature fulfilled basically scientific principles.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: in utero through lactational exposure studies
GLP compliance:
no
Remarks:
Publication
Type of method:
in vivo

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test substanceswere dicyclohexyl phthalate (Fig. 1, CAS No.: 84-61-7, Purity 99.9%, Wako Pure Chemical Industries, Ltd., Osaka, Japan) and olive oil (Maruishi Pharmaceutical Co. Ltd., Osaka, Japan) which was used as the vehicle.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Forty-eight Crl:CD (SD)male and female rats at 11 weeks of age were purchased from Charles River Japan, Inc. (Tukuba, Japan) and were mated to each other at 12 weeks of age. After detecting vaginal plugs or sperm in vaginal smears (GD 0), 40 dams were divided into 4 experimental groups consisting of 10 dams per group by a body-weight-stratified randomization method to minimize the variation in body weights between groups. The dams were kept in stainless steel cages (170mm (W)×294mm(D)×176 mm (H)) until GD 18, and then the offspring were kept with dams in stainless steel cages (340mm (W)×294mm (D)×176 mm (H)) with bedding until weaning. All off-spring were weaned at PND 21 and then housed in stainless steel wire mesh cages (170mm (W)×294mm (D)×176 mm (H)) for the remainder of the study. Water was provided automatically, and the animals were given access to a commercial diet (CRF-1,Oriental Yeast Co., Chiba, Japan) ad libitum. The animal room was maintained at a temperature of 23±2 °C with a relative humidity of 55 ± 15%. The room was artificially illuminated with fluorescent light on a 12 h light/dark cycle (08:00-20:00 h). All animals were cared for in accordance with the principles outlined in the guide for animal experimentation prepared by the Japanese Association for Laboratory Animal Science.

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
Dams were orally gavaged with 0, 20, 100, and 500mg/kg/day of the test chemical from GD 6 to PND 20. In the preliminary test, dams were orally gavaged with 0, 100, and 500mg/kg of test chemical from GD 6 to PND 20, which resulted in toxic changes and abnormal reproductive performances observed in dams and their offspring in groups receiving 100 and/or 500mg/kg. Therefore, we selected those doses. A vehicle control group was gavaged with olive oil alone. The volume of the olive oil solution containing the chemical for gavage was 2ml/kg.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration and stability of the test chemical in the vehicle were confirmed.
Duration of treatment / exposure:
Dams were orally gavaged with 0, 20, 100, and 500mg/kg/day of the test chemical from GD 6 to PND 20.
Frequency of treatment:
Once per day
Duration of test:
no data
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0
Basis:
nominal conc.
The concentration and stability of the test chemical in the vehicle were confirmed.
Remarks:
Doses / Concentrations:
20 mg/kg/day
Basis:
nominal conc.
The concentration and stability of the test chemical in the vehicle were confirmed.
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
nominal conc.
The concentration and stability of the test chemical in the vehicle were confirmed.
Remarks:
Doses / Concentrations:
500 mg/kg/day
Basis:
nominal conc.
The concentration and stability of the test chemical in the vehicle were confirmed.
No. of animals per sex per dose:
40 dams were divided into 4 experimental groups consisting of 10 dams per group by a body-weight-stratified randomization method to minimize the variation in body weights between groups.
Control animals:
yes, concurrent vehicle
Details on study design:
Animals and study designs
Forty-eight Crl:CD (SD)male and female rats at 11 weeks of age were purchased from Charles River Japan, Inc. (Tukuba, Japan) and were mated to each other at 12 weeks of age. After detecting vaginal plugs or sperm in vaginal smears (GD 0), 40 dams were divided into 4 experimental groups consisting of 10 dams per group by a body-weight-stratified randomization method to minimize the variation in body weights between groups. The dams were kept in stainless steel cages (170mm (W)×294mm(D)×176 mm (H)) until GD 18, and then the offspring were kept with dams in stainless steel cages (340mm (W)×294mm (D)×176 mm (H)) with bed ding until weaning. In order to balance provision of nutrition the litters were culled according to OECD guideline for the testing chemicals (no. 415, One-Generation
Reproduction Toxicity Study) on PND 4, keeping 4 males and 4 females per dams. Whenever the number of male or female offspring prevents having 4 of each sex per litter, the litter size was adjusted to a total of 8 offspring regardless of sex. All off-spring were weaned at PND 21 and then housed in stainless steel wire mesh cages (170mm (W)×294mm (D)×176 mm (H)) for the remainder of the study. Water was provided automatically, and the animals were given access to a commercial diet (CRF-1,Oriental Yeast Co., Chiba, Japan) ad libitum. The animal room was maintained at a temperature of 23±2 °C with a relative humidity of 55 ± 15%. The room was artificially illuminated with fluorescent light on a 12 h light/dark cycle (08:00-20:00 h). All animals were cared for in accordance with the principles outlined in the guide for animal experimentation prepared by the Japanese Association for Laboratory Animal Science.

Dams were orally gavaged with 0, 20, 100, and 500mg/kg/day of the test chemical from GD 6 to PND 20. In the preliminary test, dams were orally gavaged with 0, 100, and 500mg/kg of test chemical from GD 6 to PND 20, which resulted in toxic changes and abnormal reproductive performances observed in dams and their offspring in groups receiving 100 and/or 500mg/kg. Therefore, we selected those doses. A vehicle control group was gavaged with olive oil alone. The volume of the olive oil solution containing the chemical for gavage was 2ml/kg. The concentration and stability of the test chemical in the vehicle were confirmed. The general and nursing states of all dams were monitored daily, and their body weights were measured at GD 0, 6, 13 and 20, and PND 4, 7, 14 and 21. The gestational period of each dam was calculated. On the day of delivery, the number of live newborns and stillborns and the sex ratio of the pups were recorded, and the pups were inspected for external anomalies. Moreover, the live viability index was also calculated on PND 4 and 21 (weaning day). The dams were euthanized under ether anesthesia and necropsied the day after weaning. The following organs were weighed: liver, kidneys, ovaries and thyroid. The number of implantation sites in each uteruswas counted by immersing the incised uterus in ammonium sulfate solution. The delivery and birth indexes were calculated.
Before weaning the pups, each group was randomly assigned to two groups: a group sacrificed at 10 weeks of age and another group to evaluate the reproductive performance of the offspring (caesarean group). The general and nursing states of all offspring were monitored daily, and their body weight was measured at PND 0, 4, 7, 14 and 21, and weekly intervals thereafter until sacrifice. Ano-genital distance was measured at PND 4. At PND 13 offspring were examined for retention of thoracic and abdominal nipples. Vaginal opening was examined for all weaning female rats from PND 21, and preputial separation was examined for all male rats from PND 35 of age until it occurred.

Group sacrificed at 10 weeks of age
All pups, except for the caesarean group, were examined. Clinical signs were recorded daily, and detailed clinical observations were made of all animals. The ventral surface of each animal was examined for abnormalities, including the numbers and location of retained nipples, cleft phallus, vaginal pouch, and hypospadias. Individual body weight was recorded weekly and immediately before necropsy. The estrous cycle of the female offspring was evaluated based on vaginal cytology from 8 weeks of age and the animalswere killed in the diestrous stage after 10weeks of age. Rats were necropsied and examined internally for ectopic or atrophic testes; agenesis of the gubernaculums, epididymides and sex accessory glands; and epididymal granulomas. The following organs were weighed after necropsy: uterus, ovaries, testes, epididymides, ventral prostate, seminal vesicles with coagulation gland, levator ani and bulbocavernosus muscles, brain, liver, adrenals, kidneys, thyroids, and pituitary.

Caesarean group
Two females and 2 males per dam in each group were assigned to this group. The treated females in each group were mated with treated males at 12weeks of age without brother–sister mating, and the copulation index (%, number of copulated females/number of mated females×100) and fertility index (%, number of pregnant females/number of copulated females×100) were calculated. Caesarean sections were performed under anesthesia at GD 13, and the implantation index (%, number of implantations/number of corpora lutea×100) and loss (%, number of intrauterine deaths/number of implantation×100)were calculated. Furthermore, male rats were necropsied on the same day the caesarean section was performed on females. At the time of necropsy, the same organs described in the sacrificed group were weighed.

Histopathological examination
The organs of all male and female offspring including sacrificed and caesarean groups were examined as follows: liver, kidneys, testes, epididymides, uterus, ovaries, vagina, pituitary and thyroids.

Statistics:
Copulation, fertility, gestation indices, and histopathological findings were analyzed by chi-square test between the control and the dicyclohexyl phthalate-exposed groups. Statistical analysis of offspring before weaning was carried out using the litter as the experimental unit. All data of the birth indexes of dams, incidence of external malformations and viability of offspring for control and exposure groups were arranged in descending order, and a Kruskal–Wallis rank sum test was performed. Statistical differences in the rank means among the groups were analyzed by Dunnett’s multiple comparison tests. Other parameters were analyzed by Dunnett’s test. First, Bartlett’s test was used to test for homogeneity of variance. When the variance was homogeneous, one-way ANOVA was used. When the variance was not homogeneous, Kruskal–Wallis rank sum test was performed, by arranging all data for the control and exposure groups in descending order. Statistical differences in the means and the rank means among the groups were analyzed by Dunnett’s multiple comparison test, and the same multiple comparison test by rank, respectively. A two-sided analysis with a p-value of 0.05 was performed

Results and discussion

Effect levels

Dose descriptor:
NOAEC
Remarks on result:
not determinable
Remarks:
no NOAEC identified

Observed effects

Using the dicyclohexyl phthalate, a dam in the 500mg/kg group showed dystocia and died. The viability index of offspring on PND 4 decreased in the 500mg/kg group. Prolonged preputial separation, reduced anogenital distance, increased areolas/nipple retention, hypospadia, decreased ventral prostate and levator ani/bulbocavernosus muscle weights and decreased testicular germ cells were observed inmale offspring in the 500mg/kg group.

Any other information on results incl. tables

Dams

The absolute and relative liver weights increased in the 100 and 500mg/kg dicyclohexyl phthalate groups. A dam in the 500mg/kg dicyclohexyl phthalate group showed dystocia and died on GD 23 before delivery was completed. No abnormal bodyweight changes were detected in any group.

Offspring

The viability index on PND 4 (%, number of live pups on PND 4/number of live pups on PND 0×100) in the 500mg/kg dicyclohexyl phthalate group was lower than the control group, and no abnormalities were detected in other reproductive parameters. The body weights of male and female pups decreased significantly in the 500mg/kg dicyclohexyl phthalate group at PND 14 and/or 21 (no data shown). Hypospadia accompaniedwith small testes was detected in 2 male rats in the 500mg/kg dicyclohexyl phthalate group and one of them was sacrificed at 7 weeks of age for poor general condition. A prolonged preputial separation, a reduced anogenital distance, and an increase in areola/nipple retention were detected in the male rats in the 500mg/kg dicyclohexyl phthalate group. No abnormalities in vaginal opening day or estrous cycle were observed in any female group.

Group sacrificed at 10 weeks of age

The ventral prostate and levator ani/bulbocavernosus muscle weights decreased in the 500mg/kg dicyclohexyl phthalate group. Histologically, decreased testicular germ cells and degenerated renal proximal tubules were detected in some male rats in the 500mg/kg dicyclohexyl phthalate group.

 

Caesarean group

No changes in the reproductive parameters were detected in any dicyclohexyl phthalate-exposed group (no data shown).

Applicant's summary and conclusion

Executive summary:

Utero through lactational exposure assay using dicyclohexyl phthalate to investigate the endocrine-mediated effects of DCHP.Pregnant CD (SD) IGS rats were given dicyclohexyl phthalate orally from gestational day (GD) 6 to postnatal day (PND) 20, and the endocrine-mediated effects in dams and their offspring were examined. The reproductive performance of offspring was also examined. The doses of dicyclohexyl phthalate were 0, 20, 100, and 500mg/kg/day. Using the dicyclohexyl phthalate, a dam in the 500mg/kg group showed dystocia and died. The viability index of offspring on PND 4 decreased in the 500mg/kg group. Prolonged preputial separation, reduced anogenital distance, increased areolas/nipple retention, hypospadia, decreased ventral prostate and levator ani/bulbocavernosus muscle weights and decreased testicular germ cells were observed inmale offspring in the 500mg/kg group The endocrine-mediated effects were detected in offspring of dams given 100mg/kg dicyclohexyl phthalate.