Registration Dossier

Administrative data

Endpoint:
developmental toxicity
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:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
No declaration of guideline compliance in the article, but it seems the method was similar to the OECD guideline 414.
GLP compliance:
no
Remarks:
Publication
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
DnHP (CAS 84-75-3) and DCHP (CAS 84-61-7) were purchased from Tokyo Chemical Industry (Tokyo, Japan). Their identity was confirmed by gas chromatography mass spectrometry (GC/MS) and infrared spectroscopy. Their purity (>98 and 99%, respectively) was verified by gas chromatography.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Sprague–Dawley rats were supplied by Charles River Laboratories (Saint-Germain-sur-l’Arbresle, France).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
Pregnant females were administered DnHP or DCHP, by gavage, at 0, 250, 500 or 750 mg/kg per day. Dosing occurred once daily, in the morning, on gestational days (GD) 6-20. The dosing volume was 5 ml/kg for DnHP, and 10 ml/kg for DCHP because of its limited solubility in oil. Initial doses were based on GD 6 weight and adjusted every 3 days throughout the treatment period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing solutions were formulated in olive oil as vehicle. Olive oil complied with the quality standards of the European Pharmacopoeia. Analysis by gas chromatography indicated that the formulations were stable for up to 14 days, when stored in a dark place at room temperature.
Details on mating procedure:
After 1-2 weeks of acclimatization, primiparous female (180-200 g) Sprague-Dawley rats supplied by Charles River Laboratories (Saint-Germain-sur-l’Arbresle, France) were housed overnight with adult males from the same strain and supplier. The day sperm was detected in the vaginal smear was considered to be day 0 of gestation.
Duration of treatment / exposure:
GD 6–20; Total 15 days.
Frequency of treatment:
Dosing occurred once daily, in the morning.
Duration of test:
20 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0
Basis:
other: Control group; olive oil only
Remarks:
Doses / Concentrations:
250 mg/kg per day
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
500 mg/kg per day
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
750
Basis:
analytical conc.
mg/kg per day
No. of animals per sex per dose:
Group size ranged from 24 or 25 time-mated rats (20–25 pregnant).
Control animals:
yes, concurrent vehicle
Details on study design:
The day sperm was detected in the vaginal smear was considered to be day 0 of gestation. Mated females were singly housed in clear polycarbonate cages with stainless steel wire lids and corn cob granules as bedding. Food pellets (UAR Alimentation Villemoisson, France) and filtered tap water were available ad libitum. Animal rooms were maintained at 21 ± 2 °C, a relative humidity of 50 ± 5% and a 12 h light–dark photocycle. Mated females were randomly assigned to treatment groups by stratified randomization so that mean body weights on GD 0 did not differ among treatment groups.

Examinations

Maternal examinations:
All females were observed daily for clinical signs of toxicity. Food consumption was measured at three day intervals starting on GD 6. Maternal body weights were recorded on GD 0, 6, 9, 12, 15, 18 and 21. On GD 21, the females were killed by an intrapulmonary injection of T61® (Intervet International, GmbH, Unterschleissheim, Germany) and the uterus removed and weighed.

Satellite experiments were included to better evaluate the effects of DnHP and DCHP on the liver of the dams since it is a potential target organ. Groups of six to nine pregnant rats were given the chemicals under the same experimental conditions as in the main studies. Early on GD 21, dams were euthanized. The livers were immediately removed and weighted. The left lobe was stored in 10% neutral buffered formaldehyde for further histological examination.
Ovaries and uterine content:
Uterine contents were examined to determine the number of implantation sites, resorptions and dead and live fetuses. The number of corpora lutea in each ovary was recorded. Uteri which had no visible implantation sites were stained with ammonium sulfide (10%) to detect very early resorptions.
Fetal examinations:
Live fetuses were weighed, sexed and examined for external anomalies including those of the oral cavity. Anogenital distance (AGD) was measured using a dissecting microscope with a micrometer eyepiece. Half of the live fetuses from each litter was preserved in Bouin’s solution and examined for internal soft tissue changes. The other half were fixed in ethanol (70%), eviscerated and then processed for skeletal staining with alizarin red S for subsequent skeletal examination. The sex of all fetuses was determined by internal examination of the gonads. The degree of trans-abdominal testicular migration (TTM) was determined by measuring the distance from the bladder neck to the lower pole of the testes using a micrometer eyepiece (X10). The measurements were standardized by defining the distance between the bladder neck and the lower pole of the kidney as 100 U.
Statistics:
Whenever possible, the data were presented as means ± SD. The number of corpora lutea, implantation sites and live fetuses, various organ and body weights, serum clinical chemistry parameters and AGD were analyzed by one-way analysis of variance, followed by Dunnett’s test if differences were found. The frequency of post-implantation loss, dead fetuses, resorptions and alterations among litters was evaluated using the Kruskal-Wallis test followed by the Mann-Whitney test where appropriate. Rates of pregnancy and of litters with dead fetuses or resorptions, and incidences of fetal alterations per dose were analyzed using Fisher’s test. Where applicable, least-squares analysis was carried out. The reported level of statistical significance was P < 0.05. The litter was used as the basis for the analysis of fetal variables.
Indices:
no data
Historical control data:
no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
The corrected weight gain was lower than control at 500 mg/kg per day (although not significantly), and was significantly reduced at 750 mg/kg per day. The mean body weight change for GD 6–21, which spans the entire treatment period, was also reduced in these groups. Maternal body weight and food intake were not affected at 250 mg/kg per day. The relative liver weights were significantly different from control at 500 and 750 mg/kg per day. DCHP induced a mild elevation of hepatic peroxisomal b oxidation activity at all doses (1.7-2.1 fold, compared with the concurrent control). Histological examination of the liver did not reveal changes attributable to DCHP (data not shown).

Effect levels (maternal animals)

Key result
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: yes. Remark: Absolute AGD was significantly reduced in male fetuses at all doses. It was still significantly lower than control when fetal body weight was used as covariate for analysis, or after normalizing with the cubic root of body weight.

Details on embryotoxic / teratogenic effects:
No significant effects of DCHP were found on the number of implantations or live fetuses, on the incidence of post-implantation loss and resorptions, and on the sex ratio. There was a dose-related decrease in fetal body weight (all, males and females). It amounted to 2-3% at 500 mg/kg and reached 10-11% at the highest dose, compared with the concurrent control. Fetal weight was significantly lower than control at 750 mg kg/per day. Absolute AGD was significantly reduced in male fetuses at all doses. It was still significantly lower than control when fetal body weight was used as covariate for analysis, or after normalizing with the cubic root of body weight. This decrease was dose-related and averaged 17% at 750 mg/kg per day. AGD of female fetuses were comparable across groups. Morphological examination of fetuses revealed only few cases of malformations. Diaphragmatic hernia was seen in one control fetus. At 750 mg/kg per day, three fetuses from three different litters were malformed: one fetus had omphalocele, another had diaphragmatic hernia and a third had a thoracic vertebra malformation. These isolated findings were considered incidental and unrelated to treatment. There was no statistically significant increase in the incidence of any external, soft tissue, and skeletal variations compared to control.

Effect levels (fetuses)

Remarks on result:
other: here was no statistically significant increase in the incidence of any external, soft tissue, and skeletal variations compared to control.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, the lowest-observable-adverse-effect-level for developmental toxicity was judged to be 250 mg/kg per day.
Executive summary:

The objective of this study was to evaluate the developmental toxic potential of dicyclohexyl phthalate (DCHP) in rats. Pregnant Sprague–Dawley rats were exposed to DCHP at doses of 0 (olive oil), 250, 500 and 750 mg kg-1 per day, by gavage, on gestational days (GD) 6–20. Maternal food consumption and body weight gain were significantly reduced at 500 and 750 mg kg-1 per day of DCHP. Slight changes in liver weight associated with peroxisomal enzyme induction were seen in dams treated with DCHP. DCHP produced fetal growth retardation at 750 mg kg-1 per day, as evidenced by significant reduction of fetal weight. DCHP induced a significant and dose-related decrease in the anogenital distance of male fetuses at all doses. In conclusion, DCHP showed no clear embryolethality and teratogenicity. However, as Absolute AGD was significantly reduced in male fetuses at all doses, the lowest-observable-adverse-effect-level for developmental toxicity was judged to be 250 mg kg-1 per day.