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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Comparable to guideline with acceptable restrictions: overall, the treatment group have a good number of animals, but little less than the required for a single dose is used. No data are avaliable to determine the dosage, only the referement to other publications in which the relative potentials of various glucocorticoids to induce cleft palate in rats have been well studied. Neverthless, is possible to evaluate the gradation of the toxic effect as requested by the guideline. Prednisolone was administered subcutaneously without providing a justification. No data were reported about the observation and examination of the dams.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1995

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:
The study was performed using Sprague-Dawley rats (Charles River CD, Japan) kept under spf conditions, constant temperature (24 +/-1 °C), a relative umidity (55 +/-5 %), and a defined light-dark cycle (dark periode from 7:00 p.m. to 7:00 a.m). Food and water were provided ad libitum. Nine-week-old virgin females weighing 180-240 g, after being acclimatized for 2 weeks, were mated with males overnight and the checked for the presence of sperm in vaginal smears the following morning. Sperm-positive females were considered to be in the day 0 of gestation. These female rats were randomly assigned to one of the treatment groups. Dosage were calculated according to maternal body weight on day 7 of gestation. Prednisolone was purchased from Sigma Chemicals Co. (chemical purity of 99.9% according to the manufacturer).
Prednisolone was suspended in 0.5% carboxymethyl cellulose and administered subcutaneously in a volume of 2 ml/kg on days 14 and 15 of gestation. Untrated females were used as control. All dames were killed by cervical dislocation and hysterectomized on day 20 of gestation. The number of early and late resorptions and viable fetuses were recorded. Embryos representing death after the administration were defined as late resorption. Viable fetuses were weighed, sexed, and examined for gross external malformations. They were then fixed in 10% formalin solution for about 7 days and evaluated for palatal abnormalities under a dissecting microscope after cutting the region between the upper and lower jaws. Severe type of palatal slit; cone-shaped, triangular cleft was evaluated anso in this study in order to compare with potency of dexamethasone in inducing palatal slit.
Student's or Aspin-Welch's t-test was used for statistical analyses of mean fetal weights. Frequencies of late resorptions and malformations were evaluated by using Mann-Whitney U test. The probit analysis of the palatal slit and cleft palate response to dose was maximum likelihood procedure outlined by Finney (Finney, D.J. Probit Analysis. (1971) Third ed. Cambridge University Press, London).
GLP compliance:
not specified
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Prednisolone
- Substance type: Organic
- Analytical purity: 99.9%
- Composition of test material, percentage of components: Prednisolone 99.9%
- Other: Prednisolone was purchased from Sigma Chemical Co. and the purity is according to the manufacturer.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River CD, Japan
- Age at study initiation: 9 week-old
- Weight at study initiation: 180-240 g
- Housing: Specific Pathogen Free condictions
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1
- Humidity (%): 55 ± 5
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: To: day 20 of gestation

Administration / exposure

Route of administration:
subcutaneous
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
Prednisolone suspended in 0.5% carboxymethyl cellulose
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: mated overnight
- Length of cohabitation: one night
- Further matings after two unsuccessful attempts: no
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Prednisolone was administered subcutaneously on day 14 and 15 of gestation.
Frequency of treatment:
Daily.
Duration of test:
From day 0 to day 20 of gestation
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
12.5 mg/kg/day
Basis:
nominal conc.
Prepared suspension.
Remarks:
Doses / Concentrations:
25 mg/kg/day
Basis:
nominal conc.
Prepared suspension.
Remarks:
Doses / Concentrations:
50 mg/kg/day
Basis:
nominal conc.
Prepared suspension.
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
nominal conc.
Prepared suspension.
No. of animals per sex per dose:
12 female for the assay of 12.5 mg/kg/day
14 female for the assay of 25 mg/kg/day
11 female for the assay of 50 mg/kg/day
9 female for the assay of 100 mg/kg/day
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: no data are avaliable to determine the dosage, only the referement to other publications in which the relative potentials of various glucocorticoids to induce cleft palate in rats have been well studied. Neverthless, is possible to evaluate the gradation of the toxic effect as requested by the guideline.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: No data

POST-MORTEM EXAMINATIONS: No data
- Sacrifice on gestation day # 20
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Embryos representing death after the administration of Prednisolone were defined as late resorptions.
Fetal examinations:
- External examinations: Yes: all per litter were weighed, sexed, and examined for gross external malformations.
- Head examinations: Yes: all per litter were fixed in 10% formalin solution for about 7 days and evaluated for palatal abnormalities under dissecting microscope after cutting the region between the upper and lower jaws. Severe type of palatal slit; cone-shaped, triangular cleft was evaluated.
Statistics:
Student's or Aspin-Welch's t-test was used for statistical analyses of mean fetal weights. Frequencies of late resorptions and malformations were evaluated by using Mann-Whitney U test.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:not examined

Details on maternal toxic effects:
None of the dams in any of the groups died during the study.

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The frequencies of cleft palate, palatal slit, and late resorption among fetuses from untreated dams and from those treated with Prednisolone on day 14 and 15 of gestation are evaluated.
The frequencies of cleft palate were significantly higher in the group treated with 100 mg/kg/day prednisolone (10.6%). No significant increase in the frequencies of cleft palate was detected when pregnant rats were treated with 50 mg/kg/day prednisolone (3.4%).

The frequencies of palatal slit increased significantly when pregnant females were treated with 50 and 100 mg/kg/day prednisolone (27.5 and 77.7%, respectively), while no significant increase in the frequencies of palatal slit was detected when pregnant rats were treated with 12.5 and 25 mg/kg/day prednisolone (5.77 and 4.63%, respectively).

Significant increases in the frequency of late resorptions per litter were observed in the group treated with 100 mg/kg/day prednisolone (8.1%).

All the group treated with prednisolone showed a significant reduction in fetal body weight as compared to that of controls, suggesting a generalized growth retardation. Furthermore, the weight reduction was more pronounced with the increase in dose.

Effect levels (fetuses)

Dose descriptor:
other:
Effect level:
ca. 70 mg/kg bw/day
Based on:
act. ingr.
Remarks:
Prednisolone
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

    Cleft Palate/live fetuses  Palatal Slit/(live-CP) fetuses      Late resorptions/implants   
  Dose (mg/kg/day)   No. of dams  No.  % ± SE  No.  % ± SE  No.   % ± SE
 Control  21  0/326  0  0/326  0  1/345  0.25  ± 0.25
 Prednisolone              
 12.5  12  0/176  0  9/176  5.77  ± 6.03 0/179   0
 25  14  0/220  0  9/220  4.63  ± 2.18 6/241   2.61  ± 0.84
50   11  6/170  3.41  ± 1.85  45/164  27.51  ± 9.47**  5/188  2.23  ± 1.54
 100  9  13/123  10.59  ± 7.38 **  87/110  77.69  ± 7.25**  19/145  8.13  ± 3.72*

* Significantly different fron control by Mann-Whitney'sUtest (P<0.05).

**Significantly different fron control by Mann-Whitney'sUtest (P<0.01).

Applicant's summary and conclusion

Conclusions:
The number of foetuses with cleft palate and palatal slit are significantly increased in the groups given 50 and 100 mg/bw (up to 10.59% of cleft palate and 77.69% of palatal slit).