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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:
2000-08-28 until 2000-09-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japan/MHW: Guidelines for Taxicity Testing of Chemicals, Teratogenicity Test, MITI/MHW, 1987 (Transation), pp. 212 -213.
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-(4-Diethylamino-2-hydroxybenzoyl)- benzoesäurehexylester
- Physical state and appearance: Solid, crystalline/white-yellowish
- Analytical purity: 99.35%
- Lot/batch No.: R 323/681
- Stability under test conditions: Analytical verifications of the stability of the test substance in olive oil Ph.Eur./DAB for a period of at least 96 hours at room temperature were carried out before the study was initiated and proven by reanalysis after the in-life phase of the study.
- Date of production: 1999-10-13
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Wistar rats (CRL:WI (GLX/BRL/HAN) IGS BR) supplied by Charles River Laboratories, Germany (date of supply: 2000-08-08).
- Age at study initiation: 89 - 95 days old
- Weight at study initiation: Mean body weight on day 0 was approx. 196 g.
- Housing: The rats were housed singly from day 0 - 20 p.c. in type DK III stainless steel wire mesh cages supplied by BECKER & CO., Castrop-Rauxel, Germany
- Diet: Ground Kliba maintenance diet rat/ mouse/ hamster meal, supplied by PROVIMI KLIBA SA, Kaiseraugst, Switzerland. Food was available to the animals ad libitum throughout the study (from day of supply to day of necropsy)
- Water: Tap water ad libitum.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS:
The animals were accommodated in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature of 20 - 24°C and a range of relative humidity of 30 - 70%.
The day/night rhythm was 12 hours (12 hours Iight from 6.00 a.m. to 6.00 p.m. and 12 hours darkness from 6.00 p.m. to 6.00 a.m)


IN-LIFE DATES: From: 2000-08-28/29/30 (start of the study) To: 2000-09-18/19/20 (Due to technical reasons the study was carried out in 3 sections. Each dose group was represented in each section).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance suspensions in olive oil Ph.Eur./DAB were prepared at the beginning of the administration period and thereafter at 3-4 day intervals. For the preparation of the suspensions an appropriate amount of the test substance was weighed and subsequently suspended in olive oil Ph.Eur./DAB using a high speed sonicator. A magnetic stirrer was used to keep the suspensions homogeneous during treatment of the animals.

VEHICLE
- Concentration in vehicle (concentrations of the oily suspensions mg/100 mL) : 0 (control group), 800, 4000, 20000
- Amount of vehicle (gavage): 5 mL test substance suspensions in olive oil per kg bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test substance suspensions were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations. The samples which were taken for the first concentration control analysis at the beginning of the administration period were also used to verify the homogeneity for the samples of the low and the high concentrations (40 and 1,000 mg/kg body weight/ day). 3 samples (from the top, middle and bottom) were taken for each of these concentrations from the beaker with a magnetic stirrer running. The test substance suspensions were analysed by HPLC.
Details on mating procedure:
1-3 untreated female rats were mated with one untreated male animal of the same breed. The male mating partners were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.
Mating took place from about 4.00 p.m. to about 7.30 a.m. on the following day. If sperm were detected microscopically in the vaginal smear in the morning, the animals were considered to be fertilized (this day was designated "day 0" (beginning of the study) and the following "day 1" post coitum (p.c).
Duration of treatment / exposure:
6th (implantation day) to the 19th day post coitum (one day prior to the expected day of parturition)
Frequency of treatment:
Once a day by gavage
Duration of test:
From days 0 (a day after mating, when sperm was microscopically detected in the vaginal smear) to day 20, when all females were sacrificed.
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
nominal conc.
40; 200; 1000 mg/kg bw. Vol: 5mL/ kg bw
No. of animals per sex per dose:
25 female animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The following doses were chosen for the present full-scale toxicity study in Wistar rats:
40 mg/kg body weight/day: as the expected no observed adverse effect level
200 mg/kg body weight/day: as intermediate dose level
1,000 mg/kg body weight/day: as the dose level that shows signs of maternal toxicity and possible substance-related developmental toxicity

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS:
- Time schedule:
The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited (days 0 - 20 p.c.).

MORTALITY:
A check was made twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0 - 20 p.c.).

BODY WEIGHT:
- Time schedule for examinations:
All animals were weighed on days 0, 1, 3, 6, 8, 10, 13,15, 17, 19 and 20 p.c.. The body weight change of the animals was calculated from these results.

FOOD CONSUMPTION:
With the exception of day 0, the consumption of food was determined on the same days as the body weight was measured.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 20 post coitum

OTHER:
The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on day 20 p.c. minus the weight of the unopened uterus minus body weight on day 6 p.c.).
Ovaries and uterine content:
The uterus and the ovaries were examined and the following data recorded:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
Examinations were made per litter
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
Statistics:
The DUNNETT-test (two-sided) was used for comparing the following parameters between the test and the control groups:
Food consumption, body weight, body weight change, corrected body weight gain, carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorption, number of life fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight.
The FISHER´s EXACT test (one-sided) was used for the comparison of the following parameters: Female mortality, female pregnant at terminal sacrifice, number of litters with fetal findings.
The WILCOXON-test (one-sided) was used for the comparison of the following parameters: Proportions of fetuses with malformations, variations and/ or unclassified observations in each litter.
Historical control data:
Historical control data exists from the same year.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
1000 mg/kg bw/day:
• transiently occurring salivation in 25 rats immediately after gavaging between treatment days 7-19 p.c.
• urine-smeared fur in 4 rats from day 9 p.c. onwards
• statistically significantly reduced food consumption on days 6-13 p.c. (about 7% lower than the corresponding control value, if calculated for days 6 - 19 p.c.)
• marginally decreased body weight gain (about 8% below the control value – without attaining statistical significance - if calculated for the total treatment period (days 6 -19 p.c.)
• slightly decreased corrected body weight gain (about 11% below the control value without attaining statistical significance)

200 mg/kg bw/day:
• transiently occurring salivation in 21 rats immediately after gavaging between treatment days 8 - 19 p.c.
• slightly, but statistically significantly depressed food consumption at initiation of dosing (days 6 - 8 p.c.)

40 mg/kg bw/day:
• no substance-related effects on dams

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
1000 mg/kg bw/day:
• no substance-related effects on gestational parameters or fetuses
200 mg/kg bw/day:
• no substance-related effects on gestational parameters or fetuses
40 mg/kg bw/day:
• no substance-related effects on gestational parameters or fetuses

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Developmental toxicity: no substance-related effects observed

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion