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Toxicity to reproduction: other studies

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toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
other information
Study period:
2001-01-22 until 2001-01-26
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Principles of method if other than guideline:
Evaluation of endocrine (estogenic) effects
GLP compliance:
Type of method:
in vivo

Test material

Details on test material:
- Name of test material (as cited in study report): 2-(4-Diethylamino-2-hydroxybenzoyl)-benzoesäurehexylester (DHE)
- Physical state and appearance: Crystalline / white-yellowish
- Analytical purity: >99% by GC
- Lot/batch No.: R 323/681
-The test substance is stored in room temperature.

Test animals

Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Germany
- Age of the test animals (immature females) at study initiation: 20+/- 1days
- Weight at study initiation: 30.0 - 40.0 g.
- Housing: Makrolon cages, type M-ll (Becker & Co., Castrop-Rauxel Germany). 2 animals per cage.
- Diet: Pelleted Kliba maintenance diet rat/mouse/hamster, Provimi Kiba SA, Kaiseraugst, Switzerland; ad libitum
- Water: Drinking water (tap water) ad libitum
- Acclimation period: At least one day.

- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2001-01-23 To: 2001-01-26

Administration / exposure

Route of administration:
oral: gavage
olive oil
Details on exposure:
The preparations were carried out in olive oil. For this purpose, a defined amount of the test substance was weighed into a calibrated beaker, topped up with the vehicle and then mixed with the magnetic stirrer. The test substance preparation was made once, on day of first administration.
Olive oil EP/DAB
- Concentration in vehicle: 50 mg/mL and 200 mg/mL in olive oil.
- Amount of vehicle (if gavage): 5 mL/kg bw/day
- Origin and lot/batch no.: Henry Lamotte GmbH, batch: 2047001.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Samples were diluted in acetone. Aliquots of the dilutions were used for HPLC-analysis. The results demonstrated the correctness of the homogeneity and concentrations of 2-(4-DiethyIamino-2-hydroxybenzoyI)-benzoesäurehexylester in olive oil Ph.Eur./DAB.
Duration of treatment / exposure:
3 days
Frequency of treatment:
Once daily
Duration of test:
3 days.
Doses / concentrations
Doses / Concentrations:
250 mg/kg bw, 1000 mg/kg bw
nominal conc.
No. of animals per sex per dose:
10 per dose.
Control animals:
yes, concurrent no treatment
other: A positive control, a group with 10 rats, that received 5 µg/kg bw diethylstilbestrol dipropionate (DES-DP) that induces a distinct uterotrophic effect.
Details on study design:
The study included 4 groups, 10 female rats in each group. The first group was a negative control group. The rats in this group received only olive oil by gavage. The second group was used as positive control, receiving DES-DP 5µg /kg bw in olive oil. The third group received 250 mg/kg bw of the test substance in olive oil and the forth group received 1000 mg/kg bw of the test substance in olive oil. The different treatments were given once a day by gavage for three days. Body weights and any other clinical symptoms were recorded once a day. At the end of the study all rats were sacrificed and the uterus weight was examined and histopatological examination was conducted.
The mean body and uterus weights were statistically evaluated by the Dunnett-test (two sided) or the Wilcoxon-test (two-sided) with a P <=0.05 or P<0.01.

Results and discussion

Observed effects

No clinical signs or symptoms were detected in the animals throughout the study. The body weights of the animals treated with DES-DP (positive control) and of the animals treated with DHE (test substance) were much the same as the body weights of the carrier group. There were no statistically significant differences. In the dose group that received 1,000 mg/kg bw DHE, the body weight gain was statistically significantly reduced between days 0 and 1, and the mean was reduced between day 0 and day 3 (days 0-3: about 24% below the control value). This was regarded as a sign of systemic toxicity. In the positive control, no statistically significant difference was detected as compared with the control.
The test substance showed no increased uterus weights as compared between the carrier control and the dosed groups (250 and 1,000 mg/kg bw). As was expected, the positive control DES-DP (5 µg/kg) led to a statistically significant increase in the absolute and relative uterus weights with a 4.7- and 4.9-fold increase respectively when compared with the control (olive oil). Histopathologically, no changes were detected in the uterus of the animals that received 250 and 1,000 mg/kg bw of DHE.
The positive control (5 µg/kg DES-DP) morphologically showed the expected changes in the uterus:
1. Endometrial epithelium extended and hypertrophic
2. Uterus lumen partially clearly dilated
3. Uterus wall thickened without any of the muscular layers or particularly the stroma being prominent.

Applicant's summary and conclusion