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Endocrine disrupter mammalian screening – in vivo (level 3)

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endocrine disrupter mammalian screening – in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
4 (not assignable)

Data source

Reference Type:

Materials and methods

Test material

Specific details on test material used for the study:
17a-ethinylestradiol (EE), propyl p-hydroxybenzoate (propylparaben),
fulvestrant known as ICI 182,780 or Faslodex (Howell et al., 2002;
Tang et al., 2008), and corn oil were obtained from Sigma-Aldrich, Inc. (St Louis,
MO). Butyl
p-hydroxybenzoate (butylparaben) was purchased from Sigma-Aldrich GmbH
(Steinheim, UK). Isopropyl p-hydroxybenzoate (isopropylparaben) and isobutyl
p-hydroxybenzoate (isobutylparaben) were obtained from Tokyo Kasei Kogyo
Co. Ltd (Tokyo, Japan). All stock chemicals were dissolved in ethanol.

Test animals

Details on species / strain selection:
Sprague-Dawley immature female rats were purchased from SamTaKo-Bio Korea (Chungbuk, Korea)
immature female
Details on test animals and environmental conditions:
The rats were
housed in polycarbonate cages in a controlled environment (temperature,
23C ± 2C; relative humidity, 50 ± 10%; frequent ventilation; and an
illumination schedule of 12-h light/12-h dark). The experimental animals
were given free access to food and tap water. Rats were fed a diet of soyfree
pellets with a standard laboratory diet (Samyang formula feed;
Samyang Oil & Feed Co., Ltd, Seoul, Republic of Korea). The component
phytoestrogen-free AIN-76A-purified diet was used in order to rule out
potential estrogenic effects of phytoestrogens (Mitchell et al., 1989). All
experimental procedures were approved by the Ethics Committee of
Chungbuk National University.

Administration / exposure

Route of administration:
oral: gavage
Details on oral exposure:
From postnatal days (PND) 14 to 16, immature female rats (n= 8 per each group, mean body weight [BW]: 27.47± 0.69 g) received a sc injection daily of the following: propyl-, isopropyl-,butyl-, or isobutylparaben at a dose of 62.5, 250, or 1000 mg/kg BW/day;17a-ethinylestradiol (1 mg/kg BW/day) as a positive control; or corn oil (5 ml/kg BW/day) as a negative control. Dosages were adjusted according to changes in BW. BWs, clinical signs, and abnormal behaviors were recorded daily throughout the experimental period. In the second experiment, rats were administered fulvestrant (1 mg/kg BW/day) 30 min before chemical treatments. All animals were euthanized by ethyl ether 24 h after the final treatment. The uteri were weighed and prepared for samples of total RNAs and proteins. Each exposure was performed in triplicates.
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (nominal)
propyl-, isopropyl-, butyl-, and isobutylparaben
Dose / conc.:
250 mg/kg bw/day (nominal)
propyl-, isopropyl-, butyl-, and isobutylparaben
Dose / conc.:
1 000 mg/kg bw/day (nominal)
propyl-, isopropyl-, butyl-, and isobutylparaben


Data were analyzed by one-way ANOVA, followed
by Tukey’s test for multiple comparisons of columns. Statistical analysis was
performed using GraphPad Prism4 for Windows Edition (GraphPad Software
Inc., La Jolla, CA). A p value of < 0.05 was considered statistically significant.

Results and discussion

Results of examinations

Details on results:
Uterotrophic Effects Induced by Parabens:
The uterotrophic assay is a standard reliable method used to detect the estrogenicity of environmental compounds in vivo (Diel et al., 2000; Kang et al., 2000; Newbold et al., 2001; Padilla-Banks et al., 2001). Female rats at PND 14 were treated with increasing doses (62.5, 250, and 1000 mg/kg BW) of parabens (propyl-, isopropyl-, butyl-, and isobutylparaben) for 3 days. The highest dosage of isopropyl-, butyl-, and isobutylparaben significantly increased uterine wet weight. EE treatment, used as a positive control, induced a significantly increased uterine weight (fourfold vs. vehicle, p <
0.05). However, no significant effect was detected in the propylparaben-treated groups.

Effects of Parabens on CaBP-9k mRNA and Protein Expression:
The effects of parabens on the uterine CaBP-9k mRNA level and protein expression were examined using an immature rat model. CaBP-9k transcripts were induced by the highest dose of isopropyl-, butyl-, and isobutylparaben (13.6-, 12.4-, and 6.9- fold increases vs. vehicle, respectively). The positive
control, EE, induced uterine CaBP-9k transcription (p < 0.05). However, all concentrations of propylparaben and the middle and lowest concentration of the other parabens did not affect CaBP-9k gene expression.Similar to that observed for mRNA expression, uterine CaBP-9k protein levels were upregulated by EE and parabens, with the exception of propylparaben.

Applicant's summary and conclusion

Executive summary:

no significant effects on uterine weight were detected for any dose of propylparaben.