Effects of oral exposure to the phthalate substitute acetyl tributyl citrate on female reproduction in mice

Abstract

Acetyl tributyl citrate (ATBC), is a phthalate substitute used in food and medical plastics, cosmetics and toys. Although systemically safe up to 1000 mg kg^-1 day^-1 , its ability to cause reproductive toxicity in females at levels below 50 mg kg^-1 day^-1 has not been examined. This study evaluated the effects of lower ATBC exposures on female reproduction using mice. Adult CD-1 females (n = 7-8 per treatment) were dosed orally with tocopherol-stripped corn oil (vehicle), 5 or 10 mg kg^-1 day^-1 ATBC daily for 15 days, and then bred with a proven breeder male. ATBC exposure did not alter body weights, estrous cyclicity, and gestational and litter parameters. Relative spleen weight was slightly increased in the 5 mg kg^-1 day^-1 group. ATBC at 10 mg kg^-1 day^-1 targeted ovarian follicles and decreased the number of primordial, primary and secondary follicles present in the ovary. These findings suggest that low levels of ATBC may be detrimental to ovarian function, thus, more information is needed to understand better the impact of ATBC on female reproduction. Copyright © 2016 John Wiley & Sons, Ltd.

Keywords: acetyl tributyl citrate; female; fertility; ovarian follicle; ovary; phthalate substitute; plasticizer.

Figure 1. Experimental Design

Body weight and estrous cyclicity were monitored in female CD-1 mice for 20 days prior to dosing. Animals were then separated into vehicle, 5, and 10 mg/kg/day ATBC and dosed prior to housing with a proven breeder male as described in Section 2.2. On the day of parturition, the dams and pups were euthanized and litter data collected. Ovaries and major organs were excised, weighed, and their gross morphology evaluated. Ovaries were processed for histological evaluation as described in Section 2.7.

Figure 2. Effect of oral exposure to ATBC on body weight

CD-1 female mice were exposed to ATBC as described in Section 2.2. Individual daily body weight values were normalized to baseline (first day of dosing or gestation as applicable) and averaged per treatment to obtain mean body weight percent change ± SEM (n= 7–8 mice per treatment) during dosing (A) and during gestation (B). Data were analyzed using General Linear Model Repeated Measures test with significance set at p≤0.05. Asterisks (*) indicate significantly different from control.

Figure 3. Effect of oral exposure to ATBC on estrous cycle length

CD-1 female mice were exposed to ATBC and vaginal smears taken prior to and during dosing as described in Section 2.2. Data are presented as average number of days per cycle ± SEM (n=7–8 mice per treatment) and were analyzed using General Linear Model Repeated Measures test with significance set at p≤0.05. Asterisks (*) indicate significantly different from values prior to the start of dosing.

Figure 4. Effect of oral exposure to ATBC on estrous cyclicity

CD-1 female mice were exposed to ATBC and vaginal smears taken prior to and during dosing as described in Section 2.2. Data are presented as mean percentage of time spent in each stage of proestrus, estrus, and diestrus ± SEM (n=7–8 mice per treatment) prior (A) and during ATBC dosing (B) and were analyzed using General Linear Model Repeated Measures test with significance set at p≤0.05.

Figure 5. Effect of oral exposure to ATBC on total follicle numbers

CD-1 female mice were exposed to ATBC as described in Section 2.2. Ovaries were dissected and processed for histological evaluation as described in Section 2.7. Data are presented as mean number of follicles ± SEM (n=7–8 mice per treatment) and were analyzed using One-Way ANOVA followed by Fisher’s LSD post hoc test with significance set at p≤0.05. Asterisks (*) indicate significantly different from control.

Figure 6. Effect of oral exposure to ATBC on the number of ovarian follicles in the different developmental stages

CD-1 female mice were exposed to ATBC as described in Section 2.2. Ovaries were dissected and processed for histological evaluation as described in Section 2.7. Data are presented as mean number of follicles ± SEM (n=7–8 mice per treatment) for healthy (A) and atretic (B) follicles separately and were analyzed using One-Way ANOVA followed by Dunnett’s post hoc test with significance set at p≤0.05. Asterisks (*) indicate significantly different from control.