Reproductive and neurobehavioural effects of bis(2-ethylhexyl) phthalate (DEHP) in a cross-mating toxicity study of mice

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Abstract

Bis(2-ethylhexyl) phthalate (DEHP) was given in the diet to provide levels of 0% (C) or 0.03% (T) from 5 weeks of age of the F0 generation to 9 weeks of age of the F1 generation in mice. At 9 weeks of age, each female was paired with one male from the same or another treatment groups (cross-mating: C/C, T/C, C/T, T/T), for a period of 5 days. The selected reproductive and neurobehavioural parameters were measured. There were no adverse effects of DEHP on either litter size, litter weight and sex ratio at birth. The average body weight of female offspring was significantly affected in group IV (T/T) at PND 14. In behavioural developmental parameters, swimming direction at PND 4 was significantly accelerated in group III (C/T) in female offspring. In movement activity of exploratory behaviour at 3 weeks of age, number of movement of male offspring was significantly affected in group IV (T/T). The dose level of DEHP in the present cross-mating study produced few adverse effects in reproductive and neurobehavioural parameters in mice.

Introduction

The plasticizer, bis(2-ethylhexyl) phthalate (DEHP) is used in polyvinyl chloride (PVC) and other plastic production. Flexible PVC is normally employed for the production of floor tiles, various types of furnishing, waterproof clothing, industrial tubing, food packaging materials, and a variety of medical devices (catheters, blood storage bags, etc). The estimated amount of DEHP manufactured in 1998 was approximately 260,529 t in Japan. The tolerable daily intake (TDI) of human is presumed as 40–140 μg/kg/day (Inoue, 2000).

In toxicological studies of DEHP, the acute intraperitoneal LD50 were 20 and 50 g/kg in mice and rats, respectively (Thomas et al., 1978). Poon et al. (1997) reported that the no-observed-adverse-effect level (NOAEL) of DEHP was 50 ppm (3.7 mg/kg/day) for rats in 13 weeks toxicity study. Klimisch et al. (1992) found that the no-observed-effect level (NOEL) of DEHP was 0.05 mg/l in 28 days inhalation toxicity study of rats. David et al. (2000) found that the NOAEL of DEHP was 500 ppm (28.9–36.1 mg/kg/day) in chronic toxicity study of rats. Kluwe et al. (1982) reported that hepatocellular carcinomas were caused by DEHP in more than 12,000, 6000 and 3000 mg/kg/day for female rats, male mice and female mice, respectively. Agarwal et al. (1985) found that DEHP caused dominant lethal mutation effects in mice (1–10 ml/kg).

As regards reproductive system toxicity studies, Seth et al. (1976) reported that DEHP caused testicular damage in rats. Gray et al. (1977) found that DEHP caused testicular damage and increase of liver weight in 17 weeks toxicity study of rats (0.2–2.0% in the diet, approximately 140–1400 mg/kg/day). Tandon et al. (1991) found that DEHP affected the normal development of testes of rats in utero exposure (1000 mg/kg/day). Parmar et al. (1995) found that the destructive changes of testes were induced by DEHP in more than 250 mg/kg/day in 30 days toxicity study of rats. Oishi (1993) reported that the CD-1 mice were more sensitive for testicular atrophy induced by DEHP than other strain of mice. Davis et al. (1994) found that DEHP induced hypoestrogenic anovulatory cycles and polycystic ovaries in adult female rats (2 g/kg/day, 12 days).

As regards reproductive and developmental toxicity studies, Nakamura et al. (1979) found that the non-effective maximum levels of DEHP in mice were 0.065, 0.8 and 0.68 ml/kg for fetal death, gross and skeletal abnormalities, respectively. Shiota et al. (1980) reported that the maximum no effect level on mouse fetuses of DEHP was 70 mg/kg/day throughout gestation. Yagi et al. (1980) found that the gross and skeletal abnormalities in the live fetuses occurred with 2.5 or 7.5 ml/kg of DEHP given orally on days 7 or 8 of gestation respectively. Tomita et al. (1982) reported that the non-fetolethal maximum dose level of DEHP was 64 mg/kg. Shiota and Mima (1985) found that DEHP induced malformations in 1000 mg/kg per os but no significant effects showed in intraperitoneal administration in mice. Lamb et al. (1987) reported that DEHP induced adverse effects on reproductive parameters in more than 0.1% in the diet in mice. Melnick et al. (1987) reported that the NOEL of DEHP in developmental toxicity was 0.025% for mice in studies by National Toxicology Program. Tyl et al. (1988) found that the NOELs of DEHP in developmental toxicity studies were 0.5%, 0.5%, 0.05% and 0.025% (approximately 360, 360, 91, 44 mg/kg/day) for rat dams, rat fetuses, mouse dams and mouse fetuses, respectively. Arcadi et al. (1998) reported that DEHP damaged offspring kidney, liver and testes in reproductive toxicity study.

As regards reproductive and neurobehavioural toxicity studies, Tanaka (2002) reported that DEHP produced dose-related delays on surface righting in male offspring, but no adverse effects on litter size, litter weight, offspring weight or sex ratio (0.01–0.09% in the diet). Nevertheless, James (2003) mentioned that to guard against the possibility that DEHP treatment has opposite effects on the sex ratios of offspring of male and female mice, they need to know the offspring sex ratios of matings of treated females with untreated males, and of treated males with untreated females.

In the previous studies, DEHP showed no significant effects on sex ratio of offspring in individual offspring base analysis (Tanaka, 2002) or litter base analysis (Tanaka, 2003a). In both studies, offspring sex ratios of DEHP 0.03% group were slightly different from controls (not significant). Tanaka (2003b) reported that offspring sex ratios of DEHP 0.03% group showed no significant effects in a cross-mating method.

Tanaka (2002) reported that surface righting at PND 4 was significantly delayed in 0.01% and 0.03% groups in female offspring, and surface righting at PND 7 was significantly depressed in 0.09% group in male offspring and in 0.01% group in female offspring. In females, DEHP produced inconsistent effects on surface righting. These results might be caused by the possibility that DEHP treatment has opposite effects on the behavioural development of offspring of male and female mice.

Nevertheless, there were few studies on reproductive and neurobehavioural toxicity of DEHP in a cross-mating method. The single-dose method was used in a cross-mating method since the multidoses method was too complicated to analyze the results. As the middle-dose of DEHP 0.03% in the previous study produced any adverse effects on surface righting, the dose level was selected as 0.03% in the diet. Therefore, the present study was designed to evaluate reproductive and neurobehavioural effects of DEHP in a cross-mating method of mice.

Section snippets

Materials

Bis(2-ethylhexyl) phthalate (DEHP) was obtained from Wako Pure Chemical Industries, Ltd., Osaka, Japan (Lot No. ELH6895). The purity of the chemical was more than 97.0%.

Animals and maintenance

Male and female mice (Crj: CD-1, 4 weeks of age) were purchased from Charles River Japan Inc., Kanagawa, Japan. They were individually housed in polycarbonate solid-floored cages with wood flakes, and kept in a temperature controlled room maintained at 25 ± 1 °C with relative humidity of 50 ± 5% on a 12 h light/dark cycle. They were

Food and chemical intake

There were no significant effects of DEHP on the average food intake during any periods (Table 1). Therefore, the chemical intakes of males were similar between group II (T/C) and IV (T/T) during the preconception period, and those of females were similar between group III (C/T) and IV (T/T) in the F0 generation (Table 1). The chemical intakes of F1 generation were similar between group III (C/T) and IV (T/T) after weaning (Table 1).

F0 generation

The average body weight of male and female mice showed no

Discussion

In the present study, DEHP showed few significant adverse effects on reproductive and neurobehavioural parameters. The average sex ratio of group IV (T/T) was higher (51.7%) than other groups, but that was closer to the theoretical sex ratio approximately 50% than other groups. Also, sex ratio variations were very large in all groups (I (C/C): 23.1–54.5%, II (T/C): 28.6–66.7%, III (C/T): 0.83–63.6%, IV (T/T): 38.5–78.6%). It seems that these apparent differences of sex ratio were influenced by

References (49)

  • S. Oishi

    Strain differences in susceptibility to di-2-ethylhexyl phthalate-induced testicular atrophy in mice

    Toxicology Letters

    (1993)
  • G. Pantaleoni et al.

    Effects of maternal exposure to polychlorobiphenyls (PCBs) on F1 generation behavior in the rat

    Fundamental and Applied Toxicology

    (1988)
  • R. Poon et al.

    Subchronic oral toxicity of di-n-octyl phthalate and di(2-ethylhexyl) phthalate in the rat

    Food and Chemical Toxicology

    (1997)
  • P.K. Seth et al.

    Effect of di-2-ethylhexyl phthalate (DEHP) on rat gonads

    Environmental Research

    (1976)
  • K. Shiota et al.

    Embryotoxic effects of di-2-ethylhexyl phthalate (DEHP) and di-n-butyl phthalate (DBP) in mice

    Environmental Research

    (1980)
  • T. Tanaka

    Reproductive and neurobehavioral effects of imazalil administered to mice

    Reproductive Toxicology

    (1995)
  • T. Tanaka

    Reproductive and neurobehavioural toxicity study of bis(2-ethylhexyl) phthalate (DEHP) administered to mice in the diet

    Food and Chemical Toxicology

    (2002)
  • T. Tanaka

    Offspring sex ratio in laboratory mice

    Food and Chemical Toxicology

    (2003)
  • T. Tanaka

    Effects of bis(2-ethylhexyl) phthalate (DEHP) on secondary sex ratio of mice in a cross-mating study

    Food and Chemical Toxicology

    (2003)
  • T. Tanaka et al.

    Reproductive and neurobehavioural effects in three generation toxicity study of piperonyl butoxide administered to mice

    Food and Chemical Toxicology

    (1992)
  • J.A. Thomas et al.

    A review of the biological effects of di-(2-ethylhexyl) phthalate

    Toxicology and Applied Pharmacology

    (1978)
  • R.W. Tyl et al.

    Developmental toxicity evaluation of dietary di(2-ethylhexyl) phthalate in Fischer 344 rats and CD-1 mice

    Fundamental and Applied Toxicology

    (1988)
  • A.P. Van Wezel et al.

    Environmental risk limits for two phthalates, with special emphasis on endocrine disruptive properties

    Ecotoxicology and Environmental Safety

    (2000)
  • D.K. Agarwal et al.

    Antifertility and mutagenic effects in mice from parental administration of di-2-ethylhexyl phthalate (DEHP)

    Journal of Toxicology and Environmental Health

    (1985)
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