Toxicology 205 (2004) 131–137 Effect of oral intake of dibutyl phthalate on reproductive parameters of Long Evans rats and pre-pubertal development of their offspring Veronica Salazar, Carmen Castillo, Carmen Ariznavarreta, Roc´ ıo Camp ´ on, Jes ´ us A.F. Tresguerres * Laboratory of Experimental Endocrinology, Department of Physiology, School of Medicine, Complutense University of Madrid, Avda. Complutense s/n, 28040 Madrid, Spain Available online 3 August 2004 Abstract To investigate the influence of dibutyl phtalate (DBP) given in a soy-free rat chow on pre-pubertal development, 46 Long Evans female rats 2-month-old were divided into three experimental groups and fed three different chows: (1) control; (2) DP 0.61 g/kg chow (12 mg/kg rat/day); (3) DP 2.5 g/kg chow (50 mg/kg rat/day) for 2 months. While under this treatment, they were mated and their offspring studied. Litter size and female:male ratio were recorded. At 14 days of age 6, male pups of each group were sacrificed and testis and thymus were excised and weighed. Pups were weaned at 22 days of age and continued into three experimental groups according to diet. From day 22 onwards, vaginal opening, occurrence of first estrous, and pre-putial separation were recorded. Results: The percent of pregnancies showed a marked decrease in group 3, while no difference was observed between groups 1 and 2. Sex prevalence and litter size were not affected by the different diets. Pup survival showed a decrease when mothers were fed diet 2, but it was similar in diets 1 and 3. Pup weights on day 2 showed an evident (P < 0.05) reduction in groups 2 and 3, the decrease being more marked (P < 0.001) in group 3. On day 6, pups of group 2 showed lower weights (P < 0.01) as compared with the other groups. Weight gain was significantly higher in pups of group 3. Eye opening was not affected by the different diets. Fourteen-day-old male pups’ relative weight of thymus and testis showed a decrease in animals whose mothers had been fed diets 2 and 3. Vaginal opening and occurrence of first estrous showed an evident delay (P < 0.05; P < 0.01) in females fed diets 2 and 3. Significant differences (P < 0.001) in pre-putial separation were observed between treated and untreated groups. Conclusion: Offspring pre-pubertal development seems to be affected by oral intake of DBP by their mothers during pregnancy, the effects being more evident in the reproductive development of male pups. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: di-n-butyl phthlate (DBP); Maternal exposure; Embryonic loss; Male reproductive tract malformation; Antiandrogenic effect; Rat * Corresponding author. Tel.: +349 13941484; fax: +349 13941628. E-mail address: guerres@med.ucm.es (J.A.F. Tresguerres). 1. Introduction Dibutyl phthalate (DBP) is a phthalic acid ester (PAEs) with extensive use in industry in such products 0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2004.06.045