Document ID: EPA-HQ-OPPT-2007-0531-0821
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2014-12-29T05:00Z

Dr. Sylvia Jacobi
Corporate Toxicology Director
Parc Scientifique Einstein Rue de Bosquet 9B-1348 Louvain-la Neuve Sud 
Belgium
Phone: +3210481768
Fax: +3210481768
e-mail: Sylvia.jacobi@albemarle.com

July 29, 2014
Diethylmethylbenzene diamine (DEDTA) Developmental Toxicity Study in Rats, TNO-Triskelion Study Report No. V20193, Project No. 093.20418, 
Summary
A developmental toxicity study was conducted with Diethylmethylbenzene diamine (DEDTA) accoring to OECD TG 414 (2001) and Council Regulation EC No. 440/2008 B.31 (2008). Groups of 24 pregnant rats received DEDTA by continuous oral administration in the diet from gestation day (GD) 0 until GD 20. The test substance was given at constant dietary nominal concentrations of 0 (control), 50 (low-dose), 150 (mid-dose) and 500 mg/kg diet (high-dose). These doses corresponded to 0, 2.63, 7.83 and 20.45 mg/kg bw per day interpolated actual test substance intake. The doses were chosen on the basis of two dose range finding studies. During the in-life phase clinical signs, maternal body weight and food consumption were recorded. Ophthalmoscopic examination was performed pre-treatment and at gestation day 20. At Caesarean section, females and fetuses of all groups were macroscopically examined and blood was collected for haematology and clinical chemistry. Fetuses, placentas and reproductive organs were weighed. Histopathology of the pancreas as the primary target organ for DEDTA toxicity was performed in the dams. Fetuses were further processed for fetopathological examination. 
Upon analysis of the experimental diets, DETDA was found to be homogeneously distributed at all dose levels and stable in diets after storage in a freezer (<= - 18ºC). However, the initial measured content of DETDA was 10-16% lower than intended, and storage in the animal room for 5 days (in an open container) resulted in 32-41% loss. Although the measured concentrations were lower than the intended nominal concentrations in diets, the achieved exposure levels did result in maternal toxicity at the high dose level. Therefore the guideline requirements of maternal toxicity at the high dose level were met and the study was considered valid at relevant dose levels. 
Feed was removed and completely replaced by feed from the freezer twice weekly at three or four day intervals. The actual test substance intake was assessed taking into account the measured initial levels and the (interpolated) loss during storage for 3 or 4 days in the animal room. 
In the high dose group maternal toxicity was observed as represented by a body weight loss during the first three days of gestation, followed by a decreased mean body weight gain and decreased food consumption during gestation, reduced ovary weight and induction of acinar cell apoptosis and mononuclear cell inflammation in the pancreas in most of the animals of the high dose group (13 animals with minimal to moderate apoptosis of the pancreatic acinar cells, 15 animals with minimal to mild mononuclear cell inflammation). In the mid dose group minimal induction of acinar cell apoptosis was observed in two animals and minimal mononuclear cell inflammation in one animal. Although this finding was of low incidence and severity in this group, it could be regarded as a first indication of an effect on the target organ. Consequently, the low dose level was considered a No Observed Effect Level (NOEL) for maternal toxicity.. 
  
Developmental toxicity was observed in the high dose group, as evidenced by a decreased number of implantation sites and live fetuses. In addition mean placenta weight and mean fetus weight were decreased, which was confirmed by an increase in incidence of small fetuses.  A slight retardation in ossification in the fetuses of the high dose group was related to the lower fetus weight. 
  
Fetal observations with single incidences including one foetus showing a hernia ventralis, one fetus being too small and one fetus showing dilated ventricles of the brain were considered not treatement related based on the low (single) incidence and the absence of dose relationship. 
  
In absence of developmental effects in the mid-dose group, the NOAEL for developmental toxicity was placed at the mid-dose level of 7.83 mg/kg bw/day 
  
Effects on fetus weight, placenta weight and ossification are considered to be related to maternal toxicity as outlined in Waalkens-Berendsen et al., 2002 and 2004, Wolterbeek et al., 2004 and Meiling et al., 2004. In this study overt maternal toxicity was observed at the high dose level. An onset of the target organ effects in the dams was already observed in some animals of the mid dose group, but not accompanied by fetal toxicity. Consquently, the effects on embryo-fetal development that were observed at the high dose level were considered to be secondary to maternal toxicity 
 
References: 
Waalkens-Berendsen D.H., Kuilman-Wahls M.E.M., Wolterbeek A.P.M. Maternal toxicity and fetal development: effects of decreased food intake. 
Reproductive Toxicology 16, 425, 2002. 
  
  
Wolterbeek APM, Tegelenbosch-Schouten MM, Waalkens-Berendsen DH and Dijkstra A. 
Impact of maternal toxicity on pre- and postnatal development of rats. 
Reproductive Toxicology 18, 753, 2004. 
  
Meiling J, Wolterbeek APM, Waalkens-Berendsen DH, Tegelenbosch-Schouten MM and Dijkstra A. Impact of maternal toxicity on pre- and postnatal development of rats. 
Toxicology and Applied Pharmacology 197, 211, 2004. 
  
Waalkens-Berendsen ID, Tegelenbosch-Schouten MM, Dijkstra A and Wolterbeek APM. 
Impact of maternal toxicity on pre- and postnatal development of rats. 
The Toxicologist 78, Suppl. 1, 221, 2004