Perinatal exposure studies include the range finding study, perinatal exposure prior to the 13-week study and perinatal exposure prior to the 2-year study. The range finding study shall determine whether there is maternal toxicity and/or toxicity to the pups in order to provide a basis for determining the doses for the subsequent toxicity study (13-week or 2-year study). The animals shall be exposed to the test article during in utero development, through their mother's milk, and via dosed feed, dosed water, or gavage administration until PND21.
Perinatal Exposure Phase
- Gestation Period
Timed-pregnant, approximately gestation day 2 (GD2), female Harlan Sprague Dawley rats shall be 11-12 weeks of age upon receipt. Each female shall be housed individually and provided NIH-07 diet. All dams and pups shall remain in quarantine status during the study. On GD5 dams shall be randomly assigned to each of five (range finding study and perinatal exposure prior to the 13-week study) or three (perinatal exposure prior to the 2-year study) treatment and control groups and uniquely identified by tail tattoo. Starting on GD6 and continuing until postnatal day (PND) 21 the dams shall be gavaged daily (including weekends and holidays) or provided test article in feed or drinking water ad libitum. For gavage studies on the morning of the expected day of delivery if dams are not in the process of delivering, the dams shall be dosed; if in the process of delivery then wait 24 hours. Dams shall be weighed on GD5 prior to dosing, and then on GD6, 9, 12, 15, 18 and 21 for dosed water and dosed feed studies. Starting on GD9 food or water consumption shall be measured every three days during gestation. For gavage studies dams shall be weighed on GD5 prior to dosing and then daily throughout the gestation period. All dams shall be observed two times daily, once in the early morning and once in the late afternoon, at least six hours apart (before 10:00 AM and after 2:00 PM), including holidays and weekends, for signs of delivery, moribundity, and death. During the gestation and lactation exposure periods, any unexpected toxicity (such as mortality, body weight change, or clinical signs in dams and pups) that may impact the continuation of the study shall be reported immediately. Dams that do not deliver within 3-4 days of anticipated delivery date shall be euthanized, examined for uterine implantations/resorptions, and these findings recorded.
Pregnant Dams Treatment Groups Total Range Finding Study Treatment 5 x 5 = 25 Controls 5 x 1 = 5 Total 30 Perinatal Exposure Prior to the 13-week Study Treatment 5 x 5 = 25 Controls 5 x 1 = 5 Total 30 Perinatal Exposure Prior to the 2-year Study Treatment 25 x 3 = 75 Controls 25 x 1 = 25 Total 100
- Lactation Period
The lactation period includes the period from delivery until PND21, when the animals are weaned. PND21 is defined as 21 days post delivery of the last litter delivered. For dosed water or dosed feed studies dams shall be weighed on PND1, 4, 7, 14 and 21 and individual pups shall be weighed on PND4, 7, 14, and 21. Food or water consumption measurements shall be made on PND4, 7, 14, and 21 for the periods covered. For gavage studies dams shall be weighed daily throughout the lactation period and individual pups shall be weighed on PND4, 7, 12, and every three days thereafter until PND21. All dams and pups shall be observed two times daily, once in the early morning and once in the late afternoon, at least six hours apart (before 10:00 AM and after 2:00 PM), including holidays and weekends, for moribundity, death, or abnormal maternal behavior.
The number, sex, and general appearance of the pups shall be recorded the day after delivery (PND1). Body weights shall be recorded for dams and litter weights for pups by sex and total.
Litters and pups shall be culled on PND4. An acceptable litter shall have eight pups per litter (randomly selected to 4 males and 4 females if possible with no litter having less than 2 male and 2 female pups). Each selected pup shall be tattooed (on a limb) for unique identification.
Pups Treatment Groups Sex Total Range Finding Study Treatment 20 x 5 x 2 = 200 Controls 20 x 1 x 2 = 40 Total 240 Perinatal Exposure Prior to the 13-week Study Treatment 20 x 5 x 2 = 200 Controls 20 x 1 x 2 = 40 Total 240 Perinatal Exposure Prior to the 2-year Study Treatment 100 x 3 x 2 = 600 Controls 100 x 1 x 2 = 200 Total 800
For gavage studies, pups shall begin dosing at the same dose as the dams on PND12 and daily thereafter until PND21 (including weekends and holidays).
PND21 is defined as 21 days after the last litter was delivered. For the range finding study, dams and pups shall be sacrificed on PND21. For perinatal exposure prior to the 13-week or 2-year study, litters shall be weaned on PND21. Two male and two female pups from each liter shall be randomly selected and assigned to the 13-week or 2-year study. For the 2-year study, sentinels shall be randomly chosen from control pups not selected. Rats chosen to continue in the 13-week or 2-year study shall be tail tattooed using unique consecutive numbers and records shall be maintained to track the dam number for each selected pup. PND21 shall be designated Day 1 for the 13-week or 2-year study. Selected pups shall be provided NTP-2000 diet and are to be either placed on dosed feed or dosed water or dosed by gavage. Dams and pups not selected for continuation to the 13-week study or the 2-year study are to be euthanized.