Document jyE0EXBdq7eVXX3mDw4j0OKL2

AR226-1307-5 Summary * APFO reproductive effects, 8/23/2002 Created By:Nancy S Selzer on 10/07/2002 at 01:47 PM Document Category: Mammalian Toxicology Testing Sub Category : PFOA Consortium Products Additional Authors: Nancy S Selzer/AE/DuPont Li APFO Reproductive effects summary.d 0 .Edit History: Editor Nancy S Selzer/AE/DuPont Edit Date 10/07/2002 01:48:54 PM *Onlypast five adits are shown 000050 PTDB00OB30 EID613678 DuPont Summary - August 23,2002 Oral (Gavagc) Two-Generation (One Litter Per Generation) Reproduction Study of Ammonium Perfluorooctanoate (APFO) in Rats 3M study conducted at Argus (Argus Study #T-6889.6) The multigeneration reproduction study with APFO has been reviewed and DuPont's position on the study is reported below, in addition to some comments on potential endocrine effects of APFO. In general, we agree with the conclusions stated in the study report with the 2 exceptions noted below. After reviewing the report, Haskell contacted the Argus Study Director on May 31, 2002 to discuss our differences in data interpretation. The study director was in agreement with the recommended changes stated below. Overall, the data from the multigeneration reproduction study illustrate that the parental animals are more severely affected than the offspring, and hence, APFO is not a reproductive toxin. However, there are effects (i.e., decreased pup weights, increased pup mortality, altered estrous cyclicity, delayed puberty) that could be suggestive of reproductive effects, and 30 mg/kg/day could represent the beginning ofthe dose-response curve based on the effects observed in the reproduction study. In particular, the effects on pup viability, estrous cyclicity, and attainment of puberty (vaginal opening and preputial separation) are suggestive, although not indicative, of reproductive effects. As a result ofthe small alterations in these endpoints, which were only observed at 30 mg/kg/day, the biological significance of the effects is unclear. Clearly, these effects do not compromise the reproductive success (i.e., mating and fertility) ofthe animals at dosages of 30 mg/kg/day under the conditions of this study. Due to the toxicity of APFO to the males, testing for reproductive effects at dosages higher than 30 mg/kg/day may be problematic. The summary of fee findings of the study are summarized below; PI Generation: The paternal no-observable-adveise-effect-level (NOAEL) for APFO is less than 1 mg/kg/day based on fee significant increases in organ weights (liver and kidney) that were observed at 1 mg/kg/day and above. The maternal NOAEL is 10 mg/kg/day based on fee significant decreases in kidney weights that were observed at 30 mg/kg/day. The reproductive NOAEL in males is greater than 30 mg/kg/day since there were no affects on mating or fertility at 30 mg/kg/day. The reproductive NOAEL in females is 10 mg/kg/day based on the decreased pup weights and pup survival at 30 mg/kg/day (see discussion below). FI Generation: The paternal no-observaWe-adverse-effect-level (NOAEL) for APFO is less than 1 mg/kg/day based on fee significant alterations in body and organ weights feat were observed at 1 mg/kg/day and above. 000051 PTDB000831 EID613679 The maternal NOAEL is 1mg/kg/day based on the significant decreases in pituitary weights that were observed at 3 mg/kg/day and above. The reproductive NOAEL in males and females is greater than 30 mg/kg/day since there were no affects on mating, fertility, or maternal delivery parameters at 30 mg/kg/day. 000052 PTDB000832 EID613680 Potential endocrine-related effects were as follows: In both male and female FI weanlings, there were alterations in attainment of puberty [delayed age ofpreputial separation (PPS; androgen-dependent process) in males and delayed age at vaginal opening (VO; estrogen-dependent process) in females]. While these alterations can be attributed to perturbations in endocrine homeostasis, the pattern of the effects is inconsistent with know endocrine mechanisms. Typically, compounds that will attenuate androgen levels will delay PPS while not affecting VO, whereas compounds that attenuate estrogen levels will delay VO while not affecting PPS. A general delay in both can occur in cases ofbroad steroid depletion where both estrogens and androgens are attenuated. While there is evidence that APFO has the potential to induce aromatase in rodents (the enzyme that converts testosterone to estradiol), there is no evidence that APFO possesses broad steroid biosynthesis inhibition properties that could account for the effects that were observed. General delays in PPS and VO can also be attributed to non-specific effects on body weight In the current study, it was concluded that the delays in sexual maturation in male and female rats may be due to die decreased gestational age in the animals at puberty. For this reason, it is not suggestive ofan endocrine-mediated effect In FI females there was a slight increase in the number of estrous cycles per 21 days (4.7 versus 5.4 stages), which is outside of the historical control for the testing laboratory. While this may represent a compound-related effect, it does not manifest in altered reproductive success. The following modifications to the original report were discussed with Argus and the Study Director was in agreement with Haskell's recommendations: 1. The P I generation MaternalReproductive NOAEL: The study report states that the PI generation female's offspring have decreased pup body weights on lactation days 1,5 and 8 in the 30 mg/kg/day dosage group. Yet the report states the PI generation Maternal Reproductive NOAEL is greater than 30 mg/kg/day. In addition, the significantly increased number of pup deaths in the 30 mg/kg/day was considered by the Study Director to be unrelated to the test article because the deaths did not affect the indices of pup viability, although the lactation index value in the 30 mg/kg/day was significantly decreased (and considered by the Study Director to be unrelated to the test article because the value was within historical control limits). We agree that while no other measures of pup viability were affected, the decreased pup body weights and the failure to thrive that was noted as a cause of death for several of these animals would lead us to conclude that this was possibly a treatment-related effect All factors taken together, including the slightly higher number of FI pup deaths during lactation, the small reduction in mean pup weight on days 1,5, and8 (approx 90% of control), and toe post-weaning FI pup deaths (due to M ure to thrive) at 30 mg/kg/day suggest that there was a compound related effect on FI pup growth and survival. The significant reduction in lactation index is also probably compound-related since it is consistent with toe increased overall pup mortality in the FI generation at 30 mg/kg/day. 000053 PTDB000833 EID613681 This strengthens the determination to revise down the PI generation Maternal Reproductive NOAEL from greater than 30 mg/kg/day to 10 mg/kg/day. A comprehensive evaluation of pup mortality is not made in the report, which is why this conclusion may have been overlooked. The data for the FI pups are summarized below. Dose (mg/kg/day APFO) Dead pups day 1 Dead pups days 2-5 Dead pups days 6-22 Dead pups days 2-22 Dead pups post-weaning (male ft female) Dead pups post-weaning (males) No of above attributed to M ure to thrive Dead pups post-weaning (females) No of above attributed to M ure to thrive Total dead pups pro- and postweaning Mean pup weights: Day 1 Day 5 Day 8 Day 15 Day 22 ' Number ofpupi (litten). 0 1 3 10 30 2 230 3 7(6)` 6(5) 7(6) 5(5) 11(9)* KD 3(3) 7(3) 5(3) 12(7)* 8(7) 9(8) 14(8) 10(6) 23(11)* 3 5 4 3 13 3 3 3 2 7* 1 12 0 4 0 2 l 1 6* 0 11l 4 13 16 21 12 39 6.3 6.0 62 62 5.7* 9.4 8.8 9.5 9.3 8.5* 133 12.7 13.5 13.0 11.9* 25.0 242 26.4 24.8 22.9 37.4 36.7 39.7 38.8 35.7 2. The F I generation Maternal NOAEL: The study report further states that the F1 generation females have decreased absolute and relative pituitary weights at 3 mg/kg/day and higher. The Study Director may have overlooked this finding when setting the FI generation Maternal NOAEL at 3 mg/kg/day. The FI generation Maternal NOAEL should be revised to 1 mg/kg/day [the 3 mg/kg/day and higher doses caused decreased absolute and relative pituitary weight (3 mg/kg/day and higher), postweaning mortality (30 mg/kg/dayX decreased preweaning body weight and gain (30 mg/kg/day), decreased feed consumption (30 mg/kg/day), increased number of estrous cycles per 21 days (30 mg/kg/day), and delayed sexual maturation possibly due to decreased gestational age at acquisition (30 mg/kg/day)]. 000054 PTDB000834 EID613682 The following tables summarize the statistically significant results o f this study. PI Generation Male Rats Parameter Mortality (Treatment Related) Clinical Observations Body Weight/Gains Feed Consumption (Absolute) Feed Consumption (Relative) Mating Fertility Necropsy Observations Terminal Body Weight Organ Weights (Absolute) Epididymides, seminal vesicles, prostate, pituitary, adrenals, spleen, thymus Liver Kidney Organ Weights (Relative to Body) Epididymides, testes, seminal vesicles Brain Kidneys Adrenal Organ Weights (Relative to Brain) Epididymides, prostate, pituitary, spleen, thymus Liver Kidney Sperm Quality Histopathology (Reproductive Organs) Histopathology (Other Organs) General Toxicity (NOAEL <1 mkd) Reproductive Toxicity 1 0 - - - - - o o - o - - o o - - LOAEL Mg/Kg/day (mkd) APFO 3 10 00 -- <= <= -- <=> o -- --- c= e= 30 1 o <= <= <=> - <= o o o oo o <= -- o oo oo -- o CO <=> -- <= oo o <=> -m -- o <= - - Adrenal Adrenal NOAEL 000055 PTDB000835 EID613683 PI Generation Female Rats Parameter Mortality (Treatment Related) Clinical Observations Body Weight/Gains Feed Consumption (Absolute) Feed Consumption (Relative) Mating Fertility Necropsy Observations Pregnancy Terminal Body Weight Organ Weights (Absolute kidney) Organ Weights (Relative kidney) Estrous Cyclicity Histopathology (Reproductive Organs) Histopathology (Other Organs) General Toxicity Reproductive Toxicity l 0 - - - - Mg/1Cg/day APFO 3 10 00 ------- ---- -- -.- -" -- NOAEL NOAEL 30 0 - I - <= <= - - 000056 PTDB000836 EID613684 FI Generation Male Rats Parameter Mortality (post lactation; control=3) Clinical Observations Body Weight/Gains Feed Consumption (Absolute) Feed Consumption (Relative) Preputial Separation Mating Fertility Necropsy Observations Terminal Body Weight Organ Weights (Absolute) Liver Spleen Kidney Thymus Prostate, brain, adrenals Organ Weights (Relative to Body) Seminal vesicles, liver, kidneys Testes Epididymides, brain Organ Weights (Relative to Brain) Seminal vesicles, liver Kidney Testes Spleen Thymus Sperm Quality Histopalhology (Reproductive Organs) Histopalhology (Other Organs) General Toxicity (NOAEL <1 mkd) Reproductive Toxicity 1 3 o <= - - - c= o c= o - - o - co o - c= - . LOAEL Mg/Kg/day APFO 3 10 32 <=> <=> c= c= - c= - co -- -- -- o co c= c= 30 7 co <= c= co Delayed - - CO <= co <=> c= c= co e= c= -- CO c= c= - co co co co - co CO CO CO co CO co co o co c= c= - <= -- -- CO - CO c= <= - - Liver Liver Liver/Adrenal NOAEL 000057 PTDB000837 EID613685 FI Generation Female Rats Parameter Mortality (post lactation; control=0) Clinical Observations Body Weight/Gains Feed Consumption (Absolute) Feed Consumption (Relative) Vaginal Patency Mating Fertility Necropsy Observations Pregnancy Terminal Body Weight Organ Weights (Absolute pituitary) Organ Weights (Relative pituitary) Estrous Cyclicity Histopathology (Reproductive Organs) Histopathology (Other Organs) General Toxicity Reproductive Toxicity 1 2 - - - NOAEL Mg/ICg/day APFO 3 10 11 -- -- --- -- ---- --- es c= c= <= -- - -- 30 6 - c= - Delayed - - - <= <=> - - NOAEL 000058 PTOB000838 EID613686 F2 Generation Rats (pups') Parameter Viability/mortality Litter size Clinical Observations Body Weight Terminal body weight Necropsy Observations Organ Weights (brain, spleen, thymus) Anogenital distance 1 - - Summary o f F2 pup mortality Dose (tng/kg/day APFO) Dead pups day 1 Dead pups days 2-5 Dead pupa days 6-22 Dead pups days 2-22 Mg/Kg/day APFO 3 10 ------- -- 30 - - - 0 1 3 10 30 0 0 5* 4* 1 2 553 3 5 453 5 7 9 10 6 S Document Revision Dates: Original document: August 19,2002 l " Revision: August 23,2002 000059 PTDB000839 EID613687