Document Mk6dXR0r47Z7EX9o7BrdDYb7

flRr& 'O 5G\ Summary PFOS Rat Two-Generation Reproduction Study Study Numbers: 3M T-6295.9, Argus 418-008 (in-life), FACT-TOX-012 (analytical). Compound & Lot: PFOS (Perfluorooctanesulfonate) - Lot 217, 98.4% pure (SMD Analytical Request 53030). CAS No. 2795-39-3 Study Title: Combined Oral (Gavage) Fertility, Developmental and Perinatal/Postnatal Reproduction Toxicity Study of PFOS in Rats. Report Date: 10 June 1999 (in-life); 19 April 1999 (analytical). Study Year and GLP status: 1997-1999 and GLP study Procedures: Groups of male and female rats (35/sex/group) were administered (daily oral intubation) PFOS at dose levels of 0, 0.1, 0.4, 1.6, and 3.2 mg/kg/day. Dosing started when the animals were = 70 days old. The male and female rats (Fo generation) were dosed for 42 days prior to mating. After mating, dosing continued in the mated females throughout gestation. At day 10 of gestation, Caesarean section was done on 10 females/group to check for any effects in early gestation. The remaining 25 females/group were allow to deliver their litters. Dosing of the females continued during the 21-day lactation period. At weaning, 2 males and 2 females per litter were randomly selected for continuation on the study (Fi generation). Note: Because of post-natal effects at high doses (see below for details) only three groups of Fi animals continued the study, namely 0, 0.1 and 0.4 mg/kg/day. Daily dosing began in the Fi pups the day after weaning and continued throughout their growth to sexual maturity. At 24 days of age the Fi rats were tested in a passive avoidance paradigm. The animals were also tested in a water-filled M-maze when they were 70 days old. Cohabitation for mating began when the Fi animals were 90 days old littermate matings were avoided. Dosing continued in the females through gestation and 21-day lactation after delivery of F2 pups. The study ended upon weaning of the F2pups. Results: No deaths occurred in any of the Fo animals and there were no compound related clinical signs. Dose related reduced weight body weights occurred in both male and females at the higher two dose levels (3.2 & 1.6 mg/kg/day). Reduced food consumption correlated with these body weight effects. The 0.4 mg/kg/day males also had a slight, but statistically significant, reduced body weight gain during the pre-mating period. Dosing of PFOS as high as 3.2 mg/kg/day had no effect on estrous cycling; all mating and fertility parameters of the Fo rats were unaffected. There were no significant differences at any dose level in regard to litter averages for corpora lutea, implanations, viable embryos or nonviable embryos from the day 10 of gestation Caesarean section females. Upon littering, post-natal survival was reduced at the higher two dose levels (1.6 & 3.2 mg/kg/day). The post-natal survival effect was manifested by increased number of 001161 apparent still-births and increased pup deaths during the first 24 to 72 hours after birth. Necropsy of dead pups revealed many had not nursed - no milk curd in the stomach. Reflex and physical development (surface righting, pinna unfolding, eye opening, acoustic startle reflex, air right reflex, pupil constriction) was not affected in the 0.1 & 0.4 mg/kg/day Fi pups. Some of these parameters were slightly delayed in the 1.6 mg/kg/day Fi pups and they were not evaluated at the high dose (3.2 mg/kg/day) as no pups survived beyond four days post-natal. Because of adverse effects in the higher two dose levels (1.6 & 3.2 mg/kg/day) Fi pups, reduced survival and/or reduced growth, the decision was made, in conjunction with the attending laboratory veterinarian, not to continue these two dose levels into the second generation. It had been determined that dose of 1.6 mg/kg/day or higher produced compound toxicity. Continuance of these dose levels would not result in any additional useful information and would subject the animals to unnecessary stress. Death did not occur post-weaning in the Fi animals nor were there any compound related clinical signs. The male and female Fj rats in the 0.4 mg/kg/day group had somewhat reduced body weight and food consumption; at some points in time (weeks of growth) the differences from controls were statistically significant. The appearance of external evidence of sexual development (preputial separation in males & vaginal patency in females) was not affected at either dose level. Also, the passive avoidance and water maze testing did not reveal any differences in regard to learning, short-term retention nor long-term memory. Likewise, none of the mating or reproductive performance measurements (parameters) were adversely affected in the 0.1 & 0.4 mg/kg/day Fi male and female rats when they were mated and allowed to deliver litters. There were no significant differences in regard to F2 pup survival or pup weights at weaning. Conclusions: The results of the study indicated the following NOEL levels. The Fo maternal and paternal NOEL is 0.1 mg/kg/day. [higher dose levels had reduced body weight and food consumption] The Fo reproductive NOEL is > 3.2 mg/kg/day. [no effect on reproductive performance even at highest dose level] The F] post-natal NOEL is 0.4 mg/kg/day. [higher dose levels had reduced pup viability and growth] The Fj maternal and paternal NOEL is 0.1 mg/kg/day. [0.4 mg/kg/day animals had reduced body weight and food consumption] The Fi reproductive NOEL is > 0.4 mg/kg/day. [no effect on reproductive performance at either 0.1 or 0.4 mg/kg/day] The F2 post-natal NOEL is 0.4 mg/kg/day. [no important toxicological effect on pup survival or growth at either 0.1 or 0.4 mg/kg/day] 001162 Compound Level Samples: At the terminal sacrifice of the Fo males (after mating was completed), serum and liver samples were collected from 5 males in each of the dose groups. Serum samples were collected from 5 Fo females in all the dose groups except 3.2 mg/kg/day group at the terminal sacrifice the day after weaning the Fi litters. Liver samples were collected from the same Fo females and liver samples were also collected from the weaned Fi pups of the same dams. Analysis of the samples for PFOS (3M Analytical Report TOX012) yielded the following mean PFOS concentrations. Male F0 Serum: control - 0.024 ppm, 0.1 mg/kg/day - 10.5 ppm, 0.4 mg/kg/day - 45.4 ppm, 1.6 mg/kg/day - 152 ppm, 3.2 mg/kg/day - 273 ppm. Male Fo Liver: control - 0.665 ppm, 0.1 mg/kg/day - 84.9 ppm, 0.4 mg/kg/day - 176 ppm, 1.6 mg/kg/day - 323 ppm, 3.2 mg/kg/day - 1357 ppm. Female Fo Serum: control - 0.037 ppm, 0.1 mg/kg/day - 5.28 ppm, 0.4 mg/kg/day - 18.9 ppm, 1.6 mg/kg/day - 82.0 ppm. Female Fo Liver: control - 0.171 ppm, 0.1 mg/kg/day -14.8 ppm, 0.4 mg/kg/day - 58.0 ppm, 1.6 mg/kg/day - 184 ppm. Pup Fi Liver: control - 0.051 ppm, 0.1 mg/kg/day - 6.19 ppm, 0.4 mg/kg/day - 57.6 ppm, 1.6 mg/kg/day - 70.4 ppm. Note: Serum and liver reflect PFOS concentrations of F0 dams and Fi pups at the end of 21-day lactation, i.e. when the samples were collected. Compound levels at parturition when reduced pup survival was observed could differ and those are being measured in a follow-up PK study. Data Evaluation: A well conducted GLP study following established testing guidelines (OECD 416). Study evaluated reproductive parameters and post-natal pup development including neurological parameters. The study established valid NOEL values. C01IG3 Laboratory Report FACT-TOX-012 Serum and Liver Samples: At the terminal sacrifice of the Fo males (after mating was completed), serum and liver samples were collected from 5 males in each of the dose groups. Serum samples were collected from 5 Fo females in all the dose groups except 3.2 mg/kg/day group at the terminal sacrifice the day after weaning the Fi litters (day 21 of lactation). Liver samples were collected from the same Fo females. Liver samples were also collected from the weaned Fj pups (liver samples were pooled by litter) of the same dams. Milk Curd Samples: On day 4 of lactation, all litters, as stated in the protocol, were reduced to 8 pups per litter. At this time stomach contents (milk curd) samples, pooled by litter, were collected from 4 or 5 litters from three dose groups - control, low dose (0.1 mg/kg/day) and high dose (3.2 mg/kg/day). A validated analytical method for milk curd matrix has not been developed and these samples have not been analyzed. C0HG4 Jrf . T ) - n T*A y r T ' ' P \ T / ^ A Arqus Division : 9 05 Sheeny Drive, Bldq. A i Horsham , PA 19044 Tel 2 1 5 . 4 4 3 . 8 7 1 0 | Fax 2 1 5 . 4 4 3 . 8 5 8 7 April 13, 2000 Marvin T. Case, D.V.M., Ph.D. 3M Toxicology Services 3M Center, Building 220-2E-02 St. Paul, Minnesota 55144-1000 Telephone: (612) 733-5180 Telefax: (612)733-1773 RE: Final Report 418-008 - Combined Oral (Gavage) Fertility, Developmental and Perinatal/Postnatal Reproduction Toxicity of PFOS in Rats Sponsor's Study Number: 6295.9 Dear Dr. Case: Enclosed is one copy of the final report Amendment 1 to the above-referenced report. If you have any questions, please do not hesitate to contact me. RGY:kgp Attach. Associated Director of Research and Study Director 0011S5 Primedica C orporation j www.prim edica.com O Primedica Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, PA 19044 Telephone: (215) 443-871 0 Telefax: (215) 443-8587 REPORT AMENDMENT 1 13 APRIL 2000 PROTOCOL 418-008 SPONSOR'S STUDY NUMBER: 6295.9 COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS FINAL REPORT 1. Page 1-9, paragraph 4 has been revised to: Pregnancy occurred in 22 (95.6%), 21 (84.0%) and 24 (96.0) of the 23, 25 and 25 female rats assigned to cohabitation in the 0 (Vehicle), 0.1 and 0.4 mg/kg/day dosage groups, respectively. All pregnant dams delivered litters. The gestation index was comparable across the three dosage groups. Viability and growth of the second generation offspring (F2 pups) to weaning were also unaffected by the highest dosage tested, 0.4 mg/kg/day. There were no toxicologically important differences from the control group values. No clinical or necropsy observations in the F2 generation pups were attributable to dosages of the test article as high as 0.4 mg/kg/day. 2. Page I-10, paragraph 4 has been revised to: The FI generation reproductive NOEL is greater than a dosage of 0.4 mg/kg/day; no effects on mating or fertility occurred. The NOEL for viability and growth in the F2 generation offspring is also 0.4 mg/kg/day. There were no toxicologically important effects on pup survival or growth at the highest dosage tested, 0.4 mg/kg/day. 3. Page V-8, paragraph 3 has been revised to: The viability of the second generation offspring (F2 pups) was unaffected at the highest dosage tested, 0.4 mg/kg/day. Small increases in the number of dams with stillborn pups and in the number ofpup deaths after DL 2 in the 0.4 mg/kg/day dosage group were not toxicologically important because: 1) the values were not significantly differentfrom the control group values; 2) the average for live litter sizes delivered and surviving to DL 21 were greater than or comparable to the control group values; and 3) neither the viability or lactation indices for the 0.4 mg/kg/day dosage group remarkably differed from the control group values. 001166 4. V-8, paragraph 4 has been revised to: 418-008 FINAL REPORT AMENDMENT 1 PAGE 2 Pup body weights of the second generation offspring (F2 pups) were unaffected at the highest dosage tested, 0.4 mgfkgJday. Small reductions in pup body weights in the 0.1 and 0.4 mg/kg/day dosage groups were not toxicologically important because: 1) the small differences between the pup body weights for the control and 0.1 mg/kg/day dosage groups were not statistically significant and were associated with the larger live litter sizes of the 0.1 mg/kg/day dosage group on DLs 1 through 4, as compared with the control group values, and the random selection ofpups fo r continued observations on DL 4; 2) the small reductions in pup body weights in the 0.4 mg/kg/day dosage group were associated with a minimally larger live litter size at birth (DL 1) and on DL 4 preculling, as compared with the control group values, and the random selection of pups fo r continued observation on DL 4; the statistically significant reductions (p<0.05 andp<0.01, respectively) present on DLs 7 and 14, as compared to the control group values, were transient and disappeared by DL 21. 5. Page V-9: Revisions to paragraph 4 on page V-8 caused it to extend onto page V-9. These revisions were made to clarify the interpretation of the observations reported and the NOEL for the second generation offspring (F2 generation). R^yfnond G. York, Ph/D,, DABT Associate Director of/Research and Study Director Date Quality Assurance Manager 418-008:PAGE I-9 REVISED PAGE feed consumption values were reduced during lactation in the 0.4 mg/kg/day dosage group. Dosages of the test article as high as 0.4 mg/kg/day did not affect the average day of vaginal patency in the F1 generation female rats. There were no biologically important differences in the values for learning, short-term retention, long-term retention or response inhibition in the F1 generation female rats, as evaluated by performance in a passive avoidance or watermaze performance paradigm. Dosages of the test article as high as 0.4 mg/kg/day did not affect any mating and fertility parameters evaluated in the F1 generation female rats. All necropsy observations in the F1 generation female rats were considered unrelated to the test article. Pregnancy occurred in 22 (95.6%), 21 (84.0%) and 24 (96.0) of the 23, 25 and 25 female rats assigned to cohabitation in the 0 (Vehicle), 0.1 and 0.4 mg/kg/day dosage groups, respectively. All pregnant dams delivered litters. The gestation index was comparable across the three dosage groups. Viability and growth of the second generation offspring (F2 pups) to weaning were also unaffected by the highest dosage tested, 0.4 mg/kg/day. There were no toxicologically important differences from the control group values. No clinical or necropsy observations in the F2 generation pups were attributable to dosages of the test article as high as 0.4 mg/kg/day tv >MlG8 418-008:PAGE 1-10 REVISED PAGE C. Conclusions On the basis of these data, the Fo generation maternal and paternal noobservable-effect-level (NOEL) of PFOS is 0.1 mg/kg/day (0.4 mg/kg/day and higher dosages caused reductions in body weight gain and reduced feed consumption values). The Fo generation reproductive NOEL is greater than 3.2 mg/kg/day; no effects on mating, fertility or estrous cycling occurred. The NOEL for viability and growth in the F1 generation offspring is 0.4 mg/kg/day (1.6 mg/kg/day and higher dosages caused preimplantation loss and reductions in litter size, pup viability, growth and survival). The F1 generation maternal and paternal NOEL of PFOS is 0.1 mg/kg/day (0.4 mg/kg/day dosage caused reductions in body weight gain and reduced feed consumption values). The F1 generation reproductive NOEL is greater than a dosage of 0.4 mg/kg/day; no effects on mating or fertility occurred. The NOEL for viability and growth in the F2 generation offspring is also 0.4 mg/kg/day. There were no toxicologically important effects on pup survival or growth at the highest dosage tested, 0.4 mg/kg/day. Mildred S. Christian, Ph.D., Fellow, ATS Date 418-008:PAGE V-8 REVISED PAGE B.8. Necropsy Observations (Summary - Table E18: individual Data Table E34) All necropsy observations in the F1 generation female rats were considered unrelated to the test article because: 1) the observations occurred in rats in the control group; and 2) the observation commonly occurs in this strain of rat. These observations included rough and/or pitted cortex of the kidneys with calculi in the kidney and/or urinary bladder of two control group rats. One of these rats also had a tan granular material in the renal cortex and thickened walls of the urinary bladder. Necropsy observations for the 0.4 mg/kg/day dosage group dam that died as the result of an intubation error were described previously. B.9. Natural Delivery and Litter Observations (Summaries - Tables E19 and E20; Individual Data - Tables E35 through E38) Pregnancy occurred in 22 (95.6%), 21 (84.0%) and 24 (96.0%) of the 23, 25 and 25 female rats assigned to cohabitation in the 0 (Vehicle), 0.1 and 0.4 mg/kg/day dosage groups, respectively. All pregnant dams delivered litters. The gestation index (the percentage of pregnant rats with live offspring) was comparable across the three dosage groups. The viability of the second generation offspring (F2 pups) was unaffected at the highest dosage tested, 0.4 mg/kg/day. Small increases in the number of dams with stillborn pups and in the number of pup deaths after DL 2 in the 0.4 mg/kg/day dosage group were not toxicologically important because: 1) the values were not significantly different from the control group values; 2) the average for live litter sizes delivered and surviving to DL 21 were greater than or comparable to the control group values; and 3) neither the viability or lactation indices for the 0.4 mg/kg/day dosage group remarkably differed from the control group values. Pup body weights of the second generation offspring (F2 pups) were unaffected at the highest dosage tested, 0.4 mg/kg/day. Small reductions in pup body weights in the 0.1 and 0.4 mg/kg/day dosage groups were not toxicologically important because: 1) the small differences between the pup body weights for the control and 0.1 mg/kg/day dosage groups were not statistically significant and were associated with the larger live litter sizes of the 0.1 mg/kg/day dosage group on DLs1 through 4, as compared with the control group values, and the random selection of pups for continued observations on DL 4; 2) the small reductions in pup body weights in the 0.4 mg/kg/day dosage group were associated with a minimally larger live litter size at birth (DL 1) and on DL 4 preculling, as compared with the control group values, and the random selection of pups for continued observation on DL 4; the statistically significant reductions 0013 70 418-008:PAGE V-9 REVISED PAGE (p<0.05 and p<0.01, respectively) present on DLs 7 and 14, as compared to the control group values, were transient and disappeared by DL 21. Administration of the test article at dosages as high as 0.4 mg/kg/day did not adversely affect any other parameter evaluated at natural delivery or during the 21-day lactation period (duration of gestation, averages for implantations and live litter sizes, numbers of dams with all pups dying during lactation, viability and lactation indices, surviving pups per litter and pup sex ratios). B.10. Clinical Observations from Birth to Day 21 Postpartum and Necropsy Observations (Summaries - Tables E21 and E22; Individual Data - Tables E39 and E40) No clinical or necropsy observations were attributable to dosages of the test article as high as 0.4 mg/kg/day because: 1) the incidences were not dosagedependent; and/or 2) the observation occurred in only one or two pups. These clinical observations included one control group pup that was not nesting or nursing, had decreased motor activity and was cold to the touch, one pup in each of the control and 0.4 mg/kg/day dosage groups that had a missing tip of tail and one 0.4 mg/kg/day dosage group pup that had an umbilical hernia. No milk in stomach occurred in 2, 4 and 4 pups that were found dead in the three respective dosage groups. One control group pup had hydrocephaly and one 0.1 mg/kg/day dosage group pup had a raised tan area on the median and left lateral lobes of the liver at necropsy on DL 21. 001171