Document GmDVXdMp9M5G2Lpw7VbpYa45x

BACK TO MAIN FETAX - FROG EMBRYO TERATOGENESIS ASSAY - XENOPUS TEST SUBSTANCE_________________________________ Identity: Perfluorooctanesulfonate; may also be referred to as PFOS, U2723 or FC-95. (1 -Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafluoro-, potassium salt, CAS # 2795-39-3) Remarks: Sample obtained from 3M production lot number 217. The test substance is a white powder. Purity determined to be 86.9% by LC/MS, 1H-HMR, 19F-NMR and elemental analyses techniques. METHOD Method: ASTM E1439-91 Test type: Static renewal GLP: In-life phase - no; stock solution preparation and measurement of test concentrations - yes Year completed: 2001 Number of studies: 3 Study 1 Study 2 Study 3 Start date: 5/15/00 5/22/00 5/22/00 End date: 5/19/00 5/26/00 5/26/00 (Study 2 and 3 set up concurrently with common stock solutions) Analytical monitoring: PFOS measured at 0 and 96-hours Species: Xenopus laevis Source: Breeding colonies at the University of Maryland Wye Research and Education Center (UMD/WREC), Queenstown, Maryland. Test organisms laboratory culture: Mating pairs were bred in the dark in 23.5 + 0.5C UMD/WREC non-chlorinated well water at ~ 70 day intervals by injecting 400 and 800 I. U. of human chorionic gonadotropin (HCG) in the dorsal lymph sac of the males and females, respectively. Amplexus occurred 4-6 hours after injecting HCG; egg deposition occurred 9-12 hours following HCG injection. Age at test initiation: Embryos; normal stage 8 blastula to normal stage 11 gastrula Loading: 25 embryos/10 mL Pretreatment: Embryos de-jelled in a 2% L-cysteine solution, then rinsed and re-suspended in FETAX solution prior to introduction to test chambers. Element basis: mortality, malformations (via the atlas of Bantle et al., 1991), growth Exposure period: 96-hours BACK TO MAIN Test Conditions (all 3 studies): Dilution water: ASTM (1998) FETAX solution Test temperature: 24.0 + 0.2C Light levels: 60-85 foot candle fluorescent lights Photoperiod: 12-hour light:12-hour dark Stock and test solution preparation: A primary stock solution was prepared in FETAX medium (supplied by UM-WREC) by Wildlife International, Ltd. at 48 mg PFOS/L. The primary stock solution was mixed by sonication and stirring. After mixing, the primary stock solution was proportionally diluted with FETAX medium to prepare the six test concentrations. The six test concenetration solutions were delivered to UM-WREC prior to the start of each study. Reference substance: 6-aminonicotinamide Stock and reference substance solution preparation: as outlined in the ASTM (1998) protocol Exposure vessels: Covered 60 mm glass Petri dishes containing 10 mL test solution Number of replicates: controls - 4, treatments - 2 Number of embryos per replicate: 25 Number of concentrations: six plus a negative control plus an abiotic control at the highest concentration tested, plus two reference substance concentrations. Renewal frequency: every 24 hours Stability of the test chemical solutions: Extremely stable Water chemistry during all 3 studies: pH range (0 - 96 hours) 7.1 - 7.7 (control exposure) 7.0 - 7.6 (24 mg/L nominal exposure) Dissolved oxygen range (0-96 hours) 7.3 - 8.4 mg/L (control exposure) 7.0 - 8.5 mg/L (24 mg/L nominal exposure) Method of calculating mean measured concentrations: arithmetic mean RESULTS Nominal PFOS concentrations: Negative control, 1.82, 3.07, 5.19, 8.64, 14.4 and 24.0 mg/L plus 24.0 mg/L abiotic control. BACK TO MAIN Nominal 6-aminonicotinamide concentrations: 5.5 and 2500 mg/L Mean measured PFOS concentrations: Study 1: <LOQ, 2.00, 2.83, 4.73, 7.90, 14.7, 24.6 mg/L; abiotic control = 23.7 mg/L Study 2: < LOQ, 1.91,3.04, 4.82, 7.97, 13.3, 23.1 mg/L; abiotic control = 23.9 mg/L Study 3: < LOQ, 1.93, 3.27, 5.25, 8.26, 14.0, 23.9 mg/L; abiotic control = 24.1 mg/L PFOS element va ues and 95% confidence intervals, mg/L Study 96-Hr LC50 96-Hr EC50 Minimum conc. to Teratogenic Number Inhibit Growth Index (TI) (MCIG) 1 13.8 12.1 (12.4 -15.3) (10.0 - 14.6) Not calculable 1.1 2 17.6 17.6 (15.5 - 20.0) (13.5 - 22.9) 7.97 1.0 3 15.3 16.8 (13.1 - 17.8) (12.4 - 22.8) 8.26 0.9 All element values based on mean measured concentrations Statistical methods: The Trimmed Spearman-Karber statistical procedure was used to determine the 96-hour LC50 for mortality and 96hour EC50 for malformations. The MCIG was determined by Bonferroni's T-Test. All statistical tests were performed using Toxstat (WEST and Gulley, 1994). A minimum probability level of 0.05 was used. The teratogenic index (TI) was calculated by dividing the LC50 by the EC50. Analytical Methodology: Analyses of test solutions were performed at Wildlife International Ltd., Easton, MD using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). When determining the concentration of the test substance in the test solutions, the same and most prominent peak response for perfluorooctanesulfonate was used. No attempt was made to quantify on the basis of individual isomeric components. The LOQ (limit of quantitation) was 0.240 mg/L in these studies. The mean percent recovery of matrix fortifications analyzed concurrently during sample analysis was 97.2%. Samples collected at test initiation had measured values from 112 to 141% of nominal in the first study, and in the second and third studies, from 95.8 to 117% of nominal. Measured values for samples taken at 96-hours ranged from 54.7 to 98.6% of nominal in the first study and 80.7 to 112% of nominal in the second and third studies. The samples from the abiotic 24.0 mg/L treatment group was comparable to samples from the 24.0 mg/L treatment group with the embryos present. Summary of analytical chemistry data: Study 1__________________________ N o m in a l T e s t C o n c e n tra tio n , m g /L M e a su re d V a lu e s at 0 and 9 6 -h o u rs R e s p e c tiv e ly , m g /L M ean M easured C o n ce n tra tio n , m g /L N e g a tiv e C o n tro l 1 .8 2 3 .0 7 5 .1 9 A ll < LO Q 2.58, 1.42 3.94, 1.72 6.62, 2 .8 4 <LO Q 2 .0 0 2 .8 3 4 .7 3 8 .6 4 1 4.4 10.7, 5.0 9 7 .9 0 1 4 .7 2 4.0 2 4 .0 (a b io tic ) 26.9, 2 2.3 not a n a ly z e d , 23 .7 2 4.6 2 3.7 P e rcen t of N o m in a l 110 92 .2 9 1 .1 91 .4 102 103 98 .6 co co d LO LO co LO Study 2 N o m in a l T e s t C o n c e n tra tio n , m g /L M easured V a lu e s at 0 an d 9 6 -h o u rs R e s p e c tiv e ly , m g /L N e g a tiv e C o n tro l 1 .8 2 3 .0 7 5 .1 9 8 .6 4 1 4.4 2 4.0 2 4 .0 (a b io tic ) A ll < LO Q 1.77, 2 .0 4 3.59, 2.49 8 .4 3 , 7.51 1 4 .5 , 12.1 2 3 .0 , 23.1 not a n a ly z e d , 23 .9 M ean M easured C o n ce n tra tio n , m g /L <LO Q 1.91 3 .0 4 4 .8 2 7 .9 7 1 3 .3 2 3 .1 2 3.9 P e rcen t of N o m in a l 105 99 .0 92 .9 92 .2 92 .4 96 .3 99 .6 Study 3 N o m in a l T e s t C o n c e n tra tio n , m g /L M e a su re d V a lu e s at 0 and 9 6 -h o u rs R e s p e c tiv e ly , m g /L N e g a tiv e C o n tro l 1 .8 2 3 .0 7 5 .1 9 8 .6 4 1 4.4 2 4.0 2 4 .0 (a b io tic ) LO CO LO~ A ll < LO Q 1.77, 2.0 8 3.59, 2 .9 4 5.45, 5.05 8.43, 8.09 23.0, 2 4.7 no t a n a ly z e d , 24.1 M ean M easured C o n ce n tra tio n , m g /L <LO Q 1 .9 3 3 .2 7 5 .2 5 8 .2 6 1 4 .0 2 3.9 2 4 .1 P e rcen t of N o m in a l 106 107 101 95 .6 97 .2 99 .6 100 BACK TO MAIN BACK TO MAIN Biological observations after 96-hours: Mortality and Malformations Test 1 Test 2 Nominal Percent Percent Percent Percent Concentration*, Mortality Malformations Mortality Malformations mg/L NegativeControl 1.0 4.0 1.0 4.0 Test 3 Percent Percent Mortality Malformations 0 2.0 1.82 2.0 8.2 0 8.0 0 6.0 3.07 4.0 15 10 4.4 0 4.0 5.19 10 22 8.0 11 0 6.0 8.64 12 25 10 20 14 14 14.4 38 65 30 37 44 39 24.0 100 _ 70 67 78 73 *Nominal concentrations used for ease of comparison table Malformations: The most common types of malformations noted were improper gut coiling, edema, notochord abnormalities and facial abnormalities. Growth - Mean length (mm) after 96-hours Exposure Nominal Concentration*, mg/L Test 1 Test 2 Test 3 NegativeControl 8.59 8.88 9.47 1.82 8.29 8.45 9.10 3.07 8.80 8.57 9.28 5.19 8.51 8.72 9.28 8.64 8.71 7.93** 8.51** 14.4 8.08 7.51** 8.11** 24.0 - (total mortality) 7.39** 7.80** *Nominal concentrations used for ease of comparison table ** Significantly different at alpha = 0.05 (Bonferroni T-Test) Control response: satisfactory. Reference substance response: satisfactory at low concentration (5.5 mg/L). Did not meet ASTM (1998) criteria for high concentration (2,500 mg/L). However, results obtained at high concentration were consistent and not at variance with previous experience in this testing laboratory. BACK TO MAIN Observations: Majority of embryo mortality appeared to be caused by the gut coiling through the body wall at the two highest test concentrations. CONCLUSIONS______________________________________________ The potassium perfluorooctanesulfonate 96-hour LC50 range for FETAX was determined to be 13.8 - 17.6 mg/L. The 96-hour EC50 range was 12.1 - 17.6 mg/L. The range for Maximum Concentration to Inhibit Growth (MCIG) was 7.97 to >14.7 mg/L. The Teratogenic Index (TI) was found to be 0.9 - 1.1. This TI range indicates that potassium perfluorooctanesulfonate has a low potential to be a developmental hazard. Submitter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY_____________________________________________ Reliability: Klimisch ranking = 2 Although these were well-conducted studies, the in-life phases were not conducted in accordance with Good Laboratory Practices. REFERENCES_____________________________________________ These studies were conducted at the University of Maryland Wye Research and Education Center (UM-WREC) in Queenstown, Maryland and at Wildlife International Ltd., Easton, MD at the request of the 3M Company. Lab Request number U2723 OTHER__________________________________________________ Last changed: 6/12/01