### SAT Report PMN Number: P-13-0839 SAT Date: 8/27/2013 Print Date: 9/30/2013

Related cases :

Concern levels : Type of Concern: Health Eco Comments Level of Concern: 1-2 3 Health: ; Eco: significant mitigation of toxicity expected, i.e., >110x

Persistence Bioaccum Toxicity Comments 3 11

Exposure Based Review : Health: N o Ecotox: N o
Routes of exposure : Health: Drinking Water Inhalation Ecotox: All releases to water Fate: ;

P 2 Rec Comments : Comment: N o Commen t

Keywords : Keywords: LUNG UNCERT ACUTE AQUATOX-A,C
Summary of Assessment :

Fate : Fate Summary: P-13-0839 FATE: with MP < 25 C (E)S > 10 g/L at 25 °C (E) VP < 1.0E-6 torr at 25 C (E) BP > 400 C (E) H < 1.00E-8 (E) POTW removal (%) = 0-50 via sorption and biodeg; OECD111(Hydrolysis): t1/2( p H 4,7,9): <10%/5d. Time for complete ultimate aerobic biodeg ≥ mo Sorption to soils/sediments = moderate PBT Potential: P3B1 *CEB FATE: Migration to ground water = moderate

Health : Health Summary: Absorption is nil all routes, based on physical/chemical
p roperties. There is concern for lung toxicity from cationic binding to lung membranes. There is also uncertain concern for acute toxicity, based on submitted test data. Test Data: Submitted with the PMN:

N egative in the Salmonella assay with and without activation; N egative in the E. coli reverse mutation assay with and without activation; Rat (F) acute (15D) oral (gavage) toxicity LD50 300-2000 mg/kg

Ecotox :

Test Organism Test Type Test End Point Predicted Measured Comments

fish 96-h LC50 0.28 3.9 daphnid 48-h LC50 0.10 5.1 green algal 96-h EC50 0.040 0.015 fish

_ chronic value 0.016 daphnid

_ chronic value 0.007

algal

_ chronic value 0.020 0.011

Sewage Sludge 3-h EC50

_ Sewage Sludge

_ Chronic Value _

Ecotox Values Comments: Predictions are based on SARs for polycationic polymers; SAR chemical class = p olyme r -cationic-
p H7; effective concentrations based on 100% active ingredients and nominal concentrations; hardness <150.0 mg/L as CaCO3; and TOC <2.0 mg/L;

mitigation of toxicity expected in the presence of 10 mg TOC/L, i.e., >110x times

Ecotoxicity Test Data Results for P-13-0839:Acute fish, daphnia, and algae toxicity studies were completed for PMN P-13-0839 in 2013 by The PMN material is described as a in the neat form, as a solution with 42% PMN material in processing, a solid with the PMN material The molecular weight is Based on an estimated water solubility of >10 g/L and an estimated this PMN is expected to nonvolatile and highly soluble in water. The PMN material is used as a Studies followed the respective guidelines of OECD 203 (Acute Toxicity for Fish), OECD 202 (Daphnia sp., Acute Immobilization Test) and OECD 201 (Freshwater Alga Growth Inhibition Test) and were GLP compliant. Study methods concurred with methods specified in the respective guidelines except where specified below.

Fish Ecotoxicity Test:

conducted a 96-hour fish toxicity study in rainbow trout (Oncorhynchus mykiss) with P-13-0839 (60% p urity, PMN material considered to be the active ingredient) under semi-static conditions with 24-hour renewal. This study was reported to follow OECD guideline No. 203, “Fish, Acute Toxicity Test” and Method C.1 of Commission Regulation (EC) N o. 440/2008. Following a preliminary range-finding test, single replicates of seven O. mykiss were exposed to a dilution water control (dechlorinated tap water) or test substance at nominal concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg/L. Corresponding mean measured concentrations of the test substance were NA (not analyzed), 2.1, 3.5, 6.1 and 11 mg/L, as determined via HPLC-MS (LOQ = 0.28 mg/L). To prepare the test solutions, 1000 mg of the test substance was dissolved in dechlorinated tap water and the volume was adjusted to 1 L to give a 1000 mg/L stock solution. Aliquots (20, 36, 64, 112 and 200 mL) of the 1000 mg/L stock solution were then each added separately to final volumes of 20 L of dechlorinated tap water to give the nominal test concentrations. The stock solution was inverted several times, and the prepared concentrations were stirred using a flat-bladed mixer for approximately 1 minute to ensure adequate mixing and homogeneity. The test vessels were covered to reduce evaporation. The test solutions appeared to be clear and colorless for the duration of the test. Over the course of the study, temperature ranged from 13

– 14 °C, p H ranged fro m 7.8

– 8.3 and dissolved oxygen ranged from 9.0

– 11.3 mg/L. Dilution water hardness was 140 mg/L as CaCO3. A loading rate of 0.41 g fish/L was reported. The 96-hour cumulative % mortality at nominal concentrations of 0 (control), 1.0, 1.8, 3.2, 5.6 and 10 mg/L were 0, 0, 0, 14%, 43% and 86%, respectively. N o sub-lethal effects were observed during the test. Based on mean measured concentrations, the 96-hour LC50 was 6.5 mg/L. Taking into account the purity of the test substance (60% a.i.), the corrected 96-hour LC50 was 3.9 mg/L. This test was considered to be adequate. 96-hour LC50 = 3.9 mg/L (a.i.)

Daphnid Ecotoxicity Test:

conducted a 48-hour acute immobilization test in water flea (Daphnia magna) with P-13-0839 (60% p urity, PMN material considered to be the active ingredient) understatic conditions. This study was reported to follow OECD guideline No. 202, “Daphnia sp., Acute Immobilisation Test” and Method C.2 of Commission Regulation (EC) N o. 440/2008. Following a preliminary range-finding test, four replicates of five daphnids were exposed to a dilution water control (reconstituted water) or test substance at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L. Corresponding mean measured concentrations of the test substance were NA (not analyzed), 3.01, 9.51, 31.2 and 98.1 mg/L, as determined via HPLC-MS method (LOQ = 0.053 mg/L). The test substance (500 mg) was dissolved in reconstituted water and the volume adjusted to 5 L to give the 100 mg/L test concentration. Aliquots (20, 64, 200 and 640 mL) of the 100 mg/L test concentration were then each added separately to a final volume of 2 L of reconstituted water to give the nominal test concentrations. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity. The test solutions appeared to be clear and colorless for the duration of the test. Over the course of the study, temperature ranged from 20

– 21 °C, p H ranged fro m 7.8

– 8.1 and dissolved oxygen ranged from 8.6

– 9.1 mg/L. Reconstituted water hardness was approximately 250 mg CaCO3/L meeting and exceeding the maximum recommended water hardness of 250 mg CaCO3/L according to OECD 203 guidelines and 180 mg CaCO3/L according OPPTS 850.1010 guidelines. A loading rate of 25 daphnids/L was calculated. The 48-hour cumulative % immobilization at concentrations of 0.0, 1.0, 3.2, 10, 32 and 100 mg/L were 0, 0, 0, 65%, 100% and 100%, respectively. N o sub-lethal effects were observed during the test. Based on nominal test concentrations, the 48-hour EC50 was 8.5 mg/L. Taking into account the purity of the test substance (60% a.i.), the corrected 48-hour EC50 was 5.1 mg/L. Results should have been presented based on mean measured concentrations since the test concentrations were measured and reported. The water hardness was above OPPTS 850.1010 guidelines; however, this test was considered to be adequate. 48-hour EC50 = 5.1 mg/L (a.i.)

Algal Ecotoxicity Test:

conducted a 72-hour growth inhibition test in green algae (Pseudokirchneriella subcapitata) with P-13-0839 (60% p urity, PMN material considered to be the active ingredient) under static conditions. This study was reported to follow OECD guideline No. 201, “Freshwater Alga and Cyanobacteria, Growth Inhibition Test” referenced as Method C.3 of Commission Regulation (EC) N o. 761/2009. Following a range-finding study, three replicates of P. subcapitata (5 x 103 cells/mL) were exposed to the test substance at nominal concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. Corresponding mean measured concentrations of the test substance were < LOQ, < LOQ, < LOQ, 0.23 and 0.80 mg/L, as determined via HPLC-MS method (LOQ = 0.084 mg/L). Six replicates of P. subcapitata were exposed to a culture medium control. The algae were illuminated with a light intensity of approximately 7000 lux with constant shaking. To prepare the test solution, 50 mg of the test substance was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution. From this stock solution, a series of dilutions was made to give further stock solutions of 10, 1.0, 0.32, 0.10, 0.032 and 0.010 mg/L. An aliquot (900 mL) of each of the nominal test concentrations was separately inoculated with algal suspension (14.3 mL) to give the required test concentrations. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. The flasks were
p lugged with polyurethane foam bungs. At the start of the test, all control and test cultures wereobserved to be clear and colorless solutions. After the 72-hour test period, all control and 0.010 mg/L test cultures were observed to be green dispersions. The 0.032 mg/L test cultures were observed to be pale green dispersions, the 0.10 mg/L test cultures were observed to be very pale green dispersions, the 0.32 mg/L test cultures were observed to be extremely pale green dispersions and the 1.0 mg/L test cultures were observed to be clear and colorless solutions. HPLC-MS analysis (LOQ = 0.084 mg/L) of the test preparations at 0 and 72 hours for this study showed variable and inconsistent results. From a chemical perspective, the test item is a water soluble poly-quaternary ammonium salt with potential for the polymer to bind to any receptive surface, the effects of which were more notable/ p ronounced at the low test concentrations employed in this study. Given that the test item was known to be stable in other studies (acute fish toxicity and acute daphnid toxicity) it was considered that the algae were exposed to near nominal concentrations throughout the duration of the test. N o intact cells and cell debris was observed to be present in the test cultures at 0.32 and 1.0 mg/L. Over the course of the study, temperature was maintained at 24 ± 1°C and pH ranged from 7.6

– 8.6. The cell density in the control cultures increased by a factor of 126 within 72 hours. Based on nominal concentrations, the 72-hour EC50 values for inhibition of growth rate and yield were 0.068 mg/L and 0.025 mg/L, respectively. The 72-hour NOEC and LOEC for both growth rate and yield were 0.010 mg/L and 0.032 mg/L, respectively. The 72-hour ChV was 0.018 mg/L. Taking into account the
p urity of the test substance (60% a.i.), the corrected 72-hour LC50 values for inhibition of growth rate and yield were 0.041 mg/L and 0.15 mg/L, respectively. The purity corrected 72-hour NOEC and LOEC for both growth rate and yield were 0.006 mg/L and 0.019 mg/L, respectively. This test was considered to be adequate. 72-hour ErC50 (growth rate) = 0.041 mg/L (a.i.) 72-hour EyC50 (yield) = 0.015 mg/L (a.i.) 72-hour NOEC = 0.006 mg/L (a.i.) 72-hour LOEC = 0.019 mg/L (a.i.) 72-hour ChV = 0.011 mg/L (a.i.)

The 96-hour fish (LC50 = 3.9 mg/L a.i.), 48-hour daphnid (EC50 = 5.1 mg/L a.i.) and 72-hour algal (ErC50 = 0.041 mg/L and EyC50 = 0.015 mg/L; N OEC = 0.006 mg/L and LOEC = 0.019 mg/L a.i.) toxicity tests were adequate for P-13-0839. For comparative purposes, the 96-hour fish LC50 of 0.28 mg/L, the 48-hour daphnid LC50 of 0.10 mg/L, the 96-hour algae EC50 of 0.040 mg/L, and the fish, daphnid, and algae chronic values (ChV) of 0.016, 0.007 and 0.020 mg/L, respectively were predicted using the SAR for polycationic polymers. Available experimental data indicate algae to be the most sensitive species for calculating the acute and chronic concentrations of concern (CoC). Based on the experimental 72-hour algae EyC50 of 0.015 mg/L (15 p pb), and the application of an assessment factor of 4, the acute CoC for P-13-0839 is 3.75 p pb. The ChV for P-13-0839 is calculated by determining the geometric mean of the NOEC and LOEC to yield a ChV of 0.011 mg/L (11 p pb). The chronic CoC for P-13-0839 is then derived by dividing the ChV of 0.011 mg/L (11 p pb) by a factor of 10 (uncertainty factor) yielding a chronic CoC of 1.1 p pb.

Acute CoC = 4 p pb Chronic CoC = 1 p pb

Ecotox Study Reviewer: K. Eisenreich September 3, 2013Factors Values Comments Assessment Factor 10 Concentration of Concern ( p pb) 1

SARs p olycationic polymers SAR Class p olyme r -cationicEcotox Category Polycationic Polymers

Ecotox Factors Comments:

SAT Chair: J. Kwiat