### SAT Report PMN Number: P-14-0072 SAT Date: 11/19/2013

Print Date: 4/1/2014

Related cases :

Concern levels :
Type of Concern: Health Eco Comments Level of Concern: 2 3

Persistence Bioaccum Toxicity Comments 1 2 2 PMN 1 1 1 Hyd Pdt Acid 1 1 1 Hyd Pdt Alcohol

Exposure Based Review : Health: Yes Ecotox: Yes

Routes of exposure : Health: Dermal Drinking Water Inhalation Ecotox: All releases to water Fate: ; Hyd Pdt Acid rapid; Hyd Pdt Alcohol rapid

P 2 Rec Comments : Comment: Do Not Forward: Similar Toxicity Concerns

Keywords : Keywords: SEVR IRR- S IRR-E,L SENS-S BLOOD SPLEEN LIVER SYSTOX REPRO DEVEL MUTA ONCOAQUATOX-A.C

Summary of Assessment :

Fate : Fate Summary: P-14-0072 FATE: Liquid with MP < -82 C (M) log Kow = 4.90 (M) S = Reacts / 4.25 mg/L at 25 C (E) Hydrolysis half-life = hr VP = 2.3 torr at 25 C (M) BP = Dec. 57 C (DSC) H = 7.55E-3 (E) log Koc = 2.56 (E) log Fish BCF = 2.90 (E) log Fish BAF = 3.03 (E) POTW removal (%) = PMN 90-99 via hydrolysis, then Hyd Pdt Acid 90% via biodeg; Hyd Pdt Alcohol 0%; OECD 301 (Closed Btl): 69%/28d. Time for complete ultimate aerobic biodeg = Hyd Pdt Acid wk-mo Sorption to soils/sediments = Hyd Pdt Acid low; Hyd Pdt Alcohol low Volatilization half-life from a standard river = 2 hrs Volatilization half-life from a standard lake = 6 da Atmospheric Oxidation Half-life = 47 hr via OH radical PBT Potential: PMN P1B2; Hyd Pdt Acid P1B1; Hyd Pdt Alcohol P1B1 *CEB FATE: Migration to ground water = Hyd Pdt Acid rapid; Hyd Pdt Alcohol rapid

Health : Health Summary: Absorption is moderate with reaction all routes, based on
p hysical/chemical properties and analogs. There are concerns for severe irritation to the skin and irritation to the eye and lung, based on reactivity and submitted test data. Also, there are concerns for dermal sensitization, effects to the blood, spleen, and liver, reproductive and developmental toxicities, sytemic toxicity, mutagenicity, and oncogenicity, based on analogs and submitted test data. Test Data: Submitted with the PMN:

Positive in Salmonella with activation, negative without activation; Positive in E. coli with activation, negative without activation; Rat acute (8D) oral (gavage) LD0 > 2000 mg/kg; Rat acute (15D) dermal LD50 > 2000 mg/kg; Severe dermal irritant in male rabbits; Slight eye irritant in male rabbits; Extreme dermal sensitizer in female guinea pigs; Rat repeated dose (28D) oral (gavage) N OAEL 15 mg/kg, with blood, spleen, liver, kidney, and other systemic effects, as well as forestomach thickening and hyperplasia. The kidney effects were considered species-specific and thus not relevant to humansSubmitted with

Rat oral repeat dose toxicity with reproductive/developmental screen: reduced pup weights, incresed pup mortality, and reduced numbers of viable pups at 310 mg/kg

Submitted with

Rat 14D oral toxicity study: various organ/systemic effects (blood, kidney, thymus, testes,
p rostate, lymph system) at 250 and 750 mg/kg

Ecotox :

Test Organism Test Type Test End Point Predicted Measured Comments

fish 96-h LC50 0.74 <1.5 supportive due to questionable study design daphnid 48-h LC50 0.80 ~0.751 supportive due to questionable study design green algal 96-h EC50 0.11 ≤ 3.2 supportive due to questionable study design fish

_ chronic value 0.074 daphnid

_ chronic value 0.15

algal

_ chronic value 0.034 ≤ 0.595 supportive due to questionable study design Sewage Sludge 3-h EC50

_ Sewage Sludge

_ Chronic Value _

Ecotox Values Comments: Predictions are based on SARs for peroxy esters; SAR chemical class = p eroxy ester; MW 230; log Kow = 4.65 (EPI), 4 90 (M); liquid with mp unknown (P); S < 0.6 mg/L at 20 C (P); p H7; effective concentrations based on 100% active ingredients and mean measured concentrations; hardness <150.0 mg/L as CaCO3; and TOC <2.0 mg/L

Ecotoxicity Test Data Results for P-14-0072: Propaneperoxoic acid, 2,2-dimethyl-, 1,1,3,3-tetramethylbutyl ester (CASRN: 22288-41-1; Trade Name: Trigonox 425).

Three acute ecotoxicity studies were conducted on the PMN (P-14-0072) in 1999 by NOTOXB.V. with fish, daphnia, and algae. EPA classified the PMN as a peroxy ester and considered the test substance to be discreet but unstable. All submitted aquatic toxicity studies were conducted using what the submitter referred to as a water accommodated fraction (WAF), a method developed for complex mixtures of varying low water solubility that is unnecessary for this substance; however, based on review of the studies, methods employed by the laboratory were actually those for low water soluble substances. Since water solubility is reported as < 0.6 mg/L, this may be justifiable, but results should have been consistently in terms of mean measured concentrations since nominal tested concentrations for some tests greatly exceeded the water solubility limit. The instability of the test substance complicates review of the studies though, especially considering reports that an undefined floating layer was observed following mixing in the absence of a solvent. In addition, tests carried out in the presence of solvent may have artificially enhanced toxicity, but effects were observed (i.e., daphnid) in the absence of solvents.

Conclusion: Given noted concerns with the study designs (see endpoint sections below), the use of nominal concentrations for certain effect determinations, limited reporting of measured concentrations, and concerns with control response in the algae study, data is considered supportive only. The fish 96-hour LC50 is considered to be <1.5 mg/L based on the second limit test. The daphnid EC50 should be close to 0.7-0.9 mg/L considering the 15-35% effects observed around these concentrations. The algae study had control growth issues and reported only initial measured concentrations, but did establish an EC50 and ChV of 3.2 mg/L and 0.595 mg/L, respectively, that suggest effects at or below these levels. The CoCs are as follows:

Acute CoC: 28 p pb Chronic CoC: 3 p pb

N oted instability by the submitter and demonstrated in the studies suggest that any future work should focus on defining the fate of this substance in water (i.e., hydrolysis testing). Considering the instability described in the below studies, chronic hazard based on the PMN (as reported above) is unlikely; long term exposure to acid and alcohol hydrolysis products is more likely

Fish Ecotoxicity Test: A 96-hour acute toxicity study was conducted in carp (Cyprinus carpio) with P-14-0072 (87.7%
p urity). The study consisted of two limit tests, p erformed under static conditions, and a final LC50 test performed under semi-static conditions with 48-hour renewal. This study was reported to follow procedures based on the EEC directive 92/69; Part C, Publication No. L383, December 1992, C.1. “Acute toxicity for fish”, and the OECD guideline No. 203: “Fish Acute Toxicity Test”, Adopted 17 July, 1992.

In the first limit test, single replicates of seven C. carpio were exposed to a blank control or nominal concentration of 100 mg/L. Preparation for the first limit test started with a supersaturated solution prepared at a nominal concentration of 100 mg/L. This turbid emulsion was stirred for 2 days. Then, the emulsion was transferred to a separation funnel and left to stabilize for approximately 23 hours. This resulted in phase separation between water and test substance (floating layer). Subsequently, the water phase was separated from the test substance
p hase and used for testing. The nominal concentration of 100 mg/L was clear and colorless. N o detectable test concentrations were found in any of the samples taken during the first limit test.Water quality parameters were not monitored during the test. A loading of 0.84 g fish/L was calculated. N o mortality was observed during the test.

In the second limit test, single replicates of seven C. carpio were exposed to a solvent (acetone) control or an emulsion of 100 mg/L test substance in acetone. Acetone was chosen as a
p re-solvent in the second limit test since an analytical pre-test showed that detectable levels in water could be obtained when using the more toxic pre-solvent acetonitril. A weighed amount of test substance was mixed with acetone at a 1:1 ratio. The mixture was clear and colorless after shaking. The mixture was quantitatively transferred to a volumetric flask and filled up to 5000 mL with ISO medium. Hence, the final concentration of acetone was 100 mg/L. The acetone control contained the same concentration of acetone. Overnight stirring resulted in a turbid homogenous emulsion. The emulsion was transferred to a separation funnel and left to stabilize for approximately 3 hours. Subsequently, the water phase was separated from the test substance
p hase and used for testing. The test solution appeared to be a turbid homogeneous emulsion. The initial measured concentration of test substance was 18.5 mg/L, as determined via HPLC analysis (LOD = 0.24 mg/L, taking into account a dilution factor of 2). At 96-hours, the actual concentration had decreased below the limit of detection. An average exposure concentration of 1.5 mg/L was approximated. Water quality parameters were not monitored during testing. A loading of 0.77 g fish/L was calculated. All fish were found dead or killed in extremis within the first 24 hours of exposure. Fish were observed snapping at the surface after 2 hours of exposure and fins were already damaged. After approximately 20 hours of exposure, dead and surviving fish showed scattered hemorrhages in mouth area, skin and in fin roots. N o effects were observed in the acetone control. EPA suspects that the peroxy ester moiety prepared as a slightly more soluble emulsion may have resulted in greater reactivity that prompted observed effects in fish.

In the final LC50 test, single replicates of seven C. carpio were exposed to a blank control, a solvent (acetone) control or nominal concentration of 2.2, 4.6, 10, 28 or 46 mg/L in acetone (100 mg/L). Prior to the start of exposure, the test solutions were stirred overnight before stabilization and phase separation. After 48 hours of exposure, the test solutions were renewed. To prevent loss of actual test concentrations, the solutions were stirred for approximately 2 hours before stabilization and separation. In the freshly prepared solutions at test initiation, test substance was detected only at the nominal 46 mg/L (measured concentration of 0.180 mg/L). In the freshly
p repared 48-hour renewal solutions, measured concentrations were 0.729, 1.22, 1.32, 3.69 and 6.62 mg/L, respectively, as determined by HPLC analysis (LOD = 0.08 mg/L, taking into account a dilution factor of 2). Using half of the limit of detection to represent those concentrations in which no test substance was analyzed, the initial mean measured concentrations were determined to be 0.221, 0.230, 0.384, and 1.1 mg/L; however, given that analyzed concentrations at study termination were less than the limit of detection in the second limit test, average exposure concentrations were likely less. Over the course of testing, temperature ranged from 20.1

– 21.1  ̊ C, p H ranged fro m 7.3

– 7.9 and dissolved oxygen ranged from 6.5

– 9.1 mg/L. Dilution water hardness was 250 mg CaCO3/L. A loading of 0.72 g fish/L was calculated. N o mortality or other sub-lethal effects were observed up to the initial mean measured concentration of 1.1 mg/L.

It is clear the substance can result in extreme toxicity if prepared as an emulsion based on the second limit test in fish. In addition, analogous substances suggest similar high toxicity values.The first limit tests suggests that toxicity may be limited by water solubility though. Given the uncertainty combined with results from other similar substances, the 96-hour LC100 of 1.5 reported in the second limit test will be used to estimate hazard. This value could under- or over-estimate hazard depending on the conditions of the test. Given the extreme nature of the effects observed in the second limit test any releases should be via a POTW and should allow for time to hydrolyze/react. If additional information becomes available about the fate of the PMN, hazard levels may be reconsidered.

96-hour LC100 = 1.5 mg/L 96-hour LC50 < 1.5 mg/L

Daphnid Ecotoxicity Test: A 48-hour acute toxicity study was conducted in Daphnia magna with P-14-0072 (87.7% p urity) under static conditions. The study consisted of two limit tests and a final test. This study was reported to follow procedures based on ISO International Standard 6341: “Water quality – Determination of the inhibition of the mobility of Daphnia magna Straus – Acute toxicity test, Third edition, 1996-04-01. In addition, the procedures were designed to meet the following guidelines: EEC Directive 92/69, Part C: Methods for the determination of ecotoxicity, Publication No. L383, December 1992, C.2 “Acute Toxicity for Daphnia” and OECD guidelines for Testing of Chemicals, guideline No. 202 Part I: “Daphnia sp., Acute Immobilization Test”, Adopted April 4, 1984.

In the first limit test, two replicates of ten D. magna were exposed to a blank control or a nominal concentration of 100 mg/L. Analytical monitoring was not conducted. The project was started with a supersaturated solution prepared at nominal 100 mg/L. This solution was stirred for 2 days in the dark. Then, the solution was transferred to a separation funnel, followed by a stabilization period of approximately 22 hours. Lower concentrations were prepared by subsequent dilutions of the 100 mg/L test solution. Water quality parameters were not monitored during exposure. N o immobility of daphnids was observed during the test. Daphnids were observed trapped at the surface in the treated solutions. However, observations did not include a test substance film or foam at the surface. The trapped daphnids were re-immersed in the test medium before recording of mobility. Given the nature of peroxy esters to react with organic material, the trapped daphnid are considered an adverse effect; however, in the absence of analytical monitoring, the effect level cannot be determined.

In the second limit test, two replicates ten D. magna were exposed to a solvent (acetone) control or a nominal concentration of 100 mg/L in acetone. Acetone was chosen as a pre-solvent in the second limit test since an analytical pre-test showed that detectable levels in water could be obtained when using the more toxic pre-solvent acetonitril. A weighed amount of test substance was mixed with acetone at a 1:1 ratio. This mixture was clear and colorless after shaking. Test solutions were prepared individually. Preparations included overnight stirring followed by phase separation for 3.5 hours. The final concentration of acetone in the solutions was 100 mg/L. All final test solutions prepared with acetone as a pre-solvent were clear and colorless. For all tests, dilution water hardness was 250 mg CaCO3/L and daphnid loading was 125 daphnids/L. The initial measured concentration was 18.5 mg/L, as determined via HPLC analysis (LOD = 0.086 mg/L). During the exposure period, the measured concentration decreased below the limit ofdetection. An average exposure concentration of 0.891 mg/L was approximated. Water quality
p arameters were not monitored during the study. At 48-hours, the % immobilization in the solvent control and test group was 0% and 35%, respectively.

In the final EC50 test, two replicates of ten D. magna were exposed to a blank control, a solvent (acetone) control or nominal concentrations of 18, 32, 56, 100 and 180 mg/L in acetone using a static test system. Corresponding initial measured concentrations of test substance were 5.5, 7.9, 13.1, 11.9 and 12.8 mg/L, respectively, as determined via HPLC analysis. Given the analytic results in the second limit test at study termination, recoveries were likely low at study termination due to instability of the test substance. Over the course of testing, temperature ranged from 20.5

– 20.7oC, p H ranged fro m 7.7

– 8.0 and oxygen concentrations ranged from 8.7

–

9.3 mg/L. At initial measured concentrations of 0 (blank control), 0 (solvent control), 5.5, 7.9, 13.1, 11.9, and 12.8 mg/L, the % immobilization values were 0%, 0%, 0%, 0%, 0%, 25% and 15%, respectively. The 48-hour EC50 exceeded the initial measured concentration of 13.1 mg/L and the likely average measured concentration of 0.751 mg/L.

Considering the instability of the test substance, flow-through testing is generally recommended to maintain test concentrations. The observation of a film supports the need for some type of test concentration renewal to maintain exposure concentrations. Based on the study results, 15%, 25%, and 35% immobilization was observed between approximate average concentrations of 0.7 and 0.9 mg/L. Given the many concerns with the study design (i.e., no analytic monitoring of test concentrations at study termination, use of a static test system for an unstable substance, solvent use) and the reporting of effects close to the predicted effect level for daphnid, the predicted effect level of 0.8 mg/L will be maintained for risk determination.

Algal Ecotoxicity Test: A 72-hour toxicity test in green algae (Selenastrum capricornutum) was conducted with P-14-0072 (87.7% p urity) under static conditions. This study was reported to follow procedures based on EEC Directive 92/69, Publication No. L383 Part C-3 adopted December, 1992; OECD guideline No. 201, Adopted June 7, 1984; and ISO Standard 8692, First edition, 15 N ovembe r 1989. Following a range-finding test, three replicates of S. capricornutum (1 x 104 cells/mL) were exposed to nominal concentrations of 10, 18, 32, 56, 100 and 180 mg/L in acetone. Corresponding initial measured concentrations were 0.274, 1.29, N D, 16.5 and 11.5 mg/L. Six replicates of S. capricornutum were exposed to a blank control or a solvent (acetone) control. The algae were illuminated at a light intensity of 3500

– 4000 lux, with constant shaking. Test solutions were prepared individually using acetone as a pre-solvent. The preparation procedure further included overnight stirring followed by phase separation for 3.5 hours. The final concentration of acetone in the test solutions was 100 mg/L. After preparation, volumes of 50 mL were added to each replicate of the respective concentration. Subsequently, a volume of 0.15 mL of an algal suspension was added to each replicate. Over the course of testing, temperature of the test medium ranged from 22.0

– 23.5  ̊ C and pH ranged from 6.1

– 8.5. Average cell density in solvent control cultures increased by a factor of 30.55 within 72 hours; however, due to little growth during the first 24 hours of the test, the % coefficient of variation for section-by-section growth greatly exceeds the OECD maximum acceptable level of 35%. In addition, results of the blank control were not provided for the definitive test. The EC50 was approximated to be 3.2 mg/L, but exact determination was complicated by no analytically determine concentration thatcorresponded to the nominal 56 mg/L group. The NOEC and LOEC based on area under the curve analysis was 0.274 mg/L and 1.29 mg/L, respectively, resulting in a ChV of 0.595 mg/L. Results based on initial measured concentration likely under represent toxicity. In addition, concerns with control growth were noted for this test system.

72-hour EC50 = 3.2 mg/L (initial measured, approximate) 72-hour NOEC = 0.274 mg/L (initial measured, area under curve) 72-hour LOEC = 1.29 mg/L (initial measured, area under curve) 72-hour ChV = 0.595 mg/L(initial measured, area under curve)

Conclusion: Given noted concerns with the study designs (see endpoint sections below), the use of nominal concentrations for certain effect determinations, limited reporting of measured concentrations, and concerns with control response in the algae study, data is considered supportive only. The fish 96-hour LC50 is considered to be <1.5 mg/L based on the second limit test. The daphnid EC50 should be close to 0.7-0.9 mg/L considering the 15-35% effects observed around these concentrations. The algae study had control growth issues and reported only initial measured concentrations, but did establish an EC50 and ChV of 3.2 mg/L and 0.595 mg/L, respectively, that suggest effects at or below these levels. The CoCs are as follows:

Acute CoC: 28 p pb Chronic CoC: 3 p pb

N oted instability by the submitter and demonstrated in the studies suggest that any future work should focus on defining the fate of this substance in water (i.e., hydrolysis testing). Considering the instability described in the below studies, chronic hazard based on the PMN (as reported above) is unlikely; long term exposure to acid and alcohol hydrolysis products is more likely

Reviewer: K.Moran

Factors Values Comments Assessment Factor 10 Concentration of Concern ( p pb) 3

SARs p eroxy esters SAR Class p eroxy ester Ecotox Category Peroxides

Ecotox Factors Comments:

SAT Chair: J. Kwiat, 564-7653