SRC TR

- 1 4

- 055

HPV 2 TEST RULE DATA ADEQUACY REVIEW : NOVEMBER 2014 ( FINAL )

Castor oil, sulfated, sodium salt, CASRN 68187

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- 8

Prepared by:

Amy Nguyen, Megan Nelson, Justine Von Runnen, Ashlee Aldridge, William L. Richards, and Teresa Manyin

Defense and Environmental Solutions Division

SRC, Inc. 7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP

- W

- 09

- 027 Task FG007. 4 .009.001

Submitted to:

OCSPP/OPPT/RAD (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue Washington, D.C. 20460

Bryan Lobar , Project Officer David Brooks , Work Assignment Manager Amy Benson , Alternate Work Assignment Manager

November 2 5

, 201 4Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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i TABLE OF CONTENTS

A. SOURCE MATERIAL USED FOR ASSESSMENT

................................ .............................. 1

B. EVALUATING DATA ADEQUACY

................................ ................................ ..................... 4
C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

........ 4

D. DATA FOR SIDS ENDPOINTS

................................ ................................ .............................. 5

D

- 1. Acute Toxicity t o Fish

................................ ................................ ................................ ....... 5

D

- 2. Acute Toxicity to Aquatic Invertebrates

................................ ................................ .......... 11

D

- 3. Toxicity to Aquatic Plants

................................ ................................ ............................... 16

D

- 4. Acute Toxicity

................................ ................................ ................................ .................. 23

D

- 5. Repeated

- Dose, Reproductive, and Developmental Toxicity

................................ .......... 26

D

- 6. Genetic Toxicity

– Gene Mutation

................................ ................................ ................... 37

D

- 7. Genetic Toxicity

– Chromosomal Aberrations

................................ ................................ 43
E. DATA ADEQUACY SUMMARY TABLE

................................ ................................ ........... 51Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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1 A. SOURCE MATERIAL USED FOR ASSESSMENT

This d ocument evaluates toxicological data for castor oil, sulfated, sodium salt (CASRN 68187

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- 8) that were submitted by the Sulfated Castor Oil Consortium (SCO), which is affiliated with the Society of Chemical Manufacturers & Affiliates (SOCMA)

, to EPA in response to the final test rule, “Testing of Certain High Production Volume Chemicals; Second Group of Chemicals” (76 FR 1067, January 7, 2011; referred to herein as HPV2; available online at http://www. regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0579 as of November 25

, 2014 ).

The final HPV2 test rule required the following toxicological tests for CASRN 68187

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- 8 : C1 ( aquatic toxicity, consisting of Test Group 1 [ acute toxicity to fish, acute toxicity to Daphnia

, and toxicity to algae tests ] if log K ow < 4.2 or Test Group 2 [ chronic toxicity to Daphnia and toxicity to algae tests ] if log K ow ≥ 4.2), D (acute mammalian toxicity test), E1 (bacterial reverse mutation test), E2 (chromosomal aberration s or micronucleus test), and F1 (combined repeated

dose toxicity study with the reproduction/developmental toxicity screening test)

.

Under the HPV2 test rule, a determination of whether to per form aquatic toxicity testing under Test Group 1 or Test Group 2 is ordinarily made by a consideration of the log K ow for the test substance , using a threshold log K ow value of 4.2 as indicated above . However, as stated in the test rule (p. 1076) , “EPA ma y allow an alternative threshold or method to be used for determining whether acute or chronic aquatic toxicity testing must be performed for a specific substance ” if the sponsor requests a modification of the test rule and provides an acceptable supportin g rationale . In a previously submitted test plan (dated October 18, 2011) 1 , SCO proposed that the estimated bioconcentration factor (BCF) for CASRN 68187

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- 8 be used in lieu of a log K ow . In its October 18, 2011 submission, SCO provided an estimated BCF (obtained from the EPI Suite modeling program 2 ) of 71, which indicates relatively low bioaccumulation in aquatic organisms. Based on the relatively low estimated BCF value, EPA determined 3 that SCO’s recommendation to conduct Test Group 1 toxicity te sting was acceptable.

In its most recent submission, SCO submitted the following seven study reports for toxicological
tests in response to the testing requirements in the final HPV2 test rule:

Bouwman L.M. 2014a. 96

- Hour acute toxicity study in fath ead minnow with castor oil, sulfated, sodium salt, 75% (static). WIL Research Europe B.V., The Netherlands.

1 Letter addressed from John DiLoreto of SCO to the Office of Pollution Prevention and Toxics (OPPT) of EPA, dated October 18, 2011

, “Re: Testing of Certain High Production Volume Chemicals; Second Group of Chemicals, Federal Register 76 (5)

, 1067

- 1093, January 7, 2011; Castor Oil, sulfated, sodium Salt, CAS No. 68187

- 76

- 8”. Available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0642 as of November 25 , 2014.

2 U.S. EPA. 2012. Estimation Programs Interface SuiteTM (EPI Suite) for Microsoft® Windows, v4.11. U.S

. Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/ exposure/pubs/episuitedl.htm as of November 25 , 2014.
3 HPV2 Toxicity Test Pl an Review: Castor oil, sulfated, sodium salt, CASRN 68187

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- 8. SRC Technical Report #11

- 082, dated October 26 , 201 1

.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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- 8

2 Project No. 503337. Sponsor: Sulfated Castor Oil Consortium, Society of Chemical Manufacturers & Affiliates.

Bouwman, L.M. 2014b. Acute toxici ty study in Daphnia magna with castor oil, sulfated, sodium salt, 75% (static). WIL Research Europe B.V., The Netherlands. Project No. 503336. Sponsor: Sulfated Castor Oil Consortium, Society of Chemical Manufacturers & Affiliates.

Bouwman, L.M. 2014c. Fresh water algal growth inhibition test with castor oil, sulfated, sodium salt, 75% . WIL Research Europe B.V., The Netherlands. Project No. 503338. Sponsor: Sulfated Castor Oil Consortium, Society of Chemical Manufacturers & Aff iliates.

van Huygevoort, A.H.B.M. 2013. Assessment of acute oral toxicity with castor oil, sulfated, sodium salt, 75% in the rat (up and down procedure). WIL Research Europe B.V., The Netherlands. Project No. 503339. Sponsor: Sulfated Castor Oil Cons ortium, Society of Chemical Manufacturers & Affiliates.

d e Raaf

- Beekhuijzen, M.E.W. 2014. Combined 28

- day repeated dose toxicity study with the reproduction/developmental toxicity screening test of castor oil, sulfated, sodium salt, 75% i n rats b y oral gavage. WIL Research Europe B.V.

, The Netherlands. Project No. 503342. Sponsor: Sulfated Castor Oil Consortium, Society of Chemical Manufacturers & Affiliates.

Verspeek

- Rip, C.M. 2014. Evaluation of the mutagenic activity of castor oil, sulfated, sod ium salt, 75% in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat). WIL Research Europe B.V., The Netherlands. Project No. 503340. Sponsor: Sulfated Castor Oil Consortium, Society of Chemical Manufacturers & Affiliates.

Buskens, C.A.F. 2014. Evaluation of the ability of castor oil, sulfated, sodium salt, 75% to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiment). WIL Research Europe B .V., The Netherlands. Project No. 503341. Sponsor: Sulfated Castor Oil Consortium, Society of Chemical Manufacturers & Affiliates.

These newly submitted data are evaluated for adequacy herein. No other toxicity data are evaluated in this report.

The HPV2 test rule classifies CASRN 68187

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- 8 as a Class 2 chemical substance, for which testing may be conducted on “ any representative form of the chemical substance ”

. The initial test plan submitted by SCO to EPA ( submission dated October 18, 2011 ) 1 proposed the use of a

1 Letter addressed from John DiLoreto of SCO to the Office of Pollution Prevention and Toxics (OPPT) of EPA, dated October 18, 2011

, “R e: Testing of Certain High Production Volume Chemicals; Second Group of Chemicals, Federal Register 76 (5)

, 1067

- 1093, January 7, 2011; Castor Oil, sulfated, sodium Salt, CAS No. 68187

- 76

- 8”.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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- 8

3 commercial product consisting of an aqueous solution of the test rule substance ( 85% CASRN 68187

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- 8 in water) for toxicity testing since a neat dry form of the material was not commercially available and could be prepared only with difficulty . In its review of the test plan 1

, EPA specified that SCO was permitted to use a commercial product of no less than 85% CASRN 68187

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- 8 and 15% water. EPA also requested that SCO provide further information regarding the formulation of the co mmercial product proposed for toxicity testing, including the identification of all added ingredients.

In a revised test plan (submission dated April 6, 2012) 2

, SCO propos ed to perform toxicity testing on a 75% aqueous solution of CASRN 68187

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- 8 since a higher purity material was not commercially available

. In response, EPA continued to request further information regarding the formulation of the commercial product, including the identity of any added ingredients, such as surfactants, solvents, and st abilizers. 3 EPA deferred a decision regarding the acceptability of the proposed test substance for toxicity testing until such further information was provided by the
sponsor.

SCO submitted a second revised test plan (submission dated February 11, 2013) 4 , which contained compositional data for samples of commercial products from three different manufacturers

. Data provided by SCO included percentages of water, Na 2 SO 4 , SO 3 , glycerides (including diglycerides and monoglycerides) , and fatty acids in commercial products. SCO noted that the products proposed for HPV testing are stable dispersions containing no surfactants, solvents

, or stabilizers. In EPA’s review of the second revised test plan 5 , EPA approved the use of the proposed commercial produc t s for conduct ing the toxicity testing required by the HPV2 test rule

. The substance tested in the seven toxicity studies submitted by SCO and reviewed herein was a commercial product that contained 74.4% active ingredient ( CASRN 68187

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- 8). The perce nt active ingredient (a.i.) was calculated by subtracting % water and % sodium sulfate from 100% (i.e., 100%

- [24.3% water + 1.25% Na 2 SO 4 ] ≈ 74.4% castor oil, sulfated, sodium salt ). 6

Available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0642 as of November 25 , 2014.

1 HPV2 Toxicity Test Plan Review: Castor oil, sulfated, sodium salt, CASRN 68187

- 76

- 8. SRC Technical Repor t #11

- 082, dated October 26, 2011.

2 Letter addressed from John DiLoreto of SCO to the Office of Pollution Prevention and Toxics (OPPT) of EPA, dated April 6, 2012, “Re: Revised Test Plan; Testing of Certain High Production Volume Chemicals; Second Group of Chemicals, Federal Register 76 (5) , 1067

- 1093, January 7, 2011; Castor Oil, sulfated, sodium salt, CAS No. 68187

- 76

- 8”.

3 HPV2 Review of Revised (April 2012) Test Plan: Castor oil, sulfated, sodium salt, CASRN 68187

- 76

- 8. SRC Technical Report #12

- 044, dated June 13, 2012.

4 Letter addr essed from John DiLoreto of SCO to the Office of Pollution Prevention and Toxics (OPPT) of EPA, dated February 11, 2013. “Re: Second Revised Test Plan; Testing of Certain High Production Volume Chemicals; Second Group of Chemicals, Federal Register 76 (5) , 1067

- 1093, January 7, 2011; Castor Oil, sulfated, sodium salt, CAS No. 68187

- 76

- 8”. Available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OP PT

2007

- 0531

- 0707 as of November 25 , 2014.
5 HPV2 Review of Second Revised (February 2013) Test Plan: Castor oil, sulfated, sodium salt, CASRN 68187

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- 8. SRC Technical Report #13

- 011, dated March 22, 2013.

6 Although the batch of commercial product tha t was tested in the seven submitted toxicity studies contained 74.4% a.i., the test concentrations and doses were prepared using information from the sponsor that indicated a purity of 70.7% a.i., which was later determined to be inaccurate. The correct p urity was 74.4% a.i.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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4 B. EVALUATING DATA ADEQUACY

The submitted toxicological data are evaluated for adequacy in Section D. Each study was summarized according to a standard format and assigned a Klimisch reliability code based on its scientific merit and conduct, using professional judgment. To determine whether data were adequate to satisfy SIDS endpoints, study details were compared to current standard test guidelines to establish whether the study met current testing requirements. If a study was determined to be inadequate, a basis for that conclusion is supplied. A table summariz ing data availability/adequacy for each SIDS endpoint is included on the last page of this document (Section E ).

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

Data submitted by SCO are adequate to satisfy the acute toxicity to fish (comp onent of C1)

, acute toxicity to Daphnia (component of C1) , toxicity to algae (component of C1) , acute mammalian toxicity (D) , repeated

- dose toxicity (component of F1) , gene mutation (E1)

, chromosomal aberrations (E2) , reproductive toxicity (component of F1 ) , and developmental toxicity (component of F1) testing requirements for CASRN 68187

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- 8 promulgated in the final HPV2 test rule

. There are no outstanding HPV2 test rule requirements for toxicity testing of CASRN 68187

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- 8.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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5 D. DATA FOR SIDS ENDPOINTS

D

- 1. Acute Toxicity to Fish

Summary of endpoint: Adequate

Study 1: Acute toxicity study in Pimephales promelas (Bouwman, 2014a)

Title: 96

- Hour acute toxicity study in fathead minnow with castor oil, sulfated, sodium salt, 75% (static)

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Castor oil, sulfated, sodium salt, CAS RN 68187

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Remarks: Composition: 74.4% castor oil, sulfated, sodium salt (CASRN 68187

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- 8) 1 , 24.3% water, and 1.25% sodium sulfate; Clear, amber liquid ; Batch No. R

- 10135

- 064; referred to as “castor oil, sulfated, sodium salt, 75%” in the study repor t.

METHOD

Method/guideline followed: OPPTS 850.1075 (1996); OECD TG 203 (1992); Method C.1 of Commission Regulation (EC) No. 440/2008; ISO International Standard 7346

- 1 (1996)

Test type: Acute Toxicity to Fish

– Static

GLP compliant?: Yes

Year study performed: 2013

Test species: Fathead minnow ( Pimephales promelas )

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: Combined Limit/Range

- Finding Test: Nominal concentrations: 0 (control), 1.4, 14, and 141 m g test substance/L (equivalent to ~ 0, 1.0, 10 and 105 mg active ingredient (a.i.)/L) 2

1 The percentage of CASRN 68187

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- 8 in the test substance was calculated by subtracting % water and % sodium sulfate from 100%. (100%

- [24.3% + 1.25%] ≈ 74.4%)

2 Concentrations of active ingredient (CASRN 68187

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- 8) were calculated using a correction factor of 1.34, which was based on a test substance purity of 74.4%. (Correction factor = 1 ÷ 0.744 ≈ 1.34) The concentration of active ingredient is equal to the concentration of test substance divided by 1.34.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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6 Definitive Test: Nominal concentrations: 0 (control), 6.5, 14, 31, 65, and 141 mg test substance/L (equivalent to ~ 0, 4.9, 10, 23, 49, and 105 mg a.i./L); Average measu red concentrations: 0, 2.6, 5.1, 20, 45, and 119 mg test substance/L (equivalent to ~ 0, 1.9, 3.8, 15, 34, and 89 mg a.i./L)

Test method/conditions remarks: General Test Conditions : Test Organisms and Acclimation: Fathead minnows ( Pimephales promelas ) were obtained from an in

- house culture. Prior to the start of the test, fish were held in a tank containing medium with water quality parameters that were similar to those used during the toxicity test. Differences of pH and temperature between holding a nd test tank media were less than 0.5 unit and 0.5 °C, respectively. During holding until one day before the start of the test, the fish were fed with a pelleted fish diet. Mortality during acclimation was less than 5%. Fish were not fed during the test period.

For the combined limit/range

- finding test, the mean body length of the fish was 2.7 ± 0.3 cm and the mean wet body weight was 0.60 ± 0.22 g . In the definitive test, the mean body length of the fish was 2.9 ± 0.3 cm and the mean wet body weight w as 0.66 ± 0.22 g.

Test Medium: The test medium was adjusted ISO medium formulated using RO

- water (tap

- water purified by reverse osmosis). The prepared test medium had a hardness of 180 mg CaCO 3 /L and pH of 7.7 ± 0.3.

Preparation of Test Item Concentrations:

“ The standard test procedures required generation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. 1 The testing of concentrations that disturb the test system should be prevented (e.g. film of the test substance on the water surface). A correction was made for the purity/composition of the test substance. A correction factor of 1.41 was used [when preparing test solutions] . This factor was based on the information known during testing. Afterwards, the information on purity/composition of the test substance was changed [such that a correction factor of 1.34 was used to determine concentrations of active ingredient] 2 ... As a consequence slightly hig her concentrations than necessary were tested.” [Bouwman 2014a, p. 9]

“ Preparation of test solutions started with a nominal concentration of 141 mg [test substance]/l containing 105 mg of the active ingredient (a.i.) per litre. This concentration was mag netically stirred for 15 minutes... The lower test concentrations were prepared by subsequent dilutions of the 141 [mg test substance/L] concentration in test medium. The final test solutions were clear and colourless up to nominal concentrations of 31 mg test substance per litre. Higher

1 Due to the low solub ility of CASRN 68187

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- 8 in water (estimated by EPISuite TM to be 4.3 x 10

- 14 mg/L) and its surfactant nature, the prepared test solutions ( with average measured concentrations up to 89 mg a.i. /L ) are deemed to be dispersions.

2 During testing, a correctio n factor of 1.41 was used based on an incorrect purity of ~ 71% . After toxicity testing was complete, concentrations of active ingredient were recalculated using a correction factor of 1.34 that was based on the correct purity of 74.4%.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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7 concentrations became increasingly hazy and formed homogeneous dispersions. ” [ Bouwman 2014a, p. 9]

Combined Limit/Range

- Finding Test: Prior to conducting the definitive test, a combined limit/range

- finding test was perfo rmed. Fourteen fish (two replicates of seven fish per replicate) were exposed to a nominal concentration of 141 mg test substance/L. Three fish (one replicate) per concentration were exposed to nominal concentrations of 1.4 and 14 mg test substance/L. Seven fi sh (one replicate) were used as a control.

During the combined limit/range

- finding test, dissolved oxygen concentrations, pH, and temperature were only measured in the control and the highest test concentration with surviving fish. Aeration was begun a fter 48 hours of exposure because the dissolved oxygen concentration had dropped below 5 mg/L (i.e., dissolved oxygen was 4.75 mg/L at 48 hours); thereafter the dissolved oxygen concentration was > 5 mg/L for the remainder of the test. This deviation from protocol did not result in any mortalities or other adverse effects. All other test procedures and conditions were similar to those applied in the definitive test.

Analytical monitoring of test substance concentrations (by TOC measurement) was conducted for the nominal concentrations of 14 and 141 mg test substance/L. Samples were taken at 0, 48, and 96 hours; at each time point, one sample was taken for the nominal concentration of 14 mg test substance/L and two samples (one from each replicate) were t aken for the nominal concentration of 141 mg test substance/L. Details of sample handling and analytical monitoring were not provided but are assumed, based on the study author’s statement that the combined limit/range

- finding test procedures and conditio ns were “similar to those applied in the final test” [Bouwman 2014a, p. 10], to be comparable to those described below in the Analytical Monitoring section for the definitive test.

Definitive Test: The exposure vessels were 9.8

- L glass vessels contain ing 9 L of test medium. Within 35 minutes of test media preparation, 14 fish per concentration (two replicates of seven fish per replicate) were placed in test substance

- containing vessels at nominal concentrations of 6.5, 14, 31, 65, and 141 mg test subs tance/L. The control consisted of one replicate of seven fish in test medium without test substance. Fish loading was 0.51 g fish/L. Illumination was provided as a daily 16

- hour photoperiod. The test vessels were aerated to keep the dissolved oxygen co ncentration above 5 mg/L.

The fish were observed for mortality and other toxic effects at 5, 24, 48, 72, and 96 hours. “In addition, [test vessels were checked] every afternoon from day 0 and every morning from day 1 to observe for any dead or severely distressed fish. Dead fish were removed when observed.” [ Bouwman 2014a, p. 11] Beginning on day 0, temperature, pH, and dissolved oxygen were monitored daily in all test vessels with surviving fish.

Analytical Monitoring: During the definitive test, sa mples (40 mL) for analysis of test substance concentrations were taken at 0, 48, and 96 hours from the control vessels and from all test vessels with surviving fish.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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8 At 24 hours, samples were also taken from vessels with nominal concentrations of 31 and 6 5 mg test substance/L. Samples were stored in a refrigerator until analysis. Concentrations of test substance were determined by total organic carbon (TOC) analysis. TOC measurements were corrected for the measured amount of TOC in the control (when TOC measurements were available for the control). In order to calculate test substance concentrations, TOC measurements were adjusted for the TOC content of the test substance, which was experimentally determined to be 54.48% in a separate study (WIL Researc h Project 503334)

. 1 For nominal concentra tions of 6.5 and 14 mg test substance/L, average measured concentrations were calculated as the arithmetic mean of the geometric means of the concentrations at 0 and 48 hours and the concentrations at 48 and 96 hou rs. 2 For nominal concentrations of 31 and 65 mg test substance/L (for which all fish had died by 24 hours), average measured concentrations were calculated as the geometric mean of the concentrations at 0 and 24 hours. 3 For the nominal concentration of 1 41 mg test substance/L (for which all fish had died by 5 hours), only the measured concentration at 0 hours was used.

Statistical Analyses: “The LC 50 - values were calculated with the Weibull analysis from the probits of the percentages of affected fish and the logarithms of the corresponding average test substance concentrations using linear maximum likelihood regression.” [Bouwman 2014a, p. 12]

RESULTS

Endpoint value(s): Based on average measured concentrations: 96

- hour LC 50 = 7.3 mg test substance /L (

~ 5.4 mg a.i./L) 96

- hour LOAEC = 5.1 mg test substance /L (

~ 3. 8 mg a.i./L) 96

- hour NOAEC = 2.6 mg test substance/L (~1.9 mg a.i./L)

Results remarks: Combined L im it/Range

- Finding Test :

“ Within 4 hours of exposure all fish exposed to the highest test concentration [nominal concentration of 141 mg test substance/L] died. No mortality or clinical effects were observed at any of the lower test concentrations [nominal concentrations of 1.4 and 14 mg test substance/L] and the control during the test period . From 48 hours onwards the nominal 14 mg [test substance]/l concentration was observed to be slightly hazy.” [Bouwman 2014a, pp. 12

- 13] Based on the results of the combined limit/range

- finding test, the LC 50 was expected to be between the nominal test s ubstance concentrations of 14 and 141 mg test substance/L.

“Temperature and pH were maintained within the limits prescribed by the protocol [pH: 6.0

- 8.5, constant within 1 unit; temperature 20

- 24°C, constant within 2°C]. The oxygen concentration in the c ontrol slightly decreased below the optimum range after 48 hours of exposure [i.e.,

1 For sampling time s of 0, 48, and 96 hours, test substance concentration = [(TOC in treated flask)

– (TOC in control flask)]/0.5448. For the sampling time of 24 hours, test substance concentration = TOC in treated flask/0.5448 since the control was not sampled for analysis at 24 hours. 2 Average measured concentration = [√(C t=0 x C t=48 ) + √(C t=48 x C t=96 )]/2, where C t=x is the measured concentration at time = x hours.

3 Average measured concentration = √(C t=0 x C t=24 ), where C t=x is the measured concentration at time = x hours.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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9 dissolved oxygen was 4.75 mg/L at 48 hours]. Therefore, aeration was introduced and maintained until the end of the test.” [Bouwman 2014a, p. 13] No further details regar ding temperature, pH, and dissolved oxygen measurements during the combined limit/range

- finding test were provided in the study report.

“Samples taken from nominal [concentrations of] 14 and 141 mg test substance per litre were analysed for TOC. The conc entration of test substance in the aquatic samples was calculated using the TOC content of pure test substance... The initial concentrations were 13 and 118/119 1 mg [test substance]/l, respectively, i.e. 93 and 84% of nominal. Both concentrations remained stable during the first 24 hours of exposure (90

- 100% of initial). The highest test concentration remained also stable during the remainder of the test (85/87% 2 of initial) while [the] nominal [concentration of] 14 mg [test substance]/l slightly decreased to 73% of initial at the end of the test.” [Bouwman 2014a, p. 13]

Definitive Test: After 96 hours of exposure, the percent mortality values were 0%, 0%, 29%, 100%, 100%, and 100% at nominal concentrations of 0, 6.5, 14, 31, 65, and 141 mg test substance/ L, respectively. “Within 5 hours of exposure all fish exposed to the highest test concentration [nominal concentration of 141 mg test substance/L] died. Within 24 hours all fish exposed to nominal concentrations of 31 and 65 mg [test substance]/l died an d [within 48 hours] 29% of the fish exposed to a nominal concentration of 14 mg [test substance]/l died. No mortality or clinical effects were observed at the lowest [nominal] test concentration [6.5 mg test substance/L] and the control during the test pe riod. After 24 hours the nominal 31 mg [test substance]/l concentration was observed to be hazy. After 48 hours the nominal 14 mg [test substance]/l concentration was observed to be slightly hazy.” [Bouwman 2014a, p. 14]

During the definitive test, the pH ranged from 7.3 to 8.1, the temperature ranged from 20.0 to 21.2°C, and the dissolved oxygen concentrations ranged from 5.8 to 10.0 mg/L. “Aeration was introduced after 2 hours of exposure, in order to keep the oxygen concentration above 5 mg/l.” [Bouw man 2014a, p. 15] “All test conditions remained within the ranges prescribed by the protocol (pH: 6.0

- 8.5, constant within 1 unit; temperature 20

- 24°C, constant within 2°C; oxygen > 60% of air saturation).” [Bouwman 2014a, p. 15]

Based on TOC measurement s: “The mean initial [measured] concentrations in samples taken from nominal [concentrations of] 6.5, 14, 31, 65 and 141 mg [test substance]/l were calculated to be 3.1, 8.8, 25, 54 and 119 mg [test substance]/l, respectively, i.e. 45

- 85% of nominal. Most concentrations decreased during the test period. Effect parameters were based on the average [measured] exposure concentrations for the four lowest test concentrations and on the initial [measured] concentration for the highest test concentration: 2.6, 5 .1, 20, 45 and 119 mg test substance per litre, containing 1.9, 3.8, 15, 34 and 89 mg a.i. per litre.” [Bouwman 2014a, p. 13]

1 Measurements of 118 and 119 mg test substance/L were provided for the two replicates at the nominal concentration of 141 mg test substance/L at 0 hours.

2 Values of 85% and 87% of initial concentrations were provided for the two replicates at the nominal concentration of 141 mg test substance/L at 96 hours.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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10 CONCLUSIONS

Based on average measured concentrations, the 96

- hour LC 50 in Pimephales promelas was 7.3 mg test substance/L (95% CI: 5.2

- 12 mg test substance/L), containing ~ 5.4 mg a.i./L (95% CI: 3.9

- 9.0 mg a.i./L). Since no fish died after 48 hours, the 48

- hour LC 50 was identical to the 96

- hour LC 50

.

STUDY RELIABILITY

[2] Reliable with restric tions. Concentrations of active ingredient were determined by using a

non

- specific analytical method (TOC measurement), rather than a method specific for CASRN 68187

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- 8. Although the report notes test concentrations for which “clinical effects” were no t seen, data for abnormal responses of the fish were not provided for all test concentrations as required by OECD TG 203.

REFERENCE

Bouwman L.M. 2014a. 96

- Hour acute toxicity study in fathead minnow with castor oil, sulfated, sodium salt, 75% (static ). WIL Research Europe B.V., The Netherlands. Project No. 503337. Sponsor: Sulfated Castor Oil Consortium, Society of Chemical Manufacturers & Affiliates.

Text in quotes is taken directly from Bouwman (2014a).Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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11 D

- 2. Acute Toxicity to Aquatic Invertebrates

Summary of endpoint: Adequate

Study 1: Acute toxicity study in Daphnia magna (Bouwman, 2014b)

Title: Acute toxicity study in Daphnia magna with castor oil, sulfated, sodium salt, 75% (static)

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Castor oil, sulfated, sodium salt, CASRN 68187

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- 8

Remarks: Composition: 74.4% castor oil, sulfated, sodium salt (CASRN 68187

- 76

- 8) 1 , 24.3% water, and 1.25% sodium sulfate; Clear, amber liquid ; Batch No. R

- 10135

- 064; referred to as “castor oil, sulfated, sodium salt, 75%” in the study report.

METHOD

Method/guide line followed: OPPTS 850.1010 (1996); OECD TG 202 (2004); Method C.2 of Commission Regulation (EC) No. 440/2008; ISO International Standard 6341 (1996)

Test type: Acute toxicity to Daphnia

– Static

GLP compliant?: Yes

Year study performed: 2013

Te st species: Water fleas ( Daphnia magna )

Analytical monitoring: Yes

Exposure duration: 48 hours

Concentrations tested: Combined Limit/Range

- Finding Test: Nominal concentrations: 0 (control), 0.14, 1.4, 14, and 141 mg test substance/L (equivalent to ~ 0, 0.10, 1.0, 10, and 105 mg active ingredient (a.i.)/L) 2 Definitive Test: Nominal concentrations: 0 (control), 1.4, 2.8, 5.6, 11, 23, 45, and 90 mg test substance/L ( equivalent to ~ 0, 1.0, 2.1, 4.2, 8.2, 17, 34, and 67 mg a.i./L); Average measured

1 The percentage of CASRN 68187

- 76

- 8 in the test substance was calculated by subtracting % water and % sodium sulfate from 100%. (100%

- [24.3% + 1.25%] ≈ 74.4%)

2 Concentrations of active ingredient (CASRN 68187

- 76

- 8) were calculated using a correction factor of 1.34, which

was based on a test substance purity of 74.4%. (Correction factor = 1 ÷ 0.744 ≈ 1.34) The concentration of active ingredient is equal to the concentration of test substance divided by 1.34.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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12 conc entrations: 2.3, 5.3, 13, and 33 mg test substance/L (equivalent to ~ 1.7, 4.0, 9.7, and 25 mg a.i./L) for nominal concentrations of 5.6, 11, 23, and 45 mg test substance/L, respectively

Test method/conditions remarks: General Test Conditions: Test Organ isms: Daphnia magna were obtained from an in

- house laboratory culture by acyclical parthenogenesis. Daphnids were less than 24 hours old at the start of the study and were selected from the 2 nd to 5 th brood of parental daphnids that were more than two weeks old. The Daphnia were derived from a healthy stock, showing no signs of stress, presence of males, ephippia, or discolored animals.

Test Medium: The test medium was adjusted ISO medium formulated using RO

- water (tap

- water purified by reverse osmosis).

Preparation of Test Item Concentrations: “The standard test procedures required generation of test solutions, which should contain completely disso lved test substance concentrations or stable and homogeneous mixtures or dispersions. 1 The testing of concentrations that disturb the test system should be prevented (e.g. film of the test substance on the water surface). A correction was made for the
pu rity/composition of the test substance. A correction factor of 1.41 was used [when preparing test solutions]. This factor was based on the information known during testing. Afterwards, the information on purity/composition of the test substance was chan ged [such that a correction factor of 1.34 was used to determine concentrations of active ingredient] 2 ... As a consequence slightly higher concentrations than necessary were tested.” [Bouwman 2014b, p. 10]

“Preparation of test solutions started with a nom inal concentration of 141 mg [test substance]/l containing 105 mg of the active ingredient (a.i.) per litre. This concentration was magnetically stirred for 15 minutes... The lower test concentrations were prepared by subsequent dilutions of the 141 mg [te st substance]/l concentration in test medium. The final test solutions were clear and colourless up to nominal concentrations of 45 mg test substance per litre. Higher concentrations became increasingly hazy and formed homogeneous dispersions.” [Bouwman 2014b, p. 10]

Combined Limit/Range

- Finding Test: Prior to conducting the definitive test, a combined limit/range

- finding test was performed. Twenty daphnids per concentration (4 replicates, 5 daphnids per replicate) were exposed to nominal concentrati ons of 0 (control) and 141 mg test substance/L. Ten daphnids per

1 Due to the low solubility of CASRN 68187

- 76

- 8 in water (estimated by EPISuite TM to be 4.3 x 10

- 14 mg/L) and its surfactant nature, the prepared test solutions (with average measured concentrations as high as 25 mg a.i./L) are deemed to be dispersions.

2 During testing, a correction factor of 1.41 was used based on an incorrect purity of ~71% . After toxicity tes ting was complete, concentrations of active ingredient were recalculated using a correction factor of 1.34 based on the correct purity of 74.4%.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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13 concentration (2 replicates, 5 daphnids per replicate) were exposed to nominal concentrations of 0.14, 1.4, and 14 mg test substance/L.

During the combined limit/range

- finding test, dissolv ed oxygen concentrations and pH were only measured for the control and the highest test concentration. All other test procedures and conditions were similar to those applied in the definitive test.

Analytical monitoring of test substance concentrations (by TOC measurement) was conducted for the nominal concentration of 141 mg test substance/L at 0 and 48 hours and for the nominal concentration of 1.4 mg test substance/L at 48 hours only. Details of sample handling and analytical monitoring were not prov ided but are assumed, based on the study author’s statement that the combined limit/range

- finding test procedures and conditions were “similar to those applied in the final test” [Bouwman 2014b, p. 10], to be comparable to those described below in the Anal ytical Monitoring section for the definitive test.

Definitive Test: The exposure vessels were 100

- mL glass vessels containing 80 mL of test medium. Within 45 minutes of test media preparation, 20 daphnids per concentration (4 replicates, 5 daphnids per replicate) were placed in the control vessels or test substance

- containing vessels (nominal concentrations of 1.4, 2.8, 5.6, 11, 23, 45, and 90 mg test substance/L ) . Daphnid loading was 62.5 daphnids/L. Food was withheld th roughout the test. Illumination was provided as a daily 16

- hour photoperiod and the test vessels were not aerated.

The daphnids were observed for immobility and mortality at 24 and 48 hours. Temperature was monitored continuously in a temperature contr ol vessel. For all test concentrations and the control, the pH and dissolved oxygen concentration were measured at the beginning and end of the test. Oxygen levels were also measured at 24 hours in the highest test concentration immediately after countin g the immobilized daphnids.

Analytical Monitoring: During the definitive test, samples (40 mL) for analysis of test substance concentrations were collected from each replicate vessel at test initiation (0 hours) and at 48 hours. 1 Samples were stored in a refrigerator until analysis. “At the end of the exposure period, the replicates were pooled at each concentration before sampling .” [Bouwman 2014b, p. 11] Concentrations of test substance were determined by total organic carbon (TOC) analysis. TOC mea surements were corrected for the measured amount of TOC in the control (when TOC measurements were available for the control). In order to calculate test substance concentrations, TOC measurements were divided by the TOC content of the test substance, whi ch had been determined to be 54.48% in a separate study (WIL Research Project 503334). 2 Average measured concentrations were calculated as geometric means of the concentrations measured at 0 and 48 hours. 3

1 In the study report, analytical monitoring data from the definitive test included only measurements for nomin al concentrations of 0, 5.6, 11, 23, and 45 mg test substance/L. No measurements were provided for nominal concentrations of 1.4, 2.8, and 90 mg test substance/L.

2 Test substance concentration = [(TOC in treated flask)

– (TOC in control flask)]/0.5448

3 Average measured concentration = √(C t=0 x C t=48 ), where C t=x is the measured concentration at time = x hours.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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14 Statistical Analyses: “The 24 and 48h

- EC 50 - value was calculated with the Weibull analysis from the probits of the percentages of affected daphnids and the logarithms of the corresponding average test substance concentrations using linear maximum likelihood regression.” [Bouwman 2014b, p. 12]

RE SULTS

Endpoint value(s): Based on average measured concentrations: 48

- hour EC 50 = 5.7 mg test substance/L (~ 4.3 mg a.i./L)

Results remarks: Combined Limit/Range

- Finding Test: After 24 hours of exposure, all daphnids exposed to the highest test concentration (nominal concentration of 141 mg test substance/L) were immobilized. After 48 hours, 70% of the daphnids exposed to the nominal concentration of 14 mg test substance/L were immobilized. No immobility was observed at 0.14 and 1.4 mg test sub stance/L. After 48 hours, 10% of the control daphnids were immobilized. According to OECD TG 202, a maximum of 10% immobilization is acceptable for the control. Based on results of the combined limit/range

finding test, the EC 50 was expected to be betwe en the nominal test substance concentrations of 1.4 and 14 mg/L.

“Samples taken from [the] nominal [concentration of] 141 mg test substance per litre were analysed for TOC. The concentration of test substance in the aquatic samples was calculated using t he TOC content of pure test substance... The initial concentration was 119 mg [test substance]/l, i.e. 84% of nominal and this concentration remained stable during the test period (82% of initial at the end of the test). In addition the sample taken at the end of the test from [the] nominal [concentration of] 1.4 mg [test substance]/l was analysed and the measured concentration was calculated to be 4.5 mg [test substance]/l, which was above nominal. This relatively high concentration can be related to the fact that measurements could not be corrected for the control [because no TOC measurements from the controls were available].” [Bouwman 2014b, p. 13]

Definitive Test: After 24 hours of exposure, the percentages of immobilized daphnids were 25%, 95%, and 1 00% at nominal concentrations of 23, 45, and 90 mg test substance/L, respectively; no immobilization was observed at nominal concentrations ≤ 11 mg test substance/L at 24 hours. After 48 hours of exposure, the percentages of immobilized daphnids were 45%, 100%, 100%, and 100% at nominal concentrations of 11, 23, 45, and 90 mg test substance/L, respectively; no immobilization was observed at nominal concentrations ≤ 5.6 mg test substance/L at 48 hours.

During the definitive test, the pH measurements ranged from 7.6 to 8.0 and the dissolved oxygen concentrations ranged from 9.1 to 9.7 mg/L. “These test conditions remained within the limits prescribed by the protocol (pH: 6.0

- 8.5, not varying by more than 1.5 units; oxygen: ≥ 3 mg/l at the end of the test).” [Bouwman 2014b, p. 15]Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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15 “The temperature continuously measured in a temperature control vessel varied between 18.8 and 19.3°C during the test, and complied with the requirements as laid down in the protocol (18

- 22°C, constant within 2°C). ” [Bouwman 2014b, p. 15]

Based on TOC measurements : “The initial [measured] concentrations in samples taken from [the] nominal [concentrations of] 5.6, 11, 23 and 45 mg [test substance]/l were calculated to be 3.0, 5.9, 15 and 35 mg [test substance]/l, i.e. 54

- 78% of nomi nal, respectively. The three highest test concentrations remained stable during the exposure period (80

- 91% of initial), while the lowest [measured] test concentration [at the nominal concentration of 5.6 mg test substance/L] decreased to 60% of initial [ at 48 hours]. Given these results, effect parameters were based on the average [measured] exposure concentrations: 2.3, 5.3, 13 and 33 mg test substance per litre, containing 1.7, 4.0, 9.7 and 25 mg a.i. per litre.” [Bouwman 2014b, p. 14]

The definitive test was considered to have met the criteria for acceptability, as defined in OECD TG 202, based on the following: “1. In the control, no daphnids became immobilised. 2. The oxygen concentration at the end of the test was ≥ 3 mg/l in control and test vessels.” [Bouwman 2014b, p. 12]

CONCLUSIONS

Based on average measured concentrations, the 48

- hour EC 50 for immobilization in Daphnia
magna was 5.7 mg test substance/L (95% CI: 4.7

- 7.1 mg test substance/L), containing ~ 4.3 mg a.i./L (95% CI: 3.5

- 5.3 mg a.i./L)

.

The 24

- hour EC 50 was 19 mg test substance/L (95% CI: 15

- 23 mg test substance/L), containing

~ 14 mg a.i./L (95% CI: 11

- 17 mg a.i./L)

.

STUDY RELIABILITY

[2] Reliable with restrictions. Concentrations of active ingredient were determined by usin g a

non

- specific analytical method (TOC measurement), rather than a method specific for CASRN 68187

- 76

- 8.

REFERENCE

Bouwman, L.M. 2014b. Acute toxicity study in Daphnia magna with castor oil, sulfated, sodium salt, 75% (static). WIL Research Europe B.V., The Netherlands. Project No. 503336

. Sponsor: Sulfated Castor Oil Consortium, Society of Chemical Manufacturers & Affiliates

. Text in quotes is taken directly from Bouwman (2014b).Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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16 D

- 3. Toxicity to Aquatic Plants

Summary of endpoint: Adequate

Study 1: Toxicity to Pseudokirchneriella subcapitata (Bouwman, 2014c)

Title: Fresh water algal growth inhibition test with castor oil, sulfated, sodium salt, 75%

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Castor oil, sulfated, sodium salt, CASRN 68187

- 76

- 8

Remarks: Composition: 74.4% castor oil, sulfated, sodium salt (CASRN 68187

- 76

- 8) 1 , 24.3% water, and 1.25% sodium sulfate; Clear, amber liquid ; Batch No. R

- 10135

- 064; referred to as “castor oil, sulfated, sodium salt, 75%” in the study report.

METHOD

Method/guide line followed: OCSPP 850.4500 (2012) “Algal Toxicity” and OECD TG 201 (2011) “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”

Test type: Toxicity to plants

– algae (static)

GLP compliant?: Yes

Year study performed: 2013

- 2014

Test species : Green algae ( Pseudokirchneriella subcapitata ); strain: NIVA CHL 1; obtained from an in

- house laboratory culture

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: Combined Limit/Range

- Finding Test: Nominal concentrations: 0 (control), 0.14, 1.4, 14, and 141 mg test substance/L (equivalent to ~ 0, 0.10, 1.0, 10, and 105 mg active ingredient (a.i.)/L) 2 Definitive Test: Nominal concentrations: 0 (control), 1.4, 4.9, 17, 61, and 212 mg test substance/L ( equivalent to ~ 0, 1.0, 3.7, 13, 46, and 158 mg a.i./L); Average measured

1 The percentage of CASRN 68187

- 76

- 8 in the test substance was calculated by subtracting % water and % sodium sulfate from 100%. (100%

- [24.3% + 1.25%] ≈ 74.4%)

2 Concentrations of active ingredient (CASRN 68187

- 76

- 8) were calculated using a correction factor of 1.34, which

was based on a test substance purity of 74.4%. (Correction factor = 1 ÷ 0.744 ≈ 1.34) The concentration o f active ingredient is equal to the concentration of test substance divided by 1.34.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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17 concentrations: 0, 0.089, 2.4, 13, 51, and 140 mg test substance/L ( equivalent to ~ 0, 0.066, 1.8, 9. 7 , 38, and 104 mg a.i./L)

Test method/conditions remarks: General Test Condi tions: Test Medium: Culture medium for the study (M2 medium) was prepared according to OECD TG 201, using tap water purified by reverse osmosis (Milli

- RO water). The test medium had a hardness of 24 mg CaCO 3 /L and pH of 8.1 ± 0.2.

Algal Pre

- Culture: Th ree days before the start of the study, cells from a stock culture (maintained in M1 medium that was prepared according to Nederlandse Praktijk Richtlijn no. 6505) were inoculated in M2 medium at a cell density of 1 x 10 4 cells/mL and maintained under the same conditions as used in the test.

Preparation of Test Item Concentrations:

“ The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or st able and homogeneous mixtures or dispersions. 1 The testing of concentrations that would disturb the test system was prevented as much as possible (e.g. film of the test substance on the water surface). A correction was made for the purity/composition of the test substance. A correction factor of 1.41 was used [when preparing test solutions]. This factor was based on the information known during testing. Afterwards, the information on purity/composition of the test substance was changed [such that a cor rection factor of 1.34 was used to determine concentrations of active ingredient] 2 ... As a consequence slightly higher concentrations than necessary were tested.” [Bouwman 2014c, p. 10]

“Preparation of test solutions started with a nominal concentration o f 141 mg [test substance]/l containing 105 mg of the active ingredient (a.i.) per litre for the combined limit/range

- finding test and with a nominal concentration of 212 mg [test substance]/l containing 158 mg of the active ingredient (a.i.) per litre for the final [definitive] test. These concentrations were magnetically stirred for 15 minutes... The lower test concentrations were prepared by subsequent dilutions of the 141 or 212 mg [test substance]/l concentrations in test medium. The final test solutio ns were clear and colourless.” [Bouwman 2014c, p. 10]

Inoculation of Test Cultures and Environmental Conditions: Test vessels were 125

- mL glass containers, containing 50 mL of test solution. Immediately after preparation of the test solutions, 1 mL of a n algal suspension was added to each test vessel to provide an initial cell density of 10 4 cells/mL. Test vessels were capped and were randomly distributed in the incubator, with daily repositioning. During the 96

- hour exposure period, algae

1 Due to the low solubility of CASRN 68187

- 76

- 8 in water (estimated by EPISuite TM to be 4.3 x 10

- 14 mg/L) and its surfactant nature, the prepared test solutions (with aver age measured concentrations up to ~ 104 mg a.i./L) are deemed to be dispersions.

2 During testing, a correction factor of 1.41 was used based on an incorrect purity of ~71% . After toxicity testing was complete, concentrations of active ingredient were rec alculated using a correction factor of 1.34 based on the correct purity of 74.4%.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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- 8

18 cultures wer e constantly shaken and were maintained under continuous illumination using TLD lamps with a light intensity of 60

- 62 μE.m

- 2 .s - 1

. Combined Limit/Range

- Finding Test: Prior to conducting the definitive test, a combined limit/range

- finding test was performed. Six replicates of exponentially growing algae were exposed to nominal concentrations of 0 (control) or 141 mg test substance/L. Three replicates per concentration were exposed to nominal concentrations of 0.14, 1.4, and 14 mg test substance/L.

“After 24 hours of exposure cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength = 20 mm). Algal medium was used as blank and one extra test vessel per concentration without algae was used as background. Quantification of cell densities was based on a calibration curve.” [Bouwman 2014c, p. 10]

During the combined limit/range

- findi ng test, the pH was measured only in the control and the highest test concentration. Algistatic/algicidal effects were not determined. All other test procedures and conditions were similar to those applied in the definitive test.

Analytical monitoring o f test substance concentrations (by TOC measurement) was conducted for the nominal concentrations of 1.4 and 141 mg test substance/L at 0, 24, and 96 hours. Details of sample handling and analytical monitoring were not provided but are assumed, based on the study author’s statement that the combined limit/range

- finding test procedures and conditions were “similar to those applied in the final test” [Bouwman 20 14c, p. 10], to be comparable to those described below in the Analytical Monitoring section for the definitive test.

The following protocol deviation was noted for the combined limit/range

- finding test: The vessels for the pre

- culture were not kept sha king during the first 2 days of incubation (out of a total of 3 days). However, the study author noted that algal growth in the pre

- culture proved to be sufficient.

Definitive Test: Freshwater algae (4 replicates/concentration) were exposed to nominal c oncentrations of 0 (control), 1.4, 4.9, 17, 61 and 212 mg test substance/L for 96 hours. Four additional replicates of the control and test concentrations without algae were employed for measurements of test substance concentration.

The pH of test soluti ons was measured at the beginning and end of the test. Temperature was monitored continuously in a temperature control vessel. At the end of exposure, algal cells from each test concentration were examined microscopically for abnormalities in appearance. Cell density was determined every 24 hours in each replicate using a microscope and a counting chamber. To determine whether effects of the test substance were algistatic or algicidal, a subsample was removed from each replicate at the highest test conc entration at the end of exposure and cultured in M2 medium without test substance for a period of up to 9 days.

Analytical Monitoring:Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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19 Samples (40 mL) for analysis of test substance concentrations were taken during the definitive test at 0, 24, and 96 h ours and stored in a refrigerator until analysis. “At the end of the exposure period, the replicates without algae were pooled at each concentration before sampling.” [Bouwman 2014c, p. 11] Concentrations of test substance were determined by total organi c carbon (TOC) analysis. TOC measurements were corrected for the measured amount of TOC in the control (when TOC measurements were available for the control). In order to calculate test substance concentrations, TOC measurements were divided by the TOC c ontent of the test substance, which had been determined to be 54.48% in a separate study (WIL Research Project 503334). 1 Average measured concentrations were calculated as time

- weighted averages of the concentrations measured at 0, 24, and 96 hours. 2 Whe n TOC concentrations were below the limit of detection or quantification, a value of one

- half of the respective limit was used in calculating the average measured concentration. The limit of detection was 0.01 mg/L. The limit of quantification was not de fined.

Statistical Analyses: Statistically significant inhibition of growth rate, yield, and biomass 3 in treated cultures, relative to controls, was determined using analysis of variance followed by multiple comparisons using the Bonferroni method. The E C 50 values were determined using a log

- linear regression analysis of percentage inhibition of growth rate, yield, and biomass versus the logarithm of the average measured test substance concentration.

RESULTS

Endpoint value(s): Based on average measured concentrations: 96

- hour EC 50 (growth rate) = 62 mg test substance/L (~ 46 mg a.i./L) 96

- hour EC 50 (yield) = 40 mg test substance/L (~ 30 mg a.i./L) 96

- hour EC 50 (biomass) = 39 mg test substance/L (~ 29 mg a.i./L) 96

- hour NOEC (growth, yield, and biomass) = 13 mg test substance/L (~ 9.7 mg a.i./L) 96

- hour LOEC (growth, yield, and biomass) = 51 mg test substance/L (~ 38 mg a.i./L)

Results remarks: Combined Limit/Range

- Finding Test: At 96 hours, the mean cell densities at nominal concentrations of 0, 0.14 , 1.4, 14 and 141 mg test substance/L were 244.3, 246.9, 257.8, 188.3 and 10.4 x 10 4 cells/mL, respectively.

Mean percentage values for inhibition of growth rate, yield, and biomass relative to the control at 96 hours were as follows for each nominal test substance concentration (mg test substance/L): 0.14 mg/L:

- 0.2% (growth rate inhibition),

- 1.1% (yield inhibition),

- 2.2% (biomass inhibition); 1.4 mg/L:

- 1.0% (growth rate inhibition),

- 5.5% (yield inhibition),

- 4.1% (biomass inhibition); 14 mg/L: 4.7% (growth rate inhibition), 23.0% (yield inhibition), 20.2% (biomass inhibition); 141 mg/L: 57.4% (growth rate inhibition), 96.1% (yield inhibition), 90.8% (biomass inhibition).

1 Test substance concentration = [(TOC in treated flask)

– (TOC in control flask)]/0.5448

2 Average measured concentration = ([24 x √(C t=0 x C t=24 )] + [72 x √(C t=24 x C t=96 )])/96, where C t=x is the measured concentration at time = x hours.
3 Biomass was calculated as area under the growth curve. The study author referred to this parameter as “cell growth”.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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20 Based on results of the combined limit/range

- finding test, the EC 50 val ues for inhibition of growth rate, yield, and biomass were expected to be between the nominal concentrations of 14 and 141 mg test substance/L.

“After 48 hours of exposure test concentrations of 1.41 mg [test substance]/l and higher became increasingly ha zy with increasing concentration. After 72 and 96 hours of exposure no haziness was observed at 1.41 and 14 mg [test substance]/l but haziness, test substance particles and a precipitate were observed at the highest test concentration, indicating that par t of the test substance came out of solution at this concentration.” [Bouwman 2014c, p. 14]

“The initial [measured] concentrations were 7.1 and 101 mg [test substance]/l, respectively, i.e. 507 and 72% of nominal [at nominal concentrations of 1.4 and 14 1 mg test substance/L]. Both concentrations remained stable during the first 24 hours of exposure (83

- 94% of initial) but decreased to 44

- 70% of initial at the end of the test. The relatively high concentration at [the] nominal [concentration of] 1.4 mg [test substance]/l can be related to the fact that measurements could not be corrected for the control [because no TOC measurements from the controls were available].” [Bouwman 2014c, p. 14]

Definitive Test: At 96 hours, the mean cell densities at nomina l concentrations of 0, 1.4, 4.9, 17, 61 and 212 mg test substance/L were 254, 245, 294, 265, 70, and 2 x 10 4 cells/mL, respectively.

Mean percentage values for inhibition of growth rate, yield, and biomass relative to the control at 96 hours were as follows for each nominal test substance concentration (mg test substance/L): 1.4 mg/L: 0.8% (growth rate inhibition), 3.4% (yield inhibition), 1.4% (biomass inhibition); 4.9 mg/L:

- 2.6% (growth rate inhibition),

- 15.9% (yield inhibition),

- 13.4% (biomas s inhibition); 17 mg/L:

- 0.6% (growth rate inhibition),

- 4.5% (yield inhibition),

- 0.5% (biomass inhibition); 61 mg/L: 23.9% (growth rate inhibition), 72.9% (yield inhibition), 72.1% (biomass inhibition); 212 mg/L: 91.0% (growth rate inhibition), 99.7% (yield inhibition), 97.7% (biomass inhibition).

Growth rate, yield, and biomass at nominal concentrations of ≥ 61 mg test substance/L were statistically significantly inhibited (p < 0.05), relative to the control, at 96 hours. Growth rate, yield, and bio mass at nominal concentrations ≤ 17 mg test substance/L were statistically similar to control values.

“Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.” [Bouwman 20 14c, p. 17]

The pH measurements ranged from 7.8 to 8.6 during the definitive test. “ The pH was within the limits prescribed by the protocol (6.0

- 9.0, preferably not varying by more than 1.5 unit). The temperature of the test medium was 23.1°C at the sta rt of the test. During the exposure period the temperature measured in the incubator was maintained between 24.0 and 24.8°C. Temperature remained within the limits prescribed by the protocol (22

- 26°C, constant within 2°C). ” [Bouwman 2014c, p. 17]Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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21 Based on TOC measurements, corrected for TOC in the control and adjusted for TOC content of the test substance (54.48%): “The initial concentrations in samples taken from nominal [concentrations of] 1.4, 4.9, 17, 61 and 212 mg [test substance]/l were calculated to be 0.30, 3.6, 15, 48 and 132 mg [test substance]/l, respectively, i.e. 21

- 88% of nominal. The three highest test concentrations remained stable during the exposure period (100

- 113% of initial at the end of the test) which was not expected based on the results of the range

- finding test. However, no explanation could be given for this contradiction. The lower test concentrations decreased to below the LOD for the lowest test concentration and to 72% of initial for [the] nominal [concentration of] 4.9 mg [test substance]/l [at 96 hours]. Given these results, effect parameters were based on the average [measured] exposure concentrations: 0.089, 2.4, 13, 51 and 140 mg test substance per l itre, containing 0.066, 1.8, [9.7 1 ] , 38 and 104 mg a.i. per litre.” [ Bouwman 2014c, p. 15]

“ After 72 hours of exposure the two highest test concentrations became increasingly hazy with increasing concentration. This haziness was not observed after 96 hours of exposure.” [Bouwman 2014c, p. 16]

The control growth was consi dered to have met the criteria for acceptability, as defined in OCSPP 850.4 5 00, based on the following: “1. In the controls, cell density increased by an average factor of >100 within 96 hours. 2. The coefficient of variation of average specific growth ra tes during the whole test period in replicate control cultures did not exceed 15%. 3. The coefficient of variation of average yield during the whole test period in replicate control cultures did not exceed 15%.” [Bouwman 2014c, p. 14]

Algistatic/algicida l effects: Subsamples that had been taken from each replicate of the nominal test substance concentration of 212 mg test substance/L at 96 hours and cultured in control medium demonstrated cell growth. After a 120

- hour period of recovery, cell density ha d reached 101 to 127 x 10 4 cells/mL. “ The subculture test was discontinued after 120 hours of exposure but cell density had already increased by an average factor of >16 after 96 hours of exposure. This result showed that the inhibitory effect of Castor Oil, Sulfated, Sodium Salt, 75% was algistatic.” [Bouwman 2014c, p. 18]

CONCLUSIONS

Based on average measured concentrations, the 96

- hour EC 50 value for inhibition of growth rate was 62 mg test substance/L (95% CI: 23

- 170 mg test substance/L), containing ~ 46 mg a.i./L (95% CI: 17

- 130 mg a.i./ L ).

1 The study authors incorrectly presented a value of 9.9 within this quote. However, the correct value here is 9.7, i.e., 13/1.34 (1.34 is the correction factor that is based on the correct test substance purity of 74.4%). Within the study report, the erroneous value of 9.9 is presented twice (p. 6, paragraph 6 of Summar y and p. 15, end of paragraph at bottom of page) but is correctly presented as 9.7 in the NOEC statements on the bottom of p. 6, in Table 6 on p. 17, and in block 5 of the CONCLUSION on p. 18.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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22 The 96

- hour EC 50 for inhibition of yield was 40 mg test substance /L (95% CI: 19

- 83 mg test substance / L ), containing ~ 30 mg a.i./L ( 95% CI: 14

- 62 mg a.i./ L ).

The 96

- hour EC 50 for inhibition of biomass was 39 mg test substance / L (95% CI: 22

- 68 mg test substance / L ), containing ~ 29 mg a.i./L (95% CI: 16

- 51 mg a.i./ L ).

The 96

- hour NOEC for inhibition of growth rate, yield, and biomass was 13 mg test substance / L
, containing ~ 9.7 mg a.i./ L ; the 96

- hour LOEC was 51 mg test substance / L , containing ~ 38 mg a.i./ L

.

STUDY RELIABILITY

[2] Reliable with restrictions. Concentrations of active ingredient were determined by using a

non

- specific analytical method (TOC measurement), rather than a method specific for CASRN 68187

- 76

- 8. In addition, concentrations were only measured in replicates that did not contain algae a nd, therefore, may not be an accurate representation of the concentrations to which algae were exposed.

REFERENCE

Bouwman, L.M. 2014c. Fresh water algal growth inhibition test with castor oil, sulfated, sodium salt, 75% . WIL Research Europe B.V., The Netherlands. Project No. 503338. Sponsor: Sulfated Castor Oil Consortium, Society of Chemical Manufacturers & Affiliates.

Text in quotes is taken directly from Bouwman (2014c).Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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23 D

- 4. Acute Toxicity

Summary of endpoint: Adequate

Study 1: Acute oral toxicity study in rats ( van Huygevoort , 2013)

Title: Assessment of acute oral toxicity with castor oil, sulfated, sodium salt, 75% in the rat (up and down procedure)

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Castor oil, sulfated, sodium salt, CASRN 68187

- 76

- 8

Remarks: Composition 1 : 74.4% castor oil, sulfated, sodium salt (CASRN 68187

- 76

- 8) 2 , 24.3% water, and 1.25% sodium sulfate; Clear amber liquid; Batch No. R

- 10135

- 064; referred to as “castor oil, sulfated, sodium salt, 75%” in the study report.

METHOD

Method/guideline followed: OECD TG 425 (2001 and 2008) “Acute Oral Toxici ty

– Up and Down Procedure” and OPPTS 870.1100 (2002) “Acute Oral Toxicity”

Test type: Acute Oral Toxicity

GLP compliant?: Yes

Year study performed: 2013

Test species: Wistar Crl:WI strain rats; obtained from Charles River Deutschland, Sulzfeld, Germany; age at initiation: approximately 8 weeks old; body weight at initiation: 154

- 187 g.

Number of animals/sex/dose: 5 females

Route of administration: Oral (gavage)

1 This compositional information was obtained from the study report for WIL Research Project 503335 (van Doormalen

- Schaap, I. 201 4 . Determination of physico

- chemical properties of castor oil, sulfated, sodium salt, 75%. WIL Research Europe B.V., The Netherlands. Project No. 503335. Sponsor: Sulfated Castor Oil Consortium, Society of Chemical Manufacturers & Affiliates.) and is consistent with information provided in the study reports for the gene mutation, combined repeated

- dose toxicity with reproduction/developmental toxicity screening, and aquatic toxicity st udies, which were conducted with the same batch of this chemical (R

- 10135

- 064). A certificate of analysis provided in Appendix 1 of the acute oral toxicity study report apparently provided incorrect information ( 70.7% castor oil, sulfated, sodium salt; 26 .4% water; and 2.9% sodium sulfate).

2 The percentage of CASRN 68187

- 76

- 8 in the test substance was calculated by subtracting % water and % sodium sulfate from 100%. (100%

- [24.3% + 1.25%] ≈ 74.4%)Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

- 76

- 8

24 Vehicle: None

Doses/concentrations tested: 2105 mg active ingredient (a.i.)/kg

- bw 1

Statistical methods used: None

Test method/conditions remarks: A single dose (2105 mg a.i./kg

- bw) of the undiluted test material was administered to each of five rats at a dosing volume of 2.697 mL/kg

- bw 2 . The first rat was treated and observed fo r mortality before the remaining rats were treated. Animals were acclimated under laboratory conditions for a period of at least 5 days prior to the start of treatment. Animals were fasted overnight prior to dosing and until 3 to 4 hours following admini stration of the test substance; water was available ad libitum . The animals were group

housed in Makrolon cages during the study.

The animals were observed twice daily for mortality for 14 days after test substance administration. Clinical signs were recorded at periodic intervals on the first day of dosing and once daily thereafter. Individual animal body weights were recorded on day 1 (pre

administration), day 8, and day 15. The animals were sacrificed on day 15 and examined for gross abnormalities.

RESULTS

Endpoint value(s): LD 50 > 2105 mg a.i./kg

- bw

Results remarks: No mortalities were observed. All animals gained weight. The study author noted that body weight gains were considered to be normal throughout the study period, but did not provide laboratory reference control data. Four animals displayed hunched post ure (between days 1 and 3) and piloerection (between days 1 and 2). The other animal showed no clinical signs. Macroscopic post

- mortem examination of the animals revealed no abnormalities.

CONCLUSIONS

The acute oral LD 50 for rats was > 2105 mg a.i./k g

- bw.

STUDY RELIABILITY

[2] Reliable with restrictions. The dose of 2000 mg a.i./kg

- bw reported by the study author was based on a test substance purity of 70.7% active ingredient (CASRN 68187

- 76

- 8) as shown in the certificate of analysis provided in Appendix 1 of the acute oral toxicity study report; however, a later report (WIL Project 503335) showed that the purity of Batch No. R

- 10135

- 064

1 The dose, expressed in units of active ingredient (CAS RN 68187

- 76

- 8), was calculated by the reviewer based on a test substance purity of 74.4%. The dose provided in the study report was 2000 mg a.i./kg

- bw, which was calculated by the study author using an incorrect purity of 70.7%.

2 The first animal was ina dvertently dosed with 2.679 mL/kg

- bw instead of 2.697 mL/kg

- bw. Th e study author stated that this deviation had no effect on the dosed volume (0.41 mL). This deviation from protocol was not considered to affect the integrity of the study.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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25 was 74.4%. The dose provided in the review above has been corrected to 2105 mg a.i./kg

- bw based on the more r ecent purity of 74.4%.

REFERENCE

van Huygevoort, A.H.B.M. 2013. Assessment of acute oral toxicity with castor oil, sulfated, sodium salt, 75% in the rat (up and down procedure). WIL Research Europe B.V., The Netherlands. Project No. 503339. Sponsor: Sulfated Castor Oil Consortium, Society of Chemical Manufacturers & Affiliates.

Text in quotes is taken directly from van Huygevoort (2013).Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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- 8

26 D

- 5. Repeated

- Dose , Reproductive , and Developmental Toxicity

Summary of repeated

- dose toxicity endpoint: Adequate Summary of reproductive toxicity endpoint: Adequate Summary of developmental toxicity endpoint: Adequate

Study 1 : Combined repeated

- dose oral toxicity study with the reproduction/developmental toxic ity screening test in rats (de Raaf

- Beekhuijzen, 2014

)

Title: Combined 28

- day repeated dose toxicity study with the reproduction/developmental toxicity screening test of castor oil, sulfated, sodium salt, 75% in rats by oral gavage

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity : Castor oil, sulfated, sodium salt, 75% CAS RN 68187

- 76

- 8

Remarks : Composition: 74.4% castor oil, sulfated, sodium salt (CASRN 68187

- 76

- 8) 1 , 24.3% water, and 1.25% sodium sulfate; Clear amber liquid; Batch N o. R

- 10135

- 064; referred to as “castor oil, sulfated, sodium salt, 75%” in the study report

.

METHOD

Me thod/guideline followed: OECD TG 422, “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (1996) ; OPPTS 870.3650, “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (2000)

Test type: Combined Repeated

- Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

GLP compliant?: Yes

Year study performed: 2013

Test species: Crl:WI(Han) strain Wistar rats; obtained from Charles River Deutschland, Sulzfeld, Germany; Age at initiation: ~ 11 weeks old; Body weight at initiation: 294

- 334 g (males), 186

- 213 g (females)

Number of animals/sex/dose: 10/sex/dose

Route of administration: Oral ( gavage )

1 The percent age of CASRN 68187

- 76

- 8 in the test substance was calculated by subtracting % water and % sodium sulfate from 100%. (100%

- [24.3% + 1.25%] ≈ 74.4%)Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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- 8

27 Vehicle: Water

Exposure duration: Males: 29 days; Females: 4 1

- 4 7 days (throughout 14 days of pre

- mating, mating, and gestation, and through at least postpartum/lactation day 4)

Doses/concentrations tested: Nominal dose levels: 0 (control), 10 5 , 3 16, and 10 52 mg active ingredient (a.i.) /kg

- bw/day 1

, 2

Statistical methods used : “ The following statistical methods were used to analyze the data:

- If the variables could be assumed to follow a normal distribution, the Dunnett

- test (many

- to

one t

- test) based on a pooled variance estimate was applied for the comparison o f the treated groups and the control groups for each sex.

- The Steel

- test (many

- to

- one rank test) was applied if t he data could not be assumed to follow a normal distribution.

- The Fisher Exact

- test was applied to frequency da ta.

- The Kruskal

- Wallis nonparametric ANOVA test was ap plied to motor activity data to determine intergroup differences. In case intergroup differenc es were seen, the Wilcoxon test was applied to compare the treated groups to the control group .” [de Raaf

- Beekhuijzen 2014, p. 21]

“All tests were two

- sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.” [de Raaf

- Beekhuijzen 2014, p. 21]

The standard unit of assessment for analyses of pup data was the litter.

Test method/conditions remarks: Test Item Prepara tion: “Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity/density of the test substance (1.049). A correction was made for the purity/compos ition of the test substance.” [de Raaf

- Beekhuijzen 2014, p. 11 ]

1 The doses of active ingredient (CASRN 68187

- 76

- 8) provided in this review are based on a test substance purity of 74.4% (as provided in the certificate of analysis in Appendix 5 and determined in WIL Research Project 503335 [van Doormalen

- Schaap, I. 2014. Determination of physico

- chemical properties of castor oil, sulfated, sodium salt, 75%. WIL Research Europe B.V., The Netherlands. Project No. 503335. Sponsor: Sulfated Castor Oil Consortium, Society of Chemical Manufacturers & Affiliates.]. The stud y author notes that this purity information was not available when preparing doses for the combined repeated

- dose toxicity study with the reproduction/developmental toxicity screening te st; instead, the target doses were based on a purity value of 70.7%,

w hich had been supplied by the sponsor. Throughout the study report, the doses are provided as 100, 300, and 1000 mg a.i./kg

- bw/day, which are based on the purity of 70.7%. The study author notes only on p. 12 of the study report that dose levels calculat ed using the “updated” purity of 74.4% would be 105, 316, and 1052 mg a.i./kg

- bw/day.

2 Dose levels were selected based on the results of a 10

- day range

- finding study in female rats (3/dose) exposed to the test substance for 10 days at dose levels of 500 o r 1000 mg/kg

- bw/day, which reported clinical signs, reduced body weight gain, and increased absolute and/or relative liver weights at both dose levels (study summary is provided in Appendix 6 of the study report).Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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- 8

28 Analyses of Test Item Formulations: The formulations used for dosing the animals during the study were not analytically verified due to the absence of an appropriate analytical method at th e time of the study. When an analytical method became available, formulations were prepared in a similar manner as used during the study and the formulations were analyzed for accuracy of concentration (all concentrations) and homogeneity (highest and low est concentrations).

“For quantitative analysis of the test substance in formulation, Total Organic Carbon (TOC) analysis was used

... Stability could not be determined since TOC analysis is a general method that does not distinguish original substance fr om hydrolysis product(s); therefore results on stability assessment were not reported.” [de Raaf

- Beekhuijzen 2014, p. 11]

“ Accuracy of preparation was considered acceptable if mean measured concentrations were 90

- 110% of target concentration [s]

. Homogene ity was demonstrated if the coefficient of variation was ≤ 10%. ” [de Raaf

- Beekhuijzen 2014, p. 12]

Dosing and Environmental Conditions : Groups of sexually mature rats (10/sex/group) acclimated for at least 5 days were administered the test item daily via gavage at dose levels of 0 (vehicle only), 10 5 , 3 16 , or 10 52 mg a.i. /kg

bw/day at a constant dosing volume of 5 mL/kg

- bw. During dosing, the formulations were stirred with a magnetic stirrer. Food and wat er were available ad libitum throughout the study. Exposure continued throughout the pre

- mating (14 days), mating, and post

- mating periods (through at least 4 days of lactation in females with pups) . After the mating period, exposure continued until the day prior to euthanasia

, for a total of 2 9 consecutive days of exposure for males and 4 1 to 4 7 consecutive days of exposure for females.

Parental animals were housed in groups of 5 / sex prior to mating and in pairs (one male: one female) during mating. Animals were paired until evidence of mating (i.e., the presence of sperm in a vaginal lavage or the appearance of an intravaginal copulatory plug ) was observed. The day of mating was designated as day 0 post

- coitum. After mating, males were returned to their original cages and females were housed individually (or with their litters after parturition) throughout gestation and lactation. During locomotor activity monitoring, animals were housed individually.

Mortality and Clinical Observations: The a nima ls were observed at least once daily during dosing for mortality and clinical signs of toxicity. Detailed clinical observations were performed weekly during the dosing period outside of the cage in a standard arena

. Body Weight and Food/Water Consumption: Individual body weights were recorded on the first day of exposure, and then at least weekly thereafter. Mated females were weighed on post

- coitum days 0, 4, 7, 11

, 14, 17, and 20, and on lactation days 1 and 4. Terminal body weights of all animals were recorded at necropsy . Food consumption was determined at least weekly throughout the study period, except when males and females were housed together during mating and for females without evidence of mating. Food consumption was determined f or mated females on post

- coitum days 0, 4, 7, 11, 14, 17,Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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- 8

29 and 20, and on lactation days 1 and 4. Water intakes were monitored by visual inspection of water bottles on a regular basis throughout the study, but intakes were not quantitatively measured.

Neu rological Assessments: F unctional observations were performed on 5 animals/sex/group during week 4 of treatment for males and before the end of the lactation period for females (all before blood sampling). The following tests were performed: hearing abili ty, pupillary reflex, static righting reflex, grip strength, and locomotor activity (based on the total movements and ambulations).

Hematology and Clinical Chemistry Analyses: Blood samples for hematology and clinical chemistry evaluations were collected from 5 animals/sex/dose at the end of treatment on the day of scheduled necropsy

. All males and the selected 5 females/dose were deprived of food overnight (maximum of 24 hours) prior to blood collection. Hematological parameters that were evaluated incl uded hemoglobin, red blood cell count, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red blood cell distribution width, reticulocyte count, white blood cell count, differential leukocyte count (including counts for neutrophils, lymphocytes, monocytes, eosinophils, and basophils), and platelet count . Coagulation parameters determined from blood plasma included activate d partial thromboplastin time and prothrombin time.

The following clinica l chemistry parameters were determined from plasma collected from each blood sample: urea, glucose, total protein, albumin, sodium, potassium, chloride, calcium, inorganic phosphate, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatas e, creatinine, c holesterol, and total bilirubin. Bile acids were determined from blood serum, not plasma.

Gross Necropsy , Organ Weights, and Histopathology : Parental animals were sacrificed on the day following the last exposure (day 30 for males and lac tation days 5 to 7 for females with pups). Females that did not deliver a litter were sacrificed on post

- coitum days 25 to 27. A complete necropsy was performed on all animals, with special attention being paid to the reproductive organs. T he following o rgans were weighed for 5 animals/sex/dose : adrenals, brain, heart, kidneys, liver, spleen, thymus, thyroid glands with parathyroid, prostate gland, ovaries, seminal vesicles (with coagulating glands), and uterus (including cervix)

. In addition, the teste s and epididymides of all males, with the exception of one male in the control group, were weighed.

Histological examination was performed on the following tissues collected from 5 animals/sex /dose in the control and high

- dose groups: adrenals , sternum with bone marrow, femur including joint, brain (cerebellum, mid

- brain, and cortex), eyes, female mammary gland area, heart, kidneys, cecum, colon

, rectum, liver, lungs, axillary lymph nodes, mandibular lymph nodes, mesenteric lymph nodes, pituitary gland, sciatic nerve, skeletal muscle, large intestine

, small intestine ( duodenum, jejunum

, and ileum with Peyer's patches ) , spinal cord (cervical, midthoracic, and lumbar) , spleen, femorotibial joint, bulbourethral glands, stomach, thymus, thyroid glands includi ng parathyroid glands if detectable , trachea, and urinary bladder, as well as all gross lesions. In addition, the heart from 5 males/dose in the low

- and mid

- dose groups andFinal HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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- 8

30 the adrenal glands from 5 females/dose in the low

- and mid

- dose groups were exami ned based on possible treatment

- related changes observed at the high dose. The severity of histological findings was graded on a scale of 1 to 4 (grade 1 = minimal; grade 2 = slight; grade 3 = moderate; grade 4 = marked).

Reproductive and Developmental T oxicity: During mating, the following parameters were recorded: numbers of females paired, number of females mated, number pregnant, and time until mat ing

. Pregnant animals were observed daily and allowed to litter. “ Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.” [de Raaf

- Beekhuijzen 2014, p. 12]

“Pregnant females were examined to det ect signs of difficult or prolonged parturition

, and cage debris of pregnant females was examined to detect signs of abortion or premature birth

. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left
scatter ed and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.” [de Raaf

- Beekhuijzen 2014, p. 1 5 ]

The following pregnancy and littering parameters were recorded: duration of gestation, number of females producing a li ve litter, number of implantation sites per pregnancy, number of corpora lutea sites per pregnancy, total number of pups born per litter, number of live pups born per litter

, number of dead pups born per litter, and postnatal loss. All pups were examined for clinical signs at least once daily. Live pups were weighed on days 1 and 4 of lactation. Individual pup body weights were used to calculate mean pup weight per litter. Mean pup weight per litter was presented for males only, females only, and males and females combined. Pup sex determinations were recorded on days 1 and 4 of lactation. Surviving pups were sacrificed on lactation days 5 to 7 and examined for external abnormalities. Calculated fertility, parturition, and offspring parameters include d fertility index

, mati ng index, conception index, gestation index , viability index, percentage of live male pups at first litter check, percentage of live female pups at first litter check, and percentage of postnatal loss.

At necropsy, the numbers of implantation sites and corpora lutea were recorded for all paired females. If no implantation sites were macroscopically visible, non

- gravid uteri were stained in order to detect any former implantation sites. Spermatogenesis staging of the testes was
pe rformed on 5 males/dose from the control and high

- dose groups, as well as for all males suspected of being infertile or that died before mating. Reproductive organs were weighed, as described above. Histological examinations were performed on the followi ng reproductive tissues collected from 5 animals/sex/dose in the control and high

- dose groups: clitoral gland

, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, coagulating gland, testes, uterus, cervix, and vagina. The reproductiv e organs of all males that failed to sire a litter and all females that failed to deliver healthy pups were also examined microscopically.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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31 RESULTS

Endpoint value(s): Systemic Toxicity: LOAEL (males) = 1052 mg a.i. /kg

- bw/day, based on histological effects, including increased severity of mononuclear inflammati on in the heart, increased incidence and severity of myofib er degeneration in the heart, and increased incidence of hyaline droplets in the kidney 1 , and increased relative kidney w eight; NOAEL (males) = 316 mg a.i. /kg

- bw/day

. LOAEL (females) = 1052 mg a.i./kg

- bw/day, based on histological effects, including increased incidence of vacuolation of the zona glomerulosa in the adrenal glands, increased incidence of hemosiderin pigmentat ion in the spleen, and increased incidence of hematopoietic cell foci in the liver; NOAEL (females) = 316 mg a.i. /kg

- bw/day

. Reproductive Toxicity: LOAEL (males and females) = 1052 mg a.i./kg

- bw/day, based on increased absolute and relative seminal vesicle weights and increased absolute and relative uterus weights 2 ; NOAEL (males and females) = 316 mg a.i./kg

- bw/day.

Developmental Toxicity: NOAEL = 10 52 mg a.i. /kg

- bw/day , based on no adverse effects at the highest dose tested

.

Results remarks: Analyses of Test Item Formulations: The mean accuracies of prepared formulations were 92%, 93%, and 93% at the low

- , mid

- , and high

- dose levels, respectively. No test substance was detected in the control group formulation. “The concentrations analyzed in the formulations of... [the low

- , mid

- , and high

- dose levels] were in agreement with target concentrations... The formulations of... [the low

- and high

- dose levels] were homogeneous (i.e., coefficient of variation ≤ 10%).” [de Raaf

- Beekhuijzen 2014, p. 22 ]

Mortality and Clinical Observations: No mortalities occurred during the study. Clinical signs included piloerection in one animal (1 female) at the low

- dose level and rales in two animals (1/sex) at the high

- dose level. These findings were not considered to be treatment

- related due to the relatively low incidence.

1 The increased incidence of hyaline droplets in the kidneys of male rats at 1052 mg a.i./kg

- bw/day is suggestive of alpha 2u - globulin nephropathy, a species

- specific effect that has been described exclusively in male rats and does not appear to be relevant to humans. EPA’s Risk Assessment Fo rum has outlined the key events and the data that are necessary to demonstrate this mode of action (Alpha 2u - Globulin: Association with Chemically Induced Renal Toxicity and Neoplasia in the Rat, EPA/625/3

- 91/019F). One of the key events, alpha 2u - globulin accumulation, has not been demonstrated in this study. In the absence of direct measurement of alpha 2u - globulin, there is no conclusive evidence that the nephropathy observed in male rats was the result of an alpha 2u - globulin

- mediated mechanism; therefor e, the nephropathy observed in this study is assumed to be relevant to human health and has been used as one of the bases for setting the LOAEL for male rats.

2 According to EPA’s Guidelines for Reproductive Toxicity Risk Assessment ( EPA/630/R

- 96/009 ), a s ignificant change in seminal vesicle weight may constitute an adverse reproductive effect in males; likewise, an alteration in uterine weight may be considered an indication of female reproductive organ toxicity.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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32 Body Weight and Food/Water Consumption: A significant decrease (p < 0.05) in body weight gain was observed in males at the low

- dose level relative to the control group during the last two wee ks of treatment (reductions of 14

- 24%). This finding was not considered to be toxicologically relevant since similar changes were not observed in the other dose groups. No other changes in body weight or body weight gain were observed. Food consumption was similar among the treatment and control groups. No
treatment

- related changes in water consumption were mentioned in the study report.

Neurological Assessments: A significant increase (p < 0.05) in the mean ambulations (increase of 59.8%) was observed in male rats at the low

- dose level relative to the control group at week 4. This finding was not considered to be toxicologically relevant since similar changes were not observed in the other dose groups. No changes in any of the other neurological para meters examined were observed.

“ Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals

... All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.” [de Raaf

- Beekhuijzen 2014, p. 22 ]

Hematology and Clinical Chemistry Analyses: Mean prothrombin time was significantly decreased (p < 0.05) in males at the mid

- dose level (reduction of 6.7%) relative to the control group. This finding was not considered to be toxicologically relevant since similar changes were not observed at the high

- dose level. No other

c hanges in hematology or clinical chemistry parameters were observed at any dose level.

Gross Necropsy , Organ Weights, and Histopathology : An enlarged liver was observed in one female at the mid

- dose level during macroscopic examination. No other macroscopic observations considered to be of relevance to the treatment were noted. Mean relative kidney weight was significantly increased (p < 0 .01) in males at the mid

- and high

- dose levels (increases of 7.9% and 14.3%, respectively) relative to the control group. Additionally, mean absolute liver weight was significantly decreased (p < 0.05) in males at the low

- dose level (reduction of 10.3%) r elative to the control group. The reduction in absolute liver weight in males was not considered to be treatment

- related since similar changes were not observed in the other dose groups.

An increase in the severity of mononuclear inflammati on of the hear t was observed in males at the high

- dose level. Although the incidence was 1/5 males in all dose groups and the control group, the severity was grade 2 at the high dose, compared to a severity of grade 1 in all other groups. The increase in severity at t he high dose was considered by the study author to be potentially related to treatment

.

Increases in the i ncidence and severity of myofiber degeneration of the heart were observed in males at the high

- dose level and were considered by the study author to be potentially related to treatment; incidences were 0/5, 0/5, 1/5 (g rade 1), and 2/5 ( g rade 1 in one animal and g rade 2 in the other animal ) for the control, low

- , mid

- , and high

- dose males , respectively.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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- 8

33 An increase in the i ncidence of vacuolation of th e zona glomerulosa of the adrenal gland was observed in females at the high

- dose level and was considered by the study author to be potentially related to treatment; incidences of this lesion (all g rade 1 in severity) were 1/5, 1/5, 1/5, and 4/5 for the co ntrol, low

- , mid

- , and high

- dose females, respectively.

It is noteworthy to mention that vacuolation of the zona glomerulosa of the adrenal glands of females occurred unilaterally in the affected control animal (1/5), but bilaterally in all of the affecte d treated animals (1/5, 1/5, and 4/5 at the low

- , mid

- , and high

- dose levels, respectively).

Other histological findings that are considered to be of possible toxicological relevance occurred, but were not addressed by the author in the results section of the study report. These included histological changes in the kidneys (males), spleen (female s), and liver (females). In males, bilateral tubular hyaline droplets in the kidneys were observed at the high

- dose level (2/5 males; grade 1) but not in controls. The kidneys of males exposed to lower dose levels were not histologically examined; theref ore, it is unknown if males in other dose groups also had hyaline droplets in the kidneys. No treatment

- related histopathological changes were observed in the kidneys of females.

In females, hemosiderin pigmentation in the spleen was observed at the hi gh

- dose level (3/5 females; grade 1), but not in controls. The spleen of females exposed to lower dose levels was not histologically examined; therefore, it is unknown if females in other dose groups also had hemosiderin pigmentation in the spleen. An in creased incidence of females with hematopoietic cell foci in the liver was observed at the high

- dose level (4/5 females; grade 1) compared to controls (1/5 females; grade 1). The liver of only one female from the mid

- dose group was examined and also found to have hematopoietic cell foci (grade 1). However, the liver of no other females was examined; therefore, it is unknown if other females also had hematopoietic cell foci in the liver.

Reproductive and Developmental Toxicity: All pairs mated and the average time to mating (mean pre

- coital time), fertility index, and conception index were similar among the groups. The following number of females became pregnant and were observed to have living pups at first litter check at the control, low

- , mid

- and high

- dose levels: 9/10, 9/10, 8/10, and 9/10, respectively. The fertility indices were 90%, 90%, 80%, and 90% at the control, low

- , mid

- and high

- dose levels, respectively. On the day of scheduled sacrifice , one female at the mid

- dose level cannibalized one of her pups during transfer to the necropsy room.

A relatively low number of corpora lutea, implantation sites, and/or living pups at first litter check were observed in two females at the mid

- dose level, but the group means were not statistically significantly different from the control values. Since these anomalies were not significantly different from the control values and similar changes were not observed at the high

dose level, they were not considered to be treatment

- related. No effects rel ated to the treatment were observed for spermatogenic

- staging profiles, number of corpora lutea, and number of implantation sites.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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34 Absolute and relative seminal vesicle weights were significantly increased (p < 0.01) at the high

dose level (increases of 4 0% and 46%, respectively) relative to the control group. In addition, absolute and relative uterus weights were significantly increased (p < 0.01) at the high

- dose level (increases of 32% and 38%, respectively) relative to the control group. There were n o other changes in reproductive organ weights and no histological changes in the reproductive organs that were considered to be of relevance to the treatment.

For developmental toxicity, two pups at the low

- dose level and one pup at the mid

- dose level

wer e found dead or were missing during the lactation period. The study author noted that the missing pups were most likely cannibalized. Given the low incidence, this finding was not considered to be related to the treatment. Clinical signs in pups were li mited to blue spots on the body in two males at the low

- dose level and in one female at the mid

- dose level, and scabbing of the snout in two females at the mid

- dose level and in one female at the high

- dose level; however, these anomalies were not considere d to be related to the treatment due to the relatively low incidence and the absence of a dose

- related response.

A significant (p < 0.01) reduction in the sex ratio was observed in pups at the mid

- dose level (29% males/71% females) compared to the contr ol group (48% males/52% females). This finding was not considered to be treatment

- related since similar changes were not observed at the high

- dose level. Pup body weights were similar among all groups, including controls. No effects were noted for the f ollowing endpoints: gestation index and duration, parturition/maternal care, number of dead and living pups at first litter check, postnatal loss, viability index, pup body weight, and external macroscopy of pups

.

Determination of the NOAEL and LOAEL Values for Systemic and Reproductive Toxicity: The microscopic findings in the heart of males at the high

- dose level, including mononuclear inflammati on and myofib er degeneration, were considered to be possible evidence of a
treatment

- related effect. Ther efore, these changes were considered in the basis for determining the LOAEL value for systemic toxicity in male rats.

The increase d incidence of bilateral tubular hyaline droplets in the kidneys of male rat s at the high

- dose level (i.e., 1052 mg a.i./kg

- b w/day) is suggestive of alpha 2u - globulin nephropathy, a species

- specific effect that has been described exclusively in male rats and does not appear to be relevant to humans. 1 However, in the absence of direct measurement of alpha 2u - globulin, there is no conclusive evidence that the nephropathy observed histologically in high

- dose males, in conjunction with an increase in relative kidney weight at the high dose, was the result of an alpha 2u - globulin

- mediated mechanism; as a result, t he nephropathy observed in this study is assumed to be relevant to human health. Therefore, both hyaline droplet formation and increased relative kidney weight are included in the basis for determining the LOAEL value for systemic toxicity in male rats.

1 EPA. 1991. Alpha 2u - Globulin: Associati on with Chemically Induced Renal Toxicity and Neoplasia in the Male Rat. Risk Assessment Forum, U.S. Environmental Protection Agency. EPA/625/3

- 91/019F (NTIS PB92143668). September, 1991. Available online at http://www.epa.gov/raf/publications/alpha2u

- globulin.htm as of November 25, 2014.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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35 The study author did not consider the increased incidence of microscopic changes in the adrenal glands of females at the high

- dose level (incidence of 4/5 at the high dose vs. incidence of 1/5 in controls), including vacuolation of the zona glomerulosa, to be adverse based on the minimal severity and the absence of other test item

- related findings in females. The reviewer disagrees with this conclusion and considers this finding to be potentially adverse. Therefore, these changes were considered in the ba sis for determining the LOAEL value for systemic toxicity in female rats.

The increased incidence s of microscopic changes in the spleen and liver of females at the high

dose level, including hemosiderin pigmentation in the spleen (incidence of 3/5 at the high dose vs. incidence of 0/5 in controls) and hematopoietic cell foci (incidence of 4/5 at the high dose vs. incidence of 1/5 in controls), were considered to be related to test substance exposure

. Therefore, these changes in the spleen and liver were c onsidered in the basis for determining the LOAEL value for systemic toxicity in female rats.

The increases in absolute and relative seminal vesicle weight at the high

- dose level were considered to be evidence of an adverse effect on reproduction 1 and are included in the basis of the LOAEL determination for reproductive toxicity in male rats. Likewise, the increases in absolute and relative uterus weight at the high

- dose level were considered to be evidence of an adverse effect on reproduction 1 and are inc luded in the basis of the LOAEL determination for reproductive toxicity in female rats.

CONCLUSIONS

T he LOAEL for systemic toxicity in male rats was 10 52 mg a.i./kg

- bw/day, based on histological effects, including increased severity of mononuclear inflammation in the heart, increased incidence and severity of myofiber degeneration in the heart

, and increased incidence of hyaline droplets in the kidney, and increased relative kidney weight . The NOAEL was 316
mg a.i./kg

- bw/day.

The LOAEL for system ic toxicity in female rats was 1052 mg a.i./kg

- bw/day, based on histological effects, including increased incidence of vacuolation of the zona glomerulosa in the adrenal glands, increased incidence of hemosiderin pigmentation in the spleen, and increased i ncidence of hematopoietic cell foci in the liver. The NOAEL was 316 mg a.i./kg

- bw/day.

The LOAEL for reproductive toxicity in male and female rats was 1052 mg a.i./kg

- bw/day, based on increased absolute and relative seminal vesicle weights and increased absolute and relative uterus weights. The NOAEL was 316 mg a.i./kg

- bw/day.

The NOAEL for developmental toxicity was 1052 mg a.i./kg

- bw/day, based on no adverse effects at the highest dose tested.

1 EPA. 1996. Guidelines for Reproductive Toxicity Risk Assessment. Risk Assessment Forum, U.S. Environmental Protection Agency. EPA/630/R

- 96/009. Published on October 31, 1996 in the Federal Register 61(212):56274

- 56322. Available online at http://www.epa.gov/ra f/publications/guidelines

- reproductive

- tox

- risk

assessment.htm as of November 25, 2014.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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36 STUDY RELIABILITY

[ 2 ] Reliable with restrictions. Concentrations of active ingredient in dosing formulations were determined by using a non

- specific analytical method (TOC measurement), rather than a method specific for CASRN 68187

- 76

- 8

. In addition, analytical determinations were not conducted on dosing formulations prepared during the study but, rather, were conducted only o n formulations that were prepared ( using methods similar to those used during the study ) after the study had ended

. Furthermore, a ccording to OECD TG 422 and TSCA 799.9365 (the guideline specified by the test rule for conducting a combined repeated

- dose toxicity study with the reproduction/developmental toxicity screening test), at least 5 animals/dose level should be evaluated for hematology parameters. The study report indicat es in the methods section that blood was collected for hematological analysis from 5 animals/sex/dose; however, data are only provided for 4 males at the high

- dose level for some of the hematological parameters required by the test guidelines, including re d blood cell count, hematocrit, mean corpuscular hemoglobin concentration, and platelet count. Additionally, data were only provided for 4 males at the low

dose level for white blood cell count. For females, data were only provided for 4 females at the

m id

- dose level for white blood cell count and for 4 females at the low

- dose level for prothrombin time and activated p artial t hromboplastin . The study author did not provide an explanation for these deviations from the test guideline requirements.

REFEREN CE

d e Raaf

- Beekhuijzen, M.E.W. 2014. Combined 28

- day repeated dose toxicity study with the reproduction/developmental toxicity screening test of castor oil, sulfated, sodium salt, 75% i n rats b y oral gavage. WIL Research Europe B.V.

, The Netherlands. Project No. 503342. Sponsor: Sulfated Castor Oil Consortium, Society of Chemical Manufacturers & Affiliates.

Text in quotes is taken directly from d e Raaf

- Beekhuijzen (2014).Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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37 D

- 6 . Genetic Toxicity

– Gene Mutation

Summary of endpoint: Adequate

Study 1: Mutagenicity study in Salmonella typhimurium and Escherichia coli (Verspeek

Rip, 2014)

Title: Evaluation of the mutagenic activity of castor oil, sulfated, sodium salt, 75% in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat)

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Castor oil, sulfated, sodium salt, CASRN 68187

- 76

- 8

Remarks: Composition 1 : 74.4% castor oil, sulfated, sodium salt (CASRN 68187

- 76

- 8) 2 , 24.3% water, and 1.25% sodium sulfate; Clear amber liquid; Batch No. R

- 10135

- 064; referred to as “castor oil, sulfated, sodium salt, 75%” in the study report.

METHOD

Method/guideline followed: OECD TG 471 (1997) “Bacterial Reverse Mutation Test” and Method B.13/14 of Commission Regulation (EC) No. 440/2008, “Mutagenicity: Reverse Mutation Test using Bacteria”

Test type: Bacterial Reverse Mutation Test

GLP compliant?: Yes

Year study performed: 2013

Test species: Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537; Escherichia coli strain WP2 uvrA; obtained from Trinova Biochem GmbH, Germany (master

1 This compositional information was obtained from the study report for WIL Research Project 503335 (van Doormalen

- Schaap, I. 201 4 . Determination of physico

- chemical properties of castor oil, sulfated, sodium salt, 75%. WIL Research Europe B.V., The Netherlands. Project No. 503335. Sponsor: Sulfated Castor Oil Consortium, Society of Chemical Manufacturers & Affiliates.) and is consistent with information provided in the study reports for the combined repeated

- dose toxicity with reproduction/developmental toxicity screening and aquatic toxicity studies, which were conducted with the same batch of this chemical (R

- 10135

- 064). A certificate of analysis provided in Appe ndix 6 of the gene mutation study report apparently provided incorrect information ( 70.7% castor oil, sulfated, sodium salt; 26.4% water; and 2.9% sodium sulfate). The study report notes that the purity was originally determined to be 70.7%, but was later determined to be 74.4%.

2 The percentage of CASRN 68187

- 76

- 8 in the test substance was calculated by subtracting % water and % sodium sulfate from 100%. (100%

- [24.3% + 1.25%] ≈ 74.4%)Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

- 76

- 8

38 cultures were obtained from Dr. Bruce N. Ames [ S. typhimurium ] and the Na tional Collections of Industrial and Marine Bacteria, Aberdeen, UK [ E. coli ]).

Metabolic activation: With and without rat liver S9

- mix (5% or 10% v/v)

Concentrations tested: ~ 0 (solvent control), 105, 350, 1050, 3500, and 5250 μg active ingredient (a.i.)/plate 1

Vehicle: Ethanol

Statistical methods used: No statistical analyses provided

Test method/conditions remarks: Preparation of Test Concentrations: Test concentrations were prepared in the solvent (ethanol) and used within 2 hours of preparation. “At concentrations of 10 mg/ml and higher [the test substance]... formed a hazy suspension in ethanol. At concentrations of 3.33 mg/ml and lower the test substanc e was dissolved in ethanol.” [Verspeek

- Rip 2014, p. 9]

Preliminary Range

- Finding Test Procedure: In a range

- finding test, S. typhimurium strain TA100 and E. coli strain WP2 uvrA were exposed to the test substance at concentrations of ~ 0 (solvent control ), 3, 10, 35, 105, 350, 1050, 3500, or 5250 μg a.i./plate 2 both in the absence and presence (S9

- mix, 5% v/v) of metabolic activation

. Plates were incubated at 37°C in the dark. Three plates per test concentration were used for the
test.

Definitive Mutat ion Assay Procedure: Experiment 1: In the first mutation assay, S. typhimurium strains TA98, TA1535, and TA1537 were exposed to the test substance at concentrations of ~ 0 (solvent control), 105, 350, 1050, 3500, and 5250 μg a.i./plate both in the absence and presence (S9

- mix, 5% v/v) of metabolic activation. Three plates per test concentration were used for the test.

Experiment 2: In a second independent mutation assay, S. typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2 uvrA were exposed to the same concentrations, in triplicate, as in the first mutation assay (Experiment 1) both in the absence and presence (S9

- mix, 10% v/v) of metabolic activation.

1 The concentrations of active ingredient (CASRN 68187

- 76

- 8) provide d in this review were calculated by the reviewer based on a test substance purity of 74.4%. In the study report, the concentrations provided for the mutation assays were 100, 333, 1000, 3330, and 5000 μg a.i. /plate , which were calculated by the study auth or using an incorrect purity of 70.7%. The study author states: “The purity of [the test substance]... originally determined by the Sponsor was 70.7%. All concentrations were therefore corrected by using a correction factor of 1.41, so that all doses menti oned in this report are based on the purity of the test substance. In the course of the development it has been identified that the test substance had a purity of 74.4%. Therefore, the actual concentrations were 5.2% higher than stated in the report. Th e conclusion of this report remains unchanged.” [Verspeek

- Rip 2014, p. 9]

2 In the study report, the concentrations provided for the range

- finding test were 3, 10, 33, 100, 333, 1000, 3330, and 5000 μg a.i. /plate. These concentrations were calculated by t he study author using an incorrect purity of 70.7%. The concentrations provided above were calculated by the reviewer based on a test substance purity of 74.4%.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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39 “Top agar in top agar tubes was melted by heating to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10 9 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in ethanol and either 0.5 ml S9

- mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non

- activation assays). The ingredients were mixed on a Vor tex and the content[s] of the top agar tube w[ere] poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies histidine independent (His + ) for Salmonella typhimurium bacteria and tryptophan independent (Trp + ) for Escherichia coli were counted.” [Verspeek

- Rip 2014, p. 12]

Colony Counting: Revertant colonies were counted in the range

- finding and definitive mutagenicity tests. “The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colon y counting were counted manually and evidence of test article precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.” [Verspeek

- Rip 2 014, p. 12]

Negative (Solvent) Control: Ethanol

Positive Controls: Without metabolic activation: TA98: 2

- Nitrofluorene (10 μg/plate) TA100: Methylmethanesulfonate (650 μg/plate) TA1535: Sodium azide (5 μg/plate) TA1537: ICR

- 191 (2.5 μg/plate) WP2 uvrA: 4

- Nitroquinoline N

- oxide (10 μg/plate)

With metabolic activation: TA1535: 2

- Aminoanthracene (2.5 μg/plate with 5% and 10% S9) TA1537: 2

- Aminoanthracene (2.5 μg/plate with 5% S9; 5 μg/plate with 10% S9) TA98: 2

- Aminoanthracene (1 μg/plate with 5% and 10% S 9) TA100: 2

- Aminoanthracene (1 μg/plate with 5% S9; 2.5 μg/plate with 10% S9) WP2 uvrA: 2

- Aminoanthracene (15 μg/plate with 5% and 10% S9)

All positive control substances were dissolved in dimethyl sulfoxide with the exception of sodium azide, which was d issolved in saline.

Metabolic Activation System: “Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that ha[d] been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and β

- naphthoflavone (100 mg/kg), except in the second experiment in tester strain WP 2 uvrA. In this part of the experiment, rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany...” [Verspeek

- Rip 2014, p. 11]Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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40 For the in

- house preparation of S9 (used with W P2 uvrA in the second experiment), rats were dosed orally for three consecutive days with phenobarbital (80 mg/kg

- bw) and β

- naphthoflavone (100 mg/kg

- bw). Twenty

- four hours after the final exposure, the livers of rats were collected, washed, minced in a b lender, and homogenized in 3 volumes of phosphate buffer. The homogenate was centrifuged at 9000 g and supernatants (S9 fraction) were transferred to sterile tubes and stored in liquid nitrogen for a maximum of 1 year.

“Before use, all S9 batches wer e characterized with the mutagens Benzo

- (a)

- pyrene... and 2

- aminoanthracene..., which require metabolic activation, in tester strain TA98 at concentrations of 5 μg/plate and 1 μg/plate, respectively.” [Verspeek

- Rip 2014, p. 12]

S9

- mix was prepared immediately before use and kept on ice. Each 10 mL of S9

- mix contained: 30 mg NADP and 15.2 mg glucose

- 6

- phosphate in 5.5 or 5.0 mL Milli

- Q water (first and second experiment, respectively); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl 2 solution; and 1 mL 0.33 M KCl solution. “To 9.5 ml of S9

- mix components 0.5 ml S9

- fraction was added (5% (v/v) S9

- fraction) to complete the S9

- mix in the first experiment and to 9.0 ml of S9

- mix components 1.0 ml S9

- fraction was added (10% (v/v) S9

- fraction) to complet e the S9

- mix in the second experiment.” [Verspeek

- Rip 2014, p. 12]

Acceptability of the Assay: “A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria: a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical [control] range for each tester strain... b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean... c) The selected dose range should include a clearly toxic concentratio n or should exhibit limited solubility as demonstrated by the preliminary toxicity range

- finding test or should extend to 5 mg/plate.” [Verspeek

- Rip 2014, p. 13]

Data Evaluation: “A test substance is considered negative (not mutagenic) in the test if: a) Th e total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP 2 uvrA is not greater than three (3) times the concurrent vehicle co ntrol. b) The negative response should be reproducible in at least one independently repeated experiment.” [Verspeek

- Rip 2014, p. 13]

“A test substance is considered positive (mutagenic) in the test if: a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP 2 uvrA is greater than three (3) times the concurrent vehicle control.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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41 b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment. The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.” [Verspeek

- Rip 2014, p. 13]

Cytotoxicity was determined based on a reduction in the bacterial background lawn and/or a biologically relevant decrease in the number of revertant colonies, compared to historical solvent controls.

RESULTS

Genotoxicity result: Negative with and wit hout metabolic activation

Cytotoxic concentration (if applicable): ≥ 3500 μg a.i./plate with and without metabolic activation

Results remarks: Mutagenicity Response: The test substance showed no indication of induction of mutations with or without met abolic activation in any of the five strains tested.

Precipitation and Toxicity of Test Substance: Preliminary Range

- Finding Test: Precipitation was observed at concentrations of 3500 and 5250 μg a.i./plate at the start and end of the incubation per iod. Cytotoxicity was only observed in strain TA100 in the absence and presence of S9

- mix at concentrations of 3500 and 5250 μg a.i./plate. No cytotoxicity was observed in strain WP2 uvrA.

Definitive Mutation Assay: Experiment 1: Test substance preci pitation was observed at the highest concentration (5250 μg a.i./plate) at the start of the incubation period, but not at the end of the incubation period. Cytotoxicity was observed in strains TA1535 and TA1537 at 5250 μg a.i./plate in the absence of S9

- m ix and in strain TA1537 at 5250 μg a.i./plate in the presence of S9

- mix. No cytotoxicity was observed in strain TA98 with or without S9

- mix or in strain TA1535 in the presence of S9

- mix.

Experiment 2: Precipitation of the test substance was observed at the start of the incubation period at 3500 and 5250 μg a.i./plate for all strains and at the end of the incubation period at 5250 μg a.i./plate for strains TA1535 and TA1537 (in the absence of S9

- mix) and in strain TA100 (in the absence and presence of S9

- mix). Cytotoxicity was observed in all strains except WP2 uvrA , both in the absence of S9

- mix (at ≥ 3500 μg a.i./plate for TA100, TA1535, and TA1537 and at 5250 μg a.i./plate for TA98) and in the presence of S9

- mix (at ≥ 3500 μg a.i./plate for TA100 and at 5250 μg a.i./plate for TA98 and TA1537).Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

- 76

- 8

42 Response of Controls: “The negative control values were within the laboratory historical control data ranges, except for TA1535 in the first experiment. However, since these values were just outside the limit of the range, the validity of the test was considered to be not affected. The strain

- specific positive control values were at least three times the concurrent vehicle control group mean indicating that the test conditions were adequate.” [Verspeek

- Rip 20 14, p. 6]

CONCLUSIONS

“Based on the results of this study it is concluded that [the test substance]... is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.” [Verspeek

- Rip 2014, p. 7]

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE

Verspeek

- Rip, C.M. 2014. Evaluation of the mutagenic activity of castor oil, sulfated, sodium salt, 75% in the Salmonella typhimurium reverse mutation assay and the Escherichia coli rev erse mutation assay (with independent repeat). WIL Research Europe B.V., The Netherlands. Project No. 503340. Sponsor: Sulfated Castor Oil Consortium, Society of Chemical Manufacturers & Affiliates.

Text in quotes is taken directly from Verspeek

- Rip (2 014).Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

- 76

- 8

43 D

- 7 . Genetic Toxicity

– Chromosomal Aberrations

Summary of endpoint: Adequate

Study 1: Chromosome aberration assay in human lymphocytes ( Buskens, 2014

)

Title: Evaluation of the ability of castor oil, sulfated, sodium salt, 75% to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiment)

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Castor oil, sulfated, sodium salt, CASRN 68187

- 76

- 8

Remarks: Composition 1 : 74.4% castor oil, sulfated, sodium salt (CASRN 68187

- 76

- 8) 2 , 24.3% water, and 1.25% sodium sulfate; Clear amber liquid; Batch No. R

- 10135

- 064; referred to as “castor oil, sulfated, sodium salt, 75%” in the study report.

METHOD

Method/guideline followed: OECD TG 473 “ In Vitro Mammalian Chromosome Abe rration Test (1997)” and Method B.10 of Commission Regulation (EC) No. 440/2008, “Mutagenicity

- In Vitro Mammalian Chromosome Aberration Test”

Test type: Mammalian Chromosome Aberration Test ( in vitro )

GLP compliant?: Yes

Year study performed: 2013

Test species: Human peripheral blood lymphocytes, cultured from adult male volunteers

Metabolic activation: With and without rat liver S9

- mix (1.8% v/v)

1 This compositional information was obtained from the study report for WIL Research Project 503335 (van Doormalen

- Schaap, I. 201 4 . Determination of physico

- chemical properties of castor oil, sulfated, sodium salt, 75%. WIL Research Europe B.V., The Neth erlands. Project No. 503335. Sponsor: Sulfated Castor Oil Consortium, Society of Chemical Manufacturers & Affiliates.) and is consistent with information provided in the study reports for the gene mutation, combined repeated

- dose toxicity with reproducti on/developmental toxicity screening, and aquatic toxicity studies, which were conducted with the same batch of this chemical (R

- 10135

- 064). A certificate of analysis provided in Appendix 6 of the chromosomal aberrations study report apparently provided in correct information ( 70.7% castor oil, sulfated, sodium salt; 26.4% water; and 2.9% sodium sulfate).

2 The percentage of CASRN 68187

- 76

- 8 in the test substance was calculated by subtracting % water and % sodium sulfate from 100%. (100%

- [24.3% + 1.25%] ≈ 74.4%)Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

- 76

- 8

44 Concentrations tested 1 : Experiment 1: ~105, 350, and 700 μg active ingredient (a.i.)/mL (with and without S9

- mix) Experiment 2: ~10, 31, 105, 210, 315, 420, and 525 μg a.i./mL (without S9

- mix); ~105, 315, and 525 μg a.i./mL (with S9

- mix)

Vehicle: Ethanol

Statistical methods used: “The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi

- square sta tistics...” [Buskens 2014, p. 13] A one

- tailed test was used and a p

- value < 0.05 was considered to indicate a statistically significant difference from the control group.

Test method/conditions remarks: Preparation of Test Concentrations: Due to the lim ited solubility of the test substance in the solvent (absolute ethanol), the study author considered the test substance to be suspended in solution at concentrations of ~40 mg/mL and above, but to be dissolved in solution at concentrations of ~20 mg/mL and below. Test concentrations were used within 2.5 hours after preparation. “The final concentration of the solvent in the culture medium was 0.5

- 1.0% (v/v).” [Buskens 2014, p. 9]

Preliminary Range

- Finding Test Procedure: Lymphocytes were exposed to the test substance at concentrations of ~105, 350, 1050, 3500, or 5250 μg a.i./mL 2 culture medium in the absence or presence of S9

- mix (1.8% v/v). The cells were incubated at 37oC for 3 or 24 hours (24

- h fixation time) or 48 hours (48

- h fixation time) in the absence of S9

- mix or for 3 hours (24

- h fixation time) in the presence of S9

- mix. Negative (solvent) controls were tested concurrently.

“After 3 h exposure to [the test substance]... in the absence or presence of S9

- mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g ). The supernatant was removed and cells were rinsed with 5 ml HBSS [Hank's Balanced Salt Solution]. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20

- 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9

- mix were not rinsed after exposure but were fixed immediately (24 h and 48 h fixation time).” [Buskens 2014, p. 11] Cytotoxicity was determined based on the mitotic index.

1 The concentrations of active ingredient (CASRN 68187

- 76

- 8) provided in this review were calculated by the reviewer based on a test substance purity of 74.4%. The study report provided the following concentrations, which were calculated by the st udy author using an incorrect purity of 70.7% (correction factor of 1.41): Experiment 1: 100, 333, and 666 μg a.i. / mL (with and without S9

- mix) Experiment 2: 10, 30, 100, 200, 300, 400, and 500 μg a.i./mL (without S9

- mix); 100, 300, and 500 μg/mL (with S9

- mix ).

2 In the study report, the concentrations provided for the range

- finding test were 100, 333, 1000, 3330, and 5000 μg a.i./mL . These concentrations were calculated by the study author using an incorrect purity of 70.7%. The concentrations provided above were calculated by the reviewer based on a test substance purity of 74.4%.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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45 “Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays [Experiments 1 and 2] considering the highest dose level was determined by the solub ility (3 h exposure time) or had an inhibition of the mitotic index of 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control.” [Buskens 2014, p. 11]

Definitive Chromosomal Aberrations Assay Procedure: In two independent assays, lymphocytes were exposed to the test substance in the absence or presence of metabolic activation (S9

- mix, 1.8% v/v). In Experiment 1, test substance concentrations of ~105, 350, and 700 μg a.i./mL c ulture medium were tested with and without S9

- mix. In Experiment 2, test substance concentrations of ~10, 31, 105, 210, 315, 420, and 525 μg a.i./mL culture medium were tested without S9

- mix and concentrations of ~105, 315, and 525 μg a.i./mL culture medi um were tested with S9

- mix . Two replicates were tested per concentration. Negative (solvent) and positive controls were tested concurrently. “The cytogenetic assay was carried out as described by Evans, 1984... with minor modifications.” [Buskens 2014, p. 11]

Experiment 1: The cells were incubated with the test substance at 37oC for 3 hours (24

- h fixation time) in the absence or presence of S9

- mix. “After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g ). T he supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20

- 22 h (24 h fixation time).” [Buskens 2014, p. 1 1]

“Based on the mitotic index of the dose range finding test and the first cytogenetic assay and on the solubility of [the test substance]... in the culture medium appropriate dose levels were selected for the second cytogenetic assay.” [Buskens 2014, pp. 11

- 12]

Experiment 2: The cells were incubated with the test substance at 37°C for 24 hours (24

- h fixation time) or 48 hours (48

- h fixation time) in the absence of S9

- mix or for 3 hours (48

- h fixation time) in the presence of S9

- mix. “After 3 h exposure, the cells exposed to [the test substance]... in the presence of S9

- mix were separated from the exposure medium by centrifugation (5 min, 365 g ). The supernatant was removed and the cells were rinsed once with 5 ml of HBSS and incubated in 5 ml culture mediu m for another 44

- 46 h (48 h fixation time).” [Buskens 2014, p. 12]

“The cells that were treated for 24 h and 48 h in the absence of S9

- mix were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time).” [Bus kens 2014, p. 12]

Cells and Culture Conditions: Lymphocytes were collected by venipuncture from healthy adult, non

- smoking male volunteers (21 to 36 years old) and had an average generation time of 12.4 to 13.5 hours. Immediately after blood collection, lymphocyte cultures were started in culture medium (RPMI 1640 medium supplemented with 20% v/v heat

- inactivated fetal calf serum, 2 mM L

- glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin, and 30 U/mL heparin). For the range

- finding test andFinal HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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46 cytogenetic assays, lymphocytes were cultured by adding 0.4 mL whole blood (treated with heparin) to 5 mL or 4.8 mL culture medium in the absence or presence of metabolic activation, respectively, and 0.1 mL (9 mg/mL) phytohemagglutinin for 48 ± 2 hours; thereafter, lymphocytes were exposed to the test substance.

“All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80

- 100% (actual range 48

- 90%), containing 5.0 ± 0.5% CO 2 in air, at a t emperature of 37.0 ± 1.0°C (actual range 35.6

- 37.6°C). Temperature and humidity were continuously monitored throughout the experiment. The CO 2 percentage was monitored once on each working day. Temporary deviations from the temperature (in the range

o f 35.6

- 36.0°C), humidity (with a maximum of 30%) and CO 2 percentage (with a maximum of 1%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 2 hour[s]. Based on laboratory historical data these deviations are considered not to affect the study integrity.” [Buskens 2014, p. 10]

Preparation of Chromosomes for Microscopic Examination: During the final 2.5

- 3 hours of the culture period, cell division was arrested by the addition of colc hicine (0.5 μg/mL). At the end of the culture period, the cells were isolated by centrifugation (5 min, 365 g ) and treated for 5 minutes in a hypotonic solution (0.56% w/v potassium chloride) at 37°C. The cells were then fixed with three changes of a 3:1 (v/v) mixture of methanol: acetic acid. At least two slides of the fixed cells were prepared per culture. Slides were allowed to dry and were then stained for 10

- 30 minutes with 5% (v/v) Giemsa solution in a buffer at pH 6.8. Slides were rinsed, allo wed to dry, embedded in a 1:10 mixture of xylene/pertex, and mounted with a coverslip.

Scoring of Slides: “The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%). At least three analysable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed from those cultures with an inhibition of the mitotic index of about 50% or above whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations. In case [the test substance]... was not cytotoxic and/or difficult to dissolve in aqueous solutions, the highest concentration analysed at the 3 h exposure time was determined by the solubility in the culture medium.” [Buskens 2014, p. 12]

“To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored... One hundred metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.” [Buskens 2014, pp. 12

- 13]

Negative (Solvent) Control: EthanolFinal HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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47 Positive Controls: Without metabolic activation (

- S9

- mix): Mitomycin C 3 hours (24

- h fixation) : 0.5 and 0.75 μg/mL 24 hours (24

- h fixation) : 0.2 and 0.3 μg/mL 48 hours (48

- h fixation) : 0.1 and 0.15 μg/mL With metabolic activation (+S9

- mix): Cyclophosphamide 3 hours (24

- h fixation): 10 μg/mL

All positive control substances were dissolved in HBSS, without calcium and magnesium.

Metabolic Activation System: “Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats..., which were obtained from Charles River (Sulzfeld, Ger many). Rat liver microsomal enzymes in the second cytogenetic assay were obtained from Trinova Biochem GmbH, Giessen, Germany and w[ere] prepared from male Sprague Dawley rats that ha[d] been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and β

- naphthoflavone (100 mg/kg).” [Buskens 2014, p. 10]

For the in

- house preparation of S9, rats were dosed orally for three consecutive days with phenobarbital (80 mg/kg

- bw) and β

- naphthoflavone (100 mg/kg

- bw). Twenty

- four hours after the fin al exposure, the livers of rats were collected, washed, minced in a blender, and homogenized in 3 volumes of phosphate buffer. The homogenate was centrifuged at 9000 g and supernatants (S9 fraction) were transferred to sterile tubes and stored in liquid n itrogen for a maximum of 1 year.

S9

- mix was prepared immediately before use and kept on ice. Each 1 mL of S9

- mix contained the following: 1.63 mg MgCl 2 ∙6H 2 O, 2.46 mg KCl, 1.7 mg glucose

- 6

- phosphate, 3.4 mg NADP, and 4 μmol HEPES. “To 0.5 ml S9

- mix compo nents 0.5 ml S9

- fraction was added (50% (v/v) S9

- fraction) to complete the S

- 9 mix. Metabolic activation was achieved by adding 0.2 ml S9

- mix to 5.3 ml of a lymphocyte culture (containing 4.8 ml culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) phytohaema gglutinin). The concentration of the S9

- fraction in the exposure medium was 1.8% (v/v).” [ Buskens 2014, p. 11 ]

“The S9 batch was characterized in a bacterial reverse mutation assay in Salmonella typhimurium tester strain TA98 with the mutagens Benzo

- (a)

- pyrene... and 2

- aminoanthracene... at concentrations of 5 μg/plate and 1 μg/plate, respectively. These mutagens require metabolic activation for exerting their mutagenic activity.” [ Buskens 2014, p. 10 ]

Acceptability of the Assay: “A chromosome aberration test is considered acceptable if it meets the following criteria: a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range... b) The positive control substances should p roduce a statistically significant (Chi

- square test,

one

- sided, p < 0.05) increase in the number of cells with chromosome aberrations. c) A homogeneous response between the replicate cultures is observed.Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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48 d) A possible precipitate present on the slides should no t interfere with the scoring of chromosome aberrations.” [Buskens 2014, p. 13]

Data Evaluation: “A test substance was considered positive (clastogenic) in the chromosome aberration test if: a) It induced a dose

- related statistically significant (Chi

- square t est, one

- sided, p < 0.05) increase in the number of cells with chromosome aberrations. b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a cle ar dose

- response relationship.” [Buskens 2014, p. 13]

“A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi

- square test, one

- sided, p < 0 .05) increase in the number of cells with chromosome aberrations.” [Buskens 2014, p. 13]

“The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.” [Buskens 2014, p. 13]

RESULTS

Genotoxicity res ult: Negative with and without metabolic activation

Cytotoxic concentration (if applicable): Based on > 50% inhibition of mitotic index compared to solvent control: ≥ 315 μg a.i./mL in 24

- or 48

- hour exposures without metabolic activation ≥ 1050 μg a.i./mL in 3

- hour exposure without metabolic activation ≥ 3500 μg a.i./mL in 3

- hour exposure with metabolic activation

Results remarks: Preliminary Range

- Finding Test: Precipitation of the test substance was observed at concentrations ≥ 1050 μg a.i./mL. For the 3

- hour exposure (24

- h fixation) in the absence of metabolic activation, the mitotic index was 71%, 71%, 44%, 0%, and 0% of the control at 105, 350, 1050, 3500, and 5250 μg a.i./mL, respectively. For the 24

- hour exposure (24

- h fixation) in the absence of metabolic activation, the mitotic index was 80%, 48%, 0%, 0%, and 0% of the control at 105, 350, 1050, 3500, and 5250 μg a.i./mL, respectively. For the 48

- hour exposure (48

- h fixation) in the absence of metabolic activation, th e mitotic index was 56%, 42%, 0%, 0%, and 0% of the control at 105, 350, 1050, 3500, and 5250 μg a.i./mL, respectively. For the 3

- hour exposure (24

- h fixation) in the presence of metabolic activation, the mitotic index was 79%, 79%, 59%, 0%, and 0% of the control at 105, 350, 1050, 3500, and 5250 μg a.i./mL, respectively.

Definitive Chromosomal Aberrations Assay: Experiment 1: The test substance precipitated at a concentration of 700 μg a.i./mL both with and without metabolic activation. In the absence of metabolic activation (3

- h exposure, 24

- h fixation), the mitotic index was 91%, 95%, and 98% of the control at 105, 350, and 700 μg a.i./mL, respectively. In the presence of metabolic activation (3

- hour exposure, 24

- h fixation),Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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49 the mitotic index was 9 1%, 84%, and 75% of the control at 105, 350, and 700 μg a.i./mL, respectively.

“All dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9

- mix, [the test substance]... did not induce a statistically signif icant or biologically relevant increase in the number of cells with chromosome aberrations...” [Buskens 2014, p. 14]

“Both in the absence and presence of S9

- mix, [the test substance]... did not increase the number of polyploid cells and cells with endoredupli cated chromosomes.” [Buskens 2014, p. 14]

Experiment 2: The test substance precipitated at a concentration of 525 μg a.i./mL both with metabolic activation (3

- h exposure) and without metabolic activation (24

- h and 48

- h exposures). For the 24

- hour exposur e (24

- h fixation) in the absence of metabolic activation, the mitotic index was 100%, 93%, 95%, 89%, 41%, 26%, and 0% of the control at 10, 31, 105, 210, 315, 420, and 525 μg a.i./mL, respectively. For the 48

- hour exposure (48

- h fixation) in the absence o f metabolic activation, the mitotic index was 83%, 67%, 70%, 54%, 16%, 0%, and 0% of the control at 10, 31, 105, 210, 315, 420, and 525 μg a.i./mL, respectively. In the presence of metabolic activation (3

- h exposure, 48

- h fixation), the mitotic index was 66%, 57%, and 68% of the control at 105, 315, and 525 μg a.i./mL, respectively. The following concentrations were selected for scoring of chromosome aberrations:

Without S9

- mix: 10, 210, and 315 μg a.i./mL (24

- h exposure time, 24

- h fixation time) 10, 1 05, and 210 μg a.i./mL (48

- h exposure time, 48

- h fixation time) With S9

- mix: 105, 315, and 525 μg a.i./mL (3

- h exposure time, 48

- h fixation time)

“Both in the absence and presence of S9

- mix, [the test substance]... did not induce a statistically significan t or biologically relevant increase in the number of cells with chromosome aberrations...” [Buskens 2014, p. 15]

“Both in the absence and presence of S9

- mix, [the test substance]... did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.” [Buskens 2014, p. 15]

Control Responses: “The number of cells with chromosome aberrations foun d in the solvent control cultures was within the laboratory historical control data range... The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control range... The p ositive control chemicals... both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9

- mix) functioned properly.” [ Bus kens 2014, pp. 15

- 16]Final HPV 2 Test Rule Data Adequacy Review (November 2014) CAS RN 68187

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50 CONCLUSIONS

The test substance did not induce chromosomal aberrations in human lymphocytes under the experimental conditions.

STUDY RELIABILITY

[2] Reliable with restrictions. The concentrations reported by the study author were based on a test substance purity of 70.7% active ingredient (CASRN 68187

- 76

- 8) as shown in the certificate of analysis provided in Appendix 6 of the chromosomal aberrations study report; however, WIL Project 503335 showed that the purity of Batch No. R

- 10 135

- 064 was 74.4%. The concentrations provided in the review above have been corrected based on the purity of 74.4%.

REFERENCE

Buskens, C.A.F. 2014. Evaluation of the ability of castor oil, sulfated, sodium salt, 75% to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiment). WIL Research Europe B.V., The Netherlands. Project No. 503341. Sponsor: Sulfated Cast or Oil Consortium, Society of Chemical Manufacturers & Affiliates.

Text in quotes is taken directly from Buskens (2014)

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51 E . DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information Available Acceptable Data Gap

Aquatic Effects Acute Toxicity to Fish Yes Yes No Acute Toxicity to Aquatic Invertebrates Yes Yes No Toxicity to Aquatic Plants Yes Yes No Health Effects

Acute Toxicity Yes Yes No Repeated

- Dose Toxicity Yes Yes No Gene Mutation Yes Yes No Chromosomal Aberrations Yes Yes No Reproductive Toxicity Yes Yes No Developmental Toxicity Yes Yes No